# Contributions of DNA Sequencing Technologies to the Integrative Monitoring of Karstic Caves
## 4 NGS Taxonomic Assessment of Cave Microorganisms by Metabarcoding
### Metabarcoding-Based Knowledge of Caves
However, studying both the body- and gut-associated microbiota present is rather difficult due to sampling and contamination risks. Many studies focused on bats and guano in caves, particularly for microorganisms involved in zoonoses (viruses such as the European bat lyssavirus [87]), epizootides (_Pseudogymtoascus destructans_ responsible of white-nose syndrome [88]), or human pathologies (_Bartonella_[89]). Bat gut samples hosted diverse bacterial phyla, dominated by _Proteobacteria_ followed by _Firmicutes_[90]. Anthropization favors cave colonization by arthropods corresponding to Collembola [91]; _Folsonia candida_ present on black stains in Lascaux Cave exhibited gut darkening (as did _F. candida_ raised in vitro on plates where black fungi were grown), suggesting fungal consumption by these arthropods [8]. Accordingly, fungal conidia can be present in their feces [8], which points to the ability of collembolans to disseminate bacteria and fungi [50]. Metabarcoding confirmed these observations, with a total of 470 bacterial OTUs (including the endosymbiont _Wolbachia_) and 148 fungal OTUs (including _Ochroconis_ black fungi) associated to collembolans sampled in Lascaux Cave, which included 369 of the 382 bacterial OTUs and 21 of the 24 fungal OTUs found in the black stains underneath [86]. Metabarcoding approaches therefore make it possible to investigate the microbiota of arthropods and chiroptera in caves, but do not necessarily allow the identification of disseminated pathogens at the species level (Figure 1) [50; 90; 92]. Metabarcoding has been carried out to characterize the microorganisms of rock walls in karstic caves. The study of cave walls is often completed to address conservation issues. Indeed, many Paleolithic caves show wall alterations mainly due to ecosystem anthropization, which can cause, for example, the abnormal proliferation of microorganisms and the formation of visual stains on the walls (e.g., Lascaux Cave, Altamira Cave, etc.) [93; 8; 11; 94]. Many microorganisms potentially involved in these alterations have been identified, but the microbial diversity studies often missed comparisons with neighboring control areas, making such comparisons uncertain. The black stains and dark zones of Lascaux, the yellow and gray spots of Altamira, and the red spots of the Cave of Bats are partly due to the growth of black fungi (e.g., _Ochroconis_, _Exophilata_, _Acremonium_, etc. ), _Gammaproteobacteriaa_ from the order _Xanthomonadales_, and _Betaproteobacteria_ from the genus _Thuera_, respectively [95; 8; 11; 96; 97]. [PERSON] et al. (2018) showed contrasted fungal communities in different caves [94]. Most of the time, cave walls, whether anthropized or not, are mainly colonized by _Proteobacteria_, _Actinobacteria_, _Thuamarchaeota_, and _Ascomycota_ (Figure 1). Moreover, previous studies indicated that the anthropization of caves lead to a higher relative abundance of _Beteroide_ and a lower abundance of _Nitrospirate_ (Figure 1), suggesting their use as a bioinformatics of disturbance in karstic environments [98; 99; 26; 100]. A study comparing eight Chinese caves showed that temperature and sampling distance from the entrance have a significant influence on fungal communities on cave walls [65]. Indeed, fungal communities were more similar between the outside environment and the proximal entrance zone, while, when entering deeper into the cave, fungal communities were more dissimilar compared with the outside [65].