A toolbox of engineered mosquito lines to study salivary gland biology and malaria transmission

Abstract

Mosquito saliva is a vehicle for the transmission of vector borne pathogens such as Plasmodium parasites and different arboviruses. Despite the key role of the salivary glands in the process of disease transmission, knowledge of host-pathogen interactions taking place within this organ is very limited. To improve the experimental tractability of the salivary glands, we have generated fluorescent reporter lines in the African malaria mosquito Anopheles coluzzii using the salivary gland-specific promoters of the anopheline antiplatelet protein (AAPP), the triple functional domain protein (TRIO) and saglin (SAG) coding genes. Promoter activity was specifically observed in the distal-lateral lobes or in the median lobe of the salivary glands. Besides a comparison of the expression patterns of the selected promoters, the fluorescent probes allowed us to evaluate the inducibility of the selected promoters upon blood feeding and to measure intracellular redox changes. We also combined the aapp-DsRed fluorescent reporter line with a pigmentation-deficient yellow(-) mosquito mutant to assess the feasibility of in vivo microscopy of parasitized salivary glands. This combination allowed locating the salivary gland through the cuticle and imaging of individual sporozoites in vivo, which facilitates live imaging studies of salivary gland colonization by Plasmodium sporozoites. Author summary Over the course of evolution, a number of pathogens have developed the ability to replicate within and to be transmitted by mosquitoes. An important adaptation of pathogens to be successfully transmitted is their ability to colonize mosquito slivary glands, as the timing and efficiency with which pathogens enter the glands directly affects their transmission. Despite the importance of salivary glands in disease transmission, host-pathogen interactions in this organ are poorly understood. To facilitate biological studies, we developed fluorescent reporter lines in the malaria mosquito Anopheles coluzzii by placing fluorescent probes under the control of the salivary gland-specific promoters of the anopheline antiplatelet protein (AAPP), triple functional domain protein (TRIO), and saglin (SAG) genes. We used these lines to characterize inducibility by blood meals and spatial expression of the different promoters, as well as intracellular redox changes in salivary glands after sporozoite invasion. In addition, we combined the aapp-DsRed fluorescent reporter line with a pigmentation-deficient yellow(-) mosquito mutant to evaluate the feasibility of in vivo microscopy of parasitized salivary glands. Our data suggest that salivary gland invasion is an active process that requires Plasmodium sporozoites to actively move through the tissues of the mosquito to reach the salivary glands.