Anopheles–malaria interactions

Colloque organisé par Fotis C. Kafatos, Michael Ashburner et Louis Miller du 12 au 17 septembre 1995

Participants

Michael Ashburner, organisateur (University of Cambridge, UK), Carolina Barillas-Mury (European Molecular Biology Laboratory (EMBL), Heidelberg, Germany), Yves Carton (Centre National de Recherche Scientifique (CNRS), Gif-sur-Yvette, France), Frank Collins (Centers for Disease Control, Chamblee, USA), Andrea Crisanti (Università di Roma – La Sapienza, Italy), Ronald Davis (Stanford University, USA), Karen Day Karen (University of Oxford, UK), William Dietrich (Whitehead Institute/Massachusetts, Institute of Technology (MIT), Cambridge, USA), Jules Hoffmann (Institut de biologie moléculaire et cellulaire, Strasbourg, France), Fotis C. Kafatos, organisateur (EMBL, Germany), Dominic Kwiatkowski (John Radcliffe Hospital, Oxford, UK), Christos Louis (Research Center of Crete, Heraklion, Greece), Louis Miller, organisateur (National Institute of Health (NIH), Bethesda, USA), Geneviève Milon (Institut Pasteur, Paris, France), Victor Nussenzweig (NYU Medical Center, New York, USA), Linda Partridge (University College of London, UK), Susan Paskewitz (University of Wisconsin, USA), Jean-Marc Reichhart (Université Louis-Pasteur, CNRS, Strasbourg, France), Adam Richman (EMBL, Germany), Robert Sinden (Imperial College of Science and Technology, London, UK), Isabelle Tardieux (Institut Pasteur, Paris, France), Kenneth Vernick (NIH, Bethesda, IJSA), Liangbiao Zheng (EMBL, Germany)

Review

Following is a summary of the meeting and also the text of Robert E. Sinden’s contribution.

Introduction

The unique environment of the Fondation des Treilles was the setting for an interdisciplinary meeting focused upon interactions between Anopheline mosquitoes and Plasmodium parasites. Malaria poses one of the most serious threats to human health in much of the tropical and subtropical parts of the world today. In the absence of an effective vaccine against the Plasmodium parasite and with the emergence of drug-resistant parasite strains and insecticide-resistant mosquito vectors, it is realistic to predict a continuing worsening of this problem. Human malaria transmission occurs via vector mosquitoes of the genus Anopheles, and effective malaria control efforts have historically focused upon mosquito control. Novel strategies for manipulating mosquito populations have been proposed, using transgenic methods to alter the insect’s susceptibility to Plasmodium infection. The design and implementation of such an approach will require increased understanding of the complex interactions between insect and parasite during the mosquito stages of the Plasmodium life cycle. Heritable mechanisms of refractoriness of the vector that can interrupt several steps of parasite development have been documented, but only recently have modern cell biological, molecular, and genetic approaches been brought to bear on this important topic.

The conference began with some comments by Fotis C. Kafatos. He dedicated the meeting to the memory of Annette Gruner Schlumberger, the founder and inspiration of Les Treilles, and recalled that she had specifically asked him to organise this meeting during their last encounter at Les Treilles. He also summarized the goals of the meeting and underlined the unique opportunity for informal, intense, and interdisciplinary discussions.

In this beautiful, relaxed setting highly selected people who normally do not all meet together and who represent different scientific generations can learn from each other, and without time pressure can lay the foundations for closer collaborations. Lively discussion was indeed stimulated by the presentations, from experts in the fields of parasitology, malariology, vector biology, insect molecular biology, genomics, insect immunity, child health, evolutionary biology, and population genetics.

The conference has produced two reports, which are here printed back-to-back. Robert E. Sinden’s oral presentation was an overview of current knowledge and questions concerning the interactions that occur between vector and parasite during the development of Plasmodium in Anopheles. This overview, presented here as a written report accompanied by key references from the literature, was a valuable starting point for our discussions and provides a helpful introduction to the field. The other written report was a communal effort, begun as draft summaries prepared by the other participants in the meeting and edited and integrated by Adam Richman and Fotis C. Kafatos. It presents the diverse issues considered by the meeting, organized into thematic sessions, in the order in which they took place. Each of the seven session write-ups begins with an overall abstract followed by a summary of individual presentations.

Session I: The Plasmodium Parasite in the Vertebrate Host

The Plasmodium parasite is adapted for growth and development within diverse physiological milieux, most strikingly the environments of the vertebrate host and insect vector. The dearth of molecular insights into mosquito interactions, and our need for such insights should be clear from the accompanying summary of the parasite life history in the mosquito (Robert E. Sinden). This is contrasted with more extensive knowledge of the vertebrate host. An important question in malaria pathogenesis concerns the initial invasion of hepatocytes by the sporozoite stage parasites that are introduced during mosquito blood feeding. This specific cell invasion process has been analyzed in some detail at the molecular level, a degree of understanding in sharp contrast to a profound lack of knowledge concerning the earlier populating of mosquito salivary gland cells by hemocoel stage sporozoites. Dominic Kwiatkowski presented findings on the regulation of TNF synthesis during Plasmodium infection of humans as a consequence of ‘malaria toxins’ released during parasite growth. TNF signalling has important consequences for malaria pathogenesis.

Sporozoite receptors in liver (Victor Nussenzweig)

Malaria sporozoites invade hepatocytes in vivo with high efficiency. The parasite ligand for hepatocyte recognition is contained in the region II-plus of the circumsporozoite protein, the major surface protein of sporozoites. The recognition of region II-plus is dependent on the presence of positively charged amino acids, which are bound by negatively charged glycosaminoglycan chains of heparin sulfate proteoglycans on the surface of hepatocytes. Dr. Photini Sinnis has obtained suggestive evidence that the same highly sulfated glycosaminoglycan chains on hepatocyte microviili recognize region II-plus, and remnants of chylomicrons and very low-density lipoprotein.

The regulation of malaria parasite density within the host (Dominic Kwiatkowski)

Malaria lever is an innate host response that occurs when billions of parasites burst out of erythrocytes to release their progeny. In the process they shed toxins which stimulate monocytes and macrophages to produce tumor necrosis factor (TNF), a cytokine that causes fever through its action on the thermoregulatory center of the hypothalamus. TNF induces a variety of immunological effector mechanisms, including the elevation of temperature, that act to suppress parasite growth. Since this response is density dependent, it contributes to the regulation of parasite population within the host.

An excessive TNF response can have consequences fatal for the host, such as cerebral malaria. The amount of TNF produced during an infection depends on both parasite and host: parasite strains vary widely in their ability to induce TNF, while humans differ in their TNF-responsiveness to malarial toxins. At least part of this host variation is genetically determined, and polymorphism of the human TNF promoter region is associated with susceptibility to severe malaria. Such genetic diversity may have arisen because the optimal level of TNF response differ for each of the many life-threatening infections to which human populations are exposed.

Session II: Population biology and Epidemiology of Plasmodium Transmission

This session focused on aspects of insect life histories of relevance to potential vector control efforts, Plasmodium population/transmission dynamics, and phylogenetic relationships among members of the Anopheles gambiae species complex. Life history studies in Drosophila melanogaster can highlight ways in which genetic manipulation as well as insect rearing conditions may influence the success of vector control strategies involving, for example, the release of sterile males. Studies of the genetic structure of Plasmodium parasite populations indicate that levels of inbreeding vary according to transmission rates. Such variability may have important consequences for the spread of multi drug resistance. It was pointed out in addition that population studies of parasite strains are needed for understanding transmission dynamics with reference to implementation of vaccination programs against malaria. The last talk of this session described efforts to characterize the phylogenetic relationships within the Anopheles gambiae complex with special emphasis on An. gambiae and An. arabiensis. Will genes introduced into one species move into another? The evidence from mitochondrial genes suggests such gene flow but the time frame of the flow is unknown. It was emphasized that An. funestus is also important for transmission in many areas harboring the An. gambiae complex. Therefore, any strategy for gene introduction to field populations must include these non An. gambiae mosquitoes in terms of impact on vector populations.

Insect life histories and their relevance to vector control (Linda Partridge)

A life history is the combination of age-specific survival probabilities and fertility characteristic of an organism. Two main constraints on life history evolution are well documented: bet ween survival to adulthood and subsequent performance, and between reproductive rate and subsequent survival and fecundity (the cost of reproduction). In Drosophila, larger adults survive longer and have higher fertility. However, increased adult size correlates with an increase in the duration of larval development and a correlated drop in larval survival. In Drosophila females mating is an important cause of mortality mediated by protein components of the male seminal fluid.

Various aspects of life history evolution are of relevance to control of fruitfly pests and, perhaps, to vector control. Body size is a highly polygenic trait, the genetic basis of which, even in Drosophila, is poorly understood. Powerful techniques now exist for investigating these traits in Drosophila, and could be used to identify genes involved in control of homologous traits of interest in mosquitoes. A drop in mating success of males in sterile release programs with fruitflies has frequently been reported. Part of the reason is probably that factory strains are pushed for rapid development, with a consequent drop in adult size. This problem can be avoided with appropriate culture of the factory strain. The cost of mating to female Drosophila is probably an evolutionary side-effect of seminal fluid molecules that act in the male interest. It might be possible to engineer the relevant compounds to render the female permanently unreceptive or dead, and such males would be more effective in sterile release.

Finally, work on chromosomes carrying inversions in Drosophila suggests that naturally occurring inversions could be useful for driving single or linked groups of genes into natural mosquito populations.

Transmission of Plasmodium falciparum in Papua New Guinea (Karen Day)

A) The genetic structure of malaria parasite populations of Papua New Guinea

Description of the genetic structure of malaria parasite populations is central to an understanding of the spread of multi-locus drug and vaccine resistance. Plasmodium falciparum mating patterns from Madang, Papua New Guinea, where intense transmission of malaria occurred, were described. A high level of inbreeding occurred, in the absence of detectable linkage disequilibrium. This contrasts with another study from Tanzania where malaria transmission is ten-fold higher than Papua New Guinea. These data indicate that the genetic structure of malaria parasite populations is neither clonal nor panmitic but will vary according to the transmission characteristics of the region.

Geographical variation in the extent of inbreeding may have consequences for the success of potential global malaria control strategies. Mathematical models have demonstrated an effect of recombination and degree of outbreeding on the development of multi-drug resistance: inbreeding may accelerate the buildup of drug resistance. One would expect variability in the evolution of multi-locus phenotypes such as virulence, drug, and polyvalent vaccine resistance to occur in places of differing malaria transmission characteristics purely as a result of parasite mating patterns. The relatively slow spread of chloroquine resistance in Tanzania compared to Papua New Guinea lends weight to this prediction, as high degrees of inbreeding may accelerate the spread of multi-locus drug resistance. Furthermore, inbreeding rates may influence the rate of development of any strain-specific component of immunity.

B) Transmissibility of P. falciparum

The transmissibility of P. falciparum can be defined mathematically as the basic reproductive rate or number R0, (i.e. the number of secondary cases to arise during the infections period of a primary case in the absence of constraints on parasite population growth). The question “What is the R0 of malaria?” is particularly relevant today as we are thinking of new methods of malaria control such as vaccines. R0 is important in the development of malaria vaccination programmes since it can be related to the proportion of the population that needs to be immunised with a vaccine to block transmission. In order to estimate the potential of a transmission blocking vaccine for malaria to achieve eradication at levels of vaccine coverage feasible in endemic areas we need to know the R0 of malaria.

Gupta and Day propose a theoretical framework to calculate R0 which takes on board the antigenic diversity of Plasmodium falciparum. To calculate R0 we need to define a strain. Assuming that an independently transmitted antigen type or strain can be defined by the pattern of expression of variant surface antigens (VSAs) we measured age-specific immune responses to calculate values for individual strains. R0 estimates for malaria were calculated to be at least one to two orders of magnitude lower than calculated using a previously defined method based on vectorial capacity data. This is extremely good news for malaria vaccination if the definition of strains holds true. Population genetic studies of VSAs and a detailed understanding of this system is essential to defining the transmission system.

Population genetics and evolution of the An. gambiae complex (Frank Collins)

This presentation reported Nora Besansky’s work on the use of sequences from the mitochondrial DNA and an intron of the X-linked white gene to examine the phylogenetic relationships among 5 of the 6 members of the An. gambiae complex. The two sequences lead to different pictures of the evolutionary relationships among these species: the white intron sequence supports placement of An. gambiae and An. arabiensis into two different clades while the mtDNA sequence analysis clusters An. gambiae and An. arabiensis in the same clade. Furthermore, the mtDNA sequences of both field collected and laboratory specimens of An. gambiae md An. arabiensis do not cluster into species specific clades but are intermixed, in a manner that suggests mtDNA introgression between these two important vector species.

Session III: Plasmodium and the Anopheles Midgut

This session focused upon molecular and biochemical aspects of Plasmodium invasion of the mosquito midgut and midgut gene expression. Louis Miller presented the work of Mohammed Shahabuddin, who has demonstrated a number of very specific developmental mosquito parasite interactions that occur within the first 24 hours of sporogonic development. Of particular importance is the activation of a secreted parasite prochitinase by mosquito midgut trypsins, an event necessary for penetration of the peritrophic membrane barrier. Subsequently, the parasite appears to bind specifically to a subset of midgut epithelial cells described in earlier literature as endocrine cells. The suggestion was raised that invasion of these cells by P. falciparum may involve interaction of the parasite surface proteins Pfs25 and Pfs28 with specific epithelial cell proteins, possibly mediated by an interaction of parasite protein EGF domains and mosquito protein EGF receptors, since antibodies to these ookinete proteins inhibit ookinete invasion of midgut epithelial cells.

Christos Louis showed that even before transformation is achieved, mosquito transcriptional regulatory elements can be analyzed by interspecific transgenic studies. He reported that the promoter regions of three An. gambiae trypsin genes are capable of regulating midgut expression of reporter constructs in transformed Drosophila melanogaster, with the same tissue and developmental stage specificity seen for the endogenous gene in An. gambiae.

Biology of malaria parasite development in mosquito gut (Louis Miller)

The peritrophic membrane is a potential barrier to malaria parasite access to the mosquito midgut. If this membrane is compromised by introduced fungal chitinase, invasion of midgut cells and subsequent parasite development occur normally. In normal infection the parasite produces a chitinase that breaches the peritrophic membrane. The parasite proenzyme requires activation by mosquito trypsins in the midgut lumen. Inhibition of these trypsins inhibits parasite invasion.

A second question on midgut invasion relates to the cells invaded. There are two types of cells in the mosquito midgut: epithelial cells and endocrine cells. In preliminary studies, the parasite was observed to invade the endocrine cells, not the epithelium. Further analysis is required to follow the cell biology of invasion, to determine if the parasite can only invade endocrine cells, and to identify relevant cell surface receptors.

Expression of An. gambiae trypsin genes in transgenic Drosophila (Christos Louis)

Control of malaria transmission may be facilitated by development of strategies to block midgut penetration of An. Gambiae by Plasmodium parasites. Such strategies will require greater understanding of mosquito gut physiology, interactions between parasite and mosquito tissues, as well as the generation of suitable blocking reagents. Drosophila melanogaster was transformed with constructs that permit analysis of the promoter regions of two gut-specific late trypsin genes of An. gambiae. Deletion analysis has identified sequences sufficient to confer tissue specific gene expression in this heterologous species. Although the expression level of the reporter constructs is modulated by the feeding regimen of the transformed flies to a level of -25%, attempts to induce the Anopheles promoters using blood feeding, ecdysone, and juvenile hormone have not proven successful. Nevertheless, transformation and (issue specific gene expression in Drosophila will facilitate further studies of blood meal digestion, and may be useful in the design of anti-parasite constructs.

Session IV: Mosquito Refractoriness to Plasmodium

Transmission of malaria depends upon the ability of Anopheline mosquitoes to support growth and development of Plasmodium parasites. Several examples of incompatible mosquito-Plasmodium combinations, however, clearly indicate that some species and strains of Anopheles can resist Plasmodium invasion through active processes, in some cases reminiscent of classically observed insect immune responses (addressed in a later session). Here, Ken Vernick described a selected strain of An. gambiae in which parasites undergo lysis within cells of the midgut. Susan Paskewitz described another form of parasite killing in a different selected strain of An. gambiae. In this case, the parasite-refractory mosquito strain encapsulates all or most ookinetes as they complete passage through the midgut epithelial cells. Liangbiao Zheng described the development of a relatively high density microsatellite map for An. gambiae, now being used to define the genetic loci that contribute to the forms of Plasmodium refractoriness described by Ken Vernick and Susan Paskewitz.

Parasite killing in midgut epithelium (Ken Vernick)

A strain of An. gambiae has been developed in which ookinetes of the avian parasite, P. gallinaceum are lysed intracellularly shortly after penetration of the midgut epithelial cells. In an An. gambiae strain selected for parasite susceptibility, no ookinete lysis is observed and parasites develop normally. Preliminary genetic studies suggest that parasite lysis is regulated by one genetic locus and the allele conferring the lytic phenotype is dominant. Genetic mapping of this locus relative to microsatellite markers (in collaboration with Liangbiao Zheng, see below) suggests that the gene responsible for parasite lysis is associated with the right arm of chromosome 3.

Encapsulation of malaria ookinetes in midguts of An. gambiae (Susan Paskewitz)

Two strains of An. gambiae were genetically selected for refractoriness and susceptibility to many species of malaria. Refractory mosquitoes melanotically encapsulate parasite ookinetes. This immune response is also mimicked when the foreign agent is a negatively charged Sephadex CM-25 bead, since refractory mosquitoes melanize beads but susceptible mosquitoes do not. Genetic studies showed that the basis for the melanization of P. cynomolgi B and CM-25 beads is likely due to the action of the same major gene on chromosome 2. Injection with a variety of ion exchange beads of differing charged moieties showed that general features of negativity are more important than a specific biochemical moiety. The appearance of the differential response to negatively charged beads with a non carbohydrate matrix also suggested that lectins are not critical in recognition of these surfaces. Transfer of beads between strains showed that beads become protected from melanization when transferred to refractory mosquitoes after a 6-12 hr. incubation in the susceptible strain. However, melanization of untreated beads occurs within 2 6 hrs. in the refractory strain, suggesting that protection is separate from the primary biochemical difference of encapsulation that distinguishes the two strains. In addition to being of potential value in mapping at least one of the genes involved in encapsulation of malaria parasites, this bead assay may allow direct investigation of the mosquito proteins that actually interact with the bead surface.

Genetic mapping of An. gambiae refractoriness (Liangbiao Zheng)

An integrated genetic map of the African malaria vector, An. gambiae, has been constructed based on simple sequence repeats (or microsatellite) polymorphisms. The map consists of 46, 61, and 31 microsatellite markers on the X (spanning 48.9 cM), the second (spanning 72.4 cM), and third chromosomes (spanning 93.7 cM), respectively. The map also integrates five morphological markers: pink eye, white eye on the X; collarless, lunate, and the biochemical dieldrin resistance (Dl) trait on the second; and red eye on the third chromosome. The cytogenetic locations of 46 of these markers have been determined, thus making this an integrated cytogenetic, genetic, and molecular map. The map was used to search for the gene(s) controlling the lytic trait of An. gambiae against the ookinetes of P. gallinaceum. Genotyping was performed on 50 offspring from five backcross families, obtained by crossing hybrid F1 to the susceptible strain of An. gambiae. Preliminary results suggested that a dominant locus on the right arm of the third chromosome determines the lytic refractoriness against P. gallinaceum.

Session V: Input from Studies of Other Organisms: Genome Analysis and Mapping

Michael Ashburner addressed the question of whether the enormous amount of molecular and genetic data available for Drosophila could help further studies of Anopheles mosquitoes. Or in brief, what is the value of Drosophila as a model for Anopheles? Other well-established and rapidly advancing systems may also offer useful tools and insights for the development of mosquito molecular biology. William Dietrich discussed the use of molecular mapping techniques in analysis of genes conferring disease-related phenotypes in the laboratory mouse. Ronald Davis presented innovative instrumentation developed for rapid and efficient analysis of whole genomes.

Drosophila – a model system for mosquitoes (Michael Ashburner)

Despite being separated by a large evolutionary distance (>150 myr) Drosophila provides the best model for the genetic and molecular analysis of mosquitoes. Many of the techniques necessary for mosquito research have already been developed in the Drosophila model; polytene chromosome analysis and transformation to name but two. There is no reason why “technology transfer” from Drosophila to mosquitoes will not continue in the future.

Drosophila genes provide a rich source for studies on mosquitoes-witness the trypsin encoding genes. Comparative studies of sequence and gene organization will be fruitful. The developing study of the Drosophila immune system will have a major impact on mosquito research. Drosophila is a good model for the properties and behavior of polytene chromosome inversions in natural populations, as well as being an essential model for the development of (population) replacement strategies, whether based on chromosomal elements or symbiotic bacteria. Finally, at the level of logistic support: Drosophila databases, the development of community resources for Drosophila biology and the discovery of ways to cryopreserve Dipteran eggs will be very important models for the mosquito community.

Application of mouse genomic maps to the study of infectious disease susceptibility (William Dietrich)

Genetics can be used to study variation in disease-related phenotypes amongst inbred mouse strains. In the last several years a dense genetic map of the mouse genome has been developed, which currently consists of 6348 highly polymorphic simple sequence repeat (or microsatellite) markers. This map, with average marker spacing of about 0.25 cM, makes it possible to very accurately map genes segregating in a cross. A project is underway to use the markers in this map as a scaffold to build a marker-content based physical map of the mouse genome.

The genetic map has been used to study host genes responsible for differences in susceptibility to infectious disease, using susceptibility to Legionella pneumophila infection as a model system. A critical feature is that Legionella pathogenesis relies on the bacterium’s ability to replicate intracellularly in macrophages. While most inbred mouse strain macrophages are non-permissive for Legionella replication, A/J macrophages are permissive. This phenotypic difference segregates as a single gene trait in crosses between permissive and non-permissive strains. The gene responsible (Lgn1) has been mapped to chromosome 13 and attempts to clone this gene based on its position are underway.

New instrumentation in genomics (Ronald W. Davis)

Whole genome DNA sequencing provides a large amount of information. It also provides new means to conduct molecular biological analysis on the whole genome such as gene function and gene expression. However, whole genome sequencing and other analysis procedures are currently very expensive, which can preclude their application to many organisms. New instrumentation and automation has been developed to decrease the cost and increase the analysis rate. An automated DNA sequencing system is being developed that consists of a series of integrated modules: instruments that automatically produce random cloned libraries, pick clones into microtiter plates, produce DNA sequencing templates, conduct sequencing reactions, and determine the sequence by gel electrophoresis. When finished this system should decrease the cost by 100 fold. New techniques are being developed to allow parallel analysis of yeast phenotype for all gene disruptions and gene expression levels.

Session VI: Parasite and Host Cell Biology

This session addressed cellular mechanisms of both host and parasite, influencing successful interactions (in terms of parasite reproduction). Genevieve Milon addressed aspects of vertebral phagocytic cell functions in the context of parasite-host interactions, and suggested possible directions for study of mosquito hemocytes. Isabelle Tardieux presented observations on actin-associated proteins of Plasmodium. Such studies can have important implications for understanding target cell invasion by parasites. Andrea Crisanti discussed developmentally acquired infectivity of P. falciparum for the vertebrate host, and observations suggesting that the TRAP surface molecule of the sporozoites may play an important role in invasion of hepatocytes.

From mononuclear phagocytes of vertebrates to invertebrate hemocytes in the context of the biology of parasitism (Genevieve Milon)

For a “successful life cycle”, parasites depend upon other organisms (e.g. vertebrate hosts, invertebrate vectors) in which they must find a niche that they can occupy for the time required for optimal reproduction. In such a context, parasite physiology must match that of their carriers. They must be programmed (generally developmentally programmed) to co-opt the components of their carriers, components whose synthesis can be either constitutive or regulated indirectly by the parasites or parasite molecules. When focusing on mononuclear phagocytes in such an interaction it is important to dissect the “evolutionary rationale” behind these processes. Since Metchnikoff, macrophages are recognized through a function, namely phagocytosis, starting with binding of the “object” to be internalized to the final outcome within the cell (killing and destruction, or survival and even growth for those microorganisms which belong to the category of intracellular infectious/pathogenic organisms).

Important characteristics of the mononuclear phagocyte system (a lineage of hematopoietic origin) are its distribution in every tissue, its heterogeneity, its versatility. In steady state conditions, this system is now tractable to phenotypic and functional analysis in situ, at least in the laboratory mouse. Relevant screening with monoclonal antibodies has led to recognition of genes exclusively expressed within mononuclear phagocytes (e.g. the Nramp gene, Naturel resistance associated membrane protein, more often identified under the abbreviation of Bcg/Lsh/Ity locus). With such tools in hand, it is possible to define more and more precisely the different characteristics (generally a unique combination of so-called “markers”) of a given mononuclear phagocyte population within a given tissue. A point was made bearing on the changes in these properties with location of the mononuclear phagocytes. Are invertebrate hemocytes able to exert the functions of vertebrate mononuclear phagocytes? This theoretical question was the starting point to briefly address the main characteristics of pattern recognition molecules like CD14, scavenger receptors, mannose receptors, as well as other carbohydrate recognition molecules belonging to the C-type lectins. Homologs of these molecules might be components of the vector that are subverted or exploited for a successful parasite life cycle.

Actin dynamics in Plasmodium merozoites: an activity that inhibits actin polymerization by capping filaments (Isabelle Tardieux)

In non-muscle cells, actin filament formation is a highly dynamic and polarized process that occurs in response to a wide variety of extracellular and intracellular stimuli. Apicomplexan parasites such as Plasmodium actively invade non phagocytic target cells in a multi-step sequential process. Penetration of the parasite into target cells has been shown to require polymerization of parasite actin, based on pharmacological studies. Attempts are underway to identify actin-associated proteins from Plasmodium knowlesi which regulate actin dynamics within the parasite and thus might play a key role during invasion.

Actin affinity chromatography made it possible to isolate and identify from P. knowlesi merozoites the heat shock protein 70 kDa as it cosediments with exogenous F-actin in the presence of a doublet of 31-35 kDa. The complex inhibits the polymerization of pyrene-labeled actin in vitro by capping barbed end filaments. A complex of proteins formed by the heat shock protein 70 kDa and a heterodimer 32-34 cap has been shown to control actin filament elongation in Dictyostelium discoideum. The genes encoding the heterodimer are in the process of being cloned, and it will soon be known whether the members belong to a conserved family of capping actin binding proteins.

Malaria sporozoite development in Anopheles mosquitoes (Andrea Crisanti)

Mosquito stage Plasmodium sporozoites isolated from oocysts, hemocoel, and salivary glands exhibit differences in several biological parameters, including infectivity for vertebrate hosts and expression of cell surface proteins. In P. falciparum, expression of the TRAP (thrombospondin related adhesive protein) and the CS (circumsporozoite) proteins may be correlated with hepatocyte invasion and thus with vertebrate infectivity. TRAP is expressed in all salivary gland sporozoites, very few hemocoel sporozoites, and no oocyst stage parasites. CS protein is found in all stages. TRAP expression therefore correlates with the developmental acquisition of vertebrate infectivity (salivary gland stage). Experiments furthermore indicate that recombinant TRAP can bind to sulfated glycoconjugates on the basolateral cell surface of hepatocytes.

Session VII: Insect Immunity

Insects have long been known to possess immunity mechanisms, rendering them particularly resistant to microbial infections. An important question in regard to mosquito-Plasmodium interactions is the role of innate immune mechanisms in rendering the host insect susceptible or resistant to the protozoan parasite. Jules Hoffmann and Jean-Marc Reichhardt presented an overview of insect immune mechanisms elucidated thus far as responses to bacterial and/or fungal infection challenge. Louis Miller presented preliminary findings from his laboratory showing an effect of exogenously administered insect defensin on P. gallinaceum in Ae. aegypti. Adam Richman and Carolina Barillas-Mury discussed ongoing efforts to obtain molecular markers to study the An. gambiae immune response, ultimately focusing upon the reaction during Plasmodium infection. Studies in D. melanogaster have provided multiple important insights into the regulation of insect immune responses. In the final presentation, Yves Carton discussed the identification and characterization of Drosophila strains exhibiting resistance and susceptibility to parasitoid infestation.

Molecular and cellular aspects of the insect host defense (Jules Hoffmann and Jean-Marc -Reichhart)

Insects are particularly resistant to microbial infections. The host defense of insects is an innate, non-adaptive reaction. Its hallmark is the rapid and transient synthesis of a battery of potent antimicrobial peptides, which exhibit a broad spectrum of activity against bacteria and/or fungi. The structures of approx. 100 peptides have now been elucidated: they are either cyclic peptides, containing one or several disulfide bridges, or linear peptides, such as the cecropins. Interestingly, several induced antimicrobial insect peptides show sequence homologies with mammalian (cecropins), frog skin (thanatin), or plant (drosomycin) defense peptides. The molecular analysis of the immune-induced expression of the genes encoding these antimicrobial peptides in insects has revealed the existence of several cis-regulatory elements within their promoters, which share sequence similarities with regulatory motifs in the promoters of acute phase response genes in the mammalian innate host defense. This is in particular the case for NF-KB response elements, for NF-IL6 response elements and for Interferon Consensus Regulatory Elements. The use of transgenic Drosophila lines with various mutated reporter constructs has established that these motifs actually play a pivotal role in the induction of transcription of antimicrobial genes. Several Drosophila mutants point to the existence of at least two distinct pathways, for induction of the antifungal peptide and the antibacterial peptide genes, respectively. The pathway leading to the immune induced activation of the antifungal genes clearly relies on the same intracellular signalling cascade as the regulation of dorso-ventral patterning in early embryonic development. For the induction of the antibacterial genes, a different pathway is used, which is blocked in a new recessive mutant, immune deficiency (imd). It is surmised that the cloning of the imd gene will shed light on this pathway. Proteins of the rel-family play a role in transactivating the immune-inducible genes, although their exact role awaits further clarification. In some cases, transactivation of the antimicrobial genes can clearly occur in the absence of the hitherto known Drosophila Rel proteins. The receptors for non-self which alert the insect of injury and invasion by microorganisms have long remained elusive. Several candidate receptors have now been cloned (Drosophila scavenger receptor; the CD36 homologue croquemort) and new data have established that the transmembrane receptor Toll plays a role in the induction of the antifungal response.

The effect of immune peptides on malaria parasite development in the mosquito (Louis Miller)

A synthetic defensin based on that identified in the dragonfly Aeschna cyanea was tested for anti-Plasmodium activity in vivo (following injection into Ae. aegypty and in vitro against sporozoites. Defensin was found to kill oocysts in vivo at 5-days post infection, but was not active against zygote, ookinete, or 3-day oocyst stages. Sporozoites were killed in in vitro assays. However, a number of other insect antibacterial proteins were tested (metchnikovin, thanatin, and drosocin) and shown to have no effect upon P. gallinaceum in these assays.

The immune response in An. gambiae (Adam Richman)

In a collaboration between the laboratories of Jules Hoffmann and Fotis C. Kafatos, infection-inducible antibacterial factors were identified biochemically in larvae of An. gambiae. At least 10 induced activities could be separated by HPLC chromatography. In addition, a number of protein species were increased as a consequence of infection challenge, some of which correspond to antibacterial activity. One positive fraction was found to contain an insect defensin. Analysis of large scale larval and adult stage immunizations is in progress.

A cDNA clone representing An. gambiae preprodefensin was isolated using PCR with degenerate oligonucleotide primers based upon conserved amino acid sequences of dipteran defensins. The mature defensin peptide of 40 amino acids is most similar to the defensin isoforms of Aedes aegypti. Defensin expression is inducible at both the RNA and protein levels.

The mRNA differential display technique was used to identify genes expressed as a consequence of immune challenge in larvae. Pilot experiments produced one full-length cDNA, which bears similarity to the C-type lectin domain of L-selectin. Differences outside this domain indicate that this clone does not, however, represent a mosquito homologue of L selectin. The gene is expressed in early embryos and first instar larvae at very low levels. Expression increases significantly in late stage larvae and pupae. This expression pattern is independent of immune challenge. No expression has been detected in adults. It is at present unclear whether expression is linked both to immune challenge and development, or if the gene’s detection by differential display is an artefact of developmentally regulated expression in larvae.

Immune response in the mosquito An. Gambiae: the first rel-family member is identified (Carolina Barillas-Mury)

Several developmental stages in the life cycle of the malaria parasite take place within the mosquito vector. For malaria transmission to take place, the parasite must develop without eliciting an effective immune response. Insects, including mosquitoes, have the ability to induce the synthesis of antibiotic peptides in response to infection (humoral immune response), a process analogous to the acute phase response in mammals. Transcription factors regulating this induction have been identified in mammals and insects, which possess a conserved region, the rel-domain. A gene corresponding to a new member of this group (rel-family) has been cloned recently from the mosquito An. Gambiae and named Gambif1 (gambiae immune factor 1). The goal of this project is to develop molecular markers for activation of the immune system in the mosquito, and use them to study the process during malaria infection in vivo in both susceptible and refractory strains. These experiments could give us a better understanding of the insect parasite interactions that make malaria transmission possible.

Genetics of Drosophila cellular immunity to a parasitoid: isolation of a gene for immune recognition? (Yves Carton)

Larvae of Drosophila melanogaster produce a hemocytic reaction against eggs of the parasitoid Leptopilina boulardi, leading to the formation of a multi-cellular capsule surrounding the foreign object. The heredity of the capacity to encapsulate was analyzed by comparing 16 reciprocal crosses using selected inbred resistant (R) and susceptible (S) strains. The results showed that differences in the encapsulation capacity of D. melanogasler are inherited autosomally, with the reactive phenotype showing complete dominance over the non-reactive one. The results of all crosses suggest a single major segregating locus with two alleles and complete dominance of the resistant allele. Chromosome exchanges between the R and S strains (with the balanced strains technique) show that the locus for immune capacity is located on the second chromosome. Investigations are in progress to localize more precisely the gene, using the recombinant technique with two mutants (ltd and bw) which can be recognized at the larval stage. Previous cytogenetic analysis of the second chromosome of the resistant line demonstrated that its right arm presented no rearrangements or changes. Therefore, there was no bias in evaluating the rate of recombination. The R gene was mapped cytogenetically to 55D-55F region.

The melanization process with the intermediate catecholamine substrates and all the specific enzymes involved are integral parts of the immune system in insects. Investigations using HPLC with electrochemical detection allowed monitoring of the different biogenic amines derived from tyrosine and enzyme activity. Parasitoid infestation induced enhancement of hemolymph phenol oxidase activity, only in larvae of the resistant strain. These data indicate that the phenol oxidase system of the immune reactive strain is activated during parasitisation and results in the synthesis of some precursors which ultimately produce melanin. The mean rate of monophenoloxidase activity in infested resistant larvae was 10 times higher than in the infested larvae of the susceptible strain at 24 hrs post infection. This rate returned to more normal levels at 30 hrs post infection.

These different data are consistent with the observation that early synthesis (<18 hr) of electron dense material, deposited on the surface of the egg chorion as a thin layer and only detectable at the ultrastructural level, seems to precede the attachment of the first hemocytes. This synthesis is concomitant with high monophenoloxidase activity, only observed in infested resistant larvae. So it can be suggested that this black layer corresponds to eumelanin synthesis which appears to be necessary at early stages of the immune cellular reaction.

Dopa, 5-6 dihydroxyindole and N-acetyl arterenone have been detected by electrochemical methods in the hemolymph of immune reactive larvae of D. melanogaster, following parasitisation by Leptopilina. The presence of 5,6-dihydroxyindole unequivocally establishes the eumelanin pathway in the immune response of Drosophila.

To address the crucial question concerning the necessity of early melanin synthesis in the cellular immune process, a temperature sensitive dopa decarboxylase (Ddc) mutant of D. melanogaster was utilised. At the permissive temperature larvae are highly immune reactive exhibiting melanotic encapsulation, whereas the same larvae at the restrictive temperature have an immune capacity significantly reduced (from 80.1 % to 8.5%). It appears that when melanin synthesis is blocked or depressed, the cellular immune reaction does not occur or is very low.

The problem was to determine at what step of the immune reaction the resistant gene interferes. It was interesting to know if the immune incompetence exhibited by the S-strain was due to an inadequate immune recognition system, a deficiency in the prophenoloxidase activating system and/or some defect in the eumelanin effector response. To investigate the basis for susceptibility in the S-strain, a second species of parasitic wasp, Asobara tabida, was used. Against this wasp, both R and S strains were found to be highly immune reactive, exhibiting encapsulation rates of approximately 90 and 95%, respectively. Other data indicated that the immune reaction against A. tabida is similar to that made against L. boulardi not only at the cellular level, but also at the biochemical level. Thus the S-strain has a functional and highly competent immune system. The gene that distinguishes the reactive from the susceptible strain affects either the recognition process or the immune response mechanism. A provisional hypothesis is that this gene concerns the recognition process, which should be highly specific.

Malaria in the mosquito – an overview (Robert E. Sinden)

Transmission of malaria parasites through their insect vectors requires the interactions of at least three organisms; the parasite, the vector, and the vertebrate host, and may on occasion be further modulated by other pathogens of either insect or vertebrate hosts.

In the mosquito vector (a female anopheline in the case of mammalian malaria) these interactions take place in three fundamentally different tissue locations: in the bloodmeal, inside cells of the midgut epithelium and salivary glands; and in the haemocoele.

Mosquito transmission is mediated exclusively by the sexual stages of Plasmodium, the gametocytes. The parasite undergoes rapid differentiation in tire vector, beginning with the explosive differentiation of gametes from gametocytes in the bloodmeal (10-60 mins post ingestion (RI.)). This is followed by fertilisation, zygote formation and meiotic division (1-5 hours P.I.). Subsequently the motile ookinete differentiates (5-36 hours P.I.), then migrates from the bloodmeal through the peritrophic layer and midgut epithelium and comes to rest undcr the basal lamina where it transforms into an oocyst (24-36 bouts P.I.). The growth of each vegetative oocyst over the next 10-25 days is followed by the release of 8-10,000 motile sporozoites (Pringle, 1965). The oocyst expands from 1 to 40 60 µm in diameter and stretches the basal lamina such that daughter sporozoites emerging through the oocyst wall may now have direct access to the haemocoele (Sinden, 1974). The released sporozoites are poorly infectious to vertebrates and undergo continued development as they traverse the haemocoele. Some 15% of P. berghei sporozoites invade the salivary glands and collect in the acinar cells of the distal lateral and central lobes. The salivary gland sporozoites are more infectious to the vertebrate host, and persist in the glands for as long as 70 days (Barber, 1936). A few sporozoites pass through the secretory vesicles and in Anopheles enter the non-chitinised region of the salivary duct, where they accumulate. At each subsequent bloodmeal on vertebrate hosts the female anopheline injects 10-50 sporozoites into the host tissue completing the transmission process.

It is apparent that this complex phase of the malarial parasite life cycle is at present very poorly understood by comparison to the events in the vertebrate host, nonetheless recent exciting advances have highlighted numerous subject areas that are either of fundamental scientific interest or of critical importance to the success of parasite transmission. It should never be forgotten that previous control strategies and current mathematical models suggest that control of Plasmodium in the mosquito vector is one of the more practicable strategies to limit the spread of the disease. This may be brought into perspective when it is remembered how much of a population bottle neck transmission of the parasites through individual mosquito vectors represents. Realistic estimates from in vivo data suggest 30 < 300 > 3000 gametocytes of P. falciparum are ingested into the bloodmeal; 1-2% of these form viable ookinetes, and 1-20% of ookinetes form an oocyst (Vaughan, Narum & Azad, 1991; Vaughan, Noden & Beier, 1992). The average field infection of P. falciparum has 1-4 oocysts, each the product of a single fertilisation event. Each oocyst produces 8-10,000 sporozoites of which 15% reach the salivary glands. Assuming each mosquito bites 10 people and delivers 50 sporozoites per bite only 5% of these salivary gland sporozoites are delivered back to the host. The enormous losses incurred include both ‘normal’ losses in any sexual cycle of development, combined with the losses incurred by the parasite vector interactions. These interactions should be considered as falling into two major groups: a) those on which the parasite is dependent (i.e. classical inducers) and b) those that result from antiparasitic vector defence mechanisms. These interactions occur in 4 environments: 1) the bloodmeal; 2) the midgut epithelium; 3) the haemocoele and 4) the salivary glands.

The Bloodmeal

The bloodmeal contains all normal blood components (including viable leukocytes); intraerythrocytic asexual and sexual parasites; products of interactions between parasite and vertebrate host (e.g. antibodies, complement and cytokines); mosquito ‘factors’ including unspecified gametocyte activating factors and enzymes from the saliva and the midgut; products of interaction between mosquito and parasites, and mosquito and vertebrate blood (Sinden et al. 1995). Interactions currently identified include: the induction of gametogenesis (Sinden & Croll, 1975; Nijhout, 1979); the activation of parasite prochitinase from the ookinete by mosquito trypsin (Shahabuddin & Kaslow, 1994b), and the destruction of some parasites by mosquito enzymes (Gass & Yeates, 1979; Yeates & Steiger, 1981).

The regulation of gametogenesis is only partly understood. It is triggered by parallel induction mechanisms of temperature shock (fall) and either the presence of undefined mosquito derived gametocyte activating factors or of a rise in gut pH (Sinden et al. 1995). Recognising that exflagellation of Plasmodium spp may on occasion only be triggered in susceptible hosts (Sinden et al. 1995) the identification of mosquito species specific activating factors could provide interesting insights to the artificial modulation of parasite infectivity. Secondary messengers involved in gametogenesis include the PI-PLC, inositol/calcium/calmodulin pathway; cGMP and regulation of pH. Cellular events include 3 rounds of DNA replication each followed by mitosis, and the intracytoplasmic assembly of 8 axonemes each 22µm long, all completed within 8-10 minutes. Initial assembly of the kinetosomal flagellar templates from the amorphous microtubule organising centre takes only 15 seconds. Understanding these unprecedented rates of cell replication could be of broad cell biological interest.

Immediate meiosis in the zygote reinstates the haploid organisation of the genome. Thereafter the mature ookinete penetrates the peritrophic layer. P. falciparum and P. gallinaoeum secrete a pro-chitinase which, when processed by mosquito trypsin is responsible for the disorganisation of the peritrophic layer permitting parasite migration (Shahabuddin & Kaslow, 1994a; Sieber et al. 1991). Inhibition of the chitinase by chemical or immunological means could be considered a potential transmission blocking strategy by preventing escape from the bloodmeal (Shahabuddin et al. 1993; Shahabuddin, 1995). Mosquito trypsin activity is concentrated at the periphery of the bloodmeal and ookinetes are killed if exposed to the enzymes over extended periods (Yeates & Steiger, 1981; Gass & Yeates, 1979).

Other established transmission-blocking strategies include the development of vaccines against the surface molecules on the gametes (e.g. Pfs230) and the zygote/ookinete (eg. Pbs21/P£s25). The former would block fertilisation by gamete agglutination, and early zygote development by complement mediated lysis. The latter block zygote development by opsonisation, block ookinete motility (by Fab), and early oocyst development (by intact IgG) (Sinden, 1994).

The Midgut Epithelium

Interaction of the ookinete with the midgut epithelium is, by analogy with the merozoite and sporozoite (and in the absence of any data), likely to be mediated by multiple ligands that trigger specific events in the ookinete (e.g. the secretion of membrane active molecules to form the transient parasitophorous vacuole). Clearly this is an area worthy of further study. Recognising that the ookinete (like the sporozoite) is always motile, it should not be assumed that any specific changes in parasite motility are triggered during midgut invasion. Having reached its intracytoplasmic location, the ookinete will need to escape the midgut cell. This necessitates crossing the basement membrane “backwards” suggesting either that entering and leaving the cell are mediated by different cellular mechanisms, or that the same mechanisms are induced by different or commonplace stimuli. Suggestions that the ookinete has an obligatory intercellular pathway through the midgut are to this author less logical than the more conservative option that the ookinete (like the merozoite and sporozoite) uses an intracellular path, (the latter does not exclude the possibility that the ookinete interacts with the intercellular junctions after invasion). Having escaped through the basement membrane, the ookinete contacts the basal lamina of the midgut, ceases movement, and thereafter rapidly differentiates into an oocyst.

During its passage through the midgut epithelium losses in ookinete number occur. Even in fully susceptible mosquitoes, estimates vary from only 1/5 to 1/60 ookinetes successfully forming oocysts. Refractory An. Gambiae lines have been described in which both intracytoplasmic and intravacuolar ookinetes of P. gallinaceum are lysed. The responsible gene acts as an autosomal recessive allele (Vernick et al. 1995). Collins et al. (1986) have also described a refractory An. gambiae line in which the ookinete is melanised in a location that suggests that rupture of the basement membrane is necessary to trigger the insect response, which is achieved by localised conversion of DOPA to melanin without the direct involvement of haemocytes. Observations on the melanisation of growing oocysts suggests that melanisation attacks primarily the membranes (both plasma membrane and endoplasmic reticulum) of the parasite (Sinden & Garnham, 1973).

Differentiation to the oocyst involves the loss of motility, resorption of the apical complex and initiation of a rapid trophic phase with ensuing endomitosis of the haploid nucleus. In vivo observation, ex vivo and in vitro experimentation all indicate that molecules in the basal lamina trigger this development. Recent studies suggest ookinetes are able to bind to type IV collagen and laminin, both components of the midgut basal lamina (Gare & Billingsley, unpublished). The growing oocysts (if in high number) place a measurable burden on the female mosquito. In the laboratory significant reductions in fecundity and flight performance have been recorded (Hurd, 1994; Hogg & Hurd, 1995; Schiefer, Ward & Eldridge, 1977). Whether such losses are induced by the ‘normal’ field infection for P. falciparum and P. vivax i.e.1-4 oocysts, contrasted to laboratory infections averaging 10-100 oocysts needs to be confirmed. One might predict such powerful negative evolutionary pressures would rapidly result in selection for refractory mechanisms, yet Plasmodium is still transmitted by a wide range of vectors, suggesting it is not very deleterious to the vector. Conversely it could equally be argued that many mosquito species are refractory to transmission of selected Plasmodia. Early ‘cross-transplantation’ work observing oocyst development in normally refractory mosquitoes (Weathersby, 1985; Weathersby & McCall, 1968) and EM studies (Sinden & Garnham, 1973) suggest that oocysts may ‘die’ without melanisation. These oocysts become highly vacuolated and the nuclei rounded with highly condensed chromatin suggesting a necrotic event, subsequently these oocysts sometimes become melanised.

The oocyst initially is 2-3µm in diameter surrounded by a simple plasmalemma, as it grows to a mature size of 40 60µm diameter a wall is produced, possibly by release of parasite materials though exocytotic vesicles. The exact nature of this wall is unknown. The growing oocyst disrupts the basal lamina which is stretched forming a fish-net like layer over the oocyst. Sporozoites when released easily pass through this disrupted matrix (Sinden, 1975). Dependent upon the external temperature, oocysts mature in 6-21 days. In some species (whose naturel environment is cooler e.g. in highland regions) there is an upper limit to growth of 21°C (P. berghei) vs. a usual optimum of ca. 26° (P. falciparum, Pyoelii, inter alia). Elevated temperatures are not well tolerated by developing oocysts whereas lower temperatures are and simply delay maturation. Midway through maturation the polyploid cell undergoes cytoplasmic division producing sporoblasts with a resultant significant increase in surface area of the parasite. Sporozoites then bud from this surface in a manner typical of the Apicomplexa (Sinden, 1978). Membrane discs, the microtubule organising centre (MTOC) and subpellicular microtubules all lie directly beneath growing protuberances, the MTOC is connected to the spindle plaque of a mitotic nucleus. This whole assembly extends (on elongating microtubules) from the cell surface. One mitochondrion and (chloro) plastid are also drawn into the bud after the nucleus. At this stage 2-5 large electron dense ‘rhoptries’ lie between the nucleus and the apical complex, and are connected to the anterior pole of the sporozoite (Sinden & Garnham, 1973). Immunogold studies suggest these rhoptries secrete CS protein (Stewart & Vanderberg, 1991). The rigid sporozoite then buds from the surface.

The Haemocoele

Thereafter the sporozoite is motile and moves in a curvilinear manner, reminiscent of (directed by?) the distribution of the subpellicular microtubules. Sporozoites emerge through (make?) holes in the oocyst capsule into the haemocoele where they, and free CS protein, can adhere to the basal lamina on the haemocoelomic surface of the midgut wall. 15 % of the sporozoites reach the salivary glands, some invade the cells of the midgut (presumably, they ‘escaped’ form oocyst surfaces in contact with the epithelium), others can be found in the haemocoele of the palps and feet suggesting they are pumped passively through the haemolymph by muscular activity of the host. Rarely are there records of melanised sporozoites (Barber, 1936), presumably therefore the majority of sporozoites are either actively destroyed by mosquito lytic peptides or haemocytes, or passively die in the haemolymph and are then removed by host defence mechanisms. The exact role of the insect immune system in producing this significant bottleneck of parasite transmission is not understood.

The Salivary Glands

Sporozoites recognise the basal lamina of the salivary gland, but there is as yet no convincing evidence that a positive attractant gradient in the haemolymph is recognised by the parasite (Touray et al. 1992). It is improbable that a stable gradient could be maintained in such a turbulent medium. Directed movement of sporozoites along the basal lamina following random attachment is however entirely feasible. Invasion of the salivary gland by the sporozoites requires penetration of the basal lamina (we do not know how this is achieved) and the basement membrane of the salivary acinar cells. Sporozoites from the haemocoele are able to invade the salivary gland, they express CSP but not TRAP (Robson et al. 1995), have rhoptries (Sinden & Garnham, 1973) and infect hosts poorly (Vanderberg, 1975). Conversely sporozoites from the salivary gland have abundant micronemes, express both TRAP and CSP, can invade hepatocytes but not salivary glands (Touray et al. 1992). The mechanism of sporozoite invasion is similar to that of ookinete invasion. A vacuole is induced in the plasma membrane of the acinar cell which the parasite enters, this vacuole degrades and the sporozoite comes to lie directly in the cell cytoplasm, they then migrate into the secretory vesicle where initially they appear to be membrane bound, but then free in the matrix in massive tightly adherent rafts (Pimenta, Touray & Miller, 1994). Sporozoites can remain in this location for very extended periods (up to 70 days) (Barber, 1936), but are concentrated in the distal lateral lobes and the central lobes, where in Anopbeles the salivary duct is not (or is less) chitinised. Some of these sporozoites enter the duct from whence they are ejected in small numbers (12-100), into the blood of the host (Rosenberg, 1992; Ponnudurai et al. 1991). In Aedes the entire salivary duct is chitinised, it would be interesting therefore to determine whether sporozoites–like ookinetes secrete a chitinase enzyme at this phase in their development. Old reports state that sporozoites induce visible pathology of the mosquito salivary glands (Barber, 1936), more recent studies have shown that the feeding efficiency of mosquitoes with salivary gland infection is compromised (Rossignol, Ribeiro & Spielman, 1984). This results in mosquitoes taking more, and smaller feeds, which is potentially to the advantage of parasite transmission to a new vertebrate host.

Contributions

• Michael Ashburner – Drosophila – A model for mosquitoes
• Carolina Barillas-Mury – Immune response in An. gambiae: the first rel-family member is identified
• Yves Carton – Genetics of Drosophila cellular immunity to a parasitoid: isolation of a gene for immune recognition?
• Frank Collins – Population genetics and evolution of the Anopheles gambiae complex
• Andrea Crisanti – Malaria sporozoite development in Anopheles mosquitoes
• Ronald Davis – Automation in genomics
• Karen Day – Antigenic diversity and transmission dynamics of Plasmodium falciparum
• William Dietrich – Application of mouse genomic maps to the study of infectious disease susceptibility
• Jules Hoffmann – Drosophila immunity (with Jean-Marc Reichhart)
• Fotis C. Kafatos – Introduction: the purpose of the meeting
• Dominic Kwiatkowski – The regulation of malaria parasite density within the host
• Christos Louis – Expression of An. gambiae trypsin genes in transgenic Drosophila
• Geneviève Milon – From mononuclear phagocytes of vertebrates to invertebrate hemocytes
• Victor Nussenzweig – Sporozoite receptors in liver
• Linda Partridge – Insect life histories and their relevance to vector control
• Susan Paskewitz – Encapsulation of malaria in midguts of Anopheles gambiae
• Jean-Marc Reichhart – Drosophila immunity (with Jules Hoffmann)
• Adam Richman – Immune response in Anopheles gambiae
• Robert E. Sinden – Biology of the parasite in the mosquito
• Isabelle Tardieux – Cell biology and parasitology
• Kenneth Vernick – Parasite killing in midgut epithelium
• Liangbiao Zheng – Genetic mapping of Anopheles gambiae refractoriness to Plasmodia

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