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.

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.

Genetic adaptation, occurring over a long evolutionary time, enables host-specialized herbivores to develop novel resistance traits and to efficiently counteract the defenses of a narrow range of host plants. In contrast, physiological acclimation, leading to the suppression and/or detoxification of host defenses, is hypothesized to enable broad-generalists to shift between plant hosts. However, the host adaptation mechanisms used by generalists composed of host-adapted populations are not known. Two-spotted spider mite (Tetranychus urticae) is an extreme generalist herbivore whose individual populations perform well only on a subset of potential hosts. We combined experimental evolution, Arabidopsis thaliana genetics, mite reverse genetics, and pharmacological approaches to examine mite host adaptation upon the shift of a bean (Phaseolus vulgaris)-adapted population to Arabidopsis. We showed that cytochrome P450 monooxygenases are required for mite adaptation to Arabidopsis. We identified activities of two tiers of P450s: general xenobiotic-responsive P450s that have a limited contribution to mite adaptation to Arabidopsis and adaptation-associated P450s that efficiently counteract Arabidopsis defenses. In ∼25 generations of mite selection on Arabidopsis plants, mites evolved highly efficient detoxification-based adaptation, characteristic of specialist herbivores. This demonstrates that specialization to plant resistance traits can occur within the ecological timescale, enabling the two-spotted spider mite to shift to novel plant hosts.

Classification: Biological Sciences, Evolutionary Biology 1 2The generalist herbivore Tetranychus urticae (Koch) adapts to novel plant hosts through rapid 3 evolution of metabolic resistance 4 5Abstract 20Genetic adaptation, occurring over long evolutionary time, enables host-specialized herbivores 21to develop novel resistance traits and to counteract the defenses of a narrow range of host 22plants. In contrast, physiological acclimation, leading to the suppression and/or detoxification of 23 host defenses is hypothesized to enable generalists to shift between plant hosts. Here, we 24 examined the long-term response of an extreme generalist, the two-spotted spider mite, 25Tetranychus urticae Koch (TSSM), to the shift to the non-preferred and novel host plant 26Arabidopsis thaliana. We identified the key requirement of two tiers of cytochrome P450 27 monooxygenases for TSSM adaptation to Arabidopsis: general xenobiotic-responsive P450s that 28 have a limited contribution to mite adaptation to Arabidopsis and adaptation-associated P450s 29 that efficiently counteract Arabidopsis defenses, illustrating that in about 25 generations of 30 TSSM selection on Arabidopsis plants mites evolved metabolic resistances characteristic of both 31 generalist and specialist herbivores. 32 33

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. By contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. T. urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified β-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and β-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its β-cyanoalanine synthase activity. Consistent with the β-cyanoalanine synthase role in detoxification of hydrogen cyanide, we discovered that upon mite herbivory, Arabidopsis plants release hydrogen cyanide. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the β-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite’s ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.