The Eriocheir sinensis calcium/calmodulin-dependent protein kinase II activates apoptosis to resist Spiroplasma eriocheiris infection.

Calcium/calmodulin-dependent protein kinase II is a downstream mediator of calcium signalling and participates in the regulation of various cellular physiological functions. In previous studies, the expression of Eriocheir sinensis CaMKII (EsCaMKII) was significantly decreased in the thoracic ganglion after Spiroplasma eriocheiris infection, as shown using TMT-based quantitative proteomic analysis; however, the specific functions of EsCaMKII are still unclear. In this study, the full-length cDNA of EsCaMKII was 3314 bp long, consisting of a 1605 bp open reading frame encoding a protein of 535 amino acids, including a 258 aa serine/threonine protein kinase catalytic domain (EsCaMKII-CD). EsCaMKII is highly transcribed in haemocytes, nerves (thoracic ganglion), gills, and muscles, but lowly transcribed in the hepatopancreas, heart, and intestines. The transcription levels of EsCaMKII were altered in E. sinensis haemocytes after S. eriocheiris infection. After the over-expression of EsCaMKII-CD in RAW264.7 cells, the apoptosis rate of RAW264.7 cells was significantly increased. After the over-expression of EsCaMKII-CD, the morphology of RAW264.7 cells became worse after being infected with S. eriocheiris. Meanwhile, the copy number of S. eriocheiris in RAW264.7 cells was significantly decreased. From 48 h to 96 h after EsCaMKII RNA interference, the transcription levels of EsCaMKII decreased significantly. The transcription of apoptosis genes and cell apoptosis were also inhibited in haemocytes after EsCaMKII RNAi. The knockdown of EsCaMKII by RNAi resulted in significant increases in the copy number of S. eriocheiris and in the mortality of crabs during S. eriocheiris infection. These results indicate that EsCaMKII could promote the apoptosis of E. sinensis and enhance its ability to resist S. eriocheiris infection.

Dual proteomics of Drosophila melanogaster hemolymph infected with the heritable endosymbiont Spiroplasma poulsonii.

Insects are frequently infected with heritable bacterial endosymbionts. Endosymbionts have a dramatic impact on their host physiology and evolution. Their tissue distribution is variable with some species being housed intracellularly, some extracellularly and some having a mixed lifestyle. The impact of extracellular endosymbionts on the biofluids they colonize (e.g. insect hemolymph) is however difficult to appreciate because biofluid composition can depend on the contribution of numerous tissues. Here we investigate Drosophila hemolymph proteome changes in response to the infection with the endosymbiont Spiroplasma poulsonii. S. poulsonii inhabits the fly hemolymph and gets vertically transmitted over generations by hijacking the oogenesis in females. Using dual proteomics on infected hemolymph, we uncovered a weak, chronic activation of the Toll immune pathway by S. poulsonii that was previously undetected by transcriptomics-based approaches. Using Drosophila genetics, we also identified candidate proteins putatively involved in controlling S. poulsonii growth. Last, we also provide a deep proteome of S. poulsonii, which, in combination with previously published transcriptomics data, improves our understanding of the post-transcriptional regulations operating in this bacterium.

Novel Spiroplasma sp. Isolated From CWD Is an Extreme Bacterial Thermoacidophile That Survives Autoclaving, Boiling, Formalin Treatment, and Significant Gamma Irradiation.

Rapid spreading of chronic wasting disease (CWD) in wildlife and captive cervid populations has exposed lack of progress in dealing with the transmissible spongiform encephalopathies (TSE) of man and animals. Since the TSE transmissible agent was resistant to extremes in environmental and chemical treatments, focus was on an unconventional agent including the prion theory. Recent breakthrough research has revealed consistent isolation of a novel Spiroplasma sp. from TSE-affected tissues that propagates in cell-free media and on agar. Here, we developed a live culture assay to test whether the CWD spiroplasma isolate possessed unconventional biologic properties akin to those of the transmissible agent of TSE. The CWD spiroplasma isolate survived boiling for 1 hour, standard liquid autoclaving, 10% formalin treatment overnight, and gamma irradiation of 20 kGy. The CWD spiroplasma isolate is an acidophile, growing best at pH 2. The biologic resistance of the CWD spiroplasma isolate may be due to unusual phage-like viruses found in the bacterial pellet or to DNA-protein binding. Because the CWD spiroplasma isolate has biologic properties consistent with the causal agent of the TSEs, TSE research focus should be redirected to development of diagnostic tests and preventive vaccines for control of CWD based upon the bacterium.

Spiroplasma eriocheiris Invasion Into Macrobrachium rosenbergii Hemocytes Is Mediated by Pathogen Enolase and Host Lipopolysaccharide and ß-1, 3-Glucan Binding Protein.

Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and ß-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.

Spatio-temporal distribution of Spiroplasma infections in the tsetse fly (Glossina fuscipes fuscipes) in northern Uganda.

Tsetse flies (Glossina spp.) are vectors of parasitic trypanosomes, which cause human (HAT) and animal African trypanosomiasis (AAT) in sub-Saharan Africa. In Uganda, Glossina fuscipes fuscipes (Gff) is the main vector of HAT, where it transmits Gambiense disease in the northwest and Rhodesiense disease in central, southeast and western regions. Endosymbionts can influence transmission efficiency of parasites through their insect vectors via conferring a protective effect against the parasite. It is known that the bacterium Spiroplasma is capable of protecting its Drosophila host from infection with a parasitic nematode. This endosymbiont can also impact its host's population structure via altering host reproductive traits. Here, we used field collections across 26 different Gff sampling sites in northern and western Uganda to investigate the association of Spiroplasma with geographic origin, seasonal conditions, Gff genetic background and sex, and trypanosome infection status. We also investigated the influence of Spiroplasma on Gff vector competence to trypanosome infections under laboratory conditions. Generalized linear models (GLM) showed that Spiroplasma probability was correlated with the geographic origin of Gff host and with the season of collection, with higher prevalence found in flies within the Albert Nile (0.42 vs 0.16) and Achwa River (0.36 vs 0.08) watersheds and with higher prevalence detected in flies collected in the intermediate than wet season. In contrast, there was no significant correlation of Spiroplasma prevalence with Gff host genetic background or sex once geographic origin was accounted for in generalized linear models. Additionally, we found a potential negative correlation of Spiroplasma with trypanosome infection, with only 2% of Spiroplasma infected flies harboring trypanosome co-infections. We also found that in a laboratory line of Gff, parasitic trypanosomes are less likely to colonize the midgut in individuals that harbor Spiroplasma infection. These results indicate that Spiroplasma infections in tsetse may be maintained by not only maternal but also via horizontal transmission routes, and Spiroplasma infections may also have important effects on trypanosome transmission efficiency of the host tsetse. Potential functional effects of Spiroplasma infection in Gff could have impacts on vector control approaches to reduce trypanosome infections.

Male-killing toxin in a bacterial symbiont of Drosophila.

Several lineages of symbiotic bacteria in insects selfishly manipulate host reproduction to spread in a population 1 , often by distorting host sex ratios. Spiroplasma poulsonii2,3 is a helical and motile, Gram-positive symbiotic bacterium that resides in a wide range of Drosophila species 4 . A notable feature of S. poulsonii is male killing, whereby the sons of infected female hosts are selectively killed during development1,2. Although male killing caused by S. poulsonii has been studied since the 1950s, its underlying mechanism is unknown. Here we identify an S. poulsonii protein, designated Spaid, whose expression induces male killing. Overexpression of Spaid in D. melanogaster kills males but not females, and induces massive apoptosis and neural defects, recapitulating the pathology observed in S. poulsonii-infected male embryos5-11. Our data suggest that Spaid targets the dosage compensation machinery on the male X chromosome to mediate its effects. Spaid contains ankyrin repeats and a deubiquitinase domain, which are required for its subcellular localization and activity. Moreover, we found a laboratory mutant strain of S. poulsonii with reduced male-killing ability and a large deletion in the spaid locus. Our study has uncovered a bacterial protein that affects host cellular machinery in a sex-specific way, which is likely to be the long-searched-for factor responsible for S. poulsonii-induced male killing.

Common and unique strategies of male killing evolved in two distinct Drosophila symbionts.

Male killing is a selfish reproductive manipulation caused by symbiotic bacteria, where male offspring of infected hosts are selectively killed. The underlying mechanisms and the process of their evolution are of great interest not only in terms of fundamental biology, but also their potential applications. The two bacterial Drosophila symbionts, Wolbachia and Spiroplasma, have independently evolved male-killing ability. This raises the question whether the underlying mechanisms share some similarities or are specific to each bacterial species. Here, we analyse pathogenic phenotypes of D. bifasciata infected with its natural male-killing Wolbachia strain and compare them with those of D. melanogaster infected with male-killing Spiroplasma We show that male progeny infected with the Wolbachia strain die during embryogenesis with abnormal apoptosis. Interestingly, male-killing Wolbachia infection induces DNA damage and segregation defects in the dosage-compensated chromosome in male embryos, which are reminiscent of the phenotypes caused by male-killing Spiroplasma in D. melanogaster By contrast, host neural development seems to proceed normally unlike male-killing Spiroplasma infection. Our results demonstrate that the dosage-compensated chromosome is a common target of two distinct male killers, yet Spiroplasma uniquely evolved the ability to damage neural tissue of male embryos.

Infection Function of Adhesin-Like Protein ALP609 from Spiroplasma melliferum CH-1.

Spiroplasma melliferum is the causative agent of spiroplasmosis in honeybees. During infection, adhesion of spiroplasmas to the host cells through adhesion factors is a crucial step. In this study, we identified an adhesin-like protein (ALP609) in S. melliferum CH-1 and investigated its role in the infection. To determine whether ALP609 is an adhesion factor, we performed indirect immunofluorescence microscopy to visualize its adhesion properties. Subsequently, an infection model of S. melliferum CH-1 was established using primary midgut cells of Apis mellifera to examine the adhesion and invasion of spiroplasma using anti-ALP609 antibodies inhibition assays and competition assays with recombinant ALP609 in vitro. We found that anti-ALP609 antibodies could inhibit the adhesion and invasion of spiroplasma to the midgut cells of A. mellifera and reduce midgut cell invasion on increased exposure to recombinant ALP609. To the best of our knowledge, this is the first report identifying adhesion-related factors in S. melliferum. Our results suggested that ALP609 is an adhesin-like protein critical for invasion of S. melliferum CH-1 into midgut cells of A. mellifera.

Immune responses in the haemolymph and antimicrobial peptide expression in the abdomen of Apis mellifera challenged with Spiroplasma melliferum CH-1.

Spiroplasma melliferum generally parasitizes honeybees and is one of main pathogens causing 'bee creeping disease' in China. Spiroplasma melliferum can be spread through honeybee pollination, which causes severe economic losses to apiculture. The design of this study was based on previous studies that utilized an in vitro bioassay to investigate the effects of S. melliferum CH-1 infection. We identified invasive S. melliferum CH-1 within Apis mellifera using transmission electron microscopy and investigated the immune response of honeybees infected with S. melliferum CH-1 by assaying the cellular immune response of the haemocytes, the plasma level of phenoloxidase activity and the transcript levels of 5 antimicrobial peptides, including the Abaecin, Apidaecin, Defensin 1, Defensin 2, and Hymenoptaecin gene products. The percentage of granulocytes in the haemolymph of infected honeybees was significantly higher than those of the controls during the early phase of infection, but the percentage of plasmatocytes was significantly higher than those of the controls at the fifth day post-infection. The phenoloxidase activity of the infected honeybees reached a maximum at the second day, and then decreased continuously. Moreover, the transcript levels of the 5 evaluated antimicrobial peptide genes were significantly increased during the early phase of infection and all 5 antimicrobial peptides were significantly decreased during the middle phase of infection. During the late phase of infection, only Defensin 2 and Hymenoptaecin showed significantly increased transcription. These results suggest that the honeybee immune responses could be activated by S. melliferum CH-1 during the early phase of infection and that S. melliferum CH-1 is also capable of circumventing the host defensive mechanisms to complete its life cycle within the honeybee during the middle phase of infection.

Evaluation of the Biocontrol Potential of Purpureocillium lilacinum QLP12 against Verticillium dahliae in Eggplant.

A fungus with broad spectrum antifungal activity was isolated from the soil in Qinling Mountain, Shaanxi Province, in China. The fungus was identified as Purpureocillium lilacinum based on ITS rDNA gene analysis. The strain, coded as QLP12, showed high inhibition activity on fungal mycelium growth in vitro, especially to Mucor piriformis, Trichothecium roseum, Rhizoctonia solani, and Verticillium dahliae, and its potential for biocontrol efficacy of eggplant. Verticillium wilt disease caused by Verticillium dahliae among 10 fungal species tested was explored. In greenhouse experiments, QLP12 showed an excellent growth-promoting effect on eggplant seed germination (76.7%), bud growth (79.4%), chlorophyll content (47.83%), root activity (182.02%), and so on. QLP12 can colonize the eggplant interior and also develop in rhizosphere soil. In greenhouse, the incidence of Verticillium wilt decreased by 83.82% with pretreated QLP12 fermentation broth in the soil. In the field, QLP12 showed prominent biocontrol effects on Verticillium wilt by reducing the disease index over the whole growth period, a decline of 40.1%. This study showed that the strain QLP12 is not only an effective biocontrol agent for controlling Verticillium wilt of eggplant, but also a plant growth-promoting fungus that deserves to be further developed.

Identification of lysophospholipase protein from Spiroplasma eriocheiris and verification of its function.

Spiroplasma eriocheiris is known to cause tremor disease in the Chinese mitten crab Eriocheir sinensis; however, the molecular characterization of this pathogen is still unclear. S. eriocheiris has the ability to invade and survive within mouse 3T6 cells. The invasion process may require causing damage to the host cell membrane by chemical, physical or enzymatic means. In this study, we systematically characterized a novel lysophospholipase (lysoPL) of S. eriocheiris TDA-040725-5T. The gene that encodes lysoPL in S. eriocheiris (SE-LysoPL) was cloned, sequenced and expressed in Escherichia coli BL21 (DE3). Enzymatic assays revealed that the purified recombinant SE-LysoPL hydrolysed long-chain acyl esterases at pH 7 and 30 °C. SE-LysoPL was detected in the membrane and cytoplasmic protein fractions using the SE-LysoPL antibody in Western blot. The virulence ability of S. eriocheiris was effectively reduced at the early stage of infection (m.o.i.=100) by the SE-LysoPL antibody neutralization test. To the best of our knowledge, this is the first study to identify and characterize a gene from S. eriocheiris encoding a protein exhibiting lysoPL and esterase activities. Our findings indicate that SE-LysoPL plays important roles in the pathogenicity of S. eriocheiris.

Comparative Genomic Analysis of Drechmeria coniospora Reveals Core and Specific Genetic Requirements for Fungal Endoparasitism of Nematodes.

Drechmeria coniospora is an obligate fungal pathogen that infects nematodes via the adhesion of specialized spores to the host cuticle. D. coniospora is frequently found associated with Caenorhabditis elegans in environmental samples. It is used in the study of the nematode's response to fungal infection. Full understanding of this bi-partite interaction requires knowledge of the pathogen's genome, analysis of its gene expression program and a capacity for genetic engineering. The acquisition of all three is reported here. A phylogenetic analysis placed D. coniospora close to the truffle parasite Tolypocladium ophioglossoides, and Hirsutella minnesotensis, another nematophagous fungus. Ascomycete nematopathogenicity is polyphyletic; D. coniospora represents a branch that has not been molecularly characterized. A detailed in silico functional analysis, comparing D. coniospora to 11 fungal species, revealed genes and gene families potentially involved in virulence and showed it to be a highly specialized pathogen. A targeted comparison with nematophagous fungi highlighted D. coniospora-specific genes and a core set of genes associated with nematode parasitism. A comparative gene expression analysis of samples from fungal spores and mycelia, and infected C. elegans, gave a molecular view of the different stages of the D. coniospora lifecycle. Transformation of D. coniospora allowed targeted gene knock-out and the production of fungus that expresses fluorescent reporter genes. It also permitted the initial characterisation of a potential fungal counter-defensive strategy, involving interference with a host antimicrobial mechanism. This high-quality annotated genome for D. coniospora gives insights into the evolution and virulence of nematode-destroying fungi. Coupled with genetic transformation, it opens the way for molecular dissection of D. coniospora physiology, and will allow both sides of the interaction between D. coniospora and C. elegans, as well as the evolutionary arms race that exists between pathogen and host, to be studied.

Arthropod-Spiroplasma relationship in the genomic era.

The genus Spiroplasma comprises wall-less, low-GC bacteria that establish pathogenic, mutualistic and commensal symbiotic associations with arthropods and plants. This review focuses on the symbiotic relationships between Spiroplasma bacteria and arthropod hosts in the context of the available genomic sequences. Spiroplasma genomes are reduced and some contain highly repetitive plectrovirus-related sequences. Spiroplasma's diversity in viral invasion susceptibility, virulence factors, substrate utilization, genome dynamics and symbiotic associations with arthropods make this bacterial genus a biological model that provides insights about the evolutionary traits that shape bacterial symbiotic relationships with eukaryotes.

Rapid spread of the defensive endosymbiont Spiroplasma in Drosophila hydei under high parasitoid wasp pressure.

Maternally transmitted endosymbionts of insects are ubiquitous in nature and play diverse roles in the ecology and evolution of their hosts. To persist in host lineages, many symbionts manipulate host reproduction to their advantage (e.g. cytoplasmic incompatibility and male-killing), or confer fitness benefits to their hosts (e.g. metabolic provisioning and defense against natural enemies). Recent studies suggest that strains of the bacterial genus Spiroplasma protect their host (flies in the genus Drosophila) against parasitoid attack. The Spiroplasma-conferred protection is partial and flies surviving a wasp attack have reduced adult longevity and fecundity. Therefore, it is unclear whether protection against wasps alone can counter Spiroplasma loss by imperfect maternal transmission and any possible fitness costs to harboring Spiroplasma. To address this question, we conducted a population cage study comparing Spiroplasma frequencies over time (host generations) under conditions of high wasp pressure and no wasp pressure. A dramatic increase of Spiroplasma prevalence was observed under high wasp pressure. In contrast, Spiroplasma prevalence in the absence of wasps did not change significantly over time; a pattern consistent with random drift. Thus, the defensive mechanism may contribute to the high prevalence of Spiroplasma in host populations despite imperfect vertical transmission.

The case for involvement of spiroplasma in the pathogenesis of transmissible spongiform encephalopathies.

Spiroplasma biofilm formation explains the role of these wall-less bacteria in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Spiroplasma embedded in the biofilm polysaccharide matrix are markedly resistant to physical and chemical treatment, simulating the biologic properties of the TSE agent. Microcolonies of spiroplasma embedded in biofilm bound to clay are the likely mechanism of lateral transmission of scrapie in sheep and chronic wasting disease in deer via soil ingestion. Spiroplasma in biofilm bound to the stainless steel of surgical instruments may also cause iatrogenic transmission of Creutzfeldt-Jakob disease. Sessile spiroplasma in biofilm attach to the surface by curli-like fibrils, a functional amyloid that is important for spiroplasma entering cells. Curli fibers have been shown to interact with host proteins and initiate formation of a potentially toxic amyloid that multiplies by self-assembly. In TSE, this mechanism may explain how spiroplasma trigger the formation of prion amyloid. This possibility is supported by experiments that show spiroplasma produce α-synuclein in mammalian tissue cultures. The data linking spiroplasma to neurodegenerative diseases provide a rationale for developing diagnostic tests for TSE based on the presence of spiroplasma-specific proteins or nucleic acid. Research efforts should focus on this bacterium for development of therapeutic regimens for Creutzfeldt-Jakob disease.

RNA-Seq analysis reveals candidate genes for ontogenic resistance in Malus-Venturia pathosystem.

Ontogenic scab resistance in apple leaves and fruits is a horizontal resistance against the plant pathogen Venturia inaequalis and is expressed as a decrease in disease symptoms and incidence with the ageing of the leaves. Several studies at the biochemical level tried to unveil the nature of this resistance; however, no conclusive results were reported. We decided therefore to investigate the genetic origin of this phenomenon by performing a full quantitative transcriptome sequencing and comparison of young (susceptible) and old (ontogenic resistant) leaves, infected or not with the pathogen. Two time points at 72 and 96 hours post-inoculation were chosen for RNA sampling and sequencing. Comparison between the different conditions (young and old leaves, inoculated or not) should allow the identification of differentially expressed genes which may represent different induced plant defence reactions leading to ontogenic resistance or may be the cause of a constitutive (uninoculated with the pathogen) shift toward resistance in old leaves. Differentially expressed genes were then characterised for their function by homology to A. thaliana and other plant genes, particularly looking for genes involved in pathways already suspected of appertaining to ontogenic resistance in apple or other hosts, or to plant defence mechanisms in general. IN THIS WORK, FIVE CANDIDATE GENES PUTATIVELY INVOLVED IN THE ONTOGENIC RESISTANCE OF APPLE WERE IDENTIFIED: a gene encoding an "enhanced disease susceptibility 1 protein" was found to be down-regulated in both uninoculated and inoculated old leaves at 96 hpi, while the other four genes encoding proteins (metallothionein3-like protein, lipoxygenase, lipid transfer protein, and a peroxidase 3) were found to be constitutively up-regulated in inoculated and uninoculated old leaves. The modulation of the five candidate genes has been validated using the real-time quantitative PCR. Thus, ontogenic resistance may be the result of the corresponding up- and down-regulation of these genes.

Protection against a fungal pathogen conferred by the aphid facultative endosymbionts Rickettsia and Spiroplasma is expressed in multiple host genotypes and species and is not influenced by co-infection with another symbiont.

Many insects harbour facultative endosymbiotic bacteria, often more than one type at a time. These symbionts can have major effects on their hosts' biology, which may be modulated by the presence of other symbiont species and by the host's genetic background. We investigated these effects by transferring two sets of facultative endosymbionts (one Hamiltonella and Rickettsia, the other Hamiltonella and Spiroplasma) from naturally double-infected pea aphid hosts into five novel host genotypes of two aphid species. The symbionts were transferred either together or separately. We then measured aphid fecundity and susceptibility to an entomopathogenic fungus. The pathogen-protective phenotype conferred by the symbionts Rickettsia and Spiroplasma varied among host genotypes, but was not influenced by co-infection with Hamiltonella. Fecundity varied across single and double infections and between symbiont types, aphid genotypes and species. Some host genotypes benefit from harbouring more than one symbiont type.

Density dynamics of diverse Spiroplasma strains naturally infecting different species of Drosophila.

Facultative heritable bacterial endosymbionts can have dramatic effects on their hosts, ranging from mutualistic to parasitic. Within-host bacterial endosymbiont density plays a critical role in maintenance of a symbiotic relationship, as it can affect levels of vertical transmission and expression of phenotypic effects, both of which influence the infection prevalence in host populations. Species of genus Drosophila are infected with Spiroplasma, whose characterized phenotypic effects range from that of a male-killing reproductive parasite to beneficial defensive endosymbiont. For many strains of Spiroplasma infecting at least 17 species of Drosophila, however, the phenotypic effects are obscure. The infection prevalence of these Spiroplasma vary within and among Drosophila species, and little is known about the within-host density dynamics of these diverse strains. To characterize the patterns of Spiroplasma density variation among Drosophila we used quantitative PCR to assess bacterial titer at various life stages of three species of Drosophila naturally-infected with two different types of Spiroplasma. For naturally infected Drosophila species we found that non-male-killing infections had consistently lower densities than the male-killing infection. The patterns of Spiroplasma titer change during aging varied among Drosophila species infected with different Spiroplasma strains. Bacterial density varied within and among populations of Drosophila, with individuals from the population with the highest prevalence of infection having the highest density. This density variation underscores the complex interaction of Spiroplasma strain and host genetic background in determining endosymbiont density.

Dynamics of the continent-wide spread of a Drosophila defensive symbiont.

Facultative symbionts can represent important sources of adaptation for their insect hosts and thus have the potential for rapid spread. Drosophila neotestacea harbours a heritable symbiont, Spiroplasma, that confers protection against parasitic nematodes. We previously found a cline in Spiroplasma prevalence across central Canada, ending abruptly at the Rocky Mountains. Resampling these populations 9 years later revealed that Spiroplasma had increased substantially across the region, resembling a Fisherian wave of advance. Associations between Spiroplasma infection and host mitochondrial DNA indicate that the increase was due to local increase of Spiroplasma-infected flies. Finally, we detected Spiroplasma west of the Rocky Mountains for the first time and showed that defence against nematodes occurs in flies with a western genetic background. Because nematode infection is common throughout D. neotestacea's range, we expect Spiroplasma to spread to the Pacific coast.