Jun Ding

Transcriptome Sequencing and Characterization of Japanese Scallop Patinopecten yessoensis from Different Shell Color Lines

Shell color is an important trait that is used in breeding the Japanese scallop Patinopecten yessoensis, the most economically important scallop species in China. We constructed four transcriptome libraries from different shell color lines of P. yessoensis: the left and right shell mantles of ordinary strains of P. yessoensis and the left shell mantles of the ‘Ivory’ and ‘Maple’ strains. These four libraries were paired-end sequenced using the Illumina HiSeq 2000 platform and contained 54,802,692 sequences, 40,798,962 sequences, 74,019,262 sequences, and 44,466,166 sequences, respectively. A total of 214,087,082 expressed sequence tags were assembled into 73,522 unigenes with an average size of 1,163 bp. When the data were compared against the public Nr and Swiss-Prot databases using BlastX, nearly 30.55% (22,458) of the unigenes were significantly matched to known unique proteins. Gene Ontology annotation and pathway mapping analysis using the Kyoto Encyclopedia of Genes and Genomes categorized unigenes according to their diverse biological functions and processes and identified candidate genes that were potentially involved in growth, pigmentation, metal transcription, and immunity. Expression profile analysis was performed on all four libraries and many differentially expressed genes were identified. In addition, 5,772 simple sequence repeats were obtained from the P. yessoensis transcriptomes, and 464,197, 395,646, and 310,649 single nucleotide polymorphisms were revealed in the ordinary strains, the ‘Ivory’ strain, and the ‘Maple’ strain, respectively. These results provide valuable information for future genomic studies on P. yessoensis and improve our understanding of the molecular mechanisms involved in the growth, immunity, shell coloring, and shell biomineralization of this species. These resources also may be used in a variety of applications, such as trait mapping, marker-assisted breeding, studies of population genetics and genomics, and work on functional genomics.

Embryotoxicity and teratogenicity of pesticide indoxacarb to sea urchin (Strongylocentrotus intermedius)

Sperm cell and embryo toxicity tests using the sea urchin Strongylocentrotus intermedius (S. intermedius) were performed to assess the toxicity of indoxacarb, a new widely used insecticide. New toxicity data for indoxacarb expressed as median effective concentration (EC(50)) were reported for the sea urchin species. When sperms and cells were exposed to the pesticide before fertilization, no significant inhibition in the fertilization success of S. intermedius (up to 40 mg/L) was observed. Developmental toxicity of the pesticide displayed a significant dose-related increase of larval malformations and differentiation arrest at concentrations of 0.1 mg/L to 40.0 mg/L at each cleavage, including the 2-cell stage, 4-cell, blastula, gastrula, prism and 4-arm pluteus stages. It seems that 4-arm pluteus is the most sensitive to indoxacarb with the EC(50) of 3.73 mg/L, two times less than that of the first cleavage stage. All these results indicate that more attentions should be paid to the potential marine pollutions caused by this pesticide indoxacarb.

Effects of high temperature on the immuno-enzymetic activity in coelomic fluid of Mizuhopecten yessoensis

In this study,we tested the high temperature mutation tolerance,survival and immune enzyme activity under different high temperature levels of Mizuhopecten yessoensis.There are two stages in our laboratory experiments.Firstly,we transferred M.yessoensis from the rearing temperature(15 ℃,control temperature)to 20,22,24and 26 ℃,and then we tested the survival and related immune indicators of M.yessoensis.The survival of M.yessoensis was not affected by temperature from 15 ℃ to 22 ℃ with survival rate exceeding 85.21%,and there is no significant difference in different groups(P0.05),M.yessoensis exposed to 26 ℃ were significantly affected by temperature,with a survival rate of 26.33%.With the temperature rising,the T-AOC and MDA content in the body cavity fluid of M.yessoensis significantly changed(P0.05).The CAT vitality would rise after the initial drop with the rise of temperature.There were no significant differences among different groups,SOD vitality(P0.05).Secondly,we abruptly transferred scallops from the rearing temperature(15 ℃,control temperature)to 20,22,24 and 26 ℃ levels,and examined related immune indexes at 1,2,4,8,12,24,48 and 96 h.The results showed that,the survival of M.yessoensis was not affected by temperature from 15 ℃ to 24 ℃ during the 96-h exposure periods with survival rate exceeding 82.29%.M.yessoensis exposed to 26 ℃ were significantly affected by temperature,with a survival rate of 0% after 12 h.The 8,12,24,48 and 96 h lethal temperature for 50%(LT50)were 27.52,24.41,24.37,24.24 and 23.81 ℃ respectively.

SLAF-based high-density genetic map construction and QTL mapping for major economic traits in sea urchin Strongylocentrotus intermedius

Sea urchin (Strongylocentrotus intermedius) has long been a model species for developmental and evolutionary research, but only a few studies have focused on gene mapping. Here, we reported a high-density genetic map containing 4,387 polymorphism specific-length amplified fragment (SLAF) markers spanning 21 linkage groups (LG) for sea urchin. Based on this genetic map and phenotyping data for eight economic traits, 33 potentially significant QTLs were detected on ten different LGs with explanations ranging from 9.90% to 46.30%, partly including 10 QTLs for test diameter, six QTLs for body weight and eight QTLs for Aristotle’s lantern weight. Moreover, we found a QTL enrichment LG, LG15, gathering QTLs for test diameter, body weight, gonad weight, light orange-yellow color difference (≥E1) and light yellow color difference (≥E2). Among all QTLs, we genotyped four QTLs for test diameter, Aristotle’s lantern weight and body weight using High Resolution Melting (HRM) technology. Finally, we used the verified SNP marker (detected using SLAF sequencing) to explore their marker-assisted selection (MAS) breeding application potential and found that SNP-29 associated tightly with body weight and that heterozygous genotype was a dominant genotype, indicating that SNP-29 was a promising marker for MAS.

Molecular characterization and expression of NLRP10 in the antibacterial host defense of the sea cucumber (Apostichopus japonicus)

The nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs) can regulate the innate immune process and is an important part of inflammatory body. In this study, we use transcriptome sequencing and the rapid amplification of cDNA ends approach to identify a novel NLRP gene in Apostichopus japonicus. We designated the gene as AjNLRP10. The full-length of AjNLRP10 is 4509 bp. The putative open reading frame comprising 3489 bp encodes a polypeptide with 1162 amino acid residues. The predicted molecular mass of AjNLRP10 is 132.87 kDa and its theoretical pI is 5.60. AjNLRP10 comprises a signal peptide with two Ig superfamily (IgSF) domains and a NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] domain. Spatial distribution expression analysis detected AjNLRP10 in all of the tissues tested, but with higher expression in the coelomocytes, medium expression in the intestine and respiratory tree, and slightly weaker expression in the body wall, tube feet, and longitudinal muscle. The expression levels of AjNLRP10 in the respiratory tree and intestines of sea cucumbers with skin ulceration syndrome were increased by 4-fold and 2.7-fold compared with those in healthy sea cucumbers, respectively. We investigated expression profiles of AjCasepase-1 (Cysteinyl aspartate specific proteinase-1) and AjMMP37 (mitochondrial protein-37) after AjNLRP10 knock-down and discovered that AjCasepase-1 was raised by 2.60-fold and AjMMP37 was raised by 3.84-fold. The study showed that AjNLRP10 has inhibitory effect in the immune process. In conclusion, this study showed that the AjNLRP10 protein found in the sea cucumber involved with the innate immune responses against bacterial infection. It has a similar structure and biological function to that in other organisms, where it appears to be involved with these results provide insights into the innate immune mechanism in the sea cucumber as well as suggesting new strategies for disease prevention, molecular therapy, and the development of novel drugs for sea cucumbers.

Transcriptome analysis of male and female mature gonads of Japanese scallop Patinopecten yessonsis

The Japanese scallop Patinopecten yessoensis is an important commercial culture shellfish in China and Japan. In addition to its commercial interest, it has also attracted much attention because of its value in studying sex determination and differentiation mechanisms. Herein, two transcriptome libraries from male and female gonads at maturing stage were constructed. The two libraries were paired-end sequenced using Illumina sequencing techniques. A total of 81,986,898 cleaned reads were obtained, and assembled into 29,897 unigenes by using Trinity software and removing redundancy. Compared with the public Swiss-Prot and NR databases by BlastX, 9354 unigenes were significantly matched to known unique proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes uncovered diverse biological functions and processes. After comparing the two transcriptome libraries, 8652 up-regulated and 2973 down-regulated genes were identified in the male gonads. According to annotation information, at least 30 genes related to sex determination and differentiation, such as Dmrt1, Sox9, fem1 and vasa, were identified and characterized. The expression patterns of the random eight genes were then validated by quantitative real-time PCR (qRT-PCR) suggesting the high reliability of RNA-Seq results. The study provides an archive for further studies of molecular mechanisms of bivalve sex determination and differentiation.