Almost there: transmission routes of bacterial symbionts between trophic levels

Abstract

Many intracellular microbial symbionts of arthropods are strictly vertically transmitted and manipulate their host's reproduction in ways that enhance their own transmission. Rare horizontal transmission events are nonetheless necessary for symbiont spread to novel host lineages. Horizontal transmission has been mostly inferred from phylogenetic studies but the mechanisms of spread are still largely a mystery. Here, we investigated transmission of two distantly related bacterial symbionts--Rickettsia and Hamiltonella--from their host, the sweet potato whitefly, Bemisia tabaci, to three species of whitefly parasitoids: Eretmocerus emiratus, Eretmocerus eremicus and Encarsia pergandiella. We also examined the potential for vertical transmission of these whitefly symbionts between parasitoid generations. Using florescence in situ hybridization (FISH) and transmission electron microscopy we found that Rickettsia invades Eretmocerus larvae during development in a Rickettsia-infected host, persists in adults and in females, reaches the ovaries. However, Rickettsia does not appear to penetrate the oocytes, but instead is localized in the follicular epithelial cells only. Consequently, Rickettsia is not vertically transmitted in Eretmocerus wasps, a result supported by diagnostic polymerase chain reaction (PCR). In contrast, Rickettsia proved to be merely transient in the digestive tract of Encarsia and was excreted with the meconia before wasp pupation. Adults of all three parasitoid species frequently acquired Rickettsia via contact with infected whiteflies, most likely by feeding on the host hemolymph (host feeding), but the rate of infection declined sharply within a few days of wasps being removed from infected whiteflies. In contrast with Rickettsia, Hamiltonella did not establish in any of the parasitoids tested, and none of the parasitoids acquired Hamiltonella by host feeding. This study demonstrates potential routes and barriers to horizontal transmission of symbionts across trophic levels. The possible mechanisms that lead to the differences in transmission of species of symbionts among species of hosts are discussed.

Figure 1. A diagram illustrating the design of experiment 7, transmission of symbionts from B. tabaci to parasitoids, and 8, vertical transmission of symbionts in parasitoids.

Infection status is indicated either by red “+” sign or blue “−” sign. R = Rickettsia, H = Hamiltonella. TRT = treatment. Whitefly hosts are illustrated as small yellow ovals on the (green) leaf disks. To test transmission of symbionts from B. tabaci 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.

Figure 2. FISH of Er. emiratus stained with Rickettisa specific probe (blue).

Left panel-Rickettsia 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 Rickettsia 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.

Figure 3. FISH of En. pergandiella stained with Rickettisa specific probe (blue).

Left panel-Rickettsia probe fluorescent channel; right panel- overlay of fluorescent and brightfield channels. A- parasitoid larvae, arrow points to specific signal inside the larva body. B- parasitoid pupa, arrows pointing to the meconia deposited outside the parasitoid's body.

Figure 4. Rickettsia (white arrows) in Er. eremicus follicular epithelial cell (FC).

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.

Figure 5. Rickettsia (bordered) outside Er. eremicus ovary envelope, the tunica propria (TP).

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

Figure 6. Rickettsia (white arrows) in Er. eremicus germarium area, between nuclei of stem/pre-follicle/nurse cells as well as outside the ovary, next to the Tunica propria (TP).

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

Figure 7. Horizontal transmission (from R⁺ whiteflies to wasps) and vertical transmission (from R⁺wasps to progeny) of Rickettsia to males and females of Er. emiratus (top), Er. eremicus (middle) and En. pergandiella (bottom).

‘P’ are R⁻ wasps that were exposed to R⁺ whiteflies for 24 hrs (horizontal transmission via host feeding and/or honeydew), ‘F₁’ are their resulting progeny that developed in R⁺ hosts (also horizontal transmission), and ‘F₂’ are progeny of F₁ that were exposed to R⁻ hosts (vertical transmission). The numbers above the columns are the sample size, n, from which the proportion of infected wasps was calculated. See also Fig. 1 for this experiment's set-up.