Azadirachtin,ecdysteroids and ecdysis in Locusta migratoria

1. The injection of azadirachtin into eight-day-old 5th instar nymphs of Locusta migratoria elicits different morphological effects dependent upon the timing of the injection relative to the major haemolymph ecdysteroid peak.2. Ecdysteroid levels can be drastically reduced, delayed and extended or not significantly affected by azadirachtin treatment dependent upon the timing of injection.3. Air swallowing to inflate the gut during the moult is significantly impaired in azadirachtin-treated insects resulting in both a reduced rate of inflation and a reduction in total air volume.4. The significance of azadirachtin treatment to the attainment of a “critical body mass”, the release of eclosion hormone and successful ecdysis is discussed.     

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.     

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.     

Impact of azadirachtin on vine weevil (Coleoptera: Curculionidae) reproduction

Abstract 1 The dose–response of azadirachtin on vine weevil, Otiorhynchus sulcatus (Fabricius), reproduction is investigated by confining adults to feed on treated Taxus × media leaves, and by counting and evaluating development in the resulting eggs. 2 A dosage‐dependent reduction in oviposition is discovered for foliar surface residues of azadirachtin, with an EC₅₀ of 25–50 parts per million (p.p.m) and 99.2% inhibition of viable egg production with 100 p.p.m. 3 Switching weevils from treated to untreated foliage allows reproductive capability to be restored for weevils that cease egg laying after azadirachtin exposure of 50 p.p.m. Weevils that had already started laying eggs in untreated groups soon cease oviposition once switched to azadirachtin‐treated foliage. 4 A transovarial effect results in a decrease in the percentage of viable eggs as the azadirachtin concentration increases. 5 The amount of feeding on foliage does not appreciably decrease at these hormonally effective concentrations, and adult weevil mortality is only slightly greater in the azadirachtin‐treated groups. Therefore, the overall effect of azadirachtin on weevil populations in the field is difficult to assess, except by collecting weevils to determine whether they are able to lay viable eggs.     

Moult cycle and its chronology in Mysis mixta and Neomysis integer (Crustacea, Mysidacea): implications for growth assessment

The moult-staging technique was used to determine the main moult stages (MS) and their duration in laboratory-reared juveniles and subadults of Mysis mixta and Neomysis integer. The relative duration of each stage was similar for both species with premoult occupying the major part of the moult cycle (51% and 44% for M. mixta and N. integer, respectively), followed by postmoult (26% and 34%) and intermoult (23% and 22%). Effects of temperature and feeding regimes on the chronology of the moult cycle were investigated. When the duration of the moult cycle (MCD) was extended by manipulating the feeding regime, animals prolonged their late postmoult and early premoult stages. At 12 and 5 °C, no specific moult stage varied in relative duration as long as food supply was high. Field application of moult staging for growth assessment was tested using wild-caught M. mixta. The MCD estimated via moulting experiments was compared to that obtained by analyzing moult stage distribution combined with the experimentally obtained data on stage duration. Close correspondence between two methods of MCD assessment was observed with moult-staging technique being particularly useful in situations when conducting of experiments immediately after collection is not feasible.     

Effects of azadirachtin on the moulting cycle,endocrine system,and ovaries in last-instar larvae of the milkweed bug,Oncopeltus fasciatus

Azadirachtin, injected into newly moulted last-instar larvae of Oncopeltus fasciatus, induces a variety of effects, which are dose-dependent. It can be used as a tool for studying some processes of the moulting cycle and their endocrine control, as well as regulation of egg maturation, since azadirachtin-induced permanent larvae exhibit adult ovarian development.Low doses of azadirachtin merely prolong the intermoult stage, apparently due to a delayed ecdysteroid peak. Medium and high doses suppress adult ecdysis, and the larvae become permanent larvae, the longevity of which increases with rising doses. Although medium doses prevent ecdysis, apolysis and secretion of adult cuticle takes place. The ecdysteroid peak is further delayed in these larvae and is somewhat lower than in controls. Permanent larvae induced by high azadirachtin doses show neither ecdysis nor apolysis. However, the epidermis engages in secretory activity which may correspond to adult procuticle secretion. These larvae also show an ecdysteroid peak, which is considerably delayed and distinctly lower than in the controls. Thus, treatment with different azadirachtin doses allows some dissection of the moulting cycle into different steps, in which the hormonal regulation can be studied independently.Adult ovarian development begins in a number of female permanent larvae induced with high azadirachtin doses, in some cases leading to chorionated eggs. The corpora allata are enlarged in a number of permanent larvae. It is suggested that the last-larval ecdysteroid peak sets a clock for activation of the corpora allata, i.e. its gonadotropic function, regardless of whether the adult moult takes place or not.Neurosecretion appears to be affected by azadirachtin, however, the exact mode of action remains to be determined.     

Carbohydrate metabolism in Manduca sexta during late larval development

The carbohydrate metabolism in Manduca sexta underwent significant changes during late larval development. Approximately 10% of fat body glycogen phosphorylase was active during the feeding period of the 5th instar, pharate-pupal development and after the pupal moult; it is concluded that glycogen synthesis prevailed. During the last larval and the pupal moult, as well as the wandering stage the percentage of active phosphorylase was significantly increased indicating that fat body glycogen stores were broken down to supply substrates to meet the demands of carbohydrate metabolism. In the course of the last larval moult and the wandering stage the fat body glycogen content decreased significantly from about 300 to about 200 μg mg⁻¹ dry mass substantiating that carbohydrates were released from the fat body. Prior to phosphorylase activation, the concentrations of total haemolymph sugars decreased significantly from about 12 to about 6 mg trehalose equivalents ml⁻¹ (last larval moult) and from about 18 to about 12 mg ml⁻¹ (wandering stage), and increased again slightly when phosphorylase was activated. The haemolymph glucose concentration decreased significantly from about 1.1 to 0.3 mg ml⁻¹ (last larval moult) and in the course of the 5th-instar feeding period from about 1.1 to 0.2 mg ml⁻¹, and remained at this level until the beginning of adult development. The amount of chitosan present in the cuticle increased steadily during the feeding period of the 5th instar from about 10 to 110 mg. It appears that fat body glycogen might be broken down during the last larval moult and the wandering period to provide substrates for chitin synthesis. A dramatic decrease in the amount of chitosan was observed prior to the pupal moult.     

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.     

Effects of azadirachtin on ecdysis of Rhodnius prolixus

The effects of azadirachtin on the development of 4th-instar nymphs of Rhodnius prolixus were studied. Given through a blood meal, a dose-response relationship of azadirachtin was established using antifeedant effect and ecdysis inhibition as effective parameters. The effective dose (ED₅₀) was 25.0 μg/ml and 4 × 10⁻⁴ μg/ml of blood, respectively, for antifeedant and ecdysis inhibition effects. Feeding inhibition is an indirect effect due to an interference of azadirachtin with the endocrine system rather than through the inhibition of chemoreceptors. Ecdysone given orally (5.0 μg/ml) and juvenile hormone analogue (70 μg/insect) counteracted the ecdysis inhibition as induced by azadirachtin.     

Haemolymph constituents and osmolality as functions of moult stage,body weight,and feeding status in marron,Cherax cainii () and yabbies,Cherax destructor (Clark, 1936)

The study investigates the change in osmolality and haemolymph constituents in marron Cherax cainii and yabbies Cherax destructor associated with moult stages, body weights and their feeding status. A total of 582 haemolymph samples from 5 moult stages (postmoult-AB, intermoult-C, and premoult stages – D₀, D₁, D₂), two body weight classes (2–15 g and 61–75 g) and nutritional status were used for analysis of osmolality, protein, glucose, and ionic concentrations of potassium and chloride following the standard biochemical procedures. The haemolymph protein, glucose, potassium and chloride levels were highest at intermoult and early premoult stages, and lowest at postmoult in both crayfish species. Except protein, no significant differences were seen in analyzed parameters between various weight classes and two species. Haemolymph osmolality, protein and glucose were significantly higher in fed crayfish, whereas no variations in haemolymph potassium and chloride concentrations were observed between the fed and unfed crayfish. Maximum osmolality was recorded at 7–8 h after feeding in both crayfish species. The results showed that the biochemical changes in the haemolymph of marron and yabbies are related to moult stages, body weight and feeding and thus can be used as tools for determining suitable diets.     

Control of moulting in the house cricket, Acheta domesticus

Last-instar Acheta domesticus moulted 7–8 days after the beginning of feeding, following ecdysis, or following starvation. Two full days of feeding were required for commitment to moult, and the minimal food consumption was estimated at 80–110 mg. All day-0 larvae weighing 200 mg, that achieved a critical weight of 280–300 mg after 48 hr of feeding, moulted. Two kinds of experiments indicated that an approximate 50% increase in wet-weight gain was required for a commitment to moult, which occurred at the end of the second day of feeding. Neck ligatures indicated that a head factor, presumably prothoracicotropic hormone, (PTTH), was released during the third day after the beginning of feeding provided the critical weight was achieved. Neither PTTH release nor ecdysis appeared to be under photoperiodic control in Acheta.     

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.     

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.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

The influence of azadirachtin on the feeding behaviour of cereal aphids and slugs

The probing behaviour of Rhopalosiphum padi (L.) and Sitobion avenae (F.) and the feeding behaviour of several slug species, (Deroceras reticulatum (Müller), Arion distinctus Mabille, Agriolimax caruanae Pollonera, Maximus sp.) were assessed on seedlings of winter barley (Hordeum marinum) treated with different concentrations of azadirachtin. Settling behaviour of both aphid species was strongly biased towards the untreated seedlings or those treated with low concentrations of azadirachtin. Concentrations of <500 ppm were effective with topical application and probing activity was reduced for at least 4 days after application. Systemic activity of azadirachtin against cereal aphids was also demonstrated. Feeding behaviour of the slug species, as seen by the amount of leaf eaten compared to the controls, was not affected by the presence of azadirachtin at those concentrations which deterred aphids from feeding. The relevance of these results to crop protection is discussed.     

Compatibility of the entomopathogenic fungus Beauveria bassiana with flufenoxuron and azadirachtin against Tetranychus urticae

Laboratory studies were developed to evaluate the compatibility of flufenoxuron and azadirachtin with Beauveria bassiana against Tetranychus urticae larvae along with the required Probit analysis of the involved chemicals on all of the life stages of this mite. Flufenoxuron displayed parallel regression lines for the mortality of eggs, deutonymphs and adults. Larvae and protonymphs were the most susceptible life stages. Protonymphs were 35 times more sensitive than eggs and adults. Azadirachtin gave equal mortality on proto- and deutonymphs. The response of eggs and adults was equivalent when treated with azadirachtin. The regression lines for proto- and deutonymphs were parallel to those of adults and eggs yet three times more sensitive. The effects of separate combinations of the entomopathogenic fungus Beauveria bassiana at its LC₂₀ with flufenoxuron and azadirachtin at their corresponding LC₄₀ were evaluated on mite larvae. The application of flufenoxuron with B. bassiana revealed a clear synergy. While the combination of azadirachtin and B. bassiana had an additive effect. These combinations with B. bassiana could improve mite control by contributing to a decline in the likelihood of resistance so often described in the literature.     

Mycopesticidal effects of characterized extracts of Penicillium isolates and purified secondary metabolites (including mycotoxins) on Drosophila melanogaster and Spodoptora littoralis

The characterized crude extracts of seven Penicillium isolates were bioassayed against Spodoptora littoralis larvae for weight reduction and mortality. A total of 15 metabolites were detected in the extracts, 13 of which were named. Five extracts caused significant reductions in weight and four of these caused significant increases in mortality, both at the P < 0.05 level. Seven purified secondary metabolites selected from those present in the crude extracts were bioassayed at 10 ppm against Drosophila melanogaster for feeding inhibition and S. littoralis for feeding inhibition and mortality. Significant effects (P < 0.05) were observed in all three assays for ochratoxin A, brevianamide A, citrinin, and penicillic acid. Viomellein significantly (P < 0.05) reduced feeding in D. melanogaster and survival in S. littoralis. Cyclopenol significantly (P < 0.05) inhibited feeding in both insects but not survival of S. littorolis. Significant effects were not observed for patulin (P > 0.05). High levels of feeding inhibition were obtained for brevianamide A (84.4 ± 18.21%) and penicillic acid (95.1 ± 1.21 %) against S. littoralis. Cyclopenol and brevianamide A were found to be antagonistic, and this is the first valid toxicity data ascribed to these metabolites. The implications of these results with respect to potential mycopesticides are considered.     

Influence of starvation on larval development of Carcinus maenas L. (Decapoda : Portunidae)

Lethal and sublethal effects of particular starvation events were investigated in larvae of Carcinusmaenas L. Mean survival times of continuously starved zoeae-1 were approximately twice the normal stage duration (12, 18, 25°C), and both increased with falling temperatures. At 6°C zoea-1 was unable to develop to stage-2. No larva retained the ability for successful further development if starved for half the stage duration time and was then refed. The zoea-1 larvae had to feed for at least 20 % of the normal stage duration for some larvae to moult to zoea-2. Some initial feeding was necessary to start zoea-1 development. Beyond a certain point of energy and accumulation of reserves development of the larvae seems to continue regardless of feeding rates. The demands for larval feeding correspond very well with the larval moulting cycle. Larvae of C. maenas proved to be well adapted to natural shortage of food.     

Bioinsecticide-Predator Interactions: Azadirachtin Behavioral and Reproductive Impairment of the Coconut Mite Predator Neoseiulus baraki

Synthetic pesticide use has been the dominant form of pest control since the 1940s. However, biopesticides are emerging as sustainable pest control alternatives, with prevailing use in organic agricultural production systems. Foremost among botanical biopesticides is the limonoid azadirachtin, whose perceived environmental safety has come under debate and scrutiny in recent years. Coconut production, particularly organic coconut production, is one of the agricultural systems in which azadirachtin is used as a primary method of pest control for the management of the invasive coconut mite, Aceria guerreronis Keifer (Acari: Eriophyidae). The management of this mite species also greatly benefits from predation by Neoseiulus baraki (Athias-Henriot) (Acari: Phytoseiidae). Here, we assessed the potential behavioral impacts of azadirachtin on the coconut mite predator, N. baraki. We explored the effects of this biopesticide on overall predator activity, female searching time, and mating behavior and fecundity. Azadirachtin impairs the overall activity of the predator, reducing it to nearly half; however, female searching was not affected. In contrast, mating behavior was compromised by azadirachtin exposure particularly when male predators were exposed to the biopesticide. Consequently, predator fecundity was also compromised by azadirachtin, furthering doubts about its environmental safety and selectivity towards biological control agents.