Salamander Embryos Research Articles

Pre-Embryonic Period Observation Shows a Unique Reproductive Strategy of the Critically Endangered Anji Salamander (Hynobius amjiensis).

Hynobius amjiensis, also known as the Anji salamander, is an amphibian species currently categorized as endangered due to its limited geographical distribution, primarily in China. To address the critical conservation status of this species, artificial breeding is essential for population expansion. However, progress in artificial breeding efforts has been hindered by the scarcity of research on the reproductive biology of the Anji salamander. In this study, we identified 25 distinct early stages of embryo development. Additionally, we observed that Anji salamander embryos contain a lesser amount of yolk compared to other salamanders or frogs. We further discovered that the Anji salamander employs a highly competitive reproductive strategy, producing a smaller number of high-quality offspring. This strategy aims to generate adaptive individuals through intense intraspecific competition, driven by three factors: extremely confined breeding habitats, a substantial number of eggs, and a reduced yolk content. We introduce the term "mass escape" to describe this strategy, which provides a novel perspective on cannibalization, focusing on the consumption of specific body parts rather than a single-individual activity. This study offers valuable insights into artificial breeding techniques designed to mitigate inherent intraspecific competitive pressure, thereby improving metamorphosis and survival rates. Additionally, it provides a comprehensive table detailing the pre-embryonic developmental stages of the Anji salamander.

Egg predators improve the hatching success of salamander eggs.

A common challenge that oviparous animals face is securing survivorship during the vulnerable embryonic stage. One of the parental investment strategies to improve survivorship is providing physical structures to protect the embryos. In amphibians, there is a notable diversity in jelly-layer structures surrounding eggs. Previous studies show that these jelly layers provide eggs with protection against egg predators, egg pathogens, and desiccation. However, few studies examined the cost-benefit relationship of the jelly-layer structures. By using the predator-prey interaction between wood frog (Lithobates sylvaticus) tadpoles and spotted salamander (Ambystoma maculatum) eggs as a model system, we tested three hypotheses: (1) having the outer jelly layers would be costly to the embryos, (2) the relative benefit of the structural egg defense would become apparent and increase as the intensity of egg predation increases, and (3) a certain degree of predation would increase the hatching success of salamander embryos by mechanically thinning the thick outer jelly layers and increasing oxygen diffusion throughout an egg mass. To test these hypotheses, we conducted a factorial experiment in which we crossed four egg-predation levels with two jelly-layer conditions, intact or removed. We found that the jelly layers were essential in protecting spotted salamander embryos from wood frog tadpoles but that the associated cost was apparent in no-predation treatments. The differential survivorship between intact eggs and eggs without jelly layers showed that the fitness advantage of jelly layers increased as the level of predation increased. Finally, the hatching success of intact egg masses was highest under the high predation conditions. These results imply that the evolution of the jelly-layer thickness occurred under constant egg-predation pressure. Given this predator-prey coevolution, egg predators may play a critical role in improving the hatching success of salamander embryos under certain conditions.

The role of Wnt signaling in Xenopus neural induction.

Development of the central nervous system in amphibians has called attention from scientists for over a century. Interested in the matter of embryonic inductions, Hans Spemann and Hilde Mangold found out that the dorsal blastopore lip of the salamander's embryo has organizer properties. Such an ectopic graft could induce structures in the host embryo, including a neural tube overlying the notochord of a perfect secondary body axis. A couple of decades later, the frog Xenopus laevis emerged as an excellent embryological experimental model and seminal concepts involving embryonic inductions began to be revealed. The so-called primary induction is, in fact, a composition of signaling and inductive events that are triggered as soon as fertilization takes place. In this regard, since early 1990s an intricate network of signaling pathways has been built. The Wnt pathway, which began to be uncovered in cancer biology studies, is crucial during the establishment of two signaling centers in Xenopus embryogenesis: Nieuwkoop center and the blastula chordin noggin expression center (BCNE). Here we will discuss the historical events that led to the discovery of those centers, as well as the molecular mechanisms by which they operate. This chapter highlights the cooperation of both signaling centers with potential to be further explored in the future. We aim to address the essential morphological transformation during gastrulation and neurulation as well as the role of Wnt signaling in patterning the organizer and the neural plate.

Whole-Mount In Situ Hybridization (WISH) for Salamander Embryos and Larvae.

Whole-mount in situ hybridization (WISH) is widely used to visualize transcribed gene sequences (mRNA) in developing embryos, larvae, and other nucleotide probe permeable tissue samples. This methodology involves the hybridization of an antisense nucleotide probe to the target mRNA, followed by chromogen or fluorescence-based detection. Here we describe a protocol for the spatiotemporal analysis of mRNA transcripts in axolotl embryos/larvae using digoxigenin-labeled riboprobes, anti-digoxigenin alkaline phosphatase, Fab fragments antibody, and NBT/BCIP chromogen detection.

Expression of rhodopsin and opsin in late‐stage epigean and hypogean salamander embryos

Evolutionarily, sensory systems have undergone extreme diversifications that have allowed vertebrates to survive diverse environments. The salamander clade Eurycea of Central Texas has branched out of its epigean ancestral roots and has successfully invaded subterranean environments. The San Marcos salamander (E. nana) and the Barton Springs salamander (E. sosorum) live above ground in springs and streams whereas the Texas blind salamander (E. rathbuni) occupies caves and recesses in the Edwards Aquifer. From an evolutionary perspective, it is thought that the hypogeal state is derived, but what remains a mystery is the developmental adaption of its sensory system to life under ground. The habitational broadening of the Eurycea salamander has been associated with the phenotype of reduced eyes exhibited in the Texas blind salamander. It is not clear whether this phenotype represents incomplete development, regression, or a combination of both. The clade of Eurycea is a prime model for comparing the molecular mechanisms that are involved in diverging neural systems, since species within the same clade present two extremes of phenotypes that are associated with their environment. Retinal development in embryonic E. rathbuni has been observed; however, the extent to which the retina develops is unknown. We identify retinal rod and cone photoreceptor cells along with their associated visual proteins (rhodopsin and opsin, respectively) in blind species and in epigean salamanders with fully developed eyes. Immunohistochemistry followed by confocal microscopy was applied for the investigation of sections of late embryonic stages of these salamander species. The results suggest opsin is expressed in the retina of E. nana and E. sosorum, and rhodopsin is expressed in all three species. Based on previous work indicating absence of visual pigments in adult Texas blind salamanders, we conclude expression of rhodopsin is transient or variable in this species. In future studies we will examine the expression of visual pigments at other stages to get a clearer picture of the time course and species‐level variability in expression.

A ball microscope for viewing the entire surface of amphibian embryos

Waves on the surface of developing eggs/embryos need to be viewed from all sides of their 3D tissue. The ball microscope will enable tracking of cellular waves and determine their interactions with the cells on the surface. Nine microscopes are arrayed in a spherical formation around an imaging stage to create whole surface images of objects anywhere from 0.5 mm3 to 60 mm3 in size. The 3D printed ball-based microscope is made using nine, Opti-Tekscope OT-HD Digital USB Microscope Camera Magnifiers. Eight of the microscope cameras fit into the ball at 90° angles to each other and one bottom microscope is used for a base to hold the stage. The base will support a customised cuvette to hold the embryo in water. The microscopes are the size of a pen (13 cm long and 1 cm in diameter) and each have a ring light around their diameter for self illumination. The nine microscopes can be attached to a microcontroller for time-lapse automated imaging. This microscope will be compared to other microscopes developed for the same purpose. The microscope can be used for time lapse imaging of the surface of small 3D objects and can be used to view Axolotl salamander embryo development as the Axolotl embryos are 2 mm in diameter. Other amphibian eggs can also be imaged using this technique.

Heterotrophic Carbon Fixation in a Salamander-Alga Symbiosis.

The unique symbiosis between a vertebrate salamander, Ambystoma maculatum, and unicellular green alga, Oophila amblystomatis, involves multiple modes of interaction. These include an ectosymbiotic interaction where the alga colonizes the egg capsule, and an intracellular interaction where the alga enters tissues and cells of the salamander. One common interaction in mutualist photosymbioses is the transfer of photosynthate from the algal symbiont to the host animal. In the A. maculatum–O. amblystomatis interaction, there is conflicting evidence regarding whether the algae in the egg capsule transfer chemical energy captured during photosynthesis to the developing salamander embryo. In experiments where we took care to separate the carbon fixation contributions of the salamander embryo and algal symbionts, we show that inorganic carbon fixed by A. maculatum embryos reaches 2% of the inorganic carbon fixed by O. amblystomatis algae within an egg capsule after 2 h in the light. After 2 h in the dark, inorganic carbon fixed by A. maculatum embryos is 800% of the carbon fixed by O. amblystomatis algae within an egg capsule. Using photosynthesis inhibitors, we show that A. maculatum embryos and O. amblystomatis algae compete for available inorganic carbon within the egg capsule environment. Our results confirm earlier studies suggesting a role of heterotrophic carbon fixation during vertebrate embryonic development. Our results also show that the considerable capacity of developing A. maculatum embryos for inorganic carbon fixation precludes our ability to distinguish any minor role of photosynthetically transferred carbon from algal symbionts to host salamanders using bicarbonate introduced to the egg system as a marker.

Physiological benefits and latent effects of an algal-salamander symbiosis

Embryos of the salamander Ambystoma maculatum (Shaw) and the uni-cellular green alga Oophila amblystomatis (Lambert ex Wille) have evolved a resource exchange mutualism. Whereas some of the benefits of the symbiosis to embryos are known, the physiological limitations of the relationship to embryos and carry over or latent effects on larvae are not. To determine the impact of the relationship across life history stages, we measured the growth, survival, and metabolic rate in response to hypoxia of salamander embryos reared under 0-h light (algae absent), 14-h light (control - algae present, fluctuating light conditions) and 24-h light (algae present, chronic light conditions) and the resulting larvae two-weeks post hatch. Embryos reared under 0-h light demonstrated decreased growth and survival compared to 14- and 24-h light, with no effect on metabolic rates or the response of metabolic rates to declining oxygen partial pressure (pO2). Conversely, larvae from embryos reared under 0-h light exhibited compensatory growth during the two-week larval rearing period, with body sizes matching those from the 14-h light treatment. Larvae from embryos reared under 24-h light had lower wet body mass and LT50 values upon starvation compared to those reared under 14-h light. Coupled with the lowest metabolic rates under normoxic pO2 levels, this indicates the presence of negative latent effects. We discuss the findings in relation to the effect of the symbiotic relationship on hypoxia tolerance and larval fitness with respect to the presence of compensatory growth and negative latent effects.

Acquisition and reconstruction of 4D surfaces of axolotl embryos with the flipping stage robotic microscope

We have designed and constructed a Flipping Stage for a light microscope that can view the whole exterior surface of a 2 mm diameter developing axolotl salamander embryo. It works by rapidly inverting the bottom-heavy embryo, imaging it as it rights itself. The images are then montaged to reconstruct the whole 3D surface versus time, for a full 4D record of the surface. Imaging early stage axolotl development will help discover how cell differentiation and movement takes place in the early embryo. For example, the switch from ectodermal to neural plate cells takes place on the top, animal surface portion the egg/embryo and can be observed using the flipping stage microscope. Detailed pictures of the whole surface need to be obtained so that cell tracking and event histories, such as cell divisions and participation in differentiation waves, of individual cells can be recorded. Imaging the whole exterior of the eggs/embryos will allow for the analysis of cell behavior and the forces the cells experience in their natural setting in the intact or manipulated embryo. This will give insights into embryogenesis, development, developmental disruptions, birth defects, cell differentiation and tissue engineering.

When can embryos learn? A test of the timing of learning in embryonic amphibians.

Learning is crucial to the survival of organisms across their life span, including during embryonic development. We set out to determine when learning becomes possible in amphibian development by exposing spotted salamander (Ambystoma maculatum) embryos to chemical stimuli from a predator (Ambystoma opacum), nonpredator (Lithobates clamitans), or control at developmental stages 16–21 or 36–38 (Harrison 1969). Once exposures were completed and embryos hatched, we recorded the number of movements and time spent moving of individuals in both groups and all treatments. There was no significant difference in number of movements or time spent moving among any of the treatments. The groups that were exposed to predator stimuli and a blank control at stages 36–38 were also tested to determine whether there was a difference in refuge preference or difference in survivorship when exposed to a predator (marbled salamander). There was no difference in survival or refuge preference between individuals; however, all individuals preferred vegetated over open areas regardless of treatment type. We discuss hypotheses for the absence of embryonic learning in this species and suggest it may be the result of the intensity of the predator–prey interaction between the predator, large marbled salamander larvae, and the prey, spotted salamander larvae.

Generation of aneurogenic larvae by parabiosis of salamander embryos.

Limb regeneration of salamanders is nerve dependent, and the removal of the nerves in early stages of limb regeneration severely curtails the proliferation of the blastemal cells and growth of the regenerate. The removal of the neural tube from a developing salamander embryo results in an aneurogenic larva and the aneurogenic limb (ANL) develops independently without innervation. Paradoxically, the limb in an ANL is capable of regeneration in a nerve-independent manner. Here, we describe a detailed method for the generation of ANL in the spotted salamander, Ambystoma maculatum, for regeneration studies.

Optimization of culturing conditions for toxicity testing with the alga Oophila sp. (Chlorophyceae), an amphibian endosymbiont.

Eggs of the yellow-spotted salamander (Ambystoma maculatum) have a symbiotic relationship with green algae. It has been suggested that contaminants that are preferentially toxic to algae, such as herbicides, may impair the symbiont and, hence, indirectly affect the development of the salamander embryo. To enable testing under near-standard conditions for first-tier toxicity screening, the authors isolated the alga from field-collected eggs and identified conditions providing exponential growth rates in the apparent asexual phase of the alga. This approach provided a uniform, single-species culture, facilitating assessment of common toxicity end points and comparison of sensitivity relative to other species. Sequencing of the 18s ribosomal DNA indicated that the isolated alga is closely related to the recently described Oophila amblystomatis but is more similar to other known Chlamydomonas species, suggesting possible biogeographical variability in the genetic identity of the algal symbiont. After a tiered approach to culturing method refinement, a modified Bristol's media with 1 mM NH4 (+) as nitrogen source was found to provide suitable conditions for toxicity testing at 18 °C and 200 µmol m(-2) s(-1) photosynthetically active radiation (PAR) on a 24-h light cycle. The validity of the approach was demonstrated with Zn(2+) as a reference toxicant. Overall, the present study shows that screening for direct effects of contaminants on the algal symbiont without the presence of the host salamander is possible under certain laboratory conditions.

The roles of oxygen and ammonia in the symbiotic relationship between the spotted salamander Ambystoma maculatum and the green alga Oophila amblystomatis during embryonic development

Some of the positive benefits of the symbiotic relationship between the yellow spotted salamander Ambystoma maculatum and the green alga Oophila amblystomatis on salamander embryonic development have been well documented. However, the fundamental basis of the relationship, in terms of the exchange of metabolic and photosynthetic products, is under debate, especially as it relates to newly discovered endosymbiotic aspects of the relationship. We developed a novel experimental approach in which we liberated egg capsules from egg masses and compared de-capsulated embryos to capsulated embryos. We were able to discern the contribution of salamander embryo respiration and ammonia excretion and algal photosynthesis to oxygen and ammonia dynamics within the egg capsules under light and dark conditions. Algal photosynthesis drove oxygen production under light conditions, while salamander respiration caused hypoxia during dark conditions. Ammonia was consumed to such an extent that it may be the limiting factor for algal growth. Algal cells actively penetrated the developing gut of embryos through the blastopore from an early developmental stage, and some algae came to reside in the vicinity of liver primordia towards later embryonic development, perhaps explaining the lack of detectable ammonia production by de-capsulated embryos. We suggest that oxygen and ammonia may influence A. maculatum growth and O. amblystomatis population dynamics. We have also demonstrated the extent to which algal cells penetrate internal spaces of embryos, which may be the precursor to endosymbiosis between these two species.

Response of the green alga Oophila sp., a salamander endosymbiont, to a PSII‐inhibitor under laboratory conditions

In a rare example of autotroph-vertebrate endosymbiosis, eggs of the yellow-spotted salamander (Ambystoma maculatum) are colonized by a green alga (Oophila sp.) that significantly enhances salamander development. Previous studies have demonstrated the potential for impacts to the salamander embryo when growth of the algae is impaired by exposure to herbicides. To further investigate this relationship, the authors characterized the response of the symbiotic algae (Oophila sp.) alone to the photosystem II (PSII) inhibitor atrazine under controlled laboratory conditions. After extraction of the alga from A. maculatum eggs and optimization of culturing conditions, 4 toxicity assays (96 h each) were conducted. Recovery of the algal population was also assessed after a further 96 h in untreated media. Average median effective concentration (EC50) values of 123 µg L(-1) (PSII yield), 169 µg L(-1) (optical density), and 299 µg L(-1) (growth rate) were obtained after the 96-h exposure. Full recovery of exposed algal populations after 96 h in untreated media was observed for all endpoints, except for optical density at the greatest concentration tested (300 µg L(-1) ). Our results show that, under laboratory conditions, Oophila sp. is generally less sensitive to atrazine than standard test species. Although conditions of growth in standard toxicity tests are not identical to those in the natural environment, these results provide an understanding of the tolerance of this alga to PSII inhibitors as compared with other species.

Acid Water Interferes with Salamander–Green Algae Symbiosis during Early Embryonic Development

The inner egg capsule of embryos of the yellow-spotted salamander (Ambystoma maculatum) are routinely colonized by green algae, such as Oophila amblystomatis, that supply O(2) in the presence of light and may consume nitrogenous wastes, forming what has been proposed to be a mutualistic relationship. Given that A. maculatum have been reported to breed in acidic (pH <5.0) and neutral lakes, we hypothesized that low water pH would negatively affect these symbiotic organisms and alter the gradients within the jelly mass. Oxygen gradients were detected within jelly masses measured directly in a natural breeding pond (pH 4.5-4.8) at midday in full sunlight. In the lab, embryo jelly masses reared continuously at pH 4.5 had lower P(O)₂and higher ammonia levels relative to jelly masses held at pH 8.0 (control). Ammonia and lactate concentrations in embryonic tissues were approximately 37%-93% higher, respectively, in embryos reared at water pH 4.5 compared with pH 8.0. Mass was also reduced in embryos reared at pH 4.5 versus pH 8.0. In addition, light conditions (24 h light, 12L : 12D, or 24 h dark) and embryonic position (periphery vs. center) in the jelly mass affected P(O)₂but not ammonia gradients, suggesting that algal symbionts generate O(2) but do not significantly impact local ammonia concentrations, regardless of the pH of the water. We conclude that chronic exposure to acidic breeding ponds had a profound effect on the microenvironment of developing A. maculatum embryos, which in turn resulted in an elevation of potentially harmful metabolic end products and inhibited growth. Under acidic conditions, the expected benefit provided by the algae to the salamander embryo (i.e., high O(2) and low ammonia microenvironment) is compromised, suggesting that the A. maculatum-algal mutualism is beneficial to salamanders only at higher water pH values.

Intracapsular algae provide fixed carbon to developing embryos of the salamanderAmbystoma maculatum

Each spring, North American spotted salamander (Ambystoma maculatum) females each lay hundreds of eggs in shallow pools of water. Eggs are surrounded by jelly layers and are deposited as large gelatinous masses. Following deposition, masses are penetrated by a mutualistic green alga, Oophila amblystomatis, which enters individual egg capsules, proliferates and aggregates near the salamander embryo, providing oxygen that enhances development. We examined the effects of population density of intracapsular O. amblystomatis on A. maculatum embryos and show that larger algal populations promote faster embryonic growth and development. Also, we show that carbon fixed by O. amblystomatis is transferred to the embryos, providing the first evidence of direct translocation of photosynthate from a symbiont to a vertebrate host.

The aneurogenic limb identifies developmental cell interactions underlying vertebrate limb regeneration

The removal of the neural tube in salamander embryos allows the development of nerve-free aneurogenic limbs. Limb regeneration is normally nerve-dependent, but the aneurogenic limb regenerates without nerves and becomes nerve-dependent after innervation. The molecular basis for these tissue interactions is unclear. Anterior Gradient (AG) protein, previously shown to rescue regeneration of denervated limbs and to act as a growth factor for cultured limb blastemal cells, is expressed throughout the larval limb epidermis and is down-regulated by innervation. In an aneurogenic limb, the level of AG protein remains high in the epidermis throughout development and regeneration, but decreases after innervation following transplantation to a normal host. Aneurogenic epidermis also shows a fivefold difference in secretory gland cells, which express AG protein. The persistently high expression of AG in the epithelial cells of an aneurogenic limb ensures that regeneration is independent of the nerve. These findings provide an explanation for this classical problem, and identify regulation of the epidermal niche by innervation as a distinctive developmental mechanism that initiates the nerve dependence of limb regeneration. The absence of this regulation during anuran limb development might suggest that it evolved in relation to limb regeneration.

Symbioses between salamander embryos and green algae

The symbiosis between Ambystoma maculatum (spotted salamander) embryos and green algae was initially described over 120 years ago. Algae populate the egg capsules that surround individual A. maculatum embryos, giving the intracapsular fluid a characteristic green hue. Early work established this symbiosis to be a mutualism, while subsequent studies sought to identify the material benefits of this association to both symbiont and host. These studies have shown that salamander embryos benefit from increased oxygen concentrations provided by their symbiotic algae. The algae, in turn, may benefit from ammonia excreted by the embryos. All of these early studies considered the association to be an ectosymbiotic mutualism. However our recent work has shown that algae invade both embryonic salamander cells and tissues during development. The unexpected invasion of algal cells into a salamander host changes our understanding of this symbiosis. This review will summarize the earlier research on this association in the context of these recent findings. It will also emphasize gaps in our understanding of this and other amphibian embryo-algal interactions and suggest various research avenues to address these unanswered questions.

The effects of atrazine on spotted salamander embryos and their symbiotic alga

Worldwide amphibian declines have been a concern for biologists for the past several decades. The causes of such declines may include habitat loss, invasive species, pathogens, and man-made chemicals. Agricultural herbicides, in particular, are known to interfere with reproduction in amphibians and are likely contributing to population declines. We tested the effects of the herbicide atrazine on developing spotted salamanders (Ambystoma maculatum) and their symbiotic green alga Oophila amblystomatis. We exposed spotted salamander egg masses to atrazine at concentrations of 0 microg/L (control), 50, 100, 200, and 400 microg/L. Algae were eliminated in all atrazine treatments. Hatching success was significantly lower for atrazine-treated egg masses than for the controls, and was inversely related to atrazine concentration. The highest developmental stage reached by the embryos was significantly lower in the atrazine treatments than in the controls, and was inversely related to atrazine concentration. These results indicate that atrazine exposure affected spotted salamanders both directly by causing pathologies and mortality in embryos and indirectly by eliminating their symbiotic alga.

Embryonic origin of gustatory cranial sensory neurons

Cranial nerves VII, IX and X provide both gustatory (taste) and non-gustatory (touch, pain, temperature) innervation to the oral cavity of vertebrates. Gustatory neurons innervate taste buds and project centrally to the rostral nucleus of the solitary tract (NTS), whereas neurons providing general epithelial innervation to the oropharynx project to non-gustatory hindbrain regions, i.e., spinal trigeminal nucleus. In addition to this dichotomy in function, cranial ganglia VII, IX and X have dual embryonic origins, comprising sensory neurons derived from both cranial neural crest and epibranchial placodes. We used a fate mapping approach to test the hypothesis that epibranchial placodes give rise to gustatory neurons, whereas the neural crest generates non-gustatory cells. Placodal ectoderm or neural crest was grafted from Green Fluorescent Protein (GFP) expressing salamander embryos into unlabeled hosts, allowing us to discern the postembryonic central and peripheral projections of each embryonic neuronal population. Neurites that innervate taste buds are exclusively placodal in origin, and their central processes project to the NTS, consistent with a gustatory fate. In contrast, neural crest-derived neurons do not innervate taste buds; instead, neurites of these sensory neurons terminate as free nerve endings within the oral epithelium. Further, the majority of centrally directed fibers of neural crest neurons terminate outside the NTS, in regions that receive general epithelial afferents. Our data provide empirical evidence that embryonic origin dictates mature neuron function within cranial sensory ganglia: specifically, gustatory neurons derive from epibranchial placodes, whereas neural crest-derived neurons provide general epithelial innervation to the oral cavity.