How Insects Overcome Two-Component Plant Chemical Defence: Plant Β-Glucosidases As the Main Target for Herbivore Adaptation

Biol. Rev. (2013), pp. 000–000. 1 doi: 10.1111/brv.12066 How insects overcome two-component plant chemical defence: plant β-glucosidases as the main target for herbivore adaptation

Stefan Pentzold, Mika Zagrobelny, Fred Rook and Søren Bak∗ Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, Copenhagen Dk-1871, Denmark

ABSTRACT

Insect herbivory is often restricted by glucosylated plant chemical defence compounds that are activated by plant β-glucosidases to release toxic aglucones upon plant tissue damage. Such two-component plant defences are widespread in the plant kingdom and examples of these classes of compounds are alkaloid, benzoxazinoid, cyanogenic and iridoid glucosides as well as glucosinolates and salicinoids. Conversely, many insects have evolved a diversity of counter- adaptations to overcome this type of constitutive chemical defence. Here we discuss that such counter-adaptations occur at different time points, before and during feeding as well as during digestion, and at several levels such as the insects’ feeding behaviour, physiology and metabolism. Insect adaptations frequently circumvent or counteract the activity of the plant β-glucosidases, bioactivating enzymes that are a key element in the plant’s two-component chemical defence. These adaptations include host plant choice, non-disruptive feeding guilds and various physiological adaptations as well as metabolic enzymatic strategies of the insect’s digestive system. Furthermore, insect adaptations often act in combination, may exist in both generalists and specialists, and can act on different classes of defence compounds. We discuss how generalist and specialist insects appear to differ in their ability to use these different types of adaptations: in generalists, adaptations are often inducible, whereas in specialists they are often constitutive. Future studies are suggested to investigate in detail how insect adaptations act in combination to overcome plant chemical defences and to allow ecologically relevant conclusions.

Key words: insect herbivore-plant interactions, two-component plant chemical defence, β-glucosidases, β-glucosides, insect adaptations, feeding guild, gut pH, sequestration, generalists and specialists.

CONTENTS I. Introduction ...... 2 (1) Overview: insect herbivores and two-component plant chemical defence ...... 2 (2) Specialist versus generalist insect herbivores ...... 2 (3) The role of plant β-glucosidases and β-glucosides in two-component plant chemical defence ...... 4 II. How non-adapted insect herbivores are affected by two-component plant chemical defence ...... 6 (1) Alkaloid glucosides ...... 6 (2) Benzoxazinoid glucosides ...... 6 (3) Cyanogenic glucosides ...... 8 (4) Glucosinolates ...... 8 (5) Iridoid glucosides ...... 8 (6) Salicinoids ...... 9 III. From feeding to digestion: targets for insect herbivore adaptations to two-component plant chemcial defence in a temporal context ...... 9 (1) Before feeding: recognition, switching and selection of host plants ...... 9 (2) During feeding: impact of the feeding guild ...... 10

* Author for correspondence (Tel: +0045 3533 3346; E-mail: [email protected]).

(a) Piercing-sucking ...... 10 (b) Leaf-snipping versus leaf-chewing ...... 10 (c) Leaf-mining ...... 11 (3) During digestion: physiological and metabolic adaptations to counteract two-component defence .... 12 (a) An alkaline gut pH inhibits the plant β-glucosidase ...... 12 (b) Reduction of endogenous insect β-glucosidase activity in the gut ...... 13 (c) Specialized enzyme activity of insects ...... 14 ( i ) Before hydrolysis by plant β-glucosidases ...... 14 ( ii ) After hydrolysis by plant β-glucosidases ...... 14 (d) Sequestration: spatial separation of plant β-glucosidase and β-glucoside in the insect ...... 15 (e) Single amino acids counteract plant β-glucosidase activity ...... 16 (4) Do generalists and specialists have different types of adaptations? ...... 17 IV. Conclusions ...... 17 V. Acknowledgements ...... 18 VI. References ...... 18

I. INTRODUCTION counter-adaptations to overcome this conditional toxicity (Fig. 1B, Table 1). The permanent presence of a specific class (1) Overview: insect herbivores and two-component of β-glucosidase-activated defence compound in a particular plant chemical defence plant species is a predictable characteristic that may have facilitated herbivorous insects to evolve adaptations. This Insect herbivores account for more than one quarter of raises the questions: (i) have insect adaptations evolved all living species on Earth (Scudder, 2009), and the co- for each specific class of defence compound, or are there evolution of phytophagous insects and their food plants has general applicable mechanisms that allow insect herbivores continued for more than 350 million years (Chaloner et al., to adapt to all classes of two-component defence, (ii)do 1991; Sinclair & Hughes, 2010). Although insect herbivores generalist and specialist herbivores differ in this respect, (iii) potentially have an abundance of plant species available do adaptations of insect herbivores mainly target activity of for feeding, herbivory is often restricted by the physical and the key enzyme, the plant β-glucosidase, to avoid generation chemical defence mechanisms plants have evolved to fend off of toxic aglucones or are there also other ‘targets’, and (iv)do insect attacks. Whereas mechanical structures like cuticular insect herbivores combine several adaptations to overcome waxes, prickles and thorns provide plants with a physical two-component plant chemical defence? defence, toxic chemical compounds provide an additional Several excellent reviews have described how insect effective defensive barrier (Chen, 2008; Mithofer¨ & Boland, herbivores adapt to toxic chemicals in general, including 2012). To fend off insect herbivores, more than 200,000 behavioural, physiological and metabolic adaptations to specialized metabolites, with toxic, growth-reducing or anti- many different classes of natural plant chemical defence nutritive effects, are known to be produced by plant species compounds as well as synthetic insecticides (Brattsten, 1988; (Zhu-Salzman, Luthe & Felton, 2008; Mithofer¨ & Boland, Hoy, Head & Hall, 1998; Despres,´ David & Gallet, 2007; 2012). Chemical defence compounds can be constitutively Schowalter, 2011). Here we focus more specifically on two- present in the plant, i.e. pre-exist in anticipation of an component chemical defence, as accumulating evidence insect attack (phytoanticipins) or their biosynthesis may be suggests that insect herbivores are able to interfere with inducible (phytoalexins) (VanEtten et al., 1994; Chen, 2008; either one or both components (Boeckler, Gershenzon & Mithofer¨ & Boland, 2012). Unsicker, 2011; Dobler et al., 2011; Winde & Wittstock, 2011; Constitutive plant defence compounds are often stored in Zagrobelny & Møller, 2011). In particular species from the the form of non-active and non-toxic glucosides in the plant Lepidoptera (butterflies and moths), Coleoptera (beetles), and are spatially separated from bioactivating β-glucosidases. Hemiptera (e.g. aphids), Hymenoptera (e.g. sawflies), Well-known classes of these compounds are alkaloid, Orthoptera (e.g. locusts and grasshoppers) and Diptera (true benzoxazinoid, cyanogenic and iridoid glucosides as well as flies) have evolved a remarkable diversity of adaptations. glucosinolates and salicinoids (Halkier & Gershenzon, 2006; Morant et al., 2008; Dobler, Petschenka & Pankoke, 2011; (2) Specialist versus generalist insect herbivores Winde & Wittstock, 2011). Upon insect herbivore attack and tissue damage these glucosylated defence compounds The range of host plant species an insect feeds on defines its come into contact with plant β-glucosidases, resulting in the degree of dietary specialization. When feeding is restricted release of toxic aglucones (Fig. 1A). Such a binary system to a few, often closely related plant species, herbivores are of components that are chemically inert when separated considered specialists (Ali & Agrawal, 2012). A narrow range is referred to as two-component plant chemical defence of host plant species is thought to enable high optimal (Wittstock et al., 2004; Bak et al., 2006; Morant et al., performance on the host plant and reduce interspecific 2008). In response, many insect herbivores have evolved competition, but dietary specialization may also increase

(A) Two-component plant chemical defence and activation by β-glucosidases after herbivory

BGD BGD DC DC-glc DC-glc

Undamaged plant: Herbivory causes tissue damage: Spatial separation of both components Mixing of both components in the plant cell or tissue and release of toxic aglucones

(B) Adaptations of insect herbivores to overcome two-component plant chemical defence

Feeding Digestion

haemolymph 1. Host-plant switching digestive tract and selection

7. Single amino acids 2. Non-disruptive 3. Alkaline 5. Insect enzyme activity feeding guild gut lumen before (a) and after (b) hydrolysis by plant BGDs 4. Reduction of insect 6. Sequestration β-glucosidase activity in special tissues

1. Spilosoma virginica 3. Hyphantria cunea 5a. Athalia spp. 6. Chrysomela populi

2. Myzus persicae 4. Epilachna varivestis 5b. Zygaena filipendulae 7. Brahmaea wallichii Fig. 1. Two-component plant chemical defence, its β-glucosidase-mediated activation into toxic aglucones after herbivory and the diverse counter-adaptations of insect herbivores. (A) Spatial separation of both components in undamaged plants ensures that the defence compound stays glucosylated (DC-glc) and hence non-toxic, since the corresponding plant β-glucosidase (BGD) has no access to its substrate. After insect herbivory that causes tissue damage, both components come into contact, and the BGD hydrolyses the glucosylated defence compound into a toxic aglucone (DC). (B) Insect herbivores have evolved different adaptations to counteract and overcome two-component plant chemical defence. These adaptations occur at different stages of herbivory in a temporal context: before feeding (1), during feeding (2), during digestion (3–7). Examples of insect species that have at least one of these adaptations are pictured below and numbered according to their adaptation. For more details see Table 1. Photo credits: 1. Whitney Cranshaw, Colorado State University, Bugwood.org (S. virginica); 2. David Cappaert, Michigan State University, Bugwood.org (M. persicae); 3. Gyorgy Csoka, Hungary Forest Research Institute, Bugwood.org (H. cunea); 4. Whitney Cranshaw, Colorado State University, Bugwood.org (E. varivestis); 5a. Merle Shepard, Gerald R. Carner, and P.A.C Ooi, Insects and their Natural Enemies Associated with Vegetables and Soybean in Southeast Asia, Bugwood.org (Athalia spp.); 5b. Stefan Pentzold, University of Copenhagen (Z. filipendulae); 6. Gyorgy Csoka, Hungary Forest Research Institute, Bugwood.org (C. populi); 7. Alessandra & Rocco Marciano, Acremar Photos (B. wallichii). intraspecific competition and reduce the capacity to exploit considered a continuum and includes intermediates such as new host plants (Schowalter, 2011; Barrett & Heil, 2012). oligophagous species that feed on several plant species within Since specialists encounter a limited number of toxic plant one family (Ali & Agrawal, 2012). Generalists are insects that chemicals, specialized enzymes for detoxification may be feed on a broad range of plant species, often from more one strategy. It should be noted that dietary specialization is than one plant family. Greater resource availability and a

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 4 Stefan Pentzold and others higher capacity to exploit new hosts are the advantages, but glucosinolates and salicinoids (Fig. 2). Spatial separation can generalists usually have a lower optimal performance on any occur at different levels, and varies among defence pathways particular plant species than specialists (Mody, Unsicker & or plant species. On a cellular level, β-glucosidases that Linsenmair, 2007; Barrett & Heil, 2012). The possibility of activate cyanogenic glucosides are generally localized in the host-plant switching may improve insect development by chloroplast in monocots, or in the apoplast and chloroplast diluting the exposure to any single toxic plant chemical and in dicots, while their substrates are stored in the vacuole balancing nutrient intake (Bernays & Minkenberg, 1997; or cytosol (Morant et al., 2008; Sanchez-P´ erez´ et al., 2009). Behmer, 2009). Since generalists encounter a variety of toxic On a tissue level, activating β-glucosidases may be stored in plant chemicals, detoxification enzymes may be less efficient special cell types such as myrosin-cells as in the glucosinolate- (Barrett & Heil, 2012). myrosinase defence system, while glucosinolates are mainly Both generalist and specialist insects are able to suppress stored in sulphur-rich S-cells nearby (Koroleva et al., 2000). the toxicity of their food plants. Whereas generalists benefit β-glucosidases are generally very stable enzymes due to from suppressing any level of toxicity from plant defence their compactly folded core structure, and have pH optima in compounds, enabling at least short-term feeding, specialists slightly acidic conditions in plants as well as insects (Ruuhola, often suppress only high levels of toxicity and benefit Julkunen-Tiitto & Vainiotalo, 2003; Byeon et al., 2005; from the presence of low to intermediate levels of plant Ketudat, Cairns & Esen, 2010; Pankoke et al., 2012; Terra & defence compounds (Ali & Agrawal, 2012). Sequestering Ferreira, 2012). Most plant and insect β-glucosidases belong specialists, i.e. those that selectively take up and accumulate to the glycoside hydrolase (GH) family 1. GHs comprise plant chemicals in their own body, benefit from any level more than 100 described glycosyl hydrolase families, and of defensive chemicals, since the sequestered compounds these families are mainly classified based on structure and protect the insect against predators (Nishida, 2002; Ali & amino acid sequence similarities (Davies & Sinnott, 2008). Agrawal, 2012). Some insect herbivores can also inhibit the GH1 β-glucosidases catalyze the hydrolysis of a glucosidic production of plant chemicals for example by modulating the bond between two carbohydrates or between a carbohydrate plant’s salicylic acid and jasmonic acid signalling pathway moiety and an aryl or alkyl aglucone moiety (Cantarel (Musser et al., 2005; Zarate, Kempema & Walling, 2007; et al., 2009). They are retaining enzymes, i.e. the anomeric Sarmento et al., 2011). However, for insect herbivores configuration of the glucose is retained during hydrolysis confronted with two-component chemical defence, the (Vocadlo & Davies, 2008; Davies, Planas & Rovira, 2011). inhibition of one component, for example the β-glucosidase From an evolutionary point of view, GH1 β-glucosidases or the modification of the free aglucones once released, will constitute a monophyletic multigene family. In plants, be more effective strategies. there are 48 putative β-glucosidase genes in the Arabidopsis thaliana (Brassicaceae) genome (Xu et al., 2004), and 40 (3) The role of plant β-glucosidases and in the rice (Oryza sativa, Poaceae) genome (Opassiri et al., β-glucosides in two-component plant chemical 2006). Substrate specificity of plant β-glucosidases may defence differ from a broad range as seen for the Zea mays To be most effective against a wide range of attacking (Poaceae) β-glucosidase ZmGlu1 (Brzobohaty et al., 1993) organisms, defence compounds affect basic metabolic to the narrow specificity reported for the Sorghum bicolor processes or evolutionarily conserved cellular mechanisms. (Poaceae) β-glucosidase SbDhr1, which hydrolyses only Thus, constitutive plant defence compounds may also be dhurrin (Verdoucq et al., 2004). Insect genomes also contain toxic to the plant itself. One strategy that limits self- GH1s, and as in plants their substrate specificity range also toxicity is to store constitutive defence compounds in an differs, but insect genomes contain far fewer β-glucosidases. inactive form as β-d-glucopyranosides and to separate them For example, five putative GH1 β-glucosidases are found from their activating β-glucosidases. Glucosylation of the in the transcriptome of the termite Coptotermes formosanus plant defence compound and its storage separate from β- (Isoptera, Rhinotermitidae) (Zhang et al., 2012). A blast glucosidases, renders it chemically inert, which prevents search against the fully sequenced insect genomes Anopheles cellular damage, increases stability and solubility, facilitates gambiae, Drosophila melanogaster (both Diptera) and Apis mellifera storage and enables selective intra- and intercellular transport (Hymenoptera) showed that their genomes each harbour of the compound (Bak et al., 2006; Discher et al., 2009; between 5 and 15 β-glucosidases. The differences in number Nour-Eldin et al., 2012). After tissue damage by herbivory, of GH1 genes between plant and insect genomes may be mixing of glucosylated plant defence compounds and plant due to the fact that plant β-glucosidases are involved in β-glucosidases results in hydrolysis of the defence compounds many physiological processes such as cell-wall lignification and a fast release of toxic aglucones that serve to fend off and degradation, phytohormone activation and activation non-adapted herbivores (Fig. 1A) (Wittstock & Gershenzon, of chemical defence compounds (Morant et al., 2008), while 2002; Morant et al., 2008; Frey et al., 2009; Hopkins, van Dam insect β-glucosidases mainly have a digestive role (Ferreira, & van Loon, 2009; Boeckler et al., 2011; Dobler et al., 2011; Torres & Terra, 1998; Terra & Ferreira, 2012). The evolution Pankoke, Bowers & Dobler, 2012). This strategy applies to of insect β-glucosidases might have been driven by the need many classes of plant defence compounds such as alkaloid, to hydrolyse nutritive plant β-glucosides encountered during benzoxazinoid, cyanogenic and iridoid glucosides as well as feeding. Insect β-glucosidases with broad substrate specificity

Table 1. Overview of the various adaptations of insect herbivores to β-glucosidase-activated plant defence compounds. Entries are arranged in alphabetical order of defence compound class. Examples where adaptations occur at different levels, including the behavioural, physiological and metabolic level are indicated by *. For comparison of insect adaptations to further defence compound classes see Table 1 in Despres´ et al. (2007)

Defence Insect herbivore Dietary compound species specialisation class Adaptations of insect herbivores References Callosobruchus Generalist AG Decreased expression of a gene encoding a Desroches et al. (1995, 1997) maculatus* vicine BGD results in reduced activity of endogenous BGDs in the midgut Lack of endogenous insect BGD activity in the haemolymph Sequestration of intact AGs Mythimna separata Generalist BX UGT activity in the midgut and excretion of Sasai et al. (2009) non-toxic glucosides Ostrinia furnacalis Generalist BX UGT activity in the midgut and excretion of Kojima et al. (2010) non-toxic glucosides Spodoptera frugiperda*, Generalists BX An alkaline midgut reduces plant BGD activity Berenbaum (1980), S. littoralis* High UGT activity in the gut Dutartre et al. (2011), Excretion of BXs Glauser et al. (2011) and CNG S. frugiperda: endogenous gut BGDs lack activity Marana et al. (2000) towards CNGs Agromyzidae species Specialists CNG Leaf-mining Schappert & Shore (1999) Selection of T. ulmifolia populations with low CNG content Cyrtomenus bergi Specialist CNG Piercing-sucking of mature bugs beyond the McMahon et al. (1995) outer layer of cassava roots lacking CNGs Diatraea saccharalis Generalist CNG Selective and inducible reduction of the activity Azevedo et al. (2003) and of the endogenous insect BGD βGly2 Ferreira et al. (1997) Epilachna varivestis Specialist CNG Reduction of endogenous insect BGD activity Ballhorn et al. (2010) in the gut Heliconius sara* Specialist CNG Metabolic replacement of the nitrile group by a Engler et al. (2000) thiol Sequestration Hyphantria cunea Generalist CNG Highly alkaline midgut Fitzgerald (2008) Schistocerca gregaria* Generalist CNG Leaf-snipping Ballhorn et al. (2010) Zygaena filipendulae* Specialist CNG Detoxification by β-cyanoalanine synthase Zagrobelny & Møller (2011) Sequestration Athalia rosae* Specialist GSL Sulfatases and sulfotransferases in the gut and Opitz et al. (2011) haemolymph Sequestration Brevicoryne brassicae*, Specialists GSL Piercing-sucking Barth & Jander (2006), Lipaphis erysimi* Sequestration of intact GSLs Bridges et al. (2002) and Spatial separation of sequestered GSLs and Kazana et al.(2007) endogenous insect BGDs in the body Myzus persicae* Generalist GSL Piercing-sucking Barth & Jander (2006), Excretion of intact GSLs Francis et al. (2005) and Inducible glutathione S-transferase activity IG Piercing-sucking Gange & West (1994) Pieris rapae Specialist GSL Detoxification by nitrile specifier protein and Wittstock et al. (2004) and β-cyanoalanine synthase Stauber et al. (2012) Plutella xylostella Specialist GSL Constitutive sulfatase activity in the larval gut Ratzka et al. (2002) S. gregaria* Generalist GSL Host-plant switching Bernays et al. (1994), Falk & Inducible sulfatases in the gut Gershenzon (2007) and Inducible reduction of endogenous insect BGD Mainguet et al. (2000) activity in the midgut S. exigua, S. littoralis, Generalists GSL Glutathione S-transferase activity, probably in Schramm et al. (2012) T. ni, M. brassicae, the gut H. armigera Spilosoma virginica*, Generalists IG Inducible reduction of endogenous insect BGD Pankoke et al. (2010, 2012) Grammia incorrupta* activity in the midgut Host-plant switching

Table 1. (cont.)

Defence Insect herbivore Dietary compound species specialisation class Adaptations of insect herbivores References Amphipyra monolitha Generalist IG Inducible secretion of high levels of β-alanine Konno et al. (2010) into the midgut Artopoetes pryeri Specialist IG Secretion of high levels of GABA into the Konno et al. (2010) midgut Brahmaea wallichii, Specialists IG Secretion of high levels of free glycine into the Konno et al. (1997) Dolbina tancrei anterior part of the midgut Chrysomela populi *, Specialists SA Lack of endogenous insect BGD activity in the Discher et al. (2009) and Phratora vitellinae* haemolymph Kuhn et al. (2004, 2007) Speciﬁc transporters Sequestration of intact SAs Lymantria dispar*, Generalists SA Inducible reduction of endogenous insect Hemming & Lindroth Malacosoma disstria* BGD activity in the midgut (2000) Inducible glutathione-S-transferase in the midgut Operophtera brumata Generalist SA Highly alkaline gut Ruuhola et al. (2003) Papilio glaucus Specialist SA Inducible reduction of endogenous insect Lindroth (1988) canadensis BGD activity in the midgut Phyllonorycter Specialist SA Leaf-mining Auerbach & Alberts (1992) salicifoliella Selective feeding on poplar species with low levels of SAs

AG, alkaloid glucoside; BGD, β-glucosidase; BX, benzoxazinoid glucoside; CNG, cyanogenic glucoside; GABA, γ -aminobutyric acid; GSL, glucosinolate; IG, iridoid glucoside; SA, salicinoid; UGT, UDP-glucosyltransferase. may also activate glucosylated plant defence compounds the case of vicine and convicine, the main alkaloid glucoside during digestion. However, this process is likely to be less defence compounds in the cotyledons of the broad bean Vicia efficient in deterring insect feeding than the immediate faba (Fabaceae), pyrimidine is linked to a β-d-glucopyranose release of high amounts of toxic aglucones by specific and (Desroches et al., 1995, 1997). When bruchid beetle larvae highly active plant β-glucosidases upon tissue disruption Callosobruchus maculatus (Coleoptera, Chrysomelidae) feed on by herbivores. Moreover, insects may also reduce their V. faba, the ingested vicine and convicine are activated endogenous β-glucosidase activity to limit the release of by endogenous C. maculatus β-glucosidases into the highly toxic aglucones during digestion (see Section III.3b). reactive and free-radical-generating aglucones divicine or isouramil (Fig. 2A) (Hegazy & Marquardt, 1984; Desroches et al., 1997; Brimer, 2011). These aglucones inhibit the activity of glucose-6-phosphate dehydrogenases causing II. HOW NON-ADAPTED INSECT HERBIVORES adverse metabolic effects such as lipid peroxidation, which ARE AFFECTED BY TWO-COMPONENT PLANT CHEMICAL DEFENCE results in high mortality rates of C. maculatus larvae feeding on V. faba (Desroches et al., 1995; McMillan, Bolchoz & Jollow, 2001). The chemical compounds from the various classes of two- component defence differ in their chemical structure (Fig. 2), effect on non-adapted insect herbivores and distribution (2) Benzoxazinoid glucosides within land plant species. Whereas some classes are limited to specific orders and families, others are found in most Benzoxazinoid glucosides are found in numerous species of groups of land plants (Bak et al., 2006; Halkier & Gershenzon, the monocot family Poaceae, including major crops such as 2006). Many of the aglucones inhibit enzymes and proteins maize Z. mays,wheatTriticum aestivum and rye Secale cereale unspecifically, but others may be targeted to specific enzymes. (Frey et al., 2009). In addition, a few species of the eudicot In the following sections, compounds from several classes of orders Ranunculales and Lamiales produce benzoxazinoid two-component plant chemical defence are described as well glucosides following convergent evolution of both the as their effects on non-adapted insect herbivores. biosynthetic pathway and the activating β-glucosidase (Dick et al., 2012). The biosynthetic pathway of benzoxazinoid glucosides in maize is fully characterized. Indole-3-glycerol (1) Alkaloid glucosides phosphate is converted mainly by cytochrome P450- Alkaloid glucosides are characterised by nitrogen-containing dependent monooxygenases into cyclic hydroxamic acid, heterocyclic aglucones, such as pyrimidine for example. In which is glucosylated with a β-d-glucopyranose before

(A) Alkaloid glucosides Glc = β-D-glucopyranose NH NH2 2 H OH HO O H O Glc N BGD* N HO HO OH H OH N N O NH2 O H NH2 H H H Vicine Divicine

(B) Benzoxazinoid glucosides Insect enzymes O O O HO O O Plant enzymes Glc BGD N O N O UGT OH OH

DIMBOA-glc DIMBOA

CN Glc O BGD* b-CAS CN HO N C H β-cyanoalanine BGD

Linamarin unstable HCN unknown aglucone substitution by -SH group

(D) Glucosinolates

- - SO SO3 GST 3 BGD* RNCS GSH-ITC N O N O BGD - Isothiocyanate S S b-CAS Glc R R N C H β-cyanoalanine NSP unstable R C N R = aromatic HCN aglucone Sulfatase Nitrile N OH R = aliphatic excretion S Sulfotransferase Glc R desulfo-glucosinolate desulfo-glucosinolate-3-sulfate

(E) Iridoid glucosides

Glc HO HO O OH

BGD* O O BGD

OH OH Aucubin reactive aglucone

(F) Salicinoids R

O OH OH

O O Esterase BGD* HO Glc Glc or alkaline pH BGD

SalicortinSalicin Saligenin Fig. 2. Chemical structure of different classes of glucosylated plant defence compounds and their activation by β-glucosidases (BGDs) into toxic aglucones and further products. Activation can be due to activity of BGDs from the plant (green) or due to insect BGDs (orange). Importantly, adapted insects reduce the activity of their endogenous BGDs to reduce the formation of aglucones (marked with *). Activity of further more specialized enzymes from insects convert plant defence compounds: either before their hydrolysis by plant BGDs, so that the product can no longer be activated by plant BGDs into toxic aglucones, or after their hydrolysis by plant BGDs via conjugating or detoxifying insect enzymes. Toxicity of a compound is indicated by a skull. Abbreviations: b-CAS, β-cyanoalanine synthase; DIMBOA, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one; GSH-ITC, glutathione-isothiocyanate; GST, glutathione-S-transferase; NSP, nitrile speciﬁer protein; UGT, UDP-glucosyltransferase.

final addition of a methoxy group into 2,4-dihydroxy-7- (4) Glucosinolates methoxy-1,4-benzoxazin-3-glucoside (DIMBOA-glucoside), Similar to cyanogenic glucosides, glucosinolates are a major benzoxazinoid glucoside in Poaceae (Fig. 2B) synthesized from amino acids, but consist of a β-d- et al. et al. et al. (Jonczyk , 2008; Frey , 2009; Dick , 2012; thioglucopyranoside, a sulfonated oxime and a variable Dutartre, Hilliou & Feyereisen, 2012). Once activated by side chain (Halkier & Gershenzon, 2006; Hopkins et al., β plant -glucosidases, the toxic aglucone DIMBOA depletes 2009). More than 140 different structures of glucosinolates cellular glutathione levels and this leads to irreversible are known, but they are almost exclusively restricted to the inactivation of enzymes with cysteine residues in their active order Brassicales which includes many crop species such as site (Dixon et al., 2012). For example, when DIMBOA is oilseed rape (Brassica napus), broccoli (B. oleraceae), mustard added to artificial diets, survival, mass gain and reproduction (Sinapsis alba) and the model plant A. thaliana (Halkier & of several aphid species is strongly reduced (Escobar, Gershenzon, 2006). Activation of glucosinolates is carried out Sicker & Niemeyer, 1999). Similarly, when larvae of the by β-thioglucosidases, so-called myrosinases. Myrosinases silkworm Bombyx mori (Lepidoptera, Bombycidae) were are closely related to β-glucosidases and are known from reared on a DIMBOA-containing diet, the larvae died within plants and insects (Husebye et al., 2005). Myrosinase 3 days (Sasai et al., 2009). Another benzoxazinoid aglucone activity releases an unstable aglucone, which dissociates from maize, 2-hydroxy-4, 7-dimethoxy-1, 4-benzoxazin-3- mainly into toxic isothiocyanates, but also into nitriles one (HDMBOA), was shown strongly to deter generalist and related compounds (Fig. 2D) (Halkier & Gershenzon, herbivores such as the Egyptian armyworm Spodoptera littoralis 2006). The aglucone produced depends on the presence and the fall armyworm Spodoptera frugiperda (both Lepidoptera, of epithiospecifier proteins, the structure of the side chain Noctuidae) (Glauser et al., 2011). group and factors such as pH and the presence of ferrous ions (Halkier & Gershenzon, 2006). Isothiocyanates for example react with amino and sulfhydryl groups of proteins (3) Cyanogenic glucosides (Halkier & Gershenzon, 2006), which makes them toxic to Cyanogenic glucosides are synthesized from amino acids e.g. the springtail Folsomia fimetaria (Collembola, Isotomidae) and consist of an α-hydroxy nitrile linked to a β-d- even at low concentrations (Jensen et al., 2010). Similarly, glucopyranose (Bak et al., 2006; Møller, 2010). They are generalist herbivores like the cabbage looper Trichoplusia ni present in more than 2650 plant species distributed among (Lepidoptera, Noctuidae) or the tobacco hornworm Manduca sexta 130 families within ferns, gymnosperms and angiosperms, (Lepidoptera, Sphingidae) significantly decrease in body mass when feeding on A. thaliana containing glucosinolates and have independently evolved in different plant lineages and myrosinases, in comparison to feeding on A. thaliana by recruiting biosynthetic genes from similar gene families knock-out mutants that lack myrosinases (Barth & Jander, (Takos et al., 2011). Despite their wide distribution, only 2006). Adverse effects of the glucosinolate-myrosinase about 50 different structures of cyanogenic glucosides two-component defence are also known for the beet are known (Bak et al., 2006; Bjarnholt et al., 2008). armyworm Spodoptera exigua (Lepidoptera, Noctuidae) (Müller Cyanogenic glucosides are also found within a few arthropod et al., 2010). species (Zagrobelny, Bak & Møller, 2008). For example, Zygaena filipendulae six-spot burnet moth larvae (Lepidoptera, Zygaenidae) are able to carry out both de novo biosynthesis (5) Iridoid glucosides and sequestration of cyanogenic glucosides for use in defence, Iridoid glucosides are widespread plant defence compounds and also as a nuptial gift during mating (Zagrobelny et al., that can be found in more than 50 different plant families 2007). The biosynthetic pathways have been elucidated of the Asteridae (Dobler et al., 2011). They are derived from in both plants and insects, and this has revealed that monoterpenes and form a large group of several hundred the ability to synthesize cyanogenic glucosides has evolved different compounds (Fig. 2E) (Dobler et al., 2011). All iridoid convergently in the two kingdoms (Jensen et al., 2011). During glucosides have a similar skeleton with a cyclopentane ring herbivory, the plant tissue is damaged, and the cyanogenic connected to an oxygenated heterocyclohexane, where a β- glucosides come into contact with plant β-glucosidases. d-glucopyranose is attached at the C1 atom. Various epoxy This releases an unstable aglucone that dissociates either groups and sugars determine their structural diversity. In spontaneously or enzymatically, liberating highly toxic insects, iridoid glucosides are mainly activated in the gut, hydrogen cyanide in a process called cyanogenesis (Fig. either by co-ingested plant β-glucosidases or by endogenous 2C) (Zagrobelny et al., 2008). Hydrogen cyanide specifically insect β-glucosidases (Pankoke et al., 2012). The released inhibits cytochrome c oxidase, a key enzyme in the aglucones are highly unstable and reactive: the pyran ring mitochondrial respiratory pathway (Blom et al., 2011), which of the aglucone opens and forms irreversible bonds with causes cell and tissue death within a short time, and strongly proteins, which causes unspecific crosslinks in proteins, deters various non-adapted herbivores (Møller, 2010). For inhibits enzymes and deters non-adapted insects (Kim et al., example, fall armyworm larvae quickly die when fed on 2000; Dobler et al., 2011). The aglucone of the seco- an artificial diet containing cyanide (Hay-Roe, Meagher & iridoid glucoside oleuropein from the privet tree (Ligustrum Nagoshi, 2011). obtusifolium, Oleaceae) covalently binds to lysine residues in

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society Insect adaptations to plant chemical defence 9 proteins due to its electrophilic glutaraldehyde-like structure (1) Before feeding: recognition, switching and (Konno, Hirayama & Shinbo, 1997; Konno et al., 1999). selection of host plants This leads to denaturing and crosslinking of proteins, which Insect herbivores encounter both nutrients and plant defence decreases the nutritive value of dietary proteins and deters compounds in their food, often resulting in a trade- insect herbivores. off between nutrient intake and ingestion of harmful substances (Singer, Bernays & Carriere,` 2002; Behmer, (6) Salicinoids 2009). Importantly, plant defence compounds often have Salicinoids are phenolic glucosides consisting of a salicyl a pronounced spatial and temporal variation at all levels, alcohol linked to a β-d-glucopyranose (Boeckler et al., 2011). i.e. varying concentrations in different organs and within Salicin, the simplest phenolic glucoside, can be modified with and among populations as well as throughout developmental different organic acids resulting in at least 20 more complex stages (Hoy et al., 1998; Gebrehiwot & Beuselinck, 2001). structures (Fig. 2F) (Boeckler et al., 2011). Salicinoids are This heterogeneity in the plant puts a selection pressure on particularly abundant in species of the Salicaceae (e.g. poplars herbivores to evolve sensory systems for the recognition and Populus spp. or willows Salix spp.) where they can reach avoidance of plant defence compounds to avoid ingestion concentrations of up to 30% of plant dry mass (Donaldson of lethal doses. Underlying mechanisms are genetically et al., 2006), but are also known from the pine family determined and can be inherited, but may also be learned (Pinaceae) and oaks (Quercus spp., Fagaceae) (Delvas et al., (Chapman, 2003; Despres´ et al., 2007; Schowalter, 2011). 2011). As a two-component defence, toxicity of salicinoids Recognition of potentially toxic substances allows insect requires activation by β-glucosidases to form aglucones, herbivores to generate a beneficial behavioural response. which may be oxidized in the gut lumen of non-adapted Whereas non-adapted herbivores benefit from avoiding the herbivores into tissue-damaging and protein-crosslinking ingestion of toxins by feeding on toxin-free organs or at reactive oxygen species such as quinones (Ruuhola et al., developmental stages where toxins are absent (Hoy et al., 2003; Boeckler et al., 2011; Delvas et al., 2011). For example, 1998; Despres´ et al., 2007), adapted herbivores may respond in white spruce needles (Picea glauca, Pinaceae) the salicinoids differently (Hoy et al., 1998). picein and pungenin are activated by β-glucosidases into Insect herbivores that are adapted to low to medium levels piceol and pungenol (MacKay, 2012). These aglucones harm of defence compounds can regularly switch to other host the spruce budworm Choristoneura fumiferana (Lepidoptera, plants to avoid ingestion of lethal doses by dietary mixing - Tortricidae) by increasing its larval mortality, retarding its a feeding behaviour mainly exhibited by generalists. Host- development and reducing its pupal mass (Delvas et al., plant switching may dilute excessive detrimental effects of 2011). Needles that are susceptible to budworms do not any single plant defence compound. For example, larvae express β-glucosidase genes (MacKay, 2012). Thus, non- of the lepidopteran generalists yellow woolly bear Spilosoma resistant needles contain mainly intact salicinoids and lack virginica and tiger moth Grammia incorrupta (both Lepidoptera, the ability to release toxic aglucones (Delvas et al., 2011). Arctiidae) can feed on high iridoid-glucoside-containing plants like Plantago lanceolata (Plantaginaceae) (Pankoke, Bowers & Dobler, 2010; Pankoke et al., 2012). Both species were shown to compensate fitness costs from feeding on III. FROM FEEDING TO DIGESTION: TARGETS toxic P. lanceolata by regular host-plant switching under FOR INSECT HERBIVORE ADAPTATIONS TO field conditions (Singer et al., 2002). Several experiments TWO-COMPONENT PLANT CHEMCIAL with generalist grasshopper species have shown that host- DEFENCE IN A TEMPORAL CONTEXT plant switching and dietary mixing results in better growth, survival and fecundity than when feeding is limited to a single As discussed above, non-adapted insects will be strongly plant species (summarized in Bernays & Minkenberg, 1997). affected in different ways by toxic aglucones released from Grasshoppers also seem behaviourally to select for dietary glucosylated defence compounds by plant β-glucosidase mixing and nutritional balance if given the opportunity activity. However, other insect herbivores, including (Bernays & Minkenberg, 1997). For example, the growth rate generalists and specialists, have evolved a range of diverse of the desert locust Schistocerca gregaria (Orthoptera, Acrididae) adaptations to overcome the deleterious effects of two- was significantly higher on a mixed diet containing kale (B. component plant chemical defence, and this enables them to oleraceae, containing glucosinolates), cotton (Gossypium hirsutum, feed on these plants. These adaptive processes occur before Malvaceae) and basil (Ocimum basilicum, Lamiaceae) than on and during feeding as well as mainly during digestion of any one of the single plant species (Bernays et al., 1994). the plant material. Behavioural, physiological and metabolic Specialists that are adapted to high levels of a certain adaptations may be combined, often target the plant β- plant defence compound and even require them for their glucosidase activity and apply to all classes of two-component development, benefit from identifying plants with high plant chemical defence discussed here (Table 1, Figs 1 and concentrations. In feeding-choice experiments, larvae of 2). Finally, we discuss that adaptations in generalists are the six-spot burnet moth were shown to identify and often inducible, whereas those in specialists seem to be to prefer Lotus corniculatus (Fabaceae) food plants with constitutive. high contents of cyanogenic glucosides over plants with

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 10 Stefan Pentzold and others low contents (Zagrobelny et al., 2007). Larvae reared on 1999; Walling, 2008). Piercing-sucking minimizes tissue plants with low cyanogenic glucoside content showed a disruption, and thereby minimizes the plant β-glucosidase- decelerated development since they need to expend energy mediated activation of glucosylated defence compounds in synthesizing cyanogenic glucosides. By contrast, larvae (Fig. 3A). reared on plants with high levels of cyanogenic glucosides In the case of the glucosinolate-myrosinase defence system had normal development since they are able to accumulate in Arabidopsis plants, piercing-sucking aphids were shown to cyanogenic glucosides in their body by sequestration, which prevent toxic hydrolysis of glucosinolates. This allows the is less costly than biosynthesis (Zagrobelny et al., 2007). specialist cabbage aphid Brevicoryne brassicae (Aphididae) to Behavioural adaptation through host-plant switching in accumulate intact glucosinolates in its body, often for use in generalists and selection of toxic plants in specialists also its own defence (Bridges et al., 2002). Likewise, the generalist involves trade-offs and fitness costs. Insect herbivores need green peach aphid Myzus persicae (Aphididae) is able to ingest to invest time and energy to search for a suitable host intact glucosinolates, and excretes them in their non-toxic (Despres´ et al., 2007). Investment costs differ in generalists form via the honeydew (Barth & Jander, 2006). Green peach and specialists and mainly seem to depend on the level of aphids are also able to feed and develop on P. lanceolata plants glucosylated plant defence compound, but also to a high with a high content of iridoid glucosides (Gange & West, degree on the level of activating plant β-glucosidases. The 1994). Moreover, different aphid species are the dominant more of both components are present in the plant, the more insect taxa found in plant populations of Turnera ulmifolia often generalists need to switch host plants, and the longer (Turneraceae) that exhibit high concentrations of cyanogenic they need to search for suitable host plants, which increases glucosides of up to 640 μg HCN per g dry mass (Schappert costs. By contrast, specialists need to invest less time, energy & Shore, 1999). This shows that the non-disruptive feeding and thus costs in this case. guild of piercing-sucking efficiently minimizes mixing of plant β-glucosidase and glucosylated defence compound (2) During feeding: impact of the feeding guild and hence the formation of toxic aglucones among different classes of two-component defences. Herbivory inevitably results in tissue damage. The more plant Even the length of the stylus of piercing-sucking insects tissue is damaged, the more toxic aglucones are released seems important in the avoidance of activation of two- due to mixing of plant β-glucosidases and glucosylated component plant chemical defence. For example, immature plant defence compound (Bernays & Janzen, 1988; Barth & cassava bugs Cyrtomenus bergi (Hemiptera, Cydnidae) have a Jander, 2006). The extent of damage, however, differs among short stylus and are deterred from feeding on the outer layer herbivore species and mainly depends on their feeding guild of cyanogenic-glucoside-containing roots of cassava Manihot (Simberloff & Dayan, 1991; Gleadow & Woodrow, 2002; esculenta (Euphorbiaceae) (McMahon, White & Sayre, 1995). Textor & Gershenzon, 2009). A feeding guild is a group By contrast, fully mature cassava bugs have a longer stylet of species that use the same environmental resources in and are able to feed on cassava roots by penetrating beyond a similar way, i.e. the technique insects use to feed on the outer layer. plants. In general there are different guilds of feeding such as piercing-sucking, leaf-snipping, leaf-chewing and leaf- (b) Leaf-snipping versus leaf-chewing mining (Bernays & Janzen, 1988; Sinclair & Hughes, 2010; Ali & Agrawal, 2012). Importantly, these insect feeding Insect herbivores may snip leaves into quite large pieces guilds impact on the effectiveness of two-component plant as shown for some lepidopteran caterpillars biting off leaf chemical defence, because the amount of tissue damage and discs up to 0.6 mm2 (Bernays & Janzen, 1988). Such leaf- hence the release of aglucones will vary depending on the snipping reduces potential tissue damage and the formation feeding strategy. Whereas the non-disruptive feeding guilds of toxic aglucones markedly (Bernays & Janzen, 1988; of piercing-sucking and leaf-snipping efficiently prevent Barbehenn, 1992). Lepidopteran species that have short tissue damage (Fig. 3A, B), leaf-mining is potentially more and simple mandibles and lack molars, and locusts that have disruptive (Fig. 3C) and leaf-chewing extensively damages relatively large mandibles can all exhibit leaf-snipping (Fig. plant tissue (Fig. 3D). 3B) (Bernays & Janzen, 1988; Barbehenn, 1992; Ballhorn, Kautz & Lieberei, 2010). The specialist Mexican bean beetle Epilachna varivestis (a) Piercing-sucking (Coleoptera, Coccinellidae) and the generalist desert locust Insects belonging to the order Hemiptera such as both feed on cyanogenic-glucoside-containing lima beans aphids (Aphidae), whiteflies (Aleyrodidae) and leafhoppers (Phaseolus lunatus, Fabaceae). Although the Mexican bean (Cicadellidae) use their stylets, highly modified mouthparts, beetles have a reduced activity of endogenous β-glucosidases to pierce the cuticle, epidermis and mesophyll and suck from in comparison to locusts (see Section III.3b), more cyanogenic single phloem sieve elements, a rich source of high levels glucosides are hydrolysed during feeding by the beetles than of sucrose and other nutrients (Douglas, 2006; Kehr, 2006; by the locusts (Ballhorn et al., 2010). This difference is due to Walling, 2008). Aphids inject watery saliva containing Ca2+- their different feeding guilds: beetles with their rather small binding proteins that counteract the influx of Ca2+ and mandibles chew leaves and tend to crush the plant tissue prevent closure of sieve elements by callose formation (Miles, (leaf-chewing). By contrast, locusts with their quite large

Insect feeding Plant tissue Plant cell Molecular Tissue damage, Inhibition of guild level level level aglucone formation aglucone formation and toxicity and toxicity

(A) Piercing- No mixing No aglucones 0 % 100 % sucking BGD DC-glc

(B) Leaf- Low Minimal snipping mixing aglucone formation BGD DC DC-glc

(D) Leaf- Complete High chewing mixing aglucone formation BGD DC DC-glc 100 % 0 %

Fig. 3. Insect feeding guilds and their impact on the activation of glucosylated plant defence compounds by β-glucosidases. Feeding guild, potential damage to the plant on the tissue level, potential enzyme and substrate mixing upon tissue damage on the cellular level, and formation of toxic aglucones on the molecular level are shown. Feeding by (A) piercing-sucking insects like aphids and (B) leaf-snipping caterpillars or locusts causes minimal tissue damage, resulting in low mixing of glucosylated plant defence compound (DC-glc) and β-glucosidases (BGDs) in the cell, and thus minimal formation of toxic aglucones (DC). Leaf-mining (C) by dipteran larvae, for example, results in medium levels of tissue damage and intermediate levels of aglucone formation, whereas leaf-chewing by beetles (D) extensively damages plant tissue. This results in complete mixing of glucosylated plant defence compound and β-glucosidase and consequently high levels of aglucones released. Drawings of insects with permission and copyright by D. G. Mackean. mandibles snip leaves into larger pieces (leaf-snipping), and noted that leaf-snipping saturniid caterpillars are mainly consequently ingest a high percentage of intact plant tissue, generalists that feed on a variety of plants, including species thereby limiting hydrolysis of cyanogenic glucosides (Fig. 3B) not protected by two-component chemical defence (Janzen, (Ballhorn et al., 2010). During digestion, plant tissue and cells 1984). The evolution of leaf-snipping and piercing-sucking may be disrupted in the gut, but physiological conditions morphologies and feeding guilds thus is probably not a might favour stabilization or detoxification of plant defence specific adaptation to two-component defence; insects with compounds as discussed later. a minimally disruptive feeding morphology may simply Bernays & Janzen (1988) compared mandible morphology have been pre-adapted to feed on plants protected by two- of caterpillars between two lepidopteran families and component chemical defence. correlated morphological differences to their different feeding guilds. Mandibles of Saturniidae, a leaf-snipping family, were (c) Leaf-mining short and simple. This morphology minimizes tissue damage, Leaf-mining insects feed inside the leaf lamina, mostly on enables ingestion of large plant pieces and minimizes the parenchymous or epidermal tissues, causing channels, mines amount of activated plant defence compound. By contrast, or blotches (Sinclair & Hughes, 2010). These herbivores are the mandibles of Sphingidae, a leaf-chewing family, were protected from external factors such as ultraviolet radiation long, toothed and ridged. This morphology enables crushing or externally applied insecticides, although they are often of plant material, ingestion of small pieces and absorption highly susceptible to parasitoids (Connor & Taverner, 1997; of maximal amounts of nutrients (Bernays & Janzen, 1988). Loch, Matthiessen & Floyd, 2004). Most leaf-mining species Thus, leaf-snipping enables insect herbivores to feed on are found within the Coleoptera, Diptera, Hymenoptera potentially toxic plants, but may be less efficient in terms and Lepidoptera (Connor & Taverner, 1997). Because their of nutrient extraction. Conversely, if more plant material feeding is restricted to specific leaf tissues, the total tissue is crushed, more nutrients become available, but also more damage by leaf-mining is lower than the damage resulting plant defence compounds are activated, which potentially from leaf-chewing, but potentially higher than from piercing- limits leaf-chewers’ feeding on toxic plants. It should be sucking or leaf-snipping (Fig. 3C) (Schappert & Shore,

1999). Several studies indicate that intermediate amounts of β-glucosidases and prevent activation of ingested defence glucosylated defence compounds are activated during leaf- compounds. mining and that, according to the selective feeding hypothesis Although caterpillars of the generalist fall webworm (Scheirs, De Bruyn & Verhagen, 2001), it is adaptive for Hyphantria cunea (Lepidoptera, Arctiidae) are vulnerable to leaf-miners behaviourally to select and feed on plants or hydrogen cyanide, they can feed on leaves of black cherry tissues with low levels of chemical defence. (Prunus serotina, Rosaceae) containing cyanogenic glucosides Dipteran (Agromyzidae) and lepidopteran (Gelechiidae) without any adverse effects (Fitzgerald, 2008). A high leaf-mining species were only found in T. ulmifolia populations alkaline midgut lumen at pH 11 was shown to prevent with low concentrations of cyanogenic glucosides and were the hydrolysis of cyanogenic glucosides into toxic hydrogen absent from populations with high concentrations (Schappert cyanide during passage of the plant parts through the gut & Shore, 1999). The aspen blotch miner Phyllonorycter (Fitzgerald, 2008), most likely due to inhibition of plant β- salicifoliella (Lepidoptera, Gracillariidae) avoids exposure to glucosidases. A direct link between an alkaline midgut and high levels of salicinoids by selectively feeding on poplar reduced plant β-glucosidase activity towards benzoxazinoid species with low levels of salicinoids such as Populus tremuloides glucosides was shown in the generalist fall armyworm feeding (Auerbach & Alberts, 1992). Recent studies show that the on maize containing DIMBOA-glucosides (Dutartre et al., extent of damage by aspen blotch miners is indeed negatively 2011). The larval midgut lumen with a pH of 10 was correlated to the total salicinoid concentration of P. tremuloides shown to reduce plant β-glucosidase activity by more than leaves (Young et al., 2010a, b). Leaf-mining Scaptomyza ﬂava 80%, which strongly reduced the release of toxic aglucones. (Diptera, Drosophilidae) larvae exhibit signiﬁcantly reduced Caterpillars of the generalist winter moth Operophtera brumata growth rates on glucosinolate-containing wild-type A. thaliana (Lepidoptera, Geometridae) feed on Salix species that contain relative to larvae reared on plants with low levels of the salicinoid salicortin. In the midgut lumen, plant esterase glucosinolates (Whiteman et al., 2012). The two dipteran activity or an alkaline pH of 9.5 convert salicortin into grass miners Chromatomyia milii and C. nigra prefer to feed on salicin (Fig. 2F) (Berenbaum, 1980; Ruuhola, Tikkanen & the leaf mesophyll tissue and never feed on the epidermal Tahvanainen, 2001). However, hydrolysis of salicin by plant tissue of Holcus lunatus (Poaceae) (Scheirs et al., 2001). In β-glucosidases to toxic saligenin is markedly reduced at an another grass species such as barley Hordeum vulgare (Poaceae) alkaline pH, as Salix β-glucosidases function optimally at the cyanogenic glucoside epiheterodendrin accumulates in around pH 5 (Ruuhola et al., 2003). The alkaline midgut the epidermal cells (Nielsen et al., 2002; Li et al., 2011). It prevents formation of toxic saligenin and enables larvae to can be imagined that leaf-mining insects that do not feed on ingest salicortin and to excrete non-toxic salicin in their frass epidermal cells would avoid exposure to such epidermally (Fig. 2F) (Ruuhola et al., 2001). localized chemical defence compounds. It is important to note that numerous insect herbivores with an alkaline midgut are known to feed on plants that (3) During digestion: physiological and metabolic are not protected by two-component chemical defences, but adaptations to counteract two-component defence by diverse other toxic plant chemicals (Berenbaum, 1980). Thus, a highly alkaline midgut probably did not arise as (a) An alkaline gut pH inhibits the plant β-glucosidase an evolutionary response to two-component plant chemical Differences in pH may affect enzymatic reactions by defences. Insect herbivores with an alkaline midgut simply changing the charge of certain amino acids, affecting protein may have been pre-adapted to feed on plants protected by conformation and their catalysis (Johnson & Felton, 1996; two-component chemical defences. Harrison, 2001). Whereas the haemolymph of insects is In a classical study that employed tannins, polyphenol mostly neutral with pH ranging from 6.4 to 7.5, the glucosides that do not require β-glucosidases to exert pH of the midgut lumen varies from a strongly acidic toxicity, it was shown that generalist caterpillars of the pH 3.1 to extremely alkaline pH 12.4 among different gypsy moth Lymantria dispar (Lepidoptera, Erebidae) adjusted insect orders (Berenbaum, 1980; Dow, 1984; Schultz & the pH of their midgut according to the amount of ingested Lechowicz, 1986; Johnson & Felton, 1996; Appel & Joern, tannin from slightly to highly alkaline within a few hours 1998; Harrison, 2001; Cristofoletti et al., 2003; Fitzgerald, (Schultz & Lechowicz, 1986). This alkalinization leads to re- 2008; Terra & Ferreira, 2012). Regulation of midgut pH dissociation of harmful tannin–protein complexes and makes mainly involves H+ V-ATPases that are located in the tannins non-toxic (Schultz & Lechowicz, 1986). It would be apical membrane of goblet cells in the midgut (Wieczorek interesting to know if such an inducible alkalinization of the et al., 2003). Many larvae of Lepidoptera, Diptera and midgut is also found in insects that feed on plants defended scarab beetles (Coleoptera) have highly alkaline midguts, by two-component chemical defences or if these herbivores whereas larval Orthoptera, Hemiptera and most coleopteran have a constitutive alkaline gut. families usually have slightly acidic to neutral midguts. Evolutionary costs associated with having an alkaline gut Consequently, lepidopteran digestive enzymes like amylases may be related to entomopathogenic bacteria such as Bacillus are evolutionarily adapted to function in an alkaline gut thuringiensis that produce insecticidal δ-endotoxins forming (Pytelkova´ et al., 2009). However, unfavourable highly lytic pores in the cell membrane of the midgut epithelium (de alkaline pH conditions in the midgut lumen may inhibit plant Maagd, Bravo & Crickmore, 2001; Bravo, Gill & Soberon,´

2007). Solubilization and activation of δ-endotoxin protoxins of insect β-glucosidase activity is not complete and some requires mainly alkaline conditions in addition to specific aucubin is still hydrolyzed. Further adaptations for long-term proteolytic enzymes (de Maagd et al., 2001; Bravo et al., 2007). feeding may include dietary mixing by host-plant switching Consequently, lepidopteran and dipteran larvae with their (see Section III.1) (Pankoke et al., 2012) alkaline midguts are often more vulnerable to B. thuringiensis When feeding on a diet with an increasing concentration δ-endotoxins than coleopteran larvae with a neutral gut of the salicinoid salicortin, the generalists gypsy moth environment that keeps protoxins insoluble and prevents and forest tent caterpillar Malacosoma disstria (Lepidoptera, their activation (Lambert et al., 1992; Bradley et al., 1995; de Lasiocampidae) adaptively reduced the activity of their Maagd et al., 2001). endogenous β-glucosidases in the midgut (Hemming & Lindroth, 2000). This inducible reduction in β-glucosidase (b) Reduction of endogenous insect β-glucosidase activity in the gut activity prevents formation of toxic aglucones and enables feeding, but it may also reduce the efficiency of digestion, β Since endogenous insect -glucosidases are mainly digestive which could explain why both species show slightly decreased enzymes, they may also activate two-component plant growth rates on this diet (Hemming & Lindroth, 2000). chemical defence after ingestion of plant material, which Even at an intraspecific level, activity of endogenous insect renders them a target for insect adaptation. Indeed, several β-glucosidases may differ. A subspecies of the lepidopteran specialists and generalists were shown to reduce activity eastern tiger swallowtail Papilio glaucus canadensis (Lepidoptera, of their endogenous β-glucosidases, efficiently reducing Papilionidae) is a specialist adapted to salicinoids and feeds formation of toxic aglucones during digestion. extensively on poplars and willows (Lindroth, 1988). By Bruchid beetles C. maculatus are generalists that develop on contrast, the subspecies P. g. glaucus is susceptible to salicinoids seeds of the cow pea Vigna unguiculata (Fabaceae) (Desroches and avoids feeding on those plants. Dietary exposure of both et al., 1997). Seeds of the broad pea Vicia faba, however, exert subspecies to salicin and salicortin reduced the activity of a strong toxicity to C. maculatus larvae caused by activation specific β-glucosidase in P. g. canadensis, while the activity was of the alkaloid glucoside vicine by larval endogenous β- P. g. glaucus glucosidases (Fig. 2A) (Desroches et al., 1995). Interestingly, increased in (Lindroth, 1988). The lower activity β some larvae of C. maculatus are able to develop on vicine- of endogenous midgut -glucosidases in P. g. canadensis containing V. faba seeds (Desroches et al., 1995). This explains their adaptation to dietary intake of plant salicinoids. tolerance is due to decreased expression of a gene encoding An inducible reduction of endogenous midgut β- a β-glucosidase that is active towards vicine (Desroches glucosidase activity was shown for desert locusts when et al., 1997). The decreased expression of this β-glucosidase feeding on Schouwia purpurea (Brassicaceae), a plant which results in lowered β-glucosidase activity in the midgut of the contains glucosinolate concentrations tenfold higher than in larvae and strongly reduces formation of toxic aglucones, most other crucifers, in comparison to feeding on non-toxic even when feeding on seeds containing vicine at 1% dry plants (Mainguet et al., 2000). Such a reduction of midgut mass. Only concentrations higher than 1.5% vicine in the β-glucosidase activity enabled S. gregaria to feed without any experimental diet caused lethal effects on feeding larvae, but adverse effects for at least 1 week. Feeding for more than such concentrations are not found naturally in V. faba seeds 3 weeks on S. purpurea resulted in slower mass gain (Mainguet (Desroches et al., 1997). et al., 2000), which could reflect lower digestion rates. The Mexican bean beetle is a specialist that feeds on Inducible and selective reduction of the endogenous insect lima beans containing cyanogenic glucosides without any β-glucosidase most active on glucosylated plant defence adverse effects (Ballhorn et al., 2010). It was found that compounds avoids intoxication while maintaining efficient bean beetles strongly decrease the activity of their own digestion of ingested plant material. Generalist caterpillars endogenous β-glucosidases in the gut relative to gut β- of the sugar cane borer Diatraea saccharalis (Lepidoptera, glucosidase activity from desert locusts feeding on the same Crambidae) have three β-glucosidases, βGly1–3,intheir plant species (Ballhorn et al., 2010). After ingestion the midgut (Ferreira, Parra & Terra, 1997). Dietary exposure cyanogenic glucosides are not hydrolysed in the bean beetle of the sugar cane borer to the cyanogenic di-glucoside and can be excreted intact with the frass. amygdalin results in degradation to the cyanogenic mono- The generalist caterpillars yellow woolly bear and tiger glucoside prunasin due to βGly1 and βGly3 activity. moth feed on iridoid-glucoside-containing plants such as P. However, activity of βGly2, which hydrolyses prunasin lanceolata. Both species reduce the activity of endogenous further into toxic aglucones, was strongly reduced (Azevedo, β-glucosidases in their midgut in response to increasing Terra & Ferreira, 2003). Moreover, general digestion is levels of the iridoid glucoside aucubin (Fig. 2E) (Pankoke not affected since the remaining two β-glucosidases, which et al., 2010, 2012). A decrease of endogenous midgut β- hydrolyse plant oligosaccharides, are still active (Azevedo glucosidase activity enables both herbivore species to feed et al., 2003). on toxic aucubin-containing plants at least for short periods Adaptation may also be due to a lack of endogenous (Pankoke et al., 2010, 2012). However, larvae feeding on insect β-glucosidases able to hydrolyse glucosylated plant plants with high concentrations of aucubin weighed less and defence compounds. This was shown for fall armyworm developed more slowly compared to larvae feeding on plants larvae that can be reared on an artificial diet containing with low aucubin concentrations, probably since reduction amygdalin without any adverse effects or any reduction in

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 14 Stefan Pentzold and others insect β-glucosidase activity (Ferreira et al., 1997). Amygdalin (Fig. 2C) (Engler, Spencer & Gilbert, 2000). Moreover, may be hydrolysed to the mono-glucosylated prunasin, but after catabolism of the cyanogenic glucoside epivolkenin, the the two digestive β-glucosidases present in the midgut of the released nitrogen is used in the insect’s primary metabolism. fall armyworm are unable to further hydrolyse prunasin into Diamondback moth Plutella xylostella (Lepidoptera, toxic aglucones (Marana, Terra & Ferreira, 2000). General Plutellidae) caterpillars are specialized feeders on crucifer digestive processes are not affected since both β-glucosidases plants, which are primarily defended by the glucosinolate- hydrolyse cellulose from the ingested plant material. myrosinase two-component system. To prevent activation Adverse costs at a trophic level for insect endogenous of glucosinolates, larvae possess a sulfatase in their gut that β-glucosidase activity were shown for the cabbage white converts all major classes of glucosinolates into desulfo- butterfly Pieris rapae (Lepidoptera, Pieridae) feeding on glucosinolates, which can no longer be activated by cabbage plants containing glucosinolates (Mattiacci, Dicke myrosinases (Fig. 2D) (Ratzka et al., 2002). The insect & Posthumus, 1995). Regurgitate of P. rapae contains β- sulfatase directly competes with the plant myrosinase for glucosidase activity that elicits a blend of volatiles from the glucosinolates, and furthermore the released sulfate inhibits plant that was highly attractive to parasitic wasps—natural myrosinases (Ratzka et al., 2002). Sulfatase expression is enemies of P. rapae. By contrast, the plant β-glucosidase from under tight developmental and tissue-specific control, since the cabbage leaf extract was not as efficient as the insect transcripts are constitutively present in the larval gut, the β-glucosidase in releasing the volatiles. only stage and organ in the diamondback moth life cycle In many cases, except in that of an alkaline gut (see that is exposed to glucosinolates, but sulfatase transcripts Section III.3a), it is unclear how the activity of endogenous are absent in other tissues and developmental stages (Ratzka β-glucosidases is reduced. It may be that transcription of et al., 2002). Desert locusts that are generalists also possess the gene or translation of its mRNA is decreased, that the a sulfatase with broad substrate specificity in their gut. enzyme is inhibited by other factors than pH or that its Sulfatase activity enables feeding even on S. purpurea,a substrate specificity is altered. It would be interesting to plant with very high concentrations of glucosinolates (Falk & analyse how reduction in activity of endogenous insect β- Gershenzon, 2007). Following feeding on a glucosinolate-free glucosidases may affect digestive processes in the gut, how this diet, sulfatase activity increased tenfold when locusts were fed interacts with nutrient uptake and how insect development S. purpurea, and decreased when glucosinolates were removed may be influenced. from the diet. Thus, sulfatase activity in generalist desert locusts is highly inducible, whereas it seems constitutive in (c) Specialized enzyme activity of insects the specialist diamondback moth larva. Apart from β-glucosidases, insects possess a variety of other The interplay between specialized insect enzymes that and more specialized enzymes involved in counteracting are active before plant β-glucosidases and sequestration plant chemical defence. Cytochrome P450 monooxygenases as a further adaptation (see Section III.3d) was shown are important enzymes in insect metabolism and resistance in the sawfly Athalia rosae (Hymenoptera, Tenthredinidae). to many kinds of toxins such as insecticides, drugs and Sawfly larvae take up glucosinolates from the gut into specialized plant metabolites (Scott, 1999; Schuler, 2011; their haemolymph where they are degraded to desulfo- Feyereisen, 2012). However, cytochrome P450s seem to glucosinolates by sulfatases, and subsequently sulfated at the be of lesser importance in insect herbivores that are glucose moiety by sulfotransferases (Fig. 2D) (Opitz et al., adapted to two-component plant chemical defence, because 2011). Since excess glucosinolates are transported back into detoxification via cytochrome P450s has not been reported the gut and excreted via the frass, prior conversion in the on these compound classes (Schuler, 2011; Feyereisen, 2012; haemolymph is highly adaptive, since modified glucosinolates Tao et al., 2012). Instead, insect enzymes convert defence can no longer be activated by the remaining plant β- compounds either (i) before their hydrolysis by plant β- thioglucosidases in the gut (Müller, 2009; Opitz et al., 2011). glucosidases, i.e. the modified product can no longer be ( ii ) After hydrolysis by plant β-glucosidases. Even when activated by plant β-glucosidases into toxic aglucones, or (ii) plant β-glucosidases activate the corresponding glucosylated after their hydrolysis by plant β-glucosidases, via conjugating defence compounds into toxic aglucones, some insects have or detoxifying enzymes that render aglucones easier to evolved enzymes for counteraction. Conjugating enzymes excrete or less toxic (Fig. 2). add a glucose or glutathione moiety to the aglucone to ( i ) Before hydrolysis by plant β-glucosidases. Insect increase its water solubility and excretion efficiency. Others, enzymatic conversion of glucosylated defence compounds more specialized detoxification enzymes modify specific before their hydrolysis by plant β-glucosidases has been target substrates and render them non-toxic. shown in specialist caterpillars of the neotropical Sara For conjugation, glucose is activated by linkage to uridine longwing Heliconius sara (Lepidoptera, Nymphalidae) that diphosphate via a phosphoester bond. Activated glucose feed on cyanogenic-glucoside-containing Passiflora auriculata is then used to form O-glucosides, an enzymatic reaction (Passifloraceae) plants. This insect species prevents plant β- catalysed by family 1 UDP-glucosyltransferases (UGTs). Re- glucosidase activity on cyanogenic glucosides by metabolic glucosylation of toxic aglucones into non-toxic glucosides replacement of the nitrile group with a thiol group in directly in the gut is an efficient adaptation that allows the cyanogenic glucoside by an as yet unknown enzyme rapid and non-toxic excretion of the glucoside. For example,

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society Insect adaptations to plant chemical defence 15 the generalist larvae of the Egyptian armyworm and fall 2008; Müller et al., 2010), another study showed negative armyworm re-glucosylate toxic aglucones such as DIMBOA effects of P. rapae larvae feeding on plants with increased into non-toxic DIMBOA-glucoside via UGTs in the gut and glucosinolate levels, and a less efficient NSP (Kos et al., excrete them (Fig. 2B) (Glauser et al., 2011). Similarly, UGT 2012). It might be that nitriles, generated by NSP activity, activity in the rice armyworm Mythimna separata (Lepidoptera, are still toxic to P. rapae, but to a lesser extent (Burow & Noctuidae) midgut leads to formation and excretion of non- Wittstock, 2009), or that higher glucosinolate concentrations toxic DIMBOA-glucosides, which enables these generalists to impose higher expression of NSP, which is metabolically grow on a DIMBOA-containing diet (Sasai et al., 2009). UGT more costly (Kos et al., 2012). It would be interesting activity, and thus resistance to plant defence compounds, to see if NSP expression level increases with increasing can also differ between closely related species. Whereas the glucosinolate concentrations, and whether such an induction Asian corn borer Ostrinia furnacalis (Lepidoptera, Crambidae) involves higher metabolic costs. Similarly, it has been is able to feed on an DIMBOA artificial diet without adverse shown that young larvae grow more slowly with increasing effects, survival of the congener adzuki bean borer Ostrinia glucosinolate concentration on Brassica napus plants (Rotem, scapulalis is strongly affected (Kojima et al., 2010). Differences Agrawal & Kott, 2003), whereas older larvae were much in resistance were linked to a higher activity of midgut UGTs less affected by increasing glucosinolate concentrations. in O. furnacalis than in O. scapulalis. After NSP activity, nitriles from aliphatic glucosinolates are Conjugation of reduced glutathione to aglucones is excreted unmodified with the frass, whereas nitriles from another adaptation for some insects and is carried out by aromatic glucosinolates are α-hydroxylated into an unstable glutathione S-transferases (GSTs). GSTs have substrates with intermediate that decomposes spontaneously to an aldehyde an electrophilic centre, such as α/β-unsaturated carbonyl and cyanide (Fig. 2D) (Vergara et al., 2006; Agerbirk et al., compounds, that may be electrochemically mimicked 2010; Stauber et al., 2012). Cyanide in P. rapae is detoxified by isothiocyanates derived from glucosinolate-myrosinase by β-cyanoalanine synthase and rhodanese activity into non- activation (Brattsten, 1988). Hence, the generalists cabbage toxic β-cyanoalanine and rhodanide (Stauber et al., 2012). moth Mamestra brassicae, cotton bollworm Helicoverpa These detoxification enzymes might have enabled pierid armigera (both Lepidoptera, Noctuidae), fall armyworm, species to shift from the cyanogenic-glucoside-containing Egyptian armyworm and the cabbage looper, which Fabales to the aromatic-glucosinolate-containing Brassicales feed on glucosinolate-containing plants, conjugate toxic around 75 million years ago (Stauber et al., 2012). isothiocyanates with glutathione by GST activity in their Larvae of the six-spot burnet moth emit toxic gut and subsequently excrete non-toxic products with the hydrogen cyanide due to endogenous β-glucosidase activity frass (Fig. 2D) (Schramm et al., 2012). Induction of GST on sequestered or biosynthesized cyanogenic glucosides activity in response to increasing glucosinolate concentrations (Zagrobelny et al., 2008; Zagrobelny & Møller, 2011). was shown in green peach aphids (Francis, Vanhaelen Detoxification enzymes such as β-cyanoalanine synthase & Haubruge, 2005) as well as in the midgut of gypsy enable burnets to endure the permanent exposure to moth larvae and forest tent caterpillars when feeding on hydrogen cyanide (Zagrobelny et al., 2008; Zagrobelny & β plants containing salicinoids (Hemming & Lindroth, 2000). Møller, 2011). -cyanoalanine synthase activity converts cyanide with the amino acid cysteine into β-cyanoalanine These results suggest that detoxification by GST activity and subsequently into non-toxic asparagine, another amino may be widespread in generalist lepidopterans and efficient acid (Fig. 2C) (Zagrobelny et al., 2004; Zagrobelny & Møller, on different classes of two-component chemical defence 2011). In agreement with the target site of hydrogen cyanide (Schramm et al., 2012). toxicity, β-cyanoalanine synthase activity is found mainly in Detoxification enzymes are for example reported mitochondria (Watanabe et al., 2008). from the cabbage white butterfly, a specialized feeder on glucosinolate-containing crucifers. Larvae possess an endogenous nitrile specifier protein (NSP) in their midgut (d) Sequestration: spatial separation of plant β-glucosidase and that redirects glucosinolate hydrolysis towards the less toxic β-glucoside in the insect nitriles instead of the more toxic isothiocyanates (Fig. 2D) Some specialized insects are able selectively to take up (Wittstock et al., 2004). NSP may act as a cofactor binding and accumulate plant defence compounds in their body to the plant myrosinase to modify glucosinolate hydrolysis, tissues, e.g. the haemolymph or defence glands (Nishida, or it may use the aglucone directly as a substrate to generate 2002; Opitz & Müller, 2009). This so-called sequestration nitriles (Wittstock et al., 2004). Insect NSP activity seems to is an efficient adaptation, since glucosylated plant defence have evolved shortly after their host plants evolved, and to compounds are no longer accessible to enzymatic hydrolysis have enabled adaptive radiation of the Pierinae subfamily by ingested plant β-glucosidases that remain in the gut with significantly elevated species numbers in comparison lumen. Furthermore, sequestering insects become toxic to to related clades (Wheat et al., 2007). Recent studies on potential predators (Kazana et al., 2007; Zagrobelny et al., the detoxification efficiency of the NSP, however, have 2008; Discher et al., 2009). To date, more than 250 insect shown conflicting results. Whereas P. rapae larvae feeding on species from different orders have been shown to sequester plants with increased glucosinolate levels showed high NSP glucosylated and non-glucosylated plant defence compounds efficiency and no adverse effects in some studies (Gols et al., from over 40 plant families, but little is known about potential

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 16 Stefan Pentzold and others membrane carriers and their transport mechanism (Opitz & and mixing of both components. The Brassica specialists, Müller, 2009). the cabbage aphid, and the mustard aphid Lipaphis erysimi Alkaloid, benzoxazinoid, cyanogenic and iridoid gluco- (Hemiptera, Aphididae), sequester intact glucosinolates from sides as well as glucosinolates and salicinoids are polar their host plants and possess an endogenous myrosinase. compounds due to their β-d-glucopyranose moiety. Polar- These insects prevent hydrolysis of glucosinolates by storing ity prevents diffusion through the gut membrane into the their own myrosinases in crystalline microbodies in non-flight haemolymph, where plant defence compounds are no longer muscles, whereas sequestered plant glucosinolates are stored accessible to plant β-glucosidases that remain in the gut separately in the haemolymph (Bridges et al., 2002; Kazana (Müller, 2009; Opitz & Müller, 2009). Thus, transport of et al., 2007). Six-spot burnet moth larvae sequester the glucosylated compounds into the haemolymph has to be cyanogenic glucosides linamarin and lotaustralin from their specific and efficient since plant β-glucosidases immediately L. corniculatus food plants and store them in all tissues including hydrolyse them after tissue damage and this hydrolysis may the haemolymph, where endogenous β-glucosidases are continue in the gut (Desroches et al., 1997; Pankoke et al., also present (Zagrobelny et al., 2008). Non-separate storage 2012). Ingested plant β-glucosidases thus may have driven of both components leads to a continuous hydrolysis of the evolution of efficient transporters in insects (Müller & cyanogenic glucosides and emission of hydrogen cyanide Wittstock, 2005; Müller, 2009). As the midgut is the most for defence. Larvae of the Sara longwing sequester the permeable part of the digestive tract (Dow, 1986), gen- cyanogenic glucoside epivolkenin from their P. auriculata host eral transporters may enable most glucosides to enter the plants (Engler et al., 2000). Sequestration is highly selective haemolymph, and more specialized transporters may chan- since only epivolkenin is taken up and accumulated, whereas nel only specific substrates into the final tissue (Kunert et al., the other plant cyanogenic glucosides are not sequestered. 2008; Burse et al., 2009). Larvae of the leaf beetles Chrysomela populi and Phratora (e) Single amino acids counteract plant β-glucosidase activity vitellinae (both Coleoptera, Chrysomelidae) sequester the In addition to the various adaptations that often target Salix salicinoid salicin from their spp. food plants and plant β-glucosidase activity, single amino acids were transport it intact from the gut into the haemolymph shown to counteract activity of plant β-glucosidases and further on into their defence glands (Kuhn et al., mainly by reducing the toxicity of released aglucones. 2004; Burse et al., 2009). Membrane carriers with moderate Several lepidopteran and hymenopteran larvae secrete selectivity transport salicin through the gut membrane into extraordinarily high amounts of glycine, β-alanine or γ - the haemolymph (Discher et al., 2009; Opitz & Müller, 2009), aminobutyric acid (GABA) into their gut lumen when feeding whereas highly specific carriers that match the orientation of on plants containing the iridoid glucoside oleuropein (Konno the functional group of the glucopyranose ensure that only et al., 1997, 1998, 2010; Konno, Okada & Hirayama, salicin is transported into the defence glands (Kuhn et al., 2001). These single amino acids protect insect digestive 2007; Discher et al., 2009). Activity of insect β-glucosidases enzymes and dietary protein by competing with the amino in the defence glands hydrolyses salicin to the deterring residue in the lysine side chains of proteins for the protein- compound saligenin (Fig. 2F), which may be finally oxidized crosslinking activity of aglucones (Konno et al., 2010). Leaves to salicylaldehyde (Kuhn et al., 2004; Opitz & Müller, 2009). of the privet tree are defended by oleuropein, but larvae Highly evolved chrysomelina leaf beetles such as Chrysomela of the lepidopteran Brahmaea wallichii (Brahmaeidae) and lapponica are able to sequester a wide variety of glucosylated Dolbina tancrei (Sphingidae) or the hymenopteran Macrophya leaf alcohols into their defence glands (Hilker & Schulz, 1994; timida (Tenthredinidae) can feed on this plant by secreting Schulz, Gross & Hilker, 1997; Burse et al., 2009). After insect extremely high concentrations of free glycine (up to 164 mM) β-glucosidase activity, aglucones are esterified with butyric into their midgut (Konno et al., 1997, 2010). This is more acid derived from the insect’s own amino acid pool finally than 20-fold higher than seen in non-privet feeders (Konno to produce a diversity of deterring esters (Hilker & Schulz, et al., 2010). Specific glycine-transporters mediate secretion 1994; Schulz et al., 1997). in the anterior part of the midgut (Konno et al., 1998, Absence of β-glucosidase activity in the insect 2001). Secretion in the anterior midgut is adaptive, since haemolymph seems to enable bruchid beetles C. maculatus denaturation and lysine decrease begin immediately after to sequester intact plant defence compounds (Desroches leaf damage and it is beneficial to stop this reaction as soon et al., 1997). The alkaloid glucoside vicine was shown to be as possible after ingestion (Konno et al., 2001). Interestingly, transferred intact from the midgut into the haemolymph, when non-adapted privet feeders, such as caterpillars of where it is stored without chemical transformation until the the Eri silkworm Samia ricini (Lepidoptera, Saturniidae) are adult instar stage of larval development (Desroches et al., fed privet leaves together with high levels of free glycine, 1997). the toxic effects of plant β-glucosidase-activated oleuropein Sequestering insects that produce β-glucosidases that can are completely prevented and the caterpillars grow without activate sequestered defence compounds might benefit from apparent detrimental effects (Konno et al., 2009). Other separating both components spatially in their body. As in lepidoptera such as Artopoetes pryeri (Lycaenidae) secrete high plants, compartmentalization ensures that insect predators concentrations of GABA (up to 60 mM) into their midgut, are exposed to toxic aglucones only upon tissue damage or high levels of β-alanine (up to 35 mM), as shown in

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society Insect adaptations to plant chemical defence 17 caterpillars of Amphipyra monolitha (Noctuidae) (Konno et al., specialists. For instance, both groups possess a sulfatase with 2010). In this generalist, the secretion is induced only when a broad specificity in their gut (Ratzka et al., 2002; Falk feeding on privet trees, while no secretion occurs when & Gershenzon, 2007). However, specialized enzymes for feeding on a plant species devoid of oleuropein (Konno et al., detoxification such as NSP and β-cyanoalanine synthase are 2010). Since aglucones of other two-component defence most often found in specialists (Barrett & Heil, 2012), because classes also have protein-denaturing activities, secretion of such enzymes act on very specific substrates (Wittstock et al., high levels of certain amino acids into the midgut may be a 2004; Zagrobelny & Møller, 2011). In turn, broad-substrate- potential adaptation in other insect herbivores. specificity conjugating enzymes such as UGTs and GSTs are mainly found in generalists (Kojima et al., 2010; Schramm (4) Do generalists and specialists have different et al., 2012), and induction of enzyme activity such as GSTs types of adaptations? and sulfatases has also only been found in generalists so far (Francis et al., 2005; Falk & Gershenzon, 2007). Enzyme Do generalists and specialists have different types of induction that is sensitive to the level of plant defence adaptations? This question is relevant since generalists compound reduces metabolic costs of toxin processing and and specialists differ in their host range and hence is especially adaptive for generalists that encounter a variety the β-glucosidase-activated plant defence compounds they of plant defence compounds (Falk & Gershenzon, 2007). encounter. Could the exposure to several defence compounds Sequestration of plant defence compounds activated by plant as in generalists result in adaptations that are inducible by β-glucosidases is mainly found in specialists (Bridges et al., plant defence compounds or have broad effects on different 2002; Discher et al., 2009; Opitz, Jensen & Müller, 2010; classes of compounds? Conversely, for specialists, could Zagrobelny & Møller, 2011), and only in one generalist exposure to single defence compounds lead to constitutive species so far (Desroches et al., 1997). If given the opportunity, adaptations or adaptations that are highly effective, but only most such specialized insects sequester constitutively, but the on a small number of similar compounds? concentration of sequestered plant defence compounds in The adaptation of host-plant switching seems to support the insect varies during larval development, and there is a these notions, since this behaviour is mainly shown by balance between uptake and turnover (Müller & Wittstock, generalists. The frequency of host-plant switching depends 2005). In all cases, sequestration of intact glucosylated plant on the toxicity of the plant defence compound (Singer et al., defence compounds seems to require further adaptations 2002) and probably its concentration in the insect body. such as non-disruptive feeding, an alkaline gut lumen, Thus, the decision to switch host plants in generalists may reduced endogenous insect β-glucosidase activity or the be inducible by both factors. Feeding guilds such as non- presence of other insect enzymes that counteract plant β- disruptive piercing-sucking and leaf-snipping are efficient glucosidase activity as discussed in Section III.3c.Secretion adaptations found in both generalists and specialists (Bernays of high levels of single amino acids has mainly been found in & Janzen, 1988; McMahon et al., 1995; Bridges et al., 2002; specialists feeding on iridoid-glucoside-containing plants, and Barth & Jander, 2006; Ballhorn et al., 2010). However, only in a few generalists. Species from different lepidopteran from two lepidopteran families there are morphological and hymenopteran families have evolved this ability (Konno indications that generalists and specialists do differ. Mandible et al., 1997, 2010), and it seems to be a constitutive adaptation structure of generalist species within each group was similar, in these specialists. In generalists, secretion of amino acids whereas specialists had very characteristic mandibles, each may be inducible by plant iridoid glucosides as shown in the of unique design and related to the nature of its host leaf case of the caterpillar A. monolitha (Konno et al., 2010). (Bernays & Janzen, 1988). Adaptations that occur during Whereas some types of adaptation are found in both gen- digestion of plant material such as an alkaline pH in the gut eralists and specialists, others appear to be limited to either lumen were mainly shown in generalists and could have a generalists or specialists. Most striking is the observation broader adaptive effect (Ruuhola et al., 2003; Fitzgerald, that adaptations are often inducible in generalists, whereas 2008; Dutartre et al., 2011). The question of whether adaptations in specialists seem to be constitutive. alkalinity is inducible has not been answered clearly yet, because only a few studies have measured the pH of empty as well as filled guts. In addition, studies were only carried out on generalists showing that midgut lumen alkalinity IV. CONCLUSIONS is constitutive in fall webworm larvae (Fitzgerald, 2008), whereas it is inducible in gypsy moth larvae (Schultz & (1) The various adaptations of insect herbivores to Lechowicz, 1986). A reduction of endogenous midgut β- widespread two-component plant chemical defences, i.e. glucosidase activity that is sensitive to and inducible by the β-glucosidase-activated defence compounds, occur before level of two-component defence chemical is mainly found and during feeding as well as largely during digestion, and in generalists (Hemming & Lindroth, 2000; Mainguet et al., include the insect’s behaviour, physiology and metabolism. 2000; Azevedo et al., 2003; Pankoke et al., 2012), and only Moreover, adaptations from these different time points and in a few specialists (Lindroth, 1988; Ballhorn et al., 2010). levels are often combined in both generalists and specialists When comparing further enzymatic adaptations, we find as well as for the different classes of two-component chemical both similarities and differences between generalists and defence. Adaptations between generalists and specialists may

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society 18 Stefan Pentzold and others differ. In generalists, adaptations are often inducible and may for Independent Research, Technology and Production have broader effects, whereas they seem to be constitutive Sciences. We thank two anonymous reviewers for their and provide more specific adaptations in specialists. helpful suggestions. (2) Insect adaptations often target the activity of the enzymatic key component of the plant’s two-component defence, the β-glucosidase, at different time points of feeding VI. REFERENCES as well as at different levels. Host-plant switching may reduce exposure to and adverse effects of plant defence compounds Agerbirk,N.,Olsen,C.E.,Poulsen,E.,Jacobsen,N.&Hansen, P. R. (2010). by preventing the accumulation of toxic levels of aglucones. 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(Received 23 November 2012; revised 3 September 2013; accepted 5 September 2013 )