Influence of azadirachtin on development and reproduction of Nezara viridula L. (Het., Pentatomidae)

Abstract:  The potential of azadirachtin as an insect growth regulator was evaluated in Nezara viridula L. by applying different doses to fifth instar nymphs. At doses of 200–500 ng per insect almost all the individuals died during moulting to adults and the survivors showed a great number of nymphal characteristics, and also died very soon after. At lower doses, from 2 to 50 ng per insect, approximately 50% of the adults seemed normal. No effect was observed during the last nymphal instar, even at the highest dose applied. The fertility and fecundity of adults, males and females, developing from nymphs treated with different low doses of azadirachtin were also studied. When azadirachtin was applied at 20 ng per insect on fifth instar nymphs, females developing from these nymphs showed a conspicuous reduction of fecundity. This same effect was evident when untreated females mated with males obtained from treated nymphs. Finally, azadirachtin applied at 1  μ g/cm² to the oviposition surface showed a remarkable oviposition repellency.     

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.     

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.     

Growth and survival strategy of the Antarctic mite Alaskozetes antarcticus

Growth and mortality rates of three juvenile instars and adults of the oribatid mite Alaskozetes antarcticus were measured over a twelve month period using field enclosures m Its maritime Antarctic habitat A pattern was found of rapid growth during the short summer period (0 2-0 4 μg dry weight increment per day) followed by gradual weight loss overwinter Most individuals completed one instar per year, reaching a premoult resting phase by late summer, with the moult being delayed and relatively synchronised early the next summer A small proportion of proto- and deutonymphs completed two moults within one summer period After the final moult adults overwintered again before commencing oviposition, suggesting that the life cycle (egg-egg) will take at least five years Mortality was not biased towards any instar or time of year Highest individual growth rates were obtained at a constant temperature of 7°C in the laboratory However expenmentai increase of mean field temperature from 2 7 to 4 3°C by use of a plastic cloche led to decreased growth, although no change in moulting frequency or mortality rate Laboratory survival was greatest at 2°C and decreased with increasing temperature
Alaskozetes antarcticus may be descnbed as 'a' (adversity) selected, showing an exceptionally long life cycle of five to six years and extensive physiological investment in survival adaptations Moult synchronisation in early summer, involving entering winter in the inactive premoult phase, will increase overwinter survival by reducing the chance of inoculative freezing from gut contents, and may be advantageous in maximising the time available for feeding during the subsequent summer, or ensuring proximity of the sexes after the final moult     

Azadirachtin-induced effects on larval-pupal transformation of Spodoptera mauritia

Abstract. Penultimate (fifth) and last (sixth) stadium larvae of Spodoptera mauritia Boisd. (Lepidoptera: Noctuidae) of various ages were injected with 0.5 μg, 1 μg or 2 μg azadirachtin and the effects on moulting and larval-pupal transformation were analysed. Higher doses (1 μg and 2 μg) of azadirachtin induced a prolongation of the fifth stadium in larvae treated on day 0 and day 1. The resulting sixth stadium larvae failed to pupate. Sixth stadium larvae injected with 0.5 μg, 1 μg or 2 μg azadirachtin showed prolongation of sixth larval period. Azadirachtin treatments completely prevented normal pupation in 'day 0' and 'day 1' larvae even though the percentage of pupation increased in treated larvae of other age groups. Injection of 2 fig azadirachtin prevented normal pupation in larvae of all age groups. Injection of 4 μg ecdysterone to sixth stadium larvae pre-treated with 1 fig azadirachtin (on day 0) promoted normal pupation in the majority of animals.     

Age-dependent responses of last instar larvae of Spodoptera mauritia to precocene II

ABSTRACT. Last instar larvae of Spodoptera mauritia Boisd. (Lepidoptera: Noctuidae) of various ages were treated with a single dose of 80 μg or 160 μg of precocene II (PII) and the effects of larval—pupal transformation were studied. Final instar (sixth) larvae treated on day 0, 1 or 2 with 80 μg P II showed a high mortality; P II treatment during subsequent days of this stadium showed a diminishing tendency for mortality. P II prolonged the larval—pupal period in all larvae but the effect was somewhat reduced in the mid-stadium.
Treatments of 160μg P II to 0-day-old, 1-day-old, 2-day-old and 3-day-old larvae were highly toxic. Treatments of 4-day-old and 5-day-old larvae with 160 μg P II delayed pupation by 1 day, but in addition the larvae often moulted into larval—pupal intermediates. Co-application of 50μg juvenile hormone analogue (ZR 512) with 160 μg P II restored both normal morphogenesis and a normally-timed moult.     

Growth, body composition and hepatic tyrosine aminotransferase activity in cortisol-fed channel catfish, Ictalurus punctatus Rafinesque

Metabolic consequences of chronic elevation of cortisol in the diet of yearling channel catfish, Ictalurus punctatus , were studied. Cortisol was incorporated into the diet in concentrations of 1, 10, 50 and 100 μg/g of food. This diet was offered at 3% of the body weight per day for 10 weeks. Fish were individually weighed and measured at 2-week intervals and feeding rates were adjusted. Body weight, liposomatic index and condition factor were significantly lower and food conversion was significantly higher in fish fed 50 or 100 μng cortisol/g of food when compared with controls. The hepatosomatic index of fish fed cortisol at the rate of 100 μg/g of food decreased significantly. Specific activity of hepatic tyrosine aminotransferase was significantly higher at the two highest cortisol doses. Long-term cortisol administration can reduce growth and condition factor by activating gluconeogenic mechanisms in which lipids and amino acids, rather than carbohydrates, are used for energy production. The metabolic effects of exogenous cortisol in this study offer an explanation for the decreased growth of fish under conditions that activate the secretion of endogenous cortisol.     

The effects of azadirachtin on the endocrine control of moulting in Locusta migratoria

The effects of azadirachtin, a compound from the neem tree, Azadirachta indica A. Juss, were studied during the last-larval instar of Locusta migratoria. A dose-response relationship was established using moult inhibition and mortality as effective parameters. Although injected azadirachtin elicits feeding inhibition, our results prove that moult inhibition is due to an interference with the endocrine system rather than to the altered feeding behaviour. Modification and suppression of the ecdysteroid titre by azadirachtin is closely correlated with the morphogenetic effects. Inhibition of eclosion processes, however, suggest a wide-spread blockage of factors presumably located in the central nervous system.     

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.     

The effect of azadirachtin on the midgut histology of the locusts, Schistocerca gregaria and Locusta migratoria

The effects of azadirachtin on midgut cells of adult Schistocerca gregaria and Locusta migratoria were investigated by both light and electron microscopy. Pathological effects were seen in both species which could be related to dose and time after treatment. The midgut cells showed a slow necrosis, a rounding up of the cell and swelling of organelles; the nidi were reduced in size and number, the connective tissue beneath the epidermal layer became enlarged with many invading cells/nuclei and the circular and longitudinal muscles become rounded and swollen. The pathological effects were closely linked to a loss of feeding, with injections of 5, 10 and 15 mug/g azadirachtin causing an increasingly rapid onset of the effects associated with an increasingly reduced food intake. At the highest dose, full histopathological effects were seen at 8 hr and death occurred after 3 days. At 1 mug/g, however, the insects showed midgut disruption and reduced feeding by day 3 post-treatment but both feeding and gut pathology had recovered by day 7. At all times, histopathological effects were different from those in starved insects.     

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.     

Carbohydrate and lipid metabolism during the last larval moult of the tobacco hornworm, Manduca sexta

Abstract. At 25°C and with a light regime of 17 h light and 7h dark, the last larval moult of the tobacco hornworm, Manduca sexta , lasts approximately 32 h, during which profound changes of metabolism were observed. At the onset of the moult, which coincides with the cessation of feeding, the proportion of active fat body glycogen phosphorylase increased from 10 (-2h) to 25–30% (Oh). A biphasic pattern with peak activities of 45–50% after t – 12 h and again just prior to the shedding of the cuticle (32 h) was subsequently observed. Haemolymph trehalose concentration decreased significantly from c. 35 (Oh) to 20mM (8h), but then recovered to an intermediate level (30mM; 12h). After completion of the moult, the trehalose concentration was 35–40 mM. The haemolymph glucose level in feeding fourth instar larvae was 4–5 mM, but decreased sharply before the onset of the moult to c. 1 mM, followed by a slow 6-fold increase over the next 20h. Prior to the shedding of the cuticle, the glucose level dropped again dramatically. The haemolymph lipid level increased slowly from an initial level of 1.2–1.4mg/ml during the early part of the moult, reaching a maximum of 1.8mg/ml after /= 16 h. Afterwards, a decrease of c. 50% was observed until ecdysis occurred. Oxygen consumption per animal decreased steadily from 30–35 μl/min pre-moult by approximately 70% to c. 10 μl/min but started to increase about 5 h before the animals resumed feeding.     

Quantitative studies on anti-allatin and lethal effects of a synthetic precocene in different larval instars of the desert locust

The lethal and anti-allatin effects of various doses of 7-ethoxy-precocene II (PIII), topically applied to crowdedSchistocerca gregaria nymphs, 0–27 h after the previous moult, were investigated for the five nymphal instars. The LD (lethal dose) probit lines were parallel for the different instars. The ED (effective anti-allatin dose) probit lines were also parallel. But for each instar the LD and ED probit lines seemed to be convergent toward the higher doses. The LD₅₀ and the ED₅₀ (g PIII/g fresh weight) were not widely separated; the LD₅₀/ED₅₀ ratio was instar dependent and varied between 1.26 (Vth instar) and 5.35 (IIIrd instar). Thus, separation between lethal effect, anti-allatin effect and no effect was rather poor; indeed in many instances all these types of results were obtained in a group of hoppers treated with the same dose in the same instar. A low precocene susceptibility was observed for the Ist instar and for each subsequent instar the susceptibility further decreased; the LD₅₀ and the ED₅₀ were respectively 886 g/g and 328 g/g for the Ist instar, but increased to 14 307 g/g and 11 367 g/g for the Vth instar. The anti-allatin effect of the precocene induced various morphogenetic responses: (1) permanent adultiform in the instar next to that treated; (2) death in the moult from the next to the second consecutive instar; (3) permanent adultiform in the second consecutive instar. The occurrence and/or distribution of these responses were instar dependent and for the first three instars also limitedly dose dependent. The anti-allatin effect of the precocene applied to Vth (last) instar nymphs became overt by sterility and lack of yellow colour in the adult. An overt anti-allatin effect was always coupled with a response dependent increase of the duration of the instar treated and/or of the next instar.

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Etudes quantitatives des effets létaux ou antiallates d'un précocène de synthèse sur différents stades larvaires du criquet pélerin

Résumé L'étude a porté sur les effets létaux ou antiallates de différentes doses de 7-éthoxy-précocène II (PIII), déposées de 0 à 27 heures après la mue sur la surface dorsale de l'abdomen de larves groupées de chacun des 5 stades larvaires deSchistocerca gregaria. Les courbes des doses létales (LD) ont été parallèles pour les différents stades. Les courbes pour les doses antiallates (ED) étaient elles aussi parallèles. Cependent pour chaque stade, ces deux courbes semblaient converger et même se croiser vers les doses les plus fortes. Les LD₅₀ et ED₅₀ (en g de PIII par gramme de poids frais) n'étaient pas nettement séparées; le rapport LD₅₀/ED₅₀ dépendait du stade et variait entre 1,26 (5è stade) et 5,35 (3è stade). Ainsi la distinction entre effet létal, effet antiallate et absence d'effet était plutôt faible; à la vérité, dans de nombreux cas, tous ces effets ont été obtenus dans un groupe de criquets traités avec la même dose au même stade. Une sensibilité assez faible au précocène a été observée, même au ler stade, et pendant chaque stade suivant la sensibilité ultérieure diminuait; LD₅₀ et ED₅₀ ont été respectivement 886 g/g et 328 g/g pour le ler stade, mais ont augmenté à 14 307 g/g et 11 367 g/g pour le 5è stade. Les effets antiallates ont induit différentes conséquences morphologiques: (1) des adultiformes permanents lors du stade suivant immédiatement le stade traité; (2) mort pendant la mue du ler ou 2nd stade suivant le traitement; (3) des adultiformes permanents dans le 2nd stade après le traitement. L'apparition et/ou la distribution de ces réponses dépendaient du stade, et pour les 3 premiers stades étaient partiellement dépendantes de la dose. L'effet antiallate du précocène au 5è stade (dernier) devenait évident avec la stérilité et la perte de la coloration jaune de l'adulte. Un effet antiallate manifeste était toujours associé à un accroissement d dépendant de la dose de la durée du stade traité et/ou du stade suivant.

     

Effects of insect growth regulators on the hairy rose beetle, Tropinota squalida (Col., Scarabeidae)

Abstract:  Saponin extract from alfalfa roots, azadirachtin from the neem seed oil, synthetic ecdysteroid agonist RH-2485, and the juvenoid hydroprene disturb the development and reproduction of Tropinota squalida . Feeding beetles on diets containing 750 p.p.m. saponins, 7.5 p.p.m. RH-2485, and 1.13 p.p.m. azadirachtin reduces their progeny from 51 second instar larvae per female to 24, 15, and 15 larvae, respectively. When the larvae of untreated adults are fed for 1 week on dung with 75 p.p.m. saponins, 50 p.p.m. RH-2485, and 0.45 p.p.m. azadirachtin, the rate of adult emergence drops from 80% (controls) to 20, 0 and 13%, respectively. No adults emerge when the treatment is continued through the second and third larval instars. Two topical treatments of larvae with 0.2  μ g hydroprene decrease the rate of adult emergence from 90 to 11%, and treatments with 2  μ g prevent adult development in all insects. The observed effects warrant testing of azadirachtin, RH-2485, and hydroprene in the field. Several types of their application for the control of T. squalida are suggested.     

Occurrence of weight gain reduction and inhibition of metamorphosis and storage protein formation in last larval instars of the Mexican bean beetle, Epilachna varivestis, after injection of azadirachtin

Injection of the insect growth regulator azadirachtin into 40-h-old last larval instars of Epilachna varivestis (Mulsant) inhibited metamorphosis at doses lower than 0.3 g. Various higher doses (0.3–1.0 g) caused in addition weight gain reduction, whereas weight gain was almost entirely suppressed after injection of 2.5 g. Histological and electrophoretical analyses showed that storage protein formation in the fat body, necessary for pupation, did not occur in higher dose treated (1.0 g) individuals, but it was not affected by low dose (0.1 g) treatment. It is assumed that azadirachtin causes metabolic defects at higher concentrations.

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Zusammenfassung Injektion verschiedener Dosen von Azadirachtin in Larven des letzten Stadiums von Epilachna varivestis führt zu folgenden Ergebnissen: Niedrige Dosen (0.1–0.2 g) erzeugen keine Verminderung des Gewichtszuwachses, verhindern aber vollständig die Verpuppung. Bei etwas höheren Dosen (0.3–1.0 g) tritt zusätzlich eine einheitliche Verminderung der Gewichtszunahme auf, die in diesem Bereich nicht dosisabhängig ist. Es wird vermutet, daß Azadirachtin in höheren Dosen bestimmte metabolische Prozesse in einer bisher unbekannten Weise beeinflußt. Es wird weiterhin gezeigt, daß nach Injektion höherer Dosen die für die Metamorphose wichtigen Speicherproteine im Fettkörper nicht oder nur unzureichend gebildet werden.

     

Morphological colour change: Stage independent, optically induced ommochrome synthesis in larvae of the stick insect,Carausius morosus Br.

Summary The eyes of 4th, 5th, and 6th instar larvae of the stick insect,Carausius morosus, were partially covered with black varnish, and the ommochrome content of treated and untreated animals was determined throughout the instar. In untreated animals kept at 20°C in 16:8 LD the ommochrome content of the integument remains at the level found in newly hatched larvae (10 g/animal), up to the third moult, and increases only slightly (to 30 g) until the adult moult. Darkening the dorsal halves of the eyes does not influence the ommochrome content. Darkening the ventral halves, thus imitating the visual situation of an animal in light on a dark ground, causes after a lag of 3–4 days, a continuous increase of ommochrome content by about 5 g per day. The increase is equal at the beginning and during the second half of the 4th instar, and is also equal in the three instars investigated. Ommine and xanthommatine equally contribute to the increase.     

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.     

The accumulation of zinc by O-group plaice, Pleuronectes platessa (L.), from high concentrations in sea water and food

The uptake of zinc by young O-group plaice, Pleuronectes platessa was investigated at levels of high input from sea water and food ( Artemia nauplii). Zinc concentrations in the fish were strongly weight-specific and accumulation effects were distinguished by regression analysis. Sea water was estimated to contribute >10% of the total input up to levels corresponding to high environmental concentrations ( c. 0.1 μg Zn ml⁻¹) and food was the major source of zinc up to sea water concentrations of c. 0.6 μg Zn ml⁻¹. A high degree of regulation obtained along the food pathway to the fish although, in the system studied, most of this regulation was effected by the food organism. Regulation of food input explained the lack of variation in zinc levels previously observed in wild populations.     

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.