Microbiomes are increasingly recognized as key contributors to host fitness, yet their role in mediating stressor effects, especially across metamorphosis, remain poorly understood. To address this knowledge gap, we strongly reduced the gut microbiome of Ischnura elegans damselfly larvae using antibiotics followed by inoculation with a donor gut microbiome or not, and afterwards exposed the larvae to the pathogen Escherichia coli. Both host fitness and gut microbiome diversity and community composition were assessed during the larval and adult life stages. Despite marked differences in gut microbiome community composition between both life stages, partial retention of larval taxa in adult microbiota suggests incomplete microbiome turnover in these hemimetabolous insects. Furthermore, microbiome disruption significantly increased larval mortality, an effect mitigated by microbiome inoculation, underscoring the functional importance of microbial associations in damselflies. Moreover, pathogen exposure elevated larval mortality and tended to induce delayed mortality in the adult stage, revealing carry-over effects across metamorphosis. Yet, we did not find evidence of the gut microbiome mediating these carry-over effects. Together, our results highlight the critical role of the microbiome in determining host fitness, and the importance of considering both immediate and delayed stressor effects in animals with complex life cycles.

Triggered by global warming there is a surge of interest in understanding why conspecific populations differentiate along latitudinal gradients. Despite the insight that microbiomes may contribute to host traits and show geographic variation, how hosts differentially assemble their microbiomes across latitudes and how this shapes latitudinal patterns in host trait values has been understudied. This is especially true for the many animals that show a complex life cycle with aquatic larvae and terrestrial adults, and where metamorphosis may reset the microbiome. To address this knowledge gap, we examined microbiome composition along a latitudinal gradient and assessed the relative influence of host and environmental factors in shaping the microbiome of the damselfly Ischnura elegans. We collected bacterioplankton samples from high-latitude (Southern Sweden) and low-latitude (Southern France) ponds, along with larval gut and adult abdominal microbiome samples from I. elegans inhabiting these ponds. Additionally, we conducted a reciprocal translocation experiment in which larvae with a strongly reduced gut microbiome were transferred between three high- and three low-latitude ponds, enabling them to reassemble their gut microbiome from the local bacterioplankton, and tested the larvae for their fitness. We found clear latitudinal differences in the community composition of the bacterioplankton and host-associated microbiomes in adults and to a lesser extent in larvae, highlighting differences in microbiome composition across the latitudinal gradient and suggesting a strong influence of environmental factors on microbiome assembly. Moreover, our results showed that metamorphosis did not completely reset the microbiome, since larvae and adults shared part of their microbiome. The reciprocal translocation experiment revealed that microbiome reassembly in microbiome-depleted larvae was primarily shaped by the local environment, rather than the host source population or latitude, underscoring the strong influence of environmental filtering in shaping microbiome composition. Moreover, phenotypic differences in larval growth rate between high- and low-latitude populations disappeared after translocation, suggesting that local environmental conditions overruled any underlying genetic differentiation. By combining microbiome sampling across latitudes with a novel reciprocal translocation approach using microbiome-depleted larvae, our study provides key insights into how the environment shapes host-microbiome associations along geographic gradients.

Latitudinal patterns in fitness-related traits within species may inform how populations could evolve under global warming, yet are poorly understood. We investigated the novel idea that the gut microbiome drives latitudinal differences in immune function and pathogen tolerance by contrasting high- and low-latitude populations of Ischnura elegans damselflies. A reciprocal gut microbiome transplant was performed between high- and low-latitude larvae at two thermal regimes, whereafter larvae were exposed to the pathogen Escherichia coli. Pathogen exposure increased mortality, especially under warming. Our results confirmed latitude-associated thermal adaptation and a faster pace-of-life of the low-latitude larvae, and revealed this to be associated with a lower immune function and pathogen tolerance (higher E. coli body burden). Moreover, our results provide the first experimental evidence that the gut microbiome causally contributed to latitudinal differences in the host’s immune function and pathogen tolerance. As latitudinal patterns in the microbiome are widespread, this may be an important yet ignored proximate driver of latitudinal patterns in immune function and pathogen tolerance. As expected, warming reduced the immune function especially in high-latitude larvae. Notably, the integration of a gut microbiome transplant experiment with a space-for-time substitution suggested that the high-latitude host may avoid a strong immunosuppression under warming, when only its gut microbiome but not the host would converge to the current low-latitude host-microbiome combination. Taking into account how the gut microbiome shapes latitudinal patterns in fitness-related traits and integrating it into space-for-time substitutions will deepen our understanding of the evolution of host populations under warming.

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.

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 explainednot 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.

Explaining interspecific differences in pollutant sensitivity is key to increasing the predictive power of ecotoxicology. Besides species traits, the gut microbiome may provide an untested additional predictive factor since its ability to co-determine the host’s defence system against stressors. Therefore, we investigated the gut microbiome’s causal role in shaping differences in pesticide sensitivity between two congeneric damselfly species. After an antibiotic treatment, reciprocal gut microbiome transplants were performed between pesticide-sensitive Ischnura elegans and more tolerant I. pumilio larvae, followed by exposure to chlorpyrifos or a solvent control. The gut microbiome, which was determined by 16S rRNA gene amplicon sequencing, of both species included pesticide-degrading bacteria, but also showed shared and species-specific responses to the pesticide. Notably, the most pesticide-sensitive combination, with the highest pesticide-induced mortality, consisted of I. elegans larvae receiving I. elegans donor gut microbiota, whereas the least sensitive combination consisted of I. pumilio larvae receiving I. pumilio donor gut microbiota, whereby the pesticide did not increase larval mortality. The two mixed donor-recipient microbiome combinations resulted in an intermediate sensitivity. Our results provide, to our knowledge, the first proof-of-evidence that the gut microbiome causally contributes to species differences in pesticide sensitivity.