Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab Eriocheir sinensis

Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture crustacean species in China. A cDNA library was constructed from mixed tissues of E. sinensis challenged with LPS. Eight genes involved in immune response were identified from 319 single colonies. Among them, two different C-type lectin-like domain (CTLD)-containing proteins were firstly identified in Chinese mitten crab. The full-length cDNA sequences of two C-type lectin-like domain (CTLD)-containing proteins named EsCTLDcp-1 and EsCTLDcp-2 were cloned by 5' RACE. The deduced amino acid sequences of EsCTLDcp-1 and EsCTLDcp-2 possessed several conserved features of C-type lectin subfamily. The tissue distribution of EsCTLDcp-1 and EsCTLDcp-2 was examined by Real-time PCR. In the normal Chinese mitten crab, the expression of EsCTLDcp-2 was detected in all tested tissues such as haemolymph, muscle, intestine, gill, heart, gonad and hepatopancreas, whereas in muscle, intestine, gill, heart and hepatopancreas for EsCTLDcp-1. The highest expressions of EsCTLDcp-1 and EsCTLDcp-2 were both observed in hepatopancreas. LPS significantly induced the expression of EsCTLDcp-1 and EsCTLDcp-2 in the hepatopancreas at the different time points. The induced fold change of EsCTLDcp-1 and EsCTLDcp-2 increased significantly from 2 h for EsCTLDcp-1 and 4 h for EsCTLDcp-2, and reached a maximum at 12 h, then dropped at 24 h. A differential pattern was found in Chinese mitten crab challenged with Chinese mitten crab pathogen Aeromonas hydrophila. The expression of EsCTLDcp-1 increased significantly at 2 h post-challenge crabs with A. hydrophila, then decreased at 4 h and 8 h, after that increased at 12 h and 24 h. The expression of EsCTLDcp-2 was decreased at the all time points. All these data suggest a differential role of EsCTLDcp-1 and EsCTLDcp-2 in the crab innate immune response to bacterial infection.     

Genetic variation and population structure of swimming crab (Portunus trituberculatus) inferred from mitochondrial control region

Genetic variation and population structure in Portunus trituberculatus along the coast of China were revealed according to 617 bp of mitochondrial DNA control region. 90 polymorphic sites defined 53 distinct haplotypes, showing a moderately high diversity among 72 individuals sampled from eight localities. Neighbor-joining tree, statistics analyses of gene flow and genetic differentiation index indicated two populations from Beihai and Laizhou had differentiated. The population from Yingkou, Dandong, Laizhou and Beihai had smaller genetic diversity compared to that from Ningbo, Lianyungang, Qingdao and Japan according to the genetic distance. And mantel test showed significant positive correlation between genetic distance and geographic distance for P. trituberculatus. TCS parsimony network suggested that all the animals sampled were probably the result of recent divergence from a common ancestral haplotype but for Laizhou population. Moreover, the haplotype distribution appeared to correlate with a recent colonization followed by localized genetic differentiation. Mismatch distribution results suggested that Ningbo, Yingkou, Qingdao, Lianyungang and Japan populations, particularly Dandong population had experienced a sudden demographic or spatial expansion. The Pleistocene glaciations might contribute to this process.     

Biocontrol method in aquaculture for rearing the swimming crab larvae Portunus trituberculatus

The aim of this work is to control the waterenvironment for culturing larvae of the swimmingcrab, Portunus trituberculatus, using microorganisms.The bacterial strain PM-4 (Thalassobacter utilis)improved the survival rate of crab larvae andrepressed the growth of Vibrio anguillarum (bacterium)and Haliphthoros sp.(fungus) in seawater. PM-4 wascultured and added daily to seawater during the firstto third zoean growth stage of the crab with diatomsand rotifers. Numbers of PM-4 decreased in culturewater during the first 3 days, because of feeding bythe first zoean stage of larvae. The finalconcentration of PM-4 was 10⁵ to 10⁶ cellsml^–1according to the plate count method in larval rearingwater.During 1989 to 1993, we tried seed productions of aswimming crab in 200 m³ containers at TamanoStation, Japan Sea-Farming Association. In 33trials of the biocontrol methods, average survivalrate of crab larvae was 28.3% when the bacterialstrain PM-4 was added. In 42 trials in which the strainPM-4 was not added, average survival rate of crab larvae was15.6%. We conclude that thebacterial strain PM-4 is effective as a biocontrolagent.     

Multiple isoforms of immune-related genes from hemocytes and eyestalk cDNA libraries of swimming crab Portunus trituberculatus

Expressed sequence tags (ESTs) analysis has been shown to be an efficient approach not only for gene discovery, but also for gene expression profiles performance. Two full-length enriched cDNA libraries were constructed from hemocytes and eyestalk of Portunus trituberculatus, respectively, and randomly sequenced to collect genomic information and identify genes involved in immune defense response. A total of 99 unigenes including 64 unigenes (6.00% of 1066 unigenes) in hemocytes library and 35 unigens (6.86% of 510 unigenes) in eyestalk library are identified to be immune genes. These genes are categorized into six classes, viz. antimicrobial peptides, redox proteins, melanization related proteins, chaperone proteins, clottable proteins and other immune factors. The content and category of immune genes in eyestalk library indicate eyestalk might have unrecognized role in crab immunity. Five immune genes containing multiple protein isoforms are identified and characterized, including anti-lipopolysaccharide factor (PtALF1-7), crustin (PtCrustin1-3), thioredoxin (PtTrx1-2), clip domain serine proteinase (PtcSP1-5) and kazal-type proteinase inhibitor (PtKPI1-4). Sequence alignment and phylogenetic analysis reveal PtALF1-7 contain two conserved cysteine residues and might be encoded by multiple genomic loci. PtCrustin1-3 share the consensus cysteine motif and are considered as Type I crustins. PtTrx1 possesses the critical structural cysteine residue C?3 of Trx-1, while PtTrx2 has the N-terminal mitochondrial translocation signal of Trx-2. Sequence analysis shows PtcSP1-5 contain one clip domain and one partial SP catalytic triad domain. PtKPI1-4 present one typical Kazal domain consisting of six conserved cysteine residues. Some protein isoforms are tissue-specific, which might suggest they have different origins and perform diverse functions. Except PtALF1-3 and PtCrustin1, the other isoformes in this study are firstly identified from P. trituberculatus. Especially, PtTrx2 are firstly identified from crustaceans. Our research will provide useful genomic information of P. trituberculatus and be helpful in understanding the molecular mechanisms of crab immunity.     

三疣梭子蟹精子顶体反应过程中的形态和结构变化

用离子载体A2 3187和卵水人工诱导三疣梭子蟹精子的顶体反应 ,分别获得 75 33%和 84 83%的顶体反应率。应用光镜和电镜技术观察了顶体反应前后精子形态和结构的变化。未处理精子呈陀螺形 ,由顶体、核杯和 5 - 10条核辐射臂组成。顶体包括顶体囊和顶体管。顶体囊的伞形头帽拥有约 70条辐射肋。连续发生的精子顶体反应过程被人为地分为四个阶段 :(1)头帽鼓起 ;(2 )顶体囊外翻 ;(3)穿孔器前伸 ,顶体囊膜翻转 ;(4 )顶体囊膜脱落 ,顶体丝形成。直到第四阶段才观察到钉状精子的辐射臂开始收缩。探讨了辐射臂和穿孔器前冲在精子入卵中的功能     

三疣梭子蟹精子顶体反应前后胞内Ca~(2+)的变化

应用激光扫描共聚焦显微镜(LSCM)和Fluo-3/AM荧染技术对三疣梭子蟹精子顶体反应前后的胞内Ca2 变化进行了观察和检测.结果显示,在精子顶体反应过程中,胞内Ca2 主要分布在细胞核、穿孔器和胞质膜残存处,胞内Ca2 浓度([Ca2 ]I)总体上呈现先上升后下降的趋势.顶体反应前精子的平均荧光强度为35.95±5.71;穿孔器前伸、顶体囊膜翻转阶段精子的平均荧光强度为66.80±7.35;顶体囊膜脱落、顶体丝形成阶段精子的平均荧光强度为3.87±2.82;上述各阶段间精子荧光强度有极显著差异(P<0.01).顶体反应穿孔器前伸、顶体囊膜翻转阶段的精子相比顶体反应前精子,[Ca2 ]I显著提高;而在顶体囊膜脱落、顶体丝形成阶段,[Ca2 ]I则急剧下降,只在顶体丝基部胞质膜残存处有微量Ca2 存在.初步探讨了三疣梭子蟹精子顶体反应前后胞内Ca2 变化的功能.     

Characterization of 20 microsatellite loci by multiplex PCR in swimming crab, Portunus trituberculatus

The swimming crab, Portunus trituberculatus (Crustacea: Decapoda: Brachyura), is one of the most important fishery resources in East Asia. The aim of this work is to develop microsatellite multiplex PCR systems for 20 microsatellites which were isolated from a microsatellite-enriched genomic library and to explore the genetic diversity of P. trituberculatus captured in the coastal regions of the Yellow Sea in Korea. We prepared four multiplex PCR systems (two hexplex and tetraplex systems each) which could be used for the genotyping of 20 markers. Most markers in this system are highly informative with mean polymorphism information contents and observed heterozygosity of 0.913 and 0.930. The total number of alleles observed in this study was 724, and the mean allelic number per locus was 36.2. Significant deviations from the Hardy–Weinberg equilibrium (HWE) were observed at six loci in the Wonsando Island population, but all the loci deviated from the HWE in the Jeonjangpo population. The genetic differentiation between the two populations and the phylogenetic tree constructed by the unweighted pair group method with the arithmetic average suggested that there is no significant genetic difference between Wonsando Island and Jeonjangpo populations. The prepared multiplex PCR systems and population genetic data will be helpful to study phylogeographic analysis as well as to prepare strategies of stock management and aquaculture of P. trituberculatus.     

三疣梭子蟹卵附着机制及相关形态学特征

Using histological methods and scanning electron microscopy, the structure of cement gland and formation of outer egg-membrane and egg-stalk of the swimming crab Portunus trituberculatus were studied to explain the mechanism of egg-attachment and function of the cement gland. Eggs adhered to the setae of pleopodal basipodite and endopodite for hatching followed by spawning but no egg attached on the setae of exopodite. The setae of pleopodal basipodite and endopodite were smooth for egg attachment while the setae of exopodite were branched and suitable for collecting and protecting eggs. Cement gland distributed along longitudinal axis of the pleopodal basipodite and endopodite under epithelia cells. Cells of the cement gland were elliptical and its size was about 50 times of the epithelium. Some glue secreted to the surface of the pleopod along the seta, while the other glue secreted to the surface of the pleopod by the channel which across the integument of the pleopod. The first spawned egg was transferred to the setae that were far from the pore, not attached to the setae of the pleopodal basipodite and endopodite near the genital pore. And the egg attachment became nearer and nearer to the pore with further spawning. Since the eggs to glide rather than roll, there was almost no glue on the contrary surface of the egg at first: and with further secretion, the whole surface was surrounded by glue. The thick part of glue formed one short bud during the glide of the egg and gradually developed into a full egg-stalk. On the other hand, the else glue developed into an outer egg-membrane, which was “trichromatic egg membrane” originally. Each egg attached to the setae of pleopodal basipodite and endopodite with its own egg-stalk. The egg-stalk was a plane strap at the formation stage and later it rolled into a rope shape, but it could be reverted to the former by outside force. Four types of egg attachment : an egg to a seta, an egg to several setae, several eggs to a seta and several eggs to several setae were discovered. In summary, the cement gland existed in the pleopod of the swimming crab and formed outer egg membrane and egg-stalk during the egg attachment [Acta Zoologica Sinica 50 (5) : 873 - 879, 2004].     

三疣梭子蟹两种养殖模式浮游动物调查

于2012年6-12月,在宁波市象山县东盛水产养殖有限公司对主养三疣梭子蟹池塘两种混养模式（模式Ⅰ：三疣梭子蟹与日本对虾、黑鲷、菲律宾蛤仔混养;模式Ⅱ：三疣梭子蟹与日本对虾、黑鲷混养）浮游动物进行了调查与比较。结果显示：共检出浮游动物13属19种,其中,原生动物3属3种;轮虫2属5种;枝角类2属3种;桡足类6属8种。浮游动物平均密度模式Ⅰ（370 ind./L）〉模式Ⅱ（209 ind./L）;平均生物量模式Ⅰ（17.974 mg/L）〈模式Ⅱ（18.102 mg/L）。Margalef指数为0.22~0.81;Shannon-Wiener指数为1.28~3.03;Pielou均匀度指数为0.54~0.96,均值都是模式Ⅰ〉模式Ⅱ。     

Characterization of a fatty acid binding protein from the swimming crab Portunus trituberculatus and its effects on the composition of fatty acids in different tissues

Fatty acid binding proteins (FABPs) belonging to the lipocalin protein family, have an affinity for fatty acids (FAs) and participate in both the transport of FAs and lipid metabolism. In this study, a 909-bp full-length pt-FABP cDNA containing a 411-bp open reading frame (ORF) was obtained from the swimming crab Portunus trituberculatus. Quantitative real-time PCR revealed that during larval development, pt-FABP expression increased gradually from the zoea to the megalopa, and decreased in the juvenile crab. pt-FABP mRNA was detected at the highest level in the testis, followed by the muscles, with the lowest level in the hepatopancreas. The composition of fatty acids in three different tissues from crabs of different sex was detected by gas chromatography–mass spectrometry (GC-MS). The results indicated that the FA profile was in accordance with the expression of pt-FABP, which suggested that pt-FABP participates in the unsaturated fatty acid transportation and storage system of P. trituberculatus.