Haihui Ye

Experimental nursery culture of the mud crab Scylla paramamosain (Estampador) in China

For the improvement of nursery culture of the mud crab Scylla paramamosain, a special nursery facility was purposely designed and built. This facility consists of nursery ponds (areas of 20, 50, and 120xa0m2, respectively) and a seed collecting pond connected to each nursery pond. There were 140 nursery facility units in operation for large-scale juvenile crab production. A series of experiments were carried out using the facility to investigate the effects of the age of megalopae, stocking density, and culture duration of both megalopae and first-stage crabs on the survival of megalopae and early juveniles of S. paramamosain. The results showed that when megalopae were stocked at a density between 3,000 and 5,000xa0indxa0m−2 and reared to reach the first-stage crabs, a survival rate of up to 50% could be achieved. When the first-stage juvenile crabs were stocked at a density between 2,000 and 3,000xa0indxa0m−2, their survival was more than 50% after 14xa0days of culture.

Changes in progesterone levels and distribution of progesterone receptor during vitellogenesis in the female mud crab (Scylla paramamosain)

Vertebrate-type steroids, such as progesterone, have been identified in crustaceans. The physiological activity of progesterone during vitellogenesis is still not well understood. In this study, progesterone levels in the female mud crab, Scylla paramamosain, were determined by enzyme-linked immunosorbent assay. Peak levels of progesterone were detected during the previtellogenic stage in the hemolymph, ovary, and hepatopancreas, whereas the progesterone level decreased significantly in vitellogenic stage I. During vitellogenic stage II, progesterone levels rose again in the hemolymph and ovary, but continued to decrease in the hepatopancreas. By using western blotting, progesterone receptor (PR), with an apparent molecular weight of 70 kDa, was identified in the ovary during both vitellogenic stages I and II. By means of immunohistochemistry, PR was detected mainly in the follicle cells during vitellogenic stage I and in the nuclei of oocytes in vitellogenic stage II. Our results strongly suggest that progesterone promotes vitellogenesis in the mud crab, S. paramamosain via a classical genomic mechanism.

Hepatopancreas cell cultures from mud crab, Scylla paramamosain

Hepatopancreas is an important digestive and endocrine organ in crustacean. However, there are few reports on cell cultures from crabs. Here, the cell cultures of hepatopancreas from Scylla paramamosain was studied in vitro. Both the primary cell culture and subculture were grown in Leibovitz’ L-15 medium, M199 medium, or a specially designed medium for S. paramamosain (MSP). The results showed that hepatopancreas cells in vitro grew in compact clusters in 2–3xa0d. Four types of cells could be identified. They were embryo cells, fibrillar cells, resorptive cells, and blister-like cells, respectively. Some of these cells could be subcultured for three generations. The MSP supported the best survival of these hepatopancreas cells, while M199 medium was the least effective of these three media. Fetal bovine serum and crab muscle extracts as supplements stimulated growth, but the crab hemolymph inhibited cell growth. Taken together, MSP is an appropriate medium for hepatopancreas cell cultures from S. paramamosain and can support cultures through several passages.

Profiles of gonadotropins and steroid hormone-like substances in the hemolymph of mud crab Scylla paramamosain during the reproduction cycle

To elucidate the role of gonadotropins-like substances in mud crab Scylla paramamosain, hemolymph samples were measured for concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH) and steroid hormones by enzyme-linked immunosorbent assay (ELISA). Hormonal concentration data were analyzed in association with the stages of gonadal development. ELISA has shown that in the female crab, the level of FSH reaches its peak in the early stage of ovary development, while estradiol and LH peaked during the late maturing stage of the ovary. In the male crab, testosterone and FSH culminated during the spermatid stage, and the level of LH peaked during the sperm stage. These results indicated that substances resembling the vertebrate FSH and LH are present in the hemolymph of S. paramamosain, and they may be involved in the development of the gonad.

Identification of differentially expressed genes in the thoracic ganglion of the mud crab, Scylla paramamosain during ovarian maturation

Abstract The thoracic ganglion is an important neuroendocrine organ in crabs. In order to isolate and clone differentially expressed genes in the thoracic ganglion of the mud crab, Scylla paramamosain, during ovarian maturation, a forward suppression subtractive hybridization cDNA library was constructed (crabs at maturation stage were used as the ‘tester’ and those at an early developmental stage as the ‘driver’). cDNA obtained from suppression subtractive hybridization was inserted into an expression vector and was transferred into Escherichia coli. A total of 500 randomly selected clones were sequenced and 410 expressed sequence tags (ESTs) were obtained. The inserted fragment size was from 200 to 1500 bp. The insertion rate was up to 82%. This confirmed the successful construction of the library. The ESTs were recognized based on the BLAST searches in NCBI, clustered and assembled, annotated and finally categorized. Among the total of 139 genes, 124 genes (89.2%) were similar to known genes, while 15 genes (10.8%) were classified as a group of unknown function. This study accumulated a cohort of data for further research of functional genes in the thoracic ganglion.

Occurrence of follicle-stimulating hormone-like substance in the Kuruma prawn, Marsupenaeus japonicus, during ovarian maturation

Abstract The identification of vertebrate follicle-stimulating hormone (FSH) may further elucidate the reproductive mechanisms in the prawn Marsupenaeus japonicus. By immunohistochemistry, FSH-like neurons were detected in the brain and thoracic ganglia of the female kuruma prawn. By enzyme-linked immunosorbent assay (ELISA), concentrations of an FSH-like substance were determined in the brain, thoracic ganglia and haemolymph during the maturation of the ovaries. Peak levels of an FSH-like substance culminated in the brain and thoracic ganglia during the exogenous vitellogenic stage, while the maximum level of the FSH-like substance was detected in the haemolymph. Our results indicated that the vertebrate FSH-like substances are present in M. japonicus, suggesting they may be involved in the ovarian maturation.

Two transcripts and the expression profiles of cyclin A in ovary of the mud crab, Scylla paramamosain

Two transcripts of cyclin A (SPCyA) were identified and characterized from ovaries of the mud crab, Scylla paramamosain. The sequences of the two transcripts contained the same open reading frame and 5′ untranslated region (UTR), but differed in 3′ UTR. The polyadenylation signals “AATAAA” were found in the different sites, which might generate alternative polyadenalation and thus the different length of the transcripts. The dendrogram, based on comparison of cyclin A proteins in different species, agreed with the classic taxonomic structure. Reverse transcription-PCR (RT-PCR) showed that the SPCyA mRNA was expressed at the highest level in the ovary. Real-time RT-PCR revealed that the amount of SPCyA mRNA in the ovary increased from the first stage to the fourth stage, then declined at the fifth stage (pu2009<u20090.05). It implies that SPCyA is closely related to oogonial proliferation (mitosis) and oocyte meiosis.

Molecular cloning, expression profiles and subcellular localization of cyclin B in ovary of the mud crab, Scylla paramamosain

A full-length cDNA of cyclin B was isolated from ovary of the mud crab (Scylla paramamosain) in this study. This transcript encodes a polypeptide of 401 amino acids, which is highly homologous to cyclin B protein family. Reverse transcription-PCR (RT-PCR) showed that cyclin B mRNA was expressed at highest levels in ovary of the mud crab. During the ovarian maturation process, real-time RT-PCR revealed that the abundance of cyclin B mRNA increased from the second stage (early-developing stage) to the fourth stage (nearly-ripe stage) and reached the peak level at the fifth stage (ripe stage). This result indicates the identified cyclin B gene might be related to the cell proliferation in ovary, both mitotically and meiotically. Immunohistochemistry showed that cyclin B protein was localized in the cytoplasm of prophase oocytes at the second stage while enriched in the nuclei of pro-metaphase oocytes at the fourth stage. It suggests the tested cyclin B protein might play different roles in ovary at the two stages.

The identification and distribution of progesterone receptors in the brain and thoracic ganglion in the mud crab Scylla paramamosain (Crustacea: Decapoda: Brachyura)

The existence of progesterone receptors (PR) in the Scylla paramamosain (mud crab) was studied using immunological techniques. By Western blotting, PR with an apparent molecular weight of 70xa0kDa is identified in both the brain and the thoracic ganglion. By immunohistochemistry, PR immunoreactive neurons are detected mainly in the protocerebrum, the subesophageal ganglion and the leg ganglion. PR immunoreactivity is localized mainly in the nuclei of these neurons, while only a few neurons show such activities in their cytoplasm. Our results provide evidence that progesterone modulates the neuroendocrine system mainly via nucleus receptors.

Cloning, expression and functional analysis of farnesoic acid O-methyltransferase (FAMeT) in the mud crab, Scylla paramamosain

Farnesoic acid O-methyltransferase (FAMeT) is the enzyme that catalyses methylation of farnesoic acid (FA) to produce isoprenoid methyl farnesoate (MF) at the final step of the biosynthetic pathway. As the sesquiterpenoid precursor of the insect juvenile hormone III (JH III), MF has been suggested to play a vital role in regulating crustacean growth and reproduction. In this study, we report the identification of three isoforms of FAMeT transcript from the mud crab, Scylla paramamosain, which encode a peptide of 275, 278 and 280 amino acid residues respectively. Gene expression profiling by reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that FAMeT has wide tissue distribution in S. paramamosain. In female S. paramamosain, the expression levels of FAMeT are significantly up-regulated in thoracic ganglion and down-regulated in eyestalk during ovarian development. In contrast, FAMeT expression levels increased in both thoracic ganglion and eyestalk during testicular development stages. During the molting stages of the mud crab, the mRNA abundance of FAMeT increased significantly in the eyestalk of females but not of males. FAMeT expression is therefore differentially regulated in male and female mud crab, during reproductive and molting stages. These results suggest that FAMeT is involved in many important functions, especially in the regulation of the growth, reproduction and molting processes, in S. paramamosain.