Olfaction plays a pivotal role in a mosquito's lifecycle, influencing vital functions such as finding food, mates, identifying hosts, and locating sites for laying eggs. However, a detailed catalog of the olfactory genes in mosquitoes has remained elusive -- until now. In this study, we compiled the olfactory genes catalog for four key malaria vectors: two major Indian species, Anopheles stephensi and Anopheles culicifacies, along with two African species, Anopheles gambiae and Anopheles funestus. Using an extensive genome-wide approach, we uncovered crucial carrier proteins like odorant binding proteins (OBPs), chemosensory proteins (CSPs), and several receptors, including odorant receptors (ORs), ionotropic receptors (IRs), and gustatory receptors (GusRs). A particularly striking discovery was the significantly higher number of OBP, OR, and IR genes in African malaria vectors compared to their Indian counterparts, hinting at the gene gain and functional diversification in these species. The invasive A. stephensi -- which has spread from Asia to Africa -- showed closer genetic ties to A. minimus and A. gambiae than to A. culicifacies. Furthermore, when examining the expression of CSPs and SAPs in the larval stage of A. stephensi, we found that pyrethroid-resistant mosquito larvae exhibited elevated expression of SAP2 and SAP3, providing new evidence of their potential role in insecticide resistance. This study not only sheds light on the genetic basis of mosquito olfaction but also offers crucial insights into how these genes are linked to different physiological functions, paving the way for improved malaria control strategies.

Mosquitoes belonging to the Anopheline (Anopheles) subfamily are part of a large Diptera: Culicidae family found across the inhabited world. Adult females rely on blood meals, predominantly from vertebrate hosts such as birds, frogs, mammals, and snakes, to complete their egg development. Among these hosts, humans have the unfortunate distinction of being a preferred blood meal source by certain Anopheles species which can lead to the transmission of parasites harbored by these mosquitos. Anopheles mosquitoes, in particular, are recognized as highly perilous hematophagous arthropods due to their ability to transmit the malaria-causing Plasmodium parasites. Tragically, malaria causes the loss of more than 0.4 million lives each year, inflicting a catastrophic toll on a global scale. World Health Organization (WHO) Report 2024 clearly shows that there has been a steady decline in global malaria deaths between 2000 and 2019. However, in 2023, the number of malaria-related deaths rose to 0.597 million, accompanied by an increase of 11 million new cases. The incidence rate also climbed, rising from 58.6 to 60.2 cases per 1,000 individuals at risk compared to 2022.

Their coexistence ensures sustained transmission throughout the year, posing challenges for malaria control. During the rainy season, when there is an abundance of water sources, A. gambiae population thrives. These mosquitoes prefer breeding in temporary rain-dependent pools and show a peak in their activity during this period. Just after rainy season dry season arrives and the occurrence of A. gambiae declines, and A. funestus replaces A. gambiae as the predominant mosquito species involved in malaria transmission. In India, the two most dominant Anopheles species, A. culicifacies and A. stephensi act as significant malaria vectors in rural and urban settings and are responsible for the majority of malaria cases. A. stephensi is an invasive malaria vector that is originally endemic in Asia and competent in transmitting both malaria parasites Plasmodium falciparum and Plasmodium vivax. There is increasing evidence that A. stephensi is expanding its geographical range, with the type form being found in Sri Lanka in 2016 and crossing from the Arabian Peninsula into Africa where it has been reported in Djibouti City in 2012, in Ethiopia in 2016 and 2018 and in 2019, it was detected in the coastal and sub-coastal regions of the Red Sea in Sudan. A. stephensi differs from other primary malaria vectors in its remarkable ability to survive and reproduce in urban environments. This species has a high degree of ecological plasticity, enabling it to adapt quickly to diverse local environments such as deep wells under extreme heat in the dry season and is capable of breeding in saline water as well as polluted water. Therefore, the emergence of this highly adaptable super mosquito can drive a new malaria surge across Africa, as the arrival of this a city-dwelling mosquito from Asia presents a serious risk to rapidly urbanizing Africa. As a result of this, WHO has raised an alarm about the invasion and spread of A. stephensi into Africa. The establishment of A. stephensi in Africa poses a potential threat to malaria control and elimination, which further has a global importance and impact.

Mosquitoes heavily rely on their sensory abilities to carry out vital tasks for their survival and reproductive success. One crucial sense they rely on is their sense of smell, or olfaction, which plays a critical role in various aspects of their life cycle. For instance, when it comes to obtaining food from plants (nectar), mosquitoes use olfaction to detect and locate sources of sugar-rich plant juices. The sense of smell is also essential for mosquitoes to find suitable mates. Male mosquitoes rely on their olfactory receptors to sense pheromones, which empowers them to navigate scent trails, gather with other males to form swarms, and seek out potential mates for reproduction. When it comes to blood feeding, olfaction is again crucial as female mosquitoes rely on its sensitive and efficient sensory system to find vertebrate hosts. Additionally, mosquitoes are attracted to carbon dioxide (CO) that humans and other animals emit during respiration. They have the ability to sense the concentration of CO in the air, which aids them in locating potential hosts for blood meals. Furthermore, olfaction plays a vital role in oviposition, or the process of female mosquitoes laying their eggs. They can sense chemical cues from water sources, which indicate the availability of suitable breeding sites for their offspring. These cues may originate from microorganisms or organic matter, such as decaying leaves, signaling the presence of nutrients necessary for the development and survival of mosquito larvae.

Mosquitoes possess three important structures dedicated to their sense of smell: the antenna, maxillary palps, and labella. These appendages house specialized receptors that enable them to detect odors within their chemical surroundings. Among these structures, the mosquito antenna takes on the crucial role of being the primary organ responsible for smelling. Within the intricate olfactory system of mosquitoes, three primary categories of receptors play significant roles: odorant receptors (ORs), ionotropic receptors (IRs), and gustatory receptors (GusRs). These receptors are expressed by the olfactory neurons present in the sensory hairs, known as sensilla, located on the olfactory appendages. It's important to note that each of these receptor classes comprises multiple subunits, collectively forming a functional ion-gated channel. OR consists of a tuning receptor (ORx) and a conserved co-receptor (Orco). The IR consists of a tuning receptor (IRx) which confers substrate (ligand) specificity on the neuron, in addition to one or more coreceptors (Ir8a, Ir25a, and/or Ir76b) which are highly conserved. GusR complexes consist of 3 subunits (Gr22, Gr23, and Gr24) which together sense CO and sugars. On the other hand, ORs are sensitive to compounds like esters, alcohols, and ketones, while OBPs respond to various amines and acids. Besides these three classes of receptors, there are some water-soluble accessory proteins such as chemosensory proteins (CSPs), and odorant binding proteins (OBPs) expressed by the support cells near the olfactory neurons at the base of the sensilla. These proteins are involved in transporting odorants to the ORs at the dendritic interfaces. OBP-encoding genes exhibit significant variation in number across insect species, with certain mosquitoes possessing more than 100 genes while some ant species have only 13. Studies indicate that not all OBPs have the same function. Some are crucial for the olfactory system, while others have a modulatory impact or no function. Additionally, OBPs are found to be associated with various processes, such as odorant release, development, regeneration, and physiological pathways. Both OBPs and CSPs are small compact polypeptides composed mainly of α-helical domains and all these proteins possess hydrophobic binding cavity. The structure of OBPs is characterized by three disulfide bridges (interlocked) between conserved cysteine residues, in contrary CSPs have two disulfide bridges between adjacent cysteines. Interestingly it was found that some members of the CSP family, especially Sensory Appendage Proteins (SAPs) are upregulated upon insecticide exposure. A recent study on A. gambiae demonstrated that SAP2 is highly expressed in the legs of pyrethroid (commonly used insecticide of mosquito net) resistant mosquitoes. It was further observed that overexpression of the SAP2 results in resistance to pyrethroid, and knockdown of gene results in pyrethroid susceptibility, indicating a direct relation between SAP2 and pyrethroid resistance.

Apart from the adult stages, Anopheles mosquitoes also depend on olfaction during the vulnerable larval stage for navigation and survival. This is achieved through the expression of olfactory receptors in the antennae and maxillary palps, although larval olfaction is still very poorly understood. A strong aversive response to harmful compounds such as acetophenone (produced by Pseudomonas), DEET (a potent adult mosquito repellent), and mosquito larval repellent VUAA1 suggests Anopheles larvae possess chemosensory receptors. Electrophysiological studies of the larval antennae revealed the population response properties of larval olfactory neurons. The larval antenna is composed of a sensory cone and peg organ which are essential for olfaction and gustation.

Since olfactory genes serve a variety of functions in mosquitoes, identifying and understanding the structure and function of these genes presents a lucrative target for developing effective countermeasures to develop effective vector control strategies. We conducted this study to identify and catalog all olfactory genes of two major Indian malaria vectors and two African malaria vectors. Additionally, we delved into the evolutionary aspects of these genes, examining gene collinearity, domain duplication, and the selection pressure acting on these genes. Moreover, we pinpointed crucial odorant-binding proteins (OBPs) specific to different life stages, sexes, and post-plasmodium infection in the mosquito vector. We also examined the expression of SAP and CSP in both larval and adult stages of Anopheles stephensi, and our findings suggest that SAP2 is likely associated with insecticide resistance.