Cup and Pan Behavioral Assays for Assessing Anopheles coluzzii Larval Volatile Responses

Larval stage Anopheles coluzzii are highly reliant on their olfactory system to locate food sources and to avoid predators and less advantageous microenvironments within their aqueous habitats. The major larval chemosensory appendage, the antenna, is a complex organ with multiple sensory components that is responsible for both gustation and olfaction, thereby facilitating the detection and of both soluble and volatile compounds of biological relevance. Such compounds include food sources, predators, and a range of environmental toxicants. Unlike other mosquitoes, Anopheles coluzzii often position themselves parallel and just under the surface of their aqueous habitats, where they can detect and respond to volatile stimuli. We describe two assays for assessing the behavioral responses of larval anophelines in response to volatile chemicals. The first is a dual-choice, water-surface, inverted-cup assay designed to behaviorally characterize the response valences (attraction, neutral, and repulsion) of anopheline larvae by monitoring and recording the distribution of larvae proximate to chemical volatiles relative to solvent controls. Second, an aqueous-based larval pan behavior assay is designed to assess the responses of mosquito larvae to soluble compounds (as well as potential headspace volatiles) that are released from a point source within larval water. Here, the response valence (attractive, neutral, and repulsive) of mosquito larvae is assessed by quantifying the numbers of larvae in predefined zones proximate to chemical sources.