Introduction

Carotenoids are life-sustaining molecules. They play such a critical role in photosynthesis that all life in an oxygenated environment depends on them (Britton 1995a). Carotenoids are one of the most ubiquitous groups of organic molecules known, but how they function in modulating insect–environment interactions is only beginning to be understood. In general, they are long conjugated chains of carbon with rings on either end, which may contain oxygenated functional groups. Carotenoids are essential in photosynthesis to harvest light energy and protect chlorophyll in times of excess light energy by quenching reactive oxygen species that are produced during photosynthesis and plant stress. In animals, their bright yellow to red colour is employed as mating signals and aposematic colouration. Although it is just beginning to be investigated, there is growing evidence that carotenoids are important mediators of ecological interactions in insects. Further research into the roles of carotenoids and their derivatives in insect ecology promises to dramatically expand our comprehension of their varied functions and importance (Blount and McGraw 2008).

Although Czeczuga et al. have published extensively on the quality and quantity of carotenoids in a number of organisms, including nearly all major groups of arthropods (Czeczuga 1976, 1980; Czeczuga and Mironiuk 1980; Czeczuga 1981, 1982; Czeczuga and Weyda 1982; Czeczuga 1985, 1986, 1988, 1990, 1991), and some functions of carotenoids in insects have been examined elsewhere (Fox 1976; Goodwin 1986; Blount and McGraw 2008), no broad review of the ecological roles of carotenoids in insects exists. Here, we explore the natural functions of carotenoids in insects with a focus on the known and potential modulating roles that carotenoids or their derivatives play in insect multitrophic and environmental interactions. These modulating functions may contribute significantly to shaping the evolution of many insect taxa.

We begin with a brief overview of carotenoid structures, nomenclature and biosynthesis. We then briefly review the diversity and functions of carotenoids in plants and discuss their uptake by insects. We review some of the key functions of carotenoids in insects, illustrating their fundamental importance with particular focus on the varied roles of carotenoids in mediating insect–plant interactions. Finally, we consider the roles of carotenoids in mediating multitrophic interactions. Throughout the review, we highlight areas in need of more research and attempt to link the fundamental physiological (or internal) roles of carotenoids with their more ecological (or external) functions. The varied and essential functions of carotenoids in insects as well as their diet-dependent uptake may provide a series of model systems well suited to studying niche specialization and the complex and poorly understood relationship between phenotype and genotype (Badyaev 2011 and references therein).

Nomenclature and structures

There are over 700 different identified carotenoid molecules and many more if all the potential isomers are considered; for instance, there are theoretically 1,056 possible (E/Z)-isomers of lycopene and 272 for β,β-carotene (Pfander 1992). Furthermore, carotenoids often have chiral centres, which greatly increase the number of possible isomers. However, naturally occurring carotenoids are generally in the all-E form and certain chiral structures predominate.

Carotenoids are divided into two groups: carotenes and xanthophylls. The carotenes are hydrocarbons and the xanthophylls are their oxygenated derivatives. Carotenoids derive much of their diversity from the addition of a number of different functional groups, which are most commonly attached to the rings or the ends of the molecule, but rarely to the centre. Most of the tetraterpenoids (i.e. carotenoids with 40 carbon atoms) are named by adding prefixes to the name ‘carotene’. These prefixes are Greek letters corresponding to the end groups. Note that both end groups are written to be unambiguous. For example, the carotenoid generally known as β-carotene is unambiguously named as β,β-carotene (Fig. 1). Those carotenoids with fewer than 40 carbon atoms are called apocarotenoids if the loss of atoms occurs at one end of the molecule, diapocarotenoids if it occurs at both ends and norcarotenoids if it occurs within the molecule. These shortened structures are also generally referred to as norisoprenoids (Britton 2008).

Fig. 1
figure 1

Three examples of carotenoids degrading to volatile apocarotenoids (α-, β-ionone and β-cyclocitral) via carotenoid cleavage oxygenases (Ibdah et al. 2006), fungal peroxidases (Zorn et al. 2003) or singlet oxygen (Ramel et al. 2012)

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Carotenoids are orange because of the large light-absorbing chromophore in the centre of the molecule. This sequence of conjugated double bonds absorbs light at about 450 nm and dissipates the energy as heat. Higher wavelengths of light are reflected, giving them their characteristic orange colour. The extended chromophore of lycopene absorbs more of the yellow–orange light and thus reflects red light. Carotenoids vary in their absorption maxima, which is useful for identification.

Carotenoid biosynthesis and diversity in plants

In plants, carotenoids are synthesized from precursors derived from the methylerythritol-4-phosphate (MEP) pathway in plastids (reviewed by Eisenreich et al. 2001, Hirschberg 2001). Generally, 3 units of isopentenyl diphosphate (IPP) and one unit of dimethyl allyl diphosphate (DMAPP) from the MEP pathway condense to form geranylgeranyl diphosphate (GGPP, C20) in plastids; two units of GGPP condense to form phytoene (Dudareva et al. 2006). Four rounds of phytoene dehydrogenation lead to lycopene, which can undergo cyclizations, dehydrogenations and oxidations to form numerous carotenoid molecules. In plants and algae, monoterpenes, diterpenes and carotenoids are synthesized similarly in the plastids (Lichtenthaler 1999). Movement of precursors from the plastids (MEP pathway) to the cytosol (mevalonic acid pathway) also occurs (Bartram et al. 2006; Dudareva et al. 2006) and may contribute to the formation of sesquiterpenes, sterols (triterpenes) and polyterpenes in the cytosol (Lichtenthaler 1999). Functional groups may be added in the cytosol as well (Grunewald et al. 2001).

The diversity of carotenoids in plant leaves (i.e. chloroplasts) is generally low with components of the xanthophyll cycle (i.e. violaxanthin, antheraxanthin and zeaxanthin), β,β-carotene, lutein, neoxanthin, β,ε-carotene, β-cryptoxanthin and lutein 5,6-epoxide being the most commonly occurring. The carotenoids in bold above are generally found at the highest concentrations in leaves and lactucaxanthin occurs in some species such as lettuce (Britton 1995b; Britton et al. 2004). Lycopene is a precursor to all of these, but the metabolic pull is likely so strong that it does not accumulate in the leaves. However, lycopene is a common component of red fruits such as tomato and pepper where photosynthesis is low or non-existent. Carotenoids often contribute to flower colour, but interestingly the majority of flower pigments are phenolics (i.e. anthocyanins). Fruit and flower carotenoids are often xanthophylls and as such they can be and often are conjugated to fatty acids (Britton 1995b), which require saponification to release the free carotenoid for proper identification. This is frequently the case for insect xanthophylls as well.

Carotenoid derivatives

Flavour chemists have long been aware of the production of volatile chemicals from the degradation of carotenoids (Stevens 1970). Numerous in vitro studies have shown that oxygenase, peroxidase and possibly lipoxygenase (Walter and Strack 2011) enzymes from microbes and plants have the capacity to cleave carotenoids to form volatile apocarotenoids (Zorn et al. 2003; Simkin et al. 2004; Baldermann et al. 2005; Bouvier et al. 2005; Lewinsohn et al. 2005a; Lewinsohn et al. 2005b; Auldridge et al. 2006; Goff and Klee 2006; Ibdah et al. 2006; Garcia-Limones et al. 2008; Scherzinger and Al-Babili 2008; Vogel et al. 2008). In addition, singlet oxygen is capable of cleaving carotenoids on its own and the products can have hormonal properties (Ramel et al. 2012).

Lewinsohn et al. (2005b) showed that a mutant tomato plant and a watermelon cultivar, both extremely deficient in lycopene, produced much less geranial and neral (which are insect semiochemicals, Table 1) compared to high lycopene producers, suggesting that these volatiles were produced via lycopene degradation. This could be explained by a general decrease in the terpenoid pathway, since these compounds are both monoterpenes (C10). However, the reason the mutant tomato did not produce lycopene was because it had a defective phytoene synthase, which would leave the upstream portion of terpenoid synthesis intact. As another example, β-ionone is easily generated via cleavage of β,β-carotene by the oxygenase CmCCD1 (Fig. 1) and white flesh melon plants deficient in β,β-carotene substrate, but with expression of CmCCD1, lack the production of β-ionone (Ibdah et al. 2006). Additionally, the same oxygenase can generate α-ionone from the cleavage of a δ-carotene (Fig. 1), geranylacetone from phytoene and pseudoionone from lycopene (Ibdah et al. 2006). Singlet oxygen reacts with β,β-carotene to form a range of hormonally active apocarotenoids (Fig. 1; Ramel et al. 2012). Studies similar to these are numerous and have illustrated the production of a variety of apocarotenoids, which may be very important as insect semiochemicals (Table 1).

Table 1 Some apocarotenoids known to be generated by oxidative cleavage of carotenoids and have biological activity in plants, animals or microorganisms
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Carotenoid acquisition by insects

Although animals can modify carotenoids (e.g. cleave and add functional groups), they generally cannot synthesize them de novo (Kayser 1982; Walter and Strack 2011). One study reported β,β-carotene being synthesized by cockroaches (Shukolyukov and Saakov 2001); however, the authors acknowledged that the potential contribution of symbiotic microorganisms could not be ruled out. In two recent studies (Moran and Jarvik 2010; Altincicek et al. 2011), genes laterally transferred from a fungus and integrated into arthropod genomes (i.e. aphids and mites) appear responsible for the de novo biosynthesis of torulene and related carotenoids, but how widespread these genes might be in insects is not known.

Insects generally sequester carotenoids in proportion to the concentration found in the diet (Feltwell and Rothschild 1974; Ahmad and Pardini 1990) and this often results in accumulation of lutein, which is the most dominant carotenoid in angiosperms (Pogson et al. 1996). However, they can also concentrate specific carotenoids in specific tissues with the aid of carotenoid binding proteins and active transport mechanisms (Kiefer et al. 2002; Bhosale and Bernstein 2007; Sakudoh et al. 2007) and this can be under hormonal control (Starnecker 1997). Mobile insects may also selectively feed on plants or plant parts to bolster their carotenoid intake in response to environmental stress or enemy attack (Smilanich et al. 2011). Stereospecific oxidative transformation of dietary carotenoids is common in insects resulting in a diversity of final carotenoid molecules in insect tissues, but these usually have structural backbones that represent their dietary source (Kayser 1982).

Diversity of carotenoid functions in insects

Carotenoids play many important roles in insect structure, physiology and life history. They provide colouration; are involved in vision, diapause and photoperiodism; serve as antioxidants; mating signals and precursors to pheromones. In order to understand their importance in ecological interactions, we first review some of their known functions in insects and then concentrate on their specific roles in mediating multitrophic interactions.

Colouration

Many insects use carotenoids to colour various portions of their bodies, eggs or even galls (Feltwell and Rothschild 1974; Davidson et al. 1991; Inbar et al. 2010a; Inbar et al. 2010b; White 2010), but few studies have investigated their adaptive significance (Oberhauser et al. 1996). The abundance of brightly coloured, sexually dimorphic butterflies would suggest that carotenoids are important in mate choice, but no studies have shown that carotenoids are involved in butterfly wing colour (Nijhout 1991; Shawkey et al. 2009). In monarchs, male wing colour does influence mating success, but the source of the orange wing colouration has not been identified (Davis et al. 2007).

Vision, diapause and photoperiodism

As in vertebrate systems, carotenoids are important to invertebrates as precursors to visual pigment chromophores such as retinal or 3-hydroxyretinal. The involvement of carotenoids in insect vision has been known for decades, but a recent key connection was made by Von Lintig et al. (2001) who found that blindness in a mutant Drosophilia strain is due to a dysfunctional carotenoid cleavage dioxygenase that is responsible for biosynthesizing Vitamin A, the direct precursor to the visual chromophores. Many other invertebrates have also been shown to require carotenoids or diet-derived Vitamin A to biosynthesize visual pigment chromophores (Stavenga 2006). The clear physiological and likely pleiotropic genetic connection between sequestration of carotenoids for vision and the induction of carotenoid-based colour polyphenisms in some lepidopteran larvae warrants further investigation.

While the mechanism of photoperiodic induction of diapause in arthropods has not been fully elucidated, carotenoids are clearly involved. The photoperiod induction of diapause requires carotenoids (or Vitamin A) in spider mites, moths, wasps and butterflies (Veerman 2001). The photoreceptor associated with photoperiod measurement appears to be an opsin receptor that requires Vitamin A (Veerman and Veenendaal 2003), while entrainment of the circadian rhythm is independent of vitamin A or carotenoids (Veerman 2001). It is not hard to imagine how the fundamental requirement for carotenoids in vision and diapause may dramatically influence ecological and evolutionary outcomes (Fig. 2), but to our knowledge, no studies have made this potential connection.

Fig. 2
figure 2

A stylized, schematic representation of the various known and hypothesized functions of carotenoids in insects that mediate ecological interactions. See online colour version

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Antioxidants

Several studies have demonstrated that carotenoids can act as antioxidants in insects. In mammals, ultraviolet (UV) radiation can enhance oxidative stress (Jurkiewicz and Buettner 1994; Shindo et al. 1994) and both UV radiation and oxidative stress are known to be damaging to arthropods (Ahmad and Pardini 1990; Aarseth and Schram 2002; Suzuki et al. 2009). Through a combination of blocking UV light, which activates prooxidant allelochemicals, and by direct quenching of singlet oxygen generated from prooxidant allelochemicals, carotenoids can protect vital cellular components from damage (Ahmad 1992; Carroll et al. 1997; Carroll and Berenbaum 2006). The abundance and diversity of carotenoids in particular insects may relate to the UV environment in which the species has evolved. Carotenoids can also contribute to the immune response in arthropods, probably by scavenging reactive oxygen species associated with up-regulation of the immune system (Ojala et al. 2005; Babin et al. 2010; Smilanich et al. 2011).

Mate choice and signalling

Volatile apocarotenoids are found as components of short-range courtship pheromones released from structures in male butterflies called hair-pencils and as pheromone components of many hymenopterans (Table 1). Males of Pieris napi butterflies produce citral, which is a mixture of the apocarotenoids neral and geranial (Fig. 2; Table 1). Odourless male models are always rejected by females, but both citral-laced models and freshly killed males stimulate female mate-acceptance behaviour (Andersson et al. 2007). In butterflies, these volatiles may be produced within the body from sequestered carotenoid precursors, although this has not been documented.

Roles of carotenoids and their derivatives in mediating insect–plant interactions

Mediation of oxidative stress

The antioxidant functions of carotenoids are likely a key feature in modulating insect–plant interactions as many plants produce photosensitized prooxidant compounds such as acetophenones, carboline alkaloids, furanochromes, furanocoumarins, furanoquinoline alkaloids, extended quinones, isoflavonoid phytoalexins, isoquinoline alkaloids, lignans, polyacetylenes and thiophenes (Berenbaum 1987). Metabolically activated compounds such as quinones and flavonoids are also important prooxidant allelochemicals. These compounds can react with molecular oxygen to produce a range of reactive oxygen species (ROS), including superoxide anion radical (O ·−2 ), hydrogen peroxide (H2O2), hydroxyl radical (·OH), lipid hydroperoxides, peroxyl radicals and singlet oxygen (1O2). Of these, ·OH and 1O2 are the most reactive and therefore damaging to cellular components such as membranes (Ahmad and Pardini 1990). Carotenoids are most effective against 1O2 either through physical or chemical quenching (Fig. 1), the latter of which cleaves the carotenoid molecule into smaller volatile apocarotenoids (Stratton et al. 1993; Sommerburg et al. 2003; Ramel et al. 2012), but they can also scavenge peroxyl radicals (Sommerburg et al. 2003). Carotenoids can also have significant indirect effects through the protection of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), which work together to deactivate O ·−2 . SOD converts O ·−2 to H2O2, which is rapidly converted to H2O and O2 by CAT before H2O2 forms the more damaging ·OH radical. Singlet oxygen and peroxyl radicals are known to inhibit SOD and CAT activity (Escobar et al. 1996) and thus carotenoids may play key roles in protecting insects and other organisms from both exogenous (e.g. via plant allelochemicals) and endogenous (e.g. via metabolic by-products) sources of ROS (Ahmad 1992).

Carroll et al. (1997) showed that parsnip webworms avoid damaging UVA light when reared on a carotenoid-free diet, but when the diet was amended with lutein (a xanthophyll) or the caterpillars were allowed to feed on their host plant, they did not avoid UVA radiation. UVA avoidance is of particular importance in this system because host plants contain UV-activated phototoxic furanocoumarins. Subsequently, Carroll and Berenbaum (2006) found a significant correlation between larval lutein concentration (i.e. sequestration) and daily UV irradiance in wild populations of webworms collected across an altitudinal/latitudinal UV gradient. More direct studies have also shown the benefits to insects of sequestered carotenoids. For example, β-carotene-amended diets provide significant protection from topical applications of alpha-terthienyl, a phototoxic phytochemical, in Manduca sexta larvae (Aucoin et al. 1990; Aucoin et al. 1995). And, an association between sequestration of carotenes and larval success has recently been documented in which carotene-sequestering larvae were much more likely to survive feeding on a toxic host plant (Shao et al. 2011).

The antioxidant properties of carotenoids may also augment the immune response in insects and other arthropods by scavenging ROS generated during this process (Cornet et al. 2007), but this may come with ecological costs associated with conspicuous pigmentation. Van der Veen (2005) showed a plastic regulation of carotenoid content in individual copepods in response to predation risk. Copepods that down-regulated their carotenoid content in response to elevated risk of predation were also more susceptible to parasite infection, suggesting a trade-off between predation avoidance and immunocompetence.

The antioxidant function of carotenoids in arthropods is an area in need of more study, particularly with respect to their roles in influencing interactions between herbivores and their host plants (Ahmad 1992; Felton and Summers 1995). The antioxidant properties of carotenoids may be enhanced by the presence of other antioxidants such as ascorbic acid or tocopherols (Catoni et al. 2008), but studies in arthropods are lacking. The importance of carotenoids in aposematism might be augmented by this potential positive interaction, since other potent antioxidants are colourless. The use of carotenoids as protective antioxidants may be important in shaping insect niches. They may allow herbivores to expand their dietary range to phototoxin-protected plants (or to specialize on such plants for their own defence), persist under conditions with high parasite pressure (Smilanich et al. 2011) or feed in more UV-rich habitats.

Carotenoids may also be important in managing oxidative stress associated with herbivory and pathogen attack in plants. Plants are predicted to benefit from limiting the spread of oxidative stress to localized tissues in order to limit the use of valuable antioxidants and to attract herbivore enemies. The cells closer to the damaged site are more likely to be in an oxidizing state and thus more conducive to the formation of apocarotenoids (Oliveira et al. 2011 and references therein). Attack by phytophagous insects and pathogens is known to cause the induction of several defensive pathways in plants (Maleck and Dietrich 1999). Bi and Felton (1995) showed unequivocally that the oxidative status of soybean plants changes after herbivory and that herbivores are negatively affected by this onslaught of oxygen radicals. Earlier work showed that pathogen infection resulted in a similar reaction (Mehdy 1994). During herbivory, antioxidants such as ascorbic acid, total carotenoids, non-protein thiols and catalase decreased systemically in plants (Bi and Felton 1995). The breakdown of carotenoids, chlorophylls and the production of VOCs may be symptomatic of autotoxicity or the result of strategic management of ROS by plants. Alternatively, these decreases may represent a strategy employed by herbivores and pathogens to overwhelm plant defences. One would predict that the intensity of this effect should increase with proximity to the damage site, thus explaining the more prevalent decrease in carotenoids and chlorophylls locally as opposed to systemically (see citations in the ‘Tritrophic signalling’ section below).

Resource signalling: colour

The essential physiological roles that carotenoids play in photosynthesis (Britton 1995a; Telfer et al. 2008) such as harvesting light energy and preventing photo-oxidative damage via the xanthophyll cycle (Demmig-Adams and Adams 1996) suggest that changes in host-plant carotenoid content should provide information to foraging herbivorous insects about the quality of the tissue, because photosynthesis ultimately provides carbon sources and energy for nitrogen fixation and/or assimilation. Conversely, if carotenoids correlate strongly with defensive chemicals, insects may be repelled.

W. D. Hamilton proposed that temperate deciduous plants signal their defences to herbivores via brightly coloured fall foliage (Archetti 2000). This idea is consistent with the finding that aphid diversity is greater on host plants with coloured fall foliage, suggesting a co-evolutionary history (Hamilton and Brown 2001). However, it may be more likely that coloured fall foliage is simply used as a cue rather than evolving as a signal (Schaefer and Rolshausen 2006). This controversial idea (Wilkinson et al. 2002) spurred a series of papers (reviewed in Lev-Yadun and Gould 2007) that proposed alternative ecological explanations for brightly coloured fall foliage. All of these hypotheses recognize the fact that the proximate reason for fall colour is to protect the plant from photoinhibition during chlorophyll scavenging and nitrogen storage when temperatures drop but light is still relatively intense. Theoretically, carotenoids could signal honestly to herbivores that the carotenoid biosynthetic pathway, which also leads to the biosynthesis of mono- and diterpenoids (Lichtenthaler 1999), is well established or still functioning. Monoterpenoids (De Moraes et al. 1998) and possibly apocarotenoids (see section ‘Resource signalling: odour’) are important attractants for natural enemies, while diterpenoids have demonstrated anti-herbivore properties (Gebbinck et al. 2002). Alternatively, it has been argued that green autumn leaves rather than coloured foliage should be more attractive to natural enemies and less nutritious to phloem-feeding aphids (Holopainen 2008). Although many studies have confirmed the physiological link between carotenoid quality/quantity and photosynthetic capacity (Telfer et al. 2008), only one study has shown that these pigment differences directly affect the behaviour and fitness of insect herbivores. Zheng et al. (2010) used gene-silencing techniques to show that plant tissues that lacked carotenoids and became photo-bleached were both less attractive to an ovipositing butterfly and less nutritious for its offspring than tissue from fully competent plants. Future studies in this regard will benefit from focusing on the fitness consequences of intraspecific host-plant variation in carotenoids where stabilizing or directional selection will likely be most obvious and quantifiable. Manipulative experiments designed to disrupt the biosynthesis of specific carotenoid pigment molecules would also be informative.

Many fruits and flowers derive their bright colours from carotenoids. In some cases, these colours may warn potential herbivores (particularly mammals) that the fruit is toxic, such as the red colour of capsaicin-containing red peppers; but more often these colours serve to attract seed dispersers and pollinators (Tewksbury and Nabhan 2001). Almost every orange or yellow fruit and many red fruits contain carotenoids. Indeed, the red colour of tomatoes is due mainly to the accumulation of the carotenoid lycopene.

The control of flower colour by carotenoid content is evident from breeding experiments and transgenic plants. Suzuki et al. (2007) were able to add a gene to the carotenoid biosynthetic pathway of Lotus japonicus, thus changing its flower colour from a lemon yellow to a bright orange. Ohmiya et al. (2006) changed wild-type white chrysanthemum flowers to yellow via RNA interference of a carotenoid cleavage dioxygenase (CCD) enzyme. These enzymes are widespread in plants and are very important in conferring odour and flavours to fruits and flowers. CCDs cleave carotenoid molecules at double bonds producing molecules with shorter chromophores that do not absorb visible light (see section ‘Resource signalling: odour’). The colour of fruits and flowers can have dramatic ecological consequences (Whitney and Glover 2007); for instance, Bradshaw and Schemske (2003) illustrated an immense reversal in pollinator preference when they bred colour changes into the monkeyflowers Mimulus lewisii and M. cardinalis. Furthermore, mutant M. lewisii with reduced carotenoid-based nectar guides receive fewer successful bumblebee visits (Owen and Bradshaw 2011), but Dyer et al. (2007) warn of the possible confounding effects of not using completely isogenic lines. Regardless of whether isogenic lines are used, the potential for confounding effects seems likely because CCDs can change both colour and odour simultaneously.

The most obvious selective force maintaining fruit colour would seem to be seed-dispersing frugivores such as birds, but evidence for this is surprisingly scarce (Whitney and Stanton 2004). Whitney and Stanton (2004) have shown that pleiotropic effects (or possibly linkage) appear to maintain a carotenoid-derived fruit polymorphism in Acacia ligulata via selection by a heteropteran seed predator. Interestingly, carotenoids may also be important in mate signalling in this system because females develop a yellow colour on the legs at sexual maturity. A number of plants incorporate toxic secondary metabolites in their ripe fleshy fruits, an apparent contradiction to the dispersal hypothesis (Cipollini 2000). There seems to be a delicate balance between dispersal, predation and microbial degradation that must be optimized by plants over space and time (Cipollini 2000; Tewksbury et al. 2008).

Resource signalling: odour

Carotenoids may also play important roles as olfactory cues for insects and as olfactory signals by plants. Volatile apocarotenoids (i.e. derivatives of carotenoids) have a range of functions that can benefit the plant directly, indirectly or are detrimental. Many apocarotenoids are likely to be pollinator attractants, since they are highly represented in flower volatiles (Table 1), including those of orchids (El-Sayed 2011). Interestingly, many also appear to be hymenopteran pheromones (Table 1) and some act as courtship pheromones in butterflies. Apocarotenoids are also associated with the flavour and odours of ripe fruit and likely benefit plants by attracting seed dispersers (Cipollini 2000). However, they can also act inadvertently as feeding attractants (Table 1).

Apocarotenoids, such as α- and β-ionone (Fig. 1), have been shown to be strong attractants (Williams et al. 2000) or deterrents (Wei et al. 2011 and references therein) of phytophagous insects. α-ionone may act as an intraspecific aggregation cue produced by actively feeding insects, which by continuously crushing plant cells, may mix cytosolic carotenoid cleavage oxygenases (CCOs) with plastid-localized carotenoids or may directly degrade them with CCOs in their saliva (Heath et al. 2002; Rhainds et al. 2007). These CCOs are known to be much more active under high light conditions (Scherzinger and Al-Babili 2008), and diurnal phytophagous beetles require high light conditions to find mates (Heath et al. 2001). However, apocarotenoids can also be produced chemically under oxidizing conditions, that is, without the direct aid of enzymes (Stratton et al. 1993; Gessler et al. 2002; Walter and Strack 2011; Ramel et al. 2012). This is interesting as continuously damaged plant cells experience oxidizing conditions, and many plants generate singlet oxygen and other ROS with the aid of photosensitized allelochemicals (Ahmad and Pardini 1990).

Manipulation of plant chemistry with carotenoid-derived hormones

Mutualistic associations with microbes have likely played an important role in the phenomenal evolutionary and ecological success of insects (Moran 2002; Janson et al. 2008). Virtually, every insect species that has been examined closely has been found to be engaged in some form of microbial mutualism, most frequently in the form of gut-associated bacteria (e.g. Buchner 1965; Douglas 1998). However, insect–fungal symbioses are also widespread across many insect groups including beetles (Scolytinae: Bentz and Six 2006), ants (Formicidae: Mikheyev et al. 2006), moths (Tortricidae: Fermaud and Lemenn 1989) and flies (Borkent and Bissett 1985; Gagné 1989; Schiestl et al. 2006; Heath and Stireman 2010). In these associations, the insect typically benefits from using the fungus as a food source (Bissett and Borkent 1988; Cherrett et al. 1989; Farrell et al. 2001; Janson et al. 2009; Heath and Stireman 2010) in exchange for dispersing the fungus or promoting fungal outcrossing (Schiestl et al. 2006). Given the current understanding of the hormonal regulation of plant defence chemistry, the ability of microbes to synthesize plant hormones (Table 2), and the induction of certain defence pathways in plants by microbes and herbivores, the potential exists for cooperating organisms to short-circuit these pathways towards their mutual advantage. Carotenoid derivatives may provide a means for such manipulation.

Table 2 An abbreviated list of fungi known to produce carotenoids or abscisic acid
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There are six groups of plant hormones traditionally recognized. These are auxin, ethylene, cytokinins, gibberellins, abscisic acid (ABA) and brassinosteroids (Kende and Zeevaart 1997). Recently, strigolactones were added as a seventh class (Pichersky 2008). Two of these classes, ABA and strigolactones, are derived in part or in whole from carotenoid precursors, and additional uncharacterized carotenoid-derived plant hormones may also exist (Walter and Strack 2011; Ramel et al. 2012).

Strigolactones are biosynthesized via the oxidative breakdown of carotenoid precursors via carotenoid cleavage oxygenases (Matusova et al. 2005; Humphrey and Beale 2006; Humphrey et al. 2006; Lopez-Raez et al. 2008) and have been found in plant species from at least 13 different families (Awad et al. 2006; Bouwmeester et al. 2007; Steinkellner et al. 2007; Yoneyama et al. 2007a; Yoneyama et al. 2007b). Cook et al. (1972) identified strigol as a germination stimulant for the parasitic weeds in the genus Striga, and numerous subsequent studies have verified this (reviewed in Scholes and Press 2008). More recently, experiments have shown that the long sought after fungal branching factor associated with successful colonization by arbuscular mycorrhizal fungi is a strigolactone, 5-deoxystrigol (Fig. 2; Akiyama et al. 2005). Strigolactones are also the causal agent in prohibiting above-ground lateral branching in Arabidopsis, rice and pea, illustrating their importance to plant architecture (Gomez-Roldan et al. 2008; Umehara et al. 2008) and quite possibly its manipulation by galling insects.

ABA is synthesized in plants from carotenoid precursors via a carotenoid cleavage oxygenase (Schwartz and Zeevaart 2004; Wasilewska et al. 2008). Interestingly, many fungi also have the ability to produce ABA (Table 2), but it appears that they use the mevalonic acid pathway and not carotenoid precursors (Schwartz and Zeevaart 2004). ABA plays a central role in seed development, stomatal regulation and plant responses to abiotic stressors such as osmotic, drought and possibly cold stress (Assmann 2004; Finkelstein 2004; Yamaguchi-Shinozaki and Shinozaki 2006); evidence is mounting that ABA is also centrally involved in regulating responses to biotic stressors (Anderson et al. 2004).

It is well established that plants react to biotic stresses such as viruses, bacteria, fungi and herbivorous insects by up-regulating defensive pathways (Mauch-Mani and Mauch 2005). These induced defences are regulated by two major biochemical signalling pathways: the salicylic acid (SA) pathway and the jasmonic acid/ethylene (JA) pathway (Spoel et al. 2003). When a plant is attacked, either the SA or JA pathway is induced, but generally not both. This is because the pathways can be reciprocally inhibitory (Kunkel and Brooks 2002; Cipollini et al. 2004; Lorenzo and Solano 2005). Furthermore, ABA can negatively interact with both the SA and JA pathways (reviewed by Lorenzo and Solano 2005; Mauch-Mani and Mauch 2005; Fujita et al. 2006; Asselbergh et al. 2008), thus playing a central role in regulating plant responses to both abiotic and biotic plant stresses. It is known that environmental conditions that induce increases in plant ABA levels (e.g. drought) enhance disease susceptibility in some pathosystems (e.g. Ma et al. 2001; Mayek-Perez et al. 2002; Koga et al. 2004; Garrett et al. 2006). Positive relationships between drought and disease severity may be related to the negative effect of ABA on biotic stress signalling. However, this response may not be universal as the severity of pathogen attack can also be negatively correlated with drought stress (Achuo et al. 2006; Enright and Cipollini 2011). However, directly increasing plant levels of ABA (either by exogenous application or via mutant plants) has been shown to negatively affect disease resistance in a number of studies (Henfling et al. 1980; Salt et al. 1986; Ward et al. 1989; McDonald and Cahill 1999; Audenaert et al. 2002; Mohr and Cahill 2003; Thaler and Bostock 2004; Mohr and Cahill 2007).

Studies supporting a negative relationship between ABA and plant resistance continue to accumulate. This opens the possibility that microbes or other plant-feeding organisms could manipulate their hosts through hormonal interference; however, documentation that microorganisms produce ABA at the infection site or actively regulate its level in plants are lacking. To our knowledge, only Kettner and Dorffling (1995) have shown unequivocally that fungus-derived ABA can explain variation in plant levels of ABA during the infection process of tomato plants by Botrytis fungi.

One of the few studies to evaluate the effects of ABA on both pathogens and insect herbivores found that ABA-deficient tomato mutants had lower levels of disease caused by Pseudomonas syringae, but supported higher growth rates of Spodoptera exigua larvae than control plants (Thaler and Bostock 2004). The mutants also showed higher levels of the SA-inducible PR4 gene, which likely contributed to the observed increase in pathogen resistance. The potential for cross-talk among these defensive pathways provides an opportunity for the evolution of mutualistic associations between biotrophic pathogens and insects; for instance, herbivore feeding could induce the JA pathway; thus, down-regulating the SA pathway or pathogens could induce the SA pathway, down-regulating the JA pathway. Alternatively, herbivore production of ABA could modulate the interaction between the JA and SA pathways as suggested by the results of Thaler and Bostock (2004).

We have recently found carotenoids and ABA in the salivary glands of the Ambrosia galler Asteromyia carbonifera in concentrations well above those required for physiological effects on the plant (Heath et al. 2012). Many cecidomyiids, including A. carbonifera, have obligate fungal symbionts. This hormone may be instrumental in allowing growth of their symbiotic fungus, which forms the gall structure and is their primary food source (Janson et al. 2009; Heath and Stireman 2010). As with other systems in which larvae have glandular carotenoids (Eichenseer et al. 2002; Sakudoh et al. 2007), understanding the ecological trade-offs is key to ascribing a role for these compounds.

Carotenoids in insect-enemy and tritrophic interactions

Aposematic and cryptic colouration

Because carotenoids generally must be obtained from the environment and may be limiting, access to carotenoids may influence insect population dynamics and niche evolution via their effects on susceptibility to enemies and presumably mate choice (see also the section ‘Resource signalling: odour’). Carotenoid pigmentation is correlated with levels of toxic defensive compounds and can function as precursors to these compounds, such as the predator-repellent grasshopper ketone (Fig. 2; Meinwald et al. 1968). Carotenoids can underlie not only yellow, orange or red colouration, but also green (in some insects) and purple–blue (in other arthropods) when combined with specific carotenoproteins or chlorophyll degradation products such as pterobilin. Carotenoproteins anchor the carotenoids in specific configurations that change the spectral qualities of the chromophore and allow the absorption of light in uncharacteristic regions of the visible spectrum (Britton and Helliwell 2008).

Aphids are known to derive body colour from carotenoids and frequently exhibit intraspecific colour morphs (Losey et al. 1997). Most insects are thought to obtain carotenoids from their diets; however, aphids are phloem feeders and the carotenoid concentration in phloem is expected to be low (Czeczuga 1976). Several studies have shown the presence of torulene, a red carotenoid, in aphid and katydid colour morphs. Torulene is rare in plants and it is hypothesized to be biosynthesized by symbiotic microbes (Weisgraber et al. 1971; Britton et al. 1977; Jenkins et al. 1999), but may be derived from laterally transferred fungal genes in some insects (Moran and Jarvik 2010). Carotenoid-derived colours can have substantial ecological consequences in aphids and butterflies (Gerould 1921); for instance, Losey et al. (1997) found that the maintenance of a red–green colour polymorphism in the pea aphid was regulated by differential predation and parasitism. These opposing forces result in equilibrium between the red and green morphs. Frequency-dependent selection, mediated by parasitoid learning, can also result in equilibrium between the aphid colour morphs (Langley et al. 2006), and recent studies indicate that increased light can induce a much darker colour morph even within a clone (Alkhedir et al. 2010). Whether these aphid colour morphs represent some form of crypsis or aposematism has not been determined.

For signals to evolve and persist, they must be honest (Zahavi 1975) or be augmented by a high frequency of honest models in their environment. Current theory and evidence indicate that mate signalling traits should be strongly correlated with individual health and condition (Berglund et al. 1996), but not necessarily genetically linked or even under direct genetic control (Kodric-Brown and Brown 1984; Sandre et al. 2007). And, Badyaev et al. (2001) illustrate that carotenoid-based plumage in birds is actually a complex, composite representation of several aspects of individual condition, which suggests the existence of physiological or developmental trade-offs. The physiological connection between carotenoids and protection from autotoxicity, immunity, biosynthesis of defence (e.g. smaller terpenes) and antioxidant capacity may make them particularly useful for colour signalling both within and between species (Blount et al. 2009), but in insects, the expected physiological trade-offs have not been forthcoming (Sandre et al. 2007). Nevertheless, correlations between colouration and defensive traits have been found. The bright red, carotenoid-derived elytral colouration of many ladybird beetles has the potential to signal honestly the level of beetle toxicity to potential predators (Bezzerides et al. 2007). Upon attack by predators, Harmonia axyridis ladybird beetles react with reflexive bleeding and release the defensive alkaloid harmonine, the concentration of which is significantly correlated with the proportion of elytra area that is red (Bezzerides et al. 2007). Aposematic colouration is also correlated with carotenoid concentration in Leptinotarsa leaf beetles that appear to sequester carotenoids from their host plants (Poff 1976). Carotenoids have been found in whole-body and hair-tuft extracts of a number of aposematic Lepidoptera species (Sandre et al. 2007; Blount and McGraw 2008), including monarch butterflies where it is responsible for the dramatic yellow striping of caterpillars (Rothschild et al. 1978). Several aposematically coloured butterflies exhibit levels of carotenoids that correlate positively with toxic prooxidant compounds, suggesting that carotenoids may protect susceptible tissues from autotoxicity as well as provide warning colouration (Rothschild et al. 1986; Nishida et al. 1994). Interestingly, in butterfly systems involving mimicry, mimics consistently have lower carotenoid concentrations than their models. This pattern does not hold up in the classic monarch-viceroy batesian mimicry (Rothschild et al. 1986) and may have led Ritland and Brower (1991) to re-examine and ultimately reverse our understanding of this classical mimicry system (Rothschild 1991).

In insects, blue pigments such as pterobilins can combine with carotenoids to yield cryptic colouration (Rothschild and Mummery 1985). Larvae of the butterfly, Colias philodice, and the stick insect Dixippus morosus both combine carotenes with unknown blue pigments to achieve a green colour (Fox 1976). Many lepidopteran larvae are also coloured green by the physical mixing of blue chlorophyll degradation products and carotenoids (Rothschild and Mummery 1985). Furthermore, the intensity of the green colour is correlated with carotenoid concentration (Grayson et al. 1991). Interestingly, some of these cryptic larvae have elaborate polyphenisms that allow them to alter their colouration over the course of larval development to match the colour of their environment (Grayson and Edmunds 1989; Noor et al. 2008). This response can be triggered by the quality of the diet (Greene 1996) and/or by the quality of light perceived by the larvae (Grayson and Edmunds 1989; Noor et al. 2008) and appears to be adaptive (Edmunds and Grayson 1991).

These systems seem ideally suited for the investigation of genetic assimilation as some of the species in these groups appear to be relatively fixed for certain colour morphs that may have been derived from ancestors with flexible polyphenisms. Genetic assimilation purports to explain how polyphenisms can become fixed in populations and thus facilitate speciation and niche specialization. Although Grayson and Edmunds (1989) allude to this concept, they do not expound on its potential importance. The power of genetic assimilation to facilitate speciation comes from the conditional ratchet effect (reviewed by West-Eberhard 2003) that polyphenisms can have on natural selection, that is, because a particular polyphenism is generally only expressed under conditions in which it is likely to be beneficial, natural selection tends to select for a lower induction threshold for that trait. Therefore, any expressed polyphenisms with an equal or lower induction threshold (i.e. genetically controlled) are more likely to show up in the next generation. Given the right environmental conditions, this process can produce a more-or-less fixed trait (West-Eberhard 2003). Interestingly, threshold evolution has been documented in dung beetles where expression of horned males is ultimately controlled by size and proximately controlled by the terpenoid, juvenile hormone (Moczek and Nijhout 2002).

Tritrophic signalling

One interesting phenomenon observed frequently in the studies of plant–arthropod interactions is a decrease in chlorophyll and carotenoids in uninfested tissues of plants infested with phytophages. This chlorosis occurs locally at the feeding site (Mothes and Seitz 1982; Hildebrand et al. 1986b; Ni et al. 2002; Heng-Moss et al. 2003), but also within undamaged tissue remote from the feeding site (Schuster et al. 1990; McAuslane et al. 2004). Numerous studies have also demonstrated ultrastructural changes in chloroplasts associated with plant cells near feeding sites, which seems to correspond to a decrease in the quantity of pigments in these cells (Oliveira et al. 2011) as well as starch grains. This is especially true for pathogen-infected cells (Camp 1981; Rey 1992) and the nutritive cells induced by galling insects (Birch et al. 1992; Bronner 1992; Rey 1992; Westphal 1992; Oliveira et al. 2011), but also of cells near caterpillar feeding sites (Maffei et al. 2004). The ultimate fate of the chlorophyll and carotenoids associated with chlorosis and major changes in chloroplasts has not been studied. However, the higher activity of prooxidant enzymes in these cells (Hildebrand et al. 1986a) may result in significant releases of volatile apocarotenoids (Salt et al. 1986; Fig. 2).

Given that many hymenopterans respond to volatile apocarotenoids (Table 1) and that insect feeding is associated with chlorosis, it is surprising that the recent proliferation of plant studies on systemically inducible volatile organic compounds (VOCs), which can attract parasitoid wasps, (reviewed in Arimura et al. 2005) has failed to identify any obvious volatile apocarotenoids associated with insect feeding. Although systemic decreases in chlorophyll and carotenoid content have been observed in damaged plants (McAuslane et al. 2004), studies indicating local decreases (i.e. near the feeding site) are more prevalent and more intense. It is possible that volatile apocarotenoids are released at the feeding site, but that inducible, systemically released volatile organic compounds obscure detection of locally released apocarotenoids. Indeed, it is not uncommon to find parasitoid studies with lists of unidentified electroantennographically active compounds that are either masked by larger peaks or are undetectable by gas chromatography (e.g. Gouinguene et al. 2005). Most studies involving systemically induced volatile emission have measured the volatiles from whole plants; few studies have concentrated on the volatiles emitted only from the feeding site. Coleman et al. (1997) showed that the parasitoid Cotesia glomerata can distinguish between feeding sites of Pieris brassicae larvae on Brassica oleracea with stronger attraction to larvae feeding on the top most leaves. They also illustrated that the parasitoids were unable to distinguish between infested and unifested plants when the larvae were feeding on lower leaves. Tissue age and environment can influence leaf carotenoid concentration and may explain the lack of attractiveness of larvae feeding on lower leaves (Feltwell and Valadon 1974; Rothschild et al. 1986; Norman et al. 1990; Hartel and Grimm 1998). For example, B. oleracea core tissue with limited light exposure has up to 30-fold less carotenoid than light-exposed tissues (Bondi and Meyer 1946).

If apocarotenoids are in fact important volatile cues for natural enemies, they are more likely to be discovered by comparing systemically released volatiles with those emanating from local feeding sites. Theoretically, the volatiles emitted from local feeding sites are predicted to be more important in natural enemy attraction than those released systematically both from a behavioural and a plant physiological perspective. Therefore, a renewed focus on the volatiles specifically associated with the feeding site could provide better natural enemy attractants as well as test the presence of apocarotenoids.

The quantity of volatiles emitted from a local feeding site will be much lower than those released systemically simply because of the reduction in tissue involved. However, from a behavioural perspective, this is not likely to present a problem for predators and parasitoids. Insects are known to be highly sensitive to volatile compounds. Workers have shown that a single odour molecule is sufficient to evoke a nerve impulse (Kaissling 1996), and semiochemicals often have extremely high physiological activity at levels so low as to be nearly undetectable by even the most sensitive of chromatographic equipment (e.g. femtogram levels, Heath et al. 2005). Furthermore, volatiles emanating from a point source are far more useful as odour cues than broadly emitted sources. In fact, male moths flying in a homogenous cloud of female pheromone behave as if they were flying in clean air (Baker 1985). This is due to the physiological and behavioural mechanisms of odour source perception and location in insects. Insects do not locate an odour source by flying up a concentration gradient, but rather by responding stereotypically to pulses or filaments of highly concentrated odour (see Baker and Vickers 1997 for a review of optomotor anemotaxis). Therefore, the volatiles emanating from a point source (e.g. the damaged site of an actively feeding herbivore) are likely to be more important in host location than those released systemically. Furthermore, studies have found that galling insects fail to induce the systemic release of volatile organic compounds that are typically thought to attract predators and parasitoids (Tooker and De Moraes 2007; Tooker and De Moraes 2008; Tooker et al. 2008), and detailed studies have shown a much more intense reduction in pigments near the feeding sites of galling insects (Oliveira et al. 2011 and references therein). Perhaps, the high parasitism rates experienced by many galling insects are based instead on visual or other locally emitted volatile cues.

Carotenoids are a huge reserve of precursors for volatile apocarotenoids that may prove to be important in the attraction of natural enemies to actively feeding hosts. That plants have evolved the ability to induce the production of VOCs for the sole purpose of attracting natural enemies is unlikely. We are not suggesting that natural enemies do not respond to these compounds or that plants do not benefit from attracting them, but rather that natural enemy attraction has been an epiphenomenon of plant defence, including autotoxic protection from their onslaught of prooxidant species. Provided that plants enjoy a fitness benefit from attracting natural enemies, they may have fine-tuned the release of volatiles, especially from the feeding site, over evolutionary time, to serve a secondary purpose of attracting carnivores. Furthermore, we predict that some of the most important volatiles are likely to be derived from carotenoid precursors. In fact, in the rapidly radiating complex of the nominal species Asteromyia carbonifera (Stireman et al. 2008; Janson et al. 2010; Stireman et al. 2010), we have found that the quality and quantity of adult accessory gland carotenoids is consistently gall-morphotype specific and correlates nearly perfectly with attack by an egg parasitoid both within and across morphotypes (Heath et al. 2012).

Conclusions

The ubiquity, physiological necessity, and varied functions of carotenoids and their derivatives across the tree of life suggest that they may also play significant roles in insect ecology and evolution, as limiting resources, as defensive or repellent compounds, and as signals or cues. In particular, emerging evidence of the roles of carotenoids as self-protective antioxidants, as precursors to plant hormones such as strigolactones and ABA, and as precursors to volatile apocarotenoids suggests that they may be important mediators of plant–insect, insect-mutualist and tritrophic interactions. Their potential as volatile chemical cues that parasitoids and other insects could use to find their herbivorous prey within the torrent of intermingling odours in nature is promising, but largely unexplored. The co-option of hormone derivatives of carotenoids suggests a novel way in which insects may manipulate plant growth and defence signalling pathways and conspire in mutualistic associations with microbes and fungi. The many critical roles of carotenoids in colouration, vision, diapause, photoperiodism, as antioxidants and as defence compounds suggest that they may often be a limiting resource for insects and therefore represent an important dimension of the ecological niches of many insect taxa. This would also suggest that insects should have fine-tuned systems for detecting and assessing carotenoid quality and quantity of their food, but this remains largely unexplored.

Research aimed at understanding the roles that carotenoids and their derivatives play in species interactions should have a multitrophic perspective starting first at the level of the primary producer and working up the food web to higher trophic levels. With this in mind, tracking the flow of major plant carotenoids (with the use of stable isotopes or radiolabelling) through herbivores and on to higher trophic levels would provide a framework for further assessing the relative contribution of maternal, dietary, symbiotic and potential de novo sources of insect carotenoids. At the plant level, the role of carotenoids in protecting plants from their own production of potentially autotoxic, photosensitized allelochemicals or ROS is largely unexplored and depending on the degree to which autotoxicity is a factor, carotenoids may modulate trade-offs between defence and other plant systems such as growth, photosynthesis or reproduction. This work would help place the plant within the emerging concept of universal adaptive strategy theory (Grime and Pierce 2012). At the herbivore level, carotenoids may protect against photosensitized plant allelochemicals either directly or via indirect protection of antioxidant enzymes, but work in this area is lacking in insects. As mating signals, carotenoids may provide both colour directly and odour via apocarotenoids, but the potential trade-offs here have not been assessed nor the degree to which carotenoid-based sexual selection is involved in the evolution of insect life history strategies. Probably, the most interesting and practical area for further study is with regard to the third trophic level. Apocarotenoids generated by the plant at the herbivore feeding site have great potential as cues to foraging parasitoids and predators of the presence of herbivores, but to our knowledge, this has not been explored nor has the impact of carotenoids on the immune response of the herbivore once it has been parasitized or infected. Finally, whether insect parasitoids, predators or herbivorous insects select food resources on the basis of carotenoid quality or quantity has not been addressed directly, but provides a clear and open direction for future research.

Understanding the roles of carotenoids in the evolutionary ecology of insects is a potentially rich field that promises to enrich our understanding of the chemical mediation of ecological interactions in nature and may provide excellent model systems for integrating aspects of organism development with current evolutionary theory (Badyaev 2011). In turn, understanding the factors that drive such phenomena as host-plant attractiveness or repellency to herbivores, pathogens and natural enemies; pollinator and seed-disperser attraction; and regulation of microbial symbionts has numerous applied implications.