Neotenic Salamander Research Articles

Postembryonic development and aging of the appendicular skeleton in Ambystoma mexicanum.

The axolotl is a key model to study appendicular regeneration. The limb complexity resembles that of humans in structure and tissue components; however, axolotl limbs develop postembryonically. In this work, we evaluated the postembryonic development of the appendicular skeleton and its changes with aging. The juvenile limb skeleton is formed mostly by Sox9/Col1a2 cartilage cells. Ossification of the appendicular skeleton starts when animals reach a length of 10cm, and cartilage cells are replaced by a primary ossification center, consisting of cortical bone and an adipocyte-filled marrow cavity. Vascularization is associated with the ossification center and the marrow cavity formation. We identified the contribution of Col1a2-descendants to bone and adipocytes. Moreover, ossification progresses with age toward the epiphyses of long bones. Axolotls are neotenic salamanders, and still ossification remains responsive to l-thyroxine, increasing the rate of bone formation. In axolotls, bone maturation is a continuous process that extends throughout their life. Ossification of the appendicular bones is slow and continues until the complete element is ossified. The cellular components of the appendicular skeleton change accordingly during ossification, creating a heterogenous landscape in each element. The continuous maturation of the bone is accompanied by a continuous body growth.

Composition of the cutaneous bacterial community of a cave amphibian, Proteus anguinus.

The European cave salamander Proteus anguinus is a charismatic amphibian endemic to the concealed and inaccessible subterranean waters of the Dinaric Karst. Despite its exceptional conservation importance not much is known about its ecology and interactions with the groundwater microbiome. The cutaneous microbiota of amphibians is an important driver of metabolic capabilities and immunity, and thus a key factor in their wellbeing and survival. We used high-throughput 16S rRNA gene sequencing based on seven variable regions to examine the bacteriome of the skin of five distinct evolutionary lineages of P. anguinus and in their groundwater environment. The skin bacteriomes turned out to be strongly filtered subsamples of the environmental microbial community. The resident microbiota of the analyzed individuals was dominated by five bacterial taxa. Despite an indicated functional redundancy, the cutaneous bacteriome of P. anguinus presumably provides protection against invading microbes by occupying the niche, and thus could serve as an indicator of health status. Besides conservation implications for P. anguinus, our results provide a baseline for future studies on other endangered neotenic salamanders.

Demographics, reproduction, growth, and abundance of Jollyville Plateau salamanders (Eurycea tonkawae).

Insights into the ecology and natural history of the neotenic salamander, Eurycea tonkawae, are provided from eight years of capture‐recapture data from 10,041 captures of 7,315 individuals at 16 sites. Eurycea tonkawae exhibits seasonal reproduction, with peak gravidity occurring in the fall and winter. Size frequency data indicated recruitment occurred in the spring and summer. Open‐population capture‐recapture models revealed a similar seasonal pattern at two of three sites, while recruitment was dependent on flow at the third site. Females can reach sexual maturity within one year, and oviposition likely takes place below ground. The asymptotic body length of 1,290 individuals was estimated as 31.73 mm (at ca. two years of age), although there was substantial heterogeneity among growth trajectories. Longevity was approximately eight years, and the median age for a recaptured adult was 2.3 years. Abundance estimated from closed‐population and robust‐design capture‐recapture models varied widely within and among sites (range 41–834), although, surprisingly, dramatic changes in abundance were not observed following prolonged dry periods. Seasonal migration patterns of second‐year and older adults may help explain lower ratios of large individuals and higher temporary emigration during the latter half of the year, but further study is required. Low numbers of captures and recaptures precluded the use of open‐population models to estimate demographic parameters at several sites; therefore, closed‐population (or robust‐design) methods are generally recommended. Based on observations of their life history and population demographics, E. tonkawae seems well adapted to conditions where spring flow is variable and surface habitat periodically goes dry.

Antipredator response of Eurycea nana to a nocturnal and a diurnal predator: avoidance is not affected by circadian cycles of predators

Both predators and prey exhibit cyclic shifts in activity throughout the day, which should cause the threat posed by predators to change in a recurrent pattern. If the threat posed by a predator is dependent on their circadian foraging cycle, prey may respond more or less intensely to predators at different times of day, thereby maximizing the effectiveness and efficiency of avoidance behaviors. We examined whether predator-naïveEurycea nana, a federally threatened neotenic salamander, exhibits a different antipredator response to chemical cues of a diurnal predator, the green sunfish (Lepomis cyanellus), and a nocturnal predator, the red swamp crayfish (Procambarus clarkii). We predicted thatE. nanawould show more intense antipredator responses (reduced activity) to a diurnal predator during the day and to a nocturnal predator at night. We found that, although there was significant antipredator behavior ofE. nanatoward sunfish, there was no detectable response to crayfish and no effect of time of day on responses to either predator, suggesting that eitherE. nanadoes not innately exhibit circadian patterns in avoidance of these species or that those patterns were undetectable in this study. Future studies should examine whether experience with predators may cause these salamanders to be more sensitive to the diel variation in threat, as has been found with some other amphibians and fish. Due to the threatened nature of this species, understanding the factors that influence antipredator behavior are crucial for management.

Experimental Exposure of Adult San Marcos Salamanders and Larval Leopard Frogs to the Cercariae ofCentrocestus formosanus

The gill parasite Centrocestus formosanus (Trematoda: Heterophyidae) is an exotic parasite of concern in Texas because it has been shown to infect multiple threatened and endangered fish species. The purpose of this study was to determine if C. formosanus could present a threat to larval anurans, as well as threatened neotenic salamanders endemic to the spring-fed systems of Texas. We exposed adults of the San Marcos salamander Eurycea nana (Caudata: Plethodontidae) and tadpoles of the Rio Grande leopard frog Lithobates berlandieri (Anura: Ranidae) to the cercariae of C. formosanus . The San Marcos salamander showed no signs of metacercarial infection, suggesting that E. nana may be refractory to C. formosanus cercariae. Centrocestus formosanus readily infects the gills of leopard frog tadpoles, but the metacercariae apparently died prior to reaching maturity in our tadpoles.

Cranial Muscle Development in the Model Organism Ambystoma mexicanum: Implications for Tetrapod and Vertebrate Comparative and Evolutionary Morphology and Notes on Ontogeny and Phylogeny

There is still confusion about the homology of several cranial muscles in salamanders with those of other vertebrates. This is true, in part, because of the fact that many muscles present in early ontogeny of amphibians disappear during development and specifically during metamorphosis. Resolving this confusion is important for the understanding of the comparative and evolutionary morphology of vertebrates and tetrapods because amphibians are the phylogenetically most plesiomorphic tetrapods, concerning for example their myology, and include two often used model organisms, Xenopus laevis (anuran) and Ambystoma mexicanum (urodele). Here we provide the first detailed report of the cranial muscle development in axolotl from early ontogenetic stages to the adult stage. We describe different and complementary types of general muscle morphogenetic gradients in the head: from anterior to posterior, from lateral to medial, and from origin to insertion. Furthermore, even during the development of neotenic salamanders such as axolotls, various larval muscles become indistinct, contradicting the commonly accepted view that during ontogeny the tendency is mostly toward the differentiation of muscles. We provide an updated comparison between these muscles and the muscles of other vertebrates, a discussion of the homologies and evolution, and show that the order in which the muscles appear during axolotl ontogeny is in general similar to their appearance in phylogeny (e.g. differentiation of adductor mandibulae muscles from one anlage to four muscles), with only a few remarkable exceptions, as for example the dilatator laryngis that appears evolutionary later but in the development before the intermandibularis.

Expression of tropomyosin in relation to myofibrillogenesis in axolotl hearts

The anatomy, function and embryonic development of the heart have been of interest to clinicians and researchers alike for centuries. A beating heart is one of the key criteria in defining life or death in humans. An understanding of the multitude of genetic and functional elements that interplay to form such a complex organ is slowly evolving with new genetic, molecular and experimental techniques. Despite the need for ever more complex molecular techniques some of our biggest leaps in knowledge come from nature itself through observations of mutations that create natural defects in function. Such a natural mutation is found in the Mexican axolotl, Ambystoma mexicanum. It is a facultative neotenous salamander well studied for its ability to regenerate severed limbs and tail. Interestingly it also well suited to studying segmental heart development and differential sarcomere protein expression due to a naturally occurring mendelian recessive mutation in cardiac mutant gene “c”. The resultant mutants are identified by their failure to beat and can be studied for extended periods before they finally die due to lack of circulation. Studies have shown a differential expression of tropomyosin between the conus and the ventricle indicating two different cardiac segments. Tropomyosin protein, but not its transcript have been found to be deficient in mutant ventricles and sarcomere formation can be rescued by the addition of TM protein or cDNA. Although once thought to be due to endoderm induction our findings indicate a translational regulatory mechanism that may ultimately control the level of tropomyosin protein in axolotl hearts.

A New Salamander from the Late Paleocene—Early Eocene of Ukraine

A new neotenic salamander, Seminobatrachus boltyschkensis gen. et sp. nov., is described based on 14 skeletons of late Paleocene—early Eocene age preserved on drill core slabs from the Cherkassy Region, central Ukraine. The new taxon is diagnosed by the following unique combination of characters: dorsal process of premaxilla posteriorly elongate and overlaps frontal; maxilla greatly reduced in size; parietal—squamosal contact absent; vomerine tooth row long and parallel to maxillary arcade; pterygoid has long anterior process; quadrate ossified; marginal and palatal teeth pedicellate; trunk vertebrae amphicoelous, each having a subcentral keel, anterior basapophysis, and spinal nerve foramina; ribs bicipital; carpals and tarsals unossified; and phalangeal formulae of 2-2-3-2 and 2-2-3-4-2 for manus and pes, respectively. Phylogenetic analysis nests S. boltyschkensis within Urodela (i.e., crown-clade salamanders), but its exact phylogenetic position is equivocal, resolving in one of three ways: (1) in an u...

Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity

The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1 mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (∼50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the use of organophosphorus pesticides in the natural habitat of the axolotl.

Ecology of Georgetown Salamanders (Eurycea naufragia) Within the Flow of a Spring

Abstract The Georgetown salamander, Eurycea naufragia, is a permanently neotenic salamander known only from about a dozen surface springs and caves in Williamson County, Texas. Rapid urbanization places all known populations at risk and conservation strategies are hindered by a lack of information on the ecology of the species. To better understand requirements of microhabitat and spatial distribution of E. naufragia within flows of surface springs, we conducted counts of salamanders on the surface at one locality over a 12-month period. Numbers of salamanders and percentage of cover objects occupied by salamanders varied among months, with a general trend of higher abundance in spring and summer. Few juveniles were observed, and there was no strong seasonal trend in distribution of size of salamanders. Within the flow of the spring, abundance of salamanders decreased linearly with distance from origin of the spring. Salamanders were more likely to be under rocks than under other types of cover objects an...

Jeholotriton paradoxus (Amphibia: Caudata) from the Lower Cretaceous of southeastern Inner Mongolia, China

ABSTRACT A fossil salamander is described based on specimens from a Lower Cretaceous deposit near Ningcheng, Inner Mongolia, China. Recently reported as Jeholotriton paradoxus, this form represents a neotenic salamander as indicated by larval features such as external gills, tooth-bearing coronoids, pterygoids with anteromedially oriented anterior processes, and underdeveloped maxillae, in combination with adult features such as extensive medial contact of the nasals and posteriorly directed tooth rows in the palate. This taxon is distinguished from other Mesozoic salamanders by having 15–16 presacrals, proximally expanded unicapitate ribs, vomers with large tooth patches anteriorly and longitudinal dentigerous bars posteriorly, large nasals with no anterior notch, frontals with no anterolateral extension, premaxillae with distinct alary processes, short transverse processes on the vertebrae, and phalangeal formulae of 2-2-3-2 for the manus and 2-2-3-3-2 for the pes. Including Laccotriton, Liaoxitriton, Sinerpeton and Chunerpeton, five taxa of Mesozoic salamander have now been found in China. Jeholotriton has especially well-preserved impressions of articulated skeletons, a type of preservation that is uncommon in Mesozoic strata throughout the world and provides important anatomical details of this early salamander. The great diversity of fossil salamanders from the late Mesozoic of northeastern China implies that East Asia was an important center for the early evolution of urodeles.

Hepatic metallothioneins in two neotenic salamanders, Proteus anguinus and Necturus maculosus (Amphibia, Caudata)

The presence of metallothionein (MT) and the subcellular distribution of copper, zinc and cadmium were investigated in livers of two neotenic salamanders, Proteus anguinus and Necturus maculosus. In P. anguinus, caught in the wild, hepatic MTs were present as a single isoform of (Zn, Cu, Cd)-thioneins, whose molecular weight was estimated to be approximately 12 000 by size exclusion chromatography. The percentage of zinc and cadmium was higher in the cytosol and of copper in the pellet. Cytosolic cadmium was almost exclusively associated with MTs (80%), while zinc and copper were also present in the regions of higher-molecular weight proteins. In laboratory bred N. maculosus, MTs were isolated from the liver cytosol and extract of the pellet as (Cu, Zn)- and (Zn, Cu)-thioneins, respectively. According to the low amount of copper extracting from liver pellets of N. maculosus, the presence of water insoluble aggregated forms of Cu-thioneins should be checked in further investigations.

The ultrastructure of photoreceptor cells in the pineal organ of the blind cave salamander, Proteus anguinus (Amphibia, Urodela)

We studied ultrastructure of the photoreceptor cells in the pineal organ of blind, depigmented, neotenic cave salamander, Proteus anguinus. Unlike in epigean vertebrates the outer segments of most photoreceptor cells consists of concentrically arranged lamellae, however; in few cells, the outer segments contain 7-9 plasma membrane disks. In both types of photoreceptor cells the outer segments enclose lumps of vesicles of different sizes. The photoreceptor cells of Proteus anguinus are similar to those in other cavernicolous fish species.

Effects of Hunger and Predation Risk on Foraging Behavior of Graybelly Salamanders, Eurycea multiplicata

We examined the effects of predation and hunger levels on foraging behavior of adult neotenic graybelly salamanders, Eurycea multiplicata griseogaster. Hungry and satiated salamanders were exposed to chemical stimuli from a predatory fish (sculpin, Cottus carolinae) and from two nonpredatory species, golden redhorse fish (Moxostoma erythrurum) and tadpoles of leopard frogs (Rana sphenocephala). Latency to attack prey was lengthened in the presence of chemical stimuli from predators regardless of hunger levels, but hungry salamanders had shorter latency times than satiated salamanders. There was no interaction between hunger and threat levels. In addition, salamanders distinguished between chemical stimuli from predatory (sculpin) and nonpredatory (redhorse) fishes; responses to redhorse and tadpole stimuli were not different. Handling times were not affected by either predator treatment or hunger level. In summary, graybelly salamanders can (1) recognize sculpin predators based solely on chemical cues, (2) distinguish between chemical stimuli from predatory and nonpredatory fish, and (3) adjust their foraging behavior according to both hunger and predation risk.

The ultrastructure of photoreceptor cells in the pineal organ of the blind cave salamander, Proteus anguinus (Amphibia, Urodela).

We studied ultrastructure of the photoreceptor cells in the pineal organ of blind, depigmented, neotenic cave salamander, Proteus anguinus. Unlike in epigean vertebrates the outer segments of most photoreceptor cells consists of concentrically arranged lamellae, however; in few cells, the outer segments contain 7-9 plasma membrane disks. In both types of photoreceptor cells the outer segments enclose lumps of vesicles of different sizes. The photoreceptor cells of Proteus anguinus are similar to those in other cavernicolous fish species.

Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus.

Carbonic anhydrase (CA) activity in blood and other tissues and red blood cell (rbc) anion exchange were measured in the mud puppy, Necturus maculosus, in order to gain insight into the strategy for CO2 transport used by these neotenic salamanders and to further explore evolutionary relationships between rbc CA activity and anion exchange in nonmammalian vertebrates. CA activity was detectable in all of the tissues examined, but CA activity in blood was much lower than that in most vertebrates. There was no indication, however, that additional CA had been incorporated into the membrane fraction of other tissues to compensate for this low blood CA activity. In further contrast to most other animals, low levels of CA activity were also detectable in mud puppy plasma. Preliminary characterization of the rbc CA indicated that the Type II, fast-turnover enzyme was indeed present, but that there are a very low number of active sites in mud puppy rbc's. Further experiments showed that the rbc's were highly permeable to anions and that the relative rate of anion flux could be inhibited by 4, 4-diisothiocyanostilbene-2,2-disulphonic acid. Thus, the process of CO2 transport in the blood of mud puppies probably involves components of the Jacobs-Stewart cycle, as in most other vertebrates.

Saline lake ecosystems of Mexico

This paper aims to draw attention to the need to better know and understand Mexican saline lakes. It does this by outlining their distribution, abundance and location and their physical, chemical and biological characteristics. The influence of climatic factors on saline lakes is discussed. To sharpen the focus of the general discussion, a few Mexican saline lakes are discussed in more detail in each of the indicated topics. Saline lakes are found throughout Mexico, with the greatest number in the northwest and the least in the southeast, and from sea level to altitudes above 2000m. Mexican saline lakes widely fluctuate morphometrically. The majority are tiny, shallow, and temporal with small volumes. Some are large or deep, and perennial with large volumes. Polymixis is common in shallow lakes while deeper ones are monomictic. Temperature and dissolved oxygen fluctuate widely. Alkaline waters are frequent. Mirroring high primary productivity rates, water is often green and turbid but other colors (blue, sepia, purple, yellow) and clear water are also found. Most lakes are sodium chloride or alkali carbonate types. Salts resulting from the combination of sodium with chloride, carbonates and sulfates are common and, to a lesser extent, salts of calcium, potassium, magnesium, lithium and boron are present. Species richness in Mexican saline lakes decreases as salinity rises. Hyposaline lakes display a more diverse biota than hypersaline ones. Organisms commonly found in Mexican saline lakes are: blue-green algae (e.g., Spirulina geitleri, Phormidium tenue), rotifers (Brachionus spp.), anostracans (Artemia spp.), copepods (e.g., Diaptomus, Leptodiaptomus), corixid bugs (e.g., Corisella, Krizousacorixa, Trichocorixella), ephydrids (e.g., Ephydra hians), the neotenic salamander (e.g., Ambystoma spp.), atherinids (e.g., Chirostoma spp.), goodeids (e.g., Goodea spp.), cyprinodontids (e.g., Cyprinodon spp.), poecilids (e.g., Gambusia spp.), and the Caribbean pink flamingo (Phoenicopterus ruber ruber).

Saline lake ecosystems of Mexico

Abstract This paper aims to draw attention to the need to better know and understand Mexican saline lakes. It does this by outlining their distribution, abundance and location and their physical, chemical and biological characteristics. The influence of climatic factors on saline lakes is discussed. To sharpen the focus of the general discussion, a few Mexican saline lakes are discussed in more detail in each of the indicated topics. Saline lakes are found throughout Mexico, with the greatest number in the northwest and the least in the southeast, and from sea level to altitudes above 2000 m. Mexican saline lakes widely fluctuate morphometrically. The majority are tiny, shallow, and temporal with small volumes. Some are large or deep, and perennial with large volumes. Polymixis is common in shallow lakes while deeper ones are monomictic. Temperature and dissolved oxygen fluctuate widely. Alkaline waters are frequent. Mirroring high primary productivity rates, water is often green and turbid but other colors (blue, sepia, purple, yellow) and clear water are also found. Most lakes are sodium chloride or alkali carbonate types. Salts resulting from the combination of sodium with chloride, carbonates and sulfates are common and, to a lesser extent, salts of calcium, potassium, magnesium, lithium and boron are present. Species richness in Mexican saline lakes decreases as salinity rises. Hyposaline lakes display a more diverse biota than hypersaline ones. Organisms commonly found in Mexican saline lakes are: blue-green algae (e.g., Spirulina geitleri, Phormidium tenue), rotifers (Brachionus spp.), anostracans (Artemia spp.), copepods (e.g., Diaptomus, Leptodiaptomus), corixid bugs (e.g., Corisella, Krizousacorixa, Trichocorixella), ephydrids (e.g., Ephydra hians), the neotenic salamander (e.g., Ambystoma spp.), atherinids (e.g., Chirostoma spp.), goodeids (e.g., Goodea spp.), cyprinodontids (e.g., Cyprinodon spp.), poecilids (e.g., Gambusia spp.), and the Caribbean pink flamingo (Phoenicopterus ruber ruber).

A cyclic nucleotide-dependent chloride conductance in olfactory receptor neurons.

Whole-cell membrane currents were recorded from olfactory receptor neurons from the neotenic salamander Necturus maculosus. Cyclic nucleotides, released intracellularly by flash photolysis of NPE-caged cAMP or NPE-caged cGMP, activated a transient chloride current. The chloride current could be elicited at constant voltage in the absence of extracellular Ca2+ as well as in the presence of 3 mM intracellular Ca2+, suggesting that the current did not require either voltage or Ca2+ transients for activation. The current could be elicited in the presence of the protein kinase inhibitors H-7 and H-89, and in the absence of intracellular ATP, indicating that activation was independent of protein kinase A activity. These results suggest that Necturus olfactory receptor neurons contain a novel chloride ion channel that may be directly gated by cyclic nucleotides.

Differentiation trees, a junk DNA molecular clock, and the evolution of neoteny in salamanders

AbstractObligate neotenic salamanders die if forced to metamorphose. We suggest that this can be explained by assuming: 1) their “excess” DNA is “junk” DNA; 2) the “adult” specifying portion of the DNA becomes junk DNA and is available for repeated duplication. This suggests a “new” junk DNA molecular clock. We obtain remarkable agreement in “predicting” the amount of DNA per nucleus in present day non‐obligate neotene salamanders from this molecular clock. These observatons are consistent with the idea that the development of these animals is describable in terms of differentiation trees whose branches (gene cascades) corresponding to adult somatic tissues accumulate deleterious mutations over evolutionary time. We show that the amount of DNA per nucleus increases linearly with the phylogenetic age of salamander families. The lack of constraints by natural selection, on unused adult branches, may account for the large amount of so‐called “junk DNA” in obligate neotenic salamanders. The effects of this excess DNA, via increased cell size, suggest a positive feedback, ecophysiological explanation for such junk DNA: adaptation to cool water environments is enhanced by the lower metabolism associated with more DNA, larger cells and slower developmental time.