Iridoid glycosides ofChelone glabra (Scrophulariaceae) and their sequestration by larvae of a sawfly,Tenthredo grandis (Tenthredinidae)

Analysis ofChelone glabra (Scrophulariaceae) by gas chromatography showed that leaves of this plant contained primarily the iridoid glycoside catalpol, and in a few individuals some aucubin was also detected. There was no difference in the iridoid glycoside content of damaged compared to undamaged plants, nor was there a difference between plants collected from a population in Leverett, Massachusetts, and those from plants in an experimental garden in Cambridge, Massachusetts. Larvae and prepupae of the sawfly,Tenthredo grandis (Tenthredinidae) contained catalpol sequestered from the larval host plant. The exuvia also contained catalpol, whereas the frass contained only aucubin. These results indicate that larvae of this sawfly selectively sequester catalpol, eliminating the aucubin in the frass.     

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.     

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.     

Host plant utilization and iridoid glycoside sequestration byEuphydryas anicia (Lepidoptera: Nymphalidae)

The iridoid glycoside content of individual adultEuphydryas anicia butterflies from two Colorado populations was quantitatively determined. At one site (Red Hill), larval host plants wereCastilleja integra andBesseya plantaginea, while at the other site (Cumberland Pass) a single host plant,B. alpina, was used. At Red Hill, macfadienoside and catalpol were sequestered, while at Cumberland Pass, catalpol and aucubin were sequestered. Artificial diet studies showed that larvae hydrolyzed a major iridoid ofB. plantaginea, 6-isovanilIylcatalpol, to catalpol (which was sequestered) and isovanillic acid (which was excreted). Large year-to-year and individual variation in butterfly iridoid content was established as was a female-male difference in macfadienoside vs. catalpol content. Larval host plant distributions and numbers were determined at Red Hill for two years and compared with changes in butterfly populations and sequestered iridoids.     

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.     

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.     

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.     

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.     

Iridoid glycosides and host-plant specificity in larvae of the buckeye butterfly,Junonia coenia (Nymphalidae)

Larvae of the buckeye,Junonia coenia (Nymphalidae) feed primarily on plants in four families: Scrophulariaceae, Plantaginaceae, Verbenaceae, and Acanthaceae. These plant families have in common the presence of a group of plant secondary compounds, the iridoid glycosides. Larvae were reared on three plant species and two artificial diets, one with and one without iridoid glycosides.Larvae grew poorly and had low survivorship on the artificial diet without iridoid glycosides, while growth and survival on the artificial diet with iridoid glycosides was comparable to that on plants. Choice tests using artificial diets with and without iridoid glycosides showed that larvae: (1) chose diets with iridoid glycosides (in the form of a crude extract or pure compound) over a diet without; (2) showed no preference between the diet with the crude extract and that with pure iridoid glycoside, and (3) preferred the artificial diet with ground leaves of the host plant,Plantago lanceolata, over the diet with pure iridoid glycosides. The artificial diet that larvae had been reared on prior to these tests had no effect on subsequent larval preferences in the choice tests.     

Chemical variation within and between individuals ofPlantago lanceolata (Plantaginaceae)

Variation in concentrations of leaf nitrogen and iridoid glycosides was examined in replicate plants of five genotypes ofPlantago lanceolata (Plantaginaceae) grown in an experimental garden. Nitrogen concentration and iridoid glycoside concentration were affected by leaf age. New leaves had nitrogen concentrations 1.7 to 2.7 times higher than mature leaves. Catalpol concentration was highest in new and intermediate-aged leaves. The concentration of aucubin, the biosynthetic precursor to catalpol, was higher in intermediate-aged leaves than in mature leaves, in three of five genotypes. Consequently, the proportion of aucubin relative to total iridoid glycosides increased as leaves aged. Concentration of iridoid glycosides was not correlated with plant size. Plant genotype significantly affected concentration of nitrogen and iridoid glycosides, as well as plant size. Thus, major indicators of hostplant quality for insect herbivores varied considerably both within and among plant genotypes and individuals.     

Systemic,genotype-specific induction of two herbivore-deterrent iridoid glycosides in Plantago lanceolata L. in response to fungal infection by Diaporthe adunca (Rob.) Niessel

Iridoid glycosides are a group of terpenoid secondary plant compounds known to deter generalist insect herbivores. In ribwort plantain (Plantago lanceolata), the iridoid glycosides aucubin and catalpol can be induced following damage by insect herbivores. In this study, we investigated whether the same compounds can be induced following infection by the fungal pathogen Diaporthe adunca, the causal agent of a stalk disease in P. lanceolata. Significant induction of aucubin and catalpol was observed in two of the three plant genotypes used in this study following inoculation with the pathogen. In one of the genotypes, induction occurred within 6 hr after inoculation, and no decay was observed within 8 days. The highest level of induction was observed in reproductive tissues (spikes and stalks) where infection took place. In these tissues, iridoid glycoside levels in infected plants were, on average, 97% and 37% higher than the constitutive levels in the corresponding control plants, respectively. Significant induction was also observed in leaves (24%) and roots (17%). In addition to significant genotypic variation in the level of induction, we found genetic variation for the tissue-specific pattern of induction, further broadening the scope for evolutionary fine-tuning of induced responses. Recent studies have revealed a negative association between iridoid glycoside levels in P. lanceolata genotypes and the amount of growth and reproduction of D. adunca that these genotypes support. However, for the three genotypes used in the present study, differences in resistance were not related to their constitutive or induced levels of iridoid glycosides, suggesting that additional resistance mechanisms are important in this host-pathogen system. We conclude that iridoid glycosides in P. lanceolata can be induced both by arthropods and pathogenic micro-organisms. Pathogen infection could, therefore, potentially enhance resistance to generalist insect herbivores in this species.     

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.     

Euphydryas anicia (Lepidoptera: Nymphalidae) utilization of iridoid glycosides fromCastilleja andBesseya (Scrophulariaceae) host plants

Iridoid glycosides were found to be sequestered by natural populations ofEuphydryas anicia after ingestion from the host plantsBesseya alpina, B. plantaginea, andCastilleja integra. Both major iridoids ofB. alpina, cataipol and aucubin, were found in butterfly populations where this was the only host plant. The catalpol-aucubin ratio was higher in the butterflies than in the host plant. AnE. anicia population which uses bothB. plantaginea andC. integra as host plants was found to sequester cataipol as well as another iridoid, macfadienoside. Macfadienoside was the major iridoid ofC. integra, while catalpol esters were the major iridoids ofB. plantaginea. Although it was a major sequestered iridoid, catalpol was a minor constituent in both host plants. The macfadienoside-catalpol ratio in the butterflies from this population was highly variable, and there appeared to be both sex and individual variation in host plant and/or iridoid glucoside utilization byE. anicia. Although other iridoids were present in the host plants, none was sequestered in more than trace amounts.This work was supported by grant CHE-8213714 to FRS, in part by grant DEB-06961 to PRE from the National Science Foundation, and a grant from the Koret Foundation of San Francisco to PRE. Paper 5 in the series Chemistry of the Scrophulariaceae. Paper 4: Roby, M.R. and Stermitz, F.R. 1984.J. Nat. Prod. 47:854–857.     

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.     

Iridoid glycoside metabolism and sequestration byPoladryas minuta (Lepidoptera: Nymphalidae) feeding onPenstemon virgatus (Scrophulariaceae)

A bivoltine checkerspot butterfly,Poladryas minuta, is aPenstemon specialist, not known to utilize any other plant genus for oviposition and larval feeding. At several intermontane plains sites of central Colorado, the butterfly utilizesPenstemon virgatus as its sole host plant. Analysis of the host plant showed it to contain three cinnamyl-type catalpol esters (scutellarioside-II, globularin, globularicisin) and catalpol. The host plant contained an average of 10% dry weight iridoids, but some variation among individual plants and leaves within plants was noted. Field-collected butterflies contained 2.1–8.7% dry weight catalpol, but no other iridoids. Adults from larvae fedP. virgatus in the lab contained 4.2–9.0% dry weight catalpol and excreted large amounts of catalpol in the meconium. No catalpol was found in the larval frass. Larvae did not consume three alternate iridoid-containing host-plant species, and most eventually died rather than feed on the alternate plants. Larvae did consume small amounts of artificial diets containing the alternate species andP. virgatus, but most went into diapause and some died. Survival was good on artificial diet containing 10% dry weight of the iridoid esters fromP. virgatus. Only catalpol was found in pupae and adults, but it was absent from the larval frass. The cinnamic-type acids expected from larval hydrolysis of the esters were not found in larval frass, pupae, or adults. These results are contrasted with those found for another checkerspot,Euphydryas anicia, which consumes a different host-plant species but was present at one of the same sites withPoladryas minuta.Paper 15 in the series Chemistry of the Scrophulariaceae. Paper 14 Boros, C.M., Stermitz, F.R., and Harris, G.H. 1990.J. Nat. Prod. 5372–80.     

Iridoid glycoside sequestration byThessalia leanira (Lepidoptera: Nymphalidae) feeding onCastilleja integra (Scrophulariaceae)

A small population of a polyvoltine checkerspot butterfly,Thessalia leanira fulvia (also known asChlosyne leanira ssp.fulvia), was found to useCastilleja integra as a larval food plant at a localized site (Burnt Mill) southwest of Pueblo, Colorado. Field-captured adult butterflies contained the major iridoid glycosides (catalpol and macfadienoside) of theCastilleja. The content of a third iridoid glycoside, methyl shanzhiside, was also relatively high in the collected butterflies even though most individualCastilleja plants at Burnt Mill contained little or no methyl shanzhiside. Only a few plants, restricted to a small area, did contain appreciable methyl shanzhiside. Most of the plants that lacked the ester methyl shanzhiside contained shanzhiside, the corresponding free carboxylic acid.Thessalia larvae did not normally methylate the acid to produce methyl shanzhiside. Larvae that stopped feeding at an early instar, but yet survived several weeks, did contain major amounts of methyl shanzhiside. It is suggested that only larvae that overwinter or otherwise enter diapause convert shanzhiside to methyl shanzhiside. TheCastilleja food plant also contained iridoids other than catalpol and macfadienoside, sometimes in major amounts, but these were never found in larvae, pupae, or butterflies.Paper 27 in the series Chemistry of the Scrophulariaceae. Paper 26: Stermitz, F.R., Foderaro, T.A., and Li, Y.-X., 1993.Phytochemistry 32:1151     

Iridoid glycosides in the nectar ofCatalpa speciosa are unpalatable to nectar thieves

The floral nectar ofCatalpa speciosa has a chemical mechanism that limits thievery. A bioassay employing sphingid larvae,Ceratomia catalpae, shows that catalpa iridoid glycosides are present in the floral nectar. When potential nectar thieves are fed nectar, a sucrose solution of identical concentration, or a sucrose solution plus 0.4% catalpol and 0.4% catalposide (catalpa iridoids), the thieves drink significantly more of the pure sucrose solution than either of the other two sugar sources. Those thieves that drink either the nectar or the sucrose solution plus catalpa iridoids develop behavioral abnormalities including regurgitation and loss of locomotion. The response of the potential nectar thieves to nectar or the sucrose solution plus catalpa iridoids cannot be distinguished by the amount consumed or by their behavior. The legitimate diurnal pollinators ofC. speciosa are not affected by the iridoid glycosides.     

The Effects of Arbuscular Mycorrhizal Fungi on Direct and Indirect Defense Metabolites of Plantago lanceolata L.

Arbuscular mycorrhizal fungi can strongly influence the metabolism of their host plant, but their effect on plant defense mechanisms has not yet been thoroughly investigated. We studied how the principal direct defenses (iridoid glycosides) and indirect defenses (volatile organic compounds) of Plantago lanceolata L. are affected by insect herbivory and mechanical wounding. Volatile compounds were collected and quantified from mycorrhizal and non-mycorrhizal P. lanceolata plants that underwent three different treatments: 1) insect herbivory, 2) mechanical wounding, or 3) no damage. The iridoids aucubin and catalpol were extracted and quantified from the same plants. Emission of terpenoid volatiles was significantly higher after insect herbivory than after the other treatments. However, herbivore-damaged mycorrhizal plants emitted lower amounts of sesquiterpenes, but not monoterpenes, than herbivore-damaged non-mycorrhizal plants. In contrast, mycorrhizal infection increased the emission of the green leaf volatile (Z)-3-hexenyl acetate in untreated control plants, making it comparable to emission from mechanically wounded or herbivore-damaged plants whether or not they had mycorrhizal associates. Neither mycorrhization nor treatment had any influence on the levels of iridoid glycosides. Thus, mycorrhizal infection did not have any effect on the levels of direct defense compounds measured in P. lanceolata. However, the large decline in herbivore-induced sesquiterpene emission may have important implications for the indirect defense potential of this species.     

Effects of Herbivore Damage and Nutrient Level on Induction of Iridoid Glycosides in Plantago lanceolata

Damage by larvae of the buckeye butterfly (Junonia coenia) resulted in removal of 15–25% of Plantago lanceolata leaf area. Plants grown under high nutrients were larger than those grown under low nutrients. Twenty-eight days after herbivory, plants grown under high nutrients were still larger than those grown under low nutrients, and plants exposed to herbivores were significantly smaller than those not exposed to herbivores, regardless of the nutrient treatment. Damage by larvae also increased the iridoid glycoside content in the leaves and reproductive tissues of these Plantago lanceolata relative to undamaged controls. Whether damaged or undamaged, the iridoid glycoside content of P. lanceolata was highest in the reproductive tissues and lowest in the roots. Although initial concentrations of iridoid glycosides were significantly higher in plants grown under low nutrient conditions than in plants grown under high nutrient conditions, nutrient availability did not alter the phytochemical response of plants to herbivore damage. These results provide additional support for the defensive role of the iridoid glycosides in Plantago lanceolata by demonstrating that phytochemical variation is not always an incidental effect of nutrient stress but can be a direct response to damage by herbivores.     

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.