| Cherax quadricarinatus is an important freshwater crayfish culture species in China.Aeromonas veronii is a class of Gram-negative,facultative anaerobic bacilli that are widely found in the aquatic environment and can infect a variety of terrestrial and aquatic organisms,including humans and C.quadricarinatus,which can result in seriously affecting the healthy development of C.quadricarinatus culture.In this study,we propose to use high-throughput transcriptome sequencing and bioinformatics analysis techniques as the basis,and to investigate the changes of histopathological,humoral immune factors and transcription level and protein level of related genes in C.quadricarinatus infected with A.veronii by high-throughput transcriptome sequencing and bioinformatics analysis,and to explore the immune defense mechanism of C.quadricarinatus against A.veronii.Further,the study was conducted to provide valuable data for understanding the immune regulatory network of crayfish.The main studies were as follows:（1）In this study,the 24 h LD50 of C.quadricarinatus infected with A.veronii by intramuscular injection under laboratory artificial culture conditions was determined to be 4.5×10~7 CFU/m L by pre-experimentation.100μL of this concentration of A.veronii was injected into the treatment group and an equal amount of PBS was injected into the control group.After infection,samples were taken and stored at 0,6,12,24,48,72,and 96 h.The behavioral characteristics and mortality of the infected C.quadricarinatus were used to determine the susceptible group as dying individuals at 6 h post-infection and the resistant group as surviving individuals at 48 h post-infection.（2）The measurement of humoral immune factors activity showed that superoxide dismutase（SOD）and catalase（CAT）activities were significantly higher than the control group at 12-24 h（P<0.01）,and SOD activity decreased at 48-72 h compared to 12-24 h,but the activity was still significantly higher than the control group（P<0.05）;the activity of phenoloxidase（PO）showed a significant decrease at 12-24 h,after which the activity increased and was significantly higher than that of the control group（P<0.05）;the activity of lysozyme（LZM）remained elevated after infection and was significantly higher than that of the control group（P<0.05）.Changes in enzyme activity are important indicators of humoral immune response,and analysis of this changes can provide ideas and directions for subsequent screening of immune-related genes.The results of pathological analysis showed that the hepatopancreas of C.quadricarinatus showed pathological features such as myoepithelial rupture,dilatation of hepatic tubular lumen,and loss of stellate structure after 24 h of infection with A.veronii,and intestinal tissues also showed different degrees of damage.Brachychronic damage of tissues and cells caused by A.veronii can explain the significant decrease in the activity of humoral immune factors at the beginning of infection.（3）In the present study,the susceptible and resistant groups were sequenced together with the control group.After quality-controlling,the results showed a total of 99,403 Transcripts and56,370 Unigenes.All the Unigenes were annotated in 7 databases,including NR,NT,KO,Swiss Prot,PFAM,GO,and KOG.The GO annotation showed that 16,065 Unigenes were annotated in three major categories:biological processes,cellular components,and molecular functions;KOG annotation showed that 5,984 Unigenes were annotated in 26 subcategories including RNA processing and modification,chromosome structure,and dynamics;KEGG annotation showed that 6,845 Unigenes were annotated in 34 subcategories in 5 major categories.The analysis of differently expressed genes revealed a total of 4,167 and 3,200 genes were obtained in the susceptible and resistant groups,respectively,compared to the control group.These DEGs were enriched in some important immune-related pathways such as lysozyme,phagosome and FcγR-mediated phagocytosis.q RT-PCR results for 23 of these DEGs verified the accuracy of RNA-Seq data.（4）In the current study,the expression of four immune-related genes,SPI,CHH,ALF,and SOD1,at the m RNA level in different tissues over the time of infection was comparatively analyzed using q RT-PCR.The results showed that the expression of SPI was significantly higher than the control group at different time points in the tissues examined（hepatopancreas,gill,and intestine）（P<0.05）,but the expression decreased with the time of infection,the expression was significantly higher than the control group at 6-72 h after injection in muscle,and returned to normal levels at 96 h（P>0.05）.The expression trends of CHH and ALF were similar to those of SPI,but the expression in muscle was significantly lower than in the control group（P<0.05）.The expression of SOD1 was significantly lower in gill and muscle tissues than in the control group,but on the contrary,it was significantly higher in hepatopancreas and intestine tissues than in the control group（P<0.05）.The four immune-related genes showed different expression patterns in different tissues,which provided a theoretical basis and new directions and ideas for the subsequent study of how these genes functioned in time and location perspective.（5）In this study,the CDS region of phospholipase D（PLD）,a key regulatory gene in phagocytosis,was cloned,and the PLD prokaryotic expression plasmid was constructed using the p EASY-Blunt E1 plasmid.The recombinant protein of PLD was expressed in E.coli BL21.After purified,the recombinant protein was used as an antigen to immunize mice to prepare polyclonal antibodies.The expression of PLD at the protein level was analyzed using the obtained effective polyclonal antibodies,the results showed that the expression of PLD in hepatopancreatic was significantly higher than that in the control group at 12 h post-infection（P<0.01）,and the expression of PLD reached the highest level at 24 h（P<0.01）,and then gradually decreased,and was not significantly different from that in the control group at 96 h post-infection（P>0.05）;the expression of PLD in gill was significantly higher than that in the control group at 12-48 h（P<0.01）,and remained significantly higher than that in the control group at 72 h post-infection（P<0.05）.Further analysis of the m RNA expression of PLD using q RT-PCR showed that PLD was also significantly altered at the transcriptional level.The above results fully demonstrated that A.veronii infection significantly activated the expression of PLD in hepatopancreas and gill,and it was inferred that PLD was involved in the immune process of C.quadricarinatus against A.veronii.In summary,this study screened some new immune-related genes in the absence of genomic sequence information of red crayfish;it laid the foundation for further research on the molecular biological mechanism of crayfish disease resistance under pathogenic bacterial infection.The expression pattern of immune-related genes,dynamic changes of humoral immune factors,and pathological histological analysis provided new ideas and perspectives to explore the regulatory mechanisms of immune genes.In addition,this study confirmed that PLD plays an important role in the immune defense of C.quadricarinatus against A.veronii.This provides valuable information for refining the network of innate immune regulatory mechanisms in C.quadricarinatus. |
