In aquaculture, copper sulphate and trichlorfon are commonly used as disinfectants and insecticide, sometimes in combination. However, improper use can result in biotoxicity and increased ecological risks. The liver plays a crucial role in detoxification, lipid metabolism, nutrient storage, and immune function in fish. Selecting the liver as the main target organ for research helps to gain an in-depth understanding of various aspects of fish physiology, health, and adaptability. In the present study, zebrafish were exposed to Cu (0.5 mg/L) and Tri (0.5 mg/L) alone and in combination for 21 days. The results demonstrate that both Cu and Tri caused hepatocyte structure damage in zebrafish after 21 days of exposure, with the combination showing an even greater toxicity. Additionally, the antioxidant and immune enzyme activities in zebrafish liver were significantly induced on both day 7 and day 21. A transcriptome analysis revealed that Cu and Tri, alone and in combination, impacted various physiological activities differently, including metabolism, growth, and immunity. Overall, Cu and Tri, either individually or in combination, can induce tissue damage by generating oxidative stress in the body, and the longer the exposure duration, the stronger the toxic effects. Moreover, the combined exposure to Cu and Tri exhibits enhanced toxicity. This study provides a theoretical foundation for the combined use of heavy metal disinfectants and other drugs.

Bisphenol A (BPA) is one of the most common environmental endocrine chemicals, known for its estrogenic effects that can interfere with male spermatogenesis. Lipids play crucial roles in sperm production, capacitation, and motility as important components of the sperm plasma membrane. However, limited research has explored whether BPA affects lipid metabolism in the testes of male fish and subsequently impacts spermatogenesis. In this study, we employed Gobiocypris rarus rare minnow as a research model and exposed them to environmentally relevant concentrations of BPA (15 μg/L) for 5 weeks. We assessed sperm morphology and function and analyzed changes in testicular lipid composition and transcriptomics. The results demonstrated a significant increase in the sperm head membrane damage rate, along with reduced sperm motility and fertilization ability due to BPA exposure. Lipidomics analysis revealed that BPA increased the content of 11 lipids while decreasing the content of 6 lipids in the testes, particularly within glycerophospholipids, glycerolipids, and sphingolipid subclasses. Transcriptomics results indicated significant up-regulation in pathways such as cholesterol metabolism, peroxisome proliferator-activated receptor signaling, and fat digestion and absorption, with significant alterations in key genes related to lipid metabolism, including apolipoprotein A-I, apolipoprotein C-I, and translocator protein. These findings suggest that BPA exposure can induce testicular lipid metabolism disruption in rare minnows, potentially resulting in abnormalities in rare minnow spermatogenesis.

Hemoglobin (Hb) usually comprises two α and two β subunits, forming a tetramer responsible for oxygen transportation and storage. Few studies have elucidated fish hemoglobin immune functions. Megalobrama amblycephala is a freshwater-cultured fish prevalent in China. We identified two M. amblycephala hemoglobin subunits and analyzed their expression patterns and antibacterial activities. The respective full-length cDNA sequences of the M. amblycephala Hb α (MaHbα) and β (MaHbβ) subunits were 588 and 603 bp, encoding 143 and 148 amino acids. MaHbα and MaHbβ were highly homologous to hemoglobins from other fish, displaying typical globin-like domains, most heme-binding sites, and tetramer interface regions highly conserved in teleosts. In phylogenetic analyses, the hemoglobin genes from M. amblycephala and other cypriniformes clustered into one branch, and those from other fishes and mammals clustered into other branches, revealing fish hemoglobin conservation. These M. amblycephala Hb subunits exhibit different expression patterns in various tissues and during development. MaHbα is mainly expressed in the blood and brain, while MaHbβ gene expression is highest in the muscle. MaHbα expression was detectable and abundant post-fertilization, with levels fluctuating during the developmental stages. MaHbβ expression began at 3 dph and gradually increased. Expression of both M. amblycephala Hb subunits was down-regulated in most examined tissues and time points post-Aeromonas hydrophila infection, which might be due to red blood cell (RBC) and hematopoietic organ damage. Synthetic MaHbα and MaHbβ peptides showed excellent antimicrobial activities, which could inhibit survival and growth in five aquatic pathogens. Two M. amblycephala hemoglobin subunits were identified, and their expression patterns and antibacterial activities were analyzed, thereby providing a basis for the understanding of evolution and functions of fish hemoglobins.

Heat shock proteins (HSPs) play a key role in anti-stress and immune processes and are associated with autoimmune diseases. In order to explore the immunological role of HSPs from Schizothorax prenanti (S. prenanti), SpHSP60 was cloned for the first time in this study, and the gene expressions of SpHSP27, SpHSP60, SpHSP70 and SpHSP90 in the hepatopancreas, head kidney, hindgut and spleen were analyzed by quantitative real-time PCR (qPCR) after treatment with lipopolysaccharide (LPS). The open reading frame of the SpHSP60 gene (GenBank accession number ON245159) is 1728 bp. It encodes a protein of 575 amino acids. Its C-terminus is a highly conserved and repeated glycine sequence, which is an important cofactor in ATP binding. Compared with the control group, most of the SpHSPs were significantly upregulated in the tissues examined at 12 or 24 h after LPS challenge. The most abundant expression of SpHSP70 was found in the head kidney at 24 h after LPS injection, followed by SpHSP27 in the spleen at 24 h; both of these SpHSPs displayed strong expression under the LPS stresses, about 20–70 fold more than that of SpHSP60 and SpHSP90. The temporal expression patterns of the four SpHSP genes were different in the four tissues examined. Taken together, the results suggest that SpHSP27, SpHSP60, SpHSP70 and SpHSP90 participate in innate immunity stimulated by LPS, and the response intensity of the SpHSPs was organ-specific, indicating they could provide early warning information against bacterial infection. The findings in our study will contribute to better understanding the biological processes and important roles of SpHSPs involved in defending against pathogenic bacterial challenge.

Environmental DNA (eDNA) techniques have emerged as a cost-effective and non-invasive strategy for monitoring the distribution of endangered aquatic species. Despite their numerous advantages, operational uncertainty at each step of the process represents a significant challenge that can impact the reliability of results. Hence, the optimization of the eDNA analytic protocol is of utmost importance. In this study, the rare and endangered fish species Sichuan taimen was chosen as the target species to evaluate the concordance between different approaches (tissue, pond, and field water samples) for eDNA detection. The results showed that membrane filtration, collection of a 2-L water sample, and utilization of the DNeasy Tissue and Blood DNA extraction kit were suitable for the analysis of DNA in water samples. Furthermore, the mtDNA D-loop region demonstrated potential as a specific molecular marker for the precise identification of Sichuan taimen. Our results indicated that TaqMan fluorescence PCR can serve as the optimal detection method for reliable identification of target DNA even at extremely low concentrations in the field. This study established an optimized eDNA analysis protocol for Sichuan taimen detection, which will provide the basis for future resource investigations aimed at protection of this endangered aquatic species.

We identified and cloned cDNA encoding the heat shock protein (Hsp) 27 gene from Schizothorax prenanti (SpHsp27), and compared its expression with that of SpHsp60, SpHsp70, and SpHsp90 in the liver, head kidney, hindgut, and spleen of S. prenanti that were injected with polyinosinic-polycytidylic acid [Poly (I:C)]. The SpHsp27 partial cDNA (sequence length, 653 bp; estimated molecular mass, 5.31 kDa; theoretical isoelectric point, 5.09) contained an open reading frame of 636 bp and a gene encoding 211 amino acids. The SpHsp27 amino acid sequence shared 61.0–92.89% identity with Hsp27 sequences from other vertebrates and SpHsp27 was expressed in seven S. prenanti tissues. Poly (I:C) significantly upregulated most SpHsps genes in the tissues at 12 or 24 h (p < 0.05) compared with control fish that were injected with phosphate-buffered saline. However, the intensity of responses of the four SpHsps was organ-specifically increased. The expression of SpHsp27 was increased 163-fold in the head kidney and 26.6-fold SpHsp27 in the liver at 24 h after Poly (I:C) injection. In contrast, SpHsp60 was increased 0.97–1.46-fold in four tissues and SpHsp90 was increased 1.21- and 1.16-fold in the liver and spleen at 12 h after Poly (I:C) injection. Our findings indicated that Poly (I:C) induced SpHsp27, SpHsp60, SpHsp70, and SpHsp90 expression and these organ-specific SpHsps are potentially involved in S. prenanti antiviral immunity or mediate pathological process.