**4.2** *P. vivax* **infection and immune strategy of the** *Anopheles stephensi*

As described earlier, the parasite population undergoes several bottlenecks throughout their development inside the mosquito host. These bottlenecks are achieved because of the mosquito immune system [26]. Once the *Plasmodium* parasite transforms in the ookinete, midgut nitration modifies the parasite surface, which is then recognized by the hemocyte encoded pattern recognition receptors (like *TEP1*) circulating in the hemolymph [52]. Studies in the mosquito *An. gambiae* suggest that the complex of *LRIM1/APL1C* and *TEP1* bind to the parasite surface and activate the complement system, and in turn, the circulating hemocytes kill the parasite through cell lysis, phagocytosis, melanization, etc [53–55]. This whole phase is completed within 24 hours after infective blood meal uptake and is known as the

#### **Figure 2.**

*Systematic representation of events occurring during early and late phase immunity in malaria parasiteinfected mosquito: Once the ookinete invade the midgut epithelium, PRRs (pattern recognition receptors) like*  TEP1 *recognize the pathogen and activate the complement system, which further triggers the hemocytes for phagocytosis, melanization, etc.*

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

"early phase" immune response (**Figure 2**). Once the ookinetes reach the midgut epithelium, they get transformed into oocysts, and the immune system working against these transformed parasites is known as "late phase" immune response [56, 57]. Although very little is known about this phase but recent literature suggests that *LL3* mediated hemocyte differentiation, and *STAT* pathway activation, together helps in the restriction of the oocysts development [58]. Post oocysts maturation, millions of sporozoites evade the midgut lamina and circulate in the hemolymph, in order to reach and invade salivary glands for their successful transmission. Current literature suggests that among thousands of sporozoites only 19% can successfully invade the salivary gland, the rest are eliminated by the hemocyte mediated mosquito immune system [25]. But we have very limited information about this direct cell (hemocytes) cell (free circulating sporozoites) interaction and elimination mechanism [29].

Altogether this information is restricted to the model organisms, and due to problems in culturing of *P. vivax* and extraction of hemocytes the exact speciesspecific interaction biology of this neglected parasite is still unknown [29]. As hemocytes play a crucial role in immune regulation, decoding the direct or indirect immune interactions between hemocytes and *P. vivax* parasite, will help us to figure out the parasite population control strategies of the mosquito hosts.

#### *4.2.1 Hemocytes: the cellular immune army of the mosquito host*

Mosquitoes have an open circulatory system, and hemocytes are the tiny blood cells circulating across the body reaching every mosquito tissue. These are the major immune elicitors working against a diverse range of pathogens [29]. Hemocytes are the core of the mosquito immune system which can induce both cellular as well as humoral immune responses [30, 59, 60]. Mosquito hemocytes population can be discriminated on the basis of their anatomical location (circulatory and sessile), DNA content (euploid and polyploid), morphology, and functions (granulocytes, oenocytoids, and prohemocytes) [61–63]. Granulocytes are the phagocytic cells, which engulf the invaded parasite and kill them by lysozyme activity [64, 65]. Oenocytoids are the producers of the *Pro-phenoloxidases*, the rate-limiting enzyme of the melanization pathway [66]. Melanization is the systematic enzymatic process, which ultimately produces the melanin protein. When a foreign invader infects the mosquito, hemocytes cover the parasite in the melanin envelop, which will cut-off the parasite from the outside environment, nutrition, and also induces oxidative stress which results in the killing of the parasite. Prohemocytes are considered as the progenitor cells, which produce granulocytes and oenocytoids, although the actual function is not known yet about these tiny cells [64, 67]. Previous literature illustrated various hemocyte encoded molecules, like *TEP1*, *FBN30*, *LRR3*, etc. are vital for the early and late phase immune responses [55, 68–71]. Researchers have also successfully tracked the involvement of phagocytosis and melanization events for the removal of parasites [17]. But we do not have much information about the direct cell–cell interaction of the hemocytes and *P. vivax* free-circulating sporozoites (*fcSPZ*).

Recently we conducted a transcriptome based study, to understand that how hemocytes control the *P. vivax* free circulatory sporozoites (*fcSPZ*) population before salivary invasion [24]. Here we found that hemocyte encoded transcripts undergo a major shift during *P. vivax* infection. A detailed comparison of the *P. vivax* infected and uninfected hemocyte transcriptomes revealed that transcripts of organelle organization and riboprotein complex biogenesis have exclusively emerged during *P. vivax fcSPZ* infection. Altogether these findings suggested that the hemocyte population undergoes dynamic changes i.e., differentiate and increase the population in response to the *fcSPZ.* Through the immune database comparison,

we found that AMPs like *Defensin and Gambicin* were exclusively induced when *fcSPZ* were circulating in the hemolymph. These findings were further validated by the real-time based experiments and depicted that *Defensin3* and *Gambicin* may likely play a crucial role during *P. vivax* late-phase immune function against *fcSPZ* infection. Hence, conclusively current findings illustrate that hemocytes rapidly proliferate and impart humoral immune responses against the parasite to limit the *fcSPZ* population before salivary gland invasion (**Figure 3**).

Apart from global transcriptomic changes undergone by the hemocyte population to manage *P. vivax* infection, we also found the species-specific molecular differences among the hemocyte encoded immune transcripts. *FBN9* which was previously considered as the potent anti-*Plasmodium* molecule and showed multifold upregulation during *P. berghei*/ *P. falciparum* infection [71, 72] was found to be downregulated during *P. vivax* infection. Novel molecules like *FREP12* and *FREP50* were predicted to be involved in the clearance of *P. vivax* sporozoites. Furthermore, storage proteins like *ApolipophorinIII*, *Hexamerin* were also found to be highly induced during *P. vivax* oocysts development, which further supported the previous evidence of host nutrient scavenging by the maturing oocysts [73–75].
