Endocrine Mechanisms Regulating Post-Diapause Development in the Cabbage Armyworm,Mamestra brassicae

Diapause, a programmed developmental arrest at a specific stage, is common in insects and is regulated by hormones. It is well established that in pupal diapause, cessation of ecdysteroid secretion from the prothoracic glands (PGs) after pupal ecdysis leads to diapause initiation, while resumption of its secretion induces post-diapause development. However, what regulates the activity of the glands is poorly understood, especially for the glands of diapause-terminated pupae. In the present study, we investigate the mechanisms by which post-diapause development is regulated in the cabbage armyworm Mamestra brassicae. We demonstrate that the brain is necessary for the initiation of post-diapause development and that the factor in the brain responsible for the activation of the PGs is the prothoracicotropic hormone (PTTH). Further, through measuring the hemolymph PTTH titers by time-resolved fluoroimmunoassay, we show that PTTH is actually released into the hemolymph prior to the activation of the PGs. Although its peak titer is much lower than expected, this low concentration of PTTH is most likely still effective to activate the PGs of post-diapause pupae, because the responsiveness to PTTH of the glands at this stage is very high compared to that of nondiapause pupal PGs. These results strongly suggest that in M. brassicae, PTTH serves as a trigger to initiate pupa-adult development after diapause termination by stimulating the PGs to secrete ecdysteroid.     

The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpa armigera encodes multiple peptides that break, rather than induce, diapause

FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera.     

Prothoracicotropic Hormone Acts as a Neuroendocrine Switch between Pupal Diapause and Adult Development

Diapause is a programmed developmental arrest that has evolved in a wide variety of organisms and allows them survive unfavorable seasons. This developmental state is particularly common in insects. Based on circumstantial evidence, pupal diapause has been hypothesized to result from a cessation of prothoracicotropic hormone (PTTH) secretion from the brain. Here, we provide direct evidence for this classical hypothesis by determining both the PTTH titer in the hemolymph and the PTTH content in the brain of diapause pupae in the cabbage army moth Mamestra brassicae. For this purpose, we cloned the PTTH gene, produced PTTH-specific antibodies, and developed a highly sensitive immunoassay for PTTH. While the hemolymph PTTH titer in non-diapause pupae was maintained at high levels after pupation, the titer in diapause pupae dropped to an undetectable level. In contrast, the PTTH content of the post-pupation brain was higher in diapause animals than in non-diapause animals. These results clearly demonstrate that diapause pupae have sufficient PTTH in their brain, but they do not release it into the hemolymph. Injecting PTTH into diapause pupae immediately after pupation induced adult development, showing that a lack of PTTH is a necessary and sufficient condition for inducing pupal diapause. Most interestingly, in diapause-destined larvae, lower hemolymph titers of PTTH and reduced PTTH gene expression were observed for 4 and 2 days, respectively, prior to pupation. This discovery demonstrates that the diapause program is already manifested in the PTTH neurons as early as the mid final instar stage.     

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.     

Circadian orchestration of developmental hormones in the insect, Rhodnius prolixus

This review presents a new perspective on the circadian regulation and functions of insect developmental hormones. In Rhodnius prolixus (Hemiptera), the brain neuropeptide prothoracicotropic hormone (PTTH) is released with a circadian rhythm that is controlled by paired photosensitive clocks in the brain. These clocks comprise the dorsal and lateral PER/TIM clock neurons known to regulate behavioral rhythms in Drosophila. Axons of PTTH and clock cells make close contact. Photosensitive PER/TIM clocks also reside in the paired prothoracic glands (PGs), which generate rhythmic synthesis and release of the ecdysteroid molting hormones. The PG clocks are entrained by both light and PTTH. These four clocks are coupled together by both nerves and hormones into a timing system whose primary regulated output is the circadian rhythm of ecdysteroids in the hemolymph. This complex timing system appears necessary to ensure circadian organization of the gene expression that is induced in target cells by ecdysteroids via circadian cycling of the nuclear ecdysteroid receptor (EcR). This multioscillator system serves to transduce 'the day outside' into endocrine rhythms that orchestrate 'the day inside'. It has many functional similarities with vertebrate circadian systems.     

柞蚕蛹解除滞育过程中海藻糖合成酶基因的表达变化

【目的】克隆柞蚕Antheraea pernyi海藻糖合成酶(trehalose-6-phosphate synthase,TPS)基因,并对其进行组织表达分析,探讨该基因在柞蚕滞育蛹解除滞育过程中的表达规律,为阐明柞蚕滞育期间碳水化合物代谢规律与蛹滞育解除的关系提供数据支持。【方法】利用PCR及3'RACE技术从柞蚕幼虫脂肪体组织中克隆得到TPS基因,并进行生物信息学分析;RT-PCR检测该基因在柞蚕幼虫各组织中的表达分布,进一步采用Real-time PCR分析柞蚕滞育蛹解除滞育过程中该基因在脂肪体组织和血淋巴中的表达量变化。【结果】克隆获得柞蚕海藻糖合成酶基因并命名为ApTPS。其开放阅读框长2 487 bp,编码828个氨基酸,蛋白预测分子量为93.19 k D,等电点(p I)4.61;无信号肽,无跨膜区。蛋白质亚细胞定位预测该蛋白定位于细胞质中;蛋白质结构域分析表明,ApTPS有两个保守功能区:TPS(第22-497位氨基酸)和TPP(第532-772位氨基酸)。组织特异性分析表明,ApTPS基因在柞蚕幼虫脂肪体中表达量最高;柞蚕解除滞育过程中,ApTPS在脂肪体和血淋巴中的表达量均有所升高,且显著高于对照组(P0.05),但血淋巴中表达量的升高滞后于脂肪体。【结论】结果提示ApTPS参与了柞蚕蛹滞育中碳水化合物代谢调控并在其中发挥重要作用,与柞蚕蛹滞育解除关系密切。     

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.     

Termination of pupal diapause in the pine processionary moth Thaumetopoea pityocampa

Diapause development is a complex process involving several eco‐physiological phases. Understanding these phases, especially diapause termination, is vital for interpreting the life history of many insect species and for developing suitable predictive models of population dynamics. The pine processionary moth is a major defoliator of pine and a vertebrate health hazard in the Mediterranean region. This species can display either univoltine or semivoltine development, with a pupal diapause extending from a few months to several years, respectively. Although the ecological and applied importance of diapause is acknowledged, its physiological regulation in either case remains obscure. In the present study, we characterize pre‐termination, termination and post‐termination phases of pupae developing as univoltine or remaining in prolonged diapause. Changes in metabolic activity are monitored continuously using thermocouples, comprising a novel method based on direct calorimetry, and periodically by use of O₂ respirometry. The two methods clearly detect diapause termination in both types of pupae before any visible morphological or behavioural changes can be observed. Univoltine individuals are characterized by an increase in metabolic activity from pre‐termination through to termination and post‐termination, ultimately resulting in emergence. Remarkably, a synchronous termination is observed in individuals that enter prolonged diapause instead of emerging; however, in these pupae, the increased metabolic activity is only transient. The present study represents a starting point toward understanding the eco‐physiology of diapause development processes in the pupae of the pine processionary moth.     

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.     

Regulation and significance of ecdysteroid titre fluctuations in lepidopterous larvae and pupae

The last larval moult of Galleria mellonella is induced by an elevation of ecdysteroid titre to more than 200 ng/g. After ecdysis the titre remains very low until 70 hr of the last-instar when a slight elevation in ecdysteroid concentration initiates the onset of metamorphosis. An ecdysteroid peak (275 ng/g), which occurs between 108 and 144 hr, is associated with wandering and cocoon spinning. Pupal ecdysis follows about 20 hr after a large ecdysteroid peak (780 ng/g) with a maximum in slowly-mobile prepupae (160 hr of the last larval instar). The ecdysteroid decrease between the two peaks coincides with the period when the larvae exposed to unfavourable conditions enter diapause. The pupal-adult moult is initiated by a high ecdysteroid peak (1500–2500 ng/g) in early pupae and imaginal cuticle is secreted in response to a smaller peak (ca. 500 ng/g) in the middle of pupal instar.Until early pupae, the ecdysteroid content is regulated by the prothoracic glands. In decapitated larvae the glands become spontaneously active after 30–40 days and the body titre of ecdysteroids undergoes an increase; the glands revert to inactivity when the insects accomplish secretion of pupal cuticle. A similar ecdysteroid increase occurs within 10 days when the decapitated larvae receive implants of brains releasing the prothoracicotropic neurohormone (PTTH). In either case, the pupation-inducing increase of ecdysteroids is 3 times higher than the large ecdysteroid peak in the last-instar of intact larvae. This indicates that the function of prothoracic glands in intact larvae is restrained, probably by the juvenile hormone (JH). Exogenous JH suppresses the spontaneous activation of the prothoracic glands in decapitated larvae and reduces the ecdysteroid concentration in those larvae (both decapitated and intact), whose glands were activated by PTTH. Furthermore, JH influences the PTTH release from the brain in situ: depending on JH concentration and the age and size of treated larvae, the PTTH liberation is either accelerated or delayed.Neither in G. mellonella larvae, nor in the diapausing pupae of Hyalophora cecropia and Celerio euphorbiae, does JH directly activate the prothoracic glands. It is suggested that the induction of the moult by JH in decerebrate insects, which has been observed in some species, is either due to indirect stimulation of ecdysteroid production or to increased sensitivity of target tissues to ecdysteroids. In G. mellonella, a moult occurs at a 5–15 times lower than usual ecdysteroid concentration when the last-instar larvae are exposed to JH.     

Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species

The imidazole derivative KK-42 is a synthetic insect growth regulator known previously to be capable of averting embryonic diapause in several Lepidoptera, but whether it also affects diapauses occurring in other developmental stages remains unknown. In the present study, we examined the effect of KK-42 on pupal diapause in two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and in one species of Diptera, the flesh fly Sarcophaga crassipalpis. In A. pernyi, KK-42 delayed pupal diapause termination under the long day conditions that normally break diapause in this species. Likewise, in H. zea, KK-42 delayed termination of pupal diapause, a diapause that, in this species, is normally broken by high temperature. KK-42-treated pupae of these two species eventually terminated diapause and successfully emerged as adults, but the timing of diapause termination was significantly delayed. KK-42 also significantly increased the incidence of pupal diapause in H. zea and S. crassipalpis when administered to larvae that were environmentally programmed for diapause, but it was not capable of inducing pupal diapause in H. zea if larvae were reared under environmental conditions that do not normally evoke the diapause response. Experiments with H. zea showed that the effect of KK-42 on pupal diapause was dose- and stage-dependent, but not temperature-dependent. Results presented here are consistent with a link between KK-42 and the ecdysteroid signaling pathway that regulates pupal diapause.     

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.     

Regulation of capacitative Ca2+ entry by prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca2+ influx in Fura-2/AM loaded steroidogenic cells (prothoracic glands; PGs) of the silkworm, Bombyx mori showed that application of the neuropeptide prothoracicotropic hormone (PTTH) can increase the intracellular [Ca2+]i. This PTTH-mediated Ca2+ influx in PG cells had kinetic patterns and pharmacological characteristics similar to those induced by thapsigargin. Namely, it produced increases in intracellular Ca2+ levels only in the presence of extracellular Ca2+, it was blocked by Gd3+ and 2-Aminoethoxydiphenylborate (2-APB), and it was unaffected by several toxins or compounds that block voltage-activated Ca2+ channels. Moreover, the PTTH-stimulated increase of Ca2+ levels was eliminated in the presence of heparin (an IP3 receptor blocker), and by TMB-8 which also blocked any PTTH-dependent increase of ecdysteroid secretion. The PTTH-mediated increase of Ca2+ levels was not affected by the non-hydrolysable GDP analogue, GDPbetaS, an indication that a G protein is not downstream of the PTTH receptor. These results argue strongly in favor of gating by the PTTH receptor of capacitative Ca2+ entry (CCE) channels (or store-operated Ca2+ channels (SOCs)) by a mechanism that does not involve any G proteins but requires the presence of functional IP3 receptors. Because the ability of PTTH to stimulate the [Ca2+]i levels of PG cells was completely mimicked by thapsigargin and exhibited a pharmacological profile similar to CCE mechanisms, we believe that PTTH directly regulates a CCE pathway in PG cells thereby activating a plethora of downstream regulators responsible for ecdysteroid secretion by the PGs of Bombyx mori.     

The Drosophila gap gene giant regulates ecdysone production through specification of the PTTH-producing neurons

In Drosophila melanogaster, hypomorphic mutations in the gap gene giant (gt) have long been known to affect ecdysone titers resulting in developmental delay and the production of large (giant) larvae, pupae and adults. However, the mechanism by which gt regulates ecdysone production has remained elusive. Here we show that hypomorphic gt mutations lead to ecdysone deficiency and developmental delay by affecting the specification of the PG neurons that produce prothoracicotropic hormone (PTTH). The gt¹ hypomorphic mutation leads to random loss of PTTH production in one or more of the 4 PG neurons in the larval brain. In cases where PTTH production is lost in all four PG neurons, delayed development and giant larvae are produced. Since immunostaining shows no evidence for Gt expression in the PG neurons once PTTH production is detectable, it is unlikely that Gt directly regulates PTTH expression. Instead, we find that innervation of the prothoracic gland by the PG neurons is absent in gt hypomorphic larvae that do not express PTTH. In addition, PG neuron axon fasciculation is abnormal in many gt hypomorphic larvae. Since several other anteriorly expressed gap genes such as tailless and orthodenticle have previously been found to affect the fate of the cerebral labrum, a region of the brain that gives rise to the neuroendocrine cells that innervate the ring gland, we conclude that gt likely controls ecdysone production indirectly by contributing the peptidergic phenotype of the PTTH-producing neurons in the embryo.     

Serotonin Receptor B May Lock the Gate of PTTH Release/Synthesis in the Chinese Silk Moth,Antheraea pernyi; A Diapause Initiation/Maintenance Mechanism?

The release of prothoracicotropic hormone, PTTH, or its blockade is the major endocrine switch regulating the developmental channel either to metamorphosis or to pupal diapause in the Chinese silk moth, Antheraea pernyi. We have cloned cDNAs encoding two types of serotonin receptors (5HTR_{A and B}). 5HTR_A-, and 5HTR_B-like immunohistochemical reactivities (-ir) were colocalized with PTTH-ir in two pairs of neurosecretory cells at the dorsolateral region of the protocerebrum (DL). Therefore, the causal involvement of these receptors was suspected in PTTH release/synthesis. The level of mRNA^5HTRB responded to 10 cycles of long-day activation, falling to 40% of the original level before activation, while that of 5HTR_A was not affected by long-day activation. Under LD 16:8 and 12:12, the injection of dsRNA^5HTRB resulted in early diapause termination, whereas that of dsRNA^5HTRA did not affect the rate of diapause termination. The injection of dsRNA^5HTRB induced PTTH accumulation, indicating that 5HTR_B binding suppresses PTTH synthesis also. This conclusion was supported pharmacologically; the injection of luzindole, a melatonin receptor antagonist, plus 5th inhibited photoperiodic activation under LD 16:8, while that of 5,7-DHT, induced emergence in a dose dependent fashion under LD 12:12. The results suggest that 5HTR_B may lock the PTTH release/synthesis, maintaining diapause. This could also work as diapause induction mechanism.