First account of a transient intersex in spotted scat, Scatophagus argus: a marine gonochoristic fish.

This study presents the first incidence of intersex associated with testis-ova in spotted scat (Scatophagus argus) reared in a controlled environment. The testis-ova is associated with the abnormal occurrence of primary oocytes (POs) in some male testis and is referred to as ectopic primary oocytes (Ecto-PO), whiles individuals with Ecto-PO are called "Ecto-PO gonad/individuals." We investigated gonads of 129 male spotted scat aged 4-12 and 18 months after hatch (mah) by histological studies for the presence of female sexual characteristics. A total of 20 out of 88 gonads representing 22.7% of male fish aged 6-12, or 15.5% of all male fish sampled, were found to have visible Ecto-PO. At least, the Ecto-PO had an average of 7 oocytes per gonadal section, indicating high severity. The Ecto-PO appears after sex differentiation and degenerates during sexual maturation. The Ecto-PO did not significantly influence the expression pattern of male and female sex-related genes performed using qPCR. Immunofluorescence of 42sp50 specifically stained the Ecto-PO without influence from the surrounding testicular tissues. In addition, temperature did not correlate with the proliferation of the Ecto-PO, but rather gonad developmental strategy. The results show that the naturally occurring Ecto-PO might not be detrimental to testis development and could be considered a frequent-high-level incidence of natural aberration. This study highlights the intricacy of fish sex differentiation and provides a new research chapter to ascertain the mystery behind the occurrence of Ecto-PO.

Comparative transcriptome analysis of male and female gonads reveals sex-biased genes in spotted scat (Scatophagus argus).

Scatophagus argus is a new emerging aquaculture fish in East and Southeast Asia. To date, research on reproductive development and regulation in S. argus is lacking. Additionally, genetic and genomic information about reproduction, such as gonadal transcriptome data, is also lacking. Herein, we report the first gonadal transcriptomes of S. argus and identify genes potentially involved in reproduction and gonadal development. A total of 136,561 unigenes were obtained by sequencing of testes (n = 3) and ovaries (n = 3) at stage III. Genes upregulated in males and females known to be involved in gonadal development and gametogenesis were identified, including male-biased dmrt1, amh, gsdf, wt1a, sox9b, and nanos2, and female-biased foxl2, gdf9, bmp15, sox3, zar1, and figla. Serum estradiol-17ß and 11-ketotestosterone levels were biased in female and male fish, respectively. Sexual dimorphism of serum steroid hormone levels were interpreted after expression analysis of 20 steroidogenesis-related genes, including cyp19a1a and cyp11b2. This gonadal transcript dataset will help investigate functional genes related to reproduction in S. argus.

High Polymorphism in the Dmrt2a Gene Is Incompletely Sex-Linked in Spotted Scat, Scatophagus argus.

Unlike mammals and birds, many fishes have young sex chromosomes, providing excellent models to study sex chromosome differentiation at early stages. Previous studies showed that spotted scat possesses an XX-XY sex determination system. The X has a complete Dmrt3 copy (termed normal) and a truncated copy of Dmrt1 (called Dmrt1b), while the Y has the opposite (normal Dmrt1, which is male-specific, and a truncated Dmrt3 called Dmrt3△-Y). Dmrt1 is the candidate sex determination gene, while the differentiation of other sex-linked genes remains unknown. The spotted scat has proven to be a good model to study the evolution of sex chromosomes in vertebrates. Herein, we sequenced a neighbor gene of this family, Dmrt2, positioned farther from Dmrt1 and closer to Dmrt3 in the spotted scat, and analyzed its sequence variation and expression profiles. The physical locations of the three genes span across an estimated size of >40 kb. The open reading frames of Dmrt2a and its paralog Dmrt2b are 1578 bp and 1311 bp, encoding peptides of 525 and 436 amino acid residues, respectively. Dmrt2a is positioned close to Dmrt3 but farther from Dmrt1 on the same chromosome, while Dmrt2b is not. Sequence analysis revealed several mutations; insertions, and deletions (indels) on Dmrt2a non-coding regions and single-nucleotide polymorphisms (SNPs) on the Dmrt2a transcript. These indels and SNPs are sex-linked and showed high male heterogeneity but do not affect gene translation. The markers designed to span the mutation sites tested on four different populations showed varied concordance with the genetic sexes. Dmrt2a is transcribed solely in the gonads and gills, while Dmrt2b exists in the gonads, hypothalamus, gills, heart, and spleen. The Dmrt2a and Dmrt2b transcripts are profoundly expressed in the male gonads. Analyses of the transcriptome data from five other fish species (Hainan medaka (Oryzias curvinotus), silver sillago (Sillago sihama), Nile tilapia (Oreochromis niloticus), Hong Kong catfish (Clarias fuscus), and spot-fin porcupine fish (Diodon hystrix)) revealed testes-biased expression of Dmrt1 in all, similar to spotted scat. Additionally, the expression of Dmrt2a is higher in the testes than the ovaries in spotted scat and Hainan medaka. The Dmrt2a transcript was not altered in the coding regions as found in Dmrt1 and Dmrt3 in spotted scat. This could be due to the functional importance of Dmrt2a in development. Another possibility is that because Dmrt2a is positioned farther from Dmrt1 and the chromosome is still young, meaning it is only a matter of time before it differentiates. This study undeniably will aid in understanding the functional divergence of the sex-linked genes in fish.

Effects of probiotics on digestive enzymes of fish (finfish and shellfish); status and prospects: a mini review.

Digestive enzymes are found in the digestive tract of animals which assist in the breakdown of larger food molecules into more easily absorbed particles that can then be used by the body. The ability of fish to break down a diet is highly dependent on the availability of suitable digestive enzymes which mediate specific degradation pathways and on both the physical and chemical nature of food. Probiotics are known to produce helpful enzymes that aid in digestion and protect the gastrointestinal tract (GIT) of animals. When applied appropriately, probiotics improve intestinal microbial balance which also improves digestive enzyme activities, food absorption, and decrease pathogenic issues in the GIT. They work hand-in-hand with the digestive enzymes in the GIT of animals as supplements thereby improvings nutrition. This in turn leads to higher feed efficiency and growth as well as the prevention of antinutritional factors present in the ingredients, intestinal disorders, and pre-digestion. This review seeks to present summaries of the results of research findings on the application of probiotics on the activities of digestive enzymes including amylase, lipase, and protease. Further, this review points out gaps in available literature and suggests ideas that could be explored in further investigations to better understand and enhance the activities of these digestive enzymes to increase feed and nutrient utilization and the production of aquaculture species.

Expression and regulation of 42Sp50 in spotted scat (Scatophagus argus).

42Sp50 is an isoform of the eukaryotic translation elongation factor 1 A (eEF1A) and is vital for fish ovarian development. Spotted scat (Scatophagus argus) is a popular marine cultured fish species in Southern Asia and China, and its artificial reproduction is complicated, with a relatively low success ratio in practice. In this study, the 42Sp50 gene was cloned from spotted scat. Tissue distribution analysis showed that 42Sp50 was mainly expressed in the ovary. qRT-PCR showed that 42Sp50 expression levels gradually decreased insignificantly in the ovaries from phase II to IV. Western blot analysis showed that 42Sp50 was highly expressed in the ovary, while it was almost undetectable in the testis. Immunohistochemistry analysis stained 42Sp50 mainly in the cytoplasm of the previtellogenic oocytes in ovaries of normal XX-female and sex-reversed XY-female. Aside from fish and amphibians, 42Sp50 was also identified in some reptile species using genomic database searching. Analyses of the transcriptome data from four different fish species (Hainan medaka (Oryzias curvinotus), silver sillago (Sillago sihama), Nile tilapia (Oreochromis niloticus), and Hong Kong catfish (Clarias fuscus)) revealed ovaries biased expression of 42Sp50 in all, similar to spotted scat. While the neighbor genes of 42Sp50 did not show ovary biased expression in the fish species analyzed. Bisulfite Sequencing PCR (BSP) results showed that the DNA methylation level of 42Sp50 promoter was low in ovaries, testes, and muscles. The luciferase reporter assay demonstrated that Dmrt4 activated 42Sp50 expression in the presence of Sf1 or Foxh1. These results suggest that 42Sp50 may be involved in regulating the early phase oocytes development of spotted scat.

Homozygous Mutation of gsdf Causes Infertility in Female Nile Tilapia (Oreochromis niloticus).

Gonadal somatic cell-derived factor (Gsdf) is a member of the TGF-ß superfamily, which exists mainly in fishes. Homozygous gsdf mutations in Japanese medaka and zebrafish resulted in infertile females, and the reasons for their infertility remain unknown. This study presents functional studies of Gsdf in ovary development using CRISPR/Cas9 in Nile tilapia (Oreochromis niloticus). The XX wild type (WT) female fish regularly reproduced from 12 months after hatching (mah), while the XX gsdf-/- female fish never reproduced and were infertile. Histological observation showed that at 24 mah, number of phase IV oocyte in the XX gsdf-/- female fish was significantly lower than that of the WT fish, although their gonadosomatic index (GSI) was similar. However, the GSI of the XX gsdf-/- female at 6 mah was higher than that of the WT. The mutated ovaries were hyperplastic with more phase I oocytes. Transcriptome analysis identified 344 and 51 up- and down-regulated genes in mutants compared with the WT ovaries at 6 mah. Some TGF-ß signaling genes that are critical for ovary development in fish were differentially expressed. Genes such as amh and amhr2 were up-regulated, while inhbb and acvr2a were down-regulated in mutant ovaries. The cyp19a1a, the key gene for estrogen synthesis, was not differentially expressed. Moreover, the serum 17ß-estradiol (E2) concentrations between XX gsdf-/- and WT were similar at 6 and 24 mah. Results from real-time PCR and immunofluorescence experiments were similar and validated the transcriptome data. Furthermore, Yeast-two-hybrid assays showed that Gsdf interacts with TGF-ß type II receptors (Amhr2 and Bmpr2a). Altogether, these results suggest that Gsdf functions together with TGF-ß signaling pathway to control ovary development and fertility. This study contributes to knowledge on the function of Gsdf in fish oogenesis.

Fish Feed Intake, Feeding Behavior, and the Physiological Response of Apelin to Fasting and Refeeding.

Feed is one of the most important external signals in fish that stimulates its feeding behavior and growth. The intake of feed is the main factor determining efficiency and cost, maximizing production efficiency in a fish farming firm. The physiological mechanism regulating food intake lies between an intricate connection linking central and peripheral signals that are unified in the hypothalamus consequently responding to the release of appetite-regulating genes that eventually induce or hinder appetite, such as apelin; a recently discovered peptide produced by several tissues with diverse physiological actions mediated by its receptor, such as feed regulation. Extrinsic factors have a great influence on food intake and feeding behavior in fish. Under these factors, feeding in fish is decontrolled and the appetite indicators in the brain do not function appropriately thus, in controlling conditions which result in the fluctuations in the expression of these appetite-relating genes, which in turn decrease food consumption. Here, we examine the research advancements in fish feeding behavior regarding dietary selection and preference and identify some key external influences on feed intake and feeding behavior. Also, we present summaries of the results of research findings on apelin as an appetite-regulating hormone in fish. We also identified gaps in knowledge and directions for future research to fully ascertain the functional importance of apelin in fish.

The Roles of Neuropeptide Y (Npy) and Peptide YY (Pyy) in Teleost Food Intake: A Mini Review.

Neuropeptide Y family (NPY) is a potent orexigenic peptide and pancreatic polypeptide family comprising neuropeptide Y (Npy), peptide YYa (Pyya), and peptide YYb (Pyyb), which was previously known as peptide Y (PY), and tetrapod pancreatic polypeptide (PP), but has not been exhaustively documented in fish. Nonetheless, Npy and Pyy to date have been the key focus of countless research studies categorizing their copious characteristics in the body, which, among other things, include the mechanism of feeding behavior, cortical neural activity, heart activity, and the regulation of emotions in teleost. In this review, we focused on the role of neuropeptide Y gene (Npy) and peptide YY gene (Pyy) in teleost food intake. Feeding is essential in fish to ensure growth and perpetuation, being indispensable in the aquaculture settings where growth is prioritized. Therefore, a better understanding of the roles of these genes in food intake in teleost could help determine their feeding regime, regulation, growth, and development, which will possibly be fundamental in fish culture.

Characterization, expression, and regulatory effects of nr0b1a and nr0b1b in spotted scat (Scatophagus argus).

Nuclear receptor subfamily 0 group B member 1 (Nr0b1) belongs to the nuclear receptor (NR) superfamily. It plays critical roles in sex determination, sex differentiation, and gonadal development in mammals. In this study, the duplicated genes nr0b1a and nr0b1b were identified in spotted scat (Scatophagus argus). Phylogenetic and synteny analyses revealed that, unlike nr0b1a, nr0b1b was retained in several species of teleosts after an nr0b1 gene duplication event but was secondarily lost in other fish species, amphibians, reptiles, birds, and mammals. In a sequence analysis, only 1.5 LXXLL-related repeat motifs were identified in spotted scat Nr0b1a, Nr0b1b, and non-mammalian Nr0b1a/Nr0b1, different from the 3.5 repeat motifs in mammalian Nr0b1. By qPCR, nr0b1a and nr0b1b were highly expressed in testes from stages IV to V and in ovaries from stages II to IV, respectively. Male-to-female sex reversal was induced in XY spotted scat by the administration of exogenous E2. A qPCR analysis showed that nr0b1b mRNA expression was higher in sex-reversed XY fish than in control XY fish, with no difference in nr0b1a. A luciferase assay showed that spotted scat Nr0b1a and Nr0b1b did not individually activate cyp19a1a gene transcription. As in mammals, spotted scat Nr0b1a suppressed Nr5a1-mediated cyp19a1a expression, despite containing only 1.5 LXXLL-related repeat motifs in its N-terminal region, while Nr0b1b stimulated Nr5a1-mediated cyp19a1a transcription. These results demonstrated that nr0b1a and nr0b1b in spotted scat have distinct expression patterns and regulatory effects and further indicate that nr0b1b might be involved in ovarian development by regulating Nr5a1-mediated cyp19a1a expression.

A Chromosome-Level Genome Assembly of the Spotted Scat (Scatophagus argus).

The spotted scat, Scatophagus argus is a member of the family Scatophagidae found in Indo-Pacific coastal waters. It is an emerging commercial aquaculture species, particularly in East and Southeast Asia. In this study, the first chromosome-level genome of S. argus was constructed using PacBio and Hi-C sequencing technologies. The genome is 572.42 Mb, with a scaffold N50 of 24.67 Mb. Using Hi-C data, 563.28 Mb (98.67% of the genome) sequences were anchored and oriented in 24 chromosomes, ranging from 12.57 Mb to 30.38 Mb. The assembly is of high integrity, containing 94.26% conserved single-copy orthologues, based on BUSCO analysis. A total of 24,256 protein-coding genes were predicted in the genome, and 96.30% of the predicted genes were functionally annotated. Evolutionary analysis showed that S. argus diverged from the common ancestor of Japanese puffer (Takifugu rubripes) approximately 114.8 Ma. The chromosomes of S. argus showed significant correlation to T. rubripes chromosomes. A comparative genomic analysis identified 49 unique and 90 expanded gene families. These genomic resources provide a solid foundation for functional genomics studies to decipher the economic traits of this species.

Genome Survey of Male and Female Spotted Scat (Scatophagus argus).

The spotted scat, Scatophagus argus, is a species of fish that is widely propagated within the Chinese aquaculture industry and therefore has significant economic value. Despite this, studies of its genome are severely lacking. In the present study, a genomic survey of S. argus was conducted using next-generation sequencing (NGS). In total, 55.699 GB (female) and 51.047 GB (male) of high-quality sequence data were obtained. Genome sizes were estimated to be 598.73 (female) and 597.60 (male) Mbp. The sequence repeat ratios were calculated to be 27.06% (female) and 26.99% (male). Heterozygosity ratios were 0.37% for females and 0.38% for males. Reads were assembled into 444,961 (female) and 453,459 (male) contigs with N50 lengths of 5,747 and 5,745 bp for females and males, respectively. The average guanine-cytosine (GC) content of the female genome was 41.78%, and 41.82% for the male. A total of 42,869 (female) and 43,283 (male) genes were annotated to the non-redundant (NR) and SwissProt databases. The female and male genomes contained 66.6% and 67.8% BUSCO core genes, respectively. Dinucleotide repeats were the dominant form of simple sequence repeats (SSR) observed in females (68.69%) and males (68.56%). Additionally, gene fragments of Dmrt1 were only observed in the male genome. This is the first report of a genome-wide characterization of S. argus.

Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus).

Gonadal soma-derived factor (Gsdf) is critical for testicular differentiation and early germ cell development in teleosts. The spotted scat (Scatophagus argus), with a stable XX-XY sex-determination system and the candidate sex determination gene dmrt1, provides a good model for understanding the mechanism of sex determination and differentiation in teleosts. In this study, we analyzed spotted scat gsdf tissue distribution and gene expression patterns in gonads, as well as further analysis of transcriptional regulation. Tissue distribution analysis showed that gsdf was only expressed in testis and ovary. Real-time PCR showed that both gsdf and dmrt1 were expressed significantly higher in testes at different phases (phase III, IV and V) compared to ovaries at phase II, III and IV, while gsdf was expressed significantly higher in phase II ovaries than those of phase III and IV. Western blot analysis also showed that Gsdf was more highly expressed in the testis than ovary. Immunohistochemistry analysis showed that Gsdf was expressed in Sertoli cells surrounding spermatogonia in the testis, while it was expressed in the somatic cells surrounding the oogonia of the ovary. Approximately 2.7 kb of the 5' upstream region of gsdf was cloned from the spotted scat genomic DNA and in silico promoter analysis revealed the putative transcription factor binding sites of Dmrt1 and Sf1. The luciferase reporter assay, using the human embryonic kidney cells, demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1 in spotted scat. These results suggest that Gsdf could play a role in regulating the development of spermatogonia and oogonia, and also participate in male sex differentiation by acting as a downstream gene of Dmrt1 in spotted scat.