Project description:Wolbachia pipientis is a worldwide bacterial parasite of arthropods that infects host germline cells and manipulates host reproduction to increase the ratio of infected females, the transmitting sex of the bacteria. The most common reproductive manipulation, cytoplasmic incompatibility (CI), is expressed as embryonic death in crosses between infected males and uninfected females. Specifically, Wolbachia modify developing sperm in the testes by unknown means to cause a post-fertilization disruption of the sperm chromatin that incapacitates the first mitosis of the embryo. As these Wolbachia-induced changes are stable, reversible, and affect the host cell cycle machinery including DNA replication and chromosome segregation, we hypothesized that the host methylation pathway is targeted for modulation during cytoplasmic incompatibility because it accounts for all of these traits. Here we show that infection of the testes is associated with a 55% increase of host DNA methylation in Drosophila melanogaster, but methylation of the paternal genome does not correlate with penetrance of CI. Overexpression and knock out of the Drosophila DNA methyltransferase Dnmt2 neither induces nor increases cytoplasmic incompatibility. Instead, overexpression decreases Wolbachia titers in host testes by approximately 17%, leading to a similar reduction in CI levels. Finally, strength of CI induced by several different strains of Wolbachia does not correlate with levels of DNA methylation in the host testes. We conclude that DNA methylation mediated by Drosophila's only known methyltransferase is not required for the transgenerational sperm modification that causes CI. Genomic DNA was extracted from pooled samples of Drosophila melanogaster adult testes. One sample from Wolbachia-infected males and one from uninfected males. Bisulfite sequencing was used to determine whether Wolbachia infection affects host DNA methylation in the testes.
Project description:We investigated tissue-specific regulation of gene expression in a long lived Drosophila IIS mutant (dilp2-3,5) compared to its control (wDah). As in the majority of Drosophila laboratory strains, the endosymbiont Wolbachia was present; in the absence of Wolbachia, life extension through dilp2-3,5 is abrogated (Grönke et al. 2010). To control for this, we additionally included strains of the same genotypes that lacked Wolbachia (dilp2-3,5T, wDahT). We quantified gene expression concurrently on the level of the proteome and the transcriptome, in four key tissues: brain, gut, fat body, and muscle. Proteome quantification was carried out on five biological replicates per experimental group. Corresponding transcriptome quantification was carried out on three biological replicates per experimental group, and published on GEO (accession number *GSE122190*)
Project description:Wolbachia is a maternally transmitted bacterium that manipulates arthropod and nematode biology in myriad ways. The Wolbachia strain colonizing Drosophila melanogaster creates sperm-egg incompatibilities and protects its host against RNA viruses, making it a promising tool for vector control. Despite successful trials using Wolbachia-transfected mosquitoes for Dengue control, knowledge of how Wolbachia and viruses jointly affect insect biology remains limited. Using the Drosophila melanogaster model, transcriptomics and gene expression network analyses revealed pathways with altered expression and splicing due to Wolbachia colonization and virus infection. Included are metabolic pathways previously unknown to be important for Wolbachia-host interactions. Additionally, Wolbachia-colonized flies exhibit a dampened transcriptomic response to virus infection, consistent with early blocking of virus replication. Finally, using Drosophila genetics, we show Wolbachia and expression of nucleotide metabolism genes have interactive effects on virus replication. Understanding the mechanisms of pathogen blocking will contribute to the effective development of Wolbachia-mediated vector control programs.
Project description:We used microarray analysis to screen the Drosophila genome for effects of Wolbachia infection on transcript levels in wildtype adult immature ovaries. Keywords: Comparative expression analysis of infection effect
Project description:Using microarray-based comparative genome hybridizations (mCGH), the genomic content of Wolbachia pipientis wMel from Drosophila melanogaster was compared to the Wolbachia from D. innubila (wInn), D. santomea (wSan), and three strains from D. simulans (wAu, wRi, wSim).
2009-05-11 | GSE13825 | GEO
Project description:Single Cell RNA Sequencing of Drosophila Testis Tissue
Project description:Wolbachia, an endosymbiotic bacterium, is being investigated as a vector control agent in several insect species. Along with the well known classical reproductive parasitism Wolbachia employs against its host to spread within the population, it is emerging that the bacteria can protect the host against pathogens and reduced pathogen transmission. Anopheles mosquitoes, which transmit malaria, have never been found to harbour Wolbachia in nature, and despite numerous transinfection attempts, no stable line has been developed. However recently, two strains of Wolbachia, wAlbB from Aedes albopictus, and wRi from Drosophila simulans were cultured in Anopheles gambiae Sua5B cells. These cell lines provides an amenable system to study Wolbachia-Anopheles interaction in the absence of a stable transinfected line. It has been proposed that the compromised vector competence of Wolbachia infected insects is due to an up regulation of the basal immune state. We therefore completed a genome wide expression profile of Wolbachia infected Anopheles, assessing both wAlbB and wRi infected cells in parallel against uninfected Sua5B cells.
Project description:We used microarray analysis to screen the Drosophila genome for effects of Wolbachia infection on transcript levels in wildtype adult immature ovaries. Keywords: Comparative expression analysis of infection effect To generate the ovaries to be compared, we first rid a wildtype Canton-S line of Drosophila of the well characterized YW strain of Wolbachia that it harbored (Bourtzis et al., 1998, Presgraves, 2000) by raising the flies for three generations on food containing tetracycline. After the line had been raised for three more generations on food without tetracycline, we used a standard PCR assay (O'Neil et al., 1999) to confirm that the line was Wolbachia free. Genetically matched infected and uninfected females were recovered as the daughters from two reciprocal crosses between the infected and the cured Canton-S lines. Previtellogenic ovaries were harvested from these females 1-5 h after eclosion
Project description:Wolbachia, an endosymbiotic bacterium, is being investigated as a vector control agent in several insect species. Along with the well known classical reproductive parasitism Wolbachia employs against its host to spread within the population, it is emerging that the bacteria can protect the host against pathogens and reduced pathogen transmission. Anopheles mosquitoes, which transmit malaria, have never been found to harbour Wolbachia in nature, and despite numerous transinfection attempts, no stable line has been developed. However recently, two strains of Wolbachia, wAlbB from Aedes albopictus, and wRi from Drosophila simulans were cultured in Anopheles gambiae Sua5B cells. These cell lines provides an amenable system to study Wolbachia-Anopheles interaction in the absence of a stable transinfected line. It has been proposed that the compromised vector competence of Wolbachia infected insects is due to an up regulation of the basal immune state. We therefore completed a genome wide expression profile of Wolbachia infected Anopheles, assessing both wAlbB and wRi infected cells in parallel against uninfected Sua5B cells. Two strains of Wolbachia, wRi from Drosophila simulans and wAlbB from Aedes albopictus were transfered into Anopheles gambiae Sua5B cells via the shell vial technique. After over 30 passages, these Wolbachia infected cells lines were then compared, in parallel, to the original uninfected Sua5B cells using Affymetrix microarrays.
Project description:The Drosophila-Wolbachia system is being used to study the molecular nature of the interactions between a host and a symbiont. This system offers a unique opportunity for such a study since the Drosophila genome sequence is available, several Wolbachi genomes will also be available soon and there are at least three known Wolbachia strains infecting Drosophila: a) mod+ strain that induces cytoplasmic incompatibility, b) mod- strain that cannot induce cytoplasmic incompatibility, and c) popcorn strain, a virulent strain which reduces in half the adult lifespan of Drosophila due to its massive proliferation in adult brain. The Drosophila-Wolbachia interaction manifests itself in 3 main ways; first, destruction of the CNS in infected adults, second, induction of some kind of modification or imprinting in the male germ-line resulting in an early failure in embryonic development, (cytoplasmic incompatability (CI)) and third, modification of the female germ-line resulting in resistance to modified sperm. We are interested in identifying Drosophila genes with changes in expression due to Wolbachia infection. We have generated a series of isogenic fly lines (those being used in the IGF P-element project) which we have infected with Wolbachia strains, infection is readily cured by growth on medium containing tetracycline. Thus, we have equivalent genetic background with and without the parasite. We have tested all of the transgenic lines for the level of CI and find strain-specific levels ranging from 0-50%. We also have a strain of D. simulans that shows over 95% CI. Plan: For our initial experiments we wish to make 4 comparisons, in all cases 2 day old males will be collected and for each comparison we will isolate 3 independent biological replicates: Melanogaster no CI [tet] x Melanogaster no CI [+wol] Melanogaster high CI [tet] x Melanogaster high CI [+wol] We will therefore identify genes with changed expression levels in the male upon Wolbachia infection by comparing the melanogaster strains with high or no CI in the presence of tetracycline and Wolbachia. We also hope to identify similar genes in simulans (where we expect the magnitude of the effect to be larger), differences between melanogaster and simulans are controlled for in the mel v sim comparison.