Comprehensive analysis of histophysiology, transcriptome and metabolome tolerance mechanisms in black porgy (Acanthopagrus schlegelii) under low temperature stress.

Low temperature stress has adverse effects on fish growth and reproduction, causing huge economic losses to the aquaculture industry. Especially, black porgy (Acanthopagrus schlegelii) farming industry in north of Yangtze River has been severely affected by low temperature for a long time. To explore the tolerance mechanism of black porgy to low temperature stress, the experiment was designed. The liver and gill tissues of black porgy were taken from the water temperature point of 15 °C (control group named as CG), 3.8 °C (cold sensitive group named as CS) and 2.8 °C (cold tolerant group named as CT) with a cooling rate of 3 °C/d from 15 °C for histophysiology, transcriptomics and metabolomics analysis. After cold stress, the histological results showed that the nucleus of the black porgy liver tissue appeared swelling, the cell arrangement was disordered; meanwhile the gill lamellae were twisted and broken, the epidermis was detached and aneurysm appeared. In addition, the expression of antioxidant, glucose metabolism and immune-related enzymes in the liver and gill of black porgy also changed significantly after low temperature stress. By analyzing the transcriptome and metabolome dates of black porgy liver, 3474 differentially expressed genes (DEGs) and 689 differentially expressed metabolites (DEMs) involved in low temperature stress were identified, respectively. The results of the transcriptome and metabolome combined analysis showed that individuals in the CS group mainly supplied energy to the body through lipid metabolism and amino acid metabolism, and meanwhile the apoptosis pathway was activated. While, individuals in the CT group mainly through glucose metabolism and steroid hormone biosynthesis to supply energy for the body. The validation results of qPCR on eight functional genes further demonstrated the reliability of RNA-Seq data. In summary, the results provide molecular information about adaptation to climate change and genetic selection of black porgy.

Liver Transcriptome Analysis of the Black Porgy (Acanthopagrus schlegelii) under Acute Low-Temperature Stress.

High nutritional value and the development of efficient biotechnological methods of controlled production have made black porgy (Acanthopagrus schlegelii) an economically important fish in Chinese aquaculture in recent years. However, aquaculture production of the species faces multiple issues associated with reduced growth rate, low reproduction ability, and high mortality during production, which are associated with the species' limited tolerance to low temperatures. To date, comprehensive information on the genetic-based mechanisms of cold tolerance and adaptation to low temperature in the species are still unavailable. In this study, the HiSeq™2500 (Illumina) sequencing platform was used to analyze the transcriptomic profile of the liver tissue in the black porgy subjected to different extents of cold shock, including a control temperature group (AS, T = 15 °C), an intermediate temperature group (AL1, T = 10 °C), and an acute low-temperature stress group (AL2, T = 5 °C). For this purpose, three standardized cDNA libraries of AS, AL1, and AL2 were established. We obtained 43,258,908, 48,239,072, and 38,983,833 clean reads from the AS group, AL1 group, and AL2 group, respectively. After pairwise comparison, 70 differentially expressed genes (DEGs) were identified in the examined fish groups. Among them, 60 genes were found to be significantly differentially expressed after trend analysis. GO annotation and enrichment results showed that they were mainly enriched into three categories: biological processes (12 subcategories), molecular functions (7 subcategories), and cellular components (7 subcategories). KEGG analysis results indicated that all significantly differentially expressed genes were annotated to 102 signaling pathways, including biological rhythm, cholesterol metabolism, glycerolipid metabolism, animal autophagy, FoxO signaling pathway, steroid biosynthesis, and regulation of adipocyte lipolysis and apoptosis. Four of them, namely: G6PC, GPX1, GCK, and HSPE1 were randomly selected for further qRT-PCR verification of data reliability obtained by RNA-Seq technology. In this study, we found that environmental acute cold stress mainly affected the black porgy's biological processes related to metabolism, apoptosis, and signal transduction. The data that we have reported provides baseline information for further studies concerning the genetic responses of the black porgy under cold stress conditions, the improvement of its aquaculture production, and other economically important matters regarding their limited tolerance to cold shock.

The complete mitochondrial genome of the spotted knifejaw Oplegnathus punctatus.

In this study, the mitochondrial genome was sequenced in a new commercial species, spotted knifejaw (O. punctatus), using next-generation sequencing and PCR-based methods. The overall length of the female O. punctatus mitochondrial genome was 16,508 bp. It contained 13 PCGs, 2 r-RNA genes, 22 t-RNA genes, and a displacement loop locus (a control region). The total nucleotide composition was 28.75% A, 25.69% T, 29.70% C, and 15.86% G, with a total A + T content of 54.44%. The results demonstrated that the mitochondrial genome of O. punctatus has a high sequence identity with that of another species of Perciformes. This finding provides a deeper understanding of mitogenomic diversity and evolution in marine fish.

Transcriptome Analysis Provides Insights into Copulation, Fertilization, and Gestation in Sebastes schlegelii.

Among the viviparous marine teleosts of China, the black rockfish (Sebastes schlegelii Hilgendorf) is one of the most economically important. In addition to copulation and internal fertilization, it features lengthy sperm storage in the female ovary as well as a high rate of abortion. A network of gene regulation is necessary for these processes. To elucidate the mechanisms of copulation, fertilization, and gestation, it is essential to determine the genetic basis of viviparous teleost oogenesis and embryogenesis. In this study, we analyzed the transcriptome of the ovary during different developmental phases to investigate the dynamic changes that occur. We constructed 24 ovary transcriptomes. In order to investigate the regulation of embryogenesis, differentially expressed genes (DEGs) with specific expression patterns were subjected to gene ontology annotation, pathway analyses, and weighted gene co-expression network analysis (WGCNA). The up-regulated genes were significantly enriched in focal adhesion, regulation of the actin cytoskeleton, Wnt, and ECM-receptor interaction signaling pathways. As a result of our study, we provide omics evidence for copulation, fertilization, and gestation in viviparous marine teleosts. Decoding the S. schlegelii gene regulation network, as well as providing new insights into embryogenesis, is highly valuable to researchers in the marine teleost reproduction sciences.

Characterization of vasa and dnd homologs in summer flounder, Paralichthys dentatus: Expression analysis and colocalization of PGCs during embryogenesis.

Specification of primordial germ cells (PGCs) is particularly important for germline formation. Many maternal-effect genes such as vasa, dnd, and nanos have been identified. However, the research on distribution patterns of PGCs in marine fish is limited. Vasa has been widely used as a germ cell marker to identify its origination in teleosts because vasa RNA is a component of germ plasm. Dnd is known to be an RNA binding protein that protects germline-specific RNAs from degradation. In this study, we isolated full-length vasa and dnd cDNA from summer flounder to track germ cell origination and their expression patterns by RT-PCR and ISH. The results demonstrated that deduced amino acid sequence of Pdvas and Pddnd shared typically conserved motifs of their homologues and demonstrated high identities with other teleosts. Both vasa and dnd transcripts were exclusively detected in germ cells of the gonads. During embryogenesis, vasa and dnd RNA were located at the cleavage furrows of early cleavage stages, and then through proliferation and migration they eventually moved to a location at the predetermined genital ridge. Phylogenetic analysis revealed that summer flounder belongs to the Euteleostei species, but vasa/dnd transcripts localized at the cleavage furrows was similar to that in zebrafish (Osteriophysans). This suggests that germ cells differentiating at early embryogenesis have no direct relation with phylogenesis. At the same time, we found the spatio-temporal expression pattern of dnd was highly consistent with vasa during this process, which indicated the important function of dnd in keeping the target RNA from being degraded to maintain germ cell fate. These results will provide further understanding of germ plasm localization and PGC differentiation in teleosts, and facilitate germ cell manipulation in marine fishes.

Germline Specific Expression of a vasa Homologue Gene in the Viviparous Fish Black Rockfish (Sebastes schlegelii) and Functional Analysis of the vasa 3' Untranslated Region.

Germ cells play a key role in gonad development. As precursors, primordial germ cells (PGCs) are particularly important for germline formation. However, the origination and migration patterns of PGCs are poorly studied in marine fish, especially for viviparous economic species. The vasa gene has been widely used as a germ cell marker to identify a germline because vasa RNA is a component of germ plasm. In this study, we described the expression pattern of black rockfish (Sebastes schlegelii) vasa (Ssvas) in gonadal formation and development by in situ hybridization. The results showed that Ssvas failed in localization at the cleavage furrows until the late gastrula stage, when PGCs appeared and migrated to the genital ridge and formed elongated gonadal primordia at 10 days after birth. This study firstly revealed the PGCs origination and migration characteristics in viviparous marine fish. Furthermore, we microinjected chimeric mRNA containing EGFP and the 3'untranslated region (3'UTR) of Ssvas into zebrafish (Danio rerio) and marine medaka (Oryzias melastigma) fertilized eggs for tracing PGCs. We found that, although Sebastes schlegelii lacked early localization, similar to red seabream (Pagrus major) and marine medaka, only the 3'UTR of Ssvas vasa 3'UTR of black rockfish was able to label both zebrafish and marine medaka PGCs. In comparison with other three Euteleostei species, besides some basal motifs, black rockfish had three specific motifs of M10, M12, and M19 just presented in zebrafish, which might play an important role in labeling zebrafish PGCs. These results will promote germ cell manipulation technology development and facilitate artificial reproduction regulation in aquaculture.