The virulent <Emphasis Type="Italic">Wolbachia</Emphasis> strain wMelPop increases the frequency of apoptosis in the female germline cells of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

BACKGROUND: Wolbachia are bacterial endosymbionts of many arthropod species in which they manipulate reproductive functions. The distribution of these bacteria in the Drosophila ovarian cells at different stages of oogenesis has been amply described. The pathways along which Wolbachia influences Drosophila oogenesis have been, so far, little studied. It is known that Wolbachia are abundant in the somatic stem cell niche of the Drosophila germarium. A checkpoint, where programmed cell death, or apoptosis, can occur, is located in region 2a/2b of the germarium, which comprises niche cells. Here we address the question whether or not the presence of Wolbachia in germarium cells can affect the frequency of cyst apoptosis in the checkpoint. RESULTS: Our current fluorescent microscopic observations showed that the wMel and wMelPop strains had different effects on female germline cells of D. melanogaster. The Wolbachia strain wMel did not affect the frequency of apoptosis in cells of the germarium. The presence of the Wolbachia strain wMelPop in the D. melanogasterw1118 ovaries increased the number of germaria where cells underwent apoptosis in the checkpoint. Based on the appearance in the electron microscope, there was no difference in morphological features of apoptotic cystocytes between Wolbachia-infected and uninfected flies. Bacteria with normal ultrastructure and large numbers of degenerating bacteria were found in the dying cyst cells. CONCLUSIONS: Our current study demonstrated that the Wolbachia strain wMelPop affects the egg chamber formation in the D. melanogaster ovaries. This led to an increase in the number of germaria containing apoptotic cells. It is suggested that Wolbachia can adversely interfere either with the cystocyte differentiation into the oocyte or with the division of somatic stem cells giving rise to follicle cells and, as a consequence, to improper ratio of germline cells to follicle cells and, ultimately, to apoptosis of cysts. There was no similar adverse effect in D. melanogaster Canton S infected with the Wolbachia strain wMel. This was taken to mean that the observed increase in frequency of apoptosis was not the general effect of Wolbachia on germline cells of D. melanogaster, it was rather induced by the virulent Wolbachia strain wMelPop.     

Development of a physical and genetic map of the virulent Wolbachia strain wMelPop

We report here the construction of a physical and genetic map of the virulent Wolbachia strain, wMelPop. This map was determined by ordering 28 chromosome fragments that resulted from digestion with the restriction endonucleases FseI, ApaI, SmaI, and AscI and were resolved by pulsed-field gel electrophoresis. Southern hybridization was done with 53 Wolbachia-specific genes as probes in order to determine the relative positions of these restriction fragments and use them to serve as markers. Comparison of the resulting map with the whole genome sequence of the closely related benign Wolbachia strain, wMel, shows that the two genomes are largely conserved in gene organization with the exception of a single inversion in the chromosome.     

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.     

The effects of host age,host nuclear background and temperature on phenotypic effects of the virulent Wolbachia strain popcorn in Drosophila melanogaster

Because of their obligate endosymbiotic nature, Wolbachia strains by necessity are defined by their phenotypic effects upon their host. Nevertheless, studies on the influence of host background and environmental conditions upon the manifestation of Wolbachia effects are relatively uncommon. Here we examine the behavior of the overreplicating Wolbachia strain popcorn in four different Drosophila melanogaster backgrounds at two temperatures. Unlike other strains of Wolbachia in Drosophila, popcorn has a major fitness impact upon its hosts. The rapid proliferation of popcorn causes cells to rupture, resulting in the premature death of adult hosts. Apart from this effect, we found that popcorn delayed development time, and host background influenced both this trait and the rate of mortality associated with infection. Temperature influenced the impact of popcorn upon host mortality, with no reduction in life span occurring in flies reared at 19 degrees. No effect upon fecundity was found. Contrary to earlier reports, popcorn induced high levels of incompatibility when young males were used in tests, and CI levels declined rapidly with male age. The population dynamics of popcorn-type infections will therefore depend on environmental temperature, host background, and the age structure of the population.     

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.     

Genetic screening of meiotic mutations in mosaic clones of the Drosophila melanogaster female germline]

A method of screening for meiotic mutations based on genetic analysis of chromosome disjunction in germline mosaic clones of females homozygous for potential mutations is proposed. The clones are obtained at high frequency due to the use of the transgenic FLP/FRT system of mitotic recombination. This system permits obtaining homozygous clones in the first generation after mutagenesis, whereas the cultures are set up after selection for potential meiotic mutations. This significantly enhances, the efficiency of screening by the elimination of the limiting stage. Using this method, the following mutations were revealed in the 3L arm of Drosophila: ff6 leading to disturbed centriole disjunction, which results in appearance of multi-tail spermatids and three-pole spindles during male meiosis; ff3 leading to the formation of chromosome bridges in anaphase and telophase, chromosome nondisjunction, and premature chromatin condensation after metaphase; embryonic lethal ff29, with disturbed coordination between nuclear and centrosome cycles during syncytial cleavage; and a series of other mutations causing a wide spectrum of disturbances in male meiosis. Comparison of the proposed method with procedures of screening for yeast cell-cycle mutations showed that we succeeded in attaining the efficiency of screening in the Drosophila model close to that in the yeast model.     

The interplay of transposon silencing genes in the Drosophila melanogaster germline

Complexes of Piwi proteins and Piwi-interacting RNAs (piRNAs) carry out the repression of transposable elements in animal gonads. The Piwi protein clade is represented in D. melanogaster by three members: Piwi, Aub and Ago3. Piwi protein functions in the nuclei of somatic and germinal ovarian cells, whereas Aub and Ago3 are cytoplasmic proteins of germinal cells. Aub and Ago3 interact with each other in the perinuclear nuage organelle to perform piRNA amplification via the ping-pong mechanism. Previously, derepression of several transposable elements as a result of mutations in the piRNA silencing system was shown. Here we quantify the increase in expression level of an enlarged number of retrotransposons due to the mutations in the piwi gene, nuage components coding aub, mael and spn-E genes and the RNA helicase armi gene mutation that impairs Piwi nuclear localization, but not the ping-pong cycle. We reveal that piwi, armi, aub, spn-E and mael genes participate together in the repression of several transposons (HMS-Beagle, Gate and HeT-A), whereas silencing of land G elements requires the same genes except piwi. We suggest that Armi has other functions besides the localizing of Piwi protein in the nuclei. Our data suggest also a role of cytoplasmic Aub, Spn-E and Mael nuage proteins in Piwi-mediated repression of Gate and HMS-Beagle transposons in the germline nuclei. As a whole, our results corroborate the idea that genome stabilization in the germline is realized by different silencing strategies specific for different transposable elements. At the same time, our data suggest the existence of yet unknown mechanisms of interplay between nuclear and cytoplasmic components of the piRNA machinery in the germline.     

Distinct P-element excision products in somatic and germline cells of Drosophila melanogaster

The footprints remaining following somatic P-element excision from the Drosophila white locus were recovered and characterized. Two different types of footprints were observed. Over 75% of the footprints were short, composed of 4 or 7 nucleotides of the P-element inverted terminal repeat, and were similar to those found in a previously described plasmid excision assay. The remaining footprints were composed of 14-18 nucleotides of both inverted terminal repeats. These large footprints were indistinguishable from those recovered following germline P-element excision. Enhanced expression of the Drosophila homologue of the Ku70 protein did not affect the structure of the somatic footprints. Therefore, this protein is not a limiting factor for double-strand break repair by nonhomologous end-joining in Drosophila somatic cells.     

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.     

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.     

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.     

Wolbachia感染导致果蝇dHira基因表达下调

Wolbachia是广泛存在于节肢动物体内的一类共生微生物,可通过宿主卵的细胞质传递给子代.     

Drosophila female germline stem cells undergo mitosis without nuclear breakdown

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Evidence for a global Wolbachia replacement in Drosophila melanogaster

Wolbachia are maternally inherited intracellular alpha-Proteobacteria found in numerous arthropod and filarial nematode species. They influence the biology of their hosts in many ways. In some cases, they act as obligate mutualists and are required for the normal development and reproduction of the host. They are best known, however, for the various reproductive parasitism traits that they can generate in infected hosts. These include cytoplasmic incompatibility (CI) between individuals of different infection status, the parthenogenetic production of females, the selective killing of male embryos, and the feminization of genetic males. Wolbachia infections of Drosophila melanogaster are extremely common in both wild populations and long-term laboratory stocks. Utilizing the newly completed genome sequence of Wolbachia pipientis wMel, we have identified a number of polymorphic markers that can be used to discriminate among five different Wolbachia variants within what was previously thought to be the single clonal infection of D. melanogaster. Analysis of long-term lab stocks together with wild-caught flies indicates that one of these variants has replaced the others globally within the last century. This is the first report of a global replacement of a Wolbachia strain in an insect host species. The sweep is at odds with current theory that cannot explain how Wolbachia can invade this host species given the observed cytoplasmic incompatibility characteristics of Wolbachia infections in D. melanogaster in the field.     

Proviral amplification of the Gypsy endogenous retrovirus of Drosophila melanogaster involves env-independent invasion of the female germline

Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.     

Timing and targeting of P-element local transposition in the male germline cells of Drosophila melanogaster

The P element in Drosophila melanogaster preferentially transposes into nearby sites. The local insertions display a preferential orientation toward the starting element. We investigated the mechanism of the P-element local transposition by isolating and characterizing local insertions in the male germline. We designed a genetic screen employing a marker gene that is carried in the P element and is dose sensitive. This dose effect allows isolation of flies containing newly transposed P elements in the presence of the starting element. A rapid molecular screen with PCR was used to identify 45 local insertions located within an approximately 40-kb genomic region on both sides of the starting element. Our system permits the isolation of the cluster progeny derived from a single insertion event, but none was isolated. The data suggest that local transposition occurs in the meiotic cell cycle. Nearly all of the local insertions were located within the promoter regions of the genes that were active in the male germline cells, suggesting that local insertions target predominantly active promoters. Our analysis shows that local transposition of the P element is highly regulated, displaying a cell-type specificity and a target specificity.     

The compensatory response locus deletion increases the number of nucleoli in Drosophila melanogaster polytene cells

The formation of ribosomal DNA (rDNA) not associated with the nucleolar organizer (NO) regions was studied in polytene cells of Drosophila melanogaster mutants, mal¹² and bb^2rl, heterozygous for deficiencies in the NO. In the mutant X chromosome mal¹² a smaller part of the NO is deleted than in bb^2rl but it comprises the compensatory response (cr) locus, controlling the compensatory synthesis of rDNA. We found that in the polytene cells of all tissues of mutant X/Xmal¹² investigated (larval salivary gland, fat body and midgut; adult fly midgut) the number of nucleoli was increased compared with that in the corresponding cells of X/Xbb^2rl and X/X (wild type). Using in situ hybridization we showed that in the salivary gland cells of the X/Xmal¹² larvae chromosomal sites containing type I insertion sequences and scattered throughout the genome were more frequent than in the wild type and in X/Xbb^2rl mutant cells.     

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.