Phylogenetic tree based on aligned complete sequences of the <i>rrs</i> (16SrRNA) gene and constructed by the UPGMA method.

<p>The numbers in the nodes represent bootstrap values. The tree shows the position of <i>Diplorickettsia massiliensis</i> (isolate 20B) among recognized bacterial species and <i>rrs</i> genes amplified from uncultured bacteria and environmental samples (Genbank). The host (if any) or the source of amplification is indicated in the brackets; GenBank accession number is indicated at the end of the line for each sequence.</p

Strain 20B bacteria.

<p>Transmission electron microscopy; staining with uranyl acetate. The internal bacterial structure: electron-dense crystal-like (CL) structures located in the center of almost all bacteria. Multilayer sheath-like (SL) structure are alternating electron dense (6 nanometers), electron light (15 nanometers) layers (upper left).</p

Strain 20B bacteria grown in XTC-2 cells Transmission electron microscopy; staining with uranyl acetate.

<p>The bacteria in the process of division.</p

Strain 20B grown in XTC-2 cells, Gimenez staining, x1500.

<p>Strain 20B grown in XTC-2 cells, Gimenez staining, x1500.</p

Strain 20B bacteria grown in XTC-2 cells.

<p>Transmission electron microscopy; staining with uranyl acetate. The number of bacteria per vacuole (B/v) is indicated by arrows.</p

Details of <i>Eratyrus mucronatus</i> collect from 1991 to 2013 in French Guiana.

<p>¹,²,³,⁴,⁵,⁶,⁷: different areas of French Guiana. SEAG: Société Entomologique Antilles-Guyane (Entomological Society for French West-Indies and Guiana). JMB: Jean-Michel Bérenger. BH: Bernard Hermier. CNES Kourou: Centre National d’Etudes Spatiales (National Center for Spatial Studies).</p

Sequences of qPCR primers used to investigate the presence of pathogens’ DNA in the <i>E</i>. <i>mucronatus</i> samples. F: forward primer, R: reverse primer, P: qPCR probe.

<p>Sequences of qPCR primers used to investigate the presence of pathogens’ DNA in the <i>E</i>. <i>mucronatus</i> samples. F: forward primer, R: reverse primer, P: qPCR probe.</p

The questionable role of parasitic wasps in the transmission of rickettsiae, illustrated on the tick’s life cycle.

<p>Adult female <i>I</i>. <i>hookeri</i> oviposits in larvae and nymphs of ixodid ticks, but the wasp eggs start to develop only in fully engorged nymphs. The immature parasitoid wasps consume the nymph’s tissue and its ingested blood meal, causing nymph death. During the tick’s life cycle (eggs, larvae, nymphs, adults), rickettsiae can pass from stage to stage. In our experiments we successfully amplified rickettsial DNA not just in unfed nymphs but also in emerged adult wasps. More experiments will be needed to demonstrate if <i>I</i>. <i>hookeri</i> may act as a biological vector of <i>A</i>. <i>nasoniae</i> and <i>Rickettsia</i> sp.</p

Published studies on detection of <i>Arsenophonus nasoniae</i> in ticks.

<p>Published studies on detection of <i>Arsenophonus nasoniae</i> in ticks.</p

Workflow of laboratory experiments.

<p>A total of 360 <i>I</i>. <i>ricinus</i> nymphs were fed on 12 Balb/c mice. Of those, 50 engorged nymphs were parasitized by <i>I</i>. <i>hookeri</i> wasps. The obtained 96 parasitoids were subsequently screened for the presence of <i>A</i>. <i>nasoniae</i> and rickettsiae by PCR. The DNA of <i>A</i>. <i>nasoniae</i> was found in 27 wasps (28.1%), <i>Rickettsia</i> sp. in 22 wasps (22.9%). Eight wasps were positive for both bacteria—<i>A</i>. <i>nasoniae</i> and <i>Rickettsia</i> sp.</p