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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Dev Dyn. 2020 Sep 16;250(6):880–895. doi: 10.1002/dvdy.245

Characterizing the regenerative capacity and growth patterns of the Texas blind salamander (Eurycea rathbuni)

Warren A Vieira 1,*, Kelsey Anderson 2,*, Lindsay Glass Campbell 2, Catherine D McCusker 1,●
PMCID: PMC8454265  NIHMSID: NIHMS1676869  PMID: 32885536

Abstract

Background

Regeneration of complex patterned structures is well described amongst, although limited to a small sampling of, amphibians. This limitation impedes our understanding of the full range of regenerative competencies within this class of vertebrates, according to phylogeny, developmental life stage and age. To broaden the phylogenetic breath of this research, we characterized the regenerative capacity of the Texas blind salamander (Eurycea rathbuni), a protected salamander native to the Edwards Aquifer of San Marcos, Texas and colonized by the San Marcos Aquatic Resource Center. As field observations suggested regenerative abilities in this population, the forelimb stump of a live captured female was amputated in the hopes of restoring the structure, and thus locomotion in the animal. Tails were clipped from two males to additionally document tail regeneration.

Results

We show that the Texas blind salamander exhibits robust limb and tail regeneration, like all other studied Plethodontidae. Regeneration in this species is associated with wound epithelium formation, blastema formation and subsequent patterning and differentiation of the regenerate.

Conclusions

The study has shown that the Texas blind salamander is a valuable model to study regenerative processes, and that therapeutic surgeries offer a valuable means to help maintain and conserve this vulnerable species.

Keywords: non-model organism, conserved regenerative mechanism, limb regeneration, tail regeneration, breeding

1. INTRODUCTION

In the animal kingdom regenerative capabilities can range from simple cellular restoration to whole-body reconstitution; however, the more complex the structure being replaced or restored, the more restricted the regenerative ability. Whole-body regeneration is, for example, documented in only a limited number of metazoan linages, such as hydra and planaria; while basic cell turnover, cellular regeneration, is document in all animals.1–3 In between these two extremes is the ability to replace complex patterned structures, such as limbs and tails. Although scattered in presentation across phyla, even within the same lineage, amphibians are well characterized as exhibiting this type of regenerative response.

The processes and biological requirements of amphibian regeneration have been most extensively studied in the urodele limb. In response to amputation, keratinocytes migrate into and generate a wound epithelium over the plain of injury.4,5 Underlying nerves innervate this wound epithelium and convert it into a signaling hub that drives the formation of a blastema, a collection of cells derived from the underlying stump tissue that revert to a progenitor-like state and generate the missing structure.6–11 Pattern formation in the regenerate requires that cells derived from the different limb axes to be present in the blastema.12 Blastemas that lack positional diversity fail to regenerate, but regeneration can be rescued in this context by grafting cells into the wound site from a different limb position, or by exposing the blastema cells to biological molecules that provide positional cues (Retinoic acid, Sonic hedge and Fibroblast growth factor 8).12–17 Evidence suggests that issues during wound closure that minimize the contribution of cells from the different limb axes are a mechanism for regenerative failure in urodele species known to naturally exhibit robust regeneration.18 Thus, crush injuries or limb amputations with large amounts of tissue damage are likely sources of regenerative failure in these animals.

As juveniles, all studied amphibians, irrespective of class or family, demonstrate a robust limb regenerative phenotype with a high fidelity. However, this is not necessarily maintained during changes in developmental life stage and/or advancement in age. Anuran amphibians only demonstrate robust limb regeneration prior to but not proceeding metamorphosis.19 If any regenerative phenotype is documented after metamorphosis in anuran species, the resultant structure fails to recapitulate the original pattern. Instead, the regenerate is constituted by only a cartilaginous rod or nodule(s), surrounded by connective tissue and skin, and essentially lacking muscle (Figure 1).20–22

Figure 1: Illustration of the general regenerative responses to limb amputation observed for adult anuran and urodele amphibians, according to family.

Figure 1:

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Pattern is described by skeletal structure. The anuran families Bufonidae, Hylidae, Ranidae, and Scaphiopodidae fail to regenerate, with the exception of Pseudacris triseriata and Pseudacris clarkii which regenerate heteromorphic structures in response to limb amputation. The anuran families Alytidae, Bombinatoridae, and Pipidae, regenerate heteromorphic structures, constituted by cartilage, connective tissue, and skin but lack muscle. The urodele families Ambystomatidae, Plethodontidae and Salamandridae all regenerate robustly, restoring both pattern and tissue types; although metamorphosized Ambystomatidae frequently exhibit patterning defects in the regenerate. The regenerative ability of the urodele families Proteidae, Sirenidae and Amphiumidae are controversial (see text for details) but the only published studies demonstrate failure to regenerate or heteromorphic output. 20,21,30,51,62,22–29 Red line represents plane of amputation.

Amongst urodele families, all studied Salamandridae, Plethodontidae and Ambystomatidae species exhibit limb regeneration irrespective of age or metamorphic state (Figure 1).20,23–29 Metamorphosized Ambystomatidae do, however, demonstrate a significant reduction in regeneration rate and fidelity (digit loss and or carpel loss/fusion), relative to paedomorphic counterparts (Table 1).26 Data pertaining to the regenerative ability of adult Proteidae, Sirenidae and Amphiumidae species, which are also urodeles, is controversial. Original observations demonstrated that these families either fail to regenerate or generate a simple heteromorphic structure in response to amputation (figure 1).20,23,30 Later, Young et al. (1983) showed that the regenerative capacity in post-metamorphic, terrestrial adult Ambystomatidae was greatly influenced by laboratory conditions, including diet, photoperiod, temperature, and aquatic versus terrestrial habitat; and that conditions suitable for robust regeneration in aquatic life stages where detrimental to terrestrial forms. It was therefore posited that the poor or absent regenerative ability documented in adult Proteidae, Sirenidae and Amphiumidae could be a consequence of inappropriate housing conditions.27 Additionally, Young et al. (1983) and Monaghan et al. (2014) showed that adult Ambystomatidae exhibited reduced regeneration rates, requiring in excess of 100 days to complete regeneration; although, this extensive delay may be family specific as adult Notophthalmus viridescens and many Plethodontidae can regenerate in less than 60 day at equivalent temperatures.20,23,25–28 Never-the-less, it was proposed that the evaluation period post amputation in the adult Proteidae and Sirenidae study, 54 and 76 days respectively, may have been insufficient to observe a regenerative response.20,23,27 Although the points raised by Young et al. (1983) are valid, they have not be tested experimentally and the regenerative ability of adult Proteidae, Sirenidae and Amphiumidae has still not been demonstrated. This highlights the need for improved curation of regenerative capacity amongst amphibians, not only phylogenetically but also according to developmental life stage and age.

Table 1:

Limb regeneration times documented in different urodele species

Family Species Age Aquatic vs terrestrial Mean SVL Size (cm) Temperature (°C) Diet Site of amputation*** Day to reach References
Early stage blastema, unless stated otherwise Specified endpoint
Ambystomatidae Ambystoma mexicanum Larval Aquatic 3* 20 NS Stylopod 5 12 (four-digit stage) 63
Larval Aquatic 3* 20 NS Zeugopod 5 10 (four-digit stage) 63
Adult Aquatic 15.6–18.2* 21 Beef liver Stylopod 12–15 ≥ 30 (digit outgrowth) 24
Adult Aquatic 8.5−10.2 20 − 22 California blackworms (Ad libitum) Stylopod < 20 26.2 ± 2.9 (SE) (differentiation) 26
Adult Terrestrial 8.5−10.6 20 − 22 California blackworms (Ad libitum) Stylopod < 10 54.7 ± 25.4 (SE) (differentiation) 26
Ambystoma jeffersonianum complex Adult Aquatic or semi aquatic
(not directly stated)		 3.2 21 – 24 Various 1**** Zeugopod 25 (cone-shaped blastema) 40 (digit indentations) 23
Ambystoma laterale Adult Aquatic or semi aquatic
(not directly stated)		 3.2 21 – 24 Various 1**** Zeugopod 30 (hemispherical blastema) 71 (digit indentations) 23
Ambystoma maculatum Larval Aquatic 2.5 – 4 21 – 23 Brine shrimp (Ad libitum) Stylopod 5–7 25 (four-digit stage) 62
Adult Terrestrial 9.4 20 – 30 Night crawlers Zeugopod 25–50 255 – 300 (complete regeneration) 28
Ambystoma opacum Adult Not specifically stated 5.85 21 – 24 Various 2¥ Mixed NS 71 (digit stage) 20
Ambystoma annulatum Adult Terrestrial 8.5 20 – 30 Night crawlers Zeugopod 32–66 324 – 370 (complete regeneration) 28
Ambystoma texanum Adult Terrestrial 10.2 20 – 30 Night crawlers Zeugopod 20 – 40 215 – 250 (complete regeneration) 28
Ambystoma tigranum Adult Terrestrial 12.8 20 – 30 Night crawlers Zeugopod 18 – 43 155 – 180 (complete regeneration) 28
Plethodontidae Bolitoglossa ramosi Adult Terrestrial 7 – 10* 18 − 21 Fruit flies Stylopod 10 97 (digit outgrowth) 51
Desmognathus fuscus Adult Not specifically stated 6.2 21 – 24 Various 2¥ Zeugopod 14 34 (four-digit stage) 20
Desmognathus ochrophaeus Adult Not specifically stated 3.75 21 – 24 Various 2¥ Mixed 14 34 (four-digit stage) 20
Eurycea bislineata Adult Not specifically stated 3.87 21 – 24 Various 2¥ Mixed 15 (hemispherical blastema to elongated cone) 30 (four-digit stage) 20
Adult Aquatic or semi aquatic (not directly stated) 40 21 – 24 Various 1**** Mixed 12 24 – 30 days (digit indentation) 23
Eurycea rathbuni Adult Not specifically stated 4.1 21 – 24 Various 3§ Stylopod 21** 63 (complete pattern) ** Current study
Plethodon cinereus Adult Aquatic or semi aquatic
(not directly stated)		 3.2 21 – 24 Various 1**** Mixed 14 41 (four-digit stage) 23
Adult Not specifically stated 3.5 21 – 24 Various 2¥ Mixed 21 41– 50 (four-digit stage) 20
Plethodon dorsalis Adult Not specifically stated 4.5 21 – 24 Various 2¥ Mixed NS 71 (four-digit stage) 20
Plethodon glutinosus Adult Aquatic or semi aquatic
(not directly stated)		 5.4 21 – 24 Various 1**** Mixed NS 87 (four-digit stage) 23
Salamandridae Notophthalmus Viridescens Adult Aquatic 8* 25 Tubifex worms Stylopod 10–17 34 (late digit stage) 25
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NS – not stated

*

size = snout to tail tip length

**

these stages may have been temporally delayed due to initial damage to the wound epithelium.

***

mixed - some individuals within the study population were amputated at the stylopod and others at the zeugopod level of the limb, but all were assessed collectively as a single cohort.

****

various 1 - one or more of the following: chopped beef, pork heart, earthworms, Tubifex worms, Enchytraeus worms, or flour beetle larvae.

¥

various 2 – one or more of the following: Tubifex worms, Enchytraeus worms, flour beetle larva, mealworks, earthworms, or crickets.

§

various 3 – one or more of the following: frozen copepods, amphipods, ostracods, worms, blind cave shrimp (Palaemonetes antrorum) and troglobitic crustaceans (Stygobromus sp.). SE – standard error.

Another important consideration is that phylogenetically the above described analyses in anurans and urodeles is constituted by only a small sampling of all extant amphibian species. These studies may not represent the true breadth of regenerative capabilities in this class. Amongst teleosts, heart and fin regeneration are commonly observed phenotypes; however, even within the same species dramatic differences in regenerative potential have been documented. Both surface and cave dwelling members of Astyanax mexicanus can regenerate amputated dorsal fin lobes robustly; while the Pachón cave dwelling population of the species, which has undergone a variety of metabolic and biochemical changes compared to their non-cave dwelling relatives to survive in a subterranean environment, has lost the ability to regenerate heart tissue.31 Therefore, regenerative capacity should never been assumed even for closely related species; instead empirically testing should always be conducted.

One amphibian species for which no formal testing and documentation in regards to regenerative ability exists is the Texas blind salamander (Eurycea rathbuni). This is a cryptic subterranean cave-dwelling Plethodontidae species which is endemic to the Edwards Aquifer in and around San Marcos.32 Despite identification in 1896, very little is known regarding the biology of this species, including its regenerative capabilities.33 Understanding the biology of the Texas blind salamander would not only contribute to our overall understanding of amphibian regeneration but could also benefit the survival of this species. As this species is currently classified as vulnerable by the International Union for Conservation of Nature (IUCN), current conservation efforts involve the establishment and maintenance of a colony at the San Macros Aquatic Resources Center (SMARC), Texas.34 A biological understanding of this species may aid in empowering and improving conservation efforts, as well as provide a means to leverage the organisms innate healing abilities to remedy injuries identified in wild and colonized animals. Therefore, we investigated and characterized several aspects of this species biology, including regenerative capacity. The Texas blind salamander was found to exhibit robust limb and tail regeneration, following the classically described stages of development seen in other regenerating amphibians.

2. RESULTS AND DISCUSSION

2.1. Habitat and biology of the Texas blind salamander

The Texas blind salamander is a blind, subterranean cave-dwelling salamander species of the Plethodontidae family, endemic to the Edwards Aquifer in and around San Marcos, Texas (Figure 2A).32,33 In 1967, the Endangered Species Act (ESA) classified this species as endangered; however, the IUCN has subsequently re-designated the species as vulnerable, despite the ESA status remaining in place.34,35 For conservation purposes, individuals are captured live from subterranean wells that connect to their natural habitat (Figure 2B), and colonized at the SMARC. The gross morphology and anatomy of this species has previously been described but will be discussed here briefly.33,36 As represented in figure 2C, this species has a head with a protruding, shovel-shaped snout and lacks true eyes.33 This species is probably not photosensitive, although this has not been tested functionally, as the optic nerve in the adult is connected to an ocular structure that lacks an eye lens and is surrounded by melanized tissue.37 During embryogenesis, however, an eye lens vesicle does initially develop, but then regresses by unknown mechanisms.37 Throughout life three small gill branches are located on each side of the head of the animal which, in conjugation with cutaneous gas exchange, facilitate respiration in this lungless organism.33,36,38 The body, tail, and limbs are slender and covered in thick skin.33 The iridescent skin is pale in color, and while juvenile skin can have many small, pigmented melanocytes, the adults have little to no dark pigmentation in their skin.39,40

Figure 2: Habitat, appearance and growth patterns of the Texas blind salamander.

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A) Maps indicating the natural habitat of the Texas blind salamander. This species resides in the yellow highlighted region of Edwards Aquifer in San Macros, Texas. Black lines represent major road ways, blue lines represent major water ways, grey line represents 1 mile scale bar. San Marcos is located in Hays county (county marked in red in inset map, grey line represents 37 miles). B) Representative images of subterranean caves (left) and fissures sites (right), connecting to the Aquifer regions highlighted in map A, used for live capture of the Texas blind salamander. C) A representative photograph of a Texas blind salamander exhibiting the typical small external gills, the absence of eyes, protruding flattened snout, and slender abdomen and tail of this species. D) Live images demonstrating an observed increase in body size with age in the Texas blind salamander. White line represents a scale bar of 127mm. D’) Line graph showing change in total animal size (snout to vent length) over the first year of life (N = 109). Due to animal fragility, individual animals were not initially measured at hatching, but correlate roughly to 5mm in size (snout to vent length). Error bar represent standard deviation. D”) Line graph representing total size (snout to vent length) of animals at known ages, ranging from 1 to 3 years old. (N = 18, 25, 16, 7, 5 for each time point). Error bar represent standard deviation. D”’) Bar graph representing life-stage specific growth rates (change in snout to vent length) of the Texas blind salamander over a 12-month period. Growth rate decreased significantly with age. J – juveniles (N = 124); S - sub-adults (N= 15), M – sexually mature adults (N = 20), * p < 0.05 and error bars represent standard deviation. Only animals that remained in the same age class during the 12 month period were counted in this analysis.

Overall external appearance changes very little with age in this species; although, individuals do demonstrate continual growth throughout life (Figure 2D). During the first year of life after hatching, captive animals grow rapidly and within only 6 months have tripled in size (figure 2D’). This rapid level of growth, however, is not sustained and progressively decreases with age (figure 2D” and figure 2D”’), where sub-adults (estimated age 2 – 3 years old) grow at a significantly slower rate, over a 12 month period, relative to juveniles but still a significantly faster rate than sexually mature adults (estimated age greater than 3 years old). It should be noted that this assessment of size and age (figure 2D’–D”’) was conducted upon colonized animals and may not precisely account for growth in the wild, where factors such as nutrient availability and temperature, which affect amphibian growth, can fluctuate widely. Never-the-less, as growth never stops in this species, it is a useful surrogate for age. Published accounts suggests that survival in the wild is greater than 10 years, with the largest wild adults documented measuring approximately 135 mm snout to tail tip in length.41–43 However, at the SMARC, one female is estimated to be 20 years old and measured 146.5 mm snout to tail tip in 2020. Differences in growth rates and longevity between males and females of this species have not been determined, in part due to difficulties associated with sexing this organism.

Based on current and historical analysis, external sexual dimorphism is absent in the Texas blind salamander (Figure 3).44 Cloacal swelling, a characteristic of many sexually mature male salamander species including Plethodontidae species, is not observed.45–49 Limited, preliminary observations do suggest that vent shape differs between the sexes; but analysis of larger cohorts is required to validate this. Sexing is, therefore, dependent on the candling method – whereby eggs within gravid females are observed by shining light through the abdomen (Figure 3C). This procedure has limitations too though; the skin of the Texas blind salamander thickens with age which makes candling difficult on larger animals. Breeding is hypothesized to be temporally unrestricted, due to larvae being found throughout the year in the wild; however, numbers fluctuate greatly and the exact cue for the periodic influx of juveniles is unknown. At the SMARC, females become gravid at 1.5 to 2 years of age, but the presence of eggs does not necessarily result in the production of offspring. Additionally, observations at the SMARC show long periods of time between formation and maturation of eggs, up to 2 years in some individual females. Breeding in captivity has revealed that the female initiates courtship (Supplementary video 1), which is distinctly different to other salamander species, and clutch sizes are small.50 Records from 2008–2020 at the SMARC indicates an average clutch size for the Texas blind salamander is 23.8 eggs (N = 81 clutches), with variable hatch success. Documented discrepancies in hatch rates between clutches are due to factors including differences in documentation and husbandry methods utilized at the time, fungal infections, and overall clutch viability. In the past four years, clutch survival rates have improved through the immediate removal of fugal-infected eggs. Larva are very fragile for the first two months of life after hatching; but thereafter they exhibit high survivability in captivity. Although the increased age at first reproduction (compared to other Central Texas Eurycea species) and long generation time of this species has limited captive breeding, maintenance of the SMARC colony is essential as both a reassurance population and a means to study these organisms to aid their conservation in the wild.

Figure 3: Absence of external sexual dimorphism in the Texas blind salamander.

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Gross body (A) and cloacal (B) morphologies do not indicate any sexual dimorphism between sexually mature (2 or more years of age) members of the SMARC colony; however, the candling method (C) can identify eggs (enclosed by black dotted line) in the abdomen of gravid females. White scale bar (A) represents 64mm. Right panels are representative of male Texas blind salamanders, while the left panels represent females.

Extinction threatens 41% percent of amphibian species, highlighting the need for robust conservation efforts for all amphibians, including the Texas blind salamander. The value of this class of animals, however, is not limited to biodiversity and ecosystem maintenance, but extends to the understanding of complex biological processes such as epimorphic regeneration. A better understanding of regeneration in this species would aid medical science in the pursuit of achieving regeneration within the human population as well as provide direct benefit to the Texas blind species, where innate healing abilities could be leverage to remedy injuries identified in wild and colonized animals. We therefore set about to determine the regenerative abilities of the Texas blind salamander.

2.2. Limb regeneration in the Texas blind salamander

Amongst the limited literature attributed to the Texas blind salamander, there is no description or assessment of regenerative capacity.32,36,44 Limb regeneration studies have, however, been conducted on several other Plethodontidae (Plethodon cinereus, Plethodon glutinosus, Eurycea bislineata, Plethodon dorsalis, Desmognathus ochrophaeus, Desmognathus fuscus, and Bolitoglossa ramosi).20,23,51 In all cases, robust limb regeneration was documented; although great temporal variation exists in this process between these and other regenerative amphibians (table 1).20,23,51 These temporal differences may be compounded by differences in diet, temperature, and other housing conditions, which not only differ widely between various experimental studies but have been shown to significantly influence regeneration, at least in metamorphosized Ambystomatidae.27

While surveying the native Texas blind salamander population in the wild, observations of limb regeneration are exceptionally rare. To date, there has only been a single animal found exhibiting apparent limb regeneration. This captured male had smaller hindlimbs than the other size matched animals in the colony (Figure 4A). Upon closer inspection, there appeared to be differences in the basement membrane within the hindlimbs of this male, where the skin was opaque in the region closest to the body, and then abruptly became more transparent in the mid-stylopod. This difference in basement membrane deposition between the existing limb structure and the newly regenerated structure has been previously observed in the regenerative salamander model, Ambystoma mexicanum (axolotl), and thus suggests that a similar regenerative process was occurring in the hindlimbs of this male Texas blind salamander (Figure 4B). Over the following months the regenerating hindlimbs underwent accelerated growth; this is also consistent with how the axolotl completes regeneration, where at first a tiny limb structure is formed, and grows rapidly to reach the size that is proportionally appropriate to the rest of the animal. The time taken for a regenerate to reach proportionality in the Texas blind salamander is unknown but two years after capture the hindlimbs of this particular animal had not yet reached proportionality, when compared to other age- and size-matched controls. Together, these observations indicate that this male was in the advanced stages of hindlimb regeneration when it was captured.

Figure 4: Evidence of limb regeneration in the Texas blind salamander.

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A) A male of the species was captured with hindlimbs (HL) of small size (right), relative to other wild type (WT) colony members (left), and what appeared to be proximal stump (white arrow heads) and distal regenerated tissue. White scale bar represents 64mm. B) The hindlimbs of this captured male (right) underwent rapid growth over the following months, and a visible difference in the skin was still discernable between the proximal (red arrow, presumptive stump) and distal (blue arrow, presumptive regenerate) regions of the hindlimbs 17 months after capture. Live image of a regenerated axolotl forelimb (left) reveals a distinct different between the regenerated (blue arrow) and matured, stump skin (red arrow). Red dashed line indicates plane of amputation. White scale bar represents 2mm.

While the above example supports the idea that the Texas blind salamander can regenerate limbs, we also observed an example of an animal that appeared to have regenerative failure. This female was captured with what appeared to be a forelimb stump on its left side and a normal limb on its right side (Figure 5A). The stump didn’t have any signs of regeneration and was completely healed over (Figure 5B). It was possible that this apparent regenerative failure, relative to the male described above, may be due to sexual dimorphism, a phenomena that has been documented in zebrafish pectoral fin regeneration.52 Alternatively, it was also possible that a crush injury took place, which prevented the establishment of a wound epithelium and/or a positional disparity, which have been shown to be crucial for limb regeneration in other amphibians such as the axolotl.12,53,54

Figure 5: Limb regeneration in a limbless female.

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A) A female was captured from the wild in 2018 with a missing left forelimb. In 2019, live (B) and histological (C) evaluation of the stump revealed no signs of regeneration. H, E and A staining of tissue sections (10x magnification stitched images) showed an accumulation of adipocytes within the stump; (C’) inset showing adipocytes at higher (20x) magnification. Ep – epidermis, Ad – adipocyte, Ms – muscle, Hm – humerus. Double headed arrow indicates proximal (Prox) and distal (Dist) axis of stump. White scale bar in (B) represents 0.5mm. D) A time course of forelimb regeneration in the amputated female; the numbers in the figure represent days post amputation (dpa). After amputation, wound epithelium formation was proceeded by early blastema formation 21 dpa. A palette stage regenerate was formed by 49 dpa, digit indentations by 56 dpa, and the limb, although tiny in comparison to the intact right limb, was completely patterned by 63 wpa. Dark pigment spots migrated into the regenerate as early as 56 dpa, with normal pigmentation pattern restored to the entire regenerate by 91 dpa. White scale bar represents 1mm.

Given the apparent regenerative capacity of the Texas blind salamander, we speculated that if the limb stump on the female was surgically amputated, then a regenerative response could possibly be stimulated. The Texas blind salamander utilizes its limbs for locomotion, including underwater walking and climbing, and this female demonstrated impeded locomotion as a result of her stump. Therefore, regeneration of the female’s forelimb would restore locomotive abilities and improve the quality of life for this individual. Thus, permission was obtained from the SMARC field office, in conjunction with the associated veterinarian, to amputate the stump of the female as a possible regenerative therapy. This rare circumstance also provided the possibility to observe a complete regenerative response in the Texas blind salamander (Figure 5D).

The distal tip of the stump was surgically removed and collected for histological evaluation to identify potential characteristics that may have contributed to regenerative failure (Figure 5C). The excised stump tissue contained what appears to be a truncated portion of the stylopod and muscle. Additionally, the stump tissue exhibited an accumulation of adipocytes, rather than obvious scar tissue as expected (Figure 5C). While this unexpected composition may be associated with regenerative failure, it is also possible that this species normally exhibits a large abundance of this cell type in its skin. Thus, future work is required to determine the normal cellular composition of the limb in this species.

Stump amputation was sufficient to initiate a complete regenerative response, resulting in the restoration of an appropriately patterned forelimb (Figure 5D). A wound epidermis was generated within the first 24 hours post amputation, although this appeared to be damaged both 1- and 7-days post amputation (dpa) (Figure 6). This damage was likely due to the animal attempting to climb and, as a result, tank enrichment was greatly reduced to prevent further damage. By 21 dpa an early stage blastema was present; however, the initial damage to the wound epithelium may have temporally delayed blastema formation and subsequent development. It is noteworthy that in the other studied Plethodontidae the time taken to generate an initial blastema can vary from 10 to 21 days (Table 1). By 49 dpa the blastema had developed into a palate stage, by 56 dpa digit indentations were present, and by 63 dpa a completely patterned limb with all four digits was observed. The time taken to complete regeneration was within the range documented for other lungless salamanders – 30 to 97 days (Table 1). Normal pigmentation spots where fully restored within the regenerate by 91 dpa.

Figure 6: Wound epithelium documented 24 hours post amputation over limb and tail stumps.

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A) The wound epithelium, formed 24 hours post limb amputation (hpa), appeared to be damaged, with one half of the wound epithelium missing. White dotted line encloses the entire stump and divides the site in half according to which is (left) and is not (right, arrow head) covered by a wound epithelium. As the wound epithelium is generated by the circumferential migration of cells into the wound site in other salamander species, the absence of half of the wound epithelium suggested injury as opposed to incomplete formation. B) 7 days post amputation (dpa) the limb wound epithelium was also damaged, documented as lifting off of the wound site (white arrow head). C, D) Tail wound epithelium was generated in small (C) and large (D) males by 24 hours post amputation. White scale bar represents 1mm in all images.

The regenerated forelimb, although patterned and pigmented, was originally small in size relative to the intact right forelimb, and subsequently underwent rapid growth. By 210 dpa the regenerate was 0.9cm in length (right intact limb 1.33cm). The regenerate was functional, even in its tiny form. The animal utilized the regenerate for various forms of locomotion, including climbing (Supplementary video 2). As the Texas blind salamander utilizes its limbs for several necessary life processes, including feeding, evading predators, and mating; utilizing therapeutic amputation to restore damaged or lost limbs could therefore be effectively used to improve the survival of individual animals and the conservation of this vulnerable species.

Moreover, these observations show that both male and female Texas blind salamanders, like all other studied Plethodontidae, are able to regenerate complete limbs. As members of this family have diversified to occupy a variety of distinct habits, they provide a novel vantage point to consider the mechanisms by which regenerative traits have been maintained during environmental adaptation and specialization.

2.3. Tail regeneration in Eurycea rathbuni

Unlike limb regeneration, tail regeneration is poorly characterized amongst Plethodontidae; although, it has been documented in literature for a limited number of species, including Hemidactylium scutatum, Plethodon cinereus, Desmognathus fuscus, and Ensatina eschscholtzii platensis.55–58 No published cases of tail regeneration exist for the Texas blind salamander. Observation of this phenomenon in the wild are rare and never a result of naturally induced injury; instead they are the result of tail clipping, where previously captured individuals were clipped and then released back into the wild. Tail clips (roughly 3 mm) are taken from individuals to facilitate genetic analysis of the species as well as once for therapeutic purposes, to treating a fungal infection by removal of the infected tissue, and induce a robust regenerative response. To better characterize this phenomenon, we amputated one-third of the tail from two differently sized male animals from the SMARC colony.

Tail sections, collected and prepared for histological evaluation, show a similar morphology relative to other salamander species (Figure 7B).59,60 The surgery elicited a complete regenerative response (Figure 7A). By 1 dpa a wound epithelium had been generated across the plane of amputation (Figure 6). By 7 dpa, a small but apparent blastema was present on the tail of the smaller, but not the larger, animal (Figure 7A, insets). The larger animal showed delayed development relative to the smaller animal from this stage onwards. This difference is not surprising as animal size within the same species is known to affect regeneration rates (Table 1, see A. mexicanum); although, the mechanisms for these differences are unknown. In both animals, blastema formation was followed by patterning and differentiation leading to replacement of the missing tail structure. Myotome formation was observed 49 and 63 dpa for the small and large male respectively. As with the regenerating limb, the regenerated tail was smaller relative to the original and had visually distinct skin that lacked pigmentation. By 147 dpa, an indentation was still visible between stump and regenerate, and the skin of the regenerate remained transparent (indicating a thin basement membrane) and without pigment (Figure 7A).

Figure 7: Tail regeneration in the Texas blind salamander.

Figure 7:

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A) Tail tips were amputated from males of 8.5 cm (top panels) and 10.7 cm (lower panel) (snout to tail tip lengths) and were documented over a period of 147 days post amputation. Insets are of the tails 7 days post amputation (dpa) at higher magnification, when blastema formation is apparent at the tail tip of the smaller animal. White scale bars represent 2mm. B) Histological H, E and A stained sections of a tail clip sample taken on the day of amputation. Ep – epidermis, Sc – Spinal cord, Nc – notochord, Ms – muscle. C) Maturation and pigmentation are restored in regenerated tail skin. A portion of the tail was amputated from a male Texas blind salamander for health reasons in 2017. In 2020, mature, pigmented skin was clearly visible in the regenerate (rg) and was similar to the stump (st). Red dashed line represents plane of amputation, white scale bar represents 2mm. D) Slow skin maturation documented in response to tissue damage in tail after an attempted subcutaneous implantation of a PIT tag. Five months post injury (mpi), black arrows indicate region of inadequate restoration of basement membrane. Fifteen mpi, black arrows indicate basement membrane and pigmentation have been restored. White scale bar represents 1mm.

Although it takes a long amount of time after amputation, appropriate tail coloration does return in this species. Pigmentation and tissue opacity (indicative of a thick basement membrane) were restored in a regenerate, to a level observed in the stump, three years after the tail of a male Texas blind salamander was amputated to treat for a fungal infection (Figure 7C). This extended timing for skin repair was also observed in lateral wounds on the tail of this species. Tissue damage has been observed on occasion in response to implantation and subsequent rejection of subcutaneous passive integrated transponder (PIT) tags. At these sites of injury, the basement membrane was clearly absence 5 months but restored 15 months post injury. (Figure 7D).

3. CONCLUSION

Within the current study we present for the first time a new example of a urodele species that exhibits robust limb and tail regeneration; the Texas blind salamander. Similar to the axolotl and other regenerating amphibians, the Texas blind salamander generates blastemas to regenerate these structures. Therefore, this species is a valuable model to study the processes of limb and tail regeneration. The Texas blind salamander also differs from all these previously studied Plethodontidae in that they are completely aquatic and subterranean cave-dwelling. Subterranean, cave dwelling organisms exhibit a variety of metabolic and biochemical differences compared to their non-cave dwelling relatives which facilitate survival, and in some cases, such as in the Pachón cave dwelling Astyanax mexicanus population, have lost some regenerative capacity.31 The Pachón population is unable to regenerate heart tissue, relative to its surface dwelling counterpart, despite fin regeneration being robust.31 Therefore, it is possible that the cave-dwelling Texas blind salamander, even though it exhibits robust limb and tail regeneration like other studied Plethodontidae, could have limited or impaired regenerative abilities of internal structures, such as the heart. This an important question for future regeneration-related research in this species.

In addition to this information being vital to understanding and ultimately recapitulating such regenerative events successfully in the human population, the surgeries conducted as part of this study facilitated direct therapeutic rehabilitation and improved quality of life of a wild captured Texas blind salamander. Hence, therapeutic surgeries offer a viable and valuable means to help maintain and conserve this vulnerable animal population.

4. EXPERIMENTAL PROCEDURES

4.1. Animal capture and husbandry

Captive breeding for this species is limited due to low clutch size and viability (see section 2.1). Therefore, many of the Texas blind salamanders in the SMARC colony are obtained by live capture. These individuals are collected from eight locations in San Marcos, Texas, that include wells, fissures, caves, and springs (high and low flow). Minnow traps are suspended in the water at well, fissure and cave sites, and baited with unshelled pistachio nuts and potato peels, which grow biofilm, attracting invertebrates and thus salamanders to the trap. Sub-adult and adult individuals are captured at these locations. When trapping, U.S. Fish and Wildlife Service (USFWS) staff retain only a third of the individuals captured from these sites to reduce the potential of oversampling, as population size is unknown for this species. Before release, individuals are measured, heads photographed for potential visual identification via melanophore patterns, and approximately 3 mm of the tail tip is clipped for future genetic analysis. Driftnets are placed over the outflow of high-pressure springs and low-pressure spring orifices to filter any organisms displaced from the aquifer via the springs. All live Texas blind salamanders caught at these sites are retained, as there is no way to return them back down to the aquifer at these locations and it is assumed any Texas blind salamander entering a stream or lake environment would ultimately succumb to predation by fish species that they are not adapted to evade in their natural habitat. Larval and juvenile salamanders are encountered at these particular catchment sites.

Captured individuals are taken to the SMARC and immediately quarantined for at least 30 days. Juveniles are placed in individual tanks, as this procedure increases survivability for this age class in the first 30-days post-capture. After a month, these juveniles are progressively moved into larger groups as they grow. As part of the quarantine protocol, all salamanders greater than 30 mm from snout to tail tip are non-lethally cotton swabbed. Smaller salamanders are held in quarantine until they are of sufficient size to swab. Swab samples are processed at the SMARC and are used to screen for Batrachochytrium dendrobatidis (Bd), documented in the aquifer since 2004, and Batrachochytrium salamandrivorans (Bsal) prior to specimen incorporation into the general refugia population.61 Infection of the Texas blind salamander with the Bd pathogen appears to be asymptomatic and no cases of Bsal have been identified.

After quarantine, the animals are individually tagged. Visible implant elastomer (VIE) tags are used for animal identification, allowing records of pertinent information, including capture date and location, age if know, and sex, to be maintained for each member of the colony. Salamanders are held in mixed-sex group tanks to encourage courtship opportunities. To avoid aggressive or rare cannibalistic interactions, only similar-sized individuals are held together. Tanks are filled with a variety of rocks, mesh, and artificial vegetation as habitat enrichment, which are layered and stacked to mimic the natural aquifer habitat of the Texas blind salamander, which has pockets of vertical relief. Flow-through or partially-recirculating water systems serve the tanks, where water is pumped in from the Edwards Aquifer. Standard water quality parameters are 0.315 mS/cm conductivity, pH 7.15, dissolved oxygen 7.1 mg/L, dissolved oxygen saturation 77%, and total gas pressure (TGP) 100%. Water is chilled to 21°C before use. This species can survive temperatures ranging from 18 to 23°C; although, exposure to higher temperatures has been limited and is not recommended.

The Texas blind salamander is fed two to three times a week. The food type used depends on the animal’s age but includes Artemia nauplii, Daphnia, frozen Mysis shrimp, frozen copepods, amphipods, ostracods, worms, blind cave shrimp (Palaemonetes antrorum) and troglobitic crustaceans (Stygobromus sp.).

4.2. Animal age estimations

As the majority of the SMARC colony is live captured, the age of most animals is an estimation based on animal size. As these animals are indefinite growers, age estimation charts have been generated by the SMARC from repeated measurements of live captured hatchlings as they age (as depicted in figure 2), and secondary sexual characteristics. Generally speaking, animals are classified as juveniles from hatching to 2 years old (10–35 mm snout to vent length); sub-adults from 2 to 3 years old (35–50 mm snout to vent length; eggs and testes maybe be visible), and sexually mature adults from 3 years of age onwards (50 mm and above snout to vent length, eggs and testes visible).

4.3. Surgeries on the Texas blind salamander

All experimental work on the Texas blind salamander was approved by the SMARC field office, in conjunction with associated veterinarian, and conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Prior to amputation all animals were anesthetized using 0.1% solution of MS222 (Ethyl 3‑aminobenzoate methanesulfonate salt, Sigma), pH 7.0 before surgery.

A wild captured female was found to have a truncated left forelimb stump and intact right forelimb. Observations from other wild captured animals suggested that limb regenerative capacity was present in the Texas blind salamander population. Therefore, SMARC authorized the therapeutic amputation of the left forelimb of this female in the hopes of restoring the missing limb and thus quality of life to the animal. The female, with an estimated age of greater than 2 years, measured 8.1 cm snout to tail tip (4.1 cm snout to vent) on the day of surgery. An amputation was performed on the left forelimb, ensuring that the stump tip was removed but more proximal stump tissue remained. Protruding bone, resulting from amputation, was trimmed.

Tail clips are routinely performed on the Texas blind salamander for genotyping and therapeutic purposes, such as treating fungal infections. In the current study, one third of the tail was amputated from two male Texas Blind Salamanders. The older (estimated to be greater than 5 years old) was 10.7 cm snout to tail tip (5.2 cm snout to vent), while the younger (estimated to be 2 years old) was 8.5 cm snout to tail tip (4.6cm snout to vent) on the day of surgery.

All three salamanders were placed in isolated recovery tanks, after their initial surgeries, for up to 4 months before being returned to general housing with other animals. The recovery aquaria received flow-through well-water and had minimal enrichment during the first stages of regeneration. Rough, complex habitat items were not used in recovery tanks, to prevent damaging to the wound epithelial of regenerate. Enrichment could not be completely removed as this species does require this form of stimulation.

4.4. Tissue preparation and histological staining

Stump and tail clip tissue was fixed in 95% ethanol, incubated in 10% EDTA for a week, and then soaked in 30% sucrose overnight. After being embedded in OCT (Fisher Sci) and flash frozen, samples were sectioned at 10 μm onto Superfrost slides (Fisher Sci). Tissue sections were incubated in Citrisolv (Fisher Scientific) twice and then rehydrated in a series of decreasing ethanol concentrations (100%, 100%, 95%, 70% ethanol, and finally deionized water). Staining was first conducted with 0.04% Alcian blue for 45 minutes, slides subsequently rinsing with deionized water; then with Hematoxylin (Sigma-Aldrich) for 4 minutes, slides rinsed in running tap water for 15 minutes. Samples were finally stained with 0.25% Eosin Y for 3 minutes. After rinsing with deionized water, samples were dehydrated in ethanol (70%, 95%, 100%, 100% ethanol) and then incubated twice in Citrisolv before being mounted. All incubations were conducted for 5 minutes unless otherwise stated.

4.5. Imaging of the amputated limb and tails

Animals were anesthetized (see 4.3 for details) prior to evaluation and imaging of the amputation sites. All amputation sites were evaluated one day post amputation. Thereafter, tail amputations were imaged weekly for 6 weeks, and then monthly. The amputated arm was imaged weekly for 8 weeks and then monthly.

Supplementary Material

Supplemental video 1

Supplementary video 1: Texas blind salamander courtship behavior. Video documenting a female Texas blind salamander initiating courtship, resulting in spermatophore deposition. The female failed to take up the deposited spermatophore, thus this documented courtship event did not result in fertilization of a clutch.

Download video file (1.7GB, avi)
Supplemental Video 2

Supplementary video 2: The regenerated forelimb, although smaller than the intact limb, is functional and used to aid locomotion, including swimming, foraging, and underwater walking.

Download video file (177.9MB, avi)

5. Acknowledgments

The authors declare that no conflict of interest exists. The research in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the Nation Institutes of Health, under the Grant Number: 1R15HD092180-01A1; and funding from United States Fish and Wildlife Service. We would like to thank the Edwards Aquifer Authority for their funding of the Edwards Aquifer Refugia program that provided the opportunity for this research and their cooperation to allow Refugia staff to participate; they did not, however, provide direct funding. We would like to thank Dr. Jessica Whited for her insight related to this project. The findings and conclusions in this article are those of the authors and do not necessarily represent the views of the U.S. Fish and Wildlife Service.

Grant Sponsor: NIH (grant number 1R15HD092180-01A1); funding from United States Fish and Wildlife Service (USFWS).

6. References

  • 1.Bely AE, Nyberg KG. Evolution of animal regeneration : re-emergence of a field. Trends Ecol Evol. 2010;25(3):161–170. doi: 10.1016/j.tree.2009.08.005 [DOI] [PubMed] [Google Scholar]
  • 2.Vogg MC, Galliot B, Tsiairis CD. Model systems for regeneration: Hydra. Development. 2019;146(21):dev177212. doi: 10.1242/dev.177212 [DOI] [PubMed] [Google Scholar]
  • 3.Reddien PW. Review The Cellular and Molecular Basis for Planarian Regeneration. Cell. 2018;175(2):327–345. doi: 10.1016/j.cell.2018.09.021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ferris DR, Satoh A, Mandefro B, Cummings GM, Gardiner DM, Rugg EL. Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin. Dev Growth Differ. 2010;52(8):715–724. doi: 10.1111/j.1440-169X.2010.01208.x [DOI] [PubMed] [Google Scholar]
  • 5.Hay ED, Fischman DA. Origin of the blastema in regenerating limbs of the newt Triturus viridescens: An autoradiographic study using tritiated thymidine to follow cell proliferation and migration. Dev Biol. 1961;3(1):26–59. [DOI] [PubMed] [Google Scholar]
  • 6.Singer M, Inoue S. The nerve and the epidermal apical cap in regeneration of the forelimb of adult Triturus. J Exp Zool. 1964;155:105–116. [DOI] [PubMed] [Google Scholar]
  • 7.Singer M Trophic functions of the neuron. VI. Other trophic systems. Neurotrophic control of limb regeneration in the newt. Ann N Y Acad Sci. 1974;228:308–322. [DOI] [PubMed] [Google Scholar]
  • 8.Singer M THE INFLUENCE OF THE NERVE IN REGENERATION OF THE AMPHIBIAN EXTREMITY. Vol 27.; 1952. [DOI] [PubMed] [Google Scholar]
  • 9.Mullen LM, Bryant S V, Torok MA, Blumberg B, Gardiner DM. Nerve dependency of regeneration: the role of Distal-less and FGF signaling in amphibian limb regeneration. Development. 1996;122:3487–3497. http://www.ncbi.nlm.nih.gov/pubmed/8951064. [DOI] [PubMed] [Google Scholar]
  • 10.Kawakami Y, Esteban CR, Raya M, et al. Wnt/β-catenin signaling regulates vertebrate limb regeneration. Genes Dev. 2006;20(23):3232–3237. doi: 10.1101/gad.1475106 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Gerber T, Murawala P, Knapp D, et al. Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration. Science (80- ). 2018;362(6413):eaaq0681. doi: 10.1126/science.aaq0681 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Endo T, Bryant S V., Gardiner DM. A stepwise model system for limb regeneration. Dev Biol. 2004;270(1):135–145. doi: 10.1016/j.ydbio.2004.02.016 [DOI] [PubMed] [Google Scholar]
  • 13.McCusker C, Lehrberg J, Gardiner D. Position-specific induction of ectopic limbs in non-regenerating blastemas on axolotl forelimbs. Regeneration. 2014;1:27–34. doi: 10.1002/reg2.10 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Nacu E, Gromberg E, Oliveira CR, Drechsel D, Tanaka EM. FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration. Nature. 2016;533:407–410. doi: 10.1038/nature17972 [DOI] [PubMed] [Google Scholar]
  • 15.Kim WS, Stocum DL. Effects of retinoids on regenerating limbs: comparison of retinoic acid and arotinoid at different amputation levels. Roux’s Arch Dev Biol. 1986;195:455–463. doi: 10.1007/BF00375749 [DOI] [PubMed] [Google Scholar]
  • 16.Phan AQ, Lee J, Oei M, et al. Positional information in axolotl and mouse limb extracellular matrix is mediated via heparan sulfate and fibroblast growth factor during limb regeneration in the axolotl ( Ambystoma mexicanum ). Regeneration. 2015;2(4):182–201. doi: 10.1002/reg2.40 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Holder N, Tank PW, Bryant S V. Regeneration of Symmetrical Forelimbs in Axolotl, Ambystoma mexicanum. Dev Biol. 1980;74:302–314. [DOI] [PubMed] [Google Scholar]
  • 18.Bryant S V, French V, Bryant PJ. Distal regeneration and symmetry. Science (80- ). 1981;212(4498):993–1002. doi: 10.1126/science.212.4498.993 [DOI] [PubMed] [Google Scholar]
  • 19.Kollros JJ. Limb regeneration in anuran tadpoles following repeated amputations. J Exp Zool. 1984;232(2):217–229. doi: 10.1002/jez.1402320209 [DOI] [PubMed] [Google Scholar]
  • 20.Scadding SR. Limb regeneration in adult amphibia. Can J Zool. 1981;59(1):34–46. [Google Scholar]
  • 21.Goode BRP. The regeneration of limbs in adult anurans. J Embryol Exp Morphol. 1967;18(2):259–267. [PubMed] [Google Scholar]
  • 22.Dent JN. Limb regeneration in larvae and metamorphosing individuals of the South African clawed toad. J Morphol. 1962;110(1):61–77. [DOI] [PubMed] [Google Scholar]
  • 23.Scadding SR. Phylogenic distribution of limb regeneration capacity in adult Amphibia. J Exp Zool. 1977;202(1):57–67. doi: 10.1002/jez.1402020108 [DOI] [Google Scholar]
  • 24.Tank PW, Carlson BM, Connelly TG. A staging system for forelimb regeneration in the axolotl, Ambystoma mexicanum. J Morphol. 1976;150(1):117–128. doi: 10.1002/jmor.1051500106 [DOI] [PubMed] [Google Scholar]
  • 25.Iten LE, Bryant S V. Forelimb regeneration from different levels of amputation in the newt,Notophthalmus viridescens: Length, rate, and stages. Wilhelm Roux Arch Entwickl Mech Org. 1973;173(4):263–282. doi: 10.1007/BF00575834 [DOI] [PubMed] [Google Scholar]
  • 26.Monaghan JR, Stier AC, Michonneau F, et al. Experimentally induced metamorphosis in axolotls reduces regenerative rate and fidelity. Regeneration. 2014;1(1):2–14. doi: 10.1002/reg2.8 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Young HE, Bailey CF, Dalley BK. Environmental conditions prerequisite for complete limb regeneration in the postmetamorphic adult land-phase salamander, Ambystoma. Anat Rec. 1983;206(3):289–294. doi: 10.1002/ar.1092060307 [DOI] [PubMed] [Google Scholar]
  • 28.Young HE, Bailey CF, Dalley BK. Gross morphological analysis of limb regeneration in postmetamorphic adult Ambystoma. Anat Rec. 1983;206(3):295–306. doi: 10.1002/ar.1092060308 [DOI] [PubMed] [Google Scholar]
  • 29.Young HE. Anomalies of limb regeneration in the adult salamander, Ambystoma annulatum. Arkansas Acad Sci Proc. 1977;31:110–111. [Google Scholar]
  • 30.Scadding SR. Limb regeneration in Amphiuma tridactylum (Amphibia, Urodela). Can J Zool. 1978;56(11):2327–2332. doi: 10.1139/z78-314 [DOI] [Google Scholar]
  • 31.Stockdale WT, Lemieux ME, Killen AC, et al. Heart Regeneration in the Mexican Cavefish. Cell Rep. 2018;25(8):1997–2007.e7. doi: 10.1016/j.celrep.2018.10.072 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Uhlenhuth E Observations on the Distribution and Habits of the Blind Texan Cave Salamander,Typhlomolge rathbuni. Biol Bull. 1921;40(2):73–104. [Google Scholar]
  • 33.Stejneger L Description of a new genus and species of blind tailed batrachians from the subterranean waters of Texas. Proc United States Natl Museum. 1896;18:619–621. [Google Scholar]
  • 34.Hammerson G, Chippindale P. Eurycea rathbuni, Texas Blind Salamander. In: The IUCN Red List of Threatened Species 2004.; 2004:e.T39262A10173274. [Google Scholar]
  • 35.Udall SL. United States list of endangered fauna. US Dep Inter Fish Wildl Serv Act Off Endanger Species Int Not. 1967;42(48):4001. [Google Scholar]
  • 36.Emerson ET. General anatomy of Typhlomolge rathbuni. Proc Bost Soc Nat Hist. 1905;32:43–76. [Google Scholar]
  • 37.Tovar R Ocular histology in three south central Texas paedomorphic salamander species (Eurycea sosorum, Eurycea nana, and Eurycea rathbuni) and Comparative ocular development of two morphotypes. 2014. [Google Scholar]
  • 38.Vitt LJ, Caldwell JP. Chapter 6 - Water Balance and Gas Exchange. In: Vitt LJ, Caldwell JPBT-H (Fourth E, eds. Herpetology. 4th ed. San Diego: Academic Press; 2013:181–202. doi: 10.1016/B978-0-12-386919-7.00006-X [DOI] [Google Scholar]
  • 39.Mitchell RW, Reddell JR. Eurycea tridentifera, a new species of troglobitic salamander from Texas and a reclassification of Typhlomolge rathbuni. Texas J Sci. 1965;17(1):12–27. [Google Scholar]
  • 40.Williams NR. Key to the Herps of Texas. Killeen: Department of Science, Central Texas Collage; 1975. [Google Scholar]
  • 41.Longley G Status of the Texas Blind Salamander. Status of the Texas Blind Salamander. Endanger Species Rep 2. 1978:52. [Google Scholar]
  • 42.Petranka JW. Salamanders of the United States and Canada. Washington: Smithsonian Institution Press; 1998. [Google Scholar]
  • 43.Chippindale PT, Price AH. Conservation of Texas spring and cave salamanders (Eurycea). In: Lannoo M, ed. Amphibian Declines: The Conservation Status of United States Species.; 2005:193–197. [Google Scholar]
  • 44.Sever DM. Sexually Dimorphic Glands of Eurycea nana, eurycea neotenes and Typhlomolge rathbuni (Amphibia: Plethodontidae). Herpetologica. 1985;41(1):71–84. http://www.jstor.org/stable/3892131. [Google Scholar]
  • 45.Brizzi R, Calloni C. Male cloacal region of the spotted salamander, Salamandra salamandra gigliolii (Amphibia, Salamandridae). Boll di Zool. 1992;59(4):377–385. doi: 10.1080/11250009209386697 [DOI] [Google Scholar]
  • 46.Sever DM. Cloacal Anatomy of Male Salamanders in the Families Ambystomatidae, Salamandridae, and Plethodontidae. Herpetologica. 1981;37(3):142–155. http://www.jstor.org/stable/3891884. [Google Scholar]
  • 47.Williams RN, Gopurenko D, Kemp KR, Williams B, DeWoody JA. Breeding Chronology, Sexual Dimorphism, and Genetic Diversity of Congeneric Ambystomatid Salamanders. J Herpetol. 2009;43(3):438–449. doi: 10.1670/08-034R2.1 [DOI] [Google Scholar]
  • 48.Newman WB. A New Plethodontid Salamander from Southwestern Virginia. Herpetologica. 1954;10(1):9–14. http://www.jstor.org/stable/20171296. [Google Scholar]
  • 49.Solis M, Espinal M, Valle R, Óreilly C, Itgen M, Townsend J. On the taxonomy of Oedipina stuarti (Caudata: Plethodontidae), with description of a new species from suburban Tegucigalpa, Honduras. Salamandra. 2016;52:125–133. [Google Scholar]
  • 50.Bechler DL. Courtship Behavior and Spermatophore Deposition by the Subterranean Salamander, Typhlomolge rathbuni (Caudata, Plethodontidae). Southwest Nat. 1988;33(1):124–126. [Google Scholar]
  • 51.Arenas Gómez CM, Gómez Molina A, Zapata JD, Delgado JP. Limb regeneration in a direct-developing terrestrial salamander, Bolitoglossa ramosi (Caudata: Plethodontidae). Regeneration. 2017;4(4):227–235. doi: 10.1002/reg2.93 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Nachtrab G, Czerwinski M, Poss KD. Sexually Dimorphic Fin Regeneration in Zebrafish Controlled by Androgen/GSK3 Signaling. Curr Biol. 2011;21(22):1912–1917. doi: 10.1016/j.cub.2011.09.050 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Mescher AL. Effects on adult newt limb regeneration of partial and complete skin flaps over the amputation surface. J Exp Zool. 1976;195(1):117–127. doi: 10.1002/jez.1401950111 [DOI] [PubMed] [Google Scholar]
  • 54.Goss RJ. Regenerative inhibition following limb amputation and immediate insertion into the body cavity. Anat Rec. 1956;126(1):15–27. doi: 10.1002/ar.1091260103 [DOI] [PubMed] [Google Scholar]
  • 55.Vaglia JL, Babcock SK, Harris RN. Tail development and regeneration throughout the life cycle of the four-toed salamander Hemidactylium scutatum. J Morphol. 1997;233(1):15–29. doi: [DOI] [PubMed] [Google Scholar]
  • 56.Staub NL, Brown CW, Wake DB. Patterns of Growth and Movements in a Population of Ensatina eschscholtzii platensis (Caudata: Plethodontidae) in the Sierra Nevada, California. J Herpetol. 1995;29(4):593–599. doi: 10.2307/1564743 [DOI] [Google Scholar]
  • 57.Jamison JA, Harris RN. The Priority of Linear over Volumetric Caudal Regeneration in the Salamander Plethodon cinereus (Caudata: Plethodontidae). Copeia. 1992;1992(1):235–237. doi: 10.2307/1446558 [DOI] [Google Scholar]
  • 58.Jr Danstedt. RT. Local Geographic Variation in Demographic Parameters and Body Size of Desmognathus fuscus (Amphibia: Plethodontidae). Ecology. 1975;56(5):1054–1067. doi: 10.2307/1936146 [DOI] [Google Scholar]
  • 59.Vaglia JL, Fornari C, Evans PK. Posterior tail development in the salamander Eurycea cirrigera: exploring cellular dynamics across life stages. Dev Genes Evol. 2017;227(2):85–99. doi: 10.1007/s00427-016-0573-0 [DOI] [PubMed] [Google Scholar]
  • 60.Demircan T, İlhan AE, Aytürk N, Yıldırım B, Öztürk G, Keskin İ. A histological atlas of the tissues and organs of neotenic and metamorphosed axolotl. Acta Histochem. 2016;118(7):746–759. doi: 10.1016/j.acthis.2016.07.006 [DOI] [PubMed] [Google Scholar]
  • 61.Gaertner JP, Forstner MRJ, O’Donnell L, Hahn D. Detection of batrachochytrium dendrobatidis in endemic salamander species from central texas. Ecohealth. 2009;6(1):20–26. doi: 10.1007/s10393-009-0229-x [DOI] [PubMed] [Google Scholar]
  • 62.Stocum DL. Stages of Forelimb Regeneration in Ambystoma maculatum. J Exp Zool. 1979;209:395–416. [DOI] [PubMed] [Google Scholar]
  • 63.Maden M Blastemal kinetics and pattern formation during amphibian limb regeneration. J Exp Morphol. 1976;36(3):561–574. [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental video 1

Supplementary video 1: Texas blind salamander courtship behavior. Video documenting a female Texas blind salamander initiating courtship, resulting in spermatophore deposition. The female failed to take up the deposited spermatophore, thus this documented courtship event did not result in fertilization of a clutch.

Download video file (1.7GB, avi)
Supplemental Video 2

Supplementary video 2: The regenerated forelimb, although smaller than the intact limb, is functional and used to aid locomotion, including swimming, foraging, and underwater walking.

Download video file (177.9MB, avi)

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