A novel symbiosis between chemoautotrophic bacteria and a freshwater cave amphipod.

Symbioses involving animals and chemoautotrophic bacteria form the foundation of entire ecosystems at deep-sea hydrothermal vents and cold seeps, but have so far not been reported in terrestrial or freshwater environments. A rare example of a terrestrial ecosystem sustained by chemoautotrophy is found within the sulfide-rich Frasassi limestone cave complex of central Italy. In this study, we report the discovery of abundant filamentous bacteria on the exoskeleton of Niphargus ictus, a macroinvertebrate endemic to Frasassi. Using 16S rDNA sequencing and fluorescence in situ hybridization (FISH), we show that N. ictus throughout the large cave complex are colonized by a single phylotype of bacteria in the sulfur-oxidizing clade Thiothrix. The epibiont phylotype is distinct from Thiothrix phylotypes that form conspicuous biofilms in the cave streams and pools inhabited by N. ictus. Using a combination of 13C labeling, FISH, and secondary ion mass spectrometry (SIMS), we show that the epibiotic Thiothrix are autotrophic, establishing the first known example of a non-marine chemoautotroph-animal symbiosis. Conditions supporting chemoautotrophy, and the N. ictus-Thiothrix association, likely commenced in the Frasassi cave complex between 350,000 and 1 million years ago. Therefore, the N. ictus-Thiothrix symbiosis is probably significantly younger than marine chemoautotrophic symbioses, many of which have been evolving for tens to hundreds of million years.

Dominant microbial populations in limestone-corroding stream biofilms, Frasassi cave system, Italy.

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. beta-, gamma-, delta-, and epsilon-proteobacteria in sulfur-cycling clades accounted for > or = 75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating delta-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous gamma-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of epsilon-proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.