ABSTRACT
Parental RNAi (pRNAi) is a response of RNA interference in which treated insect pests progenies showed a gene silencing phenotypes. pRNAi of CmGNA gene has been studied in Cnaphalocrocis medinalis via injection. Our results showed significant reduction in ovulation per female that was 26% and 35.26% in G1 and G2 generations, respectively. Significant reduction of hatched eggs per female were observed 23.53% and 45.26% as compared to control in G1-G2 generations, respectively. We also observed the significant variation in the sex ratio between female (40% and 53%) in G1-G2 generations, and in male (65%) in G1 generation as compared to control. Our results also demonstrated the significant larval mortality (63% and 55%) and pupal mortality (55% and 41%), and significant reduction of mRNA expression level in G1 and G2 generations. Our findings have confirmed that effectiveness of pRNAi induced silencing on the CmGNA target gene in G1-G2 generations of C. medinalis. These results suggested the potential role of pRNAi in insect pest resistance management strategies.
Subject(s)
Glucosamine 6-Phosphate N-Acetyltransferase/genetics , Insect Control/methods , Moths/genetics , RNA Interference , Animals , Female , Male , Phenotype , ReproductionABSTRACT
The rice leaf folder, Cnaphalocrocis medinalis is a major pest of rice and is difficult to control. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin synthesis pathway in insects. In this study, the UAP gene from C. medinalis (CmUAP) was cloned and characterized. The cDNA of CmUAP is 1788 bp in length, containing an open reading frame of 1464 nucleotides that encodes 487 amino acids. Homology and phylogenetic analyses of the predicted protein indicated that CmUAP shared 91.79%, 87.89%, and 82.75% identities with UAPs of Glyphodes pyloalis, Ostrinia furnacalis, and Heortia vitessoides, respectively. Expression pattern analyses by droplet digital PCR demonstrated that CmUAP was expressed at all developmental stages and in 12 tissues of C. medinalis adults. Silencing of CmUAP by injection of double-stranded RNA specific to CmUAP caused death, slow growth, reduced feeding and excretion, and weight loss in C. medinalis larvae; meanwhile, severe developmental disorders were observed. The findings suggest that CmUAP is essential for the growth and development of C. medinalis, and that targeting the CmUAP gene through RNAi technology can be used for biological control of this insect.
Subject(s)
Cloning, Molecular/methods , Moths/growth & development , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Animals , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Inactivation, Metabolic , Insect Proteins/genetics , Insect Proteins/metabolism , Moths/enzymology , Moths/genetics , Open Reading Frames , Oryza/parasitology , RNA Interference , Tissue DistributionABSTRACT
Hexokinase (HK) is a key enzyme in chitin biosynthesis in insects and plays an important role in development and energy regulation. It also performs a crucial role in the synthesis of Glucose-6-phosphate and its putative functions are studied via injection of dsRNA corresponding to the hexokinase gene from Cnaphalocrocis medinalis (CmHK). This study was designed to analyze the characteristics and expression patterns of HK-related genes in various tissues of C. medinalis at different developmental stages. The CmHK ORF is a 1359 bp in length, encoding a protein of 452 amino acids, with homology and cluster analysis showing that CmHK shares an 85.11% sequence similarity with hexokinase from Ostrinia furnacalis.CmHK was highly expressed in the ovary and in the fifth instar larvae. Injection of dsCmHK significantly suppressed mRNA expression (73.6%) 120 h post-dsRNA injection as compared to a control group. The results demonstrated an increased incidence of larval and pupal mortality of 80% and 78%, respectively, with significant variation in the sex ratio between males (68.33%) and females (35%), overt larval deformities, and a reduction in average weight gain observed 120 h post-dsRNA injection. In addition, dsCmHK-injected C. medinalis showed a significant reduction in ovulation per female and larval hatching rate, along with increased larval and pupal mortality and variation in male and female emergence over three generations (G1, G2, and G3). Taken together, the outcomes of the study provide a foundation to study gene function and a new dimension to control C. medinalis by transgenic RNAi technology.
