Comprehensive Analysis of Whole-Transcriptome Profiles in Response to Acute Hypersaline Challenge in Chinese Razor Clam Sinonovacula constricta.

The Chinese razor clam (Sinonovacula constricta) is an important for Chinese aquaculture marine bivalve that naturally occurs across intertidal and estuarine areas subjected to significant changes in salinity level. However, the information on the molecular mechanisms related to high salinity stress in the species remain limited. In this study, nine gill samples of S. constricta treated with 20, 30, and 40 ppt salinity for 24 h were used for whole-transcriptome RNA sequencing, and a regulatory network of competing endogenous RNAs (ceRNAs) was constructed to better understand the mechanisms responsible for adaptation of the species to high salinity. A total of 83,262 lncRNAs, 52,422 mRNAs, 2890 circRNAs, and 498 miRNAs were identified, and 4175 of them displayed differential expression pattern among the three groups examined. The KEGG analyses of differentially expressed RNAs evidenced that amino acid synthesis and membrane transport were the dominant factors involved in the adaptation of the Chinese razor clam to acute salinity increase, while lipid metabolism and signaling played only a supporting role. In addition, lncRNA/circRNA-miRNA-mRNA regulatory networks (ceRNA network) showed clearly regulatory relationships among different RNAs. Moreover, the expression of four candidate genes, including tyrosine aminotransferase (TAT), hyaluronidase 4 (HYAL4), cysteine sulfinic acid decarboxylase (CSAD), and ∆1-pyrroline-5-carboxylate synthase (P5CS) at different challenge time were detected by qRT-PCR. The expression trend of TAT and HYAL4 was consistent with that of the ceRNA network, supporting the reliability of established network. The expression of TAT, CSAD, and P5CS were upregulated in response to increased salinity. This might be associated with increased amino acid synthesis rate, which seems to play an essential role in adaptation of the species to high salinity stress. In contrast, the expression level of HYAL4 gene decreased in response to elevated salinity level, which is associated with reduction Hyaluronan hydrolysis to help maintain water in the cell. Our findings provide a very rich reference for understanding the important role of ncRNAs in the salinity adaptation of shellfish. Moreover, the acquired information may be useful for optimization of the artificial breeding of the Chinese razor clam under aquaculture conditions.

Intrageneric Relationship of Datnioides (Lobotiformes) Inferred from the Complete Nuclear Ribosomal DNA Operon.

Tiger fish (genus Datnioides) are critical ornamental and economic fish and are valuable freshwater fish worldwide, belonging to the order Lobotiformes. Currently, there are five extant species (Datnioides campbelli, D. microlepis, D. polota, D. pulcher, and D. undecimradiatus) of Datnioides in the world, usually inhabiting in south and southeast Asia. Due to the decline of wild population sizes of tiger fish and the lack of molecular research on them, in the present study, we sequenced, assembled, and characterized the complete nuclear ribosomal DNA (nrDNA) operon of all five extant tiger fish species, in order to elucidate the phylogenetic relationship among the genus Datnioides. The nrDNA sequences of five tiger fish species were 8548-9182 bp in length, encompassing complete 18S rDNA, ITS1, 5.8S rDNA, ITS2, 28S rDNA, and IGS regions. Numerous repetitive sequences were detected, substantially influencing the sequence length of different regions in each species. We employed maximum-likelihood (ML) method and Bayesian inference (BI) method to construct phylogenetic trees for Datnioides. Phylogenetic analyses indicated that each region in nrDNA operon is not sufficiently phylogenetically informative to delineate the species in Datnioides; nevertheless, the whole operon is able to delineate five tiger fish species much better, three of five species were successfully partitioned. Particularly, regardless of employed markers, it was strongly supported that D. campbelli was considerably partitioned from the other four species, possibly due to the geographical separation. In spite of the fact that discrimination of Datnioides species requires further investigation, our study provides reference genome resources for the Lobotiformes, as well as insights into the phylogenetic position of Lobotiformes and further biological conservation.

Sequencing and analysis of the complete mitochondrial genome of Datnioides campbelli (Datnioididae).

In this study, the complete mitochondrial genome sequence of the New Guinea tiger fish Datnioides campbelli (Whitley 1938) (Lobotiformes: Datnioididae) was sequenced by next-generation sequencing method. The assembled mitochondrial genome consists of 13 protein-coding genes, 22 transfer RNA genes, and two ribosomal RNA genes, with a length of 16,416 bp. The total base composition of the mitogenome of D. campbelli was 29.31% for A, 29.02% for C, 15.14% for G and 26.54% for T. A phylogenetic tree based on 13 protein-coding genes (PCGs) provides important molecular data for further phylogeographic and evolutionary analysis of Lobotiformes.

The complete mitochondrial genome of Comaster schlegelii (Crinoidea, Comatulida).

Comaster schlegelii, belonging to the family Comatulidae, is a variable feather star distributed in the Pacific Ocean. The complete mitochondrial genome of this species was 15,887 bp in length, consisting of 13 protein-coding genes, 22 transport RNA genes, two ribosomal RNA genes and one control region. The whole mitochondrial genome of C. schlegelii had a high AT content of 72.73%. The phylogenetic relationship was reconstructed with 16 relevant echinoderms, which revealed C. schlegelii was closely clustered with Anneissia pinguis in the family Comatulidae.

Effects of graded levels of starch on the non-specific immune responses, antioxidant capacities and intestinal health in Chinese mitten crab, Eriocheir inensis.

A 9-week feeding trial was conducted to investigate the effects of graded levels of dietary starch (12%, 17%, 22%, 27% and 32%) on growth, non-specific immune responses, antioxidant capacities, immunity genes expression levels and pathogen resistance in Chinese mitten crab, Eriocheir inensis (initial body weight: 10.5 ± 0.5 g). Results showed that the highest weight gain rate of crabs was obtained in group containing 22% dietary starch. The highest activity of acid phosphatase, phenoloxidase and lysozyme in blood was found in crabs fed with 22-27% dietary starch. Additionally, 17%-27% dietary starch significantly increased the activities of superoxide dismutase and glutathione peroxidase, reduced malondinaldehyde content and then increased the total antioxidant capacities in hepatopancreas of crabs. The highest activity of alanine aminotransferase and aspartate aminotransferase was found in crabs fed with 32% dietary starch, indicating that excess starch had a negative effect on the liver function of crabs. With the dietary starch level increased, the transcription factors gene expression of the pro-inflammatory factors were significantly up-regulated, and the highest ILF2, IL-16, Relish and ADAM10 was found in crabs fed with dietary 32% starch, which may potentially promote the inflammatory response in intestines. Moreover, with the dietary starch increased, the activity of phenoloxidase and lysozyme, as well as the gene expression of crustin, were all increased in crabs after challenge against Citrobacter freundii, which demonstrated that additional dietary starch could provide immune-protection and help crabs improve their resistance against pathogens. In conclusion, these results suggest that adequate dietary starch can increase growth, enhance innate immune responses and promote disease resistance, reduce oxidative stress and inflammatory response in E. inensis. Taken together, 22-27% dietary starch (25.9-30.8% dietary carbohydrate) was suggested as a digestible energy source in crabs feed.

Simultaneous Determination of Malachite Green, Chloramphenicols, Sulfonamides, and Fluoroquinolones Residues in Fish by Liquid Chromatography-Mass Spectrometry.

A fast-analytical method using simplified extraction has been developed for the simultaneous determination of 42 compounds from 4 different classes of veterinary drugs (amphenicols, triphenylmethane, fluoroquinolones, and sulfonamides) in fish by reverse phase liquid chromatography-tandem mass spectrometry. The selection of extraction reagents was optimized using different types of microfiltration membrane, mobile phase, and LC column. Samples were extracted using 0.4% hydrochloric acid in acetonitrile and ethyl acetate and then were cleaned up using solid-phase extraction Cleanert Alumina N columns (500 mg) and Oasis hydrophilic-lipophilic balance (HLB) cartridges. The chromatographic separation was performed on a XR-ODS C8 column using a mobile phase of (A) 0.1% formic acid and 2 mM ammonium acetate and (B) 0.1% formic acid acetonitrile at a flow rate of 0.25 mL·min-1. The results indicated 67.7-112.8% recovery of 42 compounds with an intra- and interday relative standard deviations less than 10%. The limits of quantification for analytes were in the range of 0.3-1.0 µg kg-1 for samples which were satisfactory to support future surveillance monitoring. The method applicability was checked by analyzing 30 fish samples collected from local markets. Two fish samples surpassed the established MRL of 100 µg kg-1 with values of 104 µg kg-1 and 112 µg kg-1.