Alexandrium (Dinophyceae) species in Malaysian waters

A study was carried out to determine the presence of paralytic shellfish poisoning (PSP) toxin-producing dinoflagellates in the coastal waters of Peninsula Malaysia. This followed first ever occurrences of PSP in the Straits of Malacca and the northeast coast of the peninsula. The toxic tropical dinoflagellate Pyrodinium bahamense var. compressum was never encountered in any of the plankton samples. On the other hand, five species of Alexandrium were found. They were Alexandrium affine, Alexandrium leei, Alexandrium minutum, Alexandrium tamarense and Alexandrium tamiyavanichii. Not all species were present at all sites. A. tamiyavanichii was present only in the central to southern parts of the Straits of Malacca. A. tamarense was found in the northern part of the straits, while A. minutum was only found in samples from the northeast coast of the peninsula. A. leei and A. affine were found in both the north and south of the straits. Cultured isolates of A. minutum and A. tamiyavanichii were proven toxic by the receptor binding assay for PSP toxins but A. tamarense clones were not toxic. Mean toxin content for the A. tamiyavanichii and A. minutum clones were 26 and 15 fmol per cell STX equivalent, respectively. This study has provided evidence on the presence of PSP toxin-producing Alexandrium species in Malaysian waters which suggests that PSP could increase in importance in the future.     

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

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

Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group

The geographic range and bloom frequency of the toxic dinoflagellate Alexandrium minutum and other members of the A. minutum group have been increasing over the past few decades. Some of these species are responsible for paralytic shellfish poisoning (PSP) outbreaks throughout the world. The origins of new toxic populations found in previously unaffected areas are typically not known due to a lack of reliable plankton records with sound species identifications and to the lack of a global genetic database. This paper provides the first comprehensive study of minutum-group morphology and phylogeny on a global scale, including 45 isolates from northern Europe, the Mediterranean, Asia, Australia and New Zealand.Neither the morphospecies Alexandrium lusitanicum nor A. angustitabulatum was recoverable morphologically, due to large variation within and among all minutum-group clonal strains in characters previously used to distinguish these species: the length:width of the anterior sulcal plate, shape of the 1′ plate, connection between the 1′ plate and the apical pore complex, and the presence of a ventral pore. DNA sequence data from the D1 to D2 region of the LSU rDNA also fail to recognize these species. Therefore, we recommend that all isolates previously designated as A. lusitanicum or A. angustitabulatum be redesignated as A. minutum. A. tamutum, A. insuetum, and A. andersonii are clearly different from A. minutum on the basis of both genetic and morphological data.A. minutum strains from Europe and Australia are closely related to one another, which may indicate an introduction from Europe to Australia given the long history of PSP in Europe and its recent occurrence in Australia. A minutum from New Zealand and Taiwan form a separate phylogenetic group. Most strains of A. minutum fit into one of these two groups, although there are a few outlying strains that merit further study and may represent new species. The results of this paper have greatly improved our ability to track the spread of A. minutum species and to understand the evolutionary relationships within the A. minutum group by correcting inaccurate taxonomy and providing a global genetic database.     

Comparative studies on morphology, ITS sequence and protein profile of Alexandrium tamarense and A. catenella isolated from the China Sea

The Alexandrium tamarense species complex is a closely related cosmopolitan toxigenic group of morphology-based species, including A. tamarense, A. catenella and A. fundyense. This study investigated the morphology, internal transcribed spacer (ITS) sequence and protein profile of A. tamarense and A. catenella grown in the same culture conditions using a combination of scanning electronic microscope (SEM), molecular and proteomic approaches. The results showed that all Alexandrium strains had the plate formula of Po, 4′, 6″, 6C, 8S, 5″′, 2″″. The ventral pore, a key conventional morphological feature to discriminate A. tamarense and A. catenella, was usually present in the first apical plate of ten A. tamarense strains, however, it was found to be absent in some cells of one Alexandrium strain, ATGX01. A. tamarense and A. catenella shared an identical ITS sequence with a minor variation at intraspecific level. Protein profiles of A. catenella DH01 and A. tamarense DH01, isolated from the same region of the East China Sea, showed no significant difference, the similarity of protein profiles of the two species reached 99% with a few proteins unique to one or the other. The present results suggest that the ventral pore is not a consistent morphological feature in the Alexandrium genus, and that A. tamarense and A. catenella are conspecific and should be redesignated to one species.     

Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China

The composition of the paralytic shellfish toxins (PSTs) of five Alexandrium tamarense strains isolated from the coastal waters of southern China and one Alexandrium minutum strain from Taiwan Island were investigated. A. tamarense CI01 and A. tamarense Dapeng predominantly produced C2 toxin (over 90%) with trace amounts of C1 toxin (C1), gonyautoxin-2 (GTX2) and GTX3; two strains of A. tamarense HK9301 maintained in different locations produced C1-4 toxins and GTX1, 4, 5 and 6; no PSTs were found in A. tamarense NEW, while A. minutum TW produced only GTX1-4. The toxin compositions of cultured A. tamarense strains did not vary as much during different growth phases as did the toxin composition of A. minutum TW. The toxin compositions of A. tamarense HK9301-1 did not change significantly under different salinity, light intensity, and nitrate and phosphate levels in the culture medium, although the toxin productivity varied expectably. Another strain HK9301-2 maintained in a different location produced much less toxins with a considerably different toxin composition. Under similar culture maintenance conditions for 3 years, the toxin profiles of A. tamarense HK9301-1 did not change as much as did A. tamarense CI01. Our results indicate that toxin compositions of the dinoflagellate strains are strain-specific and are subject to influence by nutritional and environmental conditions but not as much by the growth phase. Use of toxin composition in identifying a toxigenic strain requires special caution.     

The Inhibitory Effects of Garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) and Diallyl Trisulfide on <Emphasis Type="Italic">Alexandrium tamarense</Emphasis> and other Harmful Algal Species

Using cell suspension ability as an indicator, we studied the inhibitory effect of garlic (Allium sativum) and diallyl trisulfide on six species of red tide causing algae. This included: the inhibition by 0.08% garlic solution of five algal species — Alexandrium tamarense, Scrippsiella trochoidea, Alexandrium catenella, Alexandrium minutum and Alexandrium satoanum; the effects of garlic concentration on the inhibition of A. tamarense, S. trochoidea and Chaetoceros sp.; the effects of inhibitory time on the rejuvenation of algal cells; and the effects of heating and preservation time on algal inhibition by garlic solution. In addition, whether or not the ingredients of garlic solution had a possible algicidal effect was studied by comparing inhibition of A. tamarense by garlic solution and man-made diallyl trisulfide. The results showed that 1) inhibition by garlic solution was significant on A. tamarense, A. satoanum, A. catenella and S. trochoidea, and the least effective was a concentration of 0.04% on A. tamarense and S. trochoidea. Moreover, the higher the concentration, the stronger was the inhibition, and a high inhibitory rate (IR) could be maintained for at least three days when the garlic concentration was above 0.04%. For A. tamarense, it was also found that the longer the inhibitory time and the higher the concentration, the lower was the rate of resumed cell activity. On the contrary, garlic solution could not inhibit A. minutum or Chaetoceros sp.; 2) The IR to A. tamarense was reduced slightly as the heating time of the garlic solution was lengthened, but the average IR was still above 80%. There was no significant difference between the IR of the supernatant and sediment of the garlic solution. Furthermore, no change of algal inhibition was found when the garlic solution was preserved at 20°C for several days; 3) As with garlic solution, diallyl trisulfide inhibited A. tamarense strongly; the IR was above 93% and was maintained for at least three days, as long as the concentration was 3.2–10.0 mg L⁻¹. Thus, diallyl trisulfide may have been the major ingredient in garlic solution which inhibited the algae but, in addition, more than one ingredient may have been inhibiting the algae. In conclusion, garlic was a good algal inhibitor with many advantages, such as being common, cheap, non toxic and with high efficiency, and diallyl trisulfide, one of the components of garlic, was similarly effective in algal inhibition. It would be useful, therefore, to further study garlic as an environmentally friendly algal inhibitor.     

Allelochemical interactions and short-term effects of the dinoflagellate Alexandrium on selected photoautotrophic and heterotrophic protists

The marine dinoflagellate genus Alexandrium (Halim) Balech contains members that produce highly potent phycotoxins (PSP toxins or spirolides) as well as lytic substances and other allelochemicals of unknown structure and ecological significance. One isolate each of six Alexandrium species (A. tamarense, A. ostenfeldii, A. lusitanicum, A. minutum, A. catenella, A. taylori), of the closely related gonyaulacoid dinoflagellate Fragilidium subglobosum, and of the peridinioid Scrippsiella trochoidea were tested in 24 h co-incubation experiments for their short-term deleterious effects on a diversity of marine protists. Both autotrophs (Rhodomonas salina, Dunaliella salina, Thalassiosira weissflogii) and heterotrophs (Oxyrrhis marina, Amphidinium crassum, Rimostrombidium caudatum) were included as target species. All donor isolates except S. trochoidea exhibited lytic effects on at least some target species. Lytic effects were observed with all Alexandrium species, for both whole cell samples and culture filtrate (<10 μm and <0.2 μm). Antibiotic treated cultures with drastically reduced bacterial numbers did not show any general reduction in lytic capacity, therefore direct involvement of extracellular bacteria in allelochemical production is unlikely. Values of EC₅₀, defined as the Alexandrium cell concentration causing lysis of 50% of target cells, differed by two orders of magnitude depending on the donor/target combination, from 3.1 × 10³ cells ml⁻¹ (A. minutum/O. marina) down to 0.02 × 10³ cells ml⁻¹ (A. catenella/D. salina). Within the array of nine donor Alexandrium/target combinations, variable ratios in EC₅₀ values between donor/target combination cannot be explained by quantitative differences in allelochemical production, but rather indicate qualitative differences in the composition of compounds produced by different Alexandrium strains. In conclusion, our study confirms the widespread lytic capacity within the genus Alexandrium, although allelochemical effects are not restricted to this genus. Allelochemical interactions mediated by such lytic substances may be significant in explaining the formation and maintenance of Alexandrium blooms through direct destructive effects on competing algae or unicellular grazers.     

Interactions between red tide microalgae and herbivorous zooplankton: effects of two bloom-forming species on the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> (O.F. Muller)

Experiments were carried out to investigate interspecific interactions between the rotifer Brachionus plicatilis and two harmful algal bloom (HAB) species using single and mixed culture methods. B. plicatilis populations and the growth of two algae were compared at different algal cell densities. The results demonstrate that B. plicatilis obtained sufficient nutrition from Alexandrium tamarense to support net population increase. When exposed to a density of 8 × 10⁴ cells ml⁻¹ A. tamarense, the number of B. plicatilis increased faster than it did when exposed to other four algal densities (16 × 10⁴, 24 × 10⁴, 32 × 10⁴, and 40 × 10⁴ cells ml⁻¹). Cell densities of A. tamarense decreased due to the grazing of B. plicatilis. In contrast, Heterosigma akashiwo had an adverse effect on the B. plicatilis population and its growth was largely unaffected by rotifer grazing. In this case, the B. plicatilis population decreased and H. akashiwo grew at a rate similar to that of a control without addition of rotifers. Mixed culture experiments showed that A. tamarense could partly counteract the effect of H. akashiwo in limiting the rate of population increase of rotifer. In addition, the effect of different initial cell densities on interspecific competition between A. tamarense and H. akashiwo in mixed culture(s) was also investigated. The results show that A. tamarense competed very successfully when the inoculation proportions of A. tamarense and H. akashiwo were 40:5 and 40:30. Handling editor: D. Hamilton     

Toxin profiles of natural populations and cultures ofAlexandrium minutum Halim from Galician (Spain) coastal waters

The toxin profiles of three isolates and natural populations of the PSP agentAlexandrium minutum from several Galician rías (NW Spain) was obtained by HPLC. The toxin content of cultures ofA. minutum is dominated by GTX4 (80–90%) and GTX4 (10–15%) with small amounts of GTX3 and GTX2 (less than 3% of each); similar results were obtained for natural populations ofAlexandrium from three different Galician rías, where a mixture ofA. lusitanicum Balech andA. minutum can occur. Important quantitative differences were found between the three isolates, one being highly and two weakly toxic. The results obtained from these isolates and natural populations ofAlexandrium were very similar to those obtained from HPLC analyses of mussels intoxicated during a PSP outbreak in Ría de Ares (Rías Altas) in 1984, confirming thatA. minutum (previously identified asGonyaulax tamarensis Lebour andAlexandrium lusitanicum) was the PSP agent during the toxic outbreak in May 1984. Toxin profiles obtained from natural populations during different PSP outbreaks in different rías and from cultures are fairly consistent and suggest that at least from the toxin point of view,A. lusitanicum andA. minutum are identical, and that the toxin profile ofA. minutum from Galicia can be used as a biochemical marker.     

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

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

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

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

Detection and quantification of cultured marine Alexandrium species by real-time PCR

The occurrence of harmful algal blooms (HABs) throughout the world has increased and poses a large threat to human health, fishery resources and tourism industries. The genus Alexandrium includes a number of toxic species associated with HABs. Therefore, it is very important to rapidly detect and monitor the harmful algae, such as Alexandrium genus. In this study, a standard curve of plasmid containing 18S rDNA-28S rDNA region from Alexandrium catenella was constructed and 5.8S rDNA sequence served as the primer of the real-time PCR. Cultured A. catenella, Alexandrium affine, Alexandrium lusitanicum and Alexandrium minutum samples were analyzed by real-time PCR using the same set of primers simultaneously. Using microscopy cells counts, 5.8S rDNA copies per cell and total DNA per cell were estimated. This assay method is promising for rapid detection of large number of Alexandrium samples.     

Development of a qualitative PCR method for the Alexandrium spp. (Dinophyceae) detection in contaminated mussels (Mytilus galloprovincialis)

Paralytic shellfish poisoning (PSP) is a syndrome caused by the consumption of shellfish contaminated with neurotoxins produced by organisms of the marine dinoflagellate genus Alexandrium. A. minutum is the most widespread species responsible for PSP in the Western Mediterranean basin. The standard monitoring of shellfish farms for the presence of harmful algae and related toxins usually requires the microscopic examination of phytoplankton populations, bioassays and toxin determination by HPLC. These procedures are time-consuming and require remarkable experience, thus limiting the number of specimens that can be analyzed by a single laboratory unit. Molecular biology techniques may be helpful in the detection of target microorganisms in field samples. In this study, we developed a qualitative PCR assay for the rapid detection of all potentially toxic species belonging to the Alexandrium genus and specifically A. minutum, in contaminated mussels. Alexandrium genus-specific primers were designed to target the 5.8S rDNA region, while an A. minutum species-specific primer was designed to bind in the ITS1 region. The assay was validated using several fixed seawater samples from the Mediterranean basin, which were analyzed using PCR along with standard microscopy procedures. The assay provided a rapid method for monitoring the presence of Alexandrium spp. in mussel tissues, as well as in seawater samples. The results showed that PCR is a valid, rapid alternative procedure for the detection of target phytoplankton species either in seawater or directly in mussels, where microalgae can accumulate.     

Induction of toxin production in dinoflagellates: the grazer makes a difference

The dinoflagellate Alexandrium minutum has previously been shown to produce paralytic shellfish toxins (PST) in response to waterborne cues from the copepod Acartia tonsa. In order to investigate if grazer-induced toxin production is a general or grazer-specific response of A. minutum to calanoid copepods, we exposed two strains of A. minutum to waterborne cues from three other species of calanoid copepods, Acartia clausi, Centropages typicus and Pseudocalanus sp. Both A. minutum strains responded to waterborne cues from Centropages and Acartia with significantly increased cell-specific toxicity. Waterborne cues from Centropages caused the strongest response in the A. minutum cells, with 5 to >20 times higher toxin concentrations compared to controls. In contrast, neither of the A. minutum strains responded with significantly increased toxicity to waterborne cues from Pseudocalanus. The absolute increase in PST content was proportional to the intrinsic toxicity of the different A. minutum strains that were used. The results show that grazer-induced PST production is a grazer-specific response in A. minutum, and its potential ecological importance will thus depend on the composition of the zooplankton community, as well as the intrinsic toxin-producing properties of the A. minutum population.     

Toxicity of Alexandrium lusitanicum to gastropod larvae is not caused by paralytic-shellfish-poisoning toxins

Laboratory grazing experiments compared ingestion of two subclones of the dinoflagellate Alexandrium lusitanicum by gastropod veliger larvae (Nassarius sp.). While the two prey subclones originated from the same monoclonal isolate of A. lusitanicum, one possessed the ability to produce paralytic-shellfish-poisoning toxins (PSTs), while the other did not. Ingestion rates on the two Alexandrium subclones were not significantly different over a range of prey concentrations (approximately 100–660 cells ml⁻¹), indicating that PSTs did not serve as a grazing deterrent for these larvae. However, ingestion rates on both subclones were low at the higher prey concentrations tested. Mortality of the predators also increased linearly with concentration of either subclone. These observations indicated that both A. lusitanicum subclones produced an unknown substance that inhibited and killed the grazers. Veliger mortality was not induced by culture filtrates or lysates, suggesting either that the substance was either highly labile or that contact with intact cells was required. Because toxic algae can produce multiple bioactive substances, experimental demonstrations of alleopathic effects of toxic species should not be assigned to known toxins without supporting evidence. In addition, the results show that the effectiveness of algal grazing deterrents can increase with cell concentration, which may have implications for bloom dynamics.     

Alga-lytic activity of Pseudomonas fluorescens against the red tide causing marine alga Heterosigma akashiwo (Raphidophyceae)

A bacterial strain, HAK-13, exhibited strongest activity against Heterosigma akashiwo and was capable of controlling this bloom forming phytoplankton. Based on 16S rDNA sequences and biochemical and morphological characteristics, the strain HAK-13 was determined to be Pseudomonas fluorescens on the basis of 99.9% similarity with reference strains in the DNA databases. The growth of H. akashiwo was strongly suppressed by HAK-13 in all growth phases, with the strongest alga-lytic activity noted against harmful bloom-forming species in the exponential stage (6–22 days). Host range tests showed that HAK-13 also significantly inhibited the growth of Alexandrium tamarense and Cochlodinium polykrikoides but could not destroy Gymnodinium catenatum. P. fluorescens HAK-13 indirectly attacked H. akashiwo by alga-lytic substances that might be located at the compartment of cytoplasmic membrane of the bacterium at a level of 45.86 units/mg of specific activity. The results indicated that P. fluorescens HAK-13 caused cell lysis and death of H. akashiwo, A. tamarense, and C. polykrikoides dramatically and Prorocentrum dentatum slightly. Therefore, P. fluorescens HAK-13 has potential for use as a selective biocontrol of harmful algal blooms.     

Seasonality in the excystment of Alexandrium minutum and Alexandrium tamarense in Irish coastal waters

Excystment experiments were carried out on cysts of Alexandrium minutum and A. tamarense (Group III) taken from Cork Harbour, Ireland. Freshly sampled cysts were isolated into well plates and their excystment was monitored over a 30 day period. A pronounced seasonality was observed in the excystment behaviour in both species when measurements were carried out seasonally. Between 80 and 100% of the isolated cysts excysted within 30 days when samples were taken between March and June. However, between only 0 and 15% excysted in samples taken between August and early February. This seasonal characteristic was observed repeatedly over a 3 year period (2004–2007). The effect was observed in cysts which had been sampled from both inter-tidal and sub-tidal locations. No endogenous clock was evident in the germination of A. tamarense and A. minutum cysts taken from Cork Harbour which had been stored cool and in the dark under anoxic conditions for up to 18 months. The seasonal effect observed was independent of water temperature, although temperature did affect the rate of excystment. Temperature also affected the maturation of cysts which had been freshly formed in the laboratory. The significance of incorporating seasonality in excystment of Alexandrium into models describing its growth is also discussed.     

Genus Alexandrium Halim, 1960 (Dinophyta) from the Pacific coast of Russia: Species composition, distribution, and dynamics

At present 8 species of Alexandrium genus have been found in seas and adjacent waters of Russia: A. acatenella, A. catenella, A. insuetum, A. margalefii, A. ostenfeldii, A. pseudogonyaulax, A. tamarense, and A. tamutum. The distribution and population density of Alexandrium species varied within the surveyed area of the Pacific: in the Sea of Japan and Sea of Okhotsk, 7 species were recorded; 3 species were recorded along the Pacific coast of Kamchatka; and 2 species were found in the Bering Sea. A. tamarense was the most widespread and abundant species over the area. A. insuetum was recorded only in the Sea of Japan, and A. catenella, in the Sea of Okhotsk (Terpeniya Bay). The highest concentration of Alexandrium spp. (2–7 million cells/l) was recorded along the Pacific coast of Kamchatka and in the Bering Sea; in the Sea of Okhotsk, a rather high concentration (51000 cells/l) was registered in Aniva Bay; in the Sea of Japan, the highest concentration was recorded in Peter the Great Bay (6000 cells/l). The distribution of cysts (spores) in surface sediments of the Pacific coast of Russia as a whole reflected the pattern of distribution of vegetative cells of Alexandrium. Cysts of Alexandrium cf. tamarense prevailed all over the area, with the maximum concentration along the Pacific coast of Kamchatka. Beyond that type of cysts, insignificant numbers of cysts of Alexandrium cf. minutum were recorded in Peter the Great Bay and Aniva Bay. Analysis of seasonal dynamics revealed that cells of Alexandrium spp. occurred in Peter the Great Bay from June up to September, and along the Pacific coast of Kamchatka from April to October. In the first region, the maximum density was recorded in August; it was provided by A. pseudogonyaulax (59% of the total density of Alexandrium), A. tamarense (35%), and A. insuetum (6%). In the second region, it was recorded in July, thanks only to development of A. tamarense.     

Phylogenetic relationship of Alexandrium monilatum (Dinophyceae) to other Alexandrium species based on 18S ribosomal RNA gene sequences

The phylogenetic relationship of Alexandrium monilatum to other Alexandrium spp. was explored using 18S rDNA sequences. Maximum likelihood phylogenetic analysis of the combined rDNA sequences established that A. monilatum paired with Alexandrium taylori and that the pair was the first of the Alexandrium taxa to diverge, followed by Alexandrium margalefii. All three are members of the Alexandrium subgenus Gessnerium Halim nov. comb.