Rnai insecticide materials and methods for ant control

Abstract

Disclosed herein are methods for producing interfering RNA biopesticides, such as microbial host organisms engineered to produce interfering RNA (iRNA) molecules. Such iRNA molecules inhibit the expression of a gene in an insect, such as an ant, by RNA interference. Also disclosed herein are polynucleotides, such as expression cassettes encoding iRNA molecules and facilitating integration, such as stable integration into the genome of a host. Further disclosed herein are compositions including the disclosed nucleotide sequences and host organisms, along with methods of using the same to control ant populations.

Description

RNAI INSECTICIDE MATERIALS AND METHODS FOR ANT CONTROLCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §199(e) of U.S. Provisional Application No. 63 / 643,859 entitled RNA INSECTICIDE MATERIALS AND METHODS FOR ANT CONTROL, filed on May 7, 2024, which is incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said Sequence Listing XML copy, created on May 6, 2025, is named "IU-2024-020-02-WO_st26" and is 24 KB in size.FIELD OF THE INVENTION

[0003] This disclosure generally relates to biorational insecticide compositions, such as yeastbased delivery systems for interfering RNA, which can be deployed to control ant populations. The disclosure further relates to methods of making and using the biorational insecticide compositions.BACKGROUND

[0004] Specific species of ants are considered pestilent. These ant species can cause significant property damage, such as damage wood and other building materials, or harm crops, and / or contaminate stored food, or inflict painful and life-threatening stings. Insecticidal sprays are often ineffective against ants and may have unwanted effects on non-target organisms. For at least these reasons, the current ant control repertoire is insufficient for combating these pestilent species of ants, and there is an urgent need to develop a new generation of environmentally safe options for ant control.

[0005] An emerging strategy involves facilitating exposure of ants to symbiotic or attractive microbes, such as yeast, engineered to produce insecticidal nucleic acid molecules, such as interfering RNA. Given its sequence dependent mechanism of action, RNA interference provides a more selective alternative to broad-spectrum insecticides, and the technology has been shown to undermine the growth, such as the fitness, and survival of targeted insect pests, such as mosquitoes. However, delivery of interfering RNA remains challenging, and various strategies include administration by injection of naked double stranded RNA (dsRNA) and ingestion, consumption, or oral administration, such as by combining insecticidal nucleic acid molecules with artificial diet. Accordingly, there exists a need for improved methods of engineering microbes to biosynthesize insecticidal nucleic acids, such as to generate microbes with advantageous capabilities, e.g., improved genetic stability and enhanced production of nucleic acids targeting ant genes. Aspects of the invention disclosed herein address these needs.INCORPORATION BY REFERENCE

[0006] Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually, and as if each is fully set forth herein. However, where such reference is made, and whether to patents, publications, non-patent literature, or other sources of information, it is for the general purpose of providing context for discussing features of the invention. Accordingly, unless specifically stated otherwise, the reference is not to be construed as an admission that the document or underlying information, in any jurisdiction, is prior art, or forms part of the common general knowledge in the art.SUMMARY OF THE INVENTION

[0007] A first aspect of the invention includes interfering ribonucleic acid (iRNA) molecules, which are capable of inhibiting the production of potassium voltage-gated channel protein Shaker in an ant by RNA interference.

[0008] A second aspect of the invention includes expression cassettes for the expression of disclosed iRNA molecules.

[0009] A third aspect of the invention includes vectors for the expression of disclosed iRNA molecules and expression cassettes containing nucleotide sequences encoding the same.

[0010] A fourth aspect of the invention includes microbial cells, such as host organisms, including any of the disclosed iRNA molecules, expression cassettes, vectors, and combinations thereof.

[0011] A fifth aspect of the invention includes compositions containing any of the disclosed iRNA molecules, expression cassettes, vectors, microbial cells, and combinations thereof.

[0012] A sixth aspect of the invention includes methods involving any of the disclosed iRNA molecules, expression cassettes, vectors, microbial cells, compositions, and combinations thereof, in the control of an ant population.

[0013] A first embodiment is an interfering ribonucleic acid including a nucleotide sequence of 20 to 30 contiguous nucleotides, where the nucleotide sequence is partially or perfectly complementary to mRNA transcribed from a target DNA sequence having 88%, 92%, 96%, or 100% identity to a portion of SEQ ID NO: 12 or to the entire length of SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:7; and where the interfering RNA inhibits the production of potassium voltage-gated channel protein Shaker in an ant by RNA interference.

[0014] A second embodiment is an interfering RNA of claim 1, where the interfering RNA is an RNA construct, a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an anti-sense oligonucleotide.

[0015] A third embodiment is an interfering RNA, where the interfering RNA is an shRNA.

[0016] A fourth embodiment is an interfering RNA, where the ant is a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a New World ant, a jet ant or a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, or a crazy ant.

[0017] A fifth embodiment is an interfering RNA, where the ant is a species of Acromyrmex, Atta, Camponotus, Cataglyphis, Cyphomyrmex, Dinoponera, Formica, Harpegnathos, Lasius, Nylanderia, Solenopsis, Monomorium, Linepithema, Odontomachus, Ooceraea, Paratrechina, Pogonomyrmex, Pseudomyrmex Tetramorium, Trachymyrmex, Temnothorax, Vollenhovia, or Wasmannia.

[0018] A sixth embodiment is an expression cassette including a regulatory sequence operably linked to a nucleotide sequence which encodes the interfering RNA molecule of any of the preceding embodiments.

[0019] A sixth embodiment is an expression cassette including a regulatory sequence operably linked to a nucleotide sequence which encodes an interfering RNA molecule including a nucleotide sequence of 20 to 30 contiguous nucleotides, where the nucleotide sequence is partially or perfectly complementary to mRNA transcribed from a DNA sequence having 88%, 92%, 96%, or 100% identity to a portion of SEQ ID NO: 12 or to the entire length of SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:7, and where the interfering RNA inhibits the production of potassium voltage-gated channel protein Shaker in an ant by RNA interference.

[0020] An eighth embodiment is an expression cassette where the nucleotide sequence of the interfering RNA molecule includes 25 nucleotides which are partially or perfectly complementary to a mRNA transcribed from the entire length of SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:7.

[0021] A ninth embodiment is an expression cassette including a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entire length of a) SEQ ID NO:2, SEQ ID NO:3, or the complement thereof; b) SEQ ID NO:5, SEQ ID NO:6, or the complement thereof; or c) SEQ ID NO:8, SEQ ID NO:9, or the complement thereof.

[0022] A tenth embodiment is an expression cassette where the regulatory sequence includes a yeast promoter.

[0023] An eleventh embodiment is an expression cassette where the GPD promoter includes a nucleotide sequence that has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entire length of SEQ ID NO: 10.

[0024] A twelfth embodiment is an expression cassette where the expression cassette is integrated into the genomic DNA of Saccharomyces cerevisiae.

[0025] A thirteenth embodiment is a vector including the expression cassette of any of the preceding embodiments.

[0026] A fourteenth embodiment is a vector, where the vector includes a nucleotide sequence that has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entire length of SEQ ID NO: 11.

[0027] A fifteenth embodiment is a microbial cell, algal cell, or plant cell including the expression cassette or the vector of any of the preceding embodiments.

[0028] A sixteenth embodiment is a microbial cell, algal cell, or plant cell where the expression cassette is integrated into the genomic DNA of the microbial cell.

[0029] A seventeenth embodiment is a microbial cell where the microbe is Saccharomyces cerevisiae.

[0030] An eighteenth embodiment is a composition including the interfering RNA of any preceding embodiment, the expression cassette of any preceding embodiment, the vector of any preceding embodiment, and the microbial cell, algal cell, or plant cell of any preceding embodiment, or a combination thereof.

[0031] A nineteenth embodiment is a composition including an expression cassette including a promoter operably linked to a DNA sequence encoding an interfering RNA molecule which is partially or perfectly complementary to a target sequence in a target gene, where the target gene encodes potassium voltage-gated channel protein Shaker in an ant; and the interfering RNA specifically inhibits expression of the target gene.

[0032] A twentieth embodiment is a composition where the interfering RNA is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an anti-sense oligonucleotide.

[0033] A twenty-first embodiment is a composition where the interfering RNA is a short hairpin RNA (shRNA).

[0034] A twenty-second embodiment is a composition where the ant is a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a New World ant, a jet ant or a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, or a crazy ant.

[0035] A twenty -third embodiment is a composition where the ant is a species of Acromyrmex,Atta, Camponotus, Cataglyphis, Cyphomyrmex, Dinoponera, Formica, Harpegnathos, Lasius, Nylanderia, Solenopsis, Monomorium, Linepithema, Odontomachus, Ooceraea, Paratrechina,Pogonomyrmex, Pseudomyrmex Tetramorium, Trachymyrmex, Temnothorax, Vollenhovia, or Wasmannia.

[0036] A twenty-fourth embodiment is a composition where the target sequence within the target gene is 88%, 92%, 96%, or 100% identical to the entire length of SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:7.

[0037] A twenty -fifth embodiment is a composition where the expression cassette includes a nucleotide sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the entire length of a) SEQ ID NO: 2, SEQ ID NO:3, or the complement thereof; b) SEQ ID NO:5, SEQ ID NO:6, or the complement thereof; or c) SEQ ID NO:8, SEQ ID NO:9, or the complement thereof.

[0038] A twenty-sixth embodiment is a composition where the expression cassette includes the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9.

[0039] A twenty- seventh embodiment is a composition where the interfering RNA includes a nucleotide sequence of at least 25 contiguous nucleotides which are partially or perfectly complementary to a portion of mRNA transcribed from SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:7; and where the interfering RNA is capable of inhibiting the expression of potassium voltagegated channel protein Shaker in the ant.

[0040] A twenty-eighth embodiment is a composition where the expression cassette is integrated into the genome of a yeast cell.

[0041] A twenty-ninth embodiment is a composition where the yeast cell is Saccharomyces cerevisiae.

[0042] A thirtieth embodiment is a composition where the yeast cell is spray-dried, heat-killed, lyophilized, or suspended in an aqueous medium.

[0043] A thirty-first embodiment is a composition further including a sugar bait.

[0044] A thirty-second embodiment is a composition, where the composition is within a trap.

[0045] A thirty-third embodiment is a method for controlling an ant population, including contacting the ant population with the interfering RNA of any of the preceding embodiments, the expression cassette of any of the preceding embodiments, the vector of any of the preceding embodiments, the microbial cell, algal cell, or plant cell of any of the preceding embodiments, or the composition of any of the preceding embodiments, where contacting the ant population includes ingestion of an ant in the ant population of the interfering RNA, the expression cassette, the vector, the microbial cell, algal cell, or plant cell, or the composition, thereby controlling the ant population, thereby controlling the ant population.

[0046] A thirty-fourth embodiment is a method for controlling an ant population, the method including contacting the ant population with an interfering RNA molecule including a nucleotide sequence that is partially or perfectly complementary to mRNA transcribed from a target sequence within a target gene and which specifically inhibits expression of the target gene in an ant in the ant population, thereby controlling the ant population, where the target gene encodes potassium voltage-gated channel protein Shaker or an ortholog thereof.

[0047] A thirty-fifth embodiment is a method where the interfering RNA is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an anti-sense oligonucleotide.

[0048] A thirty-sixth embodiment is a method where the interfering RNA is a short hairpinRNA (shRNA).

[0049] A thirty-seventh embodiment is a method where the target sequence within the target gene has at least 84%, 88%, 92%, or 96% identity to the entire length of SEQ ID NO: 1, SEQ IDNO:4, or SEQ ID NO:7.

[0050] A thirty-eighth embodiment is a method where the target sequence is SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:7.

[0051] A thirty-ninth embodiment is a method where the interfering RNA is produced by a microbial cell including an expression cassette including a regulatory sequence operably linked to a nucleotide sequence encoding the interfering RNA, where the expression cassette is integrated into the genome of the microbial cell.

[0052] A fortieth embodiment is a method where the expression cassette includes a nucleotide sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the entire length of a) SEQ ID NO:2, SEQ ID NO:3, or the complement thereof; b) SEQ ID NO:5, SEQ ID NO:6, or the complement thereof; or c) SEQ ID NO:8, SEQ ID NO:9, or the complement thereof.

[0053] A forty-first embodiment is a method where the expression cassette includes the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9.

[0054] A forty-second embodiment is a method where the method includes contacting the ant in the ant population with the microbial cell.

[0055] A forty-third embodiment is a method where the microbial cell is dead or alive.

[0056] A forty-fourth embodiment is a method where the microbial cell is a Saccharomyces cerevisiae cell.

[0057] A forty-fifth embodiment is a method where the ant population includes a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a NewWorld ant, a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, and a crazy ant, or a combination thereof.

[0058] A forty-sixth embodiment is a method where the ant population includes Acromyrmex spp., Atta spp., Camponotus spp., Cataglyphis spp., Cyphomyrmex spp., Dinoponera spp., Formica spp., Harpegnathos spp., Lasius spp., Nylanderia spp., Solenopsis spp., Monomorium spp., Linepithema spp., Odontomachus spp., Ooceraea spp., Paratrechina spp., Pogonomyrmex spp., Pseudomyrmex spp., Tetramorium spp., Trachymyrmex spp., Temnothorax spp., Vollenhovia spp., Wasmannia spp., or a combination thereof.

[0059] A forty-seventh embodiment is a method where the interfering RNA includes at least 25 contiguous nucleotides, where the nucleotide sequence is partially or perfectly complementary to a portion of mRNA transcribed from SEQ ID NO: 12, SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO:7; and where the interfering RNA is capable of inhibiting the expression of potassium voltagegated channel protein Shaker in the ant by RNA interference.

[0060] A forty-eighth embodiment is a method where the method further includes contacting the ant population with a sugar bait, a pheromone, an insecticide, or any combination thereof.BRIEF DESCRIPTION OF THE FIGURES

[0061] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments, and together with the description, serve to explain the principles of the disclosure.

[0062] FIG. 1 is a bar graph showing percent mortality of adult Pogonomyrmex harhatus female ants following consumption of attractive sugar bait (ASB), ASB containing control yeast (Control), and biopesticide yeast strains Sh.697, Sh.698, and Sh. 702. Mean results from replicate trials are shown, and error bars represent standard deviations (*** = P<0.001 vs. ASB or Control).

[0063] FIG. 2 is a bar graph showing mean gray matter values (average signal intensity over the selected area) for control adult ants and adult ants that were allowed to feed on yeast expressingSh.697 for 24 hours.DETAILED DESCRIPTION

[0064] Ants, which are among the most persistent and frequent insects located near buildings, can damage wood and other materials, harm crops, contaminate stored food, and inflict painful and sometimes life-threatening stings. While there are about 25 different ant species known to infest homes, ants can also significantly alter the dynamics, composition, functions, and structure of natural ecosystems. In another context, biotic ant invasions can occur when organisms are transported to new, often distant, ranges where their descendants proliferate, spread, and persist. Social insects, particularly ants, live in colonies that can house up to hundreds of thousands of individuals, are among the most damaging invasive alien species.

[0065] The ecological impacts of invasive ants comprise predation, hybridization, and competition with native species that changes the ecosystem processes with the biodiversity loss and upsurge of ants. The effects of invasion on native fauna in the same habitats might be catastrophic for the native community through various ecological mechanisms, e.g., habitat disturbance, resource competition, limiting the foraging activity of native species, and various other indirect mechanisms of invasive species (Siddiqui et al., Environ Sci Pollut Res Int. 2021 Oct;28(39):54362-54382).

[0066] Current means of ant control are often ineffective and can inflict harm on non-target organisms. It is therefore critical that new classes of biorational pesticides and cost-effective technologies for controlling ants, particularly species that are medical and agricultural ants, are identified. As disclosed herein, Applicant has effectively lured ants using a bait laced withinsecticidal yeast. The yeast was engineered to express RNA interfering (RNAi) pesticides targeting ant genes, thereby permitting biorational ant control. Such yeast strains engineered to express insecticidal RNAi molecules may be interchangeably referred to as “yeast-based RNAi biopesticides,” “RNAi biopesticides,” “interfering RNA biopesticides,” and the like.

[0067] Disclosed RNAi biopesticides can threaten the survival of a variety of ants, including but not limited to carpenter ants, fire ants, formicine ants, fungus-growing ants, leafcutter ants, imported ants, New World ants, jet ants or jet black ants, pharaoh ants, polygynous ants, Argentine ants, pavement ants, odorous house ants, and crazy ants. For example, disclosed RNAi biopesticides can be used to control ant populations including Acromyrmex spp., e.g., Acromyrmex echinatior, Atta spp., e.g., Atta colombica and Atta cephalotes, Camponotus spp., e.g., Camponotus pennsylvanicus and Camponotus floridanus, Cataglyphis spp., e.g., Cataglyphis hispanica, Cyphomyrmex spp., e.g., Cyphomyrmex costatus, Dinoponera spp., e.g., Dinoponera quadriceps, ormica spp., e.g., Formica exsecta, Harpegnathos spp., e.g., Harpegnathos saltator, Lasius spp., e.g., Lasius fuliginosus, Nylanderia spp., e.g., Nylanderia fulva, Monomorium spp., e.g., Monomorium pharaonic, Linepithema spp., e.g., Linepithema humile, Odontomachus spp., e.g., Odontomachus brunneus, Ooceraea spp., e.g., Ooceraea biroi, Paratr echina spp., e.g., Paratrechina longicornis, Pogonomyrmex spp., e.g., Pogonomyrmex barbatus, Pseudomyrmex spp., e.g., Pseudomyrmex gracilis, Solenopsis spp., e.g., Solenopsis invicta, Temnothorax spp., e.g., Temnothorax curvispinosus Tetramorium spp., e.g., Tetramorium immigrans, Trachymyrmex spp., e.g., Trachymyrmex cornetzi, Trachymyrmex septentrionalis, Trachymyrmex zeteki, Vollenhovia spp., e.g., Vollenhovia emeryi, Wasmannia spp., e.g., Wasmannia auropunctata, and combinations thereof

[0068] There is a need to provide biopesticides that selectively induce mortality in ants. An emerging strategy for ant control entails introducing, such as by the ant ingesting, the interfering RNA (iRNA) that is effective to inhibit the expression of genes implicated in the fitness and / or survival by RNA interference (RNAi). One theoretical advantage of iRNA pesticides, which may also be referred to as iRNA biopesticides, is target specificity. That is, inhibiting expression of an organism-specific gene can result in selective toxicity for the target organism, such as an ant, without observable impact to non-target organisms. Accordingly, such selectivity would not be expected to contribute to broad-spectrum toxicity and selection of insecticide-resistant organisms.

[0069] RNA interference (RNAi) is activated when an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. In addition to their use of Argonaute family proteins, a common thread among RNAi-related pathways is their dependence on sequencespecific binding between short interfering RNAs (approximately 20 to 30 nucleotides) and target sequences (Obbard et al., Philos. Trans. R. Soc. Lond. B. Biol. Sci. 2009; 364:99-115). Exogenous RNA constructs, such as dsRNA and small or short hairpin RNA (shRNA), are usually processed into 20-30 nucleotide duplexes by the ribonuclease III enzyme DICER. See, e.g., Kim & Rossi, Biotechniques . 2008 Apr; 44(5): 613-616 and Sheng et al., Front Bioeng Bi otechnol. 2020 Aug 7; 8: 940. These nucleotide duplexes are then incorporated into the RNA induced silencing complex (RISC) by the catalytic component Argonaute. The two strands of RNA are unwound, and one strand is used as a guide strand, which binds to complementary mRNAs. The RISC complex will cleave the mRNA when base pair matching is perfectly complementary, or the complex can bind to the intact mRNA and suppress translation when there are mismatched base pairs, thereby mediating degradation or suppression of the endogenous transcript. Degradation or suppression of transcripts that code for essential genes in the insect may result in decreased amounts of criticalgene products, reduced fitness, and increased mortality. See, e.g., Zotti & Smagghe, NeotropEntomol. 2015 Jun;44(3): 197-213 and Burand & Hunter, J Invertebr Pathol. 2013 Mar;112 Suppl: S68-74.

[0070] Various strategies have been explored to deliver iRNA biopesticides to a desired target insect ant population. However, oral administration or absorption of iRNA, such as involving consumption or ingestion of the iRNA, in contrast to, e.g., injection, has clear advantages for delivery in the field. As examples, the feasibility of delivering iRNA to a target organism has been explored using naked double stranded RNA (dsRNA), dsRNA combined with a transfection reagent, and nucleic acid molecules mixed with an artificial diet. See, e.g., Taning et al., Journal of Ant Science April 2016;89:803-814.

[0071] Additional ant control strategies may also be combined with iRNA biopesticides, including attractive, phagostimulant, and insecticidal element. Herein, baits and attractants may be referred to interchangeably. In one example, attractive targeted sugar baits (ATSBs) can be used to deliver iRNA biopesticides to provide compositions with excellent shelf life and residual activity, thereby reducing overall insecticide use. Together, disclosed are species-specific, cost- effective, scalable, user-friendly, and sustainable iRNA biopesticides targeting ants for control of an ant population.

[0072] Controlling an ant population can involve any of the management or regulation of ants to prevent them from causing harm or nuisance, limit their growth, spread, or presence, undermining the fitness and / or the survival of ants in an ant population. Controlling an ant population does not necessarily entail completely eliminating a population but may rather involve maintaining their population at a level that does not pose a significant threat to human health, safety, or the environment.

[0073] The term “iRNA” refers to ribonucleic acid (RNA) sequences and constructs that are able to operate within the RNA interference (RNAi) pathway by interfering with transcriptional or post-transcriptional gene expression resulting in reduced or inhibited expression of a specific gene. For purposes herein, the term “iRNA” refers to short interfering RNA (siRNA), short hairpin RNA (shRNA) and double stranded RNA (dsRNA) that operate within the RNAi pathway. In some instances, the iRNA is produced within a cell via a DNA construct that encodes said iRNA. The iRNA of the present invention are synthetic and can be expressed in a vector or host cell in which the iRNA is not normally expressed. For example, the siRNA may target an insect gene, e.g., an ant gene, and be expressed by an exogenous vector or expressed in a bacterial or yeast cell that does not naturally contain the target gene or target sequence to which the siRNA binds. The iRNA may be modified in a manner that facilitates exogenous expression by the host cell, e.g., the nucleic acid or the complementary sequence used to express the iRNA may be modified at its ends or incorporated into an exogenous sequence to allow for expression in the target host cell. In some embodiments, the nucleic acid encoding the iRNA is operably linked to an exogenous sequence that allows for its expression.

[0074] RNAi strategies typically employ a synthesized, non-naturally occurring “iRNA” or “iRNA molecule,” which typically comprises at least an RNA fragment against a target gene, a spacer sequence, and a second RNA fragment which is complementary to the first, so that a doublestranded RNA (dsRNA) structure can be formed. The introduced dsRNA takes advantage of the native RNAi pathways in the insect to trigger down -regulation of target genes that may lead to the cessation of feeding and / or growth, which may result in the death of the insect ant.

[0075] The target nucleotide sequence may be selected from any suitable region or nucleotide sequence of the target gene or RNA transcript thereof. For example, the target nucleotide sequencemay be located within the 5'UTR or 3'UTR of the target gene or RNA transcript or within exonic or intronic regions of the gene. The skilled person will be aware of methods of identifying the most suitable target nucleotide sequences within the context of the full-length target gene. For example, multiple dsRNAs targeting different regions of the target gene can be synthesized and tested. Alternatively, digestion of the RNA transcript with enzymes such as RNAse H can be used to determine sites on the RNA that are in a conformation susceptible to gene silencing. Target sites may also be identified using in silico approaches, for example, the use of computer algorithms designed to predict the efficacy of gene silencing based on targeting different sites within the full- length gene.

[0076] The term “siRNA,” or “small interfering RNA,” refers to short interfering RNA or silencing RNA, which are short double-stranded RNA molecules of <30 base pairs in length, for example, about 19-30 base pairs in length that operate through the RNAi pathway. Each siRNA is unwound into two single-stranded RNAs (ssRNAs), one of which is incorporated into the RNA- induced silencing complex (RISC) leading to post-transcriptional gene silencing. siRNAs can be generated in several ways. In some cases, long dsRNA is introduced to a cell, either by a virus, endogenous RNA expression (i.e., microRNA), or exogenously delivered dsRNA. The enzyme DICER cleaves the long duplex RNAs into siRNAs. Another way to introduce siRNA into cells is to express small hairpin RNA (shRNA) from plasmid vectors. Alternatively, chemically synthesized siRNA duplexes, which mimic the structure of DICER-processed products, are commonly used in research for gene silencing. Chemically synthesized siRNAs simply bypass the DICER cleavage step. In some preferred embodiments, the siRNA is about 25 bp in length. While use of longer (300-400 bp) double stranded RNA (dsRNA) molecules is one approach forproducing iRNA, the short length (21-25 bp) of custom small interfering RNAs (siRNAs) facilitates the design of highly specific iRNA.

[0077] The terms “short hairpin RNA” and “small hairpin RNA” are encompassed by the term “shRNA.” shRNAs are artificial RNAs having a secondary structure such that a portion of the RNA strand forms a hairpin loop. Expression of shRNA in cells is typically accomplished by delivery of a DNA construct to the cell, e.g., through a recombinant vector having an expression cassette facilitating transcription of the encoding DNA and production of the shRNA. shRNA is transcribed under the control of RNA Pol-II or Pol-III promoters, and folds into a structure resembling a siRNA duplex. shRNAs are then processed by DICER into siRNAs.

[0078] The term “dsRNA” (double stranded RNA) refers to long double-stranded RNA molecules that are cleaved by the enzyme DICER into short double-stranded fragments of about 20-25 nucleotide siRNAs.

[0079] RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) refers to the biological process in which RNA molecules interfere or inhibit the expression of specific genes with complementary nucleotide sequences to the iRNA (gene-specific suppression of gene expression). RNAi results in the degradation of mRNA after transcription, resulting in reduced translation and protein expression.

[0080] RNA interference techniques employ genetic constructs that encode iRNA molecules, such as dsRNA and shRNA. Typically, the RNA constructs comprise sense and anti-sense sequences which are placed in regions flanking an intron sequence in proper splicing orientation with donor and acceptor splicing sites. Alternatively, spacer sequences of various lengths can be employed to separate self-complementary regions of sequence in the construct. During processing of the gene construct transcript, intron sequences can be spliced-out, allowing sense and anti-sensesequences, as well as splice junction sequences, to bind forming double-stranded RNA. Alternatively, where secondary structure inhibits splicing machinery, the intron sequences are not spliced out and the dsRNA is supplied as a hairpin structure. When the dsRNA is expressed in a cell, ribonucleases bind to and cleave the double-stranded RNA, initiating a cascade of events leading to degradation of the target mRNA molecules, and thereby silencing such target genes. The phenomenon of RNA interference using shRNA is described in Sheng et al., Front Bioeng Biotechnol. 2020 Aug 7;8:940 and generally in Bass, Nature 411 : 428-29 (2001); Elbahir et al., Nature 411 : 494-98 (2001); and Fire et al., Nature 391 : 806-11 (1998); and WO 01 / 75164, where methods of making interfering RNA also are discussed.

[0081] The iRNAi can hybridize with the full-length mRNA encoded by the target gene or hybridize to a fragment of the target RNA or DNA (the target sequence). For example, to reduce expression of a target gene in an insect ant using RNAi, an expression cassette encoding an iRNA having the sequence of an mRNA transcribed from the target gene, or a substantially identical sequence (including those engineered not to translate the protein), or fragment thereof, is introduced into a yeast cell. The resulting yeast cell can then be fed to the ant to determine its ability to inhibit expression of the target gene and / or inhibit growth of the ant. Although the sequence of the iRNA used for RNAi need not be completely identical to mRNA transcribed from the target sequence of the target gene, it is typically substantially identical, e.g., at least 70%, 80%, 90%, 95%, 98%, or more identical to mRNA transcribed from the target sequence. It is known in the art that dsRNA molecules that are not perfectly complementary to mRNA transcribed from the target sequence (for example, having only 95% identity to mRNA transcribed from the target sequence) are effective to control insect ants (see, for example, Narva et al., U.S. Pat. No. 9,012,722).

[0082] Target genes can be selected based on a number of criteria, including gene essentiality, midgut expression level, and divergence from related species sequences. In the case of ants, suitable target genes encode, for example, potassium voltage-gated channel protein Shaker. Disclosed RNAi biopesticides target the Shaker gene and orthologs thereof in ants. Exemplary genomic sequences of potassium voltage-gated channel protein Shaker are available to one of skill in the art, e.g., by accessing tools provided by the National Center for Biotechnology (NCBI). Exemplary sequence identifiers for ant Shaker genes include LOC105204888, LOC126848243, LOC105149795, LOC108694211, LOC108727027, LOC108754362, LOC108764083,LQC108778041, LQC105255031, LOCI 14939378, LOCI 15237144. Preferably, the target gene is the potassium voltage-gated channel protein Shaker (LOC105426071) as exemplified in SEQ ID NO:12. Preferably, the iRNA is designed to be substantially identical, e.g., at least 70%, 80%, 90%, 95%, 98%, or more identical to mRNA transcribed from regions of SEQ ID NO: 12 with conserved sequence identity about known isoforms. Table 1 illustrates the conserved sequence identity of three regions of SEQ ID NO: 12 among several common ant species.

[0083] Table 1 : A comparison of three Shaker gene sequences among common ant species.

[0084] It is noted in Table 1 that the designation of “None” refers to the sequence matching known at the present time. Those of skill in the art would appreciate that as further sequencing is performed and / or published on these and additional ant species, additional Shaker genes with sequence identity to SEQ ID NO: 1, SEQ ID NO: 4 and / or SEQ ID NO: 7 may be identified.

[0085] Gene suppression” or “down-regulation of gene expression” or “inhibition or suppression of gene expression” are used interchangeably and refer to a measurable or observablereduction in gene expression or a complete abolition of detectable gene expression at the level of protein product (“gene silencing”), and / or mRNA product from the gene. In some embodiments, gene suppression results in gene silencing, referring to the ability of the iRNA to target mRNA for degradation, resulting in disrupted translation, which prevents protein expression. For example, the ability of the iRNA to suppress or down-regulate Shake can lead to the suppression or inhibition of an ant’s growth and maturation or lead to the organism’s death. The downregulation or inhibition may occur at the translational or post-translational stage of expression of the gene of interest by promoting transcript turnover, cleavage, or disruption of translation.

[0086] Inhibition of target gene expression may be quantified by measuring either the endogenous target RNA or the protein produced by translation of the target RNA and the consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism. Techniques for quantifying RNA and proteins are well known to one of ordinary skill in the art. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, spectinomycin, rifampicin, and tetracyclin, and the like.

[0087] In addition to inhibiting gene expression the provided nucleic acids, host cells, such as microbial cells, compositions, and methods may additionally reduce the production of the protein product of a target gene. Methods of quantifying proteins, such as comparing the production of a specific protein, e.g., Shaker, in an ant after exposure to a microbial cell engineered to produce disclosed iRNA targeting that specific protein compared to protein levels in an ant exposed to a microbial cell engineered to produce iRNA targeting a gene absent from the ant’s genome are known in the art and include, e.g., ELISA and Western blot analysis. See, e.g., Jay et al., Proc Natl Acad Sci USA. 2021 Oct 26; 118(43): e2107427118, Chang & Lovett, Biochem Mol Biol Educ.201 1 Jul;39(4):291-7, and Spencer et al., Biochem Biophys Res Commun . 1993 Feb 26; 191 (1 ):201 - 6.

[0088] The term “gene” refers to a polynucleotide sequence that comprises control and coding sequences necessary for production of a polypeptide (protein). The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence. A gene includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene (e.g., promoters, enhancers, etc.). A gene may be an uninterrupted coding sequence or may include one or more introns contained between splice junctions. As used herein, a gene may include variants of the gene, which include, but are not limited to, modifications such as mutations, insertions, deletions, or substitutions of one or more nucleotides. A “target gene” is the gene targeted for down-regulation or suppression by the iRNA of the present technology, such as Shaker and orthologs thereof. A “gene product” can refer to either the mRNA or protein expressed from a particular gene.

[0089] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. The monomer is typically referred to as a nucleotide. Nucleic acids can include modified nucleotides that permit correct read through by a polymerase and do not significantly alter expression of a polypeptide encoded by that nucleic acid.

[0090] The phrase “nucleic acid sequence encoding” refers to a nucleic acid, such as DNA, which is the template for transcription of a specific RNA molecule, e.g., a shRNA, a dsRNA, or an mRNA that is translated into a protein. The nucleic acid sequences include both the full-lengthnucleic acid sequences as well as non-full-length sequences derived from the full-length sequences. A coding sequence can include degenerate codons (relative to the native sequence) or sequences that provide codon preference in a specific host cell.

[0091] The term “promoter” refers to regions or sequence located upstream and / or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “yeast promoter” is a promoter capable of initiating transcription in yeast cells. A yeast promoter can be a nucleic acid sequence originally isolated from a yeast, but promoters not initially isolated from a yeast are also considered “yeast promoters” for the purposes of this disclosure.

[0092] An “expression cassette” refers to a nucleic acid construct, which when introduced into a host cell (e.g., a yeast cell), results in transcription of an RNA molecule (e.g., dsRNA or mRNA). An expression cassette typically includes a sequence to be expressed, and sequences necessary for expression of the sequence to be expressed, such as a promoter operably linked to the sequence. Generally, an expression cassette is inserted into an expression vector to be introduced into a host cell.

[0093] The words “complementary” or “complementarity” refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity can be partial, in which only some of the nucleic acids match according to base pairing, or complete, such as fully complementary or perfectly complementary, where all the nucleic acids match according to base pairing.

[0094] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. Theterms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0095] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified.

[0096] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or proteins of the invention, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST / . For example, the sequence of a dsRNA of the invention can be compared using the above techniques to the sequence of a target gene in an insect ant, taking into account the presence of uracil in the dsRNA and thymidine in the DNA. Sequences that have at least about 90% sequence identity using the methods described above are said to be “substantially identical.” This definition also refers to, and can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Optimal alignment of such sequences can be carried out by any of the publicly available algorithms or programs for determining sequence identity and alignment, e.g., BLAST.

[0097] In some embodiments, the reduction, inhibition, or suppression of expression of Shaker in the ant results in life cycle disruptions, such as reduced viability, growth, development or reproduction. Such assessments are within the grasp of one of skill in the art and described in, e.g., US11252965B2, US11117938B2, US20210054379A1, and US11198868B2. In some embodiments, the reduction, inhibition, or suppression of target gene expression is determined relative to a wild-type ant. In some embodiments, the reduction, inhibition, or suppression of target gene expression is determined relative to an ant contacted with a wild-type yeast, such as wild type Saccharomyces cerevisiae. In some embodiments, the reduction, inhibition, or suppression of target gene expression is determined relative to an ant contacted with a yeast, such as Saccharomyces cerevisiae engineered to express an iRNA that does not target an ant gene, e.g., lacks complementarity to mRNA transcribed from an ant gene. Exemplary effects include the inability of larvae to mature to pupae or adult stages, reductions in fitness, such as reproductive fitness, including reduced capacity for sexual reproduction by the insect, inhibition of differentiation and development, e.g., growth inhibition, inhibited muscle, appendage formation, and death. In some embodiments, the target gene required for maturation and / or growth refers to a gene necessary for the survival, growth, or development of larvae into an adult and may ultimately result in death. In some embodiments, the gene may inhibit the ability of the larvae to develop into pupae, of pupae from developing into adults, or any intervening developmental step. In some instances, the inhibition or suppression of the target gene results in the inability of an adult insect to survive.

[0098] In some examples, downregulation or inhibition of gene expression in cells of an ant can be confirmed by phenotypic analysis of the cell or the whole ant, for example death of larval stage, pupal stage, or adult stage ants (which can be quantitated, for example, as percent (%)mortality). Suitably, the iRNA or compositions provide a % mortality of at least about 50%, alternatively at least about 60%, alternatively at least about 70%, alternatively at least about 75%, alternatively at least about 80%, at least about 90%, alternatively at least about 95%, alternatively at least about 98%, alternatively at least about 100%, wherein each range is inclusive and including any and all numerical values and ranges in between.

[0099] Other methods of confirming downregulation of the gene expression are known in the art, and include, but are not limited to, measurement of mRNA or protein expression using molecular techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (MA), other immunoassays, or fluorescence-activated cell analysis (FACS) and the like.

[0100] In some embodiments, the effectiveness of larvicide is characterized by the lethal concentrations (LC) for mortality and inhibition of adult emergence (IE). In some embodiments, the effectiveness of the insecticide is characterized by the lethal concentration or lethal dose (LD) for an adult insecticide.

[0101] In some embodiments, the target sequences of disclosed RNAi biopesticides are conserved in multiple ant genera and species but not conserved in non-targeted species. Through the identification and use of multiple larval lethal genes and multiple target sequences to each gene, the present invention is able to reduce, inhibit or eliminate insecticide resistance arising from a point mutation in any one target sequence.

[0102] Suitably, the sequences and genes targeted by the present technology are specific to ants. Downregulation or inhibition of target gene expression is “specific” when downregulation or inhibition of the target gene occurs without resulting in any detrimental effects on other genes ofthe targeted organism or genes of other non-related organisms (e.g., humans, mammals, etc.). The targeted sequences selected were analyzed and determined to have little risk for targeting genes in humans. Methods of determining if sequences specifically target human genes are known in the art, and include, for example, assessing human risk empirically through toxicity testing on human cells in vitro and on animal models in vivo, and in silico methods to select only risk-reduced sequences for siRNA synthesis.

[0103] Interfering RNA (iRNA) and Other Nucleic Acid Molecules

[0104] In some aspects, provided herein are interfering RNA molecules (iRNA) effective to inhibit the expression of a gene in an ant, such as by RNA interference. In additional aspects, provided are nucleic acid sequences, such as DNA sequences, encoding the disclosed iRNA molecules. Also provided are nucleic acid constructs comprising the iRNA molecules and nucleic acid sequences encoding the same. In further aspects, provided are microbial host cells, such as a yeast cell, bacterial cell, plant cell, or algal cell comprising the disclosed iRNA and nucleic acid sequences encoding the same. Herein, the disclosed iRNA molecules may be referred to simply as “iRNA.”

[0105] Interfering RNA (iRNA)

[0106] In some aspects, provided is an iRNA molecule comprising at least one dsRNA, such as an shRNA, where the dsRNA is a region of double-stranded RNA comprising annealed strands that are either partially or fully complementary. In some embodiments, one strand of the dsRNA comprises a sequence of at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, atleast 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 contiguous nucleotides which is partially or fully complementary to a target nucleotide sequence within an ant target gene, such as Shaker or an ortholog thereof.

[0107] In some embodiments, one strand of the dsRNA comprises a sequence of 15 to 30 contiguous nucleotides, 20 to 30 contiguous nucleotides, 25 to 35 contiguous nucleotides, 30 to 50 contiguous nucleotides, 45 to 75 contiguous nucleotides, 50 to 100 contiguous nucleotides, 65 to 125 contiguous nucleotides, 75 to 150 contiguous nucleotides, 85 to 120 contiguous nucleotides, 100 to 150 contiguous nucleotides, 125 to 175 contiguous nucleotides, 150 to 200 contiguous nucleotides, 300 to 600 contiguous nucleotides, 450 to 600 contiguous nucleotides, 500 to 650 contiguous nucleotides, or 550 to 750 contiguous nucleotides, wherein each range is inclusive, which is partially or fully complementary to a target nucleotide sequence within an ant target gene, such as Shaker or an ortholog thereof.

[0108] In some embodiments, disclosed iRNA has at least 60% complementarity, at least 65% complementarity, at least 70% complementarity, at least 75% complementarity, at least 80% complementarity, at least 85% complementarity, at least 86% complementarity, at least 87% complementarity, at least 88% complementarity, at least 89% complementarity, at least 90% complementarity, at least 91% complementarity, at least 92% complementarity, at least 93% complementarity, at least 94% complementarity, at least 95% complementarity, at least 96% complementarity, at least 97% complementarity, at least 98% complementarity, at least 99% complementarity, or 100% complementarity to a messenger RNA transcript that has been transcribed from a target nucleotide sequence within an ant target gene, such as Shaker or anortholog thereof. Tn embodiments where the % complementarity is less than 100%, the iRNA may include one or more mismatches with the target nucleotide sequence within an ant target gene.

[0109] In some embodiments, the disclosed percent identity is to at least a 19, at least a 20, at least a 21, at least a 22, at least a 23, at least a 24, at least a 25, at least a 26, at least a 27, at least a 28, at least a 29, at least a 30, at least a 35, at least a 40, at least a 45, at least a 50, at least a 55, at least a 60, at least a 65, at least a 70, at least a 75, at least a 80, at least a 85, at least a 90, at least a 95, at least a 100, at least a 110, at least a 120, at least a 130, at least a 140, at least a 150, at least a 160, at least a 170, at least a 180, at least a 190, at least a 200, at least a 210, at least a 220, at least a 230, at least a 240, at least a 250, at least a 260, at least a 270, at least a 280, at least a 290, or at least a 300 contiguous nucleotide fragment of a target ant gene.

[0110] In some embodiments, the iRNA effective to inhibit the expression of a gene in an ant is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), double stranded RNA (dsRNA), or RNA construct. In some embodiments, the siRNA, shRNA, dsRNA, or RNA construct is encoded by a DNA construct, such as a recombinant vector comprising an expression cassette, which allows for expression of the iRNA within a host cell, such as a yeast cell.

[0111] In some embodiments, disclosed iRNA inhibits the expression of Shaker in any one or more of carpenter ants, fire ants, formicine ants, fungus-growing ants, leafcutter ants, imported ants, New World ants, jet ants or jet black ants, pharaoh ants, polygynous ants, Argentine ants, pavement ants, odorous house ants, and crazy ants. For example, disclosed RNAi biopesticides can be used to control ant populations including Acromyrmex spp., e.g., Acromyrmex e china! i or, Atta spp., e.g., Atta colombica and Atta cephalotes, Camponotus spp., e.g., Camponotus pennsylvanicus and Camponotus floridanus, Cataglyphis spp., e.g., Cataglyphis hispanica, Cyphomyrmex spp., e.g., Cyphomyrmex costatus, Dinoponera spp., e.g., Dinoponera quadriceps,Formica spp., e.g., Formica exsecta, Harpegnathos spp., e.g., Harpegnathos saltator, Lasius spp., e.g., Lasius fuliginosus, Nylanderia spp., e.g., Nylanderia fulva, Monomorium spp., e.g., Monomorium pharaonic, Linepithema spp., e.g., Linepithema humile, Odontomachus spp., e.g., Odontomachus brunneus, Ooceraea spp., e.g., Ooceraea biroi, Paratrechina spp., e.g., Paratr echina longicornis, Pogonomyrmex spp., e.g., Pogonomyrmex barbatus, Pseudomyrmex spp., e.g., Pseudomyrmex gracilis, Solenopsis spp., e.g., Solenopsis invicta, Temnothorax spp., e.g., Temnothorax curvispinosus Tetramorium spp., e.g., Tetramorium immigrans, Trachymyrmex spp., e.g., Trachymyrmex cornetzi, Trachymyrmex septentrionalis, Trachymyrmex zeteki, Vollenhovia spp., e.g., Vollenhovia emeryi, Wasmannia spp., e.g., Wasmannia auropunctata and combinations thereof.

[0112] In some embodiments, the target gene is SEQ ID NO: 12 or an ortholog thereof. In some embodiments, the target gene Shaker or an ortholog thereof, comprises a target nucleotide sequence that is at least 55%, 65%, 75%, 80%, 84%, 88%, 90%, 92%, or 96% identical to any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7. In some embodiments, the target ant gene Shaker or an ortholog thereof comprises a target nucleotide sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7. In some embodiments, the target ant gene comprises a nucleotide sequence represented by any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7

[0113] In some embodiments, an iRNA molecule effective to inhibit the expression of Shaker in an ant is partially complementary to the nucleobase sequence of any one of SEQ ID NO: 1, SEQ ID NO:4, or SEQ ID NO :7. In some embodiments, an iRNA molecule effective to inhibit the expression of Shaker in an ant is fully complementary to the nucleobase sequence of any one ofSEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO :7. Tn some embodiments, an iRNA molecule effective to inhibit the expression of Shaker in an ant is partially complementary to the mRNA transcribed from any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO :7. In some embodiments, an iRNA molecule effective to inhibit the expression of a gene in an ant is fully complementary to the mRNA transcribed from any one of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7

[0114] In some embodiments, the iRNA molecule effective to inhibit the expression of a gene in an ant comprises a sequence of 20-30, 21-29, 22-28, 23-27, or 24-26 contiguous nucleotides that is complementary to a target gene sequence, wherein each range is inclusive. In some embodiments, the iRNA molecule effective to inhibit the expression of a gene in an ant comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 contiguous nucleotides that are partially or perfectly complementary to mRNA transcribed from a target gene in an ant.

[0115] In some embodiments, the iRNA is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% complementary to a portion of mRNA transcribed from the target sequence. In some embodiments, the iRNA is about 70% to 99%, 75% to 95%, 80% to 90%, or 85% to 90% complementary to a portion of mRNA transcribed from the target sequence, wherein each range is inclusive. In some embodiments, the iRNA is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99%, more complementary to a portion of mRNA transcribed from the target sequence. In some embodiments, the iRNA is perfectly complementary to a portion of mRNA transcribed from the target sequence.

[0116] In some embodiments, the iRNA is an RNA construct, a double stranded RNA(dsRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an anti-sense oligonucleotide. In preferred embodiments, the iRNA is an shRNA.

[0117] In some embodiments, disclosed iRNA is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% complementary to a portion of mRNA transcribed from SEQ ID NO: 12. In some embodiments, disclosed iRNA is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% complementary to a portion of mRNA transcribed from any of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7. In some embodiments, disclosed iRNA is about 70% to 99%, 75% to 95%, 80% to 90%, or 85% to 90% complementary to a portion of mRNA transcribed from any of SEQ ID NO:1, SEQ ID NO: 4, or SEQ ID NO: 7, wherein each range is inclusive. In some embodiments, disclosed iRNA is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to a portion of mRNA transcribed from any of SEQ ID NO:1, SEQ ID NO: 4, or SEQ ID NO:7 In some embodiments, disclosed iRNA is perfectly complementary to a portion of mRNA transcribed from any of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7

[0118] Nucleic Acid Sequences Encoding iRNA

[0119] In some aspects, provided herein are nucleic acid sequences, such as DNA sequences, encoding iRNA effective to inhibit the expression of a target gene in an ant. In some embodiments, an expression vector comprises the DNA sequence encoding iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, a microbial cell, such as a yeast cell, comprises the expression vector. Herein, the term “expression vector” may be used interchangeably with “recombinant vector.”

[0120] Suitable DNA constructs will depend on the type of cell in which to express the RNA. In some embodiments, the DNA construct is a linear or a closed circular plasmid or expression vector. In some embodiments, the DNA constructs will be integrated into the host cell genome, for example, integrated into a yeast or bacterial cell genome.

[0121] In some embodiments, the DNA construct is a suitable expression vector. Sequences that encode the iRNA of the present technology can be inserted into a vector under the control of a suitable promoter that functions in one or more microbial hosts to drive expression of a linked coding sequence or other DNA sequence. Suitable vectors are known in the art and selecting the appropriate vector will depend on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Vectors may include one, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more selectable marker genes, terminators, enhancers and / or a constitutive or inducible promoter allowing expression of exogenous DNA. Vectors can also include viral vectors and the like.

[0122] Host cells, such as yeast cells, can be engineered to include such vectors, e.g., Saccharomyces cerevisiae. Suitable promoters for expression in yeast are well known and include, for example, the bacteriophage T7 promoter, promoters from GALI (which is induced by the presence of galactose), ADH1, the TEF1 promoter and the AOX promoter (a methanol inducible promoter), and the like. Various methods are available to transform S. cerevisiae cells with exogenous DNA and producing recombinant products. For example, transformed cells are selected by phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).

[0123] In some embodiments, auxotrophic yeast strains are transformed with exogenous DNA encoding disclosed iRNA. Use of these strains relies on marker genes that encode key enzymes invarious essential metabolic pathways. Examples include the URA, HIS3, LEU2, TRP1, and MET 15 marker genes, which encode essential enzymes for de novo synthesis of pyrimidines, 1- histidine, 1 -leucine, 1 -tryptophan, and 1 -methionine, respectively. Yeast strains are auxotrophic for the nutrient in question due to the absence of a functional chromosomal copy of the marker gene. The auxotrophic yeast strains can thus be propagated only in media that contain the appropriate nutrients. Synthetic auxotrophs may also be engineered to require particular compounds.

[0124] In some embodiments, more than one iRNA may be expressed by a single recombinant vector introduced into a host cell, such as a microbial cell. In some embodiments, more than one iRNA may be expressed by multiple recombinant vectors introduced into a host cell, such as a microbial cell. In some embodiments, the recombinant vector comprises multiple expression sites, each site able to drive the expression of a different nucleotide sequence. By this method, multiple iRNAs can be expressed in a single cell, where the multiple iRNA can either target multiple sites on a single gene or target multiple genes within at least one ant species.

[0125] In some embodiments the vector is a plasmid. Other vectors include artificial chromosomes and linear nucleic acid molecules that are distinct from linearized plasmids. In some embodiments the vector is an integrating vector. In some embodiments the vector comprises an expression control element operably linked to a nucleic acid to be transcribed, e.g., DNA encoding disclosed iRNA. Three well known plasmid systems used for recombinant expression and replication in yeast cells include integrative plasmids, low-copy -number ARS-CEN plasmids, and high-copy-number 2p plasmids. See, e.g., Christianson et al., Gene. 1992;110: 119-22; Sikorski,"Extrachromosomal cloning vectors of Saccharomyces cerevisiae" , in Plasmid, A PracticalApproach, Ed. K. G. Hardy, IRL Press, 1993; Parent, S.A., and Bostian, K.A., Recombinant DNAtechnology: yeast vectors, p. 121-178. In Wheals, A.E., et al. (eds.) The yeasts, vol. 6. Yeast genetics. Academic Press, Longon, UK (1995).

[0126] An example of integrating plasmids of use in budding yeast are Yip plasmids, which are maintained at one copy per haploid genome and inherited in Mendelian fashion. Such a plasmid, containing a nucleic acid of interest, a bacterial origin of replication and a selectable gene (typically an antibiotic- resistance marker), is typically produced in bacteria. The purified vector may be linearized and used to transform competent yeast cells. YCp plasmids, which contain the autonomous replicating sequence (ARS1) and a centromeric sequence (CEN4), are examples of low-copy- number ARS-CEN plasmids. These plasmids are usually present at 1-2 copies per cell. An example of the high-copy-number 2p plasmids are YEp plasmids, which contain a sequence approximately 1 kb in length (named the 2p sequence). The 2p sequence acts as a yeast replicon giving rise to higher plasmid copy number. These plasmids may require selection for maintenance.

[0127] In some embodiments, the recombinant vector comprises an expression cassette comprising a promoter operably linked to a DNA sequence encoding an iRNA molecule that specifically inhibits expression of a target gene in an ant, such as Shaker. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a yeast promoter.

[0128] Yeast vectors, e.g., plasmids, described herein may also contain expression control sequences, e.g., promoter sequences. A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and transcription factors, to initiate the transcription of a nucleic acid sequence. The phrase "operably linked" indicates that an expression control element, e.g., a promoter, is in an appropriatelocation and / or orientation in relation to a nucleic acid to control transcriptional initiation and / or expression of the nucleic acid.

[0129] A promoter may be one that is naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment. Alternatively, a promoter may be a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid segment in its natural environment. Such promoters may include promoters of other genes and promoters that are not naturally occurring. An expression control element may be derived from a yeast of the species or strain in which RNAi is to be used or in which the RNAi pathway is to be engineered. For example, if RNAi is to be used in S. cerevisiae, it may be desirable to use a S. cerevisiae promoter to direct expression of a dsRNA. However, any expression control element capable of directing transcription in the cell of interest may be used.

[0130] The promoters employed may be either constitutive or inducible. For example, various yeast-specific promoters may be employed to regulate the expression in yeast cells. Examples of inducible yeast promoters include GAL 1-10, GALI, GALL, GALS, TET, CUP1, VP 16 and VP 16- ER. Examples of repressible yeast promoters include Met25. Examples of constitutive yeast promoters include glyceraldehyde 3 -phosphate dehydrogenase promoter (GPD or GAP), phosphoglycerate kinase (PGK), alcohol dehydrogenase promoter (ADH), translation- elongation factor- 1 -alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1), and MRP7. Promoters containing steroid response elements (e g., glucocorticoid response element) inducible by glucocorticoid or other steroid hormones can also direct expression in yeast. Yet other yeast constitutive or inducible promoters such as those of the genes for alpha factor, phosphate pathwaygenes (e g., PH05), or alcohol oxidase may be used. In some embodiments, the vector comprises an expression control element known as an upstream activating sequence (UAS).

[0131] Additional yeast promoters which may be used in accordance with the present disclosure include RNA polymerase III promoters, e.g., SNR52 and the like. Further exemplary yeast promoters include pADHl, pTEFl, ScRNR2, pTEFl, pADHl, pTPIl, pHXT7, pTDH3, pPGKl, pPYKl, pHXT7, pGALl, and pGALlO. Such promoters are described, e.g., by Waterham et al., Gene. 1997 Feb 20;186(l):37-44, Partow et al., Yeast. 2010 Nov;27(l l):955-64, Microb Cell Fact. 2013 Sep 23: 12:82, Zha et al., PLoS One. 2013 Jul 2;8(7):e68317, Hector et al., N Biotechnol. 2019 Nov 25:53: 16-23, Sun et al., Biotechnol Bioeng. 2012 Aug;109(8):2082-92, and Silva & Srikrishnan, FEMS Yeast Res. 2012 Mar; 12(2): 197-214.

[0132] Such elements, which are considered functional equivalents of metazoan enhancers, can activate gene transcription from remote positions, e.g., up to about 1,000 - 1,200 bp from the promoter. See, e.g., Petrascheck, M, et al., Nucleic Acids Res., 33(12): 3743-3750, 2005, for discussion. The level of expression achieved using an inducible promoter can be regulated, e.g., by controlling the amount of inducing agent or the length of exposure. Further, mutant promoters that result in lower expression levels than a wild type promoter can be used. In some embodiments, an expression control element originates from a species in which the expression control element is to be used to direct expression while in other embodiments the expression control element originates from a different species.

[0133] In some embodiments, the recombinant vector is a plasmid, such as an integrating plasmid. In some embodiments, the plasmid is a pRS plasmid (e.g., pRS3O3, pRS304, pRS305 or pRS306 or other integrative plasmids). In some embodiments, the plasmid is an extrachromosomal plasmid (e.g., pRS313, pRS314, pRS315, pRS316, pRS413, pRS414, pRS415, pRS416, pRS423,pRS424, pRS425, pRS426). In some embodiments the plasmid is a member of the YES™ Vector Collection, e.g., pYES (Invitrogen, Carlsbad, CA). In some embodiments, the plasmid is a Gateway plasmid. See, e.g., Geiser. Biotechniques, 38:378-382 (2005); Van Mullem V, et al., Construction of a set of Saccharomyces cerevisiae vectors designed for recombinational cloning. See, e.g., Alberti et al., Yeast, 2007;24(10):913-9. Such vectors are described in, e.g., WO2011031319A8.

[0134] In some embodiments, the recombinant vector comprises an expression cassette comprising a yeast promoter operably linked to a DNA sequence encoding an iRNA molecule that specifically inhibits expression of a potassium voltage-gated channel protein Shaker, or a homolog or ortholog thereof in an ant.

[0135] In some embodiments, the expression cassette comprises a yeast promoter operably linked to a DNA sequence encoding an iRNA molecule targeting a nucleotide sequence that is transcribable from a DNA sequence that is at least 55%, 65%, 75%, 80%, 84%, 88%, 90%, 92%, or 96% identical to the entire length of any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7. In some embodiments, the expression cassette comprises a yeast promoter operably linked to a DNA sequence encoding an iRNA molecule targeting a nucleotide sequence having about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7. In some embodiments, the expression cassette comprises a yeast promoter operably linked to a DNA sequence encoding an iRNA molecule targeting any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7 In some embodiments, the yeast promoter is a GPD promoter, which is also known as a GADPH or TDH3 promoter. The GPD promoter is a strong constitutive yeast expression promoter from glyceraldehyde 3 -phosphage dehydrogenase.

[0136] In some embodiments, the expression vector comprising DNA nucleic acid sequences encoding iRNA effective to inhibit the expression of a gene in an ant is pRS426 GPD vector. An exemplary pRS426 sequence is represented by SEQ ID NO: 11, and a GPD promoter sequence is represented by SEQ ID NO: 10. The pRS426 GPD yeast shuttle vector permits constitutive expression of inserts cloned into the multiple cloning sites downstream of a GPD promoter and upstream of a cycl terminator. See, e.g., Mumberg & Funk, Gene. 1995 Apr 14; 156(1 ): 119-22 and Mysore et al., Methods Mol Biol. 2019; 1858: 213-231.

[0137] In some embodiments, the pRS426 GPD vector comprises an expression cassette encoding iRNA effective to inhibit the expression of potassium voltage-gated channel protein Shaker, or a homolog or ortholog thereof in an ant. In some embodiments, the pRS426 GPD vector comprises an expression cassette encoding iRNA effective to inhibit the expression of potassium voltage-gated channel protein Shaker, or a homolog or ortholog thereof in an ant.

[0138] In some embodiments, the DNA sequence encoding disclosed iRNA is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 or the complement thereof. In some embodiments, the DNA sequence encoding disclosed iRNA is about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more identical to the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 9, or the complement thereof In some embodiments, the DNA sequence encoding disclosed iRNA is 100% identical to the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 In some embodiments, a DNA construct comprising DNA encoding disclosed iRNA has double stranded DNA that is identical (100%identity) to the entire length of SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:5 and SEQ IDNO:6, or SEQ ID NO:8 and SEQ ID NO:9

[0139] Alternatively, techniques available to one of skill in the art may be used to achieve stable integration of the disclosed expression cassettes, such as stable transformation and expression into a host organism, e.g., Saccharomyces cerevisiae. Exemplary methods and techniques include transposon systems, e.g., PiggyBac, CRISPR, lithium acetate (LiAc)-based methodologies, electroporation, gene gun transformation, and protoplast transformation. Such methods are described in, e.g., Uetake & Niki, In Vitro Cell Dev Biol Anim . 2011 Dec;47(10):689- 94, Kildegaard et al., Yeast. 2019 May; 36(5): 237-247.

[0140] In some embodiments, the iRNA is produced by a host cell which can express the iRNA from a DNA construct or expression vector comprising an expression cassette comprising DNA encoding the iRNA. Suitable cells include, but are not limited to, a bacterial, algal or yeast cell engineered to produce or express the iRNA from the encoding DNA sequence. Other suitable host cells, e.g., microorganism cells or plant cells, are known in the art. In some embodiments, the host cell expresses at least two unique iRNA molecules, alternatively at least three unique iRNA molecules, alternatively at least four unique iRNA molecules. In some embodiments, the host cell expresses from 1 to 10 unique iRNA molecules.

[0141] Microbial Host Cells Containing iRNA and Nucleic Acid Sequences Encoding the Same

[0142] In some aspects, provided herein are microbial host cells comprising iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, the microbial host cell comprises an expression vector comprising nucleic acid sequences, such as DNA sequences, encoding iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, the microbial hostcell is a yeast cell, bacterial cell, algal cell, or plant cell. In some embodiments, the microbial host cell is alive. In some embodiments, the microbial host cell is dead. Herein, “microbial host cell” and “microbial cell” are used interchangeably.

[0143] In some embodiments, the microbial host cell expresses at least one iRNA which targets a gene of interest for inhibition. In some embodiments, the microbial host cell expresses at least two, three, four, or five iRNA which target a gene of interest for inhibition. In some embodiments, the microbial host cell expresses any of from 1 to 15, 1 to 10, or 1 to 5 iRNA which target a gene of interest for inhibition.

[0144] In some embodiments, the microbial cell expresses at least two unique iRNA that target a single gene, alternatively at least three unique iRNA that target a single gene, alternatively at least four RNA that target a single gene. In some embodiments, the microbial cell expresses at least two unique iRNA that target two different genes, alternatively at least three unique iRNA that target at least two different genes, alternatively at least three unique iRNA that target at least two different genes, alternatively at least three unique iRNA that target at least two different genes.

[0145] In some embodiments, a microbial cell, such as a yeast cell, expresses at least two unique iRNA molecules that target Shaker or an ortholog thereof. In some embodiments, a host cell expresses at least three unique iRNA molecules that target Shaker or an ortholog thereof. In some embodiments, a host cell expresses at least three unique iRNA molecules that target Shaker or an ortholog thereof. In some embodiments, a microbial cell expresses at least four unique iRNA that target Shaker or an ortholog thereof.

[0146] In some embodiments, the microbial host cells are stably transformed with nucleic acid sequences encoding iRNA. In some embodiments, a disclosed expression cassette is integrated into the genomic DNA of the microbial host cell, such as Saccharomyces cerevisiae. In someembodiments, DNA encoding disclosed iRNA is integrated into the genomic DNA of the microbial host cell, such as Saccharomyces cerevisiae. Stable transformants may be produced by incorporating a DNA construct comprising a nucleotide sequence encoding iRNA into the host cell genome. Methods of forming stable transformants of host cells are known in the art and include, e.g., transformation of integrative plasmids.

[0147] In some embodiments, a yeast cell comprises iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, a bacterial cell comprises iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, the bacterial cell is Escherichia coli, Bacillus thuringiensis israelensis, or Lactobacillus spp., among others. In some embodiments, an algal cell comprises iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, a plant cell comprises iRNA effective to inhibit the expression of a gene in an ant.

[0148] In some embodiments, a yeast cell comprises a DNA sequence encoding iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, a bacterial cell comprises a DNA sequence encoding iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, the bacterial cell is Escherichia coli. In some embodiments, an algal cell comprises a DNA sequence encoding iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, a plant cell comprises a DNA sequence encoding iRNA effective to inhibit the expression of a gene in an ant.

[0149] In some embodiments, a yeast cell comprises iRNA effective to inhibit the expression of a gene in an ant. In some embodiments, the yeast cell is a species of Saccharomyces. In some embodiments, the yeast cell is Saccharomyces cerevisiae. In some embodiments, a Saccharomyces cerevisiae cell comprises iRNA effective to inhibit the expression of a gene in an ant. Other suitable host cells will be evident to one of skill in the art. In some embodiments, the yeast cell isalive. In preferred embodiments, the yeast cell is dead. In additional preferred embodiments, the yeast cell is heat-killed and / or lyophilized.

[0150] In some embodiments, a yeast cell, such as Saccharomyces cerevisiae, comprises a DNA sequence encoding iRNA effective to inhibit the expression of potassium voltage-gated channel protein Shaker, or an ortholog thereof in an ant. In some embodiments, the target gene has a target sequence represented by any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7, or a sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7.

[0151] In some embodiments, Saccharomyces cerevisiae comprises an expression vector comprising DNA sequences encoding iRNA effective to inhibit the expression of potassium voltage-gated channel protein Shaker or ortholog thereof in an ant. In some embodiments, the target gene has a target sequence represented by any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7, or a sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7. In some embodiments, the expression vector comprises an expression cassette comprising a DNA sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the entire length of any one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, or the complement thereof operably linked to a promoter. In some embodiments, the expression vector comprises an expression cassette comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, or the complement thereof, operably linked to a promoter. In some embodiments, the expression vector comprises double stranded DNA that is identical (100% identity) to the entire length of SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:8 and SEQ ID NO:9 In someembodiments, the recombinant vector is a pRS426 vector. Tn some embodiments, the pRS426 vector comprises a GPD promoter. In preferred embodiments, the S. cerevisiae cell is heat-killed and / or lyophilized.

[0152] In order to avoid introducing the replicating host cells or live microorganisms into the environment, host cells may be killed or inactivated, e.g., unable to grow and / or replicate, before being incorporated into the compositions described herein. Host cells are preferably killed or inactivated in a manner that maintains the ability of the host cell to act as a larvicide, i.e., the inactivation does not disrupt the iRNA molecules contained within said host cell. In some embodiments, the iRNA can be purified from the host cell before incorporating into the compositions. Suitable methods of killing or inactivating the host cell are known in the art, and include, but are not limited to, heat-inactivation, high pressure, plasma treatment at atmospheric pressure, sonication, low-amperage electric treatment, or dense phase carbon dioxide processing. Concerns about introducing live organisms into treated sites can be ameliorated by using heat- killed microbial host cells, which retain insecticidal potency.

[0153] Compositions

[0154] In some aspects, provided herein are compositions comprising iRNA effective to inhibit the expression of a gene in an ant. In additional aspects, provided are compositions comprising nucleic acid sequences, such as DNA sequences, encoding iRNA effective to inhibit the expression of a gene in an ant. In further aspects, provided are compositions comprising microbial cells comprising disclosed iRNA and nucleic acid sequences encoding the same. Also provided are compositions further comprising an attractant, a phagostimulant, an insecticide, or a combination thereof, in addition to the disclosed iRNA, nucleic acid sequences encoding the iRNA, and microbial host cells comprising the same.

[0155] In some embodiments, a disclosed composition comprises two or more unique iRNA molecules, wherein the two or more iRNA molecules are present on, such as encoded by, the same nucleic acid construct, on different nucleic acid constructs, or any combination thereof. In some embodiments, a disclosed composition comprises two or more nucleic acid sequences, such as DNA sequences, wherein the two or more nucleic acid sequences each encode a different interfering RNA molecule.

[0156] In some embodiments, a disclosed composition comprises bacterial cells, yeast cells, algal cells, or a combination thereof. In some embodiments, a disclosed composition comprises nanoparticles, e.g., chitosan nanoparticles. In some embodiments, a disclosed composition is suitable for larval soaking. In some embodiments, a disclosed composition is suitable for sugar feeding. In some embodiments, a disclosed composition is provided in a trap, such as an ant trap. In some embodiments, a disclosed composition is provided in the form of a dried tablet. In some embodiments, a disclosed composition is suitable for topical application, such as application on areas or surfaces where ants are likely to encounter the compositions. Other suitable methods of delivery are known in the art. Thus, compositions may include the necessary components to deliver the iRNA to insects, such as ants. For example, compositions may comprise nanoparticles, bacterial cells, yeast cells, algal cells and the like that contain or express the iRNA.

[0157] Microbial Host Cell Compositions

[0158] In some aspects, provided are compositions comprising a microbial cell containing an expression cassette comprising a promoter operably linked to a DNA sequence encoding an iRNA molecule that specifically inhibits expression of a target gene in an ant. In some embodiments, the microbial cell is a yeast cell, abacterial cell, a plant cell, or an algal cell. In preferred embodiments, the microbial cell is a yeast cell.

[0159] In some embodiments, the composition comprises a yeast cell engineered to produce iRNA effective to inhibit expression of a target gene in an ant. In some embodiments, the composition comprises a bacterial cell engineered to produce iRNA effective to inhibit expression of a target gene in an ant. In some embodiments, the composition comprises an algal or plant cell engineered to produce iRNA effective to inhibit expression of a target gene in an ant, such as potassium voltage-gated channel protein Shaker or an ortholog thereof.

[0160] In some embodiments, the target gene has a target sequence represented by any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7, or a sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7

[0161] In some embodiments, any of the yeast, bacterial, algal, or plant cell is / are alive. In other embodiments, any of the yeast, bacterial, algal, or plant cell is / are dead. In other preferred embodiments, any of the yeast, bacterial, algal, or plant cell is killed by heat, such as heat-killed, and / or lyophilized. In some embodiments, any of the yeast, bacterial, algal, or plant cell is synthesized into a ready-to use dry formulation.

[0162] In some embodiments, disclosed compositions comprise Saccharomyces cerevisiae comprising an expression vector comprising DNA sequences encoding iRNA effective to inhibit the expression of the target gene potassium voltage-gated channel protein Shaker or an ortholog thereof. In some embodiments, the target gene has a target sequence represented by any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7, or a sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO:1, SEQID NO:4, and SEQ ID NO:7

[0163] In some embodiments, disclosed compositions comprise Saccharomyces cerevisiae comprising an expression vector which includes an expression cassette comprising a DNA sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, or the complement thereof operably linked to a promoter. In some embodiments, the expression vector comprises an expression cassette comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 or the complement thereof operably linked to a promoter. In some embodiments, the recombinant vector is an extrachromosomal plasmid, such as a pRS426 vector. In some embodiments, the extrachromosomal plasmid has a GPD promoter. In some embodiments, the S. cerevisiae cell is heat-killed and / or lyophilized.

[0164] In some embodiments, disclosed compositions further comprise at least one suitable carrier, excipient, or diluent, such as an agriculturally acceptable carrier, excipient, or diluent. In some embodiments, disclosed compositions further comprise an attractant, phagostimulant, or an insecticide. In preferred embodiments, disclosed compositions further comprise an attractant, e.g., a sugar bait, such as an attractive sugar targeted bait.

[0165] In some embodiments, disclosed compositions are insecticidal. In some embodiments, upon contact with an organism having target gene Shaker or an ortholog thereof, such as a target organism, the disclosed compositions result in a percent (%) mortality of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 100% mortality, including any and all numerical values and ranges in between.

[0166] In some embodiments, the target organism is a more of a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a New World ant, a jet antor a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, and a crazy ant. In some embodiments, disclosed compositions are capable of controlling a population of ants.

[0167] Exemplary members of the ant population may include and are not limited to Acromyrmex spp., e.g., Acromyrmex echmatior. Atta spp., e.g., Atta colombica and Atta cephalotes, Camponotus spp., e.g., Camponotus permsylvanicus and Camponotus floridanus, Cataglyphis spp., e.g., Cataglyphis hispanica, Cyphomyrmex spp., e.g., Cyphomyrmex costatus, Dinoponera spp., e.g., Dinoponera quadriceps, Formica spp., e.g., Formica exsecta, Harpegnathos spp., e.g., Harpegnathos saltator, Lasius spp., e.g., Lasius fuliginosus, Nylanderia spp., e.g., Nylanderia fulva, Monomorium spp., e.g., Monomorium pharaonic, Linepithema spp., e.g., Linepithema humile, Odontomachus spp., e.g., Odontomachus brunneus, Ooceraea spp., e.g., Ooceraea biroi, Paratrechina spp., e.g., Paratrechina longicornis, Pogonomyrmex spp., e.g., Pogonomyrmex barbatus, Pseudomyrmex spp., e.g., Pseudomyrmex gracilis, Solenopsis spp., e.g., Solenopsis invicta, Temnothorax spp., e.g., Temnothorax curvispinosus Tetramorium spp., e.g., Tetramorium immigrans, Trachymyrmex spp., e.g., Trachymyrmex cornetzi, Trachymyrmex septentrionalis, Trachymyrmex zeteki, Vollenhovia spp., e.g., Vollenhovia emeryi, Wasmannia spp., e.g., Wasmannia auropunctata, and combinations thereof.

[0168] Attractants, Phagostimulants, and Insecticides

[0169] In some embodiments, the disclosed compositions are effective to inhibit expression of a target gene in an ant by RNA interference (RNAi). In some embodiments, the compositions comprise any of disclosed iRNA, nucleic acid sequences encoding said iRNA, constructs comprising the iRNA and nucleic acid sequences encoding the same, expression vectors comprising the nucleic acid sequences, expression cassette comprising the nucleic acid sequencesoperably linked to a promoter, and microbial cells containing the preceding in any combination. In some embodiments, a disclosed composition further comprises an attractant, phagostimulant, insecticide, or a combination thereof. In some embodiments, a disclosed composition further comprises at least one suitable carrier, excipient, or diluent, such as an agriculturally acceptable carrier, excipient, or diluent.

[0170] In some embodiments, the compositions further comprising an attractant, phagostimulant, insecticide, or a combination thereof are insecticidal. In some embodiments, upon contact with an organism having a target gene, such as a target organism, the disclosed compositions result in a percent (%) mortality of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 100% mortality, including any and all numerical values and ranges in between.

[0171] In some embodiments, a disclosed composition further comprises a bait and / or a trap, such as a lure trap. In some embodiments, the bait comprises an attractant. In some embodiments, the attractant is sugar. In some embodiments, the bait is an attractive targeted sugar bait or an attractive toxic sugar bait (ATSB). Attractive targeted sugar baits typically contain an attractant, including a form of sugar, such as a fruit syrup, and a toxic agent, such as a chemical insecticide. See, e.g., Wongthangsiri et al., Agriculture and Natural Resources, 2018;52(4):393-398.

[0172] Lure traps are commonly used to attract and kill insect ants. Design and use of such traps are well known to those of skill in the art. A lure trap of the invention can be any device into which the recombinant yeast of the invention are placed, and that prevents the insect ant from escaping once the insect ant has come into contact with the trap. The traps can be of various sizes, shapes, colors, and materials. Traps may be designed and manufactured specifically for use as aninsect trap, or can be a container converted and adapted from other uses such as, for example, a glass Petri dish, a metal coffee can, a cardboard box, or any ordinary plastic, metal, fiberglass, composite or ceramic container.

[0173] In some embodiments, a disclosed composition further comprises an insecticide, such as a chemical insecticide. In some embodiments, the chemical insecticide includes beta- Cyfluthrinmethoprene, DEET (N,N-diethyl-meta-toluamidedeltamethrin), etofenprox, hydramethylnon, imidacloprid, indoxacarb, permethrin, piperonyl butoxide, phenothrin, malathion, pyriproxyfen, sodium tetraborate decahydrate (borax), spinosad, thiamethoxam, among other insecticides known to induce mortality in any ant species, including combinations thereof.

[0174] In some embodiments, disclosed compositions further include an attractant, such as a bait. In some embodiments, the attractant comprises sugar, an octanol, a plant extract, a pheromone, a volatile organic compound, carbon dioxide, lactic acid, ammonia, or a combination thereof. Ant baits, attractants, and insecticides that can be included in accordance with the present disclosure are described by, e.g., Vander Meer & Milne, J Econ En tomol. 2017 Apr 1;110(2): 567- 574, Yesudai et al., J Econ Entomol. 2022 Apr 13;115(2):624-630, Brightwell et al., J Econ Entomol. 2010 Oct; 103(5): 1790-6, and Hoffmann, Bull Entomol Res. 2023 Apr; 113(2): 190-195.

[0175] In some embodiments, the bait is a sugar bait, such as an attractive targeted sugar bait or an attractive toxic sugar bait (ASB). ASBs, which are known in the art and commercially available, may include a sugar bait and a toxic agent, e.g., an insecticide. ATSBs are described in, e.g., WO 1996 / 017515 Al, PCT / US1995 / 015566, US 2020 / 0323190 Al, W02020185583 Al , W02009150254A1, Hapairai et al., Insect Biochem Mol Biol. 2020 May; 120: 103359, Mysore et al., PLoS Negl Trap Dis. 2020 Jul; 14(7): e0008479, Wongthangsiri et al., Agric.Nat. Resour.2018;52(4):393-398, Khan et al., PLoSOne. 2013 Sep 24;8(9):e77225, Fraser et al., Malar J. 2021Mar 17;20(l):15.

[0176] Methods

[0177] In some aspects, provided herein are methods of using a disclosed polynucleotide, expression cassette, host organism, such as an interfering RNA biopesticide composition, or a combination thereof, in the control of ant populations. In some embodiments, disclosed methods involve contacting an ant with a disclosed polynucleotide, expression cassette, host organism, composition, or a combination thereof, such that contacting the ant leads to consumption by the ant. The terms “control,” “controlling,” and the like, refer to, e.g., preventing proliferation and / or survival of ant populations and / or preventing or treating ant infestations. In some embodiments, the method comprises contacting the ant with a microbial cell comprising the nucleic acid sequence that is or is capable of producing an iRNA effective to inhibit expression of a target gene in the ant.

[0178] Herein, reference to an ant generally includes reference to different stages of life, such as pupae, larvae, and adult. However, in specific embodiments, disclosed methods involve contacting ant larvae with a disclosed polynucleotide, expression cassette, host organism, composition, or a combination thereof. In other embodiments, disclosed methods involve contacting an adult ant with a disclosed polynucleotide, expression cassette, host organism, composition, or a combination thereof. Additionally, herein, contacting an ant can encompass consumption of the disclosed polynucleotide, expression cassette, host organism, composition, or a combination thereof by the ant or feeding of the same to the ant.

[0179] Methods and cells delivering disclosed iRNA for contact with an insect include, but are not limited to, e.g. larval soaking, nanoparticles (e.g., chitosan nanoparticles), bacterial cells, yeastcells, algal cells, ovitraps, dried tablets, sugar feeding, and topical applications, among others. Thus, compositions may include the necessary components to deliver the iRNA to the targeted ant population. For example, compositions may comprise nanoparticles, bacterial cells, yeast cells, algal cells and the like that contain or express the iRNA.

[0180] In some embodiments, the disclosed methods comprise contacting the ant with a microbial cell comprising a nucleic acid sequence that is or is capable of producing an iRNA effective to inhibit expression of a target gene in the ant. In some embodiments, the microbial cell is a yeast cell, a bacterial cell, a plant cell, or an algal cell. In preferred embodiments, the microbial cell is a yeast cell.

[0181] In some embodiments, the disclosed methods comprise contacting an ant with a yeast cell engineered to produce iRNA effective to inhibit expression of a target gene in an ant. In some embodiments, the disclosed methods comprise contacting an ant with a bacterial cell engineered to produce iRNA effective to inhibit expression of a target gene in an ant. In some embodiments, the disclosed methods comprise contacting an ant with an algal or plant cell engineered to produce iRNA effective to inhibit expression of a target gene in an ant.

[0182] In some embodiments, the ant may be a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a New World ant, a jet ant or a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, or a crazy ant. Disclosed methods can be used to control ant populations including but not limited to the following representative ant genera and species: Acromyrmex spp., e.g., Acromyrmex echinatior, Atta spp., e.g., Atta colombica and Atta cephalotes, Camponotiis spp., e.g., Camponotiis pennsylvanicus and Camponotiis floridanus, Cataglyphis spp., e.g., Cataglyphis hispanica, Cyphomyrmex spp., e.g., Cyphomyrmex costatus, Dinoponera spp., e.g., Dinoponeraquadriceps, Formica spp., e.g., Formica exsecta, Harpegnathos spp., e.g., Harpegnathos saltator,Lasius spp., e.g., Lasius fuliginosus, Nylanderia spp., e.g., Nylanderia fulva, Monomorium spp., e.g., Monomorium pharaonic, Linepithema spp., e.g., Linepithema humile, Odontomachus spp., e.g., Odontomachus brunneus, Ooceraea spp., e.g., Ooceraea biroi, Par air echina spp., e.g., Paratr echina longicornis, Pogonomyrmex spp., e.g., Pogonomyrmex barbatus, Pseudomyrmex spp., e.g., Pseudomyrmex gracilis, Solenopsis spp., e.g., Solenopsis invicta, Temnothorax spp., e.g., Temnothorax curvispinosus Tetramorium spp., e.g., Tetramorium immigrans, Trachymyrmex spp., e.g., Trachymyrmex cornetzi, Trachymyrmex septentrionalis, Trachymyrmex zeteki, Vollenhovia spp., e.g., Vollenhovia emeryi, Wasmannia spp., e.g., Wasmannia auropunctata and combinations thereof.

[0183] In some embodiments, the target gene has a target sequence represented by any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7, or a sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7

[0184] In some embodiments, disclosed methods comprise contacting an ant with a yeast cell comprising an expression cassette comprising a nucleotide sequence encoding iRNA. In some embodiments, the iRNA is perfectly or partially complementary to a portion of mRNA transcribable from a nucleotide sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7. In some embodiments, the iRNA is perfectly or partially complementary to a portion of mRNA transcribable from any one of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:7.

[0185] In some embodiments, the disclosed methods comprise contacting an ant with a yeast cell comprising an expression cassette comprising a DNA sequence having at least 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% identity to the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, or the complement thereof, operably linked to a promoter. In some embodiments, the disclosed methods comprise contacting an ant with a yeast cell comprising an expression cassette comprising SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, or the complement thereof operably linked to a promoter. In some embodiments, the expression cassette is integrated into the genome of the yeast cell. In some embodiments, the expression cassette is included in a recombinant vector, e.g., a pRS426 vector. The recombinant vector can include a GPD promoter.

[0186] In some embodiments, the disclosed methods comprise contacting an ant, such that contacting the ant involves consumption or ingestion by the ant or larvae, with a dead microbial host cell as disclosed herein, as a microbial host that has been spray-dried, heat-killed, lyophilized, or suspended in an aqueous medium. In some embodiments, contacting the ant comprises consumption by mouth of the microbial host cell the by the ant. In some embodiments, it is preferred that the microbial host cell is heat inactivated to reduce or eliminate the ability of the microbial host cell to grow once released into a treatment area. In some embodiments, a yeast cell for contacting an ant is spray-dried, heat-killed, lyophilized, or suspended in an aqueous medium to reduce or eliminate the ability of the yeast to grow once released into a treatment area. In some embodiments, the yeast is synthesized into a ready -to use dry formulation. In some embodiments, the yeast is suspended in an aqueous medium comprising water. In some embodiments, the yeast is S. cerevisiae.

[0187] In some embodiments, the disclosed methods further comprise contacting the ant with a bait, such as a sugar bait. In some embodiments, the bait is present within a trap, such as a lure trap. In some embodiments, the bait and / or the trap comprises any of the nucleic acids, engineeredmicrobial host cell, or compositions disclosed herein. In some embodiments, the attractant comprises any of an attractive microbe, such as an attractive symbiont microbe, sugar, or a combination thereof.

[0188] In some embodiments, the disclosed methods comprise contacting an ant with an attractant containing sugar, e.g., sucrose, fructose, glucose, or any combination thereof and / or a sugar substitute. In some embodiments, the attractant comprises any one or more of corn syrup, fruit, fruit puree, and fruit juice.

[0189] In some embodiments, the bait is a sugar bait, such as an attractive targeted sugar bait or an attractive toxic sugar bait (ATSB). In some embodiments, the sugar bait comprises sugar and any one or more of at least one pheromone, at least one attractive symbiont, and at least one insecticide. ATSB’s, which are known in the art and commercially available, may include a sugar bait and a toxic agent, e.g., an insecticide. ATSBs are described in, e.g., WO2020185583A1, W02009150254A1, Hapairai et al., Insect Biochem Mol Biol. 2020 May; 120: 103359, Mysore et al., PLoS Negl Trap Dis. 2020 Jul; 14(7): e0008479, Wongthangsiri et al., Agric. Nat. Resour. 2018;52(4):393-398, Khan et al., £o5O«e. 2013 Sep 24;8(9):e77225, Fraser et al., Malar J. 2021 Mar 17;20(l):15.

[0190] In some embodiments, disclosed methods comprise contacting an ant with disclosed nucleic acids, engineered microbial host cells, compositions, or any combination thereof, which has been dispersed onto a surface in close proximity to an ant colony. In one example, contacting an ant with any of the disclosed nucleic acids, engineered microbial host cells, compositions, or any combination thereof, dispersed onto the surface is useful to exert larvicidal activity.

[0191] In some embodiments, disclosed methods comprise contacting an ant with spray-dried, heat-killed, lyophilized, or suspended in an aqueous medium Saccharomyces cerevisiaecomprising an expression vector comprising DNA sequences encoding iRNA effective to inhibit the expression of potassium voltage-gated channel protein Shaker or an ortholog thereof.

[0192] In some embodiments, the target gene has a target sequence represented by any one of SEQ ID NO: 1, SEQ ID NO 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or a sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 or a sequence having at least 55%, 65%, 75%, 84%, 88%, 92%, or 96% sequence identity to the entire length of any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments, the expression vector comprises an expression cassette comprising SEQ ID NO:7, SEQ ID NO:8, or the complement thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the entire length of SEQ ID NO: 7, SEQ ID NO: 8, or the complement thereof.

[0193] In some embodiments, disclosed methods further comprise contacting the ant with an additional attractant, such as a bait. In some embodiments, the additional attractant comprises sugar, an octanol, a plant extract, a pheromone, a volatile organic compound, carbon dioxide, lactic acid, ammonia, or a combination thereof. Additional ant attractants and insecticides that can be used in accordance with the present disclosure are described by, e.g., Vander Meer & Milne, J Econ Entomol. 2017 Apr 1;110(2):567-574, Yesudai et al., J Econ Entomol. 2022 Apr 13; 115(2):624-630, Brightwell et al., J Econ Entomol. 2010 Oct; 103(5): 1790-6, and Hoffmann, Bull Entomol Res. 2023 Apr; 113(2): 190-195.

[0194] In some embodiments, disclosed methods further comprise contacting the ant with an insecticide, such as a chemical insecticide. In some embodiments, the insecticide contacting the ant is beta-Cyfluthrinmethoprene, DEET (N,N-diethyl-meta-toluamidedeltamethrin), etofenprox,hydramethylnon, imidacloprid, indoxacarb, permethrin, piperonyl butoxide, phenothrin, malathion, pyriproxyfen, sodium tetraborate decahydrate (borax), spinosad, thiamethoxam, or a combination thereof.

[0195] In some embodiments, the disclosed methods result in a percent (%) mortality of at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 100% mortality of ants in an ant population, including any and all numerical values and ranges in between.EXAMPLES

[0196] Example 1: Yeast RNAi strains induce mortality in P. barbatus ants

[0197] Disclosed yeast-based RNAi biopesticides were evaluated for their effect on the survival of ants. Ant Sh. 697, Ant Sh. 698, and Ant Sh. 702 hairpin expression cassettes, represented by SEQ ID NOs. 2 and 3, SEQ ID NOs. 5 and 6, and SEQ ID NOs. 8 and 9, respectively, were cloned into the pRS426 GPD vector. Yeast was prepared as described (Mysore et al., Methods Mol Biol. 2019; 1858: 213-231), as were control yeast. See Hapairai et al., Scientific Reports. 2017; 7: 13223 and Mysore et al., Methods Mol Biol. 2019; 1858: 213-231.A mosquito attractive targeted sugar bait (ATSB) protocol was modified from Mysore et al., Insects. 2021; 12(11): 986) and used to evaluate the exemplary iRNA biopesticides.

[0198] For the ant trials, 100 mg of heat-inactivated, lyophilized yeast (prepared from the control or treatment strains) was mixed with 200 pl of attractive sugar bait (ASB) in a tube. Once mixed well, the suspension was loaded onto a wick created using cotton. The cotton wick was mounted on top of a cup containing Pogonomyrmex barbatus female red harvester ants (20 individuals / test case) obtained from Carolina Biological Supply. The ants were permitted to feed ad libidum throughout a six day experimental period. Fresh ATSB was provided daily.

[0199] FIG. 1 shows the mortality rates resulting from P. barbatus consumption of the RNAi yeast strains suspended in attractive sugar bait (ASB). Although negligible ant mortality was observed following treatment with ASB alone or ASB containing control yeast (Control), consumption of biopesticide yeast strains Sh.697, Sh.698, and Sh. 702 induced significant mortality (P<0.001) in adult P. barbatus female ants.

[0200] Example 2: Yeast RNAi strains reduce expression of Shaker gene in the brains of P. barbatus ants

[0201] A riboprobe corresponding to ant Shaker was synthesized and used for in situ hybridization experiments conducted on adult brains. Adult individuals that were allowed to feed on yeast expressing Sh.697 (SEQ ID NO:1) for 24 h were fixed for in situ hybridization experiments, which were performed in duplicates. Following mounting and imaging of tissues with a Zeiss Axioimager equipped with a Spot Flex camera, mean gray values (average signal intensity over the selected area) were calculated using FIJI ImageJ software for digoxigenin-labeled transcript signals in control or experimental brains; in these studies, data were combined from two replicate experiments. The results indicate that there was decrease of 68±3% in the expression of the target gene within 24h of feeding on yeast expressing Sh.697. A paired t-test was used to statistically analyze transcript quantification data and plotted as a bar graph shown in FIG. 2.EQUIVALENTS AND SCOPE

[0202] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present invention is not intended to be limited to the above, but rather is as set forth in the appended claims.

[0203] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0204] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses and descriptive terms, from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0205] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps.

[0206] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranged can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0207] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of the ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5% or up to 1% of a given value. Alternatively, the term can mean within an order of magnitude, for example within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0208] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the method of the invention can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0209] Sequence Table

Claims

CLAIMSI claimClaim 1. An interfering ribonucleic acid comprising a nucleotide sequence of 20 to 30 contiguous nucleotides, wherein the nucleotide sequence is partially or perfectly complementary to mRNA transcribed from a target DNA sequence having 88%, 92%, 96%, or 100% identity to a portion of SEQ ID NO: 12 or to the entire length of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7; and wherein the interfering RNA inhibits the production of potassium voltage-gated channel protein Shaker in an ant by RNA interference.Claim 2. The interfering RNA of claim 1, wherein the interfering RNA is an RNA construct, a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an anti-sense oligonucleotide.Claim 3. The interfering RNA of claim 1, wherein the interfering RNA is an shRNA.Claim 4. The interfering RNA of claim 1, wherein the ant is a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a New World ant, a jet ant or a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, or a crazy ant.[00210] Claim 5. The interfering RNA of claim 1 , wherein the ant is a species of Acromyrmex, Atta, Camponotus, Cataglyphis, Cyphomyrmex, Dinoponera, Formica, Harpegnathos, Lasius, Nylanderia, Solenopsis, Monomorium, Linepithema, Odontomachus, Ooceraea, Paratrechina,Pogonomyrmex, Pseudomyrmex Tetramorium, Trachymyrmex, Temnothorax, Vollenhovia, orWasmannia.Claim 6. An expression cassette comprising a regulatory sequence operably linked to a nucleotide sequence which encodes the interfering RNA molecule of claim 1.Claim 7. An expression cassette comprising a regulatory sequence operably linked to a nucleotide sequence which encodes an interfering RNA molecule comprising a nucleotide sequence of 20 to 30 contiguous nucleotides, wherein the nucleotide sequence is partially or perfectly complementary to mRNA transcribed from a DNA sequence having 88%, 92%, 96%, or 100% identity to a portion of SEQ ID NO: 12 or to the entire length of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7, and wherein the interfering RNA inhibits the production of potassium voltage-gated channel protein Shaker in an ant by RNA interference.Claim 8. The expression cassette of claim 7, wherein the nucleotide sequence of the interfering RNA molecule comprises 25 nucleotides which are partially or perfectly complementary to a mRNA transcribed from a portion of SEQ ID NO: 12 or the entire length of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7Claim 9. The expression cassette of claim 7, comprising a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entire length of: a) SEQ ID NO:2, SEQ ID NO:3, or the complement thereof;b) SEQ ID NO:5, SEQ ID NO:6, or the complement thereof; or c) SEQ ID NO:8, SEQ ID NO:9, or the complement thereof.Claim 10. The expression cassette of claim 7, wherein the regulatory sequence comprises a yeast promoter.Claim 11. The expression cassette of claim 10, wherein the GPD promoter comprises a nucleotide sequence that has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entire length of SEQ ID NO: 10.Claim 12. The expression cassette of any one of claims 7 to 11, wherein the expression cassette is integrated into the genomic DNA of Saccharomyces cerevisiae.Claim 13. A vector comprising the expression cassette of claim 7.Claim 14. The vector of claim 13, wherein the vector comprises a nucleotide sequence that has at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the entire length of SEQ ID NO:11.Claim 15. A microbial cell, algal cell, or plant cell comprising the expression cassette or the vector of claim 7 or claim 13.Claim 16. The microbial cell, algal cell, or plant cell of claim 15, wherein the expression cassette is integrated into the genomic DNA of the microbial cell.Claim 17. The microbial cell of claim 15, wherein the microbe is Saccharomyces cerevisiae.Claim 18. A composition comprising the microbial cell, algal cell, or plant cell of any one of claims 15 to 17, or a combination thereof.Claim 19. A composition comprising an expression cassette comprising a promoter operably linked to a DNA sequence encoding an interfering RNA molecule which is partially or perfectly complementary to a target sequence in a target gene, wherein the target gene encodes potassium voltage-gated channel protein Shaker in an ant; and the interfering RNA specifically inhibits expression of the target gene.Claim 20. The composition of claim 19, wherein the interfering RNA is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an antisense oligonucleotide.Claim 21. The composition of claim 20, wherein the interfering RNA is a short hairpin RNA (shRNA).Claim 22. The composition of claim 19, wherein the ant is a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a New World ant, a jet antor a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, or a crazy ant.Claim 23. The composition of claim 19, wherein the ant is a species of Acromyrmex, Atta, Camponotus, Cataglyphis, Cyphomyrmex, Dinoponera, Formica, Harpegnathos, Lasius, Nylanderia, Solenopsis, Monomorium, Linepithema, Odontomachus, Ooceraea, Paratrechina, Pogonomyrmex, Pseudomyrmex Tetramorium, Trachymyrmex, Temnothorax, Vollenhovia, or Wasmannia.Claim 24. The composition of claim 19, wherein the target sequence within the target gene is 88%, 92%, 96%, or 100% identical to a portion of SEQ ID NO: 12 or the entire length of SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7Claim 25. The composition of claim 19, wherein the expression cassette comprises a nucleotide sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the entire length of: a) SEQ ID NO:2, SEQ ID NO:3, or the complement thereof; b) SEQ ID NO:5, SEQ ID NO:6, or the complement thereof; or c) SEQ ID NO:8, SEQ ID NO:9, or the complement thereof.Claim 26. The composition of claim 19, wherein the expression cassette comprises the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9Claim 27. The composition of claim 19, wherein the interfering RNA comprises a nucleotide sequence of at least 25 contiguous nucleotides which are partially or perfectly complementary to a portion of mRNA transcribed from SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7; and wherein the interfering RNA is capable of inhibiting the expression of potassium voltage-gated channel protein Shaker in the ant.Claim 28. The composition of any one of claims 19 to 27, wherein the expression cassette is integrated into the genome of a yeast cell.Claim 29. The composition of claim 28, wherein the yeast cell is Saccharomyces cerevisiae.Claim 30. The composition of claim 28, wherein the yeast cell is spray-dried, heat-killed, lyophilized, or suspended in an aqueous medium.Claim 31. The composition of claim 19, further comprising a sugar bait.Claim 32. The composition of claim 19, wherein the composition is within a trap.Claim 33. A method for controlling an ant population, comprising contacting the ant population with the interfering RNA of any one of claims 1 to 6, the expression cassette of any one of claims 7 to 12, the vector of claim 13 or 14, the microbial cell, algal cell, or plant cell of any one of claims 15 to 17, or the composition of any one of claims 18 to 32,wherein contacting the ant population comprises one or more ants in the ant population ingesting the interfering RNA, the expression cassette, the vector, the microbial cell, or the composition, thereby controlling the ant population.Claim 34. A method for controlling an ant population, the method comprising contacting the ant population with an interfering RNA molecule comprising a nucleotide sequence that is partially or perfectly complementary to mRNA transcribed from a target sequence within a target gene and which specifically inhibits expression of the target gene in an ant in the ant population, thereby controlling the ant population, wherein the target gene encodes potassium voltage-gated channel protein Shaker or an ortholog thereof.Claim 35. The method of claim 34, wherein the interfering RNA is a double stranded RNA (dsRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), or an anti-sense oligonucleotide.Claim 36. The method of claim 35, wherein the interfering RNA is a short hairpin RNA (shRNA).Claim 37. The method of claim 34, wherein the target sequence within the target gene has at least 84%, 88%, 92%, or 96% identity to the entire length of SEQ ID NO:1, SEQ ID NO:4, orSEQ ID NO:7Claim 38. The method of claim 34, wherein the target sequence is SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7Claim 39. The method of claim 34, wherein the interfering RNA is produced by a microbial cell comprising an expression cassette comprising a regulatory sequence operably linked to a nucleotide sequence encoding the interfering RNA, wherein the expression cassette is integrated into the genome of the microbial cell.Claim 40. The method of claim 39, wherein the expression cassette comprises a nucleotide sequence having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the entire length of: a) SEQ ID NO:2, SEQ ID NO:3, or the complement thereof; b) SEQ ID NO:5, SEQ ID NO:6, or the complement thereof; or c) SEQ ID NO:8, SEQ ID NO:9, or the complement thereof.Claim 41. The method of claim 39, wherein the expression cassette comprises the entire length of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9Claim 42. The method of claim 39, wherein the method comprises contacting the ant in the ant population with the microbial cell.Claim 43. The method of claim 39, wherein the microbial cell is dead or alive.Claim 44. The method of claim 39, wherein the microbial cell is a Saccharomyces cerevisiae cell.Claim 45. The method of any one of claims 34 to 44, wherein the ant population comprises a carpenter ant, a fire ant, a formicine ant, a fungus-growing ant, a leafcutter ant, an imported ant, a New World ant, a jet black ant, a pharaoh ant, a polygynous ant, an Argentine ant, a pavement ant, an odorous house ant, and a crazy ant, or a combination thereof.Claim 46. The method of any one of claims 34 to 44, wherein the ant population comprises Acromyrmex spp., Ata spp., Camponotus spp., Cataglyphis spp., Cyphomyrmex spp., Dinoponera spp., Formica spp., Harpegnathos spp., Lasius spp., Nylanderia spp., Solenopsis spp., Monomorium spp., Linepithema spp., Odontomachus spp., Ooceraea spp., Paratrechina spp., Pogonomyrmex spp., Pseudonryrmex spp., Tetramorium spp., Trachymyrmex spp., Temnothorax spp., Vollenhovia spp., Wasmannia spp., or a combination thereof.Claim 47. The method of claim 34, wherein the interfering RNA comprises at least 25 contiguous nucleotides, wherein the nucleotide sequence is partially or perfectly complementary to a portion of mRNA transcribed from SEQ ID NO:12, SEQ ID NO:1, SEQ ID NO:4, or SEQ ID NO:7; and wherein the interfering RNA is capable of inhibiting the expression of potassium voltage-gated channel protein Shaker in the ant by RNA interference.Claim 48. The method of claim 34, wherein the method further comprises contacting the ant population with a sugar bait, a pheromone, an insecticide, or any combination thereof.

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