Beninati, Tiziana

An intracellular bacterium with the unique ability to enter mitochondria exists in the European vector of Lyme disease, the hard tick Ixodes ricinus. Previous phylogenetic analyses based on 16S rRNA gene sequences suggested that the bacterium formed a divergent lineage within the Rickettsiales (Alphaproteobacteria). Here, we present additional phylogenetic evidence, based on the gyrB gene sequence, that confirms the phylogenetic position of the bacterium. Based on these data, as well as electron microscopy (EM), in situ hybridization and other observations, we propose the name ‘Candidatus Midichloria mitochondrii’ for this bacterium. The symbiont appears to be ubiquitous in females of I. ricinus across the tick's distribution, while lower prevalence is observed in males (44 %). Based on EM and in situ hybridization studies, the presence of ‘Candidatus M. mitochondrii’ in females appears to be restricted to ovarian cells. The bacterium was found to be localized both in the cytoplasm and in the intermembrane space of the mitochondria of ovarian cells. ‘Candidatus M. mitochondrii’ is the first bacterium to be identified that resides within animal mitochondria.

ABSTRACT Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “ Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “ Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “ Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “ Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “ Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.