Mesocosm experiments to assess the transmission of Pandora neoaphidis within simple and mixed field margins and over the crop-margin interface.

Although considerable research on the development of agri-environment schemes has focussed on the value of managed field margins as reservoirs for arthropod natural enemies, their potential as reservoirs of entomopathogenic fungi has received less attention. Whether field margins that are most beneficial for arthropod natural enemies are the same as those for entomopathogenic fungi is unknown. Here, within glasshouse mesocosms, we assessed the reproductive success of the aphid-specific entomopathogenic fungus Pandora neoaphidis on aphids in a 'simple margin' containing one plant species and on the same species of aphid in a 'mixed margin' containing seven plant species. These assessments were done in the presence of Aphidius ervi, a hymenopteran parasitoid of aphids regarded as being a key species to conserve in agri-environment schemes in the UK. When only the plants initially infested with aphids were assessed, transmission of P. neoaphidis was significantly greater (p<0.001) in the mixed margin as was parasitisation by A. ervi (p<0.05). However, when all of the plants in the mesocosms were assessed, transmission of P. neoaphidis remained greater in the mixed margin (p<0.05) whereas parasitisation by A. ervi was greater in the simple margin (p<0.05). This difference may be due to aphid dispersal which was greater in the simple margin thereby benefitting the actively foraging parasitoid whereas clustering of aphids in the mixed margin benefited the passively dispersed fungus. In a second mesocosm experiment, the movement of P. neoaphidis over the crop-margin interface was similar to that of A. ervi despite the fungus only being passively dispersed in contrast to the actively foraging parasitoid. The results presented here indicate that, although the optimal plant composition of field margins may differ for P. neoaphidis and A. ervi, both species can co-exist and reproduce in field margins and will move over the crop-margin interface. Managed field margins that benefit both key arthropod and key microbial enemies have potential for enhancing pest control in associated crops.

Order of inoculation affects the success of co-invading entomopathogenic fungi.

The effect of order of inoculation of Pandora blunckii and Zoophthora radicans co-infecting Plutella xylostella (L.) (Lepidoptera: Plutellidae) was investigated. After co-inoculation, the proportion of larvae infected by either species was greatly reduced compared to when they were inoculated singly. The order of inoculation influenced the final outcome; the isolate inoculated last always killed more larvae than the isolate inoculated first.

Intraguild interactions between the entomopathogenic fungus Pandora neoaphidis and an aphid predator and parasitoid at the population scale.

The interactions that occur between the entomopathogenic fungus Pandora neoaphidis and a predator (Coccinella septempunctata) and a parasitoid (Aphidius ervi) were assessed in microcosm and polytunnel experiments. Transmission of P. neoaphidis to the pea aphid, Acyrthosiphon pisum, was enhanced in the presence of both C. septempunctata and A. ervi in microcosm experiments done under fixed abiotic conditions. In contrast, the reproductive success of A. ervi was reduced in the presence of P. neoaphidis. Despite the increased fungal transmission in the presence of C. septempunctata, there was no additional decrease in the aphid population indicating that P. neoaphidis is functionally redundant in the presence of the coccinellid. In polytunnel experiments the reproductive success of A. ervi was not affected by P. neoaphidis. These results do not support those of the microcosm and may be due to the more natural abiotic conditions in the polytunnel reducing the competitive advantage of the fungus. Microcosms therefore provide an arena in which the interactions between fungal pathogens and other aphid-natural enemies can be assessed however, further assessments at increased spatial scales under more natural abiotic conditions are also required to accurately determine the outcome of these interactions.

Transmission of Pandora neoaphidis in the presence of co-occurring arthropods.

Transmission of the entomopathogenic fungus Pandora neoaphidis to the nettle aphid Microlophium carnosum was assessed in the presence of arthropods that co-exist with the fungus within the habitat but do not compete for aphid hosts. The presence of a parasitoid significantly enhanced transmission, and transmission rates were similar for both enemy and non-enemy parasitoids. Although herbivory of nettle leaves by Peacock butterfly (Inchis io) caterpillars indirectly reduced the number of M. carnosum by >30% due to a reduction in leaf area for feeding, the addition of I. io significantly increased transmission of P. neoaphidis in the remaining aphids. It is likely that enhanced transmission in the presence of A. rhopalosiphii and I. io is due to disturbance and subsequent movement of the aphid, resulting in contact with conidia deposited on the leaf surface. The presence and impact of co-occurring arthropods should be taken into consideration when assessing the transmission of fungal entomopathogens.

Effect of seasonal abiotic conditions and field margin habitat on the activity of Pandora neoaphidis inoculum on soil.

The ability of the aphid pathogenic fungus Pandora neoaphidis to remain active in the absence of a resting stage through a combination of continuous infection and as conidia deposited on soil was assessed alongside the potential for planted field margins to act as a refuge for the fungus. P. neoaphidis was able to infect the pea aphid, Acyrthosiphon pisum, when maintained under controlled conditions that simulated those that occur seasonally in the UK. Although there was a significant inverse relationship between temperature and time-to-kill, with death occurring after 4.2, 6.9 and 13.6 days when maintained under fluctuating summer, autumn and winter temperatures, respectively, there were no additional statistically significant effects of photoperiod. The activity of inoculum on soil was indirectly assessed by baiting with A. pisum. Under controlled conditions P. neoaphidis remained active on soil and was able to infect aphids for up to 80 days. However, the percentage of aphids that became infected decreased from 76% on day 1 to 11% on day 80. Whereas there was little difference in the activity of conidia that had been maintained at 4 degrees C and 10 degrees C, activity at 18 degrees C was considerably reduced. Under field conditions the activity of inoculum was strongly influenced by season. On day 49 there was little or no activity during spring, summer or winter. However, during autumn a mean proportion of 0.08 aphids still became infected with P. neoaphidis. Margin type did not affect the activity of conidia nor was there a difference in activity between blocks that had regenerated naturally and those that had been planted. These results suggest that P. neoaphidis can infect aphids and remain active on soil under the abiotic conditions that occur seasonally in the UK and that this fungus may be able to persist annually without a resting stage.

Food web structure of three guilds of natural enemies: predators, parasitoids and pathogens of aphids.

1. Most communities of insect herbivores are unlikely to be structured by resource competition, but they may be structured by apparent competition mediated by shared natural enemies. 2. The potential of three guilds of natural enemies (parasitoids, fungal entomopathogens and predators) to influence aphid community structure through indirect interactions is assessed. Based on the biology, we predicted that the scope for apparent competition would be greatest for the predator and least for the parasitoid guilds. 3. Separate fully quantitative food webs were constructed for 3 years for the parasitoid guild, 2 years for the pathogen guild and for a single year for the predator guild. The webs were analysed using standard food web statistics designed for binary data, and using information-theory-based metrics that make use of the full quantitative data. 4. A total of 29 aphid, 24 parasitoid, five entomopathogenic fungi and 13 aphid specialist predator species were recorded in the study. Aphid density varied among years, and two species of aphid were particularly common in different years. Omitting these species, aphid diversity was similar among years. 5. The parasitoid web showed the lowest connectance while standard food web statistics suggested the pathogen and predator webs had similar levels of connectance. However, when a measure based on quantitative data was used the pathogen web was intermediate between the other two guilds. 6. There is evidence that a single aphid species had a particularly large effect on the structure of the pathogen food web. 7. The predator and pathogen webs were not compartmentalized, and the vast majority of parasitoids were connected in a single large compartment. 8. It was concluded that indirect effects are most likely to be mediated by predators, a prediction supported by the available experimental evidence.

Effect of fungal infection on the reproductive potential of aphids and their progeny.

The effect of infection by Pandora neoaphidis and Beauveria bassiana on the reproductive potential of the pea aphid, Acyrthosiphon pisum, and their progeny was assessed. Infection by either P. neoaphidis or B. bassiana reduced the number of nymphs produced within 24 h of inoculation and over the entire infection period compared to uninfected aphids. However, infection by either P. neoaphidis or B. bassiana for 24 or 72 h did not alter the intrinsic rate of increase of the host aphid's progeny. Therefore, fungal infection appears to have no indirect effects on the fitness of the host's progeny.

Bizarre interactions and endgames: entomopathogenic fungi and their arthropod hosts.

Invertebrate pathogens and their hosts are taxonomically diverse. Despite this, there is one unifying concept relevant to all such parasitic associations: Both pathogen and host adapt to maximize their own reproductive output and ultimate fitness. The strategies adopted by pathogens and hosts to achieve this goal are almost as diverse as the organisms themselves, but studies examining such relationships have traditionally concentrated only on aspects of host physiology. Here we review examples of host-altered behavior and consider these within a broad ecological and evolutionary context. Research on pathogen-induced and host-mediated behavioral changes demonstrates the range of altered behaviors exhibited by invertebrates including behaviorally induced fever, elevation seeking, reduced or increased activity, reduced response to semiochemicals, and changes in reproductive behavior. These interactions are sometimes quite bizarre, intricate, and of great scientific interest.

Wheat containing snowdrop lectin (GNA) does not affect infection of the cereal aphid Metopolophium dirhodum by the fungal natural enemy Pandora neoaphidis.

Studies were carried out to determine if susceptibility of the cereal aphid Metopolophium dirhodum to the fungus Pandora neoaphidis was affected by wheat expressing snowdrop lectin (GNA). Aphid infection did not differ significantly between the transgenic GNA and non-transformed lines (91 and 82%, respectively). Fecundity also did not differ between aphids on the two lines, and was ca. 18 nymphs adult(-1). Time to infection was ca. 5 days for M. dirhodum on both lines in two of three assays. Our results indicate that wheat expressing GNA would not compromise the efficacy of P. neoaphidis as a biocontrol agent.

Pandora neoaphidis transmission and aphid foraging behaviour.

Pandora neoaphidis is an aphid-specific entomopathogen that produces infective conidia. As aphid movement increases, so does the likelihood of contact with conidia. Volatile distress signals released in response to aphid infestation as an indirect defence against herbivory may affect aphid foraging and, therefore, the fungus-aphid interaction. In this study, two different methods were used to investigate the effect of plant volatiles and P. neoaphidis-sporulating cadavers on (1) the colonisation of Vicia faba plants by Acyrthosiphon pisum and (2) P. neoaphidis transmission. This study indicates that A. pisum does not avoid bean plants containing P. neoaphidis and that transmission of conidia occurs during plant colonisation and, to a lesser extent, during in situ feeding. Although significantly more aphids were recovered from damaged plants compared to undamaged plants, the likelihood of infection was not affected by previous infestation by aphids.

Response of the entomopathogenic fungus Pandora neoaphidis to aphid-induced plant volatiles.

We used a model plant-aphid system to investigate whether the aphid-specific entomopathogenic fungus Pandora neoaphidis responds to aphid-induced defence by the broad-bean plant, Vicia faba. Laboratory experiments indicated that neither in vivo sporulation, conidia size nor the in vitro growth of P. neoaphidis was affected by Acyrthosiphon pisum-induced V. faba volatiles. The proportion of conidia germinating on A. pisum feeding on previously damaged plants was significantly greater than on aphids feeding on undamaged plants, suggesting a direct functional effect of the plant volatiles on the fungus. However, there were no significant differences in the infectivity of P. neoaphidis towards A. pisum feeding on either undamaged V. faba plants or plants previously infested with A. pisum. Therefore, these results provide no evidence to suggest that P. neoaphidis contributes to plant indirect defence strategies.

Study of temperature-growth interactions of entomopathogenic fungi with potential for control of Varroa destructor (Acari: Mesostigmata) using a nonlinear model of poikilotherm development.

AIMS: To investigate the thermal biology of entomopathogenic fungi being examined as potential microbial control agents of Varroa destructor, an ectoparasite of the European honey bee Apis mellifera. METHODS AND RESULTS: Colony extension rates were measured at three temperatures (20, 30 and 35 degrees C) for 41 isolates of entomopathogenic fungi. All of the isolates grew at 20 and 30 degrees C but only 11 isolates grew at 35 degrees C. Twenty-two isolates were then selected on the basis of appreciable growth at 30-35 degrees C (the temperature range found within honey bee colonies) and/or infectivity to V. destructor, and their colony extension rates were measured at 10 temperatures (12.5-35 degrees C). This data were then fitted to Schoolfield et al. [J Theor Biol (1981)88:719-731] re-formulation of the Sharpe and DeMichele [J Theor Biol (1977)64:649-670] model of poikilotherm development. Overall, this model accounted for 87.6-93.9% of the data variance. Eleven isolates exhibited growth above 35 degrees C. The optimum temperatures for extension rate ranged from 22.9 to 31.2 degrees C. Only three isolates exhibited temperature optima above 30 degrees C. The super-optimum temperatures (temperature above the optimum at which the colony extension rate was 10% of the maximum rate) ranged from 31.9 to 43.2 degrees C. CONCLUSIONS: The thermal requirements of the isolates examined against V. destructor are well matched to the temperatures in the broodless areas of honey bee colonies, and a proportion of isolates, should also be able to function within drone brood areas. SIGNIFICANCE AND IMPACT OF THE STUDY: Potential exists for the control of V. destructor with entomopathogenic fungi in honey bee colonies. The methods employed in this study could be utilized in the selection of isolates for microbial control prior to screening for infectivity and could help in predicting the activity of a fungal control agent of V. destructor under fluctuating temperature conditions.

Effect of spatial heterogeneity on the role of Coccinella septempunctata as an intra-guild predator of the aphid pathogen Pandora neoaphidis.

The foraging behavior of starved and non-starved adult and larval Coccinella septempunctata on groups of plants in the presence of Pandora neoaphidis-infected Acyrthosiphon pisum, uninfected aphids or a mixture of these two prey types was compared. In general results of these studies confirmed the results of previous work comparing foraging behavior on a smaller spatial scale in Petri dishes. However, behaviors were modified in response to spatial complexity, prey quality, and the host plant. Starved C. septempunctata adults and larvae fed for longer and consumed more aphids than non-starved coccinellids. Both larvae and adults fed on infected aphids and in some cases entirely consumed them. This was thought to be due to the ease of capture of infected (dead) aphids and the feeding stimuli provided by the presence of the host plant and, where there was a choice of prey, uninfected aphids in the environment. Both larvae and adults spent the majority of the time foraging in the upper regions of plants and visited more plants when they were not starved or when they were in the presence of less suitable, infected aphid prey.

Sporulation by Entomophthora schizophorae (Zygomycetes: Entomophthorales) from housefly cadavers and the persistence of primary conidia at constant temperatures and relative humidities.

The duration of discharge of Entomophthora schizophorae (Zygomycetes: Entomophthorales) conidia from house fly (Musca domestica, Diptera) cadavers was measured at 7, 18, and 25 degrees C. The higher the temperature, the shorter the duration of conidia discharge. Significantly more conidia were produced per cadaver at 7 degrees C over a period of 120 h than at 18 and 25 degrees C. At 25 degrees C, the initial discharge over the first 10 h was much larger than at the other temperatures, and at 7 degrees C, no peak in discharge was observed. The persistence of E. schizophorae primary conidia was measured on fabricated non-host surfaces typically found in stables (straw, wood, plaster, and glass) at 7, 18, and 25 degrees C or constant relative humidities of 45, 65, and 85%. Persistence, as measured by the subsequent ability to infect flies, was usually only a few days and depended on the temperature and type of surface. It was greatest on straw, followed by wood, glass, and plaster, and at 7 degrees C, followed by 18 and 25 degrees C. Limited transmission took place between flies exposed to conidia and previously unexposed mates.

Conflicts between a fungal entomopathogen, Zoophthora radicans, and two larval parasitoids of the diamondback moth.

Zoophthora radicans (Zygomycetes: Entomophthorales), Diadegma semiclausum (Hymenoptera: Ichneumonidae), and Cotesia plutellae (Hymenoptera: Braconidae) are all natural enemies of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Adult C. plutellae are not susceptible to Z. radicans infection but the pathogen can infect and kill adult D. semiclausum. Infection of adult D. semiclausum prior to exposure to P. xylostella host larvae significantly reduced the number of parasitoid cocoons subsequently developing from the host larvae. Although Z. radicans infection of P. xylostella larvae prior to parasitism by D. semiclausum or C. plutellae always resulted in the death of the immature parasitoids, neither species discriminated between healthy and Z. radicans-infected host larvae in an oviposition choice experiment. However, host larvae recently killed by Z. radicans were always rejected by D. semiclausum but sometimes accepted by C. plutellae. At 20 degrees C, egg to pupa development took 6.7 and 7.8 days for D. semiclausum and C. plutellae, respectively. C. plutellae parasitism significantly increased host instar duration but D. semiclausum parasitism did not. Cadavers of P. xylostella larvae parasitized 1 day prior to fungal infection showed no reduction in Z. radicans conidia yield. However, cadavers of larvae parasitized 3 days prior to fungal infection demonstrated a marked decrease in Z. radicans conidia yield. Z. radicans infection of P. xylostella larvae < or = 4 days after parasitism resulted in 100% parasitoid mortality; thereafter, the reduction in parasitoid cocoon yield decreased as the time between parasitism and initiation of fungal infection increased. The extended duration of the host larval stage induced by C. plutellae parasitism increased the availability of the parasitoid to the pathogen. Estimates of interspecific competition indicated a similar pattern for the interaction between Z. radicans and each species of parasitoid.

Implications of predator foraging on aphid pathogen dynamics.

The foraging behavior of starved and nonstarved second and fourth instar Coccinella septempunctata larvae on dead Acyrthosiphon pisum aphids, either infected with the entomopathogenic fungus Erynia neoaphidis (sporulating) or uninfected, was examined. Larvae searched for longer and fed less when presented with infected rather than uninfected A. pisum. Although no sporulating infected aphids were completely consumed, both adult and larval ladybirds can still be considered as intraguild predators. In a further study, fourth instar larvae fed on dying infected, dead infected (not sporulating), and dead uninfected aphids for similar periods of time but again the infected aphids were seldom entirely consumed. Live uninfected aphids were fed upon for significantly longer than any other prey. Infected aphids which were damaged at an early stage of infection (0, 1, or 2 days after inoculation) did not sporulate, whereas damaged moribund aphids (3 days after inoculation) did subsequently sporulate. Damaged sporulating cadavers continued to sporulate. However, damage to moribund and sporulating infected aphids, both mechanical or due to C. septempunctata feeding, reduced the number of conidia subsequently produced. Larval feeding caused the most significant reduction. Under laboratory conditions, C. septempunctata foraging on infected aphids did, therefore, reduce the pathogen density. However, conidia produced from a damaged cadaver resulted in levels of transmission to healthy aphids comparable to that resulting from an intact cadaver. Furthermore, the presence of a foraging adult ladybird resulted in a significant increase in transmission of the fungus to healthy aphids. Preliminary studies to assess the potential of other aphid natural enemies as intraguild predators illustrated that adults of the generalist carabid, Pterostichus madidus, entirely consumed sporulating cadavers. Third instar lacewing, Chrysoperla carnea, and hoverfly, Episyrphus balteatus, larvae never fed on sporulating cadavers. The ecological implications of these results are discussed.