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Abstract Acrossocheilus fasciatus is a stream-dwelling fish species of the Barbinae subfamily. It is valued for its colorfully striped appearance and delicious meat. This species is also characterized by apparent sexual dimorphism and toxic ovum. Biology and aquaculture researches of A. fasciatus are hindered by the lack of a high-quality reference genome. Here, we report chromosome-level genome assemblies of the male and female A. fasciatus. The HiFi-only genome assemblies for both female and male individuals were 899.13 Mb (N50 length of 32.58 Mb) and 885.68 Mb (N50 length of 33.06 Mb), respectively. Notably, a substantial proportion of the assembled sequences, accounting for 96.15% and 98.35% for female and male genomes, respectively, were successfully anchored onto 25 chromosomes utilizing Hi-C data. We annotated the female assembly as a reference genome and identified a total of 400.62 Mb (44.56%) repetitive sequences, 27,392 protein-coding genes, and 35,869 ncRNAs. The high-quality male and female reference genomes will provide genomic resources for developing sex-specific molecular markers, inform single-sex breeding, and elucidate genetic mechanisms of sexual dimorphism.

Carassius auratus herpesvirus (CaHV) is a pathogen isolated from crucian carp (Carassius auratus) associated with high mortality. A diagnosis method that can detect the virus at an early stage, specifically and accurately, is an urgent requirement for the prevention of CaHV transmission. In the present study, a droplet digital PCR (ddPCR) method based on the tumor necrosis factor receptor (TNFR) gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses. Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection, and positive amplification was detected on the first day by ddPCR (0.54 copies/μL), whereas the presence of CaHV was not detected by routine PCR until Day 6. Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment. The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR (qPCR), respectively. The results showed that the positive detection rate of ddPCR (100%) was higher than that of qPCR (40%). The detection limit of the ddPCR was found to be 0.52 copies/μL, which was much lower than the 50.12 copies/μL determined by qPCR. Overall, ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.

Cyprinid herpesvirus 2 (CyHV-2) is a pathogen that causes significant losses to the global aquaculture industry due to mass mortality in crucian carp and goldfish. This study demonstrates that the ORF55/ORF57 deletion mutants CyHV-2-Δ55-CP and CyHV-2-Δ57-CP obtained through homologous recombination replicate effectively within the caudal fin of Carassius auratus gibelio (GiCF) cells and exhibit morphologies similar to the CyHV-2 wild-type strain. Both mutants demonstrated a decrease in virulence, with CyHV-2-Δ57-CP exhibiting a more significant reduction. This serves as a reference for the subsequent development of recombinant attenuated vaccines against CyHV-2. Additionally, both mutants expressed the inserted RGNNV-CP (capsid protein of Redspotted grouper nervous necrosis virus) fusion protein gene, and inoculation with CyHV-2-Δ57-CP-infected GiCF cell lysates elicited an antibody response in the grouper. These results indicate that, while ORF55 and ORF57 genes of CyHV-2 are not required for viral replication in vitro, they do play a role in virulence in vivo. Additionally, expression of foreign protein in CyHV-2 suggests that the fully attenuated mutant of CyHV-2 could potentially function as a viral vector for developing subunit vaccines or multivalent recombinant attenuated vaccines.

IntroductionRibonucleotide reductase (RR) is essential for the replication of the double-stranded DNA virus CyHV-2 due to its ability to catalyze the conversion of ribonucleotides to deoxyribonucleotides, and is a potential target for the development of antiviral drugs to control CyHV-2 infection.MethodsBioinformatic analysis was conducted to identify potential homologues of RR in CyHV-2. The transcription and translation levels of ORF23 and ORF141, which showed high homology to RR, were measured during CyHV-2 replication in GICF. Co-localization experiments and immunoprecipitation were performed to investigate the interaction between ORF23 and ORF141. siRNA interference experiments were conducted to evaluate the effect of silencing ORF23 and ORF141 on CyHV-2 replication. The inhibitory effect of hydroxyurea, a nucleotide reductase inhibitor, on CyHV-2 replication in GICF cells and RR enzymatic activity in vitro was also evaluated.ResultsORF23 and ORF141 were identified as potential viral ribonucleotide reductase homologues in CyHV-2, and their transcription and translation levels increased with CyHV-2 replication. Co-localization experiments and immunoprecipitation suggested an interaction between the two proteins. Simultaneous silencing of ORF23 and ORF141 effectively inhibited the replication of CyHV-2. Additionally, hydroxyurea inhibited the replication of CyHV-2 in GICF cells and the in vitro enzymatic activity of RR.ConclusionThese results suggest that the CyHV-2 proteins ORF23 and ORF141 function as viral ribonucleotide reductase and their function makes an effect to CyHV-2 replication. Targeting ribonucleotide reductase could be a crucial strategy for developing new antiviral drugs against CyHV-2 and other herpesviruses.

The gonad specific expression of insulin-like growth factor 3 (igf3) has suggested an important role of igf3 in fish reproduction. In this study, medaka igf3 was isolated and its expression patterns were compared with igf1, and germ cell gene vasa in adult medaka ovary. Molecular cloning and sequencing showed that the open reading frame (ORF) of medaka igf3 was comprised of 501 nucleotides and encoded 166 amino acid residues. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that igf3 RNA was specifically expressed in developing embryos and adult gonads of both sexes. Real time quantitative PCR (q-PCR) indicated that igf3 expression was gradually increased during oogenesis, and reached its highest level at stage V. Using Chromogenic and fluorescent in situ hybridization, igf3 was shown to be present in the germ cells of oogonia and in oocytes at stages I-III, as well as abundant in granulosa cells and theca cells of oocytes at stages IV–V. In addition, igf3 and igf1 were expressed mutually in the outer theca cells of stage V oocytes in ovary. Collectively, we demonstrated that gonad specific igf3 could mark medaka ovarian somatic cells and germ cells. These findings highlight the importance of igf3 during ovarian development.

Diseases of grass carps often occur in juveniles but not in adults that may have established disease resistance during development. We performed both DNA bisulfite and transcriptome sequencing on liver libraries of 1- and 3-year-old grass carps. Differential DNA-methylation analysis exhibited a declined methylation level through development. Functional annotation revealed that identified differentially methylated genes (DMGs) and differentially expressed genes were enriched in immune-related pathways such as PI3K-Akt signaling pathway and its related pathways. Both differentially methylated and differentially expressed genes were clustered into the growth- and immune-related function networks. Subcellular localization analysis indicated that the DMGs localized on cell membrane were significantly enriched in calcium signaling pathway and neuroactive ligand-receptor interaction pathway, implying the importance of G protein-coupled receptors to development. These findings will broaden our understanding of the key genes and pathways that affect the immune system at different development stages and the developing protective strategies in grass carp.

Numerous studies have showed that sox3 is involved in neurogenesis and sex differentiation in vertebrates. However, the accurate expression pattern is still unclear in fish. In this study, medaka sox3 was isolated and its expression patterns were compared with germ cell gene vasa in adult gonads. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, sox3 RNA expression was detected in the brain, eyes and gonads. By chromogenic and fluorescent in situ hybridization, sox3 was present in the oogonia and early stages of oocytes as well as granulosa cells and theca cells in the later stages; in the testis, sox3 was occurred in the Sertoli cells and sperm with a specific signal in the location of chromatid body of sperm; in the embryos, sox3 was expressed in the central nervous. These results suggest that medaka sox3 gene was occurred in somatic and germ cells in adult gonads of both sexes, involved in the process of spermatogenesis, as well as the development of the nervous system. This study provides a precise expression pattern of sox3 and suggests that sox3 may be involved in gonadal and nervous system development of fish.

Germ cells of many organisms exhibit a unique perinuclear cytoplasmic cloud-like structure called the chromatoid body (CB). Specific components of the CB, such as vasa, are crucial for germline development. Herein we identify another component of the CB, the Oryzias latipes gcnf homolog (Olgcnf). By reverse transcription polymerase chain reaction (RT-PCR), Olgcnf transcript was restricted to gonads in the adult tissues and was maternally provided and persisted throughout embryogenesis. By in situ hybridization on gonadal sections, Olgcnf transcript was restricted to germ cells. In the ovary, Olgcnf was predominantly expressed in the early stages of oocytes. In the testis, Olgcnf showed dynamic expression in round spermatids and sperm, accumulated in the CB during spermatid morphogenesis and concentrated in elongated spermatids of acrosome cap region and flagellar manchette. Moreover, Olgcnf was colocalized with vasa, which is the best-characterized component of the CB. Therefore, Olgcnf is specific to germ cells of both sexes and is a component of the CB during spermiogenesis.

Aquatic viruses are naturally present in the aquatic environment and the number of viruses is staggering. Various multicellular organisms in aquatic ecosystems may be infected, cross-species transmitted, manipulated, and killed by aquatic viruses, which can lead to cascading ecological effects. The viruses in unicellular aquatic organisms can alter interactions between host individuals, and are essential in effecting or maintaining the dynamics of aquatic microbial communities, horizontal gene transfer, biodiversity, and modulating ecological processes globally. Meanwhile, hosts also impact viral abundance and diversity. Microbial diversity drives multifunctionality in ecosystems, while viruses shape complex microbial communities and are crucial for ecosystem functioning. This review focuses on molecular, genetic, evolutionary, and ecosystemic advances related to emerging and reemerging aquatic viruses, presents the contexts, novel tools, and investigative approaches pertaining to the study of aquatic virology, and discusses the mechanisms by which viruses affect aquatic ecosystems. The paper provides an efficient and broadly-based blueprint for improving understanding of aquatic viruses.

Mytilus coruscus is a significant economic species in China’s eastern coastal areas. As a dioecious species, it lacks secondary sexual characteristics, which makes it difficult for selective breeding. However, limited research is carried out on the genetic data regarding reproductive development and gender differentiation. In the current study, de novo transcriptome sequencing analyses were used to detect gonad-specific genes and miRNAs in M. coruscus. By comparing testis and ovary, 159,970 unigenes and 300 miRNAs were obtained totally, of which differentially expressed genes and miRNAs were 9,566 and 25, respectively. Analysis of qRT-PCR showed that cyp26a, dmrt4, foxl2, gdf9, 17β-hsd14, sc6a9, zar1, and zp4 were highly expressed in the ovary as compared to the testis, while sox2 showed lower expression in the ovary. Expression of miR-750-3p, novel 1, and miR-193 was higher in the ovary than that in the testis, whereas the expression of miR-9-5p, miR-9-3p, miR-317, novel 124, miR-2d and miR-263b were lower in the ovary. Furthermore, analysis of miR-317 by Targetscan and MiRanda predicted to target dmrt4 and the luciferase reporter was performed to confirm it. Our research provides a molecular basis for understanding sexual development and reproductive regulation. Further research is needed on the mechanism of gonadal maturation and differentiation in M. coruscus.

Crucian carp (Carassius auratus) is one of the major freshwater species and is also a common food fish in China. Recently, Carassius auratus herpesvirus (CaHV) could induce fatal viral disease with high mortality of crucian carp, which had caused huge economic losses. In this study, we described a rapid and simple recombinase-aid amplification (RAA) assay coupled with lateral flow dipstick (LFD), which could achieve sensitive diagnosis of tumor necrosis factor receptor (TNFR) of CaHV within 35 min at 40°C. Our RAA-LFD method had a satisfactory detection limit of 100 gene copies per reaction, which was 100-fold more sensitive than traditional PCR. In addition, no cross-reaction was observed with other viral pathogens, including koi herpesvirus (KHV), cyprinid herpesvirus 2 (CyHV-2), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Furthermore, the overall cost of the method was cut in half compared to previous studies. In conclusion, RAA-LFD assay is therefore, a promising alternative for point-of-care testing (POCT) of CaHV, which is feasible and of certain value in application of aquatic disease control.

As an economically important fish, Opsariichthys bidens has obvious sexual dimorphism and strong reproductive capacity, but no epigenetics study can well explain its phenotypic variations. In recent years, many microRNAs involved in the regulation of reproductive development have been explored. In this study, the small RNA libraries of O. bidens on the testis and ovary were constructed and sequenced. A total of 295 known miRNAs were obtained and 100 novel miRNAs were predicted. By comparing testis and ovary libraries, 115 differentially expressed (DE) miRNAs were selected, of which 53 were up-regulated and 62 were down-regulated. A total of 64 GO items (padj < 0.01) and 206 KEGG pathways (padj < 0.01) were enriched in the target gene of miRNA. After that, the expression levels of nine DE miRNAs, including let-7a, miR-146b, miR-18c, miR-202-5p, miR-135c, miR-9-5p, miR-34c-3p, miR-460-5p and miR-338 were verified by qRT-PCR. Furthermore, bidirectional prediction of DE miRNAs and sex-related genes was carried out and the targeting correlation between miR-9-5p and nanos1 was verified by Dual-Luciferase reporter assay. Our findings identified the differentially expressed miRNA and paved the way to new possibilities for the follow-up study on the mechanism of miRNA-mRNA interaction in the gonads of O. bidens.

The process by which spermatogonial stem cells (SSCs) continuously go through mitosis, meiosis, and differentiation to produce gametes that transmit genetic information is known as spermatogenesis. Recapitulation of spermatogenesis in vitro is hindered by the challenge of collecting spermatogonial stem cells under long-term in vitro culture conditions. Coilia nasus is a commercially valuable anadromous migrant fish found in the Yangtze River in China. In the past few decades, exploitation and a deteriorating ecological environment have nearly caused the extinction of C. nasus’s natural resources. In the present study, we established a stable spermatogonial stem cell line (CnSSC) from the gonadal tissue of the endangered species C. nasus. The cell line continued to proliferate and maintain stable cell morphology, a normal diploid karyotype, and gene expression patterns after more than one year of cell culture (>80 passages). Additionally, CnSSC cells could successfully differentiate into sperm cells through a coculture system. Therefore, the establishment of endangered species spermatogonial stem cell lines is a model for studying spermatogenesis in vitro and a feasible way to preserve germplasm resources.

Macrobrachium rosenbergii (M. rosenbergii), as a species of common prawn, is a delicacy that is consumed all over the world. By interacting with the target gene 3′-untranslated region (3'-UTR), microRNAs (miRNAs) regulate its expression and ultimately participate in the regulation of reproductive development. However, research focusing on miRNA regulation during gonadal development in M. rosenbergii received very little attention. To explore the association between miRNA and reproduction, we performed RNA sequencing (RNA-seq) on brain and gonad organs in male and female M. rosenbergii. A total of 494 miRNAs were obtained in RNA-seq, including 31 and 59 differentially expressed (DE) miRNAs in the brain and gonads, respectively. Furthermore, 9 DE miRNAs were randomly selected from the brain and gonads, and qRT-PCR was conducted to validate the results of RNA-seq. Interestingly, dpu-miR-133 was found to be substantially expressed in the male brain and testis but poorly expressed in the female brain, ovary, and other organs. Analysis of dpu-miR-133 by Targetscan and MiRanda predicted to target 5-HT1. Furthermore, the dual-luciferase reporter assay manifested that dpu-miR-133 can combine with 5-HT1. Overall, our research work provides basic data for further study on the miRNA-mediated regulation of brain, gonad, and reproductive development of study M. rosenbergii.

Macrobrachium rosenbergii is an important aquaculture prawn that exhibits sexual dimorphism in growth, with males growing much faster than females. However, the mechanisms controlling these complex traits are not well understood. The nervous system plays an important role in regulating life functions. In the present work, we applied PacBio RNA-seq to obtain and characterize the full-length transcriptomes of the brains and thoracic ganglia of female and male prawns, and we performed comparative transcriptome analysis of female and male prawns. A total of 159.1-Gb of subreads were obtained with an average length of 2175 bp and 93.2% completeness. A total of 84,627 high-quality unigenes were generated and annotated with functional databases. A total of 6367 transcript factors and 6287 LncRNAs were predicted. In total, 5287 and 6211 significantly differentially expressed genes (DEGs) were found in the brain and thoracic ganglion, respectively, and confirmed by qRT-PCR. Of the 435 genes associated with protein processing pathways in the endoplasmic reticula, 42 DEGs were detected, and 21/26 DEGs with upregulated expression in the male brain/thoracic ganglion. The DEGs in this pathway are regulated by multiple LncRNAs in polypeptide folding and misfolded protein degradation in the different organs and sexes of the prawn. Our results provide novel theories and insights for studying the nervous system, sexual control, and growth dimorphism.

The hook snout carp Opsariichthys bidens is an important farmed fish in East Asia that shows sexual dimorphism in growth, with males growing faster and larger than females. To understand these complex traits and improve molecular breeding, chromosome-level genome assembly of male O. bidens was performed using Illumina, Nanopore, and Hi-C sequencing. The 992.9 Mb genome sequences with a contig N50 of 5.2 Mb were anchored to 38 chromosomes corresponding to male karyotypes. Of 30,922 functionally annotated genes, 97.5% of BUSCO genes were completely detected. Genome evolution analysis showed that the expanded and contracted gene families in the male O. bidens genome were enriched in 76 KEGG pathways, and 78 expanded genes were involved in the GnRH signaling pathway that regulates the synthesis and secretion of luteinizing hormone and glycoprotein hormones, further acting on male growth by inducing growth hormone. Compared to the released female O. bidens genome, the number of annotated genes in males was much higher (23,992). The male chromosome LG06 exhibited over 97% identity with the female GH14/GH38. Male-specific genes were identified for LG06, where structural variation, including deletions and insertions, occurred at a lower rate, suggesting a centric fusion of acrocentric chromosomes GH14 and GH38. The genome-synteny analysis uncovered significant inter-chromosome conservation between male O. bidens and grass carp, the former originating from ancestral chromosome breakage to increase the chromosome number. Our results provide a valuable genetic resource for studying the regulation of sexual dimorphism, sex-determining mechanisms, and molecular-guided breeding of O. bidens.

Streptococcus agalactiae (group B streptococcus, GBS), a broad-spectrum pathogen, causes great economic losses in fish aquaculture, especially the industry of tilapia. Until now, the knowledge of the immune response mechanism against S. agalactiae infection in tilapia has been limited. In the present study, the gill transcriptome of the tilapia from the GBS and the phosphate buffered saline (PBS) groups were sequenced. The transcriptomic analysis results presented the differentially expressed genes (DEGs) at different time points (DEGs number, 6 h: 2122, 9 h: 1851, 15 h: 1791, and 18 h: 2395) after GBS injection, and significantly enriched immune-related gene ontology (GO) terms such as the innate immune response. The significantly enriched immune pathways included the Toll-like receptor signaling pathway, the nucleotide oligomerization domain (NOD)-like receptor signaling pathway, the cytosolic-DNA sensing pathway, and the intestinal immune network for Immunoglobulin A (IgA) production. Most of the DEGs in Toll-like receptor signaling, NOD-like receptor signaling, and cytosolic-DNA sensing pathways presented upregulations at 18 h, which indicated that the innate immune pathways were activated. Two immune-related pathways (phagosome and cell adhesion molecules) were significantly enriched at all time points, suggesting that these two pathways might also play important roles in the immune response against the GBS infection. The results of HE staining showed that the gills of tilapia were damaged seriously at 9 h post-infection, which might be due to the possibility of pyroptosis resulting from the changes of DEGs in the NOD-like receptor signaling pathway. This study provided new insight into the mechanisms of gill damage in fish infected with S. agalactiae.

A highly lethal freshwater crayfish Cambarus clarkii whispovirus (WSSV-Cc) was isolated from a naturally infected host. A large number of viral particles with typical nimavirus characters were observed in host cells. Genomic architecture and genetic evolution of this extremely virulent pathogen were uncovered. WSSV-Cc has a double-stranded DNA genome containing 287,179 bp, encoding 180 putative ORFs. Congruence analysis was performed between WSSV-Cc and known nimavirus genomes. The genome of WSSV-Cc is smaller than that found in isolates sampled before 2010, but is similar in size to that of recent isolates, which provided new evidence for the shrinkage of nimavirus genome size during evolution. Phylogenetic analysis with the complete genome also showed connections between genome size and evolutionary relationship. Twenty low-similarity genes and three variable regions were identified from the WSSV-Cc genome. In addition, two recombination events were detected in the WSSV-Cc genome, and analysis of the recombinant regions showed a close relationship among the three freshwater crayfish nimaviruses. These findings provide us the morphological, genomic, and genetic characters of an emerging whispovirus, which will broaden our understanding to nimaviruses and be useful for diagnosis of crayfish viral disease.

MicroRNAs (miRNAs) are regarded as key regulators in gonadal development and sex determination in diverse organisms. However, the functions of miRNAs in gonads of Acrossocheilus fasciatus, an economically important freshwater species in the south of China, are still unclear. Here, high-throughput sequencing was performed to investigate the mRNA and miRNAs on gonads of A. fasciatus. In total, 49,447 unigenes were obtained, including 11,635 differentially expressed genes (DEGs), among which 4147 upregulated genes and 7488 downregulated genes in the testis compared to the ovary, while 300 (237 known, and 63 novel) miRNAs with 36 differentially expressed miRNAs (DEMs) were identified, from which 17 upregulated and 19 downregulated DEMs. GO and KEGG enrichment analysis were performed to analyze the potential biological functions of DEGs and DEMs. Using qRT-PCR, 9 sex-related genes and 9 miRNAs were selected to verify the sequencing data. By dual-luciferase reporter assay, miR-22a-5p and miR-22b-5p interaction with piwil1, and miR-10d-5p interaction with piwil2 were identified. These findings could provide a reference for miRNA-regulated sex control of A. fasciatus and may reveal new insights into aquaculture and breeding concepts.

Spinyhead croaker (Collichthys lucidus) is an economically important fish suffering from population decline caused by overfishing and habitat destruction. Researches on the development of primordial germ cell (PGC) and reproduction biology were an emergency for the long-term conservation of the involved species. Dead end (dnd) gene plays an indispensable role in PGC specification, maintenance, and development. In the current study, we report the cloning and expression patterns of dnd in C. lucidus (Cldnd). RT-PCR analysis revealed that Cldnd was specifically expressed in both sexual gonads. In the ovary, Cldnd RNA was uniformly distributed in the oocytes and abundant in oogonia, and gradually decreased with oogenesis. A similar expression pattern was also detected in testis. Dual fluorescent in situ hybridization of Cldnd and Clvasa demonstrated that they almost had the same distribution except in oocytes at stage I, in which the vasa RNA aggregated into some particles. Furthermore, Cldnd 3′ UTR was sufficient to guide the Green Fluorescent Protein (GFP) specifically and stably expressed in the PGCs of medaka. These findings offer insight into that Cldnd is an evolutionarily conserved germline-specific gene and even a potential candidate for PGC manipulation in C. lucidus.

Coilia nasus is an important economic anadromous migratory fish of the Yangtze River in China. In recent years, overfishing and the deterioration of the ecological environment almost led to the extinction of the wild resources of C.nasus. Thus, there is an urgent need to protect this endangered fish. Recently, cell lines derived from fish have proven a promising tool for studying important aspects of aquaculture. In this study, a stable C. nasus gonadal somatic cell line (CnCSC) was established and characterized. After over one year of cell culture (>80 passages), this cell line kept stable growth. RT-PCR results revealed that the CnGSC expressed some somatic cell markers such as clu, fshr, hsd3β, and sox9b instead of germ cell markers like dazl, piwi, and vasa. The strong phagocytic activity of CnGSC suggested that it contained a large number of Sertoli cells. Interestingly, CnGSC could induce medaka spermatogonial cells (SG3) to differentiate into elongated spermatids while co-cultured together. In conclusion, we established a C. nasus gonadal somatic cell line capable of sperm induction in vitro. This research provides scientific evidence for the long-term culture of a gonadal cell line from farmed fish, which would lay the foundation for exploring the regulatory mechanisms between germ cells and somatic cells in fish.

Opsariichthys bidens belongs to the family Cyprinidae and is a small freshwater economic fish widely distributed in China. In recent years, the natural resources of O. bidens have been drastically reduced due to overfishing and the destruction of the water environment. The in vitro culture and long-term preservation of germ stem cells are the key technologies to keep genetic resources from degeneration. However, except for the establishment of the first long-term cultured medaka spermatogonia cell line (SSC) capable of producing sperm in vitro in 2004, no other long-term cultured SSC line has been found in other fish species. In this study, we successfully established another long-term-cultured spermatogonial stem cell line from Opsariichthys bidens (ObSSC). After more than 2 years of culture, ObSSC had a diploid karyotype and stable growth, with the typical gene expression patterns of SSC. Under in vitro culture, ObSSC could be induced to differentiate into sperm and other different types of somatic cells. In vivo, ObSSC could differentiate into different cells of three germ layers upon being transplanted into zebrafish embryos. Our research helps to explore the potential and regulation mechanism of fish SSC differentiation and spermatogenesis in vitro, provides a new way for solving the problem of fish genetic resource degradation and lays a foundation for further research on fish germ cell transplantation.

PCR-based DNA amplification has been one of the major methods in aquaculture research for decades, although its use outside the modern laboratory environment is limited due to the relatively complex methods and high costs. To this end, we investigated a swabbing and disc protocol for the collection of DNA samples from fish which could extract DNA from fish skin mucus by a non-invasion technique costing only $0.02 (USD) and requiring less than 30 seconds. The disc method that we chose could use the cheap filter paper to extract DNA from above 104 crucian carp blood cells, which is comparable to the commercial kit. By using skin mucus swabbing and the disc method, we can obtain amplification-ready DNA from mucus to distinguish different species from our smallest fish (medaka, ~2.5 cm and crucian carp, ~7 cm) to our biggest fish (tilapia, ~15 cm). Furthermore, the viral pathogen Carassius auratus herpesvirus (CaHV) of crucian carp was detected using our method, which would make performing molecular diagnostic assays achievable in limited-resource settings including aquafarms and aqua stores outside the laboratory environment.

In contrast to fish, amphibians inhabit both aquatic and terrestrial environments. To better understand osmoregulation in fish and amphibian, we have investigated the morphological changes in kidney cells to osmotic stress. To address this, kidney cell line isolated from the freshwater grass carp (CIK) and Chinese giant salamander (GSK) were challenged to different mediums with distinct osmotic pressures (100, 300 and 700 mOsm). Morphological alterations of the fish and amphibian cells were compared by optical and electron microscopy. Following hyposmotic treatment (100 mOsm), both CIK and GSK cells became unhealthy and show condensed chromatin, swollen mitochondria and cytoplasmic vacuole. Meanwhile, after hyperosmotic treatment (700 mOsm), shrunken CIK cells with multipolar shape, pale or lightly stained cytoplasm, condensed chromatin, vacuoles and swollen mitochondria were detected. GSK cells were seriously damaged and most were completely lysed. The results suggest that fish kidney cells show a higher degree of tolerance to hyperosmoticity by comparing to amphibians and provide novel insights on the osmoregulatory capacity and adaptability of kidney cells between the two animal groups.

Aquatic vertebrates are very abundant in the world, and they are of tremendous importance in providing global food security and nutrition. However, emergent and resurgent viruses, such as ranavirus (e.g., Rana grylio virus, RGV and Andriasd avidianus ranavirus, ADRV), herpesvirus (e.g., Carassius carassius herpesvirus, CaHV), reovirus (e.g., grass carp reovirus 109, GCRV-109, Scophthal musmaximus reovirus, SMReV and Micropterus salmoides reovirus, MsReV), and rhabdovirus (e.g., Siniper cachuatsi rhabdovirus, SCRV and Scophthal musmaximus rhabdovirus, SMRV) can cause severe diseases in aquaculture animals and wild lower vertebrates, such as frogs, giant salamanders, fish, and so on. Here, we will briefly describe the symptoms produced by the aforementioned viruses and the molecular basis of the virus–host interactions. This manuscript aims to provide an overview of viral diseases in lower vertebrates with an emphasis on visible symptomatic manifestations and pathogenesis.