Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan

The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied. A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar. In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples. Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water. In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea. In the identification of 463 isolates, 23 Vibrio spp. and 2 Listonella spp. were observed. V. harveyi was prevalent among the members of the Vibrio genus. Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group. In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region. V. vulnificus, V. fluvialis, and V. cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence. It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical composition of Laurencia spp. collected from the Seto Inland Sea of Japan

The chemical composition of three Laurencia spp., Laurencia sp., L. okamurae and L. saitoi, which were collected from the Seto Inland Sea of Japan, has been examined. Laurencia sp. collected from the coast of Matoba Park, Takehara, Hiroshima Prefecture, contained a brominated chamigrane-type sesquiterpene (1), named matobol, as the main metabolite. The structure of matobol was determined as (+)-(2R,3R,6R,10S)-2,10-dibromochamigr-7(14)-en-3-ol (1). This is the first time that the optically active 1 has been isolated from Laurencia. On the other hand, L. okamurae from the coast of Ikunoshima Island, Hiroshima Prefecture, produced laurinterol (2) that is a known cyclolaurane-type sesquiterpene characteristic to this species in Japan. L. saitoi from the coast of Matoba Park contained a known bromoallenic C₁₅-acetogenin, neolaurallene (3).     

Incidence of Salmonella spp., Clostridium botulinum, and Vibrio parahaemolyticus in an estuary.

A study of the incidence of Salmonella spp., Vibrio parahaemolyticus-like organisms, and clostridium botulinum in samples collected at five stations located in the Upper Chesapeake Bay, a major estuary on the Atlantic Coast of the United States, was conducted in December 1973 through December 1974. C. botulinum types B and E were detected in 12.3% of the total sediment samples examined. V. parahaemolyticus was recovered from 10.4% of a total of 86 water, sediment, and suspended sediment samples. Of 131 samples examined for the presence of Salmonella spp., approximately 3% were found to be positive for serologically confirmed Salmonella isolates. Shellfish examined during the investigation were also found to be free of enteric pathogens. The low frequency of occurrence of V. parahaemolyticus was attributed to the low salinities encountered at the sites included in the study. A low incidence of Salmonella spp. in the Upper Chesapeake Bay samples was found, whereas the distribution of C. botulinum appeared to be both random and autochthonous. A strong relationship between presence of potential pathogens and other generally accepted microbiological indicators of pollution was not observed.     

Blooms of tunicates Oikopleura spp. and Dolioletta gegenbauri in the Seto Inland Sea, Japan, during summer

The ecological importance of appendicularians Oikopleura dioica and O. longicauda, and a doliolid Dolioletta gegenbauri as metazoan secondary producers was assessed in summer 1997 in the Seto Inland Sea, Japan. Blooms by the appendicularians occurred twice within a month following peaks of pico-/nanoplankton biomass. The biomass of Oikopleura spp. averaged over the water column (<B> ranged 0.1 to 8.0 μgC l-¹ and temporal average of <B> over the survey period ( ) was 3.2 μgC l-¹, 70% of for calanoid copepods. Furthermore, the temporal average of the production for Oikopleura spp. was 2.4 times higher than that for calanoids, reflecting the extraordinary high growth rates of the appendicularians. D. gegenbauri developed its population following a diatom bloom and <B> reached to 37 μgC l-¹ at the peak time. Although individuals of D. gegenbauri were seldom observed during the first half of the survey, for the doliolid (8.4 μgC l-¹) nearly doubled that for calanoids. These results indicate that the gelatinous tunicates Oikopleura spp. and D. gegenbauri play important roles as metazoan secondary producers in the Seto Inland Sea during summer. This revised version was published online in September 2006 with corrections to the Cover Date.     

Studies on pathogenic Vibrio parahaemolyticus during a warm weather season in the Seto Inland Sea,Japan

Vibrio parahaemolyticus is a potentially pathogenic bacterium, occurring naturally in estuarine and marine environments throughout the world. The incidence of this organism in an aquatic environment depends upon many ecofactors. Sea water and organic material were collected during the warm weather season from a coast of the Seto Inland Sea, Japan, and analysed to determine V. parahaemolyticus densities and the occurrence of pathogenic strains, defined as those possessing tdh and/or trh genes by polymerase chain reaction (PCR), using isolated DNA from enrichment culture of the samples. About 99% of samples were positive for V. parahaemolyticus with densities of 3 to >1400 cells per 100 ml of water or 10 g of organic samples by the most-probable-number (MPN)-PCR technique, but only 76.6% were positive by the conventional MPN culture technique, with densities ranging from 3 to >1400 cells per 100 ml of water or 10 g of organics. Furthermore, the tdh and trh genes were positive in 41.5% and 8.5% of samples, respectively, by the MPN-PCR technique. No tdh and trh gene-positive strains were isolated by the conventional MPN culture procedure. The difference in detection between the MPN culture and the MPN-PCR techniques appeared to be significant and may be attributed to different detection sensitivities and other factors.     

Vertical distribution and germination ability of Alexandrium spp. cysts (Dinophyceae) in the sediments collected from Kure Bay of the Seto Inland Sea, Japan

The vertical distribution of Alexandrium tamarense/ catenella (hereinafter Alexandrium spp.) cysts was investigated with special attention to living cysts filled with fresh protoplasm and empty cysts. In addition, based on the incubation experiments of Alexandrium spp. cysts, the germination ability of the cysts was examined. A sediment core 63 cm in length, collected from Kure Bay of the Seto Inland Sea, West Japan, in September 2000, was provided for an analysis on the vertical distribution of Alexandrium spp. cysts. Samples from every 1 cm interval depth from the top down to 13 cm depth of the same core were taken to examine the germination ability of the cysts. Results show that Alexandrium spp. cysts were continuously observed from 59 to 60 cm depth to the top. The cyst densities in the upper parts of the core (from 9 to 10 cm depth to the top) were much more abundant those that in the lower parts (below 10–11 cm depth). The relationship between living and empty cysts in each depth did not reveal a positive correlation with the sediment depth. Based on the sedimentation rate of the core sediment (approximately 1.6 cm/year), Alexandrium spp. cysts have been produced since 1962, and a remarkable increase of these cysts was observed from ca 1993. Such a rapid increase of Alexandrium spp. cysts has probably been as a result of dense blooms of A. tamarense occurring since 1992 in Hiroshima Bay, including Kure Bay. The germination of Alexandrium spp. cysts was observed in samples collected from the top to 12–13 cm depth of the core. It suggests that the Alexandrium spp. cysts can keep the germination ability for more than 8 years.     

Carbon and nitrogen content of Noctiluca scintillans in the Seto Inland Sea, Japan

The carbon and nitrogen content of Noctiluca scintillans cellsfrom the Seto Inland Sea, Japan was investigated in order toestimate its biomass in natural samples. The carbon contentof N.scintillans ranged from 123 to 627 ng C cell^–1 witha mean value of 353 ng C cell^–1, or 1.12 to 2.67 fg Cµm^–3 with a mean value of 1.98 fg C µm^–3.The nitrogen content ranged from 36.0 to 232 ng N cell^–1with a mean value of 131 ng N cell^–1, or 0.499 to 0.910fg N µm^–3 with a mean value of 0.694 fg N µm^–3.Total cell carbon and nitrogen increased but the carbon andnitrogen per cell volume decreased with increasing cell volume.The C/N ratio of the cells ranged from 2.3 to 4.4, which wasrelatively low compared with the Redfield ratio. The carbonand nitrogen content was extremely low (91.2 ng C cell^–1,41.8 ng N cell^–1) for starved cells, whereas it was extremelyhigh (528 ng C cell^–1, 205 ng N cell^–1) for cellswhich had ingested the large diatom, Coscinodiscus wailesii.Our results suggest that the carbon and nitrogen content ofN.scintillans varies depending on its physiological conditionand the type of food that it has recently consumed.     

Biomass, feeding and production of Noctiluca scintillans in the Seto Inland Sea, Japan

The abundance and biomass of the large heterotrophic dinoflagellateNoctiluca scintillans, together with the changes in its potentialprey items, were monitored in the Seto Inland Sea, Japan, duringsummer 1997 (17 July-11 August). Growth and grazing rates ofNscintillans fed natural plankton populations were also measuredeight and seven times, respectively, during the survey period.The abundance and biomass of N scintillans averaged over thewater column (19 m) were in the range 1–345 cells 1^–1(temporalaverage = 93 cell1^–1) and 0.1–49.6 µg C l^–1(temporalaverage = 13.8 µg C l^–1; three times higher thanthat of calanoid copepods during the same period). Noctilucascintillans populations followed the changes in phytoplankton:N.scintillans biomass was increasing during the period of diatomblooms and was at a plateau or decreasing during periods oflow chlorophyll a. The growth rates of N.scintillans (µ)were also consistent with the wax and wane of the N.scintillanspopulation: N.scintillans showed highest growth rates duringdiatom blooms. A simple relationship between µ and chlorophylla concentration was established, and the production of N.scintillanswas estimated using this relationship and the measured biomass.The estimated production averaged over the water column wasin the range >0.1–5.2 µg C l^–1 day^–1(temporalaverage = 1.4 µg C l^–1 day^–1; 64% of the productionof calanoid copepods during the same period). Diatom clearancerates by N.scintillans were in the range 0.10–0.35 mlcell^–1 day^–1, and the phytoplankton population clearanceby N.scintillans was >12% day^–1. Thus, although thefeeding pressure of N.scintillans on phytoplankton standingstock was low, N.scintillans was an important member of themesozooplank-ton in terms of biomass and production in the SetoInland Sea during summer.     

Annual sediment respiration in estuarine sandy intertidal flats in the Seto Inland Sea, Japan

To quantify organic matter mineralization at estuarine intertidal flats, we measured in situ sediment respiration rates using an infrared gas analyzer in estuarine sandy intertidal flats located in the northwestern Seto Inland Sea, Japan. In situ sediment respiration rates showed spatial and seasonal variations, and the mean of the rates is 38.8 mg CO₂-C m⁻² h⁻¹ in summer. In situ sediment respiration rates changed significantly with sediment temperature at the study sites (r ² = 0.70, p < 0.05), although we did not detect any significant correlations between the rates and sediment characteristics. We prepared a model for estimating the annual sediment respiration based on the in situ sediment respiration rates and their temperature coefficient (Q ₁₀ = 1.8). The annual sediment respiration was estimated to be 92 g CO₂-C m⁻² year⁻¹. The total amount of organic carbon mineralization for the entire estuarine intertidal flats through sediment respiration (43 t C year⁻¹) is equivalent to approximately 25% of the annual organic carbon load supplied from the river basin of the estuary.     

Biology of the swimming acorn worm Glandiceps hacksi from the Seto Inland Sea of Japan

The enteropneust hemichordate Glandiceps hacksi inhabits the muddy bottoms of the intertidal to subtidal zones of Koguno-shima Island, located in the central part of the Seto Inland Sea of Japan. Monthly collections from October 2005 to September 2007 revealed that their spawning occurs once a year, in the latter half of May. Parameters such as density and sex ratio, as well as the type of sediment, were also examined. Worm behavior and type of burrows revealed that G. hacksi are infaunal burrowers. Autotomy and regeneration of their posterior regions, and swimming behavior were also observed in an aquarium environment. This is the first comprehensive study on the biology of G. hacksi, the swimming acorn worm.     

Abundance of Naked Amoebae in Sediments of Hiroshima Bay, Seto Inland Sea of Japan

ABSTRACT The present paper provides the first data on naked amoebae from sediments of Hiroshima Bay. Three stations in the inner part of the bay were sampled over a three-month period. Abundance of naked amoebae ranged from 1,019 to 45,561 cells/g dry sediment. Results indicate: (i) surface sediment populations in most cases were higher than subsurface populations; (ii) there was some evidence of temporal variation with counts generally increasing from March to May: and (iii) the site located near Hiroshima City had fewer amoebae on several occasions than the other two sites. There was a negative exponential relationship between acid-volatile sulfide concentration and abundance of amoebae. Most amoebae were small with the average size ranging from 6.6–14 μm. Morphotype 1, amoebae that extend lobose pseudopodia or subpseudopodia during normal locomotion, were dominant (40–100% of enumerated amoebae). Morphotypes 2 and 3 (limax amoebae) were found in lower numbers than the other two morphotypes. The proportion of amoebae occupied by Morphotype 4 (fan-shaped or discoidal-flattened amoebae) was higher at a lower total abundance.     

Reproductive seasonality of the seaweed pipefish Syngnathus schlegeli (Syngnathidae) in the Seto Inland Sea, Japan

Latitudinal variations in fitness-related traits have been reported in a variety of organisms. Intraspecific comparison of such traits among populations living under different environmental conditions is an effective approach for elucidating the cause and consequences of such latitudinal variations. In the present study, population demography, seasonal changes in somatic and reproductive conditions, and the occurrence of egg-brooding males were investigated in the seaweed pipefish, Syngnathus schlegeli, collected monthly in the Seto Inland Sea, Japan, in order to estimate the reproductive season and mating pattern. The findings were then compared with those reported for a population in Otsuchi Bay, northern Japan. We found that the reproductive season is longer in the Seto Inland Sea population (March–November) than in the Otsuchi Bay population (May–October), although reproductive activity may temporarily cease during August in the Seto Inland Sea population. Males of the Seto Inland Sea population brooded eggs that were at the same developmental stage, suggesting that males mate with only one female in a single brooding episode (i.e., monogamy)—in contrast to the Otsuchi Bay population, where a portion of males brooded eggs consisting of multiple clutches at different developmental stages, suggesting that multiple females contribute to a single brood (i.e., polygamy). Additionally, we found that the standard lengths of both males and females are approximately 30?mm smaller in the Seto Inland Sea than in the Otsuchi Bay population. These results suggest that the multiple mating by males in a single brood and the larger body size in the Otsuchi Bay population are results of fecundity selection on life history and behavioral traits to adapt to the shorter reproductive season and lower reproductive efficiency in this relatively cold environment.     

Seasonal variation in the species composition of tintinnid cilates in Hiroshima Bay, the Seto Inland Sea of Japan

Seasonal changes in the species composition of tintinnid ciliateswere examined based on time-series samples taken at 2 week intervalsover a 3 year period in Hiroshima Bay, the Seto Inland Sea ofJapan. The maximum abundance of total tintinnids over the entireperiod was 5.7 x 10³ indi viduals l^– Among 32 speciesidentified, a consistent seasonal occurrence was recognizedin 22 species. The relationships between various environmentalfactors and the abundance of each species of tintinnids wereanalyzed using principal component analysis From this analysis,the abundance of many tintinnids was revealed to be associatedwith temperature, the <20 µm size fraction of chlorophylla and water column stability, but not with the <20 µmsize fraction of chlorophyll a, nor with salinity. From theseresults, tintinnid species were divided into five associationtypes: species whose abundance increased with increasing temperature,decreasing temperature, nanophytoplankton abundance, increasingwater column mixing, or increasing water stratification coupledwith low temperature.     

Renewal of genetic composition of a lancelet, Branchiostoma belcheri, in the Seto Inland Sea, Japan

We studied renewal of genetic composition of a lancelet, Branchiostoma belcheri, at a station in the Seto Inland Sea, Japan, using random amplified polymorphic DNA (RAPD) technique. We detected a total of 49 DNA bands from 27 individuals of 5 seasonal samples. Among these, some were specific to individual or seasonal sample. There were no bands common to all individuals. We calculated the Band Sharing Index (BSI) values for each combination of individuals. BSI was in a wide range of 0.00-0.97 and the average BSI between the individuals of a sample was remarkably different from sample to sample (0.37-0.83). In cluster analysis on the basis of BSI, largely 3 clusters were constructed, which finally connected to a single cluster at as low as about 0.2 of BSI. Besides, many clusters were constructed exclusively by a seasonal sample. These results suggest a large gene pool of lancelets in this area, the remarkable differences in gene pool size among the samples and a seasonal renewal of genetic composition at a habitat.     

Photosynthetic property and primary production of phytoplankton in sublittoral sand bank area in the Seto Inland Sea,Japan

We investigated the photosynthesis–light intensity (P–I) relationships of phytoplankton collected from a sublittoral sand bank in the Seto Inland Sea, Japan, under different temperature conditions. In spite of low chlorophyll a concentration (<3 mg m⁻³), phytoplankton had considerably high photosynthetic potential (>10 mg C (mg chl a)⁻¹ h⁻¹) in the study area. Based on the P–I relationships, we conducted numerical simulation of areal primary production using published data on water temperature, chlorophyll a concentration, and irradiance. The areal primary production ranged between 159 and 187 g C m⁻² year⁻¹. This production was within the range of typical values reported previously in deeper areas of the Seto Inland Sea. The productivity in the sand bank area was discussed in relation to water current, allochthonous resource input, and fisheries.     

Occurrence of nitrogen fixation among Vibrio spp.

Virtually all Vibrio spp. known and available in culture collections and several newly isolated Vibrio sp. were tested for their ability to fix molecular nitrogen, using the acetylene reduction technique, the fixation of the heavy isotope ¹⁵N, and by growth on media devoid of combined nitrogen. Among the 27 species tested, four, including V. diazotrophicus, proved to be nitrogenase-positive. The potential of nitrogen fixation was now also discovered in V. natriegens, V. pelagius and V. cincinnatiensis. Among the 9 newly isolated strains, 4 were nitrogenase-positive. These strains were classified as V. diazotrophicus on the basis of DNA homology studies. Nitrogenase was only induced during growth under anaerobic conditions. Dissolved oxygen as low as 1 M inhibited nitrogenase completely. This inhibition at low oxygen concentration, however, was reversible. 50–100 M dissolved oxygen inhibited nitrogenase irreversibly.This work was carried out at Geomicrobiology Division, the University of Oldenburg, FRG     

Salmonella spp., Vibrio spp., Clostridium perfringens, and Plesiomonas shigelloides in Marine and Freshwater Invertebrates from Coastal California Ecosystems

The coastal ecosystems of California are highly utilized by humans and animals, but the ecology of fecal bacteria at the land–sea interface is not well understood. This study evaluated the distribution of potentially pathogenic bacteria in invertebrates from linked marine, estuarine, and freshwater ecosystems in central California. A variety of filter-feeding clams, mussels, worms, and crab tissues were selectively cultured for Salmonella spp., Campylobacter spp., Escherichia coli-O157, Clostridium perfringens, Plesiomonas shigelloides, and Vibrio spp. A longitudinal study assessed environmental risk factors for detecting these bacterial species in sentinel mussel batches. Putative risk factors included mussel collection near higher risk areas for livestock or human sewage exposure, adjacent human population density, season, recent precipitation, water temperature, water type, bivalve type, and freshwater outflow exposure. Bacteria detected in invertebrates included Salmonella spp., C. perfringens, P. shigelloides, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. Overall, 80% of mussel batches were culture positive for at least one of the bacterial species, although the pathogens Campylobacter, E. coli-O157, and Salmonella were not detected. Many of the same bacterial species were also cultured from upstream estuarine and riverine invertebrates. Exposure to human sewage sources, recent precipitation, and water temperature were significant risk factors for bacterial detection in sentinel mussel batches. These findings are consistent with the hypothesis that filter-feeding invertebrates along the coast concentrate fecal bacteria flowing from land to sea and show that the relationships between anthropogenic effects on coastal ecosystems and the environmental niches of fecal bacteria are complex and dynamic.     

Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan

Shellfish poisoning by the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech occurred for the first time in Hiroshima Bay, Japan, in 1992. Oyster culture in the bay produces as much as 60% of the total production in Japan, and it suffered severe damage. In the present study, we experimentally investigated the growth rate and phosphate uptake kinetics of A. tamarense, Hiroshima Bay strain. A short-term phosphate uptake experiment revealed that the maximum uptake rate was 1.4 pmol P cell^-1 per h and the half-saturation constant was 2.6 umol L^-1. In semicontin-uous culture, the maximum specific growth rate and the minimum phosphorus cell quota were 0.54 day^-1 and 0.56 pmol P cell^-1, respectively. These uptake rates suggest that A. tamarense is a poor phosphorus competitor compared with other species. However, the large phosphorus storage capacity (Q_pmax/q_o= 36), the surge phosphorus uptake ability (V_s/V_i= 4.1) and the low growth rate would be advantageous for surviving brief periods of phosphorus limitation which frequently occur in Hiroshima Bay.     

Clostridium botulinum type C in healthy swine in Japan.

Healthy cattle and swine bred in a district of Japan were examined for the presence of Clostridium botulinum in their liver. Liver specimens were cultivated in chopped meat-glucose medium and the cultures were examined for botulinum toxin. In cattle, none of the cultures of 100 liver specimens yielded the toxin. In swine, however, C1 or C2 toxin was demonstrated in 8 of 100 liver specimens from 36 farms. One of the five farms where the carrier-state swine were present was surveyed for about 2 years to determine whether the carrier-state was transient or resident. C. botulinum type C was found in swine livers and feces, and environmental specimens at extremely high rates during the surveillances, with 76% of specimens yielding botulinum toxin following the culture. These data suggest that it is not uncommon for healthy swine to carry C. botulinum type C in the liver and that there is a close relationship between C. botulinum carrier-state in swine and the presence of this organism in their raising environments. In 20 cattle and 20 swine suffering from parturient paresis of unknown etiology no evidence for involvement of C. botulinum type C was obtained.     

Optically active components and light attenuation in an offshore station of Harima Sound, eastern Seto Inland Sea, Japan

In the eastern Seto Inland Sea, Japan, phytoplankton abundance in the surface water has gradually declined, whereas Secchi depth has risen in recent years, particularly in offshore areas. Therefore, it may be hypothesized that phytoplankton dominate light attenuation in the offshore area, and that other constituents are less important. To test this hypothesis, we examined the roles of seawater, colored dissolved organic matter (CDOM), non-algal particles (tripton), and phytoplankton in the light attenuation at an offshore station of Harima Sound in the eastern Sea. The magnitude of light attenuation was then determined from the attenuation coefficient of photosynthetically available radiation (PAR) through the water column (K _d). During a 13-month period, K _d ranged from 0.179 to 0.507 m^?1, with a mean of 0.262 m^?1. The mean relative contributions of seawater (15%) and CDOM (13%) to K _d were small, while the most dominant K _d constituent was tripton (45%). The mean contribution of phytoplankton to K _d (27%) was consequently less than that of tripton. However, 75% of the temporal variability in K _d was attributed to phytoplankton, measured as chlorophyll a. Our results emphasize that the main component of light attenuation does not always govern the temporal variation of light attenuation in coastal regions.