*4.2.2 Mosquito salivary glands: Gatekeeper of entry and exit for the parasite*

The salivary glands are the crucial organ for the development and transmission of the *Plasmodium* to a vertebrate host. Salivary glands are paired epithelial organs that are located in the thorax, and consist of three lobes namely, two lateral and one median, where each lateral lobe is comprised of proximal and distal lobes [76, 77]. The proximal portion of the female glands produces enzymes involved in sugar metabolism, where distal lobes are shown to play roles in blood meal acquisition, *Plasmodium* invasion, and transmission. Although, studies suggest that only 10–20% hemolymph circulating sporozoites, manage to invade the salivary glands, however, the mechanism of this drastic reduction of 80–90% sporozoites is poorly

#### **Figure 3.**

*Direct interaction of free-circulatory sporozoites and hemocytes. Post oocyst maturation, sporozoites circulate freely in the hemolymph, in order to reach salivary glands for further transmission. But oocyst rupture triggers the mosquito immune system and activates the hemocyte proliferation, which leads to the sporozoite clearance by phagocytosis, melanization, and AMP (*Defensin *and* Gambicin*) production. Although the role of AMPs in hemocyte activation is still unclear.*

#### *Molecular Dynamics of Mosquito-*Plasmodium vivax *Interaction: A Smart Strategy of Parasitism DOI: http://dx.doi.org/10.5772/intechopen.96008*

known [25, 78]. Accumulating evidence highlights that sporozoite invasion into the glands is mediated by salivary specific receptor-ligand interactions [18, 79].

The sporozoites must leave (egress) the oocyst after maturation to invade the salivary gland and to be transmitted to the next vertebrate host. The egress of sporozoite is mediated by a protease named *Cysteine protease* (ECP1) which ruptures the oocyst [80, 81]. The sporozoites are released into the hemolymph and carried to the salivary glands by the circulation of hemolymph in an anterior direction from the abdomen to head, and facilitate the sporozoite invasion to the salivary gland [82]. The salivary gland epithelium forms a physical barrier that pathogens must cross, and *Plasmodium* parasites are evolved with unique proteins that drive invasion by first binding to the salivary gland specific surface receptors [83]. The salivary invasion process completion occurs in two stages, where first, sporozoite binds to invade the salivary gland basal lamina; and second, then interacts with the plasma membrane of the epithelial cells favoring sporozoite internalization. During the invasion, sporozoites attach and invade the distal and medial lobe of the salivary glands, and this attachment and invasion are highly specific to the nature of *Plasmodium* species [84, 85].

Empirical evidence showing that the salivary glands serve as an active immune organ is largely lacking, except some studies highlighting that a *Serine Protease Inhibitor* (SRPN6) produced in the salivary epithelium limits gland invasion by *Plasmodium sporozoites*, and thus *SRPN6* serves as an important salivary invasion immune-marker [86]. Several putative salivary encoded factors such as *Saglin*, *CSP* binding proteins which effectively binds with sporozoites surface antigens such as *TRAP*, *CSP* are well known salivary receptors for sporozoites invasion [87–91]. However, several other salivary factors such as *Plasmodium Responsive Salivary1* (PRS1), *ESP*, *Peptide-O-xylosyl Transferase 1* (OXT1) have also been identified to play a crucial role in parasite invasion of both midgut and salivary glands [92–95]. Once inside the salivary glands, the parasite undergoes transcriptional reprogramming before its transmission to the next mammalian host.

Transmission of many viral and protozoan parasites to a vertebrate host requires their salivary injection with the mosquito saliva during blood-feeding, and thus the migration of sporozoites needs duct for the continuation of the life cycle. Mosquito saliva has a pleiotropic property such as anti-hemostatic, vasodilator, or antiinflammatory properties and immune modulators, and basic function to facilitate blood-feeding [96–98]. However, saliva proteins can also have an indirect impact on pathogen development and transmission. For example, a recent study in mosquito *An. gambiae* shows that mosquito saliva proteins such as *AgTRIO* and *mosGILT* serve as an important mediator of the transmission of *P. falciparum*, and inhibition of this protein can reduce the parasite burden in the human host [99, 100]. Although, a major study on salivary-sporozoites interaction is restricted to *P. berghei* and/or *P. falciparum*, however, very limited information is available on the salivary-*P. vivax* interaction.

A comparative RNA-Seq analysis of uninfected and *P. vivax*-infected mosquito salivary glands suggests that salivary transcripts undergo substantial changes during *P. vivax* infection. The maturation of sporozoite seems to coincide with the change in gene expression essential for invasion and transmission. Findings of several classes of immune proteins such as *Heme-peroxidase*, *FADD*, *Gambicin*, *GNBP*, and multiple family proteins of *Serine proteases*, and *SCRC* in the *P. vivax* sporozoites invaded salivary glands highlighted their anti-*Plasmodium* immune role of salivary glands. The transcriptome of the infected salivary glands also revealed that *P. vivax* infection decreased the expression of apyrase significantly which suggests that *P. vivax* interferes with salivary secretion before probing and feeding to ensure their delivery into the next human host. These findings offer valuable new insights into the biology of malaria parasites. Manipulating tissue-specific immuno-physiology of the mosquitoes may halt the *Plasmodium vivax* development and hence the transmission (**Figure 4**).

#### **Figure 4.**

*Proposed hypothesis of salivary gland-*Plasmodium *interaction and transmission: Free circulating sporozoite in the hemolymph recognize and attach to the basal lamina of the salivary gland receptor-ligand interaction;(1) initial attachment of the sporozoite mediated by interactions of carbohydrate residues on the basal lamina with a parasite CS, SGS1, MABEL. CSP binding protein and* Saglin *bind with CSP and TRAP respectively are an important component of salivary gland invasion. (2) Sporozoite internalization: After invasion sporozoite passes into the secretory cavity and sporozoites begin to assembly there as a large bundle form. Within the salivary duct component of the mosquito immune responses* Gambicin*,* Cecropin*,* GNBP*, and* SCR *family members presumably act upon sporozoite and limit the number.*

### **5. Conclusion**

*Plasmodium* and mosquito host both are involved in the dynamic molecular relationship, where parasite tries to dodge the host immune system and utilize its nutrients for their successful proliferation/ transmission. On the contrary, the mosquito host immune system tries to restrict the parasite development and eliminate the remnants. During this ultimate battle, some host species defeat the parasite through its active immune system and become resistant but in others, the parasite smartly manipulates the host system and defends itself for successful transmission.

*P. vivax* is one of the neglected parasites which successfully manipulated the host system for its efficient transmission. *P. vivax* suppresses the microbiota proliferation to avoid nutritional competition as well as early immune responses. Different nutrient transport proteins like *Trehalase*, *Sterol Carrier*, *ApolipophorinIII*, etc. were modulated by the parasite for fulfilling its nutritional requirements. But still, mosquito hosts also developed species-specific immune effector molecules like *FREP50*, *FREP12*, *LRIM17*, etc. to block the parasite development. Likewise finding salivary-specific factors such as *Heme-peroxidase*, *SP24D* that are crucial to sporozoite invasion and survival, may further help to halt the progression of *Plasmodium* development and malaria transmission.

In summary, future functional exploration of the novel *P. vivax* specific host factors, will help in the development of transmission-blocking vaccines and the generation of new intervention techniques or modify current ones.

### **Acknowledgements**

CC, ST, SK, RKD conceptualize the idea and drafted MS. CC, ST, SK, PS, JR and RKD reviewed, edited, corrected and revised the MS. We would like to thank ICMR-NIMR and funding agencies, CSIR/UGC/DST for infrastructural and financial support to conduct the research. CC, ST, and SK are recipients of DST (DST/INSPIRE/03/2014/003463), UGC (22/12/2013(II)EU-V), and CSIR (09/905(0015)/2015-EMR-1), fellowships, respectively.

*Molecular Dynamics of Mosquito-*Plasmodium vivax *Interaction: A Smart Strategy of Parasitism DOI: http://dx.doi.org/10.5772/intechopen.96008*
