Screening And Identification Of Genes Related To Macrobrachium Nipponense Resistance To Aeromonas Veronii

Macrobrachium nipponense,commonly known as green shrimp and river shrimp,is one of the important species cultured in our freshwater shrimp farming industry.Aeromonas veronii is a group of Gram-negative,parthenogenic anaerobic rods that are widely found in freshwater lakes and estuaries,can infect humans,animals and aquatic animals.M.nipponense infected with A.veronii will show symptoms such as soft shell and limb loss,muscle edema and yellowing of the hepatopancreas.In this study,the immune defence mechanisms of M.nipponense against bacterial infections were systematically explored and dissected at four different levels:histopathology,enzyme activity,gene clone expression and transcriptome analysis,based on the effects of A.veronii on the immune system of M.nipponense,providing basic data for the study of immune regulation in M.nipponense.In this study,A.veronii was infected by manual abdominal injection,based on cumulative mortality rate after infection in M.nipponense and the Kou’s modified formula,a 24-hour LD₅₀of 1.86x10⁶ CFU/m L was obtained.Pathological histological analysis showed swelling of the tips of the gill filaments,elevation of the lamellar epithelium and disorganization of the epithelial cells and strut cells of the M.nipponense in the presence of pathogenic bacteria.The granular material inside the functioning vesicles in the hepatopancreatic tissue gradually disappears,the morphological structure of the hepatic tubules is markedly deformed and the lumen of the stellate duct disappears.The enzyme activity assay showed that the activity of alkaline phosphatase,acid phosphatase,polyphenol oxidase and lysozyme in the hepatopancreas increased and then decreased after infection with A.veronii.However,both superoxide dismutase and total antioxidant activity showed a trend of decreasing and then increasing,with the overall level of activity being lower than that of the control group and returning to the control level after 48hours.The increase in enzyme activity was apparently an important indication that the M.nipponense was responding positively to bacterial infestation,and that the suppression of enzyme activity may be the result of autoimmune cell damage caused by the virulence factors that attacked the organism.This study was followed by a high-throughput transcriptome sequencing analysis of moribund individuals within 6 hours of infection and surviving individuals after 48 hours.After QC,a total of 340,554,126 clean reads were used for transcriptome assembly,yielding 120,858Transcripts and 61,345 Unigenes,and all Unigenes were functionally annotated.GO analysis revealed that 20,679 Unigenes were clustered according to molecular function,biological processes and cellular components,and further analysis resulted in 56 functional subclasses.A total of 9,577 Unigenes were grouped into 26 functional categories based on KOG functional classification.10,922 Unigenes were mapped to the KEGG database,predicting significantly enriched pathways,including transport and catabolism,signal transduction,lipid metabolism and the immune system.232 differentially expressed genes were identified,including 80 up-regulated genes such as:copper-zinc superoxide dismutase（Cu Zn-SOD）,clamped structural domain serine proteases（CDSP）,lipocalin receptors（Adipo R）and serine proteases（SPs）.152down-regulated genes such as:chitinase 3（CHIT3）,glutathione S-transferases（GSTs）,cytochrome P450（CYP450）and histone protease（CAPL）.Alpha-2-Macroglobulin,ADP ribosylation factor and g C1q R genes were screened from the differential expressed genes and their changes in m RNA and protein expression at different times after infection were analysed using fluorescent quantitative PCR and Western Blot techniques,and the results showed that their expression after infection by A.veronii.The results showed that they were involved in the immune defence response of the M.nipponense.After functional annotation,the differential genes were mainly enriched in pathogen recognition,antigen processing and presentation,lysosomal,cytokinesis,PI3K-AKT,JAK-STAT and IMD signalling pathways related to organism metabolism and immunity.In this study,the full-length c DNA sequence of the Relish gene,a transcription factor of the IMD signalling pathway,was cloned using the RACE technique.The Mn Relish gene is5,049 bp in length and contains a 36 bp 5’untranslated region,a 3,519 bp open reading frame and a 1,494 bp 3’untranslated region.The Mn Relish open reading frame is predicted to encode1,172 amino acids.Homology analysis revealed that the Mn Relish gene encodes a protein containing a conserved N-terminal Rel homology structural domain,a C-terminal Ik B-like structural domain,and a C-terminal death structural domain.The highest amino acid sequence similarity was found between the M.nipponense Relish and the Macrobrachium rosenbergii Relish;the amino acid phylogenetic tree also showed that it clustered first with the M.rosenbergii Relish.QRT-PCR results showed that the Mn Relish gene was expressed in both hepatopancreas and gill tissues,and the expression was significantly higher in hepatopancreas tissues after infection by A.veronii,indicating that the gene was associated with the immune defense response of M.nipponense.