Microsatellite Markers in the Mud Crab (Scylla paramamosain) and their Application in Population Genetics and Marker- Assisted Selection

The mud crab (Scylla paramamosain) is a commercially important species for aquaculture and fisheries in China. In this study, a total of 302 polymorphic microsatellite markers have been isolated and characterized. The observed and expected heterozygosity ranged from 0.04 to 1.00 and from 0.04 to 0.96 per locus. The wild populations distributed along South-eastern China coasts showed high genetic diversity (HO ranged from 0.62 to 0.77) and low genetic differentiation (FST = 0.018). Meanwhile, a significant association (r2 = 0.11) was identified between genetic and geographic distance of 11 locations. Furthermore, a PCR-based parentage assignment method was successfully developed using seven polymorphic microsatellite loci that could correctly assign 95% of the progeny to their parents. Moreover, three polymorphic microsatellite loci were identified to be significantly associated with 12 growth traits of S. paramamosain, and four genotypes were considered to be great potential for marker-assisted selection. Finally, a first preliminary genetic linkage map with 65 linkage groups and 212 molecular markers was constructed using microsatellite and AFLP markers for S. paramamosain. This map was 2746 cM in length, and covered approximately 50% of the estimated genome. This study provides novel insights into genome biology and molecular marker-assisted selection for S. paramamosain

A Nonluminescent and Highly Virulent Vibrio harveyi Strain Is Associated with “Bacterial White Tail Disease” of Litopenaeus vannamei Shrimp

Recurrent outbreaks of a disease in pond-cultured juvenile and subadult Litopenaeus vannamei shrimp in several districts in China remain an important problem in recent years. The disease was characterized by “white tail” and generally accompanied by mass mortalities. Based on data from the microscopical analyses, PCR detection and 16S rRNA sequencing, a new Vibrio harveyi strain (designated as strain HLB0905) was identified as the etiologic pathogen. The bacterial isolation and challenge tests demonstrated that the HLB0905 strain was nonluminescent but highly virulent. It could cause mass mortality in affected shrimp during a short time period with a low dose of infection. Meanwhile, the histopathological and electron microscopical analysis both showed that the HLB0905 strain could cause severe fiber cell damages and striated muscle necrosis by accumulating in the tail muscle of L. vannamei shrimp, which led the affected shrimp to exhibit white or opaque lesions in the tail. The typical sign was closely similar to that caused by infectious myonecrosis (IMN), white tail disease (WTD) or penaeid white tail disease (PWTD). To differentiate from such diseases as with a sign of “white tail” but of non-bacterial origin, the present disease was named as “bacterial white tail disease (BWTD)”. Present study revealed that, just like IMN and WTD, BWTD could also cause mass mortalities in pond-cultured shrimp. These results suggested that some bacterial strains are changing themselves from secondary to primary pathogens by enhancing their virulence in current shrimp aquaculture system

Preference for Shelters at Different Developmental Stages of Chinese Mitten Crab (<i>Eriocheir sinensis</i>)

A shelter is a good habitat for aquatic organisms, which could aid in avoiding cannibalism and facilitate predation. Chinese Mitten Crab (Eriocheir sinensis) is an important aquaculture species with troglodytism and nocturnal habit. To clarify the preference for shelters at different developmental stages of E. sinensis, different shelters (mud, sand, grass and rocks) were selected for comparison. These results indicated that juvenile crabs had a significant preference for grass; button-sized crabs preferred to hide in mud; and the favorite shelters for parent crabs were rocks, followed by mud. E. sinensis in three stages all showed concealing behavior. The concealing behavior of juvenile crabs was the most significant, followed by button-sized and parent crabs. Additionally, E. sinensis held a low hiding rate at night but a high hiding rate during the daytime due to nocturnal habits. These findings will help to better understand the habits of E. sinensis and provide a reference for resource restoration, habitat construction and the restoration of E. sinensis

A New SYBR Green qRT-PCR Diagnostic Method for Screening MCRV-Free Breeding Mud Crabs

Mud crab reovirus (MCRV) is one of the most fatal pathogens of the mud crab Scylla paramamonsain. The outbreak and epidemic of MCRV has seriously affected the healthy development of the mud crab aquaculture industry. To limit MCRV transmission from breeding crabs to larva, we attempt to establish a more sensitive and practical diagnostic method for screening MCRV-free crabs. The primers of the present diagnostic methods for MCRV are based on the VP1 gene (MCRV RNA polymerase gene), and the low expression level of this gene limits the sensitivity of the diagnostic method. Therefore, it is necessary to select the target gene with the highest expression level for the detection primer to improve the sensitivity of the diagnostic method. In addition, the current diagnostic methods require gill samples for virus detection, which requires killing the mud crabs before sampling. This sampling method is obviously not suitable for screening MCRV-free breeding crabs. Therefore, it is necessary to develop a less invasive sampling method for breeding crabs. In this study, a SYBR Green fluorescent quantitative diagnostic method was developed to screen MCRV-free breeding crabs. To improve the sensitivity of the detection, we initially analyzed the relative load of MCRV in the main tissues of infected mud crabs. The viral load in the hemolymph was the highest of all the tissues. The expression levels of 13 putative genes of MCRV were detected in the hemolymph. The relative expression level of the VP11 gene was the highest. Finally, specific primers were designed based on the conserved region of the VP11 gene sequence to establish a SYBR Green qRT-PCR (quantitative reverse-transcription PCR) detection method to accurately detect 50 copies/µL of viral nucleic acid in a sample. Considering the advantages of tissue and target gene selection, the sensitivity of this method should be significantly higher than that of preexisting detection methods. This diagnostic method is very specific for MCRV and no specific amplification was observed using nucleic acid samples containing 5 different kinds of common crustacean pathogens (MCDV, WSSV, DIV1, EHP, and Vibrio parahemolyticus). Compared to other methods of extracting RNA by killing and grinding the gill tissues of crabs, we can select MCRV-free crabs by sampling very small amounts of hemolymph (as low as 20 µL). All of the healthy crabs screened by this method were able to hold eggs that hatched normally. To test the effectiveness of this method, 22 breeding crabs and 20 commercial crabs were screened for MCRV. The positive rates were 54.55% and 85.00%, respectively. In addition, we analyzed the proliferation of MCRV in the mud crabs, and found that MCRV proliferates exponentially in the early stage, then enters a plateau phase, and no crabs died during the infection period of seven days. In conclusion, this study established a highly-sensitive and practical detection method for MCRV in breeding crabs, which can meet the requirements for MCRV-free breeding crab screening with low damage to the breeders. We also investigated the pathogenic infection mechanisms

Transcriptome analysis of the mud crab (Scylla paramamosain) by 454 deep sequencing: assembly, annotation, and marker discovery.

In this study, we reported the characterization of the first transcriptome of the mud crab (Scylla paramamosain). Pooled cDNAs of four tissue types from twelve wild individuals were sequenced using the Roche 454 FLX platform. Analysis performed included de novo assembly of transcriptome sequences, functional annotation, and molecular marker discovery. A total of 1,314,101 high quality reads with an average length of 411 bp were generated by 454 sequencing on a mixed cDNA library. De novo assembly of these 1,314,101 reads produced 76,778 contigs (consisting of 818,154 reads) with 5.4-fold average sequencing coverage. The remaining 495,947 reads were singletons. A total of 78,268 unigenes were identified based on sequence similarity with known proteins (E≤0.00001) in UniProt and non-redundant protein databases. Meanwhile, 44,433 sequences were identified (E≤0.00001) using a BLASTN search against the NCBI nucleotide database. Gene Ontology (GO) analysis indicated that biosynthetic process, cell part, and ion binding were the most abundant terms in biological process, cellular component, and molecular function categories, respectively. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis revealed that 4,878 unigenes distributed in 281 different pathways. In addition, 19,011 microsatellites and 37,063 potential single nucleotide polymorphisms were detected from the transcriptome of S. paramamosain. Finally, thirty polymorphic microsatellite markers were developed and used to assess genetic diversity of a wild population of S. paramamosain. So far, existing sequence resources for S. paramamosain are extremely limited. The present study provides a characterization of transcriptome from multiple tissues and individuals, as well as an assessment of genetic diversity of a wild population. These sequence resources will facilitate the investigation of population genetic diversity, the development of genetic maps, and the conduct of molecular marker-assisted breeding in S. paramamosain and related crab species

Cross-immunity in Nile tilapia vaccinated with Streptococcus agalactiae and Streptococcus iniae vaccines

Streptococcus agalactiae and Streptococcus iniae are major bacterial pathogens of tilapia that can cause high mortality concomitant with large economic losses to aquaculture. Although development of vaccines using formalin-killed bacteria to control these diseases has been attempted, the mechanism of immunity against streptococcal infections and the cross-protective ability of these two bacteria remains unclear. To explore the immunological role of these vaccines, we compared the immune responses of tilapia after immunization with both vaccines and compared the relative percent survival (RPS) and cross-immunization protection of tilapia after separate infection with S. agalactiae and S. iniae. All results revealed that vaccinated fish had significantly higher (P &lt; 0.05) levels of specific antibodies than control fish 14 days post secondary vaccination (PSV) and 7 days post challenge. In vaccinated fish, the mRNA expression of interleukin-8 (IL-8), interleukin-12 (IL-12), caspase-3 (C-3), tumour necrosis factor (TNF), and interferon (IFN) was significantly up regulated (P &lt; 0.05) in the head kidney after immunized; similar results were found for IL-8, TNF and IFN in the posterior kidney, meanwhile the expression levels of C-3 and IFN were significantly increased (P &lt; 0.05) in the spleen of vaccinated fish. Additionally, the levels of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in vaccinated fish were improved at different degree when compared to the control fish. These results showed that vaccination with formalin-killed cells (FKCs) of either S. agalactiae or S. iniae conferred protection against infection by the corresponding pathogen in Nile tilapia, resulting in RPS values of 92.3% and 91.7%, respectively. Furthermore, cross-protection was observed, as the S. agalactiae FKC vaccine protected fish from S. iniae infection, and vice versa. These results suggested that the S. agalactiae and S. iniae FKC vaccines can induce immune responses and generate excellent protective effects in Nile tilapia.</p

The Size distribution of reads generated from 454 FLX platform pyrosequencing.

<p>The Size distribution of reads generated from 454 FLX platform pyrosequencing.</p

Characteristics of reads generated from 454 pyrosequencing.

<p>Characteristics of reads generated from 454 pyrosequencing.</p

Summary of contigs generated by de

<p>Summary of contigs generated by de</p