Interactions between TIME and PIN could play a role in the system that controls the duration of diapause development and synchronization with seasonal cycles in the silkworm Bombyx mori*

The significance of winter cold in the termination of diapause was investigated with regards to TIME and PIN in eggs of the silkworm Bombyx mori. TIME (time interval measuring enzyme) is an ATPase that can measure time intervals by exhibiting a transitory burst of activation of the enzyme in accordance with diapause development, which requires cold for resumption of embryonic development in the silkworm. The possible timer function of TIME comprises a built‐in mechanism in the protein structure. TIME is a metallo‐glycoprotein consisting of 156 amino acid residues with a unique sequence in the N‐terminal region to which a sugar chain is attached. PIN (peptidyl inhibitory needle) inhibits the ATPase activity of TIME. PIN is not a simple enzyme inhibitor, but holds the timer by forming a time‐regulatory complex with TIME. The carbohydrate moiety of TIME is essential for the assembly of a high‐affinity PIN‐binding site within the timer motif of the TIME structure. The binding interaction between TIME and PIN was much tighter (nearly 1000 times) at 25°C than that at 4°C, as measured by fluorescence polarization. Because the logEC₅₀ at 4°C was approximately 7 nmol/L, PIN must dissociate from TIME at the physiological concentration of TIME in eggs in the winter cold. Based on the results of our study, we propose that the dissociation of the TIME–PIN complex in the winter cold cues a series of conformational changes of TIME, ultimately reaching the active form of ATPase which in turn causes the completion of diapause development and initiates new developmental programs.     

The peptide PIN changes the timing of transitory burst activation of timer-ATPase TIME in accordance with diapause development in eggs of the silkworm, Bombyx mori

TIME is an ATPase that measures a time interval by exhibiting transitory burst activation in eggs of the silkworm, Bombyx mori L. PIN is a peptide that regulates the time measurement of TIME. To address the mode of action of PIN, interactions between TIME and PIN were investigated. First, TIME was mixed with PIN for various periods (days) at 25 degrees C. The longer TIME was mixed with PIN, the later the transitory burst activation of TIME ATPase activity occurred, while no such delay occurred at 5 degrees C. Second, the capacity of PIN to bind with TIME was measured at the two temperatures by fluorescence polarization. The binding interaction was much tighter (nearly 1000 times) at 25 degrees C than that at 4 degrees C. Because the log EC50 (in nM) at 4 degrees C was about 7, PIN must dissociate from TIME at low temperatures at the physiological concentration of TIME in eggs. Thus, TIME appears to be restructured into a time-measuring conformation by PIN at the high temperatures of summer, whereas the TIME-PIN complex would dissociate at the low temperatures of winter. This dissociation acts as the preliminary cue for the ATPase activity burst of TIME, which in turn causes the completion of diapause development and initiates new developmental programs.     

Demarcation of diapause development by cold and its relation to time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori

We investigated the mode of action of winter cold in the termination of diapause by investigating Time-Interval-Measuring Enzyme (TIME). First, we determined the period of cold required for the completion of diapause development. Synchronously developing egg batches of a pure strain (C108 Bombyx mori silkworm) were used to minimize variations in hatching time. Hatching occurred with only 18 days chilling at 5 degrees C when the incubation at 25 degrees C after the chilling was elongated. The 18-day period was much shorter than we expected; diapause in B. mori is known to terminate completely with about 100 days of chilling. Even in such a short period of chilling, no sporadic hatching occurred. Moreover, we determined that a temperature-insensitive stage, which we called "Neboke", followed the short cold-requiring stage. Thus, the stage of diapause development was demarcated from other stages of diapause. While the length of diapause development was elongated when chilling was delayed after oviposition, the Neboke stage length was invariant. Cold evidently exerts its effect only on diapause development. When TIME was purified from eggs and chilled in test tubes, a transitory burst of its ATPase activity occurred at a time equivalent to shortly before the completion of diapause development; this was an interval-timer activation. The mechanism by which cold activates TIME to measure the time interval may help explain in biochemical terms the insect's adaptation to its seasonal environments.     

Neuroendocrine control of diapause hormone secretion in the silkworm,Bombyx mori

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.     

Photoperiodic sensitivity and diapause induction during ovarian,embryonic and larval development of the flesh fly,Sarcophaga argyrostoma

Sensitivity to the daily photoperiod, particularly with respect to pupal diapause induction, was studied during ovarian, embryonic, and larval development of the flesh flySarcophaga argyrostoma. Large flies were shown to have a greater number of primary follicles in their ovaries and to be capable of limited ovarian maturation in the absence of exogenous protein (autogeny). Such ovarian development occurred independently of photoperiod. However, long days experienced during embryogenesis caused more rapid development, and earlier larviposition, than short days. Short days during embryonic and subsequent larval development also induced pupal diapause, whereas long days led to continuous or non-diapause development of the pupae. Pupal diapause could not be induced by photoperiods during the vitellogenic phase of ovarian development. InSarcophaga argyrostoma, a maternal effect preventing pupal diapause among the progeny of files with a diapause history was not observed.     

The effects of temperature on development of the large narcissus fly (Merodon equestris)

Eggs, larvae, pupae and adults of the large narcissus fly (Merodon equestris) were reared at a series of constant temperatures between 9–24°C. Egg development required from 37 days at 9°C to 7 days at 21.5°C. The low-temperature threshold for development was 6.7°C. Larvae reared at 1424°C were fully-grown after 18 weeks, but it took much longer for such insects to pupate, and adult flies emerged only after about 45 weeks of development. Large narcissus flies enter diapause during the larval stage and overwinter as fully-fed larvae, forming pupae in the following spring. Post-winter pupation and pupal development took from 169 days at 10°C to 36 days at 21.5°C. Of this, pupal development required from 91 days at 10°C to 19 days at 21.5°C. The low-temperature threshold for post-winter pupation and pupal development was 7.1°C, and for pupal development alone, 7.2°C. Females maintained at or below 19°C laid few eggs, whereas some females kept at or above 21.5°C laid more than 100 eggs (mean 69 ± 36). Approximately 50% of females maintained at or above 21.5°C laid less than 10 eggs during their lifetime. The mean egg-laying time was 6 to 9 days. Although temperatures at or below 19°C inhibited mating, once a female had mated, such temperatures did not prevent oviposition.     

Crystal structures of the clock protein EA4 from the silkworm Bombyx mori

Many insects pass the winter in an arrested developmental stage called diapause, either as eggs, as pupae, or even as adults. Exposure to the prolonged cold of winter is required to permit awakening from diapause in the spring. In the diapause eggs of the silkworm Bombyx mori, a metalloglycoprotein, esterase A4 (EA4), has been suggested to serve as a cold-duration clock because its characteristic ATPase activity is transiently elevated at the end of the necessary cold period. This timer property of EA4 is known to start with the dissociation of an inhibitory peptide (called “peptidyl inhibitory needle”) under cold conditions, but its time-measuring mechanism is completely unknown. Here we present the crystal structures and functional properties of EA4 with and without glycosylation. We show that EA4 is a homodimeric ATPase, with each subunit consisting of a copper-zinc superoxide dismutase fold. There is an additional short N-terminal region that is capable of binding one more copper ion, suggesting a timer mechanism in which this ion is involved. The sugar chain appears to reinforce the binding of peptidyl inhibitory needle, which may in turn stabilize the initial conformation of the N-terminal domain, explaining the requirement for glycosylation and for the peptide to set the clock.     

Control of summer and winter diapause in the leaf-mining fly Pegomyia bicolor Wiedemann (Dipt., Anthomyiidae)

Effects of photoperiod and temperature on diapause induction and termination were investigated in both aestival and hibernal pupae of Pegomyia bicolor Wiedemann under field and laboratory conditions. In the field, summer diapause had occurred already in part of the first pupal population; the proportion of diapause gradually rose as the day length and temperature increased. This fly is a short-day species with a pupal summer and winter diapause. Summer diapause was induced by both long day-lengths and mild temperatures. The whole larval life is sensitive to photoperiod. Winter diapause was induced mainly by low temperatures, especially in the first 10 days after pupation. High temperatures strongly enhanced summer diapause induction regardless of photoperiod. The diapause-averting influence of short photoperiods was fully expressed only at moderately low temperatures. High temperatures delayed diapause development, resulting in a rather long summer diapause; whereas low temperatures hastened it, leading to a short winter diapause and showing a low thermal threshold for diapause development. In the field, the post-diapause development started in January, the coldest month, suggesting that the thermal requirements for post-diapause development is also low.     

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.     

Diapause hormone in Bombyx eggs and adult ovaries

An extraction method using Triton X-100 was successful in yielding active extracts of diapause hormone both in adult ovaries and laid eggs, as well as from mature unlaid eggs. The extracts induced normal diapause eggs which underwent diapause. The extracts also exhibited activity when injected into pupae with the suboesophageal ganglion removed. The percentage of diapause eggs laid was dependent upon the amount of extract administered. The activity occurred exclusively in diapause ovaries or eggs and was not found significantly in non-diapause ovaries or eggs. By partition between butanol and water, the hormonal activity was recovered in butanol. Sephadex LH-20 chromatography located the activity in the same fraction as that of diapause hormone.These data suggest that diapause eggs and ovaries contain the diapause hormone in a bound form.     

Pupal diapause of Helicoverpa armigera (Lepidoptera: Noctuidae): sensitive stage for thermal induction in the Okayama (western Japan) population

Helicoverpa armigera (Hübner) exhibits a facultative pupal diapause, which depends on temperature and photoperiod. Pupal diapause is induced at 20 degrees C by short photoperiods and inhibited by long photoperiods during the larval stage. However, in some pupae (35% of males and 57% of females) of a non-selected field population from Okayama Prefecture (34.6 degrees N), diapause is not induced by short photoperiods. In the present experiment, the importance of temperature for diapause induction was studied in the non-diapausing strain, which was selected from such individuals reared at 20 degrees C under a short photoperiod of 10L:14D. Furthermore, the sensitive stage for thermal determination of pupal diapause was determined by transferring larvae of various instars and pupae between 20 degrees C and 15 degrees C. Diapause was induced by 15 degrees C without respect to photoperiod. When larvae or pupae reared from eggs at 20 degrees C under a short or a long photoperiod were transferred to 15 degrees C in the periods of the middle fifth instar to the first three days after pupation, the diapause induction rate was significantly reduced in both males and females, especially in females. In contrast, when larvae or pupae reared at 15 degrees C were transferred to 20 degrees C in the same periods, diapause was induced in males, but not in females. However, the diapause induction rate of pupae transferred to 20 degrees C on the fourth day after pupation was significantly increased in females. The results show that temperature is the major diapause cue in the photoperiod-insensitive strain and the periods of middle fifth larval instar to early pupal stage are the thermal sensitive stages for pupal diapause induction with some different responses to temperatures between males and females in H. armigera.     

Characteristics of summer diapause in the onion maggot,Delia antiqua (Diptera: Anthomyiidae)

Characteristics of summer diapause in the onion maggot, Delia antiqua, were clarified by laboratory experiments. Temperature was the primary factor for the induction of summer diapause in this species. The critical temperature for diapause induction was approximately 24 degrees C, regardless of the photoperiod. At 23 degrees C, the development of the diapausing pupae was arrested the day after pupariation, when about 7% of the total pupal development had occurred in terms of total effective temperature (degree-days). The most sensitive period for temperature with regard to diapause induction was estimated to be between pupariation and "pupation" (i.e., evagination of the head in cyclorrhaphous flies). Completion of diapause occurred at a wide range of temperatures (4-25 degrees C): The optimal temperature was approximately 16 degrees C, at which temperature only five days were required for diapause completion. The characteristics of summer diapause in D. antiqua are discussed in comparison with those of summer dormancy in a congener D. radicum and those of winter diapause in D. antiqua.     

Artificial light at night causes diapause inhibition and sex‐specific life history changes in a moth

Rapidly increasing levels of light pollution subject nocturnal organisms to major alterations of their habitat, the ecological consequences of which are largely unknown. Moths are well‐known to be attracted to light at night, but effects of light on other aspects of moth ecology, such as larval development and life‐history, remain unknown. Such effects may have important consequences for fitness and thus for moth population sizes. To study the effects of artificial night lighting on development and life‐history of moths, we experimentally subjected Mamestra brassicae (Noctuidae) caterpillars to low intensity green, white, red or no artificial light at night and determined their growth rate, maximum caterpillar mass, age at pupation, pupal mass and pupation duration. We found sex‐specific effects of artificial light on caterpillar life‐history, with male caterpillars subjected to green and white light reaching a lower maximum mass, pupating earlier and obtaining a lower pupal mass than male caterpillars under red light or in darkness. These effects can have major implications for fitness, but were absent in female caterpillars. Moreover, by the time that the first adult moth from the dark control treatment emerged from its pupa (after 110 days), about 85% of the moths that were under green light and 83% of the moths that were under white light had already emerged. These differences in pupation duration occurred in both sexes and were highly significant, and likely result from diapause inhibition by artificial night lighting. We conclude that low levels of nocturnal illumination can disrupt life‐histories in moths and inhibit the initiation of pupal diapause. This may result in reduced fitness and increased mortality. The application of red light, instead of white or green light, might be an appropriate measure to mitigate negative artificial light effects on moth life history.     

家蚕滞育性卵盐酸处理的靶物质

酯酶A4（EA4）是家蚕卵的滞育生物钟蛋白质。从家蚕C108品种产下后48 h的滞育性卵和盐酸活化处理卵分离纯化出EA4酶蛋白,使用合成的EA4活性多肽抑制因子PIN（氨基酸结构：SIFMTKQHSQ DDIIQHPLDY VEQQIHQQKQ KLQKQTLN）,研究了PIN对EA4酶蛋白的作用机制。滞育性卵的EA4酶蛋白和PIN在25℃混合24h后,用矩阵辅助激光解吸离子质谱法,检测到了二者的结合体,该结合体在盐酸处理后消失;盐酸活化处理蚕卵的EA4酶蛋白和合成PIN之间没有出现这种结合体。体外25℃,滞育性蚕卵EA4的ATPase特征性活性峰在6.5 h后出现,而盐酸活化处理蚕卵的EA4在1.5 h后出现活性峰值。盐酸处理可能通过解除PIN对EA4的抑制作用,在短时间内激活EA4酶蛋白,从而活化滞育性蚕卵。     

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.     

The Insect Growth Regulator Pyriproxyfen Terminates Egg Diapause in the Asian Tiger Mosquito,Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25–30 days) compared to fully embryonated eggs (71–80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.     

Neuroendocrine roles of the brain in the regulation of 20-hydroxyecdysone responsiveness in two types of diapause pupae of the cabbage armyworm, Mamestra brassicae

The cabbage armyworm, Mamestra brassicae, has winter-and aestival- diapause pupae (WD- and AD-pupae) showing differences in the strength of diapause. We tried to quantify diapause-strength by measuring the doses of 20-hydroxyecdysone (20-E) required to induce adult development in WD-, AD- and decerebrated non-diapause pupae (ND-pupae). The role of the brain in the regulation of diapause-strength was studied through the decerebration and brain-reimplantation of WD-and AD-pupae. The 20-E doses required for adult development were small within the first 2 days of pupation, and increased thereafter to reach a constant level about 10 days after pupation in AD- and decerebrated ND-pupae. The required 20-E doses in WD-pupae increased for more than 40 days after pupation. When 0-day-old WD- and 0-day-old AD-pupae were decerebrated, required 20-E doses increased after pupation and reached a constant about 10 days later. The required 20-E dose reached a constant level in decerebrated WD-pupae that was smaller than that observed for decerebrated ND- and WD-pupae. Furthermore, the required doses increased when 0-day-old WD-pupal brains were reimplanted into decerebrated WD- and decerebrated ND-pupae. In WD-, AD- and decerebrated ND-pupae, diapause-strength can be represented as the 20-E dose required for adult development. Diapause-strength is weak after pupation, increases thereafter, and reaches a constant about 10 days later in AD- and decerebrated ND-pupae. In WD-pupae, diapause-strength increases for more than 40 days after pupation and reaches a level that is twice that estimated for AD-pupae. Brains of diapausing WD-pupae may secrete a factor that suppresses the 20-E responsiveness of pupal organs, for the purpose of maintaining winter-diapause.     

Effect of larval density on development and diapause of Sesamia nonagrioides (Lef.) (Lep., Noctuidae) under laboratory conditions

The effect of larval density on development and diapause of Sesamia nonagrioides (Lef.) was examined. Larvae were grown on an artificial diet at densities of five, 10, 20, 30 and 40 larvae per box, under two environmental (diapause-including and diapause-averting) conditions. High larval densities resulted in increased mortality under both experimental conditions tested. Although crowded larvae showed a higher pupation rate than those reared under low densities, small differences occurred between mean larval developmental time at different densities under developmental condition. In spite of this, crowding proved effective on the intensity of diapause of this species and the duration of diapause was shorter for crowded larvae. Pupal weights of both sexes were significantly reduced at high densities while there were significant differences between pupal weights formed by larvae reared under diapause conditions and those formed by larvae reared under nondiapause conditions at all density treatments. The pupal period was significantly longer under diapause conditions than that under development conditions but density was not found to play a role on the time of the emergence of the adults produced by larvae reared under developmental or diapause conditions.     

Suppression of autumnal sexual morph production in spring by a seasonal timer in an aphid

Photoperiod is the major seasonal cue for phenotypic plasticity in the regulation of development and reproduction. Aphids have a peculiar mechanism in which sensitivity to the photoperiod is lost for a few months through successive generations after hatching from diapause eggs, and therefore, the mechanism responsible for the photorefractory period is termed “a seasonal timer”. However, whether the seasonal timer has adaptive significance has never been investigated. Here, we show that the photorefractory generation avoids an unseasonal sexual morph production under short days in spring in the pea aphid, Acyrthosiphon pisum (Harris). When the seasonal timer operated, the overwintered generation, stem mothers, produced only parthenogenetic progenies under natural photoperiods and temperatures in both warm and cold springs. After repeated parthenogenesis following diapause, however, aphids with an expired seasonal timer produced sexual females and males typical of autumn under natural photoperiods and temperatures in a warm spring. These sexual morphs are thought to have a reproductive disadvantage compared to parthenogenetic morphs in the following summer. We thus conclude that the seasonal timer is adaptive for avoiding sexual morph production in spring. The present study shows, for the first time, that a photorefractory period lasting over generations is a trait of ecological importance.