Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.).

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

miRNA-seq analysis of liver tissue from largemouth bass (Micropterus salmoides) in response to oxytetracycline and enzyme-treated soy protein.

The specific miRNA regulation triggered by enzyme-treated soybean protein in response to well-known stressors, such as the prophylactic use of the antimicrobial oxytetracycline, remains unknown. Hence, this study aimed to evaluate the regulatory changes of hepatic miRNAs induced by oxytetracycline and enzyme-treated soybean protein in largemouth bass dietary formulations. The experiment was designed with three groups: the normal control (NC), the oxytetracycline exposure treatment group (OTC), and the pre-treatment with enzyme-treated soybean protein before oxytetracycline exposure group (ETSP). miRNA sequencing was employed to characterize the differences between these groups. In conclusion, the NC group exhibited up-regulation of 13 host miRNAs and down-regulation of 1 miRNA compared to the OTC group, whereas the ETSP group showed an increasing trend of 36 host miRNAs and a decreasing trend of 13 host miRNAs compared to the OTC group. Nine miRNAs were identified as prudential targets for enzyme-treated soy protein, protecting the largemouth bass liver from oxytetracycline. Furthermore, gene ontology analysis revealed nine key miRNAs that mediate signaling pathways with significant differences. The cellular lipid metabolic process was identified as the most important biological process, and the propanoate metabolism pathway was highlighted as significant. These results will facilitate further exploration of the mechanism by which enzyme-treated soy protein alleviates the effects of oxytetracycline on largemouth bass in water environments.

Profiling the Physiological Roles in Fish Primary Cell Culture.

Fish primary cell culture has emerged as a valuable tool for investigating the physiological roles and responses of various cell types found in fish species. This review aims to provide an overview of the advancements and applications of fish primary cell culture techniques, focusing on the profiling of physiological roles exhibited by fish cells in vitro. Fish primary cell culture involves the isolation and cultivation of cells directly derived from fish tissues, maintaining their functional characteristics and enabling researchers to study their behavior and responses under controlled conditions. Over the years, significant progress has been made in optimizing the culture conditions, establishing standardized protocols, and improving the characterization techniques for fish primary cell cultures. The review highlights the diverse cell types that have been successfully cultured from different fish species, including gonad cells, pituitary cells, muscle cells, hepatocytes, kidney and immune cells, adipocyte cells and myeloid cells, brain cells, primary fin cells, gill cells, and other cells. Each cell type exhibits distinct physiological functions, contributing to vital processes such as metabolism, tissue regeneration, immune response, and toxin metabolism. Furthermore, this paper explores the pivotal role of fish primary cell culture in elucidating the mechanisms underlying various physiological processes. Researchers have utilized fish primary cell cultures to study the effects of environmental factors, toxins, pathogens, and pharmaceutical compounds on cellular functions, providing valuable insights into fish health, disease pathogenesis, and drug development. The paper also discusses the application of fish primary cell cultures in aquaculture research, particularly in investigating fish growth, nutrition, reproduction, and stress responses. By mimicking the in vivo conditions in vitro, primary cell culture has proven instrumental in identifying key factors influencing fish health and performance, thereby contributing to the development of sustainable aquaculture practices.

miR-125a-3p regulates the expression of FSTL1, a pro-inflammatory factor, during adipogenic differentiation, and inhibits adipogenesis in mice.

Adipogenesis is tightly regulated by various factors, including genes and microRNAs. Excessive fat deposition is the key feature of obesity, which is a low-grade chronic inflammatory disease. Follistatin-like 1 (FSTL1) has been reported to be an important mediator involved in various inflammatory diseases. However, the underlying mechanism of FSTL1 in preadipocyte differentiation and inflammatory response is still unclear. The current study was designed to explore the biological function and potential mechanism of FSTL1 in mouse subcutaneous preadipocyte differentiation. We found that FSTL1 was highly expressed in the early stage of differentiation and subsequently decreased sharply, suggesting that FSTL1 played a possible role in adipogenesis. Meanwhile, the gain- and loss-of-function assays showed that FSTL1 was not only involved in the inflammatory response by inducing the expression of pro-inflammatory factors IL-1ß and CCL2 but also significantly attenuated preadipocyte differentiation, as evidenced by the reduction of lipid accumulation and the levels of adipogenic genes, including PPARγ and FABP4. In addition, the target gene prediction and luciferase reporter assay validated that miR-125a-3p targeted the 3' UTR region of FSTL1. These results demonstrated that miR-125a-3p negatively regulated the expression of FSTL1 at the mRNA and protein levels. Furthermore, overexpressing miR-125a-3p in preadipocytes dramatically accelerated adipogenic differentiation and downregulated the levels of IL-1ß and CCL2, which were in accordance with the knockdown of FSTL1. On the contrary, treatment with miR-125a-3p inhibitors attenuated adipogenesis but induced the expression of inflammatory genes. In summary, this study suggests a positive function of FSTL1 in adipocyte-induced inflammation and negatively regulates preadipocyte differentiation. Further studies demonstrated that miR-125a-3p could reverse the effect by targeting FSTL1, which might provide a better understanding of treating obesity-related inflammatory diseases.

Pyroptosis in fish research: A promising target for disease management.

Pyroptosis is a newly discovered programmed cell death pathway that plays an essential role in the host's defense against pathogenic infections. This process is orchestrated by inflammasomes, which are intricate multiprotein complexes that orchestrate the activation of caspase and instigate the liberation of proinflammatory cytokines. Additionally, gasdermin family proteins execute their role by forming pores in the cell membrane, ultimately leading to cell lysis. In recent years, pyroptosis has emerged as a promising target for disease management in fish, particularly in the context of infectious diseases. In this review, we provide an overview of the current understanding regarding the role of pyroptosis in fish, focusing on its involvement in host-pathogen interactions and its potential as a therapeutic target. We also highlighted the latest advancements in the field development of pyroptosis inhibitors and their potential applications in fish disease management. Subsequently, we deliberate on the obstacles and future prospects for pyroptosis research in fish, emphasizing the necessity of conducting more comprehensive investigations to unravel the intricate regulatory mechanisms governing this process across diverse fish species and environmental contexts. Finally, this review will also highlight the current limitations and future perspectives of pyroptosis research in aquaculture.

Liver fibrosis in fish research: From an immunological perspective.

Liver fibrosis is a pathological process whereby the liver is subjected to various acute and chronic injuries, resulting in the activation of hepatic stellate cells (HSCs), an imbalance of extracellular matrix generation and degradation, and deposition in the liver. This review article summarizes the current understanding of liver fibrosis in fish research. Liver fibrosis is a common pathological condition that occurs in fish raised in aquaculture. It is often associated with poor water quality, stressful conditions, and the presence of pathogens. The review describes the pathophysiology of liver fibrosis in fish, including the roles of various cells and molecules involved in the development and progression of the disease. The review also covers the various methods used to diagnose and assess the severity of liver fibrosis in fish, including histological analysis, biochemical markers, and imaging techniques. In addition, the article discusses the current treatment options for liver fibrosis in fish, including dietary interventions, pharmaceuticals, and probiotics. This review highlights the need for more in-depth research in this area to better understand the mechanisms by which liver fibrosis in fish occurs and to develop effective prevention and treatment strategies. Finally, improved management practices and the development of new treatments will be critical to the sustainability of aquaculture and the health of farmed fish.

Oxytetracycline-induced oxidative liver damage by disturbed mitochondrial dynamics and impaired enzyme antioxidants in largemouth bass (Micropterus salmoides).

Oxytetracycline (OTC), a commonly used tetracycline antibiotic in aquaculture, has been found to cause significant damage to the liver of largemouth bass (Micropterus salmoides). This study revealed that OTC can lead to severe histopathological damage, structural changes at the cellular level, and increased levels of reactive oxygen species (ROS) in M. salmoides. Meanwhile, OTC impairs the activities of antioxidant enzyme (such as T-SOD, CAT, GST, GR) by suppressing the activation of MAPK/Nrf2 pathway. OTC disrupts mitochondrial dynamics and mitophagy through via PINK1/Parkin pathway. The accumulation of damaged mitochondria, combined with the inhibition of the antioxidant enzyme system, contributes to elevated ROS levels and oxidative liver damage in M. salmoides. Further investigations demonstrated that an enzyme-treated soy protein (ETSP) dietary supplement can help maintain mitochondrial dynamic balance by inhibiting the PINK1/Parkin pathway and activate the MAPK/Nrf2 pathway to counteract oxidative damage. In summary, these findings highlight that exposure to OTC disrupts mitochondrial dynamics and inhibits the antioxidant enzyme system, ultimately exacerbating oxidative liver damage in M. salmoides. We propose the use of a dietary supplement as a preventive measure against OTC-related side effects, providing valuable insights into the mechanisms of antibiotic toxicity in aquatic environments.

Profiling miRNAs of Teleost Fish in Responses to Environmental Stress: A Review.

miRNAs are a class of endogenous and evolutionarily conserved noncoding short RNA molecules that post-transcriptionally regulate gene expression through sequence-specific interactions with mRNAs and are capable of controlling gene expression by binding to miRNA targets and interfering with the final protein output. The miRNAs of teleost were firstly reported in zebrafish development, but there are recent studies on the characteristics and functions of miRNAs in fish, especially when compared with mammals. Environmental factors including salinity, oxygen concentration, temperature, feed, pH, environmental chemicals and seawater metal elements may affect the transcriptional and posttranscriptional regulators of miRNAs, contributing to nearly all biological processes. The survival of aquatic fish is constantly challenged by the changes in these environmental factors. Environmental factors can influence miRNA expression, the functions of miRNAs and their target mRNAs. Progress of available information is reported on the environmental effects of the identified miRNAs, miRNA targets and the use of miRNAs in fish.

Dietary Leucine Improves Fish Intestinal Barrier Function by Increasing Humoral Immunity, Antioxidant Capacity, and Tight Junction.

This study attempted to evaluate the possible impact and mechanism of leucine (Leu) on fish intestinal barrier function. One hundred and five hybrid Pelteobagrus vachelli ♀ × Leiocassis longirostris ♂ catfish were fed with six diets in graded levels of Leu 10.0 (control group), 15.0, 20.0, 25.0, 30.0, 35.0, and 40.0 g/kg diet for 56 days. Results showed that the intestinal activities of LZM, ACP, and AKP and contents of C3, C4, and IgM had positive linear and/or quadratic responses to dietary Leu levels. The mRNA expressions of itnl1, itnl2, c-LZM, g-LZM, and ß-defensin increased linearly and/or quadratically (p < 0.05). The ROS, PC, and MDA contents had a negative linear and/or quadratic response, but GSH content and ASA, AHR, T-SOD, and GR activities had positive quadratic responses to dietary Leu levels (p < 0.05). No significant differences on the CAT and GPX activities were detected among treatments (p > 0.05). Increasing dietary Leu level linearly and/or quadratically increased the mRNA expressions of CuZnSOD, CAT, and GPX1α. The GST mRNA expression decreased linearly while the GCLC and Nrf2 mRNA expressions were not significantly affected by different dietary Leu levels. The Nrf2 protein level quadratically increased, whereas the Keap1 mRNA expression and protein level decreased quadratically (p < 0.05). The translational levels of ZO-1 and occludin increased linearly. No significant differences were indicated in Claudin-2 mRNA expression and protein level. The transcriptional levels of Beclin1, ULK1b, ATG5, ATG7, ATG9a, ATG4b, LC3b, and P62 and translational levels of ULK1, LC3Ⅱ/Ⅰ, and P62 linearly and quadratically decreased. The Beclin1 protein level was quadratically decreased with increasing dietary Leu levels. These results suggested that dietary Leu could improve fish intestinal barrier function by increasing humoral immunity, antioxidative capacities, and tight junction protein levels.

Hypo-Osmoregulatory Roles of Vasotocinergic and Isotocinergic Systems in the Intestines of Two European Sea Bass Lineages.

European sea bass (Dicentrarchus labrax) are a major aquaculture species that live in habitats with fluctuating salinities that are sometimes higher than in seawater (SW). Atlantic and West-Mediterranean genetic lineages were compared regarding intestinal neuropeptide receptor expression in SW (36%) and following a two-week transfer to hypersalinity (HW, 55%). Phylogenetic analysis revealed seven neuropeptide receptors belonging to the arginine vasotocine (AVTR) family and two isotocin receptors (ITR). Among AVTR paralogs, the highest mRNA levels were recorded for v1a2, with a two- to fourfold upregulation in the European sea bass intestinal sections after transfer of fish to HW. Principal component analysis in posterior intestines showed that v1a2 expression grouped together with the expression and activity of main ion transporters and channels involved in solute-coupled water uptake, indicating a possible role of this receptor in triggering water absorption. v1a1 expression, however, was decreased or did not change after transfer to hypersaline water. Among ITR paralogs, itr1 was the most expressed paralog in the intestine and opposite expression patterns were observed following salinity transfer, comparing intestinal sections. Overall, different expression profiles were observed between genetic lineages for several analyzed genes which could contribute to different osmotic stress-related responses in D. labrax lineages.

Intestinal osmoregulatory mechanisms differ in Mediterranean and Atlantic European sea bass: A focus on hypersalinity.

European sea bass (Dicentrarchus labrax) migrate towards habitats where salinity can reach levels over 60‰, notably in Mediterranean lagoons. D. labrax are genetically subdivided in Atlantic and Mediterranean lineages and have evolved in slightly different salinities. We compared Atlantic and West-Mediterranean populations regarding their capacity to tolerate hypersalinity with a focus on the involvement of the intestine in solute-driven water reabsorption. Fish were analyzed following a two-week transfer from seawater (SW, 36‰) to either SW or hypersaline water (HW, 55‰). Differences among lineages were observed in posterior intestines of fish maintained in SW regarding NKA activities and mRNA expressions of nkaα1a, aqp8b, aqp1a and aqp1b with systematic higher levels in Mediterranean sea bass. High salinity transfer triggered similar responses in both lineages but at different magnitudes which may indicate slight different physiological strategies between lineages. High salinity transfer did not significantly affect the phenotypic traits measured in the anterior intestine. In the posterior intestine however, the size of enterocytes and NKA activity were higher in HW compared to SW. In this tissue, nka-α1a, nkcc2, aqp8ab and aqp8aa mRNA levels were higher in HW compared to SW as well as relative protein expression of AQP8ab. For aqp1a, 1b, 8aa and 8b, an opposite trend was observed. The sub-apical localization of AQP8ab in enterocytes suggests its role in transepithelial water reabsorption. Strong apical NKCC2/NCC staining indicates an increased Na+ and Cl- reuptake by enterocytes which could contribute to solute-coupled water reuptake in cells where AQP8ab is expressed.

The comprehensive landscape of miR-34a in cancer research.

MicroRNA-34 (miR-34) plays central roles in human diseases, especially cancers. Inactivation of miR-34 is detected in cancer cell lines and tumor tissues versus normal controls, implying its potential tumor-suppressive effect. Clinically, miR-34 has been identified as promising prognostic indicators for various cancers. In fact, members of the miR-34 family, especially miR-34a, have been convincingly proved to affect almost the whole cancer progression process. Here, a total of 512 (miR-34a, 10/21), 85 (miR-34b, 10/16), and 114 (miR-34c, 10/14) putative targets of miR-34a/b/c are predicted by at least ten miRNA databases, respectively. These targets are further analyzed in gene ontology (GO), KEGG pathway, and the Reactome pathway dataset. The results suggest their involvement in the regulation of signal transduction, macromolecule metabolism, and protein modification. Also, the targets are implicated in critical signaling pathways, such as MAPK, Notch, Wnt, PI3K/AKT, p53, and Ras, as well as apoptosis, cell cycle, and EMT-related pathways. Moreover, the upstream regulators of miR-34a, mainly including transcription factors (TFs), lncRNAs, and DNA methylation, will be summarized. Meanwhile, the potential TF upstream of miR-34a/b/c will be predicted by PROMO, JASPAR, Animal TFDB 3.0, and GeneCard databases. Notably, miR-34a is an attractive target for certain cancers. In fact, miR-34a-based systemic delivery combined with chemotherapy or radiotherapy can more effectively control tumor progression. Collectively, this review will provide a panorama for miR-34a in cancer research.

Environmental salinity influences the branchial expression of TCR pathway related genes based on transcriptome of a catadromous fish.

Environmental salinity not only affects the physiological processes such as osmoregulation and hormonal control, but also changes the immune system in fishes. Studies are limited in fish on the roles of the T cell receptor (TCR)-related genes in relation to changes in environmental salinity. A large group of salinity-challenged transcripts was obtained in gills of marbled eel (Anguilla marmorata). Moreover, bioinformatic ways were used to identify the enriched TCR pathway related genes which were significantly different expressed in fresh water (FW), brackish water (BW) and seawater (SW). Meanwhile, the RT-qPCR results were validated and consistent with the RNA-seq results. TCR a, TCR b, CD45, CD28, PI3K, LCK and LAT were up-regulated when the salinity increases in BW and SW, which connected with the related signaling pathways (Ras-MAPK and PKC pathway). CD4 and Zap70 were down-regulated when the salinity increases in BW and SW, which connected with the PLC pathway. The research offers a novel viewpoint to explore the immune pathways including the TCR pathway in fish based on transcriptome.

The influence of environmental calcium on the branchial morphology in a catadromous fish.

Eels are exposed to Ca2+ changes during migration between seawater and freshwater. The gill is the main organ of active calcium transport and has a large surface area to be particularly sensitive to environmental changes in the aquatic environment. In this research, we focused on the morphological changes of gill tissues when eels are faced with the environmental calcium challenges. Based on the results of hematoxylin and eosin (HE) staining and immunohistochemistry, compared with the control group (normal Ca2+ environment), the filament and lamella lengths and lamellar frequency (LF) appeared higher in high calcium environment and lower in deficient calcium environment, while the lamella width and filamental lamellar surface area (SAFL) decreased in high calcium environment and increased in deficient calcium environment. And there was no difference in the number filaments in first right gill arch in the three Ca2+ water environment. Transmission electron microscopy was employed to examine the ultrastructural changes in gills in different Ca2+ water environment. The nucleus and endoplasmic reticulum had a tendency to expand in calcium-deficient water, but had a tendency to shrink in high-calcium water comparing with the control group. This study provides the support that branchial surface areas are regulated in different Ca2+ waters through a list of calcium transporters including CACNB2.

The influence of Ca2+ concentration on voltage-dependent L-type calcium channels' expression in the marbled eel (Anguilla marmorata).

The catadromous species, eels, invariably exposed to variable Ca2+ concentrations circumstance i.e., lagoon or ocean. They need to maintain Ca2+ homeostasis by exchanging Ca2+ under different culture conditions. To understand the effects of environmental Ca2+ to fish, three types of genes coding for voltage-dependent L-type calcium channels (cacnb1, 2, 3) were cloned by screening an A. marmorata cDNA library. Tissue distribution analysis of Western blot showed that Cacnb1, 2, 3 had a significantly high expression in gill; while mRNA results showed the expressions of cacnb1 and cacnb3 were predominated in skin tissue but only cacnb2 was expressed in intestine. Serum osmolality and Ca2+ concentrations of A.marmorata were increased in a high calcium environment while reduced in a low calcium environment within 7 days; however, they were not significantly different among Ca2+ treatments after the eels were acclimated for 7 days. We also examined the influence of ambient Ca2+ levels on cacnbs expression of eels. With the increasing of exposure time, mRNA and protein expressions of cacnb1 were up-regulated in high level of Ca2+ (10 mM) and down-regulated in deficient Ca2+ (0 mM) compared to the control Ca2+ (2 mM). However, the opposite results were observed in cacnb2 and cacnb3. Notably, the cacnb2 expression was not significant different among Ca2+ treatments on day 7. Our study provided the insightful evidence that cacnbs play important roles in maintaining Ca2+ homeostasis of fish.

Latrophilin participates in insecticide susceptibility through positively regulating CSP10 and partially compensated by OBPC01 in Tribolium castaneum.

Latrophilin (LPH) is an adhesion G protein-coupled receptor (aGPCR) that participates in multiple essential physiological processes. Our previous studies have shown that lph is not only indispensable for the development and reproduction of red flour beetles (Tribolium castaneum), but also for their resistance against dichlorvos or carbofuran insecticides. However, the regulatory mechanism of lph-mediated insecticide susceptibility remains unclear. Here, we revealed that knockdown of lph in beetles resulted in opposing changes in two chemoreception genes, chemosensory protein 10 (CSP10) and odorant-binding protein C01 (OBPC01), in which the expression of TcCSP10 was downregulated, whereas the expression of TcOBPC01 was upregulated. TcCSP10 and TcOBPC01 were expressed at the highest levels in early pupal and late larval stages, respectively. High levels of expression of both these genes were observed in the heads (without antennae) of adults. TcCSP10 and TcOBPC01 were significantly induced by dichlorvos or carbofuran between 12 and 72 h (hrs) after exposure, suggesting that they are likely associated with increasing the binding affinity of insecticides, leading to a decrease in sensitivity to the insecticides. Moreover, once these two genes were knocked down, the susceptibility of the beetles to dichlorvos or carbofuran was enhanced. Additionally, RNA interference (RNAi) targeting of lph followed by exposure to dichlorvos or carbofuran also caused the opposing expression levels of TcCSP10 and TcOBPC01 compared to the expression levels of wild-type larvae treated with insecticides alone. All these results indicate that lph is involved in insecticide susceptibility through positively regulating TcCSP10; and the susceptibility could also further partially compensated for through the negative regulation of TcOBPC01 when lph was knockdown in the red flour beetle. Our studies shed new light on the molecular regulatory mechanisms of lph related to insecticide susceptibility.

Individual and combined effects of salinity and lipopolysaccharides on the immune response of juvenile Takifugu fasciatus.

Lipopolysaccharides (LPS) and salinity are important variables in aquatic environments. High concentration of LPS and large changes in salinity seriously threat the survival of a variety of organisms, including fish. To reveal the effects of salinity and LPS on a fish immune response, we measured the immune-related parameters (total leukocyte count, total serum protein, albumin and globulin concentrations, complement C3 concentration, and lysozyme activity) and genes (the expressions of TNF-α, IL-1ß, and SOCS1-3 at the mRNA and protein levels) of juvenile Takifugu fasciatus exposed to phosphate buffered saline (PBS) or LPS (25 µg mL-1) under different salinities (0, 15, and 30 ppt) for 24 h. Changes in key immunological indicators suggested that the LPS challenge induced considerable damage to T. fasciatus, whereas an increase in salinity mitigated the harmful effects. Moreover, although the immune responses in blood and other selected tissues (gill and kidney) were suppressed with an increase in salinity, the increased response in liver in saltwater enabled T. fasciatus to conquer large salinity variation during migration. The appropriate addition of salts appeared to be a sensible strategy to mitigate LPS-induced toxicity in the aquaculture of T. fasciatus.

The improved energy metabolism and blood oxygen-carrying capacity for pufferfish, Takifugu fasciatus, against acute hypoxia under the regulation of oxygen sensors.

Hypoxia frequently occurs in aquatic ecosystem, which is influenced by salinity, water temperature, weather, and surface water runoff. In order to shed further light on the evolutionary and adaptive mechanisms in fish under hypoxic condition, the impact of acute hypoxia (1.63 ± 0.2 mg/L) and reoxygenation (7.0 ± 0.3 mg/L) on oxygen sensors, energy metabolism, and hematological indices was evaluated in Takifugu fasciatus. Data from transcriptional level analysis show that the expressions of genes related to oxygen sensors (HIF-1α, PHD2, and VHL) were upregulated in the brain and liver under hypoxia and recovered under reoxygenation. The upregulation of GLUT2, VEGF-A, and EPO in conjugation with VEGF-A protein and hematological indices conferred the rapid adjustments of cellular glucose uptake and blood oxygen-carrying capacities in pufferfish. Higher levels of glycolysis-related mRNAs (HK, PGK1, and PGAM2), HK activity, and proteins (PGK1 and PGAM2) were detected in the brain and liver under hypoxic condition compared with control. Interestingly, the expression of MDH1 at the mRNA, enzyme activity, and protein levels was significantly increased in the brain at 0 or 2 h and in the liver at 8 h under hypoxic condition. In addition, although the enzyme activity and mRNA expression of LDH in the brain were not significantly changed, a persistent upregulation was observed in the liver during hypoxia exposure. This study demonstrated that pufferfish could counterpoise the energetic demands and hematological functional properties evoked by oxygen sensors after hypoxia. Our findings provided new insights into the molecular regulatory mechanism of hypoxia in pufferfish.

Dynamic expression of vasotocin and isotocin receptor genes in the marbled eel (Anguilla marmorata) following osmotic challenges.

To examine the physiological roles of arginine vasotocin receptor (AVTR) and isotocin receptor (ITR) in osmoregulation of a euryhaline teleost, the marbled eel (Anguilla marmorata), three different genes coding for AVTRV1a2, AVTRV2 and ITR were cloned by screening an A. marmorata cDNA library. These receptors were expressed differentially and ubiquitously in the eight tissues we examined. The changes in mRNA expression levels of AVTRV1a2, AVTRV2, and ITR were assessed in a time-course study following salinity transfer from fresh water (FW, 0‰) to fresh water (FW, 0‰), brackish water (BW, 10‰) or saline water (SW, 25‰). When eels were transferred to BW, mRNA levels underwent an adaptive period, from 0 to 24 h, and a chronic regulatory period, starting at 24 h after transfer. In the adaptive period, the relative mRNA expression of AVTRV1a2, AVTRV2, and ITR increased in BW. But after this adaptive period, the mRNA levels of the three genes were significantly decreased compared to FW (control group, 0 h). The mRNA expression levels of AVTRV1a2, AVTRV2 and ITR were low in SW. The protein level of AVTRV1a2, a key protein in the brain, was also investigated and found to be consistent with mRNA results. Our results indicated that the nonapeptide receptor system may play a role in the acute stress response induced by hyper-osmotic challenge in marbled eels.

Molecular characterization and expression of Piwil1 and Piwil2 during gonadal development and treatment with HCG and LHRH-A2 in Odontobutis potamophila.

Piwi proteins play an important regulatory role in germ cell division during gametogenesis and gonad development. In order to understand the function of Piwi genes in the reproductive process of the dark sleeper, we identified and characterized Piwil1 and Piwil2 from gonad tissue. The tissue distribution demonstrated that Piwils were highly expressed in the gonad of the dark sleeper. During gonad development, higher expression was observed in stage I of both the testes and ovaries than in subsequent stages at mRNA and protein levels. The results of immunohistochemistry demonstrated that Piwils were predominantly distributed in the spermatogonia, spermatocytes, and early oocytes. When treated with the HPG axis hormone (HCG and LHRH-A2), the expression of Piwils was significantly decreased in the testes and ovaries at mRNA and protein levels. All of these results indicated that Piwils play a vital role in gonad development and gametogenesis. Our findings provide valuable evidence to further clarify the underlying modulation mechanism of Piwils in teleosts.