Rapid, contamination-less, and efficient environmental DNA filtration system.

Due to the sporadic distribution and trace amount of environmental DNA (eDNA) in deep-sea water, in the context of biodiversity monitoring, large volumes of filtration and multiple filtration replicates are required for eDNA metabarcoding. To address issues tied to the use of multiple filtration devices and large filtration volumes (e.g., contamination, time consumption, etc.), we have developed two systems for simple, rapid, and contamination-less filtration simultaneously that allow for the processing of multiple sample replicates from large volumes of water. First, the water from a Niskin bottle was filtered directly using a solenoid pump. Second, the pumped deep-sea water, using the siphon effect, was directly filtered by a filtration device driven by water pressure. This system can process 24 replicates simultaneously without the need for expensive equipment and active driving force. Compared with conventional filtering methods, e.g., peristaltic pumps, the proposed systems reduce filtration time, minimizing contamination, and enabling the simultaneous acquisition of multiple replicates. Overall, the systems presented here provide an effective approach for eDNA metabarcoding analysis, particularly for the filtration of large volumes of water containing small amounts of eDNA, such as deep-sea water. •The present systems reduce filtration time and contamination without water having to be transferred.•Simultaneous multiple replicates improve the efficiency and reliability of biodiversity assessments.

Occurrence and levels of polybrominated diphenyl ethers (PBDEs) in deep-sea sharks from Suruga Bay, Japan.

Few studies have investigated the prevalence of polybrominated diphenyl ethers (PBDEs) in deep-sea sharks. In this study, the levels and profiles of PBDEs were determined in liver samples of eight different species of deep-sea sharks collected in Suruga Bay, Japan. Widespread contamination of PBDEs in the deep-sea environment was reconfirmed in this study as these persistent organic pollutants (POPs) were detected in all specimens analyzed. Mean ΣPBDE levels in the deep-sea sharks ranged from 7 to 517 ng/g of lipid weight. The distribution patterns of BDE homologues were similar in all species where tetra-BDEs provided the dominant contribution to total PBDEs (46%). PBDEs levels were similar to, or higher than, those seen in other deep-sea sharks from different regions. The levels of PBDEs were likely to reflect their feeding preferences as higher PBDE levels were seen in species with higher trophic positions.

Optimization of environmental DNA extraction and amplification methods for metabarcoding of deep-sea fish.

Analyses of environmental DNA (eDNA) from macroorganisms in aquatic environments have greatly advanced in recent years. In particular, eDNA metabarcoding of fish using universal PCR primers has been reported in various waters. Although pumped deep-sea water was used for eDNA metabarcoding of deep-sea fish, conventional methods only resulted in small amounts of extracted eDNA and subsequent few or no PCR amplicons. To optimize eDNA metabarcoding of deep-sea fish from pumped deep-sea water, we modified conventional procedures of eDNA extraction and PCR amplification. Here, we propose a modified eDNA extraction method, in which a filter used for eDNA sampling was shredded and incubated in microtubes for efficient lysis of eDNA sources. Total eDNA yield extracted using the modified protocol was approximately six-fold higher than that extracted by the conventional protocol. The PCR enzyme Platinum SuperFi II DNA Polymerase successfully amplified a target region of fish universal primers (MiFish) from trace amounts of eDNA extracted from pumped deep-sea water and suppressed nonspecific amplifications more effectively than the enzyme used in conventional methods. Approximately 93% of the sequence reads acquired by next generation sequencing of these amplicons were derived from fish. The improved procedure presented here provided effective eDNA metabarcoding of deep-sea fish.•A modified eDNA extraction protocol, in which a filter was shredded and incubated in microtubes, increased eDNA yields extracted from pumped deep-sea water over the conventional method.•The PCR enzyme Platinum SuperFi II DNA polymerase improved the amplification efficiency of trace amounts of MiFish objectives in eDNA extracted from pumped deep-sea water with suppressing nonspecific amplifications.•The use of Platinum SuperFi II DNA polymerase for eDNA metabarcoding using MiFish primers resulted in the acquisition of abundant sequence reads of deep-sea fish through next generation sequencing.

Structural Comparison of Diplonemid Communities around the Izu Peninsula, Japan.

Diplonemea (diplonemids) is one of the most abundant and species-rich protist groups in marine environments; however, their community structures among local and seasonal samples have not yet been compared. In the present study, we analyzed four diplonemid community structures around the Izu Peninsula, Japan using barcode sequences amplified from environmental DNA. These sequences and the results of statistical analyses indicated that communities at the same site were more similar to each other than those in the same season. Environmental variables were also measured, and their influence on diplonemid community structures was examined. Salinity, electrical conductivity, and temperature, and their correlated variables, appeared to influence the structures of diplonemid communities, which was consistent with previous findings; however, since the results obtained did not reach statistical significance, further studies are required. A comparison of each diplonemid community indicated that some lineages were unique to specific samples, while others were consistently detected in all samples. Members of the latter type are cosmopolitan candidates and may be better adapted to the environments of the studied area. Future studies that focus on the more adaptive members will provide a more detailed understanding of the mechanisms by which diplonemids are widely distributed in marine environments and will facilitate their utilization as indicator organisms to monitor environmental changes.

Discovery of a colossal slickhead (Alepocephaliformes: Alepocephalidae): an active-swimming top predator in the deep waters of Suruga Bay, Japan.

A novel species of the family Alepocephalidae (slickheads), Narcetes shonanmaruae, is described based on four specimens collected at depths greater than 2171 m in Suruga Bay, Japan. Compared to other alepocephalids, this species is colossal (reaching ca. 140 cm in total length and 25 kg in body weight) and possesses a unique combination of morphological characters comprising anal fin entirely behind the dorsal fin, multiserial teeth on jaws, more scale rows than congeners, precaudal vertebrae less than 30, seven branchiostegal rays, two epurals, and head smaller than those of relatives. Mitogenomic analyses also support the novelty of this large deep-sea slickhead. Although most slickheads are benthopelagic or mesopelagic feeders of gelatinous zooplankton, behavioural observations and dietary analyses indicate that the new species is piscivorous. In addition, a stable nitrogen isotope analysis of specific amino acids showed that N. shonanmaruae occupies one of the highest trophic positions reported from marine environments to date. Video footage recorded using a baited camera deployed at a depth of 2572 m in Suruga Bay revealed the active swimming behaviour of this slickhead. The scavenging ability and broad gape of N. shonanmaruae might be correlated with its colossal body size and relatively high trophic position.

Cryptic fungal diversity revealed in deep-sea sediments associated with whale-fall chemosynthetic ecosystems.

In this study, sediments from whale-fall chemosynthetic ecosystems (two different sites, one naturally occurring at 4200 m water depth in South Atlantic Ocean and one artificially immersed at 100 m water depth in Kagoshima Bay, Japan) were investigated by Ion Torrent PGM sequencing of the ITS region of ribosomal RNA to reveal fungal communities in these unique marine environments. As a result, a total of 107 (897 including singletons) Operational Taxonomic Units (OTUs) were obtained from the samples explored. Composition of the 107 OTUs at the phylum level among the five samples from two different whale-fall sites was assigned to Ascomycota (46%), Basidiomycota (7%), unidentified fungi (21%), non-fungi (10%), and sequences with no affiliation to any organisms in the public database (No-match) (16%). The high detection of the unidentified fungi and unassigned fungi was revealed in the whale-fall environments in this study. Some of these unidentified fungi are allied to early diverging fungi and they were more abundant in the sediments not directly in contact with whalebone. This study suggests that a cryptic fungal community exists in unique whale-fall ecosystems.

A new gastropod associated with a deep-sea whale carcass from São Paulo Ridge, Southwest Atlantic.

An unfamiliar gastropod was collected from a deep-sea whale carcass at the base of the São Paulo Ridge in the Southwest Atlantic by the manned research submersible Shinkai 6500, and is here described as a new species of the abyssochrysoidean genus Rubyspira, R. brasiliensis sp. nov., following morphological and molecular phylogenetic examinations. There are only two other known species in the genus, which occur together in the Monterey Submarine Canyon off California. The present new species was shown by the molecular analysis to be closer to one of the Californian species than the other. It was found aggregated on and around a whale carcass at a depth of 4204 m, which represents the deepest record of whale- fall ecosystems ever discovered.

New species of bone-eating worm Osedax from the abyssal South Atlantic Ocean (Annelida, Siboglinidae).

A new species of bone-eating annelid, Osedaxbraziliensis sp. n., found in a sunken whale carcass at a depth of 4,204 m at the base of the São Paulo Ridge in the South Atlantic Ocean off the Brazilian coast is described. The organism was retrieved using the human-occupied vehicle Shinkai 6500 during the QUELLE 2013 expedition. This is the 26th species of the genus and the first discovery from the South Atlantic Ocean, representing the deepest record of Osedax worldwide to date. This species morphologically resembles Osedaxfrankpressi but is distinguished by the presence of a yellow bump or patch behind the prostomium and its trunk length. Molecular phylogenetic analysis using three genetic markers (COI, 16S, and 18S) showed that O.braziliensis sp. n. is distinct from all other Osedax worms reported and is a sister species of O.frankpressi.

Aneurinibacillus tyrosinisolvens sp. nov., a tyrosine-dissolving bacterium isolated from organics- and methane-rich seafloor sediment.

A novel Gram-positive-staining, strictly aerobic and heterotrophic bacterium, designated strain LL-002T, was isolated from organics- and methane-rich seafloor sediment at a depth of 100 m in Kagoshima Bay, Kagoshima, Japan. Colonies were lustreless and translucent white in colour. The temperature, pH and salt concentration ranges for growth were 10-30 °C, pH 6.0-6.5 and 0-1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain LL-002T belongs to the genus Aneurinibacillus of the family Paenibacillaceae. 16S rRNA gene sequence similarities between strain LL-002T and the type strains of species of the genus Aneurinibacillus were 92.8-95.7 %; the highest sequence identity was with the type strain of Aneurinibacillus migulanus. The DNA G+C content of strain LL-002T was 46.2 mol%. MK-7 was the predominant menaquinone. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0, and the cell-wall peptidoglycan contained meso-diaminopimelic acid and glutamic acid, glycine and alanine in addition to muramic acid and glucosamine. The peptidoglycan type was A1γ. In DNA-DNA hybridization assays between strain LL-002T and the type strains of the other species of the genus Aneurinibacillus, the level of hybridization was 6.3-30.1 %. On the basis of its biological features and the 16S rRNA gene sequence comparison presented here, strain LL-002T is considered to represent a novel species of the genus Aneurinibacillus, for which the name Aneurinibacillus tyrosinisolvens sp. nov. is proposed; the type strain is LL-002T ( = NBRC 110097T = CECT 8536T).

Adaptive radiation of chemosymbiotic deep-sea mussels.

Adaptive radiations present fascinating opportunities for studying the evolutionary process. Most cases come from isolated lakes or islands, where unoccupied ecological space is filled through novel adaptations. Here, we describe an unusual example of an adaptive radiation: symbiotic mussels that colonized island-like chemosynthetic environments such as hydrothermal vents, cold seeps and sunken organic substrates on the vast deep-sea floor. Our time-calibrated molecular phylogeny suggests that the group originated and acquired sulfur-oxidizing symbionts in the Late Cretaceous, possibly while inhabiting organic substrates and long before its major radiation in the Middle Eocene to Early Oligocene. The first appearance of intracellular and methanotrophic symbionts was detected only after this major radiation. Thus, contrary to expectations, the major radiation may have not been triggered by the evolution of novel types of symbioses. We hypothesize that environmental factors, such as increased habitat availability and/or increased dispersal capabilities, sparked the radiation. Intracellular and methanotrophic symbionts were acquired in several independent lineages and marked the onset of a second wave of diversification at vents and seeps. Changes in habitat type resulted in adaptive trends in shell lengths (related to the availability of space and energy, and physiological trade-offs) and in the successive colonization of greater water depths.

A novel alveolate in bivalves with chemosynthetic bacteria inhabiting deep-sea methane seeps.

It has recently been unveiled that a wide variety of microbial eukaryotes (protists) occur in chemosynthetic ecosystems, such as hydrothermal vents and methane seeps. However, there is little knowledge regarding protists associated with endemic animals inhabiting these environments. In the present study, utilizing PCR techniques, we detected fragments of the small subunit ribosomal RNA gene (SSU rRNA gene) from a particular protist from gill tissues of a significant fraction of the vesicomyid clams Calyptogena soyoae and C. okutanii complex and of the mussel Bathymodiolus platifrons and B. japonicus, all of which harbor chemosynthetic endosymbiont bacteria and dominate methane seeps in Sagami Bay, Japan. Based on the phylogeny of SSU rRNA gene, the organism in question was shown to belong to Alveolata. It is noteworthy that this protist did not affiliate with any known alveolate group, although being deeply branched within the lineage of Syndiniales, for which the monophyly was constantly recovered, but not robustly supported. In addition, the protist detected using PCR followed by sequencing was localized within gill epithelial cells of B. platifrons with whole-mount fluorescence in situ hybridization. This protist may be an endoparasite or an endocommensal of Calyptogena spp. and Bathymodiolus spp., and possibly have physiological and ecological impacts on these bivalves.

Algivore or phototroph? Plakobranchus ocellatus (Gastropoda) continuously acquires kleptoplasts and nutrition from multiple algal species in nature.

The sea slug Plakobranchus ocellatus (Sacoglossa, Gastropoda) retains photosynthetically active chloroplasts from ingested algae (functional kleptoplasts) in the epithelial cells of its digestive gland for up to 10 months. While its feeding behavior has not been observed in natural habitats, two hypotheses have been proposed: 1) adult P. ocellatus uses kleptoplasts to obtain photosynthates and nutritionally behaves as a photoautotroph without replenishing the kleptoplasts; or 2) it behaves as a mixotroph (photoautotroph and herbivorous consumer) and replenishes kleptoplasts continually or periodically. To address the question of which hypothesis is more likely, we examined the source algae for kleptoplasts and temporal changes in kleptoplast composition and nutritional contribution. By characterizing the temporal diversity of P. ocellatus kleptoplasts using rbcL sequences, we found that P. ocellatus harvests kleptoplasts from at least 8 different siphonous green algal species, that kleptoplasts from more than one species are present in each individual sea slug, and that the kleptoplast composition differs temporally. These results suggest that wild P. ocellatus often feed on multiple species of siphonous algae from which they continually obtain fresh chloroplasts. By estimating the trophic position of wild and starved P. ocellatus using the stable nitrogen isotopic composition of amino acids, we showed that despite the abundance of kleptoplasts, their photosynthates do not contribute greatly to the nutrition of wild P. ocellatus, but that kleptoplast photosynthates form a significant source of nutrition for starved sea slugs. The herbivorous nature of wild P. ocellatus is consistent with insights from molecular analyses indicating that kleptoplasts are frequently replenished from ingested algae, leading to the conclusion that natural populations of P. ocellatus do not rely on photosynthesis but mainly on the digestion of ingested algae.

Extracellular and mixotrophic symbiosis in the whale-fall mussel Adipicola pacifica: a trend in evolution from extra- to intracellular symbiosis.

BACKGROUND: Deep-sea mussels harboring chemoautotrophic symbionts from hydrothermal vents and seeps are assumed to have evolved from shallow-water asymbiotic relatives by way of biogenic reducing environments such as sunken wood and whale falls. Such symbiotic associations have been well characterized in mussels collected from vents, seeps and sunken wood but in only a few from whale falls. METHODOLOGY/PRINCIPAL FINDING: Here we report symbioses in the gill tissues of two mussels, Adipicola crypta and Adipicola pacifica, collected from whale-falls on the continental shelf in the northwestern Pacific. The molecular, morphological and stable isotopic characteristics of bacterial symbionts were analyzed. A single phylotype of thioautotrophic bacteria was found in A. crypta gill tissue and two distinct phylotypes of bacteria (referred to as Symbiont A and Symbiont C) in A. pacifica. Symbiont A and the A. crypta symbiont were affiliated with thioautotrophic symbionts of bathymodiolin mussels from deep-sea reducing environments, while Symbiont C was closely related to free-living heterotrophic bacteria. The symbionts in A. crypta were intracellular within epithelial cells of the apical region of the gills and were extracellular in A. pacifica. No spatial partitioning was observed between the two phylotypes in A. pacifica in fluorescence in situ hybridization experiments. Stable isotopic analyses of carbon and sulfur indicated the chemoautotrophic nature of A. crypta and mixotrophic nature of A. pacifica. Molecular phylogenetic analyses of the host mussels showed that A. crypta constituted a monophyletic clade with other intracellular symbiotic (endosymbiotic) mussels and that A. pacifica was the sister group of all endosymbiotic mussels. CONCLUSIONS/SIGNIFICANCE: These results strongly suggest that the symbiosis in A. pacifica is at an earlier stage in evolution than other endosymbiotic mussels. Whale falls and other modern biogenic reducing environments may act as refugia for primal chemoautotrophic symbioses between eukaryotes and prokaryotes since the extinction of ancient large marine vertebrates.

Association of thioautotrophic bacteria with deep-sea sponges.

We investigated microorganisms associated with a deep-sea sponge, Characella sp. (Pachastrellidae) collected at a hydrothermal vent site (686 m depth) in the Sumisu Caldera, Ogasawara Island chain, Japan, and with two sponges, Pachastrella sp. (Pachastrellidae) and an unidentified Poecilosclerida sponge, collected at an oil seep (572 m depth) in the Gulf of Mexico, using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) directed at bacterial 16S rRNA gene sequences. In the PCR-DGGE profiles, we detected a single clearly dominant band in each of the Characella sp. and the unidentified Poecilosclerida sponge. BLAST search of their sequences showed that they were most similar (>99% identity) to those of the gammaproteobacterial thioautotrophic symbionts of deep-sea bivalves from hydrothermal vents, Bathymodiolus spp. Phylogenetic analysis of the near-full length sequences of the 16S rRNA genes cloned from the unidentified Poecilosclerida sponge and Characella sp. confirmed that they were closely related to thioautotrophic symbionts. Although associations between sponges and methanotrophic bacteria have been reported previously, this is the first report of a possible stable association between sponges and thioautotrophic bacteria.

Host-virus specificity of morbilliviruses predicted by structural modeling of the marine mammal SLAM, a receptor.

Signaling lymphocyte activation molecule (SLAM) is thought to be a major cellular receptor for high-host specificity morbilliviruses, which cause devastating and highly infectious diseases in mammals. We determined the sequences of SLAM cDNA from five species of marine mammal, including two cetaceans, two pinnipeds and one sirenian, and generated three-dimensional models to understand the receptor-virus interaction. Twenty-one amino acid residues in the immunoglobulin-like V domains of the SLAMs were shown to bind the viral protein. Notably, the sequences from pinnipeds and dogs were highly homologous, which is consistent with the fact that canine distemper virus was previously shown to cause a mass die-off of seals. Among these twenty-one residues, eight (63, 66, 68, 72, 84, 119, 121 and 130) were shared by animal groups susceptible to a particular morbillivirus species. This set of residues appears to determine host-virus specificity and may be useful for risk estimation for morbilliviruses.

Amphritea japonica sp. nov. and Amphritea balenae sp. nov., isolated from the sediment adjacent to sperm whale carcasses off Kagoshima, Japan.

Two novel species were isolated from the sediment adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of about 230 m. The isolated strains, JAMM 1866(T), JAMM 1548 and JAMM 1525(T), were Gram-negative, rod-shaped, non-spore-forming and motile by means of a single polar or bipolar flagellum. Phylogenetic analysis based on 16S rRNA gene sequences of strains JAMM 1866(T) and JAMM 1548 indicated a relationship to the symbiotic bacterial clone R21 of Osedax japonicus (100 % sequence similarity) and all three isolates were closely related to Amphritea atlantica (97.7-97.8 % similarity) within the class Gammaproteobacteria. The novel isolates were able to produce isoprenoid quinone Q-8 as the major component. The predominant fatty acids were C(16 : 0), C(16 : 1) and C(18 : 1), with C(12 : 1) 3-OH present in smaller amounts. The DNA G+C contents of the three isolated strains were about 47 mol%. Based on differences in taxonomic characteristics, the three isolated strains represent two novel species of the genus Amphritea for which the names Amphritea japonica sp. nov. (type strain JAMM 1866(T)=JCM 14782(T)=ATCC BAA-1530(T); reference strain JAMM 1548) and Amphritea balenae sp. nov. (type strain JAMM 1525(T)=JCM 14781(T)=ATCC BAA-1529(T)) are proposed.

Neptunomonas japonica sp. nov., an Osedax japonicus symbiont-like bacterium isolated from sediment adjacent to sperm whale carcasses off Kagoshima, Japan.

Novel bacterial species were isolated from sediments adjacent to sperm whale carcasses off Kagoshima, Japan, at a depth of 226-246 m. The isolated strains, JAMM 0745T, JAMM 1380, JAMM 1475 and JAMM 1610, were Gram-negative, rod-shaped, non-spore-forming and motile by means of a single polar or subterminal flagellum. Phylogenetic analysis based on 16S rRNA gene sequences of the novel isolates indicated a relationship to a symbiotic bacterial clone of the polychaete Osedax japonicus (99.6-99.9 % sequence similarity) and these bacteria were closely related to members of the genus Neptunomonas (95.6-96.0 % similarity) within the class Gammaproteobacteria. The novel strains were able to produce isoprenoid quinone Q-8 as the major quinone component. The predominant fatty acids were C16 : 0, C16 : 1 and C18 : 1, with C18 : 2 and C20 : 2 present in smaller amounts. The DNA G+C contents of the four novel strains were about 43.6-43.8 mol%. Based on the taxonomic differences observed, the four isolated strains appear to represent a novel species of the genus Neptunomonas. The name Neptunomonas japonica sp. nov. (type strain JAMM 0745T=JCM 14595T=DSM 18939T) is proposed for the novel strains.

Molecular identification of the ichthyosporean protist "Pseudoperkinsus tapetis" from the mytilid mussel Adipicola pacifica associated with submerged whale carcasses in Japan.

A protist tentatively designated "Pseudoperkinsus tapetis" belonging to the eukaryotic group Ichthyosporea (Mesomycetozoa) was previously isolated from carpet shell clams in Galicia (northwest Spain). In the present study, based on molecular data, a potential P. tapetis specimen was identified from the gill tissues of the mussel Adipicola pacifica associated with whale carcasses (generating chemosynthetic-based ecosystems) collected at shelf depths in the northwest Pacific (southwest Japan). Small subunit ribosomal DNA sequences (1751 sites) of the genotypes of P. tapetis from Spain and Japan were almost identical (only one substitution and one insertion/deletion difference). On the other hand, differences of 10 and 8 substitutions were found in two internal transcribed spacer regions of ribosomal DNA, ITS1 (288 sites) and ITS2 (251 sites) between these two genotypes, respectively, indicating that they are genetically different at the population level. These findings suggest that P. tapetis occurs worldwide and can associate with (and possibly infect) various types of bivalves. Further, a PCR method to specifically detect the P. tapetis cells in the host was also established.

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii.

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.