Antifeedant and toxic effects of sesquiterpenes fromSenecio palmensis to colorado potato beetle

A bioassay-guided fractionation of the aerial parts ofSenecio palmensis resulted in the isolation of two sesquiterpenes, 2,10-bisaboladien-1-one and 11-acetoxy-5-angeloyloxy-silphinen-3-one. The bisabolene and the silphinene represented 0.012% and 0.024% of the plant dry weight, respectively. Both compounds showed antifeedant activity againstLeptinotarsa decemlineata larvae and adults in short-term choice and no-choice bioassays. Both compounds were also tested against different species of phytopathogenic fungi. The beetles were more sensitive to these compounds in choice than in no-choice assays, with a gradient of increasing sensitivity from second instars to adults. Bisabolene was 45 times less active as an antifeedant than juglone, which was tested as a positive control. The silphinen was more active than the bisabolene, with a range of activity similar to juglone. Furthermore, exposure of fourth instars to these compounds over a 24-hr period resulted in reduced feeding and growth rates. To distinguish between antifeedant and toxic effects, growth efficiencies were calculated as the slope of the regression of relative growth rate on relative consumption rate. The comparison of these results with those of antifeedant simulation and contact toxicity bioassays indicates that feeding inhibition is the primary mode of action of the bisabolene, while the silphinene shows both antifeedant and toxic effects.     

Insect antifeedant activity associated with compounds isolated from species ofLonchocarpus andTephrosia

The antifeedant activity of a series of 21 chalcones, flavanes, and flavanones isolated from the generaLonchocarpus andTephrosia (Leguminosae) was assessed by behavioral and electrophysiological bioassays against larvae ofSpodoptera littoralis andS. exempta. The antifeedant activity is related to the molecular structure of the compounds, and possible modes of interaction with the insect taste receptors are discussed.     

Antifeedant activity againstSpodoptera litera larvae and [13C]NMR spectral assignments of the meliatoxins

The tetranortriterpene, meliatoxin A₂, induced a significant level of antifeedant activity inSpodoptera litura F. larvae, while meliatoxin B₁, which lacks the ring D epoxide, was much less active. Complete [¹³C]NMR assignments for both compounds derived from homo- and heteronuclear short- and long-range couplings are given.     

Patterns of Bioactivity and Herbivory on Nothofagus Species from Chile and New Zealand

Nothofagus species from Chile and New Zealand were surveyed in the field for invertebrate abundance and leaf feeding damage and in the laboratory for antifeedant activity against leafrollers (Ctenopsteustis obliquana, Epiphyas postvittana), deterrent activity against pea aphid (Acyrthosiphon pisum), insect growth regulatory activity (Oncopeltus fasciatus), nematicidal activity (Caenorhabditis elegans), antibiotic activity (Pseudomonas solanaciarium), and general toxicity. A data matrix indicated that N. alessandri and N. pumilio most likely have a chemical barrier to insect attack as leaves showed low faunal abundance, low herbivory, and activity in the leafroller antifeedant, aphid deterrent, and nematicidal assays. A chemical examination of N. alessandri that used the leafroller antifeedant test to guide the separation yielded an active fraction containing the flavonoid, galangin, and the stilbene, pinosylvin, which appear to act in concert to deter leafroller feeding. The discovery of the phytoalexin, pinosylvin, in the leaves, raises the possibility that Nothofagus in general, and N. alessandri in particular, may have induced chemical defense mechanisms.     

Examination of North American bison saliva for potential plant growth regulators

A series of laboratory bioassays were utilized to test for the presence of potential plant growth factors in saliva from a large native ungulate, the North American bison (Bison bison L.). Whole saliva enhancedAvena coleoptile growth at high pH, whether alone or in combination with indoleacetic acid (IAA). However, this enhancement was a result of salts in the saliva (primarily NaHCO₃) rather than of other compounds acting hormonally, enhancing IAA activity, or inhibiting IAA oxidase activity as possibly occurs with some insect salivas. Additionally, the absence of detectable cytokinins in the saliva was indicated by its failure to enhance cucumber cotyledon expansion. This suggests that biochemical control of plant growth by salivary compounds following grazing is probably not an important component of this ruminant's interactions with its food plants, as has been suggested for some herbivores.     

Antifeedant Effects of Some Novel Terpenoids on Chrysomelidae Beetles: Comparisons with Alkaloids on an Alkaloid-Adapted and Nonadapted Species

Structure–dose–feeding deterrency relationships were compared between the Colorado potato beetle, Leptinotarsa decemlineata (Say), and the western corn rootworm, Diabrotica virgifera virgifera LeConte, using 15 alkaloids, terpenoids, and phenolic derivatives. The former species, a specialist herbivore on selected alkaloid-rich Solanaceae species, was on average 100-times less sensitive to the antifeedant effects of alkaloids, but more similarly sensitive to the terpenoids and phenolics than the latter species, a generalist flower herbivore predominantly on Graminae, Cucurbitaceae, and Compositae species. Antifeedant ED₅₀ values for the potato beetle and corn rootworm, each from closely related subfamilies of Chrysomelidae, ranged over four orders of dose magnitude among the 15 compounds with major species differences in stereosensitivity to -hydrastines and analog sensitivity with the silphinenes. Extremes in sensitivity ranged from silphinene, a rare tricyclic sesquiterpene that is 53 times more active on the potato beetle to aconitine, which is 430 times more antifeedant to the corn rootworm. Among silphinene and its two hydrolysis derivatives, there was not a strong correlation between antifeedant potency and injected toxicity for the two beetle species, but there was correlation between behavioral activity and galeal taste cell electrophysiological threshold and frequency responses. That all of the established GABA- and glycinergic compounds tested were antifeedant for both species suggests a shared molecular mechanism for antifeedant taste chemoreception in these divergent Chrysomelidae species. Moreover, the wide differences in antifeedant sensitivities among these and other chrysomelids to a suite of ligand-gated ion channel antagonists implicate a common protein neuroreceptor type with extraordinary heterogeneity in beetle taste.     

Antifeedants to Armyworms, Spodoptera lituraand Pseudaletia separata,from Bitter Gourd Leaves, Momordica charantia

Bitter gourd, Momordica charantia, was less palatable to two species of armyworms, Spodoptera litura and Pseudaletia separata, than two other cucurbitaceous plants. A methanol extract of M. charantia leaves inhibited feeding of the armyworm larvae. The two most active fractions obtained by silica gel chromatography were purified by HPLC. Momordicine II, a triterpene monoglucoside, was identified as an antifeedant compound from the more active of these fractions. The second active fraction led to the isolation of a new triterpene diglucoside. Fresh leaves of M. charantia contained ca. 0.3% of momordicine II. Momordicine II showed significant antifeedant effects on P. separata at concentrations of 0.02, 0.1, and 0.5% in artificial diets. Momordicine II caused a significant feeding reduction in S. litura only at the highest concentration (0.5%) tested. The difference in feeding response of the two armyworms to momordicine II may be related to the diversity in their host range.     

Some chemical bases for gypsy moth,Lymantria dispar,larval rejection of green ash,Fraxinus pennsylvanica,foliage as food

Green ash is one of the few tree species rejected as food by larvae of the generalist gypsy moth,Lymantria dispar L. (Lepidoptera: Lymantriidae). Such rejection is based especially on chemicals present in green ash foliage. The gypsy moth larval feeding-inhibitory activity is contained in the ethyl acetate extractables of green ash foliage. Three representative columnchromatographed fractions of the extractables contained antifeedant activity. Individual fractions showed weaker antifeedant activity compared to the total ethyl acetate extractables. Acid hydrolysis of the extractables destroyed antifeedant activity and yielded feeding stimulant activity at higher concentrations. The aqueous extractables were not feeding-inhibitory. Compounds in the green ash extractables were separated by TLC, HPLC, CC, and GC.     

Structural Diversity and Defensive Properties of Norditerpenoid Alkaloids

We have tested the insect antifeedant and toxic activity of 43 norditerpenoid alkaloids on Spodoptera littoralis and Leptinotarsa decemlineata including eserine (physostigmine), anabasine, and atropine. Antifeedant effects of the test compounds were structure- and species-dependent. The most active antifeedants to L. decemlineata were 1,14-diacetylcardiopetaline (9) and 18-hydroxy- 14-O-methylgadesine (33), followed by 8-O-methylconsolarine (12), 14-O-acetyldelectinine (27), karakoline (7), cardiopetaline (8), 18-O-demethylpubescenine (13), 14-O-acetyldeltatsine (18), takaosamine (21), ajadine (24), and 8-O-methylcolumbianine (6) (EC50 < 1 microg/cm2). This insect showed a moderate response to atropine. S. littoralis had the strongest antifeedant response to 24, 18, 14-O-acetyldelcosine (19), and delphatine (29) (EC50 < 3 microg/cm2). None of the model substances affected the feeding behavior of this insect. The most toxic compound to L. decemlineata was aconitine (1), followed by cardiopetalidine (10) (% mortality > 60), 14-deacetylpubescenine (14), 18-O-benzoyl-18-O-demethyl-14-O-deacetylpubescenine (17), 14-O-acetyldelcosine (19), 14-deacetylajadine (25) and methyllycaconitine (30) (% mortality > 45). Orally injected S. littoralis larvae were negatively affected by 1, cardiopetaline (8), 10, 1,14-O-acetylcardiopetalidina (11), 12, 14, 1,18-O-diacetyl-19-oxo-gigactonine (41), olivimine (43), and eserine in varying degrees. Their antifeedant or insecticidal potencies did not parallel their reported nAChR binding activity, but did correlate with the agonist/antagonist insecticidal/antifeedant model proposed for nicotininc insecticides. A few compounds [14, tuguaconitine (38), 14-demethyldelboxine (40), 19, dehydrodelsoline (36), 18-O-demethylpubescenine (13), 41, 9, and delcosine (23)] had selective cytotoxic effects to ward insect-derived Sf9 cells. None were cytotoxic to mammalian CHO cells and none increased Trypanosoma cruzi mortality. The selective cytotoxic effects of some structures indicate that they can act on biological targets other than neuroreceptors.     

Rejection of host plant by larvae of cabbage butterfly: Diet-dependent sensitivity to an antifeedant

Garden nasturtium,Tropaeolum majus (Tropaeolaceae), is an acceptable host plant for the cabbage butterfly,Pieris rapae. Eggs are readily laid on the plant and hatching larvae feed and develop into normal pupae and adults. However, when second- to fifth-instar larvae were transferred from cabbage to nasturtium, they refused to feed and starved to death. Similar results were obtained when larvae were transferred from other host plants to nasturtium. However, larvae that were reared on nasturtium readily accepted cabbage as a new host plant. We have demonstrated the presence of strong antifeedants in nasturtium foliage and identified the most prominent active compound as chlorogenic acid. However, larvae reared on nasturtium had limited sensitivity, and larvae reared on a wheat germ diet were completely insensitive to the antifeedants. Larvae apparently develop sensitivity to the deterrent as a result of feeding on other host plants, whereas continuous exposure to the deterrent causes habituation or suppression of sensitivity development. The results demonstrate that dietary experience can dramatically affect the response of an insect to a potentially antifeedant compound in a plant.     

A new bioassay for testing plant extracts and pure compounds using red flour beetleTribolium castaneum Herbst

We designed a new bioassay to test plant extract activity against stored product pests. Plant compounds were added to feed disks composed of wheat flour and yeast and fed to the red flour bettle (Tribolium castaneum). By measuring insect mass, disk mass, and insect mortality over time it was possible to calculate a phagodepression index, an antifeedant index, the amount of treatment chemical ingested by the beetles, the mortality rate, and the efficiency of conversion of ingested food. The assay was performed for 60 hr to allow for possible habituation effects and to discriminate between phagodeterrency and physiological stress caused by treatments. - and -Pinene, eugenol, kaurenic acid, sparteine, essential oils ofMinthostachis mollis andMelaleuca quinquenervia, and extracts ofSapindus saponaria were tested. Using this assay we detected the presence of both phagodepressant and phagostimulant compounds inS. saponaria extracts, and we quantified the pronounced effects of sparteine onT. castaneum.     

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.     

NMR Metabolomics of Thrips (<Emphasis Type="Italic">Frankliniella occidentalis</Emphasis>) Resistance in <Emphasis Type="Italic">Senecio</Emphasis> Hybrids

Western flower thrips (Frankliniella occidentalis) has become a key insect pest of agricultural and horticultural crops worldwide. Little is known about host plant resistance to thrips. In this study, we investigated thrips resistance in F ₂ hybrids of Senecio jacobaea and Senecio aquaticus. We identified thrips-resistant hybrids applying three different bioassays. Subsequently, we compared the metabolomic profiles of these hybrids applying nuclear magnetic resonance spectroscopy (NMR). The new developments of NMR facilitate a wide range coverage of the metabolome. This makes NMR especially suitable if there is no a priori knowledge of the compounds related to herbivore resistance and allows a holistic approach analyzing different chemical compounds simultaneously. We show that the metabolomes of thrips-resistant and -susceptible hybrids differed considerably. Thrips-resistant hybrids contained higher amounts of the pyrrolizidine alkaloids (PA), jacobine, and jaconine, especially in younger leaves. Also, a flavanoid, kaempferol glucoside, accumulated in the resistant plants. Both PAs and kaempferol are known for their inhibitory effect on herbivores. In resistant and susceptible F ₂ hybrids, young leaves showed less thrips damage than old leaves. Consistent with the optimal plant defense theory, young leaves contained increased levels of primary metabolites such as sucrose, raffinose, and stachyose, but also accumulated jacaranone as a secondary plant defense compound. Our results prove NMR as a promising tool to identify different metabolites involved in herbivore resistance. It constitutes a significant advance in the study of plant–insect relationships, providing key information on the implementation of herbivore resistance breeding strategies in plants. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Behavioral and electrophysiological study of antifeedant mechanisms associated with polyhydroxy alkaloids

Eleven polyhydroxy alkaloids of plant origin were tested for anti-feedant effects against larvae of the lepidopteransSpodoptera littoralis, Spodoptera frugiperda, Heliothis virescens, andHelicoverpa armigera. Data from behavioral and electrophysiological investigations were correlated to reveal information on the mode of action of the antifeedants. The pyrrolidine DMDP was an effective antifeedant for all four species, whereas the piperidines fagomine and XZ-1 and the pyrrolizidine alexine were all ineffective as antifeedants. The activity of the pyrrolidines CYB-3 and DAB-1, the piperidines DNJ, DMJ, and BR1, and the bicyclic octahydroindolizine castanospermine varied among species. The investigation focuses on the structural similarities between some of the alkaloids and some common phagostimulatory sugars and illustrates a neural interaction involving the neurons that are differentially responsive to alkaloids and sugars. InS. littoralis, the neurons responding specifically to the alkaloids DMDP, DAB-1, and castanospermine and to the sugars fructose, sucrose, and glucose are more active when the compounds are applied singly than when an alkaloid and a sugar are applied together. The implications for the occurrence and functioning of different sugar receptor sites are discussed.     

Influence of trichome exudates from species ofLycopersicon on oviposition behavior ofHeliothis zea (Boddie)

Cage experiments revealed that accessions of the wild tomato speciesLycopersicon hirsutum were preferred sites for oviposition byHeliothis zea. Hexane extracts from the leaves ofL. hirsutum were also preferred sites of oviposition in choice experiments among extracts from severalLycopersicon species. Extracts ofL. hirsutum were still biologically active several days after application, indicating that the phytochemical(s) involved are relatively stable and of low volatility. Gas Chromatographic analysis of leaf hexane extracts from 12 different accessions of theL. hirsutum complex and three tomato cultivars revealed substantial qualitative and quantitative variation in the chemical composition of these extracts. Comparison of these results with extract oviposition studies implicate a group of structurally related compounds as the active agents. Mass spectroscopy has tentatively identified these compounds as sesquiterpenes with the chemical formula C₁₅H₂₂O₂. These compounds are apparently synthesized and secreted from glandular trichomes on the leaf surface. These phytochemicals did not stimulate ovipositional behavior in females of the cabbage looper,Trichoplusia ni. The existence of genetic variation for the presence and amount of kairomones that serve as cues for insect orientation and oviposition could be utilized in a breeding program to develop tomato cultivars with genetically modified allelochemic profiles that would disrupt the sequential behavioral processes of insect host-plant selection.     

Isolation and structure determination of new nematicidal triglyceride fromArgemone mexicana

The petroleum ether extract ofArgemone mexicana seeds was found to possess nematicidal activity against the plant parasitic nematodeMeloidogyne incognita. The active nematicidal compound has been isolated from the crude extract by column Chromatographic techniques and purified by TLC. Chemical structure has been determined by chemical and spectro-scopic methods to be that of a triglyceride, sn-glycerol-1-eicosa-9, 12-dien-oate-2-palmitoleate-3-linoleate.     

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.     

Action of Extracts of Apiaceae on Feeding Behavior and Neurophysiology of the Field Slug Deroceras reticulatum

A systematic examination was made of the plant family Apiaceae (Umbelliferae) in which extracts of 33 species, representing 32 genera, were screened for antifeedant activity against the field slug Deroceras reticulatum by using an electrophysiological recording assay. In this assay, the olfactory sensory epithelium of the posterior tentacle of the slug was exposed to volatile components of the plant extracts presented in an airstream, and any subsequent activity of the olfactory nerve was recorded. Extracts of 22 species elicited a range of nervous activity in the preparation. A feeding bioassay was used to measure any change in consumption when extracts were added to a standard food. Statistical analysis of data obtained from both electrophysiological traces and the feeding bioassays identified extracts of Petroselinum crispum, Conium maculatum, and Coriandrum sativum as being the most neuroactive as well as the most antifeedant.     

Factors contributing to resistance of exotic maize populations to maize weevil,Sitophilus zeamais

Factors contributing to resistance of maize to infestation by the corn weevilSitophilus zeamais were investigated in four populations of indigenous and improved maize from Belize. Resistance was related to the antifeedant properties of grain as well as sugar content and mechanical hardness. Grain extracts of all populations of maize significantly reduced insect feeding on treated artificial diets when compared to control diets. Consumption of treated diets was negatively correlated with phenolic content of the grain extract. An analysis by GC-MS indicated that ferulic acid andp-coumaric acid were the principal phenolics present in the extracts, and insect feeding was strongly deterred when pure substances were added to insect diets. Fluorescence associated with ferulic acid and related compounds in grain sections was located in the pericarp and aleurone layer and was especially intense in the most resistant grain variety.Contribution No. 838.