Isolation and identification of oviposition deterrents to cabbage butterfly,Pieris rapae,fromErysimum cheiranthoides

Avoidance of some crucifer species by the crucifer specialist,Pieris rapae, has been attributed to the presence of oviposition deterrents in these plants. Studies on one such unacceptable plant,Erysimum cheiranthoides, have resulted in the isolation ofn-butanol-soluble deterrents from the alcoholic extract of foliage. The active fraction contained three cardiac glycosides, which were isolated by reversed-phase HPLC and by open column chromatography on silica gel. Chemical and spectral evidence (UV, [¹H]NMR, and FAB-MS) led to the characterization of these compounds as erysimoside (1), erychroside (2), and erycordin (3). Erysimoside and erychroside were strongly deterrent toPieris rapae, but erycordin was inactive. Both active compounds have the same aglycone, strophanthidin (5) and the inner sugar in both cases is a 2,6-dideoxy hexose to which the outer sugar is attached at position C-4. These structural features, which are absent in the inactive compound (3), may represent specific requirements for oviposition deterrent activity.     

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.     

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.     

Relative activities of glucosinolates as oviposition stimulants forPieris rapae andP. napi oleracea

The relative activities of 10 glucosinolates in stimulating oviposition byP. rapae andP. napi oleracea were compared under the same conditions. When tested at the same concentration, the structurally different glucosinolates stimulated both butterfly species to widely varying degrees. In most cases,P. rapae was more sensitive to aromatic and indole glucosinolates than to aliphatic representatives. This species responded even less to alkyl thio and sulfinyl glucosinolates. However,P. napi oleracea responded strongly to these aliphatic and sulfur-containing members of the group, and the relative activities of aromatic and aliphatic glucosinolates did not show a clear pattern for this species.P. napi oleracea was much more sensitive to low concentrations of sinigrin than wasP. rapae. The threshold concentration for response ofP. napi oleracea to sinigrin was 10^–8 M, which was 100 times lower than forP. rapae, butP. rapae was more sensitive thanP. napi oleracea to changes in glucosinolate concentrations. For bothPieris species, an optimal concentration was reached, above which the response remained constant or tended to decrease.     

Chemical constituents of an unacceptable crucifer,Erysimum cheiranthoides,deter feeding byPieris rapae

The wild cruciferErysimum cheiranthoides was found to contain extractable constituents that deterred feeding by larvae of the crucifer specialistPieris rapae when applied to cabbage leaf disks in both choice and nochoice bioassays. High-performance liquid chromatography was used to separate the extract into several fractions, two of which retained the feeding deterrent activity of the extract. UV-absorption spectra of the fractions suggested that one contained cardenolides similar or identical to those reported to deter oviposition byP. rapae onE. cheiranthoides. The other active fraction evidently contains a compound that deters larval feeding but not adult oviposition. The results suggest that the chemical defense ofE. cheiranthoides depends on two types of compounds acting on separate developmental stages of the insect.     

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.     

Oviposition stimulants and deterrents regulating differential acceptance ofIberis amara byPieris rapae andP. napi oleracea

Iberis amara (Cruciferae) contains both stimulants and deterrents that are involved in regulating oviposition byPieris rapae andP. napi oleracea. The most active deterrents toP. rapae isolated from butanol extracts of the plant were found to be 2-O--d-glucosyl cucurbitacin I and 2-O--d-glucosyl cucurbitacin E. However,P. napi oleracea was behaviorally insensitive to these compounds and was only weakly deterred by other individual fractions of the butanol extract. Stimulant activity of the postbutanol water extract ofI. amara was associated with glucosinolates. The most abundant of these was identified as sinigrin, and a relatively minor component was shown to be glucoiberin. The isolated sinigrin was more stimulatory toP. rapae than was the glucoiberin-containing fraction, butP. napi oleracea was stimulated as strongly by the glucoiberin fraction, even though the concentration of this compound was much lower. The contrasting responses of the twoPieris species to the deterrents and stimulants inI. amara can explain the differential acceptance of the plant by these butterflies.     

Plant response to eggs vs. Host marking pheromone as factors inhibiting oviposition byPieris brassicae

Pieris brassicae L. butterflies secrete miriamides onto their eggs. These avenanthramide alkaloids are strong oviposition deterrents when sprayed onto a cabbage leaf. However, these compounds could not be detected in cabbage leaves from which egg batches had been removed two days after deposition and that still showed oviposition deterrency. It was concluded that the miriamides were not directly responsible for the avoidance by females of occupied leaves while searching for an oviposition site. Evidence was obtained that cabbage leaves themselves produce oviposition deterrents in response to egg batches. Fractions containing potent oviposition deterrents could be isolated from surface extracts of leaves from which previously laid egg batches had been removed. The term host marking pheromone that was used previously is not applicable in this case.     

Iridoid glycosides as oviposition stimulants for the buckeye butterfly,Junonia coenia (Nymphalidae)

Females ofJunonia coenia (Nymphalidae), a specialist on plants that contain iridoid glycosides, were found to use aucubin and catalpol, iridoid glycosides typical of a host plant,Plantago lanceolata (Plantaginaceae), as oviposition cues. Incorporating dried ground leaf material or pure iridoid glycosides into agar disks proved to be a very effective method of testing. In no-choice tests and choice tests, females laid more eggs on disks withP. lanceolata leaf material or iridoid glycosides, compared to agar controls. There was variation among individual females in preference for disks withP. lanceolata leaf material versus disks with iridoid glycosides. Females given a choice of three different concentrations of iridoid glycoside (0.2, 0.5, 1.0%) in the agar disks and a control laid more eggs on the disk with the highest concentration of iridoid glycoside.     

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.     

Host-marking pheromones in lepidoptera,with special reference to twoPieris spp

Adult females of several lepidopteran species avoid oviposition near conspecific eggs. Laboratory and field studies indicate that visual factors as well as chemical host markers may be involved in egg avoidance behavior. The oviposition-deterring pheromones (ODP) of two pierids,P. brassicae andP. rapae have been studied in considerable detail. The ODPs are probably produced in the female accessory glands and contain volatile and nonvolatile components. They are perceived by nonspecialized antennal or tarsal receptors, respectively. The ODP ofP. brassicae affects oviposition ofP. rapae females and vice versa. ODPs show promise for insect control programs, but more information on oviposition behavior in the presence of ODP under field conditions is required.     

Influence ofAmaranthus hybridus L. allelochemics on oviposition behavior ofSpodoptera exigua andS. eridania (Lepidoptera: Noctuidae)

Common pigweed,Amaranthus hybridus L., is a favorite host of the beet army worm (BAW),Spodoptera exigua L. Chemicals extracted from homogenized pigweed with distilled water, ethanol, or dichloromethane and sprayed back on pigweed deterred oviposition by the BAW. Similarly, water extracts of frass from conspecific larvae or southern armyworm (SAW) larvae,S. eridania (Cramer), fed pigweed leaves and sprayed back on pigweed plants also deterred BAW oviposition, thus confirming that deterrence was due to plant allelochemics rather than specific compounds associated with the metabolic or excretory products of the larvae. Confirmation of the presence of oviposition-deterring chemicals in pigweed was used to explain a previously observed seasonal displacement of BAW by SAW on pigweed in the field.Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.     

Heliothis subflexa (Gn.) (Lepidoptera: Noctuidae): Demonstration of oviposition stimulant from groundcherry using novel bioassay

Methanol extracts of homogenized groundcherry,Physalis angulata, leaves increased egg deposition byHeliothis subflexa (Gn.) (HS) on treated tobacco plants (a nonhost) 8.5-fold over untreated controls. In doseresponse tests using whole-leaf washes of groundcherry leaves, the threshold of positive response vs. no response to the chemical stimulant was within one log dose unit when compared to the controls. This response was consistent whether the chemical was evaluated on plants in greenhouse-cage tests or in an olfactometer using pieces of broadcloth as the oviposition substrate. The olfactometer used allows year-round study of the behavioral effects of plant allelochemics on insect oviposition behavior in a controlled environment.This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or the recommendation of its use by USDA.     

Female-produced oviposition deterrents of the cigarette beetle,Lasioderma serricorne (F.) (Coleoptera: Anobiidae)

Oviposition deterrents of the cigarette beetle,Lasioderma serricorne, were isolated from its adult body extract and found to be identical to (2S,3R,1'S)-2,3-dihydro-3,5-dimethyl-2-ethyl-6-(l-methyl-2-oxobutyl)-4H-pyran-4-one (-serricorone) and its lR-epimer (-serricorone) by spectroscopic evidence. Serricorone was previously found as one of the minor sex pheromone components of the same insect, and hence indicating bi-functional nature. The presence of two isomers in the body was proved by careful treatment. Each of them exhibited the same level of oviposition deterring activity, which was less potent than the crude body extract at an increased concentration.     

Field evaluation of synthetic compounds mediating oviposition inCulex mosquitoes (Diptera: Culicidae)

3-Methylindole (skatole), a compound shown to be an oviposition attractant/stimulant forCulex quinquefasciatus in laboratory tests, was evaluated against natural populations of mosquitoes in the field. In experiments using paired black plastic tubs that contained water treated with an attractant solution containing a mixture of 3-methylindole, 4-methylphenol, 4-ethylphenol, phenol, and indole or that contained only tap water, the attractantbaited traps received significantly moreCx. quinquefasciatus egg rafts than did the untreated tap water. Gravid female traps that were baited with attractant solution collected significantly moreCx. quinquefasciatus females than did traps containing only tap water. There was no significant difference in number ofCx. quinquefasciatus collected in the traps baited with the attractant solution compared to the traps that contained water with 3-methylindole alone, indicating that 3-methylindole was solely responsible for the attraction. In replicated experiments conducted in experimental ponds, ponds that were treated with 3-methylindole received significantly moreCx. quinquefasciatus, Cx. tarsalis, andCx. stigmatosoma egg rafts than did untreated ponds. Experimental ponds treated with 3-methylindole at two levels (0.12 and 0.6 mg/liter) were equally attractive to ovipositingCx. quinquefasciatus. This is the first record of 3-methylindole showing attractancy/stimulation to ovipositingCx. stigmatosoma andCx. tarsalis under field or laboratory conditions.     

Effects of cuticular duvane diterpenes from green tobacco leaves on tobacco budworm (Lepidoptera: Noctuidae) oviposition

Five cuticular chemical components isolated from the green leaves of tobacco introductions (TIs) and a commercial tobacco cultivar were tested for their effects on tobacco budworm,Heliothis virescens (F), oviposition in cage bioassays, and field experiments. These chemicals were sprayed onto budworm-resistant TI 1112 tobacco which produces low levels of most cuticular components. Individual duvane diterpenes (- and -4,8,13-duvatrien-1-ols and - and -4,8,13-duvatriene-1,3-diols) increased tobacco budworm egg laying on sprayed TI 1112 plants.cis-Abienol, docosanol, and docosanyl myristate were inactive.Mention of a company or proprietary product does not imply endorsement or recommendation by the U.S. Department of Agriculture. Paper No. 9702 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7601.     

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

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

Mating and oviposition byCotesia (=Apanteles) marginiventris (Hymenoptera: Braconidae) in presence of synthetic pheromone ofSpodoptera frugiperda (Lepidoptera: Noctuidae)

Two blends of fall armyworm,Spodoptera frugiperda (J.E. Smith), pheromonal components were evaluated in the laboratory for possible close-range effects on mating and oviposition byCotesia marginiventris (Cresson). Neither a two- nor a five-component blend had any significant effect on mating or parasitization ofS. frugiperda larvae by this parasitoid. Dosages of 200, 400, and 800 g were tested.     

Oviposition and feeding preferences of the southern pine coneworm (Lepidoptera: Pyralidae) for different host-plant materials and observations on monoterpenes as an oviposition stimulant

Bioassays were conducted with first-stage larvae and newly emerged females of the southern pine coneworm,Dioryctria amatella (Hulst), to detect feeding and ovipositional preferences for different host-plant materials collected between April and August. Correlations between measures of insect preferences and estimates of wood parameters indicated that larvae preferred to feed on host-plant materials with high moisture contents, low wood densities, and low monoterpene contents. Female moths, however, did not necessarily select those plant materials preferred by larvae for feeding, but tended to oviposit more frequently on materials with high monoterpene contents. A synthetic mixture of monoterpenes similar in composition to those present in fusiform rust galls of slash pine (Pinus elliottii) Englem. var.elliottii) elicited mating and oviposition behavior.Mention of proprietary products does not necessarily imply endorsement by USDA.     

Evaluation of organic infusions and synthetic compounds mediating oviposition inAedes albopictus andAedes aegypti (Diptera: Culicidae)

Oviposition responses of gravidAedes albopictus andAe. aegypti were evaluated to natural organic infusions (hay infusion, larval rearing water, and field-collected larval water) as well as compounds isolated from hay infusion (3-methylindole, 4-methylphenol, 4-ethylphenol, indole, and phenol) known to elicit oviposition inCulex mosquitoes. In laboratory bioassays, significant oviposition responses were obtained fromAe. albopictus, but not fromAe. aegypti, to dilutions of hay infusion and field water. Oviposition responses of both species were moderate to the synthetic compounds tested in the laboratory (0.01–100 µg/liter). Only 3-methylindole (0.1 µg/liter) and 4-ethylphenol (1.0 µg/liter) elicited significantly more oviposition byAe. albopictus than did well water. Of the synthetic compounds tested withAe. aegypti, only phenol (1.0 µg/liter) and 4-ethylphenol (0.1 µg/liter) elicited significantly more oviposition than did well water. Significant repellency or oviposition deterrence for both species occurred in response to at least one high concentration of most of the compounds tested. In field cage evaluations, oviposition responses byAe. albopictus were strongest to larval water and field water, moderate to hay infusion and 3-methylindole (100 µg/liter), and low to well water. A mixture of five synthetic compounds mimicking hay infusion was no more effective than 3-methylindole alone. ForAe. aegypti, oviposition responses were greatest to larval water and least to 3-methylindole. In an olfactometer, gravid females of both species oriented more to field water than to well water and onlyAe. albopictus oriented more to larval water or hay infusion than well water. In general, gravidAe. albopictus responded more strongly to oviposition stimuli than didAe. aegypti.