Iridoid glycoside content ofEuphydryas anicia (Lepidoptera: Nymphalidae) and its major hostplant,Besseya plantaginea (Scrophulariaceae), at a high plains colorado site

The checkerspot butterfly,Euphydryas anicia, utilizes mainlyBesseya plantaginea and only occasionallyCastilleja integra as a larval hostplant at Michigan Hill, a few kilometers from a site whereC. integra is used by over 90% of the butterflies. TheB. plantaginea leaves that are consumed contain 9–22% iridoid glycosides, composed mainly of catalpol and catalpol esters, while larvae from the same plants contain 6–18% iridoids, mainly catalpol and no esters. Field-collected adult butterflies contain 0.5–4.3% iridoids. Laboratory-reared adults secrete iridoids in the meconium upon eclosion and retain similar amounts. The adult and meconium iridoid content is considerably lower than in the larvae, and metabolism in the pupal stage may be occurring.This work was supported by grant CHE-8521382 from the National Science Foundation. Paper 13 in the series Chemistry of the Scrophulariaceae. Paper 12: G.N. Belofsky and F.R. Stermitz,J. Nat. Prod. 51:614–616, 1988.     

Quinolizidine alkaloids obtained byPedicularis semibarbata (Scrophulariaceae) fromLupinus fulcratus (Leguminosae) fail to influence the specialist herbivoreEuphydryas editha (Lepidoptera)

Pedicularis semibarbata is apparently an obligate hemiparasite of coniferous trees. It is also a facultative parasite ofLupinus fulcratus from which we find that it obtains quinolizidine alkaloids, principally -isolupanine. As a result, a single population ofP. semibarbata contains both alkaloidrich and alkaloid-free plants. The butterflyEuphydryas editha naturally oviposits on both plant types. This butterfly population, which is the principal herbivore attackingP. semibarbata at this site, is known to contain two morphs. Individuals of a specialist morph discriminate when ovipositing among individualP. semibarbata plants and produce offspring that survive better on accepted than on rejected plants. Those of a generalist morph accept allP. semibarbata plants and produce offspring that survive equally well on plants accepted or rejected by the discriminating morph. Because of the existence of this complex variation among the butterflies, the presence of naturally laid eggs on alkaloid-containing plants still leaves the possibility that the alkaloids may defend the plants against the specialist morph. In experiments on both oviposition preference and larval performance in early instars, we failed to detect any correlation between alkaloid content of a plant and either its acceptability to or suitability for the discriminating morph of the insect. Alkaloid presence in the host-plant population, achieved through root parasitism, is currently neither subject to strong insect-mediated selection nor a major cause of selection on the insects.     

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.     

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     

Flavonoid pigments in marbled white butterfly (Melanargia galathea) are dependent on flavonoid content of larval diet

Analyses of two-dimensional chromatographic flavonoid patterns of butterflies reared on different grass species have shown that the flavonoid pattern ofMelanargia galathea is dependent on the flavonoid content of the larval diet. This confirms the dietary origin of flavonoid pigments inM. galathea. The flavonoid patterns of butterflies reared on different grass species differ from each other and from the larval food plants;M. galathea reared on the same grass species have identical flavonoid patterns. Differences in the butterfly and larval food plant flavonoid patterns indicate that the ingested flavonoids are metabolized byM. galathea or its gut flora before sequestration. The distinct flavonoid patterns of butterflies reared on different larval food plants have been defined as the flavonoid fingerprint profiles for each grass species. Similarity between theFestuca rubra flavonoid fingerprint profile and the constant flavonoid pattern characteristic of wild-capturedMelanargia suggests thatMelanargia larvae are not generalist grass feeders, but are specific toF. rubra or toF. rubra and a few closely related grass species in the wild.     

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 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.     

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.     

Odor of a papilionid butterfly

Benzaldehyde, phenylacetaldehyde (major component), 2-phenylpropenal,n-heptanal, 6-methylhept-5-en-2-one, and linalool were identified as compounds responsible for the male scent ofAtrophaneura alcinous alcinous. These substances were present predominantly in the wings, and the quantity of them was largest at the inner margin of the hind wing. Female wings also contained some of them in much smaller (except a few components) amounts. The relative proportion of each component exhibited manifests sexual differences.     

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.     

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.     

Male sex pheromone of a giant danaine butterfly,Idea leuconoe

Males of a giant danaine butterfly,Idea leuconoe, display hairpencils during courtship. The females were visually attracted to and olfactorily arrested by an artificial butterfly model to which male hairpencil extracts were added. The hairpencil extracts contained a complex mixture of volatiles, including pyrrolizidine alkaloid (PA) derivatives (danaidone, viridifloric -lactone), aromatics (phenol,p-cresol, benzoic acid), terpenoids (geranyl methyl thioether, (E,E)-farnesol), a series of -lactones (6-hydroxy-4-undecanolides and its homologs), hydrocarbons [(Z)-9-tricosene, etc.], and several compounds with higher molecular weight. A mixture of the major volatiles applied to a butterfly dummy strongly elicited an abdomen-curling acceptance posture in females. Viridifloric -lactone and danaidone induced significant electroantennogram responses on the female's antennae, suggesting their principal role together with other hairpencil components as a sex pheromone to seduce females.I. leuconoe males seem to acquire the precursor for both of the PA fragments from the host plant,Parsonsia laevigata (Apocynaceae), during the larval stage; thereby they do not show pharmacophagous behavior towards PA-containing plants during the adult stage. However, males are pharmacophagously attracted to and feed on a number of simple phenolic compounds in a manner similar to other danaine species towards PAs. Wild males sequester one of the phagostimulants, (–)-mellein, in the hairpencils in varying quantities. Phenolic compounds incorporated in the hairpencils may act primarily as warning odors linked with the defensive PAs present in the body tissues.     

Effects of phenolic acids on germination and early growth of herbaceous woodland plants

Four herbaceous plant species from woodland (clearings),Deschampsia flexuosa, Scrophularia nodosa, Senecio sylvaticus, andChamaenerion angustifolium, were tested for their sensitivity to phenolic acids. Seven commonly occurring phenolic compounds were used in a germination experiment in concentrations ranging from 0.01 to 10 mM, i.e., salicylic,p-hydroxybenzoic, syringic, caffeic, vanillic,p-coumaric, and ferulic acids. Germination was delayed rather than inhibited. Radicle elongation was strongly affected; at lower concentrations stimulatory effects were observed, whereas at high concentrations radicle elongation was severely reduced. Salicylic acid was the most effective phenolic compound, whereas caffeic acid caused no effects. Early growth was studied in more detail in a second experiment withDeschampsia flexuosa andSenecio sylvaticus and the phenolic acids, ferulic and p-coumaric acid. Primary root length, number and length of secondary roots, and dry weight were stimulated at 0.01 mM but were inhibited at 10 mM of both compounds. The results are discussed in view of the allelopathic relations between trees and herbaceous understory vegetation.     

Inhibition of feeding by a generalist insect due to increased volatile leaf terpenes under nitrate-limiting conditions

Nitrogen-limited plants ofHeterotheca subaxillaris accumulate greater quantities of leaf volatile terpenes than do nitrogen-rich plants. A series of feeding trials were performed to determine if such nitrate-limited plants are better defended against generalist-feeding insect herbivores. Soybean looper (Pseudoplusia includens) larvae were fed leaves fromH. subaxillaris rosettes grown under high and low nitrate supply regimes. Larval consumption, growth, and survival declined as the leaf volatile terpene content increased. Larval consumption and growth were enhanced by higher plant nitrate supply and with increasing leafage. The results suggest that the higher quantity of volatile terpenes in the leaves of nitrate-limited plants may better defend these leaves against generalist-feeding insects.     

Effects of dietary protein and lupine alkaloids on growth and survivorship ofSpodoptera eridania

Toxic chemicals and nutrients are often positively correlated within and among plants. We studied how such correlations affect the suitability of plants as food for herbivores by assessing the growth and survivorship ofSpodoptera eridania (army worm) on artificial diets containing lupine alkaloids and casein. We found that (1) the effects of casein were determined by other dietary components: increased dietary casein led to increased larval growth only when the diet was also high in wheat germ. (2) Dietary alkaloids were effective at very low concentrations, reducing both growth and survivorship. The alkaloids lupanine and sparteine were not synergistic in their effects, and the interaction between alkaloids and casein was significant only in the low-wheat-germ diets. (3) The effects of casein and alkaloids were generally apparent only in the first instar, and the growth of fifth-instar larvae was unaffected by diet. (4) Using these artificial diet experiments, we can make simple predictions about the food quality of plants grown under various nutrient regimes. These predictions are consistant with recent ideas about optimal plant defenses.     

Differential effect of tomatine and its alleviation by cholesterol on larval growth and efficiency of food utilization inHeliothis zea andSpodoptera exigua

The effect of tomatine on larval growth ofHeliothis zea andSpodoptera exigua was assessed by rearing larvae on diets with different concentrations of the chemical added. When reared from neonates, linear dose-response relationships were obtained for both species, withS. exigua being three times more sensitive to tomatine thanH. zea. Tomatine toxicity was completely alleviated inH. zea by the addition of equimolar cholesterol into the diet; however, inS. exigua some toxicity was maintained. Larvae ofS. exigua that were started on control diet were insensitive to tomatine after five days; larvae started on diet with an EC₅₀ of tomatine and then switched to control diet after five days failed to recover from toxicosis. Larval growth ofH. zea, on the other hand, was affected both at the neonate and third-instar stage, but normal growth resumed when the larvae were transferred to control diet. Tomatine had little or no affect on food consumption, assimilation, or dietary utilization of the food by third-instar larvae ofS. exigua, except at a concentration 10 times the EC₅₀. In contrast, the efficiency of food utilization ofH. zea larvae decreased with increasing tomatine concentrations. Assimilation of the food tended to increase, although not significantly, as tomatine levels increased. Food consumption ofH. zea larvae also increased when the tomatine concentration was greater than an EC₅₀. The addition of equimolar cholesterol to diets with an EC₅₀ of tomatine restored weight gain and nutritional indices values to control values. These results are related to the utility of using tomatine in host-plant resistance programs.     

Quantitative and qualitative effects of larval diet on male scent secretions ofEstigmene acrea,Phragmatobia foliginosa,andPyrrharctia isabella (Lepidoptera: Arctiidae)

In feeding experiments with insects reared in the laboratory, the presence of the dihydropyrrolizines hydroxydanaidal and danaidal in the male scent organs (coremata) of the arctiids,Estigmene acrea (Drury),Phragmatobia fuliginosa (L.), andPyrrharctia isabella (J.E. Smith), was shown to depend on the presence of a source of pyrrolizidine alkaloids (PAs) in the larval diet.Phragmatobia males given an artificial diet supplemented with the powdered roots of the PA-containing plantSymphytum officinale L. (comfrey) produced more hydroxydanaidal than danaidal, whereas males given an artificial diet supplemented with dried whole plants of another PA-containing species,Senecio vulgaris L., produced more danaidal than hydroxydanaidal.Pyrrharctia males produced hydroxydanaidal with little if any danaidal, whether the source of PAs was comfrey orS. vulgaris. A behavioral bioassay showed that the coremata of PA-deniedPyrrharctia male progeny of PA-denied parents were pheromonally inactive, whereas those of PA-denied male progeny of PA-supplied parents (male and/or female) were often active. This indicates that a small amount of pheromone is made from PAs transferred from the female to her eggs and that males effect copulatory transfers of PAs that are, in turn, passed to the eggs by the mated female. Field observations ofPhragmatobia andPyrrharctia larvae feeding on sources of PAs were reported. The PA monocrotaline was shown to be a feeding stimulant forPyrrharctia larvae.     

Stability of phenolic and protein measures in excised oak foliage

The stability of protein and phenolic measures in excised foliage from two oak species was measured under conditions that simulated the handling and treatment of foliage during insect rearing trials. Excised foliage kept hydrated under refrigeration or insect-rearing conditions maintained stable levels of protein content, proanthocyanidins, gallotannins, total phenolics, and protein-binding capacity for up to 48 hr following field sampling. Measures of protein content, total phenolics, protein-binding capacity, and proanthocyanidins were significantly greater 48–72 hr after field sampling, followed by declines to near field levels within 120 hr.     

Allelopathic effects of phenolic mixtures on respiration of two spruce mycorrhizal fungi

Investigations were conducted to determine whether the allelopathic effect (inhibition of oxygen consumption) of mull- and mor-type humic solutions on ectomycorrhizal fungi was due to the solutions' phenolic contents. In the first experiment, two concentrations (10^–3 M and 10^–7 M) of binary equimolar phenolic mixtures were tested on the oxygen consumption ofLaccaria laccata andCenococcum graniforme. The high concentrations of most of the mixtures induced an increase of fungal respiration, whereas the 10^–7 M treatments all reduced the rate of respiration. In the second experiment, the effects on respiration of the phenolic mixture reproducing mull- and mortype humic solutions were compared with the effects of natural humic solutions. The resulting data suggest that allelopathic effect of humic solutions can at least in part be attributed to their phenolic contents.     

Effects of nitrogen and Douglas-fir allelochemicals on development of the gypsy moth,Lymantria dispar

Two experiments were conducted to examine the influence of foliar nitrogen, terpenes, and phenolics of Douglas-fir on the development of gypsy moth larvae. In the first experiment, foliar concentrations of nitrogen and allelochemicals were manipulated by fertilizing 3-year-old potted seedlings with 0 or 200 ppm nitrogen. Concentrations of foliar nitrogen (0.33–2.38%) were negatively correlated with the phenolics (15.8–24.4 mg/g). Sixth-instar larvae previously reared on current-year Douglas-fir needles were allowed to feed on these seedlings. Pupal weights (312.8–995.6 mg) were positively correlated with levels of foliar nitrogen, negatively correlated with amounts of foliar phenolics, and uncorrelated with terpene concentrations. In the second experiment, terpene and phenolic extracts from Douglas-fir foliage were incorporated at natural levels into artificial diets with high and low levels of protein nitrogen. Neonate larvae grew faster and were larger on the high nitrogen control diet (4.1–4.5%), however, fourth instars performed better on the control diet with low nitrogen levels (2.5–2.7%). Foliar terpenes incorporated into diet had little effect on neonate fitness, but may induce subtle physiological changes in later instar larvae. Phenolics, alone or in combination with terpenes, excessively suppressed growth and survival, with no individuals living through the fourth instar, regardless of the nitrogen level. Incorporating foliar phenolic extracts into artificial diet caused unnatural levels of toxicity and failed to clarify the effects of Douglas-fir phenolics on gypsy moth fitness. Foliar nitrogen is a key factor influencing gypsy moth development on Douglas fir, but may be mitigated to some degree by phenolics.