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.

First record of Biecheleria cincta (Dinophyceae) from Chinese coasts, with morphological and molecular characterization of the strains

The presence of Biecheleria cincta (=Woloszynskia cincta) in the Chinese coasts is reported for the first time. In scanning electron microscope, three to five series of vesicles and an elongated apical vesicle (EAV) were visible in the epicone, and both the hypocone and the cingulum had three series of vesicles each. Thin sections revealed that B. cincta possesses stalked pyrenoids and an unusual eyespot consisting of a stack of cisternae with brick-like materials (type E), thus supporting its transfer from Woloszynskia to Biecheleria. Spiny cysts formed spontaneously in culture, with an encystment rate of around 20%. Both large subunit ribosomal DNA (LSU rDNA) and internal transcribed spacer region (ITS) sequences in 12 strains from the Chinese coasts were determined. Phylogenetic analyses based on LSU rDNA and ITS sequences using Bayesian inference and maximum likelihood revealed two distinct ribotypes (referred to as ribotype A and B) in B. cincta. ITS region pairwise distances within B. cincta ranged from 0.024 to 0.072, suggesting the existence of a complex of cryptic species.

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.

四种海洋藻华甲藻与红色赤潮藻的化学互感作用(allelopathy)初步研究

化学互感作用或化感作用(allelopathy)在浮游植物的种群相互作用及动力学过程中起着重要的调节控制作用,已经成为有害藻华生态学研究中的重要领域。但在被研究的种类和化感作用的规律等方面还存在众多空白。本研究选取分离自中国和美国近海的重要藻华甲藻多环旋沟藻(Cochlodinium polykrikoides)、米氏凯伦藻(Karenia mikimotoi)、剧毒卡尔藻(Karlodinium veneficum)、短沟别什莱藻(Biecheleria brevisulcate),在相同的试验条件下(72 h共培养)观察其与另一种常见的藻华甲藻红色赤潮藻(Akashiwo sanguinea)的化感作用。实验结果显示化感效应随种类和特别是起始细胞密度比率而分别表现为抑制或促进共存种的生长。与起始细胞密度为500个/m L的红色赤潮藻共培养时,多环旋沟藻起始密度为250个/m L时促进红色赤潮藻生长,米氏凯伦藻苏澳株和短沟别什莱藻在细胞密度低于1 000个/m L时促进红色赤潮藻生长,同时起始密度为5 000~50 000个/m L的剧毒卡尔藻被红色赤潮藻促进生长。但是,米氏凯伦藻深圳湾株在细胞密度500个/m L以上时均抑制红色赤潮藻生长,多环旋沟藻在起始密度大于250个/m L时开始抑制红色赤潮藻生长,且抑制效应在起始密度2 500个/m L时接近100%。米氏凯伦藻的化感作用在不同株系间表现出显著差异。结果说明有害藻华甲藻之间的化感作用表现出复杂的形式,随着作用的种类和互相之间的密度比率变化而有互相促进、抑制或一方抑制(或促进)另一方等作用,这对进一步认识藻华甲藻的种群动力学具有重要意义。