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Mechanistic origins of bombardier beetle (Brachinini) explosion-induced defensive spray pulsation

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Science  01 May 2015:
Vol. 348, Issue 6234, pp. 563-567
DOI: 10.1126/science.1261166

A beetle's internal bomb

Bombardier beetles shoot a toxic pulse at potential predators and other harassers. The toxic spray is created by a chemical reaction that occurs inside the beetle's body. Although the details of the reaction are known, how the beetle is able to precisely combine the chemicals at appropriate times and release the pulse at regular intervals has remained a mystery. Arndt et al. used synchrotron x-ray imagery to observe the process as it occurs within live beetles. Expansion and contraction of an internal expansion membrane facilitate the precise cyclic injection of reactants and the subsequent ejection of toxic sprays that keep the beetle's predators at bay.

Science, this issue p. 563

Abstract

Bombardier beetles (Brachinini) use a rapid series of discrete explosions inside their pygidial gland reaction chambers to produce a hot, pulsed, quinone-based defensive spray. The mechanism of brachinines’ spray pulsation was explored using anatomical studies and direct observation of explosions inside living beetles using synchrotron x-ray imaging. Quantification of the dynamics of vapor inside the reaction chamber indicates that spray pulsation is controlled by specialized, contiguous cuticular structures located at the junction between the reservoir (reactant) and reaction chambers. Kinematics models suggest passive mediation of spray pulsation by mechanical feedback from the explosion, causing displacement of these structures.

When threatened, bombardier beetles (Fig. 1A) expel a hot spray from their pygidial glands (1, 2). The spray contains p-benzoquinones (3), chemical irritants commonly employed by arthropods (4). However, bombardier beetles are unique in using an internal explosive chemical reaction to simultaneously synthesize, heat, and propel their sprays (2, 3). The spray dynamics have been investigated by high-speed photography of the spray, spray impact force measurements, recordings of explosion sounds, and simulations (57). Species in the tribe Brachinini (brachinines) achieve spray temperatures of ~100°C (2), with ranges of several centimeters (1) and velocities of ~10 m/s via a “biological pulse jet” (5), where the spray consists of a rapid succession of pulses formed in discrete explosions. Pulse repetition rates of 368 to 735 Hz were measured from audio recordings for Stenaptinus insignis (5).

Fig. 1 Brachinus elongatulus pygidial gland morphology.

(A) Dorsal view. Dashed circle indicates location of pygidial glands. (B) Female (top) and male (bottom) pygidial glands: optical micrographs, chlorazol black staining (left) and SEM (right). Features are indicated: reservoir chamber (RSC), reaction chamber (RXC), exit channel (EC), and reflector plate (RP). (C) Female pygidial glands stained as in (B) showing rigid (highly sclerotized, brown/tan) and flexible (lightly sclerotized, stained blue) regions. Lightly sclerotized regions are identified: reaction chamber midline crease (white arrow); junction between reaction chamber and exit channel (purple arrow); exit channel dorsal membrane (yellow arrow). (D) False-color SEM showing valve muscles (VM), interchamber valve (ICV), and expansion membrane (EM). Other features labeled as in (B). Cross section shown in (E) is approximately normal to dashed line. (E) False-color SEM of cross section through interchamber region. The interchamber valve is observed in a closed conformation. Labels and colorization correspond to (D), with additional indication for the valve opening (VO).

It is well known that brachinines’ ability to produce internal explosions is facilitated by the two-chambered construction of their pygidial glands (3) (Fig. 1, B to E). Each of the beetle’s two pygidial glands comprises a reservoir chamber (RSC), reaction chamber (RXC), and exit channel (EC), which vents near the abdomen tip (Fig. 1B). The distal ends of the exit channels curve dorsally to form reflector plates (Fig. 1B, RP) used for spray aiming (8). An interchamber valve (Fig. 1, D and E, ICV) is contiguous with the walls of the reaction and reservoir chambers and separates the chambers’ contents when closed (2). The pygidial glands are constructed of cuticle, a composite of chitin, proteins, and waxes (9) that protects the beetle from the toxic chemicals, high temperatures, and high pressures during explosions. The muscle-enveloped, flexible reservoir chamber (5) stores an aqueous reactant solution of ~25% hydrogen peroxide and ~10% p-hydroquinones (3), along with ~10% alkanes as a nonreactive second liquid phase (10). Valve muscles (Fig. 1D, VM) span between the valve and the reservoir chamber to facilitate valve opening. During spray emission, reactant solution flows from the reservoir chamber into the reaction chamber, where it reacts with a solution of peroxidase and catalase enzymes (11) to form p-benzoquinones and explosively liberate oxygen gas, water vapor, and heat, propelling a hot, noxious spray out the exit channel.

The mechanism of brachinines’ spray pulsation has not been understood because previous studies, relying on external observations, have not probed internal dynamics. Here, we investigate this open question through optical and scanning electron microscopy to obtain new insights into the pygidial gland anatomy and synchrotron x-ray imaging (1216) at up to 2000 frames per second (fps) to directly observe the internal dynamics of spray pulsation in live beetles (Brachinus elongatulus) (17). These experiments provide an understanding of how explosions are initiated inside the pygidial glands and allow identification of the specific gland structures that mediate spray pulsation. An understanding of how brachinine pygidial glands produce (and survive) repetitive explosions could provide new design principles for technologies such as blast mitigation and propulsion.

Optical microscopy reveals that the reaction chamber exhibits dramatic spatial heterogeneity in cuticle sclerotization (Fig. 1C), corresponding to regions with different flexibility/rigidity (18) and, presumably, functional importance. The cuticle of most of the reaction chamber is tan or brown, implying heavy sclerotization and therefore high stiffness, which would serve to limit wall deflection and protect the beetle’s internal tissues from the explosions. However, several regions are colorless (stained blue in Fig. 1, B and C, to increase contrast) and, hence, lightly sclerotized and compliant. These regions include the reaction chamber’s dorsal midline crease and the junction between the reaction chamber and the exit channel (Fig. 1C). Similarly, the dorsal part of the exit channel is membranous and lightly sclerotized, whereas the ventral part is thick and heavily sclerotized (Fig. 1C) (6). Scanning electron microscopy (SEM) of the interchamber region in cross section (Fig. 1E) reveals that the cuticle that connects the valve to the dorsal part of the reaction chamber [hereafter called the expansion membrane (EM)] is very thin (~200 nm) and wrinkled, suggesting high flexibility.

Vapor formation during each explosion is clearly seen in the x-ray video as a bright region within the reaction chamber (Fig. 2A and movie S1). In the first pulse, vapor forms in the reaction chamber and propagates toward the exit channel. With each subsequent pulse, the vapor pocket initially expands slightly within the reaction chamber (implied by increased area) and then quickly contracts slightly as gas is ejected (Fig. 2A, first five pulses shown). Average pulsation rates calculated for 35 instances of gland activity from 18 sprays (median number of explosions, 13; range, 2 to 46) ranged from 341 to 976 Hz (median, 667 Hz; mean ± SD, 698 ± 146 Hz) (fig. S1 and table S2). A linear fit to active time versus number of pulses predicts a pulsation rate of 650 Hz (R2 = 0.88). These results are consistent with external experimental measurements of S. insignis (5) and approach the maximum rates reported for cyclic insect motions such as wing beats, measured as high as 1000 Hz for midges (19).

Fig. 2 Internal dynamics revealed by x-ray imaging.

(A) First five pulses of a spray; successive frames from 2000-fps video of a male beetle. Scale bar is 200 μm. Location of right reaction chamber (RXC) and exit channel (EC) indicated in frame 4. Right and left exit channels are open starting in frames 4 and 11, respectively. Arrows indicate dramatic displacement of the expansion membrane. Dark objects at left are external debris. (B) Reactant droplet (arrow) entering reaction chamber and exploding; successive frames from 2000-fps video of a male beetle. Scale bar is 200 μm.

Each explosion corresponds to the injection of a reactant droplet into the reaction chamber, which can sometimes be seen as a dark circle in relief against bright vapor (Fig. 2B and movie S2). Maximum diameters measured 208 ± 7 μm (mean ± SD) for four clearly visualized droplets. Assuming sphericity, the droplet volume is calculated to be 4.7 ± 0.5 nL, and the mass is estimated as 5.5 ± 0.6 μg. Based on the theoretical heat of reaction of 0.8 J/mg (2), the estimated energy release for each explosion is 4 × 10−3 J, and this energy liberates heat, boils water, and to a lesser extent provides the kinetic energy of the spray pulse. Estimating the spray pulse mass as equivalent to the droplet mass and taking 10 m/s for the spray exit velocity (5), the kinetic energy of a spray pulse is calculated to be 3 × 10−7 J. Equating this energy to work done by pressure, the average overpressure in the reaction chamber is estimated as 20 kPa, producing wall tensile stresses of ~1 MPa. For comparison, cuticle tensile strengths are typically tens to hundreds of megapascals (20). The time required to expel a pulse is estimated as 0.1 ms from the spray velocity and gland dimensions, consistent with the fact that explosions typically occur within single 2000-fps video frames (0.5 ms).

During each explosion, vapor is observed to fill a convex region between the reservoir and reaction chambers (Fig. 2A) that exceeds the dimensions of the reaction chamber indicated by microscopy (fig. S2), suggesting outward displacement of the expansion membrane driven by the explosion overpressure. Using the convex vapor shape as a proxy, the stretched expansion membrane can be modeled as a hemi-ellipsoid (fig. S2), and its maximum extension is found to be ~280% (see the supplementary text). For comparison, some insect cuticles exhibit recoverable extensions of 1000% (21). Based on the estimated overpressure and the estimated mass of the hemolymph displaced as the expansion membrane displaces into the body cavity, the expansion occurs with a maximum velocity of 6 m/s, attaining maximum displacement in 0.06 ms (supplementary text), consistent with the observation that expansion occurs within one video frame (0.5 ms). About one video frame after expansion is observed, the explosion reaction stops and vapor in the interchamber region contracts (e.g., Fig. 2A, frame 16), implying that the expansion membrane has returned to its unexpanded shape.

The exit channel of an active gland remains vapor-filled, and therefore open, throughout the entire pulse cycle (Fig. 2A and movies S1 to S3), possibly due to shape or mechanical characteristics (e.g., viscoelasticity) of its dorsal membrane, indicating that control of spray pulsation is accomplished by the reaction chamber inlet structures alone through opening and closing of the interchamber valve, as hypothesized previously (5). Typical cyclic mechanisms in insects (e.g., flapping flight and tymbal sound production) use multiple muscle sets that alternately contract or cuticular structures serving as springs (22), whereas the bombardier beetle possesses only valve-opening muscles and the valve is contiguous with flexible structures on all sides (i.e., reservoir chamber and expansion membrane). Hence, valve closure during each pulse cycle likely occurs passively due to mechanical feedback from the explosion, such as dynamic pressure from fluid (hemolymph) displaced by the expansion membrane, or impingement of the pressurized expansion membrane directly onto the valve, or a combination of both. Simple kinematics models of these scenarios incorporating valve dimensions, the vapor expansion profile, and estimated overpressure discussed above predict forces that are sufficient to close the valve (supplementary online text).

Once the spray pulse is released and the overpressure in the reaction chamber drops, the load on the valve is removed, allowing it to reopen and permit a fresh reactant droplet to enter. It is not known whether the valve-opening muscles contract continually for the duration of spraying or once per pulse cycle, but both scenarios are compatible with passive valve closure and the capabilities of insect muscles (19).

The data presented suggest the following mechanism for spray pulsation (Fig. 3): The reservoir chamber musculature contracts for the duration of spraying to apply a continuous pressure to the reactant solution, and the valve muscles also contract, opening the interchamber valve and forcing a reactant droplet into the reaction chamber (Fig. 3B). The droplet explodes upon contacting the reaction chamber enzymes (Fig. 3C), producing high-pressure vapor that propels a spray pulse out of the exit channel. Explosion overpressure displaces the expansion membrane and closes the interchamber valve, thereby interrupting the flow of reactants. After the explosion, the pressure in the reaction chamber decreases, the expansion membrane relaxes, the valve reopens, and a fresh reactant droplet enters, starting a new pulse cycle (Fig. 3D). Eventually, the reservoir and valve muscles relax, causing spraying to cease. The exit channel’s dorsal membrane relaxes and collapses into its ventral trough, and some quantity of vapor generally remains in the reaction chamber as a pocket surrounded by numerous bubbles (Fig. 3E).

Fig. 3 Mechanism of spray pulsation.

Schematics depict a sagittal section through the middle of a pygidial gland; this perspective is orthogonal to the accompanying x-ray images selected from movies S1 and S2. Scale bars are 200 μm. Reservoir chamber (RSV), reaction chamber (RXC), exit channel (EC), interchamber valve (ICV), and expansion membrane (EM) are indicated. (A) Gland is inactive. (B) Spray initiation. Reactant solution enters through valve. (C) Explosion ongoing. Displacement of expansion membrane closes the valve. A spray pulse is ejected. (D) Explosion ceases. Expansion membrane relaxes and valve reopens, permitting fresh reactant solution to enter. The process repeats C-D-C-D-C-D, and so on, with each “C-D” corresponding to one pulse cycle. (E) Spraying concluded. The exit channel closes and a vapor pocket remains in the reaction chamber.

The pulsed spray mechanism of brachinine bombardier beetles is remarkably elegant and effective, protecting these beetles from nearly all predators (and incautious humans). The passive mediation of pulsation by mechanical feedback from the explosion is advantageous because it provides automatic regulation of reactant use. Further, the evolutionary change from a continuous defensive spray (exhibited by close relatives of the brachinines) to a pulsed spray required only relatively minor changes to the reaction chamber inlet structures rather than the evolution of novel valve-closing muscles.

Supplementary Materials

www.sciencemag.org/content/348/6234/563/suppl/DC1

Materials and Methods

Supplementary Text

Figs. S1 to S3

Tables S1 and S2

Movies S1 to S3

References (2332)

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract DE-AC02-06CH11357. This work was supported in part by the U.S. Army Research Laboratory and the U.S. Army Research Office through the MIT Institute of Soldier Nanotechnologies under contract W911NF-13-D-0001 and in part by the National Science Foundation through the MIT Center for Materials Science and Engineering under contract DMR-08-19762. This research was funded in part by the U.S. Department of Defense, Office of the Director, Defense Research and Engineering, through the National Security Science and Engineering Faculty Fellowship awarded to C.O. under contract N00244-09-1-0064; in part by the National Science Foundation through funding awarded to W.M. under contract DEB-0908187; and in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract DE-SC0012704. Experiment data are available for download from DSpace@MIT (https://dspace.mit.edu); please cite this collection using the handle hdl.handle.net/1721.1/96123.
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