WO2024047148A1 - Lutte contre les insectes nuisibles au moyen de molécules d'arn - Google Patents

Lutte contre les insectes nuisibles au moyen de molécules d'arn Download PDF

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WO2024047148A1
WO2024047148A1 PCT/EP2023/073875 EP2023073875W WO2024047148A1 WO 2024047148 A1 WO2024047148 A1 WO 2024047148A1 EP 2023073875 W EP2023073875 W EP 2023073875W WO 2024047148 A1 WO2024047148 A1 WO 2024047148A1
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seq
nucleotide
interfering rna
alphitobius
dsrna
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PCT/EP2023/073875
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English (en)
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Yann Naudet
Myriam Beghyn
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Syngenta Crop Protection Ag
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/60—Isolated nucleic acids
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P7/00—Arthropodicides
    • A01P7/04—Insecticides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates generally to the control of pests that cause damage, especially in poultry houses, by their feeding activities. Specifically, the invention relates to the control of darkling beetles by interfering RNA molecules and compositions comprising said interfering RNA molecules. The invention further relates to methods of using such interfering RNA molecules and compositions comprising said interfering RNA molecules.
  • Alphitobius diaperinus is a species of beetle in the family Tenebrionidae, the darkling beetles. It is known commonly as the lesser mealworm and the litter beetle. It has a cosmopolitan distribution, occurring nearly worldwide. It is known widely as a pest insect of stored food grain products, such as flour. It has been associated with wheat, barley, rice, oat, soybean, pea, and peanuts.
  • the darkling beetle is also one of the most common pests in poultry houses. They are often found in the bedding litter material covering poultry house floors. Darkling beetles can cause vast damage to poultry facilities for a number of reasons.
  • darkling beetles can cause structural damage to almost every part of a poultry facility. They can burrow into wood structures and tunnel through insulation. They can cause damage to sill sealers and vapor barriers. They can even create spaces between walls, concrete, and flooring.
  • viral poultry diseases e.g., leucosis, Marek's disease virus, Infectious bursal disease virus (IBDV), reovirus, enterovirus, fowl pox, turkey coronavirus, astrovirus, avian influenza, and Newcastle disease virus
  • bacterial pathogens including bacteria of the genus Salmonella, Campylobacter, Escherichia (i.e., E. coli), and Staphylococcus
  • fungal pathogens including fungi of the genus Aspergillus.
  • poultry can be directly, negatively affected by darkling beetles in the facility. Aside from carrying diseases, as discussed above, darkling beetles may also pester the birds, causing excess movement and reducing feed efficiency. Poultry that feed on the pests instead of provided feed are suspect to lower bird nutrition.
  • the darkling beetles are also known to crawl on the birds when deprived of moisture, and chew at the base of the feathers.
  • the skin bites can be mistaken for skin leukosis at the processing plants. The bites also predispose the birds to certain diseases. In cases of heavy infestation, the beetles are known to kill weakened chicks in their pursuit of food and moisture.
  • RNA interference occurs when an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments of about 19-24 nucleotides in length, called small interfering RNAs (siRNAs). The siRNAs then diffuse or are carried throughout the organism, including across cellular membranes, where they hybridize to mRNAs (or other RNAs) and cause hydrolysis of the RNA. Interfering RNAs are recognized by the RNA interference silencing complex (RISC) into which an effector strand (or "guide strand") of the RNA is loaded. This guide strand acts as a template for the recognition and destruction of the duplex sequences. This process is repeated each time the siRNA hybridizes to its complementary-RNA target, effectively preventing those mRNAs from being translated, and thus "silencing" the expression of specific genes from which the mRNAs were transcribed.
  • RISC RNA interference silencing complex
  • RNAi has been found to be useful for insect control of certain insect pests.
  • RNAi strategies typically employ a synthesized, non-naturally occurring "interfering RNA", or “interfering RNA molecule” which typically comprises at least a RNA fragment against a target gene, a spacer sequence, and a second RNA fragment which is complementary to the first, so that a double-stranded RNA structure can be formed.
  • This non-natural double-stranded RNA molecule 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 and may result in the death of the insect pest.
  • RNAi strategies focused on target genes can lead to an insecticidal effect
  • the overwhelming majority of sequences complementary to corn rootworm DNAs are not lethal in species of corn rootworm when used as dsRNA or siRNA.
  • Baum et al. ((2007) Nature Biotechnology 25:1322-1326), describe the effects of inhibiting several WCR gene targets by RNAi.
  • the dosage or quantity of a given dsRNA molecule required to confer significant insecticidal activity needs to be considered for the dsRNA molecule to be of commercial value for crop protection.
  • compositions containing insecticidal active ingredients there is an ongoing need for compositions containing insecticidal active ingredients, and for methods of using such compositions, for instance for use in animal feed protection and/or insect-mediated disease control.
  • such compositions have a high toxicity and are effective when ingested orally by the target pest and/or when applied topically, and have applicability for use against all life cycle stages of the pest insect.
  • any invention which provides compositions in which any of these properties was enhanced would represent a step forward in the art.
  • the needs outlined above are, at least in part, met by the invention which, in various embodiments, provides new methods of controlling economically important insect pests.
  • the invention in part comprises a method of inhibiting expression of one or more target genes and proteins in Coleopteran insect pests.
  • the invention comprises methods of modulating expression of one or more target genes in a species of insect that causes cessation of feeding, growth, development and reproduction, and eventually results in the death of the insect, wherein the insect pest is a darkling beetle of the Alphitobiini tribe.
  • the method of the invention comprises introduction of an interfering RNA molecule comprising a double-stranded RNA (dsRNA) or its modified forms such as small interfering RNA (siRNA) sequences, into cells or into the extracellular environment, such as the midgut, within a pest insect body wherein the dsRNA or siRNA enters the cells and inhibits expression of at least one or more target genes and wherein inhibition of the one or more target genes exerts a deleterious effect upon the pest insect.
  • the interfering RNA molecule is non-naturally occurring.
  • the interfering RNA molecule of the invention may be introduced into a pest by any suitable means.
  • the interfering RNA may be introduced by contacting the pest with the interfering RNA of the invention.
  • the invention also provides interfering RNA molecules that when delivered to an insect pest inhibit, through a toxic effect, the ability of the insect pest to survive, grow, feed and/or reproduce, or to limit pest related damage or loss of animal feed.
  • the interfering RNA molecules of the invention comprise at least one dsRNA wherein the dsRNA may be a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises a sequence of at least 19 contiguous nucleotides which may be at least partially complementary to a target nucleotide sequence within a darkling beetle target gene, and (i) may be at least 85% identical to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147,
  • the insect may be a species of a genus selected from the group consisting of the genera Alphitobius, Alphitopsis, Ardoinia, Diaclina, Epipedodema, Guanobius, Hop lope It is, and Peltoides.
  • the insect may be a species of the genus Alphitobius.
  • the insect of the genus Alphitobius is selected from the group consisting of Alphitobius diaperinus (lesser mealworm), Alphitobius arnoldi, Alphitobius capitaneus, Alphitobius crenatus, Alphitobius grandis, Alphitobius hobohmi, Alphitobius karrooensis, Alphitobius kochi, Alphitobius laevigatus, Alphitobius lamottei, Alphitobius leleupi, Alphitobius limbalis, Alphitobius lucasorum, Alphitobius niger, Alphitobius parallelipennis, Alphitobius rugosulus, and Alphitobius viator, and related species.
  • the interfering molecule may comprise at least two dsRNAs, wherein each dsRNA comprises or consists of a sequence of nucleotides which may be at least partially complementary to a target nucleotide sequence within the target gene.
  • each of the dsRNAs may comprise or consist of a different sequence of nucleotides which may be complementary to a different target nucleotide sequence within the target gene.
  • the interfering RNA molecule comprises SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111- 144, 146, 147, 150, 152, 154-156, and 159-192, or the complement thereof.
  • At least of the strands of the dsRNA of the interfering RNA molecule comprises or consists of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192, or the complement thereof.
  • the invention further provides compositions comprising two or more of the interfering RNA molecules of the invention.
  • Each of the interfering RNA molecules may comprise one, two, or more of dsRNAs.
  • the two or more interfering RNA molecules each comprise a different antisense strand.
  • the invention further provides compositions comprising two or more nucleic acid constructs or nucleic acid molecules of the invention.
  • the nucleic acid molecules may encode a different RNA molecule.
  • the invention further provides insecticidal compositions for inhibiting the expression of a darkling beetle of the Alphitobiini tribe gene that comprises a dsRNA of the invention and an agriculturally acceptable carrier.
  • inhibition of the expression of a darkling beetle gene described here leads to cessation of feeding and growth and ultimately results in the death of the darkling beetle.
  • the invention further provides insecticidal compositions for controlling a darkling beetle of the Alphitobiini tribe.
  • the insecticidal compositions may comprise two or more of the interfering RNA molecules of the invention, as described hereinabove.
  • the invention also provides a method of controlling a darkling beetle of the Alphitobiini tribe pest comprising contacting the darkling beetle with a nucleic acid molecule that is or is capable of producing an interfering RNA of the invention for inhibiting expression of a gene in the darkling beetle thereby controlling the darkling beetle.
  • the target gene may comprise a coding sequence which: a) is at least 85% identical to at least a 19 nucleotide contiguous fragment of SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or a complement thereof; b) comprises at least a 19 nucleotide contiguous fragment of SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or a complement thereof; or c) comprises at least a 19 nucleotide contiguous fragment of a nucleotide sequence encoding an amino acid sequence encoded by SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53,
  • the invention provides a method of reducing a darkling beetle population by contacting the darkling beetle with a second insecticidal agent.
  • the second insecticidal agent may be for controlling a darkling beetle, and/or for controlling a different pest.
  • the invention provides a method of reducing resistance development in a darkling beetle of the Alphitobiini tribe population to an interfering RNA of the invention, the method comprising contacting the darkling beetle population an interfering RNA of the invention that may be capable of inhibiting expression of a target gene in a larval and adult darkling beetle, thereby reducing resistance development in the darkling beetle population compared to a darkling beetle population exposed to an interfering RNA capable of inhibiting expression of a darkling beetle gene described herein in only the larval stage or adult stage of a darkling beetle.
  • the invention provides a method of reducing the level of a target RNA transcribable from a darkling beetle gene described herein in a darkling beetle comprising contacting the darkling beetle with a composition comprising an interfering RNA molecule of the invention, wherein the interfering RNA molecule reduces the level of the target RNA in a cell of the darkling beetle.
  • a composition comprising an interfering RNA molecule of the invention, wherein the interfering RNA molecule reduces the level of the target RNA in a cell of the darkling beetle.
  • production of the protein encoded by the target RNA may be reduced.
  • the protein may comprise an amino acid sequence encoded by a nucleic acid sequence with at least 85% identity to SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or the complement thereof.
  • the interfering RNA may be from a transgenic organism expressing the interfering RNA.
  • the transgenic organism may a microorganism, for example bacterium, or a virus.
  • the interfering RNA may be lethal to a darkling beetle of the Alphitobiini tribe.
  • the invention provides a method of providing a farmer (for example a poultry farmer) with a means of controlling a darkling beetle of the Alphitobiini tribe pest population in a plant comprising (a) selling or providing to the farmer a pest control composition that comprises an interfering RNA molecule, a nucleic acid molecule, a nucleic acid construct, and/or a composition of any of the corresponding preceding claims; and (b) advertising to the farmer that the pest control composition controls the darkling beetle pest population.
  • the invention provides a method of identifying an orthologous target gene for using as a RNAi strategy for the control of a different Coleopteran plant pest, said method comprising the steps of: a) producing a primer pair that will amplify a target selected from the group comprising or consisting of SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or a complement thereof; b) amplifying an orthologous target gene from a nucleic acid sample of the plant pest using the primer pair of step a); c) identifying a sequence of an orthologous target gene; d) producing an interfering RNA molecule, wherein the RNA comprises at least one dsRNA, wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands, one
  • the invention provides a nucleic acid construct comprising the interfering RNA molecule.
  • the invention provides a nucleic acid molecule encoding the interfering RNA molecule of the invention.
  • the invention provides a recombinant vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes the interfering RNA molecule of the invention.
  • the invention provides a host cell comprising the nucleic acid construct, nucleic acid molecule, and/or recombinant vector.
  • nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in WIPO Standard ST.26.
  • the nucleic acids listed define molecules (i.e., polynucleotides) having the nucleotide monomers arranged in the manner described.
  • the nucleic acid sequences listed also each define a genus of polynucleotides that comprise the nucleotide monomers arranged in the manner described.
  • nucleotide sequence including a coding sequence also describes the genus of polynucleotides encoding the same polypeptide as a polynucleotide consisting of the reference sequence. It will further be understood that an amino acid sequence describes the genus of polynucleotide ORFs encoding that polypeptide.
  • RNA sequence is included by any reference to the DNA sequence encoding it.
  • SEQ ID NOs: 1-48 are DNA coding sequences of the 48 Alphitobius diaperinus target genes identified for assaying in the RNAi-based screen for insecticidal activity.
  • SEQ ID NOs: 49-96 and 289 are fragments of DNA coding sequences of Alphitobius diaperinus used to synthesize interfering RNA molecules to test for insecticidal activity in the RNAi-based screen.
  • SEQ ID NOs: 97-144 are the sense RNA sequences of the Alphitobius diaperinus DNA coding fragment sequences of SEQ ID NOs: 49-96.
  • SEQ ID Nos: 145-192 are the sense RNA sequences of the Alphitobius diaperinus DNA coding sequences of SEQ ID Nos: 1-48.
  • SEQ ID NOs: 193-240 are DNA sequences of forward primers and SEQ ID NOs: 241-288 are DNA sequences of the corresponding reverse primers for producing the fragments of SEQ ID NOs: 49-96.
  • FIGURES Figure 1 shows the effect of seven dsRNA molecules of invention on the survival of darkling beetle larvae.
  • Figure 2 shows a dose response curve of selected dsRNA molecules against darkling beetle larvae.
  • Figure 2 shows a dose response curve of selected dsRNA molecules against adult darkling beetle.
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • the term "about,” as used herein when referring to a measurable value such as an amount of a compound or agent, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified amount.
  • coding sequence is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
  • RNA is then translated in an organism to produce a protein.
  • sequence similarity or “sequence identity” of nucleotide or amino acid sequences mean a degree of identity or similarity of two or more sequences and may be determined conventionally by using known software or computer programs such as the Best-Fit or Gap pairwise comparison programs (GCG Wisconsin Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711). BestFit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of identity or similarity between two sequences. Sequence comparison between two or more polynucleotides or polypeptides is generally performed by comparing portions of the two sequences over a comparison window to identify and compare local regions of sequence similarity.
  • the comparison window is generally from about 20 to 200 contiguous nucleotides. Gap performs global alignments: all of one sequence with all of another similar sequence using the method of Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970).
  • sequence alignment program such as BestFit to determine the degree of DNA sequence homology, similarity or identity
  • the default setting may be used, or an appropriate scoring matrix may be selected to optimize identity, similarity or homology scores.
  • the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.
  • substantially identical in the context of two nucleic acids or two amino acid sequences, refers to two or more sequences or subsequences that have at least about 50% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection.
  • substantially identical sequences have at least about 60%, or at least about 70%, or at least about 80%, or even at least about 90% or 95% nucleotide or amino acid residue identity.
  • substantial identity exists over a region of the sequences that is at least about 50 residues in length, or over a region of at least about 100 residues, or the sequences are substantially identical over at least about 150 residues.
  • the sequences are substantially identical when they are identical over the entire length of the coding regions.
  • homologous in the context of the invention refers to the level of similarity between nucleic acid or amino acid sequences in terms of nucleotide or amino acid identity or similarity, respectively, i.e., sequence similarity or identity.
  • homologue, and homologous also refers to the concept of similar functional properties among different nucleic acids or proteins.
  • Homologues include genes that are orthologous and paralogous. Homologues can be determined by using the coding sequence for a gene, disclosed herein or found in appropriate database (such as that at NCBI or others) in one or more of the following ways. For an amino acid sequence, the sequences should be compared using algorithms (for instance see section on "identity” and "substantial identity”).
  • sequence of one DNA molecule can be compared to the sequence of a known or putative homologue in much the same way.
  • Homologues are at least 20% identical, or at least 30% identical, or at least 40% identical, or at least 50% identical, or at least 60% identical, or at least 70% identical, or at least 80% identical, or at least 88% identical, or at least 90% identical, or at least 92% identical, or at least 95% identical, across any substantial region of the molecule (DNA, RNA, or protein molecule).
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FAST A, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl), or by visual inspection (see generally, Ausubel et al., infra).
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • CLUSTALW vl.6 Another widely used and accepted computer program for performing sequence alignments is CLUSTALW vl.6 (Thompson, et al. Nuc. Acids Res., 22: 4673-4680, 1994).
  • the number of matching bases or amino acids is divided by the total number of bases or amino acids, and multiplied by 100 to obtain a percent identity. For example, if two 580 base pair sequences had 145 matched bases, they would be 25 percent identical.
  • the number of matches is divided by the shorter of the two lengths. For example, if there were 100 matched amino acids between a 200 and a 400 amino acid proteins, they are 50 percent identical with respect to the shorter sequence. If the shorter sequence is less than 150 bases or 50 amino acids in length, the number of matches are divided by 150 (for nucleic acid bases) or 50 (for amino acids), and multiplied by 100 to obtain a percent identity.
  • Two nucleotide sequences can also be considered to be substantially identical when the two sequences hybridize to each other under stringent conditions.
  • two nucleotide sequences considered to be substantially identical hybridize to each other under highly stringent conditions.
  • stringent conditions or “stringent hybridization conditions” include reference to conditions under which a polynucleotide will hybridize to its target sequence to a detectably greater degree than other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target polynucleotides can be identified which are 100% complementary to the probe (homologous probing).
  • stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
  • stringent conditions will be those in which the salt concentration is less than approximately 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions also may be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCI, 1% SDS (w/v; sodium dodecyl sulphate) at 37° C, and a wash in lx to 2xSSC (20xSSC - 3.0 M NaCI/0.3 M trisodium citrate) at 50 to 55° C.
  • Moderate stringency conditions detect sequences that share at least 80% sequence identity.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCI, 1% SDS at 37° C, and a wash in 0.5x to lxSSC at 55 to 60° C.
  • High stringency conditions detect sequences that share at least 90% sequence identity.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in O.lxSSC at 60 to 65° C. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
  • Tm can be approximated from the equation of Meinkoth and Wahl (Anal.
  • Tm 81.5° C+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
  • the Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe.
  • Tm is reduced by about 1° C for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with approximately 90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH.
  • Tm thermal melting point
  • Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical (e.g., due to the degeneracy of the genetic code).
  • a further indication that two nucleic acids or proteins are substantially identical is that the protein encoded by the first nucleic acid is immunologically cross reactive with the protein encoded by the second nucleic acid.
  • a protein is typically substantially identical to a second protein, for example, where the two proteins differ only by conservative substitutions.
  • a nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.
  • complementary polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A: U) in the case of RNA.
  • sequence "A-G-T” binds to the complementary sequence "T-C-A.” It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.
  • complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. Complementarity between two single-stranded molecules may be “partial,” in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • the terms “substantially complementary” or “partially complementary” mean that two nucleic acid sequences are complementary at least about 50%, 60%, 70%, 80% or 90% of their nucleotides. In some embodiments, the two nucleic acid sequences can be complementary at least at 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of their nucleotides.
  • the terms “substantially complementary” and “partially complementary” can also mean that two nucleic acid sequences can hybridize under high stringency conditions and such conditions are well known in the art.
  • dsRNA or "RNAi” refers to a polyribonucleotide structure formed either by a single self-complementary RNA strand or at least by two complementary RNA strands.
  • the degree of complementary in other words the % identity, need not necessarily be 100%. Rather, it must be sufficient to allow the formation of a double-stranded structure under the conditions employed.
  • the term “fully complementary” means that all the bases of the nucleotide sequence of the dsRNA are complementary to or 'match' the bases of the target nucleotide sequence.
  • RNA sequences with insertions, deletions and mismatches relative to the target sequence can still be effective at RNAi.
  • the dsRNA and the target nucleotide sequence of the target gene share at least 80% or 85% sequence identity, preferably at least 90% or 95% sequence identity, or more preferably at least 97% or 98% sequence identity and still more preferably at least 99% sequence identity.
  • the dsRNA may comprise 1, 2 or 3 mismatches as compared with the target nucleotide sequence over every length of 24 partially complementary nucleotides. It will be appreciated by the person skilled in the art that the degree of complementarity shared between the dsRNA and the target nucleotide sequence may vary depending on the target gene to be down-regulated or depending on the insect pest species in which gene expression is to be controlled.
  • the dsRNA may comprise or consist of a region of double-stranded RNA comprising annealed complementary strands, one strand of which, the sense strand, comprises a sequence of nucleotides at least partially complementary to a target nucleotide sequence within a target gene.
  • the target nucleotide sequence may be selected from any suitable region or nucleotide sequence of the target gene or RNA transcript thereof.
  • the target nucleotide sequence may 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 synthesised 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.
  • the % identity of a polyribonucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) using the default settings, wherein the query sequence is at least about 21 to about 23 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least about 21 nucleotides.
  • the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides.
  • the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides.
  • the query sequence corresponds to the full length of the target RNA, for example mRNA, and the GAP analysis aligns the two sequences over the full length of the target RNA.
  • the dsRNA can be produced from a single open reading frame in a recombinant host cell, wherein the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure.
  • the sense strand and antisense strand can be made without an open reading frame to ensure that no protein will be made in the transgenic host cell.
  • the two strands can also be expressed separately as two transcripts, one encoding the sense strand and one encoding the antisense strand.
  • RNA duplex formation can be initiated either inside or outside the cell.
  • the dsRNA can be partially or fully double-stranded.
  • the RNA can be enzymatically or chemically synthesized, either in vitro or in vivo.
  • the dsRNA need not be full length relative to either the primary transcription product or fully processed RNA. It is well-known in the art that small dsRNA of about 19-23 bp in length can be used to trigger gene silencing of a target gene. Generally, higher identity can be used to compensate for the use of a shorter sequence.
  • the dsRNA can comprise single stranded regions as well, e.g., the dsRNA can be partially or fully double stranded.
  • the double stranded region of the dsRNA can have a length of at least about 19 to about 23 base pairs, optionally a sequence of about 19 to about 50 base pairs, optionally a sequence of about 50 to about 100 base pairs, optionally a sequence of about 100 to about 200 base pairs, optionally a sequence of about 200 to about 500, and optionally a sequence of about 500 to about 1000 or more base pairs, up to a molecule that is double stranded for its full length, corresponding in size to a full length target RNA molecule.
  • Mao et al (2007, Nature Biotechnology, 35(11): 1307-1313) teach a transgenic plant expressing a dsRNA construct against a target gene (CYP6AE14) of an insect pest (cotton bollworm, Helicoverpa armigera). Insects feeding on the transgenic plant have small RNAs of about 19-23 bp in size of the target gene in their midgut, with a corresponding reduction in CYP6AE14 transcripts and protein. This suggests that the small RNAs were efficacious in reducing expression of the target gene in the insect pest.
  • CYP6AE14 target gene of an insect pest
  • small RNAs of about 19 bp, about 20 bp, about 21 bp, about 22 bp, about 23 bp, about 24 bp, about 25 bp, about 26 bp, about 27 bp, about 28 bp, about 29 bp, or about 30 bp may be efficacious in reducing expression of the target gene in an insect pest.
  • such small RNAs may be efficacious in reducing expression of the target gene in the insect pest when consumed by the insect pest.
  • the dsRNA may comprise a target dsRNA of at least 19 base pairs, and the target dsRNA may be within a dsRNA "carrier” or "filler" sequence.
  • a 240 bp dsRNA encompassing a target dsRNA which comprised a 21 bp contiguous sequence with 100% identity to the target sequence, had biological activity in bioassays with Southern Corn Rootworm.
  • the target dsRNA may have a length of at least 19 to about 25 base pairs, optionally a sequence of about 19 to about 50 base pairs, optionally a sequence of about 50 to about 100 base pairs, optionally a sequence of about 100 to about 200 base pairs, optionally a sequence of about 200 to about 500, and optionally a sequence of about 500 to about 1000 or more base pairs.
  • the dsRNA of the target sequence and the carrier dsRNA may have a total length of at least about 50 to about 100 base pairs, optionally a sequence of about 100 to about 200 base pairs, optionally a sequence of about 200 to about 500, and optionally a sequence of about 500 to about 1000 or more base pairs.
  • the dsRNA can contain known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiralmethyl phosphonates and 2-O-methyl ribonucleotides.
  • the term "specifically reduce the level of a target RNA and/or the production of a target protein encoded by the RNA”, and variations thereof, refers to the sequence of a portion of one strand of the dsRNA being sufficiently identical to the target RNA such that the presence of the dsRNA in a cell reduces the steady state level and/or the production of said RNA.
  • the target RNA will be mRNA, and the presence of the dsRNA in a cell producing the mRNA will result in a reduction in the production of said protein.
  • this accumulation or production is reduced at least 10%, more preferably at least 50%, even more preferably at least 75%, yet even more preferably at least 95% and most preferably 100%, when compared to a wild-type cell.
  • the interfering RNAs of the current invention may comprise one dsRNA or multiple dsRNAs, wherein each dsRNA comprises or consists of a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene and that functions upon uptake by an insect pest species to down-regulate expression of said target gene.
  • Concatemeric RNA constructs of this type are described in W02006/046148 as incorporated herein by reference.
  • the term 'multiple' means at least two, at least three, at least four, etc and up to at least 10, 15, 20 or at least 30.
  • the interfering RNA comprises multiple copies of a single dsRNA i.e.
  • the dsRNAs within the interfering RNA comprise or consist of different sequences of nucleotides complementary to different target nucleotide sequences. It should be clear that combinations of multiple copies of the same dsRNA combined with dsRNAs binding to different target nucleotide sequences are within the scope of the current invention.
  • the dsRNAs may be arranged as one contiguous region of the interfering RNA or may be separated by the presence of linker sequences.
  • the linker sequence may comprise a short random nucleotide sequence that is not complementary to any target nucleotide sequences or target genes.
  • the linker is a conditionally self-cleaving RNA sequence, preferably a pH-sensitive linker or a hydrophobic-sensitive linker.
  • the linker comprises a sequence of nucleotides equivalent to an intronic sequence.
  • Linker sequences of the current invention may range in length from about 1 base pair to about 10000 base pairs, provided that the linker does not impair the ability of the interfering RNA to down-regulate the expression of target gene(s).
  • the interfering RNA of the invention may comprise at least one additional polynucleotide sequence.
  • the additional sequence is chosen from (i) a sequence capable of protecting the interfering RNA against RNA processing, (ii) a sequence affecting the stability of the interfering RNA, (iii) a sequence allowing protein binding, for example to facilitate uptake of the interfering RNA by cells of the insect pest species, (iv) a sequence facilitating large-scale production of the interfering RNA, (v) a sequence which is an aptamer that binds to a receptor or to a molecule on the surface of the insect pest cells to facilitate uptake, or (vi) a sequence that catalyses processing of the interfering RNA within the insect pest cells and thereby enhances the efficacy of the interfering RNA. Structures for enhancing the stability of RNA molecules are well known in the art and are
  • the interfering RNA may contain DNA bases, non-natural bases or non-natural backbone linkages or modifications of the sugar-phosphate backbone, for example to enhance stability during storage or enhance resistance to degradation by nucleases.
  • the interfering RNA may be produced chemically or enzymatically by one skilled in the art through manual or automated reactions.
  • the interfering RNA may be transcribed from a polynucleotide encoding the same.
  • provided herein is an isolated polynucleotide encoding any of the interfering RNAs of the current invention.
  • the term "toxic" used to describe a dsRNA of the invention means that the dsRNA molecules of the invention and combinations of such dsRNA molecules function as orally active insect control agents that have a negative effect on an insect.
  • the result is typically death of the insect, or the insect does not feed upon the source that makes the composition available to the insect.
  • To "control” or “controlling” insects means to inhibit, through a toxic effect, the ability of one or more insect pests to survive, grow, feed, and/or reproduce, or to limit insect-related damage.
  • To "control” insects may or may not mean killing the insects, although it preferably means killing the insects.
  • a composition that controls a target insect has insecticidal activity against the target insect.
  • introduction means that the interfering RNA molecule, dsRNA and/or composition comes in contact with an insect, resulting in a toxic effect and control of the insect.
  • the introduction or delivery may be topical, such as applying a composition comprising the interfering RNA to an affected areas (locus), such a poultry house.
  • introduction or delivery may be through contacting the insect with the interfering RNA, such as when the insect feeds on a product (such as artificial insect diet and/or bait) comprising the interfering RNA, and/or by applying the interfering RNA (or a composition comprising the interfering RNA) onto the darkling beetle.
  • a product such as artificial insect diet and/or bait
  • the interfering RNA or a composition comprising the interfering RNA
  • the interfering RNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a mRNA transcribable from a target gene or a portion of a nucleotide sequence of a mRNA transcribable from a target gene of the pest insect and therefore inhibits expression of the target gene, which causes cessation of feeding, growth, development, reproduction and eventually results in death of the pest insect.
  • the invention is further drawn to nucleic acid constructs, nucleic acid molecules, recombinant vectors, and host cells that comprise or encode at least a fragment of one strand of an interfering RNA molecule of the invention.
  • the invention also provides chimeric nucleic acid molecules comprising an antisense strand of a dsRNA of the interfering RNA operably associated with a plant microRNA precursor molecule.
  • insects as used herein includes any organism now known or later identified that is classified in the animal kingdom, phylum Arthropoda, class Insecta, including but not limited to insects in the orders Coleoptera (beetles), Lepidoptera (moths, butterflies), Diptera (flies), Protura, Collembola (springtails), Diplura, Microcoryphia (jumping bristletails), Thysanura (bristletails, silverfish), Ephemeroptera (mayflies), Odonata (dragonflies, damselflies), Orthoptera (grasshoppers, crickets, katydids), Phasmatodea (walkingsticks), Grylloblattodea (rock crawlers), Mantophasmatodea, Dermaptera (earwigs), Plecoptera (stoneflies), Embioptera (web spinners), Zoraptera, Isoptera (termites), Mantodea (mantids
  • the insect may be of the order Coleoptera (beetles).
  • the insect of the order Coleoptera may be of the suborder Polyphaga.
  • the insect of the suborder Polyphaga may be of the infraorder Cucujiformia.
  • the insect of the infraorder Cucujiformia may be of the superfamily Tenebrionoidea.
  • the insect of the superfamily Tenebrionoidea may be of the family Tenebrionidae.
  • the inspect of the family Tenebrionidae may be of the Alphitobiini tribe.
  • the insect of the Alphitobiini tribe may be of the Alphitobius genus.
  • the insect of the Alphitobius genus may be selected form the group consisting of Alphitobius diaperinus, Alphitobius arnoldi, Alphitobius capitaneus, Alphitobius crenatus, Alphitobius grandis, Alphitobius hobohmi, Alphitobius karrooensis, Alphitobius kochi, Alphitobius laevigatus, Alphitobius lamottei, Alphitobius leleupi, Alphitobius limbalis, Alphitobius lucasorum, Alphitobius niger, Alphitobius parallelipennis, Alphitobius rugosulus, and Alphitobius viator.
  • the insect may be Alphitobius diaperinus.
  • the terms "insectastasis" Alphitobius
  • a "life stage of an Alphitobiini insect” or “darkling beetle life stage” means the egg, larval, pupal or adult developmental form of an insect of the Alphitobiini tribe.
  • Effective insect-controlling amount of "insecticida lly effective amount” means that concentration of dsRNA that inhibits, through a toxic effect, the ability of insects to survive, grow, feed and/or reproduce.
  • "I nsecticidally effective amount” may or may not mean a concentration that kills the insects, although it preferably means that it kills the insects. While there is no upper limit on the concentrations and dosages of a polynucleotide as described herein that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency and economy.
  • Non-limiting embodiments of effective amounts of a polynucleotide include a range from about 10 nano grams per milliliter to about 100 micrograms per milliliter of a polynucleotide in a liquid form applied (for example sprayed) to an area.
  • the area may be for example 1 square meter, 2 square meter, 3 square meter, 4 square meter, or more.
  • the area may be any area which the darkling beetle inhabits and/or feeds on.
  • effective amounts of a polynucleotide include a range from about 10 milligrams per acre to about 100 grams per acre of polynucleotide applied to an area (such as a poultry house) or from about 0.001 to about 0.1 microgram per milliliter of polynucleotide in an artificial diet for feeding the insect.
  • the artificial diet for feeding the insect may be animal feed (such as chicken feed and/or insect bait).
  • compositions as described herein are topically applied to a product (such as animal feed and/or artificial diet for feeding the insect, the concentrations can be adjusted in consideration of the volume of spray or treatment applied to said plant or product (such as animal feed and/or artificial diet for feeding the insect.
  • a useful treatment using 25-mer polynucleotides is about 1 nanomole (nmol) of polynucleotides per square meter (of an area, such as a poultry house), for example, from about 0.05 to 1 nmol polynucleotides per square meter.
  • Other embodiments per square meter (of an area, such as a poultry house) include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per square meter.
  • about 40 to about 50 nmol of a single-stranded polynucleotide as described herein are applied.
  • a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of a dsRNA (or a single-stranded 21-mer) as described herein is applied.
  • a composition of about 0.5 to about 1.5 milligrams per milliliter of a dsRNA polynucleotide as described herein of about 50 to about 200 or more nucleotides is applied.
  • about 1 nmol to about 5 nmol of a dsRNA as described herein is applied.
  • the polynucleotide composition as topically applied to an area (for example a square meter) and/or plant at least one polynucleotide as described herein at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter.
  • Very large areas and/or plants, trees, or vines can require correspondingly larger amounts of polynucleotides.
  • lower concentrations can be used.
  • Non-limiting examples of effective polynucleotide treatment regimes include a treatment of between about 0.1 to about 1 nmol of polynucleotide molecule per square meter of an area, and/or plant, or between about 1 nmol to about 10 nmol of polynucleotide molecule per square meter of an area, and/or plant, or between about 10 nmol to about 100 nmol of polynucleotide molecule per square meter of an area, and/or plant.
  • the dsRNA molecules of the invention are surprisingly effective at controlling insects, namely darkling beetles of the Alphitobiini tribe.
  • the control of such insects is particularly important as there are not many known effective agents for controlling these insects.
  • composition of the invention and/or methods of the invention are used to control insects of the tribe in particular of the Alphitobius genus, in particular of the Alphitobius genus.
  • a composition of the invention is used to control insects selected from the group consisting of Alphitobius diaperinus, Alphitobius arnoldi, Alphitobius capitaneus, Alphitobius crenatus, Alphitobius grandis, Alphitobius hobohmi, Alphitobius karrooensis, Alphitobius kochi, Alphitobius laevigatus, Alphitobius lamottei, Alphitobius leleupi, Alphitobius limbalis, Alphitobius lucasorum, Alphitobius niger, Alphitobius parallelipennis, Alphitobius rugosulus, and Alphitobius viator.
  • the compositions and/or methods of the invention may be used in the control Alphitobius diaperinus.
  • agrochemically active ingredient refers to chemicals and/or biological compositions, such as those described herein, which are effective in killing, preventing, or controlling the growth of undesirable pests, such as, plants, insects, mice, microorganism, algae, fungi, bacteria, and the like (such as pesticidally active ingredients).
  • undesirable pests such as, plants, insects, mice, microorganism, algae, fungi, bacteria, and the like (such as pesticidally active ingredients).
  • An interfering RNA molecule of the invention is an agrochemically active ingredient.
  • the present invention provides an insecticidal composition for inhibiting the expression of a darkling beetle target gene, comprising the interfering RNA of the invention and an agriculturally acceptable carrier.
  • An "agriculturally acceptable carrier” includes adjuvants, mixers, enhancers, dispersants, surfactants, additives, water, thickeners, anti-caking agents, residue breakdown products, oils, coloring agents, stabilizers, preservatives, polymers, and any combinations thereof.
  • the agriculturally acceptable carrier may be beneficial for application of an active ingredient, such as an interfering RNA molecule of the invention.
  • Agriculturally acceptable carriers such as those useful in the context of interfering RNAs, are well known in the art.
  • the insecticidal composition may be provided in the form of a spray, granules, and/or powder.
  • the insecticidal composition of the invention may be used to control a darkling beetle of the Alphitobiini tribe.
  • a method of controlling a darkling beetle of the Alphitobiini tribe comprising contacting the darkling beetle with an insecticidal composition of the invention (i.e. comprising the interfering RNA of the invention and an agriculturally acceptable carrier).
  • controlling includes applying the insecticidal composition to the darkling beetle and/or habitat of the darkling beetle.
  • the habitat may be for example a livestock building (including the surfaces of and items, such as animal feed, within the livestock building).
  • livestock building may be a poultry farm.
  • polynucleotides comprising sequences encoding the silencing element can be used to transform organisms to provide for host organism production of these components, and further used for subsequent application of the host organism to the environment of the target pest(s).
  • the combination of polynucleotides encoding the silencing element may be introduced via a suitable vector into a host cell, such as a microbial host, and said host applied to the environment, or to plants or animals.
  • an agriculturally acceptable carrier may also include non-pathogenic, attenuated strains of microorganisms, which carry the insect control agent, namely an interfering RNA molecule of the invention.
  • the microorganisms carrying the interfering RNA may also be referred to as insect control agents.
  • the microorganisms may be engineered to express a nucleotide sequence of a target gene to produce interfering RNA molecules comprising RNA sequences homologous or complementary to RNA sequences typically found within the cells of an insect. Exposure of the insects to the microorganisms result in ingestion of the microorganisms and down-regulation of expression of target genes mediated directly or indirectly by the interfering RNA molecules or fragments or derivatives thereof.
  • the present invention provides a host cell comprising the nucleic acid molecule, nucleic acid construct, and/or recombinant vector of the invention.
  • the host cell may be microbial cell, for example a bacterial cell, algae or fungi.
  • microorganisms such as bacteria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Escherichia, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agro bacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.
  • bacteria e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Escherichia, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylius, Agro bacterium, Ace
  • bacterial species such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacteria spp., Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, Clavibacter xyli and Azotobacter vinlandir, and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C.
  • expression cassettes can be constructed which include the nucleotide constructs of interest operably linked with the transcriptional and translational regulatory signals for expression of the nucleotide constructs, and a nucleotide sequence homologous with a sequence in the host organism, whereby integration will occur, and/or a replication system that is functional in the host, whereby integration or stable maintenance will occur.
  • Transcriptional and translational regulatory signals include, but are not limited to, promoters, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. Methods for the production of expression constructs comprising such regulatory signals are well known in the art; see for example Sambrook et al. (2000); Molecular Cloning: A Laboratory Manual (3rd ed.; Cold Spring Harbor Laboratory Press, Plainview, N.Y.); Davis et al. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); and the references cited therein.
  • Suitable host cells include the prokaryotes and the lower eukaryotes, such as fungi.
  • Illustrative prokaryotes both Gram-negative and Gram-positive, include Enterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella, and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae, such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio, Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such as Pseudomonas and Acetobacter; Azotobacteraceae and Nitrobacteraceae.
  • fungi such as Phycomycetes and Ascomycetes, which includes yeast such as Saccharomyces and Schizosaccharomyces; and Ba sidiomycetes yeast, such as Rhodotorula, Aureobasidium, Sporobolomyces, and the like.
  • Characteristics of particular interest in selecting a host cell for purposes of the invention include ease of introducing the coding sequence into the host, availability of expression systems, efficiency of expression, RNA stability in the host, and the presence of auxiliary genetic capabilities.
  • Characteristics of interest for use as a pesticide microcapsule include protective qualities, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; leaf affinity; lack of mammalian toxicity; attractiveness to pests for ingestion; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
  • Host organisms of particular interest include yeast, such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp., Erwinia spp., and Flavobacterium spp., and other such organisms, including Pseudomonas aeruginosa, Pseudomonas fluorescens, Saccharomyces cerevisiae, Bacillus thuringiensis, Escherichia coll, Bacillus subtilis, and the like.
  • yeast such as Rhodotorula spp., Aureobasidium spp., Saccharomyces spp., and Sporobolomyces spp.
  • phylloplane organisms such as Pseudomonas spp., Erwinia spp.,
  • An interfering RNA molecule of the invention can be fermented in a bacterial host and the resulting bacteria processed, and used as a microbial spray in the same manner that Bacillus thuringiensis strains have been used as insecticidal sprays. Any suitable microorganism can be used for this purpose.
  • Pseudomonas spp. have been used to express Bacillus thuringiensis endotoxins as encapsulated proteins and the resulting cells processed and sprayed as an insecticide (Gaertner et al. 1993. Advanced Engineered Pesticides, ed. L. Kim (Marcel Decker, Inc.).
  • E. coli is also well-known in the art for expressing molecules of interest as part during a fermentation process.
  • the resulting bacteria is processed by heat inactivation. In some embodiments, heat inactivation kills the bacteria but does not degrade the produced RNA molecules.
  • the resulting compositions may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest.
  • the components of the invention are produced by introducing heterologous genes into a cellular host. Expression of the heterologous sequences results, directly or indirectly, in the intracellular production of the silencing element. These compositions may then be formulated in accordance with conventional techniques for application to the environment hosting a target pest.
  • the transformed microorganisms carrying an interfering RNA molecule of the invention may also be referred to as insect control agents.
  • the microorganisms may be engineered to express a nucleotide sequence of a target gene to produce interfering RNA molecules comprising RNA sequences homologous or complementary to RNA sequences typically found within the cells of an insect. Exposure of the insects to the microorganisms result in ingestion of the microorganisms and down-regulation of expression of target genes mediated directly or indirectly by the interfering RNA molecules or fragments or derivatives thereof.
  • a transformed microorganism can be formulated with an acceptable carrier into separate or combined compositions that are, for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, and an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymer substances.
  • compositions comprising an interfering RNA molecule of the invention can be in a suitable form for direct application or as a concentrate of primary composition that requires dilution with a suitable quantity of water or other dilutant before application.
  • the compositions (with or without the transformed microorganisms) can be applied to the environment of an insect pest such as a darkling beetle, by, for example, spraying, atomizing, dusting, scattering, coating, pouring, or otherwise introducing into areas of livestock buildings (such as poultry houses) and/or animal feed (such as chicken feed).
  • livestock buildings such as poultry houses
  • animal feed such as chicken feed
  • the composition(s) and/or transformed microorganism(s) may be mixed with animal feed to protect the animal feed during storage.
  • livestock building refers to a building that is at least partially enclosed and/or in which livestock animals are kept (at least sometimes).
  • a livestock building may be for example a poultry house or a chicken coup.
  • Application of the compounds and/or compositions of the invention to the environment of an insect pest may be before infestation or when the pest is present.
  • Application of the compounds of the invention can be performed according to any of the usual modes of application, for example by spraying, dusting, whipping, coating, etc.
  • the compounds of the invention may be applied in combination with an attractant.
  • An attractant is a chemical that causes the insect to migrate towards the location of application. Suitable attractants may include glucose, saccharose, salt, glutamate (e.g. Aji-no- motoTM), and citric acid (e.g. OroborTM).
  • An attractant may be premixed with the compound of the invention prior to application, e.g. as a ready-mix or tank-mix, or by simultaneous application or sequential application to the plant. Suitable rates of attractants are for example 0.02kg/ha-3kg/ha.
  • compositions can conveniently contain another insecticide if this is thought necessary.
  • the compound(s) and/or composition(s) are applied to surfaces of a livestock building (for example a poultry house).
  • a livestock building for example a poultry house.
  • the phrase "surfaces of a livestock building” refers to one or more of the floor, wall, ceiling, and any items that may be found within the livestock building (such as buckets, feeders, cages, storage compartments, stalls, etc).
  • the compound(s) and/or composition(s) may be applied together with a second insecticide (which me be insecticidal against darkling beetle and/or other pests), an inert carrier, and dead cells of a Bacillus strain or live or dead cells of transformed microorganisms of the invention.
  • the interfering RNA molecules may be encapsulated in a synthetic matrix such as a polymer and applied to the surface, for example inside a livestock building. Ingestion of interfering RNA molecules by an insect permits delivery of the insect control agents to the insect and results in down-regulation of a target gene in the host.
  • a composition of the invention for example a composition comprising an interfering RNA molecule of the invention and an agriculturally acceptable carrier, may be used in conventional agricultural methods.
  • the compositions of the invention may be mixed with water and may be applied preemergence and/or postemergence to a desired locus by any means, such as airplane spray tanks, irrigation equipment, direct injection spray equipment, knapsack spray tanks, cattle dipping vats, farm equipment used in ground spraying (e.g., boom sprayers, hand sprayers), and the like.
  • the desired locus may be a habitat of the pest, for example a poultry house.
  • the compositions of the invention may be mixed with water, other insecticidal agents, and/or sanitizing agents (such as disinfectants) and applied to the locus.
  • an active ingredient to insects of the Alphitobiini tribe and/or habitat of these insects (such as livestock buildings, for example poultry houses and/or animal feed)
  • said active ingredient may be used in pure form or, more typically, formulated into a composition which includes, in addition to said active ingredient, a suitable inert diluent or carrier and optionally, a surface active agent (SFA).
  • SFAs are chemicals which are able to modify the properties of an interface (for example, liquid/solid, liquid/air or liquid/liquid interfaces) by lowering the interfacial tension and thereby leading to changes in other properties (for example dispersion, emulsification and wetting).
  • SFAs include non-ionic, cationic and/or anionic surfactants, as well as surfactant mixtures.
  • the active ingredient will be in the form of a composition additionally comprising an agriculturally acceptable carrier or diluent.
  • compositions can be chosen from a number of formulation types, including dustable powders (DP), soluble powders(SP), water soluble granules (SG), water dispersible granules (WG), wettable powders (WP), granules (GR) (slow or fast release), soluble concentrates (SL), oil miscible liquids(OL), ultra-low volume liquids (UL), emulsifiable concentrates(EC), dispersible concentrates (DC), emulsions (both oil in water (EW) and water in oil (EO)), micro-emulsions(ME), suspension concentrates (SC), aerosols, fogging/smoke formulations, capsule suspensions (CS) and seed treatment formulations.
  • the formulation type chosen in any instance will depend upon the particular purpose envisaged and the physical, chemical and biological properties of the compound of formula (I). Methods of making these formulations types are well known in the art.
  • a compound of the invention may be applied by any of the known means of applying pesticidal compounds. For example, it may be applied, formulated or unformulated, to the pests or to a locus of the pests (such as a habitat of the pests, such as a livestock building and/or animal feed).
  • a compound of the invention be injected into or topically applied (for example sprayed or dusted) onto the pest insect.
  • compositions for use as aqueous preparations are generally supplied in the form of a concentrate containing a high proportion of the active ingredient, the concentrate being added to water before use.
  • These concentrates which may include DCs, SCs, ECs, EWs, MEs, SGs, SPs, WPs, WGs and CSs, are often required to withstand storage for prolonged periods and, after such storage, to be capable of addition to water to form aqueous preparations which remain homogeneous for a sufficient time to enable them to be applied by conventional spray equipment.
  • Such aqueous preparations may contain varying amounts of a compound of the invention (for example 0.0001 to 10%, by weight) depending upon the purpose for which they are to be used.
  • the compound of the invention may be the sole active ingredient of the composition or it may be admixed with one or more additional active ingredients such as a pesticide, fungicide, synergist, herbicide or plant growth regulator where appropriate.
  • An additional active ingredient may: provide a composition having a broader spectrum of activity or increased persistence at a locus; synergize the activity or complement the activity (for example by increasing the speed of effect or overcoming repellency) of the compound of the invention; or help to overcome or prevent the development of resistance to individual components.
  • compositions of the invention include those prepared by premixing prior to application, e.g. as a readymix or tankmix, or by simultaneous application or sequential application to the locus.
  • the formulation comprises a transfection promoting agent.
  • the transfection promoting agent is a lipid-containing compound.
  • the lipid-containing compound is selected from the group consisting of; Lipofectamine, Cel Ifectin, DMRIE-C, DOTAP and Lipofectin.
  • the lipid-containing compound is a Tris cationic lipid.
  • the formulation further comprises a nucleic acid condensing agent.
  • the nucleic acid condensing agent can be any such compound known in the art. Examples of nucleic acid condensing agents include, but are not limited to, spermidine (N- [3-aminopropyl]-l,4-butanediamine), protamine sulphate, poly-lysine as well as other positively charged peptides. In some embodiments, the nucleic acid condensing agent is spermidine or protamine sulfate.
  • the formulation further comprises buffered sucrose or phosphate buffered saline.
  • “Expression cassette” as used herein means a nucleic acid sequence capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the nucleic acid sequence of interest which is operably linked to termination signal sequences. It also typically comprises sequences required for proper translation of the nucleic acid sequence.
  • the expression cassette comprising the nucleic acid sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
  • the expression of the nucleic acid sequence in the expression cassette may be under the control of, for example, a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus.
  • a "gene” is a defined region that is located within a genome and that, besides the aforementioned coding sequence, comprises other, primarily regulatory nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion.
  • a gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.
  • farmer means a person means a person or entity that is engaged in agriculture, raising living organisms, such as livestock (for example poultry).
  • a "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non- naturally occurring multiple copies of a naturally occurring nucleic acid sequence.
  • a "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.
  • Insecticidal is defined as a toxic biological activity capable of controlling insects, preferably by killing them.
  • controlling includes reducing a population of the insects by killing a portion of the population.
  • controlling includes reducing a population of the insects by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more.
  • nucleic acid molecule or nucleotide sequence or nucleic acid construct or dsRNA molecule or protein of the invention is generally exists apart from its native environment and is therefore not a product of nature.
  • An isolated nucleic acid molecule or nucleotide sequence or nucleic acid construct or dsRNA molecule or protein may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host or host cell such as a transgenic plant or transgenic plant cell.
  • a number in front of the suffix "mer” indicates a specified number of subunits. When applied to RNA or DNA, this specifies the number of bases in the molecule. For example, a 19 nucleotide subsequence of an mRNA having the sequence AGAAAUGUUGGGAAUCGGC (SEQ ID NO: 290) is a "19-mer" of SEQ ID NO: 97.
  • a darkling beetle “transcriptome” is a collection of all or nearly all the ribonucleic acid (RNA) transcripts in a darkling beetle cell.
  • Transformation is a process for introducing heterologous nucleic acid into a host cell or organism.
  • transformation means the stable integration of a DNA molecule into the genome of an organism of interest.
  • Transformed / transgenic / recombinant refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • non-transformed refers to a wildtype organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
  • the invention is based on the unexpected discovery that double stranded RNA (dsRNA) or small interfering RNAs (siRNA) designed to target a mRNA transcribable from the darkling beetle genes described herein are toxic to the darkling beetle pest and can be used to control darkling beetle or Coleopteran infestation.
  • the infestation may be in a livestock building such as poultry house, a chicken coup, or other environments in which the darkling beetle may be found in.
  • the invention provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein a nucleotide sequence of the antisense strand is complementary to a portion of a mRNA polynucleotide transcribable from a darkling beetle gene described in the present disclosure, wherein the dsRNA molecule is toxic to a darkling beetle or other Coleopteran pest.
  • dsRNA molecules that are not perfectly complementary to a target sequence (for example, having only 95% identity to the target gene) are effective to control Coleopteran pests (see, for example, Narva et al., U.S.
  • the invention provides an interfering RNA molecule comprising at least one dsRNA, where the dsRNA is a region of double-stranded RNA comprising annealed at least partially complementary strands.
  • 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, at least 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
  • the target gene may have a nucleotide sequence according to any one of SEQ ID NO: 1 to 48.
  • the interfering RNA molecule (i) has at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity, 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
  • the interfering RNA molecule comprises at least two dsRNAs, wherein each dsRNA comprises a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene.
  • each of the dsRNAs comprise a different sequence of nucleotides which is complementary to a different target nucleotide sequence within the target gene.
  • each of the dsRNAs comprise a different sequence of nucleotides which is complementary to a target nucleotide sequence within two different target genes.
  • the interfering RNA molecule comprises a dsRNA that can comprise, consist essentially of or consist of from at least 18 to about 25 consecutive nucleotides (e.g. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) to at least about 300 consecutive nucleotides. Additional nucleotides can be added at the 3' end, the 5' end or both the 3' and 5' ends to facilitate manipulation of the dsRNA molecule but that do not materially affect the basic characteristics or function of the dsRNA molecule in RNA interference (RNAi).
  • RNAi RNA interference
  • the interfering RNA molecule comprises a dsRNA which comprises an antisense strand that is complementary to 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, at least 160, at least 170, at least 180, at least
  • the portion of dsRNA comprises, consists essentially of or consists of at least from 19, 20 or 21 consecutive nucleotides to 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, at least 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 consecutive nucleotides of any one of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158
  • an interfering RNA molecule of the invention comprises a dsRNA which comprises, consists essentially of or consists of any 21-mer subsequence of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106- 108, 111-144, 146, 147, 150, 152, 154-156, and 159-192 consisting of N to N+20 nucleotides, or any complement thereof.
  • an interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 145, wherein N is nucleotide 1 to nucleotide 2340 of SEQ ID NO: 145, or any complement thereof.
  • the portion of the mRNA that is targeted comprises any of the 2340 21 consecutive nucleotide subsequences i.e. 21-mers) of SEQ ID NO: 145, or any of their complementing sequences.
  • these 2340 21 consecutive nucleotide subsequences include all possible 21 consecutive nucleotide subsequences from SEQ ID NO: 145 and from SEQ ID NO: 97, and their complements, as SEQ ID NO's 145 and 97 are all to the same target, namely Sec23. It will similarly be recognized that all 21-mer subsequences of any one of SEQ ID NO: 145-192, and all complement subsequences thereof, include all possible 21 consecutive nucleotide subsequences of SEQ ID NO: 97-144, and the complement subsequences thereof.
  • an interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 146, wherein N is nucleotide 1 to nucleotide 2715 of SEQ ID NO: 146, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 147, wherein N is nucleotide 1 to nucleotide 2877 of SEQ ID NO: 147, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 148, wherein N is nucleotide 1 to nucleotide 1519 of SEQ ID NO: 148, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 149, wherein N is nucleotide 1 to nucleotide 609 of SEQ ID NO: 149, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 150, wherein N is nucleotide 1 to nucleotide 615 of SEQ ID NO: 150, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 151, wherein N is nucleotide 1 to nucleotide 675 of SEQ ID NO: 151, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 152, wherein N is nucleotide 1 to nucleotide 747 of SEQ ID NO: 152, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 153, wherein N is nucleotide 1 to nucleotide 1131 of SEQ ID NO: 153, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 154, wherein N is nucleotide 1 to nucleotide 603 of SEQ ID NO: 154, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 155, wherein N is nucleotide 1 to nucleotide 456 of SEQ ID NO: 155, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 156, wherein N is nucleotide 1 to nucleotide 828 of SEQ ID NO: 156, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 157, wherein N is nucleotide 1 to nucleotide 2646 of SEQ ID NO: 157, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 158, wherein N is nucleotide 1 to nucleotide 654 of SEQ ID NO: 158, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 159, wherein N is nucleotide 1 to nucleotide 598 of SEQ ID NO: 159, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 160, wherein N is nucleotide 1 to nucleotide 606 of SEQ ID NO: 160, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 161, wherein N is nucleotide 1 to nucleotide 543 of SEQ ID NO: 161, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 162, wherein N is nucleotide 1 to nucleotide 2451 of SEQ ID NO: 162, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 163, wherein N is nucleotide 1 to nucleotide 351 of SEQ ID NO: 163, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 164, wherein N is nucleotide 1 to nucleotide 1539 of SEQ ID NO: 164, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 165, wherein N is nucleotide 1 to nucleotide 1674 of SEQ ID NO: 165, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 166, wherein N is nucleotide 1 to nucleotide 966 of SEQ ID NO: 166, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 167, wherein N is nucleotide 1 to nucleotide 1845 of SEQ ID NO: 167, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 168, wherein N is nucleotide 1 to nucleotide 651 of SEQ ID NO: 168, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 169, wherein N is nucleotide 1 to nucleotide 543 of SEQ ID NO: 169, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 170, wherein N is nucleotide 1 to nucleotide 4665 of SEQ ID NO: 170, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 171, wherein N is nucleotide 1 to nucleotide 537 of SEQ ID NO: 171, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 172, wherein N is nucleotide 1 to nucleotide 774 of SEQ ID NO: 172, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 173, wherein N is nucleotide 1 to nucleotide 750 of SEQ ID NO: 173, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 174, wherein N is nucleotide 1 to nucleotide 3657 of SEQ ID NO: 174, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 175, wherein N is nucleotide 1 to nucleotide 2412 of SEQ ID NO: 175, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 176, wherein N is nucleotide 1 to nucleotide 705 of SEQ ID NO: 176, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 177, wherein N is nucleotide 1 to nucleotide 618 of SEQ ID NO: 177, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 178, wherein N is nucleotide 1 to nucleotide 771 of SEQ ID NO: 178, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 179, wherein N is nucleotide 1 to nucleotide 471 of SEQ ID NO: 179, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 180, wherein N is nucleotide 1 to nucleotide 2754 of SEQ ID NO: 180, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 181, wherein N is nucleotide 1 to nucleotide 1431 of SEQ ID NO: 181, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 182, wherein N is nucleotide 1 to nucleotide 735 of SEQ ID NO: 182, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 183, wherein N is nucleotide 1 to nucleotide 477 of SEQ ID NO: 183, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 184, wherein N is nucleotide 1 to nucleotide 3885 of SEQ ID NO: 184, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 185, wherein N is nucleotide 1 to nucleotide 2721 of SEQ ID NO: 185, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 186, wherein N is nucleotide 1 to nucleotide 477 of SEQ ID NO: 186, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 187, wherein N is nucleotide 1 to nucleotide 1773 of SEQ ID NO: 187, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 188, wherein N is nucleotide 1 to nucleotide 777 of SEQ ID NO: 188, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 189, wherein N is nucleotide 1 to nucleotide 1227 of SEQ ID NO: 189, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 190, wherein N is nucleotide 1 to nucleotide 1380 of SEQ ID NO: 190, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 191, wherein N is nucleotide 1 to nucleotide 834 of SEQ ID NO: 191, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 192, wherein N is nucleotide 1 to nucleotide 765 of SEQ ID NO: 192, or any complement thereof.
  • the interfering RNA molecule of the invention comprises a dsRNA which comprises, consists essentially of or consists of SEQ ID NO: 148, 145, 151, 158, 149, 157, 153, 146, 147, 154-156, and 159 to 192, or the complement thereof.
  • the nucleotide sequence of the antisense strand of a dsRNA of the invention comprises, consists essentially of or consists of the complementary RNA sequence of any one of SEQ ID NO: 148, 145, 151, 158, 149, 157, 153, 146, 147, 154-156, and 159 to 192.
  • the nucleotide sequence of the antisense strand of a dsRNA of the invention can have one nucleotide at either the 3' or 5' end deleted or can have up to six nucleotides added at the 3' end, the 5' end or both, in any combination to achieve an antisense strand consisting essentially of any 19-mer, any 20-mer, or any 21-mer nucleotide sequence of any one of SEQ ID NO: 148, 145, 151, 158, 149, 157, 153, 146, 147, 154-156, and 159 to 192, as it would be understood that the deletion of the one nucleotide or the addition of up to the six nucleotides do not materially affect the basic characteristics or function of the double stranded RNA molecule of the invention.
  • Such additional nucleotides can be nucleotides that extend the complementarity of the antisense strand along the target sequence and/or such nucleotides can be nucleotides that facilitate manipulation of the RNA molecule or a nucleic acid molecule encoding the RNA molecule, as would be known to one of ordinary skill in the art.
  • a TT overhang at the 3' end may be present, which is used to stabilize the siRNA duplex and does not affect the specificity of the siRNA.
  • the antisense strand of the double stranded RNA of the interfering RNA molecule can be fully complementary to the target RNA polynucleotide or the antisense strand can be substantially complementary or partially complementary to the target RNA polynucleotide.
  • the dsRNA of the interfering RNA molecule may comprise a dsRNA which is a region of double-stranded RNA comprising substantially complementary annealed strands, or which is a region of double-stranded RNA comprising fully complementary annealed strands.
  • substantially or partially complementary is meant that the antisense strand and the target RNA polynucleotide can be mismatched at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide pairings.
  • Such mismatches can be introduced into the antisense strand sequence, e.g., near the 3' end, to enhance processing of the double stranded RNA molecule by Dicer, to duplicate a pattern of mismatches in a siRNA molecule inserted into a chimeric nucleic acid molecule or artificial microRNA precursor molecule of this invention, and the like, as would be known to one of skill in the art.
  • the interfering RNA comprises a dsRNA which comprises a short hairpin RNA (shRNA) molecule.
  • shRNA short hairpin RNA
  • Expression of shRNA in cells is typically accomplished by delivery of plasmids or recombinant vectors, for example in transgenic plants such as transgenic canola.
  • the invention encompasses a nucleic acid construct comprising an interfering RNA of the invention.
  • the invention further encompasses a nucleic acid molecule encoding at least one interfering molecule of the invention.
  • the invention further encompasses a nucleic acid construct comprising at least one interfering molecule of the invention or comprising a nucleic acid molecule encoding the at least one interfering molecule of the invention.
  • the invention further encompasses a nucleic acid construct wherein the nucleic acid construct is an expression vector.
  • the invention further encompasses a recombinant vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes an interfering RNA molecule of the invention.
  • a regulatory sequence may refer to a promoter, enhancer, transcription factor binding site, insulator, silencer, or any other DNA element involved in the expression of a gene.
  • the invention encompasses interfering RNA molecules, nucleic acid constructs, nucleic acid molecules or recombinant vectors comprising at least one strand of a dsRNA of an interfering RNA molecule of the invention, or comprising a chimeric nucleic acid molecule of the invention.
  • the nucleic acid construct comprises a nucleic acid molecule of the invention.
  • the nucleic acid construct is a recombinant expression vector.
  • the interfering RNA molecules of the invention have insecticidal activity on an insect, namely a darkling beetle, from the tribe Alphitobiini.
  • the coding sequence of the target gene comprises a sequence selected from the group comprising SEQ ID NO: 4, 1, 7, 14, 5, 13, 9, 2, 3, 6, 8, 10-12, and 15-48.
  • the invention encompasses a composition comprising one or more or two or more of the interfering RNA molecules of the invention.
  • the interfering RNA molecules are present on the same nucleic acid construct, on different nucleic acid constructs, or any combination thereof.
  • one interfering RNA molecule of the invention may be present on a nucleic acid construct, and a second interfering RNA molecule of the invention may be present on the same nucleic acid construct or on a separate, second nucleic acid construct.
  • the second interfering RNA molecule of the invention may be to the same target gene or to a different target gene.
  • the invention encompasses a composition comprising an interfering RNA molecule which comprises at least one dsRNA wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands.
  • 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, at least 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 at least partially complementary to a target nucleotide sequence within a darkling beetle target gene.
  • the interfering RNA molecule (i) has at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity, 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
  • the invention encompasses compositions comprising an interfering RNA molecule comprising two or more dsRNAs, wherein the two or more dsRNAs each comprise a different antisense strand. In some embodiments the invention encompasses compositions comprising at least two more interfering RNA molecules, wherein the two or more interfering RNA molecules each comprise a dsRNA comprising a different antisense strand.
  • the two or more interfering RNAs may be present on the same nucleic acid construct, on different nucleic acid constructs or any combination thereof.
  • the composition comprises a RNA molecule comprising an antisense strand consisting essentially of a nucleotide sequence comprising at least a 19 contiguous nucleotide fragment complementary to at least a 19 contiguous nucleotide fragment comprising the RNA sequence of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192, and in some embodiments may further comprise an RNA molecule comprising an antisense strand consisting essentially of a second nucleotide sequence comprising at least a 19 contiguous nucleotide fragment complementary to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158
  • RNA molecule comprising an antisense strand consisting essentially of a fifth nucleotide sequence comprising at least a 19 contiguous nucleotide fragment complementary to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192, and in some embodiments may further comprise an RNA molecule comprising an antisense strand consisting essentially of a sixth nucleotide sequence comprising at least a 19 contiguous nucleotide fragment complementary to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105
  • RNA molecule comprising an antisense strand consisting essentially of a seventh nucleotide sequence comprising at least a 19 contiguous nucleotide fragment complementary to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192.
  • the composition may comprise two or more of the nucleic acid molecules, wherein the two or more nucleic acid molecules each encode a different interfering RNA molecule. In other embodiments, the composition may comprise two or more of the nucleic acid constructs, wherein the two or more nucleic acid constructs each comprise a nucleic acid molecule encoding a different interfering RNA.
  • the composition comprises two or more nucleic acid constructs, two or more nucleic acid molecules, and/or two or more chimeric nucleic acid molecules of the invention, wherein the two or more nucleic acid constructs, two or more nucleic acid molecules, and/or two or more chimeric nucleic acid molecules each comprise a different antisense strand.
  • an acceptable agricultural carrier is a formulation useful for applying the composition comprising the interfering RNA molecule to an area (such as livestock building, for example poultry house or chicken coup).
  • the interfering RNA molecules are stabilized against degradation because of their double stranded nature and the introduction of Dnase/Rnase inhibitors.
  • dsRNA or siRNA can be stabilized by including thymidine or uridine nucleotide 3' overhangs.
  • the dsRNA or siRNA contained in the compositions of the invention can be chemically synthesized at industrial scale in large amounts. Methods available would be through chemical synthesis or through the use of a biological agent.
  • the invention further encompasses a method of controlling a Coleopteran insect or a darkling beetle comprising contacting the insect with a nucleic acid molecule that is or is capable of producing an interfering RNA molecule of the invention for inhibiting expression of a target gene in the insect thereby controlling the Coleopteran insect or the darkling beetle.
  • the target gene comprises a coding sequence (i) having at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity, 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
  • the target gene coding sequence comprises any one of SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or a complement thereof, or can hybridize under stringent conditions to a polynucleotide selected from the group consisting of SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, and the complements thereof.
  • the interfering RNA molecule of the invention is complementary to a portion of a mRNA polynucleotide transcribable from the darkling beetle target genes described herein.
  • the interfering RNA molecule of the invention comprises at least one dsRNA, wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises a sequence of at least 19 contiguous nucleotides which (i) has at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity,
  • the interfering RNA molecule comprises, consists essentially of or consists of from 18, 19, 20 or 21 consecutive nucleotides to at least about 300 consecutive nucleotides of any one of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192.
  • the interfering RNA molecule of the invention comprises a dsRNA which comprises, consists essentially of or consists of any 21-mer subsequence of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192 consisting of N to N+20 nucleotides, or any complement thereof.
  • an interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21- mer subsequence of SEQ ID NO: 145, wherein N is nucleotide 1 to nucleotide 2340 of SEQ ID NO: 145, or any complement thereof.
  • the portion of the mRNA that is targeted comprises any of the 2340 21 consecutive nucleotide subsequences i.e. 21-mers) of SEQ ID NO: 145, or any of their complementing sequences.
  • these 2340 21 consecutive nucleotide subsequences include all possible 21 consecutive nucleotide subsequences from SEQ ID NO: 145 and from SEQ ID NO: 97, and their complements, as SEQ ID NO's 145 and 97 are all to the same target, namely Sec23. It will similarly be recognized that all 21-mer subsequences of any one of SEQ ID NO: 145-192, and all complement subsequences thereof, include all possible 21 consecutive nucleotide subsequences of SEQ ID NO: 97-144, and the complement subsequences thereof.
  • an interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 146, wherein N is nucleotide 1 to nucleotide 2715 of SEQ ID NO: 146, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 147, wherein N is nucleotide 1 to nucleotide 2877 of SEQ ID NO: 147, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 148, wherein N is nucleotide 1 to nucleotide 1519 of SEQ ID NO: 148, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 149, wherein N is nucleotide 1 to nucleotide 609 of SEQ ID NO: 149, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 150, wherein N is nucleotide 1 to nucleotide 615 of SEQ ID NO: 150, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 151, wherein N is nucleotide 1 to nucleotide 675 of SEQ ID NO: 151, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 152, wherein N is nucleotide 1 to nucleotide 747 of SEQ ID NO: 152, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 153, wherein N is nucleotide 1 to nucleotide 1131 of SEQ ID NO: 153, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 154, wherein N is nucleotide 1 to nucleotide 603 of SEQ ID NO: 154, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 155, wherein N is nucleotide 1 to nucleotide 456 of SEQ ID NO: 155, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 156, wherein N is nucleotide 1 to nucleotide 828 of SEQ ID NO: 156, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 157, wherein N is nucleotide 1 to nucleotide 2646 of SEQ ID NO: 157, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 158, wherein N is nucleotide 1 to nucleotide 654 of SEQ ID NO: 158, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 159, wherein N is nucleotide 1 to nucleotide 598 of SEQ ID NO: 159, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 160, wherein N is nucleotide 1 to nucleotide 606 of SEQ ID NO: 160, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 161, wherein N is nucleotide 1 to nucleotide 543 of SEQ ID NO: 161, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 162, wherein N is nucleotide 1 to nucleotide 2451 of SEQ ID NO: 162, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 163, wherein N is nucleotide 1 to nucleotide 351 of SEQ ID NO: 163, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 164, wherein N is nucleotide 1 to nucleotide 1539 of SEQ ID NO: 164, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 165, wherein N is nucleotide 1 to nucleotide 1674 of SEQ ID NO: 165, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 166, wherein N is nucleotide 1 to nucleotide 966 of SEQ ID NO: 166, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 167, wherein N is nucleotide 1 to nucleotide 1845 of SEQ ID NO: 167, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 168, wherein N is nucleotide 1 to nucleotide 651 of SEQ ID NO: 168, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 169, wherein N is nucleotide 1 to nucleotide 543 of SEQ ID NO: 169, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 170, wherein N is nucleotide 1 to nucleotide 4665 of SEQ ID NO: 170, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 171, wherein N is nucleotide 1 to nucleotide 537 of SEQ ID NO: 171, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 172, wherein N is nucleotide 1 to nucleotide 774 of SEQ ID NO: 172, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 173, wherein N is nucleotide 1 to nucleotide 750 of SEQ ID NO: 173, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 174, wherein N is nucleotide 1 to nucleotide 3657 of SEQ ID NO: 174, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 175, wherein N is nucleotide 1 to nucleotide 2412 of SEQ ID NO: 175, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 176, wherein N is nucleotide 1 to nucleotide 705 of SEQ ID NO: 176, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 177, wherein N is nucleotide 1 to nucleotide 618 of SEQ ID NO: 177, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 178, wherein N is nucleotide 1 to nucleotide 771 of SEQ ID NO: 178, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 179, wherein N is nucleotide 1 to nucleotide 471 of SEQ ID NO: 179, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 180, wherein N is nucleotide 1 to nucleotide 2754 of SEQ ID NO: 180, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 181, wherein N is nucleotide 1 to nucleotide 1431 of SEQ ID NO: 181, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 182, wherein N is nucleotide 1 to nucleotide 735 of SEQ ID NO: 182, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 183, wherein N is nucleotide 1 to nucleotide 477 of SEQ ID NO: 183, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 184, wherein N is nucleotide 1 to nucleotide 3885 of SEQ ID NO: 184, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 185, wherein N is nucleotide 1 to nucleotide 2721 of SEQ ID NO: 185, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 186, wherein N is nucleotide 1 to nucleotide 477 of SEQ ID NO: 186, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 187, wherein N is nucleotide 1 to nucleotide 1773 of SEQ ID NO: 187, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 188, wherein N is nucleotide 1 to nucleotide 777 of SEQ ID NO: 188, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 189, wherein N is nucleotide 1 to nucleotide 1227 of SEQ ID NO: 189, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 190, wherein N is nucleotide 1 to nucleotide 1380 of SEQ ID NO: 190, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 191, wherein N is nucleotide 1 to nucleotide 834 of SEQ ID NO: 191, or any complement thereof.
  • Another interfering RNA molecule of the invention comprises a dsRNA which comprises, consist essentially of or consists of any 21-mer subsequence of SEQ ID NO: 192, wherein N is nucleotide 1 to nucleotide 765 of SEQ ID NO: 192, or any complement thereof.
  • the invention also encompasses a method of controlling a darkling beetle comprising contacting the darkling beetle with a nucleic acid molecule that is or is capable of producing the interfering RNA molecule of the invention for inhibiting expression of a target gene in the darkling beetle, and also contacting the darkling beetle with at least a second insecticidal agent for controlling the darkling beetle.
  • the invention encompasses a method of reducing the level of a target mRNA transcribable from a target gene as described herein in a Coleopteran insect or a darkling beetle comprising contacting the insect with a composition comprising the interfering RNA molecule of the invention, wherein the interfering RNA molecule reduces the level of the target mRNA in a cell of the insect.
  • the interfering RNA of the method comprises at least one dsRNA, wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises a sequence of at least 19 contiguous nucleotides which (i) has at least 80% identity, at least 85% identity, at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity, 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
  • RNA molecule has insecticidal activity against the target Coleopteran insect or a darkling beetle.
  • the contacting is achieved by the target insect feeding on the composition. In other embodiments, production of the protein encoded by the target mRNA is reduced. In other embodiments, the interfering RNA is contacted with a Coleopteran insect or a darkling beetle through a transgenic organism expressing the interfering RNA. In other embodiments, the transgenic organism is a transgenic plant, a transgenic microorganism, a transgenic bacterium or a transgenic endophyte.
  • the interfering RNA is contacted with a Coleopteran insect or a darkling beetle by topically applying an interfering RNA in an acceptable agricultural carrier to product (such as animal feed) on which the insect feeds.
  • the interfering RNA that reduces the level of a target mRNA transcribable from a target gene described herein is lethal to the Coleopteran insect or darkling beetle.
  • the darkling beetle a member of the Alphitobiini tribe.
  • the invention encompasses a method of providing a farmer with a means of controlling a darkling beetle of the Alphitobiini tribe pest population, the method comprising (a) selling or providing to the farmer a pest control composition that comprises an interfering RNA molecule, a nucleic acid molecule, a nucleic acid construct, and/or a composition of any of the corresponding preceding claims; and (b) advertising to the farmer that the pest control composition controls a darkling beetle pest population.
  • a farmer may be a poultry farmer.
  • the invention encompasses a method of identifying a target gene for using as a RNAi strategy for the control of a plant pest for RNAi in a Coleopteran plant pest, said method comprising the steps of a) producing a primer pair which can amplify a sequence that is or is orthologous to SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or a complement thereof; b) amplifying an orthologous target from a nucleic acid sample of the plant pest; c) identifying a sequence of an orthologous target gene; d) producing an interfering RNA molecule, wherein the RNA comprises at least one dsRNA, wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands, one strand of which
  • Example 1 Identification of potential RNAi gene targets in Alphitobius species
  • This example describes the cloning and sequencing of RNAi target genes and coding sequences from Alphitobius diaperinus insects.
  • Messenger RNA was isolated and a 100 bp paired-end library was prepared and sequenced according to the manufacturer's protocols on an Illumina HiSeq2000. Reads were assembled using Trinity with default settings. Open reading frames (ORFs) were predicted using Transdecoder using default parameters. The resulting ORFs were used in downstream analyses and lethal gene prediction.
  • dsRNAs based on selected targets were produced on an 96 well semi-automated library synthesis platform. Templates for the dsRNA molecules were produced based on internally developed transcriptome information for the Alphitobius diaperinus targets.
  • dsRNA samples tested were produced using primers designed using Primer3, a primer design tool, to synthetize a dsRNA fragment of around 500-600 bp based on the coding sequence of each target gene. Smaller fragments were designed if the size of the coding sequence did not allow a 500 bp fragment.
  • the list of dsRNA samples is depicted in Table 1.
  • T7 promoters were added to the 5' end of each primer so that resulting DNA templates could be immediately used in in vitro transcription reactions. Templates were synthesized by PCR on cDNA prepared from Alphitobius diaperinus at different life stages. The quality of the template material was analyzed by gel electrophoresis and spectrophotometry. Following dsRNA synthesis by in vitro T7 transcription, the dsRNA was purified, the amounts of dsRNA per target were normalized, and a final quality check of the dsRNA was performed by gel electrophoresis and spectrophotometry.
  • CDS DNA coding sequence
  • dsRNA fragment may comprise an RNA sequence that corresponds to the CDS fragment of SEQ ID NO: 289 (wherein T is substituted by U).
  • Example 2 Identification of target genes from Alphitobius diaperinus The dsRNA molecules described above were tested for toxicity against the insect pest species by injection. Briefly, synthesized dsRNA molecules were diluted to the appropriate concentration in mi I liQ. water. 30 larvae and/or adults of the darkling beetles were immobilized on a sticky tape before injection of O.lpl of dsRNA using a micro-injector device (Drummond, Nanoject III). dsRNA designed to target green fluorescent protein (GFP) was used as a negative control. Insects injected with the same volume of mi I liQ were considered controls for the injections.
  • GFP green fluorescent protein
  • insects were removed from the sticky tape and kept in a petridish of 9cm diameter, supplied with 2 chicken feed pellets as food source. The insects that did not survive the injection were removed from the assay, resulting in 20-30 insects per treatment. Each petri dish was maintained at approximately 25°C and 16:8 light:da rk photoperiod in a temperature controlled incubator. The mortality was scored at different days post-injection, with the final survival percentage calculated at 6 to 8 days. Results are depicted in Table 2 and Figure 1.
  • Table 2 and Figure 1 show the insecticidal activity of the selected targets in darkling beetle larvae. It has previously been suggested that certain genes of a given insect species can be predicted to confer an RNAi-mediated insecticidal effect based on the essential nature of the gene in insect of a different genus. However, empirical evaluation of the target genes revealed that the insecticidal effect could not be predicted (See Baum et al., 2007, Nature Biotechnology 25: 1322-1326; also U.S. Publication No. 2015/0322456).
  • a gene which has been shown to be a useful target for RNAi-mediated insect control for one insect pest is a useful target for RNAi-mediated insect control of a second insect pest of a different genus and/or family.
  • empirical evaluation of the target gene in different insect pests of different families show that a given target with very high insecticidal activity in one insect pest may not produce significant mortality or growth inhibition in a second insect pest (Knorr et al, 2018, Scientific Reports 8: 2061, DOI: 10.1038/s41598-018-20416-y). Therefore, the insecticidal activity of a dsRNA molecule against a target gene of an insect pest can only be determined empirically.
  • Example 3 Dose Response Curves of selected dsRNA molecules against darkling beetle larvae This example describes testing dsRNA molecules of the invention for biological activity against darkling beetle larvae in further injection assays.
  • the ubiquitin dsRNA molecule described above is tested for toxicity in a 10-fold dilution series starting from 20ng per insect to 200fg per insect. Results are used to generate dose response curves (DRC). Injection assays are performed as described above. Results are depicted in Figure 2.
  • Example 4 Dose Response Curves of selected dsRNA molecules against darkling beetle adults
  • This example describes testing dsRNA molecules of the invention for biological activity against darkling beetle adults in further injection assays.
  • the ubiquitin dsRNA molecule described above is tested for toxicity in a 10-fold dilution series starting from 20ng per insect to 200fg per insect. Results are used to generate dose response curves (DRC). Injection assays are performed as described above. Results are depicted in Figure 3.
  • Example 5 Testing of other dsRNA sub-fragments of selected targets against darkling beetles
  • This example describes testing other sub-fragments of dsRNA molecules of the invention for biological activity against darkling beetles. These sub-fragments are based on the coding sequence of a selection of positive targets, and are either a shorter length or are based on a different region of the coding sequence compared to the initial dsRNA fragment.
  • the dsRNA molecules are tested for toxicity against darkling beetles larvae or adults in injection assays as described above.
  • the results of SEQ ID 289, another subfragment of the same ubiquitin gene (SEQ ID 4) are depicted in the following tables for larvae (Table 3) and adults (Table 4).
  • This example describes testing dsRNA molecules of the invention for biological activity against darkling beetle larvae or adults in a feeding bio-assay.
  • the dsRNA samples produced as described above are pipetted on top of two chicken pellets (50pl/pel let). The chicken pellets are placed in a petridish of 9cm diameter. 10 insects are added to the petridish and are allowed to feed for a period of three days. The insects are then transferred to fresh chicken pellets with the same amount of dsRNA in a new petridish, and so on until the end of the assay.
  • dsRNA designed to target green fluorescent protein (GFP) was used as a negative control. Each petri dish was maintained at approximately 25°C and 16:8 light:dark photoperiod in a temperature controlled incubator. The mortality was scored at different days post-feeding until the end of the assay on day 14.
  • GFP green fluorescent protein
  • RNA interfering ribonucleic acid
  • the RNA comprises at least one dsRNA
  • the dsRNA is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises a sequence of at least 19 contiguous nucleotides which is at least partially complementary to a target nucleotide sequence within a darkling beetle target gene, and (i) is at least 85% identical to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192, or the complement thereof; (ii) comprises at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100,
  • RNA molecule of embodiment 1, wherein the darkling beetle of the Alphitobiini tribe is a species of a genus selected from the group consisting of Alphitobius, Alphitopsis, Ardoinia, Diaclina, Epipedodema, Guanobius, Hoplopeltis, and Peltoides
  • RNA molecule of embodiment 2 wherein the darkling beetle of the Alphitobius genus is a species selected from the group consisting of Alphitobius diaperinus, Alphitobius arnoldi, Alphitobius capitaneus, Alphitobius crenatus, Alphitobius grandis, Alphitobius hobohmi, Alphitobius karrooensis, Alphitobius kochi, Alphitobius laevigatus, Alphitobius lamottei, Alphitobius leleupi, Alphitobius limbalis, Alphitobius lucasorum, Alphitobius niger, Alphitobius parallelipennis, Alphitobius rugosulus, and Alphitobius viator.
  • RNA comprises at least two dsRNAs, wherein each dsRNA comprises a sequence of nucleotides which is at least partially complementary to a target nucleotide sequence within the target gene.
  • each of the dsRNAs comprise a different sequence of nucleotides which is at least partially complementary to a different target nucleotide sequence within the target gene.
  • a nucleic acid construct comprising the interfering RNA molecule of any of embodiments 1 to 8.
  • a nucleic acid construct comprising a nucleotide sequence that encodes the nucleic acid molecule of embodiment 10.
  • nucleic acid construct of any of embodiments 9 or 11 wherein the nucleic acid construct is an expression vector is an expression vector.
  • a recombinant vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes the interfering RNA molecule of any one of embodiments 1 to 8.
  • composition comprising two or more of the interfering RNA molecules of any of embodiments 1 to 8. 15. A composition of embodiment 14 wherein the two or more interfering RNA molecules are present on the same nucleic acid construct, on different nucleic acid constructs, or any combination thereof.
  • a composition comprising two or more of the nucleic acid constructs of any of embodiments 9, 11, or 12, wherein the two or more nucleic acid constructs each comprise a different interfering RNA.
  • composition comprising two or more of the nucleic acid molecules of embodiment 10, wherein the two or more nucleic acid molecules each encode a different interfering RNA molecule.
  • An insecticidal composition for inhibiting the expression of a darkling beetle target gene comprising the interfering RNA of any one of embodiments 1 to 8 and an agriculturally acceptable carrier.
  • An insecticidal composition of embodiment 18 comprising at least a second insecticidal agent for controlling a darkling beetle.
  • a method of controlling a darkling beetle of the Alphitobiini tribe comprising contacting the darkling beetle with a nucleic acid molecule that is or is capable of producing an interfering RNA molecule of embodiments 1-8 for inhibiting expression of a target gene in the darkling beetle thereby controlling the darkling beetle.
  • the target gene comprises a coding sequence which: a) is at least 85% identical to at least a 19 nucleotide contiguous fragment of SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or a complement thereof; b) comprises at least a 19 nucleotide contiguous fragment of SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or a complement thereof; or c) comprises at least a 19 nucleotide contiguous fragment of a nucleotide sequence encoding an amino acid sequence encoded by SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61,
  • the interfering RNA molecule comprises at least one dsRNA, wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises a sequence of at least 19 contiguous nucleotides which (i) is at least 85% identical to at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157, 105, 153, 98, 99, 102, 104, 106-108, 111-144, 146, 147, 150, 152, 154-156, and 159-192, or the complement thereof; or (ii) comprises at least a 19 contiguous nucleotide fragment of SEQ ID NO: 100, 148, 97, 145, 103, 151, 110, 158, 101, 149, 109, 157,
  • the darkling beetle of the Alphitobius genus is selected from the group consisting of Alphitobius diaperinus, Alphitobius arnoldi, Alphitobius capitaneus, Alphitobius crenatus, Alphitobius grandis, Alphitobius hobohmi, Alphitobius karrooensis, Alphitobius kochi, Alphitobius laevigatus, Alphitobius lamottei, Alphitobius leleupi, Alphitobius limbalis, Alphitobius lucasorum, Alphitobius niger, Alphitobius parallelipennis, Alphitobius rugosulus, and Alphitobius viator.
  • a method of controlling a darkling beetle of the Alphitobiini tribe comprising contacting the darkling beetle with a nucleic acid molecule that is or is capable of producing the interfering RNA molecule of embodiments 1-8 for inhibiting expression of a target gene in the darkling beetle, and contacting the darkling beetle with at least a second insecticidal agent for controlling the darkling beetle.
  • a method of reducing the level of a target RNA transcribed from a target gene in a darkling beetle of the Alphitobiini tribe comprising contacting the darkling beetle with a composition comprising the interfering RNA molecule of any one of embodiments 1 to 8, wherein the interfering RNA molecule reduces the level of the target RNA in a cell of the darkling beetle.
  • the protein comprises an amino acid sequence encoded by a nucleic acid sequence with at least 85% identity to SEQ ID NO: 52, 289, 4, 49, 1, 55, 7, 62, 14, 53, 5, 61, 13, 57, 9, 50, 51, 54, 56, 58-60, 63-96, 2, 3, 6, 8, 10-12, and 15-48, or the complement thereof.
  • the darkling beetle of the Alphitobiini tribe of the Alphitobius genus is selected from the group consisting of Alphitobius diaperinus, Alphitobius arnoldi, Alphitobius capitaneus, Alphitobius crenatus, Alphitobius grandis, Alphitobius hobohmi, Alphitobius karrooensis, Alphitobius kochi, Alphitobius laevigatus, Alphitobius lamottei, Alphitobius leleupi, Alphitobius limbalis, Alphitobius lucasorum, Alphitobius niger, Alphitobius parallelipennis, Alphitobius rugosulus, and Alphitobius viator.
  • a method of providing a farmer with a means of controlling a darkling beetle of the Alphitobiini tribe pest population in a plant comprising (a) selling or providing to the farmer a pest control composition that comprises an interfering RNA molecule, a nucleic acid molecule, a nucleic acid construct, and/or a composition of any of the corresponding preceding claims; and (b) advertising to the farmer that the pest control composition controls a darkling beetle pest population.
  • a method of identifying an orthologous target gene for using as a RNAi strategy for the control of a plant pest comprising the steps of: a) producing a primer pair that will amplify a target selected from the group comprising or consisting of SEQ.
  • RNA comprises at least one dsRNA, wherein the dsRNA is a region of double-stranded RNA comprising annealed complementary strands, one strand of which comprises a sequence of at least 19 contiguous nucleotides which is at least partially complementary to the orthologous target nucleotide sequence within the target gene, and e) determining if the interfering RNA molecule of step d) has insecticidal activity on the plant pest; wherein

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Abstract

L'invention concerne des molécules d'ARN double brin qui sont toxiques pour des ténébrions, en particulier les espèces de ténébrions Alphitobius diaperinus. En particulier, des molécules d'ARN interférentes capables d'interférer avec des gènes cibles nuisibles et qui sont toxiques pour le nuisible cible sont fournies. L'invention concerne en outre des procédés de fabrication et d'utilisation de l'ARN interférent, par exemple en tant que principe actif dans une composition, pour conférer une protection contre les dommages causés par les insectes.
PCT/EP2023/073875 2022-09-01 2023-08-31 Lutte contre les insectes nuisibles au moyen de molécules d'arn WO2024047148A1 (fr)

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