Release of prothoracicotropic hormone and potentiation of developmental ability during diapause in the bollworm, Heliothis zea

In Heliothis zea, pupal diapause is not due to a deficiency of the prothoracicotropic hormone (PTTH), as it is in many other insects. However, PTTH is essential for diapause termination and adult development. Removal of the pupal brain 4 hr after larval-pupal ecdysis blocks the insect's ability to initiate adult development. Transplantation of brain neurosecretory cells restores this ability, whereas other tissues such as corpora allata have no effect. In the diapausing pupa, PTTH is released from the brain within 24 hr after larval-pupal ecdysis. Subsequent removal of the brain fails to block the ability for diapause termination, because PTTH potentiates the ability for adult development. Since diapause termination is suppressed in a temperature of 21°C, the bollworm retains the ability to initiate development in 27°C whereas it remains in diapause in 21°C. Diapause continues even though pupae are supplied with additional PTTH via neurosecretory cell transplantation.Ecdysone injection and prothoracic gland-ablation experiments indicate that the prothoracic glands are the source of the prohormone α-ecdysone, and that diapause is maintained by an α-ecdysone deficiency. This evidence, in conjunction with the above results, suggests that PTTH release potentiates prothoracic gland function in the diapausing pupa which is then regulated by a temperature dependent process.     

Haemocytes of the pink bollworm, Pectinophora gossypiella, during larval development and diapause

Seven types of haemocytes were observed in the last larval instar of the pink bollworm, Pectinophora gossypiella (Saunders): prohaemocytes, plasmatocytes, granular haemocytes, spherule cells, adipohaemocytes, oenocytoids, and podocytes. Total and differential haemocyte counts made from diapausing and non-diapausing larvae showed that during diapause there was a significant reduction in the numbers of all haemocyte types. Upon termination of diapause, the haemocyte level increased. There were no significant differences in the level of haemocytes in the pharate pupae that developed from diapause or non-diapause type larvae, except in the case of adipohaemocytes, which were three times as prevalent in pharate pupae from diapausing larvae. Functional aspects of various types of haemocytes are discussed, and it is suggested that the lower haemocyte level observed during diapause is the result of lower metabolic activity.     

Temperature-sensitive mechanism regulating diapause in Heliothis zea

Pupal diapause in Heliothis zea is regulated by a temperature-sensitive mechanism which prevents ecdysone production despite the release of prothoracicotropic hormone. To determine how this mechanism functioned, donor prothoracic glands were implanted into prothoracic gland-ablated hosts to test their ability to produce ecdysone in a diapause-sustaining temperature of 19°C. Results of these experiments ruled out the possibility that ecdysis production was regulated by the nervous system or by a mechanism intrinsic to the prothoracic glands, and suggested that a humoral factor was required for diapause termination.Haemolymph injection experiments supported this humoral factor hypothesis, i.e. haemolymph from non-diapausing donor pupae terminated diapause in hosts maintained at 19°C, whereas haemolymph from diapausing donor pupae had no such effect. These findings indicate that the temperature-sensitive mechanism regulating H. zea diapause functions by controlling the availability of a humoral factor necessary for ecdysone production by the prothoracic glands.     

Control of corpus allatum activity during the imaginai diapause in females of Locusta migratoria L

In the Savio strain of Locusta migratoria an imaginai diapause is induced by long daylength. In diapausing females, the haemolymph level of juvenile hormone (JH) was undetectable during the first 3-wk of imaginai life and later rose only slightly to about 20 ng/JH₃IR per ml. Only peripheral cells of the corpora aliata (CA) were active. In nondiapausing animals, or after the termination of diapause, the JH level was high (140–200 ng/ml) and the ultrastructure of the gland exhibited signs of activity. CA severance in 3-wk-old diapausing females terminated diapause as a result of activation of the CA. CA disconnection in the fifth larval instar or at the imaginai moult in long daylength animals did not break diapause and the CA stayed inactive. The lateral cells of the protocerebrum exert a jdual effect: at the end of larval life they bring about CA maturation and render them active, whereas during the imaginai diapause they inhibit CA activity. The median neurosecretory cells of the pars intercerebralis support CA activity during vitellogenesis.     

Système endocrine et physiologie de la diapause imaginale chez le criquet égyptien, Anacridium aegyptium

The Egyptian locust, Anacridium aegyptium, has four protocerebral neurosecretory centres: the A to B neurosecretory cells of the pars intercerebralis (the A cells are rich in fuchsinophil material and the B cells are devoid of fuchsinophil neurosecretion), the voluminous C neurosecretory cells poor in neurosecretion, and the median sub-ocellar neurosecretory cells.From September to the beginning of January, imaginal diapause is characterized by an accumulation of the median neurosecretion in the pars intercerebralis-corpora cardiaca system, by small corpora allata, and, in the female, by a stop in oöcyte development although the male's sexual activity is still not altered. Allatectomy suppresses neither the male's sexual behaviour nor its fecundity. From January, the increase of the photoperiod causes a release of the median neurosecretion in both sexes, an increase of the volume of the corpora allata, and breaks ovarian diapause.In autumn, the implantation of the male's or female's corpora allata of Anacridium does not stimulate ovarian growth of diapausing females. On the contrary, the implantation of corpora allata or of pars intercerebralis or of corpora cardiaca of Locusta migratoria migratorioides (locust without diapause) causes ovarian development of the diapausing females of Anacridium. Thus, in the two sexes of the Egyptian locust, the corpora allata are inactive during the female ovarian diapause. The imaginal diapause of Anacridium affects both sexes (stocking of median neurosecretion, arrest of the corpora allata). If diapause does not seem to affect the male's development, it is because its sexual activity is free from the pars intercerebralis and corpora allata.The corpora allata of Anacridium show a sexual dimorphism in the active adult: they are smaller in the male and have more mitosis in the female. An explanation of this dimorphism is advanced.     

Ecdysteroids and diapause in pupae of Heliothis punctiger

Most pupae of H. punctiger enter diapause when reared at 19°C, 12L:12D. When pharate pupae were treated for only 12 hr at 28°C about 50% developed at 19°C. The proportion of non-diapausing pupae increased as the temperature at which the pharate pupal stage was spent increased.The quantity of injected 20-hydroxyecdysone necessary to promote development in diapausing pupae varied from about 1 μg g^?1 soon after pupation to about 4 μg g^?1 after 50 days. It fell somewhat after 150 days.Removing brains from non-diapausing pupae showed that the brain secreted its hormone at the time of pupation (or just before). However, if the pupae were kept at 19°C development did not occur unless the brain remained in situ for at least 20 hr at 28°C. Implanting brains from non-diapausing pupae into diapausing ones had no measurable effect.These results may be explained by postulating that the prothoracic gland is ‘activated’ by exposure to high temperature, but that it reverts to inactivity over a period at 19°C. The ‘active’ gland must then be stimulated by brain hormone for a long period to trigger secretion of its hormone, which results in development. Diapause is thus the result of the failure of the prothoracic gland to secrete.     

The induction of an injury reaction in cultured haemocytes from saturniid pupae

The plasmatocytes of diapausing saturniid pupae are round or spindle-shaped cells floating free in the haemolymph. Upon injury to the pupa, these haemocytes become amoeboid and adhesive. A technique is described for the isolation and short-term culture of pupal haemocytes in their inactive state and for their conversion in vitro into the active, ‘injured’ form. The activation of ‘uninjured’ haemocytes was stimulated by fragments of epidermal tissue or by plasma from previously activated blood samples. A fraction capable of stimulating the activation of haemocytes was partially purified from both plasma and epidermal tissue and has been called haemokinin. Haemokinin from either source has a molecular weight in the range of 50,000. The intensity of the haemocyte injury reaction in vitro changes systematically during diapause; the nature and significance of the changes are discussed.     

Haemolymph ecdysone concentrations in Hyalophora cecropia pupae,dauer pupae and adults

Haemolymph ecdysone concentrations were determined by radioimmunoassay in diapausing pupae, pharate adults, adults, and chilled dauer pupae. The concentration in diapausing pupae after 6 months chilling (5.35 pg/μl) increased dramatically after 3 days at 27°C (>200 pg/μl) and then decreased to low levels in adult females (1.63 pg/μl). In adult males ecdysone was undetectable in all except one animal. Dauer pupae showed a decrease from 6.1 to 1.7 pg/μl 1 day after being transferred from 6 to 27°C. Over a 3-day period the value increased to 3.19 pg/μl and remained constant for more than a year. These results suggest that diapausing pupae with and without brain neurosecretory cells maintain a low concentration of ecdysone in the haemolymph.     

Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae)

Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore, Has-SGNP I might be referred to a "diapause termination hormone" in H. assulta (Has-DTH). The maximal dose of Has-DTH for diapause termination was 1.0 microg and the half-maximal dose 0.4 microg. The time required for diapause termination of Has-DTH was 2-3 days longer than that of 20-hydroxyecdysone. During the pupal stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae were chilled at 4 degrees C, but increased rapidly and largely after being transferred to 25 degrees C. Using a competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be activated by low temperature. Has-DTH might be useful to elucidate the mechanism of diapause termination in pupal diapause species.     

Light and electron microscopic studies on the neurosecretory control of diapause incidence in Pieris rapae crucivora

The incidence of diapause was shown to be determined humorally during the larval-pupal ecdysis by means of brain extirpation experiments.On the basis of this observation, light and electron microscopic changes in the neurosecretory type II cells in the pars intercerebralis-corpus cardiacum system during pharate pupal and early pupal stages were examined in insects reared under long day-length (non-diapause individuals) and in insects reared under short day-length (diapause individuals). In the diapause individuals, neurosecretory granules in NS-II cells increased during the pupal instar and large aggregates of granules packed the cytoplasm. Thereafter, inclusion bodies showing cytoplasmic breakdown of the granules appeared.In the non-diapause individuals, on the contrary, electron micrographs suggesting the release of neurosecretory material from axon terminals were obtained just after the pupal ecdysis. There were very few granules, with many Golgi bodies and much rough ER 8 to 12 hr after the ecdysis.It is concluded that adult development is determined by the release of neurosecretory material from the axon terminals of NS-II cells at the larval-pupal ecdysis. If release does not occur, the pupae enter diapause. It is also thought that differences in day-length during the larval stages influence the activities of NS-II cells before pupation.     

Carotenoids of the pupae of the Large White butterfly (Pieris brassicae) and the Small White butterfly (Pieris rapae)

The light cues received by the larvae of Pieris brassicae which determine diapause can also influence the carotenoid distribution (and hence the colour) in the epidermis and cuticle of the diapausing pupae. Irrespective of background or light cues received during the "sensitive period" of the pharate pupa, these diapausing pupae are coloured green. They then contain more than double the concentration of carotenoids in their epidermis than the non-diapausing pupae. This green colour can be somewhat modified by switching the full grown larvae to long day regimes immediately after feeding ceases.
The Large White and Small White butterfly each has a characteristic carotenoid storage pattern, which can be demonstrated by feeding the larvae on portions of the same cabbage leaves. The braconid parasite Apanteles glomeratus mirrors the carotenoids in its host.     

Fat body stimulation of ecdysone synthesis in Heliothis zea

Prothoracic glands of Heliothis zea pupae require both a humoral factor and prothoracicotropic hormone (PTTH) to synthesize ecdysone. The humoral factor is absent when pupae are maintained at diapause-sustaining temperatures. Thus, pupae remain in diapause despite the release of PTTH at or before larval-pupal ecdysis.Tissue implantation experiments revealed that a diapause-terminating factor is present in the fat body of non-diapausing pupae. Other tissue implantation experiments showed that, when diapausing pupae were transferred from 19 to 27°C, diapause-terminating activity appeared first in the fat body and then the fat body into the haemolymph. HPLC separation of the haemolymph and fat body fractions followed by bioassay demonstrated that fractions containing diapause-terminating activity eluted from both tissues within 28–30 min. These results suggest that the factors found in the fat body and haemolymph may be the same compound.Evidence from ecdysone radioimmunoassay experiments ruled out the possibility that the diapauseterminating activity was due to either free or conjugated ecdysteroids. Corresponding in vitro experiments in which the prothoracic glands were cultured with brain extracts versus fat body and haemolymph fractions also indicated that the haemolymph/fat body factor was not PTTH.     

Effects of ablation of the pars intercerebralis on ecdysteroid quantities and yolk protein expression in the blowfly Protophormia terraenovae

Quantities of ecdysteroid are compared in the haemolymph and ovaries of the blowfly Protophormia terraenovae Robineau‐Desvoidy (Diptera: Calliphoridae) under reproductive (LD 18 : 6 h at 25 °C) and diapause (LD 12 : 12 h at 20 °C) conditions. The effects of ablation of the pars intercerebralis or ovaries on ecdysteroid quantities and of ablation of the pars intercerebralis on yolk protein expression are examined. Under reproductive conditions, the levels of ecdysteroid in vitellogenic females are high, although the levels in previtellogenic females and females with mature ovaries are low. Under diapause conditions, there are low quantities of ecdysteroid in both the haemolymph and ovaries. Ecdysteroid titres in the haemolymph are not significantly affected by the removal of the ovaries, suggesting that tissues other than the ovaries are also involved in the production of ecdysteroids. Reproductive females in which the pars intercerebralis of the brain is experimentally ablated have ecdysteroid levels that are not significantly different from sham‐operated or intact females. However, yolk protein expression in the fat body is suppressed after removal of the pars intercerebralis. These results suggest that the suppression of ecdysteroid levels in the haemolymph and ovaries is associated with reproductive diapause, and that the pars intercerebralis could play a role in yolk protein synthesis without mediating ecdysteroid production.     

Diapause-dependent changes in prothoracicotropic hormone-producing neurons of the tobacco hornworm,Manduca sexta

The prothoracicotropic hormone (PTTH), which stimulates ecdysteroid synthesis in the prothoracic glands, is produced, in the dorso-lateral protocerebrum of Manduca sexta, by paired peptidergic neurons, the lateral neurosecretory cell group III (L-NSC III). Our study revealed ultrastructural features of L-NSC III, identified by immunogold labeling, and compared developing and diapause states. In developing and early-diapause pupae, L-NSC III soma ultrastructure is similar and is characterized by numerous clusters of neurosecretory granules (NSG) and an extensive trophospongium formed by satellite-glial cells. However, as diapause progresses, the ultrastructure changes, with the NSG becoming concentrated into large clusters separated by highly organized rough endoplasmic reticulum. Most conspicuous is a substantial reduction in the number of Golgi complexes and the glial trophospongium, and the presence of stacked plasma membrane separating the glia and neuron somata. The deep-diapause soma also has abundant glycogen deposits and autophagic vacuoles. With diapause termination, this morphology reverts to the nondiapause ultrastructure within three days, i.e. just before PTTH release that evokes development to the adult. During PTTH release the abundance of NSG in the soma does not change, suggesting that NSG depletion in the perikarya is not a marker for neurosecretion by the L-NSC III.     

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.     

Comparative studies of oxidative enzyme systems in epidermis and fat body of diapausing and non-diapausing silkmoths

The activities of four oxidative enzyme systems, including NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and cytochrome c oxidase, were compared in mitochondrial-microsomal preparations from wing epidermis and fat body of diapausing Samia cynthia pupae, presumptively non-diapausing S. cynthia ricini pupae which were caused to diapause by removal of the brain, and non-diapausing S. cynthia ricini during the pupal and pharate adult period. In diapausing pupae the activities of all enzyme systems were low and presented a profile similar to that previously reported for the Cecropia silkmoth. By contrast, in non-diapausing individuals the activities showed substantially higher levels, and an essentially unchanging pattern from just after the larval-pupal ecdysis through most of adult development. These events are functionally correlated with the patterns of biosynthetic activity in diapausing and non-diapausing silkmoths and are discussed in relation to the endocrine control of diapause and development.     

Diapause and development in the tobacco budworm,Heliothis virescens: A comparison of haemolymph ecdysteroid titres

The signal to induce diapause in H. virescens comes early in development (prior to the third instar in most insects), but the signal to break diapause can come shortly after entrance into diapause at pupation. Haemolymph ecdysteroid titres in both diapause-bound and non-diapause-bound Heliothis virescens larvae were similar in the first two thirds of the last-larval instar, when similar changes in morphology and behaviour occurred. However, the number of stepwise increases in titre and the timing of the steps was different in the two groups of larvae. Haemolymph ecdysteroid titres in the last third of the instar were approx, five times higher in non-diapause than in diapause-bound larvae. In diapausing pupae, haemolymph ecdysteroid titres dropped to levels found in larvae which had completed two thirds of the last instar. When diapausing pupae were warmed to break diapause, haemolymph ecdysteroid titres rose again. However, 2 of the 4 high ecdysteroid levels detected in pupae developing after diapause break were considerably lower than those detected for non-diapause pupae.     

Changes of biogenic amine levels in haemolymph during diapausing and non-diapausing status in Pieris brassicae L

During the larval development of Pieris brassicae, photoperiod status induces either direct (non-diapausing) development or diapausing development. Biogenic amines such as dopamine and serotonin and their derivatives may be implicated in the physiological control and adaptation of these insects to different photoperiods. High performance liquid chromatography coupled with electrochemical detection was used to measure biogenic amine concentrations in insect haemolymph in different photoperiod conditions. At the end of the higher photosensitivity phase, dopamine levels were two-fold higher in diapausing (0.914 pmol/microl+/-0.025) as compared to non-diapausing insects (0.415 pmol/microl+/-0.02; P<0.001). Globally, dopamine catabolites are found in higher levels in non-diapausing insects (in prepupa, near seven times more for NADA, and 25 times more for DOPAc), thus indicating a higher dopamine turn-over. Serotonin levels were detected only at the beginning of this instar in diapausing insects but not in direct development insects. During pupal life, dopamine levels were significantly higher in diapausing than in non-diapausing insects (in 3-day pupae, dopamine was 28 times higher), and remained at high levels throughout diapausing life. At the beginning of diapause, serotonin levels were higher in diapausing insects than in non-diapausing insects (in 3-day pupae, serotonin was 13 times higher). These levels decreased in the middle of diapause period, and then increased just before its breaking. Dopamine and serotonin levels presented the typical kinetics of diapause development and may be used as an indicator of the evolution of diapause status.     

Vertebrate insulin induces diapause termination in Pieris brassicae pupae

Summary Due to its close structural homology with the 4K prothoracicotropic hormone isolated from Bombyx mori, we tested the ability of vertebrate insulin to break pupal diapause in a Lepidopteran, Pieris brassicae. Injection of 5g of bovine insulin in diapausing pupae led to diapause termination and synchronous adult eclosion; the effect of insulin was dose-dependent. Bovine insulin-A chain and B chain injected separately failed to show any biological activity suggesting that the intact structure of the molecule is required. Bovine insulin also promoted adult development of decapitated diapausing animals. We show that insulin triggers a reactivation of the neuroendocrine system leading to a neosynthesis of ecdysone beginning 6 days after treatment. This neosynthesis also occurred in beheaded animals suggesting that insulin stimulates the prothoracic glands without acting via the brain.