## Morphometrical Variables and Aspect Ratio of Wax Crystals Constituting Pruinose Surfaces in 12 Plant Species

Plant Species Organ

Acer negundo Stem

Aloe vera

Leaf Leaf Leaf Leaf

Aquilegia vulgaris Berberis vulgaris Brassica oleracea Chelidonium Leaf majus Chenopodium Leaf album Iris Leaf germanica Lactuca Leaf serriola

Prunus Fruit domestica

Trifolium Leaf montanum Vitis vinifera Fruit

Wax Projection Type

Irregular platelets Irregular platelets Irregular platelets Tubules

Crystal Shape

Ribbon

Polygonal rodlets Membraneous Plate platelets Tubules Tube

Tubules Tube

Terete rodlets Filament

Tubules

Tube Plate Plate Plate Tube Plate

Irregular platelets Membraneous Plate platelets

Variable, ^m Largest Smallest

~20 1.11 0.58 0.73 2.44 0.83 0.89 1.74 0.55 0.58 0.58 3.17

0.34 0.09 0.17 0.16 0.06 0.18 0.07 0.08 0.06 0.26 0.06 0.14

Aspect Ratio

~ 100 12.33 3.41 4.56 40.67 4.61 12.71 21.75 9.17 2.23 9.67 22.64

(A. vulgaris, T. montanum, A. vera, and B. vulgaris), adhered wax had a relatively homogeneous texture with almost unstructured or weakly structured surface, and wax crystals were not recognizable anymore (Figures 7.3H, 7.3J, 7.4D, and 7.4L). The waxes of most plants (B. oleracea, C. album, A. negundo, C. majus, and L. serriola) caused clearly structured contaminating coverages or homogeneous coverages with well-structured surfaces, where a few crystals or their contours may be identified (Figures 7.4B, 7.4H, 7.4J, 7.5D, and 7.5F). Waxes of I. germanica and V vinifera appeared on the setal tips as relatively homogeneous layers with numerous crystals visible on surfaces (Figures 7.3L and 7.5B).

In the case of A. vulgaris and B. vulgaris, the setae remained almost clean, rarely bearing a few contaminated points or single tiny spots of wax on their tips (Figures 7.3G, 7.3H, 7.4K, and 7.4L). Crystals of C. majus displayed only weak contamination appearing as several spots on the setal surface (low degree of contamination) (Figures 7.5C and 7.5D). In B. oleracea, P. domestica, and L. serriola, contaminated

FIGURE 7.3 SEM micrographs of the pad surface in the first (A and B) and the third tarsomeres (C to L) of the beetle Chrysolina fastuosa after walking on various surfaces. (A) to (D) Clean glass plate. (E) and (F) Dust particles. (G) and (H) Leaf of Aquilegia vulgaris. (I) and (J) Leaf of Trifolium montanum. (K) and (L) Leaf of Iris germanica. S1, S2, and S3 correspond to setae of the first, second, and third type, respectively (see the text). Scale bars: 50 ^m (A); 10 ^m (B); 100 ^m (C); 20 ^m (D), (E), (G), (I), and (K); 5 ^m (F), (H), (J), and (L).

FIGURE 7.3 SEM micrographs of the pad surface in the first (A and B) and the third tarsomeres (C to L) of the beetle Chrysolina fastuosa after walking on various surfaces. (A) to (D) Clean glass plate. (E) and (F) Dust particles. (G) and (H) Leaf of Aquilegia vulgaris. (I) and (J) Leaf of Trifolium montanum. (K) and (L) Leaf of Iris germanica. S1, S2, and S3 correspond to setae of the first, second, and third type, respectively (see the text). Scale bars: 50 ^m (A); 10 ^m (B); 100 ^m (C); 20 ^m (D), (E), (G), (I), and (K); 5 ^m (F), (H), (J), and (L).

spots might cover up to a half of the setal tip area (medium degree of contamination) (Figures 7.4A, 7.4B, 7.4E, 7.4F, 7.5E, and 7.5F). Waxes of T. montanum, I. germanica, A. vera, and C. album might cover the whole setal tips (strong contamination) (Figures 7.3I to 7.3L, 7.4C, 7.4D, 7. 4G, and 7.4H). In cases of A. negundo and V vinifera, wax often spread even over the setal tips' margins (very strong contamination) (Figures 7.4I, 7.4J, 7.5A, and 7.5B).

To quantify the influence of wax structure on the degree of contamination, we used the following two parameters: (1) the portion of setal tip surface covered with contaminating particles and (2) the portion of setae covered with the particles. Plant waxes studied differed greatly according to both parameters (for the portion of setal tip surface: degrees of freedom (d.f.) = 11, F = 8.963, P < 0.001, one-way ANOVA)

FIGURE 7.4 SEM micrographs of the pad surface in the third tarsomere of the beetle Chrysolina fastuosa after walking on various plant surfaces. (A) and (B) Leaf of Brassica oleracea. (C) and (D) Leaf of Aloe vera. (E) and (F) Ripe fruit of Prunus domestica. (G) and (H) Leaf of Chenopodium album. (I) and (J) Young stem of Acer negundo. (K) and (L) Leaf of Berberis vulgaris. Scale bars: 20 ^m (A), (C), (E), (G), (I), and (K); 5 ^m (B), (D), (F), (H), (J), and (L).

FIGURE 7.4 SEM micrographs of the pad surface in the third tarsomere of the beetle Chrysolina fastuosa after walking on various plant surfaces. (A) and (B) Leaf of Brassica oleracea. (C) and (D) Leaf of Aloe vera. (E) and (F) Ripe fruit of Prunus domestica. (G) and (H) Leaf of Chenopodium album. (I) and (J) Young stem of Acer negundo. (K) and (L) Leaf of Berberis vulgaris. Scale bars: 20 ^m (A), (C), (E), (G), (I), and (K); 5 ^m (B), (D), (F), (H), (J), and (L).

(Figures 7.6A and 7.6B). A positive correlation (NS) between the portion of the setal tip surface covered with particles and the portion of setae contaminated was found (linear regression: y = 10.332+0.978*, R2 = 0.574; d.f. = 1, F = 13.502, P = 0.004, one-way ANOVA) (Figure 7.6C). The positive correlation between the two parameters was observed not only in the whole set of plant species having various types of wax crystals, but also in plants with the same crystal type, e.g., platelets and tubes. Therefore, only one of these parameters, the portion of setal tip surface contaminated, was used in our further evaluations.

The portion of the setal tip surface contaminated with plant waxes depended on size of wax crystals. The aspect ratio was found to be statistically significant with

FIGURE 7.5 SEM micrographs of pad surface in the third tarsomere of the beetle Chrysolina fastuosa after walking on various plant surfaces. (A) and (B) Ripe fruit of Vitis vinifera. (C) and (D) Leaf of Chelidonium majus. (E) and (F) Leaf of Lactuca serriola. Scale bars: 20 ^m (A), (C), and (E); 5 ^m (B), (D), and (F).

FIGURE 7.5 SEM micrographs of pad surface in the third tarsomere of the beetle Chrysolina fastuosa after walking on various plant surfaces. (A) and (B) Ripe fruit of Vitis vinifera. (C) and (D) Leaf of Chelidonium majus. (E) and (F) Leaf of Lactuca serriola. Scale bars: 20 ^m (A), (C), and (E); 5 ^m (B), (D), and (F).

an increase in the largest dimension of crystals (linear regression: y = 6.112+3.955*, R2 = 0.766; d.f. = 1, F = 32.774, P < 0.001, one-way ANOVA) (Figure 7.7A). The portion of the setal tip surface contaminated correlated positively (NS) with both the largest dimension (linear regression: y = 44.013+2.509.*, R2 = 0.294; d.f. = 1, F = 4.173, P = 0.068, one-way ANOVA) and aspect ratio (linear regression: y = 41.434+0.572*, R2 = 0.313; d.f. = 1, F = 4.548, P = 0.059, one-way ANOVA) (Figures 7.7B and 7.7C, respectively).

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