Lizanne Janssens

Predation risk causes oxidative damage in prey

While there is increasing interest in non-consumptive effects of predators on prey, physiological effects are understudied. While physiological stress responses play a crucial role in preparing escape responses, the increased metabolic rates and shunting of energy away from other body functions, including antioxidant defence, may generate costs in terms of increased oxidative stress. Here, we test whether predation risk increases oxidative damage in Enallagma cyathigerum damselfly larvae. Under predation risk, larvae showed higher lipid peroxidation, which was associated with lower levels of superoxide dismutase, a major antioxidant enzyme in insects, and higher superoxide anion concentrations, a potent reactive oxygen species. The mechanisms underlying oxidative damage are likely to be due to the shunting of energy away from antioxidant defence and to an increased metabolic rate, suggesting that the observed increased oxidative damage under predation risk may be widespread. Given the potentially severe fitness consequences of oxidative damage, this largely overlooked non-consumptive effect of predators may be contributing significantly to prey population dynamics.

How does a pesticide pulse increase vulnerability to predation? Combined effects on behavioral antipredator traits and escape swimming.

An increasing number of studies have documented that sublethal pesticide exposure can change predator-prey interactions. Most of these studies have focused on effects of long-term pesticide exposure on only one type of antipredator traits and have not directly linked changes in these traits to mortality by predation. To get a better mechanistic understanding of how short-term pesticide pulses make prey organisms more vulnerable to predation, we studied effects of 24h exposure to a sublethal concentration of the insecticide endosulfan and the herbicide Roundup on the major antipredator traits and the resulting mortality by predation in larvae of the damselfly Enallagma cyathigerum. A pulse of both pesticides affected antipredator traits involved in avoiding detection by predators as well as traits involved in escape after detection. After a pesticide pulse, larvae increased activity levels and even further increased the number of walks when predation risk was present. Further, an endosulfan pulse tended to reduce escape swimming speed. In contrast, previous exposure to Roundup caused the larvae to swim faster, yet less often when attacked. Importantly, although both studied pesticides induced maladaptive changes in overall activity, only for endosulfan this resulted in an increased mortality by predation. Our study highlights that considering changed predator-prey interactions may improve ecological risk evaluations of short pesticide pulses, yet also underscores the need (1) to consider effects on all important antipredator traits of the prey as trait compensation may occur and (2) to effectively score the outcome of predator-prey interactions in staged encounters.

Fitness Effects of Chlorpyrifos in the Damselfly Enallagma cyathigerum Strongly Depend upon Temperature and Food Level and Can Bridge Metamorphosis.

Interactions between pollutants and suboptimal environmental conditions can have severe consequences for the toxicity of pollutants, yet are still poorly understood. To identify patterns across environmental conditions and across fitness-related variables we exposed Enallagma cyathigerum damselfly larvae to the pesticide chlorpyrifos at two food levels or at two temperatures and quantified four fitness-related variables (larval survival, development time, mass at emergence and adult cold resistance). Food level and temperature did not affect survival in the absence of the pesticide, yet the pesticide reduced survival only at the high temperature. Animals reacted to the pesticide by accelerating their development but only at the high food level and at the low temperature; at the low food level, however, pesticide exposure resulted in a slower development. Chlorpyrifos exposure resulted in smaller adults except in animals reared at the high food level. Animals reared at the low food level and at the low temperature had a higher cold resistance which was not affected by the pesticide. In summary our study highlight that combined effects of exposure to chlorpyrifos and the two environmental conditions (i) were mostly interactive and sometimes even reversed in comparison with the effect of the environmental condition in isolation, (ii) strongly differed depending on the fitness-related variable under study, (iii) were not always predictable based on the effect of the environmental condition in isolation, and (iv) bridged metamorphosis depending on which environmental condition was combined with the pesticide thereby potentially carrying over from aquatic to terrestrial ecosystems. These findings are relevant when extrapolating results of laboratory tests done under ideal environmental conditions to natural communities.

Extreme temperatures in the adult stage shape delayed effects of larval pesticide stress: A comparison between latitudes

Global warming and pesticide pollution are major threats for aquatic biodiversity. Yet, how pesticide effects are influenced by the increased frequency of extreme temperatures under global warming and how local thermal adaptation may mitigate these effects is unknown. We therefore investigated the combined impact of larval chlorpyrifos exposure, larval food stress and adult heat exposure on a set of fitness-related traits in replicated low- and high-latitude populations of the damselfly Ischnura elegans. Larval pesticide exposure resulted in lighter adults with a higher water content, lower fat content, higher Hsp70 levels and a lower immune function (PO activity). Heat exposure reduced water content, mass, fat content and flying ability. Importantly, both stressors interacted across metamorphosis: adult heat exposure lowered the reduction of fat content, and generated a stronger decrease in PO activity in pesticide-exposed animals. Larval pesticide exposure and larval food stress also reduced the defense response to the adult heat stress in terms of increased Hsp70 levels. In line with strong life history differences in the unstressed control situation, high-latitude animals were less sensitive to food stress (body mass and water content), but more sensitive to pesticide stress (development time and PO activity) and heat exposure (PO activity and Hsp70 levels). While low-latitude adults could better withstand the extreme temperature as suggested by the weaker increase in Hsp70, heat exposure similarly affected the delayed effects of larval pesticide exposure at both latitudes. Our study highlighted two key findings relevant for ecological risk assessment under global warming. Firstly, the delayed effects of larval pesticide exposure on adult damselflies depended upon subsequent adult heat exposure, indicating that larval pesticide stress and adult heat stress interacted across metamorphosis. Secondly, low- and high-latitude animals responded differently to the imposed stressors, highlighting that intraspecific evolution along natural thermal gradients may shape sensitivity to pesticides.

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.

Integrating ecology and evolution in aquatic toxicology: insights from damselflies

Current legislation and ecological risk assessment fails to protect aquatic biodiversity at low levels of contaminants. We addressed 3 topics embedded in general stress ecology and evolutionary ecology that are relevant to arrive at a better evaluation of the risk of low contaminant levels in aquatic systems: 1) delayed effects of contaminants, 2) interactions between contaminants and biotic interactors, and 3) vulnerability to contaminants under global warming. We developed these topics by capitalizing on the key insights obtained using damselflies as model organisms. First, delayed contaminant effects on important fitness-related effects exist during the larval stage and after metamorphosis in the adult stage. Second, synergistic interactions of contaminants with bacteria and predation risk have been demonstrated, and we present advances in the mechanistic understanding of these synergisms with biotic interactors. Third, we illustrate the strength of assessing the effect of contaminants under global warming using a space-for-time substitution approach and the need to consider temperature extremes. These studies using damselflies as model organisms highlight the relevance of considering contaminant effects after the exposure period and in the presence of natural stressors, such as predation risk and higher temperatures. They further highlight the need for spatially explicit risk-assessment and conservation tools. These insights are relevant for most aquatic taxa. Indeed most aquatic taxa have a complex life cycle, are strongly affected by predation risk and by warming, and show latitudinal gradients. Better integration of these topics in ecological risk assessment will be a major challenge for both scientists and policy makers, but of crucial importance to preserve aquatic biodiversity.

Chronic predation risk reduces escape speed by increasing oxidative damage: a deadly cost of an adaptive antipredator response.

Prey organisms evolved a multitude of plastic responses to avoid being eaten by predators. Besides the evolution of plastic morphological responses to escape predation, prey also evolved a set of physiological stress responses to avoid dying because of chronic predator stress per se due to disruption of cellular homeostasis. As physiological stress theory predicts increased energy consumption and the inhibition of essential nonemergency body functions, we tested whether chronic predation risk may increase oxidative damage thereby generating negative effects on escape performance. Specifically, we evaluated whether predation risk reduces escape swimming speed in damselfly larvae and whether this operates through stress-associated increases in oxidative damage. Counterintuitively and in contrast with many empirical studies, chronic predation risk decreased escape performance. This is however entirely consistent with the expectation of it being a long-term cost of responding to predation risk (e.g. by increasing respiration or upregulating the stress protein levels). The decreased swimming speed could be explained by an increased oxidative damage to proteins, thereby providing one of the poorly studied ecological links between oxidative damage and whole-animal performance. This likely widespread, understudied cost of chronic predation risk may provide an important pathway of non-consumptive predator effects on prey population dynamics. Moreover, it could play an evolutionary role by acting as a selective force causing prey organisms to adjust the magnitude of the physiological stress response and should be considered when evaluating life history trade-offs thought to be mediated by oxidative damage.

Exposure to a heat wave under food limitation makes an agricultural insecticide lethal: a mechanistic laboratory experiment.

Extreme temperatures and exposure to agricultural pesticides are becoming more frequent and intense under global change. Their combination may be especially problematic when animals suffer food limitation. We exposed Coenagrion puella damselfly larvae to a simulated heat wave combined with food limitation and subsequently to a widespread agricultural pesticide (chlorpyrifos) in an indoor laboratory experiment designed to obtain mechanistic insights in the direct effects of these stressors in isolation and when combined. The heat wave reduced immune function (activity of phenoloxidase, PO) and metabolic rate (activity of the electron transport system, ETS). Starvation had both immediate and delayed negative sublethal effects on growth rate and physiology (reductions in Hsp70 levels, total fat content, and activity levels of PO and ETS). Exposure to chlorpyrifos negatively affected all response variables. While the immediate effects of the heat wave were subtle, our results indicate the importance of delayed effects in shaping the total fitness impact of a heat wave when followed by pesticide exposure. Firstly, the combination of delayed negative effects of the heat wave and starvation, and the immediate negative effect of chlorpyrifos considerably (71%) reduced larval growth rate. Secondly and more strikingly, chlorpyrifos only caused considerable (ca. 48%) mortality in larvae that were previously exposed to the combination of the heat wave and starvation. This strong delayed synergism for mortality could be explained by the cumulative metabolic depression caused by each of these stressors. Further studies with increased realism are needed to evaluate the consequences of the here-identified delayed synergisms at the level of populations and communities. This is especially important as this synergism provides a novel explanation for the poorly understood potential of heat waves and of sublethal pesticide concentrations to cause mass mortality.

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.

Warming increases chlorpyrifos effects on predator but not anti-predator behaviours

Recent insights indicate that negative effects of pesticides on aquatic biota occur at concentrations that current legislation considers environmentally protective. We here address two, potentially interacting, mechanisms that may contribute to the underestimation of the impact of sublethal pesticide effects in single species tests at room temperature: the impairment of predator and antipredator behaviours and the stronger impact of organophosphate pesticides at higher temperatures. To address these issues we assessed the effects of chlorpyrifos on the predator and antipredator behaviours of larvae of the damselfly Ischnura elegans, important intermediate predators in aquatic food webs, in a common-garden warming experiment with replicated low- and high-latitude populations along the latitudinal gradient of this species in Europe. Chlorpyrifos reduced the levels of predator behavioural endpoints, and this reduction was stronger at the higher temperature for head orientations and feeding strikes. Chlorpyrifos also impaired two key antipredator behavioural endpoints, activity reductions in response to predator cues were smaller in the presence of chlorpyrifos, and chlorpyrifos caused a lower escape swimming speed; these effects were independent of temperature. This suggests chlorpyrifos may impact food web interactions by changing predator-prey interactions both with higher (predators) and lower trophic levels (food). Given that only the interaction with the lower trophic level was more impaired at higher temperatures, the overall pesticide-induced changes in food web dynamics may be strongly temperature-dependent. These findings were consistent in damselflies from low- and high-latitude populations, illustrating that thermal adaptation will not mitigate the increased toxicity of pesticides at higher temperatures. Our study not only underscores the relevance of including temperature and prey-predator interactions in ecological risk assessment but also their potential interplay and thereby highlights the complexity of contaminant effects on predator-prey interactions being differentially temperature-dependent pending on the trophic level.