The endosymbiotic bacterium <Emphasis Type="Italic">Wolbachia</Emphasis> enhances the nonspecific resistance to insect pathogens and alters behavior of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

To determine biologically important effects of the cytoplasmic endosymbiont Wolbachia, two substrains of the same Drosophila melanogaster strain have been studied, one of them infected with Wolbachia and the other treated with tetracycline to eliminate the bacterium. Females of D. melanogaster infected with Wolbachia are more resistant to the fungus Blauveria bassiana (an insect pathogen) than uninfected females; infected females also exhibited changes in oviposition substrate preference. Males infected with the bacterium are more competitive than uninfected males. The possible role of Wolbachia in the formation of alternative ecological strategies of D. melanogaster is discussed.     

Removing endosymbiotic Wolbachia specifically decreases lifespan of females and competitiveness in a laboratory strain of Drosophila melanogaster

To understand specific symbiotic relationships ensuring stable existing of the bacterium Wolbachia in laboratory strains of Drosophila melanogaster, the imago lifespan and senescence rate, as well as competitiveness, have been evaluated as components of fitness in females from the following laboratory strains: (1) inbred strain 95 infected with Wolbachia; (2) two uninfected strains obtained by tetracycline treatment that were genetically similar to strain 95; and (3) two control, uninfected, wild-type laboratory strains that were used to assess the possible effects of the antibiotic on the studied characters in the absence of Wolbachia. The results have shown that infected females have longer lifespan and competitiveness than females with the same genotype uninfected with Wolbachia. The increase in the senescence and mortality rates with age was also slower in infected females. It is noteworthy that tetracycline does not affect the lifespan of females from the two control, uninfected, wild-type strains. Therefore, the antibiotic is not the cause of the positive changes in fitness that were observed in infected females. The obtained results are the first direct evidence that the relationship s in the Wolbachia-D melanogaster symbiotic system are mutualistic rather than parasitic, at least in micropopulations adapted to laboratory conditions.     

An ancient horizontal gene transfer between mosquito and the endosymbiotic bacterium Wolbachia pipientis

The extent and biological relevance of horizontal gene transfer (HGT) in eukaryotic evolution remain highly controversial. Recent studies have demonstrated frequent and large-scale HGT from endosymbiotic bacteria to their hosts, but the great majority of these transferred genes rapidly become nonfunctional in the recipient genome. Here, we investigate an ancient HGT between a host metazoan and an endosymbiotic bacterium, Wolbachia pipientis. The transferred gene has so far been found only in mosquitoes and Wolbachia. In mosquitoes, it is a member of a gene family encoding candidate receptors required for malaria sporozoite invasion of the mosquito salivary gland. The gene copy in Wolbachia has substantially diverged in sequence from the mosquito homolog, is evolving under purifying selection, and is expressed, suggesting that this gene is also functional in the bacterial genome. Several lines of evidence indicate that the gene may have been transferred from eukaryotic host to bacterial endosymbiont. Regardless of the direction of transfer, however, these results demonstrate that interdomain HGT may give rise to functional, persistent, and possibly evolutionarily significant new genes.     

Using Drosophila melanogaster to validate metabolism-based insecticide resistance from insect pests

Identifying molecular mechanisms of insecticide resistance is important for preserving insecticide efficacy, developing new insecticides and implementing insect control. The metabolic detoxification of insecticides is a widespread resistance mechanism. Enzymes with the potential to detoxify insecticides are commonly encoded by members of the large cytochrome P450, glutathione S-transferase and carboxylesterase gene families, all rapidly evolving in insects. Here, we demonstrate that the model insect Drosophila melanogaster is useful for functionally validating the role of metabolic enzymes in conferring metabolism-based insecticide resistance. Alleles of three well-characterized genes from different pest insects were expressed in transgenic D. melanogaster : a carboxylesterase gene (αE7) from the Australian sheep blowfly Lucilia cuprina, a glutathione S-transferase gene (GstE2) from the mosquito Anopheles gambiae and a cytochrome P450 gene (Cyp6cm1) from the whitefly Bemisia tabaci. For all genes, expression in D. melanogaster resulted in insecticide resistance phenotypes mirroring those observed in resistant populations of the pest species. Using D. melanogaster to assess the potential for novel metabolic resistance mechanisms to evolve in pest species is discussed.     

The endosymbiont Wolbachia in Eurasian populations of Drosophila melanogaster

The endosymbiotic [alpha]-proteobacteria Wolbachia is widely spread among arthropods and Filariidae nematodes. This bacterium is transmitted vertically via a transovarian route. Wolbachia is a cause of several reproductive abnormalities in the host species. We analyzed the isofemale lines created using flies collected from Drosophila melanogaster natural populations for infection with the endosymbiont Wolbachia. Wolbachia were genotyped according to five variable markers: the presence of insertion sequence IS5 in two loci, the copy number of two minisatellite repeats, and an inversion. Overall, 665 isofemale lines isolated from the populations of D. melanogaster from Ukraine, Belarus, Moldova, Caucasus, Central Asia, Ural, Udmurtia, Altai, West and East Siberia, and Far East in 1974 through 2005 were used in the work. The samples from Ukrainian, Altaian, and Middle Asian populations were largest. The infection rate of D. melanogaster populations from Middle Asia, Altaian, and Eastern Europe (Ukraine, Moldavia, and Belarus) with Wolbachia amounted to 64, 56, and 39%, respectively. The D. melanogaster population from the Caucasus displayed heterogeneity in the genotypes of this cytoplasmic infection. The Wolbachia genotype wMel, detected in all the populations studied, was the most abundant. The genotype wMelCS2 was always present in the populations from Middle Asia and Altai and was among the rare variants in the D. melanogaster populations from the Eastern Europe. Single instances of the Wolbachia genotype wMelCS occurred in a few flies from the Central Asian and Altai populations, but was not found this genotype in the other regions.     

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

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

Wolbachia infection decreased the resistance of Drosophila to lead

Background

The heavy metal lead has been shown to be associated with a genotoxic risk. Drosophila melanogaster is a model organism commonly utilized in genetic toxicology testing. The endosymbionts — Wolbachia are now very common in both wild populations and laboratory stocks of Drosophila. Wolbachia may induce resistance to pathogenic viruses, filarial nematodes and Plasmodium in fruit fly and mosquito hosts. However the effect of Wolbachia infection on the resistance of their hosts to heavy metal is unknown.

Methodology/Principal Findings

Manipulating the lead content in the diet of Drosophila melanogaster, we found that lead consumption had no different effects on developmental time between Wolbachia-infected (Dmel wMel) and –uninfected (Dmel T) flies. While in Pb-contaminated medium, significantly reduced amount of pupae and adults of Dmel wMel were emerged, and Dmel wMel adults had significantly shorter longevity than that of Dmel T flies. Lead infusion in diet resulted in significantly decreased superoxide dismutase (SOD) activity in Dmel T flies (P<0.05), but not in Dmel wMel flies. Correspondingly, lead cultures induced a 10.8 fold increase in malonaldehyde (MDA) contents in Dmel T larvae (P<0.05). While in Dmel wMel larvae, it resulted in only a 1.3 fold increase. By quantitative RT-PCR, we showed that lead infused medium caused significantly increased expression level of relish and CecA2 genes in Dmel T flies (P<0.01). Lead cultures did not change dramatically the expression of these genes in Dmel wMel flies.

Conclusions/Significance

These results suggest that Wolbachia infection decreased the resistance of Drosophila to lead likely by limiting the production of peroxides resulted from lead, thus being unable to activate the immunological pathway in the host to prevent them from lead damage. This represents a novel Wolbachia–host interaction and provides information that researchers working on Drosophila toxicology should take in consideration the presence of Wolbachia in the stocks they are analyzing.     

Endophytic bacterium,Bacillus amyloliquefaciens,enhances ornamental hosta resistance to diseases and insect pests

A total of 84 bacterial endophytes were isolated from seeds of 6 cultivars of ornamental hostas, and they were identified to 5 species based on morphological characteristics and 16S rDNA sequence analysis. Among them, the strain ‘Blu-v2’, which was isolated from the seeds of cultivar ‘Blue Umbrella’ and identified to be Bacillus amyloliquefaciens, showed highest antifungal activity and capacity to deter feeding by Fall armyworms (Spodoptera fruigiperda). Lipopeptides in cultures of Blu-v2 were determined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and its antifungal activities were verified. However, the lipopeptide preparation did not show toxicity to larvae of Fall armyworms. In a greenhouse experiment, Blu-v2 was inoculated into small plantlets of hosta (cultivar ‘Rainforest Sunrise’). The leaves of plants with bacteria (endophyte-infected?=?E+) and without bacteria (endophyte-free?=?E?) were used in seven-day feeding experiments employing fourth-instar larvae of Fall armyworms. We found that there was a significant decrease in the weights of larvae fed with E+ compared to E? plants; and the mortality rate of larvae fed with E? leaves was lower (3.33%) compared to that of larvae fed with E+ leaves (30%). Based on our studies, we suggest that endophytic B. amyloliquefaciens strain Blu-v2 has potential value as a biocontrol agent to reduce damage from fungal diseases and insect pests of hosta cultivars.     

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.     

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.     

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.     

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.     

A single gene that promotes interaction of a phytopathogenic bacterium with its insect vector,Drosophila melanogaster

Insects are major vectors of plant and animal disease, and bacterial phytopathogens are often disseminated by flies. We have previously reported that some isolates of the phytopathogenic bacterial species Erwinia carotovora infect Drosophila and activate an immune response. Using a genetic screen, we have now identified two genes that are required by E. carotovora to infect Drosophila. One of these genes has a regulatory role whereas the other, evf, confers an infectious phenotype: its transfer to non-infectious Erwinia strains or to several enterobacteria improves survival in the gut and triggers the immune response. Overexpression of Erwinia virulence factor (evf) allowed bacteria to colonize the apical side of the gut epithelium and in some cases to spread to the body cavity. Our results demonstrate a specific interaction between plant pathogens and flies that promote their dissemination.     

Odor-mediated aggregation enhances the colonization ability of Drosophila melanogaster

Animal aggregation is a general phenomenon in ecological systems. Aggregations are generally considered as an evolutionary advantageous state in which members derive the benefits of mate choice and protection against natural enemies, balanced by the costs of limiting resources and intraspecific competition. Many insects use chemical information to find conspecifics and to form aggregations. In this study, we describe a spatio-temporal simulation model designed to explore and quantify the effects of the strength of chemical attraction, on the colonization ability of a fruit fly (Drosophila melanogaster) population. We found that the use of infochemicals is crucial for colonizing an area. Fruit flies subject to an Allee effect that are unable to respond to chemical information could not successfully colonize the area and went extinct within four generations. This was mainly caused by very high mortality due to the Allee effect. Even when the Allee effect did not play a role, the random dispersing population had more difficulties in colonizing the area and is doomed to extinction in the long run. When fruit flies had the ability to respond to chemical information, they successfully colonized the orchard. This happened faster, for stronger attraction to chemical information. In addition, more fruit flies were able to find the resources and the settlement on the resources was much higher. This resulted in a reduced mortality due to the Allee effect for fruit flies able to respond to chemical information. Odor-mediated aggregation thus enhances the colonization ability of D. melanogaster. Even a weak attraction to chemical information paved the way to successfully colonize the orchard.     

Population dynamics of the Wolbachia infection causing cytoplasmic incompatibility in Drosophila melanogaster.

Field populations of Drosophila melanogaster are often infected with Wolbachia, a vertically transmitted microorganism. Under laboratory conditions the infection causes partial incompatibility in crosses between infected males and uninfected females. Here we examine factors influencing the distribution of the infection in natural populations. We show that the level of incompatibility under field conditions was much weaker than in the laboratory. The infection was not transmitted with complete fidelity under field conditions, while field males did not transmit the infection to uninfected females and Wolbachia did not influence sperm competition. There was no association between field fitness as measured by fluctuating asymmetry and the infection status of adults. Infected field females were smaller than uninfecteds in some collections from a subtropical location, but not in other collections from the same location. Laboratory cage studies showed that the infection did not change in frequency when populations were maintained at a low larval density, but it decreased in frequency at a high larval density. Monitoring of infection frequencies in natural populations indicated stable frequencies in some populations but marked fluctuations in others. Simple models suggest that the infection probably provides a fitness benefit for the host in order to persist in populations. The exact nature of this benefit remains elusive.     

Passage of Wolbachia pipientis through Mutant Drosophila melanogaster Induces Phenotypic and Genomic Changes

Wolbachia pipientis is a nearly ubiquitous, maternally transmitted bacterium that infects the germ line of insect hosts. Estimates are that Wolbachia infects 40 to 60% of insect species on the planet, making it one of the most prevalent infections on Earth. However, we know surprisingly little about the molecular mechanisms used by Wolbachia to infect its hosts. We passaged Wolbachia through normally restrictive Drosophila melanogaster hosts, bottlenecking Wolbachia through stochastic segregation while simultaneously selecting for mutants that could recolonize these previously restrictive hosts. Here, we show that Wolbachia alters its behavior when passaged through heterozygous mutant flies. After only three generations, Wolbachia was able to colonize the previously restrictive hosts at control titers. Additionally, the Wolbachia organisms passaged through heterozygous mutant D. melanogaster alter their pattern of tissue-specific Wsp protein production, suggesting a behavioral response to the host genotype. Using whole-genome resequencing, we identified the mutations accumulated by these lineages of Wolbachia and confirmed the existence and persistence of the mutations through clone library Sanger sequencing. Our results suggest that Wolbachia can quickly adapt to new host contexts, with genomic mutants arising after only two generations.     

The effect of pathogens on selection against deleterious mutations in Drosophila melanogaster

In natural populations, fitness is reduced by both deleterious mutations and parasites. Few studies have examined interactions between these two factors, particularly at the level of individual genes. We examined how the presence of a bacterial pathogen, Pseudomonas aeruginosa, affected the selection against each of eight deleterious mutations in Drosophila melanogaster. We found that mutations tended to become more deleterious in the presence of disease. This increase in the average selection was primarily due to three genes with the remainder showing little evidence of change.     

The association between malathion resistance and acetylcholinesterase in Drosophila melanogaster

The relationship between the 50% survival time for flies feeding on a malathion-containing medium and the activity of acetylcholinesterase (AChE) was determined for 15 isofemale lines of Drosophila melanogaster. A significant correlation was found (r=0.28, P<0.05), with more resistant lines tending to have a lower level of AChE activity. An association between AChE and malathion resistance was also observed in a selection experiment. The AChE activity decreased in two of two populations selected for malathion resistance. AChE from these populations was altered in kinetic parameters (measured in crude head extracts) and electrophoretic mobility. Although the resistant AChE had a lower activity (V _m) on either a per milligram protein or a per individual basis, its apparent K _m for acetylthiocholine was lower than that of susceptible AChE. Recombination mapping of both low activity and fast electrophoretic mobility localized these traits to the region of the structural locus (Ace) on the third chromosome. The AChE activity of flies heterozygous for a variety of Ace lesions (kindly provided by Dr. W. M. Gelbart) was consistent with this location. The changes in AChE were suggested to have been caused by selection of alleles at the Ace locus.This work was supported by NSERC Grants A5857, G0183, and A0629.     

The parasitic bacterium Wolbachia and the origin of the eukaryotic cell

The acquisition of endosymbiotic alphaproteobacteria that gave rise to mitochondria was one of the key events in the origin of eukaryotic cell. To reconstruct this process, it is important to analyze relationships that developed later between eukaryotes and other alphaproteobacteria. Wolbachia pipientis, a bacterium that inhabits cells of numerous terrestrial invertebrates and exerts diverse effects on its hosts, is used as a model. Although Wolbachia is similar to mitochondria in many important features (basic metabolism, small molecule membrane transport, envelope structure, etc.), their relationships with the nucleocytoplasm are different. Mitochondria import most of their required proteins from the nucleocytoplasm and are controlled by the nucleocytoplasmic regulatory systems. On the contrary, Wolbachia exports its proteins into the host’s cytoplasm, thus causing dramatic aberrations in the ontogeny and reproduction of the host. This difference may be due to the fact that most of the protomitochondrial genes had been transferred into the central (nuclear) genome at the early stages of the development of the endosymbiotic system, while Wolbachia genes were not transferred into the nucleus. This fits well with the previously suggested hypothesis that there was a period of rapid lateral gene transfer in the evolution of proto-eukaryotes; the acquisition of mitochondria took place during this period. Later, eukaryotes, and especially metazoans, developed powerful mechanisms for prevention of lateral gene transfer. Therefore, the genes of the newly acquired endosymbionts cannot be transferred into the central genome, and the endosymbionts retain the capacity for selfish evolution.