Innate immune defense against malaria infection in the mosquito

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Innate immune defense against malaria infection in the mosquito George Dimopoulos*, Hans-Michael Müller, Elena A Levashina and Fotis C Kafatos† Anopheles gambiae, the most important vector of malaria, employs its innate immune system in the fight against Plasmodium. This can affect the propagative capacity of Plasmodium in the vector and, in some cases, leads to total refractoriness to the parasite. The components operating in the mosquito's innate immune system and their potential relevance to antimalarial responses are being systematically dissected. Addresses European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Immunology 2001, 13:79–88 0952-7915/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations EST expressed sequence tag FBN fibrinogen-like GNBP Gram-negative bacteria-binding protein JAK Janus kinase PO phenoloxidase PPO prophenoloxidase

Introduction The malaria parasite and its vector mosquito engage in a series of interactions involving invasion and migration by the parasite through vector epithelial tissues (reviewed in [1•]). Within the mosquito, the parasite encounters a hostile environment, where it is exposed to the mosquito’s innate defense surveillance system, which is capable of triggering a plethora of defense reactions. In some cases, these reactions can terminate the development of all parasites, leading to total refractoriness. Two resistant strains of Anopheles gambiae have been genetically selected: in one strain, all invading ookinetes are melanotically encapsulated (this process, also known as melanization, is detailed later) in the midgut epithelium; in the other, they are lysed in the midgut cells [2•,3•]. Significant parasite losses also occur in fully susceptible strains by as-yet unknown mechanisms; only a small number of ingested gametocytes develop into mature ookinetes in the midgut lumen and only a minority of ookinetes ever develop into oocysts after traversing the midgut epithelium. At the later stages of infection, when the oocyst ruptures, only a fraction of the released sporozoites end up in the salivary glands (reviewed in [1•,4]). Part of these losses is very likely to result from mosquito defense reactions. A significant body of knowledge on interactions between A. gambiae and Plasmodium has been generated, highlighting the crucial steps of malaria’s development in the vector.

Special emphasis has been placed on the study of antimalarial responses involved in limiting the extent of infection. Innate immune responses occur in A. gambiae, against various microorganisms, including malaria parasites. Several markers of immune responses have been used for monitoring temporally and spatially these defense reactions at the molecular level and have shown clear correlation of immune responses with the passage of Plasmodium through the vector [5••,6•,7••]. The mosquito has become the organism of choice for directly studying antiparasitic innate immune responses, thus complementing the studies of defense reactions against bacteria and fungi that have been made in ‘model’ organisms such as the fruitfly, moths and crustaceans. In the following sections we will review the mosquito defense system, with emphasis on responses to malaria infection.

The mosquito fights against malaria Several lines of evidence have clearly indicated that the mosquito is capable of mounting a robust innate immune response against malaria infection. It was first shown that a set of diverse immune genes is transcriptionally activated both systemically and locally in epithelial tissues in the course of Plasmodium infection [5••,6•,7••,8•]. As mentioned above, this activation of immune markers correlates with the passage of the parasites through the mosquito both temporally and spatially. At early stages, when ookinetes invade the midgut epithelium, genes of the immune system are upregulated in the midgut; at the later stages, when oocysts rupture and sporozoites invade the salivary glands, markers of immune responses are activated in the salivary glands. In parallel, systemic induction during these periods occurs primarily in the fat body [7••]. Interestingly, epithelial invasion and these two transcriptional induction peaks of immune-response markers coincide with the stages when the largest parasite losses occur in both susceptible and refractory strains [1•–3•,4]. The multiphasic, multisite immune responses engage genes that are likely to serve key steps in defense, such as recognition, signal transduction and amplification, and microbial killing. A more direct linkage between innate immune responses and parasite killing was suggested by the finding that NO — which is produced by the enzyme NO synthase (NOS), which is itself induced by infection — is involved in parasite killing in the midgut [8•]. Furthermore, parasite development is significantly restricted in mosquitoes previously challenged by bacteria [9•]. However, the known mechanisms underlying parasite killing in the mosquito are still limited to melanotic encapsulation and NO production. Other components, such as antimicrobial peptides, may also participate in controlling infection but their

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Figure 1 Anopheles mosquito

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Model illustrating Plasmodium’s lifecycle in the vector mosquito and potential defense reactions mounted against malaria infection. The route taken by Plasmodium is shown by yellow arrows; antiPlasmodium responses are shown by red arrows; and blue arrows indicate the magnification of areas in the upper part of the panel. (a) Ingested gametocytes fertilize and form a zygote, which transforms into a motile ookinete. (b) The ookinete can invade and traverse the midgut epithelial cells, reaching the basal side, where it can form an oocyst. (c) Sporozoites can develop in the oocyst and become released in the hemocoel, from where they can invade the salivary glands. Mosquito immune responses mounted against the parasite have been documented in the midgut epithelium, in the hemocoel and

in the salivary glands. In the midgut epithelium, malaria parasites can be melanotically encapsulated or lysed, respectively, in two different refractory mosquito strains. NO has also been shown to restrict parasite development in the midgut. (d) In the hemocoel, sporozoites are likely to encounter hemocytes, antimicrobial peptides and other humoral factors. Binding to soluble or surface-bound patternrecognition receptors (green) can trigger a cascade of serine proteases, which leads to the cleavage of PPO to PO; in turn, PO is involved in melanization. Serpins are involved in inactivating serine proteases. Antimicrobial peptides are also found in the salivary gland. Signaling is likely to occur between epithelial tissues, the fat body and hemocytes during these immune responses.

potential antimalarial action remains to be proven. The mosquito hemocytes are likely to contribute actively to the antimalarial defense during the later stages of infection in the hemocoel, where they may be involved in killing sporozoites (Figure 1).

As with other insects, the innate immune system of the mosquito consists of humoral and cellular defense mechanisms. During humoral immune responses, recognition of microorganisms by receptor molecules results in the activation of serine protease cascades that can activate

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melanization and coagulation reactions (this process has been extensively described in crustaceans; see [10]) as well as intracellular signaling pathways controlling expression of immune-system effector genes that may have specific activities against various classes of microorganisms (reviewed in [11,12]). Humoral immune responses can be classified as systemic, when defense components are produced by the fat body and possibly hemocytes, or local, when the epithelial tissues produce defense components that are involved in proximity to the site of infection. The cellular immune reactions are mediated by hemocytes and include phagocytosis and cellular encapsulation (reviewed in [13]). The various factors involved in humoral and/or cellular mechanisms are detailed below.

Antimicrobial peptides The transient production of antimicrobial peptides is the best-known effective defense mechanism against invading microorganisms. In most cases, the lethal action of antimicrobial peptides is exerted through a detergent-like mechanism, disrupting the integrity of the cell membrane (reviewed in [14]). Four such effector peptides have been isolated from A. gambiae: one defensin, two cecropins and a novel antimicrobial peptide, gambicin ([15,16,17•]; J Vizioli, personal communication). The expression patterns of the A. gambiae defensin, cecropin-1 and gambicin are strikingly similar, the transcripts being found to be enriched in the fat-body-containing abdomen and thorax as well as in the cardia of the anterior midgut ([7••,16]; J Vizioli, personal communication). The strong expression in the cardia, which is the valve through which ingested food enters the midgut, may control the flora of the ingested nectar or blood meal. Cecropin-2 is highly induced by immune challenge and its expression is still under study. In A. gambiae, the defensin appears to be present in specific cells of the posterior midgut (A Richman, J Vizioli, personal communication). Insect defensins are known to be mainly active against Gram-positive bacteria whereas the cecropins, which are found in both vertebrates and invertebrates, are active against both Gram-negative and Gram-positive bacteria; the A. gambiae cecropin-1 is active against both Gram-positive and Gram-negative bacteria, as well as fungi [16]. Gambicin, which was detected both as a cDNA induced by immune challenge and as an induced peptide from a cellline supernatant, is active against both bacteria (Gram-positive and Gram-negative) and fungi in in vitro assays ([17•]; J Vizioli, personal communication). Interestingly, it has been shown that two insect defensins, from Aeschna cyanea and Phormia terranovae, are toxic to the oocyst and sporozoite stages of Plasmodium in in vivo assays but are not effective against the gametocyte or ookinete stages [18].

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Immune signaling pathways Homologues of several components of the Drosophila melanogaster Toll signaling pathway — which is involved in development and immune responses — have been isolated and studied. Two Toll-like genes, encoding Ag-Toll and Ag-Trex, have been isolated from A. gambiae and belong to the D. melanogaster Toll group and Tollo/Trex group, respectively [19]. Several other Toll-related proteins are likely to exist in the mosquito — as in the case in the fruitfly, where at least nine members have been reported — and some of these are likely to be implicated in immune-response activation upon infection [20•]. Two transcription factors, Gambif1 and Ag-STAT, have been studied in A. gambiae [21,22•]. Gambif1 is a Rel family member most similar to D. melanogaster Dorsal whereas AgSTAT belongs to the STAT family and is most similar to D. melanogaster D-STAT and the vertebrate STAT5 and STAT6 moieties. Both Gambif1 and Ag-STAT are translocated to the nucleus of fat-body cells upon bacterial infection but at least Gambif1 does not respond to malaria infection. Although the exact function of these transcription factors during bacterial infection is not fully understood, this differential response pattern may point to the existence of distinct antibacterial and antimalarial immune responses in mosquitoes. Gambif1 can bind to NFκB-like DNA motifs of D. melanogaster antimicrobial gene regulatory regions, highlighting the evolutionary conservation of the regulatory mechanisms involved in innate defense [21]. Additional candidate components of signaling pathways involved in immune responses have been identified in a pilot EST (expressed sequence tag) project and include a homologue of cactus as well as homologues of other components, such as a pelle-associated protein and a κB-motif-binding protein [17•]. These mosquito components are likely to be associated with signaling pathways and to operate in immune responses. The definition and dissection of pathways controlling diverse immune responses in the mosquito is an important priority and may ultimately distinguish key antimalarial from antimicrobial defense mechanisms. Serine protease cascade components

Serine proteases are a large family in insects and, for example, they are represented by 199 genes in the Drosophila genome [23]. A subset of these are implicated in immune reactions, where they are mainly involved in signaling and amplification cascades that lead to the activation of specific defense mechanisms, such as melanization, coagulation and induction of antimicrobial peptides (reviewed in [10–12,24]). These cascades usually consist of several components and are tightly regulated by serpins, which can limit the reaction to the proximity of the site of intended action, thus preventing an uncontrolled systemic response that could be lethal to the insect ([25•]; reviewed in [26•]). An increasing number of serine proteases that are involved in immune responses are being isolated and characterized

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from A. gambiae [6•,17•,27,28•,29•,30,31,32•]. The majority of these serine proteases are present in the hemolymph (this is the fluid found in the hemocoel) and expressed by hemocytes [17•,28•,30]. This expression pattern may indicate a potential role in cascades that lead to the activation of humoral defense reactions. The main structural feature distinguishing them from the digestive proteases is a modular amino-terminal region that contains additional functional domains that are possibly involved in regulation. The most common of these is the clip domain, consisting of six cysteines with a conserved spacing (reviewed in [33•]). The function of the clip domain is not fully established but it has been proposed to be active in protein–protein and protein–extracellular-matrix interactions, for example bringing the serine protease close to a wound site. The similarity of the clip domain fold with horseshoe-crab big defensin has also suggested the possibility that the clip domain may play an antimicrobial role (reviewed in [10,33•]). A. gambiae AgSP14D1, AgSP14D2, AgSP14A and two ESTs encoding serine proteases that are induced during immune responses have the specific sequence feature of prophenoloxidase (PPO)-activating enzymes (the functions of PPO and phenoloxidase [PO] are described later): clip domains, an aspartic acid and two glycines determining trypsin specificity and two cysteines in the catalytic part [33•]. AgSP14D1, AgSP14D2 and AgSP14A are weakly induced by malaria challenge in both susceptible and refractory strains [30]. One immune responsive serine protease, SP22D, has a mosaic amino-terminal portion, consisting of putative chitin-binding domains, scavengerreceptor domains and a mucin-like domain [28•,29•]. All these types of domains are known to be involved in macromolecular interactions and may thus be of regulatory significance. Indeed, the putative chitin-binding domains have been shown to confer chitin-binding activity to SP22D, tempting the speculation that binding to chitin of the trachea, cuticle or microorganismal surface may lead to activation of the SP22D protease domain [28•]. Seven more serine proteases were identified as ESTs; three of these are induced by bacterial challenge [17•]. Another member of the family is ISPL5, a serine-protease-like molecule with a nonfunctional catalytic triad (where two of the three residues are mutated [6•]). ISPL5 contains two clip domains separated from the catalytic part by a threonine-rich spacer. A similar, hemocyte-specific, serine-protease-like molecule that is induced by immune challenge in the crayfish was shown to be involved in cell adhesion and a similar component from the coleopteran insect Holotrichia diomphalia is implicated in PPO cascade activation [34,35]. The nonfunctional catalytic domain is likely to mediate protein–protein interactions or to function as a regulatory antagonist of substrate binding to other serine proteases. Three serpins have been detected among the ESTs from immune competent A. gambiae cell lines and one is responsive to bacterial challenge in cell lines [17•].

Another serpin, which was isolated from a subtraction cDNA library derived from bacterially challenged mosquitos, is induced by malaria infection [32•]. Some of these serpins may function as regulators of serine protease cascades that are induced by immune challenge and thus be important in limiting the cascade-amplification reaction to the local site of infection. Pattern-recognition receptors

Several genes encoding proteins with domains known to bind to microorganisms have been cloned and are likely to function as pattern-recognition receptors that are involved in triggering immune responses upon microbial and parasitic recognition. Putative pattern-recognition receptors that have been identified in the mosquito are detailed below. Two putative galactose-binding lectins have been isolated from A. gambiae [17•,27]. One of these, IGALE20, is highly expressed in the midgut, where it also is induced upon microbial and malaria infection [7••]. Lectins often play roles in immunity as agglutinins and as opsonins in PPO activation [36,37]. A lectin from the Tobacco hornworm, Manduca sexta, lectin has been shown to trigger PPO activation in vitro [37]. In the mosquito midgut, lectins may play a major role in limiting bacterial growth, which has been reported to increase up to a 40-fold after blood-feeding [38]. Gram-negative bacteria-binding protein (GNBP) was initially isolated from the silkworm, Bombyx mori [39]. Similarly, D. melanogaster GNBP has been shown to bind to Gram-negative bacteria and to be highly induced upon microbial challenge. It enhances NFκB-dependent gene induction upon binding to lipopolysaccharide (LPS) and β-1,3-glucan and is thus implicated in mediating signaling during immune responses [40]. One A. gambiae homologue has been characterized extensively and another two were detected as ESTs; the studied A. gambiae homologue is highly expressed in the fat body and salivary glands [6•,7••]. ICHIT is coded by a midgut-specific gene whose expression is induced during immune responses; the gene encodes a protein with two chitin-binding domains separated by a threonine-rich mucin domain [7••]. This moiety may bind chitinous microorganismal surfaces or may be associated with the peritrophic matrix, which is highly enriched in chitin. Seven genes encoding proteins with fibrinogen-like (FBN) domains at their carboxyl termini have been isolated from immune competent A. gambiae cell lines ([17•]; G Dimopoulos unpublished data). Three of these — AgFBN-1, -2 and -5 — are highly expressed in hemocytes and fat-body tissues and AgFBN5 is also moderately expressed in the midgut (G Dimopoulos, unpublished data). The vertebrate ficolins, which also contain FBN-like domains at their carboxyl termini, can enhance phagocytosis and also activate the complement pathway in a manner

Innate immune defense against malaria in the mosquito Dimopoulos et al.

similar to the mannose-binding lectin [41,42]. Horseshoecrab homologues of the A. gambiae FBN genes have been shown to agglutinate bacteria and enhance the antimicrobial activity of big defensin [43]. Among several additional partial transcripts of putative pattern-recognition receptors that have been identified as ESTs, one encodes a putative homologue of the D. melanogaster dSR-C1 protein, which has a broad ligandbinding range; interestingly, the mosquito gene is downregulated in the cell lines by bacterial challenge [17•,44]. A second EST encodes hemomucin, which in D. melanogaster is a midgut and hemocyte immune response protein and is hypothesized to mediate immune responses [45]. A third EST encodes a putative peptidoglycan-recognition protein (PGRP) that has been isolated from several organisms and been implicated in PPO cascade activation [46,47].

Cellular immune responses Cellular defense reactions are executed by the as-yet poorly characterized mosquito hemocytes, which are found circulating in the hemocoel and attached to tissues including the fat body, trachea and midgut. The study of cellular responses has been greatly facilitated in the mosquito by the establishment of immune competent cell lines with hemocyte-like characteristics [48••]. Phagocytosis, the first innate immune response to be discovered (by Elie Metchnikoff at the end of the nineteenth century), is a complex cellular process by which blood cells recognize and destroy invading organisms. Primitive organisms use phagocytosis primarily for feeding whereas in higher organisms it is critical for the uptake and degradation of infectious agents and senescent cells. For phagocytosis of a particle to take place, several specific events should occur: binding of opsonic ligands to the particle surface, their recognition by specific receptors, activation of intracellular cascades and, ultimately, internalization of the particle by an actin-dependent mechanism (reviewed in [49•]). Descriptive studies of phagocytosis in insects had provided detailed pictures of the various stages and types of hemocytes involved in internalization of the pathogenic microorganisms and foreign particles. The first and only phagocytic receptor that has been molecularly characterized in D. melanogaster [50], Croquemort, appeared to be involved in phagocytosis of apoptotic bodies, but not of bacteria, in the early embryo [51]. Interestingly, a Croquemort homologue has been reported in A. gambiae [17•]. Another important step forward in our understanding of molecular regulation of phagocytosis has been achieved recently in A. gambiae. A novel complement-like protein, aTEP-I, has been shown to be an essential opsonin for the Gram-negative bacterium, Escherichia coli, in a mosquito hemocyte-like cell line. Depletion of aTEP-I from the

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conditioned medium by dsRNA knockout leads to a dramatic reduction in the phagocytic index of the mosquito cells. Moreover, like human complement factor C3, aTEP-I appears to be bound to the surface of E. coli through a thioester bond. Structural modeling further reveals extensive similarity of the critical thioester-containing region of the mosquito molecule with that of human C3 (EA Levashina, LF Moita, S Blandin, G Vriend, unpublished data). This conservation at both the functional and structural levels strongly suggests that primitive complement-like molecules can be traced back to dipteran insects. From the phylogenetic perspective, it will be of interest to extend comparison of this ancient opsonization system to activating cascades and to activated receptors. Six dTEPs have been recently molecularly described in D. melanogaster. Although their functions have not been elucidated as yet, three of their genes are induced in larvae and adults upon bacterial challenge [52•]. The transcriptional regulation of one of these genes, tep1, was shown to be mediated by a Janus kinase (JAK), Hopscotch. Although the JAK/STAT pathway is an established pathway in the immune system of mammals, this is the first immune-system target gene of the JAK pathway in Drosophila [52•]. Powerful genetic tools can be expected to provide more exciting insights into dTEP functions. Four additional members of the TEP family have been identified recently in the mosquito ([17•,32•]; EA Levashina, LF Moita, S Blandin, unpublished data). It is tempting to speculate that distinct TEPs are able to specifically opsonize different groups of pathogens, including malaria parasites, and thus provide a ‘missing link’ between the humoral and cellular immune responses.

A specific defense mechanism against malaria: melanotic encapsulation Melanization reactions involve sclerotization and tanning of the egg shell and cuticle, wound healing and encapsulation of infecting microorganisms (reviewed in [53•]). The multiple functions of melanization cascades are reflected in the large number of PPO genes present in insect genomes. As many as six PPOs have been identified in A. gambiae, all differing in their developmental expression patterns [48••,54,55]. Interestingly, a genetically selected, refractory laboratory strain, L3-5, melanotically encapsulates all late ookinetes and early oocysts as they reach and transverse the basal side of the midgut epithelium [2•,56]. The capsule is thicker and is first melanized on the apical end of the ookinete facing the hemolymph, while the other end of the capsule, embedded in the epithelium, is still transparent as observed by microscopy; this suggests that at least some of the key components of the melanization reaction originate in the hemolymph (G Dimopoulos, unpublished data; Figure 2). The absence of hemocytes around the melanization site suggests that this reaction is humoral [56]. Melanization that results from components present in

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Figure 2

An encapsulated parasite in the midgut epithelium of an A. gambiaerefractory mosquito strain, L3-5. The apical part of the parasite, facing the basal membrane of the midgut cell (here to the bottom left of the figure), is melanized to a greater extent. This picture was taken by G Dimopoulos, using light microscopy with a 100× lens. The ookinete’s length is about 10–15 µm. The picture has been processed using photoshop in order to enhance the basal border of the midgut cell.

the hemolymph is thought to be initiated by soluble pattern-recognition receptors that bind to the target surface and initiate enzymatic cascades that ultimately leads to cross-linking and melanization of the other proteins associated with the parasite. All six PPO mRNAs are expressed during the peak of blood-meal digestion, when melanotic encapsulation also occurs [48••]. It is unclear, however, which PPO genes may be involved in this defense reaction, as the increase in PPOs at this stage may also be linked to egg production. Moreover, the PPO genes are not transcriptionally induced upon challenge to the immune system. This is not surprising, as PPO regulation is thought to occur frequently at the translational and post-translational levels. The latter level includes proteolytic activation (e.g. the conversion of PPO to PO) (reviewed in [24]). Activated PO catalyzes the hydroxylation of tyrosine to DOPA, the oxidation of DOPA to dopachrome and the oxidation of dihydroxyindole to the melanin precursor, indolequinone. Two more enzymes, Dopachrome conversion enzyme (DCE) and DOPA decarboxylase (DDC), are involved in the synthesis of melanin (reviewed in [53•]) and have been detected in immune challenged cell lines (H-M Müller, unpublished data). The melanotic capsule consists of a proteinaceous polyquinone material surrounding the parasite. Killing of the parasite may be mediated by toxic byproducts of the reaction, such as free radicals, or by starvation within the capsule (reviewed in [57]).

The refractory trait is likely to be determined by factors such as opsonins or other putative recognition receptors, or by post-translational regulatory mechanisms. Activation of invertebrate melanization reactions upon recognition of a microorganism by a receptor molecule is known to be mediated by a cascade of serine proteases and counterbalancing protease inhibitors (serpins), leading to the cleavage of PPO to PO. Serine proteases, from moths and the coleopteran insect H. diomphalia, have been cloned and shown to be implicated in the PPO activation system [58–60] as has a serine-protease-like molecule from H. diomphalia [35]. All of them contain clip domains with one or more cysteine knots in their amino-terminal portion and share other unique features not found in other serine-protease families, such as a cysteine pair in the noncatalytic part. Six A. gambiae serine protease genes likely to be involed in PPO activation have been isolated, including AgSP14A, AgSP14D1, AgSP14D2, two ESTs and a recently cloned cDNA (H-M Müller, J Volz, unpublished data). Their potential link to melanization reactions is under study. Two moth serpins and one fall-webworm serpin inhibit melanization reactions and are likely to be involved in regulating and restricting the activating proteolytic cascade to the immediate environment of the encapsulation site [61,62]. Whether some of the cloned A. gambiae serpins (see above) are implicated in the PPO activation system is still unclear [17•,32•]. Like other innate defense mechanisms, PPO cascades are believed to be triggered by pattern-recognition receptors capable of binding to the microbial surface (reviewed in [24]). Activation of the PPO system can be stimulated by lipopolysaccharide, β-1,3-glucan and peptidoglycan. Several putative pattern-recognition receptors — including lectins, a peptidoglycan-recognition protein, a β-1,3-glucan-recognition protein and a GNBP — have been linked to PPO activation in invertebrates [36,37,46,47,63,64]. These proteins, which also exist in the mosquito (see above), may be involved in forming the proteinaceous layer that initially surrounds the parasite and then in triggering melanization. In the selected A. gambiae refractory strain, melanotic encapsulation is a general response, exhibited against all Plasmodium species capable of reaching the oocyst stage. However, a certain degree of specificity is indicated by the fact that sympatric (African) strains of P. falciparum are melanized to a lesser extent [2•]. Experiments performed with beads having different chemically modified surfaces showed that Sephadex beads were encapsulated in both susceptible and refractory mosquitoes whereas negatively charged carboxymethyl-Sephadex beads were only encapsulated in the refractory strain. Interestingly, carboxymethyl-Sephadex beads that had been preincubated in susceptible mosquitoes were not encapsulated in the refractory strain, suggesting that they had been covered with a ‘protective’ mosquito substance that may mimic the

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endogenous environment in susceptible mosquitos, making them ‘invisible’ to the PPO-activation system [65]. An interesting observation is that, in infections of some A. gambiae strains, only a few of the genetically identical ookinetes are encapsulated whereas others continue their development normally. This selective killing is probably due to differences in the concentrations of active components in the microenvironment surrounding the ookinete and the invaded cell it has traversed (FH Collins, personal communication).

Components of iron metabolism Innate insect defenses are likely to involve iron metabolism. Iron is an essential element for the propagation of microorganisms, including large parasites, and its levels in vertebrates are modulated upon infection; it is sequestered from the infection site through the use of transferrin and ferritin [66]. Transferrin is highly induced upon infection in the yellow-fever mosquito, Aedes aegypti [67]. The antimicrobial molecule NO is generally implicated in iron metabolism, influencing the translational control of these iron-binding molecules. In vertebrates, the translational regulation of ferritin and transferrin is tightly controlled by the iron-regulatory protein (IRP). Both IRP and ferritin have been cloned from A. gambiae but their function in relation to infection remains to be tested [17•]. A fragment of the A. gambiae NO synthase gene has been characterized and shown to be activated by malaria infection, as in the case of another malaria vector, A. stephensi. In the latter case, NO has been directly linked to parasite killing in the midgut [8].

Genomic approaches A promising approach towards characterization of genes that control melanotic encapsulation of Plasmodium and Sephadex beads has been initiated by genetic mapping. Three dispersed quantitative trait loci (QTL) associated with melanotic encapsulation have been located. Pen1, which maps in region 8C of the 2R chromosomal arm, is the major locus and controls more than 65% of the refractory phenotype for encapsulation of both Plasmodium and beads [68•,69•]. Ongoing work aims to identify the genetic element within the Pen1 locus that controls this trait, through positional cloning and determination of its sequence differences between the refractory and susceptible strains and ultimately germ-line transformation assays (FH Collins, FC Kafatos, unpublished data). In a pilot EST project, sequencing of ESTs from a normalized, subtracted cDNA library led to the discovery of approximately 2300 novel mosquito genes, of which 38 showed significant similarity to known innate immunity genes of other organisms [17•]. Of these, half showed transcriptional upregulation upon exposure of immune competent cell lines to bacterial challenge. Mass expression-profiling of the entire EST collection using cDNA microarrays is being used for the identification of additional, functionally related components of A. gambiae innate defense reactions (G Dimopoulos, unpublished data).

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Conclusions The fact that relatively low numbers of parasites are found in natural mosquito populations suggests the existence of a fine-tuned equilibrium between the propagative capacity of the parasite and the mosquito’s ability to control malaria infection: it is likely that a heavy infection would affect the mosquito’s fitness, leading to intense natural selection for refractory vector populations whereas a too-low infection rate would not allow transmission of the disease. Furthermore, during the developmental stages of the parasite in the mosquito, phases of rapidly decreasing parasite numbers alternate with rapid expansion of parasite numbers. Thus, it is quite plausible that the parasite load in mosquitoes is kept low by the mosquito’s innate defense mechanisms and that these mechanisms are important determinants of vector competence. The first, largely descriptive stage of characterizing innate immune responses of malaria vectors has been quite fruitful. By using the paradigms of vertebrate and fruitfly innate immunity as guides, and utilizing transcriptional upregulation as a heuristic tool, a large collection of A. gambiae genes implicated in immune responses has now been assembled. The pace of discovery of genes involved in the immune response has accelerated recently, thanks to an EST pilot project. Mass transcriptional-profiling techniques, such as cDNA microarrays, will further permit the refinement of this information. For example, cDNA microarrays will allow identification of gene cohorts that are co-regulated upon specific stimuli (for example by parasitic rather than bacterial challenge); this will allow us to begin defining the regulatory pathways that are activated by specific pathogens, as well as tissue-specific defense reactions that may be mounted by a succession of mosquito organs against the malaria parasite during its life-cycle within the vector. Mass expression-profiling of A. gambiae ESTs has already been initiated and generated promising results (G Dimopoulos, unpublished data). In our experimental system of A. gambiae and P. berghei, the malaria parasite clearly encounters robust defense reactions by the mosquito but the specific mechanisms employed in parasite killing are unknown as yet. Beyond this first descriptive stage, the field will need to develop efficient tools for functional analysis. Many of these tools will be biochemical and cell biological. The immunocompetent hemocyte-like cell lines will be invaluable in this connection. For example, a phagocytic assay has been developed recently and used to functionally characterize the complement-like, immune-inducible opsonin, aTEP-I, in A. gambiae (EA Levashina, L Moita, unpublished data). Moreover, breakthroughs have been achieved recently in developing transgenic technologies that will permit functional analysis of anopheline genes in both cultured cell lines and in the entire mosquito [70••,71••]. A century after the full life-cycle of the Plasmodium was described, we can now expect that the ‘black box’ of its development in, and interaction with,

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the mosquito vector will soon be illuminated. This should give us new ideas about novel interventions against malaria transmission and also give potentially novel insights into innate immunity [72].

Lowenberger CA, Kamal S, Chiles J, Paskewitz S, Bulet P, Hoffmann JA, Christensen BM: Mosquito-Plasmodium interactions in response to immune activation of the vector. Exp Parasitol 1999, 91:59-69. Reports the effect of mosquito immune response on malaria infection. This is the first report that parasite development is compromised in midguts of mosquitoes that have been pre-immunised with bacteria.

Acknowledgements

10. Muta T, Iwanaga S: The role of hemolymph coagulation in innate immunity. Curr Opin Immunol 1996, 8:41-47.

We acknowledge with pleasure the contributions of members of the Kafatos laboratory whose original research is reviewed here: both past colleagues (C Barillas-Mury, A Richman and L Zheng) and present members of the laboratory (D Thomasova, A Danielli, T Loukeris, L Moita, S Blandin, J Volz and R Wang). We are also grateful for the enjoyable and highly fruitful collaborations with JA Hoffmann and his colleagues and FH Collins and his colleagues as well as A Ezekowitz and C Janeway. Due to space limitations, coverage was largely limited to the innate immunity of anopheline malaria vectors but we acknowledge the rapid growth of the broader field of innate immunity in medically important vectors. The work in our laboratory has been supported by grants and fellowships from the Human Frontiers Science Program, the European Commission, the National Institute of Health, the World Health Organization, the John D and Catherine MacArthur Foundation, the Deutsche Forschungsgemeinschaft SFB544 and European Molecular Biology Organization (EMBO).

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest •• of outstanding interest 1. •

Ghosh A, Edwards MJ, Jacobs-Lorena M: The journey of the malaria parasite in the mosquito: hopes for the new century. Parasitol Today 2000, 16:196-201. A recent, comprehensive review. 2. •

Collins FH, Sakai RK, Vernick KD, Paskewitz S, Seeley DC, Miller LH, Collins WE, Campbell CC, Gwadz RW: Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae. Science 1986, 234:607-610. See annotation to [3•]. Vernick KD, Fujioka H, Seeley DC, Tandler B, Aikawa M, Miller LH: Plasmodium gallinaceum: a refractory mechanism of ookinete killing in the mosquito, Anopheles gambiae. Exp Parasitol 1995, 80:583-595. References [2•,3•] report the characterization of a two A. gambiae refractory strains that melanotically encapsulate and lyse, respectively, malaria parasites in the midgut epithelium.

9. •

11. Hoffmann JA, Reichhard J-M, Hetru C: Innate immunity in higher insects. Curr Opin Immunol 1996, 8:8-13. 12. Hoffmann JA, Reichhart J-M: Drosophila immunity. Trends Cell Biol 1997, 7:309-316. 13. Gillespie JP, Kanost MR, Trenczek T: Biological mediators of insect immunity. Annu Rev Entomol 1997, 42:611-643. 14. Cociancich S, Bulet P, Hoffmann JA: The inducible antibacterial peptides of insects. Parasitol Today 1994, 10:132-139. 15. Richman AM, Bulet P, Hetru C, Barillas-Mury C, Hoffmann JA, Kafatos FC: Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA. Insect Mol Biol 1996, 5:203-210. 16. Vizioli J, Bulet P, Charlet M, Lowenberger C, Blass C, Müller HM, Dimopoulos G, Hoffmann J, Kafatos FC, Richman A: Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae. Insect Mol Biol 2000, 9:75-84. 17. •

Dimopoulos G, Casavant TL, Chang S, Scheetz T, Roberts C, Donohue M, Schultz J, Benes V, Bork P, Ansorge W et al.: Anopheles gambiae pilot gene discovery project: identification of mosquito innate immunity genes from expressed sequence tags generated from immune-competent cell lines. Proc Natl Acad Sci USA 2000, 97:6619-6624. Reports a gene-discovery project that led to the identification of over 2200 novel A. gambiae genes. Thirty-eight putative immunity genes were detected through sequence-similarity searches of the generated ESTs. 18. Shahabuddin M, Fields I, Bulet P, Hoffmann JA, Miller LH: Plasmodium gallinaceum: differential killing of some mosquito stages of the parasite by insect defensin. Exp Parasitol 1998, 89:103-112.

3. •

19. Luo C, Zheng L: Independent evolution of Toll and related genes in insects and mammals. Immunogenetics 2000, 51:92-98.

4.

20. Tauszig S, Jouanguy E, Hoffmann JA, Imler JL: Toll-related receptors • and the control of antimicrobial peptide expression in Drosophila. Proc Natl Acad Sci USA 2000, 97:10520-10525. Reports the identification of six new D. melanogaster Toll-related genes and the implication of Toll-related genes in the activation of antifungal peptide gene expression.

Beier JC: Malaria parasite development in mosquitoes. Annu Rev Entomol 1998, 43:519-543.

5. ••

Richman A, Dimopoulos G, Seeley D, Kafatos FC: Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes. EMBO J 1997, 16:6114-6119. See annotation to [6•].

6. •

Dimopoulos G, Richman A, Müller H-M, Kafatos FC: Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc Natl Acad Sci USA 1997, 94:11508-11513. References [5••,6•] are the first reports showing systemic and epithelial transcriptional activation of a diverse set of immune marker genes upon malaria infection when the ookinetes invade the midgut epithelium. 7. ••

Dimopoulos G, Seeley D, Wolf A, Kafatos FC: Malaria infection of the mosquito Anopheles gambiae activates immune-responsive genes during critical transition stages of the parasite life cycle. EMBO J 1998, 17:6115-6123. This is the first report of temporal and spatial correlation of A. gambiae systemic and epithelial immune responses to early and late stages of malaria infection. 8. •

Luckhart S, Vodovotz Y, Cui L, Rosenberg R: The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc Natl Acad Sci USA 1998, 95:5700-5705. This is the first report of NO-mediated restriction of Plasmodium development in mosquito midgut, highlighting the significance of antimalarial molecular immune responses in controlling infection.

21. Barillas-Mury C, Charlesworth A, Gross I, Richman A, Hoffmann JA, Kafatos FC: Immune factor Gambif1, a new rel family member from the human malaria vector, Anopheles gambiae. EMBO J 1996, 15:4691-4701. 22. Barillas-Mury C, Han YS, Seeley D, Kafatos FC: Anopheles gambiae • Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection. EMBO J 1999, 18:959-967. References [21,22•] are the first reports of A. gambiae rel family and STAT family transcription factors and the implications for antibacterial immune responses. 23. Rubin GM, Yandell MD, Wortman JR, Gabor Miklos GL, Nelson CR, Hariharan IK, Fortini ME, Li PW, Apweiler R, Fleischmann W et al.: Comparative genomics of the eukaryotes. Science 2000, 287:2204-2215. 24. Söderhäll K, Cerenius L: Role of the prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol 1998, 10:23-28. 25. Levashina EA, Langley E, Green C, Gubb D, Ashburner M, • Hoffmann JA, Reichhart JM: Constitutive activation of Toll-mediated antifungal defense in serpin-deficient Drosophila. Science 1999, 285:1917-1919. The first direct evidence implying that a serpin is involved in the activation of the Toll pathway. 26. Kanost MR: Serine proteinase inhibitors in arthropod immunity. • Dev Comp Immunol 1999, 23:291-301. A recent, comprehensive review.

Innate immune defense against malaria in the mosquito Dimopoulos et al.

27.

Dimopoulos G, Richman A, della Torre A, Kafatos FC, Louis C: Identification and characterization of differentially expressed cDNAs of the vector mosquito, Anopheles gambiae. Proc Natl Acad Sci USA 1996, 93:13066-13071.

28. Danielli A, Loukeris TG, Lagueux M, Muller HM, Richman A, • Kafatos FC: A modular chitin-binding protease associated with hemocytes and hemolymph in the mosquito Anopheles gambiae. Proc Natl Acad Sci USA 2000, 97:7136-7147. See annotation to [29•]. 29. Gorman MJ, Andreeva OV, Paskewitz SM: SP22D: a multidomain • serine protease with a putative role in insect immunity. Gene 2000, 251:9-17. References [28•,29•] report the isolation and molecular characterization of an A. gambiae modular serine protease that is expressed in hemocytes; SP22D encodes domains known to be involved in binding to the surface of microorganisms. 30. Gorman MJ, Andreeva OV, Paskewitz SM: Molecular characterization of five serine protease genes cloned from Anopheles gambiae hemolymph. Insect Biochem Mol Biol 2000, 30:35-46. 31. Paskewitz SM, Reese-Stardy S, Gorman MJ: An easter-like serine protease from Anopheles gambiae exhibits changes in transcript abundance following immune challenge. Insect Mol Biol 1999, 8:329-337. 32. Oduol F, Xu J, Niare O, Natarajan R, Vernick KD: Genes identified by • an expression screen of the vector mosquito Anopheles gambiae display differential molecular immune response to malaria parasites and bacteria. Proc Natl Acad Sci USA 2000, 97:11397-11402. Reports the isolation of several novel genes that are involved in immune responses, including those encoding five serine proteases, a serpin and α-2-macroglobulin. 33. Jiang H, Kanost MR: The clip-domain family of serine proteinases • in arthropods. Insect Biochem Mol Biol 2000, 30:95-105. A recent, comprehensive review. 34. Huang TS, Wang H, Lee SY, Johansson MW, Soderhall K, Cerenius L: A cell adhesion protein from the crayfish Pacifastacus leniusculus, a serine proteinase homologue similar to Drosophila masquerade. J Biol Chem 2000, 275:9996-10001. 35. Kwon TH, Kim MS, Choi HW, Joo CH, Cho MY, Lee BL: A masquerade-like serine proteinase homologue is necessary for phenoloxidase activity in the coleopteran insect, Holotrichia diomphalia larvae. Eur J Biochem 2000, 267:6188-6196. 36. Chen C, Rowley AF, Newton RP, Ratcliffe NA: Identification, purification and properties of a beta-1,3-glucan-specific lectin from the serum of the cockroach, Blaberus discoidalis which is implicated in immune defence reactions. Comp Biochem Physiol B Biochem Mol Biol 1999, 122:309-319. 37.

Yu X-Q, Gan H, Kanost MR: Immulectin, an inducible C-type lectin from an insect, Manduca sexta, stimulates activation of plasma prophenol oxidase. Insect Biochem Mol Biol 1999, 29:585-597.

38. Pumpuni CB, Demaio J, Kent M, Davis JR, Beier JC: Bacterial population dynamics in three anopheline species: the impact on Plasmodium sporogonic development. Am J Trop Med Hyg 1999, 54:214-218. 39. Lee WJ, Lee JD, Kravchenko VV, Ulevitch RJ, Brey PT: Purification and molecular cloning of an inducible Gram-negative bacteriabinding protein from the silkworm, Bombyx mori. Proc Natl Acad Sci USA 1996, 93:7888-7893. 40. Kim YS, Ryu JH, Han SJ, Choi KH, Nam KB, Jang IH, Lemaitre B, Brey PT, Lee WJ: Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells. J Biol Chem 2000, 275:32721-32727. 41. Lu J: Collectins: collectors of microorganisms for the innate immune system. BioEssays 1997, 19:509-518. 42. Matsushita M, Endo Y, Fujita T: Complement activating complex of ficolin and mannose-binding lectin-associated serine protease. J Immunol 2000, 164:2281-2284 43. Gokudan S, Muta T, Tsuda R, Koori K, Kawahara T, Seki N, Mizunoe Y, Wai SN, Iwanaga S, Kawabata S-I: Horseshoe crab acetyl grouprecognizing lectins involved in innate immunity are structurally

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related to fibrinogen. Proc Natl Acad Sci USA 1999, 96:10086-10091. 44. Paerson A, Lux A, Krieger M: Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster. Proc Natl Acad Sci USA 1995, 92:4056-4060. 45. Theopold U, Samakovlis C, Erdjument-Bromage H, Dillon N, Axelsson B, Schmidt O, Tempst P, Hultmark D: Helix pomatia lectin, an inducer of Drosophila immune response, binds to hemomucin, a novel surface mucin. J Biol Chem 1996, 271:12708-12715. 46. Yoshida H, Kinoshita K, Ashida M: Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J Biol Chem 1996, 271:13854-13860. 47.

Ochiai M, Ashida M: A pattern recognition protein for peptidoglycan. Cloning the cDNA and the gene of the silkworm, Bombyx mori. J Biol Chem 1999, 274:11854-11858.

48. Muller HM, Dimopoulos G, Blass C, Kafatos FC: A hemocyte-like •• cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes. J Biol Chem 1999, 274:11727-11735. Reports the isolation and characterization of A. gambiae prophenoloxidase genes and the establishment of hemocyte-like cell lines that respond to immune stimuli. 49. Aderem A, Underhill DM: Mechanisms of phagocytosis in • macrophages. Annu Rev Immunol 1999, 17:593-623. A recent, comprehensive review. 50. Franc NC, Dimarcq JL, Lagueux M, Hoffmann J, Ezekowitz RA: Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 1996, 4:431-443. 51. Franc NC, Heitzler P, Ezekowitz RA, White K: Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila. Science 1999, 284:1991-1994. 52. Lagueux M, Perrodou E, Levashina EA, Capovilla M, Hoffmann JA: • Constitutive expression of a complement-like protein in Toll and JAK gain-of-function mutants of Drosophila. Proc Natl Acad Sci USA 2000, 97:427-432. The first report of insect proteins that contain a thioester site and their regulation by immune-system signaling pathways. 53. Beerntsen BT, James AA, Christensen BM: Genetics of mosquito • vector competence. Microbiol Mol Biol Rev 2000, 64:115-137. A recent, comprehensive review. 54. Lee WJ, Ahmed A, della Torre A, Kobayashi A, Ashida M, Brey PT: Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vector Anopheles gambiae. Insect Mol Biol 1998, 7:41-50. 55. Jiang H, Wang Y, Korochkina SE, Benes H, Kanost MR: Molecular cloning of cDNAs for two pro-phenol oxidase subunits from the malaria vector, Anopheles gambiae. Insect Biochem Mol Biol 1997, 27:693-699. 56. Paskewitz SM, Brown MR, Lea AO, Collins FH: Ultrastructure of the encapsulation of Plasmodium cynomolgi (B strain) on the midgut of a refractory strain of Anopheles gambiae. J Parasitol 1998, 74:432-439. 57.

Nappi AJ, Sugumaran M: Some biochemical aspects of eumelanin formation in insect immunity. In Insect Immunity. Edited by Pathak JPN. Boston: Kluwer Academic Publishers; 1993:131-148.

58. Lee SY, Cho MY, Hyun JH, Lee KM, Homma KI, Natori S, Kawabata SI, Iwanaga S, Lee BL: Molecular cloning of cDNA for pro-phenol-oxidase-activating factor I, a serine protease is induced by lipopolysaccharide or 1,3-beta-glucan in coleopteran insect, Holotrichia diomphalia larvae. Eur J Biochem 1998, 257:615-621. 59. Jiang H, Wang Y, Kanost MR: Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter. Proc Natl Acad Sci USA 1998, 95:12220-12225. 60. Satoh D, Horii A, Ochiai M, Ashida M: Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. Purification, characterization, and cDNA cloning. J Biol Chem 1999, 274:7441-7453.

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Innate immunity

61. Jiang H, Kanost MR: Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta. J Biol Chem 1997, 272:1082-1087. 62. Park DS, Shin SW, Hong SD, Park HY: Immunological detection of serpin in the fall webworm, Hyphantria cunea and its inhibitory activity on the prophenoloxidase system. Mol Cell 2000, 10:186-192. 63. Beschin A, Bilej M, Hanssens F, Raymakers J, Van Dyck E, Revets H, Brys L, Gomez J, De Baetselier P, Timmermans M: Identification and cloning of a glucan- and lipopolysaccharide-binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. J Biol Chem 1998, 273:24948-24954. 64. Lee SY, Wang R, Söderhäll K: A lipopolysaccharide- and beta-1,3 glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus. Purification, characterization, and cDNA cloning. J Biol Chem 2000, 275:1337-1343. 65. Gorman MJ, Schwartz AM, Paskewitz SM: The role of surface characteristics in eliciting humoral encapsulation of foreign bodies in Plasmodium-refractory and susceptible strains of Anopheles gambiae. J Insect Physiol 1998, 44:947-954. 66. Weiss G, Wachter H, Fuchs D: Linkage of cell-mediated immunity to iron metabolism. Immunol Today 1995, 16:495-500. 67.

Yoshiga T, Hernandez VP, Fallon AM, Law JH: Mosquito transferrin, an acute-phase protein that is up-regulated upon infection. Proc Natl Acad Sci USA 1997, 94:12337-12342.

68. Zheng L, Cornel AJ, Wang R, Erfle H, Voss H, Ansorge W, • Kafatos FC, Collins FH: Quantitative trait loci for refractoriness of Anopheles gambiae to Plasmodium cynomolgi B. Science 1997, 276:425-428. See annotation to [69•]. 69. Gorman MJ, Severson DW, Cornel AJ, Collins FH, Paskewitz SM: • Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae. Genetics 1997, 146:965-971. References [68•,69•] report genetic mapping of A. gambiae quantitative trait loci implicated in controlling melanotic encaspulation of malaria parasites and Sephadex beads. 70. Catteruccia F, Nolan T, Blass C, Muller HM, Crisanti A, Kafatos FC, •• Loukeris TG: Toward Anopheles transformation: Minos element activity in anopheline cells, and embryos. Proc Natl Acad Sci USA 2000, 97:2157-2162. See annotation to [71••]. 71. Catteruccia F, Nolan T, Loukeris TG, Blass C, Savakis C, Kafatos FC, •• Crisanti A: Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature 2000, 405:959-962. References [70••,71••] are the first reports of the use of a Minos transposable element for stable transformation of A. gambiae cell lines and A. stephensi mosquitoes. 72. Collins FH: Prospects for malaria control through the genetic manipulation of its vectors. Parasitol Today 1994, 10:370-376.