Anchialine Research Articles

Genome-centric metagenomes unveiling the hidden resistome in an anchialine cave

BackgroundAntibiotic resistance is a critical global concern, posing significant challenges to human health and medical treatments. Studying antibiotic resistance genes (ARGs) is essential not only in clinical settings but also in diverse environmental contexts. However, ARGs in unique environments such as anchialine caves, which connect both fresh and marine water, remain largely unexplored despite their intriguing ecological characteristics.ResultsWe present the first study that comprehensively explores the occurrence and distribution of ARGs and mobile genetic elements (MGEs) within an anchialine cave. Utilizing metagenomic sequencing we uncovered a wide array of ARGs with the bacitracin resistance gene, bacA and multidrug resistance genes, being the most dominant. The cave’s microbial community and associated resistome were significantly influenced by the salinity gradient. The discovery of novel β-lactamase variants revealed the cave’s potential as a reservoir for previously undetected resistance genes. ARGs in the cave demonstrated horizontal transfer potential via plasmids, unveiling ecological implications.ConclusionsThese findings highlight the need for further exploration of the resistome in unique environments like anchialine caves. The interconnected dynamics of ARGs and MGEs within anchialine caves offer valuable insights into potential reservoirs and mechanisms of antibiotic resistance in natural ecosystems. This study not only advances our fundamental understanding but also highlights the need for a comprehensive approach to address antibiotic resistance in diverse ecological settings.

Water Sample Collection Methods for 222Rn Analysis by Scuba Diving: Insights on Groundwater Flushing of Anchialine Cave Systems of the Yucatan Peninsula

AbstractRadon (222Rn) is a widely used tracer in groundwater and surface water studies. In some applications of radon, samples need to be collected from deep submerged areas or discharge points such as caves or springs, locations typically accessed by scuba diving. However, there are no established sampling methods for collecting water by scuba diving for Rn analysis. We assessed four underwater sample collection methods: (a) injection into a catheter bag followed by transfer to a bottle after the dive, (b) air purging and then filling of a bottle by allowing gas to escape, (c) air purging followed by gas escape, and then overfilling of water with a syringe, and (d) collection with a syringe followed by transfer to a bottle. The samples were collected from the anchialine caves of the Yucatan Peninsula which are the longest underwater cave systems. The samples were analyzed using the same Rn‐in‐air analyzer and protocols. Statistical comparison of sampling at the same location with different methods showed no significant differences in Rn activity. No collection method is superior in terms of measurement quality; operational simplicity is thus preferred. Statistically significant differences in activity were observed between fresh and saline samples from the same cave, with higher activities in the fresh samples, regardless of sampling method. The saline groundwater therefore has a lower residence time, suggesting vigorous landwards flow. Our assessment shows that in situ sampling of discrete water samples for Rn tracing by divers is a useful and powerful approach allowing the study of otherwise impossible sites.

Exploring the microbial community and biotechnological potential of the sponge Xestospongia sp. from an anchialine cave in the Yucatán Peninsula

Sponge-associated microorganisms are key influencers of nutrient biogeochemistry and important sources of bioactive natural products. This study provides the first insights into the taxonomic diversity of the microbial community associated with the sponge Xestospongia sp. from an anchialine cave in the coastal area of the underground river of the Yucatán Peninsula in Xcalak, Quintana Roo, Mexico, and the potential antimicrobial activity of its cultivable bacteria. High abundances of Sulfurospirillum and Desulfovibrio were detected with 16S rRNA amplicons, suggesting that the microbial community of Xestospongia sp. plays an important role in the geochemical sulfur cycle. Analysis with crude extracts of Nocardiopsis dasonvillei NCA-454 revealed antimicrobial activity against methicillin-sensitive Staphylococcus aureus ATCC 25913 (MSSA) and methicillin-​resistant ​S. aureus ATCC 43300 (MRSA). Studies of the microbiomes of sponges from the anchialine cave system in the Yucatán Peninsula can help elucidate the biogeochemical cycles of these poorly studied environments. Moreover, the microorganisms of these microbial communities represent an untapped source of secondary metabolites with biotechnological potential.

Abstract 1695 Investigating Antimicrobial Properties of Microbes Collected from Anchialine Pools

Abstract 1695 Investigating Antimicrobial Properties of Microbes Collected from Anchialine Pools

Morphology, Phylogeny, and Evolution of the Rarely Known Genus Admetella McIntosh, 1885 (Annelida, Polynoidae) with Recognition of Four New Species from Western Pacific Seamounts

The polynoid genus Admetella constitutes a deep‐sea assemblage of polychaetes, notable for their large bodies adorned with antennal scales positioned dorsally to the bases of lateral antennae. Furthermore, the genus exhibits swimming proficiencies facilitated by elongated parapodia and flattened chaetae. Despite the frequent encounters with Admetella members during various deep‐sea explorations, a substantial gap in our comprehension of their diversity, phylogeny, and evolutionary trajectories still exists. Our thorough morphological and phylogenetic investigations of specimens obtained from three seamounts located in the tropical western Pacific have unveiled six species belonging to the genus Admetella, four of these being newly identified as Admetella multiseta sp. nov., A. levensteini sp. nov., A. nanhaiensis sp. nov., and A. undulata sp. nov. The other two species of Admetella remain unidentifiable at the species level due to the loss of crucial details. Our phylogenetic analysis, grounded on 13 mitochondrial protein‐coding genes and the inclusion of 12S, 16S, 18S, 28S rRNA, and ITS1–ITS2 genes, substantiates the monophyly of Admetella. Admetella is positioned at an intermediate node within the phylogenetic tree, situated between representative shallow‐water and deep‐sea subfamilies. The independent evolution of antennal scales within Admetella among polynoids constitutes a synapomorphy for this genus. Ancestral state reconstruction (ASR) analyses suggest that deep‐sea polynoids evolved from shallow‐water ancestors that possessed lateral antennae, which were subsequently lost in members inhabiting extreme marine environments, such as deep‐sea hydrothermal vents and anchialine caves. The analysis further implicates that swimming ability independently evolved at least four times within the Polynoidae family.

Shedding light on the Ophel biome: the trans-Tethyan phylogeography of the sulfide shrimp Tethysbaena (Peracarida: Thermosbaenacea) in the Levant.

Tethysbaena are small peracarid crustaceans inhabiting extreme environments such as subterranean lakes and thermal springs, represented by endemic species found around the ancient Tethys, including the Mediterranean, Arabian Sea, Mid-East Atlantic, and the Caribbean Sea. Two Tethysbaena species are known from the Levant: T. relicta, found along the Dead Sea-Jordan Rift Valley, and T. ophelicola, found in the Ayyalon cave complex in the Israeli coastal plain, both belonging to the same species-group based on morphological cladistics. Along the biospeleological research of the Levantine subterranean fauna, three biogeographic hypotheses determining their origins were proposed: (1) Pliocenic transgression, (2) Mid-late Miocenic transgression, and (3) The Ophel Paradigm, according to which these are inhabitants of a chemosynthetic biome as old as the Cambrian. Tethysbaena specimens of the two Levantine species were collected from subterranean groundwaters. We used the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear ribosomal 28S (28S rRNA) gene to establish the phylogeny of the Levantine Tethysbaena species, and applied a molecular clock approach for inferring their divergence times. Contrary to the morphological cladistic-based classification, we found that T. relicta shares an ancestor with Tethysbaena species from Oman and the Dominican Republic, whereas the circum-Mediterranean species (including T. ophelicola) share another ancestor. The mean age of the node linking T. relicta from the Dead Sea-Jordan Rift Valley and Tethysbaena from Oman was 20.13 MYA. The mean estimate for the divergence of T. ophelicola from the Mediterranean Tethysbaena clade dated to 9.46 MYA. Our results indicate a two-stage colonization of Tethysbaena in the Levant: a late Oligocene transgression, through a marine gulf extending from the Arabian Sea, leading to the colonization of T. relicta in the Dead Sea-Jordan Rift Valley, whereas T.ophelicola, originating from the Mesogean ancestor, inhabited anchialine caves in the coastal plain of Israel during the Mid-Miocene.

Amphipods in Mediterranean Marine and Anchialine Caves: New Data and Overview of Existing Knowledge

Marine and anchialine caves host specialized faunal communities with a variable degree of endemism and functional specialization. However, biodiversity assessments on this habitat are scarce, particularly in relation to small-sized cryptic fauna (such as amphipods), which often play a key role in benthic ecosystems. The present article compiles all records of marine and brackish-water amphipods inhabiting marine and anchialine caves along the Mediterranean basin, combining information extracted from a literature review with newly acquired records. A total of 106 amphipod species has been reported (representing approximately 20% of the Mediterranean amphipod species), mostly from the North-Western Mediterranean. Examination of new material from marine caves in Greece has yielded 14 new records from the East Ionian and Aegean Sea. Most of the reported species display wide ecological amplitude in terms of habitat and substrate preferences, feeding habits as well as bathymetric and geographical distribution. In contrast, only 17 amphipod species have been reported from marine-brackish waters in anchialine caves, predominantly represented by cave specialists with a narrow spatial distribution and distinct morphological traits. Our overall knowledge on amphipods inhabiting Mediterranean caves is far from complete so that new and valuable findings are expected to occur as new caves are explored.

A new species of free-living marine nematode, Fotolaimus cavus sp. nov. (Nematoda, Oncholaimida, Oncholaimidae), isolated from a submarine anchialine cave in the Ryukyu Islands, southwestern Japan

Fotolaimus cavussp. nov. was described from a submarine anchialine cave called Akuma-no-yakata on the Shimoji Island, Miyako Island Group, Ryukyu Islands, southwestern Japan. This is the first free-living marine nematode isolated from a submarine cave in Japan, and the third species of the genus Fotolaimus. This new species differs from its congeners by its small body size, wide amphids, long buccal cavity, long conico-cylindrical tail, and proximally curved gubernaculum. We provide amended dichotomous keys to genera in the subfamily Oncholaiminae and species in Fotolaimus. We also analyzed partial DNA sequences encoding ribosomal small subunit RNA and cytochrome c oxidase subunit I from Fotolaimus cavussp. nov. and six other species of Oncholaimidae collected from Japanese waters. The phylogenetic tree based on the ribosomal small subunit RNA sequences using maximum likelihood analysis suggested a close relationship between Fotolaimus and Wiesoncholaimus as well as Oncholaimus. The topology of the tree was similar to those from previous studies; however, it suggested a new phylogenetic position of Adoncholaimus as a sister clade for Viscosia and Oncholaimus.

Anchialine pool shrimp (Halocaridina rubra) as an indicator of sewage in coastal groundwater ecosystems on the island of Hawai'i.

Groundwater is a primary pathway for wastewater and other pollutants to enter coastal ecosystems worldwide. Sewage associated pathogens, pharmaceuticals, and other emerging contaminants pose potential risks to marine life and human health. Anchialine pool ecosystems and the endemic species they support are at risk and provide an opportunity to sample for presence of contaminants prior to diffusion in the marine environment. In this study, we tested the potential use of nitrogen isotopes in the tissues of a dominant anchialine pool grazing shrimp (Halocaridina rubra), as a bioindicator for sewage in groundwater flowing through their habitats. Water quality parameters and shrimp tissue isotopes (N and C) were collected from pools exposed to a range of sewage contamination along the West Hawai'i coastal corridor from 2015 to 2017. Data were used to test for spatial and temporal variability both within and among pools and to examine the relationship between stable isotopes and water quality parameters. Within 22 pools, mean δ15N from whole tissue samples ranged between 2.74‰ and 22.46‰. Variability of isotope values was low within individual pools and within pool clusters. However, δ15N differed significantly between areas and indicated that sewage is entering groundwater in some of the sampled locations. The significant positive relationship between δ15N and dissolved nitrogen (p<0.001, R2 = 0.84) and δ15N and phosphorus (p<0.001, R2 = 0.9) support this conclusion. In a mesocosm experiment, the nitrogen half-life for H. rubra tissue was estimated to be 20.4 days, demonstrating that the grazer provides a time-integrative sample compared to grab-sample measurements of dissolved nutrients. Ubiquitous grazers such as H. rubra may prove a useful and cost-effective method for δ15N detection of sewage in conjunction with standard monitoring methods, enabling sampling of a large number of pools to establish and refine monitoring programs, especially because anchialine habitats typically support no macroalgae.

A glimpse into the dark – the bacterial and archaeal diversity of tropical anchialine cave sediments

Using ion torrent sequencing of the V4 region of the 16S rRNA gene, we investigated the sediment from four anchialine caves on Eleuthera (Bahamas). Anchialine caves are secluded, landlocked marine ecosystems, with high degrees of endemism. In the cave sediments, the bacterial and archaeal communities differ from marine communities and between caves, while often resembling those from low oxygen or anaerobic marine sediments. The waters of all caves investigated here were mostly saline and oxygenated, with Windermere Abyss differing from the other caves. This was mirrored by the bacterial and archaeal communities, with members of the wb1-A12 clade, Nitrosopumilaceae , members of the NB1-j phylum, and Actinomarinales being dominant in Preacher’s Blue Hole, Valentine’s Cave, and Bung Hole while Desulfatiglans , Bathyarchaeia, and members of the GIF3 and Sva0485 clades were dominant in Windermere Abyss. All communities showed taxa found in oxygenated and anoxic sediments, thus indicating a variety of chemoautotrophic lifestyles, including methane-, nitrogen- and sulphur cycling. The communities were different from other caves and included understudied or otherwise rare taxa, partially from deep sea sediments, implying that the isolation might be an evolutionary factor, and indicating that anchialine caves could be a window into the deep sea.

UNDERGROUND HERITAGE DOCUMENTATION: THE CASE STUDY OF GROTTA ZINZULUSA IN CASTRO (LECCE-ITALY)

Abstract. 3D documentation of the Underground Built Heritage (UBH) is always fundamental for knowledge, management, conservation, and enhancement of cultural heritage, but the challenges involved in documenting this kind of site are many and various, With the aim of providing an accurate and reliable representation of the surveyed area without employing time-consuming processes, Mobile Mapping Systems (MMSs) based on SLAM (Simultaneous Localization And Mapping) technology have now the potential to overcome these challenges, proving to be a useful alternative to traditional surveying techniques. The underground site of Grotta Zinzulusa in Castro (LE), Italy, is an anchialine cave system difficult to access and document due to its narrow passages, large rooms, and poor illumination. Within this case study, the effectiveness of using an MMS approach, employing the STONEX® X120GO SLAM Laser Scanner system, to document the cave system and integrates the data with those obtained from other traditional geomatics techniques, such as UAS (Uncrewed Aerial System) and TLS (Terrestrial Laser Scanning) surveys, has been proved. The study aims to define the best practices and operational methodologies for enhancing the speediness, usability, and cost-effectiveness of using MMSs in underground settings for 3D documentation of heritage sites to provide metrically correct and accurate products for researchers, scientists, and the wider public.

New lithistid sponge of the genus Sollasipelta (Porifera, Demospongiae,Tetractinellida, Neopeltidae) from submarine caves of the Ryukyu Islands,southwestern Japan, with redescription of S. sollasi.

A new species of 'lithistid' (rock sponge) Sollasipelta subterranea sp. nov. is described from near-shore submarine caves of Okinawa and Shimoji Islands, the Ryukyu Islands, southwestern Japan. This new species is characterized by the smooth dentate ectosomal pseudophyllotriaenes, slender choanosomal oxea/style/subtylostyles and two types of amphiasters, one of which is in two size classes. Sollasipelta subterranea sp. nov. is morphologically closest to Sollasipelta sollasi (Lévi & Lévi, 1989), known from the Philippines deep sea, and our examination of the type specimen of S. sollasi revealed that the pseudophyllotriaenes and desmas are substantially different between the two species, and both species possess two types of amphiasters, one of which has two size classes. Sollasipelta cavernicola (Vacelet & Vasseur, 1965) and S. punctata (Lévi & Lévi, 1983) are transferred to the genus Daedalopelta Sollas, 1888, based on possession of one type of amphiastes as microscleres. Sollasipelta mixta (Vacelet, Vasseur and Lévi, 1976) is formally transferred to the genus Neopelta Schmidt, 1880, based on the morphological characters. A key to species of the genus Sollasipelta Van Soest & Hooper, 2020 is also provided. Sollasipelta subterranea sp. nov. represents the first 'lithistid' sponge from submarine caves of the Western Pacific and the first species of sponge inhabiting anchialine cave environments in the Indo-West Pacific. This is also a new record of the family Neopeltidae from Japanese waters.

A transcriptome-based phylogeny for Polynoidae (Annelida: Aphroditiformia)

Polynoidae is the most diverse radiation of Aphroditiformia and one of the most successful groups of all Annelida in terms of diversity and habitats colonized. With such an unmatched diversity, phylogenetic investigations have struggled to understand their evolutionary relationships. Previous phylogenetic analyses have slowly increased taxon sampling and employed methodologies, but despite their diversity and biological importance, large genomic sampling is limited. To investigate the internal relationships within Polynoidae, we conducted the first phylogenomic analyses of the group based on 12 transcriptomes collected from species inhabiting a broad array of habitats, including shallow and deep waters, as well as hydrothermal vents, anchialine caves and the midwater. Our phylogenomic analyses of Polynoidae recovered congruent tree topologies representing the clades Polynoinae, Macellicephalinae and Lepidonotopodinae. Members of Polynoinae and Macellicephalinae clustered in well-supported and independent clades. In contrast, Lepidonotopodinae taxa were always recovered nested within Macellicephalinae. Though our sampling only covers a small proportion of the species known for Polynoidae, our results provide a robust phylogenomic framework to build from, emphasizing previously hypothesized relationships between Macellicephalinae and Lepidonotopodinae taxa, while providing new insights on the origin of enigmatic cave and pelagic lineages.

Metabetaeus lapillicola, a new species of alpheid shrimp (Decapoda: Caridea) from Japan, inhabiting unusual pebble beaches.

The alpheid shrimp genus Metabetaeus Borradaile, 1899 was previously known from two species associated with anchialine pools and caves, M. minutus (Whitelegge, 1897) and M. lohena Banner & Banner, 1960, and one species inhabiting in coral reef, M. mcphersonae Anker, 2010. In this study, a new species of the genus is described based on fourteen specimens collected from Shizuoka and Mie Prefectures, Honshu, central Japan. Metabetaeus lapillicola n. sp. inhabits interstitial spaces among small pebbles deposited in lower intertidal to upper subtidal zone, representing rather unusual habitat not only for Metabetaeus, but even for carideans. It is distinguished from three congeneric species by the presence of a pterygostomial spine on the carapace, greatly unequal chelipeds with the major one being substantially elongate, and the lack of a distinct distolateral spine on the uropodal endopod. Phylogenetic analyses using the mitochondrial 16S rRNA and COI genes support the recognition of the new species. The generic diagnosis of Metabetaeus is emended to accommodate the new species.

New sea spider species (Pycnogonida: Austrodecidae) from a submarine cave in Japan

Abstract We describe a new sea spider species, Pantopipetta hosodai sp. nov., based on one juvenile female collected from a submarine cave (‘Akumanoyakata’ Cave) in Shimojijima Island, Miyako Island Group, Ryukyu Islands, southwestern Japan. It was collected from the second slope zone of the cave, 80–100 m from the entrance, no light, low salinity and with rocky substrate. This is the first record globally of a Pantopipetta species from a submarine cave and anchialine environment. Pantopipetta hosodai sp. nov. resembles Pantopipetta auxiliata, Pantopipetta lenis and Pantopipetta oculata in having auxiliary claws, but differs from them in having a palp with three short distal articles, lateral processes without dorsodistal tubercles, coxae 1 and 3 of legs 1–3 each with one long dorsal tubercle and one dorsodistal tubercle bearing a seta on each femur. Features of the palp appear to delineate two species groups in Pantopipetta, i.e. (1) those having four small distal articles, and a small, basal palp article between the lateral cephalon process and longest palp article (eight-articulate palp) and (2) those having three small distal articles, and lacking the small basal article (six-articulate palp), but further detail examination of the described species is needed. We discuss the diagnostic characters separating Pantopipetta and Austrodecus and the generic affiliation of Austrodecus aconae. Few pycnogonids from marine or anchialine caves have been identified to species, and it is generally unknown whether cave-dwelling pycnogonids tend to be troglobites.

Carbon dioxide (CO2) gas and eDNA assessment as tools for eradicating and monitoring invasive fish in anchialine pools in Hawai‘i

Carbon dioxide (CO2) gas and eDNA assessment as tools for eradicating and monitoring invasive fish in anchialine pools in Hawai‘i

Functional eye rudiments in the anchialine crab Munidopsis polymorpha (Crustacea: Galatheidae)

Abstract The white crab Munidopsis polymorpha (Crustacea: Galatheidae) is the only one of a species-rich genus not occurring in the deep sea. It is endemic to marine caves, anchialine pools and groundwaters of the volcanic island of Lanzarote (Canary Islands). The eye derives from the superposition type possessing a clear zone formed by the proximal extensions of the rudimentary crystalline cones. The number of ommatidia ranges among the lowest found in decapods and the optic ganglia are strongly diminished in size. The crystalline cones are rudimentary or lost and facets with individual corneas are not discernible. The rhabdoms are tiny and irregularly arranged in the rhabdom layer. Some rhabdoms display an open arrangement of rhabdomers with microvilli that are not aligned to form orthogonal layers. The eye is one of the rare exceptions of traits that despite being rudimentary is still partially functional. The absence of stabilizing selection is probably the reason for variability or reduction of the traits necessary for image formation. In contrast, negative phototactic behaviour is present, because light is still perceived by the eyes of M. polymorpha.

Discovery of a new scale worm (Annelida: Polynoidae) with presumed deep-sea affinities from an anchialine cave in the Balearic Islands (western Mediterranean)

Abstract A remarkable new genus and species of scale worm (Annelida: Polynoidae) was found on the bottom sediments of an anchialine cave on the island of Mallorca (Balearic Islands, western Mediterranean). Specimens reach up to 2 cm long, lack eyes and body pigmentation except for a few scattered minute speckles and show enlarged parapodia and sensorial appendages. A red brain is visible through the translucent tegument. Morphological features resemble those of Eulagiscinae, currently comprising eight species in three genera. Phylogenetic analyses of mitochondrial and nuclear DNA sequences are not conclusive on the position of the new taxon but affinity to Eulagiscinae is not ruled out, particularly when taxa with missing data or non-homologous insertion sites are excluded from the analyses. Pollentia perezi gen. &amp; sp. nov. is characterized by a unique set of morphological features: 13 pairs of dorsal elytra; a single type of notochaetae (stout, with spinous rows and pointed tip); and two types of neurochaetae (superior flattened, spinous with tridentate tip; inferior shorter and thinner, lanceolate and pectinate). Some characteristics, such as the long parapodial appendages and swimming habits, are shared with other cave scale worms. However, the new taxon is not closely related to the other two known cave-dwelling polynoids.

Overcoming shortfalls and impediments in subterranean biology: a challenge for the future

Since the discovery and description of the first specialized cave species, subterranean fauna stimulated the scientific research of several generations of speleobiologists especially after the publication of Racovitza's classification of cave species, which is still used today, amended for non-karstic areas and groundwaters. More than 28,000 obligate subterranean species are known worldwide; however, these figures are likely to be underestimated since species richness is highly correlated with research effort (Stoch and Galassi 2010). Subterranean ecosystems are very rich in strict endemic species and taxonomic efforts are still quite low (i.e., the so-called "taxonomic impediment"), while several environments are very difficult to be explored (i.e., the "Racovitzan impediment: Ficetola et al. 2018). Furthermore, several paradigms were debated for years. Albeit constrasting hypotheses were conceived to explain the colonization of subsurface habitats, their importance is still debated (i.e., climate relicts vs. adaptive shift in colonization and speciation, dispersal vs. vicariance in shaping distributional patterns, and selective vs. neutral hypotheses in explaining regressive evolution). Moreover, the paradigm of a "truncated functional diversity" of subterranean ecosystems (Gibert and Deharveng 2002) lasted for years but was recently challenged by the discovery of chemoautotrophic ecosystems in hypogenic and anchialine caves, and the recognition that caves are not isolated environments, but they are highly interconnected with surface ecosystems. The increased importance of their conservation (like in the case of GDEs, Groundwater Dependent Ecosystems) was recently recognized, together with their provision of important ecosystem services (Boulton 2020). A deeper knowledge is required to assess biodiversity hotspots as well as to plan efficient monitoring surveys (Mammola et al. 2020). In the last decades, a growing amount of molecular data has been obtained for subterranean species, allowing some of the classical debates on colonization, evolution, and dispersal to be revisited (Bauzà-Ribot et al. 2012); moreover, novel promising techniques like metabarcoding and environmental DNA were applied in field surveys and monitoring efforts. Unfortunately, after more than one century of research in subterranean biology, large gaps remain in our knowledge of phylogeny, richness, and distribution of subterranean fauna (formalized in the so-called Darwinian, Linnean and Wallacean shortfalls), preventing the definition of large-scale sound management and protection plans. It is proposed that data from recent biomolecular techniques coupled with remotely sensed data may enhance biodiversity mapping and conservation and are promising approaches to fill our knowledge gaps. Perhaps this is the greatest challenge that tomorrow's subterranean biologists will face.

Screening of NaCl salinity sensitivity across eight species of subterranean amphipod genus Niphargus

Secondary salinization of freshwater is becoming a growing environmental problem. Currently, there is few data available on the effects of salinisation on subterranean crustaceans that are vital for the maintenance of groundwater ecosystem functioning. In this study, the sensitivity of subterranean Niphargus amphipods to NaCl was investigated. We expected that cave-dwelling species would be more sensitive as surface-subterranean boundary species. Eight ecologically different Niphargus species were tested: four live at the boundary between the surface and subterranean ecosystems (N. timavi, N. krameri, N. sphagnicolus, N. spinulifemur), three live in cave streams (N. stygius, N. scopicauda, N. podpecanus), and one species (N. hebereri) lives in anchialine caves and wells. The organisms were exposed to five concentrations of NaCl for 96 h and afterwards the immobility, mortality, and electron transfer system (ETS) activity (a measure for metabolic rate of animals) were evaluated. As expected, the most tolerant species was N. hebereri dwelling in naturally high-salinity habitat. However, contrary to our expectations, the species collected at the surface-subterranean boundary were more sensitive as cave stream species when their immobility and mortality were assessed. Interestingly, the majority of Niphargus tested were more NaCl tolerant as can be deduced from currently available data for subterranean and surface crustaceans. We could not observe a clear trend in ETS activity changes between groups of surface-subterranean boundary and cave streams species after exposure to NaCl stress, but it appears that osmotic stress-induced metabolic rate changes are species-specific. This study shows that amphipods Niphargus can be a valuable subterranean environmental research model and further ecotoxicity research is of interest.