Bad guys turned nice? A critical assessment of Wolbachia mutualisms in arthropod hosts

Wolbachia are the most abundant bacterial endosymbionts among arthropods. Although maternally inherited, they do not conform to the widespread view that vertical transmission inevitably selects for beneficial symbionts. Instead, Wolbachia are notorious for their reproductive parasitism which, although lowering host fitness, ensures their spread. However, even for reproductive parasites it can pay to enhance host fitness. Indeed, there is a recent upsurge of reports on Wolbachia‐associated fitness benefits. Therefore, the question arises how such instances of mutualism are related to the phenotypes of reproductive parasitism. Here, we review the evidence of Wolbachia mutualisms in arthropods, including both facultative and obligate relationships, and critically assess their biological relevance. Although many studies report anti‐pathogenic effects of Wolbachia, few actually prove these effects to be relevant to field conditions. We further show that Wolbachia frequently have beneficial and detrimental effects at the same time, and that reproductive manipulations and obligate mutualisms may share common mechanisms. These findings undermine the idea of a clear‐cut distinction between Wolbachia mutualism and parasitism. In general, both facultative and obligate mutualisms can have a strong, and sometimes unforeseen, impact on the ecology and evolution of Wolbachia and their arthropod hosts. Acknowledging this mutualistic potential might be the key to a better understanding of some unresolved issues in the study of Wolbachia–host interactions.     

The impacts of Wolbachia and the microbiome on mate choice in Drosophila melanogaster

Symbionts and parasites can manipulate their hosts’ reproduction to their own benefit, profoundly influencing patterns of mate choice and evolution of the host population. Wolbachia is one of the most widespread symbionts among arthropods, and one that alters its hosts’ reproduction in diverse and dramatic ways. While we are beginning to appreciate how Wolbachia's extreme manipulations of host reproduction can influence species diversification and reproductive isolation, we understand little about how symbionts and Wolbachia, in particular, may affect intrapopulation processes of mate choice. We hypothesized that the maternally transmitted Wolbachia would increase the attractiveness of its female hosts to further its own spread. We therefore tested the effects of Wolbachia removal and microbiome disruption on female attractiveness and male mate choice among ten isofemale lines of Drosophila melanogaster. We found variable effects of general microbiome disruption on female attractiveness, with indications that bacteria interact with hosts in a line‐specific manner to affect female attractiveness. However, we found no evidence that Wolbachia influence female attractiveness or male mate choice among these lines. Although the endosymbiont Wolbachia can greatly alter the reproduction of their hosts in many species, there is no indication that they alter mate choice behaviours in D. melanogaster.     

Wolbachia do not live by reproductive manipulation alone: infection polymorphism in Drosophila suzukii and D. subpulchrella

Drosophila suzukii recently invaded North America and Europe. Populations in Hawaii, California, New York and Nova Scotia are polymorphic for Wolbachia, typically with <20% infection frequency. The Wolbachia in D. suzukii, denoted wSuz, is closely related to wRi, the variant prevalent in continental populations of D. simulans. wSuz is also nearly identical to Wolbachia found in D. subpulchrella, plausibly D. suzukii's sister species. This suggests vertical Wolbachia transmission through cladogenesis (‘cladogenic transmission’). The widespread occurrence of 7–20% infection frequencies indicates a stable polymorphism. wSuz is imperfectly maternally transmitted, with wild infected females producing on average 5–10% uninfected progeny. As expected from its low frequency, wSuz produces no cytoplasmic incompatibility (CI), that is, no increased embryo mortality when infected males mate with uninfected females, and no appreciable sex‐ratio distortion. The persistence of wSuz despite imperfect maternal transmission suggests positive fitness effects. Assuming a balance between selection and imperfect transmission, we expect a fitness advantage on the order of 20%. Unexpectedly, Wolbachia‐infected females produce fewer progeny than do uninfected females. We do not yet understand the maintenance of wSuz in D. suzukii. The absence of detectable CI in D. suzukii and D. subpulchrella makes it unlikely that CI‐based mechanisms could be used to control this species without transinfection using novel Wolbachia. Contrary to their reputation as horizontally transmitted reproductive parasites, many Wolbachia infections are acquired through introgression or cladogenesis and many cause no appreciable reproductive manipulation. Such infections, likely to be mutualistic, may be central to understanding the pervasiveness of Wolbachia among arthropods.     

Few parasites,and no evidence for Wolbachia infections,in a freshwater ostracod inhabiting temporary ponds

Biological systems with asexual reproduction have often attracted research on parasites and host immune defence, because parasites are expected to be better able to exploit genetically less diverse populations. In addition, maternally inherited parasitic microorganisms such as Wolbachia can directly alter the reproductive systems of their hosts and induce parthenogenesis. In the freshwater ostracod Eucypris virens, both sexual and asexual reproduction is known, and we speculated that parasite pressures might help to explain their co‐existence. This species complex inhabits shallow, often eutrophic temporary water bodies, conditions that should provide ample opportunities for parasite infections. We surveyed natural populations of E. virens throughout its Europe‐wide range for natural parasites, and particularly tested for the presence of intracellular Wolbachia bacteria. Surprisingly, the results indicate that very few E. virens populations support parasite infections. We also found no evidence for the presence of Wolbachia in the populations screened. The results therefore show that parasitic infections do not play a role in the maintenance of sex in this system. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 207–216.     

Host–endoparasitoid–endosymbiont relationships: concealed Strepsiptera provide new twist to Wolbachia in Australian tephritid fruit flies

Wolbachia are widespread endosymbionts that affect arthropod reproduction and fitness. Mostly maternally inherited, Wolbachia are occasionally transferred horizontally. Previously, two Wolbachia strains were reported at low prevalence and titres across seven Australian tephritid species, possibly indicative of frequent horizontal transfer. Here, we performed whole-genome sequencing of field-caught Wolbachia-positive flies. Unexpectedly, we found complete mitogenomes of an endoparasitic strepsipteran, Dipterophagus daci, suggesting that Wolbachia in the flies are linked to concealed parasitization. We performed the first genetic characterization of D. daci and detected D. daci in Wolbachia-positive flies not visibly parasitized, and most but not all Wolbachia-negative flies were D. daci-negative, presumably reflecting polymorphism for the Wolbachia infections in D. daci. We dissected D. daci from stylopized flies and confirmed that Wolbachia infects D. daci, but also found Wolbachia in stylopized fly tissues, likely somatic, horizontally transferred, non-heritable infections. Furthermore, no Wolbachia cif and wmk genes were detected and very low mitogenomic variation in D. daci across its distribution. Therefore, Wolbachia may influence host fitness without reproductive manipulation. Our study of 13 tephritid species highlights that concealed early stages of strepsipteran parasitization led to the previous incorrect assignment of Wolbachia co-infections to tephritid species, obscuring ecological studies of this common endosymbiont and its horizontal transmission by parasitoids.     

Ecology,Wolbachia infection frequency and mode of reproduction in the parasitoid wasp Tetrastichus coeruleus (Hymenoptera: Eulophidae)

Whereas sexual reproduction may facilitate adaptation to complex environments with many biotic interactions, simplified environments are expected to favour asexual reproduction. In agreement with this, recent studies on invertebrates have shown a prevalence of asexual species in agricultural (simplified) but not in natural (complex) environments. We investigated whether the same correlation between reproductive mode and habitat can be found in different populations within one species. The parasitoid wasp Tetrastichus coeruleus forms an ideal model to test this question, since it occurs both in natural and agricultural environments. Further, we investigated whether Wolbachia infection caused parthenogenesis in female‐biased populations. In contrast to the general pattern, in Dutch and French natural areas, we found Wolbachia‐infected, highly female‐biased populations that reproduce parthenogenetically. In contrast, populations on Dutch agricultural fields were not infected with Wolbachia, showed higher frequencies of males and reproduced sexually. However, we also found a female‐only, Wolbachia‐infected population on agricultural fields in north‐eastern United States. All Wolbachia‐infected populations were infected with the same Wolbachia strain. At this moment, we do not have a convincing explanation for this deviation from the general pattern of ecology and reproductive mode. It may be that asparagus agricultural fields differ from other crop fields in ways that favour sexual reproduction. Alternatively, Wolbachia may manipulate life history traits in its host, resulting in different fitness pay‐offs in different habitats. The fixation of Wolbachia in the United States populations (where the species was introduced) may be due to founder effect and lack of uninfected, sexual source populations.     

Wolbachia: intracellular manipulators of mite reproduction

Cytoplasmically transmitted Wolbachia (alpha-Proteobacteria) are a group of closely related intracellular microorganisms that alter reproduction in arthropods. They are found in a few isopods and are widespread in insects. Wolbachia are implicated as the cause of parthenogenesis in parasitic wasps, feminization in isopods and reproductive (cytoplasmic) incompatibility in many insects. Here we report on the widespread occurrence of Wolbachia in spider mites and predatory mites based on a PCR assay for a 730 bp fragment of the ftsZ gene with primers that are specific for Wolbachia. An additional PCR, using two primer pairs that amplify a 259 bp region of the ftsZ gene that are diagnostic for the two Wolbachia subdivisions A and B, showed that infected mites only carried type B and not type A Wolbachia. The fact that some species tested negative for Wolbachia does not mean that the entire species is uninfected. We found that natural populations of Tetranychus urticae are polymorphic for the infection. The possible effects of Wolbachia on mite reproduction and post-zygotic reproductive isolation are discussed.To whom correspondence should be addressed at: Kruislaan 320, 1098 SM Amsterdam, The Netherlands     

Fitness effects of Wolbachia and Spiroplasma in Drosophila melanogaster

Maternally inherited endosymbionts that manipulate the reproduction of their insect host are very common. Aside from the reproductive manipulation they produce, the fitness of these symbionts depends in part on the direct impact they have on the female host. Although this parameter has commonly been investigated for single infections, it has much more rarely been established in dual infections. We here establish the direct effect of infection with two different symbionts exhibiting different reproductive manipulation phenotypes, both alone and in combination, in the fruit fly Drosophila melanogaster. This species carries a cytoplasmic incompatibility inducing Wolbachia and a male-killing Spiroplasma, occurring as single or double (co-) infections in natural populations. We assessed direct fitness effects of these bacteria on their host, by comparing larval competitiveness and adult fecundity of uninfected, Wolbachia, Spiroplasma and Wolbachia–Spiroplasma co-infected females. We found no effect of infection status on the fitness of females for both estimates, that is, no evidence of any benefits or costs to either single or co-infection. This leads to the conclusion that both bacteria probably have other sources of benefits to persist in D. melanogaster populations, either by means of their reproductive manipulations (fitness compensation from male death in Spiroplasma infection and cytoplasmic incompatibility in Wolbachia infection) or by positive fitness interactions on other fitness components.     

The transmission and effects of Wolbachia bacteria in parasitoids

Wolbachia bacteria are obligatory intracellular parasites of arthropods and have been detected in about 70 species of parasitic wasps and three parasitoid flies. Wolbachia are transmitted cytoplasmically (maternally) and modify host reproduction in different ways to enhance their own transmission: parthenogenesis induction (PI), cytoplasmic incompatibility (CI), or feminization (F) of genetic males. Only PI and CI are known in parasitoids. PI-Wolbachia cause thelytoky in otherwise arrhenotokous parasitoids by generating diploid (rather than haploid) unfertilized wasp eggs. CI-Wolbachia cause incompatibility of crosses between infected males and uninfected females because the paternally derived chromosomes fail to decondense and are destroyed after syngamy. More complex situations arise when hosts harbor multiple infections, which can lead to bidirectional incompatibility and may be involved in parasitoid speciation. The relative fitness of infected and uninfected hosts is important to the population dynamics of Wolbachia, and more data are needed. Evolutionary conflict should be common between host genes, Wolbachia genes, and other "selfish" genetic elements. Wolbachia-specific PCR primers are now available for several genes with different rates of evolution. These primers will permit rapid screening in future studies of spatial and temporal patterns of single and multiple infection. Molecular phylogenies show that CI- and PI-Wolbachia do not form discrete clades. In combination with experimental transfection data, this result suggests that host reproductive alterations depend on the interaction between attributes of both Wolbachia and host. Moreover, Wolbachia isolates from closely related hosts do not usually cluster together, and phylogenies suggest that Wolbachia may have radiated after their arthropod hosts. Both results support considerable horizontal transmission of Wolbachia between host species over evolutionary time. Natural horizontal transmisson between parasitoids and their hosts, or with entomoparasitic nematodes or ectoparasitic mites, remains a tantalizing but equivocal possibility. Received: November 27, 1998 / Accepted: January 15, 1999     

The effect of Wolbachia on the lifetime reproductive success of its insect host in the field

Wolbachia is a widespread endosymbiont that induces dramatic manipulations of its host's reproduction. Although there has been substantial progress in the developing theory for Wolbachia–host interactions and in measuring the effects of Wolbachia on host fitness in the laboratory, there is a widely recognized need to quantify the effects of Wolbachia on the host fitness in the field. The wasp Anagrus sophiae, an egg parasitoid of planthoppers, carries a Wolbachia strain that induces parthenogenesis, but its effects on the fitness of its Anagrus host are unknown. We developed a method to estimate the realized lifetime reproductive success of female wasps by collecting them soon after they die naturally in the field, counting the number of eggs remaining in their ovaries and quantifying Wolbachia density in their body. We sampled from a highly infected A. sophiae population and found no evidence for Wolbachia virulence and possible evidence for positive effects of Wolbachia on realized reproductive success.     

Independent cytoplasmic incompatibility induced by Cardinium and Wolbachia maintains endosymbiont coinfections in haplodiploid thrips populations

Cardinium and Wolbachia are common maternally inherited reproductive parasites that can coinfect arthropods, yet interactions between both bacterial endosymbionts are rarely studied. For the first time, we report their independent expression of complete cytoplasmic incompatibility (CI) in a coinfected host, and CI in a species of the haplodiploid insect order Thysanoptera. In Pezothrips kellyanus, Cardinium‐induced CI resulted in a combination of male development (MD) and embryonic female mortality (FM) of fertilized eggs. In contrast, Wolbachia‐induced CI resulted in FM together with postembryonic mortality not previously reported as a CI outcome. Both endosymbionts appeared to not influence fecundity but virgins produced more offspring than mated females. In coinfected individuals, Wolbachia density was higher than Cardinium. Wolbachia removal did not impact Cardinium density, suggesting a lack of competition within hosts. Maternal transmission was complete for Wolbachia and high for Cardinium. Our data support theoretical predictions and empirical detection of high endosymbiont prevalence in field populations of the native range of this pest thrips. However, previous findings of more frequent loss of Wolbachia than Cardinium, particularly in field populations of the host's invasive range, suggest that genetic diversity or varying environmental factors between field populations also play a role in shaping host‐endosymbiont dynamics.     

Maintenance of adaptive differentiation by Wolbachia induced bidirectional cytoplasmic incompatibility: the importance of sib-mating and genetic systems

Background  

Bacteria of the genus Wolbachia are reproductive parasites widespread among arthropods. The most common effect arising from the presence of Wolbachia in a population is Cytoplasmic Incompatibility (CI), whereby postmating reproductive isolation occurs in crosses between an infected male and an uninfected female, or when a male is infected with a different strain of Wolbachia to that of the female (bidirectional CI). Previous theoretical models have demonstrated that bidirectional CI can contribute to the genetic divergence of populations in haploid and diploid organisms. However, haplodiploid organisms were not considered in these models even though they include Nasonia parasitoid wasps – the best example of the implication of Wolbachia in ongoing speciation. Moreover, previous work did not investigate inbreeding mating systems, which are frequently observed in arthropod species.     

Consistently high incidence of Wolbachia in global fig wasp communities

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High prevalence of Wolbachia infection does not explain unidirectional cytoplasmic incompatibility of Altica flea beetles

1. Wolbachia are intracellular bacteria found in a wide range of arthropods that can impact reproductive isolation of their hosts. Previous studies showed unidirectional cytoplasmic incompatibility (CI) and high infection rates by Wolbachia in the closely related leaf beetles Altica fragariae and Altica viridicyanea; however, whether this reproductive isolation was induced by Wolbachia remains unclear. 2. This study estimated the prevalence of Wolbachia in Altica beetles, assessed genetic diversity of Wolbachia strains infecting these beetles, and tested whether Wolbachia-induced CI explains reproductive isolation of A. fragariae and A. viridicyanea. 3. The results show that all of the 11 tested Altica species were infected by Wolbachia, and the infection rate was as high as 97.0% (n = 235). Multi-strain infections were common, being found in 10 of the 11 species tested and accounting for 23.0% of all screened specimens. In total, 35 Wolbachia strains were identified based on 208 wsp sequences obtained. Although a majority of A. fragariae and A. viridicyanea individuals from the Beijing population were infected with only one strain each, multi-strain infections did occur in both species. Antibiotic curing experiments did not change hatching success in either inter- or intraspecific crosses of A. fragariae and A. viridicyanea, indicating that these Wolbachia strains do not induce CI. These results were further corroborated by the lack of the Wolbachia cifB gene, which is responsible for causing CI. 4. These findings suggest that high prevalence of Wolbachia infection is unlikely to contribute to reproductive isolation and speciation in this system.     

Detection and characterization of Wolbachia infections in laboratory and natural populations of different species of tsetse flies (genus Glossina)

Background

Wolbachia is a genus of endosymbiotic α-Proteobacteria infecting a wide range of arthropods and filarial nematodes. Wolbachia is able to induce reproductive abnormalities such as cytoplasmic incompatibility (CI), thelytokous parthenogenesis, feminization and male killing, thus affecting biology, ecology and evolution of its hosts. The bacterial group has prompted research regarding its potential for the control of agricultural and medical disease vectors, including Glossina spp., which transmits African trypanosomes, the causative agents of sleeping sickness in humans and nagana in animals.

Results

In the present study, we employed a Wolbachia specific 16S rRNA PCR assay to investigate the presence of Wolbachia in six different laboratory stocks as well as in natural populations of nine different Glossina species originating from 10 African countries. Wolbachia was prevalent in Glossina morsitans morsitans, G. morsitans centralis and G. austeni populations. It was also detected in G. brevipalpis, and, for the first time, in G. pallidipes and G. palpalis gambiensis. On the other hand, Wolbachia was not found in G. p. palpalis, G. fuscipes fuscipes and G. tachinoides. Wolbachia infections of different laboratory and natural populations of Glossina species were characterized using 16S rRNA, the wsp (Wolbachia Surface Protein) gene and MLST (Multi Locus Sequence Typing) gene markers. This analysis led to the detection of horizontal gene transfer events, in which Wobachia genes were inserted into the tsetse flies fly nuclear genome.

Conclusions

Wolbachia infections were detected in both laboratory and natural populations of several different Glossina species. The characterization of these Wolbachia strains promises to lead to a deeper insight in tsetse flies-Wolbachia interactions, which is essential for the development and use of Wolbachia-based biological control methods.

     

A comparative analysis of Wolbachia‐induced host reproductive phenotypes reveals transition rate heterogeneity

The endosymbiotic bacterium Wolbachia infects a wide range of arthropods and their relatives. It is an intracellular parasite transmitted through the egg from mother to offspring. Wolbachia can spread and persist through various means of host reproductive manipulation. How these different mechanisms of host manipulation evolved in Wolbachia is unclear. Which host reproductive phenotype is most likely to be ancestral and whether evolutionary transitions between some host phenotypes are more common than others remain unanswered questions. Recent studies have revealed multiple cases where the same Wolbachia strain can induce different reproductive phenotypes in different hosts, raising the question to what degree the induced host phenotype should be regarded as a trait of Wolbachia. In this study, we constructed a phylogenetic tree of Wolbachia and analyzed the patterns of host phenotypes along that tree. We were able to detect a phylogenetic signal of host phenotypes on the Wolbachia tree, indicating that the induced host phenotype can be regarded as a Wolbachia trait. However, we found no clear support for the previously stated hypothesis that cytoplasmic incompatibility is ancestral to Wolbachia in arthropods. Our analysis provides evidence for heterogeneous transition rates between host phenotypes.     

Absence of <Emphasis Type="Italic">Wolbachia</Emphasis> in Nonfilariid Worms Parasitizing Arthropods

Wolbachia are strictly intracellular maternally inherited α-proteobacteria, largely widespread among arthropods and filariids (i.e., filarial nematodes). Wolbachia capacities to infect new host species have been greatly evidenced and the transfer of Wolbachia between arthropods and filariids has probably occurred more than once. Interestingly, among nematode species, Wolbachia infection was found in filariids but not in closely related lineages. Their occurrence in filariids has been supposed a consequence of the parasitic lifestyle of worms within Wolbachia-infected arthropods, implying that nonfilariid worms parasitizing arthropods are also likely to be infected by some Wolbachia acquired from their hosts. To further investigate this hypothesis, we have examined seven species of nonfilariid worms of Nematoda and Nematomorpha phyla, all interacting intimately with arthropods. Wolbachia infection in nonfilariid parasitic worms was never detected by polymerase chain reaction assays of the 16S rDNA and wsp genes. By contrast, some arthropod hosts are well infected by Wolbachia of the B supergroup. Then the intimate contact with infected arthropods is not a sufficient condition to explain the Wolbachia occurrence in filariids and could underline a physiological singularity or a particular evolutionary event to acquire and maintain Wolbachia infection.     

Antagonistic interaction between male-killing and cytoplasmic incompatibility induced by Cardinium and Wolbachia in the whitefly,Bemisia tabaci

Cardinium and Wolbachia are maternally inherited bacterial symbionts of arthropods that can manipulate host reproduction by increasing the fitness of infected females.Here,we report that Cardinium and Wolbachia coinfection induced male-killing and cytoplasmic incompatibility(CI)when they coexisted in a cryptic species of whitefly,Bemisia tabaci Asia II7.Cardinium and Wolbachia symbionts were either singly or simul-taneously localized in the bacteriocytes placed in the abdomen of B.tabaci nymphs and adults.Cardinium-Wolbachia coinfection induced male-killing and resulted in a higher female sex ratio in the intraspecific amphigenetic progeny of Asia II7 IcwH and IcwL lines;interestingly,male-illing induction was enhanced with increased Cardinium titer.Moreover,single infection of Wolbachia induced partial CI in the Asia II7 Iw line and resulted in reduced fecundity,higher embryonic mortality,and lower female sex ratio.The uninfected Asia II7 Iu line had significantly higher fecundity,lower embryonic and nymphal mortalities,and a lower level of CI than both the Wolbachia infected Asia II7 Iw line and the Cardinium--Wolbachia-coinfected Asia II7 IcwH line.Our findings indicate that Cardinium-Wolbachia coinfection induced male-killing,which may have had antag-onistic effects on Wolbachia-induced CI in the Asia II7 whiteflies.For the first time,our study revealed that B.tabaci Asia II7 reproduction is co-manipulated by Cardinium and Wolbachia endosymbionts.     

Survey of <Emphasis Type="Italic">Wolbachia</Emphasis> and Its Phage WO in the Uzifly <Emphasis Type="Italic">Exorista sorbillans</Emphasis> (Diptera: Tachinidae)

Wolbachia are cytoplasmically inherited alpha-proteobacteria well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts they infect. Despite their obligate intracellular lifestyle which usually protects bacteria from phage infection, Wolbachia harbor a widespread temperate phage called WO. Evidences of horizontal phage transfers indicate that this phage could promote genetic exchanges between strains leading to evolutionary changes in the genomes of Wolbachia, and could be involved in the phenotypes these bacteria induced. In this study, we report the survey of Wolbachia and WO phage infections in 20 populations of the Uzifly Exorista sorbillans, a tachinid endoparasite of silkworm Bombyx mori, collected from different geographic regions of India. Previous studies demonstrated that Wolbachia is associated with positive reproductive fitness effects in this species. Polymerase chain reaction using the ftsZ gene encoding for a Wolbachia cell division protein and the orf7 capsid protein gene of the phage showed that all flies checked were infected by Wolbachia and its phage WO. Phylogenetic analyses based on the Wolbachia surface protein gene revealed 100% of double infections by the arthropod supergroups A and B. These results can serve as a valuable basis for understanding the evolution of Wolbachia bacteria and may provide information about the dynamics of Wolbachia–host associations. This knowledge could be exploited for the use of Wolbachia for effective control strategies of the Uzifly, a serious menace of the silkworm B. mori.     

Endosymbiont costs and benefits in a parasitoid infected with both Wolbachia and Cardinium

Theory suggests that maternally inherited endosymbionts can promote their spread and persistence in host populations by enhancing the production of daughters by infected hosts, either by improving overall host fitness, or through reproductive manipulation. In the doubly infected parasitoid wasp Encarsia inaron, Wolbachia manipulates host reproduction through cytoplasmic incompatibility (CI), but Cardinium does not. We investigated the fitness costs and/or benefits of infection by each bacterium in differentially cured E. inaron as a potential explanation for persistence of Cardinium in this population. We introgressed lines infected with Wolbachia, Cardinium or both with the cured line to create a similar genetic background, and evaluated several parasitoid fitness parameters. We found that symbiont infection resulted in both fitness costs and benefits for E. inaron. The cost was lower initial egg load for all infected wasps. The benefit was increased survivorship, which in turn increased male production for wasps infected with only Cardinium. Female production was unaffected by symbiont infection; we therefore have not yet identified a causal fitness effect that can explain the persistence of Cardinium in the population. Interestingly, the Cardinium survivorship benefit was not evident when Wolbachia was also present in the host, and the reproduction of doubly infected individuals did not differ significantly from uninfected wasps. Therefore, the results of our study show that even when multiple infections seem to have no effect on a host, there may be a complex interaction of costs and benefits among symbionts.