SpALF4: A newly identified anti-lipopolysaccharide factor from the mud crab Scylla paramamosain with broad spectrum antimicrobial activity

Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with binding and neutralizing activities to lipopolysaccharide (LPS) in crustaceans. This study identified and characterized a novel ALF homolog (SpALF4) from the mud crab Scylla paramamosain. The complete cDNA of SpALF4 had 756 bp with a 381 bp open reading frame encoding a protein with 126 aa. The deduced protein contained a signal peptide and a LPS-binding domain. SpALF4 shared the highest identity with PtALF5 at amino acid level but exhibited low similarity with most of other crustacean ALFs. Furthermore, different from the previously identified three SpALF homologs and most of other ALFs, SpALF4 had a low isoelectric point (pI) for the mature peptide and the LPS-binding domain with the values of 6.93 and 6.74, respectively. These results indicate that SpALF4 may be a unique ALF homolog with special biological function in the mud crab. Similar to the spatial structure of ALFPm3, SpALF4 contains three α-helices packed against a four-strand β-sheet, and an amphipathic loop formed by a disulphide bond between two conserved cysteine residues in LPS-binding domain. SpALF4, mainly distributed in hemocytes, could be upregulated by Vibrio harveyi, Staphylococcus aureus, or white spot syndrome virus. Recombinant SpALF4 could inhibit the growth of Gram-negative bacteria (V. harveyi, Vibrio anguillarum, Vibrio alginolyticus, Aeromonas hydrophila, Pseudomonas putida), Gram-positive bacteria (S. aureus and Bacillus megaterium), and a fungus Candida albicans to varying degrees. Further study showed that it could also bind to all the aforementioned microorganisms except S. aureus. These results demonstrate that SpALF4 is a unique ALF homolog with potent antimicrobial activity against bacteria and fungi. This characteristic suggests SpALF4 plays an essential function in immune defense against pathogen invasion in mud crab.     

Polymorphisms of anti-lipopolysaccharide factors in the swimming crab Portunus trituberculatus and their association with resistance/susceptibility to Vibrio alginolyticus

Anti-lipopolysaccharide factor (ALF) is an important antimicrobial peptide (AMP) that can bind and neutralize major component of Gram-negative bacteria cell wall, lipopolysaccharide (LPS). Seven isoforms of anti-lipopolysaccharide factors (PtALF1-7) were previously identified from the swimming crab Portunus trituberculatus in our laboratory. Here, polymorphisms of PtALF1-7 were detected and their association with resistance/susceptibility to Vibrio alginolyticus (a main Gram-negative bacteria causing high mortality in P. trituberculatus) were investigated. We identified 127, 96, 103, 53 and 158 single nucleotide polymorphisms (SNPs) in genomic fragments of PtALF1-3, PtALF4, PtALF5, PtALF6 and PtALF7, respectively. Among them, totally sixteen SNPs were significantly associated with resistance/susceptibility to V. alginolyticus (P < 0.05). Of these sixteen SNPs, most were located in introns and noncoding exons, while two synonymous SNPs and one nonsynonymous SNP were in coding exons. Additionally, simple sequence repeats (SSRs) were only identified in introns and noncoding exons of PtALF4, PtALF5 and PtALF7. Although no significant difference of allele frequencies was found, these SSRs had different polymorphic alleles according to the repeat number between susceptible and resistant stocks. After further confirmation, polymorphisms investigated here might be applied as potential molecular markers for future selection of resistant strains to diseases caused by Gram-negative bacteria.     

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.     

A C-type lectin like-domain (CTLD)-containing protein (PtLP) from the swimming crab Portunus trituberculatus

C-type lectins play important roles in the non-self innate immune system of invertebrates. In this study, we isolated the full-length cDNA of the C-type lectin like-domain (CTLD)-containing protein, designated PtLP, from the hepatopancreas of the swimming crab Portunus trituberculatus, one of the most common edible crabs of East Asia. The PtLP cDNA consists of 923bp and encodes a polypeptide of 164 amino acids containing a well-conserved C-type lectin like-domain (CTLD). The deduced amino acid sequence of PtLP shows 29-36% amino acid sequence identity to other crustacean C-type lectin sequences. A phylogenetic analysis revealed that PtLP is in a large cluster together with black tiger shrimp PmAV, a gene involved in virus resistance of shrimp, and all of the C-type lectins from the various shrimps. Quantitative RT-PCR analysis showed that the PtLP mRNA was expressed highly in hepatopancreas and moderately in gills, hemocytes, and ovary of normal swimming crabs.     

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.     

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.     

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.     

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。     

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.