Leaflet surface micromorphology
There is a difference in the crystalline epicuticular wax coverage between the adaxial and abaxial leaflet surfaces in both waxy and glossy plants, and between two mutants (Table 1).
The adaxial leaflet surface in waxy plants bears a network of interconnected numerous membranous platelets, occurring in high density and covering the surface uniformly and completely (Fig. 1a, b). Although crystals vary greatly in size and shape, they are rather small and have predominantly irregular shapes with indented, often fringed outside edges (Table 1). The platelets are connected with the underlying surface through edges. Crystal planes are oriented randomly according to their longitudinal axes, but perpendicular or slightly sloped relatively to the plane of the leaflet.
In glossy plants, the wax coverage present on the adaxial side is similar to the waxy pea. However, wax crystal density on the adaxial surface of the glossy mutant is reduced by a factor of three compared to the waxy plants, and the surface is not completely covered with the crystalline wax (Table 1; Fig. 1c). The membranous platelets have similar shapes as in waxy peas, but are longer and narrower (Fig. 1d). They emerge often as aggregated crystals (rosettes) and are connected with the underlying surface through a larger edge. The crystals show no specific orientation, and their planes are either perpendicular to or more often have acute angles with the leaflet surface.
The abaxial surface of waxy plants is completely covered with the dense crystalline wax coverage, composed of crystals of three types: large elongated filaments, ribbons, and transversely ridged rodlets (Table 1; Fig. 1e). Crystals vary essentially in size and shape, emerge solitarily, are connected with the underlying surface through a small edge, show no preferred orientation and are somewhat crowded to the surface (Fig. 1f). Ribbons have slightly irregular edges and possess widened, sometimes spatulae-like tips with rather corrugated surface. Rodlets are usually bar- or ribbon-shaped, with very uneven sides and edges.
Although the density of wax crystals on the abaxial side of the glossy mutant is much higher than on all other surfaces studied here, this surface is not completely covered with the crystalline wax (Table 1; Fig. 1g, h). Crystals of two types, short ribbons and scales, are very variable in both size and shape being essentially smaller and having more regular edges than crystals on the abaxial surface of the glossy mutant. As well as latter, these crystals originate solitarily and have random orientation relatively to their longitudinal axes, but are connected with the surface through small (ribbons) or large (scales) sides and may be either erect or crowded to the substrate.
After treatment with warm chloroform, both leaflet sides of both mutants were completely free of wax crystals (Fig. 2a, c, e, g) and therefore, the microroughness was removed. However, treated surfaces became corrugated, with a noticeably increased large-scale roughness (Fig. 2b, d, f, h).
Physicochemical properties of leaflet surfaces
In all plants studied, both adaxial and abaxial leaflet surfaces were unwettable with both polar liquids, such as water (surface tension = 72.1 mN/m, dispersion component = 19.9 mN/m, polar component = 52.2 mN/m, Busscher et al. 1984) and ethylene glycol (surf. tension = 48.0 mN/m, disp. comp. = 29.0 mN/m, pol. comp. = 19.0 mN/m, Erbil 1997) (Fig. 3a). Contact angles of water were the highest compared to other liquids and seemed to be rather similar on all surfaces. However, the statistic difference was found in contact angles of water between plant surfaces studied (Kruskal–Wallis one way ANOVA on ranks, H
3,58 = 9.728, P = 0.021), where the adaxial side of the waxy mutant and the abaxial side of the glossy mutant was the only surface pair with significantly different angles (Dunn’s method carried out after Kruskal–Wallis one way ANOVA on ranks, d.f. = 28, diff. of ranks = 17.367, Q = 2.769, P < 0.05). Contact angles of ethylene glycol were significantly lower in glossy plants compared to waxy ones (one way ANOVA, F
3,59 = 24.100, P < 0.001). In both waxy and glossy mutants, no significant difference in contact angles of ethylene glycol was detected between the abaxial and adaxial leaflet sides (Table 2). Contact angles of non-polar diiodomethane (surf. tension = 50.0 mN/m, disp. comp. = 47.4 mN/m, pol. comp. = 2.6 mN/m, Busscher et al. 1984) were lower than those of both water and ethylene glycol. The abaxial surface of glossy plants was wetted with diiodomethane (contact angle = 75.3 ± 6.4º, mean ± SD, n = 15 drops, N = 3 plants). The leaflet surfaces studied showed highly significant difference in contact angles of latter liquid (one way ANOVA, F
3,58 = 111.810, P < 0.001). The abaxial side of the waxy mutant and the adaxial side of the glossy mutant was the only surface pair, where contact angles of diiodomethane did not differ significantly (Table 3).
Considering morphometrical parameters of the waxy coverage of the leaflet surfaces (average size and density of wax crystals), there was no significant correlation between contact angles and any morphometrical parameters tested (cont. angle of water versus cryst. length: R
2 = 0.118, P = 0.144; cont. angle of water versus cryst. dens.: R
2 = 0.526, P = 0.144; cont. angle of ethylene glycol versus cryst. length: R
2 = 0.488, P < 0.001; cont. angle of ethylene glycol versus cryst. dens.: R
2 = 0.082, P = 0.186; cont. angle of diiodomethane versus cryst. length: R
2 = 0.121, P = 0.011; cont. angle of diiodomethane versus cryst. dens.: R
2 = 0.023, P = 0.159).
Leaflet surfaces in both plant mutants showed rather low values of the free surface energy with the prevailing dispersion component (Fig. 3b). The lowest surface energy with the highest dispersion component was observed for the adaxial surface of waxy plants, whereas in the abaxial leaflet side of the glossy mutant, the surface energy was several times higher with markedly higher polar component. As for the abaxial surface of waxy plants and the adaxial side in glossy plants, there was no considerable difference besides slightly higher values of the total surface energy in latter surfaces.
Morphology of the beetle attachment system
The tarsus of C. montrouzieri consists of three segments (Fig. 4a, e). The distal tarsomere (T3) bears paired claws, curved ventrally, each with a tooth. Attachment pads are of the hairy type (Beutel and Gorb 2001; Gorb 2001). The ventral side of the two first proximal tarsomeres (T1 and T2) is densely covered by tiny tenent setae (Fig. 4b, f). The setae of the first tarsomere T1 are more or less uniform, with straight or slightly curved pointed tips (Fig. 4c, d). In the second tarsomere T2, setae of the two types were found: (1) with pointed tips and (2) with flattened and widened tips called spatulae (Fig. 4g, h). No sexual dimorphism in the morphology of the attachment system in C. montrouzieri was detected.
Traction forces of beetles on pea leaflet surfaces
Although the maximal traction force generated on glass by different insect individuals (0.5–11.8 mN) did not vary significantly in different experiments (Kruskal–Wallis one way ANOVA on ranks, H
7,71 = 9.597, P = 0.213), we used data, normalised to values obtained on glass, for comparison of different surfaces. For each individual, the force, obtained on a test plant surface, was compared to that on glass (considered as 100%).
For all intact plant surfaces tested, with the only exception of the abaxial side in the glossy mutant, the values of the maximal traction force of beetles differed significantly from those, produced on the glass plate (Kruskal–Wallis one way ANOVA on ranks, H
4,44 = 27.954, P < 0.001; Table 4; Fig. 5a). The comparison of different intact plant surfaces (one way ANOVA, F
3,35 = 4.681, P = 0.008) showed that on waxy plants, insects produced similar forces regardless of a leaflet surface, while higher force was measured on the abaxial side in the glossy pea (Table 5). There was no significant difference between adaxial surfaces in waxy and glossy plants, whereas on the abaxial side, the force was stronger in the glossy mutant.
Considering morphometrical parameters of the waxy coverage of the leaflet surfaces (average size and density of wax crystals) and surface wettability (contact angles of water, ethylene glycol and diiodomethane), a significant correlation was found for the traction force with both the wax crystal length and density (Fig. 6). There was no significant correlation between the force and contact angles of any liquid (force versus cont. angle of water: R
2 = 0.202, P = 0.145; force versus cont. angle of ethylene glycol: R
2 = 0.544, P = 0.014; force versus cont. angle of diiodomethane: R
2 = 0.376, P = 0.043).
Treatment of plant surfaces with chloroform resulted in a substantial increase of traction forces on both leaflet sides in both waxy and glossy plants, compared to those on intact surfaces (Table 6; Fig. 5). Compared with glass (Kruskal–Wallis one way ANOVA on ranks, H
4,44 = 12.283, P = 0.015; Fig. 5b), the abaxial leaflet side of the waxy pea was the only substrate, where the force was significantly higher (Tukey test carried out after Kruskal–Wallis one way ANOVA on ranks, d.f. = 17, diff. on ranks = 168.000, q = 4.264, P < 0.05), and no difference was found between other treated plant surfaces and glass. The comparison of treated leaflet surfaces showed no statistically significant difference in traction forces of insects between plant substrates tested (one way ANOVA, F
3,35 = 1.675, P = 0.192).