Isolation and identification of a megalocytivirus strain (SKIV-TJ) from cultured spotted knifejaw (Oplegnathus punctatus) in China and its pathogenicity analysis.

The spotted knifejaw (Oplegnathus punctatus) has recently emerged as a highly economically significant farmed fish in China. However, due to increasing environmental pollution and breeding density, a range of infectious diseases, including the iridovirus pathogen, have begun to spread widely. In this study, we isolated and identified a strain of Megalocytivirus, SKIV-TJ, from cultured spotted knifejaw in Tianjin, China. We observed significant cytopathic effects (CPE) in SKIV-TJ-infected spotted knifejaw brain (SKB) cells, and electron microscopy showed numerous virus particles in the cytoplasm of SKB cells 6 days post-infection. The annotated complete genome of SKIV-TJ (GenBank accession number ON075463) contained 112,489 bp and 132 open reading frames. Based on the multigene association evolutionary tree using 26 iridovirus core genes, SKIV-TJ was found to be most closely related to Rock bream iridovirus (RBIV). Cumulative mortality of spotted knifejaw infected with SKIV-TJ reached 100% by day 9. A transcriptomic analysis were conducted and a total of 5517 differentially expressed genes were identified, including 2757 upregulated genes and 2760 downregulated genes. The upregulated genes were associated with viral infection and immune signaling pathways. Our findings provide a valuable genetic resource and a deeper understanding of the immune response to SKIV infection in spotted knifejaw.

Tea polyphenols, astaxanthin, and melittin can significantly enhance the immune response of juvenile spotted knifejaw (Oplegnathus punctatus).

The frequent occurrence of diseases seriously hampers the sustainable development of the spotted knifejaw (Oplegnathus punctatus) breeding industry. Our previous genome-wide scan and cross-species comparative genomic analysis revealed that the immune gene family (Toll-like receptors, TLR) members of O. punctatus underwent a significant contraction event (tlr1, tlr2, tlr14, tlr5, and tlr23). To address immune genetic contraction may result in reduced immunity, we investigated whether adding different doses (0, 200, 400, 600, and 800 mg/kg) of immune enhancers (tea polyphenols, astaxanthin, and melittin) to the bait after 30 days of continuous feeding could stimulate the immune response of O. punctatus. We found that the expression of tlr1, tlr14, tlr23 genes in immune organs (spleen and head kidney) was stimulated when tea polyphenols were added at 600 mg/kg. The tlr2 (400 mg/kg), tlr14 (200 mg/kg), tlr5 (200 mg/kg), and tlr23 (200 mg/kg) genes expression of intestine were elevated in the tea polyphenol group. When the addition of astaxanthin is 600 mg/kg, it can effectively stimulate the expression of tlr14 gene in immune organs (liver, spleen and head kidney). In the astaxanthin group, the expression of the genes tlr1 (400 mg/kg), tlr14 (600 mg/kg), tlr5 (400 mg/kg) and tlr23 (400 mg/kg) reached their highest expression in the intestine. Besides, the addition of 400 mg/kg of melittin can effectively induce the expression of tlr genes in the liver, spleen and head kidney, except the tlr5 gene. The tlr-related genes expression in the intestine was not significantly elevated in the melittin group. We hypothesize that the immune enhancers could enhance the immunity of O. punctatus by increasing the expression of tlr genes, and thereby leading to increased resistance to diseases. Meanwhile, our findings further demonstrated that significant increases in weight gain rate (WGR), visceral index (VSI), and feed conversion rate (FCR) were observed at 400 mg/kg, 200 mg/kg and 200 mg/kg of tea polyphenols, astaxanthin and melittin in the diet, respectively. Overall, our study provided valuable insights for future immunity enhancement and viral infection prevention in O. punctatus, as well as offered guidance for the healthy development of the O. punctatus breeding industry.

Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos.

In this study, the toxicity of sucrose to Oplegnathus punctatus embryos was evaluated. Embryos at the 4-6 somite, tail-bud, heart formation, and heart-beating stages were exposed to 0, 0.5, 1,1.5, 2, 2.5, or 3 M sucrose for 1 h. Survival rates of embryos at the tail-bud, heart formation, and heart-beating stages after rehydration for 1 h were not affected by treatment with 2 M sucrose (the maximum concentration). Embryos at the tail-bud, heart formation, and heart-beating stages were exposed to 2 M sucrose for 0, 30, 60, 90, 120, 150, or 180 min. Long-term developmental indicators, including rates of survival, hatching, swimming, and malformation, were evaluated for 4 days after rehydration. Based on the survival rates 10 min after rehydration, the longest tolerance time for embryos at the three stages was 120 min. Based on long-term developmental indicators, the longest tolerance times were 60 min at the tail-bud, 60 min at the heart formation stage and 30 min at the heart beating stage. The malformation rates increased as the treatment time increased. The malformation rates were 100% when embryos were exposed to sucrose for ≥120 min. Malformation was divided into larval and embryonic abnormality. As the exposure time increased for tail-bud stage embryos, the rate of larval malformation increased. Treatment at heart formation and heart-beating stages resulted in higher rates of failure to hatch at exposure time. Based on these results, toxicity tests of non-permeable cryoprotectant in embryos requires the observation of development for at least 2 days after rehydration. Based on long-term observation, it was concluded that dehydration before freezing was not the direct cause of larvae deformity that hatched from frozen-thawing embryo. These results provide a reference for the singly use of representative non-permeable cryoprotectant sucrose.

Establishment and characterization of a brain tissue cell line from spotted knifejaw (Oplegnathus punctatus) and its susceptibility to several fish viruses.

Cells are important in the study of virus isolation and identification, viral pathogenic mechanisms and antiviral immunity. The spotted knifejaw (Oplegnathus punctatus) is a significant farmed fish in China that has been greatly affected by diseases in recent years. In this study, a new cell line derived from the spotted knifejaw brain (SKB) was established and characterized. SKB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28°C. Chromosome analysis revealed that modal chromosome number was 48 for SKB. SKB cells exhibit susceptibility to several fish viruses, such as a largemouth bass virus, red grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV), Singapore grouper iridovirus (SGIV) and spotted knifejaw iridovirus isolate (SKIV-TJ), as shown by cytopathic effect and increased viral titers. Electron microscopy results showed that the cytoplasm contained a large number of vacuoles, and many virus particles existed at the edge of the vacuoles in RGNNV-infected cells and numerous viral particles were scattered throughout the cytoplasm in both ISKNV- and SKIV-TJ-infected cells. These results suggest that SKB is an ideal tool for studying host-virus interactions and potential vaccine development.

Genome-wide identification, immune response profile and functional characterization of IL-10 from spotted knifejaw (Oplegnathus punctatus) during host defense against bacterial and viral infection.

Interleukin 10 (IL-10), a pleiotropic cytokine, plays an essential role in multiple immunity responses. In the current study, the sequences of IL-10 family were identified from spotted knifejaw (Oplegnathus punctatus) whole genome, and O. punctatus IL-10 (OpIL-10) was cloned and characterized. OpIL-10 encodes 187 amino acids with a typical IL-10 family signature motif and predicted α-helices. It shared high identities with Notolabrus celidotus IL-10 and Epinephelus Lanceolatus IL-10. OpIL-10 was widely detected in healthy tissues, with the abundant expression in liver and skin. It was significantly up-regulated in the six immune-related tissues (liver, spleen, kidney, intestine, gill and skin) after infection against Vibrio harveyi and spotted knifejaw iridovirus (SKIV). Dual-luciferase analysis showed that OpIL-10 overexpression could suppress the activity of NF-κB. Meanwhile, OpIL-10 knockdown caused the down-regulation of five immune-related genes in JAK2/STAT3 signaling pathway and NF-κB signaling pathway, including IL-10R2, TYK2, STAT3, NOD2, and IκB. In addition, LPS and poly I:C stimulated expression of pro-inflammatory cytokines, including IL-6, IL-1ß, IL-8, and IL-12, were lower with recombinant OpIL-10 (rOp IL-10) than the control group, indicating the anti-inflammatory roles of rOpIL-10. Taken together, these results indicated OpIL-10 as a negative regulator in the inflammatory responses of spotted knifejaw against bacterial and viral infection, which would help us better understand the role of IL-10 in teleost immunity.

[Analysis of Ciguatoxins in the Spotted Knifejaw, Oplegnathus punctatus from the Waters of Japan].

Ciguatera fish poisoning (CFP) is recognized as the most frequent seafood poisoning due to the consumption of fish containing the principal toxins, ciguatoxins (CTXs). In Japan, CFP events have been reported annually from Okinawa and Amami Islands, locating subtropical regions. In addition, there have been reported several outbreaks due to consumption of the fish caught from the Pacific coast of the Mainland and they were often caused by the matured spotted knifejaw, Oplegnathus punctatus. As part of our research on CFP in Japan, we investigated CTXs analysis by LC-MS/MS on 176 individuals of O. punctatus (weight: 100-6,350 g, standard length: 13-60 cm) from the coast of the Mainland (Honshu, Shikoku, and Kyushu), Amami, Okinawa, and Ogasawara (Bonin) Islands. CTXs were detected from only two specimens collected from Okinawa. Total CTXs levels of the two specimens were at 0.014 and 0.040 µg/kg, respectively, exceeding FDA guidance level at 0.01 µg CTX1B equivalent/kg. However, they might be little risk of CFP because consuming over 1.5 kg of flesh is needed to develop intoxication. The toxins consisted of CTX1B analogs including CTX1B, 52-epi-54-deoxyCTX1B, CTX4A, and CTX4B, and no CTX3C analogs, supporting the finding that ciguatoxic fishes in Okinawan Waters containing only CTX1B analogs.

Spotted knifejaw (Oplegnathus punctatus) MyD88: Intracellular localization, signal transduction function and immune responses to bacterial infection.

Myeloid differentiation factor 88 (MyD88) links members of the toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) superfamily to the downstream activation of NF-κB as a "bridge" molecular in response to exogenous pathogen, but the function in spotted knifejaw (Oplegnathus. punctatus), a commercial fish in China, is still unknown. We present a functional analysis of spotted knifejaw MyD88 (OppMyD88) with a typical death domain (DD) at the N-terminus and a conservative Toll/IL-1R (TIR) domain at the C-terminus and suggest that MyD88 is important for the activation of TLR-mediated NF-κB with the synergy between domains. Subcellular localization showed that OppMyD88 was distributed in the cytoplasm in a condensed form. Tissues expression profiling analysis showed that OppMyD88 ubiquitously expressed in all tested tissues with the highest expression in the liver, as determined by real-time PCR. The expression of OppMyD88 significantly upregulated in the liver, spleen, kidney and gills within 120 h post Vibrio anguillarum infection. Moreover, we further confirmed that over-expressed OppMyD88 could also induce apoptosis. These results indicate that OppMyD88 might possess important roles in defense against microbial infection and other biological processes in spotted knifejaw similar to those in mammals, which will deepen our understandings in innate immunity of spotted knifejaw.

A novel C-type lectin from spotted knifejaw, Oplegnathus punctatus possesses antibacterial and anti-inflammatory activity.

C-type lectin is a type of carbohydrate-binding protein and plays significant roles in innate immune response against pathogen infection. To date, thousands of C-type lectin had been identified in teleost. In the present study, we isolated a novel isoform of C-type lectin (OppCTL) from spotted knifejaw (Oplegnathus punctatus). The OppCTL encoded a typical Ca2+-dependent carbohydrate-binding protein, and was mainly expressed in liver in a tissue specific fashion. The expression of OppCTL was significantly up-regulated following Vibrio anguillarum infection in vivo, suggesting involvement in immune response. Hemagglutination analysis showed that the recombinant OppCTL (rOppCTL) could agglutinate erythrocyte from Mus musculus, Oplegnathus punctatus, Sebastes schlegelii and Paralichthys olivaceus. The rOppCTL could bind and agglutinate all tested bacteria. The rOppCTL possessed capacities of calcium-dependent agglutination to all tested bacteria. Sugar binding assay revealed that rOppCTL could also bind to the glycoconjugates of the bacterial surface, including lipopolysaccharide and peptidoglycan. Interestingly, Dual-luciferase analysis revealed that OppCTL could inhibit the activity of NF-κB in HEK-293T cells after OppCTL overexpression. Taken together, these results indicate that OppCTL has immune activity capable of defending invading pathogens and possesses potential immunoregulatory activity, enriching our understanding of the function of C-type lectin.

Exploration and origin studies of high levels of ß-glucosidase in carnivorous fishes spotted knifejaw (Oplegnathus punctatus).

In order to more efficiently utilize the abundant cellulose resources in nature, increase the utilization rate of cellulose in aquaculture, implement precise feeding and save aquaculture costs, we have conducted research on cellulase genes related to the spotted knifejaw (Oplegnathus punctatus). Cellulose, as the most abundant renewable resource, is a cornerstone in the intricate ecological balance of diverse ecosystems. While herbivorous fish are recognized for their utilization of proteins, sugars, and fats, the extent of cellulose utilization by carnivorous and omnivorous fish remains an enigma. Here, through field sampling and behavioural observations, O. punctatus' omnivorous diet has been demonstrated (stomach contents contain approximately several species of algae in the Bacillariophyta (1.12 %), Streptomyces (0.55 %), Chlorophyta (0.35 %), Rhodophyta (0.16 %), and Euglenophyta (0.19 %) phylum). Additionally, the high cellulase activity in the intestine of O. punctatus has been detected first discovery (enzyme activity up to 4800.15 U/g), indicating its ability to digest cellulose. By employing whole-genome scanning and high-throughput sequencing, a single cellulase gene (ß-glucosidase) within the genome of O. punctatus, suggesting the absence of a complete cellulose digestive system. However, microbiological analysis revealed the three crucial role of microorganisms, including Actinobacteria (25.80 %), Bacteroidetes (18.93 %), and Firmicutes phylum (0.82 %), were found to play a crucial role in the decomposition of plant cell walls, thereby facilitating plant material digestion to help the host to complete the process of cellulose digestion. Expression patterns and proteomic analysis of the ß-glucosidase were notably high in the gonads. In situ hybridization confirmed the expression of the ß-glucosidase gene in the intestinal contents and gonads, highlighting its role in supplying energy of gonads. These discoveries shed light on the dietary habits of O. punctatus and its cellulose utilization, offering insights that can inform the development of customized feeding strategies to enhance aquaculture sustainability and minimize resource expenditure.

Expression pattern and cellular localization of pepsinogen in early development and induced by different diets in the spotted knifejaw (Oplegnathus punctatus).

To solve the high mortality rate of early-stage larval feed conversion during aquaculture in Oplegnathus punctatus, the investigation of the structural and functional characteristics of the gastric tissue was conducted. Histological results showed that the gastric gland rudiment appeared at 17 dph. The basic structure of the stomach was fully developed between 26 and 35 dph. Two pepsinogen genes, named OpPGA1 and OpPGA2, were identified in the spotted knifejaw genome. qPCR results of developmental period showed that the two genes were low in expression during early development (5 and 15 dph). At 20 dph, the two genes started to show trace expression, and at 30 dph the mRNA expression levels of OpPGA1 and OpPGA2 reached the highest levels. Results of pepsin activity detection during the development period showed lower activity was detected 22 dph, followed by a peak at 30 dph. Under different feeding inductions, OpPGA1 showed the highest expression in the basic diet group and hard-shell group, while the expression level in the phytophagous group remained consistently low. The mRNA expression level of OpPGA2 in the phytophagous group was significantly higher than in other groups. Enzyme activity determination under different feeding inductions showed slightly higher enzyme activity in the basic diet group and crustacean group. The results of in situ hybridization showed that the mRNA of both OpPGA1 and OpPGA2 genes was both expressed in gastric gland cells. These information can contribute to the development of practical feeding methods in terms of digestive physiology for the development of larvae.

Development of DNA Insertion-specific Markers Based on the Intergenic Region of Oplegnathus punctatus Cdkn1/srsf3 for Sex Identification.

Spotted knifejaw (Oplegnathus punctatus) is a marine economic fish with high food and ecological value, and its growth process has obvious male and female sexual dimorphism, with males growing significantly faster than females. However, the current sex identification technology is not yet mature, which will limit the growth rate of O. punctatus aquaculture and the efficiency of separate sex breeding, so the development of efficient sex molecular markers is imperative. This study identified a 926 bp DNA insertion fragment in the cdkn1/srsf3 intergenic region of O. punctatus males through whole-genome scanning, comparative genomics, and structural variant analysis. A pair of primers was designed based on the insertion information of the Y chromosome intergenic region in male individuals. Agarose gel electrophoresis revealed the amplification of two DNA fragments, 1118 bp and 192 bp, in male O. punctatus individuals. The 926 bp fragment was identified as the insertion in the intergenic region of cdkn1/srsf3 in males, while only a single 192 bp DNA fragment was amplified in females. The biological sex of the individuals identified in this manner was consistent with their known phenotypic sex. In this study, we developed a method to detect DNA insertion variants in the intergenic region of O. punctatus. Additionally, we introduced a new DNA marker for the rapid identification of the sex of O. punctatus, which enhances detection efficiency. The text has important reference significance and application value in sex identification, all-male breeding, and lineage selection. It provides new insights into the regulation of variation in the intergenic region of cdkn1/srsf3 genes and the study of RNA shearing.

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.

Morphological characteristics of beak-like tooth in spotted knifejaw (Oplegnathus punctatus) and mechanisms of dental development regulation by the Wnt, BMP, FGF and SHH signalling pathways.

Oplegnathus punctatus is a fish species with beak-like tooth that feeds on algae, oysters, sea urchins, and other organisms attached to rocks. Currently, there are no research reports on the development and regulatory mechanisms of O. punctatus beak-like tooth. This present study firstly elucidated the nesting structure pattern of the beak-like tooth with dental formula (4, 15-16, 10-1) for O. punctatus. Four critical periods during early beak-like tooth development (28dph, 40dph, 50dph, 60dph) were also identified. In addition, 11 key genes (bmp2, bmpr2, smad1, wnt5a, msx, axin2, fgfr1a, fgfr2, pitx2, ptch1, cyp27a1) closely related to the development of beak-like tooth were discovered, with the highest expression levels in the initial stages of functional teeth and replacement teeth development, and expression in the mesenchymal and epithelial tissues of the teeth. Further research found that the cyp27a1 gene, related to vitamin D metabolism and calcium accumulation, was expressed in the maxilla and base of the tooth in O. punctatus. This study provides support for the biological theory of tooth development and healing and provides a reference for the adaptive evolution of tooth healing in special habitats.

Immunological characterization and function analysis of L-type lectin from spotted knifejaw, Oplegnathus punctatus.

Lily-type lectin (LTL) plays significant roles in innate immune response against pathogen infection. LTL in animals and plants has received widespread attention. In the present study, an LTL (OppLTL) was identified from spotted knifejaw Oplegnathus punctatus. The OppLTL encoded a typical Ca2+-dependent carbohydrate-binding protein containing a CRD domain. The qRT-PCR showed that it was mainly expressed in the gill and was significantly upregulated after Vibrio anguillarum challenge. The agglutination analysis showed that the recombinant OppLTL could bind and agglutinate Gram-negative and Gram-positive bacteria in a Ca2+-dependent manner. However, the binding activity was different. Meanwhile, the recombinant OppLTL could hemagglutinate mammalian and teleost erythrocytes. Subcellular localization revealed that OppLTL was mainly detected in the cytoplasm of HEK293T cells. The dual-luciferase analysis revealed that OppLTL could inhibit the activity of the NF-κB signal pathway in HEK293T cells after OppLTL overexpression. These findings collectively demonstrated that OppLTL could be involved in host innate immune response and defense against bacterial infection in spotted knifejaw.

Transcriptomic analysis reveals the gene expression profiles in the spleen of spotted knifejaw (Oplegnathus punctatus) infected by Vibrio harveyi.

As one of the most valuable maricultured species, spotted knifejaw (Oplegnathus punctatus) has high popularity in eastern Asia. In recent years, diseases caused by Vibrio harveyi have brought huge economic losses in spotted knifejaw industry. To better understand the molecular mechanisms of immune response about V. harveyi resistance in spotted knifejaw, a comparative transcriptome analysis was performed on spleen tissues at five different time points post-infection (0, 12, 24, 48 and 72 hpi). A total of 4279 differentially expressed genes (DEGs) were identified. KEGG pathways analysis showed that multiple immune-related pathways were significant regulated, including Toll-like receptor signaling pathway, ECM-receptor interaction pathway, cytokine-cytokine receptor interaction pathway and hematopoietic cell lineage pathway. Weighted gene co-expression network analysis showed that several immune-related pathways of the highest correlation with 12 hpi (cor = 0.89, P = 7e-06) were significantly enriched. In addition, 12 hpi was a turning point for 7 gene clusters out of 9 that were divided according to gene expression patterns. Therefore, we speculated that 12 hpi might be a very critical time point for spotted knifejaw against V. harveyi infection. Additionally, qRT-PCR was carried out to validate the expressions of 12 DEGs. This study provided the first systematical transcriptome analysis of spotted knifejaw against V. harveyi. The results could help us better understand the dynamic immune responses of spotted knifejaw against bacterial infection, and provide useful information for antibacterial defense in spotted knifejaw industry as well.

Identification of Male-Specific Molecular Markers by Recombination of RhoGEF10 Gene in Spotted Knifejaw (Oplegnathus punctatus).

The spotted knifejaw (Oplegnathus punctatus) is a marine economic fish with high ecological value, food value, and fishing value, and its growth has obvious sex dimorphism. The rapid identification of its sex is beneficial to the development of sex determination and breeding. In this study, the method of comparative genomics and PCR amplification was used to further establish a rapid detection method for the recombinant RhoGEF10 gene in O. punctatus, which can quickly, accurately, and efficiently identify the sex of the O. punctatus to be tested. The homologous comparison results of male and female individuals showed that the DNA fragment length of the RhoGEF10 gene on the X1 chromosome was 326 bp, and the DNA fragment length on the Y chromosome was 879 bp. Therefore, it can be concluded that there is an insert fragment of 553 bp on the Y chromosome. PCR amplification results showed that the two DNA fragments of 879 bp and 326 bp were amplified in the Y chromosome and X1 chromosome of the male O. punctatus (X1X2Y), respectively, and the 879 bp fragment was a unique marker fragment of the recombinant RhoGEF10 gene; The female O. punctatus (X1X1X2X2) only a single DNA fragment of 326 bp was amplified. At the same time, the inserted fragment of the male individual resulted in partial inactivation of the RhoGEF10 protein, which in turn resulted in a slowing of peripheral nerve conduction velocity and thinning of the myelin sheath in male O. punctatus. The method shortens the time for accurate identification of the O. punctatus RhoGEF10 gene recombination and improves the detection efficiency. It is of great significance and application value in the research of nerve conduction and myelin development, male and female sex identification, the preparation of high male seedlings, and family selection based on the RhoGEF10 gene in the O. punctatus.

Genome-wide identification, characterization and expression analysis of the BMP family associated with beak-like teeth in Oplegnathus.

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta (TGF-ß) family, are critical for the control of developmental processes such as dorsal-ventral axis formation, somite and tooth formation, skeletal development, and limb formation. Despite Oplegnathus having typical healing beak-like teeth and tooth development showing a trend from discrete to healing, the potential role of BMPs in the development of the beak-like teeth is incompletely understood. In the present study, 19 and 16 BMP genes were found in O. fasciatus and O. punctatus, respectively, and divided into the BMP2/4/16, BMP5/6/7/8, BMP9/10, BMP12/13/14, BMP3/15 and BMP11 subfamilies. Similar TGFb and TGF_ß gene domains and conserved protein motifs were found in the same subfamily; furthermore, two common tandem repeat genes (BMP9 and BMP3a-1) were identified in both Oplegnathus fasciatus and Oplegnathus punctatus. Selection pressure analysis revealed 13 amino acid sites in the transmembrane region of BMP3, BMP7, and BMP9 proteins of O. fasciatus and O. punctatus, which may be related to the diversity and functional differentiation of genes within the BMP family. The qPCR-based developmental/temporal expression patterns of BMPs showed a trend of high expression at 30 days past hatching (dph), which exactly corresponds to the ossification period of the bones and beak-like teeth in Oplegnathus. Tissue-specific expression was found for the BMP4 gene, which was upregulated in the epithelial and mesenchymal tissues of the beak-like teeth, suggesting that it also plays a regulatory role in the development of the beak-like teeth in O. punctatus. Our investigation not only provides a scientific basis for comprehensively understanding the BMP gene family but also helps screen the key genes responsible for beak-like tooth healing in O. punctatus and sheds light on the developmental regulatory mechanism.

Genome-Wide Scan Reveals Toll-Like Receptor Contraction Events in Oplegnathidae.

The striped knifejaw (Oplegnathus fasciatus) and spotted knifejaw (Oplegnathus punctatus) are prominent members of the Oplegnathidae family and are rocky reef-loving fishes with high ecological and economic value. However, the frequent occurrence of diseases in these fishes has severely restricted the development of their breeding industry. Toll-like receptors (TLRs) play an important role in resistance to pathogens as part of innate immunity. Genome-wide scans and cross-species comparative analysis revealed 10 TLRs in O. fasciatus (OfTLRs) and only 5 in O. punctatus (OpTLRs). In contrast to those of mammals and other fishes, the TLR family of Oplegnathidae underwent significant contraction events, especially in O. punctatus (only TLR1, TLR2, TLR14, TLR5, and TLR21 were retained). A phylogenetic tree divided the 10 OfTLRs into 5 subfamilies: TLR1, TLR3, TLR5, TLR7, and TLR11. The five OpTLR genes were divided into three different subfamilies: TLR1, TLR5, and TLR11. Quantitative real-time PCR revealed that all OpTLRs were expressed in the examined tissues, especially the immune system-related tissues, such as the spleen, gill, head kidney, and middle kidney. The expression of OpTLRs was high at the early stage of development (5 days posthatching [dph]) and decreased gradually until 30 dph. We speculated that maternal immunity or the developmental function of TLRs played an important protective role in the early stage. However, from 30 to 60 dph, TLR expression was low. At this time, juvenile fish are susceptible to viruses and begin to show TLR self-expression with weak immunity. Artificial immunity enhancement is needed to improve the environmental resistance of juvenile fish. In summary, our results not only provide valuable basic data for future studies of the TLR gene family in Oplegnathidae fish but also lay a solid foundation for Oplegnathidae fish research.

Identification of Male-Specific Molecular Marker and Development of PCR-Based Genetic Sex Identification Technique in Spotted Knifejaw (Oplegnathus punctatus).

Spotted knifejaw (Oplegnathus punctatus) is a marine teleost species that is economically important for aquaculture and marine pasture proliferation and shows obvious bisexual growth dimorphism, but molecular sex markers are currently lacking. A 290 bp (base pair) insertion with two fragments (230 bp and 60 bp) was identified in male individuals of O. punctatus based on whole-genome sequencing scanning and structural variation analyses. The gene annotation results showed that the insertion event occurred in the Igfn1 gene of male O. punctatus. The results of amino acid analysis further showed that the insertion event resulted in the functional variation of Igfn1 in male O. punctatus, and recombination caused the inactivation of Igfn1. According to the male-specific insertion information, we designed a PCR-based genetic amplification technique for rapid sex identification in O. punctatus. The results of agarose gel electrophoresis showed that two DNA fragments of 635 bp and 925 bp were amplified in male O. punctatus, while only a single DNA fragment of 635 bp was amplified in female individuals. The sex of individuals identified by this method was consistent with their known phenotypic sex, which will improve sex identification efficiency. This method provides a new DNA marker for rapid sex identification in O. punctatus, which has great significance and application value in monosex breeding and provides new insights for the study of Igfn1 gene recombination and inactivation in male O. punctatus.

Isolation of a Male-Specific Molecular Marker and Development of a Genetic Sex Identification Technique in Spotted Knifejaw (Oplegnathus punctatus).

Sex-specific DNA markers are very helpful for identifying genetic sex and studying sex determination mechanisms in fish. To identify the sex-specific markers of spotted knifejaw (Oplegnathus punctatus), we performed a comparative analysis of the female and male genomes. In this study, an 18 bp insertion was identified in the male genome after verification by sequencing depth and PCR. An effective and rapid method based on PCR was then developed to identify the genetic sex. A male-female-shared primer pair and a male-specific primer were designed for PCR amplification to avoid false-negative phenomena. To examine the primers in practice, we utilized hundreds of spotted knifejaw fish from different groups to identify their genetic sex, and the results were consistent with their phenotypic sex. The male-specific DNA marker would be helpful for artificial breeding, Y chromosome assembly and further study of the sex determination mechanism. This study is the first to identify an effective sex-specific marker in spotted knifejaw.