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.     

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 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.     

Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori

Recently, it was confirmed that embryos derived from diapausing eggs of the silkworm, Bombyx mori, begin their development and reach larval maturity on mulberry leaves, when the naked eggs are cultured in vitro. In this study, we found that the method of embryo culture is useful for determining the physiological regulation of diapause. We show that the development of embryos derived from diapausing eggs was strongly inhibited by the addition of either sorbitol or trehalose to the culture medium. Furthermore, this inhibitory effect disappeared when the embryos were cultured in a control medium which did not contain either sorbitol or trehalose, indicating that the inhibitory reactions caused by both substances are reversible. The minimal effective dose of either sorbitol or trehalose was approximately 0.2 M, a value similar to the in vivo concentration of sorbitol in diapausing eggs (0.2 M). Glycerol, mannitol or glucose were moderately effective for inhibition. Sorbitol present in diapausing silkworm eggs does not appear to serve as an antifreeze, but as an strong arresting factor of embryonic development. Furthermore, these results show that a decrease in sorbitol releases the embryos from diapause at the termination of diapause.     

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.     

CARBOHYDRATE METABOLISM IN THE EGGS OF THE SILKWORM, BOMBYX MORI. I. ABSENCE OF PHOSPHOFRUCTOKINASE IN THE DIAPAUSING EGG

In the diapausing eggs of the silkworm, Bombyx mori , glycogen is rapidly converted to sorbitol and glycerol, and this conversion is reversed at termination of the diapause (C hino , 1958). To elucidate the pathway leading to this polyol formation and its regulatory mechanisms, enzymes concerning carbohydrate metabolism were surveyed in diapausing as well as in developing eggs of the silkworm.
Most of the enzyme activities concerning citric acid cycle are low at the beginning of the embryogenesis and during diapause, but increase at the later stages of the development. Making an exception, reduction rate of malate and fumarate was rather high from the onset of the embryonic development. Several glycolytic enzymes were also studied. Most remarkable fact is that phosphofructokinase activity could not be demonstrated in the diapausing and also in the early stages of the developing eggs. Other enzymes, viz. α-glycerophosphate dehydrogenase, aldolase, glyceraldehyde-3-phosphate dehydrogenase were detected from the beginning of the embryogenesis.
Absence of phosphofructokinase, together with the high activity in glucose-6-phosphate dehydrogenase, suggests that predominant pathway in carbohydrate metabolism in the early stages of embryogenesis and in the diapause period is by way of pentose phosphate pathway. This supposition is confirmed by the experiments using labeled glucose. Incorporation of the label into glycerol of the diapausing eggs was three to four fold when G-6-¹⁴C was injected into pupae as compared with the case of G-1-¹⁴C injection. The above experiments provide evidence supporting the theory that glycogen is converted into sorbitol and glycerol mostly by way of the pentose phosphate pathway in the diapausing eggs.     

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.     

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.     

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.     

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.     

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.     

即时浸酸显著提高滞育性家蚕卵辅酶Ⅰ和Ⅱ含量

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 提高了其呼吸耗氧量, 抑制了山梨醇积累。本研究利用HPLC法测定了家蚕滞育卵和5 min即时浸酸滞育性卵中辅酶Ⅰ和Ⅱ含量。结果表明： 产下后24-72 h, 家蚕滞育卵中NAD, NADH, NADP和NADPH含量分别下降了30%, 37%, 50%和4%; 而即时浸酸滞育性卵中分别增加了77%, 46%, 142%和241%。不过, 即时浸酸并未显著改变滞育性家蚕卵中NADH/NAD和NADPH/NADP比值。据此推测, 即时浸酸提高滞育性家蚕卵辅酶Ⅰ含量与其呼吸耗氧量增加有关; 即时浸酸显著提高辅酶Ⅱ含量与山梨醇积累抑制无关, 而主要与生物合成加强有关。     

The origin of free glycerol accumulated in diapause eggs of Bombyx mori

ABSTRACT. The origin of free glycerol in diapausing eggs of Bombyx mori was examined using ¹⁴C-glycerol as a tracer. When ¹⁴C-glycerol was injected into 5-day-old female pharate adults to label the egg lipids, radioactivity was recovered in glycogen as well as in total lipids in newly laid eggs, but the specific radioactivity in lipid glycerol was 2.5 times higher than that in glycogen. When eggs entered diapause, radioactivity in glycogen decreased rapidly and a concomitant accumulation of sorbitol occurred, implying the conversion of glycogen into sorbitol at the initiation of diapause. Radioactivity in total lipids decreased slowly but significantly, and in free glycerol increased steadily during the diapause period. The sum of recovered radioactivity in these components remained almost constant throughout diapause. The specific radioactivity (cpm/C-atom) of free glycerol was clearly lower than that of lipid glycerol but higher than that of glycogen or sorbitol. It was calculated that about 30% of free glycerol was derived from lipid glycerol in the diapausing eggs.     

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 sorbitol utilization in diapause eggs of the silkworm,Bombyx mori

Summary A series of experiments was conducted to determine whether the interconversion of glycogen and sorbitol at the initiation and termination of diapause in eggs of the silkworm,Bombyx mori was influenced by low temperatures (5°C and 1°C). The conversion of glycogen to sorbitol and glycerol at the initiation of diapause was not affected by exposure to 5°C and 1°C. Chilling diapause eggs at 5°C for over 70 days resulted in a progressive conversion of sorbitol to glycogen. Transfer to 1°C after chilling to 5°C led to a decrease in sorbitol and glycerol conversion. While continuous chilling at 1°C completely inhibited sorbitol utilization for at least 400 days, sorbitol began to decrease immediately following transfer to 5°C. In contrast, glycerol utilization was not prevented by acclimation to 1°C, but a delayed decrease occurred. Continuous exposure to 1°C delayed hatching for a period of 440 days, whereas acclimation to 5°C resulted in 100 per cent hatching by about 100 days. A low, or high acclimation temperature 1°C or 5°C produce different effects on sorbitol utilization and termination of diapause in silkworm eggs.     

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.     

Environmental and physiological factors for diapause induction and termination in the aphid parasitoid,Aphidius ervi (Hymenoptera: Aphidiidae)

In the aphid parasitoid Aphidius ervi Haliday, the incidence of diapause around the critical photoperiod was dependent on temperature. At a photoperiod of 12 h light:12 h dark, a significantly higher proportion of diapausing individuals was found at 12 degrees C than at 15 degrees C. The second larval stage of the parasitoid was most sensitive to photoperiod and localised-illumination experiments showed that the parasitoid responded independently of the aphid host. The sex ratio of the population which emerged after rearing in short-days and/or after diapause was male biased. Topical application of the juvenile hormone-mimic, pyriproxyfen, before or during diapause delayed emergence of Aphidius ervi but did not induce diapause or influence its termination. The ecdysteroid mimic RH 5849 induced diapause in a few treated individuals but did not affect diapause termination.     

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.