Dietary Protein Optimization for Growth and Immune Enhancement in Juvenile Hybrid Sturgeon (Acipenser baerii × A. schrenckii): Balancing Growth Performance, Serum Biochemistry, and Expression of Immune-Related Genes.

This study aimed to evaluate the effects of dietary protein levels on growth performance, serum indices, body amino acid composition, and intestinal gene expression in juvenile hybrid sturgeon (Acipenser baerii × A. schrenckii). Hybrid sturgeons (initial weight 29.21 ± 2.04 g) were fed isolipidic diets containing 30%, 33%, 36%, 39%, 42% or 45% crude protein for 12 weeks (n = 18 tanks, 30 fish/tank). Results showed significant differences between treatments, where weight gain and protein efficiency ratio peaked optimally between 35.9% and 38.3% dietary protein. Serum parameters such as glucose, alanine aminotransferase, aspartate aminotransferase, superoxide dismutase, and lipid peroxidation levels varied significantly with changes in dietary protein levels. Specifically, the highest enzymatic activities and growth parameters were observed in groups fed with 33% to 39% protein, enhancing whole-body concentrations of lysine, leucine, phenylalanine, proline, and glutamic acid. Immune parameters such as immunoglobulin M and lysozyme activity also showed peak levels at higher protein concentrations, particularly notable at 42% for lysozyme and 36% for both component 3 and immunoglobulin M. Gene expression related to immune and growth pathways, including MyD88, TLR1, IL-8, IL-6, NF-κB, and IL1ß, was significantly upregulated at protein levels of 33% to 36%, with a noted peak in expression at 39% for TLR1, IL-10, and TOR signaling genes, before diminishing at higher protein levels. Overall, the dietary protein requirement for juvenile hybrid sturgeon ranges from 35.9% to 38.3% crude protein.

Physiological, Nutritional and Transcriptomic Responses of Sturgeon (Acipenser schrenckii) to Complete Substitution of Fishmeal with Cottonseed Protein Concentrate in Aquafeed.

This study estimated the effect of substituting fishmeal completely with cottonseed protein concentrate (CPC) in the diet of sturgeon (Acipenser schrenckii) on growth, digestive physiology, and hepatic gene expression. A control diet containing fishmeal and an experimental diet based on CPC was designed. The study was conducted for 56 days in indoor recirculating aquaculture systems. The results showed that weight gain, feed efficiency, and whole-body essential amino acids (EAAs) all decreased significantly in the experimental group, while whole-body non-essential amino acids (NEAAs) and serum transaminase activity increased (p < 0.05). The activity of digestive enzymes in the mid-intestine was significantly reduced (p < 0.05), and liver histology revealed fatty infiltration of hepatocytes. The hepatic transcriptome revealed an upregulation of genes linked to metabolism, including steroid biosynthesis, pyruvate metabolism, fatty acid metabolism, and amino acid biosynthesis. These findings indicate that fully replacing fishmeal with CPC harms A. schrenckii growth and physiology. This study provides valuable data for the development of improved aquafeeds and the use of molecular methods to evaluate the diet performance of sturgeon.

Effect of dietary phenylalanine on growth performance and intestinal health of triploid rainbow trout (Oncorhynchus mykiss) in low fishmeal diets.

This study aimed to investigate the effects of phenylalanine on the growth, digestive capacity, antioxidant capability, and intestinal health of triploid rainbow trout (Oncorhynchus mykiss) fed a low fish meal diet (15%). Five isonitrogenous and isoenergetic diets with different dietary phenylalanine levels (1.82, 2.03, 2.29, 2.64, and 3.01%) were fed to triplicate groups of 20 fish (initial mean body weight of 36.76 ± 3.13 g). The weight gain rate and specific growth rate were significantly lower (p < 0.05) in the 3.01% group. The trypsin activity in the 2.03% group was significantly higher than that in the control group (p < 0.05). Amylase activity peaked in the 2.64% treatment group. Serum superoxide dismutase, catalase, and lysozyme had the highest values in the 2.03% treatment group. Liver superoxide dismutase and catalase reached their maximum values in the 2.03% treatment group, and lysozyme had the highest value in the 2.29% treatment group. Malondialdehyde levels in both the liver and serum were at their lowest in the 2.29% treatment group. Interleukin factors IL-1ß and IL-6 both reached a minimum in the 2.03% group and were significantly lower than in the control group, while IL-10 reached a maximum in the 2.03% group (p < 0.05). The tight junction protein-related genes occludin, claudin-1, and ZO-1 all attained their highest levels in the 2.03% treatment group and were significantly higher compared to the control group (p < 0.05). The intestinal villi length and muscle layer thickness were also improved in the 2.03% group (p < 0.05). In conclusion, dietary phenylalanine effectively improved the growth, digestion, absorption capacity, antioxidant capacity, and intestinal health of O. mykiss. Using a quadratic curve model analysis based on WGR, the dietary phenylalanine requirement of triploid O. mykiss fed a low fish meal diet (15%) was 2.13%.

Dietary Sodium Butyrate Improves Intestinal Health of Triploid Oncorhynchus mykiss Fed a Low Fish Meal Diet.

This study aimed to determine the effects of dietary sodium butyrate (NaB) on the growth and gut health of triploid Oncorhynchus mykiss juveniles (8.86 ± 0.36 g) fed a low fish meal diet for 8 weeks, including the inflammatory response, histomorphology, and the composition and functional prediction of microbiota. Five isonitrogenous and isoenergetic practical diets (15.00% fish meal and 21.60% soybean meal) were supplemented with 0.00% (G1), 0.10% (G2), 0.20% (G3), 0.30% (G4), and 0.40% NaB (G5), respectively. After the feeding trial, the mortality for G3 challenged with Aeromonas salmonicida for 7 days was lower than that for G1 and G5. The optimal NaB requirement for triploid O. mykiss based on weight gain rate (WGR) and the specific growth rate (SGR) was estimated to be 0.22% and 0.20%, respectively. The activities of intestinal digestive enzymes increased in fish fed a NaB diet compared to G1 (p < 0.05). G1 also showed obvious signs of inflammation, but this inflammation was significantly alleviated with dietary NaB supplementation. In comparison, G3 exhibited a more complete intestinal mucosal morphology. Dietary 0.20% NaB may play an anti-inflammatory role by inhibiting the NF-κB-P65 inflammatory signaling pathway. Additionally, the relative abundance of probiotics was altered by dietary NaB. In conclusion, dietary 0.20% NaB improved the intestinal health of triploid O. mykiss fed a low fish meal diet.

Assessment of Fish Protein Hydrolysates in Juvenile Largemouth Bass (Micropterus salmoides) Diets: Effect on Growth, Intestinal Antioxidant Status, Immunity, and Microflora.

Varying dietary inclusion levels of fish protein hydrolysates (FPH) were applied in a feeding experiment with juvenile largemouth bass (Micropterus salmoides) to assess their effects on growth, intestinal antioxidant status, immunity, and microflora. FPH were added in 4 dietary levels: 0 g/kg (control group, FPH-0), 10 g/kg (FPH-10), 30 g/kg (FPH-30), and 50 g/kg (FPH-50) dry matter, respectively substituting 0, 5.3, 16.3, and 27.3% of fish meal with dietary fish meal. Quadruplicate groups of 25 juvenile largemouth bass with initial body weight 9.51 ± 0.03 g were fed during the 56-day feeding experiment. Experimental results showed that fish fed FPH-30 obtained a significantly higher weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER), and significant feed conversion rate (FCR) compared to the other three groups (P < 0.05). FPH-30 group also promoted protein synthesis and deposition, as evidenced by the higher whole-body crude protein contents, the higher expressions of GH1, IGF-1, TOR, and S6K in the liver, and SLC7A5, SLC7A8, SLC38A2, and SLC15A2 in the intestine than the other three groups. FPH-30 group could also enhance intestinal health status by increasing the activities of SOD, POD, CAT, GSH-Px, and T-AOC activities by upregulating the expressions of SOD, GSH-Px, IL1ß, and TNFß, and by reducing the MDA contents and the expressions of IL15, Caspase 3, Caspase 9, and Caspase 10 than other groups. Compared to the control group, the Actinobacteriota abundance markedly decreased in FPH treatments, while the variation tendency of the phylum Proteobacteria was opposite. The peak value of Firmicutes:Bacteroidetes ratio and the lowest of Bacteroidetes abundance were seen in largemouth bass fed FPH-30 (P < 0.05). Fish in three FPH treatments had lower abundances of opportunistic pathogens Staphylococcus and Plesiomonas than fish in the control group. In conclusion, FPH is a nutritious feed ingredient for juvenile largemouth bass, and can be added to a dietary level of 30 g/kg dry matter replacing fish meal without any negative effect on growth and feed utilization. FPH supplements could also strengthen the intestinal immune mechanisms of largemouth bass to tackle the immunodeficiency produced by fish meal replacement.

Effects of replacing fishmeal with cottonseed protein concentrate on growth performance, blood metabolites, and the intestinal health of juvenile rainbow trout (Oncorhynchus mykiss).

Cottonseed protein concentrate (CPC) is a potential non-food protein source for fishmeal replacement in fish feed. However, a high inclusion level of CPC in diets may have adverse effects on the metabolism and health of carnivorous fish. This study aimed to investigate CPC as a fishmeal alternative in the diet of rainbow trout Oncorhynchus mykiss based on growth performance, blood metabolites, and intestinal health. Five isonitrogenous (46% crude protein) and isolipidic (16% crude lipid) diets were formulated: a control diet (30% fishmeal) and four experimental diets with substitution of fishmeal by CPC at 25%, 50%, 75%, and 100%. A total of 600 fish (mean body weight 11.24g) were hand-fed the five formulated diets to apparent satiation for eight weeks. The results showed no adverse effects on growth performance when 75% dietary fishmeal was replaced by CPC. However, reduced growth and feed intake were observed in rainbow trout fed a fishmeal-free diet based on CPC (CPC100%). Changes in serum metabolites were also observed in CPC100% compared with the control group, including an increase in alanine aminotransferase (ALT), a decrease in alkaline phosphatase (ALP), alterations in free amino acids, and reductions in cholesterol metabolism. In addition, the CPC-based diet resulted in reduced intestinal trypsin, decreased villus height and width in the distal intestine, upregulated mRNA expression levels of inflammatory cytokines in the intestine, and impaired gut microbiota with reduced bacterial diversity and decreased abundance of Bacillaceae compared with the control group. The findings suggest that the optimum substitution rate of dietary fishmeal by CPC for rainbow trout should be less than 75%.

Effects of Glutathione on Growth, Intestinal Antioxidant Capacity, Histology, Gene Expression, and Microbiota of Juvenile Triploid Oncorhynchus mykiss.

This study aimed to demonstrate the effects of dietary glutathione (GSH) on growth, intestinal antioxidant capacity, histology, gene expression, and microbiota in juvenile triploid rainbow trout (Oncorhynchus mykiss). Different diets (G0-control, G100, G200, G400, and G800) containing graded levels of GSH (0, 100, 200, 400, and 800mgkg-1) were fed to triplicate groups of 30 fish (initial mean weight 4.12±0.04g) for 56days. G400 had significantly improved weight gain and feed conversion rate. Based on the broken-line regression analysis, the optimum dietary GSH level was 447.06mgkg-1. Catalase and superoxide dismutase activities were significantly higher in G200-G800. G200 had significantly lower malondialdehyde content. The height of the intestinal muscular layer in G400 was significantly higher than that of the control group. Intestinal PepT1 and SLC1A5 gene expression was significantly increased, and the highest was observed in G400. TNF-α, IL-1ß, IL-2, and IL-8 expression were significantly decreased than that of G0. Next-generation sequencing of the 16S rDNA showed a significant difference in alpha diversity whereas no differences in beta diversity. On the genus level, LefSe analysis of indicator OTUs showed Ilumatobacter, Peptoniphilus, Limnobacter, Mizugakiibacter, Chelatococcus, Stella, Filimonas, and Streptosporangium were associated with the treatment diet, whereas Arcobacter, Ferrovibrio, Buchnera, Chitinophaga, Stenotrophobacter, Solimonadaceae, Polycyclovorans, Rhodococcus, Ramlibacter, and Azohydromonas were associated with the control diet. In summary, feeding juvenile triploid O. mykiss 200-800mgkg-1 GSH improved growth and intestinal health.

Gene expression profile of the taimen Hucho taimen in response to acute temperature changes.

The endangered cold-water fish species taimen (Hucho taimen) suffer acute temperature changes in culture and wild conditions. Understanding the effects of acute temperature changes on physiological processes of this species is essential for aquaculture practices and conservation. Liver transcriptomic profiles of the taimen (n = 24) exposed to acute temperature decrease (from 20 °C to 10 °C) and acute temperature increase (from 10 °C to 20 °C) was evaluated using high-throughput RNA-Sequencing. Samples were collected at day 0, 1, 7 and 35 in both treatments. Compared to day 0, the total numbers of differentially expressed genes (DEGs) in the taimen after acute temperature decrease were 173, 226 and 42 at day 1, 7 and 35, respectively, and the total numbers of DEGs following acute temperature increase were 260, 253 and 282 at day 1, 7 and 35, respectively. Particularly, 14 key regulatory genes were commonly found between both acute temperature treatments. Functional analysis based on the commonly identified DEGs revealed important metabolic pathways related to metabolism and immune function, suggesting a specific response mechanism of taimen against cold and heat shock. The results may assist in developing management strategies for stress mediation caused by acute temperature changes in the taimen and other cold water fish.

High-throughput metabolomics method based on liquid chromatography-mass spectrometry: Insights into the underlying mechanisms of salinity-alkalinity exposure-induced metabolites changes in Barbus capito.

It is critical to investigate the adaptive development and the physiological mechanism of fish in external stimulation. In this study, the response of Barbus capito to salinity-alkalinity exposure was explored by high-throughput nontargeted and liquid chromatography-mass spectrometry-based metabolomics to investigate metabolic biomarker and pathway changes. Meanwhile, the biochemical indexes of Barbus capito were measured to discover the chronic impairment response to salinity-alkalinity exposures. A total of 29 tissue metabolites were determined to deciphering the endogenous metabolic changes of fishes during the different concentration salinity-alkalinity exposures environment, which were mainly involved in the key metabolism including the phenylalanine, tyrosine, and tryptophan biosynthesis, arachidonic acid metabolism, pyruvate metabolism, citrate cycle, and glycerophospholipid metabolism. Finally, we found the amino acid metabolism as key target was associated with the endogenous metabolites and metabolic pathways of Barbus capito to salinity-alkalinity exposures. In conclusion, metabolomics is a potentially powerful tool to reveal the mechanism information of fish in various exposure environments.

Effects of dietary myo-inositol on growth, antioxidative capacity, and nonspecific immunity in skin mucus of taimen Hucho taimen fry.

In this study, the effects of dietary myo-inositol on the skin mucosal immunity and growth of taimen (Hucho taimen) fry were determined. Triplicate groups of 500 fish (initial weight 5.58 ± 0.15 g) were fed different diets containing graded levels of myo-inositol (28.75, 127.83, 343.83, 565.81, and 738.15 mg kg-1) until satiation for 56 days. Thereafter, the nonspecific skin mucus immune parameters, antioxidative capacity, and growth performance were measured. The skin mucus protein and the activities of alkaline phosphatase were significantly higher than those in the control group (P < 0.05). However, there were no significant differences in lysozyme activity among the treatments (P > 0.05). The antimicrobial activity and minimum inhibitory concentration of the skin mucus were increased significantly by myo-inositol supplementation (P < 0.05). The superoxide dismutase, catalase, and glutathione peroxidase activities were significantly elevated in the treatment groups (P < 0.05), whereas the malondialdehyde contents were significantly decreased (P < 0.05). Low-level myo-inositol (28.75 mg kg-1) led to a significantly lower weight gain, feed efficiency, condition factor, and survival rate compared with the other treatments (P < 0.05). In conclusion, dietary myo-inositol deficiency (28.75 mg kg-1) adversely affects the skin mucus immune parameters, antioxidative capacity, and growth performance of Hucho taimen fry.

Exploring the metabolic biomarkers and pathway changes in crucian under carbonate alkalinity exposure using high-throughput metabolomics analysis based on UPLC-ESI-QTOF-MS.

The aims of this study is to explore the metabolomic biomarker and pathway changes in crucian under carbonate alkalinity exposures using high-throughput metabolomics analysis based on ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (UPLC-ESI-QTOF-MS) for carrying out adaptive evolution of fish in environmental exposures and understanding molecular physiological mechanisms of saline-alkali tolerance in fishes. Under 60 day exposure management, the UPLC-ESI-QTOF-MS technology, coupled with a pattern recognition approach and metabolic pathway analysis, was utilized to give insight into the metabolic biomarker and pathway changes. In addition, biochemical parameters in response to carbonate alkalinity in fish were detected for chronic impairment evaluation. A total of twenty-seven endogenous metabolites were identified to distinguish the biochemical changes in fish in clean water under exposure to different concentrations of carbonate alkalinity (CA); these mainly involved amino acid synthesis and metabolism, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism, pyruvate metabolism and the citrate cycle (TCA cycle). Compared with the control group, CA exposure increased the level of blood ammonia; TP; ALB; Gln in the liver and gills; GS; urea in blood, the liver and gills; CREA; CPS; Glu and LDH; and decreased the level of weight gain rate, oxygen consumption, discharge rate of ammonia, SOD, CAT, ALT, AST and Na+/K+-ATPase. At low concentrations, CA can change the normal metabolism of fish in terms of changing the osmotic pressure regulation capacity, antioxidant capacity, ammonia metabolism and liver and kidney function to adapt to the CA exposure environment. As the concentration of CA increases, various metabolic processes in crucian are inhibited, causing chronic damage to the body. The results show that the metabolomic strategy is a potentially powerful tool for identifying the mechanisms in response to different environmental exposomes and offers precious information about the chronic response of fish to CA.

A chimeric recombinant infectious hematopoietic necrosis virus induces protective immune responses against infectious hematopoietic necrosis and infectious pancreatic necrosis in rainbow trout.

Infectious pancreatic necrosis virus (IPNV) and infectious hematopoietic necrosis virus (IHNV) are two common viral pathogens that cause severe economic losses in all salmonid species in culture, but especially in rainbow trout. Although vaccines against both diseases have been commercialized in some countries, no such vaccines are available for them in China. In this study, a recombinant virus was constructed using the IHNV U genogroup Blk94 virus as a backbone vector to express the antigenic gene, VP2, from IPNV via the reverse genetics system. The resulting recombinant virus (rBlk94-VP2) showed stable biological characteristics as confirmed by virus growth kinetic analyses, pathogenicity analyses, indirect immunofluorescence assays and western blotting. Rainbow trout were immunized with rBlk94-VP2 and then challenged with the IPNV ChRtm213 strain and the IHNV Sn1203 strain on day 45 post-vaccination. A significantly higher survival rate against IHNV was obtained in the rBlk94-VP2 group on day 45 post-vaccination (86%) compared with the PBS mock immunized group (2%). Additionally, IPNV loads decreased significantly in the rBlk94-VP2 immunized group in the liver (28.6-fold to 36.5-fold), anterior kidney (21.7-fold to 44.2-fold), and spleen (14.9-fold to 22.7-fold), as compared with the PBS mock control group. The mRNA transcripts for several innate and adaptive immune-related proteins (IFN-γ, IFN-1, Mx-1, CD4, CD8, IgM, and IgT) were also significantly upregulated after rBlk94-VP2 vaccination, and neutralizing antibodies against both IHNV and IPNV were induced on day 45 post-vaccination. Collectively, our results suggest that this recombinant virus could be developed as a vaccine vector to protect rainbow trout against two or more diseases, and our approach lays the foundations for developing live vaccines for rainbow trout.

Genomic characterization of a novel virulent phage infecting the Aeromonas hydrophila isolated from rainbow trout (Oncorhynchus mykiss).

The virulent bacteriophage MJG that specifically infects Aeromonas hydrophila was isolated from a water sample from a river in Harbin, China. The genome of phage MJG was a double-stranded linear DNA with 45,057 bp, possessing 50.11% GC content. No virulence or resistance genes were found in the phage genome. Morphological observation, genomic characterization, and phylogenetic analysis indicated that MJG was closely related to phages belonging to the genus Sp6virus in the Podoviridae family. This phage is a novel member within Sp6virus that could infect and lyse A. hydrophila. This study could serve as a genomic reference of A. hydrophila phages and provide a potential agent for phage therapy.

Identification of the optimal insertion site for expression of a foreign gene in an infectious hematopoietic necrosis virus vector.

Infectious hematopoietic necrosis virus (IHNV) was developed as a vector to aid the construction of vaccines against viral diseases such as viral hemorrhagic septicemia virus, spring viremia of carp virus, and influenza virus H1N1. However, the optimal site for foreign gene expression in the IHNV vector has not been determined. In the present study, five recombinant viruses with the green fluorescence protein (GFP) gene inserted into different genomic junction regions of the IHNV genomic sequence were generated using reverse genetics technology. Viral growth was severely delayed when the GFP gene was inserted into the intergenic region between the N and P genes. Real-time fluorescence quantitative PCR assays showed that the closer the GFP gene was inserted towards the 3' end, the higher the GFP mRNA levels. Measurement of the GFP fluorescence intensity, which is the most direct method to determine the GFP protein expression level, showed that the highest GFP protein level was obtained when the gene was inserted into the intergenic region between the P and M genes. The results of this study suggest that the P and M gene junction region is the optimal site within the IHNV vector to express foreign genes, providing valuable information for the future development of live vector vaccines.

Effects of traditional Chinese medicines on immunity and culturable gut microflora to Oncorhynchus masou.

The present study was conducted to evaluate the effect of dietary traditional Chinese medicines on the growth, immunity, and composition of culturable gut microflora in Oncorhynchus masou. Diets were formulated to contain no medicine (control), antitoxic decoction (A), general antiphlogistic decoction (B), or Herbae Artemisiae Capillariae decoction (C). Fish were manually fed twice daily till apparent satiation for 30 days. Compared with that in the control group, supplementation with the three kinds of Chinese herbal medicine enhanced fish growth significantly (P < 0.05). The activities of liver superoxide dismutase and glutathione peroxidase in the treatment groups were significantly higher compared with those in the control group (P < 0.05). The quantity of intestinal microflora was higher in the treatment groups compared with that in the control group. Moreover, there were some effects of dietary Chinese herbal medicine on the composition of intestinal microflora. Microflora of Pseudomonas sp., Psychrobacter sp., Microbacterium sp., Macrococcus sp., Burkholderia sp., and Arthrobacter sp. were found in the treatment groups, whereas there were none in the control group. There was a significant increase in their amounts in the treatment groups (P < 0.05). The three kinds of traditional Chinese medicines can improve the growth and immunity of Oncorhynchus masou and affect the quantity and composition of intestinal microflora.

A transcriptome analysis focusing on splenic immune-related mciroRNAs of rainbow trout upon Aeromonas salmonicida subsp. salmonicida infection.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that can regulate the immune responses during pathogen infection. Aeromonas salmonicida (A. salmonicida) subsp. salmonicida is the causative agent of furunculosis in salmon and trout. To identify the miRNAs and investigate the specific miRNAs in rainbow trout upon A. salmonicida subsp. salmonicida infection, we performed high throughput sequencing using the spleens of rainbow trout infected with and without an A. salmonicida subsp. salmonicida clinical isolate. A total of 381 known miRNAs and 926 novel miRNAs were identified. Eleven known and 16 novel miRNAs were found to be differentially expressed upon infection. The results of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the target genes of the differentially expressed miRNAs were closely associated with immune responses and biological regulations. Additionally, over- and suppressed expression of miR-155-5p significantly enhanced and reduced the IL-2 and IL-1ß expressions in RTG-2 cells induced by A. salmonicida, respectively. To our knowledge, this is the first experimental study on the miRNAs of rainbow trout upon A. salmonicida infection. The results here might lay a foundation for the further understanding of the roles of miRNAs in the immune responses during A. salmonicida infection in rainbow trout.

Global metabolic responses of the lenok (Brachymystax lenok) to thermal stress.

High temperature is a powerful stressor for fish living in natural and artificial environments, especially for cold water species. Understanding the impact of thermal stress on physiological processes of fish is crucial for better cultivation and fisheries management. However, the metabolic mechanism of cold water fish to thermal stress is still not completely clear. In this study, a NMR-based metabonomic strategy in combination with high-throughput RNA-Seq was employed to investigate global metabolic changes of plasma and liver in a typical cold water fish species lenok (Brachymystax lenok) subjected to a sub-lethal high temperature. Our results showed that thermal stress caused multiple dynamic metabolic alterations of the lenok with prolonged stress, including repression of energy metabolism, shifts in lipid metabolism, alterations in amino acid metabolism, changes in choline and nucleotide metabolisms. Specifically, thermal stress induced an activation of glutamate metabolism, indicating that glutamate could be an important biomarker associated with thermal stress. Evidence from Hsp 70 gene expression, blood biochemistry and histology confirmed that high temperature exposure had negative effects on health of the lenok. These findings imply that thermal stress has a severe adverse effect on fish health and demonstrate that the integrated analyses combining NMR-based metabonomics and transcriptome strategy is a powerful approach to enhance our understanding of metabolic mechanisms of fish to thermal stress.

Recovery of recombinant infectious hematopoietic necrosis virus strain Sn1203 using the mammalian cell line BHK-21.

Reverse genetics systems are powerful tools for understanding the virulence mechanisms and gene functions of negative-sense RNA viruses. The reverse genetics systems commonly used for recombinant infectious hematopoietic necrosis virus (IHNV) are based on vaccinia virus infection. To avoid the potential biological safety risks associated with vaccinia virus, a recombinant IHNV virus strain Sn1203 (rIHNV-Sn1203) was rescued in this study using a mammalian cell line, BHK-21. The genome sequence authenticity of rIHNV-Sn1203 was confirmed using two silent genetic tags introduced by site-directed mutagenesis. Indirect immunofluorescence assays and transmission electron microscopy revealed that rIHNV-Sn1203 and wild-type IHNV-Sn1203 (wtIHNV-Sn1203) had identical immunogenicity and virion morphology. The virulence and pathogenicity of rIHNV-Sn1203 were assessed in vitro and in vivo. Although rIHNV-Sn1203 displayed trends toward delayed intracellular viral replication and lower virion yields compared with wtIHNV-Sn1203, statistical analyses revealed no significant differences between these two viruses. Moreover, rainbow trout challenged with rIHNV-Sn1203 and wtIHNV-Sn1203 showed indistinguishable mortality. Together, these results show that IHNV was successfully rescued using BHK-21 cells. This method is very convenient and may also be suitable for use in the recovery of other Novirhabdoviruses.

Phylogeography and evolution of infectious hematopoietic necrosis virus in China.

Infectious hematopoietic necrosis virus (IHNV) is a well-known rhabdoviral pathogen of salmonid fish. In this study, a comprehensive analysis of 40 IHNV viruses isolated from thirteen fish farms in nine geographically dispersed Chinese provinces during 2012 to 2017 is presented. Identity of nucleotide and amino acid sequences among all the complete glycoprotein (G) genes from Chinese isolates was 98.0-100% and 96.7-100%, respectively. Coalescent phylogenetic analyses revealed that all the Chinese IHN virus characterized in this study were in a monophyletic clade that had a most recent common ancestor with the J Nagano (JN) subgroup within the J genogroup of IHNV. Within the Chinese IHNV clade isolates obtained over successive years from the same salmon fish farm clustered in strongly supported subclades, suggesting maintenance and diversification of virus over time within individual farms. There was also evidence for regional virus transmission within provinces, and some cases of longer distance transmission between distant provinces, such as Gansu and Yunnan. The data demonstrated that IHNV has evolved into a new subgroup in salmon farm environments in China, and IHNV isolates are undergoing molecular evolution within fish farms. We suggest that Chinese IHNV comprises a separate JC subgroup within the J genogroup of IHNV.

Identification and analysis of differentially expressed microRNAs in rainbow trout (Oncorhynchus mykiss) responding to infectious hematopoietic necrosis virus infection.

MicroRNAs (miRNAs) are a class of regulators essential for numerous biological processes. Infectious hematopoietic necrosis virus (IHNV) is one of the most important viral pathogens in salmon and trout. In this study, the miRNA expression profiles of rainbow trout upon IHNV infection were explored. In total, 392 known miRNAs and 936 novel miRNAs were identified. Twelve known and 13 novel miRNAs were differentially expressed between infected and uninfected fish. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that certain miRNA target genes were associated with biological regulation, the immune system, and signal transduction. In addition, over- and suppressed expression of miR-146a-3p, miR-155-5p, miR-216a-5p, and miR-499b-5p could respectively increase and decrease viral gene expression in cells and viral titers. MiR-146a-3p and miR-216a-5p inhibited the expression of type-I IFN and the Mx1 gene induced by IHNV. These results provide preliminary insights into the IHNV-host interactions mediated by miRNAs.