Thermal Biology of Insect Immunity and Host-Microbe Interactions

The influence of temperature on interactions with pathogenic or symbiotic microbes is a driving force behind the survival of insects under climate change. However, we know little of how insects physiologically respond to these pressures. In temperate climates, winter dominates the thermal landscape; thus, I am particularly interested in how cold interacts with insect responses to microbes. Here I explore the thermal biology of the insect immune system and the impacts of cold on host-microbe interactions. First, I demonstrate that acute exposure to cold activates selective components of immunity in Drosophila melanogaster, as a compensatory response to trade-offs or injury. Next, I show that cold acclimation decreases immune function at low temperatures in Gryllus veletis at the same time that cold tolerance increases. I conclude that this is a trade-off between immunity and the response to cold. Third, I demonstrate that immune activity varies seasonally in insects, but that each species responds differently. These shifts were likely driven by species-specific responses to multiple overwintering pressures. Fourth, I demonstrate that thermal plasticity in both Gryllus veletis and the fungal pathogen Metarhizium brunneum contribute to the outcome of infection. Further, fluctuating temperatures produce different outcomes of infection than constant temperatures, but we can predict these outcomes based on additive thermal performance under constant conditions. Lastly, I observe that the composition of the hindgut microbiome in Gryllus veletis, containing both beneficial and pathogenic microbes, shifts irreversibly across seasons. Further, microbial shifts coincide with changes in both cold tolerance and immune activity, which indicate that there is a functional relationship between the microbiome and host survival of low temperatures. Overall, changes in temperature are inextricably linked to changes in insect responses to both pathogenic and symbiotic microbes, which has likely selected for an adaptive physiological connection between insect immunity and the response to cold. I demonstrate that the connection between physiological responses to abiotic and biotic pressures modify our interpretation of phenotype. Therefore, we cannot rely on a univariate and species-isolated understanding of how insects respond to temperature if we are to predict the impact of climate change on their fitness

Insect Immunity Varies Idiosyncratically During Overwintering.

Overwintering insects face multiple stressors, including pathogen and parasite pressures that shift with seasons. However, we know little of how the insect immune system fluctuates with season, particularly in the overwintering period. To understand how immune activity changes across autumn, winter, and spring, we tracked immune activity of three temperate insects that overwinter as larvae: a weevil (Curculio sp., Coleoptera), gallfly (Eurosta solidaginis, Diptera), and larvae of the lepidopteran Pyrrharctia isabella. We measured baseline circulating hemocyte numbers, phenoloxidase activity, and humoral antimicrobial activity, as well as survival of fungal infection and melanization response at 12°C and 25°C to capture any potential plasticity in thermal performance. In Curculio sp. and E. solidaginis, hemocyte concentrations remained unchanged across seasons and antimicrobial activity against Gram-positive bacteria was lowest in autumn; however, Curculio sp. were less likely to survive fungal infection in autumn, whereas E. solidaginis were less likely to survive infection during the winter. Furthermore, hemocyte concentrations and antimicrobial activity decreased in P. isabella overwintering beneath snow cover. Overall, seasonal changes in activity were largely species dependent, thus it may be difficult to create generalizable predictions about the effects of a changing climate on seasonal immune activity in insects. However, we suggest that the relationship between the response to multiple stressors (e.g., cold and pathogens) drives changes in immune activity, and that understanding the physiology underlying these relationships will inform our predictions of the effects of environmental change on insect overwintering success

Thermal variability and plasticity drive the outcome of a host-pathogen interaction

Variable, changing, climates may affect each participant in a biotic interaction differently. We explored the effects of temperature and plasticity on the outcome of a host-pathogen interaction to try to predict the outcomes of infection under fluctuating temperatures. We infected Gryllus veletis crickets with the entomopathogenic fungus Metarhizium brunneum under constant (6 °C, 12 °C, 18 °C or 25 °C) or fluctuating temperatures (6 °C to 18 °C or 6 °C to 25 °C). We also acclimated crickets and fungi to constant or fluctuating conditions. Crickets acclimated to fluctuating conditions survived best under constant conditions if paired with warm-acclimated fungus. Overall, matches and mismatches in thermal performance, driven by acclimation, determined host survival. Mismatched performance also determined differences in survival under different fluctuating thermal regimes: crickets survived best when fluctuating temperatures favoured their performance (6 °C to 25 °C), compared to fluctuations that favoured fungus performance (6 °C to 18 °C). Thus, we could predict the outcome of infection under fluctuating temperatures by averaging relative host-pathogen performance under constant temperatures, suggesting that it may be possible to predict responses to fluctuating temperatures for at least some biotic interactions

Can we predict the effects of multiple stressors on insects in a changing climate?

The responses of insects to climate change will depend on their responses to abiotic and biotic stressors in combination. We surveyed the literature, and although synergistic stressor interactions appear common among insects, the thin taxonomic spread of existing data means that more multi-stressor studies and new approaches are needed. We need to move beyond descriptions of the effects of multiple stressors to a mechanistic, predictive understanding. Further, we must identify which stressor interactions, and species\u27 responses to them, are sufficiently generalizable (i.e. most or all species respond similarly to the same stressor combination), and thus predictable (for new combinations of stressors, or stressors acting via known mechanisms). We discuss experimental approaches that could facilitate this shift toward predictive understanding

Comparing apples and oranges (and blueberries and grapes): fruit type affects development and cold-susceptibility of immature Drosophila suzukii

Drosophila suzukii is a cosmopolitan polyphagous pest on unripe soft-skinned fruits. We sought to determine 1) temperature treatments that could be used to kill immature D. suzukii in fruit or packaging, and 2) whether development on different fruits led to differences in cold tolerance of immature D. suzukii. We reared animals from egg on a banana-based laboratory diet and diets made of apple, blueberry, cherry, grape, orange, raspberry, or strawberry homogenate in agar, and measured development time, adult body size, and cold tolerance. Diet type had complex effects on development time; in particular, flies reared on apple- or blueberry-based diets developed more slowly to a smaller adult body size than those on other diets. Cold exposure killed eggs and both first- and second-instar larvae. Survival of 24h at +4°C by feeding third-instar larvae was lowest in blueberry and cherry. Five days at +0.6°C killed all feeding third-instar larvae; this treatment is likely sufficient for targeting D. suzukii in fruit. Two hours at -5 or -6°C killed all wandering third-instar larvae and pupae; this exposure could be sufficient for sanitation of packaging

Paradoxical acclimation responses in the thermal performance of insect immunity.

Winter is accompanied by multiple stressors, and the interactions between cold and pathogen stress potentially determine the overwintering success of insects. Thus, it is necessary to explore the thermal performance of the insect immune system. We cold-acclimated spring field crickets, Gryllus veletis, to 6 °C for 7 days and measured the thermal performance of potential (lysozyme and phenoloxidase activity) and realised (bacterial clearance and melanisation) immune responses. Cold acclimation decreased the critical thermal minimum from -0.5 ± 0.25 to -2.1 ± 0.18 °C, and chill coma recovery time after 72 h at -2 °C from 16.8 ± 4.9 to 5.2 ± 2.0 min. Measures of both potential and realised immunity followed a typical thermal performance curve, decreasing with decreasing temperature. However, cold acclimation further decreased realised immunity at low, but not high, temperatures; effectively, immune activity became paradoxically specialised to higher temperatures. Thus, cold acclimation induced mismatched thermal responses between locomotor and immune systems, as well as within the immune system itself. We conclude that cold acclimation in insects appears to preferentially improve cold tolerance over whole-animal immune performance at low temperatures, and that the differential thermal performance of physiological responses to multiple pressures must be considered when predicting ectotherms\u27 response to climate change

Does cold activate the Drosophila melanogaster immune system?

Cold exposure appears to activate aspects of the insect immune system; however, the functional significance of the relationship between cold and immunity is unclear. Insect success at low temperatures is shaped in part by interactions with biotic stressors, such as pathogens, thus it is important to understand how and why immunity might be activated by cold. Here we explore which components of the immune system are activated, and whether those components differ among different kinds of cold exposure. We exposed Drosophila melanogaster to both acute (2h, -2°C) and sustained (10h, -0.5°C) cold, and measured potential (antimicrobial peptide expression, phenoloxidase activity, haemocyte counts) and realised (survival of fungal infection, wound-induced melanisation, bacterial clearance) immunity following recovery. Acute cold increased circulating haemocyte concentration and the expression of Turandot-A and diptericin, but elicited a short-term decrease in the clearance of gram-positive bacteria. Sustained cold increased the expression of Turandot-A, with no effect on other measures of potential or realised immunity. We show that measures of potential immunity were up-regulated by cold, whereas realised immunity was either unaffected or down-regulated. Thus, we hypothesize that cold-activation of potential immunity in Drosophila may be a compensatory mechanism to maintain stable immune function during or after low temperature exposure

An invitation to measure insect cold tolerance: Methods, approaches, and workflow.

Insect performance is limited by the temperature of the environment, and in temperate, polar, and alpine regions, the majority of insects must face the challenge of exposure to low temperatures. The physiological response to cold exposure shapes the ability of insects to survive and thrive in these environments, and can be measured, without great technical difficulty, for both basic and applied research. For example, understanding insect cold tolerance allows us to predict the establishment and spread of insect pests and biological control agents. Additionally, the discipline provides the tools for drawing physiological comparisons among groups in wider studies that may not be focused primarily on the ability of insects to survive the cold. Thus, the study of insect cold tolerance is of a broad interest, and several reviews have addressed the theories and advances in the field. Here, however, we aim to clarify and provide rationale for common practices used to study cold tolerance, as a guide for newcomers to the field, students, and those wishing to incorporate cold tolerance into a broader study. We cover the \u27tried and true\u27 measures of insect cold tolerance, the equipment necessary for these measurement, and summarize the ecological and biological significance of each. Finally, we suggest a framework and workflow for measuring cold tolerance and low temperature performance in insects

Cross-tolerance and cross-talk in the cold: relating low temperatures to desiccation and immune stress in insects.

Multiple stressors, both abiotic and biotic, often are experienced simultaneously by organisms in nature. Responses to these stressors may share signaling pathways ( cross-talk ) or protective mechanisms ( cross-tolerance ). Temperate and polar insects that must survive the winter experience low temperatures accompanied by additional abiotic stressors, such as low availability of water. Cold and desiccation have many similar effects at a cellular level, and we present evidence that the cellular mechanisms that protect against cold stress also protect against desiccation, and that the responses to cold and dehydration likely evolved as cross-tolerance. By contrast, there are several lines of evidence suggesting that low temperature stress elicits an upregulation of immune responses in insects (and vice versa). Because there is little mechanistic overlap between cold stress and immune stress at the cellular level, we suggest that this is cross-talk. Both cross-talk and cross-tolerance may be adaptive and likely evolved in response to synchronous stressors; however, we suggest that cross-talk and cross-tolerance may lead to different responses to changes in the timing and severity of multiple stress interactions in a changing world. We present a framework describing the potentially different responses of cross-tolerance and cross-talk to a changing environment and describe the nature of these impacts using interaction of cold-desiccation and cold-immunity in overwintering insects as an example

Reproductive arrest and stress resistance in winter-acclimated Drosophila suzukii.

Overwintering insects must survive the multiple-stress environment of winter, which includes low temperatures, reduced food and water availability, and cold-active pathogens. Many insects overwinter in diapause, a developmental arrest associated with high stress tolerance. Drosophila suzukii (Diptera: Drosophilidae), spotted wing drosophila, is an invasive agricultural pest worldwide. Its ability to overwinter and therefore establish in temperate regions could have severe implications for fruit crop industries. We demonstrate here that laboratory populations of Canadian D. suzukii larvae reared under short-day, low temperature, conditions develop into dark \u27winter morph\u27 adults similar to those reported globally from field captures, and observed by us in southern Ontario, Canada. These winter-acclimated adults have delayed reproductive maturity, enhanced cold tolerance, and can remain active at low temperatures, although they do not have the increased desiccation tolerance or survival of fungal pathogen challenges that might be expected from a more heavily melanised cuticle. Winter-acclimated female D. suzukii have underdeveloped ovaries and altered transcript levels of several genes associated with reproduction and stress. While superficially indicative of reproductive diapause, the delayed reproductive maturity of winter-acclimated D. suzukii appears to be temperature-dependent, not regulated by photoperiod, and is thus unlikely to be \u27true\u27 diapause. The traits of this \u27winter morph\u27, however, likely facilitate overwintering in southern Canada, and have probably contributed to the global success of this fly as an invasive species