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.     

Azadirachtin inhibits secretion of trypsin in midgut of Manduca sexta caterpillars: reduced growth due to impaired protein digestion

When given by injection to tobacco hornworm caterpillars, Manduca sexta, the allelochemical azadirachtin inhibits growth without reducing food intake. The growth reducing effect of azadirachtin is therefore in this case independent of the compound's well-known antifeedant effect. The cause of this reduced rate of growth is an increase in the costs associated with growth. These increased costs are largely a consequence of a decrease in the efficiency of utilisation of dietary nitrogen. This is associated with a drastic reduction in the activity of midgut trypsin. Azadirachtin has no effect on the activity of trypsin in vitro. Thus azadirachtin directly or indirectly inhibits the production of trypsin by the enzyme-secreting cells of the midgut wall; it is suggested that this is the cause of the increased costs and reduced rate of growth. The interesting parallel between this plant defence strategy and that of direct inhibition of herbivore proteinases by allelochemical proteinase inhibitors is discussed.     

Several Antifeedants from Neem Oil,Azadirachta indica A. Juss., against Reticulitermes speratus Kolbe (Isoptera: Rhinotermitidae)

A methanol extract of neem oil indicated antifeedant activity at 200 μg/disc by a no-choice bioassay against Reticulitermes speratus Kolbe. The extract was purified by recycling HPLC to isolate 11 compounds of variable termite antifeedant potency. Deacetylgedunin was the most active compound (95% protective concentration or PC₉₅ = 113.7 μg/disc). This was followed by salannin, gedunin, 17-hydroxyazadiradione, nimbandiol, azadiradione, deacetylsalannin, and deacetylnimbin, with PC₉₅ estimates of 203.3, 218.4, 235.6, 245.4, 827.5, 1373.1, and 1581.2 μg/disc, respectively. Epoxyazadiradione, 17-epiazadiradione, and nimbin were not active (PC₉₅ estimates were beyond the bioassay limits). It could be presumed, by comparing their structures and activities, that the furan ring, αβ-unsaturated ketone and hydroxyl group each played an important role in determining the antifeedant activity.     

Induction of hairy root cultures of Azadirachta indica A. Juss. and their production of azadirachtin and other important insect bioactive metabolites

Hairy root cultures were established from stem and leaf explants of Azadirachta indica A. Juss (neem) following infection with Agrobacterium rhizogenes.Transformation was confirmed using polymerase chain reaction analysis. The cultures had a relatively fast growth rate (0.134 g dry weight day ^–1) and showed a 100-fold increase in biomass over a 4-week culture period. Both the roots and cultivation medium were antifeedant as determined by a no-choice bioassay using the desert locust, Schistocerca gregaria. Azadirachtin, nimbin, salannin, 3-acetyl-1-tigloylazadirachtinin and 3-tigloylazadirachtol were detected in the hairy roots by supercritical fluid chromatography. The cultivation medium contained 3-acetyl-1-tigloylazadirachtinin, 3-tigloylazadirachtol and desacetylsalannin.     

Bioefficacy and mode-of-action of aglaroxin A from Aglaia elaeagnoidea (syn. A. roxburghiana) against Helicoverpa armigera and Spodoptera litura

The bioefficacy of aglaroxin A from Aglaia elaeagnoidea (syn. A. roxburghiana) was assessed using the gram pod borer, Helicoverpa armigera (Hübner), and Asian armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). The compound exhibited strong growth inhibition in a diet bioassay, with 0.67 p.p.m: and 0.78 p.p.m. of the compound reducing growth by 50% in H. armigera and S. litura neonate larvae, respectively, whereas a growth inhibition of 95% was achieved at 2.36 p.p.m: and 2.41 p.p.m., respectively; this was comparable to azadirachtin treatments used as a control. Aglaroxin A was toxic to various stadia. Nutritional analysis revealed the antifeedant properties of the compound; however, nutritional indices indicated that the reduction in growth of the larvae was not entirely due to starvation, but partly due to the toxic effects of the ingested compound. This was further confirmed in topical treatments. When relative growth rate was plotted against relative consumption rate, the growth efficiency of larvae fed on a diet containing aglaroxin A was significantly less than that of control larvae. These results further indicate that aglaroxin A acts as both antifeedant and chronic toxin. Morphologically deformed or partially pupated insects were obtained after 5th instar larvae were treated with aglaroxin A. Such developmental inhibition during ecdysis was not due to depletion of the moulting hormone, as treated larvae, when provided with exogenous 20‐hydroxyecdysone, did not show any recovery from the effect. However, it is obvious from the present findings that aglaroxin A activity does not absolutely follow the pattern of azadirachtin or the more related compound rocaglamide known in lepidopterans.     

Insecticidal compound isolated from Syzygium lineare Wall. (Myrtaceae) against Spodoptera litura (Lepidoptera: Noctuidae)

A new crystal compound 2,5-diacetoxy-2-benzyl-4,4,6,6-tetramethyl-1,3-cyclohexanedione was isolated from the leaves of Syzygium lineare. The insecticidal activity of the compound was assessed against fourth instar larvae of Spodoptera litura. Its activity was better than the positive control azadirachtin. The compound was responsible for growth inhibition on S. litura. It induced larval, pupal and adult deformities even at low concentration. The compound may be useful as a botanical pesticide.     

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.     

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.     

Insect Antifeedants,Pterocarpans and Pterocarpol,in Heartwood of Pterocarpus macrocarpus Kruz.

The insect antifeedant activities of pterocarpans and a sesquiterpene alcohol from the dichloromethane extract of Pterocarpus macrocarpus Kruz. (Leguminosae) were evaluated against the common cutworm, Spodoptera litura F. (Noctuidae), and the subterranean termite, Reticulitermes speratus (Kolbe)(Rhinotermitidae). Three pterocarpans, (?)-homopterocarpin (1), (?)-pterocarpin (2), and (?)-hydroxyhomopterocarpin (3) and the sesquiterpene alcohol, (+)-pterocarpol (5), were isolated from the dichloromethane extract of the heartwood of P. macrocarpus under guidance by a biological assay. Among these natural products, the most active insect antifeedant against both S. litura and R. speratus was 1. On the other hand, sesquiterpene alcohol 5 showed less insect antifeedant activity than the other pterocarpans against both insect species. While its methylated derivative, (?)-methoxyhomopterocarpin (4), showed high biological activity, 3 showed less insect antifeedant activity in this study. Interestingly, racemic 1 did not show insect antifeedant activity against S. litura. However, all of the test pterocarpans and isoflavones showed antifeedant activity against the test termites. Additionally, since these compounds were major constituents of P. macrocarpus, these antifeedant phenolics may act as chemical defense factors in this tree. In Thailand, lumber made from this tree is used to make furniture and in building construction due to its resistance to termite attack.     

Differential thresholds of azadirachtin for feeding deterrence and toxicity in locusts and an aphid

Conclusions The importance of using azadirachtin in the field at levels causing primary antifeedancy may have been overemphasised in the past. Currently, it is recommended that commercial preparations of neem be applied at 50–100 ppm (a.i.). While this would undoubtedly have an antifeedant effect in many phytophagous insects it may also harm beneficial species (Schmutterer, 1990). By lowering the concentration of azadirachtin applied to the crop, food intake by insect pests with low chemoreceptor sensitivity to azadirachtin, e.g. aphids (Nisbet et al., 1993; 1994), would not initially be affected. However, secondary antifeedant effects, IGR and sterilant effects could rapidly manifest themselves and bring about crop protection by reducing insect pest populations without harming natural predator or parasitoid populations.     

Behavioural and sensory responses to some neem compounds by Pieris brassicae larvae

Abstract. The efficacy of a commercial product (Margosan-O) to inhibit feeding in Pieris brassicae L. (Lepidoptera: Pieridae) larvae was compared to that of three pure triterpenoids of botanical origin: azadirachtin and salannin (occurring in Melia azedurach ), and toosendanin (occurring in M. toosendan ). In dual-choice behaviour tests the order of antifeedant efficacy was: Margosan-O > toosendanin > salannin > azadirachtin. The ranking order of their capacity to stimulate the deterrent receptor located in the medial sensillum styloconicum appears to be: Margosan-O = toosendanin > salannin > azadirachtin. In addition to stimulating the medial deterrent cell, toosendanin inhibits the sugar receptor and the glucosinolate receptor, both located in the lateral sensillum styloconicum. The amino acid receptor in this sensillum, however, is not affected. In contrast, neither azadirachtin nor salannin influences the sensitivity of any of the receptor cells in this sensillum.
The adaptation rate of the deterrent receptor is low in comparison to that of the two sugar receptors.
A significant correlation is found between the antifeedant indices of the four neem compounds tested at three different concentrations, and the responses of the deterrent receptor to these compounds at similar concentrations. It is concluded that when this insect species feeds on its natural foodplant (i.e. cabbage), the deterrent effect of neem compounds is mediated solely via the medial deterrent receptor, whereas inhibitory effects on the sugar and glucosinolate receptors do not play a significant role.     

Development of the Major Triterpenoids and Oil in the Fruit and Seeds of Neem (Azadirachta indica)

In order to learn the best time for harvesting Neem (Azadirachtaindica) seeds, the amount of the five major triterpenoids, togetherwith the oil content have been determined throughout a fruitingseason in six selected trees in Sri Lanka. The triterpenoidcontent and the relative proportions of the major compoundschanged little from the hard green fruit stage to mature seeds,while the oil content increased markedly with time. The highestcontent of azadirachtin (10 mg g^-1seed kernels) was recordedin newly ripened seeds. There was some loss of salannin andazadirachtin in storage after harvesting for up to 6 months. Azadirachtin; salannin; nimbin; neem seeds; neem fruit; seasonal change; neem oil; seed storage; Azadirachta indica ; Meliaceae     

An improved leaf-disk antifeedant bioassay and its application for the screening of Hokkaido plants

In the course of our search for new insect antifeedant substances from plants, we have devised an improved antifeedant leaf-disk bioassay. Our system allows an accurate measurement of consumed disk surfaces, using a video camera interfaced with a computer. The scanned image of the leaf disks is stored, and the eaten areas are measured with the help of a video-image analysis software. This new method allows for precise quantification of insect antifeedant activity tested on leaf material. This method was applied to the screening of Hokkaido plants for antifeedant activity. Out of 206 samples, seven demonstrated significant activity against larvae of the tobacco cutworm,Spodoptera litura.     

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.     

Insect feeding deterrents from the medicinal plant Tylophora asthmatica

Alkaloid extractables of Tylophora asthmatica (Asclepiadaceae) have been tested for the antifeedant activity against Spodoptera litura by incorporating them in semisynthetic and cellulose diets. The alkaloid extractables at 0.01% concentration inhibited feeding of the larvae. Three compounds, tylophorine, tylophorinine and pergularinine, were isolated; and their EC₅₀ values were found to be 2.9, 8.6 and 12.00 ppm, respectively.     

Growth inhibitory and antifeedant activity of extracts from Melia dubia to Spodoptera litura and Helicoverpa armigera larvae

The growth inhibitory activity and deterrency of Melia dubia (Meliaceae) extracts to Spodoptera litura and Helicoverpa armigera were investigated. Artificial diet bioassays using neonate larvae of both S. litura and H. armigera indicated that dichloroethane (DCE) and methanol (Me) extracts of M. dubia inhibited growth in a dose dependent manner. DCE and Me-5II fractions also resulted in 50% deterrency at concentrations of 22.5 and 16.8 micrograms/cm2 respectively against S. litura larvae in a leaf disc-choice test. The DCE-5 fraction was found to be more toxic to larvae (LC50 of 0.65%) than the Me-5II (LC50 of 0.8%), 72 hr after topical application. Both fractions lack contact toxicity, but the deterrent effect persisted for at least 60 hr under laboratory conditions. Although salannin was isolated from the DCE fraction to show antifeedant activity, the physico-chemical characteristics of the active fractions DCE-5 and Me-5II were not identical with either salannin or azadirachtin.     

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.     

Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus

Growth and survival responses were determined for black field crickets Teleogryllus commodus (Walker) (Orthoptera: Gryllidae) on artificial diets containing a range of levels of dietary protein, and protease inhibitors (PI's) at 0.33% (weight volume, w:v). The effect on cricket gut enzyme activities of adding PI's to a high protein diet was measured. All PI's which had in vitro binding activity against either trypsin or elastase (the two major cricket gut endopeptidases) reduced growth, but those which bound to both enzymes had the greatest effect. None of the PI's acted as a source of nutritional protein. Cricket growth rate increased with the addition of casein up to 3% w:v, but not with a similar addition of wheatgerm. The impact of PI's on growth was greatest on a 1.5% casein diet. On a high protein (3% casein) diet, the gut activity of trypsin was increased by potato proteinase inhibitors 1 and 2 while the activity of elastase and leucine amino peptidase were increased by soybean trypsin inhibitor and potato proteinase inhibitor 2. Increasing dietary casein up to 3.3% improved cricket survival. The potential of PI's as plant resistance factors against crickets was confirmed.     

Cloning and expression inEscherichia coli of entomotoxic protein gene fromBacillus thuringiensis subspecieskurstaki

A plasmid borne larvicidal crystal protein gene from B.thuringiensis subspecieskurstaki was cloned inEscherichia coli using a specific 20-mer oligonucleotide probe. The gene expressed inE. coli at a high level. TransgenicE. coli cells produced large irregular bodies which looked bright under phase contrast microscopy. The phase bright bodies released by sonic disruption of cells could be pelleted by centrifugation. Toxicity trials on the larvae ofSpodoptera litura showed that the pellet was antifeedant and toxic to the larvae. The supernatant was only mildly antifeedant. Even short term feeding of larvae on the toxin delayed the onset of pupation.