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.     

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.     

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.     

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.     

Role of low ecdysteroid titre in acquisition of competence for pupal transformation in Bombyx mori

Penultimate-instar larvae of Bombyx mori were neck-ligated or ligated posterior to the prothoracic glands. Repetitive injections of 20-hydroxyecdysone every 3 or more hours elicited the gut purge in thorax-ligated animals. Single injections of 20-hydroxyecdysone up to 40 μg failed to induce the gut purge. However, a single injection of 20-hydroxyecdysone together with juvenile hormone analogue, resulted in larval moulting of thorax-ligated animals. Once the thorax-ligated larvae showed the gut purge, a single injection of 20-hydroxyecdysone was enough to provoke pupation. The change in ecdysteroid titre in those animals receiving repeated injections was compared with that observed in neck-ligated larvae that spontaneously underwent the gut purge followed by precocious pupation. These data indicate that the very low ecdysteroid titre found before the gut purge is important for the acquisition of competence to undergo the gut purge in response to a small ecdysteroid surge just before the gut purge.     

The effect of a juvenile hormone analogue on ecdysteroid titre during development and HnRNA formation in the moth,Ephestia cautella

The juvenile hormone analogue, methoprene was found to interfere with normal development of Ephestia in a manner dependent on age. Young embryos, prior to the stage of blastokinesis, and animals, shortly before and after pupation, were found to be the most sensitive to the compound. The JHA inhibited metamorphosis and produced giant larvae when it was given to immature larvae, however, when it was given to larvae 2–3 days prior to pupation or to young pupae it did not affect metamorphosis but prevented adult emergence. Comparison of the ecdysteroid titre determined in control and treated animals in the various developmental stages showed that JHA depressed the ecdysteroid titre totally only when it was given to young larvae and partially when it was applied shortly before pupation. It seems that the action of methoprene on ecdysone regulated systems and/or ecdysone producing systems in Ephestia appears to be mainly during the larval stage prior to the appearance of the small ecdysteroid peak and the formation of HnRNA in the transition period from larvae to pupae.     

Influence of a juvenile hormone analogue on vitellogenin synthesis and oögenesis in larvae of Nauphoeta cinerea

In experiments on the synthesis of the vitellogenic protein, farnesylmethylester, a juvenile hormone (JH) analogue, was injected into female Nauphoeta cinerea larvae at various stages during their development. Two and 4 days after injection, 2 μl of haemolymph were assayed in a vitellogenin immunodiffusion test. In second last and last instar larvae less than 6 days before adult ecdysis, high doses (100 μg) of farnesylmethylester are necessary to induce vitellogenin synthesis, whereas older last stage larvae and decapitated adults respond to small doses (1 μg) with the synthesis of vitellogenin. It seems that the competence to synthesize the vitellogenic protein changes at the time of induction of the moulting process. If farnesylmethylester is injected into last instar larvae with a supposedly high titre of ecdysone, the vitellogenic protein can be detected in the haemolymph of a small percentage of animals only.Oöcyte maturation can be observed in last instar larvae injected after the fifth to ninth day with farnesylmethylester. The observed volume changes of the corpora allata suggest that an absence of JH for a short time is necessary for the oöcytes to become competent to grow. Last instar larvae treated with farnesylmethylester become larval-adult intermediates with partly developed oöcytes, demonstrating a simultaneous juvenilizing and gonadotropic influence of the JH analogue. In last instar larvae injected with farnesylmethylester a partial degeneration of already maturing oöcytes is induced at the time when the ecdysone titre is supposedly high and the possible reasons for this are discussed.     

The hormonal regulation of the larval diapause in the codling moth, Laspeyresia pomonella (Lep. Tortricidae)

The hormonal control of the facultative diapause of the codling moth has been investigated. The diapause can be divided into 4 phases or periods: (1) diapause induction by short-day conditions (SD) in young larvae, (2) initiation of the diapause in the early last larval instar by a high titre of juvenile hormone, (3) onset and maintenance of diapause with inactivity of the neuroendocrine system, as evidenced by the results of neck-ligation experiments, (4)termination of diapause by the production of ecdysteroid.Diapause-induced larvae pupated after spinning the cocoon, if the state of induction was changed by injection with the anti-juvenile hormone precocene II at the beginning of the last larval instar and subsequent results of neck-ligation experiments, (4) termination of diapause by the production of ecdysteroid. treated with juvenile hormone during the first 1.5 days after the last larval moult and subsequently reared under SD. Under LD, continuous application of juvenile hormone during the last larval instar and after spinning did not prevent the insects from moulting to either a supernumerary larva, a pupa or a larval-pupal intermediate. Termination of diapause, i.e. pupation, was achieved by injecting diapausing larvae with 20-hydroxyecdysone. Although juvenile hormone was found to have a prothoractropic effect in diapausing larvae, no pupal moult could be induced by the application of the hormone. Contrary to the hormonal situation before pupation of nondiapausing larvae, no juvenile hormone could be detected before or during the pupation of larvae after diapause.     

印楝素乳油对斜纹夜蛾的生物活性及田间防效研究

在室内测定了印楝素乳油对斜纹夜蛾(Spodopteralitura)的生物活性,并进行了田间防治试验.结果表明,印楝素乳油对斜纹夜蛾具有多种生物活性,主要表现在对幼虫的拒食作用、生长发育的抑制作用和一定的毒杀活性,以及对成虫产卵的忌避作用.经印楝素处理后,幼虫平均发育历期延长、幼虫体重、蛹重、化蛹率和蛹羽化率显著低于对照,印楝素乳油12μl·L^-1浓度处理3龄幼虫5和7d后的体重分别是对照组的36.8%和14.5%,而幼虫平均发育历期比对照组延长6d左右,化蛹率和蛹羽化率分别是27.79%和37.23%,显著低于对照组85.66%和82.11%.印楝素对低龄幼虫有一定的毒杀活性,而对高龄幼虫活性则很低.田间防治试验表明,印楝素乳油对斜纹夜蛾种群有良好的控制作用,施药7d后其防治效果要优于化学农药10%除尽悬浮剂.     

The effect of Achyranthes japonica extract on larval survival and development and oviposition behavior of Plutella xylostella L. (Lepidoptera: Plutellidae)

The extract of Achyranthes japonica was tested for effects on larval survival and development and the oviposition behavior of the diamondback moth, Plutella xylostella L. Chinese cabbage dipped in A. japonica extract solution showed 51–80% antifeedant activity for 5 days against P. xylostella larvae, and more larvae were also on untreated cabbage leaves 24 h after release. The mortality of P. xylostella larvae increased proportionally to the duration of dipping time in the extract, and both pupation and emergence rates of larvae feeding only on treated cabbage were lower than those for larvae raised on untreated or with a choice of cabbage. The 20-hydroxyecdysone (20E) concentration in leaves was approximately 549, 1232, 1275, and 1426 μg/g at 6, 12, 24, and 48 h after dipping treatment, respectively. Notably, naive females laid more eggs on untreated cabbage than on treated cabbage, and females from larvae raised on treated Chinese cabbage also preferred the non-treated leaves. Our results are in contrast to those from earlier studies using various insect models that confirmed most females prefer to lay eggs on the host type that was eaten in the larval stage (Hopkins host selection principle). Cabbage dipped in the A. japonica solution for 24 h caused 59% larval mortality and inhibited both pupation and emergence rates of the larvae when exposed to plants 15 and 22 days after planting in the field, with the 20E concentration in the treated cabbage leaves at 1600.9 ± 122.36 and 1386.8 ± 24.69 μg/g, respectively. Therefore, the biological effectiveness could be attributed to the 20E in the treated cabbage leaves.     

Prothoracicotropic hormone bioassay: Bombyx larva assay

Summary

An assay for the prothoracicotropic hormone (PTTH) has been established using in situ activation of the prothoracic glands (PG) of Bombyx mori in its larva-to-larva development. The timing of PTTH release was estimated by examining developmental response of 4th instar larvae to brain removal and neck ligation, and changes in the haemolymph ecdysteroid titer and ecdysone-releasing activity of PG in vitro during the development. Injection of Bombyx brain extracts into 4th instar larvae neck-ligated shortly before full activation of PG elicited larval moulting rather than precocious pupation in headless larvae. This developmental shift was regarded as due to the action of PTTH, and the PTTH unit has been defined from a linear log dose-response relationship. Materials chromatographically fractionated from Bombyx brain extracts were examined for the presence of stage- and species-specific PTTH molecules by using this Bombyx larva assay and Bombyx and Samia pupa assays previously developed. The same fractions were active when assayed by Bombyx larva and pupa assays.     

Disruptive developmental effects of benzyl-1,3-benzodioxole derivatives on unparasitized and parasitized Manduca sexta larvae

Treatment of tobacco hornworm larvae with the benzyl-1,3-benzodioxole derivative J-2710 immediately after ecdysis to the fourth instar disrupted development either during the moult to the fifth instar or shortly thereafter. Larvae given topical applications of 100 μg J-2710 in 1 μl acetone suffered 100% mortality, often after secreting moulting fluid in large pockets between the epidermis and the cuticle later in the fourth instar. Larvae that successfully ecdysed had abnormalities of the mouthparts and cervix that interfered with normal feeding, inhibiting growth in the fifth instar. Larvae of the gregarious endoparasitic wasp Cotesia congregata (=Apanteles congregatus) frequently failed to emerge from host Manduca sexta larvae treated with high doses of J-2710, particularly when the host failed to feed normally. Less potent disruptive effects on Manduca and Cotesia were seen after treatment of larvae with the derivatives J-3370 and J-2581.No anti-juvenile hormone action of J-2710 was observed. J-2710-treated M. sexta larvae showed no precocious metamorphosis and the developmental effects of J-2710 were not prevented by co-application of the juvenile hormone analogue methoprene in doses ranging from 1 to 100 μg/larva. Moreover, J-2710 had no effect on the action of methoprene in the black larval assay for juvenile hormone-like activity, unlike results reported to occur using the Galleria wax wound assay.     

Activation of the prothoracic gland by juvenile hormone and prothoracicotropic hormone in Mamestra brassicae

The effects of JHA (ZR-515) application or brain implantation on metamorphosis and adult development were examined in the last instar larvae and pupae of Mamestra brassicae. When JHA was applied to neck-ligated 4- or 5-day-old larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands taken from 5-day-old larvae, the insects pupated. Dauer pupae and diapausing pupae treated with JHA showed adult development. By contrast, pupation could not be induced by the application of JHA to 2- or 3-day-old neck-ligated larvae or to the isolated abdomens of 5-day-old larvae containing implanted prothoracic glands from 0-day-old larvae. Implantation of a brain into neck-ligated 3- or 5-day-old larvae (at the beginning of gut emptying and wandering) caused pupation of the host. A similar result was obtained when both a brain and the prothoracic glands from 0- or 5-day-old larvae were implanted into the isolated abdomens of 5-day-old larvae. These results indicate that activation of the prothoracic glands by application of JHA is temporally restricted to the last part of the last larval instar and to the pupal stage, while the activation by prothoracicotropic hormone (PTTH) can occur throughout the last larval instar and the pupal stage. In addition, the implantation of brains or application of JHA to neck-ligated 5-day-old larvae 25 days after ligation seldom induced pupation of the hosts, a result which suggests that larval prothoracic glands maintained under juvenile hormone (JH) or PTTH-free conditions for long periods of time may become insensitive to reactivation by both hormones.     

Developmental and behavioural responses of larval Trichoplusia ni to parasitization by an imported braconid parasite Chelonus sp

ABSTRACT. Larval Trichoplusia ni (Hübner) (Noctuidae) parasitized by Chelonus sp. (near curvimaculatus ) (Braconidae) precociously initiated pupation during the penultimate fourth instar. The temporal sequence of developmental markers exhibited by parasitized T. ni closely matched the temporal sequence in normal, pupating larvae. The parasitized larvae did not complete pupation, but consistently stopped development at a stage recognizable by a certain set of markers. This halt was observed in hosts from which parasites emerged and from hosts which had been stung but from which no parasites emerged. Weight gain and food consumption by parasitized hosts were significantly lower than normal, although most reached the fourth instar at the same time as normal larvae. Measurement of head capsule widths indicated that the width in precociously pupating larvae was less than the critical width associated with attainment of the pupation instar of normal larvae.     

Evidence of hormonal control of the nucleolar activity in Sciara ocellaris.

The nucleolus organizing region in the salivary gland of Sciara ocellaris becomes gradually less active as the larvae approach pupation. The cytological visible condensation of the nucleolar puff is paralleled by a decrease in the incorporation of [3H]-uridine into the ribosomal RNA. Alterations in the nucleolar behavior were brought about by extirpation of the neuroendocrine glands at six stages of development. There is a 'critical stage' during the 7th day of the 4th instar when the hormonal stimulus for pupation occurs. If the ligature is performed before this stage the NOR remains expanded and the larval-pupal ecdysis is inhibited; if done later on the NOR condenses at a normal rate and the ligated larvae mount to head-less pupae. These results suggest that inhibition of rRNA synthesis and consequent condensation of the NOR is under the control of the moulting hormone.     

Effects of ingested phytoecdysteroids on the growth and development of two Lepidopterous larvae

The effects of ingested or injected 20-hydroxyecdysone on silkworm larvae (Bombyx mori) including death without moulting, death following completion of promoted moulting, death during promoted moulting (ecdysis inhibition) and inhibition in growth with and without effects on moulting, are dependent upon the concentration of exogenous hormone, the precise developmental stage of the treated larvae, and the duration of exposure to the exogenous ecdysteroid. Comparisons of 20-hydroxyecdysone with other phytoecdysteroids in the silkworm and pink bollworm, Pectinophora gossypiella, show a similar but more potent effect induced by ponasterone A, while cyasterone causes an ‘antiecdysone’ effect.     

Lepidopteran anti-juvenile hormones: Effects on development of Apanteles congregatus in Manduca sexta

Parasitism by the braconid wasp Apanteles congregatus decreases the effectiveness of the anti-juvenile hormone agents ETB (ethyl 4-[2-{ittert-butyl carbonyloxy}bytoxy]benzoate) and fluoromevalonolactone (FMev) in inducing precocious metamorphosis of Manduca sexta larvae. Topical application of 1–200 μg ETB to parasitized third-instar larvae had no effect on either host or parasite development, whereas doses of 50μg or more ETB applied to unparasitized third-instar larvae caused formation of larval-pupal intermediates after the fourth instar. Parasitism also decreased the effectiveness of 100–200 μg FMev in causing metamorphosis at the moult following its application. In contrast to ETB, FMev disrupted development of the parasitoids. No wasps emerged when preterminal stage hosts were treated with FMev and the hosts formed larval-pupal intermediates. After treatment of terminal stage hosts with FMev, the number of emerging parasitoids was reduced by one-third. Precocene II (100 μg per larvae) had no effect on development of either M. sexta or A. congregatus.     

Compatibility of Spinosad, Tebufenozide and Azadirachtin with Eggs and Pupae of the Predator Chrysoperla carnea (Stephens) Under Laboratory Conditions

Under laboratory conditions, the toxicity of three novel insecticides, spinosad (Tracer ® ), tebufenozide (Mimic ® ) and azadirachtin (Align ® ), was tested against eggs and pupae of the predator Chrysoperla carnea (Stephens). In a first series of assays, eggs were dipped in an aqueous concentration and no ovicidal activity was scored for the three insecticides. In the second, when females were ovipositing on treated substrate for 24 h, fecundity and hatching percentages were similar as compared to controls and the offspring developed normally until the adult stage. However, spinosad, at the highest concentrations tested, caused a slight, significant reduction in the adult life span and fecundity. In a third series of experiments, pupae developed into normal adults after topical treatment for the three insecticides. Herewith, a pharmacokinetic study indicated low accumulation in the body after pupal cuticle penetration when administrating 14 C-labelled insecticide. Fourthly, pupation of last-instar larvae in treated substrate was normal for spinosad and tebufenozide. Only azadirachtin caused a slight reduction in the number of pupae and adults; however, fecundity and fertility of surviving adults was normal. In conclusion, the current results indicate that the three insecticides are not toxic to eggs and pupae of C. carnea .