What’s inside a Niphargus?

Host-associated microbiomes play a pivotal role in maintaining the health and fitness of diverse animal and plant species. While certain microbiomes exhibit phylogenetic relationships (phylosimbiosis) driven by evolutionary relatedness and ecological constraints, others show high flexibility, primarily influenced by environmental conditions. Furthermore, not all animals require a microbiome, underscoring the complexity of host-microbe relationships. Despite recent mainstream interest in microbiome research, our knowledge of host-microbe interactions in subterranean environments remains incomplete. Recent studies on subterranean species have highlighted the crucial role of gut microbiota in adapting to subterranean environments. However, we still don’t know to which extent phylogenetic relatedness and environmental parameters influence the microbiome composition of groundwater animals. In this study, we addressed these questions using groundwater crustaceans of the amphipod genus Niphargus, alongside detecting signals of human disturbance within the microbiome. We analysed the microbiomes of 46 individuals from three caves in Abruzzo (central Italy). These caves cover a gradient of human disturbance, ranging from minimal human access (Val Cordora cave) to moderate speleological activity (Grotta a Male), and finally to a show cave (Stiffe cave) with daily visits (ca. 45,000 tourists a year) and seasonal infiltration of wastewater. Our results showed high variability in microbiome diversity across samples and no evidence of phylosymbiosis. We found that caves and microhabitats significantly affect microbial community composition, but accounting for just 11.9% and 8.8% of the variance, respectively. These findings suggest that environmental factors play a stronger role in shaping the microbiome. Despite the variability in overall microbiome diversity, we identified a stable 'core' (a set of taxa present at ≥1% abundance in ≥75% of samples) within the microbial communities across the caves. Pseudomonas was the most dominant genus (70% of the core), followed by an unidentified genus within the order Rickettsiales (15.6% of the core). Seven other genera were also part of the core but at lower abundances, collectively contributing less than 20%. Notably, a specific Amplicon Sequence Variant (ASV) within the genus Pseudomonas, affiliated with the species P. koreensis, was a consistent core member, dominating all samples. It constituted approximately 25% of the reads and 60% of the core, highlighting its ecological importance in driving host-microbe interactions in these subterranean environments. The dominance of a single taxon in the microbiota of Niphargus of three different caves suggests a strong habitat filtering for specialists. We further investigated the occurrence of the class 1 integrons (intI1/16S gene), a marker known as a proxy for anthropogenic pollution and antimicrobial resistance genes. The abundance of intI1 was lower in the individuals of Val Cordora Cave, confirming a limited disturbance. This study highlights the potential for examining subterranean animals from 'their inside' to gain novel insights into the essential role of microbes in contributing to their fitness. Furthermore, it suggests the possibility of analysing host-associated microbial communities to detect potential human-induced alterations. However, our current understanding of these dynamics remains limited, akin to opening a Pandora's box that could change our perspective on studying subterranean species.