Cloning And Functional Characterization Of Eight Vacuolar ATPase Subunit Genes In Henosepilachna Vigintioctopunctata

With the rapid development of insecticide resistance,Henosepilachna vigintioctopunctata has become increasingly harmful to Solanaceae and Cucurbitaceae plants in many Asian countries,resulting in great economic losses of crops.The National Agricultural Technology Center in China has listed the ladybird as one of the ten most damaging potato pests and diseases.Developing novel management strategies is urgent.Vacuolar（H⁺）-ATPase（vATPase）is a highly conserved enzyme and widely distributed in the intracellular membrane system of prokaryotic and eukaryotic organisms.In insects,the vATPaseis located in the apical membrane of epithelial cells of Malpighian tubules,midgut,hindgut,salivary gland and sensory ganglion.It can produce transmembrane electrochemical proton gradient.The electrochemical proton gradient then motivates transmembrane transport of othr ions and chemicals.Therefore,vATPaseplays critical roles in many physiological processed,it is essential during larval growth and metamorphosis.In this survey,we identified eight vATPasesubnit genes from the transcriptome data of H.vigintioctopunctata.Using Escherichia coli HT115（DE3）-produced ds RNA,we intended to explore the physiological importance of vATPasesubunits in H.vigintioctopunctata.Moreover,we planed to evaluate whether these subunit genes are potential targets for the exploitation of RNAi biological pesticides.The main results are listed as follows.1.Molecular cloning and expression pattern analysis of vATPasesubunit genesBased on the annotation information of transcripts in the transcriptome data of H.vigintioctopunctata,we obtained the candidate gene sequences of the eight subunits of vATPase.Using the full-instar c DNA as the template,the complete ORF sequence of each subunit gene was cloned.Multiple alignment of amino acid sequences ande phylogenetic analysis revealed that these vATPasesubunit genes encoded vATPaseB,vATPaseC,vATPaseE,vATPaseF,vATPaseG,vATPaseH in the V₁ complex,and vATPasea and vATPased in V₀ complex.Real-time fluorescence quantitative PCR（q RT-PCR）was used to detect the expression of the eight vATPasesubunit transcripts in different developing stages and various tissues of H.vigintioctopunctata.The results showed that the expression peaks of all the eight vATPasesubunit transcripts occurred in the final instar larvae.Moreover,each vATPasesubunit transcript was abundantly expressed in the foregut,midgut,hindgut and Malpighian tubule.Therefore,disturbing the function of vATPasesubunits may have a significant impact on the growth and development in the H.vigintioctopunctata larvae.2.Silencing vATPasegene delays larval growth and impairs ecdysisContinuous feeding E.coli-produced ds RNA for three days successfully lowered the target m RNA.RNAi of each of the eight vATPasesubunit transcripts reduced consumption of potato foliage in H.vigintioctopunctata.When silencing the target vATPasesubnit gene at the final-instar stage,the fresh weights were significantly decreased.Moreover,RNAi for vATPasesubunit transcripts impaired larva-pupa-adult transformation.When silencing HvvvATPasea,HvvvATPased,and HvvvATPaseC in the third instar larva,the mortality rates were 100%.When silencing HvvATPased and HvvATPaseE in the final instar,the mortality rate reached 100%.When scilencing the other vATPasesubunit genes,more than half of the resultant insects failed to pupate,or failed to emerge as adult.The others became deformed adults and finally died one week later.3.The toxic modes of silencing vATPasesubnit genes in H.vigintioctopunctataIn order to explore the the toxic modes of silencing vATPasesubnit transcripts in H.vigintioctopunctata,the contents of main nutrients in the final instar larvae were measured after scilencing either HvvATPaseF or HvvATPaseG.The results showed that,glycogen content increased significantly,while glucose,trehalose,triglyceride,protein and total amino acid content content decreased significantly compared with the control group.Moreover,the transcription of Hv TRE1a,Hv TRE1b and Hv TRE2 were significantly downregulated in hypomorphs.The expression level of the chitin synthesis gene UAP was decreased;the depth of cuticle in the head capsule was reduced.We examined the intestinal integrity of the resuntant larvae after silencing either HvvATPaseF or HvvATPaseG.Compared with the control group,after 3-4 days of treatment,the length and width of the guts were significantly decreased.HE staining showed that the principal cells of the midgut were sparse,with abnormal forms.Many dying cells separated from the midgut epithelium and moved to the lumens.Meanwhile,TUNEL positive and the red Ed U-positive cells were detected in the midguts.This indicated that RNAi of vATPasesubnit genes greatly triggered the apoptosis of the midgut epithelium cells.In order to restore the basic functions of the midgut,intestinal stem cells actively divide to replace dying absorptive and secretory cells.Even so,the integrity of the midgut cannot be restored.In addition,the peritrophic membrane in the midgut disappeared in the HvvATPaseF or HvvATPaseG RNAi beetles.Microscopic observation showed that the Malpighian tubules were more transparent and slender than the control group after 3-4 days of the ds RNA injestion.HE staining showed that the Malpighian tubules in the control group were filled with urine,while most of the Malpighian tubules were hollow in the HvvATPaseF or HvvATPaseG RNAi beetles.Given that vATPasedrives K⁺accumulation to promote urine formation,knockdown of either HvvATPaseF or HvvATPaseG can inhibit the formation of primary urine and lead to hollow Malpighian tubules.Our results here also demonstrated that vATPasesubunit genes are potential targets for development of novel insecticides.Moreover,the findings imply the feasibility of RNAi as an alternative method for controlling this critical potato pest.