Masaru Kawato

A New Species of Osedax (Annelida: Siboglinidae) Associated with Whale Carcasses off Kyushu, Japan

Abstract A new whale-bone-eating polychaete species of the genus Osedax was found on sperm whale carcasses submerged off Cape Nomamisaki, Kyushu, Japan, at a depth of approximately 200 m. The new species, Osedax japonicus, is the fourth known species of the genus Osedax and the first species from the western Pacific. Female O. japonicus specimens (1) form dense clusters on whale carcasses; (2) have a body composed of crown, trunk, and root structure; (3) lack a digestive tract; and (4) have bacterium-like particles in the tissue of the root structure. Osedax japonicus shares all these characteristics with O. rubiplumus and O. frankpressi, and items (1) to (3) with O. mucofloris, Osedax japonicus is easily distinguished from the other three known species by oviduct morphology, body length, and palp coloration in females. No males of O. japonicus have yet been found.

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.

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.

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.

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.

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.

Epibiotic association between filamentous bacteria and the vent-associated galatheid crab, Shinkaia crosnieri (Decapoda: Anomura)

The galatheid crab Shinkaia crosnieri , is the sole member of the subfamily Shinkaiinae. It is abundant and forms dense beds around active hydrothermal vents in the Okinawa Trough. Thousands of filamentous bacteria attached to the plumose setae on the ventral surface of this crab were observed using field-emission scanning electron microscopy and transmission electron microscopy. Nucleic acids were extracted from the filamentous bacteria, and the phylotypes of 16S rRNA genes were identified from 81 clones. These phylotypes were divided into three groups: Epsilonproteobacteria (74%); Gammaproteobacteria (20%); and Bacteroidetes (6%). Gamma- and major phylotypes of Epsilonproteobacteria were also detected using fluorescence in situ hybridization analysis. These Epsilon- and Gammaproteobacteria were closely related to cultured and uncultured bacteria from hydrothermal vent fields including episymbionts of vent-associated invertebrates such as Rimicaris exoculata, Alvinella pompejana , the scaly-foot snail, Kiwa hirsuta etc. The carbon isotopic compositions of the muscle of S. crosnieri and in filamentous bacteria were similar. The muscle of S. crosnieri contained monounsaturated C 16 and C 18 fatty acids, which are known to be characteristic of sulphur-oxidizing bacteria in H 2 S-rich marine habitats. Through the video images transmitted by a submersible and a remotely operated vehicle, S. crosnieri was observed to comb out its ventral setae using the third maxilliped and appeared to consume the contents. These evidences suggest the epibiotic association between S. crosnieri and the filamentous bacteria attached to the ventral setae of the crab, but the details of role and function are still unclear at the present study.

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.

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.

Chimeric Structure of omp2 of Brucella from Pacific Common Minke Whales (Balaenoptera acutorostrata)

In the Pacific common minke whale (Balaenoptera acutorostrata), a new variant of Brucella has been detected using the polymerase chain reaction. Detailed analysis of the porin protein genes, omp2a and omp2b from the whale Brucella showed that these two genes have some motifs in common with Atlantic marine strains in the 5′‐terminal one‐third region. On the other hand, the nucleotide sequences in the 3′‐terminal two‐thirds region of the two genes were almost identical to the respective genes of terrestrial strains. Thus, Pacific whale Brucella omp2 genes are chimeras between marine and terrestrial strains.