ecological impacts of virus insecticides: host range ...

JENNY S. CORY

ECOLOGICAL IMPACTS OF VIRUS INSECTICIDES: HOST RANGE AND NON-TARGET ORGANISMS

1. INTRODUCTION Insects are infected by a wide range of DNA and RNA viruses from at least thirteen faniilies, with several more groups as yet unclassified (for a review see HunterFujita et al. 1998), However, in the majority of cases we know little about these viruses and representatives of only a few groups have been assessed for their insecticidal potential. The vast majority of insect viruses developed for pest control are baculoviruses, a group of occluded DNA viruses, although representatives from two other groups of occluded viruses, the entomopoxviruses (EPVs) and the cytoplasmic polyhedrosis viruses (cypovinises or CPVs) have also been assessed for the control of specific pests. For example, the efficacy of the EPV from the migratory grasshopper, Melanoplus sanguinipes, has been investigated in field trials in the USA (Woods et al. 1992), and CPVs have been tested against the pine caterpillar Dendrolimus spectabilis in Japan (Kunimi 1998) and against the pine processionary moth, Thaumatopoea pityocampa in Europe (Grison 1960), although in none of these cases has the virus been developed past the initial stages. Non-occluded viruses have been isolated from many pest species: limacodid moths in south-east Asia appear to have a particularly rich diversity of non-occluded viruses, including densoviruses, picomaviruses and Nudaurelia P viruses (in addition to baculoviruses and CPVs) and some of these have been applied in spray trials in oil palm plantations (Jones et al. 1998). A particularly successful example of pest control using viruses has been the application of the non-occluded and nonassigned virus isolated from the rhinoceros beetle Oryctes rhinoceros. This virus has been widely used to control populations of rhinoceros beedes in the western Pacific and the Maldives (e.g., Gorick 1980; Jacob 1996; Zelazny et al. 1989) and other dynastid beetles such as O. monoceros in the Seychelles and east Africa (e.g,, Lomer 1986). However, in general, application of viruses other than baculoviruses has not progressed, due to a paucity of ecological, biological and molecular information about the alternative groups, a lack of detailed safety testing, problems with separating individual viruses from naturally occurring mixtures and in some instances due to their slower and more chronic effects on the target pest. For these reasons this review will concentrate on baculoviruses. H.M.T. Hokkanen & A.E. Hajek (eds.), Environmental Impacts of Microbial Insecticides, 73—92. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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2. BACULOVIRUSES 2.1. Baculoviruses as pest control agents Baculoviruses are DNA viruses that have only been isolated from arthropods. They have been recorded from a wide range of hosts, primarily insects, however they have only definitively been identified from Lepidoptera, hymenopteran sawflies and Diptera. The Family Baculoviridae is currently divided into two genera, singlyoccluded Granulovirus (GV), which have only been isolated from Lepidoptera, and multiply-occluded Nucleopolyhedrovirus (NPV), which have been identified from a wider range of hosts, although again dominated by the Lepidoptera. However, further phylogenetic studies may well separate the NPVs into genera that follow the different insect orders (Hemiou, pers. comm.). Baculoviruses are primarily used as inundative control agents on pests in glasshouses, horticulture and forestry (see Hunter-Fujita et al. 1988; Moscardi 1999 for reviews). Baculoviruses which have been registered for commercial use on a wide range of horticultural crops include codling moth, Cydia pomonella, GV, in North America and Europe, beet armyworm, Spodoptera exigua, NPV, in Europe, North America and south east Asia and Helicoverpa spp. NPVs in North America, China, Eastern Europe, south-east Asia and Australasia. In forestry baculoviruses have been registered to control both lepidopteran and hymenopteran forest pests including, the gypsy moth, Lymantria dispar, the pine sawfly, Neodiprion sertifer and the fall webworm, Hyphantria cunea, mainly in temperate regions. Although alternative control strategies have been discussed and occasionally used (Cory & Bishop 1997; Moscardi 1999) in virtually all instances these baculovirus control agents are applied as sprays onto high density pest populations with the aim of short term pest suppression. 2.2. Baculovirus biology and ecology For a more detailed review of baculovirus ecology see Cory et al. (1997), Rothman & Myers (2000) and Cory & Myers (2003), however some of the key features of baculoviruses in relation to assessing their ecological impact are discussed below. 2.2.1. Productivity Baculovirus infection can only be introduced naturally in the larval stage, with infection usually being initiated via the caterpillar ingesting contaminated food. Susceptibility to baculoviruses tends to decrease as the larvae age, although there is increasing evidence that there is also considerable variation in resistance within instars. Most baculovirus infections are polyorganotrophic, resulting in high virus yields. However, the occlusion bodies (OBs) are not usually released until the host dies. An exception to this occurs among the baculoviruses that only infect the gut, such as the NPVs infecting sawflies. In these cases virus is continually shed into the gut lumen and released throughout the infection. Virus productivity is related to

ECOLOGICAL IMPACTS OF VIRUS INSECTICIDES

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insect weight and a final instar noctuid caterpillar can produce up to 1O'° OBs. This gives baculoviruses huge potential for increase. 2.2.2. Persistence The occluded nature of both NPVs and GVs endows them with the capacity to survive outside their host. In addition, the proteinaceous coat provides the stability that allows them to be formulated and sprayed as insecticides. Environmental persistence is thought to be the most important route for baculovirus survival with OBs originating from the insect cadavers responsible for horizontal transmission of the virus. Where the OBs are protected from ultraviolet (UV) irradiation they can survive for considerable periods of time, in more temperate climates from one year to the next (e.g., Carruthers et al 1988). OBs are most protected in the soil or in crevices in plants, but whether such virus reservoirs are biologically important and play a role in virus maintenance or the initiation of epizootics has not been elucidated empirically. However, recent studies have shown that the behaviour of the insect host could play a significant role in virus acquisition. For example, the ballooning behaviour demonstrated by many lymantriid larvae can enhance contact with the soil or plant understorey thereby bringing the insect in contact with the persistent virus reservoir and re-introducing infection into the host population (Richards ef a/. 1999a). 2.2.3. Vertical transmission While horizontal transmission of baculoviruses has received the greatest attention, there is also considerable evidence that baculoviruses can be transmitted vertically from adult to offspring (Fuxa & Richter 1991; Kukan 1999). The conditions which favour vertical transmission are poorly understood although intuitively it might be predicted that it would be beneficial when host densities are often low and unpredictable or where the host is highly mobile and unlikely to re-encounter virus reservoirs. Current evidence indicates that the prevalence of vertical transmission (of overt disease) varies considerably from species to species, however, it does appear to be higher in species which are migratory (e.g. armyworms such as Mythimna separata and various Spodoptera species) and cryptic (e.g. stemborers such as Sesamia nonagroides and Chilo infuscatellus) (Figure 1). Studies on vertical transmission have primarily addressed the transmission of overt disease, however, there is also evidence that baculoviruses can be transmitted at sublethal levels from adult to offspring. The possible presence of latent baculovirus infections has been discussed for a considerable period of time. The use of modem molecular techniques is starting to shed light on this issue and the presence of persistent infections has been established in some species (Hughes et al. 1997), although their role and the conditions under which they may be converted into an active infection are still unclear. Most of these studies have taken place in the laboratory and little is

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JENNY S. CORY

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30 20-

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Figure 1. Vertical transmission of overt baculovirus infection in a range of species. Larvae were treated with virus inoculum and the survivors retained and mated, and the level of baculovirus infection was monitored in the next generation. Data taken from Kukan (1999), plus J. S. Cory unpubl data.

known about their presence or relevance in field populations. However, recent studies on the African armyworm, Spodoptera exempta and its NPV have indicated that the proportion of adults supporting sublethal levels of virus in field populations can be extremely high (Vilaplana and Cory, unpubl. data). It is quite possible that sublethal or persistent baculovirus infections are far more widespread than had originally been thought. Cross-infection with a low dose of a heterologous virus is one means by which persistent virus is thought to be activated (see 5.1.3). Thus triggering of persistent baculovirus infections in field populations of non-target Lepidoptera could be a factor that needs to be considered in relation to field application of baculovirus insecticides. However as discussed below, there is no evidence that baculovirus application has ever produced an epizootic by this route and it is perhaps unlikely that sustained virus epizootics will be produced in species which do not naturally succumb to baculovirus outbreaks. It is also possible that genetically modified baculoviruses could produce persistent infections in the species they come into contact with. As yet it is not known whether specific features are required for a virus to develop a persistent infection: persistent viruses should have reduced virulence thus a genetically modified virus, or the genes they contain, would


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not appear to be ideal candidates. However this is likely to be a complex process depending on both the genetic makeup of the host and the potential (and likely) presence of competing pathogens of both an overt and a chronic nature in addition to the phenotype ofthe virus. 2.2.4. Dispersal Baculoviruses disperse rapidly from points of introduction (e.g., Entwistle et al. 1983; Fuxa & Richter 1994). Various possible mechanisms have been implicated in this dispersal, including the host itself (for example via ballooning larvae, Dwyer and Elkinton 1995) and passive dispersal by both invertebrate and vertebrate predators (e.g., Lautenschlager et al. 1990; Entwistle et al. 1993, Vasconcelos et al. 1996) and parasitoids (Fuxa 1991). While it has been clearly demonstrated that virus can be carried by these routes, their relative importance in the observed rapid dispersal of baculoviruses is still unclear. 3. VIRUS ISSUES 3.1. Regulatory issues An important issue with all microbial insecticides is how they are viewed from a regulatory point of view. Because they are introduced in large numbers into high density pest populations, usually with the aim of short term pest control, they occupy a middle ground between classical biological control agents and chemical insecticides. To some extent the way they are dealt with by the regulatory authorities reflects this and in most countries, registration requirements have evolved from those originally designed for chemical pesticides. The testing procedures still mainly focus on the requirement for experimental data on toxicity, teratogenicity, infectivity and allergenicity to vertebrates (see Hunter-Fujita et al. 1998 for a review). Although baculoviruses have no recorded effects on vertebrates, and most of this repetitive and expensive testing is unnecessary from the baculovirus point of view, the regulations are such that it is still required for the active ingredients in the formulation as well as the virus. To avoid unnecessary repeat testing, some countries, notably the USA, are developing fast track procedures for microbial control agents with good records of safe use. However, the more ecotoxicological approaches do not capture the biological nature of microbial pesticides. Thus additional tests need to be included to adequately assess the environmental or ecological impact of insect pathogens. This primarily means the effect on non-target hosts, which is the central issue of this chapter. Some host range testing is usually included in the registration packages for microbial insecticides, but again it tends to focus on groups of invertebrates which have repeatedly been shown not to be susceptible to baculoviruses, rather than careful scrutiny of more closely related potential hosts. However, the non-target effects of

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macro biological control agents are increasingly coming under scrutiny due to several well-publicised instances where introduced predators and herbivores have moved onto native species (Howarth 1991; Simberloff & Stiling 1996; Thomas & Willis 1998; Cory & Myers 2000). Whilst there have been no instances of negative environmental effects resulting from the wide-scale application of microbial insecticides that I am aware of, we should be aware of the potential need to scrutinize the release of insect pathogens in a manner consistent with other biological control agents. We must therefore expect to be able to answer more wide-reaching questions on the fate of these organisms after their release into the environment, particularly regarding their effect on susceptible non-target species. 3.2. Virus species The application of molecular tools, in particular restriction endonuclease (REN) analysis of virus DNA, means that different baculovirus isolates can be easily compared and characterized. The now almost routine use of REN analysis has shown that there is a huge diversity in baculovirus isolates, Baculoviruses isolated from the same insect species in different geographical regions vary in their DNA profile (e.g., Gettig & McCarthy 1982; Vickers et al. 1991; Laitinen et al. 1996). This leads us to the question of how different do baculoviruses need to be to be defined as a species? Baculoviruses are named after the host from which they were isolated, this is potentially a very confusing and misleading system as it is possible that very different isolates from the same species will be given the same name or identical isolates from different species will be given different names. The widespread use of REN profiling is helping to reduce such occurrences and baculovirus taxonomy is also moving towards having more detailed numerical descriptors for different isolates. The increasing generation of DNA sequence data has allowed us to assess the relatedness among different baculovirus isolates using phylogenetic trees based on whole genome sequences (Herniou et al. 2003) and more expansive trees based on single gene phylogenies (e.g,, Baldo & McClure 1999; Bulach et al. 1999). While phylogenetic studies should help to identify similar isolates, the procedure for naming baculovirus species still needs to be consolidated. For example, baculoviruses isolated from the same host species in different areas can be more distantly related to each other than they are to isolates from other species, at least in terms of single gene phylogenies (Herniou & Cory, unpubl. data). More detailed analysis of baculovirus field isolates using a variety of cloning techniques has also shown that they are frequently composed of a range of different genotypes (e.g,. Smith & Crook 1988; Stiles & Himmerich 1998) and even that multiple genotypes can be isolated from an individual caterpillar (Cory & Green, unpubl. data). Thus many baculovirus isolates currently used in field pest control programmes are likely to be mixtures of genotypes, which may or may not differ in biological activity. However, whilst there are still areas for improvement in baculovirus taxonomy, techniques for accurately identifying virus isolates are


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available allowing characterization of different strains, confirmation of purity and monitoring for potential changes during production and after field release. 4. ECOLOGICAL IMPACT A variety of terms are used to describe the process of assessing the consequences of releasing particular biotic or abiotic agents into the environment. Risk assessment is a commonly used term, however it tends to have a precise meaning which relates to the probabilistic likelihood of a specified event happening and thus is often too rigid to describe the type of assessments which are carried out, particularly where effects on non-target species are concerned. More appropriate terms are environmental or ecological impact assessment, with environmental being a broader term and ecological more usually used to refer to the effects on wildlife (Evans & Miller, pers. comm.). What are the possible ecological impacts of baculovirus insecticides? The studies that have been carried out as part of the registration package of numerous baculovirus insecticides have clearly demonstrated that their host range is extremely limited. They are not infective for man, other vertebrates or plants. They have only ever been isolated from arthropods (primarily insects) and have only been confirmed from Lepidoptera, hymenopteran sawflies and a few species of Diptera. They are not infective for predatory insects, parasitoids, cockroaches, lacewings, honeybees or other non-phytophagous species (e.g., Doyle et al. 1990, Huang et al. 1997). Furthermore, baculoviruses isolated from insects within one insect order e.g,. Lepidoptera, are not infective for insects outside that order. Baculoviruses have been isolated from over 600 species of insects and cause regular epizootics in species of Lepidoptera and sawflies as widespread as armyworms in Africa and forest insects in the South Pacific, North America and Europe. They are thus a common component of many ecosystems. Given their limitation to certain groups of insects, the main potential ecological risk of applying baculovirus insecticides is their impact on susceptible non-target species. These effects may be localized and transient, i.e. the direct effect (mortality) of spray application on non-target species in and around the crop. However, more importantly they may also initiate a longer term effect by establishing in non-target species and causing environmental perturbation. 5. HOST RANGE 5.7. Host range studies 5.1.1. Can we define natural host range? Knowledge of the host range of a baculovirus is the first step in ecological impact assessment. As with macro biological control agents, application of a baculovirus infective for a single (target) host is unlikely to present a major risk. However, one problem that is perhaps particularly pertinent with pathogens is what constitutes

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their natural host range and distribution? Without knowing this it is impossible to say whether a pathogen has invaded new hosts or has expanded its natural range. With larger organisms their natural distribution is more obvious and any changes in this distribution have often been documented, but to follow a pathogen or parasite the hosts or potential hosts need to be known and need to have been monitored for some time. This tends not to be the case for most insects except perhaps those of commercial importance. The spread of these insect pests themselves is also likely to have aided the spread of the pathogens they carry and this will make these pathogens the most difficult to ascribe a natural host range to. More comprehensive DNA sequencing of well-catalogued samples and the use of sophisticated phylogenetie techniques may aid in unravelling the temporal and spatial evolution of different baculoviruses. However, interpretation of host range data needs to be treated with care. 5.1.2. Laboratory host range studies The choice of insects selected for host range testing as part of registration packages tends to be fairly arbitrary and also tends to err towards species of commercial rather than ecological interest. In order to assess any potential ecological impact host range testing should include a broad range of non-targets including both species of conservation interest and those which are representative of the area of anticipated release from within the Order from which the original baculovirus isolate was collected. There is a paucity of published detailed host range data for baculoviruses from which generalisations can be made, in part due to the difficulty of obtaining large numbers of field-collected insects and the time and labour involved in studies of this type. Many host range studies only look at a limited number of species. These studies usually involve infecting early instar larvae with one or more doses of virus; detailed dose-mortality (LD50) analyses are rarer, except on species maintained in the laboratory, again due to the difficulty of obtaining sufficient numbers of larvae at a similar stage of development and the resources involved. Host range testing of baculoviruses needs to be carried out in a much more standardized manner both in terms of the bioassay method and the doses administered and the number, range and age of species tested. Most host range studies have concentrated on baculoviruses isolated from two lepidopteran families, the Noctuidae and the Lymantriidae. Studies on the Lymantriidae indicate that NPVs isolated from this family are very narrow in their host range, in most cases being restricted to the species from which they were originally isolated. This group includes the NPV from the gypsy moth, Lymantria dispar, (Barber et al. 1993), the brown-tail moth, Euproctis chrysorrhoea, (Cory et al. 2000) and the vapourer moth, Orgyia antiqua (Richards et al. 1999b). These host range studies are unusual in that they assess pathogenicity in 47, 73 and 23 species of Lepidoptera, respectively. The NPV from the douglas fir tussock moth, Orgyia pseudotsugata is reported to be infective to two other members of the genus

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Orgyia (Hughes 1976), but otherwise there appear to be no other hosts. It is therefore likely that all NPVs isolated from this group will be equally narrow in their host range. The host range of NPVs isolated from the noctuids appears to be much more variable. Some again are apparently very narrow, if not restricted to a single host, such as the NPVs isolated from the Spodoptera genus (e.g., Gelemter & Federici 1986). Other baculoviruses can infect a range of species from different genera, for example, Anticarsia gemmatalis NPV (Camer et al. 1979), although in this study only seven species were tested. There are also several examples of NPVs that can infect a relatively wide range of Lepidoptera such as the cabbage moth, Mamestra brassicae NPV (Doyle et al. 1990), celery looper, Anagrapha falcifera NPV (Hostetter & Puttier 1991) and alfalfa looper, Autographa califomica NPV (AcMNPV) (Payne 1986; Bishop et al. 1995). A. falcifera NPV was found to be infective for 30 out of 38 species of Lepidoptera in 10 families (Hostetter & Puttier 1991) and M. brassicae NPV was found to be infective for 32 out of 66 Lepidoptera in four families (Doyle et al. 1990). The data for AcMPNV are more scattered but at least 95 species from 15 families are susceptible to the virus (Payne 1986; J. S. Cory et al, unpubl. data).

Tineoidae Yponomeutoidea Sesioidae Gelechioidea Pyraloidea Bombycoidea Cossoidea Tortricoidea Noctuoidea

Notodonidae Noctuidae

Arctiidae Lymantriidae Geometroidea Papilionoidea

Ail Some None

- Lasiocampidae - Saturniidae " Sphingidae Pantheinae Plusiinae Acronictiinae • Amphipyrinae . Cuciiiinae ~ Hadeninae • Noctuinae • Heiiothinae

Papiiionidae Pieridae Nymphaiidae Lycaenidae

Figure 2. Autographa califomica MNPV host range in relation to host taxonomy (Cory, Possee & Hirst, unpubl data). Shading indicates the proportion of UK species tested from each family or sub-family that were susceptible to AcMNPV. Not all species within each grouping were tested, a positive score gives no indication of relative susceptibility. A. califomica is a member ofthe Plusiinae.

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A common assumption in host range testing is that susceptible species will follow a taxonomic pattern with those most closely related to the species of isolation being most susceptible. Protocols for host range testing often suggest this as a rationale for selecting species to test. A closer look at the AcMNPV data shows that this might not always be appropriate. AcMNPV was isolated from a member of the subfamily Plusiinae within the Noctuidae. Other members of the Plusiinae and other subgroups within the Noctuidae are susceptible to the virus, however, species in other families such as Satumid moths and Nymphalid butterflies are also susceptible (Figure 2). On current information there appears to be no obvious pattern, either in terms of taxonomy, geography or life history that would distinguish susceptible species from the rest. Therefore it is not possible to predict the host range of the wider host range NPVs on the data that are currently available. Data on granuloviruses (GVs) are more sparse, but indications are that their host ranges are uniformly narrow. It is quite possible that the wider host range baculoviruses, which are NPVs, are relatively uncommon. However, this assumption needs to be supported by more exhaustive and systematic host range testing. Additionally if this information is linked with a more detailed understanding of the phylogeny of baculoviruses, we might begin to establish patterns whereby we can begin to predict wider host range viruses from their genomic data. 5.7.5. Susceptibility and 'latent' viruses The section above has discussed baculovirus host range purely in terms of the numbers of species that can or cannot be fatally infected. An important point to remember is that even with wider host range viruses not all species are equally susceptible. Degree of susceptibility will be extremely important in terms of trying to assess whether a particular non-target species is likely to be vulnerable in the field and underlines the value of detailed information on the host-mortality relationship. For example, taking the host range data for AcMNPV, we could make an arbitrary cut off point for species defined as susceptible as those in which at least 50% mortality was induced by 1000 virus OBs in the second instar. This value would be considered on the high side for an LD50 for most baculoviruses in their natural hosts. The remaining species in which there were some virus deaths at this or higher doses would be considered as semi-permissive and the rest non-permissive at the virus doses used (Table 1). In the case of AcMNPV this means that less than 10% of species are highly susceptible to the virus, with most species falling into the intermediate, semi-permissive category. Thus, even under ideal laboratory conditions, a high dose is needed to infect even a small proportion of many of the species tested. Additionally, that proportion will decrease as the insect ages and increases in resistance. A second important point to consider with host range testing is that true crossinfection must be confirmed by REN or similar techniques. In most, if not all, of the


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earlier host range studies the progeny virus will not have been confirmed and this is still the case in some more recent studies. Ideally host range testing should adopt a step-wise approach in which virus-killed larvae are first identified using hght microscopy. Any positives are then confirmed using techniques such as dot blotting DNA and then the virus can be characterized using REN profiling (Doyle et al. 1990). There are frequent mentions in the literature to the stressing out or triggering of 'latent' baculoviruses. While neither the true state of the virus in wild populations or the process of triggering are understood, there is certainly evidence that inoculating an insect with one virus can result in death by another, usually the host's own virus (e.g., Hughes et al. 1993). Closer examination of several host range studies show that up to 10% of the virus positive species were not necessarily killed by the virus they were challenged with (Doyle et al. 1990; Cory et al. 2000). Thus the true level of cross-infection may have heen overestimated in some of the studies. Table 1. Susceptibility of 71 species of UK Lepidoptera to Autographa califomica MNPV. Species in which mortality with 1000 occlusion bodies (OBs) was more than 50% are classified as permissive. Insects were tested at 1& and l(f OBs per second instar larva. (data from Cory, Possee & Hirst, unpubl. data).

9% permissive 64% semi-permissive 27% non-permissive

5.1.4. Sublethal effects So far the discussion on ecological impact assessment and host range testing has focussed on direct lethal effects on potential non-target species. There is also the possibility that a baculovirus could have sublethal effects on non-target species. There is considerable evidence that baculoviruses can exert sublethal effects on Lepidoptera which survive virus challenge. Suhlethal effects can include reduced fecundity, lower pupal weights and altered development rates (Rothman & Myers 1996). How these effects are brought about is still not known - they may be a result of a persistent virus infection but they could also be a cost of fighting off the initial virus challenge. It has recently been demonstrated in the Indian meal moth, Plodia interpunctella, that a significant proportion of insects that survive baculovirus inoculation support a persistent infection which can be passed on to the next generation (Burden et al. 2002). However this has not been linked to the observed sublethal effects in this system. As discussed above (2.2.3), there is increasing evidence that persistent baculovirus infections may be widespread in field populations and possibly maintained with no or minimal cost. However, to date there has heen very little research on whether suhlethal effects or infection can occur

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in the less permissive non-target species. Sublethal effects and infection may be higher as a greater proportion of insects will survive infection or they could be lower as the infection develops more slowly and the host is better adapted to fight it off. 5.2. Host range in the field All the discussion and data pertaining to baculovirus host range so far result from laboratory bioassays. They thus relate to an ideal situation for infection where the insects are not subject to any competing mortality agents and are supplied with food ad libitum under ideal growth conditions. Additionally, each species is usually tested as a single, usually young, age cohort (instar) that does not refiect the more diverse age structure that is found in many field populations. If the test insects originate from laboratory cultures they are also likely to have reduced heterogeneity (in susceptibility) compared to those found in the field (Dwyer et al. 1997). Thus laboratory host range assays represent a worse case scenario. A major issue with all ecological impact testing is how individual level responses shown in the laboratory can be linked to population level processes in the field and even to landscape level events. This is a major research area in ecotoxicology although much of the data relate to aquatic systems where chemical concentrations are much easier to measure and manipulate (e.g., Ak9akaya 2001; Forbes & Calow 2002). With microbial insecticides in general the chances of infection in the field will be considerably reduced compared to those measured in the laboratory. This will be the result of numerous factors including behavioural differences, density/ threshold population effects, spatial and temporal segregation, host plant effects, and so on, in addition to the heterogeneity in age structure and susceptibility mentioned above. Disease transmission is usually a density-dependent process and thus, not surprisingly, baculovirus epizootics only tend to be seen in the field in high density insect populations. These often occur on monocultures and thus natural baculovirus epizootics are most commonly observed in outbreaking pest populations in agricultural crops and commercial forests, although baculovirus epizootics are also common in several polyphagous temperate forest insect species. Baculovirusinfected larvae have been collected in other circumstances but these are often small numbers of insects or even single larvae. In order to generate more accurate and meaningful data on the likelihood of baculovirus transmission in the field it is necessary to carry out experiments under more realistic conditions. As baculoviruses need to be ingested to initiate infection a key part of the process of virus acquisition is related to the behaviour of the insect itself. It is therefore important to carry out studies in which potential host insects can move naturally. Spray applications introduce the baculovirus initially into the population but to assess whether a virus could invade and persist in a non-target population other experiments need to be designed which more accurately mimic natural transmission. Additionally, spray applications introduce relatively little


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virus into the environment (lO" to lO'^ OBs per hectare) compared to natural baculovirus epizootics in which a single late instar larva can release lO' to 1O'° OBs. Field-based transmission experiments are still relatively rare and usually involve releasing pre-infected larvae onto plants within small field plots or releasing insects into bags on trees, although it is possible to get some indication of the likelihood of natural transmission using cut foliage in the laboratory or greenhouse. In each case an approximation of the transmission rate can be obtained by allowing the preinfected insects to die in a natural position in situ, then introducing a cohort of uninfected larvae which are collected at one or more intervals and reared to ascertain what proportion have acquired virus infection (e.g., Goulson et al. 1995; Dwyer et al. 1997). However, it should be noted that any experiment in which the insect is constrained in terms of movement could impact its likelihood of virus acquisition. For example, young larvae of some species, particularly lymantriids, balloon off trees and can travel some distance. This type of behaviour has been shown to increase the likelihood of virus acquisition particularly if virus has accumulated in protected reservoirs under the plant (Richards et al. 1999a). Although several experiments have estimated baculovirus transmission, most of these have been directed towards understanding the factors that influence the transmission process. Only one experiment has studied transmission in a less permissive host under field conditions and this was primarily focussed toward assessing the impact of recombinant baculoviruses on altemative hosts (Hails et al. 2002). However, the data from this experiment and related laboratory studies (Hemandez-Crespo et al. 2001) showed some interesting points. As would be predicted from the laboratory bioassays, risk of infection was considerably lower in the less susceptible species in the field. The yield of virus from the less susceptible species chosen was in fact greater than that of the highly susceptible target host (in part due the fact that infection took longer in the less susceptible species). However, this did not alter the likelihood of transmission to other hosts (Hails et al. 2002). This indicates that the quantity of virus in a cadaver does not necessarily alter the risk of infection, at least within certain limits. This example is an isolated study of baculovirus transmission in potential non-target hosts. More experiments are needed which investigate transmission in non-target hosts of varying susceptibility. However, this is perhaps the first step in the process and data is also needed on whether less susceptible species actually support virus infection in the field and whether they play a role in maintenance of virus in natural populations. 5.3. Models The issues that are relevant for the ecological impact assessment of baculovirus insecticides can draw some parallels from the problems faced when trying to assess the impact of chemicals and other potential environmental pollutants. In both instances there is a need to link the response of individuals to the response of populations. Some of the tools used are the same, in particular, the individual

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laboratory-based sensitivity assessments that tend to revolve around LD50 testing. While they are not directly comparable in what they measure, they provide some indication of the mortality effect exerted by a specific dose, with appropriate confidence intervals. The next issue is what this means in field populations and from this point of view ecotoxicological testing is well ahead of research with microbial insecticides. This has primarily resulted from the greater need for this type of assessment with chemicals that frequently have both fatal and sublethal effects on a wide range of organisms. However, as biological pest control in general comes under greater scrutiny it is perhaps wise to adopt some of these approaches and to be able to answer questions relating to non-target impact after widescale baculovirus release. There is a considerable literature in eeotoxicology on the use of mathematical models to extrapolate from laboratory toxicity testing to population level effects focussing on population growth models, extinction curves and meta-population models for landscape effects (e.g., Tanaka & Nakanishi 2001; Akfakaya 2001; Wang et al. 2001). Mathematical modelling could also be of value in assessing the ecological impact of microbial pesticides but has the added complexity that the pathogen population also has its own dynamics. The mathematical modelling of host-pathogen interactions has been a growth area (see Briggs et al. 1995 for a review). However few of the models have been developed in conjunction with empirical studies and many of their underlying assumptions remain untested (but see Dwyer et al. 1997, 2000). One approach is to use these models to estimate the basic reproductive rate, Ro, of the virus in a range of hosts. For a pathogen Ro is usually described as the number of secondary infections resulting from a single pathogeninfected host. For a pathogen to spread, Ro must be more than 1 and if it is less than 1, the pathogen is failing to reproduce enough of itself to survive and will die out. Thus any host species that produces an Ro of less than 1 for the virus is unlikely to be a viable host in the field. Depending on the model used, Ro can be estimated from the ecological parameters which comprise the model, for example, the virus decay rate, speed of kill, yield and the transmission parameter. This type of approach and the ecological parameters that need to be estimated are discussed in Cory (2000). These models deal only with two species systems (one host, one pathogen); a more accurate refiection of the situation in the field would be a model that deals with one or more host species. A few such models exist (e.g., Begon et al. 1992), in particular in relation to the potential threat of reservoir hosts to endangered species (e.g., McCallum & Dobson 1995). The accuracy and usefulness of this type of approach will only be as good as the models that underpin it and there is probably some way to go before appropriate models are developed for testing multi-host systems in agricultural situations. One option may be to combine the more analytical mathematical approach with the development of generic endpoints for particular systems, which would endow the process with more definable goals (Suter 2000).


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6. CONCLUSIONS AND RESEARCH NEEDS In the long history of baculovirus use there has been no documented negative ecological effects from their wide-scale release. It could be argued that detailed, long term ecological impact testing has not been carried out, but equally it is quite likely that major perturbations to local fauna, particularly in well-monitored groups such as butterflies and moths, are more likely to be noticed than effects on other groups of non-target invertebrates. Baculovirus epizootics appear to be rare in the majority of Lepidoptera, although baculovirus isolates have been recorded from hundreds of lepidopteran species and no doubt this number will increase as further species are studied. In the past many of these isolates were not kept or characterised so it is not known whether they are distinct species or represent a chance crossinfection event in a susceptible alternative host. What we do know from many attempts to suppress pest populations with baculoviruses is that reapplication the following year or next generation is usually needed, despite the large quantity of inoculum generated as the result of spray applications. Thus even with pest species, trying to maintain an ongoing supply of baculovirus inoculum to infect in future generations is difficult. Where problems with non-target Lepidoptera could potentially occur is where the behaviour of the insect means that populations are locally concentrated, such as in gregarious species or where a species occurs on a rare and locally concentrated food plant. However, no evidence has pointed to either of these scenarios occurring. While current evidence does not indicate that baculovirus insecticides present an ecological threat, there are still areas of baculovirus ecology that are not fully understood, and we still know very little about their behaviour in less susceptible species. These gaps in our knowledge need to be filled so that we can develop a sounder theoretical framework for ecological impact assessment. There are several areas which would benefit from more detailed investigation, although in most cases the reasons they have not been addressed before is related to the fact tbat they require long term study and wide-scale field testing, both of which tend to be prohibitively costly for biological control programmes. Firstly we need to understand what causes epizootics and why some species are more prone to baculovirus epizootics than others. Secondly, possible ecological impacts need to be monitored in specific targeted ecosystems. As with all biological control programmes, a crucial part of this process is gathering sufficient data on the site(s) before release, in addition to post-release monitoring. This should ideally focus on both direct and indirect effects in a broad range of non-target species. Both of these issues are not insignificant tasks; the first may come from greater ecological knowledge from a wide range of systems combined with the use of mathematical modelling; the second is perhaps less likely as it would require considerable input to possibly demonstrate no effect. We also need to carry out these studies over a suitable time frame to monitor for any evolutionary change in the baculovirus. However, perhaps tbe most important message is not to over-regulate. It is easy to

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find gaps in our current knowledge of baculovirus ecology, but it is important to remember that baculoviruses are common components of many ecosystems and virus control programmes are unlikely to release more virus than natural epizootics. In addition, haculoviruses have been used on many crops for several decades without any reported negative side effects.

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