Effects Of Aeromonas Hydrophil And Its Combination With Fipronil On Oxidative Stress,Immunity,Autophagy And Apoptosis Of Common Carp

With the rapid development of aquaculture,more and more problems have emerged.Among them,the bacterial septicemia caused by Aeromonas hydrophilis has attracted extensive attention because of its strong infectivity and incidence.At the same time,phenylpyrazoles insecticides,mainly fipronil,are widely used for pest control in crops and household hygiene,resulting in their residues often detected on the surface of water,soil and vegetables,while drugs in the water environment may cause certain toxicity to aquatic animals.In this paper,using carp as an animal model,we explored the toxicological mechanism of Aeromonas hydrophila and its combination with fipronil on carp oxidative stress,immunity,autophagy and apoptosis.1.The diseased carp was obtained from the aquaculture base of Henan Normal University,and its liver,intestines,gills and other tissues were isolated and purified to obtain two strains of pathogenic bacteria.It was identified by 16S r DNA as Aeromonas hydrophila and Pseudomonas putida.In this study,Aeromonas hydrophila and fipronil purchased from beihuan farmers’market in xinxiang city were selected as exogenous pollutants through preliminary experiments.The 96h semi-lethal concentrations of Aeromonas hydrophila and fipronil were respectively 8.21×10~6CFU/m L and 0.37mg/L.The experimental fish were divided into five groups,the first group was the control group,and the last four groups were the treatment group.Among them,the second and third groups were treated with 0.074 mg/L fipronil for one week,and then the second,fourth and third,fifth groups were injected with Aeromonas hydrophile bacterial suspensions with a concentration of 7.55×105CFU/m L and 4.87×107CFU/m L,respectively.Serum and tissue were collected and protein and gene levels were detected at 1,3,5 and 7 days after the injection.Name the treatment groups L1,L2,C1,and C2,respectively.2.In order to understand the effect of Aeromonas hydrophila and its combined action with fipronil on the antioxidant and non-specific immunity of carp,the changes of corresponding indexes in liver,intestine,gill and serum were detected.The results showed that in serum,intestines and gills,compared with the control group,the activity of glutathione peroxidase(microreduced glutathione(GSH))in the high concentration group firstly decreased and then increased,while its activity in the liver showed the opposite change.The level of Superoxide Dismutase(SOD)was firstly increased and then decreased.The content of malondialdehyde(MDA)in the liver,intestines and gills was significantly increased compared with the control group.Except that the AKP activity of the serum increased significantly throughout the whole infection period compared with the control group,the changes of ACP,AKP,and LZM were basically similar:their levels are generally up-regulated and then down-regulated.3.In order to understand the effect of Aeromonas hydrophila and its combination with fipronil on autophagy and apoptosis of carp,the expression of autophagy related genes atg5,atg12,atg16L,LC3-II,BECN1 and m TOR in liver,kidney and brain of treated carp was detected in this paper.The results showed that the expression of atg5,atg12,LC3-II and BECN1 in liver was significantly promoted and then significantly inhibited,while the change of m TOR was completely reversed.The expression of atg5,atg12,atg16L and m TOR in the kidney and brain after C2 treatment was generally higher than that after L1 and L2 treatment.Through the WB experiment,we found that the LC3B protein in the liver significantly increased the ratio of LC3B-II to LC3B-I at 3 and 5 days after treatment.Apoptosis-related gene Bcl-2increased significantly at the initial of exposure and then continuously decreased to the control level,while caspase3 was significantly inhibited during the entire treatment period compared to the control group.Although the expression of caspase3 increased on the seventh day compared with other time points,it was still lower than that of the control group.In summary,our results indicate that treatment with Aeromonas hydrophila and fipronil may cause oxidative stress in carps,stimulate non-specific immune responses,lead to autophagy,and might induce apoptosis in the later stages of infection.The results of this study provide new insights into the toxic mechanism of common carp infected with Aeromonas hydrophila and fipronil.