Figure 7 Hamiltonella and Arsenophonus FISH of T. vaporariorum eggs, nymphs and adults. Secondary symbiont-specific probes for Hamiltonella (green) and Arsenophonus (yellow) were used. A, D and G: FISH of Hamiltonella alone in eggs (A), nymphs (D) and adults (G). B, E and H: FISH of Arsenophonus alone in eggs (B), nymphs (E) and adults (H). C, F and I: double FISH of Hamiltonella and Arsenophonus in eggs (C), nymphs (F) and adults (I). Cardinium showed a dual localization pattern, outside and inside the bacteriocyte, with Portiera in the Saracatinib same B. tabaci individuals (Figure 8). Cardinium, like all symbionts that are confined to the bacteriocyte, is transovarially transferred from the mother to the offspring though the egg.

Thus in the egg’s early

developmental stages, it is confined to the bacteriocyte; however, in older eggs (5-7 days), it is also observed outside the bacteriocyte (not shown), and in later nymphal and adult stages, it occupies most of the body tissues, including the bacteriocyte (Figure 8). Cardinium was not detected in T. vaporariorum. Cardinium has been shown by TEM to localize to the bacteriocytes of the A and Jatropha biotypes of B. tabaci [24]. Our PCR screening assay revealed co-localization of Cardinium in B. tabaci populations (in 15 out of a total 236 individuals tested), mostly with Hamiltonella (10 of the 15 Cardinium-containing individuals also harbored Hamiltonella–66% co-localization). In some cases, multiple infections of Cardinium with two (Wolbachia and Rickettsia) or three (ABT-263 in vivo Rickettsia, Wolbachia and Hamiltonella) AZD2014 symbionts were observed. The localization pattern of Cardinium as seen by FISH was different Benzatropine from that of the other symbionts that co-localized with it. Localization of Hamiltonella and Cardinium has also been demonstrated in the bacteriocytes of the A biotype together with Portiera, as shown

here. TEM has revealed the presence of Cardinium in the spermatid cytoplasm, residual bodies, and cyst cell cytoplasm of B. tabaci males [25]. Studies on other hosts have reported the presence of Cardinium in a diverse array of tissues, including the reproductive tract [26], fat bodies, and salivary glands [27, 28], as well as inside bacteriocytes surrounded by oogonia in the apical region of the ovary [29]. Figure 8 Portiera and Cardinium FISH of B. tabaci eggs, nymphs and adults. Portiera-specific probe (red) and Cardinium-specific probe (blue) were used. A, C and G: double FISH of Portiera and Cardinium in eggs (A), nymphs (D) and adults (G) under dark field. B, E and H: double FISH of Portiera and Cardinium in eggs (B), nymphs (E) and adults (H) under bright field. C, F and I are shown only with Cardinium probe to emphasize its location inside the bacteriosome. Wolbachia has been previously shown to localize at the circumference of and inside the bacteriocytes. In adults, Wolbachia can also be seen in the abdomen outside the bacteriocyte [22].