Endosymbiont DNA in endobacteria-free filarial nematodes indicates ancient horizontal genetic transfer

Abstract

Background: Wolbachia are among the most abundant symbiotic microbes on earth; they are present in about 66% of all insect species, some spiders, mites and crustaceans, and most filarial nematode species. Infected filarial nematodes, including many pathogens of medical and veterinary importance, depend on Wolbachia for proper development and survival. The mechanisms behind this interdependence are not understood. Interestingly, a minority of filarial species examined to date are naturally Wolbachia-free.

Methodology/principal findings: We used 454 pyrosequencing to survey the genomes of two distantly related Wolbachia-free filarial species, Acanthocheilonema viteae and Onchocerca flexuosa. This screen identified 49 Wolbachia-like DNA sequences in A. viteae and 114 in O. flexuosa. qRT-PCR reactions detected expression of 30 Wolbachia-like sequences in A. viteae and 56 in O. flexuosa. Approximately half of these appear to be transcribed from pseudogenes. In situ hybridization showed that two of these pseudogene transcripts were specifically expressed in developing embryos and testes of both species.

Conclusions/significance: These results strongly suggest that the last common ancestor of extant filarial nematodes was infected with Wolbachia and that this former endosymbiont contributed to their genome evolution. Horizontally transferred Wolbachia DNA may explain the ability of some filarial species to live and reproduce without the endosymbiont while other species cannot.

Figure 1. Mapping transferred fragments to a sequenced Wolbachia genome.

Black circle represents the 1.1 Mbp genome of the Wolbachia endosymbiont of B. malayi. Tick marks in the colored outer rings indicate where a transferred DNA fragment found in the indicated species would align to the Wolbachia genome. Fragments found in the B. malayi genome were previously described by Dunning Hotopp et al. .

Figure 2. Schematic of genomic DNA fragments containing Wolbachia homologs.

Figure outlines the structure of four genomic sequence fragments identified in this study. The exact annotation and coordinates of each of the homologs depicted can be found in Tables S1 and S2. Blue blocks represent regions of homology to nematode sequences while red blocks represent regions homologous to Wolbachia sequences. Horizontal arrows represent inverted repeats in the DNA sequence. Inverted repeat segments in Of70 share 88% identity with one another while the repeated segments in Of71 share 82.8% identity.

Figure 3. In situ hybridization of adult filarial worms.

A–H are stained with probes made from an O. flexuosa sequence with homology to Wolbachia 2- methylthioadenine synthase (2-MAS, wOf53). A, C, E and G are stained with the sense probe (negative control), while B, D, F and H show matching consecutive sections stained with the antisense probes. 2-MAS probe labels lateral cords, intrauterine stretched microfilaria and uterine and intestinal epithelium of female B. malayi (B), the oocytes and uterus of female A. viteae (D), the spermatogonia in the periphery of the testes of male A. viteae (F), and lateral chords and different stages of sperm development in the testes of male O. flexuosa (H). I–L are stained with probes made from an O. flexuosa sequence with homology to Wolbachia DNA polymerase I (pol I, wOf88). I and K are stained with sense probes (negative control) while H and L are the matching consecutive sections stained with antisense probes. pol I probe labels ovaries and granular structures resembling Wolbachia (arrows) in the lateral chords of female B. malayi (J) and oocytes in female A. viteae (L). Abbreviations: m, musculature; i, intestine; lc, lateral chord; cu, cuticle; hy, hypodermis; t, testes; ut, uterus; ov, ovary. Scale bar 40 µm.