PARENTAL RNAI SUPPRESSION OF CHROMATIN REMODELING GENES TO CONTROL COLEOPTERAN PESTS

Abstract

This disclosure concerns nucleic acid molecules and methods of use thereof for control of coleopteran pests through RNA interference-mediated inhibition of target coding and transcribed non-coding sequences in coleopteran pests. The disclosure also concerns methods for making transgenic plants that express nucleic acid molecules useful for the control of coleopteran pests, and the plant cells and plants obtained thereby.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62/092,768, filed Dec. 16, 2014, and U.S. Provisional Patent Application No. 62/170,076, filed Jun. 2, 2015, the disclosures of each of which are hereby incorporated herein in their entirety by this reference.

FIELD OF THE DISCLOSURE

[0002] The present invention relates generally to genetic control of plant damage caused by coleopteran pests. In particular embodiments, the present disclosure relates to identification of target coding and non-coding polynucleotides, and the use of recombinant DNA technologies for post-transcriptionally repressing or inhibiting expression of target coding and non-coding polynucleotides in the cells of a coleopteran pest to provide a plant protective effect.

BACKGROUND

[0003] The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is one of the most devastating corn rootworm species in North America and is a particular concern in corn-growing areas of the Midwestern United States. The northern corn rootworm (NCR), Diabrotica barberi Smith and Lawrence, is a closely-related species that co-inhabits much of the same range as WCR. There are several other related subspecies of Diabrotica that are significant pests in the Americas: the Mexican corn rootworm (MCR), D. virgifera zeae Krysan and Smith; the southern corn rootworm (SCR), D. undecimpunctata howardi Barber; D. balteata LeConte; D. undecimpunctata tenella; D. speciosa Germar; and D. u. undecimpunctata Mannerheim. The United States Department of Agriculture has estimated that corn rootworms cause $1 billion in lost revenue each year, including $800 million in yield loss and $200 million in treatment costs.

[0004] Both WCR and NCR are deposited in the soil as eggs during the summer. The insects remain in the egg stage throughout the winter. The eggs are oblong, white, and less than 0.004 inches in length. The larvae hatch in late May or early June, with the precise timing of egg hatching varying from year to year due to temperature differences and location. The newly hatched larvae are white worms that are less than 0.125 inches in length. Once hatched, the larvae begin to feed on corn roots. Corn rootworms go through three larval instars. After feeding for several weeks, the larvae molt into the pupal stage. They pupate in the soil, and then they emerge from the soil as adults in July and August. Adult rootworms are about 0.25 inches in length.

[0005] Corn rootworm larvae complete development on corn and several other species of grasses. Larvae reared on yellow foxtail emerge later and have a smaller head capsule size as adults compared to larvae reared on corn. Ellsbury et al. (2005) Environ. Entomol. 34:627-34. WCR adults feed on corn silk, pollen, and kernels on exposed ear tips. Adults will quickly shift to preferred silks and pollen when they become available. NCR adults also feed on reproductive tissues of the corn plant. WCR females typically mate once. Branson et al. (1977) Ann. Entom. Soc. America 70(4):506-8.

[0006] Most of the rootworm damage in corn is caused by larval feeding. Newly hatched rootworms initially feed on fine corn root hairs and burrow into root tips. As the larvae grow larger, they feed on and burrow into primary roots. When corn rootworms are abundant, larval feeding often results in the pruning of roots all the way to the base of the corn stalk. Severe root injury interferes with the roots' ability to transport water and nutrients into the plant, reduces plant growth, and results in reduced grain production, thereby often drastically reducing overall yield. Severe root injury also often results in lodging of corn plants, which makes harvest more difficult and further decreases yield. Furthermore, feeding by adults on the corn reproductive tissues can result in pruning of silks at the ear tip. If this "silk clipping" is severe enough during pollen shed, pollination may be disrupted.

[0007] Control of corn rootworms may be attempted by crop rotation, chemical insecticides, biopesticides (e.g., the spore-forming gram-positive bacterium, Bacillus thuringiensis (Bt)), transgenic plants that express Bt toxins, or a combination thereof. Crop rotation suffers from the disadvantage of placing restrictions upon the use of farmland. Moreover, oviposition of some rootworm species may occur in crop fields other than corn or extended diapause results in egg hatching over multiple years, thereby mitigating the effectiveness of crop rotation practiced with corn and other crops.

[0008] Chemical insecticides are the most heavily relied upon strategy for achieving corn rootworm control. Chemical insecticide use, though, is an imperfect corn rootworm control strategy; over $1 billion may be lost in the United States each year due to corn rootworm when the costs of the chemical insecticides are added to the costs of yield loss from the rootworm damage that may occur despite the use of the insecticides. High populations of larvae, heavy rains, and improper application of the insecticide(s) may all result in inadequate corn rootworm control. Furthermore, the continual use of insecticides may select for insecticide-resistant rootworm strains, as well as raise significant environmental concerns due to their toxicity to non-target species.

[0009] RNA interference (RNAi) is a process utilizing endogenous cellular pathways, whereby an interfering RNA (iRNA) molecule (e.g., a double stranded RNA (dsRNA) molecule) that is specific for all, or any portion of adequate size, of a target gene results in the degradation of the mRNA encoded thereby. In recent years, RNAi has been used to perform gene "knockdown" in a number of species and experimental systems; for example, Caenorhabditis elegans, plants, insect embryos, and cells in tissue culture. See, e.g., Fire et al. (1998) Nature 391:806-11; Martinez et al. (2002) Cell 110:563-74; McManus and Sharp (2002) Nature Rev. Genetics 3:737-47.

[0010] RNAi accomplishes degradation of mRNA through an endogenous pathway including the DICER protein complex. DICER cleaves long dsRNA molecules into short fragments of approximately 20 nucleotides, termed small interfering RNA (siRNA). The siRNA is unwound into two single-stranded RNAs: the passenger strand and the guide strand. The passenger strand is degraded, and the guide strand is incorporated into the RNA-induced silencing complex (RISC). Micro ribonucleic acids (miRNAs) are structurally very similar molecules that are cleaved from precursor molecules containing a polynucleotide "loop" connecting the hybridized passenger and guide strands, and they may be similarly incorporated into RISC. Post-transcriptional gene silencing occurs when the guide strand binds specifically to a complementary mRNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout some eukaryotic organisms despite initially limited concentrations of siRNA and/or miRNA, such as plants, nematodes, and some insects.

[0011] Only transcripts complementary to the siRNA and/or miRNA are cleaved and degraded, and thus the knock-down of mRNA expression is sequence-specific. In plants, several functional groups of DICER genes exist. The gene silencing effect of RNAi persists for days and, under experimental conditions, can lead to a decline in abundance of the targeted transcript of 90% or more, with consequent reduction in levels of the corresponding protein. In insects, there are at least two DICER genes, where DICER1 facilitates miRNA-directed degradation by Argonaute1. Lee et al. (2004) Cell 117(1):69-81. DICER2 facilitates siRNA-directed degradation by Argonaute2.

[0012] U.S. Pat. No. 7,612,194 and U.S. Patent Publication Nos. 2007/0050860, 2010/0192265, and 2011/0154545 disclose a library of 9112 expressed sequence tag (EST) sequences isolated from D. v. virgifera LeConte pupae. It is suggested in U.S. Pat. No. 7,612,194 and U.S. Patent Publication No. 2007/0050860 to operably link to a promoter a nucleic acid molecule that is complementary to one of several particular partial sequences of D. v. virgifera vacuolar-type Ht ATPase (V-ATPase) disclosed therein for the expression of anti-sense RNA in plant cells. U.S. Patent Publication No. 2010/0192265 suggests operably linking a promoter to a nucleic acid molecule that is complementary to a particular partial sequence of a D. v. virgifera gene of unknown and undisclosed function (the partial sequence is stated to be 58% identical to C56C10.3 gene product in C. elegans) for the expression of anti-sense RNA in plant cells. U.S. Patent Publication No. 2011/0154545 suggests operably linking a promoter to a nucleic acid molecule that is complementary to two particular partial sequences of D. v. virgifera coatomer beta subunit genes for the expression of anti-sense RNA in plant cells. Further, U.S. Pat. No. 7,943,819 discloses a library of 906 expressed sequence tag (EST) sequences isolated from D. v. virgifera LeConte larvae, pupae, and dissected midguts, and suggests operably linking a promoter to a nucleic acid molecule that is complementary to a particular partial sequence of a D. v. virgifera charged multivesicular body protein 4b gene for the expression of double-stranded RNA in plant cells.

[0013] No further suggestion is provided in U.S. Pat. No. 7,612,194, and U.S. Patent Publication Nos. 2007/0050860, 2010/0192265, and 2011/0154545 to use any particular sequence of the more than nine thousand sequences listed therein for RNA interference, other than the several particular partial sequences of V-ATPase and the particular partial sequences of genes of unknown function. Furthermore, none of U.S. Pat. No. 7,612,194, and U.S. Patent Publication Nos. 2007/0050860 and 2010/0192265, and 2011/0154545 provides any guidance as to which other of the over nine thousand sequences provided would be lethal, or even otherwise useful, in species of corn rootworm when used as dsRNA or siRNA. U.S. Pat. No. 7,943,819 provides no suggestion to use any particular sequence of the more than nine hundred sequences listed therein for RNA interference, other than the particular partial sequence of a charged multivesicular body protein 4b gene. Furthermore, U.S. Pat. No. 7,943,819 provides no guidance as to which other of the over nine hundred sequences provided would be lethal, or even otherwise useful, in species of corn rootworm when used as dsRNA or siRNA. U.S. Patent Application Publication No. U.S. 2013/040173 and PCT Application Publication No. WO 2013/169923 describes the use of a sequence derived from a Diabrotica virgifera Snf7 gene for RNA interference in maize. (Also disclosed in Bolognesi et al. (2012) PLoS ONE 7(10): e47534. doi:10.1371/journal.pone.0047534).

[0014] The overwhelming majority of sequences complementary to corn rootworm DNAs (such as the foregoing) do not provide a plant protective effect from species of corn rootworm when used as dsRNA or siRNA. For example, Baum et al. (2007) Nature Biotechnology 25:1322-1326, describe the effects of inhibiting several WCR gene targets by RNAi. These authors reported that 8 of the 26 target genes they tested were not able to provide experimentally significant coleopteran pest mortality at a very high iRNA (e.g., dsRNA) concentration of more than 520 ng/cm.sup.2.

[0015] The authors of U.S. Pat. No. 7,612,194 and U.S. Patent Publication No. 2007/0050860 made the first report of in planta RNAi in corn plants targeting the western corn rootworm. Baum et al. (2007) Nat. Biotechnol. 25(11):1322-6. These authors describe a high-throughput in vivo dietary RNAi system to screen potential target genes for developing transgenic RNAi maize. Of an initial gene pool of 290 targets, only 14 exhibited larval control potential. One of the most effective double-stranded RNAs (dsRNA) targeted a gene encoding vacuolar ATPase subunit A (V-ATPase), resulting in a rapid suppression of corresponding endogenous mRNA and triggering a specific RNAi response with low concentrations of dsRNA. Thus, these authors documented for the first time the potential for in planta RNAi as a possible pest management tool, while simultaneously demonstrating that effective targets could not be accurately identified a priori, even from a relatively small set of candidate genes.

[0016] Another potential application of RNAi for insect control involves parental RNAi (pRNAi). First described in Caenorhabditis elegans, pRNAi was identified by injection of dsRNA into the body cavity (or application of dsRNA via ingestion), causing gene inactivity in offspring embryos. Fire et al. (1998), supra; Timmons and Fire (1998) Nature 395(6705):854. A similar process was described in the model coleopteran, Tribolium castaneum, whereby female pupae injected with dsRNA corresponding to three unique genes that control segmentation during embryonic development resulted in knock down of zygotic genes in offspring embryos. Bucher et al. (2002) Curr. Biol. 12(3):R85-6. Nearly all of the offspring larvae in this study displayed gene-specific phenotypes one week after injection. Although injection of dsRNA for functional genomics studies has been successful in a variety of insects, uptake of dsRNA from the gut environment through oral exposure to dsRNA and subsequent down-regulation of essential genes is required in order for RNAi to be effective as a pest management tool. Auer and Frederick (2009) Trends Biotechnol. 27(11):644-51.

[0017] Parental RNAi has been used to describe the function of embryonic genes in a number of insect species, including the springtail, Orchesella cincta (Konopova and Akam (2014) Evodevo 5(1):2); the brown plant hopper, Nilaparvata lugens; the sawfly, Athalia rosae (Yoshiyama et al. (2013) J. Insect Physiol. 59(4):400-7); the German cockroach, Blattella germanica (Piulachs et al. (2010) Insect Biochem. Mol. Biol. 40:468-75); and the pea aphid, Acyrthosiphon pisum (Mao et al. (2013) Arch Insect Biochem Physiol 84(4):209-21). The pRNAi response in all these instances was achieved by injection of dsRNA into the hemocoel of the parental female.

SUMMARY OF THE DISCLOSURE

[0018] Disclosed herein are nucleic acid molecules (e.g., target genes, DNAs, dsRNAs, siRNAs, shRNAs, miRNAs, and hpRNAs), and methods of use thereof, for the control of coleopteran pests, including, for example, D. v. virgifera LeConte (western corn rootworm, "WCR"); D. barberi Smith and Lawrence (northern corn rootworm, "NCR"); D. u. howardi Barber (southern corn rootworm, "SCR"); D. v. zeae Krysan and Smith (Mexican corn rootworm, "MCR"); D. balteata LeConte; D. u. tenella; D. speciosa Germar; and D. u. undecimpunctata Mannerheim. In particular examples, exemplary nucleic acid molecules are disclosed that may be homologous to at least a portion of one or more native nucleic acids in a coleopteran pest. In some embodiments, coleopteran pests are controlled by reducing the capacity of an existing generation to produce a subsequent generation of the pest. In certain examples, delivery of the nucleic acid molecules to coleopteran pests does not result in significant mortality to the pests, but reduces the number of viable progeny produced therefrom.

[0019] In these and further examples, the native nucleic acid may be a target gene, the product of which may be, for example and without limitation: involved in a metabolic process; involved in a reproductive process; and/or involved in embryonic and/or larval development. In some examples, post-transcriptional inhibition of the expression of a target gene by a nucleic acid molecule comprising a polynucleotide homologous thereto may result in reduced viability, growth, and/or reproduction of the coleopteran pest. In specific examples, a chromatin remodeling gene is selected as a target gene for post-transcriptional silencing. In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica brahma (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7). In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica mi-2 (SEQ ID NO:79 and SEQ ID NO:164). In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica iswi-1 (SEQ ID NO:81 and SEQ ID NO:165). In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica chd1 (SEQ ID NO:83). In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica iswi-2 (SEQ ID NO:85 and SEQ ID NO:166). In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica iswi30 (SEQ ID NO:87). In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica ino80 (SEQ ID NO:89). In particular examples, a target gene useful for post-transcriptional inhibition is the novel chromatin remodeling gene referred to herein as Diabrotica domino (SEQ ID NO:91).

[0020] An isolated nucleic acid molecule comprising the polynucleotide of SEQ ID NO:1; the complement of SEQ ID NO:1; SEQ ID NO:3; the complement of SEQ ID NO:3; SEQ ID NO:5; the complement of SEQ ID NO:5; SEQ ID NO:7; the complement of SEQ ID NO:7; SEQ ID NO:79; the complement of SEQ ID NO:79; SEQ ID NO:81; the complement of SEQ ID NO:81; SEQ ID NO:83; the complement of SEQ ID NO:83; SEQ ID NO:85; the complement of SEQ ID NO:85; SEQ ID NO:87; the complement of SEQ ID NO:87; SEQ ID NO:89; the complement of SEQ ID NO:89; SEQ ID NO:91; the complement of SEQ ID NO:91; SEQ ID NO:164; the complement of SEQ ID NO:164; SEQ ID NO:165; the complement of SEQ ID NO:165; SEQ ID NO:166; the complement of SEQ ID NO:166; and/or fragments of any of the foregoing (e.g., SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NOs:101-106) is therefore disclosed herein.

[0021] Also disclosed are nucleic acid molecules comprising a polynucleotide that encodes a polypeptide that is at least about 85% identical to an amino acid sequence within a target chromatin remodeling gene product (for example, the product of a brahma, mi-2, iswi-1, chd1, iswi-2, iswi30, ino80, or domino gene). For example, a nucleic acid molecule may comprise a polynucleotide encoding a polypeptide that is at least 85% identical to a polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:9 (Diabrotica BRAHMA); an amino acid sequence within a product of Diabrotica brahma (e.g., SEQ ID NO:9); SEQ ID NO:80 (Diabrotica MI-2); an amino acid sequence within a product of Diabrotica mi-2; SEQ ID NO:82 (Diabrotica ISWI-1); an amino acid sequence within a product of Diabrotica iswi-1; SEQ ID NO:84 (Diabrotica CHD1); an amino acid sequence within a product of Diabrotica chd1; SEQ ID NO:86 (Diabrotica ISWI-2); an amino acid sequence within a product of Diabrotica iswi-2; SEQ ID NO:88 (Diabrotica ISWI30); an amino acid sequence within a product of Diabrotica iswi30; SEQ ID NO:90 (Diabrotica IN080); an amino acid sequence within a product of Diabrotica ino80; SEQ ID NO:92 (Diabrotica DOMINO); and an amino acid sequence within a product of Diabrotica domino. Further disclosed are nucleic acid molecules comprising a polynucleotide that is the reverse complement of a polynucleotide that encodes a polypeptide at least 85% identical to an amino acid sequence within a target chromatin remodeling gene product.

[0022] Also disclosed are cDNA polynucleotides that may be used for the production of iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecules that are complementary to all or part of a coleopteran pest target gene, for example, a chromatin remodeling gene. In particular embodiments, dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be produced in vitro or in vivo by a genetically-modified organism, such as a plant or bacterium. In particular examples, cDNA molecules are disclosed that may be used to produce iRNA molecules that are complementary to all or part of mRNA transcribed from Diabrotica brahma (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7); Diabrotica mi-2 (SEQ ID NO:79 and SEQ ID NO:164); Diabrotica iswi-1 (SEQ ID NO:81 and SEQ ID NO:165); Diabrotica chd1 (SEQ ID NO:83); Diabrotica iswi-2 (SEQ ID NO:85 and SEQ ID NO:166); Diabrotica iswi30 (SEQ ID NO:87); Diabrotica ino80 (SEQ ID NO:89); and/or Diabrotica domino (SEQ ID NO:91).

[0023] Further disclosed are means for inhibiting expression of an essential gene in a coleopteran pest, and means for protecting a plant from a coleopteran pest. A means for inhibiting expression of an essential gene in a coleopteran pest is a single- or double-stranded RNA molecule consisting of a polynucleotide selected from the group consisting of SEQ ID NO:141 and SEQ ID NO:142; and the complements thereof. Functional equivalents of means for inhibiting expression of an essential gene in a coleopteran pest include single- or double-stranded RNA molecules that are substantially homologous to all or part of mRNA transcribed from a WCR gene encoding a ATP-dependent remodeling enzyme, such as mRNAs comprising SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166. A means for protecting a plant from a coleopteran pest is a DNA molecule comprising a polynucleotide encoding a means for inhibiting expression of an essential gene in a coleopteran pest operably linked to a promoter, wherein the DNA molecule is capable of being integrated into the genome of a maize plant.

[0024] Disclosed are methods for controlling a population of a coleopteran pest, comprising providing to a coleopteran pest an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecule that functions upon being taken up by the pest to inhibit a biological function within the pest, wherein the iRNA molecule comprises all or part of (e.g., at least 15 contiguous nucleotides of) a polynucleotide selected from the group consisting of: SEQ ID NO:135; the complement of SEQ ID NO:135; SEQ ID NO:136; the complement of SEQ ID NO:136; SEQ ID NO:137; the complement of SEQ ID NO:137; SEQ ID NO:138; the complement of SEQ ID NO:138; SEQ ID NO:139; the complement of SEQ ID NO:139; SEQ ID NO:140; the complement of SEQ ID NO:140; SEQ ID NO:141; the complement of SEQ ID NO:141; SEQ ID NO:142; the complement of SEQ ID NO:142; SEQ ID NO:143; the complement of SEQ ID NO:143; SEQ ID NO:144; the complement of SEQ ID NO:144; SEQ ID NO:145; the complement of SEQ ID NO:145; SEQ ID NO:146; the complement of SEQ ID NO:146; SEQ ID NO:147; the complement of SEQ ID NO:147; SEQ ID NO:148; the complement of SEQ ID NO:148; SEQ ID NO:149; the complement of SEQ ID NO:149; SEQ ID NO:150; the complement of SEQ ID NO:150; SEQ ID NO:151; the complement of SEQ ID NO:151; SEQ ID NO:152; the complement of SEQ ID NO:152; SEQ ID NO:153; the complement of SEQ ID NO:153; SEQ ID NO:154; the complement of SEQ ID NO:154; SEQ ID NO:155; the complement of SEQ ID NO:155; SEQ ID NO:156; the complement of SEQ ID NO:156; SEQ ID NO:157; the complement of SEQ ID NO:157; SEQ ID NO:158; the complement of SEQ ID NO:158; SEQ ID NO:159 the complement of SEQ ID NO:159; SEQ ID NO:160; the complement of SEQ ID NO:160; SEQ ID NO:161; the complement of SEQ ID NO:161; SEQ ID NO:162; the complement of SEQ ID NO:162; SEQ ID NO:163; the complement of SEQ ID NO:163; SEQ ID NO:167; the complement of SEQ ID NO:167; SEQ ID NO:168; the complement of SEQ ID NO:168; SEQ ID NO:169; the complement of SEQ ID NO:169; a polynucleotide that hybridizes to a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising all or part of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166; and the complement of a polynucleotide that hybridizes to a native coding polynucleotide of a Diabrotica organism comprising all or part of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166.

[0025] Also disclosed herein are methods wherein dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be provided to a coleopteran pest in a diet-based assay, or in genetically-modified plant cells expressing the dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs. In these and further examples, the dsRNAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be ingested by a coleopteran pest. Ingestion of dsRNAs, siRNA, shRNAs, miRNAs, and/or hpRNAs of the invention may then result in RNAi in the pest, which in turn may result in silencing of a gene essential for a metabolic process; a reproductive process; and/or larval development. Thus, methods are disclosed wherein nucleic acid molecules comprising exemplary polynucleotide(s) useful for parental control of coleopteran pests are provided to a coleopteran pest. In particular examples, the coleopteran pest controlled by use of nucleic acid molecules of the invention may be WCR, SCR, NCR, MCR, D. balteata, D. undecimpunctata tenella, D. speciosa, or D. u. undecimpunctata. In some examples, delivery of the nucleic acid molecules to coleopteran pests does not result in significant mortality to the pests, but reduces the number of viable progeny produced therefrom. In some examples, delivery of the nucleic acid molecules to a coleopteran pest results in significant mortality to the pests, and also reduces the number of viable progeny produced therefrom.

[0026] The foregoing and other features will become more apparent from the following Detailed Description of several embodiments, which proceeds with reference to the accompanying Figures.

BRIEF DESCRIPTION OF THE FIGURES

[0027] FIG. 1A includes a depiction of the strategy used to generate dsRNA from a single transcription template with a single pair of primers, and FIG. 1B includes a depiction of the strategy used to generate dsRNA from two transcription templates.

[0028] FIG. 2 includes a phylogenetic tree representation of ClustalW alignment of ATP-dependent remodelers from Diabrotica virgifera virgifera (WCR), Euschistus heros (BSB), Drosophila, human BRAHMA and S. cerevisiae SNF2. The alignment was performed in Clustal X 2.1, a windows interface for the ClustalW multiple sequence alignment program. The phylogram was rendered in TreeView (Win16) 1.40 software.

[0029] FIGS. 3A-3F include representations of the domain architecture of ATP-dependent chromatin remodeling enzymes of Diabrotica virgifera virgifera (WCR), Euschistus heros (BSB) and Drosophila melanogaster (Dme). The graphical representation is of Pfam output, with domains shaded and labeled. The proteins are organized by families and aligned with respect to SNF2 domain. "Squiggly" lines represent truncation/discontinuity for representation purposes. The protein families include SWI2NSF2: brahma, FIG. 3A; ISWI: Iswi, FIG. 3B; CHD subfamily I: Chd1; FIG. 3C; CHD subfamily II: Mi-2; FIG. 3D; CHD subfamily III: kismet, FIG. 3E; Other SNF2-containing proteins, FIG. 3F.

[0030] FIG. 4 includes an alignment of WCR dsRNA (query) to iswi-1 transcript (subject), showing 93% sequence identity. The alignment was performed using BLAST-2-Seq.

[0031] FIG. 5A includes representative photographs of WCR eggs dissected to examine embryonic development under different experimental conditions. Eggs that were oviposited by females treated with GFP dsRNA show normal development. Eggs oviposited by females treated with brahma, iswi-30, and mi-2 dsRNA (FIG. 5B-5D) show no embryonic or larval development.

[0032] FIG. 6A includes a summary of data showing the relative expression of brahma in eggs collected from WCR females exposed to dsRNA in a treated artificial diet, relative to GFP and water controls. Also shown is the relative expression of brahma (FIG. 6B), mi-2 (FIG. 6C), and iswi30 (FIG. 6D) in adult females exposed to dsRNA in a treated artificial diet, relative to GFP and water controls. Bars followed by the same letter are not significantly different (P>0.05; N=3 biological replications of 10 eggs, larvae, or adults; replication with 2 technical replications/sample).

[0033] FIG. 7 includes a summary of modeling data showing the relative magnitude of the pRNAi effect on female WCR adults emerging from a "refuge patch" (i.e., that did not express insecticidal iRNAs or recombinant proteins in a transgenic crop) on the rate of increase in allele frequencies for resistance to an insecticidal protein (R) and RNAi (Y) when non-refuge plants express the insecticidal protein and parental active iRNA.

[0034] FIG. 8 includes a summary of modeling data showing the relative magnitude of the pRNAi effect on female WCR adults emerging from a "refuge patch" (i.e., that did not express insecticidal iRNAs or recombinant proteins in a transgenic crop of plants comprising corn rootworm larval-active interfering dsRNA in combination with the corn rootworm-active insecticidal protein in the transgenic crop) on the rate of increase in allele frequencies for resistance to an insecticidal protein (R) and RNAi (Y) when non-refuge plants express the insecticidal protein and both larval active and parental active iRNA molecules.

[0035] FIG. 9A illustrates a summary of data showing the number of eggs recovered per female and FIG. 9B illustrated results of the percent total larvae that hatched, respectively, after exposure to 2 .mu.g of brahma or GFP dsRNA six times before mating, immediately after mating, and six days after mating. Comparisons performed with Dunnett's test, * indicates significance at p<0.1, ** indicates significance at p<0.05, *** indicates significance at p<0.001.

[0036] FIG. 10 illustrates a summary of data showing the relative brahma expression measured after exposure to 2 .mu.g of brahma or GFP dsRNA six times before mating, immediately after mating, and six days after mating. Comparisons performed with Dunnett's test, * indicates significance at p<0.1, ** indicates significance at p<0.05, *** indicates significance at p<0.001.

[0037] FIG. 11A illustrates a summary of data showing the effect of the duration of exposure to insects exposed to 2 .mu.g of brahma or GFP dsRNA 1, 2, 4 or 6 times (brm-T1, -T2, -T4 or -T6). FIG. 11B illustrates the relative brahma expression measured on day 12 after the first exposure to 2 .mu.g of brahma or GFP dsRNA. Comparisons performed with Dunnett's test, * indicates significance at p<0.1, ** indicates significance at p<0.05, *** indicates significance at p<0.001.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0038] The nucleic acid sequences identified in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. .sctn.1.822. The nucleic acid and amino acid sequences listed define molecules (i.e., polynucleotides and polypeptides, respectively) having the nucleotide and amino acid monomers arranged in the manner described. The nucleic acid and amino acid sequences listed also each define a genus of polynucleotides or polypeptides that comprise the nucleotide and amino acid monomers arranged in the manner described. In view of the redundancy of the genetic code, it will be understood that a nucleotide sequence including a coding sequence also describes the genus of polynucleotides encoding the same polypeptide as a polynucleotide consisting of the reference sequence. It will further be understood that an amino acid sequence describes the genus of polynucleotide ORFs encoding that polypeptide.

[0039] Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. As the complement and reverse complement of a primary nucleic acid sequence are necessarily disclosed by the primary sequence, the complementary sequence and reverse complementary sequence of a nucleic acid sequence are included by any reference to the nucleic acid sequence, unless it is explicitly stated to be otherwise (or it is clear to be otherwise from the context in which the sequence appears). Furthermore, as it is understood in the art that the nucleotide sequence of an RNA strand is determined by the sequence of the DNA from which it was transcribed (but for the substitution of uracil (U) nucleobases for thymine (T)), an RNA sequence is included by any reference to the DNA sequence encoding it. In the accompanying sequence listing:

[0040] SEQ ID NO:1 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as brahma-c4465 rc.

[0041] SEQ ID NO:2 shows the amino acid sequence of a Diabrotica BRAHMA polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0042] SEQ ID NO:3 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as brahma-8089.

[0043] SEQ ID NO:4 shows the amino acid sequence of a further Diabrotica BRAHMA polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0044] SEQ ID NO:5 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as brahma-525.

[0045] SEQ ID NO:6 shows the amino acid sequence of a further Diabrotica BRAHMA polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0046] SEQ ID NO:7 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as contig[000]_brahma_949-1126, containing "brahma variant 1" (or "brahma var 1").

[0047] SEQ ID NO:8 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as brahma reg-352.

[0048] SEQ ID NO:9 shows the amino acid sequence of a Diabrotica BRAHMA polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0049] SEQ ID NO:10 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as brahma variant 1 (or brahma var 1), which is used in some examples for the production of a dsRNA.

[0050] SEQ ID NO:11 shows a segment of an exemplary YFP gene, which is used in some examples for the production of a dsRNA.

[0051] SEQ ID NO:12 shows the nucleotide sequence of a T7 phage promoter.

[0052] SEQ ID NOs:13-20 show primers used to amplify gene regions of a Diabrotica brahma gene or a YFP gene.

[0053] SEQ ID NO:21 shows a DNA sequence of annexin region 1.

[0054] SEQ ID NO:22 shows a DNA sequence of annexin region 2.

[0055] SEQ ID NO:23 shows a DNA sequence of beta spectrin 2 region 1.

[0056] SEQ ID NO:24 shows a DNA sequence of beta spectrin 2 region 2.

[0057] SEQ ID NO:25 shows a DNA sequence of mtRP-L4 region 1.

[0058] SEQ ID NO:26 shows a DNA sequence of mtRP-L4 region 2.

[0059] SEQ ID NOs:27-52 show primers used to amplify gene regions of annexin, beta spectrin 2, mtRP-L4, and YFP for dsRNA synthesis.

[0060] SEQ ID NO:53 shows a segment of an exemplary GFP gene, which is used in some examples for the production of a dsRNA.

[0061] SEQ ID NOs:54 and 55 show primers used for PCR amplification of a GFP sequence, used in some examples for dsRNA production.

[0062] SEQ ID NO:56 shows an exemplary DNA comprising an ST-LS1 intron.

[0063] SEQ ID NO:57 shows an exemplary DNA encoding a Diabrotica chromatin remodeling gene-targetting hairpin-forming RNA; containing brahma v1 sense polynucleotides, a loop polynucleotide (underlined) including an intron, and brahma v1 antisense polynucleotide (bold font):

TABLE-US-00001 GCGCCCTACAGACTCCTGCTTACTGGTACTCCCCTACAAAATAAATTACC AGAATTATGGGCCTTGTTGAATTTCTTGTTGCCTTCGATTTTCAAGAGTT GCTCCACTTTTGAACAATGGTTCAATGCGCCATTCGCAACAACAGGAGAA AAGGTTGAGTTAAACGAAGAAGAAACTATCCTTATCATCCGTCGTCTTCA CAAAGTACTCAGGCCGTTTCTCCTGAGACGTCTCAAGAAAGAAGTCGAAT CTCAGCTTCCAGACAAAGTGGAATATATCATAAAGTGTGACATGTGACTA GTACCGGTTGGGAAAGGTATGTTTCTGCTTCTACCTTTGATATATATATA ATAATTATCACTAATTAGTAGTAATATAGTATTTCAAGTATTTTTTTCAA AATAAAAGAATGTAGTATATAGCTATTGCTTTTCTGTAGTTTATAAGTGT GTATATTTTAATTTATAACTTTTCTAATATATGACCAAAACATGGTGATG TGCAGGTTGATCCGCGGACATGTCACACTTTATGATATATTCCACTTTGT CTGGAAGCTGAGATTCGACTTCTTTCTTGAGACGTCTCAGGAGAAACGGC CTGAGTACTTTGTGAAGACGACGGATGATAAGGATAGTTTCTTCTTCGTT TAACTCAACCTTTTCTCCTGTTGTTGCGAATGGCGCATTGAACCATTGTT CAAAAGTGGAGCAACTCTTGAAAATCGAAGGCAACAAGAAATTCAACAAG GCCCATAATTCTGGTAATTTATTTTGTAGGGGAGTACCAGTAAGCAGGAG TCTGTAGGGCGC

[0064] SEQ ID NO:58 shows a further exemplary DNA encoding a Diabrotica chromatin remodeling gene-targetting hairpin-forming RNA; containing brahma v2 sense polynucleotides, a loop polynucleotide (underlined) including an intron, and brahma v2 antisense polynucleotide (bold font):

TABLE-US-00002 CATATAAAAGAACGAAGCGACAGGGTCTAAAAGAATCGAGAGCTACAGAG AAGTTAGAAAAACAACAGAAGTTAGAAGCAGAAAGAAAGAGAAGACAGAA GAACCAAGAATTTTTGAATGCTGTATTGAACAATGGAAAAGAATTCAAGG AATTCCACAAGCAGAATCAAGCGAAATTAGCTAAGATTAATAAAGCTGTT ATTAATTATCACGCTAATGCTGAAAGAGAGCAAAAGAAAGAAGCAGAAAG GAGAGAGAAGGAACGTATGATCAGATTGATGGCAGAAGATGAAGAAGGTT GACTAGTACCGGTTGGGAAAGGTATGTTTCTGCTTCTACCTTTGATATAT ATATAATAATTATCACTAATTAGTAGTAATATAGTATTTCAAGTATTTTT TTCAAAATAAAAGAATGTAGTATATAGCTATTGCTTTTCTGTAGTTTATA AGTGTGTATATTTTAATTTATAACTTTTCTAATATATGACCAAAACATGG TGATGTGCAGGTTGATCCGCGGAACCTTCTTCATCTTCTGCCATCAATCT GATCATACGTTCCTTCTCTCTCCTTTCTGCTTCTTTCTTTTGCTCTCTTT CAGCATTAGCGTGATAATTAATAACAGCTTTATTAATCTTAGCTAATTTC GCTTGATTCTGCTTGTGGAATTCCTTGAATTCTTTTCCATTGTTCAATAC AGCATTCAAAAATTCTTGGTTCTTCTGTCTTCTCTTTCTTTCTGCTTCTA ACTTCTGTTGTTTTTCTAACTTCTCTGTAGCTCTCGATTCTTTTAGACCC TGTCGCTTCGTTCTTTTATATG

[0065] SEQ ID NO:59 shows the nucleotide sequence of a T20VN primer oligonucleotide.

[0066] SEQ ID NOs:60-64 show primers and probes used for dsRNA transcript expression analyses.

[0067] SEQ ID NO:65 shows a nucleotide sequence of a portion of a SpecR coding region used for binary vector backbone detection.

[0068] SEQ ID NO:66 shows a nucleotide sequence of an AADJ coding region used for genomic copy number analysis.

[0069] SEQ ID NOs:67-78 show the nucleotide sequences of DNA oligonucleotides used for gene copy number determinations and binary vector backbone detection.

[0070] SEQ ID NO:79 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as mi-2.

[0071] SEQ ID NO:80 shows the amino acid sequence of a Diabrotica MI-2 polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0072] SEQ ID NO:81 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as iswi-1.

[0073] SEQ ID NO:82 shows the amino acid sequence of a Diabrotica ISWI-1 polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0074] SEQ ID NO:83 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as chd1.

[0075] SEQ ID NO:84 shows the amino acid sequence of a Diabrotica CHD1 polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0076] SEQ ID NO:85 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as iswi-2.

[0077] SEQ ID NO:86 shows the amino acid sequence of a Diabrotica ISWI-2 polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0078] SEQ ID NO:87 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as iswi-30 or iswi-3.

[0079] SEQ ID NO:88 shows the amino acid sequence of a Diabrotica ISWI-30 ("ISWI-3") polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0080] SEQ ID NO:89 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as ino80.

[0081] SEQ ID NO:90 shows the amino acid sequence of a Diabrotica IN080 polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0082] SEQ ID NO:91 shows a further exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as domino.

[0083] SEQ ID NO:92 shows the amino acid sequence of a Diabrotica DOMINO polypeptide encoded by an exemplary Diabrotica chromatin remodeling gene DNA.

[0084] SEQ ID NOs:93-96 show exemplary DNAs encoding dsRNA sequences for targeting SNF2-Helicase regions of insect (e.g., Diabrotica, Tribolium, Euschistus heros, and Drosophila melanogaster) chromatin remodeling gene DNA.

[0085] SEQ ID NOs:97-100 show exemplary DNAs encoding dsRNA sequences for targeting chromatin remodeling domains (Chromodomain, Bromodomain, or HAND-SLIDE regions) of insect (e.g., Diabrotica, Tribolium, Euschistus heros, and Drosophila melanogaster) chromatin remodeling gene DNA.

[0086] SEQ ID NO:101 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as mi-2 region1 ("mi2_5146").

[0087] SEQ ID NO:102 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as iswi-30 region1 ("iswi_3074").

[0088] SEQ ID NO:103 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as iswi-2 region1 ("SNF2_c18929").

[0089] SEQ ID NO:104 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as KISMET_2388 region1.

[0090] SEQ ID NO:105 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as CHD1 region1 ("Helicase_16208").

[0091] SEQ ID NO:106 shows an exemplary Diabrotica chromatin remodeling gene DNA, referred to herein in some places as ETL1 region1 ("SWI_SNF_Irc2582").

[0092] SEQ ID NOs:107-134 show primers used to amplify gene regions of chromatin remodeling genes.

[0093] SEQ ID NOs:135-163 show exemplary RNAs transcribed from nucleic acids comprising exemplary chromatin remodeling gene polynucleotides and fragments thereof.

[0094] SEQ ID NO:164 shows the open reading frame of an exemplary Diabrotica mi-2 chromatin remodeling gene DNA.

[0095] SEQ ID NO:165 shows the open reading frame of an exemplary Diabrotica iswi-1 chromatin remodeling gene DNA.

[0096] SEQ ID NO:166 shows the open reading frame of an exemplary Diabrotica iswi-2 chromatin remodeling gene DNA.

[0097] SEQ ID NOs:167-169 show further exemplary RNAs transcribed from nucleic acids comprising exemplary chromatin remodeling gene polynucleotides and fragments thereof.

DETAILED DESCRIPTION

I. Overview of Several Embodiments

[0098] RNA interference (RNAi) was developed as a tool for insect pest management, using one of the most likely target pest species for transgenic plants that express dsRNA; the western corn rootworm. Thus far, most genes proposed as targets for RNAi in rootworm larvae do not achieve their purpose, and those useful targets that have been identified involve those that cause lethality in the larval stage. Herein, we describe RNAi-mediated knockdown of chromatin remodeling genes (e.g., brahma, mi-2, iswi-1, chd1, iswi-2, iswi30, ino80, and domino) in the western corn rootworm, which is shown to disrupt embryonic development when, for example, iRNA molecules are delivered via chromatin remodeling gene-targeting dsRNA fed to adult females. There was almost complete absence of hatching in the eggs collected from females exposed to chromatin remodeling gene-targeting dsRNA. In embodiments herein, the ability to deliver chromatin remodeling gene-targeting dsRNA by feeding to adult insects confers a pRNAi effect that is very useful for insect (e.g., coleopteran) pest management. Furthermore, the potential to affect multiple target sequences in both larval and adult rootworms may increase opportunities to develop sustainable approaches to insect pest management involving RNAi technologies.

[0099] Disclosed herein are methods and compositions for genetic control of coleopteran pest infestations. Methods for identifying one or more gene(s) essential to the life cycle of a coleopteran pest (e.g., gene(s) essential for normal reproductive capacity and/or embryonic and/or larval development) for use as a target gene for RNAi-mediated control of a coleopteran pest population are also provided. DNA plasmid vectors encoding an RNA molecule may be designed to suppress one or more target gene(s) essential for growth, survival, development, and/or reproduction. In some embodiments, the RNA molecule may be capable of forming dsRNA molecules. In some embodiments, methods are provided for post-transcriptional repression of expression or inhibition of a target gene via nucleic acid molecules that are complementary to a coding or non-coding sequence of the target gene in a coleopteran pest. In these and further embodiments, a coleopteran pest may ingest one or more dsRNA, siRNA, shRNA, miRNA, and/or hpRNA molecules transcribed from all or a portion of a nucleic acid molecule that is complementary to a coding or non-coding sequence of a target gene, thereby providing a plant-protective effect.

[0100] Some embodiments involve sequence-specific inhibition of expression of target gene products, using dsRNA, siRNA, shRNA, miRNA and/or hpRNA that is complementary to coding and/or non-coding sequences of the target gene(s) to achieve at least partial control of a coleopteran pest. Disclosed is a set of isolated and purified nucleic acid molecules comprising a polynucleotide, for example, as set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; and fragments thereof. In some embodiments, a stabilized dsRNA molecule may be expressed from these polynucleotides, fragments thereof, or a gene comprising one of these polynucleotides, for the post-transcriptional silencing or inhibition of a target gene. In certain embodiments, isolated and purified nucleic acid molecules comprise all or part of any of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164, SEQ ID NO:165; and SEQ ID NO:166.

[0101] Some embodiments involve a recombinant host cell (e.g., a plant cell) having in its genome at least one recombinant DNA encoding at least one iRNA (e.g., dsRNA) molecule(s). In particular embodiments, the dsRNA molecule(s) may be produced when ingested by a coleopteran pest to post-transcriptionally silence or inhibit the expression of a target gene in the pest or progeny of the pest. The recombinant DNA may comprise, for example, any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166; fragments of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166 (e.g., SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NOs:101-106); and a polynucleotide consisting of a partial sequence of a gene comprising one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, and/or complements thereof.

[0102] Some embodiments involve a recombinant host cell having in its genome a recombinant DNA encoding at least one iRNA (e.g., dsRNA) molecule(s) comprising all or part of any of SEQ ID NOs:139-142 and 167-169 (e.g., at least one polynucleotide selected from the group consisting of SEQ ID NOs:143-146); all or part of SEQ ID NO:147 (e.g., SEQ ID NO:162); all or part of SEQ ID NO:148; all or part of SEQ ID NO:149 (e.g., SEQ ID NO:166); all or part of SEQ ID NO:150; all or part of SEQ ID NO:151 (e.g., SEQ ID NO:163); all or part of SEQ ID NO:152; all or part of SEQ ID NO:153; all or part of SEQ ID NO:167; all or part of SEQ ID NO:168; and all or part of SEQ ID NO:169. When ingested by a coleopteran pest, the iRNA molecule(s) may silence or inhibit the expression of a target chromatin remodeling gene (e.g., a DNA comprising all or part of a polynucleotide selected from the group consisting of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; SEQ ID NO:92; SEQ ID NO:94, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166) in the pest or progeny of the pest, and thereby result in cessation of reproduction in the pest, and/or growth, development, and/or feeding in progeny of the pest.

[0103] In some embodiments, a recombinant host cell having in its genome at least one recombinant DNA encoding at least one RNA molecule capable of forming a dsRNA molecule may be a transformed plant cell. Some embodiments involve transgenic plants comprising such a transformed plant cell. In addition to such transgenic plants, progeny plants of any transgenic plant generation, transgenic seeds, and transgenic plant products, are all provided, each of which comprises recombinant DNA(s). In particular embodiments, an RNA molecule capable of forming a dsRNA molecule may be expressed in a transgenic plant cell. Therefore, in these and other embodiments, a dsRNA molecule may be isolated from a transgenic plant cell. In particular embodiments, the transgenic plant is a plant selected from the group comprising corn (Zea mays) and plants of the family Poaceae.

[0104] Some embodiments involve a method for modulating the expression of a target gene in a coleopteran pest cell. In these and other embodiments, a nucleic acid molecule may be provided, wherein the nucleic acid molecule comprises a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule. In particular embodiments, a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule may be operatively linked to a promoter, and may also be operatively linked to a transcription termination sequence. In particular embodiments, a method for modulating the expression of a target gene in a coleopteran pest cell may comprise: (a) transforming a plant cell with a vector comprising a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule; (b) culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture comprising a plurality of transformed plant cells; (c) selecting for a transformed plant cell that has integrated the vector into its genome; and (d) determining that the selected transformed plant cell comprises the RNA molecule capable of forming a dsRNA molecule encoded by the polynucleotide of the vector. A plant may be regenerated from a plant cell that has the vector integrated in its genome and comprises the dsRNA molecule encoded by the polynucleotide of the vector.

[0105] Also disclosed is a transgenic plant comprising a vector having a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule integrated in its genome, wherein the transgenic plant comprises the dsRNA molecule encoded by the polynucleotide of the vector. In particular embodiments, expression of an RNA molecule capable of forming a dsRNA molecule in the plant is sufficient to modulate the expression of a target gene in a cell of a coleopteran pest that contacts the transformed plant or plant cell (for example, by feeding on the transformed plant, a part of the plant (e.g., root) or plant cell) or in a cell of a progeny of the coleopteran pest that contacts the transformed plant or plant cell (for example, by parental transmission), such that reproduction of the pest is inhibited. Transgenic plants disclosed herein may display tolerance and/or protection from coleopteran pest infestations. Particular transgenic plants may display protection and/or enhanced protection from one or more coleopteran pest(s) selected from the group consisting of: WCR; NCR; SCR; MCR; D. balteata LeConte; D. speciosa Germar; D. u. tenella; and D. u. undecimpunctata Mannerheim.

[0106] Also disclosed herein are methods for delivery of control agents, such as an iRNA molecule, to a coleopteran pest. Such control agents may cause, directly or indirectly, an impairment in the ability of a coleopteran pest population to feed, grow or otherwise cause damage in a host. In some embodiments, a method is provided comprising delivery of a stabilized dsRNA molecule to a coleopteran pest to suppress at least one target gene in the pest or its progeny, thereby causing parental RNAi and reducing or eliminating plant damage in a pest host. In some embodiments, a method of inhibiting expression of a target gene in a coleopteran pest may result in cessation of reproduction in the pest, and/or growth, development, and/or feeding in progeny of the pest. In some embodiments, the method may significantly reduce the size of a subsequent pest generation in an infestation, without directly resulting in mortality in the pest(s) that contact the iRNA molecule. In some embodiments, the method may significantly reduce the size of a subsequent pest generation in an infestation, while also resulting in mortality in the pest(s) that contact the iRNA molecule.

[0107] In some embodiments, compositions (e.g., a topical composition) are provided that comprise an iRNA (e.g., dsRNA) molecule for use with plants, animals, and/or the environment of a plant or animal to achieve the elimination or reduction of a coleopteran pest infestation. In some embodiments, compositions are provided that include a prokaryote comprising a DNA encoding an iRNA molecule; for example, a transformed bacterial cell. In particular examples, such a transformed bacterial cell may be utilized as a conventional pesticide formulation. In particular embodiments, the composition may be a nutritional composition or resource, or food source, to be fed to the coleopteran pest. Some embodiments comprise making the nutritional composition or food source available to the pest. Ingestion of a composition comprising iRNA molecules may result in the uptake of the molecules by one or more cells of the coleopteran pest, which may in turn result in the inhibition of expression of at least one target gene in cell(s) of the pest or its progeny. Ingestion of or damage to a plant or plant cell by a coleopteran pest infestation may be limited or eliminated in or on any host tissue or environment in which the pest is present by providing one or more compositions comprising an iRNA molecule in the host of the pest.

[0108] The compositions and methods disclosed herein may be used together in combinations with other methods and compositions for controlling damage by coleopteran pests. For example, an iRNA molecule as described herein for protecting plants from coleopteran pests may be used in a method comprising the additional use of one or more chemical agents effective against a coleopteran pest, biopesticides effective against a coleopteran pest, crop rotation, recombinant genetic techniques that exhibit features different from the features of RNAi-mediated methods and RNAi compositions (e.g., recombinant production of proteins in plants that are harmful to a coleopteran pest (e.g., Bt toxins)), and/or recombinant expression of non-parental iRNA molecules (e.g., lethal iRNA molecules that result in the cessation of growth, development, and/or feeding in the coleopteran pest that ingests the iRNA molecule).

II. Abbreviations



[0109] dsRNA double-stranded ribonucleic acid

[0110] GI growth inhibition

[0111] GFP green fluorescent protein

[0112] NCBI National Center for Biotechnology Information

[0113] gDNA genomic deoxyribonucleic acid

[0114] iRNA inhibitory ribonucleic acid

[0115] ISWI Imitation SWI/imitation switch

[0116] ORF open reading frame

[0117] RNAi ribonucleic acid interference

[0118] miRNA micro ribonucleic acid

[0119] siRNA small inhibitory ribonucleic acid

[0120] hpRNA hairpin ribonucleic acid

[0121] shRNA short hairpin ribonucleic acid

[0122] pRNAi parental RNA interference

[0123] UTR untranslated region

[0124] WCR western corn rootworm (Diabrotica virgifera virgifera LeConte)

[0125] NCR northern corn rootworm (Diabrotica barberi Smith and Lawrence)

[0126] MCR Mexican corn rootworm (Diabrotica virgifera zeae Krysan and Smith)

[0127] PCR Polymerase chain reaction

[0128] qPCR quantitative polymerase chain reaction

[0129] RISC RNA-induced Silencing Complex

[0130] RH relative humidity

[0131] SCR southern corn rootworm (Diabrotica undecimpunctata howardi Barber)

[0132] SEM standard error of the mean

[0133] YFP yellow fluorescent protein

III. Terms

[0134] In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

[0135] Coleopteran pest: As used herein, the term "coleopteran pest" refers to pest insects of the order Coleoptera, including pest insects in the genus Diabrotica, which feed upon agricultural crops and crop products, including corn and other true grasses. In particular examples, a coleopteran pest is selected from a list comprising D. v. virgifera LeConte (WCR); D. barberi Smith and Lawrence (NCR); D. u. howardi (SCR); D. v. zeae (MCR); D. balteata LeConte; D. u. tenella; D. speciosa Germar; and D. u. undecimpunctata Mannerheim.

[0136] Contact (with an organism): As used herein, the term "contact with" or "uptake by" an organism (e.g., a coleopteran pest), with regard to a nucleic acid molecule, includes internalization of the nucleic acid molecule into the organism, for example and without limitation: ingestion of the molecule by the organism (e.g., by feeding); contacting the organism with a composition comprising the nucleic acid molecule; and soaking of organisms with a solution comprising the nucleic acid molecule.

[0137] Contig: As used herein the term "contig" refers to a DNA sequence that is reconstructed from a set of overlapping DNA segments derived from a single genetic source.

[0138] Corn plant: As used herein, the term "corn plant" refers to a plant of the species, Zea mays (maize). The terms "corn plant" and "maize" are used interchangeably herein.

[0139] Expression: As used herein, "expression" of a coding polynucleotide (for example, a gene or a transgene) refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., gDNA or cDNA) is converted into an operational, non-operational, or structural part of a cell, often including the synthesis of a protein. Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof. Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, northern blot, RT-PCR, western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0140] Genetic material: As used herein, the term "genetic material" includes all genes, and nucleic acid molecules, such as DNA and RNA.

[0141] Inhibition: As used herein, the term "inhibition," when used to describe an effect on a coding polynucleotide (for example, a gene), refers to a measurable decrease in the cellular level of mRNA transcribed from the coding polynucleotide and/or peptide, polypeptide, or protein product of the coding polynucleotide. In some examples, expression of a coding polynucleotide may be inhibited such that expression is approximately eliminated. "Specific inhibition" refers to the inhibition of a target coding polynucleotide without consequently affecting expression of other coding polynucleotides (e.g., genes) in the cell wherein the specific inhibition is being accomplished.

[0142] Isolated: An "isolated" biological component (such as a nucleic acid or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs (i.e., other chromosomal and extra-chromosomal DNA and RNA, and proteins), while effecting a chemical or functional change in the component (e.g., a nucleic acid may be isolated from a chromosome by breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome). Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically-synthesized nucleic acid molecules, proteins, and peptides.

[0143] Nucleic acid molecule: As used herein, the term "nucleic acid molecule" may refer to a polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, gDNA, and synthetic forms and mixed polymers of the above. A nucleotide or nucleobase may refer to a ribonucleotide, deoxyribonucleotide, or a modified form of either type of nucleotide. A "nucleic acid molecule" as used herein is synonymous with "nucleic acid" and "polynucleotide." A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. By convention, the nucleotide sequence of a nucleic acid molecule is read from the 5' to the 3' end of the molecule. The "complement" of a nucleic acid molecule refers to a polynucleotide having nucleobases that may form base pairs with the nucleobases of the nucleic acid molecule (i.e., A-T/U, and G-C).

[0144] Some embodiments include nucleic acids comprising a template DNA that is transcribed into an RNA molecule that is the complement of an mRNA molecule. In these embodiments, the complement of the nucleic acid transcribed into the mRNA molecule is present in the 5' to 3' orientation, such that RNA polymerase (which transcribes DNA in the 5' to 3' direction) will transcribe a nucleic acid from the complement that can hybridize to the mRNA molecule. Unless explicitly stated otherwise, or it is clear to be otherwise from the context, the term "complement" therefore refers to a polynucleotide having nucleobases, from 5' to 3', that may form base pairs with the nucleobases of a reference nucleic acid. Similarly, unless it is explicitly stated to be otherwise (or it is clear to be otherwise from the context), the "reverse complement" of a nucleic acid refers to the complement in reverse orientation. The foregoing is demonstrated in the following illustration:

TABLE-US-00003 ATGATGATG polynucleotide TACTACTAC "complement" of the polynucleotide CATCATCAT "reverse complement" of the polynucleotide

Some embodiments of the invention may include hairpin RNA-forming RNAi molecules. In these RNAi molecules, both the complement of a nucleic acid to be targeted by RNA interference and the reverse complement may be found in the same molecule, such that the single-stranded RNA molecule may "fold over" and hybridize to itself over a region comprising the complementary and reverse complementary polynucleotides.

[0145] "Nucleic acid molecules" include all polynucleotides, for example: single- and double-stranded forms of DNA; single-stranded forms of RNA; and double-stranded forms of RNA (dsRNA). The term "nucleotide sequence" or "nucleic acid sequence" refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex. The term "ribonucleic acid" (RNA) is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), shRNA (small hairpin RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNAs, whether charged or discharged with a corresponding acylated amino acid), and cRNA (complementary RNA). The term "deoxyribonucleic acid" (DNA) is inclusive of cDNA, gDNA, and DNA-RNA hybrids. The terms "polynucleotide" and "nucleic acid," and "fragments" thereof will be understood by those in the art as a term that includes both gDNAs, ribosomal RNAs, transfer RNAs, messenger RNAs, operons, and smaller engineered polynucleotides that encode or may be adapted to encode, peptides, polypeptides, or proteins.

[0146] Oligonucleotide: An oligonucleotide is a short nucleic acid polymer. Oligonucleotides may be formed by cleavage of longer nucleic acid segments, or by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred bases in length. Because oligonucleotides may bind to a complementary nucleic acid, they may be used as probes for detecting DNA or RNA. Oligonucleotides composed of DNA (oligodeoxyribonucleotides) may be used in PCR, a technique for the amplification of DNAs. In PCR, the oligonucleotide is typically referred to as a "primer," which allows a DNA polymerase to extend the oligonucleotide and replicate the complementary strand.

[0147] A nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.

[0148] As used herein with respect to DNA, the term "coding polynucleotide," "structural polynucleotide," or "structural nucleic acid molecule" refers to a polynucleotide that is ultimately translated into a polypeptide, via transcription and mRNA, when placed under the control of appropriate regulatory elements. With respect to RNA, the term "coding polynucleotide" refers to a polynucleotide that is translated into a peptide, polypeptide, or protein. The boundaries of a coding polynucleotide are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. Coding polynucleotides include, but are not limited to: gDNA; cDNA; EST; and recombinant polynucleotides.

[0149] As used herein, "transcribed non-coding polynucleotide" refers to segments of mRNA molecules such as 5'UTR, 3'UTR and intron segments that are not translated into a peptide, polypeptide, or protein. Further, "transcribed non-coding polynucleotide" refers to a nucleic acid that is transcribed into an RNA that functions in the cell, for example, structural RNAs (e.g., ribosomal RNA (rRNA) as exemplified by 5S rRNA, 5.8S rRNA, 16S rRNA, 18 S rRNA, 23 S rRNA, and 28S rRNA, and the like); transfer RNA (tRNA); and snRNAs such as U4, U5, U6, and the like. Transcribed non-coding polynucleotides also include, for example and without limitation, small RNAs (sRNA), which term is often used to describe small bacterial non-coding RNAs; small nucleolar RNAs (snoRNA); microRNAs; small interfering RNAs (siRNA); Piwi-interacting RNAs (piRNA); and long non-coding RNAs. Further still, "transcribed non-coding polynucleotide" refers to a polynucleotide that may natively exist as an intragenic "linker" in a nucleic acid and which is transcribed into an RNA molecule.

[0150] Lethal RNA interference: As used herein, the term "lethal RNA interference" refers to RNA interference that results in death or a reduction in viability of the subject individual to which, for example, a dsRNA, miRNA, siRNA, shRNA, and/or hpRNA is delivered.

[0151] Parental RNA interference: As used herein, the term "parental RNA interference" (pRNAi) refers to a RNA interference phenotype that is observable in progeny of the subject (e.g., a coleopteran pest) to which, for example, a dsRNA, miRNA, siRNA, shRNA, and/or hpRNA is delivered. In some embodiments, pRNAi comprises the delivery of a dsRNA to a coleopteran pest, wherein the pest is thereby rendered less able to produce viable offspring. A nucleic acid that initiates pRNAi may or may not increase the incidence of mortality in a population into which the nucleic acid is delivered. In certain examples, the nucleic acid that initiates pRNAi does not increase the incidence of mortality in the population into which the nucleic acid is delivered. For example, a population of coleopteran pests may be fed one or more nucleic acids that initiate pRNAi, wherein the pests survive and mate but produce eggs that are less able to hatch viable progeny than eggs produced by pests of the same species that are not fed the nucleic acid(s). In one mechanism of pRNAi, parental RNAi delivered to a female is able to knock down zygotic gene expression in offspring embryos of the female. Bucher et al. (2002) Curr. Biol. 12(3):R85-6.

[0152] Genome: As used herein, the term "genome" refers to chromosomal DNA found within the nucleus of a cell, and also refers to organelle DNA found within subcellular components of the cell. In some embodiments of the invention, a DNA molecule may be introduced into a plant cell, such that the DNA molecule is integrated into the genome of the plant cell. In these and further embodiments, the DNA molecule may be either integrated into the nuclear DNA of the plant cell, or integrated into the DNA of the chloroplast or mitochondrion of the plant cell. The term "genome," as it applies to bacteria, refers to both the chromosome and plasmids within the bacterial cell. In some embodiments of the invention, a DNA molecule may be introduced into a bacterium such that the DNA molecule is integrated into the genome of the bacterium. In these and further embodiments, the DNA molecule may be either chromosomally-integrated or located as or in a stable plasmid.

[0153] Sequence identity: The term "sequence identity" or "identity," as used herein in the context of two polynucleotides or polypeptides, refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window.

[0154] As used herein, the term "percentage of sequence identity" may refer to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences or polypeptide sequences) of a molecule over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference sequence is said to be 100% identical to the reference sequence, and vice-versa.

[0155] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-10.

[0156] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST.TM.; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, Md.), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the "help" section for BLAST.TM.. For comparisons of nucleic acid sequences, the "Blast 2 sequences" function of the BLAST.TM. (Blastn) program may be employed using the default BLOSUM62 matrix set to default parameters. Nucleic acids with even greater sequence similarity to the sequences of the reference polynucleotides will show increasing percentage identity when assessed by this method.

[0157] Specifically hybridizable/Specifically complementary: As used herein, the terms "Specifically hybridizable" and "Specifically complementary" are terms that indicate a sufficient degree of complementarity such that stable and specific binding occurs between the nucleic acid molecule and a target nucleic acid molecule. Hybridization between two nucleic acid molecules involves the formation of an anti-parallel alignment between the nucleobases of the two nucleic acid molecules. The two molecules are then able to form hydrogen bonds with corresponding bases on the opposite strand to form a duplex molecule that, if it is sufficiently stable, is detectable using methods well known in the art. A polynucleotide need not be 100% complementary to its target nucleic acid to be specifically hybridizable. However, the amount of complementarity that must exist for hybridization to be specific is a function of the hybridization conditions used.

[0158] Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method of choice and the composition and length of the hybridizing nucleic acids. Generally, the temperature of hybridization and the ionic strength (especially the Na.sup.+ and/or Mg.sup.++ concentration) of the hybridization buffer will determine the stringency of hybridization, though wash times also influence stringency. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are known to those of ordinary skill in the art, and are discussed, for example, in Sambrook et al. (ed.) Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989, chapters 9 and 11; and Hames and Higgins (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985. Further detailed instruction and guidance with regard to the hybridization of nucleic acids may be found, for example, in Tijssen, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," in Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, N Y, 1993; and Ausubel et al., Eds., Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, N Y, 1995.

[0159] As used herein, "stringent conditions" encompass conditions under which hybridization will only occur if there is less than 20% mismatch between the sequence of the hybridization molecule and a homologous polynucleotide within the target nucleic acid molecule. "Stringent conditions" include further particular levels of stringency. Thus, as used herein, "moderate stringency" conditions are those under which molecules with more than 20% sequence mismatch will not hybridize; conditions of "high stringency" are those under which sequences with more than 10% mismatch will not hybridize; and conditions of "very high stringency" are those under which sequences with more than 5% mismatch will not hybridize.

[0160] The following are representative, non-limiting hybridization conditions. High Stringency condition (detects polynucleotides that share at least 90% sequence identity): Hybridization in 5.times.SSC buffer at 65.degree. C. for 16 hours; wash twice in 2.times.SSC buffer at room temperature for 15 minutes each; and wash twice in 0.5.times.SSC buffer at 65.degree. C. for 20 minutes each.

[0161] Moderate Stringency condition (detects polynucleotides that share at least 80% sequence identity): Hybridization in 5.times.-6.times.SSC buffer at 65-70.degree. C. for 16-20 hours; wash twice in 2.times.SSC buffer at room temperature for 5-20 minutes each; and wash twice in 1SSC buffer at 55-70.degree. C. for 30 minutes each.

[0162] Non-stringent control condition (polynucleotides that share at least 50% sequence identity will hybridize): Hybridization in 6.times.SSC buffer at room temperature to 55.degree. C. for 16-20 hours; wash at least twice in 2.times.-3.times.SSC buffer at room temperature to 55.degree. C. for 20-30 minutes each.

[0163] As used herein, the term "substantially homologous" or "substantial homology," with regard to a nucleic acid, refers to a polynucleotide having contiguous nucleobases that hybridize under stringent conditions to the reference nucleic acid. For example, nucleic acids that are substantially homologous to a reference nucleic acid of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166 are those nucleic acids that hybridize under stringent conditions (e.g., the Moderate Stringency conditions set forth, supra) to the reference nucleic acid of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:164, SEQ ID NO:165, and/or SEQ ID NO:166. Substantially homologous polynucleotides may have at least 80% sequence identity. For example, substantially homologous polynucleotides may have from about 80% to 100% sequence identity, such as 79%; 80%; about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; and about 100%. The property of substantial homology is closely related to specific hybridization. For example, a nucleic acid molecule is specifically hybridizable when there is a sufficient degree of complementarity to avoid non-specific binding of the nucleic acid to non-target polynucleotides under conditions where specific binding is desired, for example, under stringent hybridization conditions.

[0164] As used herein, the term "ortholog" refers to a gene in two or more species that has evolved from a common ancestral nucleic acid, and may retain the same function in the two or more species.

[0165] As used herein, two nucleic acid molecules are said to exhibit "complete complementarity" when every nucleotide of a polynucleotide read in the 5' to 3' direction is complementary to every nucleotide of the other polynucleotide when read in the 3' to 5' direction. A polynucleotide that is complementary to a reference polynucleotide will exhibit a sequence identical to the reverse complement of the reference polynucleotide. These terms and descriptions are well defined in the art and are easily understood by those of ordinary skill in the art.

[0166] Operably linked: A first polynucleotide is operably linked with a second polynucleotide when the first polynucleotide is in a functional relationship with the second polynucleotide. When recombinantly produced, operably linked polynucleotides are generally contiguous, and, where necessary to join two protein-coding regions, in the same reading frame (e.g., in a translationally fused ORF). However, nucleic acids need not be contiguous to be operably linked.

[0167] The term, "operably linked," when used in reference to a regulatory genetic element and a coding polynucleotide, means that the regulatory element affects the expression of the linked coding polynucleotide. "Regulatory elements," or "control elements," refer to polynucleotides that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding polynucleotide. Regulatory elements may include promoters; translation leaders; introns; enhancers; stem-loop structures; repressor binding polynucleotides; polynucleotides with a termination sequence; polynucleotides with a polyadenylation recognition sequence; etc. Particular regulatory elements may be located upstream and/or downstream of a coding polynucleotide operably linked thereto. Also, particular regulatory elements operably linked to a coding polynucleotide may be located on the associated complementary strand of a double-stranded nucleic acid molecule.

[0168] Promoter: As used herein, the term "promoter" refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter may be operably linked to a coding polynucleotide for expression in a cell, or a promoter may be operably linked to a polynucleotide encoding a signal peptide which may be operably linked to a coding polynucleotide for expression in a cell. A "plant promoter" may be a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue-preferred". Promoters which initiate transcription only in certain tissues are referred to as "tissue-specific". A "cell type-specific" promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An "inducible" promoter may be a promoter which may be under environmental control. Examples of environmental conditions that may initiate transcription by inducible promoters include anaerobic conditions and the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is a promoter which may be active under most environmental conditions or in most tissue or cell types.

[0169] Any inducible promoter can be used in some embodiments of the invention. See Ward et al. (1993) Plant Mol. Biol. 22:361-366. With an inducible promoter, the rate of transcription increases in response to an inducing agent. Exemplary inducible promoters include, but are not limited to: Promoters from the ACEI system that respond to copper; In2 gene from maize that responds to benzenesulfonamide herbicide safeners; Tet repressor from Tn10; and the inducible promoter from a steroid hormone gene, the transcriptional activity of which may be induced by a glucocorticosteroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:0421).

[0170] Exemplary constitutive promoters include, but are not limited to: Promoters from plant viruses, such as the 35S promoter from Cauliflower Mosaic Virus (CaMV); promoters from rice actin genes; ubiquitin promoters; pEMU; MAS; maize H3 histone promoter; and the ALS promoter, Xba1/NcoI fragment 5' to the Brassica napus ALS3 structural gene (or a polynucleotide similar to said Xba1/NcoI fragment) (International PCT Publication No. WO96/30530).

[0171] Additionally, any tissue-specific or tissue-preferred promoter may be utilized in some embodiments of the invention. Plants transformed with a nucleic acid molecule comprising a coding polynucleotide operably linked to a tissue-specific promoter may produce the product of the coding polynucleotide exclusively, or preferentially, in a specific tissue. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to: A seed-preferred promoter, such as that from the phaseolin gene; a leaf-specific and light-induced promoter such as that from cab or rubisco; an anther-specific promoter such as that from LAT52; a pollen-specific promoter such as that from Zm13; and a microspore-preferred promoter such as that from apg.

[0172] Transformation: As used herein, the term "transformation" or "transduction" refers to the transfer of one or more nucleic acid molecule(s) into a cell. A cell is "transformed" by a nucleic acid molecule transduced into the cell when the nucleic acid molecule becomes stably replicated by the cell, either by incorporation of the nucleic acid molecule into the cellular genome, or by episomal replication. As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al. (1986) Nature 319:791-3); lipofection (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al. (1978) Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA uptake; and microprojectile bombardment (Klein et al. (1987) Nature 327:70).

[0173] Transgene: An exogenous nucleic acid. In some examples, a transgene may be a DNA that encodes one or both strand(s) of an RNA capable of forming a dsRNA molecule that comprises a polynucleotide that is complementary to a nucleic acid molecule found in a coleopteran pest. In further examples, a transgene may be an antisense polynucleotide, wherein expression of the antisense polynucleotide inhibits expression of a target nucleic acid, thereby producing a parental RNAi phenotype. In still further examples, a transgene may be a gene (e.g., a herbicide-tolerance gene, a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desirable agricultural trait). In these and other examples, a transgene may contain regulatory elements operably linked to a coding polynucleotide of the transgene (e.g., a promoter).

[0174] Vector: A nucleic acid molecule as introduced into a cell, for example, to produce a transformed cell. A vector may include genetic elements that permit it to replicate in the host cell, such as an origin of replication. Examples of vectors include, but are not limited to: a plasmid; cosmid; bacteriophage; or virus that carries exogenous DNA into a cell. A vector may also include one or more genes, including ones that produce antisense molecules, and/or selectable marker genes and other genetic elements known in the art. A vector may transduce, transform, or infect a cell, thereby causing the cell to express the nucleic acid molecules and/or proteins encoded by the vector. A vector optionally includes materials to aid in achieving entry of the nucleic acid molecule into the cell (e.g., a liposome, protein coating, etc.).

[0175] Yield: A stabilized yield of about 100% or greater relative to the yield of check varieties in the same growing location growing at the same time and under the same conditions. In particular embodiments, "improved yield" or "improving yield" means a cultivar having a stabilized yield of 105% or greater relative to the yield of check varieties in the same growing location containing significant densities of the coleopteran pests that are injurious to that crop growing at the same time and under the same conditions, which are targeted by the compositions and methods herein.

[0176] Unless specifically indicated or implied, the terms "a," "an," and "the" signify "at least one," as used herein.

[0177] Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in, for example, Lewin's Genes X, Jones & Bartlett Publishers, 2009 (ISBN 10 0763766321); Krebs et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers R. A. (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. All temperatures are in degrees Celsius.

IV. Nucleic Acid Molecules Comprising a Coleopteran Pest Polynucleotide

[0178] A. Overview

[0179] Described herein are nucleic acid molecules useful for the control of coleopteran pests. Described nucleic acid molecules include target polynucleotides (e.g., native genes, and non-coding polynucleotides), dsRNAs, siRNAs, shRNAs, hpRNAs, and miRNAs. For example, dsRNA, siRNA, miRNA, shRNA, and/or hpRNA molecules are described in some embodiments that may be specifically complementary to all or part of one or more native nucleic acids in a coleopteran pest. In these and further embodiments, the native nucleic acid(s) may be one or more target gene(s), the product of which may be, for example and without limitation: involved in a reproductive process or involved in larval development. Nucleic acid molecules described herein, when introduced into a cell (e.g., through parental transmission) comprising at least one native nucleic acid(s) to which the nucleic acid molecules are specifically complementary, may initiate RNAi in the cell, and consequently reduce or eliminate expression of the native nucleic acid(s). In some examples, reduction or elimination of the expression of a target gene by a nucleic acid molecule specifically complementary thereto may result in reduction or cessation of reproduction in the coleopteran pest, and/or growth, development, and/or feeding in progeny of the pest. These methods may significantly reduce the size of a subsequent pest generation in an infestation, for example, without directly resulting in mortality in the pest(s) that contact the iRNA molecule.

[0180] In some embodiments, at least one target gene in a coleopteran pest may be selected, wherein the target gene comprises a chromatin remodeling polynucleotide (e.g., a gene). In particular examples, such a chromatin remodeling gene in a coleopteran pest is selected, wherein the target gene comprises a polynucleotide selected from among Diabrotica brahma (SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7); Diabrotica mi-2 (SEQ ID NO:79 and SEQ ID NO:164); Diabrotica iswi-1 (SEQ ID NO:81 and SEQ ID NO:165); Diabrotica chd1 (SEQ ID NO:83); Diabrotica iswi-2 (SEQ ID NO:85 and SEQ ID NO:166); Diabrotica iswi30 (SEQ ID NO:87); Diabrotica ino80 (SEQ ID NO:89); and Diabrotica domino (SEQ ID NO:91). For example, a target gene in certain embodiments comprises a chromatin remodeling polynucleotide selected from among SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164; SEQ ID NO:165; SEQ ID NO:166; and fragments of any of the foregoing (e.g., SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NOs:101-106).

[0181] In some embodiments, a chromatin remodeling polynucleotide encodes a member of the group of "ATP-dependent remodeling enzymes," a class of ATPases that contain a SNF2 domain (sucrose non-fermenting, originally identified in Saccharomyces cerevisiae). ATP-dependent remodeling enzymes include, for example and without limitation, BRAHMA and its orthologs; MI-2 and its orthologs; ISWI-1 and its orthologs; CHD1 and its orthologs; ISWI-2 and its orthologs; ISWI30 and its orthologs; IN080 and its orthologs; and DOMINO and its orthologs. Chromatin remodelers (e.g., ATP-dependent remodeling enzymes) exert lasting epigenetic effects by mobilizing nucleosomes and thus changing the access of the transcriptional machinery to DNA.

[0182] ATP-dependent remodeling enzymes share the same functional domains and sequence-level conservation. In Pfam (pfam.sanger.ac.uk) searches, ATP-dependent remodeling enzymes can be identified by a combination of SNF2 family N-terminal and Helicase conserved C-terminal (SNF2-Helicase) domains. Thus, RNAi target sites can be designed within the conserved SNF2 family N-terminal and Helicase C-terminal domains (here referred to as SNF2-Helicase) that are common to all chromatin remodelers, as well as chromatin binding or other functional domains that are conserved within each family, which include but are not limited to bromodomain, chromodomain, and HAND-SLIDE domains.

[0183] In some embodiments, a target gene may be a nucleic acid molecule comprising a polynucleotide that can be reverse translated in silico to a polypeptide comprising a contiguous amino acid sequence that is at least about 85% identical (e.g., at least 84%, 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or 100% identical) to the amino acid sequence of a protein product of a chromatin remodeling gene. A target gene may be any nucleic acid in a coleopteran pest, the post-transcriptional inhibition of which has a deleterious effect on the capacity of the pest to produce viable offspring, for example, to provide a protective benefit against the pest to a plant. In particular examples, a target gene is a nucleic acid molecule comprising a polynucleotide that can be reverse translated in silico to a polypeptide comprising a contiguous amino acid sequence that is at least about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 100% identical, or 100% identical to the amino acid sequence that is the in silico translation product of a brahma, mi-2, iswi-1, chd1, iswi-2, iswi30, ino80, or domino gene. Examples of such translation products include, for example and without limitation: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:9; SEQ ID NO:80; SEQ ID NO:82; SEQ ID NO:84; SEQ ID NO:86; SEQ ID NO:88; SEQ ID NO:90; and SEQ ID NO:92.

[0184] Provided in some embodiments are DNAs, the expression of which results in an RNA molecule comprising a polynucleotide that is specifically complementary to all or part of a native RNA molecule that is encoded by a coding polynucleotide in a coleopteran pest. In some embodiments, after ingestion of the expressed RNA molecule by a coleopteran pest, down-regulation of the coding polynucleotide in cells of the pest, or in cells of progeny of the pest, may be obtained. In particular embodiments, down-regulation of the coding polynucleotide in cells of the coleopteran pest may result in reduction or cessation of reproduction and/or proliferation in the pest, and/or growth, development, and/or feeding in progeny of the pest.

[0185] In some embodiments, target polynucleotides include transcribed non-coding RNAs, such as 5'UTRs; 3'UTRs; spliced leaders; introns; outrons (e.g., 5'UTR RNA subsequently modified in trans splicing); donatrons (e.g., non-coding RNA required to provide donor sequences for trans splicing); and other non-coding transcribed RNA of target coleopteran pest genes. Such polynucleotides may be derived from both mono-cistronic and poly-cistronic genes.

[0186] Thus, also described herein in connection with some embodiments are iRNA molecules (e.g., dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs) that comprise at least one polynucleotide that is specifically complementary to all or part of a target nucleic acid in a coleopteran pest. In some embodiments an iRNA molecule may comprise polynucleotide(s) that are complementary to all or part of a plurality of target nucleic acids; for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids. In particular embodiments, an iRNA molecule may be produced in vitro or in vivo by a genetically-modified organism, such as a plant or bacterium. Also disclosed are cDNAs that may be used for the production of dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary to all or part of a target nucleic acid in a coleopteran pest. Further described are recombinant DNA constructs for use in achieving stable transformation of particular host targets. Transformed host targets may express effective levels of dsRNA, siRNA, miRNA, shRNA, and/or hpRNA molecules from the recombinant DNA constructs. Therefore, also described is a plant transformation vector comprising at least one polynucleotide operably linked to a heterologous promoter functional in a plant cell, wherein expression of the polynucleotide(s) results in an RNA molecule comprising a string of contiguous nucleobases that are specifically complementary to all or part of a target nucleic acid in a coleopteran pest.

[0187] In particular examples, nucleic acid molecules useful for the control of coleopteran pests may include: all or part of a native nucleic acid isolated from Diabrotica comprising a chromatin remodeling gene polynucleotide (e.g., any of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:10; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:101; SEQ ID NO:102; SEQ ID NO:103; SEQ ID NO:104; SEQ ID NO:105; SEQ ID NO:106; SEQ ID NO:164; SEQ ID NO:165; and SEQ ID NO:166); DNAs that when expressed result in an RNA molecule comprising a polynucleotide that is specifically complementary to all or part of a native RNA molecule that is encoded by chromatin remodeling gene; iRNA molecules (e.g., dsRNAs, siRNAs, miRNAs, shRNAs, and hpRNAs) that comprise at least one polynucleotide that is specifically complementary to all or part of an RNA molecule encoded by a chromatin remodeling gene; cDNAs that may be used for the production of dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules, and/or hpRNA molecules that are specifically complementary to all or part of an RNA molecule encoded by a chromatin remodeling gene; and recombinant DNA constructs for use in achieving stable transformation of particular host targets, wherein a transformed host target comprises one or more of the foregoing nucleic acid molecules.

[0188] B. Nucleic Acid Molecules

[0189] The present invention provides, inter alia, iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules that inhibit target gene expression in a cell, tissue, or organ of a coleopteran pest; and DNA molecules capable of being expressed as an iRNA molecule in a cell or microorganism to inhibit target gene expression in a cell, tissue, or organ of a coleopteran pest.

[0190] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NOs:1, 3, 5, and 7; the complement of any of SEQ ID NOs:1, 3, 5, and 7; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of any of SEQ ID NOs:1, 3, 5, and 7 (e.g., SEQ ID NO:8 and SEQ ID NO:10); the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:1, 3, 5, and 7; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising any of SEQ ID NOs:1, 3, 5, and 7; the complement of a native coding polynucleotide of a Diabrotica organism comprising any of SEQ ID NOs:1, 3, 5, and 7; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising any of SEQ ID NOs:1, 3, 5, and 7; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising any of SEQ ID NOs:1, 3, 5, and 7. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0191] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:79; SEQ ID NO:164; the complement of SEQ ID NO:79; the complement of SEQ ID NO:164; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of SEQ ID NO:79 or SEQ ID NO:164 (e.g., SEQ ID NO:104); the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:79 or SEQ ID NO:164; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:79 or SEQ ID NO:164; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:79; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:79 or SEQ ID NO:164; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:79 or SEQ ID NO:164. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0192] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:81; SEQ ID NO:165; the complement of SEQ ID NO:81; the complement of SEQ ID NO:165; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of SEQ ID NO:81 or SEQ ID NO:165; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:81 or SEQ ID NO:165; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:81 or SEQ ID NO:165; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:81 or SEQ ID NO:165; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:81 or SEQ ID NO:165; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:81 or SEQ ID NO:165. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0193] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:83; the complement of SEQ ID NO:83; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of SEQ ID NO:83 (e.g., SEQ ID NO:105); the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:83; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:83; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:83; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:83; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:83. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0194] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:85; SEQ ID NO:166; the complement of SEQ ID NO:85; the complement of SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of SEQ ID NO:85 or SEQ ID NO:166 (e.g., SEQ ID NO:103); the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:85 or SEQ ID NO:166; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:85 or SEQ ID NO:166; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:85 or SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:85 or SEQ ID NO:166; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:85 or SEQ ID NO:166. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0195] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:87; the complement of SEQ ID NO:87; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of SEQ ID NO:87 (e.g., SEQ ID NO:102); the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:87; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:87; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:87; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:87; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:87. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0196] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:89; the complement of SEQ ID NO:89; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of SEQ ID NO:89; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:89; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:89; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:89; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:89. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0197] Some embodiments of the invention provide an isolated nucleic acid molecule comprising at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:91; the complement of SEQ ID NO:91; a fragment of at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of SEQ ID NO:91; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:91; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:91; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:91; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:91; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:91. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest.

[0198] In some embodiments, an isolated nucleic acid molecule of the invention may comprise at least one (e.g., one, two, three, or more) polynucleotide(s) selected from the group consisting of: SEQ ID NO:139; the complement of SEQ ID NO:139; SEQ ID NO:140; the complement of SEQ ID NO:140; SEQ ID NO:141; the complement of SEQ ID NO:141; SEQ ID NO:142; the complement of SEQ ID NO:142; SEQ ID NO:143; the complement of SEQ ID NO:143; SEQ ID NO:144; the complement of SEQ ID NO:144; SEQ ID NO:145; the complement of SEQ ID NO:145; SEQ ID NO:146; the complement of SEQ ID NO:146; SEQ ID NO:147; the complement of SEQ ID NO:147; SEQ ID NO:148; the complement of SEQ ID NO:148; SEQ ID NO:149; the complement of SEQ ID NO:149; SEQ ID NO:150; the complement of SEQ ID NO:150; SEQ ID NO:151; the complement of SEQ ID NO:151; SEQ ID NO:152; the complement of SEQ ID NO:152; SEQ ID NO:153; the complement of SEQ ID NO:153; SEQ ID NO:154; the complement of SEQ ID NO:154; SEQ ID NO:155; the complement of SEQ ID NO:155; SEQ ID NO:156; the complement of SEQ ID NO:156; SEQ ID NO:157; the complement of SEQ ID NO:157; SEQ ID NO:158; the complement of SEQ ID NO:158; SEQ ID NO:159; the complement of SEQ ID NO:159; SEQ ID NO:160; the complement of SEQ ID NO:160; SEQ ID NO:161; the complement of SEQ ID NO:161; SEQ ID NO:162; the complement of SEQ ID NO:162; SEQ ID NO:163; the complement of SEQ ID NO:163; SEQ ID NO:167; the complement of SEQ ID NO:167; SEQ ID NO:168; the complement of SEQ ID NO:168; SEQ ID NO:169; the complement of SEQ ID NO:169; a native polyribonucleotide transcribed in a Diabrotica organism from a gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; the complement of a native polyribonucleotide transcribed in a Diabrotica organism from a gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of a native polyribonucleotide transcribed in a Diabrotica organism from a gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; and the complement of a fragment of at least 15 contiguous nucleotides of a native polyribonucleotide transcribed in a Diabrotica organism from a gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166. In particular embodiments, contact with or uptake by a coleopteran pest of the isolated polynucleotide inhibits the growth, development, reproduction and/or feeding of the pest. In some embodiments, contact with or uptake by the insect occurs via feeding on plant material or bait comprising the iRNA. In some embodiments, contact with or uptake by the insect occurs via spraying of a plant comprising the insect with a composition comprising the iRNA.

[0199] In some embodiments, a nucleic acid molecule of the invention may comprise at least one (e.g., one, two, three, or more) DNA(s) capable of being expressed as an iRNA molecule in a cell or microorganism to inhibit target gene expression in a cell, tissue, or organ of a coleopteran pest. Such DNA(s) may be operably linked to a promoter that functions in a cell comprising the DNA molecule to initiate or enhance the transcription of the encoded RNA capable of forming a dsRNA molecule(s). In one embodiment, the at least one (e.g., one, two, three, or more) DNA(s) may be derived from the polynucleotide of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166. Derivatives of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166 includes fragments of these polynucleotides. In some embodiments, such a fragment may comprise, for example, at least about 15 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166, or a complement thereof. Thus, such a fragment may comprise, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 or more contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166, or a complement thereof. In some examples, such a fragment may comprise, for example, at least 19 contiguous nucleotides (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166, or a complement thereof.

[0200] Some embodiments comprise introducing partially- or fully-stabilized dsRNA molecules into a coleopteran pest to inhibit expression of a target gene in a cell, tissue, or organ of the coleopteran pest. When expressed as an iRNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) and taken up by a coleopteran pest, polynucleotides comprising one or more fragments of any of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164; SEQ ID NO:165; SEQ ID NO:166; and the complements thereof, may cause one or more of death, developmental arrest, growth inhibition, change in sex ratio, reduction in brood size, cessation of infection, and/or cessation of feeding by a coleopteran pest. In particular examples, polynucleotides comprising one or more fragments (e.g., polynucleotides including about 15 to about 300 nucleotides) of any of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164; SEQ ID NO:165; SEQ ID NO:166; and the complements thereof, cause a reduction in the capacity of an existing generation of the pest to produce a subsequent generation of the pest.

[0201] In certain embodiments, dsRNA molecules provided by the invention comprise polynucleotides complementary to a transcript from a target gene comprising SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and/or SEQ ID NO:166, and/or polynucleotides complementary to a fragment of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and/or SEQ ID NO:166, the inhibition of which target gene in a coleopteran pest results in the reduction or removal of a polypeptide or polynucleotide agent that is essential for the pest's or the pest's progeny's growth, development, or other biological function. A selected polynucleotide may exhibit from about 80% to about 100% sequence identity to SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and/or SEQ ID NO:166, a contiguous fragment of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and/or SEQ ID NO:166, or the complement of either of the foregoing. For example, a selected polynucleotide may exhibit 79%; 80%; about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94% about 95%; about 96%; about 97%; about 98%; about 98.5%; about 99%; about 99.5%; or about 100% sequence identity to SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and/or SEQ ID NO:166, a contiguous fragment of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and/or SEQ ID NO:166, or the complement of any of the foregoing.

[0202] In some embodiments, a DNA molecule capable of being expressed as an iRNA molecule in a cell or microorganism to inhibit target gene expression may comprise a single polynucleotide that is specifically complementary to all or part of a native polynucleotide found in one or more target coleopteran pest species, or the DNA molecule can be constructed as a chimera from a plurality of such specifically complementary polynucleotides.

[0203] In some embodiments, a nucleic acid molecule may comprise a first and a second polynucleotide separated by a "linker." A linker may be a region comprising any sequence of nucleotides that facilitates secondary structure formation between the first and second polynucleotides, where this is desired. In one embodiment, the linker is part of a sense or antisense coding polynucleotide for mRNA. The linker may alternatively comprise any combination of nucleotides or homologues thereof that are capable of being linked covalently to a nucleic acid molecule. In some examples, the linker may comprise an intron (e.g., as ST-LS1 intron).

[0204] For example, in some embodiments, the DNA molecule may comprise a polynucleotide coding for one or more different RNA molecules, wherein each of the different RNA molecules comprises a first polynucleotide and a second polynucleotide, wherein the first and second polynucleotides are complementary to each other. The first and second polynucleotides may be connected within an RNA molecule by a linker. The linker may constitute part of the first polynucleotide or the second polynucleotide. Expression of an RNA molecule comprising the first and second nucleotide polynucleotides may lead to the formation of a dsRNA molecule of the present invention, by specific intramolecular base-pairing of the first and second nucleotide polynucleotides. The first polynucleotide or the second polynucleotide may be substantially identical to a polynucleotide native to a coleopteran pest (e.g., a target gene, or transcribed non-coding polynucleotide), a derivative thereof, or a complementary polynucleotide thereto.

[0205] dsRNA nucleic acid molecules comprise double strands of polymerized ribonucleotides, and may include modifications to either the phosphate-sugar backbone or the nucleoside. Modifications in RNA structure may be tailored to allow specific inhibition. In one embodiment, dsRNA molecules may be modified through a ubiquitous enzymatic process so that siRNA molecules may be generated. This enzymatic process may utilize an RNase III enzyme, such as DICER in eukaryotes, either in vitro or in vivo. See Elbashir et al. (2001) Nature 411:494-8; and Hamilton and Baulcombe (1999) Science 286(5441):950-2. DICER or functionally-equivalent RNase III enzymes cleave larger dsRNA strands and/or hpRNA molecules into smaller oligonucleotides (e.g., siRNAs), each of which is about 19-25 nucleotides in length. The siRNA molecules produced by these enzymes have 2 to 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini. The siRNA molecules generated by RNase III enzymes are unwound and separated into single-stranded RNA in the cell. The siRNA molecules then specifically hybridize with RNAs transcribed from a target gene, and both RNA molecules are subsequently degraded by an inherent cellular RNA-degrading mechanism. This process may result in the effective degradation or removal of the RNA encoded by the target gene in the target organism. The outcome is the post-transcriptional silencing of the targeted gene. In some embodiments, siRNA molecules produced by endogenous RNase III enzymes from heterologous nucleic acid molecules may efficiently mediate the down-regulation of target genes in coleopteran pests.

[0206] In some embodiments, a nucleic acid molecule of the invention may include at least one non-naturally occurring polynucleotide that can be transcribed into a single-stranded RNA molecule capable of forming a dsRNA molecule in vivo through intermolecular hybridization. Such dsRNAs typically self-assemble, and can be provided in the nutrition source of a coleopteran pest to achieve the post-transcriptional inhibition of a target gene. In these and further embodiments, a nucleic acid molecule of the invention may comprise two different non-naturally occurring polynucleotides, each of which is specifically complementary to a different target gene in a coleopteran pest. When such a nucleic acid molecule is provided as a dsRNA molecule to a coleopteran pest, the dsRNA molecule inhibits the expression of at least two different target genes in the pest.

[0207] C. Obtaining Nucleic Acid Molecules

[0208] A variety of polynucleotides in coleopteran pests may be used as targets for the design of nucleic acid molecules of the invention, such as iRNAs and DNA molecules encoding iRNAs. Selection of native polynucleotides is not, however, a straight-forward process. Only a small number of native polynucleotides in the coleopteran pest will be effective targets. For example, it cannot be predicted with certainty whether a particular native polynucleotide can be effectively down-regulated by nucleic acid molecules of the invention, or whether down-regulation of a particular native polynucleotide will have a detrimental effect on the growth, viability, proliferation, and/or reproduction of the coleopteran pest. The vast majority of native coleopteran pest polynucleotides, such as ESTs isolated therefrom (e.g., the coleopteran pest polynucleotides listed in U.S. Pat. No. 7,612,194), do not have a detrimental effect on the growth, viability, proliferation, and/or reproduction of the pest. Neither is it predictable which of the native polynucleotides that may have a detrimental effect on a coleopteran pest are able to be used in recombinant techniques for expressing nucleic acid molecules complementary to such native polynucleotides in a host plant and providing the detrimental effect on the pest upon feeding without causing harm to the host plant.

[0209] In some embodiments, nucleic acid molecules of the invention (e.g., dsRNA molecules to be provided in the host plant of a coleopteran pest) are selected to target cDNAs that encode proteins or parts of proteins essential for coleopteran pest reproduction and/or development, such as polypeptides involved in metabolic or catabolic biochemical pathways, cell division, reproduction, energy metabolism, embryonic development, larval development, transcriptional regulation, and the like. As described herein, ingestion of compositions by a target organism containing one or more dsRNAs, at least one segment of which is specifically complementary to at least a substantially identical segment of RNA produced in the cells of the target pest organism, can result in failure or reduction of the capacity to mate, oviposit, or produce viable progeny. A polynucleotide, either DNA or RNA, derived from a coleopteran pest can be used to construct plant cells resistant to infestation by the pests. The host plant of the coleopteran pest (e.g., Z. mays), for example, can be transformed to contain one or more of the polynucleotides derived from the coleopteran pest as provided herein. The polynucleotide transformed into the host may encode one or more RNAs that form into a dsRNA structure in the cells or biological fluids within the transformed host, thus making the dsRNA available if/when the pest forms a nutritional relationship with the transgenic host. This may result in the suppression of expression of one or more genes in the cells of the pest, and ultimately inhibition of reproduction and/or development.

[0210] Thus, in some embodiments, a gene is targeted that is essentially involved in the growth, development and reproduction of a coleopteran pest. Other target genes for use in the present invention may include, for example, those that play important roles in coleopteran pest viability, movement, migration, growth, development, infectivity, and establishment of feeding sites. A target gene may therefore be a housekeeping gene or a transcription factor. Additionally, a native coleopteran pest polynucleotide for use in the present invention may also be derived from a homolog (e.g., an ortholog), of a plant, viral, bacterial or insect gene, the function of which is known to those of skill in the art, and the polynucleotide of which is specifically hybridizable with a target gene in the genome of the target coleopteran pest. Methods of identifying a homolog of a gene with a known nucleotide sequence by hybridization are known to those of skill in the art.

[0211] In some embodiments, the invention provides methods for obtaining a nucleic acid molecule comprising a polynucleotide for producing an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule. One such embodiment comprises: (a) analyzing one or more target gene(s) for their expression, function, and phenotype upon dsRNA-mediated gene suppression in a coleopteran pest; (b) probing a cDNA or gDNA library with a probe comprising all or a portion of a polynucleotide or a homolog thereof from a targeted coleopteran pest that displays an altered (e.g., reduced) reproduction or development phenotype in a dsRNA-mediated suppression analysis; (c) identifying a DNA clone that specifically hybridizes with the probe; (d) isolating the DNA clone identified in step (b); (e) sequencing the cDNA or gDNA fragment that comprises the clone isolated in step (d), wherein the sequenced nucleic acid molecule comprises all or a substantial portion of the RNA or a homolog thereof; and (f) chemically synthesizing all or a substantial portion of a gene, or an siRNA, miRNA, hpRNA, mRNA, shRNA, or dsRNA.

[0212] In further embodiments, a method for obtaining a nucleic acid fragment comprising a polynucleotide for producing a substantial portion of an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule includes: (a) synthesizing first and second oligonucleotide primers specifically complementary to a portion of a native polynucleotide from a targeted coleopteran pest; and (b) amplifying a cDNA or gDNA insert present in a cloning vector using the first and second oligonucleotide primers of step (a), wherein the amplified nucleic acid molecule comprises a substantial portion of a siRNA, miRNA, hpRNA, mRNA, shRNA, or dsRNA molecule.

[0213] Nucleic acids of the invention can be isolated, amplified, or produced by a number of approaches. For example, an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule may be obtained by PCR amplification of a target polynucleotide (e.g., a target gene or a target transcribed non-coding polynucleotide) derived from a gDNA or cDNA library, or portions thereof. DNA or RNA may be extracted from a target organism, and nucleic acid libraries may be prepared therefrom using methods known to those ordinarily skilled in the art. gDNA or cDNA libraries generated from a target organism may be used for PCR amplification and sequencing of target genes. A confirmed PCR product may be used as a template for in vitro transcription to generate sense and antisense RNA with minimal promoters. Alternatively, nucleic acid molecules may be synthesized by any of a number of techniques (See, e.g., Ozaki et al. (1992) Nucleic Acids Research, 20: 5205-5214; and Agrawal et al. (1990) Nucleic Acids Research, 18: 5419-5423), including use of an automated DNA synthesizer (for example, a P.E. Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNA Synthesizer), using standard chemistries, such as phosphoramidite chemistry. See, e.g., Beaucage et al. (1992) Tetrahedron, 48: 2223-2311; U.S. Pat. Nos. 4,980,460, 4,725,677, 4,415,732, 4,458,066, and 4,973,679. Alternative chemistries resulting in non-natural backbone groups, such as phosphorothioate, phosphoramidate, and the like, can also be employed.

[0214] An RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA molecule of the present invention may be produced chemically or enzymatically by one skilled in the art through manual or automated reactions, or in vivo in a cell comprising a nucleic acid molecule comprising a polynucleotide encoding the RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA molecule. RNA may also be produced by partial or total organic synthesis; any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. An RNA molecule may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase). Expression constructs useful for the cloning and expression of polynucleotides are known in the art. See, e.g., International PCT Publication No. WO97/32016; and U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693. RNA molecules that are synthesized chemically or by in vitro enzymatic synthesis may be purified prior to introduction into a cell. For example, RNA molecules can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, RNA molecules that are synthesized chemically or by in vitro enzymatic synthesis may be used with no or a minimum of purification, for example, to avoid losses due to sample processing. The RNA molecules may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and/or stabilization of dsRNA molecule duplex strands.

[0215] In embodiments, a dsRNA molecule may be formed by a single self-complementary RNA strand or from two complementary RNA strands. dsRNA molecules may be synthesized either in vivo or in vitro. An endogenous RNA polymerase of the cell may mediate transcription of the one or two RNA strands in vivo, or cloned RNA polymerase may be used to mediate transcription in vivo or in vitro. Post-transcriptional inhibition of a target gene in a coleopteran pest may be host-targeted by specific transcription in an organ, tissue, or cell type of the host (e.g., by using a tissue-specific promoter); stimulation of an environmental condition in the host (e.g., by using an inducible promoter that is responsive to infection, stress, temperature, and/or chemical inducers); and/or engineering transcription at a developmental stage or age of the host (e.g., by using a developmental stage-specific promoter). RNA strands that form a dsRNA molecule, whether transcribed in vitro or in vivo, may or may not be polyadenylated, and may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.

[0216] D. Recombinant Vectors and Host Cell Transformation

[0217] In some embodiments, the invention also provides a DNA molecule for introduction into a cell (e.g., a bacterial cell, a yeast cell, or a plant cell), wherein the DNA molecule comprises a polynucleotide that, upon expression to RNA and ingestion by a coleopteran pest, achieves suppression of a target gene in a cell, tissue, or organ of the pest. Thus, some embodiments provide a recombinant nucleic acid molecule comprising a polynucleotide capable of being expressed as an iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecule in a plant cell to inhibit target gene expression in a coleopteran pest. In order to initiate or enhance expression, such recombinant nucleic acid molecules may comprise one or more regulatory elements, which regulatory elements may be operably linked to the polynucleotide capable of being expressed as an iRNA. Methods to express a gene suppression molecule in plants are known, and may be used to express a polynucleotide of the present invention. See, e.g., International PCT Publication No. WO06/073727; and U.S. Patent Publication No. 2006/0200878 A1).

[0218] In specific embodiments, a recombinant DNA molecule of the invention may comprise a polynucleotide encoding an RNA that may form a dsRNA molecule. Such recombinant DNA molecules may encode RNAs that may form dsRNA molecules capable of inhibiting the expression of endogenous target gene(s) in a coleopteran pest cell upon ingestion. In many embodiments, a transcribed RNA may form a dsRNA molecule that may be provided in a stabilized form; e.g., as a hairpin and stem and loop structure.

[0219] In some embodiments, one strand of a dsRNA molecule may be formed by transcription from a polynucleotide which is substantially homologous to the RNA encoded by a polynucleotide selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166; the complement of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; a native coding polynucleotide of a Diabrotica organism (e.g., WCR) comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, or SEQ ID NO:166.

[0220] In particular embodiments, a recombinant DNA molecule encoding an RNA that may form a dsRNA molecule may comprise a coding region wherein at least two polynucleotides are arranged such that one polynucleotide is in a sense orientation, and the other polynucleotide is in an antisense orientation, relative to at least one promoter, wherein the sense polynucleotide and the antisense polynucleotide are linked or connected by a linker of, for example, from about five (.about.5) to about one thousand (.about.1000) nucleotides. The linker may form a loop between the sense and anti sense polynucleotides. The sense polynucleotide or the antisense polynucleotide may be substantially homologous to an RNA encoded by a target gene (e.g., a chromatin remodeling gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166) or fragment thereof. In some embodiments, however, a recombinant DNA molecule may encode an RNA that may form a dsRNA molecule without a linker. In embodiments, a sense coding polynucleotide and an anti sense coding polynucleotide may be different lengths.

[0221] Polynucleotides identified as having a deleterious effect on coleopteran pests or a plant-protective effect with regard to coleopteran pests may be readily incorporated into expressed dsRNA molecules through the creation of appropriate expression cassettes in a recombinant nucleic acid molecule of the invention. For example, such polynucleotides may be expressed as a hairpin with stem and loop structure by taking a first segment corresponding to an RNA encoded by a target gene polynucleotide (e.g., a chromatin remodeling gene comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, and fragments thereof); linking this polynucleotide to a second segment linker region that is not homologous or complementary to the first segment; and linking this to a third segment, wherein at least a portion of the third segment is substantially complementary to the first segment. Such a construct forms a stem and loop structure by intramolecular base-pairing of the first segment with the third segment, wherein the loop structure forms comprising the second segment. See, e.g., U.S. Patent Publication Nos. 2002/0048814 and 2003/0018993; and International PCT Publication Nos. WO94/01550 and WO98/05770. A dsRNA molecule may be generated, for example, in the form of a double-stranded structure such as a stem-loop structure (e.g., hairpin), whereby production of siRNA targeted for a native coleopteran pest polynucleotide is enhanced by co-expression of a fragment of the targeted gene, for instance on an additional plant expressible cassette, that leads to enhanced siRNA production, or reduces methylation to prevent transcriptional gene silencing of the dsRNA hairpin promoter.

[0222] Embodiments of the invention include introduction of a recombinant nucleic acid molecule of the present invention into a plant (i.e., transformation) to achieve coleopteran pest-protective levels of expression of one or more iRNA molecules. A recombinant DNA molecule may, for example, be a vector, such as a linear or a closed circular plasmid. The vector system may be a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of a host. In addition, a vector may be an expression vector. Nucleic acids of the invention can, for example, be suitably inserted into a vector under the control of a suitable promoter that functions in one or more hosts to drive expression of a linked coding polynucleotide or other DNA element. Many vectors are available for this purpose, and selection of the appropriate vector will depend mainly on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components depending on its function (e.g., amplification of DNA or expression of DNA) and the particular host cell with which it is compatible.

[0223] To impart protection from a coleopteran pest to a transgenic plant, a recombinant DNA may, for example, be transcribed into an iRNA molecule (e.g., an RNA molecule that forms a dsRNA molecule) within the tissues or fluids of the recombinant plant. An iRNA molecule may comprise a polynucleotide that is substantially homologous and specifically hybridizable to a corresponding transcribed polynucleotide within a coleopteran pest that may cause damage to the host plant species. The coleopteran pest may contact the iRNA molecule that is transcribed in cells of the transgenic host plant, for example, by ingesting cells or fluids of the transgenic host plant that comprise the iRNA molecule. Thus, expression of a target gene is suppressed by the iRNA molecule within coleopteran pests that infest the transgenic host plant. In some embodiments, suppression of expression of the target gene in the target coleopteran pest may result in the plant being tolerant to attack by the pest.

[0224] In order to enable delivery of iRNA molecules to a coleopteran pest in a nutritional relationship with a plant cell that has been transformed with a recombinant nucleic acid molecule of the invention, expression (i.e., transcription) of iRNA molecules in the plant cell is required. Thus, a recombinant nucleic acid molecule may comprise a polynucleotide of the invention operably linked to one or more regulatory elements, such as a heterologous promoter element that functions in a host cell, such as a bacterial cell wherein the nucleic acid molecule is to be amplified, and a plant cell wherein the nucleic acid molecule is to be expressed.

[0225] Promoters suitable for use in nucleic acid molecules of the invention include those that are inducible, viral, synthetic, or constitutive, all of which are well known in the art. Non-limiting examples describing such promoters include U.S. Pat. No. 6,437,217 (maize RS81 promoter); U.S. Pat. No. 5,641,876 (rice actin promoter); U.S. Pat. No. 6,426,446 (maize RS324 promoter); U.S. Pat. No. 6,429,362 (maize PR-1 promoter); U.S. Pat. No. 6,232,526 (maize A3 promoter); U.S. Pat. No. 6,177,611 (constitutive maize promoters); U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196 (CaMV 35S promoter); U.S. Pat. No. 6,433,252 (maize L3 oleosin promoter); U.S. Pat. No. 6,429,357 (rice actin 2 promoter, and rice actin 2 intron); U.S. Pat. No. 6,294,714 (light-inducible promoters); U.S. Pat. No. 6,140,078 (salt-inducible promoters); U.S. Pat. No. 6,252,138 (pathogen-inducible promoters); U.S. Pat. No. 6,175,060 (phosphorous deficiency-inducible promoters); U.S. Pat. No. 6,388,170 (bidirectional promoters); U.S. Pat. No. 6,635,806 (gamma-coixin promoter); and U.S. Patent Publication No. 2009/757,089 (maize chloroplast aldolase promoter). Additional promoters include the nopaline synthase (NOS) promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-9) and the octopine synthase (OCS) promoters (which are carried on tumor-inducing plasmids of Agrobacterium tumefaciens); the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-24); the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-2; the figwort mosaic virus 35S-promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84(19):6624-8); the sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-8); the R gene complex promoter (Chandler et al. (1989) Plant Cell 1:1175-83); the chlorophyll a/b binding protein gene promoter; CaMV 35S (U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196); FMV 35S (U.S. Pat. Nos. 6,051,753, and 5,378,619); a PC1SV promoter (U.S. Pat. No. 5,850,019); the SCP1 promoter (U.S. Pat. No. 6,677,503); and AGRtu.nos promoters (GenBank.TM. Accession No. V00087; Depicker et al. (1982) J. Mol. Appl. Genet. 1:561-73; Bevan et al. (1983) Nature 304:184-7).

[0226] In particular embodiments, nucleic acid molecules of the invention comprise a tissue-specific promoter, such as a root-specific promoter. Root-specific promoters drive expression of operably-linked coding polynucleotides exclusively or preferentially in root tissue. Examples of root-specific promoters are known in the art. See, e.g., U.S. Pat. Nos. 5,110,732; 5,459,252 and 5,837,848; and Opperman et al. (1994) Science 263:221-3; and Hirel et al. (1992) Plant Mol. Biol. 20:207-18. In some embodiments, a polynucleotide or fragment for coleopteran pest control according to the invention may be cloned between two root-specific promoters oriented in opposite transcriptional directions relative to the polynucleotide or fragment, and which are operable in a transgenic plant cell and expressed therein to produce RNA molecules in the transgenic plant cell that subsequently may form dsRNA molecules, as described, supra. The iRNA molecules expressed in plant tissues may be ingested by a coleopteran pest so that suppression of target gene expression is achieved.

[0227] Additional regulatory elements that may optionally be operably linked to a nucleic acid molecule of interest include 5'UTRs located between a promoter element and a coding polynucleotide that function as a translation leader element. The translation leader element is present in the fully-processed mRNA, and it may affect processing of the primary transcript, and/or RNA stability. Examples of translation leader elements include maize and petunia heat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus coat protein leaders, plant rubisco leaders, and others. See, e.g., Turner and Foster (1995) Molecular Biotech. 3(3):225-36. Non-limiting examples of 5'UTRs include GmHsp (U.S. Pat. No. 5,659,122); PhDnaK (U.S. Pat. No. 5,362,865); AtAnt1; TEV (Carrington and Freed (1990) J. Virol. 64:1590-7); and AGRtunos (GenBank.TM. Accession No. V00087; and Bevan et al. (1983) Nature 304:184-7).

[0228] Additional regulatory elements that may optionally be operably linked to a nucleic acid molecule of interest also include 3' non-translated elements, 3' transcription termination regions, or polyadenylation regions. These are genetic elements located downstream of a polynucleotide, and include polynucleotides that provide polyadenylation signal, and/or other regulatory signals capable of affecting transcription or mRNA processing. The polyadenylation signal functions in plants to cause the addition of polyadenylate nucleotides to the 3' end of the mRNA precursor. The polyadenylation element can be derived from a variety of plant genes, or from T-DNA genes. A non-limiting example of a 3' transcription termination region is the nopaline synthase 3' region (nos 3'; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7). An example of the use of different 3' nontranslated regions is provided in Ingelbrecht et al., (1989) Plant Cell 1:671-80. Non-limiting examples of polyadenylation signals include one from a Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. (1984) EMBO J. 3:1671-9) and AGRtu.nos (GenBank.TM. Accession No. E01312).

[0229] Some embodiments may include a plant transformation vector that comprises an isolated and purified DNA molecule comprising at least one of the above-described regulatory elements operatively linked to one or more polynucleotides of the present invention. When expressed, the one or more polynucleotides result in one or more RNA molecule(s) comprising a polynucleotide that is specifically complementary to all or part of a native RNA molecule in a coleopteran pest. Thus, the polynucleotide(s) may comprise a segment encoding all or part of a polyribonucleotide present within a targeted coleopteran pest RNA transcript, and may comprise inverted repeats of all or a part of a targeted pest transcript. A plant transformation vector may contain polynucleotides specifically complementary to more than one target polynucleotide, thus allowing production of more than one dsRNA for inhibiting expression of two or more genes in cells of one or more populations or species of target coleopteran pests. Segments of polynucleotides specifically complementary to polynucleotides present in different genes can be combined into a single composite nucleic acid molecule for expression in a transgenic plant. Such segments may be contiguous or separated by a linker.

[0230] In some embodiments, a plasmid of the present invention already containing at least one polynucleotide(s) of the invention can be modified by the sequential insertion of additional polynucleotide(s) in the same plasmid, wherein the additional polynucleotide(s) are operably linked to the same regulatory elements as the original at least one polynucleotide(s). In some embodiments, a nucleic acid molecule may be designed for the inhibition of multiple target genes. In some embodiments, the multiple genes to be inhibited can be obtained from the same coleopteran pest species, which may enhance the effectiveness of the nucleic acid molecule. In other embodiments, the genes can be derived from different insect (e.g., coleopteran) pests, which may broaden the range of pests against which the agent(s) is/are effective. When multiple genes are targeted for suppression or a combination of expression and suppression, a polycistronic DNA element can be engineered.

[0231] A recombinant nucleic acid molecule or vector of the present invention may comprise a selectable marker that confers a selectable phenotype on a transformed cell, such as a plant cell. Selectable markers may also be used to select for plants or plant cells that comprise a recombinant nucleic acid molecule of the invention. The marker may encode biocide resistance, antibiotic resistance (e.g., kanamycin, Geneticin (G418), bleomycin, hygromycin, etc.), or herbicide tolerance (e.g., glyphosate, etc.). Examples of selectable markers include, but are not limited to: a neo gene which codes for kanamycin resistance and can be selected for using kanamycin, G418, etc.; a bar gene which codes for bialaphos resistance; a mutant EPSP synthase gene which encodes glyphosate tolerance; a nitrilase gene which confers resistance to bromoxynil; a mutant acetolactate synthase (ALS) gene which confers imidazolinone or sulfonylurea tolerance; and a methotrexate resistant DHFR gene. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, streptomycin and tetracycline, and the like. Examples of such selectable markers are illustrated in, e.g., U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708; and 6,118,047.

[0232] A recombinant nucleic acid molecule or vector of the present invention may also include a screenable marker. Screenable markers may be used to monitor expression. Exemplary screenable markers include a .beta.-glucuronidase or uidA gene (GUS) which encodes an enzyme for which various chromogenic substrates are known (Jefferson et al. (1987) Plant Mol. Biol. Rep. 5:387-405); an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al. (1988) "Molecular cloning of the maize R-nj allele by transposon tagging with Ac." In 18.sup.th Stadler Genetics Symposium, P. Gustafson and R. Appels, eds. (New York: Plenum), pp. 263-82); a .beta.-lactamase gene (Sutcliffe et al. (1978) Proc. Natl. Acad. Sci. USA 75:3737-41); a gene which encodes an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al. (1986) Science 234:856-9); an xylE gene that encodes a catechol dioxygenase that can convert chromogenic catechols (Zukowski et al. (1983) Gene 46(2-3):247-55); an amylase gene (Ikatu et al. (1990) Bio/Technol. 8:241-2); a tyrosinase gene which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-14); and an .alpha.-galactosidase.

[0233] In some embodiments, recombinant nucleic acid molecules, as described, supra, may be used in methods for the creation of transgenic plants and expression of heterologous nucleic acids in plants to prepare transgenic plants that exhibit reduced susceptibility to coleopteran pests. Plant transformation vectors can be prepared, for example, by inserting nucleic acid molecules encoding iRNA molecules into plant transformation vectors and introducing these into plants.

[0234] Suitable methods for transformation of host cells include any method by which DNA can be introduced into a cell, such as by transformation of protoplasts (See, e.g., U.S. Pat. No. 5,508,184), by desiccation/inhibition-mediated DNA uptake (See, e.g., Potrykus et al. (1985) Mol. Gen. Genet. 199:183-8), by electroporation (See, e.g., U.S. Pat. No. 5,384,253), by agitation with silicon carbide fibers (See, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), by Agrobacterium-mediated transformation (See, e.g., U.S. Pat. Nos. 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; and 6,384,301) and by acceleration of DNA-coated particles (See, e.g., U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and 6,403,865), etc. Techniques that are particularly useful for transforming corn are described, for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616; and International PCT Publication WO95/06722. Through the application of techniques such as these, the cells of virtually any species may be stably transformed. In some embodiments, transforming DNA is integrated into the genome of the host cell. In the case of multicellular species, transgenic cells may be regenerated into a transgenic organism. Any of these techniques may be used to produce a transgenic plant, for example, comprising one or more nucleic acids encoding one or more iRNA molecules in the genome of the transgenic plant.

[0235] The most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. The Ti (tumor-inducing)-plasmids contain a large segment, known as T-DNA, which is transferred to transformed plants. Another segment of the Ti plasmid, the Vir region, is responsible for T-DNA transfer. The T-DNA region is bordered by terminal repeats. In modified binary vectors, the tumor-inducing genes have been deleted, and the functions of the Vir region are utilized to transfer foreign DNA bordered by the T-DNA border elements. The T-region may also contain a selectable marker for efficient recovery of transgenic cells and plants, and a multiple cloning site for inserting polynucleotides for transfer such as a dsRNA encoding nucleic acid.

[0236] Thus, in some embodiments, a plant transformation vector is derived from a Ti plasmid of A. tumefaciens (See, e.g., U.S. Pat. Nos. 4,536,475, 4,693,977, 4,886,937, and 5,501,967; and European Patent No. EP 0 122 791) or a Ri plasmid of A. rhizogenes. Additional plant transformation vectors include, for example and without limitation, those described by Herrera-Estrella et al. (1983) Nature 303:209-13; Bevan et al. (1983) Nature 304:184-7; Klee et al. (1985) Bio/Technol. 3:637-42; and in European Patent No. EP 0 120 516, and those derived from any of the foregoing. Other bacteria such as Sinorhizobium, Rhizobium, and Mesorhizobium that interact with plants naturally can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria can be made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector.

[0237] After providing exogenous DNA to recipient cells, transformed cells are generally identified for further culturing and plant regeneration. In order to improve the ability to identify transformed cells, one may desire to employ a selectable or screenable marker gene, as previously set forth, with the transformation vector used to generate the transformant. In the case where a selectable marker is used, transformed cells are identified within the potentially transformed cell population by exposing the cells to a selective agent or agents. In the case where a screenable marker is used, cells may be screened for the desired marker gene trait.

[0238] Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. In some embodiments, any suitable plant tissue culture media (e.g., MS and N6 media) may be modified by including further substances, such as growth regulators. Tissue may be maintained on a basic medium with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration (e.g., at least 2 weeks), then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoots are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturation.

[0239] To confirm the presence of a nucleic acid molecule of interest (for example, a DNA encoding one or more iRNA molecules that inhibit target gene expression in a coleopteran pest) in the regenerating plants, a variety of assays may be performed. Such assays include, for example: molecular biological assays, such as Southern and northern blotting, PCR, and nucleic acid sequencing; biochemical assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISA and/or western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and analysis of the phenotype of the whole regenerated plant.

[0240] Integration events may be analyzed, for example, by PCR amplification using, e.g., oligonucleotide primers specific for a nucleic acid molecule of interest. PCR genotyping is understood to include, but not be limited to, polymerase-chain reaction (PCR) amplification of gDNA derived from isolated host plant callus tissue predicted to contain a nucleic acid molecule of interest integrated into the genome, followed by standard cloning and sequence analysis of PCR amplification products. Methods of PCR genotyping have been well described (for example, Rios, G. et al. (2002) Plant J. 32:243-53) and may be applied to gDNA derived from any plant species (e.g., Z. mays) or tissue type, including cell cultures.

[0241] A transgenic plant formed using Agrobacterium-dependent transformation methods typically contains a single recombinant DNA inserted into one chromosome. The polynucleotide of the single recombinant DNA is referred to as a "transgenic event" or "integration event". Such transgenic plants are heterozygous for the inserted exogenous polynucleotide. In some embodiments, a transgenic plant homozygous with respect to a transgene may be obtained by sexually mating (selfing) an independent segregant transgenic plant that contains a single exogenous gene to itself, for example a T.sub.0 plant, to produce T.sub.1 seed. One fourth of the T.sub.1 seed produced will be homozygous with respect to the transgene. Germinating T.sub.1 seed results in plants that can be tested for heterozygosity, typically using an SNP assay or a thermal amplification assay that allows for the distinction between heterozygotes and homozygotes (i.e., a zygosity assay).

[0242] In particular embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different iRNA molecules are produced in a plant cell that have a coleopteran pest-protective effect. The iRNA molecules (e.g., dsRNA molecules) may be expressed from multiple nucleic acids introduced in different transformation events, or from a single nucleic acid introduced in a single transformation event. In some embodiments, a plurality of iRNA molecules are expressed under the control of a single promoter. In other embodiments, a plurality of iRNA molecules are expressed under the control of multiple promoters. Single iRNA molecules may be expressed that comprise multiple polynucleotides that are each homologous to different loci within one or more coleopteran pests (for example, the loci defined by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166), both in different populations of the same species of coleopteran pest, or in different species of coleopteran pests.

[0243] In addition to direct transformation of a plant with a recombinant nucleic acid molecule, transgenic plants can be prepared by crossing a first plant having at least one transgenic event with a second plant lacking such an event. For example, a recombinant nucleic acid molecule comprising a polynucleotide that encodes an iRNA molecule may be introduced into a first plant line that is amenable to transformation to produce a transgenic plant, which transgenic plant may be crossed with a second plant line to introgress the polynucleotide that encodes the iRNA molecule into the second plant line.

[0244] The invention also includes commodity products containing one or more of the polynucleotides of the present invention. Particular embodiments include commodity products produced from a recombinant plant or seed containing one or more of the polynucleotides of the present invention. A commodity product containing one or more of the polynucleotides of the present invention is intended to include, but not be limited to, meals, oils, crushed or whole grains or seeds of a plant, or any food product comprising any meal, oil, or crushed or whole grain of a recombinant plant or seed containing one or more of the polynucleotides of the present invention. The detection of one or more of the polynucleotides of the present invention in one or more commodity or commodity products contemplated herein is de facto evidence that the commodity or commodity product is produced from a transgenic plant designed to express one or more of the polynucleotides of the present invention for the purpose of controlling plant pests using dsRNA-mediated gene suppression methods.

[0245] In some aspects, seeds and commodity products produced by transgenic plants derived from transformed plant cells are included, wherein the seeds or commodity products comprise a detectable amount of a nucleic acid of the invention. In some embodiments, such commodity products may be produced, for example, by obtaining transgenic plants and preparing food or feed from them. Commodity products comprising one or more of the polynucleotides of the invention includes, for example and without limitation: meals, oils, crushed or whole grains or seeds of a plant, and any food product comprising any meal, oil, or crushed or whole grain of a recombinant plant or seed comprising one or more of the nucleic acids of the invention. The detection of one or more of the polynucleotides of the invention in one or more commodity or commodity products is de facto evidence that the commodity or commodity product is produced from a transgenic plant designed to express one or more of the iRNA molecules of the invention for the purpose of controlling coleopteran pests.

[0246] In some embodiments, a transgenic plant or seed comprising a nucleic acid molecule of the invention also may comprise at least one other transgenic event in its genome, including without limitation: a transgenic event from which is transcribed an iRNA molecule targeting a locus in a coleopteran pest other than the ones defined by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166; a transgenic event from which is transcribed an iRNA molecule targeting a gene in an organism other than a coleopteran pest (e.g., a plant-parasitic nematode); a gene encoding an insecticidal protein (e.g., a Bacillus thuringiensis insecticidal protein); a herbicide tolerance gene (e.g., a gene providing tolerance to glyphosate); and a gene contributing to a desirable phenotype in the transgenic plant, such as increased yield, altered fatty acid metabolism, or restoration of cytoplasmic male sterility. In particular embodiments, polynucleotides encoding iRNA molecules of the invention may be combined with other insect control and disease traits in a plant to achieve desired traits for enhanced control of plant disease and insect damage. Combining insect control traits that employ distinct modes-of-action may provide protected transgenic plants with superior durability over plants harboring a single control trait, for example, because of the reduced probability that resistance to the trait(s) will develop in the field.

V. Target Gene Suppression in a Coleopteran Pest

[0247] A. Overview

[0248] In some embodiments of the invention, at least one nucleic acid molecule useful for the control of coleopteran pests may be provided to a coleopteran pest, wherein the nucleic acid molecule leads to RNAi-mediated gene silencing in the pest. In particular embodiments, an iRNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) may be provided to the coleopteran pest. In some embodiments, a nucleic acid molecule useful for the control of coleopteran pests may be provided to a pest by contacting the nucleic acid molecule with the pest. In these and further embodiments, a nucleic acid molecule useful for the control of coleopteran pests may be provided in a feeding substrate of the pest, for example, a nutritional composition. In these and further embodiments, a nucleic acid molecule useful for the control of a coleopteran pest may be provided through ingestion of plant material comprising the nucleic acid molecule that is ingested by the pest. In certain embodiments, the nucleic acid molecule is present in plant material through expression of a recombinant nucleic acid introduced into the plant material, for example, by transformation of a plant cell with a vector comprising the recombinant nucleic acid and regeneration of a plant material or whole plant from the transformed plant cell.

[0249] B. RNAi-Mediated Target Gene Suppression

[0250] In embodiments, the invention provides iRNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) that may be designed to target essential native polynucleotides (e.g., essential genes) in the transcriptome of a coleopteran (e.g., WCR or NCR) pest, for example by designing an iRNA molecule that comprises at least one strand comprising a polynucleotide that is specifically complementary to the target polynucleotide. The sequence of an iRNA molecule so designed may be identical to that of the target polynucleotide, or may incorporate mismatches that do not prevent specific hybridization between the iRNA molecule and its target polynucleotide.

[0251] iRNA molecules of the invention may be used in methods for gene suppression in a coleopteran pest, thereby reducing the level or incidence of damage caused by the pest on a plant (for example, a protected transformed plant comprising an iRNA molecule). As used herein the term "gene suppression" refers to any of the well-known methods for reducing the levels of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA, including the reduction of protein expression from a gene or a coding polynucleotide including post-transcriptional inhibition of expression and transcriptional suppression. Post-transcriptional inhibition is mediated by specific homology between all or a part of an mRNA transcribed from a gene targeted for suppression and the corresponding iRNA molecule used for suppression. Additionally, post-transcriptional inhibition refers to the substantial and measurable reduction of the amount of mRNA available in the cell for binding by ribosomes.

[0252] In embodiments wherein an iRNA molecule is a dsRNA molecule, the dsRNA molecule may be cleaved by the enzyme, DICER, into short siRNA molecules (approximately 20 nucleotides in length). The double-stranded siRNA molecule generated by DICER activity upon the dsRNA molecule may be separated into two single-stranded siRNAs; the "passenger strand" and the "guide strand". The passenger strand may be degraded, and the guide strand may be incorporated into RISC. Post-transcriptional inhibition occurs by specific hybridization of the guide strand with a specifically complementary polynucleotide of an mRNA molecule, and subsequent cleavage by the enzyme, Argonaute (catalytic component of the RISC complex).

[0253] In embodiments of the invention, any form of iRNA molecule may be used. Those of skill in the art will understand that dsRNA molecules typically are more stable during preparation and during the step of providing the iRNA molecule to a cell than are single-stranded RNA molecules, and are typically also more stable in a cell. Thus, while siRNA and miRNA molecules, for example, may be equally effective in some embodiments, a dsRNA molecule may be chosen due to its stability.

[0254] In particular embodiments, a nucleic acid molecule is provided that comprises a polynucleotide, which polynucleotide may be expressed in vitro to produce an iRNA molecule that is substantially homologous to a nucleic acid molecule encoded by a polynucleotide within the genome of a coleopteran pest. In certain embodiments, the in vitro transcribed iRNA molecule may be a stabilized dsRNA molecule that comprises a stem-loop structure. After a coleopteran pest contacts the in vitro transcribed iRNA molecule, post-transcriptional inhibition of a target gene in the pest (for example, an essential gene) may occur.

[0255] In some embodiments of the invention, expression of an iRNA from a nucleic acid molecule comprising at least 15 contiguous nucleotides (e.g., at least 19 contiguous nucleotides) of a polynucleotide are used in a method for post-transcriptional inhibition of a target gene in a coleopteran pest, wherein the polynucleotide is selected from the group consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; SEQ ID NO:3; the complement of SEQ ID NO:3; SEQ ID NO:5; the complement of SEQ ID NO:5; SEQ ID NO:7; the complement of SEQ ID NO:7; SEQ ID NO:79; the complement of SEQ ID NO:79; SEQ ID NO:81; the complement of SEQ ID NO:81; SEQ ID NO:83; the complement of SEQ ID NO:83; SEQ ID NO:85; the complement of SEQ ID NO:85; SEQ ID NO:87; the complement of SEQ ID NO:87; SEQ ID NO:89; the complement of SEQ ID NO:89; SEQ ID NO:91; the complement of SEQ ID NO:91; SEQ ID NO:164; the complement of SEQ ID NO:164; SEQ ID NO:165; the complement of SEQ ID NO:165; SEQ ID NO:166, the complement of SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:3; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:3; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:5; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:5; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:7; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:7; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:79; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:79; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:81; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:81; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:83; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:83; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:85; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:85; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:87; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:87; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:91; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:91; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:164; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:164; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:165; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:165; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:166; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:166; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:1; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:1; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:3; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:3; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:5; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:5; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:7; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:7; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:79; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:79; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:81; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:81; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:83; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:83; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:85; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:85; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:87; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:87; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:89; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:89; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:91; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:91; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:164; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:164; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:165; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:165; a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:166; the complement of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:1; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:1; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:3; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:3; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:5; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:5; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:7; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:7; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:79; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:79; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:81; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:81; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:83; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:83; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:85; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:85; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:87; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:87; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:89; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:89; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:91; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:91; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:164; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:164; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:165; the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:165; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:166; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising SEQ ID NO:166. In certain embodiments, expression of a nucleic acid molecule that is at least about 80% identical (e.g., 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, and 100%) with any of the foregoing may be used. In these and further embodiments, a nucleic acid molecule may be expressed that specifically hybridizes to an RNA molecule present in at least one cell of a coleopteran pest.

[0256] It is an important feature of some embodiments herein that the RNAi post-transcriptional inhibition system is able to tolerate sequence variations among target genes that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. The introduced nucleic acid molecule may not need to be absolutely homologous to either a primary transcription product or a fully-processed mRNA of a target gene, so long as the introduced nucleic acid molecule is specifically hybridizable to either a primary transcription product or a fully-processed mRNA of the target gene. Moreover, the introduced nucleic acid molecule may not need to be full-length, relative to either a primary transcription product or a fully processed mRNA of the target gene.

[0257] Inhibition of a target gene using the iRNA technology of the present invention is sequence-specific; i.e., polynucleotides substantially homologous to the iRNA molecule(s) are targeted for genetic inhibition. In some embodiments, an RNA molecule comprising a polynucleotide with a nucleotide sequence that is identical to that of a portion of a target gene may be used for inhibition. In these and further embodiments, an RNA molecule comprising a polynucleotide with one or more insertion, deletion, and/or point mutations relative to a target polynucleotide may be used. In particular embodiments, an iRNA molecule and a portion of a target gene may share, for example, at least from about 80%, at least from about 81%, at least from about 82%, at least from about 83%, at least from about 84%, at least from about 85%, at least from about 86%, at least from about 87%, at least from about 88%, at least from about 89%, at least from about 90%, at least from about 91%, at least from about 92%, at least from about 93%, at least from about 94%, at least from about 95%, at least from about 96%, at least from about 97%, at least from about 98%, at least from about 99%, at least from about 100%, and 100% sequence identity. Alternatively, the duplex region of a dsRNA molecule may be specifically hybridizable with a portion of a target gene transcript. In specifically hybridizable molecules, a less than full length polynucleotide exhibiting a greater homology compensates for a longer, less homologous polynucleotide. The length of the polynucleotide of a duplex region of a dsRNA molecule that is identical to a portion of a target gene transcript may be at least about 25, 50, 100, 200, 300, 400, 500, or at least about 1000 bases. In some embodiments, a polynucleotide of greater than 20-100 nucleotides may be used; for example, a polynucleotide of 100-200 or 300-500 nucleotides may be used. In particular embodiments, a polynucleotide of greater than about 200-300 nucleotides may be used. In particular embodiments, a polynucleotide of greater than about 500-1000 nucleotides may be used, depending on the size of the target gene.

[0258] In certain embodiments, expression of a target gene in a coleopteran pest may be inhibited by at least 10%; at least 33%; at least 50%; or at least 80% within a cell of the pest, such that a significant inhibition takes place. Significant inhibition refers to inhibition over a threshold that results in a detectable phenotype (e.g., cessation of reproduction, feeding, development, etc.), or a detectable decrease in RNA and/or gene product corresponding to the target gene being inhibited. Although in certain embodiments of the invention inhibition occurs in substantially all cells of the pest, in other embodiments inhibition occurs only in a subset of cells expressing the target gene.

[0259] In some embodiments, transcriptional suppression is mediated by the presence in a cell of a dsRNA molecule exhibiting substantial sequence identity to a promoter DNA or the complement thereof to effect what is referred to as "promoter trans suppression." Gene suppression may be effective against target genes in a coleopteran pest that may ingest or contact such dsRNA molecules, for example, by ingesting or contacting plant material containing the dsRNA molecules. dsRNA molecules for use in promoter trans suppression may be specifically designed to inhibit or suppress the expression of one or more homologous or complementary polynucleotides in the cells of the coleopteran pest. Post-transcriptional gene suppression by antisense or sense oriented RNA to regulate gene expression in plant cells is disclosed in U.S. Pat. Nos. 5,107,065; 5,759,829; 5,283,184; and 5,231,020.

[0260] C. Expression of IRNA Molecules Provided to a Coleopteran Pest

[0261] Expression of iRNA molecules for RNAi-mediated gene inhibition in a coleopteran pest may be carried out in any one of many in vitro or in vivo formats. The iRNA molecules may then be provided to a coleopteran pest, for example, by contacting the iRNA molecules with the pest, or by causing the pest to ingest or otherwise internalize the iRNA molecules. Some embodiments of the invention include transformed host plants of a coleopteran pest, transformed plant cells, and progeny of transformed plants. The transformed plant cells and transformed plants may be engineered to express one or more of the iRNA molecules, for example, under the control of a heterologous promoter, to provide a pest-protective effect. Thus, when a transgenic plant or plant cell is consumed by a coleopteran pest during feeding, the pest may ingest iRNA molecules expressed in the transgenic plants or cells. The polynucleotides of the present invention may also be introduced into a wide variety of prokaryotic and eukaryotic microorganism hosts to produce iRNA molecules. The term "microorganism" includes prokaryotic and eukaryotic species, such as bacteria and fungi.

[0262] Modulation of gene expression may include partial or complete suppression of such expression. In another embodiment, a method for suppression of gene expression in a coleopteran pest comprises providing in the tissue of the host of the pest a gene-suppressive amount of at least one dsRNA molecule formed following transcription of a polynucleotide as described herein, at least one segment of which is complementary to an mRNA within the cells of the coleopteran pest. A dsRNA molecule, including its modified form such as an siRNA, miRNA, shRNA, or hpRNA molecule, ingested by a coleopteran pest in accordance with the invention may be at least from about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identical to an RNA molecule transcribed from a chromatin remodeling gene DNA molecule, for example, comprising a polynucleotide selected from the group consisting of SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164; SEQ ID NO:165; and SEQ ID NO:166. Isolated and substantially purified nucleic acid molecules including, but not limited to, non-naturally occurring polynucleotides and recombinant DNA constructs for providing dsRNA molecules of the present invention are therefore provided, which suppress or inhibit the expression of an endogenous coding polynucleotide or a target coding polynucleotide in the coleopteran pest when introduced thereto.

[0263] Particular embodiments provide a delivery system for the delivery of iRNA molecules for the post-transcriptional inhibition of one or more target gene(s) in a coleopteran plant pest and control of a population of the plant pest. In some embodiments, the delivery system comprises ingestion of a host transgenic plant cell or contents of the host cell comprising RNA molecules transcribed in the host cell. In these and further embodiments, a transgenic plant cell or a transgenic plant is created that contains a recombinant DNA construct providing a stabilized dsRNA molecule of the invention. Transgenic plant cells and transgenic plants comprising nucleic acids encoding a particular iRNA molecule may be produced by employing recombinant DNA technologies (which basic technologies are well-known in the art) to construct a plant transformation vector comprising a polynucleotide encoding an iRNA molecule of the invention (e.g., a stabilized dsRNA molecule); to transform a plant cell or plant; and to generate the transgenic plant cell or the transgenic plant that contains the transcribed iRNA molecule.

[0264] To impart protection from coleopteran pests to a transgenic plant, a recombinant DNA molecule may, for example, be transcribed into an iRNA molecule, such as a dsRNA molecule, a siRNA molecule, a miRNA molecule, a shRNA molecule, or a hpRNA molecule. In some embodiments, an RNA molecule transcribed from a recombinant DNA molecule may form a dsRNA molecule within the tissues or fluids of the recombinant plant. Such a dsRNA molecule may be comprised in part of a polynucleotide that is identical to a corresponding polynucleotide transcribed from a DNA within a coleopteran pest of a type that may infest the host plant. Expression of a target gene within the coleopteran pest is suppressed by the dsRNA molecule, and the suppression of expression of the target gene in the coleopteran pest results in the transgenic plant being resistant to the pest. The modulatory effects of dsRNA molecules have been shown to be applicable to a variety of genes expressed in pests, including, for example, endogenous genes responsible for cell division, chromosomal remodeling, and cellular metabolism or cellular transformation, including housekeeping genes; transcription factors; molting-related genes; and other genes which encode polypeptides involved in cellular metabolism or normal growth and development.

[0265] For transcription from a transgene in vivo or an expression construct, a regulatory region (e.g., promoter, enhancer, silencer, and polyadenylation signal) may be used in some embodiments to transcribe the RNA strand (or strands). Therefore, in some embodiments, as set forth, supra, a polynucleotide for use in producing iRNA molecules may be operably linked to one or more promoter elements functional in a plant host cell. The promoter may be an endogenous promoter, normally resident in the host genome. The polynucleotide of the present invention, under the control of an operably linked promoter element, may further be flanked by additional elements that advantageously affect its transcription and/or the stability of a resulting transcript. Such elements may be located upstream of the operably linked promoter, downstream of the 3' end of the expression construct, and may occur both upstream of the promoter and downstream of the 3' end of the expression construct.

[0266] In embodiments, suppression of a target gene (e.g., a chromatin remodeling gene) results in a parental RNAi phenotype; a phenotype that is observable in progeny of the subject (e.g., a coleopteran pest) contacted with the iRNA molecule. In some embodiments, the pRNAi phenotype comprises the pest being rendered less able to produce viable offspring. In particular examples of pRNAi, a nucleic acid that initiates pRNAi does not increase the incidence of mortality in a population into which the nucleic acid is delivered. In other examples of pRNAi, a nucleic acid that initiates pRNAi also increases the incidence of mortality in a population into which the nucleic acid is delivered.

[0267] In some embodiments, a population of coleopteran pests is contacted with an iRNA molecule, thereby resulting in pRNAi, wherein the pests survive and mate but produce eggs that are less able to hatch viable progeny than eggs produced by pests of the same species that are not provided the nucleic acid(s). In some examples, such pests do not oviposit eggs or produce fewer eggs than what is observable in pests of the same species that are not contacted with the iRNA molecule. In some examples, the eggs oviposited by such pests do not hatch or hatch at a rate that is significantly less than what is observable in pests of the same species that are not contacted with the iRNA molecule. In some examples, the larvae that hatch from eggs oviposited by such pests are not viable or are less viable than what is observable in pests of the same species that are not contacted with the iRNA molecule.

[0268] Transgenic crops that produce substances that provide protection from insect feeding are vulnerable to adaptation by the target insect pest population reducing the durability of the benefits of the insect protection substance(s). Traditionally, delays in insect pest adaptation to transgenic crops are achieved by (1) the planting of "refuges" (crops that do not contain the pesticidal substances, and therefore allow survival of insects that are susceptible to the pesticidal substance(s)); and/or (2) combining insecticidal substances with multiple modes of action against the target pests, so that individuals that are resistant to one mode of action are killed by a second mode of action.

[0269] In some examples, iRNA molecules (e.g., expressed from a transgene in a host plant) represent new modes of action for combining with Bacillus thuringiensis insecticidal protein technology and/or lethal RNAi technology in Insect Resistance Management gene pyramids to mitigate against the development of insect populations resistant to either of these control technologies.

[0270] Parental RNAi may result in some embodiments in a type of pest control that is different from the control obtained by lethal RNAi, and which may be combined with lethal RNAi to result in synergistic pest control. Thus, in particular embodiments, iRNA molecules for the post-transcriptional inhibition of one or more target gene(s) in a coleopteran plant pest can be combined with other iRNA molecules to provide redundant RNAi targeting and synergistic RNAi effects.

[0271] Parental RNAi (pRNAi) that causes egg mortality or loss of egg viability has the potential to bring further durability benefits to transgenic crops that use RNAi and other mechanisms for insect protection. pRNAi prevents exposed insects from producing progeny, and therefore from passing on to the next generation any alleles they carry that confer resistance to the pesticidal substance(s). pRNAi is particularly useful in extending the durability of insect-protected transgenic crops when it is combined with one or more additional pesticidal substances that provide protection from the same pest populations. Such additional pesticidal substances may in some embodiments include, for example, dsRNA; larval-active dsRNA; insecticidal proteins (such as those derived from Bacillus thuringiensis or other organisms); and other insecticidal substances. This benefit arises because insects that are resistant to the pesticidal substances occur as a higher proportion of the population in the transgenic crop than in the refuge crop. If a ratio of resistance alleles to susceptible alleles that are passed on to the next generation is lower in the presence of pRNAi than in the absence of pRNAi, the evolution of resistance will be delayed.

[0272] For example, pRNAi may not reduce the number of individuals in a first pest generation that are inflicting damage on a plant expressing an iRNA molecule. However, the ability of such pests to sustain an infestation through subsequent generations may be reduced. Conversely, lethal RNAi may kill pests that already are infesting the plant. When pRNAi is combined with lethal RNAi, pests that are contacted with a parental iRNA molecule may breed with pests from outside the system that have not been contacted with the iRNA, however, the progeny of such a mating may be non-viable or less viable, and thus may be unable to infest the plant. At the same time, pests that are contacted with a lethal iRNA molecule may be directly affected. The combination of these two effects may be synergistic; i.e., the combined pRNAi and lethal RNAi effect may be greater than the sum of the pRNAi and lethal RNAi effects independently. pRNAi may be combined with lethal RNAi, for example, by providing a plant that expresses both lethal and parental iRNA molecules; by providing in the same location a first plant that expresses lethal iRNA molecules and a second plant that expresses parental iRNA molecules; and/or by contacting female and/or male pests with the pRNAi molecule, and subsequently releasing the contacted pests into the plant environment, such that they can mate unproductively with the plant pests.

[0273] Some embodiments provide methods for reducing the damage to a host plant (e.g., a corn plant) caused by a coleopteran pest that feeds on the plant, wherein the method comprises providing in the host plant a transformed plant cell expressing at least one nucleic acid molecule of the invention, wherein the nucleic acid molecule(s) functions upon being taken up by the pest(s) to inhibit the expression of a target polynucleotide within the pest(s), which inhibition of expression results in reduced reproduction, for example, in addition to mortality and/or reduced growth of the pest(s), thereby reducing the damage to the host plant caused by the pest. In some embodiments, the nucleic acid molecule(s) comprise dsRNA molecules. In these and further embodiments, the nucleic acid molecule(s) comprise dsRNA molecules that each comprise more than one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell. In some embodiments, the nucleic acid molecule(s) consist of one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell.

[0274] In some embodiments, a method for increasing the yield of a corn crop is provided, wherein the method comprises introducing into a corn plant at least one nucleic acid molecule of the invention; and cultivating the corn plant to allow the expression of an iRNA molecule comprising the nucleic acid, wherein expression of an iRNA molecule comprising the nucleic acid inhibits coleopteran pest damage and/or growth, thereby reducing or eliminating a loss of yield due to coleopteran pest infestation. In some embodiments, the iRNA molecule is a dsRNA molecule. In these and further embodiments, the nucleic acid molecule(s) comprise dsRNA molecules that each comprise more than one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell. In some embodiments, the nucleic acid molecule(s) consists of one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell.

[0275] In some embodiments, a method for increasing the yield of a plant crop is provided, wherein the method comprises introducing into a female coleopteran pest (e.g, by injection, by ingestion, by spraying, and by expression from a DNA) at least one nucleic acid molecule of the invention; and releasing the female pest into the crop, wherein mating pairs including the female pest are unable or less able to produce viable offspring, thereby reducing or eliminating a loss of yield due to coleopteran pest infestation. In particular embodiments, such a method provides control of subsequent generations of the pest. In similar embodiments, the method comprises introducing the nucleic acid molecule of the invention into a male coleopteran pest, and releasing the male pest into the crop (e.g., wherein pRNAi male pests produce less sperm than untreated controls). For example, given that WCR females typically mate only once, these pRNAi female and/or males can be used in competition to overwhelm native WCR insects for mates. In some embodiments, the nucleic acid molecule is a DNA molecule that is expressed to produce an iRNA molecule. In some embodiments, the nucleic acid molecule is a dsRNA molecule. In these and further embodiments, the nucleic acid molecule(s) comprise dsRNA molecules that each comprise more than one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell. In some embodiments, the nucleic acid molecule(s) consists of one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell.

[0276] In some embodiments, a method for modulating the expression of a target gene in a coleopteran pest is provided, the method comprising: transforming a plant cell with a vector comprising a polynucleotide encoding at least one iRNA molecule of the invention, wherein the polynucleotide is operatively-linked to a promoter and a transcription termination element; culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture including a plurality of transformed plant cells; selecting for transformed plant cells that have integrated the polynucleotide into their genomes; screening the transformed plant cells for expression of an iRNA molecule encoded by the integrated polynucleotide; selecting a transgenic plant cell that expresses the iRNA molecule; and feeding the selected transgenic plant cell to the coleopteran pest. Plants may also be regenerated from transformed plant cells that express an iRNA molecule encoded by the integrated nucleic acid molecule. In some embodiments, the iRNA molecule is a dsRNA molecule. In these and further embodiments, the nucleic acid molecule(s) comprise dsRNA molecules that each comprise more than one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell. In some embodiments, the nucleic acid molecule(s) consists of one polynucleotide that is specifically hybridizable to a nucleic acid molecule expressed in a coleopteran pest cell.

[0277] iRNA molecules of the invention can be incorporated within the seeds of a plant species (e.g., corn), either as a product of expression from a recombinant gene incorporated into a genome of the plant cells, or as incorporated into a coating or seed treatment that is applied to the seed before planting. A plant cell comprising a recombinant gene is considered to be a transgenic event. Also included in embodiments of the invention are delivery systems for the delivery of iRNA molecules to coleopteran pests. For example, the iRNA molecules of the invention may be directly introduced into the cells of a pest(s). Methods for introduction may include direct mixing of iRNA into the diet of the coleopteran pest (e.g., by mixing with plant tissue from a host for the pest), as well as application of compositions comprising iRNA molecules of the invention to host plant tissue. For example, iRNA molecules may be sprayed onto a plant surface. Alternatively, an iRNA molecule may be expressed by a microorganism, and the microorganism may be applied onto the plant surface, or introduced into a root or stem by a physical means such as an injection. As discussed, supra, a transgenic plant may also be genetically engineered to express at least one iRNA molecule in an amount sufficient to kill the coleopteran pests known to infest the plant. iRNA molecules produced by chemical or enzymatic synthesis may also be formulated in a manner consistent with common agricultural practices, and used as spray-on or bait products for controlling plant damage by a coleopteran pest. The formulations may include the appropriate adjuvants (e.g., stickers and wetters) required for efficient foliar coverage, as well as UV protectants to protect iRNA molecules (e.g., dsRNA molecules) from UV damage. Such additives are commonly used in the bioinsecticide industry, and are well known to those skilled in the art. Such applications may be combined with other spray-on insecticide applications (biologically based or otherwise) to enhance plant protection from coleopteran pests.

[0278] All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference to the extent they are not inconsistent with the explicit details of this disclosure, and are so incorporated to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

[0279] The following Examples are provided to illustrate certain particular features and/or aspects. These Examples should not be construed to limit the disclosure to the particular features or aspects described.

EXAMPLES

Example 1

Materials and Methods

[0280] Sample Preparation and Bioassays for Diabrotica Larval Feeding Assays.

[0281] The template preparation for dsRNA including RNA extraction, cDNA synthesis, and PCR with T7-containing primers is included in Example 4. Samples were tested for activity in bioassays conducted with neonates on artificial insect diet. WCR eggs were obtained from CROP CHARACTERISTICS, INC (Farmington, Minn.).

[0282] The bioassays were conducted in 128-well plastic trays specifically designed for insect bioassays (C-D INTERNATIONAL, Pitman, N.J.). Each well contained approximately 1.0 mL of a diet designed for growth of coleopteran insects. A 60 .mu.L aliquot of dsRNA sample was delivered by pipette onto the 1.5 cm.sup.2 diet surface of each well (40 .mu.L/cm.sup.2). dsRNA sample concentrations were calculated as the amount of dsRNA per square centimeter (ng/cm.sup.2) of surface area in the well. The treated trays were held in a fume hood until the liquid on the diet surface evaporated or was absorbed into the diet.

[0283] Within a few hours of eclosion, individual larvae were picked up with a moistened camel hair brush and deposited on the treated diet (one or two larvae per well). The infested wells of the 128-well plastic trays were then sealed with adhesive sheets of clear plastic, and vented to allow gas exchange. Bioassay trays were held under controlled environmental conditions (28.degree. C., .about.40% Relative Humidity, 16:8 (Light:Dark)) for 9 days, after which time the total number of insects exposed to each sample, the number of dead insects, and the weight of surviving insects were recorded. Percent mortality, average live weights, and growth inhibition were calculated for each treatment. Stunting was defined as a decrease in average live weights. Growth inhibition (GI) was calculated as follows:

GI=[1-(TWIT/TNIT)/(TWIBC/TNIBC)],

[0284] where TWIT is the Total Weight of live Insects in the Treatment;

[0285] TNIT is the Total Number of Insects in the Treatment;

[0286] TWIBC is the Total Weight of live Insects in the Background Check (Buffer control); and

[0287] TNIBC is the Total Number of Insects in the Background Check (Buffer control).

[0288] The GI.sub.50 is determined to be the concentration of sample in the diet at which the GI value is 50%. The LC.sub.50 (50% Lethal Concentration) is recorded as the concentration of sample in the diet at which 50% of test insects are killed. Statistical analysis was done using JMP.TM. software (SAS, Cary, N.C.).

Example 2

Identification of Candidate Target Genes from Diabrotica

[0289] Insects from multiple stages of WCR (Diabrotica virgifera virgifera LeConte) development were selected for pooled transcriptome analysis to provide candidate target gene sequences for control by RNAi transgenic plant insect protection technology.

[0290] In one exemplification, total RNA was isolated from about 0.9 gm whole first-instar WCR larvae; (4 to 5 days post-hatch; held at 16.degree. C.), and purified using the following phenol/TRI REAGENT.TM.-based method (MOLECULAR RESEARCH CENTER, Cincinnati, Ohio).

[0291] Larvae were homogenized at room temperature in a 15 mL homogenizer with 10 mL of TRI REAGENT.RTM. until a homogenous suspension was obtained. Following 5 min. incubation at room temperature, the homogenate was dispensed into 1.5 mL microfuge tubes (1 mL per tube), 200 .mu.L of chloroform was added, and the mixture was vigorously shaken for 15 seconds. After allowing the extraction to sit at room temperature for 10 min, the phases were separated by centrifugation at 12,000.times.g at 4.degree. C. The upper phase (comprising about 0.6 mL) was carefully transferred into another sterile 1.5 mL tube, and an equal volume of room temperature isopropanol was added. After incubation at room temperature for 5 to 10 min, the mixture was centrifuged 8 min at 12,000.times.g (4.degree. C. or 25.degree. C.).

[0292] The supernatant was carefully removed and discarded, and the RNA pellet was washed twice by vortexing with 75% ethanol, with recovery by centrifugation for 5 min at 7,500.times.g (4.degree. C. or 25.degree. C.) after each wash. The ethanol was carefully removed, the pellet was allowed to air-dry for 3 to 5 min, and then was dissolved in nuclease-free sterile water. RNA concentration was determined by measuring the absorbance (A) at 260 nm and 280 nm. A typical extraction from about 0.9 gm of larvae yielded over 1 mg of total RNA, with an A.sub.260/A.sub.280 ratio of 1.9. The RNA thus extracted was stored at -80.degree. C. until further processed.

[0293] RNA quality was determined by running an aliquot through a 1% agarose gel. The agarose gel solution was made using autoclaved 10.times.TAE buffer (Tris-acetate EDTA; 1.times. concentration is 0.04 M Tris-acetate, 1 mM EDTA (ethylenediamine tetra-acetic acid sodium salt), pH 8.0) diluted with DEPC (diethyl pyrocarbonate)-treated water in an autoclaved container. 1.times.TAE was used as the running buffer. Before use, the electrophoresis tank and the well-forming comb were cleaned with RNaseAway.TM. (INVITROGEN INC., Carlsbad, Calif.). Two .mu.L of RNA sample were mixed with 8 .mu.L of 1E buffer (10 mM Tris HCl pH 7.0; 1 mM EDTA) and 10 .mu.L of RNA sample buffer (NOVAGEN.RTM. Catalog No 70606; EMD4 Bioscience, Gibbstown, N.J.). The sample was heated at 70.degree. C. for 3 min, cooled to room temperature, and 5 .mu.L (containing 1 .mu.g to 2 .mu.g RNA) were loaded per well. Commercially available RNA molecular weight markers were simultaneously run in separate wells for molecular size comparison. The gel was run at 60 volts for 2 hr.

[0294] A normalized cDNA library was prepared from the total RNA of whole larvae by a commercial service provider (EUROFINS MWG Operon, Huntsville, Ala.), using random priming. The normalized larval cDNA library was sequenced at 1/2 plate scale by GS FLX 454 Titanium.TM. series chemistry at EUROFINS MWG Operon, which resulted in over 600,000 reads with an average read length of 348 bp. 350,000 reads were assembled into over 50,000 contigs. Both the unassembled reads and the contigs were converted into BLASTable databases using the publicly available program, FORMATDB (available from NCBI).

[0295] Total RNA and normalized cDNA libraries were similarly prepared from materials harvested at other WCR developmental stages. A pooled transcriptome library for target gene screening was constructed by combining cDNA library members representing the various developmental stages.

[0296] Candidate genes for RNAi targeting were selected using information regarding lethal effects of particular genes in other insects such as Drosophila and Tribolium. For example, the brahma gene (ATP-dependent helicase brm) was selected based on the genetic analysis of the Drosophila brahma gene. Brizuela et al. (1994) Genet. 137:803-13. Once the sequence was identified, existing transcriptome sequences were searched using a stand-alone BLAST algorithm to identify western corn rootworm sequences that exhibited significant similarity to the brahma gene. More complete characterization of the western corn rootworm brahma-like sequences revealed a number of domains characteristic of chromatin remodeling proteins. Therefore, a search was completed on existing transcriptome resources for sequences with similar domains and activity. These genes (mi-2, iswi-1, iswi-2, iswi-3, chd1, ino80, and domino) were hypothesized to be essential for survival and growth in coleopteran insects. Selected target gene homologs were identified in the transcriptome sequence database as described below. Full-length or partial sequences of the target genes were amplified by PCR to prepare templates for double-stranded RNA (dsRNA) production as described below.

[0297] TBLASTN searches using candidate protein coding sequences were run against BLASTable databases containing the unassembled Diabrotica sequence reads or the assembled contigs. Significant hits to a Diabrotica sequence (defined as better than e.sup.-20 for contigs homologies and better than e.sup.-10 for unassembled sequence reads homologies) were confirmed using BLASTX against the NCBI non-redundant database. The results of this BLASTX search confirmed that the Diabrotica homolog candidate gene sequences identified in the TBLASTN search indeed comprised Diabrotica genes, or were the best hit available in the Diabrotica sequences to the non-Diabrotica candidate gene sequence. In most cases, Tribolium candidate genes which were annotated as encoding a protein gave an unambiguous sequence homology to a sequence or sequences in the Diabrotica transcriptome sequences. In a few cases, it was clear that some of the Diabrotica contigs or unassembled sequence reads selected by homology to a non-Diabrotica candidate gene overlapped, and that the assembly of the contigs had failed to join these overlaps. In those cases, SEQUENCHER.TM. v4.9 (GENE CODES CORPORATION, Ann Arbor, Mich.) was used to assemble the sequences into longer contigs.

[0298] Additional transcriptome sequencing of D. v. virgifera has been previously described. Eyun et al. (2014) PLoS One 9(4):e94052. In another exemplification, using Illumina.TM. paired-end as well as 454 Titanium sequencing technologies, a total of .about.700 gigabases were sequenced from cDNA prepared from eggs (15,162,017 Illumina.TM. paired-end reads after filtering), neonates (721,697,288 Illumina.TM. paired-end reads after filtering), and midguts of third instar larvae (44,852,488 Illumina.TM. paired-end reads and 415,742 Roche 454 reads, both after filtering). De novo transcriptome assembly was performed using Trinity (Grabherr et al. (2011) Nat. Biotechnol. 29(7):644-52) for each of three samples as well as for the pooled dataset. The pooled assembly resulted in 163,871 contigs with an average length of 914 bp. The amino acid sequence of BRAHMA was used as a query sequence to search the rootworm transcriptome and genome database (unpublished) with tBLASTN using a cut-off E value of 10.sup.-5. The deduced amino acid sequences were aligned with ClustalX.TM. and edited with GeneDoc.TM. software.

[0299] A candidate target gene was identified that may lead to coleopteran pest mortality or inhibition of growth, development, or reproduction in WCR, including brahma transcript SEQ ID NO:1 (with subsequence SEQ ID NO:8); brahma transcript SEQ ID NO:3 (with subsequences SEQ ID NO:8 and SEQ ID NO:10); brahma transcript SEQ ID NO:5 (with subsequence SEQ ID NO:10); brahma transcript SEQ ID NO:7 (with subsequence SEQ ID NO:10); mi-2 transcript SEQ ID NO:79 (with subsequence SEQ ID NO:104); mi-2 open reading frame SEQ ID NO:164; iswi-1 transcript SEQ ID NO:81; iswi-1 open reading frame SEQ ID NO:165; chd1 transcript SEQ ID NO:83 (with subsequence SEQ ID NO:105); iswi-2 transcript SEQ ID NO:85 (with subsequence SEQ ID NO:103); iswi-2 open reading frame SEQ ID NO:166; iswi30 transcript SEQ ID NO:87 (with subsequence SEQ ID NO:102); ino80 transcript SEQ ID NO:89; and domino transcript SEQ ID NO:91. These genes encode SNF2-type chromatin remodeler proteins, which are subunits of the chromatin remodeling complexes that play global roles in mobilizing nucleosomes. See, for example, Brizuela et al. (supra); Kal et al. (2000) Genes Devel. 14:1058-71; and Tamkun et al. (1992) Cell 68:561-72. Although they share a SNF2-Helicase domain, most chromatin remodelers within each species have non-redundant functions that are conferred by the additional domains they comprise. These characteristics present chromatin remodeling ATPases as attractive targets for multi-generational/parental RNAi.

[0300] The SWI2/SNF2 (mating type switch/sucrose non-fermenting) family of the ATP-dependent remodeling enzymes contains a bromodomain, which binds acetylated histones. While yeasts and vertebrates contain several SWI2/SNF2 proteins, only one SWI2/SNF2 protein, BRAHMA, has been identified in Drosophila. BRAHMA is well-conserved, and yet distinct, from other insect SNF2-containing proteins, with the putative WCR ortholog clustering closely to other chromatin remodeling complexes on a phylogenetic tree. FIG. 2. The human BRAHMA (BRM) as well as the Saccharomyces cerevisiae SNF2 protein cluster together with insect BRAHMAs. Furthermore, the WCR and Euschistus heros (BSB) orthologs of the Drosophila BRAHMA maintain overall protein domain conservation including the SNF2 ATPase/helicase, the bromodomain as well as additional domains: conserved Gln, Leu, Gln motif domain (QLQ), DNA-binding HSA domain, and BRK (brahma and kismet) domain. FIG. 3A.

[0301] BRAHMA is known to incorporate into BAP (Brahma Associated Proteins) and PBAP (Polybromo-associated BAP) chromatin remodeling complexes. The loss of Drosophila brahma impairs overall transcription by RNA polymerase II (Pol II), suggesting a broad function for the BRAHMA complexes. In Drosophila, the maternal contribution of brahma is needed for early embryogenesis, while the zygotic brahma expression is necessary for late embryonic development. In addition to embryogenesis, Drosophila brahma is involved in gametogenesis.

[0302] The ISWI (Imitation SWI/imitation switch) family is defined by histone-biding domain that comprises the HAND, SANT, and SLIDE domains in a HAND-SANT-SLIDE architecture. In Drosophila, the ISWI family of ATP-dependent remodeling enzymes has only one member, ISWI. The Drosophila ISWI can confer multiple functions by integrating into various complexes that include ATP-dependent chromatin assembly and remodeling factor (ACF), nucleosome remodeling factor (NURF), and chromatin accessibility complex (CHRAC). Loss of ISWI in Drosophila results in dramatic chromosome condensation defects.

[0303] Disclosed herein are iswi orthologs in WCR, and additional iswi homologs with partial sequences. The complete WCR ISWI proteins contain the SNF2 ATPase/helicase, HAND-SANT-SLIDE (identified as HAND and SLIDE by Pfam) and DNA-binding domain (DBINO). FIG. 3B. The partial sequence of WCR ISWI, ISWI-2, consists of only SNF2 domain. FIG. 3B. This sequence has high homology to the other ISWI proteins; 91% identity to WCR ISWI-1 over the entire length of ISWI-2, and 93% identity over the region of a dsRNA targeted against iswi-2. FIG. 3B; FIG. 4. Thus, the parental RNAi effect of WCR iswi-2 can be attributed to the function of complete iswi-2 sequence, or to the ability of iswi-2-targeted dsRNA (FIG. 4) to "knock down" iswi-1. Table

TABLE-US-00004 TABLE 1 Effect of dsRNA from brahma-like sequences on total number of WCR egg produced and egg viability after 11 days of ingestion on artificial diet. Means were separated using Dunnett's test. Rep iswi-1 iswi-2 chd1 mi-2 kis etl1 Water GFP Total number of eggs 1 131.60 55.80 106.00 41.50 211.33 63.80 72 202.60 2 39.60 114.83 112.67 63.80 140.67 135.17 273.17 213.83 3 12.40 211.20 171.60 34.00 119.00 55.67 104.60 139.50 Average 61.20* 127.28 130.09 46.43* 157.00 84.88 149.92 185.31 SEM.dagger. 36.07 45.29 20.84 8.95 27.88 25.25 62.34 23.13 Percent egg hatch 1 0.00 0.00 16.77 0.00 21.55 41.54 -- 18.54 2 3.34 0.00 23.40 0.00 50.52 58.75 46.17 39.42 3 0.00 0.62 34.20 0.00 26.44 20.69 23.62 36.08 Average 1.11* 0.21* 24.79 0.00* 32.84 40.33 34.90 31.35 SEM.dagger. 1.11 0.21 5.08 0.00 8.95 11.00 9.20 6.47 *Indicates significance at p-value <0.05. .dagger.SEM--Standard Error of the Mean.

[0304] Proteins of the CHD (chromodomain helicase DNA-binding) family of ATP-dependent remodeling enzymes contain two amino-terminal chromodomains [chromatin organization modifier]. FIG. 3C. The Drosophila CHD proteins include CHD1, MI-2, CHD3, and KISMET. The CHD family is further subdivided into three subfamilies, herein referred to as subfamilies I, II, and III. The Drosophila CHD1 belongs to CHD subfamily I, which has a C-terminal DNA-binding domain. FIG. 3C (DUF4208). In Drosophila, CHD1 protein shows similar distribution patterns to BRAHMA, yet chd1 mutant flies are viable. Interestingly, the Drosophila chd1 is needed for gametogenesis. WCR females subjected to chd1 RNAi show a decrease in offspring viability. Table 1.

[0305] MI-2 and CHD3 belong to subfamily II. Enzymes of the CHD subfamily II have no DNA-binding domain, but have Zn-finger-like domains called PHD (plant homeodomain) fingers. The WCR ortholog of MI-2 mirrors the Drosophila domain arrangement, and includes the SNF2 ATPase/helicase domain, the double chromodomain, PHD fingers, and CHDNT domain that is associated with PHD finger-containing chromodomain helicases, as well as other conserved domains of unknown functions, DUF1087 and DUF1086. FIG. 3D. The Drosophila MI-2 is known to associate with the NuRD (Nucleosome Remodeling Deacetylase) and dMec (Drosophila MEP-1 containing complex) complexes. Maternal expression of mi-2 is necessary for gametogenesis. Mi-2 RNAi-treated female WCR produce no viable eggs. Table 1.

[0306] The third subfamily of CHD proteins is represented by KISMET in Drosophila; in humans this subfamily comprises CHD5-98. Like other CHD proteins, KISMET contains an SNF2 domain and a chromodomain. FIG. 3E. Unlike other CHD subfamilies, KISMET has characteristics of both CHD and SWI2/SNF2 proteins, in that it has a BRK domain that is common to both BRAHMA and KISMET. Although BRK is a well-established feature of Drosophila KISMET, a standard Pfam analysis did not identify this domain in Drosophila. FIG. 3E. Loss of either maternal or zygotic function of kismet causes defects during Drosophila embryogenesis and the insects die during early larval stages, while oogenesis is unaffected. The putative WCR ortholog of the Drosophila kismet produces no oviposition or hatch defects in response to parental RNAi. Table 1.

[0307] Additional SNF2-containing genes are present in Drosophila; the functions of most of these have not been defined. For example, the WCR transcriptome contains an etl1-like transcript. FIG. 3F. The Etl1 (Enhancer Trap Locus 1) SNF2-containing gene was first described in mice. The mouse etl1 has been described as having developmental effects, but being nonessential. Parental RNAi that targets WCR etl1 shows no oviposition or egg viability defects. Table 1.

[0308] The identified polynucleotides and their encoded polypeptides are novel. The sequences are not provided in public databases, and are not disclosed in WO/2011/025860; U.S. Patent Application No. 20070124836; U.S. Patent Application No. 20090306189; U.S. Patent Application No. US20070050860; U.S. Patent Application No. 20100192265; or U.S. Pat. No. 7,612,194. The Diabrotica brahma (SEQ ID NO:1) is somewhat (72% identity) related to a fragment of a sequence from Metaseiulus occidentalis (GENBANK Accession No. XM_003742362.1). The closest homolog of the Diabrotica BRAHMA amino acid sequence (SEQ ID NO:2) is a Dendroctonus ponderosae protein having GENBANK Accession No. ENN80791.1 (86% similar; 75% identical over the homology region). The Diabrotica brahma (SEQ ID NO:3 and SEQ ID NO:5) are somewhat (74% identity) related to a fragment of a sequence from Nasonia vitripennis (GENBANK Accession No. XM_001607119.3). The closest homolog of the Diabrotica BRAHMA amino acid sequence (SEQ ID NO:4) is a Dendroctonus ponderosae protein having GENBANK Accession No. ENN80791.1 (86% similar; 78% identical over the homology region). The closest homolog of the Diabrotica BRAHMA amino acid sequence (SEQ ID NO:6) is a Dendroctonus ponderosae protein having GENBANK Accession No. ENN80791.1 (86% similar; 75% identical over the homology region). There was no significant homologous nucleotide sequence found with a search in GENBANK for Diabrotica brahma (SEQ ID NO:7). There was no significant homologous nucleotide sequence found with a search in GENBANK for Diabrotica brahma (SEQ ID NO:8). The closest homolog of the Diabrotica BRAHMA amino acid sequence (SEQ ID NO:9) is a Dendroctonus ponderosae protein having GENBANK Accession No. ENN80791.1 (95% similar; 92% identical over the homology region). The Diabrotica mi-2 (SEQ ID NO:79) is somewhat (72% identity) related to a fragment of a sequence from Aedes aegypti (GENBANK Accession No. XM 001663273.1). The closest homolog of the Diabrotica MI-2 amino acid sequence (SEQ ID NO:80) is a Tribolium castaneum protein having GENBANK Accession No. XP_001812556.1 (85% similar; 77% identical over the homology region). The Diabrotica iswi-1 (SEQ ID NO:81) is somewhat (73% identity) related to a fragment of a sequence from Python bivittatus (GENBANK Accession No. XM 007428840.1). The closest homolog of the Diabrotica ISWI-1 amino acid sequence (SEQ ID NO:82) is a Dendroctonus ponderosae protein having GENBANK Accession No. ENN80673 0.1 (95% similar; 89% identical over the homology region). The Diabrotica chd1 (SEQ ID NO:83) is somewhat (74% identity) related to a fragment of a sequence from Pediculus humanus corporis (GENBANK Accession No. XM_002428164.1). The closest homolog of the Diabrotica CHD-1 amino acid sequence (SEQ ID NO:84) is a Tribolium castaneum protein having GENBANK Accession No. XP_970343.3 (82% similar; 73% identical over the homology region). The Diabrotica iswi-2 (SEQ ID NO:85) is somewhat (75% identity) related to a fragment of a sequence from Tetrapisispora blattae (GENBANK Accession No. XM_004179654.1). The closest homolog of the Diabrotica ISWI-2 amino acid sequence (SEQ ID NO:86) is a Dendroctonus ponderosae protein having GENBANK Accession No. ERL83291.1 (95% similar; 88% identical over the homology region). The Diabrotica iswi-30 (SEQ ID NO:87) is somewhat (73% identity) related to a fragment of a sequence from Python bivittatus (GENBANK Accession No. XM_007428840.1). The closest homolog of the Diabrotica ISWI-30 amino acid sequence (SEQ ID NO:88) is a Dendroctonus ponderosae protein having GENBANK Accession No. ENN80673.1 (95% similar; 89% identical over the homology region). The Diabrotica ino80 (SEQ ID NO:89) is somewhat (77% identity) related to a fragment of a sequence from Bos mutus (GENBANK Accession No. XM_005903961.1). The Diabrotica domino (SEQ ID NO:91) is somewhat (76% identity) related to a fragment of a sequence from Acyrthosiphon pisum (GENBANK Accession No. XM_008181422.1).

[0309] Full-length or partial clones of sequences of Diabrotica candidate chromatin remodelers containing SNF2 genes were used to generate PCR amplicons for dsRNA synthesis. dsRNA was also amplified from a DNA clone comprising the coding region for a yellow fluorescent protein (YFP) (SEQ ID NO:11; Shagin et al. (2004) Mol. Biol. Evol. 21:841-850).

Example 3

Amplification of Target Genes from Diabrotica

[0310] Primers were designed to amplify portions of coding regions of each target gene by PCR. See Table 2. Where appropriate, a T7 phage promoter sequence (TAATACGACTCACTATAGGG (SEQ ID NO:12)) was incorporated into the 5' ends of the amplified sense or antisense strands. See Table 2. Total RNA was extracted from WCR, and first-strand cDNA was used as template for PCR reactions using opposing primers positioned to amplify all or part of the native target gene sequence.

TABLE-US-00005 TABLE 2 Primers and Primer Pairs used to amplify portions of coding regions of exemplary chromatin remodelers containing SNF2 target genes and YFP target genes. Gene (Region) Primer_ID Sequence Pair 1 brahma-Var2 BrahmaR2_FT7 TTAATACGACTCACTATAGGGAGAATGAGGGTCATC GTATGAAAAACC (SEQ ID NO: 13) BrahmaR2_R TGTCCTTAGATCCCCTTCCTTTAC (SEQ ID NO: 14) Pair 2 brahma-Var2 BrahmaR2_F ATGAGGGTCATCGTATGAAAAACC (SEQ ID NO: 15) BrahmaR2_RT7 TTAATACGACTCACTATAGGGAGATGTCCTTAGATC CCCTTCCTTTAC (SEQ ID NO: 16) Pair 3 brahma Reg-352 Brahma352_FT7 TAATACGACTCACTATAGGGAACCTTCTTCATCTTC TG (SEQ ID NO: 17) Brahma352_RT7 TAATACGACTCACTATAGGGTTGAACTGTATTAGGA GAG (SEQ ID NO: 18) Pair 4 mi-2 Mi2.T7.F TAATACGACTCACTATAGGGAAGAAGGCATAGAACA GA (SEQ ID NO: 107) Mi2.T7.R TAATACGACTCACTATAGGGTCAGAATGGTAATCAG AGA (SEQ ID NO: 108) Pair 5 iswi-30 ISWI30.T7.F TAATACGACTCACTATAGGGTGAATCAGTCTACCAA TT (SEQ ID NO: 109) ISWI30.T7.R TAATACGACTCACTATAGGGGGTTCTGACTCATCTA TT (SEQ ID NO: 110) Pair 6 iswi-2 ISWI2.T7.F TAATACGACTCACTATAGGGTTGCTCAATCCTACAT ACA (SEQ ID NO: 111) ISWI2.T7.R TAATACGACTCACTATAGGGGAATACCAACAGGCTA CT (SEQ ID NO: 112) Pair 7 ksmt KSMT.T7.F TAATACGACTCACTATAGGGGATCAAATTCAAGCAA CT (SEQ ID NO: 113) KSMT.T7.R TAATACGACTCACTATAGGGTTCTTCCTAAACCATG TT (SEQ ID NO: 114) Pair 8 chd1 CHD1.T7.F TAATACGACTCACTATAGGGTTTGCTTCCTTCTTTC AA (SEQ ID NO: 115) CHD1.T7.R TAATACGACTCACTATAGGGCTTCTTTGTTAAACGG ATT (SEQ ID NO: 116) Pair 9 etl1 ETL1.T7.F TAATACGACTCACTATAGGGACTTATCTAAAGGGAT GCTA (SEQ ID NO: 117) ETL1.T7.R TAATACGACTCACTATAGGGGTAGAGAGTCGTCTTC TG (SEQ ID NO: 118) Pair 10 YFP YFP-F_T7 TTAATACGACTCACTATAGGGAGACACCATGGGCTC CAGCGGCGCCC (SEQ ID NO: 19) YFP-R_T7 TTAATACGACTCACTATAGGGAGAAGATCTTGAAGG CGCTCTTCAGG (SEQ ID NO: 20) Pair 11 GFP GFP-F_T7 TAATACGACTCACTATAGGGGGTGATGCTACATACG GAAAG (SEQ ID NO: 54) GFP-R_T7 TAATACGACTCACTATAGGGTTGTTTGTCTCCGTGA T (SEQ ID NO: 55)

Example 4

RNAi Constructs

[0311] Template Preparation by PCR and dsRNA Synthesis.

[0312] The strategies used to provide specific templates for chromatin remodelers containing SNF2 target gene dsRNA production are shown in FIG. 1A and FIG. 1B. Template DNAs intended for use in dsRNA synthesis were prepared by PCR using Primer Pair 1 and Primer Pair 2 respectively (Table 2) and (as PCR template) first-strand cDNA prepared from total RNA. For the selected target gene regions, two separate PCR amplifications were performed. FIGS. 1A and 1B. The first PCR amplification introduced a T7 promoter sequence at the 5' end of the amplified sense strands. The second reaction incorporated the T7 promoter sequence at the 5' ends of the antisense strands. The two PCR amplified fragments for each region of the target genes were then mixed in approximately equal amounts, and the mixture was used as transcription template for dsRNA production. FIGS. 1A and 1B.

[0313] For the YFP negative control, a single PCR amplification was performed. FIG. 1B. The PCR amplification introduced a T7 promoter sequence at the 5' ends of the amplified sense and antisense strands. The two PCR amplified fragments for each region of the target genes were then mixed in approximately equal amounts, and the mixture was used as transcription template for dsRNA production. FIG. 1B. dsRNA for the negative control YFP coding region (SEQ ID NO:11) was produced using Primer Pair 10 (Table 2) and a DNA clone of the YFP coding region as template. A GFP negative control was amplified from the pIZT/V5-His expression vector (Invitrogen) using Primer Pair 11 (Table 2). The PCR product amplified for chromatin remodelers containing SNF2 target genes and GFP were used as a template for in vitro synthesis of dsRNAs using the MEGAscript high-yield transcription kit (Applied Biosystems Inc., Foster City, Calif.). The synthesized dsRNAs were purified using the RNeasy Mini kit (Qiagen, Valencia, Calif.) or an AMBION.RTM. MEGAscript.RTM. RNAi kit essentially as prescribed by the manufacturer's instructions. dsRNA preparations were quantified using a NANODROP.TM. 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, Del.) or equivalent means and analyzed by gel electrophoresis to determine purity.

Example 5

Screening of Candidate Target Genes in Diabrotica Larvae

[0314] Replicated bioassays demonstrated that ingestion of synthetic dsRNA preparations derived from the brahma-Var1 target gene sequence identified in EXAMPLE 2 caused mortality and growth inhibition of western corn rootworm larvae when administered to WCR in diet-based assays. Table 3 and Table 4.

TABLE-US-00006 TABLE 3 Results of diet-based feeding bioassays of WCR larvae following 9-day exposure to a single dose of dsRNAs. ANOVA analysis found some significance differences in Mean % Mortality (Mort.). Means were separated using the Tukey-Kramer test. *Mean % Dose No. Rows Mortality +/- *Mean GI +/- Sample Name (ng/cm.sup.2) (Replications) SEM SEM brahma-Var1 500 4 52.62 .+-. 7.84 (A) 0.10 .+-. 0.42 (A) brahma Reg-352 500 5 39.76 .+-. 16.72 (AB) 0.54 .+-. 0.19 (AB) TE buffer** 0 9 12.56 .+-. 3.70 (BC) 0.00 .+-. 0.00 (AB) Water 0 9 11.45 .+-. 4.08 (BC) 0.01 .+-. 0.01 (AB) YFP dsRNA*** 500 9 10.09 .+-. 4.16 (C) -0.15 .+-. 0.19 (B) *SEM = Standard Error of the Mean. Letters in parentheses designate statistical levels. Levels not connected by same letter are significantly different (p < 0.05). **TE = Tris HCl (1 mM) plus EDTA (1 mM) buffer, pH7.2. ***YFP = Yellow Fluorescent Protein

TABLE-US-00007 TABLE 4 Results of diet-based feeding bioassays of WCR larvae following 9-day exposure to a range of doses of dsRNAs. Sample Name LC.sub.50 (ng/cm.sup.2) LC.sub.50 Range (ng/cm.sup.2) brahma-Var1 839 432-1000+

[0315] It has previously been suggested that certain genes of Diabrotica spp. may be exploited for RNAi-mediated insect control. See U.S. Patent Publication No. 2007/0124836, which discloses 906 sequences, and U.S. Pat. No. 7,614,924, which discloses 9,112 sequences. However, it was determined that many genes suggested to have utility for RNAi-mediated insect control are not efficacious in controlling Diabrotica. It was also determined that brahma-Var1 provided surprising and unexpected control of Diabrotica, compared to other genes suggested to have utility for RNAi-mediated insect control.

[0316] For example, Annexin, Beta Spectrin 2, and mtRP-L4 were each suggested in U.S. Pat. No. 7,614,924 to be efficacious in RNAi-mediated insect control. SEQ ID NO:21 is the DNA sequence of Annexin Region 1 and SEQ ID NO:22 is the DNA sequence of Annexin Region 2. SEQ ID NO:23 is the DNA sequence of Beta Spectrin 2 Region 1 and SEQ ID NO:24 is the DNA sequence of Beta Spectrin 2 Region 2. SEQ ID NO:25 is the DNA sequence of mtRP-L4 Region 1 and SEQ ID NO:26 is the DNA sequence of mtRP-L4 Region 2.

[0317] Each of the aforementioned sequences was used to produce dsRNA by the dual Primer Pair methods of EXAMPLE 4 (FIGS. 1A and 1B), and the dsRNAs were each tested by the diet-based bioassay methods described above. A YFP sequence (SEQ ID NO:11) was also used to produce dsRNA as a negative control. Table 5 lists the sequences of the primers used to produce the Annexin, Beta Spectrin 2, mtRP-L4, and YFP dsRNA molecules. Table 6 presents the results of diet-based feeding bioassays of WCR larvae following 9-day exposure to these dsRNA molecules. Replicated bioassays demonstrated that ingestion of these dsRNAs resulted in no mortality or growth inhibition of western corn rootworm larvae above that seen with control samples of TE buffer, YFP dsRNA, or water.

TABLE-US-00008 TABLE 5 Primers and Primer Pairs used to amplify portions of coding regions of genes. Gene Region Primer ID Sequence Pair 12 Annexin Ann-F1_T7 TTAATACGACTCACTATAGGGAGAGCTCCAACAGTGG Region 1 TTCCTTATC (SEQ ID NO: 17) Annexin Ann-R1 CTAATAATTCTTTTTTAATGTTCCTGAGG Region 1 (SEQ ID NO: 18) Pair 13 Annexin Ann-F1 GCTCCAACAGTGGTTCCTTATC (SEQ ID NO: 19) Region 1 Annexin Ann-R1_T7 TTAATACGACTCACTATAGGGAGACTAATAATTCTTT Region 1 TTTAATGTTCCTGAGG (SEQ ID NO: 20) Pair 14 Annexin Ann-F2_T7 TTAATACGACTCACTATAGGGAGATTGTTACAAGCTG Region 2 GAGAACTTCTC (SEQ ID NO: 21) Annexin Ann-R2 CTTAACCAACAACGGCTAATAAGG Region 2 (SEQ ID NO: 22) Pair 15 Annexin Ann-F2 TTGTTACAAGCTGGAGAACTTCTC Region 2 (SEQ ID NO: 23) Annexin Ann-R2T7 TTAATACGACTCACTATAGGGAGACTTAACCAACAAC Region 2 GGCTAATAAGG (SEQ ID NO: 24) Pair 16 Beta-Spect2 Betasp2- TTAATACGACTCACTATAGGGAGAAGATGTTGGCTGC Region 1 F1_T7 ATCTAGAGAA (SEQ ID NO: 25) Beta-Spect2 Betasp2-R1 GTCCATTCGTCCATCCACTGCA Region 1 (SEQ ID NO: 26) Pair 17 Beta-Spect2 Betasp2-F1 AGATGTTGGCTGCATCTAGAGAA Region 1 (SEQ ID NO: 27) Beta-Spect2 Betasp2- TTAATACGACTCACTATAGGGAGAGTCCATTCGTCCA Region 1 R1_T7 TCCACTGCA (SEQ ID NO: 28) Pair 18 Beta-Spect2 Betasp2- TTAATACGACTCACTATAGGGAGAGCAGATGAACACC Region 2 F2_T7 AGCGAGAAA (SEQ ID NO: 29) Beta-Spect2 Betasp2-R2 CTGGGCAGCTTCTTGTTTCCTC (SEQ ID NO: 30) Region 2 Pair 19 Beta-Spect2 Betasp2-F2 GCAGATGAACACCAGCGAGAAA (SEQ ID NO: 31) Region 2 Beta-Spect2 Betasp2- TTAATACGACTCACTATAGGGAGACTGGGCAGCTTCT Region 2 R2_T7 TGTTTCCTC (SEQ ID NO: 32) Pair 20 mtRP-L4 L4-F1_T7 TTAATACGACTCACTATAGGGAGAAGTGAAATGTTAG Region 1 CAAATATAACATCC (SEQ ID NO: 33) mtRP-L4 L4-R1 ACCTCTCACTTCAAATCTTGACTTTG Region 1 (SEQ ID NO: 34) Pair 21 mtRP-L4 L4-F1 AGTGAAATGTTAGCAAATATAACATCC Region 1 (SEQ ID NO: 35) mtRP-L4 L4-R1_T7 TTAATACGACTCACTATAGGGAGAACCTCTCACTTCA Region 1 AATCTTGACTTTG (SEQ ID NO: 36) Pair 22 mtRP-L4 L4-F2_T7 TTAATACGACTCACTATAGGGAGACAAAGTCAAGATT Region 2 TGAAGTGAGAGGT (SEQ ID NO: 37) mtRP-L4 L4-R2 CTACAAATAAAACAAGAAGGACCCC Region2 (SEQ ID NO: 38) Pair 23 mtRP-L4 L4-F2 CAAAGTCAAGATTTGAAGTGAGAGGT Region 2 (SEQ ID NO: 39) mtRP-L4 L4-R2_T7 TTAATACGACTCACTATAGGGAGACTACAAATAAAAC Region 2 AAGAAGGACCCC (SEQ ID NO: 40) Pair 24 YFP YFP-F_T7 TTAATACGACTCACTATAGGGAGACACCATGGGCTCC AGCGGCGCCC (SEQ ID NO: 41) YFP YFP-R AGATCTTGAAGGCGCTCTTCAGG (SEQ ID NO: 42) Pair 25 YFP YFP-F CACCATGGGCTCCAGCGGCGCCC (SEQ ID NO: 43) YFP YFP-R_T7 TTAATACGACTCACTATAGGGAGAAGATCTTGAAGGC GCTCTTCAGG (SEQ ID NO: 44)

TABLE-US-00009 TABLE 6 Results of diet feeding assays obtained with western corn rootworm larvae. Mean weight Mean Dose per insect Mean % Growth Gene Name (ng/cm.sup.2) (mg) Mortality Inhibition annexin-region 1 1000 0.545 0 -0.262 annexin-region 2 1000 0.565 0 -0.301 beta spectrin2 region 1 1000 0.340 12 -0.014 beta spectrin2 region 2 1000 0.465 18 -0.367 mtRP-L4 region 1 1000 0.305 4 -0.168 mtRP-L4 region 2 1000 0.305 7 -0.180 TE buffer 0 0.430 13 0.000 Water 0 0.535 12 0.000 YFP 1000 0.480 9 -0.386

Example 6

Sample Preparation and Bioassays for Diabrotica Adult Feeding Assays

[0318] Parental RNA interference (RNAi) in western corn rootworms was conducted by feeding dsRNA corresponding to the segments of chromatin remodelers containing SNF2 target gene sequence to gravid adult females. Adult rootworms (<48 hr after emergence) were obtained from CROP CHARACTERISTICS, Inc. (Farmington, Minn.). Adults were reared at 23.+-.1.degree. C., relative humidity of >75%, and Light:Dark periods of 8 hr:16 hr for all bioassays. The insect rearing diet was adapted from Branson and Jackson (1988, J. Kansas Entomol. Soc. 61:353-35). Dry ingredients were added (48 gm/100 mL) to a solution comprising double distilled water with 2.9% agar and 5.6 mL of glycerol. In addition, 0.5 mL of a mixture comprising 47% propionic acid and 6% phosphoric acid solutions was added per 100 mL of diet to inhibit microbial growth. The agar was dissolved in boiling water and the dry ingredients, glycerol, and propionic acid/phosphoric acid solution were added, mixed thoroughly, and poured to a depth of approximately 2 mm. Solidified diet plugs (about 4 mm in diameter by 2 mm height; 25.12 mm.sup.3) were cut from the diet with a No. 1 cork borer. Six adult males and females (24 to 48 hrs old) were maintained on untreated artificial diet and were allowed to mate for 4 days in 16 well trays (5.1 cm long.times.3.8 cm wide.times.2.9 high) with vented lids.

[0319] On day five, males were removed from the container, and females were fed on artificial diet plugs surface treated with 3 .mu.L gene-specific SNF2 dsRNA representing the different chromatin remodelers (2 .mu.g/diet plug; approximately 79.6 ng/mm.sup.3). Control treatments consisted of gravid females exposed to diet treated with the same concentration of GFP dsRNA (SEQ ID NO:53) or the same volume of water. GFP dsRNA was produced as described above using opposing primers having a T7 promoter sequence at their 5' ends (SEQ ID NOs:54 and 55). Fresh artificial diet treated with dsRNA was provided every other day throughout the experiment. On day 11, females were transferred to oviposition cages (7.5 cm.times.5.5 cm.times.5.5 cm) (ShowMan box, Althor Products, Wilton, Conn.) containing autoclaved silty clay loam soil sifted through a 60-mesh sieve (Jackson (1986) Rearing and handling of Diabrotica virgifera and Diabrotica undecimpunctata howardi. Pages 25 to 47 in J. L. Krysan and T. A. Miller, eds. Methods for the study of pest Diabrotica. Springer-Verlag, New York). Females were allowed to oviposit for four days and the eggs were incubated in soil within the oviposition boxes for 10 days at 27.degree. C. and then removed by washing the oviposition soil through a 60-mesh sieve. Eggs were treated with a solution of formaldehyde (500 .mu.L formaldehyde in 5 mL double distilled water) and methyl-(butycarbamoy)-2-benzimidazole carbamate (0.025 g in 50 mL double distilled water) to prevent fungal growth. Females removed from the oviposition boxes and subsamples of eggs from each treatment were flash frozen in liquid nitrogen for subsequent expression analyses by quantitative RT-PCR (See EXAMPLE 7). The dishes were photographed with Dino-Lite Pro digital microscope (Torrance, Calif.) and total eggs counted using the cell counter function of Image J software (Schneider et al. (2012) Nat. Methods 9:671-5). Harvested eggs were held in Petri dishes on moistened filter paper at 28.degree. C. and monitored for 15 days to determine egg viability. Six replications, each comprising three to six females, were run on separate days. The number of larvae hatching from each treatment was recorded daily until no further hatching was observed.

[0320] Ingestion of brahma Reg-352 dsRNA molecules by adult WCR females was demonstrated to a have surprising, dramatic and reproducible effect on egg viability. The mated females exposed to brahma dsRNA produced a lower number of eggs to females exposed to untreated diet or diet treated with GFP dsRNA. Table 7. However, eggs collected from females that were exposed to brahma dsRNA were not viable. Table 7. Eggs collected from adult females exposed to brahma dsRNA did not hatch. Ingestion of brahma Reg-352 dsRNA molecules by adult WCR females was demonstrated to have dramatic and reproducible effects on both egg production and viability.

TABLE-US-00010 TABLE 7 Effect of brahma dsRNA on WCR egg production and egg viability after 11 days of ingestion on treated artificial diet. Means were separated using Dunnett's test. brahma Reg-352 dsRNA GFP dsRNA Water Eggs per female Rep1 49 44.5 108 Rep2 67 61.7 103 Rep3 31.3 137 79.5 Averages.sup..dagger. 49.1 .+-. 10.3 81.1 .+-. 28.4 96.9 .+-. 8.8 Percent egg hatch Rep1 0 41.9 56 Rep2 0 71.1 67.3 Rep3 0 53.3 42 Averages.sup..dagger. 0** 55.4 .+-. 8.5 55.1 .+-. 7.3 .sup..dagger..+-.SEM--Standard Error of the Mean. **Indicates significance at p-value < 0.05.

[0321] Ingestion of mi-2 (SEQ ID NO:101), iswi-30 (SEQ ID NO:102), and iswi-2 (SEQ ID NO:103) dsRNA molecules by adult WCR females was demonstrated to a have surprising, dramatic and reproducible effect on egg viability. The mated females exposed to iswi-30 and mi-2 dsRNA produced a lower number of eggs to females exposed to untreated diet or diet treated with GFP dsRNA. Table 8 and Table 9. However, eggs collected from females that were exposed to mi-2, iswi-30, and iswi-2 dsRNA were not viable. Table 8 and Table 9. Adult females exposed to mi-2 dsRNA had no eggs hatch.

TABLE-US-00011 TABLE 8 Effect of dsRNA from target gene sequences and controls on WCR egg production and egg viability after 11 days of ingestion on artificial diet. Means were separated using Dunnett's test. Eggs per Female Rep chd1 ksmt iswi-2 etl1 iswi mi-2 Water GFP 1 41.75 77.33 25.4 32.5 16.6 11.17 85.2 2 47.00 47.83 40.33 80 65.8 25 89.17 52 3 69.6 73.75 64.6 29 0.4 41.75 50.8 48.5 Average 52.78 66.31 43.44 47.17 27.6 25.97 69.98 61.9 SEM.dagger. 8.54 9.29 11.42 16.45 19.67 8.84 15.66 11.69 p-value 0.32 0.829 0.133 0.192 0.02** 0.02** 0.636 **Indicates significance at p-value .ltoreq.0.05. .dagger.SEM--Standard Error of the Mean.

TABLE-US-00012 TABLE 9 Effect of iswi-30 dsRNA (7 replications) and mi-2 dsRNA (6 replications) from target gene sequences and controls on WCR egg production and egg viability after 11 days of ingestion on artificial diet. A total of 7 replications (consisting of 35 females) were completed for the GFP and water controls. Means were separated using Dunnett's test. iswi-30 mi-2 Water GFP Eggs per Female Average 32.52 26.26 71.33 66.87 SEM.sup..dagger. 16.11 4.40 10.46 7.85 p-value 0.0044** 0.0014** 0.9525 Percent Hatch Rep Average 0.562 0 60.782 58.123 SEM.sup..dagger. 0.471 0 8.835 10.391 p-value 0.003** 0.0037** 0.767 **Indicates significance at p-value .ltoreq. 0.05. .sup..dagger.SEM--Standard Error of the Mean.

[0322] Unhatched eggs were dissected to examine embryonic development and to determine phenotypic responses of parental RNAi (pRNAi). The eggs deposited by WCR females treated with GFP dsRNA showed normal development. FIG. 5A. In contrast, eggs deposited by females treated with brahma Reg-352, mi-2 and iswi-30 dsRNA showed no embryonic development within the egg and, when dissected, had no indications of larval development. FIGS. 5B-5D. It is thus an unexpected and surprising finding of this invention that ingestion of brahma, mi-2, and iswi-30 dsRNA has a lethal or growth inhibitory effect on WCR eggs and larvae. It is further surprising and unexpected that brahma, mi-2, and iswi-30 dsRNA ingestion by gravid adult WCR females dramatically impacts egg production and egg viability, while having no discernible effect on the adult females themselves.

[0323] Brahma and its orthologs, as well as mi-2 and other chromatin remodelers and their orthologs, share the same functional domains and sequence-level conservation. RNAi target sites were designed within the conserved SNF2 family N-terminal and Helicase C-terminal domains (here referred to as SNF2-Helicase) that are common to all chromatin remodelers, as well as chromatin binding and other functional domains that are conserved within each family (including bromodomain, chromodomain, and HAND-SLIDE domains). RNAi target sequences that are common to Diabrotica virgifera virgifera, Euchistus heros, Tribolium castaneum, and Drosophila melanogaster were designed. The DNA nucleotides and RNAi nucleotides are listed according to the standard IUPAC code:

[0324] A=Adenine

[0325] C=Cytosine

[0326] G=Guanine

[0327] T=Thymine

[0328] R=A or G

[0329] Y=C or T

[0330] S=G or C

[0331] W=A or T

[0332] K=G or T

[0333] M=A or C

[0334] B=C or G or T

[0335] D=A or G or T

[0336] H=A or C or T

[0337] V=A or C or G

[0338] N=A or C or G or T

[0339] dsRNA encoding sequences targeting SNF2-Helicase regions (SEQ ID NOs:93-96) and chromatin remodeling domains (SEQ ID NOs:97-101) were designed by aligning the amino acid sequences for each target protein from four species, Diabrotica virgifera virgifera, Euchistus heros, Tribolium castaneum, and Drosophila melanogaster, using Vector NTI Align X (Invitrogen, Grand Island, N.Y.). Highly homologous regions of the amino acid sequence containing at least 8 amino acids within the SNF2 domain or chromatin remodeling domain specific to each target protein were selected. The corresponding nucleotide sequence for each species from each target was then aligned using the Align X program. Where there was a misalignment across the four species the nucleotides were replaced with nucleotides as shown above. Finally, the sequence was aligned against the nucleotide sequence from Apis melifera to determine if the sequence would also target that species. If the sequence could also target the protein from A. melifera either new regions were chosen or the sequence was shortened to at least 21 bases, which did not target A. melifera proteins.

[0340] Ingestion of dsRNA molecules encoding sequences targeting SNF2-Helicase regions (SEQ ID NOs:93-96) and chromatin remodeling domains (SEQ ID NOs:97-101) by adult WCR females is demonstrated to a have surprising, dramatic and reproducible effect on egg viability. The mated females exposed to dsRNA produce a lower number of eggs than females exposed to untreated diet or diet treated with GFP dsRNA.

[0341] The foregoing results clearly document the systemic nature of RNAi in western corn rootworm larvae and adults, and the potential to achieve a parental effect where genes associated with zygotic and/or embryonic development are knocked down in the eggs of females that are exposed to dsRNA. Importantly, this is the first report of a pRNAi response to ingested dsRNA in western corn rootworms. A systemic response is indicated based on the observation of knock down in tissues other than the alimentary canal where exposure and uptake of dsRNA is occurring. Because insects in general, and rootworms specifically, lack the RNA-dependent RNA polymerase that has been associated with systemic response in plants and nematodes, our results confirm that the dsRNA can be taken up by gut tissue and translocated to other tissues (e.g., developing ovarioles).

[0342] The ability to knock down the expression of genes involved with embryonic development such that the eggs do not hatch, offers a unique opportunity to achieve and improve control of western corn rootworms. Because adults readily feed on above-ground reproductive tissues (such as silks and tassels), adult rootworms can be exposed to iRNA control agents by transgenic expression of dsRNA to achieve root protection in the subsequent generation by preventing eggs from hatching. Delivery of the dsRNA through transgenic expression of dsRNA in corn plants, or by contact with surface-applied iRNAs, provides an important stacking partner for other transgenic approaches that target larvae directly and enhance the overall durability of pest management strategies.

Example 7

Real-Time PCR Analysis

[0343] Total RNA was isolated from the whole bodies of adult females, males, larvae, and eggs using RNeasy mini Kit (Qiagen, Valencia, Calif.) following the manufacturer's recommendations. Before the initiation of the transcription reaction, the total RNA was treated with DNase to remove any gDNA using Quantitech reverse transcription kit (Qiagen, Valencia, Calif.). Total RNA (500 ng) was used to synthesize first strand cDNA as a template for real-time quantitative PCR (qPCR). The RNA was quantified spectrophotometrically at 260 nm and purity evaluated by agarose gel electrophoresis. Primers used for qPCR analysis were designed using Beacon designer software (Premier Biosoft International, Palo Alto, Calif.). The efficiencies of primer pairs were evaluated using 5 fold serial dilutions (1:1/5:1/25:1/125:1/625) in triplicate. Amplification efficiencies were higher than 96.1% for all the qPCR primer pairs used in this study. All primer combinations used in this study showed a linear correlation between the amount of cDNA template and the amount of PCR product. All correlation coefficients were larger than 0.99. The 7500 Fast System SDS v2.0.6 Software (Applied Biosystems) was used to determine the slope, correlation coefficients, and efficiencies. Three biological replications, each with two technical replications were used for qPCR analysis. qPCR was performed using SYBR green kit (Applied Biosystems Inc., Foster City, Calif.) and 7500 Fast System real-time PCR detection system (Applied Biosystems Inc., Foster City, Calif.). qPCR cycling parameters included 40 cycles each consisting of 95.degree. C. for 3 sec, 58.degree. C. for 30 sec, as described in the manufacturer's protocol (Applied Biosystems Inc., Foster City, Calif.). At the end of each PCR reaction, a melt curve was generated to confirm a single peak and rule out the possibility of primer-dimer and non-specific product formation. Relative quantification of the transcripts were calculated using the comparative 2.sup.-.DELTA..DELTA.CT method and were normalized to ft-actin.

[0344] FIG. 6A shows the relative expression of brahma in eggs collected from females exposed to dsRNA in treated artificial diet relative to GFP and water controls. FIG. 6B shows the relative expression of brahma in adult females exposed to dsRNA in treated artificial diet relative to GFP and water controls. There is a reduction in transcript levels in female adults and eggs. FIG. 6C shows the relative expression of mi-2 in adult females exposed to dsRNA in treated artificial diet relative to GFP and water controls. FIG. 6D. Relative expression of iswi-30 in adult females exposed to dsRNA in treated artificial diet relative to GFP and water controls.

Example 8

Construction of Plant Transformation Vectors

[0345] Entry vectors harboring a target gene construct for dsRNA hairpin formation comprising segments of brahma-c4465 rc (SEQ ID NO:1), brahma-8089 (SEQ ID NO:3), brahma-525 (SEQ ID NO:5), Contig[0001]_brahma_949-1126 (SEQ ID NO:7), mi-2 (SEQ ID NO:79), iswi-1 (SEQ ID NO:81), chd1 (SEQ ID NO:83), iswi-2 (SEQ ID NO:85), iswi30 (SEQ ID NO:87), ino80 (SEQ ID NO:89), domino (SEQ ID NO:91), mi-2 (SEQ ID NO:164), iswi-1 (SEQ ID NO:165), and/or iswi-2 (SEQ ID NO:166) are assembled using a combination of chemically synthesized fragments (DNA2.0, Menlo Park, Calif.) and standard molecular cloning methods. Intramolecular hairpin formation by RNA primary transcripts is facilitated by arranging (within a single transcription unit) two copies of a target gene segment in opposite orientation to one another, the two segments being separated by an ST-LS1 intron sequence (SEQ ID NO:56; Vancanneyt et al. (1990) Mol. Gen. Genet. 220:245-50). Thus, the primary mRNA transcript contains the two brahma gene segment sequences as large inverted repeats of one another, separated by the intron sequence. A copy of a maize ubiquitin 1 promoter (U.S. Pat. No. 5,510,474) is used to drive production of the primary mRNA hairpin transcript, and a fragment comprising a 3' untranslated region from a maize peroxidase 5 gene (ZmPer5 3'UTR v2; U.S. Pat. No. 6,699,984) is used to terminate transcription of the hairpin-RNA-expressing gene.

[0346] An entry vector comprises a brahma v1 hairpin-RNA construct (SEQ ID NO:57) that comprises a segment of brahma-8089 (SEQ ID NO:3) and brahma-525 (SEQ ID NO:5).

[0347] An entry vector comprises a brahma v2 hairpin-RNA construct (SEQ ID NO:58) that comprises a segment of brahma-c4465 rc (SEQ ID NO:1) and brahma-8089 (SEQ ID NO:3) distinct from that above.

[0348] Entry vectors described above are used in standard GATEWAY.RTM. recombination reactions with a typical binary destination vector to produce brahma hairpin RNA expression transformation vectors for Agrobacterium-mediated maize embryo transformations.

[0349] A negative control binary vector, which comprises a gene that expresses a YFP hairpin dsRNA, is constructed by means of standard GATEWAY.RTM. recombination reactions with a typical binary destination vector and entry vector. An entry vector comprises a YFP hairpin sequence (SEQ ID NO:138) under the expression control of a maize ubiquitin 1 promoter (as above) and a fragment comprising a 3' untranslated region from a maize peroxidase 5 gene (as above).

[0350] A binary destination vector comprises a herbicide tolerance gene (aryloxyalknoate dioxygenase; AAD-1 v3) (U.S. Pat. No. 7,838,733, and Wright et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:20240-5) under the regulation of a sugarcane bacilliform badnavirus (ScBV) promoter (Schenk et al. (1999) Plant Molec. Biol. 39:1221-30). A synthetic 5'UTR sequence, comprised of sequences from a Maize Streak Virus (MSV) coat protein gene 5'UTR and intron 6 from a maize Alcohol Dehydrogenase 1 (ADH1) gene, is positioned between the 3' end of the SCBV promoter segment and the start codon of the AAD-1 coding region. A fragment comprising a 3' untranslated region from a maize lipase gene (ZmLip 3'UTR; U.S. Pat. No. 7,179,902) is used to terminate transcription of the AAD-1 mRNA.

[0351] A further negative control binary vector, which comprises a gene that expresses a YFP protein, is constructed by means of standard GATEWAY.RTM. recombination reactions with a typical binary destination vector and entry vector. The binary destination vector comprises a herbicide tolerance gene (aryloxyalknoate dioxygenase; AAD-1 v3) (as above) under the expression regulation of a maize ubiquitin 1 promoter (as above) and a fragment comprising a 3' untranslated region from a maize lipase gene (ZmLip 3'UTR; as above). An entry vector comprises a YFP coding region under the expression control of a maize ubiquitin 1 promoter (as above) and a fragment comprising a 3' untranslated region from a maize peroxidase 5 gene (as above).

[0352] SEQ ID NO:57 presents a brahma v1 hairpin-forming sequence.

[0353] SEQ ID NO:58 presents a brahma v2 hairpin-forming sequence.

Example 9

Transgenic Maize Tissues Comprising Insecticidal dsRNAs

[0354] Agrobacterium-mediated Transformation. Transgenic maize cells, tissues, and plants that produce one or more insecticidal dsRNA molecules (for example, at least one dsRNA molecule including a dsRNA molecule targeting a gene comprising segments of brahma-c4465 rc (SEQ ID NO:1), brahma-8089 (SEQ ID NO:3), brahma-525 (SEQ ID NO:5), Contig[0001]_brahma_949-1126 (SEQ ID NO:7), mi-2 (SEQ ID NO:79 and SEQ ID NO:164), iswi-ID NO:81 and SEQ ID NO:165), chd1 (SEQ ID NO:83), iswi-2 (SEQ ID NO:85 and SEQ ID NO:166), iswi30 (SEQ ID NO:87), ino80 (SEQ ID NO:89), and/or domino (SEQ ID NO:91) through expression of a chimeric gene stably integrated into the plant genome are produced following Agrobacterium-mediated transformation. Maize transformation methods employing superbinary or binary transformation vectors are known in the art, as described, for example, in U.S. Pat. No. 8,304,604, which is herein incorporated by reference in its entirety. Transformed tissues are selected by their ability to grow on Haloxyfop-containing medium and are screened for dsRNA production, as appropriate. Portions of such transformed tissue cultures may be presented to neonate corn rootworm larvae for bioassay, essentially as described in EXAMPLE 1.

[0355] Agrobacterium Culture Initiation.

[0356] Glycerol stocks of Agrobacterium strain DAt13192 cells (WO 2012/016222A2) harboring a binary transformation vector as described above (EXAMPLE 7) are streaked on AB minimal medium plates (Watson et al. (1975) J. Bacteriol. 123:255-264) containing appropriate antibiotics and are grown at 20.degree. C. for 3 days. The cultures are then streaked onto YEP plates (gm/L: yeast extract, 10; Peptone, 10; NaCl 5) containing the same antibiotics and were incubated at 20.degree. C. for 1 day.

[0357] Agrobacterium Culture.

[0358] On the day of an experiment, a stock solution of Inoculation Medium and acetosyringone is prepared in a volume appropriate to the number of constructs in the experiment and pipetted into a sterile, disposable, 250 mL flask. Inoculation Medium (Frame et al. (2011) Genetic Transformation Using Maize Immature Zygotic Embryos. IN Plant Embryo Culture Methods and Protocols: Methods in Molecular Biology. T. A. Thorpe and E. C. Yeung, (Eds), Springer Science and Business Media, LLC. pp 327-341) contained: 2.2 gm/L MS salts; 1.times.ISU Modified MS Vitamins (Frame et al. (2011)) 68.4 gm/L sucrose; 36 gm/L glucose; 115 mg/L L-proline; and 100 mg/L myo-inositol; at pH 5.4.) Acetosyringone is added to the flask containing Inoculation Medium to a final concentration of 200 .mu.M from a 1 M stock solution in 100% dimethyl sulfoxide and the solution is thoroughly mixed.

[0359] For each construct, 1 or 2 inoculating loops-full of Agrobacterium from the YEP plate are suspended in 15 mL of the Inoculation Medium/acetosyringone stock solution in a sterile, disposable, 50 mL centrifuge tube, and the optical density of the solution at 550 nm (OD.sub.550) is measured in a spectrophotometer. The suspension is then diluted to OD.sub.550 of 0.3 to 0.4 using additional Inoculation Medium/acetosyringone mixture. The tube of Agrobacterium suspension is then placed horizontally on a platform shaker set at about 75 rpm at room temperature and shaken for 1 to 4 hours while embryo dissection is performed.

[0360] Ear Sterilization and Embryo Isolation.

[0361] Maize immature embryos are obtained from plants of Zea mays inbred line B104 (Hallauer et al. (1997) Crop Science 37:1405-1406) grown in the greenhouse and self- or sib-pollinated to produce ears. The ears are harvested approximately 10 to 12 days post-pollination. On the experimental day, de-husked ears are surface-sterilized by immersion in a 20% solution of commercial bleach (ULTRA CLOROX.RTM. GERMICIDAL BLEACH, 6.15% sodium hypochlorite; with two drops of TWEEN 20) and shaken for 20 to 30 min, followed by three rinses in sterile deionized water in a laminar flow hood. Immature zygotic embryos (1.8 to 2.2 mm long) are aseptically dissected from each ear and randomly distributed into microcentrifuge tubes containing 2.0 mL of a suspension of appropriate Agrobacterium cells in liquid Inoculation Medium with 200 .mu.M acetosyringone, into which 2 .mu.L of 10% BREAK-THRU.RTM. 5233 surfactant (EVONIK INDUSTRIES; Essen, Germany) had been added. For a given set of experiments, embryos from pooled ears are used for each transformation.

[0362] Agrobacterium Co-Cultivation.

[0363] Following isolation, the embryos are placed on a rocker platform for 5 minutes. The contents of the tube are then poured onto a plate of Co-cultivation Medium, which contains 4.33 gm/L MS salts; 1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH (3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid); 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 200 .mu.M acetosyringone in DMSO; and 3 gm/L GELZAN.TM., at pH 5.8. The liquid Agrobacterium suspension is removed with a sterile, disposable, transfer pipette. The embryos are then oriented with the scutellum facing up using sterile forceps with the aid of a microscope. The plate is closed, sealed with 3M.TM. MICROPORE.TM. medical tape, and placed in an incubator at 25.degree. C. with continuous light at approximately 60 .mu.mol m.sup.-2s.sup.-1 of Photosynthetically Active Radiation (PAR).

[0364] Callus Selection and Regeneration of Transgenic Events.

[0365] Following the Co-Cultivation period, embryos are transferred to Resting Medium, which is composed of 4.33 gm/L MS salts; 1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH; 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic Hydrolysate; 15 mg/L AgNO.sub.3; 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate; PHYTOTECHNOLOGIES LABR.; Lenexa, Kans.); 250 mg/L Carbenicillin; and 2.3 gm/L GELZAN.TM.; at pH 5.8. No more than 36 embryos are moved to each plate. The plates are placed in a clear plastic box and incubated at 27.degree. C. with continuous light at approximately 50 .mu.mol m.sup.-2s.sup.-1 PAR for 7 to 10 days. Callused embryos are then transferred (<18/plate) onto Selection Medium I, which is comprised of Resting Medium (above) with 100 nM R-Haloxyfop acid (0.0362 mg/L; for selection of calli harboring the AAD-1 gene). The plates are returned to clear boxes and incubated at 27.degree. C. with continuous light at approximately 50 .mu.mol m.sup.-2s.sup.-1 PAR for 7 days. Callused embryos are then transferred (<12/plate) to Selection Medium II, which is comprised of Resting Medium (above) with 500 nM R-Haloxyfop acid (0.181 mg/L). The plates are returned to clear boxes and incubated at 27.degree. C. with continuous light at approximately 50 .mu.mol m.sup.-2s.sup.-1 PAR for 14 days. This selection step allows transgenic callus to further proliferate and differentiate.

[0366] Proliferating, embryogenic calli are transferred (<9/plate) to Pre-Regeneration medium. Pre-Regeneration Medium contains 4.33 gm/L MS salts; 1.times.ISU Modified MS Vitamins; 45 gm/L sucrose; 350 mg/L L-proline; 100 mg/L myo-inositol; 50 mg/L Casein Enzymatic Hydrolysate; 1.0 mg/L AgNO.sub.3; 0.25 gm/L MES; 0.5 mg/L naphthaleneacetic acid in NaOH; 2.5 mg/L abscisic acid in ethanol; 1 mg/L 6-benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L GELZAN.TM.; and 0.181 mg/L Haloxyfop acid; at pH 5.8. The plates are stored in clear boxes and incubated at 27.degree. C. with continuous light at approximately 50 .mu.mol m.sup.-2s.sup.-1 PAR for 7 days. Regenerating calli are then transferred (<6/plate) to Regeneration Medium in PHYTATRAYS.TM. (SIGMA-ALDRICH) and incubated at 28.degree. C. with 16 hours light/8 hours dark per day (at approximately 160 .mu.mol m.sup.-2s.sup.-1 PAR) for 14 days or until shoots and roots develop. Regeneration Medium contains 4.33 gm/L MS salts; 1.times.ISU Modified MS Vitamins; 60 gm/L sucrose; 100 mg/L myo-inositol; 125 mg/L Carbenicillin; 3 gm/L GELLAN.TM. gum; and 0.181 mg/L R-Haloxyfop acid; at pH 5.8. Small shoots with primary roots are then isolated and transferred to Elongation Medium without selection. Elongation Medium contains 4.33 gm/L MS salts; 1.times.ISU Modified MS Vitamins; 30 gm/L sucrose; and 3.5 gm/L GELRITE.TM.: at pH 5.8.

[0367] Transformed plant shoots selected by their ability to grow on medium containing Haloxyfop are transplanted from PHYTATRAYS.TM. to small pots filled with growing medium (PROMIX BX; PREMIER TECH HORTICULTURE), covered with cups or HUMI-DOMES (ARCO PLASTICS), and then hardened-off in a CONVIRON.TM. growth chamber (27.degree. C. day/24.degree. C. night, 16-hour photoperiod, 50-70% RH, 200 .mu.mol m.sup.-2s.sup.-1 PAR). In some instances, putative transgenic plantlets are analyzed for transgene relative copy number by quantitative real-time PCR assays using primers designed to detect the AAD1 herbicide tolerance gene integrated into the maize genome. Further, RNA qPCR assays are used to detect the presence of the linker sequence in expressed dsRNAs of putative transformants. Selected transformed plantlets are then moved into a greenhouse for further growth and testing.

[0368] Transfer and Establishment of T.sub.0 Plants in the Greenhouse for Bioassay and Seed Production.

[0369] When plants reach the V3-V4 stage, they are transplanted into IE CUSTOM BLEND (PROFILE/METRO MIX 160) soil mixture and grown to flowering in the greenhouse (Light Exposure Type: Photo or Assimilation; High Light Limit: 1200 PAR; 16-hour day length; 27.degree. C. day/24.degree. C. night).

[0370] Plants to be used for insect bioassays are transplanted from small pots to TINUS.TM. 350-4 ROOTRAINERS.RTM. (SPENCER-LEMAIRE INDUSTRIES; Acheson, Alberta, Canada;) (one plant per event per ROOTRAINER.RTM.). Approximately four days after transplanting to ROOTRAINERS.RTM., plants are used in bioassays.

[0371] Plants of the T.sub.1 generation are obtained by pollinating the silks of T.sub.0 transgenic plants with pollen collected from plants of non-transgenic elite inbred line B104 or other appropriate pollen donors, and planting the resultant seeds. Reciprocal crosses are performed when possible.

Example 10

Adult Diabrotica Plant Feeding Bioassay

[0372] Transgenic corn foliage (V3-4) expressing dsRNA for parental RNAi targets and GFP controls is lyophilized and ground to a fine powder with mortar and pestle and sieved through a 600 .mu.M screen in order to achieve a uniform particle size prior to incorporation into artificial diet. The artificial diet is the same diet described previously for parental RNAi experiments except that the amount of water is doubled (20 mL ddH.sub.2O, 0.40 g agar, 6.0 g diet mix, 700 .mu.L glycerol, 27.5 .mu.L mold inhibitor). Prior to solidification, lyophilized corn leaf tissue is incorporated into the diet at a rate of 40 mg/ml of diet and mixed thoroughly. The diet is then poured onto the surface of a plastic petri dish to a depth of approximately 4 mm and allowed to solidify. Diet plugs are cut from the diet and used to expose western corn rootworm adults using the same methods described previously for parental RNAi experiments.

[0373] For plant feeding bioassays pRNAi T.sub.0 or T.sub.1 events are grown in the greenhouse until the plants produce cobs, tassel and silk. A total of 25 newly emerged rootworm adults are released on each plant, and the entire plant is covered to prevent adults from escaping. Two weeks after release, female adults are recovered from each plant and maintained in the laboratory for egg collection. Depending on the parental RNAi target and expected phenotype, parameters such as number of eggs per female, percent egg hatch and larval mortality are recorded and compared with control plants.

Example 11

Diabrotica Larval Root-Feeding Bioassay of Transgenic Maize

[0374] Insect Bioassays.

[0375] Bioactivity of dsRNA of the subject invention produced in plant cells is demonstrated by bioassay methods. One is able to demonstrate efficacy, for example, by feeding various plant tissues or tissue pieces derived from a plant producing an insecticidal dsRNA to target insects in a controlled feeding environment. Alternatively, extracts are prepared from various plant tissues derived from a plant producing the insecticidal dsRNA and the extracted nucleic acids are dispensed on top of artificial diets for bioassays as previously described herein. The results of such feeding assays are compared to similarly conducted bioassays that employ appropriate control tissues from host plants that do not produce an insecticidal dsRNA, or to other control samples.

[0376] Insect Bioassays with Transgenic Maize Events.

[0377] Two western corn rootworm larvae (1 to 3 days old) hatched from washed eggs are selected and placed into each well of the bioassay tray. The wells are then covered with a "PULL N' PEEL" tab cover (BIO-CV-16, BIO-SERV) and placed in a 28.degree. C. incubator with an 18 hr/6 hr light/dark cycle. Nine days after the initial infestation, the larvae are assessed for mortality, which is calculated as the percentage of dead insects out of the total number of insects in each treatment. The insect samples are frozen at -20.degree. C. for two days, then the insect larvae from each treatment are pooled and weighed. The percent of growth inhibition is calculated as the mean weight of the experimental treatments divided by the mean of the average weight of two control well treatments. The data are expressed as a Percent Growth Inhibition (of the Negative Controls). Mean weights that exceed the control mean weight are normalized to zero.

[0378] Insect bioassays in the greenhouse. Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) eggs are received in soil from CROP CHARACTERISTICS (Farmington, Minn.). WCR eggs are incubated at 28.degree. C. for 10 to 11 days. Eggs are washed from the soil, placed into a 0.15% agar solution, and the concentration is adjusted to approximately 75 to 100 eggs per 0.25 mL aliquot. A hatch plate is set up in a Petri dish with an aliquot of egg suspension to monitor hatch rates.

[0379] The soil around the maize plants growing in ROOTRANERS.RTM. is infested with 150 to 200 WCR eggs. The insects are allowed to feed for 2 weeks, after which time a "Root Rating" is given to each plant. A Node-Injury Scale is utilized for grading, essentially according to Oleson et al. (2005) J. Econ. Entomol. 98:1-8. Plants which pass this bioassay are transplanted to 5-gallon pots for seed production. Transplants are treated with insecticide to prevent further rootworm damage and insect release in the greenhouses. Plants are hand pollinated for seed production. Seeds produced by these plants are saved for evaluation at the T.sub.1 and subsequent generations of plants.

[0380] Greenhouse bioassays include two kinds of negative control plants. Transgenic negative control plants are generated by transformation with vectors harboring genes designed to produce a yellow fluorescent protein (YFP) or a YFP hairpin dsRNA (See EXAMPLE 4). Non-transformed negative control plants are grown from seeds of line B104. Bioassays are conducted on two separate dates, with negative controls included in each set of plant materials.

Example 12

Molecular Analyses of Transgenic Maize Tissues

[0381] Molecular analyses (e.g., RNA qPCR) of maize tissues are performed on samples from leaves and roots that are collected from greenhouse grown plants on the same days that root feeding damage is assessed.

[0382] Results of RNA qPCR assays for the Per5 3'UTR are used to validate expression of hairpin transgenes. (A low level of Per5 3'UTR detection is expected in non-transformed maize plants, since there is usually expression of the endogenous Per5 gene in maize tissues.) Results of RNA qPCR assay for intervening sequence between repeat sequences (which is integral to the formation of dsRNA hairpin molecules) in expressed RNAs are used to validate the presence of hairpin transcripts. Transgene RNA expression levels are measured relative to the RNA levels of an endogenous maize gene.

[0383] DNA qPCR analyses to detect a portion of the AAD1 coding region in gDNA are used to estimate transgene insertion copy number. Samples for these analyses are collected from plants grown in environmental chambers. Results are compared to DNA qPCR results of assays designed to detect a portion of a single-copy native gene, and simple events (having one or two copies of the transgenes) are advanced for further studies in the greenhouse.

[0384] Additionally, qPCR assays designed to detect a portion of the spectinomycin-resistance gene (SpecR; harbored on the binary vector plasmids outside of the T-DNA) are used to determine if the transgenic plants contain extraneous integrated plasmid backbone sequences.

[0385] Hairpin RNA Transcript Expression Level: Per 5 3'UTR qPCR.

[0386] Callus cell events or transgenic plants are analyzed by real time quantitative PCR (qPCR) of the Per 5 3'UTR sequence to determine the relative expression level of the full length hairpin transcript, as compared to the transcript level of an internal maize gene (for example, GENBANK Accession No. BT069734), which encodes a TIP41-like protein (i.e. a maize homolog of GENBANK Accession No. AT4G34270; having a tBLASTX score of 74% identity). RNA is isolated using an RNAEASY.TM. 96 kit (QIAGEN, Valencia, Calif.). Following elution, the total RNA is subjected to a DNAse1 treatment according to the kit's suggested protocol. The RNA is then quantified on a NANODROP 8000 spectrophotometer (THERMO SCIENTIFIC) and concentration is normalized to 25 ng/.mu.L. First strand cDNA is prepared using a HIGH CAPACITY cDNA SYNTHESIS KIT (INVITROGEN) in a 10 .mu.L reaction volume with 5 .mu.L denatured RNA, substantially according to the manufacturer's recommended protocol. The protocol is modified slightly to include the addition of 10 .mu.L of 100 T20VN oligonucleotide (IDT) (TTTTTTTTTTTTTTTTTTTTVN, where V is A, C, or G, and N is A, C, G, or T; SEQ ID NO:59) into the 1 mL tube of random primer stock mix, in order to prepare a working stock of combined random primers and oligo dT.

[0387] Following cDNA synthesis, samples are diluted 1:3 with nuclease-free water, and stored at -20.degree. C. until assayed.

[0388] Separate real-time PCR assays for the Per5 3' UTR and TIP41-like transcript are performed on a LIGHTCYCLER.TM. 480 (ROCHE DIAGNOSTICS, Indianapolis, Ind.) in 10 reaction volumes. For the Per5 3'UTR assay, reactions are run with Primers P5U76S (F) (SEQ ID NO:60) and P5U76A (R) (SEQ ID NO:61), and a ROCHE UNIVERSAL PROBE.TM. (UPL76; Catalog No. 4889960001; labeled with FAM). For the TIP41-like reference gene assay, primers TIPmxF (SEQ ID NO:62) and TIPmxR (SEQ ID NO:63), and Probe HXTIP (SEQ ID NO:64) labeled with HEX (hexachlorofluorescein) are used.

[0389] All assays include negative controls of no-template (mix only). For standard curves, a blank (water in source well) is also included in the source plate to check for sample cross-contamination. Primer and probe sequences are set forth in Table 10. Reaction components recipes for detection of the various transcripts are disclosed in Table 11, and PCR reactions conditions are summarized in Table 12. The FAM (6-Carboxy Fluorescein Amidite) fluorescent moiety is excited at 465 nm and fluorescence is measured at 510 nm; the corresponding values for the HEX (hexachlorofluorescein) fluorescent moiety are 533 nm and 580 nm.

TABLE-US-00013 TABLE 10 Oligonucleotide sequences used for molecular analyses of transcript levels in transgenic maize. SEQ ID Target Oligonucleotide NO. Sequence Per5 3'UTR P5U76S (F) 60 TTGTGATGTTGGTGGCGTAT Per5 3'UTR P5U76A (R) 61 TGTTAAATAAAACCCCAAAGATCG Per5 3'UTR Roche UPL76 NAv** Roche Diagnostics Catalog Number 488996001 (FAM-Probe) TIP41 TIPmxF 62 TGAGGGTAATGCCAACTGGTT TIP41 TIPmxR 63 GCAATGTAACCGAGTGTCTCTCAA TIP41 HXTIP (HEX- 64 TTTTTGGCTTAGAGTTGATGGTGTACTGATGA Probe) *TIP41-like protein. **NAv Sequence Not Available from the supplier.

TABLE-US-00014 TABLE 11 PCR reaction recipes for transcript detection. Per5 3'UTR TIP-like Gene Component Final Concentration Roche Buffer 1.times. 1.times. P5U76S (F) 0.4 .mu.M 0 P5U76A (R) 0.4 .mu.M 0 Roche UPL76 (FAM) 0.2 .mu.M 0 HEXtipZM F 0 0.4 .mu.M HEXtipZM R 0 0.4 .mu.M HEXtipZMP (HEX) 0 0.2 .mu.M cDNA (2.0 .mu.L) NA NA Water To 10 .mu.L To 10 .mu.L

TABLE-US-00015 TABLE 12 Thermocycler conditions for RNA qPCR. Per5 3'UTR and TIP41-like Gene Detection Process Temp. Time No. Cycles Target Activation 95.degree. C. 10 min 1 Denature 95.degree. C. 10 sec 40 Extend 60.degree. C. 40 sec Acquire FAM or HEX 72.degree. C. 1 sec Cool 40.degree. C. 10 sec 1

[0390] Data are analyzed using LIGHTCYCLER.TM. Software v1.5 by relative quantification using a second derivative max algorithm for calculation of Cq values according to the supplier's recommendations. For expression analyses, expression values are calculated using the .DELTA..DELTA.Ct method (i.e., 2-(Cq TARGET-Cq REF)), which relies on the comparison of differences of Cq values between two targets, with the base value of 2 being selected under the assumption that, for optimized PCR reactions, the product doubles every cycle.

[0391] Hairpin Transcript Size and Integrity: Northern Blot Assay.

[0392] In some instances, additional molecular characterization of the transgenic plants is obtained by the use of Northern Blot (RNA blot) analysis to determine the molecular size of the brahma hairpin RNA in transgenic plants expressing a brahma hairpin dsRNA.

[0393] All materials and equipment are treated with RNaseZAP (AMBION/INVITROGEN) before use. Tissue samples (100 mg to 500 mg) are collected in 2 mL SAFELOCK EPPENDORF tubes, disrupted with a KLECKO.TM. tissue pulverizer (GARCIA MANUFACTURING, Visalia, Calif.) with three tungsten beads in 1 mL of TRIZOL (INVITROGEN) for 5 min, then incubated at room temperature (RT) for 10 min. Optionally, the samples are centrifuged for 10 min at 4.degree. C. at 11,000 rpm and the supernatant is transferred into a fresh 2 mL SAFELOCK EPPENDORF tube. After 200 .mu.L chloroform are added to the homogenate, the tube is mixed by inversion for 2 to 5 min, incubated at RT for 10 minutes, and centrifuged at 12,000.times.g for 15 min at 4.degree. C. The top phase is transferred into a sterile 1.5 mL EPPENDORF tube, 600 .mu.L of 100% isopropanol are added, followed by incubation at RT for 10 min to 2 hr, and then centrifuged at 12,000.times.g for 10 min at 4.degree. C. to 25.degree. C. The supernatant is discarded and the RNA pellet is washed twice with 1 mL 70% ethanol, with centrifugation at 7,500.times.g for 10 min at 4.degree. C. to 25.degree. C. between washes. The ethanol is discarded and the pellet is briefly air dried for 3 to 5 min before resuspending in 50 .mu.L of nuclease-free water.

[0394] Total RNA is quantified using the NANODROP8000.RTM. (THERMO-FISHER) and samples are normalized to 5 .mu.g/10 .mu.L. 10 .mu.L of glyoxal (AMBION/INVITROGEN) are then added to each sample. Five to 14 ng of DIG RNA standard marker mix (ROCHE APPLIED SCIENCE, Indianapolis, Ind.) are dispensed and added to an equal volume of glyoxal. Samples and marker RNAs are denatured at 50.degree. C. for 45 min and stored on ice until loading on a 1.25% SEAKEM GOLD agarose (LONZA, Allendale, N.J.) gel in NORTHERNMAX 10.times. glyoxal running buffer (AMBION/INVITROGEN). RNAs are separated by electrophoresis at 65 volts/30 mA for 2 hours and 15 minutes.

[0395] Following electrophoresis, the gel is rinsed in 2.times.SSC for 5 min and imaged on a GEL DOC station (BIORAD, Hercules, Calif.), then the RNA is passively transferred to a nylon membrane (MILLIPORE) overnight at RT, using 10.times.SSC as the transfer buffer (20.times.SSC consists of 3 M sodium chloride and 300 mM trisodium citrate, pH 7.0). Following the transfer, the membrane is rinsed in 2.times.SSC for 5 minutes, the RNA is UV-crosslinked to the membrane (AGILENT/STRATAGENE), and the membrane is allowed to dry at room temperature for up to 2 days.

[0396] The membrane is prehybridized in ULTRAHYB buffer (AMBION/INVITROGEN) for 1 to 2 hr. The probe consists of a PCR amplified product containing the sequence of interest, (for example, the antisense sequence portion of SEQ ID NO:57 or SEQ ID NO:58, as appropriate) labeled with digoxigenin by means of a ROCHE APPLIED SCIENCE DIG procedure. Hybridization in recommended buffer is overnight at a temperature of 60.degree. C. in hybridization tubes. Following hybridization, the blot is subjected to DIG washes, wrapped, exposed to film for 1 to 30 minutes, then the film is developed, all by methods recommended by the supplier of the DIG kit.

[0397] Transgene Copy Number Determination.

[0398] Maize leaf pieces approximately equivalent to 2 leaf punches are collected in 96-well collection plates (QIAGEN). Tissue disruption is performed with a KLECKO.TM. tissue pulverizer (GARCIA MANUFACTURING, Visalia, Calif.) in BIOSPRINT96 AP1 lysis buffer (supplied with a BIOSPRINT96 PLANT KIT; QIAGEN) with one stainless steel bead. Following tissue maceration, gDNA is isolated in high throughput format using a BIOSPRINT96 PLANT KIT and a BIOSPRINT96 extraction robot. gDNA is diluted 2:3 DNA:water prior to setting up the qPCR reaction.

[0399] qPCR Analysis.

[0400] Transgene detection by hydrolysis probe assay is performed by real-time PCR using a LIGHTCYCLER.RTM.480 system. Oligonucleotides to be used in hydrolysis probe assays to detect the linker sequence (e.g. ST-LS1; SEQ ID NO:56), or to detect a portion of the SpecR gene (i.e. the spectinomycin resistance gene borne on the binary vector plasmids; SEQ ID NO:65; SPC1 oligonucleotides in Table 13), are designed using LIGHTCYCLER.RTM. PROBE DESIGN SOFTWARE 2.0. Further, oligonucleotides to be used in hydrolysis probe assays to detect a segment of the AAD-1 herbicide tolerance gene (SEQ ID NO:66; GAAD1 oligonucleotides in Table 13) are designed using PRIMER EXPRESS software (APPLIED BIOSYSTEMS). Table 13 shows the sequences of the primers and probes. Assays are multiplexed with reagents for an endogenous maize chromosomal gene (Invertase; GENBANK Accession No: U16123; referred to herein as IVR1), which serves as an internal reference sequence to ensure gDNA was present in each assay. For amplification, LIGHTCYCLER.RTM.480 PROBES MASTER mix (ROCHE APPLIED SCIENCE) is prepared at 1.times. final concentration in a 10 .mu.L volume multiplex reaction containing 0.4 .mu.M of each primer and 0.2 .mu.M of each probe (Table 14). A two-step amplification reaction is performed as outlined in Table 15. Fluorophore activation and emission for the FAM- and HEX-labeled probes are as described above; CY5 conjugates are excited maximally at 650 nm and fluoresce maximally at 670 nm.

[0401] Cp scores (the point at which the fluorescence signal crosses the background threshold) are determined from the real time PCR data using the fit points algorithm (LIGHTCYCLER.RTM. SOFTWARE release 1.5) and the Relative Quant module (based on the .DELTA..DELTA.Ct method). Data are handled as described previously (above; RNA qPCR).

TABLE-US-00016 TABLE 13 Sequences of primers and probes (with fluorescent conjugate) used for gene copy number determinations and binary vector plasmid backbone detection. SEQ ID Name NO: Sequence ST-LS1-F 67 GTATGTTTCTGCTTCTACCTTTGAT ST-LS1-R 68 CCATGTTTTGGTCATATATTAGAAAAGTT ST-LS1-P (FAM) 69 AGTAATATAGTATTTCAAGTATTTTTTTC AAAAT GAAD1-F 70 TGTTCGGTTCCCTCTACCAA GAAD1-R 71 CAACATCCATCACCTTGACTGA GAAD1-P (FAM) 72 CACAGAACCGTCGCTTCAGCAACA IVR1-F 73 TGGCGGACGACGACTTGT IVR1-R 74 AAAGTTTGGAGGCTGCCGT IVR1-P (HEX) 75 CGAGCAGACCGCCGTGTACTTCTACC SPC1A 76 CTTAGCTGGATAACGCCAC SPC1S 77 GACCGTAAGGCTTGATGAA TQSPEC (CY5*) 78 CGAGATTCTCCGCGCTGTAGA CY5 = Cyanine-5

TABLE-US-00017 TABLE 14 Reaction components for gene copy number analyses and plasmid backbone detection. Final Component Amt. (.mu.L) Stock Concentration 2.times. Buffer 5.0 2.times. 1.times. Appropriate Forward Primer 0.4 10 .mu.M 0.4 Appropriate Reverse Primer 0.4 10 .mu.M 0.4 Appropriate Probe 0.4 5 .mu.M 0.2 IVR1-Forward Primer 0.4 10 .mu.M 0.4 IVR1-Reverse Primer 0.4 10 .mu.M 0.4 IVR1-Probe 0.4 5 .mu.M 0.2 H.sub.2O 0.6 NA* NA gDNA 2.0 ND** ND Total 10.0 *NA = Not Applicable **ND = Not Determined

TABLE-US-00018 TABLE 15 Thermocycler conditions for DNA qPCR. Genomic copy number analyses Process Temp. Time No. Cycles Target Activation 95.degree. C. 10 min 1 Denature 95.degree. C. 10 sec 40 Extend & Acquire 60.degree. C. 40 sec FAM, HEX, or CY5 Cool 40.degree. C. 10 sec 1

Example 13

Transgenic Zea mays Comprising Coleopteran Pest Sequences

[0402] Ten to 20 transgenic T.sub.0 Zea mays plants are generated as described in EXAMPLE 8. A further 10-20 T.sub.1 Zea mays independent lines expressing hairpin dsRNA for an RNAi construct are obtained for corn rootworm challenge. DNAs expressing hairpin dsRNA forming polynucleotides may be derived from a sequence as set forth in SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164; SEQ ID NO:165; SEQ ID NO:166; and fragments of the foregoing (e.g., SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NOs:101-106). Additional hairpin dsRNAs may be derived, for example, from coleopteran pest sequences such as, for example, Caf1-180 (U.S. Patent Application Publication No. 2012/0174258), VatpaseC (U.S. Patent Application Publication No. 2012/0174259), Rho1 (U.S. Patent Application Publication No. 2012/0174260), VatpaseH (U.S. Patent Application Publication No. 2012/0198586), PPI-87B (U.S. Patent Application Publication No. 2013/0091600), RPA70 (U.S. Patent Application Publication No. 2013/0091601), RPS6 (U.S. Patent Application Publication No. 2013/0097730), Brahma (USSN), and Kruppel (USSN). These are confirmed through RT-PCR or other molecular analysis methods. Total RNA preparations from selected independent T.sub.1 lines are optionally used for RT-PCR with primers designed to bind in the linker of the hairpin expression cassette in each of the RNAi constructs. In addition, specific primers for each target gene in an RNAi construct are optionally used to amplify and confirm the production of the pre-processed mRNA required for siRNA production in planta. The amplification of the desired bands for each target gene confirms the expression of the hairpin RNA in each transgenic Zea mays plant. Processing of the dsRNA hairpin of the target genes into siRNA is subsequently optionally confirmed in independent transgenic lines using RNA blot hybridizations.

[0403] Moreover, RNAi molecules having mismatch sequences with more than 80% sequence identity to target genes affect corn rootworms in a way similar to that seen with RNAi molecules having 100% sequence identity to the target genes. The pairing of mismatch sequence with native sequences to form a hairpin dsRNA in the same RNAi construct delivers plant-processed siRNAs capable of affecting the growth, development, reproduction, and viability of feeding coleopteran pests.

[0404] In planta delivery of dsRNA, siRNA or miRNA corresponding to target genes and the subsequent uptake by coleopteran pests through feeding results in down-regulation of the target genes in the coleopteran pest through RNA-mediated gene silencing. When the function of a target gene is important at one or more stages of development, the growth, development, and reproduction of the coleopteran pest is affected, and in the case of at least one of WCR, NCR, SCR, MCR, D. balteata LeConte, D. u. tenella, and D. u. undecimpunctata Mannerheim, leads to failure to successfully infest, feed, develop, and/or reproduce, or leads to death of the coleopteran pest. The choice of target genes and the successful application of RNAi is then used to control coleopteran pests.

[0405] Phenotypic Comparison of Transgenic RNAi Lines and Nontransformed Zea mays.

[0406] Target coleopteran pest genes or sequences selected for creating hairpin dsRNA have no similarity to any known plant gene sequence. Hence it is not expected that the production or the activation of (systemic) RNAi by constructs targeting these coleopteran pest genes or sequences will have any deleterious effect on transgenic plants. However, development and morphological characteristics of transgenic lines are compared with non-transformed plants, as well as those of transgenic lines transformed with an "empty" vector having no hairpin-expressing gene. Plant root, shoot, foliage and reproduction characteristics are compared. There is no observable difference in root length and growth patterns of transgenic and non-transformed plants. Plant shoot characteristics such as height, leaf numbers and sizes, time of flowering, floral size and appearance are similar. In general, there are no observable morphological differences between transgenic lines and those without expression of target iRNA molecules when cultured in vitro and in soil in the glasshouse.

Example 14

Transgenic Zea mays Comprising a Coleopteran Pest Sequence and Additional RNAi Constructs

[0407] A transgenic Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets an organism other than a coleopteran pest is secondarily transformed via Agrobacterium or WHISKERS.TM. methodologies (See Petolino and Arnold (2009) Methods Mol. Biol. 526:59-67) to produce one or more insecticidal dsRNA molecules (for example, at least one dsRNA molecule including a dsRNA molecule targeting a gene comprising SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164; SEQ ID NO:165; SEQ ID NO:166; and fragments of any of the foregoing (e.g., SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NOs:101-106)). Plant transformation plasmid vectors prepared essentially as described in EXAMPLE 7 are delivered via Agrobacterium or WHISKERS.TM.-mediated transformation methods into maize suspension cells or immature maize embryos obtained from a transgenic Hi II or B104 Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets an organism other than a coleopteran pest.

Example 15

Transgenic Zea mays Comprising an RNAi Construct and Additional Coleopteran Pest Control Sequences

[0408] A transgenic Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets a coleopteran pest organism (for example, at least one dsRNA molecule including a dsRNA molecule targeting a gene comprising SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:79; SEQ ID NO:81; SEQ ID NO:83; SEQ ID NO:85; SEQ ID NO:87; SEQ ID NO:89; SEQ ID NO:91; SEQ ID NO:164; SEQ ID NO:165; SEQ ID NO:166; and fragments of any of the foregoing (e.g., SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NOs: 101-106)) is secondarily transformed via Agrobacterium or WHISKERS.TM. methodologies (see Petolino and Arnold (2009) Methods Mol. Biol. 526:59-67) to produce one or more insecticidal protein molecules, for example, Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C insecticidal proteins. Plant transformation plasmid vectors prepared essentially as described in EXAMPLE 7 are delivered via Agrobacterium or WHISKERS.TM.-mediated transformation methods into maize suspension cells or immature maize embryos obtained from a transgenic B104 Zea mays plant comprising a heterologous coding sequence in its genome that is transcribed into an iRNA molecule that targets a coleopteran pest organism. Doubly-transformed plants are obtained that produce iRNA molecules and insecticidal proteins for control of coleopteran pests.

Example 16

pRNAi-Mediated Insect Protection

[0409] Parental RNAi that causes egg mortality or loss of egg viability brings further durability benefits to transgenic crops that use RNAi and other mechanisms for insect protection. A basic two-patch model was used to demonstrate this utility.

[0410] One patch contained a transgenic crop expressing insecticidal ingredients, and the second patch contained a refuge crop not expressing insecticidal ingredients. Eggs were oviposited in the two-modeled patches according to their relative proportions. In this example, the transgenic patch represented 95% of the landscape, and the refuge patch represented 5%. The transgenic crop expressed an insecticidal protein active against corn rootworm larvae.

[0411] Corn rootworm resistance to the insecticidal protein was modeled as monogenic, with two possible alleles; one (S) conferring susceptibility, and the other (R) conferring resistance. The insecticidal protein was modeled to cause 97% mortality of homozygous susceptible (SS) corn rootworm larvae that feed on it. There was assumed to be no mortality of corn rootworm larvae that are homozygous for the resistance allele (RR). Resistance to the insecticidal protein was assumed to be incompletely recessive, whereby the functional dominance is 0.3 (there is 67.9% mortality of larvae that are heterozygous (RS) for resistance to the protein that feed on the transgenic crop).

[0412] The transgenic crop also expressed parentally active dsRNA that, through RNA-interference (pRNAi), causes the eggs of adult female corn rootworms that are exposed to the transgenic crop to be non-viable. Corn rootworm resistance to the pRNAi was also considered to be monogenic with two possible alleles; one (X) conferring susceptibility of the adult female to RNAi, and the other (Y) conferring resistance of the adult female to RNAi. Assuming a high level of exposure to the dsRNAs, the pRNAi was modeled to cause 99.9% of eggs produced by a homozygous susceptible (XX) female to be non-viable. The model assumed that pRNAi has no effect on the viability of eggs produced by homozygous resistant (YY) females. Resistance to the dsRNA was assumed to be recessive, whereby the functional dominance is 0.01 (98.9% of eggs produced by a female that is heterozygous (XY) for resistance to dsRNA are non-viable).

[0413] In the model, there was random mating among surviving adults and random oviposition across the two patches in accordance with their relative proportions. The genotypic frequencies of viable offspring followed Mendelian genetics for a two-locus genetic system.

[0414] The effect of pRNAi required the adult females to feed on plant tissue expressing parental active dsRNA. The interference with egg development may be lower for adult females emerging from the refuge crop than from the transgenic crop; corn rootworm adults are expected to feed more extensively in the patch in which they emerged following larval development. Therefore, the relative magnitude of the pRNAi effect on female corn rootworm adults emerging from the refuge patch was varied, with the proportion of the pRNAi effect ranging from 0 (no effect of pRNAi on adult females emerging from the refuge patch) to 1 (same effect of pRNAi on adult females emerging from the refuge patch as on adult females emerging from the transgenic patch).

[0415] This model could be easily adjusted to demonstrate the situation when the effect of pRNAi is also or alternatively achieved by feeding of adult males on plant tissue expressing parental active dsRNA.

[0416] Frequencies of the two resistance alleles were calculated across generations. The initial frequencies of both of the resistance alleles (R and Y) were assumed to be 0.005. Results were presented as the number of insect generations for the frequencies of each of the resistance alleles to reach 0.05. To examine the resistance delay caused by the pRNAi, simulations that included pRNAi were compared to simulations that did not include pRNAi, but were identical in every other way. FIG. 7.

[0417] The model was also modified to include corn rootworm larval-active interfering dsRNA in combination with the corn rootworm-active insecticidal protein in the transgenic crop. Therein, the larval RNAi was assigned an effect of 97% larval mortality for homozygous RNAi-susceptible corn rootworm larvae (genotype XX), and no effect on corn rootworm larvae that are homozygous RNAi-resistant (YY). There was 67.9% mortality of corn rootworm larvae that were heterozygous for RNAi-resistance (XY). It was assumed that the same mechanism of resistance applied to both larval active RNAi and pRNAi in corn rootworms. As before, the pRNAi effect on adult females emerging from the refuge patch relative to the effect on adult females emerging from the transgenic patch was varied from 0 to 1. As before, to examine the resistance delay caused by the pRNAi, simulations that included pRNAi were compared to simulations that did not include pRNAi, but were identical in every other way (including larval RNAi). FIG. 8.

[0418] A clear resistance management benefit of pRNAi was observed when the magnitude of the pRNAi effect on egg viability for female corn rootworm adults emerging from the refuge patch was reduced compared with magnitude of the effect for adults emerging from the transgenic patch. The transgenic crops that produced parental active dsRNA in addition to an insecticidal protein were much more durable compared with transgenic crops that produced only an insecticidal protein. Similarly, transgenic crops that produced parental active dsRNA in addition to both an insecticidal protein and a larval active dsRNA were much more durable compared with transgenic crops that produced only an insecticidal protein and a larval active dsRNA. In the latter case, the durability benefit applied to both the insecticidal protein and the insecticidal interfering dsRNA.

Example 17

Parental RNAi Effects on WCR Males

[0419] Newly emerged virgin WCR males received from CROP CHARACTERISTICS (Farmington, Minn.) were exposed to artificial diet treated with dsRNA for pRNAi (brahma) for 7 days with continuous dsRNA feeding. The surviving males were then paired with virgin females and allowed to mate for 4 days. Females were isolated into oviposition chambers and maintained on untreated diet to determine if mating was successful, based on egg viability. In addition, the females were dissected to determine the presence of spermatophores after 10 days of oviposition. Controls of GFP dsRNA and water were included.

[0420] Three replicates of 10 males and 10 females per treatment per replication were performed. Replicates were completed with newly emerged adults on 3 different days. Each treatment per replicate contained 10 males per treatment per replication and were placed in one well of a tray. Each well included 12 diet plugs treated with water or dsRNA (GFP or brahma). Each diet plug was treated with 2 .mu.g dsRNA in 3 .mu.L water. Trays were transferred to a growth chamber with a temperature of 23.+-.1.degree. C., relative humidity >80%, and L:D 16:8. Males were transferred to new trays with 12 treated diet plugs in each well on days 3, 5, and 7. On day 7, three males per replication per treatment were flash frozen for qPCR analysis, as described in EXAMPLE 7. On day 8, ten females and ten treated males were placed together in a container to allow mating. Each container included 22 untreated diet plugs. Insects were transferred to new trays with 22 untreated diet plugs on day 10. Males were removed on day 12 and used to measure sperm viability using fluorescent staining techniques. Females were transferred to a new tray with 12 untreated diet plugs every other day until day 22. On day 16, females were transferred to egg cages containing autoclaved soil for oviposition. On day 22, all females were removed from the soil cages and frozen to check for the presence of spermatophores. The soil cages were transferred to a new growth chamber with a temperature of 27.+-.1.degree. C., relative humidity >80%, and 24 h dark. On day 28, the soil was washed using a sieve #60 to collect eggs from each cage. Eggs were treated with a solution of formaldehyde (500 .mu.L formaldehyde in 5 mL double distilled water) and methyl-(butycarbamoy)-2-benzimidazole carbamate (0.025 g in 50 mL double distilled water) to prevent fungal contamination, and were placed in small petri dishes containing filter paper. Photographs were taken of each petri dish for egg counting using the cell counter function of the ImageJ Software (Schneider et al. (2012) Nat. Methods 9:671-5). Petri dishes with eggs were transferred to a small growth chamber with a temperature of 27.+-.1.degree. C., relative humidity >80%, and 24 h dark. Larval hatch was monitored daily from days 29-42.

[0421] Sperm Viability.

[0422] Virgin western corn rootworm males were exposed to artificial diet treated with dsRNA for 7 days with the parental RNAi gene brahma. Treated diet was provided every other day. Four males per treatment per replication were used to test for sperm viability using a fluorescent technique to discriminate between living and dead sperm as described by Collins and Donoghue (1999). The Live Dead Sperm Viability Kit.TM. (Life Technologies, Carlsbad Calif.) contains SYBR 14, a membrane-permeant nucleic acid stain, and propidium iodine, which stains dead cells.

[0423] WCR males were anesthetized on ice, testes and seminal vesicles were dissected, placed in 10 .mu.L buffer (HEPES 10 mM, NaCl 150 mM, BSA 10%, pH 7.4,) and crushed with an autoclaved toothpick. Sperm viability was immediately assessed using the Live Dead Sperm Viability Kit.TM.. A 1 .mu.L volume of SYBR 14 (0.1 mM in DMSO) was added and incubated at room temperature for 10 minutes, followed by addition of 1 .mu.L propidium iodine (2.4 mM), and incubated again at room temperature for 10 minutes. A 10 .mu.L volume of sperm stained solution was transferred to a glass microslide and covered with a cover slip. Samples were evaluated using a Nikon.TM. Eclipse 90i microscope with a Nikon A1 confocal and MS-Elements Software. Samples were visualized at 10.times. with 488 excitation, a 500-550 nm band pass for live sperm (SYBR 14) and 663-738 nm band pass for dead sperm (propidium iodine) simultaneously. Digital images were recorded for five fields of view per sample. The number of live (green) and dead (red) sperm was evaluated using the cell counter function of ImageJ Software (Schneider et al. (2012) Nat. Methods 9:671-5)).

[0424] Males fed brahma dsRNA for 7 days produced less total sperm, less live sperm, and less dead sperm than males ingesting GFP dsRNA or water alone. Table 16. The average number of live sperm was significantly different between the treatments. There was no statistical difference in percent egg hatch from females that had mated with males that had ingested dsRNA treatments. Table 17.

TABLE-US-00019 TABLE 16 Effect of brahma dsRNA on WCR adult male sperm production and viability after 7 days of ingestion on treated artificial diet. Means were separated using Dunnett's test. Average dead Average live Average total Treatment sperm .+-. SEM.sup..dagger. sperm .+-. SEM.sup..dagger. sperm .+-. SEM.sup..dagger. brahma 46.89 .+-. 8.83** 121.88 .+-. 15.43** 152.29 .+-. 24.78** GFP 74.79 .+-. 14.17 222.74 .+-. 38.89* 288.73 .+-. 43.18** Water 68.5 .+-. 12.26 164.7 .+-. 31.87 233.2 .+-. 22.33 .sup..dagger.SEM--Standard Error of the Mean. *Indicates significance at p-value .ltoreq. 0.1. **Indicates significance at p-value .ltoreq. 0.05.

TABLE-US-00020 TABLE 17 Effect of brahma dsRNA on WCR egg production and egg viability after 7 days of ingestion dsRNA treated artificial diet by males only. Means were separated using Dunnett's test. Egg numbers per female beetle Percent egg hatch brahma brahma Reg352 GFP Reg352 GFP dsRNA dsRNA Water dsRNA dsRNA Water Average 61.55* 58.08 38.52 62.85 82.93 76.24 SEM.dagger. 21.46 11.38 15.94 12.83 2.56 5.31 *Indicates significance at p-value .ltoreq.0.1. .dagger.SEM--Standard error of the mean.

[0425] Virgin males were treated as described above except that the exposure to dsRNA was increased to a total of 6 times. Males were transferred to new trays with 12 treated diet plugs in each well on days 3, 5, 7, 9, and 11. The surviving males were then paired with virgin females and allowed to mate for 4 days. Females were isolated into oviposition chambers and maintained on untreated diet to determine if mating was successful based on egg viability.

TABLE-US-00021 TABLE 18 Effect of brahma dsRNA on WCR egg production and egg viability after 7 days of ingestion dsRNA treated artificial diet by males only. Means were separated using Dunnett's test. Egg numbers per female beetle Percent egg hatch brahma brahma Reg352 GFP Reg352 GFP dsRNA dsRNA Water dsRNA dsRNA Water Average 61.01 61.20 51.14 22.27* 32.80 35.41 SEM.dagger. 5.99 14.83 8.28 3.99 6.89 7.5 *Indicates significance at p-value .ltoreq.0.1. .dagger.SEM--Standard error of the mean.

[0426] Relative expression in males was determined as described in Example 7.

TABLE-US-00022 TABLE 19 Relative expression of brahma in adult males exposed to dsRNA in treated artificial diet relative to GFP and water controls. There is a reduction in transcript levels in male adults. Means were separated using Dunnett's test. Relative Treatment expression SEM.sup..dagger. p-value brahma 0.138 0.028 <0.0001 GFP 0.964 0.132 0.836 Water 1.023 0.075 .sup..dagger.SEM--Standard error of the mean.

Example 18

Effective Concentration

[0427] Mated females were exposed to 4 exposure conditions of brahma dsRNA to determine the effective concentrations. Newly emerged (<48 hours) adult males and females were obtained from CROP CHARACTERISTICS (Farmington, Minn.). Treatments included 2, 0.2, 0.02, and 0.002 .mu.g brahma dsRNA per diet plug. GFP at 2 .mu.g and water served as the controls. Ten males and 10 females were placed together in one well containing 20 pellets of untreated artificial diet. Trays were transferred to a growth chamber and maintained at 23.+-.1.degree. C., relative humidity >80%, and 16:8 L:D photoperiod. Males were removed from the experiment on day 5. Freshly treated diet was provided every other day until day 13. On day 14 females were transferred to egg cages containing autoclaved soil and new treated artificial diet was provided (11 plugs per cage). Egg cages were placed back in the growth chamber. On day 16 new treated diet was provided as described above. All females were removed from the soil cages on day 18 and flash frozen for qPCR. Soil cages were transferred to a new growth chamber with a temperature of 27.+-.1.degree. C., relative humidity >80% and 24 h dark. On day 24 the soil was washed using a #60 sieve to collect eggs from each cage. Eggs were treated with a solution of formaldehyde (500 .mu.l formaldehyde in 5 ml of double distilled water) and methyl-(butycarbamoy)-2-benzimidazole carbamate (0.025 g in 50 ml of double distilled water) to prevent fungal contamination and placed in small petri dishes containing filter paper. Photographs were taken of each petri dish for egg counting using the cell counter function of Image J software (Schneider et al. (2012) Nat. Methods 9:671-5). Petri dishes with eggs were transferred to a small growth chamber with a temperature of 27.+-.1.degree. C., relative humidity >80%, and 24 h dark. Larval hatching was monitored daily through 15 days. Larvae were counted and removed from the Petri dish each day.

[0428] There was significantly reduced egg hatch at the 2 and 0.2 .mu.g/diet plug treatments (Table 20), but there was no difference in the number of eggs oviposited per female between any of the doses tested and the controls.

TABLE-US-00023 TABLE 20 Effect of brahma dsRNA concentrations on WCR egg production and egg viability after ingestion of treated artificial diet. Means were separated using Dunnett's test. Avg. no. eggs per female Percent Dose beetle egg hatch Treatment (.mu.g) Average SEM.sup..dagger. Average SEM.sup..dagger. brahma Reg352 dsRNA 2 23.22 5.96 0.23** 0.25 brahma Reg352 dsRNA 0.2 80.05 9.86 7.09** 4.45 brahma Reg352 dsRNA 0.02 76.85 17.51 29.03 10.71 brahma Reg352 dsRNA 0.002 71.16 18.9 45.26 4.01 GFP dsRNA 2 64.87 28.64 32.58 10 Water 0 70.71 20.18 39.41 3.92 .sup..dagger.SEM--Standard Error of the Mean. **Indicates significance at (p-value < 0.05).

Example 19

Timing of Exposure

[0429] Females were exposed 6 times to 2 .mu.g of brahma dsRNA starting at three different times to determine the timing of exposure necessary to generate a parental RNAi effect. Females were exposed to dsRNA 6 times before mating, 6 times immediately after mating, and 6 days after mating. Three replications of 10 females and 10 males per replication were completed for each exposure time. Adult WCRs were received from CROP CHARACTERISTICS (Farmington, Minn.).

[0430] dsRNA Feeding Before Mating:

[0431] Ten females were placed in one well with 11 pellets of treated artificial diet (2 .mu.g dsRNA per pellet). Trays were transferred to a growth chamber with a temperature of 23.+-.1.degree. C., relative humidity >80%, and 16:8 L:D photoperiod. Females were transferred to trays containing fresh treated diet every other day for 10 days. On day 12 females were paired with 10 males and 22 plugs of untreated diet were provided. Males were removed after 4 days. Fresh untreated diet was provided every other day for 8 days. On day 22 females were transferred to egg cages containing autoclaved soil with 11 plugs of untreated artificial diet. Egg cages were placed back in the growth chamber and the diet was replaced on day 24. On day 26 females were removed from the soil cages and flash frozen for qPCR. Soil cages were transferred to a growth chamber with temperature 27.+-.1.degree. C., relative humidity >80% and 24 h dark. After 4 days the soil was washed using a #60 sieve to collect eggs from each cage. Eggs were treated with a solution of formaldehyde (500 .mu.l formaldehyde in 5 ml of double distilled water) and methyl-(butycarbamoy)-2-benzimidazole carbamate (0.025 g in 50 ml of double distilled water) to prevent fungal contamination and placed in small petri dishes containing filter paper. Photographs were taken of each petri dish for egg counting using the cell counter function of Image J software (Schneider et al. (2012) Nat. Methods 9:671-5). Petri dishes with eggs were transferred to a small growth chamber with a temperature of 27.+-.1.degree. C., relative humidity >80%, and 24 h dark. Larval hatching was monitored daily for 15 days. Results of the percent total larvae that hatched are shown in FIG. 9B and the percent of total larvae that hatched is shown in FIG. 9A. Relative brahma expression of females was measured after receiving 6 times dsRNA and is shown in FIG. 10.

[0432] dsRNA Feeding Immediately after Mating:

[0433] Methods similar to those described above were used except that 10 males and 10 females were placed together in one well with 22 pellets of untreated artificial diet at the start of the study. Trays were transferred to growth chamber as described above. Fresh untreated diet was provided on day 3 and males were removed on day 5. Females were then transferred to treated artificial diet and maintained in the growth chamber. Fresh treated diet was provided every other day for 6 days. On day 12 females were transferred to egg cages containing autoclaved soil with 11 plugs of treated artificial diet. Egg cages were placed back in the growth chamber and fresh treated diet was provided on day 14. On day 16 all females were removed from the soil cages and flash frozen for qPCR. Soil cages and egg wash was conducted after 6 days as described above. Photographs were taken of each petri dish for egg counting. Larval hatching was monitored daily for 15 days. Results of eggs per female are shown in FIG. 9A and results of the percent total larvae that hatched are shown in FIG. 9B. Relative brahma expression of females was measured after receiving 6 times dsRNA and is shown in FIG. 10.

[0434] dsRNA Feeding Six Days after Mating:

[0435] Methods similar to those described above for dsRNA feeding immediately after mating except that insects received untreated artificial diet every other day until day 11 when females were transferred to treated diet. On day 12 females were transferred to egg cages containing autoclaved soil with 11 plugs of treated artificial diet. Egg cages were placed back in the growth chamber. Fresh treated diet was provided every other day from days 12-20. At day 22, all females were removed from the soil cages and flash frozen for qPCR. Soil cages and egg wash was conducted after 6 days as described above. Photographs were taken of each petri dish for egg counting. Larval hatching was monitored daily for 15 days. Larvae were counted and removed from the Petri dish each day. Results of eggs per female are shown in FIG. 9A and results of the percent total larvae that hatched are shown in FIG. 9B. Relative brahma expression was measured and is shown in FIG. 10.

[0436] Female mortality was recorded every other day for all treatments throughout the study.

Example 20

Duration of Exposure

[0437] Virgin males and females were paired for a period of 4 days with untreated diet after which the mated females were exposed to 2 .mu.g brahma dsRNA. To evaluate the effect of the duration of exposure insects were exposed to brahma or GFP dsRNA 1, 2, 4 or 6 times (shown as brm-T1, -T2, -T4 or -T6 in FIG. 11A and FIG. 11B). Four replications of 10 females and 10 males were completed per treatment. Adult males and females were received from CROP CHARACTERISTICS (Farmington, Minn.). Ten males and 10 females were placed together in one well with 20 pellets of untreated artificial diet. Trays were maintained in a growth chamber with a temperature of 23.+-.1.degree. C., relative humidity >80%, and 16:8 L:D photoperiod. New untreated artificial diet was provided on day 3. Males were removed on day 5 and females were transferred to a new tray containing 11 diet plugs per well with the respective treatment. On day 7, females were transferred to trays with new treated artificial diet and mortality was recorded. Females from 1 time (T1) of exposure treatment were transferred to untreated diet. On day 10 and 12 females were transferred to new trays with new treated artificial diet and mortality was recorded. Females from T1 and T2 were transferred to untreated diet. On day 14 females were transferred to egg cages containing autoclaved soil and new treated artificial diet was provided. Females from T1, T2 and T4 were provided untreated diet. On day 16, old diet was removed and new treated diet (11 plugs per cage) was added. Females from 1, 2 and 4 times of exposure were provided untreated diet. After 18 days all females were removed from the soil cages and flash frozen for qPCR. Soil cages were transferred to a growth chamber with a temperature of 27.+-.1.degree. C., relative humidity >80% and 24 h dark. Eggs were washed and photographs were taken of each petri dish as indicated for the timing of exposure. Hatched larvae were counted and removed from each Petri dish every day for 15 days. Results of the percent total larvae that hatched are shown in FIG. 11A. Relative brahma expression of females was measured and is shown in FIG. 11B.

Example 21

Ovarian Development

[0438] D. v. virgifera ovarian development was evaluated in females exposed to artificial diet treated with brahma dsRNA before mating and immediately after mating as described for the timing of exposure. Females were exposed to 2 .mu.g brahma or GFP dsRNA, or water 6 times. Five females per treatment were collected one day after the last dsRNA exposure and stored in 70% ethanol for subsequent ovary dissections. Ovary dissections for all surviving females were performed under a stereomicroscope. Images were acquired with an Olympus SZX16 microscope, Olympus SDF PLAPO 2.times.PFC lens and the Olympus CellSens Dimensions software (Tokyo, Japan).

[0439] D. v. virgifera ovary dissections revealed no apparent differences in ovary development between females treated with water, GFP or brahma dsRNA; this was true for both unmated females as well as those dissected immediately after mating.

Sequence CWU 1

1

16914768DNADiabrotica virgifera 1caagtggcca tggcatgcca cagggtcccc ctggacaacc aggtcagcaa caccaaggcc 60gaactgctga taatttacat gccttacaaa aagcaataga tacaatggaa gaaaaaggta 120tgcaagaaga tcagaggtat tcacagttac tggcgttacg tgctagatcc agtggtcaac 180catctaacgg agttcttaca ccgctgcaaa tgaatcaact tagaaatcaa attatggcat 240acaggtgcct agcgaggagc caaccaattc ctccttcaat aatgttgggg ctgcaaggaa 300agaggcctga cggttcacca cagtttccta cacctccgtc aagtccgttt caaccacaag 360gacctggtgc accccctggt ccggaacaac caccagctaa tgcagaaaac gtagcagagc 420cagcagcacc agtaggaccg caaggtgcac aaggacctcc taaccaacag agagctcaaa 480ctagccagtt agtccccaat aagcaaactc gtttcactac catgcccaaa ccatctggac 540tagatccact agttcttctt caagagaggg aaactagggt ggcagctaga atcgctgcta 600gaatagaaca atgtagtaac ttacctacca atctttcaga caaagtccgc atgcaagcac 660agatagaatt gagagctttg cggtgcctta atttccaaag gcaactaaga agcgaaattt 720tgaactgtat taggagagat ataacgcttg aatctgctgt aaattttaaa gcatataaaa 780gaacgaagcg acagggtcta aaagaatcga gagctacaga gaagttagaa aaacaacaga 840agttagaagc agaaagaaag agaagacaga agaaccaaga atttttgaat gctgtattga 900acaatggaaa agaattcaag gaattccaca agcagaatca agcgaaatta gctaagatta 960ataaagctgt tattaattat cacgctaatg ctgaaagaga gcaaaagaaa gaagcagaaa 1020ggagagagaa ggaacgtatg atcagattga tggcagaaga tgaagaaggt tatagacagt 1080tgatcgatca aaagaaagac aaacgtctag cgttcttgct ttcccaaaca gatgaatata 1140tcagtaactt aacagagatg gtgaaaatgc acaaagtcga acaaagtaac aagaagcggg 1200aagaagaacg acggaagaga aggcaagaca aaatgcagca gcctgacagg aaagtcacag 1260ttatcgaaat ggctactggg aataaggtta gtggagaaaa cgctccgact gtccaggaac 1320ttcctgaatg gttacagact catcctggtt gggagatgat agatacagaa gacgaggacg 1380agaatgacga atatagaatg gacgattatg aagaaaataa tcaagtcgat gctacagaaa 1440tcattcagaa agccaaggtt gaggatgacg aatatcacaa gaatgccaca gaggaacaga 1500cgtactacgg tattgcacat acagtgagcg agtcagtatc agaacaggcc tccattatga 1560taaacggtga actgaaagag taccaggtca aaggactgga atggatggta tccttgtaca 1620acaacaatct taatggtatc ctagcagacg agatgggttt gggtaagact attcaaacca 1680ttggcctgat cacctacttg atggagaaaa aaaagttgaa tgggccattt ttgatcattg 1740tgccgttatc cactatatct aattggatgt tggagttcga aaaatgggct ccttctgttg 1800tggtcgtctc ctacaaaggc tcacctggtc acaggaaatt gcttcagggt cagatgaagt 1860cagcaaaatt caatgttctt cttactactt atgaatatat cattaaagat aagggaattc 1920tttcaaaagt accgtttaag tatatgatcg tggacgaggg tcacagaatg aagaaccatc 1980attgcaagtt gacccagact ttgaacactc actacgcagc tcctttccgc cttctcttaa 2040ccggtactcc tctacaaaac aaactaccag aactgtgggc gttgcttaac ttcttacttc 2100cgtctatttt caagagttgt tccactttcg agcaatggtt caacgcccct ttcgcaacca 2160cgggagaaaa ggttgaactt aacgaagaag aaaccatcct tatcatccga cgtcttcaca 2220aagtcctgcg acctttcctc ttaagacgtc tcaaaaagga agtagagtct cagcttcccg 2280acaaagtcga atacattatc aaatgcgaga tgtccggttt gcaaaaagtg ttgtaccaac 2340acatgcagag caagggagtt ctgctcaccg acgggtccga aaagggtaat aggggccgag 2400gtggagctaa ggctatcatg aataccatca tgcaactgcg gaagctgtgt aatcatcctt 2460tcatgttcca aatgatcgaa gaaaagtatt gtgaatatgt aggcatgggt gggggactca 2520catcagggcc ggatatatac agatcttctg gtaaatttga acttctggat cgggtattgc 2580caaagctcaa ggcgactgac cacagagtcc tactgttctg tcaaatgacg acgttgatga 2640acatcatgga agactacttc atttggagag gttacaaata tcttcgtctg gatggtatgg 2700taaaagcgga agatcgggcg gaactactca agaagttcaa tgacaaacaa agcgaatatt 2760ttgtgtttct attgtcaaca agagcaggag gtcttggact caacttgcaa agtgctgata 2820ctgttatcat ctttgattct gactggaatc ctcaccagga tttacaagct caagatcgtg 2880cccatcgtat aggccagcaa aatgaagtca gggtcctacg tttaatgaca gttaattcag 2940tggaagaaag aatcttagct gcagctaaat acaaacttat aatggacgag aaagtaatcc 3000aagctggtat gttcgatcag aagtctacag gctcagagag acatcagttt ttgcagagta 3060ttttacacca tgacggaagc gacgaagaag aggaaaacga agttcctgat gacgaaacag 3120tgaaccagat gttggcccga agggaaaacg aatttcagct tttccagaag atggatcagg 3180aaagaaagga agaagatgaa aagaccggaa agtcgcgact tattcaagaa agcgaattgc 3240ccgaatggct gttgaagcaa gacgatgaaa tctactcgtg gggccttgat gatccagatg 3300ctgttttagg aaggggtagt aggcaaagaa aagaagttga ttatgttgac agcctgacgg 3360agaaagagtg gcttaaggct attgacgaag agggagaatt tgaggaagaa caagaaggtg 3420ataaagaagg tctcagaaag aaaagaggga ggaagaggaa gaagcgcgat gatgacgaag 3480aggcaagcca aattaagaga agaaaggtgc atctagccga gatcaagatg aagaaaaaga 3540tgaagaggct tatggaagtt gttgtgaact acagggatag ggatggtaga gtattgagcg 3600aaccgtttat gaaacttcca tcaaagaagg agttacctga atattacgat acgattaaga 3660aacctattga tattgaaaaa gtcgttgcca acgtagaaga aggaaaatat ttcacgatgc 3720acgatttgga aagagatttc gacttgctgt gccaaaacgc ccaacaatac aacgaagaag 3780actccatgat ctacgaggac agcctcgttc ttcgacaggt gtttagaagc gcgagggaaa 3840agatcgacgg tacctcagac cacgacgaca acgccgatgg accggcggtg gctcagatca 3900aacgacctcg tggtagacct cgaaaacaca agagacccga agagatcgag gccgaagcgg 3960cggctcagaa agctatggag gaggcatcga agctgagagc tcaagctgag gcggaagagc 4020ttagatctaa ggtggaggag gcatctcaga gagccaaaga ggaagcgaaa gcaagggagg 4080aagccaaagc tagggaagaa gccgaaatcg agaacatgga ggagattccc acaagcacat 4140gatctataga gcaaccggaa acaaaaaggc aaaaaagaaa tattatatag aaaagatgta 4200catgttcaat ggagatacat tttcgctgag ttacaacggg taatgctttt acaacggata 4260ttttgacgta tgaatgttga cgttcagatg aagtatattt ataaaataat ccagaccttt 4320acgttttggt tgatttgttt tctgtattgt tcagtttatt gaacaaccat taatagcagc 4380ttacctaaat gatttagaaa agcatctgag ttatttagat aagttttgag attatattta 4440ttaactttaa tattactatc tttattatag catattgtaa ttattttttc ctgtccttct 4500ttcgttgtgt ggtagataat ccgagagtca acagttataa gcaaatgaaa ttcagttaaa 4560cctcaaatgt acaaaatgat caaattaatg tttacaattt atttttttac cacgcacatc 4620cactattact attgtcagtc attgagatat cattttatat agctccatgt ctgtcttcct 4680caatttacag agaagcaatt agacaagtaa tgacataata tggtgctgaa ataatgtgct 4740tgatagtgat gttgaaaaag taactatt 476821375PRTDiabrotica virgifera 2Met Pro Gln Gly Pro Pro Gly Gln Pro Gly Gln Gln His Gln Gly Arg 1 5 10 15 Thr Ala Asp Asn Leu His Ala Leu Gln Lys Ala Ile Asp Thr Met Glu 20 25 30 Glu Lys Gly Met Gln Glu Asp Gln Arg Tyr Ser Gln Leu Leu Ala Leu 35 40 45 Arg Ala Arg Ser Ser Gly Gln Pro Ser Asn Gly Val Leu Thr Pro Leu 50 55 60 Gln Met Asn Gln Leu Arg Asn Gln Ile Met Ala Tyr Arg Cys Leu Ala 65 70 75 80 Arg Ser Gln Pro Ile Pro Pro Ser Ile Met Leu Gly Leu Gln Gly Lys 85 90 95 Arg Pro Asp Gly Ser Pro Gln Phe Pro Thr Pro Pro Ser Ser Pro Phe 100 105 110 Gln Pro Gln Gly Pro Gly Ala Pro Pro Gly Pro Glu Gln Pro Pro Ala 115 120 125 Asn Ala Glu Asn Val Ala Glu Pro Ala Ala Pro Val Gly Pro Gln Gly 130 135 140 Ala Gln Gly Pro Pro Asn Gln Gln Arg Ala Gln Thr Ser Gln Leu Val 145 150 155 160 Pro Asn Lys Gln Thr Arg Phe Thr Thr Met Pro Lys Pro Ser Gly Leu 165 170 175 Asp Pro Leu Val Leu Leu Gln Glu Arg Glu Thr Arg Val Ala Ala Arg 180 185 190 Ile Ala Ala Arg Ile Glu Gln Cys Ser Asn Leu Pro Thr Asn Leu Ser 195 200 205 Asp Lys Val Arg Met Gln Ala Gln Ile Glu Leu Arg Ala Leu Arg Cys 210 215 220 Leu Asn Phe Gln Arg Gln Leu Arg Ser Glu Ile Leu Asn Cys Ile Arg 225 230 235 240 Arg Asp Ile Thr Leu Glu Ser Ala Val Asn Phe Lys Ala Tyr Lys Arg 245 250 255 Thr Lys Arg Gln Gly Leu Lys Glu Ser Arg Ala Thr Glu Lys Leu Glu 260 265 270 Lys Gln Gln Lys Leu Glu Ala Glu Arg Lys Arg Arg Gln Lys Asn Gln 275 280 285 Glu Phe Leu Asn Ala Val Leu Asn Asn Gly Lys Glu Phe Lys Glu Phe 290 295 300 His Lys Gln Asn Gln Ala Lys Leu Ala Lys Ile Asn Lys Ala Val Ile 305 310 315 320 Asn Tyr His Ala Asn Ala Glu Arg Glu Gln Lys Lys Glu Ala Glu Arg 325 330 335 Arg Glu Lys Glu Arg Met Ile Arg Leu Met Ala Glu Asp Glu Glu Gly 340 345 350 Tyr Arg Gln Leu Ile Asp Gln Lys Lys Asp Lys Arg Leu Ala Phe Leu 355 360 365 Leu Ser Gln Thr Asp Glu Tyr Ile Ser Asn Leu Thr Glu Met Val Lys 370 375 380 Met His Lys Val Glu Gln Ser Asn Lys Lys Arg Glu Glu Glu Arg Arg 385 390 395 400 Lys Arg Arg Gln Asp Lys Met Gln Gln Pro Asp Arg Lys Val Thr Val 405 410 415 Ile Glu Met Ala Thr Gly Asn Lys Val Ser Gly Glu Asn Ala Pro Thr 420 425 430 Val Gln Glu Leu Pro Glu Trp Leu Gln Thr His Pro Gly Trp Glu Met 435 440 445 Ile Asp Thr Glu Asp Glu Asp Glu Asn Asp Glu Tyr Arg Met Asp Asp 450 455 460 Tyr Glu Glu Asn Asn Gln Val Asp Ala Thr Glu Ile Ile Gln Lys Ala 465 470 475 480 Lys Val Glu Asp Asp Glu Tyr His Lys Asn Ala Thr Glu Glu Gln Thr 485 490 495 Tyr Tyr Gly Ile Ala His Thr Val Ser Glu Ser Val Ser Glu Gln Ala 500 505 510 Ser Ile Met Ile Asn Gly Glu Leu Lys Glu Tyr Gln Val Lys Gly Leu 515 520 525 Glu Trp Met Val Ser Leu Tyr Asn Asn Asn Leu Asn Gly Ile Leu Ala 530 535 540 Asp Glu Met Gly Leu Gly Lys Thr Ile Gln Thr Ile Gly Leu Ile Thr 545 550 555 560 Tyr Leu Met Glu Lys Lys Lys Leu Asn Gly Pro Phe Leu Ile Ile Val 565 570 575 Pro Leu Ser Thr Ile Ser Asn Trp Met Leu Glu Phe Glu Lys Trp Ala 580 585 590 Pro Ser Val Val Val Val Ser Tyr Lys Gly Ser Pro Gly His Arg Lys 595 600 605 Leu Leu Gln Gly Gln Met Lys Ser Ala Lys Phe Asn Val Leu Leu Thr 610 615 620 Thr Tyr Glu Tyr Ile Ile Lys Asp Lys Gly Ile Leu Ser Lys Val Pro 625 630 635 640 Phe Lys Tyr Met Ile Val Asp Glu Gly His Arg Met Lys Asn His His 645 650 655 Cys Lys Leu Thr Gln Thr Leu Asn Thr His Tyr Ala Ala Pro Phe Arg 660 665 670 Leu Leu Leu Thr Gly Thr Pro Leu Gln Asn Lys Leu Pro Glu Leu Trp 675 680 685 Ala Leu Leu Asn Phe Leu Leu Pro Ser Ile Phe Lys Ser Cys Ser Thr 690 695 700 Phe Glu Gln Trp Phe Asn Ala Pro Phe Ala Thr Thr Gly Glu Lys Val 705 710 715 720 Glu Leu Asn Glu Glu Glu Thr Ile Leu Ile Ile Arg Arg Leu His Lys 725 730 735 Val Leu Arg Pro Phe Leu Leu Arg Arg Leu Lys Lys Glu Val Glu Ser 740 745 750 Gln Leu Pro Asp Lys Val Glu Tyr Ile Ile Lys Cys Glu Met Ser Gly 755 760 765 Leu Gln Lys Val Leu Tyr Gln His Met Gln Ser Lys Gly Val Leu Leu 770 775 780 Thr Asp Gly Ser Glu Lys Gly Asn Arg Gly Arg Gly Gly Ala Lys Ala 785 790 795 800 Ile Met Asn Thr Ile Met Gln Leu Arg Lys Leu Cys Asn His Pro Phe 805 810 815 Met Phe Gln Met Ile Glu Glu Lys Tyr Cys Glu Tyr Val Gly Met Gly 820 825 830 Gly Gly Leu Thr Ser Gly Pro Asp Ile Tyr Arg Ser Ser Gly Lys Phe 835 840 845 Glu Leu Leu Asp Arg Val Leu Pro Lys Leu Lys Ala Thr Asp His Arg 850 855 860 Val Leu Leu Phe Cys Gln Met Thr Thr Leu Met Asn Ile Met Glu Asp 865 870 875 880 Tyr Phe Ile Trp Arg Gly Tyr Lys Tyr Leu Arg Leu Asp Gly Met Val 885 890 895 Lys Ala Glu Asp Arg Ala Glu Leu Leu Lys Lys Phe Asn Asp Lys Gln 900 905 910 Ser Glu Tyr Phe Val Phe Leu Leu Ser Thr Arg Ala Gly Gly Leu Gly 915 920 925 Leu Asn Leu Gln Ser Ala Asp Thr Val Ile Ile Phe Asp Ser Asp Trp 930 935 940 Asn Pro His Gln Asp Leu Gln Ala Gln Asp Arg Ala His Arg Ile Gly 945 950 955 960 Gln Gln Asn Glu Val Arg Val Leu Arg Leu Met Thr Val Asn Ser Val 965 970 975 Glu Glu Arg Ile Leu Ala Ala Ala Lys Tyr Lys Leu Ile Met Asp Glu 980 985 990 Lys Val Ile Gln Ala Gly Met Phe Asp Gln Lys Ser Thr Gly Ser Glu 995 1000 1005 Arg His Gln Phe Leu Gln Ser Ile Leu His His Asp Gly Ser Asp 1010 1015 1020 Glu Glu Glu Glu Asn Glu Val Pro Asp Asp Glu Thr Val Asn Gln 1025 1030 1035 Met Leu Ala Arg Arg Glu Asn Glu Phe Gln Leu Phe Gln Lys Met 1040 1045 1050 Asp Gln Glu Arg Lys Glu Glu Asp Glu Lys Thr Gly Lys Ser Arg 1055 1060 1065 Leu Ile Gln Glu Ser Glu Leu Pro Glu Trp Leu Leu Lys Gln Asp 1070 1075 1080 Asp Glu Ile Tyr Ser Trp Gly Leu Asp Asp Pro Asp Ala Val Leu 1085 1090 1095 Gly Arg Gly Ser Arg Gln Arg Lys Glu Val Asp Tyr Val Asp Ser 1100 1105 1110 Leu Thr Glu Lys Glu Trp Leu Lys Ala Ile Asp Glu Glu Gly Glu 1115 1120 1125 Phe Glu Glu Glu Gln Glu Gly Asp Lys Glu Gly Leu Arg Lys Lys 1130 1135 1140 Arg Gly Arg Lys Arg Lys Lys Arg Asp Asp Asp Glu Glu Ala Ser 1145 1150 1155 Gln Ile Lys Arg Arg Lys Val His Leu Ala Glu Ile Lys Met Lys 1160 1165 1170 Lys Lys Met Lys Arg Leu Met Glu Val Val Val Asn Tyr Arg Asp 1175 1180 1185 Arg Asp Gly Arg Val Leu Ser Glu Pro Phe Met Lys Leu Pro Ser 1190 1195 1200 Lys Lys Glu Leu Pro Glu Tyr Tyr Asp Thr Ile Lys Lys Pro Ile 1205 1210 1215 Asp Ile Glu Lys Val Val Ala Asn Val Glu Glu Gly Lys Tyr Phe 1220 1225 1230 Thr Met His Asp Leu Glu Arg Asp Phe Asp Leu Leu Cys Gln Asn 1235 1240 1245 Ala Gln Gln Tyr Asn Glu Glu Asp Ser Met Ile Tyr Glu Asp Ser 1250 1255 1260 Leu Val Leu Arg Gln Val Phe Arg Ser Ala Arg Glu Lys Ile Asp 1265 1270 1275 Gly Thr Ser Asp His Asp Asp Asn Ala Asp Gly Pro Ala Val Ala 1280 1285 1290 Gln Ile Lys Arg Pro Arg Gly Arg Pro Arg Lys His Lys Arg Pro 1295 1300 1305 Glu Glu Ile Glu Ala Glu Ala Ala Ala Gln Lys Ala Met Glu Glu 1310 1315 1320 Ala Ser Lys Leu Arg Ala Gln Ala Glu Ala Glu Glu Leu Arg Ser 1325 1330 1335 Lys Val Glu Glu Ala Ser Gln Arg Ala Lys Glu Glu Ala Lys Ala 1340 1345 1350 Arg Glu Glu Ala Lys Ala Arg Glu Glu Ala Glu Ile Glu Asn Met 1355 1360 1365 Glu Glu Ile Pro Thr Ser Thr 1370 1375 35147DNADiabrotica virgifera 3tcacgtgcct ccacaaggcc atgttcctcc acagggtcac gtgcctccac aaggccatgt 60tcctccacag ggacatcttc ctccacaggg ccatattcca ccacagggtc atggtccaac 120gcagggccac atacctcctc aggggcatgt tccaccacaa ggacatatac ctcctcaagg 180gcatgcaccc ccacaaggac atgcaccacc acaggggcat cctggcgttc ctcctggtca 240tcagagtcat cctcaagggc atccacaaac accagggcat cctggacctg gacatatacc 300acctggtgga gcaatgcacc cagggcatta tccaagtggc catggcatgc cacagggtcc 360ccctggacaa ccaggtcagc aacaccaagg ccgaactgct gataatttac atgccttaca 420aaaagcaata gatacaatgg aagaaaaagg tatgcaagaa gatcagaggt attcacagtt 480actggcgtta cgtgctagat ccagtggtca accatctaac ggagttctta caccgctgca 540aatgaatcaa cttagaaatc aaattatggc atacaggtgc ctagcgagga gccaaccaat 600tcctccttca ataatgttgg ggctgcaagg aaagaggcct gacggttcac cacagtttcc 660tacacctccg tcaagtccgt ttcaaccaca aggacctggt gcaccccctg gtccggaaca 720accaccagct aatgcagaaa acgtagcaga gccagcagca ccagtaggac cgcaaggtgc 780acaaggacct cctaaccaac agagagctca aactagccag ttagtcccca ataagcaaac 840tcgtttcact accatgccca aaccatctgg actagatcca ctagttcttc ttcaagagag 900ggaaactagg gtggcagcta gaatcgctgc tagaatagaa caatgtagta acttacctac 960caatctttca gacaaagtcc gcatgcaagc acagatagaa ttgagagctt tgcggtgcct 1020caatttccaa aggcaactaa gaagcgaaat tttgaactgt attaggagag

atataacgct 1080tgaatctgct gtaaatttta aagcatataa aagaacgaag cgacagggtc taaaagaatc 1140gagagctaca gagaagttag aaaaacaaca gaagttagaa gcagaaagaa agagaagaca 1200gaagaaccaa gaatttttga atgctgtatt gaacaatgga aaagaattca aggaattcca 1260caagcagaat caagcgaaat tagctaagat taataaagct gttattaatt atcacgctaa 1320tgctgaaaga gagcaaaaga aagaagcaga aaggagagag aaggaacgta tgatcagatt 1380gatggcagaa gatgaagaag gttatagaaa gctcattgat caaaagaaag acaaacgtct 1440agcgttcttg ctttcgcaaa cagatgagta tataactaac ctcacggaga tggtaaagca 1500acacaagttg gaacaaacca ataaaaagaa agaggaggaa aaacgcaaga agaagcagca 1560gaaaatgcaa caaccagata ggaaagttac agttctggaa actgcaacag gtaaaaaagt 1620aacaggagag gctgctccta cactgcgaca agttcaagaa tggttaatcc aacatcctgg 1680atgggagatg gtcgatacag atgatgagga tgatgaaaac ggggagaaga gggatgatga 1740ctatgatgaa aatcaagaag tggatgatgc aaaagaagtt attaaaaaag ctaaagttga 1800agatgacgaa tatcacaaaa acacaaaaga agaacagact tactacagta ttgctcacac 1860tgttcatgaa gtggtaacag aacaagcatc cattctggtt aatggaaagc ttaaggaata 1920tcaaattaga gggttagaat ggatggtgtc tttgtacaat aacaatctga atggtattct 1980agcagatgag atgggtctag gtaaaaccat tcaaacgatt ggcttgttga cctatttgat 2040ggaaaaaaag aagataaatg gaccgttttt gatcatagtg ccactttcaa ccatttctaa 2100ttggatgttg gaatttcaaa agtgggcccc tactgtagtt gtcatttcat acaaaggctc 2160tcctgtggtt agaaaagtga tccagagcca gttaaaagct gctaaattca atgtgcttct 2220cactacctac gagtacatta ttaaggacaa gggtgtatta gcaaaaatcc catttaaata 2280tatgatcata gatgagggtc atcgtatgaa aaaccaccac tgcaaattga ctcaagtcct 2340gaatacgcac tatttggcgc cctacagact cctgcttact ggtactcccc tacaaaataa 2400attaccagaa ttatgggcct tgttgaattt cttgttgcct tcgattttca agagttgctc 2460cacttttgaa caatggttca atgcgccatt cgcaacaaca ggagaaaagg ttgagttaaa 2520cgaagaagaa actatcctta tcatccgtcg tcttcacaaa gtactcaggc cgtttctcct 2580gagacgtctc aagaaagaag tcgaatctca gcttccagac aaagtggaat atatcataaa 2640gtgtgacatg tcgggcctac aaaaggttct ctatgcacac atgcagagca agggtgtgtt 2700acttaccgat ggttccgaga agggcagtaa aggaagggga tctaaggcac tgatgaacac 2760cattatgcag ctgaggaaac tgtgcaatca tccgtttatg ttccaaaata tcgaagagaa 2820atattgtgat catgttggta ttgctggtgg agtggtttct ggacccgaca cttatagggt 2880atctggtaag tttgagctct tggacagaat attgcccaaa atgaaagcaa ctaaccatag 2940gattcttctt ttctgtcaaa tgactcaatt aatgaccatc atggaagatt atctaaattg 3000gagaggattc aaatatcttc gtcttgatgg tacaatcaaa tcagaagatc gcggggacct 3060attatcgaaa tttaatgata aaaatagtga atattttttg tttttgctat ctacacgggc 3120tggaggtctg ggacttaatt tgcagacagc tgatactgtg attatcttcg attccgattg 3180gaatcctcat caggatttac aagctcagga tcgagctcat cgtattggac agcaaaatga 3240ggtccgagtt ttgcgtttga tgactgttaa ctctgttgag gaacgaattt tagctgcagc 3300taaatacaag cttactatgg acgaaaaggt cattcaagct ggtatgttcg atcagaagtc 3360tacgggatct gaaaggcagc agtttcttca gagtatttta cacaatgatg gtagtgatga 3420agaagaggaa aatgaagtgc ctgatgacga aaccgtcaac caaatgatag ctaggacaga 3480ggatgagttt cagctcttcc aaaaaatgga tacggaaaga aaagaggaga atgaaaaact 3540tggtcagcat aaaaagtcgc gattggttca agaatgtgaa cttccggatt ggctgacaaa 3600gccagatgaa gatgatggct ggggtgatga ttatactgaa gcgctattgg gcagaggaac 3660caggcagcga aaggaagttg attatgctga tagtttaaca gaaaaggaat ggttaaaggc 3720tatcgatgaa gacggagact acgatgaaga agaagaggaa gaaaaagtac aaaagaagag 3780gggtaggaag agaaggaagc gtgacgattc tgacgatgac accagcagtt ctacgagaag 3840gcgtaagcta ccccaaagtc aggtagaagc taggctaaag agaaaaatga aaaagttgat 3900gaacatagtt acaaattata aagacaggga tggacgacag cttagcgatc aattcattaa 3960attgcctcca aggaaagagt atccagacta ttatactatt attaaaaagc ctatagatat 4020tagcaagata ttaaattata tagatgatgg aaagtactct gatttctccg atctggaacg 4080agacttcatg cttctctgcc agaatgctca aatctataac gaagaagcgt cgttgattca 4140cgaagacagc atcgtactgc agtcggtctt ttcgagtgcc aagcagaaga tcgaagcctc 4200cccggattcg gacgacgaaa aagatgacaa taattccgat gtagaaactc ctaagaataa 4260aaataaacct ggtaaaggca agagacgacc cggcaggcct aggaggtcgg cgaaaaaata 4320catttcggac gacgatgacg acgactgaag agtttaggtg taagagaaat gagaatgaaa 4380ttcttattgc aaaagttgta catatgaagg tgttgttatt ctttaccaaa gctggtaaat 4440gtttgattta aagggaaact ggaacttttc tcttggtttt agatagtact atagataggt 4500tttgataggg aataacaggt tcaagattcg ccccagttga aatttgctta aatgatcaaa 4560gagtacttaa gtataatgaa tcacgattgt ttaaatttta acttgcactt agtgacaaaa 4620aaataacagc ttataaataa atttacgtag caagaatgat cctattgatt aagaatgaat 4680gagcctctcc aaagatagtc caaaaagtgt ttcagggtga aaaggagttg tgaaaaatac 4740aagatggtct gctaagcaac cttctttgta acaaaaatca tgtttttcca gaaatttttt 4800tttattttat taatatagtc ctcttttatc tcaatacaac gcgattccag cgattttcca 4860aacttcttta tgccgtcgga ataaggcccc acctagattg gatccgtatt tctcctatca 4920gatccgatcc gacgtcggat tgaaagcaaa ctcaaggtat taaggtatgg ctgcacttac 4980attggatccc catcctccga tccgatatag ggcgattgat aggagaagct acagcagaga 5040agcagttcga cgtcggccga tatcggatcg gtcttctcac tacagtgtag gcactgcgct 5100ttaatacctt ccctaataac actaaacatt ccatgtatgt tcctaga 514741344PRTDiabrotica virgifera 4Met His Pro Gly His Tyr Pro Ser Gly His Gly Met Pro Gln Gly Pro 1 5 10 15 Pro Gly Gln Pro Gly Gln Gln His Gln Gly Arg Thr Ala Asp Asn Leu 20 25 30 His Ala Leu Gln Lys Ala Ile Asp Thr Met Glu Glu Lys Gly Met Gln 35 40 45 Glu Asp Gln Arg Tyr Ser Gln Leu Leu Ala Leu Arg Ala Arg Ser Ser 50 55 60 Gly Gln Pro Ser Asn Gly Val Leu Thr Pro Leu Gln Met Asn Gln Leu 65 70 75 80 Arg Asn Gln Ile Met Ala Tyr Arg Cys Leu Ala Arg Ser Gln Pro Ile 85 90 95 Pro Pro Ser Ile Met Leu Gly Leu Gln Gly Lys Arg Pro Asp Gly Ser 100 105 110 Pro Gln Phe Pro Thr Pro Pro Ser Ser Pro Phe Gln Pro Gln Gly Pro 115 120 125 Gly Ala Pro Pro Gly Pro Glu Gln Pro Pro Ala Asn Ala Glu Asn Val 130 135 140 Ala Glu Pro Ala Ala Pro Val Gly Pro Gln Gly Ala Gln Gly Pro Pro 145 150 155 160 Asn Gln Gln Arg Ala Gln Thr Ser Gln Leu Val Pro Asn Lys Gln Thr 165 170 175 Arg Phe Thr Thr Met Pro Lys Pro Ser Gly Leu Asp Pro Leu Val Leu 180 185 190 Leu Gln Glu Arg Glu Thr Arg Val Ala Ala Arg Ile Ala Ala Arg Ile 195 200 205 Glu Gln Cys Ser Asn Leu Pro Thr Asn Leu Ser Asp Lys Val Arg Met 210 215 220 Gln Ala Gln Ile Glu Leu Arg Ala Leu Arg Cys Leu Asn Phe Gln Arg 225 230 235 240 Gln Leu Arg Ser Glu Ile Leu Asn Cys Ile Arg Arg Asp Ile Thr Leu 245 250 255 Glu Ser Ala Val Asn Phe Lys Ala Tyr Lys Arg Thr Lys Arg Gln Gly 260 265 270 Leu Lys Glu Ser Arg Ala Thr Glu Lys Leu Glu Lys Gln Gln Lys Leu 275 280 285 Glu Ala Glu Arg Lys Arg Arg Gln Lys Asn Gln Glu Phe Leu Asn Ala 290 295 300 Val Leu Asn Asn Gly Lys Glu Phe Lys Glu Phe His Lys Gln Asn Gln 305 310 315 320 Ala Lys Leu Ala Lys Ile Asn Lys Ala Val Ile Asn Tyr His Ala Asn 325 330 335 Ala Glu Arg Glu Gln Lys Lys Glu Ala Glu Arg Arg Glu Lys Glu Arg 340 345 350 Met Ile Arg Leu Met Ala Glu Asp Glu Glu Gly Tyr Arg Lys Leu Ile 355 360 365 Asp Gln Lys Lys Asp Lys Arg Leu Ala Phe Leu Leu Ser Gln Thr Asp 370 375 380 Glu Tyr Ile Thr Asn Leu Thr Glu Met Val Lys Gln His Lys Leu Glu 385 390 395 400 Gln Thr Asn Lys Lys Lys Glu Glu Glu Lys Arg Lys Lys Lys Gln Gln 405 410 415 Lys Met Gln Gln Pro Asp Arg Lys Val Thr Val Leu Glu Thr Ala Thr 420 425 430 Gly Lys Lys Val Thr Gly Glu Ala Ala Pro Thr Leu Arg Gln Val Gln 435 440 445 Glu Trp Leu Ile Gln His Pro Gly Trp Glu Met Val Asp Thr Asp Asp 450 455 460 Glu Asp Asp Glu Asn Gly Glu Lys Arg Asp Asp Asp Tyr Asp Glu Asn 465 470 475 480 Gln Glu Val Asp Asp Ala Lys Glu Val Ile Lys Lys Ala Lys Val Glu 485 490 495 Asp Asp Glu Tyr His Lys Asn Thr Lys Glu Glu Gln Thr Tyr Tyr Ser 500 505 510 Ile Ala His Thr Val His Glu Val Val Thr Glu Gln Ala Ser Ile Leu 515 520 525 Val Asn Gly Lys Leu Lys Glu Tyr Gln Ile Arg Gly Leu Glu Trp Met 530 535 540 Val Ser Leu Tyr Asn Asn Asn Leu Asn Gly Ile Leu Ala Asp Glu Met 545 550 555 560 Gly Leu Gly Lys Thr Ile Gln Thr Ile Gly Leu Leu Thr Tyr Leu Met 565 570 575 Glu Lys Lys Lys Ile Asn Gly Pro Phe Leu Ile Ile Val Pro Leu Ser 580 585 590 Thr Ile Ser Asn Trp Met Leu Glu Phe Gln Lys Trp Ala Pro Thr Val 595 600 605 Val Val Ile Ser Tyr Lys Gly Ser Pro Val Val Arg Lys Val Ile Gln 610 615 620 Ser Gln Leu Lys Ala Ala Lys Phe Asn Val Leu Leu Thr Thr Tyr Glu 625 630 635 640 Tyr Ile Ile Lys Asp Lys Gly Val Leu Ala Lys Ile Pro Phe Lys Tyr 645 650 655 Met Ile Ile Asp Glu Gly His Arg Met Lys Asn His His Cys Lys Leu 660 665 670 Thr Gln Val Leu Asn Thr His Tyr Leu Ala Pro Tyr Arg Leu Leu Leu 675 680 685 Thr Gly Thr Pro Leu Gln Asn Lys Leu Pro Glu Leu Trp Ala Leu Leu 690 695 700 Asn Phe Leu Leu Pro Ser Ile Phe Lys Ser Cys Ser Thr Phe Glu Gln 705 710 715 720 Trp Phe Asn Ala Pro Phe Ala Thr Thr Gly Glu Lys Val Glu Leu Asn 725 730 735 Glu Glu Glu Thr Ile Leu Ile Ile Arg Arg Leu His Lys Val Leu Arg 740 745 750 Pro Phe Leu Leu Arg Arg Leu Lys Lys Glu Val Glu Ser Gln Leu Pro 755 760 765 Asp Lys Val Glu Tyr Ile Ile Lys Cys Asp Met Ser Gly Leu Gln Lys 770 775 780 Val Leu Tyr Ala His Met Gln Ser Lys Gly Val Leu Leu Thr Asp Gly 785 790 795 800 Ser Glu Lys Gly Ser Lys Gly Arg Gly Ser Lys Ala Leu Met Asn Thr 805 810 815 Ile Met Gln Leu Arg Lys Leu Cys Asn His Pro Phe Met Phe Gln Asn 820 825 830 Ile Glu Glu Lys Tyr Cys Asp His Val Gly Ile Ala Gly Gly Val Val 835 840 845 Ser Gly Pro Asp Thr Tyr Arg Val Ser Gly Lys Phe Glu Leu Leu Asp 850 855 860 Arg Ile Leu Pro Lys Met Lys Ala Thr Asn His Arg Ile Leu Leu Phe 865 870 875 880 Cys Gln Met Thr Gln Leu Met Thr Ile Met Glu Asp Tyr Leu Asn Trp 885 890 895 Arg Gly Phe Lys Tyr Leu Arg Leu Asp Gly Thr Ile Lys Ser Glu Asp 900 905 910 Arg Gly Asp Leu Leu Ser Lys Phe Asn Asp Lys Asn Ser Glu Tyr Phe 915 920 925 Leu Phe Leu Leu Ser Thr Arg Ala Gly Gly Leu Gly Leu Asn Leu Gln 930 935 940 Thr Ala Asp Thr Val Ile Ile Phe Asp Ser Asp Trp Asn Pro His Gln 945 950 955 960 Asp Leu Gln Ala Gln Asp Arg Ala His Arg Ile Gly Gln Gln Asn Glu 965 970 975 Val Arg Val Leu Arg Leu Met Thr Val Asn Ser Val Glu Glu Arg Ile 980 985 990 Leu Ala Ala Ala Lys Tyr Lys Leu Thr Met Asp Glu Lys Val Ile Gln 995 1000 1005 Ala Gly Met Phe Asp Gln Lys Ser Thr Gly Ser Glu Arg Gln Gln 1010 1015 1020 Phe Leu Gln Ser Ile Leu His Asn Asp Gly Ser Asp Glu Glu Glu 1025 1030 1035 Glu Asn Glu Val Pro Asp Asp Glu Thr Val Asn Gln Met Ile Ala 1040 1045 1050 Arg Thr Glu Asp Glu Phe Gln Leu Phe Gln Lys Met Asp Thr Glu 1055 1060 1065 Arg Lys Glu Glu Asn Glu Lys Leu Gly Gln His Lys Lys Ser Arg 1070 1075 1080 Leu Val Gln Glu Cys Glu Leu Pro Asp Trp Leu Thr Lys Pro Asp 1085 1090 1095 Glu Asp Asp Gly Trp Gly Asp Asp Tyr Thr Glu Ala Leu Leu Gly 1100 1105 1110 Arg Gly Thr Arg Gln Arg Lys Glu Val Asp Tyr Ala Asp Ser Leu 1115 1120 1125 Thr Glu Lys Glu Trp Leu Lys Ala Ile Asp Glu Asp Gly Asp Tyr 1130 1135 1140 Asp Glu Glu Glu Glu Glu Glu Lys Val Gln Lys Lys Arg Gly Arg 1145 1150 1155 Lys Arg Arg Lys Arg Asp Asp Ser Asp Asp Asp Thr Ser Ser Ser 1160 1165 1170 Thr Arg Arg Arg Lys Leu Pro Gln Ser Gln Val Glu Ala Arg Leu 1175 1180 1185 Lys Arg Lys Met Lys Lys Leu Met Asn Ile Val Thr Asn Tyr Lys 1190 1195 1200 Asp Arg Asp Gly Arg Gln Leu Ser Asp Gln Phe Ile Lys Leu Pro 1205 1210 1215 Pro Arg Lys Glu Tyr Pro Asp Tyr Tyr Thr Ile Ile Lys Lys Pro 1220 1225 1230 Ile Asp Ile Ser Lys Ile Leu Asn Tyr Ile Asp Asp Gly Lys Tyr 1235 1240 1245 Ser Asp Phe Ser Asp Leu Glu Arg Asp Phe Met Leu Leu Cys Gln 1250 1255 1260 Asn Ala Gln Ile Tyr Asn Glu Glu Ala Ser Leu Ile His Glu Asp 1265 1270 1275 Ser Ile Val Leu Gln Ser Val Phe Ser Ser Ala Lys Gln Lys Ile 1280 1285 1290 Glu Ala Ser Pro Asp Ser Asp Asp Glu Lys Asp Asp Asn Asn Ser 1295 1300 1305 Asp Val Glu Thr Pro Lys Asn Lys Asn Lys Pro Gly Lys Gly Lys 1310 1315 1320 Arg Arg Pro Gly Arg Pro Arg Arg Ser Ala Lys Lys Tyr Ile Ser 1325 1330 1335 Asp Asp Asp Asp Asp Asp 1340 55134DNADiabrotica virgifera 5acagttaaat attgaaaatg gcctggtgtt ttgataaaac ggaagaggcg aatttctagt 60agcattttaa ggtttcattt gcatttaaaa caaattcatg tattataaaa tgtaggatac 120gtttcctcgt atccatctac ttaatttagg ataacaataa agggtgtgag acagttaaat 180attgaaaatg gccagtgctt cattattacc caaaactttc acttctattg gtggcaaagc 240cctacctacc aactcacaac aaaacattca gtcaaaattt aaagagatta cagttccacc 300aggaaatact cctcaagatg ttaaagaagg ccccagtcac caatcaaatc caaaccattt 360ggcttctctt caaaaggcca ttgaaactat ggaagagaag ggcttacaag ctgatcctag 420atattcacag ttacttgcat tgcgagctag cattcctggg gcagaagaaa atggttctcc 480cttctcaaac aaccaaatca agcaattaag aaaccaaata atggcttaca ggtgtttggc 540aagaaatcaa cctgttccaa acaatttagt attaggtttg catggaaaaa ctcctgaaaa 600agttccacat attgtacctc caccgcaacc tcaagaagta cctaatgggg gcgatccagg 660accttcaaca agttctgctg ctgctgtagc tcctagaaca ccacaaaagc tgccagcaaa 720accaattgag gctcagcttg tcaacagaga accaagagtc actactttat ctaaaccatc 780ttccatagac cctgttgttc tattacaaga acgagaaaac agggtagcag ctcgtatagc 840agcgaggatt gaacaagtca gtaatctgcc gactgatatg tctgaggcat tacgtattcg 900ggcacaaata gaactcagag ctttgagatg tctaaacctc cagagacaac ttcgtagtga 960ggttttgagc tgtattcgac gggacacaac attagaaaca gcagtaaatg taaaagcgtt 1020taaacggacc aaacgtcaag gtcttcgaga agctagagca acagaaaaac ttgagaaaca 1080acaaaagctg gaagcagaga gaaagaaacg ccagaagaac caagagttct taaacaatgt 1140gatggcacac gctaaagatt tcaaagaatt ccacaggcag aaccaagcaa aactttctaa 1200acttaataaa gctattctta cttatcacgc taatgcggag agagaacaaa agaaggaaca 1260agagagaaga gaaaaggaac gtatgaagaa attgatggca gaagatgaag aaggttatag 1320acagttgatc gatcaaaaga aagacaaacg tctagcgttc ttgctttcgc aaacagatga 1380gtatataact aacctcacgg agatggtaaa gcaacacaag ttggaacaaa ccaataaaaa 1440gaaagaggag gaaaaacgca agaagaagca gcagaaaatg caacaaccag ataggaaagt 1500tacagttctg gaaactgcaa caggtaaaaa agtaacagga gaggctgctc ctacactgcg 1560acaagttcag gaatggttaa tccaacatcc tggatgggag atggtcgata cagatgatga 1620ggatgatgaa aacggggaga agagggatga tgactatgat gaaaatcaag aagtggatga 1680tgcaaaagaa gttattaaaa aagctaaagt tgaagatgac gaatatcaca aaaacacaaa 1740agaagaacag acttactaca gtattgctca cactgttcat gaagtggtaa cagaacaagc 1800atccattctg gttaatggaa agcttaagga atatcaaatt agagggttag aatggatggt 1860gtctttgtac aataacaatc tgaatggtat tctagcagat gagatgggtc taggtaaaac 1920cattcaaacg attggcttgt tgacctattt gatggaaaaa aagaagataa atggaccgtt 1980tttgatcata gtgccacttt caaccatttc taattggatg

ttggaatttc aaaagtgggc 2040ccctactgta gttgtcattt catacaaagg ctctcctgtg gttagaaaag tgatccagag 2100ccagttaaaa gctgctaaat tcaatgtgct tctcactacc tacgagtaca ttattaagga 2160caagggtgta ttagcaaaaa tcccatttaa atatatgatc atagatgagg gtcatcgtat 2220gaaaaaccac cactgcaaat tgactcaagt cctgaatacg cactatttgg cgccctacag 2280actcctgctt actggtactc ccctacaaaa taaattacca gaattatggg ccttgttgaa 2340tttcttgttg ccttcgattt tcaagagttg ctccactttt gaacaatggt tcaatgcgcc 2400attcgcaaca acaggagaaa aggttgagtt aaacgaagaa gaaactatcc ttatcatccg 2460tcgtcttcac aaagtactca ggccgtttct cctgagacgt ctcaagaaag aagtcgaatc 2520tcagcttcca gacaaagtgg aatatatcat aaagtgtgac atgtcgggcc tacaaaaggt 2580tctctatgca cacatgcaga gcaagggtgt gttacttacc gatggttccg agaagggcag 2640taaaggaagg ggatctaagg cactgatgaa caccattatg cagctgagga aactgtgcaa 2700tcatccgttt atgttccaaa atatcgaaga gaaatattgt gatcatgttg gtattgctgg 2760tggagtggtt tctggacccg acacttatag ggtatctggt aagtttgagc tcttggacag 2820aatattgccc aaaatgaaag caactaacca taggattctt cttttctgtc aaatgactca 2880attaatgacc atcatggaag attatctaaa ttggagagga ttcaaatatc ttcgtcttga 2940tggtacaatc aaatcagaag atcgcgggga cctattatcg aaatttaatg ataaaaatag 3000tgaatatttt ttgtttttgc tatctacacg ggctggaggt ctgggactta atttgcagac 3060agctgatact gtgattatct tcgattccga ttggaatcct catcaggatt tacaagctca 3120ggatcgagct catcgtattg gacagcaaaa tgaggtccga gttttgcgtt tgatgactgt 3180taactctgtt gaggaacgaa ttttagctgc agctaaatac aagcttacta tggacgaaaa 3240ggtcattcaa gctggtatgt tcgatcagaa gtctacaggc tcagagagac atcagttttt 3300gcagagtatt ttacaccatg acggaagcga cgaagaagag gaaaacgaag ttcctgatga 3360cgaaacagtg aaccagatgt tggcccgaag ggaaaacgaa tttcagcttt tccagaagat 3420ggatcaggaa agaaaggaag aagatgaaaa gaccggaaag tcgcgactta ttcaagaaag 3480cgaattgccc gaatggctgt tgaagcaaga cgatgaaatc tactcgtggg gccttgatga 3540tccagatgct gttttaggaa ggggtagtag gcaaagaaaa gaagttgatt atgttgacag 3600cctgacggag aaagagtggc ttaaggctat tgacgaagag ggagaatttg aggaagaaca 3660agaaggtgat aaagaaggtc tcagaaagaa aagagggagg aagaggaaga agcgcgatga 3720tgacgaagag gcaagccaaa ttaagagaag aaaggtgcat ctagccgaga tcaagatgaa 3780gaaaaagatg aagaggctta tggaagttgt tgtgaactac agggacaggg atggtagagt 3840attgagcgaa ccgtttatga aacttccatc aaagaaggag ttacctgagt attacgatac 3900gattaagaaa cctattgata ttgaaaaagt cgttgccaac gtagaagaag gaaaatattt 3960cacgatgcac gatttggaaa gagatttcga cttgctgtgc caaaacgccc aacaatacaa 4020cgaagaagac tccatgatct acgaggacag cctcgttctt cgacaggtgt ttagaagcgc 4080gagggaaaag atcgacggta cctcagacca cgacgacaac gccgatggac cggcggtggc 4140tcagatcaaa cgacctcgtg gtagacctcg aaaacacaag agacccgaag agatcgaggc 4200cgaagcggcg gctcagaaag ctatggagga ggcatcgaag ctgagagctc aagctgaggc 4260ggaagagctt agatctaagg tggaggaggc atctcagaga gccaaagagg aagcgaaagc 4320aagggaggaa gccaaagcta gggaagaagc cgaaatcgag aacatggagg agattcccac 4380aagcacatga tctatagagc aaccggaaac aaaaaggcaa aaaagaaata ttatatagaa 4440aagatgtaca tgttcaatgg agatacattt tcgccgagtt acaacgggta atgcttttac 4500aacggatatt ttgacgtatg aatgttgacg ttcagatgaa gtatatttat aaaataatcc 4560agacctttac gttttggttg atttgttttc tgtattgttc agtttattga acaaccatta 4620atagcagctt acctaaatga tttagaaaag catctgagtt atttagataa gttttgagat 4680tatatttatt aactttaata ttactatctt tattatagca tattgtaatt attttttcct 4740gtccttcttt cgttgtgtgg tagataatcc gagagtcaac agttataagc aaatgaaatt 4800cagttaaacc tcaaatgtac aaaatgatca aattaatgtt tacaatttat ttttttacca 4860cgcacattca ctattactat tgtcagtcat tgagatatca ttttatatag ctccatgtct 4920gtcttcctca atttacagag aagcaattag acaagtaatg acataatatg gtgctgaaat 4980aatgtgcttg atagtgatgt tcacaaagta actattcgtt acaaagtact cgttacttac 5040aaataccgaa actaacgatt actatacaga gaggcaaatc gttactttga ttacactgat 5100tacttcgtat caatcgtatc agagcgagta acga 513461400PRTDiabrotica virgifera 6Met Ala Ser Ala Ser Leu Leu Pro Lys Thr Phe Thr Ser Ile Gly Gly 1 5 10 15 Lys Ala Leu Pro Thr Asn Ser Gln Gln Asn Ile Gln Ser Lys Phe Lys 20 25 30 Glu Ile Thr Val Pro Pro Gly Asn Thr Pro Gln Asp Val Lys Glu Gly 35 40 45 Pro Ser His Gln Ser Asn Pro Asn His Leu Ala Ser Leu Gln Lys Ala 50 55 60 Ile Glu Thr Met Glu Glu Lys Gly Leu Gln Ala Asp Pro Arg Tyr Ser 65 70 75 80 Gln Leu Leu Ala Leu Arg Ala Ser Ile Pro Gly Ala Glu Glu Asn Gly 85 90 95 Ser Pro Phe Ser Asn Asn Gln Ile Lys Gln Leu Arg Asn Gln Ile Met 100 105 110 Ala Tyr Arg Cys Leu Ala Arg Asn Gln Pro Val Pro Asn Asn Leu Val 115 120 125 Leu Gly Leu His Gly Lys Thr Pro Glu Lys Val Pro His Ile Val Pro 130 135 140 Pro Pro Gln Pro Gln Glu Val Pro Asn Gly Gly Asp Pro Gly Pro Ser 145 150 155 160 Thr Ser Ser Ala Ala Ala Val Ala Pro Arg Thr Pro Gln Lys Leu Pro 165 170 175 Ala Lys Pro Ile Glu Ala Gln Leu Val Asn Arg Glu Pro Arg Val Thr 180 185 190 Thr Leu Ser Lys Pro Ser Ser Ile Asp Pro Val Val Leu Leu Gln Glu 195 200 205 Arg Glu Asn Arg Val Ala Ala Arg Ile Ala Ala Arg Ile Glu Gln Val 210 215 220 Ser Asn Leu Pro Thr Asp Met Ser Glu Ala Leu Arg Ile Arg Ala Gln 225 230 235 240 Ile Glu Leu Arg Ala Leu Arg Cys Leu Asn Leu Gln Arg Gln Leu Arg 245 250 255 Ser Glu Val Leu Ser Cys Ile Arg Arg Asp Thr Thr Leu Glu Thr Ala 260 265 270 Val Asn Val Lys Ala Phe Lys Arg Thr Lys Arg Gln Gly Leu Arg Glu 275 280 285 Ala Arg Ala Thr Glu Lys Leu Glu Lys Gln Gln Lys Leu Glu Ala Glu 290 295 300 Arg Lys Lys Arg Gln Lys Asn Gln Glu Phe Leu Asn Asn Val Met Ala 305 310 315 320 His Ala Lys Asp Phe Lys Glu Phe His Arg Gln Asn Gln Ala Lys Leu 325 330 335 Ser Lys Leu Asn Lys Ala Ile Leu Thr Tyr His Ala Asn Ala Glu Arg 340 345 350 Glu Gln Lys Lys Glu Gln Glu Arg Arg Glu Lys Glu Arg Met Lys Lys 355 360 365 Leu Met Ala Glu Asp Glu Glu Gly Tyr Arg Gln Leu Ile Asp Gln Lys 370 375 380 Lys Asp Lys Arg Leu Ala Phe Leu Leu Ser Gln Thr Asp Glu Tyr Ile 385 390 395 400 Thr Asn Leu Thr Glu Met Val Lys Gln His Lys Leu Glu Gln Thr Asn 405 410 415 Lys Lys Lys Glu Glu Glu Lys Arg Lys Lys Lys Gln Gln Lys Met Gln 420 425 430 Gln Pro Asp Arg Lys Val Thr Val Leu Glu Thr Ala Thr Gly Lys Lys 435 440 445 Val Thr Gly Glu Ala Ala Pro Thr Leu Arg Gln Val Gln Glu Trp Leu 450 455 460 Ile Gln His Pro Gly Trp Glu Met Val Asp Thr Asp Asp Glu Asp Asp 465 470 475 480 Glu Asn Gly Glu Lys Arg Asp Asp Asp Tyr Asp Glu Asn Gln Glu Val 485 490 495 Asp Asp Ala Lys Glu Val Ile Lys Lys Ala Lys Val Glu Asp Asp Glu 500 505 510 Tyr His Lys Asn Thr Lys Glu Glu Gln Thr Tyr Tyr Ser Ile Ala His 515 520 525 Thr Val His Glu Val Val Thr Glu Gln Ala Ser Ile Leu Val Asn Gly 530 535 540 Lys Leu Lys Glu Tyr Gln Ile Arg Gly Leu Glu Trp Met Val Ser Leu 545 550 555 560 Tyr Asn Asn Asn Leu Asn Gly Ile Leu Ala Asp Glu Met Gly Leu Gly 565 570 575 Lys Thr Ile Gln Thr Ile Gly Leu Leu Thr Tyr Leu Met Glu Lys Lys 580 585 590 Lys Ile Asn Gly Pro Phe Leu Ile Ile Val Pro Leu Ser Thr Ile Ser 595 600 605 Asn Trp Met Leu Glu Phe Gln Lys Trp Ala Pro Thr Val Val Val Ile 610 615 620 Ser Tyr Lys Gly Ser Pro Val Val Arg Lys Val Ile Gln Ser Gln Leu 625 630 635 640 Lys Ala Ala Lys Phe Asn Val Leu Leu Thr Thr Tyr Glu Tyr Ile Ile 645 650 655 Lys Asp Lys Gly Val Leu Ala Lys Ile Pro Phe Lys Tyr Met Ile Ile 660 665 670 Asp Glu Gly His Arg Met Lys Asn His His Cys Lys Leu Thr Gln Val 675 680 685 Leu Asn Thr His Tyr Leu Ala Pro Tyr Arg Leu Leu Leu Thr Gly Thr 690 695 700 Pro Leu Gln Asn Lys Leu Pro Glu Leu Trp Ala Leu Leu Asn Phe Leu 705 710 715 720 Leu Pro Ser Ile Phe Lys Ser Cys Ser Thr Phe Glu Gln Trp Phe Asn 725 730 735 Ala Pro Phe Ala Thr Thr Gly Glu Lys Val Glu Leu Asn Glu Glu Glu 740 745 750 Thr Ile Leu Ile Ile Arg Arg Leu His Lys Val Leu Arg Pro Phe Leu 755 760 765 Leu Arg Arg Leu Lys Lys Glu Val Glu Ser Gln Leu Pro Asp Lys Val 770 775 780 Glu Tyr Ile Ile Lys Cys Asp Met Ser Gly Leu Gln Lys Val Leu Tyr 785 790 795 800 Ala His Met Gln Ser Lys Gly Val Leu Leu Thr Asp Gly Ser Glu Lys 805 810 815 Gly Ser Lys Gly Arg Gly Ser Lys Ala Leu Met Asn Thr Ile Met Gln 820 825 830 Leu Arg Lys Leu Cys Asn His Pro Phe Met Phe Gln Asn Ile Glu Glu 835 840 845 Lys Tyr Cys Asp His Val Gly Ile Ala Gly Gly Val Val Ser Gly Pro 850 855 860 Asp Thr Tyr Arg Val Ser Gly Lys Phe Glu Leu Leu Asp Arg Ile Leu 865 870 875 880 Pro Lys Met Lys Ala Thr Asn His Arg Ile Leu Leu Phe Cys Gln Met 885 890 895 Thr Gln Leu Met Thr Ile Met Glu Asp Tyr Leu Asn Trp Arg Gly Phe 900 905 910 Lys Tyr Leu Arg Leu Asp Gly Thr Ile Lys Ser Glu Asp Arg Gly Asp 915 920 925 Leu Leu Ser Lys Phe Asn Asp Lys Asn Ser Glu Tyr Phe Leu Phe Leu 930 935 940 Leu Ser Thr Arg Ala Gly Gly Leu Gly Leu Asn Leu Gln Thr Ala Asp 945 950 955 960 Thr Val Ile Ile Phe Asp Ser Asp Trp Asn Pro His Gln Asp Leu Gln 965 970 975 Ala Gln Asp Arg Ala His Arg Ile Gly Gln Gln Asn Glu Val Arg Val 980 985 990 Leu Arg Leu Met Thr Val Asn Ser Val Glu Glu Arg Ile Leu Ala Ala 995 1000 1005 Ala Lys Tyr Lys Leu Thr Met Asp Glu Lys Val Ile Gln Ala Gly 1010 1015 1020 Met Phe Asp Gln Lys Ser Thr Gly Ser Glu Arg His Gln Phe Leu 1025 1030 1035 Gln Ser Ile Leu His His Asp Gly Ser Asp Glu Glu Glu Glu Asn 1040 1045 1050 Glu Val Pro Asp Asp Glu Thr Val Asn Gln Met Leu Ala Arg Arg 1055 1060 1065 Glu Asn Glu Phe Gln Leu Phe Gln Lys Met Asp Gln Glu Arg Lys 1070 1075 1080 Glu Glu Asp Glu Lys Thr Gly Lys Ser Arg Leu Ile Gln Glu Ser 1085 1090 1095 Glu Leu Pro Glu Trp Leu Leu Lys Gln Asp Asp Glu Ile Tyr Ser 1100 1105 1110 Trp Gly Leu Asp Asp Pro Asp Ala Val Leu Gly Arg Gly Ser Arg 1115 1120 1125 Gln Arg Lys Glu Val Asp Tyr Val Asp Ser Leu Thr Glu Lys Glu 1130 1135 1140 Trp Leu Lys Ala Ile Asp Glu Glu Gly Glu Phe Glu Glu Glu Gln 1145 1150 1155 Glu Gly Asp Lys Glu Gly Leu Arg Lys Lys Arg Gly Arg Lys Arg 1160 1165 1170 Lys Lys Arg Asp Asp Asp Glu Glu Ala Ser Gln Ile Lys Arg Arg 1175 1180 1185 Lys Val His Leu Ala Glu Ile Lys Met Lys Lys Lys Met Lys Arg 1190 1195 1200 Leu Met Glu Val Val Val Asn Tyr Arg Asp Arg Asp Gly Arg Val 1205 1210 1215 Leu Ser Glu Pro Phe Met Lys Leu Pro Ser Lys Lys Glu Leu Pro 1220 1225 1230 Glu Tyr Tyr Asp Thr Ile Lys Lys Pro Ile Asp Ile Glu Lys Val 1235 1240 1245 Val Ala Asn Val Glu Glu Gly Lys Tyr Phe Thr Met His Asp Leu 1250 1255 1260 Glu Arg Asp Phe Asp Leu Leu Cys Gln Asn Ala Gln Gln Tyr Asn 1265 1270 1275 Glu Glu Asp Ser Met Ile Tyr Glu Asp Ser Leu Val Leu Arg Gln 1280 1285 1290 Val Phe Arg Ser Ala Arg Glu Lys Ile Asp Gly Thr Ser Asp His 1295 1300 1305 Asp Asp Asn Ala Asp Gly Pro Ala Val Ala Gln Ile Lys Arg Pro 1310 1315 1320 Arg Gly Arg Pro Arg Lys His Lys Arg Pro Glu Glu Ile Glu Ala 1325 1330 1335 Glu Ala Ala Ala Gln Lys Ala Met Glu Glu Ala Ser Lys Leu Arg 1340 1345 1350 Ala Gln Ala Glu Ala Glu Glu Leu Arg Ser Lys Val Glu Glu Ala 1355 1360 1365 Ser Gln Arg Ala Lys Glu Glu Ala Lys Ala Arg Glu Glu Ala Lys 1370 1375 1380 Ala Arg Glu Glu Ala Glu Ile Glu Asn Met Glu Glu Ile Pro Thr 1385 1390 1395 Ser Thr 1400 7538DNADiabrotica virgifera 7agtgtattag caaaaatccc atttaaatat atgatcatag atgagggtca tcgtatgaaa 60aaccaccact gcaaattgac tcaagtcctg aatacgcact atttggcgcc ctacagactc 120ctgcttactg gtactcccct acaaaataaa ttaccagaat tatgggcctt gttgaatttc 180ttgttgcctt cgattttcaa gagttgctcc acttttgaac aatggttcaa tgcgccattc 240gcaacaacag gagaaaaggt tgagttaaac gaagaagaaa ctatccttat catccgtcgt 300cttcacaaag tactcaggcc gtttctcctg agacgtctca agaaagaagt cgaatctcag 360cttccagaca aagtggaata tatcataaag tgtgacatgt cgggcctaca aaaggttctc 420tatgcacaca tgcagagcaa gggtgtgtta cttaccgatg gttccgagaa gggcagtaaa 480ggaaggggat ctaaggacaa ctagatgaac accattatgc agctgaggaa actgtgct 5388352DNADiabrotica virgifera 8ttgaactgta ttaggagaga tataacgctt gaatctgctg taaattttaa agcatataaa 60agaacgaagc gacagggtct aaaagaatcg agagctacag agaagttaga aaaacaacag 120aagttagaag cagaaagaaa gagaagacag aagaaccaag aatttttgaa tgctgtattg 180aacaatggaa aagaattcaa ggaattccac aagcagaatc aagcgaaatt agctaagatt 240aataaagctg ttattaatta tcacgctaat gctgaaagag agcaaaagaa agaagcagaa 300aggagagaga aggaacgtat gatcagattg atggcagaag atgaagaagg tt 3529178PRTDiabrotica virgifera 9Ser Val Leu Ala Lys Ile Pro Phe Lys Tyr Met Ile Ile Asp Glu Gly 1 5 10 15 His Arg Met Lys Asn His His Cys Lys Leu Thr Gln Val Leu Asn Thr 20 25 30 His Tyr Leu Ala Pro Tyr Arg Leu Leu Leu Thr Gly Thr Pro Leu Gln 35 40 45 Asn Lys Leu Pro Glu Leu Trp Ala Leu Leu Asn Phe Leu Leu Pro Ser 50 55 60 Ile Phe Lys Ser Cys Ser Thr Phe Glu Gln Trp Phe Asn Ala Pro Phe 65 70 75 80 Ala Thr Thr Gly Glu Lys Val Glu Leu Asn Glu Glu Glu Thr Ile Leu 85 90 95 Ile Ile Arg Arg Leu His Lys Val Leu Arg Pro Phe Leu Leu Arg Arg 100 105 110 Leu Lys Lys Glu Val Glu Ser Gln Leu Pro Asp Lys Val Glu Tyr Ile 115 120 125 Ile Lys Cys Asp Met Ser Gly Leu Gln Lys Val Leu Tyr Ala His Met 130 135 140 Gln Ser Lys Gly Val Leu Leu Thr Asp Gly Ser Glu Lys Gly Ser Lys 145 150 155 160 Gly Arg Gly Ser Lys Asp Asn Met Asn Thr Ile Met Gln Leu Arg Lys 165 170 175 Leu Cys 10459DNADiabrotica virgifera 10atgagggtca tcgtatgaaa aaccaccact gcaaattgac tcaagtcctg aatacgcact 60atttggcgcc ctacagactc ctgcttactg gtactcccct acaaaataaa ttaccagaat 120tatgggcctt gttgaatttc ttgttgcctt cgattttcaa gagttgctcc acttttgaac 180aatggttcaa tgcgccattc gcaacaacag gagaaaaggt tgagttaaac gaagaagaaa 240ctatccttat catccgtcgt cttcacaaag tactcaggcc gtttctcctg agacgtctca 300agaaagaagt cgaatctcag cttccagaca aagtggaata tatcataaag tgtgacatgt 360cgggcctaca aaaggttctc tatgcacaca tgcagagcaa gggtgtgtta cttaccgatg 420gttccgagaa gggcagtaaa ggaaggggat

ctaaggaca 45911503DNAArtificial SequenceYFP coding region 11caccatgggc tccagcggcg ccctgctgtt ccacggcaag atcccctacg tggtggagat 60ggagggcaat gtggatggcc acaccttcag catccgcggc aagggctacg gcgatgccag 120cgtgggcaag gtggatgccc agttcatctg caccaccggc gatgtgcccg tgccctggag 180caccctggtg accaccctga cctacggcgc ccagtgcttc gccaagtacg gccccgagct 240gaaggatttc tacaagagct gcatgcccga tggctacgtg caggagcgca ccatcacctt 300cgagggcgat ggcaatttca agacccgcgc cgaggtgacc ttcgagaatg gcagcgtgta 360caatcgcgtg aagctgaatg gccagggctt caagaaggat ggccacgtgc tgggcaagaa 420tctggagttc aatttcaccc cccactgcct gtacatctgg ggcgatcagg ccaatcacgg 480cctgaagagc gccttcaaga tct 5031220DNAArtificial SequenceT7 phage promoter 12taatacgact cactataggg 201348DNAArtificial SequencePrimer BrahmaVar2_FT7 13ttaatacgac tcactatagg gagaatgagg gtcatcgtat gaaaaacc 481424DNAArtificial SequencePrimer BrahmaVar2_R 14tgtccttaga tccccttcct ttac 241524DNAArtificial SequencePrimer BrahmaVar2_F 15atgagggtca tcgtatgaaa aacc 241648DNAArtificial SequencePrimer BrahmaVar2_RT7 16ttaatacgac tcactatagg gagatgtcct tagatcccct tcctttac 481738DNAArtificial SequencePrimer Brahma352_FT7 17taatacgact cactataggg aaccttcttc atcttctg 381839DNAArtificial SequencePrimer Brahma352_RT7 18taatacgact cactataggg ttgaactgta ttaggagag 391947DNAArtificial SequencePrimer YFP-F_T7 19ttaatacgac tcactatagg gagacaccat gggctccagc ggcgccc 472047DNAArtificial SequencePrimer YFP-R_T7 20ttaatacgac tcactatagg gagaagatct tgaaggcgct cttcagg 4721218DNADiabrotica virgifera 21tagctctgat gacagagccc atcgagtttc aagccaaaca gttgcataaa gctatcagcg 60gattgggaac tgatgaaagt acaatmgtmg aaattttaag tgtmcacaac aacgatgaga 120ttataagaat ttcccaggcc tatgaaggat tgtaccaacg mtcattggaa tctgatatca 180aaggagatac ctcaggaaca ttaaaaaaga attattag 21822424DNADiabrotica virgiferamisc_feature(393)..(395)n is a, c, g, or t 22ttgttacaag ctggagaact tctctttgct ggaaccgaag agtcagtatt taatgctgta 60ttctgtcaaa gaaataaacc acaattgaat ttgatattcg acaaatatga agaaattgtt 120gggcatccca ttgaaaaagc cattgaaaac gagttttcag gaaatgctaa acaagccatg 180ttacacctta tccagagcgt aagagatcaa gttgcatatt tggtaaccag gctgcatgat 240tcaatggcag gcgtcggtac tgacgataga actttaatca gaattgttgt ttcgagatct 300gaaatcgatc tagaggaaat caaacaatgc tatgaagaaa tctacagtaa aaccttggct 360gataggatag cggatgacac atctggcgac tannnaaaag ccttattagc cgttgttggt 420taag 42423397DNADiabrotica virgifera 23agatgttggc tgcatctaga gaattacaca agttcttcca tgattgcaag gatgtactga 60gcagaatagt ggaaaaacag gtatccatgt ctgatgaatt gggaagggac gcaggagctg 120tcaatgccct tcaacgcaaa caccagaact tcctccaaga cctacaaaca ctccaatcga 180acgtccaaca aatccaagaa gaatcagcta aacttcaagc tagctatgcc ggtgatagag 240ctaaagaaat caccaacagg gagcaggaag tggtagcagc ctgggcagcc ttgcagatcg 300cttgcgatca gagacacgga aaattgagcg atactggtga tctattcaaa ttctttaact 360tggtacgaac gttgatgcag tggatggacg aatggac 39724490DNADiabrotica virgifera 24gcagatgaac accagcgaga aaccaagaga tgttagtggt gttgaattgt tgatgaacaa 60ccatcagaca ctcaaggctg agatcgaagc cagagaagac aactttacgg cttgtatttc 120tttaggaaag gaattgttga gccgtaatca ctatgctagt gctgatatta aggataaatt 180ggtcgcgttg acgaatcaaa ggaatgctgt actacagagg tgggaagaaa gatgggagaa 240cttgcaactc atcctcgagg tataccaatt cgccagagat gcggccgtcg ccgaagcatg 300gttgatcgca caagaacctt acttgatgag ccaagaacta ggacacacca ttgacgacgt 360tgaaaacttg ataaagaaac acgaagcgtt cgaaaaatcg gcagcggcgc aagaagagag 420attcagtgct ttggagagac tgacgacgtt cgaattgaga gaaataaaga ggaaacaaga 480agctgcccag 49025330DNADiabrotica virgifera 25agtgaaatgt tagcaaatat aacatccaag tttcgtaatt gtacttgctc agttagaaaa 60tattctgtag tttcactatc ttcaaccgaa aatagaataa atgtagaacc tcgcgaactt 120gcctttcctc caaaatatca agaacctcga caagtttggt tggagagttt agatacgata 180gacgacaaaa aattgggtat tcttgagctg catcctgatg tttttgctac taatccaaga 240atagatatta tacatcaaaa tgttagatgg caaagtttat atagatatgt aagctatgct 300catacaaagt caagatttga agtgagaggt 33026320DNADiabrotica virgifera 26caaagtcaag atttgaagtg agaggtggag gtcgaaaacc gtggccgcaa aagggattgg 60gacgtgctcg acatggttca attagaagtc cactttggag aggtggagga gttgttcatg 120gaccaaaatc tccaacccct catttttaca tgattccatt ctacacccgt ttgctgggtt 180tgactagcgc actttcagta aaatttgccc aagatgactt gcacgttgtg gatagtctag 240atctgccaac tgacgaacaa agttatatag aagagctggt caaaagccgc ttttgggggt 300ccttcttgtt ttatttgtag 3202746DNAArtificial SequencePrimer Ann-F1_T7 27ttaatacgac tcactatagg gagagctcca acagtggttc cttatc 462829DNAArtificial SequencePrimer Ann-R1 28ctaataattc ttttttaatg ttcctgagg 292922DNAArtificial SequencePrimer Ann-F1 29gctccaacag tggttcctta tc 223053DNAArtificial SequencePrimer Ann-R1_T7 30ttaatacgac tcactatagg gagactaata attctttttt aatgttcctg agg 533148DNAArtificial SequencePrimer Ann-F2_T7 31ttaatacgac tcactatagg gagattgtta caagctggag aacttctc 483224DNAArtificial SequencePrimer Ann-R2 32cttaaccaac aacggctaat aagg 243324DNAArtificial SequencePrimer Ann-F2 33ttgttacaag ctggagaact tctc 243448DNAArtificial SequencePrimer Ann-R2T7 34ttaatacgac tcactatagg gagacttaac caacaacggc taataagg 483547DNAArtificial SequencePrimer Betasp2-F1_T7 35ttaatacgac tcactatagg gagaagatgt tggctgcatc tagagaa 473622DNAArtificial SequencePrimer Betasp2-R1 36gtccattcgt ccatccactg ca 223723DNAArtificial SequencePrimer Betasp2-F1 37agatgttggc tgcatctaga gaa 233846DNAArtificial SequencePrimer Betasp-R1_T7 38ttaatacgac tcactatagg gagagtccat tcgtccatcc actgca 463946DNAArtificial SequencePrimer Betasp2-F2_T7 39ttaatacgac tcactatagg gagagcagat gaacaccagc gagaaa 464022DNAArtificial SequencePrimer Betasp2-R2 40ctgggcagct tcttgtttcc tc 224122DNAArtificial SequencePrimer Betasp2-F2 41gcagatgaac accagcgaga aa 224246DNAArtificial SequencePrimer Betasp2-R2_T7 42ttaatacgac tcactatagg gagactgggc agcttcttgt ttcctc 464351DNAArtificial SequencePrimer L4-F1_T7 43ttaatacgac tcactatagg gagaagtgaa atgttagcaa atataacatc c 514426DNAArtificial SequencePrimer L4-R1 44acctctcact tcaaatcttg actttg 264527DNAArtificial SequencePrimer L4-F1 45agtgaaatgt tagcaaatat aacatcc 274650DNAArtificial SequencePrimer L4-R1_T7 46ttaatacgac tcactatagg gagaacctct cacttcaaat cttgactttg 504750DNAArtificial SequencePrimer L4-F2_T7 47ttaatacgac tcactatagg gagacaaagt caagatttga agtgagaggt 504825DNAArtificial SequencePrimer L4-R2 48ctacaaataa aacaagaagg acccc 254926DNAArtificial SequencePrimer L4-F2 49caaagtcaag atttgaagtg agaggt 265049DNAArtificial SequencePrimer L4-R2_T7 50ttaatacgac tcactatagg gagactacaa ataaaacaag aaggacccc 495123DNAArtificial SequencePrimer YFP-R 51agatcttgaa ggcgctcttc agg 235223DNAArtificial SequencePrimer YFP-F 52caccatgggc tccagcggcg ccc 2353370DNAArtificial SequenceGFP partial coding region 53aagtgatgct acatacggaa agcttaccct taaatttatt tgcactactg gaaaactacc 60tgttccatgg ccaacacttg tcactacttt ctcttatggt gttcaatgct tttcccgtta 120tccggatcat atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca 180ggaacgcact atatctttca aagatgacgg gaactacaag acgcgtgctg aagtcaagtt 240tgaaggtgat acccttgtta atcgtatcga gttaaaaggt attgatttta aagaagatgg 300aaacattctc ggacacaaac tcgagtacaa ctataactca cacaatgtat acatcacggc 360agacaaacaa 3705443DNAArtificial SequencePrimer GFP_T7F 54taatacgact cactataggg aaggtgatgc tacatacgga aag 435538DNAArtificial SequencePrimer GFP_T7R 55taatacgact cactataggg ttgtttgtct gccgtgat 3856222DNASolanum tuberosum 56gactagtacc ggttgggaaa ggtatgtttc tgcttctacc tttgatatat atataataat 60tatcactaat tagtagtaat atagtatttc aagtattttt ttcaaaataa aagaatgtag 120tatatagcta ttgcttttct gtagtttata agtgtgtata ttttaattta taacttttct 180aatatatgac caaaacatgg tgatgtgcag gttgatccgc gg 22257812DNAArtificial SequenceBrahma v1 hpRNA encoding sequence 57gcgccctaca gactcctgct tactggtact cccctacaaa ataaattacc agaattatgg 60gccttgttga atttcttgtt gccttcgatt ttcaagagtt gctccacttt tgaacaatgg 120ttcaatgcgc cattcgcaac aacaggagaa aaggttgagt taaacgaaga agaaactatc 180cttatcatcc gtcgtcttca caaagtactc aggccgtttc tcctgagacg tctcaagaaa 240gaagtcgaat ctcagcttcc agacaaagtg gaatatatca taaagtgtga catgtgacta 300gtaccggttg ggaaaggtat gtttctgctt ctacctttga tatatatata ataattatca 360ctaattagta gtaatatagt atttcaagta tttttttcaa aataaaagaa tgtagtatat 420agctattgct tttctgtagt ttataagtgt gtatatttta atttataact tttctaatat 480atgaccaaaa catggtgatg tgcaggttga tccgcggaca tgtcacactt tatgatatat 540tccactttgt ctggaagctg agattcgact tctttcttga gacgtctcag gagaaacggc 600ctgagtactt tgtgaagacg acggatgata aggatagttt cttcttcgtt taactcaacc 660ttttctcctg ttgttgcgaa tggcgcattg aaccattgtt caaaagtgga gcaactcttg 720aaaatcgaag gcaacaagaa attcaacaag gcccataatt ctggtaattt attttgtagg 780ggagtaccag taagcaggag tctgtagggc gc 81258822DNAArtificial SequenceBrahma v2 hpRNA encoding sequence 58catataaaag aacgaagcga cagggtctaa aagaatcgag agctacagag aagttagaaa 60aacaacagaa gttagaagca gaaagaaaga gaagacagaa gaaccaagaa tttttgaatg 120ctgtattgaa caatggaaaa gaattcaagg aattccacaa gcagaatcaa gcgaaattag 180ctaagattaa taaagctgtt attaattatc acgctaatgc tgaaagagag caaaagaaag 240aagcagaaag gagagagaag gaacgtatga tcagattgat ggcagaagat gaagaaggtt 300gactagtacc ggttgggaaa ggtatgtttc tgcttctacc tttgatatat atataataat 360tatcactaat tagtagtaat atagtatttc aagtattttt ttcaaaataa aagaatgtag 420tatatagcta ttgcttttct gtagtttata agtgtgtata ttttaattta taacttttct 480aatatatgac caaaacatgg tgatgtgcag gttgatccgc ggaaccttct tcatcttctg 540ccatcaatct gatcatacgt tccttctctc tcctttctgc ttctttcttt tgctctcttt 600cagcattagc gtgataatta ataacagctt tattaatctt agctaatttc gcttgattct 660gcttgtggaa ttccttgaat tcttttccat tgttcaatac agcattcaaa aattcttggt 720tcttctgtct tctctttctt tctgcttcta acttctgttg tttttctaac ttctctgtag 780ctctcgattc ttttagaccc tgtcgcttcg ttcttttata tg 8225922DNAArtificial SequenceT20VN primermisc_feature(22)..(22)n is a, c, g, or t 59tttttttttt tttttttttt vn 226019DNAArtificial SequencePrimer P5U76S(F) 60tgtgatgttg gtggcgtat 196124DNAArtificial SequencePrimer P5U76A(R) 61tgttaaataa aaccccaaag atcg 246220DNAArtificial SequencePrimer TIPmxF 62gagggtaatg ccaactggtt 206324DNAArtificial SequencePrimer TIPmxR 63gcaatgtaac cgagtgtctc tcaa 246432DNAArtificial SequencePrimer HXTIP 64tttttggctt agagttgatg gtgtactgat ga 3265151DNAEscherichia coli 65gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc 60ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga 120cgacatcatt ccgtggcgtt atccagctaa g 1516669DNAArtificial SequenceAAD1 partial coding region 66tgttcggttc cctctaccaa gcacagaacc gtcgcttcag caacacctca gtcaaggtga 60tggatgttg 696725DNAArtificial SequencePrimer ST-LS1-F 67gtatgtttct gcttctacct ttgat 256829DNAArtificial SequencePrimer ST-LS1-R 68ccatgttttg gtcatatatt agaaaagtt 296934DNAArtificial SequenceProbe ST-LS1-P 69agtaatatag tatttcaagt atttttttca aaat 347020DNAArtificial SequencePrimer GAAD1-F 70tgttcggttc cctctaccaa 207122DNAArtificial SequencePrimer GAAD1-R 71caacatccat caccttgact ga 227224DNAArtificial SequenceProbe GAAD1-P 72cacagaaccg tcgcttcagc aaca 247318DNAArtificial SequencePrimer IVR1-F 73tggcggacga cgacttgt 187419DNAArtificial SequencePrimer IVR1-R 74aaagtttgga ggctgccgt 197526DNAArtificial SequenceProbe IVR1-P 75cgagcagacc gccgtgtact tctacc 267619DNAArtificial SequencePrimer SPC1A 76cttagctgga taacgccac 197719DNAArtificial SequencePrimer SPC1S 77gaccgtaagg cttgatgaa 197821DNAArtificial SequenceProbe TQSPEC 78cgagattctc cgcgctgtag a 21795146DNADiabrotica virgifera 79ttgagaacga gaacacgaac gagtgtatcg tcgtgtttct ttttctttgg ttattgtgta 60aattaattac aaacgtgtta aaatttactt aaagttagtg atttgtgtat ttatagtttg 120taagtgatgg catcagatga agaagtggag gattctttcg ccggggagga agatgccccc 180gacgatacgg ctgaacaaat agataacgat cctgattctg aagatggtgt tcctaaagga 240ggggaagaag atgatgatta tgaaccagaa gattccagaa agaaaaagaa gggaaagaaa 300agaaaagcca ggggagaaga aaagaaaggc aagaaaaaga agaaaaagcg aaagaatgat 360agtggggatg aaagtgactt tggagaagat gataatggag gtggggactc agattatgca 420agcagtagta aaagaggaag gaaaaagggt tctactaaac actcttctgc atcatcaaca 480ccaacaccag ctagtgactc tggcacagga ggcatgccca ccatcgagca agtttgttca 540acatttggtt taactgatgt cgagcttgac tattcagatg ctgatatgca aaacttgacc 600acctataagt tgttccaaca gcatgtgaga ccgctccttg ctaaggaaaa tccaaaggtt 660cctatgtcaa agttgatgat gttggttgct gcaaaatggc gcgaattttc taattcaaac 720cccaatctgc aaagcgaaaa tgaaccgtct gctgcaactt caaccacatc tgaagaaagt 780tatccaaaaa ctaatcgttc gagagcatcc aaggaagcag cacaaaagat agtagaggct 840gactctgagc catatgatga cgaatttgat gacgaagacg aggaggaaaa agaagagaaa 900ggaaagaaaa aaaagagtaa tagaggaagg cctagtaaaa agaaggctac taaagtacca 960actttaaaga ttaaactagg aaagaggaag cgtggaagtt cggatgaaga gggcgatctt 1020agtggaggtg gctctgatcg cgattctgat gctgagtttg agcagatgct acaagaagct 1080gaagaaccaa aatccaacaa atctaccact ggtgaagaat ccgcacagcc atcagaatca 1140cctgcagatg aaaatccacc accaaaacgc aaagcgaaaa ccaaaattgg ttgcaaaaca 1200aagagaaaga agaaaacaaa gagtggtaaa cctgaagatg aaaattatga acatcaagat 1260tactgcgagg tatgtcaaca aggtggagaa attatcctct gtgatacttg ccctagagct 1320taccacttgg tttgcctgga acctgaatta gaagaagccc ctgaaggaaa gtggagttgc 1380cctcattgtg agaatgaagg tccggctgaa caagatgatg acgagcatca agaattctgc 1440agggtttgca aagatggtgg cgaacttttg tgttgcgatt cctgtacatc tgcgtaccac 1500acgcactgtc ttaacccgcc acttcccgaa atacctgacg gcgattggaa atgtcctagg 1560tgcggttgtc cgcctcttgt gggcaaagtt gcgaaaattc ttacgtggaa atgggttgat 1620gatcctccta aaaagaagga caatggtgaa gaggagcctc ctacacgaca tagagagtac 1680tttgttaagt ggcatgagct atcatattgg cattgtagtt ggataaccga gcttcaattg 1740gatgtatatc atcctctcat gtttcgaagt tattcaagaa agtgggacat ggaagagcct 1800cctaaacttg aagaacctat ggatgaagct gacactagat gtagcagatt cctgaaaatg 1860ggtggaaaca acaacgacga tgaactcgaa gagaagtatt acagatacgg aataaaacca 1920gaatggctaa tagtccatcg tgtcatcaac caccgtacga tgcgagacgg aagaactttg 1980tacttagtaa aatggcgaga gctaacttac gatcaagcta cctgggaaga agattctgac 2040gatatcccag ccctaaagtc tgccatcgaa tattacacag attcaagagc tgctaattta 2100tccggagctg gaggtaagct aaagaagaaa gttggaagga agccgaaagc taaagaactt 2160atcgatgacg acgatagaaa cggtcctcgc agatatactc caccgccaga taagccctgc 2220agtgatctga agaagaaact agacaaacaa ccctcatatt tggacgagag tggattgctt 2280cacgagtacc aactagaggg tcttaactgg cttcgttatt cgtgggccaa cggtatagac 2340actatcttag ccgacgagat gggtctcggt aaaaccattc aaaccattgt cttcttgtat 2400tcgctctaca aggaaggtca ctgcaaaggt ccgtttctaa ttagtgtccc actttcaacg 2460atcatcaatt gggagagaga attcgaaaat tgggcacctg atttttattg tattacatat 2520gttggtgaca aggactgcag agccgtgatt cgtgagaacg aactcagttt cgaagatggt 2580gctgtcagag gaggtcgagc ttcgagaatc agagccggtt ccatcaagtt taacgttttg 2640ttgaccagct acgaattaat ttcgatcgat tcggcatgtc tcggttctat cgaatgggcc 2700gttttggtag tcgatgaagc tcatagattg aaaagcaatc aatcaaaatt cttcaaaatc 2760ttaaacgctt ataatatagc ttataaactc ctcttgaccg gaacaccgct tcaaaacaac 2820ctcgaagaat tgttccattt gttgaacttc ctcaacggtc agaaattcaa cgatctccaa 2880aacttccaag ccgaattcgc cgacatttcg aaagaagacc aagtgaagaa attgcacgag 2940atgttgggac ctcatatgct gcgtcgtctc aaggccgatg tgctgaagag catgccttcg 3000aaatctgaat ttatcgtcag agtcgaatta tcgcccatgc agaagaaata ttataaatat 3060attttgacga ggaactttga agctttaaat cctaaaggag gcggacagtc ggtatcttta 3120cttaacatta tgatggatct caagaaatgt tgcaaccatc cctatctttt cccagccgcc 3180tcggaagaag ctccgctggg tccccatggt aattgggatg taggtcattt gattaaggct 3240tcaggaaagt tggtgctatt agcgaagatg ttgaagatcc ttagagaaca gggtcacaga 3300gtgttgatct

tctcgcaaat gacgaagatg ttggatataa tggaagattt tcttgaagga 3360gaagggtata aatacgaacg tattgatggg gctattactg gtaatctccg tcaagaagct 3420atcgataggt ttaacgctcc aggtgctccc cagtttgttt tccttttgtc cactaaagct 3480ggtggtttgg gcatcaatct tgctacagca gatactgtaa tcatctatga ttccgattgg 3540aatccccaca atgatattca ggcattctcc agagctcatc gtatcggtca agccaacaag 3600gtgatgatct accgttttgt aacacgtaac agtgtggaag aacgtgttac gcaagtagcc 3660aagcggaaaa tgatgttaac tcacttggta gtcagacctg gaatgggcgg aaaaggtgcc 3720aactttacta agcaagagtt ggacgatatt ctcagattcg gtactgaaga attgtttaaa 3780gaaagtgaag gcaaagaaga cgaagccatt cactacgacg acaaagctgt gggagaattg 3840ctggatcgtt ctaaagaagg catagaacag aaagagagtt gggcgaacga atatctcagt 3900tcatttaaag tggctagtta tgttacaaaa gaaggggaag ttgaggaaga agttgacact 3960gagattatta aacaagaagc ggaaaatacc gatccggcct actggatcaa gctgcttaga 4020catcattatg agcaacaaca ggaagatata gctaggacgt taggaaaagg caaaagagtg 4080aggaaacagg ttaattataa tgacggagga atgacaactg acacacgaga agatacgaca 4140tggcaagaaa atctctctga ttaccattct gacttttctg cgggatcgga tgaggataag 4200gaagacgata aggaagacga tgattcgatg agaagaacga cgccgattta agcagaagga 4260gtcgaagaaa gatggaaagg aaagacgaga aggatcgtcc tttaccaccg ttactagcca 4320gagttggcgg caatattgaa gtactcggtt ttaatgccag gcagcgtaaa gcgttcctta 4380atgctattat gcgctacgga atgccaccac aagacgcttt caattcacag tggctggtga 4440gagatcttcg aggaaaatct gagaagatat tcaaggctta cgtgtctctc tttatgaggc 4500atctttgcga acctggtgca gataatgctg atacgtttgc ggacggtgtg ccgagggaag 4560gactgagtag gcaacatgtt ttgacaagga ttggtgtgat gtcacttata agaaagaagg 4620ttcaggagtt cgaacacatc aacggcgagt atagcatgcc ggaagtaatc aaaaagagca 4680ttatggatca aaataaaatc aatgccgccg gcaccgccac cacaagcgaa gcagaaacgc 4740ctaaaagtgc tactaccagt actagtgcta cgccagctac aagtgctgct cccagtcccg 4800ctcccacaca aggagaagat aaagataagg ataaagattc cgttcagagt gacgaaaata 4860aagataaaga agtggttaat aaaacggaaa ccgaagatga agagaagaaa acgggagaat 4920cttcaacaga aaagccgaaa actgaaccgg aagaagtgaa agaagcttct ccgaaaaccg 4980aaattcctga agctagttcc gaagctgata aatctgagat caaatccgaa gtcgatacct 5040cgtctgtaac cagcgaggaa aagaaagaag agaaagagga agaggccaaa aaagaagaac 5100ccgaagagac caaaatggaa atacaggagg aggaacttgt taaaga 5146801974PRTDiabrotica virgifera 80Met Ala Ser Asp Glu Glu Val Glu Asp Ser Phe Ala Gly Glu Glu Asp 1 5 10 15 Ala Pro Asp Asp Thr Ala Glu Gln Ile Asp Asn Asp Pro Asp Ser Glu 20 25 30 Asp Gly Val Pro Lys Gly Gly Glu Glu Asp Asp Asp Tyr Glu Pro Glu 35 40 45 Asp Ser Arg Lys Lys Lys Lys Gly Lys Lys Arg Lys Ala Arg Gly Glu 50 55 60 Glu Lys Lys Gly Lys Lys Lys Lys Lys Lys Arg Lys Asn Asp Ser Gly 65 70 75 80 Asp Glu Ser Asp Phe Gly Glu Asp Asp Asn Gly Gly Gly Asp Ser Asp 85 90 95 Tyr Ala Ser Ser Ser Lys Arg Gly Arg Lys Lys Gly Ser Thr Lys His 100 105 110 Ser Ser Ala Ser Ser Thr Pro Thr Pro Ala Ser Asp Ser Gly Thr Gly 115 120 125 Gly Met Pro Thr Ile Glu Gln Val Cys Ser Thr Phe Gly Leu Thr Asp 130 135 140 Val Glu Leu Asp Tyr Ser Asp Ala Asp Met Gln Asn Leu Thr Thr Tyr 145 150 155 160 Lys Leu Phe Gln Gln His Val Arg Pro Leu Leu Ala Lys Glu Asn Pro 165 170 175 Lys Val Pro Met Ser Lys Leu Met Met Leu Val Ala Ala Lys Trp Arg 180 185 190 Glu Phe Ser Asn Ser Asn Pro Asn Leu Gln Ser Glu Asn Glu Pro Ser 195 200 205 Ala Ala Thr Ser Thr Thr Ser Glu Glu Ser Tyr Pro Lys Thr Asn Arg 210 215 220 Ser Arg Ala Ser Lys Glu Ala Ala Gln Lys Ile Val Glu Ala Asp Ser 225 230 235 240 Glu Pro Tyr Asp Asp Glu Phe Asp Asp Glu Asp Glu Glu Glu Lys Glu 245 250 255 Glu Lys Gly Lys Lys Lys Lys Ser Asn Arg Gly Arg Pro Ser Lys Lys 260 265 270 Lys Ala Thr Lys Val Pro Thr Leu Lys Ile Lys Leu Gly Lys Arg Lys 275 280 285 Arg Gly Ser Ser Asp Glu Glu Gly Asp Leu Ser Gly Gly Gly Ser Asp 290 295 300 Arg Asp Ser Asp Ala Glu Phe Glu Gln Met Leu Gln Glu Ala Glu Glu 305 310 315 320 Pro Lys Ser Asn Lys Ser Thr Thr Gly Glu Glu Ser Ala Gln Pro Ser 325 330 335 Glu Ser Pro Ala Asp Glu Asn Pro Pro Pro Lys Arg Lys Ala Lys Thr 340 345 350 Lys Ile Gly Cys Lys Thr Lys Arg Lys Lys Lys Thr Lys Ser Gly Lys 355 360 365 Pro Glu Asp Glu Asn Tyr Glu His Gln Asp Tyr Cys Glu Val Cys Gln 370 375 380 Gln Gly Gly Glu Ile Ile Leu Cys Asp Thr Cys Pro Arg Ala Tyr His 385 390 395 400 Leu Val Cys Leu Glu Pro Glu Leu Glu Glu Ala Pro Glu Gly Lys Trp 405 410 415 Ser Cys Pro His Cys Glu Asn Glu Gly Pro Ala Glu Gln Asp Asp Asp 420 425 430 Glu His Gln Glu Phe Cys Arg Val Cys Lys Asp Gly Gly Glu Leu Leu 435 440 445 Cys Cys Asp Ser Cys Thr Ser Ala Tyr His Thr His Cys Leu Asn Pro 450 455 460 Pro Leu Pro Glu Ile Pro Asp Gly Asp Trp Lys Cys Pro Arg Cys Gly 465 470 475 480 Cys Pro Pro Leu Val Gly Lys Val Ala Lys Ile Leu Thr Trp Lys Trp 485 490 495 Val Asp Asp Pro Pro Lys Lys Lys Asp Asn Gly Glu Glu Glu Pro Pro 500 505 510 Thr Arg His Arg Glu Tyr Phe Val Lys Trp His Glu Leu Ser Tyr Trp 515 520 525 His Cys Ser Trp Ile Thr Glu Leu Gln Leu Asp Val Tyr His Pro Leu 530 535 540 Met Phe Arg Ser Tyr Ser Arg Lys Trp Asp Met Glu Glu Pro Pro Lys 545 550 555 560 Leu Glu Glu Pro Met Asp Glu Ala Asp Thr Arg Cys Ser Arg Phe Leu 565 570 575 Lys Met Gly Gly Asn Asn Asn Asp Asp Glu Leu Glu Glu Lys Tyr Tyr 580 585 590 Arg Tyr Gly Ile Lys Pro Glu Trp Leu Ile Val His Arg Val Ile Asn 595 600 605 His Arg Thr Met Arg Asp Gly Arg Thr Leu Tyr Leu Val Lys Trp Arg 610 615 620 Glu Leu Thr Tyr Asp Gln Ala Thr Trp Glu Glu Asp Ser Asp Asp Ile 625 630 635 640 Pro Ala Leu Lys Ser Ala Ile Glu Tyr Tyr Thr Asp Ser Arg Ala Ala 645 650 655 Asn Leu Ser Gly Ala Gly Gly Lys Leu Lys Lys Lys Val Gly Arg Lys 660 665 670 Pro Lys Ala Lys Glu Leu Ile Asp Asp Asp Asp Arg Asn Gly Pro Arg 675 680 685 Arg Tyr Thr Pro Pro Pro Asp Lys Pro Cys Ser Asp Leu Lys Lys Lys 690 695 700 Leu Asp Lys Gln Pro Ser Tyr Leu Asp Glu Ser Gly Leu Leu His Glu 705 710 715 720 Tyr Gln Leu Glu Gly Leu Asn Trp Leu Arg Tyr Ser Trp Ala Asn Gly 725 730 735 Ile Asp Thr Ile Leu Ala Asp Glu Met Gly Leu Gly Lys Thr Ile Gln 740 745 750 Thr Ile Val Phe Leu Tyr Ser Leu Tyr Lys Glu Gly His Cys Lys Gly 755 760 765 Pro Phe Leu Ile Ser Val Pro Leu Ser Thr Ile Ile Asn Trp Glu Arg 770 775 780 Glu Phe Glu Asn Trp Ala Pro Asp Phe Tyr Cys Ile Thr Tyr Val Gly 785 790 795 800 Asp Lys Asp Cys Arg Ala Val Ile Arg Glu Asn Glu Leu Ser Phe Glu 805 810 815 Asp Gly Ala Val Arg Gly Gly Arg Ala Ser Arg Ile Arg Ala Gly Ser 820 825 830 Ile Lys Phe Asn Val Leu Leu Thr Ser Tyr Glu Leu Ile Ser Ile Asp 835 840 845 Ser Ala Cys Leu Gly Ser Ile Glu Trp Ala Val Leu Val Val Asp Glu 850 855 860 Ala His Arg Leu Lys Ser Asn Gln Ser Lys Phe Phe Lys Ile Leu Asn 865 870 875 880 Ala Tyr Asn Ile Ala Tyr Lys Leu Leu Leu Thr Gly Thr Pro Leu Gln 885 890 895 Asn Asn Leu Glu Glu Leu Phe His Leu Leu Asn Phe Leu Asn Gly Gln 900 905 910 Lys Phe Asn Asp Leu Gln Asn Phe Gln Ala Glu Phe Ala Asp Ile Ser 915 920 925 Lys Glu Asp Gln Val Lys Lys Leu His Glu Met Leu Gly Pro His Met 930 935 940 Leu Arg Arg Leu Lys Ala Asp Val Leu Lys Ser Met Pro Ser Lys Ser 945 950 955 960 Glu Phe Ile Val Arg Val Glu Leu Ser Pro Met Gln Lys Lys Tyr Tyr 965 970 975 Lys Tyr Ile Leu Thr Arg Asn Phe Glu Ala Leu Asn Pro Lys Gly Gly 980 985 990 Gly Gln Ser Val Ser Leu Leu Asn Ile Met Met Asp Leu Lys Lys Cys 995 1000 1005 Cys Asn His Pro Tyr Leu Phe Pro Ala Ala Ser Glu Glu Ala Pro 1010 1015 1020 Leu Gly Pro His Gly Asn Trp Asp Val Gly His Leu Ile Lys Ala 1025 1030 1035 Ser Gly Lys Leu Val Leu Leu Ala Lys Met Leu Lys Ile Leu Arg 1040 1045 1050 Glu Gln Gly His Arg Val Leu Ile Phe Ser Gln Met Thr Lys Met 1055 1060 1065 Leu Asp Ile Met Glu Asp Phe Leu Glu Gly Glu Gly Tyr Lys Tyr 1070 1075 1080 Glu Arg Ile Asp Gly Ala Ile Thr Gly Asn Leu Arg Gln Glu Ala 1085 1090 1095 Ile Asp Arg Phe Asn Ala Pro Gly Ala Pro Gln Phe Val Phe Leu 1100 1105 1110 Leu Ser Thr Lys Ala Gly Gly Leu Gly Ile Asn Leu Ala Thr Ala 1115 1120 1125 Asp Thr Val Ile Ile Tyr Asp Ser Asp Trp Asn Pro His Asn Asp 1130 1135 1140 Ile Gln Ala Phe Ser Arg Ala His Arg Ile Gly Gln Ala Asn Lys 1145 1150 1155 Val Met Ile Tyr Arg Phe Val Thr Arg Asn Ser Val Glu Glu Arg 1160 1165 1170 Val Thr Gln Val Ala Lys Arg Lys Met Met Leu Thr His Leu Val 1175 1180 1185 Val Arg Pro Gly Met Gly Gly Lys Gly Ala Asn Phe Thr Lys Gln 1190 1195 1200 Glu Leu Asp Asp Ile Leu Arg Phe Gly Thr Glu Glu Leu Phe Lys 1205 1210 1215 Glu Ser Glu Gly Lys Glu Asp Glu Ala Ile His Tyr Asp Asp Lys 1220 1225 1230 Ala Val Gly Glu Leu Leu Asp Arg Ser Lys Glu Gly Ile Glu Gln 1235 1240 1245 Lys Glu Ser Trp Ala Asn Glu Tyr Leu Ser Ser Phe Lys Val Ala 1250 1255 1260 Ser Tyr Val Thr Lys Glu Gly Glu Val Glu Glu Glu Val Asp Thr 1265 1270 1275 Glu Ile Ile Lys Gln Glu Ala Glu Asn Thr Asp Pro Ala Tyr Trp 1280 1285 1290 Ile Lys Leu Leu Arg His His Tyr Glu Gln Gln Gln Glu Asp Ile 1295 1300 1305 Ala Arg Thr Leu Gly Lys Gly Lys Arg Val Arg Lys Gln Val Asn 1310 1315 1320 Tyr Asn Asp Gly Gly Met Thr Thr Asp Thr Arg Glu Asp Thr Thr 1325 1330 1335 Trp Gln Glu Asn Leu Ser Asp Tyr His Ser Asp Phe Ser Ala Gly 1340 1345 1350 Ser Asp Glu Asp Lys Glu Asp Asp Asp Phe Asp Glu Lys Asn Asp 1355 1360 1365 Ala Asp Leu Ser Arg Arg Ser Arg Arg Lys Met Glu Arg Lys Asp 1370 1375 1380 Glu Lys Asp Arg Pro Leu Pro Pro Leu Leu Ala Arg Val Gly Gly 1385 1390 1395 Asn Ile Glu Val Leu Gly Phe Asn Ala Arg Gln Arg Lys Ala Phe 1400 1405 1410 Leu Asn Ala Ile Met Arg Tyr Gly Met Pro Pro Gln Asp Ala Phe 1415 1420 1425 Asn Ser Gln Trp Leu Val Arg Asp Leu Arg Gly Lys Ser Glu Lys 1430 1435 1440 Ile Phe Lys Ala Tyr Val Ser Leu Phe Met Arg His Leu Cys Glu 1445 1450 1455 Pro Gly Ala Asp Asn Ala Asp Thr Phe Ala Asp Gly Val Pro Arg 1460 1465 1470 Glu Gly Leu Ser Arg Gln His Val Leu Thr Arg Ile Gly Val Met 1475 1480 1485 Ser Leu Ile Arg Lys Lys Val Gln Glu Phe Glu His Ile Asn Gly 1490 1495 1500 Glu Tyr Ser Met Pro Glu Val Ile Lys Lys Ser Ile Met Asp Gln 1505 1510 1515 Asn Lys Ile Asn Ala Ala Gly Thr Ala Thr Thr Ser Glu Ala Glu 1520 1525 1530 Thr Pro Lys Ser Ala Thr Thr Ser Thr Ser Ala Thr Pro Ala Thr 1535 1540 1545 Ser Ala Ala Pro Ser Pro Ala Pro Thr Gln Gly Glu Asp Lys Asp 1550 1555 1560 Lys Asp Lys Asp Ser Val Gln Ser Asp Glu Asn Lys Asp Lys Glu 1565 1570 1575 Val Val Asn Lys Thr Glu Thr Glu Asp Glu Glu Lys Lys Thr Gly 1580 1585 1590 Glu Ser Ser Thr Glu Lys Pro Lys Thr Glu Pro Glu Glu Val Lys 1595 1600 1605 Glu Ala Ser Pro Lys Thr Glu Thr Pro Glu Ala Ser Ser Glu Ala 1610 1615 1620 Asp Lys Ser Glu Ile Lys Ser Glu Val Asp Thr Ser Ser Val Thr 1625 1630 1635 Ser Glu Glu Lys Lys Glu Glu Lys Glu Glu Glu Ala Lys Lys Glu 1640 1645 1650 Glu Pro Glu Glu Thr Lys Met Glu Ile Gln Glu Glu Glu Leu Val 1655 1660 1665 Lys Glu Glu Lys Lys Glu Glu Glu Asp Asp Lys Lys Lys Glu Glu 1670 1675 1680 Val Lys Lys Glu Val Glu Lys Lys Glu Glu Asp Asp Val Met Val 1685 1690 1695 Ile Asp Asp Asp Lys Asp Lys Lys Asp Lys Lys Glu Ile Asp Leu 1700 1705 1710 Glu Ala Lys Lys Arg Phe Met Phe Asn Ile Ala Asp Gly Gly Phe 1715 1720 1725 Thr Glu Leu His Thr Leu Trp Leu Asn Glu Glu Lys Ala Ala Ser 1730 1735 1740 Pro Gly Arg Glu Tyr Glu Ile Trp His Arg Arg His Asp Tyr Trp 1745 1750 1755 Leu Leu Ala Gly Ile Val Thr His Gly Tyr Gly Arg Trp Gln Asp 1760 1765 1770 Ile Gln Ala Asp Ala Arg Phe Ala Ile Ile Asn Glu Pro Phe Lys 1775 1780 1785 Met Asp Val Gly Lys Gly Asn Phe Leu Glu Ile Lys Asn Lys Phe 1790 1795 1800 Leu Ala Arg Arg Phe Lys Leu Leu Glu Gln Ala Leu Val Ile Glu 1805 1810 1815 Glu Gln Leu Arg Arg Ala Ala Tyr Leu Asn Leu Thr Gln Asp Pro 1820 1825 1830 Asn His Pro Ala Met Ser Leu Asn Ala Arg Phe Ala Glu Val Glu 1835 1840 1845 Cys Leu Ala Glu Ser His Gln His Leu Ser Lys Glu Ser Leu Ala 1850 1855 1860 Gly Asn Lys Pro Ala Asn Ala Val Leu His Lys Val Leu Asn Gln 1865 1870 1875 Leu Glu Glu Leu Leu Ser Asp Met Lys Ser Asp Val Ser Arg Leu 1880 1885 1890 Pro Ala Thr Leu Ala Arg Ile Pro Pro Val Ala Gln Arg Leu Gln 1895 1900 1905 Met Ser Glu Arg Ser Ile Leu Ser Arg Leu Ala Ala Thr Ser Ser 1910 1915 1920 Ser Asn Thr Gln Ser Thr Thr Gln Val Met Ser Gln Phe Pro Pro 1925 1930 1935 Gly Phe Ser Ala Gly Ser Leu Pro Gly Phe Thr Gly Ser Thr Gly 1940 1945 1950 Asn Phe Ser Asn Phe Arg Pro Gln Tyr Ser Val Pro Gly Gln Pro 1955 1960 1965

Pro Ala Gly Phe Pro Ser 1970 813076DNADiabrotica virgifera 81agcggcggca gcacgcagca ggcaacactg gcaacagcag tttttttaac gcgcggtggc 60tgagaattga gaatgctgtt gtaaatttct ttgttaatca aataaaactt tgtttcaaca 120tattgcaaaa ttcatctaaa cgttcaacat gtcacaaact gaaggctcga cagaggcgag 180cgtaagtgcc tcagaaccaa tggaagaagc agagaactcg gaattggctc aaaatgaaga 240atcttcttca gatactacct ctaagggtga agagttcgag gtcaaagtgg cttctgacag 300aggaaaaaga tttgactact tgttgaaaca gactgaaatc ttttcacatt ttatgaacca 360aacaaaatct cccagtaaac caaaaactgg gaggcctaaa aaagagaaga gtgatacatc 420tgatttaaga catcgtaaaa ctgaacaaga agaagatgaa gaacttttag cagaaaccaa 480ccttaaaaca aagactacaa ctcgttttga tgcctcacca ccctacatca aacatgggga 540aatgagagat tatcaagtcc gtggtttgaa ctggatgatt tctttgtatg aacatggcat 600caatggtatt ttagcagatg agatgggttt gggtaaaact ttacaaacca tatctctgct 660tggatatatg aagcactata aaagtacacc tggtcctcat attgtcattg ttcctaaatc 720taccttatca aactggatga atgagttcga gaagtggtgt ccaaccttga gagccgtttg 780tctcattggt gatcaagagg ctaggagctc atttatcaga gatacgatga tgcctggtga 840atgggatgtt tgtgtaacct cgtacgaaat gtgtattaaa gaaaaatctg tatttaaaaa 900gttcaactgg agatatatgg tcattgacga agctcatcgt ataaaaaatg aaaaatctaa 960gctttccgaa attctcaggg agttcaagac tactaacagg ctactgctaa caggtactcc 1020attacaaaac aatttacacg aactctgggc tcttctcaac ttcttactgc cagatgtttt 1080caactcatcg gatgatttcg atgcctggtt caacaccagt caatgtctgg gagacaacgc 1140cttggtcgag agattgcatg ctgtattaaa accattcttg cttagaagat tgaaagctga 1200agtggagaaa cggctaaaac ccaagaagga gttaaaagtg tatgtaggat tgagcaagat 1260gcaacgagaa tggtatacca aagtgctgat gaaggatatt gatatagtga atggtgcagg 1320aaaggtagaa aaaatgcgac tacagaatat tctcatgcag ttaagaaaat gcacaaatca 1380cccctacctt tttgatggcg ctgagcccgg accaccttac acaaccgatg aacatctcgt 1440gtacaattgc ggtaaaatgg tgttgctgga taaactgctt cccaaattga aggaacagga 1500atctcgtgta cttatcttct ctcagatgac ccgtatgttg gatatacttg aagattattg 1560tcattggcga cagtaccaat attgtcgttt ggatggtcaa accccacacg aagacagaca 1620gagacaaatc aacgagtata acgaagacaa tagccaaaag tttatcttta tgttgtcaac 1680tagagccggt ggattgggta tcaatttggc cacagctgat gtagttatta tatatgattc 1740ggattggaat ccccagatgg atctgcaagc catggacaga gcgcatagaa ttggtcagaa 1800gaaacaagtc agagttttca ggtttattac cgaaaacact gtggaagaaa aaatcgtcga 1860aagagctgaa gtaaaattac gtttagacaa attagttatc cagcagggtc gtttagccga 1920ttccaaagca cagactctaa acaaagacga aatgttgaac atgatccggc acggtgccaa 1980ccacgtattt gcttctaagg attccgaaat aacagatgaa gatatcgata gtatattgga 2040aaagggagaa atgaagaccg ctcagctagc tcagaagatg gaaaccatgg gcgaatcgtc 2100acttcgcaac ttcacagtcg aaacacccac tgaatcagtc taccaattcg aaggagaaga 2160ttatcgtgag aagcagaaaa ccatcggctt gagcaactgg atagaacctc ccaaaagaga 2220aaggaaggcc aactatgccg tcgatgctta cttcagagaa gctttaaggg tttctgagcc 2280taaagcgcct aaggctccaa gaccaccaaa acagcccatc gtacaagatt tccagttttt 2340cccgccgaga ttattcgaac ttttggacca ggagatctac ttttacagga aatctttggg 2400atataaggtt ccgaaaaact tagaacttgg acctgacgcg tccaagcaac agaaagaaga 2460gcaaagaaaa atagatgagt cagaaccgct caccgaagac gaacagcaag aaaaagaaaa 2520cttgttaacg caaggtttca ccaattggag taaacgcgat ttcaatcagt tcatcaaagc 2580caacgagaaa tatggtaggg acgatattga gaacatcgcc aaggatgttg aaggcaaaac 2640gcctgaagaa gttatggaat attctgcggt gttttgggaa agatgtcatg aattacagga 2700tattgataga ataatggccc agattgagag aggagaaact aaaatacaaa gaagagctag 2760tattaagaag gcacttgatg ctaaaatggc aagatatcgt gcaccattcc atcagctgag 2820aatttcttac ggcaccaaca aaggcaagaa ctacatggag gacgaagaca ggtttttggt 2880gtgtatgttg cacaagttgg gtttcgatag agaaaacgtt tatgaagagt taagagcagc 2940tgtacgtgcg tcaccacaat tcagatttga ttggttctta aaatcgagaa ctgccatgga 3000gctgcaaagg agatgcaaca cattgataac gttaatagaa agagaaaatg ctgaattgga 3060ggaaagagaa aaaatt 307682976PRTDiabrotica virgifera 82Met Ser Gln Thr Glu Gly Ser Thr Glu Ala Ser Val Ser Ala Ser Glu 1 5 10 15 Pro Met Glu Glu Ala Glu Asn Ser Glu Leu Ala Gln Asn Glu Glu Ser 20 25 30 Ser Ser Asp Thr Thr Ser Lys Gly Glu Glu Phe Glu Val Lys Val Ala 35 40 45 Ser Asp Arg Gly Lys Arg Phe Asp Tyr Leu Leu Lys Gln Thr Glu Ile 50 55 60 Phe Ser His Phe Met Asn Gln Thr Lys Ser Pro Ser Lys Pro Lys Thr 65 70 75 80 Gly Arg Pro Lys Lys Glu Lys Ser Asp Thr Ser Asp Leu Arg His Arg 85 90 95 Lys Thr Glu Gln Glu Glu Asp Glu Glu Leu Leu Ala Glu Thr Asn Leu 100 105 110 Lys Thr Lys Thr Thr Thr Arg Phe Asp Ala Ser Pro Pro Tyr Ile Lys 115 120 125 His Gly Glu Met Arg Asp Tyr Gln Val Arg Gly Leu Asn Trp Met Ile 130 135 140 Ser Leu Tyr Glu His Gly Ile Asn Gly Ile Leu Ala Asp Glu Met Gly 145 150 155 160 Leu Gly Lys Thr Leu Gln Thr Ile Ser Leu Leu Gly Tyr Met Lys His 165 170 175 Tyr Lys Ser Thr Pro Gly Pro His Ile Val Ile Val Pro Lys Ser Thr 180 185 190 Leu Ser Asn Trp Met Asn Glu Phe Glu Lys Trp Cys Pro Thr Leu Arg 195 200 205 Ala Val Cys Leu Ile Gly Asp Gln Glu Ala Arg Ser Ser Phe Ile Arg 210 215 220 Asp Thr Met Met Pro Gly Glu Trp Asp Val Cys Val Thr Ser Tyr Glu 225 230 235 240 Met Cys Ile Lys Glu Lys Ser Val Phe Lys Lys Phe Asn Trp Arg Tyr 245 250 255 Met Val Ile Asp Glu Ala His Arg Ile Lys Asn Glu Lys Ser Lys Leu 260 265 270 Ser Glu Ile Leu Arg Glu Phe Lys Thr Thr Asn Arg Leu Leu Leu Thr 275 280 285 Gly Thr Pro Leu Gln Asn Asn Leu His Glu Leu Trp Ala Leu Leu Asn 290 295 300 Phe Leu Leu Pro Asp Val Phe Asn Ser Ser Asp Asp Phe Asp Ala Trp 305 310 315 320 Phe Asn Thr Ser Gln Cys Leu Gly Asp Asn Ala Leu Val Glu Arg Leu 325 330 335 His Ala Val Leu Lys Pro Phe Leu Leu Arg Arg Leu Lys Ala Glu Val 340 345 350 Glu Lys Arg Leu Lys Pro Lys Lys Glu Leu Lys Val Tyr Val Gly Leu 355 360 365 Ser Lys Met Gln Arg Glu Trp Tyr Thr Lys Val Leu Met Lys Asp Ile 370 375 380 Asp Ile Val Asn Gly Ala Gly Lys Val Glu Lys Met Arg Leu Gln Asn 385 390 395 400 Ile Leu Met Gln Leu Arg Lys Cys Thr Asn His Pro Tyr Leu Phe Asp 405 410 415 Gly Ala Glu Pro Gly Pro Pro Tyr Thr Thr Asp Glu His Leu Val Tyr 420 425 430 Asn Cys Gly Lys Met Val Leu Leu Asp Lys Leu Leu Pro Lys Leu Lys 435 440 445 Glu Gln Glu Ser Arg Val Leu Ile Phe Ser Gln Met Thr Arg Met Leu 450 455 460 Asp Ile Leu Glu Asp Tyr Cys His Trp Arg Gln Tyr Gln Tyr Cys Arg 465 470 475 480 Leu Asp Gly Gln Thr Pro His Glu Asp Arg Gln Arg Gln Ile Asn Glu 485 490 495 Tyr Asn Glu Asp Asn Ser Gln Lys Phe Ile Phe Met Leu Ser Thr Arg 500 505 510 Ala Gly Gly Leu Gly Ile Asn Leu Ala Thr Ala Asp Val Val Ile Ile 515 520 525 Tyr Asp Ser Asp Trp Asn Pro Gln Met Asp Leu Gln Ala Met Asp Arg 530 535 540 Ala His Arg Ile Gly Gln Lys Lys Gln Val Arg Val Phe Arg Phe Ile 545 550 555 560 Thr Glu Asn Thr Val Glu Glu Lys Ile Val Glu Arg Ala Glu Val Lys 565 570 575 Leu Arg Leu Asp Lys Leu Val Ile Gln Gln Gly Arg Leu Ala Asp Ser 580 585 590 Lys Ala Gln Thr Leu Asn Lys Asp Glu Met Leu Asn Met Ile Arg His 595 600 605 Gly Ala Asn His Val Phe Ala Ser Lys Asp Ser Glu Ile Thr Asp Glu 610 615 620 Asp Ile Asp Ser Ile Leu Glu Lys Gly Glu Met Lys Thr Ala Gln Leu 625 630 635 640 Ala Gln Lys Met Glu Thr Met Gly Glu Ser Ser Leu Arg Asn Phe Thr 645 650 655 Val Glu Thr Pro Thr Glu Ser Val Tyr Gln Phe Glu Gly Glu Asp Tyr 660 665 670 Arg Glu Lys Gln Lys Thr Ile Gly Leu Ser Asn Trp Ile Glu Pro Pro 675 680 685 Lys Arg Glu Arg Lys Ala Asn Tyr Ala Val Asp Ala Tyr Phe Arg Glu 690 695 700 Ala Leu Arg Val Ser Glu Pro Lys Ala Pro Lys Ala Pro Arg Pro Pro 705 710 715 720 Lys Gln Pro Ile Val Gln Asp Phe Gln Phe Phe Pro Pro Arg Leu Phe 725 730 735 Glu Leu Leu Asp Gln Glu Ile Tyr Phe Tyr Arg Lys Ser Leu Gly Tyr 740 745 750 Lys Val Pro Lys Asn Leu Glu Leu Gly Pro Asp Ala Ser Lys Gln Gln 755 760 765 Lys Glu Glu Gln Arg Lys Ile Asp Glu Ser Glu Pro Leu Thr Glu Asp 770 775 780 Glu Gln Gln Glu Lys Glu Asn Leu Leu Thr Gln Gly Phe Thr Asn Trp 785 790 795 800 Ser Lys Arg Asp Phe Asn Gln Phe Ile Lys Ala Asn Glu Lys Tyr Gly 805 810 815 Arg Asp Asp Ile Glu Asn Ile Ala Lys Asp Val Glu Gly Lys Thr Pro 820 825 830 Glu Glu Val Met Glu Tyr Ser Ala Val Phe Trp Glu Arg Cys His Glu 835 840 845 Leu Gln Asp Ile Asp Arg Ile Met Ala Gln Ile Glu Arg Gly Glu Thr 850 855 860 Lys Ile Gln Arg Arg Ala Ser Ile Lys Lys Ala Leu Asp Ala Lys Met 865 870 875 880 Ala Arg Tyr Arg Ala Pro Phe His Gln Leu Arg Ile Ser Tyr Gly Thr 885 890 895 Asn Lys Gly Lys Asn Tyr Met Glu Asp Glu Asp Arg Phe Leu Val Cys 900 905 910 Met Leu His Lys Leu Gly Phe Asp Arg Glu Asn Val Tyr Glu Glu Leu 915 920 925 Arg Ala Ala Val Arg Ala Ser Pro Gln Phe Arg Phe Asp Trp Phe Leu 930 935 940 Lys Ser Arg Thr Ala Met Glu Leu Gln Arg Arg Cys Asn Thr Leu Ile 945 950 955 960 Thr Leu Ile Glu Arg Glu Asn Ala Glu Leu Glu Glu Arg Glu Lys Ile 965 970 975 834137DNADiabrotica virgifera 83atggaaggct cagagtcgga aaattcagct tctggttccg gttcagaaaa tgagagcaaa 60agtgattcca gtaacaactc tggaagcgct tctggtagtt cttcttcgga cggttccgat 120acaggcgaag aaactgtaga aaatggatcg gccaataaaa gtagtagtaa aagtgacgga 180gaagaagtac ttgaagaaag taacgggtgt agccaagatt caaactctat acctcctgct 240agtccagata gttcgagtaa attcgatact accaaagatt taagttctga taggagttcg 300gatccctcta gcatcagacg atcagttagg tcgcgaagag agccggaaag acttcagagt 360aaagacagtg atagggcttc gagtgataaa agcaacaaaa gtgctgaaga ttggaaatac 420aatgacgcta gttcgtcaga gtctgaacca gaagtaaaag aacgcccccc acctagtaaa 480cgcgtaggtg ccagagcgcg aacgactgtg ataaaaaaga aaaagtccaa gaaaagaagc 540caatacagtt cagaagatga ggaaacgagc gacgaaagcg atgaggatag taggagagct 600gtgtccagaa ggaaggctac tacagttagt tacaaggaag aaagtgagga tgagaaaacg 660gattccgagg atttgctaga agttgataat aatgaaccgg tagaacctgt cccggaagaa 720aaatgtgaaa caatagaaag aattttggca acgagaagag gaaaaattgg agttaccgga 780aacattacta cagtctacta tgtagaagaa aatggtgatc cgaatgaagg agttgatgaa 840aaggatttag atagtacaga agatcagtat ctaatcaaat ggaaagattg ggctcatatt 900cacaacacat gggaatcaga caaaagttta cgagaacaga aagtaaaggg gatgaaaaaa 960ttggaaaatt atatcaaaaa agaagtcgaa attcaacagt ggcttaaata ttctactcct 1020gaggatgtgg aatattatga atgtcaaatg gagttatctc aggatctttt gaagagtttc 1080aacgaggtcg agaggataat agcaaagtac aataagcctg atgggggtaa agattattat 1140attaaatggc aaagtcttcc atatgctgaa tcgacttggg aagattcagt tctaattcaa 1200cgaaaatggc ctgaccaaat aaatgaattc gaagctaggg agcaatcaag tatgacccca 1260acgagacact gtaaagtact caaacataga cccaaattcc acgaagtcaa gacccagcct 1320gaatatatga tgggcaaaga acagactttg atactgcgtg attaccaaat acatggtctc 1380aactggatga tacattcctg gtcaaaagaa aactctgtta tattagcaga cgagatgggg 1440ctcggtaaaa cgattcagac aatttgcttt ctatactatc tcttcaatac tcaccacctc 1500cacggaccat ttttgtgtgt tgtgcccctt tctacaatga cgtcgtggca gagggaaatg 1560acacagtggg cacccgactt gaactttgtc acatacttgg gagatgttca gtccagagat 1620acgattcgcc aatatgaatg gtgctttgaa gggtcaaaaa ggctaaagtt caatgcaatt 1680ctcacaacgt atgaaattgt tttgaaggat aaagcatttt taggaagtct cagctgggct 1740gtgttactag tagatgaagc tcacaggttg aaaaacgatg attctttgtt gtacaaagct 1800ttaatggaat ttgacactaa tcacaggctt cttattactg gtactccttt acaaaatagt 1860ttaaaagaac tttgggcgct gctacatttt atcatgcccg ctaagtttga aacatgggac 1920gaattcaaaa gagaacacga aaacaccaca aactccacaa actataccaa actccacaaa 1980caacttgaac cgtttatttt aagacgggta aagaaagatg tagaaaaatc tctccccgct 2040aaagtagaac aaattcttag ggtagagatg acgtctatcc agaaacaata ctataagtgg 2100atattaacaa aaaattataa tgccttgaga agaggagtca aaggatccac aacaaccttc 2160ttaaatattg tgatagaact gaagaaatgt tgtaaccatt cgtctttgat caagccccca 2220gatattgaaa cacaatacaa tcaacacgac gttttgcagc aacttctcag aggttcggga 2280aaattagtgc ttctggataa attgcttatc cgtttgcgca atactggcca tagagtacta 2340atcttctcgc agatggtccg gatgttggac attcttgccg aatatttgca gcttcgacat 2400ttcccgtttc agaggctaga tggtggcatc aagggagagc tgcgacgtca agccttagat 2460catttcaatg ctgaagggtc tcaagatttt tgcttccttc tttcaactcg cgcagggggc 2520ttgggcatta atttagctac tgctgatact gtgataattt ttgattcgga ctggaatcct 2580caaaacgatc ttcaagcgca ggcaagagct cataggatcg gtcaaaagaa ccaagtcaac 2640atttataggt tagttactgc tagatctgta gaggaagaaa ttgtagaaag ggcaaaacaa 2700aaaatggtac tggatcatct tgtaattcag agaatggaca cgacgggaag aaccgttttg 2760gacaaaaagg ggtcttctaa taataatccg tttaacaaag aagatctgac ggcgattttg 2820aaatttggag ctgaggaatt atttaaagat gaagatgacg atgaggaacc aaactgtgat 2880attgacgaaa ttcttcgacg agctgagacc agagatgaag ctccttcatt ggttggagat 2940gaactacttt cggcatttaa agtagcaagt ttcgccgctt ttgacgaaga tgccgagccc 3000tcaccagtca acaatgttgt taacgacgat gaaagtaaag actgggatga aattattcca 3060gaaaaacttc gtatcaaggc agaggaagag gaaaagaaca aggaaatgga agatctttat 3120cttcctccgc gaagtcgaaa aactcttcaa cagattaatc aatctgaaag tgacggggaa 3180gaaggcaaag gtaggaagaa aacgaagaaa gatggagatg aatcgggagg ttccagtggc 3240gatgatgaca ctgacgagga aaaacctaaa aaacgaggaa ggccaccagc aaaccccaga 3300gaaaagttca agaacttcac tgatgctgag attagaaggt ttataaaaag ttataagaaa 3360tttagtggac ccttaaagcg attagaggca gttgcttgtg atgctgaatt gcaagaaaaa 3420ccattagctg agttacggaa attgggagaa cttcttcatg agaggtgcag ggcatttatg 3480aatgaacaag ctaaagaaaa tacagagtct aacactcaag acgaacccaa aggtcgcaaa 3540agaggaccat cgtttaaaat tggaggagtg tctgtaaatg ccaaaacgat gatggcttgt 3600gaggaagagt tagaaccatt agatgaagtc attccagctg atccaaatga acgattacgt 3660tgggtttttg atgtaaaaac gaagtcgtct cactttgatg tggactgggg tatggaagag 3720gacactaagt tattgaaagg aatttatcac tatggtcttg gctcatggga gcaaataaaa 3780ttggatccat tattaggcat tggtgataaa attttcctta ataatgaaga taaaaagccg 3840caggctaaac atcttttatc aagagcagaa tacttattaa agattatgaa aaagcaatta 3900gatctaaaga agggggttca aaaaccaaaa agacagagga aaaaagaaca aaaagttctt 3960actaaggaaa ttattgatga cgatgaaagc tcaaatgatg tttcatcatt accaagttcc 4020gctccagtta cagtatcagt agctccggtt gttaaaaagg taaagaaaga agtgaaaaaa 4080gaaaaggagg ataaagaaga atcctcgccc gagaaaaagg aaaaaaaaga aggataa 4137841724PRTDiabrotica virgifera 84Met Glu Gly Ser Glu Ser Glu Asn Ser Ala Ser Gly Ser Gly Ser Glu 1 5 10 15 Asn Glu Ser Lys Ser Asp Ser Ser Asn Asn Ser Gly Ser Ala Ser Gly 20 25 30 Ser Ser Ser Ser Asp Gly Ser Asp Thr Gly Glu Glu Thr Val Glu Asn 35 40 45 Gly Ser Ala Asn Lys Ser Ser Ser Lys Ser Asp Gly Glu Glu Val Leu 50 55 60 Glu Glu Ser Asn Gly Cys Ser Gln Asp Ser Asn Ser Ile Pro Pro Ala 65 70 75 80 Ser Pro Asp Ser Ser Ser Lys Phe Asp Thr Thr Lys Asp Leu Ser Ser 85 90 95 Asp Arg Ser Ser Asp Pro Ser Ser Ile Arg Arg Ser Val Arg Ser Arg 100 105 110 Arg Glu Pro Glu Arg Leu Gln Ser Lys Asp Ser Asp Arg Ala Ser Ser 115 120 125 Asp Lys Ser Asn Lys Ser Ala Glu Asp Trp Lys Tyr Asn Asp Ala Ser 130 135 140 Ser Ser Glu Ser Glu Pro Glu Val Lys Glu Arg Pro Pro Pro Ser Lys 145 150 155 160 Arg Val Gly Ala Arg Ala Arg Thr Thr Val Ile Lys Lys Lys Lys Ser 165 170 175 Lys Lys Arg Ser Gln Tyr Ser Ser Glu Asp Glu Glu Thr Ser Asp Glu 180 185

190 Ser Asp Glu Asp Ser Arg Arg Ala Val Ser Arg Arg Lys Ala Thr Thr 195 200 205 Val Ser Tyr Lys Glu Glu Ser Glu Asp Glu Lys Thr Asp Ser Glu Asp 210 215 220 Leu Leu Glu Val Asp Asn Asn Glu Pro Val Glu Pro Val Pro Glu Glu 225 230 235 240 Lys Cys Glu Thr Ile Glu Arg Ile Leu Ala Thr Arg Arg Gly Lys Ile 245 250 255 Gly Val Thr Gly Asn Ile Thr Thr Val Tyr Tyr Val Glu Glu Asn Gly 260 265 270 Asp Pro Asn Glu Gly Val Asp Glu Lys Asp Leu Asp Ser Thr Glu Asp 275 280 285 Gln Tyr Leu Ile Lys Trp Lys Asp Trp Ala His Ile His Asn Thr Trp 290 295 300 Glu Ser Asp Lys Ser Leu Arg Glu Gln Lys Val Lys Gly Met Lys Lys 305 310 315 320 Leu Glu Asn Tyr Ile Lys Lys Glu Val Glu Ile Gln Gln Trp Leu Lys 325 330 335 Tyr Ser Thr Pro Glu Asp Val Glu Tyr Tyr Glu Cys Gln Met Glu Leu 340 345 350 Ser Gln Asp Leu Leu Lys Ser Phe Asn Glu Val Glu Arg Ile Ile Ala 355 360 365 Lys Tyr Asn Lys Pro Asp Gly Gly Lys Asp Tyr Tyr Ile Lys Trp Gln 370 375 380 Ser Leu Pro Tyr Ala Glu Ser Thr Trp Glu Asp Ser Val Leu Ile Gln 385 390 395 400 Arg Lys Trp Pro Asp Gln Ile Asn Glu Phe Glu Ala Arg Glu Gln Ser 405 410 415 Ser Met Thr Pro Thr Arg His Cys Lys Val Leu Lys His Arg Pro Lys 420 425 430 Phe His Glu Val Lys Thr Gln Pro Glu Tyr Met Met Gly Lys Glu Gln 435 440 445 Thr Leu Ile Leu Arg Asp Tyr Gln Ile His Gly Leu Asn Trp Met Ile 450 455 460 His Ser Trp Ser Lys Glu Asn Ser Val Ile Leu Ala Asp Glu Met Gly 465 470 475 480 Leu Gly Lys Thr Ile Gln Thr Ile Cys Phe Leu Tyr Tyr Leu Phe Asn 485 490 495 Thr His His Leu His Gly Pro Phe Leu Cys Val Val Pro Leu Ser Thr 500 505 510 Met Thr Ser Trp Gln Arg Glu Met Thr Gln Trp Ala Pro Asp Leu Asn 515 520 525 Phe Val Thr Tyr Leu Gly Asp Val Gln Ser Arg Asp Thr Ile Arg Gln 530 535 540 Tyr Glu Trp Cys Phe Glu Gly Ser Lys Arg Leu Lys Phe Asn Ala Ile 545 550 555 560 Leu Thr Thr Tyr Glu Ile Val Leu Lys Asp Lys Ala Phe Leu Gly Ser 565 570 575 Leu Ser Trp Ala Val Leu Leu Val Asp Glu Ala His Arg Leu Lys Asn 580 585 590 Asp Asp Ser Leu Leu Tyr Lys Ala Leu Met Glu Phe Asp Thr Asn His 595 600 605 Arg Leu Leu Ile Thr Gly Thr Pro Leu Gln Asn Ser Leu Lys Glu Leu 610 615 620 Trp Ala Leu Leu His Phe Ile Met Pro Ala Lys Phe Glu Thr Trp Asp 625 630 635 640 Glu Phe Lys Arg Glu His Glu Asn Thr Thr Asn Ser Thr Asn Tyr Thr 645 650 655 Lys Leu His Lys Gln Leu Glu Pro Phe Ile Leu Arg Arg Val Lys Lys 660 665 670 Asp Val Glu Lys Ser Leu Pro Ala Lys Val Glu Gln Ile Leu Arg Val 675 680 685 Glu Met Thr Ser Ile Gln Lys Gln Tyr Tyr Lys Trp Ile Leu Thr Lys 690 695 700 Asn Tyr Asn Ala Leu Arg Arg Gly Val Lys Gly Ser Thr Thr Thr Phe 705 710 715 720 Leu Asn Ile Val Ile Glu Leu Lys Lys Cys Cys Asn His Ser Ser Leu 725 730 735 Ile Lys Pro Pro Asp Ile Glu Thr Gln Tyr Asn Gln His Asp Val Leu 740 745 750 Gln Gln Leu Leu Arg Gly Ser Gly Lys Leu Val Leu Leu Asp Lys Leu 755 760 765 Leu Ile Arg Leu Arg Asn Thr Gly His Arg Val Leu Ile Phe Ser Gln 770 775 780 Met Val Arg Met Leu Asp Ile Leu Ala Glu Tyr Leu Gln Leu Arg His 785 790 795 800 Phe Pro Phe Gln Arg Leu Asp Gly Gly Ile Lys Gly Glu Leu Arg Arg 805 810 815 Gln Ala Leu Asp His Phe Asn Ala Glu Gly Ser Gln Asp Phe Cys Phe 820 825 830 Leu Leu Ser Thr Arg Ala Gly Gly Leu Gly Ile Asn Leu Ala Thr Ala 835 840 845 Asp Thr Val Ile Ile Phe Asp Ser Asp Trp Asn Pro Gln Asn Asp Leu 850 855 860 Gln Ala Gln Ala Arg Ala His Arg Ile Gly Gln Lys Asn Gln Val Asn 865 870 875 880 Ile Tyr Arg Leu Val Thr Ala Arg Ser Val Glu Glu Glu Ile Val Glu 885 890 895 Arg Ala Lys Gln Lys Met Val Leu Asp His Leu Val Ile Gln Arg Met 900 905 910 Asp Thr Thr Gly Arg Thr Val Leu Asp Lys Lys Gly Ser Ser Asn Asn 915 920 925 Asn Pro Phe Asn Lys Glu Asp Leu Thr Ala Ile Leu Lys Phe Gly Ala 930 935 940 Glu Glu Leu Phe Lys Asp Glu Asp Asp Asp Glu Glu Pro Asn Cys Asp 945 950 955 960 Ile Asp Glu Ile Leu Arg Arg Ala Glu Thr Arg Asp Glu Ala Pro Ser 965 970 975 Leu Val Gly Asp Glu Leu Leu Ser Ala Phe Lys Val Ala Ser Phe Ala 980 985 990 Ala Phe Asp Glu Asp Ala Glu Pro Ser Pro Val Asn Asn Val Val Asn 995 1000 1005 Asp Asp Glu Ser Lys Asp Trp Asp Glu Ile Ile Pro Glu Lys Leu 1010 1015 1020 Arg Ile Lys Ala Glu Glu Glu Glu Lys Asn Lys Glu Met Glu Asp 1025 1030 1035 Leu Tyr Leu Pro Pro Arg Ser Arg Lys Thr Leu Gln Gln Ile Asn 1040 1045 1050 Gln Ser Glu Ser Asp Gly Glu Glu Gly Lys Gly Arg Lys Lys Thr 1055 1060 1065 Lys Lys Asp Gly Asp Glu Ser Gly Gly Ser Ser Gly Asp Asp Asp 1070 1075 1080 Thr Asp Glu Glu Lys Pro Lys Lys Arg Gly Arg Pro Pro Ala Asn 1085 1090 1095 Pro Arg Glu Lys Phe Lys Asn Phe Thr Asp Ala Glu Ile Arg Arg 1100 1105 1110 Phe Ile Lys Ser Tyr Lys Lys Phe Ser Gly Pro Leu Lys Arg Leu 1115 1120 1125 Glu Ala Val Ala Cys Asp Ala Glu Leu Gln Glu Lys Pro Leu Ala 1130 1135 1140 Glu Leu Arg Lys Leu Gly Glu Leu Leu His Glu Arg Cys Arg Ala 1145 1150 1155 Phe Met Asn Glu Gln Ala Lys Glu Asn Thr Glu Ser Asn Thr Gln 1160 1165 1170 Asp Glu Pro Lys Gly Arg Lys Arg Gly Pro Ser Phe Lys Ile Gly 1175 1180 1185 Gly Val Ser Val Asn Ala Lys Thr Met Met Ala Cys Glu Glu Glu 1190 1195 1200 Leu Glu Pro Leu Asp Glu Val Ile Pro Ala Asp Pro Asn Glu Arg 1205 1210 1215 Leu Arg Trp Val Phe Asp Val Lys Thr Lys Ser Ser His Phe Asp 1220 1225 1230 Val Asp Trp Gly Met Glu Glu Asp Thr Lys Leu Leu Lys Gly Ile 1235 1240 1245 Tyr His Tyr Gly Leu Gly Ser Trp Glu Gln Ile Lys Leu Asp Pro 1250 1255 1260 Leu Leu Gly Ile Gly Asp Lys Ile Phe Leu Asn Asn Glu Asp Lys 1265 1270 1275 Lys Pro Gln Ala Lys His Leu Leu Ser Arg Ala Glu Tyr Leu Leu 1280 1285 1290 Lys Ile Met Lys Lys Gln Leu Asp Leu Lys Lys Gly Val Gln Lys 1295 1300 1305 Pro Lys Arg Gln Arg Lys Lys Glu Gln Lys Val Leu Thr Lys Glu 1310 1315 1320 Ile Ile Asp Asp Asp Glu Ser Ser Asn Asp Val Ser Ser Leu Pro 1325 1330 1335 Ser Ser Ala Pro Val Thr Val Ser Val Ala Pro Val Val Lys Lys 1340 1345 1350 Val Lys Lys Glu Val Lys Lys Glu Lys Glu Asp Lys Glu Glu Ser 1355 1360 1365 Ser Pro Glu Lys Lys Glu Lys Lys Lys Asp Lys Lys Lys Glu Lys 1370 1375 1380 Lys Thr Ser Gly Pro Met His Phe Ser Thr Asn Glu Pro Val Ala 1385 1390 1395 Leu Asn Val Leu Gly Asp Leu Asp Pro Leu Ile Phe Asn Glu Cys 1400 1405 1410 Lys Glu Lys Met Arg Pro Val Lys Lys Ala Leu Lys Ala Leu Asp 1415 1420 1425 Asn Pro Asp Glu Ser Leu Pro Glu Ala Glu Gln Val Gln His Thr 1430 1435 1440 Arg Asp Cys Leu Leu Gln Ile Gly Glu Gln Ile Asn Thr Cys Leu 1445 1450 1455 Leu Lys Tyr Thr Asp Pro Glu Lys Ile Lys Glu Trp Arg Ser Asn 1460 1465 1470 Leu Trp Tyr Phe Val Ser Lys Phe Thr Glu Tyr Asp Ala Lys Lys 1475 1480 1485 Leu Tyr Lys Leu Tyr Lys Lys Ala Cys Lys Lys Thr Asp Lys Ile 1490 1495 1500 Glu Thr Lys Lys Glu Lys Lys Ala Glu Lys Arg Ala Glu His Leu 1505 1510 1515 Glu Lys Glu Arg Glu Glu Thr Ala Ser Thr Ser Ala Asp Lys Val 1520 1525 1530 Lys Lys Ile Lys Ile Pro Arg Thr Glu Lys Lys Asp Ala Lys Glu 1535 1540 1545 His Lys Arg Lys His Asp Ser Asp Ser Glu Glu Ser Pro Lys Lys 1550 1555 1560 His Arg Ser Glu Lys Lys Glu Arg Arg Ala Lys Glu Lys Lys Arg 1565 1570 1575 Ser Arg Asp Glu Thr Ser Glu Asp Asp Gln Glu Tyr Arg Phe Asn 1580 1585 1590 Arg His Arg Lys Pro Gly Asn Tyr Arg His Asp Gln His Ala Pro 1595 1600 1605 Gln Asp Arg Trp Ser Gly Gly Gln Gln Glu Arg Phe Ser Gly Asp 1610 1615 1620 His Lys Arg Gln Gln Asp Tyr His Arg Gly Ala Gly Phe His Arg 1625 1630 1635 Asp Arg Asp Tyr Gln Arg Tyr Asp Lys Gly Ser Pro Glu Lys Asn 1640 1645 1650 Asn Asp Trp Arg Gln Tyr Pro Arg Gly Arg Glu Ile Met Pro Pro 1655 1660 1665 Met Pro Gly Gly Gln Pro Met Gly Met Gly Gly Gly Tyr Tyr Pro 1670 1675 1680 Pro Asn Tyr Asn Gln Gly Gln Gly Tyr Ala Pro Glu Gln Pro Phe 1685 1690 1695 Pro Pro Arg Asp Arg Phe Pro Leu Gly Asp Trp Arg Pro Pro Asp 1700 1705 1710 Arg Gly Gly Tyr Arg Tyr Asp Arg Arg Gln Gln 1715 1720 85767DNADiabrotica virgifera 85tacgagatgt tcatcggttg tgtaaggtgg tccgggctca gcgccatcaa aaaggtaggg 60gtgattggtg cattttctca actgcatgaa aatattctgt agccgcattt ttgccacctg 120tgcaccattc agtatatcaa tatccttcat tagcactttg gtattaccat tctcgctgca 180ttttgctcaa tcctacatac acttttaact ccttcttggg ttttagccct ttctctactt 240caattttcaa tcttctaagc aagaatggtt ttaatacagc atgcagcctc tccaccatgg 300agttgcctcc cagacattga ctggtgttaa accaggcatc gaaatcatca gatgagttaa 360aaacgtctgg cagtaagaag ttgagaagag accagagttc atgtaaattg ttttgtaatg 420gagtacctgt tagcagtagc ctgttggtat tcttgaattc cctgagaatt tcggaaagct 480tagtcttttt cattttttat acgatgagct tcatcaacga ccagatatct ccacttgaac 540ttttttaata gagatttttc tttaatacac atttcatacg aggttataca gacatcccat 600tcaccaggca tcatcgtatc tctgataatt gagttcctag cctcttgatc gccaatgaga 660caaacagctc tcaaggttgg acaccactgc tggaactcat tcatccaatt tgataaggta 720gatttaggaa caacgacaaa tatgaggacc aggtgtactt ttatagt 76786110PRTDiabrotica virgifera 86Lys Met Lys Lys Thr Lys Leu Ser Glu Ile Leu Arg Glu Phe Lys Asn 1 5 10 15 Thr Asn Arg Leu Leu Leu Thr Gly Thr Pro Leu Gln Asn Asn Leu His 20 25 30 Glu Leu Trp Ser Leu Leu Asn Phe Leu Leu Pro Asp Val Phe Asn Ser 35 40 45 Ser Asp Asp Phe Asp Ala Trp Phe Asn Thr Ser Gln Cys Leu Gly Gly 50 55 60 Asn Ser Met Val Glu Arg Leu His Ala Val Leu Lys Pro Phe Leu Leu 65 70 75 80 Arg Arg Leu Lys Ile Glu Val Glu Lys Gly Leu Lys Pro Lys Lys Glu 85 90 95 Leu Lys Val Tyr Val Gly Leu Ser Lys Met Gln Arg Glu Trp 100 105 110 873074DNADiabrotica virgifera 87ctttgttaat caaataaaac tttgtttcaa catattgcaa aattcatcta aacgttcaac 60atgtcacaaa ctgaaggctc gacagaggcg agcgtaagtg cctcagaacc aatggaagaa 120gcagagaact cggaattggc tcaaaatgaa gaatcttctt cagatactac ctctaagggt 180gaagagttcg aggtcaaagt ggcttctgac agaggaaaaa gatttgacta cttgttgaaa 240cagactgaaa tcttttcaca ttttatgaac caaacaaaat ctcccagtaa accaaaaact 300gggaggccta aaaaagagaa gagtgataca tctgatttaa gacatcgtaa aactgaacaa 360gaagaagatg aagaactttt agcagaaacc aaccttaaaa caaagactac aactcgtttt 420gatgcctcac caccctacat caaacatggg gaaatgagag attatcaagt ccgtggtttg 480aactggatga tttctttgta tgaacatggc atcaatggta ttttagcaga tgagatgggt 540ttgggtaaaa ctttacaaac catatctctg cttggatata tgaagcacta taaaagtaca 600cctggtcctc atattgtcat tgttcctaaa tctaccttat caaactggat gaatgagttc 660gagaagtggt gtccaacctt gagagccgtt tgtctcattg gtgatcaaga ggctaggagc 720tcatttatca gagatacgat gatgcctggt gaatgggatg tttgtgtaac ctcgtacgaa 780atgtgtatta aagaaaaatc tgtatttaaa aagttcaact ggagatatat ggtcattgac 840gaagctcatc gtataaaaaa tgaaaaatct aagctttccg aaattctcag ggagttcaag 900actactaaca ggctactgct aacaggtact ccattacaaa acaatttaca cgaactctgg 960gctcttctca acttcttact gccagatgtt ttcaactcat cggatgattt cgatgcctgg 1020ttcaacacca gtcaatgtct gggagacaac gccttggtcg agagattgca tgctgtatta 1080aaaccattct tgcttagaag attgaaagct gaagtggaga aacggctaaa acccaagaag 1140gagttaaaag tgtatgtagg attgagcaag atgcaacgag aatggtatac caaagtgctg 1200atgaaggata ttgatatagt gaatggtgca ggaaaggtag aaaaaatgcg actacagaat 1260attctcatgc agttaagaaa atgcacaaat cacccctacc tttttgatgg cgctgagccc 1320ggaccacctt acacaaccga tgaacatctc gtgtacaatt gcggtaaaat ggtgttgctg 1380gataaactgc ttcccaaatt gaaggaacag gaatctcgtg tacttatctt ctctcagatg 1440acccgtatgt tggatatact tgaagattac tgtcattggc gacagtacca atattgtcgt 1500ttggatggtc aaaccccaca cgaagacaga cagagacaaa tcaacgagta taacgaagac 1560aatagccaaa agtttatctt tatgttgtca actagagccg gtggattggg tatcaatttg 1620gccacagctg atgtagttat tatatatgat tcggattgga atccccagat ggatctgcaa 1680gccatggaca gagcgcatag aattggtcag aagaaacaag tcagagtttt caggtttatt 1740accgaaaaca ctgtggaaga aaaaatcgtc gaaagagctg aagtaaaatt acgtttagac 1800aaattagtta tccagcaggg tcgtttagcc gattccaaag cacagactct aaacaaagac 1860gaaatgttga acatgatccg gcacggtgcc aaccacgtat ttgcttctaa ggattccgaa 1920ataacagatg aagatatcga tagtatattg gaaaagggag aaatgaagac cgctcagcta 1980gctcagaaga tggaaaccat gggcgaatcg tcacttcgca acttcacagt cgaaacaccc 2040actgaatcag tctaccaatt cgaaggagaa gattatcgtg agaagcagaa aaccatcggc 2100ttgagcaact ggatagaacc tcccaaaaga gaaaggaagg ccaactatgc cgtcgatgct 2160tacttcagag aagctttaag ggtttctgag cctaaagcgc ctaaggctcc aagaccacca 2220aaacagccca tcgtacaaga tttccagttt ttcccgccga gattattcga acttttggac 2280caggagatct acttttacag gaaatctttg ggatataagg ttccgaaaaa cttagaactt 2340ggacctgacg cgtccaagca acagaaagaa gagcaaagaa aaatagatga gtcagaaccg 2400ctcaccgaag acgaacagca agaaaaagaa aacttgttaa cgcaaggttt caccaattgg 2460agtaaacgcg atttcaatca gttcatcaaa gccaacgaga aatatggtag ggacgatatt 2520gagaacatcg ccaaggatgt tgaaggcaaa acgcctgaag aagttatgga atattctgcg 2580gtgttttggg aaagatgtca tgaattacag gatattgata gaataatggc ccagattgag 2640agaggagaaa ctaaaataca aagaagagct agtattaaga aggcacttga tgctaaaatg 2700gcaagatatc gtgcaccatt ccatcagctg agaatttctt acggcaccaa caaaggcaag 2760aactacatgg aggacgaaga caggtttttg gtgtgtatgt tgcacaagtt gggtttcgat 2820agagaaaacg tttatgaaga gttaagagca gctgtacgtg cgtcaccaca attcagattt 2880gattggttct taaaatcgag aactgccatg gagctgcaaa ggagatgcaa cacattgata 2940acgttaatag aaagagaaaa tgctgaattg gaggaaagag aaaaaattga taaaaagaaa 3000aaagtttcca aatcttcaaa tcttggaggt attcctgccc aaatcagctc gaaatcttca 3060cagaaacgga agaa 307488987PRTDiabrotica virgifera 88Met Glu Glu Ala Glu Asn Ser Glu Leu Ala Gln Asn Glu Glu Ser Ser 1

5 10 15 Ser Asp Thr Thr Ser Lys Gly Glu Glu Phe Glu Val Lys Val Ala Ser 20 25 30 Asp Arg Gly Lys Arg Phe Asp Tyr Leu Leu Lys Gln Thr Glu Ile Phe 35 40 45 Ser His Phe Met Asn Gln Thr Lys Ser Pro Ser Lys Pro Lys Thr Gly 50 55 60 Arg Pro Lys Lys Glu Lys Ser Asp Thr Ser Asp Leu Arg His Arg Lys 65 70 75 80 Thr Glu Gln Glu Glu Asp Glu Glu Leu Leu Ala Glu Thr Asn Leu Lys 85 90 95 Thr Lys Thr Thr Thr Arg Phe Asp Ala Ser Pro Pro Tyr Ile Lys His 100 105 110 Gly Glu Met Arg Asp Tyr Gln Val Arg Gly Leu Asn Trp Met Ile Ser 115 120 125 Leu Tyr Glu His Gly Ile Asn Gly Ile Leu Ala Asp Glu Met Gly Leu 130 135 140 Gly Lys Thr Leu Gln Thr Ile Ser Leu Leu Gly Tyr Met Lys His Tyr 145 150 155 160 Lys Ser Thr Pro Gly Pro His Ile Val Ile Val Pro Lys Ser Thr Leu 165 170 175 Ser Asn Trp Met Asn Glu Phe Glu Lys Trp Cys Pro Thr Leu Arg Ala 180 185 190 Val Cys Leu Ile Gly Asp Gln Glu Ala Arg Ser Ser Phe Ile Arg Asp 195 200 205 Thr Met Met Pro Gly Glu Trp Asp Val Cys Val Thr Ser Tyr Glu Met 210 215 220 Cys Ile Lys Glu Lys Ser Val Phe Lys Lys Phe Asn Trp Arg Tyr Met 225 230 235 240 Val Ile Asp Glu Ala His Arg Ile Lys Asn Glu Lys Ser Lys Leu Ser 245 250 255 Glu Ile Leu Arg Glu Phe Lys Thr Thr Asn Arg Leu Leu Leu Thr Gly 260 265 270 Thr Pro Leu Gln Asn Asn Leu His Glu Leu Trp Ala Leu Leu Asn Phe 275 280 285 Leu Leu Pro Asp Val Phe Asn Ser Ser Asp Asp Phe Asp Ala Trp Phe 290 295 300 Asn Thr Ser Gln Cys Leu Gly Asp Asn Ala Leu Val Glu Arg Leu His 305 310 315 320 Ala Val Leu Lys Pro Phe Leu Leu Arg Arg Leu Lys Ala Glu Val Glu 325 330 335 Lys Arg Leu Lys Pro Lys Lys Glu Leu Lys Val Tyr Val Gly Leu Ser 340 345 350 Lys Met Gln Arg Glu Trp Tyr Thr Lys Val Leu Met Lys Asp Ile Asp 355 360 365 Ile Val Asn Gly Ala Gly Lys Val Glu Lys Met Arg Leu Gln Asn Ile 370 375 380 Leu Met Gln Leu Arg Lys Cys Thr Asn His Pro Tyr Leu Phe Asp Gly 385 390 395 400 Ala Glu Pro Gly Pro Pro Tyr Thr Thr Asp Glu His Leu Val Tyr Asn 405 410 415 Cys Gly Lys Met Val Leu Leu Asp Lys Leu Leu Pro Lys Leu Lys Glu 420 425 430 Gln Glu Ser Arg Val Leu Ile Phe Ser Gln Met Thr Arg Met Leu Asp 435 440 445 Ile Leu Glu Asp Tyr Cys His Trp Arg Gln Tyr Gln Tyr Cys Arg Leu 450 455 460 Asp Gly Gln Thr Pro His Glu Asp Arg Gln Arg Gln Ile Asn Glu Tyr 465 470 475 480 Asn Glu Asp Asn Ser Gln Lys Phe Ile Phe Met Leu Ser Thr Arg Ala 485 490 495 Gly Gly Leu Gly Ile Asn Leu Ala Thr Ala Asp Val Val Ile Ile Tyr 500 505 510 Asp Ser Asp Trp Asn Pro Gln Met Asp Leu Gln Ala Met Asp Arg Ala 515 520 525 His Arg Ile Gly Gln Lys Lys Gln Val Arg Val Phe Arg Phe Ile Thr 530 535 540 Glu Asn Thr Val Glu Glu Lys Ile Val Glu Arg Ala Glu Val Lys Leu 545 550 555 560 Arg Leu Asp Lys Leu Val Ile Gln Gln Gly Arg Leu Ala Asp Ser Lys 565 570 575 Ala Gln Thr Leu Asn Lys Asp Glu Met Leu Asn Met Ile Arg His Gly 580 585 590 Ala Asn His Val Phe Ala Ser Lys Asp Ser Glu Ile Thr Asp Glu Asp 595 600 605 Ile Asp Ser Ile Leu Glu Lys Gly Glu Met Lys Thr Ala Gln Leu Ala 610 615 620 Gln Lys Met Glu Thr Met Gly Glu Ser Ser Leu Arg Asn Phe Thr Val 625 630 635 640 Glu Thr Pro Thr Glu Ser Val Tyr Gln Phe Glu Gly Glu Asp Tyr Arg 645 650 655 Glu Lys Gln Lys Thr Ile Gly Leu Ser Asn Trp Ile Glu Pro Pro Lys 660 665 670 Arg Glu Arg Lys Ala Asn Tyr Ala Val Asp Ala Tyr Phe Arg Glu Ala 675 680 685 Leu Arg Val Ser Glu Pro Lys Ala Pro Lys Ala Pro Arg Pro Pro Lys 690 695 700 Gln Pro Ile Val Gln Asp Phe Gln Phe Phe Pro Pro Arg Leu Phe Glu 705 710 715 720 Leu Leu Asp Gln Glu Ile Tyr Phe Tyr Arg Lys Ser Leu Gly Tyr Lys 725 730 735 Val Pro Lys Asn Leu Glu Leu Gly Pro Asp Ala Ser Lys Gln Gln Lys 740 745 750 Glu Glu Gln Arg Lys Ile Asp Glu Ser Glu Pro Leu Thr Glu Asp Glu 755 760 765 Gln Gln Glu Lys Glu Asn Leu Leu Thr Gln Gly Phe Thr Asn Trp Ser 770 775 780 Lys Arg Asp Phe Asn Gln Phe Ile Lys Ala Asn Glu Lys Tyr Gly Arg 785 790 795 800 Asp Asp Ile Glu Asn Ile Ala Lys Asp Val Glu Gly Lys Thr Pro Glu 805 810 815 Glu Val Met Glu Tyr Ser Ala Val Phe Trp Glu Arg Cys His Glu Leu 820 825 830 Gln Asp Ile Asp Arg Ile Met Ala Gln Ile Glu Arg Gly Glu Thr Lys 835 840 845 Ile Gln Arg Arg Ala Ser Ile Lys Lys Ala Leu Asp Ala Lys Met Ala 850 855 860 Arg Tyr Arg Ala Pro Phe His Gln Leu Arg Ile Ser Tyr Gly Thr Asn 865 870 875 880 Lys Gly Lys Asn Tyr Met Glu Asp Glu Asp Arg Phe Leu Val Cys Met 885 890 895 Leu His Lys Leu Gly Phe Asp Arg Glu Asn Val Tyr Glu Glu Leu Arg 900 905 910 Ala Ala Val Arg Ala Ser Pro Gln Phe Arg Phe Asp Trp Phe Leu Lys 915 920 925 Ser Arg Thr Ala Met Glu Leu Gln Arg Arg Cys Asn Thr Leu Ile Thr 930 935 940 Leu Ile Glu Arg Glu Asn Ala Glu Leu Glu Glu Arg Glu Lys Ile Asp 945 950 955 960 Lys Lys Lys Lys Val Ser Lys Ser Ser Asn Leu Gly Gly Ile Pro Ala 965 970 975 Gln Ile Ser Ser Lys Ser Ser Gln Lys Arg Lys 980 985 894998DNADiabrotica virgifera 89tttcaaataa aatttgttcc ttgataataa aaaaacaaag aaaatggagg ttaatccaat 60aattagaact cgacatattg taaaattaat gaaaaatggc cgcaaaatat tatgtactaa 120gattgccctg attctacaat atctcgagtt aaaattattg gattaaagtt tagtaaacgt 180cattttgttt gttttttgtt caaggaacaa attttatttg aaaattcttt ttatctcttt 240tagtttacga actagaacct tataaataat tcaattgttc agcagccaaa aatacataaa 300aaatttgaaa aggtcacagt gtctagctga cccctagatt cagtttaaaa attaaaaata 360ttattaatcg tcgggataat cgtagaacga ataacttgaa tactgcgcgt tactatttac 420attagagcct aggttggtgc catctgaagg taacgggacc ggcaaaggcc ttgatggttt 480gcgaggtcgg tatgttcctc tagaaccggc gtttgacggg ccacctggtt ttaaacgggg 540tctgccagga ccccgccgca ttgatggggt ttgagcagta gaactgctag cttgagcagc 600tccagaaact tccaaaaagc agtgggacgg tgatgaatgc atcgaagctg cagacatagt 660agtagaatct ggagacggtt gatccgttaa aaagatacta ccaggtcccg gtgatgtaga 720gacagaagaa ctattagata tctggcggcc aagccctgtc gtacggcttc gagatcctct 780aggccgtccc cttcgagatg accctcttcc aggaagcctt ggtttgacag ttgtgccgta 840tacattgtat acagtatatt tattcgtaat tgccgaatca gattcatcat tgtaaatgtc 900tacgccatca tctatcagaa tgtcgttaga attgacactt aatggactgt tgggcccact 960ttcgacgaca gtactagtag cttcttctaa tgtaactgat tccagtttag cttttttggg 1020ttccatgcca actggtattg tcttatattg taattttcgt ttcctgtcta actcttcgga 1080agtattatcg attttcaaat gatatttttg taccaattca tcatcatcca acagcaagga 1140caccacttcc ttgggcttga gtgtatccgg tttaaagtta ccaccgctaa tgaccaattt 1200ttgaatctcg ctcttttctc tagccctttg taagatgcgt tcttcaatag aacctttaca 1260aattaatctg tacaccgtca cctgcttggt ctgacccaac cgatgggccc tgtccatagc 1320ctgctggtcc acagtcgggt tccaatcact gtcgtagaaa attacagtat ctgcagcagt 1380taaattgata ccaagtccac cagctcttgt cgacagtaga aaaacaaaaa tgtctgctct 1440ggcttggaaa tcagcaacca tatcccttcg ttctgatatt tttgatgaac catctaacct 1500catatactta tgatgcctgt gccacatgta ttcttctaaa agatcaatca tctttgtcat 1560ctgcgaatat attaaaacgc gatgtccttc ctctttgagt cttttaagga gtccatcaag 1620taccgacagt tttccagaat cggtaactaa actctcctta tctggtatca caatattcga 1680aaaaccgtta acaggtctca aattatcaac tgcattaaac ggtcgaggat gtaaagtttc 1740tgctgattta taattcaatt tattgttggt cgctttcgac cagtaagaat taagagagtt 1800gaaactaaat tcgtctagat gacgctgtag atcccacgct gctctacgag aataacaata 1860taagccgagt ggggccgccg ataccctagg catacaataa aaaagaaagg cgggaatttc 1920agtctttgta cattggaaca cctgagttaa cctctttatg taaggaaaat cagtcaatat 1980attttgtatc tggttcactc tatggtcatt aactcttaaa agtgcatcat ttcctgcgtt 2040aagttcttta gtttttaatc taatattttt atgttcaaca gtttctggag tggaatgaaa 2100cacatgatcg gtatgtgtgt aaaacactct atttcctttg ttttgttcag taaaaactaa 2160atcaccaaat acatcactac ttcgaatctg atattgattt aaacggaaaa aattcctcaa 2220ctgtaatgtc ggccgttttc taaaggatat gtcccatagt tttctataat ataatatttc 2280attggatttt tggcattcgt aataatgttt ccacctgtgc aaaatattcc cttgaaatat 2340cctaaaaaca tcttcagctg ataaacccaa aaagtgacaa aaattaaaaa tggtgggatg 2400caaaccatct tgaatagcat cttttatgtc tcctggtttg aaaatgaagt gtttcctaat 2460tttctccatt aaaatttctc gaacattgaa atcataaatt tgataaggaa ctgtgtactg 2520caaagaactt attctaatgg gagatttggc gtctcttctc tcaaaaagtt cgggatggtt 2580gcaaaccttt ctaaactgca tcaccaaatt catcaaattt gaagtaaaat tcttatctac 2640agtgtgagaa tctcccccgc caactgtgta attcaagaga tcttcaattt tgattttttg 2700ttttagagcc aaatacaata aattctgtct tgtggtcagt ggacagtaga ccattacttc 2760tattttatca gacagttcat tttcaacatc tttcttgatt ctcctcaaca taaaaggttt 2820taaaatcata tgtaaacgag ataagtgttt ttcatcgata ccagttttgt tttcagcatg 2880gctttcaatg tcttttgaaa accattcgtt gaactcctca tgtgagtcaa agagtgttgg 2940cataataaaa tgcaataagg cccatagctc cgccatactg ttttgtattg gggtaccact 3000caacaacaat ctgttcctgc aactaaaacc taatagagtc ttccatctca ttgaactagt 3060acttttgata gcctgtgctt catctagtat catgtattgc cattttattc tattaaagta 3120ttttatatct gttataacta tttggtagga agttaccaca atgtgaaaac tagcgtcttt 3180tgtatacatg tctttcaaat cccaaaactg ccttaaaatt tttctttcgt ttggatttcc 3240ccaatagggc accactttaa aatccggcac aaacttggcc acttcttgct gccaattgtg 3300cagggttgag gccggagata ttatcaaaaa agggccccag acagaatatt tttcagctat 3360gtgacaaaga aacgcaatac tttgaacagt tttgcctaga cccatttcat cagctagaat 3420accactgatt ccctgagagt ataaatttgc caaccaattc atccctctta gttgatatcc 3480ttttaatttg cctctaaaca tattaggttg tggctgttca ccctctccat tgggaaattc 3540attaagacac gaattggctt gttggtcaaa atgtctagtc cttgcttttt cactctgaaa 3600tgcatctaat gcattctttt tggccatttc cttcatactt tcactatcgt aagtatcaca 3660acttaatttt attgaacttt cctcgtctag ctggcttaaa attagcaact gttcttcagg 3720agaagcctgt cccaatttct tggacatgaa atgagcatac agctcagtct gagtaataag 3780aaagtttaat ttcctctgtt gtcgcttggc ttctaccaac tcgtggtcaa tttttctttg 3840ttcttctgct tctttctcta accgtttctt aacttctcta tcaaaacgcc gtgagcgttt 3900ccagtaagca atattctcac gtgataacct cttcatcctc catggctgtt ctttgactat 3960tcgagcgctt tggagagctt tttgacgtgc atatttaaca caatgtgatg ccactcgttt 4020gcactgcagt agcatttcct tgtgtttgtt aatttttgat ctatgttgct ttccaatttc 4080ttttttgact atattagtaa ataattttcg ccgctttatg gtcatcatat cttcccattt 4140ccgtctggac tcttcatctt tgaatttttt tgtttttttc tttggtatat gaagtcctaa 4200aggaggagga ggtagagact gatttggaat gacaagtcca tattcagatg gcaaatactt 4260atcagctata tcgtcgatgc tttggaaatc cattccattt tggagattca aatctaaaca 4320tggttcatcc gaaatagcat ccaaaatatc aggatggtat agtggagact taaatgtgtc 4380ttctatttta attttatttt tcttgacagg tactggtaga tttttttcta catgtggata 4440aagatctact gcactgatca atccagctcc atagtatgca taattatcaa aattctcttt 4500tctttttctt aaggattttt gatatgaatg caacctcata acatgcttat attgtacatc 4560atccggacta ctatctgaac tttcttgtag aacatcagca agccatttcc tcctagttga 4620aacagtttta agattgtaat actcattggt agagtgaggt tctgaatctt ttaaaataga 4680atctatattg acatcactca gattgcccat gccatcatca gaatcgtcag agtcgctact 4740taactgtaat tggttgtgta catctcgtaa aaacatatta gtgtcgacag cttgctcaac 4800tcttcttaca tgaactggtt tagctgtata tgatctatta gtttgatcaa ggtagctgtt 4860ggtgggtaat attttgtatg atttatccct gccgttccac attttttcta aatgtaataa 4920actgttttca gaatatctat ataaattaag ctaatataat tttacattac attacattct 4980caattagttc atttttta 4998901512PRTDiabrotica virgifera 90Met Trp Asn Gly Arg Asp Lys Ser Tyr Lys Ile Leu Pro Thr Asn Ser 1 5 10 15 Tyr Leu Asp Gln Thr Asn Arg Ser Tyr Thr Ala Lys Pro Val His Val 20 25 30 Arg Arg Val Glu Gln Ala Val Asp Thr Asn Met Phe Leu Arg Asp Val 35 40 45 His Asn Gln Leu Gln Leu Ser Ser Asp Ser Asp Asp Ser Asp Asp Gly 50 55 60 Met Gly Asn Leu Ser Asp Val Asn Ile Asp Ser Ile Leu Lys Asp Ser 65 70 75 80 Glu Pro His Ser Thr Asn Glu Tyr Tyr Asn Leu Lys Thr Val Ser Thr 85 90 95 Arg Arg Lys Trp Leu Ala Asp Val Leu Gln Glu Ser Ser Asp Ser Ser 100 105 110 Pro Asp Asp Val Gln Tyr Lys His Val Met Arg Leu His Ser Tyr Gln 115 120 125 Lys Ser Leu Arg Lys Arg Lys Glu Asn Phe Asp Asn Tyr Ala Tyr Tyr 130 135 140 Gly Ala Gly Leu Ile Ser Ala Val Asp Leu Tyr Pro His Val Glu Lys 145 150 155 160 Asn Leu Pro Val Pro Val Lys Lys Asn Lys Ile Lys Ile Glu Asp Thr 165 170 175 Phe Lys Ser Pro Leu Tyr His Pro Asp Ile Leu Asp Ala Ile Ser Asp 180 185 190 Glu Pro Cys Leu Asp Leu Asn Leu Gln Asn Gly Met Asp Phe Gln Ser 195 200 205 Ile Asp Asp Ile Ala Asp Lys Tyr Leu Pro Ser Glu Tyr Gly Leu Val 210 215 220 Ile Pro Asn Gln Ser Leu Pro Pro Pro Pro Leu Gly Leu His Ile Pro 225 230 235 240 Lys Lys Lys Thr Lys Lys Phe Lys Asp Glu Glu Ser Arg Arg Lys Trp 245 250 255 Glu Asp Met Met Thr Ile Lys Arg Arg Lys Leu Phe Thr Asn Ile Val 260 265 270 Lys Lys Glu Ile Gly Lys Gln His Arg Ser Lys Ile Asn Lys His Lys 275 280 285 Glu Met Leu Leu Gln Cys Lys Arg Val Ala Ser His Cys Val Lys Tyr 290 295 300 Ala Arg Gln Lys Ala Leu Gln Ser Ala Arg Ile Val Lys Glu Gln Pro 305 310 315 320 Trp Arg Met Lys Arg Leu Ser Arg Glu Asn Ile Ala Tyr Trp Lys Arg 325 330 335 Ser Arg Arg Phe Asp Arg Glu Val Lys Lys Arg Leu Glu Lys Glu Ala 340 345 350 Glu Glu Gln Arg Lys Ile Asp His Glu Leu Val Glu Ala Lys Arg Gln 355 360 365 Gln Arg Lys Leu Asn Phe Leu Ile Thr Gln Thr Glu Leu Tyr Ala His 370 375 380 Phe Met Ser Lys Lys Leu Gly Gln Ala Ser Pro Glu Glu Gln Leu Leu 385 390 395 400 Ile Leu Ser Gln Leu Asp Glu Glu Ser Ser Ile Lys Leu Ser Cys Asp 405 410 415 Thr Tyr Asp Ser Glu Ser Met Lys Glu Met Ala Lys Lys Asn Ala Leu 420 425 430 Asp Ala Phe Gln Ser Glu Lys Ala Arg Thr Arg His Phe Asp Gln Gln 435 440 445 Ala Asn Ser Cys Leu Asn Glu Phe Pro Asn Gly Glu Gly Glu Gln Pro 450 455 460 Gln Pro Asn Met Phe Arg Gly Lys Leu Lys Gly Tyr Gln Leu Arg Gly 465 470 475 480 Met Asn Trp Leu Ala Asn Leu Tyr Ser Gln Gly Ile Ser Gly Ile Leu 485 490 495 Ala Asp Glu Met Gly Leu Gly Lys Thr Val Gln Ser Ile Ala Phe Leu 500 505 510 Cys His Ile Ala Glu Lys Tyr Ser Val Trp Gly Pro Phe Leu Ile Ile 515 520 525 Ser Pro Ala Ser Thr Leu His Asn Trp Gln Gln Glu Val Ala Lys Phe 530 535 540 Val Pro Asp Phe Lys

Val Val Pro Tyr Trp Gly Asn Pro Asn Glu Arg 545 550 555 560 Lys Ile Leu Arg Gln Phe Trp Asp Leu Lys Asp Met Tyr Thr Lys Asp 565 570 575 Ala Ser Phe His Ile Val Val Thr Ser Tyr Gln Ile Val Ile Thr Asp 580 585 590 Ile Lys Tyr Phe Asn Arg Ile Lys Trp Gln Tyr Met Ile Leu Asp Glu 595 600 605 Ala Gln Ala Ile Lys Ser Thr Ser Ser Met Arg Trp Lys Thr Leu Leu 610 615 620 Gly Phe Ser Cys Arg Asn Arg Leu Leu Leu Ser Gly Thr Pro Ile Gln 625 630 635 640 Asn Ser Met Ala Glu Leu Trp Ala Leu Leu His Phe Ile Met Pro Thr 645 650 655 Leu Phe Asp Ser His Glu Glu Phe Asn Glu Trp Phe Ser Lys Asp Ile 660 665 670 Glu Ser His Ala Glu Asn Lys Thr Gly Ile Asp Glu Lys His Leu Ser 675 680 685 Arg Leu His Met Ile Leu Lys Pro Phe Met Leu Arg Arg Ile Lys Lys 690 695 700 Asp Val Glu Asn Glu Leu Ser Asp Lys Ile Glu Val Met Val Tyr Cys 705 710 715 720 Pro Leu Thr Thr Arg Gln Asn Leu Leu Tyr Leu Ala Leu Lys Gln Lys 725 730 735 Ile Lys Ile Glu Asp Leu Leu Asn Tyr Thr Val Gly Gly Gly Asp Ser 740 745 750 His Thr Val Asp Lys Asn Phe Thr Ser Asn Leu Met Asn Leu Val Met 755 760 765 Gln Phe Arg Lys Val Cys Asn His Pro Glu Leu Phe Glu Arg Arg Asp 770 775 780 Ala Lys Ser Pro Ile Arg Ile Ser Ser Leu Gln Tyr Thr Val Pro Tyr 785 790 795 800 Gln Ile Tyr Asp Phe Asn Val Arg Glu Ile Leu Met Glu Lys Ile Arg 805 810 815 Lys His Phe Ile Phe Lys Pro Gly Asp Ile Lys Asp Ala Ile Gln Asp 820 825 830 Gly Leu His Pro Thr Ile Phe Asn Phe Cys His Phe Leu Gly Leu Ser 835 840 845 Ala Glu Asp Val Phe Arg Ile Phe Gln Gly Asn Ile Leu His Arg Trp 850 855 860 Lys His Tyr Tyr Glu Cys Gln Lys Ser Asn Glu Ile Leu Tyr Tyr Arg 865 870 875 880 Lys Leu Trp Asp Ile Ser Phe Arg Lys Arg Pro Thr Leu Gln Leu Arg 885 890 895 Asn Phe Phe Arg Leu Asn Gln Tyr Gln Ile Arg Ser Ser Asp Val Phe 900 905 910 Gly Asp Leu Val Phe Thr Glu Gln Asn Lys Gly Asn Arg Val Phe Tyr 915 920 925 Thr His Thr Asp His Val Phe His Ser Thr Pro Glu Thr Val Glu His 930 935 940 Lys Asn Ile Arg Leu Lys Thr Lys Glu Leu Asn Ala Gly Asn Asp Ala 945 950 955 960 Leu Leu Arg Val Asn Asp His Arg Val Asn Gln Ile Gln Asn Ile Leu 965 970 975 Thr Asp Phe Pro Tyr Ile Lys Arg Leu Thr Gln Val Phe Gln Cys Thr 980 985 990 Lys Thr Glu Ile Pro Ala Phe Leu Phe Tyr Cys Met Pro Arg Val Ser 995 1000 1005 Ala Ala Pro Leu Gly Leu Tyr Cys Tyr Ser Arg Arg Ala Ala Trp 1010 1015 1020 Asp Leu Gln Arg His Leu Asp Glu Phe Ser Phe Asn Ser Leu Asn 1025 1030 1035 Ser Tyr Trp Ser Lys Ala Thr Asn Asn Lys Leu Asn Tyr Lys Ser 1040 1045 1050 Ala Glu Thr Leu His Pro Arg Pro Phe Asn Ala Val Asp Asn Leu 1055 1060 1065 Arg Pro Val Asn Gly Phe Ser Asn Ile Val Ile Pro Asp Lys Glu 1070 1075 1080 Ser Leu Val Thr Asp Ser Gly Lys Leu Ser Val Leu Asp Gly Leu 1085 1090 1095 Leu Lys Arg Leu Lys Glu Glu Gly His Arg Val Leu Ile Tyr Ser 1100 1105 1110 Gln Met Thr Lys Met Ile Asp Leu Leu Glu Glu Tyr Met Trp His 1115 1120 1125 Arg His His Lys Tyr Met Arg Leu Asp Gly Ser Ser Lys Ile Ser 1130 1135 1140 Glu Arg Arg Asp Met Val Ala Asp Phe Gln Ala Arg Ala Asp Ile 1145 1150 1155 Phe Val Phe Leu Leu Ser Thr Arg Ala Gly Gly Leu Gly Ile Asn 1160 1165 1170 Leu Thr Ala Ala Asp Thr Val Ile Phe Tyr Asp Ser Asp Trp Asn 1175 1180 1185 Pro Thr Val Asp Gln Gln Ala Met Asp Arg Ala His Arg Leu Gly 1190 1195 1200 Gln Thr Lys Gln Val Thr Val Tyr Arg Leu Ile Cys Lys Gly Ser 1205 1210 1215 Ile Glu Glu Arg Ile Leu Gln Arg Ala Arg Glu Lys Ser Glu Ile 1220 1225 1230 Gln Lys Leu Val Ile Ser Gly Gly Asn Phe Lys Pro Asp Thr Leu 1235 1240 1245 Lys Pro Lys Glu Val Val Ser Leu Leu Leu Asp Asp Asp Glu Leu 1250 1255 1260 Val Gln Lys Tyr His Leu Lys Ile Asp Asn Thr Ser Glu Glu Leu 1265 1270 1275 Asp Arg Lys Arg Lys Leu Gln Tyr Lys Thr Ile Pro Val Gly Met 1280 1285 1290 Glu Pro Lys Lys Ala Lys Leu Glu Ser Val Thr Leu Glu Glu Ala 1295 1300 1305 Thr Ser Thr Val Val Glu Ser Gly Pro Asn Ser Pro Leu Ser Val 1310 1315 1320 Asn Ser Asn Asp Ile Leu Ile Asp Asp Gly Val Asp Ile Tyr Asn 1325 1330 1335 Asp Glu Ser Asp Ser Ala Ile Thr Asn Lys Tyr Thr Val Tyr Asn 1340 1345 1350 Val Tyr Gly Thr Thr Val Lys Pro Arg Leu Pro Gly Arg Gly Ser 1355 1360 1365 Ser Arg Arg Gly Arg Pro Arg Gly Ser Arg Ser Arg Thr Thr Gly 1370 1375 1380 Leu Gly Arg Gln Ile Ser Asn Ser Ser Ser Val Ser Thr Ser Pro 1385 1390 1395 Gly Pro Gly Ser Ile Phe Leu Thr Asp Gln Pro Ser Pro Asp Ser 1400 1405 1410 Thr Thr Met Ser Ala Ala Ser Met His Ser Ser Pro Ser His Cys 1415 1420 1425 Phe Leu Glu Val Ser Gly Ala Ala Gln Ala Ser Ser Ser Thr Ala 1430 1435 1440 Gln Thr Pro Ser Met Arg Arg Gly Pro Gly Arg Pro Arg Leu Lys 1445 1450 1455 Pro Gly Gly Pro Ser Asn Ala Gly Ser Arg Gly Thr Tyr Arg Pro 1460 1465 1470 Arg Lys Pro Ser Arg Pro Leu Pro Val Pro Leu Pro Ser Asp Gly 1475 1480 1485 Thr Asn Leu Gly Ser Asn Val Asn Ser Asn Ala Gln Tyr Ser Ser 1490 1495 1500 Tyr Ser Phe Tyr Asp Tyr Pro Asp Asp 1505 1510 918026DNADiabrotica virgifera 91cgtcgctatg tcatataaga ccaatgcgtc atttttgctg aacgcgtaaa atgtagaaaa 60caggtctatt ttgcaaattt ttaagttacg tggactacct aattgtatat taaactatta 120gaaagacgat aatcatattt tttactgcgg atgattaacc ttaatgtgaa tattttgtga 180taacatgatg agaattcgaa gatgagtgac gaaactccgg gggtagggca gggtgctctg 240cccccgagag agggccaaag ccttcgactc cttgcagagc gccctactag tggtgctacg 300gtaagggtag cccaggttgt tggtggtcag tatgtgttga caacacaatc acatggtatg 360ccagctctag cacagattgc tgctgggaat cccaacgtca ctcgtctgat aagcatcagt 420ccaactagag gtggacaatc gtccccacta aggcccagtt tggccaatca gtcgatcgtt 480aatgttctta ccaagtctcg accaaatcct aatgtacgct tacagttatt tcaatccggt 540gagagttctg gaaacaatgt ccagcatagt ccccattcga gatcactcaa aaggccgttg 600tcttccacgg gtgaaaaaaa agatagctac gcttctaagc tgcaacatgt aatgaaccat 660cgcattgttc gctcaaaatt aatgaaagaa aagtataatg aacatcttct agaggcctat 720tatttagaga ccggaaataa cattctagat ttatatcaat ttgccaaaag acctaaaacc 780caagcgtatc tagcttatct taaggaacat gccatcgatc ctcgagatta tcctgaactt 840cagactacaa caactgttac tgtaccgcaa acgacgccta atacaccaac tgctacctct 900gtcagctctc tgcccggtat ctctcatagt tacgccatcc agactaccag ttctactgta 960acgacaccag aaagtaacag taacacctct acgccgaaat cagtatctgt caaagtgaag 1020tctacatcac ttccgaatac tgttagtcaa gagatgattg tagagaaggc gaaacaagaa 1080gcgtacgtgg tccaacgaat agccgattta cagaaggaag gaatatggtc tgaaaggaga 1140cttcccaaag tgcaagagat gcctcggcct aaagcgcatt gggacttttt gatcgaagaa 1200atggtctggt tggcagctga ttttgcgcag gaacgcaaat ggaagaaagc cgccgcgaaa 1260aaatgtgcca gaatggtaca gaagtatttc caagacaagg cgctcgccgc ccaaaaagcg 1320gaaaaggctc acgaacaaaa tcttagaagg atagccgcgt tctgtgcaaa ggagattaag 1380atcttttgga acaacgtcga aaaactcgtc gagtataaac agaatacaat cttggaggag 1440aagcggaaaa aggcgctcga tcagcagctt agttttatcg tggatcagac tgagaagtat 1500tcgcagttgc ttgccgaagg aatgaataag accgcagaac agcctcctag ttcagcgcca 1560tctcgatcag tgtctcgaac gcagtctgat acagaattcg atccggatct tcagagcgat 1620gaagacgacg aagagaccat tgctcgagaa gaagctttag gtaacgaagg acataaagaa 1680gaaatcgaag cgctacagaa agaatctcag atggaattag acgatttact agaggacgat 1740ttcctgaggg attacctttt aaatcgagac acgatccgat tcagcgaatc cgaagattcg 1800gacgatgaca cggactcgaa aaaagaatct ttcaaaggcg acaaagaaca gtctgacgat 1860tctgaatctt ctaaagaaga agatacggaa gacgagagcg aagatgaatc tatgaaggtt 1920actgaatcgg ttgttaaaga agaacacgac gagttgaaaa tattagtaga agattctcaa 1980aaagagggag aaattaagac tgaacaagat acaaaagacg accttatcaa tgacgcagct 2040gctatagctg aaagtatcca acctaaagga aacaccctgt cttctactaa tgtatcaaca 2100aatataccat ttttattaaa atatacgcta agagaatacc agcacatcgg tttggattgg 2160ctggtaacta tgttcgacag aaaactgaac ggtatattag cagatgaaat gggtttaggc 2220aaaacaatac aaacgatagc tcttctagca cacttggcgt gcgagaagga aaactggggc 2280ccccatctga tagtagtccc cacttctgtg atgctgaatt gggaaatgga atgcaaaaag 2340tggtgtccgg cttttaaaat tctaacgtat tacggaacgc agaaggaaag aaaatttaaa 2400cggataggat ggacgaagcc taacgcgttt cacatatgca ttacttcgta caagctagtc 2460attcaggacc accagagttt caggaggaaa aagtggaagt atctgatact ggacgaggcc 2520caaaacatca agaatttcaa gtcgcaacga tggcagctgt tgttaaattt tcaaactcaa 2580caacgtctgc tgttgactgg tacacctttg cagaacaacc tcatggaatt gtggtccctt 2640atgcattttt taatgccgaa cgtatttcag tcgcatagag agttcaaaga atggttttcg 2700aatccggtga caggaatgat tgaaggaaat tctgaataca acgaaagtat tatcaagaga 2760ctgcacaagg tattaagacc atttcttcta aggcggttga aaagcgaagt ggaaaaacaa 2820atgccaaaaa agtacgaaca cgtggtcatg tgtaggctat ccaaacgaca gaggtatttg 2880tatgatgact acatgtcccg agcaaaaacg agggaaactt taactactgg aaatctgttg 2940agtgttataa atgtgctgat gcagctgagg aaagtgtgta atcatccgaa tctatttgaa 3000attagaccaa cgacatcgcc ttttcagtgt gacaacatcc ggcttcatat tccatccatt 3060gtatattcag ctttagatta cgatcctgat aagcacgtga accttcaagc tttaaatctt 3120ctactaatca tgcaagaaat ccactttggt tcgtaccagt gttaccggat gagacaatca 3180agaaattcca agaaaatttt cgaaatggaa acgaattcta gcaaaaatcc accaccttgt 3240ccgccatgta agttagccat gcgagttcta acagacaaac cttcagccac tgacgaaaag 3300aatgagaaga aagacatgca agctttaagt cagccgcctc cattgcaagt taaaggaatg 3360agccagccta acatgaagat gaaggtttct ggagtgcaat ttgttccaca gagcatactt 3420aaatcaattc cagtagtgaa catatcacaa ggggctacag gtcaaatcgg agcacctgtt 3480agtgtgacat ctgtattaaa accacaagac aaaatatctg ctagttttgc acagctagtt 3540caaacgtcta ctggcaaaca cttgttacta acgtcgaatc ccaacattac gacgagccca 3600gtgacaacta caacaccagg tggacaaaaa ttgaccttcc tatcgaagca gcccgtttct 3660acgattggta atgcgggcca tgctgtaacg aaagcttatg tcaaatttca gttaacgtct 3720gttacgacag catcaacttt cacaacagtt acgacagtca actccaatac gatatctgta 3780gctaaaagtg aagataacaa agggatgcga atgtctgttg gtaatgatta tataggtaaa 3840ctttattcta aacaaaatag cctggacgtg cgatggaata gcggcgaaaa acatttaggt 3900ttaacaaatg aagacgatcc caaaggtgaa cgaaagaaga gactttccct catgtcacgc 3960atcaacaaga tccgttgctc agctcttcca ctgtacggcc gagatttcca agaagctgtc 4020aaaatatata cgcctaacca gctggatgtt tggaacgggg gtcatattca ctgcttgaac 4080acactgtaca ataaggatgc caggaatgaa acgacggatt gtctccaaga cgcgttgttt 4140aatcctgaaa gaagattgga agctctaaaa gatacttttg atcgatttat attctatgta 4200ccttctgtga aagtggcgga acccgaactg caagtgtggc atcctccacc gagtaaatat 4260tggggccaaa aacacgagaa acaacttata cagaaactat tcctaaaacc tgcaacacct 4320cttcatagta tagcatctgc aatggtaacg cagtttccag atcctaggct tattcaatat 4380gactgtggga agttacaaac tctggatata ctattgagga agttaaaact gggaagtcat 4440cgagtattga tcttcacgca gatgacgaaa atgttggatg tactagaggc atttttgaat 4500taccacggtc atatatatct taggttagat ggtaccacaa aagttgatca aagacaagtg 4560ttgatggaga ggttcaacgg tgataaacgt attttcgctt ttattttgtc cacgcgttcc 4620gggggcgtgg gtgtaaattt aaccggagcg gatactgtga tattttatga ttccgattgg 4680aatccaacta tggacgcgca agcgcaagat cgttgtcacc gaatcggtca aacgagagac 4740gtacacattt acaggctagt tagcgagcga acgatagagg agaatatatt gaagaaggcc 4800aatcagaaac gattgctcgg agatctggcg atcgaagggg gtaatttcac aacggcgtac 4860ttcaagagtt cgacgattca ggacttattc aacatagacc aaaacgaaga aagcgcatct 4920gcccgaatgt cagaagttgt cgaactgaga aaagaaagag agaaggccct cagcacagac 4980ctggttcatt ctgctgacga taaagccacc gtcggtgctc tcgaaaatgc tttcgaagca 5040tgcgaggacg accaggacgt ccaagccgcg aaaacggcca aagccgaagc tgttgcagat 5100cttgcagagt ttgatgaaaa cattcctctg gatgatcaag agaaagaacc tgagatcagc 5160aaggcggaac aggaaattaa taatattata gaaaagttaa ctcccataga aaaatacgcc 5220atgaatttca tcgaggcaac agaatctgcg tggtctgcag aacagcttgc agctgctgca 5280agagagatcg aagaacagaa aaaggaatgg gagcagaacc gtctggcggc gatgcgagaa 5340gaggaggaac gtcgtgctcg agagttagaa gaagaatctg atatcatcac gtattcaaga 5400gatgacgcca ccaaccaggt tagctcaaaa aacaaaaaaa tcaataggta taataaaatt 5460ttaagtaata aaagggttag gctcaaaaaa gatggagatg aagacgttga gaaaaaagat 5520gacgtcgaga aaaaagatgg agttgaaaaa aggttgaaga agactaggac acgaaggctg 5580tctcaaaaaa gcaaagatgt agaggtcgaa gaaccggatg cttgtgaatc acaagaagaa 5640tctcaaatta acggagggga tacggataat agtgatagtg attctgattc tgatagcgaa 5700tcttcatctt ccatggaatc taaaactacc ttaaaccacg ttgatccaaa ttcacctaga 5760actaggtcta ggggcacagt ggctattaac ctttggacac tcgatgtcag cccgattttg 5820cctggagaaa aaccgatgaa aaaatacggg gagaaccata gaaaaaatat taagagggtt 5880agatctgtgt ctgaaaacga taacgatgga gataaagacg gtagaaagcg attgaggagg 5940aaatacccca ccagtttaga aacatcagaa gaagaaaaca gtaaccagtc aagagaaaaa 6000tctactaaga aacgtgccaa ggtagcaccc aaagggaaaa cttgtaaagt aattttaagt 6060aatatactga acgataaacg atttaaagtc aacttaaaag aagacattga gatttcagtg 6120agcacacaaa ttaatgagac ttccaccagc tcaaaccaga atcaaaccaa agattgcgag 6180tctagtcaac atgagaatag caatttagat gaacagaatg attctcttga caatacagaa 6240gttacctcat ccgaacttag taaattaact ggttgtactg aaatagataa taacgaaagt 6300agtaaacagg aaaatgaaga attagacgaa tctatactcg aagataaata tgatgaagat 6360ttcattacaa acaaaaatga agacatagat gaagaaacac tccttgaaga agataatcag 6420atagagcagg ttgaaaataa aaatattgat agcacgaaag atgaaaaaca gggtgatgac 6480agtaatgttt cagatgtagg tcatttaagt aaagataacg ataatgagga aaaaatggaa 6540gtaacagaaa gtgttgacga ggaaaatggt gatatcaata agaaagtaga tgaagatgag 6600agcgttaaag ataaaagaga aaggagaaaa ggtaatgagg aagatgataa tacagacaat 6660gaagaaaaca tccaaaagtc agaaaatgat gagggcgata ttaaaaagca aggaaatcaa 6720gatgaagaag tagaagagaa aaccttagga aattctactg aatcagttaa cgaaatagca 6780aatgaaataa gtagatgcaa acctttaaat gagcagcaca acgaattggt agatgaagta 6840gtaaatgaca caagtaatat ggatgaatat ataaaaaaat cggaaaattc caaagttgta 6900gaaaagacaa gtgaagaaat actattcaac gataggggaa atcaagattc atcgtcccag 6960gatgtaaaag atgaagagat atcatcccac aacagaggag atgaaaaggt gtcattccac 7020gataggatag ataaagaggt attacctgaa tgtaggaaag aggaagagaa acacaataga 7080aaaaatgaag tactatcaca aactataaaa gatgaagagg cacagtccca caataggaaa 7140gatgaatcgg gtacatttcc aaacgtagca gatatagaaa atagacttaa caacaaagtt 7200cctcacgtcg ataatggtca taccgaaacg gtcagaatgt ctaaattggt aacgtctaat 7260agaaatgcca atttcaggtc tcctgaaacg agaagaagct tcagaaagtg tggtaaaatt 7320tcgaacaatc agactttaga cggttgggtg aaacggtcgc ctgtattacc tgtcgaagct 7380gctaaagtaa acgataactc aaaatataaa aatgttggtt cgcccgagtt atagggagat 7440ttgactgtgg aaagaggaga aatttaaaag tttttgatag ttaaaaaagt gtttattgat 7500caatgtacac tgcaataagg taataactca aaaataattt atttctttac agttttcttc 7560ttcatccact cctggacaaa ggcctcacga agtgttttac aagtttcttt gataacgggt 7620aaaaccaaaa aatagtaaag ttcgcataac cttctggcta atgtctagtc ttagggttca 7680agtcgcgcaa gcgcccagac cgaccgcttt acgtgccttc cgaatgacgg cggcggctca 7740gaacaatttc ttaatgtcag tgccaggaat ctaacctaga tcctctagct tgttaagtca 7800gtaataaaca ttagttcaac ggacatattg tcactttatt gcgcatgcgt ttatcattac 7860tgagtcttga gttctgccta gccgtaccgt tgacatattc gtaaagattg tagtaataag 7920gcatgcatca gatctttata gatttcaaac atgcttatga ctcagttaca attcgtccaa 7980gactatggaa tggtatagtt gagctggaca tacctaagaa attagc 8026922410PRTDiabrotica virgifera 92Met Ser Asp Glu Thr Pro Gly Val Gly Gln Gly Ala Leu Pro Pro Arg 1 5 10 15 Glu Gly Gln Ser Leu Arg Leu Leu Ala Glu Arg Pro Thr Ser Gly Ala 20 25 30 Thr Val Arg Val Ala Gln Val Val Gly Gly Gln Tyr Val Leu Thr Thr 35 40 45 Gln Ser His Gly Met Pro Ala Leu Ala Gln Ile Ala Ala Gly Asn Pro 50 55 60 Asn Val Thr Arg Leu Ile Ser Ile Ser Pro Thr Arg Gly Gly Gln Ser 65

70 75 80 Ser Pro Leu Arg Pro Ser Leu Ala Asn Gln Ser Ile Val Asn Val Leu 85 90 95 Thr Lys Ser Arg Pro Asn Pro Asn Val Arg Leu Gln Leu Phe Gln Ser 100 105 110 Gly Glu Ser Ser Gly Asn Asn Val Gln His Ser Pro His Ser Arg Ser 115 120 125 Leu Lys Arg Pro Leu Ser Ser Thr Gly Glu Lys Lys Asp Ser Tyr Ala 130 135 140 Ser Lys Leu Gln His Val Met Asn His Arg Ile Val Arg Ser Lys Leu 145 150 155 160 Met Lys Glu Lys Tyr Asn Glu His Leu Leu Glu Ala Tyr Tyr Leu Glu 165 170 175 Thr Gly Asn Asn Ile Leu Asp Leu Tyr Gln Phe Ala Lys Arg Pro Lys 180 185 190 Thr Gln Ala Tyr Leu Ala Tyr Leu Lys Glu His Ala Ile Asp Pro Arg 195 200 205 Asp Tyr Pro Glu Leu Gln Thr Thr Thr Thr Val Thr Val Pro Gln Thr 210 215 220 Thr Pro Asn Thr Pro Thr Ala Thr Ser Val Ser Ser Leu Pro Gly Ile 225 230 235 240 Ser His Ser Tyr Ala Ile Gln Thr Thr Ser Ser Thr Val Thr Thr Pro 245 250 255 Glu Ser Asn Ser Asn Thr Ser Thr Pro Lys Ser Val Ser Val Lys Val 260 265 270 Lys Ser Thr Ser Leu Pro Asn Thr Val Ser Gln Glu Met Ile Val Glu 275 280 285 Lys Ala Lys Gln Glu Ala Tyr Val Val Gln Arg Ile Ala Asp Leu Gln 290 295 300 Lys Glu Gly Ile Trp Ser Glu Arg Arg Leu Pro Lys Val Gln Glu Met 305 310 315 320 Pro Arg Pro Lys Ala His Trp Asp Phe Leu Ile Glu Glu Met Val Trp 325 330 335 Leu Ala Ala Asp Phe Ala Gln Glu Arg Lys Trp Lys Lys Ala Ala Ala 340 345 350 Lys Lys Cys Ala Arg Met Val Gln Lys Tyr Phe Gln Asp Lys Ala Leu 355 360 365 Ala Ala Gln Lys Ala Glu Lys Ala His Glu Gln Asn Leu Arg Arg Ile 370 375 380 Ala Ala Phe Cys Ala Lys Glu Ile Lys Ile Phe Trp Asn Asn Val Glu 385 390 395 400 Lys Leu Val Glu Tyr Lys Gln Asn Thr Ile Leu Glu Glu Lys Arg Lys 405 410 415 Lys Ala Leu Asp Gln Gln Leu Ser Phe Ile Val Asp Gln Thr Glu Lys 420 425 430 Tyr Ser Gln Leu Leu Ala Glu Gly Met Asn Lys Thr Ala Glu Gln Pro 435 440 445 Pro Ser Ser Ala Pro Ser Arg Ser Val Ser Arg Thr Gln Ser Asp Thr 450 455 460 Glu Phe Asp Pro Asp Leu Gln Ser Asp Glu Asp Asp Glu Glu Thr Ile 465 470 475 480 Ala Arg Glu Glu Ala Leu Gly Asn Glu Gly His Lys Glu Glu Ile Glu 485 490 495 Ala Leu Gln Lys Glu Ser Gln Met Glu Leu Asp Asp Leu Leu Glu Asp 500 505 510 Asp Phe Leu Arg Asp Tyr Leu Leu Asn Arg Asp Thr Ile Arg Phe Ser 515 520 525 Glu Ser Glu Asp Ser Asp Asp Asp Thr Asp Ser Lys Lys Glu Ser Phe 530 535 540 Lys Gly Asp Lys Glu Gln Ser Asp Asp Ser Glu Ser Ser Lys Glu Glu 545 550 555 560 Asp Thr Glu Asp Glu Ser Glu Asp Glu Ser Met Lys Val Thr Glu Ser 565 570 575 Val Val Lys Glu Glu His Asp Glu Leu Lys Ile Leu Val Glu Asp Ser 580 585 590 Gln Lys Glu Gly Glu Ile Lys Thr Glu Gln Asp Thr Lys Asp Asp Leu 595 600 605 Ile Asn Asp Ala Ala Ala Ile Ala Glu Ser Ile Gln Pro Lys Gly Asn 610 615 620 Thr Leu Ser Ser Thr Asn Val Ser Thr Asn Ile Pro Phe Leu Leu Lys 625 630 635 640 Tyr Thr Leu Arg Glu Tyr Gln His Ile Gly Leu Asp Trp Leu Val Thr 645 650 655 Met Phe Asp Arg Lys Leu Asn Gly Ile Leu Ala Asp Glu Met Gly Leu 660 665 670 Gly Lys Thr Ile Gln Thr Ile Ala Leu Leu Ala His Leu Ala Cys Glu 675 680 685 Lys Glu Asn Trp Gly Pro His Leu Ile Val Val Pro Thr Ser Val Met 690 695 700 Leu Asn Trp Glu Met Glu Cys Lys Lys Trp Cys Pro Ala Phe Lys Ile 705 710 715 720 Leu Thr Tyr Tyr Gly Thr Gln Lys Glu Arg Lys Phe Lys Arg Ile Gly 725 730 735 Trp Thr Lys Pro Asn Ala Phe His Ile Cys Ile Thr Ser Tyr Lys Leu 740 745 750 Val Ile Gln Asp His Gln Ser Phe Arg Arg Lys Lys Trp Lys Tyr Leu 755 760 765 Ile Leu Asp Glu Ala Gln Asn Ile Lys Asn Phe Lys Ser Gln Arg Trp 770 775 780 Gln Leu Leu Leu Asn Phe Gln Thr Gln Gln Arg Leu Leu Leu Thr Gly 785 790 795 800 Thr Pro Leu Gln Asn Asn Leu Met Glu Leu Trp Ser Leu Met His Phe 805 810 815 Leu Met Pro Asn Val Phe Gln Ser His Arg Glu Phe Lys Glu Trp Phe 820 825 830 Ser Asn Pro Val Thr Gly Met Ile Glu Gly Asn Ser Glu Tyr Asn Glu 835 840 845 Ser Ile Ile Lys Arg Leu His Lys Val Leu Arg Pro Phe Leu Leu Arg 850 855 860 Arg Leu Lys Ser Glu Val Glu Lys Gln Met Pro Lys Lys Tyr Glu His 865 870 875 880 Val Val Met Cys Arg Leu Ser Lys Arg Gln Arg Tyr Leu Tyr Asp Asp 885 890 895 Tyr Met Ser Arg Ala Lys Thr Arg Glu Thr Leu Thr Thr Gly Asn Leu 900 905 910 Leu Ser Val Ile Asn Val Leu Met Gln Leu Arg Lys Val Cys Asn His 915 920 925 Pro Asn Leu Phe Glu Ile Arg Pro Thr Thr Ser Pro Phe Gln Cys Asp 930 935 940 Asn Ile Arg Leu His Ile Pro Ser Ile Val Tyr Ser Ala Leu Asp Tyr 945 950 955 960 Asp Pro Asp Lys His Val Asn Leu Gln Ala Leu Asn Leu Leu Leu Ile 965 970 975 Met Gln Glu Ile His Phe Gly Ser Tyr Gln Cys Tyr Arg Met Arg Gln 980 985 990 Ser Arg Asn Ser Lys Lys Ile Phe Glu Met Glu Thr Asn Ser Ser Lys 995 1000 1005 Asn Pro Pro Pro Cys Pro Pro Cys Lys Leu Ala Met Arg Val Leu 1010 1015 1020 Thr Asp Lys Pro Ser Ala Thr Asp Glu Lys Asn Glu Lys Lys Asp 1025 1030 1035 Met Gln Ala Leu Ser Gln Pro Pro Pro Leu Gln Val Lys Gly Met 1040 1045 1050 Ser Gln Pro Asn Met Lys Met Lys Val Ser Gly Val Gln Phe Val 1055 1060 1065 Pro Gln Ser Ile Leu Lys Ser Ile Pro Val Val Asn Ile Ser Gln 1070 1075 1080 Gly Ala Thr Gly Gln Ile Gly Ala Pro Val Ser Val Thr Ser Val 1085 1090 1095 Leu Lys Pro Gln Asp Lys Ile Ser Ala Ser Phe Ala Gln Leu Val 1100 1105 1110 Gln Thr Ser Thr Gly Lys His Leu Leu Leu Thr Ser Asn Pro Asn 1115 1120 1125 Ile Thr Thr Ser Pro Val Thr Thr Thr Thr Pro Gly Gly Gln Lys 1130 1135 1140 Leu Thr Phe Leu Ser Lys Gln Pro Val Ser Thr Ile Gly Asn Ala 1145 1150 1155 Gly His Ala Val Thr Lys Ala Tyr Val Lys Phe Gln Leu Thr Ser 1160 1165 1170 Val Thr Thr Ala Ser Thr Phe Thr Thr Val Thr Thr Val Asn Ser 1175 1180 1185 Asn Thr Ile Ser Val Ala Lys Ser Glu Asp Asn Lys Gly Met Arg 1190 1195 1200 Met Ser Val Gly Asn Asp Tyr Ile Gly Lys Leu Tyr Ser Lys Gln 1205 1210 1215 Asn Ser Leu Asp Val Arg Trp Asn Ser Gly Glu Lys His Leu Gly 1220 1225 1230 Leu Thr Asn Glu Asp Asp Pro Lys Gly Glu Arg Lys Lys Arg Leu 1235 1240 1245 Ser Leu Met Ser Arg Ile Asn Lys Ile Arg Cys Ser Ala Leu Pro 1250 1255 1260 Leu Tyr Gly Arg Asp Phe Gln Glu Ala Val Lys Ile Tyr Thr Pro 1265 1270 1275 Asn Gln Leu Asp Val Trp Asn Gly Gly His Ile His Cys Leu Asn 1280 1285 1290 Thr Leu Tyr Asn Lys Asp Ala Arg Asn Glu Thr Thr Asp Cys Leu 1295 1300 1305 Gln Asp Ala Leu Phe Asn Pro Glu Arg Arg Leu Glu Ala Leu Lys 1310 1315 1320 Asp Thr Phe Asp Arg Phe Ile Phe Tyr Val Pro Ser Val Lys Val 1325 1330 1335 Ala Glu Pro Glu Leu Gln Val Trp His Pro Pro Pro Ser Lys Tyr 1340 1345 1350 Trp Gly Gln Lys His Glu Lys Gln Leu Ile Gln Lys Leu Phe Leu 1355 1360 1365 Lys Pro Ala Thr Pro Leu His Ser Ile Ala Ser Ala Met Val Thr 1370 1375 1380 Gln Phe Pro Asp Pro Arg Leu Ile Gln Tyr Asp Cys Gly Lys Leu 1385 1390 1395 Gln Thr Leu Asp Ile Leu Leu Arg Lys Leu Lys Leu Gly Ser His 1400 1405 1410 Arg Val Leu Ile Phe Thr Gln Met Thr Lys Met Leu Asp Val Leu 1415 1420 1425 Glu Ala Phe Leu Asn Tyr His Gly His Ile Tyr Leu Arg Leu Asp 1430 1435 1440 Gly Thr Thr Lys Val Asp Gln Arg Gln Val Leu Met Glu Arg Phe 1445 1450 1455 Asn Gly Asp Lys Arg Ile Phe Ala Phe Ile Leu Ser Thr Arg Ser 1460 1465 1470 Gly Gly Val Gly Val Asn Leu Thr Gly Ala Asp Thr Val Ile Phe 1475 1480 1485 Tyr Asp Ser Asp Trp Asn Pro Thr Met Asp Ala Gln Ala Gln Asp 1490 1495 1500 Arg Cys His Arg Ile Gly Gln Thr Arg Asp Val His Ile Tyr Arg 1505 1510 1515 Leu Val Ser Glu Arg Thr Ile Glu Glu Asn Ile Leu Lys Lys Ala 1520 1525 1530 Asn Gln Lys Arg Leu Leu Gly Asp Leu Ala Ile Glu Gly Gly Asn 1535 1540 1545 Phe Thr Thr Ala Tyr Phe Lys Ser Ser Thr Ile Gln Asp Leu Phe 1550 1555 1560 Asn Ile Asp Gln Asn Glu Glu Ser Ala Ser Ala Arg Met Ser Glu 1565 1570 1575 Val Val Glu Leu Arg Lys Glu Arg Glu Lys Ala Leu Ser Thr Asp 1580 1585 1590 Leu Val His Ser Ala Asp Asp Lys Ala Thr Val Gly Ala Leu Glu 1595 1600 1605 Asn Ala Phe Glu Ala Cys Glu Asp Asp Gln Asp Val Gln Ala Ala 1610 1615 1620 Lys Thr Ala Lys Ala Glu Ala Val Ala Asp Leu Ala Glu Phe Asp 1625 1630 1635 Glu Asn Ile Pro Leu Asp Asp Gln Glu Lys Glu Pro Glu Ile Ser 1640 1645 1650 Lys Ala Glu Gln Glu Ile Asn Asn Ile Ile Glu Lys Leu Thr Pro 1655 1660 1665 Ile Glu Lys Tyr Ala Met Asn Phe Ile Glu Ala Thr Glu Ser Ala 1670 1675 1680 Trp Ser Ala Glu Gln Leu Ala Ala Ala Ala Arg Glu Ile Glu Glu 1685 1690 1695 Gln Lys Lys Glu Trp Glu Gln Asn Arg Leu Ala Ala Met Arg Glu 1700 1705 1710 Glu Glu Glu Arg Arg Ala Arg Glu Leu Glu Glu Glu Ser Asp Ile 1715 1720 1725 Ile Thr Tyr Ser Arg Asp Asp Ala Thr Asn Gln Val Ser Ser Lys 1730 1735 1740 Asn Lys Lys Ile Asn Arg Tyr Asn Lys Ile Leu Ser Asn Lys Arg 1745 1750 1755 Val Arg Leu Lys Lys Asp Gly Asp Glu Asp Val Glu Lys Lys Asp 1760 1765 1770 Asp Val Glu Lys Lys Asp Gly Val Glu Lys Arg Leu Lys Lys Thr 1775 1780 1785 Arg Thr Arg Arg Leu Ser Gln Lys Ser Lys Asp Val Glu Val Glu 1790 1795 1800 Glu Pro Asp Ala Cys Glu Ser Gln Glu Glu Ser Gln Ile Asn Gly 1805 1810 1815 Gly Asp Thr Asp Asn Ser Asp Ser Asp Ser Asp Ser Asp Ser Glu 1820 1825 1830 Ser Ser Ser Ser Met Glu Ser Lys Thr Thr Leu Asn His Val Asp 1835 1840 1845 Pro Asn Ser Pro Arg Thr Arg Ser Arg Gly Thr Val Ala Ile Asn 1850 1855 1860 Leu Trp Thr Leu Asp Val Ser Pro Ile Leu Pro Gly Glu Lys Pro 1865 1870 1875 Met Lys Lys Tyr Gly Glu Asn His Arg Lys Asn Ile Lys Arg Val 1880 1885 1890 Arg Ser Val Ser Glu Asn Asp Asn Asp Gly Asp Lys Asp Gly Arg 1895 1900 1905 Lys Arg Leu Arg Arg Lys Tyr Pro Thr Ser Leu Glu Thr Ser Glu 1910 1915 1920 Glu Glu Asn Ser Asn Gln Ser Arg Glu Lys Ser Thr Lys Lys Arg 1925 1930 1935 Ala Lys Val Ala Pro Lys Gly Lys Thr Cys Lys Val Ile Leu Ser 1940 1945 1950 Asn Ile Leu Asn Asp Lys Arg Phe Lys Val Asn Leu Lys Glu Asp 1955 1960 1965 Ile Glu Ile Ser Val Ser Thr Gln Ile Asn Glu Thr Ser Thr Ser 1970 1975 1980 Ser Asn Gln Asn Gln Thr Lys Asp Cys Glu Ser Ser Gln His Glu 1985 1990 1995 Asn Ser Asn Leu Asp Glu Gln Asn Asp Ser Leu Asp Asn Thr Glu 2000 2005 2010 Val Thr Ser Ser Glu Leu Ser Lys Leu Thr Gly Cys Thr Glu Ile 2015 2020 2025 Asp Asn Asn Glu Ser Ser Lys Gln Glu Asn Glu Glu Leu Asp Glu 2030 2035 2040 Ser Ile Leu Glu Asp Lys Tyr Asp Glu Asp Phe Ile Thr Asn Lys 2045 2050 2055 Asn Glu Asp Ile Asp Glu Glu Thr Leu Leu Glu Glu Asp Asn Gln 2060 2065 2070 Ile Glu Gln Val Glu Asn Lys Asn Ile Asp Ser Thr Lys Asp Glu 2075 2080 2085 Lys Gln Gly Asp Asp Ser Asn Val Ser Asp Val Gly His Leu Ser 2090 2095 2100 Lys Asp Asn Asp Asn Glu Glu Lys Met Glu Val Thr Glu Ser Val 2105 2110 2115 Asp Glu Glu Asn Gly Asp Ile Asn Lys Lys Val Asp Glu Asp Glu 2120 2125 2130 Ser Val Lys Asp Lys Arg Glu Arg Arg Lys Gly Asn Glu Glu Asp 2135 2140 2145 Asp Asn Thr Asp Asn Glu Glu Asn Ile Gln Lys Ser Glu Asn Asp 2150 2155 2160 Glu Gly Asp Ile Lys Lys Gln Gly Asn Gln Asp Glu Glu Val Glu 2165 2170 2175 Glu Lys Thr Leu Gly Asn Ser Thr Glu Ser Val Asn Glu Ile Ala 2180 2185 2190 Asn Glu Ile Ser Arg Cys Lys Pro Leu Asn Glu Gln His Asn Glu 2195 2200 2205 Leu Val Asp Glu Val Val Asn Asp Thr Ser Asn Met Asp Glu Tyr 2210 2215 2220 Ile Lys Lys Ser Glu Asn Ser Lys Val Val Glu Lys Thr Ser Glu 2225 2230 2235 Glu Ile Leu Phe Asn Asp Arg Gly Asn Gln Asp Ser Ser Ser Gln 2240 2245 2250 Asp Val Lys Asp Glu Glu Ile Ser Ser His Asn Arg Gly Asp Glu 2255 2260 2265 Lys Val Ser Phe His Asp Arg Ile Asp Lys Glu Val Leu Pro Glu 2270 2275 2280 Cys Arg Lys Glu Glu Glu Lys His Asn Arg Lys Asn Glu Val Leu 2285 2290 2295 Ser Gln Thr Ile Lys Asp Glu Glu Ala Gln Ser His Asn Arg Lys 2300 2305 2310 Asp Glu

Ser Gly Thr Phe Pro Asn Val Ala Asp Ile Glu Asn Arg 2315 2320 2325 Leu Asn Asn Lys Val Pro His Val Asp Asn Gly His Thr Glu Thr 2330 2335 2340 Val Arg Met Ser Lys Leu Val Thr Ser Asn Arg Asn Ala Asn Phe 2345 2350 2355 Arg Ser Pro Glu Thr Arg Arg Ser Phe Arg Lys Cys Gly Lys Ile 2360 2365 2370 Ser Asn Asn Gln Thr Leu Asp Gly Trp Val Lys Arg Ser Pro Val 2375 2380 2385 Leu Pro Val Glu Ala Ala Lys Val Asn Asp Asn Ser Lys Tyr Lys 2390 2395 2400 Asn Val Gly Ser Pro Glu Leu 2405 2410 93240DNAArtificial SequenceSNF2-helicase degenerate dsRNA sequencemisc_feature(216)..(216)n is a, c, g, or tmisc_feature(222)..(222)n is a, c, g, or t 93cgsythctyy tmacsggyac hcctctvcar aayaarctwc chgarytstg ggcbytdcth 60aayttyytvc tbccstcbat yttyaarwsb tgytcbacdt tygarcartg gttcaaygcv 120cchttygcha cmacbggmga raargtygar ytdaaygarg argaracvat yytkatyaty 180mgdcgtytdc ayaargtyyt kcgwccktty ytvytnmgdc gnytvaaaaa rgargtmgar 2409427DNAArtificial SequenceSNF2-helicase degenerate dsRNA sequencemisc_feature(21)..(21)n is a, c, g, or t 94mghgcygtbt gyythatygg ngaycar 279560DNAArtificial SequenceSNF2-helicase degenerate dsRNA sequence 95tayaarctyc tvytsacmgg machccgytb caraacaayc tmgargaryt rttycatytr 609661DNAArtificial SequenceSNF2-helicase degenerate dsRNA sequence 96garttygaya cbaaycaymg rctkcthath acwggbacyc ckytvcaraa ywskytdaar 60g 619723DNAArtificial SequenceBromodomain degenerate dsRNA sequence 97ytswsygaac crttyatgaa ryt 239865DNAArtificial SequenceHAND-SLIDE degenerate dsRNA sequence 98gchgtvgatg cytayttymg vgargcwytv mgdgtytchg arccyaargc dccdaargch 60cchmg 659936DNAArtificial SequenceChromodomain degenerate dsRNA sequencemisc_feature(33)..(33)n is a, c, g, or t 99mghaartrbg ayatggavga rvvdccbaar ytngar 3610056DNAArtificial SequenceChromodomain degenerate dsRNA sequencemisc_feature(29)..(29)n is a, c, g, or t 100bhggdaarad dggrkkbryb ggmaaymwna chacdrtsta ykmhrtagar gaaaay 56101322DNADiabrotica virgifera 101aagaaggcat agaacagaaa gagagttggg cgaacgaata tctcagttca tttaaagtgg 60ctagttatgt tacaaaagaa ggggaagttg aggaagaagt tgacactgag attattaaac 120aagaagcgga aaataccgat ccggcctact ggatcaagct gcttagacat cattatgagc 180aacaacagga agatatagct aggacgttag gaaaaggcaa aagagtgagg aaacaggtta 240attataatga cggaggaatg acaactgaca cacgagaaga tacgacatgg caagaaaatc 300tctctgatta ccattctgac tt 322102401DNADiabrotica virgifera 102tcacagtcga aacacccact gaatcagtct accaattcga aggagaagat tatcgtgaga 60agcagaaaac catcggcttg agcaactgga tagaacctcc caaaagagaa aggaaggcca 120actatgccgt cgatgcttac ttcagagaag ctttaagggt ttctgagcct aaagcgccta 180aggctccaag accaccaaaa cagcccatcg tacaagattt ccagtttttc ccgccgagat 240tattcgaact tttggaccag gagatctact tttacaggaa atctttggga tataaggttc 300cgaaaaactt agaacttgga cctgacgcgt ccaagcaaca gaaagaagag caaagaaaaa 360tagatgagtc agaaccgctc accgaagacg aacagcaaga a 401103271DNADiabrotica virgifera 103ttgctcaatc ctacatacac ttttaactcc ttcttgggtt ttagcccttt ctctacttca 60attttcaatc ttctaagcaa gaatggtttt aatacagcat gcagcctctc caccatggag 120ttgcctccca gacattgact ggtgttaaac caggcatcga aatcatcaga tgagttaaaa 180acgtctggca gtaagaagtt gagaagagac cagagttcat gtaaattgtt ttgtaatgga 240gtacctgtta gcagtagcct gttggtattc t 271104372DNADiabrotica virgifera 104gatcaaattc aagcaactag cgaaattgga aaaaaaatca gacgaagaac ttactgaata 60ttacaaacat ttcgttatga tgtgcaagaa gcagacaggc atgaacatag aagacagcaa 120ctatgacaat accatcgaac atatctcaga agaaaaggca cgaaggacat tggaaaggct 180ggagctgttg tcgaggatca gagaagaaat tttaacccat cctaaactcg acgaaagatt 240gagggtgtgc attacttcgg ctgatatgcc tgaatggtgg attgccggca aacacgacaa 300ggatctcttg ttgggggtcg ccaaacatgg tttaggaaga accgactact accttctgaa 360cgatcctgat ct 372105315DNADiabrotica virgifera 105tttgcttcct tctttcaact cgcgcagggg gcttgggcat taatttagct actgctgata 60ctgtgataat ttttgattcg gactggaatc ctcaaaacga tcttcaagcg caggcaagag 120ctcataggat cggtcaaaag aaccaagtca acatttatag gttagttact gctagatctg 180tagaggaaga aattgtagaa agggcaaaac aaaaaatggt actggatcat cttgtaattc 240agagaatgga cacgacggga agaaccgttt tggacaaaaa ggggtcttct aataataatc 300cgtttaacaa agaag 315106449DNADiabrotica virgifera 106acttatctaa agggatgcta gctgagttcg atgtcatact cacaacgtat acgctggttg 60gaaatagttc agaagagaga aaaatgttcc gagtgacaag gatgcattat gtaatcttcg 120atgaagcaca tatgttgaaa aatatgaata ctcttcggta tgaaaattta attaagataa 180acgctaaaca taggatactg ttaaccggca ctccgttaca aaataattta ttagaactaa 240tgtcgctgtt gatatttgtg atgccgaata tattcgctga aaaaaggtgg acttgaaaaa 300cttattccaa aaaaattcta aaaaagcaga agacgactct ctacctacct tcgaaaagga 360gcaaattgaa caagccaaaa gaattatgaa acctttcctt ttgcgaagac tgaaatgtga 420cgtccttcgg gatcttccca agaaaacgg 44910738DNAArtificial SequencePrimer Mi2.T7.F 107taatacgact cactataggg aagaaggcat agaacaga 3810839DNAArtificial SequencePrimer Mi2.T7.R 108taatacgact cactataggg tcagaatggt aatcagaga 3910938DNAArtificial SequencePrimer ISWI30.T7.F 109taatacgact cactataggg tgaatcagtc taccaatt 3811038DNAArtificial SequencePrimer ISWI30.T7.R 110taatacgact cactataggg ggttctgact catctatt 3811139DNAArtificial SequencePrimer ISWI2.T7.F 111taatacgact cactataggg ttgctcaatc ctacataca 3911238DNAArtificial SequencePrimer ISWI2.T7.R 112taatacgact cactataggg gaataccaac aggctact 3811338DNAArtificial SequencePrimer KSMT.T7.F 113taatacgact cactataggg gatcaaattc aagcaact 3811438DNAArtificial SequencePrimer KSMT.T7.R 114taatacgact cactataggg ttcttcctaa accatgtt 3811538DNAArtificial SequencePrimer CHD1.T7.F 115taatacgact cactataggg tttgcttcct tctttcaa 3811639DNAArtificial SequencePrimer CHD1.T7.R 116taatacgact cactataggg cttctttgtt aaacggatt 3911740DNAArtificial SequencePrimer ETL1.T7.F 117taatacgact cactataggg acttatctaa agggatgcta 4011838DNAArtificial SequencePrimer ETL1.T7.R 118taatacgact cactataggg gtagagagtc gtcttctg 3811918DNAArtificial SequencePrimer Mi2.qPCR.F 119agagtgagga aacaggtt 1812021DNAArtificial SequencePrimer Mi2.qPCR.R 120aagtcagaat ggtaatcaga g 2112120DNAArtificial SequencePrimer Mi2.qPCR.F3 121agttgggcga acgaatatct 2012220DNAArtificial SequencePrimer Mi2.qPCR.R3 122cggtattttc cgcttcttgt 2012322DNAArtificial SequencePrimer ISWI30.qPCR.F 123aggaaatctt tgggatataa gg 2212418DNAArtificial SequencePrimer ISWI.qPCR.R 124ttcttgctgt tcgtcttc 1812520DNAArtificial SequencePrimer ISWI30.qPCR.F 125tcacagtcga aacacccact 2012620DNAArtificial SequencePrimer ISWI.qPCR.R 126tggccttcct ttctcttttg 2012720DNAArtificial SequencePrimer ISWI2.qPCR.F 127gcagtaagaa gttgagaaga 2012819DNAArtificial SequencePrimer ISWI2.qPCR.R 128agaataccaa caggctact 1912918DNAArtificial SequencePrimer KSMT.qPCR.F 129cacgaaggac attggaaa 1813018DNAArtificial SequencePrimer KSMT.qPCR.R 130gcacaccctc aatctttc 1813124DNAArtificial SequencePrimer CHD1.qPCR.F 131ttataggtta gttactgcta gatc 2413218DNAArtificial SequencePrimer CHD1.qPCR.R 132tcgtgtccat tctctgaa 1813320DNAArtificial SequencePrimer ETL1.qPCR.F 133tgatatttgt gatgccgaat 2013418DNAArtificial SequencePrimer ETL1.qPCR.R 134agagtcgtct tctgcttt 181354768RNADiabrotica virgifera 135caaguggcca uggcaugcca cagggucccc cuggacaacc aggucagcaa caccaaggcc 60gaacugcuga uaauuuacau gccuuacaaa aagcaauaga uacaauggaa gaaaaaggua 120ugcaagaaga ucagagguau ucacaguuac uggcguuacg ugcuagaucc aguggucaac 180caucuaacgg aguucuuaca ccgcugcaaa ugaaucaacu uagaaaucaa auuauggcau 240acaggugccu agcgaggagc caaccaauuc cuccuucaau aauguugggg cugcaaggaa 300agaggccuga cgguucacca caguuuccua caccuccguc aaguccguuu caaccacaag 360gaccuggugc acccccuggu ccggaacaac caccagcuaa ugcagaaaac guagcagagc 420cagcagcacc aguaggaccg caaggugcac aaggaccucc uaaccaacag agagcucaaa 480cuagccaguu aguccccaau aagcaaacuc guuucacuac caugcccaaa ccaucuggac 540uagauccacu aguucuucuu caagagaggg aaacuagggu ggcagcuaga aucgcugcua 600gaauagaaca auguaguaac uuaccuacca aucuuucaga caaaguccgc augcaagcac 660agauagaauu gagagcuuug cggugccuua auuuccaaag gcaacuaaga agcgaaauuu 720ugaacuguau uaggagagau auaacgcuug aaucugcugu aaauuuuaaa gcauauaaaa 780gaacgaagcg acagggucua aaagaaucga gagcuacaga gaaguuagaa aaacaacaga 840aguuagaagc agaaagaaag agaagacaga agaaccaaga auuuuugaau gcuguauuga 900acaauggaaa agaauucaag gaauuccaca agcagaauca agcgaaauua gcuaagauua 960auaaagcugu uauuaauuau 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4380uuaccuaaau gauuuagaaa agcaucugag uuauuuagau aaguuuugag auuauauuua 4440uuaacuuuaa uauuacuauc uuuauuauag cauauuguaa uuauuuuuuc cuguccuucu 4500uucguugugu gguagauaau ccgagaguca acaguuauaa gcaaaugaaa uucaguuaaa 4560ccucaaaugu acaaaaugau caaauuaaug uuuacaauuu auuuuuuuac cacgcacauc 4620cacuauuacu auugucaguc auugagauau cauuuuauau agcuccaugu cugucuuccu 4680caauuuacag agaagcaauu agacaaguaa ugacauaaua uggugcugaa auaaugugcu 4740ugauagugau guugaaaaag uaacuauu 47681365147RNADiabrotica virgifera 136ucacgugccu ccacaaggcc auguuccucc acagggucac gugccuccac aaggccaugu 60uccuccacag ggacaucuuc cuccacaggg ccauauucca ccacaggguc augguccaac 120gcagggccac auaccuccuc aggggcaugu uccaccacaa ggacauauac cuccucaagg 180gcaugcaccc ccacaaggac augcaccacc acaggggcau ccuggcguuc cuccugguca 240ucagagucau ccucaagggc auccacaaac accagggcau ccuggaccug gacauauacc 300accuggugga gcaaugcacc cagggcauua uccaaguggc cauggcaugc cacagggucc 360cccuggacaa ccaggucagc aacaccaagg ccgaacugcu gauaauuuac augccuuaca 420aaaagcaaua gauacaaugg 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agggcaguaa aggaagggga ucuaaggcac ugaugaacac 2760cauuaugcag cugaggaaac ugugcaauca uccguuuaug uuccaaaaua ucgaagagaa 2820auauugugau cauguuggua uugcuggugg agugguuucu ggacccgaca cuuauagggu 2880aucugguaag uuugagcucu uggacagaau auugcccaaa augaaagcaa cuaaccauag 2940gauucuucuu uucugucaaa ugacucaauu aaugaccauc auggaagauu aucuaaauug 3000gagaggauuc aaauaucuuc gucuugaugg uacaaucaaa ucagaagauc gcggggaccu 3060auuaucgaaa uuuaaugaua aaaauaguga auauuuuuug uuuuugcuau cuacacgggc 3120uggaggucug ggacuuaauu ugcagacagc ugauacugug auuaucuucg auuccgauug 3180gaauccucau caggauuuac aagcucagga ucgagcucau cguauuggac agcaaaauga 3240gguccgaguu uugcguuuga ugacuguuaa cucuguugag gaacgaauuu uagcugcagc 3300uaaauacaag cuuacuaugg acgaaaaggu cauucaagcu gguauguucg aucagaaguc 3360uacgggaucu gaaaggcagc aguuucuuca gaguauuuua cacaaugaug guagugauga 3420agaagaggaa aaugaagugc cugaugacga aaccgucaac caaaugauag cuaggacaga 3480ggaugaguuu cagcucuucc aaaaaaugga uacggaaaga aaagaggaga augaaaaacu 3540uggucagcau aaaaagucgc gauugguuca 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ugcaagagau gccucggccu aaagcgcauu gggacuuuuu gaucgaagaa 1200auggucuggu uggcagcuga uuuugcgcag gaacgcaaau ggaagaaagc cgccgcgaaa 1260aaaugugcca gaaugguaca gaaguauuuc caagacaagg cgcucgccgc ccaaaaagcg 1320gaaaaggcuc acgaacaaaa ucuuagaagg auagccgcgu ucugugcaaa ggagauuaag 1380aucuuuugga acaacgucga aaaacucguc gaguauaaac agaauacaau cuuggaggag 1440aagcggaaaa aggcgcucga ucagcagcuu aguuuuaucg uggaucagac ugagaaguau 1500ucgcaguugc uugccgaagg aaugaauaag accgcagaac agccuccuag uucagcgcca 1560ucucgaucag ugucucgaac gcagucugau acagaauucg auccggaucu ucagagcgau 1620gaagacgacg aagagaccau ugcucgagaa gaagcuuuag guaacgaagg acauaaagaa 1680gaaaucgaag cgcuacagaa agaaucucag auggaauuag acgauuuacu agaggacgau 1740uuccugaggg auuaccuuuu aaaucgagac acgauccgau ucagcgaauc cgaagauucg 1800gacgaugaca cggacucgaa aaaagaaucu uucaaaggcg acaaagaaca gucugacgau 1860ucugaaucuu cuaaagaaga agauacggaa gacgagagcg aagaugaauc uaugaagguu 1920acugaaucgg uuguuaaaga agaacacgac gaguugaaaa uauuaguaga agauucucaa 1980aaagagggag aaauuaagac 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5400gaugacgcca ccaaccaggu uagcucaaaa aacaaaaaaa ucaauaggua uaauaaaauu 5460uuaaguaaua aaaggguuag gcucaaaaaa gauggagaug aagacguuga gaaaaaagau 5520gacgucgaga aaaaagaugg aguugaaaaa agguugaaga agacuaggac acgaaggcug 5580ucucaaaaaa gcaaagaugu agaggucgaa gaaccggaug cuugugaauc acaagaagaa 5640ucucaaauua acggagggga uacggauaau agugauagug auucugauuc ugauagcgaa 5700ucuucaucuu ccauggaauc uaaaacuacc uuaaaccacg uugauccaaa uucaccuaga 5760acuaggucua ggggcacagu ggcuauuaac cuuuggacac ucgaugucag cccgauuuug 5820ccuggagaaa aaccgaugaa aaaauacggg gagaaccaua gaaaaaauau uaagaggguu 5880agaucugugu cugaaaacga uaacgaugga gauaaagacg guagaaagcg auugaggagg 5940aaauacccca ccaguuuaga aacaucagaa gaagaaaaca guaaccaguc aagagaaaaa 6000ucuacuaaga aacgugccaa gguagcaccc aaagggaaaa cuuguaaagu aauuuuaagu 6060aauauacuga acgauaaacg auuuaaaguc aacuuaaaag aagacauuga gauuucagug 6120agcacacaaa uuaaugagac uuccaccagc ucaaaccaga aucaaaccaa agauugcgag 6180ucuagucaac augagaauag caauuuagau gaacagaaug auucucuuga caauacagaa 6240guuaccucau ccgaacuuag uaaauuaacu gguuguacug aaauagauaa uaacgaaagu 6300aguaaacagg aaaaugaaga auuagacgaa ucuauacucg aagauaaaua ugaugaagau 6360uucauuacaa acaaaaauga agacauagau gaagaaacac uccuugaaga agauaaucag 6420auagagcagg uugaaaauaa aaauauugau agcacgaaag augaaaaaca gggugaugac 6480aguaauguuu cagauguagg ucauuuaagu aaagauaacg auaaugagga aaaaauggaa 6540guaacagaaa guguugacga ggaaaauggu gauaucaaua agaaaguaga ugaagaugag 6600agcguuaaag auaaaagaga aaggagaaaa gguaaugagg aagaugauaa uacagacaau 6660gaagaaaaca uccaaaaguc agaaaaugau gagggcgaua uuaaaaagca aggaaaucaa 6720gaugaagaag uagaagagaa aaccuuagga aauucuacug aaucaguuaa cgaaauagca 6780aaugaaauaa guagaugcaa accuuuaaau gagcagcaca acgaauuggu agaugaagua 6840guaaaugaca caaguaauau ggaugaauau auaaaaaaau cggaaaauuc caaaguugua 6900gaaaagacaa gugaagaaau acuauucaac gauaggggaa aucaagauuc aucgucccag 6960gauguaaaag augaagagau aucaucccac aacagaggag augaaaaggu gucauuccac 7020gauaggauag auaaagaggu auuaccugaa uguaggaaag aggaagagaa acacaauaga 7080aaaaaugaag uacuaucaca aacuauaaaa gaugaagagg cacaguccca caauaggaaa 7140gaugaaucgg guacauuucc aaacguagca gauauagaaa auagacuuaa caacaaaguu 7200ccucacgucg auaaugguca uaccgaaacg gucagaaugu cuaaauuggu aacgucuaau 7260agaaaugcca auuucagguc uccugaaacg agaagaagcu ucagaaagug ugguaaaauu 7320ucgaacaauc agacuuuaga cgguugggug aaacggucgc cuguauuacc ugucgaagcu 7380gcuaaaguaa acgauaacuc aaaauauaaa aauguugguu cgcccgaguu auagggagau 7440uugacugugg aaagaggaga aauuuaaaag uuuuugauag uuaaaaaagu guuuauugau 7500caauguacac ugcaauaagg uaauaacuca aaaauaauuu auuucuuuac aguuuucuuc 7560uucauccacu ccuggacaaa ggccucacga aguguuuuac aaguuucuuu gauaacgggu 7620aaaaccaaaa aauaguaaag uucgcauaac cuucuggcua augucuaguc uuaggguuca 7680agucgcgcaa gcgcccagac cgaccgcuuu acgugccuuc cgaaugacgg cggcggcuca 7740gaacaauuuc uuaaugucag ugccaggaau cuaaccuaga uccucuagcu uguuaaguca 7800guaauaaaca uuaguucaac ggacauauug ucacuuuauu gcgcaugcgu uuaucauuac 7860ugagucuuga guucugccua gccguaccgu ugacauauuc guaaagauug uaguaauaag 7920gcaugcauca gaucuuuaua gauuucaaac augcuuauga cucaguuaca auucguccaa 7980gacuauggaa ugguauaguu gagcuggaca uaccuaagaa auuagc 8026150240RNAArtificial SequenceSNF2/Helicase degenerate dsRNA sequencemisc_feature(216)..(216)n is a, c, g, or umisc_feature(222)..(222)n is a, c, g, or u 150cgsyuhcuyy umacsggyac hccucuvcar aayaarcuwc chgaryusug ggcbyudcuh 60aayuuyyuvc ubccsucbau yuuyaarwsb ugyucbacdu uygarcarug guucaaygcv 120cchuuygcha cmacbggmga raarguygar yudaaygarg argaracvau yyukauyauy 180mgdcguyudc ayaarguyyu kcgwcckuuy yuvyunmgdc gnyuvaaaaa rgargumgar 24015127RNAArtificial SequenceSNF2/Helicase degenerate dsRNA sequencemisc_feature(21)..(21)n is a, c, g, or u 151mghgcygubu gyyuhauygg ngaycar 2715260RNAArtificial SequenceSNF2/Helicase degenerate dsRNA sequence 152uayaarcuyc uvyusacmgg machccgyub caraacaayc umgargaryu ruuycauyur 6015361RNAArtificial SequenceSNF2/Helicase degenerate dsRNA sequence 153garuuygaya cbaaycaymg rcukcuhauh acwggbacyc ckyuvcaraa ywskyudaar 60g 6115423RNAArtificial SequenceBromodomain degenerate dsRNA sequence 154yuswsygaac cruuyaugaa ryu 2315565RNAArtificial SequenceHAND-SLIDE degenerate dsRNA sequence 155gchguvgaug cyuayuuymg vgargcwyuv mgdguyuchg arccyaargc dccdaargch 60cchmg 6515636RNAArtificial SequenceChromodomain degenerate dsRNA sequencemisc_feature(33)..(33)n is a, c, g, or u 156mghaarurbg ayauggavga rvvdccbaar yungar 3615756RNAArtificial SequenceChromodomain degenerate dsRNA sequencemisc_feature(29)..(29)n is a, c, g, or u 157bhggdaarad dggrkkbryb ggmaaymwna chacdrusua ykmhruagar gaaaay 56158322RNADiabrotica virgifera 158aagaaggcau agaacagaaa gagaguuggg cgaacgaaua ucucaguuca uuuaaagugg 60cuaguuaugu uacaaaagaa ggggaaguug aggaagaagu ugacacugag auuauuaaac 120aagaagcgga aaauaccgau ccggccuacu ggaucaagcu gcuuagacau cauuaugagc 180aacaacagga agauauagcu aggacguuag gaaaaggcaa aagagugagg aaacagguua 240auuauaauga cggaggaaug acaacugaca cacgagaaga uacgacaugg caagaaaauc 300ucucugauua ccauucugac uu 322159401RNADiabrotica virgifera 159ucacagucga aacacccacu gaaucagucu accaauucga aggagaagau uaucgugaga 60agcagaaaac caucggcuug agcaacugga uagaaccucc caaaagagaa aggaaggcca 120acuaugccgu cgaugcuuac uucagagaag cuuuaagggu uucugagccu aaagcgccua 180aggcuccaag accaccaaaa cagcccaucg uacaagauuu ccaguuuuuc ccgccgagau 240uauucgaacu uuuggaccag gagaucuacu uuuacaggaa aucuuuggga uauaagguuc 300cgaaaaacuu agaacuugga ccugacgcgu ccaagcaaca gaaagaagag caaagaaaaa 360uagaugaguc agaaccgcuc accgaagacg aacagcaaga a 401160271RNADiabrotica virgifera 160uugcucaauc cuacauacac uuuuaacucc uucuuggguu uuagcccuuu cucuacuuca 60auuuucaauc uucuaagcaa gaaugguuuu aauacagcau gcagccucuc caccauggag 120uugccuccca gacauugacu gguguuaaac caggcaucga aaucaucaga ugaguuaaaa 180acgucuggca guaagaaguu gagaagagac cagaguucau guaaauuguu uuguaaugga 240guaccuguua gcaguagccu guugguauuc u 271161372RNADiabrotica virgifera 161gaucaaauuc aagcaacuag cgaaauugga aaaaaaauca gacgaagaac uuacugaaua 60uuacaaacau uucguuauga ugugcaagaa gcagacaggc augaacauag aagacagcaa 120cuaugacaau accaucgaac auaucucaga agaaaaggca cgaaggacau uggaaaggcu 180ggagcuguug ucgaggauca gagaagaaau uuuaacccau ccuaaacucg acgaaagauu 240gagggugugc auuacuucgg cugauaugcc ugaauggugg auugccggca aacacgacaa 300ggaucucuug uugggggucg ccaaacaugg uuuaggaaga accgacuacu accuucugaa 360cgauccugau cu 372162315RNADiabrotica virgifera 162uuugcuuccu ucuuucaacu cgcgcagggg gcuugggcau uaauuuagcu acugcugaua 60cugugauaau uuuugauucg gacuggaauc cucaaaacga ucuucaagcg caggcaagag 120cucauaggau cggucaaaag aaccaaguca acauuuauag guuaguuacu gcuagaucug 180uagaggaaga aauuguagaa agggcaaaac aaaaaauggu acuggaucau cuuguaauuc 240agagaaugga cacgacggga agaaccguuu uggacaaaaa ggggucuucu aauaauaauc 300cguuuaacaa agaag 315163449RNADiabrotica virgifera 163acuuaucuaa agggaugcua gcugaguucg augucauacu cacaacguau acgcugguug 60gaaauaguuc agaagagaga aaaauguucc gagugacaag gaugcauuau guaaucuucg 120augaagcaca uauguugaaa aauaugaaua cucuucggua ugaaaauuua auuaagauaa 180acgcuaaaca uaggauacug uuaaccggca cuccguuaca aaauaauuua uuagaacuaa 240ugucgcuguu gauauuugug augccgaaua uauucgcuga aaaaaggugg acuugaaaaa 300cuuauuccaa aaaaauucua aaaaagcaga agacgacucu cuaccuaccu ucgaaaagga 360gcaaauugaa caagccaaaa gaauuaugaa accuuuccuu uugcgaagac ugaaauguga 420cguccuucgg gaucuuccca agaaaacgg 4491644125DNADiabrotica virgifera 164atggcatcag atgaagaagt ggaggattct ttcgccgggg aggaagatgc ccccgacgat 60acggctgaac aaatagataa cgatcctgat tctgaagatg gtgttcctaa aggaggggaa 120gaagatgatg attatgaacc agaagattcc agaaagaaaa agaagggaaa gaaaagaaaa 180gccaggggag aagaaaagaa aggcaagaaa aagaagaaaa agcgaaagaa tgatagtggg 240gatgaaagtg actttggaga agatgataat ggaggtgggg actcagatta tgcaagcagt 300agtaaaagag gaaggaaaaa gggttctact aaacactctt ctgcatcatc aacaccaaca 360ccagctagtg actctggcac aggaggcatg cccaccatcg agcaagtttg ttcaacattt 420ggtttaactg atgtcgagct tgactattca gatgctgata tgcaaaactt gaccacctat 480aagttgttcc aacagcatgt gagaccgctc cttgctaagg aaaatccaaa ggttcctatg 540tcaaagttga tgatgttggt tgctgcaaaa tggcgcgaat tttctaattc aaaccccaat 600ctgcaaagcg aaaatgaacc gtctgctgca acttcaacca catctgaaga aagttatcca 660aaaactaatc gttcgagagc atccaaggaa 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acccaagaag 1080gaguuaaaag uguauguagg auugagcaag augcaacgag aaugguauac caaagugcug 1140augaaggaua uugauauagu gaauggugca ggaaagguag aaaaaaugcg acuacagaau 1200auucucaugc aguuaagaaa augcacaaau caccccuacc uuuuugaugg cgcugagccc 1260ggaccaccuu acacaaccga ugaacaucuc guguacaauu gcgguaaaau gguguugcug 1320gauaaacugc uucccaaauu gaaggaacag gaaucucgug uacuuaucuu cucucagaug 1380acccguaugu uggauauacu ugaagauuau ugucauuggc gacaguacca auauugucgu 1440uuggaugguc aaaccccaca cgaagacaga cagagacaaa ucaacgagua uaacgaagac 1500aauagccaaa aguuuaucuu uauguuguca acuagagccg guggauuggg uaucaauuug 1560gccacagcug auguaguuau uauauaugau ucggauugga auccccagau ggaucugcaa 1620gccauggaca gagcgcauag aauuggucag aagaaacaag ucagaguuuu cagguuuauu 1680accgaaaaca cuguggaaga aaaaaucguc gaaagagcug aaguaaaauu acguuuagac 1740aaauuaguua uccagcaggg ucguuuagcc gauuccaaag cacagacucu aaacaaagac 1800gaaauguuga acaugauccg gcacggugcc aaccacguau uugcuucuaa ggauuccgaa 1860auaacagaug aagauaucga uaguauauug gaaaagggag aaaugaagac cgcucagcua 1920gcucagaaga uggaaaccau gggcgaaucg ucacuucgca acuucacagu cgaaacaccc 1980acugaaucag ucuaccaauu cgaaggagaa gauuaucgug agaagcagaa aaccaucggc 2040uugagcaacu ggauagaacc ucccaaaaga gaaaggaagg ccaacuaugc cgucgaugcu 2100uacuucagag aagcuuuaag gguuucugag ccuaaagcgc cuaaggcucc aagaccacca 2160aaacagccca ucguacaaga uuuccaguuu uucccgccga gauuauucga acuuuuggac 2220caggagaucu acuuuuacag gaaaucuuug ggauauaagg uuccgaaaaa cuuagaacuu 2280ggaccugacg cguccaagca acagaaagaa gagcaaagaa aaauagauga gucagaaccg 2340cucaccgaag acgaacagca agaaaaagaa aacuuguuaa cgcaagguuu caccaauugg 2400aguaaacgcg auuucaauca guucaucaaa gccaacgaga aauaugguag ggacgauauu 2460gagaacaucg ccaaggaugu ugaaggcaaa acgccugaag aaguuaugga auauucugcg 2520guguuuuggg aaagauguca ugaauuacag gauauugaua gaauaauggc ccagauugag 2580agaggagaaa cuaaaauaca aagaagagcu aguauuaaga aggcacuuga ugcuaaaaug 2640gcaagauauc gugcaccauu ccaucagcug agaauuucuu acggcaccaa caaaggcaag 2700aacuacaugg aggacgaaga cagguuuuug guguguaugu ugcacaaguu ggguuucgau 2760agagaaaacg uuuaugaaga guuaagagca gcuguacgug cgucaccaca auucagauuu 2820gauugguucu uaaaaucgag aacugccaug gagcugcaaa ggagaugcaa cacauugaua 2880acguuaauag aaagagaaaa ugcugaauug gaggaaagag aaaaaauu 2928169330RNADiabrotica virgifera 169augaaaaaga cuaagcuuuc cgaaauucuc agggaauuca agaauaccaa caggcuacug 60cuaacaggua cuccauuaca aaacaauuua caugaacucu ggucucuucu caacuucuua 120cugccagacg uuuuuaacuc aucugaugau uucgaugccu gguuuaacac cagucaaugu 180cugggaggca acuccauggu ggagaggcug caugcuguau uaaaaccauu cuugcuuaga 240agauugaaaa uugaaguaga gaaagggcua aaacccaaga aggaguuaaa aguguaugua 300ggauugagca aaaugcagcg agaaugguaa 330

Claims

1. An isolated nucleic acid comprising at least one chromatin remodeling gene polynucleotide, wherein the polynucleotide is operably linked to a heterologous promoter.

2. The polynucleotide of claim 1, wherein the polynucleotide is selected from the group consisting of: SEQ ID NOs:1, 3, 5, and 7; the complement of any of SEQ ID NOs:1, 3, 5, and 7; a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:1, 3, 5, and 7; the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:1, 3, 5, and 7; a native coding sequence of a Diabrotica organism comprising any of SEQ ID NOs:1, 3, 5, and 7; the complement of a native coding sequence of a Diabrotica organism comprising any of SEQ ID NOs:1, 3, 5, and 7; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:139 and/or SEQ ID NO:140; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:139 and/or SEQ ID NO:140; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:8 and/or SEQ ID NO:10; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:8 and/or SEQ ID NO:10; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:139 and/or SEQ ID NO:140; the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:139 and/or SEQ ID NO:140; SEQ ID NO:79 or SEQ ID NO:164; the complement of SEQ ID NO:79 or SEQ ID NO:164; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:79 or SEQ ID NO:164; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:79 or SEQ ID NO:164; a native coding sequence of a Diabrotica organism comprising SEQ ID NO:79 or SEQ ID NO:164; the complement of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:79 or SEQ ID NO:164; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:143 or SEQ ID NO:167; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:143 or SEQ ID NO:167; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:101; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:101; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:158; the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:158; SEQ ID NO:81 or SEQ ID NO:165; the complement of SEQ ID NO:81 or SEQ ID NO:165; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:81 or SEQ ID NO:165; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:81 or SEQ ID NO:165; a native coding sequence of a Diabrotica organism comprising SEQ ID NO:81 or SEQ ID NO:165; the complement of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:81 or SEQ ID NO:165; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:144 or SEQ ID NO:168; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:144 or SEQ ID NO:168; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:144 or SEQ ID NO:168; the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:144 or SEQ ID NO:168; SEQ ID NO:83; the complement of SEQ ID NO:83; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:83; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:83; a native coding sequence of a Diabrotica organism comprising SEQ ID NO:83; the complement of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:83; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:145; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:145; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:105; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:105; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:162; the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:162; SEQ ID NO:85 or SEQ ID NO:166; the complement of SEQ ID NO:85 or SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:85 or SEQ ID NO:166; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:85 or SEQ ID NO:166; a native coding sequence of a Diabrotica organism comprising SEQ ID NO:85 or SEQ ID NO:166; the complement of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:85 or SEQ ID NO:166; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:146 or SEQ ID NO:169; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:146 or SEQ ID NO:169; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:103; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:103; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:160; the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:160; SEQ ID NO:87; the complement of SEQ ID NO:87; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:87; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:87; a native coding sequence of a Diabrotica organism comprising SEQ ID NO:87; the complement of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:87; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:147; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:147; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:102; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:102; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:159; the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:159; SEQ ID NO:89; the complement of SEQ ID NO:89; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:89; a native coding sequence of a Diabrotica organism comprising SEQ ID NO:89; the complement of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:89; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:148; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:148; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:148; the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:148; SEQ ID NO:91; the complement of SEQ ID NO:91; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:91; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:91; a native coding sequence of a Diabrotica organism comprising SEQ ID NO:91; the complement of a native coding sequence of a Diabrotica organism comprising SEQ ID NO:91; a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:149; the complement of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:149; a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:149; and the complement of a fragment of at least 15 contiguous nucleotides of a native non-coding sequence of a Diabrotica organism that is transcribed into a native RNA molecule comprising SEQ ID NO:149.

3. A plant transformation vector comprising the polynucleotide of claim 1.

4. The polynucleotide of claim 1, wherein the organism is selected from the group consisting of D. v. virgifera LeConte; D. barberi Smith and Lawrence; D. u. howardi; D. v. zeae; D. balteata LeConte; D. u. tenella; D. speciosa Germar; and D. u. undecimpunctata Mannerheim.

5. A ribonucleic acid (RNA) molecule transcribed from the polynucleotide of claim 1.

6. A double-stranded ribonucleic acid molecule produced from the expression of the polynucleotide of claim 1.

7. The double-stranded ribonucleic acid molecule of claim 6, wherein contacting the polynucleotide sequence with a coleopteran pest inhibits the expression of an endogenous nucleotide sequence specifically complementary to the polynucleotide.

8. The double-stranded ribonucleic acid molecule of claim 7, wherein contacting said ribonucleotide molecule with a coleopteran pest kills or inhibits the growth, reproduction, and/or feeding of the pest.

9. The double stranded RNA of claim 6, comprising a first, a second and a third RNA segment, wherein the first RNA segment comprises the polynucleotide, wherein the third RNA segment is linked to the first RNA segment by the second polynucleotide sequence, and wherein the third RNA segment is substantially the reverse complement of the first RNA segment, such that the first and the third RNA segments hybridize when transcribed into a ribonucleic acid to form the double-stranded RNA.

10. The RNA of claim 5, selected from the group consisting of a double-stranded ribonucleic acid molecule and a single-stranded ribonucleic acid molecule of between about 15 and about 30 nucleotides in length.

11. A plant transformation vector comprising the polynucleotide of claim 1, wherein the heterologous promoter is functional in a plant cell.

12. A cell transformed with the polynucleotide of claim 1.

13. The cell of claim 12, wherein the cell is a prokaryotic cell.

14. The cell of claim 12, wherein the cell is a eukaryotic cell.

15. The cell of claim 14, wherein the cell is a plant cell.

16. A plant transformed with the polynucleotide of claim 1.

17. A seed of the plant of claim 16, wherein the seed comprises the polynucleotide.

18. A commodity product produced from the plant of claim 16, wherein the commodity product comprises a detectable amount of the polynucleotide.

19. The plant of claim 16, wherein the at least one polynucleotide is expressed in the plant as a double-stranded ribonucleic acid molecule.

20. The cell of claim 15, wherein the cell is a Zea mays cell.

21. The plant of claim 16, wherein the plant is Zea mays.

22. The plant of claim 16, wherein the at least one polynucleotide is expressed in the plant as a ribonucleic acid molecule, and the ribonucleic acid molecule inhibits the expression of an endogenous polynucleotide that is specifically complementary to the at least one polynucleotide when a coleopteran pest ingests a part of the plant.

23. The polynucleotide of claim 1, further comprising at least one additional polynucleotide that encodes an RNA molecule that inhibits the expression of an endogenous pest gene.

24. The polynucleotide of claim 23, wherein the additional polynucleotide encodes an iRNA molecule that results in a parental RNAi phenotype.

25. The polynucleotide of claim 24, wherein the additional polynucleotide encodes an iRNA molecule that inhibits the expression of a hunchback or kruppel gene.

26. The polynucleotide of claim 23, wherein the additional polynucleotide encodes an iRNA molecule that results in decreased growth and/or development and/or mortality in a coleopteran pest that contacts the iRNA molecule (lethal RNAi).

27. A plant transformation vector comprising the polynucleotide of claim 23, wherein the additional polynucleotide(s) are each operably linked to a heterologous promoter functional in a plant cell.

28. A method for controlling a coleopteran pest population, the method comprising providing an agent comprising a ribonucleic acid (RNA) molecule that functions upon contact with the coleopteran pest to inhibit a biological function within the coleopteran pest, wherein the RNA is specifically hybridizable with a polynucleotide selected from the group consisting of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169; the complement of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169; a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169; the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169; a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166; the complement of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166; a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166; and the complement of a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166.

29. The method according to claim 28, wherein the agent is a double-stranded RNA molecule.

30. A method for controlling a coleopteran pest population, the method comprising: introducing into a coleopteran pest, a ribonucleic acid (RNA) molecule that functions upon contact with the coleopteran pest to inhibit a biological function within the coleopteran pest, wherein the RNA is specifically hybridizable with a polynucleotide selected from the group consisting of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169, the complement of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169, a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169, the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169, a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, the complement of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, and the complement of a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, thereby producing a coleopteran pest having a pRNAi phenotype.

31. The method according to claim 30, wherein the RNA is introduced into a male coleopteran pest.

32. The method according to claim 30, wherein the RNA is introduced into a female coleopteran pest, the method further comprising releasing the female coleopteran pest having the pRNAi phenotype into the pest population, wherein mating between the female coleopteran pest having the pRNAi phenotype and male pests of the population produces fewer viable offspring than mating between other female pests and male pests of the population.

33. A method for controlling a coleopteran pest population, the method comprising: providing an agent comprising a first and a second polynucleotide sequence that functions upon contact with the coleopteran pest to inhibit a biological function within the coleopteran pest, wherein the first polynucleotide sequence comprises a region that exhibits from about 90% to about 100% sequence identity to from about 19 to about 30 contiguous nucleotides of SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:167, SEQ ID NO:168, or SEQ ID NO:169, and wherein the first polynucleotide sequence is specifically hybridized to the second polynucleotide sequence.

34. The method according to claim 33, wherein the ribonucleic acid molecule is a double-stranded ribonucleic acid molecule.

35. The method according to claim 33, wherein the coleopteran pest population is reduced relative to a population of the same pest species infesting a host plant of the same host plant species lacking the transformed plant cell.

36. A method for controlling a coleopteran pest population, the method comprising: providing in a host plant of a coleopteran pest a transformed plant cell comprising the polynucleotide of claim 1, wherein the polynucleotide is expressed to produce a ribonucleic acid molecule that functions upon contact with a coleopteran pest belonging to the population to inhibit the expression of a target sequence within the coleopteran pest and results in decreased reproduction of the coleopteran pest or pest population, relative to reproduction of the same pest species on a plant of the same host plant species that does not comprise the polynucleotide.

37. The method according to claim 36, wherein the ribonucleic acid molecule is a double-stranded ribonucleic acid molecule.

38. The method according to claim 36, wherein the coleopteran pest population is reduced relative to a coleopteran pest population infesting a host plant of the same species lacking the transformed plant cell.

39. A method of controlling coleopteran pest infestation in a plant, the method comprising providing in the diet of a coleopteran pest a ribonucleic acid (RNA) that is specifically hybridizable with a polynucleotide selected from the group consisting of: SEQ ID NOs:135-163 and SEQ ID NOs:167-169, the complement of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169, a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169, the complement of a fragment of at least 15 contiguous nucleotides of any of SEQ ID NOs:135-163 and SEQ ID NOs:167-169, a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, the complement of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166, and the complement of a fragment of at least 15 contiguous nucleotides of a transcript of any of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, and SEQ ID NO:91, SEQ ID NO:164, SEQ ID NO:165, and SEQ ID NO:166.

40. The method according to claim 39, wherein the diet comprises a plant cell transformed to express the polynucleotide.

41. The method according to claim 39, wherein the specifically hybridizable RNA is comprised in a double-stranded RNA molecule.

42. A method for improving the yield of a corn crop, the method comprising: introducing the nucleic acid of claim 1 into a corn plant to produce a transgenic corn plant; and cultivating the corn plant to allow the expression of the at least one polynucleotide; wherein expression of the at least one polynucleotide inhibits coleopteran pest reproduction or growth and loss of yield due to coleopteran pest infection.

43. The method according to claim 42, wherein expression of the at least one polynucleotide produces an RNA molecule that suppresses at least a first target gene in a coleopteran pest that has contacted a portion of the corn plant.

44. A method for producing a transgenic plant cell, the method comprising: transforming a plant cell with a vector comprising the nucleic acid of claim 1; culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture comprising a plurality of transformed plant cells; selecting for transformed plant cells that have integrated the at least one polynucleotide into their genomes; screening the transformed plant cells for expression of a ribonucleic acid (RNA) molecule encoded by the at least one polynucleotide; and selecting a plant cell that expresses the RNA.

45. The method according to claim 44, wherein the RNA molecule is a double-stranded RNA molecule.

46. A method for providing protection against a coleopteran pest to a transgenic plant, the method comprising: providing the transgenic plant cell produced by the method of claim 44; and regenerating a transgenic plant from the transgenic plant cell, wherein expression of the ribonucleic acid molecule encoded by the at least one polynucleotide is sufficient to modulate the expression of a target gene in a coleopteran pest that contacts the transformed plant.

47. A method for producing a transgenic plant cell, the method comprising: transforming a plant cell with a vector comprising a means for protecting a plant from a coleopteran pest; culturing the transformed plant cell under conditions sufficient to allow for development of a plant cell culture comprising a plurality of transformed plant cells; selecting for transformed plant cells that have integrated the means for protecting a plant from a coleopteran pest into their genomes; screening the transformed plant cells for expression of a means for inhibiting expression of an essential gene in a coleopteran pest; and selecting a plant cell that expresses the means for inhibiting expression of an essential gene in a coleopteran pest.

48. A method for producing a coleopteran pest-protected transgenic plant, the method comprising: providing the transgenic plant cell produced by the method of claim 47; and regenerating a transgenic plant from the transgenic plant cell, wherein expression of the means for inhibiting expression of an essential gene in a coleopteran pest is sufficient to modulate the expression of a target gene in a coleopteran pest that contacts the transformed plant.

49. The nucleic acid of claim 1, further comprising a polynucleotide encoding a polypeptide from Bacillus thuringiensis.

50. The nucleic acid of claim 49, wherein the polypeptide from B. thuringiensis is selected from a group comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

51. The cell of claim 15, wherein the cell comprises a polynucleotide encoding a polypeptide from Bacillus thuringiensis.

52. The cell of claim 51, wherein the polypeptide from B. thuringiensis is selected from a group comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

53. The plant of claim 16, wherein the plant comprises a polynucleotide encoding a polypeptide from Bacillus thuringiensis.

54. The plant of claim 53, wherein the polypeptide from B. thuringiensis is selected from a group comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

55. The method according to claim 42, wherein the transformed plant cell comprises a nucleotide sequence encoding a polypeptide from Bacillus thuringiensis.

56. The method according to claim 55, wherein the polypeptide from B. thuringiensis is selected from a group comprising Cry1B, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A, and Cyt2C.

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