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Cefas contract report C5525 A review of potential methods to control and eradicate the invasive gammarid, Dikerogammarus villosus from UK waters Paul Stebbing, Stephen Irving, Grant Stentiford and Nicola Mitchard For Defra, Protected Species and Non-native Species Policy Group Commercial in confidence Executive Summary The killer shrimp, Dikerogammarus villosus (Dv) is a large gammarid of Ponto-Caspian origin Dv has invaded and spread over much of mainland Europe where it has out-competed a number of native species. Dv was discovered at Grafham Water, Cambridgeshire, England, in September 2010 and subsequently in Wales in Cardiff Bay and Eglwys Nunydd near Port Talbot. In early 2012 it was found in the Norfolk Broads, the full extent of its distribution in the area is still being determined. The main objective of this work was to review the potential approaches for the control/eradication of invasive Dv populations in the UK. The approaches reviewed include physical removal (e.g. trapping), physical control (e.g. drainage, barriers), biological control (e.g. predation, disease), autocides (e.g. male sterilization and pheromone control) and biocides (the use of chemical pesticides). It should be noted that there have been no specific studies looking at the control and/or eradication of this particular species. The examples presented within this study are therefore primarily related to control of other invasive/pest species or are speculative. Recommendation made and potential applications of techniques are therefore based on expert opinion, but are limited by a relative lack of understanding of the basic life history of D. villosus within its invasive range. Following a review of the literature directly associated with Dv, and broader, including the control of other invasive aquatic invertebrates, we highlight the following areas that may form effective control and/or eradication mechanisms: 1. The examination of the effects of electricity on Dv 2. Investigation of methods to increase trap effectiveness 3. Exploration of approaches for the use of habitat modification as a method of reducing population size 4. Investigation into the potential for population control using Dv pathogens currently absent from UK populations 5. Investigate delivery methods for six biocides that have been shortlisted due to their suitability. It is unlikely that a single control mechanism will result in the eradication of Dv. It is more likely that a range of mechanisms will be required, especially given the variety of environments that Dv are found. 2 Contents Executive Summary................................................................................................................................. 2 1.0. Introduction ................................................................................................................................ 4 1.2. Objectives..................................................................................................................................... 5 2.0. Dikerogammarus villosus life history, behaviour and habitat preference ................................. 7 2.1. Growth and reproduction ............................................................................................................ 7 2.2. Habitat preference ....................................................................................................................... 8 2.3. Feeding ......................................................................................................................................... 9 3.0. Physical removal ....................................................................................................................... 10 3.1. Bank side applications........................................................................................................... 10 3.2. Trapping ................................................................................................................................ 10 4.0. Physical Control......................................................................................................................... 13 4.1. Habitat modification ............................................................................................................. 13 4.2. Physical Barriers .................................................................................................................... 13 4.3. Chemical and electrical barriers............................................................................................ 14 4.4. De-watering .......................................................................................................................... 15 5.0. Biological control ...................................................................................................................... 16 5.1. Predation............................................................................................................................... 17 5.2. Pathogens ............................................................................................................................. 17 6.0. Autocidal ................................................................................................................................... 23 6.1. Semiochemicals and pheromones ........................................................................................ 23 6.2. Male sterilisation. ................................................................................................................. 29 7.0. 7.1. 8.0. Biocidal control ......................................................................................................................... 33 Compounds warranting further evaluation .......................................................................... 36 Synopsis and Recommendations .............................................................................................. 44 Appendix 1 ............................................................................................................................................ 48 References ............................................................................................................................................ 54 3 1.0. Introduction The killer shrimp, Dikerogammarus villosus (Dv) is a large gammarid of Ponto-Caspian origin (Tricarico et al. 2010). Dv exhibits several biological characteristics which contribute to its environmental impact: long reproductive period, early sexual maturity, short generation time, high growth rates, short duration of embryonic development, large number of eggs, large reproductive capacity, highly predatory and tolerant of a wide range of environmental conditions (Dick and Platvoet, 2000; Devin et al. 2004; Kley and Maier, 2006; Pockel 2009). These biological characteristics have made Dv an effective invasive species with only a few individuals required to establish new populations in recipient ecosystems (Devin et al., 2004). Dv has invaded and spread over much of mainland Europe where it has out-competed a number of native species (Van den Brink and Van der Velde 1991; Dick and Platvoet 2000; Kinzler and Maier 2003; Kley and Maier 2003; Grabowski et al. 2007) including Britain (MacNeil et al. 2010). Ponto-Caspian macro-invertebrate species entering mainland Europe has mainly been facilitated by the interconnection of river basins through man-made canals and intentional introductions (Bij de Vaate et al. 2002). A southern corridor connecting the Danube and Rhine rivers is likely to have been the route of spread of D. villosus via the hulls of ships or in ballast water (Casellato et al. 2007). The reopening of the Main– Danube Canal in 1992 matches with the recorded spread of this species across Europe, with water supplied from the Danube basin flowing into the Rhine facilitating migration. In its natural range, D. villosus is not the most abundant species of amphipod and does not behave as aggressively as it does in areas it has invaded. Wattier et al. (2007) stated that all major harbours of western continental Europe are likely colonized by D. villosus (including Rotterdam, Le Havre, Nantes and Marseille), and that further expansion in the range by commercial shipping activity is only a matter of time (Bollache et al. 2004). Dv was discovered at Grafham Water, Cambridgeshire, England, in September 2010 and subsequently in Wales in Cardiff Bay and Eglwys Nunydd near Port Talbot. In early 2012 it was found in the Norfolk Broads, the full extent of its distribution in the area is still being determined. The prevention of the species further spread has been one of the main priorities of the Science and Technical Advisory Group (STAG), which was established to address immediate containment, associated risks, and long term risk management of Dv. All of the invaded sites in the UK are used for a number of recreational activities including sailing and angling, with members of the public using equipment at these sites that may subsequently be used at other freshwater venues in Great Britain. Dv has been found to readily attach to equipment that is used in water, such as sailing vessels, wetsuits, and fishing nets. These fomites (inanimate objects capable of carrying organisms and hence transferring them between water bodies) pose the 4 potential risk of spreading Dv to un-invaded ecosystems. Given the ease with which Dv can be spread, and the impact that the species has on invaded environments, there is a requirement to investigate methods by which it can be controlled or eradicated. Given the locations in which Dv are currently found in Great Britain, potential options also need to be considered, taking into account the following issues associated with potential eradication methods: 1. Safety of use in the vicinity of drinking water sources (non-harmful to humans); 2. Effect on the functionality of treated waters – including any necessary periods of isolation etc; 3. Effect on or risk to other species and environmental impact (e.g. invertebrates, fish, aquatic mammals, birds, dogs, farm animals, etc.); 4. If a chemical method is selected, issues concerning degradation of the chemical in the environment or safe removal/neutralising in water treatment process; 5. If a chemical method is selected, regulatory issues concerning use of the selected product; 6. Practical aspects concerning effective delivery/application in large bodies of water (and potentially running water systems) – including need for repeat treatments, time intervals etc; This review of the existing knowledge, tools and techniques that could potentially be applied to the control of Dv provides a strong basis from which to assess the feasibility of eradication and/or long term control of this species. 1.2. Objectives The main objective of this work was to consider whether there is likely to be an effective method for controlling/eradicating populations of Dikerogammarus villosus (Dv) in the UK. This was determined by undertaking an extensive bibliographic review of both published and ‘grey’ literature. The following techniques and tools commonly used in the control and eradication of pest species were reviewed: 1). Physical removal (e.g. trapping) 2). Physical control (e.g. drainage, barriers). 3). Biological control (e.g. predation, disease). 4). Autocidal (e.g. male sterilization and pheromone control). 5). Biocidal (the use of chemical pesticides). 5 All aspects of the life history of Dv were assessed to determine where potential control and eradication methods could be applied. Factors limiting the species distribution in its natural range, such as disease and predation, were also be considered. Information will be given, where possible, on overall likelihood of success, and recommendations for future work. It should be noted that there have been no specific studies looking at the control and/or eradication of this particular species. The methods presented within this study have therefore had to be assessed in relation to their use to control other invasive or pest species. Recommendation made and potential applications of techniques are therefore based on expert opinion, but are limited by a relative lack of understanding of the basic life history of D. villosus within its invasive range. 6 2.0. Dikerogammarus villosus life history, behaviour and habitat preference Invasive Alien species are often characterised (when compared to related native species) by a combination of large brood size, high partial fecundity, early maturation and a higher number of generations per year. They may also display higher tolerance towards severe environmental conditions, i.e. elevated salinity and human degradation of the environment. All of these traits have been observed in D. villosus when compared to native gammarids and other macro invertebrates. These features have facilitated the colonisation of new areas and competition with native species of D. villosus (Pöckl 2007, 2009). The spread of D. villosus has resulted in the replacement of indigenous gammarids over much of Europe (Dick and Platvoet 2000; Van Riel et al. 2006, 2007; Casellato et al. 2008; Leuven et al. 2009; Van der Velde et al. 2009), changing the composition of the invaded communities and damages to the food webs of the invaded water bodies (Dick et al. 2002; Haas et al. 2002; Van der Velde et al. 2002). D.villosus is known to be a highly mobile species with estimates of its potential for upstream spread to be up to 40km/year (Josens et al. 2005). However, their ability to colonise is limited by certain environmental constraints and preferences such as habitat preference. There is limited understanding of many aspects of Dv behaviour such as mate selection, feeding, and habitat selection of this species. All of the information provided in this section is based on existing knowledge of D. villosus outside of its native range, mainly from information from northern Europe. As there are many parallels between Northern Europe and the climate and water systems found in GB, then unless stated it is assumed that populations of Dv in GB will function in a similar manner. 2.1. Growth and reproduction D. villosus produces three reproductive peaks approximating to April, August and November per annum (Devin et al. 2004), although it will breed all year round in warmer climates (Casellato et al. 2006; Grabowski et al. 2007a; Pöckl 2009). However, ovigerous females have been found in December from populations in England (Cefas unpublished data) suggesting that temperature is not the limiting factor. Juveniles are present all year round, with a highest number in October, with adults becoming less common with the death of overwintering adults in April where there is an increase in the frequency of juveniles (Devin et al. 2004; Tricarico et al. 2010). There is no evidence available on how mate finding or selection functions in this species, but once pairs are formed the female is carried on the back of the male precopular. There is extensive use of kairomones (chemical signals) by crustacea, and it has been suggested that D. villosus use kairomones to avoid predators (Hesselschwerdt et al, 2007) and identify other heterospecifics. It would therefore not be improbable that a sex pheromone exists, but given the high densities that D. villousus occurs and frequency of breeding then chance encounters may suffice. 7 Most ovigerous females are found in October (Devin et al. 2004; Tricarico et al. 2010). Females as small as 6mm have been found to be ovigerous, however the majority of ovigerous females are >9mm (Devin et al. 2004). Each pair produces an average of 27 eggs, but up to 50 eggs can be released into the ventral brood chamber, although as many as 160 was reported by Kley (2003). Juvenile animals become sexually mature in 4 to 8 weeks (Devin et al. 2004). They are fast growing, during winter increasing by 1.3–2.9 mm in length per month and by 2.0–2.6 mm every two weeks in spring (Devin et al 2004). Populations are predominantly female (Casellato et al. 2006; Pöckl 2009), but this ratio seems to varies throughout the year with females representing 60%-70% of the population (Devin et al. 2004). Similar sex ratio fluctuations are observed in other crustacean populations as gender specific moulting is observed at certain stages of the life cycle e.g. females moulting after spawning. As animals tend to stay in shelter during the moulting process is avoid predation sampling techniques such as trapping and kick sampling would result in such biases being observed. There is no evidence to suggest that this ratio changes with resource availability or ecosystem degradation. D. villosus can grow up to 30 mm in length. It varies in appearance, with some specimens being striped, and some not. 2.2. Habitat preference D. villosus has a preference for rocky substrate where it takes advantage of crevices to avoid predation (Devin et al. 2003). Artificial habitats including concrete structures and large boulders appear to attract higher numbers of D.villosus (Van Riel et al. 2006). MacNeil et al. (2010) also found that D.villosus avoided silty areas but was frequently found in areas where boulders and pebbles were the dominant substrata. Dv is not found in soft clay substrates or watercourses with reed margins and fine sediment (Keenan and Rutt, 2012). It is also not found in pristine waters that are low in nutrients or in fast flowing water. D. villosus can colonise many types of habitat as it is able to tolerate a wide range of temperatures (0–35 °C) with optimal temperatures between 20 and 23°C, low oxygen concentrations, and salinity up to 24‰. It is found in lakes, canals and rivers where there is suitable habitat. DV has been closely associated with the presence of Dreissena polymorpha, the zebra mussel (Tricarico et al. 2010). It is thought that zebra mussels change habitats by increasing the amount of benthic organic matter, which benefits D. villosus helping them to out-compete other species. When given a choice, D. villosus spend more time feeding around zebra mussel shells than a bare natural substrate (Bruijs et al. 2001; Bij de Vaate et al. 2002; Devin et al. 2003; Brooks et al. 2009). It is considered that invasion by D. polymorpha has facilitated the establishment of D. villosus both as a form of introduction and as a preferred form of habitat (Gonzales and Downing 1999; Simberloff and Von Holle 1999; Jazdzewski and Konopacka 2002). Gergs R and Rothhaupt (2008) showed that D. 8 villosus have a preference for the odour of D. polymorpha shells with biodeposotion, suggesting that habitat and food availability dictates where Dv settle. Habitat is physically modified by the presence of D. polymorpha to the benefit to D. villosus, providing shelter as well as food in the form of faeces. It has been suggested that the stripes on D. polymorpha and Dv are of the same width and angle which may contribute to the selection of D. polymorpha as shelter by Dv. 2.3. Feeding D. villosus is omnivorous and feeds on a variety of invertebrates, including other amphipods, small fish and their eggs. Often it kills prey but does not eat it. It kills by biting prey with its large mandibles and then shreds it before eating it (Dick et al. 2002; Devin et al. 2003; Casellato et al. 2007; Platvoet et al. 2009). Dv are coprophages, not only consuming their own faeces, but also consuming mussel faeces and psedofaeces biodeposition (Gergs and Rothaupt 2008). This production of food material also provides food for detritivorous macroinvertebrates on which D.villosus actively predates (Ricciardi et al. 1997, Gonzales and Downing 1999). 9 3.0. Physical removal Trapping is effective for catching mobile animals that can be attracted to or will naturally enter traps at a high frequency e.g. crabs and lobsters. Alternatively traps can be effective when they are moved to intercept relatively less mobile animals e.g. trawling for fish. Trapping is less effective as a control measure where the capture range of the trap is small relative to the scale of the population. Where the scale of the population is significantly larger than that of the trap range a large number of traps need to be deployed to attempt a control or eradication programme. It is therefore important to try and increase the maximum range of individual traps to make them more effective and increase the traps CPUE (catch per unit effort). Attempts to eradicate an invasive population by physical removal can sometimes require considerable time, cost and manpower, especially if the species has become established. Furthermore success in the complete eradication of populations using this method has rarely been cited. Methods of physical removal have been enhanced e.g. by the use of pheromones, to greatly increase the efficiency of traps (see section 6). However employing eradication techniques in the early stages of distribution, if successful, is less costly than undertaking prolonged control programmes (Hyatt 2004). 3.1. Bank side applications Employing kick sampling techniques to disturb substrate prior to intercepting individuals with the use of sweep nets is a common sampling technique used by ecologists. The benefit of hand removal ensures elimination of all size classes which is rarely achieved through other techniques (Peay and Hiley 2001). Given the size and abundance of D.villosus the potential application of hand removal is considered impractical, however studies have been conducted which combine sweep netting with electrofishing to successfully catch crayfish. Unlike trapping this method has yielded greater success in catching all size groups (Westman et al. 1978). The limitation of this method is that its efficacy is reduced in deeper, more turbid waters. Westman et al (1978) recommended electrofishing by night when crayfish are most active in order to achieve higher catch rates. The efficacy of electrofishing on D.villosus needs to be established, but this method could be employed in areas of suitable habitat if proven effective. It may be possible to use electricity to kill off large areas of D. villosus with the correct application. 3.2. Trapping Recent investigations by Environment Agency staff in the UK studying the spread of D.villosus across England and Wales have used specially designed traps baited with cat food and fish food. These traps also contain coarse substrate offering animals both shelter and food attractants, demonstrating that Dv can be attracted to traps and in large (800+) numbers. Traps have been used 10 to capture invasive species,such as those used widely in an attempt to control the North American crayfish in Europe (Gherardi 2011). Coarse fish, liver and mammal carcasses have been cited as particularly effective baits used to catch crayfish (Holdich et al. 1999). Nine hundred trap nights were required in an attempt to reduce a population of P.leniusculus from 4000 to 1500 in carp ponds in England (Holdich et al. 1999). Despite this effort, within a couple of breeding seasons, numbers returned to their former levels demonstrating that trapping has to be applied long term. This highlights the need to commit to for long term trapping programmes if this is a technique of control applied. There have been attempts to try and increase trap CPUE, making them more effective at removing target species. It has been suggested that traps could be baited with female sex pheromones to attract male crayfish (Aquiloni and Gherardi 2010). Traps preliminary conditioned by mature females have been successfully used to collect P.leniusculus to the same level of success as food baited traps (Stebbing et al. 2004). Furthermore Aquiloni and Gherardi (2010) had success in using traps containing live mature female P.clarkii specimens in attracting heterospecific males. The potential application of this technique is discussed in more detail in section 6. In Indonesia, light traps have been used to catch the freshwater shrimp Macrobrachium sp (Ahmadi 2012). A range of collapsible traps including box-shape, wire-stage, bamboo-stage and minnow nets were used with the addition of LED and incandescent lamps. The most effective method involved the use of the minnow nets (with both LED and incandescent light). Using specific colours and intensities contributed to the efficacy of this method. Macrobrachium sp, was particularly attracted to the yellow incandescent light trap yielding higher catch numbers. Wetzel (2001) attributes this to less waveband attenuation when compared to other colours e.g. blue and red. Using light to collect freshwater and marine invertebrates is thought to exploit the positive phototropism exhibited by certain species (Ruck 1975). This method, while not requiring the use of bait does have its limitations. In turbid conditions the effectiveness may be reduced due to increasing light attenuation (Lindquist and Shaw 2005). The effect of light on D.villosus as a nocturnal attractant has not been examined to date. Many studies have commented on the association of D.villosus with the zebra mussel Dreissena polymorpha. D.polymorpha is known to be broadly distributed in Great Britain with populations discovered in East Anglia, Wales, West Sussex and as far north as Scotland (Aldridge 2010). Interestingly both species display markings consisting of stripes of the same angle and relative colour. It is speculated that using this striped pattern on traps may assist in trapping D.villosus through visual association with D.polymorpha. In an experiment conducted by Roqqeplo et al. (1995) both mesh size and colour were observed as important factors in catching both juvenile and adult 11 P.clarkii where black traps caught a larger number of individuals than white traps. Trap designs that take into account not only colour but also cryptic patterning may prove more effective at trapping D.villosus. Limitations of using the trap method include high manpower cost, the need for bait and the large variability in efficacy of trap used, whereby certain size and age classes may dominate the catch. Fjalling (1995) found that crayfish catch abundance differed greatly between seven types of traps used in Sweden. Moreover studies have shown that the size of specimens caught varies greatly with the type of trap used. Large male specimens have been found in higher abundances compared to other size groups using certain types of trap (Bills and Marking 1988; Guan and Wiles 1996). This may attributed to smaller crayfish avoiding the cannibalistic nature of larger adult specimens (Holdich et al. 1999). Furthermore Rogers (1996) states that juvenile and ovigerous female crayfish may be shy of entering traps and a more successful method might involve the use of seine and fyke nets instead while Lowery (1988) proposes ovigerous female crayfish are generally less active than males. Edsman and Soderback (1999) have suggested the best time of day to deploy traps intended to catch the invasive crayfish is at dusk, collecting them in the early hours of the morning. Spring represents the start of the D.villosus breeding season and therefore signifies a crucial time to carry out field work. However, little work has been done to examine different trap designs and how these may affect catch composition of D.villosus. Holdich et al. (1999) stated that due to the difficulty in trapping juveniles (crayfish) this method is only effective on a portion of the population, however this principle may not apply to D.villosus. Long-term trapping is undoubtedly more effective than short-term trapping and even then this may only serve to suppress a population rather than eradicate it. Currently our knowledge on trapping of Dv is limited. We know how to attract the species to traps, but this has not been optimised. We are still uncertain on the most effective manner to attract all life stages or how to make specific traps to attract all life stages. There is also no information on the affective range of traps and therefore if they could be used to eradicate populations. Despite this there are several ways in which traps could be developed to assist in removal of this species. 12 4.0. Physical Control Control is an integral aspect of an eradication process, ensuring the target species is maintained within a prescribed area, thereby preventing further dispersal. Various methods have been trialled to confine freshwater organisms to date with varying degrees of success. Hyatt (2004) suggests crustacean impoundment could be achieved though barrier construction, river diversion and drainage of ponds however the efficacy of these methods with regard to freshwater invasive crustaceans is not widely known. 4.1. Habitat modification Davidson et al. (2008) suggested that in order to control the invasive Australasian isopod Sphaeroma quoyanum (Milne Edwards) in North America, an effective removal method could involve outplanting the preferred substrate of decayed wood to encourage colonisation before removing the substrate for collection. It is widely accepted that D.villosus displays a preference for coarse substrates (Hesselschwerdt et al. 2008; Van Riel et al. 2006; MacNeil et al. 2010). Deliberately depositing boulders in order to encourage congregation of D.villosus may assist in limiting the distribution of Dv in certain location. This habitat could be then subsequently removed, either displacing or removing large numbers of Dv. Habitat could also be modified to make it less suitable for Dv, by the inclusion of fine particle substrates such as sand. The habitat preference of Dv may itself form an effective barrier to natural colonisation allowing native gammarids the chance to flourish where D.villosus is absent (Keenan and Rutt 2012). Removal of boulders and large structures may serve to reduce numbers of Dv by both increasing their vulnerability to predation by fish and also increasing cannibalism within the invasive population. Furthermore other gammarid species such as G.tigrinusi are more likely to predate upon the invasive shrimp following removal of its natural refuge. Even though D.villosus is considered to be a stronger intraguild predator than G.tigrinus, Platvoet et al. (2009) found that G.tigrinus actively predates upon juvenile D.villosus. In addition, intraguild predation and cannibalism occurred between juveniles of both species moderated by habitat. It was concluded that within certain habitat types, predation of G.tigrinus upon juvenile D.villosus may reduce the impact of the latter. The planting of shorelines with (native) macrophytes may help to modify habitat sufficiently to displace Dv to the point where is moves elsewhere or is present in low numbers. 4.2. Physical Barriers There is little documentation referring to the method of isolating invasive crustaceans by diverting rivers to separate channels which can then be pumped out at a later date (Hyatt 2004) however this technique should not be ruled out. Lacan (2012) identified a number of barriers to dispersal of D.villosus from Cardiff Bay following the discovery of the species in November 2010. These include 13 Cardiff Barrage which represents an initial major barrier to migration alongside additional concrete structures such as a number of weirs, feeders and gauging stations on the River Ely and Taff systems. It is thought that D.villosus generally avoids fast flowing sections of river but may take advantage of these conditions to drift to new locations. Furthermore the presence of fish and elver passes present on Cardiff Barrage and some of the weirs may assist the migration of this species. The exact migration route this species may take is difficult to predict however sections of the rivers studied pass through areas which have a high level of public access, increasing the risk of transport via human vectors. It is thought this species will migrate in time upstream of Cardiff Bay even though these barriers to migration exist. The erection of a barrier in the River Buåa at the border between Sweden and Norway was unsuccessful in preventing migration of P.leniusculus to the Norwegian part of the river (Johnsen et al. 2008), however unlike D.villosus, crayfish are able to spend considerable time out of water and can travel distances over land, thus negating such barriers. At Grafham Water reservoir measures have been taken to shut off the flow of water from the reservoir to the local brook, with mesh screens and a dam having been constructed to try to prevent further spread. So far this intervention appears to have been successful as shown by survey results. Since D.villosus has been discovered in Eglwys Nunydd reservoir, Port Talbot, three weirs have been installed to prevent the natural dispersal from this site to nearby ditches. Of particular concern was that individuals would migrate to the adjacent Site of Special Scientific Interest, Margam Moors, designated in part to protect several rare invertebrate species (Keenan and Rutt 2012). To date these interventions appear to have been effective. 4.3. Chemical and electrical barriers Hänfling and Edwards (2011) suggested the restriction of movement of biota through canals may be achieved through use of electric barriers, air bubble curtains, or salinity locks and these efforts may yield success in rivers also. Gollasch (2006) refers to anecdotal evidence of the prevention of the Chinese mitten crab (Eriocheir sinesis) to migrate upstream in several German rivers using electrical screens installed on riverbeds in the 1930’s and 1940’s. An electric barrier has been in operation in the Chicago Sanitary and Ship Canal since 2002 to prevent the spread of Asia silver carp and bighead carp to the Great Lakes. As with electrofishing the impact of electricity on D.villosus is not fully understood. Air Bubble curtains have been used at water intake points to minimise the entrainment of fish, however the use of air bubble curtains to control invasive crustacean species warrants further investigation (Gollasch 2011). Stebbing et al. (2011) identified the significant affect that carbon dioxide has on D.villosus. The method of exposing D.villosus to high levels of carbon dioxide could potentially be used for control purposes; however the long term impact of high levels of carbon dioxide would have to be taken into consideration. 14 Kestrup and Ricciardi (2010) found the Ponto-Caspian invader Echinogammarus ischnus is tolerant of short exposure to low conductivity but may be weakened in the environment because of it. Furthermore previous studies by Kestrup and Riccardi (2009) found that where low conductivity occurred, E. ischnus was particularly vulnerable to predation by native Gammarus fasciatus. Zehmer et al. (2002) cites calcium concentration as a limiting factor in the distribution of the amphipod Gammarus pseudolimnaeus and suggests animals able to survive in a low conductivity environment during their inter-moult phase may experience higher mortality later on. However D.villosus appears to be able to tolerate low conductivity even though it originated in relatively ion-rich water in the Ponto-Caspian region. Boets et al. (2010) concluded from a range of laboratory and field tests that one of the key factors in habitat preference of D.villosus was low conductivity. The use of salinity locks could be trialled as a method to reduce D.villosus numbers however it appears this species has relatively large salinity tolerance range compared to other gammarids. Furthermore experiments conducted by Santagata et al. (2008), found significant mortality in amphipods including D.villosus only occurred when full strength seawater was the minimum concentration over prolonged (24hrs) period. While the erection of physical barriers to control invasive species may be impractical as they pose navigational and recreational constraints on river users, Beyer et al. (2011) suggested this problem could be avoided using heat treatment on boats and fishing gear to control invasive spread. Stebbing et al. (2011) demonstrated that high water temperatures were lethal to Dv when applied as a bath treatment. Field trials are required to test the application of hot water treatments for the removal of DV from boats and other equipment using bath and other methods of application. Tests on the acute thermal tolerance limits of the zebra mussel and quagga mussels (Dreissena rostriformis bugensis) and spiny water fleas (Bythotrephes longimanus) were conducted. Beyer et al. (2011), who reported that 100% mortality in all three species was achieved by immersing specimens at 43°C for at least 5 minutes. This method may prove effective in areas where boat and or fishing activity is high and represents a major route of translocation of Dv. Furthermore, heat treatment is both environmentally sound and potentially more cost effective than other control methods. 4.4. De-watering Although there have been no attempt to eradicate Dv my dewatering it is included as a potential form of control as it has been demonstrated to have some degree of success in controlling other species. Kozak and Policar (2003) describe how signal crayfish were removed following repeated dewatering of a small pond. However despite leaving the pond empty over winter crayfish were once again present in the spring when the pond was filled. This was considered to be partly due to the burrowing behaviour of the crayfish. Kozak and Policar (2003) concluded that this method may 15 yield greater efficacy when used alongside another eradication technique such as the application of chemicals. Under certain circumstances the draining of waters containing Dv may assist in their removal or at least in making habitat less available. De-watering followed by treatment with quicklime would be an effective method of treatment of small enclosed water bodies for the eradication of Dv. Its application to wider areas would be logistically challenging and prohibitively expensive. 5.0. Biological control Biological control agents include predators and pathogens of the target organism. Although there have been a number of cases in the terrestrial environment where predators and pathogens have 16 been used in attempt to control invasive species (e.g. Australia contains many examples) there have been few cases where it has been attempted in the aquatic environment. 5.1. Predation Aquiloni et al. (2010) describes how positive results were found when a predacious fish was introduced to control populations of P.clarkii in Italy. However rather than being directly predated upon, the cause of a reduction in numbers was instead attributed to a behavioural change in P.clarkii whereby crayfish spent an increasing amount of time in shelter thereby reducing feeding activity. It has been suggested that the introduction of brown trout may help to control localised populations of Dv (Platvoet 2010). However other studies have shown that while both D.villosus and native gammarids are eaten by fish in the absence of shelter, in environments where coarse substrate dominates, native gammarids are consumed in greater quantities than invasives (Kinzler and Maier 2006). This is thought to be because invasive gammarids like D.villosus have a higher substrate affinity and lower activity. Kinzler and Maier (2006) found that D.villosus only left the substrate occasionally whereas the native gammarids left the substrate more frequently. Furthermore they suggest that the increase in the natives’ activity may be due to native invasive interaction whereby the invasives force out the natives leaving them more vulnerable to predation. Therefore in waterbodies where coarse substrate is dominant, introducing fish to predate upon D.villosus may not be as effective if native gammarids are present. This change in behaviour in the presence of fish predators as also been observed in crayfish (Blake and Hart 1995a, b). The use of predators should be investigated as a means of controlling D.villosus population density however further research is required in this area with regard to this species. There is an increasing consensus that the strategic combination of using different control methods may lead to higher success rates against invasive populations. For instance in Sparkling Lake (Wisconsin, USA), an attempt to control a population of the crayfish O.rusticus was carried out by combining mechanical removal of specimens with restriction of the harvest of fish species known to prey on the crayfish. As a result, invasive crayfish catch rates declined by 95% from 11 crayfish per trap per day in 2002 to 0.5 crayfish in 2005 (Hein et al. 2007). The major limitation when using biological means to control populations lies in the lack of certainty regarding the level of control that will be achieved. Also, there may be lengthy delays until full impact of the introduced agents takes effect. However Wittenberg and Cock (2001) suggest that using biological agents represents the safest and cheapest method of control. 5.2. Pathogens A small minority of NNIS can become invasive, thriving in their new environment and becoming pests carrying disease, predating native species and damaging structures and environments. Damage 17 caused by NNIS is often a result of the higher densities and larger sizes they attain compared to when they are native (Torchin et al. 2002). In addition to direct and indirect effects on physical and biological structuring of native habitats, the introduction of NNIS has been shown to threaten biological diversity by providing a means for the introduction of novel pathogens (Stewart 1991; Bartley et al. 2006). Amongst the crustaceans, crayfish plague (Aphanomyces astaci) is perhaps the publicised example of this within European aquatic habitats; following the inadvertent release of the North American crayfish species Pacifastacus leniusculus (an asymptomatic carrier for Aphanomyces astaci) (Edgerton et al. 2002; Edgerton et al. 2004). The introduction of this pathogen to Europe has been catastrophic for various species of European crayfish including the endangered white clawed crayfish (Austropotamobius pallipes), to which A. astaci is highly pathogenic, causing up to 100% mortality (Edgerton et al. 2002; Svardson 1992; Vey et al. 1983; Cerenius et al. 1987). Recently, Stentiford et al. (2011) have reported the introduction of a microsporidian pathogen (Hepatospora eriocheir) with invasive populations of Chinese mitten crab (Eriocheir sinensis) in the River Thames, London. In this instance, the pathogen is the same as that found in Asian populations within their native range (Wang and Chen, 2007). In other aquatic hosts, the consequences of introduced pathogens can be similarly severe; Bonamia ostreae, a pathogen of Ostreae edulis, was first reported in Europe in 1979 following its importation into France via infected spat from the USA (Grizel et al. 1988). Via further animal movements, the pathogen spread rapidly and threatened the viability of the French flat oyster industry (Grizel 1983) and the wider industry for this species across Europe (Laing 2004). Despite the direct threat of disease introduction from NNIS, it is the relative ‘escape’ from their natural enemies that is frequently cited to explain their success within their non-native range. The so-called ‘enemy release’ hypothesis states that species are more likely to become invasive when they are ‘released’ from population-control by natural enemies and pests (Clay 2003). In this context, Torchin et al. (2003) reported that the number of parasite species found in native populations is twice that found in NNIS species (on average, 16 parasite species recorded from native populations and only 4 species accompanying the host into its introduced range). Furthermore, they demonstrated that even following acquisition of some parasites from hosts residing in the native range, NNIS were afflicted by approximately half the number of parasite species than similar native species. The European shore crab (Carcinus maenas) provides an excellent example of this phenomenon; the species is considered NNIS in several global regions where it has become a major pest. Within its native range, C. maenas is host to a number of pathogens that are known to affect growth, mortality and reproduction. However when European populations were compared to those considered NNIS, the latter were less parasitized and larger 18 indicating they were either surviving longer or growing faster than crabs in Europe (Torchin et al. 2001). Several authors have reported on the presence of pathogens in D. villosus from Continental Europe. Some of the most prevalent protistan associations are with the gregarines (Apicomplexa). In a comprehensive study of gregarine parasites in a NNIS population of D. villosus (and other native gammarids) sampled from Poland, Ovcharenko et al. (2009) described infections by Cephaloidophora similis, C. mucronata, Uradiophora longissim and Uradiophora ramose. Two of these gregarines (C. mucronata, C. similis) are only though to infect Ponto-Caspian gammarids, whereas U. longissima is a wide spread parasite of fresh and brackish water hosts, excluding species of Ponto-Caspian origin. Ovcharenko et al. (2009) summarise by stating that in the case of Poland, NNIS populations of D. villosus are accompanied by their parasites. Furthermore, the lack of ‘enemy release’ in the NNIS D. villosus population is likely explained by continuous migration of this species through the artificial canal network of the sampled region. Studies in the Ukraine (Dnieper Basin, Dniester estuary and the lower Danube basin) revealed the presence of a novel species of peritrich ciliate (Lagenophrys pontocaspica) on the gills and oostegites of various Ponto-Caspian amphipods, including D. villosus, at a prevalence of up to 80% (Boshko, 1996). The most significant pathogens of D. villosus (as observed for other gammarids) are represented by the Microsporidia. Microsporidians described infecting D. villosus from sites across continental Europe include: Cucumispora dikerogammari (=Nosema dikerogammari Ovcharenko and Kurandina, 1987)(Ovcharenko et al. 2010), Nosema granulosis, Dictyocoela roeselum, Dictyocoela Muelleri and Dictyocoela berillonum (Wattier et al. 2007). Wattier et al. (2007) surveyed 34 NNIS D. villosus populations (n=1436) along the Danube river and four river drainages in France and Benelux (Rhine, Rhone, Seine and Loire) in 2002. The study proposed that D. villosus played host to 4 different microsporidian pathogens. Three of them closely related to parasites of other amphipods (Nosema granulosis, Dictyocoela mulleri and D. sp. roeselum) with the fourth a new species provisionally named Microsporidium sp. D. Microsporidium sp. D was found at high prevalence and elicited similar clinical signs of infection to N. dikerogammari (Wattier et al. 2007). A subsequent study by Ovcharenko et al. (2009) utilising a combination of morphology and molecular phylogenetics demonstrated that N. dikerogammari and Microsporidium sp. D were in fact the same pathogen. Due to the genetic dissimilarity to extant members of the genus Nosema, the pathogen was named Cucumispora dikerogammari Ovcharenko et al. (2010). Wattier et al. (2007) investigated the applied aspects of D. villosus establishment as a NNIS by testing whether population establishment is based upon the maintenance of host genetic diversity (allowing the species to adapt to new environments) or whether the absence of natural enemies 19 (such as parasites) drives establishment. By studying the whole invasion range across continental Europe, Wattier et al. (2007) found no evidence for genetic bottlenecks in D. villosus, and no evidence for significant parasite loss (microsporidians), across the invasive range. The data suggest that the invasion within continental Europe was massive and/or recurrent, leading to concurrent establishment of a pathogen fauna observed within the native range for D. villosus. The prevalence of Microsporidium sp. (=Cucumispora dikerogammari Ovcharenko et al. 2010) at sites across the invasive range within continental Europe varied between <5% (Loire) to >25% (Seine). The prevalences observed correspond to low prevalence in recently-colonized sites, high prevalence in sites colonized for approximately five years and a medium-level prevalence in sites colonized for long periods, or from the native range. According to Wattier et al. (2007), these dynamics fit better with a horizontal transmission pathway, with density dependent transmission, rather than a vertical one. Interestingly, Wattier et al. (2007) use this data to reject the invasion of continental Europe by D. villosus as a case of ‘‘enemy release’’ by parasite loss (as outlined by Torchin et al. 2003). These studies on pathogens of D. villosus in the continental NNIS range provide an insight into the carriage of pathogens to novel locations with the spread of NNIS. However, in the case of continental spread, it appears likely that introductions have been occurred on multiple occasions over extended time periods. To date, few studies have focussed on the recently established NNIS populations of D. villosus in the United Kingdom (Grafham Water, Cardiff Bay, Eglyws Nunydd reservoir and the Norfolk Broads). Given the fact that the United Kingdom is an island, it may be envisaged that the introduction pathway and frequency to these locations differs somewhat from NNIS range sites on the continent. To this end, it could also be hypothesized that the pathogen profile of the United Kingdom populations may also differ from those NNIS and native populations of D. villosus studied to date. This is hinted at in the recent paper by Wilkinson et al. (2011) who sampled (amongst other species and sites), D. villosus from Grafham water, in order to investigate the presence of the gammarid-feminising microsporidian pathogens of the genus Dictyocoela. The genus Dictyocoela are unusual amongst the Microsporidia in that they cause infected embryos of the host to develop as females, regardless of environmental conditions or genetic predisposition (Ironside et al. 2003; Terry et al. 2004). Interestingly, in the case of the survey by Wilkinson et al. (2011), Dictyocoela was not detected in D. villosus from Grafham Water but was detected in the congener (Dikerogammarus haemobaphes), another Ponto-Caspian invader in animals sampled from rivers and lakes in Poland. The absence of Dictyocoela from Grafham Water contrasts the survey of microsporidians from the continental range of invasion for D. villosus where it has been demonstrated that perhaps three species of Dictyocoela can cause infections (Wattier et al. 2007). This provides the first evidence that United Kingdom NNIS populations of D.villosus may have a 20 different pathogen profile even to those NNIS populations in continental Europe, and to animals sampled from the native range. A current Defra-funded pilot project at the Cefas Weymouth Laboratory is investigating the pathogen fauna of D. villosus, sampled monthly over a whole year, from Grafham Water. The study is also gathering comparative material, on an ad hoc basis, from other sites within the United Kingdom, and from specific sites within the NNIS range for D. villosus in continental Europe (e.g. the Rhine River in Chalampe, France). Material is being processed through the ISO 17025 accredited Pathology laboratory at Cefas and is being investigated for the presence of known and novel pathogens (ranging from RNA viruses, to DNA viruses, bacterial and fungal infections, to protists and metazoans). The study is providing the first comprehensive survey of D. villosus pathogens outside of its continental range and is adding to the list of pathogens known from the continental NNIS range. Despite the screening of approximately 1000 individual D. villosus, sampled monthly from Grafham Water over the period July 2011 to February 2012, we have not observed any examples of microsporidian infection (C. dikerogammari, Nosema granulosis or Dictyocoela sp.). In contrast, an ad hoc sample collected on our behalf from the Rhine River in Chalampe, France (continental NNIS range for D. villosus) displayed 13% prevalence of C. dikerogammari (n = 40/293), many of which had advanced infections, implicating the majority of the host musculature. In contrast, a high prevalence (>50%) of samples of D. villosus collected from Grafham Water are infected with a currently unidentified gregarine pathogen in the intestinal lumen and epithelial cells. A similarly high prevalence of gregarine infection was observed in the French sample (62.5%). Lower prevalence pathologies have also been observed in samples collected from the United Kingdom and from France. Peritrich ciliates are observed attached to the gills and carapace in up to 22.5% of animals. Rare cases of digenean trematode infection (possibly Plagioporus skrjabini Chernogorenko et al. 1978) have been observed in samples from Grafham water. Incidental findings of a presumed oligochaete infection were recorded in samples obtained from France. An isolated case of a nematode infection was observed in one animal sampled from Grafham Water. Whilst not confirmed, we have also observed pathology consistent with an RNA virus infection in the hepatopancreas of D. villosus sampled from France. In these cases, hepatopancreatic epithelial cells become apoptotic and are sloughed into the hepatopancreatic lumen. This was observed in 8% of D. villosus sampled from France. Similarly, an isolated case of pathology consistent with a DNA virus infection was observed in a single animal collected from France. The pathology involved the B-cells of hepatopancreatic epithelia, in which an inclusion (consistent with that of a DNA virus) was observed within the epithelial nuclei. 21 Comparison of the general life history parameters for the subsample of D. villosus collected from France in November 2011 with that sampled from the United Kingdom in the same month, we observed a female biased sex ratio (2:1) in the French sample and an even (1:1) ratio in the United Kingdom sample. Whether this relates to the endemicity of the feminising microsporidian Dictyocoela in the continental NNIS range requires further investigation. In conclusion: 1. Early observations suggest that NNIS populations of D. villosus in the United Kingdom are relatively pathogen-poor compared to NNIS populations of D. villosus from continental Europe and from its native range. 2. At least two important pathogens, the horizontally transmitted microsporidian Cucumispora dikerogammari and the vertically transmitted microsporidian Dictyocoela sp. are absent from the samples obtained from Grafham Water to date. 3. Any direct mortality associated with C. dikerogammari, and any distortion of population sex ratio due to Dictylocoela sp., would be absent from the Grafham Water population. 4. The absence of key pathogens in D. villosus from the United Kingdom supports the concept of ‘enemy release’ for the UK NNIS population (despite the fact that similar was not observed for the continental NNIS population). 5. The differences observed between continental and UK populations is suggestive of continuous/frequent introduction events in the former and discontinuous/infrequent introduction events in the latter. 6. The absence of key pathogens in the UK NNIS populations of D. villosus may have contributed to their relative success compared to native fauna, although this is difficult to prove or disprove. 22 6.0. Autocidal 6.1. Semiochemicals and pheromones Most, if not all animals, that have been studied use chemical signals to trigger behavioural responses. Examples range from ciliated protozoans (Alimenti, C et al 2011), insects (Heinbockel,T, et al 2004), molluscs (Cummins, SF, et al 2006], fish (Li,W, et al 2002), crustaceans (Hardege JD et al 2011, Zhang, D et al 2011) amphibians ( Kikuyama,S et al 1995) These behavioural eliciting chemicals are called semiochemcials. Semiochemicals are defined by the Health and Safety Directorate in their pesticide glossary : Semiochemicals are chemicals emitted by plants, animals and other organisms - and synthetic analogues of such substances - that evoke a behavioural or physiological response in individuals of the same or other species.( Products marketed for use as traps are pesticide products and require an approval by the CRD before they can be marketed. For detailed information on which products do or do not fall within the scope of the relevant legislation Directive 91/414/EEC please see Regulatory Update 10/2006.) When the semiochemical concerned affects an individual of the same species it is called a pheromone. When the semiochemicals affects an individual of a different species it is called an allelochemical. There are clearly major advantages in using a chemical with high species specificity to control an invasive alien species. Pheromones have been extensively used in invertebrate pest control and relevant examples of such use will be discussed in this section together with the advantages and disadvantages of the strategy and some possible approaches which might be used for Dikerogammarus villosus. Semiochemical use in invertebrate control. Semiochemicals in the form of pheromones are commonly used in the management of insect (and specifically lepidopteran and coleropteran) pest populations. The importance of insects as a pest invertebrate order has led to this order being extensively studied in order to identify effective control strategies. The importance of semiochemicals has grown as entomologists have moved more to integrated control strategies. The advent of synthetic insecticides in the 1940’s led to their widespread use and the belief that insect pests could now be controlled indefinitely using chemical pesticides. Over a decade later, widespread insect resistance to the commonly used insecticide classes were reported, as well as dramatic environmental impacts. This led to a stringent regulatory 23 system protecting the consumer and the environment and a better perception by economic entomologists of the ecosystem within which insect pests were living. Insecticides have had enormous benefits on human health, food production and food storage but an integrated approach, using biological, cultural and chemical methods, is now preferred and this has led to surge in research into alternative control methods for insects. This helps explain the domination of insect examples in the following section. A rational approach to insect control has lead to application of treatments only when the pest is present (rather than calendar treatments) and to the development of a control mechanism which is specific for the pest species or that family of pests (Rosell et al 2008). This approach ensures that any beneficial insects (which play an important part in any integrated control strategy) remain active to control any other pests that might remain uncontrolled after chemical treatment. The development of the “perfect” control agent has to be balanced against the economic reality that any new insecticide will cost c $200M to develop (cited on Crop Life International internet site) and consequently the economic benefit of eradicating the target pest must be sufficient to guarantee a return on the investment in drug development. In reality therefore, commercial insecticides are never active against a single species. Pheromones, which affect a single species, may be developed as their regulatory route is shorter (equals cost of development) in some countries, recognising their “biorational” design. Pheromones can cover a range of behavioural responses. Examples include: Aggregation: A substance produced by one or both sexes and bringing both sexes together for feeding and reproduction. By aggregating, individuals gain advantages as a group that they would not have as individuals. These include defence against predators and ability to overcome host resistance and increasing the efficacy of mate selection. Such pheromones have been found in a range of insect species. Alarm: A substance produced to repel and disperse other heterospecifcs in the area. Some communal species release volatiles when attacked by a predator. This can then trigger escape responses from other members of the community (in the case of ants, bees, termites). This clearly has advantages for the survival of the community rather than the individual. A well characterised example exists in aphids (Vandermoten et al 2012). Other minor pheromone types: • Signal territory, • Demarcate a trail by laying down volatile hydrocarbons – used extensively by ants. 24 • Behavioural modification: a number of behaviours in bees, are pheromone triggered, as befits a complex communal insect species. Sex: emitted by females to attract males for mating. Such sex pheromones exist also in other orders e.g. male copepods can follow a three dimensional trail laid down by a swimming female. (Yen, J. et al. 2011). Sex pheromones are used in control strategies for pest insect species, particularly lepidopterans (see below). Sex pheromones can be used in pest control in two modes. 1. They can be used to monitor insect populations using pheromone dispensers linked to traps which allow numbers to be counted. Such a sample of the population allows the populations to be estimated- particularly for time comparisons e.g. the relative catches from the same trap in the same location at the same time of year. 2. The use of pheromones to control insect pest populations. These can be divided into two strategies: a) Mating disruption (“sexual confusion”) whereby pheromone dispensers release plumes of pheromones which saturate the canopy of a crop, so saturating the environment such that the natural release of pheromone by females to attract males is masked. In this situation, males will be unable to located females and mating will not occur, with the consequent impact on the pest population. b) Attract and kill whereby the pheromone dispenser lures males to a trap, removing them from the population and therefore preventing them from mating and thereby controlling the population in the subsequent generation (El-Sayed,AM et al 2009). Pheromones attract males so using a pheromone to assess the population has disadvantages (as females produce the next generation). Examples of effective population monitoring and control using pheromones include: 25 Species pheromone Bark Beetle (Pityogenes chalcographus) (2s,5R)-chalcogram, methyl (2E,4Z0-deca-2,4dienoate.(aggregation pheromone) (Tomlin 2009) Olive fly (Bactrocera oleae) 1,7-dioxaspiro-[5.5]-undecane (sex pheromone) (Tomlin 2009) Grape berry moth (Lobesia botrana) (7E,pZ)-dodeca-7,9-dien-1-yl acetate (sex pheromone) (Tomlin 2009) Pea moth (Cydia nigricana) (E,E)dodeca-8,10-dien-1-yl acetate (sex pheromone) (Tomlin 2009) Rice stem borer (Chilo suppressalis) (Z)-hexadec-11-enal, (Z)-hexadec-9-enal, (Z)-octadec-13-enal in ration 50:5:6 (sex pheromone) (Tomlin 2009) Codling moth (Cydia pomonella) (E,E)-dodeca-8,10-dien-1-ol (mating disruption pheromone) (Tomlin2009) Helicoverpa armigera (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) (Tomlin 2009) Spodoptera exigua (1), Z9-14:Ac (2), Z11-16:Ac (3), Z9,E12-14:OH (4), Z9-14:OH (5), and Z11-16:OH (6) in a ratio of 26:11:1:22:31:9. (Tomlin 2009) Heliothis virescens (Z)-11-hexadecenal (Z11-16:Ald), (Z)-9-tetradecenal (Z914:Ald),(Z)-9-hexadecenal (Z9-16:Ald), (Z)-11-hexadecenyl acetate (Z9-16:OAc), and (Z)-11-hexadecen-1-ol (Z11-16:OH) (Wang,G et al 2011) The above examples are given to illustrate the chemical complexity commonly found in pheromones. The ratios of pheromone components help preserve species integrity and perhaps strain selection by varying the ratios of pheromone components. Such complexity, however, adds to the difficulty in using such mixtures of pheromone components in a control strategy. Aside from the complexities of synthesis (which are considerable and expensive) the different components of the pheromone mix need to be released from the pheromone dispenser at a rate such that the final ratio of components perceived by the male elicits a response. The technology of pheromone release of complex mixtures is problematic (see, for example Mottus E et al 1997). All known pheromones are stereo specific. The stereo specificity of the components adds another level of complexity and potential cost to any pheromone based control strategy. 26 Advantages of using pheromone based control strategies. 1. Totally species specific providing an environmentally benign control mechanism. 2. The specificity of the compounds used ensures low toxicity to humans and other occupants of the ecosystem. 3. Any natural predators or parasites are unaffected and remain to control any residual population left after the use of pheromones 4. The development of resistance to pheromones is likely to be slow (see for example Xu Zhen et al 2010) 5. Small quantities of pheromone can affect behaviour of males kilometres away. Disadvantages of using pheromone based control strategies 1. Expense: a. The stereo specificity of the compounds used and the possibility of potentially complex mixtures makes production synthesis expensive. b. The low potential use (as control is restricted to a single species) means that economies of scale (reducing per unit cost) are difficult. 2. Works best on large areas which can be isolated- this may fit well with the aquatic environment. 3. Success is not guaranteed. 4. Requires a detailed understanding of the biology of the pest species. 5. Requires precise timing when individuals are at the correct physiological state to respond to these behavioural cues. 6. Reduced efficacy at high populations (where male and female can find each other by chance and behaviour cues are not required) and low populations (where individuals may be at the periphery of the canopy where pheromone concentrations are sub optimal.) 7. Due to the high fecundity of pest species (r strategists) models indicate that for insects, 90% of males need to be controlled before a population can be reduced (Van Emden 1974). 8. Use of pheromone in any control strategy would require an authorisation for chemical use in the environment. At present this could be expensive and time consuming. Discussion of use of semiochemicals for control and eradication of Dikerogammarus villosus We do not know the chemical identity of any pheromone system used by D.villosus. Consequently, any use of pheromones as a strategy would be years in the future. The precise details of the life cycle (mating attraction, sex ratios, environmental factors affecting sex ratios) are also not known. 27 An additional complication is that there are few examples of pheromones being used in a control strategy in the aquatic environment. There are clearly major differences between terrestrial and aquatic pheromone strategies including: 1. Impact of water current on pheromone release and transduction. 2. There is no useful precedent in the aquatic environment for slow release technology with the exception of the use of pheromones for lamprey control in the great lakes in the USA (Li ,W et al 2007) Such pheromone dispensers are well commercialised in the terrestrial environment and deliver long term controlled release. Transposing this technology of release into air to release into water probably requires a different technological underpin, from release using volatility -based on vapour pressure- to release based on water solubility. Progress with this technology will require research. On a more fundamental level, pheromones have been shown to be very effective tools for the monitoring of populations and in the control of populations of some species below economic levels. Fundamental principles indicate that pheromone (sexual confusion) strategies will never completely eradicate a population (Witzgall P et al 2010) and so their use in the present scenario is limited. There are observation that D. villosus appears to like to aggregate on surfaces. Such aggregation may imply that an aggregation pheromone is implicit in this behaviour. Isolation of such an aggregation pheromone would have immediate implications for control and even eradication as populations could be attracted into cages/ traps from where they could be removed or killed. Pheromone identification could offer some real advantages to the specificity and ecological impact of the control strategy. Discovery and identification of pheromones is essentially unchanged since the first pheromones were chemically identified. The process involves identification of a useful behavioural bioassay, fractionisation of samples to identify the active fraction, re fractionisation to obtain the active chemical followed by chemical identification with validation of the identification by proven biological activity of the synthesised chemical. It is interesting to speculate that if the resources put towards quicker more effective methods of DNA sequencing had been placed at the disposal of pheromone characterisation, then faster more effective methods might be available for quickly identifying behaviour modifying chemicals (semiochemicals) from new species. Because few (if any) semiochemicals are proteins, most semiochemicals cannot be easily identified from DNA sequences making this traditional route of biochemical identification closed to those seeking to elucidate the structure of pheromones. 28 Summary 1. Semiochemicals provide a selective, environmentally benign method for controlling invasive invertebrate species. 2. Further research would be needed to identify pheromones from D.villosus and to determine an effective release mechanism and formulation. 3. Any method based on pheromones is likely to be expensive because of the nature of the chemistry involved. 4. The mechanism of action of pheromones together with their past record in invertebrate pest control indicates that their use for any eradication strategy would be unsuccessful. 5. A sighter study is recommended to determine if D.villosus uses an aggregation pheromone and if its structure could be easily determined. By removing the substrate on which D.villosus has aggregated and using this to elicit aggregating behaviour, a readily available bioassay can be simply used. The substrate can then be used as a basis for elution and chemical fractionisation using eg HPLC. 6. Identification and synthesis of such an aggregation pheromone could lead to an effective attract and kill strategy, effective against both sexes. 6.2. Male sterilisation. Male sterilisation is the technique whereby a population of a pest invertebrate (again- precedent is mainly from insects) is reared in the laboratory, and typically exposed to a radiation source which induces genetic aberrations in the individual males. The males are then mass released whereupon they mate with wild females, producing non viable progeny. The method is associated with population control to a level where eradication of the pest is possible. The famous example of its use as a technique was in the successful elimination of the screw worm (Cochliomyia hominivorax) from N America starting in the 1950s (Knipling, E. F. 1960). This technique has been used successfully against a number of pest species such as Mediterranean fruit fly Ceratitis capitata (Wiedemann), melon fly Bactrocera cucurbitae (Coquilett), pink bollworm Pectinophora gossypiella (Saunders), codling moth Cydia pomonella (L.) and tsetse fly Glossina austeni Newstead (Wyss, JH 2000; Hendrichs et al. 2005; Klassen & Curtis 2005). It has also been suggested as a method for the control of invasive alien crayfish (Aquiloni, L et al 2009). The technique is species specific (almost by definition) and is inverse density dependant. As the wild type field population declines, the percentage control increases provided the numbers of sterile males released is maintained constant. This makes it an ideal method for eradication of invasive alien species (Franz,G and Robinson,AS 2011). 29 This inverse density dependence is shown from the following simplified equation: F1=P x (1-S) x R Where F1 is the population size in the filial generation, P is the parental generation size, R is the net population growth rate per generation, and S is the sterility induced by the released sterilized insects. Thus with increasing S as a proportion of the total population, the size of F1 decreases. Historically “male sterility” was originally induced by chemo-sterilants but the inherent danger in the use of these chemicals for operators, and the risk of release of the chemicals into the environment has driven a move to the use of irradiation as the method of choice for insect sterilization (Parker, A and Mehta, K 2007). The dose of irradiation is critical as the resulting males must not have their motor or sensory functions compromised if they are to compete successfully with wild type males for females if the strategy is to be effective. In practice, a number of steps would be required for the sterile male technique to be a viable option for the control of D.villosus: 1. A simple mass rearing programme of D.villosus must be achievable which is not labour intensive or expensive. In practice this is probably achievable given the speed with which laboratory cultures have been established. Biosecurity principles must be rigidly enforced for all staff and protocols involved in the rearing of D.villosus to minimize the risk of escapes. The biosecurity processes need to have a systems approach, covering all inputs and outputs to the culture. 2. The most susceptible life stage to irradiation needs to be identified. In insects, pupae are often the most susceptible life stage and this probably reflects the susceptibility of the developing (and therefore vulnerable) early stage of morphogenesis of the male and females germ cells. We know little about the morphogenesis of germ cells in D.villosus but this could be easily investigated using histological techniques. 3. In conventional mass rearing, all the females are wasted as they cannot be used in this technique. Development of a strain which only expresses males would therefore be an enormous advantage in male mass rearing (at best doubling the number available for sterilization). The speed of sequencing now available (since the inception of “second generation sequencing” techniques) could rapidly make the entire genetic sequence of D.villosus available. This is probably sensible (given the low cost of such work) in informing any method of control. With knowledge of the genetic sequence, specific mutations of the 30 genome may be possible, making the production of a male biased population theoretically feasible. 4. A source of irradiation needs to be identified. This source must produce radiation that is adjustable and reproducible. In practice there are a number of potential sources of ionizing radiation (Universities, facilities which study the effects of ionizing radiation). Logistics dictate that the source of the irradiation should be close to the mass rearing programme to prevent the risks involved in the transport of large of invasive alien species. 5. The threshold dose and most susceptible life stage then needs to be identified. This is an iterative approach of varying doses and life stages. Resultant males then need to be assessed for their ability to compete with unsterilized wild type males for mating with females. From this process the optimal dose and life stage can be determined. It is probable that more essential information will become available during this process on sex ratios, degree days for individual life stages and mating behaviour. Such information is of long term value in the control of D.villosus. 6. Peak production by mass rearing needs to be timed to coincide with the life cycle of wild D.villosus. In order to achieve this, baseline field sampling of D.villosus must be made in order to identify times when receptive females are present in the population. Such times are usually synchronized across a population, brought about by pheromone release and the shared number of degree days for that generation. Mass release of sterilized males across the eradication area must then be achieved. In order to assess the efficiency to treatments, ongoing population assessments must be made in the treated area using reproducible and cost effective sampling regimes. If no means of separating males from females is found (either by genetic manipulation or by physical means) then an assessment would need to be made as to the impact of releasing irradiated females. This is less than ideal as there is the probable scenario of irradiated males mating with irradiated females which would have no impact on the wild unirradiated population. 7. Such releases must be achieved for every subsequent generation in order to minimize the time needed to control/eradicate the population. It is recommended that one water system is used as a pilot and other waters not treated until there is clear evidence of population declines- at which point the programme can be increased to cover all known areas of D.villosus colonisation. Advantages i. Intrinsically selective 31 ii. Proven track record of its use as an eradication technique in insects iii. Enabling research needed is not excessive for such a selective method (in comparison with pheromone research) iv. Will yield considerable information on population dynamics and biology of D.villosus. v. Does not require authorization for release of a chemical Disadvantages i. Success is not guaranteed ii. Requires use by staff of ionizing radiation which has its own system of regulatory control and safety processes. iii. The thick cuticle of D.villosus may prove to be an effective shield against ionizing radiation iv. If males and females cannot be separated (or a male only strain produced) this could severely affect the efficacy of the resulting programme. v. Mass rearing needs to be done adjacent to a source of ionizing radiation. Summary 1. Male sterilization provides a strategy for eradication of invasive alien species which has been used successfully in insects. 2. A reproducible source of irradiation close to facilities to mass rear D.villosus in a biosecure environment are required. 3. Given the other potential options, this represents a real possibility for eradication. 4. The methodology used could be readily applied to other invasive alien arthropods. 32 7.0. Biocidal control Dikerogammarus villosus is an arthropod crustacean and therefore has many biochemical and physiological processes in common with the common agricultural and public health pests of the order Insecta. It is therefore conceivable to envisage compounds that were originally active against agricultural pests having the potential to control D.villosus. The particular situation of D.villosus in water systems, whose ecological purity needs to remain uncontaminated, creates particular problems for their control. However, it is possible to construct a series of criteria which would need to be met to address these particular environmental questions. 1. Low mammalian toxicity. Clearly any chemical must not present a risk to the person applying the treatment, nor constitute a risk from disposal of any excess treatment. One would propose to use the WHO classification and only select compounds classified 111 or U. 2. Environmental impact: ideally one would want a compound that was relatively hydrolytically labile, so ensuring that the chemical did not remain in the aquatic environment causing issues because of its persistence. (see 3). The important point is that the compound should not be persistent and remain bioavailable in the aquatic environment whether the mechanism responsible is from hydrolytic degradation or sorption onto organic material / sediment. 3. Specificity to non target organisms: the biggest impact would be if a non discriminating broad spectrum chemical was added to the aquatic environment, thereby causing lasting damage to that environment- particularly to fish. Clearly no compound has (yet) been discovered which is selective to D.villosus. However, by taking compound which is intrinsically selective to arthropods one can administer it to give selectivity by matching the application technique to the behaviour of the alien invasive. 4. Availability: only compounds presently registered as agricultural or public health pesticides are available in sufficient quantities to allow their consideration. The mode of action of different insect control agents are listed by IRAC (insecticide resistance action committee) and are listed on the label as a way of encouraging good resistance management practises. A table listing these classifications is given in appendix 1. Groups 1 (acetyl cholinesterase inhibitors). This mode of action will affect most if not all animals and therefore does not meet criteria 2 or 3. Similarly, this mode of action tends to have high mammalian toxicity and so does not pass criteria 1. 33 Group 2 (GABA gated chloride channel antagonists). This group contains compounds which have substantial environmental impact (criteria 2) and the mode of action is not specific (criteria 1 and 3) Group 3 (sodium channel modulators). This group contains a diverse grouping, many of which would not pass criteria 2 or 3. However, the natural pyrethrins, being intrinsically unstable, may have utility if the intrinsic lack of selectivity (criteria 3) could be addressed by other means. The intrinsic high toxicity of pyrethroids and pyrethrum to fish and other aquatic life removes them from first priority consideration (criteria 2 and 4). Group 4 (nicotinic acetylcholine receptor agonists and antagonists). This mode of action fails criteria 1. The neonicitinoids may have some utility however, their intrinsic stability and hydrophilcity present high risks in the aquatic environment (criteria 2) and lack of intrinsic selectivity (criteria 3) so other candidates should be given a higher priority for evaluation. Group 5 (nicotinic acetylcholine receptor agonists – allosteric). As group 4. Group 6 (chloride channel activators): This mode of action is not selective and has reasonable persistence (criteria 2) although examples of this group are already used in the aquatic environment (emamectin benzoate). Its evaluation is therefore of low priority. Group 7 (Juvenile hormone mimics). This is an attractive mode of action as, by affecting the juvenile hormone system of arthropoda, a high level of specificity is built into the compounds. The compounds Hydroprene, Methoprene, are not now registered in the EU and are so not considered further. Fenoxycarb and Pyriproxifen occupy niche markets and this reflects their intrinsic selectivity within Insecta, probably because of pharmacodynamic considerations. Fenoxycarb will be discussed further below. Pyriproxifen is used in wet environments for the control of midges and mosquitoes and should be evaluated for its potential to control D.villosus. Its properties are evaluated below. Group 8 (compounds of unknown or non specific mode of action – fumigants). These fail all criteria. Group 9 (compounds of unknown or non specific mode of action – feeding blockers). These tend to be very selective even within Insecta and their action is to prevent feeding (good for crop protection) but this is not a useful mode of action for control of D.villosus. Group 10 (compounds of unknown or non specific mode of action- mite growth inhibitors). These tend to be very selective (Acaria) and so they will probably not be useful in the control of D.villosus. Group 11 (microbial disruptors of insect midgut membranes). There is no reason to believe these Bacillus thurengiensis strains will be active on D.villosus. Group 12 (Inhibitors of oxidative phosphorylation and inhibitors of ATP formation). This is intrinsically a non specific mode of action which therefore fails criteria 1 and 3. Group 13 and 14 (Uncouplers of oxidative phosphorylation via disruption of proton gradient). As for group 12. 34 Group 15 (Inhibitors of chitin biosynthesis, type 0, Lepidopteran): This mode of action has intrinsic selectivity to arthropoda (criteria 1) and some benzoylurea analogs exhibit low environmental impact. Although listed as type 0 lepidoptera, the compounds show widespread activity against other insect classes. This group would warrant further investigation (see below). Group 16 (Inhibitors of chitin biosynthesis, type 1,Homopteran) as 15. Group 17 (Moulting disruptor, Dipteran). As for group 15, these compounds have intrinsic selectivity although criteria 2 may remove these from consideration. They are discussed in more detail below. Group 18 (Ecdysone agonists / moulting disruptors). As with groups 15-17, this mode of action provides intrinsic selectivity coupled with low environmental impact. Their use will be discussed in more detail below. Group 19 (Octopaminergic agonists): This mode of action should be selective to invertebrates and, by affecting octopaminergic systems, have a fundamental effect on arthropod behaviour. Only one example of this class now exists and although early studies indicated toxicological issues (failing criteria 1) more recent work has reclassified the compound as an unrestricted or general use pesticide (US EPA use category 111). It is not registered , however, for use in the EU as an insecticide, acaricide or for biocidal use and its use will not therefore be further discussed. Groups 20 and 21 (mitochondrial [complex 3 and complex 1] electron transport inhibitors). With the exception of the METI acaricides, this mode of action is slow acting and some examples have environmental issues (criteria 2). The METI acaricides although possessing some attractive properties, are selective for mites (mainly Tetranychids) and so the probably of their showing activity against D.villosus is slight. They are therefore judged not sufficiently attractive for further discussion. Group 22 (Voltage-dependent sodium channel blockers): The example of this mode of action relies on activation (it is a propesticide) so the probability of being active against D.villosus having the right pharmacodynamic enzyme proportions make this a low priority for further evaluation. Group 23 (Inhibitors of lipid synthesis) : These compounds are selective for mites so their evaluation is not of high priority (same argument as groups 10 and 21). Group 24 (Mitochondrial complex IV electron transport inhibitors). This mechanism is non specific and does not meet criteria 1, 2 or 3. Group 25 (Neuronal inhibitors [unknown mode of action]). A mite specific so discounted for same reasons as Group 23, 21 and 10. Group 26 (Aconitase inhibitors): Fails all criteria. Group 27 (synergists). These by definition increase the activity of other compounds and are therefore not suitable for the control of D.villosus on their own. 35 Group 28 (Ryanodine receptor modulators). This recently discovered group exhibits some outstanding properties and the more active chlorantraniliprole (rynaxypyr) should be further evaluated. 7.1. Compounds warranting further evaluation In this section PPDB refers to the Pesticide Properties Database (PPDB). The PPDB is a comprehensive relational database of pesticide physicochemical and ecotoxicological data developed by the Agriculture and Environment Research Unit (AERU) at the University of Hertfordshire. Juvenile Hormone Mimics (Group 7) Pyriproxifen Toxicology: acute oral LD50 (rats) >5000mg/kg. WHO acute hazard rating: “U” unlikely to be hazardous. Environmental impact: Pyriproxyfen shows strong persistence in water (resistant to aqueous hydrolysis) and moderately fast aqueous photolysis at pH7. It has moderate toxicity to aquatic invertebrates but high toxicity to aquatic crustaceans but effects were sometimes reversible. ( PPDB pyriproxifen). Target specificity: There is not data on the activity of pyriproxyfen on D.villosus but if laboratory tests showed the intrinsic activity of this molecule, one could envisage conferring selectivity by applying as a bait. (see below). Application to mosquito larvae (also in the aquatic environment), provides control for 2 months duration. Active on some diptera, coleoptera and some homoptera (Tomlin 2009). Availability: Pyriproxifen is Annex 1 listed under 1107/2009 and this exclusion expires on 31.12.2018. The product is not registered in the UK. Fenoxycarb Toxicity: WHO classification: U – unlikely to represent an acute hazard. 36 Environmental impact: water solubility 7.9mg/l with logP of 4.07. Aqueous hydrolysis gives very persistent and aqueous photolysis is slow. The compound has moderate toxicity to fish and aquatic invertebrates. (PPDB Fenoxycarb) Species specificity: active on Lepidoptera, homoptera, coleoptera, dictyoptera, diptera and hymenoptera (Tomlin 2009). Conferring selectivity by the use of baits is discussed below. Availability: Annex 1 listed under 1107/2009 until 31.5.21. Registered for use in the UK. Inhibitors of chitin biosynthesis Lepidoptera (group 15) Early examples of this class of chemistry suffered from long environmental persistence and were less active: the following will therefore concentrate on more recent analogs. Flufenoxuron Mammalian toxicology: rated as WHO category 111 (slightly hazardous) Environmental impact: Aqueous photolysis at pH7 DT50 at 6 days (moderately fast) although classed as persistent in absence of light (little hydrolysis at pH7 at 20deg). DT50 in water sediment at 53 days classed as moderately fast. It binds to organic material and is not mobile. Classed as high risk to fish and aquatic invertebrates and persistent. (PPDP Fenfuroxuron) As of 9 February 2012 under 2012/77/EU the impact on the aquatic environment and its persistence have led to the authorisation of flufenoxuron being removed and its authorisation for sale in the EU is revoked effective 1.8.12. (annex 1 listing of 98/8/EC) This compound will therefore not be further considered in this review. Hexaflumuron This molecule is not registered in the EU for annex 1 listing, so it will not be further considered in this review. 37 Lufenuron Toxicology: Rated as “unlisted “ in WHO classification Environmental impact: Classed as non persistent in soil, with fast aqueous photolysis at pH7. Classed as “very persistent” to aqueous hydrolysis with slow degradation in water sediment. The compound has low leachability potential because of its high lipophillicity, It has high toxicity to aquatic invertebrates but moderate toxicity to fish (PPDB Lufenuron). Target specificity: There is not data on the activity of lufenuron on D.villosus but if laboratory tests showed the intrinsic activity of this molecule, one could envisage conferring selectivity by applying as a bait. (see below). Active on coleopteran, diptera, hemiptera, hymonoptera, lepidoptera and acari (Tomlin 2009) Availability: Lufenuron is Annex 1 listed under 1107/2009, with registration until 2019. Also has a registration as a veterinary medicine. It is not, however, registered as an insecticide in the UK. Triflumaron The authorisation in the EU for annex 1 listing was revoked from 16.9.2009. Under Directive 91/414/ EEC. This compound will not therefore be further considered. Inhibitors of chitin biosynthesis homoptera (Group 16) Buprofezin Authorisation for Annex 1 listing was withdrawn from 30.3.2009. Under 91/414/EEC. This molecule will therefore not be further considered. 38 Moulting disruptor dipteran (group 17) Cyromazine Toxicology: WHO classification 111 (slightly hazardous) Environmental impact: It is stable and very persistent to aqueous hydrolysis and photolysis, showing persistence in sediment. Its low lipophilicity means it is moderately mobile in soil and its high water solubility (13g/l) would increase the risk of this molecule moving in the aquatic environment. It has moderate to low toxicity to aquatic invertebrates and crustaceans (PPDB Cyromazine). Target specificity: Active on diptera in agricultural and veterinary applications (Tomlin 2009). There is not data on the activity of cyromazine on D.villosus but if laboratory tests showed the intrinsic activity of this molecule, one could envisage conferring selectivity by applying as a bait. (see below). Availability: registered under Annex 1 until 31/12/2019 under 1107/2009. The product is not registered in the UK. Ecdysone agonsists/ moulting disruptors (group 18A) Diacylhydrazines This group contains 4 molecular analogs. Tebufenozide Toxicity: WHO classification U (unlikely to present an acute hazard) Environmental impact: is classed as stable and persistent to aqueous photolysis and hydrolysis : relative persistence in sediment. Has moderate toxicity to fish, aquatic invertebrates and crustacean (PPDB Tebufenozide) Species specificity: Active mainly against Lepidoptera by stomach action (Tomlin 2009). Activity on D.villosus could be conferred by stomach application (see below) Availability: Annex 1 listed under 1107/2009 until 31.5.2021. The compound is not registered in the UK. 39 Halofenozide Is not registered in the EU and will not be further considered. Methoxyfenozide Toxicity: WHO classification U (unlikely to present an acute hazard) Environmental profile: compound is classed as very persistent in the aquatic environment. It has a high (calculated) logP and water solubility of 3.3 mg/l. It has moderate toxicity to fish, aquatic invertebrates and crustacea. (PPDB methoxyfenoxide) Species specificity: Used in the control of Lepidoptera (Tomlin 2009) Availability: Annex 1 listed under 1107/2009 until 31.3.15. The compound is registered in the UK. Chromafenozide Is not registered in the EU and will not be further considered. Ryanodine receptor modulators (group 28) Chlorantraniliprole (Rynaxypyr) Toxicity: WHO classification 111 (unlikely to present an acute hazard) 40 Environmental impact: With a logP of 2.86 and water solubility of 1.02 mg/l the compound will likely move in the aquatic environment. However, it is fast degraded by aqueous photolysis. Fish toxicity is moderate whilst toxicity to aquatic invertebrates is high. Target specificity: active on Lepidoptera, coleopteran, diptera and homoptera (Tomlin 2009). Activity on D.villosus unknown. Selectivity could be conferred by application as a bait (see below) Availability: inclusion in Annex 1 pending – has been granted extended provisional authorisations until 30.6.2012. Control by ingestion of baits. D.villosus is characterised by its voracity and non specific diet. In order to minimise effects on the surrounding fauna and environmental issues, targeting applications of chemicals in food will capitalise on this voracious appetite of D.villosus. Treatment can be easily effected by administration to preferred food followed by an oil coating to seal in the active ingredient. Alternatively, the chemical can be mixed into pellets with feed and then administered. Laboratory tests can be designed to maximise ingestion and minimise food not being ingested and thus being available to other non target organisms. Limiting the bait applied can maximise the quantity ingested. Rates needed for control will be identified from laboratory studies (see below). Environmental risks from this procedure are: 1. Compound leaks out of bait before it is ingested by D.villosus. This risk can be mitigated by clear identification of the physical properties of the chosen compound and the appropriate desorption control (oil coating, gelatine binding etc). 2. Compound is excreted by D.villosus. It is likely that only small quantities of the compound will be excreted by any individual at any one time. In comparison with the synchronised quantity of material applied at dosing, the quantities excreted will not be synchronised and so will represent low concentrations over the remaining life span of the D.villosus. Normal dilution effects found within the aquatic environment are likely to minimise this impact. In order to confirm these assumptions the release and resultant water concentration could be modelled. 3. Death of individual D.villosus which are then eaten carrion eaters and predators. This food chain effect is real but its severity will be limited by: a. The bioavailability of the compound within the body of the D.villosus (the more lipophilic molecules may be bound to tissues, rendering them not bioavailable to a predator when eaten) 41 b. The stability of the molecule in aquatic environment (whether it is prone to aqueous hydrolysis). c. It is possible that D.villosus may eat individuals killed by chemical treatment, so prolonging the effective chemical control (the palatability of the chemical would need to tested) This food chain risk can only be adequately quantified once the chosen compound has been identified. The use of microencapsulated formulation has been suggested for the control of D.villosus. The use of such a formulation in the aquatic environment for arthropod control would seem to offer few benefits. The active ingredient of the microencapsulated formulation would need to be effective and there is little to suggest that the high levels of potassium salts suggested (US patent number 7,378,104 B2)) would provide adequate levels of control and the ingestion of microcaps would not be encouraged by any behavioural response of D.villosus (in comparison with molluscs where their natural feeding will aid in the sequestration of the microcap). If the active ingredient is a pesticide, there is a real environmental risk of low ingestion of the microcaps causing more extensive environmental impact than from more conventional bait. Our recommendation would therefore be for use of a bait formulation rather than use of a microencapsulated formulation. Compound selection and further action On the basis of the above analysis the following compounds merit further evaluation: Pyriproxyfen,fenoxycarb, lufenuron, cypromazine, methoxyfenozide, chlorantraniliprole. All these compounds are available in the EU. It is recommended that: 1. The intrinsic activity of these 6 compounds is evaluated upon D.villosus in the laboratory. Levels of efficacy can then be determined for each molecule. It is recommended that the compounds are administered as bait, capitalising on their ingested activity (see reasoning above). 2. Once selection has been made (efficacy vs. impact), the HSE needs to be approached for emergency licensing of the compound as a biocide. See below from HSE web site on emergency registration. “Under certain circumstances it is possible for Member States to authorise the use of a plant protection product for a period not exceeding 120 days, for a limited and controlled use where such a measure is necessary because of a danger which cannot be contained by any other means as set out in Article 53 of Regulation (EC) 1107/2009. When issuing such emergency authorisations the MS concerned must inform the 42 other MSs and the Commission of the authorisation given, detailed information about the situation and any measures taken to ensure consumer safety. If necessary the Commission will take a decision as to whether the MS can extend or repeat the emergency authorisation or not or whether the authorisation must be amended or withdrawn. Applications for emergency authorisation cannot be made for plant protection products containing or composed of genetically modified organisms unless such release has been accepted in accordance with Directive 2001/18/EC. Agreement from the Advisory Committee on Pesticides (ACP) would be required before authorisation could be agreed within the UK. Authorisation would be given for a maximum period of 120 days and as such it is a temporary solution to a pest problem for which a more permanent solution must be found” 3. The application proposed is not, however, for plant protection and so an authorisation under the Biocidal Products Directive under Product Type 18 (Insecticides, acaricides and to control other arthropods) where its use would need to be reviewed. It is not clear if there is an equivalent emergency licensing under the Biocides Directive. However, use of a biocidal material in any experiment or test in UK which may involve or result in the release of that product/active substance into the environment can be authorised by application for a Biocidal Products Research Application. 4. With approval from the HSE, the company who market the product should be approached. Without explicit approval by the company for this off label use, practical use in the field will be difficult. The company will be concerned with risk to the perception of the product and the perception of the company from a use which is not economic (from the companies’ perspective). This could be mitigated by emphasising partnership between Defra and the company for the benefit of the UK (doing the right thing, not just for profit etc). They may wish to audit the stewardship principles in the proposed use. 43 8.0. Synopsis and Recommendations From this review there it is clear that there are several potential mechanisms that could effectively be employed against Dv to achieve either control or eradication. Many of the techniques discussed have not been applied to Dv and therefore are speculative; despite this an attempt has been made to indicate where success may be possible and provide an estimate of time of development. One main over arching area where a greater understanding is required for effective implementation of control and eradication methods is the life history of Dv, of which there is large gaps in our knowledge. It should be noted that it is unlikely that there is a ‘silver’ bullet to remove this species and that a combination of control mechanisms will need to be applied to achieve eradication. Each of the control methods are also assessed against the criteria below as discussed in the introduction to this document. 1. Safety of use in the vicinity of drinking water sources (non-harmful to humans); 2. Effect on the functionality of treated waters – including any necessary periods of isolation etc; 3. Effect on or risk to other species and environmental impact (e.g. invertebrates, fish, aquatic mammals, birds, dogs, farm animals, etc.); 4. If a chemical method is selected, issues concerning degradation of the chemical in the environment or safe removal/neutralising in water treatment process; 5. If a chemical method is selected, regulatory issues concerning use of the selected product; 6. Practical aspects concerning effective delivery/application in large bodies of water (and potentially running water systems) – including need for repeat treatments, time intervals etc; Physical removal The effects of electricity should be examined as a possible method of quickly eradicating Dv from areas of preferred habitat. Experiments to determine effectiveness could be conducted very easily and quickly. Although limited in application this could be an easily applied control mechanism if proven to be effective. It is safe to use in drinking water and the function of treating water, there may be some impact on other species, but this could be minimised by its application. Given that Dv will mainly congregate in areas where there is suitable habitat then electrical treatments could be targeted to these areas on the margins of water bodies. It has already been demonstrated that Dv can be removed from waters using traps; if this is a mechanism sufficient for the control and/or eradication of Dv will need to be tested. However, it is 44 one of the most easily applied methods discussed within this document and further development of trap designs is still considered possible by using different attractants. For example, further studies to investigate different food types as attractants, sex or aggregation pheromones (as discussed later), the possible use of light and colour as attractants, as well as the phycial nature of the trap itself to make it species specific and attractive. Other aspects of trapping will also need to be considered such as placement, and timing. Trapping is one of the more environmentally benign methods discussed within this document, and is therefore considered safe to use with minimal risk to the environment. Physical control Barriers have already been used in the UK to prevent further spread of Dv. However the application is limited without causing disruption to the wider use of water ways. The technique is therefore ideal for certain circumstances, but not applicable to every known populations. It poses no risk to water supplies and has little impact to the environment, although there is potential for migratory animals to be impacted, such as salmonids. Although speculative, there is evidence to suggest that habitat may be made less suitable for Dv, thus exposing the population to increased predation leading to an overall reduction in numbers. As Dv does not like fine sediment the spreading of sand or other fine sediment over preferred habitat could significantly reduce availability for Dv, this could also be achieved through the planting of macrophytes and other aquatic plants, increasing the settlement of fine sediment. This may just cause the population to move elsewhere within a water system, but could help in biosecurity by removing habitat that is close to areas of high use e.g. slip ways. This techniques would not affect the treatment or use of a water system, and in the case of the planting of aquatic plants would likely lead to an increase in biodiversity. While physical barriers have been shown to be effective in certain circumstances the use of chemical or electrical barriers do not have significant wider application in comparison to the input required for their development. The development of such systems is therefore not recommended. Draining down could help to reduce available habitat, and even eradicate whole populations, however, this has very limited application. In contrast it is simple to apply and quick. This treatment would affect the use of any water system, but for a limited period. Biological control There are no specific predators of Dv that have been identified, it is therefore unlikely that introduction of generalist predators would result in control and/or eradication on their own. 45 However, the use of predatory control in addition to other methods, such as habitat modification, could result in a much reduced population size. At least two important pathogens, the horizontally transmitted microsporidian Cucumispora dikerogammari and the vertically transmitted microsporidian Dictyocoela sp. are absent from the samples obtained from Grafham Water to date. These have been associated with mortalities and distortion of sex ratios respectively in Dv populations in mainland Europe. The introduction of these pathogens to Dv populations in GB may result in their control. This could be coupled with other methods such as habitat modification to enhance density and therefore transmission of disease. Some examination on the specificity of the pathogens would be required, but these will be relatively straight forward to conduct. The application of these pathogens would not affect the use of waters (e.g. as drinking water), and once tested, are unlikely pose a threat to the wider environment. Autocidal control The use of pheromones as a potential control mechanisms have a long history of success in the terrestrial environment, but none in aquatic systems. Further research would need to be conducted to determine the presence and nature of pheromones that could potentially be used for the control of Dv. This would be potentially time consuming and expensive, but is likely to produce and effective mechanism in the long run. Male sterilisation could be a viable tool, but require mass rearing of Dv. While this is a strong possibility and has been shown to be an effective tool in the control of other invasive (terrestrial) species. The further examination of this technique is not seen as a priority due to the capital cost of setting up such systems. Biocidal control Despite initial studies on the effects of various chemicals (Stebbing et al. 2011) suggesting that Dv are robust against generalist biocides, there are a number of chemicals that have not been tested against Dv which show strong potential for their control and/or eradication. From the review 6 chemicals have been short listed as showing the greatest potential. There is already some degree of specificity of these chemicals e.g. effect chitin formation. These show the most potential for the development of a short term eradication programme. Testing of these chemical could be conducted in a short time period, the development of a delivery system would be required, but given the feeding habits of the species this would seem relatively straight forward. How the chemical is deployed and where will help in making the mechanisms specific to Dv, reducing the wider impact 46 on the environment. Further discussions are required with HSE to determine how these chemicals could be employed for the control of this and potentially other invasive species. 47 Appendix 1 48 49 50 51 52 53 References Ahmadi G (2012) An Introduction of Light Traps for Sampling Freshwater Shrimp and Fish in the Barito River, South Kalimantan. Journal of Fisheries and Aquatic Science, 7: 173-182. Aldridge DC, Elliot P, and Moggridge GD (2006) Microencapsulated biobullets for the control of biofouling zebra mussels. Environmental Science and Technology. 40: 975-979. Aldridge DC (2010) The zebra mussel in Britain: history of spread and impacts. In Zebra Mussels in Europe (Eds. Van der Velde, G & Rajagopal, S.). Backhuys Publishers, Leiden. pp. 79-92. 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