Conor J. McMeniman

Insects and terrestrial fungi have lived on planet earth together for over 400 My. Different insect species are commonly associated with distinct mycobiomes. These insect–fungal associations are thought to provide a major dispersal mechanism for fungi in the environment. The Rhodotorula genus of pigmented basidiomycetous yeasts is commonly found in association with insects. Here, we demonstrate that yeasts of Rhodotorula spp. are highly attractive to the vinegar fly Drosophila melanogaster and the African malaria mosquito Anopheles gambiae . We characterized strains of the environmental yeast Rhodotorula taiwanensis that emit simple scent signatures predominated by the common microbial volatile 3-methyl-1-butanol (isoamyl alcohol) alongside other characteristic volatiles including acetone and acetaldehyde. Insect odorant receptors are required for attraction to R. taiwanensis and promote yeast dispersal to new growth niches. In addition to scent, dispersal is also likely mediated by the adhesive qualities of these yeasts to insect cuticles via hydrophobic interactions, as well as consumption. Strikingly, Rhodotorula dispersal was also facilitated by An. gambiae , although mosquitoes often died after becoming stuck to slimy lawns of these yeasts. Our work reveals that scent and adhesion drive insect dispersal of these environmental yeasts.

The zoophilic and exophilic traits of outdoor-biting Anopheles have led to this group largely being overlooked for their role in malaria transmission, despite several species now recognized as locally important in regions of sub-Saharan Africa. Given the current limitations with identification of these understudied species, it is crucial to accurately correlate morphological features to molecular data. Here, we produced high quality reference sequence data for representative understudied anopheline species to better understand the phylogenetic relationships between under- and well-studied vectors of malaria. For mitochondrial genome assembly, shallow shotgun sequencing was implemented on single mosquito specimens and phylogenetic analyses were performed on the concatenated protein coding genes of the mitogenomes using a Bayesian approach. This study generated 10 complete mitogenomes focusing on less studied taxa with an average length 15,380 bp and A-T content of 77.4% consistent with other anophelines containing 37 genes. Bayesian inference analysis yielded four main clades with molecular dating indicating that well-studied malaria vectors diverged from outdoor-biting species more than 63 million years ago. These findings support the taxonomic grouping of mosquitoes belonging to the Anopheles genus based on morphological characteristics and can provide molecular diagnostics for species identification enabling more precise and adept interventions for malaria control.

The zoophilic and exophilic traits of outdoor-biting Anopheles has led to this group largely being overlooked for their role in malaria transmission, despite several species now recognized as locally important in regions of sub-Saharan Africa. Given the current limitations with identification of these understudied species, it is crucial to accurately correlate morphological features to molecular data. Here, we produced high quality reference sequence data for representative understudied anopheline species to better understand the phylogenetic relationships between under- and well-studied vectors of malaria. For mitochondrial genome assembly, shallow shotgun sequencing was implemented on single mosquito specimens and phylogenetic analyses were performed on the concatenated protein coding genes of the mitogenomes using a Bayesian approach. This study generated 10 complete mitogenomes focusing on less-studied taxa with an average length 15,383 bp and A-T content of 77.1% consistent with other anophelines containing 37 genes. Bayesian inference analysis yielded four main clades with molecular dating indicating that well-studied malaria vectors diverged from outdoor-biting species more than 65 million years ago. These findings support the taxonomic grouping of mosquitoes belonging to the Anopheles genus based on morphological characteristics and can provide molecular diagnostics for species identification enabling more precise and adept interventions for malaria control.

The mosquito Aedes aegypti is an emerging model insect for invertebrate neurobiology. We detail the application of a dual transgenesis marker system that reports the nature of transgene integration with circular donor template for CRISPR–Cas9-mediated homology-directed repair at target mosquito chemoreceptor genes. Employing this approach, we demonstrate the establishment of cell-type-specific T2A-QF2 driver lines for the A. aegypti olfactory co-receptor genes Ir8a and orco via canonical homology-directed repair and the CO2 receptor complex gene Gr1 via noncanonical homology-directed repair involving duplication of the intended T2A-QF2 integration cassette separated by intervening donor plasmid sequence. Using Gr1+ olfactory sensory neurons as an example, we show that introgression of such T2A-QF2 driver and QUAS responder transgenes into a yellow cuticular pigmentation mutant strain facilitates transcuticular calcium imaging of CO2-evoked neural activity on the maxillary palps with enhanced sensitivity relative to wild-type mosquitoes enveloped by dark melanized cuticle. We further apply Cre-loxP excision to derive marker-free T2A-QF2 in-frame fusions to clearly map axonal projection patterns from olfactory sensory neurons expressing these 3 chemoreceptors into the A. aegypti antennal lobe devoid of background interference from 3xP3-based fluorescent transgenesis markers. The marker-free Gr1 T2A-QF2 driver facilitates clear recording of CO2-evoked responses in this central brain region using the genetically encoded calcium indicators GCaMP6s and CaMPARI2. Systematic application of these optimized methods to different chemoreceptors stands to enable mapping A. aegypti olfactory circuits at peripheral and central levels of olfactory coding at high resolution.

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating—processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar fly Drosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in the Drosophila genus, its receptor, sex peptide receptor (SPR), is the widely conserved myoinhibitory peptide (MIP) receptor. To test the functional role of SPR in mediating postmating responses in a non-Drosophila dipteran, we generated 2 independent Spr-knockout alleles in the yellow fever mosquito, Aedes aegypti. Although SPR is needed for postmating responses in Drosophila and the cotton bollworm Helicoverpa armigera, Spr mutant Ae. aegypti show completely normal postmating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate from D. melanogaster into virgin female mosquitoes did not elicit these postmating responses. Our results demonstrate that Spr is not required for these canonical postmating responses in Ae. aegypti, indicating that other, as yet unknown, signaling pathways are likely responsible for these behavioral switches in this disease vector.

Anopheles gambiaeis a highly anthropophilic (human-preferring) malaria vector that prefers to blood feed frequently and selectively on humans. This mosquito species exhibits a strong innate olfactory preference to seek out human scent over other animals, and certain humans over others—key behavioral traits with the potential to drive heterogeneity in biting risk and malaria transmission. Here, we describe the application of a large-scale, semi-field system in Zambia for the quantification ofAn. gambiaeolfactory preferences toward whole body odor sourced from individual humans. We detail steps for modifying one-person canvas tents to duct odor from sleeping humans into a central, semi-field flight cage arena that is securely screened. Using this system, we describe a protocol to perform two-choice olfactory preference assays with two human volunteers using laboratory-rearedAn. gambiaeand odor-guided thermotaxis assays that leverage infrared video tracking to quantify mosquito landings on heated targets baited with each body odor sample. This multichoice olfactory preference assay has the potential to be applied with expanded cohorts of humans to define the chemosensory basis ofAn. gambiaehost preference and interindividual differences in human attractiveness to mosquitoes and to be used to quantify the effects of protective measures such as personal and spatial repellents on mosquito landing behavior.

The African malaria mosquitoAnopheles gambiaeis strongly attracted to human body odor and skin temperature. Quantitative behavioral assays suitable for use in semi-field environments with this nocturnal mosquito species are essential to gain improved insights intoAn. gambiaesensory biology, the mechanistic basis of mosquito attraction to humans, and host preference. In this protocol, we describe steps for engineering equipment for a novel behavioral assay forAn. gambiae, which we have termed the odor-guided thermotaxis assay (OGTA). The OGTA uses infrared videography to quantify landings of femaleAn. gambiaeon an aluminum platform heated to human skin temperature that can be baited with volatile odorants such as carbon dioxide or human whole body odor. The OGTA facilitates high-content recordings ofAn. gambiaelanding behavior during odor-guided thermotaxis under naturalistic semi-field conditions without the requirement for domestic power.

The most dangerous mosquito species for human health are those that blood feed preferentially and frequently on humans (anthropophilic mosquitoes). These include prolific disease vectors such as the African malaria mosquitoAnopheles gambiaeand yellow fever mosquitoAedes aegypti. The chemosensory basis for anthropophilic behavior exhibited by these disease vectors, as well as the factors that drive interindividual differences in human attractiveness to mosquitoes, remain largely uncharacterized. Here, we concisely review established methods to quantify mosquito interspecific and intraspecific host preference in the laboratory, as well as semi-field and field environments. Experimental variables for investigator consideration during assays of mosquito host preference across these settings are highlighted.

Summary: The African malaria mosquito Anopheles gambiae exhibits a strong innate sensory drive to seek out human scent. To detect human odorants, An. gambiae uses olfactory sensory neurons (OSNs) that can be divided into different classes by unique repertoires of chemoreceptor gene expression. We applied CRISPR-Cas9-mediated T2A-In Frame Fusions and the QF2/QUAS system to gain genetic access to specific OSN subsets in An. gambiae expressing the chemoreceptor genes Ir25a , Ir76b , Gr22 and orco . We first optimized methods to generate cell-type specific QF2 driver and QUAS responder lines to map expression patterns of these chemoreceptors across mosquito sensory appendages. We next applied transcuticular calcium imaging to record neurophysiological responses to select human-related odorants for each OSN class. This neurogenetic toolkit tiling OSN subsets in An. gambiae , including those responsive to CO ₂ , stands to support systematic efforts to decode olfaction in this prolific disease vector at high-resolution to combat malaria.