Infection with the endosymbiotic bacteria Wolbachia is widespread in filarial nematodes. Previous studies have suggested concordance between the phylogeny of Wolbachia with that of their nematode hosts. However, there is only one published molecular phylogenetic study of filarial species, based on the 5S rRNA gene spacer. The phylogeny proposed by this study is partially incongruent with previous classifications of filarial nematodes, based on morphological characters. Furthermore, both traditional classifications and molecular phylogenies are, in part, inconsistent with the phylogeny of Wolbachia. Here we report mitochondrial cytochrome oxidase I (COI) gene sequences for 11 species of filaria and for another spirurid nematode which was included as an outgroup. In addition, 16S rRNA, wsp and ftsZ gene sequences were generated for the Wolbachia of several filarial species, in order to complete the available data sets and further resolve the phylogeny of Wolbachia in nematodes. We used these data to evaluate whether nematode and Wolbachia phylogenies are concordant. Some of the possible phylogenetic reconstructions based on COI gene were congruent with the phylogeny of Wolbachia and supported the grouping of the rodent filaria Litomosoides sigmodontis with the lymphatic filariae (i.e. Brugia spp. and Wuchereria spp.) and the sister group relationship of Dirofilaria spp. and Onchocerca spp. However, the placement of the Wolbachia-free filaria Acanthocheilonema viteae is ambiguous and dependent on the phylogenetic methods used.

Obligate intracellular bacteria of the genus Wolbachia (Class Alphaproteobacteria, Order Rickettsiales) are currently divided into four taxonomic supergroups on the basis of clustering patterns in ftsZ-based phylogenetic trees (Werren, Zhang, and Guo 1995<$REFLINK> ; Bandi et al. 1998<$REFLINK> ). Supergroups A and B are found only in arthropods, whereas C and D are found only in filarial nematodes. The term supergroup has recently been employed to avoid confusion with designation of more closely related groups based on wsp sequences (Zhou, Rousset, and O'Neill 1998<$REFLINK> ). Wolbachia have generated substantial interest in recent years (Zimmer 2001), primarily because of the effects they have on their arthropod hosts, which include induction of cytoplasmic incompatibility (CI), parthenogenesis, feminization, and male-killing (reviewed in Stouthammer, Breeuwer, and Hurst 1999<$REFLINK> ). Estimation of the phylogenetic relationships within each supergroup has provided useful information about the evolution and biology of these bacteria. The phylogenies of both A and B members have been found to be incongruent with that of their hosts, strongly suggesting horizontal transfer (Werren, Zhang, and Guo 1995<$REFLINK> ). Recently, direct evidence for this phenomenon was found for parasitic wasps sharing a common food source (Huigens et al. 2000). Unlike the case of arthropods, the phylogeny of each nematode Wolbachia supergroup (C and D) appears to match that of their hosts (Casiraghi et al. 2001a<$REFLINK> ), although further gene sequencing studies are required to confirm this. Such phylogenetic congruence would suggest a strictly dependent relationship, and this idea is supported by evidence that removal of Wolbachia using antibiotics has negative effects on the filariae they reside in (Bandi et al. 1999<$REFLINK> ; Langworthy et al. 2000<$REFLINK> ).

More than 150 million humans in tropical countries are infected by filarial nematodes which harbor intracellular bacterial endosymbionts of the genus Wolbachia (Rickettsiales). These bacteria have been implicated in adverse effects of drug treatment in filariasis. The present study provides evidence that purified major Wolbachia surface protein (rWSP) acts as an inducer of the innate immune system through TLR2 and TLR4: 1) recombinant, stringently purified rWSP elicited the release of TNF-α, IL-12, and IL-8 from cultured blood cells of both Onchocerca volvulus-infected and uninfected people; 2) the inflammatory response to rWSP challenge was TLR2- and TLR4-dependent as demonstrated with TLR-transfected fibroblastoid cells, as well as macrophages and dendritic cells from functional TLR-deficient mice; 3) blood cells of onchocerciasis patients exposed to rWSP also generated down-regulating mediators IL-10 and PGE2 after 6 days of culture; 4) furthermore, rWSP-reactive IgG1 Abs were present in sera of O. volvulus-infected people but not in those of uninfected Europeans. The lack of rWSP-reactive IgE and IgG4 in serum indicated a bias toward a Th1-type adaptive immune response. Abs against rWSP stained endobacteria in living and degenerating adult O. volvulus filariae, tissue microfilariae and host tissue macrophages that apparently had engulfed microfilariae. Thus, filarial helminths, through products of their endobacteria such as WSP, acquire characteristics of a typical microbial pathogen inducing immune responses via TLR2 and TLR4.

“ Candidatus Midichloria mitochondrii” is an intramitochondrial bacterium of the order Rickettsiales associated with the sheep tick Ixodes ricinus . Bacteria phylogenetically related to “ Ca . Midichloria mitochondrii” (midichloria and like organisms [MALOs]) have been shown to be associated with a wide range of hosts, from amoebae to a variety of animals, including humans. Despite numerous studies focused on specific members of the MALO group, no comprehensive phylogenetic and statistical analyses have so far been performed on the group as a whole. Here, we present a multidisciplinary investigation based on 16S rRNA gene sequences using both phylogenetic and statistical methods, thereby analyzing MALOs in the overall framework of the Rickettsiales . This study revealed that (i) MALOs form a monophyletic group; (ii) the MALO group is structured into distinct subgroups, verifying current genera as significant evolutionary units and identifying several subclades that could represent novel genera; (iii) the MALO group ranks at the level of described Rickettsiales families, leading to the proposal of the novel family “ Candidatus Midichloriaceae.” In addition, based on the phylogenetic trees generated, we present an evolutionary scenario to interpret the distribution and life history transitions of these microorganisms associated with highly divergent eukaryotic hosts: we suggest that aquatic/environmental protista have acted as evolutionary reservoirs for members of this novel family, from which one or more lineages with the capacity of infecting metazoa have evolved.

The initiation of the intracellular symbiosis that would give rise to mitochondria and eukaryotes was a major event in the history of life on earth. Hypotheses to explain eukaryogenesis fall into two broad and competing categories: those proposing that the host was a phagocytotic proto-eukaryote that preyed upon the free-living mitochondrial ancestor (hereafter FMA), and those proposing that the host was an archaebacterium that engaged in syntrophy with the FMA. Of key importance to these hypotheses are whether the FMA was motile or nonmotile, and the atmospheric conditions under which the FMA thrived. Reconstructions of the FMA based on genome content of Rickettsiales representatives—generally considered to be the closest living relatives of mitochondria—indicate that it was nonmotile and aerobic. We have sequenced the genome of Candidatus Midichloria mitochondrii, a novel and phylogenetically divergent member of the Rickettsiales. We found that it possesses unique gene sets found in no other Rickettsiales, including 26 genes associated with flagellar assembly, and a cbb₃-type cytochrome oxidase. Phylogenomic analyses show that these genes were inherited in a vertical fashion from an ancestral α-proteobacterium, and indicate that the FMA possessed a flagellum, and could undergo oxidative phosphorylation under both aerobic and microoxic conditions. These results indicate that the FMA played a more active and potentially parasitic role in eukaryogenesis than currently appreciated and provide an explanation for how the symbiosis could have evolved under low levels of oxygen.