Genetics of anti-parasite resistance in invertebrates.

This review summarizes and compares available data on genetic and molecular aspects of resistance in four well-described invertebrate host-parasite systems: snail-schistosome, mosquito-malaria, mosquito-filarial worm, and Drosophila-wasp associations. It underlies that the major components of the immune reaction, such as hemocyte proliferation and/or activation, and production of cytotoxic radicals are common to invertebrate hosts. Identifying genes responsible for naturally occurring resistance will then be helpful to understand the mechanisms of invertebrate immune defenses and to determine how virulence factors are used by parasites to overcome host resistance. Based on these four well-studied models, invertebrate resistance appears as generally determined by one major locus or a few loci, displaying at least partial dominance. Interestingly, specificity of resistance is highly variable and would involve processes other than simple recognition mechanisms. Finally, resistance was shown to be generally costly but is nevertheless observed at high frequencies in many natural populations, suggesting a high potential for host parasite coevolution.

A RhoGAP protein as a main immune suppressive factor in the Leptopilina boulardi (Hymenoptera, Figitidae)-Drosophila melanogaster interaction.

To protect its eggs, the endoparasitoid wasp Leptopilina boulardi injects immune suppressive factors into Drosophila melanogaster host larvae. These factors are localized in the female long gland and reservoir. We analyzed the protein content of these tissues and found that it strongly differed between virulent and avirulent parasitoid strains. Four protein bands present in virulent long glands were eluted and their immune suppressive effect was assessed in vivo, allowing demonstrating a major effect of one of these proteins. The corresponding cDNA encodes a predicted 30 kDa subunit containing a Ras homologous GTPase Activating Protein (RhoGAP) domain, suggesting a possible involvement in the regulation of actin cytoskeleton changes. Using Western-blot experiments, we showed that this protein is abundant in virulent female long glands but is undetectable in virulent females deprived of long glands or in long glands from avirulent wasps. Its potential role in modifying the morphology and the adhesive properties of the host lamellocytes, involved in Drosophila cellular immune responses, is discussed.

Haemocyte changes in D. Melanogaster in response to long gland components of the parasitoid wasp Leptopilina boulardi: a Rho-GAP protein as an important factor.

The hymenopteran wasp Leptopilina boulardi (Figitidae) is a larval solitary parasitoid of Drosophila larvae of the melanogaster sub-group. The factors used by parasitoid females to prevent encapsulation of their eggs by the host are localized in the female long gland and reservoir. We report here the physiological effects of these factors on host haemocytes using in vivo injection experiments. The total number of haemocytes, the number of plasmatocytes and the number of crystal cells were not modified by injection of long gland extracts. In contrast, long gland extracts either from virulent or avirulent strains had a significant effect on the lamellocyte number. Compared to the Ringer control, the avirulent long gland products induced an increase of the lamellocyte number while virulent extracts induced a drastic decrease together with an alteration of the morphology of these cells. Interestingly, changes in the lamellocyte morphology were also observed following injection of the P4 protein, a major component of L. boulardi female long glands that displays a strong immune suppressive effect on Drosophila larvae. The implication of the P4 protein in suppressing the host cellular immunity is discussed in correlation with its predicted molecular function as a Rho-GAP protein.

Statistical analysis of the individual variability of 1D protein profiles as a tool in ecology: an application to parasitoid venom.

Understanding the forces that shape eco-evolutionary patterns often requires linking phenotypes to genotypes, allowing characterization of these patterns at the molecular level. DNA-based markers are less informative in this aim compared to markers associated with gene expression and, more specifically, with protein quantities. The characterization of eco-evolutionary patterns also usually requires the analysis of large sample sizes to accurately estimate interindividual variability. However, the methods used to characterize and compare protein samples are generally expensive and time-consuming, which constrains the size of the produced data sets to few individuals. We present here a method that estimates the interindividual variability of protein quantities based on a global, semi-automatic analysis of 1D electrophoretic profiles, opening the way to rapid analysis and comparison of hundreds of individuals. The main original features of the method are the in silico normalization of sample protein quantities using pictures of electrophoresis gels at different staining levels, as well as a new method of analysis of electrophoretic profiles based on a median profile. We demonstrate that this method can accurately discriminate between species and between geographically distant or close populations, based on interindividual variation in venom protein profiles from three endoparasitoid wasps of two different genera (Psyttalia concolor, Psyttalia lounsburyi and Leptopilina boulardi). Finally, we discuss the experimental designs that would benefit from the use of this method.

Drosophila resistance genes to parasitoids: chromosomal location and linkage analysis.

Insect hosts can survive infection by parasitoids using the encapsulation phenomenon. In Drosophila melanogaster the abilities to encapsulate the wasp species Leptopilina boulardi and Asobara tabida each involve one major gene. Both resistance genes have been precisely localized on the second chromosome, 35 centimorgans apart. This result clearly demonstrates the involvement of at least two separate genetic systems in Drosophila resistance to parasitoid wasps. The resistance genes to L. boulardi and A. tabida are not clustered as opposed to many plant resistance genes to pathogens cloned to date.

Active suppression of D. melanogaster immune response by long gland products of the parasitic wasp Leptopilina boulardi.

To develop inside their insect hosts, endoparasitoid wasps must either evade or overcome the host's immune system. Several ichneumonid and braconid wasps inject polydnaviruses that display well-studied immune suppressive effects. However, little is known about the strategies of immunoevasion used by other parasitoid families, such as figitid wasps. The present study provides experimental evidence, based on superparasitism and injection experiments, that the figitid species Leptopilina boulardi uses an active mechanism to suppress the Drosophila melanogaster host immune response, i.e. the encapsulation of the parasitoid eggs. The immune suppressive factors are localised in the long gland and reservoir of the female genital tractus, where virus-like particles (VLPs) have been observed. Parasitism experiments using a host tumorous strain indicate that these factors do not destroy host lamellocytes but that they impair the melanisation pathway. Interestingly, they are not susceptible to heating and are not depleted with prolonged oviposition experience, in contrast to observations reported for L. heterotoma, another figitid species. The mechanisms that prevent encapsulation of eggs from L. boulardi and L. heterotoma differ in several respects, suggesting that different physiological strategies of immunosuppression might be used by specialised and generalist parasitoids.

Polydnavirus genome: integrated vs. free virus.

Polydnaviruses are unique because of their obligatory association with thousands of parasitoid wasp species from the braconid and ichneumonid families of hymenopterans. PDVs are injected into the parasitized hosts and are essential for parasitism success. However, polydnaviruses are also unique because of their genome composed of multiple dsDNA segments. Cytological evidence has recently confirmed the results of genetic and molecular analyses indicating that PDV segments were integrated in the wasp genome. Moreover a phylogenetic study performed using the age of available fossils to calibrate the molecular clock indicated that the polydnaviruses harboured by braconid wasps have resided within the wasp genome for approximately 70 million years. In the absence of horizontal transmission, the evolution of the PDV genomes has been driven exclusively by the reproductive success they have offered the wasps. The consequences of this particular selection pressure can be observed in the gene content of certain PDV genomes from which increasing sequence data are available. Molecular mechanisms already identified could be involved in the acquisition and loss of genes by the PDV genomes and lead us to speculate on the definition of the virus genome.

The role of melanization and cytotoxic by-products in the cellular immune responses of Drosophila against parasitic wasps.

The cellular innate immune response of several species of Drosophila terminates with the encasement of large foreign objects within melanotic capsules comprised of several layers of adhering blood cells or hemocytes. This reaction is manifested by various Drosophila hosts in response to infection by endoparasitic wasps (i.e., parasitoids). Creditable assessments of the factor(s) causing, or contributing to, parasite mortality have long been considered as cytotoxic elements certain molecules associated with enzyme-mediated melanogenesis. However, observations that warrant additional or alternative considerations are those documenting parasitoid survival despite melanotic encapsulation, and those where parasitoids are destroyed with no evidence of this host response. Recent studies of the production of some reactive intermediates of oxygen and nitrogen during infection provide a basis for proposing that these molecules constitute important components of the immune arsenal of Drosophila. Studies of the virulence factors injected by female wasps during oviposition that suppress the host response will likely facilitate identification of the cytotoxic molecules as well as the cell-signaling pathways that regulate their synthesis.

Local, geographic and phylogenetic scales of coevolution in Drosophila-parasitoid interactions.

In this chapter, we describe the geographically widespread genetic fixation of traits involved in Drosophila-parasitoid immune interactions and the situations where such fixation is not observed. We then discuss how the three classes of coevolutionary dynamics that can occur at the local scale (coevolutionary escalation, coevolutionary alternation and coevolutionary polymorphism), the geographic mosaic of selection, and the phylogenetic constraints may explain such evolutionary patterns and drive diversification in the interactions. Most Drosophila parasitoid traits involved in virulence are host-species specific. Directional selection (coevolutionary escalation) on such traits can lead to their fixation or on the contrary maintain their polymorphism if these traits are associated with fitness costs. When hosts targeted by different host-specific virulence systems coexist, fluctuations in selective pressures on these systems, together with the ability of Drosophila parasitoids to select the most susceptible host for parasitization, can lead to coevolutionary alternation. Finally, we discuss the potential for parasitoid diversification in relation with the fact that most observed geographic situations, for different parasitoid clades, correspond to coevolutionary cold spots, due to fixation of virulence in parasitoid taxa.

Variation of Leptopilina boulardi success in Drosophila hosts: what is inside the black box?

Interactions between Drosophila hosts and parasitoid wasps are among the few examples in which occurrence of intraspecific variation of parasite success has been studied in natural populations. Such variations can originate from three categories of factors: environmental, host and parasitoid factors. Under controlled laboratory conditions, it is possible to focus on the two last categories, and, using specific reference lines, to analyze their respective importance. Parasitoid and host contributions to variations in parasite success have largely been studied in terms of evolutionary and mechanistic aspects in two Drosophila parasitoids, Asobara tabida and, in more details, in Leptopilina boulardi. This chapter focuses on the physiological and molecular aspects of L. boulardi interactions with two Drosophila host species, while most of the evolutionary hypotheses and models are presented in Chapter 11 of Dupas et al.

Genetic interactions between the parasitoid wasp Leptopilina boulardi and its Drosophila hosts.

Coevolutionary arms races between hosts and parasites would not occur without genetic variation for traits involved in the outcome of parasitism. Genetic variations in resistance and virulence have only rarely been described in pairwise host-parasitoid interactions and have never been analysed in multi-species interactions, in contrast to well-characterized plant-pathogen interactions. This paper reports genetic variation in resistance of Drosophila yakuba to the parasitoid wasp Leptopilina boulardi. The genetic basis and geographic distribution of resistance is analysed. On the basis of these and previous findings, we demonstrate that there are different resistance patterns to the parasitoid species L. boulardi in D. melanogaster and D. yakuba, as well as different specificity levels in the parasitoid species, suggesting complex ecological interactions in the field. This first description of resistance-virulence genetic interactions between a parasitoid and its two host species provides empirical data showing that multi-species interactions may greatly influence coevolutionary processes.

Mapping candidate genes for Drosophila melanogaster resistance to the parasitoid wasp Leptopilina boulardi.

Drosophila melanogaster resistance against the parasitoid wasp Leptopilina boulardi is under the control of a single gene (Rlb), with two alleles, the resistant one being dominant. Using strains bearing deletions, we previously demonstrated that the 55E2-E6; 55F3 region on chromosome 2R is involved in the resistance phenomenon. In this paper, we first restricted the Rlb containing region by mapping at the molecular level the breakpoints of the Df(2R)Pc66, Df(2R)P34 and Df(2R)Pc4 deficiencies, using both chromosomal in situ hybridization and Southern analyses. The resistance gene was localized in a 100 kb fragment, predicted to contain about 10 different genes. Male recombination genetic experiments were then performed, leading to identification of two possible candidates for the Rlb gene. Potential involvement of one of this genes, edl/mae, is discussed.

Identification of two distinct amplifications of the esterase B locus in Culex pipiens (L.) mosquitoes from Mediterranean countries.

Two new highly active esterases were detected by starch electrophoretic studies in Culex pipiens mosquitoes from the area of Montpellier (France) and from Cyprus. We demonstrate here that both the French and the Cyprus esterases B are overproduced due to amplification of the coding gene. The production of the esterase B is approximately 50- and 500-fold higher in mosquitoes from France and Cyprus, respectively, than in susceptible insects, whereas the number of gene copies is about 25 and 250. Differences of about 7- and 95-fold were also found in the degree of chlorpyrifos resistance. RFLP comparison of the amplified region containing the esterase B gene revealed large differences between French and Cyprus mosquitoes. It thus appears that two distinct haplotypes with an esterase B gene coding an enzyme with identical electrophoretic mobility have been amplified. We therefore named the haplotypes in mosquitoes from France and Cyprus B4 and B5, respectively. The estimated genetic distance between these two haplotypes is not smaller than those observed in all pair comparisons of other known esterase B haplotypes. These results are discussed in the context of amplification phenomena.

Genetic dimension of the coevolution of virulence-resistance in Drosophila -- parasitoid wasp relationships.

Variations observed in parasite virulence and host resistance may be the outcome of coevolutionary processes. Recent theoretical developments have led to a 'geographic mosaic theory' of coevolution according to which there are some localities where reciprocal selection occurs (hot spots) and others where it is strongly reduced (cold spots). Studies of host-parasitoid systems back this up, revealing a geographical variation of traits subjected to antagonistic selection governed by variations in the strength of the ecological interactions. A more detailed analysis of the genetic basis of these geographic variations in a model system -- the interaction between Drosophila melanogaster and its specific parasitoid Leptopilina boulardi -- suggests that cold spots and hot spots are also driven by the amount of genetic variation available for the trait considered. Our approach, based on isolating reference strains, has been found to predict the result of sympatric interactions and it will be helpful in identifying the selective forces responsible for the coevolution. In this model, host resistance to a standardised reference strain is a weak predictor of the outcome of interactions in the field, and the main parameter accounting for the geographic variations is the number of host species available, with less parasitoid virulence towards D. melanogaster being found in areas displaying a more diversified host community.

A sex-specific number of germ cells in embryonic gonads of Drosophila.

Male first instar larvae possess more germ cells in their gonads than female larvae of the same stage. To determine the earliest time point of sexual dimorphism in germ cell number, we have counted the germ cells of sexed embryos at different developmental stages. We found no difference in germ cell number of male and female embryos at the blastoderm and early gastrulation stage, or when germ cells are about to exit the midgut pocket. We find, however, that males have significantly more germ cells than females as soon as the germ cells are near the places where the gonads are formed and in all later stages. Our results show that germ cells are subject to a sex-specific control mechanism that regulates the number of germ cells already in embryos.

Insect glutathione S-transferases. Biochemical characteristics of the major forms from houseflies susceptible and resistant to insecticides.

Two classes of glutathione transferases have been identified and purified from Musca domestica. The first, designated as GST1, migrates as a single band of 28 kDa in SDS-gel electrophoresis, and the second, designated as GST2, migrates as a 32-kDa band. Antisera prepared against each class have no immunological cross-reactivity, and heterodimeric associations between the two classes have not been detected. Each class is composed of several isoforms: GST1 is composed of forms with isoelectric points from 4 to 9, whereas all the forms of GST2 have acidic pI values. Screening of cDNA libraries yielded clones coding for GST1, and the gene was sequenced and expressed in Escherichia coli. The high activity found in an insecticide-resistant strain (Cornell R) is correlated with high level of GST1 transcript.

Genetic localization of a Drosophila melanogaster resistance gene to a parasitoid wasp and physical mapping of the region.

Drosophila melanogaster larvae usually react against eggs of the parasitoid wasp Leptopilina boulardi by surrounding them with a multicellular melanotic capsule. The genetic determinism of this response has been studied previously using susceptible (non-capsule-forming) and resistant (capsule-forming) strains. The results suggest that differences in their encapsulation response involve a single gene, resistance to Leptopilina boulardi (Rlb), with two alleles, the resistant one being dominant. Rlb confers specific protection against Leptopilina boulardi and is thus probably involved in parasitoid recognition. Recent studies have localized this gene on the right arm of the second chromosome and our aim was to precisely determine its genetic and molecular location. Using strains bearing deletions, we demonstrated that resistance to Leptopilina boulardi is conferred by the 55C; 55F3 region and that the 55E2-E6; F3 region is particularly involved. A physical map of the 55C; 56A region was then constructed, based on a set of overlapping cosmid and P1 phage clones. Using single and double digests, cross hybridization of restriction fragments, and location of genetically mapped genes and STSs, a complete, five-enzyme restriction map of this 830-kb region was obtained.

Polydnavirus replication: the EP1 segment of the parasitoid wasp Cotesia congregata is amplified within a larger precursor molecule.

Polydnaviruses are unique viruses: they are essential for successful parasitism by tens of thousands of species of parasitoid wasps. These viruses are obligatorily associated with the wasps and are injected into the host during oviposition. Molecular analyses have shown that each virus sequence in the segmented polydnavirus genome is present in the wasp DNA in two forms: a circular form found in the virus particles and an integrated form found in the wasp chromosomes. Recent studies performed on polydnaviruses from braconid wasps suggested that the circular forms were excised from the chromosome. The different forms of the EP1 circle of Cotesia congregata polydnavirus during the pupal-adult development of the parasitoid wasp were analysed. Unexpectedly, an off-size fragment formerly used to diagnose the integration of the EP1 sequence into wasp genomic DNA was found to be amplified in female wasps undergoing virus replication. The EP1 sequence is amplified within a larger molecule comprising at least two virus segments. The amplified molecule is different from the EP1 chromosomally integrated form and is not encapsidated into virus particles. These findings shed light on a new step towards EP1 circle production: the amplification of virus sequences preceding individual circle excision.