Effect of CO2 Concentrations on Entomopathogen Fitness and Insect-Pathogen Interactions.

Numerous insect species and their associated microbial pathogens are exposed to elevated CO2 concentrations in both artificial and natural environments. However, the impacts of elevated CO2 on the fitness of these pathogens and the susceptibility of insects to pathogen infections are not well understood. The yellow mealworm, Tenebrio molitor, is commonly produced for food and feed purposes in mass-rearing systems, which increases risk of pathogen infections. Additionally, entomopathogens are used to control T. molitor, which is also a pest of stored grains. It is therefore important to understand how elevated CO2 may affect both the pathogen directly and impact on host-pathogen interactions. We demonstrate that elevated CO2 concentrations reduced the viability and persistence of the spores of the bacterial pathogen Bacillus thuringiensis. In contrast, conidia of the fungal pathogen Metarhizium brunneum germinated faster under elevated CO2. Pre-exposure of the two pathogens to elevated CO2 prior to host infection did not affect the survival probability of T. molitor larvae. However, larvae reared at elevated CO2 concentrations were less susceptible to both pathogens compared to larvae reared at ambient CO2 concentrations. Our findings indicate that whilst elevated CO2 concentrations may be beneficial in reducing host susceptibility in mass-rearing systems, they may potentially reduce the efficacy of the tested entomopathogens when used as biological control agents of T. molitor larvae. We conclude that CO2 concentrations should be carefully selected and monitored as an additional environmental factor in laboratory experiments investigating insect-pathogen interactions.

Thermal ecology shapes disease outcomes of entomopathogenic fungi infecting warm-adapted insects.

The thermal environment is a critical determinant of outcomes in host-pathogen interactions, yet the complexities of this relationship remain underexplored in many ecological systems. We examined the Thermal Mismatch Hypothesis (TMH) by measuring phenotypic variation in individual thermal performance profiles using a model system of two species of entomopathogenic fungi (EPF) that differ in their ecological niche, Metarhizium brunneum and M. flavoviride, and a warm-adapted model host, the mealworm Tenebrio molitor. We conducted experiments across ecologically relevant temperatures to determine the thermal performance curves for growth and virulence, measured as % survival, identify critical thresholds for these measures, and elucidate interactive host-pathogen effects. Both EPF species and the host exhibited a shared growth optima at 28 °C, while the host's growth response was moderated in sublethal pathogen infections that depended on fungus identity and temperature. However, variances in virulence patterns were different between pathogens. The fungus M. brunneum exhibited a broader optimal temperature range (23-28 °C) for virulence than M. flavoviride, which displayed a multiphasic virulence-temperature relationship with distinct peaks at 18 and 28 °C. Contrary to predictions of the TMH, both EPF displayed peak virulence at the host's optimal temperature (28 °C). The thermal profile for M. brunneum aligned more closely with that of T. molitor than that for M. flavoviride. Moreover, the individual thermal profile of M. flavoviride closely paralleled its virulence thermal profile, whereas the virulence thermal profile of M. brunneum did not track with its individual thermal performance. This suggests an indirect, midrange (23 °C) effect, where M. brunneum virulence exceeded growth. These findings suggest that the evolutionary histories and ecological adaptations of these EPF species have produced distinct thermal niches during the host interaction. This study contributes to our understanding of thermal ecology in host-pathogen interactions, underpinning the ecological and evolutionary factors that shape infection outcomes in entomopathogenic fungi. The study has ecological implications for insect population dynamics in the face of a changing climate, as well as practically for the use of these organisms in biological control.

A Novel Lepidoptera bioassay analysed using a reduced GUTS model.

Lepidopteran species can be both pests and also beneficial pollinators for agricultural crops. However, despite these important roles, the effects of pesticides on this diverse taxa are relatively understudied. To facilitate the assessment of pesticides and other chemical hazards on this taxa, we present a novel bioassay capable of testing chemical sensitivity to lepidopteran larvae through dietary exposure. We used Mamestra brassicae caterpillars as a model lepidopteran and tested their sensitivity for the organophosphate insecticide chlorpyrifos. We exposed larvae to an artificial diet spiked with chlorpyrifos and monitored survival over time, as well as weight change over a 96-hour exposure period. To test the repeatability and reliability of the developed bioassay, the experiment was repeated three times. The survival in time data collected enabled analysis with the General Unified Threshold of Survival (GUTS) model, recently recognized by EFSA as a ready-to-use tool for regulatory purposes. The GUTS modelling was used to derive a set of relevant toxicokinetic and toxicodynamic parameters relating to the larval response to exposure over time. We found that across the three repeats studies there was no more than a threefold difference in LC50 values (13.1, 18.7 and 8.1 mg/Kg) at 48 h and fourfold difference at 96 h, highlighting the repeatability of the bioassay. We also highlighted the potential of the method to observe sub-lethal effects such as changes in weight. Finally, we discuss the applications of this new bioassay method to chemical risk assessments and its potential for use in other scenarios, such as mixture or pulsed exposure testing.

Bacillus thuringiensis impacts on primary and secondary baculovirus transmission dynamics in Lepidoptera.

Synergistic interactions between entomopathogenic micro-organisms can potentially be exploited to improve biological control of invertebrate pests but empirical data at the population level describing multiple-pathogen transmission dynamics is lacking. We examined how co-inoculation of Bacillus thuringiensis subsp. kurstaki (Btk) and the baculovirus Panolis flammea nucleopolyhedrovirus (PaflNPV) in an experimental field population of Lepidopteran Mamestra brassicae larvae impacted on viral transmission dynamics. We determined how the presence of Btk influenced primary and secondary PaflNPV transmission. When Btk was co-inoculated with PaflNPV, there was increased proportional viral mortality in primary transmission studies compared to plots with virus alone. A delay of up to 4days between applications of Btk and PaflNPV did not impact on primary viral mortality, indicating that a lag between inoculations was unlikely to affect the biocontrol potential of the two pathogens. Viral yields from cadavers in plots with Btk present were significantly lower than those from plots with virus only, and secondary cycling to introduced secondary transmission larvae was significantly reduced. Baculovirus transmission (in terms of the proportion of uninfected larvae in different treatments) was described by a 'refuge' model that allowed for heterogeneity in susceptibility and pathogen exposure. We discuss how transmission may be potentially affected by factors such as host feeding rate, spatial distribution of virus and interactions between pathogens within the insect host. This study improves understanding of the impact of pathogens within host populations and how mixtures of pathogens may be exploited for biocontrol of insect pests.

Improved control of Trialeurodes vaporariorum using mixture combinations of entomopathogenic fungi and the chemical insecticide spiromesifen.

Greenhouse whitefly (Trialeurodes vaporariorum) is a major global pest, causing direct damage to plants and transmitting viral plant diseases. Management of T. vaporariorum is problematic because of widespread pesticide resistance, and many greenhouse growers rely on biological control agents to regulate T. vaporariorum populations. However, these are often slow and vary in efficacy, leading to subsequent application of chemical insecticides when pest populations exceed threshold levels. Combining chemical and biological pesticides has great potential but can result in different outcomes, from positive to negative interactions. In this study, we evaluated co-applications of the entomopathogenic fungi (EPF) Beauveria bassiana and Cordyceps farinosa and the chemical insecticide spiromesifen in laboratory bioassays. Complex interactions between the EPFs and insecticide were described using an ecotoxicological mixtures model, the MixTox analysis. Depending on the EPF and chemical concentrations applied, mixtures resulted in additivity, synergism, or antagonism in terms of total whitefly mortality. Combinations of B. bassiana and spiromesifen, compared to single treatments, increased the rate of kill by 5 days. Results indicate the potential for combined applications of EPF and spiromesifen as an effective integrated pest management strategy and demonstrate the applicability of the MixTox model to describe complex mixture interactions.

Environment-host-parasite interactions in mass-reared insects.

The mass production of insects is rapidly expanding globally, supporting multiple industrial needs. However, parasite infections in insect mass-production systems can lower productivity and can lead to devastating losses. High rearing densities and artificial environmental conditions in mass-rearing facilities affect the insect hosts as well as their parasites. Environmental conditions such as temperature, gases, light, vibration, and ionizing radiation can affect productivity in insect mass-production facilities by altering insect development and susceptibility to parasites. This review explores the recent literature on environment-host-parasite interactions with a specific focus on mass-reared insect species. Understanding these complex interactions offers opportunities to optimise environmental conditions for the prevention of infectious diseases in mass-reared insects.

A Rapid Method for Measuring In Vitro Growth in Entomopathogenic Fungi.

Quantifying the growth of entomopathogenic fungi is crucial for understanding their virulence and pathogenic potential. Traditional methods for determining growth, such as biomass determination or colony growth area, are time-consuming and quantitatively and spatially limited in scope. In this study, we introduce a high-throughput method for rapidly measuring fungal growth using spectrophotometry in small-volume, liquid media cultures in 96-well microplates. Optical density (OD) changes were directly correlated with dry weight of samples for six isolates from three species of the genus Metarhizium to validate spectrophotometric growth measurements, and investigate species- and isolate-specific effects. We quantified fungal biomass from the microcultures by extracting, drying, and weighing mycelial mats. From the relationship established between OD and biomass, we generated standard curves for predicting biomass based on the OD values. The OD measurements clearly distinguished growth patterns among six isolates from three Metarhizium species. The logistic growth phase, as captured by the OD measurements, could be accurately assessed within a span of 80 h. Using isolates of M. acridum, M. brunneum, and M. guizhouense, this technique was demonstrated to be an effective, reproducible, and simple method for rapidly measuring filamentous fungal growth with high precision. This technique offers a valuable tool for studying the growth dynamics of entomopathogenic fungi and investigating the factors that influence their growth.

Exploring sub-lethal effects of exposure to a nucleopolyhedrovirus in the speckled wood (Pararge aegeria) butterfly.

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.

Maternal effects, flight versus fecundity trade-offs, and offspring immune defence in the speckled wood butterfly, Pararge aegeria.

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 standardised bioassay method using a bench-top spray tower to evaluate entomopathogenic fungi for control of the greenhouse whitefly, Trialeurodes vaporariorum.

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.

Viral exposure effects on life-history, flight-related traits, and wing melanisation in the Glanville fritillary butterfly.

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.

Comparing bee species responses to chemical mixtures: Common response patterns?

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.

Comparative toxicity of pesticides and environmental contaminants in bees: Are honey bees a useful proxy for wild bee species?

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 240h 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.