Detection of Spiroplasma from the mite Macrocheles sp. (Acari; Macrochelidae) ectoparasitic to the fly Drosophila hydei (Diptera; Drosophilidae): a possible route of horizontal transmission?

Some members of the genus Spiroplasma are vertically transmitted endosymbionts of insects. Among them, Spiroplasma sp. Dhd, a member of the Spiroplasma poulsonii clade, is highly prevalent among worldwide populations of Drosophila hydei. Here we found that 53 out of 3,763 wild-caught D. hydei (1.4 %) were ectoparasitized by the mite that belong to the genus Macrocheles. Many of the ectoparasitized flies (79 %) had a single mite, but some flies had up to five mites. Among 59 mites subjected to Spiroplasma-specific PCR, 15 individuals were found to be positive. Infection status of Spiroplasma in flies and the associated mites were incongruent. Partial nucleotide sequences of the Spiroplasma P58 gene suggest that some of the mites are infected with a Spiroplasma, which is identical or closely related to Spiroplasma sp. Dhd. This finding provides a potential route of horizontal Spiroplasma transmission between D. hydei individuals in natural populations. In addition, a Spiroplasma strain that does not form a monophyletic group with S. poulsonii was also found from a mite individual.     

Male Killing and Incomplete Inheritance of a Novel <Emphasis Type="Italic">Spiroplasma</Emphasis> in the Moth <Emphasis Type="Italic">Ostrinia zaguliaevi</Emphasis>

Bacteria of the genus Spiroplasma are widely found in plants and arthropods. Some of the maternally transmitted Spiroplasma endosymbionts in arthropods are known to kill young male hosts (male killing). Here, we describe a new case of Spiroplasma-induced male killing in a moth, Ostrinia zaguliaevi. The all-female trait caused by Spiroplasma was maternally inherited for more than 11 generations but was spontaneously lost in several lineages. Antibiotic treatment eliminated the Spiroplasma infection and restored the 1:1 sex ratio. The survival rates and presence/absence of the W chromosome in the embryonic and larval stages of O. zaguliaevi showed that males were selectively killed, exclusively during late embryogenesis in all-female broods. Based on phylogenetic analyses of 16S rRNA, dnaA and rpoB gene sequences, the causative bacteria were identified as Spiroplasma belonging to the tick symbiont Spiroplasma ixodetis clade. Electron microscopy confirmed bacterial structures in the follicle cells and follicular sheath of adult females. Although many congeneric Ostrinia moths harbor another sex ratio-distorting bacterium (Wolbachia), only O. zaguliaevi harbors Spiroplasma.     

Wolbachia Infect Ovaries in the Course of Their Maturation: Last Minute Passengers and Priority Travellers?

Wolbachia are widespread endosymbiotic bacteria of arthropods and nematodes. Studies on such models suggest that Wolbachia''s remarkable aptitude to infect offspring may rely on a re-infection of ovaries from somatic tissues instead of direct cellular segregation between oogonia and oocytes. In the terrestrial isopod Armadillidium vulgare, Wolbachia are vertically transmitted to the host offspring, even though ovary cells are cyclically renewed. Using Fluorescence in situ hybridization (FISH), we showed that the proportion of infected oocytes increased in the course of ovary and oocyte maturation, starting with 31.5% of infected oocytes only. At the end of ovary maturation, this proportion reached 87.6% for the most mature oocytes, which is close to the known transmission rate to offspring. This enrichment can be explained by a secondary acquisition of the bacteria by oocytes (Wolbachia can be seen as last minute passengers) and/or by a preferential selection of oocytes infected with Wolbachia (as priority travellers).     

No seasonal trend in infection of the pale grass blue butterfly, Zizeeria maha (Lepidoptera: Lycaenidae), by Wolbachia

Wolbachia are Gram-negative bacteria that cause intracellular inherited infections in many invertebrates. They are extremely common, with 20–75 % of all insects being infected. Wolbachia belong to taxa of the Anaplasmataceae family, alpha proteobacteria. Because previous studies have shown that Wolbachia generally disappear from the host insect’s body under high-temperature conditions in laboratories, we investigated seasonal changes in infection of the pale grass blue butterfly, Zizeeria maha (Kollar, 1848) by Wolbachia, for 7 months of the year. Total annual infection was 86.7 % (n = 15) of females and 96.3 % (n = 81) of males. Statistical analysis showed that monthly infection was not significantly different among months. In addition, no significant difference was found between Wolbachia densities in cells of summer and winter butterfly samples by use of real-time PCR during the months examined. The results suggest that Wolbachia infection is not affected by seasonal factors, at least for Z. maha.     

Male killing Spiroplasma protects Drosophila melanogaster against two parasitoid wasps

Maternally transmitted associations between endosymbiotic bacteria and insects are diverse and widespread in nature. Owing to imperfect vertical transmission, many heritable microbes have evolved compensational mechanisms to enhance their persistence in host lineages, such as manipulating host reproduction and conferring fitness benefits to host. Symbiont-mediated defense against natural enemies of hosts is increasingly recognized as an important mechanism by which endosymbionts enhance host fitness. Members of the genus Spiroplasma associated with distantly related Drosophila hosts are known to engage in either reproductive parasitism (i.e., male killing) or defense against natural enemies (the parasitic wasp Leptopilina heterotoma and a nematode). A male-killing strain of Spiroplasma (strain Melanogaster Sex Ratio Organism (MSRO)) co-occurs with Wolbachia (strain wMel) in certain wild populations of the model organism Drosophila melanogaster. We examined the effects of Spiroplasma MSRO and Wolbachia wMel on Drosophila survival against parasitism by two common wasps, Leptopilina heterotoma and Leptopilina boulardi, that differ in their host ranges and host evasion strategies. The results indicate that Spiroplasma MSRO prevents successful development of both wasps, and confers a small, albeit significant, increase in larva-to-adult survival of flies subjected to wasp attacks. We modeled the conditions under which defense can contribute to Spiroplasma persistence. Wolbachia also confers a weak, but significant, survival advantage to flies attacked by L. heterotoma. The host protective effects exhibited by Spiroplasma and Wolbachia are additive and may provide the conditions for such cotransmitted symbionts to become mutualists. Occurrence of Spiroplasma-mediated protection against distinct parasitoids in divergent Drosophila hosts suggests a general protection mechanism.     

A Veritable Menagerie of Heritable Bacteria from Ants,Butterflies, and Beyond: Broad Molecular Surveys and a Systematic Review

Maternally transmitted bacteria have been important players in the evolution of insects and other arthropods, affecting their nutrition, defense, development, and reproduction. Wolbachia are the best studied among these and typically the most prevalent. While several other bacteria have independently evolved a heritable lifestyle, less is known about their host ranges. Moreover, most groups of insects have not had their heritable microflora systematically surveyed across a broad range of their taxonomic diversity. To help remedy these shortcomings we used diagnostic PCR to screen for five groups of heritable symbionts—Arsenophonus spp., Cardinium hertigii, Hamiltonella defensa, Spiroplasma spp., and Wolbachia spp.—across the ants and lepidopterans (focusing, in the latter case, on two butterfly families—the Lycaenidae and Nymphalidae). We did not detect Cardinium or Hamiltonella in any host. Wolbachia were the most widespread, while Spiroplasma (ants and lepidopterans) and Arsenophonus (ants only) were present at low levels. Co-infections with different Wolbachia strains appeared especially common in ants and less so in lepidopterans. While no additional facultative heritable symbionts were found among ants using universal bacterial primers, microbes related to heritable enteric bacteria were detected in several hosts. In summary, our findings show that Wolbachia are the dominant heritable symbionts of ants and at least some lepidopterans. However, a systematic review of symbiont frequencies across host taxa revealed that this is not always the case across other arthropods. Furthermore, comparisons of symbiont frequencies revealed that the prevalence of Wolbachia and other heritable symbionts varies substantially across lower-level arthropod taxa. We discuss the correlates, potential causes, and implications of these patterns, providing hypotheses on host attributes that may shape the distributions of these influential bacteria.     

Detection of Spiroplasma and Wolbachia in the Bacterial Gonad Community of Chorthippus parallelus

We have recently detected the endosymbiont Wolbachia in multiple individuals and populations of the grasshopper Chorthippus parallelus (Orthoptera: acrididae). This bacterium induces reproductive anomalies, including cytoplasmic incompatibility. Such incompatibilities may help explain the maintenance of two distinct subspecies of this grasshopper, C. parallelus parallelus and C. parallelus erythropus, which are involved in a Pyrenean hybrid zone that has been extensively studied for the past 20 years, becoming a model system for the study of genetic divergence and speciation. To evaluate whether Wolbachia is the sole bacterial infection that might induce reproductive anomalies, the gonadal bacterial community of individuals from 13 distinct populations of C. parallelus was determined by denaturing gradient gel electrophoresis analysis of bacterial 16S rRNA gene fragments and sequencing. The study revealed low bacterial diversity in the gonads: a persistent bacterial trio consistent with Spiroplasma sp. and the two previously described supergroups of Wolbachia (B and F) dominated the gonad microbiota. A further evaluation of the composition of the gonad bacterial communities was carried out by whole cell hybridization. Our results confirm previous studies of the cytological distribution of Wolbachia in C. parallelus gonads and show a homogeneous infection by Spiroplasma. Spiroplasma and Wolbachia cooccurred in some individuals, but there was no significant association of Spiroplasma with a grasshopper’s sex or with Wolbachia infection, although subtle trends might be detected with a larger sample size. This information, together with previous experimental crosses of this grasshopper, suggests that Spiroplasma is unlikely to contribute to sex-specific reproductive anomalies; instead, they implicate Wolbachia as the agent of the observed anomalies in C. parallelus.     

Infection densities of three Spiroplasma strains in the host Drosophila melanogaster

Maternally transmitted endosymbiotic bacteria of the genus Spiroplasma associate with numerous insect species, including the genus Drosophila. Among the Spiroplasma strains associated with Drosophila, several manipulate their host??s reproduction by killing the male offspring of the infected females. Although the male-killing mechanism is not well understood, previous studies of non-native strains transferred to D. melanogaster (strain Oregon-R) indicate that the male-killing strain achieves higher densities than two non-male-killing strains. Whether this pattern of higher male-killing strain densities occurs in other host-symbiont strain combinations is not known. Herein, we used quantitative PCR to examine infection densities of one non-male-killing strain native to D. hydei (Hyd1), and two male-killing strains; one native to D. nebulosa (NSRO), and one native to D. melanogaster (MSRO; recently discovered), upon artificial transfer to D. melanogaster (strain Canton-S). Infection densities were examined at four weekly intervals in adult flies, across three consecutive generations following artificial transfer. Infection densities of the non-male-killing strain were significantly lower than those of the two male killers immediately after adult emergence. At later time points, however, the non-male-killing strain (Hyd1) is capable of proliferating to densities similar to those of the two male-killing strains (NSRO and MSRO) in D. melanogaster (Canton-S). We also examined the effect of co-infection by the heritable bacterium Wolbachia, on Spiroplasma densities and male-killing ability. Wolbachia had little to no effect of Spiroplasma densities, but the male-killing ability of MSRO was lower in the presence of Wolbachia. Generation post-infection had little effect on Spiroplasma densities, but affected the male-killing ability.     

Diversity of Wolbachia in Natural Populations of Spider Mites (genus Tetranychus): Evidence for Complex Infection History and Disequilibrium Distribution

Wolbachia are endosymbiotic bacteria that commonly infect arthropods and cause reproductive disorders in host. Within several Tetranychus species, Wolbachia have been detected and shown to affect their reproduction. However, little is known about their transmission and distribution patterns in natural populations of Tetranychus species. Here, we used multilocus sequence typing to confirm Wolbachia infection status and examined the relationship between Wolbachia infection status and host phylogeny, mitochondrial diversity, and geographical range in five Tetranychus species (Tetranychus truncatus, Tetranychus urticae, Tetranychus pueraricola, Tetranychus phaselus, and Tetranychus kanzawai) from 21 populations in China. The prevalence of Wolbachia within the five Tetranychus species ranged from 31.4 to 100 %, and the strains were remarkably diverse. Together, these observations indicate that Wolbachia was introduced to these populations on multiple separate occasions. As in other arthropods, the same Tetranychus species can accommodate very different strains, and identical Wolbachia occasionally infect different species. These observations suggest that Wolbachia are transmitted both vertically and horizontally. Horizontally, transmission is probably mediated by the host plants. The distribution patterns of Wolbachia were quite different among populations of the same species, suggesting that the dynamics of Wolbachia in nature may be affected by ecological and other factors.     

Phylogenetic analysis of the pathogenic bacteria Spiroplasma penaei based on multilocus sequence analysis

A pathogenic Spiroplasma penaei strain was isolated from the hemolymph of moribund Pacific white shrimp, Penaeus vannamei. The shrimp sample originated from a shrimp farm near Cartagena, Colombia, that was suffering from high mortalities in ponds with very low salinity and high temperatures. This new emerging disease in a marine crustacean in the Americas is described as a systemic infection. The multilocus phylogenetic analysis suggests that S. penaei strain has a terrestrial origin. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S rDNA and 23S-5S rDNA intergenic spacer region, were reconstructed using the distance-based Neighboring-Joining (NJ) method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborate the observations by other investigators using the 16S rDNA gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to Spiroplasma insolitum and distantly related to Spiroplasma sp. SHRIMP from China.     

Natural variation in population densities and vertical transmission rates of a Spiroplasma endosymbiont in Drosophila hydei

A bacterium belonging to the genus Spiroplasma, an endosymbiont of the fly Drosophila hydei, is vertically transmitted through host egg cytoplasm. To infer vertical transmission rates of Spiroplasma in natural populations of D. hydei, the infection status of Spiroplasma was examined for offspring produced by Spiroplasma-positive females that were collected in two geographical populations. In both populations, nearly half of the broods consisted of only infected offspring. Infection frequencies of the rest of the broods ranged from 0.364 to 0.975. Quantitative PCR demonstrated that the Spiroplasma titers in the whole body of wild-caught females were highly variable (1.81?×?10⁶–5.60?×?10⁸ cells per insect). Contrary to our expectations, however, the Spiroplasma titers did not account for the variation in infection frequencies among offspring (i.e., vertical transmission rates). These results suggest that the spatial distribution of Spiroplasma, particularly in somatic tissues and germ tissues, is highly variable among host individuals, which may be caused by environmental stochasticity or some unknown effects.     

Asymmetrical Interactions between Wolbachia and Spiroplasma Endosymbionts Coexisting in the Same Insect Host

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.     

Interspecific transmission of endosymbiotic Spiroplasma by mites

The occurrence of closely related strains of maternally transmitted endosymbionts in distantly related insect species indicates that these infections can colonize new host species by lateral transfer, although the mechanisms by which this occurs are unknown. We investigated whether ectoparasitic mites, which feed on insect haemolymph, can serve as interspecific vectors of Spiroplasma poulsonii, a male-killing endosymbiont of Drosophila. Using Spiroplasma-specific primers for PCR, we found that mites can pick up Spiroplasma from infected Drosophila nebulosa females and subsequently transfer the infection to Drosophila willistoni. Some of the progeny of the recipient D. willistoni were infected, indicating successful maternal transmission of the Spiroplasma within the new host species. However, the transmission rate of the infection from recipient flies to their offspring was low, perhaps due to low Spiroplasma density in the recipient flies.     

Sharing cells with Wolbachia: the transovarian vertical transmission of Culex pipiens densovirus

Culex pipiens densovirus (CpDV), a single stranded DNA virus, has been isolated from Culex pipiens mosquitoes but differs from other mosquito densoviruses in terms of genome structure and sequence identity. Its transmission from host to host, the nature of its interactions with both its host and host's endosymbiotic bacteria Wolbachia are not known. Here, we report the presence of CpDV in the ovaries and eggs of Cx. pipiens mosquitoes in close encounters with Wolbachia. In the ovaries, CpDV amount significantly differed between mosquito lines harbouring different strains of Wolbachia and these differences were not linked to variations in Wolbachia densities. CpDV was vertically transmitted in all laboratory lines to 17%–20% of the offspring. For some females, however, the vertical transmission reached 90%. Antibiotic treatment that cured the host from Wolbachia significantly decreased both CpDV quantity and vertical transmission suggesting an impact of host microbiota, including Wolbachia, on CpDV transmission. Overall our results show that CpDV is transmitted vertically via transovarian path along with Wolbachia with which it shares the same cells. Our results are primordial to understand the dynamics of densovirus infection, their persistence and spread in populations considering their potential use in the regulation of mosquito vector populations.     

Association between Wolbachia and Spiroplasma within Drosophila neotestacea: an emerging symbiotic mutualism?

Interspecific mutualism can evolve when specific lineages of different species tend to be associated with each other from one generation to the next. Different maternally transmitted endosymbionts occurring within the same cytoplasmic lineage fulfil this requirement. Drosophila neotestacea is infected with maternally transmitted Wolbachia and Spiroplasma, which are cotransmitted at high frequency in natural populations. Molecular phylogenetic evidence indicates that both endosymbionts have been present in D. neotestacea for considerable evolutionary periods. Thus, conditions are suitable for the evolution of mutualism between them. In support of this possibility, there is a significant positive association between Wolbachia and Spiroplasma infection in many samples of D. neotestacea from natural populations. Theoretically, such a positive association can result from either mutualism between these endosymbionts or recent spread. Collections from present‐day populations suggest that recent spread and mutualism have both operated to generate the positive association between Wolbachia and Spiroplasma. If selection acts on the combination of these two endosymbionts, they may be in the early stages of evolution of a more complex, cooperative association.     

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales.     

Molecular evolution of the actin-like MreB protein gene family in wall-less bacteria

The mreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthesis and often exists in one to three copies in the genomes of non-spherical bacteria. Intriguingly, while most wall-less bacteria do not have this gene, five to seven mreB homologs are found in Spiroplasma and Haloplasma, which are both characterized by cell contractility. To investigate the molecular evolution of this gene family in wall-less bacteria, we sampled the available genome sequences from these two genera and other related lineages for comparative analysis. The gene phylogenies indicated that the mreB homologs in Haloplasma are more closely related to those in Firmicutes, whereas those in Spiroplasma form a separate clade. This finding suggests that the gene family expansions in these two lineages are the results of independent ancient duplications. Moreover, the Spiroplasma mreB homologs can be classified into five clades, of which the genomic positions are largely conserved. The inference of gene gains and losses suggests that there has been an overall trend to retain only one homolog from each of the five mreB clades in the evolutionary history of Spiroplasma.     

Selenium and Topiramate Attenuates Blood Oxidative Toxicity in Patients with Epilepsy: A Clinical Pilot Study

It is well known that oxidative stress plays an important role in the etiology of epilepsy. We investigated effects of selenium (Se) and topiramate (TPM) combination supplementation on antioxidant and oxidant values in control and patients with epilepsy and refractory epilepsy. For the aim, we used control (n?=?19), epilepsy + TPM (n?=?19), epilepsy + TPM + Se (n?=?15) groups. We also used control (n?=?15), refractory epilepsy (n?=?15), and refractory epilepsy + Se (n?=?8) groups. TPM (0.2 mg/daily) and Se, as sodium selenite (twice daily with 0.1 mg doses), were orally supplemented to the patients for 45 days. Erythrocyte lipid peroxidation levels were higher in refractory epilepsy groups than in control although its level and seizure numbers were decreased in TPM and TPM + Se supplemented groups of the patients. The erythrocyte reduced glutathione (GSH), glutathione peroxidase (GSH-Px), plasma total antioxidant status (TAS), and vitamin E concentration in refractory epilepsy group were lower than in control. However, the erythrocyte and plasma TAS, erythrocyte GSH and GSH-Px, and plasma vitamins A and C values were increased either by Se or Se + TPM in epilepsy and refractory epilepsy groups. There were no effects of TPM and Se on plasma β-carotene values in the groups. In conclusion, TPM and selenium caused protective effects on the epilepsy and refractory epilepsy-induced oxidative injury by inhibiting free radical production and supporting antioxidant redox system.     

The Bacterial Symbiont Wolbachia Induces Resistance to RNA Viral Infections in Drosophila melanogaster

Wolbachia are vertically transmitted, obligatory intracellular bacteria that infect a great number of species of arthropods and nematodes. In insects, they are mainly known for disrupting the reproductive biology of their hosts in order to increase their transmission through the female germline. In Drosophila melanogaster, however, a strong and consistent effect of Wolbachia infection has not been found. Here we report that a bacterial infection renders D. melanogaster more resistant to Drosophila C virus, reducing the load of viruses in infected flies. We identify these resistance-inducing bacteria as Wolbachia. Furthermore, we show that Wolbachia also increases resistance of Drosophila to two other RNA virus infections (Nora virus and Flock House virus) but not to a DNA virus infection (Insect Iridescent Virus 6). These results identify a new major factor regulating D. melanogaster resistance to infection by RNA viruses and contribute to the idea that the response of a host to a particular pathogen also depends on its interactions with other microorganisms. This is also, to our knowledge, the first report of a strong beneficial effect of Wolbachia infection in D. melanogaster. The induced resistance to natural viral pathogens may explain Wolbachia prevalence in natural populations and represents a novel Wolbachia–host interaction.     

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.