Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria)

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 μg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.     

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.     

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori

Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp) genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis.     

Ecdysteroid and juvenile hormone changes in Bombyx mori eggs, related to the initiation of diapause

We describe a method for the routine determination of changes in juvenile hormone levels in insect eggs. The hormones are first converted into their diol derivatives, then they are purified from other lipids and separated by high-performance liquid chromatography (HPLC). The radioimmunoassay of the fractions was then determined. The method permits the simultaneous assay of ecdysteroids, and it was used for determining the hormonal changes in Bombyx eggs during the pre-diapause development. Our major finding is that the hormonal content of eggs dramatically increased prior to the initiation of diapause. This hormonal rise included ecdysone, 20-OH-ecdysone and 3 juvenile hormones. The HPLC retention time of the latter corresponded to JH₁ JH₂ and JH₃. Subsequently, the embryos entered diapause and the hormonal content of eggs was reduced to traces of ecdysteroids. These dramatic changes in juvenile hormone levels during early embryogenesis raise a number of issues which are developed in the discussion.     

Diapause hormone in Bombyx eggs and adult ovaries

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

Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm,Bombyx mori

Summary Diapause eggs of the silkworm, Bombyx mori, exposed to 5°C and 0.5°C from 2 or 30 days after oviposition, were examined for changes in contents of glycogen, sorbitol and glycerol. Cold acclimation did not alter the profile of accumulation of sorbitol from that in eggs kept continuously at 25°C. However, acclimation at 5°C resulted in conversion of sorbitol to glycogen, while acclimation at 0.5°C was not accompanied by the utilization of sorbitol. NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) activity was examined in the cold-acclimated eggs. The activity was induced by acclimation at 5°C but not at 0.5°C. Incubation at 0.5°C suppressed any further increase in the activity that had been induced. Temperature-directed changes in NAD-SDH activity paralleled those in sorbitol content. Hatching of the diapause eggs was monitored after cold acclimation for various periods of time and subsequent transfer to 25°C. Incubation at 0.5°C was less effective than 5°C at breaking diapause. The time required for the eggs to hatch in synchrony after acclimation at 5°C coincided with that required for the induction of NAD-SDH activity. These results show that different effects result from acclimation at 5°C and near 0°C with respect to the control of NAD-SDH activity, that utilization of sorbitol is controlled by NAD-SDH activity, and that induction of this activity is temperature-dependent. Furthermore, induction of NAD-SDH activity is involved in the termination of diapause in B. mori.Abbreviations DH diapause hormone - NAD nicotinamide-adenine-dinucleotide - NAD-SDH NAD-sorbitol-dehydrogenase     

Induction of non-diapause eggs in Bombyx mori by a trehalase inhibitor

The effect of validoxylamine A (VAA), a potent and specific trehalase inhibitor, on the induction of non-diapause in Bombyx mori was examined. The VAA induced non-diapause eggs and prevented the glucogen accumulation in the eggs. Trehalase activity of the pupal ovary was effectively inhibited by the VAA injection.     

Polyol metabolism related to diapause in Bombyx eggs: Different behaviour of sorbitol from glycerol during diapause and post-diapause

Metabolic relationships between glycogen, sorbitol and glycerol were studied through the entire period of diapause in silkworm eggs which had been labelled with ¹⁴C-glucose injected during oögenesis. Modified instant thin layer chromatography was suitable for the separation of the carbohydrate components by its rapidity and simplicity and it was confirmed that glycogen, sorbitol and glycerol were then main carbohydrate components of the eggs.At the initiation and termination of diapause an intimate interconversion between glycogen and sorbitol was demonstrated by the coincident and complementary changes and the comparable specific radioactivity. However, the temporal changing pattern of glycerol was different from that of sorbitol: a delayed accumulation occurred through diapause and a prolonged persistence remained after diapause termination. Furthermore, radioactivities per C-atom of glycerol were shown to be constant throughout the entire period of diapause but slightly lower than those of sorbitol.From these results the metabolic correlation and physiological roles of sorbitol and glycerol are discussed in relation to diapause phenomenon in silkworm eggs.     

Dual control of midgut trehalase activity by 20-hydroxyecdysone and an inhibitory factor in the bamboo borer Omphisa fuscidentalis Hampson

During larval diapause lasting 9 months from September to May, trehalase activity in the midgut of the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae) was low from December to April, followed by a fourfold increase in May that remained high during the pupal stage in July. An application of juvenile hormone analog (JHA) produced increases in the ecdysteroid titer, while trehalase activity was increased by both JHA and 20-hydroxyecdysone (20E) injection. The trehalase activity in the midgut of diapausing larvae was doubled by incubating the midgut with 20E for 48h. During diapause as well as after JHA application, expression of two ecdysone receptor isoform genes (EcR-A and EcR-B1) in the midgut increased simultaneously with the increase in hemolymph ecdysteroid titer, followed by an increase in trehalase activity. The hemolymph of diapausing larvae contained a trehalase inhibitor and inhibitory activity was high during diapause. After 20E injection, trehalase inhibition decreased as midgut trehalase activity increased. Taken together, at least two factors may participate in the change in midgut trehalase activity: increase in trehalase activity and decrease in trehalase inhibitor activity, both of which may be induced by 20E.     

Diapause development in Bombyx eggs in relation to ‘esterase A’ activity

Diapause is broken by hydrochloric acid treatment and also terminated by long chilling of eggs in the silkworm, Bombyx mori. One of esterases in silkworm eggs named ‘esterase A’ is closely related to diapause of this insect.Hydrochloric acid treatment of diapause eggs induced a prompt elevation of esterase A activity. The elevation was observed within 30 min after treatment. This HCl treatment effectively stimulated the eggs to hatch. This indicates that the increase of esterase A activity is correlated with an active resumption of morphogenesis.The question was examined of whether chilling also increases esterase A activity or not. It was found that chilling also caused an increase of esterase A activity. This increase occurred before the re-appearance of glycogen in eggs, which indicates the termination of diapause in this insect. In fact, the establishment of hatchability in chilled eggs was observed after esterase A activity has reached the maximum level. Thus the increase of esterase A activity could be regarded as associated with the termination of diapause per se but not with the subsequent process of post-diapause development. This change in esterase A activity was observed only in chilled diapause eggs and was not observed in diapause eggs without chilling and non-diapause eggs.These results suggest that the increase of esterase A activity during chilling may be a kind of activity that occurs during the diapause stage in preparation for resumption of morphogenesis or diapause development.     

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.     

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.     

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.     

Diapause hormone action in silkworm ovaries incubated in vitro; 14C-trehalose incorporation into glycogen

Developing ovaries from pharate adults of the silkworm, Bombyx mori, were incubated in a medium containing ¹⁴C-trehalose or ¹⁴C-glucose, and the effects of diapause hormone on the incorporation of these isotopes into ovary glycogen were studied. The rates of incorporation of ¹⁴C-trehalose remained unchanged in ovaries incubated for 36 hr when the medium was renewed at intervals of 12 hr, and showed saturation kinetics against the concentration of the sugar in the medium, giving apparent K_m values of 6·0 mM for trehalose.There was no difference in ¹⁴C-glucose incorporation by ovaries grown in the presence (+SG) and absence (?SG) of the suboesophageal ganglion (SG). However, when ¹⁴C-trehalose was used as a substrate for glycogen synthesis, there was a marked difference in the incorporation between them, i.e. the incorporation was more than 60 per cent higher in +SG ovaries than that in ?SG ovaries. Increased incorporation of ¹⁴C-trehalose was also observed in ovaries from SG-removed pharate adults which received an injection of diapause hormone preparations. Maximum stimulation rates (about twofold) appeared 36 hr after the injection. Further, comparable effects on ¹⁴C-trehalose incorporation were observed in ovaries which were incubated with diapause hormone preparations added in vitro.These data are discussed in relation to the hormonal regulation of trehalase activity in ovaries.     

即时浸酸显著降低滞育家蚕卵的还原型与氧化型谷胱甘肽比值

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 显著提高了家蚕卵H₂O₂含量。还原型谷胱甘肽（reduced glutathione, GSH）与氧化型谷胱甘肽（oxidized glutathione, GSSG）的比值是一种氧化胁迫状态的动态指标。为了调查即时浸酸是否造成滞育家蚕卵氧化胁迫, 本研究利用分光光度法分别测定了滞育家蚕卵和5 min即时浸酸滞育家蚕卵中GSH和GSSG含量以及谷胱甘肽转移酶（glutathione-S-transferase, GST）活性。结果表明: 处理后24 h, 即时浸酸处理家蚕卵的总谷胱甘肽（GSH+2GSSG）含量、 GSH含量、 GSSG含量、 GSH/GSSG比值和GST活性分别相当于同期滞育家蚕卵的204%, 78%, 550%, 14%和97%。据此推测, 即时浸酸在阻止滞育发动的同时, 可能通过促进GSH氧化为GSSG, 而显著降低了GSH/GSSG比值, 使家蚕卵处于过氧化状态。     

海藻糖酶基因TRE2-like和TRE2在异色瓢虫羽化过程中的表达与功能

【目的】异色瓢虫Harmonia axyridis是一种重要的捕食性天敌昆虫,海藻糖在异色瓢虫的变态发育、羽化等整个生命过程都起着重要的作用。本研究以前期获得的类似膜结合型海藻糖酶(TRE2-like)与膜结合型海藻糖酶(TRE2)基因为基础,探讨在异色瓢虫羽化阶段这两个海藻糖酶的潜在功能,为阐明异色瓢虫从蛹发育到成虫时海藻糖代谢机制提供参考。【方法】根据TRE2-like和TRE2基因序列设计双链RNA(dsRNA)区域片段并合成对应的dsRNA,通过RNAi将其注射到异色瓢虫2日龄蛹中。采用实时荧光定量PCR(RT-qPCR)检测RNAi处理后羽化第1天的异色瓢虫成虫糖代谢相关基因的表达;同时采用蒽酮比色法、酶标法等分别测定RNAi处理后羽化第1天的异色瓢虫成虫主要糖类物质含量及TRE活性变化,并观察异色瓢虫羽化后的表型变化。【结果】结果表明,与对照组(dsGFP注射组)相比,异色瓢虫2日龄蛹被注射TRE2-like或TRE2 dsRNA后,其新羽化成虫体内TRE2-like和TRE2表达量均极显著下调,且少数个体出现了蜕皮与翅形成困难等畸形表型。可溶性海藻糖酶活性在注射dsTRE2-like后显著降低,膜结合型海藻糖酶活性在注射dsTRE2后显著降低;注射dsTRE2后糖原含量显著下降,注射dsTRE2-like后糖原和海藻糖含量显著下降,注射dsTRE2-like+dsTRE2后糖原和葡萄糖含量显著下降,且海藻糖含量极显著下降。注射dsTRE2-like, dsTRE2和dsTRE2-like+dsTRE2后可溶性海藻糖酶基因TRE1-1和TRE1-2表达下降或显著下降,而TRE1-5表达上升或显著上升,海藻糖合成酶(trealose-6-phosphate synthase, TPS)、糖原磷酸化酶(glycogen phosphorylase, GP)、糖原合成酶(glycogen synthase, GS)基因的表达均显著下调。【结论】TRE2-like和TRE2基因表达被抑制后,异色瓢虫海藻糖等代谢受到影响。研究结果为探究异色瓢虫体内膜结合型海藻糖酶的潜在功能和调控机制奠定了基础。     

Expression of metabolic enzyme genes and heat-shock protein genes during embryonic development in diapause and non-diapause egg of multivoltine silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

The expression of metabolic enzyme genes and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm Bombyx mori was quantified by semi-quantitative RT-PCR. The trehalase gene (Tre) was expressed in non-diapause eggs up-to nine days, while in diapause eggs was not up regulated. The glycogen phosphorylase gene (GPase) was expressed in non-diapause eggs, whereas in diapause eggs a high level was observed in early stage, but down regulated in later stage. The phosphofructokinase gene (PFK) and sorbitol dehyrogenase-2 gene (SDH-2) expression was fluctuated in non-diapause eggs, whereas in diapause eggs these were expressed only at early stage and not observed in later stage. The glucose-6-phosphate dehydrogenase gene (G6P-DH) in non-diapause eggs was highly expressed during the differentiation phase and decreased in the organogenesis phase. In contrast to this, expression in diapause eggs was of low level during differentiation phase and of high level observed in the organogenesis phase. In the tissues, PFK and SDH-2 were selectively expressed in cuticle and midgut, whereas Tre expression was high in midgut and ovary of larvae incubated at 15°C. The Hsp (20.4, 20.8, 40, 70, and 90) were expressed in both diapause and non-diapause eggs. Their expression was, however, selective in tissues with Hsp20.4 in midgut and ovary, Hsp40 in head, Hsp70 in cuticle and Hsp90 in ovary and head in high amounts at 15°C. These results suggest that the metabolic enzyme genes studied except Hsp play a major role during embryogenesis of diapause and non-diapause silkworm.     

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.     

Metabolism of hydrogen peroxide between diapause and non‐diapause eggs of the silkworm,Bombyx Mori during chilling at 5°C

When diapause and non‐diapause eggs of the same bivoltine strain of Bombyx mori were chilled at 5°C for more than 30 days, the hatchability of diapause eggs increased while that of non‐diapause eggs decreased, respectively. To investigate the relationship between effects of chilling on the hatchability and the metabolism of hydrogen peroxide (H₂O₂), content of H₂O₂ and activities of superoxide dismutase (SOD), xanthine oxidase (XO), and catalase (CAT) between diapause and non‐diapause eggs were determined during the chilling at 5°C. The significant enhancement of H₂O₂ occurred prior to the quick increase of the hatchability in diapause eggs and coincided with the quick decline of the hatchability in non‐diapause eggs, respectively. Diapause eggs contained significantly higher H₂O₂ and XO activity and lower CAT activity compared to non‐diapause eggs. Our results showed that there were significant differences in the metabolism of H₂O₂ between diapause and non‐diapause eggs during chilling and that significant enhancement of H₂O₂ may be involved in the diapause termination of diapause eggs and the cell damage of non‐diapause eggs. © 2010 Wiley Periodicals, Inc.