Predator-induced transgenerational phenotypic plasticity in the cotton aphid

Transgenerational phenotypic changes, whereby offspring have an altered trait or a distinct alternate phenotype, frequently occur in response to increased maternal predation risk. The cotton aphid, Aphis gossypii (Glover), is unique, however, as offspring consist of four distinct phenotypes (normal light green apterae, normal dark green apterae, dwarf yellow apterae, and alatae), all with divergent life history patterns and resulting population dynamics. Here, we show that increased predation risk induces transgenerational phenotypic changes in cotton aphids. When exposed to search tracks from larval or adult convergent ladybird beetles, Hippodamia convergens Guérin-Méneville, cotton aphids produced greater numbers of winged offspring. In a subsequent experiment, apterous and alate individuals on clean plants were found to have primarily normal and dwarf offspring, respectively. We suggest that elevated predation risk may cause phenotypic changes in aphids over multiple generations, resulting in a more precipitous decline in herbivore populations than could be explained solely by increased predation rates.     

Shaped by the past,acting in the present: transgenerational plasticity of anti‐predatory traits

Phenotypic expression can be altered by direct perception of environmental cues (within‐generation phenotypic plasticity) and by the environmental cues experienced by previous generations (transgenerational plasticity). Few studies, however, have investigated how the characteristics of phenotypic traits affect their propensity to exhibit plasticity within and across generations. We tested whether plasticity differed within and across generations between morphological and behavioral anti‐predator traits of Physa acuta, a freshwater snail. We reared 18 maternal lineages of P. acuta snails over two generations using a full factorial design of exposure to predator or control cues and quantified adult F₂ shell size, shape, crush resistance, and anti‐predator behavior – all traits which potentially affect their ability to avoid or survive predation attempts. We found that most morphological traits exhibited transgenerational plasticity, with parental exposure to predator cues resulting in larger and more crush‐resistant offspring, but shell shape demonstrated within‐generation plasticity. In contrast, we found that anti‐predator behavior expressed only within‐generation plasticity such that offspring reared in predator cues responded less to the threat of predation than control offspring. We discuss the consequences of this variation in plasticity for trait evolution and ecological dynamics. Overall, our study suggests that further empirical and theoretical investigation is needed in what types of traits are more likely to be affected by within‐generational and transgenerational plasticity.     

Non‐additive effects of simulated heat waves and predators on prey phenotype and transgenerational phenotypic plasticity

Understanding the effects of extreme climatic events on species and their interactions is of paramount importance for predicting and mitigating the impacts of climate change on communities and ecosystems. However, the joint effects of extreme climatic events and species interactions on the behaviour and phenotype of organisms remain poorly understood, leaving a substantial gap in our knowledge on the impacts of climatic change on ecological communities. Using an aphid–ladybeetle system, we experimentally investigated the effects of predators and heat shocks on prey body size, microhabitat use, and transgenerational phenotypic plasticity (i.e., the asexual production of winged offspring by unwinged mothers). We found that (i) aphids were smaller in the presence of predators but larger when exposed to frequent heat shocks; (ii) frequent heat shocks shifted aphid distribution towards the plant's apex, but the presence of predators had the opposite effect and dampened the heat‐shock effects; and (iii) aphids responded to predators by producing winged offspring, but heat shocks strongly inhibited this transgenerational response to predation. Overall, our experimental results show that heat shocks inhibit phenotypic and behavioural responses to predation (and vice versa) and that such changes may alter trophic interactions, and have important consequences on the dynamics and stability of ecological communities. We conclude that the effects of extreme climatic events on the phenotype and behaviour of interacting species should be considered to understand the effects of climate change on species interactions and communities.     

Cues of Predation Risk Induce Instar‐ and Genotype‐Specific Changes in Pea Aphid Colony Spatial Structure

Deploying collective antipredator behaviors during periods of increased predation risk is a major determinant of individual fitness for most animal groups. Pea aphids, Acyrthosiphon pisum, which live in aggregations of genetically identical individuals produced via asexual reproduction warn nearby conspecifics of pending attack by secreting a volatile alarm pheromone. This alarm pheromone allows clone‐mates to evade predation by walking away or dropping off the host plant. Here, we test how a single alarm pheromone emission influences colony structure and defensive behavior in this species. Relative to control colonies, groups exposed to alarm pheromone exhibited pronounced escape behavior where many individuals relocated to adjacent leaves on the host plant. Alarm pheromone reception, however, also had subtle instar‐specific effects: The proportion of 1st instars feeding nearest the leaf petiole decreased as these individuals moved to adjacent leaves, while the proportion of 2nd–3rd instars feeding nearest the leaf petiole remained constant. Fourth instars also dispersed to neighboring leaves after pheromone exposure. Lastly, alarm pheromone reception caused maternal aphids to alter their preferred feeding sites in a genotype‐specific manner: Maternal aphids of the green genotype fed further from the petiole, while maternal aphids of the pink genotype fed closer to the petiole. Together, our results suggest that aphid colony responses to alarm pheromone constitute a diversity of nuanced instar‐ and genotype‐specific effects. These behavioral responses can dramatically change the spatial organization of colonies and their collective defensive behavior.     

Maternal body condition influences magnitude of anti-predator response in offspring

Organisms exhibit plasticity in response to their environment, but there is large variation even within populations in the expression and magnitude of response. Maternal influence alters offspring survival through size advantages in growth and development. However, the relationship between maternal influence and variation in plasticity in response to predation risk is unknown. We hypothesized that variation in the magnitude of plastic responses between families is at least partly due to maternal provisioning and examined the relationship between maternal condition, egg provisioning and magnitude of plastic response to perceived predation risk (by dragonfly larvae: Aeshna spp.) in northern leopard frogs (Lithobates pipiens). Females in better body condition tended to lay more (clutch size) larger (egg diameter) eggs. Tadpoles responded to predation risk by increasing relative tail depth (morphology) and decreasing activity (behaviour). We found a positive relationship between morphological effect size and maternal condition, but no relationship between behavioural effect size and maternal condition. These novel findings suggest that limitations imposed by maternal condition can constrain phenotypic variation, ultimately influencing the capacity of populations to respond to environmental change.     

Maternal predator-exposure has lifelong consequences for offspring learning in threespined sticklebacks

Learning is an important form of phenotypic plasticity that allows organisms to adjust their behaviour to the environment. An individual''s learning performance can be affected by its mother''s environment. For example, mothers exposed to stressors, such as restraint and forced swimming, often produce offspring with impaired learning performance. However, it is unclear whether there are maternal effects on offspring learning when mothers are exposed to ecologically relevant stressors, such as predation risk. Here, we examined whether maternal predator-exposure affects adult offsprings’ learning of a discrimination task in threespined sticklebacks (Gasterosteus aculeatus). Mothers were either repeatedly chased by a model predator (predator-exposed) or not (unexposed) while producing eggs. Performance of adult offspring from predator-exposed and unexposed mothers was assessed in a discrimination task that paired a particular coloured chamber with a food reward. Following training, all offspring learned the colour-association, but offspring of predator-exposed mothers located the food reward more slowly than offspring of unexposed mothers. This pattern was not driven by initial differences in exploratory behaviour. These results demonstrate that an ecologically relevant stressor (predation risk) can induce maternal effects on offspring learning, and perhaps behavioural plasticity more generally, that last into adulthood.     

Transgenerational phenotypic plasticity under future atmospheric conditions

Organisms often exhibit transgenerational phenotypic changes in response to an increased risk of parasitism or predation. Shifts in global atmospheric composition could modify these phenotypic effects through changes in either nutrient quantity/quality or altered interactions with higher trophic levels. Here we show that future atmospheric conditions alter a natural enemy‐induced wing polyphenism in aphids. Winged offspring production by Uroleucon nigrotuberculatum aphids on goldenrod (Solidago canadensis var. scabra) does not differ in enriched CO₂ and/or O₃ atmospheres. However, proportionally more winged offspring are produced in response to search cues from both coccinellid predators (Coccinella septempunctata) and hymenopteran parasitoids (Aphidius polygonaphis) relative to plants not searched by natural enemies. Moreover, the magnitude of this response differs under enriched CO₂ and O₃ environments. Aphids produce more winged offspring in response to predators under elevated CO₂, but produce more winged offspring in response to parasitoids under elevated O₃. Thus, global atmospheric changes influence natural enemy‐mediated phenotypic expression, with potentially far‐reaching consequences for trophic dynamics.     

Transgenerational plasticity of inducible defences: Combined effects of grand‐parental,parental and current environments

Phenotypic plasticity can occur across generations (transgenerational plasticity) when environments experienced by the previous generations influenced offspring phenotype. The evolutionary importance of transgenerational plasticity, especially regarding within‐generational plasticity, is a currently hot topic in the plasticity framework. How long an environmental effect can persist across generations and whether multigenerational effects are cumulative are primordial—for the evolutionary significance of transgenerational plasticity—but still unresolved questions. In this study, we investigated how the grand‐parental, parental and offspring exposures to predation cues shape the predator‐induced defences of offspring in the Physa acuta snail. We expected that the offspring phenotypes result from a three‐way interaction among grand‐parental, parental and offspring environments. We exposed three generations of snails without and with predator cues according to a full factorial design and measured offspring inducible defences. We found that both grand‐parental and parental exposures to predator cues impacted offspring antipredator defences, but their effects were not cumulative and depended on the defences considered. We also highlighted that the grand‐parental environment did alter reaction norms of offspring shell thickness, demonstrating an interaction between the grand‐parental transgenerational plasticity and the within‐generational plasticity. We concluded that the effects of multigenerational exposure to predator cues resulted on complex offspring phenotypic patterns which are difficult to relate to adaptive antipredator advantages.     

Can transgenerational plasticity contribute to the invasion success of annual plant species?

Adaptive transgenerational plasticity (TGP), i.e., significantly higher fitness when maternal and offspring conditions match, might contribute to the population growth of non-native species in highly variable environments. However, comparative studies that directly test this hypothesis are lacking. Therefore, we performed a reciprocal split-brood experiment to compare TGP in response to N and water availability in single populations of two invasive (Amaranthus retroflexus, Galinsoga parviflora) and two congeneric non-invasive introduced species (Amaranthus albus, Galinsoga ciliata). We hypothesized that the transgenerational effect is adaptive: (1) in invasive species compared with non-invasive adventives, and (2) in stressful conditions compared with resource-rich environments. The phenotypic variation among offspring was generated, in large part, by our experimental treatments in the maternal generation; therefore, we demonstrated a direct TGP effect on the offspring’s adult fitness. We found evidence, for the first time, that invasive and non-invasive adventive species differ regarding the expression of TGP in the adult stage, as adaptive responses were found exclusively in the invasive species. The manifestation of TGP was more explicit under resource-rich conditions; therefore, it might contribute to the population dynamics of non-native species in resource-rich sites rather than to their ecological tolerance spectra.     

The effect of microhabitat feeding site selection on aphid foraging and predation risk

Organisms attempt to optimize foraging by maximizing resource acquisition while minimizing predation risk. Aphids (group-living, phloem-feeding insects) routinely change feeding positions and interact with predators and parasites at the single-leaf scale. Here, we assess the life history and predation risk consequences of within-leaf feeding site choices in pea aphids in response to different natural enemies. First, three-chambered clip cages were used to isolate first instar aphids anterior and posterior to a centrally feeding adult on the underside of a single broad bean leaf. Development time to adulthood did not differ between feeding sites, nor did fecundity within the first 24 h of reproduction. Second, we recorded the frequency and latency of natural enemy attacks on aphids adhered to three leaf sites, matching those of the clip cage experiment, on the underside of a single leaf. Aphids feeding nearest the leaf petiole were at greatest risk of predation by a foliar foraging coccinellid predator, Hippodamia convergens, but not by a parasitoid wasp, Aphidius ervi. Thus, feeding nearer the leaf petiole provided no individual life history benefits and exposes the aphid to increased predation risk. We further discuss the notion that feeding at these sites may provide inclusive fitness benefits for colony mates via alarm signaling and subsequent decreased predation success.     

Maternal exposure to predation risk decreases offspring antipredator behaviour and survival in threespined stickleback

1. Adaptive maternal programming occurs when mothers alter their offspring's phenotype in response to environmental information such that it improves offspring fitness. When a mother's environment is predictive of the conditions her offspring are likely to encounter, such transgenerational plasticity enables offspring to be better-prepared for this particular environment. However, maternal effects can also have deleterious effects on fitness.2. Here, we test whether female threespined stickleback fish exposed to predation risk adaptively prepare their offspring to cope with predators. We either exposed gravid females to a model predator or not, and compared their offspring's antipredator behaviour and survival when alone with a live predator. Importantly, we measured offspring behaviour and survival in the face of the same type of predator that threatened their mothers (Northern pike).3. We did not find evidence for adaptive maternal programming; offspring of predator-exposed mothers were less likely to orient to the predator than offspring from unexposed mothers. In our predation assay, orienting to the predator was an effective antipredator behaviour and those that oriented, survived for longer.4. In addition, offspring from predator-exposed mothers were caught more quickly by the predator on average than offspring from unexposed mothers. The difference in antipredator behaviour between the maternal predator-exposure treatments offers a potential behavioural mechanism contributing to the difference in survival between maternal treatments.5. However, the strength and direction of the maternal effect on offspring survival depended on offspring size. Specifically, the larger the offspring from predator-exposed mothers, the more vulnerable they were to predation compared to offspring from unexposed mothers.6. Our results suggest that the predation risk perceived by mothers can have long-term behavioural and fitness consequences for offspring in response to the same predator. These stress-mediated maternal effects can have nonadaptive consequences for offspring when they find themselves alone with a predator. In addition, complex interactions between such maternal effects and offspring traits such as size can influence our conclusions about the adaptive nature of maternal effects.     

Maternal effects in the soft scale insect Saissetia coffeae (Hemiptera: Coccidae)

Effects of maternal environment on offspring performance have been documented frequently in herbivorous insects. Despite this, very few cases exist in which exposure of parent insects to a resource causes the phenotype of their offspring to be adjusted in a manner that is adaptive for that resource, a phenomenon called adaptive transgenerational phenotypic plasticity. I performed a two-generation reciprocal cross-transplant experiment in the field with the soft scale insect Saissetia coffeae (Hemiptera: Coccidae) on two disparate host plant species in order to separate genetic effects from possible transgenerational plasticity. Despite striking differences in quality between host species, maternal host had no effect on overall offspring performance, and I detected no "acclimatization" to the maternal host species. However, there was a significant negative association between maternal and offspring development times, with potentially adaptive implications. Furthermore, offspring of mothers reared in an environment where scale densities were higher and scales were more frequently killed by fungi were significantly less likely to suffer from fungal attack than were offspring of mothers reared in an environment where densities were low and fungal attack was rare. Although S. coffeae does not appear to alter offspring phenotype to increase offspring fitness on these two distinct plant species, it does appear that offspring phenotype may be responding to some subtler aspects of maternal environment. In particular, the possibility of induced transgenerational prophylaxis in S. coffeae deserves further investigation.     

Transduction of high‐density signals across generations in aphid wing polyphenism

Wing polyphenism in aphids represents an outstanding example of adaptive phenotypic plasticity. During summer, parthenogenic mother aphids alter the developmental fate of their embryos to produce wingless or winged adult forms in response to high population density (i.e. crowded conditions). Although this maternal effect is well known, the mechanisms underlying transgenerational winged‐morph determination remain largely unresolved. In the present study, the effects of different high‐density treatment durations are tested on the vetch aphid Megoura crassicauda Mordvilko aiming to investigate how and when the density signals detected by mothers are transmitted to embryos. The duration of density treatment shows additive effects on both the number of crowded females producing winged aphids (winged‐producers) and the number of winged progeny. In addition, even when high‐density treatment is stopped, the production of winged offspring continues for several days and depends on the duration of treatment. The results indicate that mother aphids retain high‐density signals for a period after removal of the stimulus. Furthermore, observations of the progeny sequence (i.e. the order in which the offspring are born) and the embryonic stages developing in the mothers reveal that high‐density information may affect embryonic fate at the late embryonic stage immediately before cuticle formation.     

Bigger mothers are better mothers: disentangling size-related prenatal and postnatal maternal effects

Despite a vast literature on the factors controlling adult size, few studies have investigated how maternal size affects offspring size independent of direct genetic effects, thereby separating prenatal from postnatal influences. I used a novel experimental design that combined a cross-fostering approach with phenotypic manipulation of maternal body size that allowed me to disentangle prenatal and postnatal maternal effects. Using the burying beetle Nicrophorus vespilloides as model organism, I found that a mother''s body size affected egg size as well as the quality of postnatal maternal care, with larger mothers producing larger eggs and raising larger offspring than smaller females. However, with respect to the relative importance of prenatal and postnatal maternal effects on offspring growth, only the postnatal effects were important in determining offspring body size. Thus, prenatal effects can be offset by the quality of postnatal maternal care. This finding has implications for the coevolution of prenatal and postnatal maternal effects as they arise as a consequence of maternal body size. In general, my study provides evidence that there can be transgenerational phenotypic plasticity, with maternal size determining offspring size leading to a resemblance between mothers and their offspring above and beyond any direct genetic effects.     

Empirical patterns of environmental variation favor adaptive transgenerational plasticity

Effects of parental environment on offspring traits have been well known for decades. Interest in this transgenerational form of phenotypic plasticity has recently surged due to advances in our understanding of its mechanistic basis. Theoretical research has simultaneously advanced by predicting the environmental conditions that should favor the adaptive evolution of transgenerational plasticity. Yet whether such conditions actually exist in nature remains largely unexplored. Here, using long‐term climate data, we modeled optimal levels of transgenerational plasticity for an organism with a one‐year life cycle at a spatial resolution of 4 km² across the continental United States. Both annual temperature and precipitation levels were often autocorrelated, but the strength and direction of these autocorrelations varied considerably even among nearby sites. When present, such environmental autocorrelations render offspring environments statistically predictable based on the parental environment, a key condition for the adaptive evolution of transgenerational plasticity. Results of our optimality models were consistent with this prediction: High levels of transgenerational plasticity were favored at sites with strong environmental autocorrelations, and little‐to‐no transgenerational plasticity was favored at sites with weak or nonexistent autocorrelations. These results are among the first to show that natural patterns of environmental variation favor the evolution of adaptive transgenerational plasticity. Furthermore, these findings suggest that transgenerational plasticity is likely variable in nature, depending on site‐specific patterns of environmental variation.     

Transgenerational effect alters the interspecific competition between two dominant species in a temperate steppe

One of the key aims of global change studies is to predict more accurately how plant community composition responds to future environmental changes. Although interspecific relationship is one of the most important forces structuring plant communities, it remains a challenge to integrate long‐term consequences at the plant community level. As an increasing number of studies have shown that maternal environment affects offspring phenotypic plasticity as a response to global environment change through transgenerational effects, we speculated that the transgenerational effect would influence offspring competitive relationships. We conducted a 10‐year field experiment and a greenhouse experiment in a temperate grassland in an Inner Mongolian grassland to examine the effects of maternal and immediate nitrogen addition (N) and increased precipitation (Pr) on offspring growth and the interspecific relationship between the two dominant species, Stipa krylovii and Artemisia frigida. According to our results, Stipa kryloii suppressed A. frigida growth and population development when they grew in mixture, although immediate N and Pr stimulated S. kryloii and A. frigida growth simultaneously. Maternal N and Pr declined S. krylovii dominance and decreased A. frigida competitive suppression to some extent. The transgenerational effect should further facilitate the coexistence of the two species under scenarios of increased nitrogen input and precipitation. If we predicted these species'' interspecific relationships based only on immediate environmental effects, we would overestimate S. krylovii''s competitive advantage and population development, and underestimate competitive outcome and population development of A. frigida. In conclusion, our results demonstrated that the transgenerational effect of maternal environment on offspring interspecific competition must be considered when evaluating population dynamics and community composition under the global change scenario.     

All Clone-mates are not Created Equal: Fitness Discounting Theory Predicts Pea Aphid Colony Structure

As many animals form aggregations, group-living is believed to be adaptive. It is not clear, though, if clonal aggregations should have spatial structure, as protecting clone-mates is the genetic equivalent of protecting self. ‘Fitness discounting’ theory states that immediate reproductive opportunities are of greater value than are delayed opportunities. Thus, we hypothesized that spatial structure should exist in colonies of unequal-aged, clonal organisms like aphids. We predicted that, compared to reproductive (5^th instar) individuals, young (2^nd and 3^rd instar) juveniles (i.e., the youngest instars capable of emitting an alarm signal) should occupy the most dangerous feeding positions. As individuals approach reproductive maturity and alarm signals decline (4^th instar), they should occupy increasingly safer feeding positions. We tested these predictions by documenting the spatial distribution of two (green and pink) pea aphid, Acyrthosiphon pisum, asexual lineages (“clones”) at 1, 3, 6, 24, 48, 72, 96, and 120 h after host plant colonization. Confirming our hypothesis, we found that early (2^nd and 3^rd) instar aphids occupied feeding positions with the highest predation risk. Upon reaching the penultimate (4^th) instar, individuals dispersed from the colony to colonize other leaves. Thus, pea aphid colonies are not random aggregations; aphid colony structure can be explained by fitness discounting theory.     

Paternal epigenetic effects of population density on locust phase‐related characteristics associated with heat‐shock protein expression

Many species exhibit transgenerational plasticity by which environmental cues experienced by either parent can be transmitted to their offspring, resulting in phenotypic variants in offspring to match ancestral environments. However, the manner by which paternal experiences affect offspring plasticity through epigenetic inheritance in animals generally remains unclear. In this study, we examined the transgenerational effects of population density on phase‐related traits in the migratory locust Locusta migratoria. Using an experimental design that explicitly controls genetic background, we found that the effects of crowd or isolation rearing on phase plasticity could be inherited to the offspring. The isolation of gregarious locusts resulted in reduced weight in offspring eggs and altered morphometric traits in hatchlings, whereas crowding of solitarious locusts exhibited opposite effects. The consequences of density changes were transmitted by both maternal and paternal inheritance, although the expression of paternal effects was not as pronounced as that of maternal effects. Prominent expression of heat‐shock proteins (Hsps), such as Hsp90, Hsp70 and Hsp20.6, could be triggered by density changes. Hsps were significantly upregulated upon crowding but downregulated upon isolation. The variation in parental Hsp expression was also transmitted to the offspring, in which the pattern of inheritance was consistent with that of phase characteristics. These results revealed a paternal effect on phase polyphenism and Hsp expression induced by population density, and defined a model system that could be used to study the paternal epigenetic inheritance of environmental changes.     

Soldiers with large weapons in predator-abundant midsummer: phenotypic plasticity in a eusocial aphid

Eusocial aphids produce sterile individuals (“soldiers”) that specialize behaviorally and morphologically to protect their colony from predators, while production of soldiers can negatively affect colony growth because of reproductive allocation and opportunity cost. Hence, a cost-saving soldier production strategy is expected to be favored. Here, we hypothesize that, to save the cost, a eusocial aphid Ceratovacuna japonica produces soldiers with smaller weapon in the season when predators are not abundant. The abundance of two specialist lepidopteran predators (i.e., Taraka hamada and Atkinsonia ignipicta) of C. japonica dramatically increased, and aphid colony size significantly decreased, from July to August. In line with these, the soldiers in August had larger weapons (i.e., frontal horns) than those in June, indicating a correlational increase in weapon size with predation pressure. We predict that a reliable prospective signal indicating the coming of midsummer (environmental temperature) induces mother aphids to produce soldiers with larger weapons. Experiments clarified that soldiers produced at 20 °C (typical temperature of July to August) had larger weapons than those produced at 15 °C (typical temperature of May to July). Such phenotypic plasticity appears to be adaptive to maximize the fitness of C. japonica under a temporally variable but predictable predation environment. These results indicate that C. japonica aphids not merely have distinctive reproductive—and soldier castes, but also produce differentially armed soldiers in a habitat with temporally changing predation risks.     

Reproduction in Risky Environments: The Role of Invasive Egg Predators in Ladybird Laying Strategies

Reproductive environments are variable and the resources available for reproduction are finite. If reliable cues about the environment exist, mothers can alter offspring phenotype in a way that increases both offspring and maternal fitness (‘anticipatory maternal effects’—AMEs). Strategic use of AMEs is likely to be important in chemically defended species, where the risk of offspring predation may be modulated by maternal investment in offspring toxin level, albeit at some cost to mothers. Whether mothers adjust offspring toxin levels in response to variation in predation risk is, however, unknown, but is likely to be important when assessing the response of chemically defended species to the recent and pervasive changes in the global predator landscape, driven by the spread of invasive species. Using the chemically defended two-spot ladybird, Adalia bipunctata, we investigated reproductive investment, including egg toxin level, under conditions that varied in the degree of simulated offspring predation risk from larval harlequin ladybirds, Harmonia axyridis. H. axyridis is a highly voracious alien invasive species in the UK and a significant intraguild predator of A. bipunctata. Females laid fewer, larger egg clusters, under conditions of simulated predation risk (P+) than when predator cues were absent (P-), but there was no difference in toxin level between the two treatments. Among P- females, when mean cluster size increased there were concomitant increases in both the mass and toxin concentration of eggs, however when P+ females increased cluster size there was no corresponding increase in egg toxin level. We conclude that, in the face of offspring predation risk, females either withheld toxins or were physiologically constrained, leading to a trade-off between cluster size and egg toxin level. Our results provide the first demonstration that the risk of offspring predation by a novel invasive predator can influence maternal investment in toxins within their offspring.