Convergent evolution of alternative developmental trajectories associated with diapause in African and South American killifish

Annual killifish adapted to life in seasonally ephemeral water-bodies exhibit desiccation resistant eggs that can undergo diapause, a period of developmental arrest, enabling them to traverse the otherwise inhospitable dry season. Environmental cues that potentially indicate the season can govern whether eggs enter a stage of diapause mid-way through development or skip this diapause and instead undergo direct development. We report, based on construction of a supermatrix phylogenetic tree of the order Cyprinodontiformes and a battery of comparative analyses, that the ability to produce diapause eggs evolved independently at least six times within African and South American killifish. We then show in species representative of these lineages that embryos entering diapause display significant reduction in development of the cranial region and circulatory system relative to direct-developing embryos. This divergence along alternative developmental pathways begins mid-way through development, well before diapause is entered, during a period of purported maximum developmental constraint (the phylotypic period). Finally, we show that entering diapause is accompanied by a dramatic reduction in metabolic rate and concomitant increase in long-term embryo survival. Morphological divergence during the phylotypic period thus allows embryos undergoing diapause to conserve energy by shunting resources away from energetically costly organs thereby increasing survival chances in an environment that necessitates remaining dormant, buried in the soil and surrounded by an eggshell for much of the year. Our results indicate that adaptation to seasonal aquatic environments in annual killifish imposes strong selection during the embryo stage leading to marked diversification during this otherwise conserved period of vertebrate development.     

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.     

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.     

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.     

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.     

Intrinsic variability in the frequency of embryonic diapauses of the annual fish Nothobranchius korthausae,regulated by light:dark cycle and temperature

Synopsis The influence of the light:dark cycle and temperature on the embryonic development, especially diapause, of the annual fish Nothobranchius korthausae was investigated. The variability of the frequency of diapause II during constant L:D cycle and temperature, but at different times of the year, was also studied. In agreement with previous studies it appeared that diapause I does practically not occur in N. korthausae (and other Nothobranchius species), even under sub-optimal conditions which are known to induce diapause II and III. Only at very low temperatures, a first developmental arrest could be induced during the dispersed phase, as well as during the reaggregation phase. Diapause II and III can be induced more easily, by exposing the embryos to low (sub-optimal) temperatures and short light periods (or constant darkness). Both diapauses are inhibited or terminated at high temperature and long light periods (12L:12D). The occurrence of an indirect light:dark response via the adult fishes could not be demonstrated. At moderate temperatures and in constant darkness (or short light periods) considerable variability in diapause II-frequency was observed. This could be an intrinsic feature of annual fish development, constituting a strategy for better survival of the species.     

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.     

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.     

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.     

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.     

Naturally occurring diblastodermic eggs in the annual fish Cynolebias: Implications for developmental regulation and determination

Annual fish development differs from that of other teleosts because a phase of blastomere dispersion-reaggregation spatially and temporally separates epiboly from embryogenesis. The fate of dispersed blastomeres was assessed in diblastodermic eggs of the annual fishes Cynolebias whitei and C. nigripinnis. In typical teleosts, blastomere determination and the events of primary embryonic induction occur prior to or during epiboly, so diblastodermic eggs produce partially or completely duplicated embryos. In the diblastodermic eggs of Cynolebias, the two blastoderms are completely separate from the one cell stage to the high blastula. Blastoderm fusion begins during midepiboly. By the end of epiboly, blastoderm fusion has been completed, and the deep, embryo-forming blastomeres of both blastoderms have completely dispersed and intermingled to form a single cell population. A typical annual fish dispersed blastomere phase ensues. Blastomeres reaggregate into a single mass, in which one embryo develops. When hatched, the young fish have no obvious structural or functional abnormalities. We suggest that the dispersed blastomeres of annual fish eggs are equivalent and that induction or determination takes place within the reaggregate. Alternatively, dispersed cells are partially determined but highly regulative, so that, when two populations fuse, the cells sort out according to tissue type and form a single embryo. In either instance, the formation of a single, normal embryo seems to corroborate the hypothesis that the dispersed cell phase of annual fishes is an adaptation that prevents environmentally induced developmental defects. © 1993 Wiley-Liss, Inc.     

Identification of the glycerol kinase gene and its role in diapause embryo restart and early embryo development of Artemia sinica

Glycerol kinase (GK) catalyzes the rate-limiting step in glycerol utilization by transferring a phosphate from ATP to glycerol, yielding glycerol 3-phosphate, which is an important intermediate for both energy metabolism and glycerolipid production. Artemia sinica has an unusual diapause process under stress conditions of high salinity, low temperature and lack of food. In the process, diapause embryos of A. sinica (brine shrimp) accumulate high concentrations of glycerol as a cryoprotectant to prevent low temperature damage to embryos. Upon embryo restart, glycerol is converted into glucose and other carbohydrates. Therefore, GK plays an important role in the diapause embryo restart process. However, the role of GK in diapause termination of embryo development in A. sinica remains unknown.     

Sex determination in annual fishes: Searching for the master sex-determining gene in Austrolebias charrua (Cyprinodontiformes,Rivulidae)

Evolution of sex determination and differentiation in fishes involves a broad range of sex strategies (hermaphroditism, gonochorism, unisexuality, environmental and genetic sex determination). Annual fishes inhabit temporary ponds that dry out during the dry season when adults die. The embryos exhibit an atypical developmental pattern and remain buried in the bottom mud until the next rainy season. To elucidate genomic factors involved in the sex determination in annual fish, we explored the presence of a candidate sex-specific gene related to the cascade network in Austrolebias charrua. All phylogenetic analyses showed a high posterior probability of occurrence for a clade integrated by nuclear sequences (aprox. 900 bp) from both adults (male and female), with partial cDNA fragments of A. charrua from juveniles (male) and the dsx D. melanogaster gene. The expressed fragment was detected from blastula to adulthood stages showing a sexually dimorphic expression pattern. The isolated cDNA sequence is clearly related to dsx D. melanogaster gene and might be located near the top of the sex determination cascade in this species.     

Metazoan parasites of African annual killifish (Nothobranchiidae): abundance,diversity, and their environmental correlates

Estimates of biodiversity and its global patterns are affected by parasite richness and specificity. Despite this, parasite communities are largely neglected in biodiversity estimates, especially in the tropics. We studied the parasites of annual killifish of the genus Nothobranchius that inhabit annually desiccating pools across the African savannah and survive the dry period as developmentally arrested embryos. Their discontinuous, non‐overlapping generations make them a unique organism in which to study natural parasite fauna. We investigated the relationship between global (climate and altitude) and local (pool size, vegetation, host density and diversity, and diversity of potential intermediate hosts) environmental factors and the community structure of killifish parasites. We examined metazoan parasites from 21 populations of four host species (Nothobranchius orthonotus, N. furzeri, N. kadleci, and N. pienaari) across a gradient of aridity in Mozambique. Seventeen parasite taxa were recorded, with trematode larval stages (metacercariae) being the most abundant taxa. The parasites recorded were both allogenic (life cycle includes non‐aquatic host; predominantly trematodes) and autogenic (cycling only in aquatic hosts; nematodes). The parasite abundance was highest in climatic regions with intermediate aridity, while parasite diversity was associated with local environmental characteristics and positively correlated with fish species diversity and the amount of aquatic vegetation. Our results suggest that parasite communities of sympatric Nothobranchius species are similar and dominated by the larval stages of generalist parasites. Therefore, Nothobranchius serve as important intermediate or paratenic hosts of parasites, with piscivorous birds and predatory fish being their most likely definitive hosts.     

Changing patterns of growth in a changing planet: How a shift in phenology affects critical life‐history traits in annual fishes

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Hatching date influences winter habitat occupancy: Examining seasonal interactions across the full annual cycle in a migratory songbird

Birds experience a sequence of critical events during their life cycle, and past events can subsequently determine future performance via carry‐over effects. Events during the non‐breeding season may influence breeding season phenology or productivity. Less is understood about how events during the breeding season affect individuals subsequently in their life cycle. Using stable carbon isotopes, we examined carry‐over effects throughout the annual cycle of prairie warblers (Setophaga discolor), a declining Nearctic–Neotropical migratory passerine bird. In drier winters, juvenile males that hatched earlier at our study site in Massachusetts, USA, occupied wetter, better‐quality winter habitat in the Caribbean, as indicated by depleted carbon isotope signatures. For juveniles that were sampled again as adults, repeatability in isotope signatures indicated similar winter habitat occupancy across years. Thus, hatching date of juvenile males appears to influence lifetime winter habitat occupancy. For adult males, reproductive success did not carry over to influence winter habitat occupancy. We did not find temporally consecutive “domino” effects across the annual cycle (breeding to wintering to breeding) or interseasonal, intergenerational effects. Our finding that a male''s hatching date can have a lasting effect on winter habitat occupancy represents an important contribution to our understanding of seasonal interactions in migratory birds.     

Reproductive cycle and maternal–embryonic nutritional relationship of shovelnose guitarfish Pseudobatos productus in the Gulf of California

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

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Rapid growth and large body size in annual fish populations are compromised by density-dependent regulation

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