Effects of azadirachtin on the nutrition and development of the tobacco budworm,Heliothis virescens (Fabr.) (Noctuidae)

The plant chemical azadirachtin was administered, either in artificial diet or by oral injection, to fifth instar larvae of the tobacco budworm, Heliothis virescens (Fabr.). At a dietary concentration of 0.03125 ppm, azadirachtin significantly reduced the amount of diet consumed and the weight gained by the larvae. Higher dietary concentrations (0.25 and 0.5 ppm) were necessary to reduce the efficiency of larval conversion of digested and ingested food, respectively. However, the approximate digestibility increased at the dietary concentration of 0.25 ppm.Orally injected azadirachtin (0.25 and 0.5 μg) delayed moulting to the pupal stage, produced defective pupae or adults, and inhibited development to the adult stage. Higher doses (5.0 and 10.0 μg) reduced the pre-pupal weight loss normally associated with pupation, and completely inhibited pupation. At the critical dose of 1.0 μg (the minimal dose that disrupted development to the pupal stage), azadirachtin had less of an effect on older than on younger larvae. Larvae injected on the first day of the fifth instar failed to pupate, whereas approx 40% of those injected on subsequent days pupated.The results suggest that azadirachtin affects H. virescens in a manner similar to other tested species of insects. The significance of these results, especially regarding hormonal events in the insects, is discussed.     

Systemic antifeedant effects of azadirachtin on the peach-potato aphidMyzus persicae

The electrical penetration graph (EPG) method was used to analyse the feeding behaviour of apterous, adultMyzus persicae (Sulzer) (Homoptera: Aphididae) onNicotiana clevelandii (Gray) seedlings, treated systemically with azadirachtin. A preliminary experiment showed that the effects of tethering aphids for EPG recording were minimal. The percentage of the 9 h recording period devoted to non-penetration activities, and to stylet pathway patterns increased as the azadirachtin concentration in the root treatment increased. The number of probes initiated, and the numbers of sieve tube penetrations also increased with increased azadirachtin concentration. The mean time elapsing between the initiation of the first probe to reach a sieve element and contact with this tissue was not significantly altered by azadirachtin treatment. However, azadirachtin treatment significantly reduced the percentage of probes that reached sieve elements and increased non-penetration activity before and after the first perid of ingestion from the sieve elements. The percentage of the recording period spent in the EPG pattern associated with sieve tube penetration was significantly reduced by an azadirachtin concentration of 300 ppm, and the duration of each individual penetration was significantly reduced by an azadirachtin concentration of 100 ppm. When the total EPG was split into 3 h periods, significant interactions were seen between time period and azadirachtin concentration for the duration of non-penetration, pathway, and sieve tube penetration patterns.     

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.     

Azadirachtin: its effect on gut motility, growth and moulting in Locusta

ABSTRACT. The biological effect of azadirachtin on fifth instar nymphs of Locusta migratoria migratorioides (R & F) have been studied in detail. Azadirachtin injection at the beginning of the instar results in a dose-dependent range of developmental aberrations. Low concentrations ( c. 1.7/μg/g body weight) result in adults with curled wing tips and reduced longevity; higher concentrations ( c. 2.9μg/g) result in death during the imaginal moult; doses of c. 6.5μg/g cause death immediately prior to the moult; and doses of c. 7.3μg/g induce a greatly extended instar. Such doses are related to a proportionately slower growth rate of the insect and a significantly reduced food intake, as assessed by wet weight and faeces production. Doses of 80μg/g result in death within 24 h. Experiments in vivo and in vitro demonstrate a significant reduction with azadirachtin treatment in the rate of passage of food through the gut, and in gut motility. The significance of this direct effect on gut motility is discussed in relation to the mode of action of azadirachtin on growth and moulting.     

A COMPREHENSIVE STUDY ON APOPTOSIS INDUCTION BY AZADIRACHTIN IN Spodoptera frugiperda CULTURED CELL LINE Sf9

The induction of apoptosis by azadirachtin, a well‐known botanical tetranortriterpenoid isolated from the neem tree (Azadirachta indica A. Juss) and other members of the Meliaceae, was investigated in Spodoptera frugiperda cultured cell line (Sf9). Morphological changes in Sf9 cells treated by various concentrations of azadirachtin were observed at different times under light microscopy. Morphological and biochemical analysis indicated that Sf9 cells treated by 1.5 μg/mL azadirachtin showed typical morphological changes, which were indicative of apoptosis and a clear DNA ladder. The flow cytometry analysis showed the apoptosis rate reached a maximum value of 32.66% at 24 h with 1.5 μg/mL azadirachtin in Sf9 cells. The inhibition of Sf9 cell proliferation suggested that the effect of azadirachtin was dose dependent and the EC₅₀ at 48 and 72 h was 2.727 × 10⁻⁶ and 6.348 × 10⁻⁹ μg/mL, respectively. The treatment of azadirachtin in Sf9 cells could significantly increase the activity of Sf caspase‐1, but showed no effect on the activity of Topo I, suggesting that the apoptosis induced by azadirachtinin Sf9 cells is through caspase‐dependent pathway. These results provided not only a series of morphological, biochemical, and toxicological comprehensive evidences for induction of apoptosis by azadirachtin, but also a reference model for screening insect cell apoptosis inducers from natural compounds.     

Insect growth regulating and antifeedant effects of neem extracts and azadirachtin on two aphid species of ornamental plants

Leaf disc choice test bioassay demonstrated that formulated neem seed extracts were highly deterrent and growth regulatory to rose aphid,Microsiphum rosae (L.) and Chrysanthemum aphid,Macrosiphoniella sanbornii (Gillete). Effective concentrations to produce 50% feeding deterrence was 0.80 and 0.84% respectively for 2nd instar nymphs irrespective of bioassay duration. The disruption of aphid feeding was related to the presence of azadirachtin concentration in the extract. The toxicity on contact from the leaf surface or via topical application due to azadirachtin was significantly different and topical treatment was at least 7 times more effective for both species. Thus growth regulatory effects of azadirachtin were influenced by the host plant and the stage of treatment. Field evaluation with formulated neem extracts revealed the effect to be more of growth regulatory nature thereby showing that azadirachtin is a physiological toxin for aphid species. Neem seed extracts reduced the population of aphid on respective host plants significantly, EC₅₀ values being 0.88 and 0.96% forM. rosae andM. sanbornii respectively.     

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.     

非嗜食植物提取物对桔全爪螨产卵的驱避性测定

The oviposition repellency of the alcohol extracts from 50 species of non-preferable plants and azadirachtin against citrus red mite ( Panonychus cirri ) was determined using laboratory bioassays. In choice tests, the extracts from 42 of the 50 plant species and 1% azadirachtin (2000×) significantly reduced oviposition 1d after treatment.The repellency effect of the extracts from the 42 plant species was better than that of azadirachtin. Mikania micrantha extract had the best result, with an Interference Index of Population Control (IIPC) of 0.087 1 d after treatment, and significantly reduced oviposition for 9 d, while the extracts from Sesbania cannabina, Allium tuberosum , Paederia scandens , Duranta erecta and Dicranopteris pedata also had good effects, with an oviposition repellency of over 70 % 1 d after treatment, and significantly reduced oviposition for 4 - 6 d. The effect became weaker as time went on. None of the extracts showed significant oviposition attraction.     

Effect of orally administered azadirachtin on non-specific immune parameters of goldfish Carassius auratus (Linn. 1758) and resistance against Aeromonas hydrophila

Modulation of the immune responses using active bio-ingredients as a possible prophylaxis measure has been novel prospect for aquaculture. The present study evaluated the effects of azadirachtin EC 25% on non-specific immune responses in goldfish Carassius auratus and resistance against pathogenic bacteria Aeromonas hydrophila. The experimental trial for effects of azadirachtin on immuno-haematoloical parameters in goldfish was conducted by feeding the various levels of azadirachtin as control T₀ (without azadirachtin), T₁ (0.1%), T₂ (0.2%), T₃ (0.4%), T₄ (0.8%) and T₅ (1.6%) for a period of 28 days. Fishes were challenged with A. hydrophila 28 days post feeding and relative percentage survival (%) was recorded over 14 days post infection. Immuno-haematoloical (total erythrocyte count, total leukocyte count, haemoglobin, packed cell volume, NBT activity, phagocytic activity, serum lysozyme activity, myeloperoxidase activity, total immunoglobulin) and serum biochemical parameters (serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and blood glucose) of fishes were examined at 14 and 28 days of feedings. Fish fed with azadirachtin, showed significantly (p < 0.05) enhanced TEC, TLC, Total Ig, total protein, NBT activity, serum lysozyme activity and myeloperoxidase level in different treatment groups in comparison with control group. Similarly, SGOT, SGPT and blood glucose level were found to be significantly (p < 0.05) high but PCV and Hb did not differ significantly (p > 0.05) in the treatment groups compared to control groups. Azadirachtin at the concentration of 4 g kg^?1 showed significantly (p < 0.05) higher relative percentage survival (42.60%) when compared with the control against A. hydrophila infection. This study indicated that azadirachtin EC 25% (4 g kg^?1) showed higher NBT activity, serum lysozyme, protein profiles, leukocyte counts and resistance against A. hydrophila infection and thus, can be used as a potential immunostimulant in aquaculture.     

Individual Differences in Motor Timing and Its Relation to Cognitive and Fine Motor Skills

The present study investigated the relationship between individual differences in timing movements at the level of milliseconds and performance on selected cognitive and fine motor skills. For this purpose, young adult participants (N = 100) performed a repetitive movement task paced by an auditory metronome at different rates. Psychometric measures included the digit-span and symbol search subtasks from the Wechsler battery as well as the Raven SPM. Fine motor skills were assessed with the Purdue Pegboard test. Motor timing performance was significantly related (mean r = .3) to cognitive measures, and explained both unique and shared variance with information-processing speed of Raven''s scores. No significant relations were found between motor timing measures and fine motor skills. These results show that individual differences in cognitive and motor timing performance is to some extent dependent upon shared processing not associated with individual differences in manual dexterity.     

Toxicity of some insecticides and acaricides to the predatory bugDicyphus tamaninii (Het.: Miridae)

Several pesticides were tested in the laboratory for their side-effects upon the mirid bugDicyphus tamaninii, a polyphagous predator used for IPM programmes in some vegetable crops. Residual toxicity to 3rd -4th instar nymphs on tomato leaflets was checked 24, 48 hours and seven days after treatment. The acaricides bromopropylate, dicofol+tetradifon and fenpyroximate were harmless to the nymphs. The insect growth regulators azadirachtin, buprofezin, lufenuron and pyriproxyfen were also harmless to nymphs and teflubenzuron was slightly harmful seven days after treatment. Among the conventional insecticides tested, only pirimicarb and tau-fluvalinate were harmless toD. tamaninii nymphs     

Bioefficacy and mode of action of rocaglamide from Aglaia elaeagnoidea (syn. A. roxburghiana) against gram pod borer,Helicoverpa armigera (Hübner)

Abstract: Rocaglamide, a highly substituted benzofuran, was isolated and identified as the main biologically active component in Aglaia elaeagnoidea (syn. A. roxburghiana) for gram pod borer Helicoverpa armigera (Hübner). Addition of rocaglamide to an artificial diet retarded the growth of neonate larvae in a dose‐dependent manner with EC₅₀ values of 0.76 p.p.m. These values compared favourably with azadirachtin (EC₅₀ = 0.23 p.p.m.). However, azadirachtin was apparently more potent than rocaglamide in inducing growth inhibition via oral administration to these first stadium larvae. The candidate compound was found to have LD₅₀ and LD₉₅ values of 0.40 and 1.02 μg per larva, respectively, in topical application against third instar larvae 96 h post‐treatment. However, these values for azadirachtin were 8.16 and 25.8 μg per larva for the same period. This shows that azadirachtin was less effective against third instar H. armigera larvae in inducing acute toxicity via topical treatment in comparison with rocaglamide. However, severe morphological larval deformities were observed in such azadirachtin‐treated larvae during the process of ecdysis. The cytotoxic nature of rocaglamide was established by evaluating dietary utilization and the results did not implicate any antifeedant effect but the toxicity‐mediated effect due to reduced efficiency of conversion of ingested food. It was obvious that feeding deterrence is not the primary mode of action but a centrally mediated effect, which could be due to the induced cytotoxicity at non‐specific cellular levels.     

Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Immunostimulatory effect of azadirachtin in Oreochromis mossambicus (Peters)

The effect of azadirachtin, a triterpenoid derived from Azadirachta indica on the immune response was studied in the freshwater teleost, O. mossambicus. Bovine serum albumin (BSA) and sheep erythrocytes (SRBC) were used as antigens to evoke immune response. The immune responses in fish were measured by quantifying antibodies produced and counting the peripheral blood leucocytes in control and experimental fish. In general, azadirachtin significantly enhanced the antibody response and leucocyte count in a dose dependent manner. An inverse relationship was observed between the dose of azadirachtin and the degree of immunostimulation. Timing of azadirachtin administration in relation to immunization revealed that the maximum enhancement of antibody response was observed when the stimulant was given two days prior to immunization. The observed immunostimulatory property of azadirachtin has an implication in the maintenance of finfish health in freshwater intensive aquaculture practices.     

Azadirachtin affects growth and endocrine events in larvae of the tobacco hornworm, Manduca sexta

Injection of azadirachtin in freshly emerged last-instar larvae of Manduca sexta elicited different reactions according to the dose administered. At low doses, pupation occurred in most of the cases, but the resulting pupae were defective for the most part. Individuals treated with higher doses usually did not fully complete development, moulting to supernumerary larvae or dying as larvae (sometimes at the wandering stage) after varying periods of survival. The haemolymph ecdysteroid titre of individuals treated with 2 μg azadirachtin/g bodyweight showed characteristic changes which are presumed to cause the disorders in the last stages that normally lead to pupation. Injection of moulting hormone in azadirachtin-treated individuals at certain times during the penultimate stage elicited no reduction of the azadirachtin-induced effects. It is shown that azadirachtin is able to inhibit development even when individuals performed a complete moult after the treatment.     

Effects of insect growth regulators on citrus mealybug parasitoid Leptomastix dactylopii (Hymenoptera: Encyrtidae)

In this study, we measured the effects of three commonly used insecticides classified as insect growth regulators, on the encyrtid parasitoid Leptomastix dactylopii (Howard) when parasitizing citrus mealybug, Planococcus citri (Risso). Kinoprene, pyriproxyfen, and azadirachtin were evaluated in both petri dish and a cage experiment at label-recommended rates to measure their effects on the mortality, parasitization rate, and sex ratio of L. dactylopii. Insecticides were applied to petri dishes and plants either immediately before, 24 h before, or 48 h before release of the parasitoid. Kinoprene applied 24 h before parasitoid release caused 100% mortality of L. dactylopii in petri dishes within 48 h. Mortality rates for L. dactylopii exposed to azadirachtin and pyriproxyfen did not exceed 5% regardless of release time. There were no release time x insecticide interactions on L. dactylopii parasitization rate. The insecticide alone, however, did significantly affect parasitization rates of L. dactylopii on P. citri; the kinoprene treatment significantly reduced L. dactylopii parasitization rates compared with azadirachtin and pyriproxyfen. In a cage experiment with coleus, Solenostemon scutellaroides (L.) Codd, applications of both pyriproxyfen and kinoprene resulted in fewer P. citri parasitized by L. dactylopii than azadirachtin or the control. The sex ratio was equivalent in the petri dish experiment, whereas in the cage experiment the sex ratio was biased toward males, particularly for the kinoprene treatment. Based on the results from this study, we suggest that kinoprene is not compatible with releases of L. dactylopii to control citrus mealybugs.     

乙基多杀菌素和印楝素对西花蓟马生长发育及繁殖的亚致死效应

为探讨乙基多杀菌素和印楝素对西花蓟马的亚致死效应,采用浸叶法确定了它们对西花蓟马2龄若虫的亚致死浓度(LC₂₅),并研究了该浓度下两种生物源农药对西花蓟马生长发育和繁殖力的影响.结果表明: 两种生物源农药亚致死浓度处理对西花蓟马均有不同程度的影响.在两性生殖方式下,乙基多杀菌素对西花蓟马产卵前期、雌成虫寿命和产卵量均无显著影响,但雄成虫寿命显著缩短;印楝素处理后,西花蓟马产卵量显著降低,产卵前期明显延长.在孤雌生殖方式下,不论乙基多杀菌素和印楝素处理,西花蓟马产卵前期均明显延长,但只有印楝素使雌成虫寿命缩短和产卵量显著降低.两种生物源农药对西花蓟马次代各虫态的发育历期影响不同,无论在何种生殖方式下,乙基多杀菌素处理后西花蓟马的未成熟期均明显短于印楝素处理.乙基多杀菌素处理后西花蓟马的内禀增长率(r_m)和周限增长率(λ)均高于对照,而印楝素较对照低.以上结果说明,两种杀虫剂对西花蓟马生长发育和繁殖影响不同,乙基多杀菌素亚致死浓度对西花蓟马种群的增长有一定的刺激作用,而印楝素有明显的抑制作用.两种杀虫剂的影响程度还与西花蓟马的生殖方式有关.     

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.     

Efficacy of hCG and GnRH for inducing ovulation in the jenny

Knowledge about management of ovulation in the donkey is limited compared to that in the horse. This experiment was designed to evaluate the efficacy of injecting single doses of lecirelin (a GnRH-analogue) or of hCG to induce ovulation in the jenny and to determine whether effects are dependent upon follicular diameter at time of injection. Ovarian activity and follicular growth were monitored by rectal ultrasonography. Jennies were randomly allotted to the following groups: Group GnRH, treated with 100 microg lecirelin; Group hCG, treated with 2500 IU hCG; Group C, untreated and monitored for spontaneous ovulation. Animals were also categorized into subgroups depending upon follicular diameter: 30-35 mm (GnRH-1, hCG-1 and C-1) or 36-40 mm (GnRH-2, hCG-2 and C-2). Jennies in the two hormone treatment groups did not differ significantly for time from treatment to ovulation, but there was a significant reduction in time to ovulation as follicle size at treatment increased. Jennies treated with either lecirelin or hCG had significantly smaller follicle size at ovulation than jennies in the Control groups that underwent spontaneous ovulation. Treatment groups did not differ significantly in the proportion of jennies that ovulated within 48 h of injection or between 25 and 48 h following injection. These results highlight the usefulness of lecirelin for induction and synchronization of ovulation in the jenny, particularly since it would avoid the risk of reduced hCG response in reproductive management programs in which that hormone was repeatedly used.