Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens.

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that Haoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont's genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

A highly divergent Wolbachia with a tiny genome in an insect-parasitic tylenchid nematode.

Wolbachia symbionts are the most successful host-associated microbes on the planet, infecting arthropods and nematodes. Their role in nematodes is particularly enigmatic, with filarial nematode species either 100% infected and dependent on symbionts for reproduction and development, or not at all infected. We have discovered a highly divergent strain of Wolbachia in an insect-parasitic tylenchid nematode, Howardula sp., in a nematode clade that has not previously been known to harbour Wolbachia. While this nematode is 100% infected with Wolbachia, we did not detect it in related species. We sequenced the Howardula symbiont (wHow) genome and found that it is highly reduced, comprising only 550 kilobase pairs of DNA, approximately 35% smaller than the smallest Wolbachia nematode symbiont genomes. The wHow genome is a subset of all other Wolbachia genomes and has not acquired any new genetic information. While it has lost many genes, including genes involved in cell wall synthesis and cell division, it has retained the entire haem biosynthesis pathway, suggesting that haem supplementation is critical. wHow provides key insights into our understanding of what are the lower limits of Wolbachia cells, as well as the role of Wolbachia symbionts in the biology and convergent evolution of diverse parasitic nematodes.

Autosomal suppression and fitness costs of an old driving X chromosome in Drosophila testacea.

Driving X chromosomes (XD s) bias their own transmission through males by killing Y-bearing gametes. These chromosomes can in theory spread rapidly in populations and cause extinction, but many are found as balanced polymorphisms or as "cryptic" XD s shut down by drive suppressors. The relative likelihood of these outcomes and the evolutionary pathways through which they come about are not well understood. An XD was recently discovered in the mycophagous fly, Drosophila testacea, presenting the opportunity to compare this XD with the well-studied XD of its sister species, Drosophila neotestacea. Comparing features of independently evolved XD s in young sister species is a promising avenue towards understanding how XD s and their counteracting forces change over time. In contrast to the XD of D. neotestacea, we find that the XD of D. testacea is old, with its origin predating the radiation of three species: D. testacea, D. neotestacea and their shared sister species, Drosophila orientacea. Motivated by the suggestion that older XD s should be more deleterious to carriers, we assessed the effect of the XD on both male and female fertility. Unlike what is known from D. neotestacea, we found a strong fitness cost in females homozygous for the XD in D. testacea: a large proportion of homozygous females failed to produce offspring after being housed with males for several days. Our male fertility experiments show that although XD male fertility is lower under sperm-depleting conditions, XD males have comparable fertility to males carrying a standard X chromosome under a free-mating regime, which may better approximate conditions in wild populations of D. testacea. Lastly, we demonstrate the presence of autosomal suppression of X chromosome drive. Our results provide support for a model of XD evolution where the dynamics of young XD s are governed by fitness consequences in males, whereas in older XD systems, both suppression and fitness consequences in females likely supersede male fitness costs.

Generality of toxins in defensive symbiosis: Ribosome-inactivating proteins and defense against parasitic wasps in Drosophila.

While it has become increasingly clear that multicellular organisms often harbor microbial symbionts that protect their hosts against natural enemies, the mechanistic underpinnings underlying most defensive symbioses are largely unknown. Spiroplasma bacteria are widespread associates of terrestrial arthropods, and include strains that protect diverse Drosophila flies against parasitic wasps and nematodes. Recent work implicated a ribosome-inactivating protein (RIP) encoded by Spiroplasma, and related to Shiga-like toxins in enterohemorrhagic Escherichia coli, in defense against a virulent parasitic nematode in the woodland fly, Drosophila neotestacea. Here we test the generality of RIP-mediated protection by examining whether Spiroplasma RIPs also play a role in wasp protection, in D. melanogaster and D. neotestacea. We find strong evidence for a major role of RIPs, with ribosomal RNA (rRNA) from the larval endoparasitic wasps, Leptopilina heterotoma and Leptopilina boulardi, exhibiting the hallmarks of RIP activity. In Spiroplasma-containing hosts, parasitic wasp ribosomes show abundant site-specific depurination in the α-sarcin/ricin loop of the 28S rRNA, with depurination occurring soon after wasp eggs hatch inside fly larvae. Interestingly, we found that the pupal ectoparasitic wasp, Pachycrepoideus vindemmiae, escapes protection by Spiroplasma, and its ribosomes do not show high levels of depurination. We also show that fly ribosomes show little evidence of targeting by RIPs. Finally, we find that the genome of D. neotestacea's defensive Spiroplasma encodes a diverse repertoire of RIP genes, which are differ in abundance. This work suggests that specificity of defensive symbionts against different natural enemies may be driven by the evolution of toxin repertoires, and that toxin diversity may play a role in shaping host-symbiont-enemy interactions.

Population biology of a selfish sex ratio distorting element in a booklouse (Psocodea: Liposcelis).

Arthropods harbour a variety of selfish genetic elements that manipulate reproduction to be preferentially transmitted to future generations. A major ongoing question is to understand how these elements persist in nature. In this study, we examine the population dynamics of an unusual selfish sex ratio distorter in a recently discovered species of booklouse, Liposcelis sp. (Psocodea: Liposcelididae) to gain a better understanding of some of the factors that may affect the persistence of this element. Females that carry the selfish genetic element only ever produce daughters, although they are obligately sexual. These females also only transmit the maternal half of their genome. We performed a replicated population cage experiment, varying the initial frequency of females that harbour the selfish element, and following female frequencies for 20 months. The selfish genetic element persisted in all cages, often reaching very high (and thus severely female-biased) frequencies. Surprisingly, we also found that females that carry the selfish genetic element had much lower fitness than their nondistorter counterparts, with lower lifetime fecundity, slower development and a shorter egg-laying period. We suggest that differential fitness plays a role in the maintenance of the selfish genetic element in this species. We believe that the genetic system in this species, paternal genome elimination, which allows maternal control of offspring sex ratio, may also be important in the persistence of the selfish genetic element, highlighting the need to consider species with diverse ecologies and genetic systems when investigating the effects of sex ratio manipulators on host populations.

A ribosome-inactivating protein in a Drosophila defensive symbiont.

Vertically transmitted symbionts that protect their hosts against parasites and pathogens are well known from insects, yet the underlying mechanisms of symbiont-mediated defense are largely unclear. A striking example of an ecologically important defensive symbiosis involves the woodland fly Drosophila neotestacea, which is protected by the bacterial endosymbiont Spiroplasma when parasitized by the nematode Howardula aoronymphium. The benefit of this defense strategy has led to the rapid spread of Spiroplasma throughout the range of D. neotestacea, although the molecular basis for this protection has been unresolved. Here, we show that Spiroplasma encodes a ribosome-inactivating protein (RIP) related to Shiga-like toxins from enterohemorrhagic Escherichia coli and that Howardula ribosomal RNA (rRNA) is depurinated during Spiroplasma-mediated protection of D. neotestacea. First, we show that recombinant Spiroplasma RIP catalyzes depurination of 28S rRNAs in a cell-free assay, as well as Howardula rRNA in vitro at the canonical RIP target site within the α-sarcin/ricin loop (SRL) of 28S rRNA. We then show that Howardula parasites in Spiroplasma-infected flies show a strong signal of rRNA depurination consistent with RIP-dependent modification and large decreases in the proportion of 28S rRNA intact at the α-sarcin/ricin loop. Notably, host 28S rRNA is largely unaffected, suggesting targeted specificity. Collectively, our study identifies a novel RIP in an insect defensive symbiont and suggests an underlying RIP-dependent mechanism in Spiroplasma-mediated defense.

Dissecting the interface between apicomplexan parasite and host cell: Insights from a divergent AMA-RON2 pair.

Plasmodium falciparum and Toxoplasma gondii are widely studied parasites in phylum Apicomplexa and the etiological agents of severe human malaria and toxoplasmosis, respectively. These intracellular pathogens have evolved a sophisticated invasion strategy that relies on delivery of proteins into the host cell, where parasite-derived rhoptry neck protein 2 (RON2) family members localize to the host outer membrane and serve as ligands for apical membrane antigen (AMA) family surface proteins displayed on the parasite. Recently, we showed that T. gondii harbors a novel AMA designated as TgAMA4 that shows extreme sequence divergence from all characterized AMA family members. Here we show that sporozoite-expressed TgAMA4 clusters in a distinct phylogenetic clade with Plasmodium merozoite apical erythrocyte-binding ligand (MAEBL) proteins and forms a high-affinity, functional complex with its coevolved partner, TgRON2L1. High-resolution crystal structures of TgAMA4 in the apo and TgRON2L1-bound forms complemented with alanine scanning mutagenesis data reveal an unexpected architecture and assembly mechanism relative to previously characterized AMA-RON2 complexes. Principally, TgAMA4 lacks both a deep surface groove and a key surface loop that have been established to govern RON2 ligand binding selectivity in other AMAs. Our study reveals a previously underappreciated level of molecular diversity at the parasite-host-cell interface and offers intriguing insight into the adaptation strategies underlying sporozoite invasion. Moreover, our data offer the potential for improved design of neutralizing therapeutics targeting a broad range of AMA-RON2 pairs and apicomplexan invasive stages.

Evolution and Diversity of Inherited Spiroplasma Symbionts in Myrmica Ants.

Microbial partners play important roles in the biology and ecology of animals. In insects, maternally transmitted symbionts are especially common and can have host effects ranging from reproductive manipulation to nutrient provisioning and defense against natural enemies. In this study, we report a genus-wide association of Myrmica ants with the inherited bacterial symbiont Spiroplasma We screen Myrmica ants collected from the wild, including the invasive European fire ant, Myrmica rubra, and find an extraordinarily high prevalence of this symbiont-8 of 9 species, 42 of 43 colonies, and 250 of 276 individual workers harbored Spiroplasma-only one host species was uninfected. In our screens, each host species carried a distinct Spiroplasma strain, and none were infected with more than one strain. All symbionts belong to the citri clade, allied most closely with pathogenic strains of Spiroplasma infecting corn crops and honeybees, and there is strong evidence of host-symbiont persistence across evolutionary time scales. Genome sequencing of two Spiroplasma symbionts revealed candidate genes that may play a part in the symbiosis, a nutrient transporter absent from other Spiroplasma strains, and a ribosome-inactivating protein previously implicated in parasite defense. These results together suggest long-term, likely mutualistic, relationships atypical of Spiroplasma-insect associations with potential significance for broad ecological interactions with MyrmicaIMPORTANCE Animal-associated microbial symbionts can dramatically affect the biology of their hosts. The identification and characterization of these intimate partnerships remain an essential component of describing and predicting species interactions, especially for invasive host species. Ants perform crucial ecological functions as ecosystem engineers, scavengers, and predators, and ants in the genus Myrmica can be aggressive resource competitors and reach high densities in their native and invaded habitats. In this study, a novel symbiosis is identified between Myrmica ants and the facultative bacterial symbiont Spiroplasma Broad host distribution, high frequencies of infection, and host-symbiont codivergence over evolutionary time scales, an uncommon feature of Spiroplasma associations, suggest an important likely mutualistic interaction. Genome sequencing identified highly divergent gene candidates that may contribute to Spiroplasma's role as a possible defensive or nutritional partner in Myrmica.

Maternal transmission, sex ratio distortion, and mitochondria.

In virtually all multicellular eukaryotes, mitochondria are transmitted exclusively through one parent, usually the mother. In this short review, we discuss some of the major consequences of uniparental transmission of mitochondria, including deleterious effects in males and selection for increased transmission through females. Many of these consequences, particularly sex ratio distortion, have well-studied parallels in other maternally transmitted genetic elements, such as bacterial endosymbionts of arthropods. We also discuss the consequences of linkage between mitochondria and other maternally transmitted genetic elements, including the role of cytonuclear incompatibilities in maintaining polymorphism. Finally, as a case study, we discuss a recently discovered maternally transmitted sex ratio distortion in an insect that is associated with extraordinarily divergent mitochondria.

An exceptional family: Ophiocordyceps-allied fungus dominates the microbiome of soft scale insects (Hemiptera: Sternorrhyncha: Coccidae).

Hemipteran insects of the suborder Sternorrhyncha are plant sap feeders, where each family is obligately associated with a specific bacterial endosymbiont that produces essential nutrients lacking in the sap. Coccidae (soft scale insects) is the only major sternorrhynchan family in which obligate symbiont(s) have not been identified. We studied the microbiota in seven species from this family from Israel, Spain and Cyprus, by high-throughput sequencing of ribosomal genes, and found that no specific bacterium was prevalent and abundant in all the tested species. In contrast, an Ophiocordyceps-allied fungus sp.-a lineage widely known as entomopathogenic-was highly prevalent. All individuals of all the tested species carried this fungus. Phylogenetic analyses showed that the Ophiocordyceps-allied fungus from the coccids is closely related to fungi described from other hemipterans, and they appear to be monophyletic, although the phylogenies of the Ophiocordyceps-allied fungi and their hosts do not appear to be congruent. Microscopic observations show that the fungal cells are lemon-shaped, are distributed throughout the host's body and are present in the eggs, suggesting vertical transmission. Taken together, the results suggest that the Ophiocordyceps-allied fungus may be a primary symbiont of Coccidae-a major evolutionary shift from bacteria to fungi in the Sternorrhyncha, and an important example of fungal evolutionary lifestyle switch.

Olfactory Preferences of the Parasitic Nematode Howardula aoronymphium and its Insect Host Drosophila falleni.

Many parasitic nematodes have an environmental infective stage that searches for hosts. Olfaction plays an important role in this process, with nematodes navigating their environment using host-emitted and environmental olfactory cues. The interactions between parasitic nematodes and their hosts are also influenced by the olfactory behaviors of the host, since host olfactory preferences drive behaviors that may facilitate or impede parasitic infection. However, how olfaction shapes parasite-host interactions is poorly understood. Here we investigated this question using the insect-parasitic nematode Howardula aoronymphium and its host, the mushroom fly Drosophila falleni. We found that both H. aoronymphium and D. falleni are attracted to mushroom odor and a subset of mushroom-derived odorants, but they have divergent olfactory preferences that are tuned to different mushroom odorants despite their shared mushroom environment. H. aoronymphium and D. falleni respond more narrowly to odorants than Caenorhabditis elegans and Drosophila melanogaster, consistent with their more specialized niches. Infection of D. falleni with H. aoronymphium alters its olfactory preferences, rendering it more narrowly tuned to mushroom odor. Our results establish H. aoronymphium-D. falleni as a model system for studying olfaction in the context of parasite-host interactions.

Immune genes and divergent antimicrobial peptides in flies of the subgenus Drosophila.

BACKGROUND: Drosophila is an important model for studying the evolution of animal immunity, due to the powerful genetic tools developed for D. melanogaster. However, Drosophila is an incredibly speciose lineage with a wide range of ecologies, natural histories, and diverse natural enemies. Surprisingly little functional work has been done on immune systems of species other than D. melanogaster. In this study, we examine the evolution of immune genes in the speciose subgenus Drosophila, which diverged from the subgenus Sophophora (that includes D. melanogaster) approximately 25-40 Mya. We focus on D. neotestacea, a woodland species used to study interactions between insects and parasitic nematodes, and combine recent transcriptomic data with infection experiments to elucidate aspects of host immunity. RESULTS: We found that the vast majority of genes involved in the D. melanogaster immune response are conserved in D. neotestacea, with a few interesting exceptions, particularly in antimicrobial peptides (AMPs); until recently, AMPs were not thought to evolve rapidly in Drosophila. Unexpectedly, we found a distinct diptericin in subgenus Drosophila flies that appears to have evolved under diversifying (positive) selection. We also describe the presence of the AMP drosocin, which was previously thought to be restricted to the subgenus Sophophora, in the subgenus Drosophila. We challenged two subgenus Drosophila species, D. neotestacea and D. virilis with bacterial and fungal pathogens and quantified AMP expression. CONCLUSIONS: While diptericin in D. virilis was induced by exposure to gram-negative bacteria, it was not induced in D. neotestacea, showing that conservation of immune genes does not necessarily imply conservation of the realized immune response. Our study lends support to the idea that invertebrate AMPs evolve rapidly, and that Drosophila harbor a diverse repertoire of AMPs with potentially important functional consequences.

Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches.

UNLABELLED: By combining genomics and isotope imaging analysis using high-resolution secondary ion mass spectrometry (NanoSIMS), we examined the function and evolution of Bacteroidales ectosymbionts of the protist Barbulanympha from the hindguts of the wood-eating cockroach Cryptocercus punctulatus In particular, we investigated the structure of ectosymbiont genomes, which, in contrast to those of endosymbionts, has been little studied to date, and tested the hypothesis that these ectosymbionts fix nitrogen. Unlike with most obligate endosymbionts, genome reduction has not played a major role in the evolution of the Barbulanympha ectosymbionts. Instead, interaction with the external environment has remained important for this symbiont as genes for synthesis of transporters, outer membrane proteins, lipopolysaccharides, and lipoproteins have been retained. The ectosymbiont genome carried two complete operons for nitrogen fixation, a urea transporter, and a urease, indicating the availability of nitrogen as a driving force behind the symbiosis. NanoSIMS analysis of C. punctulatus hindgut symbionts exposed in vivo to (15)N2 supports the hypothesis that Barbulanympha ectosymbionts are capable of nitrogen fixation. This genomic and in vivo functional investigation of protist ectosymbionts highlights the diversity of evolutionary forces and trajectories that shape symbiotic interactions. IMPORTANCE: The ecological and evolutionary importance of symbioses is increasingly clear, but the overall diversity of symbiotic interactions remains poorly explored. In this study, we investigated the evolution and nitrogen fixation capabilities of ectosymbionts attached to the protist Barbulanympha from the hindgut of the wood-eating cockroach Cryptocercus punctulatus In addressing genome evolution of protist ectosymbionts, our data suggest that the ecological pressures influencing the evolution of extracellular symbionts clearly differ from intracellular symbionts and organelles. Using NanoSIMS analysis, we also obtained direct imaging evidence of a specific hindgut microbe playing a role in nitrogen fixation. These results demonstrate the power of combining NanoSIMS and genomics tools for investigating the biology of uncultivable microbes. This investigation paves the way for a more precise understanding of microbial interactions in the hindguts of wood-eating insects and further exploration of the diversity and ecological significance of symbiosis between microbes.

The role of host phylogeny varies in shaping microbial diversity in the hindguts of lower termites.

The hindguts of lower termites and Cryptocercus cockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts of Cryptocercus punctulatus and 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of the Bacteroidetes, Elusimicrobia, Tenericutes, Spirochaetes, and Synergistes were structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.

Microbial associates of the vine mealybug Planococcus ficus (Hemiptera: Pseudococcidae) under different rearing conditions.

Sap-feeding insects harbor diverse microbial endosymbionts that play important roles in host ecology and evolution, including contributing to host pest status. The vine mealybug, Planococcus ficus, is a serious pest of grapevines, vectoring a number of pathogenic grape viruses. Previous studies have shown that virus transmission is abolished when mealybugs are raised in the laboratory on potato. To examine the possible role of microbial symbionts in virus transmission, the archaeal, bacterial, and fungal microbiota of field and laboratory P. ficus were characterized using molecular and classical microbiological methods. Lab and field colonies of P. ficus harbored different microbiota. While both were dominated by the bacterial obligate nutritional symbionts Moranella and Tremblaya, field samples also harbored a third bacterium that was allied with cluster L, a lineage of bacterial symbionts previously identified in aphids. Archaea were not found in any of the samples. Fungal communities in field-collected mealybugs were dominated by Metschnikowia and Cladosporium species, while those from laboratory-reared mealybugs were dominated by Alternaria and Cladosporium species. In conclusion, this study has identified a diverse set of microbes, most of which appear to be facultatively associated with P. ficus, depending on environmental conditions. The role of various members of the mealybug microbiome, as well as how the host plant affects microbial community structure, remains to be determined.

Bacterial associates of seed-parasitic wasps (Hymenoptera: Megastigmus).

BACKGROUND: The success of herbivorous insects has been shaped largely by their association with microbes. Seed parasitism is an insect feeding strategy involving intimate contact and manipulation of a plant host. Little is known about the microbial associates of seed-parasitic insects. We characterized the bacterial symbionts of Megastigmus (Hymenoptera: Torymidae), a lineage of seed-parasitic chalcid wasps, with the goal of identifying microbes that might play an important role in aiding development within seeds, including supplementing insect nutrition or manipulating host trees. We screened multiple populations of seven species for common facultative inherited symbionts. We also performed culture independent surveys of larvae, pupae, and adults of M. spermotrophus using 454 pyrosequencing. This major pest of Douglas-fir is the best-studied Megastigmus, and was previously shown to manipulate its tree host into redirecting resources towards unfertilized ovules. Douglas-fir ovules and the parasitoid Eurytoma sp. were also surveyed using pyrosequencing to help elucidate possible transmission mechanisms of the microbial associates of M. spermotrophus. RESULTS: Three wasp species harboured Rickettsia; two of these also harboured Wolbachia. Males and females were infected at similar frequencies, suggesting that these bacteria do not distort sex ratios. The M. spermotrophus microbiome is dominated by five bacterial OTUs, including lineages commonly found in other insect microbiomes and in environmental samples. The bacterial community associated with M. spermotrophus remained constant throughout wasp development and was dominated by a single OTU - a strain of Ralstonia, in the Betaproteobacteria, comprising over 55% of all bacterial OTUs from Megastigmus samples. This strain was also present in unparasitized ovules. CONCLUSIONS: This is the first report of Ralstonia being an abundant and potentially important member of an insect microbiome, although other closely-related Betaproteobacteria, such as Burkholderia, are important insect symbionts. We speculate that Ralstonia might play a role in nutrient recycling, perhaps by redirecting nitrogen. The developing wasp larva feeds on megagametophyte tissue, which contains the seed storage reserves and is especially rich in nitrogen. Future studies using Ralstonia-specific markers will determine its distribution in other Megastigmus species, its mode of transmission, and its role in wasp nutrition.

Transcriptional responses in a Drosophila defensive symbiosis.

Inherited symbionts are ubiquitous in insects and can have important consequences for the fitness of their hosts. Many inherited symbionts defend their hosts against parasites or other natural enemies; however, the means by which most symbionts confer protection is virtually unknown. We examine the mechanisms of defence in a recently discovered case of symbiont-mediated protection, where the bacterial symbiont Spiroplasma defends the fly Drosophila neotestacea from a virulent nematode parasite, Howardula aoronymphium. Using quantitative PCR of Spiroplasma infection intensities and whole transcriptome sequencing, we attempt to distinguish between the following modes of defence: symbiont-parasite competition, host immune priming and the production of toxic factors by Spiroplasma. Our findings do not support a model of exploitative competition between Howardula and Spiroplasma to mediate defence, nor do we find strong support for host immune priming during Spiroplasma infection. Interestingly, we recovered sequence for putative toxins encoded by Spiroplasma, including a novel putative ribosome-inactivating protein, transcripts of which are up-regulated in response to nematode exposure. Protection via the production of toxins may be a widely used and important mechanism in heritable defensive symbioses in insects.

Factors affecting the strength of Cardinium-induced cytoplasmic incompatibility in the parasitic wasp Encarsia pergandiella (Hymenoptera: Aphelinidae).

Bacteria that cause cytoplasmic incompatibility (CI) are among the most common maternally transmitted parasites of insects. In CI, uninfected females produce few or no offspring when they mate with infected males and, as a result, are often at a reproductive disadvantage relative to infected females. Two different bacteria are known to cause CI, Wolbachia and Cardinium. CI Cardinium was discovered more recently and has been little studied. Here, factors that could influence the reduction in reproductive output in a CI cross, or CI "strength," were explored in the parasitic wasp Encarsia pergandiella. Cardinium in this wasp exhibits variable CI strength. Experiments tested the effect of male age, male size, male host species, Cardinium density, and male development time on CI strength. We found a striking effect of male development time, with males that took longer to develop exhibiting stronger CI when mated to uninfected females. Male age had little effect; although in one experiment, the oldest males exhibited stronger CI. Male size, host species, and bacterial density had no effect on the strength of CI. Identifying the factors that control CI are crucial for understanding the dynamics of infection, as well as the success of strategies that aim to use CI microbes to control insect pests and disease vectors.

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.

Novel Rickettsiella bacterium in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae).

Bacteria in the genus Rickettsiella (Coxiellaceae), which are mainly known as arthropod pathogens, are emerging as excellent models to study transitions between mutualism and pathogenicity. The current report characterizes a novel Rickettsiella found in the leafhopper Orosius albicinctus (Hemiptera: Cicadellidae), a major vector of phytoplasma diseases in Europe and Asia. Denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to survey the main symbionts of O. albicinctus, revealing the obligate symbionts Sulcia and Nasuia, and the facultative symbionts Arsenophonus and Wolbachia, in addition to Rickettsiella. The leafhopper Rickettsiella is allied with bacteria found in ticks. Screening O. albicinctus from the field showed that Rickettsiella is highly prevalent, with over 60% of individuals infected. A stable Rickettsiella infection was maintained in a leafhopper laboratory colony for at least 10 generations, and fluorescence microscopy localized bacteria to accessory glands of the female reproductive tract, suggesting that the bacterium is vertically transmitted. Future studies will be needed to examine how Rickettsiella affects host fitess and its ability to vector phytopathogens.