Storage and release of hydrogen cyanide in a
chelicerate (Oribatula tibialis)
a,1
b,1
a
c
d
Adrian Brückner , Günther Raspotnig , Katja Wehner , Reinhard Meusinger , Roy A. Norton ,
a,2
and Michael Heethoff
a
c
b
Ecological Networks, Technische Universität Darmstadt, 64287 Darmstadt, Germany; Institute of Zoology, University of Graz, 8010 Graz, Austria;
d
Clemens–Schöpf–Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, 64287 Darmstadt, Germany; and College of
Environmental Science and Forestry, State University of New York, Syracuse, NY 13210
Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved February 17, 2017 (received for review November 4, 2016)
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 pre-
vention 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.
Results and Discussion
Oil gland secretions of undisturbed O. tibialis contained
β-pinene, octanoyl hexanoate, MNH, and an unknown compound
of molecular weight M = 162 g/mol (Fig. 1A). Of these com-
r
pounds, only the aromatic ester MNH is involved in cyanogenesis.
After gentle mechanical disturbance, specimens of O. tibialis
showed a reduced amount of MNH and the presence of an ad-
ditional compound, not detected in undisturbed mites: benzoyl
cyanide (Fig. 1A). In intensely disturbed specimens, only traces of
MNH were measured, but equimolar amounts of benzoyl cyanide
and a mixture of benzoic acid and hexanoic acid were detected.
Direct contact with moisture resulted in hydrolysis of MNH to
hexanoic acid, benzaldehyde, and HCN (Fig. 1B).
chemical defense
| cyanogenesis | Oribatida | Oribatula tibialis | toxin
Release of Hydrogen Cyanide by MNH Degradation. The detected
compounds indicate that HCN is released, and we propose the fol-
lowing two degradation pathways of mandelonitrile hexanoate (Fig. 2).
Pathway 1. Mechanical disturbance of O. tibialis results in the
active expulsion of oil gland contents, including MNH. MNH is
cleaved and catalytically oxidized to benzoyl cyanide and hex-
anoic acid on the mite’s body surface. These two products are
measurable quickly after disturbance in mite whole-body ex-
tracts, indicating rapid and spontaneous chemical reactions. The
mechanism of this breakdown is unknown, but it has been shown
that mites possess surface-associated enzymes that contribute
to previously unsuspected chemical reactions (29–31). Finally,
when exposed to water in the humid environment, benzoyl cya-
nide hydrolyzes to benzoic acid and HCN (32, 33).
hemical substances are of utmost importance in biotic in-
C
teractions among plants and their herbivores/pathogens as
well as among animals and their predators/parasites (1, 2). Many
of these semiochemicals are emitted for defense, and one of the
most deterring and toxic biogenic substances known is hydrogen
cyanide (HCN, also known as hydrocyanic or prussic acid). This
asphyxiant poison inhibits the cytochrome oxidase enzyme,
resulting in the inability of organisms to use oxygen (3).
Biosynthesis and liberation of HCN (known as cyanogenesis)
is widespread among plants, but in animals it is relatively rare.
Whereas the earliest reports of HCN in arthropods are from the
late 19th century (4), it was only in the early 1960s (5–8) that
comprehensive chemical ecology studies began to reveal cyano-
genesis as a defensive strategy of a few mandibulate arthropods,
including certain species of myriapods, beetles, true bugs, and
butterflies (2, 9–12). More recently, the genomic basis of cya-
nogenesis has been explored (13, 14).
Significance
Hydrogen cyanide (HCN) is highly volatile and among the most
toxic substances known, being lethal to humans at a dosage of
The rarity of cyanogenesis in mandibulate arthropods (2) and
its supposed absence in the other speciose arthropod subphylum,
Chelicerata, may relate to the evolutionary challenge posed by
using a universal toxin in defense: self-poisoning must be prevented
by storing the highly volatile HCN as a safe carrier molecule or
storage molecule. In case of threat or attack, the cyanogenic com-
pounds are discharged and must be quickly degradable to release
HCN. Known HCN storage compounds of mandibulate arthro-
pods include aromatic or aliphatic glycosides, lipids, and cya-
nohydrins (e.g., mandelonitrile) (2, 12, 15).
1
–2 mg/kg body weight. HCN blocks the respiratory chain and
prevents aerobic organisms from using oxygen. In nature, HCN
is produced by numerous plants that store it mainly as glyco-
sides. Among animals, cyanogenesis is a defensive strategy
that has seemed restricted to a few mandibulate arthropods
(
certain insects, millipedes, and centipedes), which evolved
ways to store HCN in the form of stable and less volatile mol-
ecules. We found an instance of cyanogenesis in the phyloge-
netically distant group Chelicerata (“spider-like” arthropods),
involving an aromatic ester for stable HCN storage and two
degradation pathways that release HCN.
Although no cyanogenic species has been known among Cheli-
cerata, chemical defense is widespread in the group, particularly
among arachnids such as whip scorpions (16, 17), harvestmen (18,
Author contributions: A.B., G.R., and M.H. designed research; A.B., G.R., K.W., and R.M.
performed research; A.B., G.R., K.W., R.M., and M.H. analyzed data; and A.B., G.R., K.W.,
R.M., R.A.N., and M.H. wrote the paper.
1
9), certain spiders (20), and mites (21, 22). Here, we demonstrate
cyanogenesis in a mite of the order Oribatida, a diverse and mostly
soil-dwelling group of decomposers that discharge myriad defense-
related semiochemicals from a pair of large exocrine opisthonotal
oil glands (22–28). The common and widespread species Oribatula
tibialis stores HCN as the natural product mandelonitrile hex-
anoate (MNH) and releases HCN upon disturbance via two
different chemical pathways.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
A.B. and G.R. contributed equally to this work.
2
PNAS Early Edition
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