Ecdysteroid Content Research Articles

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.

Ecdysteroid levels during the larval development of the spider crab Hyas araneus

Ecdysteroid levels were determined by radioimmunoassay during the larval development of the spider crab Hyas araneus L. In each of the three larval instars (zoea I, zoea II, megalopa) a minimum in ecdysteroid levels was found during early postmoult and a maximum during premoult. The amounts of ecdysteroids increased during development and reached maxima of 93, 137, and 165 pg ecdysone equivalents per larva in zoea I, zoea II, and megalopa, respectively. Ecdysteroid concentrations per unit of body dry weight or carbon increased during each moulting cycle, but the average values showed decreasing tendency from the first to the last instar. The main ecdysteroid in all larval instars and during the moulting cycle was 20-OH-ecdysone, and practically no ecdysone could be detected by HPLC-RIA.

Effect of temperature on the rate of pupal-adult development of the noctuid moth,Mamestra configurata Wlk.: evidence for differential effects on the initiation of development and subsequent metamorphic development.

Moths eclosed earlier from pupae of the bertha armyworm,Mamestra configurata, that were exposed briefly (1 to 5 days) to a warm temperature (15 or 20°C) at the beginning of postdiapause pupal-adult metamorphosis and then incubated at 10 or 12.5°C than from pupae incubated at 10 or 12.5°C throughout metamorphosis. The differences were greater than could be explained by the additional thermal units received at the higher temperature. Analyses of the times of peak concentrations of ecdysteroids (insect growth and development hormones) in metamorphosing pupae and of moth eclosion after exposure to various combinations of temperatures indicated that the 'warm termperature effect' was not on the rate metamorphic development but on an earlier neuroendocrine process concerned with the initiation of development.The magnitude of the difference in eclosion time between pupac receiving a brief warm temperature "trigger" and the control suggests that the differential effect of temperature on the initiation of development and subsequent metamorphic development is of biological significance and should be considered in the construction of models of insect development under natural conditions.

Ecdysteroid hormone levels in the pupa of the bertha armyworm, Mamestra configurata Wlk., during spermatogenesis

Maturation of the spermatocysts in the testes of post-diapause pupae of M. configurata from the round form to the elongated form began about 5 days (0.15 units) after the initiation of pupal-adult development at 20°C. Spermatocyst maturation coincided with rising levels of 20-hydroxyecdysone in the developing pupa and occurred well after the rise and fall in the level of ecdysone. At the time of peak 20-hydroxyecdysone concentration at day 12 (0.34 units) of pupal-adult development, about 50–60% of the spermatocysts were in the elongated form and by the time (day 16, 0.49 units) 20-hydroxyecdysone had fallen to low levels all of the spermatocysts were elongated. Experimental suppression of ecdysone and 20-hydroxyecdysone to low levels had no effect on spermatocyst maturation. Evidently, high concentrations of these ecdysteroids are not required for spermatogenesis. Further, since male and female pupae show similar patterns of ecdysone and 20-hydroxyecdysone production during pupal-adult development, it seems unlikely that the high concentrations of ecdysteroids are produced solely to regulate spermatogenesis. It is hypothesized that the temporary production of high concentration of 20-hydroxyecdysone during pupal-adult development of M. configurata ultimately serves as an effective feedback mechanism to ensure that production of 20-hydroxyecdysone is terminated in preparation for adult eclosion.

Ecdysteroids in the dorsal bodies of pulmonates (gastropoda): Synthesis and release of ecdysone

1. 1. The mediodorsal bodies (MDB) of the pulmonate snails Lymnaea stagnalis and Helix pomatia as well as the ovotestis of H. pomatia contain ecdysteroids. 2. 2. After in vitro incubation of isolated MDB of H. pomatia considerable amounts of immunoreactive ecdysteroids (IE) were detected in the incubation medium whereas in MDB tissue the ecdysteroid content showed no striking increase compared with controls. 3. 3. Immunoreactive ecdysteroids released by MDB into the culture medium were analyzed by HPLC and RIA and identified as ecdysone. 4. 4. We conclude that MDB have the capability of ecdysone biosynthesis and release. Thus, for the first time a gland secreting ecdysone has been found in an invertebrate species other than arthropods.

Ecdysteroid titres during larval life and metamorphosis of Galleria mellonella

The ecdysteroid titre in Galleria larvae normally follows either a short larval cycle, which is characterized by a single peak on day 2, or a long metamorphic cycle, which includes a small peak on day 6 and a large one on day 7. Intermediate cycles and moult induction with reduced ecdysteroid concentrations occur under experimental conditions. The cycles and peaks are initiated and, until the brain critical period, driven by prothoracicotropic hormone (PTTH), while juvenile hormone (JH) titre determines which type of cycle will be activated. In larval development JH stimulates PTTH release but the metamorphic programme of neuroendocrine regulations involves blocking PTTH production by JH. This hormone then causes an interruption of the ecdysteroid cycle and extension of the last larval instar.

Ecdysteroids in the Camel Tick, Hyalomma Dromedarii (Acari: Ixodidae), and Comparison with Sex Pheromone Activity1

The ecdysteroid 20-hydroxyecdysone (20-OH ecdysone) and 2 other radioimmunoassay (RIA) positive ecdysteroids were found in unfed and feeding adult Hyalomma dromedarii ticks. Increases in total ecdysteroids per tick occurred during female feeding, especially following mating and repletion. Ecdysteroid content in unfed and feeding males remained approximately the same (estimated by RIA) or declined (estimated by HPLC). Changes in concentration of 20-OH ecdysone resembled the changes in total ecdysteroid content in most cases. High-performance liquid chromatography (HPLC) gave substantially higher estimates of total ecdysteroid content in the various tick life stages or physiological states than did RIA. Increases in ecdysteroid concentrations during the first few days following female eclosion may indicate a regulatory role for stimulation of sex pheromone production. No apparent relationship was observed between ecdysteroid concentrations and sex pheromone secretion during feeding. The greatest increases in all ecdysteroids, including 20-OH ecdysone, occurred following mating and during repletion.

Hormonal regulation of phase polymorphism and storage-protein fluctuation in the common cutworm, Spodoptera litura

Three storage proteins are synthesised by Spodoptera litura last-instar larvae as detected by an antiserum against pupal fat body proteins. The putative pupal storage proteins 1 and 2, appear in the haemolymph of the last-instar larvae 36 h after ecdysis under crowded rearing conditions: they appear 1 day later in isolated conditions. The appearance of these proteins in the haemolymph is prevented by juvenile hormone treatment and enhanced by allatectomy. Injection of 20-hydroxyecdysone into ligatured larvae does not induce appearance of these 2 proteins. Accumulation of protein 3 that reacts with Bombyx mori arylphorin antiserum is not blocked by juvenile hormone and is similar in both phases. It also accumulates to a small extent in the haemolymph during the moult to the final-larval instar and then disappears at ecdysis. One-hundred ng/ml ecdysteroid caused the sequestration of these proteins by the fat body, but a higher concentration of ecdysteroid (200 ng/ml) produced pupal cuticle in the isolated abdomens, suggesting that different ecdysteroid concentrations are necessary for these two events.

Environmental control of larval behaviour and its consequences for ecdysteroid content and pupation in Ephestia kuehniella

ABSTRACT. The behaviour of postfeeding larvae of Ephestia kuehniella Zell. (Lepidoptera, Pyralidae) depends on environmental conditions. Long day and high larval density (LG) stimulate larvae to spin the outer envelope of the pupal cocoon which, in turn, accelerates pupal ecdysis. Constant darkness and isolation in a limited space (DS) inhibit spinning of the outer envelope, and cause extension of the wandering stage and a delay in pupation. This behaviour was also observed in larvae confined in the diet.In postfeeding LG larvae, an increase in ecdysteroid content coincides with spinning of the outer envelope. In DS larvae, which fail to spin the outer envelope, the ecdysteroid content remains low during the wandering stage, and rises to a prepupal peak after a considerable delay. Wandering larvae inserted into empty outer envelopes showed a premature rise of ecdysteroids, and it is thus concluded that sensory stimulation from the outer envelope is a cue promoting further development.

Ecdysteroids in the American dog tick, Dermacentor variabilis (Acari: Ixodidae), during different periods of tick development.

Ecdysteroids occur in all life stages of the American dog tick, Dermacentor variabilis . 20-hydroxyecdysone (20-OH ecdysone) and 2 other radioimmunoassay (RIA) positive fractions were found in engorged nymphs and adult ticks. 20-OH ecdysone and at least 1 RIA-positive fraction were found in the embryos and engorged larvae. No 20-OH ecdysone was found in the unfed larvae or unfed nymphs. Increases in total ecdysteroid content per tick occurred during ecdysial periods, adult feeding, and especially during female repletion and oviposition. In contrast, the total concentration declined gradually in fasting adults. In most cases, changes in the concentration of 20-OH ecdysone resembled the changes in total ecdysteroid content. With 1 exception, high-performance liquid chromatography (HPLC) gave substantially higher estimates of total ecdysteroid content in the various tick life stages or physiological states than did RIA. No relationship between the changes in the concentrations of ecdysteroids and sex pheromone (2,6-dichlorophenol) were observed. However, the foveal (pheromone) glands appeared to respond to exogenous 20-OH ecdysone; ultrastructural changes characteristic of actively secreting glands were found in unfed females following exposure to this hormone.

Excretion of ecdysteroids by schistosomes as a marker of parasite infection

Ecdysteroids produced by schistosomes are released in biological fluids of infected hosts. In the sera, the concentration of ecdysteroids correlates with the perimissiveness of the host to schistosome infection and its detection is available in the absence of positive parasitological tests. In the urine, ecdysteroid concentration decreases markedly after chemotherapy. 20-Hydroxyecdysone and its epimer were identified in the urine of infected patients using mass spectrometry. These data demonstrate for the first time that ecdysteroids are released by organisms. Moreover, they are potent molecules of parasite infection and can be used for parasite diagnosis.

Cytophysiological correlations between prothoracic gland activity and hemolymph ecdysteroid concentrations in Rhodnius prolixus during the fifth larval instar: Further studies in normal and decapitated larvae

Hemolymph ecdysteroid titers in fifth instar larvae of Rhodnius prolixus were determined by radioimmunoassay, and their prothoracic glands were excised and examined by electron microscopy. During the last larval instar, the titer of ecdysteroid increased between the head-critical period until Day 13, at which time the peak titer was 3100 pg 20-hydroxyecdysone equivalents/μl. The activation of secretory cells at the time of the second period of prothoracicotropic hormone release was correlated with the development of major cellular organelles. The smooth endoplasmic reticulum first appeared at the head-critical period and the proliferated in close relation to the increase in ecdysteroid titer until Day 13, after which time it disappeared. Mitochondria expand and develop tubular cristae. They are closely associated with the smooth endoplasmic reticulum. When insects were decapitated, hemolymph ecdysteroid titer remained below 10 pg/μl and the prothoracic gland cells failed to develop smooth endoplasmic reticulum. We conclude that in the prothoracic gland cells as well as other steroidogenic tissues the smooth endoplasmic reticulum in association with mitochondria is involved in ecdysone biosynthesis.

ECDYSTEROID TITERS DURING THE MOLT CYCLE OF THE BLUE CRAB RESEMBLE THOSE OF OTHER CRUSTACEA

Caiinectes sapidus is the only true crab (brachyuran) whose pattern of ecdyste roid titers has been described as departing from the pattern seen in other decapods. While ecdysteroids in other crabs reach a peak just prior to ecdysis, those of C. sapidus were claimed to reach their maxima after ecdysis. The data reported here challenge these findings. We have measured ecdysteroids in hemolymph, ovaries, and whole animal extracts of blue crabs using a radioimmunoassay. In hemolymph and whole animals, ecdysteroid levels rose during premolt to a maximum at stage D3 . Ecdysteroids declined rapidly from late premolt stage D4 through postmolt stage A2, increased slightly at postmolt stage B, and returned to low levels where they remained during intermolt stage C. Ecdysteroid levels in males and immature fe males were not significantly different but mature females, having reached a terminal anecdysis, had significantly lower ecdysteroid levels. Ovaries of mature females ac cumulated ecdysteroids during vitellogenesis while the concentration of ecdysteroids in hemolymph was low.

Changes in ecdysteroid and juvenile hormone levels in developing eggs of Bombyx mori

It is well-known that insect eggs can contain very high concentrations of ecdysteroids, which undergo drastic changes during embryogenesis. We found that this is equally valid for juvenile hormones. Three juvenile hormone-immunoreactive compounds were observed in developing Bombyx mori eggs. They were assumed to be juvenile hormones 1, 2 and 3 according to their retention time in HPLC. These hormones underwent drastic and sudden changes. In the space of one day their concentration was seen to rise rapidly from an undetectable level up to as high as 4 × 10 −6 micromoles per mg of eggs. Their presence was detected as early as the first day of embryonic development, as well as during the blastokinesis period (day 5 to day 9) and in late embryos (day 12 to day 14). Their relative concentrations varied greatly. On two occasions, day 1 and day 8, all three hormones were simultaneously present. Moreover, juvenile hormone 3 was present during the blastokinesis period, either alone or in combination with hormone 2. The latter was the only hormone present in late embryos, before hatching. Thus, with regard to both ecdysteroids (ecdysone and 20-hydroxyecdysone) and juvenile hormones, each day of embryonic development displayed a different hormonal pattern. These patterns undoubtedly constitute a “hormonal code” of embryogenesis control. While 20-hydroxyecdysone can be assumed to trigger cuticulogenesis in embryos as it does in larvae, the effects of the other hormones as well as their possible interactions are questionable.

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.

Relations between ecdysteroid levels and pupal development in the ecd-1 temperature-sensitive mutant of Drosophila melanogaster

Relations between ecdysteroid levels and pupal development were studied in the temperature-sensitive mutant ecd-1, shifted from 20°C to 29°C at different stages. Ecdysteroid titration, using radioimmunoassays, revealed that the mid-pupal peak of ecdysteroids was not affected when the shift was performed at pupariation although, in this condition, pupae died at emergence. A qualitative study of ecdysteroid content using HPLC failed to show the lack of any specific product. If the shift was performed 24 hr before pupariation, the ecdysteroid level decreased significantly while animals died before emergence but after a mid-pupal peak (around 75% of pupal life). However, double-shift experi-experiments showed that, even though a partial rescue of pupal development occurred (7% emergence), a normal mid-pupal peak was not restored. Furthermore, the temperature-sensitive periods for the decrease in the ecdysteroid mid-pupal peak and for the lack of emergence were not the same. Altogether, these results suggest that a decrease in ecdysteroids might not be the primary effect of the ecd-1 mutation, as the developmental anomalies observed at the end of pupariation seemed independent of the ecdysteroid levels.

Neutral sterols of representatives of two groups of hemiptera and their correlation to ecdysteroid content

AbstractThe sterols of four species of Pentatomomorpha—Oncopeltus fasciatus (Dallas), Nezara viridula (L.), Dysdercus cingulatus (F.), and Podisus maculiventris (Say)—and threé species of Cimicomorpha—Rhodnius prolixus Stal, Arilus cristatus (L), and Cimex lectularius (L.)—were isolated and examined in order to compare neutral sterol utilization and content with the known ecdysteroids of these species. In the phytophagous Pentatomomorpha, O. fasciatus, N. viridula, and D. cingulatus, the low content of cholesterol and the high content of C28 and C29 phytosterols (< 1% and > 99% of the tissue sterols, respectively) indicated that these species are unable to dealkylate the C‐24 position of sterols. These insects appear to have adapted to the challenge of both insufficient dietary cholesterol and inability to dealkylate phytosterols by evolving the ability to produce a C28 ecdysteroid (makisterone A). The secondarily predacious P. maculiventris has adequate cholesterol available for C27 ecdysteroid production, but appears to have retained the ecdysteroid biosynthetic pathways of its phytophagous ancestors because it produces a C28 ecdysteroid. Cholesterol was the major sterol of each of the three species of Cimicomorpha, and these insects are only able to produce C27 ecdysteroids.

The role of the Y-organ and cephalic gland in ecdysteroid production and the control of molting in the crayfish,Orconectes limosus

The production of ecdysteroids (monitored by RIA) by Y-organs and cephalic glands in vitro was measured and hemolymph ecdysteroid levels were determined in the crayfish,Orconectes limosus, both after eyestalk ablation and as a function of time during natural premolt. Y-organ synthesis of ecdysteroid increased in parallel with a rise in hemolymph ecdysteroid concentrations under both conditions, peaking in substage D2 of premolt. Y-organ ecdysteroid output after eyestalk ablation was 3–4 times higher. Thus, removal of the inhibiting system of the eyestalk effectively removes not only the principal control but also any modulation of ecdysteroid secretion by the Y-organs. Ecdysteroid levels remained low in Y-organ-ectomized crayfish, although premolt was initiated in some animals. The cephalic gland does not appear to contribute to the regulation of molting inOrconectes limosus. The Y-organs, on the other hand, are a principal source of ecdysteroids which regulate the major synthetic activities of premolt.

Free ecdysteroids in adult male crickets, Gryllus bimaculatus

ABSTRACT. Ecdysteroid titres were determined in testes, fat body, muscles, haemolymph, carcass tissue, spermatophores, and faeces of males of the Mediterranean field cricket, Gryllus bimaculatus de Geer, throughout its adult life span. Considerable amounts of free ecdysteroids are concentrated in the testes and the fat body. The ecdysteroid titres were only slightly influenced by environmental temperature. In all tissues except the fat body, ecdysone and 20‐hydroxyecdysone were the predominant ecdysteroids present. In faeces, highest ecdysteroid concentrations were found at the time of lowest levels in tissues.

Chironomus tentans epithelial cell lines sensitive to ecdysteroids, juvenile hormone, insulin and heat shock

A technique for the isolation of Chironomus tentans embryo cells and a medium for their in vitro cultivation are described. For survival and proliferation cells require insulin and undefined supplements like fetal calf serum (FCS), yeast extract components, bactopeptone, and/or Drosophila extract. In such cultures epithelial cells proliferate, to form closed, floating vesicles or sheets attached to the dish. Permanent, diploid, epithelial cell lines readily develop. They respond morphologically and by the cessation of proliferation to physiological concentrations of ecdysteroids. The effect of ecdysteroids is prevented or reduced in the simultaneous presence of juvenile hormone. Heat-shocked epithelial cell lines stop the synthesis of most proteins and initiate the production of about ten new proteins.