Review of Control Options for Suppression Or Elimination of the Yellow-Legged Asian Hornet, Vespa Velutina Nigrithorax in the UK

Review of control options for suppression or elimination of the Yellow-legged Asian hornet, Vespa velutina nigrithorax in the UK.

Project PH0530

Ben Jones, Kirsty Stainton and Maureen Wakefield

March 2017

Contents

1. Control of invasive species ...... 3 2. Vespa velutina nigrithorax ...... 4 3. Monitoring, trapping and mechanical control ...... 6 4. Chemical control of Vespa velutina nigrithorax ...... 7 4.1 Chemical control agents ...... 7 4.2 Formulation types available ...... 9 4.3 Pheromones ...... 9 4.4 Toxic baits against Vespids ...... 9 5. Biological control of Vespa velutina nigrithorax...... 14 6. Novel control techniques for control of Vespa velutina nigrithorax ...... 16 6.1 Sterile Insect Technique...... 16 6.2. RNA interference ...... 21 6.3 Genetic transformation and Genome editing ...... 24 7. Conclusion and recommendations ...... 28 8. References ...... 31

1. Control of invasive species

When a new species is introduced into an area where they are not indigenous, they may be referred to as ‘non-native’, ‘exotic’ or ‘alien’ (Manchester, 2000). Only approximately 10% of introduced species will become established in their new territory and only 0.1% will become invasive (Manchester, 2000). Invasiveness refers to the ability of a non-native species to be detrimental to the economy or ecosystem into which it has been introduced and may be used synonymously with the term ‘pest’.

The chances of eradicating, or suppressing, an invasive population once it has become established in its new territory is very low. Once established a pest population can be time-consuming and costly to control (Manchester, 2000).

There are 34 recorded species of introduced Vespidae around the world, the most invasive of which are eusocial, like the yellow-legged Asian hornet Vespa velutina nigrithorax (Beggs et al., 2011). Vespidae are generalist feeders with a high dispersal potential and fast reproductive rate which accounts for the success of the invasive Vespids, such as Vespula germanica and Vespula vulgaris, across North America, New Zealand and Australia (Beggs et al., 2011).

Traditional control methods can be broadly characterised as physical/mechanical, chemical or biological in nature. Physical/ mechanical control may involve trapping or manual destruction of an invasive pest, through hunting for example. Chemical control may involve the use of broad spectrum insecticides. Biological control usually refers to the introduction of biological agents, such as predators, parasites or pathogens of the target species. In the past two decades, molecular techniques have also been developed and deployed for pest control; this includes genetic modification, para- transgenesis and the use of RNA interference (RNAi).

This review will include an evaluation of the techniques described above and their applicability for use against the yellow-legged Asian hornet, Vespa velutina nigrithorax. The review will conclude with a summary of these techniques and the feasibility of using them, and a recommendation of the likely most effective techniques as well as a description of further research that may be required to improve the suite of control options available.

2. Vespa velutina nigrithorax

Vespa velutina is a species of hornet native to East Asia. It belongs to the order Hymenoptera which includes bees, wasps and sawflies; hornets are closely related to wasps, which all belong to the family Vespidae (Figure 1). There are 12 subspecies of V. velutina across its native range, which can be identified by their thoracic and abdominal colourations (Perrard et al., 2014). The dark subspecies nigrithorax was discovered in southern France in 2004 after an accidental introduction and was found to originate from a hornet population from eastern China using genetic analyses (Arca et al., 2015). These analyses determined that the incursion was initiated by very few or perhaps even a single female queen hornet that had mated with multiple males (Arca et al., 2015). Despite the strong population bottleneck experienced by this invasive species and its resulting restricted genetic diversity, V. velutina nigrithorax has gone on to colonise approximately three quarters of France (Villemant and Rome, 2016), northern Spain in 2010, Portugal and Belgium in 2011, Italy in 2012 (Darrouzet et al., 2015 and Lopez, 2011), Germany in 2014 (Orlov, 2016). At the time of writing of this report (March 2017) there had been a single incursion of V. velutina nigrithorax in the UK. A nest was subsequently found and destroyed.

Figure 1: Basic representation of relatedness of hornets to bees and wasps.

A review of control techniques will not be complete without an introduction to the biology of V. velutina; aspects of the biology must be understood when considering the merit of each control option. Some pest control strategies involve targeting the reproductive individuals and one of the crucial differences in V. velutina biology that sets them apart from other pest insects is its sex determination system. The haplo- diploid species V. velutina has a single-locus complementary sex determination system; females are heterozygous at a sex-determining locus, while males are homozygous (diploid) or hemizygous (haploid) (Darrouzet et al., 2015). A colony of V. velutina is comprised mainly of non-reproductive female workers. The only reproductive female in the colony is the queen (although workers may be able to lay haploid eggs). Hornet nests only produce reproductive individuals in autumn, the queen will first begin by laying eggs destined to become males followed, weeks later, by female eggs destined to become the future queens or gynes. In France, males begin emerging in late August and virgin queens in early/mid-September, they mate and the females disperse (this may occur just before or just after hibernation) while the males die (Darrouzet et al., 2015). Three times as many males as virgin queens are produced by a nest (Monceau et al., 2013a).

In early spring (late February onwards), mated queens emerge to find sites for nest- building (Villemant and Rome, 2015). Between 90 and 99% of V. velutina queens die of natural causes before making a nest due to over-wintering losses and fierce competition among queens for nesting sites. Even if an intervention is able to remove the majority of new queens from an environment (i.e. through trapping) the effects of density dependent competition for this species are yet to be ascertained so the effectiveness of control at this stage is unknown (Monceau and Thiéry, 2016).

Vespa velutina has a long-distance dispersal potential with a rate of spread of 78 km per year in France; this is in part due to their excellent flight ability, as queens can travel up to 200 km in 10 days (Robinet et al., 2016). However, their spread is also hastened by the inadvertent transport of queens via human activity (Robinet et al., 2016). This makes prediction and prevention of novel incursions extremely difficult.

Vespa velutina is a univoltine species, i.e. it has only a single generation each year. The successful queen will create her primary nest and rear her first set of brood alone; when this brood emerges they abscond from the primary nest and build a secondary nest. The secondary nest is managed by the workers and a single nest may produce up to 13,000 workers in a season, with approximately 1800 individuals present in the nest at a given time (Villemant and Rome, 2015). In France 70% of secondary nests are found above 10m and they may be found equally in urban (49%) or agricultural and forested areas (50%) (Villemant and Rome, 2015). The majority of nests will be located in trees and largely undetectable by sight. The difficulty involved in detecting nests is a significant obstacle for control. Detection methods have recently been reviewed in the Defra funded project PH0524 (Review of the potential detection and control options for nests of the Asian hornet (Vespa velutina nigrithorax)) and will not be considered further in the current review.

When considering the methods for control of an organism, an understanding of the life history and reproductive biology of that organism is crucial and any attempt to control will be unsuccessful without it. This will be especially true for Vespa velutina nigrithorax, as its life cycle, reproductive strategy and biology differ somewhat from the majority of canonical ‘pest’ insects. Most pest insects belong to the orders Diptera (i.e. mosquitoes, Medfly, Screwworm), Coleoptera (i.e. Colorado potato beetle, boll weevil) and Lepidoptera (i.e. codling moth, pink bollworm). Comparatively, there are few pest insects belonging to the Hymenoptera, the order to which Vespa velutina belongs, and this is reflected in the pest control literature.

Current controls for V. velutina nigrithorax involve the use of pesticides, manual trapping and mechanical destruction. Application of such methods is labour intensive and highly unlikely to result in the elimination of an invasive population, even if combined with biological control (Robinet et al., 2016).

3. Monitoring, trapping and mechanical control

Monitoring

The potential establishment of V. velutina nigrithorax poses a problem with regard to detection. A single mated queen hornet will seek out a sheltered location to overwinter and may inadvertently be transported in a shipping container or within luggage. When she emerges, she will establish her nest, but her presence may go undetected. This was the case with the original invasion of France with V. velutina nigrithorax (Arca et al., 2015) and was also the case for the eusocial wasp Vespula pensylvanica and its arrival into Hawaii (Beggs et al., 2011). Routine cargo inspections and pyrethroid treatment of shipments are only partially effective at preventing further introductions of alien invertebrate species (Beggs et al., 2011) so constant monitoring needs to be in place alongside the development of controls.

Trapping

Vespa velutina nigrithorax trapping has proven ineffective for control. Trapping the sexuals is ineffective due to the large numbers of gynes produced. High levels of natural mortality occur in overwintering gynes and this has little effect on establishment of new colonies. Trapping can also impact upon non-target species, catching large numbers of bees, flies, Hemiptera and spiders in the process of trapping V. velutina nigrithorax (Rome et al., 2011).

Brown et al. (2013) investigated the use of V. vulgaris sex pheromones as an attractant for use in spring trapping to capture emerging queens. However, sex pheromones are produced by gynes to attract males during the mating period. Attraction of males to traps would be unlikely to have a significant effect for controlling hornet populations; only a few males need escape such a strategy to successfully inseminate virgin queens.

In France, trapping is routinely used around apiaries. Due to the lack of more sophisticated pheromone based bait, the favoured baits in France are sugar-rich or protein-rich baits dispensed appropriately for the season. The ultimate problem of trapping a species like V. velutina nigrithorax is that removal of workers/foragers from the nest is unlikely to impact significantly upon its reproductive output, as this is determined by the queen who is unlikely to leave the nest to be trapped. Therefore, trapping is more useful in terms of monitoring the population.

Nest destruction/mechanical control

Locating and destroying nests of V. velutina nigrithorax has proven ineffective in France for controlling populations due to the ability of a nest to produce large numbers of new foundress queens and the difficulty in locating nests (Rome et al., 2011). 4. Chemical control of Vespa velutina nigrithorax

4.1 Chemical control agents

Insecticides for the treatment of public health pests, including wasps, are regulated by the Biocidal Products Regulation (Regulation EU 528/2012), which replaced the Biocidal Products Directive (BPD; 98/8/EC) on 1st September 2013. Approval of active substances is at EU level, whilst the authorisation of biocidal products is at the Member State level. In the UK, biocidal products are regulated by the Health and Safety Executive (HSE). Active substances that are classed as existing substances (i.e. were on the market on 14th May 2000) were subject to a Review Programme (Regulation (EU) No 1062/2014) and under transitional provisions an active substance included in the Review Programme can be made available on the market and used, subject to national rules, up to three years after the date of their approval.

In the UK many of the biocidal product types including insecticides are regulated under the Control of Pesticides Regulations 1986 (COPR). Products will be regulated in the UK under these regulations until the EU BPR active substance review is completed and a decision made as to whether the active substance should be approved. A list of products that are currently approved under COPR is available on the HSE website (http://www.hse.gov.uk/biocides/copr/approved.htm). This list was used to determine the actives that are currently approved for treatment of wasps in the UK (Table 1). Whilst some of the actives are now approved under the BPR, currently there are no products containing these actives approved under the BPR in the UK (http://webcommunities.hse.gov.uk/connect.ti/pesticides/view?objectId=6020; accessed 16/01/07).

Table 1. Actives approved for use against wasps in the UK and their current status under the BPR

Active Approval date Expiry date Pyrethrins Under review Cypermethrin Under review Tetramethrin Under review Alphacypermethrin 01/07/2016 01/07/2026 Permethrin 01/05/2016 01/05/2026 Bendiocarb 01/02/2014 01/02/2024 Phenothrin (always in 01/09/2015 01/09/2025 combination with tetramethrin) Azamethiphos Under review

For each of these actives several products are available and these may be for professional or amateur use. Some of the products are specific for the treatment of wasp nests, whilst others have a more general use against crawling or flying insects where wasps are listed in the recommendations for use. The main group of actives that are approved for control of wasps are synthetic pyrethroids and pyrethrins. This group of chemicals act by contact and are sodium channel modulators. Although the toxicity of individual compounds will vary, they are classed as toxic or highly toxic to aquatic organisms and to bees. They would therefore need to be used with caution in apiaries. Hornet nests tend to be built near water sources (Monceau et al., 2012; Monceau et al., 2013a) and therefore consideration would need to be given when using these chemicals in these locations due to the highly toxic effect on aquatic organisms. Synthetic pyrethroids are also associated with the development of insecticide resistance.

From the published literature on Vespa velutina there are no studies that have examined the effectiveness of chemical insecticides on any life stage of the Asian hornet. Direct application of insecticides is problematic for V. velutina nigrithorax due to the nature of finding and accessing nests. In the UK bendiocarb (Ficam D) is recommended for treatment of any Asian hornet nest found in the UK. Ficam D is a dust formulation containing 1.25% w/w bendiocarb and is approved for use by professional operators only. It is approved for the control of wasps and feral bees and the product label states that it does not repel or excite insects.

Ficam D (active ingredient: bendiocarb) and Whitmire PT515 Waspfreeze (active ingredients: pyrethrins) were tested on Vespula pensylvanica in Hawaii through direct application. Both compounds rarely destroyed nests due to difficulty to expose individuals inside colonies. Furthermore, both compounds are toxic to non-target organisms, predators attacking the treated colonies exposed themselves to pesticide and increasing the potential of the pesticides to move up the food chain (Gambino & Loope, 1992).

The limited amount of information available on the efficacy of chemical insecticides on hornets and wasps would make this an area of priority for research to ensure that the most effective products are used for the eradication and/or containment of V. velutina nigrithorax in the UK.

It is likely that extension of use of actives and products that are currently used for the control of wasps for the control of V. velutina would be a simpler process than approval of actives that have no known efficacy against wasps/ hornets or for actives and products that are used as agricultural insecticides and are therefore not covered for public health insects under the BPR.

Other actives with products currently registered under the BPR in the UK are dinotefuran (neonicotinoid), indoxacarb (oxadiazine), spinosad (spinosyn), fipronil (phenylpyrazole) and imidacloprid (neonicotinoid). Fipronil has been used in toxic baiting studies for control of invasive wasp species (see Toxic baiting section below). It is, however, highly toxic to honeybees and is currently under review by EFSA. The other actives have also shown toxicity against bees. As such they may have activity against V. velutina nigrithorax as they are closely related species. However, their use would have to be carefully considered and assessed for potential negative effects on non-target species such as bees and wasps prior to further research on their efficacy.

4.2 Formulation types available

Available formulations for the treatment of wasp nests include dusts, sprayable formulations and foams. Dust formulations such as Ficam D are appropriate for treatment of inaccessible areas e.g. service ducts and roof voids as spraying in such areas is not practical. Dust formulations are also appropriate for spraying near electrical installations (Ficam D; product information leaflet). However, dusts can drift during application, the residue can be moved by air currents and it may be difficult to achieve an even distribution.

Liquid formulations (e.g. wettable powders and emulsifiable concentrates, ULV sprays) may provide good adherence to surfaces, but can be difficult to use in confined areas. As with dust application, it is also possible to get drift during the application of liquid formulations.

There are also some foam applications available for the treatment of wasp nests. If used when there are a high number of foragers outside the nest, these may prevent individuals returning to the nest.

4.3 Pheromones

Pheromones in Hymenoptera are well described and have varying functions; sexual behaviour, brood maintenance, alarm and communication and thermoregulation (Howse, 1998). In honeybees, the queen bee (the focus of the hive) produces a queen pheromone which is distributed around the colony. The presence of queen pheromone suppresses ovarian function in workers, prevents the colony from swarming and making new queen cells, it is also highly attractive to drones/males (Howse, 1998). Although it is an extremely persistent compound in the environment, chemically synthesized versions did not elicit a reaction in drones (Howse, 1998). Alarm pheromones have been identified in V. velutina nigrithorax (Cheng et al., 2017), but other pheromones remain to be identified.

4.4 Toxic baits against Vespids

Toxic baiting involves the addition of a toxic compound to a protein rich food source that foragers collect and take back to their nest in order to feed brood. Baits should be desirable to the invasive organism while minimising the impact on native fauna. Toxic baiting offers a degree of specificity without the need to locate nests (Beggs et al., 2011, Gentz, 2009). The effect of toxic baits has been examined in species of invasive wasps (Vespidae), which are more closely related to hornets than any other pest organism. Numerous studies have demonstrated successful suppression of various invasive Vespula species in New Zealand, Australia, the United States and Argentina using toxic baiting (reviewed in Beggs et al. 2011, Sackman & Corley, 2007 and Ward, 2014, summarised in Table 2). The most extensive efforts have taken place in New Zealand against the non-native wasp species, V. germanica, V. pensylvanica and V. vulgaris. These invasive species cause significant economic and ecological damage, as well as being a pest to humans (Beggs, 2001, Brown et al., 2014, Fordham, 1991, MacIntyre & Hellstrom, 2015). Invasive wasps can out-compete native fauna for food sources and are responsible for increased predation on native invertebrates (Beggs, 2001, Brown et al., 2014). High densities of V. vulgaris are responsible for removing up to 99% of honeydew from an area. Honeydew is an important source of sugars for birds, insects and microbes. This predation is at its highest during the 4 months of the year when the wasps are at their peak (Beggs et al., 2011). The total annual cost of the effects of wasps in New Zealand has been estimated to be $133 million (MacIntyre & Hellstrom, 2015).

Bait location and density

The impact of low and high density baiting on V. pensylvanica in Hawaii with a variety of toxins was tested; microencapsulated diazinon (organophosphate), boric acid (a ‘natural’ acid), amidinohydrazone and the insect growth regulators (IGRs) fenoxycarb and avermectin were tested, using tuna cat food as bait (Chang, 1988). Baits were placed at densities of either one per 20 ha for three months (low density) or one per five ha for one week (high density). Amidinohydrazone was the most effective toxin, reducing wasp numbers within one month. When used at low density, baiting must start early in the season and continue until October to effectively reduce V. pensylvanica numbers when using acute toxins. IGR’s were slow acting, with reductions in wasp numbers observed after seven weeks. High density baiting resulted in a 94% reduction in wasp numbers one week after treatment with non-IGRs.

There is a demonstrated reduction in efficacy of toxic baiting on wasps (V. vulgaris) with increasing distance from the nest (Spurr, 1991a). High density baiting (40 baits per ha) with canned sardine cat food and sodium monofluoroacetate (1080) was conducted in forest clearings and road tracks in areas of 1.5-3 ha. Pre-baiting was carried out three days before toxic baiting in order to attract wasps to the area before toxic baiting. This resulted in a 95% reduction in wasp numbers after one day, and a 94% reduction of nests within 100 m of the toxic bait after 12 weeks. Wasp numbers recovered four weeks after the initial poisoning and only 6% of nests were killed more than 500 m from the toxic bait. Concentrations of 0.5-1% of sodium monofluoroacetate (1080) were needed to effectively reduce wasp numbers (Spurr, 1991) but sodium monofluoroacetate is highly toxic to mammals so its use for pest control is contra- indicated.

Fipronil versus Sulfuramid

Fipronil is a chloride channel blocking pesticide, it is a stable compound that has high levels of persistence in the environment and can be broken down into other active products (Stenerson, 2000). Fiprinol blocks chloride channels in the central nervous system of insects more effectively than in mammals and offers a greater degree of specificity than the slower acting but less toxic insecticide sulfuramid.

In New Zealand, the effects of 1% sodium monoflouracetate was compared to the substantially less toxic sulfuramid (1%) and the two were found to be comparable, reducing Vespula vulgaris colonies in 30 ha sites by 82-100% (Beggs et al., 1998). Therefore, the less toxic insecticide is preferable for large scale use.

Fiprinol was compared with sulfuramid in laboratory and large scale field trails (300 ha) against Vespula species in New Zealand. Fiprinol treatment resulted in higher mortality than treatment with sulfuramid in both laboratory and field trails, with a 99.9% reduction of wasp activity with fipronil (Harris & Etheridge, 2001). In contrast, nest activity was not significantly reduced in sulfuramid treated areas of similar bait density.

Subsequent work further demonstrated successful reductions in Vespula populations using fiprinol baits, with reports of near total reductions of nest activity. Fiprinol (0.1%) was used successfully to reduce colonies of V. germanica in Argentina; wasp numbers were reduced by 87% compared to untreated control sites (Sackmann et al., 2001). A combination of fipronil and a long range wasp attractant, heptyl butyrate was used to reduce the abundance of the invasive wasp, V.pensylvanica in Hawaii by 97% within one week of poisoning in the treated areas compared to an untreated control area (Hanna et al., 2012). In addition, a significant reduction in the abundance of V. pensylvanica over three consecutive years was reported. The use of XtinguishTM (Fiprinol 0.1%) on V. vulgaris in New Zealand reduced nest traffic by 80% within 113 m and increased with distance from bait clusters; large colonies were more susceptible, possibly due to being able to recruit foragers to bait stations (Harper et al., 2016).

Baits

A major obstacle of toxic baiting is the non-specificity of meat-based baits, which can attract non-target organisms such as birds and other Vespidae. The efficiency and specificity of baiting can be improved by combining specific meat baits with an attractant that targets the desired species such as heptyl butyrate (Hanna et al., 2012). Vespula preferences have been demonstrated to differ with the type of meat bait used, the length of time the meat is exposed to the environment, the presence of other foragers such as ants, moisture content and texture differences that occur between dried, frozen or canned meat (Pereira et al., 2013, Unelius et al., 2014, Wood et al., 2006). Previous work has focussed on attempting to identify specific compounds to attract wasps. A multi-component lure that contained volatiles from both carbohydrate and protein based food sources (honeydew, fermented brown sugar, honey and green-lipped mussels) was found to be highly attractive to wasps (Unelius et al., 2015). Brown et al. (2014) identified three compounds in fermented brown sugar (3-methyl- 1-butanol, 3-methylbutyl acetate, and ethyl hexanoate) that are attractive to wasps. However, the lure was also attractive to a small number of non-target flying insects. Essential oils have been investigated for their capacity to repel herbivorous insects for use in a ‘push-pull’ system of attractive baits and repellents. Trails with V. germanica have proved promising, but this concept is currently in initial stages (Buteler, 2016).

In conclusion, differences in the efficacy of fiprinol toxic baiting may have resulted from differences in the quantity of bait dispensed, the density of wasp colonies and bait densities between trials. However, previous work using fiprinol baits have achieved effective reductions in wasp numbers and toxic baiting therefore may have a part to play in integrated control programs for Vespa velutina nigrithorax. The numbers of wasps in an area must be sufficient for enough bait to be taken back to the colony (Hopkins, 2002). Densities of V. velutina in France have been reported as 12.26 nests/km2; much lower than those reported in previous work demonstrating successful reductions of Vespula spp. with toxic baiting in New Zealand; 75 wasp nests/ha (Spurr, 1991b), 16-34 wasp nests/ha (Beggs et al., 1998), 3-4 nests per ha (Sackmann et al., 2001), 11.2 nests/ha (Harris & Etheridge, 2001). It is worth noting that this figure represents only urban areas due to the difficulty in locating nests in rural areas (Monceau & Thiéry, 2016). In addition, it is important to note that the foraging range of V. velutina nigrithorax is not currently known (Monceau et al., 2013a) and therefore optimum deployment of bait stations around a nest site would require greater knowledge of the foraging behaviour of this species. We therefore recognise that direct treatment of colonies with a pesticide is preferable if colonies can be located. Further work is needed to assess the threshold necessary for toxic baiting to be effective for V. velutina and to investigate lures specific to V. velutina. Current monitoring techniques for V. velutina in the UK involve the use of traps containing carbohydrate based lures, which are non-specific. Therefore, the impacts on non-target insects from toxic baiting in areas of new incursions or localised populations may be less than the impact of monitoring.

Although toxic baiting can reduce wasp species locally, re-invasion from surrounding areas is consistently reported. Eradication of widespread, established populations is unlikely, but eradication of new incursions or local populations may be possible (Beggs et al., 2011). Various toxic compounds have been investigated and success is dependent on the type of bait used, the timing of baiting, the dosage, the density of baits and the distance between the bait and the wasp nest. Reference Year Vespula spp. Country Toxic compound Grant et al. 1968 V. pensylvanica USA (California) Chlordance Wagner & Reirson 1969 V. pensylvanica USA (California) Mirex Perrott 1975 V.germanica New Zealand Mirex Ennik 1979 V.pensylvanica USA (California) Diazinon Chang 1988 V. pensylvanica USA (Hawaii) Diazinon, boric acid, amidinohydrazone, fenoxycarb, avermectin Spurr 1991a V. vulgaris New Zealand Hydramethylnon Spurr 1991b V.vulgaris New Zealand Sodium monofluoroacetate Gambino & Loope 1992 V. pensylvanica USA (Hawaii) Bendiocarb & Pyrethrins Spurr et al. 1993 V. germanica, V.vulgaris New Zealand Sulfluramid Beggs et al. 1998 V. vulgaris New Zealand Sodium monofluoroacetate and sulfluramid Harris & Etheridge 2001 Vespula spp. New Zealand Fiprinol & Sulfluramid Sackmann et al. 2001 V. germanica Argentina Fiprinol Austin & Hopkins 2002 V. germanica Australia Fiprinol Sackman & Corley 2007 V. germanica Argentina Hydramethylnon, permethrin & chlorpyrifos Foote et al. 2011 V. pensylvanica USA (Hawaii) Fiprinol Hanna et al. 2012 V. pensylvanica USA (Hawaii) Fiprinol Harper et al. 2016 V. vulgaris New Zealand Fiprinol Table 2. Attempts at toxic baiting to control Vespula spp. P a g e | 14

5. Biological control of Vespa velutina nigrithorax

5.1 Introduction and history of biological control against invasive pests

In informal terms, biological control refers to the introduction of a species that acts as a natural enemy of the invading pest. It is intended that the introduced enemy exerts sufficient pressure on its host to control its population, meanwhile having no unintended side-effects on the invaded ecosystem and its inhabitants. Unfortunately, introducing natural enemies to control pest populations has a varied track record. The main issues concern 1) the unintended effects of a species introduced to control another species (the control agent may become as invasive as the species it was intended to control) and 2) whether controlling a population using such means is indeed effective.

In addressing the first issue, a look at the numbers might give some insight into the unintended effects of biological release programmes. In biological control programmes between 1890 and 1985, there were 533 releases where insects were used against a target pest; 175 established themselves and 15 were found to attack non-target species (Simberloff and Stirling, 1996). This seems encouraging, however, parasitoids are a common insect enemy introduced into an ecosystem to control other insects, but it appears that introduced parasitoids are implicated in causing significant declines, or even the complete demise, of non-target native species; especially on islands (Simberloff and Stirling, 1996). The measurement of “off-target” effects is not a simple one and opponents of biological control programmes claim that there are insufficient protocols in place to assess the impacts to non-target species and it is hard to anticipate these effects (Simberloff and Stirling, 1996). An anecdote that illustrates this point is provided in Simberloff and Stirling who remark upon the extinction of the large blue butterfly, Maculina arion, in the UK, due to the myxoma virus introduced to control rabbits. The control was so successful against rabbits that this reduced the underground nesting sites available for the ant, Myrmica sabuleti. Nesting sites for the ants had been provided by rabbits after considerable land use changes disrupted natural nesting sites. This led to serious declines for the ants and extinction of the butterfly, as the ant nest played host to the developing butterfly larvae (Simberloff and Stirling, 1996).

As for the second issue, attempts have been made to quantify the success of biological control programmes. In an attempt to quantify the success of arthropod introductions worldwide, it was found that establishment occurred in 34% of cases and “some degree” of control was found in 60% of cases (Hall et al., 1980). Another opinion estimates success in only 10% of biological control cases (Gurr and Wratten, 2000). The problem with measuring success involves not only the effect on suppressing the target population but attempting to quantify the economic benefit as well as considering the ecological, environmental and social factors involved.

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5.2 Pests

In New Zealand wasps are not native, but they are now widespread due to invasion, primarily by the European wasp species V. germanica and V. vulgaris, and a lack of natural predators. In response, the authorities in New Zealand have tested the parasitoid, Sphecophaga vesparum, to control wasp populations (Ward, 2014). The parasitoid wasp Sphecophaga vesparum vesparum was released in an attempt to control invasive Vespula species. It was deemed that this parasitoid was species specific and highly productive, and it established at three sites. Despite this, there was no evidence that this had any impact on wasp populations (Beggs et al., 2011). The parasitoids Sphecophaga vesparum burra and Sphecophaga orientalis were also released in New Zealand, but failed to establish (Beggs et al., 2011)

Villemant et al., (2015) reported that the thick headed fly (family Conopidae) is a parasitoid of adult wasps and may be an enemy of V. velutina nigrithorax. However, Conops vesicularis are active during June to September and are unlikely to infect the reproductive individuals; only the foragers which are not reproductive (Beggs et al., 2011).

5.3 Pathogens

The pathogenicity of entomopathogenic fungi was assessed in laboratory trails against the wasp species V. germanica and V. vulgaris. Aspergillus flavus reduced emergence rates of V. germanica and V. vulgaris to 0.8 and 1.3% respectively (Glare et al., 1996). Aspergillus flavus is capable of infecting humans and is therefore unlikely to be developed as a control agent. The impact of a range of fungi was tested against V. vulgaris and demonstrated high mortality and successful transfer of spores to uninfected nest mates at high spore concentrations. Metarhizium anisopliae, Beauveria bassiana, and Aspergillus flavus were found to be pathogenic while Cladosporium sp., and Paecilomyces sp. were not pathogenic (Harris et al., 2000). However, fluctuations in temperature and relative humidity in the field may reduce efficacy of fungal pathogens (Harris et al., 2000).

There is potential for a lure and kill system to be developed using entomopathogenic fungi whereby the workers are attracted to a source where they become coated with the fungus, which they then carry back to the nest. In addition to understanding the efficacy of the fungus within the nest and the amount of fungus that would needed to be transported back to the nest, a greater understanding of hornet grooming behaviour and the removal of diseased individuals (larvae, pupae and adults) is required to determine the likelihood of success of such an approach.

The mermithid nematode Pheromermis was discovered in V. velutina nigrithorax in France in 2012. This parasite requires two hosts to complete its life-cycle, it infects social wasps and Vespa crabro. When assessing its use as a biological control agent, modelling the effects of the nematode on the host predicted that even at an 80% rate of infection of individuals in the nest, colonies can still produce reproductive individuals P a g e | 16

(Villemant et al., 2015). It has also been deemed unsuitable for use as a biocontrol agent against wasps.

Unfortunately, nematodes and parasitoids are only able to act upon individuals of a colony and are therefore highly unlikely to be effective at disrupting an entire colony sufficiently to prevent reproduction. Viral pathogens, however, are very effective at disrupting the close-knit lifestyle observed in social insects. Very few viruses have been documented that cause death in Vespidae. Sacbrood virus and cricket paralysis virus injected into V. germanica pupae caused 100% mortality (Rose et al., 1999). Other viruses found to infect Hymenoptera include the baculovirus, nuclear polyhedroses, which can infect sawflies (Federici BA, 1993) and cricket paralysis virus, which infects Vespa crabro, Dolichovespula media, Vespula germanica and Vespula vulgaris (Rose et al., 1999). Research would be necessary to determine the susceptibility of V. velutina nigrithorax to a range of viruses and the potential off-target effects to native fauna, but this mode of control has the potential to be self-replicating and disruptive to sexual stages.

To date, there are no reports of successful use of biological control against V. velutina nigrithorax.

6. Novel control techniques for control of Vespa velutina nigrithorax

6.1 Sterile Insect Technique

6.1.1 An Introduction to SIT The sterile insect technique (SIT) is a species-specific method of insect control developed in the 1940s. SIT is the only technology available that can consistently eradicate insect populations on an area-wide basis (Alphey et al., 2010). It is species specific, causes no harm to the environment (on the contrary, use of pesticides is often reduced) and has no negative implications for human or animal health. SIT involves the mass rearing of a pest insect species, followed by radiation sterilisation and release of the sterilised individuals. Successful SIT has been used against New World screwworm (Cochliomyia hominvorax), codling moth (Cydia pomonella), the Mediterranean fruit fly (Ceratitis capitata) (Alphey et al., 2010). Wild females mated to released, sterilised males will have a significantly reduced number of progeny or no progeny. For this technique to be effective, sterile males must be continually released into an area to reduce the population. Successive application over many generations can lead to the local eradiation of populations (Klassen, 2009). The most successful SIT programmes release only males, as releasing females reduces the rate at which the released males will seek out and mate wild females, decreasing the overall effectiveness of the SIT programme. Released females are of no use to the control programme, although time and cost is exerted into their rearing. Additionally, they may be detrimental as agricultural pest females will still damage crops despite being infertile and infertile female mosquitoes still bite. To remove females from a target population, mosquito control programmes sort the sexes by pupae size (Klassen, P a g e | 17

2009) and medfly control programmes use a translocation based sexing system (Robinson, 2002). Considerable cost and effort may need to be incurred to develop a system of sex sorting and to sort sexes on a large scale. SIT programmes may release millions, or billions, of sterilised males per week so sorting sexes manually is no trivial matter.

Insects are most commonly sterilised using gamma radiation from Cobalt 60 or Caesium 137 (Consultants group meeting, 2000) this induces damage in chromosomes resulting in damage to sperm. It should be noted that while the sperm are damaged, the males still produce somewhat viable sperm, semen and seminal proteins capable of inseminating a female. This is an important point because it is the presence of sperm that will induce females to become refractory to further mating. However, the process of irradiation also damages somatic cells resulting in a reduced overall quality of the insect, i.e. reduced lifespan, reduced male competitiveness and reduced flight ability (Lance and McInnis, 2005). Therefore, the radiation dose must be restricted to a level and life stage whereby the males are effectively sterilised without significant deleterious effects on insect quality.

The most successful example of the application of SIT is the case of the New World Screwworm Cochliomyia hominivorax, a serious livestock pest in Central America. In the late 1950s, the USA began producing sterile male screwworm in mass-rearing facilities for irradiation and release; they were able to eliminate screwworm from the USA by 1966 and later in Mexico, Panama, Guatemala, El Salvador, Honduras, Nicaragua and Costa Rica (Klassen and Curtis, 2005). Currently, release of radiation sterilised screwworm along the Columbian border prevents re-invasion of these areas. In 1988, screwworm were discovered in Libya and SIT was used to prevent screwworm from spreading across North Africa and the Middle East. Despite ideal conditions for screwworm dispersal throughout the Middle East, the incursion was halted by 1992 through release of sterile screwworms that were shipped from an SIT facility in Mexico.

Most examples of successful SIT programmes are found against Dipterans; this does not necessarily mean that they are intrinsically more amenable to SIT control programmes; only that many pest species belong to this order (Lance and McInnis, 2005). The codling moth, Cydia pomonella, belongs to the order Lepidoptera and is the primary pest of apples and pears across the world. In Canada, a SIT programme against Codling moth resulted in a 91% reduction in codling moth damage in orchards as well as an 82% reduction in organophosphate pesticide usage (Bloem et al., 2005). Many agricultural insect pests belong to the order Coleoptera and these too have been the target of SIT programmes. Success has been variable, with localised elimination of one species of Coleoptera from an island in Japan (the sweetpotato weevil, Cylus formicarius) but limited success in another species, the bollweevil, Anthonomus grandis grandis. Table 3 summarises the effectiveness of a small number of SIT programmes. P a g e | 18

Table 3. Effectiveness of area-wide SIT programmes against a selection of insect pests

Target species Insect Order Result against target population Successfully eradicated from US and Mexico Cochliomyia Diptera and contained an introduction in Europe homnivorax/Screwworm (Klassen and Curtis, 2005). Successfully suppressed in Burkina Faso and Glossina Nigeria but failure to implement an area-wide Diptera morsitans/Tsetse programme led to re-infestation (Klassen and Curtis, 2005). Anastrepha Eradicated from Mexico and Texas in 1974 Diptera ludens/Mexfly (Klassen and Curtis, 2005). Eradicated from USA and Mexico in 1982 Ceratitis capitata/Medfly Diptera (Klassen and Curtis, 2005). Bactrocera Eradicated from the whole of in Japan in 1993 Diptera cucurbitae/Melonfly (Klassen and Curtis, 2005). Aedes aegypti/ Yellow Unsuccessful due to political turmoil (Klassen Diptera fever mosquito and Curtis, 2005). Cydia pomonella/Codling Population suppressed in Canada (Bloem et al., Lepidoptera moth 2005). Euscepes postfasciatus/ SIT programmes currently ongoing in Japan Coleoptera sweetpotato weevil (Ichinose et al., 2016). Cylus formicarius / Eradicated from Kume Island in Japan (Ichinose Coleoptera sweetpotato weevil et al., 2016). Anthonomus grandis Limited success; used in conjunction with Coleoptera grandis/ Boll weevil pesticides (Klassen and Curtis, 2005).

6.1.2 Developing SIT for control of Vespa velutina nigrithorax

There are certain characteristics of insect mating systems that make a given species more or less amenable to SIT, this is summarised in Table 4 alongside a comment on each given trait in V. velutina. Lance and McInnis (2005) define the favourable characteristics for SIT as those species with a simple mating system (i.e. no elaborate courtship rituals), species where females produce pheromones to attract males rather than males attracting females, species where males are long lived and actively dispersed (rather than aggregated species that mate in swarms) and species where the female only mates once. The more elaborate the mating system, the more difficult and expensive it would be to implement an SIT programme. Unfortunately, a lot of information about the reproductive habits of V. velutina remains to be clarified P a g e | 19 including most aspects of courtship behaviour, the identification of pheromones and post-mating sperm selection.

From what is known, there are a few potential problems for developing SIT against V. velutina. Eusocial insects are potentially problematic to control with SIT (Lance and McInnis, 2005). Vespa velutina are haplo-diploid, which often presents a problem for deployment of SIT as females can still give rise to progeny in the absence of viable sperm. In the case of V. velutina however, healthy sperm are crucial for the production of the female workers that maintain the colony so perhaps this particular concern is unwarranted. Vespa velutina only have one reproductive generation per year; this may also be a problem as SIT against a univoltine species has only been performed experimentally (Alphey et al., 2010) so its success cannot be predicted. Further research into the reproductive characteristics of Vespa velutina would certainly need to be performed before an SIT programme could be fully considered.

Table 4. Aligning reproductive characteristics beneficial and detrimental for SIT (from Klassen and Curtis, 2005) with the biological characteristics of Vespa velutina nigrithorax

Optimal mating characteristics for SIT Is characteristic present in Vespa velutina? Females attract males for mating Unknown Some evidence of multiple mating Females mate with one or few males (Monceau et al., 2013a) Males are long lived Yes Homogenous distribution No No complex courtship ritual Unknown Post copulatory sperm selection Unknown Characteristics that would severely limit

SIT Parthenogenesis Yes; but only males produced Synchronous, aggregated mating system No Migratory behaviour No Sterilised males themselves a pest No

A further problem of developing SIT against V. velutina is that a system for mass- rearing is crucial. Small insects, like many Dipterans, are easy to mass rear; they are generally small insects with short generation times and have limited mobility as pupae, which makes them easy to irradiate, transport and distribute (Lance and McInnis, 2005). Vespa velutina are neither small nor do they have fast life cycles so a mass- rearing system would be more difficult to develop. In V. velutina the developmental P a g e | 20 stages are tightly controlled inside larval cells and the developing larvae are fed by adult workers, replicating this in an artificial situation would be difficult. Although there is currently no mass rearing protocol for V. velutina, researchers have attempted to propagate them under laboratory conditions. Unfortunately, the success of this attempt was limited and the hornets were not viable long term (Monceau et al., 2013b). This is a major obstacle for implementation of SIT and would need to be in place before an SIT programme could be effective.

SIT is particularly suitable for new introductions, provided the response is rapid, as SIT is at its most effective at low population densities such as those involved in a new invasion as released males actively seek out the wild females, which are still comparatively rare at this point (Alphey et al., 2010). Following elimination, sterilised insects can be continually released forming a permanent barrier to re-invasion. However, the cost of implementation is considerable, and a strong economic case would need to be made for its use. SIT programmes require significant investment: $40 million was spent for SIT against codling moth in Canada across 14 years (Bloem et al., 2005) and to eradicate medfly from Queensland, the cost was $33 million across 9 years (Mumford, 2005), For the medfly in Queensland, an SIT project ran at cost for 6 years, although the later returns justified the investment by money saved in reduced pesticide use, reduced losses, reduced pesticide residues on commodities and lower costs for quarantine and emergency control programmes (Mumford, 2005)

In the case of V. velutina, the economic impact is restricted to the loss of honeybee colonies. One study cites that the losses of honeybee colonies due to V. velutina is approximately 5%, however this is the result of a survey rather than a systematic analysis of economic outputs (Monceau et al., 2013a). The value of the UK apiculture industry is approximately £225 million per annum from honey production and pollination services (DEFRA, 2009) and in France it is estimated to be approximately 550 million Euros per annum (The Food and Agriculture Organization of the United Nations, 2006). The research needed to gain sufficient information on the feasibility of an SIT programme against V. velutina includes gaining more information on mating systems, developing and optimising a mass-rearing protocol (diet and environment must be considered, removal of females might be a consideration) and optimisation of sterilisation procedure (radiation dose required to elicit sterility, effects of radiation on lifespan and male competitiveness) – and this is before considering the cost of a mass- rearing facility, sex separation and transport and release of insects. It remains to be understood whether the economic benefits would justify these costs.

Sterile insect technique does not release any genetic changes into the environment and it has been used in Europe before. In Italy SIT was used to target Aedes albopictus in a pilot mass rearing programme, rearing 20,000 male mosquitoes per week for release and significantly reduced the population (Bellini et al., 2007).

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6.2. RNA interference

6.2.1 An introduction to RNAi RNA interference (RNAi) is a method of gene regulation that silences the expression of genes of an organism or the expression of genes from invading pathogens. The presence of double stranded RNA (dsRNA) in the cell, elicits a response which cleaves all RNA fragments possessing that sequence. This endogenous pathway can be manipulated by the introduction of in-vitro synthesised dsRNA or short interfering RNAs (siRNA), which are homologous to a target gene or genes and knock out its function. This method has been used for over 15 years to silence genes for experimental and practical purposes (Huvenne and Smagghe, 2010) and its use as a control agent for invertebrates has been explored.

The proposed mechanism of action for insect control is to design a synthetic dsRNA against an essential gene with a unique sequence in the target species allowing a species-specific lethality to be induced. The use of dsRNA to silence gene expression has been successful across many insect orders including Diptera, Lepidoptera, Coleoptera, Isoptera, Hymenoptera, Orthoptera and Hemiptera (Burand and Hunter, 2013); a small number of examples are shown in Table 5. Many agricultural pests have been targeted with RNAi in an attempt to reduce crop damage. One example is the use of dsRNA against the Lepidopteran pest, Western corn rootworm, Diabrotica virgifera virgifera LeConte, a pest of corn. By feeding dsRNA targeting an enzyme called V-ATPase (a proton-pump transmembrane protein), researchers were able to induce a systemic silencing of this gene, which resulted in death of the larvae (Baum et al., 2007).

Table 5: Application of dsRNA in a small selection of pest insects summarising method of application and success of gene knockdown (aWalshe et al., 2008, bPridgeon et al., 2008, cBaum et al., 2007, d Mohamed and Kim, 2011, eAraujo et al., 2006, f Amdam et al. 2006, g Campbell et al., 2010.)

Species Order Method Success Glossina morsitans Diptera Microinjection Successful knockdown of a target morsitansa Feeding gene in midgut over 20 days Unsuccessful on fat-body gene Aedes aegypti b Diptera Microinjection Topical application less effective Topical than microinjection

Diabrotica virgifera Coleoptera Feeding Lethal to beetle larvae; reduced virgifera c damage to corn roots

Plutella xylostella d Lepidoptera Feeding Suppression of target gene 24 -72 hours post feeding, 100% host lethality after 5 days at high dose P a g e | 22

Rhodnius prolixus e Hemiptera Feeding Feeding and injection equally Microinjection effective at reducing levels of anticoagulant proteins in 48 hrs Apis mellifera f Hymenoptera Microinjection Significant knockdown of Vitellogenin by microinjection of dsRNA Varroa destructor g Parasitiformes Soaking Soaking in dsRNA effective at (Arachnida) Microinjection knocking down target transcript by 87%

The primary consideration for the application of RNAi technology is the mode of application. The dsRNA is administered to the insect via a route that must be determined as being effective and practical for the target species. Delivery of dsRNA to insects can be done in a variety of ways including direct microinjection, ingestion and absorption. Response to administration may depend upon the species being tested, with some being more amenable to a certain mode of delivery than others (Whangbo and Hunter, 2008). Microinjection is not an option for large scale administration to an insect pest. Oral administration is more appropriate for large scale administration, but some species are less amenable to oral delivery due to host enzymes or microbes in the digestive tract which may affect the efficacy (Palli, 2014). Absorption has been shown to work in a limited number of species, for example dsRNA is effective when administered in 0.9% NaCl to Varroa destructor, which are subsequently immersed in the solution (Campbell et al., 2010), but is not an effective means of administration for many species. Absorption may be less effective than other modes of delivery. For example, in the Yellow Fever virus vector, Aedes aegypti, it was demonstrated that mosquitoes could be killed with microinjection of dsRNA products against apoptotic inhibitor genes, however, when the product was administered through topical application it was almost 50% less effective (Pridgeon et al., 2008).

RNA is not stable in the environment, often breaking down within hours (Zhang et al., 2010). This is a benefit from an environmental perspective provided there is an effective means of delivery. Large scale administration of dsRNA to insects in the field has had some success, with administration of IAPV (Israeli Acute Paralysis virus) dsRNA to 20 colonies of honeybees in the USA resulting in improved colony performance. The dsRNA was administered through the addition of 10 mg dsRNA in 500 ml of sugar water, per colony, and resulted in a reduction in death due to IAPV (Hunter et al., 2010). When designing the dsRNA, the US Environmental Protection Agency regulations stipulated that the sequence must not be identical to any other bee sequences to rule out off-target effects (Maori et al., 2009). Off-target effects (defined as disrupting the effect of any other genes in the target organism, or genes in non- target organisms) must be considered for any field application of dsRNA, especially in the knowledge that hornets closely interact with pollinators. Off-target effects of RNAi P a g e | 23 have been reported to occur. Recommendations to avoid such effects include the use of siRNAs rather than longer dsRNAs, the use of multiple siRNAs and performing a rescue of the mutant phenotype (Moffat et al., 2007). Vespa velutina are closely related to honeybees, therefore careful consideration of gene targets will be required as there may be considerable sequence similarities.

One consideration for the use of dsRNA is the cost of synthesizing dsRNA in large quantities. Using laboratory research kits for synthesis of dsRNA for field use is certainly not cost effective. Due to commercialisation of dsRNA technologies, techniques for large scale production of dsRNA is increasing and cost is decreasing; in 2008 it was $12,500 per gram of dsRNA (de Andrade and Hunter, 2016) and it can currently be purchased online for as little as $48 per gram (AgroRNA, 2016). dsRNA can also be synthesized to high levels by bacteria; this was demonstrated in dsRNAs which were synthesized by Escherichia coli bacteria that has been transfected with a dsRNA vector (Cedeño et al., 2015). Bees were fed with these bacteria and found to express the dsRNA at 7 days post-feeding; Varroa feeding from these bees also acquired and expressed the dsRNA (Cedeño et al., 2015). This strategy has also been effective in the fruit fly, Bactrocera dorsalis, (Li et al., 2011) and the beet armyworm, Spodoptera exigua (Tian et al., 2009). Recombinant bacteria are not expensive or time-consuming to create and can be cultivated in large volumes.

6.2.2 Developing RNAi for control of Vespa velutina nigrithorax

The development of RNAi in Vespa velutina nigrithorax would involve a careful selection of target genes, followed by a screen of their effectiveness and off-target effects. Careful design of dsRNA is critical, the selected gene should be specific to the target species, and it should silence a gene that is crucial for life function where the insect does not possess similar genes which may compensate for the deficit in expression of the other. Without a full, annotated genome sequence for Vespa velutina nigrithorax, before designing dsRNA, there would be a need to identify genes of interest and compile expression profiles for each one. Genes of interest that might be explored are those required for normal metabolic functions, flight, reproduction and feeding. Direct lethality isn’t necessarily the end-point, any disruption that eventually leads to the death of an individual or colony would be just as useful.

A mode of delivery must be examined specifically for V. velutina. In the case of V. velutina, in vivo studies would have to be performed systematically on an ideal method and concentration for dsRNA delivery. If dsRNA could be administered to hornets through feeding, a possibility would be the use of bait or lures designed to specifically attract V. velutina to the feeding site. However, no such lures have been identified as yet and if they did exist, hornets could be trapped and killed without the need for dsRNA. Alternatively, apiaries could be fed with dsRNA (similar to the IAPV trial) so that hornets predating on honeybees would receive a dose of hornet specific dsRNA. It would be difficult to determine if honeybees alone comprise a large enough proportion of the Asian hornet diet to result in a strong effect. It is known that the P a g e | 24 protein rich meals collected by hornets are taken back to the nest to feed developing larvae Approximately 37% of the Asian hornet diet is comprised of honeybees (this is higher in urban areas at 66%) and this protein rich meal will be collected by the workers and taken back to the nest to feed the developing larvae (Robinet et al., 2016, Villemant et al., 2011). One idea could be to test if dsRNA administered to hornets through feeding, could be taken back to the nest and expressed in developing larvae. If this were the case, dsRNA against developmental genes might result in the collapse of a nest through disruption of developing brood or the prevention of sexual development in the developing larvae destined to be reproductives.

RNAi is not constitutively expressed, it evokes only a transient RNAi response (Whitten et al., 2016) and it may take a number of days for gene silencing to take effect; it can be introduced through feeding but is unlikely to be effective from a single feed. The studies cited perform either continuous or repeated feeding time courses to elicit knockdown of the target gene(s): 24 hours continuously in bees (Cedeño et al., 2015), daily for 4 days in Anopheles gambiae (Zhang et al., 2010) and 5 or 6 consecutive feeds over a 2-3 week period (Hunter et al., 2010. This would need to be considered when testing feeding regimens for dsRNA induced lethality.

As with any control mechanism, the risk of resistance must be considered. Similar to pesticide resistance, mutations in genes that are a target of dsRNA can arise. So far there is evidence of this in the nematode Caenorhabditis elegans (Tabara et al., 1999) and some viruses have been shown to escape RNAi strategies due to their high mutation rate, HIV (Das et al., 2004). Designing multiple target genes may help to reduce the risk of resistance arising, but it is unlikely to reduce the risk to zero. A risk assessment would need to be performed before dsRNA could be used in the field, assessing off-target effects, environmental persistence and risk of resistance (Ana María Vélez et al., 2016). However, dsRNA is not regulated for use in the UK, although it has gained approval for field testing in the USA (Hunter et al., 2010), its use in the UK would set a precedent.

6.3 Genetic transformation and genome editing

6.3.1 An introduction to germline transformation Genetic modification refers to the deliberate introduction of genetic changes into the genome of a target organism. The term genetic modification could be used to encompass organisms whose genomes have been altered over time using selective breeding, or through induced translocations, or organisms that have been deliberately trans-infected with another biological agent which induces a genetic change (such as Wolbachia). For the purposes of this review, the term ‘transgenic’ will be used and refer only to the directed integration of artificial genetic elements and non-native genes into the host genome.

Most transgenic organisms have been created through the use of mobile genetic elements termed ‘transposable elements’ that are used to insert genes into an P a g e | 25 organism. These elements are sequences of DNA which flank genes of interest such as genes for identification (i.e. fluorescent proteins), conditional lethality (to kill the target organism), parasite inhibition (i.e. to kill malaria in the mosquitoes that harbour it) or to knock out the function of certain genes (as used in research studies). However, more recently, technology has advanced such that these changes can be introduced without the need for transposable elements. The use of the “clustered regularly interspaced short palindromic repeats” or CRISPR system allows for site-directed introduction of engineered sequences, removal or manipulation of endogenous genes (Hammond et al., 2016).

These foreign DNA elements, termed ‘constructs’, are introduced into the genome of insects through germline transformation. Germline transformation involves the injection of the DNA constructs into the posterior pole of very early developing embryos of the target insect. The posterior pole of the embryo is the site at which germ cells develop; the aim of the procedure is to introduce the constructs into the DNA of these germ cells. When this occurs, the organism with transgenic germ cells will produce progeny that possess both somatic and germ cells that are transgenic.

Many insect species have been transformed such as agricultural pests; medfly (Fu et al., 2007), pink bollworm, Tribolium castaneum; beneficial insects: Apis mellifera, Bombyx mori (silkworm); and vector insects: Aedes aegypti, Anopheles gambiae (Braig and Guiyun, 2002). Reasons for transformation may include the creation of genetic sexing strains for use in SIT (Fu et al., 2007), increasing yield and quality of silks from silkworm (Xu and O’Brochta, 2015) or to induce sterility in a pest population to induce its collapse (Harris et al., 2012). The primary obstacle for creation of a new transgenic is the development of a method to transform the target species, not all species are equally amenable to genetic manipulations. Honeybees, for example, are not easily transformed; germline integration has not worked well in the laboratory as workers can detect damaged embryos and remove/ignore them. Additionally, rearing embryos to adulthood is very difficult due to issues with honeybee survival under artificial rearing conditions (Robinson et al., 2003). While honeybees might be amenable to genetic modification, their rearing system is not. Germline integration in possible in another Hymenopteran, the sawfly (Sumitani et al., 2003) but sawflies can be removed from their natural rearing environment and cultivated in the laboratory.

Para-transgenesis is the transformation of commensal organisms that reside within a target insect, for example bacterial symbionts (Rhodococcus rhodnii) from Rhodnius prolixus (the Chagas disease vector) were genetically modified to express dsRNA against vitellin, a gene essential for egg production. The insects that were fed the modified bacteria had lower levels of egg production and were confirmed to have no detectable levels of the gene transcript (Whitten et al., 2016). A similar strategy was applied to bacterial symbionts of thrips, Frankliniella occidentalis (Whitten et al., 2016), Anopheles gambiae and Rhodnius prolixus (Dotson, 2003). This process would necessitate a large-scale analysis of the gut microbiota followed by the creation of a transgenic strain of bacterium and its reintroduction into the host. If it were to be P a g e | 26 applied to Vespa velutina nigrithorax, it would be prudent to examine gut microbiota of honey bees and other pollinators to understand the probability of horizontal transfer of GM gut bacteria.

6.3.2 Developing genetic techniques for control of Vespa velutina nigrithorax

Germline transformation has not been tried and tested on V. nigrithorax and development of germline transformation for a control programme would require research testing of a variety of techniques for transformation; and, as with SIT, a method for mass rearing would be required for release of the modified individuals. However, before even attempting such a task, one would first have to determine whether genetic modification would be an appropriate strategy against V. velutina nigrithorax. The purpose of genetic modification, in this context, would be to introduce a genetic mechanism to suppress the next generation. For example, genetically modified males carrying a lethal/disrupting gene could be released to mate with any virgin queens, potentially suppressing the formation of nests by those females.

Not only does this require the development of a technique to genetically modify and mass-rear Vespa velutina, it requires an understanding of when and where to release modified insects, the likelihood that these insects will mate with wild insects and a determination of how many insects need to be released to disrupt the population (through mathematical modelling, which requires an understanding of host biology and quantification of the effectiveness of the induced lethality). In addition to this and the regulatory issues involved in releasing genetically modified arthropods, the feasibility of using genetic modification for control of Vespa velutina, seems very low.

6.3.3 Regulatory issues for use of genetic pest control technologies

The problems regarding the use of genetically modified organisms are more insoluble than the biological, logistical and cost/benefit considerations of other techniques. Genetic editing technology in the UK is deeply complicated by regulation, politics and a poor public image. While the science moves forward at a fast pace, developing safer and more tractable genetic tools; regulation has stalled and is failing the development of GM technology. To date, there have been no releases of GM insects within the EU, although the technology exists to do so. The current framework in the UK involving the release of GM insects is underpinned by EU directives. The House of Lords, Science and Technology Select committee reported upon the issue of the release of GM insects and concluded that the EU regulatory system is failing the development of potentially beneficial technology (House of Lords: Science and Technology Select Committee, 2015).

The UK GM insect company Oxitec applied for permission to conduct field trials with genetically modified Olive fly (Bactrocera oleae) in 2012 and in 2015 to the Spanish National Authorities; both requests were refused despite Oxitec addressing the issues that caused the first rejection (House of Lords: Science and Technology Select P a g e | 27

Committee, 2015). Oxitec has conducted field trials releasing GM mosquitoes in other countries including the Cayman Islands (a British Overseas Territory), Malaysia, Panama and Brazil (Oxitec, 2016). The House of Lords: Science and Technology Select Committee conclusions included a recommendation that the Government actively pursued and invested in GM insect field trials. A similar conclusion was reached by the WHO, with the recommendation that there should be pilot trials of transgenic mosquitoes for vector control (World Health Organisation, 2016).

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7. Conclusion and recommendations

Conclusion

There is no single method of control likely to result in elimination of an invading species. An approach, tailored to the specific biology and life history of the target species is required utilising a range of strategies for integrated pest management (IPM). For example, SIT in codling moth (Cydia pomonella) is practiced alongside good tree husbandry and orchard management, chemical sprays applied at critical life stages of the moth, mating disruption with sex pheromones and pheromone traps for population monitoring (Whitten and Mahon, 2005). Combining strategies can also help reduce off-target effects, for example development of an effective attractant for a pest can make the usually broad-spectrum action of an insecticide more species specific (Alphey et al., 2010).

In addition to an integrated approach, for an effective control programme there must be a system of monitoring/surveillance of the population. In France, prevention, monitoring and management of Vespa velutina is not mandatory (Robinet et al., 2016). However, they do have a citizen science project for identification and reporting sightings and the location of Asian hornets, which is then entered into a database at the French Museum of Natural History (Robinet et al., 2016). Locating hornet’s nests is very difficult so a method of nest detection should be investigated alongside development of control mechanisms.

Key findings for control options for V. velutina nigrithorax have shown:

 There is no published information on the most effective chemical control agents for V. velutina nigrithorax  There are no chemicals licenced for use as toxic baits against Vespa velutina nigrithorax.  There is no biological control agent that has been demonstrated to successfully decrease Vespa velutina nigrithorax populations.  In the immediate term, there is insufficient information on the biology and reproduction to develop a novel control technique against Vespa velutina nigrithorax.  Any control strategy is contingent on a fast and robust system of detection for nests of Vespa velutina nigrithorax.

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Recommendations: Short term

At low levels, individual nests are more easy to detect and can be controlled with direct application of insecticides, as was done with the nest in Tetbury. The efficacy of actives currently approved for the treatment of wasp nests in the UK should be evaluated against V. velutina nigrithorax. Other insecticidal actives currently approved under the BPR should also be considered within this screen.

The potential of a lure and kill system using a chemical or biological agent should be investigated.

An early warning system is crucial for preventing the establishment of Vespa velutina nigrithorax. Given the difficulty locating individuals and nests and the absence of lures or technology to do so, the most cost-effective and widespread resource available may be members of the public. The use of ‘citizen science’ has been successful in Italy for assessing the use of a formic acid treatment against Varroa destructor called Varterminator (Nanetti et al., 2016). Citizen science could be used to involve beekeepers and the wider public to deploy hand-made hornet traps and out-reach activities emphasizing the risks of invasive species could aid in the detection of Vespa velutina nigrithorax. More professional monitoring activities could be set up in areas determined as invasion ‘hot-spots’.

Recommendations: Medium term

In the worst-case scenario that Vespa velutina nigrithorax becomes established in the United Kingdom, the chances of eradication are virtually nil. At this point, the only option is management and a more extensive range of control options would be required for this, ideally based on lure and kill approaches owing to the challenge of the detection of nests, especially as nests reach higher densities.

Recommendations: Long term

A lot of the information on Vespa velutina biology is extrapolated from other eusocial hymenoptera. However, species can differ considerably, particularly when it comes to reproductive traits. Techniques such as SIT, application of dsRNA and transgenesis all require further understanding of many aspects of the biology of Vespa velutina nigrithorax before they can be recommended. Research into the reproductive biology and methods for population surveillance would be the first step towards developing these techniques. This would be a long term endeavour and would require significant investment, personnel and specialised facilities.

The most obvious questions that need answering for the development of effective control strategies include the specifics on reproduction in Vespa velutina nigrithorax (how many males does female mate with? Where do they mate? Is there sperm competition? Are they radiation sensitive? Would a sterile male have any effect on P a g e | 30 female reproduction?), queen behaviour (How far do they migrate? Do they hibernate directly after mating? How far from their originating nest do they hibernate? Where do they like to hibernate? Can we locate and exterminate hibernating queens?), foraging behaviour and range. This data could feed into mathematical modelling systems for determining the outcome of pest control strategies and predicting how, where and when to apply control. Even given an appropriate method of control, no model can predict the outcome of control attempts without more information on the biology of the organism.

P a g e | 31

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