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Background: Malaria is the most important vector-borne disease in the world. Epidemiological and ecological studies of malaria traditionally utilize detection of Plasmodium sporozoites in whole mosquitoes or salivary glands by microscopy or serological or molecular assays. However, these methods are labor-intensive, and can over- or underestimate mosquito transmission potential. To overcome these limitations, alternative sample types have been evaluated for the study of malaria. It was recently shown that Plasmodium could be detected in saliva expectorated on honey-soaked cards by Anopheles stephensi, providing a better estimate of transmission risk. We evaluated whether excretion of Plasmodium falciparum nucleic acid by An. stephensi correlates with expectoration of parasites in saliva, thus providing an additional sample type for estimating transmission potential. Mosquitoes were exposed to infectious blood meals containing cultured gametocytes, and excreta collected at different time points post-exposure. Saliva was collected on honey-soaked filter paper cards, and salivary glands were dissected and examined microscopically for sporozoites. Excreta and saliva samples were tested by real time polymerase chain reaction (RT-rtPCR)., Results: Plasmodium falciparum RNA was detected in mosquito excreta as early as four days after ingesting a bloodmeal containing gametocytes. Once sporogony (the development of sporozoites) occurred, P. falciparum RNA was detected concurrently in both excreta and saliva samples. In the majority of cases, no difference was observed between the C t values obtained from matched excreta and saliva samples, suggesting that both samples provide equally sensitive results. A positive association was observed between the molecular detection of the parasites in both samples and the proportion of mosquitoes with sporozoites in their salivary glands from each container. No distinguishable parasites were observed when excreta samples were stained and microscopically analyzed., Conclusions: Mosquito saliva and excreta are easily collected and are promising for surveillance of malaria-causing parasites, especially in low transmission settings or in places where arboviruses co-circulate.

Aedes aegypti (L.) (Diptera: Culicidae) is a vector of viruses causing dengue, Zika, chikungunya, and yellow fever and subsequently pose a significant global threat to public health. While sampling live mosquitoes is useful for surveillance purposes, most traps targeting Aedes kill captured mosquitoes. The Biogents Sentinel (BGS) trap, the gold standard for capturing Ae. aegypti, is one such trap. In our study, we modified the BGS trap to increase the survival of captured Ae. aegypti by replacing the trap's catch bag with a catch pot that protects mosquitoes from desiccation by airflow. A sucrose-soaked sponge or nucleic acid preservative card can also be placed inside the pot to enhance mosquito survival and augment arbovirus detection. These modifications to the BGS significantly increased the longevity of mosquitoes captured with weekly survivals of 93% in a semifield structure and 86% in the field. These high survival rates resulted in 3.5 times more alive Ae. aegypti captured weekly in the modified BGS compared to the original BGS, despite 40% lower overall catch rates. These cheap and simple trap modifications facilitate easier specimen identification as well as experiments requiring live field-collected samples such as virus detection from mosquito saliva and excreta.

Surveillance is critical for the prevention and control of mosquito-borne arboviruses. Detection of elevated or emergent virus activity serves as a warning system to implement appropriate actions to reduce outbreaks. Traditionally, surveillance of arboviruses has relied on the detection of specific antibodies in sentinel animals and/or detection of viruses in pools of mosquitoes collected using a variety of sampling methods. These methods, although immensely useful, have limitations, including the need for a cold chain for sample transport, cross-reactivity between related viruses in serological assays, the requirement for specialized equipment or infrastructure, and overall expense. Advances have recently been made on developing new strategies for arbovirus surveillance. These strategies include sugar-based surveillance, whereby mosquitoes are collected in purpose-built traps and allowed to expectorate on nucleic acid preservation cards which are submitted for virus detection. New diagnostic approaches, such as next-generation sequencing, have the potential to expand the genetic information obtained from samples and aid in virus discovery. Here, we review the advancement of arbovirus surveillance systems over the past decade. Some of the novel approaches presented here have already been validated and are currently being integrated into surveillance programs. Other strategies are still at the experimental stage, and their feasibility in the field is yet to be evaluated.