Author ORCiD
Date of Award
2026
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Leslie B. Vosshall
Keywords
aedes aegypti, taste, ATP, Ir100c, stylet, chemosensation
Abstract
Blood feeding is fundamental for female Aedes aegypti mosquitoes, which require this protein-rich meal for egg development. After being attracted to a human host by sensory cues such as body odor, body heat, and carbon dioxide exhaled in human breath, the mosquito lands and probes under the skin to feed on blood. Probing uses the stylet, a sharp appendage ensheathed in the mosquito labium that punctures the skin and is highly specialized for blood ingestion. Previous work from the lab with GCaMP calcium imaging of stylet neurons showed that approximately 50% of stylet neurons respond to blood, and that these same neurons respond to a mixture of saline and ATP. However, ATP alone activates only a small subset of 3-7 stylet neurons compared to those activated by blood or saline and ATP. Despite this initial view into what activates feeding using the stylet, the mechanism by which ATP stimulates mosquito feeding, whether the stylet encounters ATP in vivo during feeding, and what receptor and what neurons respond to ATP remains unknown. This work addresses each of these questions. We confirmed classic findings that ATP is a potent feeding stimulant and showed that brief exposure is sufficient to cause sustained ingestion of an ATP-free saline solution that is normally not appetitive. This suggests that ATP is a trigger of feeding and is not required for sustained intake. To ask if the stylet encounters ATP in the course of probing for blood under the skin, we adapted an in vivo mouse skin feeding assay in which we visualized the Aedes aegypti mosquito stylet inside the skin, searching for a blood vessel in real time. We found that the female stylet is highly effective in locating subdermal blood capillaries and that most feeding directly targets the capillaries, a process called “capillary feeding.” In some cases, the stylet obtains blood that is released locally from damaged blood vessels, a process called “pool feeding.” We expressed a genetically-encoded ATP sensor in mouse skin and discovered that ATP is released from skin cells in close proximity to where the stylet is probing inside the skin. This suggests that ATP is released in vivo and has the potential to activate the mosquito stylet neurons during probing. This raises the question of what receptor detects ATP in stylet neurons. Since the Aedes aegypti genome does not contain any orthologs of vertebrate extracellular P2X or P2Y ATP receptors, we focused on ionotropic receptors (IRs), a large family of chemosensory receptors in insects that has previously been shown to respond to stimuli ranging from olfactory to taste to thermal cues. IRs form heteromeric ligand-gated ion channels composed of a ligand-selective receptor subunit and a ligand-insensitive co-receptor. We showed that mosquitoes lacking either of the two major IR co-receptors, Ir25 and Ir76b, show profound defects in feeding. Previous GCaMP calcium imaging work was conducted by monitoring activity in all neurons in the stylet. To narrow down the neurons that respond to ATP to smaller subsets of neurons, we carried out GCaMP imaging in strains in which either Ir25a or Ir76b neurons expressed GCaMP. In such experiments, we found that Ir25a neurons respond to ATP, while Ir76b cells show little or no response. This suggests that the ATP receptor is likely comprised of a ligand-selective IR in complex with Ir25a. We next looked for candidate ligand-selective IRs that could be responsible for ATP sensing. With this candidate gene approach, we identified Ir100c as a putative ATP receptor in female stylet neurons. We generated single nucleus RNA-sequencing (snRNA-seq) data from the stylet and found a small number of chemosensory neurons that express Ir100c. To evaluate the expression and distribution of Ir100c in the female stylet, we performed RNA fluorescence in situ hybridization and found 3-7 Ir100c-positive cells, in agreement with the number of cells that respond to ATP in the calcium imaging experiments. Using gene-targeted mosquito lines that express fluorescent markers either in all neurons or only Ir25a or only Ir76b neurons, we found that Ir100c is expressed in neurons and that 96.9% of Ir100c-positive cells co-express Ir25a, hinting that Ir100c may assemble with Ir25a as a functional ion channel. Finally, we worked in collaboration to develop AlphaFold3 structure predictions that are consistent with the model that a heterotetramer composed of Ir100c and Ir25a is a potential extracellular ATP receptor. This work demonstrates the importance of ATP sensing for mosquito blood-feeding behavior, identifies a minimal subset of neurons in the stylet that respond to ATP, provides evidence that Ir100c+Ir25a is a novel candidate ATP receptor, and establishes ATP signaling as a potential pathway for manipulating blood-feeding arthropods to prevent disease transmission.
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Villegas, Katherine Adriana Rosas, "Dining in the Dark ATP Sensing in the Female Aedes Aegypti Mosquito" (2026). Student Theses and Dissertations. 850.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/850
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy