Leptospiral outer membrane protein LMB216 is involved in enhancement of phagocytic uptake by macrophages.

Leptospira interrogans is responsible for the zoonotic disease leptospirosis. The pathogenic mechanisms of this spirochaete remain poorly understood; however, virulence has been correlated with increased phagocytic uptake and survival within macrophages. Leptospiral outer membrane proteins are thought to be responsible for persistence in vivo via interaction with specific host components. In this study, we analysed the transcriptional profile of a virulent strain and its culture-attenuated derivative strain to identify bacterial factors that may be involved in pathogenesis. Two outer membrane proteins, LMB216 and LigB (leptospiral immunoglobulin-like protein B) were downregulated more than 10-fold in the culture-attenuated strain. We show that both proteins play a role in leptospiral uptake by macrophages and that LMB216, as well as LigB, enhances the binding of leptospires to fibronectin. Taken together, our results indicate that LMB216 plays a role in pathogen interaction with host molecule/s, which may contribute to pathogenesis of leptospirosis.

Diversity, composition and potential roles of sedimentary microbial communities in different coastal substrates around subtropical Okinawa Island, Japan.

BACKGROUND: Marine benthic prokaryotic communities play crucial roles in material recycling within coastal environments, including coral reefs. Coastal sedimentary microbiomes are particularly important as potential reservoirs of symbiotic, beneficial, and pathogenic bacteria in coral reef environments, and therefore presumably play a core role in local ecosystem functioning. However, there is a lack of studies comparing different environments with multiple sites on the island scale, particularly studies focusing on prokaryotic communities, as previous investigations have focused mainly on a single site or on specific environmental conditions. In our study, we collected coastal sediments from seven sites around Okinawa Island, Japan, including three different benthic types; sandy bottoms, seagrass meadows, and hard substratum with living scleractinian corals. We then used metabarcoding to identify prokaryotic compositions and estimate enzymes encoded by genes to infer their functions. RESULTS: The results showed that the three substrata had significantly different prokaryotic compositions. Seagrass meadow sites exhibited significantly higher prokaryotic alpha-diversity compared to sandy bottom sites. ANCOM analysis revealed that multiple bacterial orders were differentially abundant within each substratum. At coral reef sites, putative disease- and thermal stress-related opportunistic bacteria such as Rhodobacterales, Verrucomicrobiales, and Cytophagales were comparatively abundant, while seagrass meadow sites abundantly harbored Desulfobacterales, Steroidobacterales and Chromatiales, which are common bacterial orders in seagrass meadows. According to our gene-coded enzyme analyses the numbers of differentially abundant enzymes were highest in coral reef sites. Notably, superoxide dismutase, an important enzyme for anti-oxidative stress in coral tissue, was abundant at coral sites. Our results provide a list of prokaryotes to look into in each substrate, and further emphasize the importance of considering the microbiome, especially when focusing on environmental conservation. CONCLUSION: Our findings prove that prokaryotic metabarcoding is capable of capturing compositional differences and the diversity of microbial communities in three different environments. Furthermore, several taxa were suggested to be differentially more abundant in specific environments, and gene-coded enzymic compositions also showed possible differences in ecological functions. Further study, in combination with field observations and temporal sampling, is key to achieving a better understanding of the interactions between the local microbiome and the surrounding benthic community.

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.

Extensive gene flow among populations of the cavernicolous shrimp at the northernmost distribution margin in the Ryukyu Islands, Japan.

Marine cave habitats in the Ryukyu Islands, Indo-West Pacific, are located at the northern edge of the distribution of many cave-dwelling species. At distribution margins, gene flow is often more restricted than that among core populations due to the smaller effective population size. Here, we used high-throughput sequencing technology to investigate the gene flow pattern among three sampling sites of a marine cave-dwelling species at the margin of its distribution range. We collected individuals of the barbouriid shrimp Parhippolyte misticia from three marine caves in the Ryukyu Islands and performed population genetic analyses by means of multiplexed inter-simple sequence repeat genotyping by sequencing. Based on 62 single-nucleotide polymorphism markers, no clear population structure or directional gene flow pattern was found among the three sites. These results were unexpected because previous studies of other stygobitic shrimps in this region did find significant population genetic structures and northward directional gene flow patterns. Together, these inconsistent findings imply that marine cave-dwelling species in the region have different mechanisms of larval dispersal. Future studies on larval ecology and the biotic and abiotic factors influencing gene flow patterns are needed to clarify the mechanisms underlying the population dynamics of marine cave-dwelling species.

Comparison of Symbiodiniaceae diversities in different members of a Palythoa species complex (Cnidaria: Anthozoa: Zoantharia)-implications for ecological adaptations to different microhabitats.

In this study we compared genotypes of zoantharian host-associating algal symbionts among Palythoa species, which are among the dominant benthic reef organisms in the Ryukyu Archipelago, Japan, and evaluated Symbiodiniaceae diversities of closely related congeneric Palythoa species. We targeted a species complex of the zoantharian genus Palythoa (P. tuberculosa, P. sp. yoron, P. mutuki) living among different microhabitats in a narrow reef area of Tokunoshima Island. For phylogenetic analyses, we used two DNA marker regions; nuclear internal transcribed spacer (ITS) and plastid mini-circle non-coding region (psbAncr), both of which have previously been used to determine Symbiodiniaceae genotypes of zoantharian species. Our results showed that all Palythoa species hosted symbionts of the genus Cladocopium, with genotypic compositions of this genus showing some variations among the three different Palythoa species. Additionally, we found that the Cladocopium genotypic composition was statistically different among Palythoa species, and among P. tuberculosa specimens in different microhabitats. Our results suggest that ecological divergence among these three Palythoa species may be related to differing Symbiodiniaceae diversities that may in turn contribute to eco-physiological adaptation into different microhabitats on coral reefs.

Environmental DNA metabarcoding to detect pathogenic Leptospira and associated organisms in leptospirosis-endemic areas of Japan.

Leptospires, which cause the zoonotic disease leptospirosis, persist in soil and aqueous environments. Several factors, including rainfall, the presence of reservoir animals, and various abiotic and biotic components interact to influence leptospiral survival, persistence, and pathogenicity in the environment. However, how these factors modulate the risk of infection is poorly understood. Here we developed an approach using environmental DNA (eDNA) metabarcoding for detecting the microbiome, vertebrates, and pathogenic Leptospira in aquatic samples. Specifically, we combined 4 sets of primers to generate PCR products for high-throughput sequencing of multiple amplicons through next-generation sequencing. Using our method to analyze the eDNA of leptospirosis-endemic areas in northern Okinawa, Japan, we found that the microbiota in each river shifted over time. Operating taxonomic units corresponding to pathogenic L. alstonii, L. kmetyi, and L. interrogans were detected in association with 12 nonpathogenic bacterial species. In addition, the frequencies of 11 of these species correlated with the amount of rainfall. Furthermore, 10 vertebrate species, including Sus scrofa, Pteropus dasymallus, and Cynops ensicauda, showed high correlation with leptospiral eDNA detection. Our eDNA metabarcoding method is a powerful tool for understanding the environmental phase of Leptospira and predicting human infection risk.

Speciation among sympatric lineages in the genus Palythoa (Cnidaria: Anthozoa: Zoantharia) revealed by morphological comparison, phylogenetic analyses and investigation of spawning period.

Zoantharians are sessile marine invertebrates and colonial organisms possessing sexual and asexual reproductive ability. The zooxanthellate zoantharian genus Palythoa is widely distributed in coral reef ecosystems. In the Ryukyu Archipelago, Japan, sympatric Palythoa tuberculosa and P. mutuki are the dominant species of this genus in the intertidal zone. Previous phylogenetic analyses have shown that these two species are closely related, and additionally revealed a putative sympatric hybrid species (designated as Palythoa sp. yoron). In this study, we attempted to delineate Palythoa species boundaries and to clarify the relationships among these three groups plus another additional putative sympatric species (P. aff. mutuki) by multiple independent criteria. The morphology of these four lineages was clearly different; for example the number of tentacles was significantly different for each species group in all pairwise comparisons. From observations of gonadal development conducted in 2010 and 2011, P. sp. yoron and P. aff. mutuki appear to be reproductively isolated from P. tuberculosa. In the phylogenetic tree resulting from maximum likelihood analyses of the ITS-rDNA sequence alignment, P. tuberculosa and P. sp. yoron formed a very well supported monophyletic clade (NJ = 100%, ML = 95%, Bayes = 0.99). This study demonstrates that despite clear morphological and/or reproductive differences, P. tuberculosa and P. sp. yoron are phylogenetically entangled and closely related to each other, as are P. mutuki and P. aff. mutuki. Additionally, no single molecular marker was able to divide these four lineages into monophyletic clades by themselves, and a marker that has enough resolution to solve this molecular phylogenetic species complex is required. In summary, the morphological and reproductive results suggest these lineages are four separate species, and that incomplete genetic lineage sorting may prevent the accurate phylogenetic detection of distinct species with the DNA markers utilized in this study, demonstrating the value of morphological and reproductive data when examining closely related lineages.

Disappearance and Return of an Outbreak of the Coral-killing Cyanobacteriosponge Terpios hoshinota in Southern Japan.

Masashi Yomogida, Masaru Mizuyama, Toshiki Kubomura, and James Davis Reimer (2017) Terpios hoshinota is cyanobacteriosponge that can cause serious damage to coral reef ecosystems by undergoing rapid breakouts in which it smothers and encrusts hard substrates, killing living sessile benthic organisms and reducing biodiversity of the a ected area. The reasons for these outbreaks are still unclear, as are long-term prognoses of affected reefs. Some reports have suggested outbreaks may not be permanent, but very little long-term monitoring information exists. In this study, we report on a T. hoshinota outbreak (~24% coverage) at Yakomo, Okinoerabu-jima Island, Kagoshima, Japan between 2010 to 2014. In this period, the existing outbreak was seen to almost completely disappear (~0%) after unusually severe Typhoon Songda passed by in early June 2011. After this, macroalgae and a cyanobacterial bloom became the dominant benthos, but by September 2014, T. hoshinota coverage had recovered to approximately half of its pre-typhoon coverage, suggesting the conditions that had caused the outbreak still persisted at Yakomo. While the conditions promoting T. hoshinota growth at this site remain uncertain, it appears that subtropical typhoons could play an important role in the dynamics of T. hoshinota outbreaks and disappearances.

Effect of phase shift from corals to Zoantharia on reef fish assemblages.

Consequences of reef phase shifts on fish communities remain poorly understood. Studies on the causes, effects and consequences of phase shifts on reef fish communities have only been considered for coral-to-macroalgae shifts. Therefore, there is a large information gap regarding the consequences of novel phase shifts and how these kinds of phase shifts impact on fish assemblages. This study aimed to compare the fish assemblages on reefs under normal conditions (relatively high cover of corals) to those which have shifted to a dominance of the zoantharian Palythoa cf. variabilis on coral reefs in Todos os Santos Bay (TSB), Brazilian eastern coast. We examined eight reefs, where we estimated cover of corals and P. cf. variabilis and coral reef fish richness, abundance and body size. Fish richness differed significantly between normal reefs (48 species) and phase-shift reefs (38 species), a 20% reduction in species. However there was no difference in fish abundance between normal and phase shift reefs. One fish species, Chaetodon striatus, was significantly less abundant on normal reefs. The differences in fish assemblages between different reef phases was due to differences in trophic groups of fish; on normal reefs carnivorous fishes were more abundant, while on phase shift reefs mobile invertivores dominated.

Effects of causeway construction on environment and biota of subtropical tidal flats in Okinawa, Japan.

Okinawa, Japan is known for its high marine biodiversity, yet little work has been performed on examining impacts of numerous large-scale coastal development projects on its marine ecosystems. Here, we examine apparent impacts of the construction of the Kaichu-Doro causeway, which was built over 40 years ago. The causeway is a 4.75 km long embankment that divides a large tidal flat and has only two points of water exchange along its entire length. We employed quadrats, transects, sampling, visual surveys, and microbial community analyses combined with environmental, water quality data, and 1m cores, at five stations of two paired sites each (one on each side of Kaichu-Doro) to investigate how the environment and biota have changed since the Kaichu-Doro was built. Results indicate reduction in water flow, and site S1 was particularly heavily impacted by poor water quality, with low diversity and disturbed biotic communities.

Typhoon damage on a shallow mesophotic reef in Okinawa, Japan.

Little is known about effects of large storm systems on mesophotic reefs. This study reports on how Typhoon 17 (Jelawat) affected Ryugu Reef on Okinawa-jima, Japan in September 2012. Benthic communities were surveyed before and after the typhoon using line intercept transect method. Comparison of the benthic assemblages showed highly significant differences in coral coverage at depths of 25-32 m before and after Typhoon 17. A large deep stand of Pachyseris foliosa was apparently less resistant to the storm than the shallower high diversity area of this reef. Contradictory to common perception, this research shows that large foliose corals at deeper depths are just as susceptible to typhoon damage as shallower branching corals. However, descriptive functional group analyses resulted in only minor changes after the disturbance, suggesting the high likelihood of recovery and the high resilience capacity of this mesophotic reef.