No-cost meals might not exist for insects feeding on toxic plants

Predators are constant agents of natural selection that regulate ecosystems and sustain biodiversity on local and global scales. Predation risk affects the evolution of prey species through unique evolutionary processes that offer protection against predators. In general, insects protect themselves from their natural antagonists in several ways, including chemical, physical, morphological, or behavioural, although they are not exclusive to each other. For example, some insects use chemical defensive toxins (Eisner et al., 2005; Pokharel et al., 2020), hair (Sugiura and Yamazaki, 2014), and spines (Ito et al., 2016; Murphy et al., 2009). As defensive warning signals, many insects masquerade and mimic using cryptic and/or aposematic colours and patterns (Ruxton et al., 2019). Furthermore, numerous insects exhibit defensive behaviours, such as death-feigning (Humphreys and Ruxton, 2018), kicking (Catania, 2018; Gnatzy and Heusslein, 1986), stinging (Schmidt, 2016), and producing sounds (Bura et al., 2016).

Herbivorous insects have evolved various tolerance and resistance strategies to overcome plants' defences known as plant secondary/specialised metabolites (PSM). Furthermore, insects can accumulate PSMs while feeding on plants, using them for defence against biological antagonists, a common phenomenon observed in more than 275 insect species called sequestration, a syndrome of selective uptake, transport, modification, storage, and deployment of PSMs as a chemical defence (Beran and Petschenka, 2022). In contrast, many insects can synthesise their chemical defences, as observed in chrysomelid beetles (Pasteels et al., 1988) and Heliconius spp. butterflies (Brown and Francini, 1990). However, toxins are sequestered in tissues and have a physiological impact on internal targets. For example, cardenolide-sequestering insects of at least seven different orders have evolved target site insensitivity (TSI) in Na⁺/K⁺-ATPases (a ubiquitous animal enzyme that drives various essential physiological functions) where cardenolides bind (Dobler et al., 2012; Karageorgi et al., 2019). TSI is also observed in other specific receptors (for example sodium channels, neurotransmitters, and microtubules) for toxins, such as pyrethrins and alkaloids (Wink, 2009). Furthermore, some plants possess two-component defences as inactive glycosylated storage forms, which must be activated by β-glucosidases from the plant, insect, or both. In this category, several insects sequester toxins, including benzoxazinoids, cyanogenic glycosides, glucosinolates, iridoid glycosides, and salicinoids (Opitz and Müller, 2009). Nevertheless, defensive chemicals are highly complex mixtures that can be acquired from the environment or biosynthesised, which are widespread and exhibit high levels of variation even within an insect species (Blum, 2012). The answer to the critical question of whether insect individuals pay a price for chemical defence remains unresolved, and this Review focuses solely on the potential underlying costs of insect defensive chemicals.

If an insect produces defensive compounds, there are costs associated with expressing the requisite enzymes for toxin synthesis, storing toxins, and avoiding autotoxicity. Some of these costs are avoided in sequestration by deriving PSMs directly from host plants, but other related costs remain, such as toxin transport through the gut and metabolism (biotransformation). In addition to these costs, there is also the cost of resistance to toxins, usually linked to the specific molecular mechanisms of resistance. Despite its relevance, little is known about the cost-benefit analysis of various processes involved in chemical defence. It is assumed that there is minimal or no cost for chemical defences (Zvereva and Kozlov, 2016). Alternatively, costs are not always easy to detect or estimate (Lindstedt et al., 2010; Ruxton, 2014). Therefore, it is important to ask: 1) Do chemical defences come free (or at a very low cost)? 2) Is it particularly challenging to identify the cost of chemical defences (Fig. 1)?

To begin, a very weak or no cost of defence rattles the central and recurring theme in ecology and evolution: the life history trade-off (Fig. 1A). Life history theory focuses on how evolution, a natural selection imposed by ecological challenges, drives organisms to maximise their fitness (Roff, 1992; Stearns, 1992). Fitness is the sum of the reproductive success of an organism, and life history traits are the main fitness parameters. Investments in physiological and ecological traits such as defence, maintenance, growth, and reproduction cause trade-offs owing to the restriction imposed by genetic, developmental, physiological, and phylogenetic limits. Therefore, ecophysiological trade-offs are caused by competitive resource allocation, i.e. resources invested in one trait cannot be invested into another (Stearns, 1992). Each trait is anticipated to manifest differently depending on environmental conditions (Clark and Harvell, 1992). In this context, significant empirical evidence and several theoretical models have been presented (Houston et al., 1993; McPeek, 2004; Steiner and Pfeiffer, 2007; Stoks, 2001). Optimal time and resource allocation strategies differ in various situations, such as benign environmental situations (low predator densities and high resources) and harsh environmental situations (high predator densities and low resources).

Many theories have been formulated to understand how interacting species affect each other's evolution (Abrams, 2000; Thompson, 2005). Most importantly, theoretical models of victim-exploiter relationships (such as plant-insect, prey-predator, and host-pathogen interactions) assume the costs of exploiters' adaptations to overcome victims' defences (Bergelson et al., 2001). Plants have a cost for the biosynthesis, storage, and maintenance of PSMs (Koricheva, 2002; Neilson et al., 2013). Regardless of their deployment in constitutive or inducible defences, plants need to invest in PSMs. Zero investment by insect herbivores in resistance and tolerance to PSMs establishes a coevolutionary asymmetry between plant and insect interactions (Fig. 1B). Furthermore, the evolutionary arms race between plants and insects (in terms of PSM sequestration) is not an evolutionary dead end but rather a driver of the escalation of symmetrical coevolution (Petschenka and Agrawal, 2016).

The evolution of chemical defences is considered in association with the trade-offs between acquired benefits through protection against natural enemies and the possible defence costs. The costs of possessing chemical defences are assumed to be compensated by increased protection against predators and parasites (Bowers, 1992; Camara, 1997). In other words, the costs of chemical defences are often outweighed by their benefits (Fig. 1C) and such costs are not always easy to detect and estimate (Lindstedt et al., 2010). The complications in detecting and estimating the costs may be due to experimental designs that cannot control for all factors and manipulate only one variable (Stearns, 1992). Furthermore, natural selection may favour cost-effective traits, making it challenging to detect costs (Fry, 2003).

Chemical defences can be acquired through sequestration, de novo biosynthesis, or a hybrid of these two strategies. Sequestration is the phenomenon of toxin uptake from host plants (i.e. first trophic level) by insects (i.e. second trophic level) to protect themselves against predators and parasites (i.e. third trophic level). Chemical defence is often associated with warning colours and patterns (called aposematism), and sequestration and aposematism are the prominent suites of defences in many species. In an ecophysiological framework, the costs of chemical defences are expected to be seen in many aspects, including oxidative stress, reduced immune defence, impaired growth, and reduction in fecundity (Fig. 2).

Recent literature shows that the immune response (Smilanich and Muchoney, 2022) and toxin sequestration (Beran and Petschenka, 2022) are two of the vital physiological links between plant-insect-antagonist (here, biological antagonists such as pathogens and predators) interactions. Empirical studies have shown that some toxin-sequestering insects feeding on toxic host plants are protected against pathogens. However, this may be costly. Some insects compromise their immune systems while feeding on toxic dietary resources (Lampert, 2012; Smilanich et al., 2009). Although in some cases toxin-sequestering insects have a weak immune system, they are less likely to be deterred by predators as they are laden with toxins. If this is the case, then the insect host is vulnerable to parasitisation due to its weak immune system. However, the insect host would be a safe haven for parasites. Therefore, both the host and the parasite are protected (Gentry and Dyer, 2002; Harvey et al., 2005; Reudler et al., 2011).

Increasing amounts of iridoid glycosides (IG) sequestered by Buckeye caterpillars Junonia coenia have been linked to a compromised immune response (Camara, 1997; Smilanich et al., 2009). A negative effect on the immune response of J. coenia caterpillars was observed when they were fed diets containing higher IG levels and sequestered these toxins compared to caterpillars fed diets containing lower IG (Smilanich et al., 2009). In addition to laboratory studies, a negative association was observed between IG sequestration and the immune response in the Baltimore checkerspot caterpillar Euphydryas phaeton in a field-based study, implying that toxin sequestration suppresses cellular immune responses (Muchoney et al., 2022).

Contrary to these findings, in Grammia incorrupta, the concentration of IGs did not affect immune responses, when caterpillars were fed IG-containing plants (Smilanich et al., 2011). Similarly, the amount of cardenolides present in different host plants did not affect the immune response of cardenolide-sequestering monarch butterflies Danaus plexippus (Adams et al., 2021). Furthermore, gene expression analysis demonstrated that monarch butterflies did not show differential immune responses in monarch butterflies to the common protozoan parasite Ophryocystis elektroscirrha infection (Tan et al., 2019). However, feeding on milkweed plants containing large amounts of cardenolides reduced O. elektroscirrha infection and growth in monarchs (Gowler et al., 2015; Sternberg et al., 2012; Tan et al., 2018). Interestingly, withanolides had a positive effect on specialist Heliothis subflexa larvae that fed on Physalis spp. by increasing larval growth and immune system activity, but this positive effect was not observed in the closely related H. virescens (Barthel et al., 2016). Enzymes such as phenoloxidase (Liu et al., 2009) and glutathione (McMillan et al., 2018) are important for both immune functions and toxin detoxification. In an ecophysiological framework, the costs of chemical defence may exist in a competing energy demand between sequestration and immunity, which differs among insect groups sequestering different classes of PSMs.

The discrepancy in the response to immunity could also be due to the different experimental methods used to measure the immunity. In summary, insects have innate immune systems composed of cellular and humoral components. The cellular components include phagocytes, which engulf and destroy invading pathogens, and haemocytes, which are specialised immune cells that circulate throughout the body of the insect. The humoral components include antimicrobial peptides, which can kill pathogens, and the melanisation response, which involves the formation of a melanin pigment around invading microorganisms to keep them isolated from the body. Insects can be negatively influenced by a variety of factors, such as poor nutrition, exposure to extreme temperatures, pesticides, pollutants, and other environmental toxins, which can make them susceptible to infection. In addition, some parasites and pathogens have evolved mechanisms to suppress or evade the insect immune system (Beckage, 2011). Therefore, to conclusively establish an immune response, multiple methods that measure both cell-mediated and humoral immunity should be performed in tandem (Adamo, 2004). A cell-mediated immune response can be tested by melanisation [using dextran beads and nylon filament (Laurentz et al., 2012; Smilanich et al., 2009)] and haemocyte attributes [such as enlargement, agglutination, denucleation, shape distortion, and abnormal staining of the cells (Muchoney et al., 2022; Perveen and Ahmad, 2017)]. Similarly, a humoral immune response can be estimated using biochemical assays [phenoloxidase activity (Barthel et al., 2016), relative protein concentration (Rahman et al., 2004), lysozyme-like activity (Charles and Killian, 2015)] and zone of inhibition assays [using lipopolysaccharide (a cell wall component of bacteria) in the form of antimicrobial growth (Adams et al., 2021)]. Therefore, for assessing the immune response, the best would be to combine the methods such as bioassays, genetic analyses, microscopy, and molecular techniques.

Some insects such as Heliconius species have evolved to biosynthesise cyanogenic glucosides, while they already possess mechanisms to tolerate autotoxicity and have evolved to sequester from their host plant Passiflora spp. to reduce the energetic costs of biosynthesis (Zagrobelny et al., 2008). Empirical evidence regarding the metabolic costs of toxin sequestration is scarce. For example, the respiration rate (i.e. CO₂ production) of J. coenia caterpillars was negatively correlated with catalpol (an IG) present in their diet (Smilanich et al., 2009). Similarly, in Ceratomia catalpae sphinx caterpillars, respiration rate was negatively correlated with the amount of sequestered catalpol (Lampert, 2020). It is important to note that the respiration rate and catalpol concentration per dry mass of caterpillars were positively correlated (Lampert, 2020). Recent evidence suggests that cardenolide sequestration is costly and affects the flight energetics of monarch butterflies (Pocius et al., 2022). We can speculate that in some insect species, the resource pool is allocated according to metabolic (energetic) demands, which is also observed beyond insects (Box 1).

Insect adaptations to host-plant toxins involve mechanisms that metabolise toxins and prevent autotoxicity (Agrawal et al., 2022). In cardenolide-sequestering Aphis nerii, gene expression analysis (Birnbaum et al., 2017) showed the expression of a variety of classical detoxification gene families, such as cytochrome P450s, UDP-glucuronosyltransferases (UGTs), and ATP-binding cassette transporters (Heckel, 2014), when the insects were fed on cardenolide-producing plants. Similarly, in D. plexippus, which sequesters cardenolides, transcriptome analysis showed upregulation of glutathione S-transferase (GST) and carboxyl esterase genes in the gut tissues of caterpillars fed plants with higher toxicity (Tan et al., 2019). It is very important to note that A. nerii and D. plexippus are cardenolide-sequestering specialists; however, A. nerii lacks cardenolide-resistant Na⁺/K⁺-ATPases (but see Karageorgi et al., 2019) and D. plexippus possesses cardenolide-resistant Na⁺/K⁺-ATPases that mediate resistance to cardenolides via target-site insensitivity (Zhen et al., 2012). Although possessing or not possessing the resistance mechanism to toxins, both insects were challenged by the presence of dietary toxins. Therefore, the above findings indicate that metabolic costs exist in sequestering species and warrant further investigation to test the possible crosstalk between toxins and specific genes that are directly involved in their modification and transport.

When the capacity of the antioxidant defence and repair mechanisms of an organism is exceeded, oxidative stress is triggered by a rapid increase in the generation of reactive oxygen species (ROS) (Finkel and Holbrook, 2000; Metcalfe and Alonso-Alvarez, 2010; Monaghan et al., 2009). ROS are natural by-products of metabolic activities, and unless suppressed by antioxidants, they can damage a variety of biomolecules (such as DNA, proteins, and lipids) because of their instability and high reactivity (Balaban et al., 2005; Dowling and Simmons, 2009). In the early 1990s, Aucoin et al. published extensively on the oxidative stress in insect herbivores caused by phototoxins – toxic plant chemicals that are activated by the absorption of light (Aucoin et al., 1995, 1990, 1991). The impact of Anthropocene activities on insects has generated a large body of literature in which researchers have investigated different markers of oxidative stress associated with environmental pollution by heavy metals (Abdelfattah and El-Bassiony, 2022; Kafel et al., 2021; Renault et al., 2016) and pesticides (Hamama and Fergani, 2019; Palma Onetto et al., 2021). However, little is known about the possible oxidative stress in insect herbivores when dealing with plant toxins.

Theoretically, resistance and tolerance strategies to deal with toxins can damage molecules, such as lipids, proteins, and DNA, due to oxidative stress. Therefore, antioxidant defence systems must exist to combat ROS and repair or mitigate damage which is presumably very costly. Different levels of oxidative stress can occur and accumulate throughout an individual's lifespan (Beckman and Ames, 1998), which suggests that oxidative stress in organisms is a potential driver of life history trade-offs (Costantini, 2008; Dowling and Simmons, 2009; Isaksson et al., 2011; Metcalfe and Alonso-Alvarez, 2010; Monaghan et al., 2009; Speakman and Garratt, 2014). Although ROS are usually associated with oxidative stress and cellular damage, some studies have suggested that ROS can also possess antioxidant properties. For example, certain ROS, such as hydrogen peroxide, can act as signalling molecules that prompt protective responses within cells (Veal and Day, 2011). Low levels of ROS can stimulate the organism's antioxidant defences by producing enzymes such as superoxide dismutase and catalase, which can help neutralise excess ROS and prevent oxidative damage (Bhattacharya, 2015). While low levels of ROS may have beneficial effects, high levels can be damaging. The redox signalling hypothesis postulates that there is a trade-off between ROS production and ROS regulation. Both processes can alter vital physiological functions that influence life history traits in organisms (Costantini, 2019).

Estimating the cost of oxidative stress is difficult, because an individual insect is often regarded as a uniform entity. In reality, an individual is a complex assembly of tissues/organs involved in distinct physiological roles with different characteristics, including the antioxidant system and cellular turnover (Costantini, 2019). Similarly, several physiological mechanisms are involved in dealing with toxins where costs can occur (Fig. 3). Insects exhibit tissue-specific antioxidant profiles. For example, the peritrophic matrix (a membrane that surrounds the food bolus and gut) can serve as a functional antioxidant (Summers and Felton, 1996), protecting the intestinal epithelium from oxidative damage caused by PSM ingestion (Barbehenn and Stannard, 2004). Interestingly, studies suggest that the oxidative radicals and antioxidant enzymes that deal with PSM are compartmentalised in the digestive tract, with higher amounts of superoxide anions and hydrogen peroxide in the foregut and higher enzyme activities of superoxide dismutase and catalase in the midgut (Krishnan and Sehnal, 2006).

Empirical studies on insects have shown that antioxidant molecules play a dual role in the detoxification of toxins (Enayati et al., 2005) and colour pigments (Shamim et al., 2014). Similarly, pigment molecules, including melanins, pterins, carotenoids, and flavonoids, can act as antioxidants (McGraw, 2005). Some insects, such as Trichoplusia ni, sequester carotenoids at concentrations up to 20 times higher than those found in their host plants (Nguyen et al., 2019). Carotenoids in invertebrates have a dual role; they can act as antioxidants and play a role in the expression and regulation of immune genes (see review Tan et al., 2020). Interestingly, a well-studied plant toxin, catalpol (an IG), which is sequestered by many caterpillars (see above), can act as an antioxidant (Bhattamisra et al., 2020). Recently, a study showed a physiological association in D. plexippus between oxidative stress (measured by oxidative lipid damage), cardenolide sequestration, and warning colouration (Blount et al., 2023). Interestingly, in Oncopeltus fasciatus, the amount of sequestered cardenolides depleted the redox state (measured by the total glutathione level) and antioxidant availability was traded off with brightness and chroma signals (Heyworth et al., 2023). Although very few studies have supported the physiological cost of toxin sequestration in aposematic insects, there is still a lack of evidence that toxin sequestration, warning signals (pigments), and oxidative stress are interlinked.

Empirical evidence on the costs of chemical defences in life history (such as growth, fecundity, and lifespan) is equivocal, as different studies show contrasting outcomes. Cardenolide-sequestering insects grew faster with a larger adult body size when they were fed and raised on a cardenolide-rich diet (Blakley and Goodner, 1978; Chaplin and Chaplin, 1981; Isman, 1977; Krueger et al., 2021; Pokharel et al., 2021; Rothschild et al., 1975). Similarly, while feeding on plants producing higher concentrations of IGs, IG-sequestering caterpillars survived better, gained more weight, and developed faster into butterflies (Bowers and Puttick, 1988, 1989; Harvey et al., 2005; Saastamoinen et al., 2007). In contrast, insect species that sequester cardenolides did not show measurable differences in growth when fed plants containing a different amount of cardenolides (Erickson, 1973; Petschenka and Agrawal, 2015). However, recent research indicates that processing a highly potent cardenolide (vorusharin) into other cardenolides and sequestering them came at a cost in terms of growth for monarch butterflies (Agrawal et al., 2021).

Positive correlations between protective and nutritive phytochemical constituents, as well as the high degrees of adaptation in specialists to utilise plant compounds as nutrients, may be the cause of the non-appearance of costs in toxin sequestration. Interestingly, specialist insects such as Pieris rapae and arctiid moths (such as Utetheisa spp. and Tyria spp.) show a predilection for their host plants with glucosinolates (Renwick and Lopez, 1999) and pyrrolizidine alkaloids (Rothschild et al., 1979), respectively, which have been suggested to be addicted to plant chemistry (Wink, 2018). Specialists may suffer from the absence of dietary toxins due to selection for physiological homeostasis under continuous exposure to toxins. An ‘evolutionary addiction’ might be stated as the positive impact of toxins on the life history of specialist insects (Pokharel et al., 2021). Alternatively, several studies on pest insects have shown positive effects on fitness under insecticide stress, presumably due to hormetic effects (Celestino et al., 2014; Piiroinen et al., 2014). In terms of its functional rationale and potential fitness effects, insecticide-induced hormesis in arthropods is still puzzling, yet an emerging topic (Sebastiano et al., 2022).

It is commonly acknowledged that sequestered plant toxins effectively protect insects from both vertebrate (such as birds) and invertebrate (such as spiders) predators (Nishida, 2002). However, the literature suggests that the chemical defence of prey is not universal against all predators, implying additional ecological costs together with the associated physiological costs. For example, the sequestration of cardenolides by a milkweed bug Lygaeus equestris protects against insectivorous birds but not against predatory lacewing larvae (Petschenka et al., 2022; Pokharel et al., 2020). The predatory effectiveness of insect-sequestered toxins depends strongly on the chemistry of the host plant (Pokharel et al., 2020). The deterrence potential of different toxins from various plant species with a range of different amounts and diversity against predators (both invertebrates and vertebrates) should be investigated to improve our understanding of the ecological implications of sequestered PSMs. Nevertheless, the evolution of chemical defences is linked to trade-offs between the acquired benefits (through protection) and the costs of acquiring chemicals. Since chemical defences directly increase the survival of well-defended prey against predators, ecological costs are not easily apparent.

No-cost meals might not exist for insects that feed on toxic plants. Theoretically, the costs of chemical defence can be estimated by examining at the tissue/cellular level (i.e. each step of toxin detoxification/sequestration) and organism level (i.e. the universality of defence) (Fig. 3). However, it is difficult to measure empirically because various traits of an organism interact depending upon physiological conditions (e.g. age and fecundity) and ecological dynamics (e.g. foraging and predation). The trade-offs may only appear in a species (or even within the same species) if a specific set of circumstances is met (Reznick, 1985). However, we can attempt to estimate using a manipulative and reductionist approach. For the most accurate and revealing information, one should think about using a single-factor manipulation (Stearns, 1992). In agricultural systems, research on the costs of pesticide resistance will help us better understand how evolution works and will provide useful data for pest management (Box 2).

We recently reviewed methods to study the interaction between PSM and insect herbivores and recommend them for further reading (Jeckel et al., 2022). Briefly, transgenic approaches can be employed to create insect lines to measure the true costs of resistance and sequestration. Here, I review and suggest where costs could be detected in an ecophysiological framework. Both the aspects of chemical defence, the multifaceted effects on insect physiology (i.e. physiological costs) and the context-dependent effect against predators (i.e. ecological costs) are important for our better understanding of the ecology and evolution of plant-insect-antagonist interaction. I hope this Review will stimulate further interest in unanswered questions about the costs of chemical defences.

This work was originally initiated from my doctoral dissertation (Pokharel, 2023). I thank Georg Petschenka for his valuable input while writing this manuscript. I sincerely appreciate the two anonymous reviewers for their comments, which helped in improving the quality of the manuscript.