Shaojing Li

Occurrence of additional Zoea-VI larvae in the mud crab, Scylla paramamosain (Estampador), reared in the laboratory

Mud crabs, Scylla spp. , are commercially important in many Indo-Pacific countries. The larval development of mud crabs has been reported previously as five zoeal and one megalopal stages. This paper reports larval rearing experiments that revealed variability in larval developmental stages in the mud crab Scylla paramamosain, one of four mud crab species. In addition to normal five zoeal stages, an alternative pathway of developing through six zoeal stages was observed for the crab. There were evidences suggested that the appearance of the additional Zoea-VI larvae was associated with unfavourable dietary conditions, including poor quality of diet, inadequate quantity of dietary supply and a period of starvation for newly hatched larvae. Based on exuviae and larval specimens, the morphology of the additional Zoea-VI larvae was described.

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.

Ecdysone receptor in the mud crab Scylla paramamosain: a possible role in promoting ovarian development

In arthropods, it is known that ecdysteroids regulate molting, limb regeneration, and reproduction through activation of the ecdysone receptor (EcR). However, the ecdysteroid signaling pathway for promotion of ovarian development in crustaceans is still unclear. In this study, three cDNA isoforms of EcR were cloned from the mud crab Scylla paramamosain. qRT-PCR revealed that the SpEcR mRNA was abundant in the eyestalk, ovary and epidermis. During ovarian development, the SpEcR transcripts increased from stage I (undeveloped stage) and reached a peak at stage IV (late vitellogenic stage) before dropping to a lower level at stage V (mature stage). Meanwhile, levels of 20-hydroxyecdysone (20E) in the hemolymph, detected by HPLC-MS, displayed a similar pattern of increase with ovarian development. Results from in situ hybridization indicated that SpEcR mRNA was present in the follicular cells during vitellogenesis. Results from in vivo experiments revealed that 20E at 0.2 μg/g body weight significantly stimulated the expression of SpEcR and vitellogenin (SpVg) in female crabs during the early vitellogenic stage but not during the previtellogenic stage. This was confirmed by results from in vitro experiments which indicated that SpEcR and SpVg expression levels were significantly upregulated in early vitellogenic ovarian explants incubated with 5.0 μM 20E at 3 and 6 h but not in previtellogenic ovarian explants. Finally, results from in vitro gene silencing experiments indicated that the expression of SpEcR and SpVg in the ovary was significantly inhibited by SpEcR dsRNA. All these results together indicated that in S. paramamosain, 20E, and SpEcR, located in the follicular cells, play important roles in the promotion of ovarian development via regulating the expression of SpVg.

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.

The retinoid X receptor from mud crab: new insights into its roles in ovarian development and related signaling pathway.

In arthropods, retinoid X receptor (RXR) is a highly conserved nuclear hormone receptor. By forming a heterodimeric complex with the ecdysone receptor (EcR), RXR is known to be vital importance for various physiological processes. However, in comparison to EcR, the RXR signaling pathway and its roles in crustacean reproduction are poorly understood. In the present study, the RXR mRNA was detected in the ovarian follicular cells of mud crab Scylla paramamosain (SpRXR) and during ovarian maturation, its expression level was found to increase significantly. In vitro experiment showed that both SpRXR and vitellogenin (SpVg) mRNA in the ovarian explants were significantly induced by 20-hydroxyecdysone (20E) but not methyl farnesoate (MF). However, differing from the in vitro experiment, injection of MF in in vivo experiment significantly stimulated the expressions of SpRXR and SpVg in female crabs at early vitellogenic stage, but the ecdysone and insect juvenile hormone (JH) signaling pathway genes were not induced. The results together suggest that both MF and SpRXR play significant roles in regulating the expression of SpVg and ovarian development of S. paramamosain through their own specific signaling pathway rather than sharing with the ecdysone or the insect JH.

Immune responses of prophenoloxidase in the mud crab Scylla paramamosain against Vibrio alginolyticus infection: in vivo and in vitro gene silencing evidence.

Phenoloxidase (PO) plays an important role in arthropod melanization. In the present study, a proPO gene was obtained from the mud crab Scylla paramamosain, then we localized the proPO mRNA in hemocytes and detected the expression of proPO after bacterial challenge. In vivo and in vitro gene silencing mediated by dsRNA was also used to investigate the function of proPO in innate immune. The full-length of the proPO cDNA was 2600 bp and the predicted ORF encoded a protein of 673 amino acids with a predicted molecular mass of 77.3 kDa. The deduced amino acid and the main functional domain of proPO shared a high similarity to the mud crab Scylla serrata. In situ hybridization assay showed that the proPO mRNA was localized in the granular and semi-granular cells. The expression level of proPO in hemocytes showed a clear time-dependent pattern during the 96 h course after stimulated by Vibrio alginolyticus. In this study, high expression levels were observed at 3, 12, 24 and 48 h, respectively and the highest expression level was observed at 12 h, and this suggested that proPO was induced by bacteria and involved in immune response. In vivo proPO and GFP dsRNA treatment experiments showed that, proPO mRNA transcript was reduced to 39%, but the PO activity showed no significant difference (P > 0.05). Results indicated that the expression of proPO could be inhibited by dsRNA, and the enzyme activity may be influenced by incomplete knockdown of proPO, or hemocyanin, and other proPO isoforms as well. In vitro proPO-silenced experiments showed that the levels of proPO were decreased by 36%, 64% and 77% at 8, 16 and 32 h, respectively. Meanwhile, the quantity of bacteria was significantly larger in proPO dsRNA treatment than that in control at 3 h, calculated by 4,6-diamino-2-phenylindole staining (P < 0.01). These data demonstrated that the proPO gene plays an important role in the control of systemic bacterial infections and could help us to elucidate the defense role of the proPO-activating system in crabs. In addition, in vitro gene silencing operation mediated by dsRNA was expected to be a new tool for investigating the function of genes in crustaceans in the case of lacking cell line.

The mechanism of regulation of ovarian maturation by red pigment concentrating hormone in the mud crab Scylla paramamosain

In this study a full-length cDNA (Sp-RPCH) was cloned from the eyestalk ganglia of the mud crab Scylla paramamosain. Sp-RPCH is 660 base pairs in length and its open reading frame encodes a precursor that is predicted to be processed into a 25-residue signal peptide, a mature red pigment concentrating hormone (RPCH, an octapeptide), and a 75-residue precursor-related peptide. Phylogenetic analysis indicates that it clusters with other crustacean RPCHs and belongs to the adipokinetic hormone/RPCH peptide superfamily. Sp-RPCH gene expression was detected, using an end-point polymerase chain reaction (PCR), not only in the eyestalk ganglia but also in the brain and thoracic ganglia. Quantified using a real-time PCR, Sp-RPCH gene expression levels in the three tissues fluctuated along a cycle of ovarian maturation, with the levels progressively increased from stages I to IV, after which the expression levels decreased (although they remained significantly higher than stage I levels) when the ovary reached the mature stage (stage V). It was demonstrated using a patch clamp analysis that synthetic RPCH was able to evoke a Ca(2+) current in dissociated brain neurons and synthetic RPCH significantly increased the mean oocyte diameter of the ovarian tissues co-cultured with the eyestalk ganglia, brain, or thoracic ganglia; the stimulatory effect of RPCH was absent when the nervous tissues were not included in the ovarian incubation. Animals administrated with RPCH had significantly higher levels of gonad-somatic index, hepatopancreas-somatic index, and vitellogenin gene expression, when compared to control animals receiving a saline injection. The combined results clearly show that RPCH is involved in ovarian maturation in the mud crab; the stimulatory effects of RPCH are likely mediated by its actions on the release from the nervous tissues of factor(s) that directly regulate vitellogenesis in the ovary and hepatopancreas.

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.

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.