Characterization of new microsatellites selected from EST resources of Chinese mitten crab,Eriocheir sinensis

Sixteen new microsatellites were identified by screening 7533 expressed sequence tags of Chinese mitten crab, Eriocheir sinensis from GenBank data we published. They were polymorphic with the PIC value ranged from 0.349 to 0.957, the number of alleles ranged from 22 to 48, and the observed and expected heterozygosities ranged from 0.375 to 1.000 and 0.366 to 0.983, respectively. Five loci could be applicable to genetic diversity and population structure of E. sinensis.     

Eleven polymorphic microsatellites isolated from red swamp crayfish, Procambarus clarkii

Eleven microsatellites were isolated from a genomic DNA library enriched for CA- and GA-repeats, and were characterized in 48 individuals of red swamp crayfish Procambarus clarkii. All 11 microsatellites were polymorphic with an average of allele number of 6.6 per locus. Genotypic distributions of these 11 markers were found to be in conformance with the expectations of Hardy-Weinberg equilibrium. All 11 markers were unlinked. These markers are being used to study the invasion routine, genetic diversity and population structure of the species P. clarkii.     

Development and characterization of new microsatellite loci from Chinese mitten crab (Eriocheir sinensis)

A set of new microsatellite loci were isolated from Chinese mitten crab and characterized in two Chinese mitten crab populations. The number of the alleles ranged from 3 to 8 and observed and expected heterozygosity varied from 0.0870 to 0.9565 and 0.3092 to 0.8367 in each population, respectively. Loci HLJEsin1, HLJEsin4, HLJEsin30 in CLN population and HLJEsin6 in CJS population presented significant deviation from Hardy–Weinberg equilibrium (PHWE < 0.001). These markers will be useful for population studies.     

Isolation and characterization of polymorphic microsatellites in the Potato Tuber Moth Tecia solanivora (Povolny, 1973) (Lepidoptera: Gelechiidae)

Nine polymorphic microsatellite markers were isolated from Tecia solanivora, one of the most serious pests of potato tubers in Central and South America. As found in other studies of Lepidoptera, development of microsatellites is a difficult task: in our case, despite the large number of clones sequenced (796), of which 70 were unique, only nine loci were found to be both variable, and in Hardy-Weinberg equilibrium, No null alleles were detected. The loci were tested in three other co-occurring Gelechiidae species, one of which was variable. These loci will be used to provide a greater understanding of the genetic changes occurring during the invasive process in this species.     

A set of new microsatellite loci isolated from Chinese mitten crab,Eriocheir sinensis

Eriocheir sinensis has higher commercial value as food source than any other species of Eriocheir in China. To protect its germplasm resources and evaluate impact on local ecology, molecular tools to characterize the population structure and identify kinships from different water systems are highly needed. We have newly identified a set of 18 microsatellite markers with the observed number of alleles at each locus ranges from 8 to 30, the observed heterozygosity ranges from 0.140 to 0.900. These newly isolated markers increase the available molecular resources that can be used to analyse population structure of Chinese mitten crab.     

Isolation and characterization of microsatellites in Chinese soft‐shelled turtle,Pelodiscus sinensis

Fifteen polymorphic microsatellite loci were developed for the Chinese soft‐shelled turtle (Pelodiscus sinensis) from the (GT)_n microsatellite‐enriched genomic library, using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol. The polymorphism of all 15 loci ranged from two to seven alleles with observed heterozygosities ranging from 0.03 to 0.98 (mean 0.43) in one population of 40 individuals. These novel loci will be helpful for understanding the population structure at genetic level and marker‐assisted breeding of this vulnerable species.     

Effects of atrazine on osmoregulation in the Chinese mitten crab,Eriocheir sinensis

This study integrates results from acute contamination with atrazine of isolated perfused gills, and from in vivo chronic contamination of euryhaline Chinese mitten crabs, Eriocheir sinensis, acclimated to freshwater. Atrazine 1 mg/l in contact with the basolateral membrane (IN) increases the transepithelial potential difference (TEP) from -20.8 +/- 4.9 to -29.7 +/- 3.8 mV in isolated perfused posterior gills (P < 0.01). This effect is only partially explained by a modification of Na(+) and Cl(-) active influxes. No TEP modification is detected when atrazine is added (OUT) indicating that molecular mechanisms located on the basolateral membrane are likely to be the only ones affected. Another explanation would be that cuticular barrier prevents atrazine penetration into the gill. Haemolymph osmolarity, Na(+) and Cl(-) concentrations of crabs living in freshwater contaminated with atrazine 1 mg/l during 14 days are not significantly modified. We conclude that although atrazine can disturb osmoregulatory mechanisms of isolated gills, this pollutant would be of minor importance in affecting osmoregulatory capacities of the Chinese mitten crab in natural conditions.     

Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab, Eriocheir sinensis

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection.     

饥饿胁迫对中华绒螯蟹(Eriocheir sinensis)仔蟹的影响

在26.2 ～28.4℃水温条件下,研究了饥饿对中华绒螯蟹(Eriocheir sinensis)仔蟹的形态、行为、存活及体重损失的影响,同时确定了仔Ⅱ的营养饱和储存点(PRS)和不可恢复点(PNR).结果表明:饥饿胁迫下中华绒螯蟹仔Ⅰ、仔Ⅱ、仔Ⅲ的初次死亡时间(T1)分别为8.0、14.0和20.3 d,50％死亡时间(Ts0)分别为11.4、16.0和25.5 d,100％死亡时间(T100)分别为15.0、22.0和32.3 d,耐饥饿能力为仔Ⅲ＞仔Ⅱ＞仔Ⅰ;饥饿期间中华绒螯蟹体内水分含量持续升高,干重量降低显著,干重损失速率也随时间延长逐渐减小;先饱食后饥饿的给饵模式中仔Ⅱ蜕皮率随初始饱食时间延长而升高,50％个体完成蜕皮所需饱食时间(PRS50)为2.10d,各处理组仔Ⅱ的蜕皮周期与持续饱食组均无显著差异(P＞0.05),但只有饱食3d以上才能达到持续饱食饲养蟹的增重率;先饥饿后饱食的给饵模式中,仔Ⅱ的蜕皮率随着初始饥饿时间的延长而下降,其中仔Ⅱ50％不能蜕皮的初始饥饿时间(PNR50)和100％不能蜕皮的初始饥饿时间(PNR100)分别为10 d和14 d,并且蜕皮周期相对延长,延长时间约等于初始饥饿时间,不存在额外的摄食时间来弥补饥饿期间损失的能量,各处理组蜕皮后仔蟹与对照组体重无显著差异(P＞0.05).     

Isolation and characterization of 17 polymorphic microsatellites in grass carp

Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp (Ctenopharyngodon idellus) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy–Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks.     

Isolation and characterization of 12 polymorphic microsatellite markers in the tropical house gecko (Hemidactylus mabouia)

We present 12 variable microsatellite loci isolated from the invasive tropical house gecko, Hemidactylus mabouia. Polymerase chain reaction (PCR) primers were tested on 39 individuals from two locations in Miami, Florida. Heterozygote deficiency was detected for four loci, and we attribute this to possible null alleles or population substructure. Some loci successfully amplified PCR products in several congeners, indicating their potential for use in other geckos.     

Genetic population structure and contemporary dispersal patterns of a recent European invader, the Chinese mitten crab, Eriocheir sinensis

Genetic studies of recently established populations are challenging because the assumption of equilibrium underlying many analyses is likely to be violated. Using microsatellites, we investigated determinants of genetic structure and migration among invasive European-Chinese mitten crab populations, applying a combination of traditional population genetic analyses and nonequilibrium Bayesian methods. Consistent with their recent history, invasive populations showed much lower levels of genetic diversity than a native Chinese population, indicative of recent bottlenecks. Population differentiation was generally low but significant and especially pronounced among recently established populations. Significant differentiation among cohorts from the same geographical location (River Thames) suggests the low effective population size and associated strong genetic drift that would be anticipated from a very recent colonization. An isolation-by-distance pattern appears to be driven by an underlying correlation between geographical distance and population age, suggesting that cumulative homogenizing gene flow reduces founder bottleneck-associated genetic differentiation between longer-established populations. This hypothesis was supported by a coalescent analysis, which supported a drift + gene flow model as more likely than a model excluding gene flow. Furthermore, admixture analysis identified several recent migrants between the UK and Continental European population clusters. Admixture proportions were significantly predicted by the volume of shipping between sites, indicating that human-mediated transport remains a significant factor for dispersal of mitten crabs after the initial establishment of populations. Our study highlights the value of nonequilibrium methods for the study of invasive species, and also the importance of evaluating nonequilibrium explanations for isolation by distance patterns.     

Uptake of cadmium through isolated perfused gills of the Chinese mitten crab, Eriocheir sinensis

Using the perfusion method, we compared cadmium accumulation and influx across the gills of the euryhaline Chinese mitten crab Eriocheir sinensis, exposed to 4.8 microM cadmium in the incubation medium (OUT). Cadmium influx was not observed across posterior gills while it ranged from 0.15 to 6.82 nmol Cd g(-1) gill w.w. h(-1) across anterior ones. For these respiratory gills, a strong increase (40 times) was observed when calcium was removed in both incubation and perfusion media while the lack of sodium in the perfusion medium resulted in a 46 times decrease. For crabs acclimated 15 days to artificial seawater, cadmium influx across anterior gills showed a 21 times decrease when compared with freshwater acclimated ones. On the other hand, after 3 h of perfusion, we detected cadmium accumulation in both types of gills, ranging from 3.8 to 68 nmol Cd g(-1) gill w.w. in anterior gills and from 2.1 to 39 nmol Cd g(-1) gill w.w. in posterior ones. Such accumulations represent between 61.3 and 100% of the total uptake of cadmium through the gills. From these results, we suggest that cadmium can penetrate more easily into the hemolymph space through the 'respiratory' type epithelium present in the anterior gills but absent in the posterior ones. This metal uptake is likely to occur at least in part through the same pathways as calcium. On the contrary, cadmium seems to be sequestered inside the posterior gills, perhaps in the cuticle of the salt-transporting type epithelium.     

Gene expression pattern of myosin Va during spermatogenesis of Chinese mitten crab, Eriocheir sinensis

Myosin Va is an F-actin dependent molecular motor with multiple functions that are essential for acrosome formation in mouse spermiogenesis. The spermatozoon of the crab has a complicated acrosome surrounded by a cup-shaped nucleus. In the present study, the myosin Va cDNA was cloned from the testis of the Chinese mitten crab Eriocheir sinensis using degenerate PCR and rapid amplification of cDNA ends (RACE). The myosin Va cDNA consists of a 125bp 5'-untranslated region (5' UTR), a 5331bp open reading frame (ORF) and a 590bp 3' UTR. The putative myosin Va protein contains the head domain, neck domain and tail domain. Multiple alignment and phylogenetic tree showed that E. sinensis myosin Va is more closely related to the vertebrate myosin Va than to the invertebrate myosin V. E. sinensis myosin Va was expressed in various tissues. In situ hybridization demonstrated that myosin Va mRNA is located in the entire process of spermatogenesis. Quantitative real-time PCR indicated that the expression level at the mitotic and meiotic phases is higher than the spermiogenesis phase. Taken together, our work suggests that myosin Va may function in E. sinensis spermatogenesis.     

KIFC1 participates in acrosomal biogenesis, with discussion of its importance for the perforatorium in the Chinese mitten crab Eriocheir sinensis

Spermatogenesis is a complicated process during which spermatogonia undergo proliferation and divisions leading, after a series of dramatic changes, to the production of mature spermatozoa. Many molecular motors are involved in this process. KIFC1, a C-terminal kinesin motor, participates in acrosome biogenesis and nuclear shaping. We report here the expression profile of KIFC1 during spermatogenesis in the Chinese mitten crab, Eriocheir sinensis. KIFC1 mainly localizes around the nucleus but is also present within the nucleus of the spermatogonium and spermatocyte. At the early spermatid stage, KIFC1 begins to be distributed on the nuclear membrane at the region where the proacrosomal vesicle is located. By the late spermatid stage, KIFC1 is found on the acrosome. Immunocytochemical and ultrastructural analyses have shown that KIFC1 localizes on the perforatorium, which is composed of an apical cap and an acrosomal tubule. We demonstrate that, during spermatogenesis in E. sinensis, KIFC1 probably plays important roles in the biogenesis of the acrosome and in its maintenance. KIFC1 may also be essential for the eversion of the acrosome during fertilization. This work was supported in part by the following projects: the National Natural Science Foundation of China (nos. 30671606 and 40776079) and the National Basic Research Program of China (973 Program; grant no. 2007CB948104).     

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis.