Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well.     

Isolation and characterization of venom from nematocysts of jellyfish Rhopilema esculentum Kishinouye

The present work is the first report of the biochemical characterization of the venom from nematocysts of the jellyfish Rhopilema esculentum Kishinouye. The nematocysts were isolated by autolysis and centrifugation and separated by flow cytometry. Four types of nematocysts were identified: mastigophores, euryteles, and atrichous and holotrichous isorhiza. SDS-PAGE and amino acid analyses demonstrated that most of the proteins in the nematocyst extract were between 10 kDa and 40 kDa, and that glutamic acid was the main amino acid. A hemolytic activity assay showed that the activity of the nematocyst venom (RNV) was strongest in Tris-HCl buffer (50 mmol/L, pH 7.8, 5% glycerol, 0.5 mmol/L EDTA, 0.1 mol/L NaCl). The hemolytic activity was related to protein concentration and the HU50 against chicken erythrocytes was 0.91 μg/mL.     

胡子鲇3个养殖群体遗传结构的AFLP分析

应用AFLP方法,从64对引物中筛选出5对用于分析胡子鲇(Claris fuscus)厦门(XM)、江门(JM)、玉林(YL)等3个养殖群体共90个个体群体内、群体间遗传多样性。扩增共得到289条清晰谱带,引物多态性介于47.8%～63.8%,平均多态性比率为56.4%。胡子鲇群体总遗传杂合度(Ht)、遗传分化系数(Hs)、遗传分化度(Gst)和基因流(Nm)分别为0.161 1、0.153 6、0.051 1和8.501 8,表明胡子鲇多态性处于中等偏下水平,群体内部遗传分化系数较高,三个群体间无较大分化,群体间基因流动较好。其中,JM群体多态性比率、有效等位基因数Ne、Nei’s指数和Shannon指数都较XM群体和YL群体高,种内分化程度较好。对群体进行两两比较发现,XM群体和YL群体间分化程度最大,XM群体和JM群体次之,JM群体和YL群体最小。基于杂交优势,推断XM和YL群体繁殖所产生的后代将具有比亲代更为优良的性状。     

Inbreeding and genetic diversity analysis in a hatchery release population and clones of Rhopilema esculentum based on microsatellite markers

Ten microsatellite markers were used to analyze the levels of genetic diversity and inbreeding in a hatchery release population of Rhopilema esculentum Kishinouye(Scyphozoa: Rhizostomatidae). A total of 85 alleles were detected in 600 individuals. Within-population levels of observed( H o) and expected( H e) heterozygosity ranged from 0.152 to 0.839(mean=0.464) and from 0.235 to 0.821(mean=0.618), respectively. The polymorphism information content(PIC) of each marker ranged from 0.207 to 0.795 with an average of 0.580, indicating that the hatchery population maintained a high level of genetic diversity. Inbreeding levels were estimated in the hatchery population and the inbreeding coefficient was 0.203. This result revealed that a certain level of inbreeding occurred within the population. Meanwhile, we also determined genetic diversity at the clone level. Several polyps from the same scyphistomae were genotyped at the ten microsatellite loci and there was virtually no difference in their genotypes. Furthermore, we calculated the probabilities of exclusion. When both parents were known, the average exclusion probability of ten loci was 99.99%. Our data suggest that the ten microsatellite markers can not only be used to analyze the identity of individuals but they can also be applied to parentage identification. Our research provides a theoretical basis and technical support for genetic diversity detection and reasonable selection of R. esculentum hatchery populations. These findings support the use of releasing studies and conservation of R. esculentum germplasm resources.     

军曹鱼群体遗传结构的AFLP分析

筛选出10对扩增片段长度多态分析技术(Amplified fragment length polymorphism,AFLP)的选择性扩增引物(E-aga/M-cgt、E-aga/M-cga、E-agc/M-cga、E-agg/M-cga、E-act/M-cgc、E-aag/M-cgc、E-aca/M-cga、E-aac/M-cgc、E-aac/M-cac和E-aac/M-cat),分析了4个军曹鱼(Rachycentron canadum)全人工繁育群体海南1(HN1)、海南2(HN2)、湛江(ZJ)和流沙湾(LS)的遗传结构。结果表明:总体多态位点比例(83.9%)、分化指数(0.427 8)和Shannon信息指数(0.614 9)均处于较高水平,遗传多样性丰富;主坐标分析和邻位连接树中LS-HN2支与ZJ-HN1支明显分离,各群体内多样性水平存在差异(遗传多样性大小依次为HN1、HN2、LS、ZJ),但总体基因流仍然较大(Nm=10.327),分子方差分析(AMOVA)获得的群内方差(53%)也略大于群间(47%)。     

Analysis of the genetic diversity and differentiation of <Emphasis Type="Italic">Fenneropenaeus penicillatus</Emphasis> populations using AFLP technology

Fenneropenaeus penicillatus (redtail shrimp) is an important marine commercial animal in China. Recently, its resources have been depleted rapidly as a result of, for example, over-exploitation and environmental degradation of spawning grounds. Therefore, we analyzed the genetic diversity and differentiation of nine wild populations of F. penicillatus of China (Ningde, Lianjiang, Putian, Xiamen, Quanzhou, Zhangpu, Dongshan, Nanao, and Shenzhen populations) by amplified fragment length polymorphism (AFLP) technology, to provide genetic information necessary for resource protection, rejuvenation, artificial breeding, and sustainable use of the resource. Eight AFLP primer pairs were used for amplification, and 508 bands were detected among the populations. The results show that the percentage of polymorphic loci (P) ranged from 41.34% to 63.58%; the Nei’s gene diversity (H) of the populations was 0.119 4-0.230 5; and Shannon’s Information Index (I) was 0.184 1-0.342 5. These genetic data indicate that the genetic diversity of F. penicillatus was high. The genetic differentiation coefficient (GST=0.216 2) and gene flow (Nm=1.812 4) show that there was a high level of genetic differentiation and a moderate level of gene flow among populations. More studies on the genetic differentiation mechanism of F. penicillatus along the south-eastern coast of China need to be conducted to find more effective scientific protection strategies for the conservation of F. penicillatus genetic resources.     

Preliminary analysis of population genetic diversity of cultivated <Emphasis Type="Italic">Laminaria japonica</Emphasis> sporophyte via AFLP technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng, China. Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis. The parameters of the method were optimized. Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer. Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers. A total of 21 loci were obtained and 17 of them were polymorphic. The mean percent age of polymorphic loci of this population was 80.95%. The Nei’s gene diversity (H) within this population was 0.3028 and the average Shannon’s Information index (I) was 0.4498. A genetic distance matrix among different individuals was constructed as well. Through this study, an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established. The results of this research also revealed a high level of genetic diversity within the studied population.     

AFLP analysis of genetic variation among three natural populations of horseshoe crab Tachypleus tridentatus along Chinese coast

The AFLP(amplified fragment length polymorphism) technique was used to analyze and compare the genetic diversity of Tachypleus tridentatus from three south-eastern coastal sites of China(Pingtan,Hong Kong and Beihai).Eight pairs of primers generated 361 loci,including 285 polymorphic loci.The ratio of polymorphic loci was 96.97%.Nei's genetic diversity index was 0.420 8 and the Shannon information index was 0.607 5,both of which were higher than that reported for many other arthropods.These results show tha...     

Genetic diversity in two Japanese flounder populations from China seas inferred using microsatellite markers and COI sequences

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea (Qingdao population, QD) and East China Sea (Zhoushan population, ZS) using 10 polymorphic microsatellite loci and cytochrome c oxidase subunit I (COI) sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data (F_st =0.048 7, P<0.001) and mitochondrial DNA data (F_st =0.128, P<0.001) both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.     

Comparative analysis of methods for concentrating venom from jellyfish Rhopilema esculentum Kishinouye

In this study, several methods were compared for the efficiency to concentrate venom from the tentacles of jellyfish Rhopilema esculentum Kishinouye. The results show that the methods using either freezing-dry or gel absorption to remove water to concentrate venom are not applicable due to the low concentration of the compounds dissolved. Although the recovery efficiency and the total venom obtained using the dialysis dehydration method are high, some proteins can be lost during the concentrating process. Comparing to the lyophilization method, ultrafiltration is a simple way to concentrate the compounds at high percentage but the hemolytic activities of the proteins obtained by ultrafiltration appear to be lower. Our results suggest that overall lyophilization is the best and recommended method to concentrate venom from the tentacles of jellyfish. It shows not only the high recovery efficiency for the venoms but high hemolytic activities as well. Supported by the Award Foundation of Scientific Research for Excellent Young and Middle-age Scientist of Shandong Province (No. 2006BS07003) and the Knowledge Innovation Project of Chinese Academy of Sciences (KZCX2-YW-R-104)     

Genetic analysis of floating Enteromorpha prolifera in the Yellow Sea with AFLP marker

Extremely large accumulation of green algae Enteromorpha prolifera floated along China’coastal region of the Yellow Sea ever since the summer of 2008.Amplified Fragment Length Polymorphism (AFLP) analysis was applied to assess the genetic diversity and relationships among E. prolifera samples collected from 9 affected areas of the Yellow Sea.Two hundred reproducible fragments were generated with 8 AFLP primer combinations,of which 194 (97%) were polymorphic. The average Nei’s genetic diversity, the coefficiency of genetic differentiation (Gst), and the average gene flow estimated from Gst in the 9 populations were 0.4018, 0.6404 and 0.2807 respectively. Cluster analysis based on the unweighed pair group method with arithmetic averages (UPGMA) showed that the genetic relationships within one population or among different populations were all related to their collecting locations and sampling time. Large genetic differentiation was detected among the populations.The E. prolifera originated from different areas and were undergoing a course of mixing.     

Genetic diversity and population structure of Eleutheronema rhadinum in the East and South China Seas revealed in mitochondrial COI sequences

Eleutheronema rhadinum is a potential commercial fisheries species and is subject to intense exploitation in China. Knowledge on the population structure of E. rhadinum in Chinese coastal waters, which is important for sustainable exploitation and proper resource management, is lacking. In the present study, the genetic diversity and population structure of E. rhadinum were evaluated using a 564-base pair fragment of the mitochondrial cytochrome c oxidase subunit I （COl） gene. A total of 76 specimens were collected from three localities around the East （Qidong and Zhoushan） and South China Seas （Zhuhai）. Among these individuals, nine polymorphic sites were detected and 1 l distinct haplotypes were defined. High levels ofhaplotype diversity （h=0.759i0.035） and low levels of nucleotide diversity （re=0.001 98i0.003 26） were observed in these populations, Hierarchical analysis of molecular variance （AMOVA） indicated that 96.72% of the genetic variation occurred within the populations, whereas 3.28% occurred among populations. No significant genealogical branches or clusters were recognized on the neighbor-joining tree. Intra-group variation among populations was significant （~0~t=0.032 85, P〈0.01）. These results suggest that E. rhadinum populations in the East and South China Seas have developed divergent genetic structures. Tests of neutral evolution and mismatch distribution suggest that E. rhadinum may have experienced a population expansion. The present study provides basic information for the conservation and sustainable exploitation of this species.     

Genetic diversity analysis with ISSR PCR on green algae Chlorella vulgaris and Chlorella pyrenoidosa

In the present study, genetic polymorphism and diversity in unicellular clones of Chlorella vulgaris Beijerinck and Chlorella pyrenoidosa Chick were studied with Inter Simple Sequence Repeats PCR (ISSR PCR). Samples including four clones of C. vulgaris and three clones of C. pyrenoidosa were purified by single-clone-choice method. For four C. vulgaris unicellular clones, the total number of the bands scored for 18 primers was 298; and the number of the polymorphic bands was 118, of which 39.6% were polymorphic. The size of PCR products ranged from 200 to 2 500 bp. The total number of bands scored for 18 primers, the number of polymorphic bands and the percentage of three C. pyrenoidosa unicellular clones was 194.83 and 30.8%, respectively. POPGENE analysis show that the average Nei genetic diversity (h *) and Shannon index of diversity (I *) in the four C. vulgaris unicellular clones was 0.2181 and 0.3208, respectively, which is slightly higher than those of the three C. pyrenoidosa unicellular clones (0.190 3 and 0.274 8), which agreed with the percentage of polymorphic bands in the mixed samples of the two species. The results suggest that ISSR is a useful method to Chlorella for intra-species genetic analysis.     

Genetic analysis of four populations of redlip mullet (Chelon haematocheilus) collected in China seas

Horizontal starch gel electrophoresis was used to investigate genetic structures of four populations (Dandong, Rizhao, Ningbo and Guangzhou) of redlip mullet (Chelon haematocheilus). Fourteen loci of ten enzymes (G3PDH, IDHP, LAP, LDH, MDH, MPI, PGDH, PGM, SDH, and SOD) were identified. The proportions of polymorphic loci varied from 0.07 to 0.14. The average observed and expected heterozygosities ranged from 0.02 to 0.05 and from 0.02 to 0.04 respectively among the 4 populations. The average number of efficient alleles of each locus varied from 1.02 to 1.06. The genetic distance among populations was from 0.0004 to 0.0021. The genetic distances between Dandong, Rizhao and Ningbo populations were small, while those between Guangzhou population and other three populations were relatively high. The result indicated the possible divergence of redlip mullet between the South China Sea and the other sea areas of China due to geographic isolation.     

Genetic Variation and Differentiation in Wide Ranging Populations of Razor Clam （Sinonovacula constricta） Inferred from AFLP Markers

The genetic variation and differentiation of the razor clam Sinonovacula constricts distributed along the coast of China were studied through amplified fragment length polymorphism(AFLP)analysis. Six primer combinations generated 193 fragments. The He values varied from 0. 322 to 0. 463 and the percentage of polymorphic loci ranged from 74. 1% to 98. 4%, which indicates a high level of genetic diversity. Cluster analysis by Nei's pairwise distance grouped all specimens by geographical origins. AMOVA consistently showed that genetic variation among populations was 8. 71%, and most of the variation came from the genetic variation within populations(91.29%). Genetic differentiation among the six populations was moderate; pairwise FST ranged from 0. 0282 to0. 1480, which indicated that S. Constricts populations along the coast of China are genetically connected. Among all the six populations, the Beihai population is the mostly differentiated from the others, suggesting that Hainan Island and Leizhou Peninsula act as barriers to gene flow. All populations abide isolation by distance model as indicated by Mantel test, except for ZS(Zhoushan)and YQ(Yueqing)populations. Information obtained in this study will provide guidelines for conservation and fishery management of this species in the future.     

Genetic differentiation of Japanese sardinella (Sardinella zunasi) populations in the Northwest Pacific revealed by ISSR analysis

Knowledge of population genetic structure plays an important role in fisheries management.In this research,In-ter-Simple-Sequence-Repeat(ISSR)markers were employed to evaluate the genetic structure of Japanese sardinella(Sardinella zun-asi)populations in the Northwest Pacific.Eighty seven individuals from 5 locations were screened using 4 highly polymorphic prim-ers.A total of 173 polymorphic loci were detected out of 191 loci amplified.Small but significant genetic differentiation was de-tected between the Chinese and Japanese populations by both AMOVA and pairwise FST analyses,which was further supported by cluster analysis.We consider that climate change during glaciations should be responsible for the genetic differentiation.Isolation by geographic distance among populations was observed,indicating that the distance might also lead to the genetic differentiation.However,no genetic structure was found within the populations off both the Chinese and Japanese coasts,indicating a high-level along-coast gene flow,which might result from ocean current transport and common ground for over-wintering.     

Isolation, cloning and sequencing of AFLP markers related to disease-resistance traits in Fenneropenaeus chinensis

Amplified fragment length polymorphisms （AFLP） technique was used to analyze the fingerprinting of four successive generations of Fenneropenaeus chinensis to reveal their disease-resistance traits. Some loci showed quite different genetic frequencies due to artificial selection, which implied that these fragments were putative markers related to the disease-resistance trait. We developed a simple and effective method to further characterize these AFLP fragments. Specific AFLP bands were cut directly from polyacrylamide gels, re-amplified, cloned and sequenced. Eight putative genetic markers were sequenced and their sizes ranged from 63 to 209 bp. The sequences were submitted to dbGSS （database of Genome Sequence Survey）; and the BLAST analysis showed low similarity to the function genes, indicating these markers were tightly linked to a disease-resistance trait but were not functional genes.     

GENETIC DIVERSITY OF THE WILD AND REARED PSEUDOSCIAENA CROCEA

The genetic diversity of both wild and reared Pseudosciaena crocea (Richardson) col-lected from Guan-Jing-Yang in Ningde, China in May 1999 was investigated by random amplified poly-morphic DNA (RAPD) in the present study. The polymorphism and mean difference of the wild popula-tion as revealed by RAPD were 18.9% and 0.0960 respectively, and those of the reared stocks were rel-atively lower, with 16.7% in polymorphism and 0.0747 in mean difference. The genetic distance be-tween the two stocks was 0.0041. From the comprehensive investigation, the main reasons for the loss of genetic diversity were probably overilshing, small number of parents as broodstocks and the debatable arti-ficial ranching. Results from this study also showed that the large yellow croaker populations distributed along Fujian coastal waters including Guan-Jing-Yang still potentially wide genetic variability. It is sug-gested that genetic management and prevention should be scientifically conducted in order to maintain and improve the genetic diversity of the P. crocea population.