US 20140348893A1 (19) United States (12) Patent Application Publication (10) Pub. N0.: US 2014/0348893 A1 KALETTA (43) Pub. Date: NOV. 27, 2014

(54) RNAI FOR THE CONTROL OF AND A01N57/16 (2006.01) ARACHNIDS C12P 19/34 (2006.01) (52) US. Cl. (71) Applicant Devgen NV, Ghent-Zwijnaarde (BE) CPC ...... C12N15/113 (2013.01); A01N 57/16 _ _ (2013.01); C12P 19/34 (2013.01);A01N25/34 (73) Assignee:Inventor: DEVGENTltllS Jan NV, Ghent-ZWijnaardeNieder-Olm (BE) (201301) CNN 2310/1421 USPC 424/417; 536/245; 435/320.1; 435/252.3; (21) Appl' NO" 14/287’614 514/44 A; 435/913; 424/84; 424/93.2; 43/121 (22) Filed: May 27, 2014 (57) ABSTRACT Related US. Application Data _ _ _ _ _ The present invention describes a neW non-compound based (62) DWISIOn Of apphcanon NO' 11/921,370, ?led 911 Q“ approach for and/or arachnid control. The present 1’ 2009’ HOW Pat' NO' 83759306, ?led as apphcanon inventors have identi?ed for the ?rst time novel targets for NO' PCT/ 1132006/ 002360 on May 31’ 2006' RNAi, Which can effectively control insect and/or arachnid (60) Provisional application No. 60/685,765, ?led on May P€St POPUIations- Accordingly, the invention PTOVid?S bOth 31, 2005 nucleotide and amino acid sequences for the novel targets. Also provided are RNA constructs including double stranded Publication Classi?cation RNA regions for mediating RNAi in insects, DNA constructs, expression vectors, host cells and compositions for control (51) Int. Cl. ling insects and/ or arachnids using RNAi. Finally, the inven C12N 15/113 (2006.01) tion also provides for the use of the constructs, vectors, host A01M 1/20 (2006.01) cells and compositions in control of insects and/or arachnids A01N 25/34 (2006.01) populations and suitable kits for use in an RNAi based A01M 1/10 (2006.01) method of controlling insect and/ or arachnid pests. Patent Application Publication Nov. 27, 2014 Sheet 1 0f 13 US 2014/0348893 A1

Effect of 1% dsRNA on mortality 100— —&— 59033 80_ + 89001 + Bg003 60- + 89004 Mortality(%) —I—— 89005 40— —l— lmidacloprid 20_ —9— Solvent

0 I l I I l I I I I 0 5 10 15 20 25 30 3 4O 4 Days

120

100 : : : . . . . . I 80 /1/] it> Mortality(%) 60

0 3 610131720242731343841454852 Days + BgOO1 + 89001 concatemer2 —-l— Hexaflumuron FIG. 2 —6— Solvent Patent Application Publication Nov. 27, 2014 Sheet 2 0f 13 US 2014/0348893 A1

Month1 Month2

23 a?

89032 in LB 89032 in MO

FIG. 3

100 90 80 7O 60 Mortality(%) 50 40 3O 20 1O 0 O 3 610131720242731343841 Days + 59001 —+— -ve control (miscellaneous dsRNA) -9- -ve control (solvent) FIG. 4 Patent Application Publication Nov. 27, 2014 Sheet 3 0f 13 US 2014/0348893 A1

SEQ ID NO 1; DNA; Blattella germanica taaggcatggatgttggacaagctcggtggagtgtatgctccaagaccaagcacaggacctcacaagttacgagagagtctgc cccttgtaatatttcttcgtaataggctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttg atggaaaagtcagaacagaccccaactatccagctggttttatggatgttgttacaattgaaaaaactggagaatttttccgtctgat ttatgacgtgaaaggacgtttcaccattcacagaataactgctgaagaagccaagtataaactgtgcaaggtaaagagagtgca gactgggcccaagggtattccattcttggtgacccatgatggtagaactcttagatatcctgatcctgtcatcaaagttaatgataca gttcaacttgacatcgctacttccaagattatggatagcatcaaatttgataatggtaatctctgtatgattactggaggccgtaacttg ggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcctttgacattgtgcatgttaaagattcacaaggacacacatttgc taccagattgaa

SEQ ID NO 2; PRT; Blattella germanica Lys Ala Trp Met Leu Asp Lys Leu Gly Gly Val Tyr Ala Pro Arg Pro Ser Thr Gly Pro His Lys Leu Arg Glu Ser Leu Pro Leu Val lle Phe Leu Arg Asn Arg Leu Lys Tyr Ala Leu Thr Asn Cys Glu Val Lys Lys lle Val Met Gln Arg Leu lle Lys Val Asp Gly Lys Val Arg Thr Asp Pro Asn Tyr Pro Ala Gly Phe Met Asp Val Val Thr lle Glu Lys Thr Gly Glu Phe Phe Arg Leu lle Tyr Asp Val Lys Gly Arg Phe Thr lle His Arg lle Thr Ala Glu Glu Ala Lys Tyr Lys Leu Cys Lys Val Lys Arg Val Gln Thr Gly Pro Lys Gly lle Pro Phe Leu Val Thr His Asp Gly Arg Thr Leu Arg Tyr Pro Asp Pro Val lle Lys Val Asn Asp Thr Val Gln Leu Asp lle Ala Thr Ser Lys lle Met Asp Ser lle Lys Phe Asp Asn Gly Asn Leu Cys Met lle Thr Gly Gly Arg Asn Leu Gly Arg Val Gly Thr Val Val Asn Arg Glu Arg His Pro Gly Ser Phe Asp lle Val His Val Lys Asp Ser Gln Gly His Thr Phe Ala Thr Arg Leu Asn

SEQ ID NO 3; DNA; Blattella germanica; degenerate primer; w is a or t; r is a or g; m is a or c; y is c ort catttgaagc gtttwrmygc ycc

SEQ ID NO 4; DNA; Blattella germanica; speci?c primer gtgcccttgc caatgatgaa cacgttg

SEQ 10 NO 5; DNA; speci?c T7 primer cgctaatacg actcactata ggggagtgta tgctccaaga ccaag

SEQ ID NO 6; DNA; Blattella germanica; speci?c primer caatctggta gcaaatgtgt gtcc

SEQ ID NO 7; DNA; Blattella germanica; speci?c primer ggagtgtatg ctccaagacc aag

SEQ ID NO 8; DNA; synthetic T7 primer cgctaatacg actcactata ggcaatctgg tagcaaatgt gtgtcc

SEQ ID NO 9; RNA; Blattella germanica; u can be t (for DNA complement) ggaguguaug cuccaagacc aagcacagga ccucacaagu uacgagagag ucugccccuu FIG. 5

Patent Application Publication Nov. 27, 2014 Sheet 7 0f 13 US 2014/0348893 A1

SEQ ID NO 31; DNA; Blattella gemianica; ggcttgatcccaatgaaataaacgaaatlgcaaataccaattccagacaaaatattcgtaaactgattaaagatggtcttatcatc aagaagcccgtagctgtacactcaagggcccgtgttcgcaagaacaccgaagcaagaagaaaaggacgtcactgcggttttg gcaaaaggaagggtacggcaaatgcccgtatgccacagaaggtcttgtggattaatcgcatgcgtgttctgagaaggcttctca agaagtacagggaagcaaagaagatcgacagacatctataccaccagctglacatgaaggccaagggtaacgtgttcaaga acaagcgtglcctgatggagtlcatccacaagaagaaggctgagaaggccaggacaaagatgcttaacgac

SEQ ID NO 32; PRT; BIatleIIa germanica; Leu Asp Pro Asn Glu IIe Asn Glu IIe Ala Asn ThrAsn Ser Arg Gln Asn Ile Arg Lys Leu Ile Lys Asp Gly Leu He He Lys Lys Pro Val Ala Val His Ser Arg Ala Arg Val Arg Lys Asn Thr Glu Ala Arg Arg Lys Gly Arg His Cys Gly Phe Gly Lys Arg Lys Gly Thr Ala Asn Ala Arg Met Pro Gln Lys Val Leu Trp Ile Asn Arg Mat Arg Val Leu Arg Arg Leu Leu Lys Lys Tyr Arg Glu Ala Lys Lys Ile Asp Arg His Leu Tyr His Gln Leu Tyr Met Lys Ala Lys Gly Asn Val Phe Lys Asn Lys Arg Val Leu Met Glu Phe IIe His Lys Lys Lys Ala Glu Lys Ala Arg Thr Lys Met Leu Asn Asp

SEQ ID NO 33; DNA; Blatlella germanica; degenerative primer forward; r is a org; b is c org

ort tgcgatgcgg caaraaraag gtbtgg

SEQ ID NO 34; DNA; Blattella germanica; degenerative primer reverse; y is c or I; r is a or g gtcggcgagc ytcrgcytg

SEQ ID NO 35; DNA; speci?c T7 primer fonlvard cgctaalacg actcactata ggggcttgat cccaatgaaa taaacg

SEQ ID NO 36; DNA; Blattella germanica; speci?c primer reverse gtcgttaagc atctltgtcc tggc

SEQ ID NO 37; DNA; Blattella germanica; speci?c primer forward ggcttgatcc caatgaaata aacg

SEQ ID NO 38; DNA; speci?c T7 primer reverse cgctaatacg actcactata gggtcgttaa gcalctltgt cctggc

SEQ ID NO 39; RNA; Blattella germanica; u can be t (for DNA complement)

ggcuugaucc caaugaaaua aacgaaauug caaauaccaa uuccagacaa aauauucgua aacugauuaa agauggucuu aucaucaaga agcccguagc uguacacuca agggcccgug

FIG. 5CONT’D

Patent Application Publication Nov. 27, 2014 Sheet 13 0f 13 US 2014/0348893 A1

SEQ ID NO 66; DNA; synthetic BgOO1 fragment cttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcct

SEQ ID NO 67; DNA; synthetic BgOO1 concatemer 1 tatgctccaagaccaagcacaggacctcacaagttacgagagagtctgcctatgctccaagaccaagcacaggacctcacaa gttacgagagagtctgcctatgctccaagaccaagcacaggacctcacaagttacgagagagtctgcctatgctccaagacca agcacaggacctcacaagttacgagagagtctgcctatgctccaagaccaagcacaggacctcacaagttacgagagagtct gcc

SEQ ID NO 68; DNA; synthetic BgOO1 concatemer 2 cttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcctcttgggtcgtgttggaactgtagttaatcgagaacgtcat cctggttcctcttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcctcttgggtcgtgttggaactgtagttaatcga gaacgtcatcctggttcctcttgggtcgtgttggaactgtagttaatcgagaacgtcatcctggttcct

SEQ ID NO 69; DNA; synthetic BgOO1 fragment gctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttgatggaaaagtcagaacagacc ccaactatccagct

SEQ ID NO 70; DNA; synthetic BgOO1 concatemer 3 gctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttgatggaaaagtcagaacagacc ccaactatccagctgctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttattaaggttgatggaaaa gtcagaacagaccccaactatccagctgctgaaatatgcattaaccaactgtgaggttaagaaaattgttatgcagcgccttatta aggttgatggaaaagtcagaacagaccccaactatccagct

FIG. 5CONT'D US 2014/0348893 A1 Nov. 27, 2014

RNAI FOR THE CONTROL OF INSECTS AND less toxic insecticides, there is an increased probability that ARACHNIDS the insect may become resistant over time. Insecticides act by binding to a certain insect protein, such as RELATED APPLICATION INFORMATION an acethylcholine receptor for example, and cause death of [0001] This is a divisional application ofU.S. patent appli the pest species by either deactivating or over-activating the cation Ser. No. 11/921,370 ?led on Oct. 1, 2009, which is a protein. Insecticides have been developed to be safe at certain 371 of International Application No. PCT/IB2006/002360, concentration, but can and do impact on human health when ?led May 31, 2006, which claims bene?t to US. Provisional incorporated at higher dosages or over long periods. In con Application No. 60/685,765, ?led May 31, 2005, the contents trary to agrochemicals, household insecticides are applied in of which are incorporated herein by reference herein. places where food is stored or prepared and food contamina tion and contact to humans cannot be avoided. STATEMENT REGARDING ELECTRONIC [0008] One alternative to chemical pesticides is to utilise SUBMISSION OF A SEQUENCE LISTING biological agents. Over the last few years, downregulation of genes (also referred to as “gene silencing”) in multicellular [0002] A Sequence Listing in ASCII text format, submitted organisms by means of RNA interference or “RNAi” has under 37 CPR. §1.821, entitled 803 84-US-REG-ORG-P-1_ become a well-established technique. SEQUENCE_LISTING 80.1 kb in size, generated on Nov. [0009] In general, RNAi comprises contacting the organ 30, 2007 and ?led via EFS-Web is provided in lieu of a paper ism with a double stranded RNA fragment or “dsRNA” (gen copy. This Sequence Listing is hereby incorporated by refer erally either as two annealed complementary single strands of ence into the speci?cation for its disclosures. RNA or as a hairpin construct) that comprises a nucleotide sequence that corresponds to (at least part of) the nucleotide FIELD OF THE INVENTION sequence of the gene to be downregulated (the “target gene”). [0003] The present invention relates to the ?eld of double Reference is inter alia made to the International application stranded RNA (dsRNA) mediated gene silencing in insect WO 99/32619 (Carnegie Institute of Washington), the Inter species. More particularly, the present invention relates to national application W0 99/ 53050 (CSIRO), the Intema genetic constructs designed for the expression of dsRNA tional application WO 00/01846 (Devgen) and to Fire et al., corresponding to novel targets identi?ed for the ?rst time Nature, Vol. 391, pp. 806-811, February 1998. herein. These constructs are particularly useful in dsRNA [0010] In nematodes, RNAi can be performed by feeding mediated insect pest control, especially control of household the nematode with the dsRNA fragment as such, or altema insects or arachnids, for instance . tively with a bacterial strain that either contains the dsRNA fragment or that upon ingestion by the nematode is capable of BACKGROUND TO THE INVENTION expressing the dsRNA fragment. For this so-called “RNAi by [0004] Pest control and particularly insect and/or arachnid feeding”, reference is inter alia made to the International control, especially control of household insects, ecto-para application WO 00/01846 by applicant, and to WO 99/32619 sites and insects relevant for public health and hygiene (e.g. cited above, in which the nematode C. elegans is used. urban protection) such as cockroaches, ?eas, ants, termites, [0011] Many dsRNA constructs have been described in the earwigs, mosquitos, ?ies and house crickets is an important art. A classic dsRNA is produced from a DNA construct ?eld. The presence of insects in locations such as at home, in comprising two convergent promoters ?anking the sequence o?ices, restaurants, hospitals or warehouses undoubtedly complementary to the target sequence which needs to be causes distress because there is a common public perception downregulated (see for example W000/01846 (Devgen)). As that insects such as cockroaches or ?ies live in places that are the technology of dsRNA mediated gene silencing advanced, dirty and not well kept. new constructs were designed to improve the dsRNA for [0005] These insects do not only causes distress but also various purposes. contaminate food and eating utensils, destroy fabric and [0012] In order to produce the dsRNA more e?iciently, a paper products and impart stains and unpleasant odours to stem-loop-stem structure or “hairpin” was developed. As surfaces they contact. Furthermore, these insects can pose described in, for example, document W0 99/ 53050 (CSIRO), health risks as carriers for bacteria. For example, cockroaches this hairpin allows the formation of dsRNA from one single may transmit bacteria that cause food poisoning (Salmonella RNA transcript. The RNA transcript comprises the sense and spp. and Shigella spp.). German cockroaches are believed anti-sense version of the complementary sequence, separated capable of transmitting disease-causing organisms such as by a non-complementary loop structure allowing the RNA Staphylococcus spp., Streptococcus spp., hepatitis virus and transcript to fold back and to base pair into a dsRNA stem coliform bacteria. They also have been implicated in the portion. spread of typhoid and dysentery. Some people, especially [0013] DsRNA gene silencing ?nds application in many those with asthma, are sensitive to the allergens produced by different areas, such as for example dsRNA mediated gene these cockroaches. silencing in clinical applications (WO2004/001013) and in [0006] There are various chemical insecticides and captur plants. In plants, dsRNA constructs useful for gene silencing ing devices developed and commercially available for ?ght have also been designed to be cleaved and to be processed into ing household pests. However, increasing e?icacy of these Short interfering RNAs (siRNAs). means is usually linked with increased health risk. Insecti [0014] RNAi has also been proposed as a means of protect cides may contaminate food which is nearly unavoidable in ing plants against plant parasitic nematodes, i.e. by express places such as kitchens, restaurants or food storages and ing in the plant (e. g. in the entire plant, or in a part, tissue or incorporation may cause health risks to humans. cell of a plant) one or more nucleotide sequences that form a [0007] The solution to this problem of contamination has dsRNA fragment that corresponds to a target gene in the plant been to use less toxic insecticides. However, when applying parasitic nematode that is essential for its growth, reproduc US 2014/0348893 A1 Nov. 27, 2014

tion and/ or survival. Reference may be made to the Interna target gene limits the insect’ s and/or arachnids ability to feed, tional application WO 00/01846 by the present applicant, grow, or survive. Examples of insect and/or arachnid genes US. Pat. No. 6,506,559 (based on WO 99/32619), and to that may be employed in the practice of the invention include International applications WO 01/96584, WO 01/37654 and essential genes, genes involved in processes such as develop WO 03/052110 for a description of such techniques. ment, metabolism, or neurotransmission, and genes whose [0015] Elbashir et al. (Nature, 411, 494-498, 2001) have products are targets of existing insecticides and/ or arachnids. demonstrated effective RNAi-mediated gene silencing in In a preferred embodiment of the invention, the target is part mammalian cells using dsRNA fragments of 21 nucleotides of pathways required for cellular function such as transcrip in length (also termed small interfering RNAs or siRNAs). tion, translation, the cytoskeleton, cell-cycle, metabolism [0016] WO 03/004644 describes delivery of dsRNA to (anabolism or catabolism), endocytosis, intracellular and in general terms and is incorporated herein by intercellular transport, calcium binding, nucleus import and reference. WO 03/004644 details down regulation of the export, nucleic acid binding, signal peptidase-protein bind reporter gene GUS (Clonetech) using RNAi in Drosophila ing, the proteasome, vesicle transport, neuro-transmission, melanogasler and down regulation of the vATPase gene in H. water-balance, ion-balance, splicing, mitosis, meiosis, chro armigera. mosome organisation, stability or integrity, micro RNAs, siR [0017] WO 01/34815 relates to baculovirus expression NAs, posttranslational protein modi?cations, electron trans vectors which produce dsRNA and the use of these vectors in port, apoptosis, membrane integrity, and cell adhesion. pest control. [0028] The novel target genes identi?ed in the present [0018] Although the technique of RNAi has been generally invention comprise: known in the art in plants, nematodes and mammalian cells [0029] A) structural proteins, for instance tropomyosin 1 for some years, to date little is known about the use of RNAi (GenBank AF260897) (SEQ ID NOs 41 and 42), actin 5C to down-regulate gene expression in insects and/or arachnids. (GenBank AY004248) (SEQ ID NOs 57 and 58), and In addition, little is known on the application of RNAi to homologous or heterologous proteins having the same bio control pest species such as household insects, ecto-parasites logical function in the same or in other insect and/or arachnid and insects and/or arachnids relevant for public health and species; hygiene. [0030] B) metabolic enzymes, for instance the HMG Coen [0019] Constructs suitable and ef?cient for dsRNA medi zyme A synthase (GenBank X73679) (SEQ ID NO 49 and ated pest control, should meet at least some of the following 50), and homologous or heterologous proteins having the requirements same biological function in the same or in other insect and/or [0020] (1) the dsRNA must be taken up by the pest organ arachnid species; isms [0031] C) enzymes involved in ion/pH homeostasis, such as [0021] (2) the dsRNA must have good stability in the pest V-ATPase and homologous or heterologous proteins having organisms the same biological function in the same or in other insect [0022] (3) the dsRNA must be effective in the pest organ and/or arachnid species; ism to control its viability, growth and/or development [0032] D) enzymes involved in the transcriptional/transla and/or tional machinery, such as for instance [0023] (4) the dsRNA must guarantee maximized safety [0033] Ribosomal protein S4 homolog (SEQ ID NOs 1 and and minimized environmental impact. 2) [0024] It is now the purpose of the present invention to [0034] Ribosomal protein S9 homolog (SEQ ID NOs 11 provide dsRNA constructs, which meet the above-mentioned and 12) requirements. [0035] Ribosomal protein L9 homolog (SEQ ID NOs 21 and 22) DESCRIPTION OF THE INVENTION [0036] Ribosomal protein L19 homolog (SEQ ID NOs 31 [0025] The present invention describes a new non-com and 32) pound based approach for insect and/or arachnid control. The [0037] Accordingly, according to a ?rst aspect there is pro active ingredient is a nucleic acid, a double-stranded RNA vided a nucleic acid molecule comprising the nucleotide (dsRNA), which can be used as an insecticidal or arachnicidal sequence as set forth in any one of SEQ ID NOs 1, 4, 6, 7, 9, formulation (for example in baits or gel applications). The 10, 65 to 70, 71 to 79,11,16,17,19,20,80to 87, 21, 26, 27, sequence of the dsRNA matches a part of an essential insect 29, 30, 88 to 93, 31, 36, 37, 39, 40, 94 to 108, 180 and 181, or gene and causes down-regulation of the insect target via RNA an orthologous nucleotide sequence from an insect and/or interference (RNAi). As a result of the down-regulation of arachnid species, wherein the orthologous nucleotide mRNA, the dsRNA prevents expression of the corresponding sequence has at least 70%, preferably at least 75%, 80%, insect protein and hence causes death, growth arrest or steril 85%, 90%, more preferably at least about 95% and even more ity of the insect and/ or arachnid. preferably at least about 96%, 97%, 98%, most preferably at least 99% sequence identity with the nucleotide sequence of Targets any one of SEQ ID NOs 1, 11, 21 and 31. Preferred ortholo gous sequences comprise, or if being used according to the [0026] The present inventors have identi?ed for the ?rst methods of the invention include, sequences from household time novel targets for RNAi, which can effectively control insects, ecto-parasites and insects and/or arachnids relevant insect or arachnid pest populations. for public health and hygiene such as, by way of example and [0027] For the avoidance of doubt, a target is de?ned herein not limitation, ?ies, spider mites, thrips, ticks, red poultry as a gene whose protein product is required for the insect mite, ants, cockroaches, termites, crickets including house and/or arachnid to maintain its normal physiological and crickets, silver?sh, booklice, beetles, earwigs, mosquitos and biochemical functions. Inhibition of the expression of the ?eas. More preferred orthologous sequences are from cock US 2014/0348893 A1 Nov. 27, 2014

roaches () such as but not limited to Blalella spp. share at least about 70%, 75%, 80%, 85%, 90%, more pref (e.g. Blalella germanica (gerrnan cockroach)), Periplanela erably at least about 95% and even more preferably at least spp. (e.g. Periplanela americana () and about 96%, 97%, 98% or 99% sequence identity with the Periplanela auslraliasiae (Australian cockroach)), Blalla nucleotide sequence as set forth in any one of SEQ ID NOs 1, spp. (e. g. Blalla orientalis (Oriental cockroach)) and Supella 11, 21, 31, 41, 49 or 57. spp. (e.g. Supella longipalpa (brown-banded cockroach); [0044] According to another embodiment, the invention ants (Formicoidea), such as but not limited to Solenopsis spp. encompasses target genes which are insect or arachnidae (e.g. Solenopsis invicla (Red Fire Ant)), Monomorium spp. orthologues of a gene comprising, consisting essentially of, (e.g. Monomorium pharaonis (Pharaoh Ant)), Camponolus or consisting of a nucleotide sequence as represented in any of spp. (e.g. Camponolus spp (CarpenterAnts)), lasius spp. (e.g. SEQ IDNOs 1, 4, 6, 7, 9, 10, 65 to 70, 71 to 79,11,16,17,19, lasius niger (Small Black Ant)), Telramorium spp. (e.g. Tel 20, 80 to 87, 21, 26, 27, 29, 30, 88 to 93, 31, 36, 37, 39, 40, 94 ramorium caespilum (Pavement Ant)), Myrmica spp. (e.g. to 108, 180, 181, 41,43,44,47, 48, 109 to 147, 182 to 187,49, Myrmica rubra (Red Ant)), Formica spp (wood ants), Cre 51, 52, 55, 56, 148 to 150, 57, 63, 64, 151 to 179, and 188 to malogasler spp. (e.g. Cremalogasler lineolala (Acrobat 200. By way of example, orthologous may comprise a nucle Ant)), Iridomyrmex spp. (e.g. Iridomyrmex humilis (Argen otide sequence as represented in any of SEQ ID NOs 71 to tine Ant)), Pheidole spp. (Big HeadedAnts), and Dasymulilla 200, or a fragment ofat least 17, 18, 19, 20, 21, 22, 23, 24, 25, spp. (e.g. Dasymulilla occidentalis (Velvet Ant)); termites 26 or 27 nucleotides thereof. A non-limiting list of insect or (Isoptera and/ or Termilidae) such as but not limited to Amit arachnid orthologous genes or sequences comprising at least ermes spp. (e.g. Amilermes?oridensis ( dark-winged a fragment of 17 nucleotides of one of the sequences of the subterranean termite)), Reliculilermes spp. (e.g. Reliculiler invention is given in Tables 4 and 5. The sequences presented mes ?avipes (the eastern subterranean termite), Reliculiler in Tables 4 and 5 are intended to form part of the present mes hesperus (Western Subterranean Termite)), Coplolermes invention. Thus, orthologues comprise, consist essentially of spp. (e.g. Coplolermesformosanus (Formosan Subterranean or consist of any of the sequences set forth in Tables 4 and 5. Termite)), Incisilermes spp. (e.g. Incisilermes minor (West [0045] According to another aspect, the invention thus ern Drywood Termite)) and Neolermes spp. (e.g. Neolermes encompasses any of the methods described herein for con connexus (Forest Tree Termite)). trolling insect and/or arachnid infestation or infection, com [0038] According to another aspect there is provided a prising contacting insects and/or arachnids with a double nucleic acid molecule comprising, consisting essentially of, stranded RNA, wherein the double-stranded RNA comprises or consisting of the nucleotide sequence as set forth in any one annealed complementary strands, one of which has a nucle ofSEQ ID NOs 1, 4, 6, 7, 9, 10, 65 to 70, 71 to 79,11,16,17, otide sequence which is complementary to at least part of the 19, 20, 80 to 87, 21, 26, 27, 29, 30, 88 to 93, 31,36, 37, 39, 40, nucleotide sequence of a target gene comprising a fragment of 94to 108, 180, 181, 41, 43, 44, 47, 48, 109to 147, 182to 187, at least 17, 18, 19, 20 or 21 nucleotides of any of the 49, 51, 52, 55, 56, 148 to 150, 57, 63, 64, 151 to 179, and 188 sequences as represented in SEQ ID NOs 71 to 200, whereby to 200. the double-stranded RNA is taken up by the insect and/or [0039] SEQ ID NO’s 4, 6, 7, 16, 27, 26, 27, 36 and 37 all arachnid and thereby controls growth, kills or prevents infes represent the nucleotide sequences of speci?c primers which tation or infection by the insect and/ or arachnid. Said insect were utilised to identify the novel sequences of the invention and/or arachnid may comprise, consist essentially of or con in a selective and speci?c manner. sist of any target organisms/ species described herein. [0040] The novel identi?ed genes all represent components [0046] Related nucleic acid molecules encompassed by the of the transcriptional/translational machinery of Blalella ger invention may also be de?ned in terms of hybridisation to a manica. By inhibiting expression of these genes or by inhib nucleic acid molecule comprising the nucleotide sequence of iting expression of the novel identi?ed target genes, through any one ofSEQ ID NOs 1, 4, 6, 7, 9, 10, 65 to 70, 71 to 79,11, RNAi, an important pest may be controlled. 16, 17, 19, 20, 80 to 87, 21, 26, 27,29, 30, 88to 93, 31, 36, 37, [0041] It is predicted, and would be understood by the 39, 40, 94 to 108, 180 and 181. Preferably, the hybridisation skilled person, that also orthologues of these novel target conditions are moderate stringency hybridisation conditions genes represent further targets for down-regulation in the and even more preferably high stringency hybridisation con control of other insect and/or arachnid species. Thus, ortho ditions. Such conditions of moderate and high stringency logues of the novel nucleic acid molecules of the present would be immediately familiar to one of skill in the art. For invention are also contemplated. example, a hybridization reaction incubated at 42° C. in a [0042] Protein or nucleotide sequences are likely to be solution comprising 50% forrnamide, 5>

arachnid species. Thus, orthologues of the novel protein mol [0058] (3) Anoplura: lice, such as Pediculus humanus ecules of the present invention are also contemplated. (human body louse) [0050] In one embodiment, the orthologue will share at [0059] (4) Blattodea: cockroaches including German least about 40% amino acid sequence identity with the amino cockroach (Blalella germanica), of the genus Periplan acid sequence as set forth in any one of SEQ ID NOs 2, 12, 22 eta, including American cockroach (Periplanela ameri or 32. Preferably, the orthologue will share at least about cana) and Australian cockroach (Periplanela auslralia 40%, 50%, 60%, 65%, 70%, 80%, more preferably at least siae), of the genus Blalla, including Oriental cockroach about 90% and even more preferably at least about 96%, 97%, (Blalla orientalis) and of the genus Supella, including 98% or 95% amino acid sequence identity with the amino brown-banded cockroach (Supella longipalpa). A most acid sequence as set forth in any one of SEQ ID NOs 2, 12, 22 preferred target is German cockroach (Blalella ger or 32. manica). [0051] In another embodiment, the invention also provides [0060] (5) Coleoptera: beetles, examples include: the for a nucleic acid encoding a protein comprising the amino family of Powderpost beetle (family of Bostrichoidea); acid sequence as set forth in any one of SEQ ID NOs 2, 12, 22 Dendroclonus spp. (Black Turpentine Beetle, Southern or 32. The nucleic acid molecules encompassed by this aspect Pine Beetle, IPS Engraver Beetle); Carpet Beetles (An of the invention also include those which are functionally Zhrenus spp, Allagenus spp); Old House Borer (family of equivalent in that they encode the same protein molecule. Cerambycidae: Hylolrupes bajulus); Anobium punc Thus, all nucleic acid molecules which are possible due to the Zalum; Tribolium spp (?our beetle); Trogoderma grana degeneracy of the genetic code are intended to fall within the rium (Khapra Beetle); Oryzaephilus sarinamensis scope of this aspect of the invention. (Toothed Grain Beetle) etc. (Bookworm) [0061] (6) Dermaptera: family of earwigs Target Organisms/ Species [0062] (7) Diptera: mosquitoes (Culicidae) and ?ies [0052] The “target species” as used in the present invention, (Brachycera), examples are: Anophelinae such as may be any insect or arachnid species which represents a pest. Anopheles spp. and Culicinae such as Aedes fulvus; The term also relates to the insect or arachnid at any stage in Tabanidae such as Tabanuspunclifer (Horse Fly), Glos its development. Because insects have a non-living exoskel sina morsilans morsilans (tsetse ?y), drain ?ies (Psy eton, they cannot grow at a uniform rate and rather grow in chodidae) and Calyptratae such as Musca domestica stages by periodically shedding their exoskeleton. This pro (House ?y), ?esh ?ies (family of Sarcophagidae) etc. cess is referred to as moulting or ecdysis. The stages between [0063] (8) Heteroptera: bugs, such as Cimex leclularius moults are referred to as “instars” and this stage may be (bed bug) targeted according to the invention. Also, insect eggs or live [0064] (9) Hymenoptera: wasps (Apocrita), including young may also be targeted according to the present inven ants (Formicoidea), bees (Apoidea): Solenopsis invicla tion. All stages in the developmental cycle, which includes (Red Fire Ant), Monomorium pharaonis (Pharaoh Ant), metamorphosis in the pterygotes, may be targeted by RNAi Camponolus spp (Carpenter Ants), Iasius niger (Small according to the present invention. Thus, individual stages Black Ant), letramorium caespilum (Pavement Ant), such as larvae, pupae, nymph etc stages of development may Myrmica rubra (Red Ant), Formica spp (wood ants), all be targeted. Cremalogasler lineolala (Acrobat Ant), Iridomyrmex [0053] The target species may be any insect or arachnid, humilis (Argentine Ant), Pheidole spp. (Big Headed meaning any organism or species belonging to the Kingdom Ants, Dasymulilla occidentalis (Velvet Ant) etc. , more speci?cally to the Phylum Arthropoda, and to [0065] (10) Isoptera: termites, examples include: Amit the Class Insecta or the Class Arachnida. The methods of the ermes ?oridensis (Florida dark-winged subterranean invention are applicable to all insects and arachnids that are termite), the eastern subterranean termite (Reliculiler susceptible to gene silencing by RNA interference and that mes ?avipes), the R. hesperus (Western Subterranean are capable of intemalising double-stranded RNA from their Termite), Coplolermes formosanus (Formosan Subter surrounding environment. ranean Termite), Incisilermes minor (Western Drywood [0054] In one embodiment of the invention, the insect or Termite), Neolermes connexus (Forest Tree Termite) arachnid may belong to the following orders: Acari, Arach and Termilidae nida, Anoplura, Blattodea, Coleoptera, Collembola, Der [0066] (11) Lepidoptera: moths, examples include: maptera, Dictyoptera, Diplura, Diptera, Embioptera, Tineidae & Oecophoridae such as Hneola bisselliella Ephemeroptera, Grylloblatodea, Hem iptera, Heteroptera, (Common Clothes Moth), and Pyralidae such as Pyralis Homoptera, Hymenoptera, Isoptera, Lepidoptera, Mallo farinalis (Meal Moth) etc phaga, Mecoptera, Neuroptera, Odonata, Orthoptera, Phas [0067] (12) Psocoptera: booklice (Psocids) mida, Phithiraptera, Plecoptera, Protura, Psocoptera, Sipho [0068] (13) Siphonaptera: ?eas such as Pulex irritans naptera, Siphunculata, Thysanura, Sternorrhyncha, [0069] (14) Sternorrhyncha: aphids (Aphididae) Strepsiptera, Thysanoptera, Trichoptera, Zoraptera and [0070] (15) Zygentoma: silver?sh, examples are: Ther Zygentoma. mobia domestica and Lepisma saccharina [0055] In preferred, but non-limiting, embodiments of the [0071] Preferred target insects or arachnids include house invention the insect or arachnid is chosen from the group hold insects, ecto-parasites and insects and/or arachnids rel consisting of: evant for public health and hygiene such as, by way of [0056] (1) Acari: mites including Ixodida (ticks) example and not limitation, ?ies, spider mites, thrips, ticks, [0057] (2) Arachnida:Araneae (spiders) and Opiliones red poultry mite, ants, cockroaches, termites, crickets includ (harvestman), examples include: Lalrodeclus maclans ing house-crickets, silver?sh, booklice, beetles, earwigs, (black widow) and Loxosceles recluse (Brown Recluse mosquitos and ?eas. More preferred targets are cockroaches Spider) (Blattodea) such as but not limited to Blalella spp. (e. g. Bla US 2014/0348893 A1 Nov. 27, 2014

Zella germanica (german cockroach)), Periplanela spp. (e.g. red poultry mite, ants, cockroaches, termites, crickets includ Periplanela americana (American cockroach) and Periplan ing house-crickets, silver?sh, booklice, beetles, earwigs, eta auslraliasiae (Australian cockroach)), Blalla spp. (e.g. mosquitos and ?eas. Blalla orientalis (Oriental cockroach)) and Supella spp. (e.g. [0075] Most preferred targets are derived from cock Supella longipalpa (brown-banded cockroach); ants (Formi roaches, for example cockroaches of the genus Blalella, coidea), such as but not limited to Solenopsis spp. (e.g. Sole including German cockroach (Blalella germanica), of the nopsis invicla (Red FireAnt)), Monomorium spp. (e.g. Mono genus Periplanela, including American cockroach (Periplan morium pharaonis (Pharaoh Ant)), Camponolus spp. (e.g. eta americana) and Australian cockroach (Periplanela aus Camponolus spp (Carpenter Ants)), lasius spp. (e.g. lasius Zraliasiae), of the genus Blalla, including Oriental cockroach niger (Small Black Ant)), Telramorium spp. (e.g. Telramo (Blalla orientalis) and of the genus Supella, including brown rium caespilum (Pavement Ant)), Myrmica spp. (e.g. Myr banded cockroach (Supella longipalpa). The most preferred mica rubra (Red Ant)), Formica spp (wood ants), Cremato target is German cockroach (Blalella germanica), in which gasler spp. (e.g. Cremalogasler lineolala (Acrobat Ant)), the novel targets have been identi?ed. Iridomyrmex spp. (e.g. Iridomyrmex humilis (Argentine [0076] It has been previously reported that the formation of Ant)), Pheidole spp. (Big Headed Ants), and Dasymulilla short interfering RNAs (siRNAs) of about 21 bp is desirable spp. (e.g. Dasymulilla occidentalis (Velvet Ant)); termites for effective gene silencing. However, in applications of (Isoptera and/or Termilidae) such as but not limite to Amiler applicant it has been shown that the minimum length of mes spp. (e.g. Amilermes ?oridensis (Florida dark-winged dsRNA preferably is at least about 80-100 bp in order to be subterranean termite)), Reliculilermes spp. (e.g. Reliculiler e?iciently taken up by certain pest organisms. There are indi mes ?avipes (the eastern subterranean termite), Reliculiler cations that in invertebrates such as the free living nematode mes hesperus (Western Subterranean Termite)), Coplolermes C. elegans or the plant parasitic nematode Meloidogyne spp. (e.g. Coplolermesformosanus (Formosan Subterranean incognila these longer fragments are more effective in gene Termite)), Incisilermes spp. (e.g. Incisilermes minor (West silencing, possibly due to a more e?icient uptake of these long ern Drywood Termite)), Neolermes spp. (e. g. Neolermes con dsRNA by the invertebrate. nexus (Forest Tree Termite)). More preferred targets are cock roaches. A most preferred target is German cockroach [0077] It has also recently been suggested that synthetic (Blalella germanica). RNA duplexes consisting of either 27-mer blunt or short hairpin (sh) RNAs with 29 bp stems and 2-nt 3' overhangs are RNA Constructs more potent inducers of RNA interference than conventional 21-mer siRNAs (see Williams, Nature Biotechnology Vol 23, [0072] By “complementary” is meant that the RNA strand 2, February 2005, 181 and Kim et al, Nature Biotechnology represents the RNA equivalent of the speci?ed sequence if Vol 23, 2, February 2005, 222-229 and Siolas et al, Nature that sequence is a DNA sequence or the RNA equivalent of Biotechnology Vol 23, 2, February 2005, 227-231 which ref the complement of the DNA sequence. erences are incorporated herein in their entirety). Thus, mol [0073] The present invention relates to additional targets ecules based upon the targets identi?ed above and being for RNAi mediated down regulation of gene expression. For either 27-mer blunt or short hairpin (sh) RNA’s with 29-bp all targets identi?ed herein, there is provided in a further stems and 2-nt 3' overhangs are also included within the scope aspect of the invention an RNA construct comprising a double of the invention. stranded RNA region, at least one strand of which comprises [0078] Therefore, in one embodiment, the RNA construct a nucleotide sequence that is complementary to a portion of has a double stranded RNA region which has a length of at the nucleotide sequence of any one of any one of (i) the target least about 17 bp, preferably at least about 21 bp, more pref nucleic acid molecules de?ned in any one of SEQ ID NOs 1, erably between about 20-1500 bp, even more preferably 4, 6, 7, 9, 10, 65 to 70, 71 to 79, 11, 16, 17, 19, 20, 80 to 87, between about 80-1000 bp and most preferably between 21,26, 27, 29,30, 88to 93, 31, 36, 37, 39, 40, 94 to 108, 180 about 17-27 bp or between about 80-250 bp; such as double and 181; or (ii) the nucleic acid molecules comprising the stranded RNA regions of about 17 bp, 18 bp, 20 bp, 21 bp, 22 nucleotide sequence as set forth in any one of SEQ ID NOs bp, 23 bp, 24 bp, 25 bp, 26 bp, 27 bp, 50 bp, 80 bp, 100 bp, 150 41, 43, 44, 47, 48, 109 to 147, 182 to 187, 49, 51, 52, 55, 56, bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 148 to 150, 57, 63, 64, 151 to 179, and 188 to 200, or an 550 bp, 600 bp, 650 bp, 700 bp, 900 bp, 100 bp, 1100 bp, 1200 orthologous nucleotide sequence thereof from an insect and/ bp, 1300 bp, 1400 bp or 1500 bp. or arachnid species. As described above, the orthologue may share at least about 50% nucleotide sequence identity with the [0079] The upper limit on the length of the double-stranded nucleotide sequence as set forth in any one of SEQ ID NO’s RNA may be dependent on i) the requirement for the dsRNA 1, 4, 6, 7, 11, 16, 17, 21, 26, 27, 31, 36, 37, 41, 43, 44, 49, 51, to be taken up by the insect and/ or arachnid and ii) the require 52 and 57. Preferably, the orthologue will share at least about ment for the dsRNA to be processed within the relevant cell 70%, 75%, 80%, 85%, 90%, more preferably at least about into fragments that direct RNAi. The chosen length may also 95% and even more preferably at least about 96%, 97%, 98% be in?uenced by the method of synthesis of the RNA and the or 99% sequence identity with the nucleotide sequence as set mode of delivery of the RNA to the cell. Preferably the forth in any one ofSEQ ID NO’s 1, 4, 6, 7, 11, 16, 17, 21, 26, double-stranded RNA to be used in the methods of the inven 27,31,36,37,41,43,44,49, 51,52 and 57. tion will be less than 10,000 bp in length, more preferably [0074] As aforementioned, the orthologues of targets iden 1000 bp or less, more preferably 500 bp or less, more prefer ti?ed herein in Blalella germanica are considered to be viable ably 300 bp or less, more preferably 100 bp or less. targets in other insect and/or arachnid species, including [0080] E?icacy in terms of pest control may be increased household insects and/or arachnids, ecto-parasites and by targeting multiple target genes with a single RNA con insects relevant for public health and hygiene such as, by way struct. Thus, the pest is less likely to survive and acquire of example and not limitation, ?ies, spider mites, thrips, ticks, resistance because there will be multiple double stranded US 2014/0348893 A1 Nov. 27, 2014

RNA’s mediating RNA interference, possibly all at the same or RNA construct comprises, consists essentially of or con time or possibly in a cascaded manner. sists of one or at least two nucleotide sequences indepen [0081] The methods of the invention encompass the simul dently chosen from any of SEQ ID NOs 1, 4, 6, 7, 9, 10, 65 to taneous or sequential provision of two or more different 70, 71 to 79, 11, 16, 17, 19, 20, 80 to 87, 21, 26, 27, 29, 30, 88 double-stranded RNAs or RNA constructs to the same insect to 93, 31, 36, 37, 39, 40, 94 to 108, 180, 181, 41, 43, 44, 47, and/or arachnid, so as to achieve down-regulation or inhibi 48, 109to 147, 182to 187,49, 51, 52, 55, 56, 148to 150, 57, tion of multiple target genes or to achieve a more potent 63, 64, 151 to 179, and 188 to 200, preferably any of SEQ ID inhibition of a single target gene. NOs 65 to 70. [0082] According to a further embodiment, the RNA con [0086] Thus, in one embodiment of the invention, the RNA structs according to the invention comprise at least one construct comprises multiple dsRNA regions, at least one double stranded RNA region, at least one of which comprises strand of each dsRNA region comprising a nucleotide a nucleotide sequence that is complementary to a portion of sequence that is complementary to a portion of the nucleotide any of the nucleotide sequences described herein, wherein the sequence of a target gene from the insect and/or arachnid complementarity of said nucleotide sequence comprises at species. According to the invention, the dsRNA regions in the least 70%, preferably at least 75%, 80%, 85%, 90%, more RNA construct may be complementary to the same or to preferably at least about 95% and even more preferably at different target genes; the dsRNA regions may be comple least about 96%, 97%, 98% or 99% sequence identity with (i) mentary to targets from the same or from different insect the portion of the nucleotide sequence of the nucleic acid species. molecules as set forth in any of SEQ ID NOs 1, 4, 6, 7, 9, 10, [0087] The dsRNA regions may be combined as follows: 65 to 70, 71 to 79, 11, 16, 17, 19, 20, 80 to 87, 21, 26, 27, 29, 30, 88 to 93, 31, 36, 37, 39, 40, 94 to 108, 180 and 181 or (ii) [0088] a) when multiple dsRNA regions targeting a the nucleic acid molecules comprising the nucleotide single target gene are combined, they may be combined in the original order (ie the order in which the fragments sequence as set forth in any one ofSEQ ID NOs 41, 43, 44, 47, appear in the target gene) in the RNA construct, 48, 109 to 147, 182 to 187, 49, 51, 52, 55, 56, 148 to 150, 57, 63, 64, 151 to 179, and 188 to 200, or an orthologous nucle [0089] b) alternatively, the original order of the frag otide sequence thereof from an insect and/or arachnid spe ments may be ignored so that they are scrambled and cies, wherein the percentage sequence identity is calculated combined randomly or deliberately in any order into the over the same length. RNA construct, [0083] With “over the same length” is meant that when % [0090] c) alternatively, one single fragment may be identity is calculated between sequences, this is done over the repeated several times, for example from 1 to 10 times, corresponding stretch of nucleotideds in both sequences. e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times in the RNA [0084] Alternatively, multiple target genes are down regu construct, or lated by the provision of one double-stranded RNA that acts [0091] d) the dsRNA regions (targeting a single or dif against multiple target sequences. Alternatively, a single tar ferent target genes) may be combined in the sense or get may be more ef?ciently inhibited by the presence of more antisense orientation. than one copy of the double stranded RNA fragment corre [0092] In addition, the target gene(s) to be combined may sponding to the target gene. Thus, in one embodiment of the be chosen from one or more of the following categories of invention, the double-stranded RNA construct comprises genes (including all possible combinations threof, as appro multiple dsRNA regions, at least one strand of each dsRNA priate): region comprising a nucleotide sequence that is complemen tary to at least part of a target nucleotide sequence of an insect [0093] e) “essential” genes or “pathogenicty genes” and/or arachnid target gene. According to the invention, the encompass genes that are vital for one or more target dsRNA regions in the RNA construct may be complementary insects and/or arachnids and result in a lethal or severe to the same or to different target genes and/ or the dsRNA (e.g. movement, feeding, paralysis, drinking, fertility, regions may be complementary to target genes from the same reproduction, growth) phenotype when silenced. The or from different insect and/or arachnid species. choice of a strong lethal target gene results in a potent RNAi effect. In the RNA constructs of the invention, [0085] Accordingly, the invention provides an isolated multiple dsRNA regions targeting the same or different double stranded RNA or RNA construct of the invention (very effective) lethal genes can be combined to further which comprises at least two double stranded RNA regions, at increase the potency, e?icacy or speed of the RNAi least one strand of each of which comprises, consists essen effect in insect and/or arachnid control. tially of, or consists of a nucleotide sequence that is comple mentary to a portion of the nucleotide sequence of any one of [0094] f) “weak” genes encompass target genes with a SEQ IDNOs 1, 4, 6, 7, 9, 10, 65 to 70, 71 to 79,11,16,17,19, particularly interesting function in one of the cellular 20, 80 to 87, 21, 26, 27, 29, 30, 88 to 93, 31, 36, 37, 39, 40, 94 pathways described herein, but which result in a weak to 108, 180, 181, 41, 43,44,47,48, 109to 147, 182to 187,49, phenotypic effect when silenced independently. In the 51, 52, 55, 56, 148 to 150, 57, 63, 64, 151 to 179, and 188 to RNA constructs of the invention, multiple dsRNA 200, or an orthologous nucleotide sequence from an insect regions targeting a single or different weak gene(s) may and/ or arachnid species, wherein the orthologous nucleotide be combined to obtain a stronger RNAi effect. sequence has at least 70%, 80%, 85%, 87.5%, 90%, 95% or at [0095] g) “insect and/or arachnid speci?c” genes encom least 99% sequence identity with at least the relevant portion pass genes and portions of genes that have no substan of the nucleotide sequence of any one of SEQ ID NOs 1, 11, tially homologous counterpart in non-pest organisms as 21 and 31 or the nucleic acid molecules comprising the nucle can be determined by bioinformatics homology otide sequence as set forth in any one of SEQ ID NOs 41, 43, searches, for example by BLAST searches. The choice 44, 49, 51, 52 and 57. Preferably aside double stranded RNA of an insect and/ or arachnid speci?c target gene or por