Field Validation of a Transcriptional Assay for the Prediction of Age of Uncaged Aedes aegypti Mosquitoes in Northern Australia

Abstract

New strategies to eliminate dengue have been proposed that specifically target older Aedes aegypti mosquitoes, the proportion of the vector population that is potentially capable of transmitting dengue viruses. Evaluation of these strategies will require accurate and high-throughput methods of predicting mosquito age. We previously developed an age prediction assay for individual Ae. aegypti females based on the transcriptional profiles of a selection of age responsive genes. Here we conducted field testing of the method on Ae. aegypti that were entirely uncaged and free to engage in natural behavior. We produced “free-range” test specimens by releasing 8007 adult Ae. aegypti inside and around an isolated homestead in north Queensland, Australia, and recapturing females at two day intervals. We applied a TaqMan probe-based assay design that enabled high-throughput quantitative RT-PCR of four transcripts from three age-responsive genes and a reference gene. An age prediction model was calibrated on mosquitoes maintained in small sentinel cages, in which 68.8% of the variance in gene transcription measures was explained by age. The model was then used to predict the ages of the free-range females. The relationship between the predicted and actual ages achieved an R² value of 0.62 for predictions of females up to 29 days old. Transcriptional profiles and age predictions were not affected by physiological variation associated with the blood feeding/egg development cycle and we show that the age grading method could be applied to differentiate between two populations of mosquitoes having a two-fold difference in mean life expectancy. The transcriptional profiles of age responsive genes facilitated age estimates of near-wild Ae. aegypti females. Our age prediction assay for Ae. aegypti provides a useful tool for the evaluation of mosquito control interventions against dengue where mosquito survivorship or lifespan reduction are crucial to their success. The approximate cost of the method was US$7.50 per mosquito and 60 mosquitoes could be processed in 3 days. The assay is based on conserved genes and modified versions are likely to support similar investigations of several important mosquito and other disease vectors. Once infected with dengue virus, a female Aedes aegypti mosquito must survive longer than twelve days before it can transmit the virus to an uninfected person. New dengue control strategies therefore aim to circumvent dengue transmission using entomopathogenic microorganisms that shorten mosquito lifespan. Accurate methods to determine the age of individual mosquitoes are required for these and other mosquito control interventions. We have previously shown that mosquito age can be predicted from the transcription of specific genes. Here we demonstrate that this can be achieved under natural conditions when mosquitoes are uncaged and free to engage in natural behavior. To do this, we produced “free-range” female mosquitoes by releasing 8007 mosquitoes at an isolated location and recapturing the females of known ages. We developed an age prediction model from gene transcription measures of mosquitoes maintained in small “sentinel cages” maintained onsite. We then used this model to predict the ages of the free-range mosquitoes, based on their own transcription measures. Age predictions were robust to physiological changes associated with blood feeding and egg development. We show that the technique could be applied to identify a 50% reduction in mosquito population survival that is expected from infection with entomopathogenic Wolbachia bacteria.