Manabu Asakawa

Dinoflagellate Alexandrium tamarense as the source of paralytic shellfish poison (PSP) contained in bivalves from Hiroshima Bay, Hiroshima Prefecture, Japan

In April 1993, a phytoplankton dinoflagellate was isolated from Hiroshima Bay, Hiroshima Prefecture, Japan, and unambiguously identified as Alexandrium tamarense on the basis of the morphological characteristics. The dinoflagellates, cultures in modified SW-2 medium at 15 degrees C for 15 days, showed a specific toxicity of 30.7 x 10-6 MU/cell. HPLC analysis demonstrated that the toxin was composed mainly of gonyautoxin-4 (GTX4) and protogonyautoxin-2 (PX2 or GTX8)(27.6 and 37.0 mole%, respectively). Total toxin concentration of this strain was 39.5 fmole/cell. Short-necked clams, mussels, and oysters contaminated by the dinoflagellate showed a more complicated composition, with GTX1 as the major component (61.8 mole% for short-necked clams, 60.5 mole% for mussels, 42.5 mole% for oysters), and PX2 was only present in trace amounts.

Developmental stages and growth of Pseudocaligus fugu Yamaguti, 1936 (Copepoda: Siphonostomatoida: Caligidae) host-specific to Puffer

The post-embryonic development of the copepod Pseudocaligus fugu is described. This sea louse only parasitizes toxic pufferfish, and causes commercial loss of cultured Takifugu rubripes in Japan. Two naupliar, one copepodid and four chalimus stages preceding the adult are recognized in the species. The development pattern is similar to that of Caligus except for the suppression of leg 4 in the early chalimus phase. Pseudocaligus fugu lacks any preadult stage, as in Caligus, but contrasts with Lepeophtheirus species in which two preadult stages are reported. The nature of the metamorphic changes in size and even in degree of expression of articulations can occur without moulting. The validity of some chalimus stages recognized in Lepeophtheirus species is questioned. Growth and egg production in P. fugu were obtained in the laboratory. Pseudocaligus fugu attained adulthood 9 days after the infective copepodid attached to the host.

Structure of the toxin isolated from carp (Cyprinus carpio) bile

Attempts were made to elucidate the structure of the toxin isolated from the bile of carp Cyprinus carpio, which is possibly responsible for carp poisoning. By fast atom bombardment (FAB) mass spectrometry, along with 1H- and 13C-NMR, a molecular formula of C27H48O8S containing a sulfate ester group was deduced. Those and other analytical data allowed us to conclude the structure of carp toxin to be 5 alpha-cholestane-3 alpha, 7 alpha, 12 alpha, 26, 27-pentol 26-sulfate, which agreed essentially with that of 5 alpha-cyprinol, an alcohol specific to carp bile, in which the sulfate ester at the C-26 position is lacking.

Highly Toxic Ribbon Worm Cephalothrix simula Containing Tetrodotoxin in Hiroshima Bay, Hiroshima Prefecture, Japan

In 1998, during a toxicological surveillance of various marine fouling organisms in Hiroshima Bay, Japan, specimens of the ribbon worm, Cephalothrix simula (Nemertea: Palaeonemertea) were found. These ribbon worms contained toxins with extremely strong paralytic activity. The maximum toxicity in terms of tetrodotoxin (TTX) was 25,590 mouse units (MU) per gram for the whole worm throughout the monitoring period. The main toxic component was isolated and recrystallized from an acidified methanolic solution. The crystalline with a specific toxicity of 3520 MU/mg was obtained and identified as TTX by high performance liquid chromatography (HPLC)-fluorescent detection (FLD) (HPLC-FLD), electrospray ionization-mass spectrometry (ESI-MS), infrared (IR), nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC-MS). The highest toxicity of C. simula exceeded the human lethal dose per a single worm. A toxicological surveillance of C. simula from 1998 to 2005 indicated approximately 80% of the individuals were ranked as “strongly toxic” (≥1000 MU/g). Forty-eight percent of the specimens possessed toxicity scores of more than 2000 MU/g. Seasonal variations were observed in the lethal potency of C. simula. Specimens collected on January 13, 2000 to December 26, 2000 showed mean toxicities of 665–5300 MU/g (n = 10). These data prompted a toxicological surveillance of ribbon worms from other localities with different habitats in Japan, including Akkeshi Bay (Hokkaido) under stones on rocky intertidal beaches, as well as Otsuchi (Iwate) among calcareous tubes of serpulid polychaetes on rocky shores. Within twelve species of ribbon worms examined, only C. simula possessed extremely high toxicity. Therefore, C. simula appears to show generally high toxicity irrespective of their locality and habitat.

Occurrence of paralytic shellfish poison in the starfish Asterias amurensis in Kure Bay, Hiroshima Prefecture, Japan

In May 1996, during surveillance on the toxicity of invertebrates such as bivalves inhabiting the coasts of Hiroshima Bay, the starfish Asterias amurensis collected in the estuary of the Nikoh River was found to contain toxins which showed strong paralytic action in mice; the maximum toxicity (as paralytic shellfish poison, PSP) was 8.0 MU/g for whole body and 28.7 MU/g for viscera throughout the monitoring period, March to July 1996. Attempts were made to identify the paralytic toxins in the starfish. They were extracted with 80% ethanol acidified with acetic acid, followed by defatting with dichloromethane. The aqueous layer obtained was treated with activated charcoal and then applied to a Sep-Pak C18 cartridge. The unbound toxic fraction was analyzed by high-performance liquid chromatography techniques. The starfish toxin was rather unexpectedly identified as PSP. It was comprised of high toxic components (gonyautoxin-1; GTX1, GTX2, GTX3, GTX4, decarbamoyl-GTX3; dcGTX3 and dcSTX) as the major components, which accounted for approximately 77 mole% of all components, along with protogonyautoxin-1, 2, 3 and 4 (PX1-4), which are N-sulfocarbamoyl derivatives. Of the high toxic components, GTX1 was present in the largest amounts. It was concluded that the toxin of starfish collected in the estuary of Nikoh River in May 1996 consisted of PSP, which supposedly came via the food chain from toxic bivalves living in the same area. To our knowledge, this is the first report of the occurrence of PSP in starfish.

Occurrence of paralytic shellfish poison (PSP) in the starfish Asterina pectinifera collected from the Kure Bay, Hiroshima Prefecture, Japan

Assays were made for paralytic toxicity of marine invertebrates inhabiting at the coasts of Hiroshima Bay, where the infestation of bivalves such as cultured oysters with paralytic shellfish poison (PSP) has been occurred. The starfish Asterina pectinifera collected at the estuary of Nikoh River, Hiroshima Bay, was found to contain moderate levels of paralytic toxicity. Its highest toxicities as PSP found on July 30, 1999 were 12.5 MU/g for whole body, 11.0 MU/g for integument tissues and 3.9 MU/g for viscera, respectively. The toxicity of integument was changed from 3.6 to 11.0 MU/g in 1 year. Its paralytic toxin principles were identified as PSP toxins, composing mainly from saxitoxin (STX) group toxins such as carbamoyl-N-hydroxy neosaxitoxin (hyneoSTX), and STX, by HPLC and LC-MS, accounting for over 90 mol%. The PSP toxins contained in the starfish A. pectinifera considered to be transferred from bivalves or detritus living in the same area, which were contaminated with PSP. However, the involved pathway may be different from that of Asterias amurensis which was infested directly through food chain from its food bivalves, for its toxin pattern.

Occurrence of PSP-producing dinoflagellate Alexandrium tamiyavanichii in Bingo-Nada, the central coastal water of the Seto Inland Sea, Hiroshima Prefecture, Japan

During surveillance of the distribution of the paralytic shellfish poison (PSP)-producing dinoflagellate in 2003, 2004 and 2005 along the coastlines of the Seto Inland Sea, Hiroshima Prefecture, Japan, some species of toxic phytoplankton were isolated from the eastern coasts, Bingo-Nada, the central regions of the Seto Inland Sea. It was rather unexpectedly revealed from the basis of the morphological characteristics that they were unambiguously identified as Alexandrium tamiyavanichii and Alexandrium catenella. Two strains (ATY041106, ATY051018) of A. tamiyavanichii showed a specific toxicity of 38.7 x 10(-6) and 111.5 x 10(-6)MU/cell, respectively. These values seemed to be several times or much higher than that of A. catenella (AC030816, AC040614), having a specific toxicity of 4.5 x 10(-6) and 4.1 x 10(-6)MU/cell, respectively, isolated in the same area. From the results of HPLC-furuorometric analysis, it revealed that the toxins in ATY041106 exist almost exclusively as beta-epimers (C2, GTX3, GTX4), which accounted for 72.7 mol%. The toxin profiles of this strain are featured by the presence of a large amount of GTX3 (59.1 mol%) and a small amount (20.6%) of C1 and 2 in comparison with the PSP compositions of A. tamarense, which is isolated as the main responsible species in Hiroshima Bay, a western part of coastal sea in Hiroshima Prefecture. On the other hand, it revealed that the toxin profiles of two strains (AC030816, AC040614) of A. catenella exist almost exclusively as beta-epimers (C2, GTX3, GTX4), which accounted for 81.8 and 56.5 mol%, as the same manner. The toxin profiles of these two strains are featured by the presence of a large amount of C2 (80.5 and 46.3 mol%) in comparison with the PSP compositions of A. tamiyavanichii. To our knowledge, this is the first record to show the distribution and harmful influence of A. tamiyavanichii and A. catenella in Bingo-Nada in Hiroshima Prefecture. Though contamination of bivalves with these PSP-producing planktons in this area has not occurred yet so far, attention should be paid to this species as well as the other causative dinoflagellate from the stand point of public health and food hygiene.

Taxonomic Identity of a Tetrodotoxin-Accumulating Ribbon-worm Cephalothrix simula (Nemertea: Palaeonemertea) : A Species Artificially Introduced from the Pacific to Europe

We compared the anatomy of the holotype of the palaeonemertean Cephalothrix simula (Iwata, 1952) with that of the holotypes of Cephalothrix hongkongiensis Sundberg, Gibson and Olsson, 2003 and Cephalothrix fasciculus (Iwata, 1952), as well as additional specimens from Fukue (type locality of C. simula) and Hiroshima, Japan. While there was no major morphological discordance between these specimens, we found discrepancies between the actual morphology and some statements in the original description of C. simula with respect to supposedly species-specific characters. Our observation indicates that these three species cannot be discriminated by the anatomical characters so far used to distinguish congeners. For objectivity of scientific names, topogenetypes of the mitochondrial cytochrome c oxidase subunit I (COI) sequences are designated for C. simula, C. hongkongiensis, and C. fasciculus. Analysis of COI sequence showed that the Hiroshima population can be identified as C. simula, which has been found in previous studies from Trieste, Italy, and also from both the Mediterranean and Atlantic coasts of the Iberian Peninsula, indicating an artificial introduction via (1) ballast water, (2) ship-fouling communities, or (3) the commercially cultured oyster Crassostrea gigas (Thunberg, 1793) brought from Japan to France in 1970s. Cephalothrix simula is known to be toxic, as it contains large amounts of tetrodotoxin (TTX). We report here that the grass puffer Takifugu niphobles (Jordan and Snyder, 1901)—also known to contain TTX— consumes C. simula. We suggest that the puffer may be able to accumulate TTX by eating C. simula.

Toxicity Assessment of the Xanthid Crab Demania cultripes from Cebu Island, Philippines.

Several cases of poisoning resulting in human fatalities and stemming from the ingestion of coral reef crabs have been reported from the Indo-Pacific region. We assessed the toxicity of the unidentified xanthid crab collected from the Camotes Sea off the eastern coast of Cebu Island, central Visayas region of Philippines from the food hygienic point of view. All seven specimens, which were identified with Demania cultripes, collected in 2006 were toxic to mice irrespective of the season of collection and induced paralytic symptoms typical of tetrodotoxin (TTX) and paralytic shellfish poison (PSP). The activity was expressed in mouse unit (MU) being defined as the amount of TTX to kill a 20 g ddY male mice in 30 min after i.p. injection. Toxicity scores for viscera and appendages of specimens were 18.2 ± 16.0 (mean ± S.D.) and 4.4 ± 2.6 MU/g, respectively. The highest individual toxicity scores observed for viscera and appendages were 52.1 and 7.7 MU/g, respectively. The frequency of toxic samples was 100%. Toxin profiles as determined by high-performance liquid chromatography-fluorescent detection analysis (HPLC-FLD) revealed that TTX was the main toxic principle accounting for about 90% of the total toxicity along with 4-epi TTX and 4,9-anhydroTTX. Furthermore, gas chromatography-mass spectrometry (GC-MS) analysis revealed mass fragment ion peaks at m/z 376, 392 and 407, which were characteristic of the quinazoline skeleton (C9-base) specific to TTX. In addition, only a small amount of PSP containing gonyautoxins1–4 and hydroxysaxitoxin was detected. To our knowledge, this is the first report presenting evidence of occurrence of TTX and PSP in the xanthid crab D. cultripes inhabiting waters surrounding Cebu Island. From food hygienic point of view, people in coastal areas should be warned of the potential hazard of this crab in order to prevent its intentional or accidental consumption.

Paralytic Shellfish Poison (PSP)–Producing Dinoflagellate and PSP-Infested Organisms

Paralytic shellfish poison (PSP) is produced by numerous microalgae species, mainly toxic marine dinoflagellates species of the genera Alexandrium, Gymnodinium, and Pyrodinium, and is accumulated in many species of filter-feeding organisms such as bivalve mollusks through the food chain. Besides these toxic dinoflagellates, certain freshwater cyanobacteria such as Anabaena circinarlis and Aphanizomenon flos-aquae also produce PSP. PSP is transferred and bioaccumulates throughout aquatic food webs, and can be vectored to terrestrial biota. Fishery closures and food poisoning due to PSP have been documented in several vectors. Traditionally, only filter-feeding mollusks that concentrate these toxic algae are considered in monitoring programs for PSP; however, increasing attention is being paid to higher-order predators. Besides commercially important bivalves, other PSP-bearing organisms such as carnivorous gastropods and crustaceans have been reported. From the food hygiene point of view, it is important to summarize the role of marine organisms as vectors of PSP and discuss the need for surveillance to protect public health and ensure the quality of seafood. This chapter shows several case studies pertaining to management actions to prevent food-poisoning incidents by PSP accumulation in filter-feeding (traditional) and non-filter-feeding (non-traditional) vectors of PSP.