FISH of <i>Bemisia tabaci</i> nymphs parasitized by <i>Eretmocerus mundus</i>.

<p>The procedure was performed using <i>Portiera</i>-specific probe (red) and <i>Rickettsia-</i>specific probe (blue). (<b>A</b>) Scattered (S) localization pattern. White arrows indicate bacteriocytes. Blue-speckled area is the whitefly hemocoel, and the dark, clear area corresponds to the outline of the roughly spherical <i>Eretmocerus</i> larva. Bright blue area (black arrow) shows the wasp larval gut. (<b>B</b>) Confined (C) localization pattern. (<b>B1</b>) Red area (white arrows) shows <i>Portiera</i> in the bacteriocytes (<b>B2</b>) Blue area (white arrows) shows <i>Rickettsia</i> in the bacteriocytes. The pictures of <i>Rickettsia</i> and <i>Portiera</i> are presented separately because of the faint signal seen by the former. Other blue and red areas in the pictures are due to autofluorescence of the whitefly nymph and the shell of the wasp's hatched egg (white dashed arrow).</p

PCR primer sets used in this study.

a<p>Internal primers.</p

<i>Bemisia tabaci</i> lines studied.

a<p>Data from Chiel et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021096#pone.0021096-Chiel1" target="_blank">[18]</a>, Gottlieb et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021096#pone.0021096-Gottlieb1" target="_blank">[20]</a>.</p

Horizontal transmission (from <i>R⁺</i> whiteflies to wasps) and vertical transmission (from <i>R⁺</i>wasps to progeny) of <i>Rickettsia</i> to males and females of <i>Er. emiratus</i> (top), <i>Er. eremicus</i> (middle) and <i>En. pergandiella</i> (bottom).

<p>‘P’ are <i>R⁻</i> wasps that were exposed to <i>R⁺</i> whiteflies for 24 hrs (horizontal transmission via host feeding and/or honeydew), ‘F₁’ are their resulting progeny that developed in <i>R⁺</i> hosts (also horizontal transmission), and ‘F₂’ are progeny of F₁ that were exposed to <i>R⁻</i> hosts (vertical transmission). The numbers above the columns are the sample size, <i>n</i>, from which the proportion of infected wasps was calculated. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004767#pone-0004767-g001" target="_blank">Fig. 1</a> for this experiment's set-up.</p

<i>Rickettsia</i> (white arrows) in <i>Er. eremicus</i> germarium area, between nuclei of stem/pre-follicle/nurse cells as well as outside the ovary, next to the Tunica propria (TP).

<p>Note mature oocyte on the bottom left corner area. N-nucleus; EnC- endochorion; ExC- Exochorion; VE- Vitellin envelope.</p

<i>Rickettsia</i> (bordered) outside <i>Er. eremicus</i> ovary envelope, the tunica propria (TP).

<p>FC- follicular epithelial cell; EnC- endochorion; ExC- Exochorion; VE- Vitellin envelope; Tr- Trachea.</p

FISH of <i>Er. emiratus</i> stained with <i>Rickettisa</i> specific probe (blue).

<p>Left panel-<i>Rickettsia</i> probe fluorescent channel; right panel- overlay of fluorescent and brightfield channels. Arrows pointing to parasitoid gut. A- parasitoid larva (dark, ovoid sphere in the center of the host). Note <i>Rickettsia</i> in the parasitoid gut, as well in the whitefly's body remnants, surrounding the parasitoid. B- parasitoid pre-pupa. C- parasitoid pupae (note the autofluorescence of the anus and mouthpart); 1C, right image- brightfield channel only. D- parasitoid adult abdomen.</p

A diagram illustrating the design of experiment 7, transmission of symbionts from <i>B. tabaci</i> to parasitoids, and 8, vertical transmission of symbionts in parasitoids.

<p>Infection status is indicated either by red “+” sign or blue “−” sign. R = <i>Rickettsia</i>, H = <i>Hamiltonella</i>. TRT = treatment. Whitefly hosts are illustrated as small yellow ovals on the (green) leaf disks. To test transmission of symbionts from <i>B. tabaci</i> to parasitoids, one female parasitoid was introduced to each leaf disk for 24 h, after which they were tested by PCR. From the emerging F₁, one or two females from each replicate were used to continue to the vertical transmission experiment, while the rest of the cohort was tested by PCR (two-five from each cohort). The emerging F₂ were all collected and two-five from each cohort were tested by PCR.</p

<i>Rickettsia</i> (white arrows) in <i>Er. eremicus</i> follicular epithelial cell (FC).

<p>The gap between the follicular epithelial cell and the oocyte (the transition zone - TZ) is due to oocyte resorption. N-nucleus; EnC- endochorion; ExC- Exochorion; VE- Vitellin envelope.</p

Additional file 3:

Annotations for the Cardinium genome from four platforms (IMG/M, Kbase, Rast, MG-Rast) were compared with the manual annotation conducted by Santos-Garcia et al. 2014. The JGI platform had both the absolute highest number of Enzyme Commission (EC) predictions as well as the highest overlap with the manual annotation. Hence, it was selected as the standard annotation tool for all symbionts. (DOCX 113 kb