Guizhong Wang

Isolation and characterization of ten new polymorphic microsatellite loci in the mud crab, Scylla paramamosain

We developed polymerase chain primers for ten microsatellite loci in the mud crab, Scylla paramamosain. All markers were obtained from a (CA)15 and (CT)15-enrichment DNA library, and characterized in 30 individuals from one wild population. The number of alleles per locus varies between 8 and 18, and the observed and expected heterozygosities ranged from 0.6207 to 0.9333 and from 0.5886 to 0.9243, respectively. These polymorphic loci provide a valuable tool for population genetic analysis and parentage determination in this species.

Heavy metal exposure reduces hatching success of Acartia pacifica resting eggs in the sediment

The potential effects of three heavy metals (Cu, Pb, and Cd) on hatching success of Acartia pacifica resting eggs in the sediment of Xiamen Bay were experimentally investigated. The number ofA. pacifica nauplii hatched from the sediment sharply decreased with the increase of metal concentration and exposure time from 3 to 30 d. An increase of the Cu concentration from 34.8 to 348 mg/kg, reduced the number of hatched nauplii by 46.6%-100%. An increase of the Pb concentration from 75.2 to 752 mg/kg, reduced the number of hatched nauplii by 21.4%-78.9%. An increase of the Cd concentration from 0.68 to 6.8 mg/kg, reduced the number of hatched nauplii by 31.6%-94.7%. The number of nauplii also significantly decreased with the increase of mixed-metal concentration and exposure time in the mixed-metal test. Trimmed Spearman-Karber analysis gave sediment metal 72-h LC50 values of 1.25 mmol Cu/kg, 1.73 mmol Pb/kg, and 0.054 mmol Cd/kg, which suggested that Cd was the most toxic to A. pacifica resting eggs in the three tested metals. The results indicate that heavy metals with higher concentrations can reduce recruitment of A. pacifica nauplii from benthic resting eggs to planktonic population.

Differential gene expression profile of the calanoid copepod, Pseudodiaptomus annandalei, in response to nickel exposure

To better understand the underlying mechanisms of reactions of copepods exposed to elevated level of nickel, the suppression subtractive hybridization (SSH) was used to elucidate the response of the copepod Pseudodiaptomus annandalei to nickel exposure at the gene level. P. annandale is one of a few copepod species that can be cultured relatively easy under laboratory condition, and it is considered to be a potential model species for toxicity study. In the present study, P. annandalei were exposed to nickel at a concentration of 8.86 mgL(-1) for 24h, after which the RNA was prepared for SSH using unexposed P. annandalei as drivers. A total of 474 clones on the middle scale in the SSH library were sequenced. Among these genes, 129 potential functional genes were recognized based on the BLAST searches in NCBI and Uniprot databases. These genes were then categorized into nine groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 129 genes, 127 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains (CD) and proteins using the CD-Search service and BLASTp. Among 129 genes, 119 (92.2%) were annotated to be involved in different biological processes, while 10 genes (7.8%) were classified as an unknown-function gene group. To further confirm the up-regulation of differentially expressed genes, the quantitative real time PCR were performed to test eight randomly selected genes, in which five of them, i.e. α-tubulin, ribosomal protein L13, ferritin, separase and Myohemerythrin-1, exhibited clear up-regulation after nickel exposure. In addition, MnSOD was further studied for the differential expression pattern after nickel exposure and the results showed that MnSOD had a time- and dose-dependent expression pattern in the copepod after nickel exposure. To the best of our knowledge, this is the first attempt to investigate the toxicity effects of nickel on a copepod at molecular level.

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.

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.

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.

The expression pattern of scygonadin during the ontogenesis of Scylla paramamosain predicting its potential role in reproductive immunity.

The antimicrobial peptide scygonadin (Scy) was first isolated from the gonad of Scylla serrata and its gene is predominantly expressed in the ejaculatory duct of adult males. Thus, its function was predicted to be associated with reproductive immunity, but this is still unclear and needs further investigation. In our study, the expression pattern of Scy at different developmental stages of both male and female S. paramamosain was investigated, so that the potential function of this peptide could be examined. Using real-time quantitative PCR, Scy mRNA transcripts were demonstrated obviously in the vulnerable embryos and larvae-zoea I but very weakly detected in the larvae-zoea III, megalops and juveniles. The gene expression pattern showed a decreasing trend during the early developmental stages. The Scy gene had low expression in the ejaculatory duct of small and medium crabs (100g and 200g in weight) whose gonads were underdeveloped. However, the level of Scy expression was significantly increased in large crabs (300g in weight), which had normally become sexually mature at this size. It was further observed that the numbers of Scy mRNA transcripts in sexually mature crabs were significantly more abundant than in immature ones. In addition, the Scy gene was significantly expressed in the ejaculatory duct of mature male crabs during the mating period (April and May) and reached their highest expression in May. Using immunohistochemistry, the Scy protein was strongly detected in the testis and seminal vesicle of small crabs. However, in large crabs, Scy protein was intensively present in more tissues than in small crabs, including the ejaculatory duct, posterior ejaculatory duct, gill and muscle of males, and also in the spermatheca, gill and muscle of females. It is also interesting to note that Scy mRNA transcripts were detected in other crab species and showed similar expression pattern to those in S. paramamosain. This study extended our knowledge concerning the antimicrobial peptide scygonadin, which has its function principally in the ejaculatory duct of males but which may also play a role at different developmental stages of S. paramamosain from embryogenesis to maturation, and is also widely distributed in other crabs.

Molecular cloning and functional analysis of the fatty acid-binding protein (Sp-FABP) gene in the mud crab (Scylla paramamosain)

Intracellular fatty acid-binding proteins (FABPs) are multifunctional cytosolic lipid-binding proteins found in vertebrates and invertebrates. In this work, we used RACE to obtain a full-length cDNA of Sp-FABP from the mud crab Scylla paramamosain. The open reading frame of the full length cDNA (886 bp) encoded a 136 amino acid polypeptide that showed high homology with related genes from other species. Real-time quantitative PCR identified variable levels of Sp-FABP transcripts in epidermis, eyestalk, gill, heart, hemocytes, hepatopancreas, muscle, ovary, stomach and thoracic ganglia. In ovaries, Sp-FABP expression increased gradually from stage I to stage IV of development and decreased in stage V. Sp-FABP transcripts in the hepatopancreas and hemocytes were up-regulated after a bacterial challenge with Vibrio alginnolyficus. These results suggest that Sp-FABP may be involved in the growth, reproduction and immunity of the mud crab.

An approach to the study of copepod egg banks based on efficient DNA extraction from individual copepod eggs

Abstract A method of extracting DNA from individual copepod eggs was introduced for this study which included a modified proteinase K procedure and an efficient DNA sedimentation process. DNA was extracted from egg samples including freshly spawned eggs from the three copepod species Apocyclops borneoensis, Centropages tenuiremis and Calanus sinicus, together with 18 resting eggs separated from different sediment layers. A short fragment of the 28S rDNA (~300 bp) sequence that varied between copepod species was amplified and sequenced. These sequences were used to construct a UPGMA tree which helped to assess species composition and the distribution of copepods buried in the sediments. The results showed that C. tenuiremis and Acartia pacifica were closely clustered in the tree with egg samples from deeper sediment layers, whereas A. borneoensis and C. sinicus were grouped with surface egg samples. Species composition in the sediments varied between sediment layers and sampling locations. The DNA extraction method was valid for analysing individual copepod eggs with different egg-spawning types and sizes and the results helped us to reconstruct the copepod egg composition and distribution in the sediments. We believe that the technique has a wide usage in analysing the copepod egg bank in sediments, and possibly even for other zooplankton.

Feeding in the megalopae of the mud crab (Scylla paramamosain): mechanisms, plasticity, role of chelipeds and effect of prey density

We used microscopic video records to analyse the behaviour of mud crab megalopae (Scylla paramamosain) fed on rotifers (Brachiomus spp.), Artemia sp. or copepods (Schmackeria dubin). The megalopae were able to capture prey whose sizes ranged from no larger than Artemia nauplii to no smaller than adult Artemia. The megalopae employed three feeding modes: (1) Ambush-Prey mode, (2) Swim-Suspension-Feed mode and (3) Sit-Sweep mode. These involved raptorial feeding, suspension feeding and an in-between raptorial-suspension feeding mode, depending on the size of the prey and their density in the surrounding water. The chelipeds played an important role in feeding. Megalopae used the chelipeds to grip or sweep prey items and their movement generated eddies that can increase feeding efficiency. To verify the contribution of the chelipeds to feeding efficiency, we observed and compared the animals under three cheliped treatment regimes: (1) Autotomized – chelipeds removed by induced autotomy; (2) Glued – chelipeds glued at the dactylus-propodus joint to eliminate their ability to grip; and (3) Control – normal chelipeds. The feeding rates of the autotomized and glued treatments were lower than those of the controls, demonstrating that the chelipeds assist in feeding but that the megalopae can still feed without them. The density of prey also affected feeding efficiency. Feeding rate and prey density were positively correlated. When prey density was high, megalopae were able to feed in excess of their nutritional requirements. The study shows that mud crab megalopae respond flexibly to variations in feeding conditions, such as damaged chelipeds, as well as prey size and density. We postulate that this plasticity evolved in response to the dilute and patchy prey conditions of the estuarine environment. All the analysed behaviours are illustrated with video sequences.