Molecular Cloning And Functional Analysis Of Arylsulfatase B Gene In External Immunity Of Rhynchophorus Ferrugineus

Insects will prevent the pathogens in the environment from affecting themselves through the epidermal barrier or secretion when they are under microbial stress.This is an immune defense mechanism that is different from internal immune of insects.It is called external immune defense strategy.Many insects are able to synthesize and secrete chemicals with special purpose that play an important role in the survival,reproduction and spread of insects.Red palm weevil?RPW?,Rhynchophorus ferrugineus,is a major invasive pest in China,mainly harming palm plants and causing huge losses to palm planting in southern China.At present,the control of red palm weevil mainly depends on the use of chemical pesticides,but the effect is very limited.Quinones are an important part of the external immune secretion of the red flour beetle Tribolium castaneum,while arylsulfatase B can regulate the synthesis of red flour beetle and affect the antimicrobial activity of the secretions.By studying the effects of ARSB gene on the active substance of red palm weevil secretion,p-benzoquinone and antibacterial activity,the related research on its immune adaptability was introduced.By reducing its external immune potency and reducing its tolerance to pathogens in the environment,we can use biological control methods to control red palm weevil better.In this paper,the secretion of red palm weevil was induced by Metarhizium anisopliae.After that,we determined the antimicrobial activity and the content of p-benzoquinone,and analyzed the expression level of the arylsulfatase B gene.The RNA interference technique was used to interfere with the ARSB gene,and the antimicrobial activity of secretion of the red palm weevil and the effect on the change of p-benzoquinone content were detected.The following results were obtained.1.The three ARSB genes of the red palm weevil are highly expressed in the salivary glands.The relative expression of ARSB-0311 gene,ARSB-11581 gene,and ARSB-14322 gene in salivary glands were more than 5times,6 times,and 8 times higher than that of other tissues?head and integument?,respectively.2.After 24 hours of treat on the larvae of Metarhizium anisopliae,the oral secretion of larvae significantly enhanced the inhibitory activity against Escherichia coli and Staphylococcus aureus.The growth of E.coli and Staphylococcus aureus within 24 hours decreased by 20%and 14%compared with that before treat,respectively.The inhibition zone diameter of Metarhizium anisopliae was significantly greater than that of the control group.This indicates that the red palm weevil will enhance the antimicrobial activity of its own secretions after being stressed by pathogens,while the content of p-benzoquinone in the secretions has no significant change.3.After 24 hours of stress on the red palm weevil with Metarhizium anisopliae,the relative expression of ARSB-0311 gene in the head and epidermis increased to 33 times and 16 times than that before stress,respectively.There was no significant difference in the relative expression of other tissues.The relative expression of ARSB-11581 gene in the head and epidermis increased to 3 times and 9 times than that before stress,respectively.And the relative expression in salivary glands decreased by63%.The relative expression of ARSB-14322 gene in salivary glands decreased by 94%,while the expression in fat body decreased by 92%,and there was no significant change in the relative expression of other tissues.This indicates that the stress of pathogens can significantly affect the expression of ARSB gene,and the ARSB gene may be associated with the external immunity of red palm weevil.4.After 12 hours of interference,the secretion of the ARSB-14322gene was significantly increased compared with the control group,and the OD₆₀₀ value of the bacterial solution decreased by 45%in 24hours.There was no significant change in the secretion inhibitory activity of the silenced ARSB-0311 gene and ARSB-11581 gene.The secretions of the silenced ARSB-0311 gene significantly enhanced the inhibitory activity against Staphylococcus aureus,and the OD₆₀₀00 value of Staphylococcus aureus was reduced by 25%in 24 hours.After interfering with the ARSB-14322 gene,the OD₆₀₀ value of the bacterial solution decreased by 48%in 24 hours.There was no significant change in the secretion inhibitory activity after interference with the ARSB-11581 gene.Interference with ARSB-14322gene of the red palm weevil will enhance the antimicrobial activity of its own secretions,which lead to the reduction of p-benzoquinone content in the secretions.From the above results,it can be known that pathogen stress can cause changes in the expression level of ARSB gene.The changes in the expression level of the ARSB-14322 gene will regulate the antimicrobial activity of the secretions and the content of p-benzoquinone.This indicates that the ARSB gene can regulate the external immune response of the red palm weevil,which is of great significance for studying its external immune mechanism and prevention and control strategies.However,the regulation mechanism of ARSB gene is still unclear and needs to be further study.