An estimation of optimum dietary concentration of soy bean meal for carps (Catla catla, Labeo rohita and Cirhinus mrigala) / Uma estimativa da concentração dietética ideal de farinha de soja para carpas (Catla catla, Labeo rohita e Cirhinus mrigala)

Abstract Soybean meal is an inexpensive plant origin protein which has been used in practical diets as a replacement of animal protein such as fish meal or chicken meal, due to the uneconomical price of animal protein diets. Consequently, a research study was conducted on some commercial species of Indian major carps i.e. Catla (Cattla cattla), Rohu (Labeo rohita) and Mrigala (Cirhinus mrigala) (Hamilton, 1822) to estimate optimum dietary protein requirement of soy bean meal in diet in an intensive polyculture. Three different diets (SBM I, SBM II and SBM III) were formulated by 80%, 50% and 20% replacement of fish meal with soybean meal from a 45% fish meal diet (control).Highest monthly mean weight gain was obtained by SBM II (with 35% CP and about 50% substitution of fish meal), while SBM III (45% Crude Protein and about 20% substitution of fish meal) was stood second. All tested diets respond enormously by producing high yield as compare to control diet, though SBM II generated highest yield among all. On the bases of the following research, it was revealed that the SBM can surrogate even50% fish meal without any augmentation of other amino acids in the diet of Indian major carps.

Resumo O farelo de soja é uma proteína de origem vegetal de baixo custo que tem sido usada em dietas práticas como um substituto da proteína animal, como farinha de peixe ou farinha de frango, devido ao preço não econômico das dietas com proteína animal. Consequentemente, um estudo/pesquisa foi realizado com algumas espécies comerciais de carpas principais indianas, ou seja, Catla (Cattla cattla), Rohu (Labeo rohita) e Mrigala (Cirhinus mrigala) (Hamilton, 1822), para estimar a necessidade ideal de proteína dietética de farelo de soja na dieta em uma policultura intensiva. Três dietas diferentes (SBM I, SBM II e SBM III) foram formuladas por 80%, 50% e 20% de substituição de farinha de peixe por farelo de soja de uma dieta de 45% de farinha de peixe (controle). O maior ganho de peso médio mensal foi obtido por SBM II (com 35% PB e cerca de 50% de substituição de farinha de peixe), enquanto SBM III (45% de proteína bruta e cerca de 20% de substituição de farinha de peixe) ficou em segundo lugar. Todas as dietas testadas respondem enormemente produzindo alto rendimento em comparação com a dieta controle, embora SBM II tenha gerado o maior rendimento entre todas. Com base na pesquisa a seguir, foi revelado que o SBM pode substituir até 50% da farinha de peixe sem qualquer aumento de outros aminoácidos na dieta das carpas principais indianas.

Fatty acid profile variations after exposure to textile industry effluents in Indian Major Carps / Variações do perfil de ácidos graxos após exposição a efluentes da indústria têxtil nas principais carpas indianas

Abstract Indian major carps are the widely consumed fish species of Pakistan, being a cheap source of proteins and unsaturated fatty acids, they are good for cardiovascular health. Water pollution due to discharge of untreated industrial waste water into water bodies contaminates this precious source of nutrients. The present study therefore, was aimed to assess deterioration of fatty acid profile of three Indian major carp species due to different concentrations of industrial wastes. The water samples were collected from the river Chenab at the site where it receives industrial wastewater via Chakbandi drain. After exposure to 1.5%, 3.0%, and 4.5% dilutions of collected water in different aquaria it was observed that proportion of unsaturated fatty acids in selected fish species were decreased significantly as the intensity of the dose increased (P < 0.05). Conversely the level of saturated fatty acids increased with the increasing dose of treatment (P < 0.05). These findings suggest that untreated wastewater not only deteriorate the fatty acid profile of aquatic animals but also these toxic substances can reach human body through fish meat and pose further health hazards. Therefore, it is highly recommended that industrial effluents should be treated before they are dumped into water bodies.

Resumo As carpas indianas são as espécies de peixes mais consumidas no Paquistão, sendo uma fonte barata de proteínas e de ácidos graxos insaturados e boa para a saúde cardiovascular. A poluição da água por causa do descarte de resíduos industriais não tratados em corpos d'água contamina essa preciosa fonte de nutrientes. Portanto, o presente estudo teve como objetivo avaliar a deterioração do perfil de ácidos graxos de três principais espécies de carpas indianas em diferentes concentrações de resíduos industriais. As amostras de água foram coletadas do rio Chenab no local onde recebe esgoto industrial via dreno de Chakbandi. Após a exposição a diluições de 1,5%, 3% e 4,5% da água coletada em diferentes aquários, foi observado que a proporção de ácidos graxos insaturados em espécies de peixes selecionadas diminuiu significativamente com o aumento da intensidade da dose (P < 0,05). Por outro lado, o nível de ácidos graxos saturados aumentou com a elevação da dose de tratamento (P < 0,05). Essas descobertas sugerem que águas residuais não tratadas não apenas deterioram o perfil de ácidos graxos dos animais aquáticos, mas também essas substâncias tóxicas podem atingir o corpo humano por meio da carne de peixe e representar mais riscos à saúde. Portanto, é recomendável que os efluentes industriais sejam tratados antes de serem despejados em corpos d'água.

Percentage Incidences of Bacteria in Mahseer (Tor putitora), Silver carp (Hypophthalmichthys molitrix) Fish Collected from Hatcheries and Local Markets of District Malakand and Peshawar of Khyber Pakhtunkhwa, Pakistan / Incidências percentuais de bactérias em Mahseer (Tor putitora), carpa-prateada (Hypophthalmichthys molitrix), peixes coletados em incubatórios e mercados locais do distrito de Malakand e Peshawar de Khyber Pakhtunkhwa, Paquistão

Abstract Fish is the main source of animal protein for human diet. The aim of this study was to find out prevalence of pathogenic bacteria of two selected economically important fish of Pakistan namely Mahseer (Tor putitora) and Silver carp (Hypophthalmichthys molitrix). Live fish samples from hatcheries and dead fish samples from different markets of study area were randomly collected. The fish samples were analyzed for isolation, identification and prevalence of bacteria. The isolated bacteria from study fish were identified through biochemical test and about 10 species of pathogenic bacteria were identified including the pathogenic bacteria to human and fish namely, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. and Salmonella spp. The bacterial percentage frequency of occurrence in Silver carp and Mahseer fish showed Pseudomonas aeruginosa 21.42%, Staphylococcus epidermidis 17.85%, Escherichia coli 11.90%, Staphylococcus aureus 9.52%, Citrobacter spp. 9.52%, Serratia spp. 8.33%, Streptococcus iniae 7.14%, Stenotrophomonas spp. 5.95%, Bacillus spp. 4.76% and Salmonella spp. 3.57%. The study revealed that Fish samples of Mahseer and Silver carp that were collected from markets have found more isolates (10 bacterial species) than did the fresh fish pond samples (03 bacterial species) of hatcheries. The occurrence of pathogenic bacteria in study fish showed risk factor for public health consumers.

Resumo O peixe é a principal fonte de proteína animal para a alimentação humana. O objetivo deste estudo foi descobrir a prevalência de bactérias patogênicas de dois peixes economicamente importantes selecionados do Paquistão, nomeadamente Mahseer (Tor putitora) e carpa prateada (Hypophthalmichthys molitrix). Amostras de peixes vivos de incubatórios e amostras de peixes mortos de diferentes mercados da área de estudo foram coletadas aleatoriamente. As amostras de peixes foram analisadas quanto ao isolamento, identificação e prevalência de bactérias. As bactérias isoladas dos peixes do estudo foram identificadas através de testes bioquímicos e cerca de 10 espécies de bactérias patogênicas foram identificadas incluindo as bactérias patogênicas para humanos e peixes, nomeadamente, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus iniae, Serratia spp. Citrobacter spp. Stenotrophomonas spp. Bacillus spp. e Salmonella spp. A porcentagem de freqüência de ocorrência bacteriana em carpa prateada e peixes Mahseer mostrou Pseudomonas aeruginosa 21,42%, Staphylococcus epidermidis 17,85%, Escherichia coli 11,90%, Staphylococcus aureus 9,52%, Citrobacter spp. 9,52%, Serratia spp. 8,33%, Streptococcus iniae 7,14%, Stenotrophomonas spp. 5,95%, Bacillus spp. 4,76% e Salmonella spp. 3,57%. O estudo revelou que as amostras de peixes de Mahseer e carpa prateada coletadas nos mercados encontraram mais isolados (10 espécies bacterianas) do que as amostras de peixes frescos (03 espécies bacterianas) de incubatórios. A ocorrência de bactérias patogênicas nos peixes do estudo apresentou fator de risco para consumidores de saúde pública.

Enhanced hydrophobic interaction between fish (Cyprinus carpio L.) scale gelatin and curcumin: Mechanism study.

To enhance the solubility of hydrophobic nutrients, the hydrophobicity of fish scale gelatin hydrolysate (FSGH) was increased with moderate acid or alkali hydrolysis. Acid-induced FSG hydrolysate (AcFSGH) at 3 h showed a superior curcumin loading efficiency (18.30 ± 0.38 µg/mL) among all FSGHs. Compared with FSG, the proportion of hydrophobic amino acids (from 41.1% to 46.4%) and the hydrophobic interaction (from 12.72 to 20.10 mg/mL) was significantly increased in the AcFSGH. Meanwhile, the transformation of the α-helix (from 12.8% to 4.9%) to the ß-sheet (from 29.0% to 42.8%) was also observed in the AcFSGH. Based on the observation in the molecular weight and morphological analysis, AcFSGH acquired the best hydrophobic interaction with curcumin, presumably due to the formation of the flexible structure of the linear hydrolyzates. The above results call for an investigation of the role of FSG hydrolysate in the synthesis of nanoparticles loaded with bioactive lipophilic compounds.

Component stabilizing mechanism of membrane-separated hydrolysates on frozen surimi.

This study investigated the cryoprotective mechanism of ultrafiltration membrane-separated fractions (>10 kDa, UF-1; 3-10 kDa, UF-2; and <3 kDa, UF-3) derived from silver carp hydrolysates on frozen surimi. The surimi gel incorporating UF-3 exhibited a compact, continuous structure with uniform pores, even after undergoing six freeze-thaw (F-T) cycle, with the minimal reduction in entrapped water (from 95.1 % to 91.1 %) and least increase in free water (from 4.5 % to 6.6 %) as revealed by SEM and LF-NMR analysis. Through molecular docking analysis, three major peptides in UF-3 were identified to form robust interactions with the myosin head pocket, facilitated by hydrogen bonds, electrostatic forces, and hydrophobic interactions. Furthermore, molecular dynamics simulations demonstrated that the three peptides effectively prevented myosin from unfolding and aggregating by tightly binding to basic amino acids (Arg, Lys) and hydrophobic amino acids (Phe, Leu, Ile, Met, and Val) residues in the myosin head pocket, primarily governed by electrostatic energies (-156.95, -321.38, and -267.53 kcal/mol, respectively) and van der Waals energies (-395.05, -347.46, and -319.16 kcal/mol, respectively). Notably, the key action site was identified as Lys599 on myosin. The hydrophilic and hydrophobic hotspot residues of the peptides worked synergistically to stabilize the myosin structure in frozen surimi.

Texture maintenance and degradation mechanism of ice-stored grass carp (Ctenopharyngodon idella): A scope of intramuscular connective tissue.

Although intramuscular connective tissue (IMCT) is low in fish, its impact on texture cannot be ignored due to its special location. Therefore, this study was aimed to investigate the contribution of IMCT degradation to fish softening and its mechanism induced by endogenous proteases. Results showed that IMCT honeycomb-like structure collapsed entirely on the 10th day of ice storage, along with a decrease of shear force by 36.5%. Meanwhile, IMCT and myofibrils (MF) degradation accelerated softening by 25.1% and 15.3% during 10 days of ice storage, respectively. Next, IMCT deterioration was indicated to be highly correlated with decorin degradation (0.956**), followed by elastin (0.928**) and collagen (0.904**). Ulteriorly, endogenous collagenase was shown to degrade IMCT crucial components, while endogenous cathepsins had little effect. In conclusion, this study confirmed that IMCT played an essential role in maintaining fish texture and was mainly degraded by endogenous collagenase.

Ultrastructural characteristics of the accumulation of iron nanoparticles in the intestine of Cyprinus carpio (Linnaeus, 1758) under aquaculture.

During the development of nanotechnology, the production of many substances containing nanoparticles leads to the release of various nanoparticles into the environment, including the water ecosystem. The main goal of the current research was to study the ultrastructural characteristics of the entry and bioaccumulation of Fe3O4 nanoparticles in the small intestine of Cyprinus carpio (Linnaeus, 1758), as well as the pathomorphological changes in the fish organism. Two different doses (10 and 100 mg) of Fe3O4 nanoparticles were fed to fingerlings for 7 days and then intestinal samples were taken and studied. It was found that the extent of damages was boosted within the increment of nanoparticle concentration. The sequence and bioaccumulation of Fe3O4 nanoparticles in the small intestine of fish occurred as below: firstly, the nanoparticles passed into microvilli located in the apical part of enterocytes in the mucosa layer, from there into the cytoplasm of the epithelial cells, including cytoplasmatic organelles (nucleus, mitochondria, lysosomes, fat granules), and then into a lamina propria of the mucosa of the small intestine and passed into the endothelium of the blood vessels and to the erythrocytes of the vessels which located in the lumen. It was determined that although the nanoparticles were up to 30 nm in size, only particles with a maximum size of 20 nm could penetrate the intestinal wall. Thus, the release of Fe3O4 nanoparticles into the environment in high doses has a negative effect on the living ecosystem, including the body of fish living in the water.

Microplastic ingestion by invasive Prussian carp (Carassius gibelio) used in fishmeal production in Türkiye.

MP contamination in the gastrointestinal tract (GIT) of Prussian carp (Carassius gibelio) used in fishmeal production was examined. The samples were collected from Balik Lake, Karabogaz Lake, and Liman Lake located in Kizilirmak Delta, Türkiye. The overall average abundance was 3.20±0.18 MP per fish and 0.036±0.002 MP per g. No significant correlation was determined between the MP abundance in the fish, fish length, and fish weight. The polymers were composed of 56.91% PET, 25.20% PP, 9.76% PE, and 8.13% PA. The determined shapes were fiber (78.0%), fragment (13.46%), and film (8.54%). The size ranged between 112 and 4775 µm with an average of 1341.41±66.07 µm. Depending on the occurrence frequency and abundance, it can be concluded that Carassius gibelio can be considered a MP vector in fishmeal. The results provide an essential baseline for future MP studies on the potential effects of MPs on aquatic organisms, fate, and impact on the fishmeal production process, aquaculture practices, and human health.

Disruption of the intestinal barrier by avermectin in carp involves oxidative stress and apoptosis and leads to intestinal inflammation.

Avermectin (AVM) is a widely used insecticide. Due to its sensitive toxicity to aquatic organisms, the toxicology of AVM on fish intestines remains unclear. Here, we established a 96 h AVM acute toxicity model to explore the effects of AVM on the intestinal tract of carp. The 96 h LC50 of carps exposed to AVM was 24.04 µg/L, 12.02 µg/L was selected as the high-dose group and 3.005 µg/L was selected as the low-dose group. After 96 h of exposure, intestinal tissues were collected and subsequently analyzed for histopathology, the activities of antioxidant oxidases (CAT, SOD, GSH-Px), and the expression of mRNA associated with oxidative stress, inflammation, and apoptosis. Our study showed that AVM exposure caused intestinal damage in carp, decreased the expression of the tight junction protein gene, activated oxidative stress, induced apoptosis, and induced intestinal inflammation in carp. Therefore, we demonstrated that AVM exposure compromised the integrity of the intestinal barrier in carp, activated oxidative stress, induced endogenous apoptosis, and induced intestinal inflammatory responses. These results indicate that AVM, as a drug-sensitive to aquatic organisms, has a much more complex toxic effect on the fish intestinal tract, which provides a new perspective for studying the toxicology of AVM on the fish intestinal tract.

Spoilage markers for freshwater fish: A comprehensive workflow for non-targeted analysis of VOCs using DHS-GC-HRMS.

Changes of volatile organic compounds (VOCs) patterns during 6 days of storage at +4 °C were investigated in different freshwater fish species, namely carp and trout, using dynamic headspace gas chromatography time-of-flight mass spectrometry (DHS-GC-TOFMS). DHS parameters were systematically optimized to establish optimum extraction and pre-concentration of VOCs. Moreover, different sample preparation methods were tested: mincing with a manual meat grinder, as well as mincing plus homogenization with a handheld homogenizer both without and with water addition. The addition of water during sample preparation led to pronounced changes of the volatile profiles, depending on the molecular structure and lipophilicity of the analytes, resulting in losses of up to 98 % of more lipophilic compounds (logP > 3). The optimized method was applied to trout and carp. Trout samples of different storage days were compared using univariate (Mann-Whitney U test, fold change calculation) and multivariate (OPLS-DA) statistics. 37 potential spoilage markers were selected; for 11 compounds identity could be confirmed via measurement of authentic standards and 10 compounds were identified by library spectrum match. 22 compounds were also found to be statistically significant spoilage markers in carp. Merging results of the different statistical approaches, the list of 37 compounds could be narrowed down to the 14 most suitable for trout spoilage assessment. This study comprises a systematic evaluation of the capabilities of DHS-GC coupled to high-resolution (HR) MS for studying spoilage in different freshwater fish species, including a comprehensive data evaluation workflow.

Enrofloxacin hydrochloride toxicological effects on crucian carp reflected by serological changes and neurotoxicity.

Due to its water solubility and wide applicability, enrofloxacin hydrochloride (EH) may enter aquatic ecosystems and cause negative effects on aquatic organisms. This study aimed to explore toxicological effects via serological changes and neurotoxicity, which were induced by EH exposure in crucian carp (Carassius auratus var. Pengze). The drug residues in brain tissue and protein content in serum were determined to analyze serological changes. Alterations in brain tissue structure and function, cerebral microvessels permeability, and the expressions of gene and protein regarding blood-brain barrier (BBB) were studied to reflect the neurotoxicity. Employing a validated high-performance liquid chromatography (HPLC) method, EH residues could be detected at various time-points throughout the experiment. Enzyme-linked immunosorbent assay (ELISA) showed that EH increased the levels of S100B, NSE and GFAP proteins in serum. Additionally, there was a significant positive correlation between serum S100B, NSE protein contents and EH residues (P < 0.05). Hematoxylin and eosin (H&E) staining revealed brain damage from EH exposure by the formation of vacuoles in brain glial cells, pyknosis of the nucleus, and a decrease in cell population density. Transmission electron microscope (TEM) revealed morphological changes in microvessels and condensation of astrocyte nucleus. Evans blue (EB) permeability test visualized an obvious increase in cerebral microvessels leakage. The real-time quantitative PCR (qPCR) results indicated that EH up-regulated the mRNA expression levels of S100B, NSE and GFAP, down-regulated the mRNA expression levels of P-gp, ZO-1, Occludin and Claudin-5. The Western blot (WB) results demonstrated increased NSE and GFAP protein expressions, decreased P-gp and Occludin protein expressions following EH exposure in brain, in consistent with the gene expressions, respectively. In conclusion, these findings indicated that EH brought about marked rise in serum biomarker levels and disrupted the central nervous system (CNS) of crucian carp. These data would help elucidate the mechanism underlying EH-induced neurotoxicological effects.

Mulberrin alleviates triclocarban induced hepatic apoptosis and inflammation by regulating the ROS/NF-κB pathway in grass carp.

Triclocarban (TCC) is commonly used in household, personal care and industrial products and has been frequently detected in different aquatic ecosystems. Mulberrin (Mul) is a key component of the traditional Chinese medicine Romulus Mori with antioxidant and anti-inflammatory properties. The present study aimed to investigate the hepatotoxic effects of TCC in aquatic organisms and explore the protective roles of Mul. Herein, we found that exposure to TCC at environmentally realistic concentrations (5 µg/L) could impair liver function, along with impaired antioxidant defense and infiltration of inflammatory cells. Additionally, we found that TCC increased the ratio of TUNEL staining positive cells, accompanied by upregulation of pro-apoptotic protein (Bax, caspase3 and caspase9), and downregulation of anti-apoptotic proteins (Bcl2). In contrast, Mul supplementation reversed the hepatic pathological damage, ROS elevation, and apoptosis induced by TCC, likely due to hyperactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Additionally, Mul supplementation suppressed the mRNA levels of proinflammatory factors (TNF-α, IL-1ß, IFN-γ, IL-6 and IL-8) and enhanced the mRNA levels of anti-inflammatory factors (TGFß1, TGFß2, IL4, IL10 and IL11) in the liver of carp. We also discovered that Mul supplementation suppressed TCC-induced nuclear nuclear factor κB (NF-κB) elevation. In conclusion, Mul enhances Nrf2 signaling cascades and counteracts the NF-κB inflammatory program to rescue hepatotoxicity induced by TCC, providing new insights into the hepatotoxic effects of TCC and potential protection strategies for heart injury induced by TCC.

Insight into Ionic Strength-Induced Solubilization of Myofibrillar Proteins from Silver Carp (Hypophthalmichthys molitrix): Structural Changes and 4D Label-Free Proteomics Analysis.

In this study, changes in the physical, structural, and assembly characteristics of silver carp myofibrillar proteins (MPs) at different ionic strength (I) values were investigated. Moreover, the differential proteomic profile of soluble MPs was analyzed using 4D proteomics based on timsTOF Pro mass spectrometry. Solubility of MPs significantly increased at high I (>0.3), and the increase in I enhanced the apparent viscosity, fluorescence intensity, surface hydrophobicity, and α-helix content of MPs solution. Particle size and sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns also supported the solubility profiles. Transmission electron microscopy and atomic force microscopy observations revealed the morphological assembly and disassembly of MPs under different I conditions. Finally, proteomic analysis revealed the evolution law of salt-induced solubilization of MPs and the critical molecular characteristics in different I environments. The number of differentially abundant proteins (DAPs) decreased with the increase of I, and most DAPs related to the muscle filament sliding, contraction and assembly, actinin binding, and actin filament binding. The soluble abundance of myosin and some structural proteins was dependent on I, and structural proteins in the Z-disk and M-band might contribute to the solubilization of myosin. Our findings provide insightful information about the impact of common I on the solubility pattern of MPs from freshwater fish.

Development of an indirect ELISA for detection of the adaptive immune response of black carp (Mylopharyngodon piceus).

Black carp (Mylopharyngodon piceus) is an important fishery resource and the main breeding target in China. Due to the lack of an assay of immunoglobulin M (IgM) antibodies in black carp, there is no effective method to evaluate adaptive immune response, which limits immunological studies and vaccine development. The present study used mAbs (monoclonal antibodies) against serum IgM of grass carp as capture antibodies. The results of Western blot analysis indicated that these antibodies had strong affinity and specificity to IgM heavy chain in black carp serum and were used to detect the antibody titer, optimize the conditions, perform a sensitivity test, and develop an indirect ELISA (enzyme-linked immunosorbent assay) to detect specific IgM antibodies in the serum. This detection method has good specificity and is effective only for grass carp (Ctenopharyngodon idella) and black carp and not for crucian carp (Carassius aumtus), silver carp (Hypophthalmichthys molitrix), bighead carp (Hypophthalmichthys nobilis), mandarin fish (Siniperca chuatsi), black bream (Megalobrama skolkovii), or yellow catfish (Pseudobagrus fulvidraco). The lowest antigen detection level was 0.05 µg/ml. The error of experimental repetition in the same sample was 1.61-4.61%. The levels of specific IgM in black carp serum were steadily increased after immunization, peaked on day 28, and then slowly decreased. Indirect ELISA can be applied to detect the changes in specific antibodies in black carp serum. Moreover, indirect ELISA provides a convenient and reliable serological detection method for immunological research and evaluation of immune effects of a vaccine in black carp.

Temperature-associated virulence, species susceptibility and interspecies transmission of a Lactococcus petauri strain from rainbow trout.

Lactococcus petauri is an important emergent aquaculture pathogen in the USA. To better understand environmental conditions conducive to piscine lactococcosis and the susceptibility of fish species, laboratory-controlled challenges were used as models of infection. Rainbow trout Oncorhynchus mykiss maintained at 13 or 18°C were challenged by intracoelomic (ICe) injection with 101, 103 or 105 colony-forming units per fish (CFU fish-1) and monitored for 21 d. At 13°C, trout experienced mortalities of 7, 7 and 0%, and bacterial persistence of 0, 20 and 0% in survivors, respectively. When exposed to the same bacterial doses, trout maintained at 18°C experienced mortalities of 59, 84 and 91%, and bacterial persistence of 60, 66 and 0% in survivors, confirming a significant role of temperature in the pathogenesis of lactococcosis. Additionally, the susceptibility of rainbow trout, Chinook salmon Oncorhynchus tshawytscha, white sturgeon Acipenser transmontanus, Nile tilapia Oreochromis niloticus, and koi Cyprinus carpio to infection by L. petauri was compared using ICe challenges at 18°C. Trout and salmon experienced 96 and 56% cumulative mortality, respectively, and 17% of surviving salmon remained persistently infected. There were no mortalities in the other fish species, and no culturable bacteria recovered at the end of the challenge. However, when surviving fish were used in further cohabitation trials, naïve trout housed with previously exposed tilapia exhibited 6% mortality, demonstrating that non-salmonids can become sub-clinical carriers of this pathogen. The data obtained provide useful information regarding temperature-associated virulence, fish species susceptibility, and potential carrier transmission of L. petauri that can be used in the development of better management practices to protect against piscine lactococcosis.

Impact of dietary piperine on growth performance, immune response, antioxidant status, and immune-related gene expression in pathogen-aggravated Cyprinus carpio.

This study investigated the effects of dietary piperine (PIP) on growth performance, digestive enzymes, serum biochemical parameters, antioxidant and immune responses, and gene expression in Cyprinus carpio challenged with Aeromonas hydrophila. Six diets were prepared with PIP doses of 0, 0.5, 1.0, 2.0, 3.0, and 4.0 g/kg, corresponding with the control, PR50, PR100, PR200, PR300, and PR400, respectively. Fish were challenged with Aeromonas hydrophila after 8 weeks of feeding with the respective diets. Weight gain (PWG) and specific growth rate (SGR) were significantly enhanced, whereas feed conversion ratio (FCR) was lowered in PR200. The cumulative post-challenge survival was improved to 68.43% in the PR200 group compared with 28.08% in the control. Serum total protein and albumin levels were significantly enhanced in the PR200 group compared to the control. However, dietary PIP up to 3 g/kg had no significant effect on serum glucose, cortisol, aspartate aminotransferase, or alkaline phosphatase activities; however, the alanine aminotransferase level was lower (P < 0.05) in the PR200 group than in the control. Intestinal amylase, lipase, and protease activities increased in PR300, and intestinal amylase and lipase increased in the PR100 group (P < 0.05). The serum immunological indices (lysozyme, alternative complement pathway, phagocytic activity, and respiratory burst activity) were higher (P < 0.05) in the PR200 group than in the control group. Serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were significantly intensified in PR200-PR300 than in the control group, with the highest activity observed in the PR200 group. Malondialdehyde was significantly lower in the PR200 group than in the control group. Furthermore, SOD, CAT, and Nrf2 expression was strongly upregulated in the liver tissue of the PR200 and PR300 groups compared to that in the control. The transcript levels of pro-inflammatory cytokines viz. IL-1ß and TNF-α were significantly upregulated in the kidneys of the PR100 and PR200 post-challenged. In contrast, the anti-inflammatory cytokine IL-10 was significantly downregulated in the kidneys of PR200. The expression of HSP70 was upregulated only in the PR400. Quadratic regression analysis showed that the optimal dietary PIP level was estimated as 2.07-2.13 g/kg to maximize growth performance. Overall, these results indicate that dietary PIP at an appropriate level can improve immunity, cytokine gene expression, and disease resistance in C. carpio.

The Biological Functions and Intestinal Inflammation Regulation of IL-21 in Grass Carp (Ctenopharyngodon idella) during Infection with Aeromonas hydrophila.

Interleukin (IL) 21 is a pleiotropic cytokine that plays an important role in regulating innate and adaptive immune responses. In fish, the biological functions and cell source of IL-21 remain largely unknown. In this study, we performed qRT-PCR, Western blotting and immunofluorescent microscopy to examine the expression of IL-21 at the mRNA and protein levels. We found that il21 expression was induced in the primary head kidney leukocytes of grass carp (Ctenopharyngodon idella) by heat-inactivated Aeromonas hydrophila (A. hydrophila) and LPS and in tissues after infection with A. hydrophila. Recombinant IL-21 protein produced in the CHO-S cells was effective in elevating the expression of antibacterial genes, including ß-defensin and lysozyme, and, interestingly, inhibited the NF-κB signaling pathway. Furthermore, we investigated the response of the IL-21 expressing cells to A. hydrophila infection. Immunofluorescent assay showed that IL-21 protein was detected in the CD3γ/δ T cells and was markedly accumulated in the anterior, middle and posterior intestine. Collectively, the results indicate that IL-21 plays an important role in regulating the intestinal inflammation induced by bacterial infection in grass carp.

Quercetin attenuates avermectin-induced cardiac injury in carp through inflammation, oxidative stress, apoptosis and autophagy.

As an important antibiotic, avermectin (AVM) has been widely used in China, but its unreasonable application has caused serious harm to the water environment. In view of the various pharmacological effects of quercetin (QUE), such as anti-inflammatory and antioxidant, the scientific hypothesis that "QUE may cause carp poisoning by inhibiting AVM" was proposed in this study. However, its protective effect in AVM -induced heart damage has not been reported. QUE reduced the symptoms of AVM toxicity and decreased the levels of creatine kinase, lactate dehydrogenase, and creatine kinase in the serum of carp. By histological observation, QUE was found to significantly reduce cardiac fiber swelling in carp. A DHE fluorescence probe study showed that QUE was able to inhibit AVM -induced accumulation of reactive oxygen species (ROS) in carp myocardium. We found that QUE significantly increased the intracellular antioxidant enzymes CAT, T-AOC and GSH enzyme activity and reduced intracellular MDA content. In addition, QUE significantly increased il-10 and tgf-ß1 expression, and significantly down-regulated tnf-α, il-6, il-1ß and inos expression. Tunel assay showed that QUE attenuated AVM -induced apoptosis, significantly decreased the transcript levels of pro-apoptosis-related genes, and increased the expression of anti-apoptosis-related genes. We also detected the protein expression of LC3 in the AVM group and QUE + AVM group, and found that the expression of LC3 was significantly increased in both groups compared with the Control group, but after adding QUE, the expression of LC3 was significantly decreased compared with the AVM group. In addition, the transcript levels of p62 and atg5 were also detected by qPCR. QUE significantly increased the expression of p62 and decreased the expression of atg5, suggesting that QUE could attenuate AVM -induced cardiac autophagy in carp. This study will provide preliminary evidence of the principle of QUE attenuating AVM -induced myocardial injury in carp from four aspects, including oxidative stress, inflammatory response, apoptosis and autophagy, and provide a theoretical basis for its prevention and treatment.

Genotyping tool for salmonid gill pox virus (SGPV) obtained from farmed and wild Atlantic salmon (Salmo salar).

Poxviruses are common viruses found in vertebrate species. In 2006, the first poxvirus associated with salmon, salmonid gill poxvirus (SGPV), was identified during an outbreak of gill disease at a smolt production site in northern Norway and at two marine farms in western Norway. Poxviruses had previously been detected in ayu (Plecoglossus altivelis) and koi carp (Cyprinus carpio). In all three fish species, poxviruses are associated with gill disease. It has not been possible to culture SGPV from Norway, and little is known about its virulence. However, the association between SGPV and gill disease in salmon has shown the need for molecular tools to identify reservoirs and transmission routes. Sequencing the genome of a second isolate of SGPV has made it possible to compare variable regions between two strains of the virus, showing the presence of a large number of variable regions that exhibit both variable numbers of tandem repeats and intra-ORF variation. We present eight regions that are suitable for distinguishing strains of SGPV and determining their phylogenetic relationship, and these were used to compare SGPV isolates obtained from both farmed and wild salmon in fresh and sea water. The prevalence of the virus was found to be higher in wild salmon in rivers than in returning wild salmon collected from traps in Norwegian fjords. Genotyping based on the eight selected variable regions, suggests the presence of geographically distinct isolates in freshwater among both farmed and wild salmon, while SGPV from marine farms shows high local diversity and a wide geographical distribution of similar strains of the virus.

Distribution, bioaccumulation, and health risk assessment of organochlorines across the riverine ecosystem of Punjab Province, Pakistan.

This study was designed to assess the distribution of organochlorines (OCs) in fish species, their spatio-temporal variations, bioaccumulation potential, and associated human health risks via dietary intake. The levels of twenty-three organochlorine pesticides (OCPs) and thirty-five polychlorinated biphenyls (PCBs) were analyzed in six fish species collected from the riverine ecosystem of Punjab Province, Pakistan. The results indicated that the mean levels of Σ23OCPs were 74.1 ng/g ww and 184 ng/g ww, and for Σ35PCBs the levels were 38.8 ng/g ww and 74.8 ng/g ww in herbivorous and carnivorous fish species, respectively. The most abundant contaminants in all fish species were DDTs (65%) and HCHs (14%) among OCPs and heavier PCB congeners (62%) among PCBs. As for dioxin-like PCBs, the WHO toxic equivalency values (ng TEQ/g ww) were in the range of 0.21 (Cyprinus Carpio) to 2.38 (Rita Rita), exceeding the maximum allowable limit of 0.004 ng TEQ/g, ww by the European Commission. Spatio-temporal analysis indicated relatively higher OC levels in winter season with elevated concentrations in fish samples from industrial zone. The bioconcentration factor (L/kg) values ranged from 723 to 2773 for PCBs and 315 to 923 for OCPs in all fish species, with higher levels were reported in carnivorous species. The human health risk assessment at both 50th and 95th percentiles revealed the absence of any significant non-carcinogenic risk as calculated HR was less than 1. However, the critical carcinogenic risk was found to be associated for most of the contaminants, signifying the dietary exposure to OCPs and PCBs might pose the public health concern.