RNA Inferference For Chitin Deacetylase Gene Impairs Larvel Development In Henosepilachna Vigintioctopunctata

Henosepilachna vigintioctopunctata is a major pest of Solanaceae and Cucurbitaceae vegetables in Asia.It is necessary to develop new management methodsto control the beetle.RNA interference(RNAi)could be a powerful tool for pest control.Identification of potential amenable target genes is the first step.Chitin is the main component of the insect epidermis and midgut perimetrophic membrane.Precise control of the synthesis and degradation of chitin is critical for insect growth and metamorphosis.In the present paper,we focused on chitin deacetylase(CDA)genes.Three CDA genes,CDA2 b,CDA3 and CDA4,were identified in H.vigintioctopunctata.Using RNAi,the effects of the three CDA genes on the metamorphosis of H.vigintioctopunctata were compared.Moreover,the feasibility of foliar spraying dsRNA on the control of H.vigintioctopunctata was evaluated.The major findings were summarized as follows.1.Identification and expression determination of putative CDA genesBased on transcriptome data and phylogenetic analysis,three CDA genes were identified in H.vigintioctopunctata.According to their partners from other insect species,the three CDA genes were named as CDA2 b,CDA3 and CDA4.Their temporal expression profiles were determined by RT-q PCR.The results showed that the three CDA genes were expressed at all developmental stages,from egg to adult.The expression peak of CDA2 b appeared in the prepupal and the pupal stages.The expression of CDA3 peaked in the newly-hatched neonates and the 3rd day of the 4th instar larvae.CDA4 was highly expressed at the 1st day of 4th instar,pupal and adult stages.Their tissue expression patterns were also measured.The three CDA genes were expressed in the foregut,midgut,hindgut,Malpighian tubules,fat body and epidermis of the 4th instar larvae.The highest expression level of CDA2 b was in the hindgut and epidermis,followed by those in the foregut and midgut,and the lowest level was recorded in the fat body.The expression level of CDA3 was higher in the foregut,hindgut and epidermis,followed by those in the midgut and Malpighian tubules,and the lowest content was in fat body.CDA4 was highly expressed in the epidermis,intermediately transcribed in the midgut and hindgut,and lowly expressed in the foregut and Malpighian tubules.2.Knockdown of CDA2 b impairs larval-pupal-adult transformationThe RNAi efficacies of two dsRNA delivery methods,injection and ingestion,were compared,using in vitro biosynthesis and Escherichia coli HT115(DE3)-produced dsRNA respectively.Introduction of dsRNA derived from CDA2 b,CDA3 or CDA4 by injection significantly reduced corresponding transcript.However,the negative RNAi effects were observed only in the CDA2 b hypomorphs.Compared with the control group,the development duration was significantly prolonged,and the pupation and adult emergence rates were significantly decreased in the CDA2 b deplted beetles.Fifteen days after treatment,100% of the resultant larvae failed to pupate,and died as prepupae.As for the larvae having ingested E.coli-produced dsCDA2 b for 3 days,the target m RNA level was significantly decreased.Moreover,consumption of E.coli-produced dsCDA2 b delayed larval development,and impaired pupation and adult emergence.More than 85% of the resulting beetles died.3.Foliar spray of dsCDA2 b causes similar defective larval phenotypesTo detect the RNAi effciency of foliar-sprayed dsRNA on the growth and development of H.vigintioctopunctata larvae,a solution containing E.coli-produced dsCDA2 b was sprayed to potato seedlings and the H.vigintioctopunctata larvae were transferred to the treated plants.The results showed that the mortality of ladybird larvae increased by 80% compared with the control group 15 days after spraying.Larval development was inhibited and the defective phenotypes were similar to those in the bioassays introducing dsCDA2 b by injection or ingestion method.Therefore,foliar spraying dsCDA2 b displayed a good control effect on H.vigintioctopunctata larvae.Hv CDA2 b is a potential amenable target gene for RNAi-based control strategies in H.vigintioctopunctata.