Innate immunity in vertebrates plays a fundamental role inpathogen recognition and subsequent activation of the adaptiveimmune response, which is characterized by a highly diverse,somatically generated repertoire of antigen receptors. Theinterplay between adaptive and innate immunity orchestratesthe defensive responses against a variety of invaders such asviruses, bacteria, fungi or parasites (Medzhitov and Janeway,1997a). Invertebrates, including insects, lack an adaptiveimmune system but utilize innate immunity for their defence.The successful evolution of insects as the most species-richgroup of terrestrial animals testifies to the efficiency andflexibility of innate immunity in dealing with a diverse arrayof pathogens.

In insects, the peritrophic membrane (PM) of the midgut, thecuticle of the exoskeleton and the lining of the trachealrespiratory system constitute physical barriers against

invaders. The PM is a sleeve-like extracellular layersurrounding the food bolus and is composed of chitin, proteinsand proteoglycans (Wang and Granados, 2001). Ingestedookinetes of the malaria parasite Plasmodiummust penetratethe midgut wall of the Anophelesmosquito vector to developfurther into oocysts on the haemocoel basal side of the midgutepithelium (Fig.·1). This process is facilitated by the secretionof chitinases that disrupt the PM chitin (Huber et al., 1991;Langer et al., 2000). Midgut invasion was completely blockedwhen mosquitoes were fed on infected blood containing thechitinase inhibitor allosamidin (Shahabuddin et al., 1993).Similarly, knockout of the Plasmodium falciparumchitinasegene induced a marked reduction in the number of developingoocysts in the midguts of Anopheles freebornimosquitoes(Tsai et al., 2001). Microorganisms that successfully overcomeany of these physical barriers encounter an array of host innateimmune responses within the underlying epithelia orsystemically in internal immune tissues such as haemocytes(insect blood cells) and the fat body (liver analogue), which

2551The Journal of Experimental Biology 207, 2551-2563Published by The Company of Biologists 2004doi:10.1242/jeb.01066

The resurgence of malaria is at least partly attributed tothe absence of an effective vaccine, parasite resistance toantimalarial drugs and resistance to insecticides of theanopheline mosquito vectors. Novel strategies are neededto combat the disease on three fronts: protection(vaccines), prophylaxis/treatment (antimalarial drugs) andtransmission blocking. The latter entails either killing themosquitoes (insecticides), preventing mosquito biting(bednets and repellents), blocking parasite developmentin the vector (transmission blocking vaccines), geneticmanipulation or chemical incapacitation of the vector.During the past decade, mosquito research has beenenergized by several breakthroughs, including thesuccessful transformation of anopheline vectors, analysisof gene function by RNAi, genome-wide expressionprofiling using DNA microarrays and, most importantly,sequencing of the Anopheles gambiaegenome. These

breakthroughs helped unravel some of the mechanismsunderlying the dynamic interactions between the parasiteand the vector and shed light on the mosquito innateimmune system as a set of potential targets to blockparasite development. In this context, putative patternrecognition receptors of the mosquito that act as positiveand negative regulators of parasite development have beenidentified recently. Characterizing these molecules andothers of similar function, and identifying their ligands onthe parasite surface, will provide clues on the nature of theinteractions that define an efficient parasite–vector systemand open up unprecedented opportunities to control thevectorial capacity of anopheline mosquitoes.

Key words: malaria, innate immunity, Anopheles gambiae,genomics, disease control, pattern recognition receptor.

Summary

Review

Innate immunity in the malaria vector Anopheles gambiae: comparative andfunctional genomics

Mike A. Osta, George K. Christophides, Dina Vlachou and Fotis C. Kafatos*European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany

*Author for correspondence (e-mail: dg-office@embl.de)

Accepted 29 April 2004

Introduction

This paper constitutes a review for inclusion with the 2003 ‘ExperimentalBiology of Malaria and its Vectors’ special issue [J. Exp. Biol.206(21)].

are known to release immune effectors into the opencirculatory system, the haemolymph (the insect blood).Examples of such responses are phagocytosis, secretion ofantimicrobial peptides, nodule formation, agglutination,encapsulation and melanization.

Studies on Drosophilainnate immunity have had a majorimpact on this field, both in vertebrates and invertebrates,leading to key discoveries and fundamental concepts on howorganisms efficiently fight pathogens even in the absence ofthe clonal system of recognition that is central to adaptiveimmunity (Hoffmann, 2003; Hoffmann and Reichhart, 2002).What made Drosophilatractable for such studies was thepowerful genetic and molecular genetic tools available in thefruitfly, including a fully sequenced genome. The mosquitoAnopheles gambiae, the major vector of malaria in Africa,also became a suitable model for the study of innate immunityrecently, when its genome was sequenced (Holt et al., 2002),accompanied by comparative genomic analysis withDrosophila (Zdobnov et al., 2002) and the establishment ofpowerful tools for gene discovery (Dimopoulos et al., 2002),functional analysis (Blandin et al., 2002; Levashina et al.,

2001) and transgenesis (Grossman et al., 2001). As themost important vector for transmission of the malariaparasite, A. gambiae offers the advantage of assessingimmune reactions in a species of major importance tohuman health. A. gambiae mounts efficient local andsystemic immune responses against Plasmodiuminfection(Dimopoulos et al., 1997, 1998; Richman et al., 1997; Fig.·1).However, in spite of the hostile environment encounteredand major losses in parasite numbers inside the vector,some parasite species or strains successfully complete theirsexual life cycle, indicating that they have found ways toescape or subvert to some extent the vector immuneresponses. Other vector–parasite combinations are eitherpoor (Plasmodium gallinaceum/Anopheles stephensi) orincompatible (Plasmodium berghei/Aedes aegypti),suggesting that key molecular and cellular interactions are aprerequisite for a vector–parasite system to becomeestablished and subsequently co-evolve (Alavi et al., 2003).In this review, we describe our current knowledge of A.gambiae innate immunity and its impact on Plasmodiumdevelopment.

Recognition of non-selfImmune reactions are initiated when

molecules of microbial origin aredetected and recognized as ‘non-self’.This recognition step involves patternrecognition receptors (PRRs) thatrecognize and bind to so-calledpathogen-associated molecular patterns(PAMPs) that are shared by variousmicroorganisms but absent fromeukaryotic cells (Medzhitov andJaneway, 1997b, 2002). PAMPs that areknown to be potent immune elicitorsinclude lipopolysaccharides (LPS),peptidoglycan (PGN) and β-1,3-glucans. Various PRRs have beenidentified and isolated from vertebrates(Holmskov et al., 2003; Takeda et al.,2003) and invertebrates (Gobert et al.,2003; Hoffmann, 2003; Wilson etal., 1999; Yu et al., 2002). The best-studied invertebrate PRRs are thepeptidoglycan recognition proteins(PGRPs) and the Gram-negativebacteria-binding proteins (GNBPs).PGRPs are soluble or transmembraneproteins containing a domain similar tothe bacterial amidase domain, which isinvolved in recycling bacterial cell wallfragments. PGRPs have been isolatedfrom both invertebrates and vertebrates(Dziarski et al., 2003; Wang et al.,2003). The first was isolated from the

M. A. Osta and others2552

Midgut

Fat body

Haemolymph

Salivarygland

1

4

56

a

b c d

2 3

LRIM1 TEP1 CTL4 CTLMA2LRIM1

POs

Haemocytes

Fig.·1. Schematic view of Plasmodiumsporogonic (sexual) cycle and mosquito defencereactions during midgut invasion. (1) Exflagellation of microgametocytes soon after ingestionof infectious blood, giving rise to eight flagellated microgametes; (2) fertilization ofmacrogametes to form the zygote; (3) zygote development to a motile ookinete that invadesthe midgut epithelium at approximately 24·h post infection; (4) ookinete invasion arrests at thebasal lamina where the parasite rounds up to form the non-motile oocyst. Several mitoticdivisions within the oocyst give rise to thousands of sporozoites dramatically amplifying theparasite load; (5) oocyst rupture, at approximately 12·days post infection, and release ofsporozoites into the haemolymph; (6) sporozoite migration through the haemolymph andinvasion of the salivary gland. The sporozoites reside in the salivary gland lumen from wherethey are injected into the vertebrate host during the next bite. (a) Major parasite losses occurduring the first 24·h post infection; (b) killing and subsequent melanization of ookinetes in arefractory strain, a process involving several mosquito factors including POs, LRIM1 andTEP1; (c) killing of ookinetes inside the cytoplasm of midgut epithelial cells through TEP1-and possibly LRIM1-mediated lysis; (d) ookinetes protected from killing by mosquito factors,including CTL4 and CTLMA2, successfully reach the basal lamina and develop into oocysts.

haemolymph of the moth Bombyx mori, where it is involvedin activating the prophenoloxidase (PPO) cascade (Yoshidaet al., 1996). In Drosophila, three PGRPs were identified asbona fidePRRs. The soluble PGRP-SA activates the Tollpathway in response to Gram-positive bacterial infection(Gobert et al., 2003; Michel et al., 2001) in concert withanother PRR, GNBP1. By contrast, PGRP-LC (Choe et al.,2002; Gottar et al., 2002; Ramet et al., 2002) and PGRP-LE(Takehana et al., 2002) are involved in activating the immunedeficiency (IMD) pathway in response to Gram-negativebacterial infections. Among the seven identified putativeAnophelesPGRPs (Christophides et al., 2002), PGRPLCseems to play a central role in defence against bacterialinfection (G. K. Christophides, unpublished data). Theorthologous genes in both Drosophila (Werner et al., 2003)and Anopheles (Christophides et al., 2002) undergoalternative splicing resulting in at least three distinctisoforms. Interestingly, the Anopheles PGRPLCisoforms aredifferentially regulated upon immune challenge, suggestingthat splicing can be regulated by the immune signal(Christophides et al., 2002). On the other hand, PGRPLBistranscriptionally upregulated following Plasmodiuminfection of adult mosquitoes (Dimopoulos et al., 2002) andmaintains an elevated expression throughout the parasite’sentire life cycle (Christophides et al., 2002).

GNBPs were first described in Bombyx mori(Lee et al.,1996) and share significant sequence similarity with thecatalytic region of bacterial β-1,3- and β-1,3,1,4-glucanases.BmGNBP binds strongly to the surface of Gram-negativebacteria and shows pronounced transcriptional upregulationfollowing bacterial challenge (Lee et al., 1996). TheDrosophilaGNBP1 binds with high affinity to LPS and β-1,3-glucan (Kim et al., 2000) and, in concert with PGRP-SA,activates the Toll pathway upon infection with Gram-positivebacteria (Gobert et al., 2003). In A. gambiae, six putativeGNBPs have been identified (Christophides et al., 2002).Among them, GNBPB1and GNBPA1 are upregulatedfollowing Plasmodium infection, while only GNBPB1isresponsive to bacteria (Christophides et al., 2002; Dimopouloset al., 2002). Other putative AnophelesPRRs include thethioester-containing proteins (TEPs), leucine-rich immuneproteins (LRIMs) and C-type lectins (CTLs). Members of theseprotein families were recently implicated in the regulation ofPlasmodiumdevelopment in the mosquito vector and arediscussed later in this review.

Signal modulation and transductionSerine protease cascades

Recognition of non-self usually activates a proteolyticcascade of serine proteases that amplify the signal and triggerdownstream effector responses, leading to the killing of theinvader. Key components of such cascades are clip-domainserine proteases (CLIPs), which have been implicated inseveral defence mechanisms in insects and crustaceans, suchas the activation of signalling pathways leading to the synthesis

of antimicrobial peptides (AMPs) (Ligoxygakis et al., 2002a),haemolymph agglutination (Kawabata et al., 1996) andmelanization (Kanost et al., 2001), discussed also in a latersection. The role of the clip-domain is not yet known but isbelieved to include regulation of enzymatic activity or proteinlocalization. In Drosophila, some CLIPs are key componentsof the dorsoventral pattern formation system (e.g. Easter andSnake), but others, such as Persephone, are also involved inthe activation of the Toll pathway upon fungal infection(Ligoxygakis et al., 2002a). The horseshoe crab clotting systeminvolves serine protease zymogens that are activated in acascade manner, leading to the transformation of coagulogento insoluble coagulum gel and subsequent clotting of thehaemolymph (Miyata et al., 1984a,b; Nakamura et al., 1986).The clot is effective in immobilizing pathogens, which are theneliminated by other host effector mechanisms such as secretionof AMPs.

Melanization requires the enzymatic processing of inactivePPO to active phenoloxidases (PO) by activating serineproteases, referred to in the literature as prophenol-activatingproteinase (PAP) or prophenoloxidase-activating enzyme(PPAE). In the tobacco hornworm Manduca sexta, severalPAPs have been described and shown biochemically to beinvolved in PPO activation: PAP1 has one clip-domain (Jianget al., 1998) while PAP2 (Jiang et al., 2003a) and PAP3 (Jianget al., 2003b) contain two clip-domains. Interestingly, theManduca PAPs require non-proteolytic serine proteasehomologues as cofactors for the efficient activation of PPO(Jiang et al., 2003a,b; Yu et al., 2003). Two A. gambiaeCLIPs(CLIPB14 and CLIPB15) were found to be responsive tobacterial and Plasmodiuminfections: CLIPB14 showedpersistent upregulation in Plasmodium-infected mosquitoeswhile CLIPB15 showed only transient upregulation, preciselyduring midgut invasion (Christophides et al., 2002). The roleof these two genes in the activation of PPO has beendemonstrated recently (J. Volz, unpublished data).

Signal amplification by the serine protease cascade is undertight regulation by serpins (serine protease inhibitors), whichinhibit serine proteases by acting as irreversible suicidesubstrates that covalently bind to the active centre of theenzyme. The Drosophilaserpin Spn43Ac (Necrotic)downregulates the Toll pathway in response to fungalinfections by inhibiting cleavage of the Toll receptor ligand,the cytokine-like polypeptide Spaetzle (Levashina et al.,1999). The involvement of serpins in the regulation ofmelanization has been described in D. melanogaster (DeGregorio et al., 2002; Ligoxygakis et al., 2002b), M. sexta(Zhu et al., 2003) and A. gambiae(K. Michel, unpublisheddata). A recently characterized Anophelesserpin, SRPN10,encodes four alternatively spliced inhibitory isoforms(Danielli et al., 2003). Interestingly, two of these formsare specifically upregulated in female mosquitoes in responseto midgut invasion by P. bergheiookinetes, making SRPN10an excellent cell-autonomous marker of invasion (A.Danielli, T. G. Loukeris and C. Barillas-Mury, unpublisheddata).

Molecular genetics of innate immunity in A. gambiae 2553

Immune signalling pathwaysImmune signalling pathways transmit the signal originating

from PAMP-associated PRRs to the effector genes. In a nowclassical series of studies, two signal transduction pathways,the Toll and IMD pathways (Fig.·2), have been identified inDrosophilaand linked to innate immunity (Hoffmann, 2003).Fungal or Gram-positive bacterial infections activate the Tollpathway by inducing the proteolytic cleavage of Spaetzle,which binds directly to and activates the transmembranereceptor Toll (Weber et al., 2003). Hence, Toll does not appearto be a direct sensor of microbial compounds, unlike themammalian Toll-like receptors (TLRs) that recognize and bindto several microbial ligands (Takeda et al., 2003). Toll has anintracytoplasmic TIR (Toll, IL-1R) domain, which uponactivation recruits three death domain proteins, MyD88, Tubeand Pelle, the first two of which are considered as adaptorproteins. The Toll receptor–adaptor complex signals to twocytoplasmic Rel/NFκB transcription factors, Dorsal and Dif,causing their dissociation from Cactus, an ankyrin repeatinhibitory protein, and subsequent translocation to the nucleuswhere they activate the transcription of AMPs. Whereas Difhas a crucial role in the immune response (Ip et al., 1993;Petersen et al., 1995), Dorsal is believed to be mainly engagedin transcriptional activation of genes involved in thedorsoventral patterning (Morisato and Anderson, 1995).Interestingly, no orthologue of Dif was found in the Anophelesgenome. Gambif1 (now called REL1), the mosquitoorthologue of Dorsal, has been previously characterized andshown to translocate to the nucleus following bacterial but notPlasmodiuminfection (Barillas-Mury et al., 1996). Ten Toll

and six Spaetzle-like proteins have been identified in thegenome of A. gambiae, but their phylogenetic relationshipswith the respective Drosophilahomologues are unclear(Christophides et al., 2002). However, identification of themosquito orthologues of MyD88, Tube and Pelle indicates thatthe Toll pathway in the mosquito is at least partially conserved(Christophides et al., 2002).

The second immune signalling pathway in Drosophila, theIMD pathway, is activated following Gram-negative bacterialinfection leading to the cleavage of another Rel/NFκB familyprotein, Relish, through the recently proposed proteolyticaction of the caspase Dredd (Stoven et al., 2003); this causesthe release of the Rel-homology domain of Relish fromits inhibitory carboxy-terminal ankyrin domain. Thetransmembrane receptor of the IMD pathway is as yetunclear; however, a number of studies point to a role forPGRP-LC in this process (Choe et al., 2002; Gottar et al.,2002). Intracellular activation of the pathway commenceswith recruitment of IMD, a death domain protein sharingsimilarities with the mammalian TNF-α receptor interactingprotein, RIP. The sequence of events between IMD activationand the cleavage of Relish is not fully characterized;however, genetic data point to several other factorsdownstream of IMD, including the protein kinase dTAK1,another death-domain protein, dFADD, and the Drosophilahomologues of the mammalian signalosome equivalentcomprising IKK-β and IKK-γ. Interestingly, all theaforementioned components of the IMD pathway areconserved in Anopheles(Christophides et al., 2002), andpreliminary data indicate that REL2, the Anophelesorthologue of Relish, is indeed involved in anti-bacterialdefence (G. K. Christophides, unpublished data). The Ae.aegyptiRelish gene has three alternatively spliced transcriptsencoding three different proteins: Relish, IκB-type, whichlacks the Rel-homology domain, and the Rel-type in whichthe amino-terminal transactivation domain and the carboxy-terminal ankyrin repeats are missing (Shin et al., 2002). Theinvolvement of AedesRelish in the regulation of immuneresponse to bacterial challenge has been shown usingtransgenic mosquitoes carrying the Rel-type transgene drivenby the bloodmeal-inducible vitellogenin promoter (Shin et al.,2003a). In these mosquitoes, the overexpression of Rel-typetransgene following a blood meal resulted in severely reducedexpression levels of defensin and cecropin genes and a strongsusceptibility to Gram-negative bacterial infections. Theinterference of Rel-type protein with endogenous Relish wassuggested to involve competitive binding to the κB motif.

Little is known about the role of the JNK and JAK/STATpathways in antimicrobial defence in insects. The DrosophilaJAK/STAT pathway has one STAT component and is involvedin many developmental processes (Luo and Dearolf, 2001). Arecent study based on microarray analysis on Drosophilacelllines revealed that the IMD pathway branches downstream ofdTAK1: one branch controlling the synthesis of antimicrobialpeptides through Relish, and the other the synthesis ofcytoskeletal proteins through the JAK/STAT (Boutros et al.,

M. A. Osta and others2554

Fig.·2. Control of the expression of Drosophilaantimicrobial peptidegenes is mediated by the Toll and immuno deficiency (IMD)pathways. Colour-coding separates the components of each pathway.For explanations, refer to main text.

Toll IMD

Cytoskeletal genes

Fungi

bial genesAntimicro

MyD88FADD

IKKγ/IKKβDREDDRelish

JNK

TAK1

_

+Bacteria

Tube

Pelle

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Cactus

2002). Based on these data, a close link between cytoskeletalremodelling and antimicrobial defence was suggested (Boutroset al., 2002). Two members of the STAT family (STAT1 andSTAT2) have been identified in the mosquito genome(Christophides et al., 2002). The observed STAT1 (previouslycalled AgSTAT) translocation into the nucleus of mosquito fatbody cells following bacterial infection has provided the firstevidence for the involvement of insect STATs in immunedefence (Barillas-Mury et al., 1999).

Pathogen eliminationAntimicrobial peptides (AMPs)

Immune responses in insects can be divided into humoraland cellular. The synthesis of AMPs is the final step ofinducible humoral immune responses. Following pathogenrecognition, AMPs are rapidly produced by the fat body andsecreted into the haemolymph, where they accumulate in largeconcentrations (Meister et al., 1997). AMPs are also locallyproduced by barrier epithelia of several insect species such asB. mori (Brey et al., 1993), A. gambiae(Brey et al., 1993;Dimopoulos et al., 2002; Richman et al., 1997) and D.melanogaster(Ferrandon et al., 1998; Tingvall et al., 2001).To date, several hundred AMPs have been describedin immune-challenged insects, where they exhibit wideand complementary spectra of activity against variousmicroorganisms (Bulet et al., 1999). In Drosophila, the AMP-encoding genes are regulated by the finely tuned activity of theIMD and Toll pathways (Bulet et al., 1999; Hoffmann, 2003;Hoffmann and Reichhart, 2002). IMD controls the expressionof anti-Gram-negative peptides such as diptericins anddrosocins, while Toll induces the expression of the anti-fungalpeptide drosomycin by the fat body cells. Interestingly, thelocal expression of drosomycin by barrier epithelia is Toll-independent, suggesting that local and systemic expressions ofAMPs might be regulated by different signalling pathways(Ferrandon et al., 1998). Signals from both Toll and IMD seemto control jointly the induction of cecropins, attacins anddefensins in Drosophila(Hoffmann et al., 1996; Meister et al.,1997). In Ae. aegypti, defensin levels were shown to bestrongly upregulated following bacterial challenge, reachinga concentration of approximately 45·µmol·l–1 in thehaemolymph at 24·h post-inoculation (Lowenberger et al.,1999b). Another AMP, cecropin A, was isolated from thehaemolymph of bacteria-challenged adult Ae. aegyptimosquitoes. The protein was active against a broad spectrumof Gram-negative bacteria but less so against Gram-positivebacteria and fungi (Lowenberger et al., 1999a). The Anophelesgenome encodes four defensin genes (DEFs), four cecropins(CECs), one attacin and one gambicin (GAM1) (Christophideset al., 2002). The anti-Gram-positive effect of AnophelesDEF1(previously called defensin A) has been demonstrated in vitrorecently (Vizioli et al., 2001b). GAM1 is active against bothGram-positive and Gram-negative bacteria but is onlymarginally active against P. bergheiookinetes (Vizioli et al.,2001a).

Phagocytosis

Phagocytosis is a hallmark of the classical cellular immuneresponses of insects whereby haemocytes engulf targetpathogens but also apoptotic bodies. Three types ofhaemocytes have been characterized in Drosophila: theplasmatocytes that are responsible for the disposal ofmicroorganisms and apoptotic cells, the lamellocytes thatencapsulate large invaders, and the crystal cells that areinvolved in melanization (Meister and Lagueux, 2003). In theyellow fever mosquito, Ae. aegypti, four different types ofhaemocytes have been distinguished: the granulocytes, theoenocytoids, the adipohaemocytes and the thrombocytoids(Hillyer and Christensen, 2002). Phagocytosis in Drosophilaisusually receptor mediated, involving either soluble ormembrane-bound PRRs, including PGRP-LC, which isinvolved in the phagocytosis of Gram-negative bacteria(Ramet et al., 2002), and Croquemort, a membrane-boundreceptor mediating phagocytosis of apoptotic corpses (Franc etal., 1996). A soluble thioester-containing protein, TEP1,identified in A. gambiae, acts as a complement-like opsoninpromoting the phagocytosis of Gram-negative bacteria in amosquito haemocyte-like cell line (Levashina et al., 2001).TEP1 activation seems to result from its infection-inducibleproteolytic cleavage and exposure of the highly reactivethioester bond, apparently involved in a nucleophilic attackleading to the covalent binding of TEP1 to the surface ofmicroorganisms.

Cellular encapsulation

The mechanisms promoting cellular encapsulation in insectsare not well understood, and only a few examples of this innatecellular reaction have been reported. Encapsulation is a processby which insect lamellocytes form a multilayered capsulearound large invaders such as parasitoids in the haemocoel,resulting in their isolation, immobilization and subsequentkilling by asphyxiation, oxidation or melanization (Gotz,1986). Drosophila larvae encapsulate and then melanize theeggs of a parasitoid wasp in their haemocoel through theconcerted action of lamellocytes and crystal cells (Lanot et al.,2001; Sorrentino et al., 2002). Hemese, a recently identifiedtransmembrane receptor, is expressed on the surface of allDrosophila haemocytes and acts as a negative regulator of theencapsulation response (Kurucz et al., 2003). Knockout ofHemese stimulates the proliferation of lamellocytes followingparasitoid infection, resulting in an enhanced cellular response(Kurucz et al., 2003). The mechanisms through whichlamellocytes are alerted to the presence of a large invader andthe signals involved in their proliferation remain unknown.

Melanization

Melanization is a prime humoral immune reaction of insects,being involved in wound healing and sequestration of invadersin a dense melanin coat. In contrast to encapsulation, it doesnot require the direct involvement of haemocytes (Soderhalland Cerenius, 1998). Melanization requires the proteolyticactivation of the inactive PPO zymogens to the active POs, a

Molecular genetics of innate immunity in A. gambiae 2555

step tightly regulated, as described above, by the balancedaction of CLIPs and their inhibitors, serpins. POs oxidizephenolic substances such as tyrosine, DOPA and dopamine tomelanine and serve several tasks including wound healing,cuticle pigmentation and sclerotization, and melanization ofinvading pathogens (Soderhall and Cerenius, 1998). PPOs areproduced by haemocytes and released into the haemolymph(Ashida, 1971; Durrant et al., 1993; Muller et al., 1999), fromwhere they can also be transported to the cuticle through theunderlying cuticular epithelium to facilitate defence againstmicrobial invasion or abrasion of the cuticle (Asano andAshida, 2001; Ashida and Brey, 1995). There are nine PPO-encoding genes (PPO1–9) in A. gambiae(Christophides et al.,2002) that show overlapping developmental expressionprofiles (Jiang et al., 1997; Lee et al., 1998; Muller et al., 1999).PPO5 and PPO6 are mainly expressed in adult mosquitoes,whereas PPO1–4 are expressed in pre-adult stages. Somegenes such as PPO2, PPO3and PPO9are induced followingblood feeding (H. M. Muller, unpublished data).

A. gambiae: an emerging model for the study of innateimmunity

Anophelesimmune responses

A. gambiaeis the major African vector for transmission ofmalaria, a disease killing approximately two million peopleevery year, largely children between the age of one and five,in sub-Saharan Africa (WHO, 1999). The present alarmingprevalence of malaria is attributed, at least in part, to theabsence of a protective vaccine (Richie and Saul, 2002) andthe rapid spread of drug-resistant parasites (Olliaro, 2001) andinsecticide-resistant mosquitoes. Novel strategies to controlmalaria are badly needed. This has generated a growinginterest in understanding the complex interactions between themosquito vector and the Plasmodiumparasite (Varmus et al.,2003). Of particular interest are the vector immune responses,which are associated with parasite killing. Initial attemptsmonitoring the immune responses of A. gambiaeto infectionswith bacteria and P. berghei, a model rodent malaria parasite,were based on the use of a small number of mRNA immunemarkers, isolated by differential display techniques orhomology cloning (Dimopoulos et al., 1997, 1998; Richman etal., 1997). These studies clearly showed that parasite invasionof the midgut (by ookinetes) and salivary gland epithelium (bysporozoites) induces the upregulation of several immunemarkers locally but also systemically in the abdomen. Theabdominal response most probably involves several organsincluding fat body and/or haemocytes. This has suggested localimmune responses in the infected epithelium and an immune-related signalling process propagated to other tissues (Fig.·1).Whether this process involves parasite-derived diffusibleproducts or specific mosquito factors is currently unknown.Interestingly, it has been reported that midgut invasion by P.falciparum, which is accomplished by fewer ookinetes, doesnot result in upregulation of the same immune markers as doesinvasion by P. berghei (Tahar et al., 2002). Expressed

sequence tag (EST) libraries and cDNA microarrays(Dimopoulos et al., 2000) were utilized later to analyze on agenomic scale the expression profile of Anophelesgenes inresponse to parasite and bacterial infections. A pilot studybased on a 4000 EST chip revealed that Gram-negative andGram-positive bacterial challenges upregulate overlapping setsof genes, many of which belong to the immunity class, and thatthe response to Plasmodiumpartially overlaps with thisresponse (Dimopoulos et al., 2002). The documentedupregulated genes included, among others, several putativePRRs belonging to the CTL, PGRP, GNBP, TEP and LRIMgene families, as well as specific serpins, serine proteases andimmune signalling molecules such as Cactus. Functionalcharacterization of some of these genes has provided valuableclues concerning the direct involvement of specific immunitygenes in the regulation of Plasmodiumdevelopment in thevector, as discussed below.

TheA. gambiaegenome: a comparative analysis withDrosophila

Anophelesis the first insect vector whose genome has beensequenced (Holt et al., 2002). A comparative genome analysisof Drosophila and Anopheles, which are thought to havediverged approximately 250 million years ago, has revealedthat nearly half the genes are 1:1 orthologues (Zdobnov et al.,2002). Analysis of 18 gene families that include some innateimmune-related genes revealed a twofold deficit in orthologouspairs relative to the genomes as a whole (Christophides et al.,2002). The orthologue-poor families included putative patternrecognition, signal modulation and effector molecules.Interestingly, immunity gene families, in particular thoseinvolved in pattern recognition, signal modulation and effectormechanisms, showed substantial species-specific expansions.Genes belonging to immune signalling pathways are highlyconserved, and this is probably attributed to the multiplefunctions they serve, many of which involve developmentalprocesses (Christophides et al., 2002; Zdobnov et al., 2002).The expansion and diversification of the PRRs may reflect astrong selective pressure, leading to faster evolution in the faceof distinct microbial floras prevailing in the ecological habitatsof the different species. An extreme example of thisexpansion/diversification is the FBN-lectin gene family, whichunderwent two independent large expansions: one inAnopheles, resulting in 52 genes, and the second inDrosophila, resulting in 11 genes; in these species, only twoorthologous pairs persist!

Gene discovery and functional analysis in A. gambiae

The A. gambiae genome provides unprecedentedopportunities for mosquito research. For example, wholegenome expression analysis using microarrays is now feasibleand is expected to provide new insights into potential functionsof the mosquito immune system during its interactions withvarious pathogens, including Plasmodium. Microarraytechnology is a very powerful tool for gene discovery; aspreviously mentioned, a microarray-based study allowed the

M. A. Osta and others2556

identification of numerous genes induced by both bacterial andPlasmodiumchallenge (Dimopoulos et al., 2002); several ofthese are currently under in-depth analysis. In the frameworkof an international Mosquito Microarray Consortium (MMC)engaging a number of mosquito groups, our laboratory hasconstructed a 20·000 EST microarray (MMC1), and full-genome microarrays based on unique genomic amplicons havebeen designed (G. K. Christophides, unpublished data).

Direct and heritable reverse genetics

Gene discovery and generation of hypotheses based onexpression patterns must be complemented with other toolsthat permit direct assessment of gene function. For thispurpose, a direct reverse genetics method based on injection ofdsRNA into adult mosquitoes has been established (Blandin etal., 2002), in addition to using the dsRNA treatment of A.gambiaehaemocyte-like cell lines (Levashina et al., 2001). Forexample, injection of dsRNA corresponding to the Anophelesdefensin gene (DEF1) in the body cavity of adult mosquitoesefficiently and reproducibly silenced the expression of thisgene at the mRNA and protein levels (Blandin et al., 2002).Analysis of the knockout (KO) mosquitoes revealed thatdefensin is necessary to combat Gram-positive but not Gram-negative bacterial infections in vivo; however, the developmentof P. bergheiwas not affected by the absence of defensin. Theinjected dsRNA is detectable for at least 12·days post-injectionand, hence, is stable enough to mediate a long-lasting effect.To date, the RNAi approach has been utilized to assayefficiently the function of several A. gambiaegenes in responseto malaria infection, as described below. The RNAi silencingtechnique was also established in adult D. melanogasterfliesand used to efficiently silence components of the Toll pathway(Goto et al., 2003).

The replacement of wild mosquito populations withgenetically modified strains refractory to Plasmodiumdevelopment has been widely postulated as a potential strategyto control malaria transmission. Towards this aim, substantialeffort has been invested to genetically transform Anophelesspecies. The early single event of A. gambiaetransformation(Miller et al., 1987), using the transposable element (TE) Pfrom Drosophila, proved serendipitous and not transposonmediated. Routine genetic manipulation methods awaitedutilization of TEs that were truly mobile in mosquitoes andutilization of effective dominant selectable markers. Thesetools were developed over the next decade with encouragementfrom the success achieved in the medfly, Ceratitis capitata(Loukeris et al., 1995), and the mosquito Ae. aegypti(Coateset al., 1998; Jasinskiene et al., 1998). The achievement ofefficient transformation of the Asian malaria vector, A.stephensi(Catteruccia et al., 2000), was followed by that of A.gambiae (Grossman et al., 2001) and, more recently, A.albimanus, the south American vector (Perera et al., 2002).

With the methods for transgenesis in place, anophelinemosquitoes refractory to Plasmodiumdevelopment have beengenerated. Bloodmeal-induced expression of two alternativetransgenes inA. stephensi, a short effector peptide (Ito et al.,

2002) and a bee venom phospholipase (Moreira et al., 2002),led to dramatic reduction in oocyst numbers and greatlyimpaired transmission of P. bergheito naïve mice. Preliminaryresults also suggested that transgenic lines of Ae. aegyptiwererendered resistant to the development of P. gallinaceumthrough the bloodmeal-induced, systemic expression ofdefensin A (Shin et al., 2003b).

Although these results illustrate the potential of transgenictechnology to study mosquito–parasite interactions, furtherrefinements are vital to the development of this field, especiallyin view of the negative impact of transformation on mosquitofitness (Catteruccia et al., 2003). Clearly, the identification ofanopheline promoters that drive transgene expression in astage- and tissue-specific manner is a major challenge. Thesetools will be essential not only for the expression of effectormolecules targeted against the critical stages of parasitedevelopment in the midgut and salivary gland but also for thesilencing of mosquito genes that act as positive regulators ofparasite development. To date, only a few promoters have beencharacterized and used to drive gene expression in mosquitoes.These include Aedesvitellogenin (Shin et al., 2003a),Drosophila actin5C (Brown et al., 2003) and Anophelescarboxypeptidase promoter (Ito et al., 2002). Thecharacterization of mosquito promoter sequences could begreatly aided by utilization of the Cre–LoxP system, which wasshown recently to be active in Ae. aegypti(Jasinskiene et al.,2003). This system would allow precise functional comparisonof alternative promoters if it is used to target integration oftransgenes into the same chromosomal location: vagaries inexpression caused by position effects of the transgene insertionsite would be eliminated, and promoter activity would be easilycompared between transgenic lines.

These approaches will be complemented by theidentification of inducible promoters and conditionalexpression systems permitting temporal and tissue-specificcontrol of transgene expression or gene silencing (usingdsRNA-producing transgenes). Such tools will make itpossible to finely monitor effector mechanisms and genefunction and to dissect in detail immune signalling pathwaysin the vector. Indeed, anopheline strains have been producedrecently that conditionally express transgene in the adultmidgut and haemocytes (G. Lycett and T. G. Loukeris,personal communication). In these lines, tissue-specificexpression is directed by a promoter of a serpin gene, whileconditional regulation is achieved by using the tetracyclinetransactivator (Gossen and Bujard, 1992). Transgeneexpression can be switched on or off by the supply oftetracycline analogues to the mosquito. With this system,potentially any stage- or tissue-specific promoter can be made‘inducible’.

Anophelesand Plasmodium: an interplay of immune attackand evasion?

Vector immune responses are believed to account, at leastin part, for the major parasite losses during sporogonicdevelopment. In extreme cases, all Plasmodiumparasites are

Molecular genetics of innate immunity in A. gambiae 2557

killed in genetically selected refractory mosquitoes. Anophelesdirus mosquitoes selected for refractoriness completely blockthe development of P. yoeliiookinetes by melanoticencapsulation (Somboon et al., 1999), and refractory A.gambiae mosquitoes cause the lysis of P. gallinaceumookinetes in the cytosol of infected midgut cells (Vernick etal., 1995). The best-studied case of refractoriness is melanoticencapsulation of Plasmodiumookinetes (Collins et al., 1986)in an A. gambiaerefractory strain (L35). Melanization takesplace in the extracellular space, between the midgut epithelialcells and the basal lamina. Genetic mapping of the L35phenotype revealed one major (Pen1) and two minor (Pen2andPen3) quantitative trait loci (QTL) implicated in this responseagainst P. cynomolgiB (Zheng et al., 1997). A recent studyrevealed that refractoriness of L35 mosquitoes to P. cynomolgiCeylon, a different but related species, is controlled by at leastthree QTLs (Pcen2R, 3Rand 3L) (Zheng et al., 2003).Interestingly, while Pcen2Rand 3Lmap near Pen3and Pen2,respectively, and may actually be Pen3and Pen2, Pcen3Rrepresents a novel QTL unrelated to Pen1, suggesting thatdifferent genetic loci may be involved in responses to differentmalaria parasites. Sequencing of 528·kb of DNA from thePen1 region revealed a remarkable number of sequencepolymorphisms that constitute two alternative haplotypes overat least 121·kb (Thomasova et al., 2002). The significance ofthese haplotypes, and more generally the molecular basis of thecomplete melanotic phenotype, is not yet fully understood,although L35 mosquitoes show a high level of reactive oxygenspecies, which is further enhanced by bloodfeeding (Kumar etal., 2003). Similarly, the molecular basis of P. gallinaceumlysis in a refractory strain of A. gambiae(Vernick et al., 1995)is still unknown. However, recent studies showed that themelanotic response of L35 mosquitoes can be reversed bysilencing specific A. gambiaeimmunity genes (Blandin et al.,2004; G. K. Christophides, unpublished data).

Melanization of P. falciparumookinetes is rare in infectedfield-caught mosquitoes (Niare et al., 2002; Schwartz andKoella, 2002). Rather, these mosquitoes are characterized by ahigh natural frequency of segregating resistance alleles thatapparently attenuate the intensity of infection in the vector(Niare et al., 2002). This suggests that P. falciparuminducesa strong selective pressure on Anopheles. Therefore, theparasite and its vector most likely represent a co-evolvingsystem in dynamic equilibrium. The fact that the L35 strain ofA. gambiaemelanizes several species of malaria parasites,including P. berghei, P. gallinaceum, P. cynomolgiB andallopatric but not sympatric strains of P. falciparum(Collinset al., 1986), adds further support to this hypothesis. Severalquestions arise concerning mosquito factors that mayspecifically protect sympatric ookinetes and the means bywhich the parasite may evade or subvert the mosquito immuneresponses. A recent high-throughput proteomic approach hasrevealed that P. falciparumsporozoites express severalproteins of the vargene family and other surface receptors(Florens et al., 2002) that were initially thought to be restrictedto the mature asexual stages of the parasite. The fact that var

genes are involved in immune evasion in the vertebrate hostmakes it tempting to explore whether varor other specificparasite surface proteins can mediate immune evasion in thevector as well as the vertebrate host.

Protozoan pathogens have evolved several mechanisms toevade the immune responses of the vertebrate host, includingantigenic variation, shedding of surface proteins, antigenicmimicry, hiding inside cells and modulation of the hostimmune responses (Zambrano-Villa et al., 2002). Untilrecently, the molecular mechanisms that control the number ofthese parasites in their mosquito vectors, thus facilitating theirtransmission to vertebrates, remained uncharacterized. Theycould only be proposed by analogy to antibacterial immunity,by in vitro studies or by inference from descriptions of geneexpression patterns. However, application of the dsRNA-mediated gene silencing technique in vivohas changed thissituation radically. Recently, specific mosquito gene products,which act as antagonists of parasite development, have beenidentified in living mosquitoes, as well as others that act asagonists protecting the parasite against antagonists. Theantagonists identified to date are the thioester-containingprotein TEP1 and a leucine-rich protein, LRIM1. TEP1 is anacute-phase haemocyte-specific protein previously shown tobind and opsonize bacteria in a thioester-dependent manner(Levashina et al., 2001). Recently, TEP1 was also shown to beinvolved in the killing of P. bergheiookinetes as they cross themidgut epithelium of A. gambiae (Blandin et al., 2004). Thiswas supported by several lines of evidence: (1) TEP1 knockoutinduces a fivefold increase in ookinete survival in a suceptible(G3) strain of A. gambiaeand permits the successfuldevelopment of P. bergheiin a refractory (L35) strain of thesame species; (2) TEP1 binds to the surface of ookinetes afterthey cross the midgut epithelium in both strains, but the timingof binding differs between these strains, and (3) TEP1-associated ookinetes display degeneration, evidenced byparasite blebbing, loss of the vital fluorescent marker GFP,nuclear abnormalities and fragmentation, and perturbations inthe distribution of the ookinete-specific surface protein P28.Interestingly, 100% of ookinetes are killed in the L35 strain ascompared with 80% in the G3 strain. The identification ofTEP1 staining on live, morphologically normal ookinetes hassuggested that TEP1 first binds to the ookinetes and then leadsto their degeneration. Two polymorphic alleles of TEP1wereidentified: TEP1r is associated with the L35 strain and TEP1swith the susceptible strain. However, conclusive evidence isstill missing as to whether these polymorphic alleles areassociated with the faster binding of TEP1 and more efficientkilling of ookinetes in the L35 strain.

In parallel studies (Osta et al., 2004), LRIM1 was identifiedas a new parasite antagonist whose absence induces a dramatic,nearly fourfold, increase in the number of parasites insusceptible mosquitoes. LRIM1 is also predominantlyexpressed in the carcass (mosquito remnant following midgutisolation) as compared with the midgut and is specificallyupregulated in the carcass of infected mosquitoes as comparedwith non-infected mosquitoes. Interestingly, LRIM1

M. A. Osta and others2558

expression in the midgut is strong and transient: upregulationoccurs at 24–28·h post-infection, coinciding with the period ofookinete invasion of the midgut epithelium. Additionalexperiments revealed that two A. gambiae C-type lectins,CTL4 and CTLMA2, act as agonists protecting the parasitefrom mosquito immune responses (Osta et al., 2004). Silencingeither lectin gene by RNAi induces massive melanization ofookinetes in the susceptible A. gambiaeG3 strain: the CTL4KO results in melanization of nearly all ookinetes, while partialmelanization is observed in CTLMA2 KO mosquitoes. Bothlectins are predominantly expressed in the mosquito carcass(possibly in fat body and/or haemocytes) as compared with themidgut and are specifically upregulated in the carcass duringookinete invasion of the midgut epithelium, suggesting someimmune signalling between tissues. These results highlight thepotential use of CTL genes or proteins as targets to blockPlasmodiumtransmission in the vector.

Genetic epistasis analysis revealed that the melanizationresponse induced in the absence of CTLs requires LRIM1function: the double KO of LRIM1 with either lectin genecompletely abolishes the melanization phenotype and inducesa fourfold increase in oocyst numbers, a phenotype similar tothat of the single LRIM1KO. Further research is beingconducted to address the detailed mechanisms by whichLRIM1 dramatically limits the parasite load in the vector,while CTLs protect the parasite. For now, our understandingcan be summarized as in Fig.·3.

It is clear that parasite transmission depends upon complexmolecular and cellular interactions acting at different levels ofthe parasite’s life cycle in the vector (Alavi et al., 2003).

Deciphering these interactions and identifying the molecules(defence or non-defence) that negatively and positivelyregulate parasite development in the vector will providevaluable information that can be exploited in designing noveltransmission-blocking strategies for vector-borne pathogens. Itwill obviously be a matter of considerable importance todetermine whether TEP1, LRIM1, CTL4 and CTLMA2 areinduced by and act on P. falciparumin the same manner as onP. berghei.

Concluding remarks and perspectivesA decade ago, dissection of Anophelesinnate immunity and

the molecular interactions occurring between the vector and theparasite was a distant dream. Now, there is conclusive evidencethat the Anophelesimmune system is a determining factor ofvectorial capacity, and our knowledge of the specific moleculesthat are involved is advancing rapidly. Several breakthroughsunderlie this remarkable progress: the development of tools fortransformation of both the Plasmodiumparasite and itsAnophelesvector, the establishment of a direct reverse genetictechnique (RNAi) in the mosquito for rapid investigation ofgene function, the development of microarrays for genediscovery and genome-wide expression profiling and, mostimportantly, the sequencing of the genomes of A. gambiae(Holt et al., 2002), the human malaria parasite, P. falciparum(Gardner et al., 2002), and the model rodent malaria parasite,P. yoelii yoelii(Carlton et al., 2002). The genome sequence ofthe widely used model rodent malaria parasite P. bergheiwillalso be available soon. With these tools in hand, the scientificcommunity can now decipher the key interactions occurringbetween the parasite and the mosquito during the differentstages of parasite development. Of particular interest is the in-depth characterization of the Anophelesimmunity proteins thatcontrol parasite development in the vector and the immunesignalling pathways responsible for regulated production ofthese proteins. Identification of both parasite antagonists andagonists in the vector is an important conceptual advance thatsets the stage for dissecting the molecular mosquito–parasiteinteractions in detail. It also suggests a novel avenue forpotential control of the malaria parasite in the mosquito.Establishment of the infection in Anophelesis essential fordisease transmission and undoubtedly depends on multipleinteractions of the parasite with other bloodmeal componentsand with multiple mosquito tissues that it encounterssequentially. Of these interactions, the ones that occur duringthe transition from ookinete to oocyst, when parasite numbersare at a minimum, are especially important and most probablyreflect a fine balance between positive and negative mosquitofactors. Such a balance may prove a favourable point forchemical intervention to reduce malaria transmission. Futuredetailed understanding of these molecular interactions maypermit development of antimalarial ‘smart sprays’: chemicalsthat are delivered like pesticides but are designed to disruptinteractions protective of the parasite or to reinforce others thatare antagonistic to the parasite.

Molecular genetics of innate immunity in A. gambiae 2559

Fig.·3. Schematic model of LRIM1 and CTL (CTL4 and CTLMA2)protein action during Plasmodiumdevelopment in the mosquitomidgut. During or soon after invasion of the midgut epithelium (fourdownward-oriented arrows), three out of four invading ookinetes areeliminated, partly through the antagonistic action of LRIM1 (upward-oriented arrows). However, CTL4 and, to a lesser extent, CTLMA2protect the remaining ookinetes from the melanization response(slanted black bars); melanization also requires LRIM1 activity(horizontal arrow).

LRIM1

Lumen

Epithelium

Basal space

Melanization

Killing

CTL4

CTLMA2

List of abbreviationsAMP antimicrobial peptideCLIP clip-domain serine proteasesCTL C-type lectinGNBP Gram-negative bacteria-binding proteinIMD immune deficiencyKO knockoutLPS lipopolysaccharideLRIM leucine-rich immune proteinPAMP pathogen-associated molecular patternPAP prophenol-activating proteinasePGN peptidoglycanPGRP peptidoglycan recognition proteinPM peritrophic membranePO phenoloxidasePPAE prophenoloxidase-activating enzymePPO prophenoloxidasePRR pattern recognition receptorQTL quantitative trait locusTE transposable elementTEP thioester-containing proteinTLR Toll-like receptor

We thank previous and current members of the Kafatoslaboratory, as well as our collaborators in the MosquitoImmunity Consortium who contributed to the worksummarized in this review, and especially those whoauthorized the references to unpublished data. We are gratefulto Gareth Lycett for critical reading. M.A.O. is supported by aMarie Curie Intra-European Fellowship. D.V. was initiallysupported by a European Commission Marie CurieFellowship. The work on innate immunity in the laboratorywas funded by the EMBL, NIH, MacArthour Foundation,SFB and European Commission.

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