Gene-editing technologies have revolutionized the field of mosquito sensory biology. These technologies have been used to knock in reporter genes in-frame with neuronal genes and tag specific mosquito neurons to detect their activities using binary expression systems. Despite these advances, novel tools still need to be developed to elucidate the transmission of olfactory signals from the periphery to the brain. Here, we propose the development of a set of tools, including novel driver lines as well as sensors of neuromodulatory activities, which can advance our knowledge of how sensory input triggers behavioral outputs. This information can change our understanding of mosquito neurobiology and lead to the development of strategies for mosquito behavioral manipulation to reduce bites and disease transmission.

Insects house humidity-sensing neurons in the antenna, which is presumed to be important for a variety of behaviors and survival since water is a crucial component of the environment. Here we use the simple olfactory system of the Asian Citrus Psyllid (ACP), a citrus pest that transmits a deadly bacterium, to identify volatile amines that significantly inhibited humidity-induced activation of antennal neurons. The inhibition of action potentials is observed by single sensillum recordings and mixing these odorants with humid air abolished the humidity avoidance behavior of ACP. The inhibition is conserved in the humidity-sensing coeloconic neurons of dipteran Drosophila melanogaster that are known to detect humidity, but it is not seen in other coeloconic neurons that are not sensitive to humidity. Dipteran mosquitoes Aedes aegypti and Anopheles gambiae oviposit in water, and the addition of the humidity-inhibiting odorants in a two-choice oviposition assay significantly reduces oviposition. Our results demonstrate that a naturally occurring volatile compound can effectively "mask" detection of an important environmental cue and modify behavior of important vectors of plant and human disease pathogens. Odorants targeting the conserved humidity sensing system of insects, therefore, offer a novel strategy for modifying their behavior.

Exhaled CO₂ is an important host-seeking cue for Anopheles mosquitoes, which is detected by a highly conserved heteromeric receptor consisting of three 7-transmembrane proteins Gr22, Gr23, and Gr24. The CO₂ receptor neuron has been shown to also respond sensitively to a variety of odorants in Aedes aegypti. The detection of CO₂ is important for upwind navigation and for enhancing the attraction to body heat as well as to skin odorants. The orthologs of the CO₂ receptor proteins are present in malaria-transmitting mosquitoes like Anopheles coluzzii and Anopheles sinensis. Activators and inhibitors of the CO₂-neuron were tested on the maxillary palps in these two species by single-sensillum electrophysiology. The electrophysiological testing of three prolonged-activator odorants identified originally in Aedes aegypti also showed varying ability to reduce the CO₂-ellicited increase in spikes. These findings provide a foundation for comparing the functional conservation with the evolutionary conservation of an important class of odorant receptor. The identification of a suite of natural odorants that can be used to modify the CO₂-detection pathway may also contribute to odor-blends that can alter the behavior of these disease transmitting mosquitoes.

Background

The Asian Citrus Psyllid (ACP), Diaphorina citri, can transmit the bacterium Candidatus Liberibacter while feeding on citrus flush shoots. This bacterium causes Huanglongbing (HLB), a major disease of citrus cultivation worldwide necessitating the development of new tools for ACP surveillance and control. The olfactory system of ACP is sensitive to variety of odorants released by citrus plants and offers an opportunity to develop new attractants and repellents.

Results

In this study, we performed single-unit electrophysiology to identify odorants that are strong activators, inhibitors, and prolonged activators of ACP odorant receptor neurons (ORNs). We identified a suite of odorants that activated the ORNs with high specificity and sensitivity, which may be useful in eliciting behavior such as attraction. In separate experiments, we also identified odorants that evoked prolonged ORN responses and antagonistic odorants able to suppress neuronal responses to activators, both of which can be useful in lowering attraction to hosts. In field trials, we tested the electrophysiologically identified activating odorants and identified a 3-odor blend that enhances trap catches by ∼230%.

Conclusion

These findings provide a set of odorants that can be used to develop affordable and safe odor-based surveillance and masking strategies for this dangerous pest insect.

The resident human skin microbiome is responsible for the production of most of the human scents that are attractive to mosquitoes. Hence, engineering the human skin microbiome to synthesize less of mosquito attractants or produce repellents could potentially reduce bites and prevent the transmission of deadly mosquito-borne pathogens. In order to further characterize the human skin volatilome, we quantified the major volatiles of 39 strains of skin commensals (Staphylococci and Corynebacterium). Importantly, to validate the behavioral activity of these volatiles, we first assessed landing behavior triggered by human skin volatiles. We demonstrated that landing behavior is gated by the presence of carbon dioxide and L-(+)-lactic acid. This is similar to the combinatorial coding triggering mosquito short range attraction. Repellency behavior to selected skin volatiles and terpenes was tested in the presence of carbon dioxide and L-(+)-lactic acid. In a 2-choice landing behavior context, the skin volatiles 2- and 3-methyl butyric acids reduced mosquito landing by 62.0-81.6% and 87.1-99.6%, respectively. Similarly, the terpene geraniol was capable of reducing mosquito landing behavior by 74.9%. We also tested the potential repellency effects of terpenes in mosquitoes at short-range using a 4-port olfactometer. In these assays, geraniol reduced mosquito attraction (69-78%) to a mixture of key human kairomones carbon dioxide, L-(+)-lactic acid, and ammonia. These findings demonstrate that carbon dioxide and L-(+)-lactic acid change the valence of other skin volatiles towards mosquito landing behavior. Moreover, this study offers candidate odorants to be targeted in a novel strategy to reduce attractants or produce repellents by the human skin microbiota that may curtail mosquito bites, and subsequent mosquito-borne disease.