Localization of the Neuropeptide Arginine Vasotocin and Its Receptor in the Osmoregulatory Organs of Black Porgy, Acanthopagrus schlegelii: Gills, Kidneys, and Intestines.

The neurohypophysial hormone arginine vasotocin (avt) and its receptor (avtr) regulates ions in the osmoregulatory organs of euryhaline black porgy (Acanthopagrus schlegelii). The localization of avt and avtr transcripts in the osmoregulatory organs has yet to be demonstrated. Thus, in the present study, we performed an in situ hybridization analysis to determine the localization of avt and avtr in the gills, kidneys, and intestines of the black porgy. The avt and avtr transcripts were identified in the filament and lamellae region of the gills in the black porgy. However, the basal membrane of the filament contained more avt and avtr transcripts. Fluorescence double tagging analysis revealed that avt and avtr mRNAs were partially co-localized with α-Nka-ir cells in the gill filament. The proximal tubules, distal tubules, and collecting duct of the kidney all had positive hybridization signals for the avt and avtr transcripts. Unlike the α-Nka immunoreactive cells, the avt and avtr transcripts were found on the basolateral surface of the distal convoluted tubule and in the entire cells of the proximal convoluted tubules of the black porgy kidney. In the intestine, the avt and avtr transcripts were found in the basolateral membrane of the enterocytes. Collectively, this study provides a summary of evidence suggesting that the neuropeptides avt and avtr with α-Nka-ir cells may have functions in the gills, kidneys, and intestines via ionocytes.

A comparative transcriptomic study on developmental gonads provides novel insights into sex change in the protandrous black porgy (Acanthopagrus schlegelii).

Protandrous black porgy (Acanthopagrus schlegelii) is a popular and valuable commercial marine fish in China and East Asian countries. Controlling and managing its breeding has been an imperative step towards obtaining a sustainable supply of this fish in aquaculture production systems. Therefore, study on the molecular mechanisms of sex change in black porgy has both scientific and commercial importance. Previously, we identified some candidate genes related to sex determination and differentiation from a high-quality genome assembly of the black porgy. In the present study, transcriptome sequencing of developmental gonads (including testis, ovotestis and ovary) of black porgy was performed to further investigate the sex-change mechanisms. Our results showed that the highly expressed male-related genes (dmrt1, piwi1, piwi2, sox9, sox30 and amh) at the male phase were significantly down-regulated to a substantial degree at the intersexual stage, and the female-related genes (jnk1, vasa, wnt4, figla and foxl2) were distinctly up-regulated when the fish grows into a female adult, suggesting the potential roles of these genes in sex change of the black porgy. These data also support a previous hypothesis that the femaleness will be switched on when the testis is entering the degenerated stage through the diminished dmrt1 expression. Our transcriptome data provide a very useful genomic resource for future studies on sex change and practical aquaculture in the black porgy.

The Kiss2/GPR54 system stimulates the reproductive axis in male black porgy, Acanthopagrus schlegelii.

Although it is well established that the Kiss1/GPR54 system stimulates the reproductive axis in mammals, its functional roles, especially in male reproduction of non-mammalian species, is less clear. In this study, we have isolated the full-length kiss2 and gpr54 cDNAs from black porgy (Acanthopagrus schlegelii). The Kiss2 precursor expressed from kiss2 comprises 124 amino acids and contains a highly conserved 10-amino acid sequence, Kiss2-10 (FNFNPFGLRF). GPR54 comprises 375 amino acid residues and contains distinct characteristics of G protein-coupled receptors. Real-time PCR analysis indicated that kiss2 and gpr54 were expressed highly in the brain regions. Moreover, intraperitoneal injection of porgy Kiss2-10 could stimulate genes expression of the gpr54, gnrh1, gnrh3, fshß, lhß, p450c17, star, and ar, and the serum testerone level in male black porgy. Our findings demonstrate that the Kisspeptin stimulates the male reproductive axis in black porgy.

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.

HPLC-MS-based untargeted metabolomic analysis of differential plasma metabolites and their associated metabolic pathways in reproductively anosmic black porgy, Acanthopagrus schlegelii.

Olfaction, a universal form of chemical communication, is a powerful channel for animals to obtain social and environmental cues. The mechanisms by which fish olfaction affects reproduction, breeding and disease control are not yet clear. To evaluate metabolites profiles, plasma from anosmic and control black porgy during reproduction was analyzed by non-targeted metabolomics using ultra high-performance liquid chromatography-mass spectrometry and multivariate statistical analysis techniques, including principal component analysis and orthogonal partial least squares discriminant analysis. The metabolite profiles of anosmia and control groups were found to be significantly separated. Ten different differential metabolites, mainly including amino acids, such as isoleucine and methionine, and lipids, such as phosphatidylserine, were screened based on the combined analysis of variable importance in the projection and p values. In addition, six key differential metabolic pathways were analyzed using the Kyoto Encyclopedia of Genes and Genomes and enriched for four metabolic pathways including the citrate acid (TCA) cycle, tyrosine metabolism, arginine and proline metabolism, and arginine synthesis. The TCA cycle enhances fertility through the reduction of pyruvate kinase, and intermediate derivatives (acetyl CoA, malonyl CoA) act as signaling factors that regulate immune cell function. The tyrosine cycle can indirectly participate and promote reproduction in black porgy through melanin-concentrating hormone. Arginine and proline metabolism can promote reproduction by promoting growth hormone and enhance immunity in anosmic black porgy by stimulating T lymphocytes. Our metabolomic study revealed that anosmia in black porgy played an active role in immunity and reproduction and provided theoretical support for breeding and disease control.

Development of Single Nucleotide Polymorphism and Association Analysis with Growth Traits for Black Porgy (Acanthopagrus schlegelii).

Black porgy is an important marine aquaculture fish species whose production is at the fifth position in all kinds of marine-cultured fishes in China. In this study, Illumina high-throughput sequencing technology was used to sequence the total RNA of black porgy. Sixty-one candidate SNPs (Single Nucleotide Polymorphism) were screened out and genotyped through GATK4 (Genome Analysis ToolKit) software and MALDI-TOF MS (Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry). The experimental results showed that a total of sixty SNPs were successfully genotyped, with a success rate of 98.36%. The results of principal component analysis and correlation analysis of growth traits showed that body weight was the first principal component, with a cumulative contribution rate of 74%. There were significant correlations (p < 0.05) or extremely significant correlations (p < 0.01) between different growth traits. The results of genetic parameter analysis and association analysis showed that scaffold12-12716321, scaffold13-4787950, scaffold2-13687576 and scaffold290-11890 were four SNPs that met the requirement of polymorphic information content and conformed to the Hardy-Weinberg equilibrium. There were significant differences between their genotype and the phenotype of growth traits. The four SNP molecular markers developed in this research will lay a foundation for further exploration of molecular markers related to the growth traits of black porgy and will provide a scientific reference for the further study of its growth mechanisms. At the same time, these molecular markers can be applied to the production practices of black porgy, so as to realize selective breeding at the molecular level and speed up the breeding process.

The Effects of Continuous Acoustic Stress on ROS Levels and Antioxidant-related Gene Expression in the Black Porgy (Acanthopagrus schlegelii).

Hao-Yi Chang, Tzu-Hao Lin, Kazuhiko Anraku, and Yi Ta Shao (2018) Short-term exposure to strong underwater noise is known to seriously impact fish. However, the chronic physiological effects of continuous exposure to weak noise, i.e. the operation noise from offshore wind farms (OWF), remain unclear. Since more and more OWF will be built in the near future, their operation noise is an emerging ecological issue. To investigate the long-term physiological effects of such underwater noise on fish, black porgies (Acanthopagrus schlegelii) were exposed to two types of simulated wind farm noise-quiet (QC: 109 dB re 1 µPa / 125.4 Hz; approx. 100 m away from the wind turbine) and noisy (NC: 138 dB re 1 µPa / 125.4 Hz; near the turbine)-for up to 2 weeks. Measurement of auditory-evoked potentials showed that black porgies can hear sound stimuli under both NC and QC scenarios. Although no significant difference was found in plasma cortisol levels, the fish under NC conditions exhibited higher plasma reactive oxygen species (ROS) levels than the control group at week 2. Moreover, alterations were found in mRNA levels of hepatic antioxidant-related genes (sod1, cat and gpx), with cat downregulated and gpx upregulated after one week of QC exposure. Our results suggest that the black porgy may adapt to QC levels of noise by modulating the antioxidant system to keep ROS levels low. However, such antioxidant response was not observed under NC conditions; instead, ROS accumulated to measurably higher levels. This study suggests that continuous OWF operation noise represents a potential stressor to fish. Furthermore, this is the first study to demonstrate that chronic exposure to noise could induce ROS accumulation in fish plasma.

Morphological Development of Larvae and Juveniles of Acanthopagrus schlegeli.

Newly hatched black porgy larvae (Acanthopagrus schlegeli) swam to the surface, with the mouth and anus still closed and were 1.90-2.11 mm (mean, 2.0 mm) in total length (TL). The larvae were 2.71-2.94 mm TL (mean, 2.82 mm) on day 2 after hatching. At this time, about two-thirds of the yolk was absorbed, the bladder and intestines had formed, and the mouth and anus were open. Total length was 4.32-4.66 mm (mean, 4.45 mm) at the post-larval stage on days 5-6 after hatching, and the yolk and oil globule were almost absorbed. The end of the notochord began to flex, and 6-8 caudal fin rays were visible. The larvae were 15.37-16.1 mm TL (mean, 15.83 mm) at the juvenile stage on days 30-32 after hatching, and the number of rays in all fins was completely revealed.

Biological characteristics and probiotic effect of Leuconostoc lactis strain isolated from the intestine of black porgy fish.

A strain of lactic acid bacteria, Leuconostoc lactis, was isolated from the intestinal tract of black porgy, Sparus macrocephalus, and identified by conventional biochemical characteristics and 16S rDNA gene sequence analysis. The isolated strain had the ability of bile tolerance and resistance to low pH, and survived well in the trypsinase and pepsin solution. But the highly concentrated dose of trypsinase and pepsin affect the viability of the isolated strain. The isolate was resistant to several antibiotics, including Cephalothin, Ceftriaxone, Imipenem and Tobramycin. The isolate could auto-aggregate itself and coaggregate with other bacteria in vitro. The autoaggregation percentage increased to 23.29% after 20 h of incubation. The percentage of coaggregation were respectively 31.21%, 29.44%, 10.74%, 16.49%, 24.36%, 24.41% and 20.99% for Vibrio parahaemolyticus, Listeria monocytogenes, Escherichia coli O157, Salmonella typhimurium, Shigella, Staphylococcus aureus and Proteusbacillus vulgaris after 20 h incubation of a mixed suspension. The supernatant of the strain inhibited the growth of several pathogens, such as V.parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus, Escherichia coli O157, Salmonella typhimurium, Bacillus subtilis, Proteusbacillus vulgaris and Shigella. These results indicated that the isolate, Leuconostoc lactis, might be an attractive candidate for perspectival strain for probiotics in marine aquaculture.

Biological characteristics and probiotic effect of Leuconostoc lactis strain isolated from the intestine of black porgy fish

A strain of lactic acid bacteria, Leuconostoc lactis, was isolated from the intestinal tract of black porgy, Sparus macrocephalus, and identified by conventional biochemical characteristics and 16S rDNA gene sequence analysis. The isolated strain had the ability of bile tolerance and resistance to low pH, and survived well in the trypsinase and pepsin solution. But the highly concentrated dose of trypsinase and pepsin affect the viability of the isolated strain. The isolate was resistant to several antibiotics, including Cephalothin, Ceftriaxone, Imipenem and Tobramycin. The isolate could autoaggregate itself and coaggregate with other bacteria in vitro. The autoaggregation percentage increased to 23.29% after 20 h of incubation. The percentage of coaggregation were respectively 31.21%, 29.44%, 10.74%, 16.49%, 24.36%, 24.41% and 20.99% for Vibrio parahaemolyticus, Listeria monocytogenes, Escherichia coli O157, Salmonella typhimurium, Shigella, Staphylococcus aureus and Proteusbacillus vulgaris after 20 h incubation of a mixed suspension. The supernatant of the strain inhibited the growth of several pathogens, such as V.parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus, Escherichia coli O157, Salmonella typhimurium, Bacillus subtilis, Proteusbacillus vulgaris and Shigella. These results indicated that the isolate, Leuconostoc lactis, might be an attractive candidate for perspectival strain for probiotics in marine aquaculture.