| Cholesterol is an essential nutrient that insects must obtain from food,and NPC1 gene was found to play an important role in cholesterol transport and absorption.However,no study on NPC1 in H.vigintioctopunctata or other lepidopteran insects was reported so far.In the current study,two species of 28-spotted ladybeetles were firstly identified.Then the sequence of H.vigintioctopunctata NPC1 gene was cloned.The bioinformatic characteristics and the expression patterns in different development stages and different tissues of H.vigintioctopunctata were analyzed.Moreover,the function of NPC1 a gene was explored by RNA interference technology and the potential of NPC1 a gene in controlling H.vigintioctopunctata was evaluated.1.Gene clone,and spatial and temporal-specific expression of H.vigintioctopunctata NPC1 a geneWe confirmed that the insect we used was H.vigintioctopunctata according to the larval morphology and DNA molecular identification.H.vigintioctopunctata NPC1 gene was then screened out from the transcriptomes of the larva and adult.The open reading frame of the gene is 4020 bp,which encodes a protein of 1340 amino acids,137.44 k D.The protein has the three conserved domains of NPC family proteins: sterol-sensing domain(SSD),cholesterol-binding domain(NPC1＿N),and Patched domain.The NPC1 phylogenetic tree showed that the gene was clustered with the NPC1 a from other Coleopteran insects.Finally,q RT-PCR was used to characterize the expression of NPC1 a in different developmental stages and tissues.The results showed that H.vigintioctopunctata NPC1 a expressed at all developmental stages and different tissues.The expression level was higher in the early stage of H.vigintioctopunctata,e.g.,the eggs,the 1st instar larvae,the 2nd instar larvae,and the3 rd instar larvae,with the highest level detected in the 3rd instar.The expression gradually decreased in the 4th instar,pupal stage,and the adults.The expression in the gut was significantly higher than cuticle and head,where its expression was 12.8-fold as high as that of the cuticle,and 5.6-fold as high as the head.2.Evaluation of RNA interference against H.vigintioctopunctata using NPC1 a as the targetTwo methods of expressing dsRNA were used in this study.In the first experiment,we synthesized dsRNA in vitro.The results showed that NPC1 a transcription levels of the ds NPC1 a treatment groups fed with 50ng/μL and 500ng/μL decreased by about 43% and58%,respectively.However,there was no significant change in the survival rate and developmental time.We then used the concentration of 50ng/μl in the following experiment.We fed the 1st instar insects with 50ng/μl ds NPC1 a for 4 days and 7 days,respectively.The developmental time to adulthood of the insects treated by 7 days extended significantly by1.2 days in comparison to the control.In the second experiment,we adopted VIGS technology to knock down the tomato PDS gene.The albino phenotype due to photo-bleaching was used as an indication of successful silence and the reference for establishing a dsRNA system for transient expression of target gene.The 1st instar larvae were transferred on the leaves expressing the target gene ds NPC1 a.We found that the expression of NPC1 a gene in the insects fed on these leaves was significantly reduced by about 54% in comparison to the control after 10 days of feeding.However,no significant difference in larval weight and developmental time was detected.These results indicated that the expression of NPC1 can be significantly knocked down by RNAi but the decrease was not as high as other coleopteran insects reported.This may lead to the insignificant difference on growth and development.Moreover,a cholesterol absorption mechanism independent of the NPC1 gene may exist in insects so the deficiency in NPC1 a by RNAi may be compensated by the unidentified pathway.In the future,we can use other method to achieve loss of function of NPC1 a in H.vigintioctopunctata to clarify if the gene can be a good target for pest control. |
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