Structure of 45,46,47-trinorhomoyessotoxin, a new yessotoxin analog, from Protoceratium reticulatum which represents the first detection of a homoyessotoxin analog in Japan

Contamination of yessotoxin (YTX) and its analogs in shellfish has occurred worldwide and has seriously damaged shellfish industries. One of the sources of YTX has been identified the dinoflagellate Protoceratium reticulatum. A new analog of YTX, 45,46,47-trinorhomoYTX, was isolated from cultures of the dinoflagellate P. reticulatum collected at Yamada Bay, Iwate in Japan. Its structure was determined by analysis of MS and NMR experiments. This is the first isolation and confirmation of a homoYTX analog in Japan.     

Protoceratium reticulatum in northern Japan: environmental factors associated with seasonal occurrence and related contamination of yessotoxin in scallops

In 2003, an occurrence of the yessotoxin (YTX)-producing dinoflagellate,Protoceratium reticulatum, and resultant toxin concentrationin scallops was monitored in Okkirai Bay, northern Japan. Theoccurrence peaked after 1 July and reached a maximum density(400 cells L^–1) on 16 July. It occurred over a wide rangeof water temperature and inorganic nutrient concentrations andwas tolerant of a broad range of environmental conditions. Hydrologicalparameters were monitored, and it was obvious that P. reticulatumflourished under low salinity (30.59–32.60) and occurredat highest density in the surface layer (0–5 m depth)where effects from rainfall were greatest. In addition, dinoflagellatedensity increase and decrease was well correlated with inflowsof oceanic water into the bay. Thus, it can be assumed thatthe oceanic inflows may cause initial population increases (e.g.excystment or input of a ‘seed population’), withresultant growth in rain-affected waters and subsequent spreadthroughout the entire bay via water movement. YTX and 45-OHYTXconcentrations in scallops reached maximum levels (0.79 µgg^–1 and 0.44 µg g^–1 of digestive gland, respectively)2 weeks after the maximum cell density of P. reticulatum, andhigh levels of the toxin continued for a month. Low levels ofthe toxin were detected even during periods when cells werenot observed.     

Multiplex PCR system applied for analysing microsatellite loci of Schlegel's black rockfish,Sebastes schlegeli

Two multiplex PCR amplifications were performed to analyse six microsatellite loci of Schlegel's black rockfish, Sebastes schlegeli, an important commercial fish in the northern part of Japan and an important species for the stock enhancement program in this area. We analysed 67 wild samples from Yamada Bay, Iwate Prefecture, Japan. The observed genotype frequencies agreed with the Hardy–Weinberg expectations at all loci, and the observed heterozygosities ranged from 0.072 to 0.897.     

Resting stages of microalgae in recent marine sediments of Peter the Great Bay,Sea of Japan

Data on the qualitative and quantitative composition of resting stages of planktonic microalgae in recent marine sediments of Peter the Great Bay (Sea of Japan) over the period 2000–2007 are presented. A total of sixty one morphological forms of resting stages represented by dinoflagellate and raphidophyte cysts and diatom spores and resting cells were recorded in the sediment samples. This study revealed cysts of the potentially toxic species Alexandrium tamarense, A. cf. minutum, Alexandrium sp., Gymnodinium catenatum (PSP toxin producers), and Protoceratium reticulatum (yessotoxin producer); resting cells of Pseudo-nitzschia sp. (potential producer of domoic acid); and cysts of bloom-forming species Cochlodinium cf. polykrikoides and Heterosigma cf. akashiwo.     

Variations in dinoflagellate cyst morphology under conditions of changing salinity during the last 2000 years in the Limfjord, Denmark

Morphological variations are examined in the dinoflagellate cysts Spiniferites spp., Lingulodinium polyedrum and Protoceratium reticulatum (=Operculodinium centrocarpum) from a core taken in the Bj?rnsholm Bay, the Limfjord, Denmark. The fjord has a history of changing salinity, and unusual cyst morphotypes are found in the greatest numbers during periods of inferred low salinity. Variation occurs primarily in cyst process morphology, and the aberrant morphotypes have processes that are shorter, thicker and/or more membranous. The different morphotypes are described and compared with other varieties and forms of the three taxa and to other closely related taxa.     

Absence of sporidesmin production by twelve Texas isolates of Pithomyces spp.

Twelve isolates of Pithomyces spp. from Texas were tested for sporidesmin toxin production, using both high-performance and thin-layer chromatography techniques. None of the Texas isolates produced the toxin under the conditions used. A control toxigenic New Zealand isolate, Pithomyces chartarum strain C, was grown simultaneously under the conditions tested and was found to produce sporidesmin in all cases.     

Accelerated sulfur cycle in coastal marine sediment beneath areas of intensive shellfish aquaculture

Prokaryotes in marine sediments taken from two neighboring semi-enclosed bays (the Yamada and Kamaishi bays) at the Sanriku coast in Japan were investigated by the culture-independent molecular phylogenetic approach coupled with chemical and activity analyses. These two bays were chosen in terms of their similar hydrogeological and chemical characteristics but different usage modes; the Yamada bay has been used for intensive shellfish aquaculture, while the Kamaishi bay has a commercial port and is not used for aquaculture. Substantial differences were found in the phylogenetic composition of 16S rRNA gene clone libraries constructed for the Yamada and Kamaishi sediments. In the Yamada library, phylotypes affiliated with delta-Proteobacteria were the most abundant, and those affiliated with gamma-Proteobacteria were the second-most abundant. In contrast, the Kamaishi library was occupied by phylotypes affiliated with Planctomycetes, gamma-Proteobacteria, delta-Proteobacteria, and Crenarchaeota. In the gamma-Proteobacteria, many Yamada phylotypes were related to free-living and symbiotic sulfur oxidizers, whereas the Kamaishi phylotype was related to the genus Pseudomonas. These results allowed us to hypothesize that sulfate-reducing and sulfur-oxidizing bacteria have become abundant in the Yamada sediment. This hypothesis was supported by quantitative competitive PCR (qcPCR) with group-specific primers. The qcPCR also suggested that organisms closely related to Desulfotalea in the Desulfobulbaceae were the major sulfate-reducing bacteria in these sediments. In addition, potential sulfate reduction and sulfur oxidation rates in the sediment samples were determined, indicating that the sulfur cycle has become active in the Yamada sediment beneath the areas of intensive shellfish aquaculture.     

Toxin-producing ability among Bacillus spp. outside the Bacillus cereus group

A total of 333 Bacillus spp. isolated from foods, water, and food plants were examined for the production of possible enterotoxins and emetic toxins using a cytotoxicity assay on Vero cells, the boar spermatozoa motility assay, and a liquid chromatography-mass spectrometry method. Eight strains produced detectable toxins; six strains were cytotoxic, three strains produced putative emetic toxins (different in size from cereulide), and one strain produced both cytotoxin(s) and putative emetic toxin(s). The toxin-producing strains could be assigned to four different species, B. subtilis, B. mojavensis, B. pumilus, or B. fusiformis, by using a polyphasic approach including biochemical, chemotaxonomic, and DNA-based analyses. Four of the strains produced cytotoxins that were concentrated by ammonium sulfate followed by dialysis, and two strains produced cytotoxins that were not concentrated by such a treatment. Two cultures maintained full cytotoxic activity, two cultures reduced their activity, and two cultures lost their activity after boiling. The two most cytotoxic strains (both B. mojavensis) were tested for toxin production at different temperatures. One of these strains produced cytotoxin at growth temperatures ranging from 25 to 42 degrees C, and no reduction in activity was observed even after 24 h of growth at 42 degrees C. The strains that produced putative emetic toxins were tested for the influence of time and temperature on the toxin production. It was shown that they produced putative emetic toxin faster or just as fast at 30 as at 22 degrees C. None of the cytotoxic strains produced B. cereus-like enterotoxins as tested by PCR or by immunological methods.     

The Seasonal Accumulation of Amnesic Toxin (Domoic Acid) in Commercial Bivalves Mytilus trossulus Gould, 1850 and Mizuhopecten yessoensis Jay, 1850 in Vostok Bay,Sea of Japan

Russian Journal of Marine Biology - The seasonal accumulation of the amnesic toxin domoic acid (DA) in the tissues of two commercial species of bivalves from Vostok Bay (the Sea of Japan) was...     

Xenorhabdus japonicus sp. nov. associated with the nematode Steinernema kushidai

A new species, Xenorhabdus japonicus, is proposed as the bacterial symbiont of Steinernema kushidai isolated from field soil in Shizuoka Prefecture, Japan. Xenorhabdus japonicus could be distinguished phenotypically and genetically from other Xenorhabdus spp. The type strain of the species, SK-1, a Gram-negative, facultative anaerobe and peritrichously flagellated rod, has colonies with primary and secondary forms. The strain can be differentiated from the type strain of Xenorhabdus nematophilus by several characters, including the formation of arginine dehydrolase, phenylalanine deaminase and lysine decarboxylase, the assimilation of inosine and L-proline and acid production from inositol. The major cellular fatty acids are 16:0, cyclo 17:0 and 18:1. The ubiquinone system is Q-8. The G+C content of DNA is 45.9 mol%. The DNA of strain SK-1 has 20 to 58% homology with that of the type strains of other Xenorhabdus spp.Y. Nishimura, A. Hagiwara and T. Suzuki are with the Department of Applied Biological Science, Science University of Tokyo, Noda 278, Japan, and SDS Biotech K.K., Tsukuba Technology Centre, Tsukuba 300-26, Japan     

Comparative accumulation and composition of lipophilic marine biotoxins in passive samplers and in mussels (M. edulis) on the West Coast of Ireland

Monitoring of lipophilic marine toxins was carried out in three shellfish production sites on the West Coast of Ireland. The toxins were monitored using passive samplers (solid phase adsorption toxin tracking; SPATT) and toxin-free mussels that were replaced weekly in the selected sampling stations. The toxin profiles and concentrations obtained in the SPATT and in the transplanted mussels were compared with those observed in indigenous (native) mussels from each production site as well as with the phytoplankton that was detected in the water. Numerous lipophilic toxins were detected in the SPATT discs by ultra-performance liquid-chromatography–mass spectrometry/mass spectrometry (UPLC–MS/MS) and included okadaic acid (OA), dinophysistoxin-2 (DTX2), pectenotoxin-2 (PTX2), AZA1, AZA2, yessotoxin (YTX) and SPX-13-DesMe-C. The accumulation rate of toxins in the indigenous mussels and in the SPATT discs correlated well. Toxins were detected in all SPATT discs from all locations, even in the absence of toxin-producing phytoplankton, as observed previously by other research groups. It is quite clear from our data that the presence of okadaic acid in the water (even at high concentrations) did not induce toxicity in the transplanted mussels in the absence of phytoplankton. A severe toxic event of azaspiracids (AZAs) occurred in one of the sampling stations. The SPATT discs accumulated predominantly AZA1 and -2 suggesting that both toxins are biosynthesized by the AZA-producing organism. As opposed to the DSP event, the AZA event resulted in the contamination of the transplanted mussels for several consecutive weeks. This is the first study that reports the presence of YTX and SPX-13-DesMe-C in Irish waters. In our study conditions, the SPATT did not enable the forecasting of shellfish contamination as the increase in toxin concentration occurred at the same time in the shellfish and in the SPATT.     

Production and excretion of okadaic acid,pectenotoxin-2 and a novel dinophysistoxin from the DSP-causing marine dinoflagellate Dinophysis acuta – Effects of light,food availability and growth phase

Diarrhetic shellfish poisoning (DSP) toxins constitute a severe economic threat to shellfish industries and a major food safety issue for shellfish consumers. The prime producers of the DSP toxins that end up in filter feeding shellfish are species of the marine mixotrophic dinoflagellate genus Dinophysis. Intraspecific toxin contents of Dinophysis spp. vary a lot, but the regulating factors of toxin content are still poorly understood. Dinophysis spp. have been shown to sequester and use chloroplasts from their ciliate prey, and with this rare mode of nutrition, irradiance and food availability could play a key role in the regulation of toxins contents and production. We investigated toxin contents, production and excretion of a Dinophysis acuta culture under different irradiances, food availabilities and growth phases. The newly isolated strain of D. acuta contained okadaic acid (OA), pectenotoxins-2 (PTX-2) and a novel dinophysistoxin (DTX) that we tentatively describe as DTX-1b isomer. We found that all three toxins were excreted to the surrounding seawater, and for OA and DTX-1b as much as 90% could be found in extracellular toxin pools. For PTX-2 somewhat less was excreted, but often >50% was found extracellularly. This was the case both in steady-state exponential growth and in food limited, stationary growth, and we emphasize the need to include extracellular toxins in future studies of DSP toxins. Cellular toxin contents were largely unaffected by irradiance, but toxins accumulated both intra- and extracellularly when starvation reduced growth rates of D. acuta. Toxin production rates were highest during exponential growth, but continued at decreased rates when cell division ceased, indicating that toxin production is not directly associated with ingestion of prey. Finally, we explore the potential of these new discoveries to shed light on the ecological role of DSP toxins.     

Mathematical description of yessotoxin production by Protoceratium reticulatum in culture

The production dynamics of yessotoxin (YTX) by Protoceratium reticulatum and phosphate uptake in culture were investigated in relation to cell growth. The equations used were: the reparametrized logistic for cell production, Luedeking–Piret model for yessotoxin production and maintenance energy model for phosphate consumption. Thus, the YTX formation rate was proportional to producer biomass at the end of the asymptotic phase of culture as a secondary metabolite. Moreover, the equations proposed showed a high accuracy to predict these bioproductions in different experimental conditions and culture scales.     

Control of slugs with contact-action molluscicides

A number of iron and aluminium compounds were compared as contact-acting poisons against the slug Deroceras reticulatum, by confining slugs on treated surfaces of glass and wet soil in laboratory tests. Simple metal salts were effective poisons when applied to a glass surface, but were rapidly inactivated when applied to wet soil. Chelation of both metals with organic ligands retarded the rate of attenuation on wet soil. In a field trial D. reticulatum and Arion spp. slugs were killed by a broadcast application of the chelate iron (III) 2,4-pentanedionate applied at 40 kg a.i. ha^-1.     

Evidence of the existence of a toxic form of Cylindrospermopsis raciborskii (Nostocales,Cyanobacteria) in Japan

Cylindrospermopsis raciborskii is a common, bloom‐forming, planktonic, freshwater cyanobacterium. Toxic populations producing cylindrospermopsin can cause water‐safety problems. Although C. raciborskii is distributed worldwide, the presence of cylindrospermopsin‐producing strains of C. raciborskii was initially reported only in Australia and recently in Thailand. Here, we report the isolation of a toxic strain of C. raciborskii (ISG9) from a freshwater sample collected in Okinawa in 2008. This is the first report describing toxin expression in this species in Japan, detected from a subtropical area. The C. raciborskii species is known to produce cylindrospermopsin as a dominant toxin; however, in this new isolate, the dominant toxin expressed was deoxy‐cylindrospermopsin. The discovery of a toxic strain of C. raciborskii in southern Japan emphasizes the need for basic monitoring schemes for this species in water supplies located in the temperate regions of Japan because of its possible expansion and distribution to other geographic areas.     

Relationships between occurrences of toxic Dinophysis species (Dinophyceae) and small phytoplanktons in Japanese coastal waters

Fluctuations of the genus Dinophysis, which contained several toxic species of diarrhetic shellfish poisoning (DSP), were investigated during blooms in Hiroshima Bay, Mutsu Bay and Ise Bay, Japan. The co-occurrences of small phytoplanktons (cryptophytes, other nanophytoplanktons, cyanobacteria and eukaryotic picophytoplanktons) were investigated to search for relationships with mixotrophic Dinophysis. Cryptophytes were divided into three size-groups based on length of their chloroplasts (>10, 5–10 and <5 μm) during counting by epifluorescence microscopy. Clear relationships were not found between the occurrences of Dinophysis spp. and nanophytoplanktons, cyanobacteria and eukaryotic picophytoplanktons. However, the fluctuations of small-sized cryptophytes (<5 μm) showed a close relationship with that of D. acuminata in Hiroshima Bay. In Mutsu Bay, small-sized cryptophytes also accompanied the first occurrence peak of Dinophysis spp. In Ise Bay, peaks of the occurrences of middle- and small-sized cryptophytes were observed 2–3 weeks before the peak of D. acuminata. These cryptophytes decreased rapidly with increase in D. acuminata. These results suggest the possibility that small-sized cryptophytes may be food organisms for mixotrophic Dinophysis, with the abundance of Dinophysis dependent on these cryptophytes.     

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.     

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.     

Emergence of third-stage larvae of Umingmakstrongylus pallikuukensis from three gastropod intermediate host species

We investigated the emergence of third-stage larvae (L3) of Umingmakstrongylus pallikuukensis from the slugs Deroceras laeve, Deroceras reticulatum, and the snail Catinella sp. in the laboratory and from D. laeve on the tundra. Third-stage larvae emerged from 8 of 8 D. laeve and 8 of 8 D. reticulatum housed at 20 C in darkness and from 9 of 10 D. laeve and 5 of 5 Catinella sp. housed at 21 C with 10-12 hr of light/day. Larvae emerged from D. laeve and D. reticulatum over a wide range of infection intensities (2-179 and 20-65, respectively), and the patterns of emergence were independent of intensity. The majority of the L3 emerged from most of the Deroceras spp. by 58 or 60 days postinfection (PI). Lower rates of emergence were observed from Catinella sp. Larvae emerged from D. laeve on the tundra by 10 wk PI and were recovered from the vegetation in some experimental enclosures the following year. Third-stage larvae survived in tap and distilled water at 0-4 C for 13 mo. Emergence of L3 of U. pallikuukensis from the intermediate host may increase the temporal and spatial availability of L3 and enhance their survival and transmission.