Compositions And Methods For Controlling Insect Pests

Abstract

Disclosed herein are methods of controlling insect pests which infest crop plants, in particular Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), Euschistus heros (neotropical brown stink bug), and Plutella xylostella (diamondback moth), and methods of providing plants resistant to such pests. Also disclosed are polynucleotides and recombinant DNA molecules and constructs useful in such methods, insecticidal compositions such as topical sprays containing insecticidal double-stranded RNAs, and plants with improved resistance to infestation by these insects. Further disclosed are methods of selecting target genes for RNAi-mediated silencing and control of these insect pests.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS

[0001] This application claims priority to U. S. Provisional Patent Application No. 61/973,484 filed 1 Apr. 2014, which is incorporated by reference in its entirety herein. The sequence listing contained in the file "40-21_70001_0000_US_ST25.txt" (49 kilobytes, created on 1 Apr. 2014, filed with U.S. Provisional Patent Application No. 61/973,484 on 1 Apr. 2014), and the replacement sequence listing contained in the file "40-21_70001_0000_US_ST25.txt" (49 kilobytes, created on 4 Apr. 2014, filed as a replacement sequence listing on 4 Apr. 2014 solely to correct the listing of inventors), are incorporated by in their entirety herein. The sequence listing contained in the file "40-21_70001_0000_WO_ST25.txt" (70 kilobytes, created on 13 Mar. 2015) is filed herewith and is incorporated by reference in its entirety herein.

FIELD OF THE INVENTION

[0002] Disclosed herein are methods for controlling invertebrate pest infestations, particularly in plants, and compositions and polynucleotides useful in such methods. More specifically, this invention is related to polynucleotides and methods of use thereof for modifying the expression of genes in an invertebrate pest, particularly through RNA interference. Pest species of interest include insects that infest crop plants, e. g., Plutella xylostella (diamondback moth), Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), and Euschistus heros (neotropical brown stink bug).

BACKGROUND OF THE INVENTION

[0003] Commercial crops are often the targets of attack by invertebrate pests such as insects. Compositions for controlling insect infestations in plants have typically been in the form of chemical insecticides. However, there are several disadvantages to using chemical insecticides. For example, chemical insecticides are generally not selective, and applications of chemical insecticides intended to control insect pests in crop plants can exert their effects on non-target insects and other invertebrates as well. Chemical insecticides often persist in the environment and can be slow to degrade, thus potentially accumulating in the food chain. Furthermore the use of persistent chemical insecticides can result in the development of resistance in the target insect species. Thus there has been a long felt need for more environmentally friendly methods for controlling or eradicating insect infestation on or in plants, i. e., methods which are species-selective, environmentally inert, non-persistent, and biodegradable, and that fit well into pest resistance management schemes.

[0004] Insecticidal compositions that include Bacillus thuringiensis ("Bt") bacteria have been commercially available and used as environmentally safe and acceptable insecticides for more than thirty years. The effectiveness of these compositions is due to insecticidal proteins that are produced exclusively by Bt bacteria. The insecticidal Bt proteins do not persist in the environment, are highly selective as to the target species affected, exert their effects only upon ingestion by a target insect, and have been shown to be harmless to plants and other non-targeted organisms, including humans and other vertebrates. Transgenic plants containing one or more recombinant genes encoding insecticidal Bt proteins are also available in the art and are resistant to insect pest infestation. One positive environmental result of the use of transgenic plants expressing Bt proteins is a decrease in the amount of chemical insecticides that are applied to control pest infestation in such transgenic crop fields, resulting in decreased contamination of soil and waters by non-degraded or excess chemical insecticides. In addition, there has been a noticeable increase in the numbers of beneficial insects in fields in which Bt protein-expressing transgenic crop plants are grown because of the decrease in the use of non-selective chemical insecticides.

[0005] RNA interference (RNAi, RNA-mediated gene suppression) is another approach used for pest control. In invertebrates RNAi-based gene suppression was first demonstrated in nematodes (Fire et al., (1998) Nature, 391:806-811; Timmons & Fire (1998) Nature, 395:854). Subsequently, RNAi-based suppression of invertebrate genes using recombinant nucleic acid techniques has been reported in a number of species, including agriculturally or economically important pests from various insect and nematode taxa. such as: root-knot nematodes (Meloidogyne spp.), see Huang et al. (2006) Proc. Natl. Acad. Sci. USA, 103:14302-14306, doi:10.1073/pnas.0604698103); cotton bollworm (Helicoverpa armigera), see Mao et al. (2007) Nature Biotechnol., 25:1307-1313, doi:10.1038/nbt1352; Western corn rootworm (Diabrotica virgifera LeConte), see Baum et al. (2007) Nature Biotechnol., 25:1322-1326, doi:10.1038/nbt1359; sugar beet cyst nematode (Heterodera schachtii), see Sindhu et al. (2008) J. Exp. Botany, 60:315-324, doi:10.1093/jxb/ern289; mosquito (Aedes aegypti), see Pridgeon et al. (2008) J. Med. Entomol., 45:414-420, doi: full/10.1603/0022-2585%282008%2945%5B414%3ATAADRK%5D2.0.00%3B2 fruit flies (Drosophila melanogaster), flour beetles (Triboliutn castaneum), pea aphids (Acyrthosiphon pisum), and tobacco hornworms (Manduca sexta), see Whyard et al. (2009) Insect Biochem. Mol. Biol., 39:824-832, doi:10.1016/j.ibmb.2009.09.00; diamondback moth (Plutella xylostella), see Gong et al. (2011) Pest Manag. Sci., 67: 514-520, doi:10.1002/ps.2086; green peach aphid (Myzus persicae), see Pilino et al. (2011) PLoS ONE, 6:e25709, doi:10.1371/journal.pone.0025709; brown planthopper (Nilaparvata lugens), see Li et al. (2011) Pest Manag. Sci., 67:852-859, doi:10.1002/ps.2124; and whitefly (Bemisia tabaci), see Upadhyay et al. (2011) J. Biosci., 36:153-161, doi:10.1007/s12038-011-9009-1.

[0006] This invention is related to methods of controlling insect pests, in particular insects which infest crop plants and have previously been found to be recalcitrant to RNA-mediated gene suppression methods, e. g., Plutella xylostella (diamondback moth), Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), and Euschistus heros (neotropical brown stink bug). Double-stranded RNA (dsRNA) trigger sequences have been identified for testing on the recalcitrant insect species Plutella xylostella (diamondback moth, DBM), Spodoptera frugiperda (fall armyworm, FAW), Lygus hesperus (western tarnished plant bug, WTPB), and Euschistus heros (neotropical brown stink bug, NBSB). These triggers are designed to suppress novel target genes that are putative orthologues of genes previously demonstrated to be efficacious targets for RNAi-mediated mortality in Western corn rootworm (Diabrotica virgifera).

[0007] This invention is further related to polynucleotides and recombinant DNA molecules and constructs useful in methods of controlling insect pests. This invention is further related to insecticidal compositions, as well as to transgenic plants resistant to infestation by insect pests. This invention is also related to methods of identifying efficacious double-stranded RNA triggers for controlling insect pests, and methods for identifying target genes that are likely to represent essential functions, making these genes preferred targets for RNAi-mediated silencing and control of insect pests.

SUMMARY OF THE INVENTION

[0008] This invention is related to control of insect species, especially those that are economically or agriculturally important pests. The compositions and methods of this invention include recombinant polynucleotide molecules, such as recombinant DNA constructs for making transgenic plants resistant to infestation by insect species and RNA "triggers" that are useful, e. g., as topically applied agents for causing RNAi-mediated suppression of a target gene in a insect species and thus controlling or preventing infestation by that insect species. Another utility of this invention is a polynucleotide-containing composition (e. g., a composition containing a dsRNA trigger for suppressing a target gene) that is topically applied to an insect species or to a plant, plant part, or seed to be protected from infestation by an insect species. This invention is further related to methods for selecting preferred insect target genes that arc more likely to be effective targets for RNAi-mediated control of an insect species.

[0009] In one aspect, this invention provides a method for controlling an insect infestation of a plant including contacting with a dsRNA an insect that infests a plant, wherein the dsRNA includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of the insect, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In embodiments, the dsRNA includes an RNA strand with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the dsRNA includes an RNA strand with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the dsRNA trigger suppresses a gene in the insect and stunts or kills the insect.

[0010] In another aspect, this invention provides a method of causing mortality or stunting in an insect, including providing in the diet of an insect at least one recombinant RNA including at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the recombinant RNA by the insect results in mortality or stunting in the insect. In embodiments, the silencing element includes an RNA strand with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the silencing clement includes an RNA strand with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0011] In another aspect, this invention provides an insecticidal composition including an insecticidally effective amount of a recombinant RNA molecule, wherein the recombinant RNA molecule includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarily with a fragment of a target gene of an insect that infests a plant, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In embodiments, the recombinant RNA molecule is dsRNA. In embodiments, the recombinant RNA molecule includes at least one segment (e. g., an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the recombinant RNA molecule includes at least one segment (e. g., an RNA strand or segment of an RNA strand) with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0012] In another aspect, this invention provides a method of providing a plant having improved resistance to an insect, including expressing in the plant a recombinant DNA construct including DNA encoding at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the recombinant RNA by the insect results in mortality or stunting in the insect. In embodiments, the silencing element is ssRNA. In other embodiments, the silencing element is dsRNA. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ Ill NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0013] In another aspect, this invention provides a recombinant DNA construct including a heterologous promoter, such as a heterologous promoter functional in a bacterial cell or in a eukaryotic cell (e. g., a plant cell or an insect cell), operably linked to DNA encoding an RNA transcript including a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the RNA transcript is ssRNA. In other embodiments, the RNA transcript is dsRNA. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the silencing element includes RNA (e. g., an RNA strand or segment of an RNA strand) with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0014] In related aspects, this invention provides man-made compositions including the polynucleotide or trigger of this invention, such as dsRNA formulations useful for topical application to a plant or substance in need of protection from an insect infestation, recombinant constructs and vectors useful for making transgenic plant cells and transgenic plants, formulations and coatings useful for treating plants (including plant seeds or propagatable parts such as tubers), plant seeds or propagatable parts such as tubers treated with or containing a polynucleotide of this invention as well as commodity products and foodstuffs produced from such plants, seeds, or propagatable parts (especially commodity products and foodstuffs having a detectable amount of a polynucleotide of this invention). A further aspect of this invention are polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44; another aspect of this invention are polyclonal or monoclonal antibodies that hind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 or the complement thereof. Such antibodies are made by routine methods as known to one of ordinary skill in the art.

[0015] Other aspects and specific embodiments of this invention are disclosed in the following detailed description and working Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 depicts the results of the transfection experiments described in Example 2 for the dsRNA triggers (TOP PANEL) T34640 (SEQ ID NO:17, targetting V-ATPase A subunit), (MIDDLE PANEL) T34642 (SEQ ID NO:18, targetting COPI coatomer beta subunit), or (BOTTOM PANEL) T34644 (SEQ ID NO:19, targetting COPI coatomer beta prime subunit) in Spodoptera frugipertla (fall armyworm, FAW) SF9 cells.

[0017] FIG. 2 depicts the results of the transfection experiments described in Example 2 for the dsRNA triggers (TOP PANEL) T42017 (SEQ ID NO:21, targetting V-ATPase subunit A), (TOP PANEL) T33310 (SEQ ID NO:29, targetting V-ATPase subunit A), (MIDDLE PANEL) T32938 (SEQ ID NO:28, targetting COPI coatomer beta subunit), and (BOTTOM PANEL) T32937 (SEQ ID NO:13, targetting COPI coatomer beta prime subunit) in Plutella xylostella (diamondback moth, DBM) cells.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used and the manufacturing or laboratory procedures described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, "The American Heritage.RTM. Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the "McGraw-Hill Dictionary of Scientific and Technical Terms" (6.sup.th edition, 2002, McGraw-Hill, New York), or the "Oxford Dictionary of Biology" (6.sup.th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only.

[0019] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction. One of skill in the art would be aware that a given DNA sequence is understood to define a corresponding RNA sequence which is identical to the DNA sequence except for replacement of the thymine (T) nucleotides of the DNA with uracil (U) nucleotides. Thus, providing a specific DNA sequence is understood to define the exact RNA equivalent. A given first polynucleotide sequence, whether DNA or RNA, further defines the sequence of its exact complement (which can be DNA or RNA), i. e., a second polynucleotide that hybridizes perfectly to the first polynucleotide by forming Watson-Crick base-pairs. By "essentially identical" or "essentially complementary" to a target gene or a fragment of a target gene is meant that a polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize (generally under physiological conditions such as those found in a living plant or animal cell) to a target gene or to a fragment of a target gene or to the transcript of the target gene or the fragment of a target gene; one of skill in the art would understand that such hybridization does not necessarily require 100% sequence identity or complementarity. A first nucleic acid sequence is "operably" connected or "linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter sequence is "operably linked" to DNA if the promoter provides for transcription or expression of the DNA. Generally, operably linked DNA sequences are contiguous.

[0020] The term "polynucleotide" commonly refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to "oligonucleotides" (a polynucleotide molecule of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Polynucleotides also include molecules containing multiple nucleotides including non-canonical nucleotides or chemically modified nucleotides as commonly practiced in the art; see, e. g., chemical modifications disclosed in the technical manual "RNA Interference (RNAi) and DsiRNAs", 2011 (Integrated DNA Technologies Coralville, Iowa). Generally, polynucleotides or triggers of this invention, whether DNA or RNA or both, and whether single- or double-stranded, include at least one segment of 18 or more contiguous nucleotides (or, in the case of double-stranded polynucleotides, at least 18 contiguous base-pairs) that are essentially identical or complementary to a fragment of equivalent size of the DNA of a target gene or the target gene's RNA transcript. Throughout this disclosure, "at least 18 contiguous" means "from about 18 to about 10,000, including every whole number point in between". Thus, embodiments of this invention include compositions including oligonucleotides having a length of 18-25 nucleotides (18-mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e. g., polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target gene including coding or non-coding or both coding and non-coding portions of the target gene). Where a polynucleotide is double-stranded, such as the dsRNA triggers described in the working Examples, its length can be similarly described in terms of base pairs. Double-stranded polynucleotides, such as the dsRNA triggers described in the working examples, can further be described in terms of one or more of the single-stranded components.

[0021] The polynucleotides or triggers of this invention are generally designed to suppress or silence one or more genes ("target genes"). The term "gene" refers to any portion of a nucleic acid that provides for expression of a transcript or encodes a transcript. A "gene" can include, but is not limited to, a promoter region, 5' untranslated regions, transcript encoding regions that can include intronic regions, 3' untranslated regions, or combinations of these regions. In embodiments, the target genes can include coding or non-coding sequence or both. In other embodiments, the target gene has a sequence identical to or complementary to a messenger RNA, e. g., in embodiments the target gene is a cDNA.

Controlling Insect Infestations of a Plant by Contacting with a dsRNA

[0022] A first aspect of this invention provides a method for controlling an insect infestation of a plant including contacting with a double-stranded RNA (dsRNA) an insect that infests a plant, wherein the dsRNA includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a target gene of the insect, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In this context "controlling" includes inducement of a physiological or behavioural change in an insect (adult or larvae or nymphs) such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. "Double-stranded" refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure, generally under physiologically relevant conditions.

[0023] In various embodiments, the insect is selected from the group consisting of Spodoptera spp., Lygus spp., Euschistus spp., and Plutella spp. Insects of particular interest include Spodoptera frugiperda (fall armyworm), Lygus hesperus (western tarnished plant bug), Euschistus hews (neotropical brown stink bug), and Plutella xylostella (diamondback moth).

[0024] Various embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; wherein the insect is Lygus hesperus (western tarnished plant bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; wherein the insect is Euschistus heros (neotropical brown stink bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; and wherein the insect is Plutella xylostella (diamondback moth) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:10-12. Other embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and the dsRNA includes a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Other embodiments include those where the dsRNA has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.

[0025] The plant can be any plant that is subject to infestation by an insect that can be controlled by the polynucleotides disclosed herein. Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading "Plants".

[0026] In embodiments of the method, the dsRNA includes multiple segments each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a target gene of the insect. For example, the dsRNA can include segments corresponding to different regions of the target gene, or can include multiple copies of a segment. In other embodiments of the method, the dsRNA includes multiple segments, each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a different target gene; in this way multiple target genes, or multiple insect species, can be suppressed.

[0027] In embodiments of the method, the dsRNA is blunt-ended. In other embodiments, the dsRNA has an overhang at one or both ends (termini); the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand. The dsRNA can be chemically synthesized, or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. The dsRNA can be chemically modified, e. g., to improve stability or efficacy.

[0028] In some embodiments of the method, the contacting includes application of a composition including the dsRNA to a surface of the insect or to a surface of the plant infested by the insect. The composition can include or be in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, or seed treatment. In embodiments, the composition can be applied to a seed, e. g., by soaking the seed in a liquid composition including the dsRNA, wherein the seed imbibes or takes up the dsRNA into the seed interior or seed endosperm in an effective amount to provide improved resistance to the insect pest by the seed or a plant or seedling grown from the seed. In embodiments, the contacting includes providing the dsRNA in a composition that further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. In embodiments, the contacting includes providing the dsRNA in a composition that further includes at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

[0029] In some embodiments of the method, the contacting includes providing the dsRNA in a composition that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. Such compositions can further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. Such compositions can further include at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In embodiments, the combination of the dsRNA and the pesticidal agent provides a level of insect control that is synergistic, i. e., greater than the sum of the effects of the dsRNA and the pesticidal agent components if tested separately.

Methods of Causing Mortality or Stunting in an Insect

[0030] Another aspect of this invention provides a method of causing mortality or stunting in an insect, including providing in the diet of an insect at least one recombinant RNA including at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the recombinant RNA by the insect results in mortality or stunting in the insect. The method is applicable to insects at various life stages. In embodiments, the method causes mortality or stunting in an insect larva or nymph. In other embodiments, the method causes mortality in adult insects.

[0031] In embodiments of the method the recombinant RNA includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. Embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; wherein the insect is Lygus hesperus (western tarnished plant bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; wherein the insect is Euschistus heros (neotropical brown stink bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; and wherein the insect is Plutella xylostella (diamondback moth) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:10-12. Other embodiments of the method include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and the silencing element includes a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Other embodiments include those where the recombinant RNA has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.

[0032] In embodiments of the method, the recombinant RNA is dsRNA. In these embodiments, the dsRNA can be blunt-ended dsRNA, or can be dsRNA with an overhang at one or both ends (termini); the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand. The dsRNA can be chemically synthesized, or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. The dsRNA can be chemically modified, e. g., to improve stability or efficacy. In embodiments of the method where the recombinant RNA is dsRNA, the dsRNA includes at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0033] In some embodiments of the method, the recombinant RNA is provided in the insect's diet in the form of an ingestible composition, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. Such ingestible compositions can further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. Such ingestible compositions can further include at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photothabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In embodiments, the combination of the recombinant RNA and the pesticidal agent provides a level of insect stunting or mortality that is synergistic, i. e., greater than the sum of the effects of the recombinant RNA and the pesticidal agent components if tested separately.

Insecticidal Compositions

[0034] Another aspect of the invention provides an insecticidal composition including an insecticidally effective amount of a recombinant RNA molecule, wherein the recombinant RNA molecule includes at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a target gene of an insect that infests a plant, and wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44. By "insecticidally effective" is meant effective in inducing a physiological or behavioural change in an insect (adult or larvae or nymphs) that infests a plant such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development; in embodiments, application of an insecticidally effective amount of the recombinant RNA molecule to a plant improves the plant's resistance to infestation by the insect.

[0035] In embodiments of the insecticidal composition, the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In specific embodiments, the recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In embodiments, the recombinant RNA molecule is a dsRNA of at least 50 base pairs in length. In embodiments of the method, the recombinant RNA molecule is dsRNA. In embodiments, the recombinant RNA molecule is a dsRNA which can be blunt-ended dsRNA, or can be dsRNA with an overhang at one or both ends (termini); the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand. In embodiments, the recombinant RNA molecule is a dsRNA which can be chemically synthesized, or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. In embodiments, the recombinant RNA molecule is a dsRNA of a length greater than that which is typical of naturally occurring regulatory small RNAs (such as endogenously produced siRNAs and mature mRNAs), i. e., the polynucleotide is double-stranded RNA of al least about 30 contiguous base-pairs in length. In embodiments, the recombinant RNA molecule is a dsRNA with a length of between about 50 to about 500 base-pairs.

[0036] Embodiments of the insecticidal composition include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; wherein the insect is Lygus hesperus (western tarnished plant bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; wherein the insect is Euschistus heros (neotropical brown stink bug) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; and wherein the insect is Plutella xylostella (diamondback moth) and the target gene includes a DNA sequence selected from the group consisting of SEQ ID NOs:10-12. Other embodiments of the insecticidal composition include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarily with a sequence selected from the group consisting of SEQ ID NOs:17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and the recombinant RNA molecule includes at least one RNA strand having a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Specific embodiments of the insecticidal composition include those wherein the insect is Spodoptera frugiperda (fall armyworm) and the recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs: 17-19; wherein the insect is Lygus hesperus (western tarnished plant bug) and recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; wherein the insect is Euschistus heros (neotropical brown stink bug) and recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:23-25; and wherein the insect is Plutella xylostella (diamondback moth) and recombinant RNA molecule is a dsRNA including an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29. Other embodiments of the insecticidal composition include those where the recombinant RNA molecule has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.

[0037] In various embodiments the insecticidal composition includes an insecticidally effective amount of a recombinant RNA molecule that consists of naturally occurring ribonucleotides, such as those found in naturally occurring RNA. In certain embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides. In certain embodiments, the polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S. Patent Publication 2011/0152353, T J.S. Patent Publication 2011/0152346, and U.S. Patent Publication 2011/0160082, which are herein incorporated by reference. Illustrative examples include, but arc not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide which can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (e. g., fluorescein or rhodamine) or other label (e. g., biotin).

[0038] The recombinant RNA molecule of is provided by suitable means known to one in the art. Embodiments include those wherein the recombinant RNA molecule is chemically synthesized (e.g., by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), produced by expression in a microorganism or in cell culture (such as plant or insect cells grown in culture), produced by expression in a plant cell, or produced by microbial fermentation.

[0039] In embodiments the recombinant RNA molecule of use in this method is provided as an isolated RNA fragment (not part of an expression construct, i. e., lacking additional elements such as a promoter or terminator sequences). Such recombinant RNA molecules can be relatively short, such as single- or double-stranded RNA molecules of between about 18 to about 300 or between about 50 to about 500 nucleotides (for single-stranded polynucleotides) or between about 18 to about 300 or between about 50 to about 500 base-pairs (for double-stranded polynucleotides). Embodiments include those in which the polynucleotide is a dsRNA including a segment having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0040] In embodiments, the insecticidal composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment. In embodiments, the insecticidal composition can be applied to a seed, e. g., by soaking the seed in a liquid insecticidal composition including the dsRNA, wherein the seed imbibes or takes up the dsRNA into the seed interior or seed endosperm in an effective amount to provide improved resistance to the insect pest by the seed or a plant or seedling grown from the seed. In some embodiments, the insecticidal composition is provided in a form that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. The insecticidal compositions can further include one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. The insecticidal compositions can further include at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. In embodiments, the combination of the recombinant RNA molecule and the pesticidal agent provides a level of insect control that is synergistic, i. e., greater than the sum of the effects of the recombinant RNA molecule and the pesticidal agent components if tested separately.

[0041] A related aspect of the invention is a plant treated with an insecticidal composition as described herein, or a seed of the treated plant, wherein the plant exhibits improved resistance to the insect. In embodiments, the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not treated with the insecticidal composition.

Methods of Providing Plants with Improved Insect Resistance

[0042] Another aspect of the invention provides a method of providing a plant having improved resistance to an insect, including expressing in the plant a recombinant DNA construct including DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the RNA by the insect results in mortality or stunting in the insect.

[0043] In embodiments of the method, the silencing element has a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 45, and 46. In specific embodiments, the silencing element is RNA that forms double-stranded RNA from two separate, essentially complementary strands, wherein at least one RNA strand includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 45, and 46. In other embodiments, the silencing clement is RNA that forms double-stranded RNA from a single self-hybridizing hairpin transcript, wherein one "arm" of the hairpin includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 45, and 46. Other embodiments include those where the silencing element has a sequence modified as described in Example 5 to eliminate matches to non-target organisms, such as the modified sequences (SEQ ID NOs:30-42) disclosed in Table 6.

[0044] In embodiments of the method, the recombinant DNA construct further includes a heterologous promoter operably linked to the DNA encoding RNA that includes at least one silencing element, wherein the heterologous promoter is functional in a plant cell. Promoters functional in a plant cell include those listed under the heading "Promoters".

[0045] In embodiments of the method, the recombinant DNA construct is expressed in the plant by means of transgenic expression or transient expression. In some embodiments, the method further includes expression in the plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. The pesticidal agent can be expressed from the same recombinant DNA construct that includes the DNA encoding at least one silencing element, or from a different recombinant DNA construct.

[0046] A related aspect of the invention is a plant having improved resistance to an insect, or the seed of such a plant, wherein the plant is provided by the method including expressing in the plant a recombinant DNA construct including DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of the RNA by the insect results in mortality or stunting in the insect. In embodiments, the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not treated with the insecticidal composition. Also encompassed by the invention are fruit, seed, or propagatable parts of the plant provided by this method and exhibiting improved resistance to the insect.

[0047] A related aspect of the invention is a plant or seedling having improved resistance to an insect, wherein the plant or seedling is grown from a seed treated with a recombinant DNA construct including DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44; alternatively the plant is grown from a seed directly treated with the RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44. In embodiments, the recombinant DNA construct (or the encoded RNA that includes at least one silencing element) is applied by soaking the seed in a liquid composition including the recombinant DNA construct (or the encoded RNA that includes at least one silencing element), wherein the seed imbibes or takes up the DNA or encoded RNA into the seed interior or seed endosperm in an effective amount to provide improved resistance to the insect pest by a plant or seedling grown from the seed.

Recombinant DNA Constructs Encoding RNA for Insect Control

[0048] Another aspect of the invention provides a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript including a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0049] In specific embodiments, the RNA transcript forms double-stranded RNA from two separate, essentially complementary strands (e. g., where one strand is encoded on a separate DNA construct or where the two strands are encoded on separate sections of the DNA encoding an RNA transcript, and which are separately transcribed or made separate, for example, by the action of a recombinase or nuclease), wherein at least one RNA strand includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In other embodiments, the RNA transcript forms double-stranded RNA from a single self-hybridizing hairpin transcript, wherein one "arm" of the hairpin includes a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

[0050] Embodiments of the recombinant DNA construct include those wherein the heterologous promoter is functional for expression of the RNA transcript in a bacterium. In embodiments where the recombinant DNA construct is to be expressed in a bacterium, the bacterium is selected from the group consisting of Escherichia coli, Bacillus species, Pseudomonas species, Xenorhabdus species, or Photorhabdus species. In other embodiments, the recombinant DNA construct includes a heterologous promoter that is functional in a plant cell. In embodiments, the recombinant DNA construct is contained in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In embodiments, the recombinant DNA construct is integrated into a plant chromosome or plastid, e. g., by stable transformation.

[0051] Related aspects of the invention include a transgenic plant cell having in its genome the recombinant DNA construct, and a transgenic plant including such a transgenic plant cell. Transgenic plant cells and plants are made by methods known in the art, such as those described under the heading "Making and Using Transgenic Plant Cells and Transgenic Plants". Further aspects of the invention include a commodity product produced from such a transgenic plant, and transgenic progeny seed or propagatable plant part of the transgenic plant.

Related Information and Techniques

Plants

[0052] The methods and compositions described herein for treating and protecting plants from insect infestations are useful across a broad range of plants. Suitable plants in which the methods and compositions disclosed herein can be used include, but are not limited to, cereals and forage grasses (rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilseed crops (soybean, oilseed brassicas including canola and oilseed rape, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (common bean, cowpea, pea, faba bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupine, alfalfa, and clovers), temperate fruits and nuts (apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut), tropical and subtropical fruits and nuts (citrus including limes, oranges, and grapefruit; banana and plantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit, and persimmon), vegetable crops (solanaceous plants including tomato, eggplant, and peppers; vegetable brassicas; radish, carrot, cucurbits, alliums, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turf grasses), and forest tree species.

Additional Construct Elements

[0053] Embodiments of the polynucleotides and nucleic acid molecules of this invention can include additional elements, such as promoters, small RNA recognition sites, aptamers or ribozymes, additional and additional expression cassettes for expressing coding sequences (e. g., to express a transgene such as an insecticidal protein or selectable marker) or non-coding sequences (e. g., to express additional suppression elements). For example, an aspect of this invention provides a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript that includes a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46. In another embodiment, a recombinant DNA construct including a promoter operably linked to DNA encoding: (a) an RNA transcript that includes a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46, and (b) an aptamer, is stably integrated into the plant's genome from where RNA transcripts including the RNA aptamer and the RNA silencing element are expressed in cells of the plant; the aptamer serves to guide the RNA silencing element to a desired location in the cell. In another embodiment, inclusion of one or more recognition sites for binding and cleavage by a small RNA (e. g., by a miRNA or an siRNA that is expressed only in a particular cell or tissue) allows for more precise expression patterns in a plant, wherein the expression of the recombinant DNA construct is suppressed where the small RNA is expressed. Such additional elements are described below.

Promoters

[0054] Promoters of use in the invention are functional in the cell in which the construct is intended to be transcribed. Generally these promoters are heterologous promoters, as used in recombinant constructs, i. e., they are not in nature found to be operably linked to the other nucleic elements used in the constructs of this invention. In various embodiments, the promoter is selected from the group consisting of a constitutive promoter, a spatially specific promoter, a temporally specific promoter, a developmentally specific promoter, and an inducible promoter. In many embodiments the promoter is a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.

[0055] Non-constitutive promoters suitable for use with the recombinant DNA constructs of this invention include spatially specific promoters, temporally specific promoters, and inducible promoters. Spatially specific promoters can include organelle-, cell-, tissue-, or organ-specific promoters (e.g., a plastid-specific, a root-specific, a pollen-specific, or a seed-specific promoter for expression in plastids, roots, pollen, or seeds, respectively). In many cases a seed-specific, embryo-specific, aleurone-specific, or endosperm-specific promoter is especially useful. Temporally specific promoters can include promoters that tend to promote expression during certain developmental stages in a plant's growth cycle, or during different times of day or night, or at different seasons in a year. Inducible promoters include promoters induced by chemicals or by environmental conditions such as, but not limited to, biotic or abiotic stress (e. g., water deficit or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection). MicroRNA promoters are useful, especially those having a temporally specific, spatially specific, or inducible expression pattern; examples of miRNA promoters, as well as methods for identifying miRNA promoters having specific expression patterns, are provided in U.S. Patent Application Publications 2006/0200878, 2007/0199095, and 2007/0300329, which are specifically incorporated herein by reference. An expression-specific promoter can also include promoters that are generally constitutively expressed but at differing degrees or "strengths" of expression, including promoters commonly regarded as "strong promoters" or as "weak promoters".

[0056] Promoters of particular interest include the following examples: an opaline synthase promoter isolated from T-DNA of Agrobacterium; a cauliflower mosaic virus 35S promoter; enhanced promoter elements or chimeric promoter elements such as an enhanced cauliflower mosaic virus (CaMV) 35S promoter linked to an enhancer element (an intron from heat shock protein 70 of Zea mays); root specific promoters such as those disclosed in U.S. Pat. Nos. 5,837,848; 6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in U.S. Pat. No. 6,433,252; a promoter for a plant nuclear gene encoding a plastid-localized aldolase disclosed in U.S. Patent Application Publication 2004/0216189; cold-inducible promoters disclosed in U.S. Pat. No. 6,084,089; salt-inducible promoters disclosed in U.S. Pat. No. 6,140,078; light-inducible promoters disclosed in U.S. Pat. No. 6,294,714; pathogen-inducible promoters disclosed in U.S. Pat. No. 6,252,138; and water deficit-inducible promoters disclosed in U.S. Patent Application Publication 2004/0123347 A1. All of the above-described patents and patent publications disclosing promoters and their use, especially in recombinant DNA constructs functional in plants are incorporated herein by reference.

[0057] Plant vascular- or phloem-specific promoters of interest include a rolC or rolA promoter of Agrobacterium rhizogenes, a promoter of a Agrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RSs1 gene promoter, a Commelina yellow mottle badnavirus promoter, a coconut foliar decay virus promoter, a rice tungro bacilliform virus promoter, the promoter of a pea glutamine synthase GS3A gene, a invCD111 and invCD141 promoters of a potato invertase genes, a promoter isolated from Arabidopsis shown to have phloem-specific expression in tobacco by Kertbundit et al. (1991) Proc. Natl. Acad. Sci. U S A., 88:5212-5216, a VAHOX1 promoter region, a pea cell wall invertase gene promoter, an acid invertase gene promoter from carrot, a promoter of a sulfate transporter gene Sultr1;3, a promoter of a plant sucrose synthase gene, and a promoter of a plant sucrose transporter gene.

[0058] Promoters suitable for use with a recombinant DNA construct or polynucleotide of this invention include polymerase II ("pol II") promoters and polymerase III ("pol III") promoters. RNA polymerase II transcribes structural or catalytic RNAs that arc usually shorter than 400 nucleotides in length, and recognizes a simple run of T residues as a termination signal; it has been used to transcribe siRNA duplexes (see, e. g., Lu et al. (2004) Nucleic Acids Res., 32:e171). Pol II promoters are therefore preferred in certain embodiments where a short RNA transcript is to be produced from a recombinant DNA construct of this invention. In one embodiment, the recombinant DNA construct includes a pol II promoter to express an RNA transcript flanked by self-cleaving ribozyme sequences (e. g., self-cleaving hammerhead ribozymes), resulting in a processed RNA, such as a single-stranded RNA that binds to the transcript of the Leptinotarsa target gene, with defined 5' and 3' ends, free of potentially interfering flanking sequences. An alternative approach uses pol III promoters to generate transcripts with relatively defined 5' and 3' ends, i. e., to transcribe an RNA with minimal 5' and 3' flanking sequences. In some embodiments, Pol III promoters (e. g., U6 or H1 promoters) are preferred for adding a short AT-rich transcription termination site that results in 2 base-pair overhangs (UU) in the transcribed RNA; this is useful, e. g., for expression of siRNA-type constructs. Use of pol III promoters for driving expression of siRNA constructs has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnol., 20: 446-448. Baculovirus promoters such as baculovirus polyhedrin and p10 promoters are known in the art and commercially available; see, e. g., Invitrogen's "Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques", 2002 (Life Technologies, Carlsbad, Calif.) and F. J. Haines et al. "Baculovirus Expression Vectors", undated (Oxford Expression Technologies, Oxford, UK).

[0059] The promoter clement can include nucleic acid sequences that arc not naturally occurring promoters or promoter elements or homologues thereof but that can regulate expression of a gene. Examples of such "gene independent" regulatory sequences include naturally occurring or artificially designed RNA sequences that include a ligand-binding region or aptamer (see "Aptamers", below) and a regulatory region (which can be cis-acting). See, for example, Isaacs et al. (2004) Nat. Biotechnol., 22:841-847, Bayer and Smolke (2005) Nature Biotechnol., 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463, Davidson and Ellington (2005) Trends Biotechnol., 23:109-112, Winkler et al. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker (2004) Nature Struct. Mol. Biol., 11:29-35. Such "riboregulators" could be selected or designed for specific spatial or temporal specificity, for example, to regulate translation of DNA that encodes a silencing element for suppressing a target gene only in the presence (or absence) of a given concentration of the appropriate ligand. One example is a riboregulator that is responsive to an endogenous ligand (e. g., jasmonic acid or salicylic acid) produced by the plant when under stress (e. g., abiotic stress such as water, temperature, or nutrient stress, or biotic stress such as attach by pests or pathogens); under stress, the level of endogenous ligand increases to a level sufficient for the riboregulator to begin transcription of the DNA that encodes a silencing element for suppressing a target gene.

Recombinase Sites

[0060] hi some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding one or more site-specific recombinase recognition sites. In one embodiment, the recombinant DNA construct includes at least a pair of loxP sites, wherein site-specific recombination of DNA between the loxP sites is mediated by a Cre recombinase. The position and relative orientation of the loxP sites is selected to achieve the desired recombination; for example, when the loxP sites are in the same orientation, the DNA between the loxP sites is excised in circular form. In another embodiment, the recombinant DNA construct includes DNA encoding one loxP site; in the presence of Cre recombinase and another DNA with a loxP site, the two DNAs are recombined.

Aptamers

[0061] In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA that is processed to an RNA aptamer, that is, an RNA that binds to a ligand through binding mechanism that is not primarily based on Watson-Crick base-pairing (in contrast, for example, to the base-pairing that occurs between complementary, anti-parallel nucleic acid strands to form a double-stranded nucleic acid structure). See, for example, Ellington and Szostak (1990) Nature, 346:818-822. Examples of aptamers can be found, for example, in the public Aptamer Database, available on line at aptamer.icmb.utexas.edu (Lee et al. (2004) Nucleic Acids Res., 32(1):D95-100). Aptamers useful in the invention can, however, be monovalent (binding a single ligand) or multivalent (binding more than one individual ligand, e. g., binding one unit of two or more different ligands).

[0062] Ligands useful in the invention include any molecule (or part of a molecule) that can be recognized and be bound by a nucleic acid secondary structure by a mechanism not primarily based on Watson-Crick base pairing. In this way, the recognition and binding of ligand and aptamer is analogous to that of antigen and antibody, or of biological effector and receptor. Ligands can include single molecules (or part of a molecule), or a combination of two or more molecules (or parts of a molecule), and can include one or more macromolecular complexes (e. g., polymers, lipid bilayers, liposomes, cellular membranes or other cellular structures, or cell surfaces). Examples of specific ligands include vitamins such as coenzyme B.sub.12 and thiamine pyrophosphate, flavin mononucleotide, guanine, adenosine, S-adenosylmethionine, S-adenosylhomocysteine, coenzyme A, lysine, tyrosine, dopamine, glucosamine-6-phosphate, caffeine, theophylline, antibiotics such as chloramphenicol and neomycin, herbicides such as glyphosate and dicamba, proteins including viral or phage coat proteins and invertebrate epidermal or digestive tract surface proteins, and RNAs including viral RNA, transfer-RNAs (t-RNAs), ribosomal RNA (rRNA), and RNA polymerases such as RNA-dependent RNA polymerase (RdRP). One class of RNA aptamers useful in the invention are "thermoswitches" that do not bind a ligand but are thermally responsive, that is to say, the aptamer's conformation is determined by temperature; see, for example, Box 3 in Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol., 5:451-463.

Transgene Transcription Units

[0063] In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes a transgene transcription unit. A transgene transcription unit includes DNA sequence encoding a gene of interest, e. g., a natural protein or a heterologous protein. A gene of interest can be any coding or non-coding sequence from any species (including, but not limited to, non-eukaryotes such as bacteria, and viruses; fungi, protists, plants, invertebrates, and vertebrates. Particular genes of interest are genes encoding at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein. The transgene transcription unit can further include 5' or 3' sequence or both as required for transcription of the transgene.

Introns

[0064] In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding a spliceable intron. By "intron" is generally meant a segment of DNA (or the RNA transcribed from such a segment) that is located between exons (protein-encoding segments of the DNA or corresponding transcribed RNA), wherein, during maturation of the messenger RNA, the intron present is enzymatically "spliced out" or removed from the RNA strand by a cleavage/ligation process that occurs in the nucleus in eukaryotes. The term "intron" is also applied to non-coding DNA sequences that are transcribed to RNA segments that can be spliced out of a maturing RNA transcript, but are not introns found between protein-coding exons. One example of these are spliceable sequences that that have the ability to enhance expression in plants (in some cases, especially in monocots) of a downstream coding sequence; these spliceable sequences are naturally located in the 5' untranslated region of some plant genes, as well as in some viral genes (e. g., the tobacco mosaic virus 5' leader sequence or "omega" leader described as enhancing expression in plant genes by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638). These spliceable sequences or "expression-enhancing introns" can be artificially inserted in the 5' untranslated region of a plant gene between the promoter and any protein-coding exons. Examples of such expression-enhancing introns include, but are not limited to, a maize alcohol dehydrogenase (Zm-Adh1), a maize Bronze-1 expression-enhancing intron, a rice actin 1 (Os-Act1) intron, a Shrunken-1 (Sh-1) intron, a maize sucrose synthase intron, a heat shock protein 18 (hsp18) intron, and an 82 kilodalton heat shock protein (hsp82) intron. U.S. Pat. Nos. 5,593,874 and 5,859,347, specifically incorporated by reference herein, describe methods of improving recombinant DNA constructs for use in plants by inclusion of an expression-enhancing intron derived from the 70 kilodalton maize heat shock protein (hsp70) in the non-translated leader positioned 3' from the gene promoter and 5' from the first protein-coding exon.

Ribozymes

[0065] In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding one or more ribozymes. Ribozymes of particular interest include a self-cleaving ribozyme, a hammerhead ribozyme, or a hairpin ribozyme. In one embodiment, the recombinant DNA construct includes DNA encoding one or more ribozymes that serve to cleave the transcribed RNA to provide defined segments of RNA, such as silencing elements for suppressing a Leptinotarsa target gene.

Gene Suppression Elements

[0066] In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding additional gene suppression element for suppressing a target gene other than a Leptinotarsa target gene. The target gene to be suppressed can include coding or non-coding sequence or both.

[0067] Suitable gene suppression elements are described in detail in U. S. Patent Application Publication 2006/0200878, which disclosure is specifically incorporated herein by reference, and include one or more of: [0068] (a) DNA that includes at least one anti-sense DNA segment that is anti-sense to at least one segment of the gene to be suppressed; [0069] (b) DNA that includes multiple copies of at least one anti-sense DNA segment that is anti-sense to at least one segment of the gene to be suppressed; [0070] (c) DNA that includes at least one sense DNA segment that is at least one segment of the gene to be suppressed; [0071] (d) DNA that includes multiple copies of at least one sense DNA segment that is at least one segment of the gene to be suppressed; [0072] (e) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming double-stranded RNA and includes at least one anti-sense DNA segment that is anti-sense to at least one segment of the gene to be suppressed and at least one sense DNA segment that is at least one segment of the gene to be suppressed; [0073] (f) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming a single double-stranded RNA and includes multiple serial anti-sense DNA segments that are anti-sense to at least one segment of the gene to he suppressed and multiple serial sense DNA segments that are at least one segment of the gene to be suppressed; [0074] (g) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming multiple double strands of RNA and includes multiple anti-sense DNA segments that are anti-sense to at least one segment of the gene to be suppressed and multiple sense DNA segments that are at least one segment of the gene to be suppressed, and wherein the multiple anti-sense DNA segments and the multiple sense DNA segments are arranged in a series of inverted repeats; [0075] (h) DNA that includes nucleotides derived from a plant miRNA; [0076] (i) DNA that includes nucleotides of a siRNA; [0077] (j) DNA that transcribes to an RNA aptamer capable of binding to a ligand; and [0078] (k) DNA that transcribes to an RNA aptamer capable of binding to a ligand, and DNA that transcribes to regulatory RNA capable of regulating expression of the gene to be suppressed, wherein the regulation is dependent on the conformation of the regulatory RNA, and the conformation of the regulatory RNA is allosterically affected by the binding slate of the RNA aptamer.

[0079] In some embodiments, an intron is used to deliver a gene suppression element in the absence of any protein-coding exons (coding sequence). In one example, an intron, such as an expression-enhancing intron (preferred in certain embodiments), is interrupted by embedding within the intron a gene suppression element, wherein, upon transcription, the gene suppression element is excised from the intron. Thus, protein-coding exons are not required to provide the gene suppressing function of the recombinant DNA constructs disclosed herein.

Transcription Regulatory Elements

[0080] In some embodiments, the recombinant DNA construct or polynucleotide of this invention includes DNA encoding a transcription regulatory element. Transcription regulatory elements include elements that regulate the expression level of the recombinant DNA construct of this invention (relative to its expression in the absence of such regulatory elements). Examples of suitable transcription regulatory elements include riboswitches (cis- or trans-acting), transcript stabilizing sequences, and miRNA recognition sites, as described in detail in U. S. Patent Application Publication 2006/0200878, specifically incorporated herein by reference.

Making and Using Transgenic Plant Cells and Transgenic Plants

[0081] Transformation of a plant can include any of several well-known methods and compositions. Suitable methods for plant transformation include virtually any method by which DNA can be introduced into a cell. One method of plant transformation is microprojectile bombardment, for example, as illustrated in U.S. Pat. No. 5,015,580 (soybean), U.S. Pat. No. 5,538,880 (maize), U.S. Pat. No. 5,550,318 (maize), U.S. Pat. No. 5,914,451 (soybean), U.S. Pat. No. 6,153,812 (wheat), U.S. Pat. No. 6,160,208 (maize), U.S. Pat. No. 6,288,312 (rice), U.S. Pat. No. 6,365,807 (rice), and U.S. Pat. No. 6,399,861 (maize), and U.S. Pat. No. 6,403,865 (maize), all of which are incorporated by reference for enabling the production of transgenic plants.

[0082] Another useful method of plant transformation is Agrobacterium-mediated transformation by means of Agrobacterium containing a binary Ti plasmid system, wherein the Agrobacterium carries a first Ti plasmid and a second, chimeric plasmid containing at least one T-DNA border of a wild-type Ti plasmid, a promoter functional in the transformed plant cell and operably linked to a polynucleotide or recombinant DNA construct of this invention. See, for example, the binary system described in U.S. Pat. No. 5,159,135, incorporated by reference. Also see De Framond (1983) Biotechnology, 1:262-269; and Hoekema et al., (1983) Nature, 303:179. In such a binary system, the smaller plasmid, containing the T-DNA border or borders, can be conveniently constructed and manipulated in a suitable alternative host, such as E. coli, and then transferred into Agrobacterium.

[0083] Detailed procedures for Agrobacterium-mediated transformation of plants, especially crop plants, include procedures disclosed in U.S. Pat. Nos. 5,004,863, 5,159,135, and 5,518,908 (cotton); U.S. Pat. Nos. 5,416,011, 5,569,834, 5,824,877 and 6,384,301 (soybean); U.S. Pat. Nos. 5,591,616 and 5,981,840 (maize); U.S. Pat. No. 5,463,174 (brassicas including canola), U.S. Pat. No. 7,026,528 (wheat), and U.S. Pat. No. 6,329,571 (rice), and in U.S. Patent Application Publications 2004/0244075 (maize) and 2001/0042257 A1 (sugar beet), all of which are specifically incorporated by reference for enabling the production of transgenic plants. U.S. Patent Application Publication 2011/0296555 discloses in Example 5 the transformation vectors (including the vector sequences) and detailed protocols for transforming maize, soybean, canola, cotton, and sugarcane) and is specifically incorporated by reference for enabling the production of transgenic plants. Similar methods have been reported for many plant species, both dicots and monocots, including, among others, peanut (Cheng et al. (1996) Plant Cell Rep., 15: 653); asparagus (Bytebier et al. (1987) Proc. Natl. Acad. Sci. U.S.A., 84:5345); barley (Wan and Lemaux (1994) Plant Physiol., 104:37); rice (Toriyama et al. (1988) Bio/Technology, 6:10; Zhang et al. (1988) Plant Cell Rep., 7:379; wheat (Vasil et al. (1992) Bio/Technology,10:667; Becker et al. (1994) Plant J. , 5:299), alfalfa (Masoud et al. (1996) Transgen. Res., 5:313); and tomato (Sun et al. (2006) Plant Cell Physiol., 47:426-431). Sec also a description of vectors, transformation methods, and production of transformed Arabidopsis thaliana plants where transcription factors are constitutively expressed by a CaMV35S promoter, in U.S. Patent Application Publication 2003/0167537 A1, incorporated by reference. Transformation methods specifically useful for solanaceous plants are well known in the art. See, for example, publicly described transformation methods for tomato (Sharma et al. (2009), J. Biosci., 34:423-433), eggplant (Arpaia et al. (1997) Theor. Appl. Genet., 95:329-334), potato (Bannerjee et al. (2006) Plant Sci., 170:732-738; Chakravarly et al. (2007) Amer. J. Potato Res., 84:301-311; S. Millam "Agrobacterium-mediated transformation of potato." Chapter 19 (pp.257-270), "Transgenic Crops of the World: Essential Protocols", Ian S. Curtis (editor), Springer, 2004), and peppers (Li et al. (2003) Plant Cell Reports, 21: 785-788). Stably transgenic potato, tomato, and eggplant have been commercially introduced in various regions; see, e. g., K. Redenbaugh et al. "Safety Assessment of Genetically Engineered Fruits and Vegetables: A Case Study of the FLAVR SAVR.TM. Tomato", CRC Press, Boca Raton, 1992, and the extensive publicly available documentation of commercial genetically modified crops in the GM Crop Database; see: CERA. (2012). GM Crop Database. Center for Environmental Risk Assessment (CERA), ILSI Research Foundation, Washington D.C., available electronically at www.cera-gmc.org/?action=gm_crop_database. Various methods of transformation of other plant species are well known in the art, see, for example, the encyclopedic reference, "Compendium of Transgenic Crop Plants", edited by Chittaranjan Kole and Timothy C. Hall, Blackwell Publishing Ltd., 2008; ISBN 978-1-405-16924-0 (available electronically at mrw.interscience.wiley.com/einrw/9781405181099/htp/toc), which describes transformation procedures for cereals and forage grasses (rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilseed crops (soybean, oilseed brassicas, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (common bean, cowpea, pea, faba bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupine, alfalfa, and clovers). temperate fruits and nuts (apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut), tropical and subtropical fruits and nuts (citrus, grapefruit, banana and plantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit, and persimmon), vegetable crops (tomato, eggplant, peppers, vegetable brassicas, radish, carrot, cucurbits, alliums, asparagus, and leafy vegetables), sugar, tuber, and fiber crops (sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turf grasses), and forest tree species.

[0084] Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated recombinant DNA are preferably practiced in tissue culture on media and in a controlled environment. "Media" refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos or parts of embryos, and gametic cells such as microspores, pollen, sperm, and egg cells. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of this invention. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention (e. g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004/0216189, which are specifically incorporated by reference.

[0085] In general transformation practice, DNA is introduced into only a small percentage of target cells in any one transformation experiment. Marker genes are generally used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers which confer resistance to a selective agent, such as an antibiotic or herbicide. Any of the antibiotics or herbicides to which a plant cell is resistant can be a useful agent for selection. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene is integrated and expressed at sufficient levels to permit cell survival. Cells can be tested further to confirm stable integration of the recombinant DNA. Commonly used selective marker genes include those conferring resistance to antibiotics such as kanamycin or paromomycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of useful selective marker genes and selection agents are illustrated in U.S. Pat. Nos. 5,550,318, 5,633,435, 5,780,708, and 6,118,047, all of which are specifically incorporated by reference. Screenable markers or reporters, such as markers that provide an ability to visually identify transformants can also be employed. Examples of useful screenable markers include, for example, a gene expressing a protein that produces a detectable color by acting on a chromogenic substrate (e. g., beta glucuronidase (GUS) (uidA) or luciferase (kw)) or that itself is detectable, such as green fluorescent protein (GFP) (gfp) or an immunogenic molecule. Those of skill in the art will recognize that many other useful markers or reporters are available for use.

[0086] Detecting or measuring transcription of a recombinant DNA construct in a transgenic plant cell can be achieved by any suitable method, including protein detection methods (e. g., western blots, ELISAs, and other immunochemical methods), measurements of enzymatic activity, or nucleic acid detection methods (e. g., Southern blots, northern blots, PCR, RT-PCR, fluorescent in situ hybridization).

[0087] Other suitable methods for detecting or measuring transcription in a plant cell of a recombinant polynucleotide of this invention targetting an insect target gene include measurement of any other trait that is a direct or proxy indication of the level of expression of the target gene in the insect, relative to the level of expression observed in the absence of the recombinant polynucleotide, e. g., growth rates, mortality rates, or reproductive or recruitment rates of the insect, or measurements of injury (e. g., root injury) or yield loss in a plant or field of plants infested by the insect. In general, suitable methods for detecting or measuring transcription in a plant cell of a recombinant polynucleotide of interest include, e. g., gross or microscopic morphological traits, growth rates, yield, reproductive or recruitment rates, resistance to pests or pathogens, or resistance to biotic or abiotic stress (e. g., water deficit stress, salt stress, nutrient stress, heat or cold stress). Such methods can use direct measurements of a phenotypic trait or proxy assays (e. g., in plants, these assays include plant part assays such as leaf or root assays to determine tolerance of abiotic stress). Such methods include direct measurements of resistance to an invertebrate pest or pathogen (e. g., damage to plant tissues) or proxy assays (e. g., plant yield assays, or invertebrate pest bioassays such as the Western corn rootworm (Diabrotica virgifera virgifera LeConte) larval bioassay described in International Patent Application Publication WO2005/110068 A2 and U. S. Patent Application Publication US 2006/0021087 A1, specifically incorporated by reference, or the soybean cyst nematode bioassay described by Steeves et al. (2006) Funci. Plant Biol., 33:991-999, wherein cysts per plant, cysts per gram root, eggs per plant, eggs per gram root, and eggs per cyst are measured, or the bioassays described herein in the working Examples.

[0088] The recombinant DNA constructs of this invention can be stacked with other recombinant DNA for imparting additional traits (e. g., in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, and the like) for example, by expressing or suppressing other genes. Constructs for coordinated decrease and increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 A1, specifically incorporated by reference.

[0089] Seeds of fertile transgenic plants can be harvested and used to grow progeny generations, including hybrid generations, of transgenic plants of this invention that include the recombinant DNA construct in their genome. Thus, in addition to direct transformation of a plant with a recombinant DNA construct of this invention, transgenic plants of this invention can be prepared by crossing a first plant having the recombinant DNA with a second plant lacking the construct. For example, the recombinant DNA can be introduced into a plant line that is amenable to transformation to produce a transgenic plant, which can be crossed with a second plant line to introgress the recombinant DNA into the resulting progeny. A transgenic plant of this invention can be crossed with a plant line having other recombinant DNA that confers one or more additional trait(s) (such as, but not limited to, herbicide resistance, pest or disease resistance, environmental stress resistance, modified nutrient content, and yield improvement) to produce progeny plants having recombinant DNA that confers both the desired target sequence expression behavior and the additional trait(s).

[0090] In such breeding for combining traits the transgenic plant donating the additional trait can be a male line (pollinator) and the transgenic plant carrying the base traits can be the female line. The progeny of this cross segregate such that some of the plant will carry the DNA for both parental traits and some will carry DNA for one parental trait; such plants can be identified by markers associated with parental recombinant DNA Progeny plants carrying DNA for both parental traits can be crossed back into the female parent line multiple times, e. g., usually 6 to 8 generations, to produce a homozygous progeny plant with substantially the same genotype as one original transgenic parental line as well as the recombinant DNA of the other transgenic parental line.

[0091] Yet another aspect of this invention is a transgenic plant grown from the transgenic seed of this invention. This invention contemplates transgenic plants grown directly from transgenic seed containing the recombinant DNA as well as progeny generations of plants, including inbred or hybrid plant lines, made by crossing a transgenic plant grown directly from transgenic seed to a second plant not grown from the same transgenic seed. Crossing can include, for example, the following steps: [0092] (a) plant seeds of the first parent plant (e. g., non-transgenic or a transgenic) and a second parent plant that is transgenic according to the invention; [0093] (b) grow the seeds of the first and second parent plants into plants that bear flowers; [0094] (c) pollinate a flower from the first parent with pollen from the second parent; and [0095] (d) harvest seeds produced on the parent plant hearing the fertilized flower.

[0096] It is often desirable to introgress recombinant DNA into elite varieties, e. g., by backcrossing, to transfer a specific desirable trait from one source to an inbred or other plant that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred ("A") (recurrent parent) to a donor inbred ("B") (non-recurrent parent), which carries the appropriate gene(s) for the trait in question, for example, a construct prepared in accordance with the current invention. The progeny of this cross first are selected in the resultant progeny for the desired trait to be transferred from the non-recurrent parent "B", and then the selected progeny arc mated back to the superior recurrent parent "A". After five or more backcross generations with selection for the desired trait, the progeny can be essentially hemizygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes. The last backcross generation would be selfed to give progeny which are pure breeding for the gene(s) being transferred, i. e., one or more transformation events.

[0097] Through a series of breeding manipulations, a selected DNA construct can be moved from one line into an entirely different line without the need for further recombinant manipulation. One can thus produce inbred plants which are true breeding for one or more DNA constructs. By crossing different inbred plants, one can produce a large number of different hybrids with different combinations of DNA constructs. In this way, plants can be produced which have the desirable agronomic properties frequently associated with hybrids ("hybrid vigor"), as well as the desirable characteristics imparted by one or more DNA constructs.

[0098] In certain transgenic plant cells and transgenic plants of this invention, it is sometimes desirable to concurrently express a gene of interest while also modulating expression of a Leptinotarsa target gene. Thus, in some embodiments, the transgenic plant contains recombinant DNA further including a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct of this invention is preferably effected with concurrent transcription of the gene expression element.

[0099] In some embodiments, the recombinant DNA constructs of this invention can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage. Transgenic plants can be derived from any monocot or dicot plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood- or pulp-producing trees, vegetable plants, fruit plants, and ornamental plants. Examples of plants of interest include grain crop plants (such as wheat, oat, barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicots including alfalfa, vetch, clover, and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee; wood- or pulp-producing trees; vegetable crop plants such as legumes (for example, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, the brassicas (for example, cabbages, kales, mustards, and other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example, cucumbers, melons, summer squashes, winter squashes), edible alliums (for example, onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers, groundcherries), and edible members of the Chenopodiaceae (for example, beet, chard, spinach, quinoa, amaranth); fruit crop plants such as apple, pear, citrus fruits (for example, orange, lime, lemon, grapefruit, and others), stone fruits (for example, apricot, peach, plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado, and berries; plants grown for biomass or biofuel (for example, Miscanthus grasses, switchgrass, jatropha, oil palm, eukaryotic microalgae such as Botryococcus braunii, Chlorella spp., and Dunaliella spp., and eukaryotic macroalgae such as Gracilaria spp., and Sargassum spp.); and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental groundcovers, and ornamental grasses.

[0100] This invention also provides commodity products produced from a transgenic plant cell, plant, or seed of this invention, including, but not limited to harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts of a plant, meals, oils, extracts, fermentation or digestion products, crushed or whole grains or seeds of a plant, or any food or non-food product including such commodity products produced from a transgenic plant cell, plant, or seed of this invention. The detection of one or more of nucleic acid sequences of the recombinant DNA constructs of this invention in one or more commodity or commodity products contemplated herein is de facto evidence that the commodity or commodity product contains or is derived from a transgenic plant cell, plant, or seed of this invention.

[0101] Generally a transgenic plant having in its genome a recombinant DNA construct of this invention exhibits increased resistance to an insect infestation. In various embodiments, for example, where the transgenic plant expresses a recombinant DNA construct of this invention that is stacked with other recombinant DNA for imparting additional traits, the transgenic plant has at least one additional altered trait, relative to a plant lacking the recombinant DNA construct, selected from the group of traits consisting of:

[0102] (a) improved abiotic stress tolerance;

[0103] (b) improved biotic stress tolerance;

[0104] (c) modified primary metabolite composition;

[0105] (d) modified secondary metabolite composition;

[0106] (e) modified trace element, carotenoid, or vitamin composition;

[0107] (f) improved yield;

[0108] (g) improved ability to use nitrogen, phosphate, or other nutrients;

[0109] (h) modified agronomic characteristics;

[0110] (i) modified growth or reproductive characteristics; and

[0111] (j) improved harvest, storage, or processing quality.

[0112] In some embodiments, the transgenic plant is characterized by: improved tolerance of abiotic stress (e. g., tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or of biotic stress (e. g., crowding, allelopathy, or wounding); by a modified primary metabolite (e. g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition; a modified secondary metabolite (e. g., alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition; a modified trace element (e. g., iron, zinc), carotenoid (e. g., beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e. g., tocopherols) composition; improved yield (e. g., improved yield under non-stress conditions or improved yield under biotic or abiotic stress); improved ability to use nitrogen, phosphate, or other nutrients; modified agronomic characteristics (e. g., delayed ripening; delayed senescence; earlier or later maturity; improved shade tolerance; improved resistance to root or stalk lodging; improved resistance to "green snap" of stems; modified photoperiod response); modified growth or reproductive characteristics (e. g., intentional dwarfing; intentional male sterility, useful, e. g., in improved hybridization procedures; improved vegetative growth rate; improved germination; improved male or female fertility); improved harvest, storage, or processing quality (e. g., improved resistance to pests during storage, improved resistance to breakage, improved appeal to consumers); or any combination of these traits.

[0113] In another embodiment, transgenic seed, or seed produced by the transgenic plant, has modified primary metabolite (e. g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition, a modified secondary metabolite composition, a modified trace element, carotenoid, or vitamin composition, an improved harvest, storage, or processing quality, or a combination of these. In another embodiment, it can be desirable to change levels of native components of the transgenic plant or seed of a transgenic plant, for example, to decrease levels of an allergenic protein or glycoprotein or of a toxic metabolite.

[0114] Generally, screening a population of transgenic plants each regenerated from a transgenic plant cell is performed to identify transgenic plant cells that develop into transgenic plants having the desired trait. The transgenic plants are assayed to detect an enhanced trait, e. g., enhanced water use efficiency, enhanced cold tolerance, increased yield, enhanced nitrogen use efficiency, enhanced seed protein, and enhanced seed oil. Screening methods include direct screening for the trait in a greenhouse or field trial or screening for a surrogate trait. Such analyses are directed to detecting changes in the chemical composition, biomass, physiological properties, or morphology of the plant. Changes in chemical compositions such as nutritional composition of grain are detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch, tocopherols, or other nutrients. Changes in growth or biomass characteristics are detected by measuring plant height, stem diameter, internode length, root and shoot dry weights, and (for grain-producing plants such as maize, rice, or wheat) ear or seed head length and diameter. Changes in physiological properties are identified by evaluating responses to stress conditions, e. g., assays under imposed stress conditions such as water deficit, nitrogen or phosphate deficiency, cold or hot growing conditions, pathogen or insect attack, light deficiency, or increased plant density. Other selection properties include days to flowering, days to pollen shed, days to fruit maturation, fruit or tuber quality or amount produced, days to silking in maize, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, staying green, stalk lodging, root lodging, plant health, fertility, green snap, and pest resistance. In addition, phenotypic characteristics of harvested fruit, seeds, or tubers can be evaluated.

EXAMPLES

Example 1

[0115] This example illustrates non-limiting embodiments of coding DNA sequences useful as target genes for controlling insect species and for making compositions and plants of this invention, and identifies dsRNA trigger sequences useful for controlling insect species. Orthologues to genes previously demonstrated to be efficacious targets for RNAi-mediated mortality in western corn rootworm were identified from insect species that have not previously been reported to be susceptible to orally delivered RNA. These orthologous target genes and examples of dsRNA trigger sequences are provided in Table 1.

TABLE-US-00001 TABLE 1 Target Gene Trigger SEQ ID Target Target Gene Source Species Target Gene Trigger SEQ ID Trigger NO. Gene ID (common name) Annotation ID NO.* size (bp) 1 CG3762 Spodoptera frugiperda V-ATPase subunit A T34640 17 300 (fall armyworm) 2 F38E11 Spodoptera frugiperda COPI coatomer T34644 19 301 (fall armyworm) beta prime subunit 3 CG6223 Spodoptera frugiperda COPI coatomer T34642 18 301 (fall armyworm) beta subunit 4 CG3762 Lygus hesperus V-ATPase subunit A T34617 15 300 (western tarnished plant bug) 5 CG2746 Lygus hesperus ribosomal protein T34616 14 298 (western tarnished plant bug) L19 6 F38E11 Lygus hesperus COPI coatomer T34622 16 300 (western tarnished plant bug) beta prime subunit 7 AT20865p Lygus hesperus ubiquitin C T42772 22 257 (western tarnished plant bug) 7 AT20865p Lygus hesperus ubiquitin C T44045 26 302 (western tarnished plant bug) 8 CG10370 Euschistus heros TAT binding T43718 23 300 (neotropical brown stink bug) protein 9 AT20865p Euschistus heros ubiquitin C T43720 24 300 (neotropical brown stink bug) 9 AT20865p Euschistus heros ubiquitin C T44042 25 302 (neotropical brown stink bug) 10 CG3762 Plutella xylostella V-ATPase subunit A T42014 20 302 (diamondback moth) 10 CG3762 Plutella xylostella V-ATPase subunit A T42017 21 301 (diamondback moth) 10 CG3762 Plutella xylostella V-ATPase subunit A 133310 29 300 (diamondback moth) 11 F38E11.5 Plutella xylostella COPI coatomer T32937 13 302 (diamondback moth) beta prime subunit 12 CG6223 Plutella xylostella COPI coatomer T32938 28 302 (diamondback moth) beta subunit 43 CG6223 Lygus hesperus COPI coatomer T34619 45 301 (western tarnished plant bug) beta subunit 44 AT20865p Lygus hesperus ubiquitin C variant T42768 46 255 (western tarnished plant bug) (fragment) *Trigger sequences are provided for the sense strand of the dsRNA trigger in 5' to 3' direction.

[0116] The dsRNA trigger sequences that are confirmed to be effective in suppressing a target gene in a sequence-specific manner are useful for identifying efficacious RNA delivery agents and formulations. The insecticidal activity of formulations containing the dsRNA triggers can be optimized by various techniques, such as modifying the chemical entities in the formulation or modifying the ratio of the chemical components in the formulation. Non-limiting examples of delivery agents and formulations are provided in Examples 6 and 8.

Example 2

[0117] This example illustrates non-limiting embodiments of dsRNA trigger sequences useful for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect, and methods for validating dsRNA trigger efficacy or for causing stunting or mortality in an insect. More specifically this example illustrates use of dsRNA triggers for sequence-specific reduction of target gene mRNA transcript level in insect cells.

[0118] Cultured Spodoptera frugiperda (fall armyworm, FAW) SF9 cells were incubated with the dsRNA triggers T34640 (SEQ ID NO:17, targetting V-ATPase A subunit), T34642 (SEQ Ill NO:18, targetting COPI coatomer B (beta) subunit), or T34644 (SEQ ID NO:19, targetting COPI coatomer (beta prime) subunit), or with a control trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent protein); see Table 1. The dsRNA triggers were formulated with the commercial transfection agent Cellfectin.RTM. II (Life Technologies, Inc., Grand Island, N.Y. 14072). FIG. 1 depicts the results of the transfection experiments, demonstrating target-gene-specific suppression (reduction of target gene mRNA levels measured by Quantigene assays) by the dsRNA triggers in the insect cells. The control dsRNA trigger T35763 had no effect on the targetted insect gene mRNA levels.

[0119] Similar experiments were carried out with Plutella xylostella (diamondback moth, DBM) cells. The results demonstrate a similar target-gene-specific cytotoxic response to the DBM triggers but not to a non-specific trigger targetting green fluorescent protein. Cultured Plutella xylostella (diamondback moth) PxE-PO #5A3 cells were incubated with the dsRNA triggers T42017 (SEQ ID NO:21, targetting V-ATPase subunit A), T33310 (SEQ ID NO:29, targetting V-ATPase subunit A), T32938 (SEQ ID NO:28, targetting COPI coatomer beta subunit), and T32937 (SEQ ID NO:13, targetting COPI coatomer beta prime subunit), or with a control trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent protein); see Table 1. The dsRNA triggers were formulated with the commercial transfection agent Cellfectin.RTM. II (Life Technologies, Inc., Grand Island, N.Y. 14072). FIG. 2 depicts the results of the transfection experiments, demonstrating target-gene-specific suppression (reduction of target gene mRNA levels measured by Quantigene assays) by the dsRNA triggers in the insect cells. The control dsRNA trigger T35763 had no effect on the targetted insect gene mRNA levels. In this particular experiment, some reduction of mRNA levels was observed in all DBM-specific trigger treatments compared to cellfectin II or cellfectin II+T35763 (SEQ ID NO:27) treatments. Visual pathology was apparent for T32937 (SEQ ID NO:13, targetting COPI coatomer beta prime subunit) and T32938 (SEQ ID NO:28, targetting COPI coatomer beta subunit) treatments with some cell death, rounding, and dislodging occurring in transfected wells at 48 hours post-transfection. Treatments with T42017 (SEQ ID NO:21) and T33310 (SEQ ID NO:29) showed knockdown of the target gene, V-ATPase subunit A mRNA, but no visual pathology was apparent in cells transfected with those triggers.

Example 3

[0120] This example illustrates non-limiting embodiments of dsRNA trigger sequences useful for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect, and methods for validating dsRNA trigger efficacy or for causing stunting or mortality in an insect. More specifically this example illustrates oral delivery of dsRNA triggers for causing stunting or mortality in insects.

[0121] An assay using Euschistus heros (neotropical brown stink bug, NBSB) nymphs fed on an artificial diet was used for testing the efficacy of dsRNA triggers designed specifically to target Euschistus heros genes. Using this assay, a .about.500 base pair dsRNA trigger, T33199, which targets the E. heros ubiquitin C gene, was observed to effect a dose-dependent stunting and mortality response in E. heros nymphs. A shorter (302 bp) dsRNA trigger, T44042 (SEQ ID NO:26) was designed, based on the sequence of T33199, for formulating for oral or topical delivery.

Example 4

[0122] This example illustrates non-limiting embodiments of dsRNA trigger sequences useful for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect, and methods for validating dsRNA trigger efficacy or for causing stunting or mortality in an insect. More specifically this example illustrates embodiments of dsRNA triggers for causing stunting or mortality in insects, and demonstrates systemic RNAi efficacy of these triggers.

[0123] Table 2 provides dsRNA triggers tested by microinjection delivery in Lygus hesperus (Western tarnished plant bug) nymphs. The non-Lygus-specific trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent Protein) was used as a control.

TABLE-US-00002 TABLE 2 Amount Trigger injected per SEQ ID insect NO.* Trigger ID Lygus hesperus Target Gene (micrograms) 14 T34616 ribosomal protein rpL19 1-2 15 T34617 V-ATPase subunit A 1-2 16 T34622 COPI coatomer B' (beta prime) 1-2 subunit 22 T42772 ubiquitin C 1-2

[0124] Table 3 presents mortality results for dsRNA triggers, T34617 (SEQ ID NO:15, targetting Lygus hesperus V-ATPase subunit A), and T34622 (SEQ ID NO:16, targetting Lygus hesperus COPI coatomer beta prime subunit) tested by microinjection delivery in Lygus hesperus (Western tarnished plant hug) nymphs. Control nymphs were microinjected with T35763 (targetting green fluorescent protein) or with deionized water. Increased percent mortality by day was observed for Lygus-target-gene-specific treatment groups (T34617 and T34622) compared to negative control treatment groups over the 5-day observation period.

TABLE-US-00003 TABLE 3 Total Lygus Trigger Lygus hesperus Trigger SEQ ID hesperus Percent mortality, shown by days after injection nymphs ID NO.* Target gene 0 1 2 3 4 5 injected T34617 15 V-ATPase 12 25 58 75 88 100 24 subunit A T34622 16 COPI 8 12 67 96 96 100 24 coatomer beta prime subunit 135763 27 GFP 12 12 29 50 58 67 24 dH.sub.2O (none) (none) 0 5 20 45 55 75 20

[0125] Table 4 presents mortality results for dsRNA triggers, T34616 (SEQ ID NO:14, targetting Lygus hesperus ribosomal protein rpL19), T34622 (SEQ ID NO:16, targetting Lygus hesperus COPI coatomer beta prime subunit), and T42772 (SEQ ID NO:22, targetting Lygus hesperus ubiquitin C), tested by microinjection delivery in Lygus hesperus (Western tarnished plant bug) nymphs. Control nymphs were microinjected with T35763 (targetting green fluorescent protein). Increased percent mortality by day was observed for Lygus-target-gene-specific treatment groups (T34616, T34622, and T42772) compared to the negative control (T35763) treatment group over the 6-day observation period. Repeat activity of T34622 confirmed activity of this trigger observed in the earlier trial (Table 3).

TABLE-US-00004 TABLE 4 Total Lygus Trigger Lygus hesperus Trigger SEQ ID hesperus Percent mortality, shown by days after injection nymphs ID NO: Target gene 0 1 2 3 4 5 6 injected T34616 14 ribosomal 18 77 77 91 100 100 100 22 protein rpL19 T34622 16 COPT 12 50 83 88 100 100 100 24 coatomer beta prime subunit T42772 22 ubiquitin C 17 61 100 100 100 100 100 23 T35763 27 GFP 8 22 44 48 56 56 61 23

Example 5

[0126] This example discloses embodiments related to polynucleotide molecules having a nucleotide sequence containing specific modifications such as nucleotide substitutions. Embodiments of such modifications include modified dsRNA triggers that provide improved sequence discrimination between the intended target gene of the insect pest of interest, and genetic sequences of other, non-target species.

[0127] Table 5 identifies examples of matches between the sequence of a target gene provided in Table 1 and a sequence identified in a non-target organism (NTO), where the match is a segment of at least 19 contiguous nucleotides. Table 5 further provides examples of sequence modifications (e. g., nucleotide changes at a specified location in the original target gene sequence) to eliminate the sequence match to a non-target organism.

TABLE-US-00005 TABLE 5 Nucleotide Length (in position nucleotides) where of Nucleotide change is Nucleotide Target contiguous position of made to change made Gene segment beginning eliminate to eliminate SEQ Non-Target matching of match to match to ID Target Organism the NTO matching NTO NTO NO: Species (NTO) species sequence segment sequence sequence 1 Spodoptera Danaus plexippus 19 398 408 G-A frugiperda Danaus plerippus 23 562 575 C-T Danaus plexippus 26 604 617 A-T Danaus plexippus 20 631 642 C-T Danaus plexippus 20 661 671 A-T Danaus plexippus 28 695 710 T-A Danaus plexippus 90 727 737 T-A Danaus plexippus 20 793 802 A-G Danaus plexippus 20 847 858 C-T Apis mellifera 19 891 900 T-A Homo sapiens 21 905 900 T-A Danaus plexippus 19 968 977 T-C Danaus plexippus 23 1024 1035 G-A Danaus plexippus 48 1078 1092 C-T 1110 G-A 2 Spodoptera Danaus plexippus 90 241 250 T-C frugiperda Danaus plexippus 26 274 289 G-A Homo sapiens 19 516 526 A-G Danaus plexippus 26 1615 1628 A-G Danaus plexippus 20 1771 1780 C-T Danaus plexippus 90 1810 1820 T-C Apis mellifera 19 1870 1879 G-A Homo sapiens 20 1933 1945 G-A Apis mellifera 19 1934 1946 C-T Homo sapiens 71 1935 1946 C-T Apis mellifera 20 1936 1946 C-T Homo sapiens 20 1936 1946 C-T Homo sapiens 19 1936 1946 C-T Homo sapiens 19 1937 1946 C-T Homo sapiens 19 1939 1946 C-T Homo sapiens 19 1942 1946 C-T Apis mellifera 19 2681 2690 T-C 3 Spodoptera Danaus plexippus 19 62 72 C-T frugiperda Homo sapiens 19 503 512 C-G Homo sapiens 19 544 553 A-G Bombus impatiens 22 659 669 G-A Bombus terrestris 22 659 669 G-A Danaus plexippus 26 859 873 C-T Danaus plexippus 19 925 934 G-A Homo sapiens 19 927 934 G-A Homo sapiens 19 934 945 G-A Danaus plexippus 19 935 945 G-A Danaus plexippus 90 936 945 G-A Danaus plexippus 26 952 964 G-A Homo sapiens 19 974 983 T-A Homo sapiens 19 1461 1470 G-T Homo sapiens 19 1712 1799 G-A Homo sapiens 90 1713 1799 G-A Homo sapiens 20 2223 2232 G-A Homo sapiens 19 2242 2256 G-A Danaus plexippus 20 2245 2256 G-A Danaus plexippus 94 2307 2319 G-C Danaus plexippus 20 2332 2340 C-T Dartaus plexippus 29 2503 2517 C-T Danaus plexippus 41 2626 2633 T-C 4 Lygus Danaus plexippus 90 323 333 G-A hesperus Bombus impatiens 20 1010 1020 T-A Bombus terrestris 20 1010 1020 T-A Danaus plexippus 20 1400 1410 G-A Homo sapiens 19 1603 1612 C-T Bombus impatiens 96 1607 1699 C-T Bombus terrestris 26 1607 1627 C-T Homo sapiens 19 1968 1977 C-T Apis mellifera 19 2001 2010 C-G Apis mellifera 20 2041 2050 A-T 5 Lygus Homo sapiens 22 9 19 T-C hesperus Homo sapiens 20 11 20 T-A Homo sapiens 20 211 220 A-T 6 Lygus Boinbus terrestris 19 37 47 A-G hesperus Danaus plexippus 20 367 383 G-A Homo sapiens 19 374 383 G-A Manaus plexippus 20 637 647 A-T Danaus plexippus 26 802 815 A-T Apis mellifera 21 927 938 A-T Danaus plexippus 19 1529 1538 A-T Homo sapiens 19 2492 2501 T-C Homo sapiens 19 2493 2501 T-C 10 Plutella Danaus plexippus 20 1390 1399 T-A xylostella Danaus plexippus 19 1427 1437 C-T Danaus plexippus 23 1471 1482 G-A Manaus plexippus 20 1543 1553 T-C Homo sapiens 20 1645 1654 G-A Danaus plexippus 35 1654 1671 G-A Danaus plexippus 23 1726 1736 C-T Danaus plexippus 35 1942 1959 G-A Danaus plexippus 20 2005 2016 C-T Danaus plexippus 20 2038 2048 C-T Danaus plexippus 20 2181 2190 G-A 11 Plutella Homo sapiens 19 235 244 C-T xylostella Manaus plexippus 22 404 414 A-G Homo sapiens 19 517 526 T-A Danaus plexippus 19 690 699 T-C Danaus plexippus 20 1771 1780 C-T Danaus plexippus 20 1891 1899 C-T Homo sapiens 20 1900 1910 A-T Danaus plexippus 23 1942 1953 C-T 12 Plutella Danaus plexippus 21 184 194 T-C xylostella Danaus plexippus 19 384 393 G-A Danaus plexippus 20 613 622 C-T Danaus plexippus 32 916 931 A-T Homo sapiens 19 916 931 A-T Homo sapiens 71 935 931 A-T Homo sapiens 19 1066 1078 C-T Danaus plexippus 19 1067 1078 C-T Danaus plexippus 20 1068 1078 C-T Homo sapiens 19 1207 1219 T-C Danaus plexippus 19 1208 1219 T-C Danaus plexippus 20 1209 1219 T-C Danaus plexippus 19 1355 1365 G-A Homo sapiens 19 1474 1483 C-T Homo sapiens 70 1884 1894 G-A 1895 C-T Homo sapiens 19 2142 2151 C-T Bombus terrestris 19 2203 2213 G-A Homo sapiens 19 2391 2401 G-A Homo sapiens 19 2808 2817 A-G Danaus plexippus 21 3172 3182 C-T Danaus plexippus 20 3236 3245 C-T Homo sapiens 19 3249 3258 A-G Danaus plexippus 91 3270 3280 G-A Homo sapiens 19 3402 3411 C-T Danaus plexippus 27 3550 3564 C-T

[0128] Table 6 identifies examples of matches between the sequence of a dsRNA trigger provided in Table 1 and a sequence identified in a non-target organism (NTO), where the match is a segment of at least 19 contiguous nucleotides. Table 6 provides examples of sequence modifications (e.g., nucleotide changes at a specified location in the original dsRNA trigger sequence) which eliminate a specific sequence match of at least 19 contiguous nucleotides to a non-target organism. Table 6 further provides non-limiting embodiments of a modified trigger sequence in which all of the nucleotide changes recited in Table 6 for a given original trigger sequence have been made to eliminate the all of the recited match(es) of at least 19 contiguous nucleotides to a non-target organism sequence. For example, the modified dsRNA trigger having SEQ ID NO:31 has one nucleotide change (A.fwdarw.U) at position 213, when compared to the original dsRNA trigger having SEQ ID NO:15; this single change eliminates matches of at least 19 contiguous nucleotides to two non-target organisms, Bombus impatiens and Bombus terrestris. In another example, the modified dsRNA trigger having SEQ ID NO:35 has four nucleotide changes (at positions 42, 79, 124, and 194) , when compared to the original dsRNA trigger having SEQ ID NO:20; these changes eliminate four matches of at least 19 contiguous nucleotides to the NTO Danaus plexippus. In another example, the modified dsRNA trigger having SEQ ID NO:37 has four nucleotide changes (at positions 64, 72, 131, and 280) , when compared to the original dsRNA trigger having SEQ ID NO:20; these changes eliminate four matches of at least 19 contiguous nucleotides to the NTOs Bombus impatiens, Bombus terrestris, Homo sapiens, and Danaus plexippus.

TABLE-US-00006 TABLE 6 Nucleotide position Length (in where Nucleotide contiguous change is change nucleotides) Nucleotide made to made to Modified Original of segment position of eliminate eliminate trigger Trigger Non-Target matching the beginning match to match to sequence SEQ ID Organism (NTO) NTO of matching NTO NTO SEQ ID NO: species sequence segment sequence sequence NO: 13 Homo sapiens 19 229 238 C-U 30 15 Bombus impatiens 20 202 213 A-U 31 Bombus terrestris 20 202 213 A-U 16 Bombus terrestris 19 22 32 A-G 32 17 Danarrs plexippus 19 131 140 A-U 33 18 Homo sapiens 19 162 171 G-A 34 20 Danaus plexippus 20 33 42 U-A 35 Danaus plexippus 19 70 79 A-U Danaus plerippus 23 114 124 G-A Danaus plexippus 20 186 196 U-A 22 Honio sapiens 23 12 24 U-C 36 Homo sapiens 26 12 24 U-C Homo sapiens 20 15 26 G-A Homo sapiens 23 15 26 G-A Danaus plexippus 20 27 36 G-A Homo sapiens 23 54 71 C-U Homo sapiens 19 64 71 C-U Homo sapiens 23 126 138 G-A Homo sapiens 20 126 138 G-A Apis mellifera 20 210 220 A-G Bombus terrestris 20 210 990 A-G Danaus plexippus 20 216 220 A-G Apis mellifera 20 225 220 A-G Apis mellifera 29 228 220 A-G Apis mellifera 23 234 243 C-U 23 Bombus impatiens 29 53 64 A-G 37 Bombus terrestris 20 62 72 U-A Homo sapiens 19 199 131 G-A Danaus plexippus 20 271 280 A-G 24 Bombus impatiens 27 98 99 G-C 38 Danaus plexippus 19 102 112 U-A Bombus impatiens 20 164 176 A-U Apis mellifera 20 167 176 A-U Apis mellifera 29 167 176 A-U Danaus plexippus 20 167 176 A-U Apis mellifera 19 186 192 T-C Bombus impatiens 26 188 196 A-G Apis mellifera 26 200 204 A-U Apis mellifera 20 203 213 U-C Bombus impatiens 20 203 213 U-C Bombus ierresiris 20 203 213 U-C Danaus plexippus 26 209 224 G-U Apis mellifera 24 227 224 G-U Apis mellifera 97 718 994 G-U Bombus impatiens 19 228 224 G-U Homo sapiens 20 231 242 G-A Apis mellifera 23 251 264 G-A Bombus impatiens 20 281 291 A-U Bombus impatiens 23 47 63 A-U Bombus impatiens 20 47 63 A-U Bombus terrestris 20 50 63 A-U Danaus plexippus 20 53 63 A-U Bombus impatiens 39 86 99 G-C Bombus impatiens 29 86 99 G-C Bombus terrestris 39 86 99 G-C Bombus impatiens 30 95 99 G-C Apis mellifera 19 96 99 G-C Apis mellifera 22 96 99 G-C 25 Apis mellifera 23 53 66 C-U 39 Apis mellifera 26 68 66 C-U Bombus impatiens 26 68 66 C-U Apis mellifera 26 71 84 A-U Apis mellifera 23 71 84 A-U Bombus impatiens 20 80 84 A-U Apis mellifera 20 89 99 A-U Bombus impatiens 23 155 168 A-U Bombus impatiens 20 155 168 A-U Bombus terrestris 20 158 168 A-U Danaus plexippus 20 161 168 A-U Bombus impatiens 39 194 206 G-A Bombus impatiens 29 194 206 G-A Bombus terrestris 39 194 206 G-A Bombus impatiens 30 203 206 G-A Apis mellifera 19 204 206 G-A Apis mellifera 79 204 206 G-A Bombus impatiens 27 206 221 U-G Danaus plexippus 19 210 221 U-G Bombus impatiens 20 272 285 A-U Apis mellifera 20 275 285 A-U 26 Apis mellifera 28 275 285 A-U 40 Danaus plexippus 20 275 285 A-U Homo sapiens 19 62 77 G-A Datzaus plexippus 20 68 77 G-A Homo sapiens 20 68 77 G-A Homo sapiens 70 98 113 A-G Bombus terrestris 20 104 113 A-G Danaus plexippus 20 155 167 C-U Danaus plexippus 23 155 167 C-U Homo sapiens 23 155 167 C-U Danaus plexippus 23 197 211 U-C Danams plexippus 19 201 211 U-C Danaus plexippus 23 221 231 U-C Homo sapiens 30 271 285 A-G Homo sapiens 71 271 285 A-G 27 Homo sapiens 19 75 84 A-U 41 28 Homo sapiens 19 222 231 C-A 42 Bombus terrestris 19 283 291 U-C

Example 6

[0129] This example discloses embodiments related to polynucleotide molecules, such as dsRNA triggers designed to silence or suppress a target gene of an insect pest of interest, and techniques for determining efficacy of such molecules in suppressing a target gene or in controlling an insect pest. This example discloses a method of providing to an insect a polynucleotide, such as a recombinant RNA molecule or dsRNA trigger, in the form of an ingestible composition.

[0130] One method for determining efficacy of polynucleotide molecules in suppressing a target gene or in controlling a pest Lygus species is a sucrose feeding assay. In brief, this assay involves contacting western tarnished plant bug (Lygus hesperus) nymphs with dsRNA triggers in an ingestible composition (a sucrose solution), followed by maintenance on an artificial diet and monitoring of the nymphs' condition.

[0131] Parafilm sachets containing 2 milliliters of a 15% sucrose solution were prepared for the feeding experiments, with the treatment sachets containing dsRNA triggers (see Table 1) at either 500 micrograms/milliliter (or parts per million, ppm) or 1000 micrograms/milliliter (or parts per million, ppm). In some experiments the sucrose solution further included 5 milligrams/milliliter yeast tRNA to inhibit potential nuclease activity. Artificial diet sachets were also prepared using parafilm sachets containing 2 milliliters of a western tarnished plant bug (Lygus hesperus) artificial diet prepared by combining autoclaved, boiling water (518 milliliters) with 156.3 grams of Bio-Serv.RTM. diet F9644B (Bio-Serv, Frenchtown, N.J.) in a surface-sterilized blender.

[0132] Third-instar Lygus hesperus nymphs were anesthetized under carbon dioxide vapor and distributed among 4-ounce glass jars (40-75 individuals per treatment). A piece of tissue paper was added to each jar to absorb honeydew. Each experiment was carried out over 7 days. The nymphs were allowed to feed for a 72-hour period (3 days) on the sucrose sachets, after which the sucrose sachets were removed and replaced with artificial diet sachets for the remaining 4 days of the 7-day observation period. All insects for a single treatment group were observed in a single arena, incubated at 27 degrees Celsius, 60% relative humidity. Initial mortality at day 0 (start of the 72-hour sucrose-feeding stage) was 0 in all cases. Mortality was recorded from 3 days (i. e., the end of the 72-hour sucrose-feeding stage) up to 7 days from the start of the experiment. For gene expression studies, immediately following the end of the 72-hour sucrose-feeding stage, 12 living nymphs were removed from each jar and immediately frozen on dry ice in individual matrix tubes to serve as samples for RNA measurement assays.

[0133] A series of feeding experiments using the above protocol was performed to assess activity of the dsRNA triggers T34616 (SEQ ID NO:14), T34617 (SEQ ID NO:15), T34619 (SEQ ID NO:45), T34622 (SEQ ID NO:16), T42772 (SEQ ID NO:22), T42768 (SEQ ID NO:46), and T44045 (SEQ ID NO:26) against Lygus hesperus. The dsRNA triggers T42772 (SEQ ID NO:22) and T44045 (SEQ ID NO:26) were also tested on Lygus lineolaris. A control trigger T35763 (SEQ ID NO:27, a 300 bp dsRNA trigger targetting green fluorescent protein) was used in the assays. At 500 micrograms/milliliter (500 ppm), enhanced mortality was observed with all Lygus-specific dsRNA triggers compared to control treatments in the presence (Table 7) or absence of yeast tRNA supplement (Table 8). T34622 (SEQ ID NO:16) (targetting the putative COPI coatomer beta prime subunit gene with SEQ ID NO:6) and T42772 (SEQ ID NO:22) (targetting the putative ubiquitin C gene with SEQ ID NO:7) were consistently the best performing triggers. Triggers T42772 (SEQ ID NO:22) and T44045 (SEQ ID NO:26), both targetting a putative ubiquitin C gene (SEQ ID NO:7), exhibited similar activity in the Western tarnished plant bug Lygus hesperus (Table 9A); both triggers also appeared active (although slower to act) in a related pest species, the tarnished plant bug, Lygus lineolaris (Table 9B).

[0134] A three-replicate experiment tested the ubiquitin C triggers T44045 (SEQ ID NO:26) and T42768 (SEQ ID NO:46) on Lygus hesperus at 1000 micrograms/milliliter (1000 ppm); the results demonstrated statistically significant differences in the means for insect mortality at days 3, 4, 5, 6, and 7 due to ubiquitin C trigger treatments when compared to control treatments with 15% sucrose alone or 15% sucrose plus the control trigger T35763 (SEQ ID NO:27) at 1000 micrograms/milliliter (1000 ppm) (Table 10).

TABLE-US-00007 TABLE 7 Insect mortality (as percent of total insects in treatment), dsRNA triggers tested at 500 ppm in presence of 5 mg/mL yeast tRNA Trigger Total SEQ ID insects in Days since start of assay Treatment NO: treatment 0 3 4 5 6 7 15% sucrose -- 58 0 10 33 55 64 72 15% sucrose + 5 mg/mL tRNA -- 57 0 16 37 54 63 70 T35763 GFP (control) 27 45 0 16 31 38 60 69 T34616 rpL19 14 58 0 31 50 72 86 97 T34617 V-ATPase subunit A 15 62 0 34 52 73 87 92 T34619 COPI coatomer beta subunit 45 56 0 36 43 68 71 89 T34622 COPI coatomer beta prime subunit 16 69 0 43 58 78 90 93 T42772 ubiquitin C 22 75 0 61 81 93 97 99

TABLE-US-00008 TABLE 8 Insect mortality (as percent of total insects in treatment), dsRNA triggers tested at 500 ppm (without yeast tRNA) Trigger Total SEQ ID insects in Days since start of assay Treatment NO: treatment 0 3 4 5 6 7 15% sucrose -- 65 0 26 42 63 71 78 T35763 GOP (control) 27 38 0 29 55 68 76 76 T34616 rpL19 14 62 0 24 47 71 87 94 T34617 V-ATPase subunit A 15 71 0 35 68 83 89 92 T34619 COPI coatomer beta subunit 45 56 0 38 86 91 95 95 T34622 COPI coatomer beta prime subunit 16 64 0 62 95 100 100 100 T42772 ubiquitin C 22 30 0 63 80 90 93 100

TABLE-US-00009 TABLE 9A Lygus hesperus mortality (as percent of total insects in treatment), dsRNA triggers tested at 500 ppm in presence of 5 mg/mL yeast tRNA Trigger Total SEQ ID insects in Days since start of assay Treatment NO: treatment 0 3 4 5 6 7 15% sucrose -- 28 0 36 50 57 64 64 15% sucrose + 5 mg/mL tRNA -- 43 0 44 56 70 77 79 T35763 GFP (control) 27 32 0 50 66 78 88 91 T34622 COPI coatomer beta prime subunit 16 54 0 52 72 82 94 98 T42772 ubiquitin C 22 58 0 69 95 97 100 100 T14045 ubiquitin C 26 80 0 66 85 94 99 100

TABLE-US-00010 TABLE 9B Lygus lineolaris mortality (as percent of total insects in treatment), dsRNA triggers tested at 500 ppm in presence of 5 mg/mL yeast tRNA Trigger Total SEQ ID insects in Days since start of assay Treatment NO: treatment 0 3 4 5 6 7 15% sucrose + 5 mg/mL tRNA -- 10 0 20 40 40 40 40 T35763 GFP (control) 27 16 0 19 44 50 56 69 T42772 ubiquitin C 22 20 0 30 45 60 75 95 T44045 ubiquitin C 26 21 0 29 43 67 86 90

TABLE-US-00011 TABLE 10 Lygus hesperus mortality (as percent of total insects in treatment), dsRNA triggers tested at 1000 ppm Trigger Total SEQ ID insects in Days since start of assay Treatment NO: Replicate treatment 0 3 4 5 6 7 15% sucrose -- 1 29 0 45 45 48 48 52 2 30 0 23 33 57 67 70 3 30 0 21 28 34 41 41 15% sucrose--mortality, mean (sd*) 0 (0) 30 (13) 35 (9) 46 (11) 52 (13) 54 (14) T35763 GFP 27 1 30 0 20 30 53 53 53 (control) 2 30 0 13 17 53 63 67 3 30 0 30 40 50 57 60 T35763--mortality, mean (sd*) 0 (0) 21 (8) 29 (12) 52 (2) 58 (5) 60 (7) T44045 26 1 30 0 37 53 63 77 87 ubiquitin C 2 29 0 24 38 59 76 86 3 30 0 43 47 57 70 73 T44045--mortality, mean (sd*) 0 (0) 35 (10) 46 (8) 60 (3) 74 (4) 82 (8) T42768 46 1 29 0 17 55 90 97 97 ubiquitin C 2 30 0 50 60 80 83 87 3 30 0 40 60 70 77 80 T42769--mortality, mean (sd*) 0(0) 36 (17) 58 (3) 80 (10) 86 (10) 88 (8) *sd = standard deviation of the mean, given in percentage

[0135] Target gene suppression was assessed in Lygus hesperus by Quantigene analysis of three target genes (COPI coatomer beta prime subunit, V-ATPase subunit A, and COPI coatomer beta subunit). Sucrose feeding assays were carried out using the dsRNA triggers T34616 (SEQ ID NO:14), T34617 (SEQ ID NO:15), T34619 (SEQ ID NO:45), T34622 (SEQ ID NO:16), T42772 (SEQ ID NO:22), and T44045 (SEQ ID NO:26) tested at 500 or 100 ppm in 15% sucrose as described above. Immediately following the end of the 72-hour sucrose-feeding stage, the nymphs were individually frozen and subjected to Quantigene analysis. All values were normalized against the expression levels of two reference genes (EFlalpha and actin). The results (Tables 11A-C) demonstrated that each of the three tested target genes was specifically suppressed by the corresponding dsRNA trigger, coincident with observed increased mortality when compared to treatment with sucrose only or with the control GEP trigger T35763 (SEQ ID NO:27). The observed target gene suppression (reduction in target gene expression) was significant (p=0.05) compared to sucrose controls for five of six trigger experiments; in the sixth trigger experiment (T34622 at 1000 ppm, see Table 11A) visual suppression was observed just below the significance level. These results support the conclusion that the mortality observed in 15% sucrose feeding assays is mediated by RNAi.

TABLE-US-00012 TABLE 11A Target gene: L. hesperus COPI coatomer beta prime subunit (F38E11), SEQ ID NO: 6 % Significant change @ p = 0.05 Trigger from from concentration Treatment Target gene control control? n/a sucrose only n/a n/a n/a 1000 ppm T34617 (SEQ ID NO: 15) V-ATPase subunit A -4% no 1000 ppm T34619 (SEQ ID NO: 45) COPI coatomer beta subunit 33% no 1000 ppm T34622 (SEQ ID NO: 16) COPI coatomer beta prime -34% no subunit 1000 ppm T35763 (SEQ ID NO: 27) GFP 4% no 1000 ppm T44045 (SEQ ID NO: 26) ubiquitin C 19% no n/a sucrose only n/a n/a n/a 500 ppm T34617 (SEQ ID NO: 15) V-ATPase subunit A -15% no 500 ppm T34619 (SEQ ID NO: 45) COPI coatomer beta subunit -31% no 500 ppm T34622 (SEQ ID NO: 16) COPI coatomer beta prime -71% yes subunit 500 ppm T35763 (SEQ ID NO: 27) GFP -24% no n/a untreated (artificial diet fed) n/a 3% no

TABLE-US-00013 TABLE 11B Target gene: L. hesperus V-ATPase A subunit (CG3762), SEQ ID NO: 4 % Significant change @ p = 0.05 Trigger from from concentration Treatment Target gene control control? n/a sucrose only n/a n/a n/a 1000 ppm T34617 (SEQ ID NO: 15) V-ATPase subunit A -77% yes 1000 ppm T34619 (SEQ ID NO: 45) COPI coatomer beta subunit -21% no 1000 ppm T34622 (SEQ ID NO: 16) COPI coatomet beta prime -23% no subunit 1000 ppm T35763 (SEQ ID NO: 27) GFP -23% no 1000 ppm T44045 (SEQ ID NO: 26) ubiquitin C -25% no n/a sucrose only n/a n/a n/a 500 ppm T34617 (SEQ ID NO: 15) V-ATPase subunit A -63% yes 500 ppm T34619 (SEQ ID NO: 45) COPI coatomer beta subunit -25% no 500 ppm T34622 (SEQ ID NO: 16) COPI coatomer beta prime 7% no subunit 500 ppm T35763 (SEQ ID NO: 27) GFP -35% no n/a untreated (artificial diet fed) n/a 20% no

TABLE-US-00014 TABLE 11C Target gene: L. hesperus COPI coatomer beta subunit (CG6223), SEQ ID NO: 43 % Significant change @ p = 0.05 Trigger from from concentration Treatment Target gene control control? n/a sucrose only n/a n/a n/a 1000 ppm T34617 (SEQ ID NO: 15) V-ATPase subunit A 6% no 1000 ppm 134619 (SEQ ID NO: 45) COPI coatomer beta subunit -49% yes 1000 ppm T34622 (SEQ ID NO: 16) COPI coatomer beta prime 28% no subunit 1000 ppm T35763 (SEQ ID NO: 27) GFP 18% no 1000 ppm 144045 (SEQ ID NO: 26) ubiquitin C 21% no n/a sucrose only n/a n/a n/a 500 ppm T34617 (SEQ ID NO: 15) V-ATPase subunit A -22% no 500 ppm T34619 (SEQ ID NO: 45) COPI coatomer beta subunit -54% yes 500 ppm T34622 (SEQ ID NO: 16) COPI coatomer beta prime -21% no subunit 500 ppm T35763 (SEQ ID NO: 27) GFP -34% no n/a untreated (artificial diet fed) n/a 18% no n/a = not applicable

Example 7

[0136] The polynucleotides of this invention are generally designed to modulate expression by inducing regulation or suppression of an insect target gene and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence an insect target gene or cDNA (e. g., SEQ ID NOs:1-12 and 43-44) or to the sequence of RNA transcribed from an insect target gene, which can be coding sequence or non-coding sequence. These effective polynucleotide molecules that modulate expression are referred to herein as a "trigger", or "triggers". This example describes non-limiting techniques useful in the design and selection of polynucleotides as "triggers" to modulate expression of an insect target gene.

Selection of Polynucleotide Triggers by "Tiling"

[0137] Polynucleotides of use in the invention need not be of the full length of a target gene, and in many embodiments are of much shorter length in comparison to the target gene. An example of a technique that is useful for selecting effective triggers is "tiling", or evaluation of polynucleotides corresponding to adjacent or partially overlapping segments of a target gene.

[0138] Effective polynucleotide "triggers" can be identified by "tiling" gene targets in selected length fragments, e. g., fragments of 200-300 nucleotides in length, with partially overlapping regions, e. g., of about 25 nucleotides, along the length of the target gene. To suppress a single gene, trigger sequences are designed to correspond to (have a nucleotide identity or complementarity with) regions that are unique to the target gene; the selected region of the target gene can include coding sequence or non-coding sequence (e. g., promoter regions, 3' untranslated regions, introns and the like) or a combination of both.

[0139] Where it is of interest to design a target effective in suppressing multiple target genes, the multiple target gene sequences are aligned and polynucleotide triggers designed to correspond to regions with high sequence homology in common among the multiple targets. Conversely, where it is of interest to design a target effective in selectively suppressing one among multiple target sequences, the multiple target gene sequences are aligned and polynucleotide triggers designed to correspond to regions with no or low sequence homology in common among the multiple targets.

[0140] In a non-limiting example, anti-sense single-stranded RNA triggers are designed for each of the target genes listed in Table 1 as follows. Multiple anti-sense single-stranded RNA triggers, each of 200-300 nucleotides in length and with a sequence corresponding to (i. e., for anti-sense triggers, complementary to) a fragment of a target gene having a sequence selected from SEQ ID NOs:1-12 and 43-44 are designed so that each trigger's sequence overlaps about 25 nucleotides of the next adjacent trigger's sequence, in such a way that the multiple triggers in combination cover the full length of the target gene. (Sense triggers are designed in an analogous fashion, where the trigger sequence is identical to a fragment of the target gene. Similarly, double-stranded triggers can be designed by providing pairs of sense and anti-sense triggers, each pair of triggers overlapping the next adjacent pair of triggers.)

[0141] The polynucleotide triggers are tested by any convenient means for efficacy in silencing the insect target gene. Examples of a suitable test include the bioassays described herein in the working Examples. Another test involves the topical application of the polynucleotide triggers either directly to individual insects or to the surface of a plant to be protected from an insect infestation. One desired result of treatment with a polynucleotide of this invention is prevention or control of an insect infestation, e. g., by inducing in an insect a physiological or behavioural change such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. Another desired result of treatment with a polynucleotide of this invention is provision of a plant that exhibits improved resistance to an insect infestation.

[0142] The tiling procedure can be repeated, if desired. A polynucleotide trigger found to provide desired activity can itself be subjected to a tiling procedure. For example, multiple overlapping anti-sense single-stranded RNA triggers are designed, each of 50-60 nucleotides in length and with a sequence corresponding to (i. e., for anti-sense triggers, complementary to) the fragment of a target gene having a sequence selected from SEQ ID NOs:1-12 and 43-44 for which a single polynucleotide trigger of 300 nucleotides was found to be effective. Additional rounds of tiling analysis can be carried out, where triggers as short as 18 or 19 nucleotides are tested.

[0143] Effective polynucleotide triggers of any size can be used, alone or in combination, in the various methods of this invention. In some embodiments, a single polynucleotide trigger is used to make a composition of this invention (e. g., a composition for topical application, or a recombinant DNA construct useful for making a transgenic plant). In other embodiments, a mixture or pool of different polynucleotide triggers is used; in such cases the polynucleotide triggers can be for a single target gene or for multiple target genes. In some embodiments, a polynucleotide trigger is designed to target different regions of the target gene, e. g., a trigger can include multiple segments that correspond to different exon regions of the target gene, and "spacer" nucleotides which do not correspond to a target gene can optionally be used in between or adjacent to the segments.

Thermodynamic Considerations in Selecting Polynucleotide Triggers

[0144] Polynucleotide triggers can be designed or their sequence optimised using thermodynamic considerations. For example, polynucleotide triggers can be selected based on the thermodynamics controlling hybridization between one nucleic acid strand (e. g., a polynucleotide trigger or an individual siRNA) and another (e. g., a target gene transcript)

[0145] Methods and algorithms to predict nucleotide sequences that are likely to he effective at RNAi-mediated silencing of a target gene are known in the art. Non-limiting examples of such methods and algorithms include "i-score", described by Ichihara et al. (2007) Nucleic Acids Res., 35(18): 123e; "Oligowalk", publicly available at rna.urmc.rochester.edu/servers/oligowalk and described by Lu et al. (2008) Nucleic Acids Res., 36:W104-108; and "Reynolds score", described by Khovorova et al. (2004) Nature Biotechnol., 22:326-330.

Permitted Mismatches

[0146] By "essentially identical" or "essentially complementary" is meant that the trigger polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to the target gene or to the RNA transcribed from a target gene (e. g., the transcript) to suppress expression of a target gene (e. g., to effect a reduction in levels or activity of the target gene transcript and/or encoded protein). Polynucleotides of this invention need not have 100 percent identity or complementarity to a target gene or to the RNA transcribed from a target gene to suppress expression of the target gene (e. g., to effect a reduction in levels or activity of the target gene transcript or encoded protein, or to provide control of an insect species). In some embodiments, the polynucleotide or a portion thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In certain embodiments, an "essentially identical" polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides in either the endogenous target gene or to an RNA transcribed from the target gene. In certain embodiments, an "essentially complementary" polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity when compared to the sequence of 18 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene.

[0147] Polynucleotides containing mismatches to the target gene or transcript can be used in certain embodiments of the compositions and methods of this invention. In some embodiments, the polynucleotide includes at least 18 or at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript. In certain embodiments, a polynucleotide of 19 contiguous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript (i. e., 1 or 2 mismatches between the polynucleotide's 19 contiguous nucleotides and the segment of equivalent length in the target gene or target gene's transcript). In certain embodiments, a polynucleotide of 20 or more nucleotides that contains a contiguous 19 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 21 continuous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 22 or more nucleotides that contains a contiguous 21 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript.

[0148] In designing polynucleotides with mismatches to an endogenous target gene or to an RNA transcribed from the target gene, mismatches of certain types and at certain positions that are more likely to be tolerated can be used. In certain exemplary embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used as described by Du et cd. (2005) Nucleic Acids Res., 33:1671-1677. In some embodiments, mismatches in 19 base-pair overlap regions are located at the low tolerance positions 5, 7, 8 or 11 (from the 5' end of a 19-nucleotide target), at medium tolerance positions 3, 4, and 12-17(from the 5' end of a 19-nucleotide target), and/or at the high tolerance positions at either end of the region of complementarity, i. e., positions 1, 2, 18, and 19 (from the 5' end of a 19-nucleotide target) as described by Du et 01. (2005) Nucleic Acids Res., 33:1671-1677. Tolerated mismatches can be empirically determined in routine assays such as those described herein in the working Examples.

[0149] In some embodiments, the polynucleotides include additional nucleotides for reasons of stability or for convenience in cloning or synthesis. In one embodiment, the polynucleotide is a dsRNA including an RNA strand with a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 and further including an additional 5' G or an additional 3' C or both, adjacent to the segment. In another embodiment, the polynucleotide is a double-stranded RNA including additional nucleotides to form an overhang, for example, a dsRNA including 2 deoxyribonucleotides to form a 3' overhang.

Embedding Active Triggers in Neutral Sequence

[0150] In an embodiment, a bioactive trigger (i. e., a polynucleotide with a sequence corresponding to the target gene and which is responsible for an observed suppression of the target gene) is embedded in "neutral" sequence, i. e., inserted into additional nucleotides that have no sequence identity or complementarity to the target gene. Neutral sequence can be desirable, e. g., to increase the overall length of a polynucleotide. For example, it can be desirable for a polynucleotide to be of a particular size for reasons of stability, cost-effectiveness in manufacturing, or biological activity.

[0151] It has been reported that in another coleopteran species, Diabrotica virgifera, dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays; see Bolognesi et al. (2012) PLoS ONE 7(10): e47534. doi:10.1371/journal.pone.0047534. Thus, in one embodiment, a 21-base-pair dsRNA trigger corresponding to a target gene in Table 1 and found to provide control of an insect infestation is embedded in neutral sequence of an additional 39 base pairs, thus forming a polynucleotide of about 60 base pairs. In another embodiment, a single 21-base-pair trigger is found to be efficacious when embedded in larger sections of neutral sequence, e.g., where the total polynucleotide length is from about 60 to about 300 base pairs. In another embodiment, at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 is embedded in larger sections of neutral sequence to provide an efficacious trigger. In another embodiment, segments from multiple sequences selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 are embedded in larger sections of neutral sequence to provide an efficacious trigger.

[0152] It is anticipated that the combination of certain recombinant RNAs of this invention (e.g., the dsRNA triggers having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46, or active fragments of these triggers) with one or more non-polynucleotide pesticidal agents will result in a synergetic improvement in prevention or control of insect infestations, when compared to the effect obtained with the recombinant RNA alone or the non-polynucleotide pesticidal agent alone. Routine insect bioassays such as the bioassays described herein in the working Examples are useful for defining dose-responses for larval mortality or growth inhibition using combinations of the polynucleotides of this invention and one or more non-polynucleotide pesticidal agents (e. g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein). One of skill in the art can test combinations of polynucleotides and non-polynucleotide pesticidal agents in routine bioassays to identify combinations of bioactives that are synergistic and desirable for use in protecting plants from insect infestations.

Example 8

[0153] This example illustrates non-limiting embodiments of the use of polynucleotides of this invention in topically applied compositions for preventing or controlling insect infestations.

[0154] Compositions containing one or more polynucicotides of this invention arc useful as topical treatments of plants, animals, or environments wherein prevention or control of a Leptinotarsa species infestation is desired. In embodiments, a polynucleotide trigger for a target gene with a sequence selected from SEQ ID NOs:1-12 and 43-44, i. e., the target genes identified in Table 1, as described in the preceding examples, is included in an effective amount in a composition designed to be provided directly (e. g., by contact or ingestion) to an insect species, or a plant or environment wherein prevention or control of infestation by that insect is desired. In embodiments, a polynucleotide trigger for a target gene with a sequence selected from SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46 is included in an effective amount in such compositions. In embodiments, a dsRNA trigger with a strand having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46, or active fragments of these triggers, is included in an effective amount in such compositions. Such compositions are formulated and manufactured according to the art and can be in any convenient form, e.g., a solution or mixture of solutions, an emulsion, a suspension, a dispersible powder, a solid or liquid bait, a seed coating, or a soil drench. Embodiments of such compositions include those where the polynucleotide of this invention is provided in a living or dead microorganism such as a bacterium or fungal or yeast cell, or provided as a microbial fermentation product, or provided in a living or dead plant cell, or provided as a synthetic recombinant polynucleotide. In an embodiment the composition includes a non-pathogenic strain of a microorganism that contains a polynucleotide of this invention; ingestion or intake of the microorganism results in stunting or mortality of the insect pest; non-limiting examples of suitable microorganisms include E. coil, B. thuringiensis, Pseudomonas sp., Photorhabdus sp., Xenorhabdus sp., Serratia entomophila and related Serratia sp., B. sphaericus, B. cereus, B. laterosporus, B. popilliae, Clostridium Nfermentans and other Clostridium species, or other spore-forming gram-positive bacteria. In an embodiment, the composition includes a plant virus vector including a polynucleotide of this invention; feeding by an insect on a plant treated with the plant virus vector results in stunting or mortality of the insect. In an embodiment, the composition includes a baculovirus vector including a polynucleotide of this invention; ingestion or intake of the vector results in stunting or mortality of the insect. In an embodiment, a polynucleotide of this invention is encapsulated in a synthetic matrix such as a polymer or attached to particulates and topically applied to the surface of a plant; feeding by an insect on the topically treated plant results in stunting or mortality of the insect. In an embodiment, a polynucleotide of this invention is provided in the form of a plant cell (e. g., a transgenic plant cell of this invention) expressing the polynucleotide; ingestion of the plant cell or contents of the plant cell by an insect results in stunting or mortality of the insect.

[0155] Embodiments of the compositions optionally include the appropriate stickers and wetters required for efficient foliar coverage as well as UV protectants to protect polynucleotides such as dsRNAs from UV damage. Such additives are commonly used in the bioinsecticide industry and are known to those skilled in the art. Compositions for soil application can include granular formulations that serve as bait for insect larvae. Embodiments include a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator. In embodiments, the composition further includes at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

[0156] Such compositions are applied in any convenient manner, e. g., by spraying or dusting the insect directly, or spraying or dusting a plant or environment wherein prevention or control of infestation by that insect is desired, or by applying a coating to a surface of a plant, or by applying a coating to a seed (or seed potato) in preparation for the seed's planting, or by applying a soil drench around roots of a plant for which prevention or control of infestation by that insect is desired.

[0157] An effective amount of a polynucleotide of this invention is an amount sufficient to provide control of the insect, or to prevent infestation by the insect; determination of effective amounts of a polynucleotide of this invention are made using routine assays such as those described in the working Examples herein. While there is no upper limit on the concentrations and dosages of a polynucleotide of this invention that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency and economy. Non-limiting embodiments of effective amounts of a polynucleotide include a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of a polynucleotide in a liquid form sprayed on a plant, or from about 10 milligrams per acre to about 100 grams per acre of polynucleotide applied to a field of plants, or from about 0.001 to about 0.1 microgram per milliliter of polynucleotide in an artificial diet for feeding the insect. Where compositions of this invention are topically applied to a plant, the concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using 25-mer polynucleotides of this invention is about 1 nanomole (nmol) of polynucleotides per plant, for example, from about 0.05 to 1 nmol polynucleotides per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per plant. In certain embodiments, about 40 to about 50 nmol of a ssDNA polynucleotide of this invention are applied. In certain embodiments, about 0.5 nmol to about 2 nmol of a dsRNA of this invention is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of a dsRNA or an ssDNA (21-mer) of this invention is applied. In certain embodiments, a composition of about 0.5 to about 1.5 milligrams per milliliter of a dsRNA polynucleotide of this invention of about 50 to about 200 or more nucleotides is applied. In certain embodiments, about 1 nmol to about 5 nmol of a dsRNA of this invention is applied to a plant. In certain embodiments, the polynucleotide composition as topically applied to the plant contains at least one polynucleotide of this invention at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines can require correspondingly larger amounts of polynucleotides. When using long dsRNA molecules of this invention that can be processed into multiple oligonucleotides (e. g., multiple triggers encoded by a single recombinant DNA molecule of this invention), lower concentrations can be used. Non-limiting examples of effective polynucleotide treatment regimes include a treatment of between about 0.1 to about 1 nmol of polynucleotide molecule per plant, or between about 1 nmol to about 10 nmol of polynucleotide molecule per plant, or between about 10 nmol to about 100 nmol of polynucleotide molecule per plant.

[0158] Embodiments of compositions of this invention include a "transfer agent", i. e., an agent that, when combined with a composition including a polynucleotide of this invention that is topically applied to the surface of an organism, enables the polynucleotide to enter the cells of that organism. Such transfer agents can he incorporated as part of the composition including a polynucleotide of this invention, or can be applied prior to, contemporaneously with, or following application of the composition including a polynucleotide of this invention. In embodiments, a transfer agent is an agent that improves the uptake of a polynucleotide of this invention by an insect. In embodiments, a transfer agent is an agent that conditions the surface of plant tissue, e. g., seeds, leaves, stems, roots, flowers, or fruits, to permeation by a polynucleotide of this invention into plant cells. In embodiments, the transfer agent enables a pathway for a polynucleotide of this invention through cuticle wax barriers, stomata, and/or cell wall or membrane barriers into plant cells.

[0159] Suitable transfer agents include agents that increase permeability of the exterior of the organism or that increase permeability of cells of the organism to polynucleotides of this invention. Suitable transfer agents include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. In embodiments, application of a composition of this invention and a transfer agent optionally includes an incubation step, a neutralization step (e. g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. Suitable transfer agents can be in the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition, or can cause the polynucleotide composition to take the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition. Embodiments of transfer agents include counter-ions or other molecules that are known to associate with nucleic acid molecules, e. g., inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Embodiments of transfer agents include organic solvents such as DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents miscible with water or that dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Embodiments of transfer agents include naturally derived or synthetic oils with or without surfactants or emulsifiers, e. g., plant-sourced oils, crop oils (such as those listed in the 9.sup.th Compendium of Herbicide Adjuvants, publicly available on-line at herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0160] Embodiments of transfer agents include organosilicone preparations. For example, a suitable transfer agent is an organosilicone preparation that is commercially available as SILWET L-77.RTM. brand surfactant having CAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currently available from Momentive Performance Materials, Albany, N.Y. In embodiments where a SILWET L-77.RTM. brand surfactant organosilicone preparation is used as transfer agent in the form of a spray treatment (applied prior to, contemporaneously with, or following application of the composition including a polynucleotide of this invention) of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of a polynucleotide of this invention into plant cells from a topical application on the surface. One embodiment includes a composition that comprises a polynucleotide of this invention and a transfer agent including an organosilicone preparation such as Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent). One embodiment includes a composition that comprises a polynucleotide of this invention and a transfer agent including SILWET L-77.RTM. brand surfactant in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about 1%., by weight (wt percent).

[0161] Organosilicone compounds useful as transfer agents for use in this invention include, but are not limited to, compounds that include: (a) a trisiloxane head group that is covalently linked to, (b) an alkyl linker including, but not limited to, an n-propyl linker, that is covalently linked to, (c) a polyglycol chain, that is covalently linked to, (d) a terminal group. Trisiloxane head groups of such organosilicone compounds include, but are not limited to, heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an n-propyl linker. Polyglycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Polyglycol chains can comprise a mixture that provides an average chain length "n" of about "7.5". In certain embodiments, the average chain length "n" can vary from about 5 to about 14. Terminal groups can include, but are not limited to, alkyl groups such as a methyl group. Organosilicone compounds useful as transfer agents for use in this invention include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane. An example of a transfer agent for use in this invention is Compound I:

##STR00001##

[0162] (Compound I: polyalkyleneoxide heptamethyltrisiloxane, average n=7.5).

[0163] Organosilicone compounds useful as transfer agents for use in this invention are used, e. g., as freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g., about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent).

[0164] Embodiments of transfer agents include one or more salts such as ammonium chloride, tetrabutylphosphonium bromide, and ammonium sulfate, provided in or used with a composition including a polynucleotide of this invention. In embodiments, ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate are used at a concentration of about 0.5% to about 5% (w/v), or about 1% to about 3% (w/v), or about 2% (w/v). In certain embodiments, the composition including a polynucleotide of this invention includes an ammonium salt at a concentration greater or equal to 300 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes an organosilicone transfer agent in a concentration of about 0.015 to about 2 percent by weight (wt percent) as well as ammonium sulfate at concentrations from about 80 to about 1200 mM or about 150 mM to about 600 mM.

[0165] Embodiments of transfer agents include a phosphate salt. Phosphate salts useful in a composition including a polynucleotide of this invention include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts. In certain embodiments, the composition including a polynucleotide of this invention includes a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes a phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the composition including a polynucleotide of this invention includes sodium phosphate at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes sodium phosphate at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, the composition including a polynucleotide of this invention includes a sodium phosphate salt in a range of about 1 mM to about 25 mM or in a range of about 5 mM to about 25 mM. In certain embodiments, the composition including a polynucleotide of this invention includes a sodium phosphate salt in a range of about 10 mM to about 160 mM or in a range of about 20 mM to about 40 mM. In certain embodiments, the composition including a polynucleotide of this invention includes a sodium phosphate buffer at a pH of about 6.8.

[0166] Embodiments of transfer agents include surfactants and/or effective molecules contained therein. Surfactants and/or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organosilicone surfactants. In certain embodiments, the composition including a polynucleotide of this invention is formulated with counter-ions or other molecules that are known to associate with nucleic acid molecules. Non-limiting examples include, tetraalkyl ammonium ions, trialkyl ammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine. In certain embodiments, the composition including a polynucleotide of this invention is formulated with a non-polynucleotide herbicide e. g., glyphosate, auxin-like benzoic acid herbicides including dicamba, chloramben, and TBA, glufosinate, auxin-like herbicides including phenoxy carboxylic acid herbicide, pyridine carboxylic acid herbicide, quinoline carboxylic acid herbicide, pyrimidine carboxylic acid herbicide, and benazol in-ethyl herbicide, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides. In certain embodiments, the composition including a polynucleotide of this invention is formulated with a non-polynucleotide pesticide, e. g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

[0167] All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure. Although the materials and methods of this invention have been described in terms of preferred embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of this invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of this invention as defined by the appended claims.

Sequence CWU 1

1

4911125DNASpodoptera frugiperda 1ggtgacatgg ccaccatcca ggtatacgaa gaaacatcag gtgtaactgt aggtgacccc 60gtgctgcgta ccggcaagcc cctgtccgta gagctgggtc ctggtatcct cggctccatc 120tttgacggta tccagcggcc actgaaggac atcaacgagc tcacacagtc catctacatc 180cccaagggtg tcaacgtacc ctgccttgga cgtgatgtca cctgggaatt caaccccttg 240aatgttaagg tcggctccca catcaccgga ggagacttgt acggtatcgt acacgagaac 300acattggtta agcataagat gttgatccca cccaaggcca agggtaccgt cacctacatc 360gcgccctccg gcaactacaa agtcactgac gtagtgttgg agacggagtt cgacggcgag 420aaggagaagt acaccatgtt gcaagtatgg ccggtgcgcc agccccgccc cgtcactgag 480aagctgcccg ccaaccaccc cctgctcacc ggacagagag tgctcgactc tctcttccct 540tgtgtccagg gtggtaccac ggccatcccc ggcgccttcg gttgtggcaa gactgtcgtc 600tcacaggctc tgtccaagta ctccaactct gacgtcatca tctacgtcgg atgcggtgaa 660cgtggtaacg agatgtctga ggtactgcgt gacctccccg agctgacggt ggagatcgag 720ggcatgaccg agtccatcat gaagcgtacc gcgctcgtcg ccaacacctc caacatgcct 780gtagccgccc gagaggcttc catctacacc ggtatcaccc tctccgagta cttccgtgat 840atgggttaca acgtgtccat gatggctgac tccacctctc gttgggccga agctcttcgt 900gagatctcag gtcgtctggc tgagatgcct gccgactccg gttaccccgc ctacctggga 960gcccgtctgg cctccttcta cgagcgtgcc ggacgtgtga agtgcctggg taaccccgac 1020agggagggct ccgtgtccat cgtgggcgcc gtgtcgccgc ccggaggtga cttctccgac 1080cccgtgacgg ccgccacgct gggtatcgtg caggtgttct ggggt 112522724DNASpodoptera frugiperda 2aactatgaaa cacattcgca gatcaagaga ttcgaggtat gcgacctacc ggtgcgcgcc 60gctaagttcg tgcctcgcaa gaactgggtg attactggct ccgatgacat gcagattcgc 120gtgttcaact acaacacgct tgagagagtg cacgcctttg aggcgcattc tgactacgtc 180aggtgcatcg cgatacatcc cacacagcca tacatcctca ccagcagcga tgacctattg 240atcaagctgt ggaactggga acgcaactgg gcatgccagc aagtgttcga gggccacaca 300cattatgtga tgcagatcgt catcaaccct aaagacaaca acacattcgc tagtgctagt 360ctcgacacca ccgtcaaagt atggcagctt ggctcttcaa tttccaactt cacattagaa 420ggccacgaga aaggcgtgaa ctgcgtcgac tactaccacg gcggcgacaa gccttacctc 480ataagtggtg ccgacgatcg cctcgtcaaa atatgggact accagaataa aacatgtgtc 540cagacattgg agagtcacac gcagaatgtg acagccgtgt cgttccaccc ggagctgccg 600atcctgatga ctggctcaga ggacggcacc atcagaatat ggcacgcagg cacttacaga 660cttgaatcct cgctcaacta tggcttcgag agagtctgga ccatttcctc catgcacggc 720tctaacaacg tagctattgg ttacgacgag ggcacgatca tgatcaaggt gggccgcgaa 780gagccggcca tttccatgga cgtcaatgga ggcaagatta tttgggcgaa gcattctgag 840atgcagcaag tcaacctgaa ggcgttgcca gaaggcacag agataaaaga tggagagcgg 900gttccagtta tggcgaagga catgggatcc tgtgagattt atccacaaac tatagcacac 960aacccgaacg gtcggttcgt ggtagtgtgc ggggacggcg agtacattat ttacacggcc 1020atggcactta ggaacaaggc gttcggcact gcacaggagt tcgtctgggc gtttgatagc 1080tcagagtatg cgacacttga gaactccagc accatcaagg tgttcaagaa cttcaaagag 1140agaaagagtt tcaagcctga gtatggtgct gaaggaatat acggcggttt catgttgggc 1200gtgaagtcca tcagcggtgt ggcgttctcc ttctatgatt gggagaactt ggagttgatc 1260agacggattg agatccagcc gcggcacgtg tactggtcgg agagcggcaa cctggtgtgc 1320ctggcggctg atgactcgta ctacgtgctc aagtataatg cagctgttgt gacgcgagct 1380cgcgaaacca actccaacat cacagaagac ggcatcgaag acgcttttga ggtcgtgggt 1440gcagtgaacg aggtggtaaa gacaggacta tgggtgggcg actgcttcat ctacacgaat 1500tccttgaaca gaataaacta ctacgtcggc ggagagatcg tcaccatatc ccacctggac 1560cacaccatgt acatcctcgg atacgtcgct aaggagaaca gactgtacct aaatgacaag 1620gagttgaaca tagtgtcgta ctcgctgctg ctgtcggtac tggagtacca gacggcggtg 1680atgcgcgcag acttcgagac agcagaccgg gtgctcccca ccataccgca agagcatcgc 1740accagagtcg ctcacttcct agagaaacaa ggcttcaaac aacaagctct agcagtctct 1800acggagcctg aacaccagtt cgagctggct ctctcactgg gagaacttag aagagcaaag 1860cagttggccg aggaggcggc attggccgag ggttccacct cccgatcctc agcggcacgg 1920tggtctcggc tgggagcggc cgccgccgcc gctgcagaca cggaactcac caaggcctgc 1980tatcagagcg ctaaggacta cagtgccctc cttctattcg cagctagtac tggtgacaaa 2040gaactgttga agagtgtagc acagatgtcc tcggaggaaa acgcggagaa tatctcattc 2100gtggcctact ttatgctgaa tgatctcgaa tcctgtctga agctgctcat acagcgggac 2160aagctgcctg aagccgcctt ctttgccaga tcatacattc catcgaaaat gtcggaagtg 2220gtgaagttat ggcgcgagtc caccagcgcc accaacacga agctcggcca gtcgctcgcc 2280gacccggagc agtatgagaa tttgttcccc gagtttgcgc aatcattgga gttggagaga 2340tttcagcgcg agtatgggta cgagcagagt tgcttgctag cgaacttgcc ggtcgactcg 2400aagacttgca accttgagcg taaccttgcc accgagaagg aggaggccga gcaacgcggc 2460tttaagctga gctccgctgc agtggcgcgg tctcctgcac acagctcaaa tgatcttggt 2520cctgacgacc gagtcacaaa caatgatagt ccttcccacc cggtgccgga gagcacggtg 2580gcgaccgcgg cgcagcagga tctgaagaaa cggcgcgact ctctcgacat catggaggaa 2640ctggagcgcg agatcgaaga catcgtcctg gacggcacgg tcgagagcgt ggatctgtcg 2700gacgatgtag atttcatgga ctaa 272432838DNASpodoptera frugiperda 3atgtgtataa gagacagcta tactctgatc aacttcccga ctgattcaga gccttacaat 60gagatgcagc tcaagctgga tcttgagaaa ggygacacaa agaagaaaat agaagcatta 120aagaaggtaa taggtatyat cytktctggk gagaagatwc cyggtctatt gatgatcatc 180atccgattyg tgctgcccct gcaagaccac accatcaaga agctgttgct katcttctgg 240gaaatagtgc caaagaccac tcctgatggc aagctcatgc aggagatgat ccttgtctgt 300gatgcttaca gaaaggatct gcaacacccc aatgagttca tccgtggctc caccctgcgc 360ttcctctgta aactgaagga gcctgaactc ctcgagcctt tgatgcccgc catcagagct 420tgtcttgacc accgccactc atatgtcagg aggaatgctg ttcttgccat atttactatc 480tacaggaact ttgaattcct gatccctgat gctccagagc tagtagcaaa cttcttggag 540acagagcagg acatgtcctg caagaggaat gcatttctta tgctgctcca tgctgaccag 600gagcgggcac tgtcctacct ctcatccagg ctggataatg tacagggctt tggagatatc 660cttcagttgg ttattgtaga acttatttac aaggtttgcc acgcgaatcc atcagagagg 720tctcgtttca tccgtacagt atacggctta ctgaacgcga ccagtgccgc cgtgcggtac 780gaggctgccg gtactctcgt tacactgtct aacgcgcctg ctgctatcaa ggcggcggca 840gcatgctaca tcgacctaat agtgaaggag agcgacaaca acgtgaagct gatagtggtg 900ggtcggctgg gagcgctgcg cgcggaggcg ggcgaggcgg cggcgcgcgc gctgcccgag 960ctggccatgg acgtgctgcg cgtgctggcc gcctccgacc tggacgtgcg ccgacacacg 1020ctggcgctgg cgctcgacct cgtgtcctcc cgccacgccg aggacctcgt gcaggtgctg 1080cggaaggagg ccgcccgggc taccaacgca gaccacgatg atgctgccaa gtacaggcag 1140ctacttgtta gagctctgca ccgagctgca ctcaagttcc ccgaagtagc cggcagtgta 1200gccccggcgc tgctggagtt gctgggcgac ggcagtgagc cggccgcgca ggatgtcatg 1260ctgttcctac ggtccgctct acataccttc gtgacctacg gatcatatct accagaaact 1320gttggaggcg gtgcccggta tcaaagtggt aagatagcgc ggtctgcgct gtggctgctg 1380gcccagttcg ctgagactcc ggaacgcgcc aaggatgcct tggatgtact cgccaacgtc 1440ataccttccc ttagcggaca agaggataag gaagaatccg agtcggcagc taaggcccag 1500gacacttcag ctccacgaca gcttgtcacc agtgatggaa cttatgcttc gcagtctgct 1560tttaacttgc cagttagcca agcggctcca acccacgcgg gtctatgggc ggcactaggc 1620gagggcgaga gcttcacggc ggcgtgcgcg tgctcggcgc tgtgcaagct ggcgctgaag 1680ctgtcgggcc gggccgcgac cgccgcgctg cagctggccg cgcgcctgct ggccgcccac 1740aagctggccg ccggcctcac cgccgacgac gccgagcacg gcgcgcgatg tatactggcc 1800gcgagacacc gcccgcccgt cgtacaggaa gcgctgctgc aacgctcctc tgctgcgctc 1860gccgcgctgc tagcgctgcc tgaccgagct actaatctgc ttgatgatgc tgataaggaa 1920cgtgagccaa agaagcaaga caacaaggtg gaggtggaac aaggcatcgt gttcgcccag 1980ttggctggga actccgccgc ctccacacac cacgacatgt tcgagctatc gctcactaag 2040gcactgcaag gccgcagtac cggcgtgagc gaggagcgcg gcaagctgtg gaaggtgacg 2100cagctgaccg ggttctccga ccccgtgtac gccgaggcca tcgtcgccgt caaccagtac 2160gacatcgtgc tcgacgtact cgtcgtcaac cagaccgacg acacgctgca gaactgctgc 2220gtggagctgg cgacgctggg cgacctgcgg ctggtggagc ggcccggggc cgtggtgctg 2280gcccccaggg actacgccac catcaaggcg cacgtcaagg tcgcctccac cgagaacggc 2340atcatcttcg ggaacattgt gtacgaggta tcgggcgcgt cgatggaccg cggcgtggtg 2400gtcctgaacg acatccacat cgacatcgtg gactacatcc agcccgccgt ctgcaccgac 2460gcagacttca ggcagatgtg ggccgagttc gagtgggaga ataaggtgtc cgtgaacacg 2520aacatcacgg acctgcgcga gtacttacaa catttactag cttccacaaa catgaagtgt 2580ttgacgcctg aaaaggcatt atcgggtcaa tgcgggttca tggcagccaa cctgtacgcg 2640cggtccatat tcggcgagga cgcgctggcc aacctgagca tcgagacccc gctgcacaaa 2700cccaactcgc ccgtcgtcgg acatgtcagg atcagggcca agagccaggg tatggcgctg 2760tctctaggcg acaaaatcaa catgatgcac aagacgccgc aacagaagac cccctccaac 2820cccatccccg ccgcgtaa 283842097DNALygus hesperus 4ggttggtggt ttggttggac tggacgacat tctgcgaagt taactttgtc tacaaataac 60agattcaacc atggctttac ccagaatccg tgatgaggag aaagaatcca gatttggata 120tgtattcgcc gtttctggcc ctgtcgtcac tgcggagaag atgtcggggg ccgctatgta 180cgagctggtg cgcgtcgggt acttcgagtt ggtcggcgaa atcattcgtc ttgaaggaga 240catggccacc attcaggtct acgaagaaac atccggtgta acagttggag atcccgtgtt 300gagaactggg aaaccacttt cggtggagct cggtccgggt attatgagca gcatttttga 360cggtattcag cgacctttga aagacatttg cgagctgact cagagcatct acatccccaa 420gggagtcaac gttccagctc tgtccaggtc tattgcatgg gacttcactc cgtccaacaa 480tatcaaggtg ggagcacaca tcactggtgg tgatttgtat gccgtcgttc acgaaaacac 540gcttgtcaag caaaaaatga tcatgccggc cagaggaagg ggtaccgtga aatacatcgc 600tccccctggc aactacactg ttgatgacgt cgtaatggaa actgaattcg acggagagaa 660aactgaaatc aagatgttgc aagtttggcc tgtccgacag ccccgtccag ttgccgaaaa 720actgcctgct aactatccac tcttgactgg tcaacgagtt ttggatgccc tcttcccgtg 780tgtccaaggt ggtaccaccg ccattcccgg tgccttcggc tgtggaaaaa ctgtcatctc 840acaagctctg tccaaatact caaactctga cgtcatcatt tacgtcggat gcggtgaacg 900tggtaacgaa atgtctgagg tattgagaga tttccccgaa ctcacagttg agattgacgg 960tgtaactgag tccatcatga agcgtactgc tctggtcgcc aacacatcca acatgcctgt 1020agctgctcga gaagcttcca tttatactgg tatcacattg tccgaatact tccgtgacat 1080gggttacaac gtgtcgatga tggctgactc cacctctcga tgggccgaag ccttgagaga 1140aatttcaggt cgtctcgctg aaatgcctgc tgacagtggt taccctgcct acttgggagc 1200ccgtttggct tccttctacg agcgagctgg tcgtgtcaaa tgtcttggaa gtcccgacag 1260agagggctca gtcagtatcg tcggtgccgt gtcgcctcct ggtggtgact tttcggatcc 1320tgtcacttca gccacccttg gtatcgtaca ggtcttctgg ggtctcgaca agaaattggc 1380acaaaggaaa cacttcccct ccatcaactg gctcatctct tacagtaagt acatgagagc 1440tttggacgac ttctatgaca aacggtaccc tgaattcgtg cccctgagga ccaaggtcaa 1500ggagatcctc caggaggaag aagatttggc tgaaattgtg cagctcgtcg gtaaaggttc 1560gctggccgag tctgataaga tcacattgga aatcgctaag atcttgaaag acgatttctt 1620gcaacaaaac agctactcgc cctacgacag attctgtccg ttctacaaga cggtcggtat 1680gttgaagaac atgatctctt tctatgatct tgcgaggcac acggtggaat caacagcaca 1740aagcgacaac aagatcactt ggactgtcat caaagaaagc atgggcaaca tcctctacca 1800gctgtcctca atgaaattca aggaccccgt caaagacgga gaagccaaga tcaaaggcga 1860cttcgaacag ctccacgaag acatgcaaca agctttccgc aacctcgaag actaaacagt 1920tttctcgttc gctaccttat tgttgacaat agtggcacta cagattaact tcagtgcaat 1980ttttaacagc aaccgcaaat atcctcctcc tccccccctt gaaactcata ctatcgttac 2040acaatttgta catataaaaa cacgtctgtt gtaattacac ataattattg tatatct 20975701DNALygus hesperus 5ggtggttctc ttcgtccgac catgagttcg ctcaaactgc agaagaggct cgccgcctcg 60gtgatgagat gcggcaagaa gaaagtgtgg ttggacccta atgaaatcaa cgaaatcgcc 120aacaccaact ctaggcaaaa catccgtaag ctgatcaagg atggtttgat catcaaaaag 180cctgtggctg tccactccag agcccgcgtc cgtaaaaaca cagaagccag acggaagggt 240cgtcactgtg gcttcggtaa gaggaagggt accgccaacg ccagaatgcc tgtgaaggtc 300ctgtgggtca acagaatgag agtcctgcga cggctcctta aaaaatacag agaagccaag 360aagatcgata ggcaaatgta ccacgacctt tacatgaaag ccaaaggtaa cgtcttcaaa 420aacaagaggg tactgatgga cttcattcac aagaagaagg ctgaaaaggc gagatcaaag 480atgttgaagg accaggcaga ggcgagacgt ctcaaggtca aggaggcgaa gaagaggcgc 540gaggagagga tcgccaccaa gaagcaagag atcatgcagg cgtacgcccg agaagacgag 600gctgccgtca aaaagtgatc tcgccccctc cgtttttaaa ttttaaacaa aaaacgtatt 660ttgtacaaaa atctacaaaa aaattacaaa agagaaaact t 70162817DNALygus hesperus 6atgcctctca aattggacat caagagaaag ctgtctgctc gatcagaccg tgtgaaatgt 60gtcgatctgc acccaactga gccctggatg ttggcttctc tctacaacgg aaacgttcac 120atttggaacc acgaaactca acagcttctg aaatccttcg aagtatgcga gcttccaatc 180agggctgcag ttttcgtacc gaggaagaac tgggtggtca caggctcgga cgacatgcac 240gttcgtgtct tcaactacaa cactctcgag cgtgtacatt ccttcgaggc ccattctgat 300tatttgagat gcatcatcgt acatcctaca cagccttaca tattgacgtg cagcgatgac 360atgctgatca agctgtggaa ctgggaaaaa aactggctgt gccagcaagt cttcgaaagc 420cacacccatt acgtcatgca gatcgtgctg aacccgaagg ataacaacac tttcgcctct 480gcctcgctcg accacacgct caaagtgtgg cagttgggct cagcagcggc caacttcact 540ttggacggac acgaaaaagg agtgaactgc gtcgactact accacggagg agataagcct 600tatctcatct ctggcgcgga cgatcacatg gtcaaaatat gggattacca gaacaaaacg 660tgcgtccaga ctttggaggg acacgctcaa aatataactg cagtttgctt ccacactgaa 720ctaccaatcg caattactgg ctcggaagat ggaaccgttc gcttgtggca ctcagccacc 780tatcggttgg aatcgtcctt gaactacggc tttgaaagag tatggaccat acgctgtctc 840aaaggctcaa accacattgc tcttgggtac gacgagggtt ccattatggt caaagttggt 900cgagaagaac cggccatttc catggatgta aatggagaaa aaattgtttg ggctcgacat 960tctgaaatcc agcaggtcaa tttgaagtct ctcatgactg acgagagtga aattcgcgat 1020ggggagaaac tcccagtagc agctaaagac atgggtccct gcgaagtttt cccgcaaagc 1080atcgcccaca accccaatgg aagatttgtg gttgtttgcg gtgatggaga atacatcatc 1140tacactgcca tggctttgcg taataaaagt ttcggttccg cgcaagagtt cgtctgggcg 1200caggactcgt ccgactacgc catccgcgaa gggacgtcta cggtccgact tttcaggcag 1260ttcaaggaaa ggaagaactt caagcctgaa tttggagctg aaggtatttt tgggggacag 1320cttctcggag ttaggactgt aactggactg tccctctacg actgggaaac tttggagttg 1380atcagaagca tcgacattca agcgaaagcg ctgtactggt ccgaagcagg gcatctcttg 1440gcaatcgtta ctgacgacag ttactatctc ttgaaattcg accagagcgc catctcgacg 1500tccacccctg gaactgacgg ctacgaagat gcctttgagc tcgtcggtga agtcaatgat 1560actgtcaaga ccggattgtg ggttggtgac tgtttcatct acacaaacgc cgtttgtcgg 1620atcaactact acgtaggtgg tgagatcgtc accgtggctc acctcgacac tacaatgtac 1680ctcctaggat acgtggcccg tcagaacctg ctgtacctgt gcgacaagca tcataacatc 1740atttgttaca cgttgcttct gtctgtcctc gaatatcaga ctgctgtgat gaggagagac 1800tttgaaactg ctgaccgagt tttgcccact attcctgttc agcatcgctc aagagttgct 1860catttcctgg agaaacaggg cttcaaaagg caagctctgg ctgtgtccac ggatgccgag 1920cacaagtttg aacttgcgct tcagctcagt gatttggaag cagcagtcgg cctagcgagg 1980gaaatcggca gcaaagccaa gtgggttcag gtcgccgagt tggcgatgtc agaggccaag 2040ctcggactcg ctcagatgtg cttgcatcag gcacagcact acggaggact tctgctcctg 2100tcaacttctg ccggaaatgt ggacatgatg gagaaactgg ccgaaagctc gctgtccgat 2160ggcaaaaaca acgtctcgtt cctcacttac ttcctgatgg gtaacgtgga aaagtgtctc 2220caaatcctca tcgatactgg aagaattccg gaagcagctt tcttcgcccg gacctatatg 2280cctaaagaag tgtcccgcgt ggtcgacatg tggaaaactc tttctaagga caagacgggg 2340caatcgctcg ctgacccagc ccaatacccg aatctattcc ccaagcacac cgaggctctg 2400aaagccgaac agttcatgaa gaaggaattg actcaaagga ttcccgcctc gtcgcacaag 2460gatataaaac ccaactacga aaggaatgcc attgaagaaa tgaaagaagc cgaagcaaac 2520ggtctgttca cgtatgatcc tccagtggct cctgccagta tcaacaatct aattgatgtt 2580tctgaaccgg cgaatcgatc tgagcccagc ccgtccgaaa tcttctccga agcgcccgcc 2640gtgtccaaga tgaccagcga cgctcggccg ctggtcgcgc cagttccgcc tgccgcgaga 2700cctcaaaaac ggccgtcggc cttcgatgat gacgacctcg aattggaaat cgaaaatatg 2760aatttggatg acatcgatgc tagtgatttg aacgaagaag acctccttat agattag 281775621DNALygus hesperus 7actcgttcta gatcgcgacg gacgcgtgga cgagaaacga gaacgagcta cgttgagcat 60caagagcttt tgtactattg aaattgtgaa aaatgcagat cttcgttaaa actttgactg 120ggaagaccat caccctcgag gtcgagcctt ctgataccat tgaaaacgtg aaggcgaaaa 180ttcaggataa agaaggcatc cccccagatc agcagaggtt gatctttgcc ggcaagcagt 240tggaagacgg acgtactttg tctgactaca acatccaaaa agaatccact ctccacctgg 300tcttgagatt gagaggtggc atgcagatct tcgtgaagac cctcacagga aagaccatca 360ctcttgaggt cgagccttct gacaccatcg aaaacgtcaa ggctaaaatt caagacaagg 420aaggtattcc tccagatcag cagagattga tcttcgccgg caaacaactc gaagatggcc 480gtaccctctc tgactacaat attcaaaaag agtccaccct tcacttggtg ttgagattgc 540gtggaggtat gcaaatcttt gtcaaaacat tgactggaaa gaccatcacc cttgaagtcg 600aaccctccga caccatcgaa aatgtcaagg ccaagatcca ggacaaggaa ggcatccccc 660cagatcagca gaggttgatt ttcgctggca aacaacttga agacggacgt accctctcgg 720actacaacat ccagaaggag tcgaccctcc atcttgtcct ccgtctgcgt ggtggtatgc 780agatttttgt caaaactctg actggcaaga caatcaccct tgaagtagag ccctctgaca 840ccatcgaaaa tgtcaaggcg aaaatccagg acaaagaagg catcccccca gatcagcaga 900ggttgatctt tgccggtaag cagcttgaag acggccgcac cctctcggac tacaacatcc 960agaaggagtc cacccttcat cttgtcctac gtctgcgtgg tggtatgcag attttcgtaa 1020agaccttgac tggcaagacc atcactcttg aggttgagcc ctctgacacc atcgaaaacg 1080tcaaggccaa gatccaggac aaggaaggta tccccccaga tcagcagagg ttgatcttcg 1140ctggcaagca gctcgaagat ggtcgtaccc tctcggacta caacatccag aaagagtcca 1200cccttcatct tgtcctccgt ctgcgtggtg gtatgcagat tttcgtaaag accttgactg 1260gcaagaccat cactcttgag gtcgagccct ctgacaccat tgaaaacgtc aaggccaaga 1320tccaggacaa ggaaggtatc cccccagatc agcagaggtt gatcttcgcc ggtaagcaac 1380ttgaagacgg ccgtaccctc tcggactaca acatccagaa ggagtccacc cttcatcttg 1440tcctccgtct gcgtggtggt atgcagatat tcgtaaagac cttgactggc aagaccatca 1500ctcttgaggt cgagccctct gacacccttg aaaaccgcaa ggccaagatc caggacaagg 1560aaggtatccc cccagatcag cagaggtgtg atcttcgccg gtaagcaact tgaagacggc 1620cgtaccctct cggactacaa catccagaag gagtccaccc ttcatcttgt cctccgtctg 1680cgtggtggta tgcagatatt cgtaaagacc ttgactggca agaccatcac tcttgaggtc 1740gagccctctg acaccattga aaacgtcaag gccaagatcc aggacaagga aggtatcccc 1800ccagatcagc agaggttgat cttcgctggc aagcagctcg aagatggtcg taccctctcg 1860gactacaaca tccagaaaga gtccaccctt catcttgtcc tccgtctgcg tggtggtatg 1920cagattttcg taaagacctt gactggcaag accatcactc ttgaggtcga gccctctgac 1980accattgaaa acgtcaaggc caagatccag gacaaggaag gtatcccccc agatcagccg 2040aggttgatct tcgctggcaa gcagctcgaa gatggtcgta ccctctcgga ctacaacatc 2100cagaaagagt ccacccttca tcttgtcctc cgtctgcgtg gtggtatgca gattttcgta 2160aagaccttga ctgggaagac catcactctt gaggtcgagc cctctgacac cattgaaaac 2220gtcaaggcca agatccagga caaggaaggt atccccccag atcagcagag gcagatcttc 2280gccggtatgc aacttgaaga cggccgtacc ccgagaaacg agaacgggct acgttgagca 2340tcaagagctt ttgtactatt gaaattgtga aaaatgcaga tcttcgttaa aactttgact 2400gggaagacca tcaccctcga ggtcgaacct tctgatacca ttgaaaacgt gaaggcgaaa 2460attcaggata

aagaaggcat ccccccagat cagcagaggt tgatcttcgc cggtaagcag 2520cttgaggatg gacgtactct ctcggattac aacatccaga aggagtcaac cctccacctt 2580gtcctccgtc tgcgtggtgg tatgcagatc ttcgtcaaga ccttgactgg caagacgatc 2640actttggaag tcgagccctc tgacaccatt gagaatgtca aagccaaaat ccaagataag 2700gaaggcatcc ccccagatca gcagaggttg atcttcgccg gtaagcagct tgaagacggc 2760cgtactctct ctgattacaa catccagaag gagtcgaccc tccaccttgt cctccgtctt 2820cgtggtggta tgcagatttt cgtaaagacc ttgactggca agaccatcac ccttgaggtc 2880gagccctccg acaccatcga aaacgtcaag gccaagatcc aagataagga aggcatcccc 2940ccagatcagc agaggttgat cttcgccggt aagcagcttg aggatggacg taccctgtca 3000gactacaaca tccaaaagga gtccaccctg cacttggtgt tgagattgcg tggtggtatg 3060cagatcttcg tcaagacctt gactggcaag acgatcactt tggaagtcga gccctctgac 3120accattgaga atgtcaaagc caaaatccaa gataaggaag gcatcccccc agatcagcag 3180aggttgatct tcgctggtaa gcaacttgaa gacggccgca ccctctctga ctacaacatc 3240cagaaggagt cgaccctcca tcttgtcctc cgtctgcgtg gtggtatgca gattttcgtg 3300aagaccttga ctggcaagac catcaccctt gaagtcgagc cctctgacac cattgagaat 3360gttaaagcca agatccagga caaggaaggt atccccccag atcagcagag gttgatcttc 3420gccggtaagc agcttgaaga cggccgtact ctttcggatt acaacatcca gaaggagtcg 3480accctccacc ttgtcctccg tctgcgtggt ggtatgcaga tcttcgtcaa gaccttgaca 3540ggcaagacca tcacccttga agtcgagccc tctgacacca tcgaaaacgt caaggctaag 3600atccaggaca aggaaggcat ccccccagat cagcagaggt tgatcttcgc cggtaaacag 3660cttgaagacg gacgtaccct ctcggactac aacatccaaa aggagtccac tcttcacttg 3720gtgttgagat tgcgtggtgg tatgcagatc ttcgtcaaga ccttgacagg caagaccatc 3780acccttgaag tcgagccctc tgacacaatt gaaaacgtca aggccaagat ccaggacaag 3840gaaggtatcc ccccagatca gcagaggttg atcttcgccg gtaagcagct tgaagacggc 3900cgtactctct ctgattacaa catccagaag gagtcgaccc tccaccttgt actccgtctg 3960cgtggtggta tgcaaatttt cgtgaagacc ttgactggca agaccatcac tcttgaggtc 4020gagccctctg acaccattga aaacgtcaag gccaagatcc aggacaagga aggtatcccc 4080ccagatcagc agaggttgat cttcgccggc aagcagctcg aagacggccg tactctctct 4140gattacaaca tccagaagga gtccaccctt catcttgtcc tccgtctgcg tggtggtatg 4200cagattttcg tgaagacctt gactggcaag accatcactc ttgaggtcga gccctctgac 4260accattgaaa acgtcaaggc caagatccag gacaaggaag gtatcccccc agatcagcag 4320aggttgatct tcgctggcaa gcagctcgaa gatggtcgta ccctctcgga ctacaacatc 4380cagaaagagt ccaccctcca ccttgtcctt cgtctgcgtg gtggtatgca gattttcgta 4440aagaccttga ctggcaagac catcactctt gaggtcgagc cctctgacac cattgaaaac 4500gtcaaggcta agatccagga caaggaaggc atccccccag atcagcagag gttgatcttc 4560gccggtaagc agcttgaaga cggccgtact ctctctgatt acaacatcca gaaggagtcg 4620accctccacc ttgtcctccg tcttcgtggt ggtatgcaga ttttcgtgaa gaccttgact 4680ggcaagacca tcacccttga ggtcgagccc tccgacacca tcgaaaacgt caaggccaag 4740atccaagata aggaaggcat ccccccagat cagcagaggt tgatcttcgc cggtaagcag 4800cttgaggatg gacgtactct ctcggattat aacatccaga aggagtcaac cctccacctt 4860gtcctccgtc tgcgtggtgg tatgcagatc ttcgtcaaga ccttgactgg caagacgatc 4920actttggaag tggagccctc tgaccccatt gagaatgtca aagccaaaat ccaagataag 4980gaaggcatcc ccccagatca gcagaggttg atcttcgccg gtaagcaact tgaagacggc 5040cgcaccctct ctgactacaa catccagaag gagtccaccc tccatcttgt ccttcggctg 5100cgtggtggta tgcagatctt cgtcaagacc ttgacaggca agaccatcac ccttgaagtc 5160gagccttctg acaccatcga gaacgtcaag gccaagatcc aggacaagga aggtatccct 5220ccagatcagc aaagattgat cttcgccggc aaacagctcg aagatggccg taccctctca 5280gactacaaca ttcaaaagga gtcaactctt catctcgttc tgaggctccg tggcggtcgt 5340tattgatcac aattccaaac ttaaaaattg cattccgatt ttccttcttt atttggcaaa 5400aaatacatac cctagttaat taaaatgact tgaaatttga ttttttaaga atgcttcaaa 5460tttttttata gatggtttgt tacatagaca atacacaaca tgttgaaagc ataaaaaaaa 5520aaaaaaaaaa aaaaaaaaaa aaaaaaaaac caaaaaaaaa aaaagggggg gcccgttcaa 5580aaggaccaaa gttaacgacc gcgggatggc aacgcattac t 562181927DNAEuschistus heros 8gtttgttgta gttacaggtt ttaatttggt tcacatccta agttcggcaa ttcagatgtc 60tacttccaaa acagaagaaa agcccttgtg ggacgaaaga atggagcaag ctttaggtga 120agaaatcatg agaatgtcta cggatgaaat tgtcagtcgt attaggcttc ttgataatga 180aattaaaatc atgaaaagtg aagtcatgcg agtcagtcat gagttgcagg cacaaacaga 240aaaaattaag gaaaatacag aaaagatcaa agtgaacaaa actttaccat atttagtatc 300taatgtgata gagttgcttg atgttgatcc agaggaaact gaggaagatg gtgctgttgt 360tgaccttgat gcccgtcgta aaggaaaatg tgctgtcatt aaaacatcaa cgaggcagac 420ttattttttg ccagtaatag gcttagttga tgctgaaaag ttaaagccag gtgatctggt 480tggagtaaac aaagattctt atttaattct tgaaaccttg cctgctgagt atgatgcacg 540agttaaagca atggaagttg atgaaagacc tactgagcag tattctgata tcggtgggct 600tgataagcag attcaagaat taatagaagc cgttgttctt cctatgacac acaaagaaaa 660atttgaaaat ctaggaattc atcctcctaa aggtgtattg ctctatggac ctcctggaac 720aggaaagact ttactggcta gagcgtgtgc agctcaaact aaatcaactt ttttgaaact 780agctggaccc caacttgttc agatgtttat aggagatgga gccaaacttg tcagagatgc 840ttttgctctt gctaaggaaa aatctcctgc tattatcttt attgacgagt tggatgctat 900tggaacaaaa agatttgatt ctgaaaaagc tggtgatcga gaagttcaaa gaactatgtt 960agaactgctc aaccagttag atggatttag ctcaacagct gatattaagg ttattgcagc 1020tacaaataga gtagatattt tggatcccgc ccttttgcgt tcaggtcgac tagacagaaa 1080aattgaattc ccacatccta acgaagatgc ccgagcacgt ataatgcaaa ttcattctcg 1140taagatgaat attagtgttg atgttaattt tgaagaactt gctcgttcaa ctgatgattt 1200caatggagct caatgcaaag cggtttgtgt ggaagcaggt atgatagcat tgcgaagagg 1260agctggagtt gtcactcatg aagatttcat ggatgcaata ttagaagtgc aggcaaagaa 1320gaaagccaac ttaaactatt atgcttaaat atctattgat aaagattttt ttaataatgc 1380agaaactcac ttgcattctt cttttcggga gtgccaataa ctgtgattga ttttgtattg 1440ttattaactt gtaaatatgc cactctttga aaaacaaatg tgtaagtaaa ccaacaagta 1500aaatagcaca ttataatttt attaaattct taaagtcgca ttaactccag ttaaaaaagt 1560tttgttaatt catatatata tatatatttt tgcctgttta attttaaaaa gattatattt 1620tctgtatcaa ttattagttt gactaatttg attactagtt tgaaatgagt ttctgccaga 1680tttaatattt aatttttatt tttataatca ttttcaaaat taattatttg ttggttcaca 1740gtttattgaa tatattctaa tctcaaattt gttttttaat ttattttttc attgcattta 1800atattttttg gattaaattg aaaggagatt aaacttcctg ttgcaaaatt ttttttcagt 1860gattctttat attatttgta tatttataaa tttgggaaac atttaataat atcatgttat 1920gtttgtt 192792170DNAEuschistus heros 9ttacattaga agttgagcct tctgacacaa tagaaaatgt caaggccaaa atccaagaca 60aagaaggaat ccctccagac caacagaggt tgtatttttg ctggtaaaca gcttgaagat 120ggccgtactc tttcagatta caatattcaa aaagaatcta ctttacattt agtacttcgg 180ttgagaggag gcatgcaaat ttttgtgaag accttgactg gtaaaaccat caccttggaa 240gtggagccat ctgatacaat tgaaaatgtt aaagctaaaa ttcaagataa agaaggaatt 300cctccagacc aacagaggtt gatttttgca ggtaaacagc tcgaagatgg tcgtacattg 360tctgactata atattcaaaa ggaatctact cttcatcttg tcctacgttt aagaggaggc 420atgcagatct ttgtcaagac acttactggt aaaacaatca cacttgaagt tgagccttca 480gacacaattg aaaatgtgaa agctaaaatt caagataaag aaggaattcc tccagatcag 540cagaggttga tttttgcagg taaacagctc gaagatggtc gtacattgtc tgactataat 600attcaaaagg aatctactct gcatttggtc cttcgtctga gaggaggaat gcaaattttt 660gtaaaaacct tgactgggaa aactattaca ttagaagtgg aaccttctga cactattgaa 720aatgttaaag ctaaaattca agataaggaa ggaattccac cagatcagca gcgattgatc 780tttgctggaa aacagttgga agatggtcgt actttatctg attataacat tcagaaggaa 840tcaacactac atttagtatt acgtttaaga ggtggaatgc agatttttgt taagacgctt 900actggaaaaa ccataacttt agaagttgaa ccgtcagata ccattgaaaa tgtaaaagcc 960aaaattcaag ataaggaagg aattccgcca gatcagcaaa ggctgatctt tgcaggaaaa 1020cagttagaag atgggcgtac tctttctgat tacaacatcc aaaaggaatc tactttacat 1080cttgtactta gacttcgagg aggcatgcag atctttgtca agactttaac agggaagacc 1140atcaccttag aagttgaacc atctgatact attgagaacg taaaagcaaa aatccaggac 1200aaagaaggaa ttccaccaga ccagcaacgt ttgatctttg ctggaaaaca acttgaagat 1260ggccgtaccc tttcagatta taatattcaa aaagagtcta ctcttcatct tgtacttcgc 1320ttgagaggtg gcatgcagat ttttgtaaag actctaactg gtaagaccat aaccttggaa 1380gttgagcctt cagatactat tgagaatgtt aaagccaaaa ttcaagataa agaaggaata 1440ccaccagacc aacagaggct tatttttgct ggaaagcagc tggaagatgg ccgaactttg 1500tctgattaca acattcaaaa ggaatcaaca ctgcatttgg tgcttcgtct aagaggaggc 1560atgcagatct ttgtgaagac tttgacaggt aaaaccatta ccttggaagt agagccatct 1620gatacaattg agaatgtaaa agctaaaatt caggataaag aaggaattcc tcccgaccag 1680cagcgtttga tctttgctgg aaaacaactc gaagatggcc gtaccctttc agattataat 1740atccagaagg aatctacact acatctggtc cttcgattga gaggtggtat gcagatcttt 1800gtcaagaccc tcacaggcaa gactattacc ttagaagttg agccatctga cactattgaa 1860aatgttaaag ccaaaattca ggacaaagaa ggaatacctc cagatcagca gaggcttatt 1920tttgctggaa aacaattaga ggatggtcgt accctatctg attataacat ccagaaggaa 1980tcaactctgc atttagtgtt gcgtcttagg ggcggatatt aaggctactt atcatttcat 2040tccatcaaaa ttccatatca gtaaattgct tttgatattt ttattagcat tttcattatt 2100atatttataa ttatttcata aatacaatgt aactaaacta gaaataaaca gtttctttaa 2160gttcaaaaaa 2170102360DNAPlutella xylostella 10atgagtcacg ggttgaagag gattgccgac gaggacaatg aaacccagtt cggttatgtc 60ttcgctgtgt ccggtcccgt ggtcacagcg gagaagatgt ccggctcggc catgtacgag 120ctggtgcgcg tcggctacaa cgagctggtc ggcgagatca tccgtctgga gggggacatg 180gccaccatcc aggtgtacga agagacctca ggcgtgaccg tcggcgaccc cgtgctgcgc 240accggcaagc ctctctctgt agaactggga cccggcatcc tcggctccat cttcgacggc 300atccagcggc cgctgaagga catcaacgag ctcacgcaga gcatctacat ccccaagggg 360gggaacgtgc ccgcgctggc ccgcgacacc gagtgggagt tccacccgca gtacatcaag 420agcgatgtct cggctggtat ccgaaccccg gtctccgccg ggcgcgtcct cggctccatc 480tccgacggca tccagcggcc gctgaaggac atcaacgagc tcacgcagag catctacacc 540cccaaggggg tgaacgtgcc cgcgctggcc cgcgacaccg agtgggagtt ccacccgcag 600tacatcaaga gcgatgtctc ggccggtatt cgaaccccgg tctccgccgg gcgcgtcctc 660ggctccatct ccgacggcat ccagcggccg ctgaaggaaa tcaacgagct cacgcagagc 720atctacatcc ccaaggcggt gaacgtgccc gcgctggccc gcgacaccga gtgggagttc 780cacccgcagt acatcaagcg ttgtcctgtc tctgccggta ttcgaacccc ggtctccgcc 840gggcacgtcc tcggctccat ctccgacggc atccagcggc cgctaaagga catcaacgag 900ctcacgcaga gcatctacat ccccaagggg gtgaacgtgc ccgcgctggc ccgcgacacc 960gagtggcagt tcaacccgca gtacatcaag gtcggcaccc acatcaccgg cggagacttg 1020tacgggatcg tgcacgagaa cacgctggtg aagcaccgca tgctggtgcc gcccaaggcc 1080aagggcaccg tcacctacat cgcgcccgag gggaactaca aagtcactga cgtggtccta 1140gagaccgagt tcgacggcga gaagtcctcc tacaccatgc ttcaagtgtg gccggtgaga 1200cagccgcggc cgtgcaccga gaaactaccc gccaaccacc ccctgctgac cgggcagaga 1260gtgctcgact cactgttccc caacagtgac tgcatggaga aactagccaa tcacccgctg 1320ctgatcggcc agagagtgct cgaatcactg ttcccttgcg tccagggcgg caccacggcc 1380atccccgggg ccttcggttg cggcaagacc gtcatctcgc aggcgttgtc caagtactcc 1440aactctgacg tcatcgtcta tgtcgggtgt ggggagcgtg gtaacgagat gtccgaagta 1500ctgcgtgact tccccgagct gacagtcgag atcgacggcg tgaccgagtc catcatgaag 1560cgcacggcgc tcgtggccaa cacctccaac atgccagtgg ccgcccgaga ggcctccatc 1620tacaccggaa ttaccctatc cgaatacttc cgtgacatgg gctacaacgt gtccatgatg 1680gccgactcga cctcccgttg ggccgaagcg ctgcgtgaga tctcgggtcg tctggccgag 1740atgcccgctg actctggtta ccccgcgtac ttgggagcta gactggcgag cttctacgag 1800agggctggca gggtcaagtg tctgggtaac ccggagaggg aaggttcggt ctccatcgtg 1860ggcgccgtgt cgccgcccgg aggagacttc tctgaccccg tgacggcggc cacgctcggt 1920attgttcagg tgttctgggg gctggacaag aagctggcgc agaggaagca cttcccctcc 1980atcaactggc ttatttctta cagtaagtac atgcgcgcgc tggacgactt ctacgacaag 2040aactaccccg agttcgtgcc gctcaggacc aagacagtta ctcgaactac gaccgtttct 2100gcccgttcta caagacgacc ggcatgctga agaacatcat cacgttctac gacatgtcgc 2160gacacgccgt cgagtccacc gcgcagtccg acaacaaggt gacgtggaac acgatccgtg 2220acgccatggg acccgtgctc taccagctgt ccagcatgaa gttcaaggac cccgtgaaag 2280atggagaagc caagatcaag gctgacttcg accagatcgt cgaggacatg gccgctgcct 2340tccgtaacct agaggactaa 2360112030DNAPlutella xylostella 11atgccgttga gattggatat caagaggaag ctgacggctc gctcggaccg cgtgaagtgc 60gtggaccagc acccctcgga gccctggctg ctatgctccc tgtacaatgg ggatgtcaac 120atatggaact acgagacaca gctgcaaata aaaagatttg aggtaagtgt atgcgacttg 180ccagtccgtg cggccaagtt tgtgccgagg aagttctggg tgatcaccgg ctccgatgac 240atgcaaattc gtgtcttcaa ctacaacaca ctggaacgtg tacacaattt tgaagctcat 300tcagactatg tgaggtgtat tgcggttcac cccacccagc catacatctt gaccagcagt 360gatgatcttc tgataaagct ttggaactgg gatcgcaact ggacttgcca gcaagtgttc 420gagggacaca cgcactatgt gatgcagatt gtcatcaacc ccaaagacaa caacactttt 480gccagtgcca gtcttgacag aaccgtcaag gtatggcagc tgggctcttc catcccgaac 540ttcactctgg aaggtcacga gaagggagtc aactgcgtgg actactacca cggcggggac 600aagccgtacc tcatcagtgg agctgatgac cgcctcgtca agatatggga ctaccagaac 660aagacttgtg ttcagacctt ggaaggtcac gcccagaatg tgtcagcagt gtccttccac 720cccgagctac cgatcctgct gactggctca gaggacggca cggtgcgcat ctggcacgcc 780ggcacctacc ggctcgagag ctcgctgaac tacggcttcg agcgagtctg gactatatcc 840tccatgcatg ggtccaataa tgtcgctgtt ggttacgacg aagggactat aatgataaaa 900gtaggacggg aggagccggc tatttccatg gacgtcaacg gcggcaaaat catctgggcc 960aaacactcag agatgcaaca agtcaacttg aaagctttac cagaaggtac acacataaag 1020gatggcgaga ggctgccatt ggtggtaaaa gacatgggtt cttgcgagat ctatccacag 1080acgatagccc acaacccgaa cggcagattc gtggtagtct gcggagatgg ggaatacatc 1140atctacacag ccatggcttt gaggaataag gctttcggca atgcgcagga gtttgtgtgg 1200acgtttgaca gctcggagta tgccacactg gagaactcca gcactatcaa agtgttcaag 1260aacttcaagg agcggaagag cttcaaaccg gagtatgggg ctgaaggaat atttggcggt 1320ttcatgctgg gcgtgaagtc aatcagtggg gtttccttct ccatctacga ctgggaacat 1380ctggagctca tcagacgcat cgagatccag ccccgccacg tgttctggtc ggagagcggc 1440agtctagtct gtctggcgac ggacgaaagc tactacattc tgcgctacaa cgccgccgtc 1500gtcaccaagg cgcgcgagac taacaccaac atcacagagg acggcattga ggatgccttt 1560gaggtggtcg gcgaagtgaa cgagacagta aagacgggca tctggatcgg ggactgcttc 1620atctacacca actctctcaa cagaataaac tactatgtcg gcggcgagat cgtcacgata 1680tcgcatctcg atcacaccat gtacatactt ggatacgtag ctaaggaaaa cagactatac 1740ctgaacgaca aggagctcaa cgtggtctcc tattccctcc tgctgccggt gctggagtac 1800cagacggcgg tcatgcgagg ggacttcgag acggcggaca aggtgctgcc ggtcataccg 1860cacgagcacc ggacgagagt cgcgcacttc ctcgagaaac agggcttcaa gcagcaagcg 1920ctagccgtgt ccacggagcc ggaacaccag ttcgagctgg ccctagcttt aggcgagctc 1980cgaagagccc gccaactagc cgaggaggcg acggcggctg gaggggctcc 2030123621DNAPlutella xylostella 12atggggggcg tggagcaatc ctgttacact ctcatcaact tcccaaccga tgcagagcca 60tacaatgaac tgcaactcaa gattgacttg gagagaggtg acatcaagaa gaagattgag 120gctctaaaga aggtgattgg tatcatcctc tccggggaga agatacctgg tatcctcatg 180gtcatcatca ggtttgtgct gccgctgcag gaccatacca tcaagaagct gctactgatc 240ttctgggaga tcgtcccgaa gaccaccccc gacggcaagc tgctgcagga gatgatcctc 300gtctgtgact cgtacaggaa ggacctgcaa caccctaacg agttcatccg cggctccacc 360ctccggttcc tgtgcaagct gaaggagccc gagctgctgg agccgctgat gccggcgatc 420agggcctgcc tcgagcaccg ccactcgtat gtgcggagga acgccgtgct ggccatcttt 480accatctacc gactccggtt cctgtgcaag ctgaaggagc ccgagctgct gaagccgctg 540atgccggcga tcagggcctg cctcgagcac cggcactcgt atgtgcggag gaacgccgtg 600ctggctatat ttaccatcta ccgaaacttc gacttcctca tcccggacgc tccggaactg 660atcggcaagt tcttagagac ggaacaggac atgtcgtgca agcggaacgc tttcctgatg 720ttgctacatg ctgagcagga gcgtgcattg agctacctgg ctgatagatt ggacagtgtg 780gcgggcttcg gggatatact gcaactggtc attgtggagc ttatttataa ggtgtgccac 840gcgaacccgt ccgaacgctc gcgcttcatc cgcacagtgt atgggctgct caacgcgccc 900agcgccgcgg tccggtacga ggcggccggc acgctggtca cgctgtctac tgcgcctgcc 960gctattaagg cggcagcatc ctgctacatc gacctcatag tcaaagaatc tgacaacaac 1020gtgaagctaa tcgttctctc ccgtctcgga gccctacgac agggcgaggc ggcggcgcgc 1080gcgctgcccg cgctggctat ggacgtgctg agggtgctgc aatcggctga tttggatgtc 1140cgtgctcagg ctttacaact ggtgaagctg atagtgctct ctcggctcgg agccctacga 1200caaggcgagg cggcggcgcg cgcgctgccc gccctcgcca tggacgtgct tagggtgctg 1260cagtctgcgg atttggatgt gcgtgctcag gccctacagt tagccctgga cctcgtaacc 1320ccccggcacg cggacgagct agtacaagtg ctgcgcaagg aggcggcgcg cgccaccagc 1380gccgacagcg accacgccgc ccagtaccgc cagctgctgg tgcgcgcgct gcacaaggcc 1440gcgctgcagt tcccagaagt ggccggcagc gtggcgcccg cgctgctgga gctgctcggg 1500gacggcagcg agccggccgc gcccgacgtg ctgctgttca ctagagaggc cctgcagacc 1560ttcccggacc tccgggggca gatctaccag tgtcatatat gtctgtggtc tggtactacc 1620gatggtggtc tggtggatct gtccggtgac tatatcacac agggacagat ctgtccagta 1680ctagagccgg ccgcgcccga cgtgctgctg ttcactagag aggctctaca gaccttcccg 1740gacctccgcg ggcagatcta ccagcgtctc ctagactcga tcggcaactt caagggcaag 1800cgtctgctag actcgatcgg caacatcaag gtgggcaagg tggcccgctc agctctgtgg 1860ctactcgccc agttcgcgga cacggaggcg cgcgccgagg ccgccctggc tgccgtggcc 1920gcctgcgtgc ccactgtagg gggggagcag gagacggatg attccgcagc gaagcaagaa 1980gcgacggcgg cgccgcggca gctggtcacc agcgacggca cttacgcgtc gcagtccgcc 2040ttcaacctgc cctcgtccgc gtccaccgcg tcaacatcca ccaacaacct ccgctcagcc 2100ctcacctcgg gcgactcgtt cacggccgcc tgcgcctgct ccgcgctggc caagctgtcg 2160ctcaagcagg ccactgtgag ggacgccaac agggcgctgc acctcgctgc taggctactg 2220gctcattata agattaatgc tggcaacggc acaaccctca cagcggacga catagagcac 2280gcggctctct gcgccaggct cagctcgcga cgccccaagg ctcttctagg agccgccatc 2340gatggtagtg gggaggcgct taaagcactg ctgaaggaaa ctgcggctga tgatgtggct 2400gccaaggctc tactaggagc cgccatcgac ggcagcggag aggcgcttaa agcactgctg 2460aaggaaactg ctgctgatga tgtggctgcc aagacagcgg ttatagtaga gctagcaggt 2520ctatgtaagt ggctgagctc gcgacgcccc aaggctctac taggagccgc catcgatggt 2580agtggggagg cgcttaaagc actgctgaag gaaactgctg ctgatgacgt agctgctaag 2640gagcgcgaag cagcgtcagc caagcgcaca gtggatgtag aagaaggcat cgtgttctcg 2700caactggcgg gcgcgggcgc cgccaacacg caccacgaca tcttcgaggt gtcgctgtcc 2760aaggcactgg atggtcgttc cggccaaggc gaggacaaag gcaagctgtc caaggtgacg 2820cagctgacag gcttctccga tccagtctac gcggaggcca tcgtggccgt caaccagtac 2880gacatcgtgc tcgacgtgct ggtggttaac cagaccgacg atacgctcca gaactgcaca 2940gtggagctag ccacgctcgg agacctgcga ttggtcgagc ggcccgcgtc ggtggtgtta 3000gccccgcgag actacgccac cataaaggca cacgtcaagg tggcgtccac ggagaatggg 3060atcatatttg ggaatattgt gtacgaagtg acgggcgcat ccatggaccg

aggagtcgtg 3120gttctaaacg acatccacat cgacatcgtg gactacatcc agccggccgc gtgcagcgac 3180gccgacttcc gctccatgtg ggccgagttc gagcgggaga acaagccggc cgcgggcagc 3240gacgccgact tccgctcaat gtgggccgcg ttcgagtggg agaacaaggt ttccgtgaac 3300acaaacatca cagacctgaa ggaatacctg cagcacctcc tcgcttctac caacatgaag 3360tgtctcacgc cagaaaaggc gctttccggt caatgcggct tcatggcggc caacatgtac 3420gctagatcca tcttcggcga agacgctcta gccaacctga gcattgagcg agcgctcaac 3480aagccggacg caccggtcgt gggtcacgtc aggatacgag ctaagagcca gggtatggca 3540ttaagcctcg gcgacaagat caacatgatg cagaaggccg cccacaagcc gtcggcctcc 3600ccgccgacgc ccgccgcctg a 362113302RNAartificial sequencesynthetic construct 13gugccguuga gauuggauau caagaggaag cugacggcuc gcucggaccg cgugaagugc 60guggaccagc accccucgga gcccuggcug cuaugcuccc uguacaaugg ggaugucaac 120auauggaacu acgagacaca gcugcaaaua aaaagauuug agguaugcga cuugccaguc 180cgugcggcca aguuugugcc gaggaaguuc ugggugauca ccggcuccga ugacaugcaa 240auucgugucu ucaacuacaa cacacuggaa cguguacaca auuuugaagc ucauucagac 300uc 30214298RNAartificial sequencesynthetic construct 14ggcaaaacau ccguaagcug aucaaggaug guuugaucau caaaaagccu guggcugucc 60acuccagagc ccgcguccgu aaaaacacag aagccagacg gaagggucgu cauuguggcu 120ucgguaagag gaaggguacc gccaacgcca gaaugccugu gaagguccug ugggucaaca 180gaaugagagu ccugcgacgg cuccuuaaaa aauacagaga agccaagaag aucgauaggc 240aaauguacca cgaccuuuac augaaagcca aagguaacgu cuucaaaaac aagagggu 29815300RNAartificial sequencesynthetic construct 15ggugccuucg gcuguggaaa aacugucauc ucacaagcuc uguccaaaua cucaaacucu 60gacgucauca uuuacgucgg augcggugaa cgugguaacg aaauguuuga gguauugaga 120gauuuccccg aacucacagu ugagauugac gguguaacug aguccaucau gaagcguacu 180gcucuggucg ccaacacauc caacaugccu guagcugcuc gagaagcuuc cauuuauacu 240gguaucacau uguccgaaua cuuccgugac auggguuaca acgugucgau gauggcugac 30016300RNAartificial sequencesynthetic construct 16gacaucaaga gaaagcuguc ugcucgauca gaccguguga aaugugucga ucugcaccca 60acugagcccu ggauguuggc uucucucuac aacggaaacg uucacauuug gaaccacgaa 120acucaacagc uucugaaauc cuucgaagua ugcgagcuuc caaucagggc ugcaguuuuc 180guaccgagga agaacugggu ggucacaggc ucggacgaca ugcacguucg ugucuucaac 240uacaacacuc ucgagcgugu acauuccuuc gaggcccauu cugauuauuu gagaugcauc 30017300RNAartificial sequencesynthetic construct 17ggaggagacu uguacgguau cguacacgag aacacauugg uuaagcauaa gauguugauc 60ccacccaagg ccaaggguac cgucaccuac aucgcgcccu ccggcaacua caaagucacu 120gacguagugu uggagacgga guucgacggc gagaaggaga aguacaccau guugcaagua 180uggccggugc gccagccccg ccccgucacu gagaagcugc ccgccaacca cccccugcuc 240accggacaga gagugcucga cucucucuuc ccuugugucc agggugguac cacggccauc 30018301RNAartificial sequencesynthetic construct 18ggaucauauc uaccagaaac uguuggaggc ggugcccggu aucaaagugg uaagauagcg 60cggucugcgc uguggcugcu ggcccaguuc gcugagacuc cggaacgcgc caaggaugcc 120uuggauguac ucgccaacgu cauaccuucc cuuagcggac aagaggauaa ggaagaaucc 180gagucggcag cuaaggccca ggacacuuca gcuccacgac agcuugucac cagugaugga 240acuuaugcuu cgcagucugc uuuuaacuug ccaguuagcc aagcggcucc aacccacgcg 300c 30119301RNAartificial sequencesynthetic construct 19ggcguucucc uucuaugauu gggagaacuu ggaguugauc agacggauug agauccagcc 60gcggcacgug uacuggucgg agagcggcaa ccuggugugc cuggcggcug augacucgua 120cuacgugcuc aaguauaaug cagcuguugu gacgcgagcu cgcgaaacca acuccaacau 180cacagaagac ggcaucgaag acgcuuuuga ggucgugggu gcagugaacg aggugguaaa 240gacaggacua ugggugggcg acugcuucau cuacacgaau uccuugaaca gaauaaacua 300c 30120302RNAartificial sequencesynthetic construct 20gcguccaggg cggcaccacg gccauccccg gggccuucgg uugcggcaag accgucaucu 60cgcaggcguu guccaaguac uccaacucug acgucaucgu cuaugucggg uguggggagc 120gugguaacga gauguccgaa guacugcgug acuuccccga gcugacaguc gagaucgacg 180gcgugaccga guccaucaug aagcgcacgg cgcucguggc caacaccucc aacaugccag 240uggccgcccg agaggccucc aucuacaccg gaauuacccu auccgaauac uuccgugaca 300uc 30221301RNAartificial sequencesynthetic construct 21ggccgcuaaa ggacaucaac gagcucacgc agagcaucua cauccccaag ggggugaacg 60ugcccgcgcu ggcccgcgac accgaguggc aguucaaccc gcaguacauc aaggucggca 120cccacaucac cggcggagac uuguacggga ucgugcacga gaacacgcug gugaagcacc 180gcaugcuggu gccgcccaag gccaagggca ccgucaccua caucgcgccc gaggggaacu 240acaaagucac ugacgugguc cuagagaccg aguucgacgg cgagaagucc uccuacacca 300c 30122257RNAartificial sequencesynthetic construct 22gcaucccccc agaucagcag agguugaucu uugccggcaa gcaguuggaa gacggacgua 60cuuugucuga cuacaacauc caaaaagaau ccacucucca ccuggucuug agauugagag 120guggcaugca gaucuucgug aagacccuca caggaaagac caucacucuu gaggucgagc 180cuucugacac caucgaaaac gucaaggcua aaauucaaga caaggaaggu auuccuccag 240aucagcagag auugauc 25723300RNAartificial sequencesynthetic construct 23gcaggcacaa acagaaaaaa uuaaggaaaa uacagaaaag aucaaaguga acaaaacuuu 60accauauuua guaucuaaug ugauagaguu gcuugauguu gauccagagg aaacugagga 120agauggugcu guuguugacc uugaugcccg ucguaaagga aaaugugcug ucauuaaaac 180aucaacgagg cagacuuauu uuuugccagu aauaggcuua guugaugcug aaaaguuaaa 240gccaggugau cugguuggag uaaacaaaga uucuuauuua auucuugaaa ccuugccugc 30024300RNAartificial sequencesynthetic construct 24guugauuuuu gcagguaaac agcucgaaga uggucguaca uugucugacu auaauauuca 60aaaggaaucu acucugcauu ugguccuucg ucugagagga ggaaugcaaa uuuuuguaaa 120aaccuugacu gggaaaacua uuacauuaga aguggaaccu ucugacacua uugaaaaugu 180uaaagcuaaa auucaagaua aggaaggaau uccaccagau cagcagcgau ugaucuuugc 240uggaaaacag uuggaagaug gucguacuuu aucugauuau aacauucaga aggaaucaac 30025302RNAartificial sequencesynthetic construct 25gacacuuacu gguaaaacaa ucacacuuga aguugagccu ucagacacaa uugaaaaugu 60gaaagcuaaa auucaagaua aagaaggaau uccuccagau cagcagaggu ugauuuuugc 120agguaaacag cucgaagaug gucguacauu gucugacuau aauauucaaa aggaaucuac 180ucugcauuug guccuucguc ugagaggagg aaugcaaauu uuuguaaaaa ccuugacugg 240gaaaacuauu acauuagaag uggaaccuuc ugacacuauu gaaaauguua aagcuaaaau 300uc 30226302RNAartificial sequencesynthetic construct 26gaccuugacu ggcaagacca ucacucuuga ggucgagccc ucugacacca uugaaaacgu 60caaggccaag auccaggaca aggaagguau ccccccagau cagcagaggu ugaucuucgc 120uggcaagcag cucgaggaug gucguacccu cucggacuac aacauccaga aggaguccac 180ccuucaucuu guccuccguc ugcguggugg uaugcagauu uucgucaaga ccuugacugg 240caagacgauc acuuuggaag ucgagcccuc ugacaccauu gagaauguca aagccaaaau 300cc 30227300RNAartificial sequencesynthetic construct 27gagaagaacu cuucacugga guugguccca guucuuguug aauuagaugg cgauguuaau 60gggcaaaaau ucucugucag uggagagggu gaaggugaug caacauacgg aaaacuuacc 120cuuaauuuua uuugcacuac ugggaagcua ccuguuccau ggccaacacu ugucacuacu 180uucucuuaug guguucaaug cuucucaaga uacccagauc auaugaaaca gcaugacuuu 240uucaagagug ccaugcccga agguuaugua caggaaagaa cuauauuuuu caaagaugac 30028302RNAartificial sequencesynthetic construct 28ggcgugccua cuauaggggg ggagcaagag cagacugagg auuccgcagc gaagcaggag 60gcgacggcgg cgccgcggca gcuggucacc agcgacggga cuuacgccuc gcaguccgcc 120uucaaccugc ccucguccgc cgccaccgca ucaacaucca ccaacgggcu ccgcucagcg 180cucaccucgg gcgacucguu cacggccgcc ugcgccugcu ccgcgcuggc caagcugucg 240cucaagcagg ccacugugag ggacgccaac agggcgcugc accucgcugc uaggcuacug 300gc 30229300RNAartificial sequencesynthetic construct 29gcaucuacau ccccaagggg gugaacgugc ccgcgcuggc ccgcgacacc gagugggagu 60uccacccgca guacaucaag gucggcaccc acaucaccgg cggagacuua uacgggaucg 120ugcacgagaa cacgcuggug aagcaccgca ugcuggugcc gcccaaggcc aagggcaccg 180ucaccuacau cgcgcccgag gggaacuaca aagucacuga cgugguccua gagaccgagu 240ucgacggcga gaaguccucc uacaccaugc uucaagugug gccggugaga cagccgcggc 30030302RNAartificial sequencesynthetic construct 30gugccguuga gauuggauau caagaggaag cugacggcuc gcucggaccg cgugaagugc 60guggaccagc accccucgga gcccuggcug cuaugcuccc uguacaaugg ggaugucaac 120auauggaacu acgagacaca gcugcaaaua aaaagauuug agguaugcga cuugccaguc 180cgugcggcca aguuugugcc gaggaaguuc ugggugauca ccggcuccga ugacauguaa 240auucgugucu ucaacuacaa cacacuggaa cguguacaca auuuugaagc ucauucagac 300uc 30231300RNAartificial sequencesynthetic construct 31ggugccuucg gcuguggaaa aacugucauc ucacaagcuc uguccaaaua cucaaacucu 60gacgucauca uuuacgucgg augcggugaa cgugguaacg aaauguuuga gguauugaga 120gauuuccccg aacucacagu ugagauugac gguguaacug aguccaucau gaagcguacu 180gcucuggucg ccaacacauc caacaugccu guugcugcuc gagaagcuuc cauuuauacu 240gguaucacau uguccgaaua cuuccgugac auggguuaca acgugucgau gauggcugac 30032300RNAartificial sequencesynthetic construct 32gacaucaaga gaaagcuguc ugcucgauca ggccguguga aaugugucga ucugcaccca 60acugagcccu ggauguuggc uucucucuac aacggaaacg uucacauuug gaaccacgaa 120acucaacagc uucugaaauc cuucgaagua ugcgagcuuc caaucagggc ugcaguuuuc 180guaccgagga agaacugggu ggucacaggc ucggacgaca ugcacguucg ugucuucaac 240uacaacacuc ucgagcgugu acauuccuuc gaggcccauu cugauuauuu gagaugcauc 30033300RNAartificial sequencesynthetic construct 33ggaggagacu uguacgguau cguacacgag aacacauugg uuaagcauaa gauguugauc 60ccacccaagg ccaaggguac cgucaccuac aucgcgcccu ccggcaacua caaagucacu 120gacguagugu uggagacggu guucgacggc gagaaggaga aguacaccau guugcaagua 180uggccggugc gccagccccg ccccgucacu gagaagcugc ccgccaacca cccccugcuc 240accggacaga gagugcucga cucucucuuc ccuugugucc agggugguac cacggccauc 30034301RNAartificial sequencesynthetic construct 34ggaucauauc uaccagaaac uguuggaggc ggugcccggu aucaaagugg uaagauagcg 60cggucugcgc uguggcugcu ggcccaguuc gcugagacuc cggaacgcgc caaggaugcc 120uuggauguac ucgccaacgu cauaccuucc cuuagcggac aagaggauaa agaagaaucc 180gagucggcag cuaaggccca ggacacuuca gcuccacgac agcuugucac cagugaugga 240acuuaugcuu cgcagucugc uuuuaacuug ccaguuagcc aagcggcucc aacccacgcg 300c 30135302RNAartificial sequencesynthetic construct 35gcguccaggg cggcaccacg gccauccccg gggccuucgg uagcggcaag accgucaucu 60cgcaggcguu guccaaguuc uccaacucug acgucaucgu cuaugucggg uguggggagc 120gugauaacga gauguccgaa guacugcgug acuuccccga gcugacaguc gagaucgacg 180gcgugaccga guccaacaug aagcgcacgg cgcucguggc caacaccucc aacaugccag 240uggccgcccg agaggccucc aucuacaccg gaauuacccu auccgaauac uuccgugaca 300uc 30236257RNAartificial sequencesynthetic construct 36gcaucccccc agaucagcag agguuaaucu uugccggcaa gcaguuggaa gacggacgua 60cuuugucuga uuacaacauc caaaaagaau ccacucucca ccuggucuug agauugagag 120guggcaugca gaucuucaug aagacccuca caggaaagac caucacucuu gaggucgagc 180cuucugacac caucgaaaac gucaaggcua aaauucaagg caaggaaggu auuccuccag 240auuagcagag auugauc 25737300RNAartificial sequencesynthetic construct 37gcaggcacaa acagaaaaaa uuaaggaaaa uacagaaaag aucaaaguga acaaaacuuu 60accguauuua gaaucuaaug ugauagaguu gcuugauguu gauccagagg aaacugagga 120agauggugcu auuguugacc uugaugcccg ucguaaagga aaaugugcug ucauuaaaac 180aucaacgagg cagacuuauu uuuugccagu aauaggcuua guugaugcug aaaaguuaaa 240gccaggugau cugguuggag uaaacaaaga uucuuauuug auucuugaaa ccuugccugc 30038300RNAartificial sequencesynthetic construct 38guugauuuuu gcagguaaac agcucgaaga uggucguaca uugucugacu auaauauuca 60aauggaaucu acucugcauu ugguccuucg ucugagagca ggaaugcaaa uauuuguaaa 120aaccuugacu gggaaaacua uuacauuaga aguggaaccu ucugacacua uugaauaugu 180uaaagcuaaa acucaggaua agguaggaau ucuaccagau caguagcgau ugaucuuugc 240uagaaaacag uuggaagaug gucauacuuu aucugauuau aacauucaga uggaaucaac 30039302RNAartificial sequencesynthetic construct 39gacacuuacu gguaaaacaa ucacacuuga aguugagccu ucagacacaa uugaaaaugu 60gaaaguuaaa auucaagaua aaguaggaau uccuccaguu cagcagaggu ugauuuuugc 120agguaaacag cucgaagaug gucguacauu gucugacuau aauauucuaa aggaaucuac 180ucugcauuug guccuucguc ugagaagagg aaugcaaauu guuguaaaaa ccuugacugg 240gaaaacuauu acauuagaag uggaaccuuc ugacacuauu gaaauuguua aagcuaaaau 300uc 30240302RNAartificial sequencesynthetic construct 40gaccuugacu ggcaagacca ucacucuuga ggucgagccc ucugacacca uugaaaacgu 60caaggccaag auccagaaca aggaagguau ccccccagau cagcagaggu uggucuucgc 120uggcaagcag cucgaggaug gucguacccu cucggacuac aacaucuaga aggaguccac 180ccuucaucuu guccuccguc ugcguggugg caugcagauu uucgucaaga ccuugacugg 240caagacgauc acuuuggaag ucgagcccuc ugacaccauu gagaguguca aagccaaaau 300cc 30241300RNAartificial sequencesynthetic construct 41gagaagaacu cuucacugga guugguccca guucuuguug aauuagaugg cgauguuaau 60gggcaaaaau ucucugucag uggugagggu gaaggugaug caacauacgg aaaacuuacc 120cuuaauuuua uuugcacuac ugggaagcua ccuguuccau ggccaacacu ugucacuacu 180uucucuuaug guguucaaug cuucucaaga uacccagauc auaugaaaca gcaugacuuu 240uucaagagug ccaugcccga agguuaugua caggaaagaa cuauauuuuu caaagaugac 30042302RNAartificial sequencesynthetic construct 42ggcgugccua cuauaggggg ggagcaagag cagacugagg auuccgcagc gaagcaggag 60gcgacggcgg cgccgcggca gcuggucacc agcgacggga cuuacgccuc gcaguccgcc 120uucaaccugc ccucguccgc cgccaccgca ucaacaucca ccaacgggcu ccgcucagcg 180cucaccucgg gcgacucguu cacggccgcc ugcgccugcu ccgcgcuggc aaagcugucg 240cucaagcagg ccacugugag ggacgccaac agggcgcugc accucgcugc caggcuacug 300gc 302433005DNALygus hesperus 43agcacagctc agaaagttga agctctcaag aaaacgattc acatgatttc caacggcgag 60cgtttaccgg gtcttctgat gcatatcatc agattcattc tgccttccca ggaccacacg 120atcaaaaagt tgctgctaat attctgggag atcgttccta aaacttaccc cgatggaaaa 180ctgcttcaag aaatgatact tgtttgcgac gcctacagaa aggatttaca acaccccaat 240gagttcgtgc gcgggtcgac tttgagattc ctctgcaaat tgaaggagcc agaacttctg 300gaaccgctga tgcctgccat tcggtcgtgc ctcgagcaca gagtgtctta cgtccgcagg 360aacgctgtcc ttgccatttt cacgatctac aagaatttcg aatttctaat ccctgatgct 420cccgaactca ttgccaattt cctcgacgga gagcaagata tgtcttgcaa aaggaatgcc 480ttcttgatgc tcctccacgc tgaccaagac agagcactct cctatcttgc ttcttgtctg 540gaccaagtca ccagctttgg tgacatcctc cagcttgtca tcgttgaatt gatttacaag 600gtttgtcatg cgaatccctc agaacgttct cggttcataa ggtgcatata caacctattg 660aattcaagca gccctgctgt ccgatatgaa gctgctggta cgctgataac cctgtccaac 720gcacctactg ccatcaaggc tgctgcatca tgctacattg atctaatcat caaggaaagc 780gataacaacg tcaaactgat cgtcctcgat cgcctggtcg ccctcaaaga catcccgacg 840tacgaaagag tcttgcagga tctcgtcatg gacatcctcc gcgtcttggc cagcccggat 900atggaagtca ggaagaaggc tttgaatctc gctcttgatc ttacaacttc gcgttgtgtc 960gaagaagtag ttttgatgct gaagaaagag gttgccaaaa ctcataactt gtccgagcac 1020gaggaaacag gaaaatatag gcaactcctt gtgagaactc tgcactcttg cagtatgaaa 1080ttccctgatg tggctgcttc cgtcatccca gtgctcatgg aatttttgtc tgactccaac 1140gagctcgctt cccaagacgt ccttattttc gtaagggaag ccattcacaa atttgaaaat 1200ctgaggaaca caatcattga gaaattgctt gaagcttttc cgtccataaa gttcgtcaaa 1260gtccatcgtg ctgcgttgtg gatattagga gagtacgctg cttccatcga tgacgtcaga 1320gctgtcatga aacaaatcaa acagaatttg ggtgaggttc ctatggtgga agatgaaatg 1380aagcgggccg ctggagagaa gacagaagag tcatctgaac agaacagcgg gggtgcaatg 1440ccgtcaagcg cttccaaact agtaacgtct gatgggacct atgcttctca gtctgtgttc 1500agcactgtat ccacatccaa aaaagaggac cgaccacctt tgaggcagta tctgattgat 1560ggtgattatt ttattggctc caccatcgcg tccactttgg tgaaactttc tctgaagttt 1620gacaacttgg aatccaacac ggctgcgcag aacgaattct gcaatgaatg catgctgatc 1680atcgcctgca ccctccatct tggaagatct ggcctttgca caaagaattt gaataacgac 1740gacgctgaga ggatgctgtt ttgtcttcga gttctttggg atggaagccc aaccattgag 1800aagattttta ctcaagaatg ccgagaagct cttgcgtcta tgcttaccgc tcaacaccat 1860gaggaaatcg ccttgaataa ggccaaagaa aagaccgcac atctcatcca cgtagacgac 1920ccagtctcat tcctgcaatt atcatctctg agaaactctg aacttggttc tgaaaacgtg 1980ttcgagctaa gtcttactca ggcgcttggt ggtcccacca gtggtggctc ctccaactcg 2040gacctcttct tctctgccag caagctcaac aaagtcacgc agcttactgg cttttctgac 2100cctgtctacg ctgaagctta cgtccaagtc aaccagtatg atatcgtctt ggacgtactc 2160attgtcaacc agacagctga cactcttcaa aattgcactc tggaattggc tacacttggc 2220gacctgaaat tggtcgagaa gccgcaaccc tgcgttttgg cgcctcatga cttctgtaac 2280ataaaagcta acgtcaaagt ggcttccact gaaaacggaa ttatttttgg caacattgtt 2340tacgacgtta gtggagcagc ttccgaccga aacgtcgtcg tcctcaatga cattcacatc 2400gatattatgg actacatagt tcctgcatct tgttctgaca ctgaattccg ccaaatgtgg 2460gctgaattcg aatgggaaaa caaggtatct gtcaacacca acctcacgga cttgcacgag 2520tatttggccc atttggtcag gagcaccaac atgaagtgct tgacaccaga gaaagcgctc 2580tgcggtcaat gtgggttcat ggctgccaac atgtatgcgc gctcgatttt cggagaagat 2640gcgttggcga acctgagcat cgagaaaccc ttcaacaagc ctgatgcacc tgtcactgga 2700cacatccgca tccgagccaa aagccaggga atggcactca gtctgggaga caaaatcaac 2760atgacccaga agagaccgca gaaaatgtac ggtgcctaag ccctcataga tcccaccacc 2820tcggttcaac tctccatctc ctttgtgaga gcaccctact gcttacctgc gccacactgc 2880aagtaaactt ggcttcggcc tcctatttat catattttac ggtattcttt gttatcgaaa 2940tatttatgca tattatatta ttggtatttc gttatcccaa ttcattcaat aaatatatag 3000attaa 3005441623DNALygus hesperus 44gccattagag cttcagacga gaaacgagaa cgagctacgt tgagcatcaa gagcttttgt 60actattgaaa ttgtgaaaaa tgcagatctt cgttaaaact ttgactggga agaccatcac 120cctcgaggtc gagccttctg ataccattga aaacgtgaag gcgaaaattc aggataaaga 180aggcatcccc ccagatcagc agaggttgat ctttgccggc aagcagttgg aagacggacg 240tactttgtct gactacaaca tccaaaaaga atccactctc cacctggtct tgagattgag

300aggtggcatg cagatcttcg tgaagaccct cacaggaaag accatcactc ttgaggtcga 360gccttctgac accatcgaaa acgtcaaggc taaaattcaa gacaaggaag gtattcctcc 420agatcagcag agattgatct tcgccggcaa acaactcgaa gatggccgta ccctctctga 480ctacaatatt caaaaagagt ccacccttca cttggtgttg agattgcgtg gaggtatgca 540aatctttgtc aaaacattga ctggaaagac catcaccctt gaagtcgaac cctccgacac 600catcgaaaat gtcaaggcca agatccagga caaggaaggc atccccccag atcagcagag 660gttgattttc gctggcaaac aacttgaaga cggacgtacc ctctcggact acaacatcca 720gaaggagtcg accctccatc ttgtcctccg tctgcgtggt ggtatgcaga tttttgtcaa 780aactctgact ggcaagacaa tcacccttga agtagagccc tctgacacca tcgaaaatgt 840caaggcgaaa atccaggaca aagaaggcat ccccccagat cagcagaggt tgatctttgc 900cggtaagcag cttgaagacg gccgcaccct ctcggactac aacatccaga aggagtccac 960ccttcatctt gtcctccgtc tgcgtggtgg tatgcagatc ttcgtcaaga ccttgactgg 1020caagacgatc actttggaag tcgagccctc tgacaccatt gagaatgtca aagccaaaat 1080ccaagataag gaaggcatcc ccccagatca gcagaggttg atcttcgctg gtaagcaact 1140tgaagacggc cgcaccctct ctgactacaa catccagaag gagtcgaccc tccatcttgt 1200cctccgtctg cgtggtggta tgcagatctt cgtcaagacc ttgacaggca agaccatcac 1260ccttgaagtc gagccctctg acaccatcga aaacgtcaag gctaagatcc aggacaagga 1320aggtatcccc ccagatcagc aaagattgat cttcgccggc aaacagctcg aagatggccg 1380taccctctca gactacaaca ttcaaaagga gtcaactctt catctcgttc tgaggctccg 1440tggcggtcgt tattgatcac aattccaaac ttaaaaattg cattccgatt ttccttcttt 1500atttggcaaa aaatacatac cctagttaat taaaatgact tgaaatttga ttttttaaga 1560atgcttcaaa tttttttata gatggtttgt tacatagaca atacacaaca tgttgaaagc 1620aat 162345301RNAartificial sequencesynthetic construct 45guugcugcua auauucuggg agaucguucc uaaaacuuac cccgauggaa aacugcuuca 60agaaaugaua cuuguuugcg acgccuacag aaaggauuua caacacccca augaguucgu 120gcgcgggucg acuuugagau uccucugcaa auugaaggag ccagaacuuc uggaaccgcu 180gaugccugcc auucggucgu gccucgagca cagagugucu uacguccgca ggaacgcugu 240ccuugccauu uucacgaucu acaagaauuu cgaauuucua aucccugaug cucccgaacu 300c 30146255RNAartificial sequencesynthetic construct 46gacguacuuu gucugacuac aacauccaaa aagaauccac ucuccaccug gucuugagau 60ugagaggugg caugcagauc uucgugaaga cccucacagg aaagaccauc acucuugagg 120ucgagccuuc ugacaccauc gaaaacguca aggcuaaaau ucaagacaag gaagguauuc 180cuccagauca gcagagauug aucuucgccg gcaaacaacu cgaagauggc cguacccucu 240cugacuacaa uauuc 25547701DNALygus hesperus 47aagttttctc ttttgtaatt tttttgtaga tttttgtaca aaatacgttt tttgtttaaa 60atttaaaaac ggagggggcg agatcacttt ttgacggcag cctcgtcttc tcgggcgtac 120gcctgcatga tctcttgctt cttggtggcg atcctctcct cgcgcctctt cttcgcctcc 180ttgaccttga gacgtctcgc ctctgcctgg tccttcaaca tctttgatct cgccttttca 240gccttcttct tgtgaatgaa gtccatcagt accctcttgt ttttgaagac gttacctttg 300gctttcatgt aaaggtcgtg gtacatttgc ctatcgatct tcttggcttc tctgtatttt 360ttaaggagcc gtcgcaggac tctcattctg ttgacccaca ggaccttcac aggcattctg 420gcgttggcgg tacccttcct cttaccgaag ccacagtgac gacccttccg tctggcttct 480gtgtttttac ggacgcgggc tctggagtgg acagccacag gctttttgat gatcaaacca 540tccttgatca gcttacggat gttttgccta gagttggtgt tggcgatttc gttgatttca 600ttagggtcca accacacttt cttcttgccg catctcatca ccgaggcggc gagcctcttc 660tgcagtttga gcgaactcat ggtcggacga agagaaccac c 701481927DNAEuschistus heros 48aacaaacata acatgatatt attaaatgtt tcccaaattt ataaatatac aaataatata 60aagaatcact gaaaaaaaat tttgcaacag gaagtttaat ctcctttcaa tttaatccaa 120aaaatattaa atgcaatgaa aaaataaatt aaaaaacaaa tttgagatta gaatatattc 180aataaactgt gaaccaacaa ataattaatt ttgaaaatga ttataaaaat aaaaattaaa 240tattaaatct ggcagaaact catttcaaac tagtaatcaa attagtcaaa ctaataattg 300atacagaaaa tataatcttt ttaaaattaa acaggcaaaa atatatatat atatatgaat 360taacaaaact tttttaactg gagttaatgc gactttaaga atttaataaa attataatgt 420gctattttac ttgttggttt acttacacat ttgtttttca aagagtggca tatttacaag 480ttaataacaa tacaaaatca atcacagtta ttggcactcc cgaaaagaag aatgcaagtg 540agtttctgca ttattaaaaa aatctttatc aatagatatt taagcataat agtttaagtt 600ggctttcttc tttgcctgca cttctaatat tgcatccatg aaatcttcat gagtgacaac 660tccagctcct cttcgcaatg ctatcatacc tgcttccaca caaaccgctt tgcattgagc 720tccattgaaa tcatcagttg aacgagcaag ttcttcaaaa ttaacatcaa cactaatatt 780catcttacga gaatgaattt gcattatacg tgctcgggca tcttcgttag gatgtgggaa 840ttcaattttt ctgtctagtc gacctgaacg caaaagggcg ggatccaaaa tatctactct 900atttgtagct gcaataacct taatatcagc tgttgagcta aatccatcta actggttgag 960cagttctaac atagttcttt gaacttctcg atcaccagct ttttcagaat caaatctttt 1020tgttccaata gcatccaact cgtcaataaa gataatagca ggagattttt ccttagcaag 1080agcaaaagca tctctgacaa gtttggctcc atctcctata aacatctgaa caagttgggg 1140tccagctagt ttcaaaaaag ttgatttagt ttgagctgca cacgctctag ccagtaaagt 1200ctttcctgtt ccaggaggtc catagagcaa tacaccttta ggaggatgaa ttcctagatt 1260ttcaaatttt tctttgtgtg tcataggaag aacaacggct tctattaatt cttgaatctg 1320cttatcaagc ccaccgatat cagaatactg ctcagtaggt ctttcatcaa cttccattgc 1380tttaactcgt gcatcatact cagcaggcaa ggtttcaaga attaaataag aatctttgtt 1440tactccaacc agatcacctg gctttaactt ttcagcatca actaagccta ttactggcaa 1500aaaataagtc tgcctcgttg atgttttaat gacagcacat tttcctttac gacgggcatc 1560aaggtcaaca acagcaccat cttcctcagt ttcctctgga tcaacatcaa gcaactctat 1620cacattagat actaaatatg gtaaagtttt gttcactttg atcttttctg tattttcctt 1680aattttttct gtttgtgcct gcaactcatg actgactcgc atgacttcac ttttcatgat 1740tttaatttca ttatcaagaa gcctaatacg actgacaatt tcatccgtag acattctcat 1800gatttcttca cctaaagctt gctccattct ttcgtcccac aagggctttt cttctgtttt 1860ggaagtagac atctgaattg ccgaacttag gatgtgaacc aaattaaaac ctgtaactac 1920aacaaac 1927492170DNAEuschistus heros 49ttttttgaac ttaaagaaac tgtttatttc tagtttagtt acattgtatt tatgaaataa 60ttataaatat aataatgaaa atgctaataa aaatatcaaa agcaatttac tgatatggaa 120ttttgatgga atgaaatgat aagtagcctt aatatccgcc cctaagacgc aacactaaat 180gcagagttga ttccttctgg atgttataat cagatagggt acgaccatcc tctaattgtt 240ttccagcaaa aataagcctc tgctgatctg gaggtattcc ttctttgtcc tgaattttgg 300ctttaacatt ttcaatagtg tcagatggct caacttctaa ggtaatagtc ttgcctgtga 360gggtcttgac aaagatctgc ataccacctc tcaatcgaag gaccagatgt agtgtagatt 420ccttctggat attataatct gaaagggtac ggccatcttc gagttgtttt ccagcaaaga 480tcaaacgctg ctggtcggga ggaattcctt ctttatcctg aattttagct tttacattct 540caattgtatc agatggctct acttccaagg taatggtttt acctgtcaaa gtcttcacaa 600agatctgcat gcctcctctt agacgaagca ccaaatgcag tgttgattcc ttttgaatgt 660tgtaatcaga caaagttcgg ccatcttcca gctgctttcc agcaaaaata agcctctgtt 720ggtctggtgg tattccttct ttatcttgaa ttttggcttt aacattctca atagtatctg 780aaggctcaac ttccaaggtt atggtcttac cagttagagt ctttacaaaa atctgcatgc 840cacctctcaa gcgaagtaca agatgaagag tagactcttt ttgaatatta taatctgaaa 900gggtacggcc atcttcaagt tgttttccag caaagatcaa acgttgctgg tctggtggaa 960ttccttcttt gtcctggatt tttgctttta cgttctcaat agtatcagat ggttcaactt 1020ctaaggtgat ggtcttccct gttaaagtct tgacaaagat ctgcatgcct cctcgaagtc 1080taagtacaag atgtaaagta gattcctttt ggatgttgta atcagaaaga gtacgcccat 1140cttctaactg ttttcctgca aagatcagcc tttgctgatc tggcggaatt ccttccttat 1200cttgaatttt ggcttttaca ttttcaatgg tatctgacgg ttcaacttct aaagttatgg 1260tttttccagt aagcgtctta acaaaaatct gcattccacc tcttaaacgt aatactaaat 1320gtagtgttga ttccttctga atgttataat cagataaagt acgaccatct tccaactgtt 1380ttccagcaaa gatcaatcgc tgctgatctg gtggaattcc ttccttatct tgaattttag 1440ctttaacatt ttcaatagtg tcagaaggtt ccacttctaa tgtaatagtt ttcccagtca 1500aggtttttac aaaaatttgc attcctcctc tcagacgaag gaccaaatgc agagtagatt 1560ccttttgaat attatagtca gacaatgtac gaccatcttc gagctgttta cctgcaaaaa 1620tcaacctctg ctgatctgga ggaattcctt ctttatcttg aattttagct ttcacatttt 1680caattgtgtc tgaaggctca acttcaagtg tgattgtttt accagtaagt gtcttgacaa 1740agatctgcat gcctcctctt aaacgtagga caagatgaag agtagattcc ttttgaatat 1800tatagtcaga caatgtacga ccatcttcga gctgtttacc tgcaaaaatc aacctctgtt 1860ggtctggagg aattccttct ttatcttgaa ttttagcttt aacattttca attgtatcag 1920atggctccac ttccaaggtg atggttttac cagtcaaggt cttcacaaaa atttgcatgc 1980ctcctctcaa ccgaagtact aaatgtaaag tagattcttt ttgaatattg taatctgaaa 2040gagtacggcc atcttcaagc tgtttaccag caaaaataca acctctgttg gtctggaggg 2100attccttctt tgtcttggat tttggccttg acattttcta ttgtgtcaga aggctcaact 2160tctaatgtaa 2170

Claims

1. A method for controlling an insect infestation of a plant comprising contacting with a dsRNA an insect that infests a plant, wherein said dsRNA comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of said insect, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:1-12 and 43-44.

2. The method of claim 1, wherein said insect is selected from the group consisting of Spodoptera spp., Lygus spp., Euschistus spp., and Plutella spp.

3. The method of claim 1, wherein: (a) said insect is Spodoptera frugiperdo (fall armyworm) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; (b) said insect is Lygus hesperus (western tarnished plant bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; (c) said insect is Euschistus heros (neotropical brown stink bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; or (d) said insect is Plutella xylostella (diamondback moth) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:10-12.

4. The method of claim 1, wherein: (a) said insect is Spodoptera frugiperda (fall armyworm) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:17-19; (b) said insect is Lygus hesperus (western tarnished plant bug) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; (c) said insect is Euschistus heros (neotropical brown stink bug) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:23-25; or (d) said insect is Plutella xylostella (diamondback moth) and said dsRNA comprises a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.

5. The method of claim 1, wherein said at least one segment is multiple segments.

6. The method of claim 1, wherein said dsRNA is (a) blunt-ended, or (b) has an overhang at at least one terminus.

7. The method of claim 1, wherein said dsRNA is (a) chemically synthesized, or (b) produced by expression in a microorganism, expression in a plant cell, or by microbial fermentation.

8. The method of claim 1, wherein said dsRNA is chemically modified.

9. The method of claim 1, wherein said contacting comprises application of a composition comprising said dsRNA to a surface of said insect or to a surface of said plant infested by said insect.

10. The method of claim 9, wherein said composition comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, or seed treatment.

11. The method of claim 1, wherein said contacting comprises providing said dsRNA in a composition that further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator.

12. The method of claim 1, wherein said contacting comprises providing said dsRNA in a composition that further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

13. The method of claim 1, wherein said contacting comprises providing said dsRNA in a composition that is ingested by said insect.

14. The method of claim 13, wherein said composition that is ingested further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator.

15. The method of claim 13, wherein said composition that is ingested further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

16. A method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one recombinant RNA comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of said insect, wherein said target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of said recombinant RNA by said insect results in mortality or stunting in said insect.

17. The method of claim 16, wherein said recombinant RNA comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarily with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

18. The method of claim 16, wherein: (a) said insect is Spodoptera frugiperda (fall armyworm) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; (b) said insect is Lygus hesperus (western tarnished plant bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; (c) said insect is Euschistus heros (neotropical brown stink bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; or (d) said insect is Plutella xylostella (diamondback moth) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:10-12.

19. The method of claim 16, wherein: (a) said insect is Spodoptera frugiperda (fall armyworm) and said silencing clement comprises a sequence selected from the group consisting of SEQ ID NOs:17-19; (b) said insect is Lygus hesperus (western tarnished plant bug) and said silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; (c) said insect is Euschistus heros (neotropical brown stink bug) and said silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:23-25; or (d) said insect is Plutella xylostella (diamondback moth) and said silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.

20. The method of claim 16, wherein said recombinant RNA is dsRNA.

21. The method of claim 16, wherein said recombinant RNA is (a) blunt-ended dsRNA, or (b) dsRNA with an overhang at at least one terminus.

22. The method of claim 16, wherein said recombinant RNA is provided in a microbial or plant cell or in a microbial fermentation product.

23. The method of claim 16, wherein said recombinant RNA is chemically synthesized.

24. The method of claim 16, wherein said insect is in a larval or nymph stage.

25. The method of claim 16, wherein said insect is an adult.

26. The method of claim 20, wherein said dsRNA comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

27. An insecticidal composition comprising an insecticidally effective amount of a recombinant RNA molecule, wherein said recombinant RNA molecule comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of an insect that infests a plant, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ III NOs:1-12 and 43-44.

28. The insecticidal composition of claim 27, wherein said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

29. The insecticidal composition of claim 27, wherein said recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

30. The insecticidal composition of claim 29, wherein said dsRNA is at least 50 base pairs in length.

31. The insecticidal composition of claim 27, wherein: (a) said insect is Spodoptera frugiperda (fall armyworm) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:1-3; (b) said insect is Lygus hesperus (western tarnished plant bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:4-7, 43, and 44; (c) said insect is Euschistus heros (neotropical brown stink bug) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:8-9; or (d) said insect is Plutella xylostella (diamondback moth) and said target gene comprises a DNA sequence selected from the group consisting of SEQ ID NOs:10-12.

32. The insecticidal composition of claim 27, wherein: (a) said insect is Spodoptera frugiperda (fall armyworm) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:17-19; (b) said insect is Lygus hesperus (western tarnished plant bug) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; (c) said insect is Euschistus heros (neotropical brown stink hug) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:23-25; or (d) said insect is Plutella xylostella (diamondback moth) and said recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.

33. The insecticidal composition of claim 27, wherein: (a) said insect is Spodoptera frugiperda (fall armyworm) and said recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:17-19; (b) said insect is Lygus hesperus (western tarnished plant bug) and recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:14-16, 22, 26, 45, and 46; (c) said insect is Euschistus heros (neotropical brown stink bug) and recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:23-25; or (d) said insect is Plutella xylostella (diamondback moth) and recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:13, 20-21, and 28-29.

34. The insecticidal composition of claim 27, further comprising one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a safener, an insect attractant, and an insect growth regulator.

35. The insecticidal composition of claim 27, further comprising at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

36. The insecticidal composition of claim 27, wherein said composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment.

37. A plant treated with the insecticidal composition of claim 27, or seed of said plant, wherein said plant exhibits improved resistance to said insect.

38. A method of providing a plant having improved resistance to an insect, comprising expressing in said plant a recombinant DNA construct comprising DNA encoding RNA that includes at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of said insect, wherein said target gene sequence is selected from the group consisting of SEQ ID NOs:1-12 and 43-44, and wherein ingestion of said RNA by said insect results in mortality or stunting in said insect.

39. The method of claim 38, wherein said silencing element has a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

40. The method of claim 38, wherein said recombinant DNA construct further comprises a heterologous promoter operably linked to said DNA encoding RNA that includes at least one silencing clement, wherein said heterologous promoter is functional in a plant cell.

41. The method of claim 38, wherein said expressing is by means of transgenic expression or transient expression.

42. The method of claim 38, further comprising expression in said plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.

43. The plant having improved resistance to an insect, provided by the method of claim 38.

44. Fruit, seed, or propagatable parts of the plant of claim 43.

45. A recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:13-26, 28-29, 30-42, 45, and 46.

46. The recombinant DNA construct of claim 45, wherein said heterologous promoter is (a) functional for expression of said RNA transcript in a bacterium.

47. The recombinant DNA construct of claim 46, wherein said bacterium is selected from the group consisting of Escherichia coli, Bacillus species, Pseudomonas species, Xenorhabdus species, or Photorhabdus species.

48. The recombinant DNA construct of claim 45, wherein said heterologous promoter is functional in a plant cell.

49. A recombinant vector comprising the recombinant DNA construct of claim 45.

50. The recombinant vector of claim 49, wherein said recombinant vector is a recombinant plant virus vector or a recombinant baculovirus vector.

51. A plant chromosome or plastid comprising the recombinant DNA construct of claim 45.

52. A transgenic plant cell having in its genome the recombinant DNA construct of claim 45

53. A transgenic plant comprising the transgenic plant cell of claim 52.

54. A commodity product produced from the transgenic plant of claim 53.

55. A transgenic progeny seed or propagatable plant part of the transgenic plant of claim 53.