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.     

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.     

Developmental changes in the localization of protein kinase CK2 in non-diapause and diapause eggs of the silkworm, Bombyx mori

To analyze the role of protein kinase CK2 (CK2) during early embryogenesis in non-diapause and diapause of the silkworm, the distribution and localization of Bombyx mori CK2 (BmCK2) were investigated by an immunohistochemical technique using antibodies against the α- and β-subunits of BmCK2. Both were localized in blastoderm cells of non-diapause and diapause eggs until 24 h after oviposition. More than 24 h after oviposition, however, the distribution of BmCK2 was different in non-diapause and diapause eggs. In non-diapause eggs, BmCK2 was mainly localized in yolk cells. In contrast, in diapause eggs, the localization was mainly observed in germ-band cells. Furthermore, we confirmed that the RNA helicase-like protein that was localized together with BmCK2 in non-diapause eggs was phosphorylated by BmCK2 in vitro. These data suggest that the role of BmCK2 is different in non-diapause and diapause eggs.     

A novel RNA helicase-like protein during early embryonic development in silkworm Bombyx mori: molecular characterization and intracellular localization

In order to understand the molecular mechanism of development during early embryogenesis in diapause and non-diapause of the silkworm, mRNA from diapause and non-diapause eggs was compared using the differential display technique. We cloned the full length of a cDNA encoding a novel RNA helicase-like (RHL) protein by the RACE method using a cDNA fragment which was one of the specific cDNAs in the non-diapause eggs. A BLAST search using the predicted amino acid sequence of RHL revealed a low homology (21–25% identity of its partial length) with that of the DEAD-box RNA helicase. Gene expression of the RHL gene of the diapause and non-diapause eggs was investigated by RT-PCR until 60 h after oviposition. Amplified RHL cDNA was observed through all the stages in the non-diapause eggs, while in the diapause eggs, cDNA was found in eggs 0–12 h after oviposition but disappeared 24–60 h after oviposition. When the diapause eggs were activated by HCl treatment after chilling at 4 °C for 6 days from 48 h after oviposition (artificial diapause termination), cDNA was observed from 12 h after HCl treatment. We also investigated the immunohistochemical distribution and localization of RHL in non-diapause eggs using anti-recombinant His-tag RHL antiserum. RHL was distributed in blastoderm cells and yolk cells and was localized in the nucleus and the cytosol of yolk cells. These data suggest that RHL has an important role in the early embryo of the silkworm.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Metabolism of maternal ecdysteroid-22-phosphates in developing embryos of the desert locust,Schistocerca gregaria

The ecdysteroid composition of Schistocerca gregaria eggs at different stages of development was determined by analysis of ecdysteroids labelled maternally from [4-¹⁴C]cholesterol. At all stages studied, highly polar ecdysteroid derivatives predominated, but changes in their composition occurred between day 10 of development and hatching (day 17). During this period, polar conjugates of ecdysone-3-acetate and 3-epi-2-deoxyecdysone appeared together with ecdysteroid acids. At day 17, the polar conjugate of [¹⁴C]ecdysone-3-acetate represented 36% of the total conjugated steroids. Separate in vivo studies on the metabolism of [¹⁴C]ecdysteroid conjugates isolated from newly-laid eggs and consisting primarily of the 22-phosphates of ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone showed that ecdysteroid phosphates could be hydrolysed to give primarily free ecdysone during embryogenesis. Developing eggs can metabolize [³H]ecdysone to ecdysonoic acid, 3-acetylecdysone-2-phosphate and to a lesser extent ecdysone-22-phosphate and 20-hydroxyecdysonoic acid. A polar conjugate of 20-hydroxyecdysone-3-acetate, possibly the 2-phosphate derivative, was detected as a minor metabolite of ecdysone. A scheme of the proposed pathways involved in the metabolism of ecdysteroid-22-phosphates in the developing eggs of S. gregaria is presented.     

Possible involvement of ecdysteroids in embryonic diapause of Locusta migratoria

In the Ibaraki population (Japan) of Locusta migratoria, adult locusts produce diapause eggs under short-day (SD) conditions and non-diapause eggs under long-day (LD) conditions. The identity and titre of ecdysteroids in the ovaries and eggs from LD and SD adult females were investigated by RIA/HPLC. Maternal ecdysteroids accumulated in the developing ovaries represented about 90% polar conjugates, 5% free ecdysteroids and 5% non-hydrolyzable metabolites. Before oviposition the quantity of ecdysteroids reached 29.8±1.85 ng 20-hydroxyecdysone equiv. per mg tissue ovaries from LD females and 13.1±3.55 ng 20E equiv./mg in ovaries from SD females. The sum of RIA-positive materials in newly laid eggs was more than three times higher in non-diapause eggs than in diapause eggs. Ecdysteroids present in egg extracts comprised about 85% polar conjugates, 5% free ecdysteroids and 10% non-hydrolyzable metabolites. On the other hand, after diapause termination the amount of ecdysteroids increased drastically. Also, the composition of ecdysteroids differed from that observed during diapause and became comparable to that of non-diapause eggs. The significant differences in the ecdysteroids between non-diapause and diapause eggs may suggest the possible involvement of these compounds in the control of embryonic diapause of this locust.     

Studies on the yolk granules of the silkworm,Bombyx mori L.: The morphology of diapause and non-diapause eggs during early developmental stages

Summary The morphological features during development of diapause and non-diapause eggs of the silkworm,Bombyx mori, were investigated by means of light and electron microscopy, with special reference to eggs up to 24 h after oviposition.The blastoderm and yolk cells began to be formed about 6 and 24 h after oviposition, respectively, in both the diapause and non-diapause eggs, indicating that the diapause and non-diapause eggs develop at similar rates at least until 24 h after oviposition.Specific changes in the distribution of yolk granules were observed during early development of the diapause egg. Its yolk granules gradually aggregated into clusters from the periphery toward the inside of the egg during the period of blastoderm formation. Aggregation of yolk granules was most noticeable about 12 h after oviposition and then they dispersed again before yolk cell formation. On the other hand, yolk granules of the non-diapause eggs remained dispersed during development.     

Changes in amino acid pools in the silkworm,Bombyx mori during embryonic life: Alanine accumulation and its conversion to proline during diapause

Alanine accumulated in silkworm eggs at the onset of diapause. When the eggs were kept at 4°C during diapause, this alanine was converted to glutamate, glutamine and especially proline. On resumption of development at 25°C after diapause, proline was used as an energy source for protein synthesis. In HCl-treated diapause eggs, which develop like non-diapause eggs, most amino acids showed similar developmental changes to those in eggs in resumption of embryogenesis after diapause. However, the proline level increased until the middle of embryonic development and then decreased. Continuous incubation of diapause eggs at 25°C after day 10 of oviposition caused a decrease in alanine with increases in glutamine and proline, while the levels of most other amino acids either decreased slightly or remained unchanged until day 80, when most eggs died. These results show that diapause eggs have a metabolic complex coupled with carbohydrate and amino acid metabolism inclusive of the 2-oxoglutarate-glutamate shuttle. Under conditions when embryogenesis proceeded, the level of phosphoethanolamine decreased rapidly.     

Mechanism of hydroxylation at C-22 during the biosynthesis of ecdysteroids in the locust Schistocerca gregaria.

1. The fates of the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol during the biosynthesis of ecdysteroids in the ovaries of Schistocerca gregaria were investigated. 2. Two stereospecifically labelled cholesterol species, obtained by incubating 3R,2R- and 3R,2S-[2-14C, 2-3H]mevalonic acid with rat liver preparations, were administered, in turn, to maturing adult female locusts and the radiolabelled ecdysteroid conjugates isolated from the eggs. Enzymic hydrolysis of the conjugates yielded free ecdysteroids, from which ecdysone was purified. 3. Derivative formation and oxidation at C-22 of both ecdysone samples indicated that the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol were stereospecifically eliminated and retained respectively during ecdysteroid formation. This indicates that C-22 hydroxylation in ecdysone biosynthesis is direct and occurs with retention of configuration.     

The effects of temperature and moisture on the inception of diapause in eggs of the Australian plague locust,Chortoicetes terminifera Walker (Orthoptera: Acrididae)

Chortoicetes terminifera lays a mixture of diapause and non-diapause eggs during the autumn months of March and April. Diapause eggs cease development in late anatrepsis when kept in moist soil at 32.5°C. These conditions favour rapid embryogenesis and hatching in non-diapause eggs. Significant reductions in the incidence of diapause were obtained when newly-laid eggs were kept for 40 days at either 15.5°C or 32.5°C in conditions that prevented the uptake of water. In eggs held for a similar period in moist soil at 15.5°C, diapause was virtually eliminated. These eggs did not develop to the stage at which water uptake occurs. It is suggested that the inception of diapause is dependent upon the occurrence of warm, moist conditions at the time of laying and that these requirements are an integral part of the overall inductive process, which also involves the temperature and photoperiod at which the parent insects are reared. A flow-diagram illustrating the development of diapause eggs in relation to temperature and moisture is discussed in relation to the survival of the species.     

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

Maturing eggs of the desert locust, Schistocerca gregaria, contain a variety of ecdysteroid (insect moulting hormone) conjugates and metabolites, four of which have been previously isolated from polar extracts and identified as ecdysonoic acid, 20-hydroxyecdysonoic acid, 3-acetylecdysone 2-phosphate and ecdysone 2-phosphate. In the present study we have isolated eight additional ecdysteroids from similar late-stage eggs by high-performance liquid chromatography. The 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone, all of which were first identified as ecdysteroid components of newly-laid eggs of S. gregaria, were identified by co-chromatography with authentic compounds and by physicochemical techniques. The remaining compounds were identified as 3-acetyl-20-hydroxyecdysone 2-phosphate, 3-epi-2-deoxyecdysone 3-phosphate, 3-acetylecdysone 22-phosphate and 2-acetylecdysone 22-phosphate by fast atom bombardment mass spectrometry, p.m.r. spectroscopy and analysis of the steroid moieties after enzymic hydrolysis. The latter two compounds, after isolation, are susceptible to nonenzymic acetyl migration and deacetylation to give mixtures of ecdysone 22-phosphate and its 2- and 3-acetate derivatives. The possible role and significance of these ecdysteroid conjugates with respect to the control of hormone titres in insect eggs is discussed.     

Neuroendocrine control of diapause hormone secretion in the silkworm,Bombyx mori

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.     

Levels of ecdysteroids in diapause and non-diapause eggs of the Australian plague locust,Chortoicetes terminifera (Walker)

The levels of ecdysteroids during embryogenesis in Chortoicetes terminifera were measured by radioimmunoassay. Soon after laying, eggs laid by females reared under non-diapause conditions contained three times as much as those laid by females reared under diapause inducing conditions. In eggs incubated at 32°C which did not enter diapause there was a sharp rise in ecdysteroid levels which coincided with the formation of the first-instar cuticle, but this did not occur in eggs incubated at 20°C even though they developed and hatched normally. Comparisons are made with previous results in other locust species and the possible role of ecdysteroids in embryonic diapause is discussed.     

Isolation and identification of 3-acetylecdysone 2-phosphate, a metabolite of ecdysone, from developing eggs of Schistocerca gregaria.

A major ecdysteroid conjugate, which accumulates in the eggs of the desert locust, Schistocerca gregaria, during the later stages of embryogenesis, has been isolated by reversed-phase and anion-exchange high-performance liquid chromatography. Hydrolysis of the conjugate with a crude arylsulphatase preparation from Helix pomatia liberates mainly ecdysone 3-acetate. The compound was identified as 3-acetylecdysone 2-phosphate by phosphate analysis of an acid-hydrolysed sample, fast atom bombardment, electron impact and chemical ionization mass spectrometry and 1H and 13Cn.m.r. spectroscopy. The instability of 3-acetylecdysone 2-phosphate on storage results in the formation of ecdysone 2-phosphate, which was identified by physicochemical techniques. 3-Acetylecdysone 2-phosphate and ecdysone 2-phosphate are less susceptible than ecdysone 22-phosphate to hydrolysis in vitro by an enzyme preparation from S. gregaria embryos. The possible role of 3-acetylecdysone 2-phosphate as an inactive end product of ecdysteroid metabolism is discussed.     

The natural history ofPetrobia (Tetranychina) harti (Ewing) andPetrobia (Mesotetranychus) tunisiae Manson (Acari: Tetranychidae) in the laboratory

Petrobia harti (Ewing) developed more rapidly, deposited more eggs and lived longer onOxalis corniculata than onO. articulata. No development took place on several other recorded host plants. In the field this mite was most abundant during early summer. Males made up less than 10% of the population, and no diapause eggs were seen in the field.Petrobia tunisiae Manson developed on various winter Gramineae, produced about 17 non-diapause eggs during its first generation and mostly diapause eggs in the second. A few diapause eggs kept in the laboratory and dipped in chloroform hatched even after 3 years. A summer diapause is postulated for this species.     

RNA content and RNA synthesis in diapause and non-diapause eggs of Bombyx mori

Changes in amount of RNA and its synthesis were studied in diapause and non-diapause eggs of Bombyx mori. In non-diapause eggs, the amount of total RNA began to increase at the stage of late gastrula and increased constantly thereafter. The synthesis of total and ribosomal RNA commenced at the onset of gastrulation, and after two days it levelled off. In contrast, the amount of total RNA in diapause eggs did not change essentially, and only a low level of total and ribosomal RNA synthesis was detected. On the other hand, if diapause eggs were activated by hot HCl treatment 24 hr after oviposition, the results obtained were essentially similar to those for non-diapause eggs, except that there was a lag of two days after acid treatment. These findings indicate that a significant control of ribosomal and other RNA occurs in relation to diapause and development.     

Variation in diapause potential and strength in eggs of the Australian plague locust, Chortoicetes terminifera (Walker) (Orthoptera: Acrididae)

Samples of eggs of Chortoicetes terminifera were incubated under 3 temperature regimes which would allow non-diapause eggs to develop about 50% and so take them beyond the diapause stage. Even so, many more eggs entered diapause when reared at 20°C for 3 weeks than at 32°C for 1 week. By collecting and incubating eggs at intervals after laying in autumn, diapause potential or strength of eggs at different stages of development was estimated. For eggs in pre-diapause, the proportion in diapause after rearing was used to estimate diapause “potential”; for those in diapause, the proportion was used to estimate diapause “strength”. At laying, eggs varied greatly in their potential to enter diapause. However, those with lower potential at laying often increased their potential during the pre-diapause stage so that by the time diapause was reached diapause strength varied but over a lesser range. In all eggbeds, diapause strength decreased by 7–9 weeks after laying; little or none remained by mid-winter.These variations in diapause potential and diapause strength seem to reflect how much the temperature threshold for development during diapause is increased above that for non-diapause. Both diapause potential and strength may reflect the value of some factor whose level at laying is determined by the environment experienced by the parents but which changes subsequently.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.