The rockefeller university

Female mosquitoes differ from many other organisms in that they require large blood meals to initiate egg production. Mosquitoes can feed multiple times throughout their lifetime, making them an effective vector for transmission of deadly infectious diseases such as dengue fever, yellow fever, and malaria. Mosquitoes use various combinations of host cues during their pursuit for blood meals, including carbon dioxide (CO2), volatile compounds, and heat. Among these cues, heat is a robust inducer for host-seeking and blood-feeding behavior. Although temperatures close to human body temperature attract mosquitoes, the genes, sensory neurons, and behavioral patterns underlying mosquito thermosensation have remained enigmatic. I have discovered a novel mechanism of sensory compensation that emerges when specific olfactory receptor genes are mutated. This has led to the discovery that upregulation of Ir140 in foreleg sensory neurons may underlie increased sensitivity to heat in Orco and Ir8a mutants.

Aedes aegypti mosquitoes use two distinct feeding programs. While females require a blood meal for egg production, both female and male mosquitoes feed primarily on sugar-rich plant nectar. To procure the necessary nutrients from these distinct food sources, females employ two behaviorally and anatomically distinct feeding programs: blood-feeding and nectar-feeding. Ingested blood is directed to the midgut (the equivalent of the small intestine) for digestion while nectar is initially routed to the crop for storage. The mechanisms by which these parallel feeding pathways sense the meal and direct it to a specific ingestive organ are unknown. Using a combination of tissue-specific expression patterns, Microscopy, and genomics I am searching for the chemosensory receptors in the enteric nervous system that detect and differentiate sugar and blood meals. This approach can reveal principles of internal organs chemosensation and how gut-brain communication regulates mosquito biology.

A three step process to study the epigenetic basis of the refractory state.

Female Aedes aegypti mosquitoes mate only once during their lifetime, after which they become refractory to any additional insemination attempts. This shift in behavior is robustly maintained throughout the lifetime of the female (up to 6 weeks) by an unknown mechanism. Although crucial to the understanding of mosquito reproduction and dispersal, little is known about the behavioral or the molecular processes by which females reject males for a lifetime after a single mating event. We would like to mechanistically test the hypothesis that distinct transcriptional and epigenetic changes create the different refractory components and maintain it for a lifetime. Such mechanisms have been linked in the past to social behavior and learning in multiple species and are compelling candidates for sustaining this life-long memory. Refractoriness in female mosquitoes provides a unique system for decoding neuronal and transcriptional changes that modify behavior permanently. Furthermore, understanding of the refractory state in female mosquitoes, and ideally manipulating it, could contribute to the success of controlling the spread of the deadliest animal on the planet. I have also been characterizing the behavioral and anatomical mechanisms of female sexual receptivity in Aedes aegypti, as well as generating genomes of a number of exotic mosquitoes.

Mosquito aging is the most critical factor in viral transmission and yet has received essentially no attention in the field. Even though mosquito age is singularly the key determinant for vector competence in spreading virus and disease, we lack the tools and knowledge to define aging in this dangerous insect. While the overarching goal of contemporary aging research is to understand mechanisms of aging to increase lifespan and longevity, my research project will do the opposite and speed up aging in mosquitoes to reduce disease transmission. My postdoc work will start a new field of mosquito aging, connecting organismal behavioral phenotypes to cellular organization and quantitative dynamics in key organs of the female mosquitoes important for blood feeding and disease transmission. Revealing the driving forces and molecular mechanisms of (accelerated) aging in mosquitoes will not only open new solutions in combating viral transmission but will also add another dimension to our understanding of biological aging, a widely known and universal process found in all of nature.

The mosquito phylogeny spans millions of years of evolution, and anthropophilic species such as Aedes aegypti have adopted various phenotypic mechanisms to expertly seek out and blood feed on humans. The vast genetic diversity across this lineage has allowed mosquitoes to fill new ecological niches and rapidly colonize new regions of the world. Little is currently known about how genetic novelties in mosquito species contribute to novel physiological or behavioral phenotypes, in speciation, reproduction, sexual selection, and sexually-dimorphic behavior. I aim to identify lineage-specific and rapidly-evolving genes in Aedes aegypti that drive phenotypic innovation, especially molecular mechanisms that facilitate ecological expansion or reproductive isolation in sympatric species. Using novel genome assemblies, population genetics data, and tissue- and sex-specific expression patterns, I am interested in characterizing their evolutionary trajectories, selective pressures and effect on mosquito behavior and physiology. My current work is focused on two rapidly evolving genes, Tweedledee and Tweedledum, first discovered by former PhD student Krithika Venkataraman.

Mosquitoes are often defined as the deadliest animals on earth for their potential to transmit dangerous pathogens to humans. Aedes aegypti is the prime vector for arboviruses such as dengue, Zika and chikungunya. Arboviruses are (re)emerging diseases whose burden on public health is expected to increase concurrently with the global expansion of Aedes aegypti. Arboviruses and mosquitoes share one common target: humans. The transmission of arboviruses depends on the success of infected female mosquitoes in obtaining human blood meals, which they require to reproduce. Prior work has suggested that some parasites and viruses may manipulate insect hosts to enhance virus transmission, but this has never been mechanistically investigated in mosquitoes. Using behavioral assays, next generation sequencing and genome editing techniques I am studying the effects of dengue infection on the mosquito brain and sensory systems, under the hypothesis that dengue hijack mosquito physiology to promote a more aggressive host-seeking and blood-feeding behavior in mosquitoes, which in turn increases viral transmission.

The mosquito behaviors of host-seeking and blood-feeding spread viruses in a sexually dimorphic way, carried out only by the females. To begin understanding the molecular underpinnings of sexually dimorphic behaviors in mosquitoes, I am studying the sex-specifically spliced gene called Carrie. This previously uncharacterized gene was discovered by former PhD student Nipun Basrur to have a female-specific exon that leads to a premature stop codon and a shorter protein in females, and a larger protein in males. Both male and female isoforms contain nuclear localization signals and a novel 200-amino acid domain of unknown function, present in 1 copy in the female protein and 2 copies in the male protein. I am characterizing this gene, its transcripts and expression pattern in the brain, its subnuclear localization, its evolution across the tree of life, and current hypotheses governing its function.

A schematic of head structures related to blood-feeding

The mosquito mouthparts are highly specialized for blood-feeding. Once a female mosquito lands in a host, it inserts its proboscis into the skin looking for a suitable meal. The female stylet has on average 28 neurons. Around half of them respond directly to blood. Intriguingly, the same population of neurons can be activated by a saline mixture containing ATP, inducing engorgement in a female mosquito (Jové, et al. 2020). The host-associated ligands that activate the stylet neurons and guide it towards a blood vessel remain largely unknown. Furthermore, the relevance of ATP in vivo has not been studied. Using calcium imaging and other tools to visualize blood-feeding and ATP release, I am interested in exploring what novel cues can induce or deter a mosquito from feeding, ultimately affecting their behavior

Mosquito reproduction is periodic, relying on the intake of a blood meal, proper time to mature eggs, and a suitable egg laying site. Additionally, mosquitoes exhibit reduced attraction to hosts after a blood meal, behavior that is sustained throughout egg maturation until eggs are laid. These observations suggest that neural control may link physiological state of the reproductive system and related behaviors. Little is known about the neurons of the Aedes aegypti female’s reproductive system, or how their circuitry regulates reproduction-related behaviors. To address this gap, I am characterizing the neurons of the female mosquito reproductive nervous system based on molecular identity and anatomy. Using a combination of transcriptomics, genetic manipulation, and immunofluorescence, I am visualizing the reproductive nervous system and exploring the expression of candidate genes like neurotransmitter biosynthesis markers, ion channels, chemoreceptors, and more. The goal of my project is to contribute to knowledge of the unique reproductive cycles that blood-feeding mosquitoes undergo.

Ecdysone is a steroid hormone derived from cholesterol that is an essential regulator of arthropod development and a coordinator of insect reproductive cycles. Female Aedes aegypti, a mosquito species responsible for transmitting deadly viruses to humans, rely on ingesting vertebrate blood to initiate egg production. Triggered by this blood meal, ecdysone directs the physiology of egg development through transcriptional activation of many genes. Contrary to previous hypotheses that ecdysone passively diffuses across the cell membrane, recent work has shown that this steroid hormone requires active transport mediated by ecdysone importers in the organic anion transporting polypeptide (OATP) family. Though these proteins are present in species ranging from mosquitoes to humans and are drug targets for many approved medications, little is known experimentally about their mode of transport or structural features. This project aims to elucidate the molecular logic of ecdysone import by a divergent ecdysone importer during Ae. aegypti reproduction, using structural biology complemented by biochemical, pharmacological, and behavioral approaches.

Tracked trajectories of a mosquito exposed to human cues (on the left) and human cues plus DEET (on the right).

Female Aedes aegypti mosquitoes employ robust, seemingly “unbreakable” host-seeking behavior to detect and bite humans. Insect repellents containing the active ingredients DEET and picaridin effectively break this strong attraction and prevent bites, but their mechanism of action remains unclear. Although various hypotheses exist to explain how repellents work, the inability to disentangle their multimodal effects hinders our understanding of the underlying sensory mechanisms. Overcoming these obstacles, my project aims to reveal the behavioral and neurocomputational logic underlying repellency in Ae. aegypti. I combine supervised tracking and pose estimation to determine possible interactive effects between attractive human cues and repellents. In particular, I am interested in how DEET and picaridin disrupt the chaining of actions characterizing host-seeking behavioral sequences. I am also performing calcium imaging in the central and peripheral nervous system to determine neuronal responses to these repellents.