1. Introduction
Mosquito-borne illnesses such malaria, dengue fever, and infections caused by West Nile and other encephalitic viruses affect over 700 million people across the globe each year [1–4]. About a million of these perish and most deaths are concentrated in Africa. Worse still, our medical arsenal to guard against these illnesses is declining rapidly [5], and the burden of these diseases is only going to increase with climate change. New strategies are desperately needed in order to guard against the spread of mosquito-borne diseases. Biological vector control has attracted sizable interest in recent years [6] and is typically achieved either by reducing the population of the vector in the wild or making it resistant to the disease [7–9]. Both approaches for biological vector control have shown promising results in small-scale, narrowly focused field trials. Whether this performance can be replicated at larger, more realistic scales remains to be seen, not to mention the uncertainty about their long-term ecological impacts [10].
The transmission rate of mosquito-borne diseases can also be reduced by diminishing the number of interactions between mosquitoes and humans. To this end, the use of herbal repellents ranks as one of the oldest techniques to ward off mosquitoes. All female mosquitoes that harbor pathogens utilize their sense of smell to locate and feed on the blood of their targets [11]. Ergo, interfering with this elaborate odor sensing mechanism with the aid of attractants or repellents could offer the greatest protection against the threat of mosquito bites, consequently reducing transmission rates. Saliently, since the use of repellents and attractants does not necessitate lifestyle changes on the part of the users nor require active supervision by medical professionals, they could prove to be highly effective and resource-efficient alternatives for controlling the spread of mosquito-borne illnesses [12,13].
Chemical interference of mosquito olfaction is an established concept, and the most well-known repellent, diethyltoluamide (DEET), has been on the market for over seven decades. However, despite its effectiveness, DEET is toxic [14,15]. Safer and possibly more effective mosquito repellants are preferred, but the current methodology used in the industry for identifying promising candidates – which involves the use of an instrument known as an olfactometer – is slow, encumbered and error-prone [16,17]. As a consequence, only a handful of new repellents have been introduced to the market in decades. The development of a more accurate platform for screening superior mosquito repellents and attractants could turn the tide in the fight to check the transmission rate of mosquito-borne diseases. To this end, the discovery of mosquito behavior-modifying compounds could borrow a page from the playbook of the pharmaceutical industry, which has benefitted immensely by ‘industrializing’ drug discovery. Industrialization refers to the acceleration of drug discovery via the miniaturization of assays and reactions, all under robotic control. In the same vein, the development of an accelerated platform for assaying chemical modulators of the mosquito’s sense of smell could resuscitate a previously dormant field.
Herein, we have laid the foundations for the development of a high-throughput assay for the detection of mosquito behavior-modifying compounds by re-factoring the olfactory pathway of the malaria-carrying mosquito Anopheles gambiae into the methylotrophic yeastPichia pastoris . The latter is extensively used in the biotechnology industry for the production of proteins and is rated as a model eukaryotic chassis for synthetic biology. Olfaction in all mosquitoes occurs in their antennae and maxillary palps [11]. These organs are covered in sensory hairs known as sensilla, and each sensillum hosts multiple olfactory receptor neurons (ORNs) that extend into a distinct peripheral dendrite [18]. Olfactory transduction commences with the diffusion of odor molecules or odorants through the pores in the sheath of the sensilla. Once inside the sensillum, the odorants then bind to a class of soluble enzymes called odor binding proteins (OBPs) [19,20]. Female A. gambiae mosquitoes, which transmit malaria, express 69 unique OBPs [21]. The OBPs subsequently shuttle the odorants to receptor proteins located on the surface of the peripheral dendrites of the ORNs. Mosquitoes and other insects express a variety of odor-sensing receptor proteins, including olfactory receptors (ORs), ionotropic receptors (IRs) and CO2-sensing gustatory receptors (GRs) [22]. Of these, ORs are the most well-studied group [23]. They are seven-transmembrane-helix proteins that exhibit an inverted topology [24,25], and female A. gambiae mosquitoes express 79 ORs [26]. Activation of the ORs by the OBP-odorant complexes, in turn, induces a downstream signaling cascade that activates G-protein complexes, which subsequently interact with adenyl cyclase (AC) and phospholipase C (PLC) to produce the secondary messenger molecules, cyclic AMP (cAMP), diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP3). These messenger molecules then trigger the opening of Ca2+ ion channels, thereby generating the transduction currents that are central to the mosquito’s sense of smell. In some cases, the odorants themselves can diffuse to the ORs and activate them without the participation of the OBPs [27].