Vibrio splendidus is identified as one of the major pathogenic factors for the skin ulceration syndrome in sea cucumber (Apostichopus japonicus), which has vastly limited the development of the sea cucumber culture industry. In order to screen the immune genes involving Vibrio splendidus challenge in sea cucumber and explore the molecular mechanism of this process, the related transcriptome and gene expression profiling of resistant and susceptible biotypes of sea cucumber with Vibrio splendidus challenge were collected for analysis. A total of 319,455,942 trimmed reads were obtained, which were assembled into 186,658 contigs. After that, 89,891 representative contigs (without isoform) were clustered. The analysis of the gene expression profiling identified 358 differentially expression genes (DEGs) in the bacterial-resistant group, and 102 DEGs in the bacterial-susceptible group, compared with that in control group. According to the reported references and annotation information from BLAST, GO and KEGG, 30 putative bacterial-resistant genes and 19 putative bacterial-susceptible genes were identified from DEGs. The qRT-PCR results were consistent with the RNA-Seq results. Furthermore, many DGEs were involved in immune signaling related pathways, such as Endocytosis, Lysosome, MAPK, Chemokine and the ERBB signaling pathway.

DNA methylation is an important epigenetic modification that regulates many biological processes. The sea cucumber Apostichopus japonicus often suffers from heat stress that affects its growth and leads to significant economic losses. In this study, the mRNA expression patterns and DNA methylation characteristics in the body wall of A. japonicus under heat stress were analyzed by whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing (RNA-seq). We found that CpG was the main DNA methylation type, and heat stress caused a significant increase in the overall methylation level and methylation rate, especially in the intergenic region of the A. japonicus genome. In total, 1,409 differentially expressed genes (DEGs) and 17,927 differentially methylated genes (DMGs) were obtained by RNA-seq and WGBS, respectively. Association analysis between DNA methylation and transcription identified 569 negatively correlated genes in both DMGs and DEGs, which indicated that DNA methylation affects on transcriptional regulation in response to heat stress. These negatively correlated genes were significantly enriched in pathways related to energy metabolism and immunoregulation, such as the thyroid hormone signaling pathway, renin secretion, notch signaling pathway and microRNAs in cancer. In addition, potential key genes, including heat shock protein (hsp70), calcium-activated chloride channel regulator 1(clca1), and tenascin R (tnr), were obtained and their expression and methylation were preliminarily verified. The results provide a new perspective for epigenetic and transcriptomic studies of A. japonicus response to heat stress, and provide a reference for breeding sea cucumbers resistant to high temperatures.

MicroRNAs, as one of the post-transcriptional regulation of genes, play an important role in the development process, cell differentiation and immune defense. The sea cucumber Apostichopus japonicus is an important cold-water species, known for its excellent nutritional and economic value, which usually encounters heat stress that affects its growth and leads to significant economic losses. However, there are few studies about the effect of miRNAs on heat stress in sea cucumbers. In this study, high-throughput sequencing was used to analyze miRNA expression in the body wall of sea cucumber between the control group (CS) and the heat stress group (HS). A total of 403 known miRNAs and 75 novel miRNAs were identified, of which 13 miRNAs were identified as significantly differentially expressed miRNAs (DEMs) in response to heat stress. A total of 16,563 target genes of DEMs were predicted, and 101 inversely correlated target genes that were potentially regulated by miRNAs in response to heat stress of sea cucumbers were obtained. Based on these results, miRNA-mRNA regulatory networks were constructed. The expression results of high-throughput sequencing were validated in nine DEMs and four differentially expressed genes (DEGs) by quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, pathway enrichment of target genes suggested that several important regulatory pathways may play an important role in the heat stress process of sea cucumber, including ubiquitin-mediated proteolysis, notch single pathway and endocytosis. These results will provide basic data for future studies in miRNA regulation and molecular adaptive mechanisms of sea cucumbers under heat stress.

Increasing discoveries of new species of the genus Melanochlamys Cheeseman, 1881 in the recent years indicate that the biodiversity of this group remains underestimated. Recently, several aglajid sea slugs were collected from Laizhou Bay, Shandong Province, China. Morphological observation of their external and internal anatomy revealed that they represent an undescribed species of Melanochlamys. This new species, Melanochlamys aquilina sp. nov., is closely related to Melanochlamys fukudai Cooke, Hanson, Hirano, Ornelas-Gatdula, Gosliner, Chernyshev & Valdés, 2014 in body size and dark external coloration, but can be differentiated from the latter by features of both shell and male reproductive organ. Phylogenetic analyses of two mitochondrial (COI, 16S rRNA) and a nuclear (H3) genes using Bayesian inference, maximum likelihood, and species delimitation analysis also support the separation of Melanochlamys aquilina sp. nov. from its related congeners.

Twenty microsatellite DNA markers were developed for sea cucumber and used to investigate polymorphisms of 60 wild Apostichopus japonicus individuals collected from China. It revealed that all the markers were polymorphic. A total of 164 alleles were detected at 20 loci. The number of alleles per locus varied from 3 to 17 with an average of 8.2, and the expected heterozygosities of each locus ranged from 0.03 to 0.89 with an average of 0.64. Cross-species amplification was also conducted in Parastichopus parvimensis collected from the United States and Pathallus mollis collected from Peru. The result showed that 17 loci amplified Parastichopus parvimensis DNAs while only 4 loci could amplify Pathallus mollis DNAs. All of the polymorphic markers would be useful for future genetic breeding and the assessment of genetic variation within sea cucumbers.