**3.** *Plasmodium* **population dynamics and their immune regulation in the mosquito host**

During *Plasmodium* development inside the mosquito host, the parasite population undergoes various bottlenecks. Previous investigations demonstrated that if a female mosquito takes ~1000 gametocytes through its infected blood meal, ~100 can be transformed into ookinetes, and among them, only 1–5 can successfully form oocysts. Furthermore, these survived oocysts will form millions of sporozoites, but only 19-20% can successfully invade the salivary glands for further transmission [25]. In refractory strains, not a single ookinete could transform into oocysts [26]. In general, a substantial loss of parasite population occurs at each developmental stage of the parasite, and this major parasite loss can be attributed to both human as well as mosquito components, which are harmful to *Plasmodium*.

The human component includes cytokines, complement protein, and reactive nitrogen species that are ingested along with the gametocytes during blood meal intake, and detrimental to the parasite within the midgut lumen of vector [26]. During the parasite transition through midgut epithelium, the mosquito mounts early immune response by increasing midgut nitration and activation of the signaling pathway. The nitration process modifies the ookinetes surface, and mark them to be recognized by the mosquito complement system when they emerge toward the basal side of the midgut [27]. Signaling pathways provide varying responses to various species of *Plasmodium*, such as the IMD pathway acts more efficiently against *P. falciparum* than *P. berghei*, and the Toll pathway is more responsive against *P. berghei*, and *P. gallinaceum* [28]. The proliferation of microbiota following blood meal also exacerbated the mosquito immune response, which in turn is detrimental to parasite development. Plasmodium parasite faces population bottlenecks throughout their development (in vertebrate as well as invertebrate host) but the mosquito midgut serves as the major site of extermination, where the number of parasites is minimal during the oocyst stage which makes it the most susceptible stage to identify molecular targets to disrupt the transmission [26]. Parallel to gutimmune interaction, several factors have been identified from mosquito hemocyte and salivary glands that interact with *Plasmodium sporozoites*; a bulk of literature is available on the mosquito innate immune system against *P. berghei* and *P. falciparum*, and therefore readers may refer to many excellent reviews [29–31]. Here we update the reports on the Mosquito-*P. vivax* interactions, and highlight their relevance for future implications.

### **4. Mosquito-***P. vivax* **interaction**

Undoubtedly, advanced omics technologies, especially genome sequencing and transcriptome analysis, has now become a basic method in living organisms for the assessment of genome-scale gene identification. The expression of large scale identified genes is currently being explored to decode the molecular complexity of *P. vivax* development in the vertebrate host. Earlier, a high-density tiling microarray-based study showed the gene expression variation of *P. vivax* from human

and mosquito stages such as sporozoites, gametes, zygotes, ookinetes, and *in-vivo* asexual blood stages. Their comparison to *P. falciparum* and *P. yoelii* further reveals conserved and species-specific patterns highlighting the metabolic state of parasites growing within humans and identifies many orthologs of *P. falciparum* transcripts that are needed for exoerythrocytic development, which may also likely help in hypnozoite formation in the *P. vivax* [32].

### **4.1** *Plasmodium vivax* **strategy to adapt in the mosquito** *Anopheles stephensi*

The successful development of *P. vivax* within the midgut of a susceptible strain of *Anopheles stephensi* can be divided into two phases: pre-invasion (within midgut lumen) and post-invasion strategy i.e. development of oocyst stage which depends upon the nutrient availability within the host. During the pre-midgut invasion phase, *P. vivax* imparts an intricate mechanism to evade the mosquito immune response. It indirectly attenuates the mosquito immune response by dramatically suppressing the bacterial population, and whereas in the post- midgut invasion phase i.e. during the development of oocyst it modulates the expression of genes that are directly or indirectly involved with nutrition physiology to fulfill their nutritional requirement. We have limited information about the phases beyond oocysts maturation and their strategies to evade the mosquito immune system and promote their transmission.

### *4.1.1 Pre- invasion strategy of P. vivax*

The midgut of Anopheles mosquitoes is housed by a complex and diverse community of bacteria, protozoa, fungi, etc. collectively referred to as the microbiota, and this microbiota is believed to shape the vector competency of mosquito. The gut bacteria of Anopheles mosquitoes adversely affect the *Plasmodium* infection [33, 34]. These tripartite interactions have been studied between the mosquito, its microbiota, and the *Plasmodium* parasites, but the precise relationship between the three remains unknown.

Numerous research reports have revealed that microbiota of specific bacterial species, particularly gram-negative bacteria, in many *Anopheles* species have an inhibitory effect on various *Plasmodium* species. The elimination of midgut bacteria through antibiotic treatment enhances oocyst load and parasite prevalence in different species of Anopheles. There are two mechanisms by which microbiota interfere with the *Plasmodium* development in the midgut lumen:-(i) indirectly by triggering the immune response of the mosquito (Imd Pathway) that guides the synthesis of AMP and other immune effectors that interferes with the development of parasites, and (ii) directly by certain bacterial species producing the metabolites that interfere with *Plasmodium* development and survival [33]. Recently, we have demonstrated that *P. vivax* plays a unique strategy to steer clear off the mosquito immune response during its pre-invasive phase, by dramatic suppression of the gut-bacterial population [35] (**Figure 1**). This study hypothesizes that the parasites outcompete the midgut microbiota presumably by scavenging the iron from the blood meal which is necessary for bacterial growth [35].

#### *4.1.2 Post- invasion strategy of P. vivax (development of oocyst)*

During the *Plasmodium* transit through midgut epithelium within the susceptible strain of Anopheles, some of the ookinetes successfully manage to escape the mosquito immune response [36], and reach the basal lamina of midgut to further differentiate into oocyst, and rests there nearly for two weeks. The sessile oocyst

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

#### **Figure 1.**

*Alteration of midgut microbiota proliferation by* P. vivax*. Blood meal induces midgut microbiota proliferation within 24 hours. But during* P. vivax *infection, somehow this parasite restricts this microbiota proliferation after blood-meal, to avoid nutritional competition and immune defense exerted by the microbiota. This smart strategy of restriction helps the parasite to survive and proliferate better.*

stage is metabolically active, and follows an umpteen rounds of the nuclear division to transform into sporozoites. A single oocyst is capable of producing thousands of haploid sporozoites [37, 38]. Limited research has been undertaken on the underlying mechanism of *P. vivax* oocyst development (transition from a small oocyst of 7-8 μm to a large oocyst of 35-40 μm) in the mosquitoes. A few recent RNA-Seq analyses of *P. vivax* infected mosquitoes have been valuable to understand the ookinete and oocyst stage of *P. vivax* which reveals the alteration of several transcripts in the gut after 18 hours and 7 days post-infection in mosquito *Anopheles dirus* [39]. Notably, the authors identified several genes such as *Anoctamin 6* (ANO6; ADIR005670) and *Fibroblast Growth Factor* (FGF; ADIR008464), which may likely have immune regulation of *P. vivax* growth in the gut of the mosquito.

The parasite scavenges the nutrients from the host, and thus one of the main deciding factors of the infection outcome is likely dependent on the availability of nutritional resources of the host [40]. Our ongoing tissue-specific RNA-Seq analysis of *An. stephensi* infected with *P. vivax* oocyst identifies several unique sets of transcripts/genes, which have not yet find associated with any other *Plasmodium* infection. This study revealed the expression of genes involved in maintaining glucose homeostasis (*Trehalase*), nutrient transport (*Sterol Carrier* protein), energy, and nutrient homeostasis (*Folliculin*) during *P. vivax* infection [24]. We noticed that *P. vivax* infection modulates the *Trehalase* and *Sterol Carrier protein* expression in the midgut and salivary gland (SCP) for its own development and maturation. *Trehalase*, a glucosidase enzyme, catalyze the hydrolysis of disaccharide trehalose sugar into glucose units. Glucose is the main source of energy for the extensive proliferation of malarial parasites during both the blood and liver stages of malaria infection [41–44]. *Plasmodium* obtains the host glucose via hexose transporter. However, the role of sugar metabolism on *Plasmodium* infection in the mosquito vector remains poorly known. A multifold enriched expression of *Trehalase* transcript during early to late-stage oocysts in the gut as well as salivary glands, in addition to retrieval of *Plasmodium* hexose transcript in the midgut during oocyst stage, suggests that *Trehalase* may significantly contribute to hydrolyze the trehalose to provide glucose for the rapid proliferation of parasites, and also affect the reproductive capacity of adult female mosquito *An. stephensi* [45].

Similar to sugar requirement, *Plasmodium* also relies heavily on the host's cholesterol for its growth when maturing from small oocysts to large oocysts in the gut. Since *Plasmodium* is incapable to synthesize *de-novo* cholesterol [46], and *P. vivax* infection induces a multifold expression of SCP after seven days of infection in the gut, likely indicates its role in cholesterol transport. Currently, there is no functional correlation exists between SCP and *Plasmodium* infection, however, with the current observation of SCP enrichment in the midgut as well as salivary gland, we propose that besides a possible role of supplying cholesterol to developing oocyst, it is possible that *As-SCP* may impart an anti-*Plasmodium* immune response, as increased lipid droplets have been shown in the midgut of *Ae. aegypti* during bacterial and viral infection [47]. *Folliculin* (FLCN) is a tumor suppressor protein associated with Birt-Hogg-Dube(BHD) syndrome [48, 49]. It is involved in many biological processes including vesicular trafficking, energy, and nutrient homeostasis, and monitors E-cadherin protein level [50, 51]. Late induction of *FLCN* in response to *P. vivax* infection (unpublished) suggests that it might also play an important role in maintaining the integrity of midgut epithelial cells during oocyst bursting or acquisition of nutrients by developing oocyst, though further studies needed to support this hypothesis.
