Addicted? Reduced host resistance in populations with defensive symbionts

Heritable symbionts that protect their hosts from pathogens have been described in a wide range of insect species. By reducing the incidence or severity of infection, these symbionts have the potential to reduce the strength of selection on genes in the insect genome that increase resistance. Therefore, the presence of such symbionts may slow down the evolution of resistance. Here we investigated this idea by exposing Drosophila melanogaster populations to infection with the pathogenic Drosophila C virus (DCV) in the presence or absence of Wolbachia, a heritable symbiont of arthropods that confers protection against viruses. After nine generations of selection, we found that resistance to DCV had increased in all populations. However, in the presence of Wolbachia the resistant allele of pastrel—a gene that has a major effect on resistance to DCV—was at a lower frequency than in the symbiont-free populations. This finding suggests that defensive symbionts have the potential to hamper the evolution of insect resistance genes, potentially leading to a state of evolutionary addiction where the genetically susceptible insect host mostly relies on its symbiont to fight pathogens.     

Wolbachia Variants Induce Differential Protection to Viruses in Drosophila melanogaster: A Phenotypic and Phylogenomic Analysis

Wolbachia are intracellular bacterial symbionts that are able to protect various insect hosts from viral infections. This tripartite interaction was initially described in Drosophila melanogaster carrying wMel, its natural Wolbachia strain. wMel has been shown to be genetically polymorphic and there has been a recent change in variant frequencies in natural populations. We have compared the antiviral protection conferred by different wMel variants, their titres and influence on host longevity, in a genetically identical D. melanogaster host. The phenotypes cluster the variants into two groups — wMelCS-like and wMel-like. wMelCS-like variants give stronger protection against Drosophila C virus and Flock House virus, reach higher titres and often shorten the host lifespan. We have sequenced and assembled the genomes of these Wolbachia, and shown that the two phenotypic groups are two monophyletic groups. We have also analysed a virulent and over-replicating variant, wMelPop, which protects D. melanogaster even better than the closely related wMelCS. We have found that a ∼21 kb region of the genome, encoding eight genes, is amplified seven times in wMelPop and may be the cause of its phenotypes. Our results indicate that the more protective wMelCS-like variants, which sometimes have a cost, were replaced by the less protective but more benign wMel-like variants. This has resulted in a recent reduction in virus resistance in D. melanogaster in natural populations worldwide. Our work helps to understand the natural variation in wMel and its evolutionary dynamics, and inform the use of Wolbachia in arthropod-borne disease control.     

Pervasive effects of Wolbachia on host activity

Heritable symbionts have diverse effects on the physiology, reproduction and fitness of their hosts. Maternally transmitted Wolbachia are one of the most common endosymbionts in nature, infecting about half of all insect species. We test the hypothesis that Wolbachia alter host behaviour by assessing the effects of 14 different Wolbachia strains on the locomotor activity of nine Drosophila host species. We find that Wolbachia alter the activity of six different host genotypes, including all hosts in our assay infected with wRi-like Wolbachia strains (wRi, wSuz and wAur), which have rapidly spread among Drosophila species in about the last 14 000 years. While Wolbachia effects on host activity were common, the direction of these effects varied unpredictably and sometimes depended on host sex. We hypothesize that the prominent effects of wRi-like Wolbachia may be explained by patterns of Wolbachia titre and localization within host somatic tissues, particularly in the central nervous system. Our findings support the view that Wolbachia have wide-ranging effects on host behaviour. The fitness consequences of these behavioural modifications are important for understanding the evolution of host–symbiont interactions, including how Wolbachia spread within host populations.     

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.     

Symbiont strain is the main determinant of variation in Wolbachia‐mediated protection against viruses across Drosophila species

Wolbachia is a common heritable bacterial symbiont in insects. Its evolutionary success lies in the diverse phenotypic effects it has on its hosts coupled to its propensity to move between host species over evolutionary timescales. In a survey of natural host–symbiont associations in a range of Drosophila species, we found that 10 of 16 Wolbachia strains protected their hosts against viral infection. By moving Wolbachia strains between host species, we found that the symbiont genome had a much greater influence on the level of antiviral protection than the host genome. The reason for this was that the level of protection depended on the density of the symbiont in host tissues, and Wolbachia rather than the host‐controlled density. The finding that virus resistance and symbiont density are largely under the control of symbiont genes in this system has important implications both for the evolution of these traits and for public health programmes using Wolbachia to prevent mosquitoes from transmitting disease.     

Can maternally inherited endosymbionts adapt to a novel host? Direct costs of Spiroplasma infection,but not vertical transmission efficiency,evolve rapidly after horizontal transfer into D. melanogaster

Maternally inherited symbionts are common in arthropods and many have important roles in host adaptation. The observation that specific symbiont lineages infect distantly related host species implies new interactions are commonly established by lateral transfer events. However, studies have shown that symbionts often perform poorly in novel hosts. We hypothesized selection on the symbiont may be sufficiently rapid that poor performance in a novel host environment is rapidly ameliorated, permitting symbiont maintenance. Here, we test this prediction for a Spiroplasma strain transinfected into the novel host Drosophila melanogaster. In the generations immediately following transinfection, the symbiont had low transmission efficiency to offspring and imposed severe fitness costs on its host. We observed that effects on host fitness evolved rapidly, being undetectable after 17 generations in the novel host, whereas vertical transmission efficiency was poorly responsive over this period. Our results suggest that long-term symbiosis may more readily be established in cases where symbionts perform poorly in just one aspect of symbiosis.     

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.     

Macronutrients mediate the functional relationship between Drosophila and Wolbachia

Wolbachia are maternally inherited bacterial endosymbionts that naturally infect a diverse array of arthropods. They are primarily known for their manipulation of host reproductive biology, and recently, infections with Wolbachia have been proposed as a new strategy for controlling insect vectors and subsequent human-transmissible diseases. Yet, Wolbachia abundance has been shown to vary greatly between individuals and the magnitude of the effects of infection on host life-history traits and protection against infection is correlated to within-host Wolbachia abundance. It is therefore essential to better understand the factors that modulate Wolbachia abundance and effects on host fitness. Nutrition is known to be one of the most important mediators of host–symbiont interactions. Here, we used nutritional geometry to quantify the role of macronutrients on insect–Wolbachia relationships in Drosophila melanogaster. Our results show fundamental interactions between diet composition, host diet selection, Wolbachia abundance and effects on host lifespan and fecundity. The results and methods described here open a new avenue in the study of insect–Wolbachia relationships and are of general interest to numerous research disciplines, ranging from nutrition and life-history theory to public health.     

Wolbachia modifies thermal preference in Drosophila melanogaster

Environmental variation can have profound and direct effects on fitness, fecundity, and host–symbiont interactions. Replication rates of microbes within arthropod hosts, for example, are correlated with incubation temperature but less is known about the influence of host–symbiont dynamics on environmental preference. Hence, we conducted thermal preference (T_p) assays and tested if infection status and genetic variation in endosymbiont bacterium Wolbachia affected temperature choice of Drosophila melanogaster. We demonstrate that isogenic flies infected with Wolbachia preferred lower temperatures compared with uninfected Drosophila. Moreover, T_p varied with respect to three investigated Wolbachia variants (wMel, wMelCS, and wMelPop). While uninfected individuals preferred 24.4°C, we found significant shifts of −1.2°C in wMel- and −4°C in flies infected either with wMelCS or wMelPop. We, therefore, postulate that Wolbachia-associated T_p variation within a host species might represent a behavioural accommodation to host–symbiont interactions and trigger behavioural self-medication and bacterial titre regulation by the host.     

Coevolution of Drosophila melanogaster mtDNA and Wolbachia Genotypes

Maternally inherited microorganisms can influence the mtDNA pattern of variation in hosts. This influence is driven by selection among symbionts and can cause the frequency of mitochondrial variants in the population to eventually increase or decrease. Wolbachia infection is common and widespread in Drosophila melanogaster populations. We compared genetic variability of D. melanogaster mitotypes with Wolbachia genotypes among isofemale lines associated with different geographic locations and time intervals to study coevolution of the mtDNA and Wolbachia. Phylogenetic analysis of D. melanogaster mtDNA revealed two clades diverged in Africa, each associated with one of the two Wolbachia genotype groups. No evidence of horizontal transmission of Wolbachia between maternal lineages has been found. All the mtDNA variants that occur in infected isofemale lines are found in uninfected isofemale lines and vice versa, which is indicative of a recent loss of infection from some maternal fly lineages and confirms a significant role of Wolbachia in the D. melanogaster mtDNA pattern of variation. Finally, we present a comparative analysis of biogeographic distribution of D. melanogaster mitotypes all over the world.     

Sex-specific behavioural symptoms of viral gut infection and Wolbachia in Drosophila melanogaster

All organisms are infected with a range of symbionts spanning the spectrum of beneficial mutualists to detrimental parasites. The fruit fly Drosophila melanogaster is a good example, as both endosymbiotic Wolbachia, and pathogenic Drosophila C Virus (DCV) commonly infect it. While the pathophysiology and immune responses against both symbionts are the focus of intense study, the behavioural effects of these infections have received less attention. Here we report sex-specific behavioural responses to these infections in D. melanogaster. DCV infection caused increased sleep in female flies, but had no detectable effect in male flies. The presence of Wolbachia did not reduce this behavioural response to viral infection. We also found evidence for a sex-specific cost of Wolbachia, as male flies infected with the endosymbiont became more lethargic when awake. We discuss these behavioural symptoms as potentially adaptive sickness behaviours.     

Influence of oxidative homeostasis on bacterial density and cost of infection in Drosophila–Wolbachia symbioses

The evolution of symbioses along the continuum between parasitism and mutualism can be influenced by the oxidative homeostasis, that is the balance between reactive oxygen species (ROS) and antioxidant molecules. Indeed, ROS can contribute to the host immune defence to regulate symbiont populations, but are also toxic. This interplay between ROS and symbiosis is notably exemplified by recent results in arthropod–Wolbachia interactions. Wolbachia are symbiotic bacteria involved in a wide range of interactions with their arthropods hosts, from facultative, parasitic associations to obligatory, mutualistic ones. In this study, we used Drosophila–Wolbachia associations to determine whether the oxidative homeostasis plays a role in explaining the differences between phenotypically distinct arthropod–Wolbachia symbioses. We used Drosophila lines with different Wolbachia infections and measured the effects of pro‐oxidant (paraquat) and antioxidant (glutathione) treatments on the Wolbachia density and the host survival. We show that experimental manipulations of the oxidative homeostasis can reduce the cost of the infection through its effect on Wolbachia density. We discuss the implication of this result from an evolutionary perspective and argue that the oxidative homeostasis could underlie the evolution of tolerance and dependence on Wolbachia.     

Various <Emphasis Type="Italic">Wolbachia</Emphasis> genotypes differently influence host <Emphasis Type="Italic">Drosophila</Emphasis> dopamine metabolism and survival under heat stress conditions

Background

One of the most widespread prokaryotic symbionts of invertebrates is the intracellular bacteria of Wolbachia genus which can be found in about 50% of insect species. Wolbachia causes both parasitic and mutualistic effects on its host that include manipulating the host reproductive systems in order to increase their transmission through the female germline, and increasing the host fitness. One of the mechanisms, promoting adaptation in biological organisms, is a non-specific neuroendocrine stress reaction. In insects, this reaction includes catecholamines, dopamine, serotonin and octopamine, which act as neurotransmitters, neuromodulators and neurohormones. The level of dopamine metabolism correlates with heat stress resistance in Drosophila adults.

Results

To examine Wolbachia effect on Drosophila survival under heat stress and dopamine metabolism we used five strains carrying the nuclear background of interbred Bi90 strain and cytoplasmic backgrounds with different genotype variants of Wolbachia (produced by 20 backcrosses of Bi90 males with appropriate source of Wolbachia). Non-infected Bi90 strain (treated with tetracycline for 3 generations) was used as a control group. We demonstrated that two of five investigated Wolbachia variants promote changes in Drosophila heat stress resistance and activity of enzymes that produce and degrade dopamine, alkaline phosphatase and dopamine-dependent arylalkylamine N-acetyltransferase. What is especially interesting, wMelCS genotype of Wolbachia increases stress resistance and the intensity of dopamine metabolism, whereas wMelPop strain decreases them. wMel, wMel2 and wMel4 genotypes of Wolbachia do not show any effect on the survival under heat stress or dopamine metabolism. L-DOPA treatment, known to increase the dopamine content in Drosophila, levels the difference in survival under heat stress between all studied groups.

Conclusions

The genotype of symbiont determines the effect that the symbiont has on the stress resistance of the host insect.

     

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.     

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.     

Wolbachia effects on thermal preference of natural Drosophila melanogaster are influenced by host genetic background,Wolbachia type,and bacterial titer

Temperature plays a fundamental role in the fitness of all organisms. In particular, it strongly affects metabolism and reproduction in ectotherms that have limited physiological capabilities to regulate their body temperature. The influence of temperature variation on the physiology and behaviour of ectotherms is well studied but we still know little about the influence of symbiotic interactions on thermal preference (T_p) of the host. A growing number of studies focusing on the Wolbachia-Drosophila host-symbiont system found that Wolbachia can influence T_p in Drosophila laboratory strains. Here, we investigated the effect of Wolbachia on T_p in wild-type D. melanogaster flies recently collected from nature. Consistent with previous data, we found reduced T_p compared to an uninfected control in one of two fly strains infected with the wMelCS Wolbachia type. Additionally, we, for the first time, found that Wolbachia titer variation influences the thermal preference of the host fly. These data indicate that the interaction of Wolbachia and Drosophila resulting in behavioural variation is strongly influenced by the genetic background of the host and symbiont. More studies are needed to better understand the evolutionary significance of T_p variation influenced by Wolbachia in natural Drosophila populations.     

Mitochondria and Wolbachia titers are positively correlated during maternal transmission

Mothers provide their offspring with symbionts. Maternally transmitted, intracellular symbionts must disperse from mother to offspring with other cytoplasmic elements, like mitochondria. Here, we investigated how the intracellular symbiont Wolbachia interacts with mitochondria during maternal transmission. Mitochondria and Wolbachia may interact antagonistically and compete as each population tries to ensure its own evolutionary success. Alternatively, mitochondria and Wolbachia may cooperate as both benefit from ensuring the fitness of the mother. We characterized the relationship between mitochondria and Wolbachia titers in ovaries of Drosophila melanogaster. We found that mitochondria and Wolbachia titers are positively correlated in common laboratory genotypes of D. melanogaster. We attempted to perturb this covariation through the introduction of Wolbachia variants that colonize at different titers. We also attempted to perturb the covariation through manipulating the female reproductive tract to disrupt maternal transmission. Finally, we also attempted to disrupt the covariation by knocking down gene expression for two loci involved in mitochondrial metabolism: NADH dehydrogenase and a mitochondrial transporter. Overall, we find that mitochondria and Wolbachia titers are commonly positively correlated, but this positive covariation is disrupted at high titers of Wolbachia. Our results suggest that mitochondria and Wolbachia have likely evolved mechanisms to stably coexist, but the competitive dynamics change at high Wolbachia titers. We provide future directions to better understand how their interaction influences the maintenance of the symbiosis.     

Population Genomics of the Wolbachia Endosymbiont in Drosophila melanogaster

Wolbachia are maternally inherited symbiotic bacteria, commonly found in arthropods, which are able to manipulate the reproduction of their host in order to maximise their transmission. The evolutionary history of endosymbionts like Wolbachia can be revealed by integrating information on infection status in natural populations with patterns of sequence variation in Wolbachia and host mitochondrial genomes. Here we use whole-genome resequencing data from 290 lines of Drosophila melanogaster from North America, Europe, and Africa to predict Wolbachia infection status, estimate relative cytoplasmic genome copy number, and reconstruct Wolbachia and mitochondrial genome sequences. Overall, 63% of Drosophila strains were predicted to be infected with Wolbachia by our in silico analysis pipeline, which shows 99% concordance with infection status determined by diagnostic PCR. Complete Wolbachia and mitochondrial genomes show congruent phylogenies, consistent with strict vertical transmission through the maternal cytoplasm and imperfect transmission of Wolbachia. Bayesian phylogenetic analysis reveals that the most recent common ancestor of all Wolbachia and mitochondrial genomes in D. melanogaster dates to around 8,000 years ago. We find evidence for a recent global replacement of ancestral Wolbachia and mtDNA lineages, but our data suggest that the derived wMel lineage arose several thousand years ago, not in the 20th century as previously proposed. Our data also provide evidence that this global replacement event is incomplete and is likely to be one of several similar incomplete replacement events that have occurred since the out-of-Africa migration that allowed D. melanogaster to colonize worldwide habitats. This study provides a complete genomic analysis of the evolutionary mode and temporal dynamics of the D. melanogaster–Wolbachia symbiosis, as well as important resources for further analyses of the impact of Wolbachia on host biology.     

Symbionts Commonly Provide Broad Spectrum Resistance to Viruses in Insects: A Comparative Analysis of Wolbachia Strains

In the last decade, bacterial symbionts have been shown to play an important role in protecting hosts against pathogens. Wolbachia, a widespread symbiont in arthropods, can protect Drosophila and mosquito species against viral infections. We have investigated antiviral protection in 19 Wolbachia strains originating from 16 Drosophila species after transfer into the same genotype of Drosophila simulans. We found that approximately half of the strains protected against two RNA viruses. Given that 40% of terrestrial arthropod species are estimated to harbour Wolbachia, as many as a fifth of all arthropods species may benefit from Wolbachia-mediated protection. The level of protection against two distantly related RNA viruses – DCV and FHV – was strongly genetically correlated, which suggests that there is a single mechanism of protection with broad specificity. Furthermore, Wolbachia is making flies resistant to viruses, as increases in survival can be largely explained by reductions in viral titer. Variation in the level of antiviral protection provided by different Wolbachia strains is strongly genetically correlated to the density of the bacteria strains in host tissues. We found no support for two previously proposed mechanisms of Wolbachia-mediated protection — activation of the immune system and upregulation of the methyltransferase Dnmt2. The large variation in Wolbachia''s antiviral properties highlights the need to carefully select Wolbachia strains introduced into mosquito populations to prevent the transmission of arboviruses.     

Age,tissue, genotype and virus infection regulate Wolbachia levels in Drosophila

The bacterial symbiont Wolbachia can protect insects against viral pathogens, and the varying levels of antiviral protection are correlated with the endosymbiont load within the insects. To understand why Wolbachia strains differ in their antiviral effects, we investigated the factors controlling Wolbachia density in five closely related strains in their natural Drosophila hosts. We found that Wolbachia density varied greatly across different tissues and between flies of different ages, and these effects depended on the host–symbiont association. Some endosymbionts maintained largely stable densities as flies aged while others increased, and these effects in turn depended on the tissue being examined. Measuring Wolbachia rRNA levels in response to viral infection, we found that viral infection itself also altered Wolbachia levels, with Flock House virus causing substantial reductions in symbiont loads late in the infection. This effect, however, was virus‐specific as Drosophila C virus had little impact on Wolbachia in all of the five host systems. Because viruses have strong tissue tropisms and antiviral protection is thought to be cell‐autonomous, these effects are likely to affect the virus‐blocking phenomenon. However, we were unable to find any evidence of a correlation between Wolbachia and viral titres within the same tissues. We conclude that Wolbachia levels within flies are regulated in a complex host–symbiont–virus‐dependent manner and this trinity is likely to influence the antiviral effects of Wolbachia.