Ali Afify

Nature has long inspired engineering innovations. Recent advances in biohybrid research have taken this inspiration further by directly integrating biotic materials into engineered systems. Here we report “3D necroprinting,” a biohybrid manufacturing technique that repurposes female mosquito proboscides as high-resolution 3D printing nozzles. The mosquito proboscis, with its unique geometry, structure, and mechanics, enables printed line widths as fine as 20 μm, surpassing commercially available 36-gauge dispense tips by ~100%. The mosquito proboscis dispense tip can withstand internal pressures of approximately 60 kPa, enabling effective fluid extrusion. Demonstrated applications include high-resolution printing of complex structures such as a honeycomb structure, a maple leaf, and bioscaffolds encapsulating cancer cells and red blood cells, showcasing the versatility and capacity of 3D necroprinting. By introducing biotic materials as viable substitutes to complex engineered components, this work paves the way for sustainable and innovative solutions in advanced manufacturing and microengineering.

Drosophila melanogaster olfactory neurons have long been thought to express only one chemosensory receptor gene family. There are two main olfactory receptor gene families in Drosophila , the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs). The dozens of odorant binding receptors in each family require at least one co-receptor gene in order to function: Orco for ORs, and Ir25a , Ir8a , and Ir76b for IRs. Using a new genetic knock-in strategy, we targeted the four co-receptors representing the main chemosensory families in Drosophila ( Orco, Ir8a, Ir76b, Ir25a ). Co-receptor knock-in expression patterns were verified as accurate representations of endogenous expression. We find extensive overlap in expression among the different co-receptors. As defined by innervation into antennal lobe glomeruli, Ir25a is broadly expressed in 88% of all olfactory sensory neuron classes and is co-expressed in 82% of Orco+ neuron classes, including all neuron classes in the maxillary palp. Orco , Ir8a , and Ir76b expression patterns are also more expansive than previously assumed. Single sensillum recordings from Orco-expressing Ir25a mutant antennal and palpal neurons identify changes in olfactory responses. These results suggest co-expression of chemosensory receptors is common in olfactory neurons. Together, our data present the first comprehensive map of chemosensory co-receptor expression and reveal their unexpected widespread co-expression in the fly olfactory system.

Mosquitoes locate and approach humans (‘host-seek’) when specific Olfactory Neurons (ORNs) in the olfactory periphery activate a specific combination of glomeruli in the mosquito Antennal Lobe (AL). We hypothesize that dysregulating proper glomerular activation in the presence of human odor will prevent host-seeking behavior. In experiments aimed at ectopically activating most ORNs in the presence of human odor, we made a surprising finding: ectopic expression of an AgOr (AgOr2) in Anopheles gambiae ORNs dampens the activity of the expressing neuron. This contrasts studies in Drosophila melanogaster , the typical insect model of olfaction, in which ectopic expression of non-native ORs in ORNs confers ectopic neuronal responses without interfering with native olfactory physiology. To gain insight into this dysfunction in mosquitoes, RNA-seq analyses were performed comparing wild-type antennae to those ectopically expressing AgOr2 in ORNs. Remarkably, almost all Or transcripts were significantly downregulated (except for AgOr2 ), and additional experiments suggest that it is AgOR2 protein rather than mRNA that mediates this downregulation. Our study shows that ORNs of Anopheles mosquitoes (in contrast to Drosophila ) employ a currently unexplored regulatory mechanism of OR expression, which may be adaptable as a vector-control strategy. SIGNIFICANCE STATEMENT: Studies in Drosophila melanogaster suggest that insect Olfactory Receptor Neurons (ORNs) do not contain mechanisms by which Odorant Receptors (ORs) regulate OR expression. This has proved useful in studies where ectopic expression of an OR in Drosophila ORNs confers responses to the odorants that activate the newly expressed OR. In experiments in Anopheles gambiae mosquitoes, we found that ectopic expression of an OR in most Anopheles ORNs dampened the activity of the expressing neurons. RNA-seq analyses demonstrated that ectopic OR expression in Anopheles ORNs leads to downregulation of endogenous Or transcripts. Additional experiments suggest that this downregulation required ectopic expression of a functional OR protein. These findings reveal that Anopheles mosquitoes, in contrast to Drosophila , contain a feedback mechanism to regulate OR expression. Mosquito ORNs might employ regulatory mechanisms of OR expression previously thought to occur only in non-insect olfactory systems.

The mode of action for most mosquito repellents is unknown. This is primarily due to the difficulty in monitoring how the mosquito olfactory system responds to repellent odors. Here, we used the Q-system of binary expression to enable activity-dependent Ca ²⁺ imaging in olfactory neurons of the African malaria mosquito Anopheles coluzzii . This system allows neuronal responses to common insect repellents to be directly visualized in living mosquitoes from all olfactory organs including the antenna. The synthetic repellents DEET and IR3535 did not activate Odorant Receptor Co-Receptor (Orco) expressing olfactory receptor neurons (ORNs) at any concentration, while picaridin weakly activated ORNs only at high concentrations. In contrast, natural repellents ( i.e . lemongrass oil and eugenol) strongly activated small numbers of ORNs in the mosquito antennae at low concentrations. We determined that DEET, IR3535, and picaridin decrease the response of Orco expressing ORNs when these repellents are physically mixed with activating human-derived odorants. We present evidence that synthetic repellents may primarily exert their olfactory mode of action by decreasing the amount of activating ligand reaching ORNs. These results suggest that synthetic repellents disruptively change the chemical profile of host scent signatures on the skin surface rendering humans invisible to mosquitoes.

Fruit flies recognize hundreds of ecologically relevant odors and respond appropriately to them. The complexity, redundancy and interconnectedness of the olfactory machinery complicate efforts to pinpoint the functional contributions of any component neuron or receptor to behavior. Some contributions can only be elucidated in flies that carry multiple mutations and transgenes, but the production of such flies is currently labor-intensive and time-consuming. Here, we describe a set of transgenic flies that express the Saccharomyces cerevisiae GAL80 in specific olfactory sensory neurons (OrX-GAL80s). The GAL80s effectively and specifically subtract the activities of GAL4-driven transgenes that impart anatomical and physiological phenotypes. OrX-GAL80s can allow researchers to efficiently activate only one or a few types of functional neurons in an otherwise nonfunctional olfactory background. Such experiments will improve our understanding of the mechanistic connections between odorant inputs and behavioral outputs at the resolution of only a few functional neurons.

Fruit flies recognize hundreds of ecologically relevant odors and respond appropriately to them. The complexity, redundancy and interconnectedness of the olfactory machinery complicate efforts to pinpoint the functional contributions of any component neuron or receptor to behavior. Some contributions can only be elucidated in flies that carry multiple mutations and transgenes, but the production of such flies is currently labor-intensive and time-consuming. Here, we describe a set of transgenic flies that express the Saccharomyces cerevisiae GAL80 in specific olfactory sensory neurons ( OrX-GAL80s ). The GAL80s effectively and specifically subtract the activities of GAL4-driven transgenes that impart anatomical and physiological phenotypes. OrX-GAL80s can allow researchers to efficiently activate only one or a few types of functional neurons in an otherwise nonfunctional olfactory background. Such experiments will improve our understanding of the mechanistic connections between odorant inputs and behavioral outputs at the resolution of only a few functional neurons.

Gravid mosquitoes use chemosensory (olfactory, gustatory, or both) cues to select oviposition sites suitable for their offspring. In nature, these cues originate from plant infusions, microbes, mosquito immature stages, and predators. While attractants and stimulants are cues that could show the availability of food (plant infusions and microbes) and suitable conditions (the presence of conspecifics), repellents and deterrents show the risk of predation, infection with pathogens, or strong competition. Many studies have addressed the question of which substances can act as positive or negative cues in different mosquito species, with sometimes apparently contradicting results. These studies often differ in species, substance concentration, and other experimental details, making it difficult to compare the results. In this review, we compiled the available information for a wide range of species and substances, with particular attention to cues originating from larval food, immature stages, predators, and to synthetic compounds. We note that the effect of many substances differs between species, and that many substances have been tested in few species only, revealing that the information is scattered across species, substances, and experimental conditions.

Methyl N,N-dimethyl anthranilate (MDA), ethyl anthranilate (EA) and butyl anthranilate (BA) were previously shown to repel Aedes aegypti mosquitoes from landing on human skin. However, the effect of these compounds on the orientation of flying mosquitoes in a choice situation and their effect on mosquito oviposition are not yet known. Here, we used a modified Y-tube olfactometer to test the effect of these compounds on the orientation of Aedes aegypti flying towards skin odor (human fingers), and we tested their effect on Aedes aegypti oviposition choice in a cage assay. In both behavioral situations we compared the effect to the well-documented repellent N,N-diethyl-meta-toluamide (DEET). MDA, EA, and DEET inhibited Aedes aegypti from flying towards skin odor while BA had no such effect. Conversely, MDA had no effect on oviposition while EA, BA, and DEET deterred oviposition, with the strongest effect observed for BA. Thus, we confirm that EA and DEET are generally repellent, while MDA is repellent only in a host-seeking context, and BA is deterrent only in an oviposition context. These compounds appear of potential use in mosquito control programs.