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.     

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.     

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.     

Temperature-dependent activation of ERK/MAPK in yolk cells and its role in embryonic diapause termination in the silkworm Bombyx mori

The silkworm Bombyx mori requires 2-3 months of low temperature (5 degrees C) to terminate embryonic diapause. The molecular mechanisms, however, are unknown. Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is temperature-dependently activated in the yolk cells of diapausing eggs after 45 days at 5 degrees C, coincident with the acquisition of developmental competence of the embryos at 25 degrees C. Yolk cell granulation and dissociation also begin in diapause eggs incubated at 5 degrees C for 45 days. We used dechorionated egg culture as a model system of diapause termination and observed that both yolk cell dissociation and embryonic development are inhibited by MAPK-ERK kinase (MEK) inhibitor U0126. Therefore, we suggest that ERK in yolk cells has a role in regulating changes in yolk morphology and termination of embryonic diapause in B. mori.     

High temperature and hexane break pupal diapause in the flesh fly, Sarcophaga crassipalpis, by activating ERK/MAPK

Pupal diapause in the flesh fly, Sarcophaga crassipalpis, can be terminated by exposure to high temperatures or, artificially, with a topical application of organic solvents. To analyze the molecular mechanisms involved in diapause termination we explored the possibility that the mitogen-activated protein kinases (MAPK) are involved in this response. Levels of phospho-ERK increased within 10 min after hexane application. Extracellular signal-regulated kinase (ERK) was also activated when pupae were transferred from 20 to 25 degrees C, thus suggesting that ERK activation is a likely component of the signal transduction pathway used to initiate development in response to diapause-terminating signals. 20-Hydroxyecdysone and cyclic GMP terminate diapause in this fly, and the juvenile hormone analog methoprene shortens the diapause, but none of these agents activated ERK. ERK was readily activated in isolated abdomens treated with hexane, thus we conclude that ERK is directly activated by the hexane treatment. ERK activation was evident in the brain, epidermis, midgut and fat body, but not in the ventral nerve mass or ring gland, thus suggesting that ERK does not act directly on the ring gland to promote ecdysteroid synthesis but exerts its effect through stimulation of the brain.     

Effect of anoxia on diapause termination in eggs of the false melon beetle, Atrachya menetriesi

The effect of anoxia on diapause development in the leaf beetle Atrachya menetriesi was investigated to elucidate the role of oxygen in regulation of egg diapause. While anoxia alone had no effect on diapause termination, it decreased diapause intensity before chilling. Such an effect reached a maximum level when anoxia lasted for about 10 days. Anoxia applied during the pre-diapause stage also reduced diapause intensity. On the other hand, anoxia terminated diapause when the diapause intensity had been lowered by sufficient duration of chilling (50 days at 7.5 degrees C). The effect of anoxia was temperature dependent; the larger effect was elicited when anoxia was combined with a higher temperature. A 50-day chilling caused more than 20% of eggs to terminate diapause upon transfer to warm conditions. However, when this chilling period was interrupted on the 20th day by a 5-day exposure to a high temperature of 20, 25 or 30 degrees C, the effect of the former chilling was cancelled partially or completely, suggesting that warming reversed diapause development. This reversing effect of a high temperature, however, was not manifested when the warming was combined with anoxia. The results suggest that anoxia inhibits diapause reversal and facilitates a certain process of diapause development. The sequence of exposure to anoxia and chilling is not important.     

Effects of temperature and photoperiod on termination of pseudopupal diapause in the bean blister beetle, Epicauta gorhami

Larvae of the bean blister beetle, Epicauta gorhami (Coleoptera: Meloidae), feed on grasshopper eggs in soil and undergo hypermetamorphosis. This beetle undergoes larval diapause in the fifth instar as a pseudopupa, a form characteristic of hypermetamorphosis in meloid beetles. This pseudopupal diapause is maintained for a long period if the larvae are kept under continuous diapause-inducing short-day conditions. In the present study, the effects of temperature and photoperiod on termination of pseudopupal diapause in E. gorhami were examined using pseudopupae obtained under 12L-12D at 25°C. Diapause was terminated by exposure to low or high temperature, but not by transfer to long-day conditions. The pseudopupal stage comprises diapause and post-diapause phases despite its unchanging appearance. The optimum low temperature for diapause termination was 10-15°C, and a higher or lower temperature was less effective. There was an individual variation in the low temperature requirement for diapause termination and post-diapause development may not occur until this requirement is satisfied. Although under natural conditions pseudopupae may encounter low temperatures that are effective for diapause termination at different ages, our results show that pseudopupae at various ages respond similarly to low temperature exposure. This response may ensure that resumption of development synchronizes in a population.     

Mating prior to overwintering in the imported willow leaf beetle, Plagiodera versicolora (Coleoptera: Chrysomelidae)

Abstract. 1. We report on the mating status of overwintering adults of the imported leaf beetle, Plagiodera versicolora Laicharting. This species overwinters as adults, under the bark of willow trees.
2. Individuals were collected both in the autumn, just after initiation of diapause, and in the spring, just before natural termination of diapause. From the autumn sample, we discovered that diapause can be terminated by husbanding the adults at 4°C for 4 weeks.
3. Most females lay fertile eggs upon the termination of diapause; however, a significant number of females lay non-fertile eggs after overwintering in the field.
4. Many females mate with more than one male before entering diapause. Progeny analysis using allozyme genetic markers shows that, on average, females who are fertile upon termination of diapause utilize sperm from 1.2 males to fertilize their eggs.     

Diapause termination, post-diapause development and reproduction in the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae)

Effects of photoperiod and cold exposure on diapause termination, post-diapause development and reproduction in Loxostege sticticalis were examined. Larvae were reared at diapause inducing condition (22 °C, L:D 12:12) consistently or transferred to long day photoperiod (L:D 16:8) and darkness (L:D 0:24) respectively, after entering into diapause. Diapause was terminated in approximately 40% of the larvae after 36 days, and no significant differences were observed between photoperiods, suggesting larval diapause was terminated spontaneously without being induced by photoperiods. Cold exposure significantly hastened diapause termination. The diapause termination incidence increased significantly with peaks of 98% at both 5 °C and 0 °C exposure for 30 days, as compared to 42% in controls not exposed to cold, while the mortality and number of days required for diapause termination decreased dramatically. The optimal low temperature exposure periods under 5 °C or 0 °C were 20 days and 30 days, showing a higher termination incidence and shorter time for diapause termination. This suggests that the low temperatures in winter play an important role in diapause termination under natural conditions. The threshold temperatures for post-diapause development in prepupae and pupae were 9.13 °C and 10.60 °C respectively, with corresponding accumulations of 125 and 200 degree-days. Adults that experienced larval diapause significantly delayed their first oviposition, oviposition period was prolonged, and the lifetime number of eggs laid was decreased, however both males and females have significantly longer longevity. The field validation of diapause termination, the degree-days model, and the relationship between diapause and migration in L. sticticalis were also discussed.     

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 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.     

Egg development and diapause: ecophysiological and genetic basis of phenological polymorphism and adaptation to varied hosts in the green oak tortrix,Tortrix viridana L. (Lepidoptera: Tortricidae)

Experiments concerned 8 tortrix populations associated with varied oak species. They showed that the egg development included a phase of diapause. Completion of embryogenesis at 20 degrees C was used as a criterion for whether diapause was completed. Under semi-natural conditions diapause terminated in late autumn or early winter, then eggs developed continuously, without postdiapause winter quiescence, even in severe cold. The eggs from the populations associated with holm or cork oak completed diapause then hatched later than those from the populations associated with sessile or pubescent oak. Reciprocal crossbreedings confirmed that this phenological polymorphism was genetically determined. Under constant temperatures the physiological state of diapausing eggs, assessed by measuring their cold requirements to complete diapause by exposure to 8 degrees C, varied gradually with increasing age. This diapause development was strongly temperature-dependent. Cold requirements of diapausing eggs were much higher in a late-hatching than in an early-hatching population. This explains the phenological polymorphism of the tortrix: the more eggs need cold, the later they complete diapause in autumn, and the later they hatch in spring. Egg cold requirements varied widely within populations too, which resulted in large variations in the date of diapause end among individuals. The date of egg hatch was influenced by the temperatures occurring during diapause and postdiapause, but apparently not by photoperiod.     

Effects of temperature and photoperiod on the termination of larval diapause in Lucilia sericata (Diptera: Calliphoridae)

Larvae of the blow fly, Lucilia sericata (Meigen), enter diapause in the third instar after cessation of feeding. The effects of temperature and photoperiod on the termination of diapause were examined. The diapause terminated spontaneously under the diapause-inducing condition of 20 degrees C and LD 12:12, although pupariation was not synchronous. Diapause development proceeded under a low temperature of 7.5 degrees C. Transfer to long-day conditions of LD 16:8 or to a high temperature of 25 degrees C induced prompt and synchronous pupariation. Low temperatures in winter probably play a predominant role in the termination of diapause under natural conditions.     

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.     

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.     

Rapid cold hardening in the predatory mite Euseius (Amblyseius) finlandicus (Acari: Phytoseiidae)

A rapid cold hardening response was studied in diapause and non-diapause females of the predatory mite Euseius finlandicus. When laboratory reared diapause and non-diapause females were transferred and maintained from the rearing temperature of 20 degrees C for 2 h to -11.5 degrees C and -10 degrees C, 10 to 20% survived respectively. However, conditioning of diapause females for 4 h at a range of temperatures from 0 to 10 degrees C before their exposure for 2 h to -11.5 degrees C, increased survival to approximately 90%. Similarly, conditioning of non-diapause females for 4 h at 5 degrees C before their exposure for 2 h to -10 degrees C increased survival to 90%. A similar rapid cold hardening response in both diapause and non-diapause females was also induced through gradual cooling of the mites, at a rate of approximately 0.4 degrees C per min. The rapid increase in cold tolerance after prior conditioning of the mites to low temperatures, was rapidly lost when they returned to a higher temperature of 20 degrees C. Rapid cold hardening extended the survival time of diapause and non-diapause females at sub-zero temperatures. The cost of rapid cold hardening in reproductive potential after diapause termination was negligible. In non-diapause females, however, the increase in cold tolerance gained through gradual cooling could not prevent cold shock injuries, as both fecundity and survival were reduced.     

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.     

Differential changes in nucleolar size and ribosomal RNA synthesis during diapause break by prolonged chilling in Bombyx eggs

The activity of ribosomal RNA (rRNA) genes as judged by nucleolar size and rRNA synthesis has been shown to depend upon the phase of diapause in the eggs of Bombyx mori. In the present study, we found that nucleolar size in diapausing eggs was enlarged at a very early stage during cold treatment, a procedure necessary for the termination of diapause. In contrast, the intrinsic capacity of ribosomal RNA synthesis in the chilled eggs, as examined at 25°C by radioactive precursor incorporation into rRNA, increased much later, in parallel with the break of diapause. The early phase of cold treatment is the period when the eggs undergo some important changes (the so-called diapause development), preparing for diapause termination. Thus we infer that the above mentioned increase in nucleolar size may be one of the features of diapause development.