Horizontal transmission of the symbiotic bacterium Asaia sp. in the leafhopper Scaphoideus titanus Ball (Hemiptera: Cicadellidae)

Abstract

Background: Bacteria of the genus Asaia have been recently recognized as secondary symbionts of different sugar-feeding insects, including the leafhopper Scaphoideus titanus, vector of Flavescence dorée phytoplasmas. Asaia has been shown to be localized in S. titanus gut, salivary glands and gonoducts and to be maternally transmitted to the progeny by an egg smearing mechanism. It is currently not known whether Asaia in S. titanus is transmitted by additional routes. We performed a study to evaluate if Asaia infection is capable of horizontal transmission via co-feeding and venereal routes.

Results: A Gfp-tagged strain of Asaia was provided to S. titanus individuals to trace the transmission pathways of the symbiotic bacterium. Co-feeding trials showed a regular transfer of bacterial cells from donors to recipients, with a peak of frequency after 72 hours of exposure, and with concentrations of the administrated strain growing over time. Venereal transmission experiments were first carried out using infected males paired with uninfected females. In this case, female individuals acquired Gfp-labelled Asaia, with highest infection rates 72-96 hours after mating and with increasing abundance of the tagged symbiont over time. When crosses between infected females and uninfected males were conducted, the occurrence of "female to male" transmission was observed, even though the transfer occurred unevenly.

Conclusions: The data presented demonstrate that the acetic acid bacterial symbiont Asaia is horizontally transmitted among S. titanus individuals both by co-feeding and venereal transmission, providing one of the few direct demonstrations of such a symbiotic transfer in Hemiptera. This study contributes to the understanding of the bacterial ecology in the insect host, and indicates that Asaia evolved multiple pathways for the colonization of S. titanus body.

Figure 1

Gfp-Asaia infection rates and density within infected samples. White columns represent S. titanus individuals, and grey columns represent diets. The “donors” columns refer to the average values of donor insects in all of the trials. “24h”, “48h”, “72h”, and “96h” indicate the time of exposure to co-feeding or the time of incubation after mating with infected individuals. The “control” columns represent the values obtained from insects fed on sterile sugar diets, as well as those obtained from individuals co-housed with Gfp Asaia-infected specimens of the same sex. A-C) Percentage of insects and diets colonized by Gfp-tagged Asaia. D-F) Transformed (10 + log) number of gfp gene copies per positive sample. Bars represent the standard error of transformed data. Different letters (black for insect and grey for diet samples) indicate significantly different values (ANOVA, P<0.05).

Figure 2

Relative abundance of Gfp-Asaia within the whole Asaia populations. The relative abundance of the tagged strain in total Asaia community is calculated by the ratio between the number of gfp gene copies per sample and the number of Asaia cells (which is Asaia 16S rRNA gene copies divided by four, assuming that four rRNA gene copies per cell are present in Asaia, as reported in Crotti et al. [4]) per sample. In each graph white columns represent S. titanus individuals, and grey columns represent diets. The “donors” columns refer to average values of donor insects in all trials. “24h”, “48h”, “72h”, and “96h” indicate the time of exposure to co-feeding or the time of incubation after mating with infected individuals. The Gfp-tagged Asaia to total Asaia ratio is indicated in insects and diets submitted to co-feeding trials (A), and to venereal transmission experiments, from male to female (B) and from female to male (C), respectively. The bars on each column represent the standard error.

Figure 3

Positive and negative controls for FISH experiments targeting the gfp gene. The presence and distribution of Gfp-tagged Asaia in tissues of donor insects (positive controls) and of individuals submitted to transmission trials in absence of the tagged strain (negative controls) have been evaluated by FISH with the FITC-labelled Eu338 eubacterial probe (green), the Cy3-labelled Asaia-specific probes (magenta) and the Cy5.5-labeled probes specific for the gfp gene (yellow). A) Superposition of a CLSM image after staining with DAPI over the interferential contrast microscopy picture of a salivary gland lobe of an individual used as donor during co-feeding trials (bar = 50 µm). B,C) CLSM images after hybridization with the Cy3-tagged probes targeting the whole Asaia population (B), or with the Cy5.5-marked probes specific for the Gfp strain (C). In D-G) an ovariole of a female mated with a male which was not previously fed with the Gfp-tagged Asaia is shown. D) Interferential contrast micrograph showing the ovariole (bar = 150 µm). E-G) CLSM images of FISH with the FITC-labeled eubacterial probe (E), the Cy3-tagged probes targeting the whole Asaia population (F), and the Cy5.5-marked probes specific for the gfp gene (G). While the occurrence of bacteria (and Asaia in particular) is shown, no hybridization signal was observed with the gfp gene-specific probes.

Figure 4

Localization of horizontally-transmitted Gfp Asaia in organs of S. titanus individuals. Images of insect tissues after hybridization with the Cy3-labeled Asaia-specific probes (magenta) and the Cy5.5-labeled probes specific for the gfp gene (yellow) showing the distribution of the symbiont within the gut, the ovaries and testes of specimens after acquisition of the tagged bacterium via co-feeding or venereal transmission. A-C) Midgut portion of an individual after 48-hour acquisition during the co-feeding trial, observed by interferential contrast microscopy (A) and CLSM after hybridization with the Cy3-tagged probes targeting the whole Asaia population (B), or with the Cy5.5-marked probes specific for the gfp gene(C). D-F) Testis portion of an individual after co-feeding trial observed by interferential contrast microscopy (D), and by CLSM after hybridization with the Cy3-tagged probes targeting the whole Asaia population (E) and the Cy5.5-marked probes specific for the gfp gene (F). In G-I) ovaries of a S. titanus individual after the acquisition in venereal transmission experiments are shown. G) Interferential contrast micrograph showing a group of ovarioles. H, I) CLSM images of FISH with the Cy3-tagged probes targeting the whole Asaia population (H) and the Cy5.5-marked probes specific for the gfp gene (I). Bars = 150 µm.