Solid sand particle addition can enhance the production of resting cysts in dinoflagellates

Resting cysts are an important part of the life cycle for many harmful algal bloom-forming dinoflagellates, and play vital roles in the recurrence and geographical spread of harmful algal blooms. Numerous factors have been suggested to regulate the formation of resting cysts, although only a few have been proven to be significant. Cyst formation can be induced by adverse environmental conditions such as drastic changes in temperature, light, salinity, and nutrient levels, and by biological interactions. In this study, we evaluated the ability of an artificial factor(fine sand particles) to enhance the formation of resting cysts. Fine sand particles were added to cultures of dinoflagellates that are known to produce cysts. The addition of fine sand particles significantly increased both the production rate and final yield of cysts in cultures of S crippsiella trochoidea, Biecheleria brevisulcata, and Levanderina fissa(= Gymnodinium fissum, Gyrodinium instriatum, Gyr odinium uncatenum). The largest increase in the final yield(107-fold) of cysts as a result of sand addition was in S. trochoidea. However, addition of fine sand particles did not induce cyst formation, or barely af fected cyst formation, in A kashiwo san guinea, Cochlodinium polykrikoides and Pheopolykrikos hartmannii, which are also known to be cyst-producing species. We speculated that addition of sand significantly increased the chances of cell collision, which triggered cyst formation. However, further research is required to test this idea. Importantly, our findings indicate that the addition of fine sand particles is a useful method to obtain a large quantity of cysts in a short time for laboratory studies or tests; for example, if a cyst viability test is being used to assess the eff ectiveness of ships' ballast water treatment.

Mapping the resting cysts of dinoflagellate Alexandrium catenella along the coast of Qinhuangdao,China

Dinoflagellate Alexandrium catenella is a cosmopolitan bloom-forming species with complex life cycle,the formation and germination of resting cysts are critical for its bloom dynamics.In the coastal waters of Qinhuangdao,A.catenella has been identified as the major causative agent for paralytic shellfish poisoning,but there is little knowledge concerning its resting cysts in this region.In this study,three surveys were carried out along the coast of Qinhuangdao from 2020 to 2021 to map the distribution of A.catenella resting cysts,using a quantitative PCR(qPCR)assay specific for A.catenella.The resting cysts were detected in surface sediments during all the three surveys,and their distribution patterns were similar.High abundance of resting cysts(maximum 1300 cysts/g sediment(wet weight))were found in a region(119.62°E-119.99°E,39.67°N-39.98°N)northeast to the coastal waters of Qinhuangdao,where surface sediments were mainly composed of clay and silt(percentage above 50%).Prior to the formation of the A.catenella bloom in March 2021,the abundance of A.catenella vegetative cells in seawater had extremely significant positive correlation with the abundance of resting cysts in surface sediments,reflecting the important role of resting cysts in the initiation of A.catenella blooms.As far as we know,this is the first report on the distribution of A.catenella cysts along the coast of Qinhuangdao.The results will of fer a sound basis for the future monitoring and mitigation of toxic A.catenella blooms and paralytic shellfish poisoning events in this region.

Preliminary study on taxonomy of dinoflagellate cysts from major estuary and bays of Fujian Province, China

According to the morphology, wall structure, color, ornamentation, etc., 25 species belonging to 9 genera are identified and described from 144 sediment samples of the Xiamen Harbor, the mouth of the Minjiang River and the Sansha Bay. Among them there are 2 toxic species: Alexandrium minutun,A. tamarenes, 4 harmful species: Alexandrium affine,Lingulodinium polX drum, Scrippsiella trochoide,Gonyaulax spinifera. It shows that 11 species of dinoflagellate cysts (Alexandrium affine, A. minutum, Diplopelta cf. parva, Polykrikos cf. schwartzii, protoceratium reticulatum, Protoperidinium minutum, P. cf. minutum, P. cf americanum and Alexandrium sp., Protoperidinium sp. 1, P. sp. 2) are first recorded along the coast of Fujian Province, China. These newly discovered species might be transported to the coastal sea of Fujian Province by ballast water of international trade vessels.

Effects of modified clay on cysts of Scrippsiella trochoidea for harmful algal bloom control

We present results on the effect of modified clay on cyst formation of Scrippsiella trochoidea in harmful algal bloom （HAB）. Modified clay （in concentration of 0, 0.1, 0.5, and 1.0 g/L） were added to cultures, and observations were made on cysts of S. trochoidea under controlled laboratory conditions. Results indicate that the removal rate of algal cells reached 97.7% at the clay concentration of 1.0 g/L. The cyst formation rate increased from 4.6% to 24.6% when the concentration of clay was increased from 0 to 1.0 g/L. Two cyst metamorphs were observed： spinal calcareous cysts and smooth noncalcareous ones. The proportion of the spinal cysts decreased from 76.9% to 24.1% when clay concentration increased from 0 to 1.0 g/L. In addition, modified clay affected cyst germination. The germination rate decreased with the increases in the clay concentrations. Non-calcareous cysts had a lower germination rate with a longer germination time. We conclude that modified clay could depress algal cell multiplication and promote formation of temporal cysts of S. trochoidea, which may help in controlling HAB outbreaks.

Reviving and characterizing three species of dinoflagellate cysts dormant for about 70 years in the East China Sea:Biecheleria brevisulcata,Biecheleriopsis adriatica,and Scrippsiella donghaienis

Many marine dinoflagellates can form resting cysts as a part of their life cycle,and the cysts could be buried in sediment and remained viable for as long as over 150 years.However,only a very limited number of cyst species have been revived from long-buried sediments and investigated in regard to a possible shift in the intra-specific genetic structure of a species detected from the historical record at a particular location.Here,we report a successful germination of three species of resting cysts that were sampled from the depth dated back to 1941±18 AD from a 44-cm sediment core from the East China Sea.Seven isolates were established from germination of single cyst isolation or multi-cyst germinations.LSU rRNA gene or ITS sequences of these strains were obtained,then they were identified to be Biecheleria brevisulcata(five strains),Biecheleriopsis adriatica(one strain),and Scrippsiella donghaienis(one strain)in terms of morphology and rRNA gene sequence.Biecheleria brevisulcata strain 1,Bps.adriatica strain 21,and S.donghaienis strain 23 were examined in detail with light microscope(LM)and scanning electron microscope(SEM),and analyzed with high performance liquid chromatography(HPLC)for their pigment compositions,and genetic diversity.We also confirmed the presence of a resting cyst of Bps.adriatica in the field for the first time.The LSU rRNA gene-based genetic distances of Bps.adriatica from that obtained from water sample,single-cell PCR sequencing for the cysts isolated from the surface sediment of the same sea area and that reported from other regions during the recent years,and ITS-based genetic distances of S.donghaienis from that obtained from cysts isolated from the surface sediment of the same location and that reported from other regions during the recent years indicated that the intra-specific genetic structure of each species in the sampling area may have shifted during the last 70 years.Our work confirms that B.brevisulcata,Bps.adriatica,and S.donghaienis,all described as new species around 2010,have inhabited the East China Sea for about 70 years.The present work reports for the first time the revival of dinoflagellate resting cysts long-buried in the coastal sediments of China,which facilitates further study on the historical occurrences of other harmful dinoflagellates and their relevance to the regional climate and environmental changes in China.

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（q RT-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 q RT-PCR to the HAB-forming group. In this study, six candidate reference genes,18 S 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 q RT-PCR and three statistical algorithms（Ge Norm, Norm Finder, and Best Keeper） 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, 18 S 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, 18 S 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 q RT-PCR in gene analysis of dinoflagellates and help to design primers targeting orthologous genes in other algal species.