Colin G.H. Steel

Haemolymph ecdysteroid titres during larval-adult development in Rhodnius prolixus: Correlations with moulting hormone action and brain neurosecretory cell activity

Abstract 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.

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.

Neuroanatomical relations of prothoracicotropic hormone neurons with the circadian timekeeping system in the brain of larval and adult Rhodnius prolixus (Hemiptera)

This paper reports the localization in the Rhodnius prolixus brain of neurons producing the key neuropeptide that regulates insect development, prothoracicotropic hormone (PTTH) and describes intimate associations of the PTTH neurons with the brain circadian timekeeping system. Immunohistochemistry and confocal laser scanning microscopy revealed that the PTTH‐positive neurons in larvae are located in a single group in the lateral protocerebrum. Their number increases from two in the last larval instar to five during larval‐adult development. In adults, there are two distinct groups of these neurons composed of two cells each. A daily rhythm in content of PTTH‐positive material occurs in both the somata and the axons in both larval and adult stages. These rhythms correlate with previous evidence of a circadian rhythm of PTTH release from brains in vitro. The key circadian clock cells of Rhodnius are eight neurons, which co‐express pigment‐dispersing factor (PDF) and the canonical clock proteins PER and TIM; PDF fills the axons. Equivalent cells control behavioral rhythms in other insects. Double labeling revealed intimate associations between axons of larval PTTH neurons and clock neurons, indicating a neuronal pathway from the brain timekeeping system for circadian control of PTTH release. Additional PDF neurons appear in the adult, associated with the second group of PTTH neurons. These findings provide the first direct evidence that neurons of the insect brain timekeeping system control hormone rhythms. The range of functions regulated by this timekeeping system is quite similar to those of the vertebrate suprachiasmatic nucleus, for which the insect system is a valuable model. J. Comp. Neurol. 503:511–524, 2007.

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 circadian timing system in the brain of the fifth larval instar of Rhodnius prolixus (hemiptera)

The brain of larval Rhodnius prolixus releases neurohormones with a circadian rhythm, indicating that a clock system exists in the larval brain. Larvae also possess a circadian locomotor rhythm. The present paper is a detailed analysis of the distribution and axonal projections of circadian clock cells in the brain of the fifth larval instar. Clock cells are identified as neurons that exhibit circadian cycling of both PER and TIM proteins. A group of eight lateral clock neurons (LNs) in the proximal optic lobe also contain pigment‐dispersing factor (PDF) throughout their axons, enabling their detailed projections to be traced. LNs project to the accessory medulla and thence laterally toward the compound eye and medially into a massive area of arborizations in the anterior protocerebrum. Fine branches radiate from this area to most of the protocerebrum. A second group of clock cells (dorsal neurons [DNs]), situated in the posterior dorsal protocerebrum, are devoid of PDF. The DNs receive two fine axons from the LNs, indicating that clock cells throughout the brain are integrated into a timing network. Two axons of the LNs cross the midline, presumably coordinating the clock networks of left and right sides. The neuroarchitecture of this timing system is much more elaborate than any previously described for a larval insect and is very similar to those described in adult insects. This is the first report that an insect timing system regulates rhythmicity in both the endocrine system and behavior, implying extensive functional parallels with the mammalian suprachiasmatic nucleus. J. Comp. Neurol. 518:1264–1282, 2010.

Non-genomic ecdysone effects and the invertebrate nuclear steroid hormone receptor EcR--new role for an "old" receptor?

The ecdysteroids (Ec), invertebrate steroid hormones, elicit genomic but also non-genomic effects. By analogy to vertebrates, non-genomic responses towards Ec may be mediated not only by distinct membrane-integrated but also by membrane-associated receptors like the classical nuclear ecdysteroid receptor (EcR) of arthropods. This is supported by a comparison of physiological properties between invertebrate and vertebrate steroid hormone systems and recent findings on the subcellular localization of EcR. The measured or predicted high degree of conformational flexibility of both Ec and the ligand binding domain (LBD) of EcR give rise to a conformational compatibility model: the compatibility between conformations of the cognate receptors ligand binding domain and structures or conformations of the ligand would determine their interaction and eventually the initiation of genomic versus non-genomic pathways. This model could also explain why specific non-genomic effects are generally not observed with non-steroidal agonists of the bisacylhydrazine group.

Vitellogenesis and fertility in Drosophila females with low ecdysteroid titres; the L(3)3DTS mutation

After 5 days at the restrictive temperature (29.5°C) adult Drosophila females heterozygous for the dominant temperature-sensitive mutation, L(3)3DTS, have an ecdysteroid level of about half that in mutant females at 22°C and subsequently become completely sterile due to the inviability of progeny embryos. The lethal phase of progeny from mutant females varies depending upon the length of time DTS-3 females are kept at a sublethal temperature of 27°C. Thus, the DTS-3 mutation shows a maternal effect, and a deficiency of ecdysteroids or ecdysteroid-induced gene products may be responsible for progeny lethality. This lethality cannot be attributed to a deficit in the products of the hormonally-regulated yolk polypeptide genes however, since yolk polypeptide mRNA and protein levels are not reduced in DTS-3 females at the restrictive temperature.

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.

hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera)

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals.

Induction of rhythmicity in prothoracicotropic hormone and ecdysteroids in Rhodnius prolixus: roles of photic and neuroendocrine Zeitgebers

Abstract Circadian rhythms have been described in Rhodnius prolixus in both the release of prothoracicotropic hormone (PTTH) from the brain complex and the synthesis (and release) of ecdysteroids by the prothoracic glands (PGs). The PGs possess a circadian oscillator that is light-sensitive in vitro. The present work reports the ability of a ‘lights-off’ signal to induce rhythmicity in both PTTH and ecdysteroids in whole animals. Continuous light (LL) caused cessation of release of PTTH; rhythmic release was promptly initiated by transfer to darkness (DD). We infer a light-sensitive circadian oscillator that regulates PTTH release and discuss evidence of its location in the protocerebrum. PGs maintained in LL became arrhythmic but maintained a developmental modulation of steroidogenesis. Transfer of animals to DD initiated rhythmic steroidogenesis; thus, the ‘PG oscillator’ operates in vivo despite an overlying cuticle. The first initiated peak in steroidogenesis precedes that of PTTH by several hours and was not impaired when PTTH release was prevented by prior injection of tetrodotoxin. In normal animals (PTTH present), the phase of the induced rhythm of steroidogenesis was shifted in a single cycle to align with that of PTTH release. We conclude that both ‘brain oscillator’ and ‘PG oscillator’ are photosensitive, and the induced PTTH rhythm regulates the phase of rhythmic steroidogenesis. This neuroendocrine axis contains (at least) three photosensitive oscillators, in which classical pacemaker and slave oscillators are not obvious. Caution in the application of formal terminology to discrete tissues is urged. This multioscillator timing system appears to direct the circadian organization of development through the rhythm in haemolymph ecdysteroids that reaches ecdysone-responsive cells.