Differences in pigmentation between life cycle stages in Scrippsiella lachrymosa (dinophyceae)

Various life cycle stages of cyst‐producing dinoflagellates often appear differently colored under the microscope; gametes appear paler while zygotes are darker in comparison to vegetative cells. To compare physiological and photochemical competency, the pigment composition of discrete life cycle stages was determined for the common resting cyst‐producing dinoflagellate Scrippsiella lachrymosa. Vegetative cells had the highest cellular pigment content (25.2 ± 0.5 pg · cell^?1), whereas gamete pigment content was 22% lower. The pigment content of zygotes was 82% lower than vegetative cells, even though they appeared darker under the microscope. Zygotes of S. lachrymosa contained significantly higher cellular concentrations of β‐carotene (0.65 ± 0.15 pg · cell^?1) than all other life stages. Photoprotective pigments and the de‐epoxidation ratio of xanthophylls‐cycle pigments in S. lachrymosa were significantly elevated in zygotes and cysts compared to other stages. This suggests a role for accessory pigments in combating intracellular oxidative stress during sexual reproduction or encystment. Resting cysts contained some pigments even though chloroplasts were not visible, suggesting that the brightly colored accumulation body contained photosynthetic pigments. The differences in pigmentation between life stages have implications for interpretation of pigment data from field samples when sampled during dinoflagellate blooms.     

Alexandrium catenella and Alexandrium minutum blooms in the Mediterranean Sea: Toward the identification of ecological niches

Annual recurrent blooms of the toxic dinoflagellates Alexandrium catenella and Alexandrium minutum were detected from 2000 to 2003 in harbours along the Catalan coast. The interrelation study between the occurrence of the blooms and specific external conditions at the study sites demonstrated that different factors are required for the bloom of each Alexandrium species. Concentrations higher than 10⁵ cells l⁻¹ of A. catenella were only detected in Tarragona harbour. These blooms were associated with water surface temperature between 21 and 25 °C and salinities of around 34 psu or higher than 37 psu. A. minutum appeared widely spread along the Catalan coast, though the most intensive and recurrent blooms of this species were observed in Arenys de Mar harbour. Concentrations of millions of cells per litre of A. minutum were associated with water temperatures below 14 °C and salinities of around 34–36 psu. A. minutum cell densities showed a positive significant correlation with NO₃ but a negative correlation with NH₄. On the other hand, A. catenella blooms dominated when both NO₃ and NH₄ levels were high. The prevailing inorganic nitrogen form (NO₃ vs. NH₄) could explain why these two species rarely coincide in the same harbours. Accumulation of cysts in the sediment was found to be an important potential factor for the recurrence of these species. The 4.3 × 10³ A. catenella cysts cm⁻³ of wet sediment in Tarragona harbour and the 3.02 × 10³ A. minutum cysts cm⁻³ of wet sediment in Vilanova harbour were the highest concentrations observed from the cyst study. Confined waters such as harbours play an important role as reservoirs for the accumulation of cysts and vegetative cells, which contributes to the expansion of these dinoflagellates in the region. However, the particular environmental conditions are also decisive factors of bloom intensity.     

The toxic dinoflagellate Cochlodinium polykrikoides (Dinophyceae) produces resting cysts

While harmful algal blooms (HABs) caused by the toxic dinoflagellate Cochlodinium polykrikoides have been known to science for more than a century, the past two decades have witnessed an extraordinary expansion of these events across Asia, North America, and even Europe. Although the production of resting cysts and subsequent transport via ships’ ballast water or/and the transfer of shellfish stocks could facilitate this expansion, confirmative evidence for cyst production by C. polykrikoides is not available. Here, we provide visual confirmation of the production of resting cysts by C. polykrikoides in laboratory cultures isolated from North America. Evidence includes sexually mating cell pairs, planozygotes with two longitudinal flagella, formation of both pellicular (temporary) cysts and resting cysts, and a time series of the cyst germination process. Resting cyst germination occurred up to 1 month after cyst formation and 2–40% of resting cysts were successfully germinated in cultures maintained at 18–21 °C. Pellicular cysts with hyaline membranes were generally larger than resting cysts, displayed discernable cingulum and/or sulcus, and reverted to vegetative cells within 24 h to ∼1 week of formation. A putative armored stage of C. polykrikoides was not observed during any life cycle stage in this study. This definitive evidence of resting cyst production by C. polykrikoides provides a mechanism to account for the recurrence of annual blooms in given locales as well as the global expansion of C. polykrikoides blooms during the past two decades.     

LIFE HISTORY AND IN SITU GROWTH RATES OF ALEXANDRIUM TAYLORI (DINOPHYCEAE, PYRROPHYTA)

Alexandrium taylori Balech is a phototrophic marine dinoflagellate. It produced recurrent blooms during the summer months (July and August) of 1994 to 1997 in La Fosca beach (NW Mediterranean). In addition to a motile vegetative form, A. taylori had two benthic forms: temporary cysts and resting cysts. Temporary cysts were a temporally quiescent stage produced from the ecdysis of the vegetative cell in both natural populations and laboratory cultures. Temporary cysts may divide to form motile cells. Resting cysts had a thicker wall than the temporary cysts and had a red accumulation body. Gametes and planozygotes were also observed in laboratory cultures. Alexandrium taylori showed in situ diurnal vertical migration with an increase of vegetative cells in the water column in the morning through midday, with concentrations peaking in the afternoon followed by lower levels at night. Most vegetative cells lost their thecae and flagella, and with them their motility, turning into temporary cysts that settled in the early evening. The number of temporary cysts in the water column rose in the evening and at night. The temporary cysts gave rise to motile cells the following morning. Synthesis of DNA occurred in vegetative cells at night, and a preferential period of cell division occurred at sunrise. The estimated division rate in the field was 0.4–0.5 vegetative cells·day⁻¹. Temporary cysts had twice the DNA of a G₁ vegetative cell. The minimum in situ division rate of the temporary cysts was 0.14 day⁻¹. The role of the resting and temporary cyst population in the annual recurrence and maintenance of the A. taylori bloom is discussed.     

Viability of dinoflagellate cysts after the passage through the copepod gut

Several dinoflagellate species form nonmotile, thick-walled resting cysts in their life cycle. Cysts can be ingested by planktonic and benthic organisms, but there is scarce information concerning their survival after the passage through the digestive apparatus of the grazers. We tested the germination capability of cysts produced by two neritic dinoflagellates, Scrippsiella trochoidea (F. Stein) A.R. Loeblich and Scrippsiella ramonii Montresor, after their ingestion by four copepod species. Experiments have been carried out with four species: Acartia clausi Giesbrecht, 1889; Centropages typicus Kröyer, 1849; Temora stylifera Dana, 1849; and Clausocalanus lividus Frost and Fleminger, 1968. Copepods were fed either with motile cells or cysts, and feeding and clearance rates were estimated for A. clausi, C. lividus and T. stylifera. Grazing rates on both dinoflagellates was much higher for vegetative cells than for cysts. Resting cysts were isolated from the faecal pellets and incubated to test their germination capability. S. trochoidea cysts eaten by C. typicus and T. stylifera showed a high germination rate, while cysts of the same species were not viable after the passage through the gut of A. clausi and C. lividus. In contrast, S. ramonii cysts were never able to germinate after being ingested by copepods. The observed variation in viability among the two cyst types and the different survival rates observed for S. trochoidea cysts might be related to differences in cyst morphology and to differences in the digestive process among the tested copepod species.     

Profiles of Alexandrium catenella cysts in Puget Sound sediments and the relationship to paralytic shellfish poisoning events

The magnitude of paralytic shellfish poisoning (PSP) toxins in shellfish and the geographical scope of shellfish closures in Puget Sound have increased in recent decades. PSP, monitored by the Washington Department of Health, has spread from Sequim Bay in the 1950s into central Puget Sound in the 1970s and throughout Puget Sound by the 1990s. Alexandrium catenella, the species responsible for PSP toxins, produces a benthic resting cyst that, upon germinating, can seed blooms. This study examined whether there is a relationship between profiles of cysts in the sediment and temporal variation in PSP in shellfish and if the history of the toxin's southward expansion through Puget Sound can be seen in the cyst record. To address this question, sediment cores were collected from three Puget Sound basins, Sequim Bay, Penn Cove, and Carr Inlet, and cyst profiles were determined. Activities of ²¹⁰Pb were fitted to a depth-dependent diagenetic model to date the sediment cores and determine mixing and sediment-accumulation rates. In order to compare historical variation in PSP with cyst profiles that have been altered by bioturbation, a depth and time-dependent diagenetic model was then used to predict vertical profiles of cysts that would occur under the assumption that cyst deposition rates are proportional to PSP concentration in shellfish measured over several decades at each site. The cyst profiles predicted by the model were compared to measured cyst profiles. These comparisons suggested that Alexandrium blooms and resulting PSP concentration in shellfish are more closely linked to cyst germination and deposition at some stations than at others. Sequim Bay had relatively large numbers of cysts and it is likely that the persistent toxicity here is the result of recurrent seeding from the cyst bed. Penn Cove and Carr Inlet had few cysts despite occasional blooms, suggesting that blooms are transported into those areas, perhaps from other sites of cyst germination. Sequim Bay and Penn Cove had cysts from top to bottom of the cores so it was not possible to determine the date when cysts were first introduced into these bays, but it is likely that A. catenella has been in Penn Cove since at least 1955 or for about two decades before the WDOH PSP toxicity data would indicate. The cyst profile in Carr Inlet suggested a first appearance date of 1985 that is consistent with the first appearance of PSP in shellfish in 1988.     

Enhancement of growth and gymnodimine production by the marine dinoflagellate, Karenia selliformis

Because of its novel bioactive properties the production of gymnodimine for use as a pharmaceutical precursor has aroused interest. The dinoflagellate, Karenia selliformis produces gymnodimine when grown in bulk culture using GP + selenium medium but the growth rates (μ) and levels of gymnodimine are low (μ, 0.05 days⁻¹; gymnodimine 250 μg L⁻¹ max). We describe the effects of organic acid additions (acetate, glycolate, alanine and glutamate additions and combinations of these) in enhancing growth and gymnodimine production in axenic cultures. The most effective organic acid combinations in decreasing order were: glycolate/alanine > acetate > glycolate. Glycolate/alanine optimised gymnodimine production by prolonging growth (maximum cell yield, 1.76 × 10⁵ cells mL⁻¹; gymnodimine, 1260 μg L⁻¹; growth rate (μ), 0.2 days⁻¹) compared to the control (growth maximum cell yield, 7.8 × 10⁴ cells mL⁻¹; gymnodimine, 780 μg L⁻¹; μ, 0.17 days⁻¹). Acetate enhanced gymnodimine by stimulating growth rate (μ, 0.23 days⁻¹) and the large concentration of gymnodimine per cell (16 pg cell⁻¹ cf. 9.8 pg cell⁻¹ for the control) suggests a role for this compound in gymnodimine biosynthesis. Amending culture media with Mn²⁺ additions resulted in slightly decreased growth in control cultures and increased the gymnodimine while in glycolate/alanine cultures growth was stimulated but gymnodimine production decreased. The results suggest that the organic acid can enhance gymnodimine production by either enhancing growth maximum or the biosynthetic pathway.     

Comparison of the functional and numerical responses of resistant versus non-resistant populations of the copepod Acartia hudsonica fed the toxic dinoflagellate Alexandrium tamarense

The functional and numerical responses of grazers are key pieces of information in predicting and modeling predator–prey interactions. It has been demonstrated that exposure to toxic algae can lead to evolved resistance in grazer populations. However, the influence of resistance on the functional and numerical response of grazers has not been studied to date. Here, we compared the functional and numerical responses of populations of the copepod Acartia hudsonica that vary in their degree of resistance to the toxic dinoflagellate Alexandrium tamarense. In common environment experiments carried out after populations had been grown under identical conditions for several generations, female copepods were offered solutions containing different concentrations of either toxic A. tamarense or the non-toxic green flagellate Tetraselmis sp. ranging from 25 to 500 μgC L⁻¹, and ingestion and egg production rates were measured. Throughout most of the range of concentrations of the toxic diet, copepod populations that had been historically exposed to toxic blooms of Alexandrium exhibited significantly higher ingestion and egg production rates than populations that had little or no exposure to these blooms. In contrast, there were no significant differences between populations in ingestion or egg production for the non-toxic diet. Hence, the between population differences in functional and numerical response to A. tamarense were indeed related to resistance. We suggest that the effect of grazer toxin resistance should be incorporated in models of predator and toxic prey interactions. The potential effects of grazer toxin resistance in the development and control of Alexandrium blooms are illustrated here with a simple simulation exercise.     

Dinoflagellate resting cysts as factors in phytoplankton ecology of the North Sea

The occurrence and distribution of dinoflagellate resting cysts were investigated at 11 locations in the south-eastern part of the North Sea. Twenty-six known cyst species and 7 unknown cyst types, which may act as seed population for planktonic dinoflagellate blooms, have been recorded for the first time in the area. The most common cysts in recent sediments were those ofScrippsiella trochoidea, Zygabikodinium lenticulatum, Peridinium dalei, Scrippsiella lachrymosa, Protoceratium reticulatum, Protoperidinium denticulatum, andP. conicum. At all stations,S. trochoidea dominated the cyst assemblages with a maximal abundance of 1303 living cysts/cm³ in the uppermost half centimetre. Cysts of the potentially toxic dinoflagellatesAlexandrium cf.excavatum andA. cf.tamarense were scarce. In the upper 2-cm layer of sediment, dinoflagellate cysts were found in concentrations of 1.8 up to 682 living cysts/cm³. Empty cysts constituted 22–56% of total cyst abundance. The comparative distribution of the cysts showed a general increase in abundance from inshore sites to the offshore area, whereby sandy stations exhibited the lowest cyst abundance and diversity. The wide distribution of living and empty cysts ofScrippsiella lachrymosa suggests that its motile form, which has not been officially recorded in the area until now, is a common plankton organism in German coastal waters. The relatively high abundance of cysts in recent sediments demonstrates the potential importance of benthic resting stages for the initiation of dinoflagellate blooms in the study area.     

Effects of Acacia nilotica, A. polyacantha and Leucaena leucocephala leaf meal supplementation on performance of Small East African goats fed native pasture hay basal forages

Optimal utilisation of tannin-rich browse tree fodders including Acacia spp. foliages as crude protein (CP) supplements to ruminants in the tropics is limited by less available information on their feed nutritive potential. Two studies were conducted to: (1) determine rate and extent of ruminal dry matter (DM) degradability (DMD) and (2) investigate effect of sun-dried Acacia nilotica (NLM), A. polyacantha (PLM) and Leucaena leucocephala leaf meal (LLM) supplementation on growth performance of 20 growing (7–9 months old) Small East African male goats (14.6 ± 0.68 kg) fed on native pasture hay (NPH) basal diet for 84 days in a completely randomised design experiment in north-western Tanzania. The goats were randomised into four treatment groups consisting of five animals each. Three supplement diets: 115.3 g NLM (T₂), 125.9 g PLM (T₃) and 124.1 g LLM (T₄), which was used as a positive control, were supplemented at 20% of the expected DM intake (DMI; i.e., 3% body weight) to the three animal groups fed on NPH (basal diet) compared to the animals in a control group that were fed on NPH without browse supplementation (T₁).

NPH had significantly the lowest (P < 0.05) CP of 45.5 g kg⁻¹ DM compared to NLM, PLM and LLM (159, 195 and 187 g kg⁻¹ DM, respectively). NPH had higher (P < 0.05) fibre fractions; lower ruminal DM degradability characteristics and ME than NLM, PLM and LLM. Supplementation of the animals with browse resulted to (P < 0.05) higher average daily weight gains (ADG) of 157.1 g day⁻¹ in T₄ than the animals fed on T₂ (114.3 g day⁻¹) and T₃ (42.9 g day⁻¹), and even to those fed on T₁ (control), which lost weight (−71.4 g day⁻¹). Improved weight gains were mainly due to corrected feed nitrogen (N) or CP due to supplementation of the animals with browse fodder. Too low CP of the NPH would not meet the normal requirements of CP (80 g CP kg⁻¹ DM) for optimal rumen microbial function in ruminants. Higher ADG due to LLM (T₄) and NLM (T₂) supplementation suggest optimised weight gains due to browse supplementation (20% of expected DMI); while lower weight gains from supplementation with PLM (T₃) indicate the possible utilisation of A. polyacantha leaves to overcome weight losses especially during dry seasons.     

Life cycle of the ichthyotoxic dinoflagellate Cochlodinium polykrikoides in Korean coastal waters

Since 1995, blooms of the harmful dinoflagellate, Cochlodinium polykrikoides, have caused considerable mortality of aquatic organisms and economic loss in Korea. However, little is known about the life cycle of the species, except for the planktonic vegetative stage; therefore, the aim of this paper was to elucidate the life cycle of C. polykrikoides. Its life cycle has two morphologically different stages: an armored and an unarmored vegetative stage. Armored vegetative cells were found in seawater samples collected in late-November and developed into four-cell chained, unarmored vegetative cells under laboratory culture. In samples collected in late-May, both the armored and unarmored types (vegetative swimming stage) occurred; the former easily developed into an unarmored vegetative cell type, suggesting that the armoured–unarmored transition occurs as early as May. A presumptive resting cyst, round but folded at one side, was produced from armored type cells in laboratory conditions. It was also collected from natural bottom sediments, which suggests it is the dormant resting cyst of C. polykrikoides.     

Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Consumption of toxic cyanobacteria by Potamopyrgus antipodarum (Gastropoda, Prosobranchia) and consequences on life traits and microcystin accumulation

Among the wide range of toxins produced by cyanobacterial blooms, microcystins (MCs) are the most common and are known to accumulate in aquatic organisms. Freshwater gastropods are grazers and likely to ingest toxic cyanobacteria, particularly Planktothrix agardhii, one of the most common species in the northern hemisphere. The study examines (i) the ingestion of toxic P. agardhii by the prosobranch Potamopyrgus antipodarum, (ii) the kinetics of MC accumulation and depuration in snail tissues during and post-exposure, and (iii) the impact of MCs on their life traits (survival, growth and fecundity). We showed that P. antipodarum ingested 71% of cyanobacteria available during the first 24 h in the presence or not of non-toxic food, and accumulated 1.3% of ingested MCs during the 5-week intoxication period. Elimination of MCs was total after 3 weeks of depuration. A decrease of growth and fecundity was observed during the intoxication period, but it was reversible after the end of exposure. Results are discussed in terms of variation of the response between prosobranch and pulmonate gastropods to toxic cyanobacteria exposure, and the negative impact of toxic cyanobacteria on natural communities of freshwater gastropods.     

Temporal changes in the cyst densities of Pyrodinium bahamense var. compressum and other dinoflagellates in Manila Bay, Philippines

Temporal variation in the type and abundance of dinoflagellate cysts in Manila Bay, Philippines, is established using ²¹⁰Pb-dated sediment cores. At least 17 dinoflagellate cyst species, including those of the toxic species, Pyrodinium bahamense var. compressum, were identified. P. bahamense may have been present in the area since at least the 1920s. Total cyst density has increased beginning about 1988 to 1998 coinciding with records of P. bahamense blooms in the area. Heterotrophs have always dominated the cysts assemblage. These changes in the dinoflagellate record and the P. bahamense blooms in recent years may have been induced by the interplay of warmer temperatures, high rainfall leading to higher river discharge and less turbulent waters due to passage of few tropical cyclones.     

Coupling planktonic and benthic shifts during a bloom of Alexandrium catenella in southern Chile: Implications for bloom dynamics and recurrence

Cell abundances and distributions of Alexandrium catenella resting cysts in recent sediments were studied along time at two locations in the Chilean Inland Sea exposed to different oceanographic conditions: Low Bay, which is much more open to the ocean than the more interior and protected Ovalada Island. The bloom began in interior areas but maximum cyst concentrations were recorded in locations more open to the ocean, at the end of the Moraleda channel. Our results showed a time lapse of around 3 months from the bloom peak (planktonic population) until the number of resting cysts in the sediments reached a maximum. Three months later, less than 10% of the A. catenella cysts remained in the sediments. Maximum cyst numbers in the water column occurred one month after the planktonic peak, when no cells were present. The dinoflagellate assemblage at both study sites was dominated by heterotrophic cysts, except during the A. catenella bloom. CCA analyses of species composition and environmental factors indicated that the frequency of A. catenella blooms was associated with low temperatures, but not with salinity, chlorophyll a concentration, and predator presence (measured as clam biomass). However, resting cyst distribution was only related to cell abundance and location. The occurrence of A. catenella cysts was also associated with that of cysts from the toxic species Protoceratium reticulatum. By shedding light on the ecological requirements of A. catenella blooms, our observations support the relevance of encystment as a mechanism of bloom termination and show a very fast depletion of cysts from the sediments (<3 months), which suggest a small role for resting cyst deposits in the recurrence of A. catenella blooms in this area.     

Effects of natural and field-simulated blooms of the dinoflagellate Prorocentrum minimum upon hemocytes of eastern oysters, Crassostrea virginica, from two different populations

Oysters, Crassostrea virginica, from two populations, one from a coastal pond experiencing repeated dinoflagellate blooms (native), and the other from another site where blooms have not been observed (non-native), were analyzed for cellular immune system profiles before and during natural and simulated (by adding cultured algae to natural plankton) blooms of the dinoflagellate Prorocentrum minimum. Significant differences in hemocytes between the two oyster populations, before and after the blooms, were found with ANOVA, principal components analysis (PCA) and ANOVA applied to PCA components. Stress associated with blooms of P. minimum included an increase in hemocyte number, especially granulocytes and small granulocytes, and an increase in phagocytosis associated with a decrease in aggregation and mortality of the hemocytes, as compared with oysters in pre-bloom analyses. Non-native oysters constitutively had a hemocyte profile more similar to that induced by P. minimum than that of native oysters, but this profile did not impart increased resistance. The effect of P. minimum on respiratory burst was different according to the origin of the oysters, with the dinoflagellate causing a 35% increase in the respiratory burst of the native oysters but having no effect on that of the non-native oysters. Increased respiratory burst in hemocytes of native oysters exposed to P. minimum in both simulated and natural blooms may represent an adaptation to annual blooms whereby surviving native oysters protect themselves against tissue damage from ingested P. minimum.     

Halophila ovalis (R. Br.) Hook. f. from a submarine hot spring in southern Japan

Colonies of the seagrass Halophila ovalis are found growing adjacent to coral Acropora sp. and Seriatopora hystrix in a submarine hot spring (at 15.7 m depth, 28.6°C) at the north coast of Taketomi Island, near the southern tip of Japan. Halophila plants grow in sea water containing sulphide 930 μg S ml⁻¹ and on the substratum with fine precipitates of the submarine hot spring which have sulphide content up to 5400 μg S g⁻¹ DW. The accumulated sulphide concentration reaches as high as 8400 μg S g⁻¹ DW in under ground tissues and 5700 μg S g⁻¹ DW in above-ground tissues, respectively. It is suggested that, not the sulphide concentration but light and possibly water temperature are the limiting factors for the Halophila colonization in the submarine hot spring.     

Karlotoxin mediates grazing by Oxyrrhis marina on strains of Karlodinium veneficum

Karlodinium veneficum is a common member of temperate, coastal phytoplankton assemblages that occasionally forms blooms associated with fish kills. Here, we tested the hypothesis that the cytotoxic and ichthyotoxic compounds produced by K. veneficum, karlotoxins, can have anti-grazing properties against the heterotrophic dinoflagellate, Oxyrrhis marina. The sterol composition of O. marina (>80% cholesterol) renders it sensitive to karlotoxin, and does not vary substantially when fed different algal diets even for prey that are resistant to karlotoxin. At in situ bloom concentrations (10⁴–10⁵ K. veneficum ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 55% that observed on the non-toxic K. veneficum strain MD5. At lower prey concentrations typical of in situ non-bloom levels (<10³ cells ml⁻¹), grazing rates (cells ingested per Oxyrrhis h⁻¹) on toxic K. veneficum strain CCMP 2064 were 70–80% of rates on non-toxic strain MD5. Growth of O. marina was significantly suppressed when fed the toxic strain of K. veneficum. Experiments with mixed prey cultures, where non-toxic strain MD5 was fluorescently stained, showed that the presence of toxic strain CCMP 2064 inhibited grazing of O. marina on the co-occurring non-toxic strain MD5. Exogenous addition of a sub-lethal dose (100 ng ml⁻¹) of purified karlotoxin inhibited grazing of O. marina by approximately 50% on the non-toxic K. veneficum strain MD5 or the cryptophyte S. major. These results identify karlotoxin as an anti-grazing compound for those grazers with appropriate sterol composition (i.e., desmethyl sterols). This strategy is likely to be an important mechanism whereby growth of K. veneficum is uncoupled from losses due to grazing, allowing it to form ichthyotoxic blooms in situ.     

Application of real-time PCR assay for detection and quantification of Alexandrium tamarense and Alexandrium catenella cysts from marine sediments

The dinoflagellates Alexandrium tamarense (Lebor) Balech and Alexandrium catenella (Whedon and Kofoid) Balech (Dinophyceae) are believed to be the main species responsible for paralytic shellfish poisoning (PSP) all over the world. It is necessary to identify A. tamarense and A. catenella cysts and to monitor their distribution in sediment in order to minimize the damages caused by PSP to the economy and food quality because cysts are the seed population for blooms caused by motile vegetative cells. In this study, we developed an efficient DNA extraction method from the natural cysts present in marine sediments after they were size fractionated with a plankton net (mesh size of 20–150 μm). The 10–3000 cysts were added to the sediments collected from the Ariake Sea, and for which the primuline-staining method did not reveal any cysts. DNA was then extracted from each sample, and linear standard curves for A. tamarense and A. catenella cysts were obtained from the correlation between the Ct values by real-time PCR and the log of the initial densities of cysts. We monitored the A. tamarense and A. catenella cyst densities in the environmental samples. This assay was demonstrated to be a powerful tool for the identification, detection, and quantification of the cysts of the toxic dinoflagellates.     

VEGETATIVE REPRODUCTION AND SEXUAL LIFE CYCLE OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM FROM TASMANIA,AUSTRALIA1

The toxic, chain-forming dinoflagellate Gymnodinium catenatum Graham was cultured from vegetative cells and benthic resting cysts isolated from estuarine waters in Tasmania, Australia. Rapidly dividing, log phase cultures formed long chains of up to 64 cells whereas stationary phase cultures were composed primarily of single cells (23-41 pm long, 27-36 pm wide). Vegetative growth (mean doubling time 3-4 days) was optimal at temperatures from 14.5-20° C, salinities of 23-34% and light irradiances of 50-300 μE·m^?2·s^?1. The sexual life cycle of G. catenatum was easily induced in a nutrient-deficient medium, provided compatible opposite mating types were combined (heterothallism). Gamete fusion produced a large (59-73 μm long, 50-59 μm wide) biconical, posteriorly biflagellate planozygote (double longitudinal flagellum) which after several days lost one longitudinal flagellum and gradually became subspherical in shape. This older planozygote stage persisted for up to two weeks before encysting into a round, brown resting cyst (42-52 μm diam; hypnozygote) with microreticulate surface ornamentation. Resting cysts germinated after a dormancy period as short as two weeks under our culture conditions, resulting in a single, posteriorly biflagellate germling cell (planomeiocyte). This divided to form a chain of two cells, which subsequently re-established a vegetative population. Implications for the bloom dynamics of this toxic dinoflagellate, a causative organism of paralytic shellfish poisoning, are discussed.