WO2017030449A1 - Viruses for biocontrol of argentine ants - Google Patents

Abstract

Provided are Argentine ant viruses, nucleic acid molecules identifying them, compositions comprising them and methods of using them for biocontrol.

Description

VIRUSES FOR BIOCONTROL OF ARGENTINE ANTS

[0001] This invention claims priority from New Zealand Provisional Application number NZ 711268 filed on the 20^th of August 2016, the contents of which are hereby incorporated in their entirety.

[0002] A sequence listing has been filed in electronic form concurrently with this application. This sequence listing forms part of the disclosure of this application.

FIELD OF THE INVENTION

[0003] This invention relates to invertebrate viruses, nucleic acids characterising invertebrate viruses, biocontrol compositions, and methods of using the viruses and/or compositions in the control of invertebrate populations, in particular social insect populations such as ants and wasps. BACKGROUND OF THE INVENTION

[0004] Argentine ants (Linepithema humile) are one of the six most globally widespread, abundant and damaging invasive ant species (Holway et al. 2002). They frequently form large colonies with the interchange of workers between nests over a wide area. Such behaviours could substantially increase the probability of disease transmission and facilitate rapid epidemics (Ugelvig & Cremer 2012). Population collapse has been observed with Argentine ants and pathogens were

hypothesised as a potential mechanism (Cooling et al. 2012). However, with a few exceptions (Valles et al, 2007, 2014, 2016), no viral species have been described for Argentine ants or other invasive ants.

[0005] U.S. Patent No. 7,332,176 and U.S. Patent No. 8034333 disclose a virus effective against fire ants (Solenopsis invicta).

[0006] There remains a need for further pathogens, biocontrol and/or microbial control agents against invertebrate species, in particular against invasive ant colonies such as Argentine ants.

[0007] Further objects and advantages of the present invention will become apparent from the following description. One such object is to provide the public with a useful choice. SUMMARY OF THE INVENTION

[0008] The present invention relates to novel viruses identified in Argentine ant (Linepithema humile) populations. These viruses have been identified by analysis of the metagenome of various populations of Argentine Ants. Analysis of the RNA metagenome has in particular resulted in several nucleotide sequences which characterise viruses present in the ants. [0009] The present invention particularly relates to the virus named herein as Linepithema humile virus -1, or LHUV-1, nucleic acid sequences derived therefrom, compositions comprising LHUV-1 and methods and uses of LHUV-1.

[00010] In a first aspect the present invention relates to an isolated nucleic molecule. In one embodiment the nucleic acid molecule may comprise a sequence which is at least about 50%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5. In preferred embodiments the nucleic acid molecule is 100% identical to SEQ ID NO: 5.

[00011] In another embodiment the nucleic acid is at least about 50%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more preferably 100% identical to SEQ ID NO: 1.

[00012] In a still further embodiment the nucleic acid molecule is at least about 50%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, or more preferably 100% identical to SEQ ID NO: 6. [00013] In a second aspect the invention relates to a virus, preferably an isolated virus, comprising a nucleic acid molecule as described herein. The virus preferably infects Linepithema humile.

Preferably the virus is an NA virus, more preferably a single stranded RNA virus. The virus may be of the Dicistroviridae family. Still more preferably the virus does not infect honey bees.

[00014] The viruses described herein can be formulated for use as a biocontrol agent against invasive or undesirable invertebrates, in particular against social insects such as ants and wasps. Thus in a third aspect the invention relates to a composition comprising the virus described herein and a carrier. In one embodiment the composition comprises a bait matrix, particularly a bait matrix which is attractive to the invertebrate to be controlled and is preferably unattractive to other animals. In a particular embodiment the matrix is attractive to ants, and in particular Argentine ants but not to honey bees. In certain embodiments the matrix is Xstinguish™.

[00015] In certain embodiments the composition may comprise one or more additional active ingredient. Potential additional active ingredients include biological compounds, such as other viruses, bacteria or fungi, inorganic insecticides or organic insecticides such as fipronil.

[00016] In a fourth aspect, the present invention provides for a method of controlling or eradicating an invertebrate population comprising administering an effective amount of a virus or composition as described herein. In one embodiment the virus is administered by way of a bait system, which is preferably set in the vicinity of the invertebrate population. In particular the invertebrate population is a social insect population, such as an ant population, more particularly an Argentine ant population or a wasp population.

[00017] In a fifth aspect the present invention provides for the use of a virus or composition as described herein for controlling or eradicating an invertebrate population, in particular a social insect population. In particular the population is an ant population, more particularly an Argentine ant population.

[00018] The present invention is also directed to tools for analysing the presence and replication of the viruses described herein. Thus in a sixth aspect the invention provides for primers suitable for amplifying a nucleic acid with a sequence of SEQ ID NO: 1 and SEQ ID NO: 5. In certain embodiments the primers are of SEQ ID NO: 7, 8 , 9, 10 , 11 or 12, 13 or 14. In particular the primers may be tagged primers suitable for use in a T-PC protocol. In one embodiment the invention provides a kit comprising a forward and reverse primer according to the above. In a still further embodiment the invention provides for a virus identifiable by a primer having a sequence of SEQ ID NO: 7, 8 , 9, 10 , 11 or 12, 13 or 14.

[00019] The metagenomics analysis has also shown that other viruses are present in certain populations of ants. Thus also described herein are nucleic acid molecules, preferably isolated nucleic acid molecules, comprising a nucleotide sequence which is at least about 50% identical to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In certain embodiments the nucleotide sequence is at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. Also provided are (isolated) viruses comprising a nucleic acid molecule comprising those sequence, compositions comprising the viruses and methods and uses thereof in accordance with the descriptions provided herein.

[00020] In addition the Deformed wing virus (DWV), which is known to be present in bee populations has been shown to replicate in Argentine Ants in New Zealand. Thus also described is a method for the control or eradication of an ant population, preferably an Argentine ant population comprising administering an effective amount of DWV or a composition comprising DWV.

BRIEF DESCRIPTION OF THE DRAWINGS

[00021] FIG. 1 shows (a) the percentage of the RNA metagenome classified into each domain;

including viruses (complexity= 0.30; bit score= 50) from Argentine ants in New Zealand, (b)

Sequences from the RNA metagenome that were assigned to a viral family, (c) Maximum likelihood phylogenetic analysis of Dicistroviridae including the novel LHUV-1 virus: sP, partial structural protein; stpoly, partial structural polyprotein; capsid, partial capsid protein, (d) A maximum likelihood phylogenetic analysis of Deformed wing virus (DWV) based on nucleotide sequences (255 bases). DWV sequences amplified from Argentine ant (Linepithema humile) samples are shown in bold.* Sequences were obtained from Lester et al. (2015).

[00022] FIG. 2 shows the distribution of the honey bee DWV, the novel RNA dicistrovirus LHUV-1 (contig n6409), and the three contigs nlOOO, nl905 and nl050, in (a) Argentina, (b) Australia, and (c) New Zealand.

[00023] FIG. 3 shows the maximum likelihood tree of Linepithema humile virus -1 (LHUV-1) protein sequences after 1000 bootstraps of the WAG model with Uniform rates in MEGA 6.06. The original LHUV-1 (partial contig n6409) from the preliminary metagenomics analysis is shown uppermost. Sequences obtained from one step RT-PCRs in this study are in bold black. Sequences with* were the best hits of LHUV-1 after BLASTN search on GenBank and corresponded to two dicistroviruses, Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV). Country of origin (New Zealand- NZ, Canada, and United States- USA) and host organisms (Argentine ants- L. humile, and honey bees- Apis mellifera) are indicated for each sequence. [00024] FIG. 4 shows the maximum likelihood tree of n 1000 nucleotide sequences after 1000 bootstraps of the Tamura-3 parameter model with Uniform rates (T92) in MEGA 6.06. The original (partial) contig nlOOO from the preliminary metagenomics analysis is uppermost. Sequences obtained from one step RT-PCRs in this study are in bold black. Sequences with* were the best hits of nlOOO after BLASTN search on GenBank and corresponded to the dicistrovirus Israeli acute paralysis virus (IAPV). Country of origin (New Zealand- NZ, Australia, and Korea) and host organisms (Argentine ants- L. humile, and honey bees- Apis mellifera) are indicated for each sequence.

[00025] FIG. 5 shows the maximum likelihood tree of nl050 nucleotide sequences after 1000 bootstraps of the Tamura-3 parameter model with Uniform rates (T92) in MEGA 6.06. The original (partial) contig nl050 from the preliminary metagenomics analysis is the fourth from the top.

Sequences obtained from one step RT- PCRs in this study are in bold black. Sequence with* was the best hits of nlOOO after BLASTN search on GenBank and corresponded to the dicistrovirus Solenopsis invicta virus 2 (SINV-2). Country of origin (New Zealand- NZ, Australia, and United States- USA) and host organisms (Argentine ants- L. humile, and red imported fire ants- S. invicta) are indicated for each sequence.

[00026] Fig. 6 shows the maximum likelihood tree of RNA metagenome (partial) contig nl905 nucleotide sequences with 1000 bootstraps of the Tamura-3 parameter model with uniform rates (T92) in MEGA 6.06. The original RNA metagenome contig nl905 from the preliminary

metagenomics analysis is in bold red. Sequences obtained from one step RT-PCRs in this study are in bold black. Sequences with * were among the best hits of contig nl905 from BLASTX searches on GenBank and corresponded to the dicistrovirus Acute bee paralysis virus (ABPV). Country of origin (New Zealand - NZ, United Kingdom- UK) and host organisms (Argentine ants - L. humile, and honey bees - Apis mellifera) are indicated for each sequence.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[00027] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word

"comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated feature but not to preclude the presence or addition of further features in various embodiments of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[00028] For purposes of the present invention, the term "Argentine ant" and "Linepithema humile" are used interchangeably to describe the Argentine ant, originating in South America, but now widespread throughout the world including New Zealand.

[00029] For purposes of the present invention, the term "isolated" in the context of a virus is defined as separated from other viruses found in naturally occurring organisms. In the context of a nucleic acid molecule, the term "isolated" is defined as separated from other nucleic acid molecules and other non-nucleic acid structures found in a cell or virus.

[00030] A "nucleic acid molecule" means a biopolymer made from nucleotide monomers and includes but is not limited to DNA and RNA. Where a nucleic acid molecule is defined by sequence, it is to be understood that the well understood modifications to alter a DNA sequence to an RNA sequence or vice versa are also within the scope of the nucleic acid molecule. For example, it is to be understood that reference to a nucleic acid molecule defined by a DNA sequence also refers to an RNA molecule of the same sequence, but with uracil (u) in the place of thymine (t).

[00031] The term "effective amount" or "amount effective for" as used herein means that minimum amount of a virus or composition needed to control, reduce, or substantially eradicate ants in an ant colony when compared to the same colony or other colony which is untreated. The precise amount needed will vary in accordance with the particular virus or composition used; the colony to be treated; and the environment in which the colony is located. The exact amount of virus composition needed can easily be determined by one having ordinary skill in the art given the teachings of the present specification.

[00032] The term "carrier" when used in the context of the compositions described herein refers to any component which enables delivery of the virus to an invertebrate or invertebrate population. A carrier may be solid or liquid. A carrier may perform a dual purpose. For instance it may also serve as an attractant for the invertebrate to be controlled or eradicated, or a deterrent for species not desired to be controlled. The term "carrier" is not intended to exclude such additional purposes.

[00033] As used herein the term "identical", or "identity" as used in relation to amino acid or nucleotide sequences refers to a pairwise sequence identity (%). the relationship between two or more amino acid or nucleic acid sequences, as determined by aligning the sequences. Within the present invention, sequence identity with a particular sequence preferably means sequence identity over the entire length of said particular sequence in optimal alignment. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

[00034] Optimal alignment is the alignment in which the percentage sequence identity is the highest possible. It can be achieved in various ways using computer programs. One preferable method is the Needleman-Wunsch Global Alignment method (Needleman & Wunsch, 1970). A computer algorithm running Needle-Wunsch, such as that available from the National Center for

Biotechnology Information at https://blast.ncbi.nlm.nih.gov/Blast.cgi can be used to obtain an optimal alignment and calculate the percentage of sequence identity.

[00035] An "active ingredient", as used herein, refers to a compound which, when administered in an effective amount has a deleterious effect on an invertebrate population, thereby resulting in control or eradication of the population. Viruses

[00036] Described herein for the first time are viruses in one of the worlds most widespread, abundant and damaging invasive ants. One virus particularly described is the novel Linepithema humile virus-1 (LHUV-1). This virus is an ss NA virus of the Dicistroviridae family. The presence of LHUV-1 in Argentine ant populations has been established in New Zealand, Australia and the ant's native range. Given that New Zealand is at the end of the invasion pathway, LHUV-1 is highly likely to be present in all populations of the ant. [00037] The LHUV-1 virus has been shown to have a capsid region encoded by SEQ ID NO: 1. Since the capsid region is highly conserved it can serve for provisional taxonomic categorisation when incorporated into a phylogenetic analysis and thus is also suitable for the identification of the particular virus. T-PC has further confirmed the region of LHUV-1 encoded by SEQ ID NO: 5 Genbank ID: KT713624.2.

[00038] An extended sequence for LHUV-1 was prepared by using the Iterative Viral Assembler (Hunt et al. 2015) and is provided as SEQ ID NO: 6.

[00039] Also described are other potential RNA viral species, identified by contigs nlOOO (SEQ ID NO: 2), nl905 (SEQ ID NO: 3) and nl050 (SEQ ID NO: 4). Similar to LHUV-1, nlOOO, nl050 and nl905 have been found to be present in multiple populations (Figs 4, 5 and 6 respectively).

[00040] The full genome of LHUV-1 and the other viruses can be constructed using the RACE system (Invitrogen, Carlsbad, CA) as described by Valles et al. 2016, using the identified contigs.

[00041] While the viruses described above have been identified as being present in Argentine ants, it is expected that other ants, or indeed other invertebrates, will also be susceptible to infection with these viruses. One of the other viruses identified in the Argentine ant populations was the Deformed wing virus, which is a common virus found in insects such as honey bees, wasps and bumble bees. Thus it is expected that the RNA viruses described herein may show similar host specificity

[00042] Deformed wing virus (DWV) is a widespread bee pathogen associated with honey bee colony collapse, which has been shown for the first time to also be present in Argentine ants. Preparation and Isolation

[00043] The formulation of the viruses for administration can be undertaken by various methods known in the art. One method is to grind up known infected ants, and administer the ground up ants to the population to be controlled. The ground up ants can be included in a food media or other bait composition, which is fed to the population to be controlled. One approach is demonstrated by In Valles et al. 2013, in which viral extractions of the Solenopsis invicta virus-3 (SINV-3) were obtained by homogenising virus-positive ant colonies in a blender. The blended colonies were suspended in either a sugar or oil solution, and filtered. The filtrates were then added to either a 10% sugar solution, an insect paste consisting of homogenised crickets, and soybean oil adsorbed to defatted corn grit. These three baits were then feed to virus-negative colonies. All three baits delivered the virus and successfully infected ants.

[00044] Another method of preparing the viruses is to prepare them in an insect cell line. There are a variety of insect cell lines available that can be grown in the laboratory and commercial environment (Goblirsch et al. 2013, Boyapelle et al. 2007). Cell lines for the culture of viruses disclosed herein could be investigated using the methods of Goblirsch et al. 2013. These authors developed a honey bee cell culture, based on the collection and homogenisation of honey bee eggs. They used a basal solution of Leibovitz's L15 medium (Life Technologies, Grand Island, NY) modified to include the addition of glucose, organic acids, vitamins, trace minerals and amino acids.

[00045] Preferably, the virus is introduced into a cell line, allowed to reproduce, and the cells harvested and incorporated into a composition, which can then be spread into the environment. As many related viruses are spread between hosts by foraging on the same flowers, the virus is expected to have a reasonable viability outside of the host. [00046] Another method of preparing and isolating the viruses of the present invention is by way of transgenic expression system. In particular a baculovirus expression system is particularly suitable for expression of viruses of this type (Pal et al. 2007). Various methods of isolating the viruses are known in the art, for instance by sucrose gradient centrifugation (Krishna et al. 2003), or by isopycnic centrifugation using CsCI (Valles et al. 2016). Methods of controlling invertebrate populations

[00047] The viruses described herein can be used as biocontrol agents against invertebrate species and especially ants, honey bees, wasps, bumble bees etc. Thus the invention provides methods of controlling or eradicating an invertebrate population comprising administering an effective amount of a virus or composition as described herein. [00048] Viruses related to LHUV-1 can devastate insect populations, including those associated with colony collapse disorder in honey bees (Schroeder et al. 2012). Relatives of LHUV-1 are known to exert substantial effects in the widespread and invasive Red imported fire ant, Solenopsis invicta. Six viruses have been described from the Red imported fire ants and are currently being considered as potential biological control agents (Valles 2007, 2014). LHUV-1 and the other viruses disclosed herein are therefore possible causes for the population declines that have been observed in Argentine ants (Cooling et al. 2012) and have potential as a biological control agents.

[00049] Moreover, DWV, which is known to be associated with honey bee collapse has been shown for the first time to be present and replicating only in New Zealand Argentine ant populations. Thus it is possible that the presence of this virus is a cause of population decline in Argentine ants.

Accordingly, DWV may also have potential uses as a biological control agent against invertebrates, and in particular against ants such as Argentine ant. [00050] The collapse of ant populations is relatively common, with evidence they occur in nearly all invasive ant populations. One proposed model for invasive ant collapse is the acquisition of pathogens over time, combined with issues of low genetic diversity, which contribute to this collapse (Lester and Gruber 2016). [00051] Thus the viruses may not need to be lethal in order to be considered for biocontrol. Viruses of Solenopsis invicta (SINV) can cause direct mortality or have been associated with mortality when infected ant colonies experience stress (Valles et al. 2007, 2014). Interspecific interactions, including competition, are frequently thought to be a major driver of ant community assembly and population dynamics. Populations infected with pathogens may be less competitive, obtaining less food, and be excluded from other resources, resulting in reduced or negative colony growth rates.

[00052] Species such as Argentine ants are known to have a low competitive ability in interactions with native species, with competitive dominance achieved only when the invader is present in overwhelming numbers (Sagata & Lester 2009). Thus factors, such as a pathogen, that lower the competitive dominance of Argentine ants or other invertebrates could have a dramatic effect on ant population dynamics. This effect may be the equivalent of the 'extinction vortex' dynamics observed in conservation biology, whereby low genetic diversity results in low breeding success that leads to a further reduction in genetic diversity, and so on (Gilpin & Soule 1986).

[00053] Thus an effective amount of a virus as described herein need not be enough to kill the invertebrate population, but rather enough to reduce an invasive population's competitive advantage over other ant species present in the community. The reduced competitive ability of the infected ant population may limit the ability to acquire resources and result in reduced or negative colony growth rates.

[00054] The method may further comprise administration of one or more further active ingredients. Administration of the further active ingredient may be separate, simultaneous or sequential with the administration of the virus. These active ingredients may have a deleterious effect on the invertebrate population in their own right, or they may stimulate an improvement in the

effectiveness of the virus. For example the further active ingredient may act as a stressor which stimulates the viral replication necessary to achieve the control or eradication, thereby resulting in a synergistic effect. Suitable additional active ingredients include include Chlorfenapyr, Imidacloprid, Fipronil, Hydramethylnon, Sulfluramid, Hexaflumuron, Pyriproxyfen, methoprene, lufenuron, dimilin, Chlorpyrifos, and their active derivatives, Neem, azadiractin, and boric acid based toxins. For convenience it is preferable that the virus and the other active ingredient are combined into a single delivery composition. [00055] The mode of administration of the viruses will depend on the nature of the invertebrate to be targeted. Various modes of administration of insecticides are known in the art, including direct application to a colony, aerosol spray, bait system, incorporation into watering or irrigation systems, distribution of solid granules, etc. Known modes of administration of insecticides with formulation changes should be suitable for delivery of the viruses.

[00056] The viruses of the invention have the ability to replicate and thereby infect other members of the population. Accordingly, unlike conventional insecticides, it is only necessary that the delivery mechanism allows for infection of one or a small number of individuals who will subsequently spread it to the remainder of the population. This is particularly true for invertebrates who live in a hive- or nest- type colony where the queen is primarily responsible for reproduction but takes little or no part in foraging activities. In these circumstances infection of a worker individual can result in subsequent infection of the entire hive or nest, and especially the queen, thereby resulting in efficient control, or even eradication of the population. This makes bait delivery systems particularly suitable for this type of invertebrate. [00057] An alternative approach of pathogen delivery is to infect an insect with a pathogen and introduce it directly back into a population thereby making a 'living insect bomb' (Gumus et al.

2015). Thus, another method of delivery of the viruses of the present invention is to infect an invertebrate with the virus and reintroduce it back into the population, thereby facilitating the spread of the virus. [00058] In the case where the host range of the virus is broad, it may be necessary to tailor the mode of administration to ensure only the undesirable or invasive species is targeted. Argentine ants are typically found in massive densities that exclude other ant species, especially native ant species. Therefore, it is possible to target the Argentine ants directly, with few non-target effects.

[00059] However, Argentine ants are known to co-locate with honey bees. Thus, of particular concern, is a non-target effect on honey bee populations. Honey bees are responsible not only for a substantial apiculture industry, but in New Zealand are also important pollinators of native plant communities.

[00060] The Deformed Wing Virus is shown to be present in both Argentine ants and honey bees and has been implicated in population collapse of honey bees. DWV may thus not be suitable for biocontrol of Argentine ant populations which a co-located with honey bee populations. In contrast, the LHUV-1 virus has not been observed in honey bees, despite it being present in co-located ant populations. It is therefore likely LHUV-1 does not infect honey bees. The use of LHUV-1 in a biocontrol mechanism would therefore avoid non-target effects in honey bees. [00061] The formulation of the delivery composition will also be relevant for both effective delivery and host targeting.

Delivery Composition

[00062] The viruses described herein are formulated with a carrier into a composition. The carrier can be a liquid or a solid material and is an inert, non-repellent (to the species desired to be controlled) carrier for delivering the composition to a desired site.

[00063] In certain cases the composition comprises a bait matrix. A suitable bait matrix should be one to which the foraging insects are attracted, and which they will feed on and carry back to the nest and feed it to larvae, queens and nest mates. The bait matrix should be tailored to the target species and can comprise a balance of protein, carbohydrate and lipids suited to the target insect's seasonal food requirements. A bait matrix comprising soybean oil on corn grits has been used in against S. invicta since the 1960s (Lofgren et al. 1963; Williams et al. 2001).

[00064] An attractant in a bait matrix or composition can be targeted to the invertebrate in question and may comprise lipids such as soybean oil, proteins such as ground silkworm pupae,

carbohydrates, for example in the form of sugar water solutions, or a combination of these.

[00065] Solid or granular bait matrices are particularly suitable for large scale ground or aerial broadcast of the virus, while liquid baits are more suitable for small infestations. Liquids suitable as carriers include water, and any liquid which will not affect the viability of the viruses of the present invention. [00066] Preferably, the bait matrix is one which has been developed for Argentine ants, such as the Xstinguish^® matrix. The Xstinguish^® matrix is a wet matrix which comprises cooked egg and sucrose. Importantly, this matrix is unappealing to bees and highly appealing to Argentine ants and other species previously thought difficult to attract with baits other than sweet liquids. Species such as invasive social wasps are also attracted to this matrix. [00067] Where administration of a further active ingredient is desired, the composition may further include such active ingredient. Further active ingredients include biological compounds, organic insectides or inorganic insecticides. Thus the composition or bait matrix may also include toxins or further active ingredients such as Chlorfenapyr, Imidacloprid, Fipronil, Hydramethylnon, Sulfluramid, Hexaflumuron, Pyriproxyfen, methoprene, lufenuron, dimilin, Chlorpyrifos, and their active derivatives, Neem, azadiractin, and boric acid based toxins. In these cases the formulation of the composition will necessarily take into account the properties of the additional active ingredient. [00068] The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention as described herein.

EXAMPLE 1 - Metagenomic discovery of viruses

[00069] Argentine ant workers were collected in 2013 from two nests in Wellington, New Zealand (41.2218°S, 174.8724°E). Each sample included 30 ants homogenised using pestles in Eppendorf tubes containing 400 μί iPrep PureLink Virus kit lysis buffer (Life Technologies, Carlsbad, CA, USA). 400 μί of molecular grade water and 50 μί proteinase K was then added to each sample and incubated at 50°C for 1 hour, followed by 96°C for 5 minutes. Samples were then centrifuged at 12,000 x g for 5 minutes. The supernatant was removed and used in the extraction along with the iPrep Purelink Virus kit (Life Technologies, Carlsbad, CA, USA), eluting into 50 μί of RT-PCR molecular grade water (Ambion, Austin, TX, USA). The extractions were combined into a single sample to increase nucleic acid amount.

[00070] To detect RNA viruses, DNA was removed using DNAse treatment by Ambion DNA- free (Life Technologies) from half of the combined extractions. 8 μί of DNA-free RNA was incorporated into first-strand cDNA synthesis (Life Technologies) including RNase H digestion. To ensure > 1 μg of DNA for library preparation, the DNA and cDNA were amplified by multiple displacement amplification using the Whole Transcriptome Amplification kit (Qjagen). Sequencing libraries were then prepared with the lllumina TruSeq DNA library preparation kit followed by sequencing on an lllumina MiSeq instrument producing 250 bp paired-end reads. [00071] The quality of the sequence data was examined using Fast QC. Reads were trimmed when the average Phred score was <30. Duplicate reads were collapsed using FASTX-Toolkit 0.10.1. Velvet 1.2.07 (Zerbino & Birney, 2008) was used for de novo assembly of the trimmed sequence data with a k-mer of 75. Contigs were searched against the GenBank non-redundant nucleotide sequence database using BLASTN and BLASTX (BLAST+ 2.2.27 (Altschul et al. 1990)) with an e-value threshold of 0.001. Taxonomic assignment of BLASTN outputs was undertaken using M EGAN 4.7 (Huson et al. 2007) with a threshold criteria for inclusion of bit score >50 and contig sequence complexity >0.44.

[00072] A total of 139,943 contigs were assembled for the RNA metagenome and 8,594 for the DNA metagenome. No viruses of significance were found in the DNA metagenome (data not shown). A BLASTN search of the RNA metagenome contigs revealed 41 contigs similar to viral sequences in the Dicistroviridae family (figure la,b). Of these, four contigs were considered to be of particular interest, contig n6409 (SEQ ID No: 1), contig nlOOO (SEQ ID NO:2), contig nl905 (SEQ ID NO: 3) and contig nl050 (SEQ ID NO: 4). [00073] Table 1 gives the 10 closest matches, ordered by the highest score for each contig of interest after BLASTN.

Table 1

KBV KF956377 Virus in honey

737 737 97 0.0 89

bees (A. rnelliferd)

KBV AY787143 Vims in honey

735 735 71 0.0 98

bees (A. me.llife.ra)

KBV KC513761 Virus In honey

690 690 64 0.0 99

bees (A. rnelliferd)

KBV AF177935 Virus in honey

683 683 74 0.0 94

bees (A. mellifera)

KBV AF093457 Virus in mites

681 681 71 0.0 95 ( Varroa jacobsoni)

KBV AF034542 Virus in honey

681 681 71 0,0 95 bees (A. mellifera)

KBV AF135854 Virus in honey

675 675 71 0.0 95 bees (A. mellifera)

KBV AY275710 Virus used to infect

4e-

81.4 81.4 7 80 honey bees (A.

11 mellifera)

ABPV 6e- AY053368 Virus in honey

64.1 64.1 n 78

06 bees (A. mellifera)

ABPV 6e- AY053367 Virus in honey

64.1 64.1 7 78

06 bees (A. mellifera)

ABPV 6e- AY053366 V us in honey

64.1 64.1 n 1 78

06 bees (A. mellifera)

IAPV 2e- JX045857 Virus in honey

62.2 62.2 6 78

05 bees (A. mellifera)

Virus in bat guano

(Tadarida brasiliensis, Myotis n6409 / 2e-

ABPV 62.2 62.2 6 78 HM228893 velifer, Nycticeus LHUV- 05 humeralis, 1

Pe rimy otis subflavus)

Virus in bat guano

(Tadarida brasiliensis, Myotis

2e-

ABPV 62.2 62.2 6 78 HM228890 velifer, Nycticeus

05 humeralis,

Pe rimy otis subflavus)

IAPV 2e- EU436455 Virus in honey

62.2 62.2 6 78

05 bees (A. mellifera)

IAPV 2ε- EU436528 Vhus in honey

62.2 62.2 6 78

05 bees (A. mellifera)

IAPV 2e- EU436527 Virus in honey

62.2 62.2 6 78

05 bees (A. mellifera)

SINV-2 EF428566 Vims in fire ants nl050 56.4 56.4 6 0.002 74

(Solenopsis invicta) Acyrthosiphon XM_0G1943802 Aphids mR A

50.7 50.7 1

pisum 0.096 94

Carica papaya 48.8 48.8 ! 0.36 96 KM397499 Papayas mRNA

Shewanella CP000606 Bacteria genome

48.8 48.8 1 0.36 94

loihica

Oryzias latipes 46.8 46.8 1 1.4 100 HG 314000 Fish genome

Hymenolepis LM385892 Tapeworms diminuta 46.8 46.8 1 1.4 96 genome

Cyprinus LN594801 Common carp carpio 46.8 46.8 1 1.4 91 genome

Dictyosteliutn XM...629989 Amoeba mRNA dis oideum 46.8 46.8 1 1 .4 96

Ceratitis XM_. 004522381 Fruit flies mRNA

9 1 5.2

capitata 44.9 44. 93

Staphylococcus CP009554.1 Bacteria genome

44.9 44.9 1 5.2 96

aureus

[00074] Table 2 gives 10 closest matches, ordered by the highest score for each contig of interest after BLASTX search on Genbank. Abbreviations correspond to: Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV) and Solenopsis invicta virus-2 (SINV2). Table 2

le- Virus polymerase

IAPV 565 565 41 76 AGL33499

174 polyprotein le- Virus non-structural

IAPV 565 565 41 75 AGF84784

polyprotein

KBV 388 388 99 4e- 96 NPJ351403 Virus non-structural

121 polyprotein

KBV 383 383 99 2e~ 94 AHL83499 Virus non-structural

1 19 polyprotein

IAPV 382 382 99 4e- 94 ACD01403 Virus polymerase

1 19 polyprotein

IAPV 380 380 99 4ε- 94 ACD01401 Virus polymerase

1 18 polyprotein

IAPV 380 380 99 4e- 94 ABY57949 Virus non-structural nl905

1 18 polyprotein

IAPV 379 379 99 Se94 YP_ _.001040002 Virus polymerase l l s polyprotein

IAPV 379 379 99 Se- 93 AGL33499 Virus polymerase

1 18 polyprotein

IAPV 379 379 99 Se- 93 AEL12438 Virus polymerase

1 18 polyprotein

IAPV 379 379 99 6e- 93 ACD01399 Virus polymerase

1 18 polyprotein

KBV 393 393 93 le- 46 NPJ351404 Virus structural

123 polyprotein

IAPV 383 383 95 3e- 44 AGF84785 Virus structural

121 polyprotein

IAPV 384 384 95 3e- 44 ACD01404 Virus structural

120 polyprotein

KBV 380 380 90 4e- 46 AAR 19088 Virus structural

120 polyprotein

IAPV 384 384 95 6e- 44 ABY57950 Virus structural n6409

120 polyprotein

IAPV 383 383 95 l e- 44 ACD01400 Virus structural

1 19 polyprotein

IAPV 383 383 95 2e~ 44 ABY71757 Virus structural

1 19 polyprotein

IAPV 382 382 95 2e- 44 AGL33500 Virus structural

1 19 polyprotein

IAPV 382 382 95 3e~ 44 AEL 12439 Virus structural

1 19 polyprotein IAPV 382 382 95 3e- 44 ACD01402 Virus structural

1 19 polyprotein

SINV2 452 452 le- 50 YP_001285729 Virus non-structural

136 protein

Bovine 84.0 84.0 21 4e- 34 NP_859024 Virus non-structural kobuvirus 14 protein 2C

Salivirus 81.3 81 .3 23 3e- 32 YP_003038640 Virus non-structural

13 protein 2C

Caprine 80.9 80,9 21 4e- 31 YP...009001376 Virus non-structural kobuvirus 13 protein 2C

Porcine 80.5 80.5 21 5ε- 33 YP.002473940 Virus non-structural kobuvirus 13 protein 2C swine

nl050 Bovine 82.8 82.8 20 7e~ 35 NP_..740257 Virus hypothetical kobuvirus 13 protein

Kobuvirus 82.8 82,8 20 8e- 35 ADG03747 Virus polyprotein sheep 13

Porcine 82.4 82.4 26 9e- 31 AHY02128 Virus polyprotein kobuvirus 13

Kobuvirus 82.4 82.4 28 9e- 28 AIK67137 Virus polyprotein

13

Carrot 82.4 82.4 37 le- 29 ACJ04421 Virus polyprotein necrotic 12

dieback

virus

EXAMPLE 2 - Virus prevalence & distribution

[00075] To confirm the viral sequences in the metagenomic data and to detect their presence in natural populations, Argentine ants were collected between 2012 and 2015 in either 100% ethanol or Ambion RNAIater (Life Technologies). Samples were collected from 30 sites distributed among nine regions throughout the ant's invaded range in New Zealand, two sites in Australia, and three sites in the ant's Argentinian native range. RNA was extracted for each site from a pool of 30 ants using either an iPrep PureLink Virus kit (Life Technologies) as for example 1 above, or a GeneJET Viral DNA & RNA Purification Kit (Thermo Scientific), as described below. [00076] DNA and RNA were extracted from Argentine ant samples using a GeneJET Viral DNA & RNA Purification Kit (Thermo Scientific, Waltham, MA, USA) following modified protocol. Ants from each sample were homogenised using pestles in Eppendorf tubes containing 250 μί GeneJET Viral DNA & RNA Purification Kit lysis buffer, and then 5 μί RNA carrier and 50 μί protein K were added. Samples were incubated for 1 hour at 56°C. Samples were briefly centrifuged, before the supernatant was removed and used in the standard manufacturer's protocol. Amplicons were visualized by gel electrophoresis and purified using USB ExoSAP-IT (Affymetrix). The purified amplicons were sent for Sanger sequencing on a capillary sequencer at Massey University Genome Service (New Zealand). The duplicate extractions were then combined into a single sample to maximise nucleic acid content. [00077] Sanger sequencing was used to confirm the presence of the contigs of interest in a number of samples. The partial n6409 contig was detected in all sampling sites, with the exception of one site in New Zealand (figure 2). The partial nlOOO contig sequence was present in all sites in Argentina and Australia but in less than half the sites in New Zealand (13 out of 27 sites). The partial nl905 and nl050 contigs were only detected in Australia and New Zealand (figure 2). All Sanger-sequencing data was identical to the partial metagenomic contig, with the exception of a few nucleotides differences (< 1%).

Example 3 - Analysis for known viruses

[00078] The presence or absence of four pathogenic honey bee viruses in these ant samples were assessed using one step T-PC s: Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), Acute paralysis bee virus (APBV), Deformed wing virus (DWV); and Solenopsis invicta virus -1 and -2 (SINV- 1 and SINV-2). Four partial contigs from the metagenomic data (nlOOO, nl905 and nl050, n6409) were also assessed using RT-PCR.

[00079] Published primers were used to test for the presence of the hymenopteran viruses: IAPV, KBV, ABPV, DWV, SINV-1 and SINV-2. [00080] Table 3 shows the published primers used to assess the presence or absence of Israeli acute paralysis virus (IAPV), Kashmir bee virus (KBV), Acute bee paralysis virus (ABPV), Solenopsis invicta virus -1 and -2 (respectively SINV-1 and SINV-2), and Deformed wing virus (DWV) in Argentine ant samples from Argentina, Australia and New Zealand.

Table 3

(Francis and Reverse AAACTGAATAATACTGTGC

AKIR

Kryger, 2012) GTA (SEQ ID NO. 16)

ORF2- Forward CCAGCCGTGAAACATGTTC F8092 TTACC (SEQ ID NO. 17)

IAPV (Palacios

226 et al, 2008) ORF2- Reverse ACATAGTTGCACGCCAATA

R8318 CGAGAAC (SEQ ID NO. 18)

Forward AAACATCACAGATGCTCAG

IAPV- GGTCGAGACTATATGT (SEQ

IAPV (Yang et 01F

ID NO. 19)

al., 2013)

427

Reverse CTAGGGAGCTACGGAGCGT

IAPV- GATTCGCCTTGTAGCT (SEQ 02R

ID NO. 20)

Forward GATGAACGTCGACCTATTG

KBV-F

A (SEQ ID NO. 21)

KBV

393

(Tentcheva et Reverse TGTGGGTTGGCTATGAGTC

KBV-R

al., 2004) A (SEQ ID NO. 22)

Forward CACTCCATACAACATTTGT pl l7 AATAAAGATTTAATT (SEQ

SINV-1 (Valles

ID NO. 23)

and Strong, 154 2005) Reverse CCAATACTGAAACAACTGA

pl l8

GACACG (SEQ ID NO. 24)

Forward CTTGATCGGGCAGGACAAA pl l4

TTC (SEQ ID NO. 25)

SINV-1 (Valles

647 and Strong, Reverse GAACGCTGATAACCAATGA

pl l6

2005) GCC (SEQ ID NO. 26)

Forward ATTTGTTTGGCCACGGTCA p64

AC (SEQ ID NO. 27)

SINV-2 (Valles

and Hashimoto, Reverse GATGATACAAAGCATTAGC 318 2009) p65 GTAGGTAAACG (SEQ ID NO.

28) Forward TGCATACTCGTTGTAAACA

p548 ATCTGCTCATCT (SEQ ID

SINV-2 (Valles

NO. 29)

and Hashimoto,

717

2009) Reverse TGCCGTGACAATCCTGAAT

p555 ATCGTCAGATGTA (SEQ ID

NO. 30)

Forward GCAGCTGGAATGAATGCAG

DWVrtF 295

AGA (SEQ ID NO. 31)

[00081] Only DWV was detected. It was present in 22 of the 27 sites in New Zealand (figure 2) but was not found in the populations from Australia or Argentina. The DWV sequences grouped with other known DWV found in honey bees and wasps (figure lc). The absence of results for ABPV, IAPV, KBV, SINV-1 and SINV-2 indicates that the contigs of interest, which most closely matched these viruses as shown in tables 1 and 2, were likely to be new viruses. Example 4 - Phylogenetic Analysis

[00082] Phylogenetic analysis was conducted in MEGA 6.06 (Tamura et al. 2013) after ClustalW (Larkin et al. 2007) alignment. An evolutionary model was chosen using Bayesian Information Criterion scores (BIC) derived in MEGA 6.06. Maximum likelihood trees with 1000 bootstrap replicates were generated using the LG model with a gamma parameter of 4.65 (LG+G) (Le Suq et al. 2008) for the LHUV-1 protein sequences and of the Tamura 3-parameter model with uniform rate (T92)(Tamura et al. 2002) for the DWV nucleotide sequences. 1000 bootstrap replicates were performed to test tree topology.

[00083] RNA metagenome (partial) contig n6409 matched the structural polyprotein (capsid region) of KBV and therefore was suitable for a phylogenetic analysis for provisional taxonomic assignment of the putative virus. Phylogenetic analysis of this contig positioned it with other dicistroviruses (figure lc). This sequence is from a novel virus, provisionally named Linepithema humile virus 1 (LHUV-1).

Example 5 - Analysis of DWV and LHUV-1 replication in Argentine Ants [00084] A modified RT-PCR was used to examine for DWV and LHUV-1 replication, and thus whether there was an active infection within the ants. Tagged primers were designed to only allow reverse transcription of the negative-sense strand of the viral genome.

[00085] To specifically amplify the negative strand of DWV and LHUV-1 (contig n6409), tag primers were used in a modified one step RT-PCR as described by Yue & Genersch (2005). A pool containing an equal amount of RNA from each site from one country was created for each country. The 10- nucleotide long tags for the primers were generated by BARCRAWL (Frank, 2009) using the default settings of the program and one pair of tags was selected for each pair of primers (see electronic supplementary material). A modified one step RT-PCR was realized using a Superscript III One Step RT-PCR system with Platnium Taq DNA polymerase kit (Life Technologies). Reaction mixes consisted of a 24.5 μί mix containing ~20 ng RNA, IX Reaction mix (final concentration), 0.8 μΜ of reverse tagged primer (either tag- DWVrtR or tag-n6409-A1055R - final concentration), 5.5 μΐ ddH20 and 1 μί Superscript^® III RT/Platinum^® Taq Mix. After the reverse transcription step at 45°C for 30 min, and 0.8 μΜ forward tagged primer (tag-DWVrtF or tag-n6409-A1055R) was added to obtain a final 25.0 μί mix. The PCR thermal cycling continued with an initial denaturation step at 94^°C for 2 min, followed by 35 cycles of 94^°C for 15 s, 55^°C for 15 s and 68^°C for 30 s, with a final extension step of 68^°C for 5 min. To assess the proper reverse transcription and amplification of the sequences without interference of the tags, the amplification of the positive strain of the viruses was realized in parallel. In the first step of the RT-PCR, only the tagged forward primers (either tag-DWVrtF or tag- n6409-A1055F) were added and in the second step, the tagged reverse primers tag- DWVrtR or tag- n6409-A1055R) were added. After the RT-PCR cycles, RT-PCR products were visualized by gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Amplicons were purified using USB ExoSAP-IT (Affymetrix) and sent for Sanger sequencing on a capillary sequencer to Massey University (Genome Service, Palmerston North, New Zealand). [00086] Table 4 shows the tagged primers used to amplify the RNA negative strand of Deformed wing virus (DWV) and L. humile virus-1 (LHUV-1) using a modified one step RT-PCR protocol. The tag sequence is in lower case.

Table 4

tag-n6409- ttcttctatgGTCACCTGACTCCTT

Reverse

A1055R GCCTGATTT (SEQ ID NO. 8)

Modified from Lester et al. (2015)

[00087] Both DWV and LHUV-1 were actively replicating in Argentine ants, as samples were PCR positive using this test. This replication indicates that the viruses were likely parasitizing the ants, rather than merely being vectored as particles. Therefore these viruses may be candidates for the population declines observed in Argentine ants and may have potential for biocontrol programmes.

Example 6 - Confirmation of contig n6409 sequence by RT-PCR

[00088] The sequence of contig n6409 was subject to further confirmation by RT PCR and Sanger sequencing using the primers in table 5 which were designed to target overlapping sequences. The resulting overlapping sequences were then aligned to give SEQ ID NO: 5.

Table 5

Example 7 - Extension of LHUV-1 by IVA

[00089] Iterative Viral Assembler (Hunt et al. 2015) was used to extend the sequence information for the LHUV-1 virus by aligning metatranscriptomic RNAseq sequences generated for Argentine ants to SEQ ID NO: 1. We then aligned the SEQ ID NO: 1 and SEQ ID NO: 5 with the extended sequence in MEGA6 (Tamura et al. 2013) to confirm the sequences matched. The IVA assembly resulted in an extended sequence of 8268 nucleotides (SEQ ID NO. 6). SEQ ID NO 1 aligned with 100% identity to nucleotides 6958 - 8268 of the new sequence, and SEQ ID NO 5 aligned with 99% identity to nucleotides 7071 - 8268 of the new sequence.

[00090] It is to be understood that the scope of the invention is not limited to the described embodiments and therefore that numerous variations and modifications may be made to these embodiments without departing from the scope of the invention as set out in this specification.

[00091] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. However, reference to any prior art publication does not constitute an admission that the publication forms a part of the common general knowledge in the art.

REFERENCES

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990 Basic local alignment search tool. J. Mol. Biol. 215, 403-410. Boyapelle S, Pal N, Miller WA, Bonning BC (2007) A glassy-winged sharpshooter cell line supports replication of Rhopalosiphum padi virus (Dicistroviridae) J. Invertebr. Path. 94, 130-139

Cooling M, Hartley S, Sim D, Lester PJ. 2012 The widespread collapse of an invasive species:

Argentine ants (Linepithema humile) in New Zealand. Biol. Lett. 8, 430-433.

Francis RM, Kryger P. 2012 Single assay detection of Acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus. J. Apic. Sci. 56, 137-146.

Frank D. N. (2009). BARCRAWL and BARTAB: software tools for the design and implementation ofbarcoded primers for highly multiplexed DNA sequencing. BMC Bioinformatics, 10, 362.

doi:10.1186/1471-2105-10-362.

Gilpin ME, Soule ME. 1986 Minimum viable populations: processes of species extinction. In

Conservation Biology: The Science of Scarcity and Diversity (ed M. E. Soule), pp. 19-34. Sunderland, Mass, USA: Sinauer Associates.

Goblirsch MJ, Spivak MS, Kurtti TJ (2013) A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues. PLoS ONE 8(7): e69831.

Gumus A, Karagoz M, Shapiro-llan D, Hazir S, 2015 A novel approach to biocontrol: Release of live insect hosts pre-infected with entomopathogenic nematodes. J. Invertebr. Pathol. 130 56-60 Holway DA, Lach L, Suarez AV, Tsutsui ND, Case TJ. 2002 The causes and consequences of ant invasions. Ann. Rev. Ecol. Syst. 33, 181-233.

Hunt, M., Gall, A., Ong, S.H., Brener, J., Ferns, B., Goulder, P., Nastouli, E., Keane, J. A., Kellam, P., Otto, T.D. 2015. IVA: accurate de novo assembly of RNA virus genomes. Bioinformatics. 31(14):2374- 6

Huson DH, Auch AF, Qi J, Schuster SC. 2007 M EGAN analysis ofmetagenomic data. 196 Genome Res. 17, 377-386.

Krishna N K, Marshall D, Scheemann A 2003. Analysis of RNA packaging in wild-type and mosaic protein capsids of flock house virus using recombinant baculovirus vectors. Virology 305:10-24. Larkin MA, et al. 2007. ClustalW and ClustalX version 2. Bioinformatics 23, 2947-2948.

Lester PJ, Bosch PJ, Gruber MAM, Kapp EA, Peng L, Brenton-Rule E, Buchanan J, Stanislawek WL, Archer M, Corley JC, Masciocchi M, Van Oystaeyen A, Wenseleers T. 2015 No evidence of enemy release in pathogen and microbial communities of common wasps (Vespula vulgaris) in their native and introduced range. PLoS One 10, 201 e0121358. Lester P J and Gruber, M AM, 2016 Booms, busts and population collapses in invasive ants Biol. Invasions 18

Le SQ, Gascuel O. 2008. LG: An Improved, General Amino-Acid Replacement Matrix. Mol. Biol. Evol. 25, 1307-20.

Needleman, Saul B. & Wunsch, Christian D. (1970). A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology. 48 (3): 443-53

Palacios G, Hui J, Quan PL, Kalkstein A, Honkavuori KS, Bussetti AV, Conlan S, Evans J, Chen YP, van Engelsdorp D, Efrat H, Pettis J, Cox-Foster D, Holmes EC, Briese T, Lipkin Wl. 2008 Genetic analysis of Israel acute paralysis virus: distinct clusters are circulating in the United States. J. Virol. 82, 6209- 6217. Pal N, Boyapelle S, Beckett, R, Miller WA, Bonning, BC (2007) A Baculovirus-Expressed Dicistrovirus That Is Infectious to Aphids V. Virol. 81, 9339-9345

Sagata K, Lester PJ. 2009 Behavioural plasticity associated with propagule size, resources, and the invasion success of the Argentine ant Linepithema humile. J. Appl. Ecol. 46, 19-27.

Schroeder DC, Martin SJ. 2012 Deformed wing virus: the main suspect in unexplained honeybee deaths worldwide. Virulence 3, 589-591. Singh , Levitt AL, Rajotte EG, Holmes EC, Ostiguy N, Vanengelsdorp D, Lipkin Wl, Depamphilis CW, Toth AL, Cox-Foster DL. 2010 RNA viruses in hymenopteran pollinators: evidence of inter-taxa virus transmission via pollen and potential impact on non-Apis hymenopteran species. PloS One 5, el4357. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 30, 2725-2729.

Tamura K, Kumar. 2002. Evolutionary distance estimation under heterogeneous substitution pattern among lineages. Mol. Biol. Evol. 19, 1727-1736.

Tentcheva D, Gauthier L, Zappulla N, Dainat B, Cousserans F, Colin ME, Bergoin M. 2004 Prevalence and seasonal variations of six bee viruses inApis mellifera Land Varroa destructor mite populations in France. Appl. Environ. Microbial. 70, 7185-7191.

Ugelvig LV, Cremer S. 2012 Effects of social immunity and unicoloniality on host-parasite interactions in invasive insect societies. Func. Ecol. 26, 1300-1312.

Valles SM, Hashimoto Y. 2009 Isolation and characterization of Solenopsis invicta virus 3, a new positive-strand RNA virus infecting the red imported fire ant, Solenopsis invicta. Virology 388, 354- 361.

Valles SM, Porter SD, Choi M, Oi DH 2013 Successful transmission of Solenopsis invicta virus 3 to Solenopsis invicta fire ant colonies in oil, sugar, and cricket bait formulations J. Invertebr. Pathol. 113, 138-204 Valles SM, Strong CA. 2005 Solenopsis invicta virus-lA (SINV-1A): distinct species or genotype of SINV-1? 7. Invertebr. Pathol. 88, 32-237 ^'.

Valles SM, Strong CA, Hashimoto Y. 2007. A new positive-strand RNA virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta. Virology 365, 220 457-463.

Valles SM, Porter SD, Firth AE. 2014 Solenopsis invicta virus 3: pathogenesis and stage specificity in red imported fire ants. Virology 460, 66-71.

Valles et al. 2016; Isolation and characterization of Nylanderia fulva virus 1, a positive-sense, single- stranded RNA virus infecting the tawny crazy ant, Nylanderia fulva, Virology 496:244-254

Yang B, Peng GD, Li TB, Kadowaki T. 2013 Molecular and phylogenetic characterization of honey bee viruses, Nosema microsporidia, protozoan parasites, and parasitic mites in China. Ecol. Evol. 3, 298- 311. Yue C, Genersch E. 2005 RT-PCR analysis of Deformed wing virus in honeybees [Apis mellifera) and mites (Varroa destructor). J. Virol. 86, 3419-3424.

Zerbino DR, Birney E. 2008 Velvet: Algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821-829.

Claims

What we claim is:

1. An isolated nucleic acid molecule comprising a sequence which is at least about 50%

identical to SEQ ID NO: 5.

2. A nucleic acid molecule according to claim 1, comprising a sequence which is at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5.

3. A nucleic acid molecule according to claim 2, comprising a sequence which is about 100% identical to SEQ ID NO: 5.

4. A nucleic acid molecule according to claim 1, comprising a sequence which is at least about 50% identical to SEQ ID NO: 1.

5. The nucleic acid molecule according to claim 4, comprising a sequence which is at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.

6. The nucleic acid molecule according to claim 5, comprising a sequence which is 100%

identical to SEQ ID NO: 1.

7. A nucleic acid molecule according to any one of claims 1 to 6, comprising a sequence which is at least about 50% identical to SEQ ID NO: 6.

8. A nucleic acid molecule according to claim 7, comprising a sequence which is at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 6.

9. A nucleic acid molecule according to claim 8, comprising a sequence which is 100% identical to SEQ ID NO: 6.

10. A nucleic acid molecule according to any one of claims 1 to 9, which is NA.

11. An isolated virus comprising a nucleic acid molecule as defined in any one of claims 1 to 10.

12. A virus according to claim 11, which infects Linepithema humile.

13. A virus according to claim 11 or 12, which does not infect honey bees.

14. A virus according to claim 13, which is a single stranded RNA virus.

15. A virus according to any one of claims 11-14, which is a member of the Dicistroviridae family.

16. A composition comprising a virus as defined in any one of claims 11-15 and a carrier.

17. A composition according to claim 16, comprising a bait matrix.

18. A composition according to claim 16 or 17, comprising one or more additional active

ingredients.

19. A composition according to claim 18, wherein the one or more additional active ingredient another biological compound, an inorganic insecticide or an organic insecticide.

20. A method of controlling an invertebrate population comprising administering an effective amount of a virus according to any one of claims 11-15, or a composition according to claims 16-19.

21. A method according to claim 20, wherein the virus or composition is administered by way of bait system.

22. A method according to claim 20 or 21, wherein the invertebrate population is an ant

population.

23. A method according to claim 22, wherein the ant population is an Argentine ant population.

24. Use of a virus as defined in any one of claims 11-15 or composition according to any one of claims 16-19 for controlling or eradicating an invertebrate population.

25. An isolated primer selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.