Feeding Deterrence and Toxicity of Neem Triterpenoids

The neem tree, Azadirachta indica, produces a plethora of triterpenoids. One of these is azadirachtin, a complex compound with powerful insecticidal properties. The biosynthesis of azadirachtin starts with a steroid precursor and proceeds via two levels of structural complexity, i.e., furan ring formation and C-ring opening, after which further modifications yield azadirachtin, Antifeedant and toxic properties of azadirachtin against insects were compared with those of various compounds from the two lower levels of structural complexity. To larvae of the moth Spodoptera littoralis, several compounds from the lower structural levels showed strong antifeedant activity, comparable to that of azadirachtin. Nymphs of the locust Schistocerca gregaria in antifeedancy tests were more sensitive to azadirachtin than to the other compounds tested. To Spodoptera littoralis, Schistocerca gregaria, and the bug Oncopeltus fasciatus, azadirachtin was very toxic, exerting severe growth and molt disrupting effects. In contrast, compounds from the lower structural levels than azadirachtin showed virtually no toxicity to these three insects. Thus, although several simpler triterpenoid structures other than azadirachtin have insect antifeedant activity, the combination of strong antifeedant and toxic properties of azadirachtin may provide the more important chemical protection of the neem tree. Our data reveal no explicit link between antifeedant activity and toxicity of individual neem triterpenoids.     

Insect antifeedant and growth-regulating activities of Salannin and other c-seco limonoids from neem oil in relation to Azadirachtin

The antifeedant and insect growth-regulating activities of salannin, nimbin, and 6-deacetylnimbin, in comparison with azadirachtin-A, have been studied againstSpodoptera litura, Pericallia ricini, andOxya fuscovittata. Salannin deterred feeding, delayed molt by increasing larval duration, caused larval and pupal mortalities, and decreased pupal weights in the two lepidopterans. Salannin also caused molt delays and nymphal mortalities inOxya fuscovittata. The role of salannin and other compounds in conferring bioactivity, along with azadirachtin-A, to neem oil/neem seed extracts is emphasized.     

Antifeedant activity of extracts from neem,Azadirachta indica,to strawberry aphid,Chaetosiphon fragaefolii

Leaf disk choice test bioassays demonstrated that formulated neem seed oil (NSO) was equally deterrent to first- and third-instar nymphs and adult strawberry aphids,Chaetosiphon fragaefolii (Cockerell). Concentrations of NSO resulting in 50% feeding deterrence were approximately 1.1% for this species. The rapid disruption of aphid feeding (<1 hr) was not related to the presence of the limonoid azadirachtin, and deterrence likely results from the combined activity of several compounds. Activity toC. fragaefolii disappeared within 12–24 hr following application to strawberry in the greenhouse. NSO was deterrent to only half of the six aphid species tested. The antifeedant properties of neem do not appear to contribute significantly to the control of aphids and the viruses they transmit.     

Behavioral and Physiological Effects of Neem (Azadirachta indica) Seed Kernel Extracts on Larval Parasitoid, Bracon hebetor

An aqueous suspension and an ethanolic extract of neem seed kernel (NSK) at 0.3, 0.6, 1.2, 2.5, and 5.0% concentrations were tested for ovipositional deterrency, feeding deterrency, toxicity, sterility, and insect growth regulatory effects on a larval parasitoid, Bracon hebetor. Neither NSK extracts delivered in the food or by contact influenced the B. hebetor oviposition (parasitization). They also did not cause parasitoid sterility through feeding, but they showed feeding deterrent effects for a limited period. Parasitoid eggs and pupae also were unaffected by the extracts tested. The parasitoid larvae, however, were killed by feeding contaminated host larvae and also through contact with neem extracts. Thus, a minimum safety period is suggested for inundative releases of B. hebetor in integrated pest management.     

Systemic induction of feeding deterrents in cotton plants by feeding ofSpodoptera SPP. Larvae

Cotton,Gossypium hirsutum L., has been shown to exhibit systemic induced resistance to arthropods under certain conditions. We conducted experiments to determine the effects of previous feeding ofSpodoptera exigua Hübner andSpodoptera littoralis (Boisd.) larvae on feeding behavior, growth, and survival of larvae subsequently feeding on cotton. In one feeding choice test,S. exigua larvae preferred young leaves from undamaged control plants to undamaged young leaves from a previously damaged plant. Feeding deterrence was noticeable after only 6 hr of initial feeding damage by larvae, and there was almost complete deterrence after 30 and 54 hr of continuous feeding. In a second feeding choice test,S. littoralis larvae fed more on mature leaves from undamaged control plants than on undamaged mature leaves from previously damaged plants. In no-choice tests, third instars ofS. littoralis fed undamaged young leaves from damaged plants did not gain weight and died by the seventh day, whereas larvae fed young leaves from undamaged control plants gained weight and pupated within 11 days. Sixth instars ofS. littoralis fed either old damaged leaves, old undamaged leaves, or young undamaged leaves all from previously damaged plants gained weight slowly and took more than 12 days to pupate, whereas larvae fed young leaves from undamaged plants gained weight rapidly and pupated within five days of the beginning of the experiment.     

Influence of diel rhythm and brain hormone on pheromone production in two lepidopteran species

Heliothis armigera andSpodoptera littoralis females produced sex pheromone during their scotophase. Maximum levels of pheromone in Heliothisarmigera were extracted from 2- to 3-day-old females during their 4.5-hr scotophase. On ligation between the head and the thorax of both species, normal pheromone production was inhibited. Homogenates of brains (consisting of brains, subesophageal ganglia, corpora cardiaca, and corpora allata) from both species induced a normal level of pheromone production by ligated females. Moreover, inS. littoralis, female brain hormone induced the production of the two components (Z)-9- and (Z, E)-9,11-tetradecenyl acetate tested by gas chromatography. Partial separation of brain homogenates, using low pressure C18-silica reversed-phase columns, produced a stable and active hormone source that gave a dose-dependent response. This hormone was found in brain extracts of both male and femaleH. armigera andS. littoralis females. In addition,S. littoralis induced pheromone production byH. armigera ligated females.     

Effect of meliaceous seed extracts on growth and survival ofSpodoptera frugiperda (J.E. Smith)

Hexane and ethanol extracts of seeds from 10 plant species (including neem—Azadirachta indica A. Juss.) of the family Meliaceae were incorporated into artificial diet at various doses and fed to fall armyworm [Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)] larvae in nochoice tests. All produced significant mortality, reduced larval growth rate, increased time to pupation, or all three, at some concentration. The two highest doses of all of the ethanol extracts caused 100% mortality before pupation, but the hexane extracts tended to be less effective.Aglaia cordata Hiern. ethanol extract was as potent as the comparable neem seed extract at virtually all levels, and its hexane extract was active at much lower concentrations than the neem extract was. The sublethal effects (slower growth and increased time to pupation) were usually detectable at lower doses of extract than mortality was.Mention of firm or product names does not imply recommendation or endorsement by USDA over others not mentioned.     

Allelopathic effects ofPolygonum aviculare L.

We previously reported thatPolygonum aviculare has a strong allelopathic action againstCynodon dactylon (L.) Pers. and other test species. Moreover, we found that several phenolic compounds appeared to be important allelochemics in this activity. We have now isolated other potential inhibitors fromP. aviculare residues and soil underPolygonum stands, and none of these occurred in soil underC. dactylon stands. GC-MS analysis revealed that these additional inhibitors are long-chain fatty acids with 14–22 carbons. Nine were identified inP. aviculare residues and seven in soil underP. aviculare. Sodium salts of all the identified fatty acids inhibited seedling growth ofC. dactylon and at least some test strains of the nitrogen-fixing bacteria,Azotobacter andRhizobium.     

Identification of ant repellent allomone produced by social waspPolistes fuscatus (Hymenoptera: Vespidae)

Two active components were isolated from the ant repellent secretion produced by glandular cells on the sixth (terminal) gastral sternite ofPolistes fuscatus (F.) females. One of the components was identified as methyl palmitate. Field bioassays with synthetic methyl palmitate showed it had repellent activity againstSolenopsis geminata (F.)Forelius pruinosus (Roger), andPheidole sp. The effectiveness of the repellent was greater againstF. pruinosus andPheidole sp. than againstS. geminata. Four methyl ester homologs of methyl palmitate also were field tested for repellent activity: methyl myristate had repellent activity against all three ant species, whereas methyl stearate, methyl linoleate, and methyl linolenate exhibited no repellency.     

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.     

Biological amplification for increasing electroantennogram discrimination between two female sex pheromones ofSpodoptera littoralis (Lepidoptera: Noctuidae)

MaleSpodoptera littoralis stimulated by a mixture ofS. littoralis female sex pheromone with a positional isomer and by the pheromone alone, gave greatly amplified electroantennograms (EAGs) when the antennae were connected in series. The technique is described and examples are given of its use in screening extracts for EAG activity. Dose-response curves obtained with this technique are presented and discussed. Through biological amplification, EAG discrimination between the pheromones was increased.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 238-E, 1980 series.     

Evaluation of Synergism in the Feeding Deterrence of Some Furanocoumarins on Spodoptera littoralis

The phagodepression activity of five furanocoumarins (FC), bergapten, xanthotoxin, psoralen, imperatorin, and angelicin, has been studied against larvae of Egyptian cotton leafworm Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae) using a leaf-disk choice bioassay. The dose range used was 0–10 g/cm² for linear furocoumarins and 0–30 g/cm² for angelicin, the angular furanocoumarin. Dose–feeding deterrency activity correlations were governed by various sigmoidal functions, except in the case of imperatorin and bergapten, which had dose–response curves showing irregular traces with two maxima. All five FCs induced various degrees of phagodepression in S. littoralis; at 3 g/cm², the relative feeding deterrence was bergapten = xanthotoxin > psoralen = imperatorin = angelicin. Structure–activity correlations indicated that a methoxy group on C-5 or C-8 enhanced the activity. Comparison of experimental feeding deterrence of binary mixtures of imperatorin with xanthotoxin, bergapten, or psoralen at various relative concentrations with the calculated additive activity of each combination indicated that a proportion of 40% or more of imperatorin may exert a greater antifeedant effect on S. littoralis than the sum of individual compounds. The effect of angelicin also was examined in combination with psoralen, both of which are present in Psoralea plants. Their mixtures yielded a clear synergistic effect in the 20–80% angelicin range (coactivity coefficient = 124–133). By contrast, the effect of angelicin on xanthotoxin was only additive. In the dose–response curves of imperatorin/bergapten combinations, synergism was found at >60% imperatorin relative concentration and 1 g/cm², whereas lower proportions led to antagonism. The threshold between the two opposing effects was found to depend on the total FC concentration employed. Some natural systems contain FCs in the range of synergistic proportions recorded here and, thus, may have been produced by the host to increase its defensive effect at a lower metabolic cost.     

Phytotoxicity of cacalol and some derivatives obtained from the roots ofPsacalium decompositum (A. Gray) H. Rob. & Brettell (Asteraceae), matarique or maturin

The effect of cacalol and extracts (water and petroleum ether) derived from the roots ofPsacalium decompositum (Asteraceae) on the germination and radicle growth of two plants.Amaranthus hypochondriacus (Amaranthaceae) andEchinochloa crusgalli (Poaceae), and on the radial growth of four phytopathogenic fungi is described. The activity of two cacalol derivatives (methyl cacalol and cacalol acetate) was also investigated. Germination ofA. hypochondriacus was inhibited by almost all the treatments. The extracts and cacalol produced a significant inhibition of radicle growth ofA. hypochondriacus andE. crusgalli. Cacalol acetate showed a specific inhibition onE. crusgalli, and methyl cacalol significantly inhibited the growth ofA. hypochondriacus. In general, antifungal activity depended upon the target fungi and the concentration of each treatment. Cacalol had also effects on the morphology and coloration of the fungal mycelium. The bioactivity shown by the extracts ofPsacalium decompositum on the tested seeds and fungi is mainly due to their content in cacalol.     

Trade-Off Between Chemical and Biotic Antiherbivore Defense in the South East Asian Plant Genus Macaranga

The plant genus Macaranga is known for its manifold mutualistic associations with ants. The plants provide food for the ants and in turn get protection from herbivores. Depending on the strength of the plant–ant interaction, the plant's investment in ants and the biotic defense derived from them is more or less effective. We conducted a comparative study on tannin content in 12 Macaranga species that were selected based on their associations with ants (three nonmyrmecophytes and nine myrmecophytes, three of which start their ontogeny as nonmyrmecophytes). Different developmental stages were investigated in three Macaranga species. Extracts of every individual plant analyzed for tannins were also tested for their effects on larval growth employing larvae of the common cutworm (Spodoptera littoralis). The studied Macaranga species differed significantly in their tannin contents as well as in the effects of their leaf extracts on the growth of S. littoralis larvae. A correlation analysis shows a connection between tannin contents and larval growth. High tannin contents and, thus more effective chemical defense, were observed in nonmyrmecophytic Macaranga species associated only facultatively with ants as compared to obligate myrmecophytes. Our study supports the hypothesis of a trade-off between chemical and biotic defense in the genus Macaranga.     

Phytotoxic Catechin Leached by Seeds of the Tropical Weed <Emphasis Type="Italic">Sesbania virgata</Emphasis>

Sesbania virgata (Cav.) Pers (wand riverhemp) is a fast-growing tropical legume species that has been used for revegetation of riparian forests and rehabilitation of degraded areas and that exhibits an invasive behavior in certain regions of Brazil. Preliminary studies have shown that seed leachates inhibit the germination and development of seedlings of some crop species. In this study, we report that the seed leachates of S. virgata inhibit the growth of Arabidopsis thaliana and rice. The flavonoid (+)-catechin is found in high amounts in these leachates. It was active at concentrations of 50 μg ml⁻¹, and its effect was not distinguishable from the (+)-catechin obtained from a commercial source. We found that (+)-catechin is located in the seed coat and is rapidly released in high concentrations (235 μg per seed) at the beginning of imbibition. Quercetin was also detected in the seed coat of S. virgata, but it was not released from the seeds. Other phytotoxic compounds in the seed leachates were also detected. The fact that S. virgata releases high amounts of (+)-catechin, which also has antimicrobial activity, and other phytotoxins from its seeds at the earliest stages of its development might represent some adaptative advantage to the seedling that contributes to its invasive behavior and successful establishment in different soils.     

Meliaceae and rutaceae limonoids as termite antifeedants evaluated usingReticulitermes speratus kolbe (Isoptera: Rhinotermitidae)

Four limonoids were evaluated by a no-choice bioassay method as termite antifeedants usingReticulitermes speratus Kolbe. Obacunone showed the highest degree of antifeeding potency (PC₉₅=1133 ppm w/w), followed by nomilin (PC₉₅=4475 ppm), azadirachtin (PC₉₅=65,293 ppm), and limonin (with PC⁹⁵ estimates beyond the bioassay limits), which was the least potent. None of the four limonoids exhibited any evidence of acute toxicity towardsR. speratus, although termites fed on obacunone and nomilin at concentrations higher than their PC₉₅ estimates tend to die faster than those in the unfed treatment. Obacunone and nomilin also showed a drastic antifeedant consumption (AC) reduction effect at concentrations higher than 510 and 1360 ppm, respectively. With azadirachtin and limonin, such an effect was not observed. The present data suggest that both obacunone and nomilin have an absolute antifeeding activity towardsR. speratus.     

Altered Glucosinolate Hydrolysis in Genetically Engineered Arabidopsis thaliana and its Influence on the Larval Development of Spodoptera littoralis

The antiherbivore potential of the glucosinolate–myrosinase defense system found in plants of the order Capparales is heavily influenced by the types of hydrolysis products (e.g. isothiocyanates, nitriles) formed from the parent glucosinolates upon plant damage. However, comparison of the effects of glucosinolate hydrolysis products on insect herbivores has been hampered by the lack of suitable experimental tools for rigorous bioassays, such as intact plants differing only in the types of hydrolysis products they produce, or artificial diets that can accurately simulate glucosinolate hydrolysis. The wide array of molecular resources for Arabidopsis thaliana has facilitated the identification of several genes that play a role in glucosinolate hydrolysis. One of these encodes the epithio-specifier protein (ESP) that promotes the formation of nitriles at the expense of isothiocyanates in certain ecotypes of A. thaliana. We overexpressed the ESP cDNA from the nitrile-producing ecotype Landsberg erecta in the isothiocyanate-producing ecotype Columbia-0 to generate transgenic lines of A. thaliana that differed from wild-type plants in the type of glucosinolate hydrolysis products formed upon tissue damage, whereas parent glucosinolate profile and myrosinase activity levels, as well as plant morphology and growth habit, remained unchanged. Bioassays with the model generalist herbivore Spodoptera littoralis (Lepidoptera: Noctuidae) demonstrated that larvae reared on the nitrile-producing lines on average gained weight faster in the first larval stages than larvae that fed on isothiocyanate-producing control plants. Furthermore, larvae with medial growth rates showed a tendency to pupate earlier on the ESP-overexpressing plant lines. Together with the results of previous studies, these findings suggest that isothiocyanates are more effective defenses against insect herbivores than nitriles, and raise questions about what conditions select for nitrile formation in plants.     

Induction of volatile emissions in maize by different larval instars of Spodoptera littoralis

Maize plants under attack by caterpillars emit a specific blend of volatiles that is highly attractive to parasitic wasps. The release of these signals is induced by elicitors in the caterpillar regurgitant. Studies suggest that plants respond differently to different herbivore species and even to different herbivore stages, thus providing parasitoids and predators with specific signals. We tested if this is the case for different larval instars of the noctuid moth Spodoptera littoralis when they feed on maize plants. Cut maize plants were incubated in diluted regurgitant from second, third, or fifth instar caterpillars. There were no differences in total amount released after these treatments, but there were small differences in the release of the minor compounds phenethyl acetate and -humulene. Regurgitant of all three instars contained the elicitor volicitin. To test the effect of actual feeding by the larvae, potted plants were infested with caterpillars of one of the three instars, and volatiles were collected the following day. The intensity of the emissions was correlated with the number of larvae feeding on a plant, and with the amount of damage inflicted, but was independent of the instar that caused the damage. We also used artificial damage to mimic the manner of feeding of each instar to test the importance of physical aspects of damages for the odor emission. The emission was highly variable, but no differences were found among the different types of damage. In olfactometer tests, Microplitis rufiventris, a parasitoid that can only successfully parasitize second and early third instar S. littoralis, did not differentiate among the odors of maize plants attacked by different instar larvae. The odor analyses as well as the parasitoid's responses indicate that maize odors induced by S. littoralis provide parasitoids with poor information on the larval developmental stage. We discuss the results in the context of variability and lack of specificity in odorous plant signals.     

Variations in the composition of sterols,tocopherols and lignans in seed oils from fourSesamum species

Seeds from different collections of cultivatedSesamum indicum Linn and three related wild species [specifically,S. alatum Thonn.,S. radiatum Schum & Thonn. andS. angustifolium (Oliv.) Engl.] were studied for their oil contents and fatty acid composition of the total lipids. The oils from wild seeds were characterized by higher percentages of unsaponifiables (4.9, 2.6 and 3.7%, respectively) compared toS. indicum (1.4–1.8%), mainly due to their high contents of lignans. Total sterols accounted forca. 40, 22, 20 and 16% of the unsaponifiables of the four species, respectively. The four species were different in the relative percentages of the three sterol fractions (the desmethyl, monomethyl and dimethyl sterols) and in the percentage composition of each fraction. Campesterol, stigmasterol, sitosterol and Δ⁵-avenasterol were the major desmethyl sterols, whereas obtusifoliol, gramisterol, cycloeucalenol and citrostandienol were the major monomethyl sterols, and α-amyrin, β-amyrin, cycloartenol and 24-methylene cycloartanol were the main dimethyl sterols in all species. Differences were also observed among the four species in sterol patterns of the free sterols compared to the sterol esters.Sesamum alatum contained less tocopherols (210–320 mg/kg oil), andS. radiatum andS. angustifolium contained more tocopherols (ca. 750 and 800 mg/kg oil, respectively) than didS. indicum (490–680 mg/kg oil). The four species were comparable in tocopherol composition, with γ-tocopherol representing 96–99% of the total tocopherols. The four species varied widely in the identity and levels of the different lignans. The percentages of these lignans in the oils ofS. indicum were sesamin (0.55%) and sesamolin (0.50%).Sesamum alatum showed 1.37% of 2-episesalatin and minor amounts of sesamin and sesamolin (0.01% each).Sesamum radiatum was rich in sesamin (2.40%) and contained minor amounts of sesamolin (0.02%), whereS. angustifolium was rich in sesangolin (3.15%) and also contained considerable amounts of sesamin (0.32%) and sesamolin (0.16%).     

Influence of cnicin,a sesquiterpene lactone ofCentaurea maculosa (Asteraceae), on specialist and generalist insect herbivores

The sesquiterpene lactone cnicin was extracted fromCentaurea maculosa andCentaurea vallesiaca. We examined its effects on the ovipositional response and larval development of generalist and specialist insect herbivores associated withC. maculosa. For the oviposition trials, three plant species (C. maculosa, Achillea millefolium, andCichorium intybus), half of which were sprayed with 3% of cnicin, were exposed to the specialist mothsStenodes straminea, Agapeta zoegana, andPterolonche inspersa in field cages. All three species significantly preferredC. maculosa to other plants andP. inspersa significantly preferred cnicin-sprayed plants to untreated plants for oviposition. Tested over all species, cnicin significantly increased the number of eggs laid on a given plant. A larval diet test examined the toxicity of cnicin for larvae of the generalist noctuid mothSpodoptera littoralis. Cnicin concentrations of 3% and 6% were lethal and 1% and 0.5% seriously inhibited growth and development. The larvae of theC. maculosa specialistStenodes straminea survived at 6% cnicin, but none of the pupae hatched.Agapeta zoegana was able to survive at 1% and 3% cnicin. Both specialists had difficulties with the artificial diet, but weight increase and survival was not further reduced when cnicin was present compared with on the control diet. In conclusion, cnicin influenced host recognition by the specialist species, and larvae of the generalist did not survive on natural levels of cnicin. Growth and survival of the specialist were not influenced by cnicin but were considerably hampered on artificial diet.