IDENTIFICATION OF MARINE DINOFLAGELLATES USING FLUORESCENT LECTINS1

Toxic and nontoxic species of marine dinoflagellates were characterized using fluorescent lectins. Lectin binding was detected by epifluorescence as well as spectrofluorometry. The binding assay of fluorescent lectins readily differentiated between morphologically similar species (i.e the toxic dinoflagellate Gymnodinium catenatum and the nontoxic Gymnodinium sp.). Lectins appear to be a useful tool to distinguish among different clones of the same species and, thus, possibly as a tool in dinoflagellate identification. Moreover, the lectins used show that thecate species have more binding sites and diversity in glycan moieties than athecate species.     

Detection of bloom-forming dinoflagellates Karenia mikimotoi and Prorocentrum donghaiense using qPCR assays

Journal of Applied Phycology - The dinoflagellates Karenia mikimotoi and Prorocentrum donghaiense are both important causative species of harmful algal blooms (HABs) in the East China Sea. The...     

A survey of epiphytic dinoflagellates from the coastal waters of the island of Hawai‘i

Three hundred and sixty-nine macroalgal and non-algal samples were collected from six coastal sites around the island of Hawai‘i on a biweekly basis over a 1-year period to ascertain (1) the presence of potentially toxigenic benthic dinoflagellates, and (2) substrate and environmental preferences of the dinoflagellates. Twenty-six genera/species of dinoflagellates were encountered including the (potentially) toxigenic species Amphidinium sp., Coolia monotis, Gambierdiscus sp., Ostreopsis ovata, Prorocentrum concavum, Prorocentrum hoffmannianum, Prorocentrum lima, and Prorocentrum mexicanum. Twenty of the species are being reported for the first time as present in Hawaiian coastal waters. There was some evidence of macroalgal host preference (e.g., C. monotis on Tolypiocladia glomerulata), although host morphology preferences was greater (e.g., Gambierdiscus sp., and P. lima on filamentous turfs; O. sp. 1 on sheet-like macroblades). While some dinoflagellate groups were significantly correlated with nutrient concentrations (e.g., total dinoflagellates with nitrate + nitrite and phosphate concentrations), others were not (e.g., Gambierdiscus sp., Prorocentrum emarginatum, P. lima, and Sinophysis microcephalus). The presence of several potentially toxigenic dinoflagellate species in Hawai‘i merits future study on possible impacts of these dinoflagellates on coastal food webs and human health.     

Phagotrophic Mechanisms and Prey Selection in Free-living Dinoflagellates1

Three types of feeding mechanisms are known in dinoflagellates: pallium feeding, tube feeding, and direct engulfment. Pallium feeding has only been described for heterotrophic thecate species (Protoperidinium, Diplopsalis group). Tube feeding is commonly found among both naked and thecate species of mixotrophic and heterotrophic dinoflagellates (e.g. Amphidinium, Dinophysis, Gyrodinium, Peridiniopsis). Direct engulfment is mainly found among naked species (e.g. Gymnodinium, Gyrodinium, Noctiluca): recently, however, some thecate species have been shown to use this feeding mechanism as well. Feeding behavior in dinoflagellates involves several steps prior to actual ingestion, including precapture, capture, and prey manipulation. As feeding mechanisms allow the ingestion of relatively large prey or parts thereof, dinoflagellates are regarded as raptorial feeders. While prey size plays an important role in the ability of dinoflagellates to ingest food, this alone cannot explain observed prey preferences. Some dinoflagellate species can be very selective in their choice of prey, while others show a remarkable versatility.     

PATTERNS IN HOST RANGE FOR TWO STRAINS OF AMOEBOPHRYA (DINOPHYTA) INFECTING THECATE DINOFLAGELLATES: AMOEBOPHYRA SPP. EX ALEXANDRIUM AFFINE AND EX GONYAULAX POLYGRAMMA1

The endoparasitic dinoflagellate Amoebophrya ceratii (Koeppen) Cachon uses a number of its free‐living relatives as hosts and may represent a species complex composed of several host‐specific parasites. Two thecate host–parasite systems [Amoebophrya spp. ex Alexandrium affine (Inoue and Fukuyo) Balech and ex Gonyaulax polygramma Stein], were used to test the hypothesis that two strains of Amoebophrya have a high degree of host specificity. To test this hypothesis, a series of cross‐infection experiments were conducted, with 10 thecate and three athecate dinoflagellate species as potential hosts. Surprisingly, the two strains of Amoebophrya lacked host specificity and had wider host ranges than previously recognized. Among the host species tested, Amoebophrya sp. ex Alexandrium affine was capable of infecting only species of genus Alexandrium (Alexandrium affine, Alexandrium catenella, and Alexandrium tamarense), while the parasite from Gonyaulax polygramma infected species covering five genera (Alexandrium, Gonyaulax, Prorocentrum, Heterocapsa, and Scripsiella). In the context of previous reports, these results suggest that host specificity of Amoebophrya strains varies from extremely species‐specific to rather unspecific, with specificity being stronger for strains isolated from athecate hosts. Information on host specificity of Amoebophrya strains provided here will be helpful in assessing the possibility of using these parasites as biological control agents for harmful algal blooms, as well as in defining species of Amoebophrya in the future.     

Effects of a bacterial algicide,IRI-160AA,on dinoflagellates and the microbial community in microcosm experiments

Filtrates from the bacterium Shewanella sp. IRI-160 (termed IRI-160AA) have been shown to inhibit population growth and kill a variety of dinoflagellates grown in culture. Here we test the immediate efficacy of IRI-160AA in laboratory microcosms initiated from three natural dinoflagellate blooms (Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum). We measured target dinoflagellate abundance, total chlorophyll-a, photosystem II (PSII) photochemistry, and changes to the prokaryotic and eukaryotic community composition over 2–3 days of IRI-160AA incubation. Naked dinoflagellates were impacted more, while abundance of the thecate P. minimum was not affected. However, dinoflagellate growth inhibition was generally lower than that observed in uni-algal cultures, and took longer to occur. Eukaryotic community composition in IRI-160AA treated microcosms was significantly different from control incubations, and was driven predominantly by increases in heterotrophic protists (e.g. Euplotes sp. and Paraphysomonas sp.). Similarly, significant changes to the prokaryotic community structure were evident. Microcosms of G. instriatum with higher algicide concentrations indicated that algicidal activity was enhanced in a dose dependent manner. Furthermore, total ciliate abundance as well as a bactivorous chyrsophyte (Paraphysomonas sp.) increased in a dose dependent manner. Total diatom abundance increased at lower IRI-160AA concentrations, but increased less with increasing dose. Overall, the bio-activity of IRI-160AA on naturally occurring dinoflagellates in mixed natural microbial communities is encouraging from the applied perspective of using the active compound(s) in IRI-160AA as natural agent(s) to manage harmful dinoflagellate blooms.     

Roles of mixotrophy in blooms of different dinoflagellates: Implications from the growth experiment

Studies over the last two decades suggested that mixotrophy could be an important adaptive strategy for some bloom-forming dinoflagellates. In the coastal waters adjacent to the Changjiang River estuary in the East China Sea, recurrent blooms of dinoflagellates Prorocentrum donghaiense, Karenia mikimotoi and Alexandrium catenella started to appear from the beginning of the 21 century, but roles of mixotrophy in the formation of dinoflagellate blooms were not well understood. In the current study, mixotrophy-based growth of four selected bloom-causative dinoflagellate species, i.e. K. mikimotoi, A. catenella, P. donghaiense and Prorocentrum micans, were studied. Dinoflagellates were co-cultured with different prey organisms, including bacterium Marinobacter sp., microalgae Isochrysis galbana and Hemiselmis virescens, under a variant of nutrient conditions. It was found that growth of dinoflagellate K. mikimotoi was significantly promoted with the presence of prey organisms. Growth of P. donghaiense and P. micans was only slightly improved. For A. catenella, the addition of prey organisms has no effects on the growth, while both of the two prey microalgae I. galbana and H. virescens were killed, probably by allelochemicals released from A. catenella. There was no apparent relationship between nutrient conditions and the mixotrophy-based growth of the tested dinoflagellates. Based on the results of the growth experiment, it is implicated that mixotrophy may play different roles in the growth and bloom of the four dinoflagellate species. It can be an important competitive strategy for K. mikimotoi. For the two Prorocentrum species and A. catenella, however, the role of mixotrophy is much limited. They may depend more on other competitive strategies, such as phototrophy-based growth and allelopathic effect, to prevail in the phytoplankton community and form blooms.     

Kleptoplasty in an Antarctic dinoflagellate: caught in evolutionary transition?

Photosynthetic dinoflagellates contain a diverse collection of plastid types, a situation believed to have arisen from multiple endosymbiotic events. In addition, a number of heterotrophic (phagotrophic) dinoflagellates possess the ability to acquire chloroplasts temporarily by engulfing algae and retaining their chloroplasts in a functional state. These latter relationships typically last from a few days to weeks, at which point the chloroplasts lose function, are digested and replaced with newly acquired plastids. A novel and abundant dinoflagellate related to the icthyotoxic genera Karenia and Karlodinium was recently discovered by us in the Ross Sea, Antarctica. Sequencing of its plastid small subunit ribosomal gene indicated that it did not share evolutionary history with the plastids of Karenia or Karlodinium, but was closely related to the free-living haptophyte Phaeocystis antarctica, a species that often dominates phytoplankton blooms in the Ross Sea. Chloroplast uptake was observed to occur rapidly (within 2 days), with retention in cultures being long-lived (several months) but not permanent. The dinoflagellate was also incapable of growing indefinitely in continuous darkness with algae as prey. Our findings may indicate an emerging endosymbiotic event yielding a dinoflagellate that is presently neither purely phototrophic nor purely heterotrophic, but occupies a niche juxtaposed between these contrasting nutritional modes.     

甲藻的异养营养型

综述了甲藻的异养类型。目前已知异养营养型在甲藻中广泛存在,只有很少几种甲藻营严格自养营养方式。有近一半的甲藻物种是没有色素体的,还有很多甲藻即使具有色素体也会有异养营养需求,称为兼养营养类型。这些兼养类群不一定主要以有机物作为其获取碳的来源,而仅仅是补充一些生长必需的有机物如维生素、生物素等。兼养类群以渗透营养和腐食营养方式进行,同时也可以寄生方式和共生方式进行兼养生活。无色素体的甲藻以有机物作为碳的唯一来源,仅仅依靠异养方式生存,属于严格异养营养方式,又称有机营养型。它们是甲藻异养营养型的主体,其主要类型有寄生、渗透营养和吞噬营养。由于吞噬营养是甲藻异养的主要类型,因此论述了3种吞噬营养型:吞噬营养方式、捕食茎营养方式和捕食笼营养方式。吞噬营养方式在无甲类和具甲类甲藻中都有存在,主要通过甲藻细胞的纵沟或底部对猎物进行吞噬,也有研究发现吞噬部位为顶孔或片间带。捕食茎营养方式是通过捕食茎刺穿猎物细胞膜并吸食其细胞质来获取营养,在异养甲藻中也较常见。捕食笼营养方式只在原多甲藻属(Protoperidinium)和翼藻属(Diplopsalis)里发现,是甲藻通过鞭毛孔分泌细胞质到胞外形成捕食笼将猎物包裹并进行消化来摄食的。甲藻摄食对象尺寸范围变化较大,小至几微米,大至几百微米。有些甲藻具有摄食选择性,通过感应猎物释放的化学物质来判断猎物的位置并进行摄食,摄食完成后由于体积的增加经常会发生细胞分裂和蜕鞘。对于甲藻异养的其他形式如拦截摄食营养方式、伪足摄食营养方式、口足摄食营养方式、触手摄食营养方式等只作简单介绍。还就甲藻异养的研究方法、其生态学意义和进化学意义进行简要论述,并对相关研究进行展望。     

Sampling harmful benthic dinoflagellates: Comparison of artificial and natural substrate methods

This study compared two collection methods for Gambierdiscus and other benthic harmful algal bloom (BHAB) dinoflagellates, an artificial substrate method and the traditional macrophyte substrate method. Specifically, we report the results of a series of field experiments in tropical environments designed to address the correlation of benthic dinoflagellate abundance on artificial substrate and those on adjacent macrophytes. The data indicated abundance of BHAB dinoflagellates associated with new, artificial substrate was directly related to the overall abundance of BHAB cells on macrophytes in the surrounding environment. There was no difference in sample variability among the natural and artificial substrates. BHAB dinoflagellate abundance on artificial substrates reached equilibrium with the surrounding population within 24 h. Calculating cell abundance normalized to surface area of artificial substrate, rather than to the wet weight of macrophytes, eliminates complications related to the mass of different macrophyte species, problems of macrophyte preference by BHAB dinoflagellates and allows data to be compared across studies. The protocols outlined in this study are the first steps to a standardized sampling method for BHAB dinoflagellates that can support a cell-based monitoring program for ciguatera fish poisoning. While this study is primarily concerned with the ciguatera-associated genus Gambierdiscus, we also include data on the abundance of benthic Prorocentrum and Ostreopsis cells.     

Dinoflagellate phylogeny as inferred from heat shock protein 90 and ribosomal gene sequences

Background

Interrelationships among dinoflagellates in molecular phylogenies are largely unresolved, especially in the deepest branches. Ribosomal DNA (rDNA) sequences provide phylogenetic signals only at the tips of the dinoflagellate tree. Two reasons for the poor resolution of deep dinoflagellate relationships using rDNA sequences are (1) most sites are relatively conserved and (2) there are different evolutionary rates among sites in different lineages. Therefore, alternative molecular markers are required to address the deeper phylogenetic relationships among dinoflagellates. Preliminary evidence indicates that the heat shock protein 90 gene (Hsp90) will provide an informative marker, mainly because this gene is relatively long and appears to have relatively uniform rates of evolution in different lineages.

Methodology/Principal Findings

We more than doubled the previous dataset of Hsp90 sequences from dinoflagellates by generating additional sequences from 17 different species, representing seven different orders. In order to concatenate the Hsp90 data with rDNA sequences, we supplemented the Hsp90 sequences with three new SSU rDNA sequences and five new LSU rDNA sequences. The new Hsp90 sequences were generated, in part, from four additional heterotrophic dinoflagellates and the type species for six different genera. Molecular phylogenetic analyses resulted in a paraphyletic assemblage near the base of the dinoflagellate tree consisting of only athecate species. However, Noctiluca was never part of this assemblage and branched in a position that was nested within other lineages of dinokaryotes. The phylogenetic trees inferred from Hsp90 sequences were consistent with trees inferred from rDNA sequences in that the backbone of the dinoflagellate clade was largely unresolved.

Conclusions/Significance

The sequence conservation in both Hsp90 and rDNA sequences and the poor resolution of the deepest nodes suggests that dinoflagellates reflect an explosive radiation in morphological diversity in their recent evolutionary past. Nonetheless, the more comprehensive analysis of Hsp90 sequences enabled us to infer phylogenetic interrelationships of dinoflagellates more rigorously. For instance, the phylogenetic position of Noctiluca, which possesses several unusual features, was incongruent with previous phylogenetic studies. Therefore, the generation of additional dinoflagellate Hsp90 sequences is expected to refine the stem group of athecate species observed here and contribute to future multi-gene analyses of dinoflagellate interrelationships.     

Epiphytic dinoflagellates associated with ciguatera in the northwestern coast of Cuba

The spatial and temporal abundance of epiphytic dinoflagellates associated with ciguatera was studied over two annual cycles (March 1999 to March 2000 and March 2001 to March 2002) in the northwestern coast of Cuba. From 14 species of macroalgae (Phaeophyta, Chlorophyta, and Rhodophyta), 1340 samples were obtained identifying seven species as potentially noxious; five of them are new reports for Cuba's phytobenthos: Prorocentrum belizeanum Faust, P. concavum Fukuyo, P. mexicanum Tafall, Coolia monotis Meunier, and Ostreopsis lenticularis Fukuyo. ANOVA/MANOVA analysis showed significant spatial differences: lower cell abundance near the shoreline adjacent to a river inlet and higher cell abundance in the deepest area. Prorocentrum lima (Ehrenberg) Dodge 1975 was found mainly on Phaeophyta followed by Chlorophyta and Rhodophyta. Gambierdiscus toxicus was found mainly on Phaeophyta followed by Rhodophyta and Chlorophyta. All the species reported in the study area were mainly on Padina spp. (Phaeophyta). Acanthophora spicifera (Rhodophyta) did not host dinoflagellate species. Environmental conditions in summer (higher temperature, more nutrients, greater water transparency, and low wind intensity) are suitable for macroalgae development, which serves as a substrate for potentially harmful dinoflagellates, and possibly the main vector for spreading ciguatera along the coast of Cuba.     

春末三峡大坝首次蓄水期长江口浮游植物群集

根据2003年6月三峡大坝第1次蓄水期长江口及其邻近水域(28°-33°N,120°-125°E)34个测站采集的浮游植物水样,报道了该水域浮游植物群集特征.结果表明:调查区浮游植物以硅藻和甲藻为主,伴有少量的绿藻和金藻.浮游植物细胞丰度介于0.2～1504.2 cells·ml-1,平均为72.7 cells·ml-1,主要优势物种为骨条藻(Skeletonema sp.)、具齿原甲藻(Prorocentrum dentatum)和锥状施克里普藻(ScrippsieUa trochoidea).调查区表层两个浮游植物密集区分别位于北部的冲淡水域和西南部近岸水域.浮游植物细胞丰度最大值出现在中层(10m层),最小值出现在底层.冲淡水域的优势物种是骨条藻,主要出现在表层;而在长江冲淡水与台湾暖流、黄海混合水交汇的水域,优势物种为具齿原甲藻,主要分布在表层和中层.     

Combined heat shock protein 90 and ribosomal RNA sequence phylogeny supports multiple replacements of dinoflagellate plastids

Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny.     

A bacterium that inhibits the growth of Pfiesteria piscicida and other dinoflagellates

Toxic dinoflagellate blooms have increased in estuaries of the east coast of the United States in recent years, and the discovery of Pfiesteria piscicida has brought renewed attention to the problem of harmful algal blooms (HAB) in general. Many bacteria and viruses have been isolated that have algicidal or algistatic effects on phytoplankton, including HAB species. Twenty-two bacterial isolates from the Delaware Inland Bays were screened for algicidal activity. One isolate (Shewanella IRI-160) had a growth-inhibiting effect on all three dinoflagellate species tested, including P. piscicida (potentially toxic zoospores), Prorocentrum minimum, and Gyrodinium uncatenum. This bacterium did not have a negative effect on the growth of any of the other four common estuarine non-dinoflagellate species tested, and in fact had a slight stimulatory effect on a diatom, a prasinophyte, a cryptophyte, and a raphidophyte. Shewanella IRI-160 is the first non-microzooplankton example of a microbe with the ability to control and inhibit the growth of P. piscicida, suggesting that bacteria in the natural environment could play a role in controlling the growth and abundance of P. piscicida and other dinoflagellates. Such bacteria could also potentially be used as management tools to prevent the proliferation of potentially harmful dinoflagellates in estuaries and coastal waters.     

东海两种赤潮生物种间竞争的围隔实验

1998年5月和2002年5月在东海赤潮高发区进行了两次添加营养盐诱发赤潮的围隔实验,结果表明,1998年5月实验开始时,具齿原甲藻和中肋骨条藻分别占群落总量的85％和11％,对照围隔中,具齿原甲藻一直处于优势,高峰时所占比例接近99％．加磷围隔中,中肋骨条藻显示出很强的竞争能力,第五天的数量与具齿原甲藻相等．2002年5月用4个围隔装置进行实验,在不同营养状况下两个种的增殖速率不同,营养盐浓度高的围隔中,中肋骨条藻的增殖速率比具齿原甲藻快得多,在营养盐限制条件下,具齿原甲藻比中肋骨条藻存活的要好。表明在营养盐丰富情况下,中肋骨条藻以其快速的繁殖在竞争中取得优势地位,导致中肋骨条藻形成的赤潮持续时间较短,消亡也快,而具齿原甲藻赤潮形成期较长,但在营养盐很低的情况下,还能维持较长时问．     

Molecular organization of dinoflagellate ribosomal DNA: evolutionary implications of the deduced 5.8 S rRNA secondary structure

The 5.8 S rRNA gene of Prorocentrum micans, a primitive dinoflagellate, has been cloned and its 159 base pairs (bp) have been sequenced along with the two flanking internal transcribed spacers (ITS 1 and 2), respectively, 212 and 195 bp long. Nucleotide sequence homologies between several previously published 5.8 S rRNA gene sequences including those from another dinoflagellate, an ascomycetous yeast, protozoans, a higher plant and a mammal have been determined by sequence alignment. Two prokaryotic 5'-ends of the 23 S rRNA gene have been compared owing to their probable common origin with eucaryotic 5.8 S rRNA genes. Several nucleotides are distinctive for dinoflagellates when compared with either typical eucaryotes or procaryotes. This is consistent with an early divergence of the dinoflagellate lineage from the typical eucaryotes. The secondary structure of dinoflagellate 5.8 S rRNA molecules fits the model of Walker et al. (1983). Conserved nucleotides which distinguish dinoflagellate 5.8 S rRNA from that of other eucaryotes are located in specific loops which are assumed to play a structural role in the ribosome. A 5.8 S rRNA phylogenetic tree which is proposed, based on sequence data, supports our initial assumption of the dinoflagellates.