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Several herbivorous insects and plant-associated microorganisms control the phytohormonal balance, thus enabling them to successfully exploit the plant by inhibiting plant defenses and withdrawing plant resources for their own benefit. The leaf-mining moth Phyllonorycter blancardella modifies the cytokinin (CK) profile of mined leaf-tissues, and the insect symbiotic bacteria Wolbachia is involved in the plant manipulation to the benefit of the insect host. To gain a deeper understanding into the possible origin and dynamics of CKs, we conducted an extensive characterization of CKs in larvae and in infected apple leaves. Our results show the enhanced CK levels in mines, both on green and yellow leaves, allowing insects to control their nutritional supply under fluctuating environmental conditions. The spatial distribution of CKs within the mined leaves shows that hormone manipulation is strictly limited to the mine suggesting the absence of CK translocation from distant leaf areas toward the insect feeding site. Mass spectrometry analyses reveal that major CK types accumulating in mines and larvae are similar to what is observed for most gall-inducers, suggesting that strategies underlying the plant manipulation may be shared between herbivorous insects with distinct life histories. Results further show that CKs are detected in the highest levels in larvae, reinforcing our hypothesis that CKs accumulating in the mines originate from the insect itself. Presence of bacteriaspecific methylthio-CKs is consistent with previous results suggesting that insect bacterial symbionts contribute to the observed phenotype. Our study provides key findings toward the understanding of molecular mechanisms underlying this intricate plant-insect-microbe interaction.

Gracillariidae is the most species-rich leaf-mining moth family with over 2,000 described species worldwide. In Europe, there are 263 valid named species recognized, many of which are difficult to identify using morphology only. Here we explore the use of DNA barcodes as a tool for identification and species discovery in European gracillariids. We present a barcode library including 6,791 COI sequences representing 242 of the 263 (92%) resident species. Our results indicate high congruence between morphology and barcodes with 91.3% (221/242) of European species forming monophyletic clades that can be identified accurately using barcodes alone. The remaining 8.7% represent cases of non-monophyly making their identification uncertain using barcodes. Species discrimination based on the Barcode Index Number system (BIN) was successful for 93% of species with 7% of species sharing BINs. We discovered as many as 21 undescribed candidate species, of which six were confirmed from an integrative approach; the other 15 require additional material and study to confirm preliminary evidence. Most of these new candidate species are found in mountainous regions of Mediterranean countries, the South-Eastern Alps and the Balkans, with nine candidate species found only on islands. In addition, 13 species were classified as deep conspecific lineages, comprising a total of 27 BINs with no intraspecific morphological differences found, and no known ecological differentiation. Double-digest restriction-site associated DNA sequencing (ddRAD) analysis showed strong mitonuclear discrepancy in four out of five species studied. This discordance is not explained by Wolbachia-mediated genetic sweeps. Finally, 26 species were classified as “unassessed species splits” containing 71 BINs and some involving geographical isolation or ecological specialization that will require further study to test whether they represent new cryptic species.

Leaves represent the main resource for herbivorous insects and their performances are mainly a function of leaf nutritional quality. Two feeding strategies are known to optimize the exploitation of leaf resources: leaf-miners that selectively feed on tissues of high nutritional quality and gall-inducers that induce the development of a new tissue showing an enhanced nutritional value. Some leaf-miners are known to also manipulate their nutritional environment, but do not affect plant development. Cases of callus proliferation in leaf-mines have been reported, however, the direct role of the insect in the formation of additional plant cells and the nutritional function of this tissue have never been established. Using an experimental approach, we show that leaf-mining larvae of micromoth, Borboryctis euryae (Lepidoptera: Gracillariidae), that grow on Eurya japonica (Pentaphylacaceae), actively induce callus proliferation within their leaf-mine at the fourth instar. We experimentally demonstrated that, at this developmental stage, the larva feeds exclusively on this newly formed tissue and feeding of the tissue is essential for completing larval stage. Phenological census revealed considerable expansion and variation of fourth instar duration caused by the continuous production of callus. We propose here the “cornucopia” hypothesis which states that the newly produced callus induced by the leaf-mining larvae provides virtually unending nourishment, which in turn allows flexible larval development time. This represents the first example of a leaf-miner manipulating plant development to its benefit, like a gall-inducer. We propose to name this life style “mine-galler”.

In insects, the gall-inducing life-style has evolved independently many times. Several evolutionary pathways leading to this lifestyle have been proposed. While there is compelling evidence supporting surface-feeders and stem-borers as ancestral states of insect gall-inducers, an evolutionary pathway from leaf-miners remains hypothetical. Here we explored this question by comparing the developmental processes of two micromoths, a gall-inducer Caloptilia cecidophora (Lep., Gracillariidae), and its non-gall-inducing relative C . ryukyuensis . Like other Caloptilia , the first and second instars of C . cecidophora are leaf-miners and the gall is initiated inside the leaf mine by the third instar, thus suggesting leaf-mining as an ancestral, plesiomorphic state in this case. This is the first example of an insect species switching from leaf-mining to gall-inducing during larval development. The first two leaf-mining instars of C . cecidophora exhibit an absence of growth and a reduced time duration compared to C . ryukyuensis . The shortening of the duration of leaf-mining stages is apparently compensated in C . cecidophora by a larger egg size than C . ryukyuensis , and an additional larval instar during the gall phase.

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The morphology of insect-induced galls contributes to defences of the gall-inducing insect species against its natural enemies. In terms of gall chemistry, the only defensive compounds thus far identified in galls are tannins that accumulate in many galls, preventing damage by herbivores. Intrigued by the fruit-like appearance of the translucent oak gall (TOG; Amphibolips nubilipennis , Cynipidae, Hymenoptera) induced on red oak ( Quercus rubra ), we hypothesized that its chemical composition may deviate from other galls. We found that the pH of the gall is between 2 and 3, making it among the lowest pH levels found in plant tissues. We examined the organic acid content of TOG and compared it to fruits and other galls using high-performance liquid chromatography and gas chromatography–mass spectrometry. Malic acid, an acid with particularly high abundance in apples, represents 66% of the organic acid detected in TOGs. The concentration of malic acid was two times higher than in other galls and in apples. Gall histology showed that the acid-containing cells were enlarged and vacuolized just like fruits mesocarp cells. Accumulation of organic acid in gall tissues is convergent with fruit morphology and may constitute a new defensive strategy against predators and parasitoids.