Testis ecdysiotropin, an insect gonadotropin that induces synthesis of ecdysteroid.

Testes of lepidoptera synthesized ecdysteroid in a somewhat different temporal pattern than the prothoracic glands that release ecdysteroid to the hemolymph. Brain extracts from Heliothis virescens and Lymantria dispar induced testes to synthesize ecdysteroid, but did not affect prothoracic glands. The testis ecdysiotropin (LTE) was isolated from L. dispar pupal brains by a series of high-pressure chromatography steps. Its sequence was Ile-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH, of molecular mass 2,473 Daltons. The predominant signaling pathway for LTE was via G(i) protein, IP3, diacylglycerol and PKC; a modulating pathway, apparently mediated by an angiotensin II-like peptide, was controlled via G(s) protein, cAMP, and PKA. Testis ecdysteroid caused isolated testis sheaths to also synthesize a growth factor that induced development of the male genital tract. The growth factor appeared to be a glycoprotein similar to vertebrate alpha-1-glycoprotein. A polyclonal antibody to LTE indicated LTE-like peptide in L. dispar brain medial neurosecretory cells, the suboesophageal, and other ganglia, and also in its target organ, the testis sheath. LTE immunoreactivity was also seen in testis sheaths of Rhodnius prolixus. LTE-like immunoactivity was also detected in developing optic lobes, antennae, frontal ganglia, and elongating spermatids of developing L. dispar pupae. This may indicate that LTE has a role in development as well as stimulation of testis ecdysteroid synthesis. Published 2001 Wiley-Liss, Inc.

Daily rhythm of responsiveness to prothoracicotropic hormone in prothoracic glands of rhodnius prolixus

The synthesis of ecdysteroids by the prothoracic glands (PGs) of Rhodnius prolixus occurs with a circadian rhythm throughout most of larval-adult development. This rhythm is generated by a circadian oscillator within the PGs. The principle regulator of the PGs is the cerebral neuropeptide prothoracicotropic hormone (PTTH), which is also known to be released with a circadian rhythm, but it has not been shown that these daily releases of PTTH are detected by the PGs. The present report examines the ability of PTTH to augment ecdysteroid synthesis in rhythmic PGs at various times of day. There is a daily rhythm in the responsiveness of PGs to PTTH; high responsiveness occurs around dusk and PGs are largely unresponsive to PTTH during the daytime. This rhythm phase-leads the rhythms of PTTH release and of ecdysteroid synthesis by several hours. The data are suggestive of a daily rhythm of up- and down-regulation of PTTH receptor availability in the PGs. It is concluded that the daily releases of PTTH are detected by the PGs and participate in the regulation of daily ecdysteroid synthesis. Therefore, PTTH plays a continuing role in the regulation of PGs throughout development. Arch. Copyright 1999 Wiley-Liss, Inc.

Ecdysteroidogenic action of Bombyx prothoracicotropic hormone and bombyxin on the prothoracic glands of Rhodnius prolixus in vitro.

An in-vitro assay for ecdysteroid synthesis by the prothoracic glands (PGs) of fifth instar Rhodnius prolixus has been employed to evaluate the actions of prothoracicotropic neuropeptides from the silkmoth, Bombyx mori. Crude prothoracicotropic hormone (PTTH) extracts of recently emerged adult brain complexes of Bombyx induced a dose-dependent stimulation of ecdysteroid synthesis by Rhodnius PGs, which was similar to that obtained using crude Rhodnius PTTH. In both cases, maximum stimulation was obtained with one brain equivalent. Rhodnius PGs were then challenged with incremental doses of recombinant Bombyx PTTH and synthetic bombyxin-II. Dose-response curves for the action of both peptides on Rhodnius PGs were very similar to those obtained for their action on the pupal PGs of Bombyx in vitro. Bombyx PTTH stimulated the PGs of Rhodnius at concentrations comparable to those effective on Bombyx. The curve for Bombyx PTTH showed a steep ascending region from 3 to 8ng/ml and a sharp peak. For bombyxin, concentrations 40-fold higher were required to elicit the same amount of stimulation as obtained using Bombyx PTTH. Therefore, Rhodnius PGs possess recognition sites for both Bombyx PTTH and bombyxin. This is the first study of the ecdysteroidogenic properties of the Bombyx peptides on a heterologous species. It is suggested that the function and conformation of PTTH may be conserved between distantly related insect groups.

Circadian regulation of a daily rhythm of release of prothoracicotropic hormone from the brain retrocerebral complex of Rhodnius prolixus (hemiptera) during larval-adult development.

A daily rhythm of release of prothoracicotropic hormone (PTTH) has been reported throughout most of larval-adult development in Rhodnius prolixus. PTTH released by explanted brain-retrocerebral complexes was quantified using an in vitro bioassay in which the PTTH released into the incubation medium was assayed by its ability to stimulate ecdysteroid synthesis in arrhythmic prothoracic glands (PGs). The present article employs this assay to reveal that the daily rhythm of PTTH release is under circadian control. The rhythm free-runs in both continuous darkness (DD) and continuous light (LL) with a period length close to 24 hr, which is temperature compensated. The rhythm appears to damp out more rapidly in LL than in DD. It is argued that the circadian clock regulating PTTH release is in the brain and is entrained by extra-retinal photoreception. It is suggested that this "PTTH clock" is coupled in vivo to the clock previously described in the PGs that regulates rhythmic ecdysteroid synthesis by the circadian rhythm of release of PTTH. This coupling appears to be tight, since the rhythm of PTTH release retains close synchrony with the rhythm of ecdysteroid synthesis under both DD and LL. It is concluded that these two coupled clocks comprise a multioscillator system that drives the rhythms in ecdysteroid synthesis and the hemolymph ecdysteroid titer and consequently imposes temporal order on ecdysteroid-dependent developmental events.

The insect neuropeptide prothoracicotropic hormone is released with a daily rhythm: re-evaluation of its role in development.

Prothoracicotropic hormone (PTTH) is the central cerebral neurohormone in insect development. Its release has been believed for decades to be confined to one (or two) critical moments early in each developmental stage at which time it triggers prolonged activation of the prothoracic glands to synthesize and release the steroid molting hormones (ecdysteroids), which elicit developmental responses in target tissues. We used an in vitro assay for PTTH released from excised brains of the bug Rhodnius prolixus and report that release of PTTH does occur at the expected time on day 6, but that this release is merely the first in a daily rhythm of release that continues throughout most of the 21 days of larval-adult development. This finding, together with reports of circadian control of ecdysteroid synthesis and titer throughout this time, raises significant challenges to several features of the current understanding of the hormonal control of insect development. New questions are raised concerning the function(s) of PTTH, its relationship with the prothoracic glands, and the significance of circadian rhythmicity throughout this endocrine axis. The significance of the reported observations derives from the set of entirely new questions they raise concerning the regulation of insect development.

In vitro photosensitivity of ecdysteroid synthesis by prothoracic glands of Rhodnius prolixus (hemiptera).

The effects of light:dark transitions on in vitro ecdysteroid synthesis by prothoracic glands (PGs) of male fifth instar Rhodnius have been studied. PGs were rendered arrhythmic by prolonged maintenance of the animals in continuous light (LL). PGs were then explanted and ecdysteroid synthesis was measured hourly by radioimmunoassay. PGs were transferred from LL to darkness at various times after explanation and hourly synthesis was measured for several more hours. Transfer of PGs to darkness in vitro invariably elicited a prompt and substantial (up to fivefold) increase in ecdysteroid synthesis. This response was unaffected by the presence of tetrodotoxin in the medium at a concentration that blocks peripheral nervous activity. It is concluded that the PGs are themselves directly photosensitive. In contrast, PGs from animals raised in continuous darkness and incubated in vitro in darkness showed no response when exposed to light. It is inferred that PGs do not respond to maintained states of either light or dark, or to "lights on." Rather, it is argued that transfer from LL to darkness in vitro constitutes a "lights-off" Zeitgeber which is detected by a photosensitive circadian clock in the PGs. This is the first report of photosensitivity in a steroidogenic tissue and provides primary evidence of a photosensitive pacemaker in a nonneural endocrine tissue.

Circadian regulation of synthesis of ecdysteroids by prothoracic glands of the insect Rhodnius prolixus: evidence of a dual oscillator system.

Rhythmic synthesis of moulting hormones (ecdysteroids) by prothoracic glands (PGs) of the insect Rhodnius prolixus during the last larval instar was studied in vitro following explantations every 4-5 hr for up to 96 hr. Ecdysteroid synthesis was measured by radioimmunoassay as the quantity of ecdysteroid produced during 4 hr in vitro. A massive daily rhythm is seen, with synthesis at night being three- to fivefold higher than during the day. This rhythm of ecdysteroid synthesis by PGs free-runs in continuous darkness with a temperature-compensated period length close to 24 hr and is therefore controlled by a circadian system. This is the first report of circadian regulation of synthesis of an invertebrate hormone. The synthesis rhythm also free-runs in continuous light, but with an inverted phase and shorter period length. It is argued that the circadian system controlling synthesis comprises two oscillators which free-run in antiphase, occupy different anatomical locations, and are coupled by a humoral factor, possibly prothoracicotropic hormone. The ecdysteroid synthesis rhythm in PGs appears to drive the previously reported circadian rhythm in the haemolymph ecdysteroid titre. It is concluded that the circadian system controlling synthesis of ecdysteroids constitutes a pacemaker which drives various rhythms in the target cells of ecdysteroids via the rhythm in the haemolymph titre. Ecdysteroids are viewed as "hormonal Zeitgebers," imposing temporal order on development.

Sex differences in the profiles of prothoracic gland synthetic activity and of hemolymph ecdysteroid titers during development in the last larval instar of Rhodnius prolixus (Hemiptera).

The synthesis of ecdysteroids by prothoracic glands (PGs) of Rhodnius prolixus was measured in vitro by radioimmunoassay in both male and female larvae following initiation of development in the last larval instar by a blood meal. Neither ecdysteroid synthesis nor hemolymph titer differ between the sexes during the first few days following a blood meal. However, on Day 4 the PGs of female larvae commence a surge of increased synthesis, while in males this surge does not commence until Day 5. This 1 day difference is attributed to different times of activation of the PGs by prothoracicotropic hormone in male and female larvae. From Day 4 onward, differences between male and female larvae are described for both ecdysteroid synthesis and titer, both of which are more complex than a 1-day displacement of duplicate profiles. Both temporal and quantitative differences are detailed between males and females during Days 4-16 of development; ecdysteroid synthesis by PGs varies between males and females by up to fourfold on the same day. The hemolymph ecdysteroid titer profile closely follows that of PG synthesis for each sex. Both PG activity and titer in females decline 1 day ahead of males. The declining titer is known to influence the timing of ecdysis, and it was found that the median ecdysis time of females occurs 1 day before that of males even when males and females are synchronized with each other using a developmental marker on Day 14. We conclude that male and female PGs possess the intrinsic ability to synthesize ecdysteroids in a sex-specific pattern during development.(ABSTRACT TRUNCATED AT 250 WORDS)

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.