Seasonal variation in embryonic diapause of the striped ground cricket, Allonemobius fasciatus

ABSTRACT. The incidence and intensity of embryonic diapause in the striped ground cricket, Allonemobius fasciatus DeGeer (Gryllidae), is influenced by the date of oviposition and the incubation temperature. At 20C, embryos develop normally until the end of the appendage-formation stage when diapause occurs. The intensity of 'winter' diapause at this stage is greatest in eggs laid early in the season. Incubation at higher temperature induces a different and earlier 'summer' diapause. The higher the temperature the earlier the stage at which this developmental suppression is imposed. At 27 and 30C, some individuals develop without any interruption and hatch within 20 days. The proportion of these fast-developing eggs is low in summer but increases towards the end of the laying season. In nature, this species is univoltine and precocious hatching before winter is unlikely.
When embryos diapausing at an early stage at 27C are transferred to low temperatures, they resume development and enter winter diapause. The time required to reach the winter-diapause stage after transfer is inversely related to the temperature (30-30C). At 30C, sunmer diapause is neither induced nor maintained.     

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。     

Reversal of embryonic diapause in the Australian plague locust, Chortoicetes terminifera (Walker), by temperatures above the development threshold

Diapause was induced in embryos of Chortoicetes terminifera (Walker) by transferring adults from an L:D 15:9 regime to an L:D 12.5:11.5 regime. When incubated at 20°C all eggs in all pods entered and remained in diapause but when incubated at 26, 32 and 38°C a proportion of eggs in some pods did not. Pods incubated at 32°C for up to 6 days, when diapause intervenes and then transferred to 20°C gave the same result as pods incubated at 20°C throughout development. All eggs entered and remained in diapause. If the period at 32°C was extended to 8 days, the proportion remaining in diapause was not significantly different from that found when pods were incubated at 32°C throughout development. In eggs which broke diapause at 32°C there was a pause or slowing down of development for about 2 or 3 days around the stage at which diapause intervenes.     

Effects of temperature, sorbitol, alanine and diapause hormone on the embryonic development in Bombyx mori: in vitro tests of old hypotheses

Abstract.  An in vitro culture method is described in which embryonic development in Bombyx mori is traced at various temperatures and treatments. The results show that the induction, intensification and termination of diapause are distinct processes. Prediapause embryos, explanted from 40-h-old diapause-destined eggs and cultured in Grace's medium, continue to develop to the appendage-formation stage without arrest, which indicates that the isolated embryos have not entered diapause, whereas the development of embryos from diapausing eggs (15 days after being laid) is significantly slower. The rate of development of embryos dissected from diapause eggs increases during chilling (5 °C) and incubation (at 25 °C) gradually during chilling and dramatically at 25 °C. The in vitro experiments also reveal that sorbitol directly inhibits the development of embryos explanted from diapausing eggs but has no affect on the development of embryos from prediapause eggs. Neither alanine nor diapause hormone prevent isolated embryos from developing.     

Oviducal development in a Krefft's river turtle,Emydura krefftii gray (Chelonia: Chelidae)

A clutch of Emydura krefftii eggs hatched 20 days after oviposition, in contrast to the normal incubation period of 45–50 days for this species. This shortened incubation and the presence of stage 20–21 embryos at oviposition indicates facultative oviducal development in E krefftii.     

Determinants of embryonic stage at oviposition in the lizard Urosaurus ornatus

Relatively few squamate reptiles oviposit eggs with embryos at developmental stages greater than stage 30. To investigate potential proximate and ultimate bases of this phenomenon, we experimentally induced females of the lizard Urosaurus ornatus to retain their eggs past the normal time of oviposition (NTO). This procedure allowed us to determine whether the length of egg retention is fixed or facultative and to evaluate the effects of retention on embryos, hatchlings, and females. Females were able to retain eggs facultatively for at least 29 d past the NTO. However, retention resulted in arrested development of embryos; arrest occurred at stages 30-30.5, which is only slightly more advanced than that at the NTO (stage 29.5). Embryogenesis was reinitiated when eggs were removed from females and placed in incubation media. Hatching success of these eggs was high (87%), and incubation time was not affected by the number of days that development had been arrested. However, the snout-vent length and water content of hatchlings were negatively related to the length of retention, and they ran slower than hatchlings from control eggs obtained at the NTO. Retention of eggs past the NTO had no detectable effect on the body condition or running speeds of females. Developmental arrest and the adverse effects of retention on hatchling phenotype, if widespread among squamates, would account for the limited range of embryo stages at oviposition and act as major constraints on the evolution of viviparity.     

Demarcation of diapause development by cold and its relation to time-interval activation of TIME-ATPase in eggs of the silkworm, Bombyx mori

We investigated the mode of action of winter cold in the termination of diapause by investigating Time-Interval-Measuring Enzyme (TIME). First, we determined the period of cold required for the completion of diapause development. Synchronously developing egg batches of a pure strain (C108 Bombyx mori silkworm) were used to minimize variations in hatching time. Hatching occurred with only 18 days chilling at 5 degrees C when the incubation at 25 degrees C after the chilling was elongated. The 18-day period was much shorter than we expected; diapause in B. mori is known to terminate completely with about 100 days of chilling. Even in such a short period of chilling, no sporadic hatching occurred. Moreover, we determined that a temperature-insensitive stage, which we called "Neboke", followed the short cold-requiring stage. Thus, the stage of diapause development was demarcated from other stages of diapause. While the length of diapause development was elongated when chilling was delayed after oviposition, the Neboke stage length was invariant. Cold evidently exerts its effect only on diapause development. When TIME was purified from eggs and chilled in test tubes, a transitory burst of its ATPase activity occurred at a time equivalent to shortly before the completion of diapause development; this was an interval-timer activation. The mechanism by which cold activates TIME to measure the time interval may help explain in biochemical terms the insect's adaptation to its seasonal environments.     

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.     

Cell cycles in embryos of the silkworm,Bombyx mori: G2-arrest at diapause stage

Summary In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G₁, S and G₂ phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G₁, S and G₂ were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G₂ at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G₁ from G₂, suggesting that their G₁ phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G₁ fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G₂ at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G₁. Finally, most of the cells may be arrested in G₁, while there is only a small fraction of cells continuously cycling. Offprint requests to: T. Yaginuma     

Effects of temperature and moisture on embryonic diapause of the veiled chameleon (Chamaeleo calyptratus)

The development of lizard embryos is typically initiated at fertilization and continues until birth or hatching. In contrast, embryonic development of some chameleons is arrested at the gastrula stage, and embryos remain at this stage for months after the eggs are laid. Our research tested the hypothesis that increased temperature, moisture, or both, are associated with the resumption of development by diapausing embryos of Chamaeleo calyptratus, the veiled chameleon. After 40 days of incubation at 25 degrees C in a relatively dry substrate, eggs were subjected to: 1) no change in temperature or moisture, 2) no change in temperature but change from a dry to a wet substrate, 3) change to a warmer temperature but no change in substrate moisture, or 4) an increase in both temperature and substrate moisture. Overall, embryos initiated development after 50-60 days to 80 or more days of incubation. Neither substrate moisture nor water uptake by eggs was related to the interval when development resumed. In contrast, development was initiated about 10 days earlier for eggs in the high temperature treatment compared to eggs in the low temperature treatment. Our results suggest that neither water availability nor water uptake by eggs affect the length of diapause but that an increase in ambient temperature initiates development of diapausing embryos of C. calyptratus.     

宽翅曲背蝗卵的过冷却能力与抗寒性

采用热电偶法,在室内测定了宽翅曲背蝗卵的过冷却能力及抗寒性.结果表明： 土壤含水量对滞育前卵的含水量有显著影响, 而对卵过冷却点(SCP)的影响不显著,卵含水量随着土壤含水量的升高而上升.不同发育时期卵的SCP、含水量和脂肪含量存在显著差异.随着卵的发育,其含水量从产卵当天的51.5%下降至120 d的46.8%,脂肪含量从10.5%(鲜质量)/19.0%(干质量)上升到14.5%(鲜质量)/28.9%(干质量),而SCP从-23.5 ℃下降至-30.0 ℃;卵SCP与其含水量及脂肪含量存在显著相关关系;深度滞育卵的SCP显著低于滞育前和滞育初期卵的SCP.不同低温强度和处理时间对滞育卵的存活率有显著影响.滞育卵暴露12 h的致死温度为-27.3 ℃,在-25 ℃低温处理的致死时间为22.73 d.滞育卵的SCP与致死温度相近,说明宽翅曲背蝗卵为不耐结冰类型,且SCP是衡量其抗寒性的可靠指标.     

Effect of hydration,photoperiod and temperature on diapause termination in eggs ofPetrobia (Tetranychina) harti (Acari: Tetranychidae)

Petrobia harti (Ewing) displays a facultative summer diapause in the egg stage. An adult female will lay only either diapause or non-diapause eggs throughout her life. In the laboratory, diapause eggs are laid by females which develop on detachedOxalis articulata leaves under long-day photoperiods and a relatively low temperature of 19±1°C.Diapause occurs in a stage of advanced embryonic development, in which the embryo appears U-shaped when observed from the egg's ventral side. Embryonic development ceased at this stage, and no further growth occurred when the eggs were kept under a relative humidity of about 70% in various photoperiod and temperature conditions. However, when the eggs were hydrated by placing them on wet cotton wool, development in some embryos (apparently in those which had completed their diapause development) proceeded beyond the U-stage at a rate similar to that in non-diapause embryos and the eggs hatched.Under LD 168 and 19±1°C or 26±1°C, the later from oviposition the period of egg hydration started, the higher the percentage of diapause termination. Under LD 168 and 26±1°C, diapause termination occurred mostly during the first week of hydration, while at 19±1°C mostly during the second and third week.At 26±1°C, in eggs hydrated 15 days but not 30 days from oviposition, the percentage of diapause termination was higher under a long-day than under a short-day photoperiod.Under LD 168, when the eggs were hydrated continuously from oviposition or starting 15, 30 and 45 days from it, the percentage of diapause termination was higher at 26±1°C than at 19±1°C.The percentage of diapause-laying adult females and the intensity of egg diapause were higher when the pre-imaginal mites grew at LD 1212 and 19±1°C, than when they grew at LD 168 and 26±1°C. This maternal effect on egg diapause intensity was expressed when the eggs were maintained at LD 1212 and 19±1°C but not at LD 168 and 26±1°C.     

Interaction between maternal effects and temperature affects diapause occurrence in the cricket Allonemobius socius

The induction of diapause can be adaptive for egg survival during unfavorable conditions, while direct development can be advantageous under favorable conditions by allowing additional generations to exploit abundant resources. Therefore, the physiological capability of a female to respond to environmental cues indicative of habitat quality by producing eggs of the appropriate developmental phenotype should be under strong selection. Additionally, developing embryos may alter the developmental trajectory initiated by the female in response to changing environmental conditions. In this study, we used a cross-fostering approach to isolate the maternal effects of parental diapause history (not previously studied) and egg-laying temperature from the influence of the incubation environment experienced by the developing embryo on the proportion of diapause eggs produced by the striped ground cricket, Allonemobius socius. We found that an interaction between egg-incubation temperature and parental diapause history strongly affected the proportion of diapause eggs produced and the proportion of eggs that hatched within a 16–18 day incubation period, while egg-laying temperature and all other interactions did not. These novel results indicate that embryos can respond directly to the environmental conditions they experience during development, but that their ability to do so is influenced by maternal effects such as parental diapause history. The results of this study not only provide evidence, for the first time, of parental diapause history affecting diapause proportions, but also raise additional questions about the mechanism by which environmental information is transmitted from parent to offspring and how offspring are able to respond to conditions experienced during their own development.     

Embryonic Development and Rates of Metabolic Activity in Early and Late Hatching Eggs of the Major Malaria Vector Anopheles gambiae

Anopheles gambiae eggs generally hatch at the completion of embryo development; two-three days post oviposition. However, staggered or delayed hatching has been observed whereby a single batch of eggs shows marked variation in time-to-hatch, with some eggs hatching 18 days post oviposition or later. The mechanism enabling delayed hatch has not been clearly elucidated but is likely mediated by environmental and genetic factors that either induce diapause or slow embryo development. This study aimed to compare metabolic activity and embryonic development between eggs collected from sub-colonies of the baseline Anopheles gambiae GAH colony previously selected for early or late time-to-hatch. Egg batches from early and late hatch sub-colonies as well as from the baseline colony were monitored for hatching. For both time-to-hatch selected sub-colonies and the baseline colony the majority of eggs hatched on day two post oviposition. Nevertheless, eggs produced by the late hatch sub-colony showed a significantly longer mean time to hatch than those produced by the early hatch sub-colony. The overall proportions that hatched were similar for all egg batches. CO₂ output between eggs from early and late hatch sub-colonies showed significant differences only at 3 and 7 days post oviposition where eggs from the early hatch and the late hatch sub-colony were more metabolically active, respectively. No qualitative differences were observed in embryo development between the sub-colonies. It is concluded that all viable embryos develop to maturity at the same rate and that a small proportion then enter a state of diapause enabling them to hatch later. As it has previously been shown that it is possible to at least partially select for late hatch, this characteristic is likely to involve genetic as well as environmental factors. Delayed hatching in An. gambiae is likely an adaptation to maximise reproductive output despite the increased risk of desiccation in an unstable aquatic environment.     

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.     

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.     

Husbandry of the Annual Killifish Austrofundulus limnaeus with Special Emphasis on the Collection and Rearing of Embryos

Annual killifish development is unique compared to other teleosts and is characterized by the dispersion and subsequent reaggregation of pre-embryonic blastomeres and the occurrence of embryonic diapause. Austrofundulus limnaeus is an excellent species to use for studies of development and embryonic diapause in annual killifish. A. limnaeus has a high fecundity, reproduces readily in a laboratory environment, and has a relatively long laboratory life span compared to many other species of annual killifish. Methods are presented for rearing A. limnaeus in the laboratory with an emphasis on collecting and incubating large numbers of embryos for biochemical and physiological studies. Females produce an average of 29 eggs during a two to four hour spawning. Egg quality (% fertilization and survival) and egg production (eggs female^-1) are affected by the number of days between spawning events. Percent fertilization of eggs and survival of embryos decreases as the interval between spawning increases from two to eight days. The number of fertile embryos produced per female remains relatively constant as a function of spawning interval. Fertilization rates may be maintained at high levels by replacing aged males (1.5 years old) with younger males. An embryo medium was formulated to mimic the natural waters inhabited by A. limnaeus. The developmental rate and survival of embryos in the embryo medium was essentially equivalent when compared to Yamamoto's fish saline solution.