Head Critical Period Research Articles

Changes in the corpora allata and epidermal proliferation along the fourth instar of the Chagas disease vector Triatoma infestans

Triatoma infestans, a blood-feeding insect, synchronises physiological mechanisms leading to moult with food intake. Since the corpora allata are important in moult and metamorphosis regulation, we have studied morphological changes in 4th instar nymphs (gland size, cell density, percent of animals showing mitoses and cell size). Changes were correlated with the effect of precocene II, epidermal proliferation, and with the extent of the "head critical period". Based on morphological grounds, three stages can be defined in the gland along the 4th instar: Stage 1 (days 0-2 after feeding) showed small corpora allata, composed by a small number of cells, and in which mitoses were absent; Stage 2 (days 3-9) showed growing corpora allata, in which cell number was increasing and proliferation was apparent; and Stage 3 (days 10-13) showed no mitotic activity, and a sharply diminishing size of the gland, as a consequence of the diminishing size of their cells. The ability of precocene II to induce abnormal moulting disappeared during stage 2 correlating with the termination of the head critical period and suggesting that corpora allata are essential during days 3 to 5 to determine normal growth. Epidermal cell number was increasing as a consequence of more frequent mitotic activity, beginning after the finalization of the head critical period and after a first increment in the size of the gland.

Chroma acquires ISO 9001 certification

1.1. The prothoracic gland is the main source of ecdysteroids in larvae of the cockroach Periplaneta americana.2.2. Besides ecdysone the molting gland of Periplaneta secretes 3-dehydroecdysone and proteins.3.3. The molting gland of Periplaneta is regulated in different successive steps of cooperation of nervous and neuroendocrine activity.4.4. Neurogenic effects on the molting gland via the prothoracic gland nerves are concentrated on the period of prepeak production of ecdysteroids.5.5. Prior to the 17th day, the glands secretory activity is inhibited by GABA-ergic neuronal pathways from the subesophageal ganglion.6.6. Neurogenic disinhibition by a peptidergic brain factor elicits the competence of the gland for prepeak activity, completed by the glandotropic effect of PTTH.7.7. The 17th day of the larval stage is characterized as the head critical period, i.e., after this period the ecdysteroid secretion of the gland is independent of the prothoracicotropic hormone (PTTH) from the brain.8.8. The main peak of ecdysteroid production is regulated by prothoracicotropic neuropeptids from the brain.

Hemolymph ecdysteroid titer and ecdysteroid-dependent developmental events in the last-larval stadium of the silkworm, Bombyx mori: role of low ecdysteroid titer in larval–pupal metamorphosis and a reappraisal of the head critical period

The endocrine regulation of larval–pupal metamorphosis was studied in the silkworm, Bombyx mori, by measuring the following changes: hemolymph ecdysteroid titer, the secretory activity of prothoracic glands and the responsiveness of larvae to ecdysteroids and prothoracicotropic hormone (PTTH), with regard to developmental events such as the occurrence of spinneret pigmentation, initiation of cocoon spinning and onset of wandering stage as indicated by gut purge. These measurements were concentrated especially on the time before and after the head critical period (HCP) which falls 3–4 days before the gut purge ( Sakurai, 1984). A small increase in the hemolymph ecdysteroid titer was first found during the HCP, and then the titer increased with daily fluctuations. Small but significant titer peaks were found prior to the occurrence of both spinneret pigmentation and gut purge, indicating that an individual titer peak could possess a specific role in development. Responsiveness of larvae to exogenous 20-hydroxyecdysone (20E) after the HCP was markedly higher than that before the HCP. The sensitivity of the prothoracic gland to PTTH also changed during the HCP. The results thus showed that the HCP is not the period after which an additional PTTH release is not required for the developmental events occurring on schedule, but rather it is the period during which complex events occur not only in the endocrine glands but also in the peripheral tissues. In addition, various developmental phenomena before gut purge are brought about by the hemolymph ecdysteroid whose concentration gradually increased with daily fluctuations, and these precise changes in the titer appeared to be important for the sequential occurrence of developmental events in the larval–pupal metamorphosis.

Head critical period and juvenile hormone titre during the last larval instar of the cockroach,Periplaneta americana

From neck ligation experiments with last instar larvae of the cockroachPeriplaneta americana it was concluded that the head critical period is reached around day 17, which corresponds to 59% of the last larval stage. At the same stage the juvenile hormone III titre in the hemolymph dropped to undectable levels.

Juvenile hormone and hemolymph juvenile hormone binding protein titers and their interaction in the hemolymph of fourth stadium Manduca sexta

A driving force in the initiation of an endocrine response is the modulation of hormone titers surrounding the responsive tissue. For nonpolar hormones, titer fluctuations are, in part, regulated by specific circulatory transport proteins that bind, protect and convey the hormones to the responsive tissues. In the present study, juvenile hormone (JH), hemolymph juvenile hormone binding protein (hJHBP), and total hemolymph protein in the fourth larval stadium of Manduca sexta were quantified from similarly staged insects to assess whether JH is bound or unbound when it interacts with the target. JH levels, as determined by radioimmunoassay, were highest, 12.87 nM, 4 h after ecdysis to the fourth stadium, and declined 6-fold to the lowest titer, 2.28 nM, at 44 h after ecydsis, i.e. 8 h after head critical period. Conversely, hJHBP titers peaked at 11.95 μM 44 h after ecdysis and dropped 5-fold by the time of ecdysis to the fifth stadium. Total hemolymph protein also reached its highest level, 19.83 mg/ml, at 44 h; however, hJHBP titers did not follow fluctuations in the total hemolymph protein. Calculations of bound and unbound JH indicated that >99% of the hormone was bound by hJHBP. In contrast, <1% of the binding protein was hormone-loaded at any time during the stadium. Due to the rapid metabolism of JH in the hemol the hemolymph is improbable at certain times during development. Thus, the rate limiting step in JH uptake at these times may be the interaction of hJHBP with the target cell.

Release in vitro of prothoracicotropic hormone from the brain of male Rhodnius prolixus during larval-adult development: Identification of novel and predicted release times

This paper reports an in vitro study of the timing of release of prothoracicotropic hormone (PTTH) from isolated brain-retrocerebral complexes during larval-adult development in male Rhodnius. Brain complexes were removed on sequential days of development and tested for PTTH release by their ability to stimulate ecdysteroid synthesis in co-incubated prothoracic glands. This is the first systematic study to utilize ecdysteroid synthesis in vitro to detect PTTH release occurring naturally from intact brain complexes during development. It provides the most direct evidence to date of the times during development when PTTH is released. Brain complexes were found to release PTTH during three distinct periods of development. Brain complexes at other times and other ganglia released no significant activity. The first release is brief and occurs rapidly after a blood meal. The second release occurs at the head critical period for moulting on day 6. The existence of these two previously suspected release times is therefore confirmed. Both are associated in vivo with increases in ecdysteroid synthesis by prothoracic glands and in the haemolymph ecdysteroid titre. The third release time is novel and consisted of sporadic PTTH release spanning days 6–12. This release is not qualitatively necessary for the continuation of development. It is suggested that PTTH release may be episodic during this period and play a role in the previously reported circadian regulation of ecdysteroid synthesis in prothoracic glands. PTTH release during this period of development has not been reported previously in any insect.

Kopec revisited—Neuroendocrine regulation of metamorphosis in the gypsy moth ( Lymantria dispar): Development of a larval in vivo assay for prothoracicotropic hormone

The role of the brain as a neuroendocrine regulator of metamorphosis in the gypsy moth, Lymantria dispar L. was originally demonstrated by Kopec more than 70 years ago. We have reexamined this role by determining haemolymph ecdysteroid titres, activity and responsiveness of the prothoracic glands, and growth of female last (i.e. fifth)-instar larvae in relation to secretion of the brain prothoracicotropic hormone (PTTH), the factor known to trigger moulting and metamorphosis in insects. The head critical period for secretion of PTTH in Lymantria reared at 25°C in a 16 h light-8 h dark cycle was shown by neck ligation to occur on day 7–8 of the fifth instar. The critical period occurred shortly after a small peak of haemolymph ecdysteroid that reached 600 pg 20-hydroxyecdysone equivalents/μl, as determined by radioimmunoassay. Feeding was completed by day 7, thus neck ligation did not affect nutrient intake. Animals starved from day 5 onward completed pupation at the same time as non-starved controls and showed normal increases in haemolymph ecdysteroid titres. Animals neck-ligated on day 7 and injected 5 days later with extracts of brain, retrocerebral complex, or brain tissues containing lateral or median neurosecretory cells were stimulated to pupate within 5 days, and brain-extract-injected animals showed an increase in haemolymph ecdysteroid titre of over 4000 pg/μl. Animals neck-ligated earlier had lower haemolymph ecdysteroid titres than day 7, neck-ligated larvae and their prothoracic glands showed lower activity in vitro. Using the in vivo assay, PTTH activity was detected in pre-hatch eggs and in brains taken from day-5 last-instar larvae, day-1 pupae and day-1 adults. The assay appears specific to tissues from the brain and retrocerebral complex, since no activity was found with extracts of the subesophageal ganglion, fat body or muscle.

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.

Neurophysiological events during the head critical period for prothoracicotropic hormone release in fourth-instar larvae of Manduca sexta

The initiation of larval moulting in the tobacco hornworm, Manduca sexta, involves an endocrine cascade that begins with the release of the cerebral prothoracicotropic hormone (PTTH) from the retrocerebral corpora allata. During the fourth larva instar of Manduca when the gated release of PTTH occurs, the in situ electrical activity of the nervi corporis cardiaci (NCC I + II), the paired nerve trunks from the brain that innervate the corpora cardiaca and more distal corpora allata, undergoes a distinct change in firing pattern. The initial change in pattern, from a steady firing, was a brief, intense burst of relatively large units followed by a short period (3–10 min) of completely suppressed activity. Next, bursting activity of several different units resumed, and depending upon the preparation, this event was either long or short in duration. This unique bursting pattern occurred at about the head critical period for PTTH release and was expressed by the brain-corpus cardiacum-allatum both in situ and in vitro. The complexity of the action potential patterns suggests that a variety of neurosecretory cell types may be involved in a coordinated, transient inhibition of electrical activity of the NCC I + II that is followed by selective disinhibition of a specific set of cerebral neurones that, based upon the release of PTTH from the corpus cardiacum-allatum preparations, culminates in PTTH release. That this neurophysiological event occurs at a precise time and occurs in vitro suggests that a pattern generator for the unique bursting firing and a clock mechanism controlling this event reside within the brain-corpus cardiacum-allatum. The significance of these results in relation to the neuroendocrinology of larval mounting is discussed.

Interactions between limb regeneration and ecdysteroid titres in last larval instar Rhodnius prolixus (Hemiptera)

Leg-amputation experiments performed on populations of carefully staged last-instar Rhodnius prolixus indicate that the regeneration critical period in this species occurs 6–7 days following feeding. Decapitation experiments indicate that the regeneration critical period is also the time of the head critical period for moulting. The head critical period is associated with a rapid increase in ecdysteroid titre in response to a release of prothoracicotropic hormone by the brain. Leg amputations performed prior to the head critical period result in a temporary decrease in ecdysteroid titre lasting 24–36 h, while the same operation performed after the head critical period has no effect on ecdysteroid titre. When the period of temporary depression in titre encompasses the head critical period, the rapid increase in ecdysteroid titre associated with this time is delayed. Thus, the ability to regenerate a leg after initiation of the moult is dependent upon when the injury occurs in relation to the release of prothoracicotropic hormone at the head critical period. The mechanism responsible for the decline in ecdysteroid titre following leg amputation is unknown but does not appear to involve prothoracicotropic hormone or the prothoracic glands. Injection of tetrodotoxin at the time of a leg amputation prolongs the period for which the titre is depressed. In these animals the titre rises in conjunction with resumption of electrical activity in the corpus cardiacum, indicating that prothoracicotropic hormone release may be necessary for recovery of the titre following leg amputation. Results are discussed in relation to existing evidence concerning possible interactions between regeneration, prothoracicotropic hormone release and moulting.

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.

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 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.

In vitro synthesis, release and uptake of storage proteins by the fat body of Manduca sexta: putative hormonal control.

1. Two major proteins (P1 and P2) are synthesized by the fifth instar larval fat body of Manduca sexta and then released into the hemolymph. 2. These proteins are later sequestered by the pre-pupal fat body. 20-Hydroxyecdysone does not appear to affect the synthesis of either protein. 3. When day 2 fifth instar larvae are neck-ligated there is an excessive synthesis (supersynthesis) of P2 (arylphorin). 4. Juvenile hormone I (JH I) applications to ligated animals had no effect, but brain homogenate injections resulted in the inhibition of P2 synthesis. 5. Neck ligations of larvae between days 5 and 6 revealed a head critical period between day 5 + 12 hr and day 5 + 18 hr, after which the head is unnecessary for the sequestration of either protein by the fat body. 6. JH I and JH III applications to ligated larvae before the head critical period do not restore the ability of the fat body to sequester the storage proteins. 7. P1 and P2 appear to be synthesized differentially and P2 is sequestered by the fat body to a much lesser extent than P1. 8. P2 is the hemolymph storage protein of both larval and pupal stages, whereas P1 appears to be the storage protein of the pupal fat body. 9. The data indicate that the synthesis of arylphorin and the resorption of both proteins are controlled by a putative head factor(s).

Humoral inhibition of the corpora allata in larvae of Diploptera punctata: Role of the brain and ecdysteroids

Juvenile hormone synthesis by adult female corpora allata was inhibited following implantation into final-larval-instar males; inhibition was prevented by decapitation of the larval hosts on day 11 (prior to the head critical period for moulting), but not by decapitation on day 13. Implantation of one larval protocerebrum restored inhibition of implanted corpora allata, demonstrating that the brain releases an inhibitory factor. Corpora allata implanted into larvae decapitated on day 11 were inhibited by injections of 20-hydroxyecdysone. Since treatment of corpora allata with 20-hydroxyecdysone in vitro did not inhibit juvenile hormone synthesis, ecdysteroids probably act indirectly on the corpora allata. Juvenile hormone synthesis and haemolymph ecdysteroid concentration were measured following implantation of corpora allata along with two larval brains into larval hosts. Brain implantation did not affect ecdysteroid concentration, but did inhibit juvenile hormone synthesis, even in animals with low haemolymph ecdysteroid concentration. Incubation with farnesoic acid stimulated juvenile hormone synthesis by corpora allata from males early in the final larval stadium, but not after day 8, showing that one of the final two reactions of juvenile hormone synthesis is rate-limiting in larval corpora allata at this stage. Adult female corpora allata which had been humorally inhibited by implantation into larvae were stimulated by farnesoic acid.

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.

The Prothoracicotropes: Source of the Prothoracicotropic Hormone

SYNOPSIS. An in vitro assay for the insect prothoracicolropic hormone (PTTH) has been developed which measures the rate of ecdysone synthesized by Manduca sexta prothoracic glands (PG) stimulated in vitro by PTTH. This assay has been used to quantify PTTH in single neurosecretory cells (NSC) resulting in the identification of one NSC in each hemisphere of the brain as the prothoracicotrope, source of PTTH. The axonal and dendritic distribution of the prothoracicotrope has been determined by cobalt filling with silver intensification. From a comparison of the titers of PTTH in brains, corpora cardiaca and corpora allata during larval-pupal development, the corpus allatum has been identifiedas the neurohemal organ for PTTH. Electron microscopic analyses suggest that the acellular sheath surrounding the corpus allatum contains the axon terminals of the prothoracicotropes. There is at least one form of PTTH, ˜22,000 mol wt (big PTTH), and possibly a smaller form of about 7,000 mol wt (small PTTH). Bioassay and PTTH hemolymph titer data during the head critical period (HCP) for larval-larval development reveal that big PTTH is released as a single peak lasting ˜6 hr. By contrast, during the first HCP of the last larval instar PTTH is released over a period of ˜18 hr in three bursts, but its molecular weight has not been established with certainty. The kinetics of PG activation by these two forms suggest that big PTTH may function to activate the PG dramatically and thereby elicit molting, while small PTTH may activate the PG minimally at the time of cellular reprogramming.

Endocrine significance of critical periods during insect development: Analysis of ligation experiments withPieris brassicae last instar larvae

Ecdysone haemolymph levels have been analyzed in neck- or thorax-ligatedPieris larvae in order to explain the physiological significance of critical periods. It appeared that head critical period corresponds to an incomplete activation of prothoracic glands, while the thoracic critical period is related to the secretion of a minimal amount of ecdysone necessary for moulting. DuringPieris' last larval instar, there is no evidence for any noticeable synthesis of ecdysone in isolated abdomens.