The effect of a toxic dinoflagellate (Alexandrium tamarense) on the oxygen uptake of juvenile filter-feeding bivalve molluscs

• 1.1. Oxygen uptake and grazing rates of juvenile bivalve molluscs Mytilus edulis, Mya arenaria, Geukensia demissa, Placopecten magellanicus and Crassostrea virginica were measured following 1 hr exposure to bloom concentrations (10⁶ cells/1) of the toxic dinoflagellate Alexandrium tamarense (GT429) using a non-toxic clone of the same species (PLY 173) as control.
• 2.2. For all bivalves, prefeeding estimates of V̇O₂ were similar to postfeeding values and values recorded 24 hr after exposure to bloom conditions.
• 3.3. V̇O₂ was similar for bivalves fed on both the toxic and non-toxic strains of A. tamarense suggesting that there were no adverse effects on V̇O₂ following 1 hr exposure to toxic GT429.
• 4.4. Bivalves differed in their rates of grazing between toxic GT429 and non-toxic PLY 173. Similar grazing rates were recorded for M. edulis and G. demissa. For P. magellanicus and M. arenaria reduced rates of clearance were recorded in GT429 compared with the non-toxic strain.

     

Zooplankton interactions with toxic phytoplankton: Some implications for food web studies and algal defence strategies of feeding selectivity behaviour, toxin dilution and phytoplankton population diversity

This study focuses on the interactions between toxic phytoplankton and zooplankton grazers. The experimental conditions used are an attempt to simulate situations that have, so far, received little attention. We presume the phytoplankton community to be a set of species where a population of a toxic species is intrinsically diverse by the presence of coexisting strains with different toxic properties. The other species in the community may not always be high-quality food for herbivorous zooplankton. Zooplankton populations may have developed adaptive responses to sympatric toxic phytoplankton species. Zooplankton grazers may perform a specific feeding behaviour and its consequences on fitness will depend on the species ingested, the effect of toxins, and the presence of mechanisms of toxin dilution and compensatory feeding. Our target species are a strain of the dinoflagellate Alexandrium minutum and a sympatric population of the copepod Acartia clausi. Mixed diets were used with two kinds of A. minutum cells: non-toxic and toxic. The flagellate Rhodomonas baltica and the non-toxic dinoflagellate Alexandrium tamarense were added as accompanying species. The effect of each alga was studied in separate diets. The toxic A. minutum cells were shown to have negative effects on egg production, hatching success and total reproductive output, while, in terms of its effect on fitness, the non-toxic A. minutum was the best quality food offered. R. baltica and A. tamarense were in intermediate positions. In the mixed diets, copepods showed a strong preference for toxic A. minutum cells and a weaker one for A. tamarense cells, while non-toxic A. minutum was slightly negatively selected and R. baltica strongly negatively selected. Although the level of toxins accumulated by copepods was very similar, in both the diet with only toxic A. minutum cells and in the mixed diet, the negative effects on fitness in the mixed diet could be offset by toxin dilution mechanisms. The implications of these findings are the fact that mesozooplankton may not play an important role in phytoplankton blooms development. Phytoplankton endotoxin production does not seem to be an evolutionary stable strategy as a defence against some herbivores.     

Morphogenetic diversity and biotoxin composition of Alexandrium (Dinophyceae) in Irish coastal waters

The diversity of Alexandrium spp. in Irish coastal waters was investigated through the morphological examination of resting cysts and vegetative cells, the determination of PSP toxin and spirolide profiles and the sequence analysis of rDNA genes. Six morphospecies were characterised: A. tamarense, A. minutum, A. ostenfeldii, A. peruvianum, A. tamutum and A. andersoni. Both PSP toxin producing and non-toxic strains of A. tamarense and A. minutum were observed. The average toxicities of toxic strains for both cultured species were respectively 11.3 (8.6 S.D.) and 2.3 (0.5 S.D.) pg STX equiv. cell⁻¹. Alexandrium ostenfeldii and A. peruvianum did not synthesise PSP toxins but HPLC–MS analysis of two strains showed distinct spirolide profiles. A cyst-derived culture of A. peruvianum from Lough Swilly mainly produced spirolides 13 desmethyl-C and 13 desmethyl-D whereas one of A. ostenfeldii, from Bantry Bay, produced spirolides C and D. Species identification was confirmed through the analyses of SSU, ITS1-5.8S-ITS2 and LSU rDNA genes. Some nucleotide variability was observed among clones of toxic strains of A. tamarense, which all clustered within the North American clade. However, rDNA sequencing did not allow discrimination between the toxic and non-toxic forms of A. minutum. Phylogenetic analysis also permitted the differentiation of A. ostenfeldii from A. peruvianum. Resting cysts of PSP toxin producing Alexandrium species were found in Cork Harbour and Belfast Lough, locations where shellfish contamination events have occurred in the past, highlighting the potential for the initiation of harmful blooms from cyst beds. The finding of supposedly non-toxic and biotoxin-producing Alexandrium species near aquaculture production sites will necessitate the use of reliable discriminative methods in phytoplankton monitoring.     

Feeding interactions of the copepods Eurytemora affinis and Acartia bifilosa with the cyanobacteria Nodularia sp.

We measured ingestion and clearance rates of two Baltic Seacalanoid copepods, Eurytemora affinis and Acartia bifilosa,on toxic and non-toxic cyanobacteria Nodularia sp. using theisotope technique. Eurytemora affinis fed actively on the non-toxicstrain and moderately actively on the toxic strain, whereasA.bifilosa totally avoided feeding on both strains. This suggeststhat A.bifilosa rejected cyanobacterial filaments due to theirnutritional inadequacy or difficult manageability. The differentresponse of E.affinis to the non-toxic and toxic strains, inturn, shows that this copepod species was able to sense thepresence of the toxin in cyanobacterial filaments and thereforefed less on the toxic strain. The interaction between A.bifilosaand Nodularia sp. was further examined (with the particle countingmethod) by measuring the clearance rates of A.bifilosa on ediblegreen flagellates in the presence of cyanobacteria. The presenceor concentration of toxic Nodularia sp. did not affect grazingrates of A.bifilosa on Brachiomonas submarina. Since earlierstudies have shown that ingestion of Nodularia sp. decreasesegg production and increases mortality in E.affinis, we suggestthat the occurrence of Nodularia sp. blooms in the Baltic Seamay favour individuals of copepod species capable of selectivefeeding, such as A.bifilosa.     

Grazing on colonial and filamentous, toxic and non-toxic cyanobacteria by the zebra mussel Dreissena polymorpha

Colony forming and toxic cyanobacteria form a problem in surfacewaters of shallow lakes, both for recreation and wildlife. Zebramussels, Dreissena polymorpha, have been employed to help torestore shallow lakes in the Netherlands, dominated by cyanobacteria,to their former clear state. Zebra mussels have been presentin these lakes since they were created in the 19th century bythe excavation of peat and are usually not considered to bean invasive species. Most grazing experiments using Dreissenahave been performed with uni-cellular phytoplankton laboratorystrains and information on grazing of larger phytoplankton taxahardly exists. To gain more insight in to whether D. polymorphais indeed able to decrease cyanobacteria in the phytoplankton,we therefore performed grazing experiments with zebra musselsand two species of cyanobacteria, that greatly differ in shape:colony forming strains of Microcystis aeruginosa and the filamentousspecies Planktothrix agardhii. For both species a toxic anda non-toxic strain was selected. We found that zebra musselscleared toxic Planktothrix at a higher rate than non-toxic Planktothrix,toxic or non-toxic Microcystis. Clearance rates between theother strains were not significantly different. Both phytoplanktonspecies, regardless of toxicity, size and shape, were foundin equal amounts (based on chlorophyll concentrations) in theexcreted products of the mussels (pseudofaeces). The resultsshow that zebra mussels are capable of removing colonial andfilamentous cyanobacteria from the water, regardless of whetherthe cyanobacteria are toxic or not. This implies that the musselsmay be used as a biofilter for the removal of harmful cyanobacterialblooms in shallow (Dutch) lakes where the mussels are alreadypresent and not a nuisance. Providing more suitable substratefor zebra mussel attachment may lead to appropriate mussel densitiescapable of filtering large quantities of cyanobacteria.     

Non-toxic and toxic subclones obtained from a toxic clonal culture of Alexandrium tamarense (Dinophyceae): Toxicity and molecular biological feature

Alexandrium tamarense (Lebour) Taylor strain OF935-AT6 is a rare strain of paralytic shellfish toxin (PST)-producing dinoflagellate, in which non-toxic and toxic cells are found in an approximately 1:1 ratio, isolated in Japan. The non-toxic characteristics of UAT-014-009, an axenic non-toxic subclone of OF935-AT6, have been confirmed at the attomole per cell level. Three out of nine toxic subclones of OF935-AT6 became non-toxic over a relatively short period of time (4–6 years), while the other toxic subclones retained their toxicity and the non-toxic subclones retained to be non-toxic. Two axenic subclones from OF935-AT6, UAT-014-009 (non-toxic) and Axat-2 (toxic) are indistinguishable from one another, and from popularly known A. tamarense by rDNA sequence analysis. The most significant difference identified by subtractive hybridization of cDNA pertains to gene fragments homologous with mitochondrial cytochrome c oxidase polypeptide three (cox3) and cytochrome b (cob). Thus, the polymorphism targeting these regions was investigated by comparison of the gene length amplified by PCR using total DNA from other subclones with a range of toxicities. No direct correlation between any allele and toxicity was observed in this study.     

Response of two zooptankton grazers to an ichthyotoxic estuarine dinoflagellate

The dinoflagellate Pfiesteria piscicida (gen. et sp. nov.).a toxic ‘ambush predator’, has been implicated asa causative agent of major fish kills in estuanne ecosystemsof the southeastern USA. Here we report the first experimentaltests of interactions between P.piscicida and estuarine zooplanktonpredators. specifically the rotifer Brachionus plicatilis andthe calanoid copepod Acartia tonsa. Short-term (10 day) exposureof adult B.plicatilis to P.piscicida as a food resource, aloneor in combination with the non-toxic green algae Nannochlorisand Tetraselmis. did not increase rotifer mortality relativeto animals that were given only non-toxic greens Similarly,short-term (3 day) feeding trials using adult A.tonsa indicatedthat the copepods survived equally well on either P.piscicidaor the non-toxic diatom Thalassiosira pseudonana. Copepods giventoxic dinoflagellates exhibited erratic behavior, however, relativeto animals given diatom prey. The fecundity of B.plicatiliswhen fed the toxic dinoflagellate was comparable to or higherthan that of rotifers fed only non-toxic greens We concludethat, on a short-term basis, toxic stages of P.piscicida canbe readily utilized as a nutritional resource by these commonestuarine zooplankters. More long-term effects of P.piscicidaon zooplankton, the potential for toxin bioaccumulation acrosstrophic levels, and the utility of zooplankton as biologicalcontrol agents for this toxic dinoflagellate. remain importantunanswered questions.     

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.     

The Newly Described Heterotrophic Dinoflagellate Gyrodinium moestrupii,an Effective Protistan Grazer of Toxic Dinoflagellates

Few protistan grazers feed on toxic dinoflagellates, and low grazing pressure on toxic dinoflagellates allows these dinoflagellates to form red‐tide patches. We explored the feeding ecology of the newly described heterotrophic dinoflagellate Gyrodinium moestrupii when it fed on toxic strains of Alexandrium minutum, Alexandrium tamarense, and Karenia brevis and on nontoxic strains of A. tamarense, Prorocentrum minimum, and Scrippsiella trochoidea. Specific growth rates of G. moestrupii feeding on each of these dinoflagellates either increased continuously or became saturated with increasing mean prey concentration. The maximum specific growth rate of G. moestrupii feeding on toxic A. minutum (1.60/d) was higher than that when feeding on nontoxic S. trochoidea (1.50/d) or P. minimum (1.07/d). In addition, the maximum growth rate of G. moestrupii feeding on the toxic strain of A. tamarense (0.68/d) was similar to that when feeding on the nontoxic strain of A. tamarense (0.71/d). Furthermore, the maximum ingestion rate of G. moestrupii on A. minutum (2.6 ng C/grazer/d) was comparable to that of S. trochoidea (3.0 ng C/grazer/d). Additionally, the maximum ingestion rate of G. moestrupii on the toxic strain of A. tamarense (2.1 ng C/grazer/d) was higher than that when feeding on the nontoxic strain of A. tamarense (1.3 ng C/grazer/d). Thus, feeding by G. moestrupii is not suppressed by toxic dinoflagellate prey, suggesting that it is an effective protistan grazer of toxic dinoflagellates.     

Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

We have developed a method to identify species in the genus Alexandrium using whole-cell fluorescent in situ hybridization with FITC-labeled oligonucleotide probes that target large subunit ribosomal rRNA molecules. The probes were designed based on the sequence of the rDNA D1-D2 region of Alexandrium species. DNA probes specific for toxic A. tamarense and A. catenella and nontoxic A. affine, A. fraterculus, A. insuetum, and A. pseudogonyaulax, respectively, were applied to vegetative cells of all above Alexandrium species to test the sensitivity of the probes. Each DNA probe hybridized specifically with vegetative cells of the corresponding Alexandrium species and showed no cross-reactivity to noncorresponding Alexandrium species. In addition, no cross-reactivity of the probes was observed in experiments using concentrated natural seawater samples. The TAMAD2 probe, which is highly specific to A. tamarense, a common toxic species in Korean coastal waters, provides a simple and reliable molecular tool for identification of toxic Alexandrium species.     

Species-Specific Detection and Quantification of Toxic Marine Dinoflagellates Alexandrium tamarense and A. catenella byReal-Time PCR Assay

A Real-time polymerase chain reaction (PCR) assay was designed and evaluated for rapid detection and quantification of the toxic dinoflagellates Alexandrium catenella and A. tamarense, which cause paralytic shellfish poisoning. Two sets of PCR primers and fluorogenic probes targeting these two species were derived from the sequence of 28S ribosomal DNA. PCR specificity was examined in closely related Alexandrium spp. and many other microalgae. A. catenella–specific primers and probe detected the PCR amplification only from A. catenella strains, and nonspecific signals were not detected from any microalgae. Also, A. tamarense–specific primers and probe also detected the targeted species, suggesting the strict species specificity of each PCR. This assay could detect one cell of each species, showing its high sensitivity. Moreover, using the developed standard curves, A. tamarense and A. catenella could be quantified in agreement with the quantification by optical microscopy. The performance characteristics of species specificity, sensitivity, and rapidity suggest that this method is applicable to the monitoring of the toxic A. tamarense and A. catenella.     

Rapid detection of natural cells of Alexandrium tamarense and A. catenella (Dinophyceae) by fluorescence in situ hybridization

The marine toxic dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor that cause paralytic shellfish poisoning (PSP) are identified on the basis of morphological features in routine monitoring. Rapid and simple identification is, however, often difficult because of the morphological similarity. Fluorescent in situ hybridization (FISH) using ribosomal RNA (rRNA)-targeted probes has been studied as a method of easily identifying and enumerating species responsible for harmful algal blooms (HABs). Its application to monitoring natural populations of HAB species, however, is limited. Here, we applied the FISH method to identify and enumerate cells of A. tamarense and A. catenella in natural plankton assemblages collected from Japanese coastal waters. A. tamarense-specific (Atm1) and A. catenella-specific (Act1) probes were established based on the D2 region of the large-subunit ribosomal RNA gene (28S rDNA). With these two probes, natural cells of A. tamarense or A. catenella in field samples could easily be identified when the following three conditions were met. First, cells should be concentrated by filtration, not centrifugation, in order to avoid the loss of cells. Second, autofluorescence should be minimized; acetone was an effective decolorization reagent. Third, samples should be stored at −20 or −80 °C for long-term preservation. The results indicate that FISH is a useful tool for the rapid identification of toxic Alexandrium spp. and can facilitate the analysis of numerous natural samples.     

Probable origin and toxin profile of Alexandrium tamarense (Lebour) Balech from southern Brazil

The distribution of the toxic dinoflagellate Alexandrium tamarense Lebour has apparently expanded within the southern hemisphere during the last 2 decades. Toxic blooms of A. tamarense were recorded in Argentinean coastal waters since 1980; however, the first documented bloom in southern Brazil was in 1996. In this study, 13 strains of A. tamarense from southern Brazil were isolated and kept in culture. Phylogenetic analysis using RFLP and DNA sequences of the D1–D2 region of large subunit ribosomal DNA (rDNA) clearly indicates that Brazilian strains are most closely related to other South American strains. The strains from South America are placed firmly within a phylogenetic clade which contains strains from North America, northern Europe and northern Asia, previously called the North American clade. Possible dispersal hypotheses are discussed. The cultures were also analyzed for saxitoxin and its derivatives by high performance liquid chromatography (HPLC). The main saxitoxin groups found were the low toxicity N-sulfocarbamoyl group, C1, 2 (30–84%), followed by the high potency carbamate toxins, gonyautoxins 1, 4 (6.6–55%), gonyautoxins 2, 3 (0.3–29%), neosaxitoxin (1.4–24%) and saxitoxin (0–4.4%). The toxin composition is similar to that of other strains from South America, supporting a close relationship between A. tamarense from southern Brazil and other areas of South America. Toxicity values were variable (7.07–65.92 pg STX cell⁻¹), with the higher range falling among the most toxic values recorded for cultures of A. tamarense, indicating the significant risk for shellfish contamination and human intoxication during blooms of this species along the southern Brazilian coast.     

IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM CATENELLA AND A. TAMARENSE (DINOPHYCEAE) USING DNA PROBES AND WHOLE-CELL HYBRIDIZATION1

Fluorescent DNA probes (cCAT-F1 and cTAM-Fl) complementary to the 3′ end of ribosomal RNA (rRNA) internal transcribed spacer 1 sequences (ITS 1: positions 154–176) of toxic species of Alexandrium catenella (Whedon and Kofoid) Taylor and A. tamarense (Lebour) Taylor were applied to various cultures of the genus Alexandrium and several other phytoplankters using whole-cell fluorescent in situ hybridization. cCAT-F1 and cTAM-F1 reacted with targeted strains of A. catenella (catenella type) and A. tamarense (tamarense type), respectively, and did not react with isolates of A. affine (Inoue et Fukuyo) Balech, A. fraterculus (Balech) Balech, A. insuetum Balech, A. lusitanicum Balech, A. pseudogonyaulux (Biecheler)Horiguchi ex Yuki et Fukuyo comb. nov., nor isolates of Prorocentrum micans Ehrenberg, Amphidinium carterae Hulburt, Heterocapsa triquetra (Ehrenberg) Stein, Gymnodinium mikimotoi Miyake et Kominami ex Oda, Skeletonema costatum (Greville) Cleve, Heterosigma akashiwo (Hada) Hada, and Chattonella antiqua (Hada) Ono. DNase I and RNase A treatment showed that probes hybridized to ribosomal DNA, not rRNA. Probes were localized at the bottom of the U-shaped nucleus, a region that corresponds to the nucleolus. The probes are highly specific for particular strains of A. catenella and A. tamarense and are applicable for identifying these species collected from cultured and possibly natural populations.     

有毒亚历山大藻对卤虫存活率和摄食率的影响

研究了有毒亚历山大藻对卤虫存活率和摄食率两方面的影响,得出以下结论：在卤虫存活率实验中,有毒亚历山大藻在2000cells／ml的密度下,对卤虫具有致死效应,卤虫在24-168h内全部死亡;在摄食实验中,有毒亚历山大藻对卤虫的摄食产生明显的抑制作用,卤虫对有毒藻的平均摄食率明显低于无毒藻组和混合实验组。在加入无毒藻东海原甲藻的混合培养状态下。卤虫存活率上升,30-60min摄食率增加,东海原甲藻在一定程度上可以减轻塔玛亚历山大藻对卤虫的毒害作用。有毒藻产生的PSP毒素并非导致卤虫死亡的主要原因,毒害作用可能与出现在卤虫体外的黏附物质有关。通过对3个不同生长期卤虫的研究发现,后无节幼体卤虫对有毒亚历山大藻的毒害作用最为敏感。     

Fourteen FITC-conjugated lectins as a tool for the recognition and differentiation of some harmful algae in Chinese coastal waters

In order to test the use of lectins as a tool for the differentiation of harmful algal species, 13 species and 23 strains of algae were tested with 14 fluorescein isothiocyanate (FITC)-conjugated lectins, and the results examined using flow cytometry (FCM), epifluorescence microscopy (EFM) and spectrofluorometry (SFM). The lectin probes SBA, WGA, GSL I, DBA and PHA-E could distinguish between morphologically similar Gymnodinium-like species, such as Karenia mikimotoi (GMDH01), Takayama pulchellum (TPXM01) and Gymnodinium sp. (GspXM01), by their different binding activities. With the precise quantitative measurements of binding obtained using SFM and FCM, lectins appeared to be useful in distinguishing different strains of the same species. The results also showed that PHA-E could differentiate Alexandrium tamarense (ATDH04) from other strains of this species, and SJA could distinguish A. tamarense (ATMJ02) from other strains of this species (including ATMJ01). Similarly, PNA could identify A. tamarense (ATDH01, 02, 03); UEA I could recognize A. tamarense (ATCI01-JN, ATCI01); and RCA120 could differentiate Alexandrium sp (AspGX01) from strain AspGX02, which was shown to produce different levels of paralytic shellfish poisoning toxin. Lectin probes could also bind these target cells in mixed algal samples. Positive cells identified by FCM were clearer than negative cells thus, in EFM, both GspXM01 and TPXM01 labeled with a WGA lectin probe could be distinguished from target cells of K. mikimotoi, Prorocentrum donghaiense and P. minimum (PMDH01, PMXM01) in mixed algal samples. FCM, EFM and SFM analysis could clearly distinguish lectin-probe-bound cells from negative cells in culture.     

A type II restriction endonuclease of Streptococcus thermophilus ST117

Three separate sets of polymerase chain reaction primers were designed to specifically detect the presence of a toxin A gene fragment, a toxin B gene fragment, and the entire toxin B gene. In addition toxin gene fragments that were amplified from well characterized toxic strains were tagged fluorescently and used as hybridization probes to screen C. difficile strains. A survey of 37 toxic strains and 10 non-toxic strains demonstrated that toxic strains normally contain the genetic composition for toxin A and toxin B simultaneously; whereas, non-toxic strains typically did not contain detectable toxin determinants. The only exception found was strain 39, which had the genetic composition for toxins A and B, but was not cytotoxic under the conditions tested.     

Influence of toxic and non-toxic phytoplankton on feeding and survival of Dreissena polymorpha (Pallas) larvae

Grazing and survival of larvae of the zebra mussel, Dreissena polymorpha, on a green alga and cyanobacteria were studied in laboratory experiments. Clearance rates of the larvae were determined for Chlamydomonas reinhardtii (green alga), two non-toxic and two toxic Microcystis aeruginosa strains (Cyanobacteria). Clearance rates of larvae on non-toxic Microcystis were significantly higher than on toxic Microcystis. The clearance rate on Chlamydomonas reinhardtii was in between the clearance rates on toxic and non-toxic Microcystis strains and not significantly different from them. Effects of toxicity of Microcystis on the survival of zebra mussel larvae was investigated in a short-term experiment. Survival of larvae fed toxic Microcystis was lower than that of larvae fed non-toxic Microcystis, but higher than that of starved larvae. This may imply that, for survival of zebra mussel larvae, it is better to have bad quality (toxic) food than no food.     

Reactive oxygen species as causative agents in the ichthyotoxicity of the red tide dinoflagellate Cochlodinium polykrikoides

The underlying toxic mechanisms of the red tide dinoflagellate,Cochlodinium polykrikoides, were studied with respect to thereactive oxygen species-mediated toxic effect. Cochlodiniumpolykrikoides generates superoxide anion (O₂^–) and hydrogenperoxide (H₂O₂), as measured by the cytochrome c reduction methodand scopoletin–peroxidase method, respectively. The capabilityof C.polykrikoides to generate these oxygen radicals was relatedto the growth phase: the highest rate in the exponential phaseand a gradual decrease in the stationary phase. Other phytoplankton,such as Eutreptiella gymnastica, Heterosigma akashiwo, Prorocentrummicans, Gymnodinium sanguineum and Alexandrium tamarense, alsoproduce H₂O₂; the rate of H₂O₂ generation by these species waslower than that of C.polykrikoides. The exposure of liposomalsamples to intact or ruptured individuals of C.polykrikoidesresulted in severe membrane damage, such as liposomal lipidperoxidation. Cochlodinium polykrikoides-induced lipid peroxidationwas significantly reduced by oxygen radical scavengers, superoxidedismutase, benzoquinone, catalase and mannitol. In addition,lipid peroxidation of gill tissue of flatfish exposed to C.polykrikoidesincreased with increasing algal cell density. These resultssuggest that reactive oxygen species generated from C.polykrikoidesare responsible for oxidative damage leading to fish kills.     

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C₉H₁₆N₆O₂. This structure differed from that of decarbamoyl STX (dcSTX; C₉H₁₆N₆O₃) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.