Trends in mercury concentrations and methylation in Minamata Bay, Japan, between 2014 and 2018.

Methylmercury concentrations in Minamata Bay are high, but the cause is unclear. We conducted a basic study on the behavior of methylmercury in Minamata Bay seawater; the findings suggest that mercury methylation may occur throughout the year in Minamata Bay. Seawater temperature, salinity, and concentrations of dissolved organic carbon were the environmental factors that affected methylation, and the degree of methylation was closely related to bacterial community structure. The concentration of methylmercury in suspended particulate matter was highest 10 m below the surface and decreased with greater depths. We did not observe a correlation between methylmercury concentrations in suspended particulate matter and concentrations of dissolved methylmercury.

Mitochondria as a Potential Target for the Development of Prophylactic and Therapeutic Drugs against Schistosoma mansoni Infection.

The emergence of parasites resistant to praziquantel, the only therapeutic agent, and its ineffectiveness as a prophylactic agent (inactive against the migratory/juvenile Schistosoma mansoni), make the development of new antischistosomal drugs urgent. The parasite's mitochondrion is an attractive target for drug development, because this organelle is essential for survival throughout the parasite's life cycle. We investigated the effects of 116 compounds against Schistosoma mansoni cercaria motility that have been reported to affect mitochondrion-related processes in other organisms. Next, eight compounds plus two controls (mefloquine and praziquantel) were selected and assayed against the motility of schistosomula (in vitro) and adults (ex vivo). Prophylactic and therapeutic assays were performed using infected mouse models. Inhibition of oxygen consumption rate (OCR) was assayed using Seahorse XFe24 analyzer. All selected compounds showed excellent prophylactic activity, reducing the worm burden in the lungs to less than 15% of that obtained in the vehicle control. Notably, ascofuranone showed the highest activity, with a 98% reduction of the worm burden, suggesting the potential for the development of ascofuranone as a prophylactic agent. The worm burden of infected mice with S. mansoni at the adult stage was reduced by more than 50% in mice treated with mefloquine, nitazoxanide, amiodarone, ascofuranone, pyrvinium pamoate, or plumbagin. Moreover, adult mitochondrial OCR was severely inhibited by ascofuranone, atovaquone, and nitazoxanide, while pyrvinium pamoate inhibited both mitochondrial and nonmitochondrial OCRs. These results demonstrate that the mitochondria of S. mansoni are a feasible target for drug development.

Potential oxygen consumption and community composition of sediment bacteria in a seasonally hypoxic enclosed bay.

The dynamics of potential oxygen consumption at the sediment surface in a seasonally hypoxic bay were monitored monthly by applying a tetrazolium dye (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride [INT]) reduction assay to intact sediment core samples for two consecutive years (2012-2013). Based on the empirically determined correlation between INT reduction (INT-formazan formation) and actual oxygen consumption of sediment samples, we inferred the relative contribution of biological and non-biological (chemical) processes to the potential whole oxygen consumption in the collected sediment samples. It was demonstrated that both potentials consistently increased and reached a maximum during summer hypoxia in each year. For samples collected in 2012, amplicon sequence variants (ASVs) of the bacterial 16S rRNA genes derived from the sediment surface revealed a sharp increase in the relative abundance of sulfate reducing bacteria toward hypoxia. In addition, a notable shift in other bacterial compositions was observed before and after the INT assay incubation. It was Arcobacter (Arcobacteraceae, Campylobacteria), a putative sulfur-oxidizing bacterial genus, that increased markedly during the assay period in the summer samples. These findings have implications not only for members of Delta- and Gammaproteobacteria that are consistently responsible for the consumption of dissolved oxygen (DO) year-round in the sediment, but also for those that might grow rapidly in response to episodic DO supply on the sediment surface during midst of seasonal hypoxia.

Phylogeny and Toxin Profile of Freshwater Pufferfish (Genus Pao) Collected from 2 Different Regions in Cambodia.

The species classification of Cambodian freshwater pufferfish is incomplete and confusing, and scientific information on their toxicity and toxin profile is limited. In the present study, to accumulate information on the phylogeny and toxin profile of freshwater pufferfish, and to contribute to food safety in Cambodia, we conducted simultaneous genetic-based phylogenetic and toxin analyses using freshwater pufferfish individuals collected from Phnom Penh and Kratie (designated PNH and KTI, respectively). Phylogenetic analysis of partial sequences of three mitochondrial genes (cytochrome b, 16S rRNA, and cytochrome c oxidase subunit I) determined for each fish revealed that PNH and KTI are different species in the genus Pao (designated Pao sp. A and Pao sp. B, respectively). A partial sequence of the nuclear tributyltin-binding protein type 2 (TBT-bp2) gene differentiated the species at the amino acid level. Instrumental analysis of the toxin profile revealed that both Pao sp. A and Pao sp. B possess saxitoxins (STXs), comprising STX as the main component. In Pao sp. A, the toxin concentration in each tissue was extremely high, far exceeding the regulatory limit for STXs set by the Codex Committee, whereas in Pao sp. B, only the skin contained high toxin concentrations. The difference in the STX accumulation ability between the two species with different TBT-bp2 sequences suggests that TBT-bp2 is involved in STX accumulation in freshwater pufferfish.

Dynamics of Sulfate-Reducing Bacteria Community Structure in Surface Sediment of a Seasonally Hypoxic Enclosed Bay.

We herein report on the dynamics of a sulfate-reducing bacteria (SRB) community structure in the surface sediment of a seasonally hypoxic enclosed bay for two consecutive years (2012 and 2013). The uppermost (0-5 mm) and subsurface (5-10 mm) layers of sediment were examined with a terminal-restriction fragment length polymorphism (T-RFLP) analysis based on the dissimilatory sulfite reductase (dsrA) gene. The SRB community significantly differed between the two sediment layers over the sampling period. This difference was mainly attributed to operational taxonomic units (OTUs) that were unique to either of the sediment layers. However, nearly 70% of total OTUs were shared between the two layers, with a few predominating. Therefore, no significant shift was observed in the SRB community structure under varying dissolved oxygen (DO) conditions in bottom water overlying the sediment surface. An additional analysis of 16S rRNA gene amplicon sequences, conducted for three uppermost sediment samples (July, August, and September in 2012), revealed that Desulfococcus, a member of SRB with high tolerance to oxygen, was the predominant Deltaproteobacteria across the uppermost sediment samples. Based on the predominance of shared OTUs across the SRB community in the sediment (0-10 mm) regardless of bottom-water DO, some SRB that are physiologically tolerant of a wide range of DO conditions may have dominated and masked changes in responsive SRB to DO concentrations. These results suggest that the SRB community structure in the enclosed bay became stable under repeated cycles of seasonal hypoxia, but may be compromised if the severity of hypoxia increases in the future.

Effects of bottom-water hypoxia on sediment bacterial community composition in a seasonally hypoxic enclosed bay (Omura Bay, West Kyushu, Japan).

The bacterial community strongly drives carbon and other biogeochemical cycles in marine sediment. However, little is known about the impact of dissolved oxygen (DO) availability on bacterial community composition. To fill this gap, we examined diversity, richness and structure of the bacterial population for three consecutive years (2011-2013) in the uppermost (0-5 and 0-7 mm depth) and the subsurface layers (5-10 and 7-14 mm depth) of Omura Bay, Kyushu, Japan, a seasonally hypoxic enclosed bay. Automated ribosomal intergenic spacer analysis revealed a unimodal pattern of diversity indices with DO, peaking at the suboxic (11 µM O2) condition. Shifts in the bacterial communities were also evident in response to the availability of DO. Changes in the operational taxonomic units (OTUs) that were less abundant accounted for a large part of the community dissimilarity. It was further demonstrated that the relative abundance of OTUs affiliated with Gammaproteobacteria was correlated positively with DO, while that with Deltaproteobacteria was inversely correlated with DO. These results strongly suggest that DO availability of bottom water plays a fundamental role in shaping the bacterial community in sediment surfaces of shallow coastal areas.

Dactylodinium pterobelotum gen. et sp. nov., a new marine woloszynskioid dinoflagellate positioned between the two families Borghiellaceae and Suessiaceae.

A new marine woloszynskioid dinoflagellate Dactylodinium pterobelotum gen. et sp. nov., collected from a southern Vietnamese estuary, was described on the basis of LM, SEM, and TEM, and molecular phylogeny inferred from rDNA sequences. This species had the smallest number of amphiesmal vesicles (5 latitudinal series) in woloszynskioid dinoflagellates assigned to the Suessiaceae and Borghiellaceae. The eyespot was of type B, composed of osmiophilic globules and brick-like material, located in- and outside of the chloroplast respectively. An apical structure comprised a pair of elongate anterior vesicles (PEV). A large peduncle was conspicuous, located in the sulcal extension in the epicone, and supported by a microtubular strand of ~140 microtubules. Ultrastructural features of trichocysts represent a novel type in the Dinophyceae, bearing lateral hairs besides anterior fibers. The molecular phylogeny based on partial LSU rDNA showed the species in a basal position in the family Suessiaceae; this indicates the eyespot type B and PEV of the Borghiellaceae are ancestral states of the eyespot comprising brick-like material (type E) and an elongate apical vesicle of the Suessiaceae.

Dynamics of bacterial community structure on intertidal sandflat inhabited by the ghost shrimp Nihonotrypaea harmandi (Decapoda: Axiidea: Callianassidae) in Tomioka Bay, Amakusa, Japan.

Callianassid (ghost) shrimp has been claimed as an ecosystem engineer, as it is one of the most powerful bioturbating macrobenthos in intertidal sandflats. However, our knowledge about the relationship between areal distribution of bottom-dwelling ghost shrimps and dynamics of sediment microbial community structure remains obscured. We used automated ribosomal intergenic spacer analysis (ARISA) to reveal the bacterial community dynamics in the sediment of intertidal sandflat of Tomioka Bay, Kyushu, Japan, which is predominantly inhabited by a burrow-dwelling callianassid shrimp Nihonotrypaea harmandi. We found that the bacterial community structures of high and middle shrimp population areas were significantly differentiated from those of low population area (ANOSIM, R=0.10-0.18, p<0.01), while the former two areas were statistically indistinguishable (ANOSIM, R=-0.015, p>0.1). These results illustrated the potential importance of shrimp population density as a key factor in shaping the bacterial community structure and interpreting their dynamics in the sandflat. Furthermore, greater similarity between burrow and non-burrow communities was found in samples taken in autumn through winter than in those in summer (one-way ANOVA, p<0.05), whereas the phylotype richness was not simply differentiated by seasons. These results suggest not only that environmental variables including water temperature and salinity of the water column overlying the sandflat could exert notable impacts on the sediment bacterial community dynamics, but that the bio-irrigation and mixing by the ghost shrimp in permeable sandflat would strongly homogenize sediment particles, enhance solute transport surrounding the burrow and ambient subsurface substrate, and therefore reduce spatial differentiation of the bacterial community structure between the two sites. A comparison between present and previous studies of axiidean (former taxonomic group name, thalassinidean) ghost shrimps provides us with a comprehensive understanding of the shrimps' impacts on bacterial community dynamics, highlighting the importance of sediment permeability, a characteristic determined by the type of sediment, as a key controlling factor to shape spatial heterogeneity of bacterial community structure around burrow.

Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein.

Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490 nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max)  ≈ 490 to λ(max)  ≈ 476 nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472 nm, whereas other Vibrio strains emit light with a peak at around 482 nm. Therefore, we investigated the mechanism underlying this blue shift in V. azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V. azureus.

Increase in Alphaproteobacteria in association with a polychaete, Capitella sp. I, in the organically enriched sediment.

We conducted bioremediation experiments on the organically enriched sediment on the sea floor just below a fish farm, introducing artificially mass-cultured colonies of deposit-feeding polychaete, Capitella sp. I. To clarify the association between the Capitella and bacteria on the efficient decomposition of the organic matter in the sediment in the experiments, we tried to identify the bacteria that increased in the microbial community in the sediment with dense patches of the Capitella. The relationship between TOC and quinone content of the sediment as an indicator of the bacterial abundance was not clear, while a significant positive correlation was found between Capitella biomass and quinone content of the sediment. In particular, ubiquinone-10, which is present in members of the class Alphaproteobacteria, increased in the sediment with dense patches of the Capitella. We performed denaturing gradient gel electrophoresis (DGGE) analyses to identify the alphaproteobacterial species in the sediment with dense patches of the worm, using two DGGE fragments obtained from the sediment samples and one fragment from the worm body. The sequences of these DGGE fragments were closely related to the specific members of the Roseobacter clade. In the associated system with the Capitella and the bacteria in the organically enriched sediment, the decomposition of the organic matter may proceed rapidly. It is very likely that the Capitella works as a promoter of bacteria in the organically enriched sediment, and feeds the increased bacteria as one of the main foods, while the bacteria decompose the organic matter in the sediment with the assistance of the Capitella.

[Effects of the disclosure of homosexuality on heterosexual undergraduates' behaviors with a same-sex close friend and their attitudes toward homosexuality].

This study investigated heterosexual undergraduates' behavior with a same-sex close friend and their attitudes toward homosexuality after this friend disclosed his/her sexual orientation. The study also examined whether the heterosexual friend was regarded as a romantic love object or not. Participants were 77 male and 139 female undergraduates. Males decreased their behaviors with their close friend and adopted more positive attitudes toward gay men after they knew their friend's sexual orientation. Females decreased their behavior with their close friend more after learning that they were a romantic love object of their friend, compared to when tehy were not. Also females adopted more positive attitudes toward lesbians only after knowing they were not a romantic love object. These gender differences are discussed.

Photobacterium aquimaris sp. nov., a luminous marine bacterium isolated from seawater.

Two luminous marine bacteria, strains LC2-065(T) and LC2-102, were isolated from seawater at Sagami Bay in Japan. These bacteria were Gram-negative, oxidase-negative, catalase-positive, motile and coccoid-rods. 16S rRNA gene sequence analysis and multilocus sequence analysis (MLSA) using six loci (ftsZ, gapA, gyrB, mreB, pyrH and topA) and sequence analysis of the alpha subunit of luciferase (luxA) gene revealed that these bacteria were distinct from other species of the genus Photobacterium. These novel strains were most closely related to Photobacterium kishitanii. The DNA-DNA hybridization value between strain LC2-065(T) and Photobacterium kishitanii ATCC BAA-1194(T) was 42.1 %. The major fatty acids were C(12 : 0,) C(14 : 0), C(16 : 0), C(18 : 0) and C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c (summed feature 3). The DNA G+C contents of strains LC2-065(T) and LC2-086 were 42.2 and 42.9 mol%, respectively. The phenotypic features of the novel strains were similar to those of P. kishitanii and P. phosphoreum, but there were sufficient physiological differences for the novel strains to be easily differentiated. On the basis of these results, these new strains represent a novel species, for which the name Photobacterium aquimaris sp. nov. is proposed. The type strain is LC2-065(T) (=NBRC 104633(T)=KCTC 22356(T)).

Vibrio azureus sp. nov., a luminous marine bacterium isolated from seawater.

Two luminous marine bacterial strains, LC2-005(T) and LC2-102, were isolated from seawater at Kuroshio Region and Sagami Bay in Japan, respectively. These bacteria were Gram-negative, oxidase-positive, catalase-positive, motile and rod-shaped. On the basis of 16S rRNA gene sequence analysis, strains LC2-005(T) and LC2-102 formed a cluster within the Vibrio harveyi species group. However, multilocus sequence analysis using five loci (pyrH, ftsZ, mreB, gyrB and gapA) and DNA-DNA hybridization experiments indicated that these strains were distinct from the currently known Vibrio species. Additionally, these strains differ from related Vibrio species in utilization of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose and arabinose, production of lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, esterase (C4), lipase (C4), chymotrypsin, acid phosphatase, alpha-glucosidase, beta-glucosidase and N-acetyl-beta-glucosaminidase and the ability to reduce nitrate to nitrite. The major fatty acids were C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c, C(16 : 0), C(18 : 1)omega7c and C(14 : 0). The DNA G+C contents of strains LC2-005(T) and LC2-102 were 45.2 and 45.5 mol%, respectively. On the basis of the polyphasic taxonomic evidence presented in this study, it can be concluded that strains LC2-005(T) and LC2-102 belong to the same genospecies and represent a novel species of the genus Vibrio, for which the name Vibrio azureus sp. nov. is proposed. The type strain is LC2-005(T) (=NBRC 104587(T) =KCTC 22352(T)).

Co-inoculation of Capitella sp. I with its synergistic bacteria enhances degradation of organic matter in organically enriched sediment below fish farms.

A polychaete, Capitella sp. I has been shown to degrade organics actively in organically enriched sediment below fish farms. Our aim of the present study is to enhance the biological treatment of sediment by co-inoculation of Capitella sp. I with bacterial isolates that possess high degrading potential for organic matter. We isolated a total of 200 bacterial strains from fecal pellets, burrow lining, worm body, and sediment, and selected six of them for the degradation experiments in the sediment microcosms. With two out of the six isolates, tentatively identified as Vibrio sp. and Vibrio cyclitrophicus by 16SrDNA sequence, we found the TOC reduction rate was stimulated in sediment co-inoculated with the worms and each of the bacteria. In contrast, this was not observed in sediments inoculated only with the worms or the bacterium. These results strongly suggest that co-inoculation of Capitella sp. I with bacteria improves biodegradation.

LuxA gene of light organ symbionts of the bioluminescent fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) forms a lineage closely related to that of Photobacterium leiognathi ssp. mandapamensis.

A molecular phylogenetic analysis of luxA gene sequences of light organ symbionts of the fish Acropoma japonicum (Acropomatidae) and Siphamia versicolor (Apogonidae) revealed that the sequences were related to those of Photobacterium leiognathi ssp. mandapamensis, which is not known to occur as a light organ symbiont among bioluminescent P. leiognathi clades. The presence of another lux gene element, luxF, coding for nonfluorescent protein, provided additional support for the identity of the light organ symbionts of the fish. Cladogenesis of the light organ symbiont P. leiognathi may be influenced by the radiation of host fishes.

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis.

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.

Microbial responses using denaturing gradient gel electrophoresis to oil and chemical dispersant in enclosed ecosystems.

Microbial responses to the addition of oil with or without a chemical dispersant were examined in mesocosm and microcosm experiments by using denaturing gradient gel electrophoresis of bacterial ribosomal DNA and direct cell counting. When a water-soluble fraction of oil was added to seawater, increases in cell density were observed in the first 24h, followed by a decrease in abundance and a change in bacterial species composition. After addition of an oil-dispersant mixture, increases in cell density and changes in community structure coincided, and the amount of bacteria remained high. These phenomena also occurred in response to addition of only dispersant. Our results suggest that the chemical dispersant may be used as a nutrient source by some bacterial groups and may directly or indirectly prevent the growth of other bacterial groups.

Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting.

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.