Diversity of Free-Living Environmental Bacteria and Their Interactions With a Bactivorous Amoeba

A small subset of bacteria in soil interact directly with eukaryotes. Which ones do so can reveal what is important to a eukaryote and how eukaryote defenses might be breached. Soil amoebae are simple eukaryotic organisms and as such could be particularly good for understanding how eukaryote microbiomes originate and are maintained. One such amoeba, Dictyostelium discoideum, has both permanent and temporary associations with bacteria. Here we focus on culturable bacterial associates in order to interrogate their relationship with D. discoideum. To do this, we isolated over 250 D. discoideum fruiting body samples from soil and deer feces at Mountain Lake Biological Station. In one-third of the wild D. discoideum we tested, one to six bacterial species were found per fruiting body sorus (spore mass) for a total of 174 bacterial isolates. The remaining two-thirds of D. discoideum fruiting body samples did not contain culturable bacteria, as is thought to be the norm. A majority (71.4%) of the unique bacterial haplotypes are in Proteobacteria. The rest are in either Actinobacteria, Bacteriodetes, or Firmicutes. The highest bacterial diversity was found in D. discoideum fruiting bodies originating from deer feces (27 OTUs), greater than either of those originating in shallow (11 OTUs) or in deep soil (4 OTUs). Rarefaction curves and the Chao1 estimator for species richness indicated the diversity in any substrate was not fully sampled, but for soil it came close. A majority of the D. discoideum-associated bacteria were edible by D. discoideum and supported its growth (75.2% for feces and 81.8% for soil habitats). However, we found several bacteria genera were able to evade phagocytosis and persist in D. discoideum cells through one or more social cycles. This study focuses not on the entire D. discoideum microbiome, but on the culturable subset of bacteria that have important eukaryote interactions as prey, symbionts, or pathogens. These eukaryote and bacteria interactions may provide fertile ground for investigations of bacteria using amoebas to gain an initial foothold in eukaryotes and of the origins of symbiosis and simple microbiomes

Variable Incidence of Spiroplasma Infections in Natural Populations of Drosophila Species

Spiroplasma is widespread as a heritable bacterial symbiont in insects and some other invertebrates, in which it sometimes acts as a male-killer and causes female-biased sex ratios in hosts. Besides Wolbachia, it is the only heritable bacterium known from Drosophila, having been found in 16 of over 200 Drosophila species screened, based on samples of one or few individuals per species. To assess the extent to which Spiroplasma infection varies within and among species of Drosophila, intensive sampling consisting of 50–281 individuals per species was conducted for natural populations of 19 Drosophila species. Infection rates varied among species and among populations of the same species, and 12 of 19 species tested negative for all individuals. Spiroplasma infection never was fixed, and the highest infection rates were 60% in certain populations of D. hydei and 85% in certain populations of D. mojavensis. In infected species, infection rates were similar for males and females, indicating that these Spiroplasma infections do not confer a strong male-killing effect. These findings suggest that Spiroplasma has other effects on hosts that allow it to persist, and that environmental or host variation affects transmission or persistence leading to differences among populations in infection frequencies

The Isolation of Nucleic Acids from Fixed, Paraffin-Embedded Tissues–Which Methods Are Useful When?

Museums and pathology collections around the world represent an archive of genetic material to study populations and diseases. For preservation purposes, a large portion of these collections has been fixed in formalin-containing solutions, a treatment that results in cross-linking of biomolecules. Cross-linking not only complicates isolation of nucleic acid but also introduces polymerase “blocks” during PCR. A wide variety of methods exists for the recovery of DNA and RNA from archival tissues, and although a number of previous studies have qualitatively compared the relative merits of the different techniques, very few have undertaken wide scale quantitative comparisons. To help address this issue, we have undertaken a study that investigates the quality of nucleic acids recovered from a test panel of fixed specimens that have been manipulated following a number of the published protocols. These include methods of pre-treating the samples prior to extraction, extraction and nucleic acid purification methods themselves, and a post-extraction enzymatic repair technique. We find that although many of the published methods have distinct positive effects on some characteristics of the nucleic acids, the benefits often come at a cost. In addition, a number of the previously published techniques appear to have no effect at all. Our findings recommend that the extraction methodology adopted should be chosen carefully. Here we provide a quick reference table that can be used to determine appropriate protocols for particular aims

Understanding the distribution of the Spiroplasma heritable bacterial endosymbiont in Drosophila

Symbiosis can be a major source of evolutionary innovation and a driver of ecological diversification. Insects, in particular, partner with a diversity of heritable bacterial endosymbionts that affect them in myriad ways, ranging from mutualistic to parasitic. Species of the genus Drosophila, however, harbor only Wolbachia and Spiroplasma. While Wolbachia infections in Drosophila are well characterized, much less is known about the consequences of Spiroplasma infections. Spiroplasma is a male-killer in some species of Drosophila, and recent work has demonstrated that this bacterium can also confer protection against a nematode parasite in one Drosophila species. Many other species of Drosophila, however, are infected with Spiroplasma that do not cause male-killing, and their fitness effects are unclear. To discern the impacts of Spiroplasma infections in Drosophila, my dissertation work seeks to characterize the distribution of Spiroplasma among Drosophila by investigating the genetic diversity of Spiroplasma infecting Drosophila, examining the bacterial density dynamics of diverse Spiroplasma strains infecting different Drosophila species and exploring the fitness consequences of Spiroplasma infection in a cactophilic Drosophila species. Using a multilocus phylogenetic analysis, I show that there have been at least five introductions of four very different types of Spiroplasma into Drosophila, with a single origin of the male-killing Spiroplasma in Drosophila. Horizontal transmission has played an important role in driving the distribution of Spiroplasma among Drosophila, and I provide some evidence that it may be occurring within populations of certain Drosophila species. I detected no recombination, however, among Spiroplasma from different phylogenetic clades, suggesting that Spiroplasma may be similar to beneficial bacterial endosymbionts trapped in their host with no opportunity for recombination. Using quantitative PCR, I assessed the bacterial density dynamics of Spiroplasma, which vary among bacterial strain and Drosophila species. Although a number of facultative endosymbionts play a role in host plant specialization, this does not seem to be the case for the Spiroplasma strain infecting one population of Drosophila with high infection prevalence. This characterization of the Drosophila/Spiroplasma symbiosis lays much of the groundwork necessary to effectively explore the consequences of Spiroplasma infections in Drosophil

Spiroplasma_alignments

This file contains nexus alignment files for each individual Spiroplasma locus (16S, RpoB, ParE, FtsZ, SpoT, and FruR) as well as all of the sequences concatenated

Transmission Fidelity Assay

This excel datasheet contains the data on the lab assay of Spiroplasma transmission fidelity of different strains of Spiroplasma in D. neotestacea (fly line = genetic background). The infection status of Wolbachia (W) is included, as well as the number of offspring infected (determined by PCR screening) from each Spiroplasma-infected mother

QPCR Assay

This excel datasheet contains the data for the absolute qPCR assay (Spiroplasma copy number per fly) of the poulsonii Spiroplasma strains in D. neotestacea of two genetic backgrounds. Also included are the number of generations post-injection of the flies assayed, as well as their age, Wolbachia infection status, and the qPCR plate on which they were run

Spiroplasma_tree_files

This data file contains the tree files (in Newick tree format) for each Spiroplasma locus (16S, RpoB, ParE, FtsZ, SpoT, and FruR) as well as the phylogeny of the concatenated set of loci

Wasp Resistance Assay

This is an excel spreadsheet containing the data from a lab fitness assay for wasp resistance. The data include numbers of emerging flies and wasps from vials of individual Drosophila neotestacea of different genetic backgrounds (fly strain) injected with different Spiroplasma strains (spiro strain) and parasitized by different wasp strains (wasp strain). The experiment was run twice, and for the second experiment, the total number of pupal cases were recorded as well