The roles of ERK and P38 MAPK signaling cascades on embryonic diapause initiation and termination of the silkworm, Bombyx mori

To clarify the molecular mechanisms of Bombyx diapause, we focused on mitogen-activated protein kinases (MAPK), which are the major components of signal transduction cascades that regulate cell proliferation, differentiation, and stress responses. In the present study, cloning of Bombyx extracellular signal-related kinase (ERK), MAPK-ERK kinase (MEK), and p38 MAPK cDNA, revealed that their amino acid sequences have close similarity with those of other species. We analyzed the roles of the kinases in diapause initiation and termination by immuno-blotting with anti-phospho-kinase antibodies. Phospho-MEK levels remained consistently high in non-diapausing eggs, then declined after the diapausing stage in diapausing eggs, and began to increase 45 d after transfer to 5 degrees C upon diapause termination. The phospho-ERK and phospho-MEK profiles were similar, suggesting that ERK phosphorylation is regulated by MEK. The phospho-p38 MAPK levels declined 36 h after oviposition in diapausing eggs, and increased at 15-30 d at 5 degrees C in yolk cells, suggesting that p38 MAPK has a role in diapause initiation and termination. Phospho-ERK levels were maintained with diapause-interrupting treatment and declined with diapause-sustaining treatment. ERK phosphorylation is considered to have a role in diapause termination and in the resumption of development.     

Involvement of ERK/MAPK in regulation of diapause intensity in the false melon beetle, Atrachya menetriesi

Embryonic diapause is commonly terminated by exposure to low temperature for a certain duration. Previous studies using the silkworm, Bombyx mori, showed that extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase family, was activated by cold exposure and regulated diapause termination. The involvement of ERK in regulation of diapause termination was investigated in the false melon beetle, Atrachya menetriesi. Embryonic diapause of this beetle is terminated both by cold exposure and by mercury. Phospho-ERK levels remained high during the pre-diapause period but decreased after the eggs entered diapause. Exposure to 7.5 degrees C, which was effective for diapause termination, increased phospho-ERK levels, and these levels were maintained under 7.5 degrees C at least for 100 d. Incubation at 25 degrees C after the eggs were kept at 7.5 degrees C for 20 d, which intensified diapause, decreased the phospho-ERK level. An insufficient cold treatment, i.e., incubation at 0 degrees C for diapause termination did not activate ERK. However, incubation at 0 degrees C after cold treatment at 7.5 degrees C, which is effective for diapause termination, induced high phospho-ERK levels. Moreover, mercury treatment also increased phospho-ERK. Therefore, changes in the phospho-ERK level correlated well with diapause intensity. The results suggest that ERK plays a key role in the regulation of embryonic diapause.     

Effects of juvenile hormone analogs and 20-hydroxyecdysone on diapause termination in eggs of Locusta migratoria and Oxya yezoensis

To understand the hormonal control of embryonic diapause, juvenile hormone analogs (JHAs), methoprene and hydroprene, and 20-hydroxyecdysone (20E) were applied onto diapause eggs of Locusta migratoria and Oxya yezoensis. These insects enter diapause at the mid-stage of embryogenesis prior to blastokinesis. Topical application of JHAs significantly facilitated diapause termination in both species but JHA-treated embryos underwent abnormal morphogenesis, pigmentation and sclerotization without dorsal closure. The Locusta eggs immersed in the 20E solution for 24h terminated diapause in a dose-dependent manner. We also investigated phosphorylation of extracellular signal-regulated kinase (ERK), a member of mitogen-activated protein kinase (MAPK), during diapause-terminating process of Locusta migratoria and found that ERK was activated either by cold exposure or JHA treatment. The possible involvement of the hormones and ERK in embryonic diapause and the possibility of ecdysteroids synthesis by prothoracic glands of diapause embryo were proposed.     

Oxygen consumption in relation to sorbitol utilization at the termination of diapause in eggs of the silkworm,Bombyx mori

Rates of oxygen consumption were followed throughout the entire period of diapause in eggs of Bombyx mori. In non-diapause eggs at 25 degrees C, O(2) uptake was divisible into three phases, corresponding to morphogenetic processes. In diapause eggs at 25 degrees C, O(2) uptake showed a peak (100 &mgr;l/g eggs/h) at 1 day and then suddenly dropped to reach a level of 8-10 &mgr;l/g eggs/h at 10 days and thereafter. To break diapause, eggs were exposed to 5 degrees C for varying periods. When O(2) uptake was measured at 5 degrees C, it remained at 6 &mgr;l/g eggs/h. When eggs were chilled for increasing periods and O(2) uptake was measured immediately after warming to 25 degrees C, the rates increased after a lag phase. In HCl-treated eggs, O(2) uptake increased immediately after acid-treatment. In all cases, highly increasing O(2) uptake at 25 degrees C coincided with termination of diapause. These results were discussed in relation to sorbitol utilization at the termination of diapause.     

Samui, a novel cold-inducible gene, encoding a protein with a BAG domain similar to silencer of death domains (SODD/BAG-4), isolated from Bombyx diapause eggs.

Cellular responses to cold-acclimation have not yet been studied in depth. To explore this field, we focussed on insect diapause development. Although embryonic diapause of Bombyx mori is sustained at 25 degrees C, chilling at 5 degrees C for 2 months causes diapause termination, a transition that is marked when the sorbitol dehydrogenase gene (SDH) is activated. To clarify the relationship between this activation and incubation at 5 degrees C, we isolated a novel cold-inducible gene, Samui. Expression of Samui mRNA and protein was activated after incubation at 5 degrees C for 5-6 days, lasted for another 30 days and then weakened. Exposure to 25 degrees C suppressed both mRNA and protein expression. In nondiapause eggs incubated at 5 degrees C, Samui was also up-regulated, although the expression was weaker. Samui contained nuclear localization-signals, a ssDNA-binding motif and a BAG domain similar to that of SODD/BAG-4. Because Samui could bind to HSP70, it is a member of BAG protein family. It is proposed that Samui serves to transmit the '5 degrees C signal' for SDH expression in diapause eggs, while also protecting against cold-injures in nondiapause eggs, through binding to respective partners. This is the first report that a member of BAG protein family is up-regulated by cold.     

Effects of temperature, sorbitol, alanine and diapause hormone on the embryonic development in Bombyx mori: in vitro tests of old hypotheses

Abstract.  An in vitro culture method is described in which embryonic development in Bombyx mori is traced at various temperatures and treatments. The results show that the induction, intensification and termination of diapause are distinct processes. Prediapause embryos, explanted from 40-h-old diapause-destined eggs and cultured in Grace's medium, continue to develop to the appendage-formation stage without arrest, which indicates that the isolated embryos have not entered diapause, whereas the development of embryos from diapausing eggs (15 days after being laid) is significantly slower. The rate of development of embryos dissected from diapause eggs increases during chilling (5 °C) and incubation (at 25 °C) gradually during chilling and dramatically at 25 °C. The in vitro experiments also reveal that sorbitol directly inhibits the development of embryos explanted from diapausing eggs but has no affect on the development of embryos from prediapause eggs. Neither alanine nor diapause hormone prevent isolated embryos from developing.     

Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates) and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase (EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar H(+)-ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellin-ecdysteroid-phosphates complex and the dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.     

Injury‐induced rapid activation of MAPK signaling in dechorionated eggs and larvae of the silkworm Bombyx mori

Previous study showed that diapause in Bombyx mori eggs can be terminated by dechorionation and that activation in the mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that mechanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho‐ERK levels and that injury increased the phospho‐ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HCl. Effects of anaerobiosis on dechorionation‐stimulated phospho‐ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is involved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation‐stimulated phospho‐ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphorylation of c‐jun N‐terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in JNK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho‐ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury‐induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs.     

Phosphorylation of glycogen synthase kinase-3β in relation to diapause processing in the silkworm, Bombyx mori

Glycogen synthase kinase-3 (GSK-3) is a multifunctional protein kinase that plays important roles in regulating both glycogen synthesis and protein synthesis. In the present study, we investigated GSK-3β phosphorylation of silkworm eggs by immunoblotting with a conserved phospho-specific antibody to GSK-3β. Results showed that the temporal changes in GSK-3β phosphorylation were closely related to changes in glycogen levels previously reported by other researchers. In diapause eggs, an abrupt decrease in phosphorylation of GSK-3β was found with the onset of diapause, and phosphorylation level of GSK-3β reached a minimum level within 1 week after oviposition. However, when diapause eggs were incubated at 25 °C for 15 days and then transferred to 5 °C, a great increase in GSK-3β phosphorylation was observed 5 days after transfer to 5 °C and high levels were maintained throughout the chilling period. In both non-diapause eggs and eggs whose diapause initiation was prevented by HCl, levels of GSK-3β phosphorylation appeared to remain relatively high for several days and then greatly decreased 2 or 3 days before hatching. Moreover, GSK-3β phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, did not inhibit GSK-3β phosphorylation in dechorionated eggs, although U0126 dose-dependently inhibited ERK phosphorylation. This result showed that ERK phosphorylation is not involved in upstream signaling for GSK-3β phosphorylation and that there may be two distinct signaling pathways involved in diapause processing in Bombyx mori eggs.     

Effects of temperature during chilling and pre-chilling periods on diapause and post-diapause development in a katydid, Eobiana engelhardti subtropica

In Eobiana engelhardti subtropica, early laid eggs reach the diapause stage in early autumn. For long periods before winter, the eggs are exposed to temperatures higher than their theoretical lower threshold for development. In contrast, late-laid eggs cannot reach their diapause stage before winter. Our study showed that E. e. subtropica copes with these difficulties via the thermal response involving embryonic diapause. In this katydid, the almost fully developed embryo undergoes an obligatory diapause. When diapause eggs were maintained at a temperature of 20 degrees C or higher, diapause persisted for a long time. Diapause was effectively terminated by temperatures ranging from 1 to 11 degrees C, and hatching occurred successfully at temperatures from 11 to 15 degrees C. In addition to the chilling temperature, pre-chilling temperature modified diapause intensity and hatching time. Diapause eggs hatched earlier after chilling when the pre-chilling temperature was lower, within a range of 14.5-25 degrees C. Thus, the low-temperature requirement for diapause termination prevents early laid eggs from untimely hatching in autumn, and low temperatures before and during winter decrease diapause intensity and shorten the hatching time in the following spring. When eggs were chilled before diapause, they tolerated chilling and averted diapause. Thus, even if eggs encounter low temperatures before diapause, they can hatch in the following spring.     

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.     

Effect of temperature on the termination of prolonged larval diapause in the chestnut weevil Curculio sikkimensis (Coleoptera: Curculionidae)

Adults of the chestnut weevil Curculio sikkimensis emerged over a 3-year period under laboratory and quasi-field conditions due to a prolonged diapause that occurred at the mature larval stage. Variable proportions of the larvae remained in diapause after a single cold (5 degrees C) treatment of 120 days. Extension of the chilling period to as long as 540 days did not increase the percentage of diapause termination, and excessively long chilling actually reduced the percentage. Chilling was not indispensable to the termination of larval diapause. Diapause intensity was very high and variable, and more than 1000 days at 20 degrees C was necessary to reactivate all diapause larvae. When the diapause larvae were exposed to cycles of low (5 degrees C for 120 days) and high (20 degrees C for 240 days) temperatures, the percentage of diapause termination reached 100% after two or three such cycles. Thus, the prolonged diapause of C. sikkimensis has characteristics similar to the common short winter diapause in other insects, but has unique characteristics that ensure polymodal reactivation over several years.     

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.     

An esterase in relation to yolk cell lysis at diapause termination in the silkworm,Bombyx mori

Esterase A is one of the esterase isozymes in eggs of Bombyx mori. The effect of this esterase A on the yolk cells of diapause eggs was examined with a hanging-drop culture in order to discover the mechanism of diapause termination in silkworm eggs.The culture of yolk cells in diapause eggs shows spherical forms with dark fine grains in the central parts, large translucent granules in the outer parts, and a membrane on the exterior. When such yolk cells were incubated with yolk materials of acid-treated or diapause-terminated eggs, they were damaged and cell lysis occurred. This suggested that substance(s) causing the cell lysis were present in diapause-terminated eggs. When esterase A separated electrophoretically from non-diapause eggs and diapause-terminated eggs was added to hanging-drop cultures of yolk cells of diapause eggs, the yolk cells were also greatly affected. That is, a part of the yolk cell membrane was dissolved or disappeared, and the central dark fine grains diffused over the cell causing the whole cell to become dark. A few cells lost almost all of their contents and collapsed. Other esterase fractions and fractions without esterase activity in the electrophoresis exerted little effect on the yolk cells. Furthermore, a parallelism between esterase activity to hydrolyse 2-naphthyl acetate as substrate and the lytic activity on the yolk cell membrane was observed in this esterase A fraction from different sources.From these results it is highly probable that the substance responsible for cell lysis is the esterase A enzyme itself. Diapause termination of silkworm eggs is discussed in relation to the lysis of yolk cells.     

Reversible change in embryonic diapause intensity by mild temperature in the Chinese rice grasshopper, Oxya chinensis

The effects of temperature on maintenance and termination of embryonic diapause were investigated in Jining (35.4°N, 116.6°E) and Sihong (33.5°N, 118.2°E) strains of the Chinese rice grasshopper, Oxya chinensis Thunberg (Orthoptera: Catantopidae). Eggs of both strains entered diapause when incubated at 30, 25, or 20 °C. Chilling at 8 °C had an evident effect on diapause termination and almost all eggs chilled for 60 days ended diapause development. Chilling of eggs at 8 °C for only 20 days failed to result in any hatching at 20 °C, suggesting that such level of chilling was not enough to induce diapause termination. However, the treatment combining incubation of eggs at 30 °C for varying lengths of time with subsequent incubation to 20 °C had a distinct effect on the completion of diapause of the eggs. The results indicate that there were two temperature optima, that is, low temperature (chilling) and high temperature, for diapause development in this grasshopper species. Incubation of chilled eggs at 20 °C for 5–15 days followed by further incubation at 25 °C reduced termination of diapause significantly compared with the eggs only chilled at 8 °C. Exposure of eggs chilled at 8 °C to a pulse of 25 °C from 1 to 7 days, separated by a 20-day interval at 8 °C, resulted in a decrease in the percentage of successfully hatched eggs as the length of the pulse of 25 °C increased. The results suggest that diapause intensity may be restored at moderately high temperatures. This reversible change in diapause intensity would play an important role in maintaining diapause before winter.     

Repeated cycles of chilling and warming effectively terminate prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis (Coleoptera: Curculionidae) requires one or more years to complete its life cycle, owing to prolonged larval diapause. To compare the effects of temperature cycles and total periods of chilling on the termination of prolonged diapause, larvae were subjected to different chilling (5 degrees C) and warming (20 degrees C) cycles ranging from 30 to 720 days, and all cycles were repeated until the sum of chilling and warming periods reached 720 days. The prolonged diapause of C. sikkimensis was more effectively terminated by repeated cycles of chilling and warming than by prolonging the continuous chilling period. However, extremely short temperature cycles were not highly effective in enhancing diapause termination, even when such cycles were repeated many times. To examine the role of warming periods on diapause termination, diapause larvae were subjected to a sequence of chilling (120 days at 5 degrees C) and warming (240 days at 20 degrees C) with a warming period (0-120 days at 20 degrees C) inserted in the chilling period. Diapause larvae that were not reactivated in the first chilling period required exposure to a certain period of warming before they were able to complete diapause development in the subsequent chilling. Thus, C. sikkimensis appears to spread its reactivation times over several years in response to seasonal temperature cycles.     

Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination

For diapause eggs of the silkworm, Bombyx mori, diapause initiation is prevented with hydrochloric acid (HCl) at around 20 h post-oviposition while diapause status is terminated with chilling around 5°C. To investigate whether hydrogen peroxide (H(2)O(2)) and catalase expression are involved in diapause initiation and termination, the concentration of H(2)O(2), relatively higher levels of catalase mRNA and activity of catalase were compared between (1) 20-h-old diapause eggs and the HCl-treated diapause eggs, and (2) 10-day-old diapause eggs and the 5°C-chilled diapause eggs. Compared to diapause eggs, the HCl-treated eggs had significantly higher H(2)O(2) concentrations (up from approximately 1-3 μmol/g fresh mass to 5-8 μmol/g fresh mass), higher relative level of catalase mRNA (up from 0 to 35.2%) and higher catalase activity (up from 2.51 units/mg protein to 4.97 units/mg protein) at 96 h post-treatment. On the other hand, the 5°C chilling resulted in significant increases of H(2)O(2) concentration (up from 0.79 μmol/g fresh mass to 5.57 μmol/g fresh mass), relative level of catalase mRNA (up from 0 to 71.4%) and catalase activity (up from 0.88 units/mg protein to 3.42 units/mg protein) within 120 days. The results obtained in this work suggest that variations of H(2)O(2) and catalase expression in Bombyx eggs are involved in diapause initiation and termination.