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.

Drosophila serpin 27A is a likely target for immune suppression of the blood cell-mediated melanotic encapsulation response.

Avirulent strains of the endoparasitoid Leptopilina boulardi succumb to a blood cell-mediated melanotic encapsulation response in host larvae of Drosophila melanogaster. Virulent wasp strains effectively abrogate the cellular response with substances introduced into the host that specifically target and effectively suppress one or more immune signaling pathways, including elements that control phenoloxidase-mediated melanotic encapsulation. The present study implicates involvement of the Drosophila Toll pathway in cellular innate immunity by regulating the serine protease inhibitor Serpin 27A (Spn27A), which normally functions as a negative regulator of phenoloxidase. The introduction of Spn27A into normally highly immune competent D. melanogaster larvae significantly reduced their ability to form melanotic capsules around eggs of L. boulardi. This study confirms the role of Spn27A in the melanization cascade and establishes that this pathway and associated blood cell responses can be activated by parasitization. The activation of phenoloxidase and the site-specific localization of the ensuing melanotic response are such critical components of the blood cell response that Spn27A and the signaling elements mediating its activity are likely to represent prime targets for immune suppression by L. boulardi.

Superoxide anion generation in Drosophila during melanotic encapsulation of parasites.

Quinoid precursors of melanin and/or reactive oxygen species (ROS) generated during melanogenesis have been implicated as cytotoxic molecules in the immune responses of insects against their internal metazoan parasites. No study has yet identified the killing components produced in conjunction with melanotic encapsulation responses, or explained how cytotoxic molecules generated in the open circulatory system of an insect can selectively destroy foreign tissues. Strains of Drosophila melanogaster with differing immune capabilities against the wasp parasitoid Leptopilina boulardi were examined for superoxide anion (O2-.) formation during parasitization. Elevated levels of O2-. were produced by immune reactive (R-strain) hosts during melanotic encapsulation of the parasitoid, but not by susceptible (S-strain) hosts in which the parasitoid developed unmolested. Both a superoxide dismutase (SOD)-deficient strain (cSODn108, red/TM3/Sb Ser) and a catalase (CAT)-deficient strain (Catn1) also produced melanotic capsules and elevated levels of O2-. when infected, but these reactions were unsuccessful and the parasitoids survived, indicating that neither the quinoid precursors of melanin nor O2-. per se were cytotoxic. Immune incompetence in SOD-deficient and CAT-deficient hosts is attributed in part to defects in hydrogen peroxide (H2O2) metabolism, and/or the inability of these metalloenzyme-deficient strains to initiate the metal-mediated reductive cleavage of H2O2 required for the production of the cytotoxic hydroxyl radical (.OH). The role proposed for O2-. in Drosophila cellular immunity is one of potentiating the formation of .OH. Melanin, which contains both oxidizing and reducing components, may serve a dual role in producing O2-. and sequestering redox-active metal ions, thereby confining the production of ROS. Host-parasite susceptibility in the Drosophila-Leptopilina system may be determined by the ability of the parasitoid to modulate hemocyte activity and prevent both effective melanotic encapsulation and the generation of cytotoxic levels of ROS.

Isogenic, allogenic, and xenogenic transplants in an insect species.

Eggs of varying degrees of genetic foreigness were implanted into the hemocoel of an insect larvae (Pimpla instigator, Hymenoptera). For xenogenic implants, eggs of Drosophila melanogaster (Diptera) and eggs of Apechthis compuctor (Hymenoptera) were employed. Pimpla eggs of stock culture and Pimpla eggs of genetically defined pure strains were used for allogenic implants and isogenic implants, respectively. The host response that has been studied is that of encapsulation; it was possible to examine quantitatively the effect of genetic relationship on the degree of cellular reaction, i.e., thickness of capsule. Encapsulation occurred with increasing intensity as the degree of genetic difference increased, indicating that Pimpla larvae were able to discriminate not only between self and not-self, but also between different degrees of not-self.

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.

Encapsulation ability of Drosophila melanogaster: a genetic analysis.

Insects are able to effectively recognize parasitoid eggs or larvae and to eliminate them by formation of a hemocytic capsule. Although the cellular process is now well documented, the genetic aspects of recognition of foreignness and the encapsulation process are still poorly understood. Experiments using the isofemale-strain method showed that the encapsulating ability of Drosophila melanogaster exercised against a parasitic wasp varies within a given population and that this variability is under partial genetic control.

Alterations in the activities of tyrosinase, N-acetyltransferase, and tyrosine aminotransferase in immune reactive larvae of Drosophila melanogaster.

The activities of three enzymes, tyrosinase (monophenol oxidase, MPO), N-acetyltransferase (NAT), and tyrosine aminotransferase (TAT), were studied during eumelanotic encapsulation in host larvae of Drosophila melanogaster parasitized by the wasp, Leptopilina boulardi. At 24 h postinfection there was a tenfold increase in the MPO, whereas the activities of NAT and TAT were lower than those of nonparasitized controls. The data suggest that certain developmental processes are temporarily interrupted and alterations made in the metabolism of tyrosine to provide the metabolites necessary for a successful immune response. Two strains of D. melanogaster, R and Tyr-1, were parasitized and found to be immune reactive. The Tyr-1 strain is deficient in tyrosinase during the adult stage, but this mutation was found not to affect the immune capacity of the larvae. This is the first study to document concurrent alterations in the activities of various catecholamine-metabolizing enzymes during an immune response in an insect.

Induction of a putative serine protease transcript in immune challenged Drosophila.

In an effort to identify serine proteases involved in the insect's immune response, we used a degenerate PCR approach to amplify putative serine protease gene fragments in Drosophila. Sequencing of the cloned PCR products identified one serine protease previously isolated in D. melanogaster (SER1/SER2), as well as two novel putative serine protease gene fragments (SP2, SP3). The involvement of the corresponding genes in the immune response was examined by analyzing their expression in larval mRNA following both parasitic and bacterial exposures. The overexpression of one of the serine proteases-related mRNAs in immune challenged larvae suggests its involvement in the Drosophila immune response.

Diapause in a tropical species, Cothonaspis boulardi (Parasitic hymenoptera).

The strain of Cothonaspis boulardi originating from a field collection at Guadeloupe (16° lat. N-West Indies) exhibits a facultative diapause that occurs at the final larval instar. The diapause was induced by low temperature (17.5° C) and was inhibited at 25° C. Diapause was independent of photoperiod. The termination of diapause was hastened in about 10 days by exposing larvae to a temperature of 25° C. Although a succession of sufficient cold days for diapause occurs only rarely in the collection area (Petit-Bourg), at higher elevations within 20 km of Petit-Bourg conditions that could induce diapause occur annually.

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.

The effects of parasite-derived immune-suppressive factors on the cellular innate immune and autoimmune responses of Drosophila melanogaster.

Immune-suppressive factors (ISFs) introduced into larvae of Drosophila melanogaster during infection by virulent endoparasitic wasps effectively block the innate immune response mediated by blood cells (hemocytes) but have little influence on the autoimmune response made by a tumor strain in which the blood cells manifest a similar response but instead target and destroy endogenous tissues. Quantitative hemocyte analyses indicate that ISFs interfere with the immune effector responses downstream of nonself recognition, hemocyte activation and differentiation, because these responses were manifested by tumor hosts, in which the parasitoids developed. The data suggest that once activated to encapsulate aberrant tissues, the target specificity of the autoimmune-activated hemocytes, and the genetic program underlying tumor formation, cannot be blocked by parasitoid-derived ISFs, which effectively inhibit identical hemocyte-mediated responses during parasitization.

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.

Parasite-induced changes in nitric oxide levels in Drosophila paramelanica.

In larvae of Drosophila paramelanica, eggs and larvae of the endoparasitic wasp Leptopilina heterotoma succumb to an effective host reaction that does not involve blood cell-mediated melanotic encapsulation, a response that characterizes cellular immunity in various species of Drosophila and in many insects and other arthropods. A significant increase occurs, however, in the number of lamellocytes, a type of blood cell that functions in encapsulation reactions. The appearance of activated lamellocytes in D. paramelanica is viewed as an early response to infection, one most likely initiated by non-self-recognition processes that similarly function in other wasp-infected Drosophila. However, ensuing cytotoxic responses, about which little is presently known, are not accompanied by melanotic encapsulation in D. paramelanica. Concurrent analyses of the cell-signaling molecule nitric oxide (*NO) revealed significant alterations in the levels of this free radical during the early stages of infection, most notably a dramatic increase immediately upon infection, and precipitous decreases occurring at times when parasites were killed. Injections of a specific inhibitor of nitric oxide synthase (NOS) into the host's body cavity prior to infection significantly increased parasite survival. These observations suggest some involvement of *NO in the host immune response, either in recruiting hemocytes to sites of infection or as a component of the insect's cytotoxic arsenal, given the capacity of the radical to generate toxic molecules through interactions with various intermediates of oxygen and nitrogen.

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.

[Hemocytic reactions in lepidoptera pupae. Intensity of the reaction as a function of its localization in Pieris brassicae]. / Réactions hémocytaires chez la chrysalide des Lépidoptères. Instensité de la réaction en fonction de sa localisation chez Pieris brassicae

Quantitative studies were made on cellular capsules formed in diapause pupae of Pieris brassicae. A simple procedure is to implant pieces of nylon thread in hemocoele. Differences have been noticed in the vigour of the reaction at different regions of the pupae : the haemocytic reaction was less vigorous in the central region of pupae than in the distal ones.

Insect immunity: a genetic factor (hrtp) is essential for antibacterial peptide expression in Drosophila after infection by parasitoid wasps.

We have used a parasitoid wasp Drosophila melanogaster system to investigate the relationship between the humoral and cellular immune responses in insects. Expression of the gene encoding diptericin, an antibacterial peptide in various D. melanogaster strains parasitized by several species of parasitoid wasps, was studied by Northern blot. These strains have the capacity to encapsulate parasitoid eggs. Two strains appeared to produce diptericin mRNA after parasitoid challenge, regardless of their cellular immune reaction to the wasp species. This suggests that a specific genetic factor, or factors, here designated humoral response to parasitoid (hrtp), is present in these two strains of D. melanogaster and is implicated in the expression of the antibacterial gene after parasite infection. This hrtp genetic factor is recessively expressed and located on the second chromosome, suggesting that it is monofactorial. The transgenic strain Dipt.2.2-lacZ:1, in which the transgene is present on the first chromosome, is normally susceptible to the parasitoid wasp. The chromosome bearing the hrtp factor was transferred to this transgenic strain, which then became reactive when triggered by wasp infection. The hrtp factor appears necessary for the activation of diptericin by the parasitoid wasp. No correlation between the cellular immune capacity and the humoral response was observed, suggesting that the two components of insect immunity are regulated independently. Arch.

Genetic determinism of the cellular immune reaction in Drosophila melanogaster.

Larvae of Drosophila melanogaster produce a haemocytic reaction against eggs of the parasitoid Leptopilina boulardi, which leads to the formation of a multicellular capsule surrounding the foreign object. Melanization resulting from the conversion of phenol to o-quinones frequently accompanies the cellular reaction. Although various cytological and biochemical aspects of this reaction have been investigated, very little is known about genetic determinism of the insect immune response. The heredity of the capacity to encapsulate was analysed by comparing 16 reciprocal crosses made using inbred resistant and susceptible parental strains. We conclude that differences in the encapsulation capacity of D. melanogaster are inherited autosomally, with the reactive phenotype showing complete dominance over the non-reactive one. There were neither sex-linked nor maternal effects. The results of all crosses suggest a single major segregating locus with two alleles and complete dominance of the resistant allele, with cytoplasmic factors and minor modifying genes acting on the major locus.