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Phlebotomine sand flies and Leishmania parasites: friends or foes? Shaden Kamhawi Intracellular Parasite Biology Section, Laboratory of Parasitic Diseases, NIAID, NIH, Bethesda, MD 20892, USA

Leishmania parasites need phlebotomine sand flies to complete their life cycle and to propagate. This review looks at Leishmania–sand fly interactions as the parasites develop from amastigotes to infectious metacyclics, highlighting recent findings concerning the evolutionary adaptations that ensure survival of the parasites. Such adaptations include secretion of phosphoglycans, which protect the parasite from digestive enzymes; production of chitinases that degrade the stomodeal valve of the sand fly; secretion of a neuropeptide that arrests midgut and hindgut peristalsis; and attaching to the midgut to avoid expulsion. Introduction Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand flies. It is dependent on the presence of vector species and occurs in tropical, subtropical and temperate regions of some 88 countries. Of an estimated 400 sand fly species, only 50 are implicated in the transmission of Leishmania parasites. Susceptibility and refractoriness to Leishmania depend on the outcome of multiple interactions that take place within the digestive tract of the sand fly. This review focuses on recent studies contributing to our understanding of the factors and variations, in either sand fly or Leishmania species and strains, which confer susceptibility or resistance to infection. The life cycle of Leishmania in a natural vector sand fly Leishmania parasites, the etiological agents of leishmaniasis, cause a variety of symptoms, from contained cutaneous ulcers to fatal visceral disease. Leishmaniasis can be a zoonosis, involving domestic or wild animals as reservoir hosts, or an anthroponosis, with humans as reservoirs. The parasites alternate between intracellular amastigotes in the vertebrate host and extracellular promastigotes in phlebotomine sand flies, their biological vectors. Outside the mammalian host, the Leishmania life cycle is confined to the digestive tract of sand flies. Most Leishmania species (subgenus Leishmania) are suprapylarian parasites; that is, their development is restricted to the midgut. Members of the New World Viannia subgenus, such as Leishmania braziliensis, are peripylarian parasites: they enter the hindgut before migrating forward into the midgut. Most studies on parasite–vector interactions have focused on Old World Corresponding author: Kamhawi, S. ([email protected]) Available online 14 July 2006. www.sciencedirect.com

suprapylarian parasites. For this reason, we discuss only suprapylarian development here unless otherwise specified. Infection is initiated when sand flies ingest blood that contains macrophages infected with amastigotes. The infected bloodmeal passes to the posterior ‘abdominal’ midgut. Thereafter, Leishmania parasites differentiate into several distinct developmental stages as they migrate anteriorly from the posterior midgut to the stomodeal valve, which forms a junction with the foregut. Each of these stages is characterized by morphological and functional changes aimed at ensuring its survival in the fly. First, amastigotes differentiate into small, sluggish procyclic promastigotes with short flagella and commence the first multiplication cycle in the fly. These forms are observed in the early bloodmeal and are separated from the midgut by a type I peritrophic matrix (PM). They are also relatively resistant to the onslaught of digestive enzymes. Procyclics develop into nectomonads, large slender forms whose function is to escape the confinement of the PM, to anchor themselves to epithelial cells lining the midgut and to migrate forward towards the anterior ‘thoracic’ midgut. Leptomonads, a newly identified shorter form of the parasite [1–3], arise from nectomonads and undergo the second multiplication cycle in the fly. Finally, two stages are observed at the stomodeal valve, haptomonads and metacyclics. Haptomonads, whose precursor form is still in question (nectomonads or leptomonads), are highly specialized leaf-like parasites with short flagella that are non-motile and form a parasite plug at the stomodeal valve. Metacyclics, the infective stage, are found behind the stomodeal valve and are highly adapted for successful transmission to the mammalian host. They have a small cell body with an elongated flagellum and are rapid, free-swimming forms resistant to complement-mediated lysis. Figure 1 depicts the main developmental forms of Leishmania promastigotes in the sand fly midgut during infection. The approximate time needed for the parasites to complete their development in the sand fly is 6–9 days, depending on the species. Once in the posterior midgut, the infected bloodmeal is fully contained by a PM within 4 hours. Amastigotes released into the blood bolus (the mass of ingested blood) transform into procyclics, which undergo rapid replication for the next 24–48 hours. Transformation of procyclics to motile nectomonads takes place by day 2–3. At this point, degeneration of the PM enables the escape of nectomonads into the gut lumen, where they attach along the midgut epithelium and

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Figure 1. The life cycle of Leishmania in a competent vector, illustrating the time-dependent appearance of distinct morphological forms of promastigotes within the sand fly midgut.

migrate forward to colonize the anterior midgut. Nectomonads give rise to leptomonads by day 4 and, by day 5–7, division of leptomonads results in a massive infection at the anterior midgut. Leptomonads then differentiate into numerous non-dividing infectious metacyclic promastigotes that accumulate at the stomodeal valve, ensuring the availability of a large number of parasites for transmission. Leptomonads also produce a promastigote secretory gel, which fills the thoracic midgut, enveloping the leptomonads and metacyclics [1]. Haptomonads also appear by day 5–7 as static parasites attached to the cuticular lining of the stomodeal valve and to each other, forming concentric rings of parasites that plug the opening of the valve. In heavily infected sand flies, the stomodeal valve degenerates and a few metacyclics can pass through it into the foregut. Little is known of the effect of Leishmania infection on the longevity and fecundity of a sand fly. Boulanger et al. [4] identified a defensin with Leishmania major activity from Phlebotomus duboscqi. The only other report alluding to innate immunity in sand flies describes the identification of a differentially expressed cDNA encoding a mitogen-activated protein kinase from infected midguts of Lutzomyia longipalpis [5]. Information on this aspect of sand fly–Leishmania interactions is lacking and requires more attention. Determinants of parasite survival during digestion of the bloodmeal Ingestion of blood induces physiological responses in the sand fly midgut, including the synthesis of a PM, diuresis (secretion of urine) and secretion of digestive enzymes. The PM, a sac of proteins and glycoproteins held together by chitinous microfibrils, surrounds the bloodmeal within 4 hours, forming a barrier that protects the midgut epithelium from abrasive food particles and microbes and slows down the diffusion of digestive www.sciencedirect.com

proteases into the endoperitrophic space (i.e. the space within the PM). The earliest determinant of vector competence is the ability of the parasites to survive the onslaught of midgut proteases, which peak 18–48 hours after blood feeding. The enzymes create a hostile environment for the parasites within the bloodmeal [6–8]. Even in a compatible Leishmania–sand fly combination, such as Phlebotomus papatasi and L. major, transitional forms transforming from amastigotes to promastigotes are highly sensitive to proteolysis, and up to 50% of parasites can be killed in the first 2 days after blood feeding [1,9]. This early killing of parasites is also observed during the development of the New World species Leishmania amazonensis (a suprapylarian parasite) and L. braziliensis (a peripylarian parasite) in Lutzomyia migonei [10], and for Leishmania mexicana (a suprapylarian parasite) in L. longipalpis [1]. The PM barrier provides the time necessary for some 50% of transitional forms to differentiate into resistant flagellated promastigotes [9]. In addition, Leishmania species have evolved strategies for overcoming the harmful effects of digestive enzymes in competent vectors, inhibiting or retarding the peak activity of these enzymes [6]. So far, little is known of the regulation of the expression of sand fly digestive enzymes and how they are modulated by Leishmania infections. Ramalho-Ortigao et al. [8] characterized six serine proteases from P. papatasi. Of four trypsin-like proteases, two were downregulated and a third was upregulated following a bloodmeal. The two chymotrypsin-like enzymes were both induced by bloodfeeding. More work is needed to investigate the relative importance of these enzymes in the development of Leishmania and how their induction is modulated in response to infection in different sand fly species. Blood ingestion also induces lectin secretion in sand flies, and this is associated with bloodmeal digestion and participation in defense mechanisms, including parasite

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agglutination [aggregation of parasites by crosslinking carbohydrates (or glycosylated molecules) on their surface] [11]. Lectins have been identified from various tissues of several sand fly species, including the midgut [11,12]. After a bloodmeal, midgut lectin activity increases 2–16-fold depending on the sand fly species and protein content of the meal. In general, peak lectin activity is reached 2 days after bloodfeeding and returns to baseline levels after defecation [11]. Sand fly midgut lysates have been shown to agglutinate different Leishmania species and strains, even in natural parasite–vector combinations. Moreover, inhibition of lectin activity enhanced infection of Phlebotomus duboscqi with L. major, indicating that secreted lectins have an important role in determining the early survival of Leishmania in the sand fly midgut [7,13]. Midgut peristalsis (the contraction pattern that moves the meal along the gut) assists in the excretion of the undigested bloodmeal. Recently, Vaidyanathan [14,15] characterized a secreted myoinhibitory neuropeptide from L. major that arrested midgut and hindgut peristalsis of its vector P. papatasi and, to a lesser degree that of L. longipalpis. In addition, L. major lysates caused a 100% inhibition of the hindgut peristalsis of its natural vector P. papatasi, whereas inhibition attributed to the incompatible parasites L. braziliensis and L. donovani (Sudanese strain) was 50% and 20%, respectively [14]. An Indian strain of L. donovani had no inhibitory effect on the P. papatasi hindgut, suggesting that the inhibitory peptide is species-specific to a degree. Inhibition of gut peristalsis relaxes the sand fly midgut, which becomes less efficient at expelling the parasites, enhancing their chances of persisting in the gut. Chitinases and Leishmania development Chitinases are crucial to the successful development of Leishmania in the sand fly. Within 48–72 hours of blood feeding, the chitinous PM is degraded by chitinases, and then expelled from the fly along with the undigested bloodmeal. Both parasites and sand flies secrete chitinases [5,16–18]. The activity of sand fly chitinases peaks at 48 hours after blood feeding, which corresponds to the time nectomonads escape the confinement of the PM [18]. Several studies have demonstrated that inhibition of chitinase activity leads to loss of parasites, even in a competent vector, presumably because of their inability to escape the PM before its defecation [3,6]. In a study involving several strains of L. major, the strains that escaped more rapidly from the PM of P. papatasi developed more successfully in the fly [19]. The relative contribution of parasite and sand fly chitinases to the degradation of the PM and their significance to the release of nectomonads remains to be fully elucidated. Later on, in mature infections of 5–7 days, Leishmania chitinases, probably produced by haptomonads attached to the cuticular lining of the stomodeal valve, are responsible for degeneration of the chitinous lining of the valve [20,21]. Valve degeneration impairs the feeding dynamics of the sand fly, resulting in more probing and longer feeding periods, which in turn contribute to increased transmission efficiency. www.sciencedirect.com

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Leishmania glycoconjugates and development in the midgut Glycoconjugates of Leishmania include molecules that share a common phosphoglycan repeat unit. These phosphoglycans consist of glycoproteins, such as cell surface and released proteophosphoglycans (PPG), and glycolipids such as lipophosphoglycan (LPG). Gycoconjugates also include non-phosphoglycan containing molecules, such as the major surface glycoprotein, the metalloproteinase gp63. Secreted phosphoglycans Secreted phosphoglycans (Box 1) are implicated in the early survival of procyclics within the bloodfed midgut [22], presumably by conferring resistance to, or by modulating the activity of digestive enzymes. Perhaps a more significant effect of secreted phosphoglycans, is the accumulation of promastigote secretory gel, a gel-like matrix produced by leptomonads in the thoracic midgut of mature infections [1,3,23]. A growing body of evidence shows that this gel, which is composed primarily of filamentous proteophosphoglycan (fPPG), fills the lumen of the thoracic midgut and contributes to the observed ‘blocked fly’ effect and impaired feeding [1,23]. As a ‘blocked’ fly attempts to feed multiple times, it regurgitates an estimated 1000 metacyclic promastigotes into the vertebrate host, 90% of which originate from behind the pharynx [23]. This infective dose, estimated through voluntary feeding conditions, is 10 fold higher than previous estimates made by forced capillary feeding [23]. fPPG is also important in ensuring the successful transmission of Leishmania to a vertebrate host. In a L. Longipalpis–L. mexicana infection model, Rogers et al. [23] demonstrated that fPPG, regurgitated along with metacyclics into the vertebrate host, causes substantial exacerbation of disease, which is attributed to its glycan moieties and results in non-healing lesions in the resistant CBA/Ca strain of mice. fPPG is a fine example of how Leishmania parasites have evolved to manipulate both vector and vertebrate host to ensure their survival. Lipophosphoglycan Lipophosphoglycan (LPG) is the largest and most abundant surface glycoconjugate of promastigotes. Genetic and biochemical studies have implicated LPG in the attachment of nectomonads to the sand fly midgut, preventing their loss with the excreted bloodmeal [6,22,24,25]. LPG is also considered to be a major determinant of vector competence for Leishmania species, and it shows well defined structural polymorphisms among different Leishmania species. A conserved backbone of disaccharide repeats can be free of, or extensively modified with, side chains of sugar residues that vary in nature, frequency and length among different Leishmania species (Box 1). In earlier studies, LPG polymorphisms in Leishmania species and strains were closely associated with their success or failure in completing their development in different sand fly vectors; this gave rise to the theory of ‘permissive’ and ‘restricted’ vectors. For example, P. papatasi and Phlebotomus sergenti are restricted vectors and can support the growth of only the parasites that they transmit

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Box 1. Leishmania glycoconjugates and their function in the vertebrate host Leishmania glycoconjugates include secreted molecules such as proteophosphoglycan (sPPG) and acid phosphatase (sAP), and GPIanchored molecules such as LPG, the metalloproteinase gp63, surface proteophosphoglycans and small surface GPI lipids (GIPLs) [6,24] (Figure I). Phosphoglycans are glycoconjugates that share a conserved backbone polymer of phosphorylated galactose–mannose disaccharide repeats (-6Gal(b1-4)Man(a1)-PO4- with an oligosaccharide cap. The phosphoglycan domain is polymorphic among Leishmania species and is either unsubstituted or variably substituted with phosphoglycosylated oligosaccharide side chains (Figure I). Glycoconjugates seem to be important virulence factors when Leishmania is in the vertebrate host. However, recent studies suggest that their function varies among different Leishmania species. The role of LPG and gp63 in complement resistance is well established for L. major [34,46,47]. However, in mouse infections with L. mexicana,

LPG-deficient parasites were as virulent as LPG-expressing parasites, indicating that LPG does not protect against complement in this species [48]. Moreover, LPG does not seem to be required for attachment to, invasion of, or survival in the macrophage, or for inhibition of its signaling pathways [46,48]. In fact, new evidence suggests that LPG is detrimental to long-term survival in the vertebrate host, as it is associated with activation of dendritic cells, natural killer (NK) cells and NK T cells; this is perhaps the reason why it is rapidly downregulated by amastigotes [49–51]. However, gp63 seems to be important in binding to macrophages and intracellular survival and replication in them [47,52]. For L. major, phosphoglycans and GPI-anchored glycoconjugates have been shown to be important for acute virulence, including attachment and entry into macrophages [53,54], yet in L. mexicana, GPI-anchored proteins do not seem to be essential for entry and survival within macrophages [55].

Figure I. Schematic representation of GPI-anchored and secreted glycoconjugates of Leishmania. The polymorphic nature of the phosphoglycan domain is shown for Leishmania major (V1), L. tropica (KK27) and L. donovani (1S).

in nature, L. major and Leishmania tropica, respectively [6,26]. By contrast, Phlebotomus halepensis [27], Phlebotomus argentipes and L. longipalpis [6,26,28–30] can support the growth of several Leishmania species, including L. major and L. tropica. The restricted vector competence of P. papatasi to L. major is associated with the presence of galactose side chains on its procyclic LPG that are specific for the fly lectin PpGalec [6,31]. Dobson et al. [32] identified a family of six genes encoding L. major galactosyltransferases with galactosyltransferase activity and proposed that their differential expression could account for the variable levels of LPG galactosylation shown by different L. major strains. For example, the West African Seidman strain of L. major, with LPG devoid of galactose side chains, does not grow in P. papatasi [33,34] but does grow in P. duboscqi, a closely related species abundant in West Africa [34]. In addition, LV39, a strain isolated from the former USSR and characterized by having long poly-galactose side chains www.sciencedirect.com

[35], also survives poorly in a colony of P. papatasi that originated from Jordan, and it does not bind to PpGalec, the LPG receptor characterized from the midgut of this fly [31]. Conversely, the Friedlin (V1) strain of L. major isolated from the Jordan valley, whose LPG has short galactose chains [35], binds to PpGalec and develops well in the Jordanian strain of P. papatasi. The intraspecific variation in vector competence was also observed for another parasite species. LPGs characterized from two strains of L. tropica isolated from different vector species, P. sergenti and Phlebotomus arabicus, have intraspecific polymorphisms in the nature of terminal sugars on the side chains of their LPG repeat units [36]. The developmental modifications in LPG that occur in metacyclic L. (V.) braziliensis, the only peripylarian parasite studied so far, suggests a different pattern of midgut attachment compared to suprapylarian parasites [37]. In all suprapylarian species studied to date, metacyclic LPG does not bind to the midgut [6,24,25,30]. On the contrary, it

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is accepted that a crucial part of the function of metacyclics is to remain free from midgut attachment to be available for transmission. This is supported by biochemical evidence of stage-specific structural changes that occur in the LPG to render it refractory to midgut binding sites [6,25,30]. In contrast to suprapylarian parasites, metacyclics of Leishmania braziliensis make less LPG than procyclic stages and add glucose residues to its disaccharide phosphate repeat units [37]. Soares et al. [37] postulated that peripylarian metacyclics might have to traverse the midgut following their early development in the hindgut. By adding glucose residues to their LPG, a novel mechanism in metacyclogenesis, L. braziliensis metacyclics might be able to detach from the hindgut and attach to the midgut during their forward migration. However, given that the term ‘metacyclic’ normally refers to the detached infective stage available for transmission, this ‘metacyclic’ stage in the peripylarian life cycle should perhaps be renamed. LPG and other cell-surface phosphoglycans might also have a role in inducing the innate immune system of sand flies. The induction of a P. duboscqi defensin with antiparasitic activity against L. major was impaired in mutants deficient in cell surface phosphoglycans, including lipophosphoglycan [4]. The metalloproteinase gp63 (leishmanolysin) The gp63 protein (also called leishmanolysin) is a 63 kDa glycosylphosphatidylinositol (GPI)-anchored zinc metalloproteinase abundantly expressed on the surface of Leishmania promastigotes. Definitive studies in L. major using gp63 null mutants derived by deletion of the gp63 gene cluster, demonstrated that gp63, a virulence factor when the parasite is in the vertebrate host (Box 1), has no function in Leishmania development within the sand fly midgut [6,33,34]. Studies using sense and antisense transfectants of Leishmania chagasi gp63, derived by dominant expression of episomal constructs [38], found that two days after infection, sense transfectants that overexpress gp63 showed a high infection rate and a high density of parasites. By contrast, antisense transfectants with downregulated gp63 developed at a significantly lower rate and at a very low density [38]. The detrimental effect of gp63 downregulation was lost in late infections, suggesting that gp63 has a protective role in the early stage of L. amazonensis establishment in L. longipalpis. This is in contrast to earlier studies of L. major gp63 null mutants in their natural vector P. duboscqi that showed no adverse developmental effects in the absence of gp63 [33,34]. The discrepancy in the results could be due to technical differences: mutants generated by episomal expression generally overexpress their target and could therefore produce different phenotypes from those derived from gene deletion. Alternatively, L. amazonensis, a New World parasite, could have evolved different ways to overcome the adverse early environment in its vector. Further work is needed to address properly the role of Leishmania gp63 in the sand fly. Receptors of lipophosphoglycan in the sand fly midgut The structural polymorphism of LPG reflects the heterogeneity of putative receptors in the midgut of different www.sciencedirect.com

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Box 2. Galectins: outline of structure and function Galectins are a family of b-galactoside-binding lectins that share a conserved carbohydrate-recognition domain (CRD) with affinity for poly-N-acetyl-lactosamine enriched glycoconjugates (type I Gal(b1-3)GlcNAc or type II Gal(b1-4)GlcNAc. They have been reported from a wide range of animals, including sponges, fungi, worms, insects, fish and mammals [56,57]. Galectins include monomers with a single CRD, dimers with two identical CRDs, a chimera-type with a single CRD and a functional non-lectin domain, and tandem repeat molecules with two non-identical CRDs [57]. Despite the absence of a signal peptide, galectins can be intracellular, extracellular or localized on cell surfaces. They are believed to traffic through non-classical secretory pathways, binding extracellularly to glycoconjugates with suitable galactose-bearing oligosaccharides [56,57]. Galectins are multifunctional regulatory molecules involved in a wide array of biological activities, including differentiation and development, apoptosis, innate and adaptive immunoregulation, cell–cell and cell–matrix interactions and homeostasis [57]. Through their multivalent nature and crosslinking properties, galectins have been implicated in host-pathogen interactions [31,57,58]. In the case of Leishmania, specific binding of L. major LPG to galectin-3 and galectin-9 on the surface of macrophages and to PpGalec in the midgut of the vector Phlebotomus papatasi was recently demonstrated [31,59,60].

sand fly species. The highest complexity in the structure of Leishmania LPGs is found in parasites that naturally infect restricted vectors. By contrast, the LPG of parasites that naturally infect permissive vectors is relatively simple. This correlation might not be coincidental and suggests that LPG polymorphisms are driven by the complexity and specificity of midgut receptors. As LPG is composed primarily of sugars, with side chain residues or cap oligosaccharides implicated in midgut attachment [6], it is reasonable to predict that midgut receptors are lectin-like molecules. Although lectins have been reported from the midgut of sand flies (see earlier), their induction by blood, their secretion into the lumen and the similarity of their sugar-specificity in several Phlebotomus and Lutzomyia species [11,12] associates these molecules with bloodmeal digestion and precludes their role as midgut receptors. Midgut attachment lasts beyond bloodmeal excretion, with a residual number of parasites remaining attached to the midgut for the rest of the sand fly life span. Moreover, a

Figure 2. A cluster of midgut epithelial cells of Phlebotomus papatasi showing a strong expression of PpGalec (red). Actin and nuclei are stained green and blue, respectively.

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massive number of parasites are seen attached to the midgut epithelium in advanced infections of competent vector–parasite pairs. Therefore, functional prerequisites for a LPG midgut receptor include constitutive expression, abundance on the surface of epithelial midgut cells, and specificity to the sugar moieties present on the LPG of compatible parasites. PpGalec, a tandem repeat galectin (Box 2) identified from a midgut cDNA library of unfed P. papatasi flies, binds specifically to live parasites and to procyclic LPG of L. major (Friedlin V1 strain) [31]. PpGalec is midgut-specific, unaffected by blood feeding or infection, and is abundant on the surface of the majority of midgut cells (Figure 2). More importantly, antibodies raised against recombinant PpGalec significantly impair development of L. major in vivo in P. papatasi, demonstrating the potential of midgut receptors as transmission-blocking vaccines. When applied to anthroponotic parasites, such as L. tropica and L. donovani, this might represent a novel and practical approach to vaccination. Metacyclogenesis Metacyclic promastigotes are highly adapted for transmission and early survival in the vertebrate host. In the vector sand fly, metacyclogenesis results in masking of sugar residues involved in midgut binding and elongation of the LPG molecule [6,35]. The mechanisms that trigger metacyclogenesis in vivo are poorly known. In vitro, metacyclogenesis is induced by low pH [39], anaerobic conditions [40] and a decline in levels of tetrahydrobiopterin (a cofactor essential for the catalytic activity of nitric oxide synthase and a byproduct of biopterin reduction by pteridine reductase 1) [41]. Several genes specific to or upregulated in the metacyclic stage have been identified, but their function remains poorly defined. Some, such as the META gene cluster, have been associated with increased virulence in the vertebrate host [42]. Others, such as the small hydrophilic endoplasmic reticulum-associated protein (SHERP) proteins, are thought to function mainly in the vector as they are expressed exclusively in metacyclics, and homozygous null mutants of the hydrophilic, acylated surface protein (HASP)/SHERP proteins are as virulent as wild-type parasites in macrophage invasion and intracellular survival [43,44]. Conclusions Our understanding of the complex interactions between Leishmania parasites and vector sand flies continues to advance. However, it is clear that such interactions are highly specific, with many facets yet to be fully appreciated. The rapid progress in genomics and the recent publication of the L. major genome [45] highlight the need for a sand fly genome project. This would accelerate the identification of sand fly molecules pertinent to vector competence. Having the genome from both vector and parasite will enable us to take a global look at sand fly–Leishmania interactions at a molecular level. Acknowledgements I thank David Sacks and Phil Lawyer for reviewing the article and Sanjeev Kumar for the image of PpGalec expression in midgut epithelial cells of P. papatasi. www.sciencedirect.com

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