**3. Vector-malaria parasite interactions**

#### **3.1 Mosquito immune defenses**

While the male anopheles mosquito feeds exclusively on plant nectar, in addition to feeding on plant nectar, the female anopheles mosquito requires blood to nourish and develop her eggs. During blood feeding, she's exposed to malaria parasites (gametocytes), which must complete its complex developmental life cycle inside a mosquito host. The mosquito vector risks infection when there is physical injury to its cuticle or following cuticular degradation by the parasite. However, infection can be limited or reduced by mounting immune (innate and humoral) responses mediated by pattern recognition receptors and factors that trigger parasite killing via lysis, melanization (deposition of melanin on the surface of invading pathogens), and hemocyte-mediated phagocytosis. Further, many other mosquito molecules have also been reported to limit infection in the primary mosquito compartments which pathogens inhabit i.e. the midgut, the hemocoel, and the salivary glands.

*Vector-Parasite Interactions and Malaria Transmission DOI: http://dx.doi.org/10.5772/intechopen.105025*

#### *3.1.1 Midgut*

Upon ingestion of erythrocytes, cibarial armatures which mosquitoes use for RBC lysis are the first barriers faced by pathogens before they reach the midgut. Although the cibarial armature is effective in limiting infection by large metazoan parasites, it is not very effective at destroying protozoan parasites such as the malaria parasite [6, 7]. Transformation of ookinete to oocyst in the midgut is drastically reduced following lytic and melanization events. A number of molecules have been found to either facilitate or inhibit the parasite development within the midgut. They include the protein alanyl aminopeptidase N (AnAPN1); a surface recognition molecule which acts as a receptor for the malaria parasite in the mosquito midgut [8], a thioester-containing protein (TEP1), and leucine-rich repeat immune protein, (LRIM1) which recognizes the invading ookinetes at the basal lamina which surrounds the mosquito midgut and trigger immune responses [9, 10]. On the other hand, molecules such as C-type lectin 4 (CTL4), caspar, and cactus have been reported to negatively regulate the immune response of the mosquito, as silencing of these proteins resulted in decreased oocyst count [11, 12]. In *A. gambiae* midgut, (CTL4) and C-type lectin mannose-binding 2 (CTLMA2) negatively regulates the melanization of *Plasmodium berghei* ookinetes [13]. Further, the Serine protease inhibitor serpin 2 (SRPN2) also facilitates midgut invasion through inhibition of lysis and melanization [14, 15].

#### *3.1.2 Hemocoel*

The hemocoel is a nutrient-rich medium containing immune surveillance cells known as hemocytes. Hemocytes can be grouped into two sub-populations; granulocytes and oenocytoid. The granulocyte sub-population is capable of phagocytosing pathogens. Thioester-containing proteins (TEPs) are hemolymph proteins involved in the killing of *Plasmodium* ookinetes. The most studied (TEP) is the hemocyteproduced phagocytosis enhancer (TEP1). (TEP1) gets activated by complexing with the leucine-rich repeat containing proteins (LRIM1) and (APL1C) after which it opsonizes ookinetes for destruction by phagocytes [16]. Genetic variations in (TEP1) and (APL1C) are reported to affect mosquito immune competence against the parasite [3, 17].

Oenocytoids constitute the remaining population of the hemocytes. They are known to secrete enzymes of the melanization pathway (such as phenoloxidase and phenylalanine hydroxylase) used by mosquito to kill pathogens. Although the mechanism of pathogen killing by melanization remains unclear, it has been suggested that killing could either be the result of oxidative stress generated by unstable intermediates during melanogenesis or the result of starvation since melanization isolates the pathogen from the nutrient-rich hemocoel [18, 19]. In a literature search, Sreenivasamurthy et al. [20] identified a total of 22 molecules which play a role in melanization of ookinetes within the mosquito midgut.

#### *3.1.3 Salivary gland*

Sporozoites that successfully break through the mosquito immune defense system in the midgut lamina migrate to the salivary gland via the hemolymph. This they must do for transmission to occur. About 80–90% of sporozoites are reportedly lost during migration through the hemolymph. The mechanism by which this occurs is however

not fully understood [21]. The invasion of the mosquito's salivary gland has been reported to be triggered by effective and specific associations of sporozoite surface antigens such as thrombospondin-related anonymous protein (TRAP), with receptors such as saglin present on the salivary glands of the mosquito [14]. Using knockdown assays, Cui et al. [22] showed that four genes {AGAP006268 (peritrophin), AGAP002848 (Niemann-Pick Type C-2) (NPC-2), AGAP006972 (keratin-associated protein 16–1), and AGAP002851 (NPC-2)} play a crucial role in protecting the mosquito from parasite invasion whereas three other genes {AGAP008138 (uncharacterized), FREP1 (*fibrinogen-related protein 1)*, and HPX15 (Heme peroxidase)} facilitated *P. falciparum* transmission to mosquitoes.

#### **3.2 Parasite strategies for evading mosquito immune defenses**

### *3.2.1 Midgut invasion*

The malaria parasite must evolve mechanisms to evade the barriers put in place by the mosquito for successful completion of its life cycle which is an absolute requirement for parasite survival and effective transmission. A *Plasmodium falciparum* surface protein Pfs47 protects the parasite from the immune system of the mosquito in the midgut [23]. The result of the study by Molina-Cruz et al. [23] suggest that *Plasmodium falciparum* Pfs47 haplotypes dictate vector compatibility. The researchers demonstrated that *A. gambiae* fails to mount a proper immune response against several *P.falciparum* lines including NF54 and GB4 partly because of Pfs47 which mediates immune evasion by disrupting JNK/caspase-mediated apoptosis in the mosquito midgut [24]. Whereas, evasion of the complement-like response in *Anopheles coluzzii,* (a dominant species of the *An. gambiae* complex in West Africa) is mediated by the protein Plasmodium Infection of the Mosquito Midgut Screen 43 (PIMMS43) which is present on the surface of ookinete and sporozoite [25].

#### *3.2.2 Salivary gland invasion*

Once sporozoites are released from the oocyst, they migrate to the salivary gland via the hemocoel [21]. Salivary gland invasion is a key step in the life cycle of the parasite since changes that take place on the sporozoites surface proteins in the salivary gland enable them to invade the salivary gland of the mosquito and also to be successfully transmitted. The proteins, *Plasmodium* responsive salivary 1 (PRS1), epithelial serine protease (ESP), peptide-O-xylosyltransferase 1 (OXT1), and a serine protease inhibitor (SRPN6) have been shown to play crucial roles in parasite invasion of both midgut and salivary glands. While (SRPN6) limits salivary gland invasion by *Plasmodium* sporozoites [26], knocking down PRS1, ESP, retinoid and fatty-acid binding glycoprotein (RFABG) and (OXT1) have been reported to decrease oocyst and sporozoite numbers [27–30]. Further, malaria parasites carrying mutations in conserved region II of the circumsporozoite protein (CSP) are unable to escape the oocyst [31]. Deletion of TRAP and LIMP (a highly conserved protein in *Plasmodium* parasites) severely impairs gliding motility which is important for salivary gland invasion [32, 33]. Whereas, although deletion of rhoptry neck protein 2 (RON) does not affect parasite's gliding motility, salivary gland invasion is abolished [34].
