How trematodes cause limb deformities in amphibians

We used trematode cyst infestation to induce limb deformities in two species of frogs of the genus Rana and compared them to deformities induced by surgical limb bud rotations. The specific deformities produced by both treatments closely resemble those of wild-caught deformed amphibians and are consistent with a known developmental response to disruption of the spatial organization of cells in developing limb buds. Histological analysis showed that trematode cysts cause massive disruption and abnormal cellular growth involving the limb buds of infected individuals. Our results indicate that trematode cyst infestation causes deformities in frogs by perturbation of the positional relationships of cells in developing limb buds. The crippling effects of cyst-infection on frogs may reflect complex co-evolutionary interactions among trematodes, frogs, and other hosts in the trematode's life cycle.     

Early limb patterning in the direct‐developing salamander Plethodon cinereus revealed by sox9 and col2a1

Direct‐developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct‐developing frogs, but little attention has been given to direct‐developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct‐developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior‐to‐posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote‐like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct‐developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct‐developing salamander species.     

Environmentally induced limb malformations in mink frogs (Rana septentrionalis).

In recent years, there has been an increase in the incidence of frog deformities throughout many of the northern states of North America. The most readily noticed malformations involve the hindlimbs of peri-metamorphic animals. We have analyzed skeletal preparations of metamorphosing mink frogs (Rana septentrionalis) collected from a site in Minnesota, in order to develop a better understanding of the possible causes. In this paper we describe the categories of abnormalities found at this site. The spectrum of deformities includes missing limbs, truncated limbs, extra limbs (including extra pelvic girdles), and skin webbings. We also describe a newly recognized malformation of the proximal-distal limb axis, a bony triangle. In this abnormality, the proximal and distal ends of the bone are adjacent to one another forming the base of a triangle. The shaft of the bone is bent double and protrudes laterally, the midpoint of the bone forming the apex of the triangle. In this paper we consider several recently proposed explanations for the recent outbreak of amphibian deformities. Based on our analysis, we conclude that the spectrum of abnormalities seen in these frogs is remarkably similar to the range of abnormalities that has been reported as a result of exposure of developing vertebrates to exogenous retinoids. Given the potential implications of this possibility for the welfare of humans as well as wildlife, further studies are needed to determine whether environmental retinoids are responsible for the frog deformities at the site we have examined.     

Osseous pathologies in the lungless salamander Desmognathus fuscus (Plethodontidae)

The majority of reported pathologies in lissamphibians (salamanders, caecilians and frogs) include limb deformities such as missing limbs, multiple extra limbs and digits, or incomplete limb formation. However, comparatively little is known about congenital vertebral malformations or posttraumatic pathologies (e.g. injuries, infections) in the vertebral column of salamanders. In the present study, we describe eight vertebral deformities in three cleared and stained specimens of Desmognathus fuscus. Two specimens display developmental deformities which range from a potential non-segmented wedge vertebra to fully segmented hemivertebrae. The vertebral pathology in the third specimens possibly results from a parasitic infection. Apparently, these osseous deformities were not severe enough to prohibit survival of the specimens.     

Role of Antimicrobial Peptides in Amphibian Defense Against Trematode Infection

Antimicrobial peptides (AMPs) contribute to the immune defenses of many vertebrates, including amphibians. As larvae, amphibians are often exposed to the infectious stages of trematode parasites, many of which must penetrate the host’s skin, potentially interacting with host AMPs. We tested the effects of the natural AMPs repertoires on both the survival of trematode infectious stages as well as their ability to infect larval amphibians. All five trematode species exhibited decreased survival of cercariae in response to higher concentrations of adult bullfrog AMPs, but no effect when exposed to AMPs from larval bullfrogs. Similarly, the use of norepinephrine to remove AMPs from larval bullfrogs, Pacific chorus frogs, and gray treefrogs had only weak (gray treefrogs) or non-significant (other tested species) effects on infection success by Ribeiroia ondatrae. We nonetheless observed strong differences in parasite infection as a function of both host stage (first- versus second-year bullfrogs) and host species (Pacific chorus frogs versus gray treefrogs) that were apparently unrelated to AMPs. Taken together, our results suggest that AMPs do not play a significant role in defending larval amphibians against trematode cercariae, but that they could be one mechanism helping to prevent infection of post-metamorphic amphibians, particularly for highly aquatic species.     

Fibroblast growth factors in regenerating limbs of Ambystoma: cloning and semi-quantitative RT-PCR expression studies

Urodele amphibians (newts and salamanders) have the ability to regenerate amputated limbs throughout their life span. Because fibroblast growth factors (Fgfs) play important roles in developing limbs, we initiated studies to investigate these growth factors in regenerating limbs. Partial cDNAs of Fgf4, 8, and 10 were cloned from both the Mexican axolotl, Ambystoma mexicanum, and locally collected spotted salamander, Ambystoma maculatum, two salamanders well recognized for their regenerative capabilities. cDNAs from the two Ambystoma species were virtually identical, ranging from 97-100% nucleotide identity. Axolotl Fgf4, 8, and 10 showed nucleotide sequence identity with chick Fgf4, 8, and 10 of 79%, 83%, and 72%, respectively. RT-PCR showed that these growth factors are expressed in regenerating axolotl limbs as well as in developing salamander larvae at the three-digit forelimb stage. Fgf8 and 10 are upregulated during regeneration and thus may be involved in distal signaling similar to that of the developing chick limb. Fgf4, however, was undetectable by RT-PCR in the distal tips of regenerates, suggesting that it does not play the same role in limb regeneration that it does in limb development. We also investigated the role these Fgfs may have in the nerve-dependence of regeneration. They were expressed similarly in aneurogenic and innervated limbs, suggesting that they are not the neurotrophic factors responsible for nerve-dependence. Denervation prevented Fgf8 and 10 upregulation, suggesting Fgf pathways are downstream of nerve-dependence. These data highlight important similarities and differences in Fgf expression between limb development and limb regeneration. J. Exp. Zool. 290:529-540, 2001.     

Mitochondrial evidence on the phylogenetic position of caecilians (Amphibia: Gymnophiona)

The complete nucleotide sequence (17,005 bp) of the mitochondrial genome of the caecilian Typhlonectes natans (Gymnophiona, Amphibia) was determined. This molecule is characterized by two distinctive genomic features: there are seven large 109-bp tandem repeats in the control region, and the sequence for the putative origin of replication of the L strand can potentially fold into two alternative secondary structures (one including part of the tRNA(Cys)). The new sequence data were used to assess the phylogenetic position of caecilians and to gain insights into the origin of living amphibians (frogs, salamanders, and caecilians). Phylogenetic analyses of two data sets-one combining protein-coding genes and the other combining tRNA genes-strongly supported a caecilian + frog clade and, hence, monophyly of modern amphibians. These two data sets could not further resolve relationships among the coelacanth, lungfishes, and tetrapods, but strongly supported diapsid affinities of turtles. Phylogenetic relationships among a larger set of species of frogs, salamanders, and caecilians were estimated with a mitochondrial rRNA data set. Maximum parsimony analysis of this latter data set also recovered monophyly of living amphibians and favored a frog + salamander (Batrachia) relationship. However, bootstrap support was only moderate at these nodes. This is likely due to an extensive among-site rate heterogeneity in the rRNA data set and the narrow window of time in which the three main groups of living amphibians were originated.     

Helminth community structure of sympatric eastern American toad, Bufo americanus americanus, northern leopard frog, Rana pipiens, and blue-spotted salamander, Ambystoma laterale, from southeastern Wisconsin

One-hundred twelve amphibians, including 51 blue-spotted salamanders, Ambystoma laterale, 30 eastern American toads, Bufo americanus americanus, and 31 northern leopard frogs, Rana pipiens, were collected during April-October 1996 from Waukesha County, Wisconsin and examined for helminth parasites. The helminth compound community of this amphibian assemblage consisted of at least 10 species: 9 in American toads, 8 in leopard frogs, and 3 in blue-spotted salamanders. American toads shared 7 species with leopard frogs, and 2 species occurred in all 3 host species. Although there was a high degree of helminth species overlap among these sympatric amphibians, statistically significant differences were found among host species and percent of indirect or direct-life cycle parasites of amphibian species individual component communities (chi2 = 1,015, P < 0.001). American toads had a higher relative abundance of nematodes, 59%, than larval cestodes, 31%, and larval and adult trematodes, 10%, whereas leopard frogs had a higher relative abundance of larval cestodes, 71.3%, and larval and adult trematodes, 25.3%, than nematodes 3.4%. This is related to ecological differences in habitat and dietary preferences between these 2 anuran species. Helminth communities of blue-spotted salamanders were depauperate and were dominated by larval trematodes, 94%, and few nematodes, 6%. Low helminth species richness in this host species is related to this salamander's relatively small host body size, smaller gape size, lower vagility, and more fossorial habitat preference than the other 2 anuran species. Adult leopard frogs and toads had significantly higher mean helminth species richness than metamorphs, but there was no significant difference in mean helminth species richness among adult and metamorph blue-spotted salamanders. Considering adult helminths, the low species richness and low vagility of caudatans as compared with anurans suggest that local factors may be more important in structuring caudatan helminth communities of salamanders than of anuran hosts. Helminth species infecting salamanders may be more clumped in their geographic distribution as compared with anurans, and the role of other hosts and their parasites at the compound community level may be important in structuring helminth communities of salamanders.     

A DEVELOPMENTAL ANALYSIS OF AN EVOLUTIONARY TREND: DIGITAL REDUCTION IN AMPHIBIANS

In this study, we integrate information from phylogeny, comparative ontogeny, and experimental embryology in an attempt to elucidate the mechanisms controlling evolutionary trends towards digital reduction and loss observed in amphibians. Frogs and salamanders that have lost phalanges and even whole toes have done so in a very ordered manner, i.e., certain skeletal elements are lost prior to others. This pattern of morphological diversity is described and trends elucidated. It is concluded that the process is characterized by striking intraordinal convergences coupled with substantial differences between the trends observed in frogs as compared to urodeles. We argue that this pattern is essentially a reflection of the differences in the ontogenies of the two orders. Similarly, the convergences within urodeles and within anurans can be explained as the result of regulation of developmental parameters in a resilient developmental program. We further explore this hypothesis by experimentally perturbing the number of cells in the embryonic limb primordium to show that reduction in the number of mesenchymal cells secondarily affects the developmental process of pattern formation causing a rearrangement of the skeletal morphology of the foot. The same experimental manipulation has different effects in frogs as compared to salamanders. However, in both cases, the experimentally generated morphologies tend to parallel the phenotypes and trends observed in nature. Our conclusion is that most of the patterns of diversity in the digital morphology of amphibians can be explained as a reflection of developmental properties. In general, we present a methodology that attempts to empirically address the issue of identifying developmental constraint in morphological evolution.     

Molecular Evidence for the Early History of Living Amphibians

The evolutionary relationships of the three orders of living amphibians (lissamphibians) has been difficult to resolve, partly because of their specialized morphologies. Traditionally, frogs and salamanders are considered to be closest relatives, and all three orders are thought to have arisen in the Paleozoic (>250 myr). Here, we present evidence from the DNA sequences of four mitochondrial genes (2.7 kilobases) that challenges the conventional hypothesis and supports a salamander–caecilian relationship. This, in light of the fossil record and distribution of the families, suggests a more recent (Mesozoic) origin for salamanders and caecilians directly linked to the initial breakup of the supercontinent Pangaea. We propose that this single geologic event isolated salamanders and archaeobatrachian frogs on the northern continents (Laurasia) and the caecilians and neobatrachian frogs on the southern continents (Gondwana). Among the neobatrachian frog families, molecular evidence supports a South American clade and an African clade, inferred here to be the result of mid-Cretaceous vicariance.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

Why is limb regeneration possible in amphibians but not in reptiles,birds, and mammals?

The capacity to regenerate limbs is very high in amphibians and practically absent in other tetrapods despite the similarities in developmental pathways and ultimate morphology of tetrapod limbs. We propose that limb regeneration is only possible when the limb develops as a semiautonomous module and is not involved in interactions with transient structures. This hypothesis is based on the following two assumptions: To an important extent, limb development uses the same developmental mechanisms as normal limb development and developmental mechanisms that require interactions with transient structures cannot be recapitulated later. In amniotes limb development is early, shortly after neurulation, and requires inductive interactions with transient structures such as somites. In amphibians limb development is delayed relative to amniotes and has become decoupled from interactions with somites and other transient structures that are no longer present at this stage. The limb develops as a semi-independent module. A comparison of the autonomy and timing of limb development in different vertebrate taxa supports our hypothesis and its assumptions. The data suggest a good correlation between self-organizing and regenerative capacity. Furthermore, they suggest that whatever barriers amphibians overcame in the evolution of metamorphosis, they are the same barriers that need to be overcome to make limb regeneration possible in other taxa.     

Habitat Selection and Movement of Pond-Breeding Amphibians in Experimentally Fragmented Pine Forests

ABSTRACT Population-level responses of amphibians to forest management regimes are partly dictated by individual behavioral responses to habitat alteration. We examined the short-term (i.e., 24-hr) habitat choices and movement patterns of 3 amphibian species—southern leopard frogs (Rana sphenocephala), marbled salamanders (Ambystoma opacum), and southern toads (Bufo terrestris)—released on edges between forest habitats and recent clear-cuts in the Upper Coastal Plain of South Carolina, USA. We predicted that adult frogs and salamanders would preferentially select forest using environmental cues as indicators of habitat suitability. We also predicted that movement patterns would differ in clear-cuts relative to forests, resulting in lower habitat permeability of clear-cuts for some or all of the species. Using fluorescent powder tracking, we determined that marbled salamanders selected habitat at random, southern toads preferred clear-cuts, and southern leopard frogs initially selected clear-cuts but ultimately preferred forests. Frogs exhibited long-distance, directional movement with few turns. In contrast, toads exhibited wandering behavior and salamanders moved relatively short distances before locating cover. Southern toads and southern leopard frogs moved farther in forests, and all 3 species made more turns in clear-cuts than in forests. Habitat selection by southern toads did not vary according to body size, sex, or the environmental cues we measured. However, marbled salamanders were more likely to enter clear-cuts when soil moisture was high, and southern leopard frogs were more likely to enter clear-cuts when relative humidity and air temperature were higher in the clear-cut than in adjacent forest. Although we found evidence of reduced habitat permeability of clear-cuts for southern leopard frogs and southern toads, none of the species exhibited strong behavioral avoidance of the small (4-ha) clear-cuts in our study. Further studies of long-term habitat use and the potential physiological and other costs to individuals in altered forests are needed to understand the effects of forest management on population persistence. To reduce potentially detrimental effects of clear-cutting on amphibians in the Southeast, wildlife managers should consider the vagility and behavior of species of concern, especially in relation to the size of planned harvests adjacent to breeding sites.     

Limb ossification in the Paleozoic branchiosaurid Apateon (Temnospondyli) and the early evolution of preaxial dominance in tetrapod limb development

Despite the wide range of shapes and sizes that accompany a vast variety of functions, the development of tetrapod limbs follows a conservative pattern of de novo condensation, branching, and segmentation. Development of the zeugopodium and digital arch typically occurs in a posterior to anterior sequence, referred to as postaxial dominance, with a digital sequence of 4-3-5-2-1. The only exception to this pattern in all of living Tetrapoda can be found in salamanders, which display a preaxial dominance in limb development, a de novo condensation of a basale commune (distal carpal/tarsal 1+2) and a precoccial development of digits I and II. These divergent patterns have puzzled researchers for over a century leading to various explanatory hypotheses. Despite many advances in research on tetrapod limb development, the divergent evolution of these two pathways and its causes are still not understood. Based on an extensive ontogenetic series we investigated the pattern of limb development of the 300 Ma old branchiosaurid amphibian Apateon. This revealed a preaxial dominance in limb development that was previously believed to be unique and derived for modern salamanders. The Branchiosauridae are favored as close relatives of extant salamanders in most phylogenetic hypotheses of the highly controversial origins and relationships of extant amphibians. The findings provide new insights into the evolution of developmental pathways in tetrapod limb development, the relationships of modern amphibians with possible Paleozoic antecedents, and their initial timing of divergence.     

Wnt/beta-catenin signaling has an essential role in the initiation of limb regeneration

Anuran (frog) tadpoles and urodeles (newts and salamanders) are the only vertebrates capable of fully regenerating amputated limbs. During the early stages of regeneration these amphibians form a "blastema", a group of mesenchymal progenitor cells that specifically directs the regrowth of the limb. We report that wnt-3a is expressed in the apical epithelium of regenerating Xenopus laevis limb buds, at the appropriate time and place to play a role during blastema formation. To test whether Wnt/beta-catenin signaling is required for limb regeneration, we created transgenic X. laevis tadpoles that express Dickkopf-1 (Dkk1), a specific inhibitor of Wnt/beta-catenin signaling, under the control of a heat-shock promoter. Heat-shock immediately before limb amputation or during early blastema formation blocked limb regeneration but did not affect the development of contralateral, un-amputated limb buds. When the transgenic tadpoles were heat-shocked following the formation of a blastema, however, they retained the ability to regenerate partial hindlimb structures. Furthermore, heat-shock induced Dkk1 blocked fgf-8 but not fgf-10 expression in the blastema. We conclude that Wnt/beta-catenin signaling has an essential role during the early stages of limb regeneration, but is not absolutely required after blastema formation.     

The skull and jaw musculature as guides to the ancestry of salamanders

The fossil record provides no evidence supporting a unique common ancestry for frogs, salamanders and apodans. The ancestors of the modern orders may have diverged from one another as recently as 250 million years ago, or as long ago as 400 million years according to current theories of various authors. In order to evaluate the evolutionary patterns of the modern orders it is necessary to determine whether their last common ancestor was a rhipidistian fish, a very primitive amphibian, a labyrimhodom or a ‘lissamphibian’. The broad cranial similarities of frogs and salamanders, especially the dominance of the braincase as a supporting element, can be associated with the small size of the skull in their immediate ancestors. Hynobiids show the most primitive cranial pattern known among the living salamander families and “provide a model for determining the nature of the ancestors of the entire order. Features expected in ancestral salamanders include: (1) Emargination of the cheek; (2) Movable suspensorium formed by the quadrate, squamosal and pterygoid; (3) Occipital condyle posterior to jaw articulation; (4) Distinct prootic and opisthotic; (5) Absence ol otic notch; (6) Stapes forming a structural link between braincase and cheek. In the otic region, cheek and jaw suspension, the primitive salamander pattern (resembles most closely the microsaurs among known Paleozoic amphibians, and shows no significant features in common with either ancestral frogs or the majority of labyrinth odonts. The basic pattern of the adductor jaw musculature is consistent within both frogs and salamanders, but major differences are evident between the two groups. The dominance of the adductor mandibulae externus in salamanders can be associated with the open cheek in all members of that order, and the small size of this muscle in frogs can be associated with the large otic notch. The spread of different muscles over the otic capsule, the longus head ol the adductor mandibulae posterior in frogs and the superficial head of the adductor mandibulae internus in salamanders, indicates that fenestration of the skull posterodorsal to the orbit occurred separately in the ancestors of the two groups. Reconstruction of the probable pattern of the jaw musculature in Paleozoic amphibians indicates that frogs and salamanders might have evolved from a condition hypothesized for primitive labyrinthodonts, but the presence of a large otic notch in dissorophids suggests specialization toward the anuran, not the urodele condition. The presence of either an einarginated cheek or an embayment of the lateral surface of the dentary and the absence of an otic notch in microsaurs indicate a salamander-like distribution of die adductor jaw muscles. The ancestors of frogs and salamanders probably diverged from one another in the early Carboniferous, Frogs later evolved from small labyrinthodonts and salamanders from microsaurs. Features considered typical of lissamphibians evolved separately in the two groups in the late Permian andTriassic.     

Microsaurs as possible apodan ancestors

The specific ancestry and nature of the relationships of modern amphibians have not yet been established. Detailed comparisons of the anatomy of the skull roof, palate and braincase of living apodans and the Paleozoic microsaur Goniorhynchus demonstrate greater similarities than between apodans and any other group of amphibians, fossil or recent. Unlike any other amphibians, extensive pleurosphenoid ossifications are developed in the area of the Vth nerve, uniting the otic capsule with the sphenethmoid. Other important features that they share (although not uniquely) include the presence of all the primitive dermal elements of the palate, a solidly roofed temporal region, a row of palatal teeth parallel to the marginal dentition and a row of teeth on the medial surface of the lower jaw. The stapes has a similar configuration and position, totally different from that of frogs and salamanders. Such similarities do not necessarily prove close relationship, but indicate the necessity for considering that apodans may have an ancestry distinct from that of frogs and salamanders.     

Universal COI primers for DNA barcoding amphibians

DNA barcoding is a proven tool for the rapid and unambiguous identification of species, which is essential for many activities including the vouchering tissue samples in the genome 10K initiative, genealogical reconstructions, forensics and biodiversity surveys, among many other applications. A large‐scale effort is underway to barcode all amphibian species using the universally sequenced DNA region, a partial fragment of mitochondrial cytochrome oxidase subunit I COI. This fragment is desirable because it appears to be superior to 16S for barcoding, at least for some groups of salamanders. The barcoding of amphibians is essential in part because many species are now endangered. Unfortunately, existing primers for COI often fail to achieve this goal. Herein, we report two new pairs of primers (?, ?) that in combination serve to universally amplify and sequence all three orders of Chinese amphibians as represented by 36 genera. This taxonomic diversity, which includes caecilians, salamanders and frogs, suggests that the new primer pairs will universally amplify COI for the vast majority species of amphibians.