Longitudinal metabarcode analysis of karst bacterioplankton microbiomes provide evidence of epikarst to cave transport and community succession

Caves are often assumed to be static environments separated from weather changes experienced on the surface. The high humidity and stability of these subterranean environments make them attractive to many different organisms including microbes such as bacteria and protists. Cave waters generally originate from the surface, may be filtered by overlying soils, can accumulate in interstitial epikarst zones underground, and emerge in caves as streams, pools and droplets on speleothems. Water movement is the primary architect of karst caves, and depending on the hydrologic connectivity between surface and subsurface, is the most likely medium for the introduction of microbes to caves. Recently published metabarcoding surveys of karst cave soils and speleothems have suggested that the vast majority of bacteria residing in these habitats do not occur on the surface, calling into question the role of microbial transport by surface waters. The purpose of this study was to use metabarcoding to monitor the aquatic prokaryotic microbiome of a cave for 1 year, conduct longitudinal analyses of the cave’s aquatic bacterioplankton, and compare it to nearby surface water. Water samples were collected from two locations inside Panel Cave in Natural Tunnel State Park in Duffield, VA and two locations outside of the cave. Of the two cave locations, one was fed by groundwater and drip water and the other by infiltrating surface water. A total of 1,854 distinct prokaryotic ASVs were detected from cave samples and 245 (13.1%) were not found in surface samples. PCo analysis demonstrated a marginal delineation between two cave sample sites and between cave and surface microbiomes suggesting the aquatic bacterioplankton in a karst cave is much more similar to surface microbes than reported from speleothems and soils. Most surprisingly, there was a cave microbe population and diversity bloom in the fall months whereas biodiversity remained relatively steady on the surface. The cave microbiome was more similar to the surface before the bloom than during and afterwards. This event demonstrates that large influxes of bacteria and particulate organic matter can enter the cave from either the surface or interstitial zones and the divergence of the cave microbiome from the surface demonstrates movement of microbes from the epikarst zones into the cave.

Invisible dye tracing – an efficient technique for study of groundwater channels

We studied excitation spectra of uranine fluorescence in eluates from charcoal traps saturated in cave waters and in reference uranine solutions and found that the most appropriate excitation wavelength is 295±5 nm, because it excites fluorescence of uranine many times stronger than fluorescence of natural fluorescent compounds in groundwater. This allows us to lower the detection limit of uranine fluorescence with one order of magnitude, even below the level of natural fluorescence of karst waters at integral UV excitation.

INTERGLACIAL MARKERS FOUND IN USVINSKAYA-1 СAVE (MIDDLE URALS)

Mineral formation due to water freezing in caves causes supersaturation of the unfrozen part of the solution and deposition of some dissolved compounds in the form of minerals. Evaporation of water and degassing of the solution are associated processes in deposition of minerals. Cryogenic minerals of caves differ in morphology and isotopic composition from similar mineral cave formations not subject to glaciation. The morphology and mineralogy of cryogenic cave minerals depend on the initial chemical composition of karst water, the thickness of the freezing layer of water, and also on the freezing rate. Slow freezing of cave waters at 0°C in permafrost conditions leads to the formation of coarse cryogenic calcite. These carbonates can be used as indicators of permafrost thawing during interglacial periods. Morphological and isotopic study of samples from Usvinskaya-1 Cave (Middle Urals), as well as their 230Th / 234U dating, made it possible to conclude that the calcite is cryogenic. The calcite is represented by aggregates (up to 5 cm in size) of split crystals and spherulites from milky brown to black in color. This calcite, formed during the interglacial periods, indicates the existence of several periods of degrading permafrost in the Middle Urals, namely in the periods MIS9 (295.7 thousand years ago), MIS7 (205.1 thousand years ago), GI23 (approx. 102-104 thousand years ago) and GI22 (89.4-90.0 thousand years ago).

The diversity of annelids in subterranean waters: a case study from Poland

Not all invertebrate groups commonly occur in subterranean waters but annelids live in surface and underground habitats. The annelid species' richness in various underground waters (wells and interstitial and cave waters) and surface streams of Poland was compared, and the habitat preferences for the most frequent species were determined. Until now, 111 annelid taxa (mainly oligochaetes) had been identified in underground waters in Poland, with higher numbers (71) in the interstitial habitat than in stream bottoms (62). The number of species identified in the caves and wells was distinctly lower (54 and 29, respectively). The Correspondence Analysis did not separate the samples from various underground water types into distinct groups, and the distribution of well fauna was especially scattered (in the ordination diagram) because abiotic parameters differ strongly in studied wells. Only three stygobiontic species (Cernosvitoviella parviseta, Enchytraeus dominicae and Trichodrilus moravicus) were related to some caves. The analysis of the available data indicate that to obtain a comprehensive picture of the aquatic fauna in a given country all types of subterranean aquatic habitats should be sampled and taken into account. Moreover, to ascertain the composition of benthic invertebrates in running waters, investigation of the interstitial habitat should also be performed.

Comparative genomics analyses indicate differential methylated amine utilisation trait within members of the genusGemmobacter

Methylated amines are ubiquitous in the environment and play a role in regulating the earth’s climate via a set of complex biological and chemical reactions. Microbial degradation of these compounds is thought to be a major sink. Recently we isolated a facultative methylotroph,Gemmobactersp. LW-1, an isolate from the unique environment Movile Cave, Romania, which is capable of methylated amine utilisation as a carbon source. Here, using a comparative genomics approach, we investigate how widespread methylated amine utilisation trait is within the member of the bacterial genusGemmobacter. Five genomes of differentGemmobacterspecies isolated from diverse environments, such as activated sludge, fresh water, sulphuric cave waters (Movile Cave) and the marine environment were available from the public repositories and used for the analysis. Our results indicate that some members of the genusGemmobacter, namelyG. aquatilis, G. caeniandG. sp. LW-1 have the genetic potential of methylated amine utilisation while others (G. megateriumandG. nectariphilus) have not.