Plant-mediated interspecific horizontal transmission of an intracellular symbiont in insects

Abstract

Intracellular reproductive manipulators, such as Candidatus Cardinium and Wolbachia are vertically transmitted to progeny but rarely show co-speciation with the host. In sap-feeding insects, plant tissues have been proposed as alternative horizontal routes of interspecific transmission, but experimental evidence is limited. Here we report results from experiments that show that Cardinium is horizontally transmitted between different phloem sap-feeding insect species through plants. Quantitative PCR and in situ hybridization experiments indicated that the leafhopper Scaphoideus titanus releases Cardinium from its salivary glands during feeding on both artificial media and grapevine leaves. Successional time-course feeding experiments with S. titanus initially fed sugar solutions or small areas of grapevine leaves followed by feeding by the phytoplasma vector Macrosteles quadripunctulatus or the grapevine feeder Empoasca vitis revealed that the symbionts were transmitted to both species. Explaining interspecific horizontal transmission through plants improves our understanding of how symbionts spread, their lifestyle and the symbiont-host intermixed evolutionary pattern.

Figure 1. Morphology of the S. titanus head (including salivary glands) and occurrence of Cardinium in the salivary glands.

(A) Section of the head of S. titanus, stained with hematoxylin/eosin. b = brain; e = eye; sg = salivary glands; opl = optical lobe; s = stylet. Bar = 0.25 mm. (B) Stylet, particularly showing cibarial pump muscles. (C) Salivary glands, particularly showing the lumen (arrows). b = brain; m = medulla; sg = salivary gland. Bar = 100 μm. (D) Micrograph of Cardinium showing the microtubular-like complex with numerous tubules (asterisks) and the Gram-negative architecture of the bacterium cell wall. m = mitochondrion. (E,F) ISH of the head (E, bar = 0.25 mm), showing positive staining (red) for Cardinium probes (arrow) in the salivary gland (F, particular of the framed part in E. Bar = 50 μm). e = eye; l = salivary gland lumen; opl = optical lobe; sg = salivary gland. (G,H) Confocal laser scanning microscopy images of salivary glands after hybridization with Cy5-labelled Cardinium-specific probes (G, green) and Texas Red-labelled eubacterial probes (H, red) probes. Blue spots indicate DAPI-stained gland nuclei. Bar = 50 μm.

Figure 2. FISH experiments on feeding substrates after exposure to S. titanus.

(A,B) Video-confocal micrographs of a grapevine leaf midrib after it was used by the leafhopper for feeding (A) and a field vine leaf portion (B) after hybridization with the Cy5-labelled Cardinium probes. Arrows indicate Cardinium cells within the phloem tissues (x = xylem). (C) FISH of a sugar diet exposed to S. titanus. Signals corresponding to both the FITC-labelled eubacterial probe (green) and the Texas Red-labelled Cardinium-specific probe (red) are visible. Bars = 50 μm (A) and (B); 2.5 μm (C).

Figure 3. FISH experiments on midguts of leafhoppers sharing food substrates with S. titanus.

(A,B) Midgut portion of M. quadripunctulatus after a three-day co-feeding on the artificial diet. The cyan signal corresponds to hybridization with the Cardinium-specific probes (A), while the red signal corresponds to the eubacterial probe (B,C,D) Midgut segment of E. vitis after sharing a grapevine leaf with S. titanus for 3 days. The hybridization with the Cardinium-specific probe is marked in yellow (C), whereas the eubacterial probe signal is stained in red (D). Intestine tissues are coloured with DAPI (blue). Bars = 75 μm.