No effect of Wolbachia on resistance to intracellular infection by pathogenic bacteria in Drosophila melanogaster

Multiple studies have shown that infection with the endosymbiotic bacterium Wolbachia pipientis confers Drosophila melanogaster and other insects with resistance to infection by RNA viruses. Studies investigating whether Wolbachia infection induces the immune system or confers protection against secondary bacterial infection have not shown any effect. These studies, however, have emphasized resistance against extracellular pathogens. Since Wolbachia lives inside the host cell, we hypothesized that Wolbachia might confer resistance to pathogens that establish infection by invading host cells. We therefore tested whether Wolbachia-infected D. melanogaster are protected against infection by the intracellular pathogenic bacteria Listeria monocytogenes and Salmonella typhimurium, as well as the extracellular pathogenic bacterium Providencia rettgeri. We evaluated the ability of flies infected with Wolbachia to suppress secondary infection by pathogenic bacteria relative to genetically matched controls that had been cured of Wolbachia by treatment with tetracycline. We found no evidence that Wolbachia alters host ability to suppress proliferation of any of the three pathogenic bacteria. Our results indicate that Wolbachia-induced antiviral protection does not result from a generalized response to intracellular pathogens.     

Genetic variation in Drosophila melanogaster resistance to infection: a comparison across bacteria

Insects use a generalized immune response to combat bacterial infection. We have previously noted that natural populations of D. melanogaster harbor substantial genetic variation for antibacterial immunocompetence and that much of this variation can be mapped to genes that are known to play direct roles in immunity. It was not known, however, whether the phenotypic effects of variation in these genes are general across the range of potentially infectious bacteria. To address this question, we have reinfected the same set of D. melanogaster lines with Serratia marcescens, the bacterium used in the previous study, and with three additional bacteria that were isolated from the hemolymph of wild-caught D. melanogaster. Two of the new bacteria, Enterococcus faecalis and Lactococcus lactis, are gram positive. The third, Providencia burhodogranaria, is gram negative like S. marcescens. Drosophila genotypes vary highly significantly in bacterial load sustained after infection with each of the four bacteria, but mean loads are largely uncorrelated across bacteria. We have tested statistical associations between immunity phenotypes and nucleotide polymorphism in 21 candidate immunity genes. We find that molecular variation in some genes, such as Tehao, contributes to phenotypic variation in the suppression of only a subset of the pathogens. Variation in SR-CII and 18-wheeler, however, has effects that are more general. Although markers in SR-CII and 18-wheeler explain >20% of the phenotypic variation in resistance to L. lactis and E. faecalis, respectively, most of the molecular polymorphisms tested explain <10% of the total variance in bacterial load sustained after infection.     

The effects of temperature on host-pathogen interactions in D. melanogaster: who benefits?

Drosophila melanogaster is widely used to study immune system function in insects. However, little work has been done in D. melanogaster on the effect of temperature on the immune system. Here we describe experiments that demonstrate that cooler temperatures enhance survival after infection and alter expression of immune-related genes in flies. This effect appears to be due not only to the fact that colder temperatures slow down bacterial growth, but also to the beneficial effects of cooler temperature on immune function. We explore the possibility that heat shock proteins, and in particular, Hsp83, may improve immune function at cool temperatures. We have long known that temperature can alter immune responses against microbial pathogens in insects. The approach described here allows us to determine whether this effect is due primarily to temperature-specific effects on the host or on its pathogen. These results suggest that both may be important.     

Wolbachia-mediated antibacterial protection and immune gene regulation in Drosophila

The outcome of microbial infection of insects is dependent not only on interactions between the host and pathogen, but also on the interactions between microbes that co-infect the host. Recently the maternally inherited endosymbiotic bacteria Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens. Mosquitoes experimentally infected with Wolbachia have upregulated immune responses and are protected from a number of pathogens including viruses, bacteria, Plasmodium and filarial nematodes. It has been hypothesised that immune upregulation underpins Wolbachia-mediated protection. Drosophila is a strong model for understanding host-Wolbachia-pathogen interactions. Wolbachia-mediated antiviral protection in Drosophila has been demonstrated for a number of different Wolbachia strains. In this study we investigate whether Wolbachia-infected flies are also protected against pathogenic bacteria. Drosophila simulans lines infected with five different Wolbachia strains were challenged with the pathogenic bacteria Pseudomonas aeruginosa PA01, Serratia marcescens and Erwinia carotovora and mortality compared to paired lines without Wolbachia. No difference in mortality was observed in the flies with or without Wolbachia. Similarly no antibacterial protection was observed for D. melanogaster infected with Wolbachia. Interestingly, D. melanogaster Oregon RC flies which are naturally infected with Wolbachia showed no upregulation of the antibacterial immune genes TepIV, Defensin, Diptericin B, PGRP-SD, Cecropin A1 and Attacin D compared to paired flies without Wolbachia. Taken together these results indicate that Wolbachia-mediated antibacterial protection is not ubiquitous in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent. We suggest that the immune priming and antibacterial protection observed in Wolbachia-infected mosquitoes may be a consequence of the recent artificial introduction of the symbiont into insects that normally do not carry Wolbachia and that antibacterial protection is unlikely to be found in insects carrying long-term Wolbachia infections.     

Host condition and pathogen identity influence bacterial infection survival in the common eastern firefly,Photinus pyralis

1. The degree to which host, pathogen, and environmental characteristics determine fitness outcomes in response to infection across the tree of life is a key question in ecoimmunology. Because infection experiments require large sample sizes and careful controls, studies have generally been limited to lab-adapted organisms.
2. Photinus pyralis is a charismatic firefly that is abundant during its annual summer emergence in the Eastern United States. Here, we assess the importance of host condition, pathogen identity and dose, and pre-infection environment on survival outcomes in response to bacterial infection in wild-caught adult male P. pyralis.
3. Although survival after sterile saline injection was not statistically different from survival of uninjected controls, survival kinetics support sterile saline injection as the most appropriate control.
4. Serratia marcescens and Providencia rettgeri infections increased mortality, especially at high doses. In contrast to fruit fly studies, Providencia sneebia, Pseudomonas aeruginosa, and Enterococcus faecalis infections did not. Infection with higher doses of P. rettgeri had an increased risk of death for fireflies caught later in the season than those captured earlier, but seasonality did not significantly interact with dose to impact mortality with any of the other bacterial species.
5. These results highlight the importance of considering host, pathogen, and environmental factors when investigating infection outcomes in wild-caught, non-model systems. The differences in survival between fireflies and fruit flies emphasise that further studies in a broad array of organisms are needed to explore the diversity of infection responses across the tree of life.

     

Two non-linked genes for specific virulence of Leptopilina boulardi against Drosophila melanogaster and D. yakuba

The developmental success of Leptopilina boulardi parasitoids within host species of the melanogaster subgroup is determined mainly by their ability to suppress the host immune reaction (virulence). Host resistance and parasitoid virulence are genetically variable in both partners. A gene for specific resistance against L. boulardi (Rlb) has been identified in Drosophila melanogaster, and a gene for the immune suppression (IS) of D. melanogaster has been identified in L. boulardi. To understand the evolution of the IS gene, we determined its specificity regarding potential hosts of the melanogaster subgroup. It did not affect the virulence against any other species of the melanogaster subgroup and was called ISm for immune suppression of D. melanogaster. Another gene (ISy), non-linked to the gene ISm, was characterized for the specific immune suppression of D. yakuba. These results suggesting that natural selection for virulence against one host species does not influence the evolution of virulence against another will allow us to develop pertinent hypotheses concerning the evolution of this character which is expected to drive the evolution of the parasitoid toward narrow host specialization. This revised version was published online in July 2006 with corrections to the Cover Date.     

Limitations in the use of Drosophila melanogaster as a model host for gram-positive bacterial infection

AIMS: To examine sensitivities of various Drosophila melanogaster strains towards human pathogenic and nonpathogenic gram-positive bacteria. METHODS AND RESULTS: The D. melanogaster Oregon R strain was infected by injecting the thorax with a needle containing Escherichia coli (negative control), Listeria monocytogenes, Staphylococcus aureus (both food-borne pathogens), Listeria innocua, Bacillus subtilis, Carnobacterium maltaromaticum, Lactobacillus plantarum or Pediococcus acidilactici (all nonpathogenic bacteria). Listeria monocytogenes and S. aureus killed the host rapidly compared with the negative control. Infection with L. innocua, B. subtilis or C. maltaromaticum also resulted in a high fly mortality, whereas Lact. plantarum and P. acidilactici resulted in a slightly increased mortality. Four additional D. melanogaster lines, three of which had been selected for heat, cold and desiccation resistance respectively, were subjected to infection by L. monocytogenes, S. aureus and E. coli. Mortality rates were comparable with that of the Oregon R strain. CONCLUSIONS: Use of the injection method shows the limitation of D. melanogaster as a model host for gram-positive bacteria as opportunistic infection by nonpathogenic gram-positive bacteria results in partial or high mortality. In addition, lines of fruit flies resistant to various stress exposures did not show an increased resistance to infection by gram-positive pathogens under the conditions tested. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the inadequacy of D. melanogaster infected by the injection method in order to distinguish between virulent and nonvirulent gram-positive bacteria.     

Adaptive evolution of relish, a Drosophila NF-kappaB/IkappaB protein

NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals. Homologues of these proteins also play an important role in regulation of the Drosophila immune response. Here we present a molecular population genetic analysis of Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D. melanogaster. We find strong evidence for adaptive protein evolution in D. simulans, but not in D. melanogaster. The adaptive evolution appears to be restricted to the IkappaB domain. A possible explanation for these results is that Relish is a site of evolutionary conflict between flies and their microbial pathogens.     

Molecular Population Genetics of Drosophila Immune System Genes

A striking aspect of many vertebrate immune system genes is the exceptionally high level of polymorphism they harbor. A convincing case can be made that this polymorphism is driven by the diversity of pathogens that face selective pressures to evade attack by the host immune system. Different organisms accomplish a defense against diverse pathogens through mechanisms that differ widely in their requirements for specific recognition. It has recently been shown that innate defense mechanisms, which use proteins with broad-spectrum bactericidal properties, are common to both primitive and advanced organisms. In this study we characterize DNA sequence variation in six pathogen defense genes of Drosophila melanogaster and D. mauritiana, including Andropin; cecropin genes CecA1, CecA2, CecB, and CecC; and Diptericin. The necessity for protection against diverse pathogens, which themselves may evolve resistance to insect defenses, motivates a population-level analysis. Estimates of variation levels show that the genes are not exceptionally polymorphic, but Andropin and Diptericin have patterns of variation that differ significantly from neutrality. Patterns of interpopulation and interspecific differentiation also reveal differences among the genes in evolutionary forces.     

Female and male genetic contributions to post-mating immune defence in female Drosophila melanogaster

Post-mating reduction in immune defence is common in female insects, and a trade-off between mating and immunity could affect the evolution of immunity. In this work, we tested the capacity of virgin and mated female Drosophila melanogaster to defend against infection by four bacterial pathogens. We found that female D. melanogaster suffer post-mating immunosuppression in a pathogen-dependent manner. The effect of mating was seen after infection with two bacterial pathogens (Providencia rettgeri and Providencia alcalifaciens), though not after infection with two other bacteria (Enterococcus faecalis and Pseudomonas aeruginosa). We then asked whether the evolution of post-mating immunosuppression is primarily a ‘female’ or ‘male’ trait by assaying for genetic variation among females for the degree of post-mating immune suppression they experience and among males for the level of post-mating immunosuppression they elicit in their mates. We also assayed for an interaction between male and female genotypes to test the specific hypothesis that the evolution of a trade-off between mating and immune defence in females might be being driven by sexual conflict. We found that females, but not males, harbour significant genetic variation for post-mating immunosuppression, and we did not detect an interaction between female and male genotypes. We thus conclude that post-mating immune depression is predominantly a ‘female’ trait, and find no evidence that it is evolving under sexual conflict.     

The evolution of antifungal peptides in Drosophila

An essential component of the immune system of animals is the production of antimicrobial peptides (AMPs). In vertebrates and termites the protein sequence of some AMPs evolves rapidly under positive selection, suggesting that they may be coevolving with pathogens. However, antibacterial peptides in Drosophila tend to be highly conserved. We have inferred the selection pressures acting on Drosophila antifungal peptides (drosomycins) from both the divergence of drosomycin genes within and between five species of Drosophila and polymorphism data from Drosophila simulans and D. melanogaster. In common with Drosophila antibacterial peptides, there is no evidence of adaptive protein evolution in any of the drosomycin genes, suggesting that they do not coevolve with pathogens. It is possible that this reflects a lack of specific fungal and bacterial parasites in Drosophila populations. The polymorphism data from both species differed from neutrality at one locus, but this was not associated with changes in the protein sequence. The synonymous site diversity was greater in D. simulans than in D. melanogaster, but the diversity both upstream of the genes and at nonsynonymous sites was similar. This can be explained if both upstream and nonsynonymous mutations are slightly deleterious and are removed more effectively from D. simulans due to its larger effective population size.     

No evidence for immune priming in ants exposed to a fungal pathogen

There is accumulating evidence that invertebrates can acquire long-term protection against pathogens through immune priming. However, the range of pathogens eliciting immune priming and the specificity of the response remain unclear. Here, we tested if the exposure to a natural fungal pathogen elicited immune priming in ants. We found no evidence for immune priming in Formica selysi workers exposed to Beauveria bassiana. The initial exposure of ants to the fungus did not alter their resistance in a subsequent challenge with the same fungus. There was no sign of priming when using homologous and heterologous combinations of fungal strains for exposure and subsequent challenges at two time intervals. Hence, within the range of conditions tested, the immune response of this social insect to the fungal pathogen appears to lack memory and strain-specificity. These results show that immune priming is not ubiquitous across pathogens, hosts and conditions, possibly because of immune evasion by the pathogen or efficient social defences by the host.     

Drosophila innate immunity and response to fungal infections

The fruit fly Drosophila melanogaster is an important model for the analysis of the interaction between host immune systems and fungal pathogens. Recent experiments have extended our understanding of the Toll-based signalling pathway critical to response to fungal infections, and identified new elements involved in cellular and humoral-based defences. The fly immune system shows remarkable sophistication in its ability to discriminate among pathogens, and the powerful genetics available to researchers studying the adult fly response, and the ability to manipulate cultured phagocytic cell lines with RNAi, are allowing researchers to dissect the molecular details of the process.     

Immune response of Drosophila melanogaster to infection with the flagellate parasite Crithidia spp

Insects are able to recognize invading microorganisms and to mount an immune response to bacterial and fungal infections. Recently, the fruitfly Drosophila melanogaster has emerged as a promising invertebrate model to investigate innate immunity because of its well-characterized genetics. Insects are also vectors of numerous parasites which can trigger an immune response. We have investigated the interaction of Drosophila melanogaster with the flagellate protozoan Crithidia spp. We show that a per os parasitic infection triggers the synthesis of several antimicrobial peptides. By reverse phase HPLC and mass spectrometry, peptides were shown to be present in the hemolymph and not in the gut tissue, suggesting the presence of immune messengers between the site of the infection, namely the gut, and the fat body, the main site of synthesis for antimicrobial peptides. Interestingly, we have identified one molecule which is specifically induced in the hemolymph after infection with Crithidia, but not with bacteria, suggesting that Drosophila can discriminate between pathogens. When flagellates were injected into the hemolymph, a low synthesis of antimicrobial peptides was observed together with phagocytosis of parasites by circulating hemocytes. The data presented here suggest that Drosophila-Crithidia spp. represents an interesting model to study host defense against protozoan parasites.     

Post-mating disparity between potential and realized immune response in Drosophila melanogaster

Reproductive costs are an essential component of evolutionary theory. For instance, an increase in reproduction is generally coupled with a decrease in immunocompetence shortly after mating. However, recent work in Drosophila melanogaster suggests that the potential to mount an immune response, as measured by the levels of immune gene expression, increases after mating. These data are in contrast to previous studies, which suggest that mating can reduce a fly's ability to survive an actual bacterial challenge (realized immunity). This pattern may be driven by some aspect of mating, independent of resource limitation, which reduces immune function by inhibiting the effective deployment of immune gene products. Though several studies have examined both the potential and the realized immunity after mating, none have examined these immune measures simultaneously. Here, we examined the link between the potential and the realized immunity in a sterile mutant of D. melanogaster. Shortly after mating, we found that female immune gene expression was high, but survival against infection was low. Surprisingly, this pattern was reversed within 24 h. Thus, estimates of immunity based on gene expression do not appear to reflect an actual ability to defend against pathogens in the hours following copulation. We discuss the possible mechanisms that may account for this pattern.     

Wolbachia Infections in Drosophila Melanogaster and D. Simulans: Polymorphism and Levels of Cytoplasmic Incompatibility

Wolbachia are endosymbiotic bacteria, widespread in terrestrial Arthropods. They are mainly transmitted vertically, from mothers to offspring and induce various alterations of their hosts' sexuality and reproduction, the most commonly reported phenomenon being Cytoplasmic Incompatibility (CI), observed in Drosophila melanogaster and D. simulans. Basically, CI results in a more or less intense embryonic mortality, occurring in crosses between males infected by Wolbachia and uninfected females. In D. simulans, Wolbachia and CI were observed in 1986. Since then, this host species has become a model system for investigating the polymorphism of Wolbachia infections and CI. In this review we describe the different Wolbachia infections currently known to occur in D. melanogaster and D. simulans. The two species are highly contrasting with regard to symbiotic diversity: while five Wolbachia variants have been described in D. simulans natural populations, D. melanogaster seems to harbor one Wolbachia variant only. Another marked difference between these two Drosophila species is their permissiveness with regard to CI, which seems to be fully expressed in D. simulans but partially or totally repressed in D. melanogaster, demonstrating the involvement of host factors in the control of CI levels. The potential of the two host species regarding the understanding of CI and its evolution is also discussed.