The obligate intracellular bacterium Wolbachia pipientis infects around 20% of all insect species. It is maternally inherited and induces reproductive alterations of insect populations by male killing, feminization, parthenogenesis, or cytoplasmic incompatibility. Here, we present the 1,445,873-bp genome of W. pipientis strain w Ri that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans . A comparison with the previously sequenced genome of W. pipientis strain w Mel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated sequences that are stable in Drosophila lines transinfected with w Ri. Additionally, 450 genes with orthologs in w Ri and w Mel were sequenced from the W. pipientis strain w Uni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor . The comparison of these A-group Wolbachia strains uncovered the most highly recombining intracellular bacterial genomes known to date. This was manifested in a 500-fold variation in sequence divergences at synonymous sites, with different genes and gene segments supporting different strain relationships. The substitution-frequency profile resembled that of Neisseria meningitidis , which is characterized by rampant intraspecies recombination, rather than that of Rickettsia , where genes mostly diverge by nucleotide substitutions. The data further revealed diversification of ankyrin repeat genes by short tandem duplications and provided examples of horizontal gene transfer across A- and B-group strains that infect D. simulans . These results suggest that the transmission dynamics of Wolbachia and the opportunity for coinfections have created a freely recombining intracellular bacterial community with mosaic genomes.

The ancient fungus-growing termite (Mactrotermitinae) symbiosis involves the obligate association between a lineage of higher termites and basidiomyceteTermitomycescultivar fungi. Our investigation of the fungus-growing termiteMacrotermes natalensisshows thatBacillusstrains fromM. natalensiscolonies produce a single major antibiotic, bacillaene A (1), which selectively inhibits known and putatively antagonistic fungi ofTermitomyces. Comparative analyses of the genomes of symbioticBacillusstrains revealed that they are phylogenetically closely related toBacillus subtilis, their genomes have high homology with more than 90% of ORFs being 100% identical and the sequence identities across the biosynthetic gene cluster for bacillaene are higher between termite-associated strains than to the cluster previously reported inB. subtilis. Our findings suggest that this lineage of antibiotic-producingBacillusmay be a defensive symbiont involved in the protection of the fungus-growing termite cultivar.

It has long been proposed that the nominal species Anastrepha fraterculus is a species complex and earlier studies showed high levels of pre-zygotic

Ecological and evolutionary theories predict that parasitism and mutualism are not fixed endpoints of the symbiotic spectrum. Rather, parasitism and mutualism may be host or environment dependent, induced by the same genetic machinery, and shifted due to selection. These models presume the existence of genetic or environmental variation that can spur incipient changes in symbiotic lifestyle. However, for obligate intracellular bacteria whose genomes are highly reduced, studies specify that discrete symbiotic associations can be evolutionarily stable for hundreds of millions of years. Wolbachia is an inherited obligate, intracellular infection of invertebrates containing taxa that act broadly as both parasites in arthropods and mutualists in certain roundworms. Here, we analyze the ancestry of mutualism and parasitism in Wolbachia and the evolutionary trajectory of this variation in symbiotic lifestyle with a comprehensive, phylogenomic analysis. Contrary to previous claims, we show unequivocally that the transition in lifestyle cannot be reconstructed with current methods due to long-branch attraction (LBA) artifacts of the distant Anaplasma and Ehrlichia outgroups. Despite the use of 1) site-heterogenous phylogenomic methods that can overcome systematic error, 2) a taxonomically rich set of taxa, and 3) statistical assessments of the genes, tree topologies, and models of evolution, we conclude that the LBA artifact is serious enough to afflict past and recent claims including the root lies in the middle of the Wolbachia mutualists and parasites. We show that different inference methods yield different results and high bootstrap support did not equal phylogenetic accuracy. Recombination was rare among this taxonomically diverse data set, indicating that elevated levels of recombination in Wolbachia are restricted to specific coinfecting groups. In conclusion, we attribute the inability to root the tree to rate heterogeneity between the ingroup and outgroup. Site-heterogenous models of evolution did improve the placement of aberrant taxa in the ingroup phylogeny. Finally, in the unrooted topology, the distribution of parasitism and mutualism across the tree suggests that at least two interphylum transfers shaped the origins of nematode mutualism and arthropod parasitism. We suggest that the ancestry of mutualism and parasitism is not resolvable without more suitable outgroups or complete genome sequences from all Wolbachia supergroups.

Following cultivation-dependent and -independent techniques, we investigated the microbiota associated withBactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among whichAcetobacter tropicaliswas predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such asAnopheles stephensi(G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) orDrosophila melanogaster(C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established betweenA. tropicalisandB. oleae. Using anA. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that differentA. tropicalisstrains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.

The social Hymenoptera have distinct larval and adult stages separated by metamorphosis, which implies striking remodeling of external and internal body structures during the pupal stage. This imposes challenges to gut symbionts as existing cultures are lost and may or may not need to be replaced. To elucidate the extent to which metamorphosis interrupts associations between bacteria and hosts, we analysed changes in gut microbiota during development and traced the transmission routes of dominant symbionts from the egg to adult stage in the leaf-cutting ants Acromyrmex echinatior and Atta cephalotes, which are both important functional herbivores in the New World tropics. Bacterial density remained similar across the developmental stages of Acromyrmex, but Atta brood had very low bacterial prevalences suggesting that bacterial gut symbionts are not actively maintained. We found that Wolbachia was the absolute dominant bacterial species across developmental stages in Acromyrmex and we confirmed that Atta lacks Wolbachia also in the immature stages, and had mostly Mollicutes bacteria in the adult worker guts. Wolbachia in Acromyrmex appeared to be transovarially transmitted similar to transmission in solitary insects. In contrast, Mollicutes were socially transmitted from old workers to newly emerged callows. We found that larval and pupal guts of both ant species contained Pseudomonas and Enterobacter bacteria that are also found in fungus gardens, but hardly or not in adult workers, suggesting they are beneficial only for larval growth and development. Our results reveal that transmission pathways for bacterial symbionts may be very different both between developmental stages and between sister genera and that identifying the mechanisms of bacterial acquisition and loss will be important to clarify their putative mutualistic functions.