(12) Patent Application Publication (10) Pub. No.: US 2016/0194658 A1 Narva Et Al

US 2016O194658A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0194658 A1 Narva et al. (43) Pub. Date: Jul. 7, 2016

(54) NUCAMPHOLIN NUCLECACID Publication Classification MOLECULES TO CONTROL COLEOPTERAN INSECT PESTS (51) Int. Cl. (71) Applicants: Dow AgroSciences LLC, Indianapolis, CI2N 5/82 (2006.01) IN (US); Fraunhofer-Gesellschaft zur AOIN57/6 (2006.01) Forderung der angewandten Forschung E. V., Munchen (DE) CI2N IS/II3 (2006.01) (52) U.S. Cl. (72) Inventors: Kenneth E. Narva, Zionsville, IN (US); CPC ...... CI2N 15/8286 (2013.01); C12N 15/I 13 Sarah Worden, Indianapolis, IN (US); Meghan Frey, Greenwood, IN (US); (2013.01); A0IN 57/16 (2013.01); C12N Chaoxian Geng, Zionsville, IN (US); 23 10/14 (2013.01) Murugesan Rangasamy, Zionsville, IN (US); Kanika Arora, Indianapolis, IN (US); Balaji Veeramani, Indianapolis, (57) ABSTRACT IN (US); Premchand Gandra, Indianapolis, IN (US); Andreas This disclosure concerns nucleic acid molecules and methods Vilcinskas, Giessen (DE); Eileen of use thereof for control of insect pests through RNA inter Knorr, Giessen (DE) ference-mediated inhibition of target coding and transcribed (21) Appl. No.: 14/979,181 non-coding sequences in insect pests, including coleopteran (22) Filed: Dec. 22, 2015 pests. The disclosure also concerns methods for making Related U.S. Application Data transgenic plants that express nucleic acid molecules useful (60) Provisional application No. 62/095,487, filed on Dec. for the control of insect pests, and the plant cells and plants 22, 2014. obtained thereby. Patent Application Publication Jul. 7, 2016 Sheet 1 of 2 US 2016/O194658 A1

FIG. 1. Generation of dsRNA from a single transcription template with a single pair of primers

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FIG. 2. Generation of dsRNA from two transcription templates.

RNA polyherase

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NUCAMPHOLIN NUCLECACID adults also feed on reproductive tissues of the corn plant, but MOLECULES TO CONTROL COLEOPTERAN in contrast rarely feed on corn leaves. INSECT PESTS 0006 Most of the rootworm damage in corn is caused by larval feeding. Newly hatched rootworms initially feed on PRIORITY CLAIM fine corn root hairs and burrow into root tips. As the larvae grow larger, they feed on and burrow into primary roots. 0001. This application claims the benefit of the filing date When corn rootworms are abundant, larval feeding often of U.S. Provisional Patent Application Ser. No. 62/095,487, results in the pruning of roots all the way to the base of the filed Dec. 22, 2014, for “NUCAMPHOLIN NUCLEICACID corn stalk. Severe root injury interferes with the roots’ ability MOLECULES TO CONTROL INSECT PESTS which is to transport water and nutrients into the plant, reduces plant incorporated herein in its entirety. growth, and results in reduced grain production, thereby often drastically reducing overall yield. Severe root injury also TECHNICAL FIELD OF THE DISCLOSURE often results in lodging of corn plants, which makes harvest 0002 The present invention relates generally to genetic more difficult and further decreases yield. Furthermore, feed control of plant damage caused by insect pests (e.g., ing by adults on the corn reproductive tissues can result in coleopteran pests). In particular embodiments, the present pruning of silks at the ear tip. If this “silk clipping is severe invention relates to identification of target coding and non enough during pollen shed, pollination may be disrupted. coding polynucleotides, and the use of RNAi technologies for 0007 Control of corn rootworms may be attempted by post-transcriptionally repressing or inhibiting expression of crop rotation, chemical insecticides, biopesticides (e.g., the target coding and non-coding polynucleotides in the cells of spore-forming gram-positive bacterium, Bacillus thuringien an insect pest to provide a plant protective effect. sis), or a combination thereof. Crop rotation suffers from the significant disadvantage of placing unwanted restrictions BACKGROUND upon the use of farmland. Moreover, oviposition of some rootworm species may occur in Soybean fields, thereby miti 0003. The western cornrootworm (WCR), Diabrotica vir gating the effectiveness of crop rotation practiced with corn gifera virgifera LeConte, is one of the most devastating corn and soybean. rootworm species in North America and is a particular con 0008 Chemical insecticides are the most heavily relied cern in corn-growing areas of the Midwestern United States. upon strategy for achieving corn rootworm control. Chemical The northern corn rootworm (NCR), Diabrotica barberi insecticide use, though, is an imperfect corn rootWorm con Smith and Lawrence, is a closely-related species that co trol strategy; over S1 billion may be lost in the United States inhabits much of the same range as WCR. There are several each year due to corn rootworm when the costs of the chemi other related subspecies of Diabrotica that are significant cal insecticides are added to the costs of the rootworm dam pests in North America: the Mexican corn rootworm (MCR), age that may occur despite the use of the insecticides. High D. virgifera zeae Krysan and Smith; the Southern corn root populations of larvae, heavy rains, and improper application worm (SCR), D. undecimpunctata howardi Barber: D. bal of the insecticide(s) may all result in inadequate corn root teata LeConte; D. undecimpunctata tenella, D. speciosa Ger worm control. Furthermore, the continual use of insecticides mar; and D. u. undecimpunctata Mannerheim. The United may select for insecticide-resistant rootworm strains, as well States Department of Agriculture has estimated that corn as raise significant environmental concerns due to the toxicity rootworms cause S1 billion in lost revenue each year, includ of many of them to non-target species. ing S800 million in yield loss and S200 million in treatment 0009 European pollen beetles (PB) are serious pests in COStS. oilseed rape, both the larvae and adults feed on flowers and 0004 Both WCR and NCR are deposited in the soil as pollen. Pollen beetle damage to the crop can cause 20-40% eggs during the Summer. The insects remain in the egg stage yield loss. The primary pest species is Melligethes aeneus throughout the winter. The eggs are oblong, white, and less Fabricius. Currently, pollen beetle control in oilseed rape than 0.004 inches in length. The larvae hatch in late May or relies mainly on pyrethroids which are expected to be phased early June, with the precise timing of egg hatching varying out soon because of their environmental and regulatory pro from year to year due to temperature differences and location. file. Moreover, pollen beetle resistance to existing chemical The newly hatched larvae are white worms that are less than insecticides has been reported. Therefore, urgently needed 0.125 inches in length. Once hatched, the larvae begin to feed are environmentally friendly pollen beetle control solutions on corn roots. Cornrootworms go through three larval instars. with novel modes of action. After feeding for several weeks, the larvae molt into the pupal 0010. In nature, pollen beetles overwinter as adults in the stage. They pupate in the Soil, and then they emerge from the soil or under leaf litter. In spring the adults emerge from soil as adults in July and August. Adult rootworms are about hibernation and start feeding on flowers of weeds, and 0.25 inches in length. migrate onto flowering oilseed rape plants. The eggs are laid 0005 Corn rootworm larvae complete development on in oilseed rape flower buds. The larvae feed and develop in the corn and several other species of grasses. Larvae reared on buds and flowers. Late stage larvae find a pupation site in the yellow foxtail emerge later and have a smaller head capsule soil. The second generation adults emerge in July and August size as adults than larvae reared on corn. Ellsbury et al. (2005) and feed on various flowering plants before finding sites for Environ. Entomol. 34:627-34. WCRadults feed on corn silk, overwintering. pollen, and kernels on exposed ear tips. If WCRadults emerge 0011 RNA interference (RNAi) is a process utilizing before corn reproductive tissues are present, they may feed on endogenous cellular pathways, whereby an interfering RNA leaf tissue, thereby slowing plant growth and occasionally (iRNA) molecule (e.g., a dsRNA molecule) that is specific for killing the host plant. However, the adults will quickly shift to all, or any portion of adequate size, of a target gene results in preferred silks and pollen when they become available. NCR the degradation of the mRNA encoded thereby. In recent US 2016/0 194658 A1 Jul. 7, 2016 years, RNAi has been used to perform gene “knockdown” in sand sequences provided would be lethal, or even otherwise a number of species and experimental systems; for example, useful, in species of corn rootworm when used as dsRNA or Caenorhabditis elegans, plants, insect embryos, and cells in siRNA.U.S. Pat. No. 7,943,819 provides no suggestion to use tissue culture. See, e.g., Fire et al. (1998) Nature 391:806-11; any particular sequence of the more than nine hundred Martinez et al. (2002) Cell 110:563-74; McManus and Sharp sequences listed therein for RNA interference, other than the (2002) Nature Rev. Genetics 3:737-47. particular partial sequence of a charged multivesicular body 0012 RNAi accomplishes degradation of mRNA through protein 4b gene. Furthermore, U.S. Pat. No. 7,943,819 pro an endogenous pathway including the DICER protein com vides no guidance as to which other of the over nine hundred plex. DICER cleaves long dsRNA molecules into short frag sequences provided would be lethal, or even otherwise useful, ments of approximately 20 nucleotides, termed Small inter in species of corn rootworm when used as dsRNA or siRNA. fering RNA (siRNA). The siRNA is unwound into two single U.S. Patent Application Publication No. U.S. 2013/04.0173 Stranded RNAS: the passenger strand and the guide strand. and PCT Application Publication No. WO 2013/169923 The passenger Strand is degraded, and the guide Strand is describes the use of a sequence derived from a Diabrotica incorporated into the RNA-induced silencing complex virgifera Snf7 gene for RNA interference in maize. (Also (RISC). Micro ribonucleic acids (miRNAs) are structurally disclosed in Bolognesi et al. (2012) PLoS ONE 7(10): very similar molecules that are cleaved from precursor mol e47534. doi:10.1371/journal.pone.0047534). ecules containing a polynucleotide "loop' connecting the 0015 The overwhelming majority of sequences comple hybridized passenger and guide Strands, and they may be mentary to corn rootworm DNAS (Such as the foregoing) do similarly incorporated into RISC. Post-transcriptional gene not provide a plant protective effect from species of corn silencing occurs when the guide Strand binds specifically to a rootworm when used as dsRNA or siRNA. For example, complementary mRNA molecule and induces cleavage by Baum et al. (2007) Nature Biotechnology 25:1322-1326, Argonaute, the catalytic component of the RISC complex. describe the effects of inhibiting several WCR gene targets by This process is known to spread systemically throughout the RNAi. These authors reported that 8 of the 26 target genes organism despite initially limited concentrations of siRNA they tested were not able to provide experimentally signifi and/or miRNA in Some eukaryotes Such as plants, nematodes, cant coleopteran pest mortality at a very high iERNA (e.g., and some insects. dsRNA) concentration of more than 520 ng/cm. 0013 U.S. Pat. No. 7,612,194 and U.S. Patent Publication 0016. The authors of U.S. Pat. No. 7,612,194 and U.S. Nos. 2007/0050860, 2010/0192265, and 2011/0154545 dis Patent Publication No. 2007/0050860 made the first report of close a library of 91 12 expressed sequence tag (EST) in planta RNAi in corn plants targeting the western corn sequences isolated from D. v. virgifera LeConte pupae. It is rootworm. Baum et al. (2007) Nat. Biotechnol. 25(11): 1322 suggested in U.S. Pat. No. 7,612,194 and U.S. Patent Publi 6. These authors describe a high-throughput in vivo dietary cation No. 2007/0050860 to operably link to a promoter a RNAi system to screen potential target genes for developing nucleic acid molecule that is complementary to one of several transgenic RNAi maize. Of an initial gene pool of 290 targets, particular partial sequences of D. v. virgifera vacuolar-type only 14 exhibited larval control potential. One of the most H'-ATPase (V-ATPase) disclosed therein for the expression effective double-stranded RNAs (dsRNA) targeted a gene of anti-sense RNA in plant cells. U.S. Patent Publication No. encoding vacuolar ATPase subunit A (V-ATPase), resulting in 2010/0192265 suggests operably linking a promoter to a a rapid Suppression of corresponding endogenous mRNA and nucleic acid molecule that is complementary to a particular triggering a specific RNAi response with low concentrations partial sequence of a D. v. virgifera gene of unknown and of dsRNA. Thus, these authors documented for the first time undisclosed function (the partial sequence is stated to be 58% the potential for in planta RNAi as a possible pest manage identical to C56C10.3 gene product in C. elegans) for the ment tool, while simultaneously demonstrating that effective expression of anti-sense RNA in plant cells. U.S. Patent Pub targets could not be accurately identified a priori, even from a lication No. 2011/0154545 suggests operably linking a pro relatively small set of candidate genes. moter to a nucleic acid molecule that is complementary to two particular partial sequences of D. v. virgifera coatomer beta SUMMARY OF THE DISCLOSURE Subunit genes for the expression of anti-sense RNA in plant 0017 Disclosed herein are nucleic acid molecules (e.g., cells. Further, U.S. Pat. No. 7,943,819 discloses a library of target genes, DNAs, dsRNAs, siRNAs, miRNAs, shRNAs, 906 expressed sequence tag (EST) sequences isolated from and hpRNAs), and methods of use thereof, for the control of D. v. virgifera LeConte larvae, pupae, and dissected midguts, insect pests, including, for example, coleopteran pests, such and suggests operably linking a promoter to a nucleic acid as D. v. virgifera LeConte (western corn rootworm, “WCR): molecule that is complementary to a particular partial D. barberi Smith and Lawrence (northern corn rootworm, sequence of a D. v. virgifera charged multivesicular body “NCR); D. u. howardi Barber (southern corn rootworm, protein 4b gene for the expression of double-stranded RNA in “SCR); D. v. Zeae Krysan and Smith (Mexican corn root plant cells. worm, “MCR); D. balteata LeConte; D. u. tenella, D. spe 0014 No further suggestion is provided in U.S. Pat. No. ciosa Germar, D. u. undecimpunctata Mannerheim; and 7,612,194, and U.S. Patent Publication Nos. 2007/0050860, Melligethes aeneus Fabricius (pollen beetle, “PB). In par 2010/0192265, and 2011/0154545 to use any particular ticular examples, exemplary nucleic acid molecules are dis sequence of the more than nine thousand sequences listed closed that may be homologous to at least a portion of one or therein for RNA interference, other than the several particular more native nucleic acids in an insect pest. partial sequences of V-ATPase and the particular partial 0018. In these and further examples, the native nucleic sequences of genes of unknown function. Furthermore, none acid sequence may be a target gene, the product of which may of U.S. Pat. No. 7,612,194, and U.S. Patent Publication Nos. be, for example and without limitation: involved in a meta 2007/0050860 and 2010/0192265, and 2011/0154545 pro bolic process; involved in a reproductive process; or involved vides any guidance as to which other of the over nine thou in larval development. In some examples, post-transcrip US 2016/0 194658 A1 Jul. 7, 2016 tional inhibition of the expression of a target gene by a nucleic inhibiting expression of an essential gene in a coleopteran acid molecule comprising a polynucleotide homologous pest operably linked to a promoter, wherein the DNA mol thereto may be lethal to an insect pest or result in reduced ecule is capable of being integrated into the genome of a growth and/or development of an insect pest. In specific maize plant. examples, nucampholin (referred to herein as incm) may be 0022 Disclosed are methods for controlling a population selected as a target gene for post-transcriptional silencing. In of an insect pest (e.g., a coleopteran pest), comprising pro particular examples, a target gene useful for post-transcrip viding to an insect pest (e.g., a coleopteran pest) an iRNA tional inhibition is a novel gene referred to herein as a (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA) mol Diabroticancm (e.g., SEQID NO:1 and SEQID NO:77). In ecule that functions upon being taken up by the pest to inhibit particular examples, a target gene useful for post-transcrip a biological function within the pest, wherein the iRNA mol tional inhibition is the novel gene referred to herein as ecule comprises all or part of a polynucleotide selected from Melligethes incm (e.g., SEQID NO:84, SEQID NO:86, SEQ the group consisting of: SEQID NO:78; the complement of ID NO:88, and SEQ ID NO:93). An isolated nucleic acid SEQID NO:78; SEQID NO:79; the complement of SEQID molecule comprising the polynucleotide of SEQ ID NO:1; NO:79; SEQID NO:80; the complement of SEQID NO:80; the complement of SEQ ID NO:1; SEQ ID NO:77; the SEQID NO:81; the complement of SEQID NO:81; SEQID complement of SEQID NO:77; SEQID NO:84; the comple NO:82; the complement of SEQID NO:82; SEQID NO:83; ment of SEQID NO:84; SEQID NO:86; the complement of the complement of SEQ ID NO:83; a polynucleotide that SEQID NO:86; SEQID NO:88; the complement of SEQID hybridizes to a native coding polynucleotide of a Diabrotica NO:88: SEQID NO:93; the complement of SEQID NO:93; organism (e.g., WCR) comprising all or part of SEQID NO:1 and/or fragments of any of the foregoing (e.g., SEQID NOs: or SEQID NO:77; the complement of a polynucleotide that 3-6 and 90) is therefore disclosed herein. hybridizes to a native coding polynucleotide of a Diabrotica 0019. Also disclosed are nucleic acid molecules compris organism comprising all or part of SEQID NO:1 or SEQID ing a polynucleotide that encodes a polypeptide that is at least NO:77; SEQID NO:95; the complement of SEQID NO:95; about 85% identical to an amino acid sequence within a target SEQID NO:96; the complement of SEQID NO:96: SEQID gene product (for example, the product of ancm gene). For NO:97; the complement of SEQID NO:97; SEQID NO:98: example, a nucleic acid molecule may comprise a polynucle the complement of SEQ ID NO:98: SEQ ID NO:99; the otide encoding a polypeptide that is at least 85% identical to complement of SEQID NO:99; a polynucleotide that hybrid a Diabrotica NCM (e.g., SEQID NO:2); a Melligethes NCM izes to a native coding polynucleotide of a Melligethes organ (e.g., SEQID NO:85, SEQID NO:87, SEQID NO:89, and ism (e.g., PB) comprising all or part of any of SEQID NOs: SEQ ID NO:94); and/or an amino acid sequence within a 84, 86, 88, and 93; and the complement of a polynucleotide product of a Diabrotica incrm or a Melligethes incrm. Further that hybridizes to a native coding polynucleotide of a disclosed are nucleic acid molecules comprising a polynucle Melligethes organism comprising all or part of any of SEQID otide that is the reverse complement of a polynucleotide that NOs:84, 86, 88, and 93. encodes a polypeptide at least 85% identical to an amino acid 0023. In particular embodiments, an iRNA that functions sequence within a target gene product. upon being taken up by an insect pest to inhibit a biological 0020. Also disclosed are cDNA polynucleotides that may function within the pest is transcribed from a DNA compris be used for the production of iRNA (e.g., dsRNA, siRNA, ing all or part of a polynucleotide selected from the group shRNA, miRNA, and hpRNA) molecules that are comple consisting of: SEQ ID NO:1; the complement of SEQ ID mentary to all or part of an insect pest target gene, for NO:1; SEQID NO:3: the complement of SEQID NO:3:SEQ example, an incm gene. In particular embodiments, dsRNAS, ID NO:4; the complement of SEQID NO:4: SEQID NO:5; siRNAs, shRNAs, miRNAs, and/or hpRNAs may be pro the complement of SEQID NO:5; SEQID NO:6; the comple duced in vitro or in Vivo by a genetically-modified organism, ment of SEQ ID NO:6: SEQ ID NO:77; the complement of Such as a plant or bacterium. In particular examples, cDNA SEQID NO:77; SEQID NO:84; the complement of SEQID molecules are disclosed that may be used to produce iRNA NO:84; SEQID NO:86; the complement of SEQID NO:86: molecules that are complementary to all or part of a SEQID NO:88; the complement of SEQID NO:88: SEQID Diabroticancm (e.g., SEQID NO:1 and SEQID NO:77) or NO:93; the complement of SEQ ID NO:93; a native coding a Melligethes incm (e.g., SEQID NO:84, SEQIDNO:86, SEQ polynucleotide of a Diabrotica organism (e.g., WCR) com ID NO:88, and SEQID NO:93). prising all or part of SEQ ID NO:1 or SEQ ID NO:77; the 0021. Further disclosed are means for inhibiting expres complement of a native coding polynucleotide of a sion of an essential gene in a coleopteran pest, and means for Diabrotica organism comprising all or part of SEQID NO:1 providing coleopteran pest resistance to a plant. A means for or SEQ ID NO:77; a native coding polynucleotide of a inhibiting expression of an essential gene in a coleopteran Melligethes organism (e.g., PB) comprising all or part of any pest is a single- or double-stranded RNA molecule consisting of SEQID NOS:84, 86, 88, and 93; and the complement of a of a polynucleotide selected from the group consisting of native coding polynucleotide of a Melligethes organism com SEQ ID NOS:79-82; and the complements thereof. Func prising all or part of any of SEQID NOS:84, 86, 88, and 93. tional equivalents of means for inhibiting expression of an 0024. Also disclosed herein are methods wherein dsR essential gene in a coleopteran pest include single- or double NAs, siRNAs, shRNAs, miRNAs, and/or hpRNAs may be stranded RNA molecules that are substantially homologous provided to an insect pest in a diet-based assay, or in geneti to all or part of the complement of the RNA expression cally-modified plant cells expressing the dsRNAs, siRNAs, product of a ncin gene from an organism of the order shRNAs, miRNAs, and/or hpRNAs. In these and further coleoptera, comprising SEQID NO:1, SEQID NO:84, SEQ examples, the dsRNAs, siRNAs, shRNAs, miRNAs, and/or ID NO:86, SEQID NO:88, or SEQID NO:93. A means for hpRNAs may be ingested by the pest. Ingestion of dsRNAs, providing coleopteran pest resistance to a plant is a DNA siRNA, shRNAs, miRNAs, and/or hpRNAs of the invention molecule comprising a polynucleotide encoding a means for may then result in RNAi in the pest, which in turn may result US 2016/0 194658 A1 Jul. 7, 2016

in silencing of a gene essential for viability of the pest and - Continued leading ultimately to mortality. Thus, methods are disclosed AAGAGAAAAAGTCTTCCAAAAAGAAAGCCCACAATAGTAGAGACAGAGAT wherein nucleic acid molecules comprising exemplary poly nucleotide(s) useful for parental control of insect pests are TCATCAGAGGAAGGTTACAACCCTAAAGATTATCAGAGATACTATGGGGA provided to an insect pest. In particular examples, a AGATCGCCCAAACAGTGACAAATATTGGAATAAATATCCAAGGAAAGATA coleopteran pest controlled by use of nucleic acid molecules of the invention may be WCR, NCR, SCR. D. undecimpunc CTACCAAAGTTGGCCAAAGATACTATGATGCGGCTCCCGAAGAATCTGGC tata howardi, D. balteata, D. undecimpunctata tenella, D. speciosa, D. u. undecimpunctata, or Melligethes aeneus. AAGAAGGGGCCAGATAGAAATTCAGAGGAGAAGGAGTTACCAAAGCCAAT 0025. The foregoing and other features will become more GGAGTCTGTTCCTGATAAATCAGTCATCAAACCAAGAGAAAGAAAAACTG apparent from the following Detailed Description of several embodiments, which proceeds with reference to the accom TAGATATGTTAACATCGAGGACTGGTGGTGCTTATATTCCCCCAGCTAAG panying FIGS. 1-2. CTACGATTGTTACAAGCCAGTATTACAGACAAAACATCAGCAGCCTATCA BRIEF DESCRIPTION OF THE FIGURES GCGTATAGCATGGGAAGCCTTAAAGAAATCCGTTCATGGTTACATTAATA 0026 FIG. 1 includes a depiction of a strategy used to AAATTAACACCTCGAATATTGGCATCATCGCCAGAGAATTATTGCATGAA provide dsRNA from a single transcription template with a single pair of primers. AATATAGTAAGAGGTAGAGGTTTGCTGTGCAAGTCAATAATACAAGCACA 0027 FIG. 2 includes a depiction of a strategy used to AGCAGCATCTCCTACTTTTACAAACGTTTACGCAGCCTTAGTAGCTGTTA provide dsRNA from two transcription templates. TTAATTCGAAGTTTCCAAGTATAGGAGAGCTTTTATTGAAGAGGTTGGTT

SEQUENCE LISTING TTGCAGTTCAAAAGAGGGTTTAAACAAAATAATAAGTCTATTTGCATATC 0028. The nucleic acid sequences listed in the accompa GGCTACTACTTTCGTAGCTCATTTAGTAAATCAGAGAGTGGCACATGAAA nying sequence listing are shown using standard letter abbre viations for nucleotide bases, as defined in 37 C.F.R.S 1.822. TTTTGGCTTTGGAGATACTTACATTGTTGGTGGAGACTCCTACAGATGAT The nucleic acid and amino acid sequences listed define mol ecules (i.e., polynucleotides and polypeptides, respectively) TCTGTGGAGGTGGCCATTTCATTTTTGAAGGAATGTGGACAAAAACTGAC having the nucleotide and amino acid monomers arranged in AGAAGTTTCAAGTAGAGGTATTACTGCTATATTTGAGATGTTAAGAAACA the manner described. The nucleic acid and amino acid sequences listed also each define a genus of polynucleotides TTTTACATGAAGGCCAGCTAGAAAAAAAGAATTCAGTACATGATTGA or polypeptides that comprise the nucleotide and amino acid monomers arranged in the manner described. In view of the 0031 SEQID NO:2 shows the amino acid sequence of a redundancy of the genetic code, it will be understood that a Diabrotica NCM polypeptide encoded by an exemplary nucleotide sequence including a coding sequence also Diabrotica incm DNA: describes the genus of polynucleotides encoding the same polypeptide as a polynucleotide consisting of the reference MRGGVSDDMTSTCVOGGIRPIGRYOPNMLMEPSSPOSAWOFHPAMPKREP sequence. It will further be understood that an amino acid sequence describes the genus of polynucleotide ORFs encod WDHDGRNDSGLASGGEFISSSPGSDNSEHFSASYSSPTSCHTWISTNTYY ing that polypeptide. 0029. Only one strand of each nucleic acid sequence is PTNLRRPSOAOTSIPTHMMYTGDHNPLTPPNSEPMISPKSVLSRNNEGEH shown, but the complementary Strand is understood as OTTLTPCASPEDASVDATDSVNCDGALKKLOATFEKNAFSEGSGDDDTKS included by any reference to the displayed strand. As the complement and reverse complement of a primary nucleic DGEAEEYDEOGLRVPKWNSHGKIKTFKCKOCDFWAITKLVFWEHTKLHIK acid sequence are necessarily disclosed by the primary ADKLLKCPKCPFWTEYKHHLEYHLRNHYGSKPFKCNOCSYSCVNKSMLNS sequence, the complementary sequence and reverse comple mentary sequence of a nucleic acid sequence are included by HLKSHSNIYOYRCSDCSYATKYCHSLKLHLRKYSHKPAMVLNPDGTPNPL any reference to the nucleic acid sequence, unless it is explic itly stated to be otherwise (or it is clear to be otherwise from PIIDWYGTRRGPKMKSEOKSSEEMSPKPEQVLPFPFNOFLPOMOLPFPG the context in which the sequence appears). Furthermore, as it PLFGGFPGGIPNPLL LONLEKLARERRESMNSSERFSPAOSEOMDTDAGV is understood in the art that the nucleotide sequence of an RNA strand is determined by the sequence of the DNA from LDLSKPDDSSOTNRRKDSAYKLSTGDNSSDEEDDEATTTMFGNVEVVENK which it was transcribed (but for the substitution ofuracil (U) ELEDTSSGKOTPTSAKKDDYSCOYCOINFCDPWLYTMHMGYHGYKNPFIC nucleobases for thymine (T)), an RNA sequence is included by any reference to the DNA sequence encoding it. In the NMCGEECNDKWSFFLHIARNPHS accompanying sequence listing: 0030 SEQID NO:1 shows an exemplary Diabrotica incm 0032 SEQID NO:3 shows an exemplary Diabrotica incm DNA open reading frame: DNA, referred to herein in Some places as incinn reg1 (region 1), which is used in some examples for the production of a dsRNA: ATGCCAGATACCAAGGATGCCAAGGATACCAAGGATGCTAATTTGAGTTC

TCCTGAACGTAAAAGACGAAGAAAGAGTAGATCTAAATCTCCAGAACGAA GATGCGGCTCCCGAAGAATCTGGCAAGAAGGGGCCAGATAGAAATTCAGA US 2016/0 194658 A1 Jul. 7, 2016

- Continued 0039 SEQID NO:17 shows an exemplary DNA encoding GGAGAAGGAGTTACCAAAGCCAATGGAGTCTGTTCCTGATAAATCAGTCA a Diabrotica incin V2 RNA; containing a sense polynucle otide, a loop sequence (underlined), and an antisense poly TCAAACCAAGAGAAAGAAAAACTGTAGATATGTTAACATCGAGGACTGGT nucleotide (bold font): GGTGCTTATATTCCCCCAGCTAAGCTACGATTGTTACAAGCCAGTATTAC

AGACAAAACATCAGCAGCCTATCAGCGTATAGCATGGGAAGCCTTAAAGA GGCTGCGTAAACGTTTGTAAAAGTAGGAGATGCTGCTTGTGCTTGTATTA

AATCCGTTCATGGTTACATTAATAAAATTAACACCTCGAATATTGGCATC TTGACTTGCACAGCAAACCTCTACCTCTTACTATATTTTCATGCAATAAT

ATCGCCAGAGAATTATTGCATGAAAATATAGTAAGAGGTAGAGGTTTGCT TCTCTGGCGATGATGCCAATGAAACTAGTACCAGTCATCACGCTGGAGCG

GTGCAAGTCAATAATACAAGCACAAGCAGCATCTCCTACTTTTACAAACG CACATATAGGCCCTCCATCAGAAAGTCATTGTGTATATCTCTCATAGGGA

TTTACGCAGCC ACGAGCTGCTTGCGTATTTCCCTTCCGTAGTCAGAGTCATCAATCAGCTG 0033 SEQID NO:4 shows an exemplary Diabrotica incm CACCGTGTCGTAAAGCGGGACGTTCGCAAGCTCGTCCGCGGTTAATGGC DNA, referred to herein in some places as incm reg2 (region 2), which is used in some examples for the production of a ATCATCGCCAGAGAATTATTGCATGAAAATAAGTAAGAGGTAGAGGTTT dsRNA: GCGTGCAAGTCAATAAACAAGCACAAGCAGCATCTCCTACTTACAA

ACGTACGCAGCC CCCTTAGTAAAGAAATCTTAGGCAGTGATGGTGAGTCTGAATCAGGTTCC 0040 SEQID NO:18 shows an exemplary DNA encoding GAAGGTTCAGAAGAGGAATCTGATAATGAAAATGAGGACGAAGTCAAGGA a YFP V2:

CCAGGGAACAATTATTGACAATACTGAAACGAATTTAATTTCTCTTAGAA

GAACCATATATTTGACTATTCACGTCTAGTTTAGATTTTGAAGAATGTGCA ATGTCATCTGGAGCACTTCTCTTTCATGGGAAGATTCCTTACGTTGTGGA

CATAAGCTACTGAAGATGGAGTTGAAACCTGGACAAGAAATAGAATTGTG GATGGAAGGGAATGTTGATGGCCACACCTTTAGCATACGTGGGAAAGGCT

TCACATGTTTCTTGACTGCTGCGCAGAACAAAGAACCTACGAAAAGTTTT ACGGAGATGCCTCAGTGGGAAAGGTTGATGCACAGTTCATCTGCACAACT

ATGGTCTTTTGGCTCAAAGATTTTGTCAAATCAACAAAGTGTATATCGAG GGTGATGTTCCTGTGCCTTGGAGCACACTTGTCACCACTCTCACCTATGG

CCTTTCCAACAAATTTTTAAAGATACCTATTCTACCACTCACAGACTAGA AGCACAGTGCTTTGCCAAGTATGGTCCAGAGTTGAAGGACTTCTACAAGT

TGCTAAC CCTGTATGCCAGATGGCTATGTGCAAGAGCGCACAATCACCTTTGAAGGA 0034 SEQID NO:5 shows an exemplary Diabrotica incm GATGGCAACTTCAAGACTAGGGCTGAAGTCACCTTTGAGAATGGGTCTGT DNA, referred to herein in some places as ncm V1 (version 1), CTACAATAGGGTCAAACT CAATGGTCAAGGCTTCAAGAAAGATGGTCATG which is used in some examples for the production of a dsRNA: TGTTGGGAAAGAACTTGGAGTTCAACTTCACTCCCCACTGCCTCTACATC

TGGGGTGACCAAGCCAACCACGGTCTCAAGTCAGCCTTCAAGATCTGTCA

CAGTCATCAAACCAAGAGAAAGAAAAACTGTAGATATGTTAACATCGAGG TGAGATTACTGGCAGCAAAGGCGACTTCATAGTGGCTGACCACACCCAGA

ACTGGTGGTGCTTATATTCCCCCAGCTAAGCTACGATTGTTACAAGCCAG TGAACACTCCCATTGGTGGAGGTCCAGTTCATGTTCCAGAGTATCATCAC

TATTACAGACAAAACATCAGCAGCCTATCAGCGTATAGCATGGGAAGCCT ATGTCTTACCATGTGAAACTTTCCAAAGATGTGACAGACCACAGAGACAA

TAAAGAAATCCGTTCATGGTTACATTAA CATGTCCTTGAAAGAAACTGTCAGAGCTGTTGACTGTCGCAAGACCTACC

0035) SEQID NO:6 shows an exemplary Diabrotica incm TTTGA DNA, referred to herein in some places as ncm v2 (version 2), which is used in some examples for the production of a 0041 SEQID NO:19 shows an exemplary DNA compris dsRNA: ing a loop:

ATTGGCATCATCGCCAGAGAATTATTGCATGAAAATATAGTAAGAGGTAG AGTCATCACGCTGGAGCGCACATATAGGCCCTCCATCAGAAAGTCATTGT

AGGTTTGCTGTGCAAGTCAATAATACAAGCACAAGCAGCATCTCCTACTT GTATATCTCTCATAGGGAACGAGCTGCTTGCGTATTTCCCTTCCGTAGTC

TTACAAACGTTTACGCAGCC AGAGTCATCAATCAGCTGCACCGTGTCGTAAAGCGGGACGTTCGCAAGCT 0036 SEQ ID NO:7 shows the nucleotide sequence of a CGT T7 phage promoter. 0037 SEQID NO:8 shows an exemplary YFP gene. 0042 SEQID NO:20 shows an exemplary YFP gene. 0038 SEQ ID NOs:9-16 show primers used for PCR 0043 SEQID NO:21 shows a DNA sequence of annexin amplification of incm sequences comprising incm regl, incm region 1. reg2, incm V1, and incm V2, used in Some examples for dsRNA 0044 SEQID NO:22 shows a DNA sequence of annexin production. region 2. US 2016/0 194658 A1 Jul. 7, 2016

0045 SEQ ID NO:23 shows a DNA sequence of beta - Continued spectrin 2 region 1. 0046 SEQ ID NO:24 shows a DNA sequence of beta ACTTTCGTAGCTCATTTAGTAAATCAGAGAGTGGCACATGAAATTTTGGC spectrin 2 region 2. TTTGGAGATACTTACATTGTTGGTGGAGACTCCTACAGATGATTCTGTGG 0047 SEQID NO:25 shows a DNA sequence of mtRP-L4 region 1. AGGTGGCCATTTCATTTTTGAAGGAATGTGGACAAAAACTGACAGAAGTT 0048 SEQID NO:26 shows a DNA sequence of mtRP-L4 region 2. TCAAGTAGAGGTATTACTGCTATATTTGAGATGTTAAGAAACATTTTACA 0049 SEQ ID NOS:27-54 show primers used to amplify TGAAGGCCAGCTAGAAAAAAAGAATTCAGTACATGATTGAAGTTATGTTT gene regions of annexin, beta spectrin 2. mtRP-L4, and YFP for dsRNA synthesis. CAAATAAGGAAAGACGGATTTAAGGATCATGCTGCTGTCGTAGAAGAATT 0050 SEQ ID NO:55 shows a maize DNA sequence encoding a TIP41-like protein. AGATTTAGTAGAAGAGGAAGATCAATTCACTCATCTTATTATGTTAGATG 0051 SEQID NO:56 shows the nucleotide sequence of a ATGTTAAAGAGGCTGATGCAGAGGATATATTGAATGTGTTCAAATTTGAT T20VN primer oligonucleotide. 0052 SEQID NOS:57-61 show primers and probes used GAGAGTTATGAAGAAAATGAAGATAAATACAAAACCCTTAGTAAAGAAAT for dsRNA transcript expression analyses. CTTAGGCAGTGATGGTGAGTCTGAATCAGGTTCCGAAGGTTCAGAAGAGG 0053 SEQ ID NO:62 shows a nucleotide sequence of a portion of a SpecR coding region used for binary vector AATCTGATAATGAAAATGAGGACGAAGTCAAGGACCAGGGAACAATTATT backbone detection. GACAATACTGAAACGAATTTAATTTCTCTTAGAAGAACCATATATTTGAC 0054 SEQID NO:63 shows a nucleotide sequence of an AAD1 coding region used for genomic copy number analy TATTCAGTCTAGTTTAGATTTTGAAGAATGTGCACATAAGCTACTGAAGA S1S. 0055 SEQID NO:64 shows a DNA sequence of a maize TGGAGTTGAAACCTGGACAAGAAATAGAATTGTGTCACATGTTTCTTGAC invertase gene. TGCTGCGCAGAACAAAGAACCTACGAAAAGTTTTATGGTCTTTTGGCTCA 0056 SEQID NOs:65-73 show the nucleotide sequences of DNA oligonucleotides used for gene copy number deter AAGATTTTGTCAAATCAACAAAGTGTATATCGAGCCTTTCCAACAAATTT minations and binary vector backbone detection. TTAAAGATAC CTATTCTACCACTCACAGACTAGATGCTAACAGGTTAAGA 0057 SEQID NOs:74-76 show primers and probes used for dsRNA transcript maize expression analyses. AACGTCAGCAAATTTTTTGCGCATTTACTTTTTACGGATGCCATTGGATG 0058 SEQID NO:77 shows a contig comprising an exem plary Diabrotica incm DNA: GGAAGTCCTTGACATCATGAAATTGAATGAAGAGGATACCAATAGTTCTA GTAGGATTTTCATAAAAATCTTGTTTCAAGAATTGGCTGAATATATGGGA

ATTACCAAATGTCAATGTCACTCATTACTCATTACCAAATGTCAATGTCA CTAGGAAAATTAAACGCAAGGCTAAAGGATGAGACCCTGCAGGCTTATTT

CTGTCAGGTAACGTGCAATGCAAATTGTCAATGTCAAACTTAAAAATATT TTCAGGACTGTTTCCTAGAGATAACCCAAAGAATACCAGATTTTCTATTA

TTCCTGCAACTGCATCAAATTGTAAATTTTATTTTTTTTAAATATGCCAG ATTTTTTTAC CTCTATCGGTTTGGGAGGATTAACTGATGAACTCAGAGAA

ATACCAAGGATGCCAAGGATACCAAGGATGCTAATTTGAGTTCTCCTGAA CATTTAAAAAATATTCCAAAAATGATGGAAATGAAGTTAGCCACTAAAGA

CGTAAAAGACGAAGAAAGAGTAGATCTAAATCTCCAGAACGAAAAGAGAA AAAGGAAAGCAGTGGTAGTAGTAGTTCAGAAAGTAGTTCTGAGGAAGATA

AAAGTCTTCCAAAAAGAAAGCCCACAATAGTAGAGACAGAGATTCATCAG GTAGTGACGACAGCTCTGAAGATTCATCAAGTTCTGAAGACGATAGAGGC

AGGAAGGTTACAACCCTAAAGATTATCAGAGATACTATGGGGAAGATCGC AAAAAGAAGAAAAAAACCAAAAAGCTAGAAGAAAAAAATTCGAAAGTAAA

CCAAACAGTGACAAATATTGGAATAAATATCCAAGGAAAGATACTACCAA TTCTAAGTCTAGACCTAGATCAAAAGAGAAAGAACACGCAGACAAACCTA

AGTTGGCCAAAGATACTATGATGCGGCTCCCGAAGAATCTGGCAAGAAGG GAGACAAACATAGAAAGGAAGATAGACATAAATCTGACAAAAATCCCGAT

GGCCAGATAGAAATTCAGAGGAGAAGGAGTTACCAAAGCCAATGGAGTCT AGATCCTCGTCCAAAAAATATTCTAAAGAAGATAACAAGAGGAAGAAAGA

GTTCCTGATAAATCAGTCATCAAACCAAGAGAAAGAAAAACTGTAGATAT CTACGAATGGATGAAGAGCAGATATGAAGATGATATTAAGCAATTAAAAA

GTTAACATCGAGGACTGGTGGTGCTTATATTCCCCCAGCTAAGCTACGAT ACGATAAAAGGGTATTTGAAAAGTCTTCCAAACGACGATCAAGATCTAGA

TGTTACAAGCCAGTATTACAGACAAAACATCAGCAGCCTATCAGCGTATA GACAGGGTAAAAGTCAAGGAAGAGCGTAGACGTAGAAGCAGAGAAAGAAG GCATGGGAAGCCTTAAAGAAATCCGTTCATGGTTACATTAATAAAATTAA AAGGAGCTAAGATAATTTTTTAATAAGGATCTATGTATATTTATGTAAAC CACCTCGAATATTGGCATCATCGCCAGAGAATTATTGCATGAAAATATAG ATTTATTTAATACATGTTTTTTAAAAAAAAA TAAGAGGTAGAGGTTTGCTGTGCAAGTCAATAATACAAGCACAAGCAGCA 0059 SEQ ID NOS:78-83 show exemplary RNAs tran TCTCCTACTTTTACAAACGTTTACGCAGCCTTAGTAGCTGTTATTAATTC scribed from nucleic acids comprising exemplary Diabrotica GAAGTTTCCAAGTATAGGAGAGCTTTTATTGAAGAGGTTGGTTTTGCAGT incm polynucleotides and fragments thereof. TCAAAAGAGGGTTTAAACAAAATAATAAGTCTATTTGCATATCGGCTACT 0060 SEQID NO:84 shows a contig comprising an exem plary Melligethes aeneus incm DNA: US 2016/0 194658 A1 Jul. 7, 2016

- Continued GCAATTTCGTTTTTGAAGGAAAGTGGTCAAAAACT CACTGAGGTGTCGAG YEENEGKYKTLSKEILOSDSESGESGSEGSEEDSEDEEGEEDETKNOTII

TAAAGGTATCAATGCCATATTTGAGATGTTGAGGAATATTCTGCATGAAG DNTETNLITLRRTIYLTIOSSLDFEECAHKLMKMEIKPGOEIELCHMFLD

GACAGTTGGAGAAGAGAATACAGTACATGATTGAAGTCATGTTCCAAGTT CCAEORTYEKFFGLLSORFCOINKTFIEPFOOIFKDTYSTTHRLDANRLR

CGGAAAGATGGTTTCAAGGATCATGCTGCTGTTACTGAAGAACTAGATAT NVSKFFAHLLFTDAIGWEVLDIMKLNEEDTNSSSRIFIKILFOELSEYMG

TGTTGAAGAGGAAGATCAGTTTACT CACCTAATCACATTGGATGATGTTA LAKLNKRLKDETLOEYFAGLFPRDNPKNTRFAINFFTSIGLGGLTDELRE

AACAAGCTAACTCAGAGGATATATTGAATGTGTTTAAATTTGATGATAAA HLKNWPKHLEWMALKADSSSSSSSSSSSSNDSSSSSDSSDDEGSRKKKTK

TATGAGGAAAATGAGGGTAAATACAAAACTTTAAGTAAGGAAATTCTCCA KLKTPDKKKKOKEDEKPKKKSEDKPRNKPDYRDRRNDDREKFKKYRNNDE

GTCAGACAGTGAATCAGGCGAATCTGGTTCAGAGGGGTCTGAAGAAGACT ESHRRSREDAREKYRGHEERRSDHREEYRPREHRGRDRR CGGAAGATGAAGAAGGTGAAGAAGATGAAACCAAAAATCAAACCATTATT 0062 SEQID NO:86 shows a contig comprising an exem GATAACACAGAAACTAATTTAATCACCTTAAGGAGAACCATCTATCT CAC plary Melligethes aeneus incm DNA:

AATACAATCCAGTTTGGATTTTGAGGAATGTGCCCATAAATTGATGAAAA CCACACGAATTGACTGGTTAATTAAAAATAAGCACAAGAAACGAATAACA TGGAGATCAAACCTGGACAAGAGATTGAATTGTGTCACATGTTCCTTGAT CTACAATGGTTTGAATTTACACAAAAAAAAAAATGTAGTCACTACCATTG TGTTGCGCTGAACAGCGTACCTACGAAAAATTCTTCGGCCTCCTCTCGCA TAGTGTTATTCGTTTCTTGTGCTTATTTTTAATTAACCAGTCAATTCGTG GCGCTTCTGCCAAATAAACAAGACTTTCATCGAACCGTTCCAACAAATTT TGGTGTAGTGAACAAAGGTTATAGTTATGACGACGGATTCCGAGAGAGGT TCAAAGATAC CTATTCCACAACT CACAGACTTGACGCCAATCGATTGAGA TCCCCTACAGCTGCGGCTCCACGCAGAAGCGCCTCGAAATCGCCAGAACC AACGTTAGCAAATTCTTCGCGCATTTATTGTTCACCGACGCCATCGGCTG AAAAAAAGCAAAGTACGATAAGAAAGAGAAGGGCGATAAAGATCGCAAGA GGAAGTGCTCGATATCATGAAATTAAACGAGGAAGACACCAACAGTTCCA GGAGATCCCACAGATCCAGATCTAGATCCAGGGATAGAGACCATAGGGAC GCAGGATTTTCATCAAGATTTTGTTCCAGGAGTTGTCCGAATATATGGGA AAACATGGTGGAAAAAAACGTTACCACGACCTGGACGACCCTTCTGAAGA TTAGCGAAGTTGAATAAAAGGCTAAAGGATGAAACTTTACAGGAATATTT CTACCCAAGATATTATGGCGAGGATAGAAAACAGAACAGTGACAGATATT CGCGGGGCTATTTCCGAGGGATAACCCGAAGAACACGCGTTTCGCCATCA GGTCCAAGTACCCAAAGAAAGACAGGGACGAATATGTTATTGGTAGCCGG ATTTTTTCACGTCGATCGGTTTAGGAGGTCTAACGGACGAGTTGAGGGAG TATTATGATGTTGAGGAAAAGAAGGAGAAAAAAGAAAAAGAGGATGAAAA CACTTGAAAAACGTGCCAAAACATCTGGAAGTGATGGCTTTGAAAGCAGA TAAGGATAAATCCGTCATCACTCCAAGGGAAAGGAAAACAGTGGACTTAC TTCGAGCAGCTCTAGCAGCAGTAGCAGCAGTTCCAGTAACGATTCCAGCA TAACATCTCGAACAGGTGGGGCTTATATAC CTCCAGCTAAATTACGTATG GCAGTTCAGATTCTTCCGATGACGAGGGTTCCAGGAAGAAGAAAACAAAA ATGCAGGCTGAGATAACTGATAAATCATCAGCTGCATATCAAAGAATTGC AAATTGAAAACCCCGGACAAAAAGAAGAAACAGAAAGAAGATGAAAAACC CTGGGAAGCTTTAAAAAAGTCCATTCATGGTTACATCAACAAAATTAACA CAAAAAGAAAAGCGAGGATAAACCGAGGAACAAACCAGACTATAGAGATA CTTCCAATATTGGTCTTATTGCTAGAGAATTACTGCATGAAAACATTGTA GAAGAAACGACGACAGGGAAAAGTTTAAAAAATACAGAAACAACGACGAA AGAGGTAGAGGTTTGCTGTGTAAATCTATAATACAAGCACAGGCAGCTTC GAAAGCCACAGAAGAAGCAGAGAAGATGCAAGAGAAAAATACAGAGGTCA, CCCGACGTTCACCAATGTTTATGCAGCTTTAGTTGCAGTCATAAATTCAA CGAGGAAAGAAGAAGCGACCACAGAGAAGAATACCGGCCGAGAGAACATA AATTCCCCAACATTGGAGAACTGTTACTGAAAAGGTTGGTTTTGCAGTTT GAGGTAGAGATAGACGTTAGTTGTATAATAATGTATATTTTTTCGTATTT AAAAGGGGTTTCAAGCAGAACAACAAGTCTATCTGTATATCGGCTGCTAC AATAAAATAAATTATACATTTTATAGTGTTTCGGAGCATTCACCAAGCAA CTTTGTCGCGCATTTAGTAAACCAAAGAGTGGCCCACGAAATTTTAGCAT GGGTTTTACTTTCGGATAGCAATGGTGTAGTACGTTTTTGAAGGTGTCCA TGGAAATTCTTACTTTACTTGTTGAGTCCCCCACAGATGATTCAGTGGAA CACACACCAAGCCGGTTTTATCTAAATCTAGAGCTATCTTCCAAAAGTCT GTAGCAATTTCGTTTTTGAAGGAAAGTGGTCAAAAACT CACTGAGGTGTC TCAAAGGA GAGTAAAGGTATCAATGCCATATTTGAGATGTTGAGGAATATTCTGCATG 0061 SEQID NO:85 shows the amino acid sequence of a Melligethes aeneus NCM polypeptide encoded by an exem AAGGACAGTTGGAGAAGAGAATACAGTACATGATTGAAGTCATGTTCCAA plary Melligethes aeneus incm DNA: GTTCGGAAAGATGGTTTCAAGGATCATGCTGCTGTTACTGAAGAACTAGA

TATTGTTGAAGAGGAAGATCAGTTTACT CACCTAATCACATTGGATGATG AISFLKESGOKLTEVSSKGINAIFEMLRNILHEGOLEKRIOYMIEVMFOW TTAAACAAGCTAACTCAGAGGATATATTGAATGTGTTTAAATTTGATGAT RKDGFKDHAAVTEELDIVEEEDOFTHLITLDDWKOANSEDILNWFKFDDK AAATATGAGGAAAATGAGGGTAAATACAAAACTTTAAGTAAGGAAATTCT US 2016/0 194658 A1 Jul. 7, 2016

- Continued - Continued

CCAGTCAGACAGTGAATCAGGCGAATCTGGTTCAGAGGGGTCTGAAGAAG GGLTDELREHLKNWPKHLEWMALKADSSSSSSSSSSSSNDSSSSSDSSDD

ACTCGGAAGATGAAGAAGGTGAAGAAGATGAAACCAAAAATCAAACCATT EGSRKKKTKKLKTPDKKKKOKEDEKPKKKSEDKPRNKPDYRDRRNDDREK

ATTGATAACACAGAAACTAATTTAATCACCTTAAGGAGAACCATCTATCT FKKYRNNDEESHRRSREDAREKYRGHEERRRGREDOTRRGODOVPRFV CACAATACAATCCAGTTTGGATTTTGAGGAATGTGCCCATAAATTGATGA 0064 SEQID NO:88 shows a contig comprising an exem AAATGGAGATCAAACCTGGACAAGAGATTGAATTGTGTCACATGTTCCTT plary Melligethes aeneus incm DNA:

GATTGTTGCGCTGAACAGCGTACCTACGAAAAATTCTTCGGCCTCCTCTC AAATGTAGTCACTACCATTGTAGTGTTATTCGTTTCTTGTGCTTATTTTT GCAGCGCTTCTGCCAAATAAACAAGACTTTCATCGAACCGTTCCAACAAA AATTAACCAGTCAATTCGTGTGGTGTAGTGAACAAAGGTTATAGTTATGA TTTTCAAAGATACCTATTCCACAACT CACAGACTTGACGCCAATCGATTG CGACGGATTCCGAGAGAGGTTCCCCTACAGCTGCGGCTCCACGCAGAAGC AGAAACGTTAGCAAATTCTTCGCGCATTTATTGTTCACCGACGCCATCGG GCCTCGAAATCGCCAGAACCAAAAAAAGCAAAGTACGATAAGAAAGAGAA CTGGGAAGTGCTCGATATCATGAAATTAAACGAGGAAGACACCAACAGTT GGGCGATAAAGATCGCAAGAGGAGATCCCACAGATCCAGATCTAGATCCA CCAGCAGGATTTTTATCAAGATTTTGTTCCAGGAGTTGTCCGAATATATG GGGATAGAGACCATAGGGACAAACATGGTGGAAAAAAACGTTACCACGAC GGATTAGCGAAGTTGAATAAAAGGCTAAAGGATGAAACTTTACAGGAATA CTGGACGACCCTTCTGAAGACTACCCAAGATATTATGGCGAGGATAGAAA TTTCGCGGGGCTATTTCCGAGGGATAACCCGAAGAACACGCGTTTCGCCA ACAGAACAGTGACAGATATTGGTCCAAGTACCCAAAAGAAAGACAGGGAC TCAATTTTTTCACGTCGATCGGTTTAGGAGGTCTAACGGACGAGTTGAGG GAATATGTTATTGGTAACCGGTATTATGATGTTGAGGAAAAGAAGGAGAA GAGCACTTGAAAAACGTGCCAAAACATCTGGAAGTGATGGCTTTGAAAGC AAAGGAAAAAGAGGATGAAAATAAGGATAAATCCGTCATTACTCCAAGGG AGATTCGAGCAGCTCTAGCAGCAGTAGCAGCAGTTCCAGTAACGATTCCA AAAGGAAAACAGTGGACTTACTAACATCTCGAACAGGTGGGGCTTATATA GCAGCAGTTCAGATTCTTCCGATGACGAGGGTTCCAGGAAGAAGAAAACA CCTCCAGCTAAATTACGTATGATGCAGGCTGAGATAACTGATAAATCATC AAAAAATTGAAAACCCCGGACAAAAAGAAGAAACAGAAAGAAGATGAAAA AGCTGCATATCAAAGAATTGCCTGGGAAGCTTTAAAAAAGTCCATTCATG ACCCAAAAAGAAAAGCGAGGATAAACCGAGGAACAAACCAGACTATAGAG GTTACATCAACAAAATTAACACTTCCAATATTGGTCTTATTGCTAGAGAA ATAGGAGAAACGACGACAGGGAAAAGTTTAAAAAATACAGAAACAACGAC TTACTGCATGAAAACATTGTAAGAGGTAGAGGTTTGCTGTGTAAATCTAT GAAGAAAGCCACAGAAGAAGCAGAGAAGATGCAAGAGAAAAATACAGAGG AATACAAGCACAGGCAGCTTCCCCGACGTTCACCAATGTTTATGCAGCTT TCACGAGGAAAGAAGACGAGGCCGAGAAGACCAAACGCGAAGAGGACAAG TAGTTGCAGTCATAAATTCAAAATTCCCCAACATTGGAGAACTGTTACTG ACCAAGTTCCAAGGTTTGTGC AAAAGGTTGGTTTTGCAGTTTAAAAGGGGTTTCAAGCAGAACAACAAGTC 0063 SEQID NO:87 shows the amino acid sequence of a Melligethes aeneus NCM polypeptide encoded by an exem TATCTGTATATCGGCTGCTACCTTTGTCGCGCATTTAGTAAACCAAAGAG plary Melligethes aeneus incm DNA: TGGCTCATGAAATTTTAGCATTGGAAATTCTTACTTTACTTGTTGAGTCC

CCCACAGATGATTCAGTAGAAGTGAGCAGAACAACAAGTCTATCTGTATA MTTDSERGSPTAAAPRRSASKSPEPKKAKYDKKEKGDKDRKRRSHRSRSR TCGGCTGCTACCTTTGTCGCGCATTTAGTAAACCAAAGAGTGGCCCACGA SRDRDHRDKHGGKKRYHDLDDPSEDYPRYYGEDRKONSDRYWSKYPKKDR AATTTTAGCATTGGAAATTCTTACTTTACTTGTTGAGTCCCCCACAGATG DEYWIGSRYYDWEEKKEKKEKEDENKDKSWITPRERKTWDLLTSRTGGAY ATTCAGTGGAAGTAGCAATTTCGTTTTTGAAGGAAAGTGGTCAAAAACTC IPPAKLRMMOAEITDKSSAAYORIAWEALKKSIHGYINKINTSNIGLIAR ACTGAGGTGTCGAGTAAAGGTATCAATGCCATATTTGAGATGTTGAGGAA ELLHENIWRGRGLLCKSIICAOAASPTFTNWYAALWAVINSKFPNIGELL TATTCTGCATGAAGGACAGTTGGAGAAGAGAATACAGTACATGATTGAAG LKRLVLOFKRGFKONNKSICISAATFWAHLVNORVAHEILALEILTLLVE TCATGTTCCAAGTTCGGAAAGATGGTTTCAAGGATCATGCTGCTGTTACT SPTDDSVEVAISFLKESGOKLTEVSSKGINAIFEMLRNILHEGOLEKRIO GAAGAACTAGATATTGTTGAAGAGGAAGATCAGTTTACT CACCTAATCAC YMIEVMFOVRKDGFKDHAAVTEELDIVEEEDOFTHILITLDDVKOANSEDI ATTGGATGATGTTAAACAAGCTAACTCAGAGGATATATTGAATGTGTTTA LNWFKFDDKYEENEGKYKTLSKEILOSDSESGESGSEGSEEDSEDEEGEE AATTTGATGATAAATATGAGGAAAATGAGGGTAAATACAAAACTTTAAGT DETKNOTIIDNTETNLITLRRTIYLTIOSSLDFEECAHKLMKMEIKPGOE AAGGAAATTCTCCAGTCAGACAGTGAATCAGGCGAATCTGGTTCAGAGGG IELCHMFLDCCAEORTYEKFFGLLSORFCOINKTFIEPFOOIFKDTYSTT GTCTGAAGAAGACTCGGAAGATGAAGAAGGTGAAGAAGATGAAACCAAAA HRLDANRLRNWSKFFAHLLFTDAIGWEWLDIMKLNEEDTNSSSRIFIKIL ATCAAACCATTATTGATAACACAGAAACTAATTTAATCACCTTAAGGAGA FOELSEYMGLAKLNKRLKDETLOEYFAGLFPRDNPKNTRFAINFFTSIGL US 2016/0 194658 A1 Jul. 7, 2016

- Continued - Continued ACCATCTATCTCACAATACAATCCAGTTTGGATTTTGAGGAATGTGCCCA AACAGTTCCAGCAGGATTTTCATCAAGATTTTGTTCCAGGAGTTGTCCGA

TAAATTGATGAAAATGGAGATCAAACCTGGACAAGAGATTGAATTGTGTC ATATATGGGATTAGCGAAGTTGAATAAAAGGCTAAAGGATGAAACTTTAC

ACATGTTCCTTGATTGTTGCGCTGAACAGCGTACCTACGAAAAATTCTTC AGGAATATTTCGCGGGGCTATTTCCGAGGGATAACCCGAAGAACACGCGT

GGCCTCCTCTCGCAGCGCTTCTGCCAAATAAACAAGACTTTCATCGAACC TTCGCCATCAATTTTTTCACGTCGATCGGTTTAGGAGGTCTAACGGACGA

GTTCCAACAAATTTTCAAAGATACCTATTCCACAACT CACAGACTTGACG GTTGAGGGAGCACTTGAAAAACGTGCCAAAACATCTGGA CCAATCGATTGAGAAACGTTAGCAAATTCTTCGCGCATTTATTGTTCACC 0067 SEQ ID NOs:91-92 show primers used to amplify portions of a Melligethes aeneus incrm sequence comprising GACGCCATCGGCTGGGAAGTGCTCGATATCATGAAATTAAACGAGGAAGA incm reg1. CACCAACAGTTCCAGCAGGATTTTCATCAAGATTTTGTTCCAGGAGTTGT 0068 SEQID NO:93 shows a contig comprising an exem CCGAATATATGGGATTAGCGAAGTTGAATAAAAGGCTAAAGGATGAAACT plary Melligethes aeneus incm DNA:

TTACAGGAATATTTCGCGGGGCTATTTCCGAGGGATAACCCGAAGAACAC TTTTTGTTACCAACCAAACGGCCTAAATTTCCCTAGATAACCTAAAATAA GCGTTTCGCCATCAATTTTTTCACGTCGATCGGTTTAGGAGGTCTAACGG AAACAACACTTCGTCATTTGTGTAAATTCAAACAAATGTAGTCACTACCA ACGAGTTGAGGGAGCACTTGAAAAACGTGCCAAAACATCTGGAAGTGATG TTGTAGTGTTATTCGTTTCTTGTGCTTATTTTTAATTAACCAGTCAATTC GCTTTGAAAGCAGATTCGAGCAGCTCTAGCAGCAGTAGCAGCAGTTCCAG GTGTGGTGTAGTGAACAAAGGTTATAGTTATGACGACGGATTCCGAGAGA TAACGATTCCAGCAGCAGTTCAGATTCTTCCGATGACGAGGGTTCCAGGA GGTTCCCCTACAGCTGCGGCTCCACGCAGAAGCGCCTCGAAATCGCCAGA AGAAGAAAACAAAAAAATTGAAAACCCCGGACAAAAAGAAGAAACAGAAA ACCAAAAAAAGCAAAGTACGATAAGAAAGAGAAGGGCGATAAAGATCGCA GAAGATGAAAAACCCAAAAAGAAAAGCGAGGATAAACCGAGGAACAAAGT AGAGGAGATCCCACAGATCCAGATCTAGATCCAGGGATAGAGACCATAGG AAATGGTGATAAGAATATAGAAACAGAAGATACACAAGGAGTTGAGGACA GACAAACATGGTGGAAAAAAACGTTACCACGACCTGGACGACCCTTCTGA CAAAAAAGACTG AGACTACCCAAGATATTATGGCGAGGATAGAAAACAGAACAGTGACAGAT 0065. SEQID NO:89 shows the amino acid sequence of a Melligethes aeneus NCM polypeptide encoded by an exem ATTGGTCCAAGTACCCAAAGAAAGACAGGGACGAATATGTTATTGGTAGC plary Melligethes aeneus incm DNA: CGGTATTATGATGTTGAGGAAAAGAAGGAGAAAAAGGAAAAAGAGGATGA

AAATAAGGATAAATCCGTCATTACTCCAAGGGAAAGGAAAACAGTGGACT MLRNILHEGOLEKRIOYMIEVMFOVRKDGFKDHAAVTEELDIVEEEDOFT TACTAACATCTCGAACAGGTGGGGCTTATATACCTCCAGCTAAATTACGT HLITLDDWKOANSEDILNWFKFDDKYEENEGKYKTLSKEILOSDSESGES ATGATGCAGGCTGAGATAACTGATAAATCATCAGCTGCATATCAAAGAAT GSEGSEEDSEDEEGEEDETKNOTIIDNTETNLITLRRTIYLTIOSSLDFE TGCCTGGGAAGCTTTAAAAAAGTCCATTCATGGTTACATCAACAAAATTA ECAHKLMKMEIKPGOEIELCHMFLDCCAEORTYEKFFGLLSORFCOINKT ACACTTCCAATATTGGTCTTATTGCTAGAGAATTACTGCATGAAAACATT FIEPFOOIFKDTYSTTHRLDANRLRNVSKFFAHLLFTDAIGWEVLDIMKL GTAAGAGGTAGAGGTTTGCTGTGTAAATCTATAATACAAGCACAGGCAGC NEEDTNSSSRIFIKILFOELSEYMGLAKLNKRLKDETLOEYFAGLFPRDN TTCCCCGACATTCACCAATGTTTATGCAGCTTTAGTTGCAGTCATAAATT PKNTRFAINFFTSIGLGGLTDELREHLKNWPKHLEWMALKADSSSSSSSS CAAAATTCCCCAACATTGGAGAACTGTTACTGAAAAGGTTGGTTTTGCAG SSSSNDSSSSSDSSDDEGSRKKKTKKLKTPDKKKKOKEDEKPKKKSEDKP TTTAAAAGGGGTTTCAAGCAGAACAACAAGTCTATCTGTATATCGGCTGC RNKVNGDKNIETED TOGVEDTKKT TACCTTTGTCGCGCATTTAGTAAACCAAAGAGTGGCCCATGAAATTTTAG

0066 SEQ ID NO:90 shows an exemplary Melligethes CATTGGAAATTCTTACTTTACTTGTTGAGTCCCCCACAGATGATTCAGTG aeneus incin DNA, referred to herein in some places as incm regl (region 1), which is used in Some examples for the GAAGTAGCAATTTCGTTTTTGAAGGAAAGTGGTCAAAAACT CACTGAGGT production of a dsRNA: GTCGAGTAAAGGTATCAATGCCATATTTGAGATGTTGAGGAATATTCTGC

ATGAAGGACAGTTGGAGAAGAGAATACAGTACATGATTGAAGTCATGTTC GTTCCTTGATTGTTGCGCTGAACAGCGTACCTACGAAAAATTCTTCGGCC CAAGTTCGGAAAGATGGTTTCAAGGATCATGCTGCTGTTACTGAAGAACT TCCTCTCGCAGCGCTTCTGCCAAATAAACAAGACTTTCATCGAACCGTTC AGATATTGTTGAAGAGGAAGATCAGTTTACT CACCTAATCACATTGGATG CAACAAATTTTCAAAGATACCTATTCCACAACT CACAGACTTGACGCCAA ATGTTAAACAAGCTAACTCAGAGGATATATTGAATGTGTTTAAATTTGAT TCGATTGAGAAACGTTAGCAAATTCTTCGCGCATTTATTGTTCACCGACG GATAAATATGAGGAAAATGAGGGTAAATACAAAACTTTAAGTAAGGAAAT CCATCGGCTGGGAAGTGCTCGATATCATGAAATTAAACGAGGAAGACACC TCTCCAGTCAGACAGTGAATCAGGCGAATCTGGTTCAGAGGGGTCTGAAG US 2016/0 194658 A1 Jul. 7, 2016

- Continued - Continued

AAGACTCGGAAGATGAAGAAGGTGAAGAAGATGAAACCAAAAATCAAACC EGSRKKKTKKLKTPDKKKKOKEDEKPKKKSEDKPRNKPDYRDRRNDDREK

ATTATTGATAACACAGAAACTAATTTAATCACCTTAAGGAGAACCATCTA FKKYRNINDEESHRRSREDAREKYRGHEERRSDHREEYRPREHRGRDRR TCTCACAATACAATCCAGTTTGGATTTTGAGGAATGTGCCCATAAATTGA (0070 SEQ ID NOs:95-99 show exemplary RNAs tran scribed from nucleic acids comprising exemplary Melligethes TGAAAATGGAGATCAAACCTGGACAAGAGATTGAATTGTGTCACATGTTC incm polynucleotides and fragments thereof. CTTGATTGTTGCGCTGAACAGCGTACCTACGAAAAATTCTTCGGCCTCCT DETAILED DESCRIPTION CTCGCAGCGCTTCTGCCAAATAAACAAGACTTTCATCGAACCGTTCCAAC I. Overview of Several Embodiments AAATTTTCAAAGATACCTATTCCACAACT CACAGACTTGACGCCAATCGA (0071. We developed RNA interference (RNAi) as a tool TTGAGAAACGTTAGCAAATTCTTCGCGCATTTATTGTTCACCGACGCCAT for insect pest management, using one of the most likely CGGCTGGGAAGTGCTCGATATCATGAAATTAAACGAGGAAGACACCAACA target pest species for transgenic plants that express dsRNA; the western corn rootworm. Thus far, most genes proposed as GTTCCAGCAGGATTTTCATCAAGATTTTGTTCCAGGAGTTGTCCGAATAT targets for RNAi in rootworm larvae do not actually achieve ATGGGATTAGCGAAGTTGAATAAAAGGCTAAAGGATGAAACTTTACAGGA their purpose. Herein, we describe RNAi-mediated knock down of nucampholin (ncm) in the exemplary insect pest, ATATTTCGCGGGGCTATTTCCGAGGGATAACCCGAAGAACACGCGTTTCG western corn rootworm, which is shown to have a lethal phenotype when, for example, iRNA are molecules delivered CCATCAATTTTTTCACGTCGATCGGTTTAGGAGGTCTAACGGACGAGTTG via ingested incm dsRNA. In embodiments herein, the ability AGGGAGCACTTGAAAAACGTGCCAAAACATCTGGAAGTGATGGCTTTGAA to deliver incinn dsRNA by feeding to insects confers a RNAi effect that is very useful for insect (e.g., coleopteran) pest AGCAGATTCGAGCAGCTCTAGCAGCAGTAGCAGCAGTTCCAGTAACGATT management. By combining incm-mediated RNAi with other CCAGCAGCAGTTCAGATTCTTCCGATGACGAGGGTTCCAGGAAGAAGAAA useful RNAi targets (e.g., ROPRNAi targets, as described in U.S. patent application Ser. No. 14/577,811: RNAPII140 ACAAAAAAATTGAAAACCCCGGACAAAAAGAAGAAACAGAAAGAAGATGA RNAi targets, as described in U.S. patent application Ser. No. 14/577,854; Drea. RNAi targets, as described in U.S. patent AAAACCCAAAAAGAAAAGCGAGGATAAACCGAGGAACAAACCAGACTATA application Ser. No. 14/705,807; COPI alpha RNAi targets, as GAGATAGAAGAAACGACGACAGGGAAAAGTTTAAAAAATACAGAAACAAC described in U.S. Patent Application No. 62/063, 199: COPI beta RNAi targets, as described in U.S. Patent Application GACGAAGAAAGCCACAGAAGAAGCAGAGAAGATGCAAGAGAAAAATACAG No. 62/063.203; COPI gamma RNAi targets, as described in AGGTCACGAGGAAAGAAGAAGCGACCACAGAGAAGAATACCGGCCGAGAG U.S. Patent Application No. 62/063, 192: COPI delta RNAi targets, as described in U.S. Patent Application No. 62/063, AACATAGAGGTAGAGATAGACGTTAGTTGTATAATAATGTATATTTTTT 216) the potential to affect multiple target sequences, for example, incoleoptera (e.g., larval rootworms), may increase 0069 SEQID NO:94 shows the amino acid sequence of a opportunities to develop Sustainable approaches to insect pest Melligethes aeneus NCM polypeptide encoded by an exem management involving RNAi technologies. plary Melligethes aeneus incm DNA: 0072 Disclosed herein are methods and compositions for genetic control of insect (e.g., coleopteran) pest infestations. MTTDSERGSPTAAAPRRSASKSPEPKKAKYDKKEKGDKDRKRRSHRSRSR Methods for identifying one or more gene(s) essential to the lifecycle of an insect pest for use as a target gene for RNAi SRDRDHRDKHGGKKRYHDLDDPSEDYPRYYGEDRKONSDRYWSKYPKKDR mediated control of an insect pest population are also pro vided. DNA plasmid vectors encoding an RNA molecule may DEYWIGSRYYDWEEKKEKKEKEDENKDKSWITPRERKTWDLLTSRTGGAY be designed to suppress one or more target gene(s) essential IPPAKLRMMOAEITDKSSAAYORIAWEALKKSIHGYINKINTSNIGLIAR for growth, Survival, and/or development. In some embodi ments, the RNA molecule may be capable of forming dsRNA ELLHENIWRGRGLLCKSIICAOAASPTFTNWYAALWAVINSKFPNIGELL molecules. In some embodiments, methods are provided for post-transcriptional repression of expression or inhibition of LKRLVLOFKRGFKONNKSICISAATFWAHLVNORVAHEILALEILTLLVE a target gene via nucleic acid molecules that are complemen SPTDDSVEVAISFLKESGOKLTEVSSKGINAIFEMLRNILHEGOLEKRIO tary to a coding or non-coding sequence of the target gene in an insect pest. In these and further embodiments, a pest may YMIEVMFOVRKDGFKDHAAVTEELDIVEEEDOFTHILITLDDVKOANSEDI ingest one or more dsRNA, siRNA, shRNA, miRNA, and/or LNWFKFDDKYEENEGKYKTLSKEILOSDSESGESGSEGSEEDSEDEEGEE hpRNA molecules transcribed from all or a portion of a nucleic acid molecule that is complementary to a coding or DETKNOTIIDNTETNLITLRRTIYLTIOSSLDFEECAHKLMKMEIKPGOE non-coding sequence of a target gene, thereby providing a IELCHMFLDCCAEORTYEKFFGLLSORFCOINKTFIEPFOOIFKDTYSTT plant-protective effect. 0073. Thus, some embodiments involve sequence-specific HRLDANRLRNWSKFFAHLLFTDAIGWEWLDIMKLNEEDTNSSSRIFIKIL inhibition of expression of target gene products, using dsRNA, siRNA, shRNA, miRNA and/or hpRNA that is FOELSEYMGLAKLNKRLKDETLOEYFAGLFPRDNPKNTRFAINFFTSIGL complementary to coding and/or non-coding sequences of the GGLTDELREHLKNWPKHLEWMALKADSSSSSSSSSSSSNDSSSSSDSSDD target gene(s) to achieve at least partial control of an insect (e.g., coleopteran) pest. Disclosed is a set of isolated and US 2016/0 194658 A1 Jul. 7, 2016 purified nucleic acid molecules comprising a polynucleotide, ments, a method for modulating the expression of a target for example, as set forth in one of SEQID NOs: 1; 77: 84;86: gene in an insect pest cell may comprise: (a) transforming a 88; and 93, and fragments thereof. In some embodiments, a plant cell with a vector comprising a polynucleotide encoding stabilized dsRNA molecule may be expressed from these an RNA molecule capable of forming a dsRNA molecule; (b) polynucleotides, fragments thereof, or a gene comprising one culturing the transformed plant cell under conditions suffi of these polynucleotides, for the post-transcriptional silenc cient to allow for development of a plant cell culture com ing or inhibition of a target gene. In certain embodiments, prising a plurality of transformed plant cells; (c) selecting for isolated and purified nucleic acid molecules comprise all or a transformed plant cell that has integrated the vector into its part of any of SEQID NOs: 1:3-6: 77: 84; 86:88:90; and 93. genome; and (d) determining that the selected transformed 0.074. Some embodiments involve a recombinant host cell plant cell comprises the RNA molecule capable of forming a (e.g., a plant cell) having in its genome at least one recombi dsRNA molecule encoded by the polynucleotide of the vec nant DNA encoding at least one iRNA (e.g., dsRNA) mol tor. A plant may be regenerated from a plant cell that has the ecule(s). In particular embodiments, the dsRNA molecule(s) vector integrated in its genome and comprises the dsRNA may be provided when ingested by an insect (e.g., molecule encoded by the polynucleotide of the vector. coleopteran) pest to post-transcriptionally silence or inhibit 0078 Thus, also disclosed is a transgenic plant compris the expression of a target gene in the pest. The recombinant ing a vector having a polynucleotide encoding an RNA mol DNA may comprise, for example, any of SEQID NOS:1, 3-6, ecule capable of forming a dsRNA molecule integrated in its 77.84, 86, 88,90, and 93; fragments of any of SEQID NOS:1, genome, wherein the transgenic plant comprises the dsRNA 3-6, 77.84, 86, 88,90, and 93; and a polynucleotide consist molecule encoded by the polynucleotide of the vector. In ing of a partial sequence of a gene comprising one of SEQID particular embodiments, expression of an RNA molecule NOs:1, 3-6, 77, 84, 86, 88,90, and 93, and/or complements capable of forming a dsRNA molecule in the plant is suffi thereof. cient to modulate the expression of a target gene in a cell of an 0075 Some embodiments involve a recombinant host cell insect (e.g., coleopteran) pest that contacts the transformed having in its genome a recombinant DNA encoding at least plant or plant cell (for example, by feeding on the transformed one iRNA (e.g., dsRNA) molecule(s) comprising all or part of plant, a part of the plant (e.g., root) or plant cell). Such that SEQ ID NO:78 or SEQ ID NO:83 (e.g., at least one poly growth and/or survival of the pest is inhibited. Transgenic nucleotide selected from a group comprising SEQ ID NOs: plants disclosed herein may display resistance and/or 78-83). Some embodiments involve a recombinant host cell enhanced tolerance to insect pest infestations. Particular having in its genome a recombinant DNA encoding at least transgenic plants may display resistance and/or enhanced one iRNA (e.g., dsRNA) molecule(s) comprising all or part of tolerance to one or more coleopteran pest(s) selected from the any of SEQ ID NOs:95-97 and 99 (e.g., SEQ ID NO: 98). group consisting of WCR: NCR; SCR; MCR: D. balteata When ingested by an insect (e.g., coleopteran) pest, the iRNA LeConte; D. u. tenella, D. speciosa Germar, D. u. undecim molecule(s) may silence or inhibit the expression of a target punctata Mannerheim; and Melligethes aeneus. incm DNA (e.g., a DNA comprising all or part of a polynucle (0079 Also disclosed herein are methods for delivery of otide selected from the group consisting of SEQID NOs: 1; control agents, such as an iRNA molecule, to an insect (e.g., 3-6: 77, 84, 86, 88,90, and 93) in the pest, and thereby result coleopteran) pest. Such control agents may cause, directly or in cessation of growth, development, and/or feeding in the indirectly, an impairment in the ability of an insect pest popu pest. lation to feed, grow or otherwise cause damage in a host. In 0076. In some embodiments, a recombinant host cell hav Some embodiments, a method is provided comprising deliv ing in its genome at least one recombinant DNA encoding at ery of a stabilized dsRNA molecule to an insect pest to sup least one RNA molecule capable of forming a dsRNA mol press at least one target gene in the pest, thereby causing ecule may be a transformed plant cell. Some embodiments RNAi and reducing or eliminating plant damage in a pest involve transgenic plants comprising such a transformed host. In some embodiments, a method of inhibiting expres plant cell. In addition to Such transgenic plants, progeny sion of a target gene in the insect pest may result in cessation plants of any transgenic plant generation, transgenic seeds, of growth, Survival, and/or development, in the pest. and transgenic plant products, are all provided, each of which 0080. In some embodiments, compositions (e.g., a topical comprises recombinant DNA(s). In particular embodiments, composition) are provided that comprise an iRNA (e.g., an RNA molecule capable of forming a dsRNA molecule may dsRNA) molecule for use in plants, animals, and/or the envi be expressed in a transgenic plant cell. Therefore, in these and ronment of a plant or animal to achieve the elimination or other embodiments, a dsRNA molecule may be isolated from reduction of an insect (e.g., coleopteran) pest infestation. In a transgenic plant cell. In particular embodiments, the trans particular embodiments, the composition may be a nutritional genic plant is a plant selected from the group comprising corn composition or food source to be fed to the insect pest. Some (Zea mays), soybean (Glycine max), rapeseed (Brassica sp.), embodiments comprise making the nutritional composition and plants of the family Poaceae. or food source available to the pest. Ingestion of a composi 0.077 Some embodiments involve a method for modulat tion comprising iRNA molecules may result in the uptake of ing the expression of a target gene in an insect (e.g., the molecules by one or more cells of the pest, which may in coleopteran) pest cell. In these and other embodiments, a turn result in the inhibition of expression of at least one target nucleic acid molecule may be provided, wherein the nucleic gene in cell(s) of the pest. Ingestion of or damage to a plant or acid molecule comprises a polynucleotide encoding an RNA plant cell by an insect pest infestation may be limited or molecule capable of forming a dsRNA molecule. In particular eliminated in or on any host tissue or environment in which embodiments, a polynucleotide encoding an RNA molecule the pest is present by providing one or more compositions capable of forming a dsRNA molecule may be operatively comprising an iRNA molecule in the host of the pest. linked to a promoter, and may also be operatively linked to a 0081) RNAi baits are formed when the dsRNA is mixed transcription termination sequence. In particular embodi with food oran attractant or both. When the pests eat the bait, US 2016/0 194658 A1 Jul. 7, 2016

they also consume the dsRNA. Baits may take the form of gifera LeConte (WCR); D. barberi Smith and Lawrence granules, gels, flowable powders, liquids, or Solids. In another (NCR); D. u, howardi (SCR); D. v. zeae (MCR); D. balteata embodiment, incrm may be incorporated into a bait formula LeConte; D. u. tenella, D. speciosa Germar, D. u. undecim tion such as that described in U.S. Pat. No. 8,530,440 which punctata Mannerheim; and Melligethes aeneus Fabricius. is hereby incorporated by reference. Generally, with baits, the 0.108 Contact (with an organism): As used herein, the baits are placed in or around the environment of the insect term "contact with or "uptake by an organism (e.g., a pest, for example, WCR can come into contact with, and/or be coleopteran), with regard to a nucleic acid molecule, includes attracted to, the bait. internalization of the nucleic acid molecule into the organism, 0082. The compositions and methods disclosed herein for example and without limitation: ingestion of the molecule may be used together in combinations with other methods and by the organism (e.g., by feeding); contacting the organism compositions for controlling damage by insect (e.g., with a composition comprising the nucleic acid molecule: coleopteran) pests. For example, an iRNA molecule as and soaking of organisms with a solution comprising the described herein for protecting plants from insect pests may nucleic acid molecule. be used in a method comprising the additional use of one or 0109 Contig: As used herein the term “contig” refers to a more chemical agents effective against an insect pest, biope DNA sequence that is reconstructed from a set of overlapping sticides effective against Such a pest, crop rotation, recombi DNA segments derived from a single genetic source. nant genetic techniques that exhibit features different from 0110 Corn plant: As used herein, the term “corn plant” the features of RNAi-mediated methods and RNAi composi refers to a plant of the species, Zea mays (maize). tions (e.g., recombinant production of proteins in plants that 0111 Expression: As used herein, “expression of a cod are harmful to an insect pest (e.g., Bt toxins)). ing polynucleotide (for example, a gene or a transgene) refers to the process by which the coded information of a nucleic II. Abbreviations acid transcriptional unit (including, e.g., gDNA or cDNA) is 0083 dsRNA double-stranded ribonucleic acid converted into an operational, non-operational, or structural I0084 GI growth inhibition part of a cell, often including the synthesis of a protein. Gene I0085 NCBI National Center for Biotechnology Informa expression can be influenced by external signals; for tion example, exposure of a cell, tissue, or organism to an agent I0086 g|DNA genomic deoxyribonucleic acid that increases or decreases gene expression. Expression of a I0087 iRNA inhibitory ribonucleic acid gene can also be regulated anywhere in the pathway from I0088 ORF open reading frame DNA to RNA to protein. Regulation of gene expression 0089 RNAi ribonucleic acid interference occurs, for example, through controls acting on transcription, 0090 miRNA micro ribonucleic acid translation, RNA transport and processing, degradation of 0091 shRNA short hairpin ribonucleic acid intermediary molecules such as mRNA, or through activa 0092 siRNA small inhibitory ribonucleic acid tion, inactivation, compartmentalization, or degradation of 0093 hpRNA hairpin ribonucleic acid specific protein molecules after they have been made, or by 0094 UTR untranslated region combinations thereof. Gene expression can be measured at 0095 WCR western corn rootworm (Diabrotica virgifera the RNA level or the protein level by any method known in the virgifera art, including, without limitation, northern blot, RT-PCR, western blot, or in vitro, in situ, or in vivo protein activity 0096) LeConte) assay(s). 0097 NCR northern corn rootworm (Diabrotica barberi Smith and Lawrence) 0112 Genetic material: As used herein, the term “genetic 0098 MCRMexican cornrootworm (Diabrotica virgifera material includes all genes, and nucleic acid molecules, such zeae Krysan and Smith) as DNA and RNA. 0099 PCR Polymerase chain reaction 0113. Inhibition: As used herein, the term “inhibition.” 0100 qPCR quantitative polymerase chain reaction when used to describe an effect on a coding polynucleotide (for example, a gene), refers to a measurable decrease in the 0101 RISC RNA-induced Silencing Complex cellular level of mRNA transcribed from the coding poly 0102 SCR southern corn rootworm (Diabrotica undecim nucleotide and/or peptide, polypeptide, or protein product of punctata howardi Barber) the coding polynucleotide. In some examples, expression of a (0103 YFP yellow fluorescent protein coding polynucleotide may be inhibited Such that expression 0104 SEM standard error of the mean is approximately eliminated. “Specific inhibition” refers to 0105 PB Pollen beetle (Melligethes aeneus Fabricius) the inhibition of a target coding polynucleotide without con sequently affecting expression of other coding polynucle III. Terms otides (e.g., genes) in the cell wherein the specific inhibition 0106. In the description and tables which follow, a number is being accomplished. of terms are used. In order to provide a clear and consistent 0114 Insect: As used herein with regard to pests, the term understanding of the specification and claims, including the “insect pest” specifically includes coleopteran insect pests. Scope to be given such terms, the following definitions are 0115 Isolated: An "isolated biological component (such provided: as a nucleic acid or protein) has been Substantially separated, 0107 Coleopteran pest: As used herein, the term produced apart from, or purified away from other biological “coleopteran pest” refers to pest insects of the order components in the cell of the organism in which the compo Coleoptera, including pest insects in the genus Diabrotica, nent naturally occurs (i.e., other chromosomal and extra which feed upon agricultural crops and crop products, includ chromosomal DNA and RNA, and proteins), while effecting ing corn and other true grasses. In particular examples, a a chemical or functional change in the component (e.g., a coleopteran pest is selected from a list comprising D. v. vir nucleic acid may be isolated from a chromosome by breaking US 2016/0 194658 A1 Jul. 7, 2016 chemical bonds connecting the nucleic acid to the remaining hpRNA (hairpin RNA), tRNA (transfer RNAs, whether DNA in the chromosome). Nucleic acid molecules and pro charged or discharged with a corresponding acylated amino teins that have been "isolated include nucleic acid molecules acid), and cFNA (complementary RNA). The term “deoxyri and proteins purified by standard purification methods. The bonucleic acid” (DNA) is inclusive of cDNA, gDNA, and term also embraces nucleic acids and proteins prepared by DNA-RNA hybrids. The terms “polynucleotide' and recombinant expression in a host cell, as well as chemically “nucleic acid.” and “fragments’ thereofwill be understood by synthesized nucleic acid molecules, proteins, and peptides. those in the art as a term that includes both gldNAs, ribosomal 0116. Nucleic acid molecule: As used herein, the term RNAs, transfer RNAs, messenger RNAs, operons, and “nucleic acid molecule' may refer to a polymeric form of Smaller engineered polynucleotides that encode or may be nucleotides, which may include both sense and anti-sense adapted to encode, peptides, polypeptides, or proteins. strands of RNA, cDNA, gDNA, and synthetic forms and I0120 Oligonucleotide: An oligonucleotide is a short mixed polymers of the above. A nucleotide or nucleobase nucleic acid polymer. Oligonucleotides may be formed by may refer to a ribonucleotide, deoxyribonucleotide, or a cleavage of longer nucleic acid segments, or by polymerizing modified form of either type of nucleotide. A “nucleic acid individual nucleotide precursors. Automated synthesizers molecule' as used herein is synonymous with “nucleic acid allow the synthesis of oligonucleotides up to several hundred and “polynucleotide. A nucleic acid molecule is usually at bases in length. Because oligonucleotides may bind to a least 10 bases in length, unless otherwise specified. By con complementary nucleic acid, they may be used as probes for vention, the nucleotide sequence of a nucleic acid molecule is detecting DNA or RNA. Oligonucleotides composed of DNA read from the 5' to the 3' end of the molecule. The “comple (oligodeoxyribonucleotides) may be used in PCR, a tech ment of a nucleic acid molecule refers to a polynucleotide nique for the amplification of DNAs. In PCR, the oligonucle having nucleobases that may form base pairs with the nucleo otide is typically referred to as a “primer,” which allows a bases of the nucleic acid molecule (i.e., A-T/U, and G-C). DNA polymerase to extend the oligonucleotide and replicate 0117 Some embodiments include nucleic acids compris the complementary strand. ing a template DNA that is transcribed into an RNA molecule I0121. A nucleic acid molecule may include either or both that is the complement of an mRNA molecule. In these naturally occurring and modified nucleotides linked together embodiments, the complement of the nucleic acid transcribed by naturally occurring and/or non-naturally occurring nucle into the mRNA molecule is present in the 5' to 3' orientation, otide linkages. Nucleic acid molecules may be modified such that RNA polymerase (which transcribes DNA in the 5' chemically or biochemically, or may contain non-natural or to 3’ direction) will transcribe a nucleic acid from the comple derivatized nucleotide bases, as will be readily appreciated by ment that can hybridize to the mRNA molecule. Unless those of skill in the art. Such modifications include, for explicitly stated otherwise, or it is clear to be otherwise from example, labels, methylation, Substitution of one or more of the context, the term “complement” therefore refers to a poly the naturally occurring nucleotides with an analog, inter nucleotide having nucleobases, from 5' to 3', that may form nucleotide modifications (e.g., uncharged linkages: for base pairs with the nucleobases of a reference nucleic acid. example, methyl phosphonates, phosphotriesters, phospho Similarly, unless it is explicitly stated to be otherwise (or it is ramidates, carbamates, etc., charged linkages: for example, clear to be otherwise from the context), the “reverse comple phosphorothioates, phosphorodithioates, etc.; pendent moi ment of a nucleic acid refers to the complement in reverse eties: for example, peptides; intercalators: for example, acri orientation. The foregoing is demonstrated in the following dine, psoralen, etc.; chelators; alkylators; and modified link illustration: ages: for example, alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule' also includes any topological conformation, including single-stranded, double-stranded, ATGATGATG polynucleotide partially duplexed, triplexed, hairpinned, circular, and pad TACTACTAC complement" of the polynucleotide locked conformations. I0122. As used herein with respect to DNA, the term "cod CATCATCAT reverse complement" of the ing polynucleotide.” “structural polynucleotide, or “struc polynucleotide tural nucleic acid molecule' refers to a polynucleotide that is 0118. Some embodiments of the invention may include ultimately translated into a polypeptide, via transcription and hairpin RNA-forming RNAi molecules. In these RNAi mol mRNA, when placed under the control of appropriate regu ecules, both the complement of a nucleic acid to be targeted latory elements. With respect to RNA, the term “coding poly by RNA interference and the reverse complement may be nucleotide' refers to a polynucleotide that is translated into a found in the same molecule. Such that the single-stranded peptide, polypeptide, or protein. The boundaries of a coding RNA molecule may “fold over and hybridize to itself over polynucleotide are determined by a translation start codon at the region comprising the complementary and reverse the 5'-terminus and a translation stop codon at the 3'-termi complementary polynucleotides. nus. Coding polynucleotides include, but are not limited to: 0119) “Nucleic acid molecules' include all polynucle gDNA, cDNA; EST; and recombinant polynucleotides. otides, for example: single- and double-stranded forms of I0123. As used herein, “transcribed non-coding polynucle DNA; single-stranded forms of RNA; and double-stranded otide' refers to segments of mRNA molecules such as 5' UTR, forms of RNA (dsRNA). The term “nucleotide sequence' or 3'UTR and intron segments that are not translated into a “nucleic acid sequence” refers to both the sense and antisense peptide, polypeptide, or protein. Further, “transcribed non Strands of a nucleic acid as either individual single strands or coding polynucleotide' refers to a nucleic acid that is tran in the duplex. The term “ribonucleic acid (RNA) is inclusive scribed into an RNA that functions in the cell, for example, of iRNA (inhibitory RNA), dsRNA (double stranded RNA), structural RNAs (e.g., ribosomal RNA (rRNA) as exemplified siRNA (small interfering RNA), shRNA (small hairpin by 5S rRNA, 5.8S rRNA, 16S rRNA 18S rRNA, 23S rRNA, RNA), mRNA (messenger RNA), miRNA (micro-RNA), and 28S rRNA, and the like); transfer RNA (tRNA); and US 2016/0 194658 A1 Jul. 7, 2016

snRNAs such as U4, U5, U6, and the like. Transcribed non 90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pear coding polynucleotides also include, for example and without son et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. limitation, small RNAs (sRNA), which term is often used to (1999) FEMS Microbiol. Lett. 174:247-50. A detailed con describe small bacterial non-coding RNAs; small nucleolar sideration of sequence alignment methods and homology RNAs (snoRNA); microRNAs; small interfering RNAs calculations can be found in, e.g., Altschul et al. (1990) J. (siRNA); Piwi-interacting RNAs (piRNA); and long non Mo1. Biol. 215:403-10. coding RNAs. Further still, “transcribed non-coding poly I0129. The National Center for Biotechnology Information nucleotide' refers to a polynucleotide that may natively exist (NCBI) Basic Local Alignment Search Tool (BLASTTM: as an intragenic 'spacer” in a nucleic acid and which is Altschul et al. (1990)) is available from several sources, transcribed into an RNA molecule. including the National Center for Biotechnology Information 0124 Lethal RNA interference: As used herein, the term (Bethesda, Md.), and on the internet, for use in connection “lethal RNA interference refers to RNA interference that with several sequence analysis programs. A description of results in death or a reduction in viability of the subject how to determine sequence identity using this program is individual to which, for example, a dsRNA, miRNA, siRNA, available on the internet under the “help' section for shRNA, and/or hpRNA is delivered. BLASTTM. For comparisons of nucleic acid sequences, the 0.125 Genome: As used herein, the term “genome' refers “Blast 2 sequences' function of the BLASTTM (Blastn) pro to chromosomal DNA found within the nucleus of a cell, and gram may be employed using the default BLOSUM62 matrix also refers to organelle DNA found within subcellular com set to default parameters. Nucleic acids with even greater ponents of the cell. In some embodiments of the invention, a sequence similarity to the sequences of the reference poly DNA molecule may be introduced into a plant cell, such that nucleotides will show increasing percentage identity when the DNA molecule is integrated into the genome of the plant assessed by this method. cell. In these and further embodiments, the DNA molecule 0.130 Specifically hybridizable/Specifically complemen may be either integrated into the nuclear DNA of the plant tary: As used herein, the terms “Specifically hybridizable' cell, or integrated into the DNA of the chloroplast or mito and “Specifically complementary are terms that indicate a chondrion of the plant cell. The term “genome,” as it applies Sufficient degree of complementarity Such that stable and to bacteria, refers to both the chromosome and plasmids specific binding occurs between the nucleic acid molecule within the bacterial cell. In some embodiments of the inven and a target nucleic acid molecule. Hybridization between tion, a DNA molecule may be introduced into a bacterium two nucleic acid molecules involves the formation of an anti such that the DNA molecule is integrated into the genome of parallel alignment between the nucleobases of the two the bacterium. In these and further embodiments, the DNA nucleic acid molecules. The two molecules are then able to molecule may be either chromosomally-integrated or located form hydrogen bonds with corresponding bases on the oppo as or in a stable plasmid. site strand to form a duplex molecule that, if it is sufficiently 0126 Sequence identity: The term “sequence identity” or stable, is detectable using methods well known in the art. A “identity, as used herein in the context of two polynucle polynucleotide need not be 100% complementary to its target otides or polypeptides, refers to the residues in the sequences nucleic acid to be specifically hybridizable. However, the of the two molecules that are the same when aligned for amount of complementarity that must exist for hybridization maximum correspondence over a specified comparison win to be specific is a function of the hybridization conditions dow. used. 0127. As used herein, the term “percentage of sequence I0131 Hybridization conditions resulting in particular identity” may refer to the value determined by comparing two degrees of stringency will vary depending upon the nature of optimally aligned sequences (e.g., nucleic acid sequences or the hybridization method of choice and the composition and polypeptide sequences) of a molecule over a comparison length of the hybridizing nucleic acids. Generally, the tem window, wherein the portion of the sequence in the compari perature of hybridization and the ionic strength (especially son window may comprise additions or deletions (i.e., gaps) the Na" and/or Mg" concentration) of the hybridization as compared to the reference sequence (which does not com buffer will determine the stringency of hybridization, though prise additions or deletions) for optimal alignment of the two wash times also influence stringency. Calculations regarding sequences. The percentage is calculated by determining the hybridization conditions required for attaining particular number of positions at which the identical nucleotide or degrees of stringency are known to those of ordinary skill in amino acid residue occurs in both sequences to yield the the art, and are discussed, for example, in Sambrook et al. number of matched positions, dividing the number of (ed.) Molecular Cloning: A Laboratory Manual, 2" ed., vol. matched positions by the total number of positions in the 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Har comparison window, and multiplying the result by 100 to bor, N.Y., 1989, chapters 9 and 11; and Hames and Higgins yield the percentage of sequence identity. A sequence that is (eds.) Nucleic Acid Hybridization, IRL Press, Oxford, 1985. identical at every position in comparison to a reference Further detailed instruction and guidance with regard to the sequence is said to be 100% identical to the reference hybridization of nucleic acids may be found, for example, in sequence, and Vice-versa. Tijssen, “Overview of principles of hybridization and the 0128 Methods for aligning sequences for comparison are strategy of nucleic acid probe assays in Laboratory Tech well-known in the art. Various programs and alignment algo niques in Biochemistry and Molecular Biology—Hybridiza rithms are described in, for example: Smith and Waterman tion with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, NY. (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch 1993; and Ausubel et al., Eds., Current Protocols in Molecu (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) lar Biology, Chapter 2, Greene Publishing and Wiley-Inter Proc. Natl. Acad. Sci. U.S.A. 85:2444; Higgins and Sharp science, NY, 1995. (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 0.132. As used herein, “stringent conditions' encompass 5:151-3: Corpet et al. (1988) Nucleic Acids Res. 16:10881 conditions under which hybridization will only occur if there US 2016/0 194658 A1 Jul. 7, 2016

is less than 20% mismatch between the sequence of the is complementary to a reference polynucleotide will exhibit a hybridization molecule and a homologous polynucleotide sequence identical to the reverse complement of the reference within the target nucleic acid molecule. “Stringent condi polynucleotide. These terms and descriptions are well defined tions’ include further particular levels of stringency. Thus, as in the art and are easily understood by those of ordinary skill used herein, "moderate stringency' conditions are those in the art. under which molecules with more than 20% sequence mis 0140. Operably linked: A first polynucleotide is operably match will not hybridize; conditions of “high stringency” are linked with a second polynucleotide when the first polynucle those under which sequences with more than 10% mismatch otide is in a functional relationship with the second poly will not hybridize; and conditions of “very high stringency’ nucleotide. When recombinantly produced, operably linked are those under which sequences with more than 5% mis polynucleotides are generally contiguous, and, where neces match will not hybridize. sary to join two protein-coding regions, in the same reading 0133. The following are representative, non-limiting frame (e.g., in a translationally fused ORF). However, nucleic hybridization conditions. acids need not be contiguous to be operably linked. 0134 High Stringency condition (detects polynucleotides 0.141. The term, “operably linked, when used in reference that share at least 90% sequence identity): Hybridization in to a regulatory genetic element and a coding polynucleotide, 5xSSC buffer at 65° C. for 16 hours; wash twice in 2XSSC means that the regulatory element affects the expression of buffer at room temperature for 15 minutes each; and wash the linked coding polynucleotide. “Regulatory elements.” or twice in 0.5xSSC buffer at 65° C. for 20 minutes each. “control elements.” refer to polynucleotides that influence the 0135 Moderate Stringency condition (detects polynucle timing and level/amount of transcription, RNA processing or otides that share at least 80% sequence identity): Hybridiza stability, or translation of the associated coding polynucle tion in 5x-6xSSC buffer at 65-70° C. for 16-20 hours; wash otide. Regulatory elements may include promoters; transla twice in 2xSSC buffer at room temperature for 5-20 minutes tion leaders; introns; enhancers; stem-loop structures; repres each; and wash twice in 1xSSC buffer at 55-70° C. for 30 Sor binding polynucleotides; polynucleotides with a minutes each. termination sequence; polynucleotides with a polyadenyla 0136. Non-stringent control condition (polynucleotides tion recognition sequence; etc. Particular regulatory elements that share at least 50% sequence identity will hybridize): may be located upstream and/or downstream of a coding Hybridization in 6xSSC buffer at room temperature to 55° C. polynucleotide operably linked thereto. Also, particular regu for 16-20 hours; wash at least twice in 2x-3XSSC buffer at latory elements operably linked to a coding polynucleotide room temperature to 55° C. for 20-30 minutes each. may be located on the associated complementary strand of a 0.137 As used herein, the term “substantially homolo double-stranded nucleic acid molecule. gous' or 'substantial homology, with regard to a nucleic 0.142 Promoter: As used herein, the term “promoter' acid, refers to a polynucleotide having contiguous nucleo refers to a region of DNA that may be upstream from the start bases that hybridize under stringent conditions to the refer of transcription, and that may be involved in recognition and ence nucleic acid. For example, nucleic acids that are Sub binding of RNA polymerase and other proteins to initiate stantially homologous to a reference nucleic acid of any of transcription. A promoter may be operably linked to a coding SEQ ID NOS:1, 3-6, 17, 77, 84, 86, 88,90, and 93 are those polynucleotide for expression in a cell, or a promoter may be nucleic acids that hybridize under stringent conditions (e.g., operably linked to a polynucleotide encoding a signal peptide the Moderate Stringency conditions set forth, supra) to the which may be operably linked to a coding polynucleotide for reference nucleic acid of any of SEQID NOS:1, 3-6, 17, 77, expression in a cell. A "plant promoter” may be a promoter 84, 86, 88,90, and 93. Substantially homologous polynucle capable of initiating transcription in plant cells. Examples of otides may have at least 80% sequence identity. For example, promoters under developmental control include promoters Substantially homologous polynucleotides may have from that preferentially initiate transcription in certain tissues, about 80% to 100% sequence identity, such as 79%; 80%: Such as leaves, roots, seeds, fibers, xylem vessels, tracheids, about 81%; about 82%; about 83%; about 84%; about 85%: or sclerenchyma. Such promoters are referred to as “tissue about 86%; about 87%; about 88%; about 89%; about 90%; preferred. Promoters which initiate transcription only in cer about 91%; about 92%; about 93%; about 94% about 95%: tain tissues are referred to as “tissue-specific''. A "cell type about 96%; about 97%; about 98%; about 98.5%; about 99%; specific' promoter primarily drives expression in certain cell about 99.5%; and about 100%. The property of substantial types in one or more organs, for example, Vascular cells in homology is closely related to specific hybridization. For roots or leaves. An “inducible' promoter may be a promoter example, a nucleic acid molecule is specifically hybridizable which may be under environmental control. Examples of when there is a Sufficient degree of complementarity to avoid environmental conditions that may initiate transcription by non-specific binding of the nucleic acid to non-target poly inducible promoters include anaerobic conditions and the nucleotides under conditions where specific binding is presence of light. Tissue-specific, tissue-preferred, cell type desired, for example, under stringent hybridization condi specific, and inducible promoters constitute the class of “non tions. constitutive' promoters. A "constitutive' promoter is a pro 0.138. As used herein, the term “ortholog” refers to a gene moter which may be active under most environmental condi in two or more species that has evolved from a common tions or in most tissue or cell types. ancestral nucleic acid, and may retain the same function in the 0.143 Any inducible promoter can be used in some two or more species. embodiments of the invention. See Ward et al. (1993) Plant 0139. As used herein, two nucleic acid molecules are said Mol. Biol. 22:361-366. With an inducible promoter, the rate to exhibit “complete complementarity” when every nucle of transcription increases in response to an inducing agent. otide of a polynucleotide read in the 5' to 3’ direction is Exemplary inducible promoters include, but are not limited complementary to every nucleotide of the other polynucle to: Promoters from the ACEI system that respond to copper; otide when read in the 3' to 5' direction. A polynucleotide that In2 gene from maize that responds to benzenesulfonamide US 2016/0 194658 A1 Jul. 7, 2016

herbicide safeners; Tet repressor from Tn 10; and the induc include genetic elements that permit it to replicate in the host ible promoter from a steroid hormone gene, the transcrip cell. Such as an origin of replication. Examples of vectors tional activity of which may be induced by a glucocorticos include, but are not limited to: a plasmid, cosmid; bacterioph teroid hormone (Schena et al. (1991) Proc. Natl. Acad. Sci. age; or virus that carries exogenous DNA into a cell. A vector USA 88:0421). may also include one or more genes, including ones that 0144 Exemplary constitutive promoters include, but are produce antisense molecules, and/or selectable marker genes not limited to: Promoters from plant viruses, such as the 35S and other genetic elements known in the art. A vector may promoter from Cauliflower Mosaic Virus (CaMV); promoters transduce, transform, or infect a cell, thereby causing the cell from rice actin genes: ubiquitin promoters; pl.MU; MAS: to express the nucleic acid molecules and/or proteins encoded maize H3 histone promoter; and the ALS promoter, Xbal/ by the vector. A vector optionally includes materials to aid in NcoI fragment 5' to the Brassica napus ALS3 structural gene achieving entry of the nucleic acid molecule into the cell (e.g., (or a polynucleotide similar to said Xbal/NcoI fragment) a liposome, protein coating, etc.). (International PCT Publication No. WO96/30530). (O150 Yield: A stabilized yield of about 100% or greater 0145 Additionally, any tissue-specific or tissue-preferred relative to the yield of check varieties in the same growing promoter may be utilized in some embodiments of the inven location growing at the same time and under the same condi tion. Plants transformed with a nucleic acid molecule com tions. In particular embodiments, “improved yield' or prising a coding polynucleotide operably linked to a tissue “improving yield’ means a cultivar having a stabilized yield specific promoter may produce the product of the coding of 105% or greater relative to the yield of check varieties in polynucleotide exclusively, or preferentially, in a specific the same growing location containing significant densities of tissue. Exemplary tissue-specific or tissue-preferred promot the coleopteran pests that are injurious to that crop growing at ers include, but are not limited to: A seed-preferred promoter, the same time and under the same conditions, which are Such as that from the phaseolin gene; a leaf-specific and targeted by the compositions and methods herein. light-induced promoter Such as that from cab or rubisco; an anther-specific promoter such as that from LAT52; a pollen 0151. Unless specifically indicated or implied, the terms specific promoter Such as that from Zm13; and a microspore “a,” “an and “the signify "at least one.” as used herein. preferred promoter Such as that from apg. 0152 Unless otherwise specifically explained, all techni 0146 Brassica plant: As used herein, the terms “rape.” cal and Scientific terms used herein have the same meaning as “oilseed rape.” “rapeseed,” and “canola' are used inter commonly understood by those of ordinary skill in the art to changeably, and refer to a plant of the species Brassica; for which this disclosure belongs. Definitions of common terms example, B. napus. in molecular biology can be found in, for example, Lewin's 0147 Transformation: As used herein, the term “transfor Genes X, Jones & Bartlett Publishers, 2009 (ISBN 10 mation' or “transduction” refers to the transfer of one or more 0763766321); Krebs et al. (eds.), The Encyclopedia of nucleic acid molecule(s) into a cell. A cell is “transformed by Molecular Biology, Blackwell Science Ltd., 1994 (ISBN a nucleic acid molecule transduced into the cell when the 0-632-02182-9); and Meyers R. A. (ed.), Molecular Biology nucleic acid molecule becomes stably replicated by the cell, and Biotechnology: A Comprehensive Desk Reference, VCH either by incorporation of the nucleic acid molecule into the Publishers, Inc., 1995 (ISBN 1-56081-569-8). All percent cellular genome, or by episomal replication. As used herein, ages are by weight and all solvent mixture proportions are by the term “transformation' encompasses all techniques by Volume unless otherwise noted. All temperatures are in which a nucleic acid molecule can be introduced into Such a degrees Celsius. cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; elec IV. Nucleic Acid Molecules Comprising an Insect troporation (Fromm et al. (1986) Nature 319:791-3); lipofec Pest Sequence tion (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); microinjection (Mueller et al. (1978) Cell 0153 A. Overview 15:579-85); Agrobacterium-mediated transfer (Fraley et al. 0154 Described herein are nucleic acid molecules useful (1983) Proc. Natl. Acad. Sci. USA 80:4803-7); direct DNA for the control of insect pests. In some embodiments, the uptake; and microprojectile bombardment (Klein et al. insect pest is a coleopteran insect pest. Described nucleic acid (1987) Nature 327:70). molecules include target polynucleotides (e.g., native genes, 0148 Transgene: An exogenous nucleic acid. In some and non-coding polynucleotides), dsRNAs, siRNAs, shR examples, a transgene may be a DNA that encodes one or both NAs, hpRNAs, and miRNAs. For example, dsRNA, siRNA, strand(s) of an RNA capable of forming a dsRNA molecule miRNA, shRNA, and/or hpRNA molecules are described in that comprises a polynucleotide that is complementary to a Some embodiments that may be specifically complementary nucleic acid molecule found in a coleopteran pest. In further to all or part of one or more native nucleic acids in a examples, a transgene may be an antisense polynucleotide, coleopteran pest. In these and further embodiments, the wherein expression of the antisense polynucleotide inhibits native nucleic acid(s) may be one or more target gene(s), the expression of a target nucleic acid. In still further examples, a product of which may be, for example and without limitation: transgene may be a gene (e.g., a herbicide-tolerance gene, a involved in a metabolic process or involved in larval devel gene encoding an industrially or pharmaceutically useful opment. Nucleic acid molecules described herein, when compound, or a gene encoding a desirable agricultural trait). introduced into a cell comprising at least one native nucleic In these and other examples, a transgene may contain regu acid(s) to which the nucleic acid molecules are specifically latory elements operably linked to a coding polynucleotide of complementary, may initiate RNAi in the cell, and conse the transgene (e.g., a promoter). quently reduce or eliminate expression of the native nucleic 0149 Vector: A nucleic acid molecule as introduced into a acid(s). In some examples, reduction or elimination of the cell, for example, to produce a transformed cell. A vector may expression of a target gene by a nucleic acid molecule spe US 2016/0 194658 A1 Jul. 7, 2016

cifically complementary thereto may result in reduction or DNA constructs for use in achieving stable transformation of cessation of growth, development, and/or feeding in the particular host targets. Transformed host targets may express coleopteran pest. effective levels of dsRNA, siRNA, miRNA, shRNA, and/or 0155. In some embodiments, at least one target gene in an hpRNA molecules from the recombinant DNA constructs. insect pest may be selected, wherein the target gene com Therefore, also described is a plant transformation vector prises ancm polynucleotide. In particular examples, a target comprising at least one polynucleotide operably linked to a gene in a coleopteran pest is selected, wherein the target gene heterologous promoter functional in a plant cell, wherein comprises a polynucleotide selected from among SEQ ID expression of the polynucleotide(s) results in an RNA mol NOs: 1, 3-6, 77, 84, 86, 88,90, and 93. ecule comprising a string of contiguous nucleobases that is 0156. In some embodiments, a target gene may be a specifically complementary to all or part of a target nucleic nucleic acid molecule comprising a polynucleotide that can acid in an insect pest. be reverse translated in silico to a polypeptide comprising a 0160. In particular examples, nucleic acid molecules use contiguous amino acid sequence that is at least about 85% ful for the control of insect (e.g., coleopteran) pests may identical (e.g., at least 84%, 85%, about 90%, about 95%, include: all or part of a native nucleic acid isolated from about 96%, about 97%, about 98%, about 99%, about 100%, Diabrotica comprising a ncinn polynucleotide (e.g., any of or 100% identical) to the amino acid sequence of a protein SEQ ID NOs: 1, 3-6, and 77); all or part of a native nucleic product of ancm polynucleotide. A target gene may be any acid isolated from Melligethes aeneus comprising ancm poly incm polynucleotide in an insect pest, the post-transcriptional nucleotide (e.g., any of SEQID NOs:84, 86, 88,90, and 93: inhibition of which has a deleterious effect on the growth, DNAs that when expressed result in a RNA molecule com and/or Survival of the pest, for example, to provide a protec prising a polynucleotide that is specifically complementary to tive benefit against the pest to a plant. In particular examples, all or part of a native RNA molecule that is encoded by ncm; a target gene is a nucleic acid molecule comprising a poly iRNA molecules (e.g., dsRNAs, siRNAs, miRNAs, shRNAs, nucleotide that can be reverse translated in silico to a polypep and hpRNAs) that comprise at least one polynucleotide that is tide comprising a contiguous amino acid sequence that is at specifically complementary to all or part ofncm; c)NAs that least about 85% identical, about 90% identical, about 95% may be used for the production of dsRNA molecules, siRNA identical, about 96% identical, about 97% identical, about molecules, miRNA molecules, shRNA molecules, and/or 98% identical, about 99% identical, about 100% identical, or hpRNA molecules that are specifically complementary to all 100% identical to the amino acid sequence of SEQID NOS:2, or part of ncm; and recombinant DNA constructs for use in 85, 87, 89, and 94. achieving stable transformation of particular host targets, 0157 Provided according to the invention are DNAs, the wherein a transformed host target comprises one or more of expression of which results in an RNA molecule comprising the foregoing nucleic acid molecules. a polynucleotide that is specifically complementary to all or (0161 B. Nucleic Acid Molecules part of a native RNA molecule that is encoded by a coding 0162 The present invention provides, inter alia, iRNA polynucleotide in an insect (e.g., coleopteran) pest. In some (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) mol embodiments, after ingestion of the expressed RNA molecule ecules that inhibit target gene expression in a cell, tissue, or by an insect pest, down-regulation of the coding polynucle organ of an insect (e.g., coleopteran) pest; and DNA mol otide in cells of the pest may be obtained. In particular ecules capable of being expressed as an iRNA molecule in a embodiments, down-regulation of the coding sequence in cell or microorganism to inhibit target gene expression in a cells of the insect pest may result in a deleterious effect on the cell, tissue, or organ of an insect pest. growth and/or development of the pest. 0163 Some embodiments of the invention provide an iso 0158. In some embodiments, target polynucleotides lated nucleic acid molecule comprising at least one (e.g., one, include transcribed non-coding RNAs, such as 5'UTRs: two, three, or more) polynucleotide(s) selected from the 3'UTRs; spliced leaders; introns; outrons (e.g., 5' UTR RNA group consisting of: SEQID NOS:1, 77, 84, 86, 88, and 93: Subsequently modified in trans splicing); donatrons (e.g., the complement of SEQ ID NO:1, 77, 84, 86, 88, or 93; a non-coding RNA required to provide donor sequences for fragment of at least 15 contiguous nucleotides of SEQ ID trans splicing); and other non-coding transcribed RNA of NO:1,77, 84, 86, 88, or 93 (e.g., any of SEQID NOS:3-6 and target insect pest genes. Such polynucleotides may be derived 90); the complement of a fragment of at least 15 contiguous from both mono-cistronic and poly-cistronic genes. nucleotides of SEQ ID NO:1, 77, 84, 86, 88, or 93; a native 0159. Thus, also described herein in connection with some coding polynucleotide of a Diabrotica organism (e.g., WCR) embodiments are iRNA molecules (e.g., dsRNAs, siRNAs, comprising SEQID NO:1 or 77; the complement of a native miRNAs, shRNAs, and hpRNAs) that comprise at least one coding polynucleotide of a Diabrotica organism comprising polynucleotide that is specifically complementary to all or SEQ ID NO:1 or 77; a fragment of at least 15 contiguous part of a target nucleic acid in an insect (e.g., coleopteran) nucleotides of a native coding polynucleotide of a Diabrotica pest. In some embodiments an iRNA molecule may comprise organism comprising SEQID NO:1 or 77; and the comple polynucleotide(s) that are complementary to all or part of a ment of a fragment of at least 15 contiguous nucleotides of a plurality of target nucleic acids; for example, 2, 3, 4, 5, 6, 7, 8, native coding polynucleotide of a Diabrotica organism com 9, 10, or more target nucleic acids. In particular embodiments, prising SEQID NO:1 or 77; a native coding polynucleotide of an iRNA molecule may be produced in vitro, or in vivo by a a Melligethes organism (e.g., PB) comprising SEQID NO:84, genetically-modified organism, Such as a plant or bacterium. 86, 88, or 93; the complement of a native coding polynucle Also disclosed are cDNAs that may be used for the produc otide of a Melligethes organism comprising SEQID NO:84, tion of dsRNA molecules, siRNA molecules, miRNA mol 86, 88, or 93; a fragment of at least 15 contiguous nucleotides ecules, shRNA molecules, and/or hpRNA molecules that are of a native coding polynucleotide of a Melligethes organism specifically complementary to all or part of a target nucleic comprising SEQ ID NO:84, 86, 88, or 93; and the comple acid in an insect pest. Further described are recombinant ment of a fragment of at least 15 contiguous nucleotides of a US 2016/0 194658 A1 Jul. 7, 2016 native coding polynucleotide of a Melligethes organism com 90, and 93; a contiguous fragment of any of SEQID NOs: 1. prising SEQID NO:84, 86, 88, or 93. In particular embodi 3-6, 77.84, 86, 88,90, and 93; and the complement of any of ments, contact with or uptake by an insect (e.g., coleopteran) the foregoing. pest of an iRNA transcribed from the isolated polynucleotide 0166 In some embodiments, a DNA molecule capable of inhibits the growth, development, and/or feeding of the pest. being expressed as an iRNA molecule in a cell or microor 0164. In some embodiments, an isolated nucleic acid mol ganism to inhibit target gene expression may comprise a ecule of the invention may comprise at least one (e.g., one, single polynucleotide that is specifically complementary to two, three, or more) polynucleotide(s) selected from the all or part of a native polynucleotide found in one or more group consisting of: SEQID NO:78; the complement of SEQ target insect pest species (e.g., a coleopteran pest species), or ID NO:78; SEQ ID NO:79; the complement of SEQ ID the DNA molecule can be constructed as a chimera from a NO:79; SEQID NO:80; the complement of SEQID NO:80; plurality of Such specifically complementary polynucle SEQID NO:81; the complement of SEQID NO:81; SEQID otides. NO:82; the complement of SEQID NO:82; SEQID NO:83; 0167. In some embodiments, a nucleic acid molecule may the complement of SEQID NO:83; a fragment of at least 15 comprise a first and a second polynucleotide separated by a contiguous nucleotides of any of SEQ ID NOS:78-83; the 'spacer. A spacer may be a region comprising any sequence complement of a fragment of at least 15 contiguous nucle of nucleotides that facilitates secondary structure formation otides of any of SEQ ID NOS:78-83; a native coding poly between the first and second polynucleotides, where this is nucleotide of a Diabrotica organism comprising SEQ ID desired. In one embodiment, the spacer is part of a sense or NO:1 and/or SEQ ID NO:77; the complement of a native antisense coding polynucleotide for mRNA. The spacer may coding polynucleotide of a Diabrotica organism comprising alternatively comprise any combination of nucleotides or SEQID NO:1 and/or SEQIDNO:77; afragment of at least 15 homologues thereof that are capable of being linked contiguous nucleotides of a native coding polynucleotide of a covalently to a nucleic acid molecule. Diabrotica organism comprising SEQID NO:1 and/or SEQ 0168 For example, in some embodiments, the DNA mol ID NO:77; and the complement of a fragment of at least 15 ecule may comprise a polynucleotide coding for one or more contiguous nucleotides of a native coding polynucleotide of a different iRNA molecules, wherein each of the different Diabrotica organism comprising SEQID NO:1 and/or SEQ iRNA molecules comprises a first polynucleotide and a sec ID NO:77; SEQ ID NO:95; the complement of SEQ ID ond polynucleotide, wherein the first and second polynucle NO:95; SEQID NO:96; the complement of SEQID NO:96; otides are complementary to each other. The first and second SEQID NO:97; the complement of SEQID NO:97: SEQID polynucleotides may be connected within an RNA molecule NO:98; the complement of SEQID NO:98: SEQID NO:99; by a spacer. The spacer may constitute part of the first poly the complement of SEQID NO:99; a fragment of at least 15 nucleotide or the second polynucleotide. Expression of an contiguous nucleotides of any of SEQ ID NOs: 95-99; the RNA molecule comprising the first and second nucleotide complement of a fragment of at least 15 contiguous nucle polynucleotides may lead to the formation of a dsRNA mol otides of any of SEQ ID NOs: 95-99; a native coding poly ecule, by specific intramolecular base-pairing of the first and nucleotide of a Melligethes organism comprising SEQ ID second nucleotide polynucleotides. The first polynucleotide NO:84, 86, 88, and/or 93; the complement of a native coding or the second polynucleotide may be substantially identical to polynucleotide of a Melligethes organism comprising SEQID a polynucleotide (e.g., a target gene, or transcribed non-cod NO:84, 86, 88, and/or 93; a fragment of at least 15 contiguous ing polynucleotide) native to an insect pest (e.g., a nucleotides of a native coding polynucleotide of a Melligethes coleopteran pest), a derivative thereof, or a complementary organism comprising SEQID NO:84, 86, 88, and/or 93; and polynucleotide thereto. the complement of a fragment of at least 15 contiguous nucle 0169 dsRNA nucleic acid molecules comprise double otides of a native coding polynucleotide of a Melligethes Strands of polymerized ribonucleotides, and may include organism comprising SEQ ID NO:84, 86, 88, and/or 93. In modifications to either the phosphate-sugar backbone or the particular embodiments, contact with or uptake by a nucleoside. Modifications in RNA structure may be tailored coleopteran pest of the isolated polynucleotide inhibits the to allow specific inhibition. In one embodiment, dsRNA mol growth, development, and/or feeding of the pest. ecules may be modified through a ubiquitous enzymatic pro 0.165. In certain embodiments, dsRNA molecules pro cess so that siRNA molecules may be generated. This enzy vided by the invention comprise polynucleotides comple matic process may utilize an RNase III enzyme. Such as mentary to a transcript from a target gene comprising any of DICER in eukaryotes, either in vitro or in vivo. See Elbashir SEQID NOs: 1,77, 84, 86, 88, and 93, and fragments thereof, etal. (2001) Nature 411:494-8; and Hamilton and Baulcombe the inhibition of which target gene in an insect pest results in (1999) Science 286(5441):950-2. DICER or functionally the reduction or removal of a polypeptide or polynucleotide equivalent RNase III enzymes cleave larger dsRNA strands agent that is essential for the pest's growth, development, or and/or hpRNA molecules into Smaller oligonucleotides (e.g., other biological function. A selected target gene may exhibit siRNAs), each of which is about 19-25 nucleotides in length. from about 80% to about 100% sequence identity to any of The siRNA molecules produced by these enzymes have 2 to SEQID NOs: 1,77, 84, 86, 88, and 93; a contiguous fragment 3 nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl of SEQID NO:1, 77, 84, 86, 88, and/or 93; and the comple termini. The siRNA molecules generated by RNase III ment of any of the foregoing. For example, a selected target enzymes are unwound and separated into single-stranded gene may exhibit 79%; 80%; about 81%; about 82%; about RNA in the cell. The siRNA molecules then specifically 83%; about 84%; about 85%; about 86%; about 87%; about hybridize with RNAs transcribed from a target gene, and both 88%; about 89%; about 90%; about 91%; about 92%; about RNA molecules are subsequently degraded by an inherent 93%; about 94% about 95%; about 96%; about 97%; about cellular RNA-degrading mechanism. This process may result 98%; about 98.5%; about 99%; about 99.5%; or about 100% in the effective degradation or removal of the RNA encoded sequence identity to any of SEQID NOs: 1,3-6, 77.84, 86,88, by the target gene in the target organism. The outcome is the US 2016/0 194658 A1 Jul. 7, 2016

post-transcriptional silencing of the targeted gene. In some thus making the dsRNA available if/when the pest forms a embodiments, siRNA molecules produced by endogenous nutritional relationship with the transgenic host. This may RNase III enzymes from heterologous nucleic acid molecules result in the Suppression of expression of one or more genes in may efficiently mediate the down-regulation of target genes the cells of the pest, and ultimately death or inhibition of its in insect pests. growth or development. 0170 In some embodiments, a nucleic acid molecule may 0.174 Thus, in some embodiments, a gene is targeted that include at least one non-naturally occurring polynucleotide is essentially involved in the growth and/or development of an that can be transcribed into a single-stranded RNA molecule insect (e.g., coleopteran) pest. Other target genes for use in capable of forming a dsRNA molecule in vivo through inter the present invention may include, for example, those that molecular hybridization. Such dsRNAs typically self-as play important roles in pest viability, movement, migration, semble, and can be provided in the nutrition Source of an growth, development, infectivity, and establishment offeed insect (e.g., coleopteran) pest to achieve the post-transcrip ing sites. A target gene may therefore be a housekeeping gene tional inhibition of a target gene. In these and further embodi or a transcription factor. Additionally, a native insect pest ments, a nucleic acid molecule may comprise two different polynucleotide for use in the present invention may also be non-naturally occurring polynucleotides, each of which is derived from a homolog (e.g., an ortholog), of a plant, viral, specifically complementary to a different target gene in an bacterial or insect gene, the function of which is known to insect pest. When Such a nucleic acid molecule is provided as those of skill in the art, and the polynucleotide of which is a dsRNA molecule to, for example, a coleopteran pest, the specifically hybridizable with a target gene in the genome of dsRNA molecule inhibits the expression of at least two dif the target pest. Methods of identifying a homolog of a gene ferent target genes in the pest. with a known nucleotide sequence by hybridization are (0171 C. Obtaining Nucleic Acid Molecules known to those of skill in the art. 0172 A variety of polynucleotides in insect (e.g., 0.175. In some embodiments, the invention provides meth coleopteran) pests may be used as targets for the design of ods for obtaining a nucleic acid molecule comprising a poly nucleic acid molecules, such as iRNAs and DNA molecules nucleotide for producing an iRNA (e.g., dsRNA, siRNA, encoding iRNAs. Selection of native polynucleotides is not, miRNA, shRNA, and hpRNA) molecule. One such embodi however, a straight-forward process. For example, only a ment comprises: (a) analyzing one or more target gene(s) for Small number of native polynucleotides in a coleopteran pest their expression, function, and phenotype upon dsRNA-me will be effective targets. It cannot be predicted with certainty diated gene Suppression in an insect (e.g., coleopteran) pest: whether a particular native polynucleotide can be effectively (b) probing a cDNA org)NA library with a probe comprising down-regulated by nucleic acid molecules of the invention, or all or a portion of a polynucleotide or a homolog thereoffrom whether down-regulation of a particular native polynucle a targeted pest that displays an altered (e.g., reduced) growth otide will have a detrimental effect on the growth, develop or development phenotype in a dsRNA-mediated Suppression ment, and/or viability of an insect pest. The vast majority of analysis; (c) identifying a DNA clone that specifically hybrid native coleopteran pest polynucleotides, such as ESTs iso izes with the probe; (d) isolating the DNA clone identified in lated therefrom (for example, the coleopteran pest polynucle step (b); (e) sequencing the cDNA or gldNA fragment that otides listed in U.S. Pat. No. 7,612,194), do not have a detri comprises the clone isolated in step (d), wherein the mental effect on the growth and/or viability of the pest. sequenced nucleic acid molecule comprises all or a Substan Neither is it predictable which of the native polynucleotides tial portion of the RNA or a homolog thereof; and (f) chemi that may have a detrimental effect on an insect pest are able to cally synthesizing all or a substantial portion of a gene, or an be used in recombinant techniques for expressing nucleic siRNA, miRNA, hpRNA, mRNA, shRNA, or dsRNA. acid molecules complementary to Such native polynucle 0176). In further embodiments, a method for obtaining a otides in a host plant and providing the detrimental effect on nucleic acid fragment comprising a polynucleotide for pro the pest upon feeding without causing harm to the host plant. ducing a substantial portion of an iRNA (e.g., dsRNA, 0173. In some embodiments, nucleic acid molecules (e.g., siRNA, miRNA, shRNA, and hpRNA) molecule includes: (a) dsRNA molecules to be provided in the host plant of an insect synthesizing first and second oligonucleotideprimers specifi (e.g., coleopteran) pest) are selected to target cDNAS that cally complementary to a portion of a native polynucleotide encode proteins or parts of proteins essential for pest devel from a targeted insect (e.g., coleopteran) pest, and (b) ampli opment, such as polypeptides involved in metabolic or cata fying a cDNA or g|DNA insert present in a cloning vector bolic biochemical pathways, cell division, energy metabo using the first and second oligonucleotide primers of step (a), lism, digestion, host plant recognition, and the like. As wherein the amplified nucleic acid molecule comprises a described herein, ingestion of compositions by a target pest substantial portion of a siRNA, miRNA, hpRNA, mRNA, organism containing one or more dsRNAS, at least one seg shRNA, or dsRNA molecule. ment of which is specifically complementary to at least a 0177. Nucleic acids can be isolated, amplified, or pro substantially identical segment of RNA produced in the cells duced by a number of approaches. For example, an iRNA of the target pest organism, can result in the death or other (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) mol inhibition of the target. A polynucleotide, either DNA or ecule may be obtained by PCR amplification of a target poly RNA, derived from an insect pest can be used to construct nucleotide (e.g., a target gene or a target transcribed non plant cells resistant to infestation by the pests. The host plant coding polynucleotide) derived from a gCNA or cDNA of the coleopteran pest (e.g., Z. mays or Brassica), for library, or portions thereof. DNA or RNA may be extracted example, can be transformed to contain one or more poly from a target organism, and nucleic acid libraries may be nucleotides derived from the coleopteran pest as provided prepared therefrom using methods known to those ordinarily herein. The polynucleotide transformed into the host may skilled in the art. gldNA or cDNA libraries generated from a encode one or more RNAs that form into a dsRNA structure target organism may be used for PCR amplification and in the cells or biological fluids within the transformed host, sequencing of target genes. A confirmed PCR product may be US 2016/0 194658 A1 Jul. 7, 2016 20 used as a template for in vitro transcription to generate sense 0181. In some embodiments, the invention also provides a and antisense RNA with minimal promoters. Alternatively, DNA molecule for introduction into a cell (e.g., a bacterial nucleic acid molecules may be synthesized by any of a num cell, a yeast cell, or a plant cell), wherein the DNA molecule ber of techniques (See, e.g., Ozaki etal. (1992) Nucleic Acids comprises a polynucleotide that, upon expression to RNA and Research, 20: 5205-5214; and Agrawal et al. (1990) Nucleic ingestion by an insect (e.g., coleopteran) pest, achieves Sup Acids Research, 18: 5419-5423), including use of an auto pression of a target gene in a cell, tissue, or organ of the pest. mated DNA synthesizer (for example, a P.E. Biosystems, Inc. Thus, Some embodiments provide a recombinant nucleic acid (Foster City, Calif.) model 392 or 394 DNA/RNA Synthe molecule comprising a polynucleotide capable of being sizer), using standard chemistries, such as phosphoramidite expressed as an iRNA (e.g., dsRNA, siRNA, miRNA, chemistry. See, e.g., Beaucage et al. (1992) Tetrahedron, 48: shRNA, and hpRNA) molecule in a plant cell to inhibit target 2223-2311; U.S. Pat. Nos. 4,980,460, 4,725,677, 4,415,732, gene expression in an insect pest. In order to initiate or 4.458,066, and 4,973,679. Alternative chemistries resulting enhance expression, such recombinant nucleic acid mol in non-natural backbone groups, such as phosphorothioate, ecules may comprise one or more regulatory elements, which phosphoramidate, and the like, can also be employed. regulatory elements may be operably linked to the polynucle otide capable of being expressed as an iRNA. Methods to 0.178 An RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA molecule of the present invention may be produced express a gene Suppression molecule in plants are known, and chemically or enzymatically by one skilled in the art through may be used to express a polynucleotide of the present inven manual or automated reactions, or in vivo in a cell comprising tion. See, e.g., International PCT Publication No. WO06/ a nucleic acid molecule comprising a polynucleotide encod 073727; and U.S. Patent Publication No. 2006/0200878 A1) ing the RNA, dsRNA, siRNA, miRNA, shRNA, or hpRNA 0182. In specific embodiments, a recombinant DNA mol molecule. RNA may also be produced by partial or total ecule of the invention may comprise a polynucleotide encod organic synthesis—any modified ribonucleotide can be intro ing an RNA that may form a dsRNA molecule. Such recom duced by in vitro enzymatic or organic synthesis. An RNA binant DNA molecules may encode RNAs that may form molecule may be synthesized by a cellular RNA polymerase dsRNA molecules capable of inhibiting the expression of or a bacteriophage RNA polymerase (e.g., T3 RNA poly endogenous target gene(s) in an insect (e.g., coleopteran) pest merase, T7 RNA polymerase, and SP6 RNA polymerase). cell upon ingestion. In many embodiments, a transcribed Expression constructs useful for the cloning and expression RNA may form a dsRNA molecule that may be provided in a of polynucleotides are known in the art. See, e.g., Interna stabilized form; e.g., as a hairpin and stem and loop structure. tional PCT Publication No. WO97/32016; and U.S. Pat. Nos. 0183. In some embodiments, one strand of a dsRNA mol 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693. ecule may be formed by transcription from a polynucleotide RNA molecules that are synthesized chemically or by in vitro which is Substantially homologous to a polynucleotide enzymatic synthesis may be purified prior to introduction into selected from the group consisting of SEQID NOs: 1,77, 84. a cell. For example, RNA molecules can be purified from a 86,88, and 93; the complements of SEQIDNOs: 1,77, 84,86, mixture by extraction with a solvent or resin, precipitation, 88, and 93; a fragment of at least 15 contiguous nucleotides of electrophoresis, chromatography, or a combination thereof. any of SEQID NOS:1 and 77 (e.g., SEQID NOS:3-6), 84, 86, Alternatively, RNA molecules that are synthesized chemi 88, and 93 (e.g., SEQID NO:90); the complement of a frag cally or by in vitro enzymatic synthesis may be used with no ment of at least 15 contiguous nucleotides of any of SEQID or a minimum of purification, for example, to avoid losses due NOs: 1,77, 84, 86, 88, and 93; a native coding polynucleotide to sample processing. The RNA molecules may be dried for ofa Diabrotica organism (e.g., WCR) comprising any of SEQ storage or dissolved in an aqueous solution. The Solution may ID NOS:1, 3-6, and/or 77; the complement of a native coding contain buffers or salts to promote annealing, and/or stabili polynucleotide of a Diabrotica organism comprising any of zation of dsRNA molecule duplex strands. SEQ ID NOS:1, 3-6, and/or 77; a fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a 0179. In embodiments, a dsRNA molecule may beformed Diabrotica organism comprising any of SEQID NOS:1, 3-6, by a single self-complementary RNA strand or from two and/or 77; and the complement of a fragment of at least 15 complementary RNA strands. dsRNA molecules may be syn contiguous nucleotides of a native coding polynucleotide of a thesized either in vivo or in vitro. An endogenous RNA poly Diabrotica organism comprising any of SEQID NOS:1, 3-6, merase of the cell may mediate transcription of the one or two and/or 77; a native coding polynucleotide of a Melligethes RNA strands in vivo, or cloned RNA polymerase may be used organism (e.g., PB) comprising any of SEQID NOs:84, 86, to mediate transcription in vivo or in vitro. Post-transcrip 88, 90, and/or 93; the complement of a native coding poly tional inhibition of a target gene in an insect pest may be nucleotide of a Melligethes organism comprising any of SEQ host-targeted by specific transcription in an organ, tissue, or ID NOS:84, 86, 88,90, and/or 93; a fragment of at least 15 cell type of the host (e.g., by using a tissue-specific promoter); contiguous nucleotides of a native coding polynucleotide of a stimulation of an environmental condition in the host (e.g., by Melligethes organism comprising any of SEQID NOS:84, 86, using an inducible promoter that is responsive to infection, 88, 90, and/or 93; and the complement of a fragment of at stress, temperature, and/or chemical inducers); and/or engi least 15 contiguous nucleotides of a native coding polynucle neering transcription at a developmental stage or age of the otide of a Melligethes organism comprising any of SEQ ID host (e.g., by using a developmental stage-specific promoter). NOs:84, 86, 88,90, and/or 93. RNA strands that form a dsRNA molecule, whether tran 0184. In some embodiments, one strand of a dsRNA mol scribed in vitro or in Vivo, may or may not be polyadenylated, ecule may be formed by transcription from a polynucleotide and may or may not be capable of being translated into a that is Substantially homologous to a polynucleotide selected polypeptide by a cell's translational apparatus. from the group consisting of SEQID NOS:3-6, and 90; the 0180 D. Recombinant Vectors and Host Cell Transforma complements of SEQ ID NOS:3-6, and 90; fragments of at tion least 15 contiguous nucleotides of SEQID NOS:3-6, and 90: US 2016/0 194658 A1 Jul. 7, 2016 and the complements of fragments of at least 15 contiguous polynucleotide or other DNA element. Many vectors are nucleotides of SEQID NOS:3-6, and 90. available for this purpose, and selection of the appropriate 0185. In particular embodiments, a recombinant DNA vector will depend mainly on the size of the nucleic acid to be molecule encoding an RNA that may form a dsRNA molecule inserted into the vector and the particular host cell to be may comprise a coding region wherein at least two polynucle transformed with the vector. Each vector contains various otides are arranged Such that one polynucleotide is in a sense components depending on its function (e.g., amplification of orientation, and the other polynucleotide is in an antisense DNA or expression of DNA) and the particular host cell with orientation, relative to at least one promoter, wherein the which it is compatible. sense polynucleotide and the antisense polynucleotide are 0188 To impart insect (e.g., coleopteran) pest resistance linked or connected by a spacer of for example, from about to a transgenic plant, a recombinant DNA may, for example, five (-5) to about one thousand (~1000) nucleotides. The be transcribed into an iRNA molecule (e.g., an RNA molecule spacer may form a loop between the sense and antisense that forms a dsRNA molecule) within the tissues or fluids of polynucleotides. The sense polynucleotide or the antisense the recombinant plant. An iRNA molecule may comprise a polynucleotide may be substantially homologous to a target polynucleotide that is Substantially homologous and specifi gene (e.g., a ncm gene comprising SEQ ID NO:1, SEQ ID cally hybridizable to a corresponding transcribed polynucle NO:77, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, otide within an insect pest that may cause damage to the host and/or SEQID NO:93) or fragment thereof. In some embodi plant species. The pest may contact the iRNA molecule that is ments, however, a recombinant DNA molecule may encode transcribed in cells of the transgenic host plant, for example, an RNA that may form a dsRNA molecule without a spacer. by ingesting cells or fluids of the transgenic host plant that In embodiments, a sense coding polynucleotide and an anti comprise the iRNA molecule. Thus, in particular examples, sense coding polynucleotide may be different lengths. expression of a target gene is Suppressed by the iRNA mol 0186 Polynucleotides identified as having a deleterious ecule within coleopteran pests that infest the transgenic host effect on an insect pest or a plant-protective effect with regard plant. In some embodiments, Suppression of expression of the to the pest may be readily incorporated into expressed dsRNA target gene in a target coleopteran pest may result in the plant molecules through the creation of appropriate expression cas being resistant to attack by the pest. settes in a recombinant nucleic acid molecule of the inven (0189 In order to enable delivery of iRNA molecules to an tion. For example, such polynucleotides may be expressed as insect pest in a nutritional relationship with a plant cell that a hairpin with stem and loop structure by taking a first seg has been transformed with a recombinant nucleic acid mol ment corresponding to a target gene polynucleotide (e.g., a ecule of the invention, expression (i.e., transcription) of iRNA incm gene comprising SEQID NO:1, SEQID NO:77, SEQID molecules in the plant cell is required. Thus, a recombinant NO:84, SEQ ID NO:86, SEQ ID NO:88, and/or SEQ ID nucleic acid molecule may comprise a polynucleotide of the NO:93, and fragments of either of the foregoing); linking this invention operably linked to one or more regulatory elements, polynucleotide to a second segment spacer region that is not Such as a heterologous promoter element that functions in a homologous or complementary to the first segment; and link host cell, such as a bacterial cell wherein the nucleic acid ing this to a third segment, wherein at least a portion of the molecule is to be amplified, and a plant cell wherein the third segment is substantially complementary to the first seg nucleic acid molecule is to be expressed. ment. Such a construct forms a stem and loop structure by 0.190 Promoters suitable for use in nucleic acid molecules intramolecular base-pairing of the first segment with the third of the invention include those that are inducible, viral, syn segment, wherein the loop structure forms comprising the thetic, or constitutive, all of which are well known in the art. second segment. See, e.g., U.S. Patent Publication Nos. 2002/ Non-limiting examples describing Such promoters include 0048814 and 2003/0018993; and International PCT Publica U.S. Pat. No. 6,437.217 (maize RS81 promoter); U.S. Pat. tion NoS. WO94/O1550 and WO98/05770. A dsRNA mol No. 5,641,876 (rice actin promoter); U.S. Pat. No. 6,426,446 ecule may be generated, for example, in the form of a double (maize RS324 promoter); U.S. Pat. No. 6,429,362 (maize Stranded structure Such as a stem-loop structure (e.g., PR-1 promoter); U.S. Pat. No. 6,232,526 (maize A3 pro hairpin), whereby production of siRNA targeted for a native moter); U.S. Pat. No. 6,177,611 (constitutive maize promot insect (e.g., coleopteran) pest polynucleotide is enhanced by ers); U.S. Pat. Nos. 5,322.938, 5,352,605, 5,359,142, and co-expression of a fragment of the targeted gene, for instance 5,530,196 (CaMV 35S promoter); U.S. Pat. No. 6,433,252 on an additional plant expressible cassette, that leads to (maize L3 oleosin promoter); U.S. Pat. No. 6,429.357 (rice enhanced siRNA production, or reduces methylation to pre actin 2 promoter, and rice actin2 intron); U.S. Pat. No. 6,294, vent transcriptional gene silencing of the dsRNA hairpin pro 714 (light-inducible promoters); U.S. Pat. No. 6,140,078 moter. (salt-inducible promoters); U.S. Pat. No. 6,252,138 (patho 0187 Embodiments of the invention include introduction gen-inducible promoters); U.S. Pat. No. 6,175,060 (phospho of a recombinant nucleic acid molecule of the present inven rous deficiency-inducible promoters); U.S. Pat. No. 6,388, tion into a plant (i.e., transformation) to achieve insect (e.g., 170 (bidirectional promoters); U.S. Pat. No. 6,635,806 coleopteran) pest-inhibitory levels of expression of one or (gamma-coixin promoter); and U.S. Patent Publication No. more iRNA molecules. A recombinant DNA molecule may, 2009/757,089 (maize chloroplast aldolase promoter). Addi for example, be a vector, Such as a linear or a closed circular tional promoters include the nopaline synthase (NOS) pro plasmid. The vector system may be a single vector or plasmid, moter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84(16): or two or more vectors or plasmids that together contain the 5745-9) and the octopine synthase (OCS) promoters (which total DNA to be introduced into the genome of a host. In are carried on tumor-inducing plasmids of Agrobacterium addition, a vector may be an expression vector. Nucleic acids tumefaciens); the caulimovirus promoters such as the cauli of the invention can, for example, be suitably inserted into a flower mosaic virus (CaMV) 19S promoter (Lawton et al. vector under the control of a suitable promoter that functions (1987) Plant Mol. Biol. 9:315-24); the CaMV 35S promoter in one or more hosts to drive expression of a linked coding (Odell et al. (1985) Nature 313:810-2; the figwort mosaic US 2016/0 194658 A1 Jul. 7, 2016 22 virus 35S-promoter (Walker et al. (1987) Proc. Natl. Acad. Pisum sativum RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al. Sci. USA 84(19):6624-8); the sucrose synthase promoter (1984) EMBO J. 3:1671-9) and AGRtu.nos (GenBankTM (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA Accession No. E01312). 87:4144-8); the R gene complex promoter (Chandler et al. 0194 Some embodiments may include a plant transfor (1989) Plant Cell 1:1175-83); the chlorophyll a?b binding mation vector that comprises an isolated and purified DNA protein gene promoter: CaMV35S (U.S. Pat. Nos. 5,322,938, molecule comprising at least one of the above-described 5,352,605, 5,359,142, and 5,530,196); FMV 35S (U.S. Pat. regulatory elements operatively linked to one or more poly Nos. 6,051,753, and 5,378,619); a PC1SV promoter (U.S. nucleotides of the present invention. When expressed, the one Pat. No. 5,850,019); the SCP1 promoter (U.S. Pat. No. 6,677, or more polynucleotides result in one or more iRNA molecule 503); and AGRtu.nos promoters (GenBankTM Accession No. (s) comprising a polynucleotide that is specifically comple V00087: Depicker et al. (1982) J. Mol. Appl. Genet. 1:561 mentary to all or part of a native RNA molecule in an insect 73; Bevanet al. (1983) Nature 304:184-7). (e.g., coleopteran) pest. Thus, the polynucleotide(s) may 0191 In particular embodiments, nucleic acid molecules comprise a segment encoding all or part of a polyribonucle of the invention comprise a tissue-specific promoter. Such as otide present within a targeted coleopteran pest RNA tran a root-specific promoter. Root-specific promoters drive Script, and may comprise inverted repeats of all or a part of a expression of operably-linked coding polynucleotides exclu targeted pest transcript. A plant transformation vector may sively or preferentially in root tissue. Examples of root-spe contain polynucleotides specifically complementary to more cific promoters are known in the art. See, e.g., U.S. Pat. Nos. than one target polynucleotide, thus allowing production of 5,110,732; 5.459.252 and 5,837,848; and Opperman et al. more than one dsRNA for inhibiting expression of two or (1994) Science 263:221-3; and Hireletal. (1992) Plant Mol. more genes in cells of one or more populations or species of Biol. 20:207-18. In some embodiments, a polynucleotide or target insect pests. Segments of polynucleotides specifically fragment for coleopteran pest control according to the inven complementary to polynucleotides present in different genes tion may be cloned between two root-specific promoters ori can be combined into a single composite nucleic acid mol ented in opposite transcriptional directions relative to the ecule for expression in a transgenic plant. Such segments may polynucleotide or fragment, and which are operable in a be contiguous or separated by a spacer. transgenic plant cell and expressed therein to produce RNA 0.195. In some embodiments, a plasmid of the present molecules in the transgenic plant cell that Subsequently may invention already containing at least one polynucleotide(s) of form dsRNA molecules, as described, supra. The iRNA mol the invention can be modified by the sequential insertion of ecules expressed in plant tissues may be ingested by an insect additional polynucleotide(s) in the same plasmid, wherein the pest so that Suppression of target gene expression is achieved. additional polynucleotide(s) are operably linked to the same 0.192 Additional regulatory elements that may optionally regulatory elements as the original at least one polynucleotide be operably linked to a nucleic acid include 5' UTRs located (s). In some embodiments, a nucleic acid molecule may be between a promoter element and a coding polynucleotide that designed for the inhibition of multiple target genes. In some function as a translation leader element. The translation embodiments, the multiple genes to be inhibited can be leader element is present in fully-processed mRNA, and it obtained from the same insect (e.g., coleopteran) pest species, may affect processing of the primary transcript, and/or RNA which may enhance the effectiveness of the nucleic acid stability. Examples of translation leader elements include molecule. In other embodiments, the genes can be derived maize and petunia heat shock protein leaders (U.S. Pat. No. from different insect pests, which may broaden the range of 5.362,865), plant virus coat protein leaders, plant rubisco pests against which the agent(s) is/are effective. When mul leaders, and others. See, e.g., Turner and Foster (1995) tiple genes are targeted for Suppression or a combination of Molecular Biotech. 3(3):225-36. Non-limiting examples of expression and Suppression, a polycistronic DNA element 5'UTRs include GmHsp (U.S. Pat. No. 5,659,122); PhDnaK can be engineered. (U.S. Pat. No. 5,362,865); AtAnt1; TEV (Carrington and 0196. A recombinant nucleic acid molecule or vector of Freed (1990) J. Virol. 64: 1590-7); and AGRtunos (Gen the present invention may comprise a selectable marker that BankTM Accession No. V00087; and Bevan et al. (1983) confers a selectable phenotype on a transformed cell. Such as Nature 304:184-7). a plant cell. Selectable markers may also be used to select for 0193 Additional regulatory elements that may optionally plants or plant cells that comprise a recombinant nucleic acid be operably linked to a nucleic acid also include 3' non molecule of the invention. The marker may encode biocide translated elements, 3' transcription termination regions, or resistance, antibiotic resistance (e.g., kanamycin, Geneticin polyadenylation regions. These are genetic elements located (G418), bleomycin, hygromycin, etc.), or herbicide tolerance downstream of a polynucleotide, and include polynucleotides (e.g., glyphosate, etc.). Examples of selectable markers that provide polyadenylation signal, and/or other regulatory include, but are not limited to: a neogene which codes for signals capable of affecting transcription or mRNA process kanamycin resistance and can be selected for using kanamy ing. The polyadenylation signal functions in plants to cause cin, G418, etc.; a bar gene which codes for bialaphos resis the addition of polyadenylate nucleotides to the 3' end of the tance; a mutant EPSP synthase gene which encodes glypho mRNA precursor. The polyadenylation element can be sate tolerance; a nitrilase gene which confers resistance to derived from a variety of plant genes, or from T-DNA genes. bromoxynil; a mutant acetolactate synthase (ALS) gene A non-limiting example of a 3' transcription termination which confers imidazolinone or Sulfonylurea tolerance; and a region is the nopaline synthase 3' region (nos 3'; Fraley et al. methotrexate resistant DHFR gene. Multiple selectable (1983) Proc. Natl. Acad. Sci. USA80:4803-7). An example of markers are available that confer resistance to ampicillin, the use of different 3' non-translated regions is provided in bleomycin, chloramphenicol, gentamycin, hygromycin, Ingelbrecht et al., (1989) Plant Cell 1:671-80. Non-limiting kanamycin, lincomycin, methotrexate, phosphinothricin, examples of polyadenylation signals include one from a puromycin, spectinomycin, rifampicin, Streptomycin and tet US 2016/0 194658 A1 Jul. 7, 2016 racycline, and the like. Examples of Such selectable markers mation system of Agrobacterium. A. tumefaciens and A. are illustrated in, e.g., U.S. Pat. Nos. 5,550,318; 5.633,435: rhizogenes are plant pathogenic soil bacteria which geneti 5,780,708 and 6,118,047. cally transform plant cells. The Ti and Ri plasmids of A. 0197) A recombinant nucleic acid molecule or vector of tumefaciens and A. rhizogenes, respectively, carry genes the present invention may also include a screenable marker. responsible for genetic transformation of the plant. The Ti Screenable markers may be used to monitor expression. (tumor-inducing)-plasmids contain a large segment, known Exemplary screenable markers include a B-glucuronidase or as T-DNA, which is transferred to transformed plants. uidA gene (GUS) which encodes an enzyme for which vari Another segment of the Tiplasmid, the Vir region, is respon ous chromogenic Substrates are known (Jefferson et al. sible for T-DNA transfer. The T-DNA region is bordered by (1987) Plant Mol. Biol. Rep. 5:387-405); an R-locus gene, terminal repeats. In modified binary vectors, the tumor-in which encodes a product that regulates the production of ducing genes have been deleted, and the functions of the Vir anthocyanin pigments (red color) in plant tissues (Dellaporta region are utilized to transfer foreign DNA bordered by the et al. (1988) “Molecular cloning of the maize R-njallele by T-DNA border elements. The T-region may also contain a transposon tagging with Ac.” In 18' Stadler Genetics Sym selectable marker for efficient recovery of transgenic cells posium, P. Gustafson and R. Appels, eds. (New York: Ple and plants, and a multiple cloning site for inserting polynucle num), pp. 263-82); a B-lactamase gene (Sutcliffe et al. (1978) otides for transfer Such as a dsRNA encoding nucleic acid. Proc. Natl. Acad. Sci. USA 75:3737-41); a gene which 0201 Thus, in some embodiments, a plant transformation encodes an enzyme for which various chromogenic Sub vector is derived from a Ti plasmid of A. tumefaciens (See, strates are known (e.g., PADAC, a chromogenic cepha e.g., U.S. Pat. Nos. 4,536,475, 4,693,977, 4,886,937, and losporin); a luciferase gene (Ow et al. (1986) Science 234: 5,501,967; and European Patent No. EP 0122 791) or a Ri 856-9); an Xyl gene that encodes a catechol dioxygenase plasmid of A. rhizogenes. Additional plant transformation that can convert chromogenic catechols (Zukowski et al. vectors include, for example and without limitation, those (1983) Gene 46(2-3):247-55); an amylase gene (Ikatu et al. described by Herrera-Estrella et al. (1983) Nature 303:209 (1990) Bio/Technol. 8:241-2); a tyrosinase gene which 13; Bevan et al. (1983) Nature 304:184-7; Klee et al. (1985) encodes an enzyme capable of oxidizing tyrosine to DOPA Bio/Technol. 3:637-42; and in European Patent No. EP 0120 and dopaquinone which in turn condenses to melanin (Katz, et 516, and those derived from any of the foregoing. Other al. (1983) J. Gen. Microbiol. 129:2703-14); and an O-galac bacteria such as Sinorhizobium, Rhizobium, and Mesorhizo tosidase. bium that interact with plants naturally can be modified to 0198 In some embodiments, recombinant nucleic acid mediate gene transfer to a number of diverse plants. These molecules, as described, Supra, may be used in methods for plant-associated Symbiotic bacteria can be made competent the creation of transgenic plants and expression of heterolo for gene transfer by acquisition of both a disarmed Tiplasmid gous nucleic acids in plants to prepare transgenic plants that and a suitable binary vector. exhibit reduced Susceptibility to insect (e.g., coleopteran) 0202 After providing exogenous DNA to recipient cells, pests. Plant transformation vectors can be prepared, for transformed cells are generally identified for further culturing example, by inserting nucleic acid molecules encoding iRNA and plant regeneration. In order to improve the ability to molecules into plant transformation vectors and introducing identify transformed cells, one may desire to employ a select these into plants. able or screenable marker gene, as previously set forth, with 0199 Suitable methods for transformation of host cells the transformation vector used to generate the transformant. include any method by which DNA can be introduced into a In the case where a selectable marker is used, transformed cell. Such as by transformation of protoplasts (See, e.g., U.S. cells are identified within the potentially transformed cell Pat. No. 5,508,184), by desiccation/inhibition-mediated population by exposing the cells to a selective agentoragents. DNA uptake (See, e.g., Potrykus et al. (1985) Mol. Gen. In the case where a screenable marker is used, cells may be Genet. 199:183-8), by electroporation (See, e.g., U.S. Pat. screened for the desired marker gene trait. No. 5,384.253), by agitation with silicon carbide fibers (See, 0203 Cells that survive the exposure to the selective e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), by Agrobacte agent, or cells that have been scored positive in a screening rium-mediated transformation (See, e.g., U.S. Pat. Nos. assay, may be cultured in media that Supports regeneration of 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; and plants. In some embodiments, any Suitable plant tissue cul 6,384.301) and by acceleration of DNA-coated particles (See, ture media (e.g., MS and N6 media) may be modified by e.g., U.S. Pat. Nos. 5,015,580: 5,550,318; 5,538,880; 6,160, including further Substances, such as growth regulators. Tis 208; 6.399,861; and 6,403,865), etc. Techniques that are par Sue may be maintained on a basic medium with growth regu ticularly useful for transforming corn are described, for lators until Sufficient tissue is available to begin plant regen example, in U.S. Pat. Nos. 7,060,876 and 5,591,616; and eration efforts, or following repeated rounds of manual International PCT Publication WO95/06722. Through the selection, until the morphology of the tissue is suitable for application of techniques such as these, the cells of virtually regeneration (e.g., at least 2 weeks), then transferred to media any species may be stably transformed. In some embodi conducive to shoot formation. Cultures are transferred peri ments, transforming DNA is integrated into the genome of the odically until sufficient shoot formation has occurred. Once host cell. In the case of multicellular species, transgenic cells shoots are formed, they are transferred to media conducive to may be regenerated into a transgenic organism. Any of these root formation. Once Sufficient roots are formed, plants can techniques may be used to produce a transgenic plant, for be transferred to soil for further growth and maturation. example, comprising one or more nucleic acids encoding one 0204 To confirm the presence of a nucleic acid molecule or more iRNA molecules in the genome of the transgenic of interest (for example, a DNA encoding one or more iRNA plant. molecules that inhibit target gene expression in a coleopteran 0200. The most widely utilized method for introducing an pest) in the regenerating plants, a variety of assays may be expression vector into plants is based on the natural transfor performed. Such assays include, for example: molecular bio US 2016/0 194658 A1 Jul. 7, 2016 24 logical assays, such as Southern and northern blotting, PCR, cells are included, wherein the seeds or commodity products and nucleic acid sequencing; biochemical assays, such as comprise a detectable amount of a nucleic acid of the inven detecting the presence of a protein product, e.g., by immuno tion. In some embodiments, such commodity products may logical means (ELISA and/or western blots) or by enzymatic be produced, for example, by obtaining transgenic plants and function; plant part assays, such as leaf or root assays; and preparing food or feed from them. Commodity products com analysis of the phenotype of the whole regenerated plant. prising one or more of the polynucleotides of the invention 0205 Integration events may be analyzed, for example, by includes, for example and without limitation: meals, oils, PCR amplification using, e.g., oligonucleotide primers spe crushed or whole grains or seeds of a plant, and any food cific for a nucleic acid molecule of interest. PCR genotyping product comprising any meal, oil, or crushed or whole grain is understood to include, but not be limited to, polymerase of a recombinant plant or seed comprising one or more of the chain reaction (PCR) amplification of gldNA derived from nucleic acids of the invention. The detection of one or more of isolated host plant callus tissue predicted to contain a nucleic the polynucleotides of the invention in one or more commod acid molecule of interest integrated into the genome, fol ity or commodity products is de facto evidence that the com lowed by Standard cloning and sequence analysis of PCR modity or commodity product is produced from a transgenic amplification products. Methods of PCR genotyping have plant designed to express one or more of the iRNA molecules been well described (for example, Rios, G. et al. (2002) Plant of the invention for the purpose of controlling insect (e.g., J. 32:243-53) and may be applied to gldNA derived from any coleopteran) pests. plant species (e.g., Z. mays or Brassica napus) or tissue type, 0210. In some embodiments, a transgenic plant or seed including cell cultures. comprising a nucleic acid molecule of the invention also may 0206. A transgenic plant formed using Agrobacterium comprise at least one other transgenic event in its genome, dependent transformation methods typically contains a single including without limitation: a transgenic event from which is recombinant DNA inserted into one chromosome. The poly transcribed an iRNA molecule targeting a locus in a nucleotide of the single recombinant DNA is referred to as a coleopteran pest other than the ones defined by SEQ ID “transgenic event' or “integration event'. Such transgenic NOs: 1, 77, 84, 86, 88, and 93, such as, for example, one or plants are heterozygous for the inserted exogenous poly more loci selected from the group consisting of Caf1-180 nucleotide. In some embodiments, a transgenic plant (U.S. Patent Application Publication No. 2012/0174258), homozygous with respect to a transgene may be obtained by VatpaseC (U.S. Patent Application Publication No. 2012/ sexually mating (selfing) an independent segregant trans 0174259), Rhol (U.S. Patent Application Publication No. genic plant that contains a single exogenous gene to itself, for 2012/0174260), VatpaseH (U.S. Patent Application Publica example a To plant, to produce T seed. One fourth of the T tion No. 2012/0198586), PPI-87B (U.S. Patent Application seed produced will be homozygous with respect to the trans Publication No. 2013/009 1600), RPA70 (U.S. Patent Appli gene. Germinating T. Seed results in plants that can be tested cation Publication No. 2013/0091601), RPS6 (U.S. Patent for heterozygosity, typically using an SNP assay or a thermal Application Publication No. 2013/0097730), ROP (U.S. amplification assay that allows for the distinction between patent application Ser. No. 14/577,811), RNAPII140 (U.S. heterozygotes and homozygotes (i.e., a Zygosity assay). patent application Ser. No. 14/577,854), Drea. (U.S. patent 0207. In particular embodiments, at least 2, 3, 4, 5, 6, 7, 8, application Ser. No. 14/705,807), COPI alpha (U.S. Patent 9 or 10 or more different iRNA molecules are produced in a Application No. 62/063.199), COPI beta (U.S. Patent Appli plant cell that have an insect (e.g., coleopteran) pest-inhibi cation No. 62/063.203), COPI gamma (U.S. Patent Applica tory effect. The iRNA molecules (e.g., dsRNA molecules) tion No. 62/063,192), and COPI delta (U.S. Patent Applica may be expressed from multiple nucleic acids introduced in tion No. 62/063.216); a transgenic event from which is different transformation events, or from a single nucleic acid transcribed an iRNA molecule targeting a gene in an organ introduced in a single transformation event. In some embodi ism other thana coleopteran pest (e.g., a plant-parasitic nema ments, a plurality of iRNA molecules are expressed under the tode); a gene encoding an insecticidal protein (e.g., a Bacillus control of a single promoter. In other embodiments, a plural thuringiensis insecticidal protein); an herbicide tolerance ity of iRNA molecules are expressed under the control of gene (e.g., a gene providing tolerance to glyphosate); and a multiple promoters. Single iRNA molecules may be gene contributing to a desirable phenotype in the transgenic expressed that comprise multiple polynucleotides that are plant, such as increased yield, altered fatty acid metabolism, each homologous to different loci within one or more insect or restoration of cytoplasmic male sterility. In particular pests (for example, the loci defined by SEQID NOs: 1,77,84, embodiments, polynucleotides encoding iRNA molecules of 86, 88, and 93), both in different populations of the same the invention may be combined with other insect control and species of insect pest, or in different species of insect pests. disease traits in a plant to achieve desired traits for enhanced 0208. In addition to direct transformation of a plant with a control of plant disease and insect damage. Combining insect recombinant nucleic acid molecule, transgenic plants can be control traits that employ distinct modes-of-action may pro prepared by crossing a first plant having at least one trans vide protected transgenic plants with Superior durability over genic event with a second plant lacking Such an event. For plants harboring a single control trait, for example, because of example, a recombinant nucleic acid molecule comprising a the reduced probability that resistance to the trait(s) will polynucleotide that encodes an iRNA molecule may be intro develop in the field. duced into a first plant line that is amenable to transformation to produce a transgenic plant, which transgenic plant may be V. Target Gene Suppression in an Insect Pest crossed with a second plant line to introgress the polynucle 0211 A. Overview otide that encodes the iRNA molecule into the second plant 0212. In some embodiments of the invention, at least one line. nucleic acid molecule useful for the control of insect (e.g., 0209. In some aspects, seeds and commodity products coleopteran) pests may be provided to an insect pest, wherein produced by transgenic plants derived from transformed plant the nucleic acid molecule leads to RNAi-mediated gene US 2016/0 194658 A1 Jul. 7, 2016

silencing in the pest. In particular embodiments, an iRNA 0217. In embodiments of the invention, any form of iRNA molecule (e.g., dsRNA, siRNA, miRNA, shRNA, and molecule may be used. Those of skill in the art will under hpRNA) may be provided to a coleopteran pest. In some stand that dsRNA molecules typically are more stable during embodiments, a nucleic acid molecule useful for the control preparation and during the step of providing the iRNA mol of insect pests may be provided to a pest by contacting the ecule to a cell than are single-stranded RNA molecules, and nucleic acid molecule with the pest. In these and further are typically also more stable in a cell. Thus, while siRNA and embodiments, a nucleic acid molecule useful for the control miRNA molecules, for example, may be equally effective in of insect pests may be provided in a feeding substrate of the some embodiments, a dsRNA molecule may be chosen due to pest, for example, a nutritional composition. In these and its stability. further embodiments, a nucleic acid molecule useful for the 0218. In particular embodiments, a nucleic acid molecule control of an insect pest may be provided through ingestion of is provided that comprises a polynucleotide, which poly plant material comprising the nucleic acid molecule that is nucleotide may be expressed in vitro to produce an iRNA ingested by the pest. In certain embodiments, the nucleic acid molecule that is Substantially homologous to a nucleic acid molecule is present in plant material through expression of a molecule encoded by a polynucleotide within the genome of recombinant nucleic acid introduced into the plant material, an insect (e.g., coleopteran) pest. In certain embodiments, the for example, by transformation of a plant cell with a vector in vitro transcribed iRNA molecule may be a stabilized comprising the recombinant nucleic acid and regeneration of dsRNA molecule that comprises a stem-loop structure. After a plant material or whole plant from the transformed plant an insect pest contacts the in vitro transcribed iRNA mol cell. ecule, post-transcriptional inhibition of a target gene in the 0213 B. RNAi-mediated Target Gene Suppression pest (for example, an essential gene) may occur. 0214. In embodiments, the invention provides iRNA mol 0219. In some embodiments of the invention, expression ecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) of a nucleic acid molecule comprising at least 15 contiguous that may be designed to target essential native polynucle nucleotides (e.g., at least 19 contiguous nucleotides) of a otides (e.g., essential genes) in the transcriptome of an insect polynucleotide are used in a method for post-transcriptional pest (for example, a coleopteran (e.g., WCR, NCR, SCR, and inhibition of a target gene in an insect (e.g., coleopteran) pest, Melligethes aeneus) pest), for example by designing an iRNA wherein the polynucleotide is selected from the group con molecule that comprises at least one strand comprising a sisting of: SEQID NO:1; the complement of SEQID NO:1; polynucleotide that is specifically complementary to the tar SEQ ID NO:3: the complement of SEQ ID NO:3: SEQ ID get polynucleotide. The sequence of an iRNA molecule so NO:3: the complement of SEQID NO:3: SEQID NO:4; the designed may be identical to that of the target polynucleotide, complement of SEQ ID NO:4: SEQ ID NO:5; the comple or may incorporate mismatches that do not prevent specific ment of SEQ ID NO:5: SEQ ID NO:6; the complement of hybridization between the iRNA molecule and its target poly SEQID NO:6; SEQID NO:77; the complement of SEQ ID nucleotide. NO:77, SEQID NO:84; the complement of SEQID NO:84, 0215 iRNA molecules of the invention may be used in SEQID NO:86; the complement of SEQID NO:86, SEQID methods for gene Suppression in an insect (e.g., coleopteran) NO:88; the complement of SEQID NO:88: SEQID NO:90; pest, thereby reducing the level or incidence of damage the complement of SEQ ID NO:90; SEQ ID NO:93; the caused by the pest on a plant (for example, a protected trans complement of SEQ ID NO:93; a fragment of at least 15 formed plant comprising an iRNA molecule). As used herein contiguous nucleotides of SEQID NO:1; the complement of the term “gene suppression” refers to any of the well-known a fragment of at least 15 contiguous nucleotides of SEQ ID methods for reducing the levels of protein produced as a result NO:1; a fragment of at least 15 contiguous nucleotides of of gene transcription to mRNA and Subsequent translation of SEQID NO:3: the complement of a fragment of at least 15 the mRNA, including the reduction of protein expression contiguous nucleotides of SEQ ID NO:3: a fragment of at from a gene or a coding polynucleotide including post-tran least 15 contiguous nucleotides of SEQID NO:4; the comple Scriptional inhibition of expression and transcriptional Sup ment of a fragment of at least 15 contiguous nucleotides of pression. Post-transcriptional inhibition is mediated by spe SEQ ID NO:4; a fragment of at least 15 contiguous nucle cific homology between all or a part of an mRNA transcribed otides of SEQID NO:5; the complement of a fragment of at from a gene targeted for Suppression and the corresponding least 15 contiguous nucleotides of SEQID NO:5; a fragment iRNA molecule used for suppression. Additionally, post-tran of at least 15 contiguous nucleotides of SEQ ID NO:6; the scriptional inhibition refers to the substantial and measurable complement of a fragment of at least 15 contiguous nucle reduction of the amount of mRNA available in the cell for otides of SEQID NO:6; a fragment of at least 15 contiguous binding by ribosomes. nucleotides of SEQID NO:77; the complement of a fragment 0216. In embodiments wherein an iRNA molecule is a of at least 15 contiguous nucleotides of SEQ ID NO:77; a dsRNA molecule, the dsRNA molecule may be cleaved by the fragment of at least 15 contiguous nucleotides of SEQ ID enzyme, DICER, into short siRNA molecules (approximately NO:84; the complement of a fragment of at least 15 contigu 20 nucleotides in length). The double-stranded siRNA mol ous nucleotides of SEQID NO:84; a fragment of at least 15 ecule generated by DICER activity upon the dsRNA mol contiguous nucleotides of SEQID NO:86; the complement of ecule may be separated into two single-stranded siRNAs; the a fragment of at least 15 contiguous nucleotides of SEQ ID “passenger Strand' and the 'guide Strand’. The passenger NO:86; a fragment of at least 15 contiguous nucleotides of Strand may be degraded, and the guide Strand may be incor SEQID NO:88; the complement of a fragment of at least 15 porated into RISC. Post-transcriptional inhibition occurs by contiguous nucleotides of SEQ ID NO:88; a fragment of at specific hybridization of the guide strand with a specifically least 15 contiguous nucleotides of SEQ ID NO:90; the complementary polynucleotide of an mRNA molecule, and complement of a fragment of at least 15 contiguous nucle Subsequent cleavage by the enzyme, Argonaute (catalytic otides of SEQID NO:90; a fragment of at least 15 contiguous component of the RISC complex). nucleotides of SEQID NO:93; the complement of a fragment US 2016/0 194658 A1 Jul. 7, 2016 26 of at least 15 contiguous nucleotides of SEQ ID NO:93; a nucleotide of a Melligethes organism comprising SEQ ID native coding polynucleotide of a Diabrotica organism com NO:84; the complement of a fragment of at least 15 contigu prising SEQ ID NO:1; the complement of a native coding ous nucleotides of a native coding polynucleotide of a polynucleotide of a Diabrotica organism comprising SEQID Melligethes organism comprising SEQID NO:84; a fragment NO:1; a native coding polynucleotide of a Diabrotica organ of at least 15 contiguous nucleotides of a native coding poly ism comprising SEQ ID NO:3: the complement of a native nucleotide of a Melligethes organism comprising SEQ ID coding polynucleotide of a Diabrotica organism comprising NO:86; the complement of a fragment of at least 15 contigu SEQID NO:3: a native coding polynucleotide of a Diabrotica ous nucleotides of a native coding polynucleotide of a organism comprising SEQ ID NO:4; the complement of a Melligethes organism comprising SEQID NO:86; a fragment of at least 15 contiguous nucleotides of a native coding poly native coding polynucleotide of a Diabrotica organism com nucleotide of a Melligethes organism comprising SEQ ID prising SEQ ID NO:4; a native coding polynucleotide of a NO:88; the complement of a fragment of at least 15 contigu Diabrotica organism comprising SEQID NO:5; the comple ous nucleotides of a native coding polynucleotide of a ment of a native coding polynucleotide of a Diabrotica organ Melligethes organism comprising SEQID NO:88; a fragment ism comprising SEQ ID NO:5; a native coding polynucle of at least 15 contiguous nucleotides of a native coding poly otide of a Diabrotica organism comprising SEQID NO:6; the nucleotide of a Melligethes organism comprising SEQ ID complement of a native coding polynucleotide of a NO:90; the complement of a fragment of at least 15 contigu Diabrotica organism comprising SEQID NO:6; a native cod ous nucleotides of a native coding polynucleotide of a ing polynucleotide of a Diabrotica organism comprising SEQ Melligethes organism comprising SEQID NO:90; a fragment ID NO:77; the complement of a native coding polynucleotide of at least 15 contiguous nucleotides of a native coding poly of a Diabrotica organism comprising SEQID NO:77; a frag nucleotide of a Melligethes organism comprising SEQ ID ment of at least 15 contiguous nucleotides of a native coding NO:93; the complement of a fragment of at least 15 contigu polynucleotide of a Diabrotica organism comprising SEQID ous nucleotides of a native coding polynucleotide of a NO:1; the complement of a fragment of at least 15 contiguous Melligethes organism comprising SEQID NO:93. In certain nucleotides of a native coding polynucleotide of a Diabrotica embodiments, expression of a nucleic acid molecule that is at organism comprising SEQID NO:1; a fragment of at least 15 least about 80% identical (e.g., 79%, about 80%, about 81%, contiguous nucleotides of a native coding polynucleotide of a about 82%, about 83%, about 84%, about 85%, about 86%, Diabrotica organism comprising SEQID NO:3: the comple about 87%, about 88%, about 89%, about 90%, about 91%, ment of a fragment of at least 15 contiguous nucleotides of a about 92%, about 93%, about 94%, about 95%, about 96%, native coding polynucleotide of a Diabrotica organism com about 97%, about 98%, about 99%, about 100%, and 100%) prising SEQID NO:3: a fragment of at least 15 contiguous with any of the foregoing may be used. In these and further nucleotides of a native coding polynucleotide of a Diabrotica embodiments, a nucleic acid molecule may be expressed that organism comprising SEQ ID NO:4; the complement of a specifically hybridizes to an RNA molecule present in at least fragment of at least 15 contiguous nucleotides of a native coding polynucleotide of a Diabrotica organism comprising one cell of an insect (e.g., coleopteran) pest. SEQ ID NO:4; a fragment of at least 15 contiguous nucle 0220. It is an important feature of some embodiments otides of a native coding polynucleotide of a Diabrotica herein that the RNAi post-transcriptional inhibition system is organism comprising SEQ ID NO:5; the complement of a able to tolerate sequence variations among target genes that fragment of at least 15 contiguous nucleotides of a native might be expected due to genetic mutation, strain polymor coding polynucleotide of a Diabrotica organism comprising phism, or evolutionary divergence. The introduced nucleic SEQ ID NO:5; a fragment of at least 15 contiguous nucle acid molecule may not need to be absolutely homologous to otides of a native coding polynucleotide of a Diabrotica either a primary transcription product or a fully-processed organism comprising SEQ ID NO:6; the complement of a mRNA of a target gene, so long as the introduced nucleic acid fragment of at least 15 contiguous nucleotides of a native molecule is specifically hybridizable to either a primary tran coding polynucleotide of a Diabrotica organism comprising scription product or a fully-processed mRNA of the target SEQ ID NO:6; a fragment of at least 15 contiguous nucle gene. Moreover, the introduced nucleic acid molecule may otides of a native coding polynucleotide of a Diabrotica not need to be full-length, relative to either a primary tran organism comprising SEQID NO:77; and the complement of scription product or a fully processed mRNA of the target a fragment of at least 15 contiguous nucleotides of a native gene. coding polynucleotide of a Diabrotica organism comprising 0221) Inhibition of a target gene using the iRNA technol SEQ ID NO:77; a native coding polynucleotide of a ogy of the present invention is sequence-specific; i.e., poly Melligethes organism comprising SEQ ID NO:84; the nucleotides substantially homologous to the iRNA molecule complement of a native coding polynucleotide of a (s) are targeted for genetic inhibition. In some embodiments, Melligethes organism comprising SEQ ID NO:84; a native an RNA molecule comprising a polynucleotide with a nucle coding polynucleotide of a Melligethes organism comprising otide sequence that is identical to that of a portion of a target SEQ ID NO:86; the complement of a native coding poly gene may be used for inhibition. In these and further embodi nucleotide of a Melligethes organism comprising SEQ ID ments, an RNA molecule comprising a polynucleotide with NO:86; a native coding polynucleotide of a Melligethes organ one or more insertion, deletion, and/or point mutations rela ism comprising SEQID NO:88; the complement of a native tive to a target polynucleotide may be used. In particular coding polynucleotide of a Melligethes organism comprising embodiments, an iRNA molecule and a portion of a target SEQ ID NO:88; a native coding polynucleotide of a gene may share, for example, at least from about 80%, at least Melligethes organism comprising SEQ ID NO:90; the from about 81%, at least from about 82%, at least from about complement of a native coding polynucleotide of a 83%, at least from about 84%, at least from about 85%, at Melligethes organism comprising SEQID NO:90; a fragment least from about 86%, at least from about 87%, at least from of at least 15 contiguous nucleotides of a native coding poly about 88%, at least from about 89%, at least from about 90%, US 2016/0 194658 A1 Jul. 7, 2016 27 at least from about 91%, at least from about 92%, at least from polynucleotides of the present invention may also be intro about 93%, at least from about 94%, at least from about 95%, duced into a wide variety of prokaryotic and eukaryotic at least from about 96%, at least from about 97%, at least from microorganism hosts to produce iRNA molecules. The term about 98%, at least from about 99%, at least from about “microorganism' includes prokaryotic and eukaryotic spe 100%, and 100% sequence identity. Alternatively, the duplex cies, such as bacteria and fungi. region of a dsRNA molecule may be specifically hybridizable 0226 Modulation of gene expression may include partial with a portion of a target gene transcript. In specifically or complete Suppression of Such expression. In another hybridizable molecules, a less than full length polynucleotide embodiment, a method for Suppression of gene expression in exhibiting a greater homology compensates for a longer, less an insect (e.g., coleopteran) pest comprises providing in the homologous polynucleotide. The length of the polynucle tissue of the host of the pest a gene-suppressive amount of at otide of a duplex region of a dsRNA molecule that is identical least one dsRNA molecule formed following transcription of to a portion of a target genetranscript may beat least about 25, a polynucleotide as described herein, at least one segment of 50, 100, 200, 300, 400, 500, or at least about 1000 bases. In which is complementary to an mRNA within the cells of the some embodiments, a polynucleotide of greater than 20-100 insect pest. A dsRNA molecule, including its modified form nucleotides may be used. In particular embodiments, a poly such as an siRNA, miRNA, shRNA, or hpRNA molecule, nucleotide of greater than about 200-300 nucleotides may be ingested by an insect pest may be at least from about 80%, used. In particular embodiments, a polynucleotide of greater 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, than about 500-1000 nucleotides may be used, depending on 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about the size of the target gene. 100% identical to an RNA molecule transcribed from ancm 0222. In certain embodiments, expression of a target gene DNA molecule, for example, comprising a polynucleotide in an insect pest (e.g., a coleopteran insect pest) may be selected from the group consisting of SEQID NOS:1, 3-6, 77. inhibited by at least 10%;at least 33%;at least 50%; or at least 84, 86, 88, 90, and 93. Isolated and substantially purified 80% within a cell of the pest, such that a significant inhibition nucleic acid molecules including, but not limited to, non takes place. Significant inhibition refers to inhibition over a naturally occurring polynucleotides and recombinant DNA threshold that results in a detectable phenotype (e.g., cessa constructs for providing dsRNA molecules are therefore pro tion of growth, cessation of feeding, cessation of develop vided, which Suppress or inhibit the expression of an endog ment, induced mortality, etc.), or a detectable decrease in enous coding polynucleotide or a target coding polynucle RNA and/or gene product corresponding to the target gene otide in an insect pest when introduced thereto. being inhibited. Although, in certain embodiments of the 0227 Particular embodiments provide a delivery system invention, inhibition occurs in substantially all cells of the for the delivery of iRNA molecules for the post-transcrip pest, in other embodiments inhibition occurs only in a Subset tional inhibition of one or more target gene(s) in an insect of cells expressing the target gene. (e.g., coleopteran) plant pest and control of a population of 0223. In some embodiments, transcriptional Suppression the plant pest. In some embodiments, the delivery system is mediated by the presence in a cell of a dsRNA molecule comprises ingestion of a host transgenic plant cell or contents exhibiting substantial sequence identity to a promoter DNA of the host cell comprising RNA molecules transcribed in the or the complement thereof to effect what is referred to as host cell. In these and further embodiments, a transgenic plant “promoter trans Suppression. Gene Suppression may be cell or a transgenic plant is created that contains a recombi effective against target genes in an insect pest that may ingest nant DNA construct providing a stabilized dsRNA molecule or contact such dsRNA molecules, for example, by ingesting of the invention. Transgenic plant cells and transgenic plants or contacting plant material containing the dsRNA mol comprising nucleic acids encoding a particular iRNA mol ecules. dsRNA molecules for use in promoter trans Suppres ecule may be produced by employing recombinant DNA sion may be specifically designed to inhibit or Suppress the technologies (which basic technologies are well-known in the expression of one or more homologous or complementary art) to construct a plant transformation vector comprising a polynucleotides in the cells of the insect pest. Post-transcrip polynucleotide encoding an iRNA molecule of the invention tional gene Suppression by antisense or sense oriented RNA (e.g., a stabilized dsRNA molecule); to transform a plant cell to regulate gene expression in plant cells is disclosed in U.S. or plant; and to generate the transgenic plant cell or the Pat. Nos. 5,107,065; 5,759,829; 5,283,184; and 5,231,020. transgenic plant that contains the transcribed iRNA molecule. 0224 C. Expression of iRNA Molecules Provided to an 0228 To impart insect (e.g., coleopteran) pest resistance Insect Pest to a transgenic plant, a recombinant DNA molecule may, for 0225 Expression of iRNA molecules for RNAi-mediated example, be transcribed into an iRNA molecule, such as a gene inhibition in an insect (e.g., coleopteran) pest may be dsRNA molecule, a siRNA molecule, a miRNA molecule, a carried out in any one of many in vitro or in Vivo formats. The shRNA molecule, or a hpRNA molecule. In some embodi iRNA molecules may then be provided to an insect pest, for ments, a RNA molecule transcribed from a recombinant DNA example, by contacting the iRNA molecules with the pest, or molecule may form a dsRNA molecule within the tissues or by causing the pest to ingest or otherwise internalize the fluids of the recombinant plant. Such a dsRNA molecule may iRNA molecules. Some embodiments include transformed be comprised in part of a polynucleotide that is identical to a host plants of a coleopteran pest, transformed plant cells, and corresponding polynucleotide transcribed from a DNA progeny of transformed plants. The transformed plant cells within an insect pest of a type that may infest the host plant. and transformed plants may be engineered to express one or Expression of a target gene within the pest is Suppressed by more of the iRNA molecules, for example, under the control the dsRNA molecule, and the suppression of expression of the of a heterologous promoter, to provide a pest-protective target gene in the pest results in the transgenic plant being effect. Thus, when a transgenic plant or plant cell is consumed resistant to the pest. The modulatory effects of dsRNA mol by an insect pest during feeding, the pest may ingest iRNA ecules have been shown to be applicable to a variety of genes molecules expressed in the transgenic plants or cells. The expressed in pests, including, for example, endogenous genes US 2016/0 194658 A1 Jul. 7, 2016 28 responsible for cellular metabolism or cellular transforma termination element; culturing the transformed plant cell tion, including house-keeping genes; transcription factors; under conditions sufficient to allow for development of a molting-related genes; and other genes which encode plant cell culture including a plurality of transformed plant polypeptides involved in cellular metabolism or normal cells; selecting for transformed plant cells that have inte growth and development. grated the polynucleotide into their genomes; Screening the 0229. For transcription from a transgene in vivo or an transformed plant cells for expression of an iRNA molecule expression construct, a regulatory region (e.g., promoter, encoded by the integrated polynucleotide; selecting a trans enhancer, silencer, and polyadenylation signal) may be used genic plant cell that expresses the iRNA molecule; and feed in some embodiments to transcribe the RNA strand (or ing the selected transgenic plant cell to the insect pest. Plants Strands). Therefore, in Some embodiments, as set forth, Supra, may also be regenerated from transformed plant cells that a polynucleotide for use in producing iRNA molecules may express an iRNA molecule encoded by the integrated nucleic be operably linked to one or more promoter elements func acid molecule. In some embodiments, the iRNA molecule is tional in a plant host cell. The promoter may be an endog a dsRNA molecule. In these and further embodiments, the enous promoter, normally resident in the host genome. The nucleic acid molecule(s) comprise dsRNA molecules that polynucleotide of the present invention, under the control of each comprise more than one polynucleotide that is specifi an operably linked promoter element, may further be flanked cally hybridizable to a nucleic acid molecule expressed in an by additional elements that advantageously affect its tran insect pest cell. In some examples, the nucleic acid molecule Scription and/or the stability of a resulting transcript. Such (s) comprises a polynucleotide that is specifically hybridiz elements may be located upstream of the operably linked promoter, downstream of the 3' end of the expression con able to a nucleic acid molecule expressed in a coleopteran struct, and may occur both upstream of the promoter and pest cell. downstream of the 3' end of the expression construct. 0233 iRNA molecules of the invention can be incorpo 0230. Some embodiments provide methods for reducing rated within the seeds of a plant species (e.g., corn or canola), the damage to a host plant (e.g., a corn plant or canola plant) either as a product of expression from a recombinant gene caused by an insect (e.g., coleopteran) pest that feeds on the incorporated into a genome of the plant cells, or as incorpo plant, wherein the method comprises providing in the host rated into a coating or seed treatment that is applied to the plant a transformed plant cell expressing at least one nucleic seed before planting. A plant cell comprising a recombinant acid molecule of the invention, wherein the nucleic acid mol gene is considered to be a transgenic event. Also included in ecule(s) functions upon being taken up by the pest(s) to embodiments of the invention are delivery systems for the inhibit the expression of a target polynucleotide within the delivery of iRNA molecules to insect (e.g., coleopteran) pest(s), which inhibition of expression results in mortality pests. For example, the iRNA molecules of the invention may and/or reduced growth of the pest(s), thereby reducing the be directly introduced into the cells of a pest(s). Methods for damage to the host plant caused by the pest(s). In some introduction may include direct mixing of iRNA with plant embodiments, the nucleic acid molecule(s) comprise dsRNA tissue from a host for the insect pest(s), as well as application molecules. In these and further embodiments, the nucleic acid of compositions comprisingiRNA molecules of the invention molecule(s) comprise dsRNA molecules that each comprise to host plant tissue. For example, iRNA molecules may be more than one polynucleotide that is specifically hybridizable sprayed onto a plant surface. Alternatively, an iRNA molecule to a nucleic acid molecule expressed in a coleopteran pest may be expressed by a microorganism, and the microorgan cell. In some embodiments, the nucleic acid molecule(s) con ism may be applied onto the plant Surface, or introduced into sist of one polynucleotide that is specifically hybridizable to a root or stem by a physical means such as an injection. As a nucleic acid molecule expressed in an insect pest cell. discussed, Supra, a transgenic plant may also be genetically 0231. In some embodiments, a method for increasing the engineered to express at least one iRNA molecule in an yield of a corn crop is provided, wherein the method com amount sufficient to kill the insect pests known to infest the prises introducing into a corn plant at least one nucleic acid plant. iRNA molecules produced by chemical or enzymatic molecule of the invention; cultivating the corn plant to allow synthesis may also be formulated in a manner consistent with the expression of an iRNA molecule comprising the nucleic common agricultural practices, and used as spray-on prod acid, wherein expression of an iRNA molecule comprising ucts for controlling plant damage by an insect pest. The for the nucleic acid inhibits insect (e.g., coleopteran) pest dam mulations may include the appropriate Stickers and wetters age and/or growth, thereby reducing or eliminating a loss of required for efficient foliar coverage, as well as UV pro yield due to pest infestation. In some embodiments, the iRNA tectants to protect iRNA molecules (e.g., dsRNA molecules) molecule is a dsRNA molecule. In these and further embodi from UV damage. Such additives are commonly used in the ments, the nucleic acid molecule(s) comprise dsRNA mol bioinsecticide industry, and are well known to those skilled in ecules that each comprise more than one polynucleotide that the art. Such applications may be combined with other spray is specifically hybridizable to a nucleic acid molecule on insecticide applications (biologically based or otherwise) expressed in an insect pest cell. In some examples, the nucleic to enhance plant protection from the pests. acid molecule(s) comprises a polynucleotide that is specifi 0234 All references, including publications, patents, and cally hybridizable to a nucleic acid molecule expressed in a patent applications, cited herein are hereby incorporated by coleopteran pest cell. reference to the extent they are not inconsistent with the 0232. In some embodiments, a method for modulating the explicit details of this disclosure, and are so incorporated to expression of a target gene in an insect (e.g., coleopteran) pest the same extent as if each reference were individually and is provided, the method comprising: transforming a plant cell specifically indicated to be incorporated by reference and with a vector comprising a polynucleotide encoding at least were set forth in its entirety herein. The references discussed one iRNA molecule of the invention, wherein the polynucle herein are provided solely for their disclosure prior to the otide is operatively-linked to a promoter and a transcription filing date of the present application. Nothing herein is to be US 2016/0 194658 A1 Jul. 7, 2016 29 construed as an admission that the inventors are not entitled to 0245. The statistical analysis was done using JMPTM soft antedate such disclosure by virtue of prior invention. ware (SAS, Cary, N.C.). 0235. The following EXAMPLES are provided to illus 0246 The LCs (Lethal Concentration) is defined as the trate certain particular features and/or aspects. These dosage at which 50% of the test insects are killed. The GIs EXAMPLES should not be construed to limit the disclosure (Growth Inhibition) is defined as the dosage at which the to the particular features or aspects described. mean growth (e.g. live weight) of the test insects is 50% of the mean value seen in Background Check samples. EXAMPLES 0247 Replicated bioassays demonstrated that ingestion of particular samples resulted in a Surprising and unexpected Example 1 mortality and growth inhibition of corn rootworm larvae. Materials and Methods Example 2 0236 Sample Preparation and Bioassays. 0237. A number of dsRNA molecules (including those Identification of Candidate Target Genes corresponding to ncm reg1 (SEQID NO:3), incm reg2 (SEQ 0248 Multiple stages of WCR (Diabrotica virgifera vir ID NO:4), incm v1 (SEQ ID NO:5), and incm v2 (SEQ ID gifera LeConte) development were selected for pooled tran NO:6) were synthesized and purified using a MEGAS Scriptome analysis to provide candidate target gene CRIPTR) RNAi kit or HiScribe(R) T7 In Vitro Transcription sequences for control by RNAi transgenic plant insect resis Kit. The purified dsRNA molecules were prepared in TE tance technology. buffer, and all bioassays contained a control treatment con 0249. In one exemplification, total RNA was isolated from sisting of this buffer, which served as a background check for about 0.9 gm whole first-instar WCR larvae; (4 to 5 days mortality or growth inhibition of WCR (Diabrotica virgifera post-hatch; held at 16°C.), and purified using the following virgifera LeConte). The concentrations of dsRNA molecules phenol/TRI REAGENT-based method (MOLECULAR in the bioassay buffer were measured using a NANODROPTM RESEARCH CENTER, Cincinnati, Ohio): 8000 spectrophotometer (THERMO SCIENTIFIC, Wilm 0250 Larvae were homogenized at room temperature in a ington, Del.). 15 mL homogenizer with 10 mL of TRI REAGENTR) until a 0238 Samples were tested for insect activity in bioassays homogenous Suspension was obtained. Following 5 min. conducted with neonate insect larvae on artificial insect diet. incubation at room temperature, the homogenate was dis WCR eggs were obtained from CROP CHARACTERIS pensed into 1.5 mL microfuge tubes (1 mL per tube), 200LL TICS, INC. (Farmington, Minn.). of chloroform was added, and the mixture was vigorously 0239. The bioassays were conducted in 128-well plastic shaken for 15 seconds. After allowing the extraction to sit at trays specifically designed for insect bioassays (C-DINTER room temperature for 10 min, the phases were separated by NATIONAL Pitman, N.J.). Each well contained approxi centrifugation at 12,000xg at 4°C. The upper phase (com mately 1.0 mL of an artificial diet designed for growth of prising about 0.6 mL) was carefully transferred into another coleopteran insects. A 60 uL aliquot of dsRNA sample was sterile 1.5 mL tube, and an equal Volume of room temperature delivered by pipette onto the surface of the diet of each well isopropanol was added. After incubation at room temperature (40 uL/cm). dsRNA sample concentrations were calculated for 5 to 10 min, the mixture was centrifuged 8 min at as the amount of dsRNA per square centimeter (ng/cm) of 12,000xg (4° C. or 25° C.). surface area (1.5 cm) in the well. The treated trays were held 0251. The supernatant was carefully removed and dis in a fume hood until the liquid on the diet surface evaporated carded, and the RNA pellet was washed twice by vortexing or was absorbed into the diet. with 75% ethanol, with recovery by centrifugation for 5 min 0240. Within a few hours of eclosion, individual larvae at 7,500xg (4°C. or 25°C.) after each wash. The ethanol was were picked up with a moistened camel hairbrush and depos carefully removed, the pellet was allowed to air-dry for 3 to 5 ited on the treated diet (one or two larvae per well). The min, and then was dissolved in nuclease-free sterile water. infested wells of the 128-well plastic trays were then sealed RNA concentration was determined by measuring the absor with adhesive sheets of clear plastic, and vented to allow gas bance (A) at 260 nm and 280 nm. A typical extraction from exchange. Bioassay trays were held under controlled envi about 0.9 gm of larvae yielded over 1 mg of total RNA, with ronmental conditions (28°C., ~40% Relative Humidity, 16:8 an A/Aso ratio of 1.9. The RNA thus extracted was stored (Light:Dark)) for 9 days, after which time the total number of at -80° C. until further processed. insects exposed to each sample, the number of dead insects, 0252 RNA quality was determined by running an aliquot and the weight of Surviving insects were recorded. Average through a 1% agarose gel. The agarose gel Solution was made percent mortality and average growth inhibition were calcu using autoclaved 10xTAE buffer (Tris-acetate EDTA; 1 x lated for each treatment. Growth inhibition (GI) was calcu concentration is 0.04M Tris-acetate, 1 mM EDTA (ethylene lated as follows: diamine tetra-acetic acid sodium salt), pH 8.0) diluted with DEPC (diethyl pyrocarbonate)-treated water in an autoclaved container. 1x TAE was used as the running buffer. Before use, 0241 where TWIT is the Total Weight of live Insects in the the electrophoresis tank and the well-forming comb were Treatment; cleaned with RNaseAwayTM (INVITROGEN INC., Carlsbad, 0242 TNIT is the Total Number of Insects in the Treat Calif.). Two LL of RNA sample were mixed with 8 uI of TE ment, buffer (10 mM Tris HCl pH 7.0; 1 mM EDTA) and 10 uL of 0243 TWIBC is the Total Weight of live Insects in the RNA sample buffer (NOVAGENR) Catalog No. 70606: Background Check (Buffer control); and EMD4 Bioscience, Gibbstown, N.J.). The sample was heated 0244 TNIBC is the Total Number of Insects in the Back at 70° C. for 3 min, cooled to room temperature, and 5 uL ground Check (Buffer control). (containing 1 ug to 2 ug RNA) were loaded per well. Com US 2016/0 194658 A1 Jul. 7, 2016 30 mercially available RNA molecular weight markers were dsRNA that targets incinnare useful for preventing root feeding simultaneously run in separate wells for molecular size com damage by cornrootworm. Ncm dsRNA transgenes represent parison. The gel was run at 60 volts for 2 hrs. new modes of action for combining with Bacillus thuringien 0253) A normalized cDNA library was prepared from the sis insecticidal protein technology in Insect Resistance Man larval total RNA by a commercial service provider (EURO agement gene pyramids to mitigate against the development FINS MWG Operon, Huntsville, Ala.), using random prim of rootworm populations resistant to either of these rootworm ing. The normalized larval cDNA library was sequenced at /2 control technologies. plate scale by GS FLX 454 TitaniumTM series chemistry at EUROFINS MWG Operon, which resulted in over 600,000 0259 Full-length or partial clones of sequences of a reads with an average read length of 348 bp. 350,000 reads Diabrotica candidate gene, herein referred to as incm, were were assembled into over 50,000 contigs. Both the unas used to generate PCR amplicons for dsRNA synthesis. sembled reads and the contigs were converted into BLASTable databases using the publicly available program, Example 3 FORMATDB (available from NCBI). 0254 Total RNA and normalized cDNA libraries were similarly prepared from materials harvested at other WCR Amplification of Target Genes to Produce dsRNA developmental stages. A pooled transcriptome library for tar get gene screening was constructed by combining cDNA 0260 Primers were designed to amplify portions of cod library members representing the various developmental ing regions of each target gene by PCR. See Table 1. Where Stages. appropriate, a T7 phage promoter sequence (TTAATAC 0255 Candidate genes for RNAi targeting were selected GACTCACTATAGGGAGA: SEQ ID NO:7) was incorpo using information regarding lethal RNAi effects of particular rated into the 5' ends of the amplified sense or antisense genes in other insects Such as Drosophila and Tribolium. Strands. See Table 1. Total RNA was extracted from WCR These genes were hypothesized to be essential for survival using TRIZol(R) (Life Technologies, Grand Island, N.Y.), and growth in coleopteran insects. Selected target gene where WCR larvae and adults were homogenized at room homologs were identified in the transcriptome sequence data temperature in a 1.5 mL microfuge tube with 1 mL of TRI base as described below. Full-length or partial sequences of ZolR using a Pestle Motor Mixer (Cole-Parmer, Vernon Hills, the target genes were amplified by PCR to prepare templates Ill.) until a homogenous Suspension was obtained. Following for double-stranded RNA (dsRNA) production. 0256 TBLASTN searches using candidate protein coding 5 min. incubation at room temperature, the homogenate was sequences were run against BLASTable databases containing centrifuged to remove cell debris and 1 mL Supernatant was the unassembled Diabrotica sequence reads or the assembled transferred to a new tube. 200 uL of chloroform was added, contigs. Significant hits to a Diabrotica sequence (defined as and the mixture was vigorously shaken for 15 seconds. After better than e' for contigs homologies and better thane' allowing the extraction to sitat room temperature for 2-3 min, for unassembled sequence reads homologies) were confirmed the phases were separated by centrifugation at 12,000xg at 4 using BLASTX against the NCBI non-redundant database. C. The upper phase (comprising about 0.6 mL) was carefully The results of this BLASTX search confirmed that the transferred into another sterile 1.5 mL tube, and 500 uL of Diabrotica homolog candidate gene sequences identified in room temperature isopropanol was added. After incubation at the TBLASTN search indeed comprised Diabrotica genes, or room temperature for 10 min, the mixture was centrifuged 10 were the best hit to the non-Diabrotica candidate gene min at 12,000xg at 4° C. The supernatant was carefully sequence present in the Diabrotica sequences. In a few cases, removed and discarded, and the RNA pellet was washed it was clear that some of the Diabrotica contigs or unas twice by vortexing with 75% ethanol, with recovery by cen sembled sequence reads selected by homology to a non trifugation for 5 min at 7,500xg (4°C. or 25°C.) after each Diabrotica candidate gene overlapped, and that the assembly wash. The ethanol was carefully removed, the pellet was of the contigs had failed to join these overlaps. In those cases, allowed to air-dry for 3 to 5 min, and then was dissolved in SequencherTM v4.9 (GENE CODES CORPORATION, Ann nuclease-free sterile water. Arbor, Mich.) was used to assemble the sequences into longer 0261 Total RNA was then used to make first-strand cDNA contigs. with SuperScriptIII(R) First-Strand Synthesis System and 0257. A candidate target gene encoding Diabrotica incm manufacturers Oligo dT primed instructions (Life Technolo (SEQ ID NO:1 and SEQID NO:77) was identified as a gene gies, Grand Island, N.Y.). This first-strand cDNA was used as that may lead to coleopteran pest mortality, inhibition of template for PCR reactions using opposing primers posi growth or inhibition of development in WCR. tioned to amplify all or part of the native target gene sequence. 0258 Ncm dsRNA transgenes can be combined with other dsRNA was also amplified from a DNA clone comprising the dsRNA molecules to provide redundant RNAi targeting and coding region for a yellow fluorescent protein (YFP) (SEQID synergistic RNAi effects. Transgenic corn events expressing NO:8: Shagin et al. (2004) Mol. Biol. Evol. 21(5):841-50). TABLE 1. Primers and Primer Pairs used to amplify portions of coding regions of exemplary ncm target gene and YFP negative control gene.

Gene ID Primer ID Sequence

Pair 1 incrm reg1 Dvv incm-1- TTAATACGACT CACTATAGGGAGAGGCTGCGTA US 2016/0 194658 A1 Jul. 7, 2016 31

TABLE 1- Continued Primers and Primer Pairs used to amplify portions of coding regions of exemplary ncm target gene and YFP negative control gene. Gene ID Primer ID Sequence 411 AACGTTTGTAAAAG (SEO ID NO: 9) Dv V incrm-1- TTAATACGACT CACTATAGGGAGAGATGCGGCT 411 Rev CCCGAAGAATCTG (SEO ID NO : 10) Pair 2 incm reg2 Dvv ncm-2 - TTAATACGACT CACTATAGGGAGAGTTAGCATC 4O7. For TAGTCTGTGAGTGG (SEO ID NO : 11) Dvv incm-2 - TTAATACGACT CACTATAGGGAGACCCTTAGTA 4 O7. Rew AAGAAATC TTAGGCAG (SEQ ID NO: 12) Pair 3 incm v1 Dvv ncm-1 TTAATACGACTCACTATAGGGAGATTAATGTAA v1. For CCATGAACGGATTTC (SEO ID NO : 13) Dv V incrm-1 TTAATACGACT CACTATAGGGAGACAGTCATCA w1 Rew AACCAAGAGAAAG (SEQ ID NO: 14) Pair 4 incm v2 Dvv ncm-1 TTAATACGACTCACTATAGGGAGAGGCTGCGTA w2 For AACGTTTGTAAAAG (SEO ID NO: 15) Dv V incrm-1 TTAATACGACT CACTATAGGGAGAATTGGCATC w2 Rew ATCGCCAGAGAATTATTG (SEO ID NO: 16)

Pair 5 YFP YFP-F T7 TTAATACGACT CACTATAGGGAGACACCATGGG CTCCAGCGGCGCCC (SEO ID NO: 27) YFP-RT7 TTAATACGACT CACTATAGGGAGAAGATCTTGA AGGCGCTCTTCAGG (SEO ID NO : 3 O)

Example 4 Intramolecular hairpin formation by RNA primary transcripts is facilitated by arranging (within a single transcription unit) RNAi Constructs two copies of a target gene segment in opposite orientation to one another, the two segments being separated by a linker 10262) Template preparation by PCR and dsRNA synthe- sequence (e.g. SEQ ID NO:19). Thus, the primary mRNA S1S. transcript contains the two ncin gene segment sequences as 0263. A strategy used to provide specific templates for large inverted repeats of one another, separated by the linker incm and YFP dsRNA production is shown in FIG. 1. Tem sequence. A copy of a promoter (e.g. maize ubiquitin 1, U.S. plate DNAs intended for use in ncm dsRNA synthesis were Pat. No. 5,510,474; 35S from Cauliflower Mosaic Virus prepared by PCR using the primer pairs in Table 1 and (as (CaMV): Sugarcane bacilliform badnavirus (ScBV) pro PCR template) first-strand cDNA prepared from total RNA moter, promoters from rice actin genes; ubiquitin promoters; isolated from WCR eggs, first-instar larvae, or adults. For pEMU; MAS; maize H3 histone promoter; ALS promoter; each selected incrm and YFP target gene region, PCR amplifi phaseolin gene promoter; cab: rubisco; LAT52: Zm13; and/or cations introduced a T7 promoter sequence at the 5' ends of apg) is used to drive production of the primary mRNA hairpin the amplified sense and antisense strands (the YFP segment transcript, and a fragment comprising a 3' untranslated region was amplified from a DNA clone of the YFP coding region). for example but not limited to a maize peroxidase 5 gene The two PCR amplified fragments for each region of the (ZmPerS 3'UTR v2; U.S. Pat. No. 6,699.984), Atubi10, target genes were then mixed in approximately equal AtEf1, or StPinII is used to terminate transcription of the amounts, and the mixture was used as transcription template hairpin-RNA-expressing gene. for dsRNA production. See FIG. 1. The sequences of the 0266 Entry vectors plDAB126948 and plDAB126954 dsRNA templates amplified with the particular primer pairs comprise a ncm V2-RNA construct (SEQ ID NO:17) that were: SEQID NO:3 (ncm reg1), SEQID NO:4 (ncm reg2), comprises a segment of ncm (SEQID NO:1). SEQ ID NO:5 (ncm v1), SEQID NO:6 (ncm v2) and YFP 0267 Entry vectors described above are used in standard (SEQ ID NO:8). Double-stranded RNA for insect bioassay GATEWAYR recombination reactions with a typical binary was synthesized and purified using an AMBIONR MEGAS destination vector to produce incinn hairpin RNA expression CRIPTR) RNAi kit following the manufacturers instructions transformation vectors for Agrobacterium-mediated maize (INVITROGEN) or HiScribe(RT7 In Vitro Transcription Kit embryo transformations. following the manufacturer's instructions (New England 0268. The Binary destination vector comprises a herbicide Biolabs, Ipswich, Mass.). The concentrations of dsRNAs tolerance gene (aryloxyalknoate dioxygenase; AAD-1 V3) were measured using a NANODROPTM 8000 spectropho (U.S. Pat. No. 7,838,733, and Wright et al. (2010) Proc. Natl. tometer (THERMO SCIENTIFIC, Wilmington, Del.). Acad. Sci. U.S.A. 107:20240-5) under the regulation of a 0264 Construction of Plant Transformation Vectors plant operable promoter (e.g., Sugarcane bacilliform badnavi 0265 Entry vectors harboring a target gene construct for rus (Sc3V) promoter (Schenk et al. (1999) Plant Mol. Biol. hairpin formation comprising segments of ncm (SEQ ID 39:1221-30) or ZmUbi1 (U.S. Pat. No. 5,510.474)). A 5' UTR NO:1 and/or SEQID NO:77) are assembled using a combi and intron are positioned between the 3' end of the promoter nation of chemically synthesized fragments (DNA2.0, Menlo segment and the start codon of the AAD-1 coding region. A Park, Calif.) and standard molecular cloning methods. fragment comprising a 3' untranslated region from a maize US 2016/0 194658 A1 Jul. 7, 2016 32 lipase gene (ZmLip 3'UTR: U.S. Pat. No. 7,179,902) is used TABLE 3-continued to terminate transcription of the AAD-1 mRNA. 0269. A negative control binary vector that comprises a Summary of oral potency of nem dsRNA on WCR larvae (ng/cm). gene that expresses aYFP protein, is constructed by means of Gene standard GATEWAY(R) recombination reactions with a typi Name LCso Range GIso Range cal binary destination vector and entry vector. The binary destination vector comprises a herbicide tolerance gene (ary incm V2 2.52 221-2.85 18.19 1282-25.82 loxyalknoate dioxygenase; AAD-1 V3) (as above) under the expression regulation of a maize ubiquitin 1 promoter (as 0272. It has previously been suggested that certain genes above) and a fragment comprising a 3' untranslated region of Diabrotica spp. may be exploited for RNAi-mediated from a maizelipase gene (ZmLip3'UTR; as above). The entry insect control. See U.S. Patent Publication No. 2007/ Vector comprises a YFP coding region (SEQ ID NO:20) 0.124836, which discloses 906 sequences, and U.S. Pat. No. under the expression control of a maize ubiquitin 1 promoter 7,612,194, which discloses 9,112 sequences. However, it was (as above) and a fragment comprising a 3' untranslated region determined that many genes Suggested to have utility for from a maize peroxidase 5 gene (as above). RNAi-mediated insect control are not efficacious in control Example 5 ling Diabrotica. It was also determined that sequences incm reg1, incrm reg2, incm V1 and incm V2 each provide Surprising Screening of Candidate Target Genes and unexpected Superior control of Diabrotica, compared to other genes suggested to have utility for RNAi-mediated 0270 Synthetic dsRNA designed to inhibit target gene insect control. sequences identified in EXAMPLE 2 caused mortality and growth inhibition when administered to WCR in diet-based 0273 For example, annexin, beta spectrin2, and mtRP-L4 assays. Ncm reg1, incin reg2, incin V1 and incin V2 were were each suggested in U.S. Pat. No. 7,612,194 to be effica observed to exhibit greatly increased efficacy in this assay cious in RNAi-mediated insect control. SEQID NO:21 is the over other dsRNAs screened. DNA sequence of annexin region 1 (Reg. 1) and SEQ ID 0271 Replicated bioassays demonstrated that ingestion of NO:22 is the DNA sequence of annexin region 2 (Reg2). SEQ dsRNA preparations derived from ncm reg1, incinn reg2, incm ID NO:23 is the DNA sequence of beta spectrin 2 region 1 V1 and incm V2 each resulted in mortality and/or growth (Reg. 1) and SEQ ID NO:24 is the DNA sequence of beta inhibition of western cornrootworm larvae. Table 2 and Table spectrin 2 region 2 (Reg2). SEQ ID NO:25 is the DNA 3 show the results of diet-based feeding bioassays of WCR sequence of mtRP-L4 region 1 (Reg. 1) and SEQID NO:26 is larvae following 9-day exposure to these dsRNAs, as well as the DNA sequence of mtRP-L4 region 2 (Reg. 2). A YFP the results obtained with a negative control sample of dsRNA sequence (SEQID NO:8) was also used to produce dsRNA as prepared from a yellow fluorescent protein (YFP) coding a negative control. region (SEQID NO:8). 0274 Each of the aforementioned sequences was used to produce dsRNA by the methods of EXAMPLE 3. The strat TABLE 2 egy used to provide specific templates for dsRNA production is shown in FIG. 2. Template DNAs intended for use in Results of incm dsRNA diet feeding assays obtained with dsRNA synthesis were prepared by PCR using the primer western corn rootworm larvae after 9 days offeeding. ANOVA analysis found significance differences in Mean pairs in Table 4 and (as PCR template) first-strand cDNA % Mortality and Mean % Growth Inhibition (GI). Means prepared from total RNA isolated from WCR first-instar lar were separated using the Tukey-Kramer test. vae. (YFP was amplified from a DNA clone.) For each selected target gene region, two separate PCR amplifications Gene Dose Mean (% Mortality) + Mean (GI) + Name (ng/cm) N SEM* SEM were performed. The first PCR amplification introduced a T7 promoter sequence at the 5' end of the amplified sense strands. incm reg1 500 7 86.50 + 4.35 (A) 0.77 + 0.06 (A) The second reaction incorporated the T7 promoter sequence incm reg2 500 8 76.14 + 3.99 (A) 0.75 + 0.06 (A) incm V1 500 8 85.20 + 3.51 (A) 0.96+ 0.01 (A) at the 5' ends of the antisense strands. The two PCR amplified incm V2 500 4 87.10 + 3.40 (A) 0.95 + 0.03 (A) fragments for each region of the target genes were then mixed TE** O 10 19.29 +4.36 (B) -0.01 + 0.08 (B) in approximately equal amounts, and the mixture was used as WATER O 10 12.88 + 3.20 (B) 0.05 + 0.08 (B) transcription template for dsRNA production. See FIG. 2. YFP: 500 11 18.29 + 1.98 (B) 0.14 + 0.08 (B) Double-stranded RNA was synthesized and purified using an *SEM = Standard Error of the Mean. Letters in parentheses designate statistical levels. AMBIONR MEGAScript(R) RNAi kit following the manufac Levels not connected by same letter are significantly different (P<0.05). **TE = Tris HCl (1 mM) plus EDTA (0.1 mM) buffer, pH 7.2. turer's instructions (INVITROGEN). The concentrations of ***YFP = Yellow FluorescentProtein dsRNAs were measured using a NANODROPTM 8000 spec trophotometer (THERMO SCIENTIFIC, Wilmington, Del.) and the dsRNAs were each tested by the same diet-based TABLE 3 bioassay methods described above. Table 4 lists the sequences of the primers used to produce the annexin Reg1, Summary of oral potency of nem dsRNA on WCR larvae (ng/cm). annexin Reg2, beta spectrin 2 Regl, beta spectrin 2 Reg2, Gene mtRP-L4 Reg1, and mtRP-L4 Reg2 dsRNA molecules. YFP Name LCso Range GIso Range primer sequences for use in the method depicted in FIG.2 are incm reg1 7.04 4.87-9.96 4.2O 161-10.89 also listed in Table 4. Table 5 presents the results of diet-based incm reg2 17.32 11.46-25.99 8.2O 2.21-30.41 feeding bioassays of WCR larvae following 9-day exposure incm V1 2.39 2.05-2.73 17.00 7.60-38.00 to these dsRNA molecules. Replicated bioassays demon strated that ingestion of these dsRNAs resulted in no mortal US 2016/0 194658 A1 Jul. 7, 2016 33 ity or growth inhibition of western corn rootworm larvae above that seen with control samples of TE buffer, water, or YFP protein. TABLE 4 Primers and Primer Pairs used to amplify portions of coding regions of genes.

Gene (Region) Primer ID Sequence

Pair 5 YFP YFP-F T7 TTAATACGACT CACTATAGGGAGACACCATG GGCTCCAGCGGCGCCC (SEO ID NO: 27) YFP YFP-R AGATCTTGAAGGCGCTCTTCAGG (SEO ID NO: 28)

Pair 6 YFP YFP-F CACCATGGGCTCCAGCGGCGCCC (SEO ID NO: 29) YFP YFP-RT7 TTAATACGACT CACTATAGGGAGAAGATCTT GAAGGCGCTCTTCAGG (SEO ID NO : 3O)

Pair 7 annexin Ann-F1. T7 TTAATACGACT CACTATAGGGAGAGCTCCAA (Reg. 1) CAGTGGTTCCTTATC (SEO ID NO : 31) annexin Ann-R1 CTAATAATTCTTTTTTAATGTTCCTGAGG (Reg. 1) (SEQ ID NO: 32)

Pair 8 annexin Ann-F1 GCTCCAACAGTGGTTCCTTATC (SEQ ID (Reg. 1) NO: 33) annexin Ann-R1. T7 TTAATACGACT CACTATAGGGAGACTAATAA (Reg. 1) TTCTTTTTTAATGTTCCTGAGG (SEQ ID NO: 34)

Pair 9 annexin Ann-F2 T7 TTAATACGACT CACTATAGGGAGATTGTTAC (Reg 2) AAGCTGGAGAACTTCTC (SEQ ID NO : 35) annexin Ann-R2 CTTAACCAACAACGGCTAATAAGG (SEQ ID (Reg 2) NO: 36)

Pair 10 annexin Ann-F2 TTGTTACAAGCTGGAGAACTTCTC (SEO ID (Reg 2) NO: 37) annexin Ann-R2Tf TTAATACGACT CACTATAGGGAGACTTAACC (Reg 2) AACAACGGCTAATAAGG (SEQ ID NO: 38) Pair 11 beta-spect2 Betasp2-F1. T7 TTAATACGACTCACTATAGGGAGAAGATGTT (Reg. 1) GGCTGCATCTAGAGAA (SEQ ID NO: 39) beta- spect2 Beta sp2-R1 GTCCATTCGTCCATCCACTGCA (SEQ ID (Reg. 1) NO: 40) Pair 12 beta- spect2 Beta sp2-F1. AGATGTTGGCTGCATCTAGAGAA (SEO ID (Reg. 1) NO: 41) beta-spect2 Betasp2-R1. T7 TTAATACGACTCACTATAGGGAGAGTCCATT (Reg. 1) CGTCCATCCACTGCA (SEO ID NO : 42) Pair 13 beta-spect2 Betasp2-F2 T7 TTAATACGACTCACTATAGGGAGAGCAGATG (Reg 2) AACACCAGCGAGAAA (SEQ ID NO: 43) beta- spect2 Beta sp2-R2 CTGGGCAGCTTCTTGTTTCCTC (SEQ ID (Reg 2) NO: 44) Pair 14 beta- spect2 Beta sp2-F2 GCAGATGAACACCAGCGAGAAA (SEQ ID (Reg 2) NO: 45) beta-spect2 Betasp2-R2 T7 TTAATACGACTCACTATAGGGAGACTGGGCA (Reg 2) GCTTCTTGTTTCCTC (SEQ ID No. 46)

Pair 15 mtRP-L14 L4-F1. T7 TTAATACGACTCACTATAGGGAGAAGTGAAA (Reg. 1) TGTTAGCAAATATAACATCC (SEO ID NO: 47) mtRP-L14 L4-R1 ACCTCTCACTTCAAATCTTGACTTTG (SEQ ID (Reg. 1) NO: 48)

Pair 16 mtRP-L14 L4 - F1 AGTGAAATGTTAGCAAATATAACATCC (SEQ (Reg. 1) ID NO: 49) mtRP-L14 L4-R1. Tf TTAATACGACT CACTATAGGGAGAACCTCTC (Reg. 1) ACTTCAAATCTTGACTTTG (SEO ID NO: 5O)

Pair 17 mtRP-L14 L4-F2 T7 TTAATACGACT CACTATAGGGAGACAAAGTC (Reg 2) AAGATTTGAAGTGAGAGGT (SEQ ID NO: 51) mtRP-L14 L4-R2 CTACAAATAAAACAAGAAGGACCCC (SEQ ID (Reg 2) NO: 52) US 2016/0 194658 A1 Jul. 7, 2016 34

TABLE 4 - continued Primers and Primer Pairs used to amplify portions of coding regions of cenes.

Gene (Region) Primer ID Sequence Pair 18 mtRP-L14 L4-F2 CAAAGTCAAGATTTGAAGTGAGAGGT (SEQ ID (Reg 2) NO: 53) mtRP-L14 L4-R2 T7 TTAATACGACT CACTATAGGGAGACTACAAA (Reg 2) TAAAACAAGAAGGACCCC (SEQ ID NO: 54)

TABLE 5 (0279 Agrobacterium Culture. 0280. On the day of an experiment, a stock solution of Results of diet feeding assays obtained with Inoculation Medium and acetosyringone is prepared in a Vol western corn rootworm larvae after 9 days. ume appropriate to the number of constructs in the experi Mean Live ment and pipetted into a sterile, disposable, 250 mL flask. Gene Dose Larval Weight Mean % Mean Growth Inoculation Medium (Frame et al. (2011) Genetic Transfor Name (ng/cm) (mg) Mortality Inhibition mation Using Maize Immature Zygotic Embryos. IN Plant annexin-Reg 1 OOO O.S.45 O -0.262 Embryo Culture Methods and Protocols: Methods in Molecu annexin-Reg 2 OOO 0.565 O -O.301 lar Biology. T. A. Thorpe and E. C. Yeung, (Eds), Springer beta spectrin2 OOO O.340 12 -0.014 Reg 1 Science and Business Media, LLC. pp. 327-341) contained: beta spectrin2 OOO O465 18 -0.367 2.2 gm/L MS salts: 1XISU Modified MS Vitamins (Frame et Reg 2 al., ibid.) 68.4 gm/L Sucrose; 36 gm/L glucose; 115 mg/L mtRP-L4 Reg 1 OOO O.30S 4 -0.168 L-proline; and 100 mg/L myo-inositol; at pH 5.4.) Acetosy mtRP-L4 Reg 2 OOO O.30S 7 -018O ringone is added to the flask containing Inoculation Medium TE buffer O O430 13 O.OOO Water O 0.535 12 O.OOO to a final concentration of 200 uM from a 1 M stock solution YFP* * OOO O480 9 -0.386 in 100% dimethyl sulfoxide and the solution is thoroughly mixed. *TE = Tris HCl (10 mM) plus EDTA (1 mM) buffer, pH8. 0281 For each construct, 1 or 2 inoculating loops-full of YFP = Yellow FluorescentProtein Agrobacterium from the YEP plate are suspended in 15 mL of the Inoculation Medium/acetosyringone stock solution in a Example 6 sterile, disposable, 50 mL centrifuge tube, and the optical density of the solution at 550 nm (ODsso) is measured in a Production of Transgenic Maize Tissues Comprising spectrophotometer. The Suspension is then diluted to ODsso Insecticidal dsRNAs of 0.3 to 0.4 using additional Inoculation Medium/acetosy ringone mixture. The tube of Agrobacterium suspension is 0275 Agrobacterium-Mediated Transformation. then placed horizontally on a platform shaker set at about 75 0276 Transgenic maize cells, tissues, and plants that pro rpm at room temperature and shaken for 1 to 4 hours while duce one or more insecticidal dsRNA molecules (for embryo dissection is performed. example, at least one dsRNA molecule including a dsRNA 0282 Ear Sterilization and Embryo Isolation. molecule targeting a gene comprising incinn; SEQIDNO:1 and 0283 Maize immature embryos are obtained from plants SEQID NO:77) through expression of a chimeric gene sta of Zea mays inbred line B104 (Hallauer et al. (1997) Crop bly-integrated into the plant genome are produced following Science 37: 1405-1406) grown in the greenhouse and self- or Agrobacterium-mediated transformation. Maize transforma sib-pollinated to produce ears. The ears are harvested tion methods employing Superbinary or binary transforma approximately 10 to 12 days post-pollination. On the experi tion vectors are known in the art, as described, for example, in mental day, de-husked ears are Surface-sterilized by immer U.S. Pat. No. 8.304,604, which is herein incorporated by sion in a 20% solution of commercial bleach (ULTRA CLO reference in its entirety. Transformed tissues are selected by ROX(R) Germicidal Bleach, 6.15% sodium hypochlorite; with their ability to grow on Haloxyfop-containing medium and two drops of TWEEN 20) and shaken for 20 to 30 min, are screened for dsRNA production, as appropriate. Portions followed by three rinses insterile deionized water in a laminar of Such transformed tissue cultures may be presented to neo flow hood. Immature Zygotic embryos (1.8 to 2.2 mm long) nate corn rootworm larvae for bioassay, essentially as are aseptically dissected from each ear and randomly distrib described in EXAMPLE 1. uted into microcentrifuge tubes containing 2.0 mL of a Sus 0277 Agrobacterium Culture Initiation. pension of appropriate Agrobacterium cells in liquid Inocu 0278 Glycerol stocks of Agrobacterium strain DAt 13192 lation Medium with 200 uMacetosyringone, into which 2 ul cells (WO 2012/016222A2) harboring a binary transforma of 10% BREAK-THRUR S233 surfactant (EVONIK tion vector as described above (EXAMPLE 4) are streaked on INDUSTRIES: Essen, Germany) had been added. For a given AB minimal medium plates (Watson et al. (1975) J. Bacteriol. set of experiments, embryos from pooled ears are used for 123:255-64) containing appropriate antibiotics and are grown each transformation. at 20°C. for 3 days. The cultures are then streaked onto YEP 0284 Agrobacterium Co-Cultivation. plates (gm/L: yeast extract, 10; Peptone, 10; NaCl, 5) con 0285 Following isolation, the embryos are placed on a taining the same antibiotics and are incubated at 20° C. for 1 rocker platform for 5 minutes. The contents of the tube are day. then poured onto a plate of Co-cultivation Medium, which US 2016/0 194658 A1 Jul. 7, 2016

contains 4.33 gm/LMS salts; 1xISU Modified MS Vitamins: 0289 Transformed plant shoots selected by their ability to 30gm/L Sucrose: 700 mg/L L-proline; 3.3 mg/L Dicamba in grow on medium containing Haloxyflop are transplanted from KOH (3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxy PHYTATRAYSTM to small pots filled with growing medium benzoic acid); 100 mg/L myo-inositol; 100 mg/L Casein (PROMIX BX; PREMIERTECH HORTICULTURE), cov Enzymatic Hydrolysate; 15 mg/L. AgNO, 200 uMacetosy ered with cups or HUMI-DOMES (ARCO PLASTICS), and ringone in DMSO; and 3 gm/L GELZANTM, at pH 5.8. The then hardened-offin a CONVIRON growth chamber (27°C. liquid Agrobacterium suspension is removed with a sterile, day/24° C. night, 16-hour photoperiod, 50-70% RH, 200 disposable, transfer pipette. The embryos are then oriented umol mis' PAR). In some instances, putative transgenic with the scutellum facing up using sterile forceps with the aid plantlets are analyzed for transgene relative copy number by of a microscope. The plate is closed, sealed with 3MTM quantitative real-time PCR assays using primers designed to MICROPORETM medical tape, and placed in an incubator at detect the AAD1 herbicide tolerance gene integrated into the 25° C. with continuous light at approximately 60 umol maize genome. Further, DNA qPCR assays are used to detect m’s' of Photosynthetically Active Radiation (PAR). the presence of the linker sequence and/or target sequence in 0286 Callus Selection and Regeneration of Transgenic putative transformants. Selected transformed plantlets are Events. then moved into a greenhouse for further growth and testing. 0287. Following the Co-Cultivation period, embryos are 0290 Transfer and Establishment of to Plants in the transferred to Resting Medium, which is composed of 4.33 Greenhouse for Bioassay and Seed Production. gm/L MS salts: 1XISU Modified MS Vitamins; 30 gm/L 0291. When plants reach the V3-V4 stage, they are trans sucrose: 700 mg/L L-proline; 3.3 mg/L Dicamba in KOH: planted into IECUSTOMBLEND (PROFILE/METROMIX 100 mg/L myo-inositol; 100 mg/L Casein Enzymatic 160) Soil mixture and grown to flowering in the greenhouse Hydrolysate; 15 mg/L. AgNO, 0.5gm/L MES (2-(N-mor (Light Exposure Type: Photo or Assimilation; High Light pholino)ethanesulfonic acid monohydrate; PHYTOTECH Limit: 1200 PAR; 16-hour day length; 27° C. day/24° C. NOLOGIES LABR.: Lenexa, Kans.); 250 mg/L Carbenicil night). lin; and 2.3 gm/L GELZANTM: at pH 5.8. No more than 36 0292 Plants to be used for insect bioassays are trans embryos are moved to each plate. The plates are placed in a planted from small pots to TINUSTM 350-4 ROOTRAIN clear plastic box and incubated at 27°C. with continuous light ERS(R) (SPENCER-LEMAIRE INDUSTRIES, Acheson, at approximately 50 umol m°s' PAR for 7 to 10 days. Alberta, Canada;) (one plant per event per Callused embryos are then transferred (<18/plate) onto Selec ROOTRAINER(R). Approximately four days after trans tion Medium I, which is comprised of Resting Medium planting to ROOTRAINERS(R), plants are infested for bioas (above) with 100 nM R-Haloxyfop acid (0.0362 mg/L; for Say. selection of calli harboring the AAD-1 gene). The plates are 0293 Plants of the T generation are obtained by pollinat returned to clear boxes and incubated at 27°C. with continu ing the silks of To transgenic plants with pollen collected from ous light at approximately 50 umol ms' PAR for 7 days. plants of non-transgenic inbred line B104 or other appropri Callused embryos are then transferred (<12/plate) to Selec ate pollen donors, and planting the resultant seeds. Reciprocal tion Medium II, which is comprised of Resting Medium crosses are performed when possible. (above) with 500 nM R-Haloxyfop acid (0.181 mg/L). The plates are returned to clear boxes and incubated at 27°C. with Example 7 continuous light at approximately 50 umolm's PAR for 14 days. This selection step allows transgenic callus to further Molecular Analyses of Transgenic Maize Tissues proliferate and differentiate. 0288 Proliferating, embryogenic calli are transferred (<9/ 0294 Molecular analyses (e.g. RNA qPCR) of maize tis plate) to Pre-Regeneration medium. Pre-Regeneration Sues are performed on samples from leaves that are collected Medium contains 4.33 gm/LMS salts: 1XISU Modified MS from greenhouse grown plants on the day before or same days Vitamins; 45 gm/L sucrose; 350 mg/L L-proline; 100 mg/L that root feeding damage is assessed. myo-inositol; 50 mg/L Casein Enzymatic Hydrolysate; 1.0 0295 Results of RNA qPCR assays for the target gene are mg/L AgNO, 0.25 gm/L MES: 0.5 mg/L naphthaleneacetic used to validate expression of the transgene. Results of RNA acid in NaOH: 2.5 mg/L abscisic acid in ethanol; 1 mg/L qPCR assay for intervening sequence between repeat 6-benzylaminopurine; 250 mg/L Carbenicillin; 2.5 gm/L sequences (which is integral to the formation of dsRNA hair GELZANTM; and 0.181 mg/L Haloxyfop acid; at pH 5.8. The pin molecules) in expressed RNAs can also be used to vali plates are stored in clear boxes and incubated at 27°C. with date the presence of hairpin transcripts. Transgene RNA continuous light at approximately 50 umol m°s' PAR for 7 expression levels are measured relative to the RNA levels of days. Regenerating calli are then transferred (<6/plate) to an endogenous maize gene. Regeneration Medium in PHYTATRAYSTM (SIGMA-ALD 0296 DNA qPCR analyses to detect a portion of the RICH) and incubated at 28°C. with 16 hours light/8 hours AAD1 coding region in genomic DNA are used to estimate dark per day (at approximately 160 umolm’s' PAR) for 14 transgene insertion copy number. Samples for these analyses days or until shoots and roots develop. Regeneration Medium are collected from plants grown in environmental chambers. contains 4.33 gm/LMS salts; 1xISU Modified MS Vitamins: Results are compared to DNA qPCR results of assays 60 gm/L sucrose; 100 mg/L myo-inositol; 125 mg/L Carbe designed to detect a portion of a single-copy native gene, and nicillin; 3 gm/L GELLANTM gum; and 0.181 mg/L R-Ha simple events (having one or two copies of the transgenes) are loxyfop acid; at pH 5.8. Small shoots with primary roots are advanced for further studies in the greenhouse. Results are then isolated and transferred to Elongation Medium without compared to DNA qPCR results of assays designed to detect selection. Elongation Medium contains 4.33 gm/LMS salts: a portion of a single-copy native gene, and simple events (one 1xISU Modified MS Vitamins; 30 gm/L sucrose; and 3.5 or two copies of the transgenes) are advanced for further gm/L GELRITETM: at pH 5.8. studies. US 2016/0 194658 A1 Jul. 7, 2016 36

0297. Additionally, qPCR assays designed to detect a por (SEQ ID NO:59) and TIPmxR (SEQ ID NO:60), and Probe tion of the spectinomycin-resistance gene (SpecR; harbored HXTIP (SEQID NO:61) labeled with HEX (hexachlorofluo on the binary vector plasmids outside of the T-DNA) are used rescein) are used. to determine if the transgenic plants contained extraneous 0302 All assays include negative controls of no-template integrated plasmid backbone sequences. (mix only). For the standard curves, a blank (water in source 0298 RNA Transcript Expression Level: well) is also included in the source plate to check for sample 0299 target qPCR. Callus cell events or transgenic plants cross-contamination. Primer and probe sequences are set are analyzed by real time quantitative PCR (qPCR) of the forth in Table 7. Reaction components recipes for detection of the various transcripts are disclosed in Table 8, and PCR target sequence to determine the relative expression level of reactions conditions are summarized in Table 9. The FAM the transgene, as compared to the transcript level of an inter (6-Carboxy Fluorescein Amidite) fluorescent moiety is nal maize gene (SEQ ID NO:55: GENBANKAccession No. excited at 465 nm and fluorescence is measured at 510 nm, the BTO69734), which encodes a TIP41-like protein (i.e., a maize corresponding values for the HEX (hexachlorofluorescein) homolog of GENBANKAccession No. AT4G34270; having fluorescent moiety are 533 nm and 580 nm. TABLE 7 Oligonucleotide sequences for molecular analyses of transcript levels in transgenic maize. Target Oligonucleotide Sequence Nom HpNcm1v2 TGCTGCTTGTGCTTGTATTATTG (SEQ ID No. 57) FWD Set 1

Nom HpNCM1 v2 GGCATCATCGCCAGAGAATTA (SEO ID NO : 58 REW Set 1

Nom HpNcm1v2 PRB A56 Set 1 FAM/ACTTGCACA/ZEN/GCAAACCTCTACCTCT/3 (FAM-Probe) IABkFOA

TIP41, TIPmxF TGAGGGTAATGCCAACTGGTT (SEO ID NO. 59

TIP41, TIPmxR GCAATGTAACCGAGTGTCTCTCAA (SEO ID NO : 6O)

TIP41. HXTIP TTTTTGGCTTAGAGTTGATGGTGTACTGATGA (SEQ (HEX-Probe) ID NO: 61) *TIP41-like protein. a tBLASTX score of 74% identity). RNA is isolated using TABLE 8 Norgen BioTek Total RNA Isolation Kit (Norgen, Thorold, ON). The total RNA is subjected to an On-Column DNase1 PCR reaction recipes for transcript detection. treatment according to the kits suggested protocol. The RNA Cl TIP-like Gene is then quantified on a NANODROP8000 spectrophotometer Component Final Concentration (THERMO SCIENTIFIC) and concentration is normalized Roche Buffer 1X 1X to 50 ng/uL. First strand cDNA is prepared using a High HpNcm1 v2 FWD Set1 0.4 IM O Capacity cDNA synthesis kit (INVITROGEN) in a 10 ul HpNcm1 v2 REV Set1 0.4 IM O HpNcm1 v2 PRB Set1 (FAM) 0.2 IM O reaction volume with 5 uL denatured RNA, substantially HEXtipZMF O 0.4 M according to the manufacturer's recommended protocol. The HEXtipZM R O 0.4 M protocol is modified slightly to include the addition of 10 LIL HEXtipZMP (HEX) O 0.2M of 100 uM T20VN oligonucleotide (IDT) (SEQ ID NO:56: cDNA (2.0 L) NA NA TTTTTTTTTTTTTTTTTTTTVN, where Vis A, C, or G, and Water To 10 L To 10 L N is A, C, G, or T/U) into the 1 mL tube of random primer stock mix, in order to prepare a working stock of combined random primers and oligo dT. TABLE 9 0300 Following cDNA synthesis, samples are diluted 1:3 Thermocycler conditions for RNA qPCR. with nuclease-free water, and stored at -20°C. until assayed. Target gene and TIP41-like Gene Detection 0301 Separate real-time PCR assays for the target gene Process Temp. Time No. Cycles and TIP41-like transcript are performed on a LIGHTCY Target Activation 95° C. 10 min 1 CLERTM 480 (ROCHE DIAGNOSTICS, Indianapolis, Ind.) Denature 95° C. 10 sec 40 Extend 60° C. 40 sec in 10 LL reaction Volumes. For the target gene assay, reactions Acquire FAM or HEX 72° C. 1 Sec are run with Primers HpNcm1 v2 FWD Set 1 (SEQID NO:57) Cool 40° C. 10 sec 1 and HpNCM1 V2 REV Sea (SEQ ID NO:58), and an IDT Custom Oligo probe Hpn.cnlv2 PRB Set1, labeled with FAM and double quenched with Zen and Iowa Black quench (0303 Data is analyzed using LIGHTCYCLERTM Soft ers. For the TIP41-like reference gene assay, primers TIPmxF ware v1.5 by relative quantification using a second derivative US 2016/0 194658 A1 Jul. 7, 2016 37 max algorithm for calculation of Cq values according to the ization, the blot is subjected to DIG washes, wrapped, Supplier's recommendations. For expression analyses, exposed to film for 1 to 30 minutes, then the film is developed, expression values are calculated using the AACt method (i.e., all by methods recommended by the supplier of the DIG kit. 2-(CdTARGET-Cd REF)), which relies on the comparison 0309 Transgene Copy Number Determination. of differences of Cq values between two targets, with the base 0310 Maize leaf pieces approximately equivalent to 2 leaf value of 2 being selected under the assumption that, for opti punches are collected in 96-well collection plates mized PCR reactions, the product doubles every cycle. (QIAGENTM). Tissue disruption is performed with a 0304 Hairpin transcript size and integrity: Northern Blot KLECKOTM tissue pulverizer (GARCIA MANUFACTUR Assay. In some instances, additional molecular characteriza ING, Visalia, Calif.) in BIOSPRINT96TM AP1 lysis buffer tion of the transgenic plants is obtained by the use of Northern (supplied with a BIOSPRINT96TM PLANT KIT; Blot (RNA blot) analysis to determine the molecular size of QIAGENTM) with one stainless steel bead. Following tissue the ncm hairpin RNA in transgenic plants expressing ancm maceration, genomic DNA (gDNA) is isolated in high hairpin dsRNA. throughput format using a BIOSPRINT96TM PLANT KIT 0305 All materials and equipment are treated with and a BIOSPRINT96TM extraction robot. Genomic DNA is RNASEZAPTM (AMBION/INVITROGEN) before use. Tis diluted 1:3 DNA: water prior to setting up the qPCR reaction. sue samples (100 mg to 500 mg) are collected in 2 mL SAFE 0311 qPCRAnalysis. LOCK EPPENDORF tubes, disrupted with a KLECKOTM 0312 Transgene detection by hydrolysis probe assay is tissue pulverizer (GARCIA MANUFACTURING, Visalia, performed by real-time PCR using a LIGHTCYCLERR480 Calif.) with three tungsten beads in 1 mL TRIZOL (INVIT system. Oligonucleotides to be used in hydrolysis probe ROGEN) for 5 min, then incubated at room temperature (RT) assays to detect the target gene (e.g. incm), the linker sequence for 10 min. Optionally, the samples are centrifuged for 10 min (e.g., SEQID NO:19), and/or to detect a portion of the SpecR at 4°C. at 11,000 rpm and the supernatant is transferred into gene (i.e. the spectinomycin resistance gene borne on the a fresh 2 mL SAFELOCK EPPENDORF tube. After 200 uL binary vector plasmids: SEQ ID NO:73; SPC1 oligonucle of chloroform are added to the homogenate, the tube is mixed otides in Table 10), are designed using LIGHTCYCLER(R) by inversion for 2 to 5 min, incubated at RT for 10 minutes, PROBE DESIGN SOFTWARE 2.0. Further, oligonucle and centrifuged at 12,000xg for 15 min at 4°C. The top phase otides to be used in hydrolysis probe assays to detect a seg is transferred into asterile 1.5 mL EPPENDORF tube, 600 uL ment of the AAD-1 herbicide tolerance gene (SEQID NO:67: of 100% isopropanol are added, followed by incubation at RT GAAD 1 oligonucleotides in Table 10) are designed using for 10 minto 2 hr, then centrifuged at 12,000xg for 10 minat PRIMER EXPRESS software (APPLIED BIOSYSTEMS). 4 to 25°C. The supernatant is discarded and the RNA pellet is Table 10 shows the sequences of the primers and probes. washed twice with 1 mL of 70% ethanol, with centrifugation Assays are multiplexed with reagents for an endogenous at 7,500xg for 10 min at 4 to 25° C. between washes. The maize chromosomal gene (Invertase (SEQID NO:64: GEN ethanol is discarded and the pellet is briefly air dried for 3 to BANKAccession No: U16123; referred to herein as IVR1), 5 min before resuspending in 50 L of nuclease-free water. which serves as an internal reference sequence to ensure (0306 Total RNA is quantified using the NANODROP gDNA is present in each assay. For amplification, LIGHTCY 8000(R) (THERMO-FISHER) and samples are normalized to CLER(R480 PROBES MASTER mix (ROCHE APPLIED 5ug/10 ul. 10 uL of glyoxal (AMBION/INVITROGEN) are SCIENCE) is prepared at 1x final concentration in a 10 uL then added to each sample. Five to 14 ng of DIG RNA stan Volume multiplex reaction containing 0.4LM of each primer dard marker mix (ROCHEAPPLIED SCIENCE, Indianapo and 0.2 LM of each probe (Table 11). A two-step amplifica lis, Ind.) are dispensed and added to an equal Volume of tion reaction is performed as outlined in Table 12. Fluoro glyoxal. Samples and marker RNAs are denatured at 50° C. phore activation and emission for the FAM- and HEX-labeled for 45 min and stored on ice until loading on a 1.25% probes are as described above: CY5 conjugates are excited SEAKEM GOLD agarose (LONZA, Allendale, N.J.) gel in maximally at 650 nm and fluoresce maximally at 670 nm. NORTHERNMAX 10x glyoxal running buffer (AMBION/ 0313 Cp scores (the point at which the fluorescence signal INVITROGEN). RNAs are separated by electrophoresis at 65 crosses the background threshold) are determined from the volts/30 mA for 2 hr and 15 min. real time PCR data using the fit points algorithm (LIGHTCY 0307 Following electrophoresis, the gel is rinsed in CLER(R) SOFTWARE release 1.5) and the Relative Quant 2xSSC for 5 min and imaged on a GEL DOC station (BIO module (based on the AACt method). Data are handled as RAD, Hercules, Calif.), then the RNA is passively transferred described previously (above: RNA qPCR). to a nylon membrane (MILLIPORE) overnight at RT, using 10xSSC as the transfer buffer (20xSSC consists of 3 M TABL E 1O sodium chloride and 300 mM trisodium citrate, pH 7.0). Following the transfer, the membrane is rinsed in 2xSSC for Sequences of primers and probes (with fluorescent 5 minutes, the RNA is UV-crosslinked to the membrane conjugate) for gene copy number determinations and (AGILENT/STRATAGENE), and the membrane is allowed binary vector plasmid backbone detection. to dry at room temperature for up to 2 days. Name Sequence 0308. The membrane is pre-hybridized in ULTRAHYBTM buffer (AMBION/INVITROGEN) for 1 to 2 hr. The probe GAAD1-F TGTTCGGTTCCCTCTACCAA (SEO ID NO : 65) consists of a PCR amplified product containing the sequence GAAD1-R CAACATCCATCACCTTGACTGA (SEO ID NO : 66) of interest, (for example, the antisense sequence portion of SEQID NO:17, as appropriate) labeled with digoxygenin by GAAD1-P CACAGAACCGTCGCTTCAGCAACA (SEO ID NO : 67) means of a ROCHE APPLIED SCIENCE DIG procedure. (FAM) Hybridization in recommended buffer is overnight at a tem IVR1-F TGGCGGACGACGACTTGT (SEO ID NO : 68) perature of 60° C. in hybridization tubes. Following hybrid US 2016/0 194658 A1 Jul. 7, 2016

TABLE 1 O - continued feeding various plant tissues or tissue pieces derived from a plant producing an insecticidal dsRNA to target insects in a Sequences of primers and probes (with fluorescent conjugate) for gene copy number determinations and controlled feeding environment. Alternatively, extracts are binary vector plasmid backbone detection. prepared from various plant tissues derived from a plant pro ducing the insecticidal dsRNA, and the extracted nucleic Name Sequence acids are dispensed on top of artificial diets for bioassays as previously described herein. The results of such feeding IVR1-R AAAGTTTGGAGGCTGCCGT (SEO ID NO: 69) assays are compared to similarly conducted bioassays that IVR1-P CGAGCAGACCGCCGTGTACTTCTACC (SEO ID NO : 7 O) employ appropriate control tissues from host plants that do (HEX) not produce an insecticidal dsRNA, or to other control samples. Growth and Survival of target insects on the test diet SPC1A CTTAGCTGGATAACGCCAC (SEO ID NO : 71.) is reduced compared to that of the control group. SPC1S GACCGTAAGGCTTGATGAA (SEO ID NO: 72) 0316 Insect Bioassays with Transgenic Maize Events. TOSPEC CGAGATTCTCCGCGCTGTAGA (SEO ID NO: 73) 0317. Two western corn rootworm larvae (1 to 3 days old) (CYsk) hatched from washed eggs are selected and placed into each Loop F GGAACGAGCTGCTTGCGTAT (SEO ID NO : 74) well of the bioassay tray. The wells are then covered with a “PULL N' PEEL tab cover (BIO-CV-16, BIO-SERV) and Loop R. CACGGTGCAGCTGATTGATG (SEO ID NO : 75) placed in a 28°C. incubator with an 18 hr/6 hr light/dark Loop. FAM TCCCTTCCGTAGTCAGAG (SEQ ID NO: 76) cycle. Nine days after the initial infestation, the larvae are assessed for mortality, which is calculated as the percentage CY5 = Cyanine-5 of dead insects out of the total number of insects in each treatment. Significant mortality is observed. The insect TABLE 11 samples are frozen at -20° C. for two days, then the insect larvae from each treatment are pooled and weighed. The Reaction components for gene copy number percent of growth inhibition is calculated as the mean weight analyses and plasmid backbone detection. of the experimental treatments divided by the mean of the Amt. Final average weight of two control well treatments. The data are Component (LL) Stock Concentration expressed as a Percent Growth Inhibition (of the negative 2x Buffer S.O 2x 1x controls). Mean weights that exceed the control mean weight Appropriate Forward Primer 0.4 10 M 0.4 are normalized to Zero. Significant growth inhibition is Appropriate Reverse Primer 0.4 10 M 0.4 observed. Appropriate Probe 0.4 5 M O.2 IVR1-Forward Primer 0.4 10 M 0.4 0318 Insect Bioassays in the Greenhouse. IVR1-Reverse Primer 0.4 10 M 0.4 IVR1-Probe 0.4 5 M O.2 0319 Western corn rootworm (WCR, Diabrotica vir HO O.6 NA* NA gifera virgifera LeConte) eggs are received in soil from gDNA 2.0 ND* * ND CROP CHARACTERISTICS (Farmington, Minn.). WCR Total 1O.O eggs are incubated at 28°C. for 10 to 11 days. Eggs are *NA = Not Applicable washed from the soil, placed into a 0.15% agar solution, and NTD = Not Determined the concentration is adjusted to approximately 75 to 100 eggs per 0.25 mL aliquot. A hatch plate is set up in a Petridish with an aliquot of egg Suspension to monitor hatch rates. TABLE 12 0320. The soil around the maize plants growing in Thermocycler conditions for DNA qPCR. ROOTRANERSR) is infested with 150 to 200 WCReggs. The Genomic copy number analyses insects are allowed to feed for 2 weeks, after which time a Process Temp. Time No. Cycles “Root Rating is given to each plant. A Node-Injury Scale is utilized for grading, essentially according to Oleson et al. Target Activation 95° C. 10 min 1 Denature 95° C. 10 sec 40 (2005) J. Econ. Entomol. 98: 1-8. Plants passing this bioassay, Extend & Acquire 60° C. 40 sec showing reduced injury, are transplanted to 5-gallon pots for FAM, HEX, or CY5 seed production. Transplants are treated with insecticide to Cool 40° C. 10 sec 1 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 Example 8 T and Subsequent generations of plants. Bioassay of Transgenic Maize 0321 Transgenic negative control plants are generated by transformation with vectors harboring genes designed to pro 0314 Insect Bioassays. duce a yellow fluorescent protein (YFP). Non-transformed 0315 Bioactivity of dsRNA of the subject invention pro negative control plants are grown from seeds of parental corn duced in plant cells is demonstrated by bioassay methods. varieties from which the transgenic plants are produced. Bio See, e.g., Baum et al. (2007) Nat. Biotechnol. 25(11):1322 assays are conducted with negative controls included in each 1326. One is able to demonstrate efficacy, for example, by set of plant materials. US 2016/0 194658 A1 Jul. 7, 2016 39

Example 9 0326 Phenotypic Comparison of Transgenic RNAi Lines and Nontransformed Zea mays. Transgenic Zea Mays Comprising Coleopteran Pest 0327 Target coleopteran pest genes or sequences selected Sequences for creating hairpin dsRNA have no similarity to any known 0322 10-20 transgenic To Zea mays plants are generated plant gene sequence. Hence, it is not expected that the pro as described in EXAMPLE 6. A further 10-20T Zea mays duction or the activation of (systemic) RNAi by constructs independent lines expressing hairpin dsRNA for an RNAi targeting these coleopteran pest genes or sequences will have construct are obtained for corn rootworm challenge. Hairpin any deleterious effect on transgenic plants. However, devel dsRNA areas set forthin SEQID NO:17, or otherwise further opment and morphological characteristics of transgenic lines comprising SEQ ID NO:1 or SEQ ID NO:77. Additional are compared with non-transformed plants, as well as those of hairpin dsRNAs are derived, for example, from coleopteran transgenic lines transformed with an “empty vector having pest sequences such as, for example, Caf1-180 (U.S. Patent no hairpin-expressing gene. Plant root, shoot, foliage and Application Publication No. 2012/0174258), VatpaseC (U.S. reproduction characteristics are compared. Plant shoot char Patent Application Publication No. 2012/0174259), Rhol acteristics Such as height, leaf numbers and sizes, time of (U.S. Patent Application Publication No. 2012/0174260), flowering, floral size and appearance are recorded. In general, VatpaseH (U.S. Patent Application Publication No. 2012/ there are no observable morphological differences between 0198586), PPI-87B (U.S. Patent Application Publication No. transgenic lines and those without expression of target iRNA 2013/0091600), RPA70 (U.S. Patent Application Publication molecules when cultured in vitro and in Soil in the glasshouse. No. 2013/009 1601), RPS6 (U.S. Patent Application Publica tion No. 2013/0097730), ROP (U.S. patent application Ser. Example 10 No. 14/577,811), RNAPII140 (U.S. patent application Ser. No. 14/577,854), Drea. (U.S. patent application Ser. No. Transgenic Zea Mays Comprising a Coleopteran Pest 14/705,807), COPI alpha (U.S. Patent Application No. Sequence and Additional RNAi Constructs 62/063.199), COPI beta (U.S. Patent Application No. 62/063, 0328. A transgenic Zea may’s plant comprising a heterolo 203), COPI gamma (U.S. Patent Application No. 62/063, gous coding sequence in its genome that is transcribed into an 192), or COPI delta (U.S. Patent Application No. 62/063, iRNA molecule that targets an organism other than a 216). These are confirmed through RT-PCR or other coleopteran pest is secondarily transformed via Agrobacte molecular analysis methods. rium or WHISKERSTM methodologies (see Petolino and 0323 Total RNA preparations from selected independent Arnold (2009) Methods Mol. Biol. 526:59-67) to produce one T lines are optionally used for RT-PCR with primers or more insecticidal dsRNA molecules (for example, at least designed to bind in the linker of the hairpin expression cas one dsRNA molecule including a dsRNA molecule targeting sette in each of the RNAi constructs. In addition, specific a gene comprising SEQ ID NO:1 or SEQID NO:77). Plant primers for each target gene in an RNAi construct are option transformation plasmid vectors prepared essentially as ally used to amplify and confirm the production of the pre described in EXAMPLE 4 are delivered via Agrobacterium or processed mRNA required for siRNA production in planta. WHISKERSTM-mediated transformation methods into maize The amplification of the desired bands for each target gene Suspension cells or immature maize embryos obtained from a confirms the expression of the hairpin RNA in each trans transgenic Hi II or B104 Zea mays plant comprising a heter genic Zea mays plant. Processing of the dsRNA hairpin of the ologous coding sequence in its genome that is transcribed into target genes into siRNA is Subsequently optionally confirmed an iRNA molecule that targets an organism other than a in independent transgenic lines using RNA blot hybridiza coleopteran pest. tions. 0324 Moreover, RNAi molecules having mismatch Example 11 sequences with more than 80% sequence identity to target genes affect corn rootworms in a way similar to that seen with Transgenic Zea Mays Comprising an RNAi RNAi molecules having 100% sequence identity to the target Construct and Additional Coleopteran Pest Control genes. The pairing of mismatch sequence with native Sequences sequences to form a hairpin dsRNA in the same RNAi con struct delivers plant-processed siRNAs capable of affecting 0329. A transgenic Zea may’s plant comprising a heterolo the growth, development, and viability offeeding coleopteran gous coding sequence in its genome that is transcribed into an pests. iRNA molecule that targets a coleopteran pest organism (for 0325 In planta delivery of dsRNA, siRNA, or miRNA example, at least one dsRNA molecule including a dsRNA corresponding to target genes and the Subsequent uptake by molecule targeting a gene comprising SEQID NO:1 or SEQ coleopteran pests through feeding results in down-regulation ID NO:77) is secondarily transformed via Agrobacterium or of the target genes in the coleopteran pest through RNA WHISKERSTM methodologies (see Petolino and Arnold mediated gene silencing. When the function of a target gene is (2009) Methods Mol. Biol. 526:59-67) to produce one or important at one or more stages of development, the growth more insecticidal protein molecules, for example, Cry3, and/or development of the coleopteran pest is affected, and in Cry34 and Cry35 insecticidal proteins. Plant transformation the case of at least one of WCR, NCR, SCR, MCR, D. bal plasmid vectors prepared essentially as described in teata LeConte, D. u. tenella, D. speciosa Germar, D. u. EXAMPLE 4 are delivered via Agrobacterium or WHIS undecimpunctata Mannerheim, and Melligethes aeneus Fab KERSTM-mediated transformation methods into maize sus ricius, leads to failure to successfully infest, feed, and/or pension cells or immature maize embryos obtained from a develop, or leads to death of the coleopteran pest. The choice transgenic B104 Zea mays plant comprising a heterologous of target genes and the Successful application of RNAi are coding sequence in its genome that is transcribed into an then used to control coleopteran pests. iRNA molecule that targets a coleopteran pest organism. US 2016/0 194658 A1 Jul. 7, 2016 40

Doubly-transformed plants are obtained that produce iRNA Example 13 molecules and insecticidal proteins for control of coleopteran pests. Mortality of Pollen Beetle (Melligethes Aeneus) Following Treatment with Ncm RNAi Example 12 0338 Gene-specific primers including the T7 polymerase promoter sequence at the 5' end were used to create PCR Pollen Beetle Transcriptome products of approximate 500 bp by PCR (SEQ ID NOs:91 92). PCR fragments were cloned in the pGEMT easy vector 0330. Insects: according to the manufacturer's protocol and sent to a 0331 Larvae and adult pollen beetles were collected from sequencing company to verify the sequence. The dsRNA was fields with flowering rapeseed plants (Giessen, Germany). then produced by the T7 RNA polymerase (MEGA script(R) Young adult beetles (each per treatment group: n=20, 3 rep RNAi Kit, Applied Biosystems) from a PCR construct gen licates) were challenged by injecting a mixture of two differ erated from the sequenced plasmid according to the manu ent bacteria (Staphylococcus aureus and Pseudomonas facturer's protocol. aeruginosa), one yeast (Saccharomyces cerevisiae) and bac 0339 Injection of ~100 mL dsRNA (1 lug/LL) into larvae terial LPS. Bacterial cultures were grown at 37° C. with and adult beetles was performed with a micromanipulator agitation, and the optical density was monitored at 600 nm under a dissecting Stereomicroscope (n=10.3 biological rep (OD600). The cells were harvested at OD600-1 by centrifu lications). Animals were anaesthetized on ice before they gation and resuspended in phosphate-buffered saline. The were affixed to double-stick tape. Controls received the same mixture was introduced ventrolaterally by pricking the abdo volume of water. A negative control dsRNA of IMPI (insect men of pollen beetle imagoes using a dissecting needle metalloproteinase inhibitor gene of the lepidopteran Galleria dipped in an aqueous solution of 10 mg/ml LPS (purified E. mellonella) were conducted. All controls in all stages could coli endotoxin; Sigma, Taufkirchen, Germany) and the bac not be tested due to a lack of animals. terial and yeast cultures. Along with the immune challenged 0340 Pollen beetles were maintained in Petri dishes with beetles naive beetles and larvae were collected (n=20 per and dried pollen and a wet tissue. The larvae were reared in plastic 3 replicates each) at the same time point. boxes on inflorescence of canola in an agar/water media. 0332 RNA. Isolation: 0333 Total RNA was extracted 8 h after immunization TABLE 13 from frozen beetles and larvae using TriReagent (Molecular Research Centre, Cincinnati, Ohio, USA) and purified using Results of adult pollen beetle injection bioassay. the RNeasy Micro Kit (Qiagen, Hilden, Germany) in each % Survival Mean SD case following the manufacturers’ guidelines. The integrity of the RNA was verified using an Agilent 2100 Bioanalyzer and Treatment Day 0 Day 2 Day 4 Day 6 Day 8 a RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, Cl 1OOO 83 - 21 80 - 17 6715 60 - 17 Calif., USA). The quantity of RNA was determined using a Water 1OOO 100 - O 1OOO 1OOO 1OOO Nanodrop ND-1000 spectrophotometer. RNA was extracted from each of the adult immune-induced treatment groups, Day 10 Day 12 Day 14 Day 16 adult control groups, and larval groups individually and equal Cl 37 - 15 33 - 15 30, 17 13 6 amounts of total RNA were subsequently combined in one Water 93 - 12 90 - 10 87 - 12 80 - 10 pool per sample (immune-challenged adults, control adults and larvae) for sequencing. Standard deviation 0334 Transcriptome Information: 0335 RNA-Seq data generation and assembly Single-read TABLE 14 100-bp RNA-Seq was carried out separately on 5 ug total Results of larval pollen beetle iniection bioassay. RNA isolated from immune-challenged adult beetles, naive (control) adult beetles and untreated larvae. Sequencing was % Survival Mean + SD* carried out by Eurofins MWG Operon using the Illumina HiSeq-2000 platform. This yielded 20.8 million reads for the Treatment Day 0 Day 2 Day 4 Day 6 adult control beetle sample, 21.5 million reads for the LPS Cl 1OOO 73 - 15 SO 10 37 2S challenged adult beetle sample and 25.1 million reads for the Negative 1OOO 1OOO 97 6 73 21 larval sample. The pooled reads (67.5 million) were control assembled using Velvet/Oases assembler software (M. H. Standard deviation Schulz et al. (2012) Bioinformatics. 28:1086–92: Zerbino & Controls were performed on a different date due to the limited availability of insects. E. Birney (2008) Genome Research. 18:821-9). The tran Scriptome contained 55648 sequences. 0341 Feeding Bioassay: Beetles were kept without access to water in empty falcon tubes 24 h before treatment. A 0336 Pollen Beetle incm Identification: droplet of dsRNA (~5uL) was placed in a small Petridish and 0337 Athlastn search of the transcriptome was used to 5 to 8 beetles were added to the Petri dish. Animals were identify matching contigs. As a query the peptide sequence of observed under a stereomicroscope and those that ingested incm from Tribolium Castaneum was used (Genbank dsRNA containing diet solution were selected for the bioas XM 001811253.1). GAPS (Bonfield J K & Whitwham say. Beetles were transferred into Petri dishes with dried (2010). Bioinformatics 26: 1699-1703) was used for verifi pollen and a wet tissue. Controls received the same volume of cation of sequences. water. A negative control dsRNA of IMPI (insect metallopro US 2016/0 194658 A1 Jul. 7, 2016 teinase inhibitor gene of the lepidopteran Galleria mel regime set at 25°C., photoperiod of 16 hours light and 8 hours lonella) was conducted. All controls in all stages could not be dark for 5 days of germination. tested due to a lack of animals. 0348 Pre-Treatment: 0349. On day 5, hypocotyl segments of about 3 mm in TABLE 1.5 length are aseptically excised, the remaining root and shoot sections are discarded (drying of hypocotyl segments is pre Results of adult feeding bioassay. vented by immersing the hypocotyls segments into 10 mL of % Survival Mean SD sterile milliOTM water during the excision process). Hypo cotyl segments are placed horizontally on sterile filter paper Treatment Day 0 Day 2 Day 4 Day 6 Day 8 on callus induction medium, MSK1D1 (MS. 1 mg/L kinetin, Cl 1OOO 1OOO 1OOO 93 6 93 6 1 mg/L 2,4-D, 3.0% sucrose, 0.7% phytagar) for 3 days Negative 1OOO 935.8 90 - 10 875.8 835.8 pre-treatment in a PercivalTM growth chamber with growth control regime of 22-23°C., and a photoperiod of 16 hours light, 8 Water 1OOO 1OOO 1OOO 933.8 93 - 3.8 hours dark. Day 10 Day 12 Day 14 Day 16 0350 Co-Cultivation with Agrobacterium: 0351. The day before Agrobacterium co-cultivation, Cl 93 6 83 12 83 - 12 83 12 flasks of YEP medium containing the appropriate antibiotics, Negative 80 - 10 80 - 10 80 - 10 77 12 control are inoculated with the Agrobacterium strain containing the Water 933.8 87 - 10 80 - 13 8013 binary plasmid. Hypocotyl segments are transferred from filter paper callus induction medium, MSK1D1 to an empty Standard deviation 100x25mm PetriTM dishes containing 10 mL of liquid M-me Controls were performed on a different date due to the limited availability of insects. dium to prevent the hypocotyl segments from drying. A 0342. While the present disclosure may be susceptible to spatula is used at this stage to scoop the segments and transfer various modifications and alternative forms, specific embodi the segments to new medium. The liquid M-medium is ments have been described by way of example in detail removed with a pipette and 40 mL of Agrobacterium Suspen herein. However, it should be understood that the present sion is added to the PetriTM dish (500 segments with 40 mL of disclosure is not intended to be limited to the particular forms Agrobacterium solution). The hypocotyl segments are treated disclosed. Rather, the present disclosure is to cover all modi for 30 minutes with periodic swirling of the PetriTM dish so fications, equivalents, and alternatives falling within the that the hypocotyl segments remain immersed in the Agro scope of the present disclosure as defined by the following bacterium solution. At the end of the treatment period, the appended claims and their legal equivalents. Agrobacterium solution is pipetted into a waste beaker, auto claved and discarded (the Agrobacterium solution is com pletely removed to prevent Agrobacterium overgrowth). The Example 14 treated hypocotyls are transferred with forceps back to the original plates containing MSK1D1 media overlaid with filter Agrobacterium-Mediated Transformation of Canola paper (care is taken to ensure that the segments did not dry). (Brassica Napus) Hypocotyls The transformed hypocotyl segments and non-transformed 0343 Agrobacterium Preparation control hypocotyl segments are returned to the PercivalTM growth chamber under reduced light intensity (by covering 0344. The Agrobacterium strain containing a binary plas the plates with aluminum foil), and the treated hypocotyl mid is streaked out on YEP media (Bacto PeptoneTM 20.0 segments are co-cultivated with Agrobacterium for 3 days. gm/L and Yeast Extract 10.0 gm/L) plates containing strep tomycin (100 mg/ml) and spectinomycin (50 mg/mL) and 0352 Callus induction on selection medium: After 3 days incubated for 2 days at 28°C. The propagated Agrobacterium of co-cultivation, the hypocotyl segments are individually strain containing the binary plasmidis scraped from the 2-day transferred with forceps onto callus induction medium, streak plate using a sterile inoculation loop. The scraped MSK1D1H1 (MS. 1 mg/L kinetin, 1 mg/L 2,4-D, 0.5gm/L Agrobacterium strain containing the binary plasmid is then MES, 5 mg/L. AgNO, 300 mg/L TimentinTM, 200 mg/L car inoculated into 150 mL modified YEP liquid with streptomy benicillin, 1 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar) cin (100 mg/ml) and spectinomycin (50 mg/ml) into sterile with growth regime set at 22-26°C. The hypocotyl segments 500 mL baffled flask(s) and shaken at 200 rpm at 28°C. The are anchored on the medium but are not deeply embedded into cultures are centrifuged and resuspended in M-medium (LS the medium. salts, 3% glucose, modified B5 vitamins, 1 uM kinetin, 1 uM 0353. Selection and Shoot Regeneration: 2,4-D, pH 5.8) and diluted to the appropriate density (50Klett 0354 After 7 days on callus induction medium, the cal Units as measured using a spectrophotometer) prior to trans lusing hypocotyl segments are transferred to Shoot Regen formation of canola hypocotyls. eration Medium 1 with selection, MSB3Z1H1 (MS, 3 mg/L BAP 1 mg/L Zeatin, 0.5 gm/L MES, 5 mg/L AgNO, 300 0345 Canola Transformation mg/L TimentinTM, 200 mg/L carbenicillin, 1 mg/L Herbi 0346 Seed Germination: aceTM, 3% sucrose, 0.7% phytagar). After 14 days, the hypo (0347 Canola seeds (var. NEXERA 710TM) are surface cotyl segments which develop shoots are transferred to Sterilized in 10% CloroxTM for 10 minutes and rinsed three Regeneration Medium 2 with increased selection, times with sterile distilled water (seeds are contained in steel MSB3Z1H3 (MS, 3 mg/L BAP 1 mg/L Zeatin, 0.5gm/L strainers during this process). Seeds are planted for germina MES, 5 mg/L AgNO, 300 mg/l TimentinTM, 200 mg/L car tion on /2 MS Canola medium (/2 MS, 2% sucrose, 0.8% benicillin, 3 mg/L HerbiaceTM, 3% sucrose, 0.7% phytagar) agar) contained in PhytatraysTM (25 seeds per PhytatrayTM) with growth regime set at 22-26°C. and placed in a PercivalTM growth chamber with growth 0355 Shoot Elongation: US 2016/0 194658 A1 Jul. 7, 2016 42

0356. After 14 Days, the Hypocotyl Segments that via a PCR molecular confirmation assay. Leaf tissue is Develop Shoots are transferred from Regeneration Medium 2 obtained from the green shoots and tested via PCR for the to shoot elongation medium, MSMESH5 (MS, 300 mg/L presence of the selectable marker gene. Any chlorotic shoots TimentinTM, 5 mg/l HerbiaceTM, 2% sucrose, 0.7%TC Agar) are discarded and not subjected to PCR analysis. Samples that with growth regime set at 22-26°C. Shoots that are already are identified as positive for the presence of the selectable elongated were isolated from the hypocotyl segments and marker gene are kept and cultured on MSMEST medium to transferred to MSMESH5. After 14 days the remaining shoots continue development and elongation of the shoots and roots. which have not elongated in the first round of culturing on The samples that are identified as not containing the select shoot elongation medium are transferred to fresh shoot elon able marker gene negative according to PCR analysis are gation medium, MSMESH5. At this stage all remaining hypocotyl segments which do not produce shoots are dis discarded. carded. 0361. The transformed canola plants comprising shoots 0357 Root Induction: and roots that are PCR-positive for the presence of the select 0358. After 14 days of culturing on the shoot elongation able marker gene are transplanted into Soil in a greenhouse. medium, the isolated shoots are transferred to MSMEST After establishment of the canola plants within soil, the medium (MS, 0.5 g/L MES, 300 mg/L TimentinTM, 2% canola plants are further analyzed to quantify the copy num sucrose, 0.7%TC Agar) for root induction at 22-26°C. Any ber of the selectable marker gene expression cassette via an shoots which do not produce roots after incubation in the first InvaderTM quantitative PCR assay and Southern blotting. transfer to MSMEST medium are transferred for a second or Transgenic To canola plants which are confirmed to contain at third round of incubation on MSMEST medium until the least one copy of the selectable marker gene expression cas shoots develop roots. sette are advanced for further analysis of the seed. The seeds 0359 PCR Analysis: obtained from theses transgenic To canola plants, i.e., T. 0360 Transformed canola hypocotyl segments which canola seeds, are analyzed to detect the presences of the target regenerated into shoots comprising roots are further analyzed gene.

SEQUENCE LISTING

< 16 Os NUMBER OF SEO ID NOS: 99

<21Os SEQ ID NO 1 <211 > LENGTH: 104.7 <212> TYPE : DNA <213> ORGANISM: Diabrotica virgifera

< 4 OOs SEQUENCE: 1 atgccagata cCaaggatgc Caaggatacc aaggatgcta atttgagttc tcc tigaacgt. 6 O aaaagacgaa gaaagagtag atctaaat Ct ccagaacgaa aagagaaaaa gtct tccaaa 12 O aagaaag.ccc acaatagtag agacagagat t catcagagg alaggttacaa c cctaaagat 18O tat cagagat act atgggga agat.cgcc.ca alacagtgaca aat attggaa taalatat CCa 24 O aggaaagata ctaccaaagt tggccaaaga tactatatg cggct Cocga agaatctggc 3OO aagaaggggc Cagatagaaa ttcagaggag aaggagttac caaagccaat ggagt ctgtt 360 cctgataaat cagt catcaa accalagagaa agaaaaactg tagatatgtt alacatcgagg 42O actggtggtg cittatatt co CCC agctaag citacgattgt tacaa.gc.ca.g tattacagac 48O aaaa catcag cagcctatica gcgtatagca tgggaagcct taaagaaatc cgttcatggit 54 O taCattaata aaattalacac citcgaatatt ggcatcatcg ccagaga att attgcatgaa 6OO aatatagtaa gaggtagagg tittgctgtgc aagt caataa tacaa.gcaca agcagcatct 660 cc tact titta caaacgttta cgcagcctta gtagctgtta ttaatticgaa gtttccaagt 72 O ataggaga.gc ttittattgaa gaggttggitt ttgcagttca aaagagggitt taaacaaaat 78O aataagttcta tittgcatat c ggctact act titcgtagctic atttagtaaa t cagaga.gtg 84 O gCacatgaaa ttittggctitt ggagatactt acattgttgg tggagactic C tacagatgat 9 OO tctgttggagg tggccatttic atttittgaag gaatgtggac aaaaactgac agaagtttca 96.O agtagaggta ttactgctat atttgagatg ttaagaaaca ttitta catga aggc.ca.gcta 1 O2O gaaaaaaaga att cagtaca tgattga 104.7 US 2016/0 194658 A1 Jul. 7, 2016 43

- Continued

<210s, SEQ ID NO 2 &211s LENGTH: 573 212. TYPE: PRT <213> ORGANISM: Diabrotica virgifera <4 OOs, SEQUENCE: 2 Met Arg Gly Gly Val Ser Asp Asp Met Thr Ser Thr Cys Val Glin Gly 1. 5 1O 15 Gly Ile Arg Pro Ile Gly Arg Tyr Glin Pro Asn Met Leu Met Glu Pro 2O 25 3O Ser Ser Pro Glin Ser Ala Trp Glin Phe His Pro Ala Met Pro Lys Arg 35 4 O 45 Glu Pro Val Asp His Asp Gly Arg Asn Asp Ser Gly Lieu Ala Ser Gly SO 55 6 O Gly Glu Phe Ile Ser Ser Ser Pro Gly Ser Asp Asn Ser Glu. His Phe 65 70 7s 8O Ser Ala Ser Tyr Ser Ser Pro Thr Ser Cys His Thr Val Ile Ser Thr 85 90 95 Asn Thr Tyr Tyr Pro Thr Asn Lieu. Arg Arg Pro Ser Glin Ala Glin Thr 1OO 105 11 O Ser Ile Pro Thr His Met Met Tyr Thr Gly Asp His Asn Pro Leu. Thr 115 12 O 125 Pro Pro ASn Ser Glu Pro Met Ile Ser Pro Llys Ser Val Lieu Ser Arg 13 O 135 14 O Asn Asn Glu Gly Glu. His Glin Thr Thr Lieu. Thr Pro Cys Ala Ser Pro 145 150 155 160 Glu Asp Ala Ser Val Asp Ala Thr Asp Ser Val Asn. Cys Asp Gly Ala 1.65 17O 17s Lieu Lys Llys Lieu. Glin Ala Thr Phe Glu Lys Asn Ala Phe Ser Glu Gly 18O 185 19 O Ser Gly Asp Asp Asp Thir Lys Ser Asp Gly Glu Ala Glu Glu Tyr Asp 195 2OO 2O5 Glu Glin Gly Lieu. Arg Val Pro Llys Val Asn. Ser His Gly Lys Ile Llys 21 O 215 22O Thir Phe Lys Cys Lys Glin Cys Asp Phe Val Ala Ile Thr Lys Lieu Val 225 23 O 235 24 O Phe Trp Glu. His Thr Llys Lieu. His Ile Lys Ala Asp Llys Lieu. Lieu Lys 245 250 255 Cys Pro Llys Cys Pro Phe Val Thr Glu Tyr Lys His His Lieu. Glu Tyr 26 O 265 27 O His Lieu. Arg Asn His Tyr Gly Ser Llys Pro Phe Lys Cys Asn Glin Cys 27s 28O 285

Ser Tyr Ser Cys Val Asn Llys Ser Met Lieu. Asn. Ser His Lieu Lys Ser 29 O 295 3 OO His Ser Asn Ile Tyr Glin Tyr Arg Cys Ser Asp Cys Ser Tyr Ala Thr 3. OS 310 315 32O Llys Tyr Cys His Ser Lieu Lys Lieu. His Lieu. Arg Llys Tyr Ser His Lys 3.25 330 335

Pro Ala Met Val Lieu. Asn Pro Asp Gly Thr Pro Asn Pro Leu Pro Ile 34 O 345 35. O

Ile Asp Val Tyr Gly Thr Arg Arg Gly Pro Llys Met Lys Ser Glu Glin US 2016/0 194658 A1 Jul. 7, 2016 44

- Continued

355 360 365 Lys Ser Ser Glu Glu Met Ser Pro Llys Pro Glu Glin Val Leu Pro Phe 37 O 375 38O Pro Phe Asin Glin Phe Leu Pro Gln Met Glin Leu Pro Phe Pro Gly Phe 385 390 395 4 OO Pro Leu Phe Gly Gly Phe Pro Gly Gly Ile Pro Asn Pro Leu Lleu Lieu. 4 OS 41O 415 Glin Asn Lieu. Glu Lys Lieu Ala Arg Glu Arg Arg Glu Ser Met Asn. Ser 42O 425 43 O Ser Glu Arg Phe Ser Pro Ala Glin Ser Glu Gln Met Asp Thir Asp Ala 435 44 O 445 Gly Val Lieu. Asp Lieu. Ser Llys Pro Asp Asp Ser Ser Glin Thr Asn Arg 450 45.5 460 Arg Lys Asp Ser Ala Tyr Llys Lieu. Ser Thr Gly Asp Asn. Ser Ser Asp 465 470 47s 48O Glu Glu Asp Asp Glu Ala Thir Thr Thr Met Phe Gly Asn Val Glu Val 485 490 495 Val Glu Asn Lys Glu Lieu. Glu Asp Thir Ser Ser Gly Lys Glin Thr Pro SOO 505 51O Thir Ser Ala Lys Lys Asp Asp Tyr Ser Cys Glin Tyr Cys Glin Ile Asn 515 52O 525 Phe Gly Asp Pro Val Lieu. Tyr Thr Met His Met Gly Tyr His Gly Tyr 530 535 540 Lys Asn Pro Phe Ile Cys Asn Met Cys Gly Glu Glu. Cys Asn Asp Llys 5.45 550 555 560 Val Ser Phe Phe Lieu. His Ile Ala Arg Asn Pro His Ser 565 st O

<210s, SEQ ID NO 3 &211s LENGTH: 411 &212s. TYPE: DNA <213> ORGANISM: Diabrotica virgifera

<4 OOs, SEQUENCE: 3 gatgcggctic ccgaagaatc tigcaagaag gggccagata galaattcaga ggaga aggag 6 O ttaccaaag.c caatggagtc. togttcctgat aaatcagt catcaaaccaag agaaagaaaa 12 O actgtagata tittaa.catc gaggactggt ggtgcttata titc.ccc.ca.gc taa.gctacga 18O ttgttacaag ccagtattac agacaaaa.ca totago agcct at cagcgitat agcatgggaa 24 O gcct taaaga aatcc.gttca toggttacatt aataaaatta acacct cqaa tattggcatc 3OO atcgc.cagag aattattgca taaaatata gtaagaggta gaggtttgct gtgcaagttca 360 ataatacaag cacaag cago atctoctact tttacaaacg tttacgcagc c 411

<210s, SEQ ID NO 4 &211s LENGTH: 4. Of &212s. TYPE: DNA <213> ORGANISM: Diabrotica virgifera

<4 OOs, SEQUENCE: 4

CCCttagtaa agaaatctta ggcagtgatg gtgagtctga at Caggttcc galaggttcag 6 O aagaggaatc tataatgaa aatgaggacg aagttcaagga C cagggalaca attattgaca 12 O at actgaaac gaatttaatt tot cittagaa gaaccatata tittgactatt cagtic tagtt 18O

US 2016/0 194658 A1 Jul. 7, 2016 46

- Continued

<210s, SEQ ID NO 9 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer oligonucleotide <4 OOs, SEQUENCE: 9 ttaatacgac toactatagg gagaggctgc gtaaacgttt gtaaaag 47

<210s, SEQ ID NO 10 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer oligonucleotide <4 OOs, SEQUENCE: 10 ttaatacgac toactatagg gagagatgcg gct cocgaag aatctg 46

<210s, SEQ ID NO 11 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer oligonucleotide

<4 OOs, SEQUENCE: 11 ttaatacgac toactatagg gagagittagc atctagt Ctg tagtgg 47

<210s, SEQ ID NO 12 &211s LENGTH: 49 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer oligonucleotide

<4 OOs, SEQUENCE: 12 ttaatacgac toactatagg gag accctta gtaaagaaat cittaggcag 49

<210s, SEQ ID NO 13 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer oligonucleotide

<4 OOs, SEQUENCE: 13 ttaatacgac toactatagg gagattaatg taaccatgaa cqgatttic 48

<210s, SEQ ID NO 14 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer oligonucleotide

<4 OOs, SEQUENCE: 14 ttaatacgac toactatagg gagacagt catcaaac Caag agaaag 46

<210s, SEQ ID NO 15 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence

US 2016/0 194658 A1 Jul. 7, 2016 50

- Continued

<210s, SEQ ID NO 25 &211s LENGTH: 330 &212s. TYPE: DNA <213> ORGANISM: Diabrotica virgifera

<4 OOs, SEQUENCE: 25 agtgaaatgt tagcaaatat aac atccaag titt cqtaatt gtacttgctic agittagaaaa 6 O tatt ctdtag titt cactato ttcaa.ccgaa aatagaataa atgtagalacc ticgcgaactt 12 O gcctitt cotc caaaatat ca agaac ct cqa caagtttggit toggagagttt agatacgata 18O gacgacaaaa aattgggt at t cittgagctg catcc tdatgtttittgctac taatccaaga 24 O atagat atta tacatcaaaa tottagatgg caaagttitat atagatatgt aagctatgct 3OO

Catacaaagt caagatttga agtgagaggit 33 O

<210s, SEQ ID NO 26 &211s LENGTH: 32O &212s. TYPE: DNA <213> ORGANISM: Diabrotica virgifera <4 OOs, SEQUENCE: 26 caaagt caag atttgaagtg agaggtggag gtcgaaalacc gtggcc.gcaa aagggattgg 6 O gacgtgctic acatggttca attagaagtic cactittggag aggtggagga gttgttcatg 12 O gaccaaaatc. tccaac cc ct catttittaca tatt coatt ctacaccc.gt ttgctgggitt 18O tgactagogc actitt Cagta aaatttgcCC aagatgactit gcacgttgttg gat agtictag 24 O atctgccaac tacgaacaa agittatatag aagagctggit caaaag.ccgc titttgggggt 3OO ccttcttgtt ttatttgtag 32O

<210s, SEQ ID NO 27 &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer YFP-F T7

<4 OOs, SEQUENCE: 27 ttaatacgac toactatagg gagacaccat gggct coagc ggcgc cc 47

<210s, SEQ ID NO 28 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer YFP-R

<4 OOs, SEQUENCE: 28 agat Cttgaa ggcgct Cttic agg 23

<210s, SEQ ID NO 29 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer YFP-F

<4 OOs, SEQUENCE: 29

Caccatgggc tccagcggcg C cc 23 US 2016/0 194658 A1 Jul. 7, 2016 51

- Continued

<210s, SEQ ID NO 3 O &211s LENGTH: 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer YFP-RT7

<4 OOs, SEQUENCE: 30 ttaatacgac toactatagg gagaagat.ct taaggcgct Ctt Cagg 47

<210s, SEQ ID NO 31 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Ann-F1. T7

<4 OOs, SEQUENCE: 31 ttaatacgac toactatagg gagagcticca acagtggttc ctitatic 46

<210s, SEQ ID NO 32 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Ann-R1

<4 OOs, SEQUENCE: 32 ctaataattic titttittaatgttcct gagg 29

<210s, SEQ ID NO 33 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer Ann-F1

<4 OOs, SEQUENCE: 33 gctic caacag tdgttcctta t c 22

<210s, SEQ ID NO 34 &211s LENGTH: 53 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Ann-R1. T7

<4 OOs, SEQUENCE: 34 ttaatacgac toactatagg gagactaata attcttttitt aatgttcct g agg 53

<210s, SEQ ID NO 35 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Ann-F2 T7

<4 OOs, SEQUENCE: 35 ttaatacgac toactatagg gagattgtta caa.gctggag aacttctic 48

<210s, SEQ ID NO 36 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 2016/0 194658 A1 Jul. 7, 2016 52

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Primer Ann-R2

<4 OOs, SEQUENCE: 36 cittalaccaac aacggctaat aagg 24

<210s, SEQ ID NO 37 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer Ann-F2

<4 OO > SEQUENCE: 37 ttgttacaag ctggagaact tctic 24

<210s, SEQ ID NO 38 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer Ann-R2Tf

<4 OOs, SEQUENCE: 38 ttaatacgac toactatagg gagacittaac caacaacggc taataagg 48

<210s, SEQ ID NO 39 & 211 LENGTH 47 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F1. T7 <4 OOs, SEQUENCE: 39 ttaatacgac toactatagg gagaagatgt totgcatc tagagaa 47

<210s, SEQ ID NO 4 O &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R1 <4 OOs, SEQUENCE: 4 O gtcc attcgt. c catccactg. ca. 22

<210s, SEQ ID NO 41 &211s LENGTH: 23 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F1

<4 OOs, SEQUENCE: 41 agatgttggc tigcatctaga gaa 23

<210s, SEQ ID NO 42 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R1. T7

<4 OOs, SEQUENCE: 42 US 2016/0 194658 A1 Jul. 7, 2016 53

- Continued ttaatacgac toactatagg gagagtcc at t cqtc catcc actgca 46

<210s, SEQ ID NO 43 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F2 T7 <4 OOs, SEQUENCE: 43 ttaatacgac toactatagg gaga.gcagat galacaccagc gagaaa 46

<210s, SEQ ID NO 44 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R2 <4 OOs, SEQUENCE: 44 ctgggcagct tcttgtttico to 22

<210s, SEQ ID NO 45 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-F2 <4 OOs, SEQUENCE: 45 gCagatgaac accago.gaga aa 22

<210s, SEQ ID NO 46 &211s LENGTH: 46 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer Betasp2-R2 T7 <4 OOs, SEQUENCE: 46 ttaatacgac toactatagg gag actgggc agctt cittgt titcctic 46

<210s, SEQ ID NO 47 &211s LENGTH: 51 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer L4 -F1. T7

<4 OOs, SEQUENCE: 47 ttaatacgac toactatagg gagaagtgaa atgttagcaa atata acatc c 51

<210s, SEQ ID NO 48 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer L4 -R1

<4 OOs, SEQUENCE: 48 acct ct cact tcaaatcttg actittg 26 US 2016/0 194658 A1 Jul. 7, 2016 54

- Continued

<210s, SEQ ID NO 49 &211s LENGTH: 27 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer L4 - F1

<4 OOs, SEQUENCE: 49 agtgaaatgt tagcaaatat aac atcc 27

<210s, SEQ ID NO 50 &211s LENGTH: 50 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer L4 -R1. T7

<4 OOs, SEQUENCE: 50 ttaatacgac toactatagg gagaacct ct cacttcaaat cittgactittg SO

<210s, SEQ ID NO 51 &211s LENGTH: 50 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer L4 -F2 T7

<4 OOs, SEQUENCE: 51 ttaatacgac toactatagg gagaCaaagt Caagatttga agtgagaggit 5 O

<210s, SEQ ID NO 52 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer L4 -R2

<4 OOs, SEQUENCE: 52 ctacaaataa aacaagaagg acccc 25

<210s, SEQ ID NO 53 &211s LENGTH: 26 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer L4 -F2

<4 OOs, SEQUENCE: 53 caaagt caag atttgaagtg agaggit 26

<210s, SEQ ID NO 54 &211s LENGTH: 49 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Primer L4 -R2 T7

<4 OOs, SEQUENCE: 54 ttaatacgac toactatagg gagacitacaa ataaaacaag aaggacc cc 49

<210s, SEQ ID NO 55 &211s LENGTH: 115 O &212s. TYPE: DNA <213> ORGANISM: Zea mays

US 2016/0 194658 A1 Jul. 7, 2016 56

- Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Primer P5U76A (R)

<4 OOs, SEQUENCE: 58 tgttaaataa aaccc.caaag atcg 24

<210s, SEQ ID NO 59 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer TIPmxF

<4 OO > SEQUENCE: 59 tgaggg taat gccaactggt t 21

<210s, SEQ ID NO 60 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer TIPmixR

<4 OOs, SEQUENCE: 60 gcaatgtaac coagtgtc.tc. tcaa 24

<210s, SEQ ID NO 61 & 211 LENGTH 32 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Probe HXTIP

<4 OOs, SEQUENCE: 61 tttittggctt agagttgatg gtgtactgat ga 32

<210s, SEQ ID NO 62 &211s LENGTH: 151 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs, SEQUENCE: 62 gaccgtaagg Cttgatgaaa Caacg.cggcg agctttgatc alacgaccttt taalacttic 6 O ggct tcc cct ggagagagcg agatt Ctcc.g. c9ctgtagaa gtcaccattgttgttgcacga 12 O cgacat catt cogtggcgtt atc.ca.gctaa g 151

<210s, SEQ ID NO 63 &211s LENGTH: 69 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Partial AAD1 coding region

<4 OOs, SEQUENCE: 63 tgttcggttc cct ctaccaa goacagaacc gtc.gctt cag caacacctica gtcaaggtga 6 O tggatgttg 69

<210s, SEQ ID NO 64 &211s LENGTH: 4233 &212s. TYPE: DNA <213> ORGANISM: Zea mays

US 2016/0 194658 A1 Jul. 7, 2016 59

- Continued

SEQUENCE: 65 tgttcggttc cct ctaccaa

SEQ ID NO 66 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer GAAD1-R

SEQUENCE: 66 caa.catccat caccittgact ga 22

SEO ID NO 67 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe GAAD1-P (FAM)

SEQUENCE: 67 cacagaac cq t cqctt cago aaca 24

SEQ ID NO 68 LENGTH: 18 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer IWR1-F

SEQUENCE: 68 tggcggacga cacttgt 18

SEO ID NO 69 LENGTH 19 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer IWR1-R

SEQUENCE: 69 aaagtttgga ggctgc.cgt. 19

SEO ID NO 7 O LENGTH: 26 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Probe IVR1-P (HEX)

SEQUENCE: 7 O cgagcagacic gcc.gtgtact tct acc 26

SEO ID NO 71 LENGTH 19 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Primer SPC1A

SEQUENCE: 71 cittagotgga taacgc.cac 19 US 2016/0 194658 A1 Jul. 7, 2016 60

- Continued

<210s, SEQ ID NO 72 &211s LENGTH: 19 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer SPC1S

<4 OOs, SEQUENCE: 72 gaccgtaagg Cttgatgaa 19

<210s, SEQ ID NO 73 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe TOSPEC (CY5*)

<4 OO > SEQUENCE: 73 cgagattct c cqc.gctgtag a 21

<210s, SEQ ID NO 74 &211s LENGTH: 25 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer ST-LS1-F

< 4 OO SEQUENCE: 74 gtatgtttct gcttctacct ttgat 25

<210s, SEQ ID NO 75 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223s OTHER INFORMATION: Primer ST-LS1-R

<4 OO > SEQUENCE: 75 c catgttittg gtcatatatt agaaaagtt 29

<210s, SEQ ID NO 76 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Probe ST-LS1-P (FAM)

<4 OO > SEQUENCE: 76 agtaatatag tatttcaagt atttittitt ca aaat 34

<210s, SEQ ID NO 77 &211s LENGTH: 2681 &212s. TYPE: DNA <213> ORGANISM: Diabrotica virgifera

<4 OO > SEQUENCE: 77 attaccaaat gtcaatgtca ct cattactic attaccalaat gtcaatgtca citgtcaggta 6 O acgtgcaatig caaattgtca atgtcaaact taaaaatatt titcctgcaac togcatcaaat 12 O tgtaaattitt atttitttitta aatatgccag ataccaagga tigccaaggat accaaggatg 18O ctaatttgag ttcticctgaa cqtaaaagac gaagaaagag tagat ctaaa tot coagaac 24 O US 2016/0 194658 A1 Jul. 7, 2016 61

- Continued gaaaagagaa aaagttctt CC aaaaagaaag cccacaatag tagagacaga gatt cat cag 3OO aggaiaggitta caa.ccctaaa gattatcaga gat actatgg ggaagat.cgc ccaaacagtg 360 acaaat attg gaataaatat coaaggaaag atactaccaa agttggccaa agatact atg 42O atgcggct co Caagaatct ggcaagaagg ggc.ca.gatag aaatt cagag gagaaggagt 48O taccaaagcc aatggagt ct gttcc tigata aat cagt cat caaac caaga gaaagaaaaa 54 O Ctgtagat at gttalacat cq aggactggtg gtgcttatat tcc cc cagct aagctacgat 6OO tgttacaa.gc cagtattaca gacaaaac at Cagcagocta t cagcgtata gcatgggaag 660 ccittaaagaa atc.cgttcat ggitta catta ataaaattaa cacct cqaat attggcatca 72 O tcgc.ca.gaga attattgcat gaaaatatag taagaggtag aggtttgctg. tcaagttcaa 78O taatacaa.gc acaagcagca tot cotactt ttacaaacgt ttacgcagoc ttagtagctg 84 O ttattaattic gaagtttcca agtataggag agcttittatt galagaggttg gttittgcagt 9 OO tcaaaagagg gtttaaacaa aataataagt c tatttgcat atcggctact actitt cqtag 96.O ct catttagt aaatcagaga gtggcacatgaaattittggc tittggagata cittacattgt O2O tggtggagac toctacagat gattctgtgg aggtggc.cat tt catttittg aaggaatgtg O8O gacaaaaact gacagaagtt toaagtagag gtatt actgc tat atttgag atgttaagaa 14 O acattt taca tdaaggc.cag ctagaaaaaa agaatticagt acatgattga agittatgttt 2OO Caaataagga aagacggatt taaggatcat gctgctgtcg tagaagaatt agatttagta 26 O gaagaggaag atcaattic act catcttatt atgttagatg atgttaaaga ggctgatgca 32O gaggat at at taatgtgtt Caaatttgat gagagittatg aagaaaatga agataaatac 38O aaaaccctta gtaaagaaat Cttaggcagt gatggtgagt ctgaat Cagg ttc.cgalaggt 44 O t cagaagagg aatctgataa taaaatgag gacgaagttca aggaccaggg aacaattatt SOO gacaat actgaaacgaattit aatttct citt agaagaacca tatatttgac tatt cagtict 560 agtttagatt ttgaagaatgtcacataag Ctactgaaga tiggagttgaa acctggacaa 62O gaaatagaat ttgtcacat gtttcttgac totgcgcag aacaaagaac Ctacgaaaag 68O ttittatggtc. ttittggctica aagattttgt caaat caa.ca aagtgtatat cqagc ctitt c 74 O caacaaattt ttaaagatac ctatt ctacc act cacagac tagatgctaa caggittalaga 8OO aacgtcagca aattittittgc gcatttactt tttacggatg ccattggatg gigaagtic citt 86 O gacat catga aattgaatga agaggatacc aatagttcta gtaggattitt cataaaaatc 92 O ttgtttcaag aattggctga atatatggga citaggaaaat taaacgcaag gctaaaggat 98 O gaga.ccctgc aggct tattt ttcaggactg titt coltagagata accolaaa gaataccaga 2O4. O ttittct atta atttittttac ct citat cqgt ttgggaggat taactgatga act cagagaa 21OO catttaaaaa at attccaaa aatgatggaa atgaagittag ccactaaaga aaaggaaagc 216 O agtgg tagta gtagttcaga aagtagttct gaggaagata gtag tacga cagct Ctgaa 222 O gatt catcaa gttctgaaga catagaggc aaaaagaaga aaaaaac caa aaa.gctagaa 228O gaaaaaaatt cqaaagtaaa ttctaagttct agacctagat caaaagagaa agaac acgca 234 O gacaaaccta gagacaaaca tagaaaggaia gatagacata aatctgacaa aaatc.ccgat 24 OO agat.cct Ct c caaaaaata ttctaaagaa gataacaaga ggaagaaaga Citacgaatgg 246 O atgaagagca gatatgaaga tigatattaag caattaaaaa acgataaaag ggt atttgaa 252O

US 2016/0 194658 A1 Jul. 7, 2016 65

- Continued aatgcc at at ttgagatgtt gaggaatatt Ctgcatgaag gacagttgga gaagagaata 12 O Cagtacatga ttgaagttcat gttcCaagtt C9gaaagatg gtttcaagga t catgctgct 18O gttactgaag alactagat at tdttgaagag gaagat cagt ttact cacct aat cacattg 24 O gatgatgtta aacaagctaa citcagaggat at attgaatg tdtttaaatt tdatgataaa 3OO tatgaggaaa atgagggitaa atacaaaact ttaagtalagg aaatt ct coa gtcagacagt 360 gaat Caggcg aatctggttc agaggggtct gaagaagact cqgaagatga agaaggtgaa 42O gaagatgaaa ccaaaaatca aaccattatt gataacacag aaactaattit aat caccitta 48O aggagaac catctatot cac aatacaatcc agtttggatt ttgaggaatg togcc cataaa 54 O ttgatgaaaa tigagatcaa acctggacaa gagattgaat ttgtcacat gttcCttgat 6OO tgttgcgctgaacagogtac ctacgaaaaa ttctt cqgcc ticcitct cqca gcqcttctgc 660 caaataaa.ca agactitt cat cqaac cqttic caacaaattt toaaagatac ctatt coaca 72 O acticacagac ttgacgc.caa totgattgaga aacgttagca aattctt.cgc gcatttattg 78O ttcaccgacg C catcggctg ggaagtgctic gat at catga aattaaacga ggalagacacc 84 O alacagttcca gcaggattitt catcaagatt ttgttcCagg agttgtc.cga atatatggga 9 OO ttagcgaagt taataaaag gctaaaggat gaaactttac aggaatattt cqcggggcta 96.O titt.ccgaggg at aaccogaa gaacacgc.gt titcgc catca atttittt cac gtcgatcggit O2O ttaggagg to taacggacga gttgagggag Cacttgaaaa acgt.gc.calaa a catctggaa O8O gtgatggctt taaag caga titcgagcago totagcagca gtagcagcag titc.cagtaac 14 O gatt CC agca gcagttcaga ttctt Cogat gacgagggitt C caggaagaa gaaaacaaaa 2OO aaattgaaaa ccc.cggacaa aaagaagaaa Cagaaagaag atgaaaaac C. Caaaaagaaa 26 O agcgaggata aaccgaggaa caaaccagac tatagagata gaagaaacga cacagggaa 32O aagtttaaaa aatacagaaa Caacgacgaa gaaagccaca gaagaag cag agaagatgca 38O agagaaaaat acagaggt ca Caggaaaga agaag.cgacc acagagaaga at accggcc.g 44 O agagaacata gaggtagaga tagacgttag ttgtataata atgtatattt titt cqtattt SOO aataaaataa attata catt ttatagtgtt toggagcatt caccalagcaa goggttt tact 560 titcggatagc aatggtgtag tacgtttittg aaggtgtc.ca cacacaccala gcc.ggittitta 62O tctaaatcta gagctatott coaaaagt ct tcaaagga 658

<210s, SEQ ID NO 85 &211s LENGTH: 489 212. TYPE: PRT <213> ORGANISM: Melligethes aeneus <4 OOs, SEQUENCE: 85 Ala Ile Ser Phe Lieu Lys Glu Ser Gly Glin Llys Lieu. Thr Glu Val Ser 1. 5 1O 15

Ser Lys Gly Ile Asn Ala Ile Phe Glu Met Lieu. Arg Asn. Ile Lieu. His 2O 25 3O

Glu Gly Glin Leu Glu Lys Arg Ile Glin Tyr Met Ile Glu Val Met Phe 35 4 O 45 Glin Val Arg Lys Asp Gly Phe Lys Asp His Ala Ala Val Thr Glu Glu SO 55 6 O

Lieu. Asp Ile Val Glu Glu Glu Asp Glin Phe Thr His Lieu. Ile Thr Lieu. US 2016/0 194658 A1 Jul. 7, 2016 66

- Continued

Asp Asp Wall Lys Glin Ala Asn. Ser Glu Asp Ile Lieu. Asn Val Phe Lys 85 90 95 Phe Asp Asp Llys Tyr Glu Glu Asn. Glu Gly Lys Tyr Llys Thir Lieu. Ser 1OO 105 11 O Lys Glu Ile Lieu. Glin Ser Asp Ser Glu Ser Gly Glu Ser Gly Ser Glu 115 12 O 125 Gly Ser Glu Glu Asp Ser Glu Asp Glu Glu Gly Glu Glu Asp Glu Thir 13 O 135 14 O Lys Asn Glin Thir Ile Ile Asp Asn Thr Glu Thir Asn Lieu. Ile Thr Lieu. 145 150 155 160 Arg Arg Thir Ile Tyr Lieu. Thir Ile Glin Ser Ser Lieu. Asp Phe Glu Glu 1.65 17O 17s Cys Ala His Llys Lieu Met Lys Met Glu Ile Llys Pro Gly Glin Glu Ile 18O 185 19 O Glu Lieu. Cys His Met Phe Lieu. Asp Cys Cys Ala Glu Glin Arg Thr Tyr 195 2OO 2O5 Glu Lys Phe Phe Gly Lieu Lleu Ser Glin Arg Phe Cys Glin Ile Asn Lys 21 O 215 22O Thr Phe Ile Glu Pro Phe Glin Glin Ile Phe Lys Asp Thr Tyr Ser Thr 225 23 O 235 24 O Thir His Arg Lieu. Asp Ala Asn Arg Lieu. Arg Asn Val Ser Llys Phe Phe 245 250 255 Ala His Lieu. Lieu. Phe Thr Asp Ala Ile Gly Trp Glu Val Lieu. Asp Ile 26 O 265 27 O Met Lys Lieu. Asn. Glu Glu Asp Thr Asn. Ser Ser Ser Arg Ile Phe Ile 27s 28O 285 Lys Ile Lieu. Phe Glin Glu Lieu. Ser Glu Tyr Met Gly Lieu Ala Lys Lieu. 29 O 295 3 OO Asn Lys Arg Lieu Lys Asp Glu Thir Lieu. Glin Glu Tyr Phe Ala Gly Lieu 3. OS 310 315 32O Phe Pro Arg Asp Asn Pro Lys Asn Thr Arg Phe Ala Ile Asin Phe Phe 3.25 330 335 Thir Ser Ile Gly Lieu. Gly Gly Lieu. Thir Asp Glu Lieu. Arg Glu. His Lieu. 34 O 345 35. O Lys Asn Val Pro Llys His Lieu. Glu Val Met Ala Lieu Lys Ala Asp Ser 355 360 365

Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Asn Asp Ser Ser Ser 37 O 375 38O Ser Ser Asp Ser Ser Asp Asp Glu Gly Ser Arg Llys Llys Llys Thir Lys 385 390 395 4 OO Llys Lieu Lys Thr Pro Asp Llys Llys Llys Lys Gln Lys Glu Asp Glu Lys 4 OS 41O 415

Pro Llys Llys Llys Ser Glu Asp Llys Pro Arg Asn Llys Pro Asp Tyr Arg 42O 425 43 O

Asp Arg Arg Asn Asp Asp Arg Glu Lys Phe Llys Llys Tyr Arg Asn. Asn 435 44 O 445 Asp Glu Glu Ser His Arg Arg Ser Arg Glu Asp Ala Arg Glu Lys Tyr 450 45.5 460 Arg Gly His Glu Glu Arg Arg Ser Asp His Arg Glu Glu Tyr Arg Pro 465 470 47s 48O

US 2016/0 194658 A1 Jul. 7, 2016 68

- Continued tgaaattaaa cdaggaagac accaa.cagtt coagcaggat ttittatcaag attttgttcc 198O aggagttgtc. c.gaatatatg ggattagcga agttgaataa aaggctaaag gatgaaactt 2O4. O tacaggaata titt cqcgggg ctattitcc.ga gggataa.ccc gaagaac acg cgttt cqc.ca 21OO t caattittitt Cacgt.cgatc ggtttaggag gtctaacgga cagttgagg gag cacttga 216 O aaaacgtgcc aaaacatctg gaagtgatgg Ctttgaaagc agatt.cgagc agctictagda 222 O gcagtagcag cagttcCagt aacgatticca gcagcagttc agattct tcc gatgacgagg 228O gttcCaggaa gaagaaaa.ca aaaaaattga aaaccc.cgga caaaaagaag aaacagaaag 234 O aagatgaaaa accCaaaaag aaaag.cgagg ataaaccgag gaacaaacca gactatagag 24 OO ataggaga aa cacga Cagg gaaaagttta aaaaatacag aaacaacgac gaagaaagcc 246 O acagaagaag cagagaagat gcaa.gagaaa aatacagagg to acgaggala agaagacgag 252O gcc.gagaaga C caaacgcga agaggacaag accaagttcc alaggtttgttg C 2571.

<210s, SEQ ID NO 87 &211s LENGTH: 798 212. TYPE: PRT <213> ORGANISM: Melligethes aeneus

<4 OO > SEQUENCE: 87 Met Thr Thr Asp Ser Glu Arg Gly Ser Pro Thr Ala Ala Ala Pro Arg 1. 5 1O 15 Arg Ser Ala Ser Llys Ser Pro Glu Pro Llys Lys Ala Lys Tyr Asp Llys 2O 25 3O Lys Glu Lys Gly Asp Lys Asp Arg Lys Arg Arg Ser His Arg Ser Arg 35 4 O 45 Ser Arg Ser Arg Asp Arg Asp His Arg Asp Llys His Gly Gly Lys Llys SO 55 6 O Arg Tyr His Asp Lieu. Asp Asp Pro Ser Glu Asp Tyr Pro Arg Tyr Tyr 65 70 7s 8O Gly Glu Asp Arg Lys Glin Asn. Ser Asp Arg Tyr Trp Ser Lys Tyr Pro 85 90 95 Llys Lys Asp Arg Asp Glu Tyr Val Ile Gly Ser Arg Tyr Tyr Asp Wall 1OO 105 11 O Glu Glu Lys Lys Glu Lys Lys Glu Lys Glu Asp Glu Asn Lys Asp Llys 115 12 O 125 Ser Val Ile Thr Pro Arg Glu Arg Llys Thr Val Asp Lieu. Lieu. Thir Ser 13 O 135 14 O Arg Thr Gly Gly Ala Tyr Ile Pro Pro Ala Lys Lieu. Arg Met Met Glin 145 150 155 160 Ala Glu Ile Thr Asp Llys Ser Ser Ala Ala Tyr Glin Arg Ile Ala Trip 1.65 17O 17s Glu Ala Lieu Lys Llys Ser Ile His Gly Tyr Ile Asn Lys Ile Asn. Thir 18O 185 19 O

Ser Asn. Ile Gly Lieu. Ile Ala Arg Glu Lieu. Lieu. His Glu Asn. Ile Val 195 2OO 2O5

Arg Gly Arg Gly Lieu. Lieu. Cys Llys Ser Ile Ile Glin Ala Glin Ala Ala 21 O 215 22O

Ser Pro Thr Phe Thr Asn Val Tyr Ala Ala Leu Val Ala Val Ile Asn 225 23 O 235 24 O

Ser Llys Phe Pro Asn. Ile Gly Glu Lieu. Lieu. Lieu Lys Arg Lieu Val Lieu. US 2016/0 194658 A1 Jul. 7, 2016 69

- Continued

245 250 255 Glin Phe Lys Arg Gly Phe Lys Glin Asn. Asn Llys Ser Ile Cys Ile Ser 26 O 265 27 O Ala Ala Thir Phe Val Ala His Lieu Val Asin Glin Arg Val Ala His Glu 27s 28O 285 Ile Lieu Ala Lieu. Glu Ile Lieu. Thir Lieu. Lieu Val Glu Ser Pro Thr Asp 29 O 295 3 OO Asp Ser Val Glu Val Ala Ile Ser Phe Lieu Lys Glu Ser Gly Glin Lys 3. OS 310 315 32O Lieu. Thr Glu Val Ser Ser Lys Gly Ile Asn Ala Ile Phe Glu Met Leu 3.25 330 335 Arg Asn. Ile Lieu. His Glu Gly Glin Lieu. Glu Lys Arg Ile Glin Tyr Met 34 O 345 35. O Ile Glu Val Met Phe Glin Val Arg Lys Asp Gly Phe Lys Asp His Ala 355 360 365 Ala Val Thr Glu Glu Lieu. Asp Ile Val Glu Glu Glu Asp Glin Phe Thr 37 O 375 38O His Lieu. Ile Thr Lieu. Asp Asp Wall Lys Glin Ala Asn. Ser Glu Asp Ile 385 390 395 4 OO Lieu. Asn Val Phe Llys Phe Asp Asp Llys Tyr Glu Glu Asn. Glu Gly Lys 4 OS 41O 415 Tyr Lys Thr Lieu. Ser Lys Glu Ile Lieu. Glin Ser Asp Ser Glu Ser Gly 42O 425 43 O. Glu Ser Gly Ser Glu Gly Ser Glu Glu Asp Ser Glu Asp Glu Glu Gly 435 44 O 445 Glu Glu Asp Glu Thir Lys Asn. Glin Thir Ile Ile Asp Asn Thr Glu Thir 450 45.5 460 Asn Lieu. Ile Thr Lieu. Arg Arg Thr Ile Tyr Lieu. Thir Ile Glin Ser Ser 465 470 47s 48O Lieu. Asp Phe Glu Glu. Cys Ala His Llys Lieu Met Lys Met Glu Ile Llys 485 490 495 Pro Gly Glin Glu Ile Glu Lieu. Cys His Met Phe Lieu. Asp Cys Cys Ala SOO 505 51O Glu Glin Arg Thr Tyr Glu Lys Phe Phe Gly Lieu Lleu Ser Glin Arg Phe 515 52O 525 Cys Glin Ile Asn Lys Thr Phe Ile Glu Pro Phe Glin Glin Ile Phe Lys 53 O 535 54 O Asp Thr Tyr Ser Thir Thr His Arg Lieu. Asp Ala Asn Arg Lieu. Arg Asn 5.45 550 555 560 Val Ser Llys Phe Phe Ala His Leu Lleu Phe Thr Asp Ala Ile Gly Trp 565 st O sts

Glu Val Lieu. Asp Ile Met Lys Lieu. Asn. Glu Glu Asp Thr Asn. Ser Ser 58O 585 59 O

Ser Arg Ile Phe Ile Lys Ile Leu Phe Glin Glu Lieu Ser Glu Tyr Met 595 6OO 605

Gly Lieu Ala Lys Lieu. Asn Lys Arg Lieu Lys Asp Glu Thir Lieu. Glin Glu 610 615 62O

Tyr Phe Ala Gly Lieu. Phe Pro Arg Asp Asn Pro Lys Asn. Thir Arg Phe 625 630 635 64 O

Ala Ile Asin Phe Phe Thr Ser Ile Gly Lieu. Gly Gly Lieu. Thir Asp Glu 645 650 655 US 2016/0 194658 A1 Jul. 7, 2016 70

- Continued

Lieu. Arg Glu. His Lieu Lys Asn Val Pro Llys His Lieu. Glu Val Met Ala 660 665 67 O Lieu Lys Ala Asp Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 675 68O 685 Asn Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Asp Glu Gly Ser Arg 69 O. 695 7 OO Llys Llys Llys Thir Lys Llys Lieu Lys Thr Pro Asp Llys Llys Llys Lys Glin 7 Os 71O 71s 72O Lys Glu Asp Glu, Llys Pro Llys Llys Llys Ser Glu Asp Llys Pro Arg Asn 72 73 O 73 Llys Pro Asp Tyr Arg Asp Arg Arg Asn Asp Asp Arg Glu Lys Phe Lys 740 74. 7 O Llys Tyr Arg Asn. Asn Asp Glu Glu Ser His Arg Arg Ser Arg Glu Asp 7ss 760 765 Ala Arg Glu Lys Tyr Arg Gly His Glu Glu Arg Arg Arg Gly Arg Glu 770 775 78O Asp Glin Thir Arg Arg Gly Glin Asp Glin Val Pro Arg Phe Val 78s 79 O 79.

<210s, SEQ ID NO 88 &211s LENGTH: 251.2 &212s. TYPE: DNA <213> ORGANISM: Melligethes aeneus

<4 OOs, SEQUENCE: 88 aaatgtag to actaccattg tagtgttatt cqttt cittgt gct tatttitt aattalaccag 6 O t caatticgtg tdgtgtagt aacaaaggitt at agittatga cacggatt C cagagaggt 12 O tcc.cct acag ctg.cggct Co acgcagaa.gc gcct caaat cqc.ca.galacc aaaaaaagca 18O aagtacgata agaaagagaa gggcgataaa gat.cgcaaga ggagat.ccca Cagat.ccaga 24 O tctagat.cca gggatagaga C catagggac aaa catggtg gaaaaaaacg ttaccacgac 3OO Ctggacgacc Cttctgaaga ct accCaaga tatt atggcg aggatagaala acagaac agt 360 gacagatatt ggtc.calagta Cccaaaagaa agaCagggac gaatatgtta ttggit aaccg 42O gt attatgat gttgaggaala agaaggagaa aaaggaaaaa gaggatgaaa ataaggataa 48O atc.cgt catt act coaaggg aaaggaaaac agtggactta Ctaac at Ct c galacaggtgg 54 O ggcttatata cct coagcta aattacgitat gatgcaggct gagataact g ataaatcatc 6OO agctgcatat caaagaattig cct gggaagc tittaaaaaag to catt catg gttacat caa 660 caaaattaac act tccaata ttggit ctitat tdctagagaa ttactgcatgaaaac attgt 72 O aagagg taga ggtttgctgt gtaaatct at aatacaa.gca Caggcagctt CCC cacgtt 78O caccaatgtt tatgcagott tagttgcagt cataaattica aaatticcicca acattggaga 84 O actgtt actgaaaaggttgg ttittgcagtt taaaaggggit ttcaa.gcaga acaacaagtic 9 OO tatctgtata t cqgctgcta cctttgtc.gc gcatttagta aaccaaagag toggct catga 96.O aattittagca ttggaaattic titactittact tdttgagtcc cccacagatg attcagtaga 1 O2O agtgagcaga acaacaagtic tatctgtata t cqgctgcta cctttgtc.gc gcatttagta 108 O aaccaaagag toggcc.cacga aattittagca ttggaaattic titactitt act tdttgagt cc 114 O

Cccacagatg attcagtgga agtag caatt t cqtttittga aggaaagtgg tdaaaaactic 12 OO US 2016/0 194658 A1 Jul. 7, 2016 71

- Continued actgaggtgt cagtaaagg tat caatgcc at atttgaga tigttgaggala tattotgcat 26 O gaaggacagt tagaagag aatacagtac atgattgaag ticatgttcca agttctggaaa 32O gatggittt ca aggat catgc tigctgttact gaagaac tag at attgttga agaggaagat 38O cagttt actic acctaatcac attggatgat gttaaacaag ctaacticaga gqatatattg 44 O aatgtgttta aatttgatga taaatatgag gaaaatgagg gtaaatacaa aactittaagt SOO aaggaaattic ticcagt caga cagtgaatca ggc gaatctg gttcagaggg gtctgaagaa 560 gact cigaag atgaagaagg taagaagat gaalaccaaaa atcaaac cat tattgataac 62O acagaaacta atttaatcac cittaaggaga accatctato t cacaataca atccagtttg 68O gattittgagg aatgtgcc.ca taaattgatgaaaatggaga t caaacctgg acaagagatt 74 O gaattgtgtc. acatgttcct tdattgttgc gctgaacago gtacctacga aaaattcttic 8OO ggcct cotct cqcagogctt citgccaaata aacaagacitt to atcgalacc gttccaacaa 86 O attittcaaag atacct attc. cacaact cac agacittgacg ccaatcgatt gagaaacgtt 92 O agcaaattct tcgc.gcattt attgttcacc gacgc catcg gctgggaagit gct cqatatic 98 O atgaaattaa acgaggaaga caccalacagt to cagoagga ttitt catcaa gattttgttc 2O4. O Caggagttgt C caat at at gggattagcg aagttgaata aaaggctaala ggatgaaact 21OO ttacaggaat attitcgcggg gct attitc.cg agggatalacc Caagaacac gcgtttcgc.c 216 O atcaatttitt tdacgt.cgat cqgtttagga ggtctaacgg acgagttgag ggagc acttg 222 O aaaaacgtgc caaaac atct ggaagtgatg gctttgaaag Cagattic gag cagct ctago 228O agcagtagca gcagttcCag taacgattico agcagcagtt Cagattcttic catgacgag 234 O ggttcCagga agaagaaaac aaaaaaattgaaaac ccc.gg acaaaaagaa gaalacagaaa 24 OO gaagatgaaa aacccaaaaa gaaaag.cgag gataa accga ggaacaaagt aaatggtgat 246 O aagaatatag aaacagaaga tacacaagga gttgaggaca caaaaaagac td 2512

<210s, SEQ ID NO 89 &211s LENGTH: 424 212. TYPE: PRT <213> ORGANISM: Melligethes aeneus

<4 OOs, SEQUENCE: 89 Met Lieu. Arg Asn. Ile Lieu. His Glu Gly Glin Lieu. Glu Lys Arg Ile Glin 1. 5 1O 15 Tyr Met Ile Glu Val Met Phe Glin Val Arg Lys Asp Gly Phe Lys Asp 2O 25 3O His Ala Ala Val Thr Glu Glu Lieu. Asp Ile Val Glu Glu Glu Asp Glin 35 4 O 45 Phe Thr His Lieu. Ile Thr Lieu. Asp Asp Wall Lys Glin Ala Asn. Ser Glu SO 55 6 O

Asp Ile Lieu. Asn Val Phe Llys Phe Asp Asp Llys Tyr Glu Glu Asn. Glu 65 70 7s 8O

Gly Lys Tyr Lys Thr Lieu. Ser Lys Glu Ile Lieu. Glin Ser Asp Ser Glu 85 90 95

Ser Gly Glu Ser Gly Ser Glu Gly Ser Glu Glu Asp Ser Glu Asp Glu 1OO 105 11 O

Glu Gly Glu Glu Asp Glu Thir Lys Asn Glin Thir Ile Ile Asp Asn. Thir 115 12 O 125 US 2016/0 194658 A1 Jul. 7, 2016 72

- Continued

Glu Thir Asn Lieu. Ile Thr Lieu. Arg Arg Thr Ile Tyr Lieu. Thir Ile Glin 13 O 135 14 O Ser Ser Lieu. Asp Phe Glu Glu. Cys Ala His Llys Lieu Met Lys Met Glu 145 150 155 160 Ile Llys Pro Gly Glin Glu Ile Glu Lieu. Cys His Met Phe Lieu. Asp Cys 1.65 17O 17s Cys Ala Glu Glin Arg Thr Tyr Glu Lys Phe Phe Gly Lieu. Lieu. Ser Glin 18O 185 19 O Arg Phe Cys Glin Ile Asn Lys Thr Phe Ile Glu Pro Phe Glin Glin Ile 195 2OO 2O5 Phe Lys Asp Thir Tyr Ser Thir Thr His Arg Lieu. Asp Ala Asn Arg Lieu. 21 O 215 22O Arg Asn Val Ser Llys Phe Phe Ala His Lieu. Lieu. Phe Thr Asp Ala Ile 225 23 O 235 24 O Gly Trp Glu Val Lieu. Asp Ile Met Lys Lieu. Asn. Glu Glu Asp Thr Asn 245 250 255 Ser Ser Ser Arg Ile Phe Ile Lys Ile Leu Phe Glin Glu Lieu. Ser Glu 26 O 265 27 O Tyr Met Gly Lieu Ala Lys Lieu. Asn Lys Arg Lieu Lys Asp Glu Thir Lieu. 27s 28O 285 Glin Glu Tyr Phe Ala Gly Lieu. Phe Pro Arg Asp ASn Pro Lys Asn. Thir 29 O 295 3 OO Arg Phe Ala Ile ASn Phe Phe Thr Ser Ile Gly Lieu. Gly Gly Lieu. Thir 3. OS 310 315 32O Asp Glu Lieu. Arg Glu. His Lieu Lys Asn Val Pro Llys His Lieu. Glu Val 3.25 330 335 Met Ala Leu Lys Ala Asp Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 34 O 345 35. O Ser Ser Asn Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Asp Glu Gly 355 360 365 Ser Arg Llys Llys Llys Thr Llys Llys Lieu Lys Thr Pro Asp Llys Llys Llys 37 O 375 38O Lys Glin Lys Glu Asp Glu Lys Pro Llys Llys Llys Ser Glu Asp Llys Pro 385 390 395 4 OO Arg Asn Llys Val Asn Gly Asp Lys Asn. Ile Glu Thr Glu Asp Thr Glin 4 OS 41O 415 Gly Val Glu Asp Thr Llys Llys Thr 42O

<210s, SEQ ID NO 90 &211s LENGTH: 489 &212s. TYPE: DNA <213> ORGANISM: Melligethes aeneus

<4 OOs, SEQUENCE: 90 gttccttgat tdttgcgctgaac agcgtac ctacgaaaaa ttctitcqgcc ticct citcgca 6 O gcqcttctgc caaataaa.ca agactitt cat cqaac cqttic caacaaattt toaaagatac 12 O ctatt coaca act cacagac ttgacgc.caa togattgaga aacgittagca aattctt cqc 18O gcattt attgttcaccgacg C catcggctg ggaagtgctic gat at catga aattaaacga 24 O ggaagacacic alacagttcca gcaggattitt Cat Caagatt ttgttcCagg agttgtc.cga 3OO atatatggga ttagcgaagt taataaaag gctaaaggat gaaactttac aggaatattt 360 US 2016/0 194658 A1 Jul. 7, 2016 73

- Continued cgcggggcta titt CC9aggg at aacccgaa galacacgcgt titcgc.catca attittitt cac 42O gtcgat.cggt ttaggagg to taacggacga gttgagggag cacttgaaaa acgtgccaaa 48O a catctgga 489

<210s, SEQ ID NO 91 &211s LENGTH: 44 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: nom reg1 forward primer <4 OOs, SEQUENCE: 91 taatacgact Cactataggg agagttcCtt gattgttgcg Ctga 44

<210s, SEQ ID NO 92 &211s LENGTH: 45 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: nom reg1 reverse primer <4 OOs, SEQUENCE: 92 taatacgact cactataggg agatccagat gttittggcac gttitt 45

<210s, SEQ ID NO 93 & 211 LENGTH 2599 &212s. TYPE: DNA <213> ORGANISM: Melligethes aeneus <4 OOs, SEQUENCE: 93 ttitttgttac caaccaaacg gcc taaattt coctagataa cctaaaataa aaacaac act 6 O tcqt catttgttgtaaattica aacaaatgta gtcactacca ttgtagt gtt attcgtttct 12 O tgtgct tatt tittaattaac cagt caattic gtgtggtgta gtgaacaaag gttatagitta 18O tgacgacgga titcc.gagaga ggttc.cccta cagctg.cggc ticcacgcaga agcgc.ctica 24 O aatcgc.ca.ga accaaaaaaa gCaaagtacg ataagaaaga gaagggcgat aaagat.cgca 3OO agaggagat C C Cacagat Co agatctagat C cagggatag agaccatagg gacaaac atg 360 gtggaaaaaa acgttaccac gacctggacg acc ctitctga agact accca agatatt atg 42O gcgaggatag aaaacagaac agtgacagat attggtc.caa gtaccCaaag aaaga caggg 48O acgaatatgt tattggtagc cgg tattatg atgttgagga aaagaaggag aaaaaggaaa 54 O aagaggatga aaataaggat aaatc.cgt.ca ttact coaag ggaaaggaala acagtggact 6OO tact aa catc. tcgaac aggt gigggcttata tacct coagc taaattacgt atgatgcagg 660 ctgagataac tdataaatca totagotgcat atcaaagaat togcctgggaa gotttaaaaa 72 O agtic cattca tdgttacatc aacaaaatta acact tccaa tattggit citt attgctagag 78O aattactgca taaaacatt gtaagaggta gaggtttgct gtgtaaatct ataatacaag 84 O cacaggcago titcc.ccgaca ttcaccaatig tittatgcago tttagttgca gtcataaatt 9 OO caaaattic cc caa.cattgga gaactgttac taaaaggtt ggttittgcag tittaaaaggg 96.O gtttcaagca gaacaacaag totatctgta tat cqgctgc tacctttgtc gcgcatttag 1 O2O taaaccaaag agtggcc.cat gaaattittag cattggaaat tct tactitta cittgttgagt 108 O

CCCC cacaga tigatticagtg galagtagcaa titt cqtttitt gaaggaaagt ggtcaaaaac 114 O US 2016/0 194658 A1 Jul. 7, 2016 74

- Continued t cactgaggt gtcgagtaaa ggt at caatig C catatttga gatgttgagg aat attctgc 2OO atgaaggaca gttggagaag agaatacagt acatgattga agt catgttc Caagttcgga 26 O aagatggttt Caaggatcat gctgctgtta Ctgaagaact agatattgtt galagaggaag 32O atcagtttac to acctaatc acattggatg atgttaalaca agctaactica gaggatatat 38O tgaatgtgtt taaatttgat gataaatatg aggaaaatga gogg taaatac aaaactittaa 44 O gtaaggaaat t ct coagt ca gacagtgaat Caggcgaatc tigttcagag gggtctgaag SOO alagact cqga agatgaagaa ggtgaagaag atgaalaccala aaatcaaac C attattgata 560 acacagaaac taatttaatc accittaagga gaaccatcta t ct cacaata caatccagtt 62O tggattittga ggaatgtgcc cataa attga tigaaaatgga gatcaaacct gga caagaga 68O ttgaattgttgtcacatgttc cittgattgtt gcgctgaaca gcgtacctac gaaaaattct 74 O tcqgcct c ct citcgcagogc titctgccaaa taaacaagac titt catcgaa ccgttccaac 8OO aaattittcaa agatacct at tccacaactic acagacittga cqc caatcqa ttgagaaacg 86 O ttagcaaatt citt cqcgcat ttattgttca ccgacgc.cat cqgctgggaa gtgct cqata 92 O t catgaaatt aaacgaggaa gacaccaa.ca gttccagcag gattitt catc aagattttgt 98 O tccaggagtt gtc.cgaat at atgggattag caagttgaa taaaaggcta aaggatgaaa 2O4. O

Ctttacagga at attt cqcg gggct atttic Cagggataa ccc.gaagaac acgcgtttcg 21OO C Cat Caattt ttt Cacgt.cg atcggitttag gaggtctaac gigacgagttg agggagc act 216 O tgaaaaacgt gccaaaac at Ctggalagtga tiggctttgaa agcagatt.cg agcagcticta 222 O gcagcagtag cagcagttcc agtaacgatt C cagcagcag titcagattct tcc gatgacg 228O agggttccag gaagaagaaa acaaaaaaat taaaac ccc gga caaaaag aagaalacaga 234 O aagaagatga aaalacc caaa aagaaaag.cg aggataalacc gaggaacaaa C cagactata 24 OO gagatagaag aaacgacgac agggaaaagt ttaaaaaata Cagaaacaac gacgaagaaa 246 O gccacagaag aag cagagaa gatgcaa.gag aaaaatacag agg to acgag gaaagaagaa 252O gcgaccacag agaagaatac C9gcc.gagag alacatagagg tagagataga C9ttagttgt 2580 ataataatgt at attittitt 2599

<210s, SEQ ID NO 94 &211s LENGTH: 798 212. TYPE: PRT <213> ORGANISM: Melligethes aeneus <4 OOs, SEQUENCE: 94 Met Thr Thr Asp Ser Glu Arg Gly Ser Pro Thr Ala Ala Ala Pro Arg 1. 5 1O 15 Arg Ser Ala Ser Lys Ser Pro Glu Pro Llys Lys Ala Lys Tyr Asp Llys 2O 25 3O Lys Glu Lys Gly Asp Lys Asp Arg Lys Arg Arg Ser His Arg Ser Arg 35 4 O 45 Ser Arg Ser Arg Asp Arg Asp His Arg Asp Llys His Gly Gly Lys Llys SO 55 6 O Arg Tyr His Asp Lieu. Asp Asp Pro Ser Glu Asp Tyr Pro Arg Tyr Tyr 65 70 7s 8O

Gly Glu Asp Arg Lys Glin Asn. Ser Asp Arg Tyr Trp Ser Lys Tyr Pro 85 90 95 US 2016/0 194658 A1 Jul. 7, 2016 75

- Continued

Llys Lys Asp Arg Asp Glu Tyr Val Ile Gly Ser Arg Tyr Tyr Asp Wall 1OO 105 11 O Glu Glu Lys Lys Glu Lys Lys Glu Lys Glu Asp Glu Asn Lys Asp Llys 115 12 O 125 Ser Val Ile Thr Pro Arg Glu Arg Llys Thr Val Asp Lieu. Lieu. Thir Ser 13 O 135 14 O Arg Thr Gly Gly Ala Tyr Ile Pro Pro Ala Lys Lieu. Arg Met Met Glin 145 150 155 160 Ala Glu Ile Thr Asp Llys Ser Ser Ala Ala Tyr Glin Arg Ile Ala Trip 1.65 17O 17s Glu Ala Lieu Lys Llys Ser Ile His Gly Tyr Ile Asn Lys Ile Asn. Thir 18O 185 19 O Ser Asn. Ile Gly Lieu. Ile Ala Arg Glu Lieu. Lieu. His Glu Asn. Ile Val 195 2OO 2O5 Arg Gly Arg Gly Lieu. Lieu. Cys Llys Ser Ile Ile Glin Ala Glin Ala Ala 21 O 215 22O Ser Pro Thr Phe Thr Asn Val Tyr Ala Ala Leu Val Ala Val Ile Asn 225 23 O 235 24 O Ser Llys Phe Pro Asn. Ile Gly Glu Lieu. Lieu. Lieu Lys Arg Lieu Val Lieu. 245 250 255 Glin Phe Lys Arg Gly Phe Lys Glin Asn. Asn Llys Ser Ile Cys Ile Ser 26 O 265 27 O Ala Ala Thir Phe Val Ala His Lieu Val Asin Glin Arg Val Ala His Glu 27s 28O 285 Ile Lieu Ala Lieu. Glu Ile Lieu. Thir Lieu. Lieu Val Glu Ser Pro Thr Asp 29 O 295 3 OO Asp Ser Val Glu Val Ala Ile Ser Phe Lieu Lys Glu Ser Gly Glin Lys 3. OS 310 315 32O Lieu. Thr Glu Val Ser Ser Lys Gly Ile Asn Ala Ile Phe Glu Met Leu 3.25 330 335 Arg Asn. Ile Lieu. His Glu Gly Glin Lieu. Glu Lys Arg Ile Glin Tyr Met 34 O 345 35. O Ile Glu Val Met Phe Glin Val Arg Lys Asp Gly Phe Lys Asp His Ala 355 360 365 Ala Val Thr Glu Glu Lieu. Asp Ile Val Glu Glu Glu Asp Glin Phe Thr 37 O 375 38O His Lieu. Ile Thr Lieu. Asp Asp Wall Lys Glin Ala Asn. Ser Glu Asp Ile 385 390 395 4 OO Lieu. Asn Val Phe Llys Phe Asp Asp Llys Tyr Glu Glu Asn. Glu Gly Lys 4 OS 41O 415 Tyr Lys Thr Lieu. Ser Lys Glu Ile Lieu. Glin Ser Asp Ser Glu Ser Gly 42O 425 43 O

Glu Ser Gly Ser Glu Gly Ser Glu Glu Asp Ser Glu Asp Glu Glu Gly 435 44 O 445

Glu Glu Asp Glu Thir Lys Asn. Glin Thir Ile Ile Asp Asn Thr Glu Thir 450 45.5 460

Asn Lieu. Ile Thr Lieu. Arg Arg Thr Ile Tyr Lieu. Thir Ile Glin Ser Ser 465 470 47s 48O Lieu. Asp Phe Glu Glu. Cys Ala His Llys Lieu Met Lys Met Glu Ile Llys 485 490 495 US 2016/0 194658 A1 Jul. 7, 2016 76

- Continued Pro Gly Glin Glu Ile Glu Lieu. Cys His Met Phe Lieu. Asp Cys Cys Ala SOO 505 51O Glu Glin Arg Thr Tyr Glu Lys Phe Phe Gly Lieu Lleu Ser Glin Arg Phe 515 52O 525 Cys Glin Ile Asn Lys Thr Phe Ile Glu Pro Phe Glin Glin Ile Phe Lys 53 O 535 54 O Asp Thr Tyr Ser Thir Thr His Arg Lieu. Asp Ala Asn Arg Lieu. Arg Asn 5.45 550 555 560 Val Ser Llys Phe Phe Ala His Leu Lleu Phe Thr Asp Ala Ile Gly Trp 565 st O sts Glu Val Lieu. Asp Ile Met Lys Lieu. Asn. Glu Glu Asp Thr Asn. Ser Ser 58O 585 59 O Ser Arg Ile Phe Ile Lys Ile Leu Phe Glin Glu Lieu Ser Glu Tyr Met 595 6OO 605 Gly Lieu Ala Lys Lieu. Asn Lys Arg Lieu Lys Asp Glu Thir Lieu. Glin Glu 610 615 62O Tyr Phe Ala Gly Lieu. Phe Pro Arg Asp Asn Pro Lys Asn. Thir Arg Phe 625 630 635 64 O Ala Ile Asin Phe Phe Thr Ser Ile Gly Lieu. Gly Gly Lieu. Thir Asp Glu 645 650 655 Lieu. Arg Glu. His Lieu Lys Asn Val Pro Llys His Lieu. Glu Val Met Ala 660 665 67 O Lieu Lys Ala Asp Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 675 68O 685 Asn Asp Ser Ser Ser Ser Ser Asp Ser Ser Asp Asp Glu Gly Ser Arg 69 O. 695 7 OO Llys Llys Llys Thir Lys Llys Lieu Lys Thr Pro Asp Llys Llys Llys Lys Glin 7 Os 71O 71s 72O Lys Glu Asp Glu, Llys Pro Llys Llys Llys Ser Glu Asp Llys Pro Arg Asn 72 73 O 73 Llys Pro Asp Tyr Arg Asp Arg Arg Asn Asp Asp Arg Glu Lys Phe Lys 740 74. 7 O Llys Tyr Arg Asn. Asn Asp Glu Glu Ser His Arg Arg Ser Arg Glu Asp 7ss 760 765 Ala Arg Glu Lys Tyr Arg Gly His Glu Glu Arg Arg Ser Asp His Arg 770 775 78O Glu Glu Tyr Arg Pro Arg Glu. His Arg Gly Arg Asp Arg Arg 78s 79 O 79.

<210s, SEQ ID NO 95 &211s LENGTH: 1658 212. TYPE : RNA <213> ORGANISM: Melligethes aeneus

<4 OO > SEQUENCE: 95 gcaaluulu.cgu ulululugaagga aagugglucaa aaacuca clug aggugu.cgag lullaaaggulaulic 6 O aaugccaulau ulugagaluguu gaggaalualulu clugcaugaag gacagulugga gaagagaaua 12 O

Caguacaluga lulugaagucau guluccalagulu cqgaaagalug guuucaagga lucaugclugclu. 18O guuaculgaag aacuagaulau lugulugaagag galagalucagu uluaculcaccu. aalucacaulug 24 O gaugauguula aacaagculaa Clucagaggau alualuugaalug lugulululaalaulu ugalugaulaala 3OO ulaugaggaaa alugagggulaa aluacaaaacul lullaagua agg aaaluulcucca glucagacagul 360

US 2016/0 194658 A1 Jul. 7, 2016 81

- Continued augaaggaca guluggagaag agaaulacagul acalugauluga agucauguuc Caagulu.cgga 26 O alagalugglululu Caaggaucau gclugclugulua clugaagaacu agauauuguu galagaggaag 32O alucagulululac ulcacculaalluc acauluggalug auguulaalaca agcuaacuca gaggallalu.au 38O ugaauguguu ulaalaululugau gallaaalualug aggaaaaluga ggguaaauac aaaacuuluaia 44 O guaaggaaalu ulcuccagulca gacagugaalu Caggcgaaluc lugglulucagag gggluculgaag SOO alagacucgga agaugaagaa ggugaagaag alugaalaccala aaaucaaac C aluulaulugaula 560 acacagaaac ulaaluululaalluc accuulaagga galaccaucua luculcacalaula Caaluccagulu 62O luggaluuluuga ggaaugugcc caulaalauluga ugaaaalugga gaucaaaccu gga caagaga 68O ulugaaluugug ulcacauguuc culugauluguu go.gculgaaca gcgulaccuac gaaaaaulucu. 74 O ulcggccuccu. Cucgcagogc lulu.clugccalaa lullaaacaagac ululucaucgaa ccguluccaac 8OO aaaulululucaa agallac Cuau luccacaacuc acagacuuga CC caaucga lulugagaaacg 86 O ullagcaaalulu culu.cgc.gcau ulualuuguuca cc.gacgc.cau. C9gclugggala glugclu.cgaula 92 O ulcaugaalalulu aaacgaggaia gacac caa.ca guluccagcag gauluulucaulic aagaluuluugu 98 O luccaggagulu gluccgaaulau alugggaululag Caaguugaa lullaaaaggcula aaggaugaaa 2O4. O cuuulacagga alualuulu.cgcg gggculaululuc Cagggaluaa ccc.gaagaac acgcgululu.cg 21OO cCaucalaululu ululucacgucg aucgguuulag gagglucuaac ggacgagulug agggagcacul 216 O ugaaaaacgll gcc aaaac all cluggalaguga luggcuuugaa agcagallucg agcagclcula 2220 gcagcagulag cagcagulucci aguaiacgaluul C cagcagcag lulucagaulucu uccgalugacg 228O aggguluccag gaagaagaaa acaaaaaaau lugaaaac ccc gga caaaaag aagaalacaga 234 O aagaagaluga aaalacc caaa aagaaaag.cg aggallaalacc gaggaacaaa C cagaculaua 24 OO gagaluagaag aaacgacgac agggaaaagu lullaaaaaaua Cagaaacaac gacgaagaaa 246 O gccacagaag aag cagagaa gaugdaagag aaaaaluacag agglucacgag gaaagaagaa 252O gcgaccacag agaagaallac cqgcc.gagag alacaulagagg ulagagaluaga cquulagulugu 2580 aluaaluaalugu alualuuluuluu 2599

What may be claimed is: ing sequence of a Diabrotica organism comprising SEQ 1. An isolated nucleic acid comprising at least one poly ID NO:77; the complement of a native coding sequence nucleotide operably linked to a heterologous promoter, of a Diabrotica organism comprising SEQID NO:77; a fragment of at least 15 contiguous nucleotides of a native wherein the polynucleotide is selected from the group con coding sequence of a Diabrotica organism comprising sisting of: SEQID NO:77; the complement of a fragment of at least SEQ ID NO:1; the complement of SEQID NO:1; a frag 15 contiguous nucleotides of a native coding sequence ment of at least 15 contiguous nucleotides of SEQ ID of a Diabrotica organism comprising SEQID NO:77: NO:1; the complement of a fragment of at least 15 con SEQ ID NO:84; the complement of SEQ ID NO:84; a tiguous nucleotides of SEQ ID NO:1; a native coding fragment of at least 15 contiguous nucleotides of SEQ sequence of a Diabrotica organism comprising SEQID ID NO:84; the complement of a fragment of at least 15 NO:1; the complement of a native coding sequence of a contiguous nucleotides of SEQID NO:84; a native cod Diabrotica organism comprising SEQID NO:1; a frag ing sequence of a Melligethes organism comprising SEQ ment of at least 15 contiguous nucleotides of a native ID NO:84; the complement of a native coding sequence coding sequence of a Diabrotica organism comprising of a Melligethes organism comprising SEQID NO:84; a SEQID NO:1; the complement of a fragment of at least fragment of at least 15 contiguous nucleotides of a native 15 contiguous nucleotides of a native coding sequence coding sequence of a Melligethes organism comprising of a Diabrotica organism comprising SEQID NO:1; SEQID NO:84; the complement of a fragment of at least SEQ ID NO:77; the complement of SEQ ID NO:77; a 15 contiguous nucleotides of a native coding sequence fragment of at least 15 contiguous nucleotides of SEQ of a Melligethes organism comprising SEQID NO:84; ID NO:77; the complement of a fragment of at least 15 SEQ ID NO:86; the complement of SEQ ID NO:86; a contiguous nucleotides of SEQID NO:77; a native cod fragment of at least 15 contiguous nucleotides of SEQ US 2016/0 194658 A1 Jul. 7, 2016 82

ID NO:86; the complement of a fragment of at least 15 9. The double-stranded ribonucleic acid molecule of claim contiguous nucleotides of SEQID NO:86; a native cod 8, wherein contacting said ribonucleotide molecule with a ing sequence of a Melligethes organism comprising SEQ coleopteran pest kills or inhibits the growth, and/or feeding of ID NO:86; the complement of a native coding sequence the pest. of a Melligethes organism comprising SEQID NO:86; a 10. The double stranded RNA of claim 6, comprising a fragment of at least 15 contiguous nucleotides of a native first, a second and a third RNA segment, wherein the first coding sequence of a Melligethes organism comprising RNA segment comprises the polynucleotide, wherein the SEQID NO:86; the complement of a fragment of at least third RNA segment is linked to the first RNA segment by the 15 contiguous nucleotides of a native coding sequence second polynucleotide sequence, and wherein the third RNA of a Melligethes organism comprising SEQID NO:86: segment is substantially the reverse complement of the first SEQ ID NO:88; the complement of SEQ ID NO:88; a RNA segment, such that the first and the third RNA segments fragment of at least 15 contiguous nucleotides of SEQ hybridize when transcribed into a ribonucleic acid to form the ID NO:88; the complement of a fragment of at least 15 double-stranded RNA. contiguous nucleotides of SEQID NO:88; a native cod 11. The RNA of claim 6, selected from the group consisting ing sequence of a Melligethes organism comprising SEQ of a double-stranded ribonucleic acid molecule and a single ID NO:88; the complement of a native coding sequence stranded ribonucleic acid molecule of between about 15 and of a Melligethes organism comprising SEQID NO:88; a about 30 nucleotides in length. fragment of at least 15 contiguous nucleotides of a native 12. A plant transformation vector comprising the poly coding sequence of a Melligethes organism comprising nucleotide of claim 1, wherein the heterologous promoter is SEQID NO:88; the complement of a fragment of at least functional in a plant cell. 15 contiguous nucleotides of a native coding sequence 13. A cell transformed with the polynucleotide of claim 1. of a Melligethes organism comprising SEQID NO:88; 14. The cell of claim 13, wherein the cell is a prokaryotic cell. SEQ ID NO:93; the complement of SEQ ID NO:93; a 15. The cell of claim 13, wherein the cell is a eukaryotic fragment of at least 15 contiguous nucleotides of SEQ cell. ID NO:93; the complement of a fragment of at least 15 16. The cell of claim 15, wherein the cell is a plant cell. contiguous nucleotides of SEQID NO:93; a native cod 17. A plant transformed with the polynucleotide of claim 1. ing sequence of a Melligethes organism comprising SEQ 18. A seed of the plant of claim 17, wherein the seed ID NO:93; the complement of a native coding sequence comprises the polynucleotide. of a Melligethes organism comprising SEQID NO:93; a 19. A commodity product produced from the plant of claim fragment of at least 15 contiguous nucleotides of a native 17, wherein the commodity product comprises a detectable coding sequence of a Melligethes organism comprising amount of the polynucleotide. SEQID NO:93; and the complement of a fragment of at 20. The plant of claim 17, wherein the at least one poly least 15 contiguous nucleotides of a native coding nucleotide is expressed in the plant as a double-stranded sequence of a Melligethes organism comprising SEQID ribonucleic acid molecule. NO:93. 21. The cell of claim 16, wherein the cell is a Zea mays or 2. The polynucleotide of claim 1, wherein the polynucle Brassica napus cell. otide is selected from the group consisting of SEQID NO:1, 22. The plant of claim 17, wherein the plant is Zea mays or SEQID NO:3, SEQID NO:4, SEQID NO:5, SEQID NO:6, Brassica napus. SEQID NO:17, and SEQID NO:77, and the complements of 23. The plant of claim 17, wherein the at least one poly any of the foregoing. nucleotide is expressed in the plant as a ribonucleic acid 3. The polynucleotide of claim 1, wherein the polynucle molecule, and the ribonucleic acid molecule inhibits the otide is selected from the group consisting of SEQID NO:84, expression of an endogenous polynucleotide that is specifi SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID cally complementary to the at least one polynucleotide when NO:93, and the complements of any of the foregoing. a coleopteran pest ingests a part of the plant. 24. The polynucleotide of claim 1, further comprising at 4. A plant transformation vector comprising the polynucle least one additional polynucleotide that encodes an RNA otide of claim 1. molecule that inhibits the expression of an endogenous pest 5. The polynucleotide of claim 1, wherein the organism is gene. selected from the group consisting of D. v. virgifera LeConte; 25. A plant transformation vector comprising the poly D. barberi Smith and Lawrence; D. u. howardi. D. v. Zeae, D. nucleotide of claim 24, wherein the additional polynucleotide balteata LeConte; D. u. tenella, D. speciosa Germar, D. u. (s) are each operably linked to a heterologous promoter func undecimpunctata Mannerheim; and Melligethes aeneus Fab tional in a plant cell. 1C1US. 26. A method for controlling a coleopteran pest population, 6. A ribonucleic acid (RNA) molecule transcribed from the the method comprising providing an agent comprising a ribo polynucleotide of claim 1. nucleic acid (RNA) molecule that functions upon contact with the coleopteran pest to inhibit a biological function 7. A double-stranded ribonucleic acid molecule produced within the coleopteran pest, wherein the RNA is specifically from the expression of the polynucleotide of claim 1. hybridizable with a polynucleotide selected from the group 8. The double-stranded ribonucleic acid molecule of claim consisting of any of SEQID NOS:78-83; the complement of 7, wherein contacting the polynucleotide sequence with a any of SEQID NOS:78-83; a fragment of at least 15 contigu coleopteran pest inhibits the expression of an endogenous ous nucleotides of any of SEQ ID NOS:78-83; the comple nucleotide sequence specifically complementary to the poly ment of a fragment of at least 15 contiguous nucleotides of nucleotide. any of SEQ ID NOS:78-83; a transcript of any of SEQ ID US 2016/0 194658 A1 Jul. 7, 2016

NOs: 1, 3-6, and 77; the complement of a transcript of any of the complement of a transcript of any of SEQ ID NOS:1, SEQ ID NOS:1, 3-6, and 77; a fragment of at least 15 con 3-6, and 77: tiguous nucleotides of a transcript of SEQID NOs: 1 or 77; the a fragment of at least 15 contiguous nucleotides of a tran complement of a fragment of at least 15 contiguous nucle script of any of SEQID NOS:1, 3-6, and 77: otides of a transcript of SEQID NOS:1 or 77; SEQID NOs: the complement of a fragment of at least 15 contiguous 95-99; the complement of any of SEQ ID NOs:95-99; a nucleotides of a transcript of any of SEQID NOs: 1, 3-6, fragment of at least 15 contiguous nucleotides of any of SEQ and 77: ID NOs: 95-99; the complement of a fragment of at least 15 SEQID NOs:95-99; contiguous nucleotides of any of SEQID NOs:95-99; a tran the complement of any of SEQID NOs: 95-99; script of any of SEQ ID NOS:84, 86, 88,90, and 93; the a fragment of at least 15 contiguous nucleotides of any of complement of a transcript of any of SEQID NOS:84, 86, 88, SEQ ID NOs: 95-99; 90, and 93; a fragment of at least 15 contiguous nucleotides of the complement of a fragment of at least 15 contiguous a transcript of SEQ ID NOs:84, 86, 88,90, and 93; and the nucleotides of any of SEQID NOs:95-99; complement of a fragment of at least 15 contiguous nucle a transcript of any of SEQID NOs:78, 80, 82,90, and 93: otides of a transcript of SEQID NOS:84, 86, 88,90, and 93. the complement of a transcript of any of SEQID NOS:78, 27. The method according to claim 26, wherein the agent is 80, 82,90, and 93: a double-stranded RNA molecule. a fragment of at least 15 contiguous nucleotides of a tran 28. A method for controlling a coleopteran pest population, script of any of SEQID NOs:78,80, 82,90, and 93; and the method comprising: the complement of a fragment of at least 15 contiguous providing an agent comprising a first and a second poly nucleotides of a transcriptofany of SEQID NOS:78,80, nucleotide sequence that functions upon contact with the 82, 90, and 93. coleopteran pest to inhibit a biological function within 33. The method according to claim 32, wherein the diet the coleopteran pest, wherein the first polynucleotide comprises a plant cell transformed to express the polynucle sequence comprises a region that exhibits from about otide. 90% to about 100% sequence identity to from about 15 34. The method according to claim 32, wherein the specifi to about 30 contiguous nucleotides of a nucleic acid cally hybridizable RNA is comprised in a double-stranded selected from the group consisting of SEQ ID NO:78, RNA molecule. SEQID NO:83, SEQID NO:95, SEQID NO:96, SEQ 35. A method for improving the yield of a plant crop, the ID NO:97, and SEQ ID NO:99, and wherein the first method comprising: polynucleotide sequence is specifically hybridized to the introducing the nucleic acid of claim 1 into a plant to second polynucleotide sequence. produce a transgenic plant; and 29. A method for controlling a coleopteran pest population, cultivating the plant to allow the expression of the at least the method comprising: one polynucleotide; wherein expression of the at least providing in a host plant of a coleopteran pest a trans one polynucleotide inhibits coleopteran pest growth and formed plant cell comprising the polynucleotide of loss of yield due to coleopteran pest infection. claim 1, wherein the polynucleotide is expressed to pro 36. The method according to claim 35, wherein expression duce a ribonucleic acid molecule that functions upon of the at least one polynucleotide produces an RNA molecule contact with a coleopteran pest belonging to the popu that Suppresses at least a first target gene in a coleopteran pest lation to inhibit the expression of a target sequence that has contacted a portion of the corn plant. within the coleopteran pest and results in decreased 37. The method according to claim 35, wherein the plant is growth and/or Survival of the coleopteran pest or pest Zea mays or Brassica napus. population, relative to reproduction of the same pest 38. A method for producing a transgenic plant cell, the species on a plant of the same host plant species that does method comprising: not comprise the polynucleotide. transforming a plant cell with a vector comprising the 30. The method according to claim 29, wherein the ribo nucleic acid of claim 1: nucleic acid molecule is a double-stranded ribonucleic acid culturing the transformed plant cell under conditions Suf molecule. ficient to allow for development of a plant cell culture 31. The method according to claim 29, wherein the comprising a plurality of transformed plant cells; coleopteran pest population is reduced relative to a popula selecting for transformed plant cells that have integrated tion of the same pest species infesting a host plant of the same the at least one polynucleotide into their genomes; host plant species lacking the transformed plant cell. screening the transformed plant cells for expression of a 32. A method of controlling coleopteran pest infestation in ribonucleic acid (RNA) molecule encoded by the at least a plant, the method comprising providing in the diet of a one polynucleotide; and coleopteran pest a ribonucleic acid (RNA) that is specifically selecting a plant cell that expresses the RNA. hybridizable with a polynucleotide selected from the group 39. The method according to claim 38, wherein the RNA consisting of: molecule is a double-stranded RNA molecule. SEQ ID NOs:78-83; 40. A method for producing a coleopteran pest-resistant the complement of any of SEQID NOS:78–83: transgenic plant, the method comprising: a fragment of at least 15 contiguous nucleotides of any of providing the transgenic plant cell produced by the method SEQID NOs:78-83; of claim 38; and the complement of a fragment of at least 15 contiguous regenerating a transgenic plant from the transgenic plant nucleotides of any of SEQID NOS:78–83: cell, wherein expression of the ribonucleic acid mol a transcript of any of SEQID NOS:1, 3-6, and 77: ecule encoded by the at least one polynucleotide is suf US 2016/0 194658 A1 Jul. 7, 2016

ficient to modulate the expression of a target gene in a 46. The cell of claim 45, wherein the polypeptide from B. coleopteran pest that contacts the transformed plant. thuringiensis is selected from a group comprising Cry 1B, 41. A method for producing a transgenic plant cell, the Cry1 I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry 14, Cry 18, method comprising: Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Crys5, transforming a plant cell with a vector comprising a means Cyt1A, and Cyt2C. for providing coleopteran pest resistance to a plant; 47. The plant of claim 17, wherein the plant comprises a culturing the transformed plant cell under conditions Suf polynucleotide encoding a polypeptide from Bacillus thur ficient to allow for development of a plant cell culture ingiensis, Alcaligenes spp., or Pseudomonas spp. comprising a plurality of transformed plant cells; 48. The plant of claim 47, wherein the polypeptide from B. Selecting for transformed plant cells that have integrated thuringiensis is selected from a group comprising Cry 1B, the means for providing coleopteran pest resistance to a Cry1 I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry 14, Cry 18, plant into their genomes; Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Crys5, Screening the transformed plant cells for expression of a Cyt1A, and Cyt2C. means for inhibiting expression of an essential gene in a 49. The method according to claim 38, wherein the trans coleopteran pest, and formed plant cell comprises a nucleotide sequence encoding Selecting a plant cell that expresses the means for inhibiting a polypeptide from Bacillus thuringiensis, Alcaligenes spp., expression of an essential gene in a coleopteran pest. or Pseudomonas spp. 42. A method for producing a coleopteran pest-resistant 50. The method according to claim 49, wherein the transgenic plant, the method comprising: polypeptide from B. thuringiensis is selected from a group providing the transgenic plant cell produced by the method comprising Cry 1B, Cry1 I, Cry2A, Cry3, Cry7A, Cry8, of claim 41; and Cry9D, Cry 14, Cry 18, Cry22, Cry23, Cry34, Cry35, Cry36, regenerating a transgenic plant from the transgenic plant Cry37, Cry43, Crys5, Cyt1A, and Cyt2C. cell, wherein expression of the means for inhibiting 51. A method for improving the yield of a plant crop, the expression of an essential gene in a coleopteran pest is method comprising: Sufficient to modulate the expression of a target gene in contacting a coleopteran insect feeding on tissue of the a coleopteran pest that contacts the transformed plant. plant with a polynucleotide selected from the group 43. The nucleic acid of claim 1, further comprising a poly consisting of SEQIDNOs:78-83 and 95-99, the comple nucleotide encoding a polypeptide from Bacillus thuringien ments of SEQID NOs:78-83 and 95-99, and fragments sis, Alcaligenes spp., or Pseudomonas spp. having at least 15 nucleotides of any of the foregoing; 44. The nucleic acid of claim 43, wherein the polypeptide and from B. thuringiensis is selected from a group comprising contacting the coleopteran insect with a polynucleotide Cry1B, Cry1 I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry 14, encoding an insecticidal polypeptide from Bacillus thu Cry 18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, ringiensis, oran iRNA molecule targeting an insect gene Cry55, Cyt1A, and Cyt2C. other than incm. 45. The cell of claim 16, wherein the cell comprises a 52. The double stranded RNA of claim 10, wherein the polynucleotide encoding a polypeptide from Bacillus thur second polynucleotide comprises SEQID NO:19. ingiensis, Alcaligenes spp., or Pseudomonas spp. k k k k k