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.     

Venom from the ectoparasitoid wasp Eulophus pennicornis disrupts host ecdysteroid production by regulating host prothoracic gland activity

Abstract. Attack by the ectoparasitoid Eulophus pennicornis Nees (Hymenoptera: Eulophidae) prevents larvae of Lacanobia oleracea L. (Lepidoptera: Noctuidae) from moulting. Prothoracic glands (PGs) excised from parasitized or artificially envenomated hosts show a reduced basal level of ecdysteroid release at a time when non-parasitized caterpillars produce an ecdysteroid surge (48 h post moult to 5th stadium = penultimate stadium in non-venomated hosts). By contrast, PGs from similarly parasitized or envenomated caterpillars release comparatively high levels of ecdysteroid at 120 h post-moult. Temporary inactivation of PGs cannot be attributed solely to a parasitoid-induced reduction in cell viability, and incubation in E. pennicornis venom in vitro does not exert any direct effect on either PG cell viability or ecdysteroid release. However, inactivated PGs are not stimulated by forskolin, which may indicate that the absence of the required pre-moult ecdysteroid surge in developmentally arrested L. oleracea is due to insensitivity to a prothoracicotropic hormone. Even though parasitized caterpillars never moult, reversed-phase HPLC separations and radioimmunoassay confirm that they produce active moulting hormone (20-hydroxyecdysone) at 120 h post-moult. These results suggest that E. pennicornis arrests host development through the indirect effects on their hosts' PGs. This effect is not achieved through the destruction of gland cells, but more likely reflects the interruption of an innate cycle in PG activity, such that they lose their ability to respond to a normal cue to produce an essential hormone peak at a crucial point in development.     

Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity

A haemolymph ecdysteroid titre of the fifth (last)-larval instar of the hemipteran, Rhodnius prolixus has been determined by radioimmunoassay. During the last-larval stadium the ecdysteroid titre increases from a negligible level in the unfed insect to a detectable level within minutes following a blood meal. The titre reaches a plateau of ～50–70 ng/ml at 3–4 hr and this level is maintained until day 5–6, the time of the head-critical period in Rhodnius. At the head-critical period the titre begins to increase again, this time dramatically, reaching a peak of ～ 3500 ng/ml at day 13. From day 14 to ecdysis (day 21) the titre declines to a low level, ～ 30 ng/ml. Basal levels of ecdysteroids, ～ 15 ng/ml, were detectable in young adult males and females. A survey of haemolymph volumes during the last-larval instar indicates that the changes in the ecdysteroid titre reflect changes in the rates of ecdysteroid synthesis, and not changes in haemolymph volume. Excretion of ecdysteroids varies systematically during the instar, suggesting that control of ecdysteroid excretion may be important in regulation of the haemolymph titre. Qualitative analysis of the haemolymph ecdysteroid RIA activity revealed the presence of only ecdysone and 20-hydroxy-ecdysone. For the large peak preceding larval-adult ecdysis, 20-hydroxy-ecdysone was the predominant hormone. These results indicate that there may be two periods of release of prothoracicotropic hormone (PTTH) from the brain in Rhodnius, one immediately following the blood meal and the second on day 5 or 6. The significance of these times of PTTH release is discussed in relation to classical evidence of the timing of moulting hormone action, the response of target tissues, and with more recent findings on the timing of release of neurosecretory material from the brain of Rhodnius during moulting.     

Age dependency and tissue distribution of ecdysteroids in adult male crickets,Gryllus bimaculatus de Geer (Ensifera,Gryllidae)

Ecdysteroid titers were determined in tissues (gut plus Malpighian tubules, carcass tissue, fat body, muscles, haemolymph, accessory reproductive glands, and testes) of male adult crickets, Gryllus bimaculatus, during the first 20 days of adult life as well as in spermatophores. In all tissues, except testes, total ecdysteroid titers are high on the day of imaginal moulting and then drop more or less continuously until day 8 after moulting. Distinctly higher levels are found on day 12 and 18 as well. Freshly moulted males contain high quantities of polar ecdysteroid conjugates in the digestive tract, testes, accessory reproductive glands, and haemolymph. Apolar ecdysteroid conjugates are mainly detectable in carcass tissue and fat body, but also in the haemolymph during entire adulthood. Free ecdysteroids represent the domineering class of moulting hormones in the gut during all stages of adult life. The significance of cycling ecdysteroid concentrations during adulthood is discussed in relation to spermatophore production and development of male accessory reproductive glands.     

Synchrony of juvenile hormone-sensitive periods for internal and external development in last-instar larvae of Oncopeltus fasciatus

Breakdown of the moulting glands in Oncopeltus can be completely inhibited by topical application of a juvenile hormone analogue prior to day 2 of the fifth instar, and partially inhibited by application prior to day 4. The analogue-sensitive period for the inhibition of external metamorphosis is very similar to that for the inhibition of cell death in the moulting glands. A decline in response to the analogue between days 2 and 4 is correlated with rising ecdysteroid levels in the haemolymph. That this rise in ecdysteroids may be responsible for termination of the juvenile hormone-sensitive period is suggested by premature loss of sensitivity to the analogue by the moulting glands following premature exposure to 20-hydroxyecdysone.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.     

Makisterone A: The major ecdysteroid from the pupa of the honey bee,Apis mellifera

Makisterone $\text{A}$, a 28-carbon moulting hormone, has been identified as the major free pupal ecdysteroid in the honey bee, Apis mellifera. The pupal ecdysteroid was isolated and identified by normal and reversed-phase high performance liquid chromatography in conjunction with a radioimmune assay. The compound was further characterized physico-chemically by both mass spectrometry and nuclear magnetic resonance spectroscopy. No C₂₇ ecdysteroids (i.e. 20-hydroxyecdysone or ecdysone) were detected at this stage of development. This is the first isolation and identification of a 28-carbon ecdysteroid in an insect species from the order Hymenoptera. Utilization of dietary sterols by honey bees is also discussed.     

Ecdysteroids in nematodes

The occurrence of ecdysteroids (insect moulting hormones) in nematodes, albeit at low concentrations, has been firmly established. In addition to apparently stimulating moulting in a few species, exogenously applied ecdysteroids have now been shown to have interesting biological effects on meiotic reinitiation in oocytes and on microfilarial production in filariae. Although such effects demonstrate the feasibility of influencing nematode physiology with exogenously applied ecdysteroids, hitherto it has not been possible to demonstrate synthesis de novo of these steroids in nematodes. Thus, it remains to be established whether ecdysteroids are truly endogenous nematode hormones or merely represent compounds with strong biological activity. Nonetheless, there are indications that interference with the ecdysteroid system might be exploitable in the development of novel approaches to control of nematodes.     

棉铃虫蛹期血淋巴的蜕皮甾类

目前为止仅在少数几种昆虫中研究过蛹期的蜕皮激素。关于蜕皮甾类的性质分析,结果也颇不一致。本文采用放射免疫分析、薄层层析、高压液相色谱及质谱对棉铃虫Heliothis armigera蛹血淋巴内的蜕皮激素进行了研究。结果如下:1.物理-化学方法证明蛹血淋巴内存在二种蜕皮甾类:蜕皮酮和20-羟基蜕皮酮。2.蛹期蜕皮甾类滴度呈一宽峰,高峰出现在化蛹后的第5天(3435ng/ml)。3.在高峰时,蜕皮酮与20-羟基蜕皮酮的比例为1:3.57,说明20-羟基蜕皮酮是主要的蜕皮甾类。4.比较雌雄两性蛹的蜕皮甾类滴度,未见明显差异。研究表明在棉铃虫中影响成虫发育的主要激素是20-羟基蜕皮酮而不是蜕皮酮。     

Biotransformations of 20-hydroxyecdysone and analogues by Curvularia lunata NRRL 2178

20-Hydroxyecdysone, the arthropod moulting hormone, was biotransformed by the fungus Curvularia lunata NRRL 2178 to the rare ecdysteroid, 2-dehydro-3-epi-20-hydroxyecdysone, and the novel 3alpha,9alpha-cyclo ecdysteroid analogue, (20R,22R)-3beta,14alpha,20,22,25-pentahydroxy-3alpha,9alpha-cyclo-5beta-cholest-7-en-2,6-dione in 14 and 44% yields, respectively. Ponasterone A and pterosterone were similarly biotransformed to the corresponding 2-dehydro-3-epi- and 3alpha,9alpha-cyclo-analogues.     

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

Maturing eggs of the desert locust, Schistocerca gregaria, contain a variety of ecdysteroid (insect moulting hormone) conjugates and metabolites, four of which have been previously isolated from polar extracts and identified as ecdysonoic acid, 20-hydroxyecdysonoic acid, 3-acetylecdysone 2-phosphate and ecdysone 2-phosphate. In the present study we have isolated eight additional ecdysteroids from similar late-stage eggs by high-performance liquid chromatography. The 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone, all of which were first identified as ecdysteroid components of newly-laid eggs of S. gregaria, were identified by co-chromatography with authentic compounds and by physicochemical techniques. The remaining compounds were identified as 3-acetyl-20-hydroxyecdysone 2-phosphate, 3-epi-2-deoxyecdysone 3-phosphate, 3-acetylecdysone 22-phosphate and 2-acetylecdysone 22-phosphate by fast atom bombardment mass spectrometry, p.m.r. spectroscopy and analysis of the steroid moieties after enzymic hydrolysis. The latter two compounds, after isolation, are susceptible to nonenzymic acetyl migration and deacetylation to give mixtures of ecdysone 22-phosphate and its 2- and 3-acetate derivatives. The possible role and significance of these ecdysteroid conjugates with respect to the control of hormone titres in insect eggs is discussed.     

Prothoracic gland involution related to moulting hormone levels during the metamorphosis of Tenebrio molitor L.

Prothoracic gland (PG) of Tenebrio shows ultrastructural changes which can be correlated with ecdysteroid levels (measured by radioimmunoassay) during larval-pupal development. However, the gland cells begin to degenerate before pupal-adult ecdysis: the PG involution is completed before the moulting hormone peak which triggers pupal-adult development. These facts strongly suggest that another endocrine organ produces moulting hormone needed for adult development.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.     

Protein synthesis by mammalian cells treated with C-3-modified analogs of the 12,13-epoxytrichothecenes T-2 and T-2 tetraol.

Modification at the C-3 position of the trichothecenes T-2 and T-2 tetraol affected their ability to inhibit protein synthesis in African green monkey kidney (Vero) and mouse erythroleukemia cells. Replacement of the 3-hydroxyl of T-2 with hydrogen caused a 24-fold decrease in activity, whereas acetylation resulted in a 500-to 1,000-fold decrease. Protection of the 3-hydroxyl with a tetrahydropyranyl moiety gave an analog that was 37-fold more inhibitory to Vero than to mouse erythroleukemia cells; with the other analogs a similar effect on protein synthesis was found for both types of cells. The analogs obtained after alkaline hydrolysis were much less potent than the parent trichothecenes. The 3-tetrahydropyranyl-modified analog was equivalent in potency to T-2 tetraol, while the deoxygenated species was at least threefold less potent. All T-2 analogs caused some degree of polysome "runoff," thereby demonstrating that these species inhibit protein synthesis at the chain initiation stage when added at their 50% infective dose concentrations or lower. From these results, we suggest that the 3-hydroxyl moiety is essential for T-2 to exhibit such high activity on eucaryotic cell protein synthesis and that modification at the C-3 position decreases but does not eliminate this activity.     

Physiological aspects of nm-g mutant: An ecdysteroid-deficient mutant of the silkworm,Bombyx mori

We examined physiological and endocrinological properties of the nm-g mutant of the silkworm, whose development was arrested at 1st or 2nd larval instar. The larva of the nm-g homozygote continued to feed and grow past the stage when a normal larva moulted to the next instar. The nm-g larva attained more than twice the body weight of a normal moulting larva, but remained in the same instar with no sign of the moulting until it finally died. The ecdysteroid concentration in the haemolymph of the nm-g larva was reduced and an increase in the titre, which occurred before moulting in the normal larva, was not observed. Injection of 20-hydroxyecdysone induced the nm-g larva to moult and ecdyse to the next larval instar. From these results, we conclude that nm-g is an ecdysteroid-deficient mutant.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.     

Girard derivatization for LC-MS/MS profiling of endogenous ecdysteroids in Drosophila

Ecdysteroids are potent developmental regulators that control molting, reproduction, and stress response in arthropods. In developing larvae, picogram quantities of individual ecdysteroids and their conjugated forms are present along with milligrams of structural and energy storage lipids. To enhance the specificity and sensitivity of ecdysteroid detection, we targeted the 6-ketone group, which is common to all ecdysteroids, with Girard reagents. Unlike other ketosteroids, during the reaction, Girard hydrazones of ecdysteroids eliminated the C14-hydroxyl group, creating an additional C14-C15 double bond. Dehydrated hydrazones of endogenous ecdysteroids were detected by LC-MS/MS in the multiple reaction monitoring (MRM) mode using two mass transitions: one relied upon neutral loss of a quaternary amine from the Girard T moiety; another complementary transition followed neutral loss of the hydrocarbon chain upon C20-C27 cleavage. We further demonstrated that a combination of Girard derivatization and LC-MS/MS enabled unequivocal detection of three major endogenous hormones at the picogram level in an extract from a single Drosophila pupa.     

Effect of dietary ecdysterone on protein ingestion and copulatory behaviour of the blowfly, Sarcophaga bullata

ABSTRACT. In Sarcophaga bullata and other blowflies, the low haemolymph ecdysteroid titre is responsible for the inability of males to synthesize vitellogenin, although they have the genes coding for this protein. The high moulting hormone titre in females is of crucial importance for vitellogenesis. In this context we investigated whether the difference in protein ingestion between the sexes might also be related to differences in ecdysteroid titre. Using a two-choice system, we observed that upon addition of ecdysterone to the food, protein ingestion increased in males. Topical application of methoprene also had a stimulating effect. Prolonged treatment with ecdysterone reduced the copulatory behaviour of males.     

Inactive proteinases in silkmoth moulting gel

The moulting gel of silkmoths lacks proteolytic activity but contains an inactive form of the proteinases which are later found in the moulting fluid. These inactive enzymes are activatable in vitro by dilution, activation proceeding most rapidly at low ionic strength. Activation proceeds as a first-order process and is not autocatalytic. Approximately the full amount of proteinases ultimately found in moulting fluid are already present in the gel. Moulting gel does not inhibit the active proteinases of moulting fluid; moreover, the proteolytic activity elicited by dilution of the moulting gel does not disappear upon reconcentration. These observations suggest that the proteinases in moulting gel are not inhibited by a stable, dissociable inhibitor; they may be present either as compartmentalized active enzymes or as proenzymes. Several possible mechanisms for the in vivo activation at the time of gel to fluid transformation are discussed.