Expression of heat shock protein 70a mRNA in Bombyx mori diapause eggs

In an effort to understand whether heat shock protein 70 (Hsp70) participates in the environmental 5 °C signal reception/transduction toward breaking embryonic diapause of the silkworm Bombyx mori, we isolated a cDNA for Hsp70a and examined the expression of Hsp70a mRNA in B. mori diapause and nondiapause eggs by quantitative real-time PCR. Hsp70a mRNA gradually increased in diapause eggs continuously kept at 25 °C after oviposition to maintain diapause. When diapause eggs were exposed to the diapause-terminating condition of 5 °C beginning at 2 days post-oviposition, Hsp70a mRNA increased beginning at 5 days post-cold treatment. Even in nondiapause eggs, Hsp70a mRNA increased slightly with exposure to 5 °C. These results suggest that Hsp70a is involved in reception/transduction of the diapause-terminating (5 °C) signal via gene activation. The expression patterns of Hsp70a mRNA are discussed in relation to those of the cold-response gene Samui.     

Carbohydrate metabolism and restricted oxygen supply in the eggs of the silkworm, Bombyx mori

Increased oxygen supply to diapause eggs of the silkworm (O₂-incubation) effectively prevented diapause initiation and induced the same pattern of glycogen, polyol and lactate levels as was observed in normal non-diapause eggs. Sensitivity to oxygen decreased as embryonic development proceeded. After the termination of this sensitive period, accumulation of polyols and lactate followed.Experiments were carried out to test whether changes in the oxygen permeability of the egg membranes are involved in restricting the supply of this gas to eggs at the onset of diapause. Oxygen permeability of the chorion was measured with apparatus especially designed for this purpose. Although the chorion of the diapause egg was less permeable than that of the non-diapause egg, the oxygen permeability of the chorion does not change appreciably during the early developmental stages of the diapause eggs. The changes in rate of water loss through the egg membranes were measured during the early developmental stages of the embryos. The level of water loss decreased gradually as the formation of serosal cuticle proceeded. Moreover, it was observed that the water loss up to the time of formation of serosal cuticle was closely related to the oxygen permeability of the chorion.From these results, we suggest that the formation of the serosal cuticle may be an additional cause of the restricted oxygen supply at the onset of the 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.     

In vitro release of lipophorin from the fat body of nondiapause and diapause larvae of the Southwestern corn borer,Diatraea grandiosella

The release of lipophorin and total protein was examined from the fat body of nondiapause and diapause larvae of the southwestern corn borer, Diatraea grandiosella, incubated in vitro in Grace's medium. The characteristics of the released lipophorin were compared to those of the high-density lipophorin present in the hemolymph of nondiapause and diapause larvae. Over a 4 h incubation period, the fat body of nondiapause larvae released about 1.5 times more total protein and 2 times more lipophorin per mg dry weight than did that of diapause larvae. Lipophorin isolated from the medium in which fat bodies of nondiapause and diapause larvae had been incubated and from the plasma of nondiapause and diapause larvae had similar mean densities of 1.115, 1.112, 1.117 and 1.119 g/ml, respectively. Although the lipid classes detected in lipophorin isolated from the fat body incubation medium and hemolymph were identical, more polar lipids and less diacylglycerol were associated with lipophorin isolated from fat body incubation medium then were associated with lipophorin isolated from the hemolymph. Sterols accounted for about 11% of the total lipids of lipophorin isolated from the fat body incubation medium, whereas they accounted for about 20% of the total lipids of lipophorin from hemolymph. We conclude that the fat body of feeding nondiapause larvae and nonfeeding diapause larvae releases high-density lipophorin.     

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.     

不同催青方式对二化性家蚕过氧化氢酶基因表达的影响

25℃明催青和15℃暗催青分别诱导二化性家蚕Bombyx mori 产滞育性卵和非滞育性卵。此前我们的研究表明, 上述催青处理的二化性家蚕H₂O₂水平存在显著差异。过氧化氢酶（catalase, CAT）是昆虫清除H₂O₂的关键酶。为了进一步明确家蚕滞育过程中H₂O₂代谢的调控机制, 用RT-PCR测定了上述两种催青处理对二化性家蚕CAT基因表达的影响。结果表明：25℃明催青显著提高了滞育诱导和决定阶段的CAT mRNA 水平和CAT活性。滞育性卵的CAT mRNA水平在产后24 h形成峰值, 在72 h后消失; CAT活性在96 h前上升, 120 h后保持于低水平。非滞育性卵的CAT mRNA水平和CAT活性都随着胚胎发育而上升。可见, 25℃明催青诱导二化性家蚕子代滞育可能是通过影响CAT基因表达来调节H₂O₂水平。     

Environmental control of the seasonal life cycle of a zoophytophagous mirid, <Emphasis Type="Italic">Adelphocoris triannulatus</Emphasis> (Hemiptera: Miridae)

Adelphocoris triannulatus (Stål) (Heteroptera: Miridae) is a zoophytophagous bug that inhabits grasslands and crop fields, and is widely distributed in eastern Asia. In this study, the seasonal occurrence and environmental factors controlling the seasonal life cycle of this bug were investigated in a local population in southern Kyushu, Japan. When insects were reared under different photoperiods at 20 and 25 °C, they produced diapause or nondiapause eggs depending on the photoperiod: when reared under short-day photoperiods (≤?13 h/day), females laid diapause eggs, whereas they laid nondiapause eggs when reared under long-day photoperiods (≥?14 h/day). Because the developmental duration of the pre-adult stages and maturation (minimum duration of one generation) is estimated to be approximately 30 days at summer temperatures, these results suggest that this bug has a multivoltine seasonal life cycle. In agreement with the laboratory results, only nondiapause eggs were produced until mid-August in the field, and an increasing proportion of diapause eggs were produced from early September. In addition to these findings, field sampling from spring to autumn in 2009 indicated that three or four generations were produced in one year. These results suggest that egg diapause plays a role in preventing nymphs of A. triannulatus from developing in winter, and thereby ensures the survival of the population in adverse seasons.     

Metabolic rate in diapause and nondiapause brown locust eggs correlated with embryonic development

Insects use dormancy to survive adverse conditions. Brown locust Locustana pardalina (Walk.) eggs offer a convenient model to study dormancy (diapause and quiescence), which contributes to their survival under arid conditions. The metabolic rates of developing nondiapause, diapause and quiescent eggs are compared in the present study using closed‐system respirometry. The embryo becomes committed to continue development and hatch or to enter diapause 6 days after the eggs are placed on moist soil. The metabolic rate of nondiapause eggs increases exponentially until hatching, whereas that of diapause eggs is low and stable. The metabolic rate of diapause laboratory eggs (1.9 ± 0.6 µL CO₂ mg^?1 h^?1) is significantly higher than that of field eggs (0.5 ± 0.3 µL CO₂ mg^?1 h^?1), although the ranges of metabolic rate overlap and the embryos are all in late anatrepsis. The metabolic rate of quiescent eggs is similar to that of diapause eggs but decreases with time. Low metabolic rates during arrested development allow eggs to persist over long periods before hatching.     

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.     

Oxygen uptake in fowl eggs incubated in air and pure oxygen

1. The oxygen uptake of small (bantam hen) and large (white leghorn) domestic fowl eggs was measured during incubation in air and pure O2. 2. A 2 hr exposure to pure O2 increased the O2-uptake from the 9th day in the large eggs and from the 15th day in the small eggs. 3. On the last 4 days of the incubation period, both small and large eggs increased their O2-uptake by about 22% when transferred from air to pure O2. 4. When 1 cm2 of the eggshell above the air cell was removed, the O2-uptake of the white leghorn eggs incubated in air increased by 13%.     

Esterases in relation to embryonic diapause in the field cricket,Teleogryllus commodus

Fourteen esterase isozymes were detected by polyacrylamide gel electrophoresis in homogenates of eggs of the field cricket Teleogryllus commodus, at various stages of development. One esterase isozyme appeared during embryonic diapause termination effected by prolonged chilling of the eggs.Esterase activity towards methyl butyrate in T. commodus egg homogenates increased with time. Sonication generated the maximum levels of esterase activity observed. Methyl butyrate esterase activity in T. commodus eggs was high during the first few days of embryogenesis; fell markedly during the period of rapid water uptake, and then remained fairly constant during diapause, diapause development, and during resumed embryogenesis.     

Genome‐wide microarray screening for Bombyx mori genes related to transmitting the determination outcome of whether to produce diapause or nondiapause eggs

The bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae) exhibits a maternally controlled embryonic diapause. Maternal silkworms decide whether to lay diapause or nondiapause eggs depending on environmental factors such as the temperature and photoperiod during the egg and larval stages, and then induce diapause eggs during the pupal stage. However, little is known about the molecular mechanism that conveys the outcome of whether to produce diapause or nondiapause eggs from the egg or larval stages to the pupal stage. This study used microarray analysis to investigate differentially expressed genes in the larval brains of diapause‐ and nondiapause‐egg producers, to which bivoltine silkworms were destined by thermal or photic stimulation during the egg stage. The cytochrome P450 18a1 and Krüppel homolog 1 genes were upregulated in producers of diapause eggs compared with those of nondiapause eggs under both experimental conditions. Cytochrome P450 18a1 encodes a key enzyme for steroid hormone inactivation and Krüppel homolog 1 is an early juvenile hormone‐inducible gene that mediates the repression of metamorphosis. The upregulation of these genes during the larval stage might be involved in the signaling pathway that transmits information about the diapause program from the egg stage to the pupal stage in the silkworm.     

水浸对越北腹露蝗卵的影响

越北腹露蝗Fruhstorferiola tonkinensis Will.是广东北江流域河滩蝗虫的优势种。以不浸水的蝗卵为对照,研究滞育期蝗卵浸水5～95d和胚胎发育期蝗卵浸水1～30d对卵孵化率和历期的影响。结果表明,随浸水时间的增加,孵化率下降,卵发育延迟;滞育期蝗卵经受95d的浸水仍有8.01%存活,而胚胎发育期蝗卵浸水30d后全部死亡,滞育期蝗卵比胚胎发育期蝗卵具有更强的耐浸水能力。说明越北腹露蝗以卵滞育的形式发展了对水浸胁迫的适应。     

The amino acid pool of Bombyx mori eggs during diapause

The levels of alanine and glutamine in the amino acid pool of Bombyx mori eggs changed significantly after oviposition. In diapause eggs the alanine content increased by about 8-fold between 24 and 48 hr after oviposition to reach 42.7 μmol/g. In non-diapause eggs, however, alanine levels remained constant over a similar period. In contrast the glutamine content remained constant at less than 0.5 μmol/g in diapause eggs but increased progressively in non-diapause eggs. At the end of diapause, the alanine content decreased and the glutamine content approached the same level as in non-diapause eggs. The glutamate level did not change at the initiation or termination of diapause, but the proline content increased markedly at the latter time. These results suggest that alanine accumulation at the initiation of diapause might serve a protective function similar to that of polyols. De novo alanine synthesis may require protein breakdown to provide the needed amino groups; at the termination of diapause, however, alanine might be converted to proline.     

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.     

The role of juvenile hormone in the larval diapause of the European corn borer,Ostrinia nubilalis

The possible role of juvenile hormone (JH) in the induction and termination of larval diapause in the European corn borer, Ostrinia nubilalis, was investigated using topical applications of both JH I and a JH mimic as well as by monitoring JH titers with the Galleria bioassay. Neither JH nor the JH mimic ZR515 was capable of influencing diapause termination when administered topically. The Galleria bioassay revealed little or no JH in the hemolymph of mid diapause (>30 days) insects, indicating no demonstrable role for JH in diapause maintenance. When ZR515 was administered to nondiapause, newly ecdysed fifth instar larvae the pupal molting cycle was delayed. By use of photoperiodic regimes we were able to show that the molting delay was not equivalent to diapause induction. The Galleria bioassay showed differences in JH titer profiles between diapause and nondiapause animals during the final larval stadium. The nondiapause insects showed titers that decline rapidly to trace amounts following the molt to fifth instar then rose prior to pupation. The diapause insects had generally higher titers and exhibited a more gradual decline after the molt. No evidence was obtained to support the hypothesis that JH plays a key role in the induction, maintenance, or termination of larval diapause.     

The eggshell structure in apteryx; form,function, and adaptation

Apteryx is a genus of flightless birds endemic to New Zealand known to lay very large eggs in proportion to body weight. The eggshell of Apteryx is unusually thin and less porous than allometrically expected possibly as a compensation for a very long incubation period. Past studies have been carried out on Apteryx australis, a species which once comprised all kiwi with brown plumage, now separated into three distinct species. These species use different habitats and live at different latitudes and altitudes, therefore generating a need to revise our knowledge of the attributes of their eggshells. In this study, we measured the physical characteristics and water conductance on eggshell fragments of these three species and Great‐spotted Kiwi and relate them to the environmental conditions of their respective environments; we also measured the water vapor conductance of Brown Kiwi eggs of late stages of incubation. We found that several trade‐offs exist between incubation behavior, environmental conditions, and eggshell structure. We found differences between species in eggshell water vapor conductance seemingly related to altitude; Brown Kiwi and Rowi generally inhabiting lower altitudes had the highest conductance and Tokoeka, generally living in montane environments, the lowest. This is achieved by an increased eggshell thickness rather than a pore area reduction. Finally, the water vapor conductance late in incubation was 58% higher than infertile unincubated eggs, suggesting a drastic increase in conductance throughout the long incubation period. Using the values previously reported, we calculated the embryonic eggshell thinning to be 32.5% at the equatorial region of the eggshell. We describe several new features, such as triangular mineral particles in the cuticle, reported for the extinct Trigonoolithus amoei, and confirmed the existence of plugged pores. We suggest that these structures provide microbial protection needed by a burrow nesting species with a long incubation period.     

Regulation of juvenile hormone esterase activity in the European corn borer, Ostrinia nubilalis

The regulation of juvenile hormone esterase in last-instar diapause and nondiapause larvae of Ostrinia nubilalis was investigated using topically applied juvenile hormone I and a juvenile hormone mimic, methoprene. The influence of the head on juvenile hormone esterase was also investigated. Both juvenile hormone and methoprene caused increases in esterase levels when applied to feeding animals. Neither the hormone nor methoprene was capable of elevating nondiapause esterase activity to levels comparable to those found in untreated prediapause larvae. The esterase levels could be elevated in the larval body, without the head, during prepupal development of nondiapause larvae and in post-feeding diapause larvae. In both cases, juvenile hormone or methoprene induced juvenile hormone esterase activity in head-ligated animals. Topically applied methoprene prolonged feeding and delayed the onset of diapause. When methoprene was applied to larvae that had entered diapause, it disrupted diapause by inducing a moult.     

Haemolymph ecdysteroid titres in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis

The ecdysteroid titres of last-instar prediapausing, diapausing and nondiapausing larvae of Ostrinia nubilalis were determined by radioimmunoassay. In the nondiapause larvae a major peak of ecdysteroid activity preceded pupation by 24 h and continued through the pupal ecdysis. This peak was correlated with head and thorax critical periods as well as with changes in behaviour and physiology marking the transition from feeding larva to prepupa. Nondiapause larvae also displayed a rise in ecdysteroid titre during the feeding phase of development. This rise was approx one tenth that of the major peak and lasted 32 h. It was not correlated with any overt changes in larval physiology or behaviour. The diapause ecdysteroid profile was distinctive in that the levels measured were all lower than the lowest of the nondiapause curve. No peaks were observed in the diapause titres. Prepupal changes such as spinning and the cessation of feeding were not correlated with any increase in ecdysteroid levels. During diapause termination, under a long-day photoperiod, no increases in ecdysteroid titre were observed for the first 10 days. After 12 days individuals began to show ecdysteroids above the diapause levels. Pupation started after 16 long days.