Temperature effects on embryonic development of Paratlanticus ussuriensis (Orthoptera: Tettigoniidae) in relation to its prolonged diapause

Paratlanticus ussuriensis eggs overwinter by entering diapause, which can be prolonged to more than 1 year depending on environmental conditions. To determine temperature effects on diapause duration of P. ussuriensis eggs, the rates of embryonic development and hatching were compared at various temperatures conditions by measuring embryonic stages and egg weights. Most eggs stayed in a very young stage (blastoderm formation, stage 4) when reared at 15 and 20 °C, 10–30% eggs developed into middle or late stages when reared at 25 °C, and most embryos developed fully (stage 23/24) when reared at 30 °C. Egg weight at 30 °C was 1.5 times higher than those reared at 20 °C. Chilling induced hatching in embryos at stage 23/24. Chilling caused stage 4 embryos to develop into stage 24, but they failed to hatch in response to a second warm period. Thus, P. ussuriensis eggs can overwinter either as young embryos (initial diapause) or as fully-developed embryos (final diapause). Eggs that experience an initial diapause overwinter again the second year in a final stage diapause. The post-diapause period was shorter when embryos overwintered in a final stage diapause. The hatching rate was highest in a temperature range of 7.5–15 °C. Our results suggest that temperature is an important environmental factor for the control of prolonged diapause in P. ussuriensis and initial diapause plays an important role in the control of its life cycle.     

Development, surface exposure, and embryo behavior affect oxygen levels in eggs of the red-eyed treefrog, Agalychnis callidryas

Oxygen stress can slow development, induce hatching, and kill eggs. Terrestrial anamniote embryos face a potential conflict between oxygen uptake and water loss. We measured oxygen levels within eggs to characterize the respiratory environment for embryos of the red-eyed treefrog, Agalychnis callidryas, a Neotropical frog with arboreal egg masses and plastic hatching timing. Perivitelline oxygen partial pressure (Po2) was extremely variable both within and among eggs. Po2 increased with air-exposed surface of the egg and declined over the developmental period before hatching competence. Through the plastic hatching period, however, average Po2 was stable despite continued rapid development. Development was synchronous across a wide range of perivitelline Po2 (0.5-16.5 kPa), and hatching-competent embryos tolerated Po2 as low as 0.5 kPa without hatching. The variation in Po2 measured over short periods of time within individual eggs was as great as that measured across development or surface exposure, including sharp transients associated with embryo movements. There was also a strong gradient of Po2 across the egg from superficial to deep positions. Ciliary circulation of fluid within the egg is clearly insufficient to keep it mixed. Embryos may maintain development under hypoxic conditions by strategic positioning of respiratory surfaces, particularly external gills, to exploit the patchy distribution of oxygen within their eggs.     

Morphological changes during diapause stages in the embryonic cortex of the annual killifish <Emphasis Type="Italic">Millerichthys robustus</Emphasis> (Cyprinodontiformes: Cynolebiidae) under natural conditions

The annual killifish inhabits in extreme locations with unpredictable rainy season where survives through the massive generation of embryos resistant of drought, capable to remain in a state of metabolic dormancy (three moments of diapause during embryonic development) protected by embryonic cortical structures: perivitelline space, egg envelope and its ornamented structures (trapeze-shaped projections and filaments in Millerichthys robustus). This research describes, for the first time, changes in cortical structures during three diapause stages in embryos of annual fish M. robustus during an annual life cycle. Embryos were collected in three periods through the year in a temporal water body: flood, drought and wet. During flood period all embryos were found in diapause I (during epiboly, dispersion of the blastomeres stage) with maximum thickness in all cortical structures and presence of egg envelope filaments. During drought period all embryos were in diapause II (development during somitogenesis, before the organogenesis) and its structures reduced its thickness significantly and lost the egg envelope filaments. Interestingly, embryos in diapause II and III (embryonic development completed in a pre hatching stage) were found during wet period (an example of bet-hedging strategy) in which all structures presented a recovery tending to its original condition observed during flood period. This research demonstrates that annual fish embryos respond to their exposure to seasonal environmental variations with dynamic structural changes that are fundamental for their survival.     

The plurennial life cycles of the European Tettigoniidae (Insecta: Orthoptera)

Summary The effect of drought on embryonic development and on hatching was studied in 13 European Tettigoniidae species. Drought can affect development in three different ways: (1) Embryonic development proceeds slower than if the eggs are in contact with water; (2) it stops (for final diapause) in an earlier embryonic stage; (3) it affects maintenance and termination of the initial embryonic diapause.In many Tettigoniidae species, the initial diapause is prolonged, and may last several years. Without draught stress, between 1 and 7 cold treatments in the laboratory, and with eggs of the Tettigonia-species between 1 and 6 winters in the field were necessary to enable all eggs to complete initial diapause. In Central European species, the number of eggs maintaining initial diapause significantly increased when the eggs had no contact with water at the time when they should recover from diapause. In contrast, termination of initial diapause in Tettigonia caudata from Greece, when the environment became favorable for growth again, was highest in that group of eggs that had lost most water in a preceding period of drought. The importance of the prolonged initial diapause for the survival of unpredictable adverse conditions is discussed.     

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.     

An analysis of diapause and resistance in the egg stage of Floronia bucculenta (Araneida: Linyphiidae)

Summary Floronia bucculenta hibernates in the egg stage; the egg sacs are deposited on the leaves of grass tussocks without any shelter. The morphogenesis of the eggs was divided into 10 arbitrarily chosen stages, in order to test the dependence of embryonic development on temperature in the laboratory. The eggs developed slowly at 23°C, 16°, 12.5°; embryogenesis stopped after 70–45 days, when prosomal appendage rudiments began to form. At 10°, 7.5°, 5°, 0° complete embryogenesis was possible until the emergence of the first complete stage. The eggs developed most rapidly at 5° (mean developmental time 203 days). The egg development was normal at 5° and 0°, when compared with the timetable of the embryogenesis of the linyphiid Bathyphantes gracilis, a species which has no egg diapause. At 7.5° and 10° the embryogenesis was strongly delayed during the median phases of development (elongation of the germ band, formation of prosomal appendages); after reversion the development was accelerated (postdiapause phase). After long exposure to low temperatures (-10° to +10°) the diapause was terminated. A temperature of 0° was optimal (minimal time of exposure 8–9 weeks). The time required for embryonic development of postdiapause eggs decreased hyperbolically with increasing temperature. In the field the median phases of embryogenesis were retarded by low ambient temperatures; diapause was terminated from late December to mid-January. The spread of hatching in spring was 7–15 days.During the diapause phase the O₂-consumption of the eggs at 25° was depressed. It rose from 1.55 (in late diapause) to 4.21 ml/100 eggs·h at the onset of postdiapause, whereas O₂-utilization did not change significantly at 5° (from 0.54 to 0.61 ml/100 eggs·h just after the termination of diapause).The diapause phase was not characterized by higher resistance to cold, drought, or flooding. As compared with single eggs removed from the cocoon, the silken wall of the intact egg sac did not affect the survival of postdiapause eggs exposed to-15° (LD₅₀=28 days); it raised, however, the survival time of eggs exposed to a R.H. of 32% (at 5°) or flooding by distilled water (at 5°): from LD₅₀=37 to 68 days at drought, from LD₅₀=30 to 92 days at flooding.Diapause is important for synchronizing the life-cycle of F. bucculenta with the seasonal fluctuations of environment. The egg stage is highly tolerant to the extreme factors of the winter. Some implications of the relation of the studied spider to its habitat are discussed.     

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.     

Effects of temperature during chilling and pre-chilling periods on diapause and post-diapause development in a katydid, Eobiana engelhardti subtropica

In Eobiana engelhardti subtropica, early laid eggs reach the diapause stage in early autumn. For long periods before winter, the eggs are exposed to temperatures higher than their theoretical lower threshold for development. In contrast, late-laid eggs cannot reach their diapause stage before winter. Our study showed that E. e. subtropica copes with these difficulties via the thermal response involving embryonic diapause. In this katydid, the almost fully developed embryo undergoes an obligatory diapause. When diapause eggs were maintained at a temperature of 20 degrees C or higher, diapause persisted for a long time. Diapause was effectively terminated by temperatures ranging from 1 to 11 degrees C, and hatching occurred successfully at temperatures from 11 to 15 degrees C. In addition to the chilling temperature, pre-chilling temperature modified diapause intensity and hatching time. Diapause eggs hatched earlier after chilling when the pre-chilling temperature was lower, within a range of 14.5-25 degrees C. Thus, the low-temperature requirement for diapause termination prevents early laid eggs from untimely hatching in autumn, and low temperatures before and during winter decrease diapause intensity and shorten the hatching time in the following spring. When eggs were chilled before diapause, they tolerated chilling and averted diapause. Thus, even if eggs encounter low temperatures before diapause, they can hatch in the following spring.     

Oxygen consumption during embryonic development of the annual fish Nothobranchius korthausae with special reference to diapause

The oxygen consumption of Nothobranchius korthausae eggs in different developmental stages, including diapause II and III, was measured. Oxygen consumption increases exponentially during embryonic development. In diapause II and III there is a drop in oxygen consumption, which attains a minimal level in diapause II after 3 weeks and in diapause III after 2 weeks. During early development the embryos can escape from hypoxic stress by entering diapause I and II. During late embryogenesis embryos in diapause III can escape from hypoxic stress by hatching. We conclude that survival of annual fish embryos is enhanced during conditions of low oxygen concentration by reduced oxygen consumption rates during diapause.     

Nondormanz-Entwicklung prospektiver Diapause-Eier vonBombyx mori mit oder ohne Chorion unter Paraffinöl

Summary Prospective diapause eggs ofBombyx mori become permeable to water in or after contact with paraffin, and they die before reaching diapause. If the eggs are hindered from drying up, their germ anlagen develop to diapausing germ bands. Prospective diapause eggs opened in paraffin, always develop without egg diapause, but their degree of organogenesis depends on the chorion: When eggs are completely dechorionized most of them develop to hatching or nearly hatching larvae; when eggs are completely dechorionized, but their chorion remains in the paraffin, the degree of organogenes is issignificantly lower; and when the opened eggs keep 1/2–3/4 of the chorion, their organogenesis is significantly lower than in the two previous experiments.It is discussed that nondormancy development ofprospective diapause eggs in vitro is not a specific effect of culture media containing water but is possible in paraffin oil too. It is not only possible to stop but also to prevent egg diapause. Nothing, as yet, can be said about the prospective significance of the chorion's reducing factor in organogenesis.     

Role of maternal provisioning in controlling interpopulation variation in hatching size in the marine snail Nucella ostrina

This study examined the role of maternal provisioning in controlling interpopulation variation in hatching size in nine isolated populations of the intertidal gastropod Nucella ostrina, in which development to the early juvenile stage takes place within an egg capsule. Variation among populations was almost entirely due to the ratio of nurse eggs to embryo, which explained 65% of the variation in hatching size. Egg size was not a significant predictor of hatching size. Differences among seven of these populations in the nurse egg/embryo ratio were entirely due to the number of nurse eggs allocated per capsule; these populations allocated different numbers of nurse eggs per capsule but allocated the same number of embryos. Intriguingly, the two most wave-sheltered populations allocated significantly more nurse eggs and more embryos to each capsule than did the seven other populations, but they maintained nurse egg/embryo ratios consistent with patterns observed in the other populations. Inter- and intrapopulation variation in hatching size appears to be controlled largely by different mechanisms: within-population variation being controlled mainly by differences in allocation of embryos per capsule, whereas most among-population variation being due to differences in allocation of nurse eggs per capsule.     

Evidence for embryo–embryo interactions in controlling hatching time and synchronization in the brown marmorated stink bug,Halyomorpha halys (Hemiptera: Pentatomidae)

The mechanisms controlling egg diapause and circadian rhythms of hatching activity have been extensively studied in insects. However, relatively little attention has been paid to the mechanisms controlling synchronized hatching from an egg mass. In this study, we examined the possible involvement of embryo–embryo interaction in controlling hatching time in Halyomorpha halys (Stål). Eggs tended to hatch earlier as the egg mass size increased. Egg separation and clumping of separated eggs at various times showed that hatching synchrony was largely determined shortly before hatching. However, whether eggs were kept in a mass or separated until several hours before hatching also influenced the hatching time, indicating the presence of embryo–embryo interactions. Eggs derived from different masses and kept in physical contact with one another hatched synchronously if their ages were within approximately 8 h. In this case, both younger and older eggs advanced only in hatching time, in contrast to a case of locusts reported by others. Eggs separated by more than 7 mm hatched as synchronously as those kept in a mass when glued to the same substrate, suggesting an important role of the egg substrate in transmitting the vibrational hatching signals to neighboring sibling eggs to synchronize hatching.     

Fertilization of eggs of zebrafish, Danio rerio, by intracytoplasmic sperm injection

To evaluate the potential for fertilization by sperm injection into fish eggs, sperm from zebrafish, Danio rerio, were microinjected directly into egg cytoplasm of two different zebrafish lines. To evaluate physiological changes of gametes on the possible performance of intracytoplasmic sperm injection (ICSI), four different combinations of injection conditions were conducted using activated or nonactivated gametes. From a total of 188 zebrafish eggs injected with sperm in all treatments, 31 (16%) developed to blastula, 28 (15%) developed to gastrula, 10 (5%) developed abnormally to larval stages, and another 3 (2%) developed normally and hatched. The highest fertilization rate (blastodisc formation) was achieved by injection of activated spermatozoa into nonactivated eggs (35%). Injections were most effective when performed within the first hour after egg collection. Flow cytometric analysis of the DNA content of the developing ICSI embryos revealed diploidy, and the use of a dominant pigment marker confirmed paternal inheritance. Our study indicates that injection of a single sperm cell into the cytoplasm of zebrafish eggs allows fertilization and subsequent development of normal larvae to hatching and beyond.     

Desiccation and egg viability of the ragwort flea beetle,Longitarsus flavicornis (Coleoptera: Chrysomelidae)

A study was conducted to investigate the effect of desiccation on the survival of eggs of Longitarsus flavicornis. Eclosion of L. flavicornis eggs in laboratory trials decreased with increasing desiccation time between 0 days (93% hatching) and 42 days (no egg hatching) at 50±2% relative humidity and 23±2°C. Probit analysis indicated that 25, 50 and 99% mortality of L. flavicornis eggs occurred after 5.7, 9.3 and 50.4 days desiccation, respectively. Egg development varied between a minimum of 8 days at 7 days desiccation to a maximum of 15 days at 28 days desiccation. Hatching span did not differ between treatments with all eggs hatching within 12 days of each other. A relative humidity of 88–100% was measured under ragwort rosettes in non-drought field conditions. This would be expected to facilitate successful egg eclosion. However, the occurrence of summer drought could be detrimental to egg survival.     

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.     

Environmental and developmental effects on external gill loss in the red-eyed tree frog, Agalychnis callidryas

I examined the effects of development, hatching, and oxygen availability on external gill loss in red-eyed tree frogs, Agalychnis callidryas. Under natural conditions, the arboreal embryos maintained large external gills until hatching, which occurred from 5-8 d after oviposition. At hatching, when tadpoles entered the water, external gills began to regress. In older hatchlings this process was extremely rapid. Gill circulation was lost on average within 16 min and sometimes within 5 min. Gills often regressed completely in under 2 h. Younger hatchlings reduced gill circulation, shortened and adducted their gills, then resumed normal circulation for some time after hatching; half had completely lost external gills within 24 h. Experimentally increasing the area of egg surface exposed to the air induced loss of external gills in unhatched embryos. Older hatchlings in hypoxic water without access to air maintained their external gills. This suggests that loss of external gills is a response to increased oxygen availability, rather than a response to hatching per se. Extended maintenance of external gills by large, late-hatching embryos may facilitate continued rapid development in closely packed eggs.     

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

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

Intraclutch Hatch Synchronization in Pheasants and Mallard Ducks

Synchronization of hatching within clutches of precocial bird species can be achieved either by acceleration or retardation, i.e. by shortening or prolonging the incubation period. The ability of mallard ( Anas platyrhynchos ) and ring-necked pheasant ( Phasianus colchicus ) embryos to accelerate or retard hatching was tested by incubating separate clutches, of which three eggs had 2 d longer or shorter incubation time than the others, and observing their individual time of pipping (breaking of the shell). Mallard embryos were able to delay hatching by on average 0.6 d (43% of the eggs delayed at least 1 d), but were better at acceleration (on average 1.3 d; 91% of the eggs accelerated more than 1 d). Conversely, pheasant embryos were only able to accelerate by 0.4 d (50% accelerated more than 1 d), but were better at delaying the hatching (1.2 days; 77% delayed more than 1 d). This difference between the species may depend on different degrees of relatedness within clutches in pheasants and mallards. It may also be an effect of the more developed sensory and neuromuscular systems in galliforms; a reduction of the incubation period would mean that the development of, for example, locomotion would be insufficient at hatching.     

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.     

Development and the influence of nurse egg allotment on hatching size in Searlesia dira (Reeve, 1846) (Prosobranchia : Buccinidae)

The reproductive biology of the intertidal prosobranch Searlesia dira (Reeve, 1846) was examined with special attention given to variability in the nurse egg to embryo ratio among capsules, among clutches and among geographically isolated populations. Embryos and nurse eggs were distributed among the capsules in a manner consistent with the hypothesis that nurse eggs were genetically predetermined, that each female had a genetically defined nurse egg to embryo ratio, and that each capsule represented a random sample of that ratio. The binomial distribution of embryos and nurse eggs among the capsules resulted in some capsules receiving many more embryos per nurse egg than others. The number of nurse eggs an embryo succeeded in eating was proportional to the number of capsule-mates sharing a capsule. Embryos eating more nurse eggs hatched out at a larger size. Differences in the nurse egg to embryo ratios among capsules in the same clutch were much larger than that of the mean ratios among clutches. Among-site differences in the mean nurse egg to embryo ratios suggest that selection pressure for different mean hatching sizes may have acted on the mean nurse egg to embryo ratios.In contrast to the predictions of optimal hatching size theory, hatching size varied widely within clutches as a consequence of differences in nurse egg to embryo ratios among capsules. This variance may be adaptive for species that lay their eggs months before juveniles emerge into an unpredictable environment, or simply be a consequence of an imperfect mechanism for increasing hatching size.