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.     

Action of genotypical surrounding of host Drosophila melanogaster on biological effects of endosymbiont Wolbachia (strain wMelPop)

In this work, a comparative study of the structure of symbiotic bacteria Wolbachia (strain wMelPop decreasing the fly lifespan) in genotypically different Drosophila melanogaster, as well as the effect of the bacteria on the host cell ultrastructure was investigated out. As a result of special crossings, the Drosophila melanogaster [w]Trl ³⁶² and [w]Trl ^en82 lines, which are carried of mutations for the gene Trithorax-like, are synthesized (lines infected with Wolbachia are designated as [w]). The Drosophila melanogaster line free of Wolbachia was obtained by treatment with antibiotics of the initially infected [w]w ¹¹¹⁸ line. The complex of the used methods and approaches has allowed us to perform a comparative study of the morphology of cell structures for the first time before and after the infestation of insects with bacteria and to evaluate effect of the bacteria on viability and fertility of flies of these lines. Electron microscopy analysis has shown that the embryos of the analyzed lines contain typical Wolbachia in contact with various host cell compartments; the ultrastructural organization of the bacteria indicates the preservation of their functional activity. In the cytoplasm of embryos that are mutant for the gene Trithorax-like, morphologically atypical mitochondria were revealed, as well as Wolbachia (wMelPop) of unusual morphology with a modified form of membtane envelopes. The presence of Wolbachia in ovarian cells of the female mutant fly lines has been found to produce no effect on the amount of the female-ovipositioned eggs. It has been established for the first time that lifespans of the infected and Wolbachia-free Drosophila melanogaster mutant lines TM3 containing chromosome 3 as a balancer are equal. However, it is significantly shorter in the imago of the [w]w ¹¹¹⁸ line than in flies of the mutant lines. This has allowed us to suggest that either the chromosome-balancer TM3 or mutation of the gene Trl play an important role in the host-symbiont interactions. On checking this suggestion, it was found that the lifespan of homozygotes [w]Trl ³⁶² and [w]Trl ^en82 after the infection of flies with bacteria decreased markedly and was close to the lifespan of [w]w ¹¹¹⁸ line. The obtained data indicate that the chromosome-balancer TM3 can have a significant effect on the symbiont-host interaction.     

Wolbachia effects in Drosophila melanogaster: in search of fitness benefits

Insect endosymbionts often influence host nutrition but these effects have not been comprehensively investigated in Wolbachia endosymbionts that are widespread in insects. Using strains of Drosophila melanogaster with the wMel Wolbachia infection, we showed that Wolbachia did not influence adult starvation resistance. Wolbachia also had no effect on larval development time or the size of emerging adults from a low nutrition medium. While Wolbachia may influence the expression of heat shock proteins, we found that there was no effect on adult heat resistance when tested in terms of survival or virility following heat stress. The absence of nutrition or stress effects suggests that other processes maintain wMel frequencies in natural populations of Drosophila melanogaster.     

High temperature initiates changes in Wolbachia ultrastructure in ovaries and early embryos of Drosophila melanogaster

Electron microscopic analysis of Drosophila melanogaster (w1118) ovarian cells has shown that stressful heat treatment of flies causes the appearance of electron-dense granules and large lysosomes in the cytoplasm of ovarian cells. These changes are not due to the presence of the endosymbiotic bacteria Wolbachia, as these changes were observed in both infected and uninfected flies. Essential envelope disturbances and other structural alterations have been revealed in the bacteria present in the ovarian cell cytoplasm of the flies. Some of the fly embryos died after heat shock; however, the bacteria retain their typical morphology in survived embryos. Endosymbionts did not change their localization in ovarian cells and in early fly embryos; they closely interacted with mitochondria and endoplasmic reticulum after the heat-shock treatment of flies. The performed study has shown that the high temperature affects both the host and the endosymbiont, but does not change the character of their structural interaction. Original Russian Text M.V. Zhukova, D.A. Voronin, E.V. Kiseleva, 2008, published in Tsitologiya, vol. 50, No. 5, 2008.     

Wolbachia interactions that determine Drosophila melanogaster survival

Abstract.— We have recently described a mutualistic symbiosis in which Wolbachia bacteria were shown to improve the fitness of some Drosophila melanogaster stocks. Wolbachia did not extend longevity in all Drosophila genotypes, even though 16s rDNA sequences indicated that our Drosophila stocks were infected with the same Wolbachia strain. Here, we use reciprocal hybrid crosses between two Drosophila strains, one that lived longer with Wolbachia (Z53) and one that did not (Z2), to investigate the inheritance of the survival phenotype and its dependence on the host genotype, sex, and mating conditions. Wolbachia's positive effects were more apparent in hybrid flies than in parental flies, ruling out exclusive maternal inheritance or the dependence of the survival phenotype on Wolbachia strain differences. The Wolbachia survival effects were more apparent in single-sex cages, where courtship and mating were not permitted. In these cages, nearly all flies with Wolbachia lived longer than uninfected flies, even though strain Z2 showed no Wolbachia effect in mixed-sex mating cages. We used comparisons between single- and mixed-sex cages to estimate the cost of reproduction for both sexes. Our data suggest that Wolbachia infection may increase the inferred cost of reproduction, particularly in males. Wolbachia can even produce a positive survival effect almost as large as the negative survival effect associated with reproduction. We discuss the implications of our experiments for the study of insect symbioses.     

The endosymbiont Wolbachia increases insulin/IGF-like signalling in Drosophila

Insulin/IGF-like signalling (IIS) is an evolutionarily conserved pathway that has diverse functions in multi-cellular organisms. Mutations that reduce IIS can have pleiotropic effects on growth, development, metabolic homeostasis, fecundity, stress resistance and lifespan. IIS is also modified by extrinsic factors. For instance, in the fruitfly Drosophila melanogaster, both nutrition and stress can alter the activity of the pathway. Here, we test experimentally the hypothesis that a widespread endosymbiont of arthropods, Wolbachia pipientis, can alter the degree to which mutations in genes encoding IIS components affect IIS and its resultant phenotypes. Wolbachia infection, which is widespread in D. melanogaster in nature and has been estimated to infect 30 per cent of strains in the Bloomington stock centre, can affect broad aspects of insect physiology, particularly traits associated with reproduction. We measured a range of IIS-related phenotypes in flies ubiquitously mutant for IIS in the presence and absence of Wolbachia. We show that removal of Wolbachia further reduces IIS and hence enhances the mutant phenotypes, suggesting that Wolbachia normally acts to increase insulin signalling. This effect of Wolbachia infection on IIS could have an evolutionary explanation, and has some implications for studies of IIS in Drosophila and other organisms that harbour endosymbionts.     

Rapid evolution of Wolbachia incompatibility types

In most insects, the endosymbiont Wolbachia induces cytoplasmic incompatibility (CI), an embryonic mortality observed when infected males mate either with uninfected females or with females infected by an incompatible Wolbachia strain. Although the molecular mechanism of CI remains elusive, it is classically viewed as a modification–rescue model, in which a Wolbachia mod function disables the reproductive success of the sperm of infected males, unless eggs are infected and express a compatible resc function. The extent to which the modification–rescue model can predict highly complex CI pattern remains a challenging issue. Here, we show the rapid evolution of the mod–resc system in the Culex pipiens mosquito. We have surveyed four incompatible laboratory isofemale lines over 50 generations and observed in two of them that CI has evolved from complete to partial incompatibility (i.e. the production of a mixture of compatible and incompatible clutches). Emergence of the new CI types depends only on Wolbachia determinants and can be simply explained by the gain of new resc functions. Evolution of CI types in Cx. pipiens thus appears as a gradual process, in which one or several resc functions can coexist in the same individual host in addition to the ones involved in the self-compatibility. Our data identified CI as a very dynamic process. We suggest that ancestral and mutant Wolbachia expressing distinct resc functions can co-infect individual hosts, opening the possibility for the mod functions to evolve subsequently. This gives a first clue towards the understanding of how Wolbachia reached highly complex CI pattern in host 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     

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.     

Bacteria in ovarioles of females from maleless families of ladybird beetles Adalia bipunctata L. (Coleoptera: Coccinellidae) naturally infected with Rickettsia,Wolbachia, and Spiroplasma

Ovarioles were found to be infected with Spiroplasma, Wolbachia, and Rickettsia in Adalia bipunctata females with maleless progeny in different natural populations. Ooplasm was infected with few Wolbachia bacteria. In ooplasm infected by Rickettsia, bacteria were present in small foci. Spiroplasmas were found encapsulated into ooplasm from the wider intercellular spaces between epithelial and oocyte cells. The cytoplasm of follicular epithelia infected with Rickettsia was heavily destroyed, but the nucleus was intact and free from bacteria. The essential feature of follicular epithelium cells from Spiroplasma and Wolbachia infected A. bipunctata females was inclusions of three types: crystalline, filaments, and concentric myelin-like lamellae. Observations of smears prepared from ovaries of A. bipunctata from natural populations revealed a low concentration of bacteria within a microscopy field (less 10 bacteria) in more than 90% of specimens, and only a few ovaries were heavily infected. Two different ways of bacterial invasion of the oocyte are suggested: Spiroplasma-like, through the intercellular spaces in the epithelium and Rickettsia-like, through the cytoplasm of follicular epithelium cells. Bacteria were not found in germarium zones and we suggest that each follicle is infected from haemolymph.     

Wolbachia in the invasive European paper wasp Polistes dominulus

The European paper wasp Polistes dominulus has been expanding its North American range since its introduction in the 1970s. We screened P. dominulus from Italy and the northeastern U.S. for the presence of the intracellular reproductive symbiont Wolbachia. Infection rates among females varied from 16% to 87% among U.S. sites and from 33% to 71% in Italy. We also found infected haploid and diploid males, indicating that this is not a male-killing Wolbachia infection. Our data show that infected individuals from New York, Massachusetts, and Italy carry the same Wolbachia strain, and that some mtDNA haplotypes include both infected and uninfected individuals. We discuss possible implications of Wolbachia infection in this invasive social hymenopteran. Received 28 October 2005; revised 25 January 2006; accepted 17 February 2006.     

Many compatible Wolbachia strains coexist within natural populations of Culex pipiens mosquito

Maternally inherited Wolbachia often manipulate the reproduction of arthropods to promote their transmission. In most species, Wolbachia exert a form of conditional sterility termed cytoplasmic incompatibility (CI), characterized by the death of embryos produced by the mating between individuals with incompatible Wolbachia infections. From a theoretical perspective, no stable coexistence of incompatible Wolbachia infections is expected within host populations and CI should induce the invasion of one strain or of a set of compatible strains. In this study, we investigated this prediction on CI dynamics in natural populations of the common house mosquito Culex pipiens. We surveyed the Wolbachia diversity and the expression of CI in breeding sites of the south of France between 1990 and 2005. We found that geographically close C. pipiens populations harbor considerable Wolbachia diversity, which is stably maintained over 15 years. We also observed a very low frequency of infertile clutches within each sampled site. Meanwhile, mating choice experiments conducted in laboratory conditions showed that assortative mating does not occur. Overall, this suggests that a large set of compatible Wolbachia strains are always locally dominant within mosquito populations thus, fitting with the theoretical expectations on CI dynamics.     

Clonal analysis in hybrids between Drosophila melanogaster and Drosophila simulans

We have analysed the viability of cellular clones induced by mitotic recombination in Drosophila melanogaster/D. simulans hybrid females during larval growth. These clones contain a portion of either melanogaster or simulans genomes in homozygosity. Analysis has been carried out for the X and the second chromosomes, as well as for the 3L chromosome arm. Clones were not found in certain structures, and in others they appeared in a very low frequency. Only in abdominal tergites was a significant number of clones observed, although their frequency was lower than in melanogaster abdomens. The bigger the portion of the genome that is homozygous, the less viable is the recombinant melano-gaster/simulans hybrid clone. The few clones that appeared may represent cases in which mitotic recombination took place in distal chromosome intervals, so that the clones contained a small portion of either melanogaster or simulans chromosomes in homozygosity. Moreover, Lhr, a gene of D. simulans that suppresses the lethality of male and female melanogaster/simulans hybrids, does not suppress the lethality of the recombinant melanogaster/simulans clones. Thus, it appears that there is not just a single gene, but at least one per tested chromosome arm (and maybe more) that cause hybrid lethality. Therefore, the two species, D. melanogaster and D. simulans, have diverged to such a degree that the absence of part of the genome of one species cannot be substituted by the corresponding part of the genome of the other, probably due to the formation of co-adapted gene complexes in both species following their divergent evolution after speciation. The disruption of those coadapted gene complexes would cause the lethality of the recombinant hybrid clones.     

Embryonic cAMP and developmental potential in Drosophila melanogaster

Summary Measurements of cAMP in early embryos of Drosophila melanogaster demonstrate that the dunce gene plays a major role, and the rutabaga gene a secondary role, in maternal regulation of embryonic cAMP content. Studying the double mutant combination, we find that variability in elevated cAMP content between individual embryos is associated with a wide variability in developmental potential. Embryos with about five times the normal cAMP content define a threshold between apparently normal and abnormal development. Measurements of cAMP content in anterior and posterior halves of embryos indicate that the posterior embryonic region, which is developmentally more sensitive to the effects of elevated cAMP than the anterior region, does not contain more cAMP than the anterior region. The variety of developmental defects observed is discussed in relation to possible targets of cAMP action. Offprint requests to: J.A. Kiger, Jr     

Are we underestimating the diversity and incidence of insect bacterial symbionts? A case study in ladybird beetles

Vertically transmitted bacterial symbionts are common in arthropods. However, estimates of their incidence and diversity are based on studies that test for a single bacterial genus and often only include small samples of each host species. Focussing on ladybird beetles, we collected large samples from 21 species and tested them for four different bacterial symbionts. Over half the species were infected, and there were often multiple symbionts in the same population. In most cases, more females than males were infected, suggesting that the symbionts may be sex ratio distorters. Many of these infections would have been missed in previous studies as they only infect a small proportion of the population. Furthermore, 11 out of the 17 symbionts discovered by us were either in the genus Rickettsia or Spiroplasma, which are rarely sampled. Our results suggest that the true incidence and diversity of bacterial symbionts in insects may be far greater than previously thought.     

Variable fitness effects of Wolbachia infection in Drosophila melanogaster

Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness. Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects. Here, we have investigated the symbiosis between Wolbachia and D. melanogaster and found that Wolbachia infection can have significant survival and fecundity effects. Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal. We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences. Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility. More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males. This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare. We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D. melanogaster populations. These interactions may further explain why many D. melanogaster populations are polymorphic for Wolbachia infection. We discuss our results in the context of host-symbiont co-evolution.     

LTR Retrotransposon-Gene Associations in Drosophila melanogaster

Thirty-three percent (228/682) of all long terminal repeat (LTR) retrotransposon sequences (LRSs) present in the sequenced Drosophila melanogaster genome were found to be located in or within 1000 bp of a gene. Recently inserted LTR retrotransposons are significantly more likely to be located in or within genes than are older, fragmented LTR retrotransposon sequences, indicating that most LRS-gene associations are selected against over evolutionary time. LRSs associated with conserved genes (homologenes) are especially prone to negative selection. In contrast, fragmented LRSs that have persisted in the genome over long spans of evolutionary time are preferentially associated with genes involved in signal transduction and other newly evolved functions. Reviewing Editor: Dr. Juergen Brosius     

Interaction between host genotype and environmental conditions affects bacterial density in Wolbachia symbiosis

Regulation of microbial population density is a necessity in stable symbiotic interactions. In Wolbachia symbiosis, both bacterial and host genotypes are involved in density regulation, but environmental factors may also affect bacterial population density. Here, we studied the interaction between three strains of Wolbachia in two divergent homozygous lines of the wasp Leptopilina heterotoma at two different temperatures. Wolbachia density varied between the two host genotypes at only one temperature. Moreover, at this temperature, reciprocal-cross F1 insects displayed identical Wolbachia densities, which were intermediate between the densities in the two parental lines. While these findings confirm that the host genotype plays an important role in Wolbachia density, they also highlight its interaction with environmental conditions, making possible the evolution of local adaptations for the regulation of Wolbachia density.     

Monitoring long-term evolutionary changes following Wolbachia introduction into a novel host: the Wolbachia popcorn infection in Drosophila simulans

Wolbachia may act as a biological control agent for pest management; in particular, the Wolbachia variant wMelPop (popcorn) shortens host longevity and may be useful for dengue suppression. However, long-term changes in the host and Wolbachia genomes can alter Wolbachia spread and/or host effects that suppress disease. Here, we investigate the phenotypic effects of wMelPop in a non-native host, Drosophila simulans, following artificial transinfection approximately 200 generations ago. Long-term rearing and maintenance of the bacteria were at 19°C in the original I-102 genetic background that was transinfected with the popcorn strain. The bacteria were then introgressed into three massbred backgrounds, and tetracycline was used to create uninfected sublines. The effect of wMelPop on longevity in this species appears to have changed; longevity was no longer reduced at 25°C in some nuclear backgrounds, reflecting different geographical origin, selection or drift, although the reduction was still evident for flies held at 30°C. Wolbachia influenced productivity and viability, and development time in some host backgrounds. These findings suggest that long-term attenuation of Wolbachia effects may compromise the effectiveness of this bacterium in pest control. They also emphasize the importance of host nuclear background on Wolbachia phenotypic effects.