Largemouth Research Articles

Effects of dietary salidroside on intestinal health, immune parameters and intestinal microbiota in largemouth bass (Micropterus salmoides)

The largemouth bass has become one of the economically fish in China, according to the latest China Fishery Statistical Yearbook. The farming scale is constantly increasing. Salidroside has been found in past studies to have oxidative stress reducing and immune boosting properties. In this study, the addition of six different levels of salidroside supplements were 0、40、80、120、160 and 200 mg/kg. A 56-day feeding trial was conducted to investigate the effects of salidroside on the intestinal health, immune parameters and intestinal microbiota composition of largemouth bass. Dietary addition of salidroside significantly affected the Keap-1β/Nrf-2 pathway as well as significantly increased antioxidant enzyme activities resulting in a significant increase in antioxidant capacity of largemouth bass. Dietary SLR significantly reduced feed coefficients. The genes related to tight junction proteins (Occludin, ZO-1, Claudin-4, Claudin-5) were found to be significantly upregulated in the diet supplemented with salidroside, indicating that salidroside can improve the intestinal barrier function (p < 0.05). The dietary administration of salidroside was found to significantly reduce the transcription levels of intestinal tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) (p < 0.05). Furthermore, salidroside was observed to reduce the transcription levels of intestinal apoptosis factor Bcl-2 associated death promoter (BAD) and recombinant Tumor Protein p53 (P53) (p < 0.05). Concomitantly, the beneficial bacteria, Fusobacteriota and Cetobacterium, was significantly increased in the SLR12 group, while that of pathogenic bacteria, Proteobacteria, was significantly decreased (p < 0.05). In conclusion, the medium-sized largemouth bass optimal dosage of salidroside in the diet is 120mg/kg−1.

Hepatic Oxidative Stress and Cell Death Influenced by Dietary Lipid Levels in a Fresh Teleost.

Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation, affecting physiological and pathological processes. Fatty liver disease associated with metabolic dysfunction is a common pathological condition in aquaculture. However, the exact role and mechanism of ferroptosis in its pathogenesis and progression remains unclear. In this study, an experiment was conducted using different dietary lipid levels in the feeding of largemouth bass (Micropterus salmoides) for 11 weeks. The results revealed that the growth performance and whole-body protein content significantly increased with the elevation of dietary lipid levels up to 12%. The activities of antioxidant enzymes as well as the content of GSH (glutathione) in the liver initially increased but later declined as the lipid levels increased; the contents of MDA (malondialdehyde) and GSSG (oxidized glutathione) demonstrated an opposite trend. Moreover, elevating lipid levels in the diet significantly increased liver Fe2+ content, as well as the expressions of TF (Transferrin), TFR (Transferrin receptor), ACSL4 (acyl-CoA synthetase long-chain family member 4), LPCAT3 (lysophosphatidylcholine acyltransferase 3), and LOX12 (Lipoxygenase-12), while decreasing the expressions of GPX4 (glutathione peroxidase 4) and SLC7A11 (Solute carrier family 7 member 11). In conclusion, the optimal lipid level is 12.2%, determined by WG-based linear regression. Excess lipid-level diets can up-regulate the ACSL4/LPCAT3/LOX12 axis, induce hepatic oxidative stress and cell death through a ferroptotic-like program, and decrease growth performance.

Effects of High Dietary Starch Levels on the Growth Performance, Liver Function, and Metabolome of Largemouth Bass (Micropterus salmoides)

In this study, we conducted a 16-week feeding trial to investigate the effects of a high-cassava starch diet on growth performance, liver function, and metabolism in largemouth bass (Micropterus salmoides). We formulated five diets containing varying levels of cassava starch: 12%, 9%, 6%, 3%, and 0% (termed M12, M9, M6, M3, and M0, respectively). We distributed these diets among largemouth bass with the initial body weight of 83.33 ± 0.55 g via an in-pond “raceway” aquaculture system. Our findings suggest that high level (12%) of cassava starch dietary inclusion adversely affected growth performance metrics such as weight gain rate and specific growth rate, along with feed utilization efficiency indicators, including protein efficiency, protein deposition rate, and the apparent digestibility of dry matter and protein. This negative impact was accompanied by a decrease in intestinal amylase activity. Through further transcriptomic analysis, we identified several key genes associated with carbohydrate metabolism, which underwent changes influencing liver function. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed the involvement of these differentially expressed genes (DEGs) in the tricarboxylic acid cycle (TCA cycle). Comparative metabolomics analysis further indicated that the M9 group showed significant enrichment in pathways related to amino acid metabolism and alterations in the levels of metabolites involved in carbohydrate metabolism. In conclusion, our study demonstrates that incorporating up to 9% cassava starch in the diet can enhance growth performance in largemouth bass by stimulating digestive enzyme activities and promoting glucose utilization.

Hydrolysable tannin improves growth performance and liver health of largemouth bass (Micropterus salmoides) fed high soybean meal diets

This study provided evidence that the inclusion of hydrolysable tannin (HT) in high soybean meal (SBM) diets improved growth performance and glycolipid metabolism of largemouth bass (Micropterus salmoides). In vivo, various levels of HT were added to high SBM diets and fed to largemouth bass (initial weight: 6.00 ± 0.03 g) for 56 days. Results showed that a high level of SBM led to the reduction in growth performance, as evidenced by decreased weight gain rate and impaired hepatic function. Dietary supplementation with HT (1.0 g/kg) improved growth performance of largemouth bass, accompanied by the enhancements in hepatic antioxidant capacity and glycolipid metabolism. In vitro, HT facilitated glucose utilization in hepatocytes and positively influenced the modulation of crucial genes within the PI3K/Akt signaling pathway. Conversely, administration of LY294002 (a PI3K inhibitor) reversed the detrimental effects observed in hepatocytes exposed to high glucose levels. Overall, incorporating HT (1.0 g/kg) into the diet enhanced liver health and improved the absorption and utilization of SBM in largemouth bass, potentially achieved through modulation of the PI3K/Akt signaling pathway.

Longitudinal distribution of fish fauna and characteristics of indicator species in the East Seto Inland Ecological Region within the Japanese Archipelago

Understanding biota distribution patterns is essential for ecosystem conservation and restoration. In addition to biogeography, the distribution patterns of aquatic organisms that inhabit rivers vary longitudinally along river networks. Although longitudinal changes in distribution patterns in aquatic organisms along river networks have been studied in single river basins, it is crucial to understand these patterns at a regional ecological scale for broad-scale ecosystem conservation. This study aimed to elucidate the longitudinal distribution patterns of fish fauna and their correlation with environmental factors in several watersheds within the East Setouchi Ecological Region, renowned for its rich freshwater fish diversity in the Japanese archipelago. Data were obtained for 118 species across 10 watersheds and 85 sites, and the surveyed sites were classified into four longitudinal groups based on the similarity of the fish fauna. The lowest group comprised river estuaries, abundant in brackish-water fish, comprising primarily Gobiidae species. In lower watershed areas with low agricultural land and forest cover but high urbanization, all fish species selected as indicators were non-native. Upstream areas with a high percentage of agricultural land featured a mix of native Cyprinidae and non-native species such as Micropterus salmoides and Rhodeus ocellatus ocellatus. The most upstream groups, marked by significantly greater elevations and bed slopes, exclusively comprised native species as indicator species. Analyzing the longitudinal distribution patterns of fish fauna at multiple sites within the same ecological region enabled us to choose endemic fish species as indicators and identify important species for conservation. Additionally, the selection of sites characterized by significant urbanization and strong anthropogenic influences, along with the selection of non-native species as indicators, underscores the urgency of conserving native organisms in these areas, calling for continuous monitoring.

Using heart rate and acceleration biologgers to estimate winter activity costs in free-swimming largemouth bass

Winter is a critical period for largemouth bass (Micropterus nigricans) with winter severity and duration limiting their population growth at northern latitudes. Unfortunately, we have an incomplete understanding of their winter behaviour and energy use in the wild. More winter-focused research is needed to better understand their annual energy budget, improve bioenergetics models, and establish baselines to assess the impacts of climate warming; however, winter research is challenging due to ice cover. Implantable tags show promise for winter-focused research as they can be deployed prior to ice formation. Here, using swim tunnel respirometry, we calibrated heart rate and acceleration biologgers to enable estimations of metabolic rate (ṀO2) and swimming speed in free-swimming largemouth bass across a range of winter-relevant temperatures. In addition, we assessed their aerobic and swim performance. Calculated group thermal sensitivities of most performance metrics indicated the passive physicochemical effects of temperature, suggesting little compensation in the cold; however, resting metabolic rate and critical swimming speed showed partial compensation. We found strong relationships between acceleration and swimming speed, as well as between ṀO2 and heart rate, acceleration, or swimming speed. Jackknife validations indicated that these modeled relationships accurately estimate swimming speed and ṀO2 from biologger recordings. However, there were relatively few reliable heart rate recordings to model the ṀO2 relationship. Recordings of heart rate were high-quality during holding but dropped during experimentation, potentially due to interference from aerobic muscles during swimming. The models informed by acceleration or swimming speed appear to be best suited for field applications.

Growth, Antioxidant Capacity, and Liver Health in Largemouth Bass (Micropterus salmoides) Fed Multi-Strain Yeast-Based Paraprobiotic: A Lab-to-Pilot Scale Evaluation.

A multi-strain yeast-based paraprobiotic (MsYbP) comprising inactive cells and polysaccharides (β-glucan, mannan oligosaccharides, and oligosaccharides) derived from Saccharomyces cerevisiae and Cyberlindnera jadinii could ensure optimal growth and health in farmed fish. This study assessed the impact of an MsYbP on the growth, immune responses, antioxidant capacities, and liver health of largemouth bass (Micropterus salmoides) through lab-scale (65 days) and pilot-scale (15 weeks) experiments. Two groups of fish were monitored: one fed a control diet without the MsYbP and another fed 0.08% and 0.1% MsYbP in the lab-scale and pilot-scale studies, respectively (referred to as YANG). In the lab-scale study, four replicates were conducted, with 20 fish per replicate (average initial body weight = 31.0 ± 0.8 g), while the pilot-scale study involved three replicates with approximately 1500 fish per replicate (average initial body weight = 80.0 ± 2.2 g). The results indicate that the MsYbP-fed fish exhibited a significant increase in growth in both studies (p < 0.05). Additionally, the dietary MsYbP led to a noteworthy reduction in the liver function parameters (p < 0.05), such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP), and hepatic nuclear density, indicating improved liver health. Furthermore, the dietary MsYbP elevated the antioxidative capacity of the fish by reducing their malondialdehyde levels and increasing their levels and gene expressions related to antioxidative markers, such as total antioxidant ca-pacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), catalase (CAT), nuclear factor erythroid 2-related factor 2 (nrf2) and kelch-1ike ech-associated protein (keap1) in both studies (p < 0.05). In terms of hepatic immune responses, the lab-scale study showed an increase in inflammation-related gene expressions, such as interleukin-1β (il-1β) and transforming growth factor β1 (tgf-β1), while the pilot-scale study significantly suppressed the expressions of genes related to inflammatory responses, such as tumor necrosis factor α (tnfα) and interleukin-10 (il-10) (p < 0.05). In summary, our findings underscore the role of dietary multi-strain yeast-based paraprobiotics in enhancing the growth and liver health of largemouth bass, potentially through increased antioxidative capacity and the modulation of immune responses, emphasizing the significance of employing yeast-based paraprobiotics in commercial conditions.

Single-Cell Transcriptomic Analysis Revealed the Cell Population Changes and Cell–Cell Communication in the Liver of a Carnivorous Fish in Response to High-Carbohydrate Diet

BackgroundCarnivorous fish have a low carbohydrate utilization ability, and the physiologic and molecular basis of glucose intolerance has not been fully illustrated. ObjectivesThis study aimed to use largemouth bass as a model to investigate the possible mechanism of glucose intolerance in carnivorous fish with the help of single-nuclei RNA sequencing (snRNA-seq). MethodsTwo diets were formulated, a low-carbohydrate (LC) diet and a high-carbohydrate (HC) diet. The feeding trial lasted for 6 wk, and then, growth performance, biochemical parameters, liver histology, and snRNA-seq were performed. ResultsGrowth performance of fish was not affected by the HC diet, while liver glucolipid metabolism disorder and liver injury were observed. A total of 13,247 and 12,848 cells from the liver derived from 2 groups were isolated and sequenced, and 7 major liver cell types were annotated by the marker genes. Hepatocytes and cholangiocytes were lower and hepatic stellate cells (HSCs) and immune cells were higher in the HC group than those in the LC group. Reclustering analysis identified 7 subtypes of hepatocytes and immune cells, respectively. The HSCs showed more cell communication with other cell types, and periportal hepatocytes showed more cell communication with other hepatocyte subtypes. Cell–cell communication mainly focused on cell junction–related signaling pathways. Uncovered by the pseudotime analysis, midzonal hepatocytes were differentiated into 2 major branches—biliary epithelial hepatocytes and hepatobiliary hybrid progenitor. Cell junction and liver fibrosis–related genes were highly expressed in the HC group. HC diet induced the activation of HSCs and, therefore, led to the liver fibrosis of largemouth bass. ConclusionsHC diet induces liver glucolipid metabolism disorder and liver injury of largemouth bass. The increase and activation of HSCs might be the main reason for the liver injury. In adaption to HC diet, midzonal hepatocytes differentiates into 2 major branches—biliary epithelial hepatocytes and hepatobiliary hybrid progenitors.

Establishment of an LMBV-infection model in zebrafish larvae and its application in studying virus-host interactions

Largemouth bass virus (LMBV) causes high transmission efficiency and mortality in freshwater aquaculture industry. However, the host responses to LMBV infection, particularly the molecular mechanisms involved in vivo, remain largely uncertain. Zebrafish has been developed as a powerful model organism for the study of host inflammation and immune response upon pathogen infections. Here, we evaluated the susceptibility of LMBV to zebrafish larvae and investigated the host response upon virus infection using transcriptomic analysis. LMBV replicated well in larvae by inoculation with LMBV via the brain and the duct of Cuvier, but the later showed more severe systemic infection, evidenced by the cumulative mortality, the increased transcriptions of viral core genes, and the presence of mcp positive signal in infected larvae. After inoculation with LMBV via the duct of Cuvier, we observed numerous viral particles in the head and tail regions of infected larvae. Moreover, ROS and TUNEL positive signaling also significantly increased in LMBV infected larvae. Of note, a great deal of neutrophils migrated towards the virus infected sites during LMBV infection. Transcriptomic analysis showed that a total of 1263 genes were up-regulated and 1039 genes were down-regulated in response to LMBV infection. Enrichment analysis indicated that the majority of the enriched KEGG pathways were associated with metabolism, cell death and immune regulation, such as fatty acid metabolism, steroid hormone biosynthesis, PPAR signaling, apoptosis, necroptosis, phagosome, cytokine-cytokine receptor pathway, NOD-like receptor signaling, and toll-like receptor pathway. For instance, cell death related signaling molecules including casp9,fas,tnfα and sqstm1 were significantly up-regulated, while camk2a,sept4b and akt1 were down-regulated in response to LMBV infection. Thus, our data firstly established LMBV-infected model in zebrafish larvae which could provide an alternative means for investigation of LMBV-host interaction in vivo, and application for high throughput drug screening in the future.

Intermittent swimming and muscle power output in brook trout, Salvelinus fontinalis.

Slow and sustainable intermittent swimming has recently been described in several Centrarchid fishes, such as bluegill and largemouth bass. This swimming behavior involves short periods of body-caudal fin undulation alternating with variable periods of coasting. This aerobic muscle powered swimming appears to reduce energetic costs for slow, sustainable swimming, with fish employing a "fixed-gear" or constant tailbeat frequency and modulating swimming speed by altering the length of the coasting period. We asked if this swimming behavior was found in other fish species by examining volitional swimming by brook trout in a static swimming tank. Further, we employed muscle mechanics experiments to explore how intermittent swimming affects muscle power output in comparison to steady swimming behavior. Brook trout regularly employ an intermittent swimming form when allowed to swim volitionally, and consistently showed a tailbeat frequency of ~2 Hz. Coasting duration had a significant, inverse relationship to swimming speed. Across a range of slow, sustainable swimming speeds, tailbeat frequency increased modestly with speed. The duration of periods of coasting decreased significantly with increasing speed. Workloop experiments suggest that intermittent swimming reduces fatigue, allowing fish to maintain high power output for longer compared to continuous activity. This study expands the list of species that employ intermittent swimming, suggesting this behavior is a general feature of fishes.

Population life‐history differences and their correlates across a regional spatial scale in largemouth bass (Micropterus nigricans)

Population life‐history differences and their correlates across a regional spatial scale in largemouth bass (<i>Micropterus nigricans</i>)

Dietary threonine influences antioxidant capacity and immune status in juvenile largemouth bass (Micropterus salmoides)

As a restricted amino acid, threonine (Thr) deficiency reduces antioxidant and immune capacity in fish. In the present study, six diets containing different Thr levels (1.39 %, 1.71 %, 1.95 %, 2.28 %, 2.58 %, and 2.65 %) were fed to largemouth bass (15±0.1 g) for 60 days. The present results showed that neither growth nor whole-body composition was affected by different Thr levels. And low dietary Thr levels (1.39 %) significantly decreased serum total protein (TP) and albumin (ALB) levels, but increased alkaline phosphatase (ALP) levels. And 1.39 % Thr diet significantly decreased liver catalase (CAT) activity but increased malonaldehyde (MDA) levels, and the maximum total antioxidant capacity (T-AOC) was observed in response to the 1.95 % Thr diet. Liver proapoptotic factors (bax and caspase9) were significantly elevated in fish fed the 1.39 % Thr diet. The nuclear factor κB (nf-κb) and the NF-κB-mediated inflammatory cytokines were significantly increased in fish fed the 1.39 % Thr diet. The mRNA levels of protein kinase R (PKR)-like endoplasmic reticulum kinase (perk), eukaryotic translation initiation factor 2 (eif2α), activating transcription factor 4 (atf4), and C/EBP-homologous protein (chop), which are factors in the PERK/ATF4/CHOP pathway, one of the unfolded protein response (UPR) signaling pathways, were significantly increased by the 1.39 % Thr diet. The present study showed that low dietary Thr levels caused oxidative damage, reduced immunity, might affected PERK-ATF4-CHOP signaling to induce apoptosis, and via NF-κB signaling to trigger an inflammatory response in largemouth bass. Based on the TP, ALB, CAT, and MDA levels, the dietary Thr requirements for juvenile largemouth bass were estimated to be 2.18 %, 2.29 %, 1.92 %, and 1.88 %. Furthermore, these findings provided a new perspective that low dietary amino acid levels may trigger apoptosis and inflammatory responses in fish through the UPR signaling pathway.

Use of Fin Rays and Fin Spines in Nonlethal Age Estimation of Florida Bass

Abstract Nonlethal age-estimation methods for Florida Bass Micropterus salmoides using fin rays and spines would enhance trophy-bass research, management, and conservation. Nonlethal removal of fin structures would also expand opportunities for collecting age-related information, specifically during small-population projects as well as tournaments and citizen-science programs, for which sacrificing fish is not an option. We assessed the bias of age estimates derived from Florida Bass anal-, pelvic-, dorsal-, and pectoral-fin rays and anal-, pelvic-, and dorsal-fin spines by comparing them to age estimates derived from otoliths (i.e., sagittae), which are the standard for ageing Florida Bass. We evaluated the precision of age estimates by calculating the absolute difference between replicate reads of each ageing structure for each reader. Linear-mixed effects model selection indicated that precision was most affected by age, structure, and the age*structure interaction, in which the effect of age varied by structure. Age estimates from otoliths were the most precise whereas pelvic spines were the least precise among all structures examined. Bias was similar across all fin structures, but the effect of age on bias varied by reader. Marginal increment analysis indicated that annulus formation was completed from November to March in Florida Bass dorsal spines, 5 months earlier than in Florida Bass otoliths. Our results suggest that nonlethally removed fin structures, especially dorsal spines, have potential for use in estimating age, growth rates, mortality, and age structure of bass populations, which could be particularly beneficial where bass can reach unusual trophy sizes.

Predator presence influences survival and behavior of translocated stream fish in ponds

Abstract Small artificial impoundments such as farm ponds have recently been recognized as potential habitat for threatened native fish species. However, factors influencing translocation or colonization success into these environments, including connectivity to stream networks and interactions with existing fish community, are largely unknown. In this study we conducted a controlled experiment to quantify the influence of piscivorous Largemouth Bass Micropterus salmoides on the survival of a translocated native minnow species that we used as a surrogate for federally endangered Topeka Shiner Notropis topeka. We translocated or released 100 Bluntnose Minnow Pimephales notatus into each replicate treatment ponds with and without Largemouth Bass in the summers of 2020 and 2021. Each minnow was implanted with a passive integrated transponder (PIT) tag. Translocated populations were monitored using stationary and mobile passive integrated transponder antennas and estimates of apparent survival and probability of detection for each pond were derived from open population mark-recapture models. Apparent survival was nearly two times higher in ponds without bass suggesting predation by bass leads to higher mortality. Additionally, probability of detection was nearly 10 times higher in ponds without bass, suggesting reduced movement of translocated minnows when bass were present. While the direct effect of mortality impacts translocated populations, the indirect effect of altered behavior may also be impactful on translocation success. These results confirm that Largemouth Bass can limit the success of translocated minnow species.

Responses of Digestive, Antioxidant, Immunological and Metabolic Enzymes in the Intestines and Liver of Largemouth Bass (Micropterus salmoides) under the Biofloc Model.

To investigate the activities of intestinal digestive enzymes, liver antioxidant enzymes, immunological enzymes, and glucometabolic enzymes in largemouth bass (Micropterus salmoides) under the biofloc model, an experiment was conducted in 300-liter glass tanks. The experiment comprised a control group, which was fed a basal diet, and a biofloc group, where glucose was added to maintain a C/N ratio of 15. Each group had three parallel setups, with a stocking density of 20 fish per tank. The experiment ran for 60 days, employing a zero-water exchange aquaculture model. The results showed that at the end of the culture period, there were no significant differences between the initial weight, final weight, WGR, SGR, and SR of the biofloc group and the control group of largemouth bass (p > 0.05), whereas the lower FCR and the higher PER in the biofloc group were significant (p < 0.05); intestinal α-amylase, trypsin, and lipase activities of largemouth bass in the biofloc group were significantly increased by 37.20%, 64.11%, and 51.69%, respectively, compared with the control group (p < 0.05); liver superoxide dismutase and catalase activities, and total antioxidant capacity of largemouth bass in the biofloc group were significantly increased by 49.26%, 46.87%, and 98.94% (p < 0.05), while the malondialdehyde content was significantly reduced by 19.91% (p < 0.05); liver lysozyme, alkaline phosphatase, and acid phosphatase activities of largemouth bass in the biofloc group were significantly increased by 62.66%, 41.22%, and 29.66%, respectively (p < 0.05); liver glucokinase, pyruvate kinase, glucose-6-phosphate kinase, pyruvate kinase, glucose-6-phosphatase, and glycogen synthase activities were significantly increased by 46.29%, 99.33%, 32.54%, and 26.89%, respectively (p < 0.05). The study showed that the biofloc model of culturing largemouth bass can not only enhance digestive enzyme activities, antioxidant capacity, and immune response but can also promote the process of glucose metabolism and reduce feeding costs. This study provides data support for healthy culturing of largemouth bass in future production, provides a theoretical reference for optimizing the biofloc technology culture model, and is crucial for promoting the healthy and green development of aquaculture.

High temperature exposure causes sexual dimorphism in immune responses against largemouth bass ranavirus (LMBV) in Micropterus salmoides

Largemouth bass (Micropterus salmoides) is an important freshwater fish species in aquaculture. Largemouth bass ranavirus (LMBV) infection at high temperature results in a considerably higher mortality rate in females than that in males. This study compared the spleen transcriptomes and analyzed the impact of sexual dimorphism on immunity and metabolism of largemouth bass infected with LMBV at high temperatures to gain a comprehensive understanding of the sex-based differences in immunity and metabolism of this species. (1) Compared with the ambient temperature group (W20), the high temperature group (W30) exhibited multiple significant differences in metabolic and immune terms, with several immunity-related pathways, including phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) signaling pathway. Most identified pathways were associated with the estrogen signaling pathway, which also differed significantly. (2) Differences in glycolipid metabolism, immunity, and the endocrine and complex adaptive systems were distinctly different between male groups (W20M vs. W30M), whereas the female groups (W20F vs. W30F) were not significantly different. The male groups had a significantly higher number of enriched immunity-related KEGG pathways and differentially expressed genes (DEGs) and pathways related to the estrogen signaling pathway. (3) When comparing male and female samples infected with LMBV at the same temperature, no significant difference was observed between the ambient temperature groups (W20F vs. W20M). The high-temperature groups (W30F vs. W30M) had 18 immunity-related pathways among the top 25 enriched KEGG pathways, and gene expression correlation analysis of the DEGs showed positive correlations. This study demonstrates that the combined effects of high temperature and LMBV affect multiple metabolic and immune pathways in largemouth bass, thereby modulating immune responses by regulating the estrogen signaling pathway and influencing the expression of estrogen and its receptors. Under high-temperature conditions, male fish may have stronger estrogen signaling pathway-associated immune responses, which regulate immune pathways, including antigen processing and presentation, complement system, and phagocytosis. Consequently, males may have higher disease resistance than that of females. Our result contribute to mono-sex breeding and antiviral drug therapies available in largemouth bass, thereby markedly improving breeding efficiency.

Interleukin-1 beta (IL-1β) as adjuvant enhances the immune effects of Aeromonas veronii inactivated vaccine in largemouth bass (Micropterus salmoides)

Largemouth bass (Micropterus salmoides) has emerged as a significant economic fish species, with a rise in Aeromonas veronii infections in farming. However, research on adjuvants for vaccines against A. veronii in largemouth bass remains scarce. In present study, recombinant largemouth bass IL-1β (LbIL-1β) was expressed to explore its adjuvant effect on the A. veronii inactivated vaccine. Following vaccination with recombinant LbIL-1β (rLbIL-1β) and the inactivated A. veronii, higher serum SOD levels and lysozyme activities were observed in largemouth bass from inactivated A. veronii + rLbIL-1β vaccinated group. Furthermore, it was discovered that rLbIL-1β was able to boost the serum-specific antibody levels induced by the inactivated A. veronii. The qRT-PCR analysis revealed that rLbIL-1β also enhanced the expression of IgM, CD4, and MHC II in largemouth bass triggered by the inactivated A. veronii. After challenged with live A. veronii, the outcomes demonstrated that the relative percentage survival (RPS) for largemouth bass resulting from the inactivated A. veronii in combination with rLbIL-1β was 76.67 %, surpassing the RPS of 60 % in the inactivated A. veronii group. Collectively, these findings indicate that rLbIL-1β enhances the protective effect of the A. veronii inactivated vaccine on largemouth bass, showcasing potential as an adjuvant for further development.

Enhancing understanding of interferon-induced protein 35 (IFI35) in hybrid snakehead (Channa maculate ♀ × Channa argus ♂): Cloning, expression, and interaction with EF1α in response to SHVV infection

Interferon-induced protein 35 (IFI35) play a crucial role in the host's antiviral responses. However, research focusing on the characteristics of IFI35 genes in fish remains limited. In the present study, the IFI35 gene from hybrid snakehead was cloned and characterized. The potential interacting proteins of IFI35 to elucidate its role in disease resistance was also investigated. The open reading frame (ORF) of IFI35 was found to be 1119 bp, encoding a polypeptide with 372 amino acids. Sequence analysis revealed that the IFI35 protein in hybrid snakehead shares the highest identity with those in Micropterus salmoides and Siniperca chuatsi. Expression of the IFI35 gene was detected in all examined tissues of healthy fish, with the highest levels in the spleen, gills, and intestine. Under viral stress from snakehead vesicularvirus (SHVV), a significant induction of IFI35 expression was observed, especially in the liver and spleen. Yeast two-hybrid assays identified 21 proteins that interact with IFI35, including elongation factor 1-alpha (EF1α), ribosomal proteins, transcription factors, collagen, ferritin-like domain, cytochrome c oxidase, and short chain dehydrogenase, etc. Subsequent validation confirmed the interaction between IFI35 and EF1α by point-to-point validation. These findings suggest a significant role for IFI35 in the immune regulation of hybrid snakeheads, enhancing our understanding of its function and interaction networks, which could facilitate future explorations of IFI35 mechanisms in snakehead fish.

Protective efficacy and immune responses of largemouth bass (Micropterus salmoides) immunized with an inactivated vaccine against the viral hemorrhagic septicemia virus genotype IVa

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to the aquaculture industry, prompting the need for effective preventive measures. Here, we developed an inactivated VHSV and revealed the molecular mechanisms underlying the host's protective response against VHSV. The vaccine was created by treating VHSV with 0.05 % formalin at 16 °C for 48 h, which was determined to be the most effective inactivation method. Compared with nonvaccinated fish, vaccinated fish exhibited a remarkable increase in survival rate (99 %) and elevated levels of serum neutralizing antibodies, indicating strong immunization. To investigate the gene changes induced by vaccination, RNA sequencing was performed on spleen samples from control and vaccinated fish 14 days after vaccination. The analysis revealed 893 differentially expressed genes (DEGs), with notable up-regulation of immune-related genes such as annexin A1a, coxsackievirus and adenovirus receptor homolog, V-set domain-containing T-cell activation inhibitor 1-like, and heat shock protein 90 alpha class A member 1 tandem duplicate 2, indicating a vigorous innate immune response. Furthermore, KEGG enrichment analysis highlighted significant enrichment of DEGs in processes related to antigen processing and presentation, necroptosis, and viral carcinogenesis. GO enrichment analysis further revealed enrichment of DEGs related to the regulation of type I interferon (IFN) production, type I IFN production, and negative regulation of viral processes. Moreover, protein-protein interaction network analysis identified central hub genes, including IRF3 and HSP90AA1.2, suggesting their crucial roles in coordinating the immune response elicited by the vaccine. These findings not only confirm the effectiveness of our vaccine formulation but also offer valuable insights into the underlying immunological mechanisms, which can be valuable for future vaccine development and disease management in the aquaculture industry.

Mulberry (Morus alba) leaves improved the growth and intestinal health in largemouth bass (Micropterus salmoides) fed a cottonseed protein concentrate-based diet

Mulberry leaves (ML) have diverse biological activities and show potential as a functional additive. However, it is unclear whether ML can alleviate the adverse effects of high cottonseed protein concentrate (CPC) inclusion in carnivores. In this study, Micropterus salmoides were respectively fed six different diets over an 8-week period. Four experimental diets were formulated with varying levels of ML (0%, 2%, 4%, and 6%), designated as CG (basic diet), ML2, ML4, and ML6. Additionally, two diets replaced 75% of fishmeal (FM) protein with CPC in the CG diet, with or without 4% ML, named CPC and CM4 diet, respectively. Results showed that ML4 diet improved growth and feed utilization in largemouth bass compared with the control. The growth of fish fed with the CPC diet were significantly lower than those fed with the CG diet (P < 0.05). However, supplementing 4% ML in the CPC diet could improve the decreased growth and feed utilization, compromised intestinal barrier, and intestinal inflammation caused by high CPC in largemouth bass. Through 16S rDNA and intestinal transcriptome correlation analysis, it was revealed that ML could reduce non-homeostatic apoptosis by positively regulating intestinal microbiota to down-regulate the expression of pro-apoptosis genes in the PI3K-Akt pathway of the Apoptosis pathway.