Embryo ecology: Developmental synchrony and asynchrony in the embryonic development of wild annual fish populations

Embryo–environment interactions are of paramount importance during the development of all organisms, and impacts during this period can echo far into later stages of ontogeny. African annual fish of the genus Nothobranchius live in temporary pools and their eggs survive the dry season in the dry bottom substrate of the pools by entering a facultative developmental arrest termed diapause. Uniquely among animals, the embryos (encased in eggs) may enter diapause at three different developmental stages. Such a system allows for the potential to employ different regulation mechanisms for each diapause. We sampled multiple Nothobranchius embryo banks across the progressing season, species, and populations. We present important baseline field data and examine the role of environmental regulation in the embryonic development of this unique system. We describe the course of embryo development in the wild and find it to be very different from the typical development under laboratory conditions. Development across the embryo banks was synchronized within and across the sampled populations with all embryos entering diapause I during the rainy season and diapause II during the dry season. Asynchrony occurred at transient phases of the habitat, during the process of habitat desiccation, and at the end of the dry season. Our findings reveal the significance of environmental conditions in the serial character of the annual fish diapauses.     

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

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

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.     

The evolution of an annual life cycle in killifish: adaptation to ephemeral aquatic environments through embryonic diapause

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.     

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

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

Oxygen consumption by developing and diapausing eggs of Eupholidoptera smyrnensis (Orthoptera: Tettigoniidae)

Oxygen consumption by eggs of the Mediterranean bush-cricket Eupholidoptera smyrnesis was manometrically measured at 18, 24, 30 and 33°C. The aim was to study changes in oxygen consumption during embryonic development, and to compare a facultative initial and a facultative penultimate diapause with subitaneous developing eggs and an obligatory final diapause. In developing eggs, oxygen consumption increased as the embryo grew, but remained constant during katatrepsis. A comparison of the different diapauses revealed that in the initial diapause, oxygen consumption was lower than that of the corresponding embryonic stage in subitaneous eggs; the temperature dependence of oxygen consumption was also low. In contrast, in penultimate and in final diapause, oxygen consumption remained at the same high level as in developing embryos of the same size, and the temperature dependence of oxygen consumption was high. The results suggest that it is energetically profitable to the embryo to spend the hot season in a facultative initial and/or penultimate diapause.     

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.     

Incubation media affect the survival,pathway and time of embryo development in Neotropical annual fish Austrolebias nigrofasciatus (Rivulidae)

To analyse the survival, pathway and time of embryo development in the annual fish Austrolebias nigrofasciatus eggs were monitored in four liquid media and two damp media under experimental conditions for 130 days until their development was complete. Eggs kept in the same breeding water from oviposition remained in diapause I (DI) during all experiments. In constrast, up to the stage prior to entering diapause II (DII), the other media had no influence on development. Embryos at this stage (DII), however, show longer development time when treated in medium with water and powdered coconut shell so that about 80% of embryos remained in DII at 100 days. In contrast, all other treatments had a significantly lower proportion of embryos remaining in DII. When treated with Yamamoto's solution in humid media, embryos showed the fastest development. The first fully developed embryos (DIII) were seen at 27 days after oviposition. It took an average of 46–58 days for 50% of eggs in each treatment to reach DIII. Compared with other studies, survival in all incubation media was high at between 70 and 98%. Taken together, it can be concluded that all incubation media were found to be viable for maintaining embryos. Altering developmental trajectories through the manipulation of diapauses in different media makes this species a potential model organism for laboratory studies.     

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage,diapause III

Embryo of an annual fish (Austrolebias charrua) in the last dormancy stage, diapause III. The embryo, surrounded by a transparent vitelline envelope, is in the pre‐hatching stage. A prominent eye and part of the pigmented body and tail are apparent. Why annual fishes? Annual fishes (Order Cyprinodontiformes) are a special kind of teleost, found in Africa and South America, with developmental strategies closely related to their life cycle. These fishes inhabit temporary pools that undergo drying during summer, when all adults die. The embryos remain buried in the bottom mud and are resistant to desiccation. In the subsequent rainy season they hatch a few hours after the pool is flooded and a new reproductive cycle begins. This developmental pattern is characterized by the presence of a unique stage between cleavage and embryogenesis, dispersion‐aggregation of blastomeres and because the embryos show reversible developmental arrests (diapauses) at different stages. Annual fish embryos are transparent, large, hardy and easy to maintain in the laboratory. Adults show continuous production of eggs and juveniles reach sexual maturity a few weeks after hatching (an unusual condition in fishes). Their particular developmental features confer unique opportunities for research on cell behavior during early development, the effect of environmental factors on development, the regulation of diapauses and the mechanisms involved in sex determination, among others topics. Image provided by Nibia Berois, Universidad de la República, Montevideo, Uruguay.     

Adaptation in a variable environment: Phenotypic plasticity and bet‐hedging during egg diapause and hatching in an annual killifish

Two ways in which organisms adapt to variable environments are phenotypic plasticity and bet‐hedging. Theory suggests that bet‐hedging is expected to evolve in unpredictable environments for which reliable cues indicative of future conditions (or season length) are lacking. Alternatively, if reliable cues exist indicating future conditions, organisms will be under selection to produce the most appropriate phenotype —that is, adaptive phenotypic plasticity. Here, we experimentally test which of these modes of adaptation are at play in killifish that have evolved an annual life cycle. These fish persist in ephemeral pools that completely dry each season through the production of eggs that can remain in developmental arrest, or diapause, buried in the soil, until the following rainy season. Consistent with diversified bet‐hedging (a risk spreading strategy), we demonstrate that the eggs of the annual killifish Nothobranchius furzeri exhibit variation at multiple levels—whether or not different stages of diapause are entered, for how long diapause is entered, and the timing of hatching—and this variation persists after controlling for both genetic and environmental sources of variation. However, we show that phenotypic plasticity is also present in that the proportion of eggs that enter diapause is influenced by environmental factors (temperature and light level) that vary seasonally. In nature there is typically a large parameter zone where environmental cues are somewhat correlated with seasonality, but not perfectly so, such that it may be advantageous to have a combination of both bet‐hedging and plasticity.     

The adaptive role of facultative embryonic diapause in the grasshopper Caledia captiva (Orthoptera: Acrididae) in southeastern Australia

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     

Local variation in embryo development rate in annual fish

Extreme asynchrony in embryo development, a typical feature of annual killifish living in temporary pools, represents a bet‐hedging strategy to cope with unpredictable rainfall. African annual killifish are distributed across a large precipitation gradient, raising the potential for local adaptation in the degree of developmental asynchrony (e.g. higher in arid areas, lower in humid areas). Eight populations of two sister species, Nothobranchius furzeri and Nothobranchius kadleci, from sites along the rainfall gradient were tested and compared for asynchrony and duration of embryo development. Degree of asynchrony and mean duration of embryo development did not differ across the examined range. Despite generally high developmental variability, fish from more arid regions (where rain is more erratic) produced a significantly higher proportion of short‐developing embryos. Comparable developmental asynchrony, regardless of precipitation level, suggests that all populations tested need to cope with some level of rainfall stochasticity. By producing more short‐developing embryos, however, fish in more arid areas with relatively more erratic rains are better adapted to very short pool durations and are more likely to produce multiple offspring generations within a single rainy season.     

Asymmetric life-history decision-making in butterfly larvae

In temperate environments, insects appearing in several generations in the growth season typically have to decide during the larval period whether to develop into adulthood, or to postpone adult emergence until next season by entering a species-specific diapause stage. This decision is typically guided by environmental cues experienced during development. An early decision makes it possible to adjust growth rate, which would allow the growing larva to respond to time stress involved in direct development, whereas a last-minute decision would instead allow the larva to use up-to-date information about which developmental pathway is the most favourable under the current circumstances. We study the timing of the larval pathway decision-making between entering pupal winter diapause and direct development in three distantly related butterflies (Pieris napi, Araschnia levana and Pararge aegeria). We pinpoint the timing of the larval diapause decision by transferring larvae from first to last instars from long daylength (inducing direct development) to short daylength conditions (inducing diapause), and vice versa. Results show that the pathway decision is typically made in the late instars in all three species, and that the ability to switch developmental pathway late in juvenile life is conditional; larvae more freely switched from diapause to direct development than in the opposite direction. We contend that this asymmetry is influenced by the additional physiological preparations needed to survive the long and cold winter period, and that the reluctance to make a late decision to enter diapause has the potential to be a general trait among temperate insects.     

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.     

In search of the vertebrate phylotypic stage: a molecular examination of the developmental hourglass model and von Baer's third law

In 1828, Karl von Baer proposed a set of four evolutionary "laws" pertaining to embryological development. According to von Baer's third law, young embryos from different species are relatively undifferentiated and resemble one another but as development proceeds, distinguishing features of the species begin to appear and embryos of different species progressively diverge from one another. An expansion of this law, called "the hourglass model," has been proposed independently by Denis Duboule and Rudolf Raff in the 1990s. According to the hourglass model, ontogeny is characterized by a starting point at which different taxa differ markedly from one another, followed by a stage of reduced intertaxonomic variability (the phylotypic stage), and ending in a von-Baer-like progressive divergence among the taxa. A possible "translation" of the hourglass model into molecular terminology would suggest that orthologs expressed in stages described by the tapered part of the hourglass should resemble one another more than orthologs expressed in the expansive parts that precede or succeed the phylotypic stage. We tested this hypothesis using 1,585 mouse genes expressed during 26 embryonic stages, and their human orthologs. Evolutionary divergence was estimated at different embryonic stages by calculating pairwise distances between corresponding orthologous proteins from mouse and human. Two independent datasets were used. One dataset contained genes that are expressed solely in a single developmental stage; the second was made of genes expressed at different developmental stages. In the second dataset the genes were classified according to their earliest stage of expression. We fitted second order polynomials to the two datasets. The two polynomials displayed minima as expected from the hourglass model. The molecular results suggest, albeit weakly, that a phylotypic stage (or period) indeed exists. Its temporal location, sometimes between the first-somites stage and the formation of the posterior neuropore, was in approximate agreement with the morphologically defined phylotypic stage. The molecular evidence for the later parts of the hourglass model, i.e., for von Baer's third law, was stronger than that for the earlier parts.     

Inverting the hourglass: quantitative evidence against the phylotypic stage in vertebrate development

The concept of a phylotypic stage, when all vertebrate embryos show low phenotypic diversity, is an important cornerstone underlying modern developmental biology. Many theories involving patterns of development, developmental modules, mechanisms of development including developmental integration, and the action of natural selection on embryological stages have been proposed with reference to the phylotypic stage. However, the phylotypic stage has never been precisely defined, or conclusively supported or disproved by comparative quantitative data. We tested the predictions of the 'developmental hourglass' definition of the phylotypic stage quantitatively by looking at the pattern of developmental-timing variation across vertebrates as a whole and within mammals. For both datasets, the results using two different metrics were counter to the predictions of the definition: phenotypic variation between species was highest in the middle of the developmental sequence. This surprising degree of developmental character independence argues against the existence of a phylotypic stage in vertebrates. Instead, we hypothesize that numerous tightly delimited developmental modules exist during the mid-embryonic period. Further, the high level of timing changes (heterochrony) between these modules may be an important evolutionary mechanism giving rise to the diversity of vertebrates. The onus is now clearly on proponents of the phylotypic stage to present both a clear definition of it and quantitative data supporting its existence.     

Diapause in the pea aphid (<Emphasis Type="Italic">Acyrthosiphon pisum</Emphasis>) is a slowing but not a cessation of development

Background  

Many insects undergo a period of arrested development, called diapause, to avoid seasonally recurring adverse conditions. Whilst the phenology and endocrinology of insect diapause have been well studied, there has been comparatively little research into the developmental details of diapause. We investigated developmental aspects of diapause in sexually-produced embryos of the pea aphid, Acyrthosiphon pisum.     

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.     

Sorbitol as an arrester of embryonic development in diapausing eggs of the silkworm, Bombyx mori

Recently, it was confirmed that embryos derived from diapausing eggs of the silkworm, Bombyx mori, begin their development and reach larval maturity on mulberry leaves, when the naked eggs are cultured in vitro. In this study, we found that the method of embryo culture is useful for determining the physiological regulation of diapause. We show that the development of embryos derived from diapausing eggs was strongly inhibited by the addition of either sorbitol or trehalose to the culture medium. Furthermore, this inhibitory effect disappeared when the embryos were cultured in a control medium which did not contain either sorbitol or trehalose, indicating that the inhibitory reactions caused by both substances are reversible. The minimal effective dose of either sorbitol or trehalose was approximately 0.2 M, a value similar to the in vivo concentration of sorbitol in diapausing eggs (0.2 M). Glycerol, mannitol or glucose were moderately effective for inhibition. Sorbitol present in diapausing silkworm eggs does not appear to serve as an antifreeze, but as an strong arresting factor of embryonic development. Furthermore, these results show that a decrease in sorbitol releases the embryos from diapause at the termination of diapause.     

Physical constraints on body size in teleost embryos

All members of the subphylum "Vertebrata" display the characteristics of the vertebrate body plan. These characteristics become apparent during the phylotypic period, in which all vertebrate embryos have a similar body shape and internal organization. Phylogenetic constraints probably limit the morphological variation during the phylotypic period. Physical laws, however, also limit growth and morphogenesis in embryos. We investigated to what extent oxygen availability-as a physical constraint-might limit morphological variation during embryonic development. This paper gives an analysis of time-dependent diffusion into spherical embryos without a circulatory system. Equilibrium appeared to settle in about 1.5 min in running water and in about 10min in stagnant water. Hence, steady-state conditions were assumed and expressions for maximum body size were obtained for spherical, cylindrical and sheet-like embryos in running water and spherical embyros in stagnant water. Predictions of the model based on literature data suggest that in running water-both for spherical, cylindrical and sheet-like embryos-diffusion alone suffices to cover the oxygen needs of a teleost embryo in its phylotypic period. The size of carp (Cyprinus carpio) and African catfish (Clarias gariepinus) embryos is very close to the predicted maximum. This suggests that in these species the development of a functional circulatory system is correlated with the onset of oxygen shortage. Oxygen availability is therefore a potentially important physical constraint on embryonic morphology, though in most species the circulatory system becomes functional well in advance of the onset of oxygen shortage and other demands than oxygen delivery (e.g. nutrient distribution, waste disposal, osmoregulation) might require the development of a circulatory system.