Larval moulting hormone of trichophoran Hemiptera-Heteroptera: Makisterone A,not 20-hydroxyecdysone

The haemolymph ecdysteroids were examined in fifth-stage larvae of Nezara viridula, Podisus maculiventris and Dysdercus cingulatus (Hemiptera-Heteroptera) using high-pressure liquid chromatography to separate the ecdysteroids and a radioimmunoassay to detect the fractionated ecdysteroids. The length of the fifth stage ranged from 5 to 8 days, and a peak in ecdysteroid titre (1700–2650 ng/ml) occurred 2–3 days prior to ecdysis to the adult. An ecdysteroid matching the retention time of makisterone A (24-methyl-20-hydroxyecdysone) was clearly present in haemolymph taken at the time of peak titre in all 3 of these true bugs, whereas little, if any, ecdysone or 20-hydroxyecdysone was detected. These data, along with previously reported data for the milkweed bug Oncopeltus fasciatus, are persuasive evidence that makisterone A is the larval moulting hormone of a group of closely related Heteroptera called the Trichophora (Lygaeoida, Pentatomoidea, Pyrrhocoroidea and Coreoidea).     

Ecdysteroids in the haemolymph and the ovaries of the firebrat Thermobia domestica (Packard) (Insecta,Thysanura): Correlations with integumental and ovarian cycles

Ecdysteroids were analysed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC) in females of the apterygotous insect Thermobia domestica, which ahs overlapping moulting and reproductive cycles. During each moulting cycle, a peak in the haemolymph concentration of ecdysteroids occurs at day 9 (in the 11-day standard cycle), which can be correlated with apolysis and the beginning of new cuticle deposition. The ovaries show a peak of ecdysteroids at day 5 (i.e. one day before egg-laying), which suggests that these hormones are also involved in the reproductive cycle. In both cases, HPLC analysis combined with RIA suggests that the main ecdysteroid is 20-hydroxyecdysone. This duality in the function of ecdysteroids is discussed.     

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。     

The levels of ecdysteroids in uninjured and leg-autotomized nymphs of Blattella germanica (L.)

The levels of ecdysteroids in control and leg-autotomized first-instar nymphs of Blattella germanica were determined by radioimmunoassay from hatching to the time of the first ecdysis. Uninjured nymphs showed a distinct release of ecdysteroids half-way through the stadium, and this resulted in the commencement of the moult cycle which formed the cuticle of the second instar. Cockroaches which had legs autotomized at 48 h after hatching (i.e. before the control ecydsteroid release) had their instar duration increased by that time period. Releases of ecdysteroids and events of the moulting cycle were also postponed by the 48 h period. The titre of ecdysteroids in injured animals was double that of controls. Nymphs were also autotomized at 96 h (i.e. after the normal release of ecdysteroids) but no changes in instar duration, ecdysteroid releases, or events of the moult cycle were recorded. The effects of injury, prothoracicotropic hormone activity and ecdysteroid release are discussed.     

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.     

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.     

DNA synthesis in the imaginal wing discs of the American bollwormHelicoverpa armigera (Hübner)

The effect of two insect growth regulators of plant origin viz. plumbagin and azadirachtin and the ecdysteroids 20-hydroxyecdysone, makisterone A and a phytoecdysteroid on DNA synthesis in imaginal wing discs of day 4 final instarHelicoverpa armigera larvae was studied. DNA synthesis increased with increase in time of incubation up to 8 h and decreased later without the addition of moulting hormone. Addition of 20-hydroxyecdysone supported long term acquisition of competence for DNA synthesis in the wing discs. Both DNA synthesis and protein content were drastically reduced in plumbagin and azadirachtin-treated insects. Underin vitro conditions, plumbagin had a more pronounced inhibitory effect than azadirachtin. All the ecdysteroids tested, viz. makisterone A, 20-hydroxyecdysone and the ecdysteroidal fraction from the silver fernCheilanthes farinosa enhanced DNA synthesis     

Identification of the molting hormone of the sweet potato (Bemisia tabaci) and greenhouse (Trialeurodes vaporariorum) whitefly

In order to identify the whitefly molting hormone, whole body extracts of mature 4th instar and newly formed pharate adult Bemisia tabaci (Biotype B) and Trialeurodes vaporariorum were prepared and subjected to reverse phase high performance liquid chromatography (RPHPLC). Ecdysteroid content of fractions was determined by enzymeimmunoassay (EIA). The only detectable ecdysteroids that were present in significant amounts in whitefly extracts were ecdysone and 20-hydroxyecdysone. The concentrations of 20-hydroxyecdysone in B. tabaci and T. vaporariorum extracts, respectively, were 40 and 15 times greater than the concentrations of ecdysone. The identity of the two ecdysteroids was confirmed by normal phase high performance liquid chromatography (NPHPLC). When ecdysteroid content of RPHPLC fractions was assayed by radioimmunoassay (RIA), small amounts of polar ecdysteroids were also detected indicating that these ecdysteroids have a very low affinity for the antiserum used in the EIA. Ecdysteroid at 10.4 mM administered by feeding stimulated 2nd instar whitefly nymphs to molt. Based on our results, it appears that 20-hydroxyecdysone is the whitefly molting hormone.     

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 helminths and annelids

Summary

This review focuses on recent research developments in the past three years concerning ecdysteroid biochemistry in helminths and annelids, with emphasis on results reported at the IXth Ecdysone Workshop in Paris, France, in September 1989. During the past three years, most of the research in this area has been concentrated on nematodes, in several species of which the occurrence of ecdysteroids has been demonstrated. Interesting biological effects of exogenously applied ecdysteroids have been discovered on meiotic reinitiation in oocytes of the dog heartworm, Dirofilaria immitis, and on microfilarial production by Brugia pahangi. These effects, together with previous discoveries, demonstrate the feasibility of affecting nematode physiology with exogenously applied ecdysteroids. However, experiments with four species of nematodes in three separate laboratories have failed to demonstrate that nematodes biosynthesize ecdysteroids from cholesterol. Therefore, it remains to be proven whether ecdysteroids are truly endogenous nematode hormones or are merely compounds with strong biological activity. Research with cestodes and trematodes has similarly revealed that ecdysteroids occur in these organisms and that the compounds have possible regulatory roles, but experiments demonstrating endogenous biosynthesis have yet to be performed. Annelids, which are more closely related to insects than helminths, also contain ecdysteroids; recent research has demonstrated the C-20 hydroxylation of ecdysone to 20-hydroxyecdysone, the active moulting hormone of most insects. Experiments to demonstrate this metabolic step in helminths have failed.     

Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly,Sarcophaga peregrina

Metabolites of radioactive ecdysone or 20-hydroxyecdysone in larvae and pharate pupae of Sarcophaga peregrina were separated and identified by using thin-layer chromatography, high-performance liquid chromatography, and chemical methods. At the larval stage ecdysone was metabolized to biologically less active ecdysteroids predominantly through 20-hydroxyecydsone, at the pharate pupal stage, to other ecdysteroids which were tentatively identified as 26-hydroxyecdysone, 3-epi-26-hydroxyecdysone, and 3-epi-20,26-dihydroxyecdysone. Ecdysteroid acids were found in the polar metabolites during pharate pupal-pupal transformation, but scarcely detected in the larval metabolites. These acids were presumed to be ecdysonoic acid, 20-hydroxyecdysonoic acid, and their epimers. The conjugates of ecdysteroid that released the free ecdysteroids by enzymatic hydrolysis were produced more in larvae than in pupae, whereas the very polar ecdysteroids that were not affected by the enzyme were found more in pupae. Therefore, there are different metabolic pathways of ecdysone between these two successive developmental stages, and the alteration of the metabolic pathway may serve as one of the important factors in a regulatory mechanism of molting hormone activity which is responsible for normal development of this insect.     

An ecdysone receptor from the pentatomomorphan, Nezara viridula, shows similar affinities for moulting hormones makisterone A and 20-hydroxyecdysone

It has been suggested that Pentatomomorpha utilise the C₂₈ ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C₂₇ hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at the level of the ecdysone receptor protein, a heterodimer of nuclear receptors EcR and USP. cDNAs encoding two alternatively spliced isoforms of EcR and a single USP were isolated from a high-quality cDNA library prepared from a representative pentatomomorphan, Nezara viridula (Nv). NvEcR and NvUSP were found to group phylogenetically with heteropteran and other insect EcRs and USP/RXRs, respectively. Sequence comparison and phylogenetic analysis of these proteins found them to be distinct from those belonging to other hemipteran ecdysone receptors characterised to date. Co-expression of the His₆-tagged ligand binding regions (LBRs) of the two NvEcR variants with the FLAG-tagged LBR of NvUSP was achieved in insect cells employing appropriately constructed baculoviruses. The corresponding heterodimers, designated NvE10 and NvE11, were purified by affinity chromatography utilising the His₆ tags on their NvEcR subunits. The heterodimers displayed nanomolar affinity for [³H]ponasterone A (K_d = 6.8-7.5 nM), characteristic of ecdysone receptors. MakA has a similar affinity to 20E for both NvE10 and NvE11, consistent with MakA being a major moulting hormone in N. viridula.     

MOULTING HORMONE ACTIVITY IN THE ADULT COLORADO POTATO BEETLE,LEPTINOTARSA DECEMLINEATA SAY IN RELATION TO REPRODUCTION AND DIAPAUSE

Ecdysteroid levels in Leptinotarsa decemlineata were measured by radioimmunoassay (RIA), using an antiserum with a 10 times higher affinity for ecdysterone than for ecdysone and polyethylene glycol as a means for separating free from bound hormone. In reproducing males, raised under long day conditions (LD: 16 h photophase, 25°C) there was a peak of 54 ng/g at day 3–4, declining thereafter to very low values. In LD-females, 50 ng/g was found at day 2 (just before the onset of vitellogenesis), decreasing to 20 ng/g at day 5 (start of oviposition) and increasing again thereafter to 95 ng/g at day 14. In prediapausing animals, raised under short day conditions (SD: 10 h photophase, 25°C) the level was practically the same in both sexes and it was significantly higher than in reproducing animals. In both sexes a peak of about 96 ng/g was found from day 2 to 4, thereafter the level dropped quickly. During diapause the level increased slowly and in 9 month diapausing adults it was about 75 ng/g. This is the first time that high ecdysteroid levels are reported in relation to diapause induction in an adult insect, and the first time that high levels are found in adult males, indicating that the ovary cannot be the only site of synthesis of moulting hormone. The content during metamorphosis was also determined, mainly as a test for our RIA method. Hitherto there was no indication whatsoever that moulting hormone (MH) might be present and play a role in the adult stage in this insect. Our results make necessary a reappraisal of the hormonal regulation of oogenesis and induction of diapause in the Colorado beetle. The nature of the ecdysteroids present in the adult stage, their site of synthesis, their metabolism and physiological significance remain to be determined.     

Wound-stimulated moulting and calcification responses in the adult cirripede Balanus balanoides (L.)

Earlier studies of hormonal action in adult barnacles have involved the injection of crustecdysone into the basal mantle space through a hole drilled in the shell. The drilling and injection result in increased moulting activity and in calcification within the mantle tissue around the site of injection. A preliminary investigation of the nature and causes of these responses has been carried out. The increase in moulting activity is distinct from that induced by injected crustecdysone and can be sustained over several months, the level of stimulation depending upon whether the adults are subjected to single or multiple injections. The associated calcification extends from the shell wall and develops as a layer around an area of wound tissue at the injection site. It appears that these moulting and calcification activities are healing responses to wounding and that the processes involved may be similar to those of other arthropods. Moulting in barnacles is apparently controlled by an ecdysone hormonal system and these results suggest that such a system may be implicated in the control of calcification.     

Untersuchungen zur Häutungsphysiologie der dekapoden Krebse am Beispiel der StrandkrabbeCarcinus maenas

Under constant laboratory conditions, juvenile shore crabs moult at fixed intervals which depend upon their body size. During one moult every crab exhibits increases of the same relative amounts, independent of its absolute size. Basing on the predictable duration of the intermoult period, the morphological changes in the structure of the cuticle and the development of limb-buds, the intermoult period could be divided into 21 different stages. After studying the moulting rhythm in constant milieu, the influence of the following exogenous and endogenous factors upon the moulting rhythm and growth of normal and of eye-stalkless individuals was investigated: temperature, photoperiod, loss of pereiopods, feeding, and presence of larger specimens. From these investigations it became evident that the moulting rhythm is regulated by growth. The crabs are able to moult only after achieving a minimum of tissue growth. So long as this minimum growth is not achieved, a moult-inhibiting hormone is secreted and moulting is prevented. If the moult-inhibiting hormone is absent, moulting hormone is secreted and initiates a moult. Under dangerous conditions, the crabs are able to delay the next moult. Under unfavourable conditions they consume less food than normal. Therefore, the amount of tissue growth which is the necessary prerequisite for moulting is delayed, and continued release of moult-inhibiting hormone prevents the moult. Under conditions favourable for moulting, or demanding moult (e. g. after loss of many pereiopods) the crabs accelerate the moult. Temperature influences the moulting rhythm by indirect effects on the metabolic rate. During further investigations, the variation of the following parameters were determined quantitatively: content of moulting hormone in whole crabs; content of aminoacids, protein, glucose, Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in the hemolymph; pH and osmotic pressure in the hemolymph; and Ca⁺⁺ content in skeleton and whole crabs. All parameters mentioned — excepting pH and K⁺ content of the hemolymph — vary characteristically during the intermoult period. The titre of moulting hormone has 4 different maxima. Of all parameters, only the content of animoacids and protein in the hemolymph vary in the same way as the titre of the hormone. From these results the following conclusions are drawn: The moulting hormone not only initiates the moulting process, but controls it at several stages. Only protein metabolism seems to be under direct control of the moulting hormone which stimulates protein-synthesis. Chitin formation, regeneration, apolysis and ecdysis are indirectly controlled by the moulting hormone through protein metabolism. As in most of the other processes mentioned, the calcification of the new cuticle is not under the direct influence of the moulting hormone. The conclusion ofDigby (1966) that calcification in crabs is an electrochemical process, is confirmed.     

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.     

The influence of forskolin on the metabolism of ecdysone and 20-hydroxyecdysone in isolated fat body of the blowfly, Calliphora vicina

Rates of ecdysone and 20-hydroxyecdysone metabolism were measured by radio-assay in an in vitro system containing fat body isolated from blowfly larvae. The addition of forskolin which is known to stimulate artificially the intracellular adenylate cyclase system led to decreased rates of conversion of ecdysone into 20-hydroxyecdysone (= hormone activation) and of 20-hydroxyecdysone further to other metabolites (= hormone inactivation). The effect of forskolin was dose-dependent and reversible. Extracts prepared from larval brains were also tested. Some of them had the same effect as forskolin. It is concluded that the reactions leading from ecdysone to 20-hydroxyecdysone and further to hormonally inactive ecdysteroids are modulated by the intracellular level of cyclic nucleotides. We propose that a neurohormone is involved in the control of the reactions of the ecdysone metabolism. The observed new principle may contribute to the control of the titer of moulting hormone.     

In vitro stimulation of cell death in the moulting glands of Oncopeltus fasciatus by 20-hydroxyecdysone

The moulting glands of the milkweed bug, Oncopeltus fasciatus, normally degenerate just before the time of ecdysis to an adult (day 7 of the fifth instar). Morphologically normal cell death can be prematurely stimulated in vitro by 20-hydroxyecdysone. Breakdown is triggered by a 24-hr period of exposure to 20-hydroxyecdysone, but an additional incubation period is required before clear signs of degeneration are manifested. Glands removed after the onset of endogenous ecdysteroid secretion degenerate in vitro in the absence of added hormones. Thus, in the moulting glands of Oncopeltus, ecdysteroids appear to act as an important trigger for metamorphic cell death.     

Humoral inhibition of the corpora allata in larvae of Diploptera punctata: Role of the brain and ecdysteroids

Juvenile hormone synthesis by adult female corpora allata was inhibited following implantation into final-larval-instar males; inhibition was prevented by decapitation of the larval hosts on day 11 (prior to the head critical period for moulting), but not by decapitation on day 13. Implantation of one larval protocerebrum restored inhibition of implanted corpora allata, demonstrating that the brain releases an inhibitory factor. Corpora allata implanted into larvae decapitated on day 11 were inhibited by injections of 20-hydroxyecdysone. Since treatment of corpora allata with 20-hydroxyecdysone in vitro did not inhibit juvenile hormone synthesis, ecdysteroids probably act indirectly on the corpora allata. Juvenile hormone synthesis and haemolymph ecdysteroid concentration were measured following implantation of corpora allata along with two larval brains into larval hosts. Brain implantation did not affect ecdysteroid concentration, but did inhibit juvenile hormone synthesis, even in animals with low haemolymph ecdysteroid concentration. Incubation with farnesoic acid stimulated juvenile hormone synthesis by corpora allata from males early in the final larval stadium, but not after day 8, showing that one of the final two reactions of juvenile hormone synthesis is rate-limiting in larval corpora allata at this stage. Adult female corpora allata which had been humorally inhibited by implantation into larvae were stimulated by farnesoic acid.