Dietary influence of cardenolides on larval growth and development of the milkweed bug Oncopeltus fasciatus

Larvae of the milkweed bug Oncopeltus fasciatus were reared on the seeds of eight different species of milkweed (Asclepias), representing a wide range of cardenolide concentrations in the diet. There were few significant differences in larval developmental period, wet body weight of teneral adults, dry weight of adults, and pronotal width of adults reared on the different diets. However, the data indicate no significant correlations between cardenolide content, and body weight or size of the adult insects.There was no evidence in this study of a physiological cost or adverse effect on the larval growth and development of insects which sequestered and stored differing quantities of cardenolides. Instead, the data support a recently-proposed model of cardenolide sequestration which may be energy-independent.The validity of evidence supporting a physiological cost hypothesis for sequestration of cardenolides by the monarch butterfly is discussed in the light of these findings.     

Chemical egg defence in the large milkweed bug,Oncopeltus fasciatus,derives from maternal but not paternal diet

Many herbivorous insects sequester defensive compounds from their host‐plants and incorporate them into their eggs to protect them against predation. Here, we investigate whether transmission of cardenolides from the host‐diet to the eggs is maternal, paternal, or biparental in the large milkweed bug, Oncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae). We reared individual bugs on either milkweed seeds [MW; Asclepias syriaca L. (Apocynaceae)] that contain cardenolides, or on sunflower seeds [SF; Helianthus annuus L. (Asteraceae)] that do not contain cardenolides. We mated females and males so that all four maternal/paternal diet combinations were represented: MW/MW, MW/SF, SF/MW, and SF/SF. Using larvae of the common green lacewing, Chrysoperla (Chrysopa) carnea (Stevens) (Neuroptera: Chrysopidae), we conducted two‐choice predation trials to assess whether maternal, paternal, or biparental transmission of cardenolides into the eggs of O. fasciatus increased protection against predation. Furthermore, we used high performance liquid chromatography (HPLC) to assess putative cardenolide content of eggs from the various parental diet treatment groups. The predation trials suggested that regardless of male diet, eggs were afforded better protection when females had been raised on milkweed. However, many eggs were at least partially consumed. This suggests that although chemical defence of eggs does not guarantee protection to eggs on an individual basis, they may increase the probability that some eggs in a clutch are left intact thereby potentially conferring a fitness advantage to more offspring than if eggs are left unprotected. Based on HPLC analysis we found that maternal contribution of cardenolides was significantly greater than paternal contribution of cardenolides to the eggs, supporting the results of our predation trials that a maternal diet of milkweed makes eggs more distasteful than a paternal diet of milkweed.     

Gene duplications circumvent trade‐offs in enzyme function: Insect adaptation to toxic host plants

Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K‐ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K‐ATPase α‐subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade‐offs in gene function, we expressed the cardenolide‐sensitive Na,K‐ATPase of Drosophila melanogaster in vitro and introduced four distinct combinations of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K‐ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K‐ATPase α1C copy maintained activity similar to the wild‐type enzyme. Our results suggest that the Na,K‐ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier.     

Variation in the geometry of foreleg claws in sympatric giant water bug species: an adaptive trait for catching prey?

When giant water bugs (Heteroptera: Belostomatidae) encounter prey animals that are larger than they are themselves, they first hook the claw of their raptorial legs onto the animal, and then use all their legs to pin it. The claws of the raptorial legs in giant water bugs play an important role in catching larger prey, but the relationship between the claws, body lengths of predators, and prey size has not been fully investigated. To elucidate the functioning of claws in catching prey, we investigated prey body size relative to predator size in nymphs of two sympatric belostomatid giant water bug species, the vertebrate eater Kirkaldyia (=Lethocerus) deyrolli Vuillefroy and the invertebrate eater Appasus japonicus Vuillefroy, captured in rice fields. The younger nymphs of K. deyrolli caught preys that were larger than themselves, whereas those of A. japonicus caught preys that were smaller. Younger nymphs of K. deyrolli had claws that were curved more sharply than those of A. japonicus. The more curved claws of younger nymphs of K. deyrolli probably hook more easily onto larger vertebrates and thus this shape represents an adaptation for acquiring such prey.     

Na+/K+-ATPase resistance and cardenolide sequestration: basal adaptations to host plant toxins in the milkweed bugs (Hemiptera: Lygaeidae: Lygaeinae)

Despite sequestration of toxins being a common coevolutionary response to plant defence in phytophagous insects, the macroevolution of the traits involved is largely unaddressed. Using a phylogenetic approach comprising species from four continents, we analysed the ability to sequester toxic cardenolides in the hemipteran subfamily Lygaeinae, which is widely associated with cardenolide-producing Apocynaceae. In addition, we analysed cardenolide resistance of their Na⁺/K⁺-ATPases, the molecular target of cardenolides. Our data indicate that cardenolide sequestration and cardenolide-resistant Na⁺/K⁺-ATPase are basal adaptations in the Lygaeinae. In two species that shifted to non-apocynaceous hosts, the ability to sequester was secondarily reduced, yet Na⁺/K⁺-ATPase resistance was maintained. We suggest that both traits evolved together and represent major coevolutionary adaptations responsible for the evolutionary success of lygaeine bugs. Moreover, specialization on cardenolides was not an evolutionary dead end, but enabled this insect lineage to host shift to cardenolide-producing plants from distantly related families.     

Defence by plant toxins in milkweed bugs (Heteroptera: Lygaeinae) through the evolution of a sophisticated storage compartment

Species of the heteropteran subfamily Lygaeinae possess special subcuticular compartments to store cardiac glycosides, plant‐derived defensive compounds, which they release upon predator attack. In adults of the large milkweed bug, Oncopeltus fasciatus, these storage compartments have previously been described as a modified integument, forming a fluid‐filled dorsolateral space. Here we use three‐dimensional imaging of serial histological sections and synchrotron radiation‐based micro‐computed tomography data to reveal the morphology of these storage compartments and the mechanisms used for the release of a cardiac glycoside‐rich fluid upon attack. Our comparative analysis revealed that the morphology and release mechanism vary among the species investigated. By reconstructing these traits on a recent molecular phylogeny of the Lygaeinae, we demonstrate that the adaptations for the storage and release of cardiac glycosides have evolved in a stepwise manner.     

Chinese mantids gut toxic monarch caterpillars: avoidance of prey defence?

1. Monarch caterpillars, Danaus plexippus (Linnaeus), feed on milkweed plants in the genus Asclepias and sequester cardenolides as an anti–predator defence. However, some predators are able to consume this otherwise unpalatable prey. 2. Chinese mantids, Tenodera sinensis (Saussure), were observed consuming monarch caterpillars by ‘gutting’ them (i.e. removing the gut and associated internal organs). They then feed on the body of this herbivore without any apparent ill effects. 3. How adult T. sinensis handle and consume toxic (D. plexippus) and non–toxic [Ostrinia nubilalis (Hübner) and Galleria mellonella (Linnaeus)] caterpillars was explored. The differences in the carbon/nitrogen (C:N) ratio and cardenolide content of monarch tissue consumed or discarded by mantids were analysed. 4. Mantids gutted monarchs while wholly consuming non–toxic species. Monarch gut tissue had a higher C:N ratio than non–gut tissue, confirming the presence of plant material. Although there were more cardenolide peaks in the monarch body compared with gut tissue, the total cardenolide concentration and polarity index did not differ. 5. Although T. sinensis treated toxic prey differently than non–toxic prey, gutting did not decrease the mantid's total cardenolide intake. As other predators consume monarch caterpillars whole, this behaviour may be rooted in species–specific vulnerability to particular cardenolides or simply reflect a preference for high–N tissues.     

Adaptive change in protective coloration in adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae): test of detectability of two colour forms by avian predators

1. Protective coloration in insects may be aposematic or cryptic, and some species change defensive strategy between instars. In Sweden, the adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae) undergo a seasonal colour change from pale brown and black striation in the pre‐hibernating adults, to red and black striation in the same post‐hibernating individuals. To the human eye the pre‐hibernating adults appear cryptic against the withered late summer vegetation, whereas the red and black post‐hibernating adults appear aposematic. This suggests a possibility of a functional colour change. However, what is cryptic to the human eye is not necessarily cryptic to a potential predator. 2. Therefore we tested the effect of coloration in adult G. lineatum on their detectability for avian predators. Great tits (Parus major) were trained to eat sunflower seeds hidden inside the emptied exoskeletons of pale or red G. lineatum. Then the detection time for both colour forms was measured in a dry vegetation environment. 3. The birds required a longer time to find the pale form of G. lineatum than the red one. The pale form appears more cryptic on withered late summer vegetation than the red form, not only to the human eye but also to avian predators. The result supports the idea that the adult individuals of G. lineatum undergo a functional change from a cryptic protective coloration to an aposematic one.     

Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa – spiders and birds – to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.     

An energetic analysis of host plant selection by the large milkweed bug,Oncopeltus fasciatus

Summary The large milkweed bug, Oncopeltus fasciatus, is a specialized seed feeder that has been observed completing nymphal development in the field on only a small proportion of its potential host species within the genus Asclepias. In central Missouri only two of the six milkweed species studied, A. syriaca and A. verticillata, commonly supported nymphal O. fasciatus growth in the field. The seed of all six species, however, was equally suitable food for bugs reared in the laboratory. In laboratory preference tests, adult bugs chose to feed on the largest seeds, A. hirtella, but such a preference could not explain the observed field feeding patterns.One explanation to account for the observed host plant selection is based upon an energetic analysis. Only A. syriaca provided enough seed biomass for a clutch of O. fasciatus nymphs to develop on a single plant, and only A. verticillata grew in high enough density that a clutch could find sufficient food within the limited range of nymphal movement. These results illustrate a corollary of the resource concentration hypothesis: within a plant group whose members share similar secondary plant chemistries, the only species that will be viable hosts for a specialized herbivore are those that provide the minimal resource density necessary for the completion of nymphal development.In central Missouri, O. fasciatus has specialized on a critical resource density, not traits of individual Asclepias species. The appearance of host selection within the potential host plant spectrum is the result of a characteristic growth form, seed output, and dispersion pattern for each milkweed species that makes some species much more likely than others to produce sufficient seed resources.     

Effects of soil nutrients on the sequestration of plant defence chemicals by the specialist insect herbivore,Danaus plexippus

1. Although anthropogenic nitrogen (N) enrichment has significantly changed the growth, survival and reproduction of herbivorous insects, its effects on the defensive sequestration of secondary chemicals by insect herbivores are less well understood. Previous studies have shown that soil nutrient availability can affect sequestration directly through changing concentrations of plant defence chemicals, or indirectly through altering growth rates of herbivores. There has been less exploration of how nutrient deposition affects the consumption of secondary chemicals and subsequent sequestration efficiency. In the current study, the overall effect of soil N availability on cardenolide sequestration by the monarch caterpillar Danaus plexippus was examined. Specifically, the effects of soil nutrient availability on growth, consumption, excretion and sequestration efficiency of cardenolides by D. plexippus larvae fed on the tropical milkweed Asclepias curassavica were measured. 2. The results showed that soil N and phosphorus (P) fertilisation significantly reduced caterpillar growth rate and the sequestration efficiency of cardenolides by monarch caterpillars feeding on A. curassavica. The lowered sequestration efficiency was accompanied by higher concentrations of cardenolides in frass. Although the total cardenolide contents of caterpillars were lower under high N or P fertilisation levels, caterpillar cardenolide concentrations were constant across fertilisation treatments because of lower growth rates (and therefore lower body mass) under high fertilisation. It is concluded that anthropogenic N deposition may have multiple effects on insect herbivores, including their ability to defend themselves from predators with sequestered plant defences.     

Maternal Care in the Burrower Bug <Emphasis Type="Italic">Adomerus triguttulus</Emphasis>: Defensive Behavior

Some burrower bugs (Heteroptera: Cydnidae) show complex patterns of maternal care, including defense against predators and the provisioning of food to nymphs. Recently, the subsocial cydnid bugs have attracted the interest of researchers as model systems to study the behavioral ecology of parental investment. However, there have been few attempts to quantify the fitness benefits of maternal behavior other than provisioning. Here, we examined the maternal behavior of Adomerus triguttulus and its adaptive significance in terms of offspring survival in the field. A. triguttulus young depend on fallen nutlets of myrmecophorous mints, Lamium spp. Under field conditions, females attend offspring, from eggs to second instar nymphs, in nests on the ground under the litter. When disturbed, the females showed aggressive responses against the source of disturbance. The females often carried spherical clutches of eggs away from the nest when heavily disturbed. Female-removal experiments in the field demonstrated a defensive function of the female behavior; predators, such as ants, attacked egg clutches without females and the clutches often disappeared during the experiment. Egg clutches without females sometimes also suffered from fungal infection. Selective factors on maternal defensive behavior in A. triguttulus are discussed in terms of habitat properties possibly emerging from insect–plant associations.     

Effects of food plant and group size on predator defence: differences between two co-occurring aposematic Lygaeinae bugs

1. The role of food plant and aggregation on the defensive properties of two aposematic sympatrically occurring seed bugs, Tropidothorax leucopterus and Lygaeus equestris (Lygaeinae, Heteroptera), was investigated. Larvae reared on seeds either of their natural host plant Vincetoxicum hirundinaria (Asclepiadaceae) or of sunflower Helianthus annuus were subjected to predation by chicks. 2. The two species differ in their dependency on the host for their defence. Lygaeus equestris was better defended on its natural host plant than on the alternative food, as indicated by fewer attacks, lower mortality, and predator avoidance after experience. No such effect of food plant could be found for T. leucopterus, suggesting the existence of alternative defences in this species. 3. The number of attacks was lower when host plant‐fed larvae of both species were presented in groups. 4. The discussion concerns how major components of an aposematic syndrome, such as host plant chemistry, insect colouration, and aggregation, are integrated with other life‐history traits to form alternative lifestyles in L. equestris and T. leucopterus.     

Death feigning as an adaptive anti‐predator behaviour: Further evidence for its evolution from artificial selection and natural populations

Death feigning is considered to be an adaptive antipredator behaviour. Previous studies on Tribolium castaneum have shown that prey which death feign have a fitness advantage over those that do not when using a jumping spider as the predator. Whether these effects are repeatable across species or whether they can be seen in nature is, however, unknown. Therefore, the present study involved two experiments: (a) divergent artificial selection for the duration of death feigning using a related species T. freemani as prey and a predatory bug as predator, demonstrating that previous results are repeatable across both prey and predator species, and (b) comparison of the death‐feigning duration of T. castaneum populations collected from field sites with and without predatory bugs. In the first experiment, T. freemani adults from established selection regimes with longer durations of death feigning had higher survival rates and longer latency to being preyed on when they were placed with predatory bugs than the adults from regimes selected for shorter durations of death feigning. As a result, the adaptive significance of death‐feigning behaviour was demonstrated in another prey–predator system. In the second experiment, wild T. castaneum beetles from populations with predators feigned death longer than wild beetles from predator‐free populations. Combining the results from these two experiments with those from previous studies provided strong evidence that predators drive the evolution of longer death feigning.     

A comparison between allozyme data and phenotypic distances from defensive secretion in Oreina leaf-beetles (Chrysomelinae)

The genetic relationships between five Oreina species (Chrysomelidae, Coleoptera) were studied. Of these species, four (O. bifrons, O. gloriosa, O. speciosa, O. variabilis) feed on Apiaceae and secrete mixtures of autogenous cardenolides from defensive glands, whilst the other (O. speciosissima) feeds on Asteraceae and is able both to produce cardenolides and to sequester pyrrolizidines N-oxides (PAs). A dendrogram based on the different mixtures of cardenolides produced by the different species agreed with these genetic relationships. In other words, cardenolide mixtures are good taxonomic markers, since the clustering method based on chemical defense produces a branching pattern similar to that based on genetic relationships.     

Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core)

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na⁺/K⁺-ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.     

The indestructible insect: Velvet ants from across the United States avoid predation by representatives from all major tetrapod clades

Velvet ants are a group of parasitic wasps that are well known for a suite of defensive adaptations including bright coloration and a formidable sting. While these adaptations are presumed to function in antipredator defense, observations between potential predators and this group are lacking. We conducted a series of experiments to determine the risk of velvet ants to a host of potential predators including amphibians, reptiles, birds, and small mammals. Velvet ants from across the United States were tested with predator's representative of the velvet ants native range. All interactions between lizards, free‐ranging birds, and a mole resulted in the velvet ants survival, and ultimate avoidance by the predator. Two shrews did injure a velvet ant, but this occurred only after multiple failed attacks. The only predator to successfully consume a velvet ant was a single American toad (Anaxyrus americanus). These results indicate that the suite of defenses possessed by velvet ants, including aposematic coloration, stridulations, a chemical alarm signal, a hard exoskeleton, and powerful sting are effective defenses against potential predators. Female velvet ants appear to be nearly impervious to predation by many species whose diet is heavily derived of invertebrate prey.     

Milkweed butterfly resistance to plant toxins is linked to sequestration,not coping with a toxic diet

Insect resistance to plant toxins is widely assumed to have evolved in response to using defended plants as a dietary resource. We tested this hypothesis in the milkweed butterflies (Danaini) which have progressively evolved higher levels of resistance to cardenolide toxins based on amino acid substitutions of their cellular sodium–potassium pump (Na⁺/K⁺-ATPase). Using chemical, physiological and caterpillar growth assays on diverse milkweeds (Asclepias spp.) and isolated cardenolides, we show that resistant Na⁺/K⁺-ATPases are not necessary to cope with dietary cardenolides. By contrast, sequestration of cardenolides in the body (as a defence against predators) is associated with the three levels of Na⁺/K⁺-ATPase resistance. To estimate the potential physiological burden of cardenolide sequestration without Na⁺/K⁺-ATPase adaptations, we applied haemolymph of sequestering species on isolated Na⁺/K⁺-ATPase of sequestering and non-sequestering species. Haemolymph cardenolides dramatically impair non-adapted Na⁺/K⁺-ATPase, but had systematically reduced effects on Na⁺/K⁺-ATPase of sequestering species. Our data indicate that major adaptations to plant toxins may be evolutionarily linked to sequestration, and may not necessarily be a means to eat toxic plants. Na⁺/K⁺-ATPase adaptations thus were a potential mechanism through which predators spurred the coevolutionary arms race between plants and insects.     

Density-mediated indirect effects of a common prey tadpole on interaction between two predatory bugs: <Emphasis Type="Italic">Kirkaldyia deyrolli</Emphasis> and <Emphasis Type="Italic">Laccotrephes japonensis</Emphasis>

This paper suggests that the nymphs of a specialist predator, Kirkaldyia (=Lethocerus) deyrolli (Belostomatidae: Heteroptera), are indirectly affected by their tadpoles’ prey. K. deyrolli nymphs and the predator Laccotrephes japonensis (Nepidae: Heteroptera) adults coexist in rice paddy fields. It was predicted that the difference in tadpole density may influence the K. deyrolli nymph survival rate. We first compared survival rates of the first instar nymphs of K. deyrolli in June (high tadpole density period) and July (low tadpole density period). Secondly, we investigated the survival rate of K. deyrolli nymphs at different tadpole densities and under the presence or absence of L. japonensis adults to examine whether higher tadpole density moderates predation pressure from L. japonensis adults to K. deyrolli nymphs, e.g., density-mediated indirect effects. As a result of the comparison, the survival rate of K. deyrolli nymphs in June was higher than that in July. For the field experiment, the slopes between the survival rate of K. deyrolli nymphs and tadpole density were positive under both predator presence and absence. However, the slope under the presence of a predator was steeper than that under absence of the predator (“predator-by-tadpole density interaction” was significant). These results suggest that a higher tadpole density in June provides an abundant food resource for K. deyrolli nymphs and also moderates predation pressure from L. japonensis.