Mechanism Study On Immune Response And Bacterial Susceptibility Of Largemouth Bass (Micropterus Salmoides) Under High Temperature Stress

Aquaculture of largemouth bass(LMB,Micropterus salmoides),an economically important species,is deeply affected by the outbreak of bacterial diseases in the summer seasons.However,the impacts of heat waves on the immune functions and the mechanisms underlying the heat-induced disease susceptibility remain largely unknown.In this study,heat resistance of LMB was assessed and the critical thermal maximum(CTMax)for the fish acclimated to 28℃was measured as 39.71±0.18℃.After being exposed to a high temperature of 34℃for 1,7 and 14d,the head kidney,spleen and blood were sampled for histological and biochemical assays to explore the effects of heat exposure on tissue structure,oxidative status and immune functions.While a short-term exposure(7d)evoked certain extent of physiological plasticity,chronic exposure(14d)caused severe cell apoptosis,oxidative damage and attenuation of the immune functions.The results showed that heat stress increased the number of free melanocyte macrophages(MMs)in head kidney and spleen tissues,and the number increased with the increase of exposure time..Prolonged exposure resulted in a significant increase in the number of fragmented nuclei(FNs)in the head kidney and spleen,causing severe apoptosis in the immune organs.In addition,prolonged heat exposure led to a decrease in the activity of the antioxidant enzymes CAT and GSH-PX in the head kidney and spleen,and resulted in an increase in the MDA content in the head kidney(p<0.05).Expression of hsp70 gene was significantly induced in both tissues after heat exposure(p<0.05).Short-term(7 days)heat exposure enhanced the expression of igm gene in both tissues and activated the blood respiratory burst activity(p<0.05).However,long-term exposure decreased igm(in the spleen)and ifng(in both tissues)gene expression and inhibited blood respiratory burst activity(p<0.05).Consistently,chronic heat exposure predisposed LMB to Aeromonas hydrophila infection and significantly raised the tissue bacteria load.Furthermore,gene expression in the head kidney and spleen tissues of the fish subjected to heat exposure,Aeromonas hydrophila infection and both the stressors were analyzed by RNA sequencing.PCA results showed that samples exposed to heat exposure or Aeromonas hydrophila infection clustered closely with the control group kept at 28℃ and injected with PBS,while samples exposed to both heat exposure and Aeromonas hydrophila infection were separated from the corresponding control samples.The amounts of differential genes and enriched GO terms were significantly higher in the head kidney and spleen samples subjected to heat+infection double stress than in the other groups.After being subjected to dual stress,upregulated genes in the head kidney and spleen were highly enriched in functions such as protein folding,endoplasmic reticulum stress response,cellular processes,and metabolic processes,and downregulated genes were highly enriched in bioregulation,gas transport,oxygen transport,hydrogen peroxide catabolic processes,and cell-to-cell signaling.Furthermore,KEGG pathway enrichments showed that multiple pathways involved in protein hydrolysis,energy metabolism,and biosynthesis,such as proteasome,oxidative phosphorylation,citric acid cycle,fatty acid biosynthesis,and amino acid biosynthesis,were enhanced in the head kidney and spleen upon exposure to heat+infection,while multiple pathways that maintain cellular structure and normal cellular function in the head kidney and spleen were downregulated,such as cell adhesion molecules,ECM receptor interaction and focal adhesion,calcium signaling,MAPK signaling and FoxO signaling,etc.Weighted gene co-expression network analysis(WGCNA)was performed and gene co-expression modules and corresponding central genes(crema,dusp4,zbtb21,arf2b,arl2 and eaf2)were identified.In addition,the expression of genes ass1,acsl1a,fasn,ndufs5 and phgdh was analyzed by qPCR to validate the RNA-seq data.The results showed a significant correlation between RNA-seq and qPCR data(p<0.0001,R~2=0.8776).The above studies suggest that chronic heat exposure greatly facilitated the bacterium-elicited metabolic pathway shift in the host.Pathways involved in energy production and nutrients biosynthesis were activated in both tissues,whereas the downregulated pathways are mainly associated with host cell structures and normal cellular functions.Our data provide new insights into the mechanisms underlying the heat-induced disease susceptibility of LMB,which promise new therapeutic opportunities to ameliorate the pathology through metabolic modulations.