Effects of heat and pesticide stress on life history, physiology and the gut microbiome of two congeneric damselflies that differ in stressor tolerance.

The combined impact of toxicants and warming on organisms is getting increased attention in ecotoxicology, but is still hard to predict, especially with regard to heat waves. Recent studies suggested that the gut microbiome may provide mechanistic insights into the single and combined stressor effects on their host. We therefore investigated effects of sequential exposure to a heat spike and a pesticide on both the phenotype (life history and physiology) and the gut microbiome composition of damselfly larvae. We compared the fast-paced Ischnura pumilio, which is more tolerant to both stressors, with the slow-paced I. elegans, to obtain mechanistic insights into species-specific stressor effects. The two species differed in gut microbiome composition, potentially contributing to their pace-of-life differences. Intriguingly, there was a general resemblance between the stressor response patterns in the phenotype and in the gut microbiome, whereby both species responded broadly similar to the single and combined stressors. The heat spike negatively affected the life history of both species (increased mortality, reduced growth rate), which could be explained not only by shared negative effects on physiology (inhibition of acetylcholinesterase, increase of malondialdehyde), but also by shared effects on gut bacterial species' abundances. The pesticide only had negative effects (reduced growth rate, reduced net energy budget) in I. elegans. The pesticide generated shifts in the bacterial community composition (e.g. increased abundance of Sphaerotilus and Enterobacteriaceae in the gut microbiome of I. pumilio), which potentially contributed to the relatively higher pesticide tolerance of I. pumilio. Moreover, in line with the response patterns in the host phenotype, the effects of the heat spike and the pesticide on the gut microbiome were mainly additive. By contrasting two species differing in stress tolerance, our results suggest that response patterns in the gut microbiome may improve our mechanistic understanding of single and combined stressor effects.

Thermal and latitudinal patterns in pace-of-life traits are partly mediated by the gut microbiome.

The integration of life-history, physiological and behavioural traits into the pace-of-life generates a powerful framework to understand trait variation in nature both along environmental gradients and in response to environmental stressors. While the gut microbiome has been hypothesized as a candidate mechanism to underlie differentiation in the pace-of-life, this has been rarely studied. We investigated the role of the gut microbiome in contributing to the differentiation in pace-of-life and in thermal adaptation between populations of Ischnura elegans damselfly larvae inhabiting warmer low latitudes and colder high latitudes. We carried out a common-garden experiment, whereby we manipulated the exposure of the damselfly larvae to two key global warming factors: 4 °C warming and a 30 °C heat wave. Comparing the bacterial composition of the food source and the bacterioplankton indicated that damselfly larvae differentially take up bacteria from the surrounding environment and have a resident and functionally relevant microbiome. The gut microbiome differed between larvae of both latitudes, and this was associated with the host's latitudinal differentiation in activity, a key pace-of-life trait. Under heat wave exposure, the gut microbial community composition of high-latitude larvae converged towards that of the low-latitude larvae, with an increase in bacteria that likely are important in providing energy to cope with the heat wave. This suggests an adaptive latitude-specific shift in the gut microbiota matching the better ability of low-latitude hosts to deal with heat extremes. In general, our study provides evidence for the gut microbiome contributing to latitudinal differentiation in both the pace-of-life and in heat adaptation in natural populations.

Evolution of pesticide tolerance and associated changes in the microbiome in the water flea Daphnia magna.

Exposure to pesticides can have detrimental effects on aquatic communities of non-target species. Populations can evolve tolerance to pesticides which may rescue them from extinction. However, the evolution of tolerance does not always occur and insights in the underlying mechanisms are scarce. One understudied mechanism to obtain pesticide tolerance in hosts are shifts toward pesticide-degrading bacteria in their microbiome. We carried out experimental evolution trials where replicated experimental populations of the water flea Daphnia magna were exposed to the pesticide chlorpyrifos or a solvent control, after which we performed acute toxicity assays to evaluate the evolution of chlorpyrifos tolerance. Additionally, we quantified changes in the microbiota community composition of whole body and gut samples to assess which sample type best reflected the pesticide tolerance of the Daphnia host. As expected, chlorpyrifos-selected clones became more tolerant to chlorpyrifos as shown by the higher EC5048 h (36% higher) compared with the control clones. This was associated with shifts in the microbiome composition whereby the abundance of known organophosphate-degrading bacterial genera increased on average ~4 times in the chlorpyrifos-selected clones. Moreover, the abundances of several genera, including the organophosphate-degrading bacteria Pseudomonas, Flavobacterium and Bacillus, were positively correlated with the EC5048 h of the host populations. These shifts in bacterial genera were similar in magnitude in whole body and gut samples, yet the total abundance of organophosphate-degrading bacteria was ~6 times higher in the whole body samples, suggesting that the gut is not the only body part where pesticide degradation by the microbiome occurs. Our results indicate that the microbiome is an important mediator of the development of tolerance to pesticides in Daphnia.

Daily temperature fluctuations can magnify the toxicity of pesticides.

We review the effect of daily temperature fluctuations (DTF), a key thermal factor predicted to increase under climate change, on pesticide toxicity. The effect of DTF on pesticide toxicity may be explained by: (i) a DTF-specific mechanism (caused by Jensen's inequality) and (ii) general mechanisms underlying an increased pesticide toxicity at both higher (increased energetic costs, pesticide uptake and metabolic conversion) and lower constant temperatures (lower organismal metabolic and associated elimination rates, increased sodium channel modulated nervous system vulnerability and energetic costs). Furthermore, DTF may enhance pesticide-induced reductions in heat tolerance due to stronger effects on oxygen demand (increase) and oxygen supply (decrease). Our literature review showed considerable support that DTF increase the negative impact of pesticides on insects, especially in terms of decreased survival. Therefore, we suggest that considering DTF in ecotoxicological studies may be of great importance to better protect biodiversity in our warming world.

Higher mean and fluctuating temperatures jointly determine the impact of the pesticide chlorpyrifos on the growth rate and leaf consumption of a freshwater isopod.

There is growing evidence that both increases in mean temperature and the widespread daily temperature fluctuations (DTF) may increase pesticide toxicity. Nevertheless, the likely more stressful, realistic combination of the two warming-related stressors has rarely been considered in ecotoxicology. Moreover, we have little knowledge on whether these stressor combinations could impair ecosystem functioning. We examined the effect of the pesticide chlorpyrifos under an increased mean temperature (+4 °C, from 18 °C to 22 °C) and in the presence of DTF (constant and 8 °C) on two life-history traits (mortality and growth rate) and one ecologically important behavioural trait (feeding rate) in the freshwater isopod Asellus aquaticus. The chlorpyrifos concentration used, 0.2 µg/L, did not cause mortality in any thermal condition, nor did it cause sublethal effects at the mean temperature of 18 °C. A key finding was that growth rate was strongly reduced by the pesticide only under the combination of both a higher mean temperature and DTF, highlighting the importance of testing toxicity under this realistic thermal scenario. The leaf consumption of chlorpyrifos-exposed isopods increased at the higher mean temperature when this was kept constant, however, it lowered again towards control levels when DTF was induced, thereby contributing to the growth reduction at this most stressful condition. These alterations of growth and leaf degradation rates may impact nutrient recycling, a key ecosystem function. Our results highlight the importance of integrating both increases in mean temperature and in DTF to improve current and future ecological risk assessment of pesticides.