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.     

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).     

Characterization of microsatellite loci for the Chinese mitten crab,Eriocheir sinensis

The Chinese mitten crab, Eriocheir sinensis is an important human food source in Asia and causes considerable ecological and economical damage as a recent invader of North America and Europe. Here we report the isolation and characterization of 12 highly polymorphic microsatellite loci for E. sinensis. The number of observed alleles per locus ranged from 7 to 40 and the observed heterozygosity from 0.50 to 0.95. These markers should prove a useful tool to investigate the colonization process and its evolutionary implications.     

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.     

Isolation and characterization of polymorphic microsatellites from Chinese mitten crab,Eriocheir sinensis

Chinese mitten crab (Eriocheir sinensis) has great commercial significance in Asia, but is an invasive organism in Europe and America. We isolated five novel microsatellites for E. sinensis. All five loci were polymorphic. The average allele number was 16.8 per locus with a range of 13 to 21, while the expected heterozygosity ranged from 0.82 to 0.91 with an average of 0.88. These markers could be applicable to studies of the genetic diversity and population structure of E. sinensis.     

Phylogenetic analysis of intestinal bacteria in the Chinese mitten crab (Eriocheir sinensis)

AIMS: To identify the dominant intestinal bacteria in the Chinese mitten crab, and to investigate the differences in the intestinal bacteria between pond-raised and wild crabs. METHODS AND RESULTS: The diversity of intestinal bacteria in the Chinese mitten crabs was investigated by denaturing gradient gel electrophoresis (DGGE) fingerprinting, 16S rRNA gene clone library analysis and real-time quantitative PCR. The principal component analysis of DGGE profiles indicated that substantial intersubject variations existed in intestinal bacteria in pond-raised crab. The sequencing of 16S rRNA genes revealed that 90-95% of the phylotypes in the clone libraries were affiliated with Proteobacteria and Bacteroidetes. Some genera were identified as unique in wild crabs and in pond-raised crabs, whereas Bacteroidetes was found to be common in all sampled crab groups. Real-time quantitative PCR indicated that the abundance of Bacteroides and the total bacterial load were approximately four-to-10 times higher in pond-raised crabs than in wild crabs. A significant portion of the phylotypes shared low similarity with previously sequenced organisms, indicating that the bacteria in the gut of Chinese mitten crabs are yet to be described. CONCLUSIONS: The intestinal bacteria of pond-raised crabs showed higher intersubject variation, total diversity and abundance than that observed in wild crabs. The high proportion of the clones of Proteobacteria and Bacteroidetes in the clone library is an indication that these bacteria may be the dominant population in the gut of the Chinese mitten crab. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated obvious differences in the intestinal bacterial composition of pond-raised crabs and wild crabs. This knowledge will increase our understanding of the effects of aquaculture operations on bacterial community composition in the crab gut and provide necessary data for the development of probiotic products for crab cultivation.     

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.     

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.     

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.     

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.     

Rickettsia-like organism associated with tremor disease and mortality of the Chinese mitten crab Eriocheir sinensis

We report the discovery of a rickettsia-like organism (RLO) in cultured freshwater Chinese mitten crab Eriocheir sinensis. The RLO caused tremor disease and was apparently responsible for a mass mortality (30 to 90%) in 2 provinces in southeast China. Moribund crabs were investigated from different districts during outbreaks in 1999 and 2000. With electron microscopy, 3 different pathogens were detected in moribund crabs: a rickettsia-like organism (RLO), virus-like particles (VLP) and a microsporidian-like protozoan (MLP). Based on the high prevalence, infection intensity and cytopathological signs, the RLO was considered to be the probable cause of the high mortality. Both VLP and MLP occurred at low prevalences and were considered secondary infections. The RLO was 0.22 to 0.35 microm in diameter, granular or clavate, bounded by a cell wall and membrane, and possessed no nucleus but a nucleoid was present. When dividing, RLOs occurred in irregular shapes, such as dumbbells, awls, and crescents. The RLOs exhibited a predilection for muscle and connective tissues and were probably transported to various tissues and organs by hemocytes.     

Characterization and expression pattern of p53 during spermatogenesis in the Chinese mitten crab Eriocheir sinensis

p53, as a “Guardian of the Genome”, plays an important role in cell cycle arrest, apoptosis, DNA repair and inhibition of angiogenesis in different tissues including testis. p53 gene and its protein perform many essential roles for mammalian spermatogenesis. To explore its functions during spermatogenesis in Eriocheir sinensis, we have cloned and sequenced the cDNA (1,218 bp) of p53 from the testis by degenerating primer PCR and rapid-amplification of cDNA ends. The protein alignment of p53 shows the conserved DNA binding domain, dimerization site and zinc binding site consisted of the predicted structures. Phylogenetic analysis revealed that p53 was more closer to Marsupenaeus japonicus and Tigriopus japonicus than other examined species. Tissue expression analysis of p53 mRNA showed p53 was distinctly expressed in accessory sexual gland, muscle, gill, heart, hepatopancreas and testis. In situ hybridization revealed that the p53 mRNA was weakly distributed around the nucleus, but stronger in the invaginated acrosomal tubule at the early stage. At the middle stage, p53 mRNA signal was increased than the early stage and the signal displayed dot-like pattern on the surface of cup-like nucleus. The signal on acrosomal cap is stronger than on the acrosomal tubule, despite acrosomal tubule signal was also distinct. At the late stage, the signal was still mainly located in acrosomal cap and acrosomal tubule. Sporadic signal were found surrounding the cup-like nucleus, but they were very weak. In the mature sperm, the signal was dramatically decreased. Even though the signal on cup-like nucleus and acrosomal tubule were distinct, they were weaker than those in middle stage. Based on these results, we concluded that p53 may play an important role in formation of acrosome biogenesis and nuclear shaping during spermiogenesis of E. sinensis.