Fig 1.
Life history and phenology of Borboryctis euryae.
(A) The leaf-mines made by the five larval instars of B. euryae on Eurya japonica. First and second instars, respectively in dark and light orange and lasted 4 and 3 days, make a serpentine mine in the adaxial epidermis. The third instar (yellow) burrow a blotch mine (black arrow), visible by a brightening of leaf color and lasted 8 days. The fourth instar (green) is associated with an inflation and a progressive yellowing of the blotch mine. It lasted about five months in our rearing. The last stage (blue) lasted from 2 to 4 weeks and was linked with the apprearance of brown patches on the mine. (B) The morphology of third and fourth instar larvae. The mandibula shape, visible as the red structure on larva head, designate by its elongation a fluid-feeding habit for the third instar whereas its compactness for the fourth instar correspond to a tissue-feeder mode. Scale: 500μm. (C) B. euryae phenology. The left axis show the percentage of each instar recorded at each time point (same color code as in (A). The right axis corresponds to number of larvae at each count (dashed line). Instar abundances show the occurrence of two generation each year (G1 and G2) and the fact that fourth instar percentage do not reach zero at the end of G1 indicate that a part of the population is univoltine.
Fig 2.
Histology of Borboryctis euryae leaf-mines on Eurya japonica in normal conditions.
Cross-sections of mined leaves (left) and detail (right). From top to bottom, fixed at third instar, at one day after beginning of fourth instar, t fourteen days after beginning of fourth instar, at fifth instar. Legend: e: epidermis, p: palisade parenchyma, s: spongy parenchyma, f: larva frass, mv: mid-vein, m: mine, large green arrow: primary and secondary vein, small green arrow: other vein, yellow arrow: callus, red arrow: cut inside the mid-vein. Scale: 500μm (left), 200μm (right). Staining: Toluidine Blue O.
Fig 3.
Estimation of the mass of callus in Borboryctis euryae leaf-mines on Eurya japonica.
(A) Comparison of leaf-area density between leaves with fourth instar mines (red) and intact leaves (black), and (B) evolution of their mass in function of leaf area.
Fig 4.
Effect of detaching Eurya japonica leaves mined by Borboryctis euryae on larval viability and callus development.
(A) Last developmental stage reached after detaching a mined leaf, as a function of larval instar at leaf detachment, in percentage (Exp. 1: detached leaves experiment). (B) Viability until adulthood when mined leaf is detached at third and at fourth instar, and when larva is transferred at third and at fourth instar into a fourth instar mine, in percentage (Exp. 2: translocation experiment).
Fig 5.
Histology of Borboryctis euryae leaf-mine on Eurya japonica after detaching the leaf and killing the larva.
Cross-sections of mined leaves (left) and detail (right). From top to bottom, Fourth instar mine fixed at fourteen days after beginning of fourth instar (control), at fourteen days after beginning of fourth instar and after detaching the mined leaf at third instar (Exp. 1: detached leaves experiment), at fourteen days after killing the larvae at late third instar (Exp. 3: larval ablation experiment). Legend: e: epidermis, p: palisade parenchyma, s: spongy parenchyma, mv: mid-vein, m: mine, large green arrow: primary and secondary vein, small green arrow: other vein, yellow arrow: callus, red arrow: cut inside the mid-vein. Scale: 500μm (left), 200μm (right). Staining: Toluidine Blue O.
Fig 6.
Increase of callus tissue in the mine as a function of the duration of the interaction with fourth instar larva.
(A) Cross-sections of mined leaves at fourteen days after beginning of fourth instar and when larva is killed three, six or ten days after beginning of fourth instar. Callus is highlighted in yellow. Scale: 1mm. Staining: Toluidine Blue O. (B) Callus area per mine section measured on histology sections.