Plant green-island phenotype induced by leaf-miners is mediated by bacterial symbionts

Abstract

The life cycles of many organisms are constrained by the seasonality of resources. This is particularly true for leaf-mining herbivorous insects that use deciduous leaves to fuel growth and reproduction even beyond leaf fall. Our results suggest that an intimate association with bacterial endosymbionts might be their way of coping with nutritional constraints to ensure successful development in an otherwise senescent environment. We show that the phytophagous leaf-mining moth Phyllonorycter blancardella (Lepidoptera) relies on bacterial endosymbionts, most likely Wolbachia, to manipulate the physiology of its host plant resulting in the 'green-island' phenotype--photosynthetically active green patches in otherwise senescent leaves--and to increase its fitness. Curing leaf-miners of their symbiotic partner resulted in the absence of green-island formation on leaves, increased compensatory larval feeding and higher insect mortality. Our results suggest that bacteria impact green-island induction through manipulation of cytokinin levels. This is the first time, to our knowledge, that insect bacterial endosymbionts have been associated with plant physiology.

Figure 1.

Influence of Wolbachia on green-island formation. (a) Apple tree leaves infected with the tentiform leaf-miner Phyllonorycter blancardella in autumn. Offspring (G₁) obtained from untreated controls and antibiotic-treated adults show distinct feeding behaviours and emergence success in autumnal conditions. On the left panel, untreated individuals induce green-islands on host plants. The feeding area exhibits intact green chlorophyll-containing tissues, while the remaining leaf tissues undergo leaf senescence. The white spots on the mine are feeding windows, where all but the epidermis has been consumed by the caterpillar. On the right panel, larvae from antibiotic-treated individuals are unable to induce green-islands and consume almost the entire amount of tissues present within the mine leaving mostly non-nutritional epidermal cells only. (b) Representative PCR amplifications from offspring obtained from non-treated (left panel) and antibiotic-treated individuals (right panel) with the insect EF1-α primers, universal bacterial 16S rRNA primers and Wolbachia-specific wsp primers. 16S rRNA-amplified products correspond to Wolbachia, only a very faint signal could be detected in antibiotic-treated individuals. No wsp amplification was detected in treated individuals.

Figure 2.

Influence of the presence of endosymbionts on green-island formation. (a) Representative leaf showing the impact of symbionts on green-island induction. Second generation-cured individuals failed to produce green-islands (upper mine), while untreated insects (control) successfully maintained intact green chlorophyll-containing tissues within the mine (lower mine). (b) Cytokinin concentration in control and mined leaf tissues. Green-islands (untreated) contained high levels of cytokinins, while tissues mined by antibiotic-treated insects had very low levels of phytohormones. Homologous groups are indicated by identical letters (Kruskal–Wallis test: p < 0.01). Means ± s.e.

Figure 3.

Various effects of antibiotic treatments. (a) Impact of antibiotics on the Wolbachia hosted by leaf-miner larvae. Increased levels of antibiotics provided to G₀ females lead to a strong decrease of the wsp signal in G₂ offspring. A Wolbachia-specific signal can be detected (W+) up to a level of 0.50% of antibiotics in insect diet. When 1% antibiotic diet is provided to insects, the Wolbachia-specific signal cannot be detected anymore (W−). (b) Relative proportion of green tissues within the mine. For control insects, most tissues within the mine remain green on yellow leaves. At high antibiotic concentrations, most tissues enter senescence and turn yellow. Results shown for the tetracycline treatment. (c) Impact of antibiotics on the insect feeding behaviour. For levels of up to 0.50% of antibiotics in the parent (G₀) insect diet, less than 20% of tissues are consumed by the second generation (G₂) larva by the end of its fourth instar. One per cent of antibiotics leads to a significant increase of the feeding activity of G₂ larvae, inducing the consumption of about 70 per cent of tissues within the mine by the end of the fourth instar. Homologous groups are indicated by identical letters (Kruskal–Wallis test: p < 0.01). Means ± s.e. Results shown for the tetracycline treatment.