Interaction between honeybee mandibles and propolis

Abstract

In a biomimetic top-down process, challenging the problem of resin deposition on woodworking machine tools, an adequate biological model was sought, which hypothetically could have developed evolutionary anti-adhesive strategies. The honeybee (Apis mellifera) was identified as an analogue model since it collects and processes propolis, which largely consists of collected tree resin. Propolis is a sticky substance used by bees to seal their hive and protect the colony against pathogens. In spite of its stickiness, honeybees are able to handle and manipulate propolis with their mandibles. We wanted to know if beneficial anti-adhesive properties of bee mandibles reduce propolis adhesion. The anatomy of bee mandibles was studied in a (cryo-)scanning electron microscope. Adhesion experiments were performed with propolis on bee mandibles to find out if bee mandibles have anti-adhesive properties that enable bees to handle the sticky material. A scale-like pattern was found on the inside of the mandible. Fresh mandibles were covered with a seemingly fluid substance that was at least partially removed during the washing process. Propolis adhesion on bee mandibles was measured to be 1 J/m² and was indeed significantly lower compared to five technical materials. Propolis adhesion was higher on mandibles that were washed compared to fresh, unwashed mandibles. Results indicate that the medial surface of the mandible is covered with a fluid substance that reduces propolis adhesion. First results suggested that the surface pattern does do not have a direct effect on propolis adhesion.

Keywords: Apis mellifera; adhesion; bee mandibles; honeybee; propolis.

Figure 1

Bee propolis. (A) Raw propolis as collected from the hive. (B) Homogenised propolis powder. (C) Cone-shaped propolis sample used for adhesion tests. Scale bar: 1 cm (A, B), 500 µm (C). Figure 1 was reproduced from [1] (© 2021 Saccardi, Schiebl, Weber, Schwarz, Gorb and Kovalev, published by Frontiers, distributed under the terms of the Creative Commons Attribution 4.0 International License, https://creativecommons.org/licenses/by/4.0).

Figure 2

Head and mandible of a worker bee. (A) Frontal view of a honeybee head with an arrow pointing to the outside of the left mandible. Scale bar: 1 mm. (B) Medial surface of the left mandible of a worker bee. Proximal and distal ends are marked. Scale bar: 200 µm.

Figure 3

Adhesion experiments. (A) Experimental set-up for adhesion testing with the Basalt-01 mechanical tester (Tetra GmbH). (B) Propolis contact in the presence of fluid (mineral oil) and without fluid. (C) Schematic of a bee mandible. Adhesion measurements were conducted along the arrow, the circle represents the size of the contact area. Scale bar: 500 µm. (D) 3D profile of a propolis sample. The inscribed circle was used to estimate the tip radius. The small subimage depicts the sample topography with darker areas being higher than lighter areas. (E) Typical force–distance curve obtained from adhesion experiments. BM, bee mandible (optional); FL, fluid; FOS, fibre-optic sensor; GC, glass capillary; GS, glass slide or other substrate material; MM, micro-manipulators; MR, mirror; MS, metal spring; PS, propolis sample. Figure 3A was adapted and Figure 3B, D, and E were reproduced from [1] (© 2021 Saccardi, Schiebl, Weber, Schwarz, Gorb and Kovalev, published by Frontiers, distributed under the terms of the Creative Commons Attribution 4.0 International License, https://creativecommons.org/licenses/by/4.0).

Figure 4

Anatomy of the bee mandible. (A) Medial surface of left bee mandible. Characteristic features are labelled. (B–I) Higher magnified images of areas labelled in (A). (B) Scales in flat area. (C) Smooth surface near the sharp edge. (D) Curved bristles along the central ridge. (E) Fading structures towards the apex. (F) Scales in channel area. (G) Hair growing on the hairy edge. (H) Oblong scales on the ledge. (I) Scales on the stem of the mandible. ap, apex; br, bristles; ch, channel area; cr, central rigde; fl, flat area; fs, faded structures; hr, hair; he, hairy edge; le, ledge; se, sharp edge; sr, small ridge; st, stem. Scale bars 100 µm (A), 10 µm (B–I).

Figure 5

Profile of scales on bee mandibles. (A) Cryo-SEM micrograph of mandible cuticle. The arrowhead indicates the step between two scales. (B) 3D laser scanning microscope image of medial surface of the mandible in the channel area. (C) Profile of scales in along the red arrow in (B). (D) Profile of scales along the blue arrow in (B). Scale bars: 2 µm (A), 10 µm (B).

Figure 6

Mandibles of propolis bees. (A) Mandible tip completely covered with resin. (B) Resin residues on mandible. The arrowhead points to resin rests adhering to the hairs on the mandible. (C) SEM micrograph of central ridge and bristles covered by resin. Scale bars: 500 µm (A,B), 100 µm (C).

Figure 7

Propolis adhesion on bee mandibles. (A) Adhesion of propolis on bee mandibles compared to glass. Adhesion experiments with propolis samples were carried out on the following substrates: a dry glass surface (glass reference), a glass surface with a drop of oil (glass in oil), freshly prepared, untreated mandibles (BM fresh), defrosted and subsequently prepared mandibles (BM defrosted). (B) Adhesion on bee mandibles washed with different methods. Adhesion experiments with propolis samples were carried out on the following substrates: freshly prepared, untreated mandibles (BM untreated), freshly prepared mandibles washed with water (BM H₂O), prepared mandibles washed with chloroform and water (BM chloroform), prepared mandibles washed with acetone and water (BM acetone). All experiments were conducted using the micro-force tester Basalt-01 at room temperature (24 °C) with a maximum applied force of 5 mN (N = 5–8 propolis samples per condition, n = 10 individual measurements per sample). The ends of the boxes define the 25th and 75th percentiles, with a line at the median and error bars defining the 10th and 90th percentiles. Conditions marked with different letters differ significantly from each other (one-way ANOVA, P < 0.001 and Tukey test, P < 0.05).

Figure 8

Fractures of freshly prepared, frozen bee mandibles. (A) Overview of mandible fracture. Inside and outside are marked. (B) Cuticle in channel area. (C–F) Cuticle in the flat area. (G,H) Cuticle near the sharp edge. (H) Contact angle of surface coating on cuticle (25°). (I–K) Angled top view of flat area. (L) Cuticle on outside of mandible. ep, epicuticle; ex, exocuticle; en, endocuticle. Arrowheads indicate the surface layer on top of the cuticle. Asterisks mark the top view of the surface layer. Scale bars: 100 µm (A), 10 µm (B–D,I,J,L), 2 µm (E–H,K).

Figure 9

Bee mandibles washed with different methods. (A–C) Cryo-SEM micrographs of untreated bee mandibles (overview and close-up of flat area). (D–F) SEM micrographs of bee mandibles washed with water. (E) channel area, (F) flat area. (G–I) SEM micrographs of bee mandibles washed with acetone and water (mandible overview and close-up of flat area). (J–L) SEM micrographs of bee mandibles washed with chloroform and water (mandible overview and close-up of flat area). Scale bars: 100 µm (A,D,G,J), 10 µm (B,C,E,F,H,I,K,L).

Figure 10

Fractures of bee mandibles washed with chloroform. (A) Cryo-SEM micrograph of fractured bee mandible. (B) Cryo-SEM micrograph of scales on washed mandible. (C) Cryo-SEM micrograph of fractured cuticle. Arrowheads indicate residues on epicuticle. ep, epicuticle; ex, exocuticle. Scale bars: 100 µm (A), 10 µm (B), 2 µm (C).

Figure 11

Replica of bee mandible. (A) Bee mandible replica made of Spurr’s epoxy resin. (B) Propolis adhesion on real and replicated bee mandibles and smooth resin surface. The adhesion experiments were conducted using the micro-force tester Basalt-01 at room temperature (24 °C) with a maximum applied force of 5 mN (N = 5–8 propolis samples per condition, n = 10 individual measurements per sample). The ends of the boxes define the 25th and 75th percentiles, with a line at the median and error bars defining the 10th and 90th percentiles. Conditions marked with different letters differ significantly from each other (one-way ANOVA, P < 0.001 and Tukey test, P < 0.05). Scale bar: 500 µm (A).