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.     

Effect of gross cardiac glycoside content of seeds of common milkweed,Asclepias syriaca,on cardiac glycoside uptake by the milkweed bugOncopeltus fasciatus

Milkweed bugs,Oncopeltus fasciatus, were fed seeds of common milkweed,Asclepias syriaca, that contained differing concentrations of cardiac glycoside. Whole seeds had a mean cardiac glycoside concentration of 4.01 mg equivalents to digitoxin per g dry weight, and seed embryos had a mean concentration of 5.56 mg/g dry weight. Bugs fed these seeds concentrated cardiac glycoside: their mean concentration was 6.85 mg/g dry weight. Milkweed bugs fed seeds of lower cardiac glycoside content sequestered a greater percent of the available glycoside than bugs fed seeds of high glycoside content. The quantitative variation of cardiac glycoside content of the seeds of this single species did not significantly affect the growth of bugs. In a separate feeding preference experiment, bugs were offered seeds of both high (5.18 mg/g dry weight) and low (2.30 mg/g dry weight) cardiac glycoside content. The bugs showed no indication of selecting seeds of either high or low glycoside content.     

Variation in cardenolide content of the lygaeid bugs,Oncopeltus fasciatus andLygaeus kalmii kalmii and of their milkweed hosts (Asclepias spp.) in central California

A colorimetric assay was used to quantify the amount of cardenolides in the lygaeid bugsOncopeltus fasciatus andLygaeus kalmii kalmii and their milkweed host plants (Asclepias spp.) in central California. The cardenolide content of individual insects, determined in microgram equivalents of digitoxin, varied from zero to over 300 g per insect. Sources of variation of cardenolide content in the insects include interspecific and intraspecific differences in the content of the host plant species and also differences in the content of plant organs on which insects were feeding. This last source of variability may explain temporal variation in the cardenolide content of the insects. Adults ofO. fasciatus, which migrate into California in the late spring and early summer, and adults ofL. k. kalmii, which emerge from winter hibernacula in the early spring, contained small to immeasureable amounts of cardenolides. The colonization pattern ofO. fasciatus on species ofAsclepias in north central California suggests that this species does not maximize its opportunities to sequester large quantities of cardenolides from potential hosts. The emetic potential of lygaeids in California to vertebrate predators is briefly discussed.     

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.     

Pheromones of milkweed bugs (Heteroptera: Lygaeidae) attract wayward plant bugs: Phytocoris mirid sex pheromone

The synthetic aggregation pheromone of the large milkweed bug, Oncopeltus fasciatus (Dallas) (Lygaeinae), also attracted males of the plant bug, Phytocoris difficilis Knight (Miridae). Field testing partial blends against the six-component blend comprising the Oncopeltus pheromone showed that cross-attraction of P. difficilis males was due to synergism between (E)-2-octenyl acetate and (E,E)-2,4-hexadienyl acetate. Hexyl acetate was abundant in the metathoracic scent gland (MSG) secretion of P. difficilis males, but because female P. difficilis could not initially be found in the field, further combinatorial tests were guided by prior research on the pheromones of two Phytocoris species in the western United States. The combination of hexyl, (E)-2-hexenyl, and (E)-2-octenyl acetates was as attractive to P. difficilis males as the milkweed bug pheromone, yet no milkweed bugs were drawn to this blend. Gas chromatographic (GC)-electroantennographic detection (EAD) and GC-mass spectrometric (MS) analyses of female P. difficilis MSGs determined that their secretion contained predominantly hexyl, (E)-2-hexenyl, and (E)-2-octenyl acetates (all strongly EAD-active)—the latter two compounds found only in trace amounts from males—plus five minor female-specific compounds, three of which were EAD-active. (E,E)-2,4-Hexadienyl acetate was not detected from P. difficilis females or males. The blend of the three major components, hexyl, (E)-2-hexenyl, and (E)-2-octenyl acetates (2:1.5:1 by volume), was as attractive as the blend of all six EAD-active compounds identified from females, indicating that this ternary blend constitutes the sex pheromone of P. difficilis. Hexyl acetate with (E)-2-octenyl acetate also attracted males of another species, P. breviusculus Reuter, but addition of (E)-2-hexenyl acetate and/or (E,E)-2,4-hexadienyl acetate inhibited attraction of P. breviusculus males. Attraction of P. difficilis males occurred mainly during the first half of scotophase. The possible neurophysiological basis for this asymmetrical cross-attraction is discussed.     

Synthesis of highly active juvenile hormone analogs,juvocimene I and II,from the oil of sweet basil,Ocimum basilicum L.

Juvocimene I and II are potent juvenile hormone mimics isolated from the essential oil of sweet basil,Ocimum basilicum L. The structures given by the formula I and II have been confirmed by synthesis withtrans--ocimene andp-methoxycinnamyl chloride. Biological activity of the natural and synthetic juvocimenes was found to be identical.     

Exocrine secretions of the andromeda lace bugStephanitis takeyai (Hemiptera: Tingidae)

The four major components of the setal exudate of nymphs of the andromeda lace bug,Stephanitis takeyai, were identified. They are 7-hydroxy-3-nonylchromanone1, the novel 3,5-dihydroxy-2-dodecanoylcyclohex-2-en-1-one2,2,6-dihydroxydodecanophenone3, and its 3-hydroxy derivative4. Only three species ofStephanitis commonly occur in the United States; withS. takeyai we have now identified the major compounds secreted by each of these species. The individual species of the genus are chemotaxonomically distinct and are characterized by the oxidation states of the secreted compounds.     

Volatile compounds from the predatory insectPodisus maculiventris (Hemiptera: Pentatomidae)

In the predatory spined soldier bug,Podisus maculiventris, the dorsal abdominal glands are much smaller in adult females than males. Females produce a mixture of (E)-2-hexenal, (E)-2-octenal, (E)-2-hexenoic acid, benzaldehyde, and nonanal in these glands. The female dorsal abdominal gland secretion may be a close-range pheromone since the dorsal abdominal gland secretion from males has been shown to be a long-range aggregation pheromone in this species. The metathoracic scent gland secretions of male and female spined soldier bugs are apparently identical, and similar to that of other pentatomids, except for the presence of the monoterpene alcohol, linalool.Mention of a commercial product does not constitute an endorsement of this product by the USDA.     

Production of cardenolides versus sequestration of pyrrolizidine alkaloids in larvae ofOreina species (Coleoptera,Chrysomelidae)

Adult leaf beetles of the genusOreina are known to be defended either by autogenously produced cardenolides or by pyrrolizidine alkaloids (PAs) sequestered from the food plant, or both. In this paper we analyze larvae of differentOreina species and show that the larvae contain the same defensive toxins as the adults in quantities similar to those released in the adults' secretion. Both classes of toxins are found in the body and hemolymph of the larvae, despite their different origins and later distribution in the adults. Larvae of sequestering species differed in their PA patterns, even though they fed on the same food plants. The concentration in first-instar larvae of a PA-sequestering species was similar to that in fourth-instar larvae. In all stages examined, the amount of PAs per larva did not greatly exceed the estimated uptake of one day. Eggs of two oviparous species contained large concentrations of the adult's toxins, while neonates of a sequestering larviparous species had no PAs.     

Phenolic acetogenins secreted by rhododendron lace bug,Stephanitis rhododendri Horvath (Hemiptera: Tingidae)

Nymphs of the rhododendron lace bug produce from specialized setae a liquid secretion from which a number of related acetogenins have been identified. Two (2,6-dihydroxyphenyl)-1,3-diketones are the principal components and are accompanied by the corresponding chromones as well as by chromones bearing an additional phenolic oxygen.Mention of a company name or proprietary product does not constitute an endorsement by the U.S. Department of Agriculture.     

Pharmacodynamics of some individual milkweed cardenolides fed to larvae of the monarch butterfly (Danaus plexippus L.)

Adult monarch butterflies,Danaus plexippus L. (Lepidoptera: Danaidae), store only some of the cardenolides present in the larval milkweed (Asclepiadaceae) host. Feeding known doses of individual cardenolides to 4th instar monarch larvae led to more efficient larval tissue incorporation at low doses than at high ones, and favored storage of cardenolide glycosides over genins. A qualitative regulation also occurs during larval feeding; calactin and calotropin were stored as such but uscharidin was rapidly converted to a mixture of calactin and calotropin which were the forms stored by the larvae. Two genins, uzarigenin and digitoxigenin, were stored by larvae as polar cardenolide metabolites.Research supported by National Science Foundation grants DEB 7514266 and DEB 7514266-AO2 (U.C. Davis) and DEB 7514265 (Amherst College).     

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.     

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.)     

Identification of food volatiles attractive toGlischrochilus quadrisignatus andGlischrochilus fasciatus (Coleoptera: Nitidulidae)

Seven volatile compounds identified from the headspace of whole wheat bread dough were investigated for their role in attractingGlischrochilus quadrisignatus andG. fasciatus in the field. Traps baited with either whole wheat bread dough or a synthetic seven-component bread dough odor caught similar numbers of these beetles, suggesting that the seven-compound combination could simulate the behavioral effect of bread dough. A series of trials using traps baited with various combinations of these chemicals showed that five compounds were significantly active in attractingG. quadrisignatus andG. fasciatus, but not all were essential for maximum response. The simplest blend eliciting a level of response comparable to the seven-component combination included ethyl acetate, acetaldehyde, ethanol, and racemic 2-methylbutanol, of which ethyl acetate, ethanol, and acetaldehyde were essential and 2-methylbutanol was replaceable with 2-methylpropanol forG. quadrisignatus attraction. Ethyl acetate and ethanol were essential for comparable attraction ofG. fasciatus. The chemical mediation of food finding in G.quadrisignatus andG. fasciatus is discussed in the context of volatile blends characterized for other nitidulid species.     

Chemical investigation of aggregation behavior ofTriatoma bugs (Hemiptera: Reduviidae)

Nymphs ofTriatoma infestans andTriatoma mazzottii are weakly attracted to their feces and to extracts of feces in polar solvents, but not to nonpolar solvent extracts. The major volatile compounds identified in feces by solvent extraction and thermal desorption wereo-aminoacetophenone, 4-methylquinazoline, and 2,4-dimethylquinazoline, but these showed no attractant activity at a range of concentrations. Choice tests with a moving current of air gave no positive reaction to feces, extracts, or pure compounds.     

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.     

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.     

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.