Study On The Effect Of Areca Catechu Extract On Preventing And Controlling Enterocytozoon Hepatopenaei Infection Of Litopenaeus Vannamei And Transcriptome Analysis

Since the discovery of Enterocytozoon hepatopenaei(EHP)in Penaeus monodon cultured in Thailand in 2003,its parasitic nature,which requires energy from the host,has greatly limited the growth and development of the host,causing huge economic losses to the shrimp farming industry.In this study,we investigated the effect of Areca catechu extract on the prevention and treatment of Litopenaeus vannamei infected with EHP by the following three aspects:the physiological index trends of shrimp body mass and body length were examined by pre-infection with shrimp hepatopancreas and treatment with Areca catechu extract;the quantitative real-time PCR(q PCR)standard curve was established to quantify the changes of hepatopancreas EHP copy number before and after the administration of Areca catechu extract.The hepatopancreas pathological characteristics were observed in combination with Masson sections;the transcriptome analysis of the hepatopancreas of Litopenaeus vannamei before and after the administration of Areca catechu extract was performed to screen the differential genes and evaluate the prevention and control effect of Areca catechu extract.1.Hepatopancreas grinds containing EHP were poured into the shrimp rearing bucket and the EHP copy number was 1×10~3 copies/μg by q PCR after 48 h.At this time,the infected shrimp of uniform size were selected and divided into two groups,one group was maintained in the infected shrimp hepatopancreas state and recorded as the infected group,and one group was the shrimp infected with shrimp hepatopancreas and given Areca catechu extract after the infection and recorded as the Administration group.The healthy Litopenaeus vannamei were recorded as the control group.The effect of Areca catechu extract on the non-specific immune performance of the shrimp was analyzed by comparing the physiological response of the shrimp under different treatments by adding betel nut extract to the shrimp culture water before and after EHP infection,respectively.The results showed that the administration of Areca catechu extract significantly improved the growth performance of shrimp after 14 days.The growth rate of body weight was 13.41%and 23.37%,and the growth rate of body length was 4.07%and 9.07%in the administered group on day 7 and 14 compared to day 1 of the experiment.In contrast,the growth rate of the infected shrimp was 7.69%and 10.99%in body weight and 3.87%and 5.66%in length on day 7 and 14.Compared to the healthy control shrimp,the shrimp supplemented with betel nut extract recovered to the same body mass and body length,while the infected shrimp were significantly different from the control group.The same trend of body weight and body length was observed throughout the test period,with increasing total hemocyte counts and hemocyanin concentration in all groups of shrimp as the rearing time increased.The total hemocyte counts and hemocyanin concentrations of the shrimp in the treated group were basically restored to the level of the control group by the seventh day of the experiment,while the infected group was significantly different from the control group.The addition of Areca catechu extract to the water column of Litopenaeus vannamei after EHP infection actively mobilized non-specific immunity against EHP infection.2.Quantitative real-time PCR was performed by constructing a standard curve corresponding to the Cq value of the standard plasmid copy number,determining the recombinant plasmid DNA concentration of 109 ng/μL,and using the specific primers SWP-F514 and SWP-R514 targeting the spore wall protein gene fragment as templates to determine the infected and administered groups of van the hepatopancreas EHP copy number of the infected and administration shrimp was determined.On day 1,there was no significant difference in the copy number between the infected and control group shrimp.On days 7 and 14,the EHP copy number of infected shrimp was 17.93 and90.52 times higher than that of the administered group shrimp.rupture,and basically no EHP spore parasitism was observed in the cells after 14 days of administration of the extract.3.Illumina high-throughput sequencing analysis was performed on the healthy control,infected and administered group shrimp,56634546,52905990 and 600482292raw data were obtained,respectively.55221626,51745408 and 58425750 clean reads were obtained after data filtering and elimination of low quality bands.The 23,783obtained genes were compared with nine databases,and the number of successfully annotated genes was 21641,accounting for 90.99%.A total of 530 differentially expressed genes were screened in the control,infected and administered group shrimp hepatopancreas,of which 315 genes were up-regulated and 215 genes were down-regulated.A total of 17,946 genes were annotated to cellular component,biological process and molecular function categories in GO annotation.In the KEGG pathway analysis,there were 4397 differentially expressed genes involving 18 metabolic pathways in the control and administration groups.In contrast,there were 4559differentially expressed genes involving 17 metabolic pathways in the control and infected groups.There were 2282 differentially expressed genes involving 4 metabolic pathways in the administration and infection groups.By analyzing genes such as Tenascin and MFS transporter,Taurine and hypotaurine metabolism,ECM-receptor interaction and other pathways,it is possible to understand the response mechanism of Litopenaeus vannamei in mobilizing genes in response to EHP infection under the effect of Areca catechu extract,which can provide ideas for shrimp disease control and reduce the economic loss of farmers.