Oxygen, gills, and embryo behavior: mechanisms of adaptive plasticity in hatching.

Many species alter the timing of hatching in response to egg or larval predators, pathogens, or physical risks. This plasticity depends on separation between the onset of hatching competence and physiological limits to embryonic development. I present a framework based on heterokairy to categorize developmental mechanisms and identify traits contributing to and limiting hatching plasticity, then apply it to a case of predator-induced hatching. Red-eyed treefrogs have arboreal eggs, and tadpoles fall into ponds upon hatching. Egg and tadpole predators select for earlier and later hatching, respectively. Embryos hatch up to 30% early in predator attacks, and later if undisturbed. They maintain large external gills throughout the plastic hatching period, delaying gill regression while development otherwise continues. Rapid gill regression occurs upon hatching. Prolonged embryonic development depends on external gills; inducing gill regression causes hatching. External hypoxia retards development, kills eggs, and induces hatching. Nonetheless, embryos develop synchronously and without hatching prematurely across a broad range of perivitelline PO2, from 0.5-12.5 kPa. Embryos exploit spatial variation of PO2 within eggs by positioning gills against patches of air-exposed surface. Respiratory plasticity and oxygen-sensitive behavior appear critical for the hatching plasticity that balances a predation risk trade-off across life stages.     

Hatching timing,oxygen availability,and external gill regression in the tree frog,Agalychnis callidryas

The physiological role of the embryonic external gills in anurans is equivocal. In some species, diffusion alone is clearly sufficient to supply oxygen throughout the embryonic period. In others, morphological elaboration and environmental regulation of the external gills suggest functional importance. Since oxygen stress is a common trigger of hatching, I examined the relationships among hatching timing, oxygen stress, and external gill loss. I worked with the red-eyed tree frog, Agalychnis callidryas, a species with arboreal eggs and aquatic tadpoles in which gill regression is associated with hatching, and hatching timing affects posthatching survival with aquatic predators. Both exposure to a hypoxic gas mixture and submergence in water, a natural context in which hypoxic stress can occur, induced early hatching. Exposure to hyperoxic gas mixtures induced regression of external gills, and subsequent exposure to air induced early hatching. Prostaglandin-induced external gill regression also induced hatching, and this effect was partially ameliorated by exposure to hyperoxic gas. Together, these results suggest that external gills enhance the oxygen uptake of embryos and are necessary to extend embryonic development past the onset of hatching competence.     

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

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

Effects of hatching age on development and hatchling morphology in the red-eyed tree frog, Agalychnis callidryas

The red-eyed treefrog, Agalychnis callidryas , lays eggs on leaves overhanging ponds. Tadpoles hatch and enter the water at different ages, and late-hatched tadpoles survive aquatic predators better than do early-hatched tadpoles. Here I assess developmental consequences of hatching age through: (1) a morphological study of embryos and tadpoles through the plastic hatching period; (2) a behavioural assay for an effect of hatching age on feeding; and (3) a field experiment testing the effect of hatching age on growth to metamorphosis. Substantial development of feeding, digestive, respiratory and locomotor structures occurs in embryos over the plastic hatching period. Hatchling morphology thus varies with age, with consequences for behaviour and predation risk. Hatched tadpoles develop faster than embryos, and early-hatched tadpoles feed before late-hatched tadpoles. After all tadpoles have hatched, the effect of hatching age on size decreases. I found no evidence for an effect of hatching age on size at metamorphosis and only weak evidence for an effect on larval period. Hatching age affects the sequence of developmental change: early-hatched tadpoles lose external gills while otherwise more developed embryos maintain them. Plasticity in external gill resorption may be adaptive given differences in the respiratory environments of embryos and tadpoles. Early-hatched tadpoles also diverge from embryos in shape, growing relatively smaller tails. The study of functional morphology and developmental plasticity will contribute to understanding hatching as an ontogenetic niche shift.     

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

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

Effects of temperature experienced during embryonic development on biomass and C and N composition at hatching in Palaemon serratus (Pennant, 1777)

In decapod crustaceans, the conditions experienced during embryonic development trigger phenotypic plasticity of the larvae at hatching. The objective of this study was to test the effects of temperature during embryonic development of Palaemon serratus on the phenotypic plasticity of hatching larvae. We incubated egg-bearing females from eggs laying to hatching at four temperatures (10, 15, 18 and 20°C). Weight, carbon and nitrogen contents were measured on newly laid eggs and on freshly hatched larvae. The duration of embryonic development was negatively correlated with incubation temperature. At 20°C, all females abandoned their eggs during development. Incubation temperature had no effect on the weight and the percentage of N of the larvae at hatching, while it did affect their percentage of C and their C/N ratio. Embryos incubated at 10°C seemed to produce larvae with fewer lipid reserves than those incubated at 15 and 18°C. They probably overconsumed their lipid reserves to compensate for the metabolic losses due to the low temperature. These results provide information on the link between maternal investment per egg and larval development in P. serratus.     

版纳鱼螈胚胎和幼体鳃的退化观察

为了解版纳鱼螈(Ichthyophis bannanicus)胚胎和幼体鳃的退化特征,同窝卵中8枚在胚胎成熟阶段早期被随机取出剖产,随后余下的9枚卵在胚胎成熟阶段晚期时孵出或剖产,观察幼体或胚胎鳃的变化。成熟阶段早期的胚胎,初时其鳃色鲜红,鳃枝充盈,鳃丝与鳃轴近乎垂直;随后鳃色发白,鳃枝疲软,鳃丝卷曲,鳃丝与鳃轴夹角变小。成熟阶段晚期的胚胎和刚孵出的幼体,初时其鳃色纯白,鳃枝疲软,鳃丝卷曲,鳃丝与鳃轴夹角较小;随后鳃逐渐脱落。观察发现,版纳鱼螈胚胎或幼体鳃的退化模式不是重吸收,也不是重吸收加脱落,而仅为脱落。鳃退化过程经历外鳃供血减少-供血停止-鳃枝组织死亡和脱落3个阶段,每个阶段都有明显的外部形态特征。胚胎进入成熟阶段后,越早孵化,鳃枝留存越多,反之,鳃枝留存越少。发白后的鳃枝随机性脱落。     

Do prostaglandins regulate external gill regression in anurans?

Although the endocrinological mechanism controlling regression of the internal, larval gills of anurans (frogs and toads) is well understood, the mechanism regulating loss of the external, embryonic gills is not known. Based on the homology of the mammalian ductus arteriosus with a portion of the amphibian branchial arches, and the regulation of blood flow in the mammalian ductus by prostaglandins of the E family (PGEs), we hypothesized that anuran external gill loss is also regulated by PGEs. To test this hypothesis, we topically applied both PGE2 and a synthetic analogue of PGE1, misoprostol, to embryos and young hatchlings of the red-eyed treefrog, Agalychnis callidryas. Both agents accelerated external gill regression. Furthermore, misoprostol overrode the inhibitory effect of hypoxia on gill regression in hatchlings and induced rapid loss of external gills in embryos, which normally maintain the gills until hatching. These observations support the hypothesis that PGEs regulate anuran external gill loss. The specific site of action for prostaglandins within the gills is not known; however, PGEs are secreted in the oral mucus of tadpoles, and this could be a natural topical source for these agents. PGEs offer a tool for manipulation of external gills and should facilitate tests of the physiological importance of these structures.     

Plasticity of hatching in amphibians: evolution, trade-offs, cues and mechanisms

Many species of frogs and salamanders, in at least 12 families, alter their timing of hatching in response to conditions affecting mortality of eggs or larvae. Some terrestrially laid or stranded embryos wait to hatch until they are submerged in water. Some embryos laid above water accelerate hatching if the eggs are dehydrating; others hatch early if flooded. Embryos can hatch early in response to predators and pathogens of eggs or delay hatching in response to predators of larvae; some species do both. The phylogenetic pattern of environmentally cued hatching suggests that similar responses have evolved convergently in multiple amphibian lineages. The use of similar cues, including hypoxia and physical disturbance, in multiple contexts suggests potential shared mechanisms underlying the capacity of embryos to respond to environmental conditions. Shifts in the timing of hatching often have clear benefits, but we know less about the trade-offs that favor plasticity, the mechanisms that enable it, and its evolutionary history. Some potentially important types of cued hatching, such as those involving embryo-parent interactions, are relatively unexplored. I discuss promising directions for research and the opportunities that the hatching of amphibians offers for integrative studies of the mechanisms, ecology and evolution of a critical transition between life-history stages.     

Wasp predation and wasp-induced hatching of red-eyed treefrog eggs

Eggs often suffer high levels of predation and, compared with older animals, embryos have few options available for antipredator defence. None the less, hatchlings can escape from many predators to which eggs are vulnerable. I studied early hatching as an antipredator defence of red-eyed treefrog embryos, Agalychnis callidryas, in response to egg predation by social wasps (Polybia rejecta). Red-eyed treefrogs attach their eggs to vegetation overhanging water, where they are exposed to arboreal and aerial predators. Wasps attacked half the egg clutches and killed almost a quarter of the eggs I monitored at a natural breeding site in Panama. Hatching tadpoles fall into the water, where they face aquatic predators. As predicted from improved survival of older hatchlings with aquatic predators, most undisturbed eggs hatched relatively late. However, many younger embryos directly attacked by wasps hatched immediately. Embryos attacked by wasps hatched as much as a third younger than the peak undisturbed hatching age, and most hatching embryos escaped. Thus hatching is an effective defence against wasp predation, and plasticity in hatching stage allows embryos to balance risks from stage-specific egg and larval predators. Wasp-induced hatching is behaviourally similar to the snake-induced hatching previously described in A. callidryas, but occurs in fewer eggs at a time, congruent with the scale of the risk. Individual embryos hatch in response to wasps, which take single eggs, whereas whole clutches hatch in response to snakes, which consume entire clutches. Embryos of A. callidryas thus respond appropriately to graded variation in mortality risks. Copyright 2000 The Association for the Study of Animal Behaviour.     

Phenotypically plastic responses of green frog embryos to conflicting predation risk

Predators have been shown to alter the timing of switch points between life history stages, but few studies have addressed switch point plasticity in prey exposed simultaneously to conflicting predation pressure. We tested hatching responses of green frog (Rana clamitans) embryos subject to perceived predation risk from chemical cues released by two stage-specific predators, predicting that these predators would elicit: (1) directional hatching responses when presented independently, and (2) intermediate phenotypic responses when presented simultaneously. R. clamitans embryos in outdoor exclosures were exposed to cues from an egg predator (freshwater leeches; Nephelopsis obscura), a larval predator (dragonfly nymphs, Aeschna canadensis), and both predators in a 2 × 2 factorial experiment, and changes in hatchling size, hatchling developmental stage, and hatching time were compared to those for control embryos. Leeches alone induced embryos to hatch at a smaller size and an earlier developmental stage than controls, while dragonfly nymphs elicited a delay in egg hatching time that was associated with larger size and later developmental stage at hatching. Embryos failed to respond to simultaneous exposure to both predators, implying that responses to each occurred concurrently and were therefore dampened. Our results indicate that prey under threat from conflicting predators may manifest intermediate defensive phenotypes. Such intermediate responses may result in elevated rates of prey mortality with possible consequences at the population level.     

Ultrastructure and development of the gills in<Emphasis Type="Italic"> Rana dalmatina</Emphasis> (Amphibia,Anura)

The appearance and the modification of the gill apparatus in Rana dalmatina tadpoles have been described in the different phases of larval development. The morphology and ultrastructure have been studied using light microscopy and both scanning and transmission electron microscopy. The organization of the gills during the initial phases of development (external gills or transient gills) brings to mind the characteristics of Urodela larvae in which the gills appear to consist of three tufts of filaments supported by the gill arches III, IV and V. The cellular composition of the transient gills appears to be extremely simple and the presence of specialized cells is not noted. Basal cells, pavement cells and ciliated cells form the thin mono- or bilayered epithelium. In the persistent gills (or internal gills) of the R. dalmatina tadpole (Ortons larval type 4) the gill arches carry four rows of gill tufts branching out to the ventral region. Meanwhile, from the dorsal portion of the arch the gill filters present an axial portion from which there is much branching out, which confers a characteristic appearance on this part of the gills. The cellular composition of the gill tufts and of the filters is different: in the gill tufts basal cells, pavement cells, ciliated cells, cubic cells and mitochondria-rich cells (MRCs) have been recognized, while in the gill filters the last cellular type does not appear. The MRC has highly variable forms and dimensions and is characterized by the presence of numerous mitochondria in the cytoplasm. Often the MRCs manifest themselves grouped together, in groups of three or more. The pavement cells and the cubic cells demonstrate identical ultrastructural characteristics and have an external surface area characterized by the presence of short superficial microridges and numerous vacuoles in the apical cytoplasm.     

Pre-hatching exposure to water mold reduces size at metamorphosis in the moor frog

Developmental plasticity is increasingly recognized as important for ecological and evolutionary processes. However, few studies consider the potential for delayed effects of early environments. Here, we show that tadpoles hatching from clutches exposed to water mold (Saprolegnia) have 20% decreased mass at metamorphosis, despite no further exposure subsequent to hatching. The effects were consistent across four populations that have previously been shown to vary in their resistance to infection during embryonic development. Contrary to expectations, time to hatching or metamorphosis was not affected, suggesting that the results do not reflect an evolved escape strategy from infected waters triggered by embryonic conditions. Instead, decreased mass at metamorphosis may arise from carry-over effects of impaired embryo development. Such strong links across developmental stages have potential consequences for the evolution of plasticity and the responses of populations to emergent infections.     

The external gills of anuran amphibians: Comparative morphology and ultrastructure

The external gills of anuran amphibians are transient structures, covered by the development of the operculum and regressing soon afterwards. Their functional role has been regarded as equivocal. However, detailed morphological analysis has been limited. Analysis of 21 species from six families using scanning and transmission electron microscopy revealed diversity at the anatomical and cellular levels in extent and length of gill filaments, numbers of surface ciliated cells, width of water‐blood barrier distance, and evidence of gill motility. The most highly developed external gills were found in species with delayed hatching, such as Phyllomedusa trinitatis, or in species in which hatchlings hang from the surface film of temporary ponds, such as Phrynohyas venulosa in which gills added 26–38% to body surface area. In one family, the bufonids, all four species examined had poorly developed gills, but in other families where we examined several species, the hylids and leptodactylids, there was considerable diversity of external gills, suggesting flexible adaptation to incubation and hatching environment. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Environmental context shapes immediate and cumulative costs of risk-induced early hatching

In animals with complex life cycles, fitness trade-offs across life stages determine the optimal time for transitions between stages. If these trade-offs vary predictably, adaptive plasticity in the timing of life history transitions may evolve. For instance, embryos of many species are capable of accelerating hatching to escape from egg predation and other hazards, but for plasticity in hatching timing to be selectively maintained, early hatching must also entail costs, probably in subsequent life stages. However the post-hatching environment, which influences this cost, is variable in nature. We assessed how two elements of the post-hatching environment, predator species and age structure created by hatching age plasticity, affect costs of hatching early in red-eyed treefrogs, Agalychnis callidryas. Red-eyed treefrog embryos were induced to hatch at the onset of hatching competence or near the peak of spontaneous hatching and exposed to one of three insect predators in single or mixed hatching-age treatments. Age structure created by hatching-age plasticity did not affect tadpole survivorship or growth; however, the consequences of hatching timing depended on predator species and foraging mode. Tadpoles that were induced to hatch early experienced initially higher mortality rates only with the more actively foraging predator. Nonetheless, mortality costs of accelerated hatching were apparent with all predators once we factored in the longer duration of exposure that early hatchlings experience in nature. This study suggests that extended exposure of young larvae to predators may be a general cost of early hatching, explaining why spontaneous hatching occurs later in life across variable environmental contexts.     

Immunolocalization of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase in the branchial cavity during the early development of the crayfish <Emphasis Type="Italic">Astacus leptodactylus</Emphasis> (Crustacea,Decapoda)

The ontogeny of osmoregulation was examined in the branchial cavity of embryonic and early post-embryonic stages of the crayfish Astacus leptodactylus maintained in freshwater, at the sub-cellular level through the detection of the sodium–potassium adenosine triphosphatase (Na⁺,K⁺-ATPase). The embryonic rate of development was calculated according to the eye index (EI) which was 430–450 m at hatching. The distribution of the enzyme was identified by immunofluorescence microscopy using a monoclonal antibody IgG5 raised against the avian -subunit of the Na⁺,K⁺-ATPase. Immunoreactivity staining, indicating the presence of Na⁺, K⁺-ATPase appeared in the gills of late embryos (EI400 m), i.e. a few days before hatching time, and steadily increased throughout the late embryonic and early post-embryonic development. The appearance of the enzyme correlates with the ability to osmoregulate which also occurs late in the embryonic development at EI 410–420 m and with tissue differentiation within the gill filaments. These observations indicate that the physiological shift from osmoconforming embryos to hyper-regulating late embryos and post-hatching stages in freshwater must originate partly from the differentiation in the gill epithelia of ionocytes which are the site of ion pumping, as suggested by the location of Na⁺,K⁺-ATPase. Only the gills were immunostained and a lack of specific staining was noted in the lamina and the branchiostegites. Therefore, osmoregulation through Na⁺active uptake is likely achieved in embryos at the gill level; all the newly formed gills in embryos function in ion regulation; other parts of the branchial chamber such as the branchiostegites and lamina do not appear to be involved in osmoregulation.     

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

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

Plasticity and limitations of extended egg retention in oviparous Zootoca vivipara (Reptilia: Lacertidae)

The transition between oviparity and viviparity in reptiles is generally accepted to be a gradual process, the result of selection for increasingly prolonged egg retention within the oviduct. We examined egg retention plasticity in an oviparous strain of the lacertid lizard Zootoca vivipara, a species having both oviparous and viviparous populations. We forced a group of female Z. vivipara to retain their clutch in utero by keeping them in dry substrata, and assessed the effect on embryonic development and hatching success, along with offspring phenotype and locomotor performance. Forced egg retention for one additional week affected the developmental stage of embryos at oviposition, as well as hatchling robustness and locomotor performance. However, embryos from forced clutch retention treatment reached one stage unit more than control embryos at oviposition time. Embryos from control eggs were more developed than embryos from experimental eggs after approximately the same period of external incubation, showing that embryonic development is retarded during the period of extended egg retention, despite the high temperature inside the mother's body. Significant differences in external incubation time were only found in one of the two years of study. Hatching success was much lower in the experimental group with forced egg retention (21.1%) than in the control group (95.4%). Therefore, we conclude that there are limitations that hinder the advance of intrauterine embryonic development beyond the normal time of oviposition, and that extended egg retention does not represent clear advantages in this population of Z. vivipara. Nevertheless, the fact that some eggs are successful after forced egg retention could be advantageous for the females that are able to retain their clutch under unfavourable climatic conditions. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 75–82.     

Salinity tolerance and structure of external and internal gills in tadpoles of the crab-eating frog,Rana cancrivora

Summary Salinity tolerance and histology of gills were studied in Rana cancrivora larvae. The tadpoles at the external gill stages (W stages 21–22) were able to survive in media containing up to 40% seawater, but died in water of higher salinity. Their external gills appear to have no critical role in adaptation to seawater. However, advanced tadpoles with internal gills (T-K stages I–XVIII) were able to tolerate 50% or higher seawater. In the internal gills, there are numerous mitochondriarich cells (MR cells) scattered on the ventral and lateral epithelia of the gill arches and the gill tufts in both freshwater-and seawater-acclimated tadpoles. In freshwater-acclimated tadpoles there are three types of MR cell: (1) microplicated, (2) microvillous, and (3) apically vacuolated. In tadpoles acclimated to dilute seawater, the ratio of type-1 to type-2 cells is lower, although all three types of MR cell are present. In 60%-seawater-acclimated tadpoles, a few MR cells with a lumen and concave cytoplasm at the apical membrane (type 4) are present. The changes in MR cell morphology under ambient conditions of low or high salinity may reflect alterations in the physiological roles of the gills with regard to transport of ions.