Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.     

Influence of temperature and salinity on germination of eelgrass (Zostera marina L.) seeds

Seagrass restoration as part of ocean ecosystem protection has been launched for many years all over the world, but intensive research on this subject in China has just begun in recent years. Seed broadcasting has been widely accepted as the most potentially useful method for seagrass restoration over large areas. We examined the influence of key environmental factors on seed germination to help promote eelgrass bed restoration. Under anoxic conditions, the influence of temperature and salinity on the germination rate of eelgrass (Zostera marina L.) seeds was examined at different combinations of four temperatures (4, 9, 14, and 24°C) and nine salinities (5 to 45, increment of 5). The effect of significant interaction of temperature and salinity on germination rate was observed (ANOVA) (P<0.001). The highest germination rate (83.3 ± 3.5)% was reached in 8 weeks at 14°C and salinity 5. Higher temperature significantly increased the germination rate at salinity 5 (P<0.001) during the whole observation period except for 24°C, while lower salinity significantly increased the germination rate at 14°C (P<0.001). Although significant interaction was found between temperature and salinity (P<0.001), the influence of salinity was stronger than that of temperature for the germination of eelgrass seeds. These results provide useful information for the propagation of artificial seedlings for seagrass restoration in China.     

Fatty acid composition analyses of the DCMU resistant mutants of <Emphasis Type="Italic">Nannochloropsis oculata</Emphasis> (eustigmatophyceae)

Ultraviolet mutagenesis was applied to Nannochloropsis oculata and three mutants resistant to 3-(3, 4-dichlorophenyl)-1,1-dimethylurea (DCMU) were isolated. The cellular chlorophyll a and total lipid content of the wild are higher in the medium supplemented with DCMU than in the control without DCMU. Without DCMU, the growth rates and chlorophyll a contents of the mutants are similar to those of the wild. Significant changes of fatty acid content and composition have occurred in DCMU-resistant mutants growing in the medium supplemented with DCMU. The total lipid, palmitic acid (16:0), palmitoleic acid (16:1ω9) and oleic (18:1ω9) contents decrease significantly, while the vaccenic acid (18:1ω11) increases significantly and the EPA content of dried powder increases slightly in the mutants. The study may provide a basis to improve EPA content in Nannochloropsis oculata in the future.     

Predicting the reproduction strategies of several microalgae through their genome sequences

Documenting the sex and sexual reproduction of the microalgae is very difficult, as most of the results are based on the microscopic observation that can be heavily influenced by genetic, physiological and environmental conditions. Understanding the reproduction strategy of some microalgae is required to breed them in large scale culture industry. Instead of direct observation of sex and sexual reproduction under microscope, the whole set or the majority of core meiosis genes may evidence the sex and sexual reproduction in the unicellular algae, as the meiosis is necessary for maintaining the genomic stability and the advantages of genetic recombination. So far, the available genome sequences and bioinformatic tools(in this study, homolog searching and phylogenetic analysis) allow us to propose that at least 20 core meiosis genes(among them ≥6 must be meiosis specific) are enough for an alga to maintain its sexual reproduction. According to this assumption and the genome sequences, it is possible that sexual reproduction was carried out by Micromonas pusilla and Cyanidioschyzon merolae, while asexual reproduction was adopted by Bigelowiella natans, Guillardia theta, Nannochloropsis gaditana, N. oceanica, Chlorella variablis, Phaeodactylum tricornutum and Thalassiosira pseudonana. This understanding will facilitate the breeding trials of some economic microalgae(e.g., N. gaditana, N. oceanica, C. variablis and P. tricornutum). However, the reproduction strategies of these microalgae need to be proved by further biological experiments.     

Cryopreservation of Gametophytes of Laminaria japonica （Phaeophyta） Using Encapsulation-Dehydration with Two-Step Cooling Method

Gametophytes of Laminaria japonica were cryopreserved in liquid nitrogen using encapsulation-dehydration with two-step cooling method. Gametophytes cultured at 10℃ and under continuous irradiance of 30 μmol m-2 s-1 for 3 weeks were encapsulated in calcium alginate beads. The beads were dehydrated in 0.4 molL-1 sucrose prepared with seawater for 6 h, desiccated in an incubator set at 10℃ and 70% relative humidity for 4 h, pre-frozen at either -40℃ or -60℃ for 30 min, and stored in liquid nitrogen for ＞24 h. As high as 43% of survival rate was observed when gametophytes were thawed by placing the beads in 40℃seawater and re-hydrated in 0.05 molL-1 citrate sodium prepared using 30‰ NaC1 7 d later. More cells of male gametophytes survived the whole procedure in comparison with female gametophytes. The cells of gametophytes surviving the preservation were able to grow asexually and produce morphologically normal sporophytes.     

FloatingEscherichia coli by Expressing Cyanobacterial Gas Vesicle Genes

Gas vesicles are hollow, air-filled polyprotein structures that provide the buoyancy to cells. They are found in a variety of prokaryotes. In this study, we isolated a partial gas vesicle protein gene cluster containing gvp A and gvp C20Ψ from Planktothrix rubescens, and inserted it into an expression vector and expressed it in E. coli. The gas vesicle was developed in bacterial cells, which made bacterial cells to float on medium surface. We also amplified gvp A and gvp C20Ψ separately and synthesized an artificial operon by fusing these two genes with the standardized gene expression controlling elements of E. coli. The artificial operon was expressed in E. coli, forming gas vesicles and floating bacteria cells. Our findings verified that the whole set of genes and the overall structure of gas vesicle gene cluster are not necessary for developing gas vesicles in bacteria cells. Two genes, gvp A and gvp C20Ψ, of the gas vesicle gene cluster are sufficient for synthesizing an artificial operon that can develop gas vesicles in bacteria cells. Our findings provided a wide range of applications including easing the harvest of cultured microalgae and bacteria, as well as enriching and remediating aquatic pollutants by constructing gas vesicles in their cells.