Study On The Insecticidal Effect Of Haedoxan A On Aedes Albopictus Based On Transcriptome Analysis And RNAi

Aedes albopictus is an important vector of of virus diseases such as dengue fever and yellow fever.It is one of the most invasive mosquitoes and poses a great threat to millions of people and animals around the world.In the past,chemical control was used to control mosquitoes.However,with the increasing problem of insecticide resistance and excessive reliance on chemical insecticides,a series of negative ecological effects in the environment,it is urgent to find alternative new environment-friendly insecticides to overcome these adverse effects.Haedoxan A（HA）is a compound isolated from（Phryma leptostachya L.）with super high insecticidal activity.Its insecticidal activity against mosquitoes and flies is close to or equivalent to pyrethroid insecticides,which has the potential to be developed as a green and efficient new botanical insecticide.Previous studies of the research group found that HA may affect the transduction of excitatory signals by acting on axon conduction and neuromuscular synaptic transmission.We have also found that metabolic enzymes in insects,especially P450 enzymes,significantly affect the insecticidal activity of HA.However,the molecular mechanism of these effects is still unclear.Based on this,this paper analyzed transcriptome data of Ae.albopictus induced by the sublethal dose of HA in the previous period,and aimed at the possible insecticidal mechanism of HA on its nerve conduction and metabolic process.Three candidate genes,acetylcholine receptor nAChR-α5 gene related to HA putative target,cytochrome P450 gene CsCYP304A1 and CsCYP4C related to HA metabolism were identified.The specific expressions ofAChR-α5 and cytochrome P450genes in different developmental stages of Ae.albopictus were analyzed.The correlation action mechanism of nAChR-α5,CsCYP304A1 and CsCYP4C genes to HA was studied by RNAi.The main results are as follows:1.The transcriptome data were analyzed and RT-q PCR was used to verify the expression of related to HA nerve signal transduction and potential metabolic detoxification genes.Three nAChRs（α2:gene 9110,α5:gene 9112,α9:gene 24585）and glutamate receptor ionoalginate 2（GRIK2,gene 6359）were up-regulated in RNA-seq(log₂^{（Fold Change）}values are2.1-3.24 times),and RT-q PCR results showed that these genes were also up-regulated（1.76～19.49 times）.And the expressions of nAChR-α5 is the most significant.The expression of CsCYP4C（gene28866）,CsCYP304A1（gene34251）,carboxylesterase 5A（gene35112）and ABC transporter（gene31706）related to detoxification of HA were up-regulated in RT-q PCR（1.52～8.07 times）,and up-regulated in transcriptional group(log₂^{（Fold Change）}values are 1.25～2.47 times).These differential genes were differentially expressed in Ae.albopictus.Functional analysis showed that the genes of Aedes albopictus were obviously enriched in signal transduction and detoxification metabolism.The related neuroreceptors and detoxification genes can be regulated by HA treatment.2.The expression of Ae.albopictus genes nAChR-α5,CsCYP304A1 and CsCYP4C in different developmental stages was detected by q PCR.nAChR-α5,CsCYP304A1 and CsCYP4C genes were expressed in different developmental stages,in which the expression of nAChR-α5 reached the peak in the 4th instar larvae,followed by female adults,and that of CsCYP304A1was 3rd instar,female adult,pupa,2nd instar,1st instar and 4th instar larvae,and there was no significant difference between pupa and female adults.The expression of CsCYP4C decreased gradually during the whole developmental period.The expression level was the highest in the 1st instar,and significantly lower in the pupae and female adults than in other stages,especially in the pupa stage.3.The function of nAChR-α5 to HA insecticidal effect was verified by RNAi technique.After feeding chitosan/dsAChR-α5 nanoparticles to 2nd instar larvae of Ae.albopictus for 24 h,the interference rate was 70%.And then,the interfered Ae.albopictus larvae were treated with LC₃₀dose of HA,it was found that the mortality rate of chitosan/dsAChR-α5 was 19.78%after 24 h which was significantly lower than that of the control group（chitosan and ds GFP RNAi）about 10%.These results indicated that the sensitivity of the Ae.albopictus treated with dsAChR-α5 to HA was significantly reduced,suggesting that nAChR-α5 subunit may be a target of HA,and HA may affect the excitatory signal transduction of synapses by acting on nAChR-α5.4.The insecticidal functions of Ae.albopictus CsCYP304A1 and CsCYP4C in HA were verified by RNAi technology.The results showed that after feeding the ds RNA nanoparticles of CsCYP304A1 and CsCYP4C,the interference efficiency against the 2nd instar larvae of Ae.albopictus in the two treatment groups was 58.21%and 54%,respectively.And then,the interfered larvae were treated with LC₃₀dose of HA,the mortality rate of the test Ae.albopictus fed chitosan/ds CsCYP304A1 was 42.86%,which was significantly increased by about 10%compared with the treatment of chitosan/ds CsCYP4C and the control.This result also suggests that CsCYP304A1 may be involved in HA metabolism in Ae.albopictus,while CsCYP4C may not be directly involved in HA metabolism.In summary,we speculate that HA may act on the nAChR-α5 subunit of Ae albopictus,affect the transmission of neurotransmitters in the nervous system.CsCYP304A1 plays an important role in the detoxification of HA by Ae albopictus.It is very likely to participate in the metabolism of HA.The possibility of Ae.albopictus CsCYP4C participating in the metabolism of HA was excluded preliminarily.