Double stranded RNA methods and compositions for insecticide

Abstract

Disclosed herein are insecticidal compositions and methods for control of plant pests. The insecticidal compositions and methods comprise a recombinant RNA molecule for RNA interference-mediated inhibition of target genes in plant pests. Further provided are transgenic plants that express recombinant RNA molecule useful for the control of plant pests, and the plant cells and plants products obtained thereby.

Description

DOUBLE STRANDED RNA METHODS AND COMPOSITIONS FOR INSECTICIDECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 520,023, filed August 16, 2023, and titled “DOUBLE STRANDED RNA METHODS AND COMPOSITIONS FOR INSECTICIDE” which is incorporated by reference here in in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted in .XML format via Patent Center and is hereby incorporated by reference in its entirety. Said WIPO Sequence Listing was created on August 13, 2024, XML copy is named 066803-818366_Sequence listing.xml, and is 41 kilobytes in size.FIELD

[0003] The present disclosure relates to the field of RNA interference (RNAi)-mediated gene silencing in insect pests.BACKGROUND

[0004] Control of insect pests on agronomically significant crops is important for maximized crop yield. Insect pests which damage plants belonging to the Solanaceae family, especially potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), capsicums (Solanum capsicum), and nightshade (for example, Solanum aculeastrum, S. bulbocastanum, S. cardiophyllum, S. douglasii, S. dulcamara, S. lanceolatum, S. robustum, and S. triquetrum), include many coleopteran pests. Though progress has been made in the last few decades towards developing more efficient methods and compositions for controlling insect infestations in plants using chemical pesticides, development of resistance to compounds by the insect pests is a major issue. Biological methods of control such as RNA interference (RNAi)-based biopesticides offer an alternative and unique approach to integrated pest management.

[0005] RNAi is a natural biological process found in most eukaryotic organ isms to defend against viruses or play a role in regulation of messenger RNA (mRNA) stability and translation. In some insects, such as Colorado potato beetle (CPB) and other coleopteran species, the RNAi pathway can be trigged after insect ingestion of exogenous double-stranded RNA which potentiallyofferthis technology to control this pest. Therefore, there is an unmet need to identify new targets in the insect genome and develop RNAi based insecticides to cause death, growth arrest or sterility of the insect pests.SUMMARY

[0006] In some aspects, the disclosure encompasses an insecticidal composition comprising an insecticidally effective amountof a recombinant RNA molecule comprising a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene of an insect that infests a plant, the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID Nos: 1 , 4, 6 and 8.

[0007] In some aspects, the disclosed insecticidal composition comprises a 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 consistingof SEQ ID NOs: 3, 5, and 10. In some aspects, the recombinantRNA molecule is a double-stranded RNA (dsRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a hairpin RNA (hpRNA), or piwi-interacting RNA (pi-RNA).

[0008] In some aspects, the disclosed insecticidal composition comprises the recombinant RNA molecule is a double-stranded RNA (dsRNA) comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 10. In some aspects, the dsRNA is at least 17 base pairs in length.

[0009] In some aspects, the insect is an adult, or in a larval or a nymph stage. In some aspects, the plant-infesting insect is a Coleopteran insect. In some aspects, the plantinfesting insect is an insect from the genus Leptin otarsa. In some aspects, the plantinfesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No: 1. In some aspects, the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No: 4 or 6. In some aspects, the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No: 8. In some aspects, the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the 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 thegroup consisting of SEQ ID Nos: 3, 5, and 10. In some aspects, the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID Nos: 3, 5, and 10.

[0010] In certain aspects, the disclosed insecticidal composition further comprises at least one component selected from a carrier, excipient, diluent, 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 some aspects, the insecticidal composition furthercomprises at least one pesticidal agent. In some aspects, the pesticidal agent is selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, an insecticidal protein.

[0011] In some aspects, the disclosed insecticidal composition is in a form selected from a solid, a liquid, a powder, a suspension, an emulsion, a spray, an encapsulation, microbeads, carrier particulates, a film, a solid matrix, a soil drench, an insectdiet, insect bait, and a seed treatment.

[0012] Further disclosed herein is a plant or seed thereof treated with the disclosed insecticidal composition, wherein the plant exhibits improved resistance to the insect.

[0013] In some aspects, the disclosure further encompasses 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: 3, 5, and 10. In some aspects, the recombinant DNA construct, wherein the heterologous promoter is functional for expression of the RNA transcript in a bacterium or a fungus.

[0014] In some aspects, the heterologous promoter is functional in a plant cell. Further disclosed herein is a recombinant vector comprising the disclosed recombinant DNA construct. Further aspects, a plant chromosome or plastid comprising the disclosed recombinant DNA construct is provided herein. In some aspects, a transgenicplantcell having in its genome the disclosed recombinant DNA construct, a transgenic plant comprising the transgen icplantcell, or a crop product produced from the transgen icplant are further provided. In some aspects, a transgenic progeny seed or propagatable plant part of the disclosed transgenic plant is provided.

[0015] In further aspects, the disclosure encompasses a method for controlling an infestation of a plant by a plant-infesting insect, the method comprising contacting the insectwith a dsRNAcomprisingat least one portion of 17 or more contiguous nucleotideshaving a sequence of about 95% to about 100% complementarity with a target gene or portion thereof of the insect, wherein the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID NOs: 1 , 4. 6, and 8. In some aspects of the method, the insectis an adult, or in a larval or a nymph stage. In some aspects, the plantinfesting insect is a Coleopteran insect. In some aspects, the plant-infesting insect is an insect from the genus Leptinotarsa. In some aspects, the insect is Leptinotarsa decemlineata (Colorado potato beetle).

[0016] In some aspects of the method, the dsRNA comprises a sequence selected from the groupconsisting of SEQ ID Nos: 3, 5, and 10. In some aspects, thedsRNA comprises more than one portion of 17 or more contiguous nucleotides having a sequence of about 95% to about 100% complementarity with the target gene or portion thereof. In some aspects, the dsRNA is blunt-ended. In some aspects, the dsRNA has an overhang at least one terminus. In some aspects, the dsRNA is (a) chemically synthesized, or (b) produced by expression in a microorganism, expression in a plant cell, or by microbial fermentation. In some aspects, the dsRNA is chemically modified.

[0017] In some aspects of the method, the contacting comprises application of a composition comprising the dsRNA to a surface of the insector to a surface of the plant infested by the insect.

[0018] In some aspects of the method, the composition comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, or seed treatment. In further aspects, the contacting comprises providing the dsRNA in a composition further comprising one or more components selected from 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 some aspects, the contacting comprises providing the dsRNA in a composition further comprising at least one pesticidal agent. In some aspects, the pesticidal agent is selected from a patatin, a plant lectin, a phytoecdysteroid, and an insecticidal protein. In some aspects, the contacting comprises providingthe dsRNAin a composition ingested by the insect. In some aspects, the ingested composition further comprises one or more components selected from 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.

[0019] The disclosure further encompasses a method of causing mortality in an insect, comprising providing in thediet of an insectan insecticidal composition, orplant disclsoed herein, wherein the composition or plant causes mortality or stunting of the insect upon ingestion by the insect. In some aspects, the insect is an adult, or in a larval or a nymph stage.

[0020] A method of providing a plant having improved resistance to a plant-infesting in sect is further provided. In such aspects, the method comprises introducing to the plant expressing a recombinantDNAconstruct comprising a nucleotide sequence encoding an RNA molecule comprising a silencing element, the silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes a mRNA sequence selected from SEQ ID Nos: 1 , 4, 6 and 8, and wherein ingestion of the RNA by the insect results in mortality or stunting in the insect. In some aspects, the silencingelement has a sequence of about 95% to about 100% sequence identity or complementarity with a sequence selected from the group consisting of SEQ ID Nos: 3, 5 and 10. In some aspects, the recombinant DNA construct further comprises a heterologous promoter operably linked to the nucleotide sequence encoding the RNA molecule, and functional in a plant cell. In some aspects, the silencing element is a dsRNA, a miRNA, a small siRNA, a hpRNA, or a pi-RNA. In some aspects, the plant-infesting insect is an adult, or in a larval or a nymph stage. In some aspects, the plant-infesting insect is a Coleopteran insect. In some aspects, the plant-infesting insect is an insect from the genus Leptinotarsa. In some aspects, the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle). In some aspects, the introducing comprises transgenic expression or transient expression. In some aspects, A plant produced by the disclosed method and having improved resistance to the insect is further provided. In further aspects, Fruit, seed, or propagatable parts of the discloses plant is further provided.BRIEF DESCRIPTION OF THE FIGURES

[0021] FIG. 1 shows results from RNAi screening assays involving 95 CPB essential genes (EG). Each dsRNA was provided as 120 pl, 10 ng / pL concentration on detached leaves. Treatments that had two dsRNAs from two genes, 10 ng / pL of each dsRNAs were mixed. Appropriate concentrations of the non-target dsRNA were used. Graph shows mortality on the last day of the assays as indicated at the top of the graph. The graph shows pul led results from several assays, and assays where the non-target dsRNA(negative control) treatment had greater than 20% mortality were discarded as invalid assay.

[0022] FIG. 2A-2E show results from screening assays involving LdEG12. FIG. 2A shows mean percentage of larval mortality in Colorado potato beetle (CPB) larvae (L2) exposed to excised leaves treated with 120 pl of 10 ppm (10 ng / pL) LdEG12 dsRNA or non-target dsRNA (negative control) for 4 days in 12-ounce cups with mesh lids for ventilation. Five L2 larvae were placed into each cup. Each treatment had 6 replicates. Mortality was scored on days 4, 5, 6 and 8, one-way AN OVA was run on day 8 mortality. (Fi,w = 75.34, p < 0.001 indicated by ***). FIG. 2B shows percentage of larval mortality in CPB L2 exposed to excised leaves treated with 120 pl of 10 ppm (10 ng / pL) LdEG12 dsRNA or non -target dsRNA (negative control) for 4 days in 150 X 25 mm petri dishes. Untreated leaves were added after day 4. Five L2 larvae were placed into each dish. Each treatment had 6 replicates. Mortality was scored on day 4 and 7. One-way AN OVA was run on day 7 mean mortality percentage. (Fi,w = 375.6, p < 0.001 indicated by ***). FIG. 2C shows percentage of larval mortality in CPB larvae exposed to 24 mm diameter leaf disks treated with 24 pl of 10 ppm (10 ng / pL) LdEG12 dsRNA or non-target dsRNA (negative control) for 4 days in 32-well bioassay tray. After 4 days, untreated leaf disks were added to the tray. Mortality was scored on day 8. The experiment was replicated twice, each experiment had 4 replicates, and one-way ANOVA was run on day 8 mortality percentage. (FI,4 = 80.81 , p < 0.001 indicated by ***). FIG. 2D shows knockdown of LdEG12 gene expression following dsRNA ingestion by L2 CPB larvae. Bioassay was conducted in 32-well tray and each CPB L2 larvae were exposed to 24 pL, 10 ppm (10 ng / pL) dsRNA LdEG12 or non-target dsRNA on leaves for 4 days. After4 days, the alive larvae were collected for RNA extraction and cDNA synthesis. The cDNAswere used in qPCR using TaqMan master mix. The LdEG12 gene expression on the figure is normalized using RPL18andARF1 reference genes expression. FIG. 2E shows mortality of LdEG12 compared to three other genes all of which work in mitochondria. For this assay, bioassays were conducted in 32-well trays as described above for FIG 2C-2D. The small letters on the bars in dicate significantly different treatments, with same letter indicating no significant difference and different letters between bar / treatment indicating that the treatments produced significantly different results.

[0023] FIG. 3A-3C show results from screeningassays involving LdEG53. FIG. 3A shows larval mortality in CPB L2 exposed to excised preserved leaves treated with 120 pl of 10 ppm (10 ng / pL) LdEG53 dsRNA or non-target dsRNA (negative control) for 4 days in 150X25 mm petri dishes. Each treatment had 6 replicates. One-way AN OVA was run on day 8 mortality mean percentage (F1 ,6 = 18.44, p < 0.01 indicated by ***). FIG. 3B shows LdEG53 gene knockdown following dsRNA treatment as described for FIG. 2D above FIG. 3C shows percentage larval mortality in CPB larvae in whole plants treated with 52 ppm (52 ng / pL) LdEG53 dsRNA or untreated plants for 14 days. There were 5 cages / biological replicates per treatment. (Day M ANOVA, F1 ,3,35= 454.711 , p < 0.001 indicated by ***).

[0024] FIG. 4A-4B show results from screening assays involving LdEG81. FIG. 4A shows lethality of LdEG81 in bioassay conducted in bioassay trays. One CPB larva was placed into each well of a 32-well bioassay tray. Leaves were treated with 24 pl of 10 ppm dsRNA LdEG 81 or non-target negative control dsRNA. Each treatment had 20 insects total. FIG. 4B shows lethality of LdEG81 in whole plant assay. Whole 3-5-week-old Kennebec potato plants were treated with 52 ppm of dsRNA LdEG81 or left untreated. Ten L2 CPB larvae were placed on plants inside butterfly cages. There were 5 cages / biological replicates per treatment. (Day 14 ANOVA, FI,28 = 69.82, p < 0.001 indicated by ***).DETAILED DESCRIPTION

[0025] The present disclosure encompasses compositions and methods for controlling insect pest. The disclosure in part is based on the surprising discovery of LdEG12, LdEG53 and LdEG81 in the plant insect pest CPB, expression of which when downregulated using RNAi, can cause mortality in an insect pest (for e.g., CPB). The disclosed target genes were identified by the inventors as essential genes and recombinant RNA molecule optimized as a potential as insecticide.

[0026] All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.A. Definitions

[0027] Before the present compounds, compositions, articles, systems, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unlessotherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalentto those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0028] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure.

[0029] As used in the specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0030] The phrase “consisting essentially of” limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of” excludes any component, step, or element that is not recited in the claim.

[0031] As used herein, the terms "comprising," "including," “encompassing” and "having" are used interchangeably in this disclosure.

[0032] As used herein, when referring to any numerical value, the term ‘about’ means a value falling within a range that is ± 10% of the stated value.

[0033] Ranges can be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, a further aspect includesfromthe one particularvalueand / orto the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a numberof values disclosed herein, and th at each value is also herein disclosed as ‘about that particular value in addition to the value itself. For example, if the value ‘10’ is disclosed, then ‘about 10’ is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11 , 12, 13, and 14 are also disclosed.

[0034] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in thecomposition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weightcomponentX and 5 parts by weightcomponentY, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0035] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0036] As used herein, the terms “or” and “and / or,” are to be interpreted as inclusive or meaning anyone or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of thefollowing:“A,”“B” or“C”; “A and B”; “A and C”;“B and C”; “A, B and C.” An exception to th is definition will occuronly when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0037] The present disclosure also contemplates that in some aspects, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0038] As used herein, the terms “nucleicacid”and “nucleotide” refer to isolated, purified, natural, recombinant, synthetic deoxyri bon u cl eoti des or ribonucleotides and polymers thereof in either single- or double-stranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a mannersimilarto naturally occurring nucleotides. Unless otherwise indicated, a particularnucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the reference sequence explicitly indicated.

[0039] The term “polynucleotide” as used herein refers to a molecule comprised of two or more deoxyribonucleotides orribonucleotides, preferably more than three, and usually more than ten. There is no precise upper limit on the size of an oligonucleotide. However, in general, an oligonucleotide is shorter than about 250 nucleotides, preferably shorter than about 200 nucleotides and more preferably shorter than about 100 nucleotides. Theexact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.

[0040] As used herein the terms “recombinant RNA” and “recombinant nucleotide” refer to polynucleotide molecules that contain a genetically engineered modification through manipulation via mutagenesis, restriction enzymes, and the like.

[0041] The terms "identical" or “percent identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more biological sequences or subsequences (of nucleotides or of amino acids) that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least about 60%, preferably 65%, 70%, 75%, preferably 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Such sequences that have at least about 60% identity are then said to be "substantially identical." Th is definition also refers to the complimentof a test sequence. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length. One of skill will recognize that the polynucleotide sequence need not be identical but may be only "substantially identical" to a sequence of the gene from which it was derived. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

[0042] As used herein “complementary” refers to that the nucleotide sequence is fully complementary to the nucleotide sequence of the target over more than two nucleotides, for instance over at least 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more contiguous nucleotides. Further, the term “complementary” as used herein relates to both DNA-DNAcomplementarity as to DNA-RNA complementarity. In analogy herewith, the term “RNA equivalent” substantially means that in the DNA sequence(s), the base “T’ may be replaced by the corresponding base “U” normally present in ribonucleic acids.

[0043] As used herein, “effective amount” and “amount effective” can refer to an amount that is sufficientto achieve the desired result such as, for example, controlling a plant pest. In some aspects, the compositions and / or methods described herein, may be used curatively, preventively, or systematically to control an insect pest or to avoid insect pest growth or development or infection or infestation. Thus, the invention may allow previously susceptible organisms to develop resistance against infestation by the insect organism.

[0044] Controlling insects as used herein encompasses controlling insect progeny (such as development of eggs, for example for insect pests), inhibiting viability, growth, development, or reproduction of the insect, or decreasing pathogenicity or infectivity of the insect. In some aspects, controlling insects may inhibit a biological activity in an insect, resulting in one or more of the following attributes: reduction in feeding by the insect, reduction in viability of the insect, death of the insect, inhibition of differentiation and development of the insect, absence of or reduced capacity for sexual reproduction by the insect, muscle formation, juvenile hormone formation, juvenile hormone regulation, ion regulation and transport, maintenance of cell membrane potential, amino acid biosynthesis, amino acid degradation, sperm formation, pheromone synthesis, pheromone sensing, antennae formation, wing formation, leg formation, development and differentiation, egg formation, larval maturation, digestive enzyme formation, haemolymph synthesis, haemolymph maintenance, neurotransmission, cell division, energy metabolism, respiration, apoptosis, and any component of a eukaryotic cells' cytoskeletal structure, such as, for example, actins and tubulins.

[0045] As used herein “pest” or “insect pests” refer to organisms possessing characteristics that are considered damagingorunwanted. In some aspects, pests cause significantplantloss, plantdamage, any destruction or loss in value, usefulness, or ability resulting from an action or event associated with a pest such as an insect. Types of plant damage include, but are not limited to, the following: feeding damage occurs as a result of direct feeding on above-ground and / or below-ground plant parts, holes or notches in foliage and other plantparts, leaf skeleton izing (removal of tissue between the leaf veins), leaf defoliation, cutting plants off at the soil surface, or consumption of roots can all occur from pests with chewing mouthparts, chewing pests can also bore or tunnel into planttissue, stem-boring insectscan kill or deform individual stems or wholeplants, leaf mining insects feed between the upper and lower surfaces of leaves, creating distinctive tunnel patterns visibleas translucentlinesorblotches on leaves, pests with sucking mouth parts can suck sap from plant tissue, which may cause spotting or stippling of foliage, leaf curling and stunted or misshapen fruits, insects such as th rips have rasping mouth parts that scrape the surface of foliage or flower parts, disrupting plant cells, oviposrtion damage occurs as a result of egg laying into plant tissue, heavy oviposition into stems can cause death or dieback of stems or branches on the plant, flagging is a result of dieback of the ends of stems or branches, oviposition in fruits can result in misshapen or aborted fruits, and is sometimes called cat-facing, some insects form galls on their host plant, causing the plant to grow abnormally, depending on the insect species, the gall formation can be stimulated by feeding or by oviposition into plant tissue, pests can also cause damage by transmitting plant pathogens such as viruses, fungi, bacteria, rnollicutes, protozoa, and nematodes, the transmission can be accidental or incidental (the plant pathogen enters planttissue through feeding or oviposition wounds), phoretic or passive (the pest carries the plant pathogen from one plantto another), or active (the plant pathogen is carried within the body of the pest, and a plant is inoculated with the pathogen when the pest feeds on a plant).

[0046] As used herein, the term “insect” refers to any insect, meaning any organism belonging to the Kingdom Animals, more specific to the Phylum Arthropoda, and to the Class Insecta or the Class Arachnida. As used herein “insect” also encompasses insects of all stages of development, including egg, larval or nymphal, pupal, and adult stages. The compositions and methods disclosed herein are applicable to all insects that are susceptible to gene silencing by RNA interference and that are capable of internalizing double-stranded RNAfrom theirimmediate environment. Thecompositionsand methods are also applicable to the insect at any stage in its development, including the stages between moults referred to as “instars”. In some aspects, the disclosed compositions and methods can target insect eggs or live young. All stages in the developmental cycle, which includes metamorphosis in the pterygotes, may be targeted according to the present disclosure. Thus, individual stages such as larvae, pupae, nymph etc. stages of development may all be targeted.

[0047] In some aspects, the insect belongs to any one of the following orders: Acari, Araneae, Anoplura, Coleoptera, Collembola, Dermaptera, Dictyoptera, Diplura, Diptera, Embioptera, Ephemeroptera, Grylloblatodea, Hemiptera, Homoptera, Hymenoptera,Isoptera, Lepidoptera, Mallophaga, Mecoptera, Neuroptera, Odonata, Orthoptera, Phasmida, Plecoptera, Protura, Psocoptera, Siphonaptera, Siphunculata, Thysanura, Strepsiptera, Thysanoptera, Trichoptera, and Zoraptera.

[0048] In some aspects, an insect is a plant pest, non-limiting examples of which include Nilaparvata spp. (e.g. N. lugens (brown planthopper)); Laodelphaxspp. (e.g. L. striatellus (small brown planthopper)); Nephotettix spp. (e.g. N. virescens or N. cincticeps (green leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp. (e.g. S. furcifera (white- backed planthopper)); Blissus spp. (e.g. B. leucopterus leucopterus (chinch bug)); Scotinophora spp. (e.g. S. vermidulate (rice blackbug)); Acrosternum spp. (e.g. A. hilare (green stink bug)); Parnara spp. (e.g. P. guttata (rice skipper)); Chilo spp. (e.g. C. suppressalis (rice striped stem borer), C. auricilius (gold-fringed stem borer), or C. polychrysus (dark-headed stem borer)); Chilotraea spp. (e.g. C. polychrysa (rice stalk borer)); Sesamia spp. (e.g. S. inferens (pink rice borer)); Tryporyza spp. (e.g. T. innotata (white rice borer), or T. incertulas (yellow rice borer)); Cnaphalocrocis spp. (e.g. C. medinalis (rice leafroller)); Agromyza spp. (e.g. A. oryzae (leafminer), or A. parvicornis (corn blot leafminer)); Diatraea spp. (e.g. D. saccharalis (sugarcane borer), or D. grandiosella (southwestern corn borer)); Narnaga spp. (e.g. N. aenescens (green rice caterpillar)); Xanthodes spp. (e.g. X. transversa (green caterpillar)); Spodoptera spp. (e.g. S. frugiperda (fall armyworm), S. exigua (beet armyworm), S. littoralis (climbing cutworm) or S. praefica (western yellowstriped armyworm)); Mythimna spp. (e.g. Mythmna (Pseudaletia) seperata (armyworm)); Helicoverpa spp. (e.g. H. zea (corn earworm)); Colaspis spp. (e.g. C. brunnea (grape colaspis)); Lissorhoptrus spp. (e.g. L. oryzophilus(ricewaterweevil)); Echinocnemusspp. (e.g. E. squamos(rice plantweevil)); Diclodispa spp. (e.g. D. armigera (rice hispa)); Oulema spp. (e.g. 0. oryzae (leaf beetle); Sitophilus spp. (e.g. S. oryzae (rice weevil)); Pachydiplosis spp. (e.g. P. oryzae (rice gall midge)); Hydrellia spp. (e.g. H. griseola (small rice leafminer), or H. sasakii (rice stem maggot)); Chlorops spp. (e.g. C. oryzae (stem maggot)); Diabrotica spp. (e.g. D. virgifera virgifera (western corn rootworm), D. barberi (northern corn rootworm), D. undecimpunctata howardi (southern corn rootworm), D. virgifera zeae (Mexican corn rootworm); D. balteata (banded cucumber beetle)); Ostrinia spp. (e.g. 0. nubilalis (European corn borer)); Agrotis spp. (e.g. A. ipsilon (black cutworm)); Elasmopalpusspp. (e.g. E. lignosellus (lesser cornstalk borer)); Melanotus spp. (wireworms); Cyclocephala spp. (e.g. C. borealis (northern masked chafer), or C. immaculata (southern masked chafer)); Popillia spp. (e.g. P. japonica (Japanese beetle)); Chaetocnema spp. (e.g. C.pulicaria (corn flea beetle)); Sphenophorus spp. (e.g. S. maidis (maize billbug)); Rhopalosiphum spp. (e.g. R. maidis (corn leaf aphid)); Anuraphis spp. (e.g. A. maidiradicis (corn root aphid)); Melanoplus spp. (e.g. M. femurrubrum (redlegged grasshopper) M. differential is (differential grasshopper) or M. sanguinipes (migratory grasshopper)); Hylemya spp. (e.g. H. platura (seedcorn maggot)); Anaphothrips spp. (e.g. A. obscrurus (grass thrips)); Solenopsis spp. (e.g. S. milesta (thief ant)); or spp. (e.g. T. urticae (twospotted spider mite), T. cinnabarinus (carmine spider mite); Helicoverpa spp. (e.g. H. zea (cotton bollworm), or H. armigera (American bollworm)); Pectinophora spp. (e.g. P. gossypiella (pink bollworm)); Earias spp. (e.g. E. vittella (spotted bollworm)); Heliothis spp. (e.g. H. virescens (tobacco budworm)); Anthonomus spp. (e.g. A. grandis (boll weevil)); Pseudatomoscelis spp. (e.g. P. seriatus (cotton fleahopper)); Trialeurodes spp. (e.g. T. abutiloneus (banded-winged whitefly) T. vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B. argentifolii (silverleaf whitefly)); Aphis spp. (e.g. A. gossypii (cotton aphid)); Lygus spp. (e.g. L. lineolaris (tarnished plant bug) or L. hesperus (western tarnished plant bug)); Eusch istus spp. (e.g. E. conspersus (consperse stink bug)); Chlorochroa spp. (e.g. C. sayi (Say stinkbug)); Nezara spp. (e.g. N. viridu la (southern green stinkbug)); Thrips spp. (e.g. T. tabaci (onion thrips)); Frankliniellaspp. (e.g. F. fu sea (tobacco th rips), or F. occidentalis (western flower thrips)); Leptinotarsa spp. (e.g. L. decemlineata (Colorado potato beetle), L. jun eta (false potato beetle), or L. texana (Texan false potato beetle)); Lerna spp. (e.g. L. trilineata (three-lined potato beetle)); Epitrix spp. (e.g. E. cucumeris (potato flea beetle), E. hirtipennis (flea beetle), or E. tuberis (tuber flea beetle)); Epicauta spp. (e.g. E. vittata (striped blister beetle)); Phaedon spp. (e.g. P. cochleariae (mustard leaf beetle)); Epilachnaspp. (e.g. E. varivetis (mexican bean beetle)); Acheta spp. (e.g. A. domesticus (house cricket)); Empoasca spp. (e.g. E. fabae (potato leafhopper)); Myzus spp. (e.g. M. persicae (green peach aphid)); Paratrioza spp. (e.g. P. cockerelli (psyllid)); Conoderus spp. (e.g. C. falli (southern potato wireworm), or C. vespertinus (tobacco wireworm)); Phthorimaea spp. (e.g. P. opercu lei la (potato tuberworm)); Macrosiphum spp. (e.g. M. euphorbiae (potato aphid)); Thyantaspp. (e.g. T. pallidovirens (redshoulderedstinkbug)); Phthorimaea spp. (e.g. P. opercu lei la (potato tuberworm)); Helicoverpa spp. (e.g. H. zea (tomato fruitworm); Keiferiaspp. (e.g. K. lycopersicella (tomato pinworm)); Limoniusspp. (wireworms); Manduca spp. (e.g. M. sexta (tobacco hornworm), or M. quinquemaculata (tomato hornworm)); Liriomyza spp. (e.g. L. sativae, L. trifolli or L. huidobrensis (leafminer)); Drosophila spp. (e.g. D. melanogaster, D. yakuba, D. pseudoobscura or D.simulans); Carabus spp. (e.g. C. granulatus); Chironomus spp. (e.g. C. tentanus); Ctenocephalides spp. (e.g. C. felis (cat flea)); Diaprepes spp. (e.g. D. abbreviatus (root weevil)); Ips spp. (e.g. I. pini (pineengraver)); Triboliumspp. (e.g. T. castaneum (red floor beetle)); Glossina spp. (e.g. G. morsitans (tsetse fly)); Anopheles spp. (e.g. A. gambiae (malaria mosquito)); Helicoverpa spp. (e.g. H. armigera (African Bollworm)); Acyrthosiphon spp. (e.g. A. pisum (pea aphid)); Apis spp. (e.g. A. melifera (honey bee)); Homalodisca spp. (e.g. H. coagulate (glassy-winged sharpshooter)); Aedes spp. (e.g. Ae. aegypti (yellow fever mosquito)); Bombyxspp. (e.g. B. mori (silkworm)); Locusta spp. (e.g. L. migratoria (migratory locust)); Boophilus spp. (e.g. B. microplus (cattle tick)); Acanthoscurriaspp. (e.g. A. gomesiana (red-haired chololate bird eater)); Diplopteraspp. (e.g. D. punctata (pacific beetle cockroach)); Heliconius spp. (e.g. H. erato (red passion flower butterfly) or H. melpomene (postman butterfly)); Curculio spp. (e.g. C. glandium (acorn weevil)); Plutella spp. (e.g. P. xylostella (diamondbackmoth)); Amblyomma spp. (e.g. A. variegatum (cattle tick)); Anteraea spp. (e.g. A. yamamai (silkmoth)); and Armigeres spp. (e.g. A. subalbatus).

[0049] In other aspects, the compositions and methods disclosed herein are applicable for Leptinotarsa species. Leptinotarsa belong to the family of Chrysomelidae or leaf beetles. There are more than 30 Leptinotarsa species. Chrysomelid beetles such as Flea Beetles and Corn Rootworms and Curculionids such as Alfalfa Weevils are particularly important pests. Flea Beetles include a large number of small leaf feeding beetles that feed on the leaves of a number of grasses, cereals and herbs. Flea Beetles include a large number of genera (e.g., Attica, Apphthona, Argopistes, Disonycha, Epitrix, Longitarsus, Prodagricomela, Systena, and Phyllotreta). The Flea Beetle, Phyllotreta cruciferae, also known as the Rape Flea Beetle, is a particularly important pest. Com rootworms include species found in the genus Diabrotica (e.g., D. undecimpunctata undecimpunctata, D. undecimpunctata howardii, D. longicornis, D. virgifera and D. balteata). Corn rootworms cause extensive damage to corn and curcubits. The Western Spotted Cucumber Beetle, D. undecimpunctata undecimpunctata, is a pest of curcubits in the western U.S. Alfalfa weevils (also known as clover weevils) belong to the genus, Hypera (H. postica, H. brunneipennis, H. n igrirostris, H. punctata and H. meles), and are considered an important pest of legumes. The Egyptian alfalfa weevil, H. brunneipennis, is an important pest of alfalfa in the western U.S.

[0050] In some aspects, the disclosed plant insect pest are selected from the group consisting of Leptinotarsa spp. (e.g. L decemlineata (Colorado potato beetle), Ljuncta (false potato beetle), or L texana (Texan false potato beetle)); Nilaparvata spp. (e.g. N. lugens (brown planthopper)); Laodelphax spp. (e.g. L striatellus (small brown planthopper)); Nephotettix spp. (e.g. N. virescens or N. ci ncticeps (green leafhopper), or N. nigropictus (rice leafhopper)); Sogatella spp. (e.g. S. furcifera (white-backed planthopper)); Chilo spp. (e.g. C. suppressalis (rice striped stem borer), C. auricilius (gold-fringed stem borer), or C. polychrysus (dark-headed stem borer)); Sesamia spp. (e.g. S. inferens (pink rice borer)); Tryporyza spp. (e.g. T. innotata (white rice borer), or T. incertulas (yellow rice borer)); Diabrotica spp. (e.g. D. virgifera virgifera (western corn rootworm), / ?, barberi (northern corn rootworm), / ?. undecim punctata howardi (southern corn rootworm), D. virgifera zeae (Mexican corn rootworm); Ostrinia spp. (e.g. 0. nubilalis (European corn borer)); Anaphothrips spp. (e.g. A. obscrurus (grass th rips)); Pectinophora spp. (e.g. P. gossypiella (pink bollworm)); Heliothis spp. (e.g. H. virescens (tobacco budworm)); Trialeurodes spp. (e.g. T. abutiloneus (banded-winged whitefly) ?. vaporariorum (greenhouse whitefly)); Bemisia spp. (e.g. B. argentifolii (silverleaf whitefly)); Aphis spp. (e.g. A. gossypii (cotton aphid)); Lygus spp. (e.g. L Hneolaris (tarnished plant bug) or L hesperus (western tarnished plant bug)); Euschistus spp. (e.g. E. conspersus (con sperse stink bug)); Chlorochroa spp. (e.g. C. sayi (Say stinkbug)); Nezara spp. (e.g. N. viridula (southern green stinkbug)); Thrips spp. (e.g. T. tabaci (onion thrips)); Frankliniella spp. (e.g. F. fusca (tobacco thrips), or F. occidentalis (western flower thrips)); Myzus spp. (e.g. M. persicae (green peach aphid)); Macrosiphum spp. (e.g. M. euphorbiae (potato aphid)); B / issus spp. (e.g. B. leucopterus leucopterus (chinch bug)); Acrosternum spp. (e.g. A. hilare (green stink bug)); Chilotraea spp. (e.g. C. polychrysa (rice stalk borer)); Lissorhoptrus spp. (e.g. L oryzophi I us (rice water weevil)); Rhopalosiphum spp. (e.g. R. maidis (corn leaf aphid)); and Anuraphis spp. (e.g. A. maidiradicis (corn root aphid)).

[0051] In certain aspects, the present disclosure encompasses compositions and methods for controlling Leptinotarsa species, including Colorado Potato Beetle (Leptinotarsa decemlineata (Say) and False Potato Beetle (Leptinotarsa juncta (Say). In some aspects, the disclosed composition sand methods are used for control ling Colorado Potato Beetle (CPB).

[0052] As used herein “target host” or “host plant” is a plant that the insect pest infests. CPB is a pest of domestic potato (Solanum tuberosum), other cultivated and wild tuber bearing and non-tuberbearing potato species (e.g. S. demissum, S. phureja a.o.) andother Solanaceous (nightshades) plantspecies non-limiting examples of which include: the crop species tomato (several Lycopersicon species), eggplant(Solanum melongena), peppers (several Capsicum species), tobacco (several Nicotiana species including ornamentals) and ground cherry (Physalis species); the weed / herb species, horse nettle (S. carolinense), common nightshade(S. dulcamara), belladonna (Atropa species), thorn apple (datura species), henbane (Hyoscyamus species) and buffalo burr (S. rostratum).

[0053] As used herein “plant” encompasses any plant material that it is desired to control insect pests (e.g., treat to prevent or reduce insect growth and / or insect infestation). This includes, inter alia, whole plants, seedlings, propagation, or reproductive material such as seeds, cuttings, grafts, explants, etc. and also plant cell and tissue cultures. In some aspects, the plant material should express, or have the capability to express, the RNA molecule comprising at least one nucleotide sequence that is the RNA complement of or that represents the RNA equivalent of at least part of the nucleotide sequence of the sense strand of at least one target gene of the pest organ ism, such th at the RNA molecule is taken up by a pest upon plant-pest interaction, said RNA molecule being capable of inhibiting the target gene or down-regulating expression of the target gene by RNA interference.

[0054] As used herein, the phrases “down regulation of gene expression”, “inhibition of gene expression” and “knockdown of gene” refer to a measurable or observable reduction in gene expression or a complete abolition of detectable gene expression, at the level of protein product and / or mRNA product from the target gene. Preferably the down regulation does not substantially directly inhibitthe expression of other genes of the insect. The down-regulation effect of the recombinant RNA molecule on gene expression may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% when compared to insects not contacted with recombinant RNA molecule or compared to insects that contacted with a recombinant RNA molecule that does not have any complementary target RNA present in the insect. For e.g., the non-insect control recombinant RNA can be RNA from white spot syndrome virus (WSSV) genome. Depending on the nature of the target gene, down -regulation or inhibition of gene expression in cells of an insect can be confirmed by phenotypic analysis of the cell orthe whole insect or by measurement of mRNA or protein expression using molecular techniques such as RNA solution hybridization, PCR, Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), nuclease protection, Northern blotting, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, or fluorescence-activated cell analysis (FACS).

[0055] In some aspects, the down-regulation of the target gene causes mortality of the insect, orthe reproduction orgrowth of the insect is stopped or retarded. In some aspects, an insect contacted with disclosed composition can cause a mortality of the insects at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% more than compared to insects not contacted with disclosed compositions or contacted with a non-insect control recombinant RNA.

[0056] As used interchangeably herein, the terms “RNA interference” and “RNAi” referto degradation of mRNA or inhibition of protein synthesis through an endogenous pathway including the DICER protein complex. DICER cleaves long double stranded RNA (dsRNA) molecules into short fragments of approximately 21 nucleotides, termed smallinterfering RNA (siRNA). The siRNA is unwound into two single-stranded RNAs: the passengerstrand and theguide strand. The passengerstrand is degraded, andthe guide strand is incorporated into the RNA-induced silencing complex (RISC). Micro ribonucleic acids (miRNAs) are usually 22 nucleotides long, therefore, very similarto siRNAs in size, however, the miRNAs are cleaved from precursor molecules containing a polynucleotide "loop" connecting thehybridized passengerand guide strands, and they may be similarly incorporated into RISC. Post-transcriptional genesilencing occurswhen theguide strand binds specifically to a complementary mRNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC. One possibility to generate siRNAs in cells is the expression of a precursor RNA, called hairpin RNA (hp-RNA). Transcription of hp-RNAs is usually driven by RNA-polymerase-lll promoters like the U6 or H1 promoter or T7 RNA polymerase. RNA-mediated silencing using an inverted repeat of a nucleicacid or a part thereof (in thiscase a stretch of substantially contiguous nucleotides derived from thetarget gene, orfrom any nucleicacid capableof encoding an orthologue, paralogue, or homologue of the protein of interest), preferably capable of forming a hairpin structure. The inverted repeat is cloned in an expression vector comprising control sequences. A non-coding DNA nucleic acid sequence (a spacer, for example a matrix attachment region fragment (MAR), an intron, a polylinker, etc.) is located between the two inverted nucleic acids forming the inverted repeat. After transcription of the inverted repeat, a chimeric RNA with a self-complementary structure is formed (partial or complete). This double-stranded RNA structure is referred to as the hairpin RNA (hpRNA). The hpRNA is processed by the insects into siRNAs that are incorporated intoan RNA-induced silencing complex (RISC). The RISC subsequently cleaves the target mRNA transcripts, thereby substantially reducing the number of mRNA transcripts to be translated into polypeptides.

[0057] A “recombinant RNA molecule” may be an RNA molecule with a silencing element and encompasses an interfering RNA moleculethat can down regulate the expression of a target insect pest gene. In some aspects, a recombinant RNA molecule is specific for all, or any portion of adequate size, of a target insect gene and can result in the degradation of the mRNA encoded thereby. In some aspects, a recombinant RNA molecule can be a dsRNA, a miRNA, a siRNA, or a hpRNA or a short hairpin RNA (shRNA). In some aspects, dsRNA, siRNA, shRNA, miRNA and / or hpRNA is complementary to coding and / or non-coding sequences of the target gene in the insects and causes the sequence-specific inhibition of expression of target gene products, to achieve at least partial control of a coleopteran pest. In some aspects, dsRNAs, siRNAs, shNAs, miRNAs, and / or hpRNAs may be produced in vitro, or in vivo by a genetically- modified organism, such as a plant or bacterium.

[0058] As used herein “silencing RNA” or “silencing RNA molecule,” also referred to herein as “inhibitory RNA” or “inhibitory RNA molecule,” indicates any RNA molecule which upon introduction into a host cell (for e. g., an insect cell), reduces the expression of a target gene, particularly through transcriptional and / or post-transcriptional silencing. Such silencing RNA may for example be “antisense RNA”, whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having at least 95% sequence identity to the complement of the sequence of the target nucleic acid, preferably the coding sequence of the target gene. However, antisense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals. Silencing RNAfu rtherincludes so-called “sense RNA” whereby the RNA molecule comprises a sequence of at least 20 consecutive nucleotides having at least 95% sequence identity to the sequence of the target nucleicacid. Sense RNA may also be directed to regulatory sequences of target genes, including the promoter sequences and transcription termination and polyadenylation signals.

[0059] As used herein, “piRNA” or “Piwi-interacting RNA” is a class of small RNA about 24-31 nucleotides in length. While siRNA and miRNA both require double stranded RNA (unstructured long dsRNA form or hairpin form, etc.), piRNAs are produced fromsingle stranded precursor sequence which can be anti-sense to the target mRNA (for e.g., a target mRNA of an insect pest).

[0060] As used herein, “contacting” refers to contacting the target pest insect with a recombinant RNA molecule, a composition comprising the recombinant RNA molecule, or a cell expressing the recombinant RNA molecule, to permit ingestion of the recombinant RNA molecule by the pest insect. In some aspects, the cells expressing the recombinantRNAmolecule may be linked to a food componentof the insect pest in order to increase uptake of the recombinant RNA molecule by the insect pest. The host cells expressing the recombinant RNA molecule may also be incorporated in the medium in which the insect pest grows or in or on a material or substrate such as a plant part that is infested by an insect pest or impregnated in a substrate or material susceptible to infestation by an insect pest.

[0061] As used herein “target gene” refers to a gene of interest in the insect that can be down-regulated. In some aspects, the target genes described herein, is LdEG12, LdEG53, LdEG81 , or any combination thereof. In some aspects, the recombinant RNA molecule comprises a target region or target nucleotides in the target insect gene and may be any suitable region ornucleotide sequence within the targets, LdEG12, LdEG53, or LdEG81. The target region could comprise at least 17, at least 18 or at least 19 consecutive nucleotides of the target gene, more preferably at least 20 or at least 21 nucleotide and still more preferably at least 22, 23 or 24 nucleotides of the target gene.

[0062] As used herein “essential gene” is a gene is selected that is essentially involved in the growth, development, viability, infectivity, and / or reproduction of an insect. In some aspects, essential gene is a gene likely involved in some biological function within the insect pest that was either essential to life, or thatwas necessary for ensuring health and vitality, or were likely to be involved in cellular integrity, cell maintenance, reproductive capacity, and the like.

[0063] As used herein “Leptinotarsa decemlineata essential gene 12” or “LdEG12” refers to the gene ATP synthase, [3 subunit (ATPsyn [3). This gene is encoded by the nuclear genome and works in the mitochondria. The last step in the oxidative phosphorylation is chemiosmosis, mediated by the Complex V, also known as the ATP synthase complex or the FiFo-ATPase / synthase. The multiprotein ATP synthase complexes are also found in bacteria, and chloroplasts. The holoenzyme consists of two structural domains namely, Fi and Fo. The Fi domain harbor the catalytic core and resides outside the membrane while the hydrophobic Fo domain is a membrane embedded structural unit. These twocomplexes are linked together by a central and a peripheral stalk. The total molecular mass of the F1 complex is approximately 350 kDa and its catalytic core is constituted from three a and three [3 subunits, interestingly, the a-subun its are non-catalyticwhereas the three [3-su bun its carry out the catalytic function of the enzyme complex. In addition to the a / p subunits, three other globularproteins y, 5 ands are also part of the Fi domain. ATP synthesis in Fi domain is coupled to proton translocation via a rotatory mechanism of the central stalk.

[0064] As used herein “Leptinotarsa decemlineata essential gene53” or “LdEG53” refers to the gene Adaptor Protein complex 1 , y subunit (AP-1y). The adaptor proteins are heterotetrametric protein complexes. Five AP complexes have been identified in mammals, AP1 -5, whileAP-4 and AP-5 are absent in insects. Each AP complex has two large subunits, one each of y / a / b / s / ^ and [31 -5, respectively, one medium-sized subunit (p1-5) andone small-sized subunit(o1-5). AP-1 , AP-2 andAP-3 are cl athrin -associated complexes, whereas AP-4 and AP-5 are not. The AP-1 complex is localized at the transGolgi network (TGN and rough endoplasmicreticulum (RE) and mediates the biogenesis of secretory granule and bidirectional transport between these two membranous systems. The major function of the AP-2 complex is clathrin-dependentendocytosisfrom the plasma membrane. AP-3 is localized at the endosomes and regulates tracking between the endosomal and the lysosomal pathways. For membrane recruitment, binding to phosphoinositide i.e., binding to PI4P, is sufficientfor the AP-2 complex but not for the AP-1 complex, which in addition to the phosphoinositide binding, needs binding to Arf1 to be localized to the TGN and ER. Interaction between Arf1 and both [31 and y subunits is required for Ap-1 recruitment at the membrane and determines the subcellular localization of the complex while interaction between the [31 site and the Arf1 is important for the allosteric activation of AP-1. y subunit is responsible for interacting with PI4P.

[0065] As used herein “Leptinotarsa decemlineata essential gene81” or “LdEG81” refers to small ribonucleoprotein particle protein SmD1 (SmD1 ). The major component of the spliceosome are four small nuclear ribonucleoprotein particles (snRNPs) known as U1 , U2, U4 / U6, and U5. These snRNPsform the spliceosome structure as well as carry out the splicing reactions. Before initiating the slicing reactions, the snRNPs recognize two features on the pre-mRNA, short-conserved sequences on the exon-intron boundaries and a branch point with in the introns located around 18 to 40 nucleotides upstream of the 3' end of an intron. The snRNPs associate with undine (U) rich non-polyadenylated smallnuclear RNAs (snRNAs), which play key role in spliceosome formation and subsequent splicing of the pre-mRNAs. Among these snRNAs, U2, U5 and U6form the catalytic RNA core of an active spliceosome, the U1 snRNA recruits the spliceosome at the 5' exonintron boundary and by acting as a chaperon , U4 brings the U6 to the spliceosome. Each snRNPs has two sets of proteins, one set is specific to the snRNPs and thus regulate function ofthatparticularsnRNP,whileasecondset of proteins are common in all snRNP complexes, these shared proteins are called Sm proteins. The Sm proteins bind to the snRNAs (except the U6 and U6 atac) on a uridine-rich docking site called the Sm site. There are seven Sm proteins described in eukaryotes, B / B', D1 , D2, D3, E, F and G. The Sm proteins form a heptameric ring around the ll-rich motif, which in turn stabilizes the RNA structure and facilitate binding of other Il-specific proteins to the spliceosomal RNP as they assemble. Therefore, the Sm proteins play crucial role in spliceosome assembly.

[0066] Un less otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.B. Recombinant RNA molecule

[0067] The present disclosure encompasses a recombinant RNA molecule that down- regulates expression of a target gene of an insect that infests a plant. In some aspects, a recombinant RNA molecule comprises a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% sequence complementarity to a portion of a target gene of an insect that infests a plant. In some aspects, the target gene is LdEG12, LdEG53, LdEG81 , or any combination thereof. In some aspects, the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID Nos: 1 , 4, 6 and 8.

[0068] In some aspects, the disclosed recombinant RNA molecule comprises a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene, wherein the target gene is LdEG12. In such aspects, the target gene encodes a mRNA sequence of SEQ ID No: 1. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequence of SEQ ID No: 1. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 1 .

[0069] In some aspects, the disclosed recombinant RNA molecule comprises a nucleotide sequenceof at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene, wherein the target gene is LdEG53. In such aspects, the target gene encodes a mRNA sequence of SEQ ID No: 4, SEQ ID No: 6, or any combination thereof. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequence of SEQ ID No: 4. In some aspects, the target gene encodesa mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 4. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequence of SEQ ID No: 6. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 6.

[0070] In some aspects, the disclosed recombinant RNA molecule comprises a nucleotide sequenceof at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene, wherein the target gene is LdEG81 . In such aspects, the target gene encodes a mRNA sequence of SEQ ID No: 8. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequence of SEQ ID No: 8. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 8.

[0071] In certain aspects, the 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: 3, 5, and 10.

[0072] In some aspects, the target gene is LdEG12, and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 3. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 3. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 3.

[0073] In some aspects, the target gene is LdEG53, and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 5. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 5. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 5.

[0074] In some aspects, the target gene is LdEG81 , and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 10. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 10. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 10.

[0075] In one aspect, the recombinant RNA molecule comprises a nucleotide sequence having at least 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255,260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340,345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425,430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510,515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595,600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680,685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775,780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870,875, 880, 885, 890, 895, 900, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965,970, 975, 980, 985, 990, 995, 1000, 1010, 1015, 1120, 1125, 1130, 1135, 1140, 1145,1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215,1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275, 1280, 1285,1290, 1295, 1300, 1305, 1310, 1315, 1320, 1325, 1330, 1335, 1340, 1345, 1350, 1355,1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415, 1420, 1425,1430, 1435, 1440, 1445, 1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485, 1490, 1495,1500, 1505, 1510, 1515, 1520, 1525, 1530, 1535,1540, 1545, 1550, 1555, 1560, 1565, 1570, 1575, 1580, 1585, 1590, 1595, 1600, 1605, 1610, 1615, 1620, 1625, 1630, 1635,1640, 1645, 1650, 1655, 1660, 1665, 1670, 1675, 1680, 1685, 1690, 1695, 1700, 1715,1720, 1725, 1730, 1735, 1740, 1745, 1750, 1755, 1760, 1765, 1770, 1775, 1780, 1785,1790, 1795, 1800, 1815, 1820, 1825, 1830, 1835, 1840, 1845, 1850, 1855, 1860, 1865,1870, 1875, 1880, 1885, 1890, 1895, 1900, or 1901 of SEQ ID NO: 1 .

[0076] In another aspect, the recombinant RNA molecule comprises a nucleotide sequence having at least 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415,420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500,505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585,590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860,865, 870, 875, 880, 885, 890, 895, 900, 915, 920, 925, 930, 935, 940, 945, 950, 955,960, 965, 970, 975, 980, 985, 990, 995, 1000, 1010, 1015, 1120, 1125, 1130, 1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205, 1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1271 , or 1272 more contiguous nucleotides of SEQ ID NO: 4.

[0077] In yet another aspect, the recombinant RNA molecule comprises a nucleotide sequence having at least 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415,420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500,505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585,590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860,865, 870, 875, 880, 885, 890, 895, 900, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, 1000, 1010, 1015, 1120, 1125, 1130, 1135, 1140, 1145, 1150, 1155, 1160, 1165, 1170, 1175, 1180, 1185, 1190, 1195, 1200, 1205,1210, 1215, 1220, 1225, 1230, 1235, 1240, 1245, 1250, 1255, 1260, 1265, 1270, 1275,1280, 1285, 1290, 1295, 1300, 1305, 1310, 1315, 1320, 1325, 1330, 1335, 1340, 1345,1350, 1355, 1360, 1365, 1370, 1375, 1380, 1385, 1390, 1395, 1400, 1405, 1410, 1415,1420, 1425, 1430, 1435, 1440, 1445, 1450, 1455, 1460, 1465, 1470, 1475, 1480, 1485,1490, 1495, 1500, 1505, 1510, 1515, 1520, 1525, 1530, 1535, 1540, 1545, 1550, 1555,1560, 1565, 1570, 1575, 1580, 1585, 1590, 1595, 1600, 1605, 1610, 1615, 1620, 1625,1630, 1635, 1640, 1645, 1650, 1655, 1660, 1665, 1670, 1675, 1680, 1685, 1690, 1695,1700, 1705, 1710, 1715, 1720, 1725, 1730, 1735, 1740, 1745, 1750, 1755, 1760, 1765,1770, 1775, 1780, 1785, 1790, 1795, 1800, 1805, 1810, 1815, 1820, 1825, 1830, 1835,1840, 1845, 1850, 1855, 1860, 1865, 1870, 1875, 1880, 1885, 1890, 1995, 1900, 1905,1910, 1915, 1920, 1925, 1930, 1935, 1940, 1945, 1950, 1955, 1960, 1965, 1970, 1975,1980, 1985, 1990, 1995, 2000, 2010, 2015, 2120, 2125, 2130, 2135, 2140, 2145, 2150,2155, 2160, 2165, 2170, 2175, 2180, 2185, 2190, 2195, 2200, 2205, 2210, 2215, 2220,2225, 2230, 2235, 2240, 2245, 2250, 2255, 2260, 2265, 2270, 2275, 2280, 2285, 2290,2295, 2300, 2305, 2310, 2315, 2320, 2325, 2330, 2335, 2340, 2345, 2350, 2355, 2360,2365, 2370, 2385, 2390, 2395, 2400, 2405, 2410, 2415, 2420, 2425, 2430, 2435, 2440,2445, 2450, 2455, 2460, 2465, 2470, 2475, 2480, 2485, 2490, 2495, 2500, 2505, 2510,2515, 2520, 2525, 2530, 2535, 2540, 2545, 2550, 2555, 2560, 2565, 2570, 2575, 2580,2585, 2590, 2595, 2600, 2605, 2610, 2615, 2620, 2625, 2630, 2635, 2640, 2645, 2650,2655, 2660, 2665, 2670, 2675, 2680, 2685, 2690, 2695, 2700, 2705, 2710, 2715, 2720,2725, 2730, 2735, 2740, 2745, 2750, 2755, 2760, 2765, 2770, 2775, 2780, 2785, 2790,2795, 2800, 2805, 2810, 2815, 2820, 2825, 2830, 2835, 2840, 2845, 2850, 2855, 2860,2865, 2870, 2875, 2880, 2885, 2890, 2995, 2900, 2905, 2910, 2915, 2920, 2925, 2930,2935, 2940, 2945, 2950, 2955, 2960, 2965, 2970, 2975, 2980, 2985, 2990, 2995, 3000,3010, 3015, 3120, 3125, 3130, 3135, 3140, 3145, 3150, 3155, 3160, 3165, 3170, 3175,3180, 3185, 3190, 3195, 3200, 3205, 3210, 3215, 3220, 3225, 3230, 3235, 3240, 3245,3250, 3255, 3260, 3265, 3270, 3285, 3290, 3295, 3300, 3305, 3310, 3315, 3320, 3325,3330, 3335, 3340, 3345, 3350, 3355, 3360, 3365, 3370, 3375, 3380, 3385, 3390, 3395,3400, 3405, 3410, 3415, 3420, 3425, 3430, 3435, 3440, 3445, 3450, 3455, 3460, 3465, 3470, 3475, 3480, 3481 , or 3482 more contiguous nucleotides of SEQ ID NO: 6.

[0078] In another aspect, the recombinant RNA molecule comprises a nucleotide sequence having at least 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330,335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415,420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500,505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585,590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670,675, 680, 685, 690, 695, 700, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860,861 , or 862 more contiguous nucleotides of SEQ ID NO: 6

[0079] In some aspects, the recombinant RNA molecule is a double-stranded RNA (dsRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a hairpin RNA (hpRNA), or a piwi-interacting RNA (pi-RNA).

[0080] In some aspects, the target gene is LdEG12, and the recombinant RNA molecule is a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA. In some aspects, the target gene is LdEG53, and the recombinant RNA molecule is a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA. In some aspects, the target gene is LdEG81 , and the recombinant RNA molecule is a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA.

[0081] In one aspect, the recombinant RNA molecule is a dsRNA. dsRNA may comprise an RNA strand comprising for example, nucleotides 800-1700 of SEQ ID NO: 1 , 1000- 1700 of SEQ ID NO: 1 , 1197-1546 of SEQ ID NO: 1 , 500 to 1300 of SEQ ID NO: 4, 600 to 1200 of SEQ ID NO: 4, 700 to 1200 of SEQ ID NO: 4, 805 to 1155 of SEQ ID NO: 4, 1000 to 1800 of SEQ ID NO: 6, 1100 to 1700 of SEQ ID NO: 6, 1283 to 1633 of SEQ ID NO: 6, 100 to 600 of SEQ ID NO: 8, 200 to 500 of SEQ ID NO: 8, or 204 to 495 of SEQ ID NO: 8.

[0082] In some aspects, the recombinant RNA molecu le is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 10.

[0083] In some aspects, the target is LdEG12 and the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In someaspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0084] In some aspects, the target is LdEG53 and the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0085] In some aspects, the target is LdEG81 and the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about85%, about90%, about95%, about98%) identityto SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0086] In some aspects, the double stranded RNA (dsRNA) is preferably between about 17-1500 base pair (bp), between about 80-1000 bp and most preferably between about 17-27 bp or between about 80-250 bp; such as double stranded RNA regions of about 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp, 25 bp, 27 bp, 50 bp, 80 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 900 bp, 100 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp or 1500 bp. In some aspects, the disclosed dsRNA is at Ieast 17 bp in length. In furtheraspects, the disclosed dsRNA comprises at least 18, 19, 20, 21 , or more bp in length. In some aspects the disclosed dsRNA comprises at least 22, 23 or 24 bp in length. In certain aspects, minimum length of dsRNA preferably is at least about 80-100 bp in order to be efficiently taken up by certain insect pests.

[0087] Although the dsRNA contains a sequence which corresponds to the target region of the target gene, it is not absolutely essential forthe whole of the dsRNA to correspond to the sequence of the target region. For example, the dsRNA may contain short nontarget regions flanking the target-specific sequence, provided that such sequences do not affect performance of the dsRNA in RNA inhibition to a material extent.

[0088] In some aspects, the dsRNA may contain one or more substitute bases in order to optimize performance in RNAi. It will be apparentto the skilled reader howto vary each of the bases of the dsRNA in turn and test the activity of the resulting dsRNAs (e.g., in a suitable in vitro test system) in order to optimize the performance of a given dsRNA.

[0089] In some aspects, the dsRNA may further con tain DNA bases, non -natural bases or non-natural backbone linkages or modifications of the sugar-phosphate backbone, forexample to enhance stability during storage or enhance resistance to degradation by nucleases.

[0090] In some aspects, the double-stranded RNA may be fully (blunt-ended) or partially double-stranded. In some aspects, partially double-stranded RNAs may include short single-stranded overhangs at one or both ends of the double-stranded portion, provided that the RNA is still capable of being taken up by insects and directing RNAi. In some aspects, the double-stranded RNA may also contain internal non-complementary regions.

[0091] In some aspects, the disclosure encompasses simultaneous or sequential contacting of two or more different dsRNA to the same insect, so as to achieve down- regu lation or in h ibition of mu Itiple target genes or to ach ieve a more potent in h ibition of a single target gene. In other aspects, one dsRNA may target multiple target gene sequences. In some aspects, the dsRNA regions in the RNA construct may be complementary to the same or to different target genes and / or the dsRNA regions may be complementary to targets from the same or from different insect species.

[0092] In some aspects, the target is LdEG12 and the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0093] In some aspects, the target is LdEG53 and the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0094] In some aspects, the target is LdEG81 and the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about85%, about90%, about95%, about98%) identityto SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0095] In some aspects, siRNAs disclosed herein have a length in the range of from 19 to 25 base pairs, or from 20 to 24 base pairs. In some aspects, siRNAsmay havea length of 19, 20, 21 , 22, 23, 24 or 25 base pairs, and in some aspects 21 or 22 base pairs, corresponding to the target gene to be down -regulated.

[0096] In some aspects, siRNAs may include single-stranded overhangs at one or both ends, flanking the double-stranded portion. In some aspects, the siRNA may contain s' overhanging nucleotides, preferably two 3' overhanging thymidines (dTdT) or uridines (ULI). In some aspects, although a 3' TT or ULI overhang may also be included at the other end of the siRNA it is not essential for the target sequence downstream of the sequence included in double-stranded part of the siRNA to have AA. In such aspects, siRNAs which are RNA / DNA chimeras are also contemplated. These chimeras include, for example, the siRNAs comprising a double-stranded RNA with 3' overhangs of DNA bases (e.g., dTdT), and also double-stranded RNAs which are polynucleotides in which one or more of the RNA bases or ribonucleotides, or even all of the ribonucleotides on an entire strand, are replaced with DNA bases or deoxynucleotides.

[0097] In some aspects, the target is LdEG12 and the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0098] In some aspects, the target is LdEG53 and the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0099] In some aspects, the target is LdEG81 and the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about95%, about98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0100] The miRNA precursor is generally transcribed as a single strand of RNA. This single strand of RNA includes at least one stem-loop that can be regarded as equivalent to a naturally occurring pre-miRNA, in that it is processed to a mature miRNA. The stemloop is formed when the single strand folds back on itself and sufficient base pairing occurs to stabilize the resulting folded structure. The stem-loop includes a stem region and a loop region, all within the same single strand of RNA. The stem region includes a first segment and a second segment, which are joined through the loop region. The first segment includes at least 19 contiguous nucleotides for silencing a messenger RNAencoding the target gene. The second segment contains at least 19 contiguous nucleotides. The first segment and second segment are generally of similar length (in terms of number of contiguous nucleotides making up each segment) but are not necessarily of identical length. The loop region is located on the single strand between the first and second segments. The first and second segments hybridize to form partially double stranded RNA, wherein at least one nucleotide of the first segment is unpaired; that is, within the partially double stranded RNA, there is at least one nucleotide of the first segment that is mismatched to the corresponding position on the second segment. The mismatch results in a bulge or loop or kink in the otherwise substantially doublestranded stem region. The mismatch can be due to, for example, at least one nucleotide of the second segment that does not base-pair to the nucleotide in question of the first segment, or at least one extra or at least one missing nucleotide on the second segment at the position corresponding to the nucleotide in question of the first segment.

[0101] In some aspects, the first segment of the stem region includes at least 19 contiguous nucleotides for silencing a messenger RNA encoding the target gene. In some aspects, the nucleotide sequence of the stem region's first segment's at least 19 contiguous nucleotides is selected so that the mature miRNA processed from the stemloop is perfectly complementary to the target mRNA at nucleotide positions 2, 3, 4, 5, 6, and 7 (from the 5' end) of the mature miRNA. In some aspects, the nucleotide sequence of the stem region'sfirst segment's at least 19 contiguous nucleotides is designed so that the mature miRNA processed from the stem-loop structure has few or no G:ll wobble base pairs. The loop region of the stem-loop typically includes between about4 to about 40 nucleotides. In some preferred embodiments, the loop region includes consecutive nucleotides of a native loop sequence of the invertebrate miRNA precursor. In some embodiments, the loop region is identical to a native loop sequence of the invertebrate miRNA precursor.

[0102] In some aspects, the stem-loop is processed to a mature miRNA, typically of 21 , 22, 23, 24, 25, or 26 nucleotides in length. In other aspects, the stem-loop preferably remains relatively intact (that is, substantially uncleaved to smaller polynucleotides), but is processed to a mature miRNA (typically of 21 , 22, 23, 24, 25, or 26 nucleotides in length) in the gut or in or on a cell of an insect pest that ingests miRNA.

[0103] In some aspects, the single strand of RNA includes a single stem-loop that is processed to a mature miRNA. In other aspects, the single strand of RNA includes multiple stem-loops that are processed to mature miRNAs. Where multiple stem-loopsare present, they can consist of multiples of the same stem-loop, or multiple different stem-loops. In some aspects, the single strand of RNA includes multiple stem-loops that correspond to a group of invertebrate miRNAs that are natively transcribed in a single polycistronic transcription unit.

[0104] In some aspects, the target is LdEG12 and the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence th at comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0105] In some aspects, the target is LdEG53 and the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence th at comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0106] In some aspects, the target is LdEG81 and the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence th at comprises at least about 80% (e.g., about 85%, about 90%, about95%, about98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0107] In some aspects, hpRNA comprises an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem. The base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the target gene, and an antisense sequence that is fully or partially complementary to the sense sequence. In some aspects, the sense sequence and the antisense sequence are generally of similar lengths but may differ in length. In some aspects, these sequences may be portions or fragments of at least 10, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 70, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. In some aspects, the loop region may be at least 100, 200, 300, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length.

[0108] In some aspects, hpRNA can be formed when the sense and antisense region form a double stranded RNA region. The hpRNA may be classified as long hpRNA, having long, sense andantisenseregionswhich can be largely complementary, but neednot be entirely complementary (typically larger than about200 bp, ranging between 200- 1000 bp). hpRNA can also be rather small ranging in size from about 30 to about 42 bp, but not much longer than 94 bp.

[0109] In some aspects, the disclosed sense or antisense RNA can be about 30 nt, about 50 nt, about 100 nt, about200 nt, about300 nt, about 500 nt, about OO nt, about 2000 nt or even about 5000 nt or larger in length. The longer the sequence, the less stringent the requirement for the overall sequence identity. In some aspects, the length of the sense or antisense RNA is between 20 and 30 nucleotides. In some aspects, the length of the sense or antisense RNA is 21 , 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides. In some aspects, the length of the sense and antisense RNAs are the same or differ by up to 10% or 20%, not more.

[0110] In some aspects, a short hairpin RNA which may be optionally polyadenylated having a sense nucleotide sequence of 20-30 consecutive nucleotides at least 95% identical to the first region of 20-30 nucleotides of the target gene and an antisense nucleotide sequence of 20-30 consecutive nucleotides at least 95% identical to the complement of the first region of the target gene, the sense and antisense sequences being covalently joined by a spacer region of 3-20 nucleotides. The first and second regions of the target gene are preferably not the same, and more preferably nonoverlapping. Preferably, the short hairpin RNA is expressed from a chimeric gene having an RNA polymerase III (RNA Pol III) promoter and the miRNA molecule is transcribed from a chimericgene having an RNA Pol II promoter as a precursor RNA (pri-miRNA or pre-microRNA) which is subsequently processed in the cell.

[0111] In some aspects, the target is LdEG12 and the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0112] In some aspects, the target is LdEG53 and the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0113] In some aspects, the target is LdEG81 and the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0114] In some aspects, the pi-RNA may comprise about 26 to 30 nucleotides complementary to a target disclosed herein . In some aspects, the pi-RNA may comprise about 26, 27, 28, 29, or 30 nucleotides complementary to a target disclosed herein.

[0115] In some aspects, the recombinant RNA is provided in a microbial or plant cell that expresses the recombinant RNA, or in a microbial fermentation product, or is chemically synthesized.

[0116] In some aspects, dsRNA, siRNA, miRNA, shRNA, hpRNA, orpiRNAof the present disclosure may be produced chemically or enzymatically by one skilled in the art through manual or automated reactions, or in vivo in a cell comprising a nucleic acid molecule comprising a polynucleotide encoding the RNA, dsRNA, siRNA, miRNA, shRNA, hpRNA or piRNA molecule. RNA may also be produced by partial or total organic synthesis; any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. An RNA molecule may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3 RNA polymerase, 17 RNA polymerase, and SP6 RNA polymerase). Any expression constructs useful for the cloning and expression of polynucleotides, known in the art, can be used. RNA molecules that are synthesized chemically or by in vitro enzymatic synthesis may be purified prior to introduction into a cell. For example, RNA molecules can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof. Alternatively, RNA molecules that are synthesized chemically or by in vitro enzymatic synthesis may be used with no or a minimum of purification, for example, to avoid losses due to sample processing. The RNA molecules may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and / or stabilization of the duplex strands.

[0117] In some aspects, the disclosure also provides a DNA construct for introduction into a cell (for e.g., a bacterial cell, a fungus cell, or a plant cell), wherein the DNA construct comprises a polynucleotide that, upon expression to RNA and ingestion by an insect pest, achieves down regulation of a target gene in a cell, tissue, or organ of theinsect pest. In some aspects, further provided is a polynucleotide capable of being expressed as dsRNA, siRNA, miRNA, shRNA, hpRNA, or pi-RNA in a plantcell to down regulate target gene expression in an insect pest. In such aspects, a polynucleotide may comprise one or more regulatory elements, which may be operably linked to the polynucleotide capable of being expressed as an dsRNA, siRNA, miRNA, shRNA, hpRNA, or piRNA.

[0118] In some aspects, the disclosed recombinant RNA molecule or DNA construct comprising a polynucleotide for expressing the recombinant RNA molecule (for e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, orpi-RNA) is expressed by a prokaryotic, such as a bacterial, or eukaryotic, such as a fungus, host cell or host organism. In such aspects, any bacterium or yeast cell that is capable of expressing recombinant RNA molecule orDNA construct can be used. In some aspects, the bacterium includes Gramnegative and Gram-positive bacteria, such as, but not limited to, Escherichia spp. (e.g., E. coli), Bacillus spp. (e.g., B. thuringiensis), Rhizobium spp., Lactobacilli us spp., Lactococcus spp., Pseudomonas spp., Xenorhabdus spp., or Photorhabdus spp., etc. Non-limiting example of a fungus may include yeast (Saccharomyces cerevisiae, Pichia pastoris, and Hanseniaspora uvarum), etc.

[0119] In some aspects, contacting the insect pest can comprise the recombinant RNA provided in the diet of the insect in the form of a plant, bacterial orfungal cell containing the recombinant RNA, or as a fermentation product (e. g., a hairpin form of a dsRNA, produced in a bacterium, or fungal cell). In such aspects, the recombinant RNA is expressed in a bacterial, fungal, or plantcell andthe bacterial, fungal orplantcell is taken up or eaten by the insect pest. In some aspects, recombinant RNA producing bacteria or fungal cells can be sprayed directly onto the crops.

[0120] In some aspects, provided herein is a recombinant DNA construct comprising a heterologous promoter which is functional for expression of the RNA transcript in a bacterium or a fungus. In some aspects, the recombinant DNA construct is to be expressed in a bacterium, the bacterium selected from the group consisting of Escherichia coli, Bacillus species,Rhizobium spp., Lactobacilllus spp., Lactococcus spp., Pseudomonas spp., Xenorhabdus spp., or Photorhabdus spp., etc. In some aspects, the recombinant DNA construct is to be expressed in a fungus, the fungus is a yeast species selected from thegroup consisting of Saccharomyces cerevisiae, Pichia pastoris, and Hanseniaspora u varum.

[0121] In some aspects, the heterologous promoter is functional for expression of the RNA transcript in a bacterium comprises promoters inducible through use of an appropriate chemical, such as IPTG, a constitutive promoter, a bacteriophage promoter, such as a 17, T3, SV40 or SP6 promoter, promoters from an RNA Pol I, an RNA Pol II or an RNA Pol III polymerase, tac, trc and lac promoters, [3-lactamase promoter, E. coli phage PL and PR promoters, and . co / / galactose promoter, arabinose promoter and alkaline phosphatase promoter.

[0122] Suitable promoters for expression in yeast are also well known and include, for example, the bacteriophage T7 promoter, promoters from GAL1 (which is induced by the presence of galactose), ADH1 , the TEF1 promoter and the AOX promoter (a methanol inducible promoter) and the like. Many yeast cloning vectors have been designed and are readily available. Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant products polypeptides are also well known. Transformed cells are selected by phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).

[0123] In some aspects, the disclosure further encompasses generation of recombinant RNA molecules (for e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, or pi-RNA). In such aspects, the recombinant RNA molecule is introduced (e.g., by transformation, transfection, or injection) using an isolated nucleic acid or a recombinant (DNA) construct under conditions that allow transcription of said nucleic acid or recombinant (DNA) construct to produce the recombinant RNA.

[0124] In some aspects, one or more transcription termination sequences may also be incorporated in the recombinantconstruct disclosed herein. The transcription termination sequence may encompass a control sequence at the end of a transcriptional unit, which signals 3' processing and poly-adenylation of a primary transcript and termination of transcription. Additional regulatory elements, such as transcriptional or translational enhancers, may be incorporated in the recombinant expression construct.

[0125] In further aspects, the DNA construct may further include an origin of replication which isrequired formaintenanceand / orreplication in a specific cell type. For e.g., when an expression construct is required to be maintained in a bacterial cell as an episomal geneticelement (e.g., plasmid or cosmid molecule) in a cell. In some aspects, the origins of replication include, but are not limited to, f 1 -ori and colE1 ori.

[0126] In some aspects, the DNA con struct may optionally comprise a selectable marker gene. The selectable marker gene may include any gene, which confers a phenotype on a cell in which it is expressed to facilitate the identification and / orselection of cells, which are transfected or transformed. Non-limiting examples of selectable markers include resistance genes against ampicillin (Ampr), tetracycline (Ter), kanamycin (Kanr), phosph i nothricin, and chloramphenicol (CAT) gene. Other suitable marker genes provide a metabolic trait, for example manA. Visual marker genes may also be used and include forexample beta-glucuronidase(GUS), luciferase and Green Fluorescent Protein (GFP).

[0127] In some aspects, a virus which specifically infects the insect pest (may be used to deliver the recombinant RNA. In some aspects, non-limiting examples of viruses can include baculovirus (fore.g., Autographa californica multiple nuclear polyhedrosis virus), ntomopoxviruses (EPV) (for e.g., Amsacta moorei EPV), densoviruses (for e.g., Aedes aegypti densovirus), nudiviruses, and nodaviruses (fore.g., black beetle virus). Additional viruses of use in the present invention include viruses that infect a broad range of arthropods, including white spot shrimp virus and baculoviruses of marine shrimp, PmSNPV and PvSNPV.

[0128] In some aspects, the virus is modified using a viral transfer plasmid comprising a DNA construct encoding the recombinant RNA molecule and one or more of an enhancer, a promoter, ora transcriptional terminator. In some aspects, the DNA construct is inserted between the two promoter / terminator / enhancer complexes to produce a complete expression unit capable of directing synthesis of the recombinant RNA. The modified transfer plasmid is then utilized to introduce the expression unit into the virus genome.

[0129] In some aspects, the bacterial cell, yeast cell or virus can be inactivated before being applied on a host plant or being contacted by insect pest. Inactivation may be achieved by any means, such as by heat treatment, phenol, or formaldehyde treatment, or by mechanical treatment.

[0130] In further aspects, the disclosure comprises the steps of introducing (e.g., by transformation, or transfection) an isolated nucleic acid or a DNA construct into a bacterial, fungal, orviral cell of the disclosure under conditions thatallow transcription of said nucleic acid or DNA construct to produce the recombinant RNA molecu le. Any well know method of transformation or transfection in the art can be used for introduction of the isolated nucleic acid or a DNA construct into a bacterial, fungal, or viral cell.

[0131] In further aspects, the disclosure encompasses transformation of particular host targets (for e.g., plants) and a recombinant DNA construct for use in achieving stable transformation of target host (for e.g., Solanaceous plant). Transformed target host may express effective levels of recombinant RNA molecule (for e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, or pi-RNA) from the recombinant DNA molecules. In some aspects, further disclosed herein is a plant transformation vector comprising at least one polynucleotide operably linked to a heterologous promoter functional in a plant cell, wherein expression of the polynucleotide(s) results in a recombinant RNA molecule (for e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, pi-RNA).

[0132] In some aspects, the polynucleotide includes one or more elements selected from: (a) a promoter functional in a plant cell; (b) a transgene transcription unit; (c) a gene suppression element; and (d) a transcription regulatory / tran script stabilizing element. Promoters may be any that have promoter activity in a plant cell. In some aspects, promoters can include a constitutive promoter, a spatially specific promoter, a temporally specific promoter, a developmentally specific promoter, and an inducible promoter. Nonlimiting examples of promoters can include Spatially specific promoters such as organelle- cell-, tissue-, or organ-specific promoters (e.g., a plastid-specific, a rootspecific, a pollen -specific, or a seed-specific promoter for suppressing expression of the first target RNA in plastids, roots, pollen, or seeds, respectively), temporally specific promoters which 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 which 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 nutrientorsalt levels, high or low lightlevels, or pest or pathogen infection), an expression-specificpromoterwhich can 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”. Methods for stable transformation of recombinant DNA molecules in plants are well known and may be used to express the disclosed polynucleotide.

[0133] In some aspects, the promoter element can include nucleic acid sequences that are not naturally occurring promoters or promoter elements or homologues thereof but that can regulate expression of a gene. Examples of such regulatory sequences include naturally occurring or artificially designed RNA sequences that include a ligand-bindingregion or aptamer and a regulatory region (which can be d -acting). In some aspects, the promoter can include riboregulators 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 sufficientforthe riboregulatorto begin transcription of the DNA that encodes a silencing element for suppressing a target gene.

[0134] In some aspects, the recombinant DNA constructor polynucleotide includes DNA encoding one or more site-specific recombinase recognition sites. In one aspect, 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 aspect, the recombinant DNAconstruct includes DNAencoding one loxP site; in the presence of Cre recombinase and another DNA with a loxP site, the two DNAs are recombined.

[0135] In further aspects, provided herein is a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising a sequence at least about 80% (for e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the RNA transcript comprises 100% identity to SEQ ID NO: 3.

[0136] In some aspects, provided herein is a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising a sequence at least about 80% (for e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the RNA transcript comprises 100% identity to SEQ ID NO: 5.

[0137] In some aspects, provided herein is a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising a sequence at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the RNA transcript comprises 100% identity to SEQ ID NO: 10.

[0138] In some aspects, the recombinant DNA construct comprises a heterologous promoter is functional for expression of the RNA transcript in a bacterium. In some aspects, the recombinantDNA construct comprises a heterologous promoter is functional in a plantcell. Further provided herein is a recombinant vector comprising a recombinant DNA con struct and a plant chromosome or plastid comprising the disclosed recombinant DNA construct.

[0139] The disclosure further encompasses a transgenic plant cell having in its genome the disclosed recombinant DNA construct and a crop product, transgenic progeny seed, or propagatable plant part produced from the disclosed transgenic plant.

[0140] In some aspects, the recombinant DNA construct is expressed in the plant by means of transgenic expression or transient expression. Transgenic plant cells and plantscan be made by methods known in theart. In some aspects, the recombinantDNA constructs disclosed herein can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage. Methods for transient expression can include synthesizing the recombinant RNA in vitro, and applying the recombinant RNA to foliage by spraying, or to roots by soaking, or abrading the surface of leaves and applying a solution comprising bacterial cell expressing recombinant RNA, resulting in a transient presence of recombinant RNA within the plant tissue.

[0141] In some aspects, recombinant RNA molecule (for e.g., dsRNA, siRNA, miRNA, shRNA, hpRNA, pi-RNA) is produced by microbial fermentation. In such aspects, the disclosed recombinant DNA molecules comprises a polynucleotide express recombinant RNA molecule, is expressed in a bacterium, such as E. coli; and the resulting RNA produced in the bacterium is purified as a fermentation product.

[0142] In some aspects, the disclosed recombinant RNA contact or ingestion of which results in the down-regulation of LdEG12 expression in the insect pest by at least 30%, 40%, 50%, 60%, preferably 70%, 80% or even more preferably 90% or 95% when compared with gene expression in control insects not contacted with the recombinant RNA. In some aspects, the disclosed recombinant RNA contact or ingestion of which results in the down-regulation of LdEG53 expression in the insect pest by at least 30%, 40%, 50%, 60%, preferably 70%, 80% or even more preferably 90% or 95% when compared with gene expression in control insects not contacted with the recombinant RNA. In some aspects, the disclosed recombinant RNA contact or ingestion of which results in the down-regulation of LdEG81 expression in the insect pest by at least 30%, 40%, 50%, 60%, preferably 70%, 80% or even more preferably 90% or 95% whencompared with gene expression in control insects not contacted with the recombinant RNA. Expression of the genes can be measured by measurement of mRNA or protein expression using molecular techniques such as RNA solution hybridization, PCR, RT- qPCR, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, or fluorescence-activated cell analysis (FACS).B. Insecticidal Compositions

[0143] The present disclosure further encompasses insecticidal composition comprising the recombinantRNA molecule disclosed in Section B. In some aspects, the insecticidal compositions comprise a recombinant RNA which can down regulate expression of a target gene. In some aspects, the target gene is LdEG12, LdEG53, LdEG81 , or any combination thereof. In some aspects, the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID Nos: 1 , 4, 6 and 8.

[0144] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecu le comprising a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about95% to about 100% complementarity to a portion of a target gene of an insect that infests a plant, the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID Nos: 1 , 4, 6 and 8.

[0145] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about95% to about 100% complementarity to a portion of a target gene of an insect that infests a plant, the target gene encodes a mRNA sequence of SEQ ID No: 1. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about95%, about98%) identityto the sequence of SEQ ID No: 1. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 1 .

[0146] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about95% to about 100% complementarity to a portion of a target gene of an insect that infests a plant, the target gene encodes a mRNA sequence of SEQ ID No: 4. In some aspects, the target geneencodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about95%, about98%) identityto the sequence of SEQ ID No: 4. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 4.

[0147] In some aspects, the insecticidal composition comprises an in secticidally effective amount of a recombinant RNA molecule comprising a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about95% to about 100% complementarity to a portion of a target gene of an insect that infests a plant, the target gene encodes a mRNA sequence of SEQ ID No: 6. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about95%, about98%) identityto the sequence of SEQ ID No: 6. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 6.

[0148] In some aspects, the insecticidal composition comprises an in secticidally effective amount of a recombinant RNA molecu le comprising a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene of an insect that infests a plant, the target gene encodes a mRNA sequence of SEQ ID No: 8. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about95%, about98%) identityto the sequence of SEQ ID No: 8. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 8.

[0149] In certain aspects, the insecticidal composition comprises an in secticidally effective amount of a recombinant RNA molecule comprising 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: 3, 5, and 10.

[0150] In some aspects, the target gene is LdEG12, and the insecticidal composition comprises an insecticidally effective amountof a recombinant RNA molecule comprising at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 3. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 3. In further aspects, the recombinantRNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 3.

[0151] In some aspects, the target gene is LdEG53, and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 5. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 5. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 5.

[0152] In some aspects, the target gene is LdEG81 , and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 10. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 10. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 10.

[0153] In some aspects, the insecticidal composition comprises a recombinant RNA molecule comprising a dsRNA, miRNA, siRNA, hpRNA, and / orpi-RNA disclosed herein.

[0154] In some aspects, the target gene is LdEG12, and the insecticidal composition comprises an insecticidally effective amou nt of a recombinant RNA molecu le comprising a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA. In some aspects, the target gene is LdEG53, and the insecticidal composition comprises an insecticidally effective amountof a recombinant RNA molecule comprising a dsRNA, a miRNA, siRNA, a hpRNA, or a pi- RNA. In some aspects, the target gene is LdEG81 , and the insecticidal composition comprises an insecticidally effective amountof a recombinant RNA molecule comprising a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA.

[0155] In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 10.

[0156] In some aspects, the target is LdEG12 and the insecticidal composition comprises an insecticidal ly effective amount of a recombinant RNA molecule comprising a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0157] In some aspects, the target is LdEG53 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0158] In some aspects, the target is LdEG81 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0159] In some aspects, the target is LdEG12 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0160] In some aspects, the target is LdEG53 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0161] In some aspects, the target is LdEG81 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a siRNAcomprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0162] In some aspects, the target is LdEG12 and the insecticidal composition comprises an insecticidal ly effective amount of a recombinant RNA molecule comprising a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0163] In some aspects, the target is LdEG53 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0164] In some aspects, the target is LdEG81 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0165] In some aspects, the target is LdEG12 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a hpRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0166] In some aspects, the target is LdEG53 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a hpRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In someaspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0167] In some aspects, the target is LdEG81 and the insecticidal composition comprises an insecticidal ly effective amount of a recombinant RNA molecule comprising a hpRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0168] In some aspects, the target is LdEG12 and the insecticidal composition comprises an insecticidal ly effective amount of a recombinant RNA molecule comprising a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0169] In some aspects, the target is LdEG53 and the insecticidal composition comprises an insecticidally effective amountof a recombinant RNA molecule comprising a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0170] In some aspects, the target is LdEG81 and the insecticidal composition comprises an insecticidally effective amount of a recombinant RNA molecule comprising a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0171] The insecticidal composition disclosed herein can comprise at least one of a recombinant RNA molecule (e.g., a dsRNA, a miRNA, siRNA, a hpRNA, ora pi-RNA), at least DNA molecule comprising a polynucleotide for expression of the recombinant RNAmolecule, and / or at least one host cell (e.g., a bacterial, a yeast, a viral, or a plant cell) expressing a disclosed recombinant RNA molecule.

[0172] In certain aspects, the insecticidal composition further comprises a carrier, excipient, diluent, 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, or any combination thereof. In further aspects, insecticidal composition comprises suspension agents, agglomeration agents, bases, buffers, preservatives, propellants, thixotropic agents, and / or anti -freezing agents. In some aspects, the insecticidal composition may contain further components which serve to stabilize the recombinant RNA molecule and / or prevent degradation of the recombinant RNA molecule during prolonged storage of the insecticidal composition. In some aspects, the insecticidal composition may still further contain components which serve to preserve the viability of the target host cell during prolonged storage.

[0173] Non-limiting examples of solid carriers, or an excipient can include fillers such as kaolin, bentonite, dolomite, calcium carbonate, talc, powdered magnesia, Fuller's earth, wax, gypsum, diatomaceous earth, rubber, plastic, China clay, mineral earths such as silicas, silica gels, silicates, attaclay, limestone, chalk, loess, clay, dolomite, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials, fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, thiourea and urea, products of vegetable origin such as cereal meals, tree bark meal, wood meal and nutshell meal, cellulose powders, attapulgites, montmorillonites, mica, vermiculites, synthetic silicas and synthetic calcium silicates, or compositions of these. Examples of liquid carriers, or a excipient include but are not limited to water; alcohols, such as ethanol, butanol orglycol, as well as theirethers or esters, such as methylglycol acetate; ketones, such as acetone, cyclohexanone, methylethyl ketone, methylisobutylketone, or isophorone; alkanes such as hexane, pentane, or heptanes; aromatic hydrocarbons, such as xylenes or alkyl naphthalenes; mineral or vegetable oils; aliphatic chlorinated hydrocarbons, such as trichloroethane or methylene chloride; aromatic chlorinated hydrocarbons, such as chlorobenzenes; water-soluble or strongly polar solvents such as dimethylformamide, dimethyl sulfoxide, or N- methylpyrrolidone; liquefied gases; waxes, such as beeswax, lanolin, shellac wax, carnauba wax, fruit wax (such as bayberry or sugar cane wax) candelilla wax, other waxes such as microcrystalline, ozocerite, ceresin, or montan; salts such as monoethanolamine salt, sodium sulfate, potassium sulfate, sodium, chloride, potassium chloride, sodium acetate, ammonium hydrogensulfate, ammonium chloride, ammonium acetate, ammonium formate, ammonium oxalate, ammonium carbonate, ammonium hydrogen carbonate, ammonium thiosulfate, ammonium hydrogen diphosphate, ammonium dihydrogen monophosphate, ammonium sodium hydrogen phosphate, ammonium thiocyanate, ammonium sulfamate or ammonium carbamate, and mixtures thereof.

[0174] In some aspects, a solid diluent may include, but not limited to clay, such as diatomaceous earth, corn husk, tricalcium phosphate, cork powder, kaolin, betonies, or attapulgite etc., and a water soluble polymer. In some aspects, liquid diluents include, but not limited to water, organic diluents, water soluble polymer, or any combination thereof. In addition, the diluents may include, for example, ionic or non -ionic wetting agents, dispersion agents, or emulsifiers, or their mixtures.

[0175] Examples of useful surfactants include but not limited to sodium or lithium salts of fatty acids (such as tallow, tallow amines or phospholipids) and organosilicone surfactants. Other useful surfactant include organosilicone surfactant including nonionic organosilicone surfactant such as for e.g., Tween-20.

[0176] In some aspects, the composition may still further contain components which enhance or promote uptake of a recombinant RNA molecule by the insect pest. These may include, for example, chemical agents which generally promote the uptake of RNA into cells (e.g., lipofectamine).

[0177] In some aspects, the organosilicone is an organic silicone surfactant that contains trisiloxanes or modification trisiloxanes structure, comprising single organosilicon, ortwo kinds and above organic silicone compound or organosilicone and other auxiliary agent as a composite. In some aspects, the said auxiliary agent comprises in APES (OP-9-15), NPE phosphate (NPEP04), APES sulfosuccinate disodium (2020), EO-PO block type surfactant (EPE), castor oil polyoxyethylene ether, styrene phenol formaldehyde resin APEO, diphenyl biphenyl aldehyde APEO, cinnamicaldehydes phenol APEO (603#), the AEO (A-105) or a combination thereof.

[0178] In some aspects, the polynucleotide herbicidal molecule for e.g., comprises a herbicide tolerance gene non-limiting examples of which include, a gene providing tolerance to glyphosate, a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an acetohydroxyacid synthase, an acetolactate synthase (ALS), an acetyl-coenzyme A carboxylase (ACCase), a dihydropteroate synthase, a phytoene desaturase (PDS), a protoporphyrin IX oxygenase (PPO), a hydroxyphenylpyruvate dioxygenase (HPPD), a para-aminobenzoate synthase, a glutamine synthase (GS), a glufosinate-tolerantglutamine synthase, a 1-deoxy-D-xylulose 5-phosphate (DOXP) synthase, a dihydropteroate (DHP) synthase, a phenylalanineammonia lyase (PAL), a glutathioneS- transferase (GST), a D1 protein of photosystem II, a mono-oxygenase, a cytochrome P450, a cellulose synthase, a beta-tubulin, or a serine hydroxymethyltransferase.

[0179] Non-limiting examples of herbicides include glyphosate, dicamba, phosphinothricin, glufosinate, 2,2-dichloropropionic acid (Dalapon), acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide, bromoxynil, cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop), sulfonamide herbicides, triazine herbicides, 5- methyltryptophan, aminoethyl cysteine, pyridazinone herbicides such as norflurazon; hydroxyphenylpyruvate dioxygenase, cyclopropylisoxazole herbicides such as isoxaflutole, protoporphyrinogen oxidase inhibitors, an herbicide containing an aryloxyalkanoate moiety, phenoxy auxins (such as 2,4-D and dichlorprop), pyridyloxy auxins (such as fluroxypyr and triclopyr), aryloxyphen oxypropionates (AOPP) acetylcoenzyme A carboxylase (ACCase) inhibitors (such as haloxyfop, quizalofop, and diclofop), and 5-substituted phen oxyacetate protoporphyrinogen oxidase IX inhibitors (such as pyraflufen and flumiclorac), or any combination thereof.

[0180] In some aspects, the insecticidal composition may further comprise a pesticidal agent. In some aspects thepesticidal agent is a patatin, a plant lectin, a phytoecdysteroid, an insecticidal protein, or any combination thereof. In some aspects, the pesticidal agent is a chemical (organic) insecticide.

[0181] In some aspects, the insecticidal protein is a Bacillus f / wr / ng / ens / s insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacilluslaterosporous insecticidal protein, a Bacillus sphearicus insecticidal protein, or a combination thereof. In some aspects, insecticidal protein is a Cry1 , a Cry3, a TIC851 , a CryET170, a Cry22, a binary insecticidal protein CryET33 and CryET34, a binary insecticidal protein CryET80 and CryET76, a binary insecticidal protein TIC100 and TIC101 , or a binary insecticidal protein PS149B1.

[0182] In some aspects, a chemical insecticide include one or more of pyrethoroid or organophosphorus insecticides, including but are not limited to, cyfluthrin, permethrin, cypermethrin, bifinthrm, fenvaierate, flucythrinate, azinphosmethyl, methyl parathion, buprofezin, pyriproxyfen, flonicamid, acetamiprid, dmotefuran, clothianidin, acephate, malathion, quinolphos, chloropyriphos, profenophos, bendiocarb, bifenth rin,chiorpyrifos, cyfluthrin, diazinon, pyrethrum, fen propath rin, kinoprene, insecticidal soap or oil,neonicotinoids, diamides, avermectin and derivatives, spinosad and derivatives, azadirachtin, pyridaiyi, or any combination thereof. In some aspects, a chemical insecticide is a insecticide that target the nerve and muscle. Examples include acetylcholinesterase (AChE) inhibitors, such as carbamates (e.g., methomyl and thiodicarb) and organophosphates (e.g., ehiorpyrifos) GABA-gated chloride channel antagonists, such as cyclodiene organ ochiorines (e.g., endosuifan) and phenylpyrazoles (e.g., fipronii), sodium channel modulators, such as pyrethrins and pyrethroids (e.g., cypermethriii and A-cylialothrin), nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids (e.g., acetamiprid, tiacloprid, tliiamethoxam), nicotinic acetylcholine receptor (nAChR) allosteric modulators, such as spinosyns (e.g., spinose and spinetoram), chloride channel activators, such as avermectins and milbemycins (e.g., abamectin, emamectin benzoate), Nicotinic acetylcholine receptor (nAChR) blockers, such as bensuitap and cartap, voltage dependent sodium, channel blockers, such as indoxacarb and metaffumizone, ryanodine receptor modulator, such as diamides (e.g. dhlorantraniliproleandflubendiamide). In anotheraspects, the insecticide is a insecticide that target respiration. Examples include chemicals that uncouple oxidative phosphorylation via disruption of the proton gradient, such as chlorfenapyr, and mitochondrial complex I electron transport inhibitors. In some aspects, the chemical insecticide isa insecticide th at target growth and development. Examples includejuvenile hormone mimics, such as juvenile hormone analogues (e.g., fenoxycarb), inhibitors of chitin biosynthesis. Type 0, such as benzoylureas (e.g., flufenoxuron, lufenuron, and novaluron), and ecdysone receptor agonists, such as diacylhydrazines (e.g., methoxyfenozide and tebufenozide).

[0183] In some aspects, the insecticide is oneor more biological insecticide known to one skilled in the art. Examples of the biological insecticide include, but are not limited to, azadirachtin (neem oil), toxins from, natural pyrethrins. Bacillus thuringiencis and Beauvena bassiana, viruses (e.g., CYD-X™, CYD-X HP™, Germstar™, Madex HP™ and Spod-X™), peptides (Spear-T™, Spear-P™, and Spear-C™).

[0184] In some aspects, a safener is a compound for reducing phytotoxic actions of agrochemicals. In some aspects, non-limiting examples of a safener include (D-4) cloquintocet-mexyl, (D-5) cumyluron, (D-9) dymron, (D-11 ) dimepiperate, (D-14) fenclorim, (D-15) fenchlorazol-ethyl, (D-18) furilazole, (D-19) isoxadifen-ethyl, (D-23) mefenpyr-diethyl, (D-62) N-{[4-(cyclopropylcarbamoyl)phenyl]sulfonyl}-2-methoxybenzamide(cyprosulfamide) and (D-63) N-{[4-(cyclopropylcarbamoyl)phenyl]sulfonyl}-2-methoxy-5-methylbenzamide.

[0185] In some aspects, a phytoecdysteroid comprises for e.g., compound including triterpenoids, triterpene saponins, phytosterols, or any combination thereof.

[0186] In some aspects, the insecticidal composition may be in any suitable physical form for application to pest insect. The insecticidal composition may be in solid form such as a powder, pellet or a bait, liquid form such as a spray, gel form, or aerosol form. In some aspects, the insecticidal composition is in a form suitable for ingestion by an insect pest. In some aspects, the insecticidal composition is in a physical form selected from solid, a liquid, a powder, a suspension, an emulsion, a spray, an encapsulation, microbeads, carrier particulates, a film, a solid matrix, a soil drench, an insect diet, insect bait, and a seed treatment. In further aspects, the insecticidal composition may comprise a nanoparticle comprising a polymer matrix and a recombinant RNA molecule, DNA molecule comprising a polynucleotide for expression of the recombinant RNA molecule, and / or at least one host cell (e.g., a bacterial, a yeast, or a plant cell) expressing a disclosed recombinant RNA molecule. In a specific aspect, the insecticidal composition is formulated for use as a spray, a feeding station, or a food trap.

[0187] The nature of the excipients and the physical form of the insecticidal composition may vary depending upon the nature of the material or substrate that it is desired to treat. For example, the insecticidal composition may be a liquidthat is brushed or sprayed onto or imprinted into the material or substrate to be treated, or a coating or powder that is applied to the material or substrate to be treated. In some aspects, the insecticidal composition is in the form of a coating on a suitable surface which adheres to and is eventually ingested by an insect pest which comes into contact with the coating.

[0188] Preparation of powder formulations may comprise, for e.g., grinding a surfaceactive agent with a natural clay or diatomaceous earth solid substance such as talc, kaolin, betonies, etc. Granularformulation can be prepared by spraying active ingredient (for e.g., a recombinant RNA molecule) to allow it to adsorb on inert substance particles or apply the active ingredienton the surface of a carrier (such as sand, kaolin, or inert substance granules) with aid of an adhesive agent (such as polyvinyl alcohol, sodium polyacrylate, or mineral oil). If used with a fertilizer, the recombinant RNA molecule may be prepared in granules as in the preparation of fertilizer granules.

[0189] Accordingto some aspects, the host plant is treated againstinsectpest infestation. The insecticidal composition is then internalized or eaten by the insect, from where itcanmediate RNAi, thus controlling the insect. The particles of insecticidal composition may be of suitable size such that they adhere to the host plant to be treated or to the insect pest, for example to the exoskeleton, of the insect pest and may be absorbed therefrom. The insecticidal composition may be formulated for administration to the host plant, or to any part of the host plant, in any suitable way. For example, the composition may be formulated for administration to the leaves, stem, roots, fruit, vegetables, grains and / or pulses of the host plant. In some aspects, the insecticidal composition is formulated for administration to the leaves of the plant.

[0190] In some aspects, the insecticidal composition is in the form of a bait. The bait is designed to lure the insect pest to come into contact with the insecticidal composition. Upon coming into contact therewith, the composition is then internalized by the insect pest, by ingestion for example and mediates RNAi to thus kill the insect. Said bait may further comprise a food substance. In some aspects, the insect foods or attractants include any type of insectfood, including various sugars, proteins, carbohydrates, yeast, fats, and / oroils. The bait may depend on the species being targeted. The bait may be in any suitable form, such as a solid, paste, pellet or powdered form. In some aspects, the baits may be provided in a suitable “housing” or “trap”. Such housings and traps are commercially available and existing traps may be adapted to include the disclosed insecticidal compositions.

[0191] In some aspects, an insecticidal attractant may also be used in the composition. The attractant may be a pheromone, such as a male orfemale pheromone. The attractant acts to lure the insect pest to the insecticidal composition (for e.g., bait), and may be targeted for a particular insect pest or may attract a whole range of insects. In some aspects, pheromones or pheromone blends comprises: methyl 2,6,10- trimethyltridecanoate, (Z)-a-bisabolene, trans- and cis-l ,2-epoxides of (Z)-a-bisabolene, (E)- nerolidol, n-nonadecane, (Z)-9-tetradecenyl acetate, (Z,E)-9,12-tetradecadienyl acetate, (Z)- 11 -hexadecenal, (Z)-9-hexadecenal, (Z)-l l-hexadecenyl acetate, 4- methoxycinnamaldehyde, or any combination thereof.

[0192] In some aspects, the effective amountof insecticidal composition may contain the disclosed recombinant RNA molecule (fore.g., a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA) at about 1 ng / pl to 500 pg / ml. In some aspects, less than about 100 pg / ml of recombinantRNAmoleculeperplantmay be administered, especially less than 50 pg / ml, more especially less than about40, 30, 20, 10 or 5 pg / ml, most especially less than about 1 pg / ml or about 0.5 pg / ml of recombinant RNA molecule per plant. In some aspects,when the composition is administered to the leaf of the plant, less than about 100 pg / ml of recombinant RNA molecule per leaf may be administered to the plant, less than about 50 pg / ml, less than about40, 30, 20, 10 or 5 pg / ml, less than about 1 pg / ml or about 0.5 pg / ml of recombinant RNA molecule per leaf is administered to the plant. In some aspects, the recombinant RNA molecule may be administered to the plant, at less than about 50 ng / ml, less than about 40, 30, 20, 10 or about 5 ng / ml, or less than about 1 ng / ml per plant or per leaf of the plant. In some aspects, the disclosed recombinant RNA molecule is administered to the plant at about 10 ng / ml.

[0193] In some aspects, contacting the insect pest with the disclosed insecticidal compositions comprise application of a composition including the recombinant RNA molecule (fore. g., a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA)to a surface of the insect pest or to a surface of the plant infested by the insect pest. n some aspects, contacting results in mortality (death) or stunting (growth stunting or decrease in or cessation of metamorphosis stage development) of the insect, thereby preventing or treating infestation of the plant by the insect. In some aspects, contacting results in inducement of a physiological or behavioral change in an insect (adult or larvae or nymphs) that results in a decreased ability of the insect to infest or damage a plant, for example, a decrease in reproductive capacity, or a decrease in or cessation of feeding behavior or movement. In some aspects, application of an effective amount of the recombinant RNA, to a crop plant improves yield (e. g., increased biomass, increased seed or fruit production, or increased oil, starch, sugar, or protein content) of that crop plant, in comparison to a crop plant not treated with the polynucleotide. In some aspects, a feeding assay is used to determine efficacy of a recombinant RNA molecule trigger in causing stunting or mortality in insects pest.

[0194] In some aspects, contacting the insect pest with the insecticidal composition comprising the recombinant RNA molecule (for e.g., a dsRNA, a miRNA, siRNA, a hpRNA, ora pi-RNA) described herein results mortality of the insect pest by at least about 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to mortality of the insect pest before administration of the composition, a control insect pest not receiving the composition, or insect pest contacted with a recombinant RNA comprising a non-insect pest target (for e.g., a white spot syndrome virus gene). In some aspects, contacting the insect pest with the insecticidal composition described herein results in mortality of the insect pest by at least 25%. The mortality of insect pests can be assessed using feeding assay described in examples.

[0195] In some aspects, the insecticidal composition is applied to a plant-infesting insect, wherein the insect is a Coleopteran insect. In some aspects, the insecticidal composition is applied to an insect pest, wherein the insect is an insect of Leptinotarsa spp. In some aspects the insect is a L. decemlineata (Colorado potato beetle), L. juncta (false potato beetle), or L. texana (Texan false potato beetle). In some aspects the insect is a L. decemlineata (Colorado potato beetle). In some aspects, the insect is an adult, or in a larval or a nymph stage.

[0196] In some aspects, the insecticidal composition is applied to a plant-infesting insect wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising at least about 80% (e.g., about 85%, about90%, about95%, about98%) identity to SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 1

[0197] In some aspects, the insecticidal composition is applied to a plant-infesting insect wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 4.

[0198] In some aspects, the insecticidal composition is applied to a plant-infesting insect wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 6.

[0199] In some aspects, the insecticidal composition is applied to a plant-infesting insect wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 8.

[0200] In some aspects, the insecticidal composition is applied to a plant-infesting insect wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 3. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 3.

[0201] In some aspects, the insecticidal composition is applied to a plant-infesting insect wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 5. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 5.

[0202] In some aspects, the insecticidal composition is applied to a plant-infesting insect wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 10. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 10.C. Method of Use

[0203] Further disclosed herein is a method for controlling an insect pest infestation. In some aspects, the method comprises the application of a composition comprising the recombinant RNA molecule (fore. g., a dsRNA, a miRNA, siRNA, a hpRNA, ora pi-RNA) to a surface of the insect or to a surface of the plant infested by the insect.

[0204] In some aspects, disclosed herein is a method for inducing mortality or inhibiting growth of an insect pest, comprising contacting the insect pest with a insecticidal composition disclosed herein. In further aspects, disclosed is a method for reducing insect pest growth on a planter for reducing insect pest infestation of a plant comprising applying to the plantan effective amount of an insecticidal composition disclosed herein. In some aspects, a method for improving yield of a plant, comprising applying to a plant an effective amount of insecticidal composition, is provided herein.

[0205] In some aspects, a method for controlling an infestation of a plant by a plantinfesting insect, comprises contacting the insect pest with a recombinant RNA disclosed herein. In such aspects, the recombinant RNA comprises at least one portion of 17 or more contiguous nucleotides having a sequence of about 95% to about 100% complementarity with a portion of a target gene of an insect that infests a plant. In some aspects, the target gene is LdEG12, LdEG53, LdEG81 , or any combination thereof. Insome aspects, the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID Nos: 1 , 4, 6 and 8.

[0206] In some aspects, the disclosed method comprises contacting the insect pest with a recombinant RNA molecule comprises a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene, wherein the target gene is LdEG12. In such aspects, the target gene encodes a mRNA sequence of SEQ ID No: 1. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequence of SEQ ID No: 1. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 1 .

[0207] In some aspects, the disclosed method comprises contacting the insect pest with a recombinant RNA molecule comprises a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene, wherein the target gene is LdEG53. In such aspects, the target gene encodes a mRNA sequence of SEQ ID No: 4, SEQ ID No: 6, or any combination thereof. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequence of SEQ ID No: 4. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 4. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequenceof SEQ ID No: 6. In some aspects, the target gene encodesa mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 6.

[0208] In some aspects, the disclosed method comprises contacting the insect pest with a recombinant RNA molecule comprises a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene, wherein the target gene is LdEG81. In such aspects, the target gene encodes a mRNA sequence of SEQ ID No: 8. In some aspects, the target gene encodes a mRNA sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to the sequence of SEQ ID No: 8. In some aspects, the target gene encodes a mRNA sequence that comprises about 100% identity to the sequence of SEQ ID No: 8.

[0209] In certain aspects, the disclosed method comprises contacting the insect pest with a 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: 3, 5, and 10.

[0210] In some aspects of the method, the target gene is LdEG12, and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 3. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 3. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 3.

[0211] In some aspects of the method, the target gene is LdEG53, and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 5. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 5. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 5.

[0212] In some aspects of the method, the target gene is LdEG81 , and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 95% to about 100% identity or complementarity with a sequence of SEQ ID NO: 10. In such aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a sequence of SEQ ID NO: 10. In further aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence that comprises at least about 100% identity or complementarity with a sequence of SEQ ID NO: 10.

[0213] In some aspects of the method, the recombinant RNA molecule is a dsRNA, a miRNA, a siRNA, a hpRNA, or a pi-RNA.

[0214] In some aspects, the target gene is LdEG12, and the recombinant RNA molecule is a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA. In some aspects, the target gene is LdEG53, and the recombinant RNA molecule is a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA. In some aspects, the target gene is LdEG81 , and the recombinant RNA molecule is a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA.

[0215] In some aspects, the disclosed method comprises contacting the insect pest with a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 10.

[0216] In some aspects, the target is LdEG12 and method comprises contacting the insect pest with a recombinant RNA molecule which is a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0217] In some aspects, the target is LdEG53 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0218] In some aspects, the target is LdEG81 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a dsRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a dsRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0219] In some aspects, the target is LdEG12 and method comprises contacting the insect pest with a recombinant RNA molecule which is a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0220] In some aspects, the target is LdEG53 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a miRNA comprising an RNAstrand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0221] In some aspects, the target is LdEG81 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a miRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a miRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0222] In some aspects, the target is LdEG12 and method comprises contacting the insect pest with a recombinant RNA molecule which is a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0223] In some aspects, the target is LdEG53 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0224] In some aspects, the target is LdEG81 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a siRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a siRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0225] In some aspects, the target is LdEG12 and method comprises contacting the insect pest with a recombinant RNA molecule which is a hpRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0226] In some aspects, the target is LdEG53 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a hpRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0227] In some aspects, the target is LdEG81 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a hpRNA comprising an RNA strand having a sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a hpRNA comprising an RNA strand having a sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0228] In some aspects, the target is LdEG12 and method comprises contacting the insect pest with a recombinant RNA molecule which is a pi-RNA comprising an RNA strand havinga complementary sequence th at comprises at least about80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 3. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 3.

[0229] In some aspects, the target is LdEG53 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a pi-RNA comprising an RNA strand havinga complementary sequence th at comprises at least about80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 5. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 5.

[0230] In some aspects, the target is LdEG81 and the method comprises contacting the insect pest with a recombinant RNA molecule which is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID NO: 10. In some aspects, the recombinant RNA molecule is a pi-RNA comprising an RNA strand having a complementary sequence that comprises at least about 100% identity to SEQ ID NO: 10.

[0231] In some aspects, the method comprises contacting a plant-infesting insect, wherein the insect is a Coleopteran insect. In some aspects, the method comprises contacting an insect pest, wherein the insect is an insect of Leptinotarsa spp.. In some aspects the insectis a L. decemlineata (Colorado potato beetle), Ljuncta (falsepotato beetle), or L texana (Texan false potato beetle). In some aspects the insect is a L decemlineata (Colorado potato beetle). In some aspects, the insect is an adult, or in a larval or a nymph stage.

[0232] In some aspects, the method comprises contacting an insecticidal composition to a plant-infesting insectwherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target geneencodes an mRNA sequence comprisingat least about80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID No: 1. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 1

[0233] In some aspects, the method comprises contacting an insecticidal composition to a plant-infesting insectwherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target geneencodes an mRNA sequence comprisingat least about80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID No: 4. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 4.

[0234] In some aspects, the method comprises contacting an insecticidal composition to a plant-infesting insectwherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target geneencodes an mRNA sequence comprisingat least about80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID No: 6. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 6.

[0235] In some aspects, the method comprises contacting an insecticidal composition to a plant-infesting insectwherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target geneencodes an mRNA sequence comprisingat least about80% (e.g., about 85%, about 90%, about 95%, about 98%) identity to SEQ ID No: 8. In some aspects, the target gene encodes an mRNA sequence comprising 100% identity to SEQ ID No: 8.

[0236] In some aspects, the method comprises contacting an insecticidal composition to a plant-infesting insectwherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 80% (e.g., about 85%, about90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 3. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 3.

[0237] In some aspects, the method comprises contacting an insecticidal composition to a plant-infesting insectwherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 80% (e.g., about 85%, about90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 5. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 5.

[0238] In some aspects, the method comprises contacting an insecticidal composition to a plant-infesting insectwherein the insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecule comprises at least one RNA strand having a sequence of about 80% (e.g., about 85%, about90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 10. In some aspects, the recombinant RNA molecule comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 10.

[0239] In some aspects, the method comprises contacting the insect pest with the disclosed insecticidal compositions comprising the recombinant RNA molecule (for e.g., a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA) or a host cell expressing the recombinant RNA (for e.g., a bacterial, fungal cell or a virus) to a surface of the insect pest or to a surface of the plant infested by the insect pest. In some aspects of the method, contacting results in mortality (death) or stunting (growth stunting or decrease in or cessation of metamorphosis stage development) of the insect, thereby preventing or treating infestation ofthe plantby the insect. In some aspects of the method, contacting results in inducementofa physiological or behavioral change in an insect (adult or larvae or nymphs) that results in a decreased ability of the insect to infest or damage a plant, for example, a decrease in reproductive capacity, or a decrease in or cessation of feeding behavior or movement. In some aspects, application of an effective amount of the recombinant RNA, to a crop plant improves yield (e. g., increased biomass, increased seed or fruit production, or increased oil, starch, sugar, or protein content) of that crop plant, in comparison to a crop plant not treated with the polynucleotide. In some aspects, a feeding assay is used to determine efficacy of a recombinant RNA molecule trigger in causing stunting or mortality in insects pest.

[0240] In some aspects, the method comprising contacting the insect pest with the insecticidal composition which comprises the recombinant RNA molecule (for e.g., a dsRNA, a miRNA, siRNA, a hpRNA, or a pi-RNA), results in mortality of the insect pestby at least about 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to mortality of the insect pest before administration of the composition or a control insect pests not receiving the composition. In some aspects, the method comprises contacting the insect pest with the insecticidal composition described herein resulting in mortality of the insect pest by at least 25%. The mortality of insect pests can be assessed using any known methods in the art, for e.g., a feeding assay as described in examples.

[0241] In another aspect, the methods disclosed herein may improve one or more plant attribute, such as productivity, biomass, yield, or growth. For example, the methods disclosed herein may increase one or more of productivity, biomass, yield, and / orgrowth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to productivity, biomass, yield, and / or growth of plants not receiving the insecticidal composition of the present disclosure.

[0242] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding an RNA molecule comprising a silencing element, the silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes a mRNA sequence SEQ ID NO: 1. In such aspects the ingestion of the RNA by the in sect results in mortality or stunting in the insect.

[0243] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding an RNA molecule comprising a silencing element, the silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes a mRNA sequence SEQ ID NO: 4. In such aspects the ingestion of the RNA by the insect results in mortality or stunting in the insect.

[0244] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding an RNA molecule comprising a silencing element, the silencing element comprising a nucleotide sequence substantially identical or complementary to a portionof a target gene sequence of the insect, wherein the target gene encodes a mRNA sequence SEQ ID NO: 6. In such aspects the ingestion of the RNA by the in sect results in mortality or stunting in the insect.

[0245] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding an RNA molecule comprising a silencing element, the silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes a mRNA sequence SEQ ID NO: 8. In such aspects the ingestion of the RNA by the in sect results in mortality or stunting in the insect.

[0246] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding dsRNA, the dsRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 3. In some aspects, the dsRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 3. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0247] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding dsRNA, the dsRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 5. In some aspects, the dsRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 5. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0248] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding dsRNA, the dsRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a ofSEQ ID NO: 10. In some aspects, the dsRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 10. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0249] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding siRNA, the siRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 3. In some aspects, the siRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 3. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0250] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding siRNA, the siRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 5. In some aspects, the siRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 5. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0251] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding siRNA, the siRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 10. In some aspects, the siRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 10. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0252] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequenceencoding miRNA, the miRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 3. In some aspects, the miRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 3. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0253] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding miRNA, the miRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 5. In some aspects, the miRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 5. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0254] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding miRNA, the miRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 10. In some aspects, the miRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 10. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0255] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding hpRNA, the hpRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 3. In some aspects, the hpRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 3. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0256] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding hpRNA, the hpRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 5. In some aspects, the hpRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 5. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0257] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding hpRNA, the hpRNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 10. In some aspects, the hpRNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 10. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0258] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding pi-RNA, the pi-RNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 3. In some aspects, the pi-RNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 3. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0259] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding pi-RNA, the pi-RNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 5. In some aspects, the pi-RNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 5. In such aspectsthe ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0260] In some aspects, disclosed herein is a method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinant DNA construct comprising a nucleotide sequence encoding pi-RNA, the pi-RNA comprising a nucleotide sequence of about 80% (e.g., about 85%, about 90%, about 95%, about 98%) identity or complementarity with a of SEQ ID NO: 10. In some aspects, the pi-RNA comprises at least one RNA strand having a sequence of 100% identity or complementarity with a of SEQ ID NO: 10. In such aspects the ingestion of the RNA expressed by the plant, by the insect pest results in mortality or stunting in the insect.

[0261] In some aspects, the method of providing a plant having improved resistance to a plant-infesting insect comprises providing resistance to an insectpest, wherein the plantinfesting insect is an adult, or in a larval or a nymph stage. In some aspects, the method of providing a plant having improved resistance to a plant-infesting insect comprises providing resistance to an insect pest, wherein the plant-infesting insect is a Coleopteran insect. In such aspects, the plant-infesting insect is an insect from the genus Leptinotarsa. In some aspects, the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle).

[0262] In some aspects, the method of expressing the recombinant DNA construct in the plantcan be by means of transgen icexpression or transientexpression. Transgen icplant cells and plants can be made by methods known in the art. In some aspects, the recombinant DNA constructs disclosed herein can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage. Further provided is a plant produced by the disclosed having improved resistance to the insect, or a fruit, seed, or propagatable parts of the plant having improved resistance. In some aspects, also disclosed herein are commodity products produced from a transgen ic plant cell, plant, or seed expressing a recombinant DNA construct imparting improved resistance, 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 as disclosed herein.

[0263] In some aspects, the method of providing a plant having improved resistance to a plant-infesting insect comprises providing increased resistance to the insect pest by at least about 10%, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or above, as compared to a control plant that does not express the recombinant DNA construct. The resistance of the plants to the insect pests can be assessed using feeding assay described in examples.

[0264] In some aspects, thetransgenicplantexpresses a recombinantDNA constructfor resistance to an insect pest (for e.g., CPB) that is stacked with other recombinant DNA for imparting additional traits, the transgen icplanthas at least one additional altered trait, relative to a plant lacking the recombinant DNA construct, selected from the group of traits consisting of: improved abiotic stress tolerance; improved biotic stress tolerance; modified primary metabolite composition; modified secondary metabolite composition; modified trace element, carotenoid, or vitamin composition; improved yield; improved ability to use nitrogen, phosphate, or other nutrients; modified agronomic characteristics; modified growth or reproductive characteristics; and improved harvest, storage, or processing quality.

[0265] In some aspects, the methods disclosed herein can be further modified. As used herein, “modifying the method” can comprise modifyingorchanging oneormore features or aspects of one or more steps of a disclosed method. In an aspect, a method can be altered for e.g., by changing the amount of recombinant RNA molecule, used in a disclosed method, or by changing the frequency application of the insecticidal compositions in a disclosed method, by changing the duration of time wherein the insect pest is in contact with the insecticidal compositions, or by substituting for one or more of the disclosed components used in the compositions with a similar or equivalent component and / or reagent.D. Kits

[0266] Disclosed herein is a kit comprising at least one recombinant RNA molecule (for e.g., a dsRNA, a miRNA, siRNA, a hpRNA, ora pi-RNA), or recombinant DNA construct, or host cell, or composition as described herein fortreating insect infection in plants. The kit may be supplied with suitable instructions for use. The instructions may be printed on suitable packaging in which the other components are supplied or may be provided as a separate entity, which may be in the form of a sheet or leaflet for example. Theinstructions may be rolled or folded for example when in a stored state and may then be unrolled and unfolded to direct use of the remaining components of the kit.EXAMPLES

[0267] The Examples that follow are illustrative of specific aspects of the invention, and various uses thereof. They set forth for explanatory purposes only and are not to be taken as limiting the invention.Methods and Materials

[0268] Colorado potato beetle rearing condition: Kennebec seed potatoes were purchased from North shire seed farms (North Dakota, USA) and grown in a greenhouse at 25°C for 4-6 weeks. Adult beetles were reared on whole Kennebec potato plants in mesh Bugdorms (12x48 inches) with 75-100 adults per cage. These cages were maintained at 25-27°C with ~65-80% relative humidity. Approximately 100 new adults were reared from eggs and added to the colony every month depending on colony activity and egg laying abundance. For bioassays, eggs were collected from the adult cages, placed into 48 well insect rearing trays with 1 clutch per well, and the trays were kept in an incubator with a 16:8h L:D photoperiod, 25°C temperature and 75% RH. Newly hatched larvae were synchronized by date of emergence and transferred to fresh leaf diet until they reached second instar larvae. Bioassays were conducted using larvae in the early L2 stage, wh ich was confirmed by head capsu le width (width : 1 mm).

[0269] dsRNA production for bioassay: From the coding sequence of each gene, 300- 400 base pair (bp) region was used to synthesize dsRNA either by following a conventional in vitro transcription (IVT) method or RNAiSSANCE Ag’s proprietary technology for dsRNA production in E. coli using a fermentation protocol. The detailed fermentation protocol can be found in patent publication No. US 2022 / 0145294 A1 . For both approaches, the 300-400 bp DNA sequence was purchased from vendor (gBIocks Gene Fragments, Integrated DNATechnologies). For the IVT method, the DNA fragment was amplified in a PCR using primers that had T7 Promoter Sequence attached to either the primer or included in the gBIock gene fragment so that the PCR product had the T7 Promoter Sequence attached on both ends. This PCR product was used in an in vitro transcription (IVT) reaction using Megascript T7 transcription kit (ThermoFisher Scientific Catalog number: AMB13345) to synthesize dsRNA. The quantity of the dsRNA was measure in a Nanodrop (NanoDrop 2000 / 2000c Spectrophotometer, ThermoScientific™). Forquantification of the dsRNA produced by fermentation, an agarose gelbased method was used.

[0270] Screening of lethal genes using leaf-disk bioassay: Pesticidal efficacy of the CPB gene targets were assessed using a laboratory leaf disk bioassay protocol to evaluate how well orally delivered double-stranded RNA (dsRNA) performed the intended function of killing CPB larvae as measured by corrected insect mortality recorded over the assay period. For the screening bioassay, the target dsRNA treatment was prepared at a concentration of 10 ng / pL. When two sequencesfrom two genes were used in any assay, 10 ng / pL dsRNA from each gene were mixed as one treatment. 20 early L2 stage larvae were used in the bioassay following randomized complete block design. Each larva was placed in each cell of a white rearing 32-cell tray (RT32W, Frontier Agricultural Sciences), 4 insects constituted a pseudorep, and five such reps were used in one assay. Each replicate (4 insects) was placed on separate trays. One 50 mm diameter Whatman circularfilter paper (Cytiva, Cat No.1004-050) was placed in the cell and 300 pL distilled water was added to the filter paper to provide moisture so that the leaf disks did not dry out. Using a leaf cutter, 2 cm diameter circular leaf discs (area = 3.14 cm2) were cutfrom fully expanded excised leaves collected from 6-8-week- old potato plants (Solan urn tuberosum ‘Kennebec’). 24 pL dsRNA solution was applied to the leaf disks and the solution was spread using plastic or glass spreader. Tween-20 (0.1 %, v / v final concentration) was used as the surfactant. Once the leaf disks were completely dry, one disk was added to each cell, and one larva was placed inside the cell using a fine paint brush. Before infestation, the larvae were starved for five hours to encourage immediate feeding after they were placed inside the cells. Finally, the cells were sealed using clear sealing stickers (RTCV4, Frontier Agricultural Sciences). Bioassay trays were kept in a standard incubator / growth chamber at 25°C with a 16:8h L:D photoperiod and 60-65% humidity. The leaf disks were replaced with new dsRNA treated leaf disks on day 2 (after 48 hours) following the steps described above. The treated leaf disks were replaced with untreated leaf disk on day 4 (after 96 hours). The duration of the assays were about 8 days. On the last day of the assay, larval mortality was scored. Each assay was replicated twice.

[0271] Whole-plant bioassay: Bioassays were conducted with larvae in the early L2 stage. For whole plants bioassay, potato plants of 6-8 weeks old (var. Kennebec) were used. The bottom leaves of the plants were trimmed to get an appropriately sized plant. One plantwas used as one biological replicate, and each plant was sprayed with 10 mLE. coli encapsulated dsRNA using handheld spray bottle until the leaves and stems were dripping. The dsRNA concentration of the E coli cells was 52 ng / pL (52 ppm). The sprayed plants were let dry completely at room temperature. Following the starvation period of 5 hours, 10 early L2 stage CPB larvae were placed randomly on the plants. Total five replicates (five plants, 10 larvae on each plant) were used for the target gene, the same numberof plants / insectswere usedwithoutsprayingthe plantswith the dsRNA containing E. coli cells as untreated control. After infestation, the plants were placed inside one 12" x 20" circular butterfly cage (Restcloud, ASIN B07HMMGRR2). The total duration of the bioassay was 14 days, leaf consumption was measured on day 14 by visual inspection.

[0272] Data analysis: All data were analyzed in R. In order to detect differences in mean mortality among treatments, binomial models were created followed by a one-way ANOVA. Lethal Concentration values were created using the Survival package in R, where a probit model was created.Example 1: Pipeline for screening the target genes

[0273] Screening was undertaken in 95 genes selected from Colorado potato beetle (CPB) larvae using a screening bioassay (FIG. 1). The genes tested were selected from a broad range of essential gene function categories including but not limited to protein processing, export, and degradation, RNA synthesis, surveillance and turn -over, energy production, oxidative phosphorylation, and others. In the screening bioassay, each dsRNA was provided as 120 pL 10 ng / pL concentration on detached leaves / disk. Treatments that had two dsRNAs from two differentgenes included a mixture of 10 ng / pL of each of the dsRNAs. Appropriate concentrations of the non-target dsRNA were used as a control. Assays where the non-target dsRNA (negative control) treatment had greater than 20% mortality were discarded. FIG. 1 depicts mortality on the last day of the assay, as indicated, and shows combined results from several assays (negative control morality data not shown). From the screening bioassays, three targets LdEG12, LdEG53, LdEG81 , were chosen for further characterization using assays including whole plant bioassay, detached leaf / leaf disk bioassays, and lethal concentration assay (LC).Example 2: Characterization of biological activity of LdEG12

[0274] To characterize the biological activity of RNAi-mediated LDEG12 inhibition, CPB larvae (L2) were placed in 12-ounce cups with mesh lids for ventilation. Excised leaveswere preserved by placing stems of the leaves inside 0.5% agar microcentrifuge tubes. Th preserved excised leaves were placed into cups and were treated with 120 pl of 10 ppm (10 ng / pL) LdEG12 dsRNA or non-target Shrimp white spot syndrome virus gene VP28 dsRNA (SEQ ID NO: 11 ), as negative control. Five L2 developmental larvae were then placed into each cup. Each treatment had six biological replicates. Larvae were exposed to dsRNA treatment for 4 days after which untreated detached leaves were added to the cups. FIG. 2A shows the percent mortality of larvae. A one-way ANOVA was performed on the data observed on day 8 of the treatment. The analysis showed that LdEG12 had significantly higher mean larval mortality (F1 ,10 = 75.34, p < 0.001 indicated by ***) compared to negative control (FIG. 2A).

[0275] Further, a bioassay was conducted by placing CPB L2 larvae in 150 X 25 mm petri dishes. Excised leaves preserved by placing stems inside 0.5% agar microcentrifuge tubes were placed into the petri dishes. Leaves were treated with 120 pl of 10 ppm (10 ng / pL) dsRNA LdEG12 or non-target dsRNA, non-target dsRNA Shrimp white spot syndrome virusgeneVP28 dsRNA (SEQ ID NO: 11 ) as a negative control. Five L2 larvae were placed into each petri dish. Each treatment had six biological replicates. Larvae were exposed to dsRNA for 4 days after which, untreated detached leaves were added to the petri dishes. A one-way ANOVA was performed on the data observed on day 7, results of which showed th atLdEG12 hadsignificantlyhighermean larval mortality at day 7 (F1 ,10 = 375.6, p < 0.001 indicated by ***) when compared to negative control (FIG. 2B)

[0276] Bioassay was further performed using a 32-well tray. One CPB L2 larva was placed into each well of a 32-well bioassay tray using randomized block design where each block contained 4 larvae (pseudo-rep) in 4 wells; total 20 larvae were used for each treatment groups, therefore there were 5 true biological replicates for each treatment. Leaves were treated with 24 pl of 10 ppm (10 ng / pL) dsRNA LdEG12 or non-target dsRNA, as a negative control. Larvae were exposed to dsRNA for 4 days, and the experimentwas repeated 3 times. A one-way AN OVA was performed on the day 8 data, results of which showed that LdEG12 had significantly higher mean larval mortality (F1 ,4 = 80.81 , p < 0.001 indicated by ***) compared to negative control (FIG. 2C).

[0277] To understand if the insect mortality was in fact caused by gene silencing, bioassays were conducted following randomized block design in 32-well tray and CPB L2 larvae were exposed to 24 pL, 10 ppm (10 ng / pL) dsRNA LdEG12 or non-target dsRNA on leaves for 4 days. After 4 days, the alive larvae were collected for RNAextraction and cDNA synthesis. Total RNA was extracted using standard Trizol method and cDNA synthesis was conducted using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem™, Catalog number: 4368814). The cDNAs were used in quantitative PCR (qPCR) using Bullseye TaqProbe qPCR 2x Mastermix-Multiplex (MidSci Item# BEQPCR-PM) in a Bio-Rad CFX Opus 96 cycler. The relative gene expression of LdEG12 was calculated by comparing LdEG12 expression between the LdEG12 dsRNA or non-target dsRNA treatments and the expressions were normalized across the treatments using RPL18 and ARF1 as reference genes (primer and probe sequences of LDEG12 and RPL18 are provided in Table 4). t-test was run between control and the LdEG12 treatments. As shown in FIG. 2D, expression of LdEG12 was significantly reduced in dsRNA LdEG12 treated larvae.

[0278] Further, lethal concentration of LdEG12 dsRNA was determined. Twenty L2 developmental stage CPB larvae were placed into a 32 -well bioassay tray with one larva in each well.20 mm diameter leaf discs were treated with 0.001 ppm, 0.01 ppm, 0.1 ppm, 1 , ppm, 10 ppm, or 100 ppm of dsRNA LdEG12 per disc. Untreated leaf discs were used as controls. Larvae were exposed to dsRNA continuously for8 days by replacing the leaf disc every day with newly prepared dsRNA applied on the leaf discs. Results from the experiment is shown in T able 1. The LCso on day 8 was determined as 0.313 ppm (ng / pL) and the LC90 on day 8 was determined as 3.81 ppm. The LC were calculated using R package ecotox. The concentrations were Iog10 transformed and the estimates were fitted to 95% confidence interval using a probit model. Treatment mortality values were corrected to untreated control mortality using Handerson-Tifton formula. Mortality was defined as insect that failed to move with repeated nudging with paint brush.Table 1 : LC concentration of dsRNA LdEG12

[0279] Two additional subunits of the ATPase / synthase complex, the b subunit(LdEG70) and the d subunit (LdEG118) were also screened to assess if all ATPase / synthasesubunits are essential for CPB biology. Additionally, a gene, mitochondrial-processing peptidase subunitalpha (PMPC A) (LdEG102) that function in the mitochondria, was also compared to LdEG12 to see if any of these genes that are involved in mitochondrial functions, are essential similarto LdEG12. The PMPCA protein is not part of the oxidative phosphorylation pathway, however, it is located and function in the mitochondrial matrix by the cleaving off the leader peptides from the core or precursor proteins newly imported into the mitochondria. Most of the mitochondrial proteins are encoded by the nucleus, therefore, they must be translocated to the mitochondria. For proper localization, these proteins carry leader peptides to lead them to the mitochondria and upon arrival at the mitochondria, these leader peptides are cleaved of by the mitochondrial-processing peptidase for the proteins to be properly functional. For the bioassay conducted, the 32- well tray method was used, as described above, using dsRNA targeting LdEG12, LdEG118, LdEG70, and LdEG102 (SEQ ID NO: 25, SEQ ID NO: 27, and SEQ ID NO: 29), respectively. The graph depicted in the top panel shows percent mortality of larvae treated with respective dsRNAs, on Day 8, and the graph depicted in the bottom panel shows average leaf consumption measured as the percent of the leaf disks, consumed by each larva on Day 5. Data on both graphs were analyzed using ANOVA and Fisher's LSD post hoc with binomial model. As shown in FIG. 2E, targeting LdEG12 was most effective in killing the larvae than any of the other dsRNA tested.Table 2: CPB genes tested that are related to mitochondrial function or are key component of the ATPase / synthase complex**Tribolium gene ID and function data is extracted from iBeetle-Base hit n s : / / ib eet I e- base. uni -eoetti neen . d e / Example 3: Characterization of biological activity ofLdEG53

[0280] To characterize the biological activity of RNAi-mediated LdEG53 inhibition, CPB larvae L2 were placed in 150 X 25 mm petri dishes. Excised leaves preserved by placing stems inside 0.5% agar microcentrifuge tubes, were placed into the petri dishes. Leaves were treated with 120 pl of 10 ppm (10 ng / pL) dsRNA LdEG53 or non-target Shrimp white spot syndrome virus gene VP28 dsRNA (SEQ ID NO: 11 ), as a negative control. Five L2 larvae were placed into each petri dish. One live larva from each group was obtained at day 4, for qPCR analysis. Each treatment was replicated six times. Larvae were exposed to dsRNAfor4 and 8 days (FIG. 3A). Results from the experiments showed th at there was a significant difference among mean larval mortality at day 8 (F1 ,6 = 18.44, p < 0.01 indicated by ***) using one-way ANOVA.

[0281] To understand if the insectmortality was in fact caused because of genesilencing, bioassays were conducted for LdEG53 following the same protocol described for LdEG12 above. The qPCR primer and probe sequences of LdEG53 are provided in Table 4. t-test was run between control and the LdEG53 treatments. FIG. 3B shows that the expression of LdEG53 was indeed reduced in larvae exposed to dsRNA LdEG53.

[0282] Further, lethal concentration of dsRNA LdEG53 was identified using a similar method as described above for LdEG12. The LCso on day 8 was determined as 0.059 ppm or ng / pL and the LC90 on day 8 was identified as 0.501 ppm (T able 3).Table 3: LC concentration of dsRNA LdEG53

[0283] Additionally, activity of LdEG53 dsRNA was tested in whole plants. 3-5-week-old Kennebec potato plants were treated with 52 ppm of LdEG53 dsRNA. Untreated plantswere used as controls. Each plant was applied with 10 mL dsRNA solution using a fine mist spray bottle (Fantasea, Model# BX-FSC296), treatment was applied once. Ten L2 CPB larvae were placed on each plant inside butterfly cages. 5 cages / plants per treatment were used. Plants and larvae were kept at room temperature, at green house conditions, or in a growth tent at approximately 25°C. Mortality was recorded for up to 14 days. The experiment was replicated 4 times. Significant difference among experimental replicates were found using a two-way AN OVA (FI ,3,35 = 4.009, p <0.05). Significant difference in mean larval mortality was observed between L2 CPB larvae placed on LdEG53 treated and untreated plants on day 14 (Fi ,3,35= 454.711 , p < 0.001 indicated by

[0284] Activity of LdEG81 dsRNA was characterized by screening the lethality of LdEG81 dsRNA. One CPB larva was placed into each well of a 32-well bioassay tray. Leaves were treated with 24 pl of 10 ppm LdEG81 dsRNA or non-target negative control dsRNA (Shrimp white spot syndrome virus gene VP28 dsRNA, SEQ ID NO: 11 ). Each treatment included 20 insects. Larvae were exposed to dsRNA or non-target negative control dsRNA for 4 days and percentage mortality of CPB larva was recorded on days 2, 4, 7, 8 and 10. Application of LdEG81 dsRNA showed a significantly greater mortality in CPB larva compared to non-target negative control dsRNA (FIG. 4A).

[0285] Further, whole plantassays were conducted using LdEG81 dsRNA. 3-5-week-old whole Kennebec potato plants were treated with 52 ppm LdEG81 dsRNA. As control, untreated plants were used. Ten L2 CPB larvae were placed on plants inside butterfly cages. There were 5 cages per treatment. Plants and larvae were kept in a growth tent or greenhouse at approximately 25°C and 60-65% relative humidity. Mortality was recorded for up to 14 days. Plants treated LdEG81 showed significantly higher larval mortality at day 14 (FI ,28 = 69.82, p < 0.001 ) compared to the untreated plants (FIG. 4B).Summary of Examples

[0286] Three target genes LdEG12, LdEG53, and LdEg81 were identified and down regulation of these genes using RNAi targeting the identified genes were tested for lethality in CPB larvae. Both in vitro and whole plant assays showed that CPB larvae contacted with dsRNA targeting LdEG12, LdEG53, or LdEg81 had high mortality compared to control CPB. These results indicated that LdEG12, LdEG53, and LdEg81are essential genes in CPB and down regulation of these genes using RNAi has insecticidal applicability.Table 4: Sequences

Claims

CLAIMSWhat is claimed is:

1. An insecticidal composition comprising an insecticidally effective amount of a recombinant RNA molecule comprising a nucleotide sequence of at least 17 or more contiguous nucleotides having a sequence of at least about 95% to about 100% complementarity to a portion of a target gene of an insect that infests a plant, the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID Nos: 1 , 4, 6 and 8.

2. The insecticidal composition of claim 1 , wherein the 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: 3, 5, and 10.

3. The insecticidal composition of claim 1 , wherein the recombinant RNA molecule is a double-stranded RNA (dsRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a hairpin RNA (hpRNA), or piwi-interacting RNA (pi-RNA).

4. The insecticidal composition of claim 1 , wherein the recombinant RNA molecule is a double-stranded RNA (dsRNA) comprising an RNA strand having a sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 10.

5. The insecticidal composition of claim 1 , wherein the dsRNA is at least 17 base pairs in length.

6. The insecticidal composition of claim 1 , wherein the insect is an adult, or in a larval or a nymph stage.

7. The insecticidal composition of claim 1 , wherein the plant-infesting insect is aColeopteran insect.

8. The insecticidal composition of claim 7, wherein the plant-infesting insect is an insect from the genus Leptinotarsa.

9. The insecticidal composition of claim 8, wherein the plant-infesting insect isLeptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No: 1 .

10. The insecticidal composition of claim 1 , wherein the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No: 4 or 6.

11. The insecticidal composition of claim 1 , wherein the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the target gene encodes an mRNA sequence comprising the nucleotide sequence of SEQ ID No: 8.

12. The insecticidal composition of claim 1 , wherein the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the 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: 3, 5, and 1013. The insecticidal composition of claim 1 , wherein the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle), and the recombinant RNA molecu le is a dsRNA comprising an RNA strand having a sequence selected from the group consisting of SEQ ID Nos: 3, 5, and 10.

14. The insecticidal composition of claim 1 , furthercomprisingat least onecomponent selected from a carrier, excipient, diluent, 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 insecticidal composition of claim 1 , furthercomprising at least one pesticidal agent.

16. The insecticidal composition of claim 14, wherein the pesticidal agent is selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, an insecticidal protein.

17. The insecticidal composition of claim 1 , in a form selected from a solid, a liquid, a powder, a suspension, an emulsion, a spray, an encapsulation, microbeads, carrier particulates, a film, a solid matrix, a soil drench, an insect diet, insect bait, and a seed treatment.

18. A plant or seed thereof treated with the insecticidal composition of claim 1 , wherein the plant exhibits improved resistance to the insect.

19. 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: 3, 5, and 10.

20. The recombinant DNA construct of claim 19, wherein the heterologous promoter is functional for expression of the RNA transcript in a bacterium or a fungus.

21. The recombinant DNA construct of claim 19, wherein the heterologous promoter is functional in a plant cell.

22. A recombinant vector comprising the recombinant DNA construct of claim 19.

23. A plant chromosome or plastid comprising the recombinant DNA construct of claim 22.

24. A transgenic plant cell having in its genome the recombinant DNA construct of claim 22.

25. A transgenic plant comprising the transgenic plant cell of claim 24.

26. A crop product produced from the transgenic plant of claim 25.

27. A transgenic progeny seed or propagatable plant part of the transgenic plant of claim 25.

28. A method for controlling an infestation of a plant by a plant-infesting insect, the method comprising contacting the insect with a dsRNA comprising at least one portion of 17 or more contiguous nucleotides having a sequence of about 95% to about 100% complementarity with a target gene or portion thereof of the insect, wherein the target gene encodes a mRNA sequence selected from the group consisting of SEQ ID NOs: 1 , 4. 6, and 8.

29. The method of claim 28, wherein the insect is an adult, or in a larval or a nymph stage.

30. The method of claim 28, wherein the plant-infesting insectisa Coleopteran insect.31 . The method of claim 30, wherein the plant-infesting insect is an insect from the genus Leptinotarsa.

32. The method of claim 31 , wherein the insect is Leptinotarsa decemlineata (Colorado potato beetle).

33. The method of claim 28, wherein the dsRNA comprises a sequence selected from the group consisting of SEQ ID Nos: 3, 5, and 10.

34. The method of claim 28, wherein the dsRNA comprises more than one portion of 17 or more contiguous nucleotides having a sequence of about 95% to about 100% complementarity with the target gene or portion thereof.

35. The method of claim 28, wherein the dsRNA is blunt-ended.

36. The method of claim 28, wherein the dsRNA has an overhang at least one terminus.

37. The method of claim 28, wherein the dsRNA is (a) chemically synthesized, or (b) produced by expression in a microorganism, expression in a plant cell, or by microbial fermentation.

38. The method of claim 28, wherein the dsRNA is chemically modified.

39. The method of claim 28, wherein the contacting comprises application of a composition comprising the dsRNA to a surface of the insect or to a surface of the plant infested by the insect.

40. The method of claim 28, wherein the composition comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, or seed treatment.41 . The method of claim 28, wherein the contacting comprises providing the dsRNA in a composition further comprising one or more components selected from 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.

42. The method of claim 28, wherein the contacting comprises providing the dsRNA in a composition further comprising at least one pesticidal agent.

43. The method of claim 42, wherein the pesticidal agent is selected from a patatin, a plant lectin, a phytoecdysteroid, and an insecticidal protein.

44. The method of claim 28, wherein the contacting comprises providing the dsRNA in a composition ingested by the insect.

45. The method of claim 44, wherein the ingested composition furthercomprises one or more components selected from 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.

46. A method of causing mortality in an insect, comprising providing in the diet of an insect an insecticidal composition of any of claims 1 -17, or plant of claim 18, wherein the composition or plant causes mortality or stunting of the insect upon ingestion by the insect.

47. The method of claim 46, wherein the insect is an adult, or in a larval or a nymph stage.

48. A method of providing a plant having improved resistance to a plant-infesting insect, the method comprising introducing to the plant expressing a recombinantDNA construct comprising a nucleotide sequence encoding an RNA molecule comprising a silencing element, the silencing element comprising a nucleotide sequence substantially identical or complementary to a portion of a target gene sequence of the insect, wherein the target gene encodes a mRNA sequence selected from SEQ ID Nos: 1 , 4, 6 and 8, and wherein ingestion of the RNA by the insect results in mortality or stunting in the insect.

49. The method of claim 42, wherein the silencing element has a sequence of about 95% to about 100% sequence identity or complementarity with a sequence selected from the group consisting of SEQ ID Nos: 3, 5 and 10.

50. The method of claim 42, wherein the recombinant DNA construct further comprises a heterologous promoter operably linked to the nucleotide sequence encoding the RNA molecule, and functional in a plant cell.51 . The method of claim 42, wherein the silencing element is a dsRNA, a miRNA, a small siRNA, a hpRNA, or a pi-RNA.

52. The method of claim 42, wherein the plant-infesting insect is an adult, or in a larval or a nymph stage.

53. The method of claim 42, wherein the plant-infesting insect is a Coleopteran insect.

54. The method of claim 42, wherein the plant-infesting insect is an insect from the genus Leptin otarsa.

55. The method of claim 42, wherein the plant-infesting insect is Leptinotarsa decemlineata (Colorado potato beetle).

56. The method of claim 42, wherein the introducing comprises transgen icexpression or transient expression.

57. A plant produced by the method of claim 42 and having improved resistance to the insect.

58. Fruit, seed, or propagatable parts of the plant of claim 57.

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