Herbivore Adaptations to Plant Cyanide Defenses

4.1. Sulfur transferases

Sulfur transferases such as the rhodanese (thiosulfate:cyanidesulfurtransferase, EC 2.8.1.1; see Figure 2A), and its close relative, the 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2), are enzymes described in plants, fungi, bacteria and a wide range of animals including snails, insects, fish and mammals (see Figure 3) [52–58]. Enzymatic formation of thiocyanate, the so-called rhodanide, was first described in 1933 using vertebrate tissues as discussed by Lewis [59]. Rhodaneses from mammals have been investigated most thoroughly and most insight has been gained from the examination of human and bovine liver rhodaneses [60, 61]. These two enzymes served to uncover the first protein structure of a rhodanese which revealed two similarly folded “rhodanese domains” [62]. In contrast to the highly similar tertiary structure, the two domains differ strongly in their amino acid sequences in agreement with their divergent functions as C-terminal catalytical and N-terminal regulatory domains [62]. At the level of primary structure, rhodaneses from different organisms show little similarity apart from two conserved, 11–13 amino acids long “signature” regions at the N- and C-termini which are also present in distantly related proteins of the rhodanese superfamily such as cdc25 phosphatase and heat shock proteins [63]. This low identity at the amino acid level and the involvement of single rhodanese domains in aberrant proteins make homology-based identification of rhodaneses from further species difficult. To specify the sulfur transferases involved in cyanide detoxification among the members of the diverse superfamily, the tertiary structure has to be taken into account, classifying the true rhodaneses as tandem domain thiosulfate:cyanide sulfurtransferases [63]. This group also comprises the 3-mercaptopyruvate sulfurtransferases. Both types of enzymes have distinct substrate and product spectra, but are yet interconvertible by few amino acid substitutions [64].

Rhodaneses do not only accept thiosulfate as sulfur donor, but all sulfane anions such as organic sulfanes and persulfides [55, 65]. Next to cyanide, the sulfur atom may be accepted by other thiophilic substrates such as the amino acids cysteine and glutathione [65]. The kinetic mechanism of rhodanese was uncovered with its classical substrates cyanide and thiosulfate by Westley and coworkers [60, 66]. In a ping-pong reaction, the sulfane sulfur atom is abstracted from the donor substrate thiosulfate and bound to a cysteine residue in the active site of rhodanese [62]. This is followed by entrance of the acceptor substrate cyanide into the active site and transfer of the sulfur atom [60, 66]. In contrast, the reaction of 3-mercaptopyruvate sulfurtransferase (which can also convert cyanide to thiocyanate [67]) follows a sequential mechanism with formation of a ternary complex composed of the enzyme and both substrates (3-mercaptopyruvate and cyanide) [68, 69].

A main function of the rhodaneses in cyanide detoxification is in agreement with their subcellular localization. Rhodanese activity is predominantly detected in the mitochondria, the site of cellular respiration with the cyanide-susceptible cytochrome c oxidase [70, 71]. Nevertheless, for 3-mercaptopyruvate sulfurtransferase and rhodanese of some species, an additional localization in the cytosolic fraction has been described [55, 71, 72]. The cytosolic enzymes may serve to reduce cyanide levels in this compartment whose components (glycolysis intermediates, proteins) may also be affected by cyanide poisoning (see above) [48]. The reaction product of rhodanese-catalyzed cyanide detoxification, thiocyanate, possesses a toxic potential toward mitochondria which could be an additional reason why rhodanese isoforms are also localized in the cytosol in several species.

Although a major role of rhodaneses in cyanide detoxification seems likely based on the present knowledge, cyanide detoxification might not be their exclusive physiological function. The ubiquitous occurrence of rhodaneses in organisms and tissues with no obvious cyanide exposure as well as the low physiological concentration of their substrate thiosulfate in the mitochondria has fueled doubts about their main role in cyanide detoxification [73]. In support of a major role in cyanide detoxification in mammals, rhodanese activity is inducible in rats by exposition to cyanide or supplementation with thiosulfate [74, 75]. In vivo, sulfane substrates may be provided by the action of an enzyme involved in cysteine metabolism in animals, cystathionine-γ-lyase [76]. Further and alternative functions of rhodaneses have been discussed. As plant homologs have been shown in vitroto supply sulfide for iron sulfur clusters of the electron transport chain, this has been suggested as a general function of rhodaneses from other taxa [73, 77]. Alternatively, a general role of rhodaneses in the regulation of sulfur homeostasis has been proposed based on their low substrate specificity. The in vivofunction of 3-mercaptopyruvate sulfurtransferases in cyanide detoxification has been questioned not least because addition of 3-mercaptopyruvate led to an antidote effect only in some mammal species [78]. Additionally, it has been difficult to separate their possible direct and indirect contributions to cyanide detoxification in vivo, as 3-mercaptopyruvate sulfurtransferase can also donate sulfur for the formation of the rhodanese substrate thiosulfate [79].

4.2. β-Cyanoalanine synthase

The main enzyme of cyanide detoxification in plants and many bacteria, β-cyanoalanine synthase (EC 4.4.1.9, Figures 2B and 3), belongs to the family of β-substituted alanine synthases sharing the cofactor pyridoxal-5′-phosphate and a uniform fold [80, 81]. This family also comprises O-acetylserine thiol lyase and cystathionine-β-synthase which occur in bacteria, plants, fungi and animals and are involved in cysteine biosynthesis from O-acetylserine or serine and homocysteine, respectively [80, 82]. The amino acid sequence of these enzymes and their tertiary structure, the so-called fold II of the pyridoxal-5′-phosphate-dependent enzymes, are highly conserved. However, the members of the protein family vary in the quaternary structure, as mono-, di-, tetra- and oligomers have been reported, although dimers are most predominant [80, 83, 84]. Some members of the family can catalyze several of the above-mentioned reactions making a classification as β-cyanoalanine synthase or O-acetylserine thiol lyase difficult. Thus, plant enzymes are mainly assigned to an enzyme clade based on a comparison of kinetic characteristics for the diverse reactions catalyzed and their subcellular localization [80]. The only phyla of multicellular animals in which β-cyanoalanine synthase activity has been demonstrated up to now are Nematoda and Arthropoda (see Figure 3) [14, 85–87]. Only few animal β-substituted alanine synthases with β-cyanoalanine synthase activity have been identified to date (see below). As the protein family’s name indicates, β-substituted alanine synthases catalyze a substitution or elimination reaction at the β-carbon of proteinogenic and nonproteinogenic amino acids. Their reaction mechanism has been examined mainly on the basis of prokaryotic and plant O-acetylserine thiol lyases [88–90]. All examined β-substituted alanine synthases share a common fold and perform a similar conformational change upon substrate binding to close the active site for catalysis [90]. In the active site, a Schiff basebinding between the cofactor pyridoxal-5′-phosphate and a catalytic lysine residue awaits the substrate amino acid [90]. Upon entrance of the substrate, pyridoxal-5′-phosphate is transferred from the lysine residue to the α-amino group of the substrate as an external Schiff base. Lysine’s free amino group now acts as a base catalyst for the deprotonation of the α-carbon, inducing the α,β-elimination of sulfide, acetate or water from the substrate amino acid [88, 90]. The formed α-aminoacrylate intermediate is shared by all members of the β-substituted alanine synthase family regardless of the catalyzed reaction [88]. After formation of this intermediate, the second substrate enters the active site. It attacks the amino acid’s side chain nucleophilically. Facilitated by acid catalysis by the protonated lysine residue, a bond between the β-carbon and the second substrate is formed. The newly formed amino acid product can then be released from the active site under reformation of the internal Schiff base between pyridoxal-5′-phosphate and lysine [90]. Because of high conservation in the reaction mechanism of different β-substituted alanine synthases, an O-acetylserine thiol lyase and a β-cyanoalanine synthase from soybean were analyzed for their substrate and product spectra by mutagenesis studies [90]. This showed that the exchange of only three amino acids can switch an O-acetylserine thiol lyase to a β-cyanoalanine synthase, illustrating their close relationship and giving insight into the evolution of β-substituted alanine synthases [90].

Next to amino acid biosynthesis, β-substituted alanine synthases are involved in cellular sulfur and redox homeostasis [84, 91–93]. Cyanide detoxification by these enzymes is mainly catalyzed by β-cyanoalanine synthases, but O-acetylserine thiol lyases can also bind the toxic ion to either cysteine or O-acetylserine [81]. According to enzyme activity assays, β-cyanoalanine synthases from plants and animals are mainly localized in the mitochondria [86, 94]. However, the recently identified arthropod β-cyanoalanine synthases do not seem to possess a mitochondrial signal sequence [87, 95]. Cytosolic expression might be beneficial as cyanide detoxification by β-cyanoalanine synthases leads to the formation of equimolar amounts of sulfide which itself is an inhibitor of cytochrome c oxidase. If released in the cytosol (instead of the mitochondria), sulfide can immediately be captured by the cytosolic O-acetylserine thiol lyases. In plants, experiments with T-DNA insertion mutants have shown that cytosolic O-acetylserine thiol lyases are essential for safe and efficient disposal of cyanide [92].

β-Cyanoalanine itself may also exert harmful effects. It has been identified as a neurotoxin and can also be lethal to plants [96, 97]. In order to protect themselves from poisoning with β-cyanoalanine and to minimize costs, plants and microorganisms are able to turn over β-cyanoalanine by nitrile hydratases and nitrilases [98]. Nitrile hydratases catalyze the addition of a water molecule to β-cyanoalanine leading to the formation of the proteinogenic amino acid asparagine. Nitrilases convert β-cyanoalanine to the proteinogenic amino acid aspartate by addition of two water molecules [99]. In addition, the conversion of β-cyanoalanine to asparagine with γ-glutamyl-β-cyanoalanine as an intermediate has been described for some plants [96]. Recycling of β-cyanoalanine has also been shown in some arthropod species (see below). Due to its neurotoxic effect, β-cyanoalanine stored in the defensive droplets of the cyanogenic lepidopteran species Z. filipendulaehas also been discussed to directly act as a defensive compound for the protection of this herbivore against predators [100].

5.1. Cyanide detoxification in mammalian herbi- and omnivores

In mammals, rhodanese is generally believed to be the major enzyme for cyanide detoxification, while β-cyanoalanine synthase activity has not been detected in a mammal so far. A comparison of rhodanese activity between mammalian herbi-, omni- and carnivores shows highest activities in herbivores, especially in ruminants which feed on a broad range of plant material including plants with high cyanide potential [58]. In several mammalian species such as plant-feeding rabbits, rhodanese activity is ubiquitously distributed in the body with the highest activity in hepatocytes, the main detoxification site of e.g. xenobiotics [101, 102]. Rhodanese activity is also localized in the mammalian brain, where cyanide acts as a neuromodulator [63].

5.2. Cyanide detoxification in invertebrates

Intensive research on mammalian rhodaneses also raised the question whether these enzymes are involved in cyanide detoxification in other animals. Rhodanese activity is widely distributed in insects and occurs in snails (see Figure 3) [55, 103]. The level of activity is comparable to that of mammalian gut tissue [53]. However, activity levels are largely in the same range among herbivores which frequently or rarely encounter high cyanide levels. The basal rhodanese activity might be sufficient to capture dietary cyanide in herbivores regardless of the cyanide level in the diet. Alternatively, the uniform distribution of rhodanese activity among herbivores could indicate that arthropod rhodaneses possess an additional function unrelated to cyanide detoxification. This would likely require other mechanisms of cyanide detoxification such as β-cyanoalanine synthase activity [103]. In agreement with this, β-cyanoalanine activity has been found to be broadly distributed in arthropod herbivores (see Figure 3) [86, 95, 104]. In support of the activity data, labeled β-cyanoalanine can be detected in arthropods after feeding of or exposition to isotopically labeled cyanide [30, 105]. Further support for the relevance of β-cyanoalanine synthases comes from experiments with several cyanide-forming lepidopteran species in whose defensive glands β-cyanoalanine and its hydration product asparagine were detected [106]. In millipedes, β-cyanoalanine synthase-catalyzed detoxification of cyanide and further metabolism of β-cyanoalanine to asparagine was demonstrated by studies with radiolabeled precursors [14, 107]. For insects, a similar utilization of cyanide for the formation of proteinogenic amino acids using β-cyanoalanine as an intermediate has been discussed [18], but has only be proven for one beetle by radioactive feeding experiments so far [108]. In the beetle, the radioactive label was recovered from a polypeptide rich in aspartate, the product of nitrilase-catalyzed conversion of β-cyanoalanine [108].

In order to estimate the relevance of rhodanese vs. β-cyanoalanine synthase for the in vivodetoxification, several studies compared the enzyme activities within one species upon cyanide induction or in related species with or without cyanide specialization [53, 86]. Most of these studies are more than 20 years old, and they cover only a very limited number of species. Nevertheless, for rhodanese no induction of activity upon cyanide feeding was observed although the occurrence in Diptera species parasitizing the cyanogenic moth Z. filipendulaeindicates a connection with cyanide detoxification [109]. β-Cyanoalanine synthase activity was higher in cyanide-tolerant than in cyanide-sensitive species [86]. Three studies analyzed a broad spectrum of insect species either by measuring rhodanese enzyme activities or by determining β-cyanoalanine as an indicator of β-cyanoalanine synthase activity [103, 106, 110]. However, in addition to the difficulty to compare enzyme activity and metabolite level per se, several other factors render a statement on the in vivorelevance of β-cyanoalanine synthase and rhodanese for cyanide detoxification in insects impossible: the low number of replicates [103], the use of specimen of different developmental stages and from different origins [103, 110], quantification by thin-layer chromatography, and long-term storage of specimen before analysis despite decomposition of metabolites over time [106, 110]. Thus, conclusions on the quantitative contribution of the two pathways of cyanide detoxification in insects cannot be drawn from these experiments. Nevertheless, the studies showed that both enzymes have a broad distribution among insects and many species possess both activities [30, 55, 103, 104, 110, 111]. β-Cyanoalanine synthase seems to be most relevant to protect arthropod herbivores feeding on cyanide-defended plants.

Most β-cyanoalanine synthase activity data for animals were generated with intact, partly dried and stored animal tissues, but some enzymes have been purified and characterized [84, 104]. In the nematode Caenorhabditis elegans, identification of the first animal members of the β-substituted alanine synthase family that do not belong to the cystathionine-β-synthases was achieved at the molecular level [85]. Biochemical characterization of the enzymes proposed roles in the regulation of the metabolism and the detoxification of cyanide, sulfide and S-sulfocysteine [84]. Although the enzymes possess O-acetylserine thiol lyase activity in vitro, an in vivofunction in sulfur assimilation and cysteine biosynthesis is unlikely [84]. Further β-cyanoalanine synthases have been identified in the mite Tetranychus urticaeand larvae of P. rapae[87, 95]. These first molecular data indicate an astonishing evolutionary background. In phylogenetic analyses, the nematode enzymes group together with plant O-acetylserine thiol lyases [84, 85] indicating a common origin of β-cyanoalanine synthases of both kingdoms, that is, acquisition of β-cyanoalanine synthases by gene duplication and partial neofunctionalization of a common ancestor. In contrast, the β-cyanoalanine synthases from the mite and the butterfly show amino acid sequence similarity with bacterial sequences [87, 95]. Phylogenetic analyses group the mite and butterfly enzymes among bacterial homologs from α- and β-proteobacteria, distant to other metazoan β-substituted alanine synthases [87, 95]. Genetic analyses such as determination of the GC-nucleotide content and mapping of adjacent genomic DNA have shown that these sequences belong to the arthropods’ genome rather than to the genome of bacterial symbionts of the present species [87, 95]. As a likely explanation, the genes may have been acquired by horizontal gene transfer from bacteria, likely from symbiotic bacteria which lived in close association with ancestral arthropods [87]. Nevertheless, a common horizontal gene transfer event to arthropods seems to be unlikely as this would involve at least 13 independent gene losses each at a very specific point in the evolution [87]. Two independent gene transfer events from closely related bacterial donor species or a gene transfer from bacterium to mite followed by a second transfer event from an ancient mite to a Lepidopteran ancestor are discussed [87]. Interestingly, mites possess only one copy of the sequence in their genome, while some Lepidoptera seem to possess two or three copies [95], a phenomenon frequently observed for genes assimilated by horizontal gene transfer [112]. Horizontal gene transfer, in particular from bacteria and protists, but also from plants and fungi, has contributed gravely to metazoan evolution. Its remnants besides the β-cyanoalanine synthases can be found in tens to hundreds of examples in nematodes, arthropods and chordates and are involved in main metabolic pathways and responses to environmental influences [113].

In general, proteins involved in the adaptation of herbivores to their host plants and, in particular, those catalyzing the detoxification and transport of host plant xenobiotics are thought to be under narrow transcriptional regulation [114]. For the mite β-cyanoalanine synthase, an induction by cyanide exposure over 30 generations led to a transcriptional response allowing for the identification of the detoxification enzyme [87]. Thus, these enzymes are among the most variable ones and play a key role in the adaptation to and population of new host plants [114]. As the relationship between herbivore and host plant is close and evolution favors adapted defense of the plant in order to diminish resource and tissue loss through predation [115], transcriptional responses could be discovered also in the host plant [114]. This close coevolution between herbivores and their host plants has therefore shaped both partners and is likely to underlie the higher cyanide tolerance of specialist herbivores on cyanogenic plants [30, 115].

7.1. Occurrence of cyanogenic compounds in arthropods

Arthropods are the only phylum of animals in which biosynthesis or sequestration of cyanogenic compounds has been shown [15]. Within arthropods, the presence of cyanogenic glucosides appears to be restricted to millipedes (Diplopoda), centipedes (Chilopoda) and three orders within the Insecta (Lepidoptera, Coleoptera and Hemiptera) [128]. The Lepidoptera and Hemiptera are the only groups containing cyanogenic compounds with aliphatic side chains, while in the others groups of arthropods, cyanogenic compounds possess aromatic side chains [4, 129]. Among the most intensely studied species, larvae of Z. filipendulaespecialized on cyanogenic plants are able to de novobiosynthesize cyanogenic glucosides, but can also sequester cyanogenic glucosides from their food plants [4, 17, 130] (see Sections 7.2 and 7.3). Both de novobiosynthesis and sequestration of cyanogenic glucosides have also been reported for larvae of Euptoieta hegesia(Lepidoptera:Nymphalidae) based on the detection of cyanide after larval feeding on acyanogenic plants and an increase of cyanide formation upon transfer to cyanogenic glucoside-defended plants [131]. Species of the genus Heliconius are also able to synthesize and to sequester cyanogenic glucosides based on the pattern of compounds detected in the insects relative to those found in the food plants [124, 132]. In contrast to lepidopterans, millipedes do not store glycosides, but unglycosylated cyanogens, and do not sequester cyanide precursors from their diet as discussed in Ref. [133].

7.2. De novobiosynthesis of cyanogenic compounds in arthropods

First indications for de novobiosynthesis of cyanogenic glucosides in a herbivore came from experiments conducted by Jones and coworkers in 1962. They showed that larvae of Z. filipendulaerelease cyanide upon tissue disruption also when raised on food plants devoid of cyanogenic glucosides [109]. Feeding experiments with ¹³C-labeled valine and isoleucine showed the incorporation of the isotope label into linamarin and lotaustralin and thereby provided evidence for de novobiosynthesis of cyanogenic glucosides in Z. filipendulaeand the butterfly Heliconius melpomene(Lepidoptera:Nymphalidae) [132, 134]. The complete biosynthetic pathway of cyanogenic glucosides in Z. filipendulaewas elucidated in 2011 including pathway intermediates and involved enzymes [16, 17, 19, 134, 135]. This revealed high similarity to the biosynthesis of cyanogenic glucosides in plants with two cytochrome P450 enzymes catalyzing the conversion of the precursor amino acid to a cyanohydrin via an aldoxime and a glucosyltransferase responsible for the final glycosylation step (see Figure 4). Several butterfly species of the Papilionoidae also contain linamarin and, in part, lotaustralin in different life stages [128, 132, 136, 137] due to de novobiosynthesis rather than sequestration as the larval host plants, in most cases, do not form aliphatic cyanogenic glucosides [4].

Among arthropods, millipedes also contain cyanogenic compounds, namely cyanohydrins such as mandelonitrile, and use them as defense against predators as discussed by Shear [133]. Synthesis of cyanide and cyanohydrins such as mandelonitrile was demonstrated for different species of millipedes using feeding tests with ¹⁴C-phenylalanine and further radioactively labeled precursors [14, 107, 138]. As this resulted in labeling of phenylacetaldoxime and phenylacetonitrile as potential pathway intermediates, a biosynthesis pathway very alike the one described in plants and later in insects was proposed [14, 107, 138] (Figure 4). The unwanted release of cyanide is prevented by specifically shaped, two chamber glands where cyanide precursor and the hydrolyzing enzyme α-hydroxynitrile lyase are stored separately [107, 133]. In the α-hydroxynitrile chamber, organic acids generate low pH values to stabilize the α-hydroxynitrile [133]. Upon attack by a predator, gland secretions are mixed to generate cyanide. This mechanism allows the millipede to liberate cyanide in a controlled way, thereby economizing its chemical defense and protecting its own tissue from poisoning.

Although the presence of cyanogenic glucosides and, in part, their de novobiosynthesis has been described in selected species of the centipedes [139], millipedes and insects, and these phyla derive from a common ancestor, the absence of cyanogens in most insect species and the diversity of cyanogenic glucosides in combination with the long time span since their diversion (>390 MYA) [140] indicate that the biosynthetic pathways evolved independently in the different phyla [4]. Interestingly, insects biosynthesizing cyanogenic glucosides de novoalso express both enzymes needed for their degradation and cyanide liberation, β-glucosidase and α-hydroxynitrile lyase [141].

7.3. Sequestration of cyanogenic compounds from the food plants

Most data known on sequestration of cyanogenic compounds in insects were generated using larvae of Z. filipendulaeas a model. Z. filipendulaesequesters the cyanogenic glucosides linamarin and lotaustralin from its host plant L. corniculatusas intact glucosides, transports them into all tissues and retains them upon metamorphosis where they can be found in all tissues, too, as studies with isotopically labelled linamarin and lotaustralin have shown [142]. Interestingly, larvae of Z. filipendulaeare also able to sequester the aromatic cyanogenic glucoside prunasin which they do not come in contact with naturally [118]. This indicates a versatile transport mechanism [118] and an evolutionary origin from a more general mechanism to deal with host plant xenobiotics.

Sequestration of cyanogens has also been shown for gynocardin, a cyclic α-hydroxynitrile glucoside, in Acraea horta(Lepidoptera:Nymphalidae) [143] and for sarmentosin, a β-hydroxynitrile, in Parnassius phoebus(Lepidoptera:Papilionidae) [144]. Although the latter compound is not cyanogenic per se, enzymatic catalysis may lead to the liberation of cyanide. Alternatively, a function of the intact compound as deterrent against predators is discussed [144].

Larvae of the bug Leptocoris isolata(Hemiptera:Serinethinae), but not the adult bugs, were shown to contain cyanogenic glucosides such as cardiospermin not present in their host plant [129]. However, their host plant produces cyanolipids containing the same aglucone. Therefore, it was proposed that this species recruited enzymes of xenobiotic metabolism to transform sequestered cyanolipids into cyanogenic glucosides for use in its own defense [129] (Figure 4).

7.4. Benefit of cyanogenesis for herbivores

Cyanogenic glucosides derived from de novobiosynthesis and sequestration may play a pivotal role in herbivore defense against predators as has been demonstrated with the model species Z. filipendulae. Larvae of Z. filipendulaereact to aggression such as pricking by release of a defensive fluid from their dorsal cavities. This fluid contains, next to the cyanide detoxification product β-cyanoalanine, high amounts of the two aliphatic cyanogenic glucosides, linamarin and lotaustralin, and, among other proteins, their hydrolysis enzyme, β-glucosidase [100, 145]. Thus, cyanogenic glucosides and cyanide are used for deterrence and intoxication of potential predators. If whole larvae are ingested by a mammalian predator, another mechanism comes into effect. The larval hemolymph contains high amounts of β-glucosidase inactivated by a high pH value. Upon release of these proteins into the highly acidic environment of the predator’s stomach, the β-glucosidase gets activated and cyanogenic glucoside hydrolysis leads to cyanide poisoning of the predator (discussed in Ref. [4]).

Nevertheless, cyanide alone is not always efficient for the animal’s defense. It has been shown that in millipedes, not cyanide itself but the second product of mandelonitrile hydrolysis, benzaldehyde, is repellent to ants [49]. In contrast, for the intact cyanogenic glucoside cardiospermin a deterrent effect on ants has been shown which could not be observed for any cyanogenic glucoside before [129]. Thus, predating insects facing cyanide in their prey may have evolved a sensitive perception of substances with stronger odor or taste usually occurring alongside cyanide [49]. Alternatively, it was proposed that cyanide is the main means of defense against vertebrate predators, while benzaldehyde is used to repel arthropod enemies [133]. Phylogenetic and physiological data indicate that cyanogenesis as defense strategy has been lost and replaced by ancient phenolic defense compounds in some groups among the Polydesmida, mainly those unlikely to be targeted by vertebrate predators [133].

Next to their defensive roles, linamarin and lotaustralin are also used as nitrogen sources for chitin biosynthesis based on their turnover during metamorphosis [146]. During the formation of the pupal cuticle, cyanogenic glucosides are a key nitrogen source [146, 147]. However, mobilization through β-cyanoalanine synthase and nitrilase/nitrile hydratase leading to the formation of asparagine and aspartate similar to plants [148] has not been demonstrated in insects so far. The efficient transport of cyanogenic glucosides in Z. filipendulaeis nevertheless evident based on the occurrence of these compounds in the wings, an organ devoid of any biosynthetic activity [146]. The important role of sequestered and de novo-biosynthesized cyanogenic glucosides for the defense and metabolism of the lepidopteran specialist Z. filipendulaewas additionally proven by the finding that male adult butterflies transfer the compounds to their partners as nuptial gift upon mating [142, 147].

Based on transcriptional and metabolite analyses [149], it has been hypothesized that the biosynthesis of cyanogenic glucosides in arthropods is older than their sequestration [149]. The biosynthesis is thought to have been constitutive in the ancestors of Zygaena which did not live on cyanogenic plants (but on Celastraceae) and were, therefore, not able to receive cyanogenic glucosides from their host plants. Upon exploration of cyanogenic plants, the insects’ endogenous biosynthesis became inducible as sequestration helped to reduce metabolic costs for de novobiosynthesis [149]. Thus, the ability to handle and de novobiosynthesize cyanogenic glucosides allowed the moths to extend their host plant range and to even exploit the newly acquired host plants, cyanogenic glucoside-producing Fabaceae, to conserve energy and nutrients otherwise needed for the biosynthesis of these compounds [4, 128].