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Anopheles gambiae is a highly anthropophilic (human-preferring) malaria vector that prefers to blood feed frequently and selectively on humans. This mosquito species exhibits a strong innate olfactory preference to seek out human scent over other animals, and certain humans over others-key behavioral traits with the potential to drive heterogeneity in biting risk and malaria transmission. Here, we describe the application of a large-scale, semi-field system in Zambia for the quantification of An. gambiae olfactory preferences toward whole body odor sourced from individual humans. We detail steps for modifying one-person canvas tents to duct odor from sleeping humans into a central, semi-field flight cage arena that is securely screened. Using this system, we describe a protocol to perform two-choice olfactory preference assays with two human volunteers using laboratory-reared An. gambiae and odor-guided thermotaxis assays that leverage infrared video tracking to quantify mosquito landings on heated targets baited with each body odor sample. This multichoice olfactory preference assay has the potential to be applied with expanded cohorts of humans to define the chemosensory basis of An. gambiae host preference and interindividual differences in human attractiveness to mosquitoes and to be used to quantify the effects of protective measures such as personal and spatial repellents on mosquito landing behavior., (© 2024 Cold Spring Harbor Laboratory Press.)

Anopheles gambiae uses its sense of smell to hunt humans. We report a two-step method yielding cell-type-specific driver lines for enhanced neuroanatomical and functional studies of its olfactory system. We first integrated a driver-responder-marker (DRM) system cassette consisting of a linked T2A-QF2 driver, QUAS-GFP responder, and a gut-specific transgenesis marker into four chemoreceptor genes (Ir25a, Ir76b, Gr22, and orco) using CRISPR-Cas9-mediated homology-directed repair. The DRM system facilitated rapid selection of in-frame integrations via screening for GFP+ olfactory sensory neurons (OSNs) in G 1 larval progeny, even at genomic loci such as orco where we found the transgenesis marker was not visible. Next, we converted these DRM integrations into T2A-QF2 driver-marker lines by Cre-loxP excision of the GFP responder, making them suitable for binary use in transcuticular calcium imaging. These cell-type-specific driver lines tiling key OSN subsets will support systematic efforts to decode olfaction in this prolific malaria vector., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The African malaria mosquito Anopheles gambiae exhibits a strong innate drive to seek out humans in its sensory environment, classically entering homes to land on human skin in the hours flanking midnight. To gain insight into the role that olfactory cues emanating from the human body play in generating this epidemiologically important behavior, we developed a large-scale multi-choice preference assay in Zambia with infrared motion vision under semi-field conditions. We determined that An. gambiae prefers to land on arrayed visual targets warmed to human skin temperature during the nighttime when they are baited with carbon dioxide (CO 2 ) emissions reflective of a large human over background air, body odor from one human over CO 2 , and the scent of one sleeping human over another. Applying integrative whole body volatilomics to multiple humans tested simultaneously in competition in a six-choice assay, we reveal high attractiveness is associated with whole body odor profiles from humans with increased relative abundances of the volatile carboxylic acids butyric acid, isobutryic acid, and isovaleric acid, and the skin microbe-generated methyl ketone acetoin. Conversely, those least preferred had whole body odor that was depleted of carboxylic acids among other compounds and enriched with the monoterpenoid eucalyptol. Across expansive spatial scales, heated targets without CO 2 or whole body odor were minimally or not attractive at all to An. gambiae. These results indicate that human scent acts critically to guide thermotaxis and host selection by this prolific malaria vector as it navigates towards humans, yielding intrinsic heterogeneity in human biting risk., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)