Effects of pesticide exposure and predation risk on nutrient cycling and primary production.

Understanding how pesticides and natural stressors shape ecosystem functions remains a major challenge. A largely overlooked way how stressors may affect nutrient cycling and primary production is through effects on body stoichiometry and the egestion of elements. We investigated how exposure to the pesticide chlorpyrifos and to predation risk, an abundant natural stressor in aquatic systems, altered the stoichiometry of the bodies and the egested faecal pellets of Enallagma cyathigerum damselfly larvae and how this further cascaded into effects on primary production (algae growth). Chlorpyrifos exposure reduced egestion rates while predation risk had no effect. Chlorpyrifos exposure and predation risk affected both elemental composition of bodies and faecal pellets, and this in an additive way. Chlorpyrifos exposure increased body C(carbon), N(nitrogen), and P(phosphorous) contents, and increased the C content of the faecal pellets. Predation risk induced an increase of the N content, resulting in a decreased C:N ratio, of both the bodies and faecal pellets. The changes in the composition of the faecal pellets caused by predation risk but not by chlorpyrifos exposure increased algae growth under control conditions. This indicated that algae growth was N limited. Our results provide an important proof-of-principle how a stressor may shape nutrient cycling and subsequently primary productivity.

Latitude-associated evolution and drivers of thermal response curves in body stoichiometry.

Trait-based studies are needed to understand the plastic and genetic responses of organisms to warming. A neglected organismal trait is elemental composition, despite its potential to cascade into effects on the ecosystem level. Warming is predicted to shape elemental composition through shifts in storage molecules associated with responses in growth, body size and metabolic rate. Our goals were to quantify thermal response patterns in body composition and to obtain insights into their underlying drivers and their evolution across latitudes. We reconstructed the thermal response curves (TRCs) for body elemental composition [C (carbon), N (nitrogen) and the C:N ratio] of damselfly larvae from high- and low-latitude populations. Additionally, we quantified the TRCs for survival, growth and development rates and body size to assess local thermal adaptation, as well as the TRCs for metabolic rate and key macromolecules (proteins, fat, sugars and cuticular melanin and chitin) as these may underlie the elemental TRCs. All larvae died at 36°C. Up to 32°C, low-latitude larvae increased growth and development rates and did not suffer increased mortality. Instead, growth and development rates of high-latitude larvae were lower and levelled off at 24°C, and mortality increased at 32°C. This latitude-associated thermal adaptation pattern matched the 'hotter-is-better' hypothesis. With increasing temperatures, low-latitude larvae decreased C:N, while high-latitude larvae increased C:N. These patterns were driven by associated changes in N contents, while C contents did not respond to temperature. Consistent with the temperature-size rule and the thermal melanism hypothesis, body size and melanin levels decreased with warming. While all traits and associated macromolecules (except for metabolic rate that showed thermal compensation) assumed to underlie thermal responses in elemental composition showed thermal plasticity, these were largely independent and none could explain the stoichiometric TRCs. Our results highlight that thermal responses in elemental composition cannot be explained by traditionally assumed drivers, asking for a broader perspective including the thermal dependence of elemental fluxes. Another key implication is that thermal evolution can reverse the plastic stoichiometric thermal responses and hence reverse how warming may shape food web dynamics through changes in body composition at different latitudes.

Additive bioenergetic responses to a pesticide and predation risk in an aquatic insect.

Ignoring natural stressors such as predation risk may contribute to the failure of ecological risk assessment of pesticides to protect freshwater biodiversity. To better understand combined effects of multiple stressors, bioenergetic responses are important as these inform about the balance between energy input and consumption, and provide a unifying mechanism to integrate the impact of multiple stressors with different modes of action. We studied in Enallagma cyathigerum damselfly larvae the single and combined effects of exposure to the pesticide chlorpyrifos and predation risk on life history (survival and growth rate) and bioenergetic response variables at the organismal level (assimilation and conversion efficiency) and the cellular level (cellular energy allocation CEA, energy storage Ea, and energy consumption Ec). Chlorpyrifos exposure almost halved the survival of the damselfly larvae, while predation risk had no effect on survival. Both exposure to the pesticide and to predation risk reduced larval growth rates. This was caused by a reduced conversion efficiency under chlorpyrifos exposure, and by a reduced assimilation efficiency under predation risk. Both chlorpyrifos and predation risk reduced the CEA because of a decreased Ea, and for chlorpyrifos also an increased Ec. The lower Ea was driven by reductions in the fat and glycogen contents. Effects of the pesticide and predation risk were consistently additive and for most variables the strongest response was detected when both stressors were present. The absence of any synergisms may be explained by the high mortality and hypometabolism caused by the pesticide. Our results indicate that CEA can be a sensitive biomarker to evaluate effects of not only contaminants but also natural stressors, such as predation risk, and their combined impact on organisms.

Warming under seminatural outdoor conditions in the larval stage negatively affects insect flight performance.

Laboratory studies indicate global warming may cause changes in locomotor performance directly relevant for fitness and dispersal. Yet, this remains to be tested under seminatural settings, and the connection with warming-induced alterations in the underlying traits has been rarely studied. In an outdoor mesocosm experiment with the damselfly Ischnura elegans, 4°C warming in the larval stage decreased the flight muscle mass, which correlated with a lower flight endurance. Warming did not affect body mass, size or wing morphology. This illustrates how carry-over effects of warming under seminatural conditions during early development bridge metamorphosis and negatively impact locomotor performance through changes in a key flight-related trait.

Daily temperature variation and extreme high temperatures drive performance and biotic interactions in a warming world.

We review the major patterns on the effects of daily temperature variation (DTV) and extreme high temperatures (EXT) on performance traits and the resulting outcome of biotic interactions in insects. EXT profoundly affects the outcome of all types of biotic interactions: competitive, predator-prey, herbivore-plant, host-pathogen/parasitoid and symbiotic interactions. Studies investigating effects of DTV on biotic interactions are few but also show strong effects on competitive and host-pathogen/parasitoid interactions. EXT typically reduces predation, and is expected to reduce parasitoid success. The effects of EXT and DTV on the outcome of the other interaction types are highly variable, yet can be predicted based on comparisons of the TPCs of the interacting species, and challenges the formulation of general predictions about the change in biotic interactions in a warming world.

Beneficial effects of a heat wave: higher growth and immune components driven by a higher food intake.

While heat waves will become more frequent and intense under global warming, the ability of species to deal with extreme weather events is poorly understood. We investigated how a heat wave influenced growth rate and investment in two immune components (phenoloxidase activity and melanin content) in larvae of two damselfly species, Ischnura elegans and Enallagma cyathigerum Late instar larvae were kept at 18°C (i.e. their average natural water temperature) or under a simulated long heat wave at 30°C. To explain the heat wave effects, we quantified traits related to energy uptake (food intake and growth efficiency), energy expenditure (metabolic rate measured as activity of the electron transport system, ETS) and investment in energy storage (fat content). The two species differed in life strategy, with I. elegans having a higher growth rate, growth efficiency, ETS activity and fat content. In line with its preference for cooler water bodies, the heat wave was only lethal for E. cyathigerum However, both species benefited from the heat wave by increasing growth rate, which can be explained by the higher increase in food intake than metabolic rate. This may also have contributed to the increased investment in energy storage and immune components under the heat wave. This mediatory role of food intake indicates the critical role of food availability and behaviour in shaping the impact of heat waves. Our results highlight the importance of including behavioural and physiological variables to unravel and predict the impact of extreme climate events on organisms.

Short- and long-term behavioural, physiological and stoichiometric responses to predation risk indicate chronic stress and compensatory mechanisms.

Prey organisms are expected to use different short- and long-term responses to predation risk to avoid excessive costs. Contrasting both types of responses is important to identify chronic stress responses and possible compensatory mechanisms in order to better understand the full impact of predators on prey life history and population dynamics. Using larvae of the damselfly Enallagma cyathigerum, we contrasted the effects of short- and long-term predation risk, with special focus on consequences for body stoichiometry. Under short-term predation risk, larvae reduced growth rate, which was associated with a reduced food intake, increased metabolic rate and reduced glucose content. Under long-term predation risk, larvae showed chronic predator stress as indicated by persistent increases in metabolic rate and reduced food intake. Despite this, larvae were able to compensate for the short-term growth reduction under long-term predation risk by relying on physiological compensatory mechanisms, including reduced energy storage. Only under long-term predation risk did we observe an increase in body C:N ratio, as predicted under the general stress paradigm (GSP). Although this was caused by a predator-induced decrease in N content, there was no associated increase in C content. These stoichiometric changes could not be explained by GSP responses because, under chronic predation risk, there was no decrease in N-rich proteins or increase in C-rich fat and sugars; instead glycogen decreased. Our results highlight the importance of compensatory mechanisms and the value of explicitly integrating physiological mechanisms to obtain insights into the temporal dynamics of non-consumptive effects, including effects on body stoichiometry.

Warming reinforces nonconsumptive predator effects on prey growth, physiology, and body stoichiometry.

While nonconsumptive effects of predators may strongly affect prey populations, little is known how future warming will modulate these effects. Such information would be especially relevant with regard to prey physiology and resulting changes in prey stoichiometry. We investigated in Enallagma cyathigerum damselfly larvae the effects of a 4°C warming (20°C vs. 24°C) and predation risk on growth rate, physiology and body stoichiometry, for the first time including all key mechanisms suggested by the general stress paradigm (GSP) on how stressors shape changes in body stoichiometry. Growth rate and energy storage were higher at 24°C. Based on thermodynamic principles and the growth rate hypothesis, we could demonstrate predictable reductions in body C:P under warming and link these to the increase in P-rich RNA; the associated warming-induced decrease in C:N may be explained by the increased synthesis of N-rich proteins. Yet, under predation risk, growth rate instead decreased with warming and the warming-induced decreases in C:N and C:P disappeared. As predicted by the GSP, larvae increased body C:N and C:P at 24°C under predation risk. Notably, we did not detect the assumed GSP-mechanisms driving these changes: despite an increased metabolic rate there was neither an increase of C-rich biomolecules (instead fat and sugar contents decreased under predation risk), nor a decrease of N-rich proteins. We hypothesize that the higher C:N and N:P under predation risk are caused by a higher investment in morphological defense. This may also explain the stronger predator-induced increase in C:N under warming. The expected higher C:P under predation risk was only present under warming and matched the observed growth reduction and associated reduction in P-rich RNA. Our integrated mechanistic approach unraveled novel pathways of how warming and predation risk shape body stoichiometry. Key findings that (1) warming effects on elemental stoichiometry were predictable and only present in the absence of predation risk and that (2) warming reinforced the predator-induced effects on C:N:P, are pivotal in understanding how nonconsumptive predator effects under global warming will shape prey populations.