Alternative splicing dynamically regulates common carp embryogenesis under thermal stress.

BACKGROUND: Thermal stress is a major environmental factor affecting fish development and survival. Common carp (Cyprinus carpio) are susceptible to heat stress in their embryonic and larval phases, but the thermal stress response of alternative splicing during common carp embryogenesis remains poorly understood. RESULTS: Using RNA-seq data from eight developmental stages and four temperatures, we constructed a comprehensive profile of alternative splicing (AS) during the embryogenesis of common carp, and found that AS genes and events are widely distributed among all stages. A total of 5,835 developmental stage-specific AS (SAS) genes, 21,368 temperature-specific differentially expressed genes (TDEGs), and 2,652 temperature-specific differentially AS (TDAS) genes were identified. Hub TDAS genes in each developmental stage, such as taf2, hnrnpa1, and drg2, were identified through protein-protein interaction (PPI) network analysis. The early developmental stages may be more sensitive to temperature, with thermal stress leading to a massive increase in the number of expressed transcripts, TDEGs, and TDAS genes in the morula stage, followed by the gastrula stage. GO and KEGG analyses showed that from the morula stage to the neurula stage, TDAS genes were more involved in intracellular transport, protein modification, and localization processes, while from the optic vesicle stage to one day post-hatching, they participated more in biosynthetic processes. Further subgenomic analysis revealed that the number of AS genes and events in subgenome B was generally higher than that in subgenome A, and the homologous AS genes were significantly enriched in basic life activity pathways, such as mTOR signaling pathway, p53 signaling pathway, and MAPK signaling pathway. Additionally, lncRNAs can play a regulatory role in the response to thermal stress by targeting AS genes such as lmnl3, affecting biological processes such as apoptosis and axon guidance. CONCLUSIONS: In short, thermal stress can affect alternative splicing regulation during common carp embryogenesis at multiple levels. Our work complemented some gaps in the study of alternative splicing at both levels of embryogenesis and thermal stress in C. carpio and contributed to the comprehension of environmental adaptation formation in polyploid fishes during embryogenesis.

Effect of aqueous extract of Millettia speciosa Champ on intestinal health maintenance and immune enhancement of Cyprinus carpio.

Millettia speciosa Champ (MSP) is a natural Chinese herb that improves gastrointestinal health and enhances animal immunity. An 8-week feeding trial with different MSP levels (0, 150, 300, and 600 mg/kg) was conducted to evaluate the promotive effects of MSP in Cyprinus carpio. Results indicate that MSP improved intestinal immunity to some extent evidenced by the immuno-antioxidant parameters and the 16S rRNA in the Illumina MiSeq platform. With the analysis of transcriptome sequencing, 4685 differentially expressed genes (DEGs) were identified, including 2149 up-regulated and 2536 down-regulated. According to the GO and KEGG enrichments, DEGs were mainly involved in the immune system. Transcriptional expression of the NOD-like signaling pathway and key genes retrieved from the transcriptome database confirmed that innate immunity was improved in response to dietary MSP administration. Therefore, MSP could be used as a feed supplement that enhances immunity. This may provide insight into Chinese herb additive application in aquaculture production.

Regulation of NF-κB signaling by NLRC (NLRC3-like) gene in the common carp (Cyprinus carpio).

Among teleost NLRs, NLR-C subfamily is a large group of proteins that were teleost-specific and evolution analysis showed that NLR-Cs are most likely to evolve from NLRC3 gene (thus also called as NLRC3Ls). Presently, although there have been rich studies investigating teleost NLRC3 and NLRC3L, the data on the regulatory mechanism was limited. In this study, immune regulation of inflammatory signaling pathway mediated by common carp NLRC3L gene (CcNLRC) has been investigated. Confocal microscopy analysis showed that CcNLRC was located in cytoplasm, and in HEK293T cells, dual-luciferase reporter assay showed the regulation of NF-κB signaling by CcNLRC, in which CcNLRC could alter/decrease RIPK2-induced activation of NF-κB. These results indicated that CcNLRC may function as a negative NLR in the regulation of inflammatory response in common carp. Our data will allow to gain more insights into the molecular mechanism of teleost specific NLR (NLRC3L).

MiR-214_L-1R+4 regulate gossypol-induced immune response through MyD88-dependent signaling pathway in Cyprinus carpio.

MicroRNAs (miRNAs) have been demonstrated to act as crucial modulators with considerable impacts on the immune system. Cottonseed meal is often used as a protein source in aqua feed, cottonseed meal contains gossypol, which is harmful to animals. However, there is a lack of research on the role of miRNAs in fish exposed to gossypol stress. To determine the regulatory effects of miRNAs on gossypol toxicity, Cyprinus carpio were given to oral administration of 20 mg/kg gossypol for 7 days, and the gossypol concentration in the tissues was tested. Then, we detected spleen index, histology, immune enzyme activities of fish induced by gossypol. The results of miRNA sequencing revealed 8 differentially expressed miRNAs in gossypol group, and miR-214_L-1R+4 was found involved in immune response induced by gossypol. The potential targets of miR-214_L-1R+4 were predicted, and found a putative miR-214_L-1R+4 binding site in the 3'UTR of MyD88a. Furthermore, dual-luciferase reporter assays displayed miR-214_L-1R+4 decreased MyD88a expression through binding to the 3'UTR of MyD88a. Moreover, miR-214_L-1R+4 antagomir were intraperitoneally administered to C. carpio, down-regulated miR-214_L-1R+4 could increase MyD88a expression, as well as inflammatory cytokines and anti-inflammatory cytokines expression. These findings revealed that miR-214_L-1R+4 via the MyD88-dependent signaling pathway modulate the immune response to gossypol in C. carpio spleen.

The function of NLRP3 in anti-infection immunity and inflammasome assembly of common carp (Cyprinus carpio L.).

NLRP3 inflammasome can be activated by a variety of stimuli and plays an important role in protecting host from pathogen invasion and maintaining homeostasis. However, the activation mechanism of NLRP3 inflammasome in fish is still unclear. In the present study, the NLRP3 gene (CcNLRP3) was identified from common carp, which was 3069 bp in length and encoded a protein with five domains. Sequence analysis showed that NLRP3 was evolutionarily conserved, and CcNLRP3 was closely related to that in grass carp and zebrafish. Real-time PCR showed that CcNLRP3 was widely expressed in various immune-related tissues of healthy common carp, and significantly increased after stimulation with E. tarda, A. hydrophila and Cyprinus spring viremia virus (SVCV), suggesting that CcNLRP3 might be involved in the immune defense of common carp. The results of co-IP, spot formation, oligomerization and fluorescence localization showed that CcNLRP3 could interact with CcASC and assemble into inflammasome. The cytotoxicity assays showed that CcNLRP3 inflammasome was involved in the pyroptosis induced by CcGSDME. At the same time, CcNLRP3 could directly interact with CcCaspase-A/B and result in increased Caspase-B enzyme activity and LDH release, indicating that CcNLRP3 could also form inflammasome through ASC-independent pathway. Taken together, the results provide targets and theoretical basis for the prevention and control of infectious diseases in aquaculture.

Expression analysis and antiviral activity of koi carp (Cyprinus carpio) viperin against carp edema virus (CEV).

Viperin, also known as radical S-Adenosyl methionine domain containing 2 (RSAD2), is an IFN stimulated protein that plays crucial roles in innate immunity. Here, we identified a viperin gene from the koi carp (Cyprinus carpio) (kVip). The ORF of kVip is 1047 bp in length, encoding a polypeptide of 348 amino acids with neither signal peptide nor transmembrane protein. The predicted molecular weight is 40.37 kDa and the isoelectric point is 7.7. Multiple sequence alignment indicated that putative kVip contains a radical SAM superfamily domain and a conserved C-terminal region. kVip was highly expressed in the skin and spleen of healthy koi carps, and significantly stimulated in both natural and artificial CEV-infected koi carps. In vitro immune stimulation analysis showed that both extracellular and intracellular poly (I: C) or poly (dA: dT) caused a significant increase in kVip expression of spleen cells. Furthermore, intraperitoneal injection of recombinant kVip (rkVip) not only reduced the CEV load in the gills, but also improved the survival of koi carps following CEV challenge. Additionally, rkVip administration effectively regulated inflammatory and anti-inflammatory cytokines (IL-6, IL-1ß, TNF-α, IL-10) and interferon-related molecules (cGAS, STING, MyD88, IFN-γ, IFN-α, IRF3 and IRF9). Collectively, kVip effectively responded to CEV infection and exerted antiviral function against CEV partially by regulation of inflammatory and interferon responses.

Unlocking the potential of N-acetylcysteine: Improving hepatopancreas inflammation, antioxidant capacity and health in common carp (Cyprinus carpio) via the MAPK/NF-κB/Nrf2 signalling pathway.

N-acetylcysteine (NAC) positively contributes to enhancing animal health, regulating inflammation and reducing stress by participating in the synthesis of cysteine, glutathione, and taurine in the body. The present study aims to investigate the effects of dietary different levels of NAC on the morphology, function and physiological state of hepatopancreas in juvenile common carp (Cyprinus carpio). 450 common carps were randomly divided into 5 groups: N1 (basal diet), N2 (1.5 g/kg NAC diet), N3 (3.0 g/kg NAC diet), N4 (4.5 g/kg NAC diet) and N5 (6.0 g/kg NAC diet), and fed for 8 weeks. The results indicated that dietary 3.0-6.0 g/kg NAC reduced hepatopancreas lipid vacuoles and nuclear translocation, and inhibited apoptosis in common carp. Simultaneously, the activities of hepatopancreas alanine aminotransferase and aspartate aminotransferase progressively increased with rising dietary NAC levels. Dietary NAC enhanced the non-specific immune function of common carp, and exerted anti-inflammatory effects by inhibiting the MAPK/NF-κB signaling pathway. Additionally, dietary 3.0-6.0 g/kg NAC significantly improved the antioxidant capacity of common carp, which was associated with enhanced glutathione metabolism, clearance of ROS and the activation of Nrf2 signaling pathway. In summary, NAC has the potential to alleviate inflammation, mitigate oxidative stress and inhibit apoptosis via the MAPK/NF-κB/Nrf2 signaling pathway, thereby improving hepatopancreas function and health of common carp. The current findings provide a theoretical basis for promoting the application of NAC in aquaculture and ecological cultivation of aquatic animals.

Polluted water from a storage dam (Villa Victoria, méxico) induces oxidative damage, AChE activity, embryotoxicity, and behavioral changes in Cyprinus carpio larvae.

The Villa Victoria dam is one of the most important storage reservoirs in Mexico since it distributes water to more than 20 million inhabitants in the Metropolitan Zone of Mexico City. In this dam, the common carp (Cyprinus carpio) is an important food resource for the inhabitants, so the aim of this work was to evaluate the oxidative damage (lipoperoxidation, oxidized proteins, antioxidant enzymes activity and gene expression), AChE, embryotoxicity and behavioral changes in C. carpio embryos and larvae exposed to water from Villa Victoria dam for 24, 48, 72 and 96 h. The embryotoxicity was evaluated trough the General Morphology Score (GMS) and the teratogenic index. Behavioral changes in basal locomotor activity and thigmotaxis were evaluated in a DanioVision, Noldus ™. An increase in lipid and protein oxidation as well as modification of CAT, SOD and GPx enzymatic activity was observed during the exposure times. The GMS indicated a low development in the embryos, the teratogenic index was less than 1, however teratogenic effects as yolk edema, fin malformation, head malformation and scoliosis were observed. In parallel, an increase in AChE activity and gene expression was observed reflecting changes in distance traveled of the basal locomotor activity and thigmotaxis at the sampling points. In conclusion, pollutants in water from Villa Victoria dam caused oxidative damage, changes in SOD, CAT, GPx and AChE activity as well as embryotoxicity and modifications in the behavior of C. carpio larvae. This study demonstrates the need to implement restoration programs for this reservoir since, contamination in the Villa Victoria dam could eventually endanger aquatic life and human health.

Metabolites and metabolic pathway analysis of sulfadimidine in carp (Cyprinus carpio) based on UHPLC-Q-orbitrap HRMS.

Sulfadimidine (SM2) is an N-substituted derivative of p-aminobenzenesulfonyl structure. This study aimed to analyze the metabolism of SM2 in carp (Cyprinus carpio). The carps were fed with SM2 at a dose of 200 mg/(kg · bw) and then killed. The blood, muscle, liver, kidney, gill, other guts, and carp aquaculture water samples were collected. The UHPLC-Q-Exactive Plus Orbitrap-MS was adopted for determining the metabolites of SM2 in the aforementioned samples. Twelve metabolites, which were divided into metabolites in vivo and metabolites in vitro, were identified using Compound Discoverer software. The metabolic pathways in vivo of SM2 in carp included acetylation, hydroxylation, glucoside conjugation, glycine conjugation, carboxylation, glucuronide conjugation, reduction, and methylation. The metabolic pathways in vitro included oxidation and acetylation. This study clarified the metabolites and metabolic pathways of SM2 in carp and provided a reference for further pharmacodynamic evaluation and use in aquaculture.

A transmission electron microscopy investigation suggests that telocytes, skeletal muscles, myoblasts, and stem cells in common carp (Cyprinus carpio) respond to salinity challenges.

BACKGROUND: Telocytes are modified interstitial cells that communicate with other types of cells, including stem cells. Stemness properties render them more susceptible to environmental conditions. The current morphological investigation examined the reactions of telocytes to salt stress in relation to stem cells and myoblasts. The common carp are subjected to salinity levels of 0.2, 6, and 10 ppt. The gill samples were preserved and prepared for TEM. RESULTS: The present study observed that telocytes undergo morphological change and exhibit enhanced secretory activities in response to changes in salinity. TEM can identify typical telocytes. This research gives evidence for the communication of telocytes with stem cells, myoblasts, and skeletal muscles. Telocytes surround stem cells. Telopodes made planar contact with the cell membrane of the stem cell. Telocytes and their telopodes surrounded the skeletal myoblast. These findings show that telocytes may act as nurse cells for skeletal stem cells and myoblasts, which undergo fibrillogenesis. Not only telocytes undergo morphological alternations, but also skeletal muscles become hypertrophied, which receive telocyte secretory vesicles in intercellular compartments. CONCLUSION: In conclusion, the activation of telocytes is what causes stress adaptation. They might act as important players in intercellular communication between cells. It is also possible that reciprocal interaction occurs between telocytes and other cells to adapt to changing environmental conditions.

Variation in the gut microbiota during the early developmental stages of common carp (Cyprinus carpio L.) and its correlation with feed and pond water microflora.

BACKGROUND: Fish gut microbiota undergo dynamic changes under the influence of many factors and play an important role in the nutrition, immunity and development in fish. Although common carp (Cyprinus carpio L.) is an economically important freshwater fish, there are few reports on its gut microbiota changes at different early developmental stages. In the present study, the gut microbiota of common carp during the early developmental stages and its correlation with the feed and pond water flora were studied using the Illumina MiSeq sequencing platform. RESULTS: The results showed that the gut microbiota of common carp underwent continuous and mild changes over the development process, and the pond water environment might provide bacterial resources and have a certain influence on the changes in the gut microbiota of common carp. However, host selection pressure played a more important role in shaping the gut microbiota. Although the gut microbiota was affected by many factors, the presence of core microbiota indicated that some bacterial species adapt to the gut microenvironment of common carp and played a role in its growth process. CONCLUSIONS: The dynamic changes of gut microbiota of carp in early development stage were related to the feed, water environment and host selection. The results of this study provide a theoretical basis for healthy farming and disease prevention of common carp.

Selected micro- and macro-element associations with oxidative status markers in common carp (Cyprinus carpio) blood serum and ejaculate: a correlation study.

The aim of this study was to (1) determine complex interactions between macro- and micro-elements present in blood serum and ejaculate of common carp (Cyprinus carpio), and (2) examine the association between alterations in these macro- and micro-elements with markers of oxidative stress. Blood and ejaculate from 10 male carp were collected in the summer period on the experimental pond in Kolínany (West Slovak Lowland). Reactive oxygen species (ROS), total antioxidant capacity (TAC), protein carbonyls (PC), and malondialdehyde (MDA) levels were measured in blood serum and ejaculate using spectrophotometric methods. The amounts of elements (Ag, Al, Ba, Co, Li, Mo, Ca, K, Na, and Mg) in all samples were quantified using inductively coupled plasma optical emission spectrophotometry. Data demonstrated significant differences in elemental concentrations between blood and ejaculate, specifically significantly higher ejaculate levels were detected for Ag, Al, Ba, Co, Li, Mo, K, and Mg. Potassium was the most abundant macro-element in the ejaculate, while sodium was the most abundant in blood serum. Among the micro-elements, Al was predominant in both types of samples. It is noteworthy that oxidative status markers including ROS, TAC, and MDA were significantly higher in ejaculate indicating the presence of oxidative stress in C. carpio reproductive tissue. The positive correlations between Mg and Ca in blood serum and ejaculate suggest these elements play a functional role in metabolic and physiological processes. In contrast, the positive correlations of Ba and Al with markers of oxidative stress indicated the association of these metals with induction of oxidative stress. Our findings provide insights into the association of metals with biomarkers of physiological function as well as adverse effects in C. carpio.

Dietary exposure to 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) induces oxidative damage promoting cell apoptosis primarily via mitochondrial pathway in the hepatopancreas of carp, Cyprinus carpio.

To investigate the mechanisms of BDE-47 on hepatotoxicity in fish, this study examined the effects of dietary exposure to BDE-47 (40 and 4000 ng/g) on carp for 42 days. The results showed that BDE-47 significantly increased carp's condition factor and hepatosomatic index. Pathological results revealed unclear hepatic cord structure, hepatocytes swelling, cellular vacuolization, and inflammatory cell infiltration in the hepatopancreas of carp. Further investigation showed that ROS levels significantly increased on days 7, 14, and 42. Moreover, the activities of antioxidant enzymes SOD, GSH, CAT, and GST increased significantly from 1 to 7 days, and the transcription levels of antioxidant enzymes CAT, Cu-Zn SOD, Mn-SOD, GST, and GPX, and antioxidant pathway genes Keap1, Nrf2, and HO-1 changed significantly at multiple time-points during the 42 days. The results of apoptosis pathway genes showed that the mitochondrial pathway genes Bax, Casp3, and Casp9 were significantly upregulated and Bcl2 was significantly downregulated, while the transcription levels of FADD and PERK were significantly enhanced. These results indicate that BDE-47 induced oxidative damage in hepatopancreas, then it promoted cell apoptosis mainly through the mitochondrial pathway. This study provides a foundation for analyzing the mechanism of hepatotoxicity induced by BDE-47 on fish.

Epigenetic mechanisms of lncRNA in response to thermal stress during embryogenesis of allotetraploid Cyprinus carpio.

Embryogenesis and epigenetic mechanisms of lncRNA may play an important role in the formation of temperature tolerance in allotetraploid Cyprinus carpio. To investigate the response of lncRNA to thermal stress during embryogenesis of C. carpio, transcriptome sequencing was performed on 81 embryo or larva samples from different early development stages and temperatures. We identified 45,097 lncRNAs and analyzed transcriptome variation during embryogenesis. Stage-specific and temperature-specific DE lncRNAs and DEGs were screened. GO and KEGG analysis identified numerous pathways involved in thermal stress. Temperature-specific regulation of cis-/trans-/antisense lncRNAs was analyzed. Interaction network analysis identified 6 hub lncRNAs and many hub genes, such as cdk1 and hsf1. Decreased expression of many essential genes regulated by lncRNAs may lead to the death of embryos at 33 °C. Our findings provide new insights into the regulation of lncRNA in thermal stress response during embryogenesis and contribute to the understanding of environmental adaptation of allotetraploid species.

Identification of Isthmin-1 in common carp (Cyprinus carpio L.) and the effects on glucose metabolism in vivo and in vitro.

Isthmin-1 (Ism1) plays roles in glucose uptake in mammals as an adipokine. To investigate its role in the glucose metabolism of common carp (Cyprinus carpio. L), the Ism1 sequence was cloned, and its expression and distribution in tissues were detected. In addition, we prepared and purified the recombinant Ism1 protein using the E. coli expression system and assessed changes in the expression of key genes related to glucose metabolism through both in vivo injection experiments and primary hepatocyte experiments in vitro. The results revealed that the open reading frame of Ism1 was 1377 bp long, encoding 458 amino acids. Similarity analysis indicated that Ism1 exhibited a close evolutionary relationship with goldfish (Carassius auratus), sharing 98.35% amino acid similarity. Ism1 was expressed in all tissues of common carp, with the highest level observed in the heart, followed by the gill, head kidney, and hepatopancreas. Distinct patterns of Ism1 expression were identified during the oral glucose tolerance test and long-term high-carbohydrate and high-fat diet feeding experiments. In vivo studies demonstrated that the serum glucose concentration was reduced on treatment with Ism1, accompanied by a significant upregulation of mRNA levels for gk, hk, and pfk genes in hepatopancreas; conversely pepck and g6pase mRNA levels were significantly downregulated in the hepatopancreas under these conditions as well. Furthermore, our primary hepatocyte experiment confirmed that Ism1 could inhibit pepck and g6pase mRNA expression, while promoting gk, hk, and pfk mRNA expression levels. In conclusion, Ism1, in common carp, could participate in the glucose metabolism, which provides essential information for future studies on the function of Ism1.

Evaluation of growth, nutrient absorption, body composition and blood indices under dietary exposure of iron oxide nanoparticles in Common carp (Cyprinus carpio).

The bioavailability, small size and direct absorption in the blood, make nanoparticles (NPs) a remarkable feed additive in the aquaculture industry. Therefore, dietary iron oxide nanoparticles (Fe2 O3 -NPs) were used to examine their effects on growth, nutrient absorption, body composition and blood indices in Cyprinus carpio (Common carp) fingerlings. Healthy C. carpio fingerlings (n = 270) were fed with six canola meal based experimental diets (D1-control, D2, D3, D4, D5, D6) supplemented with 0, 10, 20, 30, 40 and 50 mg/kg Fe2 O3 -NPs respectively. A total of 15 fingerlings (average initial weight 5.51 ± 0.04 g/fish) were kept in triplicates for 70 days. The results indicated that maximum growth performance, apparent digestibility coefficient, body composition and haematological parameters were observed in 40 mg/kg Fe2 O3 -NPs supplementation. All the experimental diets were significantly improved (p < 0.05) in all the above parameters than control diet. In the present research, the recommended dosage of Fe2 O3 -NPs as dietary supplement is 40 mg/kg for improving the growth, nutrient absorption, body composition and haematological indices in C. carpio fingerlings. Hence, this study demonstrates the potential of NPs to improve the health of fish.

Potential protective effects of sodium butyrate on glycinin-induced oxidative stress, inflammatory response, and growth inhibition in Cyprinus carpio.

Investigated mitigating effects of sodium butyrate (SB) on the inflammatory response, oxidative stress, and growth inhibition of common carp (Cyprinus carpio) (2.94 ± 0.2 g) are caused by glycinin. Six isonitrogenous and isoenergetic diets were prepared, in which the basal diet was the control diet and the Gly group diet contained 80 g/kg glycinin, while the remaining 4 diets were supplemented with 0.75, 1.50, 2.25, and 3.00 g/kg SB, respectively. The feeding trial lasted for 8 weeks, and the results indicated that supplementing the diet with 1.50-2.25 g/kg of SB significantly improved feed efficiency and alleviated the growth inhibition induced by glycinin. Hepatopancreas and intestinal protease activities and the content of muscle crude protein were significantly decreased by dietary glycinin, but supplement 1.50-2.25 g/kg SB partially reversed this result. SB (1.50-2.25 g/kg) increased the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the hepatopancreas and reduced the activities of AST and ALT in the serum. Glycinin significantly reduced immune and antioxidant enzyme activities, whereas 1.50-2.25 g/kg SB reversed these adverse effects. Furthermore, compared with the Gly group, supplement 1.50-2.25 g/kg SB eminently up-regulated the TGF-ß and IL-10 mRNA, and down-regulated the IL-1ß, TNF-α, and NF-κB mRNA in hepatopancreas, mid-intestine (MI), and distal intestine (DI). Meanwhile, supplement 1.50-2.25 g/kg SB activated the Keap1-Nrf2-ARE signaling pathway and upregulate CAT, SOD, and HO-1 mRNA expression in hepatopancreas, MI, and DI. Summarily, glycinin induced inflammatory response, and oxidative stress of common carp ultimately decreased the digestive function and growth performance. SB partially mitigated these adverse effects by activating the Keap1-Nrf2-ARE signaling pathway and inhibiting the NF-κB signaling pathway.

Effects of Di-(2-Ethylhexyl) Phthalate (DEHP) on Gamete Quality Parameters of Male Koi Carp (Cyprinus carpio).

In this study, we evaluated gamete quality parameters of mature male koi carp (Cyprinus carpio) exposed to three different concentrations (1, 10, and 100 µg/L) of di-(2-ethylhexyl) phthalate (DEHP). After 60 days of exposure, there was a significant decrease in the gonadosomatic index (GSI) of males exposed to 10 and 100 µg/L of DEHP. Histological analysis of the testes revealed impaired histoarchitecture, including inflammatory cells, intratubular vacuoles, and swollen seminiferous tubules in treatment groups. Gamete quality parameters like sperm production, motility, spermatocrit, and sperm density values were significantly decreased at the 10 and 100 µg/L concentrations. Biochemical compositions, including glucose, cholesterol, and total protein levels, were significantly changed in the treatment groups. Similarly, the ionic compositions of seminal fluid (Na, K, Ca, and Mg) also varied in the treatment groups. Furthermore, the 11-ketotestosterone levels were decreased, and the 17-ß estradiol levels were increased in the DEHP-treated groups. The mRNA expression levels of reproduction-related genes, including Fshr, Lhr, Ar, Erα, and Erß, were significantly changed in the DEHP-treated males in a dose-dependent manner. In conclusion, the findings of this study confirmed that environmentally relevant exposure to DEHP may contribute to a decline in the gamete quality of male fishes.

Isolation and pathogenicity evaluation of Escherichia coli O157:H7 from common carp, Cyprinus carpio.

Escherichia coli O157:H7 is the primary serotype of enterohaemorrhagic E. coli (EHEC), which can cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. It is considered as a major health concern due to it being a zoonotic disease that is transmitted through food. In this study, a pathogenic bacterium was isolated from infected carp, which identified as E. coli O157:H7 named X21 through genetic sequencing, phylogenetic analysis, physiological and biochemical tests. In the experiment, crucian carp was used as a model to study the pathogenicity of the isolate, the pathological histological observations and cytokines expression of fish tissues were determined after bacterial challenge. The results showed that severe pathological damage observed in the liver, spleen, headkidney of fish infected with isolate X21. Besides, we found that accumulation of IgT+ B cells in the lamina propria of intestine, and up-regulation of SUCH-r, IL-1ß, IL-10, IL-11, MyD88, and TNF-α gene in various tissues. After challenged, the survivability of crucian carp infected with isolate X21 stands at a mere 14.27%. To our knowledge, this is the first report that E. coli O157:H7 infected the freshwater fish C. carpio, which indicates that this bacterium is a potential threat to public health and freshwater fish aquaculture.

The hidden world of fish fungal pathogens: molecular identification and phylogenetic analysis in common carp, Cyprinus carpio.

Fungal infections pose a significant threat to aquaculture, causing substantial economic losses and ecological disruptions. The common carp (Cyprinus carpio), as a crucial farmed fish, requires in-depth research to uncover the underlying fungal pathogens affecting its health. In this study, we analyzed 150 samples of C. carpio to identify the fungal pathogens responsible for the infections based on clinical signs and symptoms. Further, we assessed fungal diversity and prevalence in the infected fish. The infected fish exhibited varying degrees of gross pathogenicity, with fins and skin heavily affected, intermediate infection observed in the head and gills, and the least infection found in the operculum. Morphological examination revealed distinct characteristics such as necrosis, lesions on the skin, fins, and gills, as well as loss of scales, hemorrhagic lesions, and red spots. Furthermore, the presence of gray and white cottony patches on the body confirmed ascomycete and zygomycete infections, while a dark white cottony mass indicated phycomycete infection. Some fish exhibited severe fungal infections, presenting prominently curved spines and necrosis with red spots on the skin. These isolates belonged to various fungal groups, including ascomycetes, zygomycetes, phycomycetes, deuteromycetes, and basidiomycetes. Among these, Fusarium oxysporum emerged as the most prevalent fungal pathogen, followed by Fusarium solani, Saprolegnia delica, and Saprolegnia parasitica. Molecular identification based on ITS and LSU rRNA sequences confirmed the presence of Saprolegnia delica, Mucor hiemalis, Coniothyrium telephii, Rhodotorula mucilaginosa, Penicillium cellarum, and Fusarium californicum in the fish samples. Phylogenetic analysis further supported the morphological and molecular data, providing insights into the relationship between the isolated fungal strains and known species from various geographical regions. Our study enhances our understanding of the diversity and prevalence of fish fungal pathogens in common carp, emphasizing the significance of employing molecular techniques for accurate identification. These comprehensive findings offer essential insights into the impact of fungal infections on common carp populations, laying the groundwork for targeted control measures to mitigate their effects on global aquaculture.