Azadirachtin: structural requirements for reducing growth and increasing mortality in lepidopterous larvae

Spodoptera littoralis, Spodoptera frugiperda and Heliothis virescens were exposed to azadirachtin and 23 other related compounds in three bioassays: oral cannulation, haemolymph injection and topical application. Spodoptera littoralis was susceptible to a larger proportion of the compounds than either of the other species. Overall, azadirachtin, 22,23-dihydroazadirachtin and 1-detigloyl-22,23-dihydroazadirachtin were the most active compounds. The nature of the substitutes at C-1 and C-3 of the decalin ring affects the potency of the compounds, as does the addition of substitutes to C-22,23. Generally, larvae were less sensitive to compounds when they were topically applied than when they were cannulated into the gut or injected into the haemolymph.

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Résumé Spodoptera littoralis, S. frugiperda et Heliothis virescens ont été exposés à l'azadirachtine et 23 autres substances voisines dans 3 expériences: canulation orale, injection d'haemolymphe et application topique. S. littoralis est sensible à un plus grand nombre de substances que les 2 autres espèces. De toutes façons, les substances les plus actives ont été l'azadirachtine, la 22,23-dihydroazadirachtine et la 1-détigloyl-22,23-dihydroazadirachtine. La nature des substitutions en C-1 et C-3 de l'anneau décaline modifie la puissance des substances, tout comme les substitutions en C-22,23. D'une façon générale, les chenilles ont été moins sensibles aux applications topiques qu'aux canulations dans le tube digestif ou aux injections dans l'haemolymphe.

     

Food selection in adults and larvae of three species of Lepidoptera: a behavioural and electrophysiological study

Adults and larvae of Spodoptera littoralis, Heliothis virescens and Heliothis armigera were tested with a range of sugars, amino acids, sugar alcohols and allelochemics. Feeding behaviour was correlated with the electrophysiological responses of maxillary styloconic sensilla in the larvae and proboscis styloconic sensilla in the adult. The neural response was more vigorous in larvae than in adults but otherwise the spectra of responses were similar in the two life stages. Phagostimulants and antifeedants stimulated maximally different sensilla in larvae but not in adults. The responses of adult sensilla to sugars and amino acids were significantly correlated to behaviour in all three species, but only in H. armigera was there a similar correlation with the sugar alcohols and allelochemics.     

Behavioural and neurophysiological responses ofSpodoptera littoralis to azadirachtin and a range of synthetic analogues

The antifeedant activity of azadirachtin and 56 azadirachtin analogues, including 22,23-dihydroazalirachtin, against larvae ofSpodoptera littoralis was investigated using behavioural and electrophysiological bioassays. None of the analogues was as active as azadirachtin, although many showed significant antifeedant activity at high concentrations. The majority of the analogues stimulated a dose-dependent response from a neurone in the medial styloconic maxillary sensilla which correlated with the behavioural activity. Methylation of the hydroxy substitutions on the azadirachtin molecule usually resulted in a decrease in antifeedant activity, as did the addition of bulky groups to the dihydrofuran ring.     

Uptake, metabolism, and excretion of injected tritiated 22,33-dihydroazadirachtin in last instar larvae of Heliothis virescens

To evaluate its uptake, metabolism, and excretion, we studied the fate of tritiated 22,23-dihydroazadirachtin, when injected either orally or directly into the hemocoel of last instar larvae of Heliothis virescens (Fabr.). An oral injection of 1.0 g of tritiated 22,23-dihydroazadirachtin, the minimum oral dose necessary to disrupt pupation, was rapidly absorbed through the midgut such that >50% of the radioactivity was detected in the body 1 h post injection. Excretion of radioactivity following an oral injection was slow; 24 to 72 h elapsed before approximately 50% of the radioactivity was detected in the feces. In contrast, excretion of radioactivity following a hemocoel injection of 0.3 g of tritiated 22,23-dihydroazadirachtin, the minimum hemocoel dose necessary to disrupt pupation, was rapid; almost 50% of the radioactivity was detected in the feces only 1 h post injection. Tritiated 22,23-dihydroazadirachtin was metabolized to a more polar compound(s) by H. virescens larvae, as indicated by thin layer chromatography. Metabolism to a more polar form(s) occurred probably following absorption through the midgut, since hemocoel-injected larvae exhibited a more rapid rate of conversion than oral-injected larvae. Even though metabolism to a more polar form(s) should enhance its excretion, approximately 25% of the radioactivity remained in the body 72 h following either oral or hemocoel injection, and this was sufficient to disrupt pupation.

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Résumé L'étude a été réalisée avec de la 22,23-dihydroazadirachtine tritiée injectée soit oralement, soit directement dans l'hémocoele des chenilles de H. virescens Fabr. du dernier stade. La dose orale minimale de 1,0 g, nécessaire pour perturber la nymphose, a été rapidement absorbée à travers l'intestin moyen, d'une façon telle que plus de 50% de la radioactivité a été retrouvée dans le corps une heure après l'injection. L'excrétion de la radioactivité à la suite d'une injection orale a été lente; 24 à 72 heures se sont écoulées avant que 50% de la radioactivité ait été décelée dans les excréments. A l'opposé, l'excrétion de la radio-activité à la suite d'une injection hémocoelique de 0,3 g, — dose hémocoelique minimale pour perturber la nymphose-, a été rapide; presque 50% de la radioactivité a été décelée dans les excréments au bout d'une heure seulement après l'injection. La chromatographie en couche mince a montré que la 22,23-dihydroazadirachtin a été métabolisée en substance(s) plus polaire(s) par les chenilles de H. virencens. La métabolisation en forme(s) plus polaire(s) se produit vraisemblablement après l'absorption à travers l'intestin moyen, puisque les chenilles injectées directement dans l'hémocoele ont présenté un taux de conversion plus rapide que les chenilles injectées oralement. Bien que la métabolisation sous une (des) forme(s) plus polaire(s) ait augmenté l'excrétion, approximativement 25% de la radioactivité subsistait dans le corps 72 heures après une injection orale ou hémocoelique, ce qui est suffisant pour perturber la nymphose.

     

Bioefficacy and mode-of-action of some limonoids of salannin group fromAzadirachta indica A. Juss and their role in a multicomponent system against lepidopteran larvae

Biological activities of the salannin type of limonoids isolated fromAzadirachta indica A. Juss were assessed using the gram pod borerHelicoverpa armigera (Hubner) and the tobacco armywormSpodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Inhibition of larval growth was concomitant with reduced feeding by neonate and third instar larvae. All three compounds exhibited strong antifeedant activity in a choice leaf disc bioassay with 2.0, 2.3 and 2.8 (μ/cm² of 3-O-acetyl salannol, salannol and salannin, respectively deterring feeding by 50% inS. litura larvae. In nutritional assays, all three comounds reduced growth and consumption when fed to larvae without any effect on efficiency of conversion of ingested food (ECI), suggesting antifeedant activity alone. No toxicity was observed nor was there any significant affect on nutritional indices following topical application, further suggesting specific action as feeding deterrents. When relative growth rates were plotted against relative consumption rates, growth efficiency of theH. armigera fed diet containing 3-O-acetyl salannol, salannol or salannin did not differ from that of starved control larvae (used as calibration curve), further confirming the specific antifeedant action of salannin type of limonoids. Where the three compounds were co-administered, no enhancement in activity was observed. Non-azadirachtin limonoids having structural similarities and explicitly similar modes of action, like feeding deterrence in the present case, have no potentiating effect in any combination.     

Antifeedant and insecticidal properties of azadirachtin to the European Corn Borer, Ostrinia nubilalis

Azadirachtin, a triterpenoid substance from the neem tree, Azadirachta indica A. Juss., Meliaceae, was evaluated for its antifeedant and insecticidal properties to the European corn borer Ostrinia nubilalis Hübner. The 50% protective concentration for larval feeding on treated corn disks (PC₅₀) was 3.5 ppm for neonate larvae and 24 ppm for third instar larvae. Azadirachtin severely reduced larval growth at 1 and 10 ppm in a meridic diet and eventually caused 100% mortality at 10 ppm and 90% at 1 ppm. At 0.1 ppm azadirachtin showed no significant effect on larvae but altered the sex ratio in favor of male moths during adult emergence and arrested oviposition of female moths.Nutritional indices for naive and habituated third instar larvae suggested that reduction of growth was due to toxicity as shown by a lowered efficiency of conversion in the azadirachtin treated groups, rather than changes in digestibility. No clear evidence of tolerance was observed in the habituated group. The data suggest that at 10 ppm azadirachtin is an effective botanical insecticide for control of O. nubilalis.

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Résumé On a examiné les propriétés anti-appétantes et insecticides de l'azadirachtine, une substance triterpénoïde provenant d'Azadirachta indica envers la pyrale du maïs Ostrinia nubilalis. La concentration effective pour causer 50% d'inhibition de l'alimentation larvaire (PC₅₀) sur des disques foliaires a été de 3.5 ppm pour les larves de premier stade et de 24 g/8 pour celle de troisième stade. L'azadirachtine a sévèrement réduit la croissance larvaire à des concentrations de 1 et 10 ppm dans une diète méridique et, éventuellement, a causé 100% de mortalité à 10 ppm et 90% à 1 ppm. A 0.1 ppm l'azadirachtine n'a pas eu d'effet significatif sur les larves maïs a modifié l'indice de masculinité en faveur des mâles au moment de l'émergence et a empêché l'oviposition chez les femelles. Les indices nutritionnels pour les larves naïves de troisième âge et celles ayant subi une accoutumance suggèrent que la réduction de croissance est le résultat d'une intoxication comme le démontre une efficacité réduite de transformation chez les groupes traités à l'azadirachtine, plutôt que des changements de digestibilité. Il n'y a pas d'évidence claire de tolérance dans le groupe ayant subi l'accoutumance. Les résultats suggèrent qu'à 10 ppm l'azadirachtine est un insecticide botanique efficace envers O. nubilalis.

     

Insect growth regulating effects of neem extract and azadirachtin on aphids

Neem,Azadirachta indica (A. Juss.), seed oil (NSO) applied to leaf discs at a concentration of 1.0% resulted in 94% to 100% mortality of second instar nymphs of currant-lettuce aphid,Nasonovia ribis-nigri (Mosley), and green peach aphid,Myzus persicae (Sulzer), after nine days. The equivalent amount of pure azadirachtin (AZA) (≈40 ppm), the principle active ingredient of neem, was as effective as NSO. The survival of adult aphids was unaffected by NSO or AZA, but the survival of offspring from treated adultM. persicae andN. ribis-nigri was reduced significantly. The lethal concentration of AZA resulting in 50% mortality of second instar nymphs of nine species of aphids ranged from 2.4 ppm forM. persicae on pepper to 635.0 ppm for the strawberry aphid,Chaetosiphon fragaefolii (Cockerell), on strawberry. ForM. persicae, the growth regulating effect of AZA was influenced by the host plant and the nymphal instar treated.     

Antifeedant and toxic effects of drimanes on Colorado potato beetle larvae

Among the drimane compounds tested, the dialdehydes polygodial and warburganal were the most active as antifeedants against Colorado potato beetle larvae, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), in a dual-choice assay with potato, Solanum tuberosum L., leaf discs. Lactones were less effective. Direct observations showed that decreased feeding on leaf discs treated with polygodial and warburganal was accompanied by increased locomotry activity. Topical application of these two compounds on the insect's cuticle decreased food intake of untreated leaf discs, indicating that besides deterrent effects, toxic properties of these molecules influence feeding behaviour.     

Predation on eggs and larvae ofHeliothis virescens [Lep.: Noctuidae] by an adult predator complex in cage studies on cotton

Predation by a predator complex consisting of adults of the lygaeidGeocoris punctipes (Say), the nabidsNabis roseipennis Reuter and/orTropiconabis capsiformis Germar and the coccinelids,Hippodamia convergens Guerin-Meneville andColeomegilla maculata (DeGeer) onHeliothis virescens (F.) eggs, 1st-instar larvae or both on late pinhead square and early bloom stage cotton was evaluated in field cages in Mississippi. Prey densities of 4 (11, 512/ha) or 8 per cage and predator: prey (P:p) ratios of 1∶1, 2∶1, 3∶1, 4∶1 and 5∶1 were used. Prey were labeled with phosphorus-32 or carbon-14 to determine predator ingestion and effectiveness. Average percent egg predation as a function of P:p ratio ranged from 2.1 to 12.1 for a 48 h period. The average percent predation on larvae as a function of P:p ratio ranged from 5.3 to 22.0. The hemiptera fed more than the coleoptera on larvae, andG. punctipes was the best egg predator. For the range of predator densities used, the average area of discovery for the predator complex was 6.98 × 10⁻³ m/day and 2.34 × 10⁻² m/day when exposed to eggs and larvae, respectively. Publication N^o 5936-Mississippi Agricultural and Forestry Experiment Station, Mississippi State, MS 39762.     

Physiological effects of a juvenile hormone analog on larvae of Spodoptera littoralis

Topical application of JHA to Spodoptera littoralis larvae extends the duration and increases the final weight of the last larval instar. No supernumary moults occur. Respiration decreases to very low levels and glycogen and lipid stores increase. Respiration remains lower in JHA-treated pharate pupae and more polyunsaturated C₁₈ fatty acids accumulate than in normal pharate pupae. These physiological parameters are similar to those occurring during diapause but are not definitive. Diapause has not been reported in field populations of S. littoralis, nor it's artificial induction by photoperiodic manipulation.

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Résumé Le dernier stade des chenilles de Spodoptera littoralis, traitées avec JHA, est nettement prolongé par rapport aux témoins. Bien que le poids final des chenilles traitées soit plus élevé que celui des témoins, il n'y a pas de mue surnuméraire. Ces chenilles géantes s'alimentent rarement et leur respiration est sérieusement réduite. Les réserves de glycogène s'accumulent chez les chenilles traitées à la même date chronologique que chez les prénymphes non traitées. Bien que les prénymphes traitées à la JHA conservent leur profil biochimique juvénile — tout au moins dans la mesure où on considère la teneur en glycogène et en lipides totales — leur composition en acides gras saturés et non saturés diffère de celle normalement rencontrée à la fin du développement larvaire, et beaucoup plus d'acide oléique monosaturé s'accumule finalement chez les insectes traités par JHA. Ces résultats suggèrent que la JHA fournie peut avoir provoqué un état de développement de diapause naissante semblable par quelques traits fondamentaux à celle induite par la JH endogène chez les insectes enclins à la diapause. Il est cependant prématuré de définir cet état physiologique comme indiquant une vraie diapause chez Spodoptera littoralis.

     

Desensitization of fifth instar Spodoptera litura to azadirachtin and neem

The deterrence of azadirachtin, in its pure form and as a constituent of neem seed extract, to fifth instar Spodoptera litura (Fab.) larvae, was measured using cabbage, Brassica oleraceae (L.) var. capitata, leaf disc assays. Paired-choice assays, in which larvae could choose between feeding on a treated (1.3 ng azadirachtin per square cm leaf area) or an untreated leaf disc for 2 h, were conducted at 24 h intervals throughout the fifth instar. In addition, no-choice assays, in which larvae could feed on only one leaf disc (10 ng azadirachtin per square cm leaf area) for 1.5 h, were conducted consecutively over a six hour period at the beginning of the fifth instar. The effects of hunger and habituation on desensitization in our no-choice tests were partitioned. After repeated exposures, larvae became desensitized to pure azadirachtinal in both choice and no-choice tests, but did not desensitize to neem containing the same absolute amount of azadirachtin in choice tests. Hunger was responsible for approximately one third of the desensitization response in the no-choice tests. Sensitivity to azadirachtin was independent of age within the fifth instar.     

Antifeedant and toxic activity of Trichilia americana extract against the larvae of Spodoptera litura

The biological activity of a crude methanolic extract of Trichilia americana (Sesse and Mocino) Pennington (Meliaceae) was assessed using the Asian armyworm, Spodoptera litura (Fabr.) (Lepidoptera: Noctuidae). The extract exhibited strong antifeedant activity in a choice leaf disc bioassay with 0.18 g cm^–2 extract deterring feeding by 50%. In nutritional assays, the crude extract reduced growth, consumption and the utilisation of ingested and digested food in a dose-dependent manner when fed to larvae, suggesting both antifeedant and toxic activities. When relative growth rates were plotted against relative consumption rates, the growth efficiency of the S. litura fed on diet containing T. americana crude extract was significantly less than that of control larvae. This result further indicates that the extract acts as both an antifeedant and chronic toxin. Toxicity is only seen following ingestion and was not observed following topical application or injection into the hemocoel. Larvae reared initially on extract-containing diet then transferred to control diet showed nutritional indices comparable to those of larvae fed continuously on control diet. This suggests that the extract is not permanently damaging the insect's digestive tract. The mode-of-action of the extract as a chronic toxin remains unknown.     

An analytical model for genetic control of Heliothis virescens incorporating the effects of sterile males

Summary A recent computer simulation model by Levins and Parker (1983) indicated that mass releases of male-sterile Heliothis hybrid moths could cause genetic suppression of the tobacco budworm, Heliothis virescens, without the risk of significant crop damage. We present an analytical model to explore the behavior of the Levins-Parker model. Our model shows that the length of time between matings for females when they mate with wild type fertile males to that when they mate with hybrid sterile males is extremely important to the efficacy of a suppression program. Release ratios needed to suppress a natural pest population were examined across a range of biological parameters.     

Effects of azadirachtin after systemic uptake intoBrassica oleracea on larvae ofPieris brassicae

Petioles of intact cabbage leaves were placed in azadirachtin solutions for 24 h. Azadirachtin or active metabolites of this compound appeared to be translocated systemically as performance of first instar larvae ofPieris brassicae L. placed on disks from those leaves was affected. The position within a leaf from which disks were excised did not affect the extent of these effects, indicating a homogeneous distribution of active compounds in the laminar tissue. At doses ranging from 1–10 ppm of azadirachtin in the solution taken up by the leaves, gain of larval fresh weight and leaf area consumed were reduced. These effects were dose dependent up to 60–90 ppm. At 10 ppm and higher doses no larva reached the second instar within 72 h. Mortality during the 72 h period was highest at 10 ppm, due to the coincidence of lethal action with ecdysis in combination with the effect on developmental rate. Azadirachtin solutions applied directly to the surface of leaf disks in order to produce known doses on a leaf fresh weight basis gave results similar to the systemic treatment for weight gain, leaf area consumed and developmental rate, while mortality was higher in the latter treatment.     

Wound-induced changes in tomato leaves and their effects on the feeding patterns of larval lepidoptera

Several studies have shown changes in the patterns of damage from feeding insects associated with changes in palatability and overall consumption as a result of wound-induced chemical changes in plants. This paper describes how the pattern of feeding damage made by the larvae of Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) on tomato is affected by changes in palatability of the leaves. Two sorts of responses to leaves from plants that had received prior damage were observed. Larvae offered a choice of leaves tended to take fewer meals on leaves from previously-wounded plants than on control leaves, frequently rejecting the former after sampling them. On wounded plants this rejection behaviour was associated with a shift in feeding site towards the base of the plant. However, starved larvae offered only a single excised leaf readily ate leaves from wounded plants but took shorter meals on these leaves than on controls. Although it was not directly tested it is possible that this difference in response reflected changes in food selectivity with a differing level of satiation. The results are considered in relation to the adaptive significance of the plant of changes in within-plant distributions of herbivore damage.     

Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens

ScathL is a cathepsin L-like cysteine protease from the flesh fly, Sarcophaga peregrina, which digests components of the basement membrane during insect metamorphosis. A recombinant baculovirus (AcMLF9.ScathL) expressing ScathL kills larvae of the tobacco budworm Heliothis virescens significantly faster than the wild type virus and triggers melanization and tissue fragmentation shortly before death. The tissue fragmentation was assumed to be a direct consequence of basement membrane degradation by ScathL. The goal of this study was to investigate the tissue specificity of ScathL when expressed by AcMLF9.ScathL using light, transmission and scanning electron microscopy. Baculovirus expression of ScathL resulted in damage to the basement membrane overlying the midgut, fat body and muscle fibers in larvae infected with AcMLF9.ScathL, but not in larvae infected with the control virus AcMLF9.ScathL.C146A or wild type virus AcMNPV C6. Injection of recombinant ScathL and high levels of baculovirus-mediated expression of ScathL resulted in complete loss of the gut. Extensive damage to the basement membrane mediated by ScathL likely resulted in loss of viability of the underlying tissue and subsequent death of the insect. These results confirm the conclusion of an earlier study (Philip, J.M.D., Fitches, E., Harrison, R.L., Bonning, B.C., Gatehouse, J.A., 2007. Characterisation of functional and insecticidal properties of a recombinant cathepsin L-like proteinase from flesh fly (Sarcophaga peregrina), which plays a role in differentiation of imaginal discs. Insect Biochem. Mol. Biol. 37, 589-600) of the remarkable specificity of this protease.     

Functional response ofChrysopa carnea [Neur: Chrysopidae] larvae feeding onHeliothis virescens [Lep.: Noctuidae] eggs on cotton in field cages

The functional response of 3rd instarChrysopa carnea (Stephens) larvae feeding on 4 densities ofHeliothis virescens (F.) eggs was determined on caged cotton (Gossypium hirsutum L.).C. carnea larvae had a mean search rate of 1.08×10⁻⁵ ha/predator-day or 0.11 row-m/predator-day.      

The protection of barley seedlings from attack bySchistocerca gregaria using azadirachtin and related analogues

Barley seedlings were sprayed with azadirachtin or analogues and the resulting crop protection againstSchistocerca gregaria was examined. Any change of the parent azadirachtin molecule resulted in less effective insect control and hence more crop damage. Crop protection by azadirachtin and dihydroaza-dirachtin resulted from both antifeedancy and toxicity, 3-tigloylazadirachtol was more effective by direct toxicity after significant ingestion and bromoethoxyazadirachtin showed extremely low antifeedant and insecticidal properties.S. gregaria was shown to be very sensitive to change in the azadirachtin molecule.     

Daily changes in the responsiveness of taste receptors correlate with feeding behaviour in larvae of Spodoptera littoralis

Larvae of Spodoptera littoralis were monitored through the final larval stadium (6 days) for weight gain, amount eaten in bioassays and responsiveness of gustatory receptors to stimulation with sodium chloride and four concentrations of sucrose. The age of larvae within the stadium, time of day (am or pm) and interactions between these factors influenced the electrophysiological response to 0.05 M sucrose. Overall, the responsiveness of taste receptors increased in the first half of the stadium, then declined. Neural input correlated with weight gain on days 1 to 4, and with amount eaten in bioassays on days 1 to 5. This correlation between neural input and feeding behaviour indicates that the observed modulation in neural sensitivity has a causal relationship with feeding behaviour. The ways in which the modulation might occur are discussed.