Fate of iridoid glycosides in different life stages of the Buckeye,Junonia coenia (Lepidoptera: Nymphalidae)

The buckeye butterfly,Junonia coenia (Lepidoptera: Nymphalidae), specializes on plants that contain iridoid glycosides. To determine the fate of these compounds in larvae, pupae, and adults of this species, we reared larvae on artificial diets with and without iridoid glycosides, and on leaves of a host plant,Plantago lanceolata (Plantaginaceae). Quantification by gas chromatography showed that newly molted third-, fourth-, and fifth-instar larvae reared on leaves ofP. lanceolata contained means of 5.13, 2.88, and 6.83% dry weight iridoid glycoside. In contrast, the mean iridoid glycoside concentration of actively feeding fifth-instar larvae was 0.28% dry weight, that of pupae was 0.19% dry weight iridoids, and adults contained no detectable iridoids. Feeding experiments suggested that this reduction in actively feeding larvae was due to the metabolism of iridoid glycosides.P. lanceolata leaves in these experiments contained a mean of 1.00% dry weight iridoid glycoside, with a 2:1 ratio of aucubin to catalpol. Calculation of iridoid consumption and utilization indices showed that larvae fed artificial diets consumed, digested, and sequestered aucubin and catalpol in similar ways. When these indices were calculated for larvae fed leaves ofP. lanceolata, catalpol was sequestered twice as efficiently as aucubin.     

A chemical basis for differential acceptance ofErysimum cheiranthoides by twoPieris species

Wormseed mustard,Erysimum cheiranthoides, is unacceptable as a host for the cabbage butterfly,Pieris rapae. However, it is preferred for oviposition byPieris napi oleracea in the greenhouse. Isolation and identification of the oviposition stimulants toP. napi oleracea were accomplished by C₁₈ open-column chromatography, TLC, ion-exchange chromatography, HPLC, UV, and NMR spectroscopy. Glucoiberin and glucocheirolin were identified as the most active stimulants. The extracted glucoiberin was as stimulatory as glucocheirolin, although its concentration in theErysimum plants was about 10 times lower than that of glucocheirolin. These glucosinolates were only weak stimulants toP. rapae. Furthermore,P. rapae was strongly deterred by the cardenolides, erysimoside and erychroside, fromE. cheiranthoides, andP. napi oleracea was less sensitive to these compounds. No other deterrent toP. napi oleracea was detected in this plant species. The results explain the differential acceptance ofE. cheiranthoides by these twoPieris species.     

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.     

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

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

Chemical aspects of host-plant specificity in threeLarrea-feeding grasshoppers

The host-selection behavior of three species of grasshopper feeding on creosote bush,Larrea tridentata, in southern California was investigated. The species wereBootettix argentatus, which is monophagous;Ligurotettix coquilletti, oligophagous; andCibolacris parviceps, polyphagous. The monophagous species is stimulated to bite by nordihydroguaiaretic acid (NDGA), a compound that is characteristic of the host plant and that may comprise up to 10% of the dry weight of the leaf. Host specificity ofB. argentatus is enhanced by deterrent responses to compounds present in the surface waxes of all non-host-plant species. Both the oligophagous and polyphagous species are deterred by NDGA at naturally occurring concentrations. Their association withLarrea is probably based on tolerance of the plant chemicals rather than on dependence on specific chemicals. Factors other than the chemistry of the plant probably also contribute to the specificity ofB. argentatus andL. coquilletti.     

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.     

A neolignoid feeding deterrent againstLuehdorfia puziloi larvae (lepidoptera: Papilionidae) fromHeterotropa aspera,a host plant of sibling species,L. japonica

A feeding deterrent against larvae of a papilionid butterfly,Luehdorfia puziloi (Parnassiinae), a specialist onAsiasarum plants (Aristolochiaceae), was isolated from another aristolochiaceous plant,Heterotropa aspera. Antifeedant activity was exhibited by then-hexane-soluble fraction that proved to contain at least two active components. One of the deterrents was identified as a neolignan compound, asatone. The concentration of asatone inH. aspera was estimated at approximately 225 ppm, and larval feeding ofL. puziloi was significantly deterred at concentrations over 90 ppm. By contrast, asatone was not detectable (< 1 ppm, if any) in its host plant,Asiasarum sieboldii.     

Lignan analysis in seed oils from fourSesamum species: Comparison of different chromatographic methods

Different chromatographic methods, thin-layer chromatography (TLC), gas chromatography (GC), gas chromatography/mass spectrometry (GC/MS) and normal- and reversed-phase high-performance liquid chromatography (HPLC), were compared for their ability to separate the different lignans present in fourSesamum species,viz., S. indicum Linn.,S. alatum Thonn., S. radiatum Schum & Thonn. andS. angustifolium (Oliv.) Engl. The advantages and limitations of each method are discussed, and a combination of methods is suggested for qualitative analyses. Two-dimensional TLC was found to be a valuable qualitative technique and one-dimensional TLC is useful for preparative purposes. GC is a good supplement for qualitative analysis, but it had many limitations as a quantitative tool—it involves many preparative steps, no suitable internal standard was found to be commercially available and the various lignans had markedly different response factors. GC/MS is a necessary techniqee to confirm the identity of the lignans present. HPLC is a one-step technique suitable for quantitative analyses, and is fast and simple because it involves direct injection of oil solutions. Reversed-phase HPLC was unable to separate sesamolin and sesangolin, but a normal-phase silica column provided satisfactory separation for these two lignans. 2-Episesalation ofS. alatum, however, did not elute from the normalphase column. Once lignans are identified, a relevant HPLC method can be used for quantitative analyses. Sesamin was present in large amounts inS. radiatum, in considerable amounts inS. indicum andS. angustifolium, and in small amounts inS. alatum. Sesamolin occurred in considerable amounts inS. indicum andS. angustifolium, but only in small amounts in the other two wild species studied.Sesamum alatum was characterized by high amounts of 2-episesalatin, andS. angustifolium was characterized by high levels of sesangolin.     

Purification and characterization of new fatty acids with antibiotic activity produced bySporothrix flocculosa

Chloroform extracts from a culture broth of the biocontrol fungusSporothrix flocculosa were separated by thin-layer chromatography (TLC). Compounds with antifungal activity were revealed with theCladosporium cucumerinum bioassay on TLC plates and were isolated by using silica gel column, preparative TLC, and reverse-phase high-performance liquid chromatography (RP-HPLC). They were identified by standard methods of nuclear magnetic resonance (NMR), gas chromatography (GC), mass spectrometry (MS), and infrared spectrum (IR). In addition to an active molecule previously described, two new fatty acids with antibiotic activity were characterized, 9-heptadecenoic acid and 6-methyl-9-heptadecenoic acid, the latter displaying the strongest activity among all three active molecules. This high number of active metabolites produced byS. flocculosa would explain its rapid and strong activity as a biocontrol agent of powdery mildews. In this context, screening ofS. flocculosa isolates for increased production of these molecules should help in selection of candidates with superior biocontrol properties.Contribution no. 150 du Centre de recherche en horticulture.     

Allelopathic potential ofAnthoxanthum odoratum for invadingZoysia-grassland in Japan

The growth ofZoysia japonica surroundingAnthoxanthum odoratum onZoysia-grassland in Japan was investigated in June 1994. The stem density ofZ. japonica tended to decrease with short distances between twoA. odoratum plants. This showed that the growth ofZ. japonica surroundingA. odoratum was reduced. Basic, neutral, and acidic fractions extracted fromA. odoratum plants inhibited the seedling growth of lettuce. In particular the neutral fraction showed the strongest activity among the three fractions. The main inhibitory compound obtained atR _f 0.6–0.7, on the thin-layer chromatogram of the neutral fraction, was isolated and identified as coumarin by means of GC-MS. Coumarin solution inhibited seedling growth ofZ. japonica in low concentrations but, conversely, promoted seedling growth ofA. odoratum. Coumarin was contained in all parts ofA. odoratum and its concentration varied with the season and from one individual plant to another. In particular, coumarin was highly concentrated in the leaves, accounting for more than 2.5% of dry leaf weight in June. The inhibitory effect of these aqueous extracts was correlated to the amount of coumarin inA. odoratum leaves and cournarin was considered to be the main inhibitory compound.     

Hostplant Suitability and Defensive Chemistry of the Catalpa Sphinx, Ceratomia catalpae

The growth and survival of the Catalpa sphinx, Ceratomia catalpae (Sphingidae), were measured on five different species of Catalpa: C. bignonioides, C. bungeii, C. fargeseii, C. ovata, and C. speciosa. Larval growth varied significantly among these host plant species; however, survival did not differ. Quantification of the iridoid glycoside content of larvae, pupae, adults, larval frass, and leaves of the larval host plant, C. bignonioides, by gas chromatography showed that leaves contained both catalpol and catalposide; larvae, pupae, and frass contained only catalpol; and the adults contained no detectable iridoid glycosides. Amounts were highest in the larvae and declined in the pupal stage. Very small amounts of catalpol were detected in adults of the parasitoid, Cotesia congregata, and in the silken cocoons. The hemolymph in which the parasitoid larvae grew contained over 50% dry weight catalpol. Larvae of C. catalpae often regurgitate when disturbed. This may serve as a defense against predators. A comparison of the growth of larvae pinched with forceps to induce regurgitation with those that were not so treated showed that larvae that were pinched, and usually regurgitated, grew significantly more slowly than those that were not.     

Trimerization ofEarias insulana sex pheromone, (E,E)-10,12-Hexadecadienal,A phenomenon affecting trapping efficiency

The sex pheromone ofEarias insulana, (E,E)-10,12-hexadecadienal, may trimerize extensively to form a crystalline trioxane derivative. The structure of the trimer was deduced from its CI-MS and NMR spectra. Capillary GC analysis resulted in the thermal decomposition of the trimer to the monomer. This process could be studied on a 2-m packed column under specific conditions. A convenient separation between the pheromone and its trimer was achieved by TLC. The trimer was inactive in the field, and it has a harmful effect on the performance of the polyethylene dispenser. Material which contains large amounts of the trimer is unsuitable for field use, even if applied at high dosage. The pheromone should be analyzed by NMR or TLC in addition to GC in order to detect the presence of its trimer. The trimerization process is catalyzed by acid which should therefore be completely eliminated from the storing vessels.Boisduval, Lepidoptera: Noctuidae.Contribution from the Agricultural Research Organization (ARO), No. 675-E, 1983 series.     

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.     

Oviposition stimulants inBarbarea vulgaris forPieris rapae andP. napi oleracea: isolation,identification and differential activity

The closely related butterflies,Pieris rapae andP. napi oleracea, readily laid eggs onBarbarea vulgaris in greenhouse cages. When offered a choice between cabbage andB. vulgaris, P. rapae showed no preference, butP. napi oleracea preferredB. vulgaris. Bioassays of extracts ofB. vulgaris foliage revealed the presence of oviposition deterrent(s) in l-butanol extracts as well as stimulants in the postbutanol water extracts. However, the deterrent effect was apparently outweighed by the strong stimulatory effect in the whole plants. The postbutanol water extract was preferred over an equivalent cabbage extract by both species, but more significantly in the case ofP. napi oleracea. The stimulants were isolated by open column chromatography and HPLC, and the activity was associated with three glucosinolates.P. napi oleracea was more sensitive thanP. rapae to the natural concentration of compounds1 and3, whereas both species were strongly stimulated to oviposit by natural concentrations of compound2. Compounds1 and2 were identified as (2R)-glucobarbarin and (2S)-glucobarbarin, respectively, and3 was identified as glucobrassicin, on the basis of their UV, mass, and NMR spectra. When the pure compounds were tested at the same concentrations applied to bean plants, the (2R)-glucobarbarin at 0.2 mg/plant was preferred over a standard cabbage extract by both butterfly species. However, at a dose of 0.02 mg/plant,P. rapae preferred the cabbage extract whereasP. napi oleracea still preferred the (2R)-glucobarbarin. No such difference in response of the two species to the same two concentrations of (2S)-glucobarbarin was obtained. The results indicate a distinct difference in sensitivity of these butterflies to the epimers of glucobarbarin, and the differences in behavioral responses of the two butterfly species depend to a large extent on the concentration of stimulant present.     

Oviposition stimulants for the tropical swallowtail butterfly,Papilio polytes,feeding on a rutaceous plant,Toddalia asiatica

In nature, Papilio polytes utilizes a limited range of rutaceous plants as hosts. We isolated and identified oviposition stimulants for the butterfly from the foliage of its primary host plant Toddalia asiatica. Females readily deposited eggs in response to a methanolic extract of the plant. Partition of the extract with organic solvents revealed that chemicals responsible for eliciting egg-laying resided in a water-soluble fraction. Further bioassay-guided fractionation of the active fraction by column chromatography, preparative TLC, and HPLC led to the isolation of two oviposition stimulants. One was isolated from an amphoteric fraction and identified as trans-4-hydroxy-N-methyl-L-proline [(–)-(2S,4R)-4-hydroxy-1-methyl pyrrolidine-2-carboxylic acid; HMP]. The other, isolated from an acidic fraction, was identified as 2-C-methyl-D-erythronic acid [(–)-(2R,3R)-2-methyl-2,3,4-trihydroxybutanoic acid; MEA]. HMP alone evoked significant oviposition-stimulatory activity, although this was much lower than that of the original water-soluble fraction. MEA, on the other hand, alone did not elicit positive responses from females. However, HMP, when assayed in combination with MEA, markedly enhanced the female response, and the mixture was as active in stimulating oviposition as were the original water-soluble fraction and the plant foliage. We conclude that HMP is a substance crucial for host recognition by females, while MEA is a synergistic stimulant involved in host recognition and/or preference.     

Occurrence of conjugated fatty acids in the seed oil ofcouepia longipendula (chrysobalanaceae)

The seeds ofCouepia longipendula contain 74.2% oil. Gas chromatography (GC) and gas chromatography/ mass spectrometry (GC/MS) of the methyl esters and oxazoline derivatives of the fatty acids and ultraviolet (UV), infrared (IR),¹H-nuclear magnetic resonance (NMR) and¹³C-NMR spectra of the oil showed the presence of palmitic (25.2%), palmitoleic (0.9%), stearic (6.2%), oleic (26.5%), vaccenic (0.4%), linoleic (7.4%), arachidic (0.3%), α-eleostearic (11.3%) and α-licanic (21.8%) acids. Licanic acid methyl ester was isolated by thin-layer chromatography (TLC) and the¹³C-NMR and¹H-NMR data are presented.     

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.     

Oviposition stimulants of an Aristolochiaceae-feeding swallowtail butterfly,Atrophaneura alcinous

Oviposition stimulants of an Aristolochiaceae-feeding swallowtail butterfly,Atrophaneura alcinous, were isolated from the leaves ofAristolochla debilis and characterized as a mixture of aristolochic acids and sequoyitol. An artificial blend of these components applied to filter paper induced a significant oviposition response by the female butterflies, identical to that elicited by intact leaves of theAristolochia host plant.     

Seasonal patterns of juglone in soil beneathJuglans nigra (black walnut) and influence ofJ. nigra on understory vegetation

The allelopathic nature ofJ. nigra L. was investigated in several planted mixed hardwood stands located near Syracuse, New York. Concentrations of chloroform-extracted juglone from soil collected beneathJ. nigra was determined by thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC). Soil juglone concentrations were corrected based on recovery of synthetic juglone added to soil. Soil juglone levels were high in the spring, decreased during the summer, and were high again in the fall. The quantification of juglone from soil by HPLC was found to be more accurate than by TLC. Regression analysis indicated that individual tree variation in soil juglone levels could not be explained by differences in soil moisture, pH, organic matter content, and texture. The results of juglone recovery experiments suggest that chloroform-extractable juglone does not persist in soil. Juglone degradation by microorganisms could only explain a portion of the juglone decline. Ordinations revealed that the herbaceous and woody vegetation beneathJ. nigra, in comparison to vegetation beneathAcer saccharum andQuercus rubra, is distinct in only one of the four stands studied. This vegetational difference did not appear to be a consequence of any strong allelopathic influences ofJ. nigra (Scheffe's method of contrast, chi-square analysis). The allelopathic nature of juglone under these field conditions is questionable.     

Chemical stimulants and deterrents regulating acceptance or rejection of crucifers by cabbage butterflies

GravidPieris rapae butterflies oviposit on many, but not all, crucifers. Rejection ofErysimum cheiranthoides andCapsella bursa-pastoris was initially explained by the presence of chemical deterrents in the plants. Analyses and bioassays of plant extracts indicated the absence of oviposition stimulants inC. bursa-pastoris, but similar chemical separation ofE. cheiranthoides extracts revealed the presence of stimulants as well as deterrents. Choice tests illustrate how acceptance or rejection of a plant by an insect may depend on the balance of positive and negative chemical stimuli within the plant.