Molecular basis of the explosive defence response in the bombardier beetle Brachinus crepitans

Abstract

Bombardier beetles have evolved a sophisticated and unique chemical defence mechanism involving controlled explosions within their paired defensive glands, producing a hot, benzoquinone-rich defensive spray. The molecular basis of this response is not well characterized. We therefore combined the transcriptomic and proteomic analysis of different gland compartments in the bombardier beetle Brachinus crepitans (Linnaeus, 1758) (Coleoptera, Carabidae) to identify abundant transcripts and gland-specific proteins with key defensive functions, such as catalases, peroxidases and enzymes involved in hydroquinone synthesis. By combining precise dissections with protein sequence analysis, we built a comprehensive atlas of the relevant proteins and their spatio-temporal organization. We found that glucose is important as a stable precursor of hydrogen peroxide and hydroquinone. These chemicals, together with gland-specific peroxidases and catalases, then initiate the explosive defence reaction. We also present evidence that the evolution of explosive secretions involved the functional adaptation of peroxidase genes involving atypical substitutions in otherwise highly conserved protein domains.

Keywords: Brachinus crepitans; bombardier beetles; chemical defence; hydrogen peroxide; hydroquinone; omics.

Figure 1.

Explosive defence reaction of the bombardier beetle Brachinus crepitans and its dissected defensive glands. (A) The defence secretion is ejected as a jet of fine mist without visible dissipation (red arrowheads) in a rapid series of consecutive pulses. Part of the secretion was deflected during an earlier pulse onto the bottom of the plate, and is visible as a puddle (white arrowheads). (B) Higher magnification highlights the precision with which the beetle orientates the jet towards the contact point with forceps (black arrowhead). (C) Freshly dissected and untreated defensive glands of a female B. crepitans specimen. MGS = multi-lobed glandular system, CD = collection duct, RC = reservoir chamber, RXC = reaction chamber.

Figure 2.

Proposed molecular basis of defensive gland secretions in the bombardier beetle B. crepitans based on combined transcriptomic and proteomic analysis. The principal reactions in the MGS with gland (green), RC (yellow) and RXC (red) are shown in black, with candidate genes identified in the transcriptome and the corresponding proteins identified by proteomic analysis shown in blue. The circles show additional abundant proteins identified in the corresponding parts of the defensive gland. More detailed descriptions of the proposed pathways are provided in table 1.

Figure 3.

SDS-PAGE analysis of defensive gland fractions and spray proteins from the bombardier beetle B. crepitans. The defensive gland was dissected and proteins were extracted separately from the MGS, RC (Reservoir) and RXC (ExpChamb), as well as from the defensive spray (Spray). Following SDS-PAGE, each gel lane was cut into 30 segments (horizontal red lines) for tryptic digestion and proteomic analysis. M1 and M2 are protein marker sets of different sizes, with the indicated molecular masses.