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.

Rhythmic steroidogenesis by the prothoracic glands of the insect Rhodnius prolixus in the absence of rhythmic neuropeptide input: implications for the role of prothoracicotropic hormone.

Circadian rhythms have been reported both in the synthesis of the insect steroid moulting hormones (ecdysteroids) by the prothoracic glands (PGs) and in the release of the cerebral neuropeptide, prothoracicotropic hormone (PTTH). PTTH is known to activate steroidogenesis early in development, but the function of continued rhythmic release is unknown. The functional relationship between these two hormonal rhythms was examined. We report the properties of the rhythm of steroidogenesis by PGs of animals in which PTTH release was prevented by decapitation or by injection of a sublethal dose of tetrodotoxin. Rhythmic steroidogenesis by PGs was maintained in both cases; the rhythm retained entrainment to a light-dark cycle and free-ran in continuous light or darkness. It is inferred that rhythmic neuropeptide input is not required to drive rhythmic steroidogenesis and that in its absence, steroidogenesis becomes entrained by light cues. In both decapitated and paralyzed animals, the rhythm of steroidogenesis showed a reversal of phase from that of intact animals under all conditions of illumination tested. We infer that the rhythm of PTTH appears to entrain rhythmic steroidogenesis and entrainment by PTTH dominates entrainment by light in vivo. Similarities to other circadian systems are discussed, in which neurochemical agents entrain overt rhythms to a phase displaced by 12 hr from that for light. It is concluded that the function of PTTH is not confined to initial activation of steroidogenesis early in development, as previously thought, but continues throughout development as a central element in the circadian organization of the endocrine system that regulates development.

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.

Changes during the moult cycle in the bursting firing pattern of the electrical activity recorded extracellularly from the sinus gland of the terrestrial isopod, Oniscus asellus.

Ongoing electrical activity of the sinus gland (SG) of the terrestrial isopod, Oniscus asellus, was recorded extracellularly from almost intact breeding or non-breeding females to delineate the major times of neurohormone release during the moult cycle. In intermoult, SGs discharged in long bursts (10-50 s) at high frequency (10-45 Hz), and their activity ratios (total burst duration divided by total time the SG was monitored) ranged from 0.22 to 0.73. At premoult initiation when release of moult-inhibiting hormone is expected to decline, a decrease in SG activity occurred. It rose again in early premoult in parallel with increases in ecdysteroid titre; declined again in late premoult during peak ecdysteroid titres; increased again just prior to posterior ecdysis, and was very low during posterior ecdysis itself. Activity increased immediately after posterior and anterior ecdysis suggesting the release of neurohormones involved in calcification of the new cuticle. Burst duration was ca. two-fold longer in breeding compared to non-breeding females during early premoult suggesting the release of neurohormones involved in vitellogenesis, and before anterior ecdysis suggesting release of neurohormones involved in egg deposition. Thus, the release of neurohormones occurred during 4 major periods in each moult cycle, clearly demonstrating a relationship between SG activity in situ, and the physiological events dependent on SG hormones.

Induction of rhythmic modulation of haemolymph ecdysteroids in the insect Rhodnius prolixus by treatments which elicit rhythmic ecdysis.

The titer of moulting hormones (ecdysteroids) in fifth instar male Rhodnius displays a gradual decline throughout the week preceding ecdysis when animals are maintained in constant light (LL). Multiple sampling of haemolymph from the same animal reveals a constant rate of decline with no increases in titer observed. However, 20-hydroxyecdysone (20HE) provokes increases in the titer of ecdysteroids which occur between 18 and 27 hr after injection. Multiple injections of 20HE provoke regular changes in the rate of decline of the titer. When 20HE is injected into animals reared in LL, a rhythm in the titer of ecdysteroids with an initial periodicity of approximately 28 hr is provoked which is reduced to 24 hr by 72-96 hr after injection. Injection of antiserum to ecdysteroids elicits an immediate rapid oscillation in titer similar to that observed in light/dark (L/D) cycles. Therefore injections of 20HE or antiserum to ecdysteroids can provoke a rhythm in the titer of ecdysteroids in animals reared in LL. Rhythms in ecdysteroid titer would appear to result from the interactions of synthetic and catabolic systems, both of which would appear to be able to act in a timed fashion. Such rhythmic modulation of ecdysteroid titer may provide time cues to the circadian system timing ecdysis. Thus the gated ecdysis rhythm observed after 20HE or antiserum injections may be a response to induced modulation of the ecdysteroid titer.

Electrical activity of the sinus gland of the terrestrial isopod, Oniscus asellus: characteristics of identified potentials recorded extracellularly from neurosecretory terminals.

Spontaneously occurring neurosecretory action potentials recorded extracellularly from the sinus gland (SG) of the terrestrial isopod. Oniscus asellus, are of 5 types (A through E) identified by their amplitudes and patterns of discharge. Type A have the largest (200-450 microV) and type E the smallest (25-50 microV) amplitude. Types A, B and C originate from the bulb of the SG, and discharge at high frequencies (30-60 Hz) in coordinated bursts ranging from seconds to several minutes in duration. Coordination of their discharges suggests a mechanism for synchronizing bursting activity among different cell types. Types D and E originate from the lateral extension of the SG, and discharge at low frequencies (0.5-1.0 Hz) for prolonged periods (5-10 min). Their activity is not synchronized with discharges of other potentials. Following transection of the brain through the lateral part of the central protocerebral neuropile, A, B and C potentials are eliminated whereas D and E potentials remain active. This result suggests A, B and C potentials arise from neurosecretory cells (NSCs) whose cell bodies are located in the medial protocerebrum, and D and E potentials arise from NSCs identified in the optic lobe. Alterations in the appearance of action potentials following exposure to salines deficient in Na+ or Ca2+, or containing tetrodotoxin or cobalt, reveal that A and B potentials are primarily Ca2+ dependent whereas C potentials are both Ca2+ and Na+ dependent.

Recording electrophysiological data on video tape: a superior and less costly alternative to conventional tape recorders.

Electrical potentials recorded extracellularly from the sinus gland of the isopod, Oniscus asellus, were stored on video tape with the aid of a digital-audio (DA) processor and a video cassette recorder (VCR). The DA processor transforms the analog signal to digital pulses of equal amplitude and converts these pulses into a television signal for recording on video tape. In playback, the DA processor reconverts the pulses to an analog signal with negligible distortion. When viewed on the oscilloscope screen, electrical potentials reproduced by this method were indistinguishable from electrical potentials recorded 'live' from the sinus gland. However, electrical potentials recorded from the same sinus gland and reproduced by a conventional FM tape recorder were easily differentiated from the 'live' recording. The special effects inherent in the VCR (e.g. stop action, frame advance) also permitted detailed analysis of spontaneously occurring electrical potentials. Special effects were not possible with the FM tape recorder. The price, ease of operation and ability to produce extremely high quality recordings, makes the DA processor and VCR an exceptional system for storing electrophysiological data.

Circadian control of a daily rhythm in hemolymph ecdysteroid titer in the insect Rhodnius prolixus (Hemiptera).

The hemolymph levels of the insect molting hormone (ecdysteroid) during the week preceding ecdysis in fifth-instar male Rhodnius prolixus have been determined using a radioimmunoassay. When animals are kept on light-dark cycles, the titer displays massive daily increases and decreases producing a daily rhythm. This rhythm is maintained with a period of approximately 24 hr in continuous darkness. The free-running period of the rhythm was determined at 24 and 28 degrees and found to be temperature compensated. Therefore the titer of ecdysteroids is modulated by a circadian system. Since ecdysteroids are known to influence a wide variety of developmental events ranging from chromosome puffing to cuticle deposition, circadian modulation of the titer will provide information concerning time to all ecdysteroid sensitive tissues hence could function as a pacemaker for imposing developmental synchrony.

Coupling of electrical activity from contralateral sinus glands.

Bursts of electrical activity recorded extracellularly from the sinus gland (SG) of the isopod, Oniscus asellus, occur synchronously in right and left SGs. Synchronization results from the electrical activity of two physiologically identifiable neurosecretory cell (NSC) types in one SG being coupled to the electrical activity of their respective contralateral counterparts. Furthermore, the coupling mechanism which serves to coordinate hormone release from contralateral SGs appears to differ for each of the two NSC types.

Ultrastructure and distribution of identified neurosecretory terminals in the sinus gland of the terrestrial isopod Oniscus asellus.

An ultrastructural study of the sinus gland of the terrestrial isopod, Oniscus asellus, reveals that this structure consists of two regions: the bulb, which is attached by a narrow stalk to the optic lobe, and the lateral extension, which extends from the bulb along the optic tract to the compound eye. The bulb has a distal region containing only neurosecretory terminals, and a proximal region containing terminals, glial cells, and axons that give rise to the distally located terminals. In total, the sinus gland contains five types of terminals which can be distinguished by their location and the appearance of their neurosecretory granules. Three terminal types are located in the bulb and two in the lateral extension. The size of the terminals in the bulb varies among the three types, but the number of terminals is approximately the same for each type. Conversely, the two terminal types in the lateral extension are similar in size, but differ in number. Axons of two terminal types in the bulb can be traced to the central region of the protocerebrum; axons of one terminal type in the bulb and of terminals in the lateral extension can be traced to the optic lobe.

Electrical activity of neurosecretory axons from the brain of Rhodnius prolixus: relation of changes in the pattern of activity to endocrine events during the moulting cycle.

Ongoing electrical activity was recorded from the axons of neurosecretory cells from the brain of 5th instar Rhodnius prolixus throughout the moulting cycle. Dramatic changes in both the frequency and pattern of electrical activity occur at specific times during the cycle, enabling the timing of release of neurohormones from the brain to be inferred. The level of transport of stainable secretion appears to be closely coupled to the electrical activity at all times. Activity is low in unfed Rhodnius, but within minutes of the insect taking a blood meal, there is a rapid appearance of bursting activity from a number of units, in conjunction with apparently continuous firing components. The bursting activity declines at about 2 h remaining low for the following 5 days, at which time there is a resurgence in the bursting pattern for a few hours. Both peaks of bursting activity immediately precede increases in haemolymph titer of ecdysones, suggesting that release of prothoracotropic hormone occurs on these two occasions. The continuously firing components initiated at feeding maintain a high level for 5 days indicating release of other brain neurosecretions. Intense electrical activity in the form of bursting activity and high apparently continuous pattern resumes shortly before ecdysis and continues until 12 h after. The relationship of neurohormone release at this time to bursicon and ecdysis behavior is discussed.

The role of neurosecretion in the photoperiodic control of polymorphism in the aphid Megoura viciae.

The location of the photoperiodic mechanism controlling the production of the sexual and parthenogenetic morphs by apterous parents was examined by selectively injuring the brain with an R.F. microcautery. Lesions destroying the Group I neurosecretory cells (NSC) in the protocerebrum abolished the response to changed daylength. Extensive damage to other NSC Groups, to the compound eyes and optic lobes was without effect. It is concluded that the Group I NSC are the effectors, secreting a virginoparapromoting substance; in its absence only oviparae are produced. Areas slightly lateral to the group I NSC are also required for the long-day response, indicating that this is the probable site of the neuronal photoperiodic clock which regulates the release of neurosecretory material (NSM) from the Group I cells.