A novel alpha-Proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes ricinus

Abstract

An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacterium: ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.

FIG. 1.

Transmission electron micrographs showing developing oocytes from the ovary of a partially engorged adult female I. ricinus. Bacteria with the same characteristics were obtained during examination of four other tick ovaries. (A) Bacteria are present either in the cytoplasm within membrane-limited vacuoles (not clearly visible at this magnification) or inside the mitochondria. The arrowheads indicate the bacteria, and the arrows indicate mitochondria. (B) Higher magnification showing one bacterium (to the left) adjacent to a mitochondrion (M) and two other bacteria (to the right) between the outer (arrows) and inner (arrowheads) membranes of a mitochondrion. The bacterium on the left is enclosed within a cytoplasmic membrane-limited vacuole (V) and has an outer cell wall (CW) and an inner cell membrane (CM) typical of gram-negative bacteria. (C) Three bacteria enclosed within the matrix of a mitochondrion.

FIG. 2.

Phylogenetic comparison of the 16S rRNA of IricES1, an intracellular bacterium from the tick I. ricinus, with the 16S rRNAs of selected members of the alpha-proteobacterial assemblage. The tree was constructed by using TreePuzzle 5.0 with the TN+G model of substitution. A parsimony bootstrap tree estimated with PAUP* was found to have an identical topology. The GenBank accession number for each sequence is indicated. The numbers above and below each node are quartet puzzling support values and bootstrap values from parsimony analyses, respectively. The scale bar indicates the number of inferred substitutions per site. Other analyses in which various beta-, gamma-, and delta-proteobacteria were included as outgroups resulted in very similar relationships for the alpha-proteobacteria shown.

FIG. 3.

(A) Light microscopy image of a transverse section of a partially engorged I. ricinus adult female. O, ovary; Md, midgut diverticulum; Mm, muscles; Rs, rectal sac. (B) ISH with the 16S rRNA of IricES1 in ovarian tissue of a partially engorged I. ricinus adult female by using a probe mixture containing two DIG-labeled specific oligonucleotides, S-*-IrES1-0066-a-A-18 and S-*-IrES1-1410-a-A-19. The probes were detected with alkaline phosphatase anti-DIG antibody (Boehringer Mannheim) and were stained with the BCIP-nitroblue tetrazolium substrate (Vector Laboratories). (C) Same section shown in panel B, but at a higher magnification. In all panels, the arrows indicate oocytes. (D) ISH, as described above for panel B, for a section from a different tick sample. (E) Negative control hybridization with a general gamma-proteobacterial probe (DIG-Gam1019). (F) No-probe control. (G) RNase treatment control. The slides were treated with 20 μg of RNase per ml prior to the probe hybridization step.