COMPARISON OF TOXINS IN THREE ISOLATES OF GONYAULAX TAMARENSIS (DINOPHYCEAE)1

Toxicity levels and profiles of three isolates of Gonyaulax tamarensis Lebour grown under the same conditions were compared. One isolate was collected from Ipswich, Massachusetts, during the massive red tide of 1972 along the New England coast. The other two isolates were obtained from Perch Pond (Falmouth, Massachusetts) and Mill Pond (Orleans, Massachusetts) located in the southwest and south of Cape Cod, Massachusetts, respectively. All the three cultures produced toxins with variation in their toxicity levels. Toxin contents were highest in the Ipswich isolate, followed in an order by Mill Pond and Perch Pond cultures. Morphological similarity existed between Ipswich and Mill Pond cells, whereas the Perch Pond cells possessed an additional ventral pore on the l' epithecal plate.     

SEXUAL REPRODUCTION IN THE TOXIC DINOFLAGELLATE GONYAULAX MONILATA1

The sexual cycle of Gonyaulax monilata Howell was observed in stationary cultures and in nitrogen-deficient medium. The armored, isogamous gametes fuse in a characteristic manner with cingula at oblique angles. Nuclear fusion lags slightly behind cytoplasmic fusion. The zygote enlarges for several days. The dark, double-flagellated planozygote encysts within 1–3 wk. Early hypnozygotes are round to ovoid and contain lipid and one or two large golden-yellow globules. As the hypnozygote matures, the globules become smaller and the cytoplasm darkens and pulls from the wall. All cysts examined contained only one nucleus. A very dark, uninucleate post-hypnozygotic cell escapes through an archeopyle and within 24 h divides into daughter cells which divide in 24–48 h forming a small chain. The production of thick walled zygates in culture implies that such resting stages in marine sediments could serve as a source stock for blooms. This species causes toxic red tides and the existence of benthic “seed beds” consisting of hypnozygotes is now plausible.     

BEHAVIOR AND ECOLOGY OF THE ATLANTIC WHITE-SIDED DOLPHIN (LAGENORHYNCHUS ACUTUS) IN COASTAL NEW ENGLAND WATERS

Atlantic white-sided dolphins ( Lagenorhynchus acutus ) are among the most abundant, and least studied, cetaceans in coastal New England. Between April and October 1984 through 1997 we sighted 1,231 groups of Atlantic white-sided dolphins, primarily on Stellwagen Bank and Jeffreys Ledge (two shallow glacial deposits along the coasts of Massachusetts, New Hampshire, and Maine). Mean group size was 52 (≥90.9), and was significantly larger from August through October (71.9 ± 111.4) than April through June (35.0 ± 45.4). Calf sightings were uncommon until June and July, after which they were present in over 50% of groups. Combined with observations of apparent newborn calves, this confirms that early summer is an important calving period. The presence of calves did not, however, solely account for the increase in group size. Boat interaction (bow- and stern-wake riding) was the most commonly recorded behavior (47.4% of sightings), followed by traveling (31.4%), interactions with other cetacean species (27.6%), social interaction (15.5%), and feeding (9.5%). While feeding was uncommon, one observation of apparently coordinated "ball" feeding was seen with sand lance ( Ammodytes spp.) as the visible prey. Aerial behavior showed a positive correlation with group size, although it was often impossible to tell whether the same dolphins were leaping repeatedly. Eighty-eight dolphins were photo-identified using either unusual body pigment or a distinctive dorsal fin. While several individuals were reidentified between years and between areas, no reidentifications were made within a year in the same area. Unusually pigmented individuals were much more likely to be reidentified than those with distinctive dorsal fins, most likely due to higher visibility. We suggest that Atlantic white-sided dolphins are generally using the study area as transients in what appears to be a large home range.     

POTENTIAL IMPORTANCE OF BENTHIC CYSTS OF GONYAULAX TAMARENSIS AND G. EXCAVATA IN INITIATING TOXIC DINOFLAGELLATE BLOOMS1,2,3

Thick-walled, nonmotile cysts (termed hypnocysts) of two dinoflagellates were isolated from estuarine sediments in Cape Cod, Massachusetts, and germinated to produce their respective motile, thecate stages. Hypnocysts from Orleans district were identified as Gonyaulax excuvata (Braarud) Balech sensu Loeblich & Loeblich. Visually identical hypnocysts from Falmouth district were provisionally identified as Gonyaulax tamarensis Lebour. Both species were toxic. A geographic survey in September detected hypnocysts in only the sediments of locations where toxic blooms developed the preceding and following Spring. Laboratory incubation (16 C) of hypnocysts from sediment samples stored in the dark (5 C) for 6 mo initiated excystment by the temperature increase, with no appreciable effect from light regime, nutrient, or chelator concentrations. Motility of excysted germlings was optimum in highly chelated medium and in the presence of light. We conclude that hypnocysts of both tasa are important in seeding recurrent annual blooms, synchronizing early bloom development with vernal warning of seawater and increasing the geographic range of the species. We suggest that many red tides in New England and eastern Canadian waters are initiated through the displacement of motile estuarine populations into nearshore area by tidal advection and surface runoff, although the potential existence and importance of offshore cyst reservoirs cannot be discounted. Evidence is presented that hypnocysts are probable sexual zygotes whereas the thin-walled cysts readily formed in laboratory cultures (pellicle cyst) are asexual. Pellicle cysts are of limited durability, do not overwinter in nature, and therefore do not play a significant role in initiating toxic blooms.     

SEXUALITY AND CYST FORMATION IN PACIFIC STRAINS OF THE TOXIC D1NOFLAGELLATE GONYAULAX TAMARENSIS1,2

Sexuality has been established for a culture of Gonyaulax tarnarensis Lebour (strain NEPCC–71). The addition of a thick inoculum to a nitrogen–deprived medium results in the occurrence of anisogamous sexual fusion within the first three days in the new culture. Planozygotes, large “lumpy” cells recognizable by their four flagella, may persist up to 2 wk before forming a smooth–walled, oval hypnozygote. The latter resembles cysts released asexually by ecdysis but has a slightly thicker wall. Viable cysts resembling hypnozygotes (zygotic cysts), but with reduced photosynthetic pigmentation, have been isolated from natural murine sediments in Hidden Basin, British Columbia, and a culture (strain NEPCC–254) was initiated from excysted individuals. Zygotic cysts of NEPCC–71 remained encysted in the light at 17 C for 8 wk before excysting. The presence of a ventral pen with toxicity in the latter strain indicates that the taxonomy of G. tamarensis-like organisms is still in a stale of flux and the criteria for recognition of G. excavata (Braarud) Balech as a separate species are not satisfactory as presently formulated.     

MICROSATELLITE MARKERS REVEAL POPULATION GENETIC STRUCTURE OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN JAPANESE COASTAL WATERS1

This is the first report to explore the fine‐scale diversity, population genetic structure, and biogeography of a typical planktonic microbe in Japanese and Korean coastal waters and also to try to detect the impact of natural and human‐assisted dispersals on the genetic structure and gene flow in a toxic dinoflagellate species. Here we present the genetic analysis of Alexandrium tamarense (Lebour) Balech populations from 10 sites along the Japanese and Korean coasts. We used nine microsatellite loci, which varied widely in number of alleles and gene diversity across populations. The analysis revealed that Nei's genetic distance correlated significantly with geographic distance in pair‐wise comparisons, and that there was genetic differentiation in about half of 45 pair‐wise populations. These results clearly indicate genetic isolation among populations according to geographic distance and restricted gene flow via natural dispersal through tidal currents among the populations. On the other hand, high P‐values in Fisher's combined test were detected in five pair‐wise populations, suggesting similar genetic structure and a close genetic relationship between the populations. These findings suggest that the genetic structure of Japanese A. tamarense populations has been disturbed, possibly by human‐assisted dispersal, which has resulted in gene flow between geographically separated populations.     

COMPARATIVE STUDY OF LUMINESCENT AND NONLUMINESCENT STRAINS OF GONYAULAX EXCAVATA (PYRRHOPHYTA)1,2

Three of ten cultures of Gonyaulax excavata (Braarud) Balech isolated from the 1972 New England red tide are nonluminescent, Biochemical components of dinoflagellate bioluminescence were not detected in the extracts from these three isolates. Cells of the nonluminescent cultures were identical to those of luminescent cultures as compared by light microscopy, major body plate tabulation, cell size and growth. Both luminescent and nonluminescent cells were toxic as determined by using the mouse bioassay for paralytic shellfish poisoning. All the 122 clones made from one of the luminescent isolates were luminescent suggesting this feature is a stable trait. We conclude that these isolates represent luminescent and luminescent strains of G. excavata. This is the first intraspecific investigation of in vitro bioluminescent components between nonluminescent and luminescent strains of a dinoflagellate.     

PRODUCTION OF 7-DEOXY-OKADAIC ACID BY A NEW CALEDONIAN STRAIN OF PROROCENTRUM LIMA (DINOPHYCEAE)

7-Deoxy-okadaic acid and okadaic acid were identified as the major diarrhetic shellfish poisoning (DSP) toxins produced by a New Caledonian strain of Prorocentrum lima Ehrenberg. Dinophysistoxin-1 was not produced by this strain. The cellular concentrations of 7-deoxy-okadaic acid were about one tenth that of okadaic acid and were maximal (∼1.4 pg·cell ^{− 1}) during the stationary growth phase of batch culture. Autolytic hydrolysis of cell extracts did not increase the concentrations of 7-deoxy-okadaic acid, whereas okadaic acid production increased more than 4-fold, indicating that 7-deoxy-okadaic acid, unlike okadaic acid, is not directly derived from large sulfated precursors. 7-Deoxy-okadaic acid could be detected by liquid chromatography-selected reaction monitoring mass spectrometry, HPLC-fluorescence detection after derivatization with 9-anthryldiazomethane (ADAM), and inhibition of protein phosphatases. The solvent washes currently used for solid-phase clean-up of ADAM-derivatized DSP samples elute derivatized 7-deoxy-okadaic acid, indicating that the current sample clean-up protocol for HPLC-fluorescence detection would miss any contamination by this toxin.     

CHARACTERIZATION OF GYMNODINIUM MIKIMOTOI (DINOPHYCEAE) NUCLEI AND IDENTIFICATION OF THE MAJOR HISTONE-LIKE PROTEIN, HGm

A simple and rapid method for isolation of nuclei from Gymnodinium mikimotoi Miyake et Kominami ex Oda is described along with chemical characterization of the nuclei. The isolated nuclei were completely free of whole cells, 99.96% free of cytoplasmic contamination, and were collected with a yield of 40% from harvested whole cells. Each nucleus contained 47 pg of DNA and the ratio of DNA to acid-soluble proteins to acid-insoluble proteins was 1:0.25:1.21, respectively. SDS electrophoresis of acid-extracted proteins showed one histone-like protein, which we termed HGm, with an apparent molecular mass of 12 kDa. V8 protease digestion analysis of HGm, the histone-like protein from Crypthecodinium cohnii (HCc), and two histone-like proteins from Gymnodinium dorsum , showed that the HGm digestion pattern was more similar to that of HCc than to that of either of the G. dorsum histone-like proteins.     

EFFECTS OF SMALL-SCALE TURBULENCE ON PHOTOSYNTHESIS,PIGMENTATION, CELL DIVISION,AND CELL SIZE IN THE MARINE DINOFLAGELLATE GOMAULAX POLYEDRA (DINOPHYCEAE)1

Several experiments were conducted to understand better the physiological mechanisms underlying growth inhibition of the dinoflagellate Gonyaulax polyedra Stein due to small-scale turbulence shear. To measure photosynthetic ¹⁴C uptake, a “phytoplankton wheel” device for rotating cultures in closed bottles was used. Turbulence was quantified biologically in the bottles by comparing growth inhibition with that in cultures with constant shear between a fixed cylinder and an outer concentric rotating cylinder (a stable Couette flow). At saturating irradiances, particulate photosynthesis (P_sat) or photosynthesis per unit chlorophyll (P^B_sat) were not inhibited completely at the highest turbulence level (26.6 rad.s^?1), and photosynthesis was less sensitive than growth. Photosynthesis per cell (P^C_sat) was increased by turbulence. In three experiments on the effects of turbulence on photosynthesis versus irradiance curves, the slope of the curve, α, for particulate photosynthesis at limiting irradiances did not change. Photosynthesis per unit chlorophyll per unit irradiance (α^B) decreased at high (but not intermediate) turbulence levels. Photosynthesis per cell per unit irradiance, α^C, increased with turbulence, suggesting an increase in photosynthetic efficiency in turbulent cultures. In two of the three experiments, respiration rates increased with turbulence, and in one experiment excretion of photosynthetically fixed ¹⁴C was not affected by motion. Ratios of accessory pigments to chlorophyll a did not change with turbulence, but pigments per cell and per dry weight increased with turbulence. These findings suggest little or no disruption of the photosynthetic apparatus. When turbulence was applied for 1 week, β-carotene increased while peridinin and diadinoxanthin decreased, suggesting inhibition of synthesis of these latter pigments by prolonged turbulence. Since cell numbers did not increase or decreased during turbulent 72–h incubations, cell division was inhibited and also the cells were very much enlarged. Increases in α^C per cell suggest that, in the sea, photo synthetic metabolism can persist efficiently without cell division during turbulent episodes. After turbulence ceases or reaches low levels again, cells can then divide and blooms may form. Thus, blooms can come or go fairly rapidly in the ocean depending on the degree of wave- and wind-induced turbulence.     

CACHONINA ILLDEFINA SP. NOV. (DINOPHYCEAE): CHLOROPLAST TUBULES AND DEGENERATION OF THE PYRENOID1

A new species of the dinoflagellate genus Cachonina, C. illdefina sp. nov., was isolated from a red tide off El Capitan State Park, Santa Barbara County, California, in October 1973. The organism is light yellowgreen in color with deeply incised girdle and sulcal grooves. Electron microscopy of the organism, revealed a typical dinokaryotic nucleus. The chloroplasts of the organism are connected, and often contain microtubule-like elements, 25 nm diam. The pyrenoids are characterized as excluding chloroplast thylakoids and ribosomes, although containing an amorphous matrix and numerous tubular invaginations from the cytoplasm. The pyrenoids become detached from the chloroplasts and degenerate into small vesicles. C. illdefina is not bioluminescent.     

IDENTIFICATION AND TOXICITY OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN SCOTTISH WATERS1

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (～2,000 fg STX equivalents · cell^?1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.     

LSU rDNA-BASED RFLP ASSAYS FOR DISCRIMINATING SPECIES AND STRAINS OF ALEXANDRIUM (DINOPHYCEAE)1

In a previous study large-subunit ribosomal RNA gene (LSU rDNA) sequences from the marine dinoflagellates Alexandrium tamarense (Lebour) Balech, A. catenella (Whedon et Kofoid) Balech, A. fundyense Balech, A. affine (Fukuyo et Inoue) Balech, A. minutum Halim, A. lusitanicum Balech, and A. andersoni Balech were compared to assess inter- and intraspecific relationships. Many cultures compared in that study contained more than one class of LSU rDNA. Sequencing pooled clones of rDNA from single cultures revealed length heterogeneities and sequence ambiguities. This complicated sequence comparisons because multiple rDNA clones from a single culture had to be sequenced individually to document the different classes of molecules present in that culture. A further complication remained as to whether or not the observed intraculture sequence variations were reliable genetic markers or were instead artifacts of the polymerase chain reaction (PCR) amplification, cloning, and/or sequencing methods employed. The goals of the present study were to test the accuracy of Alexandrium LSU rDNA sequences using restriction fragment-length polymorphism (RFLP) analysis and to devise RFLP-based assays for discriminating among representatives of that group. Computer-assisted examination of the sequences allowed us to identify a set of restriction enzymes that were predicted to reveal species, strain, and intraculture LSU rDNA heterogeneities. All groups identified by sequencing were revealed independently and repeatedly by RFLP analysis of PCR-amplified material. Five ambiguities and one length heterogeneity, each of which ascribes a unique group of Alexandrium species or strains, were confirmed by restriction digests. Observed intraculture LSU rDNA heterogeneities were not artifacts of cloning and sequencing but were instead a good representation of the spectrum of molecules amplified during PCR reactions. Intraculture LSU rDNA heterogeneities thus serve as unique genetic markers for particular strains of Alexandrium, particularly those of A. tamarense, A. catenella, and A. fundyense. However, some of these “signature heterogeneities” represented a smaller portion of PCR product than was expected given acquired sequences. Other deviations from predicted RFLP patterns included incomplete digestions and appearance of spurious products. These observations indicate that the diversity of sequences in PCR product pools were greater than that observed by cloning and sequencing. The RFLP tests described here are useful tools for characterizing Alexandrium LSU rDNA to define the evolutionary lineage of cultures and are applicable at a fraction of the time, cost, and labor required for sequencing.     

FLUORESCENCE IN SITU HYBRIDIZATION USING rRNA‐TARGETED PROBES FOR SIMPLE AND RAPID IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM TAMARENSE AND ALEXANDRIUM CATENELLA1

The toxic marine dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor have been mainly responsible for paralytic shellfish poisoning in Japan. Rapid and precise identification of these algae has been difficult because this genus contains many morphologically similar toxic and nontoxic species. Here, we report a rapid, precise, and quantitative identification method using three fluorescent, rRNA‐targeted, oligonucleotide probes for A. tamarense (Atm1), A. catenella (Act1), and the nontoxic A. affine (Inoue et Fukuyo; Aaf1). Each probe was species specific when applied using fluorescence in situ hybridization (FISH). None of the probes reacted with three other Alexandrium spp., A. lusitanicum Balech, A. ostenfeldii (Paulsen) Balech & Tangen, and A. insuetum Balech, or with eight other microalgae, including Gymnodinium mikimotoi Miyake et Kominami ex Oda and Heterosigma akashiwo (Hada) Hara et Chihara, suggesting that the species specificity of each probe was very high. Cells labeled with fluorescein 5‐isothiocyanate–conjugated probes showed strong green fluorescence throughout the whole cell except for the nucleus. FISH could be completed within 1 h and largely eliminated the need for identifying species based on key morphological criteria. More than 80% of targeted cells of both species could be identified by microscopy and quantified during growth up to the early stationary phase; more than 70% of cells could be detected in the late stationary phase. The established FISH protocol was found to be a specific, rapid, precise, and quantitative method that might prove to be a useful tool to distinguish and quantify Alexandrium cells collected from Japanese coastal waters.     

EFFECTS OF GRAZERS AND ALGAL COMPETITORS ON FUCOID COLONIZATION IN TIDE POOLS1

In New England, fucoids are conspicuously absent from midlittoral sheltered tide pools, though abundant on adjacent emergent rocks. Experimental removal of either herbivores or potential algal competitors from mid littoral tide pools failed to result in the establishment of fucoids. However, removal of both groups together resulted in the establishment of Fucus vesiculosus L. Once established, some individuals persisted for up to three years. These grew well, were of normal, healthy appearance and reproduced. The herbivores removed were Littorina littorea, L. obtusata and Acmaea testudinalis. Algae removed were Chondrus crispus Stackhouse, Ulva lactuca L., Rhizoclonium tortuosum Kützing, Spongomorpha spinescens Kützing, Polysiphonia sp., Dumontia incrassata (O.F. Muller) O. Kuntze and Scytosiphon lomentaria (Lyngbye) Link. It is concluded that absence of fucoids from mid littoral, protected pools is due to the joint action of herbivores and algal competitors and not to possible deleterious factors associated with constant submersion.     

RELATIONSHIPS OF SEDIMENTED DIATOM SPECIES (BACILLARIOPHYCEAE) TO ENVIRONMENTAL GRADIENTS IN DILUTE NORTHERN NEW ENGLAND LAKES1

Limnological gradients of small, oligotrophic, and low conductance lakes in northern New England were defined by principal components analysis; relationships of sedimented diatom species to the gradients were investigated by correlation analysis. Diatom distributions were most strongly related to the gradient of pH and alkalinity and the covarying variables, conductance, Mg, Ca, total Al, and exchangeable Al. Weaker relationships to lake morphology, dissolved organic carbon and water color, altitude and marine aerosol inputs, and the distinctive water chemistry of some New Hampshire lakes were also present. Results for 16 taxa of importance in our studies of lake acidity are given in detail and are compared to results from other regions of eastern North America. Planktonic taxa were absent below pH 5.5, with the exception of the long form of Asterionella ralfsii var. americana Korn. The two forms of this taxon differed ecologically: the long form (>45μm) had an abundance weighted mean (AWM) pH 4.90 and occurred mostly in lakes that were deep relative to transparency; the short form (<45μm)had an AWM pH and occurred on lakes that were shallow relative to transparency. The ecological advantage of a “splitter” approach to diatom taxonomy was demonstrated by examination of other taxa as well, including Tabellaria flocculosa (Roth) Kütz. These results have important implications for paleolimnological interpretations.     

广东沿海的砂壳纤毛虫及3种国内新纪录

对广东沿海的砂壳纤毛虫进行了初步研究,涉及7属16种,其中3种确定为中国的新纪录:僧帽壳铃虫Codonella apicata Kofoid&Campbell,1929,陀螺形拟铃虫Tintinnopsis turbo Meunier,1919和膨胀细壳虫Stenosemella expansa Wailes,1925。另有3种为我国亚热带海域的新纪录:筒状拟铃虫Tintinnopsis tubu-losoides Meunier,1910,罗氏拟铃虫Tintinnopsis lohmanni(J rgensen,1927)Laackmann,1906和贪婪铃壳虫Codonella rapa Kofoid&Campbell,1929。其余10种为常见种:百系拟铃虫Tintinnopsis beroidea Stein,1867,尖底拟铃虫Tintinnopsis acuminata(Daday,1887)Kofoid et Campbell,1929,妥肯丁拟玲虫Tintinnopsis to-cantinensis Kofoid&Campbell,1929,筒状拟铃虫Tintinnopsis tubulosoides Meunier,1910,管状拟玲虫Tintinnopsis tublosa Levander,1900,长形拟铃虫Tintinnopsis clongata Daday,1887,罗氏拟铃虫Tintinnopsislohmanni(J rgensen,1927)Laackmann,1906,小领细壳虫Stenosemella parvicollis(Marshall,1934)Hada,1935,钟状网纹虫Favella campanula(Schmidt,1901)J rgensen,1924和贪婪铃壳虫Codonella rapa Kofoid&Camp-bell,1929。文章对其形态分类学做了简要描述。     

CYST FORMATION IN THE RED TIDE DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE): EFFECTS OF IRON STRESS1

The toxic red tide dinoflagellate Alexandrium tamarense (Lebour) Balech (synonymous with Protogonyaulax tamarensis (Lebour) Taylor) was subjected to iron stress in batch culture over a 24-day time course. Monitoring of life history stages indicated that iron stress induced formation of both temporary (= pellicular) and resting (= hypnozygotic) cysts. Our experimental induction of sexuality appeared to be associated with iron limitation rather than the total depletion of biologically available iron. Degenerative changes in organelle (i.e. chloroplast, mitochondrion and chromosome) ultrastructure were largely restricted to pellicular cysts, suggesting that these temporary cysts were more susceptible to short-term iron stress effects than were hypnozygotes. These results are consistent with the hypothesized ecological roles of cysts in maintaining viability over brief (pellicular cysts) and extended (hypnozygotes) exposure to adverse environmental conditions.     

HEMOLYTIC ACTIVITY OF HETEROCAPSA CIRCULARISQUAMA (DINOPHYCEAE) AND ITS POSSIBLE INVOLVEMENT IN SHELLFISH TOXICITY

Heterocapsa circularisquama Horiguchi is lethal to shellfish, particularly bivalves such as pearl oysters ( Pinctada fucata Gould). No detrimental effects of this flagellate on fish have been observed thus far. In this study, we found that H. circularisquama causes mammalian erythrocytes to lyse. Among the erythrocytes tested, rabbit erythrocytes showed the highest susceptibility, whereas erythrocytes from cattle, sheep, and human were relatively insensitive. Heterocapsa triquetra Stein, which is morphologically similar to H. circularisquama but not toxic to bivalves, showed no hemolytic activity toward rabbit erythrocytes. Culture supernatant or ultrasonic-ruptured cells of H. circularisquama showed only weak hemolytic activity. Hemolytic activity was found in the ethanol extract of H. circularisquama cells, suggesting that the hemolytic agents may be more stable in ethanol than in aqueous solution. Both an intact flagellate cell suspension and the ethanol extract caused morphological changes and eventual collapse of unfertilized eggs of Pacific oyster. Furthermore, the ethanol extract was lethal to the microzooplankton rotifer Brachionus plicatilis Müller, which is highly sensitive to H. circularisquama. Our results suggest that a hemolytic toxin produced by H. circularisquama may be one of the causative agents responsible for the shellfish toxicity.