Development and Evaluation of a PCR Based Assay for Detection of the Toxic Dinoflagellate, <Emphasis Type="Italic">Gymnodinium catenatum</Emphasis>(Graham) in Ballast Water and Environmental Samples

Gymnodinium catenatum is a bloom forming dinoflagellate that has been known to cause paralytic shellfish poisoning (PSP) in humans. It is being reported with increased frequency around the world, with ballast water transport implicated as a primary vector that may have contributed to its global spread. Major limitations to monitoring and management of its spread are the inability for early, rapid, and accurate detection of G. catenatum in plankton samples. This study explored the feasibility of developing a PCR-based method for specific detection of G. catenatumin cultures and heterogeneous ballast water and environmental samples. Sequence comparison of the large sub unit (LSU) ribosomal DNA locus of several strains and species of dinoflagellates allowed the design of G. catenatum specific PCR primers that are flanked by conserved regions. Assay specificity was validated through screening a range of dinoflagellate cultures, including the morphologically similar and taxonomically closely related species G. nolleri. Amplification of the diagnostic PCR product from all the strains of G. catenatum but not from other species of dinoflagellates tested imply the species specificity of the assay. Sensitivity of the assay to detect cysts in ballast water samples was established by simulated spiked experiments. The assay could detect G. catenatum in all ‘blank’ plankton samples that were spiked with five or more cysts. The assay was used to test environmental samples collected from the Derwent river estuary, Tasmania. Based on the results we conclude that the assay may be utilized in large scale screening of environmental and ballast water samples.     

Late Quaternary Protoperidinium cysts as indicators of paleoproductivity in the northern Arabian Sea

The reliability of organic-walled cysts of the heterotrophic dinoflagellate Protoperidinium as paleoproductivity indicators and the influence of bottom water oxygenation on cyst preservation is assessed by using Arabian Sea records of the past 125 kyr as a natural laboratory. Multidisciplinary geochemical, micropaleontological and palynological datasets are integrated to analyze the relationship between Protoperidinium cyst concentrations and other paleoproductivity proxies. Differential preservation potential is quantified in order to establish threshold oxidative degradation values for a possible application of quantitative Protoperidinium cyst records in paleoenvironmental reconstructions. Results indicate that variations in Protoperidinium cyst concentration closely correspond to other marine productivity and/or upwelling proxies. Although oxygenation will lead to significant cyst degradation, and thus decreased concentrations, down-core patterns in Protoperidinium cyst concentration still primarily reflect changes in sea surface productivity. In view of differential preservation among dinoflagellate cysts, down-core variations in relative abundance of Protoperidinium should be treated with caution.     

Using gigartinine to track the distribution of an alien species of <Emphasis Type="Italic">Gracilaria</Emphasis> in New Zealand

The presence of gigartinine has been used previously as a taxonomic marker for the positive identification in Manukau Harbour (west coast, Auckland) of Gracilaria sp., which has apparently been introduced to New Zealand waters and is easily confused morphologically with the native species, G. chilensis. Thirty two estuarine/harbour populations of Gracilaria in New Zealand were screened for the presence of gigartinine to further test the utility of gigartinine as a reliable species marker. DNA fingerprinting was used to confirm the identity of a subset of the specimens surveyed. Using genetic rather than chemical characterisation, it was discovered that Gracilaria sp. is also present in Orakei Basin (east coast, Auckland). Although a sample from the wild did not have the anticipated gigartinine content, tank cultivation of thalli from this population in an artificially elevated nitrogen environment allowed the plant to accumulate nitrogen as gigartinine. This confirmed the unusual ability of this species of Gracilaria to store nitrogen in this form, unlike the widespread, morphologically similar, G. chilensis.     

Recent Dinoflagellate cyst distribution in the North Canary Basin,NW Africa

The North Canary Basin (NW Africa) falls within a major eastern boundary upwelling system. This part of the coastal upwelling system is seasonal and is characterised by the development of large filaments migrating seawards. Hence, 16 samples from this location were selected to identify an “upwelling signal” in the composition of the dinoflagellate cyst assemblages.

Samples closest to the most intense upwelling cells are dominated by L. machaerophorum and G. catenatum and Protoperidinium spp. These make up the “upwelling signal” characteristic for the system. Moreover, the “upwelling signal” can be advected offshore, with filaments that may extend as far as 300 km. Finally, the finding of cysts from G. catenatum, a toxic dinoflagellate, raises the need for a better understanding of the relationship between its presence and distribution in the region, and the coastal upwelling system.     

Variation in scope for growth: a test of food limitation among intertidal mussels

Wellington Harbour supports large populations of the mussels Aulacomya maoriana, Mytilus galloprovincialis and Perna canaliculus that are almost entirely absent from nearby coastal locations in Cook Strait. We calculated scope for growth (SFG) using ambient Cook Strait water over a broad temporal scale and a broad range of seston conditions to determine if negative SFG explains this phenomenon. Although all three mussel species had positive mean SFG values, variation in SFG was high and negative values often occurred: A. maoriana 19.1 J g⁻¹ h⁻¹, 43% of mussels showed negative SFG; M. galloprovincialis 1.26, 52% negative SFG; P. canaliculus 45.6, 27% negative SFG. Negative SFG was most often due to negative absorption efficiency caused by metabolic faecal loss that is characteristic of mussels feeding in environments with low seston quality. From our ecophysiology data we constructed a model to estimate SFG based on physiological responses to the narrow range of seston conditions typical of Cook Strait (Model One), and a model to estimate SFG based on physiological responses of mussels to the broad range of seston conditions typical of Wellington Harbour and Cook Strait (Model Two). We used seston data collected over an 18-month period from sites in Wellington Harbour and Cook Strait to derive 159 estimates of species-specific mussel SFG from both models. Both models produced higher estimates of SFG for mussels in the Harbour compared with those at Cook Strait sites. This was consistent with elevated particulate concentrations in the Harbour than at Cook Strait sites, and in agreement with previous studies. For Cook Strait mussels, both models produced negative estimates of net energy balance for long periods of time (several months), whereas for Harbour mussels negative SFG estimates were generally short in duration. We conclude that our short-term laboratory-based determinations of SFG and our long-term bioenergetics modelling estimates do not conclusively support the hypothesis of food limitation for three coexisting taxa of mussels in the intertidal region of Cook Strait, New Zealand. Handling editor: P. Viaroli     

Palynological record (1483–1994) of Gymnodinium catenatum in Pescadero Basin, southern Gulf of California, Mexico

This paper presents the stratigraphic record of Gymnodinium catenatum during the last ~ 500 years, in Pescadero Basin, southern Gulf of California. Our aim is to help clarify the relation between abundances of cysts of G. catenatum and regional changes in the sea surface temperature and nutrient availability at a decadal scale. The record was obtained from core samples of laminated sediments dated with ¹⁴C, representing conditions in the area from 1483 to 1967 (PCM99-74C-5) and from 1907 to 1994 (PCM00-61C-4). Samples were treated with normal palynological processing, without oxidation, and using Lycopodium spores for quantification. The palynological assemblages observed contain varied and abundant terrestrial and marine components. However, we focus on the abundance of G. catenatum, due to its toxicity and the resulting impact in the area. This species is currently common in the area, and according to our results has been present there since ~ 1483. Cysts of G. catenatum are generally abundant in the 20th century, with maximum concentrations observed from 1888 to 1920, and from 1945 to 1965, but they show a steady decrease in the latter part of the century (1965–1994). Before 1830, abundances of G. catenatum were low because of increased upwelling conditions, probably related to a higher variability of the winter sea surface temperatures in the area. Both our data in the 20th century and the actual reports in the area, indicate a close relation with sea surface temperature. From 1907 to 1994 cyst abundances seem to increase during cool La Niña conditions in the area, and decrease during warmer El Niño events. They also show an evident decrease in cyst abundance from 1970 to 1994, while the sea surface temperature in the area increased during the same period. This inverse relation is also indicated by the low abundances of G. catenatum observed during red tide events, combined with high sea surface temperatures in Mazatlan. Additionally, our results do not show any relation of G. catenatum blooms and anthropogenic activity in the area.     

Global toxicology,ecophysiology and population relationships of the chainforming PST dinoflagellate Gymnodinium catenatum

Increasing scientific awareness since the 1980s of the chain-forming dinoflagellate Gymnodinium catenatum has led to this species being reported with increased frequency in a globally increasing number of countries (23 at present). G. catenatum exhibits little molecular genetic variation in rDNA over its global range, in contrast to RAPD fingerprinting which points to high genetic variation within regional populations even between estuaries 50 km apart. All Australian and New Zealand strains possess a thymine nucleotide (T-gene) near the start of the 5.8S rRNA whereas all other global populations examined to date possess cytosine-nucleotide (C-gene), except for southern Japan which harbours both C-gene and T-gene strains. Together with cyst and plankton evidence this strongly suggests that both Australian and New Zealand populations have derived from southern Japan. Global dinoflagellate populations and cultures exhibit an extraordinary variation in PST profiles (STX and 21 analogues), but consistent regional patterns are evident with regard to the production of C1,2; C3,4; B1,2; and neoSTX analogues. PST profiles of cyst-derived cultures are deemed unrepresentative. Distinct ecophysiological differences exist between tropical (21–32 °C) and warm-temperate ecotypes (12–18 °C), but these appear unrelated to ITS genotypes and PST toxin phenotypes. On current evidence, cyst germination appears to play a minimal role in the bloom dynamics of this species, while seasonal and inter-annual bloom variations result from the physical constraints (temperature and light) on the growth of the dinoflagellates in the water column. G. catenatum exhibits a capacity to utilize many forms of nitrogen. Its chain formation and strong motility allow it to undergo retrieval migrations to exploit light and nutrient resource gradients in both stratified and mixed environments. Subtle strain-level variations in micronutrient (Se, humics) requirements and interaction with associated bacterial flora may provide a partial explanation for the contrasting inshore (Tasmanian), and offshore (Spain, Mexico) bloom patterns by the same species in different geographic regions.     

Morphology and taxonomy of chain-forming species of the genus Cochlodinium (Dinophyceae)

The morphology of an unarmored chain-forming harmful dinoflagellate Cochlodinium polykrikoides and its similar species such as Cochlodinium catenatum, Cochlodinium fulvescens, and Cochlodinium convolutum was carefully observed, emphasizing the single cell stage for clarifying taxonomically important morphological features. To differentiate C. polykrikoides from C. convolutum, the shape and the position of the nucleus are useful characters. C. polykrikoides also differs from C. fulvescens in being smaller in size, possessing many rod-shaped chloroplasts and having the sulcus running just below the cingulum on the dorsal surface. Careful observation of the ichnotype of C. catenatum suggests that C. catenatum sensu Kofoid and Swezy collected from off La Jolla, CA, USA, is not identical to C. catenatum sensu Okamura and is probably a different species, in having no chloroplasts and a nucleus positioned at the center of the cell. In addition, C. polykrikoides has many morphological features in common with C. catenatum sensu Okamura except for slightly elongate cells and is probably a junior synonym of this species.     

Resting stages of microalgae in recent marine sediments of Peter the Great Bay,Sea of Japan

Data on the qualitative and quantitative composition of resting stages of planktonic microalgae in recent marine sediments of Peter the Great Bay (Sea of Japan) over the period 2000–2007 are presented. A total of sixty one morphological forms of resting stages represented by dinoflagellate and raphidophyte cysts and diatom spores and resting cells were recorded in the sediment samples. This study revealed cysts of the potentially toxic species Alexandrium tamarense, A. cf. minutum, Alexandrium sp., Gymnodinium catenatum (PSP toxin producers), and Protoceratium reticulatum (yessotoxin producer); resting cells of Pseudo-nitzschia sp. (potential producer of domoic acid); and cysts of bloom-forming species Cochlodinium cf. polykrikoides and Heterosigma cf. akashiwo.     

Dinoflagellate cyst production in one-liter containers

Methods for the production of dinoflagellate cysts in two types of 1 L containers have been developed. Using these methods, dinoflagellate cysts can be produced in amounts large enough for shellfish grazing experiments or whenever large amounts of cysts are needed. The species used were Scrippsiella lachrymosa (B-10) and toxic Alexandrium fundyense (CB501 and GTM25). Cultures of S. lachrymosa yielded 628 ± 74 cysts mL^–1 and A. fundyense cultures yielded 350 ± 98 cysts mL^–1. Findings suggest that aspects of the boundary layer between the media and the wall of the container are important for gamete mating; especially, the slope of the container wall appears to be relevant, which offers some explanation of previous observations that the shape of the container is important in the formation of dinoflagellate resting cysts. These observations may support the theory that physical interfaces in nature facilitate dinoflagellate encystment.     

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

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

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.     

Quantitative competitive (qc) RT-PCR as a tool in biomarker analysis

All inducible proteins which respond to known pollutants are potential molecular biomarkers. Quantitative competitive (qc) RT-PCR represents a uniquely sensitive tool for measuring the extent of induction of molecular biomarkers such as metallothionein, which is responsive to inducers that include a range of heavy metals. Using the yellowbelly flounder Rhombosolea leporina collected from sites in the Manukau Harbour and Hauraki Gulf (Auckland, New Zealand) as an indicator species, we describe the methodology underpinning the use of qcRT-PCR as a tool in biomarker analysis with reference to the induction of metallothionein. The results show reasonable correlation between the extent of metallothionein induction and the liver burden of Cu and Zn.     

GENETIC VARIATION AMONG STRAINS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

The toxic dinoflagellate Gymnodinium catenatum Graham has formed recurrent toxic blooms in southeastern Tasmanian waters since its discovery in the area in 1986. Current evidence suggests that this species might have been introduced to Tasmania prior to 1973, possibly in cargo vessel ballast water carried from populations in Japan or Spain, followed by recent dispersal to mainland Australia. To examine this hypothesis, cultured strains from G. catenatum populations in Australia, Spain, Portugal, and Japan were examined using allozymes and randomly amplified polymorphic DNA (RAPD). Allozyme screening detected very limited polymorphism and was not useful for population comparisons; however, Australian, Spanish, Portuguese, and Japanese strains showed considerable RAPD diversity, and all strains examined represented unique genotypes. Multidimensional scaling analysis (MDS) of RAPD genetic distances between strains showed clear separation of strains into three nonoverlapping regional clusters: Australia, Japan, and Spain/Portugal. Analysis of genetic distances between strains from the three regional populations indicated that Australian strains were almost equally related to both the Spanish/Portuguese population and the Japanese population. Analysis of molecular variance (AMOVA) found that genetic variation was partitioned mainly within populations (87%) compared to the variation between the regions (8%) and between populations within regions (5%). The potential source population for Tasmania’s introduced G. catenatum remains equivocal; however, strains from the recently discovered mainland Australian population (Port Lincoln, South Australia, 1996) clustered with Tasmanian strains, supporting the notion of a secondary relocation of Tasmanian G. catenatum populations to the mainland via a shipping vector. Geographic and temporal clustering of strains was evident among the Tasmanian strains, indicating that genetic exchange between neighboring estuaries is limited and that Tasmanian G. catenatum blooms are composed of localized, estuary-bound subpopulations.     

A fish kill of massive proportion in Kuwait Bay, Arabian Gulf, 2001: the roles of bacterial disease, harmful algae, and eutrophication

In August and September 2001, Kuwait Bay, a semi-enclosed embayment of the Arabian Gulf, experienced a massive fish kill involving over >2500 metric tons of wild mullet (Liza klunzingeri), due to the bacterium Streptococcus agalactiae. In the Bay, this event was preceded by a small fish kill (100–1000 dead fish per day) of gilthead sea bream (Sparus auratus) in aquaculture net pens associated with a bloom of the dinoflagellate Ceratium furca. Sea bream were found to be culture positive for S. agalactiae, but did not show any visible signs of disease. Unusually warm temperatures (up to 35 °C) and calm conditions prevailed during this period. As the wild fish kill progressed, various harmful algae were observed, including Gymnodinium catenatum, Gyrodinium impudicum, and Pyrodinium bahamense var. compressum. Cell numbers of G. catenatum and G. impudicum exceeded 10⁶ l⁻¹ in some locations. All fish tested below the limits of detection for paralytic shellfish poisoning (PSP) and brevetoxins. Clams (Circe callipyga) were positive for PSP but at levels below regulatory limits. Nutrient concentrations, both inorganic and organic, were highly variable with time and from site to site, reflecting inputs from sewage outfalls, the aquaculture operations, a high biomass of decomposing fish, and other sources. It is hypothesized that many factors contributed to the initial outbreak of the bacterial disease, including unusual warm and calm conditions. The same factors, as well as enriched nutrient conditions, also apparently were conducive to the subsequent HAB outbreaks. The detection of PSP, while below regulatory limits, warrants further monitoring to protect human health.     

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

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

Molecular species identification of morphologically similar mussel larvae reveals unexpected discrepancy between relative abundance of adults and settlers

Understanding the relative importance of larval supply vs. post-settlement mortality underlies studies of marine invertebrate recruitment, yet is often hampered by researchers' inability to identify species among morphologically similar larvae or early juveniles. In New Zealand, two species of co-occurring intertidal mytilid mussels have morphologically indistinguishable settlers: the blue mussel Mytilus galloprovincialis, which is often numerically dominant in the mid-zone of the rocky intertidal, and the ribbed mussel Aulacomya atra maoriana which is often much less abundant. In this study, we obtained samples of newly settled mussels from 6 sample dates April-May 2005 from the rocky intertidal in Wellington Harbour, New Zealand. We used PCR-RFLP of the cytochrome c oxidase subunit I (COI) mitochondrial gene region to identify settlers to species. Of a total of 224 settlers that could be identified, 64% were identified as Mytilus galloprovincialis and 36% as Aulocomya atra maoriana. The percentage of A. atra maoriana in the samples was unexpectedly high and ranged from 22–50% among the sample dates. This study reinforces the need to quantify larval supply at the species level to understand the relative importance of pre- and post-settlement mortality, and also demonstrates the usefulness of the COI region as a species-specific marker for identifying mussel larvae and juveniles.     

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates, Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species. Correspondence to: J. M. Bautista