Abstract
BackgroundMicroRNAs are small non-coding RNAs that are involved in various biological processes including insect development. Anopheles stephensi serves as primary vector of malaria parasite in Asia and exhibits holometabolous life cycle that involves four different stages of development. Regulation and role of mosquito miRNAs during various stages of mosquito development remain largely unknown.MethodsHigh throughput small RNA sequencing was employed for identification and profiling of miRNAs across immature and adult stages of malaria vector, which were further validated using Northern hybridization and real time PCR. Target prediction and pathway analysis was carried out to understand the role of regulated miRNAs in insect development. Degradome sequencing was employed to identify cleaved targets of some regulated miRNAs. Loss of function strategy was employed for miR-989 to understand its probable role in female reproductive process.ResultsSmall RNA sequencing and data analysis revealed 111 and 14 known and novel miRNAs respectively across all stages of Anopheles stephensi. Nine miRNAs showed gender specific regulation across different stages of mosquito development. Analysis of miRNAs revealed regulation of 24 and 26 miRNAs across different stages of male and female mosquito development respectively. mRNA targets and significant pathways targeted by regulated miRNAs were identified for each stage of mosquito development. Degradome sequencing revealed twenty nine cleaved targets of insect miRNAs. MicroRNA-989 showed significant up-regulation in the adult female as compared to adult male mosquito. Knockdown of miR-989 expression in adult female using miRNA specific antagomir affected targets playing roles in protein binding, proteolysis and nucleic acid binding in ovary tissue of female mosquito post blood feeding.ConclusionsThis is the first comprehensive effort to understand regulation of Anopheles stephensi miRNAs across developmental stages of male and female mosquito. Preliminary role of regulated miRNAs in mosquito development was revealed by target prediction and pathway analysis. MicroRNA-989 emerged to have important roles in adult female mosquitoes showing significant up-regulation which was further studied using miR-989 specific antagomir. This study provides insights into mosquito development and reproductive process and has implications for effective control of mosquito population required for reducing spread of mosquito-borne infectious diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0772-y) contains supplementary material, which is available to authorized users.
Highlights
MicroRNAs are small non-coding RNAs that are involved in various biological processes including insect development
MicroRNA dynamics was studied during metamorphosis of both male and female An.stephensi mosquito
Six small RNA libraries enriched in miRNAs were constructed from larva male (LM), larva female (LF), pupa male (PM), pupa female (PF) adult male (AM) and adult female (AF) mosquitoes
Summary
MicroRNAs are small non-coding RNAs that are involved in various biological processes including insect development. Eggs laid by blood fed female mosquitoes hatch into larvae that metamorphosis into pupae and to imago or adult mosquitoes. These stages exhibit distinctive morphological and physiological differences as depicted by their choice of different ecological niches. Larva grows and periodically sheds its old cuticle by the process of ecdysis. This process results in larval growth through successive instar to reach fourth instar stage of development. Pupa is a non feeding inactive stage during which reconstruction of histolysed tissue results in formation of organs of an adult mosquito
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