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BACKGROUND Bioassays evaluating entomopathogenic fungi (EPF) isolates for effective microbial control of whitefly are a fundamental part of the screening process for bioprotectants, but development of repeatable, robust bioassays is not straightforward. Currently, there is no readily available standardised method to test the efficacy of EPF on whitefly. Here, we describe the calibration and use of a spray tower to deliver a standardised protocol to assess EPF activity; the method was validated using 18 EPF from four genera in tests against greenhouse whitefly, Trialeurodes vaporariorum (Westwood). RESULTS At 138 kPa, the sprayer delivered 0.062 mL mm−2 (620 L ha−1) and an even deposition of spray across the central 1590 mm2 of the spray area. Average conidial deposition for all EPF was 252 conidia mm−2 and equivalent to 2.5 × 1012 conidia ha−1 at an application concentration of 1 × 107 conidia mL−1. Conidial deposition of a test Beauveria bassiana suspension increased with increasing application concentration. Egg laying by T. vaporariorum adults was restricted to 177 mm2 using clip cages specifically designed to ensure that third‐instar T. vaporariorum received a uniform spray coverage. Nymphs occupied 373 ± 5 mm2 of the leaf after migrating during the first instar. Average T. vaporariorum mortality totaled 8–89% 14 days after application of 1 × 107 conidia mL−1 of each EPF isolate. CONCLUSION Combining the calibrated sprayer and bioassay method provides a reliable, standardised approach to test the virulence of EPF against whitefly nymphs. This laboratory‐based assay is affordable, replicable and allows the user to alter the dose of conidia applied to the target., Entomopathogenic fungi (EPF) are commercially developed as microbial bioprotectants for whitefly control. Here, we describe the calibration and use of a pressurised spray tower to deliver a standardised laboratory protocol to allow 14 day assessments of EPF activity; the method was validated using 18 EPF from four genera in tests against greenhouse whitefly, Trialeurodes vaporariorum. This assay is affordable, replicable and allows the user to alter the dose of spores applied to the target area. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Invasive alien species (IAS) can drive community change through ecological interactions. Parasites and pathogens can play an important role in community function including mitigating or enhancing IAS impacts. Despite this, the degree to which pathogen pressure influences IAS impacts remains poorly understood. We quantified the predatory behaviour of the highly invasive alien harlequin ladybird (Harmonia axyridis) and two UK native species, the 7-spot (Coccinella septempunctata) and 2-spot (Adalia bipunctata) ladybirds, using comparative functional response experiments. We investigated the impacts of pathogen infection on the predatory ability of the ladybirds by exposing individuals to Beauveria bassiana, a widespread entomopathogen. Invasive H. axyridis was a more efficient predator than both the native A. bipunctata and C. septempunctata, often having higher attack and/or lower prey handling time coefficients, whereas native A. bipunctata were the least efficient predators. These differences were found in both adult and larval life-stages. Beauveria bassiana infection significantly altered the predatory efficiency of adult and larval ladybird predators. The effects of pathogenic infection differed between species and life-stage but in many cases infection resulted in a reduced predatory ability. We suggest that the interactions between IAS and pathogens are integral to determining invasion success and impact.

Graphical abstract, Highlights • Baculovirus exposure reduces larval and pupal survival. • Development and mortality was affected in a concentration-dependent manner. • Sub-lethal infection has no effect on flight-related wing morphology. • Wing melanisation increases when individuals are exposed to baculovirus., Infections represent a constant threat for organisms and can lead to substantial fitness losses. Understanding how individuals, especially from natural populations, respond towards infections is thus of great importance. Little is known about immunity in the Glanville fritillary butterfly (Melitaea cinxia). As the larvae live gregariously in family groups, vertical and horizontal transmission of infections could have tremendous effects on individuals and consequently impact population dynamics in nature. We used the Alphabaculovirus type strain Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and demonstrated that positive concentration-dependent baculovirus exposure leads to prolonged developmental time and decreased survival during larval and pupal development, with no sex specific differences. Viral exposure did not influence relative thorax mass or wing morphometric traits often related to flight ability, yet melanisation of the wings increased with viral exposure, potentially influencing disease resistance or flight capacity via thermal regulation. Further research is needed to explore effects under sub-optimal conditions, determine effects on fitness-related traits, and investigate a potential adaptive response of increased melanisation in the wings due to baculovirus exposure.

Threats to wild and managed insect pollinators in Europe are cause for both ecological and socio-economic concern. Multiple anthropogenic pressures may be exacerbating pollinator declines. One key pressure is exposure to chemicals including pesticides and other contaminants. Historically the honey bee (Apis mellifera spp.) has been used as an ‘indicator’ species for ‘standard’ ecotoxicological testing but it has been suggested that it is not always a good proxy for other types of eusocial and solitary bees because of species differences in autecology and sensitivity to various stressors. We developed a common toxicity test system to conduct acute and chronic exposures of up to 240 h of similar doses of seven chemicals, targeting different metabolic pathways, on three bee species (Apis mellifera spp., Bombus terrestris and Osmia bicornis). We compared the relative sensitivity between species in terms of potency between the chemicals and the influence of exposure time on toxicity. While there were significant interspecific differences that varied through time, overall the magnitude of these differences (in terms of treatment effect ratios) was generally comparable (< 2 fold) although there were some large divergences from this pattern. Our results suggest that A. mellifera spp. could be used as a proxy for other bee species provided a reasonable assessment factor is used to cover interspecific variation. Perhaps more importantly our results show significant and large time dependency of toxicity across all three tested species that greatly exceeds species differences (> 25 fold within test). These are rarely considered in standard regulatory testing but may have severe environmental consequences, especially when coupled with the likelihood of differential species exposures in the wild. These insights indicate that further work is required to understand how differences in toxicokinetics vary between species and mixtures of chemicals.

The rapid increase in insect production for food and feed both in Europe and elsewhere in the world has led to a need for a coordinated action to assist producers in the diagnosis and management of insect diseases in production stock. Diseases caused by viruses, bacteria, fungi, protozoa and other insect pathogens can be detrimental for reared insects and may cause significant economic loss to producers. Here, we suggest how academia, commercial companies and other insect producers can jointly develop best practice for diagnosing insect diseases early and thereby manage such diseases efficiently. First, we analyse different ways of transmission of insect diseases in closed and semi-closed production facilities. Thereafter we describe four recent cases where companies have requested advice about insect pathogens in their insect stock namely: with giant mealworm Zophobas morio, yellow mealworm Tenebrio molitor, house cricket Acheta domesticus, and with lesser mealworm Alphitobius diaperinus. Our experience dealing with these cases gave us insight to suggest how we should coordinate European activities to establish a service to diagnose and provide advice, and how different European laboratories specialised in insect pathology should collaborate. An important issue will be to educate a new generation of insect pathologists, who with a combination of classical insect pathology methods and the most modern tools can become professionals in diagnosing and managing the various types of insect pathogens.

Harmonia axyridis (Coleoptera: Coccinellidae) has been introduced widely for biological control of agricultural pests. Harmonia axyridis has established in four continents outside of its native range in Asia and it is considered an invasive alien species (IAS). Despite a large body of work on invasion ecology, establishment mechanisms of IAS and their interactions with natural enemies remain open questions. Parasites, defined as multicellular organisms that do not directly kill the host, could potentially play an important role in regulating host populations. This study presents a review of the parasites of H. axyridis, discussing their distributions and effects on host populations across the host’s native and invasive range. These parasites are: Hesperomyces virescens Thaxt. fungi, Coccipolipus hippodamiae (McDaniel and Morrill) mites, and Parasitylenchus bifurcatus Poinar and Steenberg nematodes.

Concern over reported honeybee (Apis mellifera spp.) losses has highlighted chemical exposure as a risk. Current laboratory oral toxicity tests in A. mellifera spp. use short-term, maximum 96 hour, exposures which may not necessarily account for chronic and cumulative toxicity. Here, we use extended 240 hour (10 day) exposures to examine seven agrochemicals and trace environmental pollutant toxicities for adult honeybees. Data were used to parameterise a dynamic energy budget model (DEBtox) to further examine potential survival effects up to 30 day and 90 day summer and winter worker lifespans. Honeybees were most sensitive to insecticides (clothianidin > dimethoate ≫ tau-fluvalinate), then trace metals/metalloids (cadmium, arsenic), followed by the fungicide propiconazole and herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). LC50s calculated from DEBtox parameters indicated a 27 fold change comparing exposure from 48 to 720 hours (summer worker lifespan) for cadmium, as the most time-dependent chemical as driven by slow toxicokinetics. Clothianidin and dimethoate exhibited more rapid toxicokinetics with 48 to 720 hour LC50s changes of

The use of fungal entomopathogens as microbial control agents has driven studies into their ecology in crop ecosystems. Yet, there is still a lack of understanding of the ecology of these insect pathogens in semi-natural habitats and communities. We review the literature on prevalence of fungal entomopathogens in insect populations and highlight the difficulties in making such measurements. We then describe the theoretical host-pathogen models available to examine the role that fungal entomopathogens could play in regulating insect populations in semi-natural habitats, much of the inspiration for which has been drawn from managed systems, particularly forests. We further emphasise the need to consider the complexity, and particularly the heterogeneity, of semi-natural habitats within the context of theoretical models and as a framework for empirical studies. We acknowledge that fundamental gaps in understanding fungal entomopathogens from an ecological perspective coupled with a lack of empirical data to test theoretical predictions is impeding progress. There is an increasing need, especially under current rapid environmental change, to improve our understanding of the role of fungi in insect population dynamics beyond the context of forestry and agriculture.

Over the last few decades there have been considerable advances in the fields of insect pathology and insect conservation but the two disciplines rarely meet. The potential of entomopathogens as biological control agents of pest insects is widely recognized but information on the role of pathogens in insect population regulation, more generally, is limited. For example, the role of pathogens as natural enemies of non-pest insects, including those of conservation value, is seldom considered beyond their context as ‘non-targets’ of microbial control agents. Entomopathogens are prevalent in natural systems and should receive greater attention in life history studies. There is no doubt that viruses, bacteria and fungi are major mortality agents of insects but their significance tends to be overshadowed by the attention given to predators and parasitoids. We highlight the critical function that entomopathogens could have in insect population dynamics with particular reference fragmented habitats as illustrated by the theoretical literature. However, we emphasize that there are few empirical studies to test theoretical predictions. Furthermore, we suggest that since an increase in the incidence of disease is predicted in most environmental change scenarios, it is more important than ever to turn our attention to insect pathology when we consider insect population dynamics.

This study was the second part of a project to assess the applicability of the male-produced, aggregation pheromone of the strawberry blossom weevil, Anthonomus rubi, for commercial monitoring and control. Following optimisation of pheromone lure and trap in previous work, the potential of the pheromone traps for monitoring and control of the pest was investigated. There was no obvious relationship between total pheromone trap catches and numbers of weevils on the crop determined by beating. However, good correlations were obtained at five sites during two seasons between the cumulative numbers of weevils and the amount of severing damage on the crop. Early catches of weevils preceded the first occurrence of damage by at least 1 week in several instances,providing useful information for timing the application of insecticide sprays against the adults to prevent occurrence of damage. The number of flowers severed was usually in the range of 0.5–2.0 times the cumulative number of weevils captured per trap. Adult weevil catches started at a low level in April or early May with similar numbers of males and females. The catches showed a marked increase in mid June coinciding with the emergence of new adults from the damaged flower buds, even though these are in reproductive diapause. Male weevils predominated in catches beyond this date by a factor of approximately 2:1. Two field experiments investigated the interference between traps and the effects of the traps on populations and crop damage by this pest. There was no evidence for interference between traps at spacings as close as 1.7 m, and no effect on weevil damage or distribution was observed. Replicated trials were carried out of three approaches to exploit the pheromone for control of strawberry blossom weevil damage in 0.5 ha plots in commercial strawberry crops. These involved surrounding the crop with a perimeter of lures, the same method but with a boarder spray of the pyrethroid insecticide bifenthrin or by mass trapping with sticky stake pheromone traps at a density of 1000 per ha. None of these showed any reduction in crop damage compared to untreated plots. Possible reasons for this lack of success are discussed

This study was the first part of a project to assess the applicability of the male-produced, aggregation pheromone of the strawberry blossom weevil, Anthonomus rubi, for commercial monitoring and control. An optimised pheromone lure and trap were developed. Low-cost, robust and reliable polyethylene sachet dispensers containing 100 μl of a blend of grandlures I, II and (±)-lavandulol in the naturally occurring 1:4:1 ratio were shown to have constant release rates of 0.64 and 0.96 mg/day at 20 and 27 °C, respectively and a life of over 8 weeks in the field. Field experiments showed that increasing the release rate by approximately five times marginally increased attractiveness but a four times reduction in the release rate significantly decreased attractiveness. It was concluded that the standard release rate was satisfactory. Male A. rubi weevils were shown to produce the R enantiomer of lavandulol, but it was also demonstrated that the S enantiomer is not repellent and that low-cost racemic (±)-lavandulol is equally attractive. Although (−)-germacrene-D showed a weak synergistic effect when added to the pheromone components, inclusion in a commercial lure was uneconomic. Two further experiments examined the effect of reducing the amount of grandlure I, a costly chemical, in the blend. Although the results of one of these experiments was inconclusive, in the other it was found that reducing the amount of grandlure I by a factor of four did not decrease attractiveness significantly, though the ratio of males to females decreased significantly. A four times reduction in grandlure I content gives a 40% reduction in the cost of the chemicals in the lures. Experiments were carried out to develop an effective and practical trap design. Various modifications of the sticky board trap used in the original work were compared with boll weevil, funnel, delta and sticky stake designs. Most weevils were caught with the sticky stake design, made from a pointed wooden stake inserted vertically into the ground with a band of polybutene sticker around the circumference and a plastic board fixed horizontally on top of the stake to provide protection of the sticky surface from rain. A lure was hung from one corner of the board.

Previous work showed that females of the European tarnished plant bug, Lygus rugulipennis Poppius (Heteroptera: Miridae), produced three chemicals, hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal, and that these were suspected to be components of the female sex pheromone. In field experiments, traps baited with blends of these chemicals dispensed from polyethylene vials and sachets failed to catch significant numbers of males. Here, we report more recent field experiments in which the chemicals were released from glass microcapillary tubes. A blend of hexyl butyrate and (E)-4-oxo-2-hexenal was significantly attractive to male L. rugulipennis. In addition, whereas the mixture of all three components attracted fewer L. rugulipennis males, this tertiary blend captured significantly greater numbers of males of the congeneric species Lygus pratensis than the binary mixture. The possible reasons for the success of the microcapillaries compared with other dispensers are discussed.

The potential for common black ant Lasius niger workers to vector conidia of the entomopathogenic fungus Lecanicillium longisporum to colonies of the rosy apple aphid Dysaphis plantaginea was assessed in laboratory and field experiments. Scanning electron microscope studies showed that L. niger workers which were artificially contaminated with L. longisporum conidia, carried conidia primarily on their tarsae but also on antennae and mandibles. Neither L. niger workers nor larvae were susceptible to infection with L. longisporum. Workers which were artificially contaminated with conidia of L. longisporum initiated infection in colonies of healthy D. plantaginea on apple. Mortality due to L. longisporum was recorded as 68.3, 30.8 and 3.7% of aphids under laboratory, semi-field and field conditions, respectively.

Pollinators in agricultural landscapes can be exposed to mixtures of pesticides and environmental pollutants. Existing mixture toxicity modelling approaches, such as the models of concentration addition and independent action and the mechanistic DEBtox framework have been previously shown as valuable tools for understanding and ultimately predicting joint toxicity. Here we apply these mixture models to investigate the potential to interpret the effects of semi-chronic binary mixture exposure for three bee species: Apis mellifera, Bombus terrestris and Osmia bicornis within potentiation and mixture toxicity experiments. In the potentiation studies, the effect of the insecticide dimethoate with added propiconazole fungicide and neonicotinoid insecticide clothianidin with added tau-fluvalinate pyrethroid acaricide showed no difference in toxicity compared to the single chemical alone. Clothianidin toxicity showed a small scale, but temporally conserved increase in exposure conducted in the presence of propiconazole, particularly for B. terrestris and O. bicornis, the latter showing a near three-fold increase in clothianidin toxicity in the presence of propiconazole. In the mixture toxicity studies, the dominant response patterns were of additivity, however, binary mixtures of clothianidin and dimethoate in A. mellifera, B. terrestris and male O. bicornis there was evidence of a predominant antagonistic interaction. Given the ubiquitous nature of exposures to multiple chemicals, there is an urgent need to consider mixture effects in pollinator risk assessments. Our analyses suggest that current models, particularly those that utilise time-series data, such as DEBtox, can be used to identify additivity as the dominant response pattern and also those examples of interactions, even when small-scale, that may need to be taken into account during risk assessment.

This study investigated the sub-lethal effects of larval exposure to baculovirus on host life history and wing morphological traits using a model system, the speckled wood butterfly Pararge aegeria (L.) and the virus Autographa californica nucleopolyhedrovirus. Males and females showed similar responses to the viral infection. Infection significantly reduced larval growth rate, whilst an increase in development time allowed the critical mass for pupation to be attained. There was no direct effect of viral infection on the wing morphological traits examined. There was, however, an indirect effect of resisting infection; larvae that took longer to develop had reduced resource investment in adult flight muscle mass.

Background Maternal condition can generate resource-related maternal effects through differential egg provisioning, and can greatly affect offspring performance. In the present study, the speckled wood butterfly Pararge aegeria (L.) was used to investigate whether (after controlling for egg size) maternal age, and increased flight during the oviposition period, resulted in changes in egg provisioning and whether this contributed to variation in offspring performance, i) early in development (egg stage and early post-hatching development), and ii) later in larval development after being exposed to the model viral pathogen system; the baculovirus Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV). Results Age-related changes in maternal egg provisioning were observed to influence egg stage development only. Flight-induced changes in maternal egg provisioning had direct consequences for offspring growth and survival across each life stage from egg to adulthood; offspring from forced flight mothers had lower larval masses and longer development times. Offspring with lower larval masses also had reduced survival after exposure to the viral pathogen. Conclusion The present study demonstrates that a change in maternal provisioning as a result of increased flight during the oviposition period has the potential to exert non-genetic cross-generational fitness effects in P. aegeria. This could have important consequences for population dynamics, particularly in fragmented anthropogenic landscapes.

A technically standardised bioassay method was designed, evaluated and used to assess virulence and host range of hypocrealean fungi against aphids. A track mounted sprayer was used to apply conidia because hand held versions of the same sprayer can be used for field applications, thereby allowing the outcome from laboratory experiments to predict activity in the field accurately. Eighteen fungal isolates were assessed in single concentration bioassays against the black bean aphid Aphis fabae Scopoli. Isolates comprised commercially available mycoinsecticides (based on Beauveria bassiana and Lecanicillium longisporum) and isolates of B. bassiana, Lecanicillium spp., Paecilomyces fumosoroseus and Metarhizium anisopliae. Aphid mortality was in excess of 80% for 15 isolates, and HRI 1.72 (L. longipsorum), Z11 (P. fumosoroseus), Mycotech strain GHA (B. bassiana) and ARSEF 2879 (B. bassiana) were studied further. Multiple concentration bioassays identified HRI 1.72 as the most virulent isolate against A. fabae with significantly smaller LC50 and LT50 values compared to other isolates. A precise LC50 value (2.95 × 102 conidia ml−1) was calculated for HRI 1.72 using a second multiple concentration assay with smaller concentrations of conidia. The four isolates were applied at a single concentration (1 × 108 conidia ml−1) against Myzus persicae, A. fabae, Acyrthosiphon pisum, Metopolophium dirhodum, Sitobion avenae and Rhopalosiphum padi. A ranking of aphid susceptibility was obtained, such that S. avenae > M. persicae, A. pisum, A. fabae > R. padi. Results indicate the importance of standardising bioassay methods to reduce bioassay variability without compromising the ability to use the bioassay to investigate fungus–host interactions under varying abiotic and biotic conditions.