**6.2 Neurological control over salivation**

*Sino-Nasal and Olfactory System Disorders*

is still in infancy.

**6.1 Mosquito sialome leads to feeding success**

with the salivary gland action for successful food uptake. The endocrine system of the salivary gland induces saliva secretion that gets mixed with foods and facilitates the food intake [85]. Mosquitoes have paired salivary glands in their thorax which is flanking in the esophagus. During sugar feeding saliva is mixed with the sugar, and the mixture enters into the crop where digestion commenced. During blood meal ingestion, salivary gland secretions serve in blood vessel localization [86]. The hemostatic and immune factor of the vertebrate host makes the blood meal uptake process challenging for mosquitoes [86]. Thus, salivary glands of mosquitoes are evolved and adapted with a unique ability to serve the leading function during blood meal ingestion by providing secretory salivary factors such as vasodilators, anticoagulant, antihistamines, etc. [86, 87]. Furthermore, salivary gland components not only support mosquitoes to overcome host homeostasis and defense response but also serve as the primary route for parasite transmission and maintenance of disease cycle [86, 88]. Due to the involvement of salivary gland in malaria transmission, most of the previous studies are restricted to the role of salivary gland in blood feeding and pathogen survival [89]. A recent study by Sharma et al. showed that salivary gland has a distinguished ability of gene expression switching to manage the meal-specific (sugar vs. blood meal) molecular responses [90]. But, our understanding of the regulatory mechanism of the neuro-olfactory system modulating salivary gland cocktail composition depending on the type of food

To feed on a vertebrate host, the arthropods are required to overcome a series of obstacles [86]. The saliva produced by the hematophagous insects contains bioactive molecules that counteract host defense [91]. Mosquitoes are reported to feed on arterioles and venules rather than capillaries, and they often probe multiple times at different sites to find a suitable site for feeding [86]. Initiation of feeding induces hemostatic cascade within the host including the platelet aggregation followed by collagen interaction with ADP which supports the blood coagulation pathway [87, 92]. The presence of secretory apyrase enzyme in the salivary gland of blood-feeding arthropods inhibits platelet aggregation by hydrolyzing ATP and ADP into AMP and inorganic phosphate [93]. Vasoconstriction is a common phenomenon following laceration of blood vessels due to insect bite to minimize blood flow and hence loss of blood [87]. The hematophagous insects, including *Aedes aegypti* mosquito saliva, contain sialokinins which act as a vasodilatory molecule by stimulating nitric oxide (NO) production by the endothelial cells via cGMP induction [87, 94, 95]. Except apyrase and sialokinin, salivary specific D7 family proteins have been implicated to function as a scavenger molecule of serotonin, histamine, and norepinephrine and antagonize their vasoconstrictor, plateletaggregating, and pain-inducing properties [94, 96]. Salivary peroxidases are well known for their potent function as a vasodilator, as it might act as a hydrogen peroxide-dependent destructor of serotonin and noradrenaline [97]. Furthermore,

the secretory anophelin protein is reported to inhibit thrombin activity and collagen sequestration and hence delay platelet aggregation [98]. An additional challenge arises from the immune components of the blood meal itself which have been generated during previous exposure of mosquito bites [86]. Thus, successful blood feeding is dependent on the evolution of salivary composition possessing anti-immune molecules to suppress the action of host immune factors. Antitumor necrosis factor in female salivary glands is one of the crucial molecule that may

play anti-immune function in hematophagous insects [86].

**84**

The experimental evidence about the classical conditioning of salivation in dogs was demonstrated by Pavlov in the early nineteenth century [99, 100]. By definition, classical conditioning refers to the learning procedure where a conditioned stimulus (CS), for example, the sound of a bell, is paired with an unconditioned stimulus (US), such as food which eventually triggers salivation [100], although secretion of saliva is obligatory to facilitate feeding for majority of animals from invertebrates to vertebrates. However, the knowledge of classical conditioning of salivation is restricted to mammals and invertebrate cockroaches [99]. The salivary gland and the saliva make the bridge that joins the mosquito vectors, parasite, and the host together by facilitating blood meal uptake and parasite transmission [86]. But, the cellular and molecular mechanisms underlying the classical conditioning of salivation in mosquitoes remain unknown. Considering the finicky host-seeking behavior of mosquitoes and their preference towards a certain host [47], it can be hypothesized that mosquitoes can learn during the repeated exposure of conditioned stimulus such as host odor and unconditioned stimulus, which is a reward of blood meal [47]. Reward may be appetitive when mosquitoes get benefited from the blood meal or aversive if mosquitoes experience any kind of host defensive behavior [47]. Thus, it can be speculated that mosquitoes should exhibit classical conditioning of salivation, i.e., increase saliva secretion which is tightly regulated by the neuro-olfactory system (**Figure 5**).

Our knowledge about control of insects' salivary secretion is limited to cockroaches, locusts, and blowflies, where neuronal innervation of the salivary gland or neuro-hormonal regulation was reported to play a significant role in salivation [101, 102]. Insects' salivary glands are innervated with nerves that are originated from different sources of the central nervous system [102]. Stomatogastric nervous system projects its nerves in the salivary gland of *Manduca sexta* [103]. The salivary gland of cockroaches (*Periplaneta americana*) is innervated with nerves that are projected from both the stomatogastric system and the subesophageal ganglion [102, 104, 105]. An exception to that is that the blowfly salivary glands are not innervated, but the salivary secretion is regulated by the secretion of the biogenic amine serotonin [102, 106]. Gustatory stimulation leads to the release of

#### **Figure 5.**

*Graphical illustration of conditioning of salivation in mosquitoes. Mosquitoes navigate towards vertebrate host through olfaction when they sense the odor plume emanating from the host (both appetitive and defensive). Olfaction also induces the salivary secretion (conditioning of salivation) with the aim to facilitate blood meal uptake. But the host's defensive behavior interrupts successful encountering of the mosquito with the host (redcolored human), which mosquitoes can memorize, and during consecutive exposure they probably restrict the salivation process to avoid the respective host, whereas mosquitoes get a reward from the appetitive host through successful blood-feeding without any interference. This positive memory along with olfaction further empowers the navigation process by induction of salivation.*

serotonin from the neurons into the hemolymph which acts as a neurohormone and alters the cytosolic calcium (Ca2+) and adenosine cyclic monophosphate (cAMP) concentration within the secretory cells of the salivary gland [107]. The increased calcium level consequently facilitates the movement of chloride (Cl<sup>−</sup>) ions from the hemolymph side into the lumen of the gland. On the contrary, cAMP was found to stimulate potassium (K<sup>+</sup> ) transport towards the luminal side of the salivary gland. The simultaneous induction of two different pathways leads to the activation of either phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP3)/diacylglycerol/ Ca2+ signaling pathway or cyclic AMP/adenylyl cyclase signaling cascade which

#### **Figure 6.**

*The tripartite communication of three tissues [olfactory tissue (OLF), central nervous system (CNS)/brain, and salivary gland (SG)] for successful feeding. The left picture showed the flow of signal from odor response to salivary action, which is indicated by the downward arrows. The right picture is the detailed representation of the left one. Primarily, odorants bind with their cognate receptors, present on the dendritic membrane of the olfactory receptor neurons (ORNs). Odor binding initiates the downstream signal transduction procedure, which includes the synthesis of either the second messengers (cAMP, IP3) or change in the membrane ion channel conformation which then allows the flow of ions (Na+ , Ca+ , and K+ ) and facilitates the change in membrane potential and consequently generates the action potential. This action potential rapidly moves through the axons towards the CNS (indicated as red arrow). The antennal lobe (AL) is the primary site for odor perception in mosquitoes. The axons of the ORNs expressing the same receptors which bind to a particular odor molecule merge in a single AL (indicated by orange and blue rods). From the AL, the odor signal then transmitted to higher brain centers [mushroom body (MB), and lateral horn (LH)] through projection neurons (PNs). Along with the neuromodulator-mediated regulation, nerve innervation (originating from the higher brain region) also regulates salivation of the salivary gland in insects (indicated by red zigzag lines over the salivary gland). One of the biogenic amines, the 5-hydroxy tryptamine (5-HT), and its cognate receptor (highlighted in sky blue circle and purple rods) facilitate salivation. But this receptor-mediated downstream signal transduction events and the resultant change in salivary gland membrane potential is not known in the case of mosquitoes (highlighted in red circle). The involvement of other biogenic amine receptors (BAR) and neuropeptide receptors (NPR) in saliva regulation is also yet to be explored. DL, distant lobe; ML, medial lobe of the salivary gland.*

**87**

**Acknowledgements**

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

are the potent secondary messengers found to play a significant role in insect salivation [107, 108]. Studies in blood-feeding insects are limited to *Aedes aegypti* mosquito and on tsetse flies *Glossina pallidipes*, depicting the presence of serotonergic innervation in their salivary glands [109, 110]. Together it can be stated that both the biogenic amines and neuropeptides play a crucial role in insect salivation by modulating the salivary glands' ability to alter second messenger level and ion channel conformation. Furthermore, olfactory conditioning of salivation is directly linked to long-term memory formation which is accomplished by the active involvement of NO signaling for the induction of protein synthesis required for memory signature [111]. Salivary conditioning is also suitable to monitor the activity pattern of salivary neurons located in specific regions of the brain; thus this conditioning system will be suitable for the study of molecular mechanisms of learning and

Evolution and adaptation to blood-feeding behavior in adult female mosquitoes provided a natural mechanism for their reproductive success. Here, we propose a system biology approach which defines the harmonious actions of the olfactory, the brain, and salivary glands, regulating the complex feeding behavior of mosquitoes. However, deciphering the molecular basis on how mosquitoes meet and manage the conflicting demands of sugar feeding vs. blood-feeding and how olfactory conditioning of salivation commenced may lead to the identification of crucial molecular targets including different neurohormones, biogenic amines, neuropeptides, and their receptors for genetic manipulation. Functional genomics and the advancement of electrophysiological techniques illuminate our understanding of mosquitoes' sensory systems. Although it is challenging to identify the species-specific potential olfactory factors that play a pivotal role in mosquitoes' host-seeking and blood-feeding behavior, it will be very effective for the development of novel approaches to control different mosquito populations. The efficacy of emerging genetic tools such as CRISPR/Cas9, a gene drive technology in mosquitoes, can facilitate the molecular understanding of neuronal mechanism of olfactory selection and differential learning and memory formation across different mosquito species which can be manipulated for more effective disruption of host-seeking behavior. Furthermore, unraveling the microbiome-gut-brain-axis communication mechanism during metabolic switch in mosquitoes may enlighten the innovative idea of microbiome-mediated alteration of mosquitoes' olfactory perception.

We are thankful to the Indian Council of Medical Research (ICMR), Government of India (No. 3/1/3/ICRMR-VFS/HRD/2/2016), and Tata Education and Development Trust (Health-NIMR-2017-2101-03/AP/db) for financial and infrastructural support to conduct the research at NIMR. Tanwee Das De is the recipient of the ICMR postdoctoral fellowship scheme (3/1/3/PDF(18)/2018-HRD).

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

memory formation in mosquitoes' brains (**Figure 6**).

**7. Conclusion and future direction**

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

are the potent secondary messengers found to play a significant role in insect salivation [107, 108]. Studies in blood-feeding insects are limited to *Aedes aegypti* mosquito and on tsetse flies *Glossina pallidipes*, depicting the presence of serotonergic innervation in their salivary glands [109, 110]. Together it can be stated that both the biogenic amines and neuropeptides play a crucial role in insect salivation by modulating the salivary glands' ability to alter second messenger level and ion channel conformation. Furthermore, olfactory conditioning of salivation is directly linked to long-term memory formation which is accomplished by the active involvement of NO signaling for the induction of protein synthesis required for memory signature [111]. Salivary conditioning is also suitable to monitor the activity pattern of salivary neurons located in specific regions of the brain; thus this conditioning system will be suitable for the study of molecular mechanisms of learning and memory formation in mosquitoes' brains (**Figure 6**).
