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.     

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.     

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.     

Activity of NAD-sorbitol dehydrogenase is caused by biosynthesis of enzyme protein in diapause and non-diapause eggs of the silkworm,Bombyx mori

• 1.1. Using SDS-PAGE and immunoblotting analyses with anti-sorbitol dehydeogenase (EC 1.1.1.14, SDH) serum, changes in amount of SDH protein were examined in diapause and non-diapause eggs of the silkworm, Bombyx mori.
• 2.2. When diapause eggs were exposed to 5°C from 2 days after oviposition to break the diapause gradually, SDH protein appeared after 50-day chilling, and then the amount increased along with chilling period. This changing pattern paralleled that in SDH activity.
• 3.3. In diapause eggs treated with HCl after chilling at 5°C for 30 days to break the diapause quickly, and non-diapause eggs, changing patterns in amount of SDH protein also paralleled those in SDH activity.
• 4.4. These results showed that SDH activity was caused by biosynthesis of SDH protein, independent of diapause or non-diapause eggs.
• 5.5. Occurrence of SDH correlates with the three developmental phases: diapause termination, embryonic growth, and larval differentiation. In larva, SDH was mainly localized in the fat-body.

     

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.     

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.     

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.     

中华稻蝗长沙种群的生活史及其卵滞育的进化意义

中华稻蝗Oxya chinensis为重要的水稻害虫,在我国除青海、西藏、新疆、内蒙古等未见报道外,南起海南北至东北均有分布,在许多的分布区域1年发生1代。为探索中华稻蝗长沙种群的生活史及其季节适应特征,通过野外和实验室的研究,调查了其发生代数、孵化率和卵滞育率的季节变化及越冬卵的存活率。结果显示,中华稻蝗长沙种群为1年2代和1年1代混合发生:第1代卵产卵后大部分孵化为若虫而1年完成2代,但亦有19.4% -4.1%的卵不孵化而1年只能完成1世代。第1代成虫于6月上旬至8月上旬羽化,6月下旬至8月中旬产卵;第2代若虫于7月初开始孵化,9-10月羽化为成虫,10月上旬至11月下旬产卵。在室外自然条件下,中华稻蝗长沙种群6-8月(第1代)和10-11月(第2代)所产卵块均为部分滞育,滞育率为30%左右,皆无显著差异。然而,其卵滞育率在12月以后显著降低,仅为6.6%或以下,卵滞育快速地得以解除。因此,包括非滞育卵和滞育解除卵,中华稻蝗长沙种群的越冬卵皆以非滞育状态度过其后的寒冷季节。即使是遭遇长江流域2007年末至 2008年初异常寒冷的冬季,在长沙地区越冬后其卵的存活率亦在98%以上。非滞育状态的中华稻蝗长沙种群越冬卵完全能安全地越冬,其滞育的发生并非是为了提高其耐寒性而安全度过不适环境。并探讨了中华稻蝗长沙种群卵滞育的进化意义。     

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.     

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.     

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.     

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

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

Ecdysone Metabolism in Diapause Eggs and Non-Diapause Eggs of the Silkworm, Bombyx mori

In order to compare ecdysone metabolism between diapause eggs and non-diapause eggs of the silkworm, Bombyx mori, (3)H-ecdysone and its derivatives ((3)H-3-epiecdysone and (3)H-ecdysone 22-phosphate) were injected into the eggs at various stages during early embryogenesis, and the resultant labelled metabolites were analyzed by high-performance liquid chromatography. From the quantitative and qualitative changes in the labelled metabolites between diapause eggs and non-diapause eggs, it was demonstrated that epimerization of ecdysone occurred during early embryogenesis irrespective of the embryonic stage in both diapause eggs and non-diapause eggs, and that phosphorylation of ecdysone was a major metabolic step in diapause eggs, whereas dephosphorylation of ecdysone 22-phosphate and its subsequent hydroxylation at the C-20 and C-26 positions were characteristic in non-diapause eggs.     

Influence of oviposition date and temperature upon embryonic development in Somatochlora alpestris and S. arctica (Odonata: Corduliidae)

In two bog-dwelling dragonflies, Somatochlora alpestris and S. arctica , the influence of oviposition date and temperature upon duration of embryonic development was studied. Egg diapause was facultative. With advancing season, the proportion of diapause eggs increases from 0 to 37% in S. alpestris and from 0 to 18% in S. arctica. Eggs needed at least 17 to 38 days for development. Hatching curves were temperature-independent in nearly all experiments but developmental rate increased at higher ambient temperature. In S. arctica , responses of developmental rate to temperature differed in eggs laid on different dates. In S. alpestris , duration of egg development decreased as season progressed. The duration of egg development of non-diapause eggs and proportion of diapause eggs in S. alpestris and possibly in S. arctica may also both be a function of female age at the time of oviposition. The ecological significance of the different development patterns is discussed.     

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.     

Under-sized larvae from short-day adults of the blow fly, Calliphora vicina, side-step the diapause programme

Abstract. Larvae of Calliphora vicina R.-D. (Diptera: Calliphoridae) hatching from eggs laid by adult females exposed to short day length, and then raised in darkness at 11^oC, normally enter diapause rather than undergoing prompt pupariation. However, if the feeding stage is curtailed by premature extraction of larvae from their food, or if the larvae are subjected to severe overcrowding, smaller larvae side-step the diapause programme to become miniature puparia, whereas larger larvae proceed to diapause as fully-fed ones. In addition, smaller diapausing larvae show a shorter (or less intense) diapause than full-sized larvae. Apart from the smallest individuals, flies can emerge from these miniature puparia and in some cases are capable of laying eggs; this suggests that avoidance of diapause may allow undersized individuals an opportunity to reproduce before winter sets in. Fat determinations performed on larvae and pupae of various sizes, however, showed that short-day (diapause-destined) individuals lay down the same proportion of fat as long-day (non-diapause) individuals, and the same proportion of fat across all size classes. The possible functional significance of this phenomenon is discussed.     

Effects of low temperatures on NAD-sorbitol dehydrogenase activity and morphogenesis in non-diapause eggs of the silkworm,Bombyx mori

• 1.1. The activity of NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) and levels of sorbitol were examined in non-diapause eggs of the silkworm, Bombyx mori, exposed to temperatures of 20-0.5°C from 1 day after oviposition. The morphology of embryos in the cold-acclimated eggs and the hatching of eggs after transfer to 25°C were monitored.
• 2.2. Temperatures between 15 and 0.5°C retarded the development of NAD-SDH activity at a specific embryonic stage that was comparable to diapause, and sorbitol accumulated in the eggs.
• 3.3. With the appearance of NAD-SDH activity, sorbitol was converted into glycogen, just as it is in diapause eggs. The results indicate that NAD-SDH participates in the utilization of sorbitol rather than in its formation in non-diapause eggs.
• 4.4. Distinct effects of low temperatures on the morphological development of the embryos are also discussed.

     

Selection for non-diapause in the bumblebee Bombus terrestris, with notes on the effect of inbreeding

Bumblebees are generally believed to be annual insects. However, here we will show that under laboratory conditions the bumblebee Bombus terrestris (L.) can produce a second generation without a period of cold storage (diapause) or CO₂ narcosis (a method to break diapause). It is also shown that this so-called non-diapause trait can be selected for. The percentage of non-diapausing queens increased from 8% (minimum) to 97% (maximum) in two generations of selection. However, it was not possible to maintain isofemale non-diapause lines. Colonies of the fourth and fifth generation remained small (expressed in worker number) and produced a small number of queens. Also the percentage of queens that started laying eggs (defined as the percentage non-diapause) decreased in the fourth and fifth generations. To study whether this decline of the non-diapause lines was caused by inbreeding, a control experiment was conducted. In this control experiment queens were mated with their brothers (full-sib mating) for several generations and the number of queens that start egg laying was measured. This revealed that inbreeding can have a negative effect on the egg-laying capacities of queens thus causing the decline of inbred (non-diapause) lines.     

In vitro diapause substance in the silkworm, Bombyx mori

By means of in vitro studies, in which isolated suboesophageal ganglions of the Bombyx silkworm were cultured, it was shown that at least two kinds of substances are biosynthesized and exert independent effects on determination of diapause or non-diapause in silkworm eggs. They are referred to as the diapause and non-diapause substance, respectively. Whether diapause or non-diapause eggs are laid may depend upon the different quality of these substances.