| Procambarus clarkii is one of the commonly cultivated aquatic species in China,which is commonly known as the "crayfish".It has the characteristics of feeding miscellaneous,short breeding cycle,strong environmental adaptability and has high economic value.The Procambarus clarkii culture industry has grown quickly since crayfish was introduced from Japan in 1929.It has become a pillar industry and an important economic source in some regions.However,crayfish diseases have increased in frequency and severity,as a result of the rapid deterioration of the water environment and the continuous growth of aquaculture scale.In particular,there was a large-scale death outbreak of farmed crayfish around May every year,its onset was fast and the mortality rate was high.The diseased shrimp mostly lied on the edge of the pond,the head and chest armor appeared white spots,farmers called it"Black May".White spot syndrome virus is one of main pathogens of P.clarkii.Therefore,this research investigated the pathogenicity of WSSV from 2020 to 2022,carried out pathogen detection on diseased crayfish,and identified the cause of the deaths as a mixed infection of bacteria and viruses and the biological control technology was used to study the inhibition of the occurrence and spread of crayfish disease at the source,which provided a new perspective for the comprehensive prevention and control of WSSV and provided a theoretical basis for the healthy culture of Procambarus clarkii.(1)From April 2020 to July 2022,an epidemiological investigation of P.clarkii cultured ponds was conducted in 14 counties and cities including Lianshui,Sihong,Peixian,Guannan and Jiangdu,and the occurrence of diseases was recorded in detail,while PCR detection was performed on random samples.The occurrence patterns and characteristics of WSSV in the aquaculture production process of P.clarkii in different counties and cities were also analyzed.The findings demonstrated that,although WSSV symptoms in diseased crayfish were not apparent and only a small number of cases of white pancreas and liver were discovered during dissection,the detection rate for WSSV was comparatively high.Among them,the WSSV detection rate was the highest in the main culture area of P.clarkii in 2022.In 2020,there were 10 counties and cities including Lianshui,Xinghua,Guangling and Jiangyin,and the detection rate of WSSV was 100%;In 2021,there were 8 counties and cities including Gaochun,Jiangdu,Jiangyan and Xinghua,and the WSSV detection rate was 100%;In 2022,there are 11 counties and cities including Jiangdu,Jiangyan,Donghai,Lianshui,Xinghua and Guangling,and the WSSV detection rate is 100%.(2)The dominant strain CPA2020 was isolated from the hepatopancreas of dying shrimps and identified as Aeromonas veronii through morphological observation,biochemical identification,physiology characteristics and sequencing of and gyrB gene.In challenge test,LD50 value of the strain CPA2020 to P.clarkii was 6.31×105 CFU/mL.Detection of virulence-associated genes by PCR indicated that the isolated strain carried 8 virulence genes:lip,hly,flgN,ompA,flgM,flaH,aha and flgA.Detection of virulence-associated genes by PCR indicated that the isolated strain carried 8 virulence genes:lip,hly,flgN,ompA,flgM,flaH,aha and flgA.The results of drug resistance test showed that the strain was sensitive to 13 kinds of drugs such as aztreonam,cefuroxime and levofloxacin.The results of extracellular enzyme detection revealed that the isolated strain could secrete lecithinase,gelatinase and hemolysin.Moreover,PCR testing detected WSSV in the diseased shrimp,and artificial infection experiments showed that the mortality rate of mixed infection of A.veronii and WSSV group was higher than that of the A.veronii-infected and WSSV-infected groups.Histopathology analysis of the mixed infection group revealed that hepatopancreas and intestinal epithelial cells became necrotic and damaged,accompanied by atrophy of the intestinal connective tissue and reduction of the wrinkled ridges,which was similar with those of natural infected P.clarkia.Therefore,the cause of mass mortality was a mixed infection of A.veronii and WSSV.The results provide a theoretical basis for further studying the pathogenic mechanism of bacterial and viral mixed infections.(3)Based on the experimental study of the predation effect of Trionyx sinensis on P.clarkii,it was found that a 2m×2m× 1.2m indoor cement pool could accommodate about 40 P.clarkii as the experimental unit.The hunting time of T.sinensis concentrated in 23:00-3:00.The type of predatory functional response of T.sinensis to P.clarkii is of Holling-Il model,with a statistically significant handling time of(?),which could be used as a universal model for predation by T.sinensis on P.clarkii.The equation for predation rate of T.sinensis was E=0.1073P-0.2617.The results suggest that techniques for polyculture of P.clarkii and T.sinensis can effectively prevent the spread of WSSV in P.clarkii,achieving a biological control effect. |
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