Warming, but not infection with Borrelia burgdorferi, increases off-host winter activity in the ectoparasite, Ixodes scapularis.

Warming winters will change patterns of behaviour in temperate and polar arthropods, but we know little about the drivers of winter activity in animals such as ticks. Any changes in behaviour are likely to arise from a combination of both abiotic (e.g. temperature) and biotic (e.g. infection) drivers, and will have important consequences for survival and species interactions. Blacklegged ticks, Ixodes scapularis, have invaded Atlantic Canada and high proportions (30-50%) are infected with the bacteria causing Lyme disease, Borrelia burgdorferi. Infection is correlated with increased overwintering survival of adult females, and ticks are increasingly active in the winter, but it is unclear if infection is associated with activity. Further, we know little about how temperature drives the frequency of winter activity. Here, we exposed wild-caught, adult, female Ixodes scapularis ticks to three different winter temperature regimes (constant low temperatures, increased warming, and increased warming + variability) to determine the thermal and infection conditions that promote or suppress activity. We used automated behaviour monitors to track daily activity in individual ticks and repeated the study with fresh ticks over three years. Following exposure to winter conditions we determined whether ticks were infected with the bacteria B. burgdorferi and if infection was responsible for any patterns in winter activity. Warming conditions promoted increased activity throughout the overwintering period but infection with B. burgdorferi had no impact on the frequency or overall number of ticks active throughout the winter. Individual ticks varied in their levels of activity throughout the winter, such that some were largely dormant for several weeks, while others were active almost daily; however, we do not yet know the drivers behind this individual variation in behaviour. Overall, warming winters will heighten the risk of tick-host encounters.

Cold-induced immune activation in chill-susceptible insects.

Chilling injuries in chill-susceptible insects, such as the model dipteran Drosophila melanogaster, have been well-documented as a consequence of stressful low-temperature exposures. Cold stress also causes upregulation of genes in the insect immune pathways, some of which are also upregulated following other forms of sterile stress. The adaptive significance and underlying mechanisms surrounding cold-induced immune activation, however, are still unclear. Here, we review recent work on the roles of reactive oxygen species, damage-associated molecular patterns, and antimicrobial peptides in insect immune signaling or function. Using this emerging knowledge, we propose a conceptual model linking biochemical and molecular causes of immune activation to its consequences during and following cold stress.

Female ticks (Ixodes scapularis) infected with Borrelia burgdorferi have increased overwintering survival, with implications for tick population growth.

The tick, Ixodes scapularis, vectors pathogens such as Borrelia burgdorferi, the bacterium that causes Lyme disease. Over the last few decades I. scapularis has expanded its range, introducing a novel health threat into these areas. Warming temperatures appear to be one cause of its range expansion to the north. However, other factors are also involved. We show that unfed adult female ticks infected with B. burgdorferi have greater overwintering survival than uninfected female ticks. Locally collected adult female ticks were placed in individual microcosms and allowed to overwinter in both forest and dune grass environments. In the spring we collected the ticks and tested both dead and living ticks for B. burgdorferi DNA. Infected ticks had greater overwintering survival compared with uninfected ticks every winter for three consecutive winters in both forest and dune grass environments. We discuss the most plausible explanations for this result. The increased winter survival of adult female ticks could enhance tick population growth. Our results suggest that, in addition to climate change, B. burgdorferi infection itself may be promoting the northern range expansion of I. scapularis. Our study highlights how pathogens could work synergistically with climate change to promote host range expansion.

From perplexing to predictive: are we ready to forecast insect disease susceptibility in a warming world?

Insects are critical to our ecosystems, but we do not fully understand their future in our warming world. Rising temperatures are affecting insect physiology in myriad ways, including changes to their immune systems and the ability to fight infection. Whether predicted changes in temperature will contribute to insect mortality or success, and the role of disease in their future survival, remains unclear. Although heat can enhance immunity by activating the integrated defense system (e.g. via the production of protective molecules such as heat-shock proteins) and accelerating enzyme activity, heat can also compromise the immune system through energetic-resource trade-offs and damage. The responses to heat are highly variable among species. The reasons for this variability are poorly known, and we are lagging in our understanding of how and why the immune system responds to changes in temperature. In this Commentary, we highlight the variation in insect immune responses to heat and the likely underlying mechanisms. We suggest that we are currently limited in our ability to predict the effects of rising temperatures on insect immunity and disease susceptibility, largely owing to incomplete information, coupled with a lack of tools for data integration. Moreover, existing data are concentrated on a relatively small number of insect Orders. We provide suggestions for a path towards making more accurate predictions, which will require studies with realistic temperature exposures and housing design, and a greater understanding of both the thermal biology of the immune system and connections between immunity and the physiological responses to heat.

Balsam fir (Abies balsamea) needles and their essential oil kill overwintering ticks (Ixodes scapularis) at cold temperatures.

The blacklegged tick, Ixodes scapularis, vectors Borrelia burgdorferi, a bacterium that causes Lyme Disease. Although synthetic pesticides can reduce tick numbers, there are concerns about their potential effects on beneficial insects, such as pollinators. Plant-based pest control agents such as essential oils could provide an alternative because they have low environmental persistency; however, these products struggle to provide effective control. We found a new natural acaricide, balsam fir (Abies balsamea) needles, that kill overwintering I. scapularis ticks. We extracted the essential oil from the needles, analyzed its chemical composition, and tested it for acaricidal activity. We placed ticks in tubes with substrate and positioned the tubes either in the field or in incubators simulating winter temperatures. We added balsam fir essential oil, or one of the main components of balsam fir essential oil (i.e., ß-pinene), to each tube. We found that both the oil and ß-pinene kill overwintering ticks. Whole balsam fir needles require several weeks to kill overwintering ticks, while the essential oil is lethal within days at low temperatures (≤ 4 °C). Further, low temperatures increased the efficacy of this volatile essential oil. Higher temperatures (i.e., 20 °C) reduce the acaricidal effectiveness of the essential oil by 50% at 0.1% v/v. Low temperatures may promote the effectiveness of other natural control products. Winter is an overlooked season for tick control and should be explored as a possible time for the application of low toxicity products for successful tick management.

Applied ecoimmunology: using immunological tools to improve conservation efforts in a changing world.

Ecoimmunology is a rapidly developing field that explores how the environment shapes immune function, which in turn influences host-parasite relationships and disease outcomes. Host immune defence is a key fitness determinant because it underlies the capacity of animals to resist or tolerate potential infections. Importantly, immune function can be suppressed, depressed, reconfigured or stimulated by exposure to rapidly changing environmental drivers like temperature, pollutants and food availability. Thus, hosts may experience trade-offs resulting from altered investment in immune function under environmental stressors. As such, approaches in ecoimmunology can provide powerful tools to assist in the conservation of wildlife. Here, we provide case studies that explore the diverse ways that ecoimmunology can inform and advance conservation efforts, from understanding how Galapagos finches will fare with introduced parasites, to using methods from human oncology to design vaccines against a transmissible cancer in Tasmanian devils. In addition, we discuss the future of ecoimmunology and present 10 questions that can help guide this emerging field to better inform conservation decisions and biodiversity protection. From better linking changes in immune function to disease outcomes under different environmental conditions, to understanding how individual variation contributes to disease dynamics in wild populations, there is immense potential for ecoimmunology to inform the conservation of imperilled hosts in the face of new and re-emerging pathogens, in addition to improving the detection and management of emerging potential zoonoses.

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°, 12°, 18°, or 25°C) or fluctuating (from 6° to 18°C or from 6° to 25°C) temperatures. 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 favored their performance (from 6° to 25°C), compared with fluctuations that favored fungus performance (from 6° 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.

Eco-immunology in the cold: the role of immunity in shaping the overwintering survival of ectotherms.

The effect of temperature on physiology mediates many of the challenges that ectotherms face under climate change. Ectotherm immunity is thermally sensitive and, as such, environmental change is likely to have complex effects on survival, disease resistance and transmission. The effects of temperature on immunity will be particularly profound in winter because cold and overwintering are important triggers and regulators of ectotherm immune activity. Low temperatures can both suppress and activate immune responses independent of parasites, which suggests that temperature not only affects the rate of immune responses but also provides information that allows overwintering ectotherms to balance investment in immunity and other physiological processes that underlie winter survival. Changing winter temperatures are now shifting ectotherm immunity, as well as the demand for energy conservation and protection against parasites. Whether an ectotherm can survive the winter will thus depend on whether new immune phenotypes will shift to match the conditions of the new environment, or leave ectotherms vulnerable to infection or energy depletion. Here, we synthesise patterns of overwintering immunity in ectotherms and examine how new winter conditions might affect ectotherm immunity. We then explore whether it is possible to predict the effects of changing winter conditions on ectotherm vulnerability to the direct and indirect effects of parasites.

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.

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.

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' 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.

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 'winter morph' 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 'true' diapause. The traits of this 'winter morph', however, likely facilitate overwintering in southern Canada, and have probably contributed to the global success of this fly as an invasive species.

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' response to climate change.

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 'tried and true' 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.

Parallel ionoregulatory adjustments underlie phenotypic plasticity and evolution of Drosophila cold tolerance.

Low temperature tolerance is the main predictor of variation in the global distribution and performance of insects, yet the molecular mechanisms underlying cold tolerance variation are poorly known, and it is unclear whether the mechanisms that improve cold tolerance within the lifetime of an individual insect are similar to those that underlie evolved differences among species. The accumulation of cold-induced injuries by hemimetabolous insects is associated with loss of Na(+) and K(+) homeostasis. Here we show that this model holds true for Drosophila; cold exposure increases haemolymph [K(+)] in D. melanogaster, and cold-acclimated flies maintain low haemolymph [Na(+)] and [K(+)], both at rest and during a cold exposure. This pattern holds across 24 species of the Drosophila phylogeny, where improvements in cold tolerance have been consistently paired with reductions in haemolymph [Na(+)] and [K(+)]. Cold-acclimated D. melanogaster have low activity of Na(+)/K(+)-ATPase, which may contribute to the maintenance of low haemolymph [Na(+)] and underlie improvements in cold tolerance. Modifications to ion balance are associated with both phenotypic plasticity within D. melanogaster and evolutionary differences in cold tolerance across the Drosophila phylogeny, which suggests that adaptation and acclimation of cold tolerance in insects may occur through similar mechanisms. Cold-tolerant flies maintain haemolymph osmolality despite low haemolymph [Na(+)] and [K(+)], possibly through modest accumulations of organic osmolytes. We propose that this could have served as an evolutionary route by which chill-susceptible insects developed more extreme cold tolerance strategies.

Cold hardiness and deacclimation of overwintering Papilio zelicaon pupae.

Seasonally-acquired cold tolerance can be reversed at warm temperatures, leaving temperate ectotherms vulnerable to cold snaps. However, deacclimation, and its underlying mechanisms, has not been well-explored in insects. Swallowtail butterflies are widely distributed but in some cases their range is limited by low temperature and their cold tolerance is seasonally acquired, implying that they experience mortality resulting from deacclimation. We investigated cold tolerance and hemolymph composition of Anise swallowtail (Papilio zelicaon) pupae during overwintering in the laboratory, and after four days exposure to warm temperatures in spring. Overwintering pupae had supercooling points around -20.5°C and survived brief exposures to -30°C, suggesting partial freeze tolerance. Overwintering pupae had hemolymph osmolality of approximately 920 mOsm, imparted by high concentrations of glycerol, K⁺ and Na⁺. After exposure to spring warming, supercooling points increased to approximately -17°C, and survival of a 1h exposure to -20°C decreased from 100% to 0%. This deacclimation was associated with decreased hemolymph osmolality and reduced glycerol, trehalose, Na⁺ and Ca²âº concentrations. We compared cold tolerance of pupae to weather conditions at and beyond the species' northern range boundary. Minimum temperatures at the range boundary approached the lower lethal temperature of pupae, and were colder north of the range, suggesting that cold hardiness may set northern range limits. Minimum temperatures following warm snaps were likely to cause mortality in at least one of the past three years. Cold snaps in the spring are increasing in frequency as a result of global climate change, so are likely to be a significant source of mortality for this species, and other temperate ectotherms.

Fecundity reduction in the second gonotrophic cycle of Culex pipiens infected with the apicomplexan blood parasite, Hepatozoon sipedon.

Fecundity reduction is a well-recognized phenomenon of parasite infection in insects. Reduced production of eggs might increase longevity of a host and release nutrients to both host and parasite that would otherwise be used for oogenesis. The objective of this study was to assess effects on fecundity caused by Hepatozoon sipedon, an apicomplexan blood parasite of snakes, in its invertebrate host, the mosquito Culex pipiens. In the first gonotrophic cycle, the mean number of eggs laid by mosquitoes infected with H. sipedon did not differ significantly from those laid by uninfected mosquitoes. However, in the second gonotrophic cycle infected mosquitoes laid significantly fewer eggs than did uninfected mosquitoes, and fecundity was reduced by 100% in mosquitoes with parasite burdens of more than 60 oocysts. There was a significant negative correlation between parasite burden, or the number of oocysts, and the number of eggs produced in the second gonotrophic cycle. Significantly fewer viable larvae hatched from eggs laid by infected compared to uninfected mosquitoes in the second gonotrophic cycle. These data indicate that fecundity reduction occurs in this system, although the physiological mechanisms driving this phenotype are not yet known.

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.

Influence of Hepatozoon parasites on host-seeking and host-choice behaviour of the mosquitoes Culex territans and Culex pipiens.

Hepatozoon species are heteroxenous parasites that commonly infect the blood of vertebrates and various organs of arthropods. Despite their ubiquity, little is known about how these parasites affect host phenotype, including whether or not these parasites induce changes in hosts to increase transmission success. The objectives of this research were to investigate influences of the frog blood parasite Hepatozoon clamatae and the snake blood parasite Hepatozoon sipedon on host-seeking and host-choice behaviour of the mosquitoes Culex territans and Culex pipiens, respectively. During development of H. sipedon in C. pipiens, significantly fewer infected mosquitoes fed on uninfected snakes compared to uninfected mosquitoes. When H. sipedon was mature in C. pipiens, the number of infected and uninfected C. pipiens that fed on snakes was not significantly different. Higher numbers of mosquitoes fed on naturally infected snakes and frogs compared to laboratory-reared, uninfected control animals. However, experiments using only laboratory-raised frogs revealed that infection did not significantly affect host choice by C. territans. Behaviour of C. pipiens in the presence of H. sipedon may increase transmission success of the parasite and provide the first evidence of phenotypic changes in the invertebrate host of Hepatozoon parasites.

Reciprocal Trophic Interactions and Transmission of Blood Parasites between Mosquitoes and Frogs.

The relationship between mosquitoes and their amphibian hosts is a unique, reciprocal trophic interaction. Instead of a one-way, predator-prey relationship, there is a cyclical dance of avoidance and attraction. This has prompted spatial and temporal synchrony between organisms, reflected in emergence time of mosquitoes in the spring and choice of habitat for oviposition. Frog-feeding mosquitoes also possess different sensory apparatuses than do their mammal-feeding counterparts. The reciprocal nature of this relationship is exploited by various blood parasites that use mechanical, salivary or trophic transmission to pass from mosquitoes to frogs. It is important to investigate the involvement of mosquitoes, frogs and parasites in this interaction in order to understand the consequences of anthropogenic actions, such as implementing biocontrol efforts against mosquitoes, and to determine potential causes of the global decline of amphibian species.