Selective sequestration of milkweed (Asclepias sp.) cardenolides inOncopeltus fasciatus (Dallas) (Hemiptera: Lygaeidae)

The cardenolide content of the gut, wings, and fat body ofOncopeltus fasciatus was examined. The female fat body contained 4–5% of the total cardenolide content of the insect. The cardenolide content of male fat body, and gut and wings of both sexes was below the detection limit of the cardenolide assay. Thin-layer chromatography was used to determine the cardenolide array of various tissues and secretions ofO. fasciatus reared on seeds of a single species of milkweed (A. Speciosa) and adult extracts and dorsolateral space fluid ofO. fasciatus reared on seeds of two species of milkweed with different cardenolide arrays (A. speciosa andA. syriaca). Our results indicate that cardenolides are not sequestered in the insect simply on the basis of polarity and that metabolism and differential excretion of cardenolides are involved in the sequestration of cardenolides inO. fasciatus. The similarities in the cardenolide profiles ofO. fasciatus reared on different food sources, and tissues ofO. fasciatus reared on a single food source indicates that there is regulation of the cardenolide array inO. Fasciatus.     

Cardenolide content and thin-layer chromatography profiles of monarch butterflies,Danaus plexippus L., and their larval host-plant milkweed,Asclepias asperula subsp.Capricornu (woods.) woods., in north central Texas

This paper is the second in a series on cardenolide fingerprinting of monarch butterflies and their host-plant milkweeds in the eastern United States. Spectrophotometric determinations of the gross cardenolide content ofAsclepias asperula plants in north central Texas indicated wide variation ranging from 341 to 1616 g/0.1 g dry weight. The mean plant cardenolide concentration (886 g/0.1 g) is the highest for any milkweed species on which monarch cardenolide profiles have been produced. Forty-one butterflies reared individually on these plants contained a skewed distribution of cardenolide concentrations ranging from 231 to 515 g/0. 1 g dry weight with a mean of 363g/0.1 g. The uptake of cardenolide by the butterflies was independent of plant concentration, suggesting that saturation occurs in cardenolide sequestration by monarchs when feeding on cardenolide-rich host-plants. Female monarchs contained significantly greater mean cardenolide concentrations (339 g/0.1 g) than did males (320 g/0.1 g). The mean dry weight of the male butterflies (0.211 g) was significantly greater than the female mean (0.191) so that the mean total cardenolide contents of males (675 fig) and females (754 g) were not significantly different. Butterfly size was not significantly correlated to butterfly cardenolide concentration when differences due to sex and individual host-plant concentration were removed. Thin-layer chrornatograms of 24 individual plant-butterfly pairs developed in two solvent systems resolved 22 individual spots in the plants and 15 in the butterflies.A. asperula plants appear to contain several relatively nonpolar cardenolides of the calotropagenin series which are metabolized to more polar derivatives in the butterflies. Quantitative evaluation of theR _f values, spot intensities, and probabilities of occurrence in the chloroform-methanol-formamide TLC system produced a cardenolide fingerprint clearly distinct from those previously established for monarchs reared on otherAsclepias species. Our data support the use of fingerprints to make ecological predictions concerning larval host-plant utilization.A. asperula subsp.capricornu andA. viridis Walt, are the predominant early spring milkweeds throughout most of the south central United States. Cardenolide-rich monarchs reared on these two species may be instrumental in establishing and reinforcing visual avoidance of adults by naive predators throughout their spring and summer breeding cycle in eastern North America.Lepidoptera: Danaidae.Apocynales: Asclepiadaceae.     

Cardenolide content and thin-layer chromatography profiles of monarch butterflies,danaus plexippus L., and their larval host-plant milkweed,asclepias viridis walt., in northwestern louisiana

This paper is the first in a series on cardenolide fingerprinting of monarch butterflies and their host-plant milkweeds in the eastern United States. Spectrophotometric determinations of the gross cardenolide content of 60Asclepias viridis plants in northwestern Louisiana indicate a positively skewed variation ranging from 95 to 432 g/0.1 g dry weight with a mean of 245 g/0.1 g. Butterflies reared individually on these plants contained a normal cardenolide distribution ranging from 73 to 591 g/0.1 g dry weight with a mean of 337 g/0.1 g. The uptake of cardenolide by the butterflies best fit a logarithmic function of the plant concentration. Female monarchs (385 g/0.l g) contained significantly greater mean cardenolide concentrations than did males (287 g/0.1 g). No indications of a metabolic cost for either cardenolide ingestion or storage were adduced from size or dry weight data. Thin-layer chromatograms of 24 individual plant-butterfly pairs developed in two solvent systems resolved 21 individual spots in the plants and 15 in the butterflies.A. viridis plants appear to contain several relatively nonpolar cardenolides of the calotropagenin series which are metabolized to the more polar 3'-hydroxy derivatives calactin and calotropin as well as to calotropagenin in the butterflies. The epoxy cardenolides labriformin and labriformidin were absent, although desglucosyrioside (a 3'-hydroxy derivative) appeared present in both plants and butterflies. Quantitative evaluation of theR _f values, spot intensities, and probabilities of occurrence in the chloroform-methanol—formamide TLC system produced a cardenolide fingerprint clearly distinct from those previously established for monarchs reared on otherAsclepias species, supporting the use of fingerprints to make ecological predictions concerning larval host-plant utilization.A. viridis is the predominant early spring milkweed throughout most of the south central United States and may be important in providing chemical protection to spring and early summer generation monarchs in the eastern United States.Lepidoptera: Danaidae.Apocynales: Asclepiadaceae.     

Plant-determined variation in the cardenolide content,thin-layer chromatography profiles,and emetic potency of monarch butterflies,Danaus plexippus L. Reared on milkweed plants in California: 2.Asclepias speciosa

The pattern of variation in gross cardenolide concentration of 111Asclepias speciosa plants collected in six different areas of California is a positively skewed distribution which ranges from 19 to 344 g of cardenolide per 0.1 g dry weight with a mean of 90 g per 0.1 g. Butterflies reared individually on these plants in their native habitats ranged from 41 to 547 g of cardenolide per 0.1 g dry weight with a mean of 179 g. Total cardenolide per butterfly ranged from 54 to 1279 g with a mean of 319 g. Differences in concentrations and total cardenolide contents in the butterflies from the six geographic areas appeared minor, and there were no differences between the males and the females, although the males did weigh significantly more than females. The uptake of cardenolide by the butterflies was found to be a logarithmic function of the plant concentration. This results in regulation: larvae which feed on low-concentration plants produce butterflies with increased cardenolide concentrations relative to those of the plants, and those which feed on high-concentration plants produce butterflies with decreased concentrations. No evidence was adduced that high concentrations of cardenolides in the plants affected the fitness of the butterflies. The mean emetic potencies of the powdered plant and butterfly material were 5.62 and 5.25 blue jay emetic dose fifty units per milligram of cardenolide and the number of ED₅₀ units per butterfly ranged from 0.28 to 6.7 with a mean of 1.67. Monarchs reared onA. speciosa, on average, are only about one tenth as emetic as those reared onA. eriocarpa. UnlikeA. eriocarpa which is limited to California,A. speciosa ranges from California to the Great Plains and is replaced eastwards byA. syriaca L. These two latter milkweed species appear to have a similar array of chemically identical cardenolides, and therefore both must produce butterflies of relatively low emetic potency to birds, with important ecological implications. About 80% of the lower emetic potency of monarchs reared on A. speciosa compared to those reared onA. eriocarpa appears attributable to the higher polarity of the cardenolides inA. speciosa. Thin-layer Chromatographie separation of the cardenolides in two different solvent systems showed that there are 23 cardenolides in theA. speciosa plants of which 20 are stored by the butterflies. There were no differences in the cardenolide spot patterns due either to geographic origin or the sex of the butterflies. As when reared onA. eriocarpa, the butterflies did not store the plant cardenolides withR _f values greater than digitoxigenin. However, metabolic transformation of the cardenolides by the larvae appeared minor in comparison to when they were reared onA. eriocarpa. AlthoughA. eriocarpa andA. speciosa contain similar numbers of cardenolides and both contain desglucosyrioside, the cardenolides ofA. speciosa overall are more polar. ThusA. speciosa has no or only small amounts of the nonpolar labriformin and labriformidin, whereas both occur in high concentrations inA. eriocarpa. A. speciosa plants and butterflies also contain uzarigen, syriogenin, and possibly other polar cardenolides withR _f values lower than digitoxin. The cardenolide concentration in the leaves is not only considerably less than inA. eriocarpa, but the latex has little to immeasurable cardenolide, whereas that ofA. eriocarpa has very high concentrations of several cardenolides. Quantitative analysis ofR _f values of the cardenolide spots, their intensities, and their probabilities of occurrence in the chloroform-methanol-formamide TLC system produced a cardenolide fingerprint pattern very different from that previously established for monarchs reared onA. eriocarpa. This dispels recently published skepticism about the predictibility of chemical fingerprints based upon ingested secondary plant chemicals.Lepidoptera: Danaidae.Apocynales: Asclepiadaceae.This study was supported by U.S. National Science Foundation grants DEB 75-14265 and 78-10658 to Amherst College and BSR-8119382 to the University of Florida with L.P. Brower as Principal Investigator and DEB75-14266, DEB78-15419, and DEB-81-19391 to the University of California at Davis with J.N. Seiber as Principal Investigator.     

Seasonal and intraplant variation of cardenolide content in the California milkweed,Asclepias eriocarpa,and implications for plant defense

Root, stem, leaf, and latex samples ofAsclepias eriocarpa collected from three plots in one population at 12 monthly intervals were assayed for total cardenolide content by spectroassay and for individual cardenolides by thin-layer chromatography. From May to September mean milligram equivalents of digitoxin per gram of dried plant were: latices, 56.8 stems, 6.12 > leaves, 4.0 > roots, 2.5. With the exception of the roots, significant changes in gross cardenolide content occurred for each sample type with time of collection during the growing season, whereas variation within this population was found to be small. Labriformin, a nitrogen-containing cardenolide of low polarity, predominated in the latices. Leaf samples contained labriformin, labriformidin, desglucosyrioside, and other unidentified cardenolides. In addition to most of the same cardenolides as the leaves, the stems also contained uzarigenin. The roots contained desglucosyrioside and several polar cardenolides. The results are compared with those for other cardenolide-containing plants, and discussed in relation to anti-herbivore defense based on plant cardenolide content. Arguments are advanced for a central role of the latex in cardenolide storage and deployment which maximizes the defensive qualities of the cardenolides while preventing toxicity to the plant.Research supported by National Science Foundation Grants DEB 75-14266 and DEB 78-15419 (U.C. Davis) and DEB 75-14265, DEB 78-10658, and DEB 80-40388 (U.F.)     

Plant-determined variation in cardenolide content and thin-layer chromatography profiles of monarch butterflies,Danaus plexippus reared on milkweed plants in California

Variation in gross cardenolide concentration of the mature leaves of 85Asclepias californica plants collected in four different areas of California is a positively skewed distribution ranging from 9 to 199 g of cardenolide per 0.1 g dry weight with a mean of 66 g/0.1 g. Butterflies reared individually on these plants in their native habitats contained a normal distribution of cardenolide ranging from 59 to 410 g of cardenolide per 0.1 g dry weight with a mean of 234 g. Cardenolide uptake by the butterflies was a logarithmic function of plant concentration. Total cardenolide per butterfly ranged from 143 to 823 g with a mean of 441 g and also was normally distributed. Populational variation of plant cardenolide concentrations occurs within subspecies, but the northern subspeciesA. c. greenei does not differ significantly from the southernA. c. californica. Generally higher concentrations occur in butterflies from northern populations and in females. No evidence was adduced that cardenolides in the plants adversely affected the butterflies. Low cardenolide concentrations in the leaves and the absence of cardenolides in the latex characterize bothA. californica andA. speciosa, but notA. eriocarpa. Thin-layer chromatography in two solvent systems isolated 24 cardenolide spots in the plants, of which 18 are stored by the butterflies. There was a minor difference in the cardenolide spot patterns due to geographic origin of the plants, but as in our previous studies, none in the sexes of the butterflies. UnlikeA. eriocarpa andA. speciosa, A. californica plants lack cardenolides withRf values greater than digitoxigenin. Overall, the cardenolides of bothA. californica andA. speciosa are more polar than those inA. eriocarpa. A. californica plants contain cardenolides of the calotropagenin series including calotropin, calactin, and uscharidin, and the latter is metabolically transformed by monarch larvae to calactin and calotropin. Cardenolides of this series also occur inA. vestita, andA. cordifolia from California, the neotropicalA. curassavica, and the AfricanCalotropis procera, Gomphocarpus spp., andPergularia extenso; they therefore cross established taxonomic lines.A. californica is the predominant early season milkweed in California and may be important in providing chemical protection to the spring generation of monarchs in the western United States.A. speciosa, A. eriocarpa, andA. californica each imparts distinctive cardenolide fingerprints to the butterflies, so that ecological predictions are amenable to testing.Lepidoptera: Danaidae.Apocynales: Asclepiadaceae.This study was supported by U.S. National Science Foundation grants DEB 75–14265 and 78–10658 to Amherst College; BSR-8119382 to the University of Florida with L.P. Brower as Principal Investigator; and DEB 75–14266, DEB 78–15419, and DEB 81–19391 to the University of California at Davis with J.N. Seiber as Principal Investigator.     

Interspecific Variation Within the Genus <Emphasis Type="Italic">Asclepias</Emphasis> in Response to Herbivory by a Phloem-feeding Insect Herbivore

Induced plant responses to leaf-chewing insects have been well studied, but considerably less is known about the effects of phloem-feedings insects on induction. In a set of laboratory experiments, we examined density-dependent induction by the milkweed-oleander aphid, Aphis nerii, of putative defenses in four milkweed species (Asclepias incarnata, Asclepias syriaca, Asclepias tuberosa, and Asclepias viridis). We hypothesized that high aphid density would lead to increased cardenolide expression in species with low constitutive levels of cardenolides (e.g., A. tuberosa), but that there would be no induction in high constitutive cardenolide species (e.g., A. viridis). Based on previous studies, we did not expect cardenolide induction in A. incarnata. Contrary to our predictions, we observed feeding-induced declines of cardenolide concentrations in A. viridis. Cardenolide concentrations did not respond to aphid feeding in the other three milkweed species. Aphids also caused reductions in biomass accumulation by two of four Asclepias species, A. viridis and A. incarnata. High aphid density led to a decrease in A. viridis foliar nitrogen concentration. However, aphids had no effect on the defensive chemistry, growth, or nutritional quality of either A. syriaca or A. tuberosa. Our results highlight that congeneric plant species may respond differently to the same levels of herbivore damage.     

Differences and similarities in cardenolide contents of queen and monarch butterflies in florida and their ecological and evolutionary implications

Florida queen butterflies are highly variable in cardenolide content and, in three populations studied, contained less cardenolide than did a sample of sympatric Florida monarchs. The possibility that queens stored a more potent set of cardenolides from their host plants (and therefore were as well protected as monarchs, even at lower concentrations) is refuted by Chromatographic analysis of wild butterflies, as well as controlled laboratory rearings. It therefore appears that, with respect to cardenolides, monarchs are better defended than are queens. Consequently, cardenolides are unlikely to explain the apparent shift in Florida viceroy mimicry away from resemblance of the monarch, toward mimicry of the queen. Other hypotheses to explain this mimetic phenomenon are suggested. Adult monarchs exhibit significant negative correlations between the concentration of cardenolide stored in their tissues and both body size and weight, whereas queens show no such correlations. The implications of these results for the study of metabolic costs of allelochemic storage are discussed. Chromatographic evidence is provided that monarchs do breed in south Florida during the winter months and that the likely host plant employed by the population studied wasAsclepias curassavica. This represents the first practical application of cardenolide fingerprinting to identify the larval host plants of wild danaid butterflies.     

Sequestration of cardenolides inOncopeltus fasciatus: Morphological and physiological adaptations

The morphological and physiological adaptations associated with sequestration of cardenolides by the lygaeidOncopeltus fasciatus are summarized and discussed. Cardenolides are efficiently accumulated inO. fasciatus; however, the insect does not appear to suffer any physiological cost as a result of handling large amounts of these plant toxins. Morphological adaptations of the insect include a modified integument composed of a double layered epidermis with an inner layer (the dorsolateral space) specialized for cardenolide storage. Special weak areas of the cuticle are found on both the thorax and abdomen, which rupture when the insect is squeezed, resulting in the cardenolide-rich contents of the inner epidermal layer being released onto the body surface in the form of discrete spherical droplets. Physiological adaptations include selective sequestration of food plant cardenolides, concentration of cardenolides in the dorsolateral space, passive uptake of cardenolides at the gut and dorsolateral space requiring little energy output, reabsorption of secreted cardenolides by the Malpighian tubules, high in vivo tolerance to cardenolides, and the presence of cardenolide-resistant Na,K-ATPases.Hemiptera-Heteroptera: Lygaeidae.     

Cardenolide fingerprint of monarch butterflies reared on common milkweed,Asclepias syriaca L.

Monarch butterfly,Danaus plexippus (L.), larvae were collected during August 1983 from the common milkweed,Asclepias syriaca L., across its extensive North American range from North Dakota, east to Vermont, and south to Virginia. This confirms that the late summer distribution of breeding monarchs in eastern North America coincides with the range of this extremely abundant milkweed resource. Plant cardenolide concentrations, assayed by spectrophotometry in 158 samples from 27 collection sites, were biased towards plants with low cardenolide, and ranged from 4 to 229 g/ 0.1 g dry weight, with a mean of 50 g/0.1 g. Monarch larvae reared on these plants stored cardenolides logarithmically, and produced 158 adults with a normally distributed concentration range from 0 to 792 g/0. l g dry butterfly, with a mean of 234 g/0.1 g. Thus butterflies increased the mean plant cardenolide concentration by 4.7. The eastern plants and their resultant butterflies had higher cardenolide concentrations than those from the west, and in some areas monarchs sequestered more cardenolide from equivalent plants. Plants growing in small patches had higher cardenolide concentrations than those in larger patches, but this did not influence butterfly concentration. However, younger plants and those at habitat edges had higher cardenolide concentrations than either older, shaded, or open habitat plants, and this did influence butterfly storage. There were no apparent topographical differences reflected in the cardenolides of plants and butterflies. Twenty-eight cardenolides were recognized by thin-layer chromatography, with 27 in plants and 21 in butterflies. Butterflies stored cardenolides within the more polar 46% of the plantR _d range, these being sequestered in higher relative concentrations than they occurred in the plants. By comparison with published TLC cardenolide mobilities, spots 3, 4, 9, 16, 24 or 25, 26, and 27, may be the cardenolides syrioside, uzarin, syriobioside, syriogenin, uzarigenin, labriformidin, and labriformin, respectively. Cochromatography with cardenolide standards indicated that desglucosyrioside did not occur in the plants but did occur in 70% of the butterflies, and aspecioside was in 99% of the plants and 100% of the butterflies. The polar aspecioside was the single most concentrated and diagnostic cardenolide in both plants and butterflies. ButterflyR _d values were dependent on those of the plant, and both showed remarkable uniformity over the range of areas sampled. Thus contrary to previous reports,A. syriaca has a biogeographically consistent cardenolide fingerprint pattern. The ecological implications of this for understanding the monarch's annual migration cycle are significant.     

Genetic and Environmental Sources of Variation in the Autogenous Chemical Defense of a Leaf Beetle

Chemical defense plays a central role for many herbivorous insects in their interactions with predators and host plants. The leaf beetle genus Oreina (Coleoptera, Chrysomelidae) includes species able to both sequester pyrrolizidine alkaloids and autogenously produce cardenolides. Sequestered compounds are clearly related to patterns of host-plant use, but variation in de novo synthesized cardenolides is less obviously linked to the environment. In this study, intraspecific variation in cardenolide composition was examined by HPLC–MS analysis in 18 populations of Oreina speciosa spanning Europe from the Massif Central to the Balkans. This revealed the defense secretion to be a complex blend of up to 42 compounds per population. There was considerable geographical variation in the total sample of 50 compounds detected, with only 14 found in all sites. The environmental and genetic influences on defense chemistry were investigated by correlation with distance matrices based on habitat factors, host-plant use, and genetics (sequence data from COI, COII, and 16s rRNA). This demonstrated an influence of both genetics and host-plant use on the overall blend of cardenolides and on the presence of some of the individual compounds. The implications of this result are discussed for the evolution of defense chemistry and for the use of cardenolide compounds as markers of the evolutionary history of the species. Triponez and Naisbit contributed equally to this work and are considered joint first authors. An erratum to this article can be found at     

Rapid,quantitative HPLC analysis ofAsclepias fruticosa L. andDanaus plexippus L. cardenolides

The cardenolide extracts from latex and aerial parts ofAsclepias fruticosa and ofDanaus plexippus reared onA. fruticosa orA. curassavica were purified by adsorption chromatography on silica gel. HPLC analysis on a C₁₈ reverse-phase column with an acetonitrile-water gradient as mobile phase, separated 28 compounds with a UV spectrum typical forcardenolides. Afroside and gomphoside (major components), as well as calotropagenin, calotoxin, calotropin, calactin, uscharidin, uscharin, and voruscharin, occurred as single peaks in the profiles of latex and aerial plant parts ofA. fruticosa. Calactin and calotropin were the major cardenolides inDanaus plexippus reared onA. fruticosa orA. curassavica. Quantitative data obtained with digitoxin as internal standard showed that 1.3–1.5% of the leaf cardenolides were sequestered byDanaus plexippus in which levels of 70–80g cardenolide per butterfly were measured. The calotropin from the leaves was almost completely sequestered, and 10–13% of the calactin was stored by the butterfly, assuming that no conversion occurred in larval tissues. Apocynales: Asclepiadaceae. Lepidoptera: Danaidae.     

Effectiveness of cardenolides as feeding deterrents toPeromyscus mice

I compared the feeding responses of five species ofPeromyscus mice (aztecus, polionotus, melanotis, leucopus, andmaniculatus) to three bitter-tasting cardenolides (ouabain, digoxin, and digitoxin) that differ greatly in lipophilic character.Peromyscus, like other muroid rodents, are unusual in that they can ingest relatively large amounts of cardenolides without adverse physiologic effects. In experiment 1, I determined avoidance thresholds for the three cardenolides with 48 hr, two-choice tests. Mice exhibited large interspecific differences in avoidance threshold, and the interspecific ranking of the thresholds (maniculatus=leucopus >melanotis >polionotus >aztecus) was the same for each of the cardenolides. In experiment 2, I reevaluated the avoidance thresholds, but this time monitored the pattern of intake (i.e., bout lengths) during initial feeding encounters with cardenolidelaced diets. For each cardenolide, mice were subjected to three tests. In test 1, they received a control diet; in test 2, a diet containing the cardenolide at a concentration 1 log, unit below the avoidance threshold (as determined in experiment 1); and in test 3, a diet containing the cardenolide at the avoidance threshold concentration. Results were similar across all species and cardenolide types: Bout lengths in tests 1 and 2 were statistically equal, whereas those in test 3 were significantly shorter than those in test 1. The rapid rejection of cardenolide-laced diets in test 3 is consistent with a preingestive (i.e., gustatory) mechanism underlying the avoidance thresholds. I conclude (1) thatPeromyscus species differ substantially in taste sensitivity to cardenolides and that these differences may influence each species' respective ability to eat cardenolide-laced insects; and (2) that a species' relative taste sensitivity to one cardenolide predicts its sensitivity to other cardenolides.     

Cardenolides as oviposition deterrents to twoPieris species: Structure-activity relationships

Oviposition responses ofPieris rapae andP. napi oleracea to 18 cardenolides were compared under the same conditions. Effects of different concentrations of selected cardenolides were also tested. Most of the compounds were deterrent to oviposition by both insects, but to significantly different degrees.P. rapae were strongly deterred by K-strophanthoside, K-strophanthin-, cymarin, convallatoxin, oleandrin, erysimoside, erychroside, and gitoxigenin. The most deterrent compounds forP. napi oleracea were erychroside, cymarin, erysimoside, convallatoxin, and K-strophanthoside. Strophanthidin-based glycosides were more deterrent than digitoxigenin-based ones, and the number and type of sugar substitutions can have profound effects on activity. Both similarities and contrasts were found in responses ofP. rapae andP. napi oleracea to these cardenolides. Cymarin was equally deterrent to bothPieris species at all concentrations tested. However, when compared withP. rapae, P. napi oleracea was less sensitive to most of the cardenolides.P. napi oleracea was insensitive to K-strophanthin- and oleandrin at 0.5 × 10^–4 M, which were highly deterrent toP. rapae.     

Chemical constituents ofErysimum cheiranthoides deterring oviposition by the cabbage butterfly,Pieris rapae

Avoidance ofErysimum cheiranthoides for oviposition byPieris rapae has been attributed to the presence of water-soluble deterrents. The active material was extracted inton-butanol and isolated by a series of HPLC separations. TLC of the active fraction and visualization of individual constituents with Kedde's reagent indicated that cardenolides are responsible for deterring oviposition. UV spectra were also characteristic of cardenolides. Bioassays of selected known cardenolides revealed a general lack of activity, except for cymarin, which was as strongly deterrent as the most prominent cardenolide isolated in pure form fromE. cheiranthoides. The results suggest that cardenolides in this plant can explain its escape from cabbage butterflies, but specific structural features of the glycosides are necessary for oviposition-deterring activity.     

Cardenolide sequestration by the dogbane tiger moth (Cycnia tenera; Arctiidae)

Cycnia tenera adults, reared as larvae onAsclepias humistrata, had 10 times higher cardenolide concentrations, and contained 15 times more total cardenolide, than did moths reared onA. tuberosa. Thin-layer chromatography confirmed that each individual cardenolide visualized in the adult moths reared on the former host plant corresponds to one present in the plant, thus demonstrating that the insects' cardenolides are indeed derived from the larval food. Adult weights were significantly greater when the larvae had been fed upon the higher cardenolide plant species,A. humistrata. Similar results for other milkweed-feeding insects have been interpreted by some authors as evidence against a metabolic cost of handling cardenolides. However, such interpretations confound cardenolide differences among milkweed species with other differences in plant primary and secondary chemistry that affect insect growth and development. While the cooccurrence inC. tenera of other noxious chemicals (e.g., alkaloids) is not precluded, cardenolides sequestered from larval host plants have probably contributed to the evolution of visual and auditory aposematism in this species. As the eggs are laid in large clutches and larvae are gregarious, such aposematism may have evolved via kin selection.     

Olfactory responses of a specialist and a generalist grasshopper to volatiles ofArtemisia ludoviciana nutt. (Asteraceae)

Hypochlora alba is a specialist grasshopper that lives and feeds almost exclusively onArtemisia ludoviciana, a plant that produces large amounts of allelochemics including a variety of monoterpenes. This plant is not a host for generalist grasshoppers such asMelanoplus sanguinipes. The role of olfaction in the grasshopper-plant relationship was investigated by comparing electroantennograms (EAGs) of males and females of both species generated by solvent-extracted volatiles from plant leaves and by major individual components. Volatiles ofA. ludoviciana were identified by gas chromatography-mass spectrometry. The major components were 1,8-cineole, camphor, camphene, and borneol, while minor identified compounds were - and -pinene, -thujene, myrcene,p-cymene, Artemisia ketone, -thujone, and bornyl acetate. The EAGs (mV) ofH. alba males to a range of concentrations of individual volatiles or the total plant extract were nearly double those of conspecific females or both sexes ofM. sanguinipes. However, both sexes ofM. sanguinipes were more sensitive than either sex ofH, alba to geraniol, a monoterpene that commonly occurs in many plant species but is absent or is present in only trace amounts inA. ludoviciana. The increased sensitivity ofH. alba males to the odor components of their host plant appears to be related to the greater number of certain olfactory chemoreceptors on male versus female antennae. The significance of this phenomenon is discussed.     

The Effects of Milkweed Induced Defense on Parasite Resistance in Monarch Butterflies, <Emphasis Type="Italic">Danaus plexippus</Emphasis>

Many plants express induced defenses against herbivores through increasing the production of toxic secondary chemicals following damage. Phytochemical induction can directly or indirectly affect other organisms within the community. In tri-trophic systems, increased concentrations of plant toxins could be detrimental to plants if herbivores can sequester these toxins as protective chemicals for themselves. Thus, through trophic interactions, induction can lead to either positive or negative effects on plant fitness. We examined the effects of milkweed (Asclepias spp.) induced defenses on the resistance of monarch caterpillars (Danaus plexippus) to a protozoan parasite (Ophryocystis elektroscirrha). Milkweeds contain toxic secondary chemicals called cardenolides, higher concentrations of which are associated with reduced parasite growth. Previous work showed that declines in foliar cardenolides caused by aphid attack render monarch caterpillars more susceptible to infection. Here, we ask whether cardenolide induction by monarchs increases monarch resistance to disease. We subjected the high-cardenolide milkweed A. curassavica and the low-cardenolide A. syriaca to caterpillar grazing, and reared infected and uninfected caterpillars on these plants. As expected, monarchs suffered less parasite growth and disease when reared on A. curassavica than on A. syriaca. We also found that herbivory increased cardenolide concentrations in A. curassavica, but not A. syriaca. However, cardenolide induction in A. curassavica was insufficient to influence monarch resistance to the parasite. Our results suggest that interspecific variation in cardenolide concentration is a more important driver of parasite defense than plasticity via induced defenses in this tri-trophic system.     

Specialist Weevil, Rhyssomatus lineaticollis, Does Not Spatially Avoid Cardenolide Defenses of Common Milkweed by Ovipositing into Pith Tissue

Rhyssomatus lineaticollis is a milkweed specialist whose larvae feed upon pith parenchyma in ramet stems of the common milkweed, Asclepias syriaca. Compared with other specialist insect herbivores on milkweeds, this curculionid beetle is unusual in that it is cryptically colored and does not sequester cardenolides characteristic of milkweed chemical defense. Based upon optimal defense theory, we predicted that pith tissue would be low in defensive compounds and that oviposition into the pith would spatially avoid cardenolides. We rejected this hypothesis because we found that pith tissue has a relatively high cardenolide concentration compared to cortex, epidermis, and leaf tissues. Moreover, we found total plant cardenolide concentration was lower in plants that contained the beetle eggs. Cardenolide concentrations were different among tissues in intact stems without the pith herbivore compared to stems where it was present. Furthermore, the overall polarity of the cardenolides present varied among plant tissues and between plants with and without R. lineaticollis eggs. Although we found lower concentrations of cardenolide in piths where the eggs were present, the cardenolides present in the pith contained more nonpolar forms, indicating that the plant may be responding to herbivory by increasing toxic efficacy of cardenolide defenses while lowering the total concentration. We suggest that preoviposition behavior by female beetles, which includes feeding on new leaves of the plant, is a mechanism by which females manipulate plant chemistry and assess quantitative and qualitative changes in cardenolide chemistry in response to herbivory prior to oviposition.