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Embryonic diapause is an enigmatic phenomenon that appears in diverse species. Although regulatory mechanisms have been established, there is much to be discovered. Herein, we have made the first comprehensive attempt to elucidate diapause regulatory mechanisms using a computational approach. We found transcription factors unique to promoters of genes in diapause species. From pathway analysis and STRING PPI networks, the signaling pathways regulated by these unique transcription factors were identified. The pathways were then consolidated into a model to combine various known mechanisms of diapause regulation. This work also highlighted certain transcription factors that may act as 'master transcription factors' to regulate the phenomenon. Promoter analysis further suggested evidence for independent evolution for some of regulatory elements involved in diapause.

Roe deer (Capreolus spp.) are a little odd. They are one of only a few placental mammals-and the only genus among even-toed ungulates-capable of putting embryonic development "on ice", also known as embryonic diapause (Figure 1). It would seem such an unusual trait is probably the product of natural selection, but a big question is, how does selection for important traits, such as diapause, interact with the historical demography of a species? In a 'From the Cover' article in this issue of Molecular Ecology, de Jong et al. (2020) demonstrate that selection is acting on genes associated with reproductive biology in roe deer, despite heightened genetic drift due to reduced effective population size through the Pleistocene.

It has been reported that the lawn ground cricket, Polionemobius mikado (Orthoptera: Trigonidiidae), shows variations in voltinism and life‐history traits along latitude in Japan, but whether it shows variations along altitude has not been examined. The present study aims to determine whether there is variation in voltinism, body size and the incidence of embryonic diapause among populations of P. mikado at different altitudes in Kyoto, Japan. Six populations collected from 70 to 800 m a.s.l. showed a positive relationship between the adult head width and the altitude of sampling locality. This body size cline is attributed to the increasing proportion of larger univoltine individuals from low to high altitude. The incidence of diapause of the strain from altitude 800 m was approximately 100% when reared under natural photoperiods and temperatures from spring to summer at an altitude of 70 m, and was significantly higher than that of the strain from 70 m. These differences in life‐history traits suggest that the population of P. mikado at a higher altitude has adapted to a shorter length of the season available for growth.

A female buffalo (Bubalus bubalis) of the Bulgarian Murrah breed aged 1,090 days was observed to give birth to a second newborn (normally developed male) after she had calved (normal female) 49 days earlier. This phenomenon is highly associated with her melatonin treatment within a trial for induction of puberty, the last ear implants being placed approximately 50 days before the assumed date of first mating, to which point the level of progesterone had increased dramatically. Despite none of the matings of the dam was visually witnessed to prove ovulation over an existing gestation, we take the liberty to qualify this phenomenon as superfetation, ruling out the other possible phenomena, namely embryonic diapause as it is highly unlikely to occur in any livestock species, and differentiated development of twin foetuses as it is associated with foetal malformation, which was not observed in this case.

The embryonic stage of development is defined as the period between fertilization and the establishment of most of the organ systems by the end of this period. Development in this stage is rapid. In many mammalian species, particularly in humans, the interval between fertilization and implantation is exactly determined and continuous without intermission. However, European roe deer (Capreolus capreolus) embryos undergo a reversible retardation of development. This interesting reproduction strategy is called embryonic diapause (delayed implantation). After this period of embryonic arrest, development continues without further interruption. The aim of this study was to investigate embryonic development after diapause in European roe deer. Because of the embryonic diapause and the unknown date of fertilization, it was impossible to assign the embryos to a certain gestational age (days). This study describes normal stages of embryonic development mainly based on the external morphological traits of 56 well-preserved post-implantation roe deer embryos and attempts to assign the embryos to certain development stages. Carnegie stages of human embryos were used as an orientation for staging roe deer embryos. We observed a considerable range of variation of embryonic stages investigated until the end of January. We found post-implantation stages of embryonic development already at the end of December and foetuses at the end of January. Moreover, assigning the embryos to a particular stage of development allows the comparison between pairs of twins and triplets. We showed that twins and triplets were always at the same development level, despite the discrepancy in inter-twin and inter-triplet size.

While the proximate driver behind the decline of the Western stock of Steller sea lions (Eumetopias jubatus, >80% since 1970s) is likely multifactorial, the population reduction may have been powered by a decrease in fecundity. A harvest of Steller sea lions in the 1970s and 80s revealed a 30% reduction in the proportion of pregnant females from early (October-November) to late gestation (April-May). Identification and quantification of these reproductive failures are difficult when we lack species-specific data on endocrinology associated with discrete stages of the reproductive cycle (i.e., estrus, implantation, and gestation). We tracked changes in serum estradiol and progesterone in three adult female Steller sea lions from 2011 to 2015. In all years and most females, a discrete increase in estradiol was observed during the breeding season (June-August), indicative of estrus. Estradiol concentrations from October to May in a pregnant female compared to her corresponding values when non-pregnant did not consistently differ through gestation. An elevation in progesterone was observed in all females and all years beginning approximately in June and lasting through November. This likely results from progesterone production by the corpus luteum in both pregnant and pseudopregnant females. Serum progesterone shows promise as a diagnostic tool to identify pregnancy during months 3-5 (December-February) of the 8-month active gestation following embryonic implantation. This study provides ranges of key hormones during estrus, embryonic diapause/pseudopregnancy, and gestation in pregnant and non-pregnant females for studying reproduction in Steller sea lions.

The bivoltine silkworm Bombyx mori (Lepidoptera: Bombycidae) exhibits a maternally controlled embryonic diapause. Maternal silkworms decide whether to lay diapause or nondiapause eggs depending on environmental factors such as the temperature and photoperiod during the egg and larval stages, and then induce diapause eggs during the pupal stage. However, little is known about the molecular mechanism that conveys the outcome of whether to produce diapause or nondiapause eggs from the egg or larval stages to the pupal stage. This study used microarray analysis to investigate differentially expressed genes in the larval brains of diapause- and nondiapause-egg producers, to which bivoltine silkworms were destined by thermal or photic stimulation during the egg stage. The cytochrome P450 18a1 and Kruppel homolog 1 genes were upregulated in producers of diapause eggs compared with those of nondiapause eggs under both experimental conditions. Cytochrome P450 18a1 encodes a key enzyme for steroid hormone inactivation and Kruppel homolog 1 is an early juvenile hormone-inducible gene that mediates the repression of metamorphosis. The upregulation of these genes during the larval stage might be involved in the signaling pathway that transmits information about the diapause program from the egg stage to the pupal stage in the silkworm.

Previous study showed that diapause in Bombyx mori eggs can be terminated by dechorionation and that activation in the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that mechanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho-ERK levels and that injury increased the phospho-ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HCl. Effects of anaerobiosis on dechorionation-stimulated phospho-ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is involved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation-stimulated phospho-ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphorylation of c-jun N-terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in JNK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho-ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury-induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs.

The utility of transrectal ultrasonography was tested in a field setting to characterize the reproductive tract and detect early pregnancy (embryonic vesicles

An annual life cycle is characterized by growth, maturity, and reproduction condensed into a single, short season favourable to development, with production of embryos (seeds, cysts, or eggs) capable of surviving harsh conditions which juveniles or adults cannot tolerate. More typically associated with plants in desert environments, or temperate-zone insects exposed to freezing winters, the evolution of an annual life cycle in vertebrates is fairly novel. Killifish, small sexually dimorphic fishes in the Order Cyprinodontiformes, have adapted to seasonally ephemeral water bodies across much of Africa and South America through the independent evolution of an annual life history. These annual killifish produce hardy desiccation-resistant eggs that undergo diapause (developmental arrest) and remain buried in the soil for long periods when fish have perished due to the drying of their habitat. Killifish are found in aquatic habitats that span a continuum from permanent and stable to seasonal and variable, thus providing a useful system in which to piece together the evolutionary history of this life cycle using natural comparative variation. I first review adaptations for life in ephemeral aquatic environments in killifish, with particular emphasis on the evolution of embryonic diapause. I then bring together available evidence from a variety of approaches and provide a scenario for how this annual life cycle evolved. There are a number of features within Aplocheiloidei killifish including their inhabitation of marginal or edge aquatic habitat, their small size and rapid attainment of maturity, and egg properties that make them particularly well suited to the colonization of ephemeral waters.

SUMMARY The aim of this study was to evaluate the role of adiponectin in the delayed embryonic development of Cynopterus sphinx. Adiponectin receptor (ADIPOR1) abundance was first observed to be lower during the delayed versus non-delayed periods of utero-embryonic unit development. The effects of adiponectin treatment on embryonic development were then evaluated during the period of delayed development. Exogenous treatment increased the in vivo rate of embryonic development, as indicated by an increase in weight, ADIPOR1 levels in the utero-embryonic unit, and histological changes in embryonic development. Treatment with adiponectin during embryonic diapause showed a significant increase in circulating progesterone and estradiol concentrations, and in production of their receptors in the utero-embryonic unit. The adiponectin-induced increase in estradiol synthesis was correlated with increased cell survival (BCL2 protein levels) and cell proliferation (PCNA protein levels) in the utero-embryonic unit, suggesting an indirect effect of adiponectin via estradiol synthesis by the ovary. An in vitro study further confirmed the in vivo findings that adiponectin treatment increases PCNA levels together with increased uptake of glucose by increasing the abundance of glucose transporter 8 (GLUT8) in the utero-embryonic unit. The in vitro study also revealed that adiponectin, together with estradiol but not alone, significantly increased ADIPOR1 protein levels. Thus, adiponectin works in concert with estradiol to increase glucose transport to the utero-embryonic unit and promote cell proliferation, which together accelerate embryonic development. Mol. Reprod. Dev. 81: 1086–1102, 2014. © 2014 Wiley Periodicals, Inc.

In the silkworm, Bombyx mori (Lepidoptera: Bombycidae), embryonic diapause is under maternal control and the decision between diapause and direct development in bivoltine strains depends on environmental factors such as temperature and photoperiod experienced by the preceding generation. We reared ten bivoltine silkworm strains (c10, g32, k06, n25, p21, p22, p24, p44, p50 and p63) under various thermal and light conditions and examined the incidence of embryonic diapause in the next generation to identify strains in which the incidence of diapause can be controlled within the range of 0 to 100% by adjusting a single ambient parameter. Some strains were clearly destined towards either diapause or direct development. The diapause incidence in the c10, p22 and p50 strains was controlled by temperature during the egg stage (0% at 18°C and 100% at 25°C), that in the p50 strain was also determined to be dependent on illumination during the egg stage (0% under continuous darkness and 100% under continuous illumination), and photoperiod during the larval stage regulated diapause in p44 and p50 (0% and 100% under long-day and short-day photoperiod, respectively), when all other external parameters remained constant under each experimental condition. These diapause-controllable silkworm strains might serve as model systems for studies of insect diapause, especially for the differential screening of related factors.

Numerous mammalian taxa exhibit reproductive delays, pauses in reproduction that occur between mating and fertilization, between fertilization and implantation of the embryo, or after an embryo has implanted. Of the 27 mammalian orders, 9 are known to exhibit reproductive delays, including Diptrotodontia, Dasyuromorphia, Eulipotyphyta, Cingulata, Carnivora, Rodentia, Chiroptera, Lagomorpha and Cetartiodactyla. Most researchers interested in delays have focused on their evolutionary origins. However, the consequences of these delays have not been considered fully. Given the lengthening of the period over which reproduction occurs, it is possible that this unique aspect of reproduction facilitates post-copulatory sexual selection. When considered in the context of sexual selection, delays may allow sperm competition and female manipulation of fertilization (cryptic female choice) as well as other post-copulatory processes. We investigate the potential for reproductive delays to facilitate post-copulatory sexual selection and suggest avenues for research that may further our knowledge of sexual selection. We also provide a general review of reproductive delays in mammals.

Although rats in various stages of pregnancy are routinely shipped by vendors, the effects of shipping on pregnancy outcomes have not been reported. This study examined the effects of shipping rats 1 day after mating. Two outbred stocks, (Crl:CD(SD), Crl:WI(Han)) and one inbred strain (F344/Crl) of rats (n=300/strain) were mated in a vendor barrier room at 3-month intervals five times, and either shipped the next day (total time in transit ∼24 hr) or held in the room of origin until parturition. The pregnancy status, length of gestation, number of pups born per female, sex ratio of pups born, and neonatal mortality were compared between transported and nontransported rats. These pregnancy and litter parameters were also compared among strains and examined for seasonality; no seasonal effects were observed. Neonatal mortality was negligible at less than 2% in any of the groups. All sex ratios were normal. Transportation affected pregnancy rates only in the F344/Crl, in which 81.8% of the nontransported versus 70% of the transported rats had pups (p=0.025). Overall, slightly fewer transported rats were pregnant, but they had larger litters (10.08 compared with 9.68, p=0.02, pooling across all three strains) so produced the same numbers of pups. A total of 77±8% of transported rats had gestation periods of 22 days or more compared with only 52±10% in the nontransported rats. The reason for larger litters in transported females is unclear. Longer gestation in transported females may be due to facultative embryonic diapause, which might have implications for reproductive toxicology.

Diapause in Bombyx mori eggs is induced by temperature and photoperiod at the stage of embryonic development in the maternal generation. In those diapause eggs, Esterase-A4 is suggested to serve as a diapause-termination timer (TIME-EA4), because its ATPase activity shows an interval-timer elevation after acid treatment or chilling of eggs to break diapause. To clarify whether the timed ATPase activity of TIME-EA4 is related to its gene (Ea4) expression, we analyzed Ea4 mRNA of eggs in diapause-inducing environmental conditions. Reverse transcription PCR (RT-PCR) analysis showed that the level of Ea4 mRNA was lower in 15DD than in 25LL (P 0.05). Furthermore, expressed sequence tag profile and gene microarray analysis demonstrated that Ea4 showed stage-specific and tissue-specific expression during postembryonic stages, high Ea4 mRNA in the spinning and eclosion stages, and in integument and head, but low in gonads of fifth-instar day-3 larvae. Then we analyzed the relationship between TIME-EA4 ATPase activities and Ea4 gene expression. The ATPase activities in diapause eggs laid by the resultant adults transiently elevated after treatments to break diapause, that is, at 1.5 h after common-acid treatment and day 13 after chilling at 5°C. However, these elevations of enzyme activities were not accompanied by any increases in Ea4 mRNA levels. In conclusion, the termination of the Bombyx embryonic diapause is related to TIME-EA4 but not to its gene expression.

A paucity of data on the reproductive cycle of crabs in the family Lithodidae inhibits both the development of management strategies and the formulation of hypotheses regarding the evolution of lithodid life histories. Life-history parameters of Lopholithodes foraminatus from British Columbia, Canada, were investigated based on 26 females maintained in the laboratory and supplementary observations on other living and preserved animals. The rate of embryonic development was determined by measuring the percentage area occupied by yolk in lateral views of eggs removed from brooding females throughout development. Females of L. foraminatus exhibited biennial reproduction including an 18-month brooding period. Females molted, mated, and extruded eggs in mid-summer, and did not release larvae until late winter or early spring of the second year after fertilization. Embryogenesis included a 12-month diapause at the gastrula stage. Females released larvae over a mean interval of 69 d, the longest reported for any lithodid. While the development stage of embryos was observed to be heterogeneous within a brood, no spatial gradient in development rate was observed, calling into question the oxygen limitation hypothesis of extended hatching. Biennial reproduction of individuals of L. foraminatus may be a consequence of a relatively low-quality habitat. Relative to annual reproduction, biennial reproduction halves the potential rate of increase of a population and increases vulnerability to overharvesting, suggesting that L. foraminatus is not a good candidate for commercial exploitation. The adaptive value of embryonic diapause is uncertain and warrants further research.

The effects of temperature on maintenance and termination of embryonic diapause were investigated in Jining (35.4� N, 116.6� E) and Sihong (33.5� N, 118.2� E) strains of the Chinese rice grasshopper, Oxya chinensis Thunberg (Orthoptera: Catantopidae). Eggs of both strains entered diapause when incubated at 30, 25, or 20 � C. Chilling at 8 � C had an evident effect on diapause termination and almost all eggs chilled for 60 days ended diapause development. Chilling of eggs at 8 � C for only 20 days failed to result in any hatching at 20 � C, suggesting that such level of chilling was not enough to induce diapause termination. However, the treatment combining incubation of eggs at 30 � Cf or varying lengths of time with subsequent incubation to 20 � C had a distinct effect on the completion of diapause of the eggs. The results indicate that there were two temperature optima, that is, low temperature (chilling) and high temperature, for diapause development in this grasshopper species. Incubation of chilled eggs at 20 � C for 5–15 days followed by further incubation at 25 � Cr educed termination of diapause significantly compared with the eggs only chilled at 8 � C. Exposure of eggs chilled at 8 � Ct o ap ulse of 25� C from 1 to 7 days, separated by a 20-day interval at 8 � C, resulted in a decrease in the percentage of successfully hatched eggs as the length of the pulse of 25 � C increased. The results suggest that diapause intensity may be restored at moderately high temperatures. This reversible change in diapause intensity would play an important role in maintaining diapause before winter.

To investigate geographic adaptation of the migratory locust Locusta migratoria in China, locusts were collected from six localities, ranging from 47.4°N to 19.2°N. Using offspring from the various populations, we compared embryonic diapause, reproductive traits, cold-hardiness and adult body size. The incidence of embryonic diapause was influenced by the genetic makeup, parental photoperiod, and incubation temperature of the eggs. The northern strain (47.4°N) produced diapause eggs under all photoperiodic conditions, whereas the other strains produced a higher proportion of diapause eggs when exposed to a short photoperiod. The incubation temperature greatly influenced diapause induction. At a low temperature, all eggs entered diapause, even some of those from a tropical strain (19.2°N) in which no diapause was induced at high temperatures. Photoperiodic changes during the parental generation affected the incidence of embryonic diapause. Diapause intensity decreased with decreasing original latitude. Cold hardiness was compared by exposing eggs in diapause to either -10 or -20°C for various periods; the northern strain was more cold-hardy than the southern strain, although some eggs in the tropical strain were probably not in a state of diapause. Adult body size and head width showed a complicated pattern of variation along the latitudinal gradient, whereas egg pod size (egg pod width and egg number) and hatchling weight tended to decrease with decreasing latitude. These results reveal that L. migratoria has adapted to local environments and that the latitudinal gradient appears to play an important role in shaping L. migratoria life cycle and development.

In order to better understand the relationship between dopamine and the release of diapause hormone into the blood, we cloned and characterized cDNAs encoding Bombyx mori dopamine receptor-1 and -2 (BmDopR1 and 2) from the pupal brain-suboesophageal ganglion. BmDopR1 and 2 had high similarities to group 1 (Drosophila melanogaster DOP1 and Apis mellifera DOP1) and group 2 (D. melanogaster DopR99B, A. mellifera DOP2 and Papilio xuthus DOP1), respectively. When BmDopR1 and 2 were expressed in human embryonic kidney (HEK) cells, they responded to dopamine by increasing intracellular cAMP levels, thus indicating the presence of D1-like receptors. There were no clear differences in BmDopR1 and 2 mRNA levels between brain-suboesophageal ganglion complexes of diapause and nondiapause egg producers during pupal-adult development. BmDopR1 and 2 mRNAs were concentrated in the mushroom body calyx rather than in the suboesophageal ganglion. Taking into account the results of earlier experiments on excised regions corresponding to mushroom bodies, BmDopR1 and 2 in the mushroom body apparently play a role in the release of diapause hormone.

Cold and desiccation tolerance was investigated in the eggs of the band-legged ground cricket Dianemobius nigrofasciatus in relation to embryonic diapause. Diapause eggs were more tolerant to both desiccation and cold than non-diapause eggs. In addition, diapause-destined eggs on day zero (0–12 h after being laid) already showed high tolerance to these stresses before entering diapause. This clearly indicates that stress tolerance, like diapause, is controlled by photoperiod, but is not directly associated with diapause itself. Because the acquisition of stress tolerance predates the onset of diapause, it is plausible that diapause programming during some period before the onset of diapause is involved in the acquisition of stress tolerance. Weights and sizes were nearly identical in short-day and long-day eggs until day five. Sorbitol, a major sugar alcohol in eggs of D. nigrofasciatus, was accumulated at the same level in short-day and long-day eggs on days zero and five. These results indicate that the surface-to-volume ratio as well as the accumulation of sugar alcohol is not involved in the acquisition of stress tolerance. Maternal factors are clearly involved in the acquisition of stress tolerance in D. nigrofasciatus eggs, but the physiological mechanisms underlying the tolerance are still unclear.

Environmental cues for embryonic diapause termination (EDT) were investigated in the laboratory-produced encysted dormant embryos of the brine shrimp, Artemia franciscana. The cysts were spawned and kept throughout in a 2% sea salt solution. They were activated by various temperatures of the temperate zones or by continuous dark condition (DD), resulting in a state of EDT, and were thereafter able to resume their subsequent development and hatch under appropriate conditions. The level of EDT was conveniently assayed by a hatch test observed within 2 days at 28 degrees C under continuous light condition (LL). A cold treatment of the newly spawned dormant cysts, at 4 degrees C under DD for more than 100 days, resulted in more than 95% hatch of the dormant cysts. Similar treatments of the dormant cysts but at room temperature or 28 degrees C led to significantly different results (30-40% hatch). Almost all the residual non-hatched cysts derived from the above could hatch after an additional cold treatment (at 4 degrees C under DD for about 100 days). This might prove to be latent partial bivoltine in Artemia. Meanwhile, a rearing condition (28 degrees C under LL) induced the newly spawned cysts to hatch scatteredly at and after 1 month, resulting in 22% cumulative hatch on the 92nd day after spawning. When the newly spawned dormant cysts were pretreated at 28 degrees C under DD for 5 or 14 days and then reared at 28 degrees C under LL, the cumulative hatch significantly increased (60%). These results are discussed with respect to probable diapause regulator(s) involved in EDT.

Eggs laid by adult female Dianemobius nigrofasciatus, reared under long-day (LD 16 : 8 h, 25 °C) or short-day (LD 12 : 12 h, 25 °C) conditions from the nymphal stage, are kept at several constant temperatures. At 22.5–30.0 °C, eggs laid by long-day adults show lower incidences of diapause than those laid by short-day adults. In both eggs laid by adults under long-day conditions and those under short-day conditions, the higher the temperature at which the eggs are kept, the lower the incidence of diapause. When eggs of long-day adults are exposed to a low-temperature pulse (10 °C, 24 h) on the day of deposition (day 0), the incidence of diapause increases. The low-temperature pulse on day 1 does not increase the incidence of diapause. By contrast, when the eggs of short-day adults are exposed to a high-temperature pulse (35 °C, 24 h) on day 0 or day 1, the incidence of diapause decreases. The temperature pulses on day 0 are more effective at diapause prevention. Staining of diapause eggs by the Feulgen–Rossenbeck method shows that the eggs enter diapause at the blastoderm stage, which is on day 1 or day 2 at 25 °C. The exposure of adults to long days and higher temperatures prevents the eggs from entering diapause. In D. nigrofasciatus, embryonic diapause is controlled by maternal effects, adult photoperiod and temperature, and egg temperature before or at diapause.

In bitterling Acheilognathus rhombeus, developmental arrest always occurred at stage D of the free-embryonic phase, regardless of incubation temperature. Developmental arrest was terminated only by a cold treatment at 4° C for 60-90 days, initiated 10 days post-hatching. After the termination of developmental arrest, free-embryos became larvae c. 6 months after hatching, regardless of the time of initiation and duration of the cold treatment. In hybridization experiments between A. rhombeus and several species of spring-spawning bitterlings, free-embryos became free-swimming larvae within 60 days after hatching in all experiments. Developmental arrest was not observed in any of the hybrids, regardless of parental sex. These results suggest that free-embryonic diapause in A. rhombeus is not induced by environmental factors, such as cold, but by genetic factors, which are recessive to those in spring-spawning bitterlings. Free-embryonic diapause in A. rhombeus appears to be an adaptation to winter, which might have evolved with reproduction in autumn among autumn-spawning bitterling species. This is the only report of free-embryonic diapause after hatching in fishes, and only the second example of diapause in fishes, along with annual killifishes (Rivulidae).

Cellular responses to cold-acclimation have not yet been studied in depth. To explore this field, we focussed on insect diapause development. Although embryonic diapause of Bombyx mori is sustained at 25 °C, chilling at 5 °C for 2 months causes diapause termination, a transition that is marked when the sorbitol dehydrogenase gene (SDH) is activated. To clarify the relationship between this activation and incubation at 5 °C, we isolated a novel cold-inducible gene, Samui. Expression of Samui mRNA and protein was activated after incubation at 5 °C for 5–6 days, lasted for another 30 days and then weakened. Exposure to 25 °C suppressed both mRNA and protein expression. In nondiapause eggs incubated at 5 °C, Samui was also up-regulated, although the expression was weaker. Samui contained nuclear localization-signals, a ssDNA-binding motif and a BAG domain similar to that of SODD/BAG-4. Because Samui could bind to HSP70, it is a member of BAG protein family. It is proposed that Samui serves to transmit the ‘5 °C signal’ for SDH expression in diapause eggs, while also protecting against cold-injures in nondiapause eggs, through binding to respective partners. This is the first report that a member of BAG protein family is up-regulated by cold.

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non-diapausing progeny (non-diapause type). Measurement of catecholamine concentrations in haemolymph and brain-subesophageal ganglia (Br-SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause-type than non-diapause-type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br-SGs were also much higher in diapause-type than non-diapause-type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate-instar and last-instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non-diapause-destined insects lay diapause-destined eggs at 59% and approximately 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br-SGs of non-diapause-type pupae 1 day after injection. Incubation of Br-SGs isolated from non-diapause-type day-2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br-SGs from the larval stage to early pupal stage, which results in laying of diapause-destined eggs by female adults through enhanced expression of the diapause hormone gene.

Four factors (moisture, light regime, temperature, food type) were examined for their effects on the embryonic diapause of Homichloda (Weiseana) barkeri (Jacoby) (Coleoptera: Chrysomelidae), a biocontrol agent for prickly acacia, Acacia nilotica (L.) Willdenew ex Delile (Mimosaceae). Moisture is critical for termination of diapause. A single wetting of eggs resulted in a low hatch rate while a sequence of wetting events followed by periods of dryness produced a high hatch rate. A relatively constant proportion of embryos within each batch initiated development at each wetting event, with hatching complete after the eighth wetting event in these trials. An extended interval between wetting events, tested at up to 23 days, did not result in a decreased overall hatch rate. A threshold time of exposure to moisture of between 3 to 6 h is required before development proceeds. The response of eggs to the moisture regime is seen as a strategy for taking advantage of available food after rainfall by terminating diapause, rather than merely a quiescent response to the absence of moisture. Temperature affected development time and the proportion of eggs that developed. Experimental manipulations of photoperiod and host-plant availability showed no effect on embryonic development.

Mouse blastocysts appear to be under direct inhibition from the uterine environ- ment, whereas no evidence of direct inhibition during diapause in the tammar wallaby has been observed. Normally developing (day 4) and quiescent mouse blastocysts were incubated for up to 12 hr in media supplemented with BSA, wallaby plasma, wallaby day 0 (day of removal of pouch young; RPY), day 5, or day 10 endometrial exudates at a concentration of 2 mg/ml of protein, and analyzed for rates of carbohydrate metabolism using fluorescence and radioisotopes. Rates of glu- cose uptake and lactate production by day 4 blastocysts increase after incubation with day 10 and day 5 wallaby exudates compared with rates by blastocysts incubated in BSA. Pyruvate uptake increased after 8 hr irrespective of incubation media, except for embryos incubated in day 0 exu- date, which maintained levels significantly lower than BSA-incubated embryos. Quiescent mouse embryos displayed a high ATP/ADP ratio during diapause (1.06 ± 0.24) which decreased after 4 hr incubation in all media (0.42 ± 0.05; P < 0.01) but embryos incubated in day 0 exudate media remained at a significantly higher level than embryos incubated in BSA. These results indicate that quiescent tammar endometrial exudate is not capable of initiating diapause in mouse em- bryos at the concentration used, but is able to slow the rate of reactivation of quiescent blasto- cysts. Importantly, reactivated wallaby exudate increases mouse blastocyst glucose metabolism and lactate production. It is possible that the quiescent tammar endometrial environment has an inhibitory factor necessary to maintain diapause in the tammar blastocyst. J. Exp. Zool. 283:590n 599, 1999. © 1999 Wiley-Liss, Inc. Embryonic diapause, or delayed implantation, is a state of quiescence known to be displayed in the blastocysts of about 100 eutherian and mar- supial species (Renfree and Calaby, '81). The tammar wallaby is capable of maintaining a vi- able blastocyst in diapause for up to 11 months in utero, when a pouch young is being suckled. This blastocyst does not undergo mitosis or dif- ferentiation and has low rates of metabolism throughout the period of quiescence compared with reactivating blastocysts. The blastocyst can be reactivated by removal of the sucking pouch young (RPY), and the resultant fetus is born around 26 days later. The mouse is also capable of facultative, lactational diapause. As in the tammar, the blastocyst is metabolically and de- velopmentally suspended until the sucking young are weaned or removed although this period is much shorter than that of the tammar. Although the exact mechanism acting on the blastocyst that controls diapause is not known, the endometrial environment has been shown to directly affect embryo development, during dia- pause as well as during normal development (Weitlauf, '94). Diapause can be induced in normally developing rat and mice blastocysts by transferring them to quiescent uteri (Dickman, '67), and quies- cent blastocysts of mice, rat, quokka, and wallaby can also be reactivated by transfer to an active uterus (Tyndale-Biscoe, '63, '70; Dickman, '67). Al- though the later stages of blastocyst reactivation of many eutherian mammals may depend on physi- cal stimulus between the uterus and the embryo (Blandau, '49), it is clear that as blastocysts are free-living in the uterine lumen during diapause and for some time throughout reactivation, the endometrial secretions must mediate communi- cations between embryo and endometrium (Daniel

The three investigated populations of migratory locust, Locusta migratoria L. (Orthoptera, Acrididae), reported in this paper show significant variation in diapause characteristics. The insects of the Heshuo Xinjiang population show diapause in egg stage regardless of the photoperiod of rearing condition, while the adults of the Beidagang Tianjin and the Hami Xinjiang populations produce diapause or nondiapause eggs according to the short or long photoperiod. From this research, we infer altitude plays an important role in inducing diapause. The long photoperiod which extends the duration of pre-oviposition period and reduces the oviposition frequency in the Beidagang and Hami populations shows no effect on those features of the Heshuo population. The duration at low temperature to end diapause in the Heshuo population is longer than that in the other two populations.

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice. Whether LIF plays a role in termination of embryonic diapause and initiation of implantation in carnivores, especially in species with obligate delayed implantation such as the mink, is not known. The objectives of this study were to clone the LIF coding sequence in the mink and determine its mRNA abundance in the uterus through embryonic diapause, implantation, and early postimplantation. We show that the mink LIF cDNA contains 609 nt encoding a deduced protein of 203 amino acids. The homologies are 80.6, 90, 88.2, 87.6, and 86.8% in coding sequence and 79.2, 90.1, 91, 90.1 and 85.4% in amino acid sequence with mouse, human, pig, cow, and sheep respectively. Glycosylation sites and disulfide bonds present in other species are generally conserved in the mink LIF sequence. Quantitation by polymerase chain reaction amplification indicates that LIF mRNA is expressed in mink uterus just prior to implantation and during the first two days after implantation, but not during diapause or later after implantation pregnancy. The abundance of LIF mRNA was significantly higher in the uterus at the embryo expansion stage (P < 0.05) than at days 1-2 of postimplantation. By immunohistochemical localization it was shown that LIF is expressed in the uterine epithelial glands at time of embryonic expansion and in early postimplantation. The coincidence of LIF expression with implantation in this species suggests that LIF is involved in the implantation process, and may be a maternal signal which terminates obligate embryonic diapause.

In Phodopus, all first litters are born after an 18 (or 19) day gestation. Birth of subsequent litters conceived during a postpartum estrus is routinely, but not always, delayed beyond 18 days. With an interval from first mating to birth of a second litter of as little as 36 days, Phodopus have the most compressed reproductive cycle of any eutherian mammal. Although pups of the Siberian hamster P. sungorus gain thermal independence from maternal care faster than pups of the Djungarian hamster P. campbelli, no species difference in the extent of delay to second litters was found. However, poor growth of first litter pups as a result of unintentional limitation of water bottle access increased delays before birth of a second litter in P. sungorus. Weight of pups in the first litter was a good predictor of the length of delay. In P. campbelli, females with large pups early in lactation were the only ones to deliver a second litter without delay, and short delays enhanced weaning weights for pups in the first litter. Patterns in P. sungorus were similar. The timing of the delay in second litters was investigated in P. campbelli. Delayed implantation during second pregnancies was common. Postimplantation embryonic diapause occurred in 43% of females following unilateral ovariohysterectomy on day 8 of a first pregnancy. As pup growth curves also implicated a postimplantation embryonic diapause, Phodopus may be the only known mammalian genus outside the order Chiroptera in which postimplantation diapause can occur.

The mouse, Mus musculus, can maintain blastocysts in embryonic diapause in the uterus while suckling young. This study used microfluorimetry to simultaneously examine glucose and pyruvate uptake by quiescent blastocysts, and at four hourly intervals after administration of the reactivating stimulus, oestradiol-17 beta. Following the non-invasive analysis of energy metabolism, blastocysts were incubated in colcemid (0.2 mg/ml), and mitotic activity determined. Mitoses and cell numbers in reactivated embryos increased significantly within 8 and 12 hours, respectively, after oestradiol-17 beta administration, compared to those of diapause (control) blastocysts (0.5 +/- 0.1 vs. 0.22 +/- 0.03 mitoses/embryo; P < 0.05, and 141.8 +/- 1.5 vs. 133.8 +/- 2.4 cells/ embryo; P < 0.05). Similarly, pyruvate uptake by reactivating blastocysts (9.3 +/- 1.1) was significantly higher than controls (5.8 +/- 0.8 pmol/embryo/hour; P < 0.05), within 4 hours of oestradiol-17 beta, but by 16 hours after oestradiol-17 beta administration, pyruvate uptake by reactivating blastocysts was no longer significantly different from the delayed controls. In contrast, significant differences in glucose uptake between the reactivated and control groups were not evident until 16 hours after oestradiol-17 beta (reactivating, 14.9 +/- 1.5; control, 10.6 +/- 1.7 pmol/embryo/hour; P < 0.05). These results demonstrate that pyruvate rather than glucose could supply the additional energy required during the first 12 hours of reactivation in the mouse, but from 16 hours after injection of the reactivating stimulus oestradiol-17 beta, glucose is the predominant energy source.

Embryonic diapause induction in the Senegalese grasshopper, Oedaleus senegalensis Krauss (Orthoptera: Acrididae), is influenced both by the photoperiod and the temperature experienced by females. High temperatures (40°C) and long photoperiods (LD 14:10h), which characterize the beginning of the rainy season in the Sahel, cause non-diapausing eggs to be laid. Lower temperatures (25°C) and shorter photoperiods (LD 12:12h), which occur at the end of the rains, result in the production of diapausing eggs. At 30°C and constant photoperiods, O. senegalensis exhibited a long-day-short-day response with critical photoperiods of c. 13 h and c. 20 h, only the former value being of ecological significance. The photoperiodically sensitive stages to diapause induction in females occurred from the fifth stadium onwards. Temperature also affected the coloration of both nymphs and adults. Dark-black and pale-white individuals were produced by low (25°C) and high (40°C) temperatures respectively, whereas an intermediate temperature (30°C) produced individuals which were greyish brown. These results are discussed in relation to the ecology of O. senegalensis.

In a temperate zone population of the grasshopper Caledia captiva (Fabnerus) (Orthoptera Acrididae), facultative embryonic diapause provided the basis for a number of aspects of life cycle flexibility Most offspring produced during the summer entered diapause, destined to overwinter as embryos and hatch in the spring, but a small percentage developed directly to hatching, overwintered as nymphs and reproduced in the spring The two different developmental pathways often occurred among the offspring of the same mother, and could represent a bet-hedging adaptation Offspring of the small overwintering generation were influenced by a maternal effect which greatly diminished their likelihood of entering diapause This is an adaptation to prevent an inappropriate developmental delay that would negate the fitness advantage of an extra generation Offspring produced in the autumn failed to develop to the stage at which diapause intervenes before winter and overwintered at a pre-diapause stage They did not enter diapause when development resumed in the spring Facultative diapause in this case also prevented an inappropriate developmental delay in the spring, and provided females with the option of reproducing toward the end of the growing season Life cycle flexibility appears to have evolved to exploit a climate with a short growing season but mild winter

The harbour seal (Phoca vitulina) has delayed implantation, precise annual birth timing, and significant latitudinal variation in birth timing. The birth timing patterns of four distinct groups of seals, including colonies of P. v. vitulina and colonies and captive individuals of P. v. richardsi, were examined using population-based photoperiod analysis to assess the role of photoperiod in setting annual birth timing. This analysis simultaneously determined the time, relative to birth, at which photoperiod response was likely to occur and the critical photoperiod. Despite marked differences in birth timing patterns, a high level of agreement was found among groups for the timing of photoperiod response. The two subspecies, however, demonstrated significantly divergent critical photoperiods. Eastern Atlantic harbour seals were exposed to a common critical photoperiod of 11.7 h/day on the 268th pre-partum day. Wild Pacific harbour seals were exposed to 14.3 h/day on the 283rd pre-partum day. These times corresponded to the estimated occurrence of blastocyst implantation. Using the above information, three small captive populations were subjected to artificially prolonged photoperiods during the period of embryonic diapause to test whether subsequent birth timing could be delayed. Technical difficulties invalidated results at two sites. At the third and largest colony, the mean pupping date of six individuals was significantly delayed by 10.7days. The precision and latitudinal variation of annual birth timing in the harbour seal are due to a response to photoperiod which occurs immediately prior to blastocyst implantation. The critical photoperiod, however, is divergent among subspecies and, thus, has probably evolved allowing seasonal adaptation. Similar environmental signalling has been described for California sea lions and northern fur seals and represents the likely timing mechanism for most pinniped species.

This study is the first to demonstrate the capacity of an arthropod egg, that of a tropical walking stick Extatosoma tiaratum (Macleuy), to absorb water vapour from the air. This species diapauses both as an early embryo and then again as a pharate first instar larva, and both stages are capable of absorbing water vapour. Water vapour absorption occurs at lower humidities and at a lower rate for an egg in early embryonic diapause (c. av 0.30, 0.516 mg h-1δv-1) than in the diapausing pharate first instar (c. av 0.60, 0.725 mgh-1δav-1) at 25dC. In addition to having the capacity to gain water at very low vapour activities, water is efficiently conserved as indicated by the low rate of water loss (0.015% h-1 in the early embryo and 0.046% h-1 in the pharate larva at 25dC). Eggs that have been killed lose water when held at a hydrating vapour activity, thus implying that active uptake contributes to net absorption. Wax block experiments suggest that water is absorbed over the entire chorionic surface. Eggs of five other insect species that were examined [Lymantria dispar (L.), Bombyx mori (L.), Antheraea polyphemus (Cram.), Oncopeltus fasciatus (Dallas) and Diaferomera femorata (Say)] lacked the ability to absorb atmospheric water.

The anatomy of female reproductive tracts collected from 28 Acrobates pygmaeus (Shaw) is described. The tract differs from that of other small possums of the families Burramyidae and Tarsipedidae by having a prominent septum that divides the left and right sides of the vaginal system for most of its length. A median birth canal occurs at parturition but closes within 3–4 days of birth. In south-eastern Australia, most female A. pygmaeus produce two litters within the breeding season (July to February). A post-partum oestrus follows the birth of the first litter and the resulting embryos undergo a period of embryonic diapause. The embryos grow slowly during diapause, until the unilaminar blastocysts become approximately 2.0 mm in diameter, and contain about 2,000 cells. The factors influencing reactivation after diapause remain unclear, since no embryos past the blastocyst stage were found. Post-partum oestrus may follow the birth of the second litter, but if it occurs, the ova either remain unfertilized or degenerate after fertilization. Females are anoestrus during the non-breeding season. Corpora lutea in the ovaries seldom outnumber blastocysts in the uteri or teats in the pouch. During diapause they undergo a pattern of growth similar to that of the blastocysts and enter a quiescent phase. Corpora lutea disappear within nine days of birth. The pattern of female reproduction in this species is similar to that of other small possums of the families Burramyidae and Tarsipedidae, but is distinct from those of other marsupials.

Nothobranchius guentheri is an East African annual fish found in temporary bodies of water in areas of alternating rainy and dry seasons. During the dry season the embryos may be exposed to temperature extremes. Embryos have been reported to undergo diapause (developmental arrest), and are thus able to withstand the variable durations of the dry seasons. Since the embryos appear to endure long durations in diapause, and since in diapause they may be exposed to large variations in temperature, we have attempted to determine the thermal tolerance and responsiveness of embryos at diapause and at stages exclusive of diapause. Embryos were exposed to temperatures ranging from −8-40°C for variable durations of time, and survival, abnormalities and developmental rates determined. Both Diapause I and Diapause II embryos were much less sensitive to temperature extremes than were the other stages, and Diapause II was much less sensitive than Diapause I. Abnormalities only occurred under certain temperature exposures but showed the highest frequency at the early stages of development. Short exposure of Diapause I embryos to −8 and 3.4°C eliminated Diapause II, while short exposure to 40°C enhanced its duration. Although it has been proposed that the onset and duration of the diapauses are denendent on the oxygen concentration of the water, we propose that temper ature may also be another imposrant factor.

Between August 1981 and October 1982, 76 female and 50 male of the estimated 400 Bennett's wallabies at Whipsnade were caught and examined. Sixty-four of the adult females inspected (84%) were carrying pouch young. A small captive breeding colony was established in the laboratory. Weight and growth curves were established for captive born pouch young and these curves were used to determine the approximate age of pouch young examined at Whipsnade. Eighty-four percent of births occurred in August and September, exactly a six month difference from births reported for this species in Tasmania. The mean length of pouch life of wild living wallabies was 247 days with a range of185–284 days. Young wallabies were observed accompanying their mothers for up to 204 days after emerging permanently from the pouch. Sixty-three percent of young vacated the pouch in May. Sixty-three percent of the total (104) pouch young examined in the course of this study, from animals caught or from post mortem records, were male, but teat selection showed a random distribution. The gestation period from removal of pouch young to day of birth was 27·3 days. The Bennett's wallaby showed precise seasonal breeding with embryonic diapause that may extend for up to 11 months.

The initiation and pattern of embryonic development in Leptophyes punctatissima (Bosc) (Tettigoniidae) are shown to be variously dependent on temperature. Immediate high temperature (30°C) facilitated rapid and direct embryogenesis to the stage of diapause in the fully formed embryo. Slightly lower temperature (25°C) resulted in a delay before embryogenesis started, and this delay was greatly extended as an initial diapause if incubation temperatures were decreased (20-16°C). Still lower initial temperatures (8–12°C) facilitated subsequent development at 20–30°C. These responses all increased as a function of exposure time. Once initiated, the rate of development was temperature dependent, but competence to tolerate high temperature, and ability to continue development at low temperatures, changed with age. In general, the developmental temperature range appears to be lowered with age. None of these different treatments had any effect on the late embryonic diapause. According to the temperatures prevailing at oviposition, Leptophyes could be an annual or a biennial species; the biennial pattern is normal.

Embryonic diapause of the silkworm, Bombyx mori, is generally induced by temperature and photoperiod during the egg stage of the previous generation and not in the larval stage. However, when silkworm larvae are reared on an artificial diet instead of mulberry leaves, their diapause is strongly affected by temperature and photoperiod experienced in the larval stage, with a distinct long-day response for diapause induction. Moreover when larvae which have been reared on artificial diet under long-day condition are fed mulberry leaves even for a short period of time, most of the resultant female adults lay diapause eggs. These results suggest that the photoperiodic response of larvae for diapause induction may be strongly suppressed by some components in mulberry leaves.

The transient embryonic diapause associated with delayed implantation in mice is characterized by decreases in the rates of synthesis of RNA and protein as well as a cessation of development. The present experiments were undertaken to examine the possibility that controls on protein synthesis at the level of translation of mRNA provide a regulatory mechanism in this situation. Rates of peptide chain elongation were determined in dormant embryos as well as in embryos that were reactivated either in vivo by estradiol-17 beta or by incubation in vitro. In dormant embryos the rate of peptide elongation was found to be approximately half that in active embryos. Although this change in translational efficiency appears to be sufficient to account for previously observed differences in overall rates of protein synthesis in dormant and reactivated embryos, the possibility that some changes also occur at the level of transcription during reactivation is not ruled out.

During the breeding season from January to June, female wallabies which are suckling a young animal in the pouch may carry a dormant embryo (lactational quiescence). Removal of the pouch young during this period results in a resumption of embryo development. In the latter half of the year, the embryo will not reactivate after removing the suckling young (seasonal quiescence). In this situation, development resumes spontaneously in late December or may be induced prematurely by progesterone treatment. The response of the genome of quiescent macropod blastocysts was studied during the early period of growth. Changes in the transcriptional activity of the embryo cells were measured by assay for endogenous RNA polymerases. Embryos actively synthesized RNA during both lactational and seasonal quiescence. Termination of seasonal quiescence resulted in increases in RNA polymerase activities within the nucleolus and nucleoplasm of the cell. This occurred on the day following the summer solstice, December 22, 1974, in animals captured in the wild, or within 48 hours of administration of progesterone. Embryos which were induced to resume development during the breeding season also showed increases in nucleolar and nucleo-plasmic polymerase activities within five days of removal of suckling young from the pouch. In all situations, the response of the nucleolar enzymes was greater than that of the nucleoplasmic enzymes. This is in agreement with other observations of the regulation of gene activity in growth-stimulated cells.

Bennett's wallaby (Macropus r. rufogriseus) of Tasmania give birth from late January to early August in marked contrast to the Red-necked wallaby (M. r. banksianus) of mainland south-eastern Australia which produced young in all months. Within the breeding season however, the lengths of the oestrous cycle and gestation period are similar in the two forms and did not differ by more than 0.5 days. The gestation period of about 30 days extended to almost the length of the oestrous cycle of approximately 33 days. Birth was closely followed by mating which normally resulted in fertilization and subsequent embryonic diapause. Renewed blastocyst development was initiated by removal or loss of a pouch young and birth followed about 27 days later. Unlike other macropodids with a similar breeding pattern, birth, as a result of renewed blastocyst development near the end of a large young's pouch life, did not occur within a day or two of the permanent emergence of the young, but followed 16 to 29 days later. In M. r. rufogriseus, young that left the pouch permanently in the non-breeding period were not replaced by new young until the beginning of the next breeding season two to four months later, and blastocysts resulting from mating of females without pouch young at the end of the breeding season remained quiescent until the next breeding season five to eight months later. Females of both subspecies first mated at an age of about 14 months, and males were producing mature spermatozoa by about 19 months. Young first left the pouch for short periods at about 230 days of age and permanently at about 280 days. Observations are also given on reproductive behaviour, interpretation of vaginal smears, sex ratio of young, selection of teat by pouch young, and development of morphological features in known-age young that may be used as an aid in age determination.

Ecdysteroids were studied in relation to embryonic development and diapause of the silkworm, Bombyx mori. The majority of the ecdysteroids was found to be in the conjugated form, and minor part, in the free form. In the developing eggs, 2-deoxyecdysone, 2-deoxy-20-hydroxy-ecdysone and 3-epi-ecdysone were found to have the free ecdysteroid form as well as the conjugated ecdysteroid form. Ecdysone and 20-hydroxyecdysone almost exclusively had the conjugated form. The concentration of ecdysteroids in the embryo was higher than that in yolkplasm in the early embryonic stages. During the embryonic diapause, the concentration of free ecdysteroids decreased to a low level while the conjugated form maintained the original level.

In seasonally breeding mammals, the hormone melatonin, produced at night by the pineal gland, is known to be important in transducing the effect of photoperiod in timing reproduction. In the Bennett's wallaby, an unimplanted unilaminar blastocyst is held in a state of seasonal diapause from mid-winter to mid-summer. Here we show that an implant of the hormone melatonin rapidly terminates seasonal diapause in this species. Blastocyst reactivation is not accompanied by a significant reduction in levels of the hormone prolactin, thereby refuting earlier suggestions that this hormone is responsible for maintaining seasonal embryonic diapause.

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 20dC, 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 30dC, 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 27dC 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 (30-30dC). At 30dC, sunmer diapause is neither induced nor maintained.

Environmental cues terminating embryonic diapause and a potential regulatory mechanism involving depressed pH were investigated in the encysted embroyos of the brine shrimp, Artemia. Two species, Artemia franciscana and Artemia monica, were used for this comparative study. To determine if alteration of intracellular pH (pHi) can terminate diapause, the pHi of diapause cysts was manipulated by exposure to NH3 and CO2. Alkalinization did not result in activation of cysts from either species but acidification activated A. franciscana cysts. A. monica did not respond to acidification. The pHi of aerobic diapausing embryos was determined using 31P nuclear magnetic resonance spectroscopy. During diapause, embryos from both species had pHi's similar to those reported for activated (nondiapause) embryos, indicating that diapause is not imposed by depressed pHi, as is the case during suppression of metabolism under anaerobic conditions. Diapause embryos were exposed to varying conditions of temperature, salinity, and oxygen to determine what conditions are necessary to break diapause. A. franciscana cysts were activated by cold temperature or salinites above 2.0 M NaCl, while A. monica cysts only responded to cold temperature. The availability of oxygen did not influence either population's ability to terminate diapause. The results are discussed with respect to each population's environment and potential mechanisms of diapause regulation.