Regulatory mechanisms in phenotypic plasticity of diapause and nondiapause pupal colouration of the swallowtail butterfly Papilio machaon

Nondiapause pupae of Papilio machaon L. exhibit pupal colour diphenism comprising green–yellow and brown–white types. To understand the regulatory mechanism underlying the control of pupal colouration in P. machaon, the effect of environmental cues on diapause and nondiapause pupal colouration is investigated. When larvae reared under short‐day and long‐day conditions are allowed to pupate in sites with a smooth surface and a yellow background colour, all diapause pupae exhibit a brown–white type and 89.5% of nondiapause pupae exhibit a green–yellow type, respectively. With rough‐surface pupation sites, all diapause pupae exhibit brown–white and intermediate types, whereas a large proportion of nondiapause pupae exhibit brown–white and intermediate types, although some exhibit a green–yellow type. When extracts prepared from the head‐thoracic and thoracic‐abdominal regions of larval central nervous systems are injected into the ligated abdomens of P. machaon short‐day pharate pupae, all recipients exhibit a brown–white colouration. Furthermore, when each extract is injected into the ligated abdomen of Papilio xuthus L. short‐day pharate pupae with orange‐pupa‐inducing factor activity, recipients injected with the head‐thoracic extract exhibit the brown type, whereas those injected with the thoracic‐abdominal extract exhibit an orange colour. The results indicate that the response to the environmental cues of pupation site in P. machaon changes according to the photoperiodic conditions experienced during larval stages, and that at least two hormonal factors producing brown–white pupae are located in the larval central nervous system, with the secretion of these factors being regulated by the recognition of environmental cues in long‐day larvae.     

Brain hormone carrier haemocytes in the moth Monema flavescens

The movement of neurosecretory substances released from the neurosecretory B cell in the pars intercerebralis to the haemolymph was examined with the progress of the termination of diapause in the slug moth pharate pupa, Monema flavescens.The injection of precipitates in the haemolymph of the pharate pupa just before the termination of diapause into diapausing pharate pupae reduced the numbers of days required for them to pupate. In the precipitates, seven types of haemocytes were present. The number of haemocytes, especially the granular cell, increased just before the termination of diapause. AF and CHP positive substances not detected in the haemocytes of diapausing pharate pupae appeared in the granular cells just before the termination of diapause. The period also coincided well with the releasing period of the neurosecretory B cell. Histological examination showed that granular haemocytes gathered around the pars intercerebralis at this period and exchange of neurosecretory substances occurred between granular haemocytes and neurosecretory B cells. Then granular haemocytes migrated to the region of the prothoracic gland. From digestion tests of the neurosecretory substances with rabbit serum and from the implantation tests of the neuroendocrine system, the substances detected in both the neurosecretory B cell and the granular haemocytes seemed to be the same. The dye injection caused a delay in larval-pupal ecdysis emergence. Droplets of black ink are incorporated into the granular haemocytes. This seems to be caused by blocking of the transport of neurosecretory substances released from cytoplasmic processes of the neurosecretory B cell.From these experiments, it is suggested that neurosecretory substances of the prothoracotropic hormone are transported to the prothoracic gland, along with granular haemocytes, after being released directly from the neurosecretory B cell to the haemolymph.     

Stages of diapause development in the pupa of Mamestra configurata based on the β-ecdysone sensitivity index

Four distinct stages of diapause development in pupae of Mamestra configurata held at 20°C can be recognized by means of the ‘β-ecdysone sensitivity index’. The latter refers to the ED50 of injected β-ecdysone required to break diapause in half of the treated pupae. Stage 1 begins with the newly-formed pupa, lasts about 3.5 days and is characterized by a rapidly falling ED50. Stage 2 lasts about four weeks during which the ED50 increases by almost 20-fold, from 0.27 μg/g at the beginning to 4.9 μg/g at the end. Stage 3 begins when the pupae are about 4 weeks old and lasts for about six weeks. Stage 3 is the stable diapausing state and is characterized by a virtually unchanging ED50. The onset of stage 4 occurs when the pupae are about 10 weeks old and is recognized by the beginning of a decline in the ED50. Stage 4 precedes the completion of diapause development and may signal the transition of the endocrine system to its active state.Pupal diapause deepens with time in M. configurata. The deepening process evidently occurs during stage 2 of diapause development. Pupae that were transferred to 5°C at the beginning of stage 2 failed to make the transition to stage 3 and were trapped in a very shallow diapause.     

Firing activity of "diapause hormone" producing cells in the male silkmoth,Bombyx mori

Diapause hormone (DH) originally identified to be a factor originating from neurosecretory cells in the suboesophageal ganglion acts on developing ovaries to produce diapause eggs in a female silkmoth, Bombyx mori. A male silkmoth has homologous neurosecretory cells, but little is known of the physiological nature of the cells and actions of their products. We examined the long-term firing activity of putative DH-producing neurosecretory cells and hormonal activity of their products in male pupae that had been experienced different environmental regimens for diapause induction. Firing activity patterns of male labial cells strongly depended on diapause types of pupae: cells in a diapause-type male were active throughout the pupal period, whereas the same cells in a non-diapause-type male were usually inactive during the early two-thirds of the pupal period. A male pupa with electrically active labial cells could induce diapause eggs in a female pupa connected parabiotically to that male. The firing activity of male neurosecretory cells and hormonal action of their products are qualitatively the same as in the female previously examined. We suggest that there is no evident sexual dimorphism in the physiological and biochemical nature of neurosecretory cells producing DH and the amidated peptide DH has different functions in a male.     

Crowding inhibits pupation inTribolium freemani: contact chemical and mechanical stimuli are involved

The relationship between the development ofCoccinella septempunctata brucki Mulsant (Coleoptera: Coccinellidae) and its parasitoid,Perilitus coccinellae (Schrank) (Hymenoptera: Braconidae) was studied at two photoperiods (L 16:D8 and L 12: D12) at 26°C. The development ofP. coccinellae is well synchronized with the physiological state of the host,C. septempunctata, which can be parasitized not only as adult but also as larva or pupa. The parasitoid larva completed larval development within 19 days in a non-diapausing host, while in diapausing adults as well as in pupae held at diapause-averting conditions, the parasitoid larva ceased growth at the first instar. Growth was resumed when diapause of the host terminated or by the emergence of the adult host from the pupa. About 550 spheric cells, teratocytes, were liberated into the host hemocoel when the parasitoid egg hatched. The teratocytes increased in size in the active host, while their development was arrested in the diapausing host. Application of methoprene caused diapause termination of both host and parasitoid larva. The results indicate that the development of the larva ofP. coccinellae depends on the physiological conditions of the host,C. septempunctata brucki. The host-parasite relation thus represents an ‘endogenous synchronization’ in the sense of Schoonhoven's definition.     

Dopamine is a key factor for the induction of egg diapause of the silkworm, Bombyx mori.

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non-diapausing progeny (non-diapause type). Measurement of catecholamine concentrations in haemolymph and brain-subesophageal ganglia (Br-SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause-type than non-diapause-type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br-SGs were also much higher in diapause-type than non-diapause-type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate-instar and last-instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non-diapause-destined insects lay diapause-destined eggs at 59% and approximately 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br-SGs of non-diapause-type pupae 1 day after injection. Incubation of Br-SGs isolated from non-diapause-type day-2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br-SGs from the larval stage to early pupal stage, which results in laying of diapause-destined eggs by female adults through enhanced expression of the diapause hormone gene.     

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.     

葱蝇非滞育、 冬滞育和夏滞育蛹发育和形态特征比较

昆虫非滞育、 冬滞育和夏滞育蛹具有不同的生理和发育过程。本研究以葱蝇Delia antiqua作为模式种, 以黑腹果蝇Drosophila melanogaster蛹的发育形态特征和命名为参照, 用解剖、 拍照、 长度测量和图像处理等方法系统地比较研究了非滞育、 冬滞育和夏滞育蛹的发育历期和形态变化, 重点在头外翻和滞育相关发育和形态特征, 目的在于弄清非滞育、 冬滞育和夏滞育蛹发育和形态特征差异, 为滞育发育阶段的识别、 滞育分子机理研究奠定形态学基础。冬滞育蛹的滞育前期、 滞育期和滞育后期分别为4, 85和27 d, 夏滞育蛹的滞育前期、 滞育期和滞育后期分别为2, 8和22 d。从化蛹至头外翻完成为滞育前期, 头外翻完成约10 h内复眼中央游离脂肪体可见。头外翻的完成是滞育发生的前提, 非滞育、 夏滞育和冬滞育蛹头外翻发生在化蛹后的48, 36和83 h, 在头外翻过程中发育形态没有差异。头外翻的过程为： 首先, 头囊和胸部附肢从胸腔外翻, 头部形态出现; 然后, 腹部肌肉继续收缩, 将血淋巴和脂肪体推进头部及胸部附肢。葱蝇蛹在完成蛹期有效积温约15%时进入冬滞育或夏滞育。在滞育期, 蛹的形态一直停留在复眼中央游离脂肪体可见这一形态时期, 且冬滞育和夏滞育的蛹在形态上没有区别。在体长、 体宽和体重上, 冬滞育蛹最大, 夏滞育蛹次之, 非滞育蛹最小。在滞育后期, 在黄色体出现期间, 非滞育蛹的马氏管清楚可见, 呈绿色, 而滞育蛹的马氏管几乎不可见。本研究为认知昆虫滞育生理、 从发育历期和形态上推断滞育发育进程提供了依据。     

Obligate annual and successive facultative diapause establish a bet‐hedging strategy of Rhagoletis cerasi (Diptera: Tephritidae) in seasonally unpredictable environments

To cope with temporal and spatial heterogeneity of habitats, herbivorous insects in the temperate zone usually enter diapause that facilitates synchronization of their life cycle with specific stages of host plants, such as fruit ripening. In the present study, we address those factors regulating dormancy responses as part of a ‘longer strategy’ to persist and thrive in temperate environments, focusing on Rhagoletis cerasi, a univoltine, oligophagous species, which overwinters as pupae and emerges when host fruits are available for oviposition at local scale. To ensure population survival and reproduction at habitats with ecological heterogeneity, R. cerasi has evolved a sophisticated diapause strategy based on a combination of local adaptation and diversified bet‐hedging strategies. Diapause duration is determined both by (i) the adaptive response to local host fruit phenology patterns (annual diapause) and (ii) the plastic responses to unpredictable inter‐annual (temporal) climatic variability that drives a proportion of the populations to extend dormancy by entering a second, successive, facultative cycle of prolonged diapause as part of a bet‐hedging strategy. Besides the dormant periods, post‐diapause development (which varies among populations) exerts ‘fine tune’ adjustments that assure synchronization and may correct possible errors. Adults emerging from pupae with prolonged diapause are larger in body size compared with counterparts emerging during the first year of diapause. However, female fecundity rates are reduced, followed by an extended post‐oviposition period, whereas adult longevity remains unaffected. Overall, it appears that R. cerasi populations are adapted to ecological conditions of local habitats and respond plastically to unpredictable environmental (climatic) conditions.     

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.     

One host—one parasitoid system: Seasonal life cycles ofPryeria sinica (Ledipoptera) andAgrothereutes Minousubae (Hymenoptera)

The zygaenid Pryeria sinicaMoore and the ichneumonid Agrothereutes minousubaeNakanishi form a one host—one parasitoid system in nature. Their seasonal life cycles were investigated by laboratory experiments and field observations, and the life-cycle adaptation of the parasitoid to its host was examined. The moth is univoltine. The larva hatches from mid-February to mid-March and feeds on leaf buds and young leaves of ever green Euonymus japonicusThunb . The thermal constants for completing the 1st, 2nd, 3rd, 4th larval instars and prepupal stage were 85.6, 80.5, 85.2, 177,0 and 197.6 degree-days, respectively. The prepupa and pupa vulnerable to the attack by the parasitoid occurred from mid-April to early May and from mid- to late May, respectively. Diapause in the parasitoid is facultative and occurs in the eonymphal stage. The photoperiodic response for this diapause was a long-day type with a critical photoperiod of 13 hr 40 min at 20°C, but it was not expressed at 25°C, most larvae entering diapause irrespective of photoperiod. About 19, 120, 82 and 112 degree-days above 7°C were required to complete the egg, larval, prepupal and pupal development, respectively. These data were superimposed on the photothermograph of Fukuoka, and it is predicted that the 1st adult eclosion would occur in late April and the partial 2nd adult eclosion in early June. The prediction was supported by field observations. The adult eclosion of the parasitoid synchronized well with the apperance of prepupae and pupae of the moth. The parasitoid has two types of seasonal life cycle, one generation and two generations a year. Both types have an extremely long dormant period of 10–11 months due to aestivo-hibernation. This seasonal life cycle enables the parasitoid to maintain its population when the host is in short supply.     

Control of diapause-factor secretion from the suboesophageal ganglion in the silkworm,Bombyx mori: The roles of the protocerebrum and tritocerebrum

To localize any functional areas in the brain which control the secretion of the diapause factor from the suboesophageal ganglion in the Bombyx silkworm, various parts of the brain were excised or transected surgically. It was demonstrated that cortices slightly lateral to the median line on the dorsal side of the protocerebrum (Lmdp) are closely related to the inhibitory mechanism in the pupa producing non-diapause eggs. On the other hand, cortices of anterolateral areas on the ventral side of the protocerebrum (Alvp) participate in the stimulatory mechanism in the pupa producing diapause eggs. Moreover, it is suggested that the tritocerebrum is competent to stimulate secretion of the diapause factor from the suboesophageal ganglion not only in the pupae producing diapause eggs but also in the pupae producing non-diapause eggs. From these results, we propose a possible relationship between the Alvp, Lmdp and tritocerebrum concerning regulation of diapause-factor secretion from the suboesophageal ganglion.     

Variations of biogenic amine levels in the brain of Pieris brassicae pupae during nondiapausing and diapausing development

The post-embryonic development of Pieris brassicae can either be continuous (under a long photoperiod) or interrupted at the pupal stage (induced by a short photoperiod); this phenomenon is termed facultative diapause. Several studies have indicated that certain brain mechanisms could be directly involved in the perception of variations in the photoperiod and could mediate some physiological effects particular to dormancy. Biogenic amines have been particularly implicated in the response to photoperiod variations and also in the regulation of development, especially in diapause induction and termination. High performance liquid chromatography with dual electrochemical detection has therefore been used to measure several biogenic amines in pupal nervous tissues at various stages of nondiapausing and diapausing development. During direct development, the levels of dopamine (DA) and N-acetyldopamine (NADA: a DA metabolite) in brain were relatively high in 3-day-old pupae and at the end of pupal life (on the 8th day). Dihydroxyphenylacetic acid (another metabolite of DA) showed no variation. Serotonin was mainly observed in 2–3-day-old pupae but 5-hydroxyindoleacetic acid was never detected. In young diapausing insects, similar variations of DA levels were observed even though a slight decrease of DA metabolites was noted. Serotonin appeared somewhat later (4–5 days) and attained higher levels. In late diapausing pupae, a marked increase in DA levels was observed, especially when pupae were kept at low temperature (4°C). During diapause, serotonin levels were reduced or even absent.     

Thermal responses in the citrus fruit fly,Dacus tsuneonis: evidence for a pupal diapause

Thermal responses controlling pupariation and adult eclosion in a citrus fruit fly,Dacus tsuneonis (Miyake), were studied to understand the winter biology of this species. When mature larvae were exposed to various temperature conditions, the highest percentage of pupariation was obtained at 15 °C, although the variance at this temperature was greater than at 20 °C or 25 °C. Pupariation occurred most rapidly at 20 °C and an alternating temperature with a mean of 15 °C. At constant 15 °C, pupae failed to emerge as adults. Pupae were characterized by a reduced respiration rate, which is typical of a diapausing pupa. When insects were stored at different temperatures for 45 days after pupariation, and then transferred to 25 °C, adult eclosion occurred earlier when the initial temperature was 10 °C than when it was 5 °C or 15 °C. Adult eclosion occurred most synchronously and pupal mortality was lowest when insects were stored at 15 °C for 90 days before incubation at 25 °C. These results strongly suggest thatD. tsuneonis enters a pupal diapause.     

What Makes a Fly Enter Diapause?

Diapause is a dormant state that insects may undergo as a response to changing environmental conditions. In flies, like many insects inhabiting temperate zones, diapause occurs generally during the winter months when ambient temperatures are cool and food sources scarce. Whilst the environmental factors involved in determining diapause have been known for a long time, the genes and molecular events controlling its initiation are poorly understood. Here I outline the factors that initiate diapause and highlight recent studies that implicate insulin signaling in its control.     

What makes a fly enter diapause?

Diapause is a dormant state that insects may undergo as a response to changing environmental conditions. In flies, like many insects inhabiting temperate zones, diapause occurs generally during the winter months when ambient temperatures are cool and food sources scarce. Whilst the environmental factors involved in determining diapause have been known for a long time, the genes and molecular events controling its initiation are poorly understood. Here I outline the factors that initiate diapause and highlight recent studies that implicate insulin signaling in its control.     

高温条件下棉铃虫化蛹率、夏滞育率和蛹重的变化

在预蛹期,高温处理能诱导棉铃虫蛹进入夏滞育。本实验着重就33～39℃的变温下滞育蛹和未滞育蛹的失重动态进行了对比研究,同时以常温（27℃）下蛹作为参照。研究发现： 在33～39℃的变温条件下,棉铃虫化蛹率显著低于其在常温下的化蛹率,且所化蛹中有63.2%的雄性和10.9%的雌性进入高温夏滞育,其中高温滞育蛹和未滞育蛹分别都轻于正常发育蛹。化蛹后第2日至第5日期间,高温滞育蛹失重量显著低于高温未滞育蛹和正常发育蛹的失重量,分别为3.62、13.30和5.49 mg;蛹期总失重量结果与化蛹后第2～5日间蛹失重量趋势一致,高温滞育蛹、未滞育蛹和正常发育蛹失重量分别为15.60、49.35和26.30 mg。蛹失重动态研究发现高温滞育蛹在夏滞育期间其失重曲线平缓,显著低于高温未滞育蛹和正常发育蛹;高温滞育蛹滞育解除后,其失重曲线与正常发育蛹的失重趋势基本一致。结果表明,棉铃虫夏滞育蛹能通过维持低的代谢水平来度过不利环境,具有一定的生态适应意义。     

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.