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Immunity to eukaryotic parasites in vector insects
Immunity to eukaryotic parasites in vector insects Adam Richman* and Fotis C Kafatost Mosquitoes and blackflies have been the focus of recent efforts to elucidate factors influencing the susceptibility of vector insects to metazoan and protozoan parasites of medical significance. Vector species exhibit variation in cellular and humeral immune responses, as highlighted by studies of melanotic encapsulation and components of the phenoloxidase system. Significant progress has been made in the development of genetic maps based upon molecular markers, leading to the genetic analysis of loci influencing susceptibility. The identification of specific inducible antibacterial peptides, and the cloning of genes encoding immune effector proteins as well as potential regulatory factors, open the path to fruitful studies of vector insect innate immunity and its relationship to insect-parasite interactions. Addresses *#European Molecular Biology Laboratory, Meyerhofstrasse 1,69117 Heidelberg, Germany *e-mail: [email protected] te-mail: [email protected] Current Opinion in Immunology 1995, 8:14-19 © Current Biology Ltd ISSN 0952-7915 Abbreviations QTL quantitativetrait loci RFLP restrictionfragment length polymorphism
Introduction A number of insect species act as vectors of metazoan or protozoan pathogens that are of medical importance. In most cases, the parasites undergo a complex series of growth and differentiation events within the insect to ultimately become infective to the vertebrate host [1]. Thus, the insect-parasite interaction is a critical aspect of disease transmission and a potential target for efforts to control vector-borne disease [2]. T h e complexity and importance of vector-parasite interactions is manifested by the occurrence of specific incompatibility, in which a particular parasite species is blocked from developing in a given vector beyond a certain developmental stage [3,4°]. In a number of cases, incompatibility appears to have a relatively simple genetic basis [5,6°]. Establishment and maintenance of compatibility must reflect a dynamic evolutionary adjustment of the parasite to the physiology of potential vector species. Insects present various barriers to infection, both general (non-targeted) and specific (targeted). General barriers can block opportunistic infections and include anatomical features (e.g. cuticle and cibarial armature [7]), as well as physiological processes (e.g. peritrophic membrane formation [8] and synthesis of digestive proteases in the
midgut [9]). In addition, the innate immune system of insects [10 °] presents more targeted barriers to a variety of microorganisms and macroparasites. Vector insects are known to possess both cellular and humoral defence capabilities [7,10°,11]. Thus, successful maturation of a parasite within the vector requires circumvention of the immune system, which theoretically might be due to the parasite being insensitive to, evading or suppressing the immune response [12]. Which of these possibilities operate is generally not known, although in some cases of refractoriness it is clear that immune defences are rapidly and specifically activated upon parasite invasion, are localized to the microenvironment of the parasite surface, and may be targeted to specific stages of parasite development [7,13,14]. This review focuses on selected hematophagous members of the order Diptera, which are among the most important vectors of eukaryotic parasites of medical significance and are currently favorite systems in the field of vector biology. Mosquitoes (Diptera; Culicidae) are vectors of Plasmodium spp. (the protozoan parasites causing malaria), as well as Brugia and Wucheretia (metazoan agents of filariasis). T h e blackflies (Diptera; Simuliidae) are vectors of metazoan parasites of the genus Onchocerca, responsible for river blindness disease.
Encapsulation, melanization and phenol oxidase Melanotic encapsulation of invading organisms, associated with the activity of phenoloxidase, is a frequently observed manifestation of the immune response of insects [14-16]. Filarial parasite infection of refractory mosquitoes triggers an encapsulation and melanization response that involves both cellular and humoral components of the vector immune system [14]. Hemocyte involvement is evidenced by the proximity of these cells to the parasite surface and cell lysis associated with the encapsulation reaction. Guo et al. [17 °°] have documented changes in hemocyte numbers and lectin binding binding activity in Armigeres subalbatus mosquitoes infected with Brugia parasites. T h e numbers of hemocytes were reduced at 24 hours post-infection, and this decrease was attributed to the involvement of hemocytes in encapsulation reactions. In Aedes aegypti, however, increased hemocyte numbers were detected as a consequence of parasite infection. T h e apparent discrepancy may be a result of species-specific rates of encapsulation, which peaks at 72-96 hours post-infection in Ae. aegypti, compared to 24 hours in Ar. subalbatus. Hemocyte populations may thus be increasing in Ae. aegypti at 24 hours post-infection, but sequestered in encapsulation reactions at this time in Ar. subalbatus. An additional change in hemocyte properties is associated with Brugia infection ofAr. subalbatus: following infection, an increased percentage of hemocytes bind wheat germ
Immunity to eukaryotic parasites in vector insects Richman and Kafatos
agglutinin (WGA), a phenomenon also observed in Ae. aegypti and Ae. trivitattus [ 17"]. Melanization through the action of phenoloxidase is an important aspect of insect defence against microorganisms and eukaryotic parasites [14,16]. T h e inactive precursor prophenoloxidase is converted to the active form by serine proteases, which are themselves activated in response to various elicitors, including components of bacterial and fungal cell surfaces. This cascade results in the production of melanin and associated toxic metabolic by-products (quinones), which may kill encapsulated parasites [16]. Because of the potential toxicity of the ultimate products, phenoloxidase activation is highly localized. Increased phenoloxidase activity associated with melanotic encapsulation of eukaryotic macroparasites has been detected in Ar. subalbatus and Ae. aegypti [17"']. Such observations, however, are potentially subject to variation depending upon the species of vector and parasite, methods of analysis, and other considerations. Shih and Chen [18] reported lower levels of monophenoloxidase activity in bloodfed Ar. subalbatus infected with B. pahangi compared to uninfected controls, a reduction attributed to the sequestration of the enzyme during melanization of the parasite. Phenoloxidase is also implicated in melanotic encapsulation of Plasmodium in Anopheles gambiae, but in the apparent absence of proximal hemocytes [13,19]. In this case, prophenoloxidase/phenoloxidase synthesized in the fat body, circulating hemocytes, midgut epithelial cells or salivary glands may become localized to the parasite surface as part of the innate immune recognition system ([19,20]; see below). Questions concerning the cellular origin(s) of prophenoloxidase and the regulation of its synthesis in An. gambiae may soon be resolved, as a cDNA encoding this enzyme has been cloned (P Brey, personal communication). Interestingly, blackflies (genus Simulium) do not respond to Onchocerca infection by melanization, although they do possess phenoloxidase and serine proteases and are capable of producing melanin in the context of cuticle formation [21"']. Ham and coworkers [22] speculate that prophenoloxidase/phenoloxidase may serve another immune function in blackflies, perhaps by binding to the parasite surface as a recognition factor or opsonin for hemocytes. This suggestion is based on comparisons of the amount of phenoioxidase that can be detected in the hemolymph of blackflies infected with compatible and non-compatible species of Onchocerca: reduced levels of phenoloxidase are observed in the hemolymph of S. damnosum infected with O. dukei, an incompatible parasite that is normally transmitted by a different vector species [221. Study of the blackfly immune response to Onchocerca has established that melanization is not essential for killing
15
of the parasite. Other mechanisms must therefore be responsible for the observed 'clearance' of microfilaria-stage parasites from resistant blackflies. Lehmann et al. [23] observed a rapid decrease in the number of inoculated microfilaria within 5 hours, suggesting a novel lytic response that occurs in living flies as well as in flies killed by extremes of temperature shortly before the introduction of parasites. Apparently, macroparasite killing is due in this case to the action of soluble factor(s), rather than immune cells. Whether these factors represent homologs of known inducible antimicrobial peptides (see below) remains to be determined. Recognition of non-self
In addition to components of the phenoloxidase cascade, agglutinins or lectins of vector insects represent candidate recognition factors. In Rhodnius prolixus (a non-Dipteran species) lectins exhibiting different carbohydrate specificities interact with various developmental stages of the parasite Trypanosoma cruzi [24]. Lectin-like activities have been implicated in recognition of microfilariae by Ar. subelbatus mosquitoes [25,26]. In this system, agglutinins may mediate the attachment of phenoioxidase to the surface of microfilariae, thus facilitating melanization of the parasite. Observations from the study of blackflies suggest a role for lectins in specific recognition of O. lienalis larvae by the S. ornatum vector. Here, carbohydrate specificities of hemolymph lectins undergo variation in a manner complementary to developmentally regulated changes in the sugar moieties expressed o n the parasite cuticle; this is consistent with a function of lectins as recognition molecules and/or opsonins for hemocytes [21"']. A different recognition mechanism must account for the clearance of 0. linealis microfilaria from S. vittatum, however, as this developmental stage is not believed to express available surface carbohydrate [21"]. Humphreys and Reinherz [27"] have proposed a model, in which hemocyte-mediated recognition of self is the operative principle directing invertebrate immune reactions. In this model, invertebrate pre-immunocytes are positively selected through the expression of a self-recognition receptor that is specific for a polymorphic histocompatibility antigen expressed on the surface of self cells. T h e selected immunocytes would also bear receptors for 'generic ligands' expressed on both self and nonself ceils. Immunocyte activation would be prevented when both receptors are engaged (contact with self cells). Upon contact with nonself, however, engagement of the generic ligand receptor alone would trigger hemocyte activation and initiate subsequent immune reactions. Although this model is principally formulated to account for rapid histocompatibility reactions observed in various invertebrate species, it is theoretically applicable to the recognition of macroparasites in vector insects. Janeway [28,29] has suggested that innate immune responses are activated by multiple or polyspecific receptors detecting constituents of infecting cell surfaces. Although no such
16
Innateimmunity
'pattern recognition' receptors have definitively been shown to function in this way in vector-parasite interactions, the cloning of a scavenger receptor from Drosophila melanogaster (displaying broad ligand specificity) suggests an approach that could well prove fruitful in application to vector species [30°]. In An. gamln'ae a homolog of the CD36 family of multi-ligand receptors has recently been identified (A Crisanti, personal communication), and it is possible that this molecule may function as a 'pattern recognition' receptor to facilitate host-parasite discrimination by hemocytic cells (JA Hoffmann, personal communication). Charge may play a significant role in the response to nonself in insects [31-34,35°]. A correlation has been demonstrated [35°,36°] between refractoriness to Plasmodium and differential responsiveness to negatively-charged Sephadex beads in selected strains of An. gambiae. Refractory mosquitoes melanize negatively-charged beads more avidly than do animals of the susceptible strain. Melanization is affected by gender, age, nutritional and/or reproductive status. T h e weak response of susceptible mosquitoes to negatively-charged beads may indicate that such surfaces more closely resemble self, or are covered with soluble mosquito factors that render them invisible to the immune recognition system [35°]. In this regard, Warburg and Schneider [37] have suggested that Plasmodium oocysts may become surrounded by basement membrane components produced by hemocytes of the infected mosquito. It is not yet clear whether these refractory and susceptible mosquito strains differ in recognition, or in downstream effector functions involved in melanin synthesis. For example, the charged surface could provide a microenvironment for enzyme activity. T h e observed differences between strains might then reflect differential abilities of melanizing enzymes to function within this microenvironment. Nevertheless, the use of charged beads to monitor Plasmodium refractory versus susceptible phenotypes will undoubtedly facilitate analysis of the genes and factors involved in immunity to the parasite (see below).
Genetics of vector susceptibility to parasites Genetic analysis of mosquito susceptibility to parasite infection has a long history [38]. Recent advances in the development of genetic maps based on the molecular markers [39°,40] provide powerful tools to identify genetic determinants influencing the response of the vector to the parasite. Quantitative trait loci (QTLs) influencing mosquito susceptibility to eukaryotic parasites have been localized to genomic intervals delineated by restriction fragment length polymorphism (RFLP) and simple sequence repeat (microsatellite) markers [39°1. Two Q T L s implicated in susceptibility of Ae. aegypti to P gallinaceum have been mapped [6°]. T h e one on chromosome 2 broadly coincides with a Q T L that influences susceptibility to filaria and yellow fever virus; this may represent either a single locus or a tightly-linked
cluster of genes involved in determining susceptibility to multiple disease agents. In An. gambiae, two apparently distinct systems of refractoriness to Plasmodium have been identified through selective breeding of laboratory populations. In one system, general refractoriness to diverse Plasmodiurn species is manifested by extracellular melanotic encapsulation of late stage ookinetes/early oocysts [41]. Susceptibility is determined through the inheritance of at least two loci, one of which is associated with an inversion on the left arm of chromosome 2 [5]. Through another mechanism, ookinetes of P gallinaceum are subject to lysis within midgut cells of refractory, but not susceptible, An. gambiae [42°]. Although this trait may also be influenced by multiple QTLs, a major locus has been roughly mapped to the right arm of chromosome 3 (L Zheng, personal communication).
Inducible antimicrobial factors and the regulation of immune gene expression From the above discussion, it is evident that the biochemical nature of the factors involved in immunity to parasites is largely unknown. It is tempting to speculate that, in addition to the encapsulation and melanization system, humoral antimicrobial peptides may play a role [11]. Molecular tools that can be used to examine this possibility critically are now becoming available. Major advances have been made recently in the identification and characterization of inducible insect antimicrobial peptides, and in the analysis of regulated expression of the genes encoding these proteins [10°,111. Despite the detection of antibacterial activities in vectors [7], specific factors (aside from An. gambiae lysozyme [J-Y Lee, personal communication]) have been described only recently. Defensin proteins and cDNA clones have now been isolated from Ae. aegypti [43,44] and An. gambiae (A Richman, unpublished data) following bacterial infection. T h e mature proteins are very similar in sequence to previously characterized defensins of other Diptera. Evidence currently suggests that antimicrobial factors of insects can influence the viability of some eukaryotic parasites. Exogenous administration of cecropin was reported to inhibit sporogonic development of Plasmodium in An. gambiae [45]. Synthetic cecropin inhibited the motility of Brugia microfilariae in vitro, and reduced larval numbers in Aedes aegypti when co-injected with Brugia parasites [46]. A synthetic peptide based on cecropin, Shiva-3, was active against Plasmodium berghei both in vitro and in An. albimanus [47]. Administration of synthetic defensin has been shown to affect oocyst-stage P gallinaceum in Ae. aegypti, and to kill sporozoite (haemocoel) stage parasites in vitro (M Shahabuddin, LH Miller, JA Hoffmann, C Hetru, personal communication). A number of insect genes encoding antibacterial peptides bear upstream DNA sequence elements resembling binding sites for transcription factors, including the rel family member NF-KB, associated with the acute-phase response
Immunity to eukaryotic parasites in vector insects Richman and Kafatos
of mammals [48]. A tel-domain is also found in several transcription factors of insects that may be implicated in the control of antibacterial and antifungal gene expression [10•,48]. An An. gamln'ae tel-containing protein has been identified (C Barillas-Mury, FC Kafatos, unpublished data) that can activate transcription from antibacterial gene promoters of D. melanogaster. Sequence-specific DNA binding activities that recognize the NF-KB-Iike binding site have been identified in bacterially challenged An. gambiae and are not detected in unchallenged control animals. Sequence elements matching the insect NF-KB consensus binding site have been identified in the putative upstream regulatory region of the An. gambiae defensin gene (A Richman, unpublished data). A critical question concerns the degree of specificity of the insect immune response. Recent observations in D. melanogaster suggest that genes encoding antibacterial and antifungal proteins may be regulated independently of each other, in response to fungal or bacterial infection challenge (JA Hoffmann, personal communication). Antiparasitic immune activities have not been described in vector insects. Nevertheless the cloning of defensin genes from Ae. aegypti and An. gambiae, together with the identification of a putative regulatory transcription factor from An. gambiae, offer powerful tools to investigate the possible involVement of known immune mechanisms of the antibacterial pathway in the vector response to eukaryotic parasites.
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T h e continuing development of high resolution genetic maps based upon molecular markers represents a tremendous advance towards identification of genes influencing vector immunity to metazoan and protozoan parasites. T h e positional cloning of such genes will require the construction of high resolution physical maps for the Ae. aegypti and An. gambiae genomes. A low resolution physical map, based on microdissected chromosomal regions, is already available for An. gambiae [49]; construction of maps based on large-insert DNA clones such as YACs and BACs has begun for both Ae. aegypti and An. gambiae (D Knudson, F Collins, L Zheng, personal communication). Investigations into vector immunity to parasites will be greatly facilitated by integration of the rapidly accumulating molecular, cellular, and genetic insights into the regulatory and effector mechanisms of the innate immune response in D. melanogaster and other model laboratory insects. T h e recent identification of defensin proteins, cloning of defensin genes, and the identification of immune-activated nuclear DNA binding activities in mosquitoes suggests that this integration process is under way.
Acknowledgements We are grateful to S Paskewitz for comments on the manuscript. Work in the authors' laboratory supported by grants from the John D and Catherine T MacArthur Foundation, TDR/WHO, and the Alexander von Humboldt Foundation.
References and recommended reading Conclusions Vector insect responses to protozoan or metazoan parasite infection, particularly the encapsulation and melanization observed in mosquitoes and the immune-related activation of phenoloxidase, resemble well-studied humoral and cellular immune reactions of model insect systems (although hemocyte involvement in encapsulation appears to be more limited in mosquitoes than in some other insects [14]). T h e precise molecular basis of parasite recognition as nonself remains obscure. With the exception of the recently cloned CD36 homolog, candidate 'pattern recognition' receptors have not been identified in vector insects to date. T h e lack of melanization characteristic of the blackfly response to Onchocerca, and the decreased levels of phenoloxidase activity in infected animals indicate the existence of diverse immune mechanisms in hematophagous Diptera, which may involve dual immune functions for some molecules (e.g. phenoloxidase functioning as both a recognition factor and effector pathway molecule in parasite killing). T h e clearance of microfilariae from Simulium, furthermore, suggests that novel cell-free mechanisms of parasite recognition and killing may remain to be elucidated.
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Introduction A number of insect species act as vectors of metazoan or protozoan pathogens that are of medical importance. In most cases, the parasites undergo a complex series of growth and differentiation events within the insect to ultimately become infective to the vertebrate host [1]. Thus, the insect-parasite interaction is a critical aspect of disease transmission and a potential target for efforts to control vector-borne disease [2]. T h e complexity and importance of vector-parasite interactions is manifested by the occurrence of specific incompatibility, in which a particular parasite species is blocked from developing in a given vector beyond a certain developmental stage [3,4°]. In a number of cases, incompatibility appears to have a relatively simple genetic basis [5,6°]. Establishment and maintenance of compatibility must reflect a dynamic evolutionary adjustment of the parasite to the physiology of potential vector species. Insects present various barriers to infection, both general (non-targeted) and specific (targeted). General barriers can block opportunistic infections and include anatomical features (e.g. cuticle and cibarial armature [7]), as well as physiological processes (e.g. peritrophic membrane formation [8] and synthesis of digestive proteases in the
midgut [9]). In addition, the innate immune system of insects [10 °] presents more targeted barriers to a variety of microorganisms and macroparasites. Vector insects are known to possess both cellular and humoral defence capabilities [7,10°,11]. Thus, successful maturation of a parasite within the vector requires circumvention of the immune system, which theoretically might be due to the parasite being insensitive to, evading or suppressing the immune response [12]. Which of these possibilities operate is generally not known, although in some cases of refractoriness it is clear that immune defences are rapidly and specifically activated upon parasite invasion, are localized to the microenvironment of the parasite surface, and may be targeted to specific stages of parasite development [7,13,14]. This review focuses on selected hematophagous members of the order Diptera, which are among the most important vectors of eukaryotic parasites of medical significance and are currently favorite systems in the field of vector biology. Mosquitoes (Diptera; Culicidae) are vectors of Plasmodium spp. (the protozoan parasites causing malaria), as well as Brugia and Wucheretia (metazoan agents of filariasis). T h e blackflies (Diptera; Simuliidae) are vectors of metazoan parasites of the genus Onchocerca, responsible for river blindness disease.
Encapsulation, melanization and phenol oxidase Melanotic encapsulation of invading organisms, associated with the activity of phenoloxidase, is a frequently observed manifestation of the immune response of insects [14-16]. Filarial parasite infection of refractory mosquitoes triggers an encapsulation and melanization response that involves both cellular and humoral components of the vector immune system [14]. Hemocyte involvement is evidenced by the proximity of these cells to the parasite surface and cell lysis associated with the encapsulation reaction. Guo et al. [17 °°] have documented changes in hemocyte numbers and lectin binding binding activity in Armigeres subalbatus mosquitoes infected with Brugia parasites. T h e numbers of hemocytes were reduced at 24 hours post-infection, and this decrease was attributed to the involvement of hemocytes in encapsulation reactions. In Aedes aegypti, however, increased hemocyte numbers were detected as a consequence of parasite infection. T h e apparent discrepancy may be a result of species-specific rates of encapsulation, which peaks at 72-96 hours post-infection in Ae. aegypti, compared to 24 hours in Ar. subalbatus. Hemocyte populations may thus be increasing in Ae. aegypti at 24 hours post-infection, but sequestered in encapsulation reactions at this time in Ar. subalbatus. An additional change in hemocyte properties is associated with Brugia infection ofAr. subalbatus: following infection, an increased percentage of hemocytes bind wheat germ
Immunity to eukaryotic parasites in vector insects Richman and Kafatos
agglutinin (WGA), a phenomenon also observed in Ae. aegypti and Ae. trivitattus [ 17"]. Melanization through the action of phenoloxidase is an important aspect of insect defence against microorganisms and eukaryotic parasites [14,16]. T h e inactive precursor prophenoloxidase is converted to the active form by serine proteases, which are themselves activated in response to various elicitors, including components of bacterial and fungal cell surfaces. This cascade results in the production of melanin and associated toxic metabolic by-products (quinones), which may kill encapsulated parasites [16]. Because of the potential toxicity of the ultimate products, phenoloxidase activation is highly localized. Increased phenoloxidase activity associated with melanotic encapsulation of eukaryotic macroparasites has been detected in Ar. subalbatus and Ae. aegypti [17"']. Such observations, however, are potentially subject to variation depending upon the species of vector and parasite, methods of analysis, and other considerations. Shih and Chen [18] reported lower levels of monophenoloxidase activity in bloodfed Ar. subalbatus infected with B. pahangi compared to uninfected controls, a reduction attributed to the sequestration of the enzyme during melanization of the parasite. Phenoloxidase is also implicated in melanotic encapsulation of Plasmodium in Anopheles gambiae, but in the apparent absence of proximal hemocytes [13,19]. In this case, prophenoloxidase/phenoloxidase synthesized in the fat body, circulating hemocytes, midgut epithelial cells or salivary glands may become localized to the parasite surface as part of the innate immune recognition system ([19,20]; see below). Questions concerning the cellular origin(s) of prophenoloxidase and the regulation of its synthesis in An. gambiae may soon be resolved, as a cDNA encoding this enzyme has been cloned (P Brey, personal communication). Interestingly, blackflies (genus Simulium) do not respond to Onchocerca infection by melanization, although they do possess phenoloxidase and serine proteases and are capable of producing melanin in the context of cuticle formation [21"']. Ham and coworkers [22] speculate that prophenoloxidase/phenoloxidase may serve another immune function in blackflies, perhaps by binding to the parasite surface as a recognition factor or opsonin for hemocytes. This suggestion is based on comparisons of the amount of phenoioxidase that can be detected in the hemolymph of blackflies infected with compatible and non-compatible species of Onchocerca: reduced levels of phenoloxidase are observed in the hemolymph of S. damnosum infected with O. dukei, an incompatible parasite that is normally transmitted by a different vector species [221. Study of the blackfly immune response to Onchocerca has established that melanization is not essential for killing
15
of the parasite. Other mechanisms must therefore be responsible for the observed 'clearance' of microfilaria-stage parasites from resistant blackflies. Lehmann et al. [23] observed a rapid decrease in the number of inoculated microfilaria within 5 hours, suggesting a novel lytic response that occurs in living flies as well as in flies killed by extremes of temperature shortly before the introduction of parasites. Apparently, macroparasite killing is due in this case to the action of soluble factor(s), rather than immune cells. Whether these factors represent homologs of known inducible antimicrobial peptides (see below) remains to be determined. Recognition of non-self
In addition to components of the phenoloxidase cascade, agglutinins or lectins of vector insects represent candidate recognition factors. In Rhodnius prolixus (a non-Dipteran species) lectins exhibiting different carbohydrate specificities interact with various developmental stages of the parasite Trypanosoma cruzi [24]. Lectin-like activities have been implicated in recognition of microfilariae by Ar. subelbatus mosquitoes [25,26]. In this system, agglutinins may mediate the attachment of phenoioxidase to the surface of microfilariae, thus facilitating melanization of the parasite. Observations from the study of blackflies suggest a role for lectins in specific recognition of O. lienalis larvae by the S. ornatum vector. Here, carbohydrate specificities of hemolymph lectins undergo variation in a manner complementary to developmentally regulated changes in the sugar moieties expressed o n the parasite cuticle; this is consistent with a function of lectins as recognition molecules and/or opsonins for hemocytes [21"']. A different recognition mechanism must account for the clearance of 0. linealis microfilaria from S. vittatum, however, as this developmental stage is not believed to express available surface carbohydrate [21"]. Humphreys and Reinherz [27"] have proposed a model, in which hemocyte-mediated recognition of self is the operative principle directing invertebrate immune reactions. In this model, invertebrate pre-immunocytes are positively selected through the expression of a self-recognition receptor that is specific for a polymorphic histocompatibility antigen expressed on the surface of self cells. T h e selected immunocytes would also bear receptors for 'generic ligands' expressed on both self and nonself ceils. Immunocyte activation would be prevented when both receptors are engaged (contact with self cells). Upon contact with nonself, however, engagement of the generic ligand receptor alone would trigger hemocyte activation and initiate subsequent immune reactions. Although this model is principally formulated to account for rapid histocompatibility reactions observed in various invertebrate species, it is theoretically applicable to the recognition of macroparasites in vector insects. Janeway [28,29] has suggested that innate immune responses are activated by multiple or polyspecific receptors detecting constituents of infecting cell surfaces. Although no such
16
Innateimmunity
'pattern recognition' receptors have definitively been shown to function in this way in vector-parasite interactions, the cloning of a scavenger receptor from Drosophila melanogaster (displaying broad ligand specificity) suggests an approach that could well prove fruitful in application to vector species [30°]. In An. gamln'ae a homolog of the CD36 family of multi-ligand receptors has recently been identified (A Crisanti, personal communication), and it is possible that this molecule may function as a 'pattern recognition' receptor to facilitate host-parasite discrimination by hemocytic cells (JA Hoffmann, personal communication). Charge may play a significant role in the response to nonself in insects [31-34,35°]. A correlation has been demonstrated [35°,36°] between refractoriness to Plasmodium and differential responsiveness to negatively-charged Sephadex beads in selected strains of An. gambiae. Refractory mosquitoes melanize negatively-charged beads more avidly than do animals of the susceptible strain. Melanization is affected by gender, age, nutritional and/or reproductive status. T h e weak response of susceptible mosquitoes to negatively-charged beads may indicate that such surfaces more closely resemble self, or are covered with soluble mosquito factors that render them invisible to the immune recognition system [35°]. In this regard, Warburg and Schneider [37] have suggested that Plasmodium oocysts may become surrounded by basement membrane components produced by hemocytes of the infected mosquito. It is not yet clear whether these refractory and susceptible mosquito strains differ in recognition, or in downstream effector functions involved in melanin synthesis. For example, the charged surface could provide a microenvironment for enzyme activity. T h e observed differences between strains might then reflect differential abilities of melanizing enzymes to function within this microenvironment. Nevertheless, the use of charged beads to monitor Plasmodium refractory versus susceptible phenotypes will undoubtedly facilitate analysis of the genes and factors involved in immunity to the parasite (see below).
Genetics of vector susceptibility to parasites Genetic analysis of mosquito susceptibility to parasite infection has a long history [38]. Recent advances in the development of genetic maps based on the molecular markers [39°,40] provide powerful tools to identify genetic determinants influencing the response of the vector to the parasite. Quantitative trait loci (QTLs) influencing mosquito susceptibility to eukaryotic parasites have been localized to genomic intervals delineated by restriction fragment length polymorphism (RFLP) and simple sequence repeat (microsatellite) markers [39°1. Two Q T L s implicated in susceptibility of Ae. aegypti to P gallinaceum have been mapped [6°]. T h e one on chromosome 2 broadly coincides with a Q T L that influences susceptibility to filaria and yellow fever virus; this may represent either a single locus or a tightly-linked
cluster of genes involved in determining susceptibility to multiple disease agents. In An. gambiae, two apparently distinct systems of refractoriness to Plasmodium have been identified through selective breeding of laboratory populations. In one system, general refractoriness to diverse Plasmodiurn species is manifested by extracellular melanotic encapsulation of late stage ookinetes/early oocysts [41]. Susceptibility is determined through the inheritance of at least two loci, one of which is associated with an inversion on the left arm of chromosome 2 [5]. Through another mechanism, ookinetes of P gallinaceum are subject to lysis within midgut cells of refractory, but not susceptible, An. gambiae [42°]. Although this trait may also be influenced by multiple QTLs, a major locus has been roughly mapped to the right arm of chromosome 3 (L Zheng, personal communication).
Inducible antimicrobial factors and the regulation of immune gene expression From the above discussion, it is evident that the biochemical nature of the factors involved in immunity to parasites is largely unknown. It is tempting to speculate that, in addition to the encapsulation and melanization system, humoral antimicrobial peptides may play a role [11]. Molecular tools that can be used to examine this possibility critically are now becoming available. Major advances have been made recently in the identification and characterization of inducible insect antimicrobial peptides, and in the analysis of regulated expression of the genes encoding these proteins [10°,111. Despite the detection of antibacterial activities in vectors [7], specific factors (aside from An. gambiae lysozyme [J-Y Lee, personal communication]) have been described only recently. Defensin proteins and cDNA clones have now been isolated from Ae. aegypti [43,44] and An. gambiae (A Richman, unpublished data) following bacterial infection. T h e mature proteins are very similar in sequence to previously characterized defensins of other Diptera. Evidence currently suggests that antimicrobial factors of insects can influence the viability of some eukaryotic parasites. Exogenous administration of cecropin was reported to inhibit sporogonic development of Plasmodium in An. gambiae [45]. Synthetic cecropin inhibited the motility of Brugia microfilariae in vitro, and reduced larval numbers in Aedes aegypti when co-injected with Brugia parasites [46]. A synthetic peptide based on cecropin, Shiva-3, was active against Plasmodium berghei both in vitro and in An. albimanus [47]. Administration of synthetic defensin has been shown to affect oocyst-stage P gallinaceum in Ae. aegypti, and to kill sporozoite (haemocoel) stage parasites in vitro (M Shahabuddin, LH Miller, JA Hoffmann, C Hetru, personal communication). A number of insect genes encoding antibacterial peptides bear upstream DNA sequence elements resembling binding sites for transcription factors, including the rel family member NF-KB, associated with the acute-phase response
Immunity to eukaryotic parasites in vector insects Richman and Kafatos
of mammals [48]. A tel-domain is also found in several transcription factors of insects that may be implicated in the control of antibacterial and antifungal gene expression [10•,48]. An An. gamln'ae tel-containing protein has been identified (C Barillas-Mury, FC Kafatos, unpublished data) that can activate transcription from antibacterial gene promoters of D. melanogaster. Sequence-specific DNA binding activities that recognize the NF-KB-Iike binding site have been identified in bacterially challenged An. gambiae and are not detected in unchallenged control animals. Sequence elements matching the insect NF-KB consensus binding site have been identified in the putative upstream regulatory region of the An. gambiae defensin gene (A Richman, unpublished data). A critical question concerns the degree of specificity of the insect immune response. Recent observations in D. melanogaster suggest that genes encoding antibacterial and antifungal proteins may be regulated independently of each other, in response to fungal or bacterial infection challenge (JA Hoffmann, personal communication). Antiparasitic immune activities have not been described in vector insects. Nevertheless the cloning of defensin genes from Ae. aegypti and An. gambiae, together with the identification of a putative regulatory transcription factor from An. gambiae, offer powerful tools to investigate the possible involVement of known immune mechanisms of the antibacterial pathway in the vector response to eukaryotic parasites.
17
T h e continuing development of high resolution genetic maps based upon molecular markers represents a tremendous advance towards identification of genes influencing vector immunity to metazoan and protozoan parasites. T h e positional cloning of such genes will require the construction of high resolution physical maps for the Ae. aegypti and An. gambiae genomes. A low resolution physical map, based on microdissected chromosomal regions, is already available for An. gambiae [49]; construction of maps based on large-insert DNA clones such as YACs and BACs has begun for both Ae. aegypti and An. gambiae (D Knudson, F Collins, L Zheng, personal communication). Investigations into vector immunity to parasites will be greatly facilitated by integration of the rapidly accumulating molecular, cellular, and genetic insights into the regulatory and effector mechanisms of the innate immune response in D. melanogaster and other model laboratory insects. T h e recent identification of defensin proteins, cloning of defensin genes, and the identification of immune-activated nuclear DNA binding activities in mosquitoes suggests that this integration process is under way.
Acknowledgements We are grateful to S Paskewitz for comments on the manuscript. Work in the authors' laboratory supported by grants from the John D and Catherine T MacArthur Foundation, TDR/WHO, and the Alexander von Humboldt Foundation.
References and recommended reading Conclusions Vector insect responses to protozoan or metazoan parasite infection, particularly the encapsulation and melanization observed in mosquitoes and the immune-related activation of phenoloxidase, resemble well-studied humoral and cellular immune reactions of model insect systems (although hemocyte involvement in encapsulation appears to be more limited in mosquitoes than in some other insects [14]). T h e precise molecular basis of parasite recognition as nonself remains obscure. With the exception of the recently cloned CD36 homolog, candidate 'pattern recognition' receptors have not been identified in vector insects to date. T h e lack of melanization characteristic of the blackfly response to Onchocerca, and the decreased levels of phenoloxidase activity in infected animals indicate the existence of diverse immune mechanisms in hematophagous Diptera, which may involve dual immune functions for some molecules (e.g. phenoloxidase functioning as both a recognition factor and effector pathway molecule in parasite killing). T h e clearance of microfilariae from Simulium, furthermore, suggests that novel cell-free mechanisms of parasite recognition and killing may remain to be elucidated.
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