Circadian modulation of mosquito host-seeking persistence by Pigment-Dispersing Factor impacts daily biting patterns

Background

In animals, circadian clocks impose a daily rhythmicity to many behaviors and physiological processes. In mosquitoes, biting activity follows species-specific rhythms to maximize their success, helping them find a host in their niche and avoid unfavorable conditions like exposure to repellents. This rhythm is controlled by the molecular components of circadian clock cells. The synchronization among these cells is mediated by the Pigment Dispersing Factor (PDF), a neuropeptide expressed in a subpopulation of the brain clock cells.

While the biting rhythm is cell-autonomous, it is influenced by external stimuli such as carbon dioxide (CO₂).  In mosquitoes, CO₂ serves as a key signal for finding hosts, triggering a predatory state that leads to biting. In addition, olfactory sensitivity exhibits a daily rhythm which is modulated by the clock cells in the brain and the antennae. Considering this, mosquitoes must coordinate their locomotor activity with chemosensory cues, such as CO₂ exposure, to achieve an optimal bite behavior.

What has remained unclear so far are the cellular mechanisms underlying how behavioral responses to host cues vary throughout the day—and whether these responses rely on PDF coordination within central brain clock cells. The authors of this preprint address these questions by studying female Aedes aegypti mosquitoes using a combination of genetic, cellular, and behavioral approaches.

Key Findings

• Female aegypti mosquitoes exhibited peaks of locomotor activity in the morning and evening. This bimodal pattern disappeared in pdf null mutant animals under light/dark and constant darkness conditions.
• Using a blood-feeding assay with controlled CO₂ conditions, the authors showed that wildtype mosquitoes display biting activity early in the morning and during the evening. This daily pattern was missing in mosquitoes lacking pdf.
• Genetically disruption pdf changed the shape and axon projections of a group of clock cells in the brain.
• pdf null mutant mosquitoes exhibited changes in the daily transcription rhythm of Period, a key molecular component of the clock cells.
• Under light/dark conditions with controlled CO₂ pulses, female aegypti responded strongly to CO₂ in the early morning and late evening, less around midday, and none at all at night. Mosquitoes also had a peak in response duration in the evening. This daily rhythm in responding to CO₂, was missing in pdfnull mutant mosquitoes.
• In constant darkness conditions, female aegypti still responded to CO₂, but their response was shorter and weaker compared to light/dark conditions. Interestingly, the rhythm in their inmediate response (0–2 minutes after CO₂ application) was still present in pdf mutants but not in period mutants, while the rhythm in their longer response (2–10 minutes after CO₂ application) was missing in pdf knockout mosquitoes.

One correction here – we did not test CO₂ response in DD in period mutants (the only mutants that we use in this paper are the pdf mutants). However, our model hypothesizes immediate CO₂ responses should become arrhythmic under DD in period mutants.

Why this work is relevant?

Mosquitoes are major vectors of human diseases such as Zika, malaria, and dengue. Gaining insights into the mechanisms underlying their rhythmic biting behavior could have significant implications for insect control and disease transmission. This study is the first to propose a model in which the neuropeptide PDF, produced in a specific cluster of brain clock cells, integrates daily variations in both CO₂ responsiveness and locomotor activity in female A. aegypti, ultimately promoting successful biting behavior.

Why did I choose to highlight this work?

There is a lot of evidence on the role of neuropeptides in regulating rhythmic behaviors, but in this preprint, the authors propose a mechanism where a neuropeptide is essential for coordinating the rhythmicity of two vital behaviors in mosquitoes: biting and locomotor activity. While the mosquito’s unique sensitivity to host CO₂ cues suggests that other cellular mechanism may act as modulators, it is fascinating to see how the role of PDF as a master regulator is conserved across species.

Questions for the authors

• You revealed that the daily rhythm in the acute response to CO₂ is mediated by Period but not by PDF. It would be fascinating to explore whether other neuropeptides known to regulate circadian behavior, such as small neuropeptide F (sNPF), may play a role in modulating the acute response to CO₂ exposure. Have you investigated other signaling molecules that could explain the differences in the mechanisms controlling acute versus sustained responses to CO₂?
• In other insects, such as the fruit fly Drosophila melanogaster, it is known that peripheral clocks can be influenced by PDF. While you mention a study from Paul Hardin’s lab suggesting that the antennae clock operates independently from the brain clock, it would be valuable to test whether a similar situation occurs in aegypti.
• In the discussion, you state, “Understanding the mechanisms of daily rhythms of host-seeking effectiveness can provide new targets to disrupt these and potentially lock females into a ‘low host-seeking’ state.” Considering the novel system that you designed to test the daily pattern of biting activity under controlled conditions, it would be interesting to test whether injections of synthetic PDF could alter the phase of biting activity.

Tags: circadian rhythms, neuropeptide, pdf

doi: https://doi.org/10.1242/prelights.39017

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