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

BACKGROUND: Antibiotic 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. RESULTS: We 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 ß-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. CONCLUSIONS: These 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.

Microbial consortium involved in ferromanganese and francolite biomineralization in an anchialine environment (Zinzulùsa Cave, Castro, Italy).

Tidally-influenced subterranean settings represent natural geomicrobiological laboratories, relatively unexplored, that facilitate the investigation of new biomineralization processes. The unusual water chemistry of Zinzulùsa Cave and its oligotrophic and aphotic conditions have allowed the development of a unique ecosystem in which complex bacterial activities induce rare biomineralization processes. A diversified microbial community develops on centimeter-thick crusts that form in the submerged part of the cave. The crusts are formed of Ca-phosphate minerals, mostly carbonate-fluoroapatite (francolite), covered by a black crust, few microns in thickness, composed of ferromanganiferous oxides (hematite and vernadite). Diffuse coccoidal and filamentous bacteria and amorphous organic matter are mixed with the minerals. The micromorphologies and comparative 16S rRNA gene-based metabarcoding analyses identify a "core microbiota" also common to other natural environments characterized by FeMn and Ca-phosphate mineralization. The microbiota is characterized by nitrifying, sulfide/sulfur/thiosulfate-oxidizing and sulfate/thiosulfate/sulfur-reducing bacteria. In addition, manganese-oxidizing bacteria include the recently described "Ca. Manganitrophus noduliformans" and an abundance of bacteria belonging to the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum, as well as Haliangiales (fruiting body-forming bacteria) and Hyphomicrobiales (stalked and budding bacteria) that are known to produce extracellular polymers that trap iron and manganese oxides. 16S rRNA gene metabarcoding analysis showed the presence of bacteria able to utilize many organic P substrates, including Ramlibacter, and SEM images revealed traces of fossilized microorganisms resembling "cable bacteria", which may play a role in Ca-phosphate biomineralization. Overall, the data indicate biomineralization processes induced by microbial metabolic activities for both ferromanganiferous oxide and francolite components of these crusts.

Exploring virus-host-environment interactions in a chemotrophic-based underground estuary.

BACKGROUND: Viruses play important roles in modulating microbial communities and influencing global biogeochemistry. There is now growing interest in characterising their ecological roles across diverse biomes. However, little is known about viral ecology in low-nutrient, chemotrophic-based environments. In such ecosystems, virus-driven manipulation of nutrient cycles might have profound impacts across trophic levels. In particular, anchialine environments, which are low-energy underground estuaries sustained by chemotrophic processes, represent ideal model systems to study novel virus-host-environment interactions. RESULTS: Here, we employ metagenomic sequencing to investigate the viral community in Bundera Sinkhole, an anchialine ecosystem rich in endemic species supported by microbial chemosynthesis. We find that the viruses are highly novel, with less than 2% representing described viruses, and are hugely abundant, making up as much as 12% of microbial intracellular DNA. These highly abundant viruses largely infect important prokaryotic taxa that drive key metabolic processes in the sinkhole. Further, the abundance of viral auxiliary metabolic genes (AMGs) involved in nucleotide and protein synthesis was strongly correlated with declines in environmental phosphate and sulphate concentrations. These AMGs encoded key enzymes needed to produce sulphur-containing amino acids, and phosphorus metabolic enzymes involved in purine and pyrimidine nucleotide synthesis. We hypothesise that this correlation is either due to selection of these AMGs under low phosphate and sulphate concentrations, highlighting the dynamic interactions between viruses, their hosts, and the environment; or, that these AMGs are driving increased viral nucleotide and protein synthesis via manipulation of host phosphorus and sulphur metabolism, consequently driving nutrient depletion in the surrounding water. CONCLUSION: This study represents the first metagenomic investigation of viruses in anchialine ecosystems, and provides new hypotheses and insights into virus-host-environment interactions in such 'dark', low-energy environments. This is particularly important since anchialine ecosystems are characterised by diverse endemic species, both in their microbial and faunal assemblages, which are primarily supported by microbial chemosynthesis. Thus, virus-host-environment interactions could have profound effects cascading through all trophic levels.

Response and resilience of karst subterranean estuary communities to precipitation impacts.

The impact of meteorological phenomena on ecosystem communities of karst subterranean estuaries (KSEs) remains unknown. KSEs are characterized by vertically stratified groundwater separated by a halocline and host endemic aquatic cave-adapted fauna (stygobionts). In October 2015, 8 days of heavy precipitation caused the first recorded mortality event in the KSE. This event was marked by a halocline shift 5 m deeper. The present study aimed to provide insights into resilience of KSEs faunal communities to temporal shifts in temperature and precipitation. Cave water temperature decreased on average 0.0068°C per mm of accumulated precipitation over 4 days, which can add up to, and surpass, the interannual temperature variation in cases of heavy precipitations. Biological surveys (2012-2021) conducted within cave systems El Aerolito and La Quebrada, in Cozumel, indicated that change in community structure was not detected and stygobionts were resilient; however, marine species inhabiting the caves were impacted. Overall, the faunal community at KSEs remains resilient within short-term meteorological phenomena despite shifts of non-stygobionts.

El impacto de fenómenos meteorológicos en las comunidades de los ecosistemas de estuarios subterráneos kársticos (KSE, por sus siglas en inglés) sigue siendo desconocido. Los KSE se caracterizan por aguas subterráneas estratificadas verticalmente separadas por una haloclina, y albergan fauna acuática endémica adaptada a la vida en cuevas (estigobiontes). En octubre de 2015, ocho días de fuertes precipitaciones causaron el primer evento de mortandad registrado en un KSE. Este evento estuvo marcado por un desplazamiento de la haloclina, resultando en una haloclina 5 m más profunda. El presente estudio tuvo como objetivo proveer información sobre la resiliencia de las comunidades faunísticas de los KSE a cambios temporales en la temperatura y la precipitación. La temperatura del agua de la cueva disminuyó en promedio 0.0068°C por mm de precipitación acumulado durante cuatro días, lo cual puede alcanzar, e incluso superar, la variación interanual de la temperatura debido a fuertes precipitaciones. Los estudios biológicos (2012­2021) realizados dentro de los sistemas de cuevas El Aerolito y La Quebrada, en Cozumel, indicaron que no se detectó un cambio en la estructura de la comunidad y que los estigobiontes fueron resilientes, sin embargo, las especies marinas que habitan las cuevas se vieron afectadas. En general, la comunidad de fauna de KSEs se mantiene resiliente a los fenómenos meteorológicos a corto plazo a pesar de los cambios en la fauna no estigobia.

Unravelling unknown cyanobacteria diversity linked with HCN production.

Cyanobacteria are ecologically versatile microorganisms, occupying diverse habitats, from terrestrial caves to coastal shores and from brackish lakes to thermal springs. Cyanobacteria have also been linked with hydrogen cyanide (HCN), mainly for their ability to catabolize HCN by the nitrogenase enzyme. In this context, we sampled disparate environments, spanning from Canary Islands and Iceland to Estonia and Cyprus. Eighty-one (81) strains were isolated and characterised with taxonomic indices and molecular markers (16S-23S rRNA region and cpcBA region), whilst their ability to produce HCN was evaluated. This approach resulted in the description of five new genera (Speleotes, Haliplanktos, Olisthonema, Speos, and Iphianassa) and their type species (S. anchialus, H. antonyquinny, O. eestii, S. fyssassi, I. zackieohae) representing Chroococcales, Chroococcidiopsales, Oscillatoriales, Synechococcales, and Nostocales orders, respectively. We also found unique lineages inside the genera Komarekiella, Stenomitos, Cyanocohniella, and Nodularia, describing four new species (K. chia, S. pantisii, C. hyphalmyra, N. mediterannea). We report for the first time a widespread production of HCN amongst different taxa and habitats. Epilithic lifestyle, where cyanobacteria are more vulnerable to grazers, had the largest relative frequency in HCN production. In this work, we show novel cyanobacteria diversity from various habitats, including an unexplored anchialine cave, and possible correlation of cyanobacteria chemo- with species diversity, which may have implications on strategic focusing of screening programs on underexploited taxa and/or habitats.

Microeukaryotic and Prokaryotic Diversity of Anchialine Caves from Eastern Adriatic Sea Islands.

Anchialine ecosystems in the eastern Adriatic Sea are diverse both morphologically and biologically. In this study, for the first time, we explored the microeukaryotic and prokaryotic community of anchialine caves in the Mediterranean region using high-throughput sequencing. Four anchialine caves located on nearby islands with a well-pronounced salinity gradient were sampled at the surface freshwater area, halocline area, and seawater area. Sequencing revealed a surprisingly wide diversity of the microeukaryotic and prokaryotic community with the relative abundance of major phyla differing within the salinity gradient and between the caves. Interestingly, microeukaryotic and prokaryotic communities clustered into four groups based on location, pointing out that sampled anchialine caves have different microbial community patterns and high microbial endemism. Our results indicate that even with the halocline acting as a selecting barrier, the salinity is not the only community structuring factor. Despite the short geographical distance, the isolation of anchialine caves facilitated high microbial community adaptation and endemism. Our study suggests that anchialine caves represent reservoirs of new biodiversity, maintaining unique and complex microbial diversity influenced by biotic interactions and abiotic environmental conditions.

Collecting eco-evolutionary data in the dark: Impediments to subterranean research and how to overcome them.

Caves and other subterranean habitats fulfill the requirements of experimental model systems to address general questions in ecology and evolution. Yet, the harsh working conditions of these environments and the uniqueness of the subterranean organisms have challenged most attempts to pursuit standardized research.Two main obstacles have synergistically hampered previous attempts. First, there is a habitat impediment related to the objective difficulties of exploring subterranean habitats and our inability to access the network of fissures that represents the elective habitat for the so-called "cave species." Second, there is a biological impediment illustrated by the rarity of most subterranean species and their low physiological tolerance, often limiting sample size and complicating laboratory experiments.We explore the advantages and disadvantages of four general experimental setups (in situ, quasi in situ, ex situ, and in silico) in the light of habitat and biological impediments. We also discuss the potential of indirect approaches to research. Furthermore, using bibliometric data, we provide a quantitative overview of the model organisms that scientists have exploited in the study of subterranean life.Our over-arching goal is to promote caves as model systems where one can perform standardized scientific research. This is important not only to achieve an in-depth understanding of the functioning of subterranean ecosystems but also to fully exploit their long-discussed potential in addressing general scientific questions with implications beyond the boundaries of this discipline.

New arietellid copepods (Calanoida, Arietellidae) from anchialine caves in the Eastern Adriatic Sea.

Two new species of calanoid copepods are described; Metacalanus adriaticus sp. nov. from an anchialine cave on Vis Island, and Paramisophria tvrtkovici sp. nov. from Orljak Cave, located in the lower part of River Krka estuary, near the town of Sibenik (Croatia). This is the first report of arietellid copepods found in any anchialine cave along the coast of the Adriatic Sea. In M. adriaticus sp. nov. the antennules are asymmetrical in both sexes (the female left antennule is 18-segmented, right 20-segmented; male left 16-segmented and right 20-segmented); the uniramous fifth legs of the female are 2-segmented; the terminal segment of the fifth leg in both sexes is the longest; and in the male the fifth leg exopod is 2-segmented. In P. tvrtkovici sp. nov. the antennules of both sexes are asymmetrical with the left antennule longer than the right, the female antennule is 21-segmented on both sides; the male left antennule is 19-segmented, the right 21-segmented; the armature of the terminal exopod segment of leg 1 is II, 2 ,2; the male fifth legs have a rudimentary endopod on the left leg, the third exopodal segment is smallest and bears three unequal processes on its outer margin, and the terminal spine is completely separated from the segment. On the right leg the third segment carries two short, unequal processes on its outer distal margin, as well as a long sigmoidal spine which is fused to the segment. It is inferred that, after the last glaciation, these new Arietellids moved out from their southern Adriatic refuge, colonizing first the anchialine habitats of the outer eastern Adriatic islands and then spreading along the coast.

A new species of Rangabradya (Copepoda, Harpacticoida, Ectinosomatidae) from a cave in Satun Province, southern Thailand.

A representative of the family Ectinosomatidae was discovered in a temporary pool in a cave in the Satun Province, southern Thailand. Based on the characteristics of the antennary exopod, armature of the female fifth leg, and configuration of the male sixth leg, a new species of the genus Rangabradya was identified, representing the first record of the genus in the continental waters of Thailand and in Southeast Asia. The new species can be distinguished from Rangabradya indica by the configurations of the fifth and the sixth legs in both sexes, the body ornamentation of the urosomite, and the armature of the mouthparts. These structures show a more primitive state in the new species. Accordingly, a new subgeneric rank in the genus Rangabradya, Siamorangabradya subgen. nov, was established to accommodate the Thai species and Rangabradya (Siamorangabradya) wongkamhaengaesubgen. et sp. nov. was described. Also, the key to all 23 genera of the family was updated.

A new species of the cave-fish genus Lucifuga (Ophidiiformes, Bythitidae), from eastern Cuba.

Recently, a barcoding study and a molecular phylogenetic analysis of the Cuban species of the cave-fish genus Lucifuga Poey, 1858 revealed the existence of different evolutionary lineages that were previously unknown or passed unnoticed by morphological scrutiny (i.e., cryptic candidate species). In the present study, Lucifuga gibarensis is described as a new species restricted to anchialine caves in the northeastern karst region of the main island. The species was earlier described as a variety of Lucifuga dentata, but since the name was introduced as a variety after 1960, it is deemed to be infrasubspecific and unavailable according to the International Code of Zoological Nomenclature Art. 15.2. The new species differs from L. dentata by pigmented eyes vs. eyes absent and lack of palatine teeth vs. present. Lucifuga gibarensis seems to be most similar to the Bahamian species L. lucayana by showing pigmented eyes, 13 or 14 precaudal vertebrae and ten caudal fin rays. However, differs from it by a larger size of the pigmented eye (1.1-1.9 vs. 0.9-1.0% SL) and number of posterior lateral line neuromasts (30-33 vs. 34-35).

New distribution records of subterranean crustaceans from cenotes in Yucatan (Mexico).

New records of 14 stygobiont crustacean species pertaining to six Malacostraca orders from 32 cenotes are presented, with their associated caves of the state of Yucatan, Mexico, together with an individual account for each species. Species composition of most of the investigated cenotes is examined for the first time. A thermosbaenacean and two amphipod species were not formally recorded to the cenote ecosystems of the state of Yucatan prior to our research. Distribution data of a cirolanid isopod previously known only from its type locality is also provided. Barcodes of mitochondrial cytochrome c oxidase subunit I for the reported peracarid species previously lacking this information have been included in present study as tools for species identification and a baseline of further molecular genetic analyses.

Recognizing peripheral ecosystems in marine protected areas: A case study of golden jellyfish lakes in Raja Ampat, Indonesia.

Peripheral marine ecosystems can harbor endemic diversity and attract tourism attention, yet are generally not included in conservation management plans due to their remoteness or inland positioning. A case study in Raja Ampat of seven landlocked marine lakes containing golden jellyfish (Mastigias spp.) was conducted to address the lack of fundamental insights into evolutionary, ecological and social contexts of these ecosystems. An interdisciplinary approach was taken towards identifying the jellyfish lakes as distinct management units in order to incorporate them into existing Marine Protected Areas. Mastigias papua populations showed strong genetic (ϕST: 0.30-0.86) and morphological (F = 28.62, p-value = 0.001) structure among lakes, with putative new subspecies. Risks arising from rapid increase in tourism to Raja Ampat (30-fold since 2007) warrant restrictions on jellyfish lake use. Recommendations are provided for adaptive management and science-based conservation policies for jellyfish lakes across Indonesia.

A new species of Boholina (Crustacea, Copepoda, Calanoida) and a first record for stygobiotic calanoid fauna from a cave in Thailand.

A new species of Calanoida belonging to the genus Boholina Fosshagen & Iliffe, 1989 was found in a freshwater pool within a cave of the Satun province, South Thailand. It is the first record of the genus and of a stygobiotic representative of calanoid fauna in this country. The new species is most similar to B. crassicephala Fosshagen & Iliffe, 1989, based on position of genital pores, structures of P4 and P5 in both sexes, relative length of subapical spine vestige on the male right P5, and shape of the male left P5 endopods. However, this new species is distinguished from its known congeners by: (1) relatively longer distal outer spines on the male right P5 exopods, (2) smaller endopods of the male left P5 and (3) elongated apical spines on the distal exopodal segment of the female P4 and P5. Furthermore, the distinctive characteristic of the Thai Boholina is the presence of inner minute seta on the distal segment of the male right P5 exopod. Detailed descriptions of the new species and a key to all six known species of the genus Boholina is provided.

Tossed 'good luck' coins as vectors for anthropogenic pollution into aquatic environment.

Superstition has it that tossing coins into wells or fountains brings good luck, thereby causing a potential accumulation of microbially contaminated metal particles in the water. Here, we characterized the microbiota and the resistance profile in biofilm on such coins and their surrounding sediments. The study site was a tidal marine lake within a touristic center located in a natural reserve area. Notwithstanding the fact that coin-related biofilms were dominated by typical marine taxa, coin biofilms had specific microbial communities that were different from the communities of the surrounding sediment. Moreover, the communities were different depending on whether the coin were made mainly of steel or of copper. Sequences affiliated with putative pathogens were found on every third coin but were not found in the surrounding sediment. Antibiotic resistance genes (ARGs) were detected on most of the coins, and interestingly, sediments close to the area where coins accumulate had a higher frequency of ARGs. We suggest that the surface of the coins might offer a niche for ARGs and faecal bacteria to survive, and, thus, tossed coins are a potential source and vector for ARGs into the surrounding environment.

A comprehensive taxonomic list of brittle stars (Echinodermata: Ophiuroidea) from submarine caves of the Ryukyu Islands, southwestern Japan, with a description of a rare species, Dougaloplus echinatus (Amphiuridae).

The ophiuroid species listed were collected by benthic faunal surveys from 2016 to 2018 in submarine caves of the Ryukyu Islands, southwestern Japan. The material includes 20 species from 8 families, Amphiuridae, Ophiocomidae, Ophiodermatidae, Ophiolepididae, Ophioleucidae, Ophiomyxidae, Ophionereididae and Ophiotrichidae. This is the first comprehensive report of submarine cave-dwelling ophiuroids from Japanese waters. Also provided is a redescription of a rare species, Dougaloplus echinatus (Ljungman, 1867), with a detailed description of its ossicle morphology. The species has previously been reported from Brazil, Australia, Eastern Indian Ocean to Southern China, in 11-118 m depth, and this is the first record of D. echinatus from Japanese waters. A brief discussion of the scarcity of submarine cave systems, and the need for their conservation, is provided.

A new genus and two new species of sea stars (Family Asterinidae) from Indonesian marine lakes, with notes on habitat and feeding ecology.

A new genus of asterinid asteroid, Limnasterias gen. nov. and two new species, Limnasterias oinops sp. nov. and L. estradivariae sp. nov. are described from two East Kalimantan marine lakes, Indonesia. Limnasterias is differentiated from other asterinid genera by a combination of features: high aspect body with thick tissue covering; extensive papulate area; numerous single papulae in up to five rows at ray sides; boot-shaped inferomarginal plates; appressed superactinal plates and absence of superambulacral plates. The ecology and conservation of these species and their unique habitat are discussed.

Unravelling the moons: review of the genera Paratetilla and Cinachyrella in the Indo-Pacific (Demospongiae, Tetractinellida, Tetillidae).

Paratetillabacca (Selenka, 1867) and Cinachyrellaaustraliensis (Carter, 1886) occur in a broad range of marine environments and are allegedly widely distributed species in the Indo-Pacific. We coin the term 'moon sponges' for these species as they are spherical in shape with numerous porocalices resembling the lunar surface. Both species have a complex taxonomic history with high synonymization, in particular by Burton (1934, 1959). An examination of the junior synonyms proposed by Burton (1934, 1959) was conducted to establish the validity of the names. More than 230 specimens from Naturalis Biodiversity Center were reviewed that belong to the genera Paratetilla and Cinachyrella from marine lakes, coral reefs, and mangroves in Indonesia. The aim of the current study was to untangle the taxonomic history, describe the collection of moon sponges from Indonesia, and develop a key. We extensively reviewed the taxonomic literature as well as holotypes of most of the species synonymized by Burton. The taxonomic history of Paratetilla spp. and Cinachyrellaaustraliensis showed some cases of misinterpreted synonyms, misidentifications, and lack of detailed descriptions for some species. The conclusion of the revision is that there are three valid species of Paratetilla (P.arcifera, P.bacca, and P.corrugata) and four valid species of Cinachyrella (C.australiensis, C.porosa, C.paterifera, and C.schulzei) in Indonesia. This is furthermore corroborated by molecular work from previous studies. ParatetillaarciferaWilson 1925 and C.porosa (Lendenfeld, 1888) are resurrected. A full review of taxonomic history is provided as well as a key for identification of moon sponges from Indonesia. All species are sympatric and we expect that there are undescribed species remaining within the Tetillidae from the Indo-Pacific. Our current review provides the framework from which to describe new species in the genera Paratetilla and Cinachyrella from the Indo-Pacific.

First finding of anchialine and submarine cave dwelling brittle stars from the Pacific Ocean, with descriptions of new species of Ophiolepis and Ophiozonella (Echinodermata: Ophiuroidea: Amphilepidida).

Two new species, Ophiolepis cavitata n. sp. and Ophiozonella cavernalis n. sp., are described from the specimens collected in shallow water submarine caves in the Ryukyu Islands, southwestern Japan. Ophiolepis cavitata n. sp. is distinguished from the other congeners in having prominent tubercle-shaped knobs on dorsal arm plates; two sizes of disc scales on the aboral disc, the larger surrounded by 3-8 times smaller disc scales; larger scales concave; radial shields wider than long; a trio of distinct scales present distal to each pair of radial shields; oral shields partly concave; 3 arm spines on proximal portion of the arms. Ophiozonella cavernalis n. sp. is distinguished from the other congeners in having polygonal, uniform size disc scales; oral shields almost as long as wide, shallow concave proximal sides that end in a fairly sharp point medially; arms about 9 times longer than disc diameter; 3 cylindrical arm spines, of which the oral most one is slightly larger than other 2 spines. Ophiozonella cavernalis n. sp. is the second finding of an anchialine-endemic ophiuroid and the first finding from the Pacific Ocean.

Bacterial Communities Inhabiting the Sponge Biemna fortis, Sediment and Water in Marine Lakes and the Open Sea.

Marine lakes are small bodies of landlocked seawater that are isolated from the open sea and have been shown to house numerous rare and unique taxa. The environmental conditions of the lakes are also characterised by lower pH and salinity and higher temperatures than generally found in the open sea. In the present study, we used a 16S rRNA gene barcoded pyrosequencing approach and a predictive metagenomic approach (PICRUSt) to examine bacterial composition and function in three distinct biotopes (sediment, water and the sponge species Biemna fortis) in three habitats (two marine lakes and the open sea) of the Berau reef system, Indonesia. Both biotope and habitat were significant predictors of higher taxon abundance and compositional variation. Most of the variation in operational taxonomic unit (OTU) composition was related to the biotope (42% for biotope alone versus 9% for habitat alone and 15% combined). Most OTUs were also restricted to a single biotope (1047 for B. fortis, 6120 for sediment and 471 for water). Only 98 OTUs were shared across all three biotopes. Bacterial communities from B. fortis, sediment and water samples were, however, also distinct in marine lake and open sea habitats. This was evident in the abundance of higher bacterial taxa. For example, the phylum Cyanobacteria was significantly more abundant in samples from marine lakes than from the open sea. This difference was most pronounced in the sponge B. fortis. In line with the compositional differences, there were pronounced differences in predicted relative gene count abundance among biotopes and habitats. Of particular interest was the predicted enrichment in B. fortis from the marine lakes for pathways including DNA replication and repair and the glutathione metabolism. This may facilitate adaptation of host and microbes to life in 'stressful' low pH, low salinity and/or high temperature environments such as those encountered in marine lakes.

Venomics of Remipede Crustaceans Reveals Novel Peptide Diversity and Illuminates the Venom's Biological Role.

We report the first integrated proteomic and transcriptomic investigation of a crustacean venom. Remipede crustaceans are the venomous sister group of hexapods, and the venom glands of the remipede Xibalbanus tulumensis express a considerably more complex cocktail of proteins and peptides than previously thought. We identified 32 venom protein families, including 13 novel peptide families that we name xibalbins, four of which lack similarities to any known structural class. Our proteomic data confirm the presence in the venom of 19 of the 32 families. The most highly expressed venom components are serine peptidases, chitinase and six of the xibalbins. The xibalbins represent Inhibitory Cystine Knot peptides (ICK), a double ICK peptide, peptides with a putative Cystine-stabilized α-helix/ß-sheet motif, a peptide similar to hairpin-like ß-sheet forming antimicrobial peptides, two peptides related to different hormone families, and four peptides with unique structural motifs. Remipede venom components represent the full range of evolutionary recruitment frequencies, from families that have been recruited into many animal venoms (serine peptidases, ICKs), to those having a very narrow taxonomic range (double ICKs), to those unique for remipedes. We discuss the most highly expressed venom components to shed light on their possible functional significance in the predatory and defensive use of remipede venom, and to provide testable ideas for any future bioactivity studies.