Response of generalist and specialist insects to qualitative allelochemical variation

We examined the effects of a set of four biosynthetically related iridoid glycosides, aucubin, catalpol, loganin, and asperuloside, on larvae of a generalist,Lymantria dispar (Lymantriidae), the gypsy moth, and an adapted specialist, the buckeye,Junonia coenia (Nymphalidae). In general,L. dispar grew and survived significantly less well on artificial diets containing iridoid glycoside, compared to a control diet without iridoid glycosides. In choice tests, previous exposure to a diet containing iridoid glycosides caused larvae subsequently to prefer iridoid glycoside-containing diets even though they were detrimental to growth and survival. In contrast,J coenia larvae grew and survived better on diets with aucubin and catalpol, the two iridoid glycosides found in the host plantPlantago lanceolata (Plantaginaceae), than on diets with no iridoid glycoside or with loganin and asperuloside. The results of choice tests of diets with and without iridoid glycosides and between diets with different iridoid glycosides reflected these differences as well. These results are discussed in terms of (1) differences between generalists and specialists in their response to qualitative variation in plant allelochemical content, (2) the induction of feeding preferences, and (3) the evolution of qualitative allelochemical variation as a plant defense.     

Fate of iridoid glycosides in different life stages of the Buckeye,Junonia coenia (Lepidoptera: Nymphalidae)

The buckeye butterfly,Junonia coenia (Lepidoptera: Nymphalidae), specializes on plants that contain iridoid glycosides. To determine the fate of these compounds in larvae, pupae, and adults of this species, we reared larvae on artificial diets with and without iridoid glycosides, and on leaves of a host plant,Plantago lanceolata (Plantaginaceae). Quantification by gas chromatography showed that newly molted third-, fourth-, and fifth-instar larvae reared on leaves ofP. lanceolata contained means of 5.13, 2.88, and 6.83% dry weight iridoid glycoside. In contrast, the mean iridoid glycoside concentration of actively feeding fifth-instar larvae was 0.28% dry weight, that of pupae was 0.19% dry weight iridoids, and adults contained no detectable iridoids. Feeding experiments suggested that this reduction in actively feeding larvae was due to the metabolism of iridoid glycosides.P. lanceolata leaves in these experiments contained a mean of 1.00% dry weight iridoid glycoside, with a 2:1 ratio of aucubin to catalpol. Calculation of iridoid consumption and utilization indices showed that larvae fed artificial diets consumed, digested, and sequestered aucubin and catalpol in similar ways. When these indices were calculated for larvae fed leaves ofP. lanceolata, catalpol was sequestered twice as efficiently as aucubin.     

Iridoid glycosides as oviposition stimulants for the buckeye butterfly,Junonia coenia (Nymphalidae)

Females ofJunonia coenia (Nymphalidae), a specialist on plants that contain iridoid glycosides, were found to use aucubin and catalpol, iridoid glycosides typical of a host plant,Plantago lanceolata (Plantaginaceae), as oviposition cues. Incorporating dried ground leaf material or pure iridoid glycosides into agar disks proved to be a very effective method of testing. In no-choice tests and choice tests, females laid more eggs on disks withP. lanceolata leaf material or iridoid glycosides, compared to agar controls. There was variation among individual females in preference for disks withP. lanceolata leaf material versus disks with iridoid glycosides. Females given a choice of three different concentrations of iridoid glycoside (0.2, 0.5, 1.0%) in the agar disks and a control laid more eggs on the disk with the highest concentration of iridoid glycoside.     

The role of iridoid glycosides in host-plant specificity of checkerspot butterflies

The potential role of iridoid glycosides as feeding stimulants forEuphydryas chalcedona larvae was examined in three laboratory experiments. The first experiment examined larval behavior in choice tests between an artificial diet with no additives (AD) and an artificial diet with the iridoid glycoside, catalpol, added (AD + I) in one group; and AD and AD plus a crude extract from which the iridoid glycoside catalpol was crystallized (AD + Ex) in the second group. The larvae were found more often on AD + I or AD + Ex. The second experiment quantified larval consumption of artificial diets when given a choice of AD or AD + I, and AD or AD + Ex, and showed that larvae ate significantly more AD + I or AD + Ex than AD. The third experiment compared growth and survival on six diets: AD; AD + I; artificial diet with dried, ground upScrophularia californica leaves (AD + S); artificial diet with dried, ground upPlantago lanceolata leaves (AD + P);S. californica leaves (S); andP. lanceolata leaves (P). Growth was best onS. californica leaves, and survival was highest onS. californica andP. lanceolata leaves. There were no differences in growth rate or survival between AD andAD + I. Thus, iridoid glycosides serve as feeding attractants and stimulants for larvae ofEuphydryas chalcedona and are suggested as the basis of radiation in butterflies of the genusEuphydryas.     

Effect of qualitative and quantitative variation in allelochemicals on a generalist insect: Iridoid glycosides and the southern armyworm

The behavioral and physiological effects of plant allelochemicals have been difficult to demonstrate; it is not often clear whether the compounds are deterrent, toxic, or both. In this study, we compared the qualitative and quantitative effects of several iridoid glycosides on a generalist lepidopteran herbivore,Spodoptera eridania (Noctuidae). Larval growth and survivorship and larval preference or avoidance were measured on artificial diets containing different iridoid glycosides at different concentrations. We also tested the toxicity/deterrence of these compounds. We found that iridoid glycosides retarded larval growth significantly at relatively low concentrations and that they were usually avoided in preference tests. The toxicity/ deterrence test did not always reflect the results of these other tests. The merits of using a variety of methods for determining deterrence and/or toxicity of plant allelochemicals are discussed.     

The importance of sequestered iridoid glycosides as a defense against an ant predator

We reared larvae ofJunonia coenia Hubner (Nymphalidae) on artificial diets with trace concentrations of iridoid glycosides and on leaf diets with higher concentrations of iridoid glycosides. We offered these caterpillars to predacious ants and observed the effects of the following on predation: diet (artificial vs. leaf), site (ant colonies in dry vs. wet areas), instar (early vs. late), and time (changes in predation over five days). Diet and site were consistently significant predictors of the ants' propensities to reject prey and the caterpillars' abilities to escape predation. Leaf-diet caterpillars escaped more frequently than artificial-diet caterpillars, and ants from dry sites were more likely to reject prey than ants from wet sites. The percentage of iridoid glycosides found in individual caterpillars was also a good predictor of the probability of rejection by predators and prey escape. Caterpillars with higher levels of iridoids were more likely to be rejected and to escape, suggesting that sequestered iridoid glycosides are a defense against predaceous ants.     

Incorporation of an Introduced Weed into the Diet of a Native Butterfly: Consequences for Preference, Performance and Chemical Defense

The introduction of exotic plants, animals, and pathogens into non-native ecosystems can have profound effects on native organisms. Plantago lanceolata, narrow-leaf or ribwort plantain (Plantaginaceae), is a weed that was introduced to North America from Eurasia approximately 200 years ago and that has been incorporated into the diet of a variety of native North American herbivores. Plantain contains two iridoid glycosides, aucubin and catalpol, that can be toxic or deterrent to non-specialized herbivores or herbivores that have recently incorporated this species into their diet. Anartia jatrophae (Nymphalidae), the white peacock, feeds on plants in five families including the Plantaginaceae, and was recently observed feeding on plantain; however, the effects of feeding on this novel host plant are unknown. In this study, we performed a series of experiments to assess larval preference and performance on the introduced P. lanceolata and on a native host plant that does not contain iridoid glycosides, water hyssop, Bacopa monnieri (Plantaginaceae). We also tested whether or not white peacocks were able to sequester iridoid glycosides and compared this ability with an iridoid specialist, the buckeye, Junonia coenia (Nymphalidae). White peacocks successfully developed to the adult stage on plantain; larvae grew more slowly but pupae were heavier when compared with larvae and pupae reared on the native host plant. Larvae showed induced feeding preferences for the host plant on which they were reared. Furthermore, larvae sequestered small amounts of iridoids that were also retained in pupae and adults. These results suggest that incorporation of the introduced weed, plantain, into the diet of the white peacock may have important consequences for larval performance and preference, as well as for interactions with natural enemies.     

A Comparison of Sample Preparation Techniques for Quantifying Iridoid Glycosides Sequestered by Lepidopteran Larvae

This study compared different methods of tissue preparation for extraction of iridoid glycosides sequestered by three species of lepidopteran larvae. Junonia coenia is a specialist on plant species that produce iridoid glycosides, while the arctiids Estigmene acrea and Spilosoma congrua are both polyphagous and will eat plants that produce iridoid glycosides. Larvae of all three species were reared on leaves of Plantago lanceolata, which produces two primary iridoid glycosides, aucubin and catalpol. Three methods of preparing the specimens before extraction in methanol were compared in all three species: 1) larvae were flash-frozen in liquid nitrogen, 2) larvae were macerated fresh in boiling methanol, or 3) larvae were macerated fresh in room temperature methanol. A set of J. coenia larvae was oven-dried before maceration as an additional treatment for this species only. Junonia coenia sequestered the most iridoid glycosides, while E. acrea sequestered the least, and S. congrua was intermediate. Estigmene acrea was poor at sequestering catalpol. Tissue preparation method only significantly influenced iridoid glycoside recovery from S. congrua, with maceration in room-temperature methanol being the most effective of the three methods. This study shows that treatment of insects prior to iridoid glycoside extraction can influence recovery of the compounds, and that the effects of treatment may vary among different species.     

Effects of Ingested Secondary Metabolites on the Immune Response of a Polyphagous Caterpillar <Emphasis Type="Italic">Grammia incorrupta</Emphasis>

We considered the effects of plant secondary metabolites on the immune response, a key physiological defense of herbivores against pathogens and parasitoids. We tested the effect of host plant species and ingested iridoid glycosides on the immune response of the grazing, polyphagous caterpillar, Grammia incorrupta (Arctiidae). Individuals of G. incorrupta were fed either one of three plant diets with varying secondary metabolites, or an artificial diet with high or low concentrations of iridoid glycosides. An immune challenge was presented, followed by measurement of the encapsulation response. We failed to detect a significant difference in the immune response of G. incorrupta feeding on diets with varying concentrations of iridoid glycosides, or feeding on different host plants. However, the immune response was lower in caterpillars consuming the artificial diet compared to those consuming the plant diets. When caterpillar performance was measured, pupal weights were lower when caterpillars ingested high concentrations of iridoid glycosides due to a decrease in feeding efficiency. Overall, individuals of G. incorrupta that consumed different plant diets exhibited a high immune response with low variation. We conclude that the immune response of G. incorrupta is adapted to feeding on a variety of plants, which may contribute to the maintenance of this caterpillar’s polyphagous habit.     

Host Plant Influences on Iridoid Glycoside Sequestration of Generalist and Specialist Caterpillars

The effect of diet on sequestration of iridoid glycosides was examined in larvae of three lepidopteran species. Larvae were reared upon Plantago major, or P. lanceolata, or switched from one to the other in the penultimate instar. Junonia coenia is a specialist on iridoid glycoside-producing plants, whereas the arctiids, Spilosoma congrua and Estigmene acrea, are both polyphagous and eat iridoid-producing plants. All species sequestered iridoids. The specialist J. coenia sequestered from three to seven times the amounts sequestered by the two generalist species. Junonia coenia iridoid glycoside content depended on diet, and they sequestered from 5 to 15% dry weight iridoid glycosides. Estigmene acrea iridoid glycoside sequestration was relatively low, around 2% dry weight and did not vary with diet. Spilosoma congrua sequestration varied with diet and ranged from approximately 3 to 6% dry weight.     

Effect of iridoid glycoside content on oviposition host plant choice and parasitism in a specialist herbivore

The Glanville fritillary butterfly Melitaea cinxia feeds upon two host plant species in Å land, Finland, Plantago lanceolataand Veronica spicata, both of which produce iridoid glycosides. Iridoids are known to deter feeding or decrease the growth rate of many generalist insect herbivores, but they often act as oviposition cues to specialist butterflies and are feeding stimulants to their larvae. In this study, two iridoid glycosides (aucubin and catalpol) were analyzed by micellar electrokinetic capillary chromatography. We measured the spatial and temporal variation of iridoid glycosides in natural populations of the host plants of M. cinxia. We also analyzed the aucubin and catalpol content in plants in relation to their use by ovipositing females, and in relation to the incidence of parasitism of M. cinxia larvae in natural populations. The mean concentrations of aucubin and catalpol were higher in P. lanceolata than in V. spicata, and catalpol concentrations were higher than aucubin concentrations in both host species. Plantago lanceolata individuals that were used for oviposition by M. cinxia had higher aucubin concentrations than random plants and neighboring plants. Additionally, oviposition and random plants had higher catalpol concentrations than neighboring plants, indicating that ovipositing females select for high iridoid glycoside plants or that oviposition induces iridoid glycoside production in P. lanceolata. Parasitism by the specialist parasitoid wasp Cotesia melitaearum occurred most frequently in larval groups that were feeding on plants with low concentrations of catalpol, irrespective of year, population, and host plant species. Therefore, parasitoids appear to avoid or perform poorly in host larvae with high catalpol content.     

Laboratory colonization has not reduced constitutive or induced polysubstrate monooxygenase activity in velvetbean caterpillars

We evaluated whether velvetbean caterpillars (Anticarsia gemmatalis) from a laboratory colony had reduced constitutive (basal) and/or induced activities of their polysubstrate monooxygenase (PSMO) detoxification enzyme system as a result of long-term rearing (> 100 generations) on artificial diet without introduction of field-collected individuals. Larvae from the laboratory colony and those from a recently collected field strain were fed either a standard artificial diet (control), one containing the inducing allelochemical, flavone, or foliage ofIndigofera hirsuta (a host plant of this species), and their midgut PSMO activity was assessed by measuring the in vitro rate of aldrin epoxidation. Compared with the field-strain larvae, caterpillars from the laboratory colony had 1.9-fold greater constitutive activity (standard artificial diet) and 2.3-fold greater induced activity (flavone-treated diet). In addition, the magnitude of induction was somewhat greater for the laboratory-colony larvae (induced activity was 2.0-fold greater than constitutive activity) compared with those from the field-strain (1.6-fold). In contrast, no difference in strain activity was found when larvae were fedI. hirsuta foliage. The lower PSMO activity of the field-strain larvae when fed artificial diet may have been caused by their reduced feeding and growth performance compared with laboratory-colony larvae, and it may explain their greater sensitivity to allelochemicals incorporated in the artificial diet, as we found previously. The results of this study indicate that long-term rearing of theA. gemmatalis laboratory colony on artificial diet, without the introduction of field individuals, apparently has not selected for low constitutive activity or decreased inducibility of PSMO, and thus these larvae provide a suitable model for studying xenobiotic detoxication. In addition, they suggest that using an artificial diet to evaluate resistance to pesticides or other xenobiotics in fieldcollected insects, as is frequently done, may underestimate the level of resistance if the diet, through various causes, reduces the activity of detoxification enzymes contributing to the resistance.     

Rejection of host plant by larvae of cabbage butterfly: Diet-dependent sensitivity to an antifeedant

Garden nasturtium,Tropaeolum majus (Tropaeolaceae), is an acceptable host plant for the cabbage butterfly,Pieris rapae. Eggs are readily laid on the plant and hatching larvae feed and develop into normal pupae and adults. However, when second- to fifth-instar larvae were transferred from cabbage to nasturtium, they refused to feed and starved to death. Similar results were obtained when larvae were transferred from other host plants to nasturtium. However, larvae that were reared on nasturtium readily accepted cabbage as a new host plant. We have demonstrated the presence of strong antifeedants in nasturtium foliage and identified the most prominent active compound as chlorogenic acid. However, larvae reared on nasturtium had limited sensitivity, and larvae reared on a wheat germ diet were completely insensitive to the antifeedants. Larvae apparently develop sensitivity to the deterrent as a result of feeding on other host plants, whereas continuous exposure to the deterrent causes habituation or suppression of sensitivity development. The results demonstrate that dietary experience can dramatically affect the response of an insect to a potentially antifeedant compound in a plant.     

Hostplant Suitability and Defensive Chemistry of the Catalpa Sphinx, Ceratomia catalpae

The growth and survival of the Catalpa sphinx, Ceratomia catalpae (Sphingidae), were measured on five different species of Catalpa: C. bignonioides, C. bungeii, C. fargeseii, C. ovata, and C. speciosa. Larval growth varied significantly among these host plant species; however, survival did not differ. Quantification of the iridoid glycoside content of larvae, pupae, adults, larval frass, and leaves of the larval host plant, C. bignonioides, by gas chromatography showed that leaves contained both catalpol and catalposide; larvae, pupae, and frass contained only catalpol; and the adults contained no detectable iridoid glycosides. Amounts were highest in the larvae and declined in the pupal stage. Very small amounts of catalpol were detected in adults of the parasitoid, Cotesia congregata, and in the silken cocoons. The hemolymph in which the parasitoid larvae grew contained over 50% dry weight catalpol. Larvae of C. catalpae often regurgitate when disturbed. This may serve as a defense against predators. A comparison of the growth of larvae pinched with forceps to induce regurgitation with those that were not so treated showed that larvae that were pinched, and usually regurgitated, grew significantly more slowly than those that were not.     

Effects of Quantitative Variation in Allelochemicals in <Emphasis Type="Italic">Plantago lanceolata</Emphasis> on Development of a Generalist and a Specialist Herbivore and their Endoparasitoids

Studies in crop species show that the effect of plant allelochemicals is not necessarily restricted to herbivores, but can extend to (positive as well as negative) effects on performance at higher trophic levels, including the predators and parasitoids of herbivores. We examined how quantitative variation in allelochemicals (iridoid glycosides) in ribwort plantain, Plantago lanceolata, affects the development of a specialist and a generalist herbivore and their respective specialist and generalist endoparasitoids. Plants were grown from two selection lines that differed ca. 5-fold in the concentration of leaf iridoid glycosides. Development time of the specialist herbivore, Melitaea cinxia, and its solitary endoparasitoid, Hyposoter horticola, proceeded most rapidly when reared on the high iridoid line, whereas pupal mass in M. cinxia and adult mass in H. horticola were unaffected by plant line. Cotesia melitaearum, a gregarious endoparasitoid of M. cinxia, performed equally well on hosts feeding on the two lines of P. lanceolata. In contrast, the pupal mass of the generalist herbivore, Spodoptera exigua, and the emerging adult mass of its solitary endoparasitoid, C. marginiventris, were significantly lower when reared on the high line, whereas development time was unaffected. The results are discussed with regards to (1) differences between specialist and generalist herbivores and their natural enemies to quantitative variation in plant secondary chemistry, and (2) potentially differing selection pressures on plant defense.     

Detrimental effects ofCinchona leaf alkaloids on larvae of the polyphagous insectSpodoptera exigua

YoungCinchona ledgeriana plants contain two types of alkaloid: indole alkaloids in the leaves and quinoline alkaloids in the root. FromCinchona leaves, a crude alkaloid extract was made, containing the cinchophylline type of indole alkaloids and a small amount of 5-methoxytryptamine. The leaf alkaloid extract exerted a strong detrimental effect on the growth of larvae of the polyphagous beet armyworm,Spodoptera exigua (Lepidoptera). Feeding of larvae on an artificial diet containing the leaf alkaloids at the same concentrations as those found in the plant resulted in significant growth reduction, retardation in development, and mortality of the larvae. Cinchophyllines are composed of 5-methoxytryptamine coupled to a corynantheal unit. When incorporated into the artificial diet, 5-methoxytryptamine alone had no effect on the 5.exigua larvae. Corynantheal, however, had a strong detrimental effect on growth of the larvae, its effect being comparable to that of the leaf alkaloid extract. In contrast to the indole-type leaf alkaloids, the quinolinetypeCinchona root alkaloids did not affect growth and development of the larvae. These results suggest that the indole-type alkaloids, which inCinchona plants are present at the highest concentrations in the young, vulnerable leaflets, are involved in the chemical defense of the plant against herbivorous insects.     

Do Enemies of Herbivores Influence Plant Growth and Chemistry? Evidence from a Seminatural Experiment

It is predicted that enemies of insect herbivores may influence the effects of herbivores on their host plants by affecting the choice of plant genotypes. To examine the effect of predators, we conducted two experiments, each with a different caterpillar species (Junonia coenia and Pyrrharctia isabella). Under seminatural conditions, we provided a choice between two genotypes of plantain (Plantago lanceolata) with different levels of iridoid glycosides and used Podisus maculiventris stinkbugs as predators. There were four treatments: no herbivores and no predators, low density of herbivores and no predators, high density of herbivores and no predators, and high density of herbivores plus predators. The caterpillars had little effect on plant growth but did influence the iridoid glycoside concentration. For the Junonia experiment, the concentration of iridoid glycosides was less for plots with a low density of caterpillars (with no predators) compared to the other treatments of caterpillar density. In the Pyrrharctia experiment, catalpol was induced by a high density of caterpillars (with no predators). There were no increases in total iridoid glycosides associated with either herbivore species. The presence of predators had no effect on plant growth or total iridoid glycoside pattern. The lack of effect by predators seems to reflect the relatively large variation in iridoid glycoside concentration among leaf ages, and the herbivores ability to respond to that variation, such that the difference in iridoid glycoside concentrations in the plant genotypes was less important.     

Use of chemical variation and predation as plant defenses byEncelia farinosa against a specialist herbivore

Larvae of the monophagous herbivore,Trirhabda geminata, selectively eat particular plants and plant parts of its natural host,Encelia farinosa. Measurements of leaf damage and larval positions on branches through time support this observation. Time-lapse movie photography revealed that larvae are sufficiently mobile to search most of a plant in a 48-hr period and that aggregations were the result of larval activity and not directly the result of oviposition. Experiments withT. geminata larvae on artificial diets containing a range of natural concentrations of chemical extracts fromE. farinosa leaves showed that the larvae grew significantly slower and had a lower overall survivorship at the high concentration. Combining the results of all choice tests, larvae appeared unable to distinguish between high- and low-concentration agar diets. Considered individually, larval preferences for natural production concentrations changed as the season progressed. Early-season larvae preferred low-concentration leaves, while late-season larvae preferred high-concentrations. Measurements of chemical and nitrogen content of leaves selected by larvae in the field confirmed this pattern. Percent parasitism in field-collected larvae increased with season as the larval population decreased. This combination of slowed growth and increasing parasitism and predation is a putative defense strategy ofEncelia farinosa to prevent adaptation by a specialist herbivore to the total range of compounds elaborated.     

Accumulation of Glucosinolates by the Cabbage Aphid Brevicoryne brassicae as a Defense Against Two Coccinellid Species

Brassica nigra plants, characterized by high levels of sinigrin, and artificial aphid diets to which sinigrin was selectively added were used to rear the crucifer specialist, Brevicoryne brassicae. Aphids were provided as a food source to two species of polyphagous ladybird, Adalia bipunctata and Coccinella septempunctata. First instar A. bipunctata were unable to survive when fed with B. brassicae reared on B. nigra or diets containing 0.2% sinigrin, but when fed with aphids reared on diets containing 0% sinigrin, survival rates were high. By contrast, first instar C. septempunctata were able to survive when fed with aphids reared on B. nigra or artificial diets containing up to 1% sinigrin. However, the presence of sinigrin in the aphid diet decreased larval growth and increased the time necessary for larvae to reach second instar for this species of ladybird. These results indicate that the presence of sinigrin in the diet of B. brassicae makes this aphid unsuitable as a food source for A. bipunctata but not for C. septempunctata, although for this ladybird species, there appear to be costs associated with feeding on aphids that contain this secondary metabolite.     

Tests for induction of feeding preferences in larvae of eastern spruce budworm using extracts from three host plants

Feeding preferences to extracts from three host plants were determined for sixth-instar eastern spruce budworm larvae reared on one of three hosts or on artificial diet. Preferences of foliage-reared larvae were compared to those of diet-reared, or naive, larvae. No effect of plant induction was found using amino acids and bases and chloroform extracts on test disks in three-choice experiments. Possible effects of habituation for sugars and glycosides from white spruce were shown. Organic acids contributed to a phenomenon of avoidance learning in larvae reared on all three hosts, showing that this fraction from balsam fir was the least deterrent, while that from black spruce was the most deterrent.