Storage and release of hydrogen cyanide in a chelicerate (Oribatula tibialis)

Abstract

Cyanogenesis denotes a chemical defensive strategy where hydrogen cyanide (HCN, hydrocyanic or prussic acid) is produced, stored, and released toward an attacking enemy. The high toxicity and volatility of HCN requires both chemical stabilization for storage and prevention of accidental self-poisoning. The few known cyanogenic animals are exclusively mandibulate arthropods (certain myriapods and insects) that store HCN as cyanogenic glycosides, lipids, or cyanohydrins. Here, we show that cyanogenesis has also evolved in the speciose Chelicerata. The oribatid mite Oribatula tibialis uses the cyanogenic aromatic ester mandelonitrile hexanoate (MNH) for HCN storage, which degrades via two different pathways, both of which release HCN. MNH is emitted from exocrine opisthonotal oil glands, which are potent organs for chemical defense in most oribatid mites.

Keywords: Oribatida; Oribatula tibialis; chemical defense; cyanogenesis; toxin.

Fig. 1.

GC/MS (A) chromatograms of oil gland secretions of Oribatula tibialis and HPLC (B) chromatograms of mandelonitrile hexanoate (MNH) hydrolysis. (A) Black: profile of secretions from undisturbed mites; orange: profile of slightly disturbed mites (brush stimulus); green: profile of strongly disturbed mites (vortex mixer). (B) Lilac: synthetic MNH; blue: in vitro hydrolysis of synthetic MNH; red: in vivo hydrolysis of natural MNH. Yellow bands highlight compounds involved in cyanogenesis.

Fig. 2.

Expulsion and degradation of MNH from opisthonotal oil glands in the oribatid mite, Oribatula tibialis. Pathway 1: MNH is cleaved by a catalytic oxidation to benzoyl cyanide and hexanoic acid on the mite´s body surface. Subsequently, benzoyl cyanide hydrolyzes to benzoic acid and HCN. Pathway 2: Direct hydrolysis of the ester bond in MNH in the presence of moisture, resulting in the release of HCN, benzaldehyde, and hexanoic acid.

Fig. S3.

Synthetic MNH: complete 500 MHz ¹H NMR in CDCl₃ (ref. 7.20 ppm) of MNH. *, Byproduct contaminations (hexane, carboxylic acid derivatives).

Fig. S4.

Synthetic MNH: ¹H NMR only showing expanded ranges of essential signals. *, Byproduct contaminations (hexane, carboxylic acid derivatives).

Fig. S5.

Synthetic MNH: Distortionless enhancement by polarization transfer (DEPT) 135 (A) and broadband decoupled (B) ¹³C NMR spectra (ref. CDCl₃ 77.2 ppm). The DEPT spectrum shows a negative phase for the CH₂ signals.

Fig. S6.

Synthetic MNH: 2D NMR, ¹H-¹³C-heteronuclear single quantum correlation (HSQC) (A) and ¹H-¹³C-heteronuclear multiple-bond correlation (HMBC) (B) spectra show the hydrogen–carbon connectivity over one and two or three covalent bonds, respectively.

Fig. S1.

Mass spectra of mandelonitrile hexanoate (MNH) extracted from the oil glands of Oribatula tibialis (A) and from synthetic MNH (B).

Fig. S2.

Structural formula of MNH with observed NMR shift values for ¹H (upper values) and ¹³C (lower values). Peak multiplicity: d, doublet; m, multiplet; quint, quintet; s, singlet; t, triplet. Differences between predicted and experimental chemical shifts (in ppm) based on the evaluation report from the CSEARCH database (nmrpredict.orc.univie.ac.at/c13robot/robot.php): 1 (0.1 ppm), 2 (0.1 ppm), 3 (3.2 ppm), 4 (3.0 ppm), 5 (0.7 ppm), 6 (0.7 ppm), 7 (1.6 ppm), 8 (1.3 ppm), 9 (1.2 ppm), 10 (0.1 ppm), 11 (0.3 ppm), 12 (1.0 ppm).