Toxicity And Detoxification Enzyme Genes Transcriptome Analysis Of P-Benzoquinone In Oral Secretions Produced By Rhynchophorus Ferrugineus Larvae

As an invasive pest of palm plants,red palm weevil?RPW?,Rhynchophorus ferrugineus Olivier,can secrete external secretions with p-benzoquinone as the main active ingredient.The secretion can provide an antimicrobial coating to the cuticle and resists the invasion of pathogens and other foreign substances.However,due to the toxicity of p-benzoquinone,RPW may also face the risk of self-poisoning.Therefore,RPW requires precise regulation?including synthesis and metabolism?of p-benzoquinone in oral secretions to protect itself from its toxicity.In this study,the RPW larvae were used as research object,and Metarhizium anisopliae var.anisopliae was used to induce external immunity of RPW.Upon induction,the mass of oral secretions,the concentration of p-benzoquinone and the antimicrobial activity of secretions were measured.In addition,toxicity of p-benzoquinone against third instar larvae of RPW was also evaluated.Finally,transcriptome technology was employed to screen the genes related to detoxification of p-benzoquinone and elucidate the detoxification mechanism of p-benzoquinone by the larvae.The main findings are as follows:1.M.anisopliae induces a significant amount of oral secretions from the third instar larvae of RPW.Counterpart with the control group,the oral secretions of the third instar larvae under stress with M.anisopliae for 12h and 24 h post-treatment increased by 0.56 times and 0.52 times,respectively.With the increase of treatment time,the amount of oral secretions in the stress group and the control group increased by 0.48 times and 0.19 times,respectively.In addition,the oral secretions of the seventh instar larvae treated with 0.1%tween 80 were significantly more?54.99±4.35 mg?than the third-instar larvae?13.23±0.68 mg?,indicating a positive correlation between the amount of oral secretions and age of RPW larvae.2.The concentration of p-benzoquinone in oral secretions of the third instar larvae was decrease significantly with the increase of secretions.At48 h post-treatment compared to 12 h post-treatment,the concentration of p-benzoquinone in the secretions of third instar larvae under 0.1%tween80 and M.anisopliae stress decreased by 50.71%and 24.96%,respectively.This indicated that the stress of M.anisopliae reduced the amplitude of the reduction in the p-benzoquinone concentration to enhance the host immunity.In addition,the concentration of p-benzoquinone in oral secretions of third instar larvae and seventh instar larvae treated with 0.1%tween 80 was 6.20±0.32?g/m L and 4.72±0.35?g/m L,respectively,and there was no significant difference between the two groups.3.The antimicrobial activity of oral secretions of the RPW third instar larvae increased significantly with the increase of p-benzoquinone concentration.After 24 h of M.anisopliae stress,the antibacterial activity of oral secretions of the third instar larvae enhanced significantly by32.19%.This indicated that not only the concentration of p-benzoquinone in the secretions but also the stress of M.anisopliae both affect the antimicrobial activity of oral secretions of third instar larvae.4.After the treatment with p-benzoquinone,the third instar RPW larvae lost the molting ability,part or the whole body eventually becomes blackened,and finally the individuals died.The LC₅₀ was 6.98×10³?g/m L.The concentration of p-benzoquinone in secretions of third instar larvae treated with 0.1%tween 80 and 1×10⁵ M.anisopliae spore/m L was 5.91±0.59?g/m L and 6.05±0.28?g/m L,respectively,which were both far less than LC₁₀?4.91×10³?g/m L?.This indicated that the larvae could finely regulate the concentration of p-benzoquinone in the secretion.Moreover,the transcriptome results showed that under treatment with p-benzoquinone,there were three upregulated genes in the CYP450 family with the maximum upregulated amount log₂^FCm=3.42,two down-regulated gene and seven upregulated genes in the ATP binding cassette transporter superfamily(log₂^FCm=4.29).The differential expression of these genes indicated that the third instar larvae had the detoxification and metabolism properties of p-benzoquinone in external secretion by regulating the detoxification enzyme genes of P450,GST and ABC families.In summary,The higher the secretion of the RPW larvae oral secretion,the lower the concentration of p-benzoquinone in the secretion and the lower the antibacterial activity,and by regulating the expression of cytochrome P450,glutathione sulfur transferase,ATP binding cassette transporter and other detoxification metabolic enzyme genes to metabolize the p-benzoquinone that are secreted into the environment and avoid self-poisoning.