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Micropterus salmoides rhabdovirus (MSRV) is a primary viral pathogen in largemouth bass aquaculture, which leads to tremendous economic losses yearly. Currently, there are no approved drugs for the treatment and control of this virus. Our previous studies screened the herb Magnolia officinalis from many traditional Chinese medicines, and we isolated and identified magnolol as its main active compound against multiple rhabdoviruses, including MSRV. On the basis of the structure–activity relationship and pharmacophore model of magnolol, two new magnolol derivatives, namely, hydrogenated magnolol and 2,2′-dimethoxy-magnolol, were designed and synthesized. Their anti-MSRV activities were systematically investigated both in vitro and in vivo. By comparing the half-maximal inhibitory concentration (IC50), it was found that hydrogenated magnolol possessed a higher anti-MSRV activity than magnolol and 2,2′-dimethoxy-magnolol, with an IC50 of 13.37 μM. Furthermore, hydrogenated magnolol exhibited a protective effect on the grass carp ovary (GCO) cell line by reducing the cytopathic effect induced by MSRV. Further studies revealed that hydrogenated magnolol did not directly impact virions or interfere with MSRV adsorption. It worked within the 6–8 h of the phase of virus replication. In vivo treatment of MSRV infection with magnolol and hydrogenated magnolol showed that they significantly improved the survival rate by 44.6% and 62.7%, respectively, compared to MSRV-infected groups. The viral load measured by the expression of viral glycoprotein in the organs including the liver, spleen, and kidney also significantly decreased when fish were intraperitoneally injected at a dose of 20 mg/kg. Altogether, the structural optimization of magnolol via hydrogenation of the propylene groups increased its anti-MSRV activity both in vitro and in vivo. These results may provide a valuable reference for anti-MSRV drug discovery and development in aquaculture.

The reported requirements of largemouth bass (LMB, which is native to North America) for dietary protein and lipids varied substantially among previous studies, and this fish fed current formulated diets exhibit poor growth performance and pale liver syndrome. Because amino acids and lipids are known to affect hepatic metabolism and function in mammals, it is imperative to understand the impacts of these dietary macronutrients on the growth and liver morphology of LMB. In this study, we designed six isocaloric diets to determine the effects of different dietary crude protein (CP; 40%, 45%, and 50%; dry matter basis) and lipid levels (7.5% and 10%; dry matter basis) on fat and glycogen deposits, as well as hepatosis in LMB. There were four tanks (12 fish per tank, an average initial weight of 18.4 g/fish) per dietary treatment group and the trial lasted for 8 weeks. Fish were fed to apparent satiation three times daily. Results indicated that LMB fed the 45% or 50% CP diet grew faster (P < 0.05), had less (P < 0.05) glycogen in the liver and smaller (P < 0.05) hepatocyte sizes than fish fed the 40% CP diet, but there was no difference in weight gain or feed efficiency between the 45% and 50% CP diets. The hepatic lipid content did not differ between LMB fed the 40% and 45% CP diets, and the values for these two groups were 29% lower (P < 0.05) than those for LMB fed the 50% CP diet. Compared with the 40% CP group, LMB fed the 45% or 50% CP diet had 8–12% lower content of total minerals, phosphorus, and calcium in the body. Increasing the dietary lipid level from 7.5 to 10% enhanced the weight gains (+ 15%) and feed efficiency (+ 22%), as well as the retention of dietary protein (+ 18%), energy (+ 25%), and phosphorus (+ 7.6%) in the body. No fatty liver occurred in any group of LMB (with hepatic lipid concentrations being < 2%, wet weight basis). Based on these growth, metabolic and histologic data, we recommend dietary CP and lipids levels to be 45% and 10%, respectively, for juvenile LMB.

Largemouth bass (Micropterus salmoides) are common hosts of an epizootic bacterial infection by Nocardia seriolae. We conducted transcriptome profiling of M. salmoides to understand the host immune response to N. seriolae infection, using the Illumina sequencing platform. De novo assembly of paired-end reads yielded 47,881 unigenes, the total length, average length, N50, and GC content of which were 49,734,288, 1038, 1983 bp, and 45.94%, respectively. Annotation was performed by comparison against non-redundant protein sequence (NR), non-redundant nucleotide (NT), Swiss-Prot, Clusters of Orthologous Groups (COG), Kyoto Encyclopaedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Interpro databases, yielding 28,964 (NR: 60.49%), 36,686 (NT: 76.62%), 24,830 (Swissprot: 51.86%), 8913 (COG: 18.61%), 20,329 (KEGG: 42.46%), 835 (GO: 1.74%), and 22,194 (Interpro: 46.35%) unigenes. Additionally, 8913 unigenes were classified into 25 Clusters of Orthologous Groups (KOGs) categories, and 20,329 unigenes were assigned to 244 specific signalling pathways. RNA-Seq by Expectation Maximization (RSEM) and PossionDis were used to determine significantly differentially expressed genes (False Discovery Rate (FDR) < 0.05) and we found that 1384 were upregulated genes and 1542 were downregulated genes, and further confirmed their regulations using reverse transcription quantitative PCR (RT-qPCR). Altogether, these results provide information on immune mechanisms induced during bacterial infection in largemouth bass, which may facilitate the prevention of nocardiosis.

Antimicrobial peptides (AMPs) represent an evolutionarily conserved component of innate immunity with broad-spectrum antimicrobial and antiviral activities. However, the antiviral potential of fish-specific piscidins against emerging aquatic viruses largely remains to be explored. In this study, we evaluated the antiviral properties of three piscidins (designated here as Ms Piscidin1, Ms Piscidin2 and Ms Piscidin3) identified from largemouth bass ( Micropterus salmoides ) against Micropterus salmoides rhabdovirus (MSRV), a major pathogen causing high mortality in farmed largemouth bass. Computational prediction and expression profiling revealed inducible expression of Ms Piscidins upon MSRV infection, with distinct tissue-specific patterns. Functional assays demonstrated that while Ms Piscidin1 and Ms Piscidin3 primarily modulated host antiviral responses, Ms Piscidin2 exhibited direct virucidal activity against MSRV. Molecular docking predicted potential interactions between Ms Piscidin2 and the MSRV glycoprotein, where histidine and glutamic acid residues of Ms Piscidin2 are positioned in close proximity to cysteine and methionine residues of the MSRV glycoprotein, supporting its capacity to directly target viral particles. In vitro assays further confirmed that Ms Piscidin2 significantly suppressed MSRV replication and attenuated cytopathic effects in a dose-dependent manner. Further, Ms Piscidin2 treatment conferred significant in vivo protection, delaying disease progression and improving survival rates in MSRV-infected juvenile bass. These findings provide the first evidence of piscidin-mediated antiviral defense against MSRV and highlight Ms Piscidin2 as a promising candidate for developing novel antiviral strategies in largemouth bass aquaculture.

A 90-day feeding trial was conducted to evaluate the effects of replacing dietary fishmeal (FM) with defatted black soldier fly larvae meal (BSFL) on the growth performance and fillet quality of largemouth bass (Micropterus salmoides). The largemouth bass was divided into six groups (BSFL0, BSFL15, BSFL30, BSFL45, BSFL75, and BSFL100) and fed six isonitrogenous(CP 50%, 508 g/kg) and isolipid (CL 9%, 124 g/kg) diets, in which 0, 15%, 30%, 45%, 75%, and 100% of the fishmeal was replaced with BSFL, respectively. The results showed that the final body weight (FBW) and specific growth rate (SGR) of the largemouth bass decreased with increasing BSFL content, and they were significantly lower in BSFL75 than in BSFL0. The weight gain rate (WGR) decreased with increasing BSFL content and the feed conversion ratio (FCR) of largemouth bass increased with increasing BSFL content. The saturated fatty acids (SFAs) and n-3 polyunsaturated fatty acids (PUFA) contents of the largemouth bass fillet significantly decreased, and the n-6 PUFA content of the largemouth bass fillets significantly increased with increasing dietary BSFL. The fillet b* significantly decreased with increasing BSFL content. The biological parameters, fillet proximate nutrient composition, fillet amino acid composition, skin color, and fillet texture of the largemouth bass were not affected by the replacement of BSFL. Transcriptomic analysis revealed that BSFL replacement of FM affects the immune system and metabolic processes of largemouth bass through signaling pathways such as complement and coagulation cascades, the PPAR signaling pathway, cholesterol metabolism, and fat digestion and absorption. In conclusion, a replacement level lower than 45% BSFL was suggested for the overall growth and fillet quality of largemouth bass.

Micropterus salmoides rhabdovirus (MSRV) is one of the most serious pathogens harming M. salmoides juvenile, which had brought huge economic losses to farming industry. Studies involving candidate genes to the clinical diseases, however, are limited. In this study, the viral target and clinical manifestation of MSRV on M. salmoides juvenile were analyzed, and the protective effects of a single immune enhancer and a compound immune enhancer were evaluated. The results showed that the brain, liver, intestine and muscle of M. salmoides showed obvious lesions after infection with MSRV. The relative expression levels of nucleoprotein (N) and matrix protein (M) genes showed a trend of increasing at first and then decreasing and reached the peak in each tissue at 36 h post-infection. The mortality rate of M. salmoides was over 90% after 7 days of MSRV infection. The immune enhancers containing free nucleotides and Astragalus polysaccharide added to the diet effectively inhibited the replication of N and M genes in M. salmoides and increased the survival rate by 25% to 28%. This study provided basic data and theoretical reference for the analysis of the pathological mechanism and prevention and treatment of MSRV.

This study investigated the role of cholecystokinin (cck) in the feeding regulation of largemouth bass (Micropterus salmoides) via peptide activation and antagonist inhibition. The results show that the cck gene was expressed in various tissues, with the highest expression level occurring in the brain. Feeding, continuous feeding, and refeeding after fasting could significantly improve the mRNA levels of cck in the brain. Moreover, the activation of cck via injecting an exogenous CCK peptide could inhibit feed intake by regulating the mRNA levels of anorexigenic and feed-promoting factors in the brain and intestine. Furthermore, the CCK peptide reduced feed intake; however, the presence of an antagonist (Ly225910-CCK1R and devazepide-CCK2R) could reverse this effect through regulating the mRNA levels of anorexigenic and feed-promoting factors in the brain and intestine. Treatment with devazepide + CCK (CCK2R) reversed feed intake more effectively than Ly225910 + CCK (CCK1R) treatment. In summary, cck could regulate the feed intake of largemouth bass through regulating feeding-related genes in the brain and intestine. In addition, cck required binding with the receptor to inhibit feed intake more effectively in largemouth bass, and the binding effect of CCK1R was better than that of CCK2R.

This 56-day study aimed to evaluate the effects of histidine levels on intestinal antioxidant capacity and endoplasmic-reticulum stress (ERS) in largemouth bass (Micropterus salmoides). The initial weights of the largemouth bass were (12.33 ± 0.01) g. They were fed six graded levels of histidine: 0.71% (deficient group), 0.89%, 1.08%, 1.26%, 1.48%, and 1.67%. The results showed that histidine deficiency significantly suppressed the intestinal antioxidant enzyme activities, including SOD, CAT, GPx, and intestinal level of GSH, which was supported by significantly higher levels of intestinal MDA. Moreover, histidine deficiency significantly lowered the mRNA level of nrf2 and upregulated the mRNA level of keap1, which further lowered the mRNA levels of the downstream genes sod, cat, and gpx. Additionally, histidine-deficiency-induced intestinal ERS, which was characterized by activating the PEPK-signalling pathway and IRE1-signalling pathway, including increased core gene expression of pepk, grp78, eif2α, atf4, chopα, ire1, xbp1, traf2, ask1, and jnk1. Dietary histidine deficiency also induced apoptosis and necroptosis in the intestine by upregulating the expressions of proapoptotic genes, including caspase 3, caspase 8, caspase 9, and bax, and necroptosis-related genes, including mlkl and ripk3, while also lowering the mRNA level of the antiapoptotic gene bcl-2. Furthermore, histidine deficiency activated the NF-κB-signalling pathway to induce an inflammatory response, improving the mRNA levels of the proinflammatory factors tnf-α, hepcidin 1, cox2, cd80, and cd83 and lowering the mRNA levels of the anti-inflammatory factors tgf-β1 and ikbα. Similarly, dietary histidine deficiency significantly lowered the intestinal levels of the anti-inflammatory factors TGF-β and IL-10 and upregulated the intestinal levels of the proinflammatory factor TNF-α, showing a trend similar to the gene expression of inflammatory factors. However, dietary histidine deficiency inhibited only the level of C3, and no significant effects were observed for IgM, IgG, HSP70, or IFN-γ. Based on the MDA and T-SOD results, the appropriate dietary histidine requirements of juvenile largemouth bass were 1.32% of the diet (2.81% dietary protein) and 1.47% of the diet (3.13% dietary protein), respectively, as determined by quadratic regression analysis.

Ranaviruses are promiscuous pathogens that threaten lower vertebrates globally. In the present study, two ranaviruses (SCRaV and MSRaV) were isolated from two fishes of the order Perciformes: mandarin fish (Siniperca chuatsi) and largemouth bass (Micropterus salmoides). The two ranaviruses both induced cytopathic effects in cultured cells from fish and amphibians and have the typical morphologic characteristics of ranaviruses. Complete genomes of the two ranaviruses were then sequenced and analyzed. Genomes of SCRaV and MSRaV have a length of 99, 405, and 99, 171 bp, respectively, and both contain 105 predicted open reading frames (ORFs). Eleven of the predicted proteins have differences between SCRaV and MSRaV, in which only one (79L) possessed a relatively large difference. A comparison of the sequenced six ranaviruses from the two fish species worldwide revealed that sequence identities of the six proteins (11R, 19R, 34L, 68L, 77L, and 103R) were related to the place where the virus was isolated. However, there were obvious differences in protein sequence identities between the two viruses and iridoviruses from other hosts, with more than half lower than 55%. Especially, 12 proteins of the two isolates had no homologs in viruses from other hosts. Phylogenetic analysis revealed that ranaviruses from the two fishes clustered in one clade. Further genome alignment showed five groups of genome arrangements of ranaviruses based on the locally collinear blocks, in which the ranaviruses, including SCRaV and MSRaV, constitute the fifth group. These results provide new information on the ranaviruses infecting fishes of Perciformes and also are useful for further research of functional genomics of the type of ranaviruses.

In order to evaluate the effects of the interaction between different proteins and feeding frequency on largemouth bass (Micropterus salmoides) and to provide scientific guidance for the application of novel proteins and the corresponding optimal feeding strategy, a two-factorial design (5 × 3) with five protein feeds (fishmeal (FM), Clostridium autoethanogenum protein (CAP), Tenebrio molitor (TM), Chlorella meal (ChM), cottonseed protein concentrate (CPC)), and three feeding frequency (1, 2, and 3 times/d; FF1, FF2, FF3) was designed in culturing largemouth bass (initial weight, 2.98 ± 0.22 g/fish) for 8 weeks. Z-score combined with cluster analysis was used to analyze and compare the effects of different treatments on different indicators, such as growth performance, feed utilization, antioxidant capacity, and immune response to draw a general picture of the relationship among all these massive biomarkers. The results showed that different protein sources and feeding frequencies had significant interactive effects on growth performance, feed utilization efficiency, body lipid, and health status of largemouth bass. Fish fed with ChM feed showed similar performance to that in FM group, implying its potential for complete replacement of fishmeal in largemouth bass. Fish fed with CAP, TM, and CPC feeds showed worse performance compared to FM and ChM groups, characterized by poor growth and feed utilization, enhanced stress, chronic inflammation, and varying symptoms of histological changes in the liver and intestine, which demonstrated the adverse effects of the complete replacement of fishmeal by these three proteins. In terms of feeding frequency, fish fed with FM feed in FF3 group led to liver hypertrophy, fat accumulation, and the risk of fatty liver, while inducing liver inflammation. In addition, the TM and CAP group had the higher expression levels of inflammatory factors at FF3 group, which displayed that the interactions between FM, CAP, TM feeds and feeding frequency at FF3 might aggravate the occurrence of liver inflammation and oxidative damage of hepatocytes. Overall, FF2 had higher feed efficiency, protein efficiency, antioxidant enzyme and lysozyme activities, lower MDA content, and lower gene expression of inflammatory cytokines and could be considered as the optimum feeding frequency for largemouth bass fed with different protein feeds.