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].