Dinoflagellate Akashiwo Sanguinea Research Articles

Full-length transcriptome analysis of the bloom-forming dinoflagellate Akashiwo sanguinea by single-molecule real-time sequencing.

The dinoflagellate Akashiwo sanguinea is a harmful algal species and commonly observed in estuarine and coastal waters around the world. Harmful algal blooms (HABs) caused by this species lead to serious environmental impacts in the coastal waters of China since 1998 followed by huge economic losses. However, the full-length transcriptome information of A. sanguinea is still not fully explored, which hampers basic genetic and functional studies. Herein, single-molecule real-time (SMRT) sequencing technology was performed to characterize the full-length transcript in A. sanguinea. Totally, 83.03 Gb SMRT sequencing clean reads were generated, 983,960 circular consensus sequences (CCS) with average lengths of 3,061 bp were obtained, and 81.71% (804,016) of CCS were full-length non-chimeric reads (FLNC). Furthermore, 26,461 contigs were obtained after being corrected with Illumina library sequencing, with 20,037 (75.72%) successfully annotated in the five public databases. A total of 13,441 long non-coding RNA (lncRNA) transcripts, 3,137 alternative splicing (AS) events, 514 putative transcription factors (TFs) members from 23 TF families, and 4,397 simple sequence repeats (SSRs) were predicted, respectively. Our findings provided a sizable insights into gene sequence characteristics of A. sanguinea, which can be used as a reference sequence resource for A. sanguinea draft genome annotation, and will contribute to further molecular biology research on this harmful bloom algae.

Temporal dynamics of surface ocean carbonate chemistry in response to natural and simulated upwelling events during the 2017 coastal El Niño near Callao, Peru

Abstract. Oxygen minimum zones (OMZs) are characterized by enhanced carbon dioxide (CO2) levels and low pH and are being further acidified by uptake of anthropogenic atmospheric CO2. With ongoing intensification and expansion of OMZs due to global warming, carbonate chemistry conditions may become more variable and extreme, particularly in the eastern boundary upwelling systems. In austral summer (February–April) 2017, a large-scale mesocosm experiment was conducted in the coastal upwelling area off Callao (Peru) to investigate the impacts of ongoing ocean deoxygenation on biogeochemical processes, coinciding with a rare coastal El Niño event. Here we report on the temporal dynamics of carbonate chemistry in the mesocosms and surrounding Pacific waters over a continuous period of 50 d with high-temporal-resolution observations (every second day). The mesocosm experiment simulated an upwelling event in the mesocosms by addition of nitrogen (N)-deficient and CO2-enriched OMZ water. Surface water in the mesocosms was acidified by the OMZ water addition, with pHT lowered by 0.1–0.2 and pCO2 elevated to above 900 µatm. Thereafter, surface pCO2 quickly dropped to near or below the atmospheric level (405.22 µatm in 2017; Dlugokencky and Tans, 2021; NOAA/Global Monitoring Laboratory (GML)) mainly due to enhanced phytoplankton production with rapid CO2 consumption. Further observations revealed that the dominance of the dinoflagellate Akashiwo sanguinea and contamination of bird excrements played important roles in the dynamics of carbonate chemistry in the mesocosms. Compared to the simulated upwelling, natural upwelling events in the surrounding Pacific waters occurred more frequently with sea-to-air CO2 fluxes of 4.2–14.0 mmol C m−2 d−1. The positive CO2 fluxes indicated our site was a local CO2 source during our study, which may have been impacted by the coastal El Niño. However, our observations of dissolved inorganic carbon (DIC) drawdown in the mesocosms suggest that CO2 fluxes to the atmosphere can be largely dampened by biological processes. Overall, our study characterized carbonate chemistry in nearshore Pacific waters that are rarely sampled in such a temporal resolution and hence provided unique insights into the CO2 dynamics during a rare coastal El Niño event.

Toxic Characteristics and Action Mode of the Mixotrophic Dinoflagellate Akashiwo sanguinea on Co-Occurring Phytoplankton and Zooplankton.

The mixotrophic dinoflagellate Akashiwo sanguinea frequently forms harmful algal blooms around the world and has caused massive deaths of shellfish, finfish and birds, yet its toxic mechanism is still unclear. In this study, toxic effects of A. sanguinea on co-culturing phytoplankton and zooplankton were investigated. The results showed that sonicated cultures of A. sanguinea JX13 and JX14, isolated from the Pearl River Estuary, had a significant lethal effect on the rotifer Brachionus plicatilis, with the highest mortality rate of 80%. The highest inhibition rates of A. sanguinea cultures JX13 (90%) and JX14 (80%) on R. salina were much higher than that of AS2 (20%). Toxicity varied with the growth stage, during which A. sanguinea cells in the exponential stage showed the highest toxicity (40%), while A. sanguinea filtrate had the highest toxicity (10%) in the decline stage. The action mode of A. sanguinea toxicity on plankton was explored through an osmotic membrane culture device. It was found that A. sanguinea JX13 displayed an inhibitory effect on coexisting phytoplankton, whether they had contact or not, but the inhibition rate increased by 25% with contact. A lethal effect of A. sanguinea JX13 on rotifer Brachionus plicatilis was observed only in contact treatment. This study suggests that direct contact is the key action mode to trigger the release of toxins and induce toxic effects of A. sanguinea on co-occurring plankton.

Chloroplast Ultrastructure and Photosynthetic Response of the Dinoflagellate Akashiwo sanguinea Throughout Infection by Amoebophrya sp.

The endoparasitic dinoflagellate Amoebophrya infects a number of marine dinoflagellates, including toxic and harmful algal bloom-forming species. The parasite kills its host and has been proposed to be a determining factor in the demise of dinoflagellate blooms in restricted coastal waters. Previous studies have mainly focused on the occurrence, prevalence, and diversity of Amoebophrya, while the interactions between the parasite and its host have received limited attention. Herein, an Amoebophrya sp.-Akashiwo sanguinea co-culture was established from Chinese coastal waters, and morphological, physiological, and transcriptional changes throughout an infection cycle of the parasite were systemically studied. The parasitic dinoflagellate was very infectious, resulting in an infection rate up to 85.83% at a dinospore:host ratio of 10:1. Infected host cells died eventually and released approximately 370 dinospores/cell. The host nuclear structures were rapidly degraded by Amoebophrya infection, and the chloroplasts of parasitized host cells remained intact until the parasite filled the almost entire cell structure. Nevertheless, infected cultures showed sustained but lower levels of photosynthetic performance (∼64% of control cultures), and the photosynthesis-related genes were significantly down-regulated. These findings provide a better understanding of the biological basis of the complex parasite-host interactions, which will be helpful to further elucidate the ecological significance of parasitic dinoflagellates in marine ecosystems.

Interactions between the seaweed Gracilaria and dinoflagellate Akashiwo sanguinea in an indoor co-cultivation system and the interference of bacteria

Interactions between the seaweed Gracilaria and dinoflagellate Akashiwo sanguinea in an indoor co-cultivation system and the interference of bacteria

Parasite-mediated increase in prey edibility in the predator-prey interaction of marine planktonic protists

In planktonic predator-prey interactions, predation pressure could be affected by several factors associated with prey, including cell size, shape, nutritional quality, presence of chemical deterrents, and movement behaviors. In addition, parasitism may also play an important role in predator-prey interaction by infecting one or both partners involved in the biological interaction. In this study, parasite mediation in predator-prey interactions of planktonic protists was addressed using model organisms commonly observed in temperate coastal environments, namely, a phototrophic dinoflagellate Akashiwo sanguinea as a potential host and prey, a heterotrophic dinoflagellate predator Luciella masanensis, and a dinoflagellates-infecting parasitoid Parvilucifera infectans. Parasite mediation permitted L. masanensis to exploit the prey A. sanguinea that the predator was otherwise unable to use or did not prefer. However, parasite-mediated exploitation seems to be dependent on the infection cycle of P. infectans. Although zoospores and mature sporangium produced from infected A. sanguinea were not ingested by the predator L. masanensis, both newly infected (i.e., cells that zoospores had just penetrated) and infected (those containing early to late trophocytes) A. sanguinea cells attracted numerous L. masanensis cells and were rapidly ingested by the predator, leading to the predator's positive growth. The results from mixed culture experiments of the three protists showed that the presence of P. infectans at low density led to co-occurrence of the three protists. Thus, the parasitoid seems to play the role of a mediator in either inedible prey- or predator-dominated environments, leading to co-occurrence of the three protists.

Allelopathic effects of mixotrophic dinoflagellate Akashiwo sanguinea on co-occurring phytoplankton: the significance of nutritional ecology

Blooms of Akashiwo sanguinea frequently break out around the world, causing huge economic losses to the aquaculture industry and seriously damaging coastal ecosystems. However, the formation mechanisms of A. sanguinea blooms remain unclear. We investigated the allelopathic effects of A. sanguinea on multiple phytoplankton species, explored the mode of allelochemicals action and the way of nutrient factors regulation of the allelopathic activity. Results show that strains of A. sanguinea could inhibit the growth of co-occurring phytoplankton including Scrippsiella trochoidea, Phaeocystis globosa, and Rhodomonas salina, but inhibition of Prorocentrum micans was not obvious. The inhibition rates on phytoplankton were positively correlated with the cell densities of A. sanguinea. The highest inhibition rate of 94% on R. salina was for A. sanguinea CCMA256 culture of 2 000 cells/mL at 72 h. We observed that cells of S. trochoidea, Ph. globosa, and R. salina were lysed when co-cultured with A. sanguinea, with the shortest time for S. trochoidea. Additionally, the growth rates of A. sanguinea were promoted by co-culturing with S. trochoidea, Ph. globosa, and R. salina. Four components of A. sanguinea culture were all able to inhibit growth of R. salina: the strongest inhibitory effect was found in the sonicated culture, followed by whole-cell culture, filtrates of sonicated culture, and filtrate culture. The crude extract of A. sanguinea culture also lysed cells of R. salina, and the inhibition rates on R. salina increased with the increasing dose of crude extract. It was shown that both nutrient enrichment and nitrogen:phosphorus ratio imbalance enhanced remarkably the allelopathic activity of A. sanguinea. The highest inhibition rate on R. salina of 70% occurred in A. sanguinea JX13 treatment at 2 000 cells/mL under high nutrient condition in 48 h. In JX14 treatment at 2 000 cells/mL for N:P of 10:1, the inhibition rate increased by 1.7 times of that for N:P of 20:1. In addition, the allelopathy of A. sanguinea could not only be a competitive strategy but also a nutrition strategy, playing an important role in formation and/or maintenance of blooms of the mixotrophic dinoflagellate A. sanguinea.

Variation in the photosynthetic activities of the dinoflagellate Akashiwo sanguinea during formation of resting cysts

Resting cysts represent the dormant life stage produced by sexual reproduction of dinoflagellates; it generally maintain a limited level of metabolic activities. In the present study, changes in the photosynthetic apparatus of Akashiwo sanguinea during the encystment process in different nitrogen (N) conditions were characterized, and the transcriptional responses of nine photosynthesis genes were further evaluated. The photochemical efficiency decreased with elevation of N deprivation and growth of algae, and the resting cysts maintained limited photosynthetic activity. Meanwhile, the transcription of photosystem II genes (psbA and psbD) were down-regulated, especially the levels of the psbA gene, suggesting the inactivation of photosystem II under N limitation. Moreover, genes involved in cytochrome f (petA) and ATP synthase (atpA, atpB) were also transcribed at low levels in N-deprivation cultures. In contrast, the transcription of the RubisCO large subunit gene (rbcL) was up-regulated, indicating the maintenance of carbon fixation in N-limited conditions. The transcription levels of photosystem I genes (psaA and psaB) showed no significant correlation with the N condition. Further, most of the photosynthesis genes were down-regulated in the resting cysts, which was coincided with the inactive physiological status of the dormant stage. The results demonstrated that photosynthesis genes are differentially regulated in response to varying N conditions and life cycles. The findings will provide fundamental knowledge for better understanding the physiological characteristics and molecular regulation mechanisms in the developmental life cycle of marine dinoflagellates.

Unprecedented DMSP Concentrations in a Massive Dinoflagellate Bloom in Monterey Bay, CA

AbstractThe organic sulfur compound dimethylsulfoniopropionate (DMSP) is synthesized by numerous species of marine phytoplankton, and its volatile degradation products are a major source of biogenic sulfur to the atmosphere. A massive bloom of the dinoflagellate Akashiwo sanguinea occurred in Monterey Bay, CA, USA, in the fall of 2016 and led to exceptionally high seawater DMSP concentrations that peaked at 4,240 nM. Bacterial consumption rates showed that only a small fraction of the DMSP standing stock flowed through the dissolved DMSP pool per day, contributing to the high DMSP concentrations and creating conditions conducive to production of dimethylsulfide (DMS). Conservative calculations of DMS yield from this persistent A. sanguinea bloom suggest substantial regional‐scale inputs of DMS‐sulfur to the atmosphere. Other recently reported major coastal blooms of A. sanguinea, along with indications that this species may benefit from climate change conditions, reveal a mechanism that could alter oceanic contributions to atmospheric sulfur pools.

Variation in biochemical composition during encystment of the planktonic dinoflagellate Akashiwo sanguinea in N-limited cultures

The planktonic dinoflagellate Akashiwo sanguinea is a commonly observed bloom-forming species in estuarine and coastal waters worldwide. Large scales of A. sanguinea bloom cause the mass mortality of fish, shellfish, and sea birds. The formation of resting cysts plays as a potential vector for its wide distribution and frequent recurrence of blooms; however, the biochemical characteristics of algal cells during their life cycle remain unclear. For the first time, the variations in cellular chlorophyll a (Chl a), protein, carbohydrate, and total lipid contents during encystment of A. sanguinea cultured in different nitrate concentrations, namely 0-, 25-, 50-, 100-, 200-, and 833-μM N-added treatment, were studied in the present study. The results indicated that resting cysts were formed in all the N treatments, with the highest encystment ratio of 3.34 ± 0.57% observed in the 100-μM N-added treatment, and the lowest rate of 0.80 ± 0.03% observed in the 883-μM N-added treatment. The levels of the four biochemical components varied significantly during the encystment process. The Chl a and protein levels were significantly lower in newly formed cysts than in vegetative cells; however, continuous accumulation of carbohydrates and total lipids occurred with the algal growth, particularly of carbohydrates in the resting cysts, which was more than tenfold greater than those in the vegetative cells. Low initial N concentrations were more favorable for carbohydrate accumulation in cysts than high N concentrations. The accumulated components may play vital roles for the substance of A. sanguinea during resting stage. The results provided fundamental information for an improved understanding of the physiological response of A. sanguinea during encystment.

Epibiotic bacterial community composition in red-tide dinoflagellate Akashiwo sanguinea culture under various growth conditions.

The phycosphere of phytoplankton harbors a diversity of microbes that can potentially interact with the host phytoplankton cells. In spite of increasing reports regarding the interactions between some model phytoplankton and bacteria, there remain many unknowns regarding interactions between the dinoflagellate Akashiwo sanguinea and its phycosphere bacteria. Here we used both cultivation and metagenomic sequencing methods to investigate the microbial community composition in A. sanguinea batch cultures under various growth conditions. The microbial community dynamics under different growth stages and nutrient (nitrogen and iron) depletion scenarios were determined by Illumina MiSeq sequencing of 16S rRNA gene amplicons. Rhodobacteraceae, Alteromonadaceae and Flavobacteriaceae were the main bacterial families and varied significantly under different states of the host A. sanguinea. Selective fluorescence in situ hybridization was also performed to label Rhodobacterales and Alteromonas clade bacteria to confirm their attachment to A. sanguinea cells under confocal laser scanning microscopy. Plate streaking protocol isolated 19 bacterial strains from A. sanguinea cultures, identified through 16S rRNA analysis. Most culturable strains (11 of 19) belonged to Rhodobacteraceae consistent with Illumina MiSeq sequencing data. The possible interaction pathway between these epibiotic bacteria and A. sanguinea in the phycosphere is also discussed.

Revisiting the Parvilucifera infectans / P. sinerae (Alveolata, Perkinsozoa) species complex, two parasitoids of dinoflagellates

Members of the family Parviluciferaceae (Alveolata, Perkinsozoa) are the well-known dinoflagellate parasitoids along with Amoebophrya ceratii species complex and parasitic chytrid Dinomyces arenysensis and contain six species across three genera (i.e., Parvilucifera infectans, P. sinerae, P. rostrata, and P. corolla, Dinovorax pyriformis, and Snorkelia prorocentri) so far. Among Parvilucifera species, the two species, P. infectans and P. sinerae, are very similar or almost identical each other morphologically and genetically, thereby make it difficult to distinguish between the two. The only main difference between the two species known so far is the number of sporangium wall (i.e., 2 layers in P. infectans vs. 3 layers in P. sinerae). During sampling in Masan bay, Korea during the spring season of 2015, the dinoflagellate Akashiwo sanguinea cells infected by the parasite Parvilucifera were observed and this host-parasite system was established in culture. Using this culture, its morphological and ultrastructural features with special emphasis on the variation in the number of sporangium wall over developmental times, were investigated. In addition, the sequences of rDNA regions and β-tubulin genes were determined. The result clearly demonstrated that the trophocyte at 36 h was covered with 4 layers, and then outer layer of the sporocyte gradually degraded over time, resulting in wall structure consisting of two layers, with even processes being detached from 7-day-old sporangium with smooth surface, indicating that the difference in the number of layers seems not to be an appropriate ultrastructural character for distinguishing P. infectans and P. sinerae. While pairwise comparison of the large subunit rDNA sequences showed 100% identity among P. infectans / P. sinerae species complex, genetic differences were found in the small subunit (SSU) rDNA sequences but the differences were relatively small (11–13 nucleotides) compared with those (190–272 nucleotides) found among the rest of Parvilucifera species (P. rostrata and P. corolla). Those small differences in SSU rDNA sequences of P. infectans / P. sinerae species complex may reflect the variations within inter-strains of the same species from different geographical areas. Taken together, all morphological, ultrastructural, and molecular data from the present study suggest that they are the same species. Key words: inter-strain variation; parasitism; parasitoid; sporangium; synonymy

Acute toxicity of the cosmopolitan bloom-forming dinoflagellate Akashiwo sanguinea to finfish, shellfish, and zooplankton

The unarmored dinoflagellate Akashiwo sanguinea is a well known cosmopolitan harmful alga; however, the toxic nature of this alga has yet to be examined using multiple clonal isolates. Here we examined the toxicity of 3 clonal cultures of A. sanguinea, including JX13 and JX14, isolated from Daya Bay, South China Sea, and AS2, isolated from Chesapeake Bay, USA, to multiple aquatic animals including species of finfish, shellfish, shrimp, and zooplankton. The whole-cell cultures of A. sanguinea exhibited acute lethal effects on the shrimp Litopenaeus vannamei, the bivalve Meretrix meretrix, and 2 species of fish (Mugil cephalus and Mugilidae sp.) with 72 h mortalities ranging from 20 to 100%. The sonicated and filtrated cultures were lethal to brine shrimp Artemia salina, while the filtrates of whole-cell cultures were not, suggesting that the toxins are intracellular. Boiling and freezing led to significant reductions in toxicity. A. sanguinea toxicity differed among the Chinese strains, and the hemolytic activity of 1 Chinese strain was 3-fold greater than that of the US strain. Cultures in exponential phase displayed stronger toxicity, and the greatest toxicity of A. sanguinea was observed at 20 degrees C and a salinity of 35, conditions optimal for growth of the alga. Toxicity was enhanced by increased nutrient supply, suggesting that this species could both directly (via increased growth) and indirectly (e.g. via enhanced toxin production) become more toxic in response to eutrophication. Collectively, our findings suggest that the ability to produce and release toxin(s) may promote A. sanguinea blooms by suppressing predators and competitors.

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica, a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d−1. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13ngC predator−1d−1 (0.06 cells predator−1d−1). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019ngC predator−1d−1 (266 bacteria predator−1d−1), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 107 cells ml−1 was 0.01ngC predator−1d−1 (48 Synechococcus predator−1d−1). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.

The dinoflagellate Akashiwo sanguinea will benefit from future climate change: The interactive effects of ocean acidification, warming and high irradiance on photophysiology and hemolytic activity

Due to global climate change, marine phytoplankton will likely experience low pH (ocean acidification), high temperatures and high irradiance in the future. Here, this work report the results of a batch culture experiment conducted to study the interactive effects of elevated CO2, increased temperature and high irradiance on the harmful dinoflagellate Akashiwo sanguinea, isolated at Dongtou Island, Eastern China Sea. The A. sanguinea cells were acclimated in high CO2 condition for about three months before testing the responses of cells to a full factorial matrix experimentation during a 7-day period. This study measured the variation in physiological parameters and hemolytic activity in 8 treatments, representing full factorial combinations of 2 levels each of exposure to CO2 (400 and 1000μatm), temperature (20 and 28°C) and irradiance (50 and 200μmol photons m−2s−1). Sustained growth of A. sanguinea occurred in all treatments, but high CO2 (HC) stimulated faster growth than low CO2 (LC). The pigments (chlorophyll a and carotenoid) decreased in all HC treatments. The quantum yield (Fv/Fm) declined slightly in all high-temperature (HT) treatments. High irradiance (HL) induced the accumulation of ultraviolet-absorbing compounds (UVabc) irrespective of temperature and CO2. The hemolytic activity in the LC treatments, however, declined when exposed to HT and HL, but HC alleviated the adverse effects of HT and HL on hemolytic activity. All HC and HL conditions and the combinations of high temperature*high light (HTHL) and high CO2*high temperature*high light (HCHTHL) positively affected the growth in comparison to the low CO2*low temperature*low light (LCLTLL) treatment. High temperature (HT), high light (HL) and a combination of HT*HL, however, negatively impacted hemolytic activity. CO2 was the main factor that affected the growth and hemolytic activity. There were no significant interactive effects of CO2*temperature*irradiance on growth, pigment, Fv/Fm or hemolytic activity, but there were effects on Pm, α, and Ek. If these results are extrapolated to the natural environment, it can be hypothesized that A. sanguinea cells will benefit from the combination of ocean acidification, warming, and high irradiance that are likely to occur under future climate change. It is assumed that faster growth and higher hemolytic activity and UVabc of this species will occur under future conditions compared with those the current CO2 (400μatm) and temperature (20°C) conditions.

Cryptic diversity within the harmful dinoflagellate Akashiwo sanguinea in coastal Chinese waters is related to differentiated ecological niches

Blooms of the harmful dinoflagellate Akashiwo sanguinea are responsible for the mass mortality of fish and invertebrates in coastal waters. This cosmopolitan species includes several genetically differentiated clades. Four clonal cultures were established by isolating single cells from Xiamen Harbour (the East China Sea) for morphological and genetic analyses. The cultures displayed identical morphology but were genetically different, thus revealing presence of cryptic diversity in the study area. New details of the apical structure complex of Akashiwo sanguinea were also found. To investigate whether the observed cryptic diversity was related to environmental differentiation, 634 cells were obtained from seasonal water samples collected from 2008 to 2012. These cells were sequenced by single-cell PCR. For comparison with Chinese material, additional large subunit ribosomal DNA sequences were obtained for three established strains from Malaysian and French waters. To examine potential ecological differentiation of the distinct genotypes, growth responses of the studied strains were tested under laboratory conditions at temperatures of 12°C to 33°C. These experiments showed four distinct ribotypes of A. sanguinea globally, with the ribotypes A and B co-occuring in Xiamen Harbour. Ribotype A of A. sanguinea was present year-round in Xiamen Harbour, but it only bloomed in the winter and spring, thus corresponding to the winter type. In contrast, A. sanguinea ribotype B bloomed only in the summer, corresponding to the summer type. This differentiation supports the temperature optimum conditions that were established for these two ribotypes in the laboratory. Ribotype A grew better at lower temperatures compared to ribotype B which preferred higher temperatures. These findings support the idea that various ribotypes of A. sanguinea correspond to distinct ecotypes and allopatric speciation occurred in different climatic regions followed by dispersal.

Effects of irradiance on growth, pigment content and photosynthetic efficiency on three peridinin-containing dinoflagellates

The effects of two irradiance levels on growth, cell volume, pigment content and photosynthetic efficiency were analysed in cultures of the peridinin-containing dinoflagellates Akashiwo sanguinea, Prorocentrum micans and Scrippsiella trochoidea. Strains were isolated from coastal waters in southern Brazil, and cells were grown at low (LL, 87–90μmolphotonsm−2s−1) and high (HL, 450–490μmolphotonsm−2s−1) light intensities. The composition of photosynthetic pigments was analysed by high-performance-liquid-chromatography (HPLC) in samples collected at days 2, 7 and 14 of growth, and the photosynthetic efficiency (Fv/Fm) was determined by pulse-amplitude-modulated fluorometry. On day 14, cells were stained with Nile Red and observed under the fluorescence microscope for lipid content determination. Similar growth rates were observed for A. sanguinea (μ=0,26–0,33d−1) and P. micans (μ=0,25–0,31d−1) under HL and LL, whereas S. trochoidea presented significantly higher values under HL (0,29±0.02; p<0,001) than LL (μ=0,23±0.01d−1). Increase in the cell volume was observed for P. micans along the growth curve in both irradiances while for A. sanguinea at LL only. The three species presented low concentration of photoprotective pigments under both HL and LL, whereas S. trochoidea and P. micans showed higher photosynthetic efficiency (average: Fv/Fm>0.5) compared to A. sanguinea (Fv/Fm~0.2). Microscope observation revealed that intracellular lipid concentration increased at HL in the three species, in all growth stages. In short, the three dinoflagellates are adapted to HL intensity and increased lipids might serve as a protective mechanism for the cells.

Validation of reference genes for gene expression studies in the dinoflagellate Akashiwo sanguinea by quantitative real-time RT-PCR

The accurate measurement of gene expression via quantitative real-time reverse transcription PCR (qRT-PCR) heavily relies on the choice of valid reference gene(s) for data normalization. Resting cyst is the dormant stage in the life cycle of dinoflagellate, which plays crucial roles in HAB-forming dinoflagellate ecology. However, only limited investigations have been conducted on the reference gene selection in dinoflagellates. Gap remained in our knowledge about appropriate HKGs for normalizing gene expression in different life stages, which laid obstacles for the application of qRT-PCR to the HAB-forming group. In this study, six candidate reference genes, 18S ribosomal RNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-tubulin (TUA), β-tubulin (TUB), actin (ACT) and cytochrome oxidase subunit 1 (COX1), were evaluated for their expression stability with qRT-PCR and three statistical algorithms (GeNorm, NormFinder, and BestKeeper) for the cosmopolitan, harmful algal bloom-forming dinoflagellate Akashiwo sanguinea. Expression patterns were observed across 18 biological samples, including cells at resting stages (resting cysts), different growth stages, in darkness, exposed to abscisic acid (ABA) and exposed to temperature stress. The results indicated that TUA, 18S and GAPDH were relatively stable across all tested scenarios. While the best-recommended reference genes differed across experimental groups, the pairs of ACT and TUA, 18S and GAPDH were the most reliable for cells at different growth stages and darkness treatment. The combination of TUA and TUB was the best choice for normalization in resting cysts and in ABA treatment, respectively. The pair of ACT and COX1 was suitable for temperature treatments. This study was the first to investigate the stable internal reference genes in dinoflagellates at different stages of life cycle, particularly in resting cysts. Our results provided useful information for selection of reference genes in dinoflagellates regarding quantification of gene expression at different experimental scenarios, which will facilitate more accurate and widespread use of qRT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species.

Effects of nitrogenous nutrition on growth and nitrogen assimilation enzymes of dinoflagellate Akashiwo sanguinea

The dinoflagellate Akashiwo sanguinea is a cosmopolitan species which frequently causes dense blooms in estuaries and coasts around the world, while the mechanism regulating the formation of the algal bloom remains unclear. To further explore the physiological response of A. sanguinea during algal blooms, we studied the effects of various nitrogenous nutrients on growth and nitrogen assimilation enzymes of A. sanguinea in laboratory trials. Our results indicated that A. sanguinea was able to grow well on multiple N substrates within appropriate ranges of concentrations. The dinoflagellate grew faster in NH4+, while it showed greatest affinity for urea. The intracellular enzymes of A. sanguinea were regulated to adapt for the environmental nutrient conditions and growth stages: nitrate reductase (NR) was induced when grown on NO3− while inhibited by NH4+ and urea, glutamine synthetase (GS) expressed constitutively in all N sources, and urease was up-regulated by NO3− and urea but down-regulated by NH4+. Meanwhile, the activities of the N assimilation enzymes decreased with the growth of A. sanguinea, and GS activity showed a significant positive correlation with growth rates. The urease activity of A. sanguinea was extremely high assessed by the kinetics of N assimilation, indicating its competitive advantage for urea over other phytoplankton species. The physiological activity of N assimilation enzymes in A. sanguinea facilitates its utilization of reduced N, and implies its capability of forming blooms in environments where reduced N is the common N source.

Differential expressions of an Hsp70 gene in the dinoflagellate Akashiwo sanguinea in response to temperature stress and transition of life cycle and its implications

The unarmoured dinoflagellate Akashiwo sanguinea is a cosmopolitan species frequently causing harmful algal blooms in temperate to tropical estuaries and marine coasts. While resting cyst production and wide temperature tolerance have been well documented as two adaptive strategies for HAB-forming dinoflagellates, little attention has been paid to the molecular mechanisms, such as the functional genes and their expressions relevant to these ecological processes. Here, the cloning of the gene Hsp70, a gene putatively involved in the process of temperature stress responses, in A. sanguinea and its differential expressions in response to temperature stress and transition of life cycle stages were reported. Based on homologous cloning and the rapid amplification of cDNA ends (RACE), the full-length cDNA sequence (2171 bp) of an Hsp70 gene from A. sanguinea (AsHsp70) was obtained (Accession No. KJ755185), with an open reading frame of 1950 bp. The deduced 649 amino acids (aa) possessed an ATPase domain of 399 aa, a substrate peptide binding domain of 124 aa and a C-terminus domain of 126 aa. The highly conserved motifs of Hsp70 family identified in AsHsp70 suggest conservative function of Hsp70. Real-time quantitative PCR revealed that AsHsp70 expression (20°C-maintained) was dramatically up-regulated by both low (15°C, 10°C, 5°C) and high (25°C, 30°C) temperature shocks. Rapid and significant increase of AsHsp70 expression could be detected within 10min after exposure to lower temperature stress (10°C). Compared with the stepwise increases or decreases in temperature (±5°C each step), one-step shocks (±10°C) elicited markedly higher AsHsp70 transcripts. In addition, AsHsp70 transcription varied with growth stage and life cycle, peaking in immature cysts undergoing morphogenesis and returning to lower levels in cysts when morphogenesis was completed. The results together strongly suggest that A. sanguinea is more tolerant to gradual than drastic temperature stress and that AsHsp70 may be involved in the adaptation of A. sanguinea to varying temperatures. It is also suggested that Hsp70 might play roles in the encystment of dinoflagellates, which, however, needs more solid evidence for confirmation.