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The HOST assay: an automated video-based evaluation of mosquito attraction to thermal infrared

Nature Protocols (2026)Cite this article

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Abstract

Female mosquitoes transmit pathogens to people during blood feeding, leading to ~700 million infections annually. Females navigate toward humans by integrating multiple sensory cues while flying. Here we describe a behavioral assay system using automated video analysis to measure mosquito landing and walking on target areas emitting human body temperature, elevated CO2, and human skin odor, and quantify preference indices for such behaviors. This host seeker tracking (HOST) assay can be adapted to measure landing and movement on a target of interest under different combinations and intensities of sensory stimuli. It is distinct from other host-seeking assays in that it allows the impact of thermal infrared radiation and convection to be analyzed individually. Mated female mosquitoes are transferred to specialized cages, allowed to recover, and then assayed. During each HOST experiment, an assay cage is lowered into an arena where females are presented with host-associated cues, and their landing and walking behaviors on targets of interest are video-recorded. After completing the assays, the videos are analyzed using custom scripts (V-HOST), which automatically determine landing and movement on the target areas, and preference indices. The HOST protocol uses interchangeable cages rather than direct release of mosquitoes into the behavior arena, which increases assay throughput, and prevents release of mosquitoes. The HOST assay employs commonly available materials and equipment, making it a readily accessible technique. Using this approach, an experimenter can perform ~24 individual technical replicates in a 4 h period (ZT0–ZT4) and quickly perform the video analysis (10–15 min).

Key points

  • This protocol describes an assay to study mosquito landing and walking behaviors on targets of interest in the presence of thermal infrared radiation in combination with other host-associated cues.

  • The approach provides a modular platform that researchers can adapt to their specific scientific needs and allows the roles of thermal infrared radiation and convection in host seeking to be studied independently.

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Fig. 1: Protocol.
Fig. 2: Assay modifications for testing different thermal cues.
Fig. 3: The construction of HOST assay cages.
Fig. 4: The construction of a HOST assay arena base panel and side wall panels.
Fig. 5: The construction of front and rear panels for a HOST assay.
Fig. 6: CO2 release tubing.
Fig. 7: A programmable CO2 release system.
Fig. 8: A side view of the fully assembled HOST assay arena.
Fig. 9: Representative HOST assay results with IR configuration.

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Data availability

Source data for Fig. 9 is available in the Supplementary Information.

Code availability

All source code for this publication can be found at https://github.com/Craig-Montell-Lab/V-HOST_DeBeaubien_etal_2025.

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Acknowledgements

We thank G. Meyerhof for comments on the manuscript. This work was supported by a grant to C.M. from the National Institute of Allergy and Infectious Diseases (grant no. R01-AI165575) and the US Army Research Office, which was accomplished under cooperative agreement W911NF-19-2-0026 for the Institute for Collaborative Biotechnologies.

Author information

Author notes

  1. Vincent L. Salgado

    Present address: Department of Biology, Duke University, Durham, NC, USA

Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, and the Neuroscience Research Institute, University of California, Santa Barbara, CA, USA

    Nicolas A. DeBeaubien, Avinash Chandel & Craig Montell

  2. BASF Corp, Research Triangle Park, NC, USA

    Vincent L. Salgado

Authors
  1. Nicolas A. DeBeaubien
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  2. Avinash Chandel
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  3. Vincent L. Salgado
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  4. Craig Montell
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Contributions

All authors prepared and edited the manuscript.

Corresponding author

Correspondence to Craig Montell.

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The authors declare no competing interests.

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Nature Protocols thanks Felix Hol and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references

Chandel, A. et al. Nature 633, 615–623 (2024): https://doi.org/10.1038/s41586-024-07848-5

Carr, A. L. & Salgado, V. L. PLoS One 14, e0221659 (2019): https://doi.org/10.1371/journal.pone.0221659

Source data

Source Data Fig. 9 (download XLSX )

Statistical source data.

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DeBeaubien, N.A., Chandel, A., Salgado, V.L. et al. The HOST assay: an automated video-based evaluation of mosquito attraction to thermal infrared. Nat Protoc (2026). https://doi.org/10.1038/s41596-026-01335-z

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