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Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 Biocontrol Science and Technology (2000) 10, 737 ± 752 Interactions Between Entomopathogenic Fungi and Other Natural Enemies: Implications for Biological Control HELEN E. ROY1 ,2 and JUDITH K. PELL1 1 Department of Entomology and Nematology, IACR-Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, UK; 2 Department of Life Sciences, Anglia Polytechnic University, Cambridge CB1 1PT, UK (Received for publication 11 Januar y 2000; revised manuscript accepted 30 July 2000) Pathogens and arthropod natural enemies may contribute to the suppression of insect pest population s either as individua l species or as species complexes. However, because natural enemies of insects have evolved and function in a multitrophic context it is important to assess interactions within complexes of natural enemies if they are to be exploited eVectively in pest management. Natural enemies can interact either synergistically/additively (e.g. enhanced transmission and dispersal of insect pathogens) or antagonisticall y (e.g. parasitism/infection, predation and competition). In this paper, studies assessing the potential interactions between insect and fungal natural enemies are reviewed. In general, these studies indicate the positive nature of the interactions between arthropod natural enemies and fungal pathogens with respect to the control of insect populations . More work is required to investigate further the many ways in which the natural enemy community interacts in the agroecosystem Keywords: entomopathogeni c fungi, insect natural enemies, interactions, ecological susceptibility, physiologica l susceptibility, biologica l control INTRODUCTION Natural enemies of insects have evolved and function in a complex multitrophi c environment (Vet & Dicke, 1992; Poppy, 1997). Organisms are in¯ uenced by a range of abiotic and biotic factors which aVect their physiology, ecology and behaviour. Interactions between organism s within and between trophic levels are important factors aVecting the structure of population s and communities. In this paper we review the possible eVects of such interactions in relation to biologica l control strategies which incorporate the use of multiple species of natural enemies. We largely focus on the interactions between fungal and insect natural enemies. Multiple biological control species may act synergistically, additively or antagonisticall y (Ferguson & Stiling, 1996). Synergistic interactions would result in a higher mortality than the combined individua l mortalities of the pest population . Additive mortality occurs if the natural enemies do not interact and, thus, the total level of mortality is equivalen t to the Correspondence to: H. E. Roy. Tel: + 44 1223 363 271; Fax: + 44 1223 352 979; E-mail: [email protected] ISSN 0958-3157 (print)/ISSN 1360-047 8 (online)/00/060737-16 DOI: 10.1080 /0958315002001170 8 2000 Taylor & Francis Ltd Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 738 H. E. ROY & J. K. PELL combined individua l mortalities caused by each agent. Ferguson and Stiling (1996) recognized three possible antagonistic eVects. First the natural enemies could interact, resulting in the total pest mortality being less than the additive mortality. Second, the total mortality might be less than that caused by one natural enemy but not the other. Finally the total mortality could be less than when either natural enemy acted alone. Thus, an increase in the richness of natural enemy species could result in an increase in the host populatio n if interference between the natural enemies is suYcient. Alternatively, a pest populatio n may be reduced to a greater extent by multiple rather than single natural enemies if interference between species is minimal or advantageou s to either species (Chang, 1996). ADDITIVE/SYNERGISTIC INTERACTIONS Enhanced Fungal Pathogen Transmission and Dispersal The development of an epizootic (sudden increase in disease incidence within a population ) is dependent on properties of both the host population , the pathogen populatio n and an eVective mechanism of transmission between the two. The transmission of a fungal pathogen is dependent on a number of processes: conidia production, discharge, dispersal, survival and germination (Hajek & St. Leger, 1994). The large number of conidia produced by infected cadavers partially compensates for the high probabilit y that many conidia will not actually infect a host (Shimazu & Soper, 1986). Indeed, the density and distribution of a pathogen population is one of the most important factors determining whether a disease becomes epizootic (Fuxa & Tanada, 1987; Carruthers et al., 1991). In an unmanaged host/ pathogen system, epizootics are usually host-density dependent, developin g as the host populatio n increases. However, an epizootic may develop at low host densities if the pathogen is widely distributed within the host habitat (Fuxa & Tanada, 1987). Pathogen population s are often distributed at low densities or discontinuousl y in the host habitat and, therefore, pathogen dispersal is essential (Fuxa & Tanada, 1987; Richards et al., 1994). Pathogens can be dispersed in several ways: active discharge of infective spores (local dispersal), weather factors (wind and rain) or by host and non-host dispersal (long distance dispersal) (Fuxa & Tanada, 1987; Roy, 1997). The presence of insect natural enemies may have an impact on local transmission of a fungal pathogen. The presence of a foraging adult coccinellid, for example, resulted in a substantial increase in the local transmission of the aphid pathoge n Erynia neoaphidis within a populatio n of pea aphids, Acyrthosiphon pisum on individua l bean plants in the laborator y (Roy et al., 1998). Foraging ladybird s cause an increase in aphid movement (Clegg & Barlow, 1982), although the degree to which this occurs depends on the aphid species and host plant (MuÈller, 1983; Hajek & Dahlsten, 1987). The increase in movement of aphids in the presence of foraging predators such as coccinellids would increase the probabilit y of the aphid coming into contact with the sporulating cadaver, and therefore receiving more inoculum. Increased transmission of fungal entomopathogen s between host insects at a local scale has also been observed in systems where parasitoids forage. In a laborator y study by Furlong and Pell (1996) the presence of the foraging parasitoid Diadegma semiclausum increased the movement of Plutella xylostella larvae and consequently the transmission of the entomopathogen Zoophthora radicans. In this study, larval movement was quanti® ed using video techniques; larvae foraged on by D. semiclausum moved signi® cantly further and into signi® cantly more new areas of leaf than control larvae. Interestingly, larvae foraged on by a diVerent parasitoid, Cotesia plutellae, were intermediate in their movement and were not more likely to become infected by the entomopathoge n than control larvae. This suggests that there is a threshold of movement necessary for fungal transmission to be encouraged and that this threshold was exceeded by larvae foraged on by D. semiclausum but not by larvae foraged on by C. plutellae. Clearly, these interactions are complex and diYcult to predict because they vary between insect natural enemies. In a similar study, the transmission Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 INTERACTIONS BETWEEN ENTOMOPATHOGENIC FUNGI AND OTHER NATURAL ENEMIES 739 of E. neoaphidis was increased in the presence of the parasitoid Aphidius rhopalosiph i (Fuentes-Contreras et al., 1998). For transmission to occur over a larger spatial scale, pathoge n dispersal is essential particularly if host population s are discontinuou s in the environment. Most pathogens have a limited capacity for active dispersal; however, entomophthoralea n fungi (except Massospora), produce conidiophore s that forcibly discharge primary conidia across the leaf boundar y layer (Ingold, 1971; Steinkraus et al., 1996; Hemmati, 1999). Dispersal of conidia over a greater distance requires physical factors such as rain and wind or dispersal by hosts and non-hosts. Rain can remove pathogens in splash droplets or by vibration caused by the impact of rain drops. Many studies have shown the importance of rain in the dispersal of plant pathogens (Fitt et al., 1989; McCartney, 1990; Pedersen et al., 1994; McCartney, 1994), though this may be less important for some entomopathogen s such as E. neoaphidis (Pell et al., 1997a). Wind is undoubtedly important in the long distance dispersal of many fungal entomopathogen s (Wilding, 1970; Steinkraus et al., 1996; Hemmati, 1999) with most fungal conidia being small (for example, E. neoaphidis primary conidia are approximately 25 l m in length and the conidia of Hyphomycetes are less than 10 l m) and capable of being carried by wind currents. Dispersal of entomopathogen s by weather factors, however, is random in contrast to dispersal by hosts and non-host vectors which is targeted. The movement of infected hosts is considered to be one of the most important ways in which a pathoge n is transmitted and dispersed to new habitats (Fuxa & Tanada, 1987). Pathogens can also be dispersed by non-hosts such as invertebrate natural enemies of the host which have become contaminated with the pathogen during foraging . Insect vectors are important dispersal agents of many plant pathogens (Agrios, 1988; Nemeye et al., 1990; Peng et al., 1992; Gillespie & Menzies, 1993). It has long been recognized that predators and parasitoids contribute to the dissemination of viral entomopathogen s and the development of epizootics (Smith et al., 1956; Stairs, 1965; Capinera & Barbosa, 1975; Reardon & Podgwaite, 1976; Andreadis, 1981; Levin et al., 1983), however, only a few more recent studies have assessed the transmission of fungal entomopathogen s by non-host vectors (Schabel, 1982; Poprawski et al., 1992; Pell et al., 1997b; Butt et al., 1998). In a laborator y study, Roy (1997) demonstrated that adult coccinellids (Coccinella septempunctata) which had foraged on plants with diVerent densities of sporulating cadavers became contaminated with E. neoaphidis conidia and carried these conidia to uninfected aphid populations , thereby initiatin g infection. Transmission was greatest when coccinellid adults had previously foraged on plants with high densities of infected aphids (30 cadavers per plant). However, even coccinellids which had foraged on plants with only one cadaver became contaminate d with suYcient conidia to vector to a small proportion of the uninfected aphids. Therefore, coccinellids may contribute to pathogen dispersal from aphid population s even when the pathoge n density is low. In contrast, the number of conidia produced by an individua l sporulating cadaver may not be suYcient for aerial dispersal because of its random nature. The ability of coccinellids to aggregate in areas with large aphid densities (Kareiva & Odell, 1987) would contribute to their role in pathogen dispersal by targeting the pathogen to aphid population s. This ability to vector pathogens between infected and uninfected host population s could also be manipulate d for integrated pest management (IPM) if the vector can be arti® cially contaminated with inoculum, perhaps using semiochemicals and contaminatio n stations as has been done in other systems (for example Pell et al., 1993a; Furlong et al., 1995). Pell et al. (1997b) demonstrated the potential of coccinellid adults to vector E. neoaphidis passively to uninfected pea aphid population s after arti® cial contamination . Direct exposure of aphids to adult coccinellids inoculate d with E. neoaphidis resulted in 10 per cent E. neoaphidis infection. Furthermore, a similar proportion of aphids which were placed on the plants previously foraged on by inoculated adult and larval coccinellids also succumbed to E. neoaphidis infection (Pell et al., 1997b; Roy, 1997). The potential of coccinellid adults to vector E. neoaphidis in a caged ® eld study has also been demonstrated (Roy, 1997). Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 740 H. E. ROY & J. K. PELL Inoculated coccinellids caused approximatel y ® ve per cent of the aphid population to become infected by E. neoaphidis. Although this may appear to be a low percentage of infection, under suitable conditions an epizootic could be initiated by such a level of infection (Pell et al., 1997b; Roy et al., 1998). Only a few other studies have examined the potential of arthropod natural enemies to vector fungal entomopathogen s. In the study of Furlong and Pell (1996), although the presence of a foraging parasitoid, D. semiclausum, increased the movement of P. xylostella larvae and consequently the transmission of Z. radicans, there was no evidence that the parasitoid vectored the pathogen to new P. xylostella populations. Similarly, the aphid parasitoid A. rhopalosiphi did not vector E. neoaphidis between population s of aphids (Fuentes-Contreras et al., 1998). Bene® cial insects, other than natural enemies, may provide an alternative means of pathogen dissemination . For example, the ability of honey bees (Apis mellifera) to eVectively vector the Hyphomycete fungus Metarhizium anisoplia e to pollen beetles (Meligethes aeneus) has been demonstrated (Butt et al., 1998). Although honey bees are susceptible to M. anisopliae (Ball et al., 1994), no detrimental eVects to the honey bees were observed (Butt et al., 1998). ANTAGONISTIC INTERACTIONS An increase in the richness of natural enemy species could result in an increase of the host if interference between the natural enemies is suYcient (Hochberg & Lawton, 1990). Parasitism/infection, predation, and competition are three potentially antagonisti c interactions which would result in interference between insect and fungal natural enemy species. Direct Infection of Non-target Insects by Entomopathogeni c Fungi Entomopathogeni c fungi may reduce non-target insect population s by directly infecting them. However, many entomopathogeni c fungi appear to have a limited host range and are speci® c to single orders of arthropod hosts. In general, fungi from the order Entomophthorales commonly cause epizootics in insect population s (often foliar insects) and these epizootics can eradicate population s at a local scale (Wilding & Brady, 1984; Glare & Milner, 1989). Species of fungi within this order are generally considered to be highly host speci® c (Lacey et al., 1997). For example, E. neoaphidis is the most common fungal species infecting aphids in temperate regions (Wilding & Brady, 1984; Glare & Milner, 1989) and is aphid-speci® c. Therefore, Entomophthoralea n fungi considered for pest management are unlikely to constitute a risk to bene® cial insects and other non-target organisms unless those organisms are very closely related to the target pest (Lacey et al., 1997). This was highlighte d by the concerns raised on the release of Entomophaga grylli (pathotype 3) as a classical biologica l control agent of grasshopper pests in the rangelands of the western USA (Carruthers & Onsager, 1993). This fungus, while limited to Orthoptera, has a wide host range with respect to North American grasshopper s with the potential that non-pest grasshoppers may become infected (Lockwood, 1993). However, in the ® eld, only 8 out of 20 grasshopper species were infected and these were all pest species (Carruthers et al., 1997). Although this limited host range makes them valuable potential biological control agents, most entomophthoralea n fungi are quite diYcult to mass-produce and store, therefore, use as inundativ e mycoinsecticides is currently limited. In contrast, Hyphomycete fungi are only infrequently recorded causing substantial epizootics (for example, Keller, 1986) and are generally considered to have wide host ranges (Feng et al., 1994; Lacey et al., 1997). However, they can be mass produced commercially and are, therefore, often augmented within pest insect population s as inundativ e mycoinsecticides (Vinson, 1990; Lacey et al., 1997; Jaronski et al., 1998). Due to their perceived wide host range, for example, Beauveria bassiana has been recorded infecting over 200 species of insects in nine orders (Li, 1988; Feng et al., 1994) these fungi could potentially pose a threat INTERACTIONS BETWEEN ENTOMOPATHOGENIC FUNGI AND OTHER NATURAL ENEMIES 741 Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 to non-targets. However, the pathogenicit y of fungal isolates from the same species towards diVerent insect groups varies signi® cantly (Yeo et al., 1998). There is a tendency for fungal isolates to have higher virulence to their original hosts or to closely related species than more distant relatives (Xu, 1988; Goettel et al., 1990; Hajek & Butler, 1999). Examples of host range studies of isolates from species in the Entomophthorale s and Hyphomycetes against insect natural enemies speci® cally are described below. Direct Infection of Insect Natural Enemies by Entomophthorale s Zoophthora radicans is an entomophthoralea n fungus which is considered to have a relatively broad host range because it has been recorded from a number of diVerent insect orders (Hajek & Butler, 1999). This fungus is an important natural enemy of the diamondbac k moth (P. xylostella) a crucifer pest. The parasitoids D. semiclausum and C. plutellae are also common natural enemies of this moth and could therefore be at risk from infection by Z. radicans. Laborator y studies demonstrated that C. plutellae never became infected with Z. radicans; however, D. semiclausum was susceptible to Z. radicans but only at extremely high doses of the fungus (Furlong & Pell, 1996). Diadegma semiclausum foraging in the presence of infected larvae in the laborator y did not succumb to Z. radicans infection and infected individual s were never observed in the ® eld, even during epizootics in diamondbac k moth populations. For these reasons Furlong and Pell (1996) concluded that although the parasitoid was clearly physiologicall y susceptible to Z. radicans, it was unlikely to be ecologically susceptible in the ® eld. These ® ndings are consistent with other studies on Z. radicans which have demonstrated that isolates are often unable to infect species from orders other than the one from which they were isolated (Papierok et al., 1984; McGuire et al., 1987; Magalhae s et al., 1988) and sometimes even from diVerent families within an order (Mietkiewski et al., 1986; Pell et al., 1993b). Direct Infection of Natural Enemies by Hyphomycetes As already described, fungi within the class Hyphomycetes are generally considered to have much wider host ranges than entomophthoralean fungi (Goettel & Inglis, 1997) and may infect both the target pest species and their insect natural enemies (for example Magalhaes et al., 1988). However, although many species of these fungi have been recorded from numerous diVerent insect orders, isolates or strains within a species are frequently only virulent to a few arthropod species (Feng et al., 1994; Yeo et al., 1998). Exposure of two parasitoid species (Bracon hebetor and Apoanagyru s lopezi) to eleven isolates of M. anisopliae and one isolate of B. bassiana, under development for control of grasshoppers and locusts, resulted in 100% mortality of the parasitoids. However, no infection was observed in two other non-target species, the tenebrionids Pimelia senegalensis and Trachyderma hispida, exposed to high doses of the same isolates (Danfa & Van der Valk, 1999). A commerciallyformulated isolate of B. bassiana (Naturalis ) is reported to be extremely eYcient at controlling a number of greenhouse pests, such as white¯ ies, thrips and mites but with no impact on bene® cial insects (Wright & Kennedy, 1996). In a laborator y study, Poprawski et al. (1998) assessed the pathogenicit y of B. bassiana and Paecilomyce s fumosoroseus to the coccinellid Serangium parcesetosum, an important white¯ y predator. Serangium parcesetosum were highly susceptible to B. bassiana but not to P. fumosoroseus (Poprawski et al., 1998). As a species, Verticillium lecanii has been recorded from a number of insect and mite hosts, but in glasshouse trials using target-derived isolates against aphids and white¯ y it did not infect Tetranychus urticae (red spider mite), Phytoseiulus persimilis (spider mite predator) or Encarsia formosa (white¯ y parasitoid) (Hall, 1981). Askary and Brodeur (1999) have demonstrated that V. lecanii is pathogeni c to the aphid parasitoid Aphidius nigripes but only when aphid population s are heavily infected by the fungus. Similarily P. fumosoroseus infects the aphid parasitoid, Aphelinus asychis, but only at high pathoge n doses and at high humidity (Lacey et al., 1997). Another species of Hyphomycete, Nomuraea rileyi, primarily infects lepidopteran pests Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 742 H. E. ROY & J. K. PELL but natural epizootics of this fungus have also been observed in two species of Coleoptera (Hypera punctata and Leptinotarsa decemlineata) (IgnoVo, 1981). However, various natural enemies (predators (Hippodamia convergens, Chrysoperla carnea and Podisus maculiventris) and parasitoids (Voria ruralis, Apanteles marginiventris, Campoletis sonorensis and Telenomus proditor)) did not succumb to N. rileyi infection when exposed to high concentrations of conidia (25 times higher than used in ® eld experiments to induce epizootics) in laborator y bioassays (IgnoVo, 1981). Ecological vs Physiological Susceptibility Overall, many studies have shown that entomopathogeni c fungi with broad host ranges can interact antagonisticall y with arthropod natural enemies but only under environmental conditions that are optimal for the fungus (James & Lighthart, 1994; Lacey et al., 1997; Askary & Brodeur, 1999). In these cases, `physiologica l susceptibility’ is demonstrated. However, this may not represent `ecological susceptibility’ under more realistic conditions in the ® eld which are likely to be suboptimal for the fungus. It is important to distinguish between physiologica l and ecological susceptibility of predators and parasitoids to pathogens when considering complexes of these natural enemies as biological control agents. It is recognized that laborator y bioassays do not always re¯ ect infection levels that are likely to occur in the ® eld (Goettel et al., 1990; Jaronski et al., 1998). A laborator y bioassay on one particular isolate (strain GHA) of B. bassiana demonstrated that some predators and parasitoids were susceptible, but in the ® eld the impact on the natural enemy complex was minimal (Jaronski et al., 1998). The importance of conducting realistic ® eld experiments, in addition to preliminary laborator y bioassays, has been further demonstrated through a series of studies on the interactions between B. bassiana and the convergent ladybird, H. convergens. Hippodamia convergens is a widespread, abundan t predator of aphids in the USA. Laboratory studies demonstrated that H. convergens was susceptible to three fungal pathogens currently being considered as biologica l control agents in the USA, B. bassiana, M. anisopliae and P. fumosoroseus (James & Lighthart, 1994), which are all known to have broad host ranges (Goettel et al., 1990). Field studies demonstrated that B. bassiana caused 75± 93% mortality of H. convergens early in the season but that late season application s had little impact (James et al., 1995). Late season environmenta l conditions may not favour B. bassiana mycosis in H. convergens. James et al. (1998) showed that, although fastest germination and growth rates of B. bassiana occurred at temperatures in excess of 25ë C, levels of mycosis in H. convergens decreased as temperature increased above this temperature. It appears that temperature may be signi® cantly aVecting the defence of H. convergens against B. bassiana. Indeed, some metabolic compounds induced by heat stress are known to play a role in defending an insect against pathogen invasio n (James et al., 1998). Interestingly, starvation and nutrition stresses increase the susceptibility of C. carnea to B. bassiana (Donegan and Lighthart, 1989). Prevailin g environmenta l conditions will undoubtedly aVect the physiology of the host and, hence, its susceptibility to a pathogen. A further example demonstrating the diVerence between physiologica l and ecological susceptibility comes from Entomophaga maimaga, an entomophthoralea n fungus associated with many North American and Japanese lepidopteran species. The host speci® city of E. maimaiga has been extensively studied because it is a potential biologica l control agent of the gypsy moth (Lymantria dispar), a lepidopteran pest of forests. Laboratory studies indicated that 35.6 % of the 78 lepidopteran species exposed to E. maimaiga were susceptible (Hajek et al., 1995). However, in ® eld trials only two lepidopteran non-target individual s were infected (Hajek et al., 1996). This highlight s the signi® cance of considering both physiological and ecological susceptibility of non-target insects to pathogens. Laborator y practices, such as repeated subculturing , long term storage and in vitro culturing, can result in changes in virulence which would also aVect the proportion of insects infected during bioassays (St. Leger et al., 1991; Hajek & Butler, 1999). Furthermore, there are many potential diVerences between insect hosts in the laborator y and in the ® eld Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 INTERACTIONS BETWEEN ENTOMOPATHOGENIC FUNGI AND OTHER NATURAL ENEMIES 743 (Hajek & Butler, 1999). Insects reared on arti® cial diet in the laborator y are more susceptible to fungal pathogens than insects fed on natural diets (Hajek & Butler, 1999). Insects in the ® eld can exhibit behaviours which are not possible in the laborator y and which can aVect the pathogen, for example, some insects actively manipulat e thermoregulatio n to impede development of fungal infections (Watson et al., 1993; Inglis et al., 1996; Blanford et al., 1998). The spatial and temporal separation of insects and fungi in the ® eld further contributes to the discrepancy between infection rates observed in the laborator y compared to those in the ® eld (Hajek & Butler, 1999). Therefore, when considering the potential risk to insect natural enemies of releasing a fungal pathogen, susceptibility assays should be carried out beginning with maximum challenge laborator y studies to identify isolates that have no pathogenicit y to non-targets even under optimal conditions for the fungus. Such isolates present minimal to no direct risk. Isolates that are able to infect non-targets should then pass through semi-® eld and ® eld studies under more realistic conditions to determine whether they are likely to represent an ecological risk. Interaction Between Predation and Fungal Infection Intra-guild predation is a dramatic example of interference between natural enemies, which could result in antagonism , and reduced mortality of the host. It occurs if competing predators also engage in a trophic interaction (predation or parasitism) with one another (Polis & Holt, 1992), and is prevalent within many communities of biologica l control agents (Rosenheim et al., 1995). Many studies have illustrated the occurrence of asymmetric (unidirectional ) and symmetric (bi-directional ) intra-guild predation between: (a) predators and parasitoids (Wheeler, 1977; Colfer & Rosenheim, 1995; Evans & England, 1996); (b) diVerent parasitoid species attacking the same arthropod pests (Briggs, 1993); (c) diVerent predator species (Sengonca & Frings, 1985); and (d) entomopathogen s and arthropod natural enemies (Flexner et al., 1986; Goettel et al., 1990). Studies on the interactions between entomopathogen s and arthropod natural enemies have generally concentrated on the pathogen as the `intra-guild predator’, i.e. directly able to infect insect natural enemies in the guild (Flexner et al., 1986; Goettel et al., 1990). However, the pathoge n can also be preyed upon. In non-choice laborator y experiments, adult C. septempunctata and the carabid, Pterostichus madidus, have been shown to consume aphids at a late stage of E. neoaphidis infection (Pell et al., 1997b; Roy et al., 1998) and can, therefore, be considered as intra-guild predators of the pathogen. Fourth instar C. septempunctata larvae partially consumed sporulatin g infected aphids. However, other common aphid predators (larval stages of the syrphid, Episyrphus balteatus, and the chrysopid, C. carnea) never consumed infected aphids. Therefore, it is unlikely that syrphid, chrysopid and early instar coccinellid larvae will negatively aVect the biological control potential of E. neoaphidis. Adult and fourth instar larvae of C. septempunctata and adults of P. madidus could be considered as intra-guild predators of E. neoaphidis and have the potential to have a negative impact on E. neoaphidis. However, the biology of these two predatory beetles suggest that this interaction may not be important. First, in the absence of uninfected aphids, fourth instar C. septempunctata larvae predated on aphids infected with E. neoaphidis, but often only partially consumed the cadavers. Although coccinellid larvae have an increased motivation to feed than adults (Ferran & Dixon, 1993), even fourth instar larvae starved for 24 h did not entirely consume sporulating E. neoaphidis infected A. pisum. By comparison, uninfected A. pisum were readily consumed. Hence C. septempunctata has the potential to reduce E. neoaphidis population density, but uninfected aphids are more likely to be consumed than infected aphids. Indeed, from ® eld observations it was apparent that no visibly-infecte d aphids were consumed by either adult or larval C. septempunctata (Roy, 1997). Partially-consume d cadavers produced signi® cantly fewer conidia but this did not aVect transmission rates in laborator y experiments. The same proportion of aphids became infected regardless of whether the source of inoculum was a damaged or an intact cadaver Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 744 H. E. ROY & J. K. PELL (Roy et al., 1998). Furthermore, the presence of a foraging predator greatly increased withinpopulatio n transmission, as already described, which would more than compensate for the small reduction in inoculum caused by feeding damage. Infected aphids were entirely consumed more quickly by carabids than uninfected aphids and, therefore, more infected, than uninfected, aphids were consumed over the study period. This generalist predator forages on the ground and consumes cadavers that become dislodged from the plant. These cadavers are already lost from the arena of fungal transmission on the plant and so may not contribute to the development of a local epizootic. Morgan (1994) demonstrated that E. neoaphidis conidia could persist on soil where they may be important in the survival of the fungus over short periods when conditions are unfavourable for infection. Therefore, P. madidus may not have an eVect on immediate transmission of E. neoaphidis but may have an eVect on its persistence in soil. Living infected aphids are able to produce alarm pheromone but are less able to respond to it (Roy et al., 1999), which may increase the probabilit y of them not surviving attack by a predator and thus may result in a reduction in pathogen density. Predators feed less on infected aphids and this, combined with their reduced response to alarm pheromone, may result in a greater proportion of infected aphids remaining on the plant which would bene® t local transmission of the pathogen. The reduced abilit y of late-stage infected aphids to recolonize a plant after dislodgement may increase their vulnerabilit y to ground predators, such as the carabid, P. madidus. However, of the infected aphids that responded to alarm pheromone, walking was a more common response than dislodging . A limited number of other laborator y studies have examined the predation of insects infected with fungal pathogens. Pell and Vandenberg (1998) demonstrated that the convergent ladybird, H. convergens, avoided feeding on Russian wheat aphids, Diuraphis noxia, infected with P. fumosoroseus. Sahelian grasshoppers (Acrotylus blondeli and Pyrgomorpha cognata) infected with M. anisopliae were less likely to be predated on than uninfected individual s (Arthurs, 1999). Therefore, cadavers of all these insects are likely to persist in the environment. This was in contrast to brown locusts, Locustana pardalina infected with M. anisopliae, which are more susceptible to predation than uninfected individual s and, therefore, less likely to contribute to secondary cycling (Arthurs, 1999). Polis et al. (1989) concluded that intra-guild predation incorporates aspects of exploitation , apparent and classical competition: a tendency for exclusion (either because of direct competition or because the intra-guild predator can be sustained by the intra-guild prey), possibilitie s for co-existence (if the intra-guild prey species is superior to the intra-guild predator at resource exploitation ) or an increase in the resource (as a consequence of removal of predator species by intra-guild predators). Intra-guild predation is widespread amongst communities of biological control agents and is an important consideration in the use of multiple species for biologica l control. However, the signi® cance of intra-guild predation varies in diVerent community contexts (Polis et al., 1989) and, consequently, its impact on biologica l control programmes will be variable as indicated by the studies outlined above. Competition Between Entomopathogeni c Fungi and Other Natural Enemies A pathoge n may interfere with the natural enemy complex indirectly by reducing the host population s of predators and parasitoids or by rendering the host unsuitabl e for other natural enemies (Rosenheim et al., 1995). Most interactions between parasitoids and entomopathogeni c fungi are asymmetrical in favour of the pathogen (Hochberg & Lawton, 1990), however, the relative timing of parasitism and fungal infection is often crucial to the ® nal competitive outcome. The interaction between the aphid parasitoid, A. nigripes, and the fungus, V. lecanii, is dependent on the relative timing of parasitism/infection. Development of the aphid parasitoid, A. nigripes, is impeded by V. lecanii infection; however, a high proportion of parasitoids developed successfully when aphids were exposed to V. lecanii 4 days after parasitizatio n Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 INTERACTIONS BETWEEN ENTOMOPATHOGENIC FUNGI AND OTHER NATURAL ENEMIES 745 (Askary & Brodeur, 1999). Similarily the entomopathogeni c fungus, N. rileyi, inhibits development of the braconid parasitoid, Microplitis croceipes, if larvae of the bollwor m host (Heliothis zea) are infected one day after parasitizatio n (King & Bell, 1978). The successful augmentation of these natural enemies may be impeded by this antagonism , however, careful management ensuring temporal separation of the interacting natural enemies could result in eVective biologica l control (King & Bell, 1978). The eVective introduction of antagonisticall y interacting natural enemies has also been achieved in other systems with careful management. Some parasitoids can discriminate between hosts infected with a fungal pathoge n and noninfected hosts (Brobyn et al., 1988; Fransen & van Lenteren, 1993). Greenhouse white¯ y, Trialeurode s vaporariorum, infected with the entomopathogeni c fungus, Aschersonia aleyrodis, are unsuitabl e hosts for the parasitoid, Encarsia formosa (Fransen & van Lenteren, 1993). However, the parasitoid can discriminate between infected and non-infected hosts after seven days of fungal infection. Thus, the introduction of the fungus followed seven days later by the parasitoid may result in eVective white¯ y control (Fransen & van Lenteren, 1993). Similarily the aphid parasitoid, A. rhopalosiphi, avoids ovipositin g in aphid hosts at late stages of infection (Brobyn et al., 1988). Increased mortality of pest species can result even when interference between pathogens and parasitoids is evident. The fungal pathogen Hirsutella cryptosclerotiu m and the parasitoid Gyranussoidea tebygi are both natural enemies of the mealybug Rastrococcus invadens. The pathogen reduced levels of parasitism by the parasitoid, but the overall mortality of the mealybug was still greater when both natural enemies acted together (Akalach et al., 1992). Similarly there were additive eVects with regard to aphid control, with no detrimental eVects on the percentage parasitism nor parasitoid emergence when P. fumosoroseus and the parasitoid Aphelinus asychis were used together for control of the Russian wheat aphid D. noxia under ® eld conditions (Mesquita et al., 1997). The observation of competitive interactions in the laborator y may not re¯ ect the ® eld situation. Competition has been demonstrated between two cereal aphid natural enemies: the entomopathogeni c fungus, E. neoaphidis, and the parasitoid, A. rhopalosiphi (Powell et al., 1986). The development of the parasitoid was prevented when the aphids were infected by the fungus less than four days after parasitation. Conversely, fungus development was impeded when infection occurred more than four days after parasitization . However, in the ® eld, parasitoids are often more abundant early in the season when hyperparasitoid s are scarce (Powell, 1982) and at this time fungal pathogens are usually at low levels. Thus, interference between the parasitoid and fungus is likely to be minimal. Antagonistic interactions between natural enemies may be reduced or prevented by such temporal or spatial separation. Many of the studies demonstrating natural enemy interactions are laboratory-base d with behavioural experiments being conducted in simple arenas, for example, Petri dishes. Although such arenas provide environment s in which preliminary observation s on invertebrate behaviour can be made, conclusions from such studies are limited by the context in which they were collected. The interactions between natural enemies are undoubtedly aVected by the complexitie s of the environment in which they forage (Sengonca & Frings, 1985; Chang, 1996; Fuentes-Contreras et al., 1998). The competitive interactions between A. rhopalosiphi and E. neoaphidis are aVected by host plant resistance (Fuentes-Contreras et al., 1998). Host plants which are partially-resistan t to aphid attack result in an increase in the developmental times of aphids and their parasitoids. Consequently, this aVects the third trophic level but appears to be more detrimental to the parasitoid than the fungus. Situations where insects are susceptible to more than one pathoge n are very common (Wilding, 1975; Wilding & Perry, 1980; Sivcev, 1992) and, therefore, there is also potential for mixed infection and competition between pathogens. The fungal pathogens N. fresenii and E. neoaphidis both commonly infect aphids. Dual infection of aphids by these two pathogens has been observed during epizootics in the ® eld (Sivcev, 1992). In laborator y studies, aphids infected simultaneously with both pathogen s died from N. fresenii at high Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 746 H. E. ROY & J. K. PELL temperatures (30ë C) and E. neoaphidis at low temperatures (10ë C) (Villacarlos, unpublishe d data). Therefore, these two pathogens may be spatially and temporally isolated from one another due to their diVerent temperature optima. A similar eVect was observed when B. bassiana and M. Xavoviride co-infected the migratory grasshopper Melanoplu s sanguinipe s under constant and oscillatin g temperatures. Proliferatio n of the fungi in the haemocoel was measured in diVerent environment s with the same mean daily temperature, (25ë C), but diVering in the degree to which they oscillated daily (constant 25ë C, 20± 30ë C, 15± 35ë C, or 10± 40ë C). More B. bassiana than M. anisopliae was recovered from coinfected nymphs at constant 25ë C but as the amplitude of temperature increased, more M. anisopliae than B. bassiana was recovered suggesting that temperature in¯ uenced their competitiveness (Inglis et al., 1999). SPATIAL AND TEMPORAL OCCURRENCE The importance of predators, parasitoids and pathogens in the suppression of pest populations has been the motivation for many studies to quantify ® eld population s of natural enemies. These studies have generally concentrated on either predators (Edwards et al., 1979; Coombes & Sotherton, 1986; Booij & Noorlander, 1992; Ekbom, 1994), parasitoids (Powell, 1982; MuÈller et al., 1999) or pathogens (Wilding, 1975; Ekbom & Pickering, 1990) in isolation . Assessments of the temporal and spatial occurrence of diVerent natural enemies have largely been neglected. Chambers et al. (1986) monitored population s of predators, parasitoids and pathogeni c fungi in winter wheat and concluded that the presence of multiple natural enemies impeded development of the aphid population . However, the contribution of the diVerent natural enemies varied from year to year. Aphid-speci® c predators were the major factor limiting cereal aphid population s in the ® rst year studied but parasitoids and pathogens also contributed to the decline of aphids in the following year. The importance of natural enemies acting concurrently was implicated in this study. In a recent study, Roy (1997) assessed the spatial and temporal occurrence of E. neoaphidis and C. septempunctata in ® eld crops (spring wheat and spring bean) and in a nettle patch. Monitoring of insect and fungus population s began at the beginning of May 1995 and continue d until the crops were harvested (August). Although the ® eld study represents observation s from only one year, C. septempunctata and E. neoaphidis co-occurred both spatially and temporally in crops and in the weed patch. The ® rst observations of both C. septempunctata and E. neoaphidis were in the nettle patch which provided an early source of aphids for both the pathogen and the predator. Nettle patches situated adjacent to crop ® elds may act as reservoirs for parasitoids, predators and pathogens (Perrin, 1975). However, the concept of using reservoirs (alternative hosts) to maintain and increase natural enemy population s depends on the ability of the natural enemies to transfer between diVerent host species. Some aphid parasitoids are capable of transferring between certain hosts without any apparent loss of ® tness (Wratten & Powell, 1990), but others are unable to transfer (Cameron et al., 1984). In addition, the virulence of isolates of the same fungal pathoge n can vary depending on the host species or clone (Sitch & Jackson, 1997); thus aphid species and clones are diVerentially susceptible to diVerent isolates of E. neoaphidis (Milner, 1982; Milner, 1985; Pickering & Gutierrez, 1991). Therefore, even generalists may exist as discrete population s associated with particular hosts or habitats. The potential of natural enemy reservoirs would be impeded by the evolution of distinct host biotypes. Roy (1997) collected samples of infected nettle aphids and used these to infect both A. pisum and Sitobion avenae in the laborator y, providin g evidence that the isolate of E. neoaphidis that infected the nettle aphids was infective to crop aphid species. The importance of nettles as a reservoir for both E. neoaphidis and C. septempunctata has been implicated and the movement of these natural enemies between noncrop and crop habitats is currently under investigation . INTERACTIONS BETWEEN ENTOMOPATHOGENIC FUNGI AND OTHER NATURAL ENEMIES 747 Downloaded by [CIMMYT -International Maize and Wheat Improvement Center ] at 07:45 05 September 2012 IMPLICATIONS FOR BIOLOGICAL CONTROL Manipulativ e strategies The use of entomopathogen s to control pest insects could be enhanced by manipulatin g pest populations. The process of autodisseminatio n involve s the use of host insects to introduce and disperse entomopathogen s (IgnoVo, 1978). A sex pheromone trap has been developed for the autodisseminatio n of Z. radicans by the diamond back moth, P. xylostella (Pell et al., 1993; Furlong et al., 1995). Synthetic sex pheromone is used to attract adult male P. xylostella into traps positioned throughout a cabbage crop where they are inoculated with Z. radicans conidia from a sporulatin g mycelial mat. After habituatio n to the pheromone, the moths leave the trap and disperse into the cabbage crop where they consequently die of infection releasing infective conidia into the host populatio n on the crop. Attractant traps have also been developed for the autodisseminatio n of other fungal and viral pathogens to target pests (Klein & Lacey, 1999; Vega et al., 2000). Similar approaches could be used for developin g traps for the transmission of pathogens by insect natural enemies. Conservation Strategies The potential of arthropod natural enemies to vector entomopathogen s to pest population s could be encouraged within the agroecosystem. Reservoirs (distinct patches of pathogen such as cadavers on trees or resting spores in the soil) are important in the population dynamics of many entomopathogens. For example, the overwintering resting spores of Entomophaga maimaiga have applied to form a reservoir of the pathoge n for the biological control of gypsy moth, Lymantria dispar (Hajek & Roberts, 1991). Erynia neoaphidis does not produce resting spores, but reservoirs of the pathogen could be established in the form of conidia or cadavers. Nettle aphids are often an early source of E. neoaphidis inoculum and may act as a pathogen reservoir. The movement of E. neoaphidis from such a reservoir could be increased by C. septempunctata which disperse from woodland habitats to nettle patches early in spring (Zhou & Carter, 1992; Zhou et al., 1994). Coccinellids foraging in nettle patches may become contaminate d with E. neoaphidis conidia which they may subsequently vector to crop aphids. Therefore, nettle patches within an agroecosystem may conserve both E. neoaphidis and insect natural enemies and enhance transmission to crop species. The provision of reservoirs which conserve complementary insect and fungal natural enemies have potential in other pest/ crop systems. CONCLUSIONS The importance of assessing interactions within potential complexes of natural enemies being considered for use in biologica l control is unquestionabl e (Ferguson & Stiling, 1996). Intra-guild predation among biologica l control agents could reduce the mortality of the target pest populatio n (Rosenheim et al., 1995) and it could be argued that intra-guild predators should be excluded from multi-species assemblages used for the biologica l control of pests (Polis & Holt, 1992). However, intra-guild predators are likely to be generalists (Polis & Strong, 1996) and, so, in the presence of multiple prey species, the diets of intraguild predators may only partially overlap. It is important to assess the interactions between biologica l control agents in a realistic environment. The interactions between natural enemies are often studied in simpli® ed laborator y arenas (Chang, 1996). Although such arenas indicate the potential for natural enemies to interact, they do not re¯ ect the complexities of the multidimensiona l environment of the ® eld situation. Furthermore, such studies are often limited to the examinatio n of trophic interactions, ignoring other aspects of the relationships between species, such as behaviour. From studies described in this paper, it is evident that there are many positive interactions between natural enemies of pest insects and these interactions could be manipulate d or encouraged within the agroecosytem . 748 H. E. ROY & J. K. 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