Correlation between the green-island phenotype and Wolbachia infections during the evolutionary diversification of Gracillariidae leaf-mining moths.

Internally feeding herbivorous insects such as leaf miners have developed the ability to manipulate the physiology of their host plants in a way to best meet their metabolic needs and compensate for variation in food nutritional composition. For instance, some leaf miners can induce green-islands on yellow leaves in autumn, which are characterized by photosynthetically active green patches in otherwise senescing leaves. It has been shown that endosymbionts, and most likely bacteria of the genus Wolbachia, play an important role in green-island induction in the apple leaf-mining moth Phyllonorycter blancardella. However, it is currently not known how widespread is this moth-Wolbachia-plant interaction. Here, we studied the co-occurrence between Wolbachia and the green-island phenotype in 133 moth specimens belonging to 74 species of Lepidoptera including 60 Gracillariidae leaf miners. Using a combination of molecular phylogenies and ecological data (occurrence of green-islands), we show that the acquisitions of the green-island phenotype and Wolbachia infections have been associated through the evolutionary diversification of Gracillariidae. We also found intraspecific variability in both green-island formation and Wolbachia infection, with some species being able to form green-islands without being infected by Wolbachia. In addition, Wolbachia variants belonging to both A and B supergroups were found to be associated with green-island phenotype suggesting several independent origins of green-island induction. This study opens new prospects and raises new questions about the ecology and evolution of the tripartite association between Wolbachia, leaf miners, and their host plants.

Leaf-miners co-opt microorganisms to enhance their nutritional environment.

Organisms make the best of their mother's oviposition choices and utilize specific feeding options that meet energetic requirements and cope with environmental constraints. This is particularly true for leaf-miner insects that develop enclosed in the two epidermis layers of a single leaf for their entire larval life. Cytokinins (CKs) play a central role in plant physiology - including regulation of senescence and nutrient translocation - and, as such, can be the specific target of plant exploiters that manipulate plant primary metabolism. 'Green-islands' are striking examples of a CK-induced phenotype where green areas are induced by plant pathogens/insects in otherwise yellow senescent leaves. Here, we document how the leaf-miner caterpillar Phyllonorycter blancardella, working through an endosymbiotic bacteria, modifies phytohormonal profiles, not only on senescing (photosynthetically inactive) but also on normal (photosynthetically active) leaf tissues of its host plant (Malus domestica). This leaf physiological manipulation allows the insect to maintain sugar-rich green tissues and to create an enhanced nutritional microenvironment in an otherwise degenerating context. It also allows them to maintain a nutritional homeostasis even under distinct leaf environments. Our study also highlights that only larvae harboring bacterial symbionts contain significant amounts of CKs that are most likely not plant-derived. This suggests that insects are able to provide CKs to the plant through their symbiotic association, thus extending further the role of insect bacterial symbionts in plant-insect interactions.

Predator-prey pursuit-evasion games in structurally complex environments.

Pursuit and evasion behaviors in many predator-prey encounters occur in a geometrically structured environment. The physical structures in the environment impose strong constraints on the perception and behavioral responses of both antagonists. Nevertheless, no experimental or theoretical study has tackled the issue of quantifying the role of the habitat's architecture on the joint trajectories during a predator-prey encounter. In this study, we report the influence of microtopography of forest leaf litter on the pursuit-evasion trajectories of wolf spiders Pardosa sp. attacking the wood cricket Nemobius sylvestris. Fourteen intact leaf litter samples of 1 m × 0.5 m were extracted from an oak-beech forest floor in summer and winter, with later samples having the most recently fallen leaves. Elevation was mapped at a spatial resolution of 0.5 mm using a laser scanner. Litter structuring patterns were identified by height transects and experimental semi-variograms. Detailed analysis of all visible leaf-fragments of one sample enabled us to relate the observed statistical patterns to the underlying geometry of individual elements. Video recording of pursuit-evasion sequences in arenas with flat paper or leaf litter enabled us to estimate attack and fleeing distances as a function of substrate. The compaction index, the length of contiguous flat surfaces, and the experimental variograms showed that the leaf litter was smoother in summer than in winter. Thus, weathering as well as biotic activities compacted and flattened the litter over time. We found good agreement between the size of the structuring unit of leaf litter and the distance over which attack and escape behaviors both were initiated (both ∼3 cm). There was a four-fold topographical effect on pursuit-escape sequences; compared with a flat surface, leaf litter (1) greatly reduced the likelihood of launching a pursuit, (2) reduced pursuit and escape distances by half, (3) put prey and predator on par in terms of pursuit and escape distances, and (4) reduced the likelihood of secondary pursuits, after initial escape of the prey, to nearly zero. Thus, geometry of the habitat strongly modulates the rules of pursuit-evasion in predator-prey interactions in the wild.

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

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.

Cytokinin-mediated leaf manipulation by a leafminer caterpillar.

A large number of hypotheses have been proposed to explain the adaptive significance and evolution of the endophagous-feeding mode, nutritional benefits being considered to be one of the main advantages. Leaf-mining insects should feed on most nutritional tissues and avoid tissues with high structural and/or biochemical plant defences. This selective feeding behaviour could furthermore be reinforced by manipulating the plant physiology, as suggested by the autumnal formation of 'green islands' around mining caterpillars in yellow leaves. The question we address here is how such metabolic manipulation occurs and what the nutritional consequences for the insect are. We report a large accumulation of cytokinins in the mined tissues which is responsible for the preservation of functional nutrient-rich green tissues at a time when leaves are otherwise turning yellow. The analogy with other plant manipulating organisms, in particular galling insects, is striking.