Unveiling the diet-microbiome-reproduction interconnection within two gregarious insectivorous bat hosts at the northern edge of their geographic range

The research explores the relationship between diet, microbiome, and reproductive strategies of two insectivorous bat species, Myotis emarginatus (the Geoffroy’s bat) and Rhinolophus ferrumequinum (the greater horseshoe bat), at the northernmost edge of their range in Belgium. These species experienced significant population declines in the 20th century, followed by a partial recovery in recent decades. Despite this increase, studying these bats at the limit of their range is essential because individuals in these regions might be less adapted to local environmental conditions—including food opportunities, making them less flexible in their feeding behavior. Unveiling the diet of insectivorous bats is crucial for several additional reasons. It enhances our knowledge of arthropod diversity, informs pest control strategies, provides insights into habitat use and foraging strategies, and aids in habitat management. The diet also directly influences the gut microbiome, which affects nutrient absorption and overall health. While the connection between diet and microbiome has been well-established, there has been limited research on this relationship at an individual level, particularly during the breeding season. The reproductive period is particularly energy intensive for female bats due to the physiological and ecological demands of pregnancy, lactation, and raising offspring. To meet these heightened energy requirements, bats optimize their food intake and nutrient absorption. The breeding season may thus necessitate specific dietary adaptations that are reflected in the gut microbiome. Disentangling the link between diet and microbiome during the reproductive period was the main goal of the research, as it could inform conservation efforts, particularly in habitats where insect populations are declining or where bats are under environmental stress. In addition, we hypothesized that physical proximity in multi-species bat colonies could promote bacterial transfer between species. M. emarginatus and R. ferrumequinum, which frequently share maternity roosts, were ideal candidates to test this hypothesis. To carry out this project, 378 non-invasive guano samples were collected from eight summer maternity colonies, each hosting either one or both species. Sampling was conducted during three phases of the breeding season: pregnancy, lactation, and post-lactation. The study focused on factors such as species identity, colony size, locality, and inter-species interactions in mixed species roosts. Metabarcoding techniques were selected to analyze both diet and microbiome, providing detailed insights into the consumed prey species and the bacterial communities present in the guano. In the diet of M. emarginatus, we identified 512 prey taxa, dominated by Diptera, Araneae, and Lepidoptera. For R. ferrumequinum, the 305 diet taxa mostly comprised Lepidoptera, followed by Diptera, Coleoptera, and Hymenoptera prey. Using the prey’s own ecology to infer the bats’ foraging behavior, we revealed that M. emarginatus would predominantly feed with a gleaning style while R. ferrumequinum would primarily catch prey in flight. However, both species could employ the other strategy, thus exhibiting dietary flexibility, which is crucial for their survival at the edge of their range. In both species, a seasonal June versus July and August shift was observed in their feeding behavior, with a diverse diet in June transitioning to a restricted diet in July and August. We found that M. emarginatus mostly shifted their food consumption from spiders—the species primal diet—in more forested landscapes to flies (mostly pest flies), which are superabundant around cattle. This opportunistic dietary shift likely helped female bats to meet the increased energy demands during parturition and lactation (in July and August). Contrary to M. emarginatus, R. ferrumequinum decreasingly relied on Diptera (mostly Tipulidae) over the breeding season, in favor of Lepidoptera and specific taxa of Coleoptera. In contrast to the expectation that R. ferrumequinum prefers large prey, many small-sized prey items were found in its diet. This could be due to the scarcity of other Rhinolophidae species in the area, thereby allowing R. ferrumequinum to expand its dietary niche. Finally, the diet study highlighted the role of M. emarginatus in consuming pest species—especially cattle related flies and defoliator caterpillars, making them valuable allies to farmers and forest managers. The microbiome analysis revealed that host species was the strongest predictor of guano bacterial communities’ composition, but we also demonstrated huge inter-sample variation, which could hint at individualization in the microbiota. In fact, the findings supported the hypothesis of functional redundancy, which postulates that despite high individual variation in microbial composition, metagenomic functions remained consistent across individuals. In spite of the physical proximity of the two species in shared roosts, there seemed to be limited bacterial transfer between them. In addition, the expected influence of colony size on microbial diversity was not consistently observed. Interestingly, contrary to our expectation, the diet would be more closely linked to the compositional rather than to the functional microbiome. We further report that dietary differences at the finest taxonomic level were those shaping the most microbial composition in both species. Temporal factors also influenced microbial communities, particularly in M. emarginatus, in which a June versus July and August consistent pattern mirrored that of food consumption. Finally, while we observed some synchronized variations in the diet-microbiome relationships over the breeding season, the relationships were complex and sometimes contradictory. Nevertheless, certain bacterial taxa were consistently associated with various diet items, suggesting their role as regular commensals in the gut of M. emarginatus and R. ferrumequinum. We conclude that reproductive-related changes in the microbiome of M. emarginatus females may contribute to (or alternatively, be governed by) the adjustment of their diet, which are required to meet the specific energy needs during different phases of reproduction. However, the research also emphasizes the need for further studies on prey availability across the study area to disentangle the effects of arthropod phenology from those of changing reproductive needs in terms of food habits. Overall, the present thesis highlighted that these insectivorous bats were generally flexible in their feeding strategies, hence depicting their generalist behavior. This plasticity could help them to cope with environmental changes, particularly at the northern edge of their geographic range. Ultimately, these bats also play a crucial role in sampling invertebrate biodiversity and providing pest control services, underscoring their ecological importance.