Hemolymph Ecdysteroid Titer Research Articles

Developmental and diurnal changes in ecdysteroid biosynthesis by prothoracic glands of Rhodnius prolixus (Hemiptera) in vitro during the last larval instar

The synthesis of ecdysteroids by prothoracic glands (PGs) of male last instar larvae of Rhodnius prolixus was measured in vitro by radioimmunoassay throughout the course of larval-adult development. Large and systematic changes in relative rates of synthesis occur during development. Two bursts of elevated synthetic activity were found. The first commences as soon as development is initiated by a blood meal and lasts approximately 1 day. The second commences 4 days later and increases progressively to a peak at Days 11-13 after feeding (up to 25 ng of 20-hydroxyecdysone eq. gland-1/4 hr-1). The onset of each of these bursts of activity coincides with apparent times of PG stimulation in vivo by release of the prothoracicotropic hormone from the brain. Both bursts result in increases in hemolymph ecdysteroid titer measured in the donor animals. PGs exhibit an abrupt attenuation of synthesis on Day 14, which is followed by a rapid decline in the hemolymph ecdysteroid titer. Clearly, ecdysteroid synthesis by PGs is a major factor regulating the hemolymph titer. Ecdysteroid synthesis by PGs exhibits diurnal changes in vitro. The amount of ecdysteroid synthesized by PGs from animals during the scotophase is two to five times higher than that from animals during the photophase. A corresponding rhythm is seen in the hemolymph ecdysteroid titer. The rhythm in the titer is known to be under circadian control. It is therefore suggested that ecdysteroid synthesis in PGs of Rhodnius is regulated by a circadian system, possibly located in the PGs themselves.

The relationship of testicular and hemolymph ecdysteroid titer to spermiogenesis in the common armyworm, Leucania separata

The relationship of testicular and hemolymph ecdysteroid titer to spermiogenesis in the common armyworm, <i>Leucania separata</i>

The timing of release of prothoracicotropic hormone in relation to the head critical period during larval-adult development in Rhodnius prolixus (Hemiptera): Evidence from head transplantation experiments

This paper examines the timing of release of prothoracicotropic activity from heads of various stages of last larval instar Rhodnius prolixus by their ability to stimulate the haemolymph ecdysteroid titre of a host body (decapitated on day 5 after a blood meal) onto which they were transplanted. The ecdysteroid titre of normal animals at day 5 is constant, but in decapitated controls it begins a progressive decline due to the injury, not to removal of the source of prothoracicotropic activity. Stimulation by transplanted heads is therefore measured in relation to this declining titre of decapitated controls. Activity was found to emanate from heads of female larvae on days 4 and 5 after a blood meal and from male heads on days 5 and 6. Heads of both younger and older developmental stages had no effect on the host titre. It is concluded that prothoracicotropic activity is released from the head for a period of about 48 h in both sexes, but this period occurs one day earlier in females than in males. This period of release encompasses the classical head critical period, which also occurs one day earlier in females than in males. However, activity is detectable from heads about one day prior to the beginning of the head critical period. It is argued that release of prothoracicotropic activity prior to the head critical period occurs in the most rapidly developing animals in the population and ceases in these animals at the beginning of the head critical period. In the most slowly developing animals, release ceases at the end of the head critical period. It is concluded that the head critical period for the population represents the range of times over which release of prothoracicotropic activity ceases in the population. The significance of this interpretation is discussed in relation to the classical literature and work with other insects.

Determination of Larval Instar Number in the Common Armyworm, Leucania separata (Lepidoptera : Noctuidae) : II. Timing of PTTH Secretion and Ecdysteroid Titer for Fifth Ecdysis

Larval moult before and after the penultimate instar (5th instar) in Leucania separata involves an endocrine cascade that begins with the release of PTTH. The mechanism of this process is as follows : the newly ecdysed 5th instar larvae weighting average 44.0 mg have been already destined to release PTTH at Gate II stage (just before the 3rd night), and subsequently the released PTTH activates the prothoracic glands to increase the hemolymph ecdysteroid titer. The head capsule slippage is induced by this ecdysteroid surge. The 6th instar moult is finally observed about 20 hr after the head capsule slippage. The entire process of this endocrine cascade from 5th instar larva to last instar takes 3 days.

Prothoracic glands and ecdysteroid titre in Galleria mellonella larvae

Changes in the haemolymph ecdysteroid titre during the last larval instar of Galleria are parallelled by changes in the rate of ecdysteroid production by the prothoracic glands in vitro. Extirpation of more than 90% of the prothoracic glands has no significant effect on the ecdysteroid titre in the haemolymph. It was found that the cells remaining after extirpation hypertrophy and produce in vitro comparable amounts of ecdysteroids as the whole glands from intact insects. The production is several times higher than in the corresponding non-hypertrophied cells. The results confirm that prothoracic glands are an indispensable source of ecdysteroids for the induction of pupation in Lepidoptera.

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

AbstractInjection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain‐corpora cardiaca‐corpora allata complexes were removed from intact fifth‐instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20‐hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.

Degeneration of last instar Heliothis virescens prothoracic glands by Campoletis sonorensis polydnavirus

The development of last instar Heliothis virescens larvae was experimentally arrested by ligation between the head and thorax or by injection with calyx fluid from the endoparasitoid Campoletis sonorensis. Prothoracic glands were removed from these larvae at various times after treatment and the gland cells were either measured or prepared for electron microscopy. At 4 days postligation, gland cell ultrastructure was similar to that of cells from unligated larvae and cell size was not significantly reduced until 7 days after ligation. In contrast to ligated controls, cells from calyx fluid-injected larvae were significantly smaller 24 hr after injection. Significant degeneration in gland cell ultrastructure was observed 2 days after injection and by 7 days postinjection, prothoracic glands showed gross degeneration. Similarly, injection of purified polydnavirus caused gross gland degeneration by 7 days after injection. Irradiating calyx fluid abolished its ability to cause gland degeneration. Our results indicate that C. sonorensis polydnavirus stimulates prothoracic gland degeneration. This virally induced degeneration of the prothoracic glands may account for our previously reported observation that injection of calyx fluid causes a drastic reduction in hemolymph ecdysteroid titer and consequent developmental arrest.

Ecdysteroid titres and head critical period for moulting in male larvae of the cockroach, Diploptera punctata

The regulation of moulting was studied in the cockroach, Diploptera punctata, by measuring the head critical period for moulting and ecdysteroid titres during the final two stadia of male larvae. The end of the head critical period for moulting was determined from the percentage of animals that moulted following decapitation of animals on a series of days. The day of decapitation following which more than 50% of the animals moulted was day 8 (of a 15-day stadium) for penultimate instars, and day 12 (of a 22-day stadium) for final instars. The haemolymph ecdysteroid titres remained below 1 μM in the first half of both stadia. In the second half of the penultimate stadium the ecdysteroid titre rose to a peak of 22.5 μM on day 11. In the final stadium a small peak occurred on day 10 (1.2 μM) after which the titre declined significantly and was followed by the large increase between days 13–20 with a peak of 20.1 μM on day 17. In the final stadium on days 8 and 17 the 20-hydroxyecdysone/ecdysone ratios were 2.5 and 7.5, respectively. For comparison with males, haemolymph ecdysteroid titres were also determined in female larvae during the peak periods in the penultimate and final stadia. High titres of 8.2 and 14.2 μM were found in the penultimate and final stadia respectively.

Dose‐dependent influence of Campoletis sonorensis polydnavirus on the development and ecdysteroid titers of last‐instar Heliothis virescens larvae

AbstractCampoletis sonorensis calyx fluid arrests the development of last‐instar Heliothis virescens larvae and is associated with the gross degeneration of the host's prothoracic glands. Through manipulations of ovary supernatant, Campoletis sonorensis polydnavirus (CsV) was found to be the only component of calyx fluid responsible for causing host developmental arrest. Venom from C. sonorensis had no effect on host development. Suspensions of CsV were quantified, and various doses were injected into last‐instar hosts. The percentage of larvae developmentally arrested was dose dependent. In addition, larvae not arrested by injection with CsV suspensions were developmentally delayed in a dose‐dependent manner. Hosts were delayed in the stage in which they were injected and, after recovery, developed at normal rates. Measurements by radioimmunoassay indicated that developmental delay was due to a suppression of ecdysteroid titers. After a dose‐dependent period of suppression, hemolymph ecdysteroid titers recovered and reached titers comparable to those observed in saline‐injected controls. Examination of prothoracic glands from developmentally delayed larvae revealed that partial degeneration occurred. Comparisons of the number and mean size of surviving gland cells and the length of developmental delay suggested that surviving gland cells may compensate for degenerated cells by increasing their ecdysone production.

Regulation of juvenile hormone biosynthesis by 20-hydroxyecdysone during the fourth larval instar of the tobacco hornworm, Manduca sexta

A previous study of the role of 20-hydroxyecdysone in the regulation of juvenile hormone (JH) biosynthesis during pupal commitment in the last (fifth, V) larval instar of Manduca sexta revealed that the increase in the hemolymph edysteroid titer that elicits commitment also stimulates the corpora allata (CA) to synthesize JHs I and III and/or their acids. This stimulation is exerted indirectly via the brain-corpora cardiaca and results in the postcommitment increase in the JH titer that is important for the molt to a pupa. The possibility that a similar form of interendocrine regulation of JH biosynthesis by 20-hydroxyecdysone is involved in the control of larval molting in Manduca was investigated for the molt from the fourth (IV) to the V larval instar. During the IV instar, the increase in the hemolymph ecdysteroid titer and the capacity of the CA to synthesize JHs I and III in vitro correlated temporally in a manner suggestive of CA regulation by 20-hydroxyecdysone. With an in vitro approach, physiological concentrations of 20-hydroxyecdysone (⩾ 1 μg ml −1 hemolymph) were shown to have a dose-dependent trophic effect on CA activity, specifically stimulating JH I synthesis. Since neither basally active nor stimulated CA contained significant amounts of JH I, it appears that 20-hydroxyecdysone acts at the level of biosynthesis rather than at the level of release. The specificity of 20-hydroxyecdysone stimulation of JH I synthesis was demonstrated by the inability of the biologically inactive ecdysteroids 22-isoecdysone and 5α-ecdysone to activate the glands. 20-Hydroxyecdysone's regulation of IV instar larval CA compared with its regulation of the CA during pupal commitment is discussed.

Developmental endocrinology of larval moulting in the tobacco hornworm, Manduca sexta.

A larval moult in the tobacco hornworm, Manduca sexta, involves an endocrine cascade that begins with the release of a cerebral peptide hormone, the prothoracicotropic hormone (PTTH). The release of PTTH is gated, occurs during the scotophase and appears to be developmentally cued. In fourth instar Manduca larvae, PTTH release into the haemolymph occurs as a single burst over a few hours during the head critical period, i.e. the time during which the head (brain) is needed for the initiation of the moult to the fifth (last) instar. Released PTTH activates the prothoracic glands (PGs), and within a few hours the cumulative effect of this event results in a dramatic increase in the haemolymph ecdysteroid titre, which then elicits the moult. An assessment of the capacity of the corpora allata (CA) to synthesize juvenile hormone (JH) in vitro indicates that the above sequence of endocrine events begins only when JH synthesis has reached a nadir for the instar. Since CA activity is an indirect measure of the haemolymph titre of the hormone, it is conceivable that the developmentally cued release of PTTH is permissively controlled by a decreasing haemolymph titre of JH. With the increase in the ecdysteroid titre which marks the end of this endocrine cascade, the CA again become active, presumably to cause the increase in the JH haemolymph titre which directs the larval moult. This investigation has thus established the temporal and quantitative dynamics of the PTTH-PG axis that drive larval moulting and provides insight into the interendocrine regulatory relationships that may exist between the ecdysteroids and JHs. These possible relationships and the role of the brain in their regulation are discussed.

Effects of infection with a granulosis virus on larval growth, development and ecdysteroid production in the cabbage looper, Trichoplusia ni

ABSTRACT. Granulosis virus‐infected Trichoplusia ni (Hûbner) larvae exhibited an increased larval life span with no supernumerary moult and no pupation. Weight gain was not affected. Insects infected shortly after hatching were slower in reaching the fourth and fifth stadia than were control insects. Haemolymph ecdysteroid titres were lower in virus‐infected insects than control insects, but these differences were only significant (P&lt;0.05) in the fifth stadium. Electron microscopic examination of the pro thoracic glands revealed extensive granulosis virus infection, and glands from virus‐infected insects produced no RIA‐detectable ecdysteroids in vitro. Injection of 20‐OII‐ecdysone into virus‐infected larvae at various concentrations and times did not induce pupation.

Effect of allatectomy on ecdysteroid-dependent development of Rhodnius prolixus larvae

the regulation of haemolymph titres of ecdysteroids during larval development of the bloodsucking bug, Rhodnius prolixus was studied. Corpus allatum ablation in 4th-instar larvae 1 day after feeding was reflected in an increase of the intermoult period and in a high level of ecdysial arrest. These effects could be corrected by juvenile hormone and ecdysone therapies. Comparison of the ecdysteroid titres in haemolymph determined in control and allatectomized larvae, at different intervals after feeding, showed that allatectomy drastically depressed the ecdysteroid levels. Juvenile hormone treatment reestablished ecdysteroid titres in the haemolymph of allatectomized insects. Isolated prothoracic glands from allatectomized larvae had a very low production of ecdysteroid-RIA-activity when compared with prothoracic glands from control or allatectomized larvae which received in vivo juvenile hormone treatment. The complexity of the corpus allatum-prothoracic glands interaction in Rhodnius post-embryonic development is discussed.

Effects of decapitation at the head critical period for moulting on haemolymph ecdysteroid titres in final-instar male and female Rhodnius prolixus (Hemiptera)

Decapitation experiments performed on large populations of carefully staged last-instar Rhodnius prolixus indicate that there is a 24 h sex difference in the time of the head critical period. Males pass the head critical period over a 36 h period beginning at 1200 A.Z.T. (Arbitrary Zeitgeber Time) day 6 after feeding, while females pass the head critical period over a 36 h period beginning at 1200 A.Z.T. day 5 after feeding. Radioimmunoassay determinations of haemolymph ecdysteroid titres show that there is also a 24 h sex difference in the time of the major increase in ecdysteroid titre that commences at the head critical period. These results imply that there is a sex difference in the time at which the release of prothoracicotropic hormone (PTTH) associated with the head critical period occurs. Decapitation prior to the head critical period results in a rapid, permanent decrease in ecdysteroid titre, while decapitation after the head critical period does not prevent normal increases in ecdysteroid levels. We infer that the presence of the head is essential until just prior to the head critical period and its associated increase in at the head critical period also indicates that the head critical period represents a change in the activity of the prothoracic glands indicative of a response to a significant release of PTTH at this time. The significance of these results is discussed in relation to both classical and recent evidence concerning the timing of PTTH release and changes in ecdysteroid titres in Rhodnius prolixus.

Juvenile hormone coordinates the regulation of the hemolymph ecdysteroid titer during pupal commitment in Manduca sexta

The timing and magnitude of the pupal commitment peak in the hemolymph ecdysteroid titer of fifth instar Manduca sexta larvae are controlled by the combined effects of prothoracicotropic hormone (PTTH), a prothoracic gland-stimulating factor present in the hemolymph, and the biosynthetic competence of the prothoracic glands themselves. The present data indicate those individual effects are coordinated by juvenile hormone (JH): (1) Treatment of larvae with the JH analog (7 S)-hydroprene prevents the normal precommitment drop in the titer of the stimulatory factor; (2) treatment of larvae with (7 S)-hydroprene suppresses in a dose- and time-dependent manner the biosynthetic competence of the prothoracic glands; and (3) (7 S)-hydroprene acts directly on the brain to inhibit the release of PTTH in vitro. Thus, during Manduca development, a drop in the JH titer early in the fifth instar results in a rapid drop in the titer of the stimulatory factor, the gradual acquisition by prothoracic glands of biosynthetic competence, and lastly, the gated release of PTTH into the hemolymph. The resulting increase in ecdysone synthesis by the prothoracic glands gives rise to the small peak in the ecdysteroid titer that drives pupal commitment.

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.

Regulation of the stress-responsive X-organ-Y-organ axis by 5-hydroxytryptamine in the crab, Cancer antennarius

Eyestalk-intact, but not de-eyestalked, crabs ( Cancer antennarius) given five injections of 5-hydroxytryptamine (5HT, 750 μg per injection) in 48 hr showed significant reductions in hemolymph ecdysteroid titers within the first 12 hr which continued through Hour 72; the effect was reversible (Hours 72–144). The 5HT synthesis inhibitor p-chlorophenylalanine (PCPA; 60 μg) or the 5HT receptor antagonist cyproheptadine (CPH; 100 μg) caused significant rises in serum ecdysteroids in intact, but not in de-eyestalked, crabs; with four injections over 48 hr, a rise was evident at 12 hr, continued through 72 hr, and returned to control levels within 4 days post-treatment. Stress due to handling depressed ecdysteroid levels, an effect that was reversed by PCPA and CPH; values in the treated groups returned to prestress levels within 24 hr and were significantly greater than prestress levels within 48 hr. 5HT treatment did not further decrease stress-reduced ecdysteroid titers. Normal behaviors are described in posturing and in defensive responses to stress; these were changed in both intact and de-eyestalked crabs by 5HT treatment (but not by PCPA or CPH). These findings support the hypothesis that release of molt-inhibiting hormone (MIH) from crab eyestalks is mediated by 5HT and suggest that stress induces 5HT-mediated MIH release.

Hemolymph ecdysteroid titer and midgut ecdysone 20-monooxygenase activity during the last larval stage of Diploptera punctata

Hemolymph ecdysteroid titers during the last larval stage (lasting 20–21 days) of Diploptera punctata were determined by radioimmunoassay. A small peak (1 γM on day 10) preceded a larger peak (> 10 γM on day 17). Although ecdysone was the predominant circulating ecdysteroid in the beginning of the last larval stage, the ratio of 20-hydroxyecdysone to ecdysone increased to about 7 1 at the time of the large ecdysteroid peak. Ecdysone was converted to 20-hydroxyecdysone by fat body, midgut and Malpighian tubules incubated in vitro. Conditions for the measurement of ecdysone 20-monooxygenase activity were established for midgut homogenates. Maximal activity was found on days 5–9 and activity declined in the second half of the last larval stage. By using differential centrifugation and marker enzymes, most midgut ecdysone 20-monooxygenase activity was found to be associated with microsomes. Antibodies to NADPH-cytochrome P-450 reductase inhibited microsomal ecdysone 20-monooxygenase activity, thus confirming the cytochrome P-450 nature of this enzyme. The antibodies inhibited the ecdysone 20-monooxygenase activity associated with mitochondria as strongly as they inhibited the microsomal enzyme activity, thus showing that the mitochondrial activity was a microsomal contamination and that there was little or no intrinsically mitochondrial ecdysone 20-monooxygenase in the midgut.

Juvenile hormone biosynthesis in female larvae of Diploptera punctata and the effect of allatectomy on haemolymph ecdysteroid titre

Using an in vitro radiochemical assay, rates of juvenile hormone III biosynthesis/release by the corpora allata were determined in female larvae (1st–4th instars) of the cockroach, Diploptera punctata. Higher rates were observed principally in the latter half of the 1st–3rd stadia, whereas juvenile hormone release was undetectable in the latter half of the 4th (final) stadium. Junenile hormone release was also determined for corpora allata stimulated by exogenous farnesoic acid; high rates of hormone release were observed during the entire 3rd stadium and the early portion of the 4th stadium. However, farnesoic acid did not enhance hormone production in the latter half of the 4th stadium, indicating that rate limitation in juveline hormone biosynthesis at this time may not be strictly associated with a single enzymatic step. Since all larvae allatectomized at the beginning of the 4th stadium underwent imaginal ecdyses but exhibited considerably prolonged stadium duration, haemolymph ecdysteroid titre during this stadium was measured using a radioimmunoassay. Ecdysteroid titre of allatectomized larvae was found to remain at a low level for an extended period (at least 20 days). Topical application of a juvenile hormone analogue (ZR 2006) to allatectomized larvae reversed the delay in ecdyses. A possible interaction(s) between juvenile hormone and ecdysteroid for the regulation of metamorphosis is proposed.

Critical periods for juvenile hormone sensitivity during larval life of female Diploptera punctata

The critical periods for juvenile hormone sensitivity were examined in Diploptera punctata female larvae following systematic allatectomy of 2nd, 3rd and 4th (final) instars. Second-instar larvae allatectomized at the beginning of the stadium underwent one or more additional ecdyses after the operation, but most of them retained their larval form; all of them died without completing metamorphosis. Thus, at this stage, juvenile hormone may not be morphogenetically functional. By contrast, allatectomy of 3rd-instar larvae during the first 8 days of the stadium resulted in a high incidence of precocious metamorphosis at the next ecdysis. Larvae allatectomized during this critical period showed prolonged stadium duration (≤65 days). Similarly, in 4th-instar larvae allatectomized during the first half of the stadium (≤10 days), there was a considerable prolongation of the stadium duration (≤92 days), although all larvae underwent imaginal ecdyses. The occurrence of a critical period was also examined in 4th-instar larvae following denervation of corpora allata at selected times during the stadium. A high incidence of supernumerary larval ecdyses was observed in larvae operated during the first 2 days of the stadium, whereas almost all larvae operated on day 6 or later underwent imaginal ecdyses. This switch-over in developmental programming was further examined by monitoring rates of juvenile hormone release and haemolymph ecdysteroid titre. Denervation of corpora allata on day 2 enhanced corpora allata activity. Juvenile hormone release continued up to day 20; thereafter it declined progressively. On the other hand, following denervation of corpora allata on day 6, release rates underwent cyclical fluctuation with a periodicity of 4 or 5 days. The operation considerably prolonged stadium duration (≤49 days). An elevation in ecdysteroid titre was observed only after juvenile hormone release began to decline, suggesting that the presence of juvenile hormone might influence ecdysteroid titre.