Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Vosshall Laboratory

Abstract

The ability to detect and respond to environmental cues is the fundamental start of any brain function, computation, or behavior. For my doctoral work, I studied chemosensation in the Aedes aegypti mosquito, which can detect cues including human odor, carbon dioxide, and insect repellents. Mosquitoes are human’s deadliest predator, causing 700,000 deaths every year through the transmission of malaria parasites and arboviruses including dengue. My research has focused on the molecular underpinnings of mosquito chemosensory cell types. In olfaction, research following the cloning of the first odorant receptors in1991 has shown that each mouse olfactory sensory neuron (OSN) expresses a single odorant receptor. Early work in Drosophila melanogaster flies reported a similar organization, although some key differences were noted. Insects detect odorants with three families of ionotropic ligand-gated ion channels encoded by three large multi-gene families: Odorant Receptors (ORs), Ionotropic Receptors (IRs), and Gustatory Receptors (GRs) each of which form heteromultimeric ligand-gated ion channels composed of co-receptor and ligand-selective receptor subunits. These families were thought to be mutually exclusively expressed in non-overlapping OSNs. We developed techniques to extract chemosensory neurons from the two mosquito olfactory organs, the antenna, and the maxillary palp, for single nucleus RNA-sequencing (snRNA-seq). Performing careful analysis, we found that mosquitoes co-express multiple chemosensory receptor genes within individual OSNs. In the maxillary palp, nearly all the OSNs co-expressed multiple chemo receptor subunits from at least two of the three families of chemoreceptors. In the antenna, there were at least 35 chemosensory cell types with unique receptor profiles, containing two, three, or more receptors per cell. We concluded that the mosquito olfactory system represents a departure from the canonical understanding of olfaction as first established in the mouse. These studies established a foundational molecular understanding of mosquito sensory cell types, the neurobiological substrate of human detection and host-seeking. We applied our approach to mosquito legs, which contain taste and other sensory neurons that are critical to support mutually exclusive behaviors related to important reproductive life stages of the adult female mosquito. These include assessing the skin of a human host prior to blood feeding, sensing the pheromones of a conspecific prior to copulation, and detecting freshwater appropriate for egg-laying. We characterized the neuron types in the mosquito leg and discovered that the number of neurons varies across the fore-, mid-, and hind-legs. We also showed that each of the legs contributes to contact chemo avoidance of DEET, the principal ingredient in most insect repellents. Moreover, we discovered that the avoidance of DEET is a distinct sensation from the avoidance of bitter, as demonstrated by mosquitoes not being deterred by high concentrations of bitter substances applied to skin. We were interested in the possibility that taste neuron activity is modulated to reflect the female’s physiological needs for food, mating, or depositing her offspring. Using snRNA-seq, we were able to classify leg sensory cell types and their receptors and discovered that mosquito leg neurons are polymodal, co-expressing sensory receptors that respond to diverse stimuli across sensory domains, including noxious heat, warmth, sugar, salt, water, and fatty acids. Strikingly, these cell types also express distinct profiles of biogenic amine, neuropeptide, and neurotransmitter receptors. This suggests the possibility that these sensory neurons are subject to local or top-down modulation that drives context- and state-relevant behaviors. We are now expanding on this work with the Mosquito Cell Atlas project, a large-scale snRNA-seq project of essentially every tissue from the adult Aedes aegypti mosquito. Many mosquito sensory organs are capable of gustation, including the legs, wings, mouth parts, and egg-laying organs, and taste is necessary for every stage of the mosquito’s life and reproductive cycle. Our goal is to provide a reliable cell atlas to the field, interpret the molecular mechanisms driving mosquito biology and behavior, and understand potential targets that will be invaluable for developing interventions to mitigate the spread of deadly pathogens from mosquitoes to humans.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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