Reference gene and tropomyosin expression in mud crab Scylla olivacea,Scylla paramamosain and Scylla tranquebarica

Molecular Biology Reports - Tropomyosin, a muscle tissue protein is a major allergen in most of shellfish including mud crab. Quantitative real time-PCR (qRT-PCR) using a stable reference gene is...     

Erk2 in ovarian development of green mud crab Scylla paramamosain

We identified extracellular signal-regulated kinase 2 (erk2) from green mud crab, Scylla paramamosain, in this article. It was originally identified from an expressed sequence tag fragment from a normalized gonadal cDNA library. 5' Rapid amplification of cDNA end (RACE) technique was used to obtain the 5' untranslated region (UTR). The full-length cDNA of Sp-erk2 is 1516 bp, including a 5'-terminal UTR of 19 bp, an open-reading frame of 1098 bp, and a 3'-terminal UTR of 399 bp. The translated protein is 365 amino acids in length with a predicted molecular weight of 42 kDa, which is the same as other species. It is the first time that the expression of Sp-erk2 in different stages of ovary development of crustacean was analyzed, and the result showed that the expression of Sp-erk2 increased gradually with ovarian development, with a peak in the mature phase. In situ hybridization histochemistry was used to clarify the detail of expression. Positive signals illustrated that Sp-erk2 mRNA is present in follicular cells when the ovary is in early stages, and in both follicular cells and oocytes when it is in mature phases. All above suggest that Sp-erk2 is important for ovarian development.     

Comparative profiling of ovarian and testicular piRNAs in the mud crab Scylla paramamosain

PIWI-interacting RNAs (piRNAs) are abundantly found in germ cells and involved in gametogenesis and gonadal development. Information on the regulatory roles of piRNAs in crustacean reproduction, however, is scarce. Thus, we identified gonadal piRNAs of mud crab Scylla paramamosain. Of the 115,491 novel piRNAs, 596 were differentially expressed. Subsequently, 389,887 potential piRNA-target genes were predicted. The expression of 4 piRNAs and 9 genes with high piRNA interactions were validated with the inclusion of additional immature specimens, including LRP2 that is involved in growth and reproduction, MDN1 in ribosome biogenesis pathway and gametogenesis, and PRKDC, a DNA repair gene involved in gonadal differentiation and maturation. KEGG analysis further revealed the involvement of predicted piRNA target genes in gametogenesis- and reproduction-related pathways. Our findings provide baseline information of mud crab piRNAs and their differential expression between testes and ovaries suggests that piRNAs play an essential role in regulating gametogenesis and gonadal development.     

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.     

Identification and comparative analysis of the ovary and testis microRNAome of mud crab Scylla paramamosain

The role of microRNA (miRNA) in reproductive regulation is attracting increasingly more attention. In this study, we obtained 9,643,114 and 15,498,999 raw reads from the ovary and testis library of important farmed mud crab Scylla paramamosain, respectively. After data mining, a total of 4,096,464 and 11,737,973 mappable small RNA sequences remained for analysis. By mapping to the reference genome and expressed sequence tag (EST) of Daphnia pulex and other crabs, a total of 1,417 miRNAs were identified. On the basis of 1,417 miRNAs, 514 (36.3%) unique miRNAs coexpressed in the gonad of female and male libraries, and 336 (23.7%) and 567 (40%) expressed preferentially in female and male libraries, respectively. Analysis of library sequencing data resulted in the identi?cation of 108 miRNAs (out of 1,417; 7.6%) that showed signi?cant differential expression between the two samples. Of these, 13 miRNAs were expressed only in the testis, two miRNAs were expressed only in the ovary, and 93 miRNAs were coexpressed: 57 (61.3%) were upregulated (ovary/testis) and 36 (38.7%) were downregulated (ovary/testis). To confirm the expression patterns of the predicted miRNAs, we randomly selected 14 candidate miRNAs from 108 differentially expressed miRNAs and performed stem–loop real time quantitative PCR (RT‐qPCR) assays in five ovary developing stages. Five miRNAs showed similar expression patterns in almost every stage as those revealed by identification of differentially expressed genes (IDEG6) analysis. The above five miRNAs were predicted to match the 3′‐untranslated region of the published S. paramamosain gene. Four out of five miRNA had a regulation effect on many genes, especially the genes related to gonadal development.     

Inhibitory effects of the androgenic gland on ovarian development in the mud crab Scylla paramamosain

Isolation and characterization of androgenic hormone in decapod crustaceans depend on an effective bioassay of its action. In the present study, the effect of androgenic gland on ovarian development in the mud crab Scylla paramamosain was investigated with a view to develop a bioassay for androgenic hormone. Ovarian regression with degeneration of oocytes occurred in some female crabs implanted with androgenic gland in vivo. In vitro incubation of ovarian tissues at secondary vitellogenesis in extract of androgenic gland resulted in a significant decrease in amino acid uptake by the tissues. We propose that this inhibitory effect could be established as an effective bioassay for the isolation of androgenic hormone in the mud crab.     

Parentage assignment of the mud crab (Scylla paramamosain) based on microsatellite markers

In this study, microsatellite markers were employed to identify the parentage relationship in Scylla paramamosain. The exclusion probability of loci was found to be related with the level of their heterozygosity. When no parent information or only one parent information was available, the exclusion probability ranged from 22.0% to 56.6% and from 41.2% to 73.1%, with the combined exclusion probability for ten loci being 97.0% and 99.8%, respectively. The cumulative assignment success rate was 100% when no parent information was available using seven most informative microsatellite markers. Moreover, the power of the seven microsatellite markers for parentage assignment was tested by a double-blind test, which indicated that 95% of the progeny can be correctly assigned to their parents. This study provided a microsatellite-based approach for parentage assignment in S. paramamosain that will be useful for investigation of genetic background and molecular marker-assisted selective breeding in this important crab species.     

Rapid isolation and characterization of polymorphic microsatellite loci in the mud crab, Scylla paramamosain (Portunidae)

Scylla paramamosain is a widespread and commercially important species of coastal marine crab. We identified 13 polymorphic microsatellite loci from a genome library constructed with 5'-anchored PCR method. Thirty-two S. paramamosain from the East China Sea were used to analyze the characteristics of these loci. The number of alleles per locus ranged from 3 to 8, with a mean of 5.923. Observed and expected heterozygosities ranged from 0.500 to 0.875 and from 0.500 to 0.859, respectively. Eleven of the 13 loci were highly polymorphic (polymorphic information content >0.5). All of the 13 novel loci were in Hardy-Weinberg equilibrium after Bonferroni's correction (P < 0.0038). There was no null allele, stuttering errors or evidence of allelic dropout in any of the loci analyzed by MICRO-CHECKER. According to pairwise tests, no significant linkage disequilibrium was found among the 13 loci (P < 0.0038, adjusted value). These novel developed microsatellites will be useful for studies of genetic variation, population structure, conservation genetics, and molecular-assisted selective breeding of S. paramamosain.     

Characterization and expression of calcium-activated potassium channels (Slo) in ovary of the mud crab,Scylla paramamosain

Large conductance calcium-activated potassium channels (Slo) play important roles in controlling neuronal excitability. At present, very little is known about the function of Slo channels on ovarian development. We cloned the SPSlo gene from the mud crab, Scylla paramamosain. This gene shows 91 and 93 % sequence identity to PISlo from the spiny lobster, Panulirus interruptus and CBSlo from the jonah crab, Cancer borealis, respectively. We isolated six variants of the SPSlo cDNA within S. paramamosain ovary tissue. Sequence analysis indicated that there were at least seven alternative sites in SPSlo, each with multiple alternative segments. Real-time PCR showed that the SPSlo gene was expressed in various tissues, and highly expressed in brain and ovary. In addition, the expression of SPSlo changed throughout ovarian development, highest at the early-developing stage (Stage II) followed by a slow decrease in subsequent stages. These results suggested that SPSlo channels may be implicated in the ovarian development of the mud crab.     

Molecular cloning and expression profiles of nitric oxide synthase (NOS) in mud crab Scylla paramamosain

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in vertebrates and invertebrates in recent years. However, it keeps unknown of nitric oxide (NO) system and NOS gene family in mud crab Scylla paramamosain, an important cultured commercial crustacean in China and Pacific area. In this report, the cDNA of NOS containing full-length ORF was cloned from mud crab, S. paramamosain. It was of 4424 bp, including a 5′-terminal untranslated region (UTR) of 239 bp, a 3′-terminal UTR of 540 bp, which contained two ATTTA motifs, and an open reading frame (ORF) of 3645 bp encoding a polypeptide of 1214 amino acids. Structural analysis indicated that NOS contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time PCR analysis revealed S. paramamosain NOS (SpNOS) to be expressed in all tissues examined, with the highest expression in midintestine and the weakest level in heart and eyestalk. The expression profiles of SpNOS indicated that the NOS expression levels were significantly induced in midintestine, hepatopancrease and hemocytes after challenged with Vibrio Parahaemolyticus, the synthetic double-stranded RNA polyinosinic polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). The NOS activity in hemocytes showed significant increase during at 24 h-48 h time period after immune challenges with V. Parahaemolyticus, poly I:C and LPS. Results here may suggest that the inducible NOS play an important role in mud crab’s defense against pathogenic infection.     

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.