Abstract
BackgroundTemporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes.MethodsmiRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes.ResultsThe knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults.ConclusionThis study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector.
Highlights
Temporal and stage specific expression of microRNAs in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development
Functional interventional studies Stage specific expression of three miRNAs, miR-286b, miR-2942 and miR-1891, which are up-regulated in Ae. albopictus were further evaluated in this study to explore their importance in the development of this mosquito species
Stage-specific mosquito microRNA expression Based on a previous study of microarray analysis, the functional miRNAs up-regulated or down-regulated in each of the developmental stages of mosquito expressed at female adult, male adult, larvae, pupae and embryo stages were analyzed and three important miRNAs, which were up-regulated in adults, larvae and embryo were selected for our study
Summary
Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. Despite many traditional control measures, their failure has sparked interest in several new approaches [1,2] These include the development of genetically modified mosquitoes (GMMs) designed either to reduce population sizes or to replace existing populations with vectors unable to transmit the disease [3,4]. One new approach which is in the initial stages of development has emerged with the understanding of newly discovered RNAs like microRNA (miRNA) and other short RNAs which have immense capacity to function as regulatory molecules in many essential biological functions in humans [5,6], plants [7] and all living species including insects [8,9] which are important vectors for spreading many diseases. Differential gene expression and proteomic studies have contributed significantly to our understanding of developmental, ecological and behavioral changes in insects [15,16]
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