Comparative Analysis of the Systematics and Evolution of the Pampus Genus of Fish (Perciformes: Stromateidae) Based on Osteology, Population Genetics and Complete Mitogenomes.

Pampus is a widespread species of fish in the western Pacific and Indian Oceans that has significant commercial worth. Its evolutionary history and phylogenetics are still poorly understood, and details on its intraspecific taxonomy are debatable, despite some morphological and molecular research. Here, we analyzed this species using skeletal structure data as well as nuclear (S7 gene) and mitochondrial genetic information (COI, D-loop and mitogenomes). We found that the genetic distance between P. argenteus and P. echinogaster was much smaller than that between other Pampus species, and both maximum likelihood and Bayesian phylogenetic trees yielded almost identical tree topologies. An additional and adjacent M repeat was found in the downstream region of the IQM gene cluster of P. argenteus and P. echinogaster, and the trnL2 gene of P. minor was translocated. The genus Pampus experienced early rapid radiation during the Palaeocene with major lineages diversifying within a relatively narrow timescale. Additionally, three different methods were conducted to distinguish the genus Pampus species, proving that P. argenteus and P. echinogaster are the same species, and P. liuorum is speculated to be a valid species. Overall, our study provides new insights not only into the evolutionary history of Pampus but its intraspecific taxonomy as well.

Cyp19a1a Promotes Ovarian Maturation through Regulating E2 Synthesis with Estrogen Receptor 2a in Pampus argenteus (Euphrasen, 1788).

In the artificial breeding of Pampus argenteus (Euphrasen, 1788), female fish spawn before male release sperm, which indicates rapid ovarian development. In fish, aromatase is responsible for converting androgens into estrogens and estrogen plays a crucial role in ovarian development. In this study, we aimed to investigate the potential role of brain-type and ovarian-type aromatase to study the rapid ovarian development mechanism. The results showed that cyp19a1a was mainly expressed in the ovary and could be classified as the ovarian type, whereas cyp19a1b could be considered as the brain type for its expression was mainly in the brain. During ovarian development, the expression of cyp19a1a in the ovary significantly increased from stage IV to stage V and Cyp19a1a signals were present in the follicle cells, while cyp19a1b expression in the pituitary gland decreased from stage IV to stage V. To further investigate the function of Cyp19a1a, recombinant Cyp19a1a (rCyp19a1a) was produced and specific anti-Cyp19a1a antiserum was obtained. The expressions of cyp19a1a, estrogen receptors 2 alpha (esr2a), and androgen receptor alpha (arα) were significantly upregulated in the presence of rCyp19a1a. Meanwhile, cyp19a1a was expressed significantly after E2 treatment in both ovarian and testicular tissue culture. Taken together, we found two forms of aromatase in silver pomfret. The ovarian-type aromatase might play an important role in ovarian differentiation and maturation, and participate in E2 synthesis through co-regulation with esr2a. The brain-type aromatase cyp19a1b might be involved in the regulation of both brain and gonadal development.

Comprehensive Analysis of Phylogenetic Relationship and Optimal Codons in Mitochondrial Genomes of the Genus Pseudogastromyzon.

As indicator organisms for water pollution detection, Pseudogasteromyzon species play a vital role in aquatic environment monitoring. We have successfully sequenced the mitogenomes of P. fasciatus jiulongjiangensis and P. myersi and downloaded the mitogenomes of nine other Pseudogastromyzon fish on GenBank to conduct a detailed comparative analysis of their phylogenetic relationships and evolutionary history. The findings revealed a conservation in both gene composition and gene order. Except for the trnS1 gene lacking dihydrouracil arms, the other 21 tRNAs showed the typical clover-leaf secondary structure. According to the ΔRSCU method, we identified the seven most abundant optimal codons: CUA, GUA, CCA, CAA, GAA, AGC, and GGC. The construction of maximum parsimony, maximum likelihood, and Bayes trees yielded congruent topologies, and the 11 Pseudogastromyzon species were clustered into two major clusters. Among them, one of which was composed of P. fangi, P. changtingensis changtingensis, and P. changtingensis tungpeiensis, while the remaining eight species formed another cluster, further subdivided into five smaller clusters. Distinct clusters formed between P. fasciatus jiulongjiangensis and P. meihuashanensis, P. cheni and P. peristictus, and P. laticeps and P. lianjiangensis, and the remaining two species were clustered separately, thereby enhancing our understanding of them. Furthermore, our analysis results of divergence times revealed that these 11 Pseudogasteromyzon species underwent rapid differentiation in the Pleistocene epochs. Overall, our study sheds light on the phylogenetic relationship and evolutionary history of Pseudogasteromyzon species, providing a necessary knowledge foundation for further understanding the intricacies of an ecosystem health assessment.

Change and Regulation of Nutritional Metabolism in Silver Pomfret During Compensatory Growth.

Compensatory growth (CG) in fish is heavily influenced by nutrient metabolism. However, there are limited studies examining how nutrient metabolism is regulated during this process. For silver pomfret, an important commercial marine fish, it's crucial to establish effective starvation and re-feeding strategies to ensure good water quality and fast growth. To identify the complete compensatory growth model of silver pomfret, we conducted an experiment with a control group (normal feeding) and three starvation/re-feeding groups. We observed that the recovery of weight and condition factor in the 14-day starvation and 14-day re-feeding groups was significantly faster than other groups, indicating full compensatory growth. Thus, we selected this group for the next experiment. We performed untargeted metabolomics and transcriptome analysis of muscle tissue on Day 14, 21 and 28 (CG process), and examined the key regulatory genes of nutrient metabolism on Day 0, 7, 14, 21 and 28 (starvation and re-feeding process). Our data revealed that during starvation, silver pomfret first utilized carbohydrates and short-chain lipids, followed by proteins and long-chain lipids. After re-feeding, lipids accumulated first, resulting in rapid growth, followed by the recovery of protein content in muscle. During starvation, the expression of anabolic-related genes such as TER and CALR decreased, and catabolic-related genes such as TSC2 and MLYCD increased, promoting the AMPK pathway. During re-feeding, anabolic-related gene expression increased without AMPK inhibition. Our findings provide insights into the energy utilization strategies of fish and molecular regulation during compensatory growth in fish.

Identification and characterization of toll-like receptor genes in silver pomfret (Pampus argenteus) and their involvement in the host immune response to Photobacterium damselae subsp. Damselae and Nocardia seriolae infection.

Toll-like receptors (TLRs) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLRs in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1, TLR3, TLR5, TLR7, and TLR11, indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88-dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2, PaTLR3, PaTLR5, PaTLR7, PaTLR9, and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLRs expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR-interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding.

Cold Stress Induces Apoptosis in Silver Pomfret via DUSP-JNK Pathway.

We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 ℃, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 ℃ detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 ℃, 23 ℃, 20 ℃, 17 ℃, and 14 ℃). The cell viability significantly declined from 14 to 20 ℃; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 ℃ and 14 ℃; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 ℃ and 14 ℃, and PIDD was up-regulated at 20 ℃, 17 ℃, and 14 ℃. DUSP3 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃, and PARP was down-regulated at 17 ℃ and 14 ℃. JNK2 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.

Establishment and characterization of a liver cell line from silver pomfret (Pampus argenteus) for studying fish health.

Fish cell lines have become a useful tool to study in resource conservation, genetic breeding, diseases control, and environmental pollutants detection. The silver pomfret (Pampus argenteus) is a high-valued marine fish species in aquaculture, which is seriously threatened by various fish diseases. In this study, a new cell line derived from P. argenteus liver (PaL) was established and characterized. PaL cells mainly consisted of fibroblast-like morphology and multiplied well in Leibovitz's L-15 medium supplemented with 15% foetal bovine serum and 3 ng/mL basic fibroblast growth factor at 22°C. Amplification of the Cyt b gene confirmed that the origin of PaL cells as P. argenteus. Chromosome analysis revealed that PaL cells had a diploid Karyotyp. The PaL cells were efficiently transfected with pEGFP-N3 plasmids, indicating its potential application in foreign gene manipulation studies. The PaL cells were found to be susceptible to red sea bream iridovirus (RSIV) and the expression of immune-related gene (TLR5) and apoptosis-related genes (Bax, Cyt c3, CASP9) were upregulated. Furthermore, lipopolysaccharide and palmitic acid (PA) treatments decreased cell viability and up-regulated the expression of inflammation related genes (IL-8, IL-1ß). Meanwhile, PA incubation induced cell apoptosis by Bcl-2-regulated caspase activation. In conclusion, the newly established PaL cell line will be an appropriate in vitro tool for viral propagation and immune response.

Developmental Genetic Basis of Hoxd9 Homeobox Domain Deletion in Pampus argenteus Pelvic Fin Deficiency.

Pampus argenteus is important for commercial fishery catch species and is an emerging target for aquaculture production. Notably, P. argenteus has a bizarre morphology and lacks pelvic fins. However, the reason for the lack of pelvic fins remains unclear, ultimately leading to frequent upside-down floating of P. argenteus during breeding and marked consumption of physical energy. Some lineages, including whales, fugu, snakes, and seahorse, independently lost the pelvic appendages over evolutionary time. Do different taxa employ the same molecular genetic pathways when they independently evolve similar developmental morphologies? Through analysis of the gene responsible for appendage localization, Hoxd9, it was discovered that the Hox domain was absent in the Hoxd9 gene of P. argenteus, and the Hoxd9b gene lacked the Hox9 activation region, a feature not observed in the Hoxd9 gene of other fish species. Interestingly, those distinctive characteristics are not observed in the Hoxd9 gene of other fish species. To determine the association between the Hoxd9 gene characteristics and the pelvic fin deletion in P. argenteus, the full-length cDNA of the Hoxd9a gene was cloned, and morphological observations of the species' juveniles were performed using stereomicroscopy and scanning electron microscopy. Thereafter, the tissue localization of Hoxd9a in the species was analyzed at the gene and protein levels. Based on the results, deletion of the Hoxd9a structural domain possibly leads to disruptions in the protein translation and the pelvic fin localization in P. argenteus during its early ontogenetic developmental stage, resulting in the absence of pelvic fins.

Histopathology, immunoenzyme activity and transcriptome analysis of immune response in silver pomfret infected by cryptokaryon (Cryptorchidism irritant).

Cryptorchidism irritant (CI) infection is a major problem in the culturing process of silver pomfret (Pampus argenteus), which can result in rapid and massive death. However, there is limited information available on the immune response of silver pomfret infected by CI. To address this gap, we sampled naturally infected fish and observed milky white translucent oval CI trophozoites on the gills, body surface, and fin rays. Histological analysis showed that CI infection led to vacuolation of epithelial cells and a decrease in blood cells in the gills. We also performed transcriptome profiling of the gill, kidney complex, and spleen, generating 399,616,194 clean reads that assembled into 101,228 unigenes, which were annotated based on public databases. We detected 14,369 differentially expressed genes, and selected several key immune-related genes for further validation using RT-qPCR. The Graft-versus-host pathway and Allograft rejection pathway were enriched in the gills, leading to inflammation and ulceration. CI infection activated the immune system, increasing levels of interleukin-1 beta and MHC class II antigen, and also activated innate and acquired immune genes in silver pomfret. Furthermore, we measured the activities of five immune-related enzymes (SOD, AKP, CAT, CSH and ACP), which all increased to varying degrees after CI infection. Our findings enhance our understanding of the immune response of fish to parasitic infection and may contribute to the development of strategies to prevent high mortality in CI-stimulated fish in aquaculture.

Effects of different photoperiods on growth and ovarian development and maturation of silver pomfret Pampus argenteus.

Photoperiod has a great influence on the growth and ovarian development and maturation of fishes. To analyse the effects of photoperiod on growth and ovarian development of an important marine economic fish, silver pomfret Pampus argenteus, short photoperiod group (L:D = 8:16), control group (L:D = 12:12) and long photoperiod group (L:D = 18:6) were set up for 60 days. The growth performance, ovarian development, changes in hormones and key enzyme activities in the hypothalamic-pituitary-gonadal (HPG) axis and expressions of key regulatory genes in the HPG axis were studied under different photoperiod conditions. The results showed that the final weight gain, body weight index, specific growth rate for weight, specific growth rate for length and average daily growth were the highest in the long photoperiod group, and the feed conversion rate was the lowest. Under long photoperiod condition, gonado-somatic index and hepato-somatic index were higher, ovarian maturity was better and expressions of HPG axis-related regulatory genes foxl2a, foxl2b, cyp19a1a, cyp19a1b, kiss, gpr54-2, gnrh2, fsh and lh were higher. When compared with the other two groups, in the long photoperiod group, the change trend of estradiol (E2) was consistent with those of luteinizing hormone, melatonin (MT) and kisspeptin, and the levels were higher on the 20th and 50th days. These results indicate that prolongation of the photoperiod can improve the growth performance of P. argenteus and promote ovary development and maturation. The authors speculate that photoperiod may regulate the ovarian activity of P. argenteus through MT and kisspeptin/gpr54 signalling pathways. The results show that photoperiod can regulate the ovarian development of P. argenteus, which would help in breaking the seasonal restrictions of animals and regulating an animal's reproductive cycle.

Long-term waterborne Cu2+ exposure affects collagen metabolism in fish.

Copper pollution might have a negative effect on collagen metabolism in fish. To test this hypothesis, we exposed an important economical fish, silver pomfret (Pampus argenteus), to three concentrations of Cu2+ for up to 21 days to simulate natural exposure to copper. With increasing copper exposure concentration and time, hematoxylin and eosin staining and picrosirius red staining revealed extensive vacuolization, cell necrosis, and tissue structure destruction, and a change of type and abnormal accumulation of collagen in the liver, intestine, and muscle tissues. To further study the mechanism of collagen metabolism disorder caused by copper exposure, we cloned and analyzed a key collagen metabolism regulation gene, timp, of silver pomfret. The full-length timp2b cDNA was 1035 bp with an open reading frame of 663 bp, encoding a protein of 220 amino acids. Copper treatment significantly increased the expression of akts, erks, and fgfs genes and decreased the mRNA and protein expression of Timp2b and MMPs. Finally, we constructed a silver pomfret muscle cell line (PaM) for the first time and used PaM Cu2+ exposure models (450 µM Cu2+ exposure for 9 h) to examine regulation function of the timp2b-mmps system. We knocked down or overexpressed timp2b in the model, and found that downregulation of mmps expression and upregulation of akt/erk/fgf were further aggravated in the timp2b- group (subjected to RNA interference), whereas some recovery was achieved in the timp2b+ group (overexpression). These results indicated that long-term excessive copper exposure can lead to tissue damage and abnormal collagen metabolism in fish, which might be caused by the alteration of akt/erk/fgf expression, which disrupts the effects of the timp2b-mmps system on extracellular matrix balance. The present study assessed the impact of copper on the collagen of fish and clarified its regulatory mechanism, providing a basis for toxicity of copper pollution study.

Morphological and Molecular Functional Evidence of the Pharyngeal Sac in the Digestive Tract of Silver Pomfret, Pampus argenteus.

The pharyngeal sac is a comparatively rare organ in the digestive tract among teleost fishes. However, our understanding of this remarkable organ in the silver pomfret (Pampus argenteus) is limited. In the present study, we examined the various morphological and histological characteristics of the pharyngeal sac using histochemical techniques and electron microscopy. The pharyngeal sac showed unique characteristics such as well-developed muscular walls, weakly keratinized epithelium, numerous goblet cells, and needle-like processes on the papillae. The porous cavity of the papillae contained numerous adipocytes and was tightly enveloped by type I collagen fibers. These structures might provide mechanical protection and excellent biomechanical properties for grinding and shredding prey. A comparison of gene expression levels between the pharyngeal sac and esophagus using RNA-seq showed that phenotype-associated genes (epithelial genes and muscle genes) were upregulated, whereas genes related to nutrient digestion and absorption were downregulated in the pharyngeal sac. These results support the role of the pharyngeal sac in shredding and predigesting food. Overall, these findings provide a clearer understanding of the pharyngeal sac morphology and explain the morphological adaptations of the digestive tract for feeding on gelatinous prey. To our knowledge, this is the first report on pharyngeal sac gene expression in P. argenteus.

Analyses of growth performance and realized heritability of the second generation of Indo-Pacific Pampus argenteus.

Silver pomfret (Pampus argenteus) is one of the most important fishery products in the Indo-Pacific Ocean. Nonetheless, little is known about the genetic parameters of economic traits. To determine whether the selective breeding programme would improve economic traits, the authors evaluated the growth performance of the selected group and the control group at 60, 90 and 120 days, and the growth performance parameters of the selected group were higher than those of the control group. Through Pearson correlation analysis, the authors found that the morphological traits that are highly related to the weight of P. argenteus are body length and body height. Genetic gain and realized heritability for body weight were highest at day 90, which were 12.72% and 0.23%, respectively. Therefore, the authors speculate that day 90 may be a critical time for screening. The average coefficient of variation for body weight reached over 30%. All three morphological traits reached moderate realized heritability at day 90 after hatching. These implied that selective breeding has the potential to improve the growth traits of P. argenteus. In addition, the reproductive performance of P. argenteus parent fish was obviously improved after gonadal strengthening. These results provide important information for future breeding programmes for P. argenteus.

Effects of acute low-salinity stress on osmoregulation, antioxidant capacity, and growth of the black sea bream (Acanthopagrus schlegelii).

The black sea bream (Acanthopagrus schlegelii) is an important marine economic fish found on the southeast coast of China. Because of the frequent climate change, the salinity of the waters inhabited by A. schlegelii often decreases, which interferes with the fish's physiological homeostasis. The isotonic salinity of teleosts are usually lower than that of seawater, so maximum economic benefits cannot be obtained from conventional mariculture. This study was performed to preliminarily clarify the osmotic regulation and antioxidant mechanism of juvenile A. schlegelii and find an appropriate culture salinity value. We selected 5 psu, 10 psu, 15 psu, and 25 psu (control) to conduct physiological experiments for 96 h and growth experiments for 60 days. We found that the juvenile A. schlegelii could adjust their osmotic pressure within 12 h. The growth hormone and cortisol were found to be seawater-acclimating hormones, whereas prolactin was freshwater-acclimating hormone. The activity and mRNA expression of Na+/K+-ATPase showed a U-shaped trend with the decrease of in salinity at 12-96 h. Serum ion concentration and osmotic pressure remained at a relatively stable level after being actively adjusted from 6 to 12 h. At 96 h, the osmotic pressure of the serum isotonic point of juvenile A. schlegelii was approximately equal to that of water with 14.94 salinity. The number and volume of Cl--secreting cells in the gills decreased. The glomeruli were more developed and structurally sound, with the renal tubules increasing in diameter and the medial brush border being more developed; this may indicate a decrease in salt secretion and an enhanced reabsorption function in the low salinity groups. The activities of superoxide dismutase and catalase and concentration of malondialdehyde were the lowest in the 15 psu group. In addition, the culture conditions of the 15 psu group improved the feed conversion rate without significant differences in weight gain when compared with the control group. Our results show that 15 psu salinity may be the best parameter for obtaining the maximum economic benefits.

Screening and identification of miRNAs regulating Tbx4/5 genes of Pampus argenteus.

Background: Silver pomfret (Pampus argenteus) is one of the most widely distributed and economically important pelagic fish species. However, an unique morphological feature of P. argenteus is the loss of pelvic fins, which can increase the energy requirement during food capture to some extent and is therefore not conducive to artificial culture. Tbx4/5 genes are highly conserved regulatory factors that regulate limb development in vertebrates and are in turn regulated by microRNAs (miRNAs). However, the miRNAs that directly regulate the Tbx4/5 genes in P. argenteus remain to be elucidated. Methods: The Tbx4/5 genes of P. argenteus were first cloned, and the small RNA transcriptomes were sequenced by high-throughput sequencing during the critical period of the fin development at days 1, 7, and 13 of hatching. The miRNAs regulating the Tbx4/5 genes of P. argenteus were subsequently predicted by bioinformatics analysis, and the related miRNAs were verified in vitro using a dual fluorescence reporter system. Results: A total of 662 miRNAs were identified, of which 257 were known miRNAs and 405 were novel miRNAs were identified. Compared to day 1, 182 miRNAs were differentially expressed (DE) on day 7, of which 77 and 105 miRNAs were downregulated and upregulated, respectively, while 278 miRNAs were DE on day 13, of which 136 and 142 miRNAs were downregulated and upregulated, respectively. Compared to day 13, four miRNAs were DE on day 7, of which three miRNAs were downregulated and one miRNA was upregulated. The results of hierarchical clustering of the miRNAs revealed that the DE genes were inversely expressed between days 1 and 7, and between days 1 and 13 of larval development, indicating that the larvae were in the peak stage of differentiation. However, the number of DE genes between days 7 and 13 of larval development was relatively small, suggesting the initiation of development. The potential target genes of the DE miRNAs were subsequently predicted, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of target genes were performed. The results suggested that the DE miRNAs were involved in growth, development, and signal transduction pathways, of which the Wnt and Fgfs signaling pathways are known to play important roles in the growth and development of fins. The results of dual fluorescence reporter assays demonstrated that miR-102, miR-301c, and miR-589 had a significant negative regulatory effect on the 3'-UTR of the Tbx4 gene, while miR-187, miR-201, miR-219, and miR-460 had a significant negative regulatory effect on the 3'-UTR of the Tbx5 gene. Altogether, the findings indicated that miRNAs play an important role in regulating the growth and development of pelvic fins in P. argenteus. This study provides a reference for elucidating the interactions between the miRNAs and target genes of P. argenteus in future studies.

Expression and cellular localization of insulin-like growth factor 3 in gonads of the seasonal breeding teleost silver pomfret (Pampus argenteus).

Insulin-like growth factor 3 plays an important role in gonad development in teleost fish. Previous studies found that igf3 was specifically expressed in gonads of silver pomfret (Pampus argenteus). Unlike in other fish, IGF3 is a membrane protein in silver pomfret, and its specific role in gonads is unclear. Herein, we explored the importance of IGF3 in oogenesis and spermatogenesis in silver pomfret by analyzing gene expression and cellular localization. During follicular development, igf3 was detected in ovaries at both mRNA and protein levels during the critical stages of vitellogenesis (IV-VI). Localization analysis detected igf3 mRNA and protein in somatic cells, including theca and granulosa cells around oocytes. Similar to cathepsin L and cathepsin K, igf3 was consistently expressed in ovaries during vitellogenesis, suggesting that it might play a key role in vitellogenesis of oocytes. During spermatogenesis, igf3 mRNA and protein levels were high in stages II, IV, and V, similar to sycp3 and dmc1, and the highest igf3 mRNA and protein levels were reached in stage VI. Furthermore, igf3 mRNA and protein were detected in spermatogonia, spermatocytes, spermatids, and surrounding Sertoli cells, but not in spermatozoon, indicating that IGF3 might be involved in differentiation and meiosis of spermatogonia.

Eicosapentaenoic acid mitigates palmitic acid-induced heat shock response, inflammation and repair processes in fish intestine.

Understanding the metabolic effects of fatty acids on fish intestine is critical to the substitution of fish oil with vegetable oils in aquaculture. In this study, the effects of eicosapentaenoic acid (EPA) and palmitic acid (PA) on fish intestine were evaluated in vitro and in vivo. As the first step for in vitro study, an intestinal cell line (SPIF) was established from silver pomfret (Pampus argenteus). Thereafter, the effects of EPA and PA on cell viability, prostaglandin E2 (PGE2) production, and the expression of genes related to heat shock response, inflammation, extracellular matrix (ECM) formation and degradation were examined in SPIF cells. Finally, these metabolic effects of EPA and PA on the intestine were examined in zebrafish (Danio rerio) larvae. Results showed that all tested fatty acids (PA, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, and docosahexaenoic acid) except EPA reduced SPIF viability to distinct degrees at the same concentrations. PA decreased SPIF viability accompanied by an increase in PGE2 level. Meanwhile, PA increased the expression of genes related to heat shock response (grp78, grp94, hsp70, and hsp90) and inflammation (nf-κb, il-1ß, and cox2). Furthermore, PA reduced the expression of collagen type I (col1a1a and col1a1b) and extracellular matrix (ECM) degradation-related gene mmp2, while up-regulating timp2 mRNA expression. In vivo, PA also increased hsp70, il-1ß, and cox2 mRNA levels and limited the expression of collagen type I in the larval zebrafish intestine. Interestingly, the combination of EPA and PA partially recovered the PA-induced changes in cell viability, PGE2 production, and mRNA expression in vitro and in vivo. These results suggest that PA may result in heat shock and inflammatory responses, as well as alter ECM formation and degradation in fish intestine, while EPA could at least partially mitigate these negative effects caused by PA.

Transcriptome, antioxidant enzymes and histological analysis reveal molecular mechanisms responsive to long-term cold stress in silver pomfret (Pampus argenteus).

Temperature is a major environmental factor influence fish growth, development, metabolism and physiological performance. Silver pomfret (Pampus argenteus) is an economically important fishery species, however, the molecular mechanisms responsive to long-term cold stress are still unclear. Hence, we altered water temperature from 13 °C to 8 °C, a logistic fit curve for the survival rate of P. argenteus under a gradient cold stress were thus achieved, 50% survival rate at a measured temperature of 7 °C-7.5 °C. After stimulation, the gill, liver and muscle tissues were investigated through transcriptome, antioxidant enzymes and histological observation. The results showed that antioxidant enzyme and Na+-k+ ATPase activity in gill tissue was significantly increased, tissue damage and apoptosis were observed in multi-tissues. By high-throughput sequencing, a total of 618,097,404 reads of raw data and 598,855,490 reads of clean data were obtained, containing 12,489 differently expressed genes (DEGs). KEGG pathway enrichment analysis showed that DNA replication, protein digestion and absorption, cardiac muscle contraction, adrenergic signaling in cardiomyocytes, and metabolic pathways were significantly enriched in multi-tissues. Fifteen DEGs were selected for real-time PCR (RT-qPCR) analysis, and the results were consistent with transcriptome profiling. Based on the results, we inferred that P. argenteus survived at low temperatures may be achieved by improving the ability to scavenge oxyradical substance and enhancing cell fluidity. This present study indicated that the effects of long-term cold stress on P. argenteus, which is valuable for breeding cold-tolerant P. argenteus stocks for cultivation.

The ultrastructure of spermatogenic cells and morphological evaluation of testicular development in the silver pomfret (Pampus argenteus).

The silver pomfret (Pampus argenteus) is a widely distributed and economically important marine fish in the Indo-Pacific. In this study, we acquired the second generation of wild P. argenteus by artificial breeding and further studied the testicular development and ultrastructure of spermatogenesis. The results of gonadosomatic index (GSI) showed the spawning period of this marine fish was from April to June. Besides, through morphological analysis, we found that P. argenteus had an anastomosing tubular testis surrounded by a layer of tunica albuginea, in which spermatogenesis occurred in cysts where the synchronous germ cells were completely surrounded by the cytoplasmic projection of Sertoli cells. Meanwhile, based on submicroscopic characteristics, the germ cells are classified into nine different types. During the ontogenesis of testis, both the early stage of spermatogenesis and sperm were observed in P. argenteus. At sperm maturation stage, different types of spermatozoa and activation of sperms occurred non-synchronously in the tubules. Cytoplasmic bridges also were observed among synchronous germ cells within the cysts, suggesting an interrelated and differentiated relationship among these germ cells.

Immune response of silver pomfret (Pampus argenteus) to Amyloodinium ocellatum infection.

Amyloodinium ocellatum (AO) infection in silver pomfret (Pampus argenteus) causes extensive mortality. Insufficient information exists on the molecular immune response of silver pomfret to AO infestation, so herein we simulated the process of silver pomfret being infected by AO. Translucent trophosomes were observed on the gills of AO-infected fish. Transcriptome profiling was performed to investigate the effects of AO infection on the gill, kidney complex and spleen. Overall, 404,412,298 clean reads were obtained, assembling into 96,341 unigenes, which were annotated against public databases. In total, 2730 differentially expressed genes were detected, and few energy- and immune-related genes were further assessed using RT-qPCR. Moreover, activities of three immune-related (SOD, AKP and ACP) and three energy-related (PKM, LDH and GCK) enzymes were determined. AO infection activated the immune system and increased interleukin-1 beta and immunoglobulin M heavy chain levels. Besides, the PPAR signalling pathway was highly enriched, which played a role in improving immunity and maintaining homeostasis. AO infection also caused dyspnoea, leading to extensive lactic acid accumulation, potentially contributing towards a strong immune response in the host. Our data improved our understanding regarding the immune response mechanisms through which fish coped with parasitic infections and may help prevent high fish mortality in aquaculture.