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Summary: Mosquito behavioral assays are an important component in vector research and control tool development. Here, we present a protocol for rearing Anopheles mosquitoes, performing host-seeking behavioral bioassays, and collecting 3D flight tracks in a large wind tunnel. We describe steps for setting up host-seeking landing assays, both as a non-choice and as a dual-choice assay, and analyzing flight tracks. This protocol can be applied in the research of several behavioral traits, including nectar seeking, resting, mating, and oviposition behavior.For complete details on the use and execution of this protocol, please refer to Carnaghi et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Malaria prevention relies on mosquito control interventions that use insecticides and exploit mosquito behavior. The rise of insecticide resistance and changing transmission dynamics urgently demand vector control innovation. To identify behavioral traits that could be incorporated into such tools, we investigated the flight and landing response of Anopheles coluzzii to human-like host cues. We show that landing rate is directly proportional to the surface area of thermal stimulus, whereas close-range orientation is modulated by both thermal and visual inputs. We modeled anopheline eye optics to theorize the distance at which visual targets can be detected under a range of conditions, and experimentally established mosquito preference for landing on larger targets, although landing density is greater on small targets. Target orientation does not affect landing rate; however, vertical targets can be resolved at greater distance than horizontal targets of the same size. Mosquito traps for vector control could be significantly enhanced by incorporating these features.

We used the Photonic Fence Monitoring Device (PFMD) to evaluate orientation by Musca domestica L. (Diptera: Muscidae), and Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) to the Zevo Flying Insect Trap Model 3. The PFMD's dual cameras record infrared light (IR) reflected from a wavelength-specific fabric; insects do not reflect IR at the same wavelength and are tracked in 3 dimensions as moving objects. The plug-in trap emits ultraviolet and blue light from behind an opaque shield; attracted insects enter the space between the shield and the wall and are trapped on a sticky cartridge facing the wall. An experiment (N  = 10) with replicates of approximately 40, 1- to 7-day-old mixed-sex flies of each species was conducted in a 6.1 m3 arena. Prior to turning the trap on, the fly movement was not directed toward the trap on the back wall of the arena, regardless of whether the overhead light was on or off. When the overhead light was off, the mean first catch of both species occurred within 5 min after the trap was turned on, and 33.1% and 41.8% of M. domestica and C. vicina , respectively, were caught. House flies flew toward the trap, many approaching from below, while C. vicina apparently walked or flew outside the field of view of the PFMD until they appeared on the vertical reflective surface, and then walked toward the trap from all directions. Our results show that the Zevo Trap attracts and catches flies, and that the PFMD can be used to track flying and walking flies. [ABSTRACT FROM AUTHOR]

Background: Mosquito-borne diseases are a global health problem, causing hundreds of thousands of deaths per year. Pathogens are transmitted by mosquitoes feeding on the blood of an infected host and then feeding on a new host. Monitoring mosquito host-choice behaviour can help in many aspects of vector-borne disease control. Currently, it is possible to determine the host species and an individual human host from the blood meal of a mosquito by using genotyping to match the blood profile of local inhabitants. Epidemiological models generally assume that mosquito biting behaviour is random; however, numerous studies have shown that certain characteristics, e.g. genetic makeup and skin microbiota, make some individuals more attractive to mosquitoes than others. Analysing blood meals and illuminating host-choice behaviour will help re-evaluate and optimise disease transmission models. Methods: We describe a new blood meal assay that identifies the sex of the person that a mosquito has bitten. The amelogenin locus (AMEL), a sex marker located on both X and Y chromosomes, was amplified by polymerase chain reaction in DNA extracted from blood-fed Aedes aegypti and Anopheles coluzzii. Results: AMEL could be successfully amplified up to 24 h after a blood meal in 100% of An. coluzzii and 96.6% of Ae. aegypti, revealing the sex of humans that were fed on by individual mosquitoes. Conclusions: The method described here, developed using mosquitoes fed on volunteers, can be applied to field-caught mosquitoes to determine the host species and the biological sex of human hosts on which they have blood fed. Two important vector species were tested successfully in our laboratory experiments, demonstrating the potential of this technique to improve epidemiological models of vector-borne diseases. This viable and low-cost approach has the capacity to improve our understanding of vector-borne disease transmission, specifically gender differences in exposure and attractiveness to mosquitoes. The data gathered from field studies using our method can be used to shape new transmission models and aid in the implementation of more effective and targeted vector control strategies by enabling a better understanding of the drivers of vector-host interactions. [ABSTRACT FROM AUTHOR]