Multiple mode of action of the feeding deterrent,toosendanin, on the sense of taste in Pieris brassicae larvae

Toosendanin, a tetranortriterpenoid isolated from the bark of Melia toosendan, is a feeding deterrent for larvae of Pieris brassicae. By using electrophysiological techniques, it was found that toosendanin stimulates a deterrent receptor cell located in the medial maxillary sensillum styloconicum. Toosendanin also inhibits responses of both the sugar and glucosinolate receptor cell, which are localized in the lateral sensillum styloconicum. The degree of inhibition of the sugar receptor increases with increasing sucrose concentration. The glucosinolate receptor cell shows a reversed reaction: inhibition by toosendanin decreases with increasing sinigrin concentration. Inhibitory effects occur at a toosendanin concentration as low as 10^–9 M and are dose dependent. The taste neurons that respond to amino acids or deterrents in the lateral sensillum, however, are not affected by toosendanin. It is concluded that the sensory code underlying feeding behaviour is modulated by toosendanin via several different peripheral sensory mechanisms.     

Behavioural and sensory responses to drimane antifeedants in Pieris brassicae larvae

Fifteen drimane compounds were tested for their feeding inhibiting activity in larvae of Pieris brassicae L. (Lepidoptera: Pieridae) when applied to leaf material of the host plant Brassica oleracea L. The antifeedant efficacy of the drimanes was related to their molecular structure in order to identify important functional groups. Of the drimanes tested, those with a lactone group on the B-ring were the most effective feeding inhibitors. Additionally, the sensory responses to 13 of the drimanes were measured. Neural activity was evoked in the deterrent cell in the medial sensillum styloconicum. Also, inhibition of sensory responses to feeding stimulants was found. Results of behavioural and electrophysiological tests were correlated in an attempt to elucidate the sensory code underlying feeding inhibition by drimanes in Pieris brassicae. It was concluded that the response of the deterrent cell in the medial sensillum styloconicum contributes significantly to inhibition of feeding behaviour in larvae of Pieris brassicae.     

Comparative study on the responses of maxillary sensilla styloconica of cotton bollwormHelicoverpa armigera and Oriental tobacco budwormH. assulta larvae to phytochemicals

Using the electro-physiological technique, the sensory mechanisms of maxillary sensilla styloconica to stimulants and deterrents were explored on two closely related species, the generalist Helicoverpa armigera and the specialist H. assulta. The results showed that: (i) in both species, cells sensitive to sucrose and azadirachtin were mainly in the lateral sensillum styloconicum, and those to inositol were in the medial sensillum styloconicum; (ii) sensitivity of medial sensillum styloconicum in H. assulta to inositol was higher than that in H. armigera; (iii) among 6 tested deterrents, only azadirachtin evoked high impulse discharge from the lateral sensillum styloconicum in both insects; (iv) the deterrents could disturb stimulants evoking impulse discharge from maxillary sensilla styloconica of both species in different degrees: To sucrose evoking impulses on lateral sensillum styloconicum, for H. armigera capsaicin had a strong inhibition and gossypol had a weak inhibition, while for H. assulta tann     

Comparative study on the responses of maxillary sensilla styloconica of cotton bollwormHelicoverpa armigera and Oriental tobacco budwormH. assulta larvae to phytochemicals

Using the electro-physiological technique, the sensory mechanisms of maxillary sensilla styloconica to stimulants and deterrents were explored on two closely related species, the generalistHelicoverpa armigera and the specialistH. assulta. The results showed that: (i) in both species, cells sensitive to sucrose and azadirachtin were mainly in the lateral sensillum styloconicum, and those to inositol were in the medial sensillum styloconicum; (ii) sensitivity of medial sensillum styloconicum inH. assulta to inositol was higher than that inH. armigera; (iii) among 6 tested deterrents, only azadirachtin evoked high impulse discharge from the lateral sensillum styloconicum in both insects; (iv) the deterrents could disturb stimulants evoking impulse discharge from maxillary sensilla styloconica of both species in different degrees: To sucrose evoking impulses on lateral sensillum styloconicum, forH. armigera capsaicin had a strong inhibition and gossypol had a weak inhibition, while forH. assulta tannic acid, gossypol, and tomatine all had strong inhibition except nicotine and capsaicin; to inositol evoking impulses on medial sensilum styloconicum, forH. armigera inhibition of tomatine was strong but that of gossypol was weak; and forH. assulta inhibition of gossypol was strong but that of nicotine was weak.     

The effect of neem allelochemicals on nutritional physiology of larval Spodoptera litura

Neem allelochemicals azadirachtin, salannin, nimbinene and nimbin were administered to different larval instars of the tobacco armyworm, Spodoptera litura orally in artificial diet, topically or via injection. Nutritional analyses revealed strong antifeedant and growth regulatory effects of azadirachtin which were independent of each other. While salannin and nimbinene induced concentration dependent feeding deterrence only; nimbin was inactive to the 1000 ppm level against this insect species. One of the causes of the reduced growth rate of azadirachtin treated insects was due to an increase in the costs associated with growth. This was relative to a drastic reduction in the activity of gut trypsin. Salannin and nimbinene, however, did not interfere with the trypsin activity of the gut. These results and those from nutritional studies suggest that salannin and nimbinene have no toxicity mediated effects on S. litura larvae and antifeedant activity is a result of the effects on deterrent and other chemoreceptors. The fact that azadirachtin directly or indirectly inhibits the secretion of trypsin by the enzyme-secreting cells of the gut is discussed.     

草地贪夜蛾幼虫对四种刺激物质的味觉感受和取食选择

【目的】为了筛选有效的草地贪夜蛾Spodoptera frugiperda幼虫取食激食素和抑制剂并探究其味觉感受机理,为生态防治草地贪夜蛾提供理论和实践上的依据。【方法】利用单感受器记录法测定草地贪夜蛾5龄第2天幼虫下颚外颚叶上中栓锥感器和侧栓锥感器对不同浓度的蔗糖、黑芥子苷、单宁酸和盐酸奎宁4种刺激物质的电生理反应,并采用二项叶碟法测定草地贪夜蛾幼虫对这些刺激物质的取食选择行为。【结果】草地贪夜蛾幼虫中栓锥感器和侧栓锥感器内均存在对蔗糖、黑芥子苷和单宁酸敏感的味觉受体神经元,但是神经元的活性随着刺激物的种类及浓度而变化。其中,两类感器内神经元对蔗糖和黑芥子苷的反应均呈现典型的浓度梯度反应。中栓锥感器内存在对盐酸奎宁敏感的味觉受体神经元,但是呈现逆浓度梯度的反应模式,侧栓锥感器内不存在对盐酸奎宁敏感的神经元。蔗糖显著诱导幼虫的取食行为,而盐酸奎宁、黑芥子苷和单宁酸均抑制幼虫的取食行为,且都呈现浓度梯度的抑制活性。【结论】草地贪夜蛾幼虫中栓锥感器和侧栓锥感器内均存在对取食激食素和抑制剂敏感的味觉受体神经元,但是两类感器不论在反应谱上还是敏感性上均存在差异。蔗糖可以作为取食激食素,盐酸奎宁、黑芥子苷和单宁酸可以作为取食抑制剂的有效候选物质。幼虫对蔗糖、黑芥子苷和单宁酸的味觉反应在一定程度上解释了其取食选择行为的味觉基础。本研究为草地贪夜蛾的生态防治提供了味觉信息。     

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.     

SYSTEMIC INSECTICIDAL ACTION OF AZADIRACHTIN, NEEM SEED AND CHINABERRY SEED EXTRACTS APPLIED AS SOIL DRENCHES TO POTTED PLANTS

Abstract  The systemic insecticidal activities of: 1) azadirachtin, a potent insect antifeedant and growth regulator isolated from seeds of the neem tree, Azadirachta indica , 2) a neem seed extract containing 0. 30 quantitative fraction of azadirachtin, and 3) a methanolic extract of seeds from the chinaberry tree, Melia azedarach , a close relative of neem were determined. Soil drenches of azadirachtin or the neem seed extract at 1 and 2. 5 mg to potted potato ( Solanum tuberosum ) and tomato ( Lycopersicon esculentum ), remarkably retarded the growth of Manduca sexta larvae and strongly disrupted the pupation of Leptinotarsa decemlineata larvae, respectively. However, five to ten times as much azadirachtin or neem seed extract was needed to reduce the amount of tomato and potato foliage consumed. On sorghum ( Surghum biocolor ), much higher quantity (≥25 mg) of azadirachtin or the neem seed extract was necessary to slow the population growth of Schizaphis graminum , and prolong the survival of treated plants. But methanolic extracts of chinaberry seeds ( M. azedarach ) from two sources (Guangdong, PRC and Hurricane, Utah, USA) at 150 mg per pot were ineffective in reducing aphid population growth and preventing plant damage.     

Taste cell responses in the polyphagous arctiid, Grammia geneura: towards a general pattern for caterpillars

The caterpillars of Grammia geneura are polyphagous as individuals. Electrophysiological responses of its medial and lateral galeal styloconic sensilla to 21 amino acids, 6 carbohydrates, 10 chemically diverse plant secondary compounds and two inorganic salts were examined. In the medial sensillum, a single cell responded to 8 amino acids, 3 carbohydrates, and the iridoid, catalpol, which is present in a favored hostplant. In the lateral sensillum, one cell responded to amino acids and another to fructose. Two cells in each sensillum responded to secondary compounds and it is suggested that the same cells are stimulated by inorganic salts. There was no evidence of a separate salt-sensitive cell. Phenylalanine stimulated a deterrent cell in the medial sensillum and was behaviorally deterrent. Some essential amino acids did not stimulate any cells and it is suggested that a small number of amino acids (sometimes non-essential) may serve as indicators of nutrient quality. Sugars probably serve as the primary phagostimulants because they are in relatively high concentrations in plants. It is proposed that taste receptor cells should be categorized primarily by their behavioral effects as phagostimulatory or deterrent, rather than their specific ranges of responsiveness. This would emphasize the basic similarities across taxa.     

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     

Potential of Margosan-O, an azadirachtin-containing formulation from neem seed extract, as a control agent for spruce budworm, Choristoneura fumiferana

The effects on spruce budworm larvae, Choristoneura fumiferana (Clem.), produced by ingestion of Margosan-O, a commercially available neem seed extract formulation containing 0.3% azadirachtin, were investigated. Bioassays with the test material were conducted using various instars of spruce budworm larvae, with either artificial diet, cut branches of balsam fir, Abies balsamea (L.) Mill., or small growing balsam fir trees as substrates. The dose-response data on feeding reduction, developmental retardation, and mortality (LC50, LC95, and LD50) suggest that Margosan-O has promise as a control agent for spruce budworm in an integrated pest management program.     

家蚕味觉电生理反应的个体差异

为探讨家蚕Bombyx mori人工饲料饲养发育不齐的生理原因,从同一蚕品种中挑选出对人工饲料摄食性不同的个体,用电生理学方法测定了下颚瘤状体味觉感器对4种代表性物质（蔗糖、肌醇、大豆粉提取物和柠檬酸）的电生理反应。结果表明,栓锥感器Ss-Ⅰ对蔗糖等促食物质的反应以及栓锥感器Ss-Ⅱ对大豆粉提取物等阻食物质的反应,均存在明显的个体差异。在临界浓度下,低摄食性个体的放电脉冲频率显著高于高摄食性个体。说明低摄食性蚕的味觉反应比高摄食性蚕敏感。     

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.     

Gustatory perception of phytoecdysteroids in Plodia interpunctella larvae

Phytoecdysteroids are steroidal compounds produced by various plants that disrupt growth and development of insects eating them. They exhibit an insecticidal activity on a number of insect pests such as Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). In this study, we further evaluated whether phytoecdysteroids deter larvae of this species from feeding, by using four phytoecdysteroid molecules, commonly occurring in plants: 20‐hydroxyecdysone (20E), ponasterone A (PonA), polypodine B (PolB), and makisterone A (MakA). Fourth instar P. interpunctella avoided contact with food pellets treated with these phytoecdysteroids in a dose‐dependent way (2–30 mm ). In order to test whether this avoidance was mediated by taste sensitivity, we recorded the responses of taste neurons located in the lateral and medial sensilla styloconica of the galea. At least one neuron responded to each of these compounds in both sensilla. The neuron located in the medial sensillum had a detection threshold of 10^?6 m for PonA, 10^?4 m for 20E and PolB, and 10^?3 m for MakA. The lateral sensillum neuron responded with less intensity and its detection threshold was 10 times higher than that of the medial sensillum neuron. These results indicate that phytoecdysteroids are detected as deterrent stimuli by P. interpunctella larvae and that small structural differences significantly affect their biological activity.     

An electrophysiological analysis of the effect of phagostimulant mixtures on the responses of a deterrent-sensitive cell of gypsy moth larvae, Lymantria dispar (L.)

Gypsy moth larvae, Lymantria dispar (L.), are polyphagous feeders. The medial styloconic sensillum of this species bears a taste receptor cell that responds to alkaloids and another that responds to the sugar alcohol, inositol. The lateral styloconic sensillum bears a taste receptor cell that is sensitive to the sugar, sucrose. We tested the effect of two phagostimulants, namely sucrose and inositol, on the response of the deterrent-sensitive cell and found that both phagostimulants suppressed its response, equally, while their combination was significantly more effective. We also tested the effect of two alkaloids (i.e., strychnine and caffeine), which deter feeding in this species, on the response of the inositol- and sucrose-sensitive cells. Although both of these deterrents had no effect in suppressing the response of the sucrose-sensitive cell, they both had an effect in suppressing the inositol-sensitive cell. We also found that sucrose suppressed the response of the inositol-sensitive cell, whereas inositol had no significant effect on the response of the sucrose-sensitive cell. In this paper, we examined the effect of mixtures of these compounds to determine the nature of their interaction. In the context of host–plant interactions and, for example, host recognition, whereby host plant acceptability depends on the total sensory impression acquired from responses to multiple plant components rather than the presence or absence of single stimulant or deterrent compounds, this study could have a direct bearing in the development of natural compounds (i.e., alkaloids) for pest control and crop protection. It will also contribute to our understanding of the neural basis of the feeding behavior of this insect.     

Detection of alkaloids and carbohydrates by taste receptor cells of the galea of gypsy moth larvae, Lymantria dispar (L.)

Gypsy moth larvae are polyphagous feeders. The electrophysiological responses of the medial and lateral styloconic sensilla to four secondary compounds (e.g., alkaloids), two carbohydrates, and one inorganic salt were examined using an extracellular tip-recording method. In the medial sensillum, one taste receptor cell responded to the alkaloids, strychnine, caffeine, nicotine, and aristolochic acid (i.e., deterrent-sensitive cell), while another, responded to the sugar alcohol and inositol (inositol-sensitive cell). In both medial and lateral sensilla, two taste receptor cells in each sensillum responded minimally and sporadically to 30?mM potassium chloride (KCl) (i.e., KCl-sensitive cells); one cell produced much larger amplitude action potentials than the other. In the medial sensillum, only the large-amplitude KCl-sensitive cell exhibited an increased firing rate with increasing salt concentration. When binary mixture experiments were conducted, it was confirmed that the large-amplitude KCl-sensitive cell and the deterrent-sensitive cell in the medial sensillum were one in the same cell. Only a single cell in the lateral sensillum responded to the sugar, sucrose (sucrose-sensitive cell). The temporal dynamics of responses of the deterrent-sensitive, sucrose-sensitive, and inositol-sensitive cells were compared. Concentration?Cresponse data were obtained for the deterrent-sensitive cell to various alkaloids, as well as to KCl.     

An endophytic fungus from <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. that produces azadirachtin

Azadirachtin A and its structural analogues are a well-known class of natural insecticides having antifeedant and insect growth-regulating properties. These compounds are exclusive to the neem tree, Azadirachta indica A. Juss, from where they are currently sourced. Here we report for the first time, the isolation and characterization of a novel endophytic fungus from A. indica, which produces azadirachtin A and B in rich mycological medium (Sabouraud dextrose broth), under shake-flask fermentation conditions. The fungus was identified as Eupenicillium parvum by ITS analysis (ITS1 and ITS2 regions and the intervening 5.8S rDNA region). Azadirachtin A and B were identified and quantified by LC-HRMS and LC-HRMS², and by comparison with the authentic reference standards. The biosynthesis of azadirachtin A and B by the cultured endophyte, which is also produced by the host neem plant, provides an exciting platform for further scientific exploration within both the ecological and biochemical contexts.     

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.     

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.