The troglobitic beetle, Cansiliella servadeii /HSWRGLULQL KDV VSHFLDOL]HG PRXWKSDUWV PRGL¿HG IRU EURZVLQJ DQG IHHGLQJ XQGHU percolating water on moonmilk, a speleothem formation in Grotta della Foos, Italy. Results from analyses of stable isotopes of carbon and nitrogen suggest that acquires and assimilates dissolved allochthonous organic carbon, inorganic nitrogen, and possibly phosphorus and other nutrients from the microbial fauna associated with moonmilk.

The microbial diversity of moonmilk, a hydrated calcium carbonate speleothem, was\ud evaluated from two Italian caves to provide context for the food web of highly-specialized\ud troglobitic beetles, Cansiliella spp. (Leptodirinae), with distinctive carbon and nitrogen\ud isotope values indicative of a novel food source. The moonmilk and associated percolating\ud waters had low to no extractable chlorophyll, with an average organic C:N ratio of 9,\ud indicating limited allochthonous input and a significant contribution from microbial biomass.\ud The biomass from moonmilk was estimated to be ~104 micro- and meiofaunal individuals per\ud m2 and ~108 microbial cells/ml. Betaproteobacteria dominated the 16S rRNA gene sequences\ud retrieved from the moonmilk from both caves. The distribution of other proteobacterial\ud classes and phyla in the moonmilk were statistically similar to each other, even though the\ud two caves are geographically separated from each other. Comparing the moonmilk gene\ud sequences to sequences from previously described environmental clones or cultured strains\ud revealed the uniqueness of the moonmilk habitat, as ~ 15% of all of the moonmilk sequences\ud were more closely related to each other than to sequences retrieved from any other habitat.\ud However, comparative analyses confirmed that as much as ~34% of the clones sequences\ud were also closely related to environmental clones and cultured strains derived from soil and\ud freshwater habitats, which is likely due to the fact that the putative inoculation source for the\ud moonmilk bacterial communities is from overlying soil and percolating fluids from the\ud surface. Prior to our studies of Cansiliella spp., moonmilk has not been considered a food\ud source for cave animals. Our findings provide unique insight into moonmilk microbial\ud diversity that could reveal the underpinnings of the moonmilk carbon and nitrogen cycle that\ud influences the isotopic composition and the morphological adaptations of the troglobitic\ud beetles associated with the moonmilk

BackgroundCansiliella servadeii (Coleoptera) is an endemic troglobite living in deep carbonate caves in North-Eastern Italy. The beetle constantly moves and browses in its preferred habitat (consisting in flowing water and moonmilk, a soft speleothem colonized by microorganisms) self-preens to convey material from elytra, legs, and antennae towards the mouth. We investigated its inner and outer microbiota using microscopy and DNA-based approaches.ResultsAbundant microbial cell masses were observed on the external appendages. Cansiliella’s midgut is fully colonized by live microbes and culture-independent analyses yielded nearly 30 different 16S phylotypes that have no overlap with the community composition of the moonmilk. Many of the lineages, dominated by Gram positive groups, share very low similarity to database sequences. However for most cases, notwithstanding their very limited relatedness with existing records, phylotypes could be assigned to bacterial clades that had been retrieved from insect or other animals’ digestive traits.ConclusionsResults suggest a history of remote separation from a common ancestor that harboured a set of gut-specific bacteria whose functions are supposedly critical for host physiology. The phylogenetic and coevolutionary implications of the parallel occurrences of these prokaryotic guilds appear to apply throughout a broad spectrum of animal diversity. Their persistence and conservation underlies a possibly critical role of precise bacterial assemblages in animal-bacteria interactions.