Barbs Facilitate the Helical Penetration of Honeybee (<i>Apis mellifera ligustica</i>) Stingers

<div><p>The stinger is a very small and efficient device that allows honeybees to perform two main physiological activities: repelling enemies and laying eggs for reproduction. In this study, we explored the specific characteristics of stinger penetration, where we focused on its movements and the effects of it microstructure. The stingers of Italian honeybees (<i>Apis mellifera ligustica</i>) were grouped and fixed onto four types of cubic substrates, before pressing into different substrates. The morphological characteristics of the stinger cross-sections were analyzed before and after penetration by microscopy. Our findings suggest that the honeybee stinger undergoes helical and clockwise rotation during penetration. We also found that the helical penetration of the stinger is associated directly with the spiral distribution of the barbs, thereby confirming that stinger penetration involves an advanced microstructure rather than a simple needle-like apparatus. These results provide new insights into the mechanism of honeybee stinger penetration.</p></div

Observation of the stinger cross-sections and calculation of the rotation angles.

<p>(A) The four types of cubic substrates were attached to the positioning block assembly under the microscope. (B) Marker points in the stinger cross-section. If the stinger rotated clockwise, the rotation angle was recorded as a negative angle, whereas an anticlockwise rotation was recorded as a positive angle.</p

Rotation angles of the stinger shafts.

<p>The stinger samples marked with <i>S</i> and the hair samples marked with <i>H</i> were used for comparison. The two adjacent bars show the rotation angles of the honeybee stingers (left side) and the hair samples (right side). In each type of substrate, the rotation angle of the hair was very small, which demonstrated that the instrument had no significant effect on the rotation angle during pushing. The experimental observations confirmed the existence of rotation during the stinging process. Furthermore, we found that the rotation angle was associated with the stiffness of the substrate.</p

Preparation of the stinger samples and the experiments.

<p>The stingers of worker bees were collected and separated into two groups. (1) The first group of stingers were placed onto the polymethyl methacrylate panel using drops of 15% polyvinyl alcohol (0.1 µL), and they were then placed vertically on the substrates (agar, silica gel, soft rubber and paraffin wax), before pushing the stingers into the substrates at a velocity of 6 mm mm/s using the positioner. The positioner, also called the precision position platform, is a machine that is able to push tiny appendages accurately into the substrates following the planned kinematics, for instance the preset average velocity, the total displacement even the acceleration. (2) The microstructures of the second group of stingers were observed using an environmental scanning electron microscope.</p

Environmental scanning electron microscope images of the stinger.

<p>(A) The needle-like sting, venom sac, and related glands. The stinger is activated by the muscles to penetrate the skin of the victim. (B) Barbs along the axial direction of the sting. The solid line in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103823#pone-0103823-g003" target="_blank">Figure 3(b)</a> is the axis of the sting which is obtained by connecting the tip of the stinger and the midpoint of the stinger root. The stinger of <i>Apis mellifera ligustica</i> has two rows of barbs, each of which comprises about 10 barbs. The angle between the rows of barbs and the axis of the stinger shaft was around 8–9°, according to observations based on 10 samples. The row of barbs was found to form a right-handed helix. (C) Magnified view of the barbs. Seven barbs are marked with the notations 1–1′, 2–2′, etc. Note that the angles of the tips were 90.33°, 89.62°, 80.31°, 72.13°, 72.36°, 59.63°, and 46.19°, thereby demonstrating that the barbs were relatively sharper near the tip of the stinger. (D) Magnified view of two rows of barbs. Viewed in the axial direction, the angles between the two rows of barbs were about 95°.</p

Anatomy of the honeybee’s stinger apparatus.

<p>The stinger resides in the sting chamber inside the last abdominal segment (not to scale). The sting apparatus mainly comprises the protractor/retractor muscles, the bulb, the stinger, and the venom sac. The protractor muscles drive the stinger to penetrate the wound and the retractor muscles are used in the reverse manner to pull the stinger back into the sting chamber. During penetration, the venom is pumped into the stinger from the bulb, which is also known as the venom reservoir.</p

Mechanistic model of stinging.

<p>The angle between a row of barbs and the shaft axis was about 9°. The equivalent tetrahedron model mimics the force condition of a single barb (not to scale). During penetration, the contact force on the barb (<i>F</i>) can be decomposed into the friction force (<i>F_f</i>) and the tangential force (<i>F</i>_T). The torque around the stinger shaft is generated by the tangential force, which drives the stinger to rotate clockwise in the line of sight along the stinger shaft toward the tip.</p