FIG 4.

Localization of Coxiella sp. (green) and Rickettsia sp. (yellow) in H. longicornis (A) and R. haemaphysaloides (B to D) using species specific probes. Nuclei were stained with DAPI (blue). Merged images of DAPI and Coxiella sp. staining in female H. longicornis (A1 to A4). Merged images of DAPI, Coxiella sp., and Rickettsia sp. staining in female (B1 to B4) and male (C1 to C4) R. haemaphysaloides. Spliced view of whole-mount in situ hybridization in female Malpighian tubules of R. haemaphysaloides (D1 to D3). (A1, B1, C1), midgut (MG); (A2, B2, C2), Malpighian tubules (MT); (A3, B3, C3), salivary glands (SG); (A4), ovaries of H. longicornis (reproductive tissues [RT]); (B4), ovaries of R. haemaphysaloides; (C4), testes of R. haemaphysaloides; (B5) close-up view of boxed region, showing colocalization of Rickettsia sp. and Coxiella sp. in ovaries; (C5) close-up view of panel C2, colocalization of Rickettsia sp. and Coxiella sp. in Malpighian tubules. (D2, D3) Close-up view of boxed regions in D1. Green arrows denote Coxiella sp. and yellow arrows denote Rickettsia sp. Tissues from at least 5 individual ticks were pooled and used for FISH analysis. Each image shows a single focal plane. Images are representative of three independent experiments. Bars, 100 μm.