Effects Of RNA Interference With 3,4-dihydroxyphenylacetaldehyde Synthase On Larval Development And Female Adult Survival Of Aedes Aegypti

Cuticle is an indispensable structure that protects insect against adverse environmental conditions and prevents pathogen entry.Insect cuticles can be divided into rigid and flexible cuticles according to their functions.Flexible cuticle plays an important role in body supporting and segment connecting,especially in the blood-sucking behavior.The biochemical characteristics and enzyme kinetics of 3,4-dihydroxyphenylaldehyde(DOPAL)synthase were studied in our laboratory.It was found that DOPAL synthase is associated with flexible cuticle formation in Ae.aegypti.However,the molecular function of DOPAL synthase in the development of mosquito has not been reported.Therefore,the transcription level of DOPAL synthase in different growth and developmental stages and tissues of Ae.aegypti was analyzed by qRT-PCR.The effects of DOPAL synthase on the larvae and adults were analyzed by feeding larvae with chitosan-coated DOPAL synthase dsRNA nanoparticles and injecting the dsRNA into adult mosquitoes.The results of qRT-PCR showed that the expression of DOPAL synthase is different during development and the transcriptional level reached its peak at the female white pupal stage.In addition,DOPAL synthase is also highly expressed in eggs and larvae of the first instar,and its expression in females is significantly higher than that in males.DOPAL synthase was more expressed in the cuticle and midgut than in other tissues of the adult.The results of interference with larvae fed with chitosan/dsRNA nanoparticles showed that feeding chitosan/dsRNA nanoparticles from the first instar larva,the development process from larva to pupa was strikingly slow down,even stagnating.A qRT-PCR analysis confirmed that the dsRNA-mediated transcription of the DOPAL synthase was reduced>50%in the fourth larval instar.Meanwhile,larval molt was abnormal during the development.Transmission electron microscopy results indicated that the formation of endocuticle and exocuticle was blocked,and the texture of the cuticles becomed irregular.In general,feeding larvae with chitosan/dsRNA nanoparticles for RNA interfere was an effective and relatively simple method for mosquito RNAi application.In addition,it was detected that the dsDOPAL synthase RNA caused significant mortality(60%)while injected into the female adult.The mortality rate of the control group was only about 10%.In general,these results revealed that DOPAL synthase plays a critical role in mosquito laval development and adult survival.It is suggested that DOPAL synthase could be a good candidate gene in intervention strategies using RNAi in mosquito control.