COMPOSITIONS AND METHODS FOR CONTROLLING MOSQUITOES

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/370,543, filed August 3, 2016, which is incorporated by reference herein in its entirety. The sequence listing contained in the file "P34300WO00_SEQ.txt" which is 1,624,790 bytes (measured in MS-Windows®) and created on August 2, 2017, is filed electronically herewith and is incorporated by reference in its entirety. FIELD

[0002] This invention relates to polynucleotides, compositions, and methods for mosquito control. Polynucleotides and methods of use thereof for modifying the expression of genes in a mosquito, particularly through RNA interference are disclosed. The polynucleotides, compositions, and methods of the invention are useful for controlling vectors of human diseases including the mosquito genera Aedes sp., Culex sp., and Anopheles sp.

BACKGROUND

[0003] Mosquitoes are the insect vectors for a number of important pathogen-caused diseases of humans (Table 1). These vector mosquito species require a blood meal by the female as part of the egg-laying cycle, during which transmission of the causative pathogen to the human can occur.

Table 1

[0004] Compositions for controlling mosquito infestations have typically been in the form of chemical insecticides. However, there are several disadvantages to using chemical insecticides. For example, chemical insecticides are generally not selective, and applications of chemical insecticides intended to control mosquitoes can exert their effects on non-target insects and other invertebrates as well.

Chemical insecticides often persist in the environment and can be slow to degrade, thus potentially accumulating in the food chain. Furthermore, the use of persistent chemical insecticides can result in the development of resistance in the target mosquito species. Thus, there has been a long felt need for environmentally friendly methods for controlling or eradicating mosquito infestations, / ^' . e. , methods which are species-selective, environmentally inert, non-persistent, and biodegradable, and that fit well into pest resistance management schemes.

[0005] RNA interference (RNAi, RNA -mediated gene suppression) is an approach that shows promise for environmentally friendly pest control. In invertebrates, RNAi-based gene suppression was first demonstrated in nematodes (Fire et al , ( 1998) Nature, 391 : 806-81 1; Timmons & Fire ( 1998) Nature, 395 : 854). Subsequently, RNAi-based suppression of invertebrate genes using recombinant nucleic acid techniques has been reported in a number of species, including agriculturally or economically important pests from various insect and nematode taxa. The enzymes involved in canonical RNAi pathways (e. g. , Dicers, Drosha, Argonaute proteins) and various classes of small silencing RNAs are known to exist in these vector mosquito species; see, e. g., Barnard et al. (2012), Genes, 3 :702-741. Attempts to control mosquito pests or to silence target genes in mosquitoes through the use of RNA interference or RNA -mediated gene suppression have been reported. See, e. g. , Boisson et al. (2006) FEBSLett., 580: 1988 - 1992; Pridgeon et al. (2008) J. Med Entomol, 45 : 414 - 420; Zhang et al. (2010) Insect Mol. Biol, 19:683 - 693; Gu et al. (201 1) PLoS ONE, 6:e21329; and Coy et al. (2012) J. Applied Entomol. , 136:741 - 748.

SUMMARY

[0006] The present embodiments relate to control of mosquitoes. The compositions and methods disclosed herein include recombinant polynucleotide molecules, such as recombinant DNA constructs and polynucleotide agents, such as RNAs that are useful for controlling or preventing mosquito infestation. Several embodiments described herein relate to a polynucleotide -containing composition (e. g. , a composition containing a dsRNA for suppressing a mosquito target gene) that is provided to a mosquito species or is applied to an environment to be protected from infestation by a mosquito species. Other embodiments relate to methods for selecting mosquito target genes that are likely to be effective targets for RNAi -mediated control of mosquito species.

[0007] Several embodiments relate to methods of mosquito control, including (a) contacting a mosquito with a polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene, or an RNA transcribed from the target gene; (b) contacting a mosquito with an effective amount of a polynucleotide, one strand of which is complementary to at least 21 contiguous nucleotides of a mosquito target gene, or an RNA transcribed from said target gene; (c) providing in the diet of a mosquito a polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a mosquito target gene, or an RNA transcribed from said target gene; (d) causing mortality or stunting in larvae of a mosquito by providing in the diet of said larvae at least one polynucleotide comprising at least one silencing element, wherein said silencing element comprises at least 21 contiguous nucleotides that are complementary to a mosquito target gene, or an RNA transcribed from said target gene; (e) contacting a mosquito with a

polynucleotide comprising at least one segment that is identical or complementary to at least 21 contiguous nucleotides of a mosquito target gene, or an RNA transcribed from said target gene; or (f) contacting an environment infested by said mosquito with a composition comprising a polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a mosquito target gene, or an RNA transcribed from said target gene; in embodiments of the above methods of mosquito control the target gene is at least one gene selected from the group consisting of (a) a gene identified by name in Tables 2 and 3; (b) a gene encoding a proteasome protein selected from the group consisting of: Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6; (c) a gene encoding an ATPase protein selected from the group consisting of: V-ATPase subunit A, V-ATPase subunit B, V- ATPase subunit D, V-ATPase subunit E, Actin 6; (d) a gene encoding a ribosomal protein selected from the group consisting of: Ribosomal protein Large subunit 3, Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4; (e) a gene encoding a vesicle protein selected from the group consisting of: Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmpl, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta; (f) a gene encoding an actin; (g) a gene encoding a protein related to behaviour selected from the group consisting of: Bendless, scribbler, capa receptor, diapause hormone receptor, Ebl, Ether-a-go-go, flightless-I, foraging, juvenile hormone epoxide hydrolase, neuropeptide F, pheromone biosynthesis activating neuropeptide receptor, semaphorin la, shaker, slowmo, stress-sensitive B, turtle, twitchin, myosin heavy chain, nervana, N-synaptobrevin, nuclear hormone receptor 25, syntaxin, and bent; (h) a gene encoding a glycolysis and energy metabolism protein selected from the group consisting of: BiP, citrate synthase, glycogen synthase, hexokinase, isocitrate dehydrogenase, pyruvate dehydrogenase, phosphofructokinase, GTPase activator, GTPase Ran, sarcoplasmic Ca2+ ATPase, Ca2+ ATPase, hydrogen exporting ATPase, NADH dehydrogenase, FIFO ATPase, spermatogenesis ATPase, beta- ketoacyl synthase, pyruvate kinase, and knickkopf; (i) a gene encoding a protein turnover and mitosis protein selected from the group consisting of: cyclins including cyclin A, cyclin B, cyclin B3, cyclin C, cyclin D, cyclin E, cyclin G, cyclin H, cyclin J, cyclin K, cyclin T, and cyclin Y, Ubiquitin conjugating enzyme El, chromosome segregation protein, CDC5, COP9 signalosome, and ketel; (j) a gene encoding a gene expression protein selected from the group consisting of: cleavage polyadenylation specificity factor, ElA/CREB-binding protein, RNA helicases, RNA polymerases, Arfl, Bx42, and eft2; (k) a gene encoding a protein-folding, stress, and heat shock response protein selected from the group consisting of: protein disulfide isomerase, heat shock cognate 70, chaperonin TCP1, chaperonins, carrier protein, and HSP70; and (1) a gene encoding a miscellaneous protein selected from the group consisting of: spermatogenesis ATPase, pitchoune, and insect proteins. [0008] Several embodiments relate to a mosquitocidal composition including a mosquitocidally effective amount of a recombinant polynucleotide comprising a nucleotide sequence that is

complementary to at least 21 contiguous nucleotides of a mosquito target gene, or an RNA transcribed from the target gene. In some embodiments, the target gene is selected from the group consisting of (a) a gene identified in Tables 2 and 3; (b) a gene encoding a proteasome protein selected from the group consisting of: Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6; (c) a gene encoding an ATPase protein selected from the group consisting of: V-ATPase subunit A, V-ATPase subunit B, V-ATPase subunit D, V-ATPase subunit E, Actin 6; (d) a gene encoding a ribosomal protein selected from the group consisting of: Ribosomal protein Large subunit 3, Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4; (e) a gene encoding a vesicle protein selected from the group consisting of: Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmpl, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta; (f) a gene encoding an actin; (g) a gene encoding a protein related to behaviour selected from the group consisting of: Bendless, scribbler, capa receptor, diapause hormone receptor, Ebl, Ether-a-go-go, flightless-I, foraging, juvenile hormone epoxide hydrolase, neuropeptide F, pheromone biosynthesis activating neuropeptide receptor, semaphorin la, shaker, slowmo, stress-sensitive B, turtle, twitchin, myosin heavy chain, nervana, N-synaptobrevin, nuclear hormone receptor 25, syntaxin, and bent; (h) a gene encoding a glycolysis and energy metabolism protein selected from the group consisting of: BiP, citrate synthase, glycogen synthase, hexokinase, isocitrate dehydrogenase, pyruvate dehydrogenase, phosphofructokinase, GTPase activator, GTPase Ran, sarcoplasmic Ca2+ ATPase, Ca2+ ATPase, hydrogen exporting ATPase, NADH dehydrogenase, FIFO ATPase, spermatogenesis ATPase, beta- ketoacyl synthase, pyruvate kinase, and knickkopf; (i) a gene encoding a protein turnover and mitosis protein selected from the group consisting of: cyclins including cyclin A, cyclin B, cyclin B3, cyclin C, cyclin D, cyclin E, cyclin G, cyclin H, cyclin J, cyclin K, cyclin T, and cyclin Y, Ubiquitin conjugating enzyme El, chromosome segregation protein, CDC5, COP9 signalosome, and ketel; (j) a gene encoding a gene expression protein selected from the group consisting of: cleavage polyadenylation specificity factor, ElA/CREB-binding protein, RNA helicases, RNA polymerases, Arfl, Bx42, and eft2; (k) a gene encoding a protein-folding, stress, and heat shock response protein selected from the group consisting of: protein disulfide isomerase, heat shock cognate 70, chaperonin TCP1, chaperonins, carrier protein, and HSP70; and (1) a gene encoding a miscellaneous protein selected from the group consisting of: spermatogenesis ATPase, pitchoune, and insect proteins. In some embodiments, the mosquitocidal composition is provided as a solid, liquid, powder, suspension, emulsion, colloid, spray, encapsulation, microbeads, carrier particulates, granules, film, gel, or matrix. In some embodiments, the

mosquitocidal composition is provided in the form of a bait, a trap, a sprayable liquid, a concentrate, dunks, dispersible granules, or ingestible particulates or cells. In some embodiments, the mosquitocidal composition further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a lipid, a sugar, a non-polynucleotide mosquitocide, a diptericidal Bacillus thuringiensis toxin, a safener, a mosquito attractant or pheromone, and an insect growth regulator. In some embodiments the composition further comprises a cationic carbohydrate. In some embodiments the composition further comprises a cationic starch. In some embodiments the composition further comprises a cationic guar. In some embodiments the composition further comprises a branched Polyethylenimine .

[0009] Several embodiments relate to a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript including: (a) a sequence of about 95% to about 100% identity or complementarity with a mosquito target gene; (b) at least 21 contiguous nucleotides that are complementary to a mosquito target gene, or an RNA transcribed from the mosquito target gene; (c) at least 21 contiguous ribonucleotides of a mosquito target gene RNA sequence; or (d) a strand comprising a RNA sequence complementary to at least 21 nucleotides of a mosquito target gene. In some embodiments, the RNA transcript is dsRNA, which can be encoded on a single RNA strand or on two essentially complementary RNA strands. In some embodiments, the heterologous promoter is functional for expression of the RNA transcript in a bacterium, or in a eukaryotic cell. Various other embodiments include a recombinant vector, chromosome, plastid, or transgenic cell including the recombinant DNA construct.

[0010] Several embodiments relate to man-made compositions comprising one or more

polynucleotides as described herein, such as dsRNA formulations useful for topical application to an area or surface in need of treatment for or protection from a mosquito infestation; recombinant constructs and vectors useful for making transgenic eukaryotic or prokaryotic cells and transgenic plants; and formulations and coatings useful for treating a surface or area. Several embodiments relate to polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs.:l - 105 and 897 - 1132. Several embodiments relate to polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087 or the complement thereof. Such antibodies are made by routine methods as known to one of ordinary skill in the art. In some embodiments the composition further comprises a cationic carbohydrate. In some embodiments the composition further comprises a cationic starch. In some embodiments the composition further comprises a cationic guar. In some embodiments the composition further comprises a branched Polyethylenimine.

[0011] Other aspects and specific embodiments of this invention are disclosed in the following Detailed Description and Examples. DETAILED DESCRIPTION

[0012] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms "to include" or "to include" are understood to mean "to include, but not to be limited to". Where a term is provided in the singular, the inventors also contemplate aspects of the invention described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given in this application. The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only.

[0013] Unless otherwise stated, nucleic acid sequences are given, when read from left to right, in the 5' to 3' direction. One of skill in the art would be aware that a given DNA sequence is understood to define a corresponding RNA sequence which is identical to the DNA sequence except for replacement of the thymine (T) nucleotides of the DNA with uracil (U) nucleotides. Thus, providing a specific DNA sequence is understood to define the exact RNA equivalent. A given first polynucleotide sequence, whether DNA or RNA, further defines the sequence of its exact complement (which can be DNA or RNA), which hybridizes perfectly to the first polynucleotide by forming Watson-Crick base- pairs. By "essentially identical" or "essentially complementary" to a target gene or a fragment of a target gene is meant that a polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize (generally under physiological conditions such as those found in a living plant or animal cell) to a target gene or to a fragment of a target gene or to the transcript of the target gene or the fragment of a target gene; one of skill in the art would understand that such hybridization does not necessarily require 100% sequence identity or complementarity. A first nucleic acid sequence is "operably" connected or "linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter sequence is "operably linked" to DNA if the promoter provides for transcription or expression of the DNA. Generally, operably linked DNA sequences are contiguous.

[0014] The term "polynucleotide" commonly refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to "oligonucleotides" (a polynucleotide molecule of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Polynucleotides also include molecules containing multiple nucleotides including non-canonical nucleotides or chemically modified nucleotides as commonly practiced in the art; see, e. g, chemical modifications disclosed in the technical manual "RNA Interference (RNAi) and DsiRNAs", 2011 (Integrated DNA Technologies Coralville, IA). Generally, polynucleotide as described herein, whether DNA or RNA or both, and whether single- or double -stranded, comprise at least one segment of 18 or more contiguous nucleotides (or, in the case of double-stranded polynucleotides, at least 18 contiguous base-pairs) that are essentially identical or complementary to a fragment of equivalent size of the DNA of a target gene or the target gene's RNA transcript. Throughout this disclosure, "at least 18 contiguous" means "from about 18 to about 10,000, including every whole number point in between". Thus, embodiments of this invention include compositions including oligonucleotides having a length of 18-25 nucleotides (18- mers, 19-mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300 nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e. g. , polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target gene including coding or non-coding or both coding and non-coding portions of the target gene). Where a polynucleotide is double-stranded, such as the dsRNAs described in the Examples, its length can be similarly described in terms of base pairs. Double-stranded polynucleotides, such as the dsRNAs described in the Examples, can further be described in terms of one or more of the single-stranded components.

[0015] The polynucleotides described herein can be single -stranded (ss) or double-stranded (ds). "Double-stranded" refers to the base-pairing that occurs between sufficiently complementary, anti- parallel nucleic acid strands to form a double -stranded nucleic acid structure, generally under physiologically relevant conditions. Embodiments include those wherein the polynucleotide is selected from the group consisting of sense single-stranded DNA (ssDNA), sense single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), a double-stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA; a mixture of polynucleotides of any of these types can be used. In some embodiments, the polynucleotide is double -stranded RNA of a length greater than that which is typical of naturally occurring regulatory small RNAs (such as endogenously produced siRNAs and mature miRNAs). In some embodiments, the polynucleotide is double-stranded RNA of at least about 30 contiguous base-pairs in length. In embodiments, the polynucleotide is double -stranded RNA with a length of between about 50 to about 500 base-pairs. In embodiments, the polynucleotide can include components other than standard ribonucleotides, e. g. , an embodiment is an RNA that includes terminal deoxyribonucleotides.

[0016] One or more effective polynucleotides of any size can be used, alone or in combination, in the various methods and compositions described herein. In some embodiments, a single polynucleotide is used in a mosquitocidal composition (e. g. , a composition to be provided to a mosquito or to be applied to an environment, or a recombinant DNA construct useful for making a transgenic cell). In embodiments, a mixture or pool of different polynucleotides is used; in such cases the polynucleotides can be for a single target gene or for multiple target genes, or for a single or multiple mosquito species.

[0017] In various embodiments, the polynucleotides described herein include naturally occurring nucleotides, such as those which occur in DNA and RNA. In some embodiments, the polynucleotide is a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides or synthetic polynucleotides consisting mainly of

deoxyribonucleotides but with one or more terminal dideoxyribonucleotides. In embodiments, the polynucleotide includes non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In embodiments, the polynucleotide includes chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S. Patent Publication

2011/0152353, U.S. Patent Publication 2011/0152346, and U.S. Patent Publication 2011/0160082, which are herein incorporated by reference. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide which can be partially or completely modified with phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (e. g. , fluorescein or rhodamine) or other label (e. g , biotin).

[0018] The term "recombinant", as used to refer to a polynucleotide (such as the recombinant RNA molecules or recombinant DNA constructs described herein), means that the polynucleotide is not a naturally occurring molecule, / ^' . e. , that human intervention is required for the polynucleotide to exist. A recombinant polynucleotide is produced using recombinant nucleic acid techniques, or by chemical synthesis, and can include heterologous combinations of sequences (e. g. , heterologous combinations of a promoter and a DNA encoding an RNA to be expressed, or an RNA molecule that includes concatenated segments of a target gene that do not in nature occur adjacent to one another). A recombinant polynucleotide can be biologically produced in a cell (such as a bacterial or plant or animal cell), for example, when that cell is transfected or transformed with a vector encoding the recombinant polynucleotide. A recombinant polynucleotide can include sequences of nucleotides designed in silico using appropriate algorithms.

[0019] The polynucleotides described herein are generally designed to suppress or silence one or more genes ("target genes"). The term "gene" refers to any portion of a nucleic acid that provides for expression of a transcript or encodes a transcript. A "gene" can include, but is not limited to, a promoter region, 5 ' untranslated regions, transcript encoding regions that can include intronic regions, 3' untranslated regions, coding regions, or combinations of these regions. In embodiments, the target genes can include coding or non-coding sequence or both. In other embodiments, the target gene has a sequence identical to or complementary to a messenger RNA, e. g. , in embodiments the target gene is a cDNA. [0020] As used herein, the term "isolated" refers to separating a molecule from other molecules normally associated with it in its native or natural state. The term "isolated" thus may refer to a DNA molecule that has been separated from other DNA molecule(s) which normally are associated with it in its native or natural state. Such a DNA molecule may be present in a recombined state, such as a recombinant DNA molecule. Thus, DNA molecules fused to regulatory or coding sequences with which they are not normally associated (e.g. , in a heterologous combination), for example as the result of recombinant techniques, are considered isolated, even when integrated as a transgene into the chromosome of a cell or present with other DNA molecules.

[0021] By "mosquitocidally effective" is meant effective in inducing a physiological or behavioural change in a mosquito (adult, larval, and/or egg) such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. In embodiments, application of a mosquitocidally effective amount of the polynucleotide, such as a dsRNA molecule, to an area or surface decreases the viable mosquito population of that area or surface. While there is no upper limit on the concentrations and dosages of a polynucleotide as described herein that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency and economy. Non-limiting embodiments of mosquitocidally effective amounts of a polynucleotide include a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of a polynucleotide in a sprayable liquid form, or from about 10 milligrams per acre to about 100 grams per acre of polynucleotide applied in the form of a bait or sprayable liquid or dispersible particulates to an area or surface, or from about 0.001 to about 0.1 microgram per milliliter of polynucleotide in an artificial mosquito diet. Where compositions as described herein are topically applied to an area or surface (e. g , sprayed or dusted on plants or other surfaces in an environment), the concentrations can be adjusted in consideration of the volume of spray or other treatment applied to the area or surface. In embodiments, a mosquitocidal composition contains about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of a dsRNA (or a single-stranded 21-mer) such as the polynucleotides described herein. In embodiments, a mosquitocidal composition contains about 0.5 to about 1.5 milligrams per milliliter of a dsRNA polynucleotide as described herein of about 50 to about 200 or more nucleotides. In embodiments, the mosquitocidal composition contains at least one polynucleotide as described herein at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. When using long dsRNA molecules that can be processed into multiple oligonucleotides (e. g , multiple mosquitocidal polynucleotides encoded by a single recombinant DNA molecule of this invention), lower concentrations can be used.

[0022] The present embodiments relate to methods and compositions for mosquito control, in particular mosquito genera and species that are vectors of disease. Disclosed herein are target genes identified as useful for designing polynucleotide agents for mosquito control. Also disclosed are sequences for suppressing one or more mosquito target genes. Several embodiments relate to polynucleotide agents, for example, in the form of dsRNA, that suppress mosquito target genes. In some embodiments, polynucleotides and recombinant DNA molecules and constructs useful in mosquito control. Several embodiments relate to mosquitocidal compositions. Several embodiments relate to methods of identifying mosquitocidally efficacious polynucleotide agents, for example double- stranded RNA molecules, for mosquito control, and methods for identifying mosquito genes that are likely to represent essential functions, making these genes preferred targets for RNAi-mediated silencing and control of mosquitoes.

[0023] Several embodiments relate to methods and compositions for mosquito control by inhibiting in the mosquito the expression of one or more target genes selected from the group consisting of: (a) a gene identified in Tables 2 and 3; (b) a gene encoding a proteasome protein selected from the group consisting of: Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6; (c) a gene encoding an ATPase protein selected from the group consisting of: V-ATPase subunit A, V-ATPase subunit B, V-ATPase subunit D, V-ATPase subunit E, Actin 6; (d) a gene encoding a ribosomal protein selected from the group consisting of: Ribosomal protein Large subunit 3, Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4; (e) a gene encoding a vesicle protein selected from the group consisting of: Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmpl, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta; (f) a gene encoding an actin; (g) a gene encoding a protein related to behaviour selected from the group consisting of: Bendless, scribbler, capa receptor, diapause hormone receptor, Ebl, Ether-a-go-go, flightless-I, foraging, juvenile hormone epoxide hydrolase, neuropeptide F, pheromone biosynthesis activating neuropeptide receptor, semaphorin la, shaker, slowmo, stress-sensitive B, turtle, twitchin, myosin heavy chain, nervana, N-synaptobrevin, nuclear hormone receptor 25, syntaxin, and bent; (h) a gene encoding a glycolysis and energy metabolism protein selected from the group consisting of: BiP, citrate synthase, glycogen synthase, hexokinase, isocitrate dehydrogenase, pyruvate dehydrogenase, phosphofructokinase, GTPase activator, GTPase Ran, sarcoplasmic Ca2+ ATPase, Ca2+ ATPase, hydrogen exporting ATPase, NADH dehydrogenase, FIFO ATPase, spermatogenesis ATPase, beta- ketoacyl synthase, pyruvate kinase, and knickkopf; (i) a gene encoding a protein turnover and mitosis protein selected from the group consisting of: cyclins including cyclin A, cyclin B, cyclin B3, cyclin C, cyclin D, cyclin E, cyclin G, cyclin H, cyclin J, cyclin K, cyclin T, and cyclin Y, Ubiquitin conjugating enzyme El, chromosome segregation protein, CDC5, COP9 signalosome, and ketel; (j) a gene encoding a gene expression protein selected from the group consisting of: cleavage polyadenylation specificity factor, ElA/CREB-binding protein, RNA helicases, RNA polymerases, Arfl, Bx42, and eft2; (k) a gene encoding a protein-folding, stress, and heat shock response protein selected from the group consisting of: protein disulfide isomerase, heat shock cognate 70, chaperonin TCP1, chaperonins, carrier protein, and HSP70; and (1) a gene encoding a miscellaneous protein selected from the group consisting of: spermatogenesis ATPase, pitchoune, and insect proteins. Several embodiments relate to methods and compositions for mosquito control by inhibiting in the mosquito the expression of one or more target genes selected from the group consisting of SEQ ID NOs.:l - 105 and 897 - 1132. In embodiments, inhibiting the expression of one or more target gene in the mosquito (adult, larva, and/or egg) results in stunting or mortality. In embodiments, contacting of the polynucleotide (e.g., dsRNA) to the mosquito is by oral delivery, or by non-oral contact, e. g. , by absorption through the cuticle, or through a combination of oral and non-oral delivery.

[0024] Several embodiments relate to polynucleotides of use in methods and compositions for mosquito control. In some embodiments, the mosquitocidally effective polynucleotide is a dsRNA. In embodiments, the mosquitocidally effective polynucleotide comprises an RNA strand comprising a sequence having about 95% to about 100% identity or complementarity with a mosquito target gene. In embodiments, the mosquitocidally effective polynucleotide comprises a sequence that is about 95% to about 100% identical or complementary to a sequence selected from the group consisting of SEQ ID NOs.:106 - 896. In embodiments, the mosquitocidally effective polynucleotide is a dsRNA compring an RNA strand comprising at least 21, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50 or more contiguous nucleotides that are identical or complementary to a sequence selected from the group consisting of SEQ ID NOs.:106 - 896. In embodiments, the polynucleotide is a dsRNA comprising an RNA strand comprising a sequence that is identical or complementary to a sequence selected from the group consisting of SEQ ID NOs.:106 - 896. In embodiments, the mosquitocidally effective polynucleotide is a dsRNA that suppresses a target gene in the mosquito and stunts growth, development or reproduction by the mosquito, or kills the mosquito. In embodiments, the

mosquitocidally effective polynucleotide is dsRNA which is at least 21 base pairs in length. In embodiments, the mosquitocidally effective polynucleotide is dsRNA which is at least 50 base pairs in length. In embodiments, the mosquitocidally effective polynucleotide is dsRNA which is at least 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, or more base pairs in length. In embodiments, the polynucleotide is dsRNA which is (a) is blunt-ended, or (b) has an overhang at at least one terminus, or (c) includes at least one stem -loop. In embodiments the dsRNA is chemically modified. In embodiments, the polynucleotide is dsRNA which is produced by (a) chemical synthesis, or (b) expression in a microorganism, or (c) expression in a eukaryotic cell. Various embodiments include those in which the mosquitocidally effective polynucleotide is provided in an ingestible composition such as a bait, or in a sprayable or otherwise easily dispersible composition; such compositions can include components such as a carrier agent, a surfactant, an organosilicone, a lipid, a sugar, a non-polynucleotide mosquitocide, an insecticidal bacterial toxin, a safener, a mosquito attractant, a pheromone, and an insect growth regulator.

[0025] Several embodiments relate to a mosquitocidally effective polynucleotide molecule, such as a dsRNA, which includes one or more segments including 18 or more contiguous nucleotides, for example, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides, having 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a mosquito target gene identified in Tables 2 or 3. Several embodiments relate to a mosquitocidally effective polynucleotide molecule, such as a dsRNA, which includes one or more segments including 18 or more contiguous nucleotides (for example 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides) having 100% complementarity with a fragment of a mosquito target gene selected from the group consisting of (a) a gene identified in Tables 2 and 3; (b) a gene encoding a proteasome protein selected from the group consisting of: Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6; (c) a gene encoding an ATPase protein selected from the group consisting of: V-ATPase subunit A, V-ATPase subunit B, V- ATPase subunit D, V-ATPase subunit E, Actin 6; (d) a gene encoding a ribosomal protein selected from the group consisting of: Ribosomal protein Large subunit 3, Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4; (e) a gene encoding a vesicle protein selected from the group consisting of: Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmp l, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta; (f) a gene encoding an actin; (g) a gene encoding a protein related to behaviour selected from the group consisting of: Bendless, scribbler, capa receptor, diapause hormone receptor, Ebl, Ether-a-go-go, flightless-I, foraging, juvenile hormone epoxide hydrolase, neuropeptide F, pheromone biosynthesis activating neuropeptide receptor, semaphorin la , shaker, slowmo, stress-sensitive B, turtle, twitchin, myosin heavy chain, nervana, N-synaptobrevin, nuclear hormone receptor 25, syntaxin, and bent; (h) a gene encoding a glycolysis and energy metabolism protein selected from the group consisting of: BiP, citrate synthase, glycogen synthase, hexokinase, isocitrate dehydrogenase, pyruvate dehydrogenase, phosphofructokinase, GTPase activator, GTPase Ran, sarcoplasmic Ca2+ ATPase, Ca2+ ATPase, hydrogen exporting ATPase, NADH dehydrogenase, FIFO ATPase, spermatogenesis ATPase, beta- ketoacyl synthase, pyruvate kinase, and knickkopf; (i) a gene encoding a protein turnover and mitosis protein selected from the group consisting of: cyclins including cyclin A, cyclin B, cyclin B3, cyclin C, cyclin D, cyclin E, cyclin G, cyclin H, cyclin J, cyclin K, cyclin T, and cyclin Y, Ubiquitin conjugating enzyme E l, chromosome segregation protein, CDC5, COP9 signalosome, and ketel; (j) a gene encoding a gene expression protein selected from the group consisting of: cleavage polyadenylation specificity factor, ElA/CREB-binding protein, RNA helicases, RNA polymerases, Arfl, Bx42, and eft2; (k) a gene encoding a protein-folding, stress, and heat shock response protein selected from the group consisting of: protein disulfide isomerase, heat shock cognate 70, chaperonin TCP 1, chaperonins, carrier protein, and HSP70; and (1) a gene encoding a miscellaneous protein selected from the group consisting of: spermatogenesis ATPase, pitchoune, and insect proteins. In embodiments, the mosquitocidally effective polynucleotide, such as a single -stranded RNA transcript, or one strand of a dsRNA, includes multiple segments each of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of one or more DNA sequences selected from the group consisting of SEQ ID NOs.: l - 105 and 897 - 1132. In embodiments, the mosquitocidally effective polynucleotide, such as dsRNA, includes 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides having 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs.: l - 105 and 897 - 1132. In embodiments, the mosquitocidally effective polynucleotide, such as dsRNA, includes segments corresponding to different regions of the mosquito target gene, or can include multiple copies of a segment. In other embodiments, the mosquitocidally effective

polynucleotide, such as dsRNA, includes multiple segments, each of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a different mosquito target gene; in this way multiple mosquito target genes, or multiple mosquito species, can be suppressed.

[0026] Several embodiments relate to a mosquitocidally effective polynucleotide, such as dsRNA, which inhibits the expression of one or more mosquito target selected from the group consisting of (a) a gene identified in Tables 2 and 3; (b) a gene encoding a proteasome protein selected from the group consisting of: Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6; (c) a gene encoding an ATPase protein selected from the group consisting of: V-ATPase subunit A, V-ATPase subunit B, V-ATPase subunit D, V-ATPase subunit E, Actin 6; (d) a gene encoding a ribosomal protein selected from the group consisting of: Ribosomal protein Large subunit 3, Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4; (e) a gene encoding a vesicle protein selected from the group consisting of: Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmpl, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta; (f) a gene encoding an actin; (g) a gene encoding a protein related to behaviour selected from the group consisting of: Bendless, scribbler, capa receptor, diapause hormone receptor, Ebl, Ether-a-go-go, flightless-I, foraging, juvenile hormone epoxide hydrolase, neuropeptide F, pheromone biosynthesis activating neuropeptide receptor, semaphorin la , shaker, slowmo, stress-sensitive B, turtle, twitchin, myosin heavy chain, nervana, N-synaptobrevin, nuclear hormone receptor 25, syntaxin, and bent; (h) a gene encoding a glycolysis and energy metabolism protein selected from the group consisting of: BiP, citrate synthase, glycogen synthase, hexokinase, isocitrate dehydrogenase, pyruvate dehydrogenase, phosphofructokinase, GTPase activator, GTPase Ran, sarcoplasmic Ca2+ ATPase, Ca2+ ATPase, hydrogen exporting ATPase, NADH dehydrogenase, FIFO ATPase, spermatogenesis ATPase, beta- ketoacyl synthase, pyruvate kinase, and knickkopf; (i) a gene encoding a protein turnover and mitosis protein selected from the group consisting of: cyclins including cyclin A, cyclin B, cyclin B3, cyclin C, cyclin D, cyclin E, cyclin G, cyclin H, cyclin J, cyclin K, cyclin T, and cyclin Y, Ubiquitin conjugating enzyme El, chromosome segregation protein, CDC5, COP9 signalosome, and ketel; (j) a gene encoding a gene expression protein selected from the group consisting of: cleavage polyadenylation specificity factor, ElA/CREB-binding protein, RNA helicases, RNA polymerases, Arfl, Bx42, and eft2; (k) a gene encoding a protein-folding, stress, and heat shock response protein selected from the group consisting of: protein disulfide isomerase, heat shock cognate 70, chaperonin TCP1, chaperonins, carrier protein, and HSP70; and (1) a gene encoding a miscellaneous protein selected from the group consisting of: spermatogenesis ATPase, pitchoune, and insect proteins. Several embodiments relate to a dsRNA having a length greater than that which is typical of naturally occurring regulatory small RNAs (such as endogenously produced siRNAs and mature miRNAs), / ^' . e. , the dsRNA is at least about 30 contiguous base-pairs in length. In embodiments, the dsRNA has a length of between about 50 to about 500 base- pairs. In some embodiments, the dsRNA is at least 50 base pairs in length. In embodiments, the dsRNA is formed from two separate, essentially complementary strands (e. g., where each strand is separately provided, or where each strand is encoded on a separate DNA molecule, or where the two strands are encoded on separate sections of a DNA and are separately transcribed or made separate, for example, by the action of a recombinase or nuclease), wherein at least one RNA strand includes a sequence of about 95% to about 100% (e. g, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs.:106 - 896. In embodiments, the dsRNA is blunt-ended, e. g. , two separate, equal -length strands of RNA which form the dsRNA through intermolecular hybridisation. In embodiments, the dsRNA has an overhang at one or both ends (termini), e. g. , two separate, unequal -length strands of RNA which form the dsRNA through intermolecular hybridisation; the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand. In embodiments, the dsRNA includes at least one stem-loop, e. g. , a single RNA molecule that forms a dsRNA with a "hairpin" secondary structure through intramolecular hybridization. In embodiments, the dsRNA is formed from a single self-hybridizing hairpin transcript, wherein one "arm" of the hairpin includes a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs.:106 - 896. In embodiments, the dsRNA includes multiple stem-loops, with or without spacer nucleotides between each stem-loop. The dsRNA can be chemically synthesized (e. g. , by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. The dsRNA can be chemically modified, e. g., to improve stability, ease of formulation, or efficacy. In some embodiments, the dsRNA molecule is provided in a microbial or plant cell that expresses dsRNA, or in a microbial fermentation product.

[0027] In some embodiments, mosquitocidally effective polynucleotides are generally designed to induce regulation or suppression of mosquito target genes, which can be coding sequence or non-coding sequence. Examples of such mosquito target genes include those disclosed in Tables 2 and 3. In some embodiments, mosquitocidally effective polynucleotides are designed to have a nucleotide sequence essentially identical or essentially complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a sequence of a mosquito target gene or cDNA (e. g. , SEQ ID NOs.:l - 105 and 897 - 1132) or to the sequence of RNA transcribed from a mosquito target gene.

Selection of Mosquitocidally Effective Polynucleotide by "Tiling"

[0028] Mosquitocidally effective polynucleotides need not be of the full length of a target gene, and in many embodiments are of much shorter length in comparison to the target gene. An example of a technique that is useful for selecting mosquitocidally effective polynucleotides is "tiling", or evaluation of polynucleotides corresponding to adjacent or partially overlapping segments of a target gene.

[0029] Mosquitocidally effective polynucleotides can be identified by "tiling" target genes in selected length fragments, for example, in fragments of 100 - 300 nucleotides in length, with partially overlapping regions (e. g. , of about 25 nucleotides) along the length of the target gene. In some embodiments, polynucleotide sequences are designed to correspond to (have a nucleotide identity or complementarity with) regions that are unique to the target gene; the selected region of the target gene can include coding sequence or non-coding sequence (e. g. , promoter regions, 3' untranslated regions, introns and the like) or a combination of both. In some embodiments, a set of polynucleotides is designed for each of the target genes listed in Tables 2 and 3. In some embodiments, a set of polynucleotides comprising 200 - 300 nucleotides with a sequence complementary to a fragment of a target gene having a sequence selected from SEQ ID NOs.: 1 - 105 and 897 - 1132 are designed so that each polynucleotide's sequence overlaps about 25 nucleotides of the next adjacent polynucleotide's sequence, in such a way that the set of polynucleotides in combination cover the full length of the target gene. The tiling procedure can be repeated, if desired. In some embodiments, a polynucleotide found to provide desired mosquitocidal activity can itself be subjected to a tiling procedure. For example, multiple overlapping polynucleotides are designed, each of 50 - 60 nucleotides in length and with a sequence complementary to the fragment of a target gene having a sequence selected from SEQ ID NOs.: 1 - 105 and 897 - 1132 for which a single polynucleotide of 300 nucleotides was found to be effective. Additional rounds of tiling analysis can be carried out, where polynucleotides as short as 18, 19, 20, or 21 nucleotides are tested.

[0030] In some embodiments where it is of interest to design a polynucleotide effective in suppressing multiple target genes, the multiple target gene sequences are aligned and polynucleotides designed to correspond to regions with high sequence homology in common among the multiple targets.

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

[0031] The polynucleotides are tested by any convenient means for efficacy in silencing the target gene. Examples of a suitable tests include the bioassays described herein in the Examples. Another test involves the topical application of the polynucleotide either directly to individual mosquitoes or to an environment to be protected from a mosquito infestation. One desired result of treatment with a mosquitocidally effective polynucleotide is prevention or control of a mosquito infestation, e. g. , by inducing in a mosquito a physiological or behavioural change such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development.

[0032] Mosquitocidally effective polynucleotides of any size can be used, alone or in combination, in the various compositions and methods described herein. In some embodiments, a single

mosquitocidally effective polynucleotide is used to make a composition (e. g. , a composition for topical application, or a recombinant DNA construct useful for making a transgenic plant). In other embodiments, a mixture or pool of different mosquitocidally effective polynucleotides is used; in such cases the mosquitocidally effective polynucleotides can be for a single target gene or for multiple target genes. In some embodiments, a mosquitocidally effective polynucleotide is designed to target different regions of a target gene. In some embodiments, a mosquitocidally effective polynucleotide includes multiple segments that correspond to different exon regions of a target gene, and "spacer" nucleotides which do not correspond to a target gene can optionally be used in between or adjacent to the segments.

Thermodynamic Considerations in Selecting Mosquitocidally Effective Polynucleotides

[0033] Mosquitocidally effective polynucleotides can be designed or their sequence optimised using thermodynamic considerations. For example, mosquitocidally effective polynucleotides can be selected based on the thermodynamics controlling hybridization between one nucleic acid strand (e. g. , a polynucleotide or an individual siRNA) and another (e. g. , a target gene transcript). Methods and algorithms to predict nucleotide sequences that are likely to be effective at RNAi-mediated silencing of a target gene are known in the art. Non-limiting examples of such methods and algorithms include "i- score", described by Ichihara et al. (2007) Nucleic Acids Res. , 35( 18): 123e; "Oligowalk", publicly available at rna.urmc.rochester.edu/servers/oligowalk and described by Lu et al. (2008) Nucleic Acids Res. , 36:W104-108; and "Reynolds score", described by Khovorova et al. (2004) Nature Biotechnol. , 22:326-330. Permitted mismatches

[0034] By "essentially identical" or "essentially complementary" is meant that the mosquitocidally effective polynucleotide (or at least one strand of a double -stranded polynucleotide) has sufficient identity or complementarity to the target gene or to the RNA transcribed from a target gene (e. g. , the transcript) to suppress expression of a target gene (e. g. , to effect a reduction in levels or activity of the target gene transcript and/or encoded protein). Mosquitocidally effective polynucleotides need not have 100 percent identity or complementarity to a target gene or to the RNA transcribed from a target gene to suppress expression of the target gene (e. g. , to effect a reduction in levels or activity of the target gene transcript or encoded protein, or to provide control of an insect species). In some embodiments, the mosquitocidally effective polynucleotide or a portion thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In some

embodiments, the mosquitocidally effective polynucleotide or a portion thereof is designed to be exactly identical to, or exactly complementary to, a sequence of at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In certain embodiments, an "essentially identical" polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides in either the endogenous target gene or to an RNA transcribed from the target gene. In certain embodiments, an "essentially complementary" polynucleotide has 100 percent sequence complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence

complementarity when compared to the sequence of at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides in either the target gene or RNA transcribed from the target gene.

[0035] Mosquitocidally effective polynucleotides containing mismatches to the target gene or transcript can be used in certain embodiments of the compositions and methods described herein. In some embodiments, the mosquitocidally effective polynucleotide includes 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript. In certain embodiments, a

polynucleotide of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript (/ ^' . e., 1 or 2 mismatches between the polynucleotide's contiguous nucleotides and the segment of equivalent length in the target gene or target gene's transcript). In certain embodiments, a polynucleotide of 20 or more nucleotides that contains a contiguous 19 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 21 continuous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript. In certain

embodiments, a polynucleotide of 22 or more nucleotides that contains a contiguous 21 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript.

[0036] In designing mosquitocidally effective polynucleotides with mismatches to an endogenous target gene or to an RNA transcribed from the target gene, mismatches of certain types and at certain positions that are more likely to be tolerated can be used. In certain embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used as described by Du et al.

(2005) Nucleic Acids Res., 33: 1671-1677. In some embodiments, mismatches in 19 base-pair overlap regions are located at the low tolerance positions 5, 7, 8 or 11 (from the 5' end of a 19-nucleotide target), at medium tolerance positions 3, 4, and 12-17 (from the 5' end of a 19-nucleotide target), and/or at the high tolerance positions at either end of the region of complementarity, positions 1, 2, 18, and 19 (from the 5 ' end of a 19-nucleotide target) as described by Du et al. (2005) Nucleic Acids Res. , 33: 1671-1677. Tolerated mismatches can be empirically determined in routine assays such as those described herein in the Examples.

[0037] In some embodiments, the mosquitocidally effective polynucleotides comprise additional nucleotides for reasons of stability or for convenience in cloning or synthesis. In one embodiment, the polynucleotide is a dsR A including an RNA strand with a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087 and further including an additional 5' G or an additional 3' C or both, adjacent to the segment. In another embodiment, the mosquitocidally effective polynucleotide is a double-stranded RNA including additional nucleotides to form an overhang, for example, a dsRNA including 2 deoxyribonucleotides to form a 3' overhang. Embedding Active Triggers in Neutral Sequence

[0038] In some embodiments, a sequence corresponding to the target gene and which is responsible for an observed suppression of the target gene is embedded in "neutral" sequence, e.g. , inserted into additional nucleotides that have no sequence identity or complementarity to the target gene. Neutral sequence can be desirable, e. g. , to increase the overall length of a polynucleotide. For example, it can be desirable for a mosquitocidally effective polynucleotide to be of a particular size for reasons of stability, cost-effectiveness in manufacturing, or biological activity.

[0039] In some insects, the size of a polynucleotide may affect efficacy of dietary uptake. For example, it has been reported that in the coleopteran species, Diabrotica virgifera, dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays; see Bolognesi et al. (2012) PLoS ONE 7(10): e47534. doi: 10.1371/journal.pone.0047534. Thus, in one embodiment, a 21-base-pair sequence corresponding to a mosquito target gene in Tables 2 or 3 is embedded in neutral sequence of an additional 39 base pairs, thus forming a mosquitocidally effective polynucleotide of about 60 base pairs. In another embodiment, a single 21-base-pair sequence is found to be efficacious when embedded in larger sections of neutral sequence, e. g. , where the total polynucleotide length is from about 60 to about 300 base pairs. In another embodiment, at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087 is embedded in larger sections of neutral sequence to provide a mosquitocidally effective polynucleotide. In another embodiment, segments from multiple sequences selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087 are embedded in larger sections of neutral sequence to provide a mosquitocidally effective polynucleotide.

[0040] It is anticipated that the combination of certain mosquitocidally effective polynucleotides (e. g. , dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087, or active fragments thereof) with one or more non-polynucleotide pesticidal agents will result in an improvement in prevention or control of mosquito infestations, when compared to the effect obtained with the mosquitocidally effective polynucleotide alone or the non-polynucleotide pesticidal agent alone. Routine bioassays such as the bioassays described herein in the Examples are useful for defining dose-responses for mosquito larval or adult mortality or growth inhibition using combinations of the mosquitocidally effective polynucleotide and one or more non-polynucleotide mosquitocidal agents (e. g. , an insecticidal protein or insect growth inhibitor). One of skill in the art can test combinations of mosquitocidally effective polynucleotide and non-polynucleotide mosquitocidal agents in routine bioassays to identify combinations of bioactives that are desirable for use in mosquito control.

METHODS OF MOSQUITO CONTROL [0041] Provided herein are methods of mosquito control comprising contacting a mosquito with a mosquitocidally effective polynucleotide, such as a dsRNA, designed to suppress a target gene in the mosquito.

[0042] An embodiment is a method of mosquito control including contacting a mosquito with a mosquitocidally effective polynucleotide including a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity or identity with a target gene, or an RNA transcribed from the target gene. In embodiments, the method includes contacting a mosquito with a mosquitocidally effective polynucleotide including a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of multiple target genes, or multiple alleles of a target gene, or an RNA transcribed from the target gene. In embodiments of these methods, the target gene is a gene essential to normal metabolic function or to normal reproduction in a mosquito. In embodiments, the target gene is one or more genes selected from the group consisting of: (a) a gene identified in Tables 2 and 3; (b) a gene encoding a proteasome protein selected from the group consisting of: Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6; (c) a gene encoding an ATPase protein selected from the group consisting of: V-ATPase subunit A, V- ATPase subunit B, V-ATPase subunit D, V-ATPase subunit E, Actin 6; (d) a gene encoding a ribosomal protein selected from the group consisting of: Ribosomal protein Large subunit 3,

Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4; (e) a gene encoding a vesicle protein selected from the group consisting of: Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmpl, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta; (f) a gene encoding an actin; (g) a gene encoding a protein related to behaviour selected from the group consisting of: Bendless, scribbler, capa receptor, diapause hormone receptor, Ebl, Ether-a-go-go, flightless-I, foraging, juvenile hormone epoxide hydrolase, neuropeptide F, pheromone biosynthesis activating neuropeptide receptor, semaphorin la, shaker, slowmo, stress-sensitive B, turtle, twitchin, myosin heavy chain, nervana, N-synaptobrevin, nuclear hormone receptor 25, syntaxin, and bent; (h) a gene encoding a glycolysis and energy metabolism protein selected from the group consisting of: BiP, citrate synthase, glycogen synthase, hexokinase, isocitrate dehydrogenase, pyruvate dehydrogenase, phosphofructokinase, GTPase activator, GTPase Ran, sarcoplasmic Ca2+ ATPase, Ca2+ ATPase, hydrogen exporting ATPase, NADH dehydrogenase, FIFO ATPase, spermatogenesis ATPase, beta-ketoacyl synthase, pyruvate kinase, and knickkopf; (i) a gene encoding a protein turnover and mitosis protein selected from the group consisting of: cyclins including cyclin A, cyclin B, cyclin B3, cyclin C, cyclin D, cyclin E, cyclin G, cyclin H, cyclin J, cyclin K, cyclin T, and cyclin Y, Ubiquitin conjugating enzyme El, chromosome segregation protein, CDC5, COP9 signalosome, and ketel; (j) a gene encoding a gene expression protein selected from the group consisting of: cleavage polyadenylation specificity factor, E1A/CREB -binding protein, RNA helicases, RNA polymerases, Arfl, Bx42, and eft2; (k) a gene encoding a protein-folding, stress, and heat shock response protein selected from the group consisting of: protein disulfide isomerase, heat shock cognate 70, chaperonin TCP1, chaperonins, carrier protein, and HSP70; and (1) a gene encoding a miscellaneous protein selected from the group consisting of: spermatogenesis ATPase, pitchoune, and insect proteins.

[0043] Another embodiment is a method of mosquito control including contacting a mosquito with an effective amount of a mosquitocidally effective polynucleotide, one strand of which includes a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity or identity with a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the method includes contacting a mosquito with a mosquitocidally effective amount of a polynucleotide, one strand of which is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the method results in inducing RNA interference, and mosquito mortality or failure to reproduce occurs.

[0044] Another embodiment is a method of mosquito control including providing in the diet of a mosquito a mosquitocidally effective polynucleotide including a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity or identity with a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the method includes providing in the diet of a mosquito a mosquitocidally effective polynucleotide comprising a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene.

[0045] Another embodiment is a method of mosquito control including causing mortality or stunting in larvae of a mosquito by providing in the diet of the larvae at least one mosquitocidally effective polynucleotide including at least one silencing element, wherein the silencing element includes at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity or identity with a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the method includes causing mortality or stunting in larvae of a mosquito by providing in the diet of the larvae at least one mosquitocidally effective polynucleotide including at least one silencing element, wherein the silencing element includes at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the mosquitocidally effective polynucleotide comprises multiple silencing elements; each silencing element can comprise sequence that is complementary to a single target gene (e. g , multiple copies of a single silencing element sequence, or a combination of silencing elements that are complementary to different regions of a target gene) or to different target genes or different alleles of a target gene.

[0046] Another embodiment is a method of mosquito control including contacting a mosquito with a mosquitocidally effective polynucleotide including at least one segment that is identical or

complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a target gene selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the method includes contacting a mosquito with a

mosquitocidally effective polynucleotide including at least one segment that is identical or

complementary to at least 21 contiguous nucleotides of a target gene selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In

embodiments, the mosquitocidally effective polynucleotide comprises multiple segments; each segment can include sequence that is complementary to a single target gene (e. g , multiple copies of a single segment, or a combination of segments that are complementary to different regions of a target gene) or to different target genes or different alleles of a target gene.

[0047] Another embodiment is a method of mosquito control including contacting an environment infested by the mosquito with a composition including a mosquitocidally effective polynucleotide including a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene, thereby preventing or decreasing infestation by the mosquito. In embodiments, the method includes contacting an environment infested by the mosquito with a composition including a mosquitocidally effective polynucleotide including a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. [0048] In embodiments of these methods, the mosquito is an Aedes sp., a Culex sp., or an Anopheles sp. In embodiments of these methods, (a) the mosquitoes are Aedes aegypti and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs.:l - 36 and 897 - 975; or (b) the mosquitoes are Culex quinquefasciatus and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs.:37 - 71 and 1014 and 1060 - 1132; or (c) the mosquitoes are

Anopheles gambiae or Anopheles gambiae species complex and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs.:72 - 105 and 976 - 1013 and 1015 - 1059.

[0049] In embodiments of these methods, the mosquito is an adult, pupa, larva, or egg. In

embodiments the method is effective on multiple life-stages of mosquitoes. In embodiments of these methods, contacting or ingesting the mosquitocidally effective polynucleotide, such as a dsRNA, results in mortality or stunting of the mosquito, or failure of the mosquito to reproduce, or failure of the mosquito to progress through metamorphosis. In embodiments of these methods, the mosquito is in a population of mosquitoes and the population size or reproductive fitness is reduced.

[0050] In embodiments of these methods, the mosquitocidally effective polynucleotide is provided in an ingestible composition, a sprayable composition, a particulate or powder, a solid bait, or a liquid bait. In embodiments of these methods, contacting includes oral delivery to the mosquito, or includes non-oral delivery to the mosquito, or includes a combination of oral and non-oral delivery to the mosquito. In embodiments of these methods, the contacting includes providing the mosquitocidally effective polynucleotide in a composition that is ingested or contacted by the mosquito. In

embodiments of these methods, the composition includes a solid, liquid, powder, suspension, emulsion, colloid, spray, encapsulation, microbeads, carrier particulates, granules, film, gel, or matrix. In embodiments of these methods, the composition includes a bacterial or eukaryotic cell expressing the mosquitocidally effective polynucleotide. In embodiments of these methods, the composition is provided in the form of a bait (such as a sugar bait), a trap, a sprayable liquid, a concentrate, dunks, dispersible granules, or ingestible particulates or cells. In embodiments of these methods, the composition further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a lipid (such as cationic lipids, dendrimers, neutral lipids, fatty acids, or compositions comprising lipid components such as liposomes), a sugar, a non-polynucleotide mosquitocide, a diptericidal Bacillus thuringiensis toxin (or other insecticidal bacterially produced toxin), a safener, a mosquito attractant, a pheromone, and an insect growth regulator such as a ecdysis inhibitor. Embodiments of the mosquito control methods include providing mosquitocidally effective polynucleotides in the form of attractive toxic sugar baits, or as larvicidal formulations (e. g. , dispersible powders or granules or dunks, or sprayable liquids). In some embodiments the composition further comprises a cationic carbohydrate. In some embodiments the composition further comprises a cationic starch. In some embodiments the composition further comprises a cationic guar. In some embodiments the composition further comprises a branched Polyethylenimine. In embodiments, methods utilizing a combination of a mosquitocidally effective polynucleotide designed to suppress a mosquito target gene and a insecticidal protein or an insect growth regulator provides a level of mosquito control that is greater than the effects of the polynucleotide and the insecticidal protein or an insect growth regulator components if tested separately.

[0051] In embodiments of these methods, the mosquitocidally effective polynucleotide is a double- stranded RNA (dsR A). In embodiments, the dsR A (a) is blunt-ended, or (b) has an overhang (e. g., an overhang of 1, 2, 3, or 4 nucleotides) at one terminus or both termini, and/or (c) includes at least one stem -loop. In embodiments, the dsRNA includes at least one stem -loop with additional secondary structure, such as mismatches or additional nucleotides within the "stem" of the stem-loop structure. In embodiments, the dsRNA is produced by (a) chemical synthesis, or (b) expression in a microorganism, or (c) expression in a eukaryotic cell. In embodiments, the dsRNA is chemically modified.

[0052] In embodiments of these methods, the mosquitocidally effective polynucleotide is a double- stranded RNA that includes a strand including at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides that are identical or complementary to a sequence selected from the group consisting of: SEQ ID NOs.:106 - 896. In specific embodiments of these methods, (a) the mosquitoes are Aedes aegypti and the double -stranded RNA includes one strand which is complementary to at least 21 contiguous nucleotides of a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:106 - 382 and 1133 - 1440; or (b) the mosquitoes are Culex quinquefasciatus and the double- stranded RNA includes one strand which is complementary to at least 21 contiguous nucleotides of a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:383 - 647 and 1613 - 1614 and 1762 - 2087; or (c) the mosquitoes are Anopheles gambiae or Anopheles gambiae species complex and the double -stranded RNA includes one strand which is complementary to at least 21 contiguous nucleotides of a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:648 - 896 and 1441 - 1612 and 1615 - 1761. MOSQUITOCIDAL COMPOSITIONS AND POLYNUCLEOTIDES

[0053] Provided herein are mosquitocidal compositions including a mosquitocidally effective amount of a recombinant polynucleotide, such as a dsRNA, designed to suppress a target gene in a mosquito.

[0054] An embodiment is a mosquitocidal composition including a mosquitocidally effective amount of a recombinant polynucleotide including at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g., about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a mosquito target gene, or an RNA transcribed from the mosquito target gene. In embodiments, the mosquitocidal composition includes a recombinant polynucleotide including a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a mosquito target gene, or an RNA transcribed from the mosquito target gene. In embodiments, the mosquitocidal composition includes a recombinant polynucleotide including a nucleotide sequence that is complementary to at least 21 contiguous nucleotides of a mosquito target gene, or an RNA transcribed from the mosquito target gene. In embodiments of these mosquitocidal compositions, the target gene is a gene essential to normal metabolic function or to normal reproduction in a mosquito. In embodiments, the target gene is selected from the group consisting of: (a) a gene identified by name in Tables 2 and 3; (b) a gene encoding a proteasome protein selected from the group consisting of: Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6; (c) a gene encoding an ATPase protein selected from the group consisting of: V- ATPase subunit A, V-ATPase subunit B, V-ATPase subunit D, V-ATPase subunit E, Actin 6; (d) a gene encoding a ribosomal protein selected from the group consisting of: Ribosomal protein Large subunit 3, Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4; (e) a gene encoding a vesicle protein selected from the group consisting of: Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmpl, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta; (f) a gene encoding an actin; (g) a gene encoding a protein related to behaviour selected from the group consisting of: Bendless, scribbler, capa receptor, diapause hormone receptor, Ebl, Ether-a-go-go, flightless-I, foraging, juvenile hormone epoxide hydrolase, neuropeptide F, pheromone biosynthesis activating neuropeptide receptor, semaphorin la, shaker, slowmo, stress-sensitive B, turtle, twitchin, myosin heavy chain, nervana, N-synaptobrevin, nuclear hormone receptor 25, syntaxin, and bent; (h) a gene encoding a glycolysis and energy metabolism protein selected from the group consisting of: BiP, citrate synthase, glycogen synthase, hexokinase, isocitrate dehydrogenase, pyruvate dehydrogenase, phosphofructokinase, GTPase activator, GTPase Ran, sarcoplasmic Ca2+ ATPase, Ca2+ ATPase, hydrogen exporting ATPase, NADH dehydrogenase, FIFO ATPase, spermatogenesis ATPase, beta-ketoacyl synthase, pyruvate kinase, and knickkopf; (i) a gene encoding a protein turnover and mitosis protein selected from the group consisting of: cyclins including cyclin A, cyclin B, cyclin B3, cyclin C, cyclin D, cyclin E, cyclin G, cyclin H, cyclin J, cyclin K, cyclin T, and cyclin Y, Ubiquitin conjugating enzyme El, chromosome segregation protein, CDC5, COP9 signalosome, and ketel; (j) a gene encoding a gene expression protein selected from the group consisting of: cleavage polyadenylation specificity factor, EIA/CREB -binding protein, RNA helicases, RNA polymerases, Arfl, Bx42, and eft2; (k) a gene encoding a protein-folding, stress, and heat shock response protein selected from the group consisting of: protein disulfide isomerase, heat shock cognate 70, chaperonin TCPl, chaperonins, carrier protein, and HSP70; and (1) a gene encoding a miscellaneous protein selected from the group consisting of: spermatogenesis ATPase, pitchoune, and insect proteins. In embodiments, the mosquitocidal composition includes a recombinant polynucleotide including a nucleotide sequence that is complementary to at least 18 contiguous nucleotides of multiple target genes (or their transcripts), or multiple alleles of a target gene (or its transcript), wherein the target gene is selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132.

[0055] Another embodiment is a mosquitocidal composition including a mosquitocidally effective amount of a recombinant polynucleotide including a nucleotide sequence that is complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the mosquitocidal composition includes a mosquitocidally effective amount of a recombinant polynucleotide including a nucleotide sequence that is

complementary to at least 21 contiguous nucleotides of a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the mosquitocidal composition includes a mosquitocidally effective amount of a recombinant polynucleotide including a nucleotide sequence that is

complementary to at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of multiple target genes (or their transcripts), or multiple alleles of a target gene (or its transcript), wherein the target gene is selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132.

[0056] In embodiments of these mosquitocidal compositions, mosquitocidally effective polynucleotide is provided in a microbial or eukaryotic cell that expresses the recombinant polynucleotide, or in a microbial fermentation product. In embodiments of these mosquitocidal compositions, the composition is provided as a solid, liquid, powder, suspension, emulsion, colloid, spray, encapsulation, microbeads, carrier particulates, granules, film, gel, or matrix. In embodiments of these mosquitocidal

compositions, the composition is provided in the form of a bait (such as a sugar bait), a trap, a sprayable liquid, a concentrate, dunks, dispersible granules, or ingestible particulates or cells. In embodiments of these mosquitocidal compositions, the composition further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a lipid (such as cationic lipids, dendrimers, neutral lipids, fatty acids, or compositions comprising lipid components such as liposomes), a sugar, a non-polynucleotide mosquitocide, a diptericidal Bacillus thuringiensis toxin (or other insecticidal bacterially produced toxin), a safener, a mosquito attractant or pheromone, and an insect growth regulator such as a ecdysis inhibitor. Embodiments of attractive toxic sugar baits formulated with dsRNAs are described in the Examples. Other embodiments are larvicidal formulations such as dispersible powders or granules or dunks, or sprayable liquids, which are applied to mosquito breeding areas. In some embodiments the composition further comprises a cationic carbohydrate. In some embodiments the composition further comprises a cationic starch. In some embodiments the composition further comprises a cationic guar. In some embodiments the composition further comprises a branched Polyethylenimine. In embodiments, a mosquitocidal composition including a polynucleotide designed to suppress a mosquito target gene and at least one of a insecticidal bacterially produced toxin or an insect growth regulator provides a level of insect control that is greater than the sum of the effects of the polynucleotide and the insecticidal bacterially produced toxin or an insect growth regulator components if tested separately.

[0057] In embodiments of these mosquitocidal compositions, the recombinant polynucleotide is a dsRNA. In embodiments, the dsRNA includes a strand including at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous ribonucleotides of a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087. In embodiments, the dsRNA includes a strand including at least 21 contiguous ribonucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087. In embodiments, the dsRNA includes a strand including multiple segments each at least 21 contiguous ribonucleotides of at least one RNA sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087. In embodiments, the dsRNA (a) is blunt-ended, or (b) has an overhang (e. g , an overhang of 1, 2, 3, or 4 nucleotides) at one terminus or both termini, and/or (c) includes at least one stem-loop. In embodiments, the dsRNA includes at least one stem-loop with additional secondary structure, such as mismatches or additional nucleotides within the "stem" of the stem -loop structure. In embodiments, the dsRNA is produced by (a) chemical synthesis, or (b) expression in a microorganism, or (c) expression in a eukaryotic cell. In embodiments, the dsRNA is chemically modified.

[0058] Embodiments of the mosquitocidal compositions intended for dispersal by spraying onto plants or in an outdoor environment optionally include the appropriate stickers and wetters for efficient surface coverage as well as UV protectants to protect polynucleotides, such as dsRNAs, from UV damage. Such additives are commonly used in the bioinsecticide industry and are known to those skilled in the art. Embodiments of the compositions include solid granular formulations that can be dispersed on the surface of standing water or other environments that contain mosquito larvae or where adult mosquitoes breed or feed. Embodiments of the compositions include solid baits, such as the attractive toxic sugar baits described in the Examples. In some embodiments the composition further comprises a cationic carbohydrate. In some embodiments the composition further comprises a cationic starch. In some embodiments the composition further comprises a cationic guar. In some embodiments the composition further comprises a branched Polyethylenimine.

[0059] Embodiments of mosquitocidal compositions include a "transfer agent" that, when combined with a composition including a mosquitocidally effective polynucleotide described herein that is topically applied to or comes into contact with the surface of a mosquito, enables the polynucleotide to enter the cells of the mosquito. Such transfer agents can be incorporated as part of the composition including a mosquitocidally effective polynucleotide, or can be applied prior to, contemporaneously with, or following application of the composition including a mosquitocidally effective polynucleotide as described herein. Suitable transfer agents include agents that increase permeability of the exterior of the mosquito or that increase permeability of cells of the mosquito to mosquitocidally effective polynucleotides. Examples of transfer agents include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. Suitable transfer agents can cause the polynucleotide composition to take the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition. Examples of transfer agents include counter-ions or other molecules that are known to associate with nucleic acid molecules, e. g. , cationic lipids, inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, cationic starches and other cations. Embodiments of transfer agents include organic solvents such as DMSO, DMF, pyridine, N- pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents miscible with water or that dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Embodiments of transfer agents include naturally derived or synthetic oils with or without surfactants or emulsifiers, e. g. , plant-sourced oils, crop oils (such as those listed in the 9 ^th

Compendium of Herbicide Adjuvants, publicly available on-line at herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine. Embodiments of transfer agents include organosilicone preparations. For example, a suitable transfer agent is an organosilicone preparation that is

commercially available as SILWET L-77® brand surfactant having CAS Number 27306-78-1 and EPA Number: CAL.REG.NO. 5905-50073-AA, and currently available from Momentive Performance Materials, Albany, New York. Organosilicone compounds useful as transfer agents include, but are not limited to, compounds that include: (a) a trisiloxane head group that is covalently linked to, (b) an alkyl linker including, but not limited to, an ^-propyl linker, that is covalently linked to, (c) a polyglycol chain, that is covalently linked to, (d) a terminal group.

RECOMBINANT DNA CONSTRUCTS

[0060] Provided herein are recombinant DNA constructs including a heterologous promoter operably linked to DNA encoding an RNA transcript designed to suppress a target gene in a mosquito.

[0061] An embodiment is a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript including a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs.:l - 105 and 897 - 1132.

[0062] An embodiment is a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript including at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the recombinant DNA construct includes a heterologous promoter operably linked to DNA encoding an RNA transcript including at least 21 contiguous nucleotides that are complementary to a target gene having a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene. In embodiments, the recombinant DNA construct includes a heterologous promoter operably linked to DNA encoding an RNA transcript including multiple segments each of at least 21 contiguous nucleotides that are complementary to a target gene comprising a nucleotide sequence selected from the group consisting of: SEQ ID NOs.:l - 105 and 897 - 1132, or an RNA transcribed from the target gene; each segment can include sequence that is complementary to a single target gene (e. g. , multiple copies of a single segment, or a combination of segments that are complementary to different regions of a target gene) or to different target genes or different alleles of a target gene; the segments need not be immediately contiguous in the RNA transcript.

[0063] An embodiment is a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript including at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous ribonucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087. In embodiments, the recombinant DNA construct includes a heterologous promoter operably linked to DNA encoding an RNA transcript including at least 21 contiguous ribonucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087. In embodiments, the recombinant DNA construct includes a heterologous promoter operably linked to DNA encoding an RNA transcript including multiple segments each of at least 21 contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087; in embodiments the RNA transcript includes multiple copies of a single segment, or a combination of segments that are complementary to different regions of an RNA sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087, or to different RNA sequences selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087, or different alleles of an RNA sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087; the segments need not be immediately contiguous in the RNA transcript.

[0064] An embodiment is a recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript including a strand including a sequence selected from the group consisting of: SEQ ID NOs.:106 - 896 and 1133 - 2087. In embodiments, the recombinant DNA construct includes a heterologous promoter operably linked to DNA encoding an RNA transcript including a strand consisting of a sequence selected from the group consisting of: SEQ ID NOs.:106 - 896 and 1133 - 2087.

[0065] In embodiments of these recombinant DNA constructs, the RNA transcript includes dsRNA. In embodiments, the RNA transcript is essentially completely double-stranded (e. g. , an RNA transcript that self-hybridizes to form a hairpin structure with little or no single-stranded RNA outside of the loop, or two separate, hybridized strands of RNA). In embodiments, the dsRNA is blunt-ended. In embodiments, the dsRNA has an overhang at one or both ends (termini), e. g. , two separate, unequal- length strands of RNA which form the dsRNA through intermolecular hybridisation; the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand. In embodiments, the dsRNA includes at least one stem-loop, e. g. , a single RNA molecule that forms a dsRNA with a "hairpin" secondary structure through intramolecular hybridization. In embodiments, the dsRNA includes at least one stem-loop with additional secondary structure, such as mismatches or additional nucleotides within the "stem" of the stem-loop structure. In embodiments, the dsRNA is formed from a single self-hybridizing hairpin transcript, wherein one "arm" of the hairpin includes a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087. In embodiments, the dsRNA includes multiple stem-loops, with or without spacer nucleotides between each stem-loop.

[0066] In embodiments, the RNA transcript includes both single-stranded and double -stranded regions. In embodiments, the recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript which includes dsRNA encoded on a single RNA strand (/ ^' . e. , where at least part of the RNA transcript self-hybridizes). In embodiments, the recombinant DNA construct including a heterologous promoter operably linked to DNA encoding an RNA transcript which includes dsRNA encoded on multiple RNA strands, e. g. , where the RNA transcript is further processed (for example by endonucleases) into multiple RNAs, or wherein the recombinant DNA construct includes two heterologous promoters, each operably linked to DNA encoding a RNA transcript that forms part of a dsRNA.

[0067] In embodiments, the heterologous promoter used in the recombinant DNA construct is a promoter functional for expression of the RNA transcript in a bacterium, for example, in a bacterium selected from the group consisting of Escherichia coli, Bacillus species, Pseudomonas species, Xenorhabdus species, or Photorhabdus species. In embodiments, the heterologous promoter used in the recombinant DNA construct is a promoter functional in an eukaryotic cell, for example a promoter functional in yeast cells, insect cells, or plant cells. The recombinant DNA constructs are useful for the production of mosquitocidally effective polynucleotides described in the Examples, for use in compositions and methods of controlling mosquitoes. For example, the recombinant DNA constructs are useful in in vitro transcription, or for expression of the desired RNA molecule through microbial fermentation (e. g. , by expression in E. coli or other bacterium or in a yeast culture) or in a eukaryotic cell (e. g. , a plant cell or an insect cell).

[0068] In embodiments, the recombinant DNA construct also encodes additional elements, which can be operably linked to the promoter that drives expression of the DNA encoding an RNA transcript designed to suppress a target gene in a mosquito, or can be linked to a separate promoter. Examples of such elements include DNA encoding an insecticidal protein (e. g., a. Bacillus thuringiensis insecticidal protein) or DNA encoding an ecdysis inhibitor.

EXAMPLES

Example 1 [0069] This example illustrates non-limiting embodiments of coding DNA sequences useful as target genes for controlling insect species and for making mosquitocidal compositions, and identifies mosquitocidally effective polynucleotides. More specifically, target genes identified by name

(annotation) and sequence identifier (SEQ ID NO.) for controlling mosquitoes are provided in SEQ ID NOs.:l - 105, and mosquitocidally effective polynucleotide sequences ranging in size from 218 to 276 bp and designed to suppress these target genes are provided in SEQ ID NOs.:106 - 896.

[0070] Target gene sequences with the following sequence identifiers were identified from cDNAs sourced from transcriptomes of three mosquito species: SEQ ID NOs.:l - 36, Aedes aegypti (yellow fever mosquito), SEQ ID NOs.:37 - 71, Culex quinquefasciatus (southern house mosquito), and SEQ ID NOs.:72 - 105, Anopheles gambiae (or Anopheles gambiae species complex). The mosquito target genes included genes encoding: proteasome proteins (Proteasome Alpha 2, Proteasome Beta 5, Proteasome regulatory subunit 2, Proteasome regulatory subunit 3, Proteasome regulatory subunit 8, Proteasome regulatory subunit 9, Proteasome regulatory GTPase SARI, 26S protease regulatory subunit 6), ATPase proteins (V-ATPase subunit A, V-ATPase subunit B, V-ATPase subunit D, V- ATPase subunit E, Actin 6), ribosomal proteins (Ribosomal protein Large subunit 3, Ribosomal protein Large subunit 7, Ribosomal protein Large subunit 19, Ribosomal protein Large subunit 40, Ribosomal protein Small subunit 21, Ribosomal protein Small subunit 4), vesicle proteins (Vesicle Sorting protein 2, Vesicle Sorting protein 4, Vesicle Sorting protein 16A, Vesicle Sorting protein 20, Vesicle Sorting protein 24, Vesicle Sorting protein 27, Vesicle Sorting protein 28, Vesicle Sorting protein Shrb (Snf7), Vesicle Sorting protein Chmpl, Secretory vesicle protein 6, Secretory vesicle protein 23, Coatomer Protein Alpha, Coatomer Protein Beta, Coatomer Protein Beta Prime, Coatomer Protein Delta, Coatomer Protein Epsilon, Coatomer Protein Zeta), and actin (Actin 6). For each target gene, individual siRNAs were selected using an algorithm publicly available in the software RNAstructure (Reuter and Mathews (2010) BMC Bioinformatics, 11 : 129, doi: 10.1186/ 1471 -2105 - 11 - 129) and the longer dsRNA or suppression sequences of a user-defined length and incorporating the selected siRNAs (Table 2) were designed using a proprietary algorithm and defined criteria; in general, multiple mosquitocidally effective polynucleotides were designed for a given target gene. The dsRNA or suppression sequences include SEQ ID NOs.:106 - 382, designed to target Aedes aegypti (yellow fever mosquito), SEQ ID NOs.:383 - 647, designed to target Culex quinquefasciatus (southern house mosquito), and SEQ ID NOs.:648 - 896, designed to target Anopheles gambiae (or Anopheles gambiae species complex).

Table 2

Ribosomal protein Large

subunit 3 3 113 - 122 39 397 - 406 74 664 - 671

Vesicle Sorting protein 16 A 4 123 - 132 40 407 - 417 75 672 - 682

Ribosomal protein Large

subunit 19 5 133 - 138 41 418 - 423 76 683 - 687

26 S protease regulatory subunit

6 6 139 - 148 42 424 - 434 77 688 - 696

Coatomer Protein Beta 7 149 - 157 43 435 - 445 78 697 - 701

Vesicle sorting protein Shrb

(Snf7) 8 158 - 163 44 446 - 451 79 702 - 707

Coatomer Protein Beta Prime 9 164 - 174 45 452 - 460 80 708 - 717

Ribosomal protein Small

subunit 21 10 175 46 461 81 718

Ribosomal protein Small

subunit 4 11 176 - 185 47 462 - 468 82 719 - 725

V-ATPase subunit A 12 186 - 195 48 469 - 478 83 726 - 736

Ribosomal protein Large

subunit 40 13 196 - 200 49 479 - 484 84 737 - 743

Proteasome Regulatory Subunit

2 14 201 - 210 50 485 - 495 85 744 - 753

Secretory vesicle protein 23 15 211 - 221 51 496 - 506 86 754 - 761

Proteasome Alpha 2 16 222 - 228 52 507 - 511 87 762 - 764

Actin 6 17 229 - 239 53 512 - 522 88 765 - 773

Secretory vesicle protein 6 18 240 - 250 54 523 - 531 89 774 - 781

Coatomer Protein Alpha 19 251 - 260 55 532 - 542 90 782 - 792

Coatomer Protein Delta 20 261 - 271 56 543 - 552 91 793 - 801

Coatomer Protein Epsilon 21 272 57 553 - 558 92 802 - 812

Coatomer Protein Zeta 22 273 - 275 — — — —

Ribosomal Protein Large

Subunit 7 23 276 - 282 58 559 - 566 93 813 - 822

Proteasome Regulatory Subunit

3 24 283 - 293 59 567 - 575 94 823 - 833

Proteasome Regulatory Subunit

8 25 294 - 301 60 576 - 583 95 834 - 837

Proteasome Regulatory Subunit

9 26 302 - 311 61 584 - 590 96 838 - 845

Proteasome regulatory GTPase

SARI 27 312 - 317 62 591 - 595 97 846 - 848

V-ATPase subunit B 28 318 - 327 63 596 - 606 98 849 - 858

V-ATPase subunit D 29 328 - 333 64 607 - 614 99 859 - 866

Vesicle Sorting protein 2 30 334 - 342 65 615 - 618 100 867 - 873

Vesicle Sorting protein 4 31 343 - 352 66 619 - 627 101 874 - 878

Vesicle Sorting protein 20 32 353 - 359 67 628 - 630 102 879 - 884

Vesicle Sorting protein 24 33 360 68 631 103 885

Vesicle Sorting protein 27 34 361 - 368 69 632 - 640 104 886 - 893

Vesicle Sorting protein 28 35 369 - 376 70 641 - 644 105 894 - 896

Vesicle Sorting Protein Chmpl 36 377 - 382 71 645 - 647 — — anti-sense nucleotide sequence complementary to the Target Gene (e.g., nucleotide sequence of the anti-sense strand of a dsRNA)

[0071] The mosquito target genes include genes encoding the following protein groups and subgroups:

(1) proteasome proteins including Proteasome Alpha 2 (e. g. , proteins encoded by SEQ ID

NOs.: 16, 52, 87), Proteasome Beta 5 (e. g , proteins encoded by SEQ ID NOs.:l, 37, 72), Proteasome regulatory subunit 2 (e. g. , proteins encoded by SEQ ID NOs.:14, 50, 85),

Proteasome regulatory subunit 3 (e. g. , proteins encoded by SEQ ID NOs.:24, 59, 94),

Proteasome regulatory subunit 8 (e. g. , proteins encoded by SEQ ID NOs.:25, 60, 95), Proteasome regulatory subunit 9 (e. g. , proteins encoded by SEQ ID NOs.:26, 61, 96), Proteasome regulatory GTPase SARI (e. g. , proteins encoded by SEQ ID NOs.:27, 62, 97), and 26S protease regulatory subunit 6 (e. g. , proteins encoded by SEQ ID NOs.:6, 42, 77);

(2) ATPase proteins including V-ATPase subunit A (e. g. , proteins encoded by SEQ ID NOs.: 12, 48, 83), V-ATPase subunit B (e. g. , proteins encoded by SEQ ID NOs.:28, 63, 98), V-ATPase subunit D (e. g. , proteins encoded by SEQ ID NOs.:29, 64, 99), V-ATPase subunit E (e. g. , proteins encoded by SEQ ID NOs.:2, 38, 73), Actin 6 (e. g. , proteins encoded by SEQ ID NOs.: 17, 53, 88);

(3) ribosomal proteins including (a) large ribosomal subunit proteins, e. g., Ribosomal protein Large subunit 3 (e. g. , proteins encoded by SEQ ID NOs.:3, 39 , 74), Ribosomal protein Large subunit 7 (e. g. , proteins encoded by SEQ ID NOs.:23, 58, 93), Ribosomal protein Large subunit 19 (e. g. , proteins encoded by SEQ ID NOs.:5, 41, 76), Ribosomal protein Large subunit 40 (e. g. , proteins encoded by SEQ ID NOs.:13, 49, 84); and (b) small ribosomal subunit proteins, e. g. , Ribosomal protein Small subunit 21 (e. g. , proteins encoded by SEQ ID NOs.: 10, 46, 81), Ribosomal protein Small subunit 4 (e. g. , proteins encoded by SEQ ID NOs.: ll, 47, 82);

(4) vesicle proteins including (a) vesicle sorting proteins, e. g. , Vesicle Sorting protein 2 (e. g. , proteins encoded by SEQ ID NOs.:30, 65, 100), Vesicle Sorting protein 4 (e. g. , proteins encoded by SEQ ID NOs.:31, 66, 101), Vesicle Sorting protein 16A (e. g. , proteins encoded by SEQ ID NOs.:4, 40, 75), Vesicle Sorting protein 20 (e. g. , proteins encoded by SEQ ID

NOs.:32, 67, 102), Vesicle Sorting protein 24 (e. g. , proteins encoded by SEQ ID NOs.:33, 68, 103), Vesicle Sorting protein 27 (e. g. , proteins encoded by SEQ ID NOs.:34, 69, 104), Vesicle Sorting protein 28 (e. g. , proteins encoded by SEQ ID NOs.:35, 70, 105), Vesicle Sorting protein Shrb (Snf7) (e. g. , proteins encoded by SEQ ID NOs.:8, 44, 79), Vesicle Sorting protein Chmp l (e. g. , proteins encoded by SEQ ID NOs.:36, 71); (b) secretory vesicle proteins, e. g. , Secretory vesicle protein 6 (e. g. , proteins encoded by SEQ ID NOs.: 18, 54, 89), Secretory vesicle protein 23 (e. g. , proteins encoded by SEQ ID NOs.: 15, 51, 86); and (c) coatomer proteins, e. g. , Coatomer Protein Alpha (e. g. , proteins encoded by SEQ ID

NOs.: 19, 55, 90), Coatomer Protein Beta (e. g. , proteins encoded by SEQ ID NOs.:7, 43, 78), Coatomer Protein Beta Prime (e. g. , proteins encoded by SEQ ID NOs.:9, 45, 80), Coatomer Protein Delta (e. g. , proteins encoded by SEQ ID NOs.:20, 56, 91), Coatomer Protein Epsilon (e. g. , proteins encoded by SEQ ID NOs.:21, 57, 92), Coatomer Protein Zeta (e. g. , proteins encoded by SEQ ID NO:22); and

(5) actin including Actin 6 (e. g. , proteins encoded by SEQ ID NOs.:17, 53, 88).

[0072] The polynucleotide sequences include nucleotide sequences encoding double-stranded RNA for silencing target genes that encode: (1) proteasome proteins including Proteasome Alpha 2 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:222 - 228, 507 - 511, 762 - 764), Proteasome Beta 5 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:106 - 110, 383 - 393, 648 - 658), Proteasome regulatory subunit 2 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:201 - 210, 485 - 495, 744 - 753), Proteasome regulatory subunit 3 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:283 - 293, 567 - 575, 823 - 833), Proteasome regulatory subunit 8 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:294 - 301, 576 - 583, 834 - 837), Proteasome regulatory subunit 9 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:302 - 311, 584 - 590, 838 - 845), Proteasome regulatory GTPase SARI (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:312 - 317, 591 - 595, 846 - 848), and 26S protease regulatory subunit 6 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 139 - 148, 424 - 434, 688 - 696);

(2) ATPase proteins including V-ATPase subunit A (e. g , dsRNA with an anti -sense strand

encoded by any of SEQ ID NOs.:186 - 195, 469 - 478, 726 - 736), V-ATPase subunit B (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:318 - 327, 596 - 606, 849 - 858), V-ATPase subunit D (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:328 - 333, 607 - 614, 859 - 866), V-ATPase subunit E (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:lll, 112, 394 - 396, 659 - 663), Actin 6 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:229 - 239, 512 - 522, 765 -

773);

(3) ribosomal proteins including (a) large ribosomal subunit proteins, e. g, Ribosomal protein Large subunit 3 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 113 - 112, 397 - 406, 664 - 671), Ribosomal protein Large subunit 7 (e. g , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:276 - 282, 559 - 566, 813 - 822), Ribosomal protein Large subunit 19 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 133 - 138, 418 - 423, 683 - 687), Ribosomal protein Large subunit 40 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:196 - 200, 479 - 484, 737 - 743); and (b) small ribosomal subunit proteins, e. g , Ribosomal protein Small subunit 21 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:175, 461, 718), Ribosomal protein Small subunit 4 (e. g. , dsRNA with an anti -sense strand encoded by any of SEQ ID NOs.: 176 - 185, 462 - 468, 719 - 725);

(4) vesicle proteins including (a) vesicle sorting proteins, e. g , Vesicle Sorting protein 2 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:334 - 342, 615 - 618, 867 - 873), Vesicle Sorting protein 4 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ

ID NOs.:343 - 352, 619 - 627, 874 - 878), Vesicle Sorting protein 16A (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:123 - 132, 407 - 417, 672 - 682), Vesicle Sorting protein 20 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:353 - 359, 628 - 630, 879 - 884), Vesicle Sorting protein 24 (e. g , dsR A with an anti- sense strand encoded by any of SEQ ID NOs.:360, 631, 885), Vesicle Sorting protein 27 (e. g, dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:361 - 368, 632 - 640, 886 - 893), Vesicle Sorting protein 28 (e. g, dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:369 - 376, 641 - 644, 894 - 896), Vesicle Sorting protein Shrb (Snf7) (e. g, dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:158 - 163, 446 - 451, 702 - 707), Vesicle Sorting protein Chmp 1 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:377 - 382, 645 - 647); (b) secretory vesicle proteins, e. g , Secretory vesicle protein 6 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:240 - 250, 523 - 531, 774 - 781), Secretory vesicle protein 23 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:211 - 221, 496 - 506, 754 - 761); and (c) coatomer proteins, e. g , Coatomer Protein Alpha (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:251 - 260, 532 - 542, 782 - 792), Coatomer Protein Beta (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:149 - 157, 435 - 445, 697 - 701), Coatomer Protein Beta Prime (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:164 - 174, 452 - 460, 708 - 717), Coatomer Protein Delta (e. g , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:261 - 271, 543 - 552, 793 - 801), Coatomer Protein Epsilon (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:272, 553 - 558, 802 - 812), Coatomer Protein Zeta (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:273 - 275); and

(5) actin including Actin 6 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:229 - 239, 512 - 522, 765 - 773).

[0073] The embodiments of polynucleotide sequences provided in Table 2 are generally useful for RNA-mediated suppression of the corresponding target gene identified in Table 2. These

polynucleotides are useful for controlling insects, especially mosquitoes, including the source species from which the target genes in Table 2 were identified. RNA-mediated suppression of one or more of the target genes provided in Table 2, or use of one or more of the polynucleotides provided in Table 2, is useful for causing mortality or stunting, or otherwise controlling, target mosquitoes, especially mosquito species in the following genera: Aedes, Culex, and Anopheles .

[0074] In embodiments, compositions including a mosquitocidally effective polynucleotide for suppression of one or more of the target genes provided in Table 2 (e. g , a composition including an insecticidally effective amount of one or more of the polynucleotides provided in Table 2) are useful for controlling mosquito species, including mosquito species in the following genera: Aedes, Culex, and Anopheles. For example, a composition including an insecticidally effective amount of one or more of the polynucleotides provided in Table 2 is useful for inducing growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development, in mosquito species, including mosquito species in the following genera: Aedes, Culex, and Anopheles.

Compositions include embodiments insecticidally effective on adult mosquitoes, larval mosquitoes, or mosquito eggs, or on combinations thereof.

[0075] In embodiments, RNA-mediated suppression of one or more of the target genes provided in Table 2, or use of one or more of the polynucleotides provided in Table 2, is useful for inducing growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development, in mosquito species, including mosquito species in the following genera: Aedes, Culex, and Anopheles. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs.:l - 36 is used to cause mortality or stunting in Aedes spp., such as Aedes aegypti adults or larvae, for example, by contacting Aedes aegypti adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs.:106 - 382. In embodiments, RNA-mediated suppression of one or more of the target genes comprising a sequence selected from the group consisting of SEQ ID NOs.:37 - 71 is used to cause mortality or stunting in Culex spp., such as Culex quinquefasciatus adults or larvae, for example, by contacting Culex quinquefasciatus adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs.:383 - 647. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs.:72 - 105 is used to cause mortality or stunting in Anopheles spp., such as Anopheles gambiae adults or larvae, for example, by contacting Anopheles gambiae adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs.:648 - 896.

[0076] Adult mosquitoes feed on naturally occurring plant sugars, and are attracted to sugar-containing compositions. Thus, an aspect of this invention includes mosquito bait compositions, such as attractive toxic sugar baits (ATSBs), including an insecticidally effective amount of one or more of the polynucleotides provided in Table 2. Typically, ATSBs include an aqueous solution of a sugar (such as sucrose) as a phagostimulant, an attractant such as fruit or flower scents, and an orally available toxin; other ingredients can include fruit juice or pulp, yeast, preservatives, stabilizers, and visual dyes. ATSBs can be sprayed (e. g. , on vegetation) or provided in indoor or outdoor bait stations. Examples of ATSBs which use non-polynucleotide toxins such as Spinosad, pyrethroids, boric acid, or eugenol include those described by Miiller et al. (2010) Malaria J. , 9:210 - 216; Beier et al. (2012) Malaria J. , 11 :31 - 37; Stewart et al. (2013) PLoS ONE, 8(12):e84168; and Quails et al. (2014) Acta Tropica, 131 : 104 - 110. The disadvantage of ATSBs containing non-polynucleotide toxins is that these are nonselective and are toxic to non-target species including beneficial insects such as honey bees and other pollinators and potentially toxic to non-target species that might ingest a mosquito that has fed on the ATSB. Use of a polynucleotide such as the mosquitocidally effective polynucleotides provided herein permits a species-specific approach to mosquito control. [0077] Other embodiments include larvicidal compositions including a polynucleotide such as the mosquitocidally effective polynucleotides provided herein. Larvicidal compositions formulated as a sprayable liquid or dispersible powder or granules can be deployed in and around mosquito breeding grounds such as stagnant water, cisterns, ponds, lakes, streams, and in vegetated areas.

Example 2

[0078] This example illustrates additional non-limiting embodiments of coding DNA sequences useful as target genes for controlling mosquitoes and for making mosquitocidally effective compositions, and identifies polynucleotide sequences useful for controlling mosquitoes. More specifically, embodiments of target genes identified by name (annotation) and sequence identifier (SEQ ID NO.) (Table 3) for controlling mosquitoes are provided in SEQ ID NOs.:897 - 1132, and embodiments of polynucleotide sequences ranging in size from 98 to 305 bp and designed to suppress these target genes are provided in SEQ ID NOs.: 1133 - 2087

[0079] Target gene sequences (Table 3) with the following sequence identifiers were identified from cDNAs sourced from transcriptomes of three mosquito species: SEQ ID NOs.:897 - 975, Aedes aegypti (yellow fever mosquito), SEQ ID NOs.:1014 and 1060 - 1132, Culex quinquefasciatus (southern house mosquito), and SEQ ID NOs.:976 - 1013 and 1015 - 1059, Anopheles gambiae (or Anopheles gambiae species complex). For each target gene, individual siRNAs were selected using an algorithm publicly available in the software RNAstructure (Reuter and Mathews (2010) BMC

Bioinformatics, 11: 129, doi: 10.1186/1471-2105-11-129) and the longer polynucleotide or suppression sequences of a user-defined length and incorporating the selected siRNAs (Table 3) were designed using a proprietary algorithm and defined criteria; in general, multiple polynucleotides were designed for a given target gene. The polynucleotide or suppression sequences include SEQ ID NOs.:1133 -

1440, designed to target Aedes aegypti (yellow fever mosquito), SEQ ID NOs.:1613 - 1614 and 1762 - 2087, designed to target Culex quinquefasciatus (southern house mosquito), and SEQ ID NOs.:1441 - 1612 and 1615 - 1761, designed to target Anopheles gambiae (or Anopheles gambiae species complex).

Table 3

receptor

Ebl 905 1162-1164 1115 2014-2017 993 1521-1523

Ether-a-go-go 906 1165-1168 1117 2021-2025 995 1529-1533

Flightless-I 907 1169-1172 1113 2004-2008 991 1511-1515

Foraging 908 1173-1176 1109 1989-1992 988 1494-1498 glycogen synthase 909 1177-1180 1102 1955-1959 981 1462-1466 hexokinase 910 1181-1184 1098 1936-1939 977 1446-1448 isocitrate

dehydrogenase 911 1185-1188 1099 1940-1944 978 1449-1453

Juvenile hormone

epoxide hydrolase 912 1189-1191 1118 2026-2029 997 1539-1542

Neuropeptide F 913 1192-1194 — — 1000 1550-1554

Pheromone

biosynthesis

activating

neuropeptide

receptor 914 1195-1197 1120 2035-2037 999 1548-1549 protein disulfide

isomerase 915 1198-1201 1103 1960-1964 982 1467-1470 pyruvate

dehydrogenase 916 1202-1206 1100 1945-1949 979 1454-1456 semaphorin 2a 917 1207-1209 1116 2018-2020 994 1524-1528 shaker 918 1210-1213 1108 1984-1988 987 1489-1493 slowmo 919 1214-1215 1014 1613-1614 996 1534-1538 stress-sensitive B 920 1216-1217 1111 1996-1999 — — turtle 921 1218-1221 1114 2009-2013 992 1516-1520 twitchin 922 1222-1225 1119 2030-2034 998 1543-1547 phosphofructokinase 923 1226-1227 1121 2038-2040 1002 1559-1562 cyclin A 924 1228-1229 1122 2041-2044 1003 1563-1567 cyclin B 925 1230-1232 1123 2045-2049 1004 1568-1571 cyclin B 3 926 1233-1234 1124 2050-2054 1005 1572-1576 cyclin C 927 1236 1125 2055-2058 1006 1577-1579 cyclin D 928 1237-1238 1126 2059-2063 — — cyclin E 929 1239-1241 — — 1007 1580-1583 cyclin G 930 1242-1244 1127 2064-2068 1008 1584-1588 cyclin H 931 1245-1247 1128 2069-2071 1009 1589-1593 cyclin J 932 1248-1250 1129 2072-2074 1010 1594-1598 cyclin K 933 1251-1253 1130 2075-2078 1011 1599-1603 cyclin T 934 1254-1256 1131 2079-2082 1012 1604-1608 cyclin Y 935 1257-1259 1132 2083-2087 1013 1609-1612 cleavage

polyadenylation

specificity factor 936 1260-1264 1060 1762-1767 1015 1615-1618

RNA helicase 937 1265-1270 1061 1768-1773 1016 1619-1622

RNA polymerase 938 1271-1273 1062 1774-1777 1017 1623-1626 daf21 939 1274-1279 1063 1778-1782 1018 1627-1630 nucleic acid binding

protein 940 1280-1284 1064 1783-1787 1019 1631-1634

GTPase activator — — 1020 1635-1638

RNA polymerase — — 1021 1639

RNA polymerase II 941 1285-1288 1065 1788-1789 1033 1677-1680

GTPase Ran 942 1289-1291 1066 1790-1794 1022 1640-1643 sarcoplasmic Ca2+

ATPase 943 1292-1294 1067 1796-1798 1023 1644-1647

ElA/CREB-binding

protein — — 1068 1799-1804 1024 1648-1651

N-synaptobrevin 944 1295-1296 1069 1805-1807 1025 1652 ubiquitin

conjugating enzyme

El 945 1297-1301 1070 1808-1812 1026 1653-1656 myosin heavy chain 946 1302-1305 1071 1813-1815 1027 1657-1658 eft2 947 1306-1311 1072 1816-1821 1028 1659-1662 spermatogenesis

ATPase 948 1312-1317 1073 1822-1827 1029 1663-1666 ketel 949 1318-1323 1074 1828-1833 1030 1667-1670

FIFO ATPase 950 1324-1325 1075 1834 1031 1671-1672

ABC transporter 951 1326-1330 1076 1835-1840 1032 1673-1676 bent — — — — 1034 1681-1684 beta-ketoacyl

synthase 952 1331-1336 1077 1841-1846 1035 1685-1688

Ca2+ ATPase 953 1337-1339 1078 1847-1851 1036 1689-1692 nuclear hormone

receptor 25 954 1340-1345 1079 1852-1856 1037 1693-1696 syntaxin 955 1346-1349 1080 1857-1861 1038 1697-1698 heat shock cognate

70 956 1350-1355 1081 1862-1867 1039 1699-1702 chromosome

segregation protein 957 1356-1357 1082 1868 1040 1703-1704

RNA helicase 958 1358-1361 1083 1869-1873 1041 1705-1708 chaperonin TCPl 959 1362-1367 1084 1874-1878 1042 1709-1710

Rabl l 960 1368-1370 1085 1879-1880 1043 1711-1712

RNA helicase 961 1371-1376 — — 1044 1713-1714 knickkopf 962 1377-1381 1086 1881-1886 1045 1715-1718 pitchoune 963 1382-1385 — — 1046 1719-1722

CDC5 964 1386-1391 1087 1887-1892 1047 1723-1726 chaperonin 965 1392-1396 1088 1893-1894 1048 1727-1730

RNA helicase 966 1397-1400 1089 1895-1900 1049 1731-1732 carrier protein 967 1401-1402 — — 1050 1733-1734

Bx42 968 1403-1408 1090 1901-1905 1051 1735-1738

Art! — 1091 1906 1052 1739-1740

Insect protein 969 1409-1413 1092 1907-1913 1053 1741-1742

HSP70 970 1414-1419 1093 1914-1919 1054 1743-1746

Insect protein 971 1420-1425 — — 1055 1747-1750 chaperonin 972 1426-1431 — — 1056 1751-1754 hydrogen exporting

ATPase 973 1432 1094 1920 1057 1755

NADH

dehydrogenase 974 1433-1435 1095 1921-1926 1058 1756-1757 nervana 975 1436-1440 1096 1927-1932 1059 1758-1761 pyruvate kinase — — 1097 1933-1935 976 1441-1445 anti-sense nucleotide sequence complementary to the Target Gene (e.g., nucleotide sequence of the anti-sense strand of a dsRNA)

[0080] The mosquito target genes include genes encoding the following protein groups and subgroups:

(1) genes encoding proteins related to behaviour including Bendless (e. g. , proteins encoded by

SEQ ID NOs.:897, 1110, and 989), scribbler (e. g , proteins encoded by SEQ ID NOs.:899, 1107, and 986), capa receptor (e. g , proteins encoded by SEQ ID NOs.:901, 1112, and 990), diapause hormone receptor (e. g. , proteins encoded by SEQ ID NOs.:904 and 1001), Eb l (e. g. , proteins encoded by SEQ ID NOs.:905, 1115, and 993), Ether-a-go-go (e. g. , proteins encoded by SEQ ID NOs.:906, 1117, and 995), flightless-I (e. g , proteins encoded by SEQ ID

NOs.:907, 113, and 991), foraging (e. g , proteins encoded by SEQ ID NOs.:908, 1109, and 988), juvenile hormone epoxide hydrolase (e. g. , proteins encoded by SEQ ID NOs.:912, 1118, and 997), neuropeptide F (e. g. , proteins encoded by SEQ ID NOs.:913 and 1000), pheromone biosynthesis activating neuropeptide receptor (e. g , proteins encoded by SEQ ID NOs.:914, 1120, and 999), semaphorin la (e. g , proteins encoded by SEQ ID NOs.:917, 1116, and 994), shaker (e. g , proteins encoded by SEQ ID NOs.:918, 1108, and 987), slowmo (e. g , proteins encoded by SEQ ID NOs.:919, 1014, and 996), stress-sensitive B (e. g , proteins encoded by SEQ ID NOs.:920 and 1111), turtle (e. g. , proteins encoded by SEQ ID NOs.:921, 1114, and

992), twitchin (e. g , proteins encoded by SEQ ID NOs.:922, 1119 and 998), myosin heavy chain (e. g , proteins encoded by SEQ ID NOs.:946, 1071, and 1027), nervana (e. g , proteins encoded by SEQ ID NOs.:975, 1096, and 1059), N-synaptobrevin (e. g , proteins encoded by SEQ ID NOs.:944, 1069, and 1025), nuclear hormone receptor 25 (e. g , proteins encoded by SEQ ID NOs.:954, 1079, and 1037), syntaxin (e. g , proteins encoded by SEQ ID NOs.:955,

1080, and 1038), and bent (e. g , proteins encoded by SEQ ID NO:1034);

(2) glycolysis and energy metabolism proteins including BiP (e. g , proteins encoded by SEQ ID NOs.:898, 1106, and 985), citrate synthase (e. g , proteins encoded by SEQ ID NOs.:902, 1101, and 980), glycogen synthase (e. g , proteins encoded by SEQ ID NOs.:909, 1102, and 981), hexokinase (e. g. , proteins encoded by SEQ ID NOs.:910, 1098, and 977), isocitrate dehydrogenase (e. g , proteins encoded by SEQ ID NOs.:911, 1099, and 978), pyruvate dehydrogenase (e. g , proteins encoded by SEQ ID NOs.:916, 1100, and 979),

phosphofructokinase (e. g , proteins encoded by SEQ ID NOs.:923, 1121, and 1002), GTPase activator (e. g , proteins encoded by SEQ ID NO:1020), GTPase Ran (e. g , proteins encoded by SEQ ID NOs.:942, 1066, and 1022), sarcoplasmic Ca2+ ATPase (e. g , proteins encoded by

SEQ ID NOs.:943, 1067, and 1023), Ca2+ ATPase (e. g , proteins encoded by SEQ ID NOs.:953, 1078, and 1036), hydrogen exporting ATPase (e. g , proteins encoded by SEQ ID NOs.:973, 1094, and 1057), NADH dehydrogenase (e. g , proteins encoded by SEQ ID NOs.:974, 1095, and 1058), F IFO ATPase (e. g , proteins encoded by SEQ ID NOs.:950, 1075, and 1031), spermatogenesis ATPase (e. g , proteins encoded by SEQ ID NOs.:948,

1073, and 1029), beta-ketoacyl synthase (e. g , proteins encoded by SEQ ID NOs.:952, 1077, and 1035), pyruvate kinase (e. g , proteins encoded by SEQ ID NOs.: 1097 and 976), and knickkopf (e. g , proteins encoded by SEQ ID NOs.:962, 1086, and 1045);

(3) protein turnover and mitosis proteins including cyclins— such as cyclin A (e. g , proteins encoded by SEQ ID NOs.:924, 1122, and 1003), cyclin B (e. g , proteins encoded by SEQ ID

NOs.:925, 1123, and 1004), cyclin B3 (e. g , proteins encoded by SEQ ID NOs.:926, 1124, and 1005), cyclin C (e. g , proteins encoded by SEQ ID NOs.:927, 1125, and 1996), cyclin D (e. g , proteins encoded by SEQ ID NOs.:928 and 1126), cyclin E (e. g , proteins encoded by SEQ ID NOs.:929 and 1007), cyclin G (e. g , proteins encoded by SEQ ID NOs.:930, 1127, and 1008), cyclin H (e. g , proteins encoded by SEQ ID NOs.:931, 1128, and 1009), cyclin J

(e. g , proteins encoded by SEQ ID NOs.:932, 1129, and 1010), cyclin K (e. g , proteins encoded by SEQ ID NOs.:933, 1130, and 1011), cyclin T (e. g , proteins encoded by SEQ ID NOs.:934, 1131, and 1012), and cyclin Y (e. g , proteins encoded by SEQ ID NOs.:935, 1131, and 1013), Ubiquitin conjugating enzyme El (e. g , proteins encoded by SEQ ID NOs.:945, 1070, and 1026), chromosome segregation protein (e. g. , proteins encoded by SEQ ID

NOs.:957, 1082, and 1040), CDC5 (e. g , proteins encoded by SEQ ID NOs.:964, 1087, and 1047), COP9 signalosome (e. g. , proteins encoded by SEQ ID NOs.:903, 1104, and 983), and ketel (e. g. , proteins encoded by SEQ ID NOs.:949, 1074, and 1030);

(4) gene expression proteins including cleavage polyadenylation specificity factor (e. g. , proteins encoded by SEQ ID NOs.:936, 1060, and 1015), E1A/CREB -binding protein (e. g. , proteins encoded by SEQ ID NOs.:1068 and 1024), RNA helicases (e. g. , proteins encoded by SEQ ID NOs.:937, 1061, and 1016; 958, 1083, and 1041; 961 and 1044; and 966, 1089, and 1049), RNA polymerases (e. g , proteins encoded by SEQ ID NOs.:938, 1062, and 1017; 1021; and 941, 1065, and 1033), Arfl (e. g. , proteins encoded by SEQ ID NOs.: 1091 and 1052), Bx42 (e. g , proteins encoded by SEQ ID NOs.:968, 1090, and 1051), and eft2 (e. g , proteins encoded by SEQ ID NOs.:947, 1072, and 1028);

(5) protein-folding, stress, and heat shock response proteins including protein disulfide

isomerase (e. g , proteins encoded by SEQ ID NOs.:915, 1103, and 982), heat shock cognate 70 (e. g , proteins encoded by SEQ ID NOs.:956, 1081, and 1039), chaperonin TCP 1 (e. g , proteins encoded by SEQ ID NOs.:959, 1084, and 1042), chaperonins (e. g , proteins encoded by SEQ ID NOs.:965, 1088, and 1048; 972 and 1056), carrier protein (e. g , proteins encoded by SEQ ID NOs.:967 and 1050), and HSP70 (e. g , proteins encoded by SEQ ID NOs.:970, 1093, and 1054); and

(6) miscellaneous proteins including spermatogenesis ATPase (e. g , proteins encoded by SEQ ID NOs.:948, 1073, and 1029), pitchoune (e. g , proteins encoded by SEQ ID NOs.:963 and

1046), and insect proteins (e. g , proteins encoded by SEQ ID NOs.:969, 1092, and 1053; 971 and 1055).

[0081] The polynucleotide sequences include nucleotide sequences encoding double-stranded RNA for silencing target genes that encode:

(1) genes encoding proteins related to behaviour including Bendless (e. g , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:1133 - 1135, 1993 - 1995, and 1499 - 1503), scribbler (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1140 - 1143, 1979 - 1983, and 1485 - 1488), capa receptor (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1140 - 1143, 1979 - 1983, and 1485 - 1488), diapause hormone receptor (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID

NOs.:2259 - 2261 and 1555 - 1558), Ebl (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1162 - 1164, 2014 - 2017, and 1521 - 1523), Ether-a-go-go (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1165 - 1168, 2021 - 2025, and 1529 - 1533), flightless-I (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1169 - 1172, 2004 - 2008, and 1511 - 1515), foraging (e. g , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:1173 - 1176, 1989 - 1992, and 1494 - 1498), juvenile hormone epoxide hydrolase (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1189 - 1191, 2026 - 2029, and 1539 - 1542), neuropeptide F (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1192 - 1194 and 1150 - 1554), pheromone biosynthesis activating neuropeptide receptor (e. g. , dsRNA with an anti -sense strand encoded by any of SEQ ID NOs.:1195 - 1197, 2035 - 2037, and 1548 - 1549), semaphorin la (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1207

- 1209, 2018 - 2020, and 1524 - 1528), shaker (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1210 - 1213, 1984 - 1988, and 1489 - 1493), slowmo (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1214 - 1215, 1613 - 1614, and

1534 - 1538), stress-sensitive B (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1216 - 1217 and 1996 - 1999), turtle (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1218 - 1221, 2009 - 2013, and 1516 - 1520), twitchin (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1222 - 1225, 2030 - 2034, and 1543 - 1547), myosin heavy chain (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1302 - 1305, 1813 - 1815, and 1657 - 1658), nervana (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1436 - 1440, 1927 - 1932, and 1758 - 1761), N-synaptobrevin (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1295 - 1296, 1805 - 1807, and 1652), nuclear hormone receptor 25 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1340 - 1345, 1852 - 1856, and 1693 -

1696), syntaxin (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1346

- 1349, 1857 - 1861, and 1697 - 1698), and bent (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1681 - 1684);

(2) glycolysis and energy metabolism proteins including BiP (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1136 - 1139, 1975 - 1978, and 1481 - 1484), citrate synthase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1152 - 1154, 1950 - 1954, and 1457 - 1461), glycogen synthase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1177 - 1180, 1955 -1959, and 1462 - 1466),

hexokinase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1181 - 1184, 1936 - 1939, and 1446 - 1448), isocitrate dehydrogenase (e. g. , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:1185 - 1188, 1940 - 1944, and 1449 - 1453), pyruvate dehydrogenase (e. g. , dsRNA with an anti -sense strand encoded by any of SEQ ID NOs.: 1202 - 1206, 1945 - 1949, and 1454 - 1456), phosphofructokinase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1226 - 1227, 2038 - 2040, and 1559 - 1562), GTPase activator (e. g. , dsRNA with an anti -sense strand encoded by any of SEQ ID

NOs.: 1635 - 1638), GTPase Ran (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1289 - 1291, 1790 - 1794, and 1640 - 1643), sarcoplasmic Ca2+ ATPase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1292 - 1294, 1796 - 1798, and 1644 - 1647), Ca2+ ATPase (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1337 - 1339, 1847 - 1851, and 1689 - 1692), hydrogen exporting ATPase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1432, 1920, and 1755), NADH dehydrogenase (e. g. , dsRNA with an anti -sense strand encoded by any of SEQ ID NOs.: 1433 - 1435, 1921 - 1926, and 1756 - 1757), FIFO ATPase (e. g. , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:1324 - 1325, 1834, and 1671 - 1672),

spermatogenesis ATPase (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1312 - 1317, 1822 - 1827, and 1663 - 1666), beta-ketoacyl synthase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1331 - 1336, 1841 - 1846, and 1685 - 1688), pyruvate kinase (e. g. , dsRNA with an anti -sense strand encoded by any of SEQ ID

NOs.: 1933 - 1935 and 1441 - 1445), and knickkopf (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1377 - 1381, 1881 - 1886, and 1715 - 1718);

(3) protein turnover and mitosis proteins including cyclins— such as cyclin A (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1228 - 1229, 2041 - 2044, and 1563 - 1567), cyclin B (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID

NOs.: 1230 - 1232, 2045 - 2049, and 1568 - 1571), cyclin B3 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1233 - 1234, 2050 - 2054, and 1572 - 1576), cyclin C (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1236, 2055 - 2058, and 1577 - 1579), cyclin D (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1237 - 1238 and 2059 - 2063), cyclin E (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1239 - 1241 and 1580 - 1583), cyclin G (e. g. , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:1242 - 1244, 2064 - 2068, and 1584 - 1588), cyclin H (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1245 - 1247, 2069 - 2071, and 1589 - 1593), cyclin J (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1248 - 1250, 2072 - 2074, and 1594 - 1598), cyclin K (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1251 - 1253, 2075 - 2078, and 1599 - 1603), cyclin T (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1254 - 1256, 2079 - 2082, and 1604 - 1608), and cyclin Y (e. g. , dsRNA with an anti- sense strand encoded by any of SEQ ID NOs.:1257 - 1259, 2083 - 2087, and 1609 - 1612), Ubiquitin conjugating enzyme El (e. g , dsRNA with an anti-sense strand encoded by any of

SEQ ID NOs.:1297 - 1301, 1808 - 1812, and 1653 - 1656), chromosome segregation protein (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1356 - 1357, 1868, and 1703 - 1704), CDC5 (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1386 - 1391, 1887 - 1892, and 1723 - 1726), COP9 signalosome (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1155 - 1158, 1965 - 1969, and 1471 -

1475), and ketel (e. g , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.: 1318 - 1323, 1828 - 1833, and 1667 - 1670); (4) gene expression proteins including cleavage polyadenylation specificity factor (e. g. , dsR A with an anti-sense strand encoded by any of SEQ ID NOs.:1260 - 1264, 1762 - 1767, and 1615 - 1618), EIA/CREB binding protein (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1799 - 1804 and 1648 - 1651), RNA helicases (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1265 - 1270, 1768 - 1773, and 1619 -

1622; 1371 - 1376 and 1713 - 1714; and 1397 - 1400, 1895 - 1900, and 1731 - 1732), RNA polymerases (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1271 - 1273, 1774 - 1777, and 1623 - 1626; 1639; and 1285 - 1288, 1788 - 1789, and 1677 - 1680), Arfl (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1906 and 1739 - 1740), Bx42 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1403 -

1408, 1901 - 1905, and 1735 - 1738), and eft2 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1306 - 1311, 1816 - 1821, and 1659 - 1662);

(5) protein-folding, stress, and heat shock response proteins including protein disulfide

isomerase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1198 - 1201, 1960 - 1964, and 1467 - 1470), heat shock cognate 70 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1350 - 1355, 1862 - 1867, and 1699 - 1702), chaperonin TCP1 (e. g. , dsRNA with an anti -sense strand encoded by any of SEQ ID

NOs.:1362 - 1367, 1874 - 1878, and 1709 - 1710), chaperonins (e. g. , proteins encoded by dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1392 - 1396, 1893 - 1894, and 1727 - 1730; and 1426 - 1431 and 1751 - 1754), carrier protein (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1401 - 1402 and 1733 - 1734), and HSP70 (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1414 - 1419, 1914 - 1919, and 1743 - 1746); and

(6) miscellaneous proteins including spermatogenesis ATPase (e. g. , dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1312 - 1317, 1822 - 1827, and 1663 - 1666), pitchoune (e. g., dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1382 - 1385 and 1719 - 1722), and insect proteins (e. g., dsRNA with an anti-sense strand encoded by any of SEQ ID NOs.:1409 - 1413, 1907 - 1913, and 1741 - 1742; and 1420 - 1425 and 1747 - 1750).

[0082] The embodiments of polynucleotide sequences provided in Table 3 are generally useful for RNA -mediated suppression of the corresponding target gene identified in Table 3. These

polynucleotides are useful for controlling mosquitoes, including the source species from which the target genes in Table 3 were identified. RNA-mediated suppression of one or more of the target genes provided in Table 3, or use of one or more of the polynucleotides provided in Table 3, is useful for causing mortality or stunting, or otherwise controlling, target mosquitoes, especially mosquito species in the following genera: Aedes, Culex, and Anopheles. [0083] In embodiments, compositions including a polynucleotide for suppression of one or more of the target genes provided in Table 3 (e. g. , a composition including an insecticidally effective amount of one or more of the polynucleotides provided in Table 3) are useful for controlling mosquito species, including mosquito species in the following genera: Aedes, Culex, and Anopheles. For example, a composition including an insecticidally effective amount of one or more of the polynucleotides provided in Table 3 is useful for inducing growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development, in mosquito species, including mosquito species in the following genera: Aedes, Culex, and Anopheles. Compositions include embodiments insecticidally effective on adult mosquitoes, larval mosquitoes, or mosquito eggs, or on combinations thereof.

[0084] In embodiments, R A-mediated suppression of one or more of the target genes provided in Table 3, or use of one or more of the polynucleotides provided in Table 3, is useful for inducing growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development, in mosquito species, including mosquito species in the following genera: Aedes, Culex, and Anopheles. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs.:897 - 975 is used to cause mortality or stunting in Aedes spp., such as Aedes aegypti adults or larvae, for example, by contacting Aedes aegypti adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs.:1133 - 1440. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID

NOs.:1014 and 1060 - 1132 is used to cause mortality or stunting in Culex spp., such as Culex quinquefasciatus adults or larvae, for example, by contacting Culex quinquefasciatus adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs.:1613 - 1614 and 1762 - 2087. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs.:976 - 1013 and 1015 - 1059 is used to cause mortality or stunting Anopheles spp., such as Anopheles gambiae adults or larvae, for example, by contacting Anopheles gambiae adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs.:1441 - 1612 and 1615 - 1761.

[0085] Adult mosquitoes feed on naturally occurring plant sugars, and are attracted to sugar-containing compositions. Thus, an aspect of this invention includes mosquito bait compositions, such as attractive toxic sugar baits (ATSBs), including an insecticidally effective amount of one or more of the polynucleotides provided in Table 3. Typically, ATSBs include an aqueous solution of a sugar (such as sucrose) as a phagostimulant, an attractant such as fruit or flower scents, and an orally available toxin; other ingredients can include fruit juice or pulp, yeast, preservatives, stabilizers, and visual dyes. ATSBs can be sprayed (e. g. , on vegetation) or provided in indoor or outdoor bait stations. Examples of ATSBs which use non-polynucleotide toxins such as Spinosad, pyrethroids, boric acid, or eugenol include those described by Miiller et al. (2010) Malaria J. , 9:210 - 216; Beier et al. (2012) Malaria J. , 11 :31 - 37; Stewart et al. (2013) PLoS ONE, 8(12):e84168; and Quails et al. (2014) Acta Tropica, 131 : 104 - 110. The disadvantage of ATSBs containing non-polynucleotide toxins is that these are non- selective and are toxic to non-target species including beneficial insects such as honey bees and other pollinators and potentially toxic to non-target species that might ingest a mosquito that has fed on the ATSB. Use of a mosquitocidally effective polynucleotide provided herein permits a species-specific approach to mosquito control.

[0086] Other embodiments include larvicidal compositions including a polynucleotide such as a dsRNA provided herein. Larvicidal compositions formulated as a sprayable liquid or dispersible powder or granules can be deployed in and around mosquito breeding grounds such as stagnant water, cisterns, ponds, lakes, streams, and in vegetated areas. Example 3

[0087] This example illustrates non-limiting embodiments of assaying the effectiveness of polynucleotides designed to suppress mosquito target genes such as those disclosed in Tables 2 and 3. More specifically, this example describes assays of dsRNA with an anti-sense strand encoded by a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087.

[0088] Blunt-ended, double-stranded RNAs with an anti-sense strand encoded by a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087 are produced. In embodiments, assays are carried out using species-specific dsRNAs. Efficacy of each dsRNA is evaluated by one or more approaches: microinjection studies and evaluation of phenotypic effects in adult mosquitoes, feeding assays in mosquito larvae, sugar bait feeding studies in adult mosquitoes, and routine molecular analyses to measure target gene mRNA or protein suppression (e. g. , northern or Western blots). A dsRNA designed to silence a non-relevant gene, such as a dsRNA targeting green fluorescent protein (GFP), is used as a negative control. Mosquitocidal fficacy is validated with multiple or iterative assays. For example, dsRNAs found effective in larval feeding assays are advanced to adult feeding assays using sugar bait preparations. In addition, dsRNA sequences can be modified by tiling procedures to improve efficacy.

[0089] A non-limiting example of a larval feeding assay using Aedes aegypti (yellow fever mosquito, YFM) follows. Similar assays are designed for other mosquito species, e. g. , Culex quinquefasciatus or Anopheles gambiae. Aedes aegypti eggs are obtained from a commercial source. YFM larval diet is prepared by adding 250 microliters of stock diet solution (0.5 gram of 3: 1 mixture of liver powder and brewers yeast mixed into 40 milliliters of sterile deionized water) to 100 milliliters of sterile deionized water; YFM larval diet is de-oxygenated prior to use by degassing overnight under vacuum.

Approximately 2000 YFM eggs are hatched in -100 milliliters de-oxygenated YFM larval diet. The larvae are ready for infestation after overnight incubation. Larvae are collected onto a #40 mesh sieve, gently rinsed with sterile deionized water, and transferred to a fresh container with 250 milliliters sterile deionized water. The larvae are now ready for loading into a large particle flow cytometer such as a COPAS™ instrument (Union Biometrica, Holliston, MA, USA); larvae should be used within 24 hours. Larvae are distributed into assay containers (e. g. , flat-bottom 96-well plates pre-loaded with 200 microliters YFM larval diet), followed by addition of the test or control material. Typically wells contain a single larva each; 8 - 16 wells per test sample and 16 wells for untreated controls and a positive control are used. Plates are manually sealed, with a ventilation pinhole in the sealing film for each well. Plates are maintained at 27 degrees Celsius, 0:24, light: dark cycle, at 70% relative humidity for the required period before scoring. Mortality response can be seen in less than 24 hours, and assay quality remains good for up to 5 days; therefore assay duration may be modified, e. g , scored at less than 1 day, or at 1, 2, 3, 4, or 5 days. To score mortality, wells are checked visually for larval movement; live larvae are very active and therefore inactive larvae are scored as dead.

[0090] Sugar bait feeding studies are carried out in adult mosquitoes. In embodiments, assays are carried out using species-specific polynucleotides. Sugar baits containing one or more of dsRNAs with an anti -sense strand encoded by a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and 1133 - 2087 are prepared using methods similar to those known in the art (see, e. g. , the ATSB compositions described by Miiller et al. (2010) Malaria J. , 9:210 - 216; Beier et al. (2012) Malaria J. , 11 :31 - 37; Stewart et al. (2013) PLoS ONE, 8(12):e84168; and Quails et al. (2014) Acta Tropica, 131 : 104 - 110) . In one embodiment, the sugar bait is an aqueous solution of sucrose (10% w/v) with an added attractant and the test dsRNA added at the desired concentration. The sugar bait can be provided as a liquid, e. g. , in a container or in droplets, or via a wick or other feeding device. In another embodiment, the dsRNA is provided in a sugar-containing solid or gel. The sugar bait is offered to adult mosquitoes, and mortality or other changes in phenotype are monitored. Controls include sugar bait with no dsRNA added; optionally, starved controls (no water or bait offered) are included in the event that the dsRNA reduced feeding behaviour.

Example 4

[0091] This example illustrates non-limiting embodiments of assaying the effectiveness of dsRNAs designed to suppress mosquito target genes such as those disclosed in Tables 2 and 3. More specifically, this example describes methods useful for assaying target gene transcript suppression by dsRNA with an anti-sense strand encoded by a sequence selected from the group consisting of SEQ ID NOs.:106 - 896 and SEQ ID NOs.:1133 - 2087.

[0092] Various dsRNAs were tested for their ability to suppress levels of target gene transcripts in adult Aedes aegypti mosquitoes. In embodiments, similar assays are designed for other mosquito species, e. g., Culex quinquefasciatus or Anopheles gambiae. The representative Aedes aegypti target genes selected were RpL19, Copi beta, Copi beta prime, RpS4, VATPaseA, actin 6, and vesicle sorting protein Chmpl . Aedes aegypti adults were anesthetized on a -10 degrees Celsius chill plate. 100 nanoliters of a solution containing IX PBS and dsRNA (10 milligrams/milliliter) were injected into the thorax of each insect using a Nanoject II instrument (Drummond Scientific Co., Broomall, PA); 1 microgram dsRNA was administered per insect. After 48 hours, 12 individuals were collected from each treatment, frozen on dry ice, and levels of target gene transcripts were analyzed. As a control, dsRNA designed to target green fluorescent protein (GFP) was administered in the same way.

Transcript expression data were normalized to the geometric mean of control genes Ef 1 A and RPS 13.

Table 4

[0093] The results demonstrated that all target genes tested in these experiments showed specific and statistically significant suppression of transcript levels at 48 hours after treatment; target gene transcript levels were reduced between 51 - 90%, compared to expression levels in the control GFP dsRNA injection (Table 4). These data indicated that each of the target-gene-specific dsRNAs effectively generated a systemic and gene-specific RNAi response in adult Aedes aegypti mosquitoes.

[0094] In a separate series of experiments, various dsRNAs were tested for their ability to cause species-specific mortality in a larval feeding assay using Aedes aegypti larvae. The representative Aedes aegypti target genes selected were ribosomal protein large subunit 19, vesicle sorting protein Chmpl, and coatomer protein (COPI) beta prime. The larval feeding assay was carried out on Aedes aegypti larvae using a procedure similar to that described in Example 3 above, with multiple doses of species-specific dsRNAs comprising SEQ ID NO: 133 (targeting ribosomal protein large subunit 19, "RPL19", SEQ ID NO:5), SEQ ID NO:377 (targeting vesicle sorting protein Chmpl, "Chmpl", SEQ ID NO:36), and SEQ ID NO:164 (targeting coatomer protein (COPI) beta prime, "COPI beta prime", SEQ ID NO:9). dsRNAs (either a -100 base-pair "GFP-100" dsRNA or -300 base-pair "GFP-300" dsRNA) targeting green fluorescent protein ("GFP") were used as controls. Results for a first set of dsRNA formulations are shown in Table 5. These feeding assay data demonstrate that nucleic acids targeting species-specific genes in mosquito larvae cause larval mortality at a statistically significant level. Table 5

p me

[0095] In another series of experiments, various dsRNAs were tested for their ability to cause species- specific mortality in adult Aedes aegypti by injection. The representative Aedes aegypti target genes selected were ribosomal protein large subunit 19, vesicle sorting protein Chmpl, coatomer protein (COPI) beta, coatomer protein (COPI prime) beta prime, Ribosomal protein small Subunit 4, and Vacuolar ATPase A. The insects were evaluated for target gene knock-down (KD) at 48 hr post- injection. Insects (12 insects per treatment) were maintained on a -10°C chill plate and injected in the thorax with lOOnL of solution containing lxPBS and dsRNA (lOmg/mL) using a Nanoject II instrument (Drummond Scientific Co., Broomall, PA). After 48 hr all individuals were collected from each treatment, frozen on dry ice and analyzed for target gene KD. Expression data was normalized to the geometric mean of EF1A and RPS13. All target genes showed specific transcript KD at 48 hrs ranging from 51% to 90% reduction compared to expression levels in the control GFP saRNA injection. These data indicated a potent systemic RNAi response is functional in aAvlt Aedes aegypti. However, no significant mortality compared to the GFP control was observed.

Table 6. Percent target gene knockdown (KD) in dsRNA-injected Aedes aegypti adults.

Aedes 36 377 CHMP 1 73.63% YES NO

aegypti

Aedes 12 186 vATPase 75.99% YES NO

aegypti A

[0096] In another series of experiments the retention of target gene knockdown over a period of several days for three gene targets was assessed. The gene targets tested were: Chmpl, RPL19 and vATPA. Target gene knockdown appeared to be retained over two to eight days with some slight variability (Table 7.) indicating that the selection of target gene may be important for inducing prolonged knock down.

Table 7. Time course retention of target gene kinock down (KD) in dsRNA-injected 4ei/es aegypti adults.

[0097] In order to rule out whether protein stability is a factor in preventing adult specific mortality in Aedes aegypti, Western blot analysis of two proteins (RPL7 and SNF7) was undertaken. The protein levels were monitored over several days (day 2, 5 and 7) post-injection of SEQ ID NO 133 (RPL 19) and SEQ ID NO 158 (SNF7). Very limited to no protein suppression was seen by Western blot.

Example 5.

[0098] The following example outlines a screen to identify the excipients capable of enhanced delivery and RNAi phenotypes post-feeding in Aedes aegypti larvae. A total of 1052 dsRNA formulations utilizing forty nine unique excipient mixtures were tested in feeding bioassays for dsRNA-induced mortality in Aedes aegypti larvae. Formulation compositions including cationic guar or

Polyethylenimine (PEI, branched, 10-25 kDa) and Aedes aegypti -specific dsRNAs targeting RPL19, vesicle sorting protein Chmpl, and COPI beta prime were tested for specific activity against larvae in feeding bioassays with multiple doses of dsRNA. Table 8 illustrates bioassay results against Aedes aegypti utilizing dsRNA formulations with a cationic guar and Table 9 illustrates bioassay results against Aedes aegypti larvae utilizing dsRNA formulations with branched PEI (10-25 kDa).

Formulation of dsRNA

[0099] Materials utilized in formulations included: dsRNA (10 mg/ml), PEI (10 mg/ml) in sodium acetate buffer pH 5.3 (0.05 M), Sodium acetate buffer pH 5.3 (0.05M), or cationic guar (in which case sodium acetate buffer was replaced with nuclease-free water). For the preparation of the formulations, dsRNA was added to a 1.5mL Eppendorf tube and the appropriate buffer (sodium acetate for PEI or water for cationic guar) was added to the tube followed by a brief vortexing period. Either PEI or cationic guar were then added and vortexing continued for 1 minute. Formulations were kept for a period of 10 minutes at room temperature before use. As Tables 8 and 9 illustrate, some of the formulations containing cationic guar or PEI had efficacious effects in Aedes aegypti larvae versus the GFP control. However, no obvious trend in dsRNA concentration was discernible.

Table 8. Summary of cationic guar formulation bioaassay hits against Aedes aegypti. Treatments targeting Green Fluorescent Protein are negative control for the individual test sets.

cationic guar, 1

mg/ml}

YFM- 377 Aedes 0.5 mg/mL dsR A{TRUE}; 29.17 4.17 NO 2087 aegypti dH20{TRUE};

Chmpl Adj uvant {

cationic guar, 1

mg/ml}

YFM- 377 Aedes 1.0 mg/mL dsR A{TRUE}; 83.33 8.33 YES 2087 aegypti dH20{TRUE};

Chmpl Adj uvant {

cationic guar, 1

mg/ml}

YFM- GFP 0.5 mg/mL dsR A{TRUE}; 8.33 8.33 NO

2090 NaOAc{50

mMolar} ;

pH{5.3};

Adj uvant { cationic

guar, 1.0 mg/ml}

YFM- 133 Aedes 0.5 mg/mL dsR A{TRUE}; 41.67 8.33 NO

2090 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adj uvant { cationic

guar, 1.0 mg/ml}

YFM- GFP 0.5 mg/mL dsR A{TRUE}; 8.33 4.17 NO

2090 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant

{cationic guar, 1.5

mg/ml}

YFM- 133 Aedes 0.5 mg/mL dsR A{TRUE}; 54.17 11.02 YES

2090 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adjuvant

{cationic guar, 1.5

mg/ml}

YFM- 377 Aedes 0.5 mg/mL dsR A{TRUE}; 41.67 15.02 NO

2090 aegypti NaOAc{50

Chmpl mMolar} ;

pH{5.3};

Adj uvant { cationic

guar, 1.5 mg/ml}

YFM- GFP 1.0 mg/mL dsR A{TRUE}; 29.17 4.17 NO

NaOAc{50 2090 mMolar} ;

pH{5.3};

Adj uvant { cationic

guar, 1.5 mg/ml}

YFM- 377 Aedes 1.0 mg/mL dsRNA{TRUE} ; 50.00 7.22 YES

2090 aegypti NaOAc{50

Chmpl mMolar} ;

pH{5.3};

Adj uvant { cationic

guar, 1.5 mg/ml}

Table 9. Summary of PEI 10-25 kDa (branched ) formulation bioassay hits against A edes aegypti. Treatments targeting Green Fluorescent Protein are negative control for the individual test sets.

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.2 mg/mL dsRNA{TPvUE} ; 0.00 0.00 NO 2088 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.3 mg/mL dsRNA{TPvUE} ; 0.00 0.00 NO 2088 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.4 mg/mL dsRNA{TPvUE} ; 41.67 4.17 YES 2088 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.1 mg/mL dsRNA{TPvUE} ; 50.00 0.00 YES 2088 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.15 mg/mL dsRNA{TPvUE} ; 87.50 0.00 YES 2088 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.2 mg/mL dsRNA{TPvUE} ; 75.00 0.00 YES 2088 aegypti NaOAc{50 RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.3 mg/mL dsRNA{TRUE}; 45.83 8.33 YES 2088 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.4 mg/mL dsRNA{TRUE}; 79.17 4.17 YES 2088 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.01 mg/mL dsRNA{TRUE}; 8.33 4.17 NO

2098 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.1 mg/mL dsRNA{TRUE}; 29.17 16.67 NO

2098 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.2 mg/mL dsRNA{TRUE}; 12.5 7.22 NO

2098 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.01 mg/mL dsRNA{TRUE}; 70.83 8.33 YES aegypti NaOAc{50 2098 RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.1 mg/mL dsRNA{TRUE}; 62.5 7.22 YES

2098 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 133 Aedes 0.2 mg/mL dsRNA{TRUE}; 79.17 4.17 YES

2098 aegypti NaOAc{50

RPL19 mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 377 Aedes 0.01 mg/mL dsRNA{TRUE}; 25.00 12.5 NO

2098 aegypti NaOAc{50

Chmpl mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 377 Aedes 0.1 mg/mL dsRNA{TRUE}; 37.50 7.22 NO

2098 aegypti NaOAc{50

Chmpl mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 377 Aedes 0.2 mg/mL dsRNA{TRUE}; 62.5 12.5 YES

2098 aegypti NaOAc{50

Chmpl mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.1 mg/mL dsRNA{TRUE}; 4.17 4.17 NO 2100 NaOAc{50

mMolar} ;

pH{5.3}; Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.2 mg/mL dsRNA{TRUE}; 0.00 0.00 NO 2100 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.3 mg/mL dsRNA{TRUE}; 0.00 0.00 NO 2100 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- GFP 0.4 mg/mL dsRNA{TRUE}; 0.00 0.00 NO 2100 NaOAc{50

mMolar} ;

pH{5.3};

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 164 Aedes 0.1 mg/mL dsRNA{TRUE}; 29.17 15.02 YES 2100 aegypti NaOAc{50

COPI mMolar} ;

BETA pH{5.3};

PRIME Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 164 Aedes 0.2 mg/mL dsRNA{TRUE}; 25.00 7.22 YES 2100 aegypti NaOAc{50

COPI mMolar} ;

BETA pH{5.3};

PRIME Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

YFM- 164 Aedes 0.3 mg/mL dsRNA{TRUE}; 37.50 12.5 YES 2100 aegypti NaOAc{50

COPI mMolar} ;

BETA pH{5.3};

PRIME Adjuvant {PEI

10-25 kDa, branched 1 : 1

w/w}

YFM- 164 Aedes 0.4 mg/mL dsRNA{TRUE} ; 41.67 11.02 YES 2100 aegypti NaOAc{50

COPI mMolar} ;

BETA pH{5.3};

PRIME Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

[00100] Further to the data presented in the Tables above, target gene knockdown was evaluated in larval Aedes aegypti at 5 days post-feeding on those formulations that appeared active through induced mortality in bioassays. Table 10 illustrates the significant target gene suppression observed for a select number of formulations tested in the feeding bioassays.

Table 10. Evaluation of target gene knockdown in Aedes aegypti larvae post feeding on PEI 10-25 kDa (branched) or cationic guar -based formulation. Treatments targeting green fluorescent protein are negative control dsRNA treatments for the individual testsets.

10-25 kDa,

branched 1 : 1

w/w}

BDX- 377 Aedes 0.2 mg/mL dsRNA{TRUE}; 12.85% NO

01234 aegypti NaOAc{50

Chmpl mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 164 Aedes 0.1 mg/mL dsRNA{TRUE}; 22.65% NO

01234 aegypti NaOAc{50

COPI mMolar};

BETA pH{5.3 };

PRIME Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 164 Aedes 0.2 mg/mL dsRNA{TRUE}; 3.3% NO

01234 aegypti NaOAc{50

COPI mMolar};

BETA pH{5.3 };

PRIME Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 133 Aedes 0.2 mg/mL dsRNA{TRUE}; -32.76% NO

01293 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 133 Aedes 0.2 mg/mL dsRNA{TRUE}; 7.97% NO

01293 aegypti dH20{TRUE};

RPL19 Adjuvant{DV

10496, 1 mg/ml}

BDX- 377 Aedes 0.2 mg/mL dsRNA{TRUE}; -12.86% NO aegypti NaOAc{50 01293 Chmpl mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 377 Aedes 0.2 mg/mL dsRNA{TRUE}; 14.02% NO

01293 aegypti dH20{TRUE};

Chmpl AdjuvantpV

10496, 1 mg/ml}

BDX- 164 Aedes 0.2 mg/mL dsRNA{TRUE}; -11.65% NO

01293 aegypti NaOAc{50

COPI mMolar};

BETA pH{5.3 };

PRIME Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 164 Aedes 0.2 mg/mL dsRNA{TRUE}; 7.39% NO

01293 aegypti dH20{TRUE};

COPI AdjuvantpV

BETA 10496, 1 mg/ml}

PRIME

BDX- 133 Aedes 0.02 mg/mL dsRNA{TRUE}; -10.57% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 133 Aedes 0.04 mg/mL dsRNA{TRUE}; -4.17% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 133 Aedes 0.06 mg/mL dsRNA{TRUE}; 0.99% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI 10-25 kDa,

branched 1 : 1

w/w}

BDX- 133 Aedes 0.08 mg/mL dsRNA{TRUE}; 0.81% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 133 Aedes 0.1 mg/mL dsRNA{TRUE}; -13.68% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 1 : 1

w/w}

BDX- 133 Aedes 0.01 mg/mL dsRNA{TRUE}; -33.93% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.02 mg/mL dsRNA{TRUE}; -8.78% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.04 mg/mL dsRNA{TRUE}; 6.68% NO

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.06 mg/mL dsRNA{TRUE}; 21.95% YES

01365 aegypti NaOAc{50 RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.08 mg/mL dsRNA{TRUE}; 19.07% YES

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.1 mg/mL dsRNA{TRUE}; 15.61% YES

01365 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.05 mg/mL dsRNA{TRUE}; 14.00% NO

01405 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.1 mg/mL dsRNA{TRUE}; -28.00% NO

01405 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 133 Aedes 0.15 mg/mL dsRNA{TRUE}; -14.41% NO

01405 aegypti NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w} BDX- 164 Aedes 0.05 mg/mL dsRNA{TRUE}; -0.26% NO

01405 aegypti NaOAc{50

COPI mMolar};

BETA pH{5.3 };

PRIME Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 164 Aedes 0.1 mg/mL dsRNA{TRUE}; 6.89% NO

01405 aegypti NaOAc{50

COPI mMolar};

BETA pH{5.3 };

PRIME Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 164 Aedes 0.15 mg/mL dsRNA{TRUE}; 15.98% NO

01405 aegypti NaOAc{50

COPI mMolar};

BETA pH{5.3 };

PRIME Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 377 Aedes 0.1 mg/mL dsRNA{TRUE}; 20.72% NO

01405 aegypti NaOAc{50

Chmpl mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

BDX- 377 Aedes 0.15 mg/mL dsRNA{TRUE}; -6.67% NO

01405 aegypti NaOAc{50

Chmpl mMolar};

pH{5.3 };

Adjuvant {PEI

10-25 kDa,

branched 2: 1

w/w}

Specificity of formulation activity to larval stage:

[00101] Target gene suppression was further assessed in adult Aedes aegypti mosquitoes 3 days post- feeding on dsRNA formulations adjusted for delivery in 10% fructose or 10% sucrose. Weak to no gene suppression was observed post sugar feeding in adult mosquitoes suggesting formulation-based efficacy was more potent in the aquatic larval stage of the mosquito (Table 11. below).

Table 11. Results of sugar-based dsRNA formulation feeding in adult Aedes aegypti.

Aedes 0.1 mg/mL dsRNA{TRUE -7.24% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10_%_Fructo

sc

AGA EDAC PEI 10-25

kDa, branched

1 : 1 w/w }

Aedes 0.1 mg/mL dsRNA{TRUE -0.48% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10_%_Fructo

SC

AGA EDAC PEI 10-25

kDa, branched

2:1 w/w }

Aedes 0.1 mg/mL dsRNA{TRUE -0.59% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10 % Sucros

e; PEI 10-25

kDa, branched

2:1 w/w}

Aedes 0.1 mg/mL dsRNA{TRUE -1.5% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10_%_Sucros

e;

BSA EDAC P EI 10-25 kDa,

branched 1 : 1

w/w}

Aedes 0.1 mg/mL dsRNA{TRUE 3.58% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3}; Adjuvant

{10_%_Sucros

e;

BSA EDAC P EI 10-25 kDa,

branched 2:1

w/w }

Aedes 0.1 mg/mL dsRNA{TRUE -2.5% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10_%_Sucros

e;

AGA EDAC PEI 10-25

kDa, branched

1 : 1 w/w }

Aedes 0.1 mg/mL dsRNA{TRUE 11.58% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10_%_Sucros

e;

AGA EDAC PEI 10-25

kDa, branched

2:1 w/w }

Aedes 0.1 mg/mL dsRNA{TRUE 13.48% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10 % Fructo

se_PEI_ 10-25

kDa, branched

1:1 w/w}

Aedes 0.1 mg/mL dsRNA{TRUE -4.17% NO aegypti };NaOAc{50

RPL19 mMolar};

pH{5.3};

Adjuvant

{10 % Fructo

se DOTAP/D

OPE 2:1 w/w} 133 Aedes 0.1 mg/mL dsRNA{TRUE -14.00% NO aegypti }; NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant

{ 10_%_Fructo

se}

133 Aedes 0.1 mg/mL dsRNA{TRUE 16.55% YES

aegypti }; NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant

{ 10 % Sucros

e_PEI_ 10-25

kDa, branched

1 : 1 w/w}

133 Aedes 0.1 mg/mL dsRNA{TRUE -9.37% NO

aegypti }; NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant

{ 10 % Sucros

e DOTAP/DO

PE_2: 1 w/w}

133 Aedes 0.1 mg/mL dsRNA{TRUE 6.14% NO

aegypti }; NaOAc{50

RPL19 mMolar};

pH{5.3 };

Adjuvant

{ 10 % Sucros

e}

[00102] All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure. Although the materials and methods of this invention have been described in terms of preferred embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of this invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of this invention as defined by the appended claims.