Background

Because Aedes aegypti mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, understanding the variables that influence the vector’s ability to successfully transmit these viruses will be key in preventing disease. Intrinsic factors governing vector competence are often studied in a laboratory setting, where mosquitoes can be orally infected with arboviruses via an artificial bloodmeal. Artificial membrane feeding involves supplementing defibrinated animal blood with a virus of interest, introducing the blood into a feeding system that is heated to 37°C by a water bath, and allowing the mosquitoes to feed on the blood through a membrane such as parafilm or hog’s gut. This strategy mimics the natural route of infection for a mosquito, albeit without a skin barrier that undoubtedly contains many factors that mediate the infection process. Feeding mosquitoes on infected animals overcomes this disadvantage, but animal models for many arboviruses remain limited (Reynolds et al., 2017) or cannot induce sufficient viremia for infecting mosquitoes.

Not all Ae. aegypti that are fed with virus-containing blood will become infected. Furthermore, not all Ae. aegypti that exhibit midgut infections will be able to transmit the virus to a new host. This is because Ae. aegypti exhibit natural barriers against arbovirus infections, including midgut infection and escape barriers (MIB or MEB, respectively), as well as salivary gland infection and escape barriers (SIB and SEB, respectively) (Franz et al., 2015). Only after a virus has overcome these barriers, disseminated throughout the mosquito, and infected the saliva glands along with saliva can it be transmitted to a new host. The factors that govern these tissue barriers and mediate vector competence continue to be intensely investigated. However, MIB and MEBs can also be seen as barriers against vector competence studies investigating, for example, SIB or SEBs. Indeed, the molecular mechanisms underlying SIB and SEBs are poorly understood in comparison to MIB and MEBs, perhaps because of the difficulty acquiring enough infected salivary glands to perform such studies.

To facilitate our studies of salivary gland infection and escape barriers (Sanchez-Vargas et al., 2021), we modified and adapted a protocol originally described in Gubler and Rosen (1976) for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge. Mosquitoes also become infected with a consistent dose of virus, as compared to during blood feeding, when the infectious virus titer introduced into the mosquito is governed by the volume of ingested blood. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut (for example, as a control for MIB studies), require a disseminated infection, or investigate whether a particular virus has the ability to infect a mosquito. This protocol can be adapted and used to intrathoracically inoculate other viruses of interest, including DENV, CHIKV, or even SARS-CoV-2 (Huang et al., 2020) into a wide variety of mosquito vectors.

We do not discuss the generation and quantification of ZIKV stocks in this paper as that has been detailed in Bio-protocol elsewhere (Freppel et al., 2018).