Induction of cytochrome P-450 activities by nicotine in the tobacco hornworm,Manduca sexta

The induction by dietary nicotine of a series of cytochrome P-450 enzyme activities was investigated in early fifth-instarManduca sexta larvae. At a low nicotine concentration in the diet (0.1 %), three of 12 midgut microsomal enzyme activities were significantly increased. At a higher concentration (0.75%) commonly found in plants of the genusNicotiana, nine of 12 activities were induced by 1.4- to 10.0-fold. Total cytochrome P-450, P-450 reductase activity, and midgut microsomal metabolism of nicotine were also increased by feeding 0.75% nicotine. Nicotine was metabolized by midgut microsomes to nicotine-1-N-oxide and cotinine-N-oxide. Fat body microsomal nicotine metabolism was low and unaffected by dietary nicotine. Isolated nerve cords were able to metabolize nicotine in vitro but this metabolism was not inducible by dietary nicotine. Nicotine-fed fifth-instarM. sexta larvae showed an increased tolerance to subsequent nicotine injection when compared to larvae fed a control diet. These results support the idea that induction of midgut cytochrome P-450-related metabolism is an adaptation ofManduca sexta to dietary nicotine.     

Differential activity and degradation of plant volatile elicitors in regurgitant of tobacco hornworm (Manduca sexta) larvae

Plants respond to insect herbivory by emitting volatile compounds that attract natural enemies of the herbivores. Biosynthesis of many of these volatiles in plants is induced by herbivore-produced compounds. Components of tobacco hornworm (THW) regurgitant were investigated for their efficacy as elicitors of corn seedling volatiles. Two components that elicited the strongest release of volatiles were isolated and identified as N-linolenoyl-L-glutamine (18:3-GLN) and N-linolenoyl-L-glutamic acid (18:3-GLU). The approximately 10 times more active 18:3-GLN, which also is found in the regurgitant of several other Lepidopteran larvae, was rapidly degraded when THW regurgitant was left at room temperature, while 18:3-GLU degraded at a much slower rate. Different dietary sources of THW and tobacco bud worm larvae, including both host and nonhost plants, did not affect the amino acid composition of the fatty acid–amino acid conjugates in the regurgitant.     

The role of leaf lipids in food selection by larvae of the tobacco hornworm,Manduca sexta

The role of leaf lipids in food plant selection by larvae ofManduca sexta was investigated by measuring preference responses in a two-choice preference test using glass fiber filter paper disks laced with extract (test) or water (control). The larvae respond to the petroleum ether extract of whole leaves of the host-plantLycopersicon esculentum (tomato) extract in a concentration-dependent manner. At natural concentration it is the most strongly stimulating extract or compound yet tested using the disk test. This response is affected by food plant experience of the larvae, suggesting stimulation by plant-specific compounds in the extract. The extract contains volatile compounds that attract the larvae. In contrast, it does not promote continued feeding on an agar-cellulose diet that incorporates the extract. Also stimulating are the extracts of leaf surfaces of two hosts,L. esculentum andSolanum pseudocapsicum, and two acceptable nonhosts,Brassicae napus andVigna sinensis, indicating the presence of nonpolar feeding stimulants at the leaf surface. However, similar leaf-surface extracts of the unacceptable plantCanna generalis were inactive, although the surface extraction process renders this plant acceptable. Leaf-surface extracts ofL. esculentum, S. pseudocapsicum, andB. napus evoke feeding responses that are qualitatively comparable to those of their corresponding leaves. However, no such parallel is found for surface extracts ofV. sinensis andC. generalis. Thus, nonpolar compounds at the leaf surface of host and some acceptable non-host-plant species strongly stimulate feeding and hence must play an important role in food selection by the tobacco hornworm.     

Feeding stimulatory and inhibitory chemicals from an acceptable nonhost plant forManduca sexta: Improved detection by larvae deprived of selected chemosensory organs

The chemical basis of feeding responses to the acceptable nonhost plantVigna sinensis (cowpea) by larvae ofManduca sexta was investigated using chemical isolation techniques directed by a novel chemosensory-based bioassay. The presence of feeding stimulatory and inhibitory compounds in leaves or leaf extracts was determined in a two-choice preference test using leaf disks or glass fiber filter paper disks laced with leaf extract as test substrate and filter paper disks laced with water as control. Larvae strongly prefer the control disks over leaf disks, indicating the presence of feeding inhibitory compounds in the leaf. An ethanol extract of both fresh and dried leaves neither stimulated nor inhibited feeding. The cause of this inactivity was examined by using larvae that respond strongly to either feeding stimulatory or inhibitory compounds due to selective chemosensory deprivation. Larvae having chemosensory organs remaining only on the maxillary palps are stimulated to feed by whole leaf disks and by the ethanol extracts. In contrast, larvae having only the medial and lateral maxillary sensilla styloconica and the epipharyngeal sensilla remaining are strongly inhibited by whole leaf disks and the ethanol extract of fresh leaves. Thus, the ethanol extract contains both feeding stimulatory and inhibitory compounds, which elicit opposite behavioral effects in unoperated larvae, therefore nullifying any stimulatory and inhibitory activity. These compounds can only be demonstrated by using discrimination-enhanced larvae in the choice tests. Further isolation of the feeding stimulatory principle inV. sinensis yielded two separate fractions of neutral compounds, suggesting at least two different chemicals belonging to two different classes: nonpolar and polar lipids. Feeding inhibitory chemicals have apparently polar properties because strong activity was found in the ethyl acetate and methanol extracts of dried leaves. The role of feeding stimulatory and inhibitory compounds in food selection ofM. sexta larvae is discussed.     

Electroantennographic and behavioral responses of the sphinx moth Manduca sexta to host plant headspace volatiles

Coupled gas chromatography with electroantennographic detection (GC-EAD) using antennae of adult female Manduca sexta was employed to screen for olfactory stimulants present in headspace collections from four species of larval host plants belonging to two families: Solanaceae—Lycopersicon esculentum (tomato), Capiscum annuum (bell pepper), and Datura wrightii; and Martyniaceae—Proboscidea parviflora. Headspace volatiles were collected from undamaged foliage of potted, living plants. GC–EAD revealed 23 EAD-active compounds, of which 15 were identified by GC-mass spectrometry. Identified compounds included aliphatic, aromatic, and terpenoid compounds bearing a range of functional groups. Nine EAD-active compounds were common to all four host plant species: (Z)-3-hexenyl acetate, nonanal, decanal, phenylacetaldehyde, methyl salicylate, benzyl alcohol, geranyl acetone, (E)-nerolidol, and one unidentified compound. Behavioral responses of female moths to an eight-component synthetic blend of selected tomato headspace volatiles were tested in a laboratory wind tunnel. Females were attracted to the blend. A comparison of responses from antennae of males and females to bell pepper headspace volatiles revealed that males responded to the same suite of volatiles as females, except for (Z)-3-hexenyl benzoate. EAD responses of males also were lower for (Z)- and (E)-nerolidol and one unidentified compound. Electroantennogram EAG dose–response curves for the 15 identified EAD-active volatiles were recorded. At the higher test doses (10–100 g), female antennae yielded larger EAG responses to terpenoids and to aliphatic and aromatic esters. Male antennae did respond to the higher doses of (Z)-3-hexenyl benzoate, indicating that they can detect this compound. On the basis of ubiquity of the EAD-active volatiles identified to date in host plant headspace collections, we suggest that M. sexta uses a suite of volatiles to locate and identify appropriate host plants.     

Effect of nitrogen and water treatment on leaf chemistry in horsenettle (Solanum carolinense), and relationship to herbivory by flea beetles (Epitrix spp.) and tobacco hornworm (Manduca sexta)

We studied the interaction between plants (horsenettle; Solanum carolinense) and herbivorous insects (flea beetles; Epitrix spp., and tobacco hornworm; Manduca sexta) by focusing on three questions: (1) Does variation in nitrogen availability affect leaf chemistry as predicted by the carbon-nutrient balance (CNB) hypothesis? (2) Does variation in plant treatment and leaf chemistry affect insect feeding? (3) Is there an interaction between the insect herbivores that is mediated by variation in leaf chemistry? For three successive years (1998-2001), we grew a set of clones of 10 maternal plants under two nitrogen treatments and two water treatments. For each plant in the summer of 2000, we assayed herbivory by hornworms in both indoor (detached leaf) and outdoor (attached leaf) assays, as well as ambient flea beetle damage. Estimates of leaf material consumed were made via analysis of digitized leaf images. We also assayed leaves for total protein, phenolic, and glycoalkaloid content, and for trypsin inhibitor, polyphenol oxidase, and peroxidase activity. Despite strong effects of nitrogen treatment on growth and reproduction, only total protein responded as predicted by CNB. Leaf phenolic levels were increased by nitrogen treatment, polyphenol oxidase activity was decreased, and other leaf parameters were unaffected. Neither hornworm nor flea beetle herbivory could be related to plant treatment or genotype or to variation in any of the six leaf chemical parameters. A negative relationship between flea beetle and hornworm herbivory was found, but was not apparently mediated by any of the measured leaf chemicals. Because leaf resistance was maintained in low nitrogen plants at the apparent expense of growth and reproduction, our results support the concept of a fitness cost of defense, as predicted by the optimal defense hypothesis.     

Insecticidal activity of hypericin towardsManduca sexta larvae

The toxic effect of hypericin (HYP), a photodynamic quinone that occurs in certain species of the genusHypericum, towardsManduca sexta third-instar larvae was investigated. The LD₅₀ of purified hypericin was 16 g/g larval initial fresh weight in constant light (22 W/m²). Reduced irradiance resulted in decreased mortality. Sublethal applications retarded larval growth (body fresh weight) in a dose-dependent manner. Toxicity had an absolute light dependence at the dose levels used, the active wavelengths being >500 nm. The phototoxic effect was rapidly lost when larvae were maintained in darkness (>8 hr) prior to irradiation. However, the potential for light-dependent mortality was retained if larvae were starved prior to irradiation.     

Diet-related differences in the cuticular lipids ofManduca sexta larvae

Cuticular lipid components were examined from fourth-instar larvae ofManduca sexta reared on artificial diet or growing plants. The plants used were potato, tobacco, and tomato grown in pots in a greenhouse. Twenty-eight components made up the bulk of the lipids, but there were significant differences in the proportions of them present in insects reared on the different diets. In the case of some insect cuticular lipid components, there was an obvious relationship with the surface components of the plant, but generally this relationship was weak. Nonetheless, the differences may have ecological relevance, as indicated by preliminary work on predation.     

Field tests of syntheticManduca sexta sex pheromone

In field experiments traps were baited with live females or with a two-, four-, or eight-component blend of the 16-carbon aldehydes previously identified as components of the sex pheromone emitted by femaleManduca sexta moths. The blends were formulated on rubber septa. Traps baited with a blend of all eight aldehydes captured moreM. sexta males than any other treatment. Septa loaded with 600 g of the eight-component blend were attractive to males for about seven days in the field. Septa loaded with the eight-component blend and stored in a refrigerator at 4°C for a year released the conjugated diene and triene aldehydes at the same rate as freshly prepared septa and were equally attractive in the field.     

The protective effect of antioxidants to a phototoxin-sensitive insect herbivore,Manduca sexta

Photo-activated plant secondary compounds have been shown to be toxic to many organisms including insects. Insect defenses include behavioral mechanisms such as light avoidance, as well as specific biochemical defenses such as antioxidants and antioxidant enzymes. These antioxidant defenses eliminate or quench the deleterious singlet oxygen and free radicals formed by these phototoxins. In this paper we examined the role of dietary antioxidants in protecting the phototoxin-sensitive insect herbivoreManduca sexta. Elevated dietary levels of the lipid-soluble antioxidants-carotene and vitamin E resulted in a concentration-dependent reduction in the mortality associated with treatment ofM. sexta larvae with the phototoxic thiophene-terthienyl. Elevated levels of dietary ascorbic acid had no effect, whereas reduced levels greatly increased the toxicity of-terthienyl. Tissue levels of antioxidants were shown to increase substantially in larvae fed antioxidant-supplemented diets. The results suggest that the ability to absorb and utilize plant-derived antioxidants could be an important defense against photo-activated plant secondary compounds and may have allowed some insects to exploit phototoxic plants.     

An Oviposition Stimulant for Spicebush Swallowtail Butterfly, Papilio troilus, from Leaves of Sassafras albidum

Female butterflies of the spicebush swallowtail, Papilio troilus, are specialists, ovipositing on plants in the family Lauraceae. Column chromatography and HPLC were used to isolate an oviposition stimulant from the leaves of one of its hosts, Sassafras albidum. The stimulant was identified as 3-trans-caffeoyl-muco-quinic acid on the basis of FAB-MS and ¹H NMR spectra as compared to a compound previously isolated from another plant. It was not active alone, but it increased the oviposition activity of butterflies when combined with other stimulant(s) at a concentration of 7 ng/mm² leaf surface area. Other caffeoylquinic acid isomers tested did not have this effect. This is the first report of a swallowtail contact oviposition stimulant from a plant in the family Lauraceae.     

Effect of day length and light intensity on 2-tridecanone levels and resistance inLycopersicon hirsutum f.glabratum toManduca sexta

First instarManduca sexta (L.) larvae confined on foliage fromLycopersicon hirsutum f.glabratum (accession PI 134417) plants grown under a long-day regime exhibited greater mortality than larvae on foliage from plants grown under a short-day regime. 2-Tridecanone, a toxin important in the insect resistance of PI 134417, was significantly more abundant in the foliage of plants grown under the long-than the short-day regimes. Light intensity influenced neither 2-tridecanone levels nor the expression of resistance. The density of glandular trichomes, which secrete 2-tridecanone, was influenced by an interaction between day length and light intensity.Lepidoptera: Sphingidae.Paper no. 6503 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650.     

Effects of comsumption of high and low nicotine tobacco byManduca sexta (Lepidoptera: Sphingidae) on survival of gregarious endoparasitoidCotesia congregata (Hymenoptera: Braconidae)

The significance of nicotine in the three trophic level interaction involving tobacco (Nicotiana tabacum), the tobacco hornworm (Manduca sexta), and the parasitoidCotesia congregata was investigated in field plots of two varieties of tobacco which had about a 10-fold difference in their nicotine content. WhileM. sexta mortality, rates of parasitism byC. congregata, and the total number ofC. congregata larvae produced per host were similar on each of the two varieties, the number of parasitoids reaching adult-hood on the low nicotine treatment was nearly twice that on the high nicotine treatment. This difference was due to the significantly greater proportion of parasitoid larvae which failed to emerge from the host or that died prior to pupation after emerging from hosts which fed on the high nicotine variety. A greater proportion of larvae from hosts which fed on the low nicotine tobacco died as pupae. No treatment differences occurred for either sex of the parasitoid in individual dry weight, longevity, or pupal development time, except that female pupal duration was prolonged in the high nicotine treatment. These results support the suggestion that plant allelochemicals, which may function to provide plant resistance against pest herbivores, can be detrimental to natural enemies of the pest.Scientific article No. A4148 of the Maryland Agricultural Experiment Station, Department of Entomology. Research supported by NSF grant BSR-84-00614.     

Tolerance to Thiarubrines from Ambrosia chamissonis by the Rare Moth Lasionycta wyatti

The larvae of a rare noctuid moth, Lasionycta wyatti, were found to be associated with the stems and roots of Ambrosia chamissonis, a species found along sandy beaches in western North America. High-performance liquid chromatographic (HPLC) analyses of L. wyatti larvae and their frass revealed significant amounts of thiarubrines, secondary compounds characteristic of this host plant. Artificial diet studies showed that thiarubrines were well tolerated by these larvae. These same diets, however, were toxic to larvae of Manduca sexta and inhibited larval growth of Spodoptera litura. Even in the presence of simulated sunlight, artificial diets containing thiarubrines did not affect larvae of L. wyatti. However, dietary incorporation of thiophenes, natural UV-activated breakdown products of thiarubrines, was deleterious to larvae of L. wyatti.     

Oviposition Stimulant for a Gall-Inducing Sawfly, Euura lasiolepis, on Willow is a Phenolic Glucoside

The ovipositional stimulation for the shoot galling sawfly, Euura lasiolepis, whose natural host is the North American willow, Salix lasiolepis, was studied in response to its original host, Finnish willows, poplar species, and individual phenolic glucosides and fractions. The major phenolic glucosides in the natural host were salicortin and tremulacin, which were also the major components in the Finnish S. purpurea and S. rosmarinifolia. The natural host, S. lasiolepis, was chosen over six Finnish willow species in multiple-choice tests. Multiple-choice tests with six Finnish willow species and one Finnish popular species showed that S. rosmarinifolia was significantly more attractive than any of the other species by at least 16-fold. No-choice testing with phenolic glucosides showed the tremulacin fraction to be the significant ovipositional stimulant, which elicited probes with the ovipositor at least 19 times more frequently than other glucosides. A tremulacin fraction at 1.5% elicited significantly more probes than a tremulacin fraction tested at 0.4%. In addition, tremulacin was the only glucoside to induce prolonged and repeated probing with the ovipositor, lasting a mean of 6.4 sec per bout per female. Only single probes lasting a fraction of a second were observed in response to other glucosides. This is the second study showing a direct effect of a phenolic glucoside as an ovipositional stimulant for a sawfly, and it also suggests the existence of a mechanism that enables females to respond positively in a graded way to shoot length heterogeneity in host plants and populations.     

Insect hydrocarbons

Standard mono- and dimethyl-branched hydrocarbons were synthesized from substituted thiophenes for the purpose of comparing their mass spectra to those of hydrocarbons isolated from the tobacco hornworm,Manduca sexta L. The mass spectra of the standard compounds confirmed the structures of the partially characterized natural products.     

Biochemical investigations of antibiosis material in leaf exudate of wildNicotiana species and interspecific hybrids

A model system involving severalNicotiana species containing novel nicotine alkaloids was used to study heritability and expression of alkaloid production in leaf trichomes. The three species that comprise the section Repandae (N. repanda, N. stocktonii, andN. nesophila) were hybridized with eitherN. tabacum orN. sylvestris (neither of which producesN-acylnornicotine). The progeny of the hybrid with sylvestris produced theN-acylnornicotines at a level found in the Repandae parent.Nicotiana repanda was crossed toN. tabacum, and the F₂ progeny produced the alkaloid at the same level as the original Repandae parent. Inheritance of the ability to acylate nornicotine in Repandae species is inherited in hybrids in a dominant manner These and other data obtained suggest that theN-acyltransferase that acylates nornicotine in Repandae species inherited in hybrids is in a dominant manner and that the regulatory sequence(s) for the gene is expressed in leaf trichomes when the gene is in a foreignNicotiana background.     

Floral CO2 Reveals Flower Profitability to Moths

The hawkmoth Manduca sexta (Lepidoptera: Sphingidae), an experimentally favorable Lepidopteran that is highly sensitive to carbon dioxide (CO2), feeds on the nectar of a range of flowering plants, such as Datura wrightii (Solanaceae). Newly opened Datura flowers give off dramatically elevated levels of CO2 and offer ample nectar. Thus, floral CO2 emission could indicate food-source profitability. This study documents that foraging Manduca moths prefer surrogate flowers that emit high levels of CO2, characteristic of newly opened Datura flowers. We show for the first time that CO2 may play an important role in the foraging behavior of nectar-feeding insects.     

Chemical Defenses of Cryptic and Aposematic Gastropterid Molluscs Feeding on their Host Sponge Dysidea granulosa

Numerous opisthobranchs are known to sequester chemical defenses from their prey and use them for their own defense. Information on feeding biology is critical for understanding the ecology and evolution of molluscs, yet information on feeding biology is still scarce for many groups. Gastropterid molluscs are often found on sponges, but there is controversy as to whether they are true sponge feeders. On Guam, we found the gastropterids Sagaminopteron nigropunctatum and S. psychedelicum on the sponge Dysidea granulosa. They seem to rely on contrasting defense strategies as S. psychedelicum has vivid colors, consistent with the warning coloration found in many chemically defended opisthobranchs, whereas S. nigropunctatum is highly cryptic on the sponge. S. nigropunctatum is avoided by the pufferfish Canthigaster solandri in aquarium assays. We analyzed the secondary metabolites of the two species and found that both share polybrominated diphenyl ethers (BDEs) with their host sponge D. granulosa. S. psychedelicum and S. nigropunctatum sequester the major BDE in the sponge and accumulate it in the mantle at approximately the same concentration as in the sponge (4.03 and 2.37%, respectively), and concentrate it in their parapodia at over twice the sponge concentration (7.97 and 10.10%, respectively). We also detected trace amounts in the mucus secretion of S. psychedelicum, and quantified significant amounts in the mucus (1.84%) and egg masses (2.22%) of S. nigropunctatum. Despite contrasting color patterns displayed by the two gastropterid species, they seem to share a similar chemical defense strategy, i.e., they feed on D. granulosa and accumulate the major BDE of the sponge in their tissues.