Occurrence of dinoflagellate Alexandrium tamarense, a causative organism of paralytic shellfish poisoning in Chinhae Bay, Korea

A paralytic shellfish poisoning (PSP) incident caused by consumptionof the mussel Mytilus edulis occurred for the first time inKorea in April 1986. Weekly water samplings were carried Outduring the period from 7 March to 21 April 1989 in Chinhae Bay,Korea, in order to identify the causative organism. The temperaturecharacteristics of the water column indicated three differenthydrological regimes: well mixed (up to 7 March), weakly stratified(17–31 March) and stratified (7–21 April). Toxicityof the phytoplankton was detected during the weakly stratifiedperiod, but only in the 10–50 p.m phytoplankton size fraction.This study presents the occurrence of the toxigenic dinoflagellateAlexandrium tamarense, which is a causative organism of PSP,in Korean coastal waters. Its biomass varied at different depthsin the water column, ranging from 200 to 8000 cells 1^–1in the water column. The weekly fluctuation of A.tamarense toxicitywas similar to that of mussel toxicity. ¹ Present address: Department of Biology, College of NaturalSciences, Hanyang University, Seoul 133-791, Korea     

Toxic dinoflagellates in the Mediterranean sea

Abstract

A review of the distribution of toxic dinoflagellates and related toxic events in the Mediterranean Sea is provided. Diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP) are the main seafood contaminations, that have been reported since 1987. In 1994 four lethal cases for PSP were reported in Morocco. DSP seems to be restricted in the Northern part of the basin, while PSP contamination presents a wider distribution. Potentially DSP species, belonging to the genus Dinophysis, are widely distributed in the Mediterranean Sea, suggesting a wider extension of this contamination. The lack of monitoring programs in the Southern Mediterranean could be responsible of DSP overlooking. The species responsible for PSP are Gymnodinium catenatum in the Alboran Sea and Alexandrium minutum in various parts of the basin. Ichtyotoxic effects, due to Gymnodinium and Gyrodinium species, have been sporadically reported in some Mediterranean areas.     

First report of paralytic shellfish poisoning (PSP) caused by Alexandrium tamiyavanichii in Kuantan Port,Pahang, East Coast of Malaysia

Harmful algal bloom (HAB) is a proliferation of algae, which naturally produce biotoxins and cause harmful effects to humans, the environment and organisms associated with it. Paralytic shellfish poisoning (PSP) was reported for the first time in Kuantan Port, Pahang, Malaysia, in November 2013, followed by a second episode in August 2014. The toxicity level reported during the second event was as high as 3500 μg of STX equiv./100 g shellfish. Ten people were hospitalized with PSP symptoms after consuming contaminated shellfish. This study was conducted at Kuantan Port to identify the organisms responsible for these events. Water samples were collected monthly for a period of 12 months beginning in September 2014. HAB species were identified based on their morphology using light and fluorescence microscopes, and their classification was supported by molecular evidence based on internal transcribed spacer (ITS) sequences. Monthly cell abundance of Alexandrium tamiyavanichii was measured at four sampling stations. Toxin production by three strains isolated from the area was determined using HPLC. Our results revealed the presence of several HAB species, including the PSP‐producing species A . tamiyavanichii . The highest cell density of A . tamiyavanichii was 840 cells L^?1. The presence of GTX components was detected in these strains. However, other toxin components could not be determined. This study reported, for the first time, the presence of PSP‐producing A . tamiyavanichii on the Pahang coast of east Peninsular Malaysia and confirmed that the PSP events in Kuantan Port were attributable to this species. The presence of this species further indicates that several safety measures need to be considered to safeguard public health, particularly in Pahang coastal waters.     

Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

Development of specific rRNA probes to distinguish between geographic clades of the Alexandrium tamarense species complex

The globally occurring Alexandrium tamarense/fundyense/catenellaspecies complex consists of toxic and non-toxic strains thatare morphologically difficult to distinguish. We developed fourspecific ribosomal RNA probes that can identify the entire speciescomplex, the strains of the toxic North American clade and thestrains of the two non-toxic clades from Western Europe andthe Mediterranean Sea by DNA dot blot and fluorescence in situhybridization. These probes are a first step for the developmentof an early warning system for the presence of A. tamarense.     

A toxic dinoflagellate bloom and PSP production associated with upwelling in Golfo Nuevo,Patagonia, Argentina

In 1980 Paralytic Shellfish Poison (PSP) was detected for the first time in Patagonia (Argentina), and has been recurring since during spring and summer.Human intoxicantions were recorded during the 1987/88 sumer due to ingestion of mussles. In January, concentrations of 750 000 cell l^–1 were observed, decreasing to 3100 cells l^–1 twenty days later. At the same time, values of 66 150.Mouse Unit (MU) 100 g^–1 of meat were measured in Aulacomya ater (a bivalve mollusk). A decrease in the concentration of toxic plankton resulted in a concomitant decrease in MU values. The mean detoxification time was 7.6 days. However, mussels retained toxicity up to 50 days after the disappearance of cells.The upwelling of cold waters at the beginning of summer, could have been one of the causes that favoured a bloom of Alexandrium excavatum and of Prorocentrum micans. In March, an abundance of the diatom Skeletonema costatum coincided with high concentrations of silicate.     

Gymnodinium catenatum Graham (Dinophyceae)in Europe: a growing problem?

The microreticulate resting cyst of the potentially toxic, chain-forming,unarmoured neritic dinoflagellate Gymnodinium catenalum Graham1943. the planktonic stage of which is not known from NorthEuropean waters, is reported for the first time from recentGerman coastal sediments of the North Sea and Baltic Sea. Insandy mud sediments of the German Bight, a maximum of 8 5 livingcysts cm^–3 were found. In Kiel Bight sediments G.catenalumwas found in maximum concentrations of 17.0 living cysts cm^–3.In surface waters of the German Bight resuspended G catenatumcysts were observed at concentrations of up to 3.6 cysts l^–1.Successful germination experiments conducted with natural seawatershow that the occurrence of a vegetative form of G.catenatumin northern Europe is very likely. The present study highlightsthat cyst surveys provide an important tool for the evaluationof areas with potential toxicity problems, as they may indicatethe presence of hitherto overlooked species in the water column.     

Pre-ingestive selection efficiency in two populations of the razor clam Tagelus dombeii with different histories of exposure to paralytic shellfish poisoning (PSP)

We studied toxic effects of the dinoflagellate Alexandrium catenella on filtration activity and pre-ingestive selection efficiency in Tagelus dombeii (razor clam). Samples came from two populations with different histories of exposure to paralytic shellfish poisoning (PSP): Melinka, Aysén (with frequent exposure to PSP) and Corral, Valdivia (without previous exposure to PSP). Feeding activity of T. dombeii was affected by a diet containing A. catenella, showing a reduction in individuals from Corral, Valdivia and Melinka, Aysén. Furthermore, pre-ingestive selection efficiency was significantly higher in specimens from the population of Melinka, than those from Corral. Significantly higher values of clearance rate and pre-ingestive selection efficiency from the Melinka samples may reflect adaptation to specific environmental conditions where PSP events frequently occur.     

A toxic dinoflagellate bloom of Alexandrium tamarense (Lebour) Balech in Tokyo Bay

We report here, for the first time, the occurrence of the toxigenicdinoflagellate species. Alexandrium tamarense, in Tokyo Bay.It suggests the possibility of human intoxication (paralyzingshellfish poisoning) upon consuming local shellfish.     

Transport of diatom and dinoflagellate resting spores in ships' ballast water: implications for plankton biogeography and aquaculture

Diatom and dinoflagellate species that are not endemic to aregion can be inadvertently introduced when their resistantresting stages are discharged with the ballast-tank waters andsediments of bulk cargo vessels. A survey of 343 cargo vesselsentering 18 Australian ports showed that 65% of ships were carryingsignificant amounts of sediment on the bottom of their ballasttanks. All of these samples contained diatoms, including speciesthat are not endemic to Australian waters. Diatom resting spores,especially of Chaetoceros, were also detected. Dinoflagellateresting spores (cysts) were present in 50% of the sediment samples.Of the 53 cyst species identified, 20 (including Diplopelta,Diplopsalopsis, Gonyaulax, Polykrikos, Protoperidinium, Scrippsiellaand Zygabikodinium spp.) were successfully germinated to produceviable cultures. Such diversity of diatom and dinoflagellatespecies in ships' ballast water suggests that the apparent cosmopolitanismof many coastal phytoplankton species may be due partly to theglobal transport of seawater ballast. Of considerable concernwas the detection in 16 ships of cysts of the toxic dinoflagellatesAlexandrium catenella, Alexandrium tamarense and Gymnodiniumcatenatum. One single ballast tank was estimated to contain>300 million viable A.tamarense cysts, some of which weresuccessfully germinated in the laboratory to produce toxic cultures.These toxic dinoflagellate species, which can contaminate shellfishwith paralytic shellfish poisons, pose a serious threat to humanhealth and the aquaculture industry. Ballast-water quarantinemeasures recently introduced in Australia are discussed. Mid-oceanexchange of ballast water is only partially effective in removingdinoflagellate cysts which have settled to the bottom of ballasttanks. The present work indicates that the most effective measureto prevent the spreading of toxic dinoflagellate cysts via ships'ballast water would be to avoid taking on ballast water duringdinoflagellate blooms in the water column of the world's ports.     

Which species,Alexandrium catenella (Group I) or A. pacificum (Group IV), is really responsible for past paralytic shellfish poisoning outbreaks in Jinhae-Masan Bay,Korea?

Paralytic shellfish poisoning (PSP) caused the deaths of four people in coastal area of Korea, mainly Jinhae-Masan Bay and adjacent areas, in April 1986 and in 1996. The PSP outbreaks were caused by the consumption of mussels, Mytilus edulis. The organism that caused PSP was identified, from morphological data only, as Alexandrium tamarense which is recently renamed as A. catenella, however recent studies have shown that the morphological diagnostic characteristics used to identify Alexandrium species have uncertainties and molecular tools and other criteria should be considered as well. The organism that caused past PSP outbreaks and incidents in Korea therefore need to be carefully reconsidered. The aim of this study was to re-evaluate the species really responsible for past outbreaks of PSP in Jinhae-Masan Bay, Korea. The temporal production and fluxes of the resting cysts of Alexandrium species were investigated for one year (from March 2011 to February 2012) using a sediment trap, and the morphology and phylogeny of vegetative cells germinated from the resting cysts were analysed. The production of Alexandrium species peaked in August and November, when temporal discrepancies were found in the water temperature (22.4 and 22.7 °C in August, 19.1 and 19.6 °C in November) and salinity (29.5 and 26.1 psu in August, 30.5 and 31.8 psu in November). The morphological data revealed that Alexandrium species germinated from resting cysts collected in August have a ventral pore on the 1′ plate, whereas the 1′ plate in Alexandrium species germinated from resting cysts collected in November lacks a ventral pore. Molecular phylogenetic data for the vegetative cells from the germination experiments allowed the August and November peaks to be assigned to Alexandrium catenella (Group I) and A. pacificum (Group IV), respectively. This indicates that the production of resting cysts of A. catenella can be enhanced by relatively high water temperature. This result is not consistent with those of previous studies that A. catenella responsible for PSP outbreaks was found at relatively low water temperature. In addition, large subunit ribosomal sequences data revealed that A. pacificum isolates from Korea were closely related to those from Australia, Japan and New Zealand where the PSP toxicity of shellfish and blooms occurred in the 1990s, indicating that the introduction of toxic dinoflagellates were related to ballast water from bulk-cargo shipping. Based on these results, we concluded that past PSP outbreaks in Jinhae-Masan Bay of Korea could have been caused by A. pacificum rather than by A. catenella.     

Presence of Gymnodinium catenatum (Dinophyceae) in a coastal Mediterranean lagoon

The occurrence and abundance of the toxic, chain-forming dinoflagellateGymnodinium catenatum in a Tyrrhenian coastal lagoon, the Fusaro,during an annual sampling cycle are reported. Peak abundanceswere observed from late spring until early autumn Although veryhigh cell numbers were recorded, up to 1 5 x 10⁶ cells l^–1,no monospecific bloom of this species occurred. The first observationof G.catenatum in the Mediterranean occurred in the Fusaro andthe appearance of this species in a traditional shellfish farmingarea, where no shellfish intoxication has been reported to date,is discussed in relation to human interventions in the basin.In particular, intensive dredging in recent years with resuspensionof bottom sediments may have seeded the water body with cysts.A Gymnodinium n d species, illustrated using scanning electronmicroscopy, caused a monospecific bloom in concomitance withmaximum abundances of G.catenatum, apparently outcompeting thislatter species     

Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR

The toxic dinoflagellate Alexandrium fundyense is widespread in the northeastern part of North America, including the Gulf of Maine, and is responsible for seasonal harmful algal blooms in these regions. Even at low cell densities, A. fundyense toxins can accumulate in shellfish and result in paralytic shellfish poisoning (PSP). PSP can be debilitating or lethal to humans and other shellfish consumers and is a public health concern. As a result, accurate measurements of A. fundyense distributions, particularly at low cell density, are critical to continued PSP monitoring and mitigation efforts. Towards this end we have developed a real-time quantitative PCR (qPCR) method to monitor A. fundyense. Laboratory validation indicates that the qPCR assay is sensitive enough to detect 10 cells per sample, and that it does not detect co-occurring dinoflagellates such as Alexandrium ostenfeldii. The qPCR methodology was used to quantify A. fundyense cell densities in samples collected during a spring 2003 transect in the Gulf of Maine, and the data were compared to those obtained in parallel from light microscope and DNA hybridization-based methods. Results show that A. fundyense cell density was low during this period relative to typical cell densities required for PSP contamination of local shellfish, and that qPCR values were comparable to numbers determined by independent methods.     

The effects of temperature, growth medium and darkness on excystment and growth of the toxic dinoflagellate Gymnodinium catenatum from northwest Spain

The chain-forming dinoflagellate Gynmodinium catenatum Grahamcauses recurrent outbreaks of paralytic shellfish poisoning(PSP) in the Galician Rias Bajas (northwest Spain). A sedimentsurvey in Ria de Vigo in April 1986 indicated that the highestconcentrations of cysts of this species were located in themiddle sections of the ria, with maximum abundance of 310 cystscm^–3. The effects of temperature, growth medium compositionand irradiance on the germination of laboratory-produced restingcysts were investigated. Newly formed cysts required very littletime for maturation, as excystment was possible within 2 weeksof encystment. Growth media did not affect germination success.In contrast, the excystment rate was retarded signifiantly indarkness. Germination was also strongly affected by temperature,with {small tilde}75% excystment success at 22–28°Cand little or no germination below 11°C after 1 month ofincubation. In culture, the optimum growth rate of vegetativecells was between 22 and 28°C, the highest rate being 0.53divisions day^–1 at 24°C. Growth did not occur at temperatures< 11°C or >30°C. These results are important withrespect to the different hypotheses proposed to explain theinitiation of G.catenatum blooms in the Galician Rias Bajasand Northern Portugal. The pattern of G.catenatum bloom developmentalong this coast has been related to seasonal upwelling in thearea, with major blooms occurring during the autumn as warmeroffshore surface water is transported towards the coast whenupwelling relaxes. The landward transport of established offshorepopulations of G.catenatum with the warm surface layer remainsa viable explanation for the observed blooms within the rias,but alternatively, our data suggest that cysts within the riascan provide the inoculum population at times conducive to growthand bloom formation. Even though newly formed G.catenatum cystshave a very short maturation time and can germinate in darknessacross a wide temperature range, bloom development will be significantonly during the late summer and early autumn, since in othermonths light levels at the sediment surface and temperaturesthroughout the water column are too low for significant germinationor growth.     

16S rRNA Targeted Probes for the Identification of Bacterial Strains Isolated from Cultures of the Toxic Dinoflagellate Alexandrium tamarense

Abstract Bacteria have been implicated in the production of paralytic shellfish poison (PSP) toxins, which are normally associated with bloom-forming algal species, specifically toxic dinoflagellate algae. To clarify the role that these bacteria may play in the production of PSP toxins, it is desirable to identify and localize the bacteria associated with the dinoflagellates. 16S rRNA-targeted probes offer the possibility for both, and thus, probes have been made to putatively toxigenic bacteria isolated from the PSP-related dinoflagellate Alexandrium tamarense and tested for their specificity in dot blot and in situ hybridization experiments. Received: 5 August 1999; Accepted: 19 January 2000; Online Publication: 5 May 2000;     

Enzymatic digestive activity and absorption efficiency in Tagelus dombeii upon Alexandrium catenella exposure

We analyzed absorption efficiency (AE) and digestive enzyme activity (amylase, cellulase complex, and laminarinase) of the infaunal bivalve Tagelus dombeii originating from two geographic sites, Corral-Valdivia and Melinka-Aysén, which have different long-term paralytic shellfish poisoning (PSP) exposure rates. We report the effects of past feeding history (origin) on T. dombeii exposed to a mixed diet containing the toxic dinoflagellate Alexandrium catenella and another dinoflagellate-free control diet over a 12-day period in the laboratory. Absorption efficiency values of T. dombeii individuals that experienced PSP exposure in their habitat (Melinka-Aysén) remained unchanged during exposure to toxic food in the laboratory. In contrast, T. dombeii from a non-PSP exposure field site (Corral-Valdivia) showed a significant reduction in AE with toxic exposure time. This study established that the amylase and cellulase complexes were the most important enzymes in the digestive glands of Tagelus from both sites. The temporal evolution of enzymatic activity under toxic diet was fitted to exponential (amylase and cellulase) and to a logarithmic (laminarinase) models. In all fits, we found significant effect of origin in the model parameters. At the beginning of the experiment, higher enzymatic activity was observed for clams from Corral-Valdivia. The amylase activity decreased with time exposure for individuals from Corral and increased for individuals from Melinka. Cellulase activity did not vary over time for clams from Corral, but increased for individuals from Melinka and laminarinase activity decreased over time for individuals from Corral and remained unchanged over time for Melinka. A feeding history of exposure to the dinoflagellate A. catenella was reflected in the digestive responses of both T. dombeii populations.     

Toxin profiles of natural populations and cultures ofAlexandrium minutum Halim from Galician (Spain) coastal waters

The toxin profiles of three isolates and natural populations of the PSP agentAlexandrium minutum from several Galician rías (NW Spain) was obtained by HPLC. The toxin content of cultures ofA. minutum is dominated by GTX4 (80–90%) and GTX4 (10–15%) with small amounts of GTX3 and GTX2 (less than 3% of each); similar results were obtained for natural populations ofAlexandrium from three different Galician rías, where a mixture ofA. lusitanicum Balech andA. minutum can occur. Important quantitative differences were found between the three isolates, one being highly and two weakly toxic. The results obtained from these isolates and natural populations ofAlexandrium were very similar to those obtained from HPLC analyses of mussels intoxicated during a PSP outbreak in Ría de Ares (Rías Altas) in 1984, confirming thatA. minutum (previously identified asGonyaulax tamarensis Lebour andAlexandrium lusitanicum) was the PSP agent during the toxic outbreak in May 1984. Toxin profiles obtained from natural populations during different PSP outbreaks in different rías and from cultures are fairly consistent and suggest that at least from the toxin point of view,A. lusitanicum andA. minutum are identical, and that the toxin profile ofA. minutum from Galicia can be used as a biochemical marker.     

Toxin profile of Alexandrium catenella from the Chilean coast as determined by liquid chromatography with fluorescence detection and liquid chromatography coupled with tandem mass spectrometry

The profile of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was determined from a Chilean strain of the marine dinoflagellate Alexandrium catenella. The toxin composition was compared with that of toxic shellfish, presumably contaminated by natural blooms of A. catenella from the same region in southern Chile. Ion pair-liquid chromatography with post-column derivatization and fluorescence detection (LC-FD) was employed for relative quantitative analysis of the toxin components, whereas unambiguous identification of the toxins was confirmed by tandem mass spectrometry (LC–MS/MS). In the dinoflagellate strain from Chile, the N-sulfocarbamoyl derivatives (C1/C2, B1) and the carbamoyl gonyautoxins GTX1/GTX4 comprise >90% of the total PSP toxin content on a molar basis. This toxin composition is consistent with that determined for A. catenella populations from the Pacific coast in the northern hemisphere. The characteristic toxin profile is also reflected in the shellfish, but with evidence of epimerization and metabolic transformations of C1 and C2 to GTX2 and GTX3, respectively. This work represents the first unequivocal identification and confirmation of such PSP toxin components from the Chilean coast.     

Phycotoxin accumulation in zooplankton feeding on Alexandrium fundyense--vector or sink?

Zooplankton can consume toxic Alexandrium spp. dinoflagellatesin the Gulf of Maine and retain paralytic shellfish poisoning(PSP) toxins, potentially acting as toxin vectors. We performedexperiments to determine toxin budgets for common species ofcopepods (Acartia hudsonica, Eurytemora herdmani, Centropageshamatus) feeding on toxic Alexandrium fundyense, offered asmonocultures or in mixtures of algal prey, by comparing calculatedtoxin ingestion rates and toxin content of copepod body tissueand fecal pellets. When fed monocultures, both copepod tissueand fecal pellet fractions accounted for 5% each of the calculatedingested toxin, and thus by difference 90% was lost as a dissolvedfraction into the seawater medium. The presence of alternativefood did not significantly alter the efficiency of toxin retention.Sloppy feeding or regurgitation are probable mechanisms forrelease of toxin to sea water. Experiments using varying concentrationsof A. fundyense and alternativenon-toxic species did not showsignificant effects of cell concentration on toxin retentionefficiency. Total toxin retained and efficiency of retentionvaried among copepod species. Toxin profiles (% molar composition)of dinoflagellates, copepod tissues and fecal pellets differedslightly, suggesting some metabolic transformation. Becauseof their low retention efficiency, copepod grazers can effectivelydisperse PSP toxins produced by Alexandrium spp. into the environment,where they are much less likely to be harmful—zooplanktonact as a sink for PSP toxins. Nevertheless, sufficient toxinbody burdens are attained to contribute to propagation of PSPtoxins to other trophic levels.