**4.2 Odorant receptors**

After the OBPs, the principal molecules in odor detection are odorant receptors that convert the chemical signal into electrical outputs and therefore ensure the continuous flow of information from the environment to the insect brain [31–33]. Within the insect phylum, odorant receptors (Ors) were first identified and characterized from the model insect *Drosophila melanogaster*, using an intensive bioinformatics approach [34, 35]. Insects' OR proteins consist of seven transmembrane domains with inverted topology, where N-terminus is intracellular, as compared to mammalian odorant receptors, which are the conventional G protein-coupled receptors (GPCR) [36, 37]. Further experimental evidence suggested that mosquito ORs act as ligand-gated ion channels comprising of heteromeric complexes of two subunits [38, 39]. One subunit is highly conserved and known as olfactory receptor co-receptors (Orco), and the other subunit is largely divergent in terms of number as well as amino acid sequences (Orx) [31, 40–42]. Pilot studies of the OR gene repertoire primarily in *Drosophila melanogaster* [35] and later in mosquitoes [42, 43] suggest that despite having a limited number of ORs, mosquitoes can respond to an array of varied chemicals depending on the specific demand at different life cycle stages [44]. This is possible due to the combinatorial coding mechanism of the insect's olfactory system which increases the perceived odor space of each species. Combinatorial coding increases odor sensitivity to several-fold, where each OR can respond to multiple ligands and a single ligand can activate more than one OR [45]. Moreover, a single odorant can either elicit attractive responses or activate repellent pathway depending on their quality and concentration which subsequently determine insects' behavior [46, 47].

The number of receptors present in each mosquito species is highly variable, e.g., *A. gambiae* genome contains 79 OR genes, *C. quinquefasciatus* has 117, and *A. aegypti* possesses 110 OR genes [47]. Among them the receptor protein Or8 is predominantly studied in mosquitoes because of its conserved nature and specificity towards 1-octen-3-ol, which is a crucial component of human sweat [48, 49]. Deorphanization of other olfactory receptors of mosquitoes was performed

**79**

*Neuro-Olfactory Regulation and Salivary Actions: A Coordinated Event for Successful…*

using an in vivo heterologous expression system, the "empty neuron" system, originally established in the fruit fly *Drosophila*. The "empty neuron" system is a combination of a GAL4 driver line and a mutant ORN line (UAS—"OR gene") where endogenous odorant receptor is missing and thus gives the opportunity to express and functionally characterize mosquito olfactory receptor gene repertoire. The complexes of both *Drosophila* and mosquitoes using "empty neuron" model indicated that the "odor space" of mosquito and flies is significantly distinct [50, 51]. Furthermore, over the evolutionary time scale, the sensitivity of a particular mosquito OR either increases towards certain predominant hosts or decreases if the host odor profile changes [47]. Thus, it is not difficult to predict that ORs have evolved with highly sensitive and selective property for the detection of diverse odorants which consequently facilitate mosquito adaptation in diverse ecology.

After detection of a particular odor through synergistic actions of OBPs and ORs, mosquitoes use other sensory modalities such as vision, thermosensation, and hygrosensation to make the differentiation between biting hosts [15, 47, 50]. For visualization, the photoreceptor cells expressing multiple UV-sensitive and longwavelength sensitive opsin proteins are responsible for detecting and transmitting visual information towards optic lobe (the region of mosquito brain where optic information is processed) [52]. But how mosquitoes integrate visual information with other cues to differentiate hosts remains unclear. Following visual selection, the temperature and humidity are intricately linked to make biting decision [47, 50]. The thermosensory transient receptor potential (TRP) channel protein present on the tips of the antennae of mosquitoes can sense the variation of temperature associated with vertebrate skin [53]. For hygrosensory information processing, the ionotropic receptors (IRs) are reported to play a crucial role in *Drosophila* [47]. Although the role of IRs in humidity sensing in mosquitoes remains elusive, few recent studies highlight their sensitivities against narrow range of odorants such as amines and carboxylic acids and thus have potential function in host-seeking [54]. Once the host is located by the harmonious actions of all the sensory modalities, the mosquito first lands over the host and engages in a mission of locating a proper site for probing by repeated contacting of the skin with the labellum [50]. The gustatory receptors (taste receptors) (GRs), expressing on the labellum and the tarsae (the last segment of their legs through which mosquitoes make contact with the host), may play a pivotal role in biting behavior of mosquitoes [47, 55]. While the functional characterization of mosquito OR genes are of prime focus, a significant number of studies reported that the putative gustatory receptors (Gr1, Gr3 in *A. aegypti* and *C. quinquefasciatus*; Gr22, Gr24 in *A. gambiae*) of mosquitoes are sensitive to CO2 and thus influence host-seeking behavioral activities [47, 50].

The information, i.e., hidden within the odor molecules, are amplified by activating the sensory neurons. The activation of a different subset of sensory neurons to a different degree is the basis for neuronal coding. When compared with the vertebrate OR, the insect's ORs show a high degree of variation with different topologies, which strongly suggest a different signal transduction mechanism [8]. Some previous studies highlight that olfactory signal transduction in insects involving a ligand-gated ion channel that is formed by the hetero-dimerization of diverse odorant receptor and its co-receptors [27]. This fast ionotropic response does not postulate the involvement of any G proteins and any intracellular second

*DOI: http://dx.doi.org/10.5772/intechopen.90768*

**4.3 Other sensory receptors**

**4.4 Olfactory signal transduction**

*Neuro-Olfactory Regulation and Salivary Actions: A Coordinated Event for Successful… DOI: http://dx.doi.org/10.5772/intechopen.90768*

using an in vivo heterologous expression system, the "empty neuron" system, originally established in the fruit fly *Drosophila*. The "empty neuron" system is a combination of a GAL4 driver line and a mutant ORN line (UAS—"OR gene") where endogenous odorant receptor is missing and thus gives the opportunity to express and functionally characterize mosquito olfactory receptor gene repertoire. The complexes of both *Drosophila* and mosquitoes using "empty neuron" model indicated that the "odor space" of mosquito and flies is significantly distinct [50, 51]. Furthermore, over the evolutionary time scale, the sensitivity of a particular mosquito OR either increases towards certain predominant hosts or decreases if the host odor profile changes [47]. Thus, it is not difficult to predict that ORs have evolved with highly sensitive and selective property for the detection of diverse odorants which consequently facilitate mosquito adaptation in diverse ecology.

#### **4.3 Other sensory receptors**

*Sino-Nasal and Olfactory System Disorders*

transport pheromones and different chemosensory proteins (CSPs) which are smaller in size but can bind with a broad spectrum of semiochemicals. In mosquitoes, the OBP genes are classified in three subfamilies: (i) classic OBPs that carry a conserved motif consisting of six cysteine residues; (ii) Plus-C OBPs which contain six additional cysteine residues with novel disulfide connectivity along with three classic OBP motifs; and (iii) atypical OBPs, the longest OBPs that contain a single classic OBP domain in its N-terminal which is extended by a C-terminal extension. Among these three subfamilies, Plus-C OBP class is more divergent in nature and has only been identified from Diptera *Anopheles*, *Culex*, and *Drosophila*; however, Hymenoptera and Lepidoptera did not possess these OBPs. The first OBP of mosquito origin was isolated from the antennae of female *Culex quinquefasciatus* (CquiOBP1) in the early twenty-first century [20, 23]. The availability of genome sequence of several mosquito species in the public domain facilitates the identification and characterization of this large family of OBP genes from different mosquito species, for example, the total number of 69 OBPs from *A. gambiae*, 111 OBPs from *A. aegypti*, 109 OBPs from *C. quinquefasciatus*, *and* 63 OBPs in *A. culicifacies* [20, 24, 25]. Activation of the chemosensory receptors by odorants also requires timely termination and desensitization of peripheral signaling to maintain sensitivity of ORN-based signaling [26, 27]. In this process, odorant-degrading enzymes (ODEs), particularly several esterases and cytochrome p450s, play a crucial role by terminat-

ing the odor-induced signal transduction processes [28–30].

After the OBPs, the principal molecules in odor detection are odorant receptors that convert the chemical signal into electrical outputs and therefore ensure the continuous flow of information from the environment to the insect brain [31–33]. Within the insect phylum, odorant receptors (Ors) were first identified and characterized from the model insect *Drosophila melanogaster*, using an intensive bioinformatics approach [34, 35]. Insects' OR proteins consist of seven transmembrane domains with inverted topology, where N-terminus is intracellular, as compared to mammalian odorant receptors, which are the conventional G protein-coupled receptors (GPCR) [36, 37]. Further experimental evidence suggested that mosquito ORs act as ligand-gated ion channels comprising of heteromeric complexes of two subunits [38, 39]. One subunit is highly conserved and known as olfactory receptor co-receptors (Orco), and the other subunit is largely divergent in terms of number as well as amino acid sequences (Orx) [31, 40–42]. Pilot studies of the OR gene repertoire primarily in *Drosophila melanogaster* [35] and later in mosquitoes [42, 43] suggest that despite having a limited number of ORs, mosquitoes can respond to an array of varied chemicals depending on the specific demand at different life cycle stages [44]. This is possible due to the combinatorial coding mechanism of the insect's olfactory system which increases the perceived odor space of each species. Combinatorial coding increases odor sensitivity to several-fold, where each OR can respond to multiple ligands and a single ligand can activate more than one OR [45]. Moreover, a single odorant can either elicit attractive responses or activate repellent pathway depending on their quality and concentration which subsequently deter-

The number of receptors present in each mosquito species is highly variable, e.g., *A. gambiae* genome contains 79 OR genes, *C. quinquefasciatus* has 117, and *A. aegypti* possesses 110 OR genes [47]. Among them the receptor protein Or8 is predominantly studied in mosquitoes because of its conserved nature and specificity towards 1-octen-3-ol, which is a crucial component of human sweat [48, 49]. Deorphanization of other olfactory receptors of mosquitoes was performed

**4.2 Odorant receptors**

mine insects' behavior [46, 47].

**78**

After detection of a particular odor through synergistic actions of OBPs and ORs, mosquitoes use other sensory modalities such as vision, thermosensation, and hygrosensation to make the differentiation between biting hosts [15, 47, 50]. For visualization, the photoreceptor cells expressing multiple UV-sensitive and longwavelength sensitive opsin proteins are responsible for detecting and transmitting visual information towards optic lobe (the region of mosquito brain where optic information is processed) [52]. But how mosquitoes integrate visual information with other cues to differentiate hosts remains unclear. Following visual selection, the temperature and humidity are intricately linked to make biting decision [47, 50]. The thermosensory transient receptor potential (TRP) channel protein present on the tips of the antennae of mosquitoes can sense the variation of temperature associated with vertebrate skin [53]. For hygrosensory information processing, the ionotropic receptors (IRs) are reported to play a crucial role in *Drosophila* [47]. Although the role of IRs in humidity sensing in mosquitoes remains elusive, few recent studies highlight their sensitivities against narrow range of odorants such as amines and carboxylic acids and thus have potential function in host-seeking [54].

Once the host is located by the harmonious actions of all the sensory modalities, the mosquito first lands over the host and engages in a mission of locating a proper site for probing by repeated contacting of the skin with the labellum [50]. The gustatory receptors (taste receptors) (GRs), expressing on the labellum and the tarsae (the last segment of their legs through which mosquitoes make contact with the host), may play a pivotal role in biting behavior of mosquitoes [47, 55]. While the functional characterization of mosquito OR genes are of prime focus, a significant number of studies reported that the putative gustatory receptors (Gr1, Gr3 in *A. aegypti* and *C. quinquefasciatus*; Gr22, Gr24 in *A. gambiae*) of mosquitoes are sensitive to CO2 and thus influence host-seeking behavioral activities [47, 50].

#### **4.4 Olfactory signal transduction**

The information, i.e., hidden within the odor molecules, are amplified by activating the sensory neurons. The activation of a different subset of sensory neurons to a different degree is the basis for neuronal coding. When compared with the vertebrate OR, the insect's ORs show a high degree of variation with different topologies, which strongly suggest a different signal transduction mechanism [8]. Some previous studies highlight that olfactory signal transduction in insects involving a ligand-gated ion channel that is formed by the hetero-dimerization of diverse odorant receptor and its co-receptors [27]. This fast ionotropic response does not postulate the involvement of any G proteins and any intracellular second

messengers. In contrast, another study indicated the entanglement of G protein and the synthesis of cAMP, IP3, and other secondary messengers that consequently induce the downstream effector enzymes and also affect the membrane potential through activating the co-receptor protein [27, 56]. The resultant change in the membrane potential/permeability by either process causes the generation and propagation of action potentials along the ORN axon membrane towards the antennal lobes. In contrast to the rapid ionotropic pathway, the G protein-mediated metabotropic pathway is slower. However, it plays an important role when the odor cues are present in lower concentration, whereas high concentration directly involves the ionotropic pathway [8, 27, 57].
