Abstract

Malaria continues to be one of the world’s most devastating diseases and is caused by parasites of the genus Plasmodium. In 2010, there were an estimated 219 million cases of malaria resulting in around one million fatalities, mostly in children under five years old (Murray et al. 2012Murray CJ, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D, Fullman N, Naghavi M, Lozano R, Lopez AD 2012. Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet 379: 413-431., WHO 2012WHO - World Health Organization 2012. World malaria report 2012. Available from: who.int/malaria/publications/world_malaria_report_2012/en/.
who.int/malaria/publications/world_malar... ). Increased awareness of the devastating impacts of malaria has led to a significant reduction of malaria cases and fatalities in the recent past. However, these achievements are threatened by a reduction in the distribution of insecticide treated nets, the resistance of mosquitoes to insecticides, parasite resistance to antimalarial drugs (including the 1st-line drug artemisinin) together with the levelling of funding for malaria control efforts (WHO 2012WHO - World Health Organization 2012. World malaria report 2012. Available from: who.int/malaria/publications/world_malaria_report_2012/en/.
who.int/malaria/publications/world_malar... ). For these reasons, there is an urgent need to improve current malaria control efforts and, importantly, to develop new strategies to eliminate and eventually eradicate the disease. Different from the other two major infectious disease killers, human immunodeficiency virus and tuberculosis, malaria is unique because it requires a mosquito vector for transmission to occur. Thus, the mosquito stages of the malaria parasite development have the potential to provide important targets for the control of transmission.

Parasite development in its vector starts when a mosquito ingests an infected blood meal containing Plasmodium sexual forms, known as gametocytes (Fig. 1). Within ~15 min, gametocytes round-up (in case of Plasmodium falciparum), egress from the red blood cell (RBC) and differentiate into gametes. Male gametocytes undergo a drastic transformation known as exflagellation by which the DNA replicates to 8N followed by the formation of eight haploid microgametes. Microgametes detach from the exflagellation centre and actively search for female gametes to fertilise. Fertilisation gives rise to a diploid zygote that subsequently undergoes one round of DNA replication to become tetraploid. Zygotes differentiate into motile ookinetes that migrate in the blood bolus to invade and traverse the mosquito midgut epithelium. The ookinete may traverse multiple epithelial cells before emerging from the basal side facing the haemocoel, where it lodges beneath the basal lamina and differentiates into a round oocyst. Within the next 10-14 days each oocyst grows in size and undergoes sporogony to produce thousands of sporozoites. Upon oocyst maturation, sporozoites are released into the haemolymph where they circulate with the haemolymph and specifically invade the salivary glands. Following invasion, sporozoites lodge in the lumen of the salivary gland. When an infected mosquito feeds on a human host, sporozoites are released with the saliva and deposited in the skin, thus closing the transmission cycle.

Malaria parasites undergo dramatic losses during their development in the mosquito vector (Fig. 1). Population reduction occurs at each developmental step, from the formation of gametocytes in the human host to oocyst formation, resulting in very low parasite numbers. In fact, even in high transmission areas, the majority of the mosquitoes are not infected by the parasite (Chege & Beier 1990Chege GM, Beier JC 1990. Effect of Plasmodium falciparum on the survival of naturally infected afrotropical Anopheles (Diptera: Culicidae). J Med Entomol 27: 454-458., Mbogo et al. 1993Mbogo CN, Snow RW, Kabiru EW, Ouma JH, Githure JI, Marsh K, Beier JC 1993. Low-level Plasmodium falciparum transmission and the incidence of severe malaria infections on the Kenyan coast. Am J Trop Med Hyg 49: 245-253., Mendis et al. 2000Mendis C, Jacobsen JL, Gamage-Mendis A, Bule E, Dgedge M, Thompson R, Cuamba N, Barreto J, Begtrup K, Sinden RE, Høgh B 2000. Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique. Med Vet Entomol 14: 171-180., Gouagna et al. 2010Gouagna LC, Bancone G, Yao F, Yameogo B, Dabiré KR, Costantini C, Simporé J, Ouedraogo JB, Modiano D 2010. Genetic variation in human HBB is associated with Plasmodium falciparum transmission. Nat Genet 42: 328-331.). This reduction in numbers in the mosquito midgut is mediated in part by the transition of the parasite from an intracellular (RBC) to extracellular forms, thus exposing the parasites to both human and mosquito components that are deleterious to the parasite (Figs 1-3). It has been estimated that out of the thousands of gametocytes that a female Anopheles mosquito typically ingests in a blood meal, only 50-100 develop into ookinetes and only around five survive to form oocysts (Gouagna et al. 1998Gouagna LC, Mulder B, Noubissi E, Tchuinkam T, Verhave JP, Boudin C 1998. The early sporogonic cycle of Plasmodium falciparum in laboratory-infected Anopheles gambiae: an estimation of parasite efficacy. Trop Med Int Health 3: 21-28., Sinden & Billingsley 2001Sinden RE, Billingsley PF 2001. Plasmodium invasion of mosquito cells: hawk or dove? Trends Parasitol 17: 209-212., Whitten et al. 2006Whitten MM, Shiao SH, Levashina EA 2006. Mosquito midguts and malaria: cell biology, compartmentalization and immunology. Parasite Immunol 28: 121-130.). A study performed in Anopheles gambiae mosquitoes with blood from P. falciparum-infected patients showed that on average, from 433.5 gametocytes detected in patient blood, only 12.6 round forms, 5.5 ookinetes, 1.8 early oocysts and two mid-size oocyst were detected in the mosquito (Gouagna et al. 1998Gouagna LC, Mulder B, Noubissi E, Tchuinkam T, Verhave JP, Boudin C 1998. The early sporogonic cycle of Plasmodium falciparum in laboratory-infected Anopheles gambiae: an estimation of parasite efficacy. Trop Med Int Health 3: 21-28.) (Fig. 1). More importantly, this study also shows that prevalence (the proportion of mosquitoes that carry at least 1 parasite) was only 38%, meaning that 62% of the mosquitoes that fed on the infected blood never got infected (Gouagna et al. 1998Gouagna LC, Mulder B, Noubissi E, Tchuinkam T, Verhave JP, Boudin C 1998. The early sporogonic cycle of Plasmodium falciparum in laboratory-infected Anopheles gambiae: an estimation of parasite efficacy. Trop Med Int Health 3: 21-28.). In the entire Plasmodium life cycle (in both human and mosquito hosts), parasite numbers are lowest during the oocyst stage and then quickly expand when each oocyst releases thousands of sporozoites. For this reason, the midgut stages of parasite development constitute prime targets for strategies aiming to block malaria transmission.

Here we discuss the multiple mechanisms that limit parasite survival in the mosquito, starting with events occurring in the human host, and then in the mosquito midgut lumen (Fig. 2), and finally during invasion of the mosquito midgut epithelium (Fig. 3). In addition, we describe the mechanisms that the parasite has evolved to evade some of these antiplasmodial responses. Finally, we address strategies that are under consideration to target Plasmodium development in the mosquito and discuss future challenges that need to be overcome in order to succeed in any malaria transmission-blocking (TB) strategy.

Human factors - Cytokines and reactive nitrogen species (RNS) - Evidence suggests that cytokines produced during malaria infections can mediate the killing of gametocytes circulating in the blood stream of the host (Naotunne et al. 1991Naotunne TS, Karunaweera ND, Del Giudice G, Kularatne MU, Grau GE, Carter R, Mendis KN 1991. Cytokines kill malaria parasites during infection crisis: extracellular complementary factors are essential. J Exp Med 173: 523-529., Karunaweera et al. 1992Karunaweera ND, Carter R, Grau GE, Kwiatkowski D, Del Giudice G, Mendis KN 1992. Tumour necrosis factor-dependent parasite-killing effects during paroxysms in non-immune Plasmodium vivax malaria patients. Clin Exp Immunol 88: 499-505.) (Fig. 2). Infections of Plasmodium cynomolgi in toque monkeys or Plasmodium vivax in humans cause a period known as “crisis” or clinical paroxysms that are characterised by acute fever preceded by chills and rigor that coincide with the release of asexual parasites from RBCs (Naotunne et al. 1991Naotunne TS, Karunaweera ND, Del Giudice G, Kularatne MU, Grau GE, Carter R, Mendis KN 1991. Cytokines kill malaria parasites during infection crisis: extracellular complementary factors are essential. J Exp Med 173: 523-529., Karunaweera et al. 2003Karunaweera ND, Wijesekera SK, Wanasekera D, Mendis KN, Carter R 2003. The paroxysm of Plasmodium vivax malaria. Trends Parasitol 19: 188-193.). During this period, Plasmodium gametocytes lose infectivity to mosquitoes due to an increase of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ produced by the host immune system (Naotunne et al. 1991Naotunne TS, Karunaweera ND, Del Giudice G, Kularatne MU, Grau GE, Carter R, Mendis KN 1991. Cytokines kill malaria parasites during infection crisis: extracellular complementary factors are essential. J Exp Med 173: 523-529., Karunaweera et al. 1992Karunaweera ND, Carter R, Grau GE, Kwiatkowski D, Del Giudice G, Mendis KN 1992. Tumour necrosis factor-dependent parasite-killing effects during paroxysms in non-immune Plasmodium vivax malaria patients. Clin Exp Immunol 88: 499-505.). Although not completely understood, two proposed mechanisms by which TNF-α and IFN-γ could affect Plasmodium gametocytes in the human host are through the induction of phagocytosis or by increased nitric oxide (NO) production by leukocytes (Naotunne et al. 1993Naotunne TS, Karunaweera ND, Mendis KN, Carter R 1993. Cytokine-mediated inactivation of malarial gametocytes is dependent on the presence of white blood cells and involves reactive nitrogen intermediates. Immunology 78: 555-562., Muniz-Junqueira et al. 2001Muniz-Junqueira MI, dos Santos-Neto LL, Tosta CE 2001. Influence of tumor necrosis factor-alpha on the ability of monocytes and lymphocytes to destroy intraerythrocytic Plasmodium falcipa- rum in vitro. Cell Immunol 208: 73-79.).

Similar to the effects of pro-inflammatory cytokines, the levels of RNS have also been shown to increase during the paroxysm periods of P. vivax infections (Naotunne et al. 1993Naotunne TS, Karunaweera ND, Mendis KN, Carter R 1993. Cytokine-mediated inactivation of malarial gametocytes is dependent on the presence of white blood cells and involves reactive nitrogen intermediates. Immunology 78: 555-562., Cao et al. 1998Cao YM, Tsuboi T, Torii M 1998. Nitric oxide inhibits the development of Plasmodium yoelii gametocytes into gametes. Parasitol Int 47: 157-166.). Induced by unknown parasite factors, the activation of leukocytes results in an up-regulation of NO synthase (NOS) expression and increased NO production (Naotunne et al. 1993Naotunne TS, Karunaweera ND, Mendis KN, Carter R 1993. Cytokine-mediated inactivation of malarial gametocytes is dependent on the presence of white blood cells and involves reactive nitrogen intermediates. Immunology 78: 555-562.). NO is a highly reactive molecule and its reaction products (RNS) induce damage to DNA, proteins and lipids that ultimately result in cell death. Supporting the role of NO, Naotunne et al. (1993)Naotunne TS, Karunaweera ND, Mendis KN, Carter R 1993. Cytokine-mediated inactivation of malarial gametocytes is dependent on the presence of white blood cells and involves reactive nitrogen intermediates. Immunology 78: 555-562. demonstrate that TNF-α-mediated gametocyte inactivation is dependent on leukocyte activation and the production of NO. The inhibition of NO synthesis restored the infectivity of P. falciparum and P. vivax gametocytes to mosquitoes (Motard et al. 1993Motard A, Landau I, Nussler A, Grau G, Baccam D, Mazier D, Targett GA 1993. The role of reactive nitrogen intermediates in modulation of gametocyte infectivity of rodent malaria parasites. Parasite Immunol 15: 21-26., Naotunne et al. 1993Naotunne TS, Karunaweera ND, Mendis KN, Carter R 1993. Cytokine-mediated inactivation of malarial gametocytes is dependent on the presence of white blood cells and involves reactive nitrogen intermediates. Immunology 78: 555-562.). Similarly, in mouse infections with Plasmodium yoelii, gametocyte infectivity to mosquitoes is highly impaired when mice experience crisis (4-5 days after infection) (Cao et al. 1998Cao YM, Tsuboi T, Torii M 1998. Nitric oxide inhibits the development of Plasmodium yoelii gametocytes into gametes. Parasitol Int 47: 157-166.). During this time, treatment of the mice with the NOS inhibitor L-NMMA partially restored gametocyte infectivity suggesting that RNS are involved in this inhibitory effect.

One important aspect of these studies is that serum from semi-immune patients, which present mild clinical symptoms, did not affect P. vivax gametocyte infectivity (Karunaweera et al. 1992Karunaweera ND, Carter R, Grau GE, Kwiatkowski D, Del Giudice G, Mendis KN 1992. Tumour necrosis factor-dependent parasite-killing effects during paroxysms in non-immune Plasmodium vivax malaria patients. Clin Exp Immunol 88: 499-505.). This would suggest that clinical immunity is an adaptation in which parasite-killing factors (e.g., TNF-α) are reduced in order to diminish disease pathology (Karunaweera et al. 1992Karunaweera ND, Carter R, Grau GE, Kwiatkowski D, Del Giudice G, Mendis KN 1992. Tumour necrosis factor-dependent parasite-killing effects during paroxysms in non-immune Plasmodium vivax malaria patients. Clin Exp Immunol 88: 499-505.), where lower levels of circulating TNF-α would have little effect on circulating gametocytes. This is in contrast to the high TNF-α levels and increased NO production by a non-immune malaria-infected host (e.g., a child under 5 years old), which reduces gametocyte infectivity of the mosquito (Mshana et al. 1991Mshana RN, Boulandi J, Mshana NM, Mayombo J, Mendome G 1991. Cytokines in the pathogenesis of malaria: levels of IL-I beta, IL-4, IL-6, TNF-alpha and IFN-gamma in plasma of healthy individuals and malaria patients in a holoendemic area. J Clin Lab Immunol 34: 131-139., Othoro et al. 1999Othoro C, Lal AA, Nahlen B, Koech D, Orago AS, Udhayakumar V 1999. A low interleukin-10 tumor necrosis factor-alpha ratio is associated with malaria anemia in children residing in a holoendemic malaria region in western Kenya. J Infect Dis 179: 279-282., Lyke et al. 2004Lyke KE, Burges R, Cissoko Y, Sangare L, Dao M, Diarra I, Kone A, Harley R, Plowe CV, Doumbo OK, Sztein MB 2004. Serum levels of the proinflammatory cytokines interleukin-1 beta (IL-1beta), IL-6, IL-8, IL-10, tumor necrosis factor alpha and IL-12(p70) in Malian children with severe Plasmodium falciparum malaria and matched uncomplicated malaria or healthy controls. Infect Immun 72: 5630-5637.).

In summary, these data suggest that human factors that limit gametocyte infectivity are probably the first contributors to the reduction of malaria parasite numbers in the mosquito.

Mosquito midgut lumen - Cytokines - In addition to their effects in the human host, antimalarial blood stream components can also target the parasite in the mosquito midgut lumen (Fig. 2). Blood components, including white blood cells (WBCs), cytokines, complement proteins, RNS and other factors, remain active for several hours in the mosquito midgut after blood ingestion (Lensen et al. 1997Lensen AHW, Bolmer-Van de Vegte M, Van Gemert GJ, Eling WMC, Sauerwein RW 1997. Leukocytes in a Plasmodium falciparum-infected blood meal reduce transmission of malaria to Anopheles mosquitoes. Infect Immun 65: 3834-3837., Margos et al. 2001Margos G, Navarette S, Butcher G, Davies A, Willers C, Sinden RE, Lachmann PJ 2001. Interaction between host complement and mosquito-midgut-stage Plasmodium berghei. Infect Immun 69: 5064-5071., Simon et al. 2013Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13: 29-41.). TNF-α was shown to reduce the formation of Plasmodium berghei ookinetes through the RNS-mediated reduction of exflagellating males and subsequent ookinete formation (Ramiro et al. 2011Ramiro RS, Alpedrinha J, Carter L, Gardner A, Reece SE 2011. Sex and death: the effects of innate immune factors on the sexual reproduction of malaria parasites. PLoS Pathog 7: e1001309.). Although the mechanism by which TNF-α enhances RNS inhibition of male gamete exflagellation is unknown, it has been proposed that the reduction in ookinete numbers could be the result of TNF-α-induction of leukocyte phagocytosis of sexual stage parasites (Muniz-Junqueira et al. 2001Muniz-Junqueira MI, dos Santos-Neto LL, Tosta CE 2001. Influence of tumor necrosis factor-alpha on the ability of monocytes and lymphocytes to destroy intraerythrocytic Plasmodium falcipa- rum in vitro. Cell Immunol 208: 73-79.). In support of this theory, phagocytosis of P. falciparum and P. berghei gametocytes/gametes by lymphocytes was shown to occur in vitro and in vivo in the midgut of An. gambiae mosquitoes after ingestion of an infected blood meal (Sinden & Smalley 1976Sinden RE, Smalley ME 1976. Gametocytes of Plasmodium falcipa- rum: phagocytosis by leucocytes in vivo and in vitro. Trans R Soc Trop Med Hyg 70: 344-345., Lensen et al. 1997Lensen AHW, Bolmer-Van de Vegte M, Van Gemert GJ, Eling WMC, Sauerwein RW 1997. Leukocytes in a Plasmodium falciparum-infected blood meal reduce transmission of malaria to Anopheles mosquitoes. Infect Immun 65: 3834-3837.).

RNS - RNS have been shown to affect the development of the malaria parasite inside the mosquito midgut lumen (Fig. 2). Pre-incubation of P. yoelii gametocytes with NOC5, a NO donor, inhibits gametogenesis and zygote formation (Cao et al. 1998Cao YM, Tsuboi T, Torii M 1998. Nitric oxide inhibits the development of Plasmodium yoelii gametocytes into gametes. Parasitol Int 47: 157-166.). In addition, it has been reported that up to 50% of P. berghei ookinetes developing inside the midgut of Anopheles stephensi mosquitoes show markers of apoptosis. It was hypothesised that these killing effects could be achieved in newly formed ookinetes when exposed to RNS or reactive oxygen species (ROS) donors (Ali et al. 2010Ali M, Al-Olayan EM, Lewis S, Matthews H, Hurd H 2010. Naturally occurring triggers that induce apoptosis-like programmed cell death in Plasmodium berghei ookinetes. PLoS ONE 5: e12634.). Supporting this theory, the removal of WBCs or treatment with the NOS inhibitor L-NAME significantly reduced the number of apoptotic ookinetes (Ali et al. 2010Ali M, Al-Olayan EM, Lewis S, Matthews H, Hurd H 2010. Naturally occurring triggers that induce apoptosis-like programmed cell death in Plasmodium berghei ookinetes. PLoS ONE 5: e12634.). In addition, Ramiro et al. (2011)Ramiro RS, Alpedrinha J, Carter L, Gardner A, Reece SE 2011. Sex and death: the effects of innate immune factors on the sexual reproduction of malaria parasites. PLoS Pathog 7: e1001309. showed that RNS can affect P. berghei male gamete exflagellation, fertilisation, as well as ookinete development.

Activated WBCs are believed to be one of the primary sources of NO that affect the sexual stages of the parasite in the human host and in the mosquito midgut. However, additional sources of NO and RNS come into play after a mosquito takes a blood meal.

Upon ingestion of a malaria infected blood meal, mosquito midgut levels of NO and nitrates significantly increase (Luckhart et al. 2003Luckhart S, Crampton AL, Zamora R, Lieber MJ, dos Santos PC, Peterson TM, Emmith N, Lim J, Wink DA, Vodovotz Y 2003. Mammalian transforming growth factor beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity. Infect Immun 71: 3000-3009., Peterson et al. 2007Peterson TM, Gow AJ, Luckhart S 2007. Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection. Free Radic Biol Med 42: 132-142.). Digestion of RBCs by mosquito proteases release haemoglobin which comprises ~90% of the total ingested blood mass (Briegel & Rezzonico 1985Briegel H, Rezzonico L 1985. Concentration of host blood protein during feeding by anopheline mosquitoes (Diptera: Culicidae). J Med Entomol 22: 612-618.). Oxyhaemoglobin and haeme which persist throughout blood digestion in the mosquito midgut can react with NO and ROS to produce toxic NO metabolites which in turn can affect malaria parasite development (Peterson et al. 2007Peterson TM, Gow AJ, Luckhart S 2007. Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection. Free Radic Biol Med 42: 132-142.).

In addition, the mosquito midgut epithelium can also be a source of NO. The up-regulation of NOS has been observed in midguts of An. gambiae and An. stephensi mosquitoes upon ingestion of a malaria infected blood meal (Dimopoulos et al. 1998Dimopoulos G, Seeley D, Wolf A, Kafatos FC 1998. Malaria infection of the mosquito Anopheles gambiae activates immune-responsive genes during critical transition stages of the parasite life cycle. EMBO J 17: 6115-6123., Luckhart et al. 1998Luckhart S, Vodovotz Y, Cui L, Rosenberg R 1998. The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc Natl Acad Sci USA 95: 5700-5705.). Blood components have also been shown to increase mosquito midgut NOS expression. The cytokine transforming growth factor-β1 (TGF-β1), a component of the human serum, at low concentrations induces An. stephensi NOS and reduces the mosquito parasite burden (Luckhart et al. 2003Luckhart S, Crampton AL, Zamora R, Lieber MJ, dos Santos PC, Peterson TM, Emmith N, Lim J, Wink DA, Vodovotz Y 2003. Mammalian transforming growth factor beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity. Infect Immun 71: 3000-3009.). In addition, P. falciparum glycosylphosphatidylinositol and parasite-derived haemozoin pigments also appear to induce An. stephensi NOS expression in the mosquito midgut epithelium (Lim et al. 2005Lim J, Gowda DC, Krishnegowda G, Luckhart S 2005. Induction of nitric oxide synthase in Anopheles stephensi by Plasmodium falciparum: mechanism of signaling and the role of parasite glycosylphosphatidylinositols. Infect Immun 73: 2778-2789., Akman-Anderson et al. 2007Akman-Anderson L, Olivier M, Luckhart S 2007. Induction of nitric oxide synthase and activation of signaling proteins in Anopheles mosquitoes by the malaria pigment, hemozoin. Infect Immun 75: 4012-4019.). However, the impact that each of these NO sources (especially mid- gut-derived NO) have on each parasite stage in the mid- gut lumen remains to be determined.

The two main antioxidant systems of the malaria parasite, the thioredoxin and the glutathione (GSH) redox systems, play a protective role during the parasite development in the mosquito. Interruption of the GSH redox pathway by disruption of the gamma-glutamylcysteine synthetase gene (the rate limiting enzyme in GSH biosynthesis) or alternatively, the GSH reductase gene, results in the formation of fewer ookinetes and stunted oocysts that fail to fully develop in An. stephensi mosquitoes (Vega-Rodríguez et al. 2009Vega-Rodríguez J, Franke-Fayard B, Dinglasan RR, Janse CJ, Pastrana-Mena R, Waters AP, Coppens I, Rodríguez-Orengo JF, Srinivasan P, Jacobs-Lorena M, Serrano AE 2009. The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission. PLoS Pathog 5: e1000302., Pastrana-Mena et al. 2011Pastrana-Mena R, Dinglasan RR, Franke-Fayard B, Vega-Rodríguez J, Fuentes-Caraballo M, Baerga-Ortiz A, Coppens I, Jacobs-Lorena M, Janse CJ, Serrano AE 2011. Glutathione reductase-null malaria parasites have normal blood stage growth but arrest during development in the mosquito. J Biol Chem 285: 27045-27056.). Likewise, P. berghei oocysts lacking thioredoxin-dependent 2-Cys peroxidase 1 expression, which is part of the thioredoxin redox system, produce fewer sporozoites (Yano et al. 2008Yano K, Otsuki H, Arai M, Komaki-Yasuda K, Tsuboi T, Torii M, Kano S, Kawazu S 2008. Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito. Mol Biochem Parasitol 159: 142-145.). Moreover, P. berghei ookinetes increase the expression of antioxidant peroxiredoxins after exposure to increased levels of oxidative stress (Turturice et al. 2013Turturice BA, Lamm MA, Tasch JJ, Zalewski A, Kooistra R, Schroeter EH, Sharma S, Kawazu S, Kanzok SM 2013. Expression of cytosolic peroxiredoxins in Plasmodium berghei ookinetes is regulated by environmental factors in the mosquito blood meal. PLoS Pathog 9: e1003136.). It is conceivable that the malaria parasite regulates its antioxidant defense systems to overcome the damage cause by RNS in the mosquito midgut lumen. The mosquito peritrophic matrix (PM), a cellular layer that completely surrounds the blood meal and is secreted by the midgut epithelium in response to feeding, may constitute another protective mechanism from RNS damage. Proteins from the PM have been shown to bind and consequently remove haeme produced by haemoglobin digestion, thus reducing its capacity to form additional RNS (Pascoa et al. 2002Pascoa V, Oliveira PL, Dansa-Petretski M, Silva JR, Alvarenga PH, Jacobs-Lorena M, Lemos FJ 2002. Aedes aegypti peritrophic matrix and its interaction with heme during blood digestion. Insect Biochem Mol Biol 32: 517-523., Devenport et al. 2006Devenport M, Alvarenga PH, Shao L, Fujioka H, Bianconi ML, Oli- veira PL, Jacobs-Lorena M 2006. Identification of the Aedes aegypti peritrophic matrix protein AeIMUCI as a heme-binding protein. Biochemistry 45: 9540-9549.). The protective properties of the PM have yet to be determined, but presumably this structure confers some level of protection for parasites located at the periphery of the blood bolus where active digestion is taking place.

Host complement system - The complement system is part of the innate immune defense in vertebrates and is a first-line defense against pathogens including bacteria, fungi and protozoans. After a foreign organism is detected by the vertebrate host, the complement system is activated within seconds through either the classical, lectin or alternative pathways [reviewed in Walport (2001)Walport MJ 2001. Complement. First of two parts. N Engl J Med 344: 1058-1066.]. This complex system is made up of around 30 abundant proteins in the blood plasma. Once complement has been activated, ultimate destruction of the foreign organism can be achieved by several mechanisms including ingestion by phagocytes or by the assembly of a membrane attack complex on the pathogen surface that ultimately results in its killing.

Infection of the human host by malaria parasites activates both the classical and alternative complement pathways (ACP) (Adam et al. 1981Adam C, Géniteau M, Gougerot-Pocidalo M, Verroust P, Lebras J, Gibert C, Morel-Maroger L 1981. Cryoglobulins, circulating immune complexes and complement activation in cerebral malaria. Infect Immun 31: 530-535., Wenisch et al. 1997Wenisch C, Spitzauer S, Florris-Linau K, Rumpold H, Vannaphan S, Parschalk B, Graninger W, Looareesuwan S 1997. Complement activation in severe Plasmodium falciparum malaria. Clin Immunol Immunopathol 85: 166-171., Goka et al. 2001Goka BQ, Kwarko H, Kurtzhals JA, Gyan B, Ofori-Adjei E, Ohene SA, Hviid L, Akanmori BD, Neequaye J 2001. Complement binding to erythrocytes is associated with macrophage activation and reduced haemoglobin in Plasmodium falciparum malaria. Trans R Soc Trop Med Hyg 95: 545-549., Roestenberg et al. 2007Roestenberg M, McCall M, Mollnes TE, van Deuren M, Sprong T, Klasen I, Hermsen CC, Sauerwein RW, van der Ven A 2007. Complement activation in experimental human malaria infection. Trans R Soc Trop Med Hyg 101: 643-649.). By comparing the infectivity of Plasmodium parasites to mosquitoes by feeding gametocytes in either native or in heat-inactivated serum (heat destroys complement activity), parasites in inactivated serum produced significantly more oocysts than those in native serum (Grotendorst et al. 1986Grotendorst CA, Carter R, Rosenberg R, Koontz LC 1986. Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement. J Immunol 136: 4270-4274., Tsuboi et al. 1995Tsuboi T, Cao YM, Torii M, Hitsumoto Y, Kanbara H 1995. Murine complement reduces infectivity of Plasmodium yoelii to mosquitoes. Infect Immun 63: 3702-3704., Margos et al. 2001Margos G, Navarette S, Butcher G, Davies A, Willers C, Sinden RE, Lachmann PJ 2001. Interaction between host complement and mosquito-midgut-stage Plasmodium berghei. Infect Immun 69: 5064-5071., Simon et al. 2013Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13: 29-41.). Furthermore, Grotendorst et al. (1986)Grotendorst CA, Carter R, Rosenberg R, Koontz LC 1986. Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement. J Immunol 136: 4270-4274. demonstrated that the ACP is responsible for this reduction in parasite numbers. When factor B (a specific component of the alternative pathway) is removed from serum, parasite viability is increased. Similar experiments, in which components specific to the classical pathway were also removed from serum, did not alter parasite viability. Interestingly, the early mosquito stages of the parasite (gametocytes, gametes and early zygotes) are more protected from complement attack than later stages (late zygotes and ookinetes) (Grotendorst et al. 1986Grotendorst CA, Carter R, Rosenberg R, Koontz LC 1986. Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement. J Immunol 136: 4270-4274., Tsuboi et al. 1995Tsuboi T, Cao YM, Torii M, Hitsumoto Y, Kanbara H 1995. Murine complement reduces infectivity of Plasmodium yoelii to mosquitoes. Infect Immun 63: 3702-3704., Margos et al. 2001Margos G, Navarette S, Butcher G, Davies A, Willers C, Sinden RE, Lachmann PJ 2001. Interaction between host complement and mosquito-midgut-stage Plasmodium berghei. Infect Immun 69: 5064-5071., Simon et al. 2013Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13: 29-41.), suggesting that malaria parasites may have evolved a mechanism of protection during their most vulnerable stages.

Immediately after the female mosquito feeds on blood, the midgut starts secreting digestive enzymes that degrade proteins from the serum and the RBCs. Host complement proteins are also exposed to mosquito proteases which raise the question of how long does the complement system from the human serum remain active after the mosquito has taken a blood meal? Two different studies addressed this question by measuring the products of C3 activation, which results in the formation of C3a (Simon et al. 2013Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13: 29-41.) and C3b (Margos et al. 2001Margos G, Navarette S, Butcher G, Davies A, Willers C, Sinden RE, Lachmann PJ 2001. Interaction between host complement and mosquito-midgut-stage Plasmodium berghei. Infect Immun 69: 5064-5071.). Both reports show that the peak of complement activity in the midgut is during the first hour post-blood-feeding and remains active up to 6 h after feeding. This loss of complement activity in the mosquito blood bolus also coincides with the transition of the parasite from complement-resistant stages (gametocytes, gametes and early zygotes) to those that are complement-sensitive (late zygotes and ookinetes) (Fig. 2) (Grotendorst et al. 1986Grotendorst CA, Carter R, Rosenberg R, Koontz LC 1986. Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement. J Immunol 136: 4270-4274., Tsuboi et al. 1995Tsuboi T, Cao YM, Torii M, Hitsumoto Y, Kanbara H 1995. Murine complement reduces infectivity of Plasmodium yoelii to mosquitoes. Infect Immun 63: 3702-3704., Margos et al. 2001Margos G, Navarette S, Butcher G, Davies A, Willers C, Sinden RE, Lachmann PJ 2001. Interaction between host complement and mosquito-midgut-stage Plasmodium berghei. Infect Immun 69: 5064-5071., Simon et al. 2013Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13: 29-41.). However, digestion of the blood bolus is not uniform and occurs from the outside in. Therefore, it can be predicted that the complement molecules closer to the periphery of the blood bolus will be inactivated faster and consequently that parasites located in these areas will be protected from complement attack much earlier.

Evasion of the complement system - To survive, pathogenic microorganisms have evolved different mechanisms to evade the attack from the host complement system. To date, three evasion mechanisms have been described: inactivation of complement by pathogen-derived proteases, inactivation by complement-binding (neutralising) proteins and the acquisition of complement regulatory proteins. There is evidence to suggest that Plasmodium has evolved to acquire a complement regulatory protein needed to evade complement attack during its development in the mosquito blood bolus (Fig. 2).

Parasites became sensitive to complement attack following treatment of Plasmodium gallinaceum gametes with trypsin, implying that a parasite surface protein is responsible for the protection from complement (Grotendorst et al. 1986Grotendorst CA, Carter R, Rosenberg R, Koontz LC 1986. Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement. J Immunol 136: 4270-4274.). This was further examined in a recent report showing that Plasmodium gametes and zygotes bind on their surface factor H (FH) from the host serum. FH regulates ACP activation by binding to C3b thus preventing the formation of the C3 convertase and accelerating its decay [reviewed in de Córdoba et al. (2004)de Córdoba S, Esparza-Gordillo J, de Jorge EG, Lopez-Trascasa M, Sánchez-Corral P 2004. The human complement factor H: functional roles, genetic variations and disease associations. Mol Immunol 41: 355-367.]. When mosquitoes were fed with P. falciparum gametocytes in native human serum together with anti-FH antibodies, mosquitoes were rendered resistant as parasite numbers were effectively reduced to zero (Simon et al. 2013Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13: 29-41.). Co-immunoprecipitation assays with anti-FH antibodies on protein extracts from activated gametes in native human serum identified PfGAP50 as a parasite receptor for FH (Simon et al. 2013Simon N, Lasonder E, Scheuermayer M, Kuehn A, Tews S, Fischer R, Zipfel PF, Skerka C, Pradel G 2013. Malaria parasites co-opt human factor H to prevent complement-mediated lysis in the mosquito midgut. Cell Host Microbe 13: 29-41.). However, the authors suggest that there are additional unknown FH receptors on the surface of the gametes as anti-PfGAP50 antibodies only reduced the infectivity to mosquitoes by 38-60%. This hypothesis is plausible as other pathogens including Streptococcus pyogenes and Borrelia burgdorferi use more than one surface protein to capture FH from the host and evade the ACP response [reviewed in Zipfel et al. (2007)Zipfel PF, Würzner R, Skerka C 2007. Complement evasion of pathogens: common strategies are shared by diverse organisms. Mol Immunol 44: 3850-3857.].

TB antibodies - Naturally acquired immunity against malaria asexual stages can confer partial protection against the disease [reviewed in Doolan et al. (2009)Doolan DL, Dobaño C, Baird JK 2009. Acquired immunity to malaria. Clin Microbiol Rev 22: 13-36.]. In addition to the asexual stages, gametocytes also circulate in the blood. If a gametocyte is not ingested by a mosquito during blood-feeding, it decays and evidence suggests that it is eventually removed from circulation by the host immune system resulting in the production of gametocyte-specific antibodies (Baird et al. 1991Baird JK, Jones TR, Purnomo, Masbar S, Ratiwayanto S, Leksana B 1991. Evidence for specific suppression of gametocytemia by Plasmodium falciparum in residents of hyperendemic Irian Jaya. Am J Trop Med Hyg 44: 183-190., Taylor & Read 1997Taylor LH, Read AF 1997. Why so few transmission stages? Reproductive restraint by malaria parasites. Parasitol Today 13: 135-140., Saeed et al. 2008Saeed M, Roeffen W, Alexander N, Drakeley CJ, Targett GA, Sutherland CJ 2008. Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte. PLoS ONE 3: e2280.). Such antibodies would also be ingested by the mosquito and could interfere with progression of the developmental cycle in the midgut lumen (Fig. 2).

Immune sera of individuals living in malaria endemic regions have TB activity against the mosquito midgut stages of both P. falciparum and P. vivax (Baird et al. 1991Baird JK, Jones TR, Purnomo, Masbar S, Ratiwayanto S, Leksana B 1991. Evidence for specific suppression of gametocytemia by Plasmodium falciparum in residents of hyperendemic Irian Jaya. Am J Trop Med Hyg 44: 183-190., Mulder et al. 1994Mulder B, Tchuinkam T, Dechering K, Verhave JP, Carnevale P, Meuwissen JH, Robert V 1994. Malaria transmission-blocking activity in experimental infections of Anopheles gambiae from naturally infected Plasmodium falciparum gametocyte carriers. Trans R Soc Trop Med Hyg 88: 121-125., Lensen et al. 1998Lensen AHW, Mulder L, Tchuinkam T, Willemsen L, Eling W, Sauerwein R 1998. Mechanisms that reduce transmission of Plasmodium falciparum malaria in semiimmune and nonimmune persons. J Infect Dis 177: 1358-1363., Arévalo-Herrera et al. 2005Arévalo-Herrera M, Solarte Y, Zamora F, Mendez F, Yasnot MF, Rocha L, Long C, Miller LH, Herrera S 2005. Plasmodium vivax: transmission-blocking immunity in a malaria-endemic area of Colombia. Am J Trop Med Hyg 73: 38-43., Bousema et al. 2011Bousema T, Sutherland CJ, Churcher TS, Mulder B, Gouagna LC, Riley EM, Targett GA, Drakeley CJ 2011. Human immune responses that reduce the transmission of Plasmodium falciparum in African populations. Int J Parasitol 41: 293-300.). Specifically, antibodies against the gametocyte/gamete proteins Pfs48/45 and Pfs230 show a negative correlation between antibody activity and parasite development in the mosquito (Healer et al. 1999Healer J, McGuinness D, Carter R, Riley E 1999. Transmission-blocking immunity to Plasmodium falciparum in malaria-immune individuals is associated with antibodies to the gamete surface protein Pfs230. Parasitology 119: 425-433., Drakeley et al. 2004Drakeley CJ, Eling W, Teelen K, Bousema JT, Sauerwein R, Greenwood BM, Targett GA 2004. Parasite infectivity and immunity to Plasmodium falciparum gametocytes in Gambian children. Parasite Immunol 26: 159-165., van der Kolk et al. 2006, Bousema et al. 2007Bousema JT, Drakeley CJ, Kihonda J, Hendriks JC, Akim NI, Roeffen W, Sauerwein RW 2007. A longitudinal study of immune responses to Plasmodium falciparum sexual stage antigens in Tanzanian adults. Parasite Immunol 29: 309-317., 2010). Both Pfs48/45 and Pfs230 participate in gamete fertilisation and are members of a 6-cysteine protein family containing adhesive domains (van Dijk et al. 2001van Dijk MR, Janse CJ, Thompson J, Waters AP, Braks JA, Dodemont HJ, Stunnenberg HG, van Gemert GJ, Sauerwein RW, Eling W 2001. A central role for P48/45 in malaria parasite male gamete fertility. Cell 104: 153-164., 2010, Eksi et al. 2006Eksi S, Czesny B, van Gemert GJ, Sauerwein RW, Eling W, Williamson KC 2006. Malaria transmission-blocking antigen, Pfs230, mediates human red blood cell binding to exflagellating male parasites and oocyst production. Mol Microbiol 61: 991-998.). These proteins are stored in the parasitophorous vacuole membrane of the gametocyte and relocate to the surface of the gamete during activation in the mosquito midgut (Williamson et al. 1996Williamson KC, Fujioka H, Aikawa M, Kaslow DC 1996. Stage-specific processing of Pfs230, a Plasmodium falciparum transmission-blocking vaccine candidate. Mol Biochem Parasitol 78: 161-169.). Gene disruption of P48/45 and P230 in P. berghei and Pfs230 in P. falciparum drastically reduces infectivity to mosquitoes by interfering with fertilisation (van Dijk et al. 2001van Dijk MR, Janse CJ, Thompson J, Waters AP, Braks JA, Dodemont HJ, Stunnenberg HG, van Gemert GJ, Sauerwein RW, Eling W 2001. A central role for P48/45 in malaria parasite male gamete fertility. Cell 104: 153-164., 2010, Eksi et al. 2006Eksi S, Czesny B, van Gemert GJ, Sauerwein RW, Eling W, Williamson KC 2006. Malaria transmission-blocking antigen, Pfs230, mediates human red blood cell binding to exflagellating male parasites and oocyst production. Mol Microbiol 61: 991-998.).

It appears that anti-Pfs230 antibodies hinder fertilisation by a mechanism different from interference with gamete adhesion. Early TB studies reported that antibodies against Pfs48/45, but not Pfs230 inhibited P. falciparum development in the mosquito, a finding that discouraged the use of Pfs230 as a potential TB vaccine antigen (Vermeulen et al. 1985Vermeulen AN, Roeffen WF, Henderik JB, Ponnudurai T, Beckers PJ, Meuwissen JH 1985. Plasmodium falciparum transmission blocking monoclonal antibodies recognize monovalently expressed epitopes. Dev Biol Stand 62: 91-97.). Later studies showed that anti-Pfs230 antibodies inhibit P. falciparum development in the mosquito in the presence of active serum complement (Quakyi et al. 1987Quakyi IA, Carter R, Rener J, Kumar N, Good MF, Miller LH 1987. The 230-kDa gamete surface protein of Plasmodium falciparum is also a target for transmission-blocking antibodies. J Immunol 139: 4213-4217., Read et al. 1994Read D, Lensen AH, Begarnie S, Haley S, Raza A, Carter R 1994. Transmission-blocking antibodies against multiple, non-variant target epitopes of the Plasmodium falciparum gamete surface antigen Pfs230 are all complement-fixing. Parasite Immunol 16: 511-519., Healer et al. 1997Healer J, McGuinness D, Hopcroft P, Haley S, Carter R, Riley E 1997. Complement-mediated lysis of Plasmodium falciparum gametes by malaria-immune human sera is associated with antibodies to the gamete surface antigen Pfs230. Infect Immun 65: 3017-3023.). These antibodies activate the classical complement pathway resulting in the formation of the membrane-attack complex causing lysis of the parasite (Fig. 2).

In summary, these studies highlight the possible applications of an antibody-based TB vaccine to reduce parasite development in its mosquito host and could have a profound influence on the transmission of malaria in endemic countries.

Midgut microbiota - Mosquitoes, as all higher organisms including humans, carry an intestinal microbiota that is mostly composed of bacteria and yeast [reviewed in Dillon and Dillon (2004)Dillon RJ, Dillon VM 2004. The gut bacteria of insects: nonpathogenic interactions. Annu Rev Entomol 49: 71-92.]. There is evidence to suggest that the symbiotic relationship between bacteria and the mosquito confers to the latter some protection against invading pathogens like malaria. Several studies using laboratory-reared and field-captured Anopheles mosquitoes have shown that midgut bacteria (primarily Gram-negative bacteria) have a negative effect on P. falciparum and P. vivax development in the mosquito (Seitz et al. 1987Seitz HM, Maier WA, Rottok M, Becker-Feldmann H 1987. Concomitant infections of Anopheles stephensi with Plasmodium berghei and Serratia marcescens: additive detrimental effects. Zentralbl Bakteriol Mikrobiol Hyg A 266: 155-166., Pumpuni et al. 1993Pumpuni CB, Beier MS, Nataro JP, Guers LD, Davis JR 1993. Plasmodium falciparum: inhibition of sporogonic development in Anopheles stephensi by Gram-negative bacteria. Exp Parasitol 77: 195-199., 1996Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC 1996. Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development. Am J Trop Med Hyg 54: 214-218., Straif et al. 1998Straif SC, Mbogo CN, Toure AM, Walker ED, Kaufman M, Toure YT, Beier JC 1998. Midgut bacteria in Anopheles gambiae and An. funestus (Diptera: Culicidae) from Kenya and Mali. J Med Entomol 35: 222-226., Gonzalez-Ceron et al. 2003Gonzalez-Ceron L, Santillan F, Rodriguez MH, Mendez D, Hernandez-Avila JE 2003. Bacteria in midguts of field-collected Anophe- les albimanus block Plasmodium vivax sporogonic development. J Med Entomol 40: 371-374., Dong et al. 2009Dong Y, Manfredini F, Dimopoulos G 2009. Implication of the mosquito midgut microbiota in the defense against malaria parasites. PLoS Pathog 5: e1000423., Cirimotich et al. 2011Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mulenga M, Dimopoulos G 2011. Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science 332: 855-858.). For instance, infections after removal of endogenous microbiota by antibiotic treatment lead to higher parasite numbers when compared with infections of untreated mosquitoes (Beier et al. 1994Beier MS, Pumpuni CB, Beier JC, Davis JR 1994. Effects of para-aminobenzoic acid, insulin and gentamicin on Plasmodium falciparum development in anopheline mosquitoes (Diptera: Culicidae). J Med Entomol 31: 561-565., Dong et al. 2009Dong Y, Manfredini F, Dimopoulos G 2009. Implication of the mosquito midgut microbiota in the defense against malaria parasites. PLoS Pathog 5: e1000423., Meister et al. 2009Meister S, Agianian B, Turlure F, Relógio A, Morlais I, Kafatos FC, Christophides GK 2009. Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLoS Pathog 5: e1000542.). In addition, when antisera raised against An. gambiae midgut lysates were fed to An. gambiae mosquitoes together with P. falciparum cultured gametocytes, there was an increase in the number of parasites that developed into oocysts relative to controls and this effect was attributed to antibacteria antibodies in the serum (Noden et al. 2011Noden BH, Vaughan JA, Pumpuni CB, Beier JC 2011. Mosquito ingestion of antibodies against mosquito midgut microbiota improves conversion of ookinetes to oocysts for Plasmodium falciparum, but not P. yoelii. Parasitol Int 60: 440-446.). These results are in contrast to previous reports where similar experiments resulted into a reduced parasite load due to antibodies targeting mosquito proteins required for parasite development (Srikrishnaraj et al. 1995Srikrishnaraj KA, Ramasamy R, Ramasamy MS 1995. Antibodies to Anopheles midgut reduce vector competence for Plasmodium vivax malaria. Med Vet Entomol 9: 353-357., Lal et al. 2001Lal AA, Patterson PS, Sacci JB, Vaughan JA, Paul C, Collins WE, Wirtz RA, Azad AF 2001. Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship. Proc Natl Acad Sci USA 98: 5228-5233., Suneja et al. 2003Suneja A, Gulia M, Gakhar SK 2003. Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae). Arch Insect Biochem Physiol 52: 63-70., Chugh et al. 2011Chugh M, Adak T, Sehrawat N, Gakhar SK 2011. Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: Culicidae). Indian J Exp Biol 49: 245-253.). The reasons for these discrepancies are unclear and require further investigation.

Recent studies have shown that certain variants of Serratia marcescens, a bacterium commonly found in the midgut of laboratory and field mosquitoes, are able to inhibit Plasmodium in the mosquito (Bando et al. 2013Bando H, Okado K, Guelbeogo WM, Badolo A, Aonuma H, Nelson B, Fukumoto S, Xuan X, Sagnon N, Kanuka H 2013. Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity. Sci Rep 3: 1641.). Increased Plasmodium inhibition was correlated to increased flagella length and abundance and with reduced ability of the ookinete to invade the midgut (Bando et al. 2013Bando H, Okado K, Guelbeogo WM, Badolo A, Aonuma H, Nelson B, Fukumoto S, Xuan X, Sagnon N, Kanuka H 2013. Intra-specific diversity of Serratia marcescens in Anopheles mosquito midgut defines Plasmodium transmission capacity. Sci Rep 3: 1641.). The authors hypothesise that a physical barrier imposed by the flagella may contribute to the decreased success of ookinetes to invade the midgut epithelium.

Recent evidence has also emerged that bacteria may influence Plasmodium development directly through the production of antimalarial compounds (Fig. 2). Cirimotich et al. (2011)Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mulenga M, Dimopoulos G 2011. Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science 332: 855-858. isolated an Enterobacter bacterium (Esp_Z) from Zambian populations of Anopheles arabiensis mosquitoes. Co-feeding of Esp_Z and P. falciparum-infected blood to mosquitoes reduced the parasite burden in a dose-dependent manner. The antiparasitic effect of this bacterium, which is observed at the zygote to ookinete transition, can be rescued by addition of the antioxidant vitamin C to the infectious blood meal. This result suggests that ROS secreted by Esp_Z bacteria is responsible for the reduction in parasite numbers (Cirimotich et al. 2011Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mulenga M, Dimopoulos G 2011. Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science 332: 855-858.). These findings have important implications as a naturally occurring bacterium like Esp_Z could be exploited to develop new malaria TB alternatives. However, as mentioned above, the parasite’s antioxidant systems are highly active during development in the mosquito and can be regulated according to the redox state of the environment were the parasite is developing (Yano et al. 2008Yano K, Otsuki H, Arai M, Komaki-Yasuda K, Tsuboi T, Torii M, Kano S, Kawazu S 2008. Disruption of the Plasmodium berghei 2-Cys peroxiredoxin TPx-1 gene hinders the sporozoite development in the vector mosquito. Mol Biochem Parasitol 159: 142-145., Vega-Rodríguez et al. 2009Vega-Rodríguez J, Franke-Fayard B, Dinglasan RR, Janse CJ, Pastrana-Mena R, Waters AP, Coppens I, Rodríguez-Orengo JF, Srinivasan P, Jacobs-Lorena M, Serrano AE 2009. The glutathione biosynthetic pathway of Plasmodium is essential for mosquito transmission. PLoS Pathog 5: e1000302., Pastrana-Mena et al. 2011Pastrana-Mena R, Dinglasan RR, Franke-Fayard B, Vega-Rodríguez J, Fuentes-Caraballo M, Baerga-Ortiz A, Coppens I, Jacobs-Lorena M, Janse CJ, Serrano AE 2011. Glutathione reductase-null malaria parasites have normal blood stage growth but arrest during development in the mosquito. J Biol Chem 285: 27045-27056.).

Additional evidence suggests that the midgut microbiota play a direct role in the activation of the mosquito immune response. Shortly after blood ingestion, the resident microbiota undergo dramatic proliferation (about 2 orders of magnitude) peaking at about 24 h (Pumpuni et al. 1996Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC 1996. Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development. Am J Trop Med Hyg 54: 214-218.). This strong bacterial proliferation is likely to result in an immune response independent of parasite presence. Dong et al. (2009) reported that the mosquito midgut bacteria induce a basal level of immunity that enhances the expression of antimicrobial immune genes that also have antiplasmodial activity. This is true for the induction of SRPN6 in response to Enterobacter infection, which in turn contributes to the anti-Plasmodium response of the mosquito (Eappen et al. 2013Eappen AG, Smith RC, Jacobs-Lorena M 2013. Enterobacter-activated mosquito immune responses to Plasmodium involve activation of SRPN6 in Anopheles stephensi. PLoS ONE 8: e62937.). Moreover, Rodrigues et al. (2010) reported that bacteria are necessary for immune priming.

A delicate balance exists between the commensal gut microbiota and its mosquito host to limit bacterial over-proliferation and the subsequent immune response that may have negative effects towards mosquito fitness. To reduce hyperactivation of the mosquito immune system by the midgut bacteria, the mosquito is thought to form a dityrosine network that restricts contact of bacteria with the midgut epithelium, thus reducing the antimicrobial response (Kumar et al. 2010Kumar S, Molina-Cruz A, Gupta L, Rodrigues J, Barillas-Mury C 2010. A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae. Science 327: 1644-1648.). As a result, proliferation of the normal gut flora is not impaired and malaria parasite development is unhindered (Kumar et al. 2010Kumar S, Molina-Cruz A, Gupta L, Rodrigues J, Barillas-Mury C 2010. A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae. Science 327: 1644-1648.). In addition, dual oxidase expression by the mosquito midgut epithelium likely results in ROS production, playing a similar role to that in Drosophila of limiting bacterial numbers (Ha et al. 2009Ha EM, Lee KA, Seo YY, Kim SH, Lim JH, Oh BH, Kim J, Lee WJ 2009. Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in Drosophila gut. Nature Immunol 10: 949-957.). Moreover, it was proposed that when Plasmodium ookinetes breach the PM and the protective dityrosine network lining the luminal surface of the midgut epithelium, bacteria “leak” through the open spaces thus activating an antibacterial immune response that is also harmful to invading ookinetes (Kumar et al. 2010Kumar S, Molina-Cruz A, Gupta L, Rodrigues J, Barillas-Mury C 2010. A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae. Science 327: 1644-1648., Rodrigues et al. 2010Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C 2010. Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes. Science 329: 1353-1355.).

Physical barriers to Plasmodium development - The PM - The distension of the mosquito midgut by a blood meal induces midgut epithelial cells to secrete components of an extracellular layer, known as the PM, which completely surrounds the ingested blood. The PM is composed of proteins, glycoproteins and proteoglycans that are structurally linked by chitin (Shao et al. 2001Shao L, Devenport M, Jacobs-Lorena M 2001. The peritrophic matrix of hematophagous insects. Arch Insect Biochem Physiol 47: 119-125., Dinglasan et al. 2009Dinglasan RR,Devenport M, Florens L, Johnson JR, McHugh CA, Donnelly-Doman M, Carucci DJ, Yates JR 3rd, Jacobs-Lorena M 2009. The Anopheles gambiae adult midgut peritrophic matrix proteome. Insect Biochem Mol Biol 39: 125-134.). The PM constitutes a physical barrier that prevents direct contact of commensal bacteria and of components of the blood meal with the midgut epithelium. Initially soft and fragile, the PM polymerises and gradually thickens reaching maximal rigidity at about 24 h after the blood meal (Shao et al. 2001Shao L, Devenport M, Jacobs-Lorena M 2001. The peritrophic matrix of hematophagous insects. Arch Insect Biochem Physiol 47: 119-125.). As the PM completely surrounds the blood meal, it constitutes the first significant physical barrier to Plasmodium development in the mosquito. As ookinetes mature (approximately 16-20 h after blood ingestion) they migrate to the periphery of the blood bolus, presumably guided by environmental or sensory cues. Upon contact with the PM, the ookinete secretes a chitinase (and possibly other proteases) from its micronemes to locally disrupt and penetrate the chitinous PM. Inactivation of the chitinase genes dramatically reduces the ability of the ookinete to traverse the PM (Dessens et al. 2001Dessens JT, Mendoza J, Claudianos C, Vinetz JM, Khater E, Hassard S, Ranawaka GR, Sinden RE 2001. Knockout of the rodent malaria parasite chitinase pbCHT1 reduces infectivity to mosquitoes. Infect Immun 69: 4041-4047., Tsai et al. 2001Tsai YL, Hayward RE, Langer RC, Fidock DA, Vinetz JM 2001. Disruption of Plasmodium falciparum chitinase markedly impairs parasite invasion of mosquito midgut. Infect Immun 69: 4048-4054.). In some parasite species, ookinetes secrete a chitinase precursor (or zymogen) that is activated by mosquito midgut proteases (Shahabuddin et al. 1993Shahabuddin M, Toyoshima T, Aikawa M, Kaslow DC 1993. Transmission-blocking activity of a chitinase inhibitor and activation of malarial parasite chitinase by mosquito protease. Proc Natl Acad Sci USA 90: 4266-4270., Shahabuddin & Kaslow 1994Shahabuddin M, Kaslow DC 1994. Plasmodium: parasite chitinase and its role in malaria transmission. Exp Parasitol 79: 85-88.), indicating that the parasite has adapted to the protease-rich environment of the mosquito midgut to facilitate its own development.

The midgut epithelium - After traversal of the PM it is believed that ookinetes display extensive gliding motility along the lumenal surface of the midgut epithelium. This movement may be important to initiate midgut invasion (Zieler & Dvorak 2000Zieler H, Dvorak JA 2000. Invasion in vitro of mosquito midgut cells by the malaria parasite proceeds by a conserved mechanism and results in death of the invaded midgut cells. Proc Natl Acad Sci USA 97: 11516-11521.), possibly through the interaction with one or more of the numerous glycoproteins that comprise the glycocalyx of the midgut epithelium (Shen et al. 1999Shen Z, Dimopoulos G, Kafatos FC, Jacobs-Lorena M 1999. A cell surface mucin specifically expressed in the midgut of the malaria mosquito Anopheles gambiae. Proc Natl Acad Sci USA 96: 5610-5615., Wilkins & Billingsley 2001Wilkins S, Billingsley PF 2001. Partial characterization of oligosaccharides expressed on midgut microvillar glycoproteins of the mosquito, Anopheles stephensi Liston. Insect Biochem Mol Biol 31: 937-948., Dinglasan et al. 2007aDinglasan RR, Kalume DE, Kanzok SM, Ghosh AK, Muratova O, Pandey A, Jacobs-Lorena M 2007a. Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen. Proc Natl Acad Sci USA 104: 13461-13466.). Carbohydrate moieties on the midgut epithelium seem to play an important role in ookinete binding to the midgut (Zieler et al. 1999Zieler H, Nawrocki JP, Shahabuddin M 1999. Plasmodium gallinaceum ookinetes adhere specifically to the midgut epithelium of Aedes aegypti by interaction with a carbohydrate ligand. J Exp Biol 202: 485-495., Zieler & Dvorak 2000Zieler H, Dvorak JA 2000. Invasion in vitro of mosquito midgut cells by the malaria parasite proceeds by a conserved mechanism and results in death of the invaded midgut cells. Proc Natl Acad Sci USA 97: 11516-11521.). Moreover, it appears that the interaction between mosquito sugars and parasite lectins are required to establish invasion (Zieler & Dvorak 2000Zieler H, Dvorak JA 2000. Invasion in vitro of mosquito midgut cells by the malaria parasite proceeds by a conserved mechanism and results in death of the invaded midgut cells. Proc Natl Acad Sci USA 97: 11516-11521., Dinglasan et al. 2007aDinglasan RR, Kalume DE, Kanzok SM, Ghosh AK, Muratova O, Pandey A, Jacobs-Lorena M 2007a. Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen. Proc Natl Acad Sci USA 104: 13461-13466., bDinglasan RR, Alaganan A, Ghosh AK, Saito A, van Kuppevelt TH, Jacobs-Lorena M 2007b. Plasmodium falciparum ookinetes require mosquito midgut chondroitin sulfate proteoglycans for cell invasion. Proc Natl Acad Sci USA 104: 15882-15887.). However, very little is known regarding the specific protein-protein interactions that mediate this process since these carbohydrate moieties are post-translational modifications of yet unknown proteins. Given the implications as possible TB vaccine targets, identifying these specific interactions is a high priority area of research.

Using a phage display library, a peptide termed SM1 was identified that interacts with the lumenal surface of the mosquito midgut epithelium (Ghosh et al. 2001Ghosh AK, Ribolla PE, Jacobs-Lorena M 2001. Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library. Proc Natl Acad Sci USA 98: 13278-13281.) and has greatly increased our understanding of the process of midgut invasion. The SM1 dodecapeptide strongly inhibits P. berghei ookinete invasion via competitive binding to a putative mosquito midgut receptor (Ghosh et al. 2001Ghosh AK, Ribolla PE, Jacobs-Lorena M 2001. Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library. Proc Natl Acad Sci USA 98: 13278-13281.). Transgenic mosquitoes engineered to express SM1 in the midgut following blood-feeding are impaired in the transmission of the malaria parasite (Ito et al. 2002Ito J, Ghosh A, Moreira LA, Wimmer EA, Jacobs-Lorena M 2002. Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. Nature 417: 452-455.). Further studies have identified that SM1 is a mimotope (its conformation resembles) of Plasmodium enolase, a protein secreted onto the surface of the ookinete where it acts as an invasion ligand (Ghosh et al. 2011Ghosh AK, Coppens I, Gardsvoll H, Ploug M, Jacobs-Lorena M 2011. Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut. Proc Natl Acad Sci USA 108: 17153-17158.). Ookinete surface enolase also interacts with plasminogen from the blood serum to locally promote its conversion into the proteolytically active plasmin that in turn, is required for midgut invasion (Ghosh et al. 2011Ghosh AK, Coppens I, Gardsvoll H, Ploug M, Jacobs-Lorena M 2011. Plasmodium ookinetes coopt mammalian plasminogen to invade the mosquito midgut. Proc Natl Acad Sci USA 108: 17153-17158.). Recently, the mosquito midgut receptor for SM1 and ookinete surface enolase was identified as enolase binding protein (EBP) (Vega-Rodríguez et al. 2014Vega-Rodríguez J, Ghosh AK, Kanzok SM, Dinglasan RR, Wang S, Bongio NJ, Kalume D, Kazutoyo M, Long CA, Pandey A, Jacobs-Lorena M 2014. Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut. Proc Natl Acad Sci USA doi: 10.1073/pnas.1315517111.). EBP is expressed on the lumenal surface of the mosquito midgut and is required for P. berghei ookinete midgut invasion through its interaction with surface enolase. This interaction can be disrupted through competition with excess SM1, thus inhibiting ookinete midgut invasion (Vega-Rodríguez et al. 2014Vega-Rodríguez J, Ghosh AK, Kanzok SM, Dinglasan RR, Wang S, Bongio NJ, Kalume D, Kazutoyo M, Long CA, Pandey A, Jacobs-Lorena M 2014. Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut. Proc Natl Acad Sci USA doi: 10.1073/pnas.1315517111.). However, some ookinetes still invade the mosquito midgut in the presence of excess SM1 and this served as the basis for the selection of SM1-resistant parasites that do not require EBP for midgut invasion. This is the first evidence demonstrating that mosquito midgut invasion by Plasmodium ookinetes, similar to merozoite invasion of the RBC, can occur through multiple invasion pathways (Vega-Rodríguez et al. 2014Vega-Rodríguez J, Ghosh AK, Kanzok SM, Dinglasan RR, Wang S, Bongio NJ, Kalume D, Kazutoyo M, Long CA, Pandey A, Jacobs-Lorena M 2014. Multiple pathways for Plasmodium ookinete invasion of the mosquito midgut. Proc Natl Acad Sci USA doi: 10.1073/pnas.1315517111.). While further work is needed to characterise additional midgut receptor(s), these findings suggest that the process of ookinete midgut invasion is complex and involves multiple parasite-mosquito interactions. Characterisation of these interactions remains a major goal for future research and has the potential to lead to novel intervention strategies.

The process of ookinete invasion is thought to produce severe damage to the midgut as the ookinete traverses multiple epithelial cells during its journey to the basal lamina. Invaded cells undergo dramatic cytoskeletal changes and increase the production of reactive oxygen and nitrogen species that trigger apoptosis and cell death (Han et al. 2000Han YS, Thompson J, Kafatos FC, Barillas-Mury C 2000. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J 19: 6030-6040.). During this time ookinetes are likely exposed to a highly toxic cellular environment. To ensure its survival, the ookinete must rapidly escape before it is damaged or before the damaged cell is extruded from the midgut epithelium. This evasion process occurs by invasion of neighbouring naïve cells or by exiting the epithelium to rest at its final extracellular destination between the basal side of the epithelium and the basal lamina. While it remains unclear how ookinete invasion triggers programmed cell death, this is likely a general response to remove damaged epithelial cells and may not be specific to parasite invasion (Baton & Ranford-Cartwright 2005Baton LA, Ranford-Cartwright LC 2005. How do malaria ookinetes cross the mosquito midgut wall? Trends Parasitol 21: 22-28., Okuda et al. 2007Okuda K, de Almeida F, Mortara RA, Krieger H, Marinotti O, Bijovsky AT 2007. Cell death and regeneration in the midgut of the mosquito, Culex quinquefasciatus. J Insect Physiol 53: 1307-1315.).

Recent evidence further supports the concept that midgut traversal is a critical step in the mosquito anti-Plasmodium response. Invaded cells produce high levels of NOS and peroxidases that in turn increase the levels of midgut nitration (Kumar et al. 2004Kumar S, Gupta L, Han YS, Barillas-Mury C 2004. Inducible peroxidases mediate nitration of Anopheles midgut cells undergoing apoptosis in response to Plasmodium invasion. J Biol Chem 279: 53475-53482.). Further nitration is increased by the activation of the haeme peroxidase 2 (HPX2) and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 5 (NOX5), processes that are regulated by the c-Jun N-terminal kinase (JNK) pathway (Oliveira et al. 2012Oliveira GA, Lieberman J, Barillas-Mury C 2012. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science 335: 856-859., Garver et al. 2013Garver LS, Oliveira GA, Barillas-Mury C 2013. The JNK pathway is a key mediator of Anopheles gambiae antiplasmodial immunity. PLoS Pathog 9: e1003622.). This epithelial nitration response is believed to modify ookinetes, thus “marking” them for immune recognition by the mosquito complement system (Oliveira et al. 2012Oliveira GA, Lieberman J, Barillas-Mury C 2012. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science 335: 856-859., Garver et al. 2013Garver LS, Oliveira GA, Barillas-Mury C 2013. The JNK pathway is a key mediator of Anopheles gambiae antiplasmodial immunity. PLoS Pathog 9: e1003622.). Thus, midgut nitration appears to be a major determinant of Plasmodium survival, yet many questions remain regarding how nitration mediates immune recognition. What proteins on the surface of the ookinete undergo modifications as a result of increased nitration? How are these modifications subsequently recognised by the mosquito complement system?

Components of the anti-Plasmodium immune response - A great deal of effort has been invested in the identification of components that contribute to parasite developmental success in the mosquito in the hope that this knowledge can be translated to the development of alternative strategies for parasite killing. Through the use of dsRNA-mediated gene-silencing, several genes have been described that positively or negatively influence parasite development in the mosquito (Blandin et al. 2008Blandin SA, Marois E, Levashina EA 2008. Antimalarial responses in Anopheles gambiae: from a complement-like protein to a complement-like pathway. Cell Host Microbe 3: 364-374.). While more research is required to fully understand the contributions of individual mosquito genes to anti-Plasmodium immunity, evidence suggests that parasite killing after traversal of the midgut epithelium occurs primarily during two separate stages or phases of parasite development (Fig. 3). The first phase, or “early-phase”, occurs during the ookinete to oocyst transition immediately after exiting the midgut epithelium. A second, or “late-phase”, is thought to act on developing oocysts.

The “early-phase” immune response - Approximately 18-24 h after blood-feeding, Plasmodium ookinetes invade the lumenal side of the midgut epithelium and within minutes reach the basal surface that is believed to be the major site of ookinete killing (Shiao et al. 2006Shiao SH, Whitten MM, Zachary D, Hoffmann JA, Levashina EA 2006. Fz2 and cdc42 mediate melanization and actin polymerization, but are dispensable for Plasmodium killing in the mosquito midgut. PLoS Pathog 2: e133.). At this point, ookinetes are exposed to the haemocoel and to the complement-like soluble immune proteins that circulate in the haemolymph (Blandin et al. 2004Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116: 661-670., Shiao et al. 2006Shiao SH, Whitten MM, Zachary D, Hoffmann JA, Levashina EA 2006. Fz2 and cdc42 mediate melanization and actin polymerization, but are dispensable for Plasmodium killing in the mosquito midgut. PLoS Pathog 2: e133.).

The thioester protein 1 (TEP1) was initially identified in view of its high similarity to the vertebrate complement factor C3 and to the related family of alpha-2-macroglobulins (Levashina et al. 2001Levashina EA, Moita LF, Blandin S, Vriend G, Lagueux M, Kafatos FC 2001. Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae. Cell 104: 709-718.). Similar to the role of C3 in the complement pathway of mammals, TEP1 behaves as an opsonin promoting the phagocytosis of bacteria (Levashina et al. 2001Levashina EA, Moita LF, Blandin S, Vriend G, Lagueux M, Kafatos FC 2001. Conserved role of a complement-like protein in phagocytosis revealed by dsRNA knockout in cultured cells of the mosquito, Anopheles gambiae. Cell 104: 709-718.) and binds to the ookinete surface to mediate parasite lysis or melanisation (Blandin et al. 2004Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116: 661-670.). In the haemolymph, TEP1 is expressed constitutively as full-length (inactive) and processed (active) forms that require the scaffolding proteins leucine-rich immune molecule 1 (LRIM1) and Anopheles Plasmodium-responsive leucine-rich repeat protein 1 (APL1) to direct TEP1 to pathogen surfaces and prevent self-recognition (Fraiture et al. 2009Fraiture M, Baxter RH, Steinert S, Chelliah Y, Frolet C, Quispe-Tintaya W, Hoffmann JA, Blandin SA, Levashina EA 2009. Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium. Cell Host Microbe 5: 273-284., Povelones et al. 2009Povelones M, Waterhouse RM, Kafatos FC, Christophides GK 2009. Leucine-rich repeat protein complex activates mosquito complement in defense against Plasmodium parasites. Science 324: 258-261.). Loss of TEP1, LRIM1, or APL1 prevents ookinete recognition and dramatically increases parasite numbers (Fraiture et al. 2009Fraiture M, Baxter RH, Steinert S, Chelliah Y, Frolet C, Quispe-Tintaya W, Hoffmann JA, Blandin SA, Levashina EA 2009. Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium. Cell Host Microbe 5: 273-284., Povelones et al. 2009Povelones M, Waterhouse RM, Kafatos FC, Christophides GK 2009. Leucine-rich repeat protein complex activates mosquito complement in defense against Plasmodium parasites. Science 324: 258-261.). Interestingly, members of the APL1 family (APL1A, APL1B, APL1C) appear to confer pathogen recognition specificity to the TEP1/LRIM1/APL1 complex. APL1A expression is required for protection against P. falciparum parasites, while APL1C is needed for protection against the rodent malaria parasites P. berghei and P. yoelli (Mitri et al. 2009Mitri C, Jacques JC, Thiery I, Riehle MM, Xu J, Bischoff E, Morlais I, Nsango SE, Vernick KD, Bourgouin C 2009. Fine pathogen discrimination within the APL1 gene family protects Anopheles gambiae against human and rodent malaria species. PLoS Pathog 5: e1000576.). Although it is unclear how the mosquito immune system is able to distinguish their targets, it is reasonable to assume that members of the APL1 gene family have important functions in parasite recognition as part of the mosquito complement-like pathway.

Upon binding to the ookinete, TEP1 is thought to promote parasite killing through lysis or melanisation (Blandin et al. 2004Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116: 661-670.). Recent evidence has identified that a non-catalytic protease, SPCLIP1, may function to amplify complement activation by binding to TEP1 on the pathogen surface and promote the recruitment of additional TEP1 (Povelones et al. 2013Povelones M, Bhagavatula L, Yassine H, Tan LA, Upton LM, Osta MA, Christophides GK 2013. The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae. PLoS Pathog 9: e1003623.). A similar amplification of the complement response is also seen in the vertebrate complement system where C3b, the cleavage product of C3 (equivalent to TEP1), binds factor B on the surface of the pathogen. This complex is then activated by factor D into the C3 convertase that in turn mediates the recruitment of additional C3b molecules to the pathogen surface [reviewed in Walport (2001)Walport MJ 2001. Complement. First of two parts. N Engl J Med 344: 1058-1066.]. However, our knowledge of this process in the mosquito is incomplete and additional proteins that promote parasite killing may be recruited to the ookinete surface, as is the case for the vertebrate complement system. Multiple proteins and co-factors are known to assemble onto pathogen surfaces as part of the mammalian complement pathways and further investigation of the components of the mosquito complement-like pathway remains an important goal for future investigation.

Furthermore, many questions remain regarding the mechanisms involved in directing TEP1 binding to the ookinete surface. As mentioned previously, midgut nitration appears to be a critical determinant of parasite recognition by the mosquito complement-like pathway (Oliveira et al. 2012Oliveira GA, Lieberman J, Barillas-Mury C 2012. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science 335: 856-859.). However, it is unclear if TEP1 directly recognises these protein modifications on the parasite surface or if this is mediated by other supporting molecules.

“Late-phase” immunity - Ookinetes that survive the process of midgut invasion and the early immune responses are subjected to a second or “late-phase” immune response that further limits oocyst numbers. First proposed by Gupta et al. (2009)Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507., the signal transducer and activator of transcription (STAT)-A or STAT-B significantly increases parasite survival without altering the number of early oocysts. Further experiments determined that STAT-A or STAT-B silencing increased oocyst survival through decreased production of NOS (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.).

In An. gambiae, STAT-B regulates stat-A mRNA expression and STAT-A mediates the transcriptional activation of nos in response to infection (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.). This transcriptional cascade can be manipulated through suppressor of cytokine signalling-3 (SOCS-3)-silencing (an inhibitor of STAT-A) resulting in the constitutive activation of STAT-A signalling and increased NOS expression (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.). This results in a near refractory phenotype that elevates NOS expression in the mosquito midgut and carcass (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.).

In addition to this study, there is a great deal of evidence suggesting that NOS and subsequent NO production are important determinants of oocyst development (Luckhart et al. 1998Luckhart S, Vodovotz Y, Cui L, Rosenberg R 1998. The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc Natl Acad Sci USA 95: 5700-5705., Bahia et al. 2011Bahia AC, Kubota MS, Tempone AJ, Araújo HR, Guedes BA, Orfanó AS, Tadei WP, Ríos-Velásquez CM, Han YS, Secundino NF, Barillas-Mury C, Pimenta PF, Traub-Csekö YM 2011. The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection. PLoS Negl Trop Dis 5: e1317., Vijay et al. 2011Vijay S, Rawat M, Adak T, Dixit R, Nanda N, Srivastava H, Sharma JK, Prasad GB, Sharma A 2011. Parasite killing in malaria non-vector mosquito Anopheles culicifacies species B: implication of nitric oxide synthase upregulation. PLoS ONE 6: e18400.). NOS expression appears to be induced throughout the entire mosquito in response to Plasmodium infection (Luckhart et al. 1998Luckhart S, Vodovotz Y, Cui L, Rosenberg R 1998. The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc Natl Acad Sci USA 95: 5700-5705., Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.) suggesting that NOS activation is a generalised, rather than local, epithelial response to infection (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.). Much work remains to be done to better define the mechanisms of late-phase immune response and how it interferes with oocyst development.

Immune pathways that limit parasite development - Much of our knowledge of the mosquito innate immune response stems from Drosophila research and from other insect systems. Orthologous immune signalling pathways have been described in mosquitoes that respond to a variety of pathogens and regulate anti-Plasmodium immunity. However, compared to Drosophila, there have been rapid expansions of mosquito immune gene families, suggesting a functional broadening of the mosquito defense systems (Waterhouse et al. 2007Waterhouse RM, Kriventseva EV, Meister S, Xi Z, Alvarez KS, Bartholomay LC, Barillas-Mury C, Bian G, Blandin S, Christensen BM, Dong Y, Jiang H, Kanost MR, Koutsos AC, Levashina EA, Li J, Ligoxygakis P, Maccallum RM, Mayhew GF, Mendes A, Michel K, Osta MA, Paskewitz S, Shin SW, Vlachou D, Wang L, Wei W, Zheng L, Zou Z, Severson DW, Raikhel AS, Kafatos FC, Dimopoulos G, Zdobnov EM, Christophides GK 2007. Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes. Science 316: 1738-1743.). As a result, the mosquito represents a unique model to study host-parasite interactions and the innate immune response.

In Drosophila, the Toll and immunodeficiency (IMD) pathways have traditionally been associated with the production of antimicrobial peptides (such as cecropin and defensin) in response to bacterial infection, while the JAK-STAT pathway has been implicated in antiviral immunity (Lemaitre & Hoffmann 2007Lemaitre B, Hoffmann J 2007. The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697-743.). While these pathways certainly exist in mosquitoes [reviewed by Cirimotich et al. (2010)Cirimotich CM, Dong Y, Garver LS, Sim S, Dimopoulos G 2010. Mosquito immune defenses against Plasmodium infection. Dev Comp Immunol 34: 387-395.], it appears that these immune pathways also mediate anti-Plasmodium defenses. However, given current technology that relies primarily on systemic gene-silencing, the contributions of individual mosquito tissues (midgut, haemocytes, fat body) to the immune defenses has been difficult to ascertain. Three major insect immune pathways have been described: Toll, IMD and JAK-STAT.

The Toll pathway - In Drosophila, the involvement of the Toll pathway has been well described in the host response to Gram-positive bacteria, fungi and viruses (Lemaitre & Hoffmann 2007Lemaitre B, Hoffmann J 2007. The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697-743.). Similar experiments in Anophe-les suggest that this pathway is evolutionarily conserved in mosquitoes (Frolet et al. 2006Frolet C, Thoma M, Blandin S, Hoffmann JA, Levashina EA 2006. Boosting NF-kappaB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei. Immunity 25: 677-685., Garver et al. 2009Garver LS, Dong Y, Dimopoulos G 2009. Caspar controls resistance to Plasmodium falciparum in diverse anopheline species. PLoS Pathog 5: e1000335.).

REL1, originally described as Gambif1, is an anopheline nuclear factor kappa B-like transcription factor orthologous to Drosophila Dorsal (Barillas-Mury et al. 1996Barillas-Mury C, Charlesworth A, Gross I, Richman A, Hoffmann JA, Kafatos FC 1996. Immune factor Gambif1, a new rel family member from the human malaria vector, Anopheles gambiae. EMBO J 15: 4691-4701.). Upon immune activation, Toll signalling results in the directed degradation of Cactus, a negative regulator of REL1, thus allowing translocation of REL1 to the nucleus and the expression of its downstream effector genes (Frolet et al. 2006Frolet C, Thoma M, Blandin S, Hoffmann JA, Levashina EA 2006. Boosting NF-kappaB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei. Immunity 25: 677-685.). Silencing cactus expression by dsRNA injection results in the constitutive activation of the Toll pathway, even without immune challenge (Frolet et al. 2006Frolet C, Thoma M, Blandin S, Hoffmann JA, Levashina EA 2006. Boosting NF-kappaB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei. Immunity 25: 677-685.). Frolet et al. (2006)Frolet C, Thoma M, Blandin S, Hoffmann JA, Levashina EA 2006. Boosting NF-kappaB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei. Immunity 25: 677-685. demonstrate that Toll activation significantly impairs P. berghei development, although the exact mechanism is not well understood. Recent evidence implicates mosquito haemocytes as critical mediators of this anti-Plasmodium response (Ramirez et al. 2014Ramirez JL, Garver LS, Brayner FA, Alves LC, Rodrigues J, Molina-Cruz A, Barillas-Mury C 2014. The role of hemocytes in Anopheles gambiae antiplasmodial immunity. J Innate Immun 6: 119-128.).

Further studies suggest that Toll activation may be more efficient at limiting P. berghei (a rodent parasite) development than P. falciparum (the human malaria parasite) in multiple mosquito vectors (Garver et al. 2009Garver LS, Dong Y, Dimopoulos G 2009. Caspar controls resistance to Plasmodium falciparum in diverse anopheline species. PLoS Pathog 5: e1000335.). This would imply that immune recognition of rodent and human malaria parasites may occur through different mechanisms (Dong et al. 2006Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G 2006. Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog 2: e52., Garver et al. 2009Garver LS, Dong Y, Dimopoulos G 2009. Caspar controls resistance to Plasmodium falciparum in diverse anopheline species. PLoS Pathog 5: e1000335.). How the mosquito distinguishes these two parasites is a very interesting question that remains to be answered.

The IMD pathway - The IMD pathway of mosquitoes is analogous to the TNF signalling pathway in mammals. Pathogen recognition is mediated by peptidoglycan recognition protein LC and the adaptor protein IMD, triggering the cleavage of the transcription factor REL2 (Drosophila Relish), which results in its nuclear translocation (Meister et al. 2005Meister S, Kanzok SM, Zheng XL, Luna C, Li TR, Hoa NT, Clayton JR, White KP, Kafatos FC, Christophides GK, Zheng L 2005. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc Natl Acad Sci USA 102: 11420-11425., 2009Meister S, Agianian B, Turlure F, Relógio A, Morlais I, Kafatos FC, Christophides GK 2009. Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLoS Pathog 5: e1000542., Luna et al. 2006Luna C, Hoa NT, Lin H, Zhang L, Nguyen HL, Kanzok SM, Zheng L 2006. Expression of immune responsive genes in cell lines from two different Anopheline species. Insect Mol Biol 15: 721-729.). This cascade of events is commonly referred to as the canonical IMD pathway, yet additional signalling events are activated through other IMD pathway components that include TGF-ß-activated protein kinase 1 (TAK1), which mediates the JNK signalling pathway in Drosophila (Silverman et al. 2003Silverman N, Zhou R, Erlich RL, Hunter M, Bernstein E, Schneider D, Maniatis T 2003. Immune activation of NF-kappaB and JNK requires Drosophila TAK1. J Biol Chem 278: 48928-48934., Delaney et al. 2006Delaney JR, Stöven S, Uvell H, Anderson KV, Engström Y, Mlodzik M 2006. Cooperative control of Drosophila immune responses by the JNK and NF-kappaB signaling pathways. EMBO J 25: 3068-3077.). Currently, it is unclear what role the IMD pathway plays in TAK1 signalling and mitogen-activated protein kinase (MAPK) activation in mosquitoes, however recent evidence implies that JNK activation plays a key role in the mosquito immune response to Plasmodium (Garver et al. 2013Garver LS, Oliveira GA, Barillas-Mury C 2013. The JNK pathway is a key mediator of Anopheles gambiae antiplasmodial immunity. PLoS Pathog 9: e1003622.). As a result, the Anopheles IMD pathway appears to be highly complex and further research is required to fully understand its intricacies.

Based on Drosophila research, the Anopheles IMD pathway is likely regulated at several different steps [reviewed by Cirimotich et al. (2010)Cirimotich CM, Dong Y, Garver LS, Sim S, Dimopoulos G 2010. Mosquito immune defenses against Plasmodium infection. Dev Comp Immunol 34: 387-395.]. In mosquitoes, it has been suggested that immune regulation of one IMD pathway component occurs through the differential splicing of the transcription factor REL2 (Meister et al. 2005Meister S, Kanzok SM, Zheng XL, Luna C, Li TR, Hoa NT, Clayton JR, White KP, Kafatos FC, Christophides GK, Zheng L 2005. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc Natl Acad Sci USA 102: 11420-11425., Luna et al. 2006Luna C, Hoa NT, Lin H, Zhang L, Nguyen HL, Kanzok SM, Zheng L 2006. Expression of immune responsive genes in cell lines from two different Anopheline species. Insect Mol Biol 15: 721-729.). A short form (REL2-S) lacking the inhibitory ankyrin domain and a full-length form (REL2-F) are constitutively expressed throughout development (Meister et al. 2005Meister S, Kanzok SM, Zheng XL, Luna C, Li TR, Hoa NT, Clayton JR, White KP, Kafatos FC, Christophides GK, Zheng L 2005. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc Natl Acad Sci USA 102: 11420-11425.). Whereas the short form is constitutively active and thought to be responsible for basal immune function, REL2-F is localised in the cytoplasm and thus transcriptionally inactive (Meister et al. 2005Meister S, Kanzok SM, Zheng XL, Luna C, Li TR, Hoa NT, Clayton JR, White KP, Kafatos FC, Christophides GK, Zheng L 2005. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc Natl Acad Sci USA 102: 11420-11425., Luna et al. 2006Luna C, Hoa NT, Lin H, Zhang L, Nguyen HL, Kanzok SM, Zheng L 2006. Expression of immune responsive genes in cell lines from two different Anopheline species. Insect Mol Biol 15: 721-729.). Upon immune activation, IMD signalling stimulates DREDD-dependent cleavage of REL2-F exposing its nuclear translocation signal, resulting in nuclear translocation and transcriptional activation of REL2-dependent genes (Kim et al. 2006Kim M, Lee JH, Lee SY, Kim E, Chung J 2006. Caspar, a suppressor of antibacterial immunity in Drosophila. Proc Natl Acad Sci USA 103: 16358-16363.). Due to the inability to distinguish the REL2 short and full-length forms by RNAi, their effect on parasite infection have been difficult to elucidate (Meister et al. 2005Meister S, Kanzok SM, Zheng XL, Luna C, Li TR, Hoa NT, Clayton JR, White KP, Kafatos FC, Christophides GK, Zheng L 2005. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc Natl Acad Sci USA 102: 11420-11425., Luna et al. 2006Luna C, Hoa NT, Lin H, Zhang L, Nguyen HL, Kanzok SM, Zheng L 2006. Expression of immune responsive genes in cell lines from two different Anopheline species. Insect Mol Biol 15: 721-729.). Nevertheless, the anti-Plasmodium effects are likely due to REL2-F processing (Meister et al. 2005Meister S, Kanzok SM, Zheng XL, Luna C, Li TR, Hoa NT, Clayton JR, White KP, Kafatos FC, Christophides GK, Zheng L 2005. Immune signaling pathways regulating bacterial and malaria parasite infection of the mosquito Anopheles gambiae. Proc Natl Acad Sci USA 102: 11420-11425.).

The most striking evidence that IMD signalling is involved in directing anti-Plasmodium immunity was obtained by silencing caspar, an inhibitor of IMD signalling through DREDD-dependent cleavage of REL2-F (Kim et al. 2006Kim M, Lee JH, Lee SY, Kim E, Chung J 2006. Caspar, a suppressor of antibacterial immunity in Drosophila. Proc Natl Acad Sci USA 103: 16358-16363.). Caspar-silencing renders mosquitoes refractory to malaria parasites and it appears that activation of the IMD pathway is more efficient in limiting P. falciparum infection than that of the murine malaria parasite, P. berghei (Garver et al. 2009Garver LS, Dong Y, Dimopoulos G 2009. Caspar controls resistance to Plasmodium falciparum in diverse anopheline species. PLoS Pathog 5: e1000335.). However, additional work is needed to understand the precise mechanisms of REL2 activation and the contributions made by the various mosquito immune tissues to this process.

The JAK-STAT pathway - The JAK-STAT (or STAT) pathway has been the least investigated of the three major signalling pathways in mosquitoes and as such, much of our knowledge is based on JAK-STAT signalling from vertebrate or Drosophila model systems. In Drosophila, the STAT pathway regulates several aspects of development, epithelial renewal, the immune response to bacterial and viral infections and haemocyte differentiation/proliferation (Arbouzova & Zeidler 2006Arbouzova NI, Zeidler MP 2006. JAK/STAT signalling in Drosophila: insights into conserved regulatory and cellular functions. Development 133: 2605-2616., Buchon et al. 2009Buchon N, Broderick NA, Poidevin M, Pradervand S, Lemaitre B 2009. Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe 5: 200-211.). Similar to Drosophila, the mosquito JAK-STAT pathway has also been implicated in the immune response to bacteria (Barillas-Mury et al. 1999Barillas-Mury C, Han YS, Seeley D, Kafatos FC 1999. Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection. EMBO J 18: 959-967., Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.), viruses (Souza-Neto et al. 2009) and Plasmodium parasites (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507., Bahia et al. 2011Bahia AC, Kubota MS, Tempone AJ, Araújo HR, Guedes BA, Orfanó AS, Tadei WP, Ríos-Velásquez CM, Han YS, Secundino NF, Barillas-Mury C, Pimenta PF, Traub-Csekö YM 2011. The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection. PLoS Negl Trop Dis 5: e1317.).

In An. gambiae, two STAT transcription factors (STAT-A and STAT-B) have been identified (Barillas-Mury et al. 1999Barillas-Mury C, Han YS, Seeley D, Kafatos FC 1999. Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection. EMBO J 18: 959-967., Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.), where STAT-B is thought to regulate the transcription of the stat-A gene upon activation (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.). This is in contrast to Drosophila and other mosquito species that contain only a single STAT transcription factor (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507., Souza-Neto et al. 2009, Bahia et al. 2011Bahia AC, Kubota MS, Tempone AJ, Araújo HR, Guedes BA, Orfanó AS, Tadei WP, Ríos-Velásquez CM, Han YS, Secundino NF, Barillas-Mury C, Pimenta PF, Traub-Csekö YM 2011. The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection. PLoS Negl Trop Dis 5: e1317.). Following immune activation, STAT is phosphorylated leading to its translocation to the nucleus and activation of downstream effector genes. Silencing of STAT leads to increased P. berghei and P. falciparum survival in An. gambiae (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.), as well as P. vivax in the Brazilian vector Anopheles aquasalis (Bahia et al. 2011Bahia AC, Kubota MS, Tempone AJ, Araújo HR, Guedes BA, Orfanó AS, Tadei WP, Ríos-Velásquez CM, Han YS, Secundino NF, Barillas-Mury C, Pimenta PF, Traub-Csekö YM 2011. The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection. PLoS Negl Trop Dis 5: e1317.).

STAT signalling is tightly regulated by the inhibitors suppressors of cytokine signalling (SOCS) and protein inhibitors of activated STAT, which respectively prevent STAT phosphorylation or promote degradation. Expression of SOCS is mediated by STAT activation and thus serves to shut off STAT signalling through a negative feedback loop (Gupta et al. 2009Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507.). Gupta et al. (2009)Gupta L, Molina-Cruz A, Kumar S, Rodrigues J, Dixit R, Zamora RE, Barillas-Mury C 2009. The STAT pathway mediates late-phase immunity against Plasmodium in the mosquito Anopheles gambiae. Cell Host Microbe 5: 498-507. demonstrated that SOCS-silencing dramatically reduces parasite numbers and that this response is mediated by increased levels of NOS as a result of constitutive STAT activation.

Other pathways - Although the Toll, IMD and JAK-STAT pathways have been the most investigated in mosquitoes, other less characterised pathways may also contribute to the mosquito immune response to Plasmodium.

Recently, a lipopolysaccharide-induced TNF-α factor-like transcription factor (LL3) was described in An. gambiae that mediates a potent anti-Plasmodium immune response against both P. berghei and P. falciparum parasites (Smith et al. 2012Smith RC, Eappen AG, Radtke AJ, Jacobs-Lorena M 2012. Regulation of anti-Plasmodium immunity by a LITAF-like transcription factor in the malaria vector Anopheles gambiae. PLoS Pathog 8: e1002965.). LL3 expression is strongly up-regulated in response to ookinete invasion of the midgut and directly influences the expression of SRPN6 (Smith et al. 2012Smith RC, Eappen AG, Radtke AJ, Jacobs-Lorena M 2012. Regulation of anti-Plasmodium immunity by a LITAF-like transcription factor in the malaria vector Anopheles gambiae. PLoS Pathog 8: e1002965.), a serine protease inhibitor implicated in the anti-Plasmodium immune response (Abraham et al. 2005Abraham EG, Pinto SB, Ghosh A, Vanlandingham DL, Budd A, Higgs S, Kafatos FC, Jacobs-Lorena M, Michel K 2005. An immune-responsive serpin, SRPN6, mediates mosquito defense against malaria parasites. Proc Natl Acad Sci USA 102: 16327-16332., Pinto et al. 2008Pinto SB, Kafatos FC, Michel K 2008. The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anophe- les gambiae. Cell Microbiol 10: 891-898., Eappen et al. 2013Eappen AG, Smith RC, Jacobs-Lorena M 2013. Enterobacter-activated mosquito immune responses to Plasmodium involve activation of SRPN6 in Anopheles stephensi. PLoS ONE 8: e62937.). However, because SRPN6-silencing in susceptible lines of An. gambiae does not impact infection intensity (Abraham et al. 2005Abraham EG, Pinto SB, Ghosh A, Vanlandingham DL, Budd A, Higgs S, Kafatos FC, Jacobs-Lorena M, Michel K 2005. An immune-responsive serpin, SRPN6, mediates mosquito defense against malaria parasites. Proc Natl Acad Sci USA 102: 16327-16332.), the large increase in oocyst numbers following LL3 knockdown likely extends beyond the regulation of SRPN6 in the mosquito anti-Plasmodium response. Clarification of the mechanisms of LL3 activation and the role of LL3 in the overall context of mosquito immunity are the subjects of further study.

Emerging evidence suggests that components of the ingested blood meal also affect mosquito immune function (Pakpour et al. 2013Pakpour N, Akman-Anderson L, Vodovotz Y, Luckhart S 2013. The effects of ingested mammalian blood factors on vector arthropod immunity and physiology. Microbes Infect 15: 243-254.). TGF-ß1 and insulin in the ingested blood are believed to activate insulin/insulin growth factor 1 (IGF1) signalling (IIS) and MAPK signalling cascades in the mosquito thus increasing mosquito susceptibility to parasite infection (Surachetpong et al. 2009Surachetpong W, Singh N, Cheung KW, Luckhart S 2009. MAPK ERK signaling regulates the TGF-beta1-dependent mosquito response to Plasmodium falciparum. PLoS Pathog 5: e1000366., 2011Surachetpong W, Pakpour N, Cheung KW, Luckhart S 2011. Reactive oxygen species-dependent cell signaling regulates the mosquito immune response to Plasmodium falciparum. Antioxid Redox Signal 14: 944-953., Pakpour et al. 2012Pakpour N, Corby-Harris V, Green GP, Smithers HM, Cheung KW, Riehle MA, Luckhart S 2012. Ingested human insulin inhibits the mosquito NF-κB-dependent immune response to Plasmodium falciparum. Infect Immun 80: 2141-2149.). In contrast, another blood component, human IGF1, reduces malaria parasite infection (Drexler et al. 2013Drexler A, Nuss A, Hauck E, Glennon E, Cheung K, Brown M, Luckhart S 2013. Human IGF1 extends lifespan and enhances resistance to Plasmodium falciparum infection in the malaria vector Anopheles stephensi. J Exp Biol 216: 208-217.). It appears that a number of factors contribute to IIS and MAPK signalling and that this delicate balance determines Plasmodium developmental success in the mosquito host. However, there is much more to learn on how the mosquito IIS and MAPK pathways contribute to mosquito immunity.

Recent evidence suggests that components of the IIS pathway can be manipulated in transgenic An. stephensi to reduce parasite infection and decrease mosquito lifespan through the over-expression of an activated Akt molecule in the mosquito midgut (Corby-Harris et al. 2010Corby-Harris V, Drexler A, de Jong LW, Antonova Y, Pakpour N, Ziegler R, Ramberg F, Lewis EE, Brown JM, Luckhart S, Riehle MA 2010. Activation of Akt signaling reduces the prevalence and intensity of malaria parasite infection and lifespan in Anopheles stephensi mosquitoes. PLoS Pathog 6: e1001003.). However, these effects appear to be mediated by a disruption of mitochondrial dynamics that perturb midgut homoeostasis and are independent of IIS signalling (Luckhart et al. 2013Luckhart S, Giulivi C, Drexler AL, Antonova-Koch Y, Sakaguchi D, Napoli E, Wong S, Price MS, Eigenheer R, Phinney BS, Pakpour N, Pietri JE, Cheung K, Georgis M, Riehle M 2013. Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host. PLoS Pathog 9: e1003180.).

Contributions of mosquito tissues to the anti-Plasmodium immune response - The immune responses that limit Plasmodium development are multi-faceted, involving multiple mosquito tissues that exert both individual and concerted responses over several days as the parasite journeys through the mosquito. While the midgut serves as the initial barrier and recognition site for invading ookinetes, immune responses in the fat body at the time of ookinete invasion suggest that pathogen recognition triggers a systemic humoral response (Dimopoulos et al. 1997Dimopoulos G, Richman A, Müller HM, Kafatos FC 1997. Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc Natl Acad Sci USA 94: 11508-11513.). While cellular and humoral mosquito immunity effectively limit parasite development in the mosquito, the role of each immune tissue have been difficult to ascertain due to limitations in mosquito genetics. Thus, the contribution of each mosquito immune tissue to the overall anti-Plasmodium defense is an important question that has yet to be fully addressed. Once identified, it may be possible to harness the most effective responses to limit Plasmodium development via genetic engineering or chemical inhibitors.

Although incomplete, our current understanding of the role of each respective tissue (midgut, haemocytes, fat body) is summarised below.

Midgut - The mosquito midgut is an important component of the mosquito immune response, serving as a physical barrier to parasite development and the initial site of pathogen recognition. There is evidence that the mosquito can sense the presence of malaria parasites shortly after the ingestion of the blood meal (Vlachou et al. 2005Vlachou D, Schlegelmilch T, Christophides GK, Kafatos FC 2005. Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion. Curr Biol 15: 1185-1195., Dong et al. 2006Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G 2006. Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog 2: e52.), but it appears that the primary midgut immune response is triggered in response to ookinete invasion (Vlachou et al. 2005Vlachou D, Schlegelmilch T, Christophides GK, Kafatos FC 2005. Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion. Curr Biol 15: 1185-1195., Dong et al. 2006Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G 2006. Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog 2: e52., Smith et al. 2012Smith RC, Eappen AG, Radtke AJ, Jacobs-Lorena M 2012. Regulation of anti-Plasmodium immunity by a LITAF-like transcription factor in the malaria vector Anopheles gambiae. PLoS Pathog 8: e1002965.).

Several groups have identified immune components that are up-regulated in response to Plasmodium ookinete invasion (Vlachou et al. 2005Vlachou D, Schlegelmilch T, Christophides GK, Kafatos FC 2005. Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion. Curr Biol 15: 1185-1195., Dong et al. 2006Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G 2006. Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog 2: e52., Mendes et al. 2011Mendes AM, Awono-Ambene PH, Nsango SE, Cohuet A, Fontenille D, Kafatos FC, Christophides GK, Morlais I, Vlachou D 2011. Infection intensity-dependent responses of Anopheles gambiae to the African malaria parasite Plasmodium falciparum. Infect Immun 79: 4708-4715.). However, it is unclear to what extent these immune effectors are produced in the midgut itself. It has been suggested that the increased TEP1 transcript expression in the midgut following ookinete invasion may actually be the result of increased haemocyte attachment to the midgut epithelium (haemocytes strongly express TEP1) (Blandin et al. 2004Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116: 661-670., Vlachou et al. 2005Vlachou D, Schlegelmilch T, Christophides GK, Kafatos FC 2005. Functional genomic analysis of midgut epithelial responses in Anopheles during Plasmodium invasion. Curr Biol 15: 1185-1195.). Further experiments are needed to clarify if the increased expression of other immune effectors and antimicrobial peptides are also the result of haemocyte attachment or de novo synthesis in the midgut epithelium.

Parasite invasion also induces dramatic physiological changes in cells of the midgut epithelium (Han et al. 2000Han YS, Thompson J, Kafatos FC, Barillas-Mury C 2000. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J 19: 6030-6040.). NOS is up-regulated, which in turn leads to the formation of nitrites and peroxides that promote apoptosis (Han et al. 2000Han YS, Thompson J, Kafatos FC, Barillas-Mury C 2000. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J 19: 6030-6040., Kumar et al. 2004Kumar S, Gupta L, Han YS, Barillas-Mury C 2004. Inducible peroxidases mediate nitration of Anopheles midgut cells undergoing apoptosis in response to Plasmodium invasion. J Biol Chem 279: 53475-53482., Kumar & Barillas-Mury 2005Kumar S, Barillas-Mury C 2005. Ookinete-induced midgut peroxidases detonate the time bomb in anopheline mosquitoes. Insect Biochem Mol Biol 35: 721-727., Herrera-Ortiz et al. 2011Herrera-Ortiz A, Martínez-Barnetche J, Smit N, Rodriguez MH, Lanz-Mendoza H 2011. The effect of nitric oxide and hydrogen peroxide in the activation of the systemic immune response of Anopheles albimanus infected with Plasmodium berghei. Dev Comp Immunol 35: 44-50.). This likely produces a hostile environment for the invading ookinete that it must escape before damage to itself and before the damaged cell is extruded from the midgut epithelium, to ensure its own survival (Han et al. 2000Han YS, Thompson J, Kafatos FC, Barillas-Mury C 2000. Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes. EMBO J 19: 6030-6040.). While it remains unclear to what extent the “time bomb” model limits parasite development, some have suggested that this is likely a general response to remove damaged epithelial cells and is not specific to parasite invasion (Baton & Ranford-Cartwright 2005Baton LA, Ranford-Cartwright LC 2005. How do malaria ookinetes cross the mosquito midgut wall? Trends Parasitol 21: 22-28.).

New evidence suggests that the process of ookinete invasion does not directly limit parasite numbers, but rather “marks” ookinetes for later destruction by components of the haemolymph (Oliveira et al. 2012Oliveira GA, Lieberman J, Barillas-Mury C 2012. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science 335: 856-859.). Invading ookinetes are thought to become labelled by epithelial nitration mediated by HPX2 and NADPH NOX5, thus marking the ookinete for recognition by TEP1 and ultimate lysis or melanisation (Oliveira et al. 2012Oliveira GA, Lieberman J, Barillas-Mury C 2012. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science 335: 856-859.). Recent work implicates the JNK pathway in the induction of HPX2 and NOX5 (Garver et al. 2013Garver LS, Oliveira GA, Barillas-Mury C 2013. The JNK pathway is a key mediator of Anopheles gambiae antiplasmodial immunity. PLoS Pathog 9: e1003622.), thus modulating the levels of epithelial nitration in response to midgut invasion that is required for mosquito complement recognition and subsequent parasite destruction (Oliveira et al. 2012Oliveira GA, Lieberman J, Barillas-Mury C 2012. Epithelial nitration by a peroxidase/NOX5 system mediates mosquito antiplasmodial immunity. Science 335: 856-859., Garver et al. 2013Garver LS, Oliveira GA, Barillas-Mury C 2013. The JNK pathway is a key mediator of Anopheles gambiae antiplasmodial immunity. PLoS Pathog 9: e1003622.).

Importantly, ROS balance in different tissues of the mosquito, including the midgut, haemolymph and fat body, is an important determinant of parasite survival. For example, the refractory mosquito L35 strain is in a chronic state of oxidative stress when compared to the susceptible S or G3 strains, resulting in a deleterious environment that promotes parasite killing (Kumar et al. 2003Kumar S, Christophides GK, Cantera R, Charles B, Han YS, Meister S, Dimopoulos G, Kafatos FC, Barillas-Mury C 2003. The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci USA 100: 14139-14144.). Reducing oxidative stress in refractory mosquitoes by dietary supplementation with antioxidants abrogates the ROS-mediated killing effect (Kumar et al. 2003Kumar S, Christophides GK, Cantera R, Charles B, Han YS, Meister S, Dimopoulos G, Kafatos FC, Barillas-Mury C 2003. The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci USA 100: 14139-14144.). Silencing of the ROS detoxification enzymes, catalase or oxidation resistance 1, increase oxidative stress and greatly reduce parasite survival (Molina-Cruz et al. 2008Molina-Cruz A, de Jong RJ, Charles B, Gupta L, Kumar S, Jaramillo-Gutierrez G, Barillas-Mury C 2008. Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium. J Biol Chem 283: 3217-3223., Jaramillo-Gutierrez et al. 2010Jaramillo-Gutierrez G, Molina-Cruz A, Kumar S, Barillas-Mury C 2010. The Anopheles gambiae oxidation resistance 1 (OXR1) gene regulates expression of enzymes that detoxify reactive oxygen species. PLoS ONE 5: e11168.). Taken together, evidence would suggest that the levels of ROS are tightly controlled by the mosquito to enhance parasite killing during ookinete traversal of the midgut (Kumar et al. 2003Kumar S, Christophides GK, Cantera R, Charles B, Han YS, Meister S, Dimopoulos G, Kafatos FC, Barillas-Mury C 2003. The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae. Proc Natl Acad Sci USA 100: 14139-14144., Molina-Cruz et al. 2008Molina-Cruz A, de Jong RJ, Charles B, Gupta L, Kumar S, Jaramillo-Gutierrez G, Barillas-Mury C 2008. Reactive oxygen species modulate Anopheles gambiae immunity against bacteria and Plasmodium. J Biol Chem 283: 3217-3223.).

Haemocytes - The role of haemocytes, or circulating blood cells, has been largely unexplored in the context of their contributions to mosquito innate immunity. Much like their Drosophila counterparts, mosquito haemocytes are believed to be the primary phagocytic cells that directly eliminate bacterial pathogens in the haemocoel (Lavine & Strand 2002Lavine MD, Strand MR 2002. Insect hemocytes and their role in immunity. Insect Biochem Mol Biol 32: 1295-1309.). In addition, haemocytes are thought to produce several immune components found in the mosquito haemolymph, such as TEP1 (Blandin et al. 2004Blandin S, Shiao SH, Moita LF, Janse CJ, Waters AP, Kafatos FC, Levashina EA 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116: 661-670., Frolet et al. 2006Frolet C, Thoma M, Blandin S, Hoffmann JA, Levashina EA 2006. Boosting NF-kappaB-dependent basal immunity of Anopheles gambiae aborts development of Plasmodium berghei. Immunity 25: 677-685.), which mediate the immune response and pathogen clearance. However, very little is known regarding their role in anti-Plasmodium immunity.

Transcriptional profiling of An. gambiae haemocytes reveal specific responses to Plasmodium parasites (Baton et al. 2009Baton LA, Robertson A, Warr E, Strand MR, Dimopoulos G 2009. Genome-wide transcriptomic profiling of Anopheles gambiae hemocytes reveals pathogen-specific signatures upon bacterial challenge and Plasmodium. BMC Genomics 10: 257., Pinto et al. 2009Pinto SB, Lombardo F, Koutsos AC, Waterhouse RM, McKay K, An C, Ramakrishnan C, Kafatos FC, Michel K 2009. Discovery of Plasmodium modulators by genome-wide analysis of circulating hemocytes in Anopheles gambiae. Proc Natl Acad Sci USA 106: 21270-21275.) and bacterial pathogens (Baton et al. 2009Baton LA, Robertson A, Warr E, Strand MR, Dimopoulos G 2009. Genome-wide transcriptomic profiling of Anopheles gambiae hemocytes reveals pathogen-specific signatures upon bacterial challenge and Plasmodium. BMC Genomics 10: 257.), suggesting that haemocytes may have an integral role in regulating mosquito innate immune responses. However, these studies did not distinguish among the three haemocyte sub-types (prohaemocytes, oenocytoids and granulocytes) thus far characterised in Anopheles (Castillo et al. 2006Castillo JC, Robertson AE, Strand MR 2006. Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti. Insect Biochem Mol Biol 36: 891-903.). These haemocyte populations are dynamic with changes in overall numbers and proportions in response to age (Hillyer et al. 2005Hillyer JF, Schmidt SL, Fuchs JF, Boyle JP, Christensen BM 2005. Age-associated mortality in immune challenged mosquitoes (Aedes aegypti) correlates with a decrease in haemocyte numbers. Cell Microbiol 7: 39-51., King & Hillyer 2013King JG, Hillyer JF 2013. Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection. BMC Biol 11: 55.), feeding status (Castillo et al. 2011Castillo J, Brown MR, Strand MR 2011. Blood-feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti. PLoS Pathog 7: e1002274.) and bacterial (King & Hillyer 2013King JG, Hillyer JF 2013. Spatial and temporal in vivo analysis of circulating and sessile immune cells in mosquitoes: hemocyte mitosis following infection. BMC Biol 11: 55.) or Plasmodium infection (Rodrigues et al. 2010Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C 2010. Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes. Science 329: 1353-1355., Ramirez et al. 2014Ramirez JL, Garver LS, Brayner FA, Alves LC, Rodrigues J, Molina-Cruz A, Barillas-Mury C 2014. The role of hemocytes in Anopheles gambiae antiplasmodial immunity. J Innate Immun 6: 119-128.).

Rodrigues et al. (2010)Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C 2010. Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes. Science 329: 1353-1355. have shown that the proportion of circulating granulocytes increases in response to ookinete invasion rendering the mosquito more resistant to Plasmodium infection upon further challenge. Interestingly, the haemolymph of Plasmodium-infected mosquitoes (in the presence of midgut microbiota) contains a soluble factor that promotes haemocyte differentiation and is able to confer Plasmodium resistance when transferred to naïve mosquitoes (Rodrigues et al. 2010Rodrigues J, Brayner FA, Alves LC, Dixit R, Barillas-Mury C 2010. Hemocyte differentiation mediates innate immune memory in Anopheles gambiae mosquitoes. Science 329: 1353-1355., Ramirez et al. 2014Ramirez JL, Garver LS, Brayner FA, Alves LC, Rodrigues J, Molina-Cruz A, Barillas-Mury C 2014. The role of hemocytes in Anopheles gambiae antiplasmodial immunity. J Innate Immun 6: 119-128.). Although the identity of this haemocyte differentiation factor is at present unknown, its production is independent of the major mosquito immune pathways (Toll, IMD, JAK-STAT) (Ramirez et al. 2014Ramirez JL, Garver LS, Brayner FA, Alves LC, Rodrigues J, Molina-Cruz A, Barillas-Mury C 2014. The role of hemocytes in Anopheles gambiae antiplasmodial immunity. J Innate Immun 6: 119-128.).

Several questions regarding mosquito haemocyte biology and function have yet to be addressed and is further confounded by the lack of agreement on the methodology for haemocyte isolation and analysis. However, these challenges precipitate the need for reliable cell-type specific haemocyte markers to better understand their function and identify the contributions of each cell type to anti-Plasmodium immunity.

Fat body - Based on Drosophila research, the mosquito fat body is thought to play a central role in the regulation of humoral immunity through the production of antimicrobial peptides (Lemaitre & Hoffmann 2007Lemaitre B, Hoffmann J 2007. The host defense of Drosophila melanogaster. Annu Rev Immunol 25: 697-743.). However, given the systemic nature of dsRNA mediated silencing commonly used in mosquito research, the specific contributions of the fat body to immune signalling require further examination.

Transcriptional analysis of the mosquito carcass (that includes the fat body) reveals significant changes in response to a blood meal, as well as specific transcriptional profiles characteristic of rodent and human malaria parasites (Dong et al. 2006Dong Y, Aguilar R, Xi Z, Warr E, Mongin E, Dimopoulos G 2006. Anopheles gambiae immune responses to human and rodent Plasmodium parasite species. PLoS Pathog 2: e52.).

Additional evidence also suggests that two major nutrient transporters, lipophorin and vitellogenin, produced by the fat body during the process of vitellogenesis (or egg production), also influence parasite survival (Rono et al. 2010Rono MK, Whitten MM, Oulad-Abdelghani M, Levashina EA, Marois E 2010. The major yolk protein vitellogenin interferes with the anti-Plasmodium response in the malaria mosquito Anopheles gambiae. PLoS Biology 8: e1000434.). Silencing of lipophorin reduces Plasmodium oocyst size and impairs development, while loss of vitellogenin also results in reduced parasite numbers. These results suggest that developing parasites may be able to capture nutrients circulating in the mosquito haemolymph to facilitate its own development. Moreover, it has been suggested that these proteins may protect Plasmodium against non-self recognition by the mosquito complement-like cascade (Rono et al. 2010Rono MK, Whitten MM, Oulad-Abdelghani M, Levashina EA, Marois E 2010. The major yolk protein vitellogenin interferes with the anti-Plasmodium response in the malaria mosquito Anopheles gambiae. PLoS Biology 8: e1000434.).

Mechanisms of immune evasion - There has been significant evolutionary pressure on malaria parasites to adapt to their mosquito host to ensure their survival and subsequent transmission to a new host. As a result, malaria parasites have evolved mechanisms to evade the mosquito immune response in their natural mosquito vectors, a process that may be specific to different geographical regions. Collins et al. (1986)Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, Collins WE, Campbell CC, Gwadz RW 1986. Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 234: 607-610. found that African P. falciparum parasites strains were significantly more proficient in evading the melanisation response of refractory An. gambiae than those of New World or Asian origin.

More recently, light has been cast on the mechanism for this unique phenotype by determining that African strains of P. falciparum can evade TEP1-mediated lysis by the mosquito complement-like system of refractory An. gambiae mosquitoes, while Brazilian 7G8 P. falciparum isolates are efficiently targeted and melanised by this system (Molina-Cruz et al. 2012Molina-Cruz A, de Jong RJ, Ortega C, Haile A, Abban E, Rodrigues J, Jaramillo-Gutierrez G, Barillas-Mury C 2012. Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes. Proc Natl Acad Sci USA 109: e1957-e1962.). Through quantitative trait locus analysis of genetic crosses between the African and Brazilian parasite strains, Molina-Cruz et al. (2013)Molina-Cruz A, Garver LS, Alabaster A, Bangiolo L, Haile A, Winikor J, Ortega C, van Schaijk BC, Sauerwein RW, Taylor-Salmon E, Barillas-Mury C 2013. The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system. Science 340: 984-987. identified Pfs47 as the candidate gene mediating this process. Present on the ookinete surface, Pfs47 is thought to prevent the induction of the mosquito midgut nitration responses that mark parasites for TEP1-mediated lysis by a yet unknown mechanism. The highly polymorphic nature of Pfs47 and its geographic distribution suggest that P. falciparum parasites have adapted to different Anopheles mosquitoes to ensure their survival (Molina-Cruz et al. 2013Molina-Cruz A, Garver LS, Alabaster A, Bangiolo L, Haile A, Winikor J, Ortega C, van Schaijk BC, Sauerwein RW, Taylor-Salmon E, Barillas-Mury C 2013. The human malaria parasite Pfs47 gene mediates evasion of the mosquito immune system. Science 340: 984-987.).

While details of host-parasite co-evolution are only beginning to emerge, these studies highlight the ability of the parasite to adapt to its vector host to ensure its survival. Mosquito factors have also been described that may protect parasites from immune recognition (Osta et al. 2004Osta MA, Christophides GK, Kafatos FC 2004. Effects of mosquito genes on Plasmodium development. Science 303: 2030-2032.) or have been co-opted by parasites to facilitate invasion (Rodrigues et al. 2012Rodrigues J, Oliveira GA, Kotsyfakis M, Dixit R, Molina-Cruz A, Jochim R, Barillas-Mury C 2012. An epithelial serine protease, AgESP, is required for Plasmodium invasion in the mosquito Anopheles gambiae. PLoS ONE 7: e35210.). However, the precise role of these components, and possibly others, requires future investigation.

Intervention strategies - The bottleneck in malaria parasite numbers within its mosquito host (Fig. 1) argues that the midgut is an optimal target for intervention strategies to prevent malaria transmission.

For many years, a large effort has been invested into the identification of TB vaccine antigens that target the sexual stages of the parasite in the mosquito midgut, including Pfs230, Pbs48/45, HAP2, Pfs25 and PfCHT1 [reviewed by Pradel (2007)Pradel G 2007. Proteins of the malaria parasite sexual stages: expression, function and potential for transmission blocking strategies. Parasitology 134: 1911-1929. and Blagborough and Sinden (2009)Blagborough AM, Sinden RE 2009. Plasmodium berghei HAP2 induces strong malaria transmission-blocking immunity in vivo and in vitro. Vaccine 27: 5187-5194.]. In addition, it has also been proposed that antibodies targeting mosquito components may also serve as promising TB targets (Dinglasan & Jacobs-Lorena 2008Dinglasan RR, Jacobs-Lorena M 2008. Flipping the paradigm on malaria transmission-blocking vaccines. Trends Parasitol 24: 364-370.). One major advantage of this strategy is that using a conserved mosquito TB vaccine target could be applied for all anopheline mosquitoes, thus obviating the need to develop specific targets for each parasite-host combination. Proof-of-principle experiments targeting mosquito midgut ligands involved in ookinete invasion (Dinglasan et al. 2007aDinglasan RR, Kalume DE, Kanzok SM, Ghosh AK, Muratova O, Pandey A, Jacobs-Lorena M 2007a. Disruption of Plasmodium falciparum development by antibodies against a conserved mosquito midgut antigen. Proc Natl Acad Sci USA 104: 13461-13466.) or that regulate mosquito immunity (Williams et al. 2013Williams AR, Zakutansky SE, Miura K, Dicks MD, Churcher TS, Jewell KE, Vaughan AM, Turner AV, Kapulu MC, Michel K, Long CA, Sinden RE, Hill AV, Draper SJ, Biswas S 2013. Immunisation against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes. Int J Parasitol 43: 869-874.) have been explored.

Transgenic technology has also been proposed to genetically modify mosquito vectors to render them incapable of malaria parasite transmission. Through the production of synthetic peptides, effector proteins or methods to increase the mosquito immune response, several approaches have been used to confer anti-Plasmodium resistance in laboratory and natural host-pathogen combinations [reviewed by Wang and Jacobs-Lorena (2013)Wang S, Jacobs-Lorena M 2013. Genetic approaches to interfere with malaria transmission by vector mosquitoes. Trends Biotechnol 31: 185-193.]. Although these experiments are promising, significant hurdles remain regarding release strategies and required drive-mechanisms to ensure that these resistant mosquitoes can spread into wild mosquito populations.

Wolbachia is an endosymbiont commonly found in arthropods and can spread into populations by transmission through the germ line (Werren et al. 2008Werren JH, Baldo L, Clark ME 2008. Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol 6: 741-751.). A surprising and important discovery was that when Aedes aegypti harbour Wolbachia it becomes resistant to viral and Plasmodium infections (Moreira et al. 2009Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LM, Rocha BC, Hall-Mendelin S, Day A, Riegler M, Hugo LE, Johnson KN, Kay BH, McGraw EA, van den Hurk AF, Ryan PA, O’Neill SL 2009. A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya and Plasmodium. Cell 139: 1268-1278.). In An. gambiae, somatic, non-heritable Wolbachia infections were shown to also confer similar anti-Plasmodium properties (Kambris et al. 2010Kambris Z, Blagborough AM, Pinto SB, Blagrove MS, Godfray HC, Sinden RE, Sinkins SP 2010. Wolbachia stimulates immune gene expression and inhibits Plasmodium development in Anopheles gambiae. PLoS Pathog 6: e1001143., Hughes et al. 2011Hughes GL, Koga R, Xue P, Fukatsu T, Rasgon JL 2011. Wolbachia infections are virulent and inhibit the human malaria parasite Plasmodium falciparum in Anopheles gambiae. PLoS Pathog 7: e1002043.). Recently, stable maternally-inherited Wolbachia was introduced into An. stephensi and was found to limit P. falciparum development, as well as confers cytoplasmic incompatibility (Bian et al. 2013Bian G, Joshi D, Dong Y, Lu P, Zhou G, Pan X, Xu Y, Dimopoulos G, Xi Z 2013. Wolbachia invades Anopheles stephensi populations and induces refractoriness to Plasmodium infection. Science 340: 748-751.). However, Wolbachia imposed a significant fitness load to these mosquitoes. While promising, further challenges remain regarding the mechanism by which Wolbachia confers anti-Plasmodium immunity, as well as its introduction into other anopheline vectors.

An alternative approach has recently been described involving the engineering of the mosquito microbiota to secrete antimalarial effector genes, an approach known as paratransgenesis (Wang et al. 2012Wang S, Ghosh AK, Bongio N, Stebbings KA, Lampe DJ, Jacobs-Lorena M 2012. Fighting malaria with engineered symbiotic bacteria from vector mosquitoes. Proc Natl Acad Sci USA 109: 12734-12739.). Genetically engineered Pantoea agglomerans, a common resident of the mosquito midgut, were able to confer resistance to P. falciparum and P. berghei and dramatically reduce infection prevalence independent of the anopheline vector (Wang et al. 2012Wang S, Ghosh AK, Bongio N, Stebbings KA, Lampe DJ, Jacobs-Lorena M 2012. Fighting malaria with engineered symbiotic bacteria from vector mosquitoes. Proc Natl Acad Sci USA 109: 12734-12739.). Future research must address issues of how to introduce the modified bacteria into field populations and, importantly, resolve issues relating to the release of genetically modified organisms in nature.

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