Suitable habitat shifts and ecological niche overlay assessments among benthic Oplegnathus species in response to climate change.

Climate change has had a significant impact on many marine organisms. To investigate the effects of environmental changes on deep-water benthic fishes, we selected the genus Oplegnathus and applied species distribution modeling and ecological niche modeling. From the last glacial maximum to the present, the three Oplegnathus species (O. conwayi, O. robinsoni, and O. peaolopesi) distributed in the Cape of Good Hope region of southern Africa experienced fitness zone fluctuations of 39.9%, 13%, and 5.7%, respectively. In contrast, O. fasciatus and O. punctatus, which were primarily distributed in the western Pacific Ocean, had fitness zone fluctuations of -6.5% and 11.7%, respectively. Neither the O. insignis nor the O. woodward varied by more than 5% over the period. Under future environmental conditions, the range of variation in fitness zones for the three southern African Oplegnathus species was expected to be between -30.8% and -26.5%, while the range of variation in fitness zones for the two western Pacific stonefish species was expected to remain below 13%. In addition, the range of variation in the fitness zones of the O. insignis was projected to be between -2.3% and 7.1%, and the range of variation in the fitness zones of the O. woodward is projected to be between -5.7% and -2%. The results indicated that O. fasciatus and O. punctatus had a wide distribution and high expansion potential, while Oplegnathus species might have originated in western Pacific waters. Our results showed that benthic fishes were highly adaptable to extreme environments, such as the last glacial maximum. The high ecological niche overlap between Oplegnathus species in the same region suggested that they competed with each other. Future research could explore the impacts of environmental change on marine organisms and make conservation and management recommendations.

Chromosome-level genome assembly of the silver pomfret Pampus argenteus.

Pampus argenteus (Euphrasen, 1788) is one of the major fishery species in coastal China. Pampus argenteus has a highly specialized morphology, and its declining fishery resources have encouraged massive research efforts on its aquacultural biology. In this study, we reported the first high-quality chromosome-level genome of P. argenteus obtained by integrating Illumina, PacBio HiFi, and Hi-C sequencing techniques. The final size of the genome was 518.06 Mb, with contig and scaffold N50 values of 20.47 and 22.86 Mb, respectively. The sequences were anchored and oriented onto 24 pseudochromosomes based on Hi-C data corresponding to the 24-chromatid karyotype of P. argenteus. A colinear relationship was observed between the P. argenteus genome and that of a closely related species (Scomber japonicus). A total of 24,696 protein-coding genes were identified from the genome, 98.9% of which were complete BUSCOs. This report represents the first case of high-quality chromosome-level genome assembly for P. argenteus and can provide valuable information for future evolutionary, conservation, and aquacultural research.

Prediction of suitable habitat shifts and assessment of ecological niche overlaps for three Tridentiger species with intertidal and subtidal characteristics under future climate changes.

To show how dramatic global climate change affects marine ecosystem species in different habitats. We used a joint species distribution model (SDM) and an ecological niche model (ENM) to investigate the suitable habitat shifts and ecological niche overlaps of the Tridentiger fishes. In the present study, the SDM results showed that 5 hotspots were identified for T. trigonocephalus and T. barbatus, and 4 hotspots for T. bifasciatus. The study on center-of-mass transfer revealed notable reductions in the habitual range of the three Tridentiger species with future climate change and no significant bipolar shifts in the center of mass. The ENM results indicated that T. trigonocephalus and T. barbatus exhibited the greatest ecological niche overlap with Schoener's D (D) and Hellinger-based I (I) values of 0.4719 and 0.7690, respectively. Both SDM and ENM results have suggested that T. trigonocephalus occupied a wider distribution and greater adaptability to future climate change. This study sought to measure the variations in the effects of global climate change on marine species in different habitats. Our study first found that intertidal species with specific life histories may be more resilient to environmental change.

Genome-wide investigation of the DMRT gene family sheds new insight into the regulation of sex differentiation in spotted knifejaw (Oplegnathus punctatus) with fusion chromosomes (Y).

The role of the DMRT family in male sex determination and differentiation is significant, but its regulatory role in spotted knifejaw with Y fusion chromosomes remains unclear. Through genome-wide scanning, transcriptome analysis, qPCR, FISH, and RNA interference (RNAi), we investigated the DMRT family and the dmrt1-based sex regulation network. Seven DMRTs were identified (DMRT1/2 (2a,2b)/6, DMRT4/5, DMRT3), and dmrt gene dispersion among chromosomes is possibly driven by three whole-genome duplications. Transcriptome analysis enriched genes were associated with sex regulation and constructed a network associated with dmrt1. qPCR and FISH results showed the expression dimorphism of sex-related genes in dmrt-related regulatory networks. RNAi experiments indicated a distinct sex regulation mode in spotted knifejaw. Dmrt1 knockdown upregulated male-related genes (sox9a, sox9b, dmrt1, amh, amhr2) and hsd11b2 expression, which is critical for androgen synthesis. Amhr2 is located on the heterozygous chromosome (Y) and is specifically localized in primary spermatocytes, and is extremely upregulated after dmrt1 knockdown which suggested besides the important role of dmrt1 in male differentiation, the amhr2 along with amhr2/amh system, also play important regulatory roles in maintaining high expression of the hsd11b2 and male differentiation. This study aims to further investigate sex regulatory mechanisms in species with fusion chromosomes.

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.

Improved high-quality reference genome of red drum facilitates the processes of resistance-related gene exploration.

Sciaenops ocellatus is among the most important artificially introduced farmed fish across 11 countries and regions. However, the frequent occurrence of extreme weather events and breeding escapes have placed great pressure on local marine biodiversity and ecosystems. We reported the de novo assembly and annotation with a contig N50 of 28.30 Mb using PacBio HiFi sequencing and Hi-C technologies, which resulted in a 283-fold increase in contig N50 length and improvement in continuity and quality in complex repetitive region for S. ocellatus compared to the previous version. In total, 257.36 Mb of repetitive sequences accounted for 35.48% of the genome, and 22,845 protein-coding genes associated with a BUSCO value of 98.32%, were identified by genome annotation. Moreover, 54 hub genes rapidly responding to hypoosmotic stress were identified by WGCNA. The high-quality chromosome-scale S. ocellatus genome and candidate resistance-related gene sets will not only provide a genomic basis for genetic improvement via molecular breeding, but will also lay an important foundation for investigating the molecular regulation of rapid responses to stress.

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.

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.

A Number of the N-terminal RASSF Family: RASSF7

The Ras association domain family 7 (RASSF7, also named HRC1), a potential tumor-related gene, located on human chromosome 11p15, has been identified as an important member of the N-terminal RASSF family. Whereas, the molecular biological mechanisms of RASSF7 in tumorigenesis remain to be further established. We perform a systematic review of the literature and assessment from PUBMED and MEDLINE databases in this article. RASSF7 plays a significant role in mitosis, microtubule growth, apoptosis, proliferation and differentiation. Many research literature shows that the RASSF7 could promote the occurrence and advance of human tumors by regulating Aurora B, MKK4, MKK7, JNK, YAP, MEK, and ERK, whereas, it might inhibit c-Myc and thus lead to the suppression of tumorigenesis. The pregulation of RASSF7 often occurs in various malignancies such as lung cancer, neuroblastoma, thyroid neoplasm, hepatocellular cancer, breast cancer and gastric cancer. The expression stage of RASSF7 is positively correlated with the tumor TNM stage. In this review, we primarily elaborate on the acknowledged structure and progress in the various biomechanisms and research advances of RASSF7, especially the potential relevant signaling pathways. We hope that RASSF7 , a prospective therapeutic target for human malignancies, could play an available role in future anti-cancer treatment.

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.

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.

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.

First observation of the mating, egg-laying and hatching behaviour of a captive female Kong skate, Okamejei kenojei (Müller & Henle, 1841).

This study provides a novel record of the reproductive behaviour of the Kong skate (Okamejei kenojei) in captivity. These skates were found to mate and deposit eggs at a temperature of 16.5 ± 0.5°C. The results showed that 76.13% of the eggs possessed one yolk, 0.77% of the eggs possessed two yolks and 23.11% of the eggs had no yolk (N = 1043). The deposition of non-yolk and double-yolk eggs was random. A total of 100 eggs were collected. After nearly 92 ± 5 days of incubation, 28 eggs failed to hatch, and 72 skates were successfully hatched with a female-to-male ratio of 1:1 (P > 0.05). The results enrich our knowledge of the reproduction in cartilaginous fishes and can inform management and conservation strategies for this species.

A Novel Biomarker, FKBP10, for Poor Prognosis Prediction in Patients with Clear Cell Renal Cell Carcinoma.

Objective: To screen genes associated with poor prognosis of clear cell renal cell carcinoma (CcRCC) from the public databases HPA (Human Protein Atlas), UALCAN, and GEPIA (Gene Expression Profiling Interactive Analysis) and to investigate the expression of FKBP10 in CcRCC and the effect on prognosis of the patients and the biological behavior of CcRCC cells. Methods: The tumor tissues and adjacent noncancerous tissues of 42 patients with CcRCC diagnosed and treated in our hospital were collected, and the general information of the patients was recorded. FKBP10 expression in the tissues was determined by qRT-PCR and western blot, and its relationship with general information and prognosis of patients was analyzed. Knockdown or overexpression experiments were carried out with the human proximal tubule epithelial cell line HK-2 and CcRCC cell lines Caki-1, 786-O, ACHN, and A498 to verify the relationship between FKBP10 expression and cell proliferation and adhesion ability using MTT assay and fibronectin adhesion assay, respectively. Western blot was utilized to examine the protein expression level of c-Myc, cyclin D1, and Bcl-2 in the cells. Results: FKBP10 was highly expressed in CcRCC tissues and cells and was correlated with poor prognosis. In addition, FKBP10 expression was positively correlated with CcRCC tumor size and staging and negatively correlated with tumor differentiation. Moreover, knockdown of FKBP10 significantly inhibited the proliferation of CcRCC cells, notably declined the protein expression of c-Myc, cyclin D1, and Bcl-2, and promoted cell adhesion. Conclusion: FKBP10 is highly expressed in CcRCC tissues and cells and is associated with poor prognosis in patients. FKBP10 participated in the occurrence and development of CcRCC by promoting cell proliferation and inhibiting apoptosis and adhesion.

Folic Acid-Modified miR-491-5p-Loaded ZIF-8 Nanoparticles Inhibit Castration-Resistant Prostate Cancer by Regulating the Expression of EPHX1.

Epoxide hydrolase 1 (EPHX1) has been reported to be related to the development of several tumors. However, the regulation of castration-resistant prostate cancer (CRPC) development by EPHX1 has not been reported. We used proteomic technology and found that the EPHX1 protein was highly expressed in CRPC tissues and the CRPC cell line C4-2. We performed screening and found that EPHX1 is a direct target of miR-491-5p. High miR-491-5p expression significantly reduced the EPHX1 level in C4-2 cells and inhibited C4-2 cell proliferation and migration. Zeolite imidazolate framework-8 (ZIF-8) has good thermal stability, a simple synthesis method, tumor site stability, and specific acid responsiveness. We synthesized ZIF-8 nanodrug vectors to deliver miR-491-5p into C4-2 cells. After loading miR-491-5p into ZIF-8, we modified the ZIF-8 surface with folic acid (FA) as the target group (FA@ZIF-8). Our synthesized nanodrug carrier showed less cytotoxicity to C4-2 cells even at 200 µg/ml. Modified FA could increase the efficiency of nanomaterial entry into C4-2 cells. FA@miR-491-5p@ZIF-8 could stably release miR-491-5p for a long period in both phosphate-buffered saline (pH 7.4) and acetate buffer (pH 4.8), and miR-491-5p was released faster at the beginning of the experiment in acetate buffer (pH 4.8). FA@miR-491-5p@ZIF-8 significantly reduced C4-2 cell proliferation and migration, and FA@miR-491-5p@ZIF-8 had a better effect than miR-491-5p alone. In vivo, FA@miR-491-5p@ZIF-8 significantly inhibited CRPC growth in nude mice. Overall, we verified that miR-491-4p regulated CRPC development by targeting EPHX1. The drug nanocarrier FA@miR-491-5p@ZIF-8 not only significantly reduced C4-2 CRPC cell proliferation and migration but also significantly inhibited CRPC growth. Our research provides a theoretical basis for treatment and treatment strategies for CRPC.

Chronic nitrate exposure cause alteration of blood physiological parameters, redox status and apoptosis of juvenile turbot (Scophthalmus maximus).

Nitrate (NO3-) is one of the common inorganic nitrogen compound pollutants in natural ecosystems, which may have serious risks for aquatic organisms. However, its toxicological mechanism remains unclear. In the current study, juvenile turbot (Scophthalmus maximus) were exposed to different concentrations of NO3- (CK- 3.57 ± 0.16, LN - 60.80 ± 1.21, MN - 203.13 ± 10.97 and HN - 414.16 ± 15.22 mg/L NO3-N) for 60 d. The blood biochemical assays results revealed that elevated NO3- exposure significantly increased the concentrations of plasma NO3-, NO2-, MetHb, K+, cortisol, glucose, triglyceride, lactate, while significantly decreased the concentrations of plasma Hb, Na+ and Cl-, which meant that NO3- caused hypoxic stress and further affected the osmoregulation and metabolism in fish. Besides, exposure to MN and HN induced a significant decrease in the level of antioxidants, including SOD (Point: 60th day, MN, HN v.s. CK: 258.36, 203.73 v.s. 326.95 U/mL), CAT (1.97, 1.17 v.s. 2.37 U/mL), GSH (25.38, 20.74 v.s. 37.00 µmol/L), and GPx (85.32, 71.46 v.s. 129.36 U/mL), and a significant increase of MDA (7.54, 9.73 v.s. 5.27 nmol/L), suggesting that NO3- exposure leading to a disruption of the redox status in fish. Also, further research revealed that NO3- exposure altered the mRNA levels of p53 (HN: up to 4.28 folds) and p53-regulated downstream genes such as Bcl-2 (inferior to 0.44 folds), caspase-3 (up to 2.90 folds) and caspase-7 (up to 3.49 folds), indicating that NO3- exposure induced abnormal apoptosis in the fish gills. Moreover, IBRv2 analysis showed that the toxicity of NO3- exposure to turbot was dose-dependent, and the toxicity peaked on the 15th day. In short, NO3- is an environmental toxicological factor that cannot be ignored, because its toxic effects are long-term and could cause irreversible damage to fish. These results would be beneficial to improve our understanding of the toxicity mechanism of NO3- to fish, which provides baseline evidence for the risk assessment of environmental NO3- in aquatic ecosystems.

Sperm maturation, migration, and localization before and after copulation in black rockfish (Sebastes schlegelii).

Sebastes schlegelii is a typical viviparous teleost with six months sperm storage duration from November to April. In this study, spermatozoa morphological and physiological characteristics and sperm location in the female ovary were investigated by electron microscopy, computer-assisted sperm analyzer and histologic analysis, respectively. During copulation, we observed that spermatozoa in the testis had mature structure with rod-shaped head, a short midpiece, and a long flagellum. And further verified sperm swam freely at a high speed in the ovary fluid. After copulation, we only found swimming sperm in the ovary fluid at the early storage stage (November to December) and the majority of sperm were scattered randomly in the ovary cavity and partially concentrated in the crypt between the oocyte and stalk of follicle. Thereafter, the ovarian epithelium around the oocytes proliferated rapidly and wrapping spermatozoa outside of the follicular layer and formed a lot of crypts outside of the follicular layer which served as the sperm storage site until fertilization. The present findings would be useful for further understanding the mechanism of long-term sperm storage in viviparous teleost.

Investigating the effect of nitrate on juvenile turbot (Scophthalmus maximus) growth performance, health status, and endocrine function in marine recirculation aquaculture systems.

Nitrate (NO3-), a potential toxic nitrogenous compound to aquatic animals, is distributed in aquatic ecosystems worldwide. The aim of this study was to investigate the effects of different NO3- levels on growth performance, health status, and endocrine function of juvenile turbot (Scophthalmus maximus) in recirculating aquaculture systems (RAS). Fish were exposed to 0 mg/L (control, CK), 50 mg/L (low nitrate, LN), 200 mg/L (medium nitrate, MN), and 400 mg/L (high nitrate, HN) NO3-N for 60 d in experimental RAS. Cumulative survival (CS) was significantly decreased with increasing NO3- levels in LN, MN, and HN. The lowest CS was 35% in the HN group. Growth parameters, including absolute growth rate, specific growth rate, and feed conversion rate, were significantly different in HN compared with that in the CK. Histological survey of gills and liver revealed dose-dependent histopathological damage induced by NO3- exposure and significant differences in glutamate pyruvate transaminase and glutamate oxalate transaminase in MN and HN compared with that in the CK. The hepatosomatic index in HN was significantly higher than that in the CK. Additionally, NO3- significantly increased bioaccumulation in plasma in LN, MN, and HN compared to that in the CK. Significant decreases in hemoglobin and increases in methemoglobin levels indicated reduced oxygen-carrying capacity in HN. Additionally, qRT-PCR and enzyme-linked immunosorbent assay (ELISA) were developed to investigate key biomarkers involved in the GH/IGF-1, HPT, and HPI axes. Compared with that in the CK, the abundance of GH, GHRb, and IGF-1 was significantly lower in HN, whereas GHRa did not differ between treatments. The plasma T3 level significantly decreased in LN, MN, and HN and T4 significantly decreased in HN. The CRH, ACTH, and plasma cortisol levels were significantly upregulated in HN compared with that in the CK. We conclude that elevated NO3- exposure leads to growth retardation, impaired health status, and endocrine disorders in turbot and the NO3- level for juvenile turbot culture should not exceed 50 mg/L NO3-N in RAS. Our findings indicate that endocrine dysfunction of the GH/IGF-1, HPT, and HPI axes might be responsible for growth inhibition induced by NO3- exposure.

Effects of chronic nitrate exposure on the intestinal morphology, immune status, barrier function, and microbiota of juvenile turbot (Scophthalmus maximus).

Coming along with high water reuse in sustainable and intensive recirculating aquaculture systems (RASs), the waste products of fish in rearing water is continuously accumulated. Nitrate, the final product of biological nitrification processes, which may cause aquatic toxicity to fish in different degrees when exposed for a long time. Therefore, the present study was conducted to evaluate the impact of chronic nitrate exposure on intestinal morphology, immune status, barrier function, and microbiota of juvenile turbot. For that, groups of juvenile turbot were exposed to 0 (control check, CK), 50 (low nitrate, L), 200 (medium nitrate, M), and 400 (high nitrate, H) mg L-1 nitrate-N in small-sized recirculating aquaculture systems. After the 60-day experiment period, we found that exposure to a high concentration of nitrate-N caused obvious pathological damages to the intestine; for instance, atrophy of intestinal microvilli and necrosis in the lamina propria. Quantitative real-time PCR analysis revealed a significant downregulation of the barrier forming tight junction genes like occludin, claudin-like etc. under H treatment (P < 0.05). Intestinal MUC-2 expression also decreased significantly in the nitrate treatment groups compared to that in the control (P < 0.05). Additionally, the expression of HSP70 and HSP90 heat-shock proteins, toll-like receptor-3 (TLR-3), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) significantly increased (P < 0.05), whereas that of transforming growth factor-ß (TGF-ß), lysozyme (LYS), and insulin-like growth factor-I (IGF-I) significantly decreased with H treatment (P < 0.05). The results also revealed that intestinal microbial community was changed following nitrate exposure and could alter the α-diversity and ß-diversity. Specifically, the proportion of intrinsic flora decreased, whereas that of the potential pathogens significantly increased with M and H treatments (P < 0.05). In conclusion, chronic nitrate exposure could weaken the barrier function and disturb the composition of intestinal microbiota in marine teleosts, thereby harming their health condition.