Genetic polymorphism in Gymnodinium galatheanum chloroplast DNA sequences and development of a molecular detection assay

Nuclear and chloroplast-encoded small subunit ribosomal DNA sequences were obtained from several strains of the toxic dinoflagellate Gymnodinium galatheanum. Phylogenetic analyses and comparison of sequences indicate that the chloroplast sequences show a higher degree of sequence divergence than the nuclear homologue. The chloroplast sequences were chosen as targets for the development of a 5'--3' exonuclease assay for detection of the organism. The assay has a very high degree of specificity and has been used to screen environmental water samples from a fish farm where the presence of this dinoflagellate species has previously been associated with fish kills. Various hypotheses for the derived nature of the chloroplast sequences are discussed, as well as what is known about the toxicity of the species.     

Morphological variation and phylogenetic analysis of the dinoflagellate Gymnodinium aureolum from a tributary of Chesapeake Bay

Cultures of four strains of the dinoflagellate Gymnodinium aureolum (Hulburt) G. Hansen were established from the Elizabeth River, a tidal tributary of the Chesapeake Bay, USA. Light microscopy, scanning electron microscopy, nuclear-encoded large sub-unit rDNA sequencing, and culturing observations were conducted to further characterize this species. Observations of morphology included: a multiple structured apical groove; a peduncle located between the emerging points of the two flagella; pentagonal and hexagonal vesicles on the amphiesma; production and germination of resting cysts; variation in the location of the nucleus within the center of the cell; a longitudinal ventral concavity; and considerable variation in cell width/length and overall cell size. A fish bioassay using juvenile sheepshead minnows detected no ichthyotoxicity from any of the strains over a 48-h period. Molecular analysis confirmed the dinoflagellate was conspecific with G. aureolum strains from around the world, and formed a cluster along with several other Gymnodinium species. Morphological evidence suggests that further research is necessary to examine the relationship between G. aureolum and a possibly closely related species Gymnodinium maguelonnense.     

The dinoflagellates Pfiesteria shumwayae and Luciella masanensis cause fish kills in recirculation fish farms in Denmark

Fish kills in two geographically separate fish farms in northern Denmark in 2012, one using marine, the other brackish water ‘Recirculation Aquaculture Systems’ (RAS), were found to be caused by Pfiesteria shumwayae and Luciella masanensis, two species of dinoflagellates belonging to the family Pfiesteriaceae. There were no other harmful algae present in either of the aquaculture plants. Serious fish kills in the US have been attributed to Pfiesteria during the past 20 years, but this type of mortality has not been documented elsewhere. L. masanensis, described recently from Korea and USA, has not been previously reported to be the source of fish kills. In the marine farm, the affected fish was rainbow trout, in the brackish water farm pikeperch. Light microscopy is presently insufficient to discriminate between the approx. 20 species of the family Pfiesteriaceae described. Identification of the two algal species was therefore based on molecular sequencing of nuclear-encoded LSU rDNA, confirmed by scanning electron microscopy and, eventually, also by examination of the very thin amphiesmal plates of the flagellates by calcofluor-stained cells in a fluorescence microscope.Although the two fish farms differed in light and salinity conditions, both farms used re-circulating water in closed circuit systems. The dinoflagellates were examined in detail and shown to feed on organic material such as live, damaged nematodes, as described for the single pfiesteriacean flagellate known from freshwater, Tyrannodinium edax. Algal cells were observed to attach to their prey by an attachment filament and subsequently used a peduncle to suck up the food. Fish farms utilizing water recirculation technology are gaining popularity due to their reduced effect on the environment. The two cases from Denmark are apparently the first RAS farms in which serious fish kills have been reported. In the marine farm (Luciella) fish mortality increased dramatically despite treatment of the water with peracetic acid and chloramine-T. The plant was temporarily closed down pending investigation into the cause of mortality and subsequently to determine a method of management to control the dinoflagellate and avoid future fish kills. In the brackish water farm (Pfiesteria), water was treated with chloramine-T, which caused the dinoflagellates to disappear temporarily from the water column, apparently forming temporary cysts. The treatment was repeated after a few days to a week, when the temporary cysts appeared to germinate and the dinoflagellates reappeared in the water column.     

Flagellar apparatus and nuclear chambers of the green dinoflagellate Gymnodinium chlorophorum

The green dinoflagellate Gymnodinium chlorophorum (BAH ME 100, the type culture) was reexamined with emphasis on the structure of the flagellar apparatus and nuclear envelope. Like other Gymnodinium species, G. chlorophorum possessed a nuclear fibrous connective linking the flagellar apparatus and the nucleus, albeit in a very reduced and unique form. Microtubules nucleated from the R3 flagellar root associated with the nuclear fibrous connective and terminated at the nucleus, a novel arrangement not known in any other dinoflagellate. Although overlooked by previous researchers, nuclear chambers were present in G. chlorophorum similar to those reported in Gymnodinium aureolum and Gymnodinium nolleri. In contrast to the type species of Gymnodinium, Gymnodinium fuscum, only one nuclear pore was present per chamber. The presence of a feeding tube (peduncle) suggests that G. chlorophorum is mixotrophic. Although the fine structure of G. chlorophorum revealed its affiliation to the Gymnodinium group the above discrepancies set it apart, indicating that it might belong in a different genus.     

Comparative Toxicity of Gonyaulax monilata and Gymnodinium breve to Annelids, Crustaceans, Molluscs and a Fish

SYNOPSIS. Toxicities of laboratory cultures of the dinoflagellates Gonyaulax monilata and Gymnodinium breve were compared using fish, annelids, crustaceans and molluscs. Toxicity was evaluated as mortality over a 48-hr test period in 100%, 75% 50%, 25% and 10% concentrations of dinoflagellate cultures. Responses of the assay animals indicated that the dinoflagellates do not produce the same toxins. Effects of the toxins seemed uniform within each major group of animals tested. Fish were most sensitive to both G. breve and Gonyaulax monilata; crustaceans were resistant to both; annelids and molluscs were more sensitive to G. monilata than to G. breve.     

Species resolution and global distribution of microreticulate dinoflagellate cysts

The distribution, abundance and morphology of microreticulatedinoflagellate cysts were examined from samples collected fromthe coastal waters of Australia, the Baltic Sea, Italy, HongKong and Uruguay. On the basis of a combination of size range,variation in microreticulate pattern, and cyst wall colour,the three microreticulate species Gymnodinium catenatum (36–62µm diameter), Gymnodinium nolleri (25–40 µm)and Gymnodinium microreticulatum (17–29 µm) couldbe distinguished. Only G. catenatum and G. microreticulatumwere found at Australian sites. Gymnodinium microreticulatumwas rare but widespread in sediments from Tasmania and temperateand tropical sites on mainland Australia, whereas G. catenatumwas restricted to the eastern coast of Tasmania, southern Victoria,Port Lincoln [South Australia (SA)] and the Hawkesbury Estuary[New South Wales (NSW)]. Significant variation in G. catenatummean cyst size was observed between sites, with mean diametersvarying from 40.1 µm (Hawkesbury River, NSW) to 52.3 µm(Port Lincoln, SA). Laboratory experiments suggest that cystsize may be predominantly a genetically determined, population-specificcharacter, rather than being influenced by environmental parameters.Using the species criteria refined from the dataset, existingreports of microreticulate cysts are re-examined, and the globaldistribution of microreticulate cyst species and the biogeographyof the toxic dinoflagellate G. catenatum are re-evaluated.     

Length–weight relationships of seven fish species from the upper Pelus River,Kuala Kangsar district,Perak, Malaysia

Length–weight relationships (LWR) are reported for seven fish species from the upper Pelus River, Kuala Kangsar district, Perak, Malaysia. This river is located in a remote area and supplies fishes for the aboriginal peoples. Fish samplings were conducted in three different seasons, the dry (June 2014), wet (August 2014), and moderately wet (April 2015). All samples were collected using fish electro–shocker and scoop nets with a 1 cm mesh size. A total of seven LWRs were unknown to FishBase, and four new maximum fish lengths were identified in the study.     

INTRA‐CELLULAR AND EXTRA‐CELLULAR TOXINS IN THE RED TIDE DINOFLAGELLATE,GYMNODINIUM BREVE

The marine dinoflagellate, Gymnodinium breve (Davis), produces several neurotoxins that cause neurotoxic shellfish poisoning (nsp), massive fish kills and respiratory irritation in marine mammals and humans. The common method for discerning toxic levels of G. breve for public health advisories is enumeration of live cells in a given water mass. In this study, laboratory cultures, as well as natural blooms, were added to a stirred ultra‐filtration cell concentrator to separate viable cells containing intra‐cell toxins from ambient water containing extra‐cell toxins. Methods were validated using various mixtures of lysed and whole G. breve laboratory culture. Extractions and recovery of brevetoxins were done using a C‐18 bonded‐phase glass fiber extraction disc eluted with methanol. Total PbTx toxin concentrations were quantified by HPLC/UV using a C‐18 column and an 85:15 methanol:water (1 ml min^?1) isocratic elution at 215 nm. This method of separation and extraction was subsequently applied to water samples collected during natural blooms along two different areas of the Florida Gulf coast. The results indicated that early stages of G. breve blooms contained primarily intra‐cell toxins with extra‐cell toxins increasing as the bloom progressed, even though very few viable G. breve cells were present. This suggests that enumeration of cells alone may be insufficient and additional toxin quantitation is necessary.     

Discovery of the toxic dinoflagellate Pfiesteria in northern European waters.

Several dinoflagellate strains of the genus Pfiesteria were isolated by culturing techniques from sediment samples taken in the Oslofjord region of Norway. Pfiesteria piscicida, well known as a fish killer from the Atlantic coast of America, was identified by genetic methods and light microscopy. The related species Pfiesteria shumwayae was attracted from the sediment by the presence of fish, and has proved toxic. This present survey demonstrates the wide distribution of these potentially harmful species, but so far they have not been connected with fish kills in Europe.     

基于环境DNA宏条码和底拖网的珠江河口鱼类多样性

文章采用环境DNA宏条码和底拖网对珠江河口鱼类多样性进行了研究, 并对两种方法进行了比较。利用环境DNA宏条码检测到了175种鱼类, 而利用底拖网采集到了47种鱼类, 结合两种方法共检测出179种鱼类, 隶属于15 目63科128属。其中两种方法共同识别了鱼类43种, 占总检测物种的24.02%, 基于底拖网的调查未能收集到基于环境DNA宏条码检测到的大多数物种。根据Shannon指数和Simpson指数显示, DNA宏条码所检测珠江河口鱼类群落α多样性显著高于底拖网方法(P<0.05)。两种方法的PCoA结果均显示珠江河口鱼类群落存在空间结构, 基于环境DNA宏条码的分析显示空间重叠更多。两种方法基于冗余分析均显示溶解氧和盐度是影响鱼类群落结构的主要环境因子。研究表明, 环境DNA 宏条形码是一种环保且可靠的评估方法, 将其搭载到现有调查可以更好地了解河口鱼类多样性。     

Habitat associations of fish assemblages in the Cache River, Illinois

Fish and habitat were sampled by state agencies at 48 stations throughout the Cache River watershed, Illinois between 1992 and 2009. Two distinct fish assemblages were identified, one primarily found in the lower mainstem Cache River and a second found throughout tributaries and the upper mainstem Cache River. Using a canonical correspondence analysis, the distribution of fish species was largely explained by substrate, land use, drainage area and local habitat features. Creek chub, central stoneroller, fringed darter and fantail darter are species found to be positively associated with gravel substrate and forest. In contrast, black buffalo, gizzard shad, smallmouth buffalo, freshwater drum and bigmouth buffalo were positively associated with drainage area, silt, channel width and row crops. Cobble appears to be rare habitat associated with fringed darter, freckled madtom and fantail darter. Results suggest that substrate, land use and local habitat features influence fish assemblages within the Cache River watershed. This information contributes to both understanding aquatic community structure in a highly altered yet diverse watershed as well as management activities within the Cache River watershed.     

Human health effects of exposure to Pfiesteria piscicida: a review

Since its identification, the dinoflagellate Pfiesteria piscicida has been implicated in fish kills and fish disease in the southeastern United States. Adverse health effects have been reported in researchers working with the organism and in watermen following exposure to a fish kill in Maryland. A bioactive secretion is postulated as the cause of these effects but has not yet been isolated and chemically characterized. The biology and toxicology of this organism remain the topic of debate and research.     

Selective toxic effects of polyunsaturated fatty acids derived from Ulva fasciata on red tide phyotoplankter species

Alpha-linolenic acid and linoleic acid isolated from Ulva fasciata showed toxic effects on red tide phytoplankters in a concentration-dependent manner. Among six species tested, raphidophycean flagellate Heterosigma akashiwo was the most susceptible to these fatty acids, and 50% lethal concentrations (LC50) of alpha-linolenic acid and linoleic acid were estimated to be 0.58 and 1.91 microg/ml respectively, whereas dinoflagellate Gymnodinium impudicum and Heterocapsa circularisquama were highly resistant and no significant toxic effects were observed up to 1,000 microg/ml. Both fatty acids were less toxic to fish (devil stinger), zooplankters (brine shrimp and rotifer), and mammalian cell lines (U937, HeLa, Vero, and CHO cells) than H. akashiwo.     

2004年春季长江口海域甲藻孢囊的分布研究

于2004年4月初,采集长江口(E122°～123°30',N29°～32°)10个站点0～15cm底泥样品,研究甲藻孢囊在这10个站点的水平和垂直分布情况。在30个样品中共鉴定出6大类24种甲藻孢囊。孢囊组成以异养型原多甲藻类孢囊为主,有13种,平均密度为157cysts·g^-1DW,为调查海域的最优势种群。两种产麻痹性贝类毒素(Paralytic shellfish poisoning,PSP)的孢囊,塔玛亚历山大藻和链状裸甲藻,在海区分布广泛但数量较低。10个站点甲藻孢囊的种类数在11～18种之间,平均密度为189～846cysts·g^-1DW,在远离河口的D6站点有一个明显的最高峰,位于最北部的D1站点孢囊密度最低。与其它海湾相比,属于孢囊密度较低的海区。Shannon-Weaver生物多样性指数(H')变化范围在2.57～3.27之间。甲藻孢囊的密度分布与生物多样性相关系数r=-0.72。3个不同深度的甲藻孢囊密度分别为351cysts·g^-1DW、412cysts·g^-1DW、432cysts·g^-1DW;生物多样性指数分别为3.22、2.95、2.98。     

Gymnodinium smaydae n. sp., a New Planktonic Phototrophic Dinoflagellate from the Coastal Waters of Western Korea: Morphology and Molecular Characterization

The marine phototrophic dinoflagellate Gymnodinium smaydae n. sp. is described from cells prepared for light, scanning, and transmission electron microscopy. Also, sequences of the small (SSU) and large subunits (LSU) and the internal transcribed spacer region (ITS1–5.8S–ITS2) of ribosomal DNA were analyzed. This newly isolated dinoflagellate possessed nuclear chambers, nuclear fibrous connective, an apical groove running in a counterclockwise direction around the apex, and a major accessory pigment peridinin, which are four key features for the genus Gymnodinium. The epicone was conical with a round apex, while the hypocone was ellipsoid. Cells growing photosynthetically were 6.3–10.9 μm long and 5.1–10.0 μm wide, and therefore smaller than any other Gymnodinium species so far reported except Gymnodinium nanum. Cells were covered with polygonal amphiesmal vesicles arranged in 11 horizontal rows, and the vesicles were smaller than those of the other Gymnodinium species. This dinoflagellate had a sharp and elongated ventral ridge reaching half way down the hypocone, unlike other Gymnodinium species. Moreover, displacement of the cingulum was 0.4–0.6 × cell length while in other known Gymnodinium species it is less than 0.3 × cell length. In addition, the new species possessed a peduncle, permanent chloroplasts, pyrenoids, trichocysts, pusule systems, and small knobs along the apical furrow, but it lacked an eyespot, nematocysts, and body scales. The sequence of the SSU, ITS1–5.8S–ITS2, and LSU rDNA region differed by 1.5–3.8%, 6.0–17.4%, and 9.1–17.5%, respectively, from those of the most closely related species. The phylogenetic trees demonstrated that the new species belonged to the Gymnodinium clade at the base of a clade consisting of Gymnodinium acidotum, Gymnodinium dorsalisulcum, Gymnodinium eucyaneum, etc. Based on morphological and molecular data, we suggest that the taxon represents a new species, Gymnodinium smaydae n. sp.     

PCNA-LIKE PROTEINS IN DINOFLAGELLATES

Dinoflagellate chromosomes are permanently condensed and lack nucleosomes. These features suggest that dinoflagellate chromosomes must have an altered structural arrangement when compared to other eukaryotes and some modified DNA replication machinery to accommodate it. To investigate this possibility, proliferating cell nuclear antigen (PCNA), an essential component of the DNA replication machinery, was chosen for closer examination. A protein in the dinoflagellate Crypthecodinium cohnii Biecheler was found to react specifically with two monoclonal antibodies raised against PCNA. The observed band had a size of 55 kDa, which is far in excess of what has been described previously. Another dinoflagellate, Gymnodinium catenatum Bravo, also displayed a band of this size; however, a third species Amphidinium carterae Hulburt, had a band of lower molecular weight. The putative PCNA homolog in C. cohnii showed a nonconstitutive expression pattern. A time-course western blot using cells from a synchronized G₁ population showed that protein levels peak during S phase of the cell cycle. Both C. cohnii and A. carterae displayed a strong nuclear localization as determined by immunofluorescence microscopy. The signal was present in a subpopulation of cells, supporting a cell-cycle-specific expression pattern. It is possible that the larger size of this protein in some dinoflagellates reflects the unusual cell cycle and DNA arrangement of this group.     

Gyrodiniellum shiwhaense n. gen., n. sp., a new planktonic heterotrophic dinoflagellate from the coastal waters of western Korea: morphology and ribosomal DNA gene sequence

The heterotrophic dinoflagellate Gyrodiniellum shiwhaense n. gen., n. sp. is described from live cells and from cells prepared for light, scanning electron, and transmission electron microscopy. Also, sequences of the small subunit (SSU) and large subunit (LSU) of rDNA have been analyzed. The episome is conical, while the hyposome is ellipsoid. Cells are covered with polygonal amphiesmal vesicles arranged in 16 horizontal rows. Unlike other Gyrodinium-like dinoflagellates, the apical end of the cell shows a loop-shaped row of five elongate amphiesmal vesicles. The cingulum is displaced by 0.3-0.5 × cell length. Cells that were feeding on the dinoflagellate Amphidinium carterae Hulburt were 9.1-21.6 μm long and 6.6-15.7 μm wide. Cells of G. shiwhaense contain nematocysts, trichocysts, a peduncle, and pusule systems, but they lack chloroplasts. The SSU rDNA sequence is >3% different from that of the six most closely related species: Warnowia sp. (FJ947040), Lepidodinium viride Watanabe, Suda, Inouye, Sawaguchi & Chihara, Gymnodinium aureolum (Hulburt) Hansen, Gymnodinium catenatum Graham, Nematodinium sp. (FJ947039), and Gymnodinium sp. MUCC284 (AF022196), while the LSU rDNA is 11-12% different from that of Warnowia sp., G. aureolum, and Nematodinium sp. (FJ947041). The phylogenetic trees show that the species belongs in the Gymnodinium sensu stricto clade. However, in contrast to Gymnodinium spp., cells lack nuclear envelope chambers and a nuclear fibrous connective. Unlike Polykrikos spp., cells of which possess a taeniocyst-nematocyst complex, G. shiwhaense has nematocysts but lacks taeniocysts. It differs from Paragymnodinium shiwhaense Kang, Jeong, Moestrup & Shin by possessing nematocysts with stylets and filaments. Gyrodiniellum shiwhaense n. gen., n. sp. furthermore lacks ocelloids, in contrast to Warnowia spp., Nematodinium spp., and Proterythropsis spp. Based on morphological and molecular data, we suggest that the taxon represents a new species within a new genus.     

Seasonal variation in diet and trophic relationships within the fish communities of the lower Slave River, Northwest Territories, Canada

Increased industrial activities on the Peace and Athabasca River systems have raised concerns about cumulative impacts on fish and water resources downstream, in the Slave River of Alberta and the Northwest Territories, Canada. Because very little information was available on the fish communities in this system, we examined spatial and temporal patterns of diet for nine species (four piscivores and five invertebrate feeders) from three different types of habitat along the lower Slave River system and assessed trophic relationships within the communities. All actively feeding species exhibited seasonal variations in diet within and among the study areas. Dietary overlap was generally low throughout all seasons and locations. In the lower Slave River and its major tributary, the Salt River, substantial dietary overlap between piscivores (particularly walleye, Stizostedion vitreum), and invertebrate feeders occurred in the spring. In the summer no overlap occurred as walleye shifted to a more piscivorous diet, attaining a moderate degree of overlap with northern pike, Esox lucius. Compared with the Slave River, which is a large but homogeneous system upstream of its delta at Great Slave Lake, there was a greater diversity of actively feeding invertebrate feeders in the Salt River. Three of the latter were benthic feeders exhibiting moderate degrees of diet overlap during spring and summer. During the fall, few fish were feeding. Most fishes in the lower Slave River system are generalist, opportunistic feeders, consuming a number of different prey, the importance of which varies spatially and seasonally, as the abundance of these prey varies in the environment.     

Association of Large Juvenile Red Drum, Sciaenops ocellatus, with an Estuarine Creek on the Atlantic Coast of Florida

Seasonal abundance, size distribution, year-class presence, residence duration, and migrational patterns of red drum, Sciaenops ocellatus, in an estuarine marsh creek in the northern Indian River Lagoon, Florida, were investigated during a study in which gill net samples were collected monthly from August 1991 to March 1996. A total of 282 large juvenile red drum were collected, of which 161 were tagged and released and 68 were sacrificed for age determination. Although red drum were collected in the creek throughout the year, abundance levels were negatively correlated with water temperatures and reached maximum levels during the winter months. Significant correlations between fish abundance and salinity or dissolved oxygen levels were not detected. The majority (90%) of the fish collected were large juveniles (260–450mm standard length) that were estimated to be from 10 to 26 months old. The oldest red drum we examined from the creek was estimated to be 37 months old. Tag-recapture data indicated that some fish repeatedly used or were associated with the creek for periods of up to 18 months after release. Estuarine creeks in this region provide exploitable habitat for large juvenile red drum (相似文献   

A RE-EVALUATION OF CORALLINE RED ALGAL TAXONOMY USING ULTRA-STRUCTURAL INFORMATION

The marine dinoflagellate, Gymnodinium breve (Davis), produces several neurotoxins that cause neurotoxic shellfish poisoning (nsp), massive fish kills and respiratory irritation in marine mammals and humans. The common method for discerning toxic levels of G. breve for public health advisories is enumeration of live cells in a given water mass. In this study, laboratory cultures, as well as natural blooms, were added to a stirred ultra-filtration cell concentrator to separate viable cells containing intra-cell toxins from ambient water containing extra-cell toxins. Methods were validated using various mixtures of lysed and whole G. breve laboratory culture. Extractions and recovery of brevetoxins were done using a C-18 bonded-phase glass fiber extraction disc eluted with methanol. Total PbTx toxin concentrations were quantified by HPLC/UV using a C-18 column and an 85:15 methanol:water (1 ml min⁻¹) isocratic elution at 215 nm. This method of separation and extraction was subsequently applied to water samples collected during natural blooms along two different areas of the Florida Gulf coast. The results indicated that early stages of G. breve blooms contained primarily intra-cell toxins with extra-cell toxins increasing as the bloom progressed, even though very few viable G. breve cells were present. This suggests that enumeration of cells alone may be insufficient and additional toxin quantitation is necessary.