Metabolism of uscharidin,a milkweed cardenolide,by tissue homogenates of monarch butterfly larvae,Danaus plexippus L.

Midgut and fat body homogenates of monarch butterfly larvae,Danaus plexippus L. (Lepidoptera:Danaidae), were examined for microsomal monooxygenase activity usingp-chloro-N-methylanilineN-demethylation and for the ability to metabolize a milkweed (Asclepias spp.) cardenolide (C₂₃ steroid glycoside), uscharidin. All homogenates tested had bothN-demethylation and uscharidin biotransformation activities. Both transformations required NADPH. The monooxygenase inhibitors sesamex, SKF525A, and carbon monoxide inhibitedN-demethylation but not uscharidin biotransformation. Subsequent subcellular fractionation revealed the uscharidin biotransformation occurs in the soluble fraction and not the microsomal fraction, whileN-demethylation occurs in the microsomal fraction and not the soluble fraction. The larval NADPH-dependent microsomal monooxygenase apparently is not involved in the metabolism of uscharidin.     

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.     

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.     

Pyrrolizidine alkaloids in an overwintering population of monarch butterflies (Danaus plexippus) in California

California overwintering monarch butterflies contain both pyrrolizidine alkaloids (PAs) and theirN-oxides. Analysis of 76 individual monarchs by TLC, HPLC, GLC, and GC-MS has shown the presence of three types of PAs, the saturated diester sarracine, the saturated monoester 7-angelylplatynecine, and the unsaturated dialcohol retronecine. Monarchs arriving at the overwintering site in Santa Cruz, California, showed a wide variation in both the type and amount of PA present. Those sampled after a PA-containing plant (Senecio mikanioides) had bloomed at the site showed an altered PA profile. While the plant was found to contain sarracine and 7-angelylplatynecine, which are nontoxic to mammals, the monarchs showed an increase in retronecine levels, a toxic PA, after the plant bloom. Apparently monarchs utilize PA-containing plants both en route to their overwintering site and at the site, and potentially alter those PAs to forms toxic to mammals.     

Monooxygenase activities of fat body and gut homogenates of monarch butterfly larvae,Danaus plexippus,fed four cardenolide-containing milkweeds,Asclepias spp.

Monarch butterfly larvae were examined for NADPH-dependent monooxygenase activities. Midgut and fat body homogenates catalyzed aldrin epoxidation andp-chloro-N-methylanilineN-demethylation at consistently low rates compared to many other lepidopteran larvae. Homogenates from larvae collected from four different milkweeds (Asclepias spp.) with quite different cardenolide contents had similar levels of activity. There were no detectable variations in activity due to season or year of collection.Research supported in part by a National Science Foundation grant DEB 7514266 and the Agricultural Experiment Station, University of California, Davis, California 95616.     

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 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.     

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.     

Mediation of cardiac glycoside insensitivity in the monarch butterfly (Danaus plexippus): Role of an amino acid substitution in the ouabain binding site of Na+,K+-ATPase

The Monarch butterfly (Danaus plexippus) sequesters cardiac glycosides (CG) for its chemical defense against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, whereas other Lepidoptera are sensitive and intoxicated by ouabain. Ouabain inhibits Na⁺,K⁺-ATPase by binding to its -subunit. We have amplified and cloned the DNA-sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain-sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of 12 amino acids. Starting with the CG-sensitive Na⁺,K⁺-ATPase gene fromDrosophila, we converted pos. 122 to a histidine residue as inDanaus plexippus by site-directed mutagenesis. Human embryonic kidney cells (HEK) (which are sensitive to ouabain) were transfected with the mutated Na⁺,K⁺-ATPase gene in a pSVDF-expression vector and showed a transient expression of the mutatedDrosophila Na⁺,K⁺-ATPase. When treated with ouabain, the transfected cells tolerated ouabain at a concentration of 50 mM, whereas untransformed controls or controls transfected with the unmutatedDrosophila gene, showed a substantial mortality. This result implies that the asparagine to histidine exchange contributes to ouabain insensitivity in the Monarch. In two other CG-sequestering insects, e.g.,Danaus gilippus andSyntomeida epilais, the pattern of amino acid substitution differed, indicating that the Monarch has acquired this mutation independently during evolution.     

Cardenolide connection between overwintering monarch butterflies from Mexico and their larval food plant,Asclepias syriaca

The majority (85%) of 394 monarch butterflies sampled from overwintering sites in Mexico contain the same epoxy cardenolide glycosides, including most conspicuously a novel polar glycoside with a single genin-sugar bridge (aspecioside), as occur in the milkweedsAsclepias speciosa andA. syriaca. This cardenolide commonality was established by isolating aspecioside and syriobioside from the wings of overwintering monarchs and the two plant species, and comparing Chromatographie and NMR spectrometric characteristics of the isolates. When combined with the migratory pattern of monarchs and the distribution of these two milkweed species, this chemical evidence lends strong support to the hypothesis thatA. syriaca is the major late summer food plant of monarchs in eastern North America. This finding may be of ecological importance, forA. syriaca contributes less cardenolide and cardenolides of lower emetic potency to monarchs than most milkweeds studied to date.Research supported by National Science Foundation Grants DEB 81-19391 (UC Davis) and BSR-81-19382 (Univ. of Florida).     

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.     

The Influence of Eastern North American Autumnal Migrant Monarch Butterflies (Danaus plexippus L.) on Continuously Breeding Resident Monarch Populations in Southern Florida

In Florida, the eastern North American population of the monarch butterfly exhibits geographic variability in population structure and dynamics. This includes the occurrence of migrants throughout the peninsula during the autumnal migration, occasional overwintering clusters that form along the Gulf Coast, remigrants from Mexico that breed in north-central Florida during the spring, and what have been assumed to be year-round, resident breeding populations in southern Florida. The work reported here focused on two monarch populations west of Miami and addressed four questions: Are there permanent resident populations of monarchs in southern Florida? Do these breed continuously throughout the year? Do they receive northern monarchs moving south during the autumn migration? Do they receive overwintered monarchs returning via Cuba or the Yucatan during the spring remigration from the Mexican overwintering area? Monthly collections and counts of spermatophores in the bursa copulatrices of females established that a resident population of continuously breeding monarchs exists year-round in southern Florida. It was determined through cardenolide fingerprinting that most of the butterflies had bred on the local southern Florida milkweed species, Asclepias curassavica. During the autumn migration period, however, some monarchs had fed on the northern milkweed, Asclepias syriaca. It appears that instead of migrating to Mexico, these individuals travel south through peninsular Florida, break diapause, mate with and become incorporated into the resident breeding populations. None of the monarchs captured in spring had the A. syriaca cardenolide fingerprint, which is evidence against the southern Florida populations receiving overwintered remigrants from Cuba, Central America or Mexico.     

Feeding preferences of spruce budworm (Choristoneura fumiferana Clem.) larvae to some host-plant chemicals

Single-choice and three-choice tests were used to determine the relative importance of host-plant chemical extracts in eliciting feeding by spruce bud worm larvae. Water-soluble components of the host trees are the most important and, of these, the sugar and glycoside fractions are the most stimulating. The amino acid and organic base fractions have no apparent effect. The organic acid fractions deter feeding slightly.Lepidoptera: Tortricidae.Supported by the National Sciences and Engineering Research Council of Canada, Operating Grant No. A9723 to P.J.A.     

Difference in hydroxamic acid content in roots and root exudates of wheat (Triticum aestivum L.) and rye (Secale cereale L.): Possible role in allelopathy

Hydroxamic acids (Hx) produced by some cereal crops have been associated with allelopathy. However, the release of Hx to the soil by the producing plant-an essential condition for a compound to be involved in allelopathy-has not been shown. GC and HPLC analysis of roots and root exudates of wheat (Triticum aestivum L.) and rye (Secale cereale L.) cultivars, with high Hx levels in their leaves, demonstrated the presence of these compounds in the roots of all cultivars analyzed and in root exudates of rye. Moreover, bioassays employing root exudates collected from wheat and rye seedlings demonstrated that only rye exudates inhibited root growth of wild oats,Avena fatua L., a weed whose root growth is inhibited by Hx. These results suggest that rye could potentially interfere with the growth ofAvena fatua in nature and that this interference could be due to the release of Hx to the soil by way of roots.     

In vitro metabolism of a linear furanocoumarin (8-methoxypsoralen,xanthotoxin) by mixed-function oxidases of larvae of black swallowtail butterfly and fall armyworm

Studies were made of the comparative in vitro metabolism of [¹⁴C]xanthotoxin and [¹⁴C]aldrin by homogenate preparations of midguts and bodies (carcass minus digestive tract and head) of last-stage larvae of the black swallowtail butterfly (Papilio polyxenes Fabr.) and the fall armyworm [Spodoptera frugiperda (J. E. Smith)]. The two substrates were metabolized by 10,000g supernatant microsomal preparations from both species. Evidence gained through the use of a specific inhibitor and cofactor indicated that mixed-function microsomal oxidases were major factors in the metabolism and that the specific activity of this enzyme system was considerably higher in midgut preparations fromP. polyxenes than in similar preparations fromS. frugiperda. Aldrin was metabolized 3–4 times faster byP. polyxenes, and xanthotoxin 6–6.5 times faster.     

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.     

Orientation behavior of second-instar larvae of eastern spruce budwormChoristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae) in a y-type olfactometer

A two-choice, wind-tunnel olfactometer was designed and contructed to determine whether second-instar eastern spruce budworm larvae,Christoneura fumiferana (Clem.), could detect and discriminate among host-plant volatiles. Volatiles of current year's growth ofPicea glauca were preferred over those ofP. rubens, P. mariana, or air.Abies balsamea was preferred overP. mariana or air.P. rubens andP. mariana were both preferred over air. Two-year-old growth ofP. glauca, A. balsamea, P. rubens, andP. mariana were all preferred over air. Current year's growth of these host evergreen species was usually preferred over former year's growth of the same species in each case.     

Attraction ofOryzaephilus surinamensis (L.) andOryzaephilus mercator (Fauvel) (Coleoptera: Cucujidae) to some common volatiles of food

Responses by adultOryzaephilus surinamensis (L.) andOryzaephilus mercator (Fauvel) to various food volatiles were assessed by means of a two-choice, pitfall olfactometer. The individual experimental stimuli, all potential products of lipid oxidation, had a range of attractive doses of 1000-fold over the test dose ranges of 0.001–100 gmg, or 0.01–1000 gmg. Of 13 aliphatic C₃-C₁₄ aldehydes and benzaldehyde tested forOryzaephilus spp., 10 C₃-C₁₀ aliphatic aldehydes and benzaldehyde showed some attractiveness for both species. ForO. mercator, nonanal had the lowest lower threshold for positive response at 0.01 g. The addition of small amounts of nonanal or of a 111 mixture of hexanal, octanal, and nonanal to small amounts of cucujolide aggregation pheromones enhanced response by mixed-sexO. mercator to the pheromones. Eleven aliphatic C₂-C₉ free fatty acids showed some attractiveness for bothOryzaephilus spp. Isovaleric acid and valeric acid had the lowest lower thresholds for positive response at 0.1 g forO. mercator andO. surinamensis, respectively. Four olefinic oat volatiles were found to possess various degrees of attractiveness for bothOryzaephilus spp. The data suggest that food volatiles in this study might be used byOryzaephilus spp. as host-finding kairomones in nature.Research supported by the Natural Sciences and Engineering Research Council of Canada, Strategic Grant G0958 and Operating Grants A3881 and A3785.     

Aristolochic acids fromThottea spp. (Aristolochiaceae) and the osmeterial secretions of Thottea-feeding troidine swallowtail larvae (Papilionidae)

Two aristolochiaceous plants in the genusThottea were shown to contain aristolochic acids. Larvae of two Malaysian troidine swallowtail butterflies,Troides (Troides) amphrysus andPachliopta (Losaria) neptunus, that fed onThottea leaves were found to sequester corresponding aristolochic acid analogs in the osmeterial glands.