Microsatellite Variations Among Four Populations of Eriocheir sinensis

Using microsatellite markers, we analysed genetic polymorphism in four populations of Eriocheir sinensis, sampled from Jiangsu, Anhui, Liaoning and Tianjin. Twenty-four pairs of primers were used to amplify the target fragments ranging from 80 bp to 445 bp, which included 16 pairs designed in our laboratory and eight pairs published internationally. Two to 10 alleles per locus in four populations were amplified, and there were 155 alleles in all populations. The average number of alleles per locus was 6.458. The average number of effective alleles per locus （Ne） was 4.349l to 4.7234; the average observed heterozygosity （Ho） was 0.5690 to 0.6722; and the average expected heterozygosity （He） was 0.7238 to 0.7546. Hardy-Weinberg equilibrium analysis （Z2 test, P 〈 0.05） revealed that seven loci in the four populations were in equilibrium. The genetic distances between the four populations were calculated and revealed that the Anhui, Jiangsu and Tianjin crabs belong to a Yangtse River population, while the Liaohe crabs form another branch.

Nerve Terminal Types of Sinus Gland and Neurosecretory Cell Types of X-organ in Crab Eriocheir sinensis

The ultrastructure of the X organ sinus gland complex in sexually immature Chinese mitten crab Eriocheir sinensis was examined under the electron microscope. The sinus gland (SG) is composed primarily of nerve terminals and glial cells. The terminal contains a great number of membrane bound granules, mitochondria, rough endoplasmic reticulum and electron lucent vesicles. The profiles of the terminals are sometimes exhibited finger like projections. The X organ (XO) locates at the ventral surface of the medulla terminalis ganglion in the eyestalk, oppositing to SG. According to the features of granule size, shape, electron density and cytoplasm, 6 types of terminals in SG and 7 types of neurosecretory (NS) cells in XO were identified. The release process by exocytosis of the granules in SG was observed and a tentative comparison was made for the granules between different types of terminals and NS cells. It seemed that they had a good correspondence since no change in electron density and just a slight increase of sizes was shown.

The Study on the Localization of Acid Phosphatase Activity During Spermiogenesis in Chinese Mitten-Handed Crab,Eriocheir sinensis

Ultrastructural cytochemical techniques and electron microscopy were used for localization of acid phosphatase activity during spermiogenesis in Eriocheir sinemsis. The results showed that： Acid phosphatase was synthesized in the endoplasmic reticulum in the early spermatids. The acid phosphatase was found gradually in nucleus, the membrane of acrosomal vesicle, the cytoplasmic region and the acrosomal tubule. And then the reaction product particles became thicker during the spermiogenesis. In the mature sperm, acid phosphatase was localized in the percutor organ slightly, but it was massive and compact in the acrosomal tubule.

6个不同地理湖泊中华绒螯蟹(Eriocheir sinensis)肌肉组织氨基酸组成、营养评分及其差异性分析

为探究江苏省长荡湖、固城湖、高邮湖、洪泽湖、太湖、阳澄湖6个不同地理湖泊中华绒螯蟹(Eriocheir sinensis)群体的氨基酸组成及其对应的营养品质评价,本研究采用OPA-FMOC柱前衍生化HPLC法测定中华绒螯蟹肌肉组织中氨基酸组成及含量,并根据联合国粮食及农业组织/世界卫生组织(FAO/WHO)标准对其进行营养品质评价。结果显示:所有样本均检测出17种常见氨基酸,各群体间氨基酸含量差异显著;雌雄群体各氨基酸指标均符合FAO/WHO标准模式。雌蟹中,总必需氨基酸(EAA)、总非必需氨基酸(NEAA)、总氨基酸(TAA)、必需氨基酸指数(EAAI)均为洪泽湖、固城湖群体显著高于长荡湖、太湖、阳澄湖群体,与高邮湖群体差异不显著。雄蟹中,高邮湖群体各氨基酸指标均显著高于阳澄湖、洪泽湖、太湖群体,与长荡湖,固城湖群体差异不显著;主成分分析和聚类分析结果与其氨基酸指标差异相似,雌雄群体综合判别准确率分别为79.20%、95.80%。综上,江苏6个地理湖泊中华绒螯蟹雌雄群体肌肉组织中氨基酸种类齐全,氨基酸含量存在显著性差异,且氨基酸之间的比例适宜,均为优质或良好蛋白源,基于中华绒螯蟹群体间的氨基酸含量差异可对其群体进行有效的判别。

白芍(Radix paeoniae alba)提取物对中华绒螯蟹(Eriocheir sinensis)造血组织发育及造血因子Astakine的影响

血淋巴细胞在甲壳动物免疫反应中起着非常重要的作用,其主要由造血组织(Hematopoietic tissue,HPT)生成并随着开管式循环分布到身体各处。到目前为止,还没有相关研究表明甲壳动物造血组织是否受草本植物的影响。本研究以中华绒螯蟹(Eriocheir sinensis)为对象,利用荧光定量PCR、免疫荧光和组织化学等技术,初步探索了白芍(Radix paeoniae alba)提取物对中华绒螯蟹造血组织和造血因子Astakine的影响。结果显示,在原代培养的造血组织细胞中添加终浓度为0.85 mg/mL的白芍提取物后,造血因子Astakine mRNA的表达量显著上调(P<0.05)。进一步制备含1%白芍提取物的中华绒螯蟹饲料并进行饲喂实验,结果显示饲喂含白芍提取物饲料的中华绒螯蟹造血组织的Astakine mRNA的表达量较饲喂前上调了10.5倍,要高于对照组。免疫荧光结果同样表明,饲喂含白芍提取物饲料后中华绒螯蟹造血组织的Astakine蛋白荧光信号也要强于对照组。通过造血组织的形态学观察和造血组织体重百分比的检测,我们发现白芍组中华绒螯蟹造血组织含有较多卵形小叶,且造血组织体重百分比较饲喂前增加了6.64%,而对照组仅增加1.6%。免疫保护率实验显示,在脂多糖急性刺激下,白芍添加组的中华绒螯蟹的存活率(77.5%)要高于对照组(57.5%)。综上所述,白芍提取物可以促进中华绒螯蟹造血因子的表达和造血组织的发育,从而提高中华绒螯蟹的免疫调节功能。

THE EFFECTS OF ACUTE PROGRESSIVEHYPOXIA ON THE RESPIRATIONRATE OF THE CHINESE CRABEriocheir sinensis

The effects of acute progressive hypoxia on the respiration rate of the Chinese freshwater crab, Eriocheir sinensis, acclimated at three temperatures were investigated with a closed respiromet er. E. sinensis can maintain its respiration rate down to the critical point (Pc) and from this point its respiration rate declines rapidly, reaching zero at a lower ambient oxygen concentration called the zero respiration oxygen concentration. Because of this, a new hyperbolic equation is introduced to express the relationship between respiration rate and ambient oxygen concentration. A new method for calculating the PC value is also developed. The PC values for E. sinensis at 20-35℃ range from 1.92-3.47 mg/1.

Observation on Behavior of Procambarus clarkii and Eriocheir sinensis in Polyculture System

[Objective] This study aimed to observe and explore the behavior of Procambarus clarkii and Eriocheir sinensis in polyculture system. [Method] P. clarkii and E. sinensis with the same weight were used for the experiment in a relatively small polyculture system. The agonistic behavior of P. clarkii and E. sinensis in the first 30 min of the initial encounter and the fighting results were observed and recorded; the approach-avoidance behavior of P. clarkii to bigger E. sinensis in a relatively large space （the ＂Y＂ maze） was observed and recorded. [Result] The re- sults showed that the times of first aggressions and approaches and the total times of active aggressions and approaches of P. clarkii in the initial period of polyculture experiment were all significantly more than E. sinensis （/：＇〈0.05）; after 30 d of poly- culture, P. clarkfi died in 30% of the groups while E. sinensis died in only 10% of the groups, and they coexisted in 60% of the groups. In the experiment of ap- proach-avoidance behavior of P. clarkii to bigger E. sinensis, with the increase of experiment times, the tendency of approach and avoidance of P. clarkii to the con- trol and E. sinensis became control-prefer （P〈0.05） from insignificantly different （P〉 0.05）, which indicated that the recognition ability of P. clarkii for E. sinensis was sig- nificantly improved, and the selection behavior of E. sinensis was obviously inhibited. [Conclusion] Presumably, polyculture of crayfishes and crabs can achieve better effi- ciency when under appropriate habitat conditions.

Water quality, nutrient budget, and pollutant loads in Chinese mitten crab (Eriocheir sinensis) farms around East Taihu Lake

To understand the factors causing frequent outbreaks of harmful algae blooms in the Taihu Lake, China, we studied water quality and nutrient budget in Chinese mitten crab (Eriocheir sinensis) farm ponds in the eastern part of the lake from November 2007 to December 2009. We estimated the nitrogen (N), phosphorus (P), and chemical oxygen demand (COD) loads. Materials input and output ponds, water exchange, and applied management practices of 838.5-hm2 crab ponds were surveyed using questionnaires. Water quality of 12 ponds, which were located no more than 2 km from East Taihu Lake, were monitored. The results show that water quality in the crab ponds was better than reference data. Feeds, including corn seed, commercial feed, trash fish, and gastropod, were the major sources of N and P input in the crab ponds, contributing 88.7% and 94.9%, respectively. In total, 60.5% of N and 37.3% of P were sequestered by macrophytes, and only 15.7% and 8.5% of them were discharged as effluent. The net loads of N and P in effluent were 16.43 kg/hm2/cycle and 2.16 kg/hm2/cycle, respectively, while the COD load was -17.88 kg/hm2/cycle. This indicated that crab farming caused minor negative impact on the trophic status of the lake area, which was attenuated by macrophytes. However, wastewater purification is still necessary in crab faming.

Effect of Salinity on Hemolymph Osmotic Pressure,Sodium Concentration and Na^+-K^+-ATPase Activity of Gill of Chinese Crab,Eriocheir sinensis

The effects of salinity on hemolymph osmotic pressure, Na+ concentration and Na+-K+-ATPase activity of gill of Chinese crab Eriocheir sinensis were studied. The results showed that hemolymph osmotic pressure and Na+ concentration increased signifi- cantly (P<0.05), and the Na+-K+-ATPase activity of gills decreased significantly (P<0.05) when salinity increased from 0 to 16. The hemolymph osmotic pressure and Na+ concentration in each treatment group rose remarkably at 0.125 d or 0.25 d, while the Na+-K+-ATPase activity of gill reduced gradually with increased experiment time in 3 d. Then the three parameters remained at a constant level after 0.25 d, 0.125 d and 3 d, respectively, and higher hemolymph osmotic pressure, higher Na+ concentration and lower Na+-K+-ATPase activity of gill occurred at higher salinity. The effect of salinity change on protein concentration of hemolymph was indistinct (P>0.05); However, the protein concentration decreased gradually with the increase of salinity from 0.25 d to 1 d, and then tended to be stable from day 1 to day 15.

Mercury (Hg^(2+)) effect on enzyme activities and hepatopancreas histostructures of juvenile Chinese mitten crab Eriocheir sinensis

We studied the effects of mercury (Hg2+) on antioxidant and digestive enzyme activities in terms of LC50 value and on hepatopancreas histostructures of juvenile Chinese mitten crabs Eriocheir sinensis in 40-day exposure to various concentrations of Hg2+ (0, 0.01, 0.05, 0.10, 0.20, and 0.30 mg/L). The results show that the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) significantly increased in the concentrations of 0.01 and 0.05 mg/L, while that of enzyme decreased in 0.10, 0.20 and 0.30 mg/L treatments. Meanwhile, Hg2+ disrupted the histostructures of the hepatopancreas, causing decreases in activities of pepsin, tryptase, amylase, and cellulose, which are synthesized in the hepatopancreas. Moreover, as the Hg2+ concentration increased, the survival rate of the crabs decreased, worst at 56.57% in 0.30 mg/L. Therefore, although crabs are able to tolerate low levels of mercury pollution, high levels lead to cellular injury and tissue damage in hepatopancreas, which then loses some of its vital physiological functions such as absorption, storage, and secretion.

Molecular characterization and expression profiles of four transformer-2 isoforms in the Chinese mitten crab Eriocheir sinensis

The transformer-2(tra-2) gene plays a key role in the regulatory hierarchy of sexual differentiation in somatic tissues and in the germline of Drosophila melanogaster.In this study,sequences and expression profiles of tra-2 in the Chinese mitten crab Eriocheir sinensis were characterized.Four tra-2 isoforms,designated as Estra-2a,Estra-2b,Estra-2c,and Estra-2d,were isolated.They all contained an RNA-recognition motif(RRM) and a linker region,which shared high similarity with other reported tra-2s.Sequence analysis revealed that Estra-2a,Estra-2b and Estra-2c are encoded by the same genomic locus and are generated by alternative splicing of the pre-mRNA.Compared with the other three isoforms,Estra-2d lacks the RS2 domain.Quantitative real-time PCR showed that all four isoforms were highly expressed in the fertilized egg,and in the 2-4 cell and blastula stages compared with larval stages(P<0.01),suggesting their maternal origin in early embryonic developmental stages.Notably,Estra-2a was highly expressed in male somatic tissues,while Estra-2c was significantly highly expressed in the ovary.These results suggest that Estra-2c is involved in sexual differentiation of the Chinese mitten crab.Our findings provide basic information for further functional studies of the tra-2 gene/protein in this species.

EFFECTS OF FISHES ON THE GROWTH AND SURVIVAL OF CHINESE MITTEN CRAB,ERIOCHEIR SINENSIS

This experiment on the effects of fishes ( Pelteobagrus fulvidraco, Channa argus, Cyprinus carpio ) on growth and survival of juvenile Chinese mitten crab, Eriocheir sinensis was conducted in cages placed in Bao’an Lake. T tests showed that effects of fishes on the carapace width growth were not significant as compared with the control (no fish). But the effects of these fishes on crab survival were negative. The causes of negative effects of fishes on crab survival were simply discussed on the basis of food habits.

Effects of Different Dietary Lipid Contents on Growth and Lipase Activity of Eriocheir sinensis Larvae

The effects of different dietary lipid content on the growth and lipase activity ofEriocheir sinensis larvae were studied in the paper. The results showed that the survival, metamorphic rate and weight gain of E. sinensis larvae at different stages of growth all varied significantly with lipid content （P〈0.05）. Further, the survival and metamorphosis rates were the highest during the larval phases Z3 to Z4, and the weight gain was the highest during the larval phases Z5 to M. During the first 20h after metamorphosis of every larval stage, the lipase activity increased over time at Z1, Z2, Z3 and M and declined at Z4 and Zs, and was influenced significantly by lipid content （P〈0.05）. In addition, lipase activity at each larval stage began to respond to dietary lipid contents 4h after the larvae were fed, and tended to be stable after 12 h. The diets with higher lipase activity and lower lipid content were selected to give the suitable recipe of lipid requirements at each larval stage. It was concluded that the suitable lipid requirements at Zb Z2, Z3, Z4, Z5 and M were 6%, 4% 6%, 8%, 8% and 10%, respectively.

A Comparative Study of Genetic Variation between Chinese Mitten Crab Eriocheir Sinensis and Hepu Mitten Crab E.Hepuensis

Horizontal starch gel electrophoresis was used to investigate the biochemical genetic structure of Chinese mitten crab Eriocheir sinensis and Hepu mitten crab E.hepuensis. Sixteen putative enzyme-coding loci were examined for forty-nine Chinese mitten crabs and thirty-eight Hepu mitten crabs. Nine loci, AAT-1 *, AAT-2 *, G3PDH *, GPI *, IDHP-1 *, IDHP-2 *, MDH-1 *, MDH-2 * and PGM *, are polymorphic in Chinese mitten crab, and seven, AAT-1 *, AAT-2 *, GPI *, IDHP-1 *, MDH-1 *, MDH-2 * and PGM *, are polymorphic in Hepu mitten crab. The proportion of polymorphic loci and the expected heterozygosity are 0.562 5 and 0.080 3 for Chinese mitten crab, and 0.437 5 and 0.075 4 for Hepu mitten crab. The Nei’s genetic distance between two species is 0.002 4.

Improvement of polyploidy induction in Eriocheir sinensis

An incubating temperature of 15 C is used to induce triploidy in Eriocheir sinensis through inhibition of the releaseof polar body II, and that of 18 C to induce tetraploidy through inhibition of the first cleavage. Flow cytometry isused to identify the ploidy in different developmental stages. For induction of triploidy in fertilized eggs in vitro, thehighest induction rate observed in blastula by cytochalasin B, 6-DMAP and KCl is 49.1%, 51.7% and 77.5%,respectively. In the KCl treatment of pregnant crabs with the fertilized eggs, the highest triploid induction rateobserved in the zoea is 85.3%. For induction of tetraploidy, the highest induction rate observed in the blastula bycytochaslasin B, 6-DMAP and KCl is 50.3%, 54.9% and 79.8% respectively. In the KCl treatment of pregnant crabswith the fertilized eggs, the highest induction rate in zoea is 27.3%. Through this study such difficulty as in vitroculture is overcome. Triploid zoea Eriocheir sinensis has been developed for the first time. The induction rate oftetraploid zoea has also been greatly improved.

Transcriptome of Eriocheir sinensis under air exposure

Chinese mitten crab,Eriocheir sinensis,is an economically important crab in China.Air exposure is a common stress for E.sinensis,especially during the harvest and transportation.Several studies have investigated the effects of air exposure stress on E.sinensis at physiological and molecular levels.However,the common and distinct mechanisms adopted by male and female crabs to cope with air exposure stress remain unclear.In this study,we performed a comparative transcriptome analysis from hepatopancreatic tissue of female and male Chinese mitten crabs in response to air exposure stress.In total,428 and 1322 diff erentially expressed genes(DEGs)were identifi ed in female and male crabs under air exposure,respectively.Our results showed that the transcriptional levels of several glycolysis related genes and anti-apoptotic proteins were up-regulated in both female and male crabs in response to air exposure.Moreover,our findings indicated that female E.sinensis might preferentially increase the expression of heat shock proteins(HSPs)to deal with air exposure stress,while male E.sinensis tend to resist air exposure stress via increasing antioxidant enzyme expression.Overall,this study provides novel insights into the molecular mechanisms underlying the air exposure stress response of E.sinensis.

Es-ATG7 is required for spermatogenesis of Eriocheir sinensis and modulates p53-dependent apoptosis in germ cells

Spermatogenesis is a complicated and highly regulated male gamete differentiation process that begins with the proliferation and differentiation of spermatogonia to the release of the mature spermatozoa.The autophagy-related gene atg7 has been reported as closely related to spermatogenesis and communication of Sertoli cell-germ cells in mice,including acrosome biogenesis,sperm flagellum development,and ectoplasmic specialization assembly.However,the function of es-ATG7 and its molecular regulatory mechanism during spermatogenesis in Crustacea remain largely unknown.Here,we cloned and identified es-atg7 from the testes of the Chinese mitten crabs Eriocheir sinensis and found that the expression of es-atg7 was relatively high in testes through semi-quantitative RT-PCR.The dynamic localization of es-ATG7 detected by immunofluorescence may convey information about its role in the spermatogenesis of E.sinensis.Furthermore,a knockdown of es-atg7 revealed that the malformed sperm with irregular sperm shape or loose nuclear cup and germ cell apoptosis were increased significantly.Accompanying this,we found an up-regulated expression of es-p53 during spermatogenesis in es-atg7 knockdown groups.Altogether,our results indicate that es-ATG7 plays an essential role during spermatogenesis of E.sinensis,and we demonstrated that es-ATG7 acts as an antagonist for p53-dependent apoptosis induction in this process.

EFFECTS OF TEMPERATURE AND Ca^( 2+) ON THE LARVAL DEVELOPMENT OF THE DECAPODA CRUSTACEAN: ERIOCHEIR SINENSIS

With increasing demand in China for the mitten crab larvae, understanding its survival mechanism gets more important. This research focused on the effects of temperature and Ca 2+ on the larval growth and development. Eriocheir sinensis larvae were reared in laboratory under 21 different combinations of temperature (15, 20, 25℃) and Ca 2+ content (120, 130, 140, 150, 160, 170, 180 mg/l) and constant salinity (20) and pH (8). The results suggested that the survival rate increases with temperature and Ca 2+ content. These combinations of temperature and Ca 2+ content maximized survival rate in our study and it may be the optimum water environmental conditions for culturing the larvae. To predict surviving larvae number under different water environmental conditions, 21 dynamic mathematical models were developed. This for the first time observation of the zoeal Ⅵ larvae of the Changjiang River E. sinensis population showed that they occurred under stressed water environmental conditions: temperature of 15℃ and Ca 2+ content of 120,130 mg/l.

Culture of Eriocheir sinensis Larvae in Ponds in Greenhouse

A total of 15 pools were selected from a greenhouse, and they were randomly and evenly divided into three groups.In each group, one stocking density of Eriocheir sinensis was arranged. The results showed that after 45-d culture, the crab number per kilogram reached about 300 with survival rate of about 20%; the initial stocking density had significantly effect on the body size of E. sinensis on sell, instead on the survival rate of E. sinensis larvae. Therefore, it is feasible to conduct the culture of E. sinensis larvae in a pool in greenhouse.

Residual Effect of Fipronil in Eriocheir sinensis

The residual effects of fipronil aqueous solutions(at mass concentrations of 10,30 and 50μg/L,respectively)on Eriocheir sinensis were investigated by the semi-static experiment method.The results showed that among the treatment groups with different mass concentrations,the high mass concentration group contained fipronil at a significantly higher content in the crab legs than the low-mass concentration group,and also a significantly higher content in the body than the low mass concentration group.There were no significant differences in the content of fipronil between different parts of E.sinensis.The trend of fipronil-enriched parts was from the legs to the body to the gonads.Fipronil residue in E.sinensis had no sex selectivity,but it enriched faster in female E.sinensis than males.This study provides a certain reference for dealing with related fishery pollution accidents.