Metamorphosis of the feeding mechanism in tiger salamanders (Ambystoma tigrinum): the ontogeny of cranial muscle mass

Most previous research on metamorphosis of the musculoskeletal system in vertebrates has focused on the transformation of the skeleton. In this paper we focus on the transformation of the muscles of the head during metamorphosis in tiger salamanders ( Ambystoma tigrinum ) in order (1) to provide new data on changes in myology during ontogeny, and (2) to aid in interpreting previous data on the metamorphosis of function in the head of salamanders.
The physiological cross-sectional area of nine head muscles was calculated by measuring fibre angles, fibre lengths, and muscle mass in two samples of tiger salamanders obtained just before and just after metamorphosis. The major mouth-opening muscles (rectus cervicis and depressor mandibulae) exhibit a significant decrease in estimated maximum tetanic tension (MTT) across metamorphosis of about 36%. The jaw-closing muscles (adductor mandibulae internus and externus) and the head-lifting muscles (epaxials) also decrease in MTT but not significantly. The muscles associated with tongue projection during feeding on land (the subarcualis rectus I, geniohyoideus, interhyoideus and intermandibularis) all show a slight increase in MTT at metamorphosis.
Metamorphic transformation of feeding behaviour in Ambystoma tigrinum involves changes in performance, the design of skeletal elements, changes in muscle force-generating capability, and changes in hydrodynamic design from unidirectional flow in larvae to bidirectional flow during aquatic feeding after metamorphosis. Although muscle activity patterns during aquatic feeding do not change across metamorphosis, tongue-based terrestrial feeding involves a suite of novel muscle activity patterns, morphological characters acquired at metamorphosis, and a metamorphic increase in the masses of muscles important in tongue projection.     

Instrumentally conditioned avoidance by tiger salamanders (Ambystoma tigrinum) to reagent grade odorants

Tiger salamanders (Ambystoma tigrinum) were given pairings ofair dilutions of either n-amyl acetate or d-camphor and intenselight in a stainless steel and glass chamber using a simpleflow dilution olfactometer. Avoidance responses were conditionedto presentations of amyl acetate but avoidance responses didnot condition to presentations of camphor     

Evaluation of two methods for measuring nonspecific immunity in tiger salamanders (Ambystoma tigrinum)

Study of amphibian immunotoxicology is a growing area of research, but very little information is available on how environmental contaminants affect disease resistance in urodele amphibians. Urodele amphibians lack the more highly evolved aspects of the specific immune system that are present in anurans, birds, and mammals. Instead, these animals rely more heavily on innate defense mechanisms than do anurans to provide rapid, nonspecific protection from pathogens. Thus, it is prudent that immunotoxicologic research with urodele amphibians includes an evaluation of effects of contaminant exposure on nonspecific immunity. The objectives of this study were to measure the phagocytic and oxidative-burst activity of peritoneal neutrophils collected from a urodele, the tiger salamander (Ambystoma tigrinum), and to evaluate the use of these assays in immunotoxicologic research using urodele amphibians. Using tiger salamanders collected in August 2000, phagocytosis and oxidative-burst assays modified from mammalian protocols were conducted through October 2001. Results indicated that large numbers of peritoneal neutrophils for use in immunotoxicologic tests can be collected from salamanders injected with thioglycollate. Moreover, these neutrophils readily engulfed foreign material (phagocytic activity) and produced measurable amounts of hydrogen peroxide (oxidative-burst activity). Phagocytosis was effectively inhibited by incubating cells with sodium azide (P<0.001), and quantification of phagocytosis using flow cytometry was well correlated with manual counts (r=0.84, P<0.001). Dexamethasone treatment reduced phagocytic activity as measured by manual counts (P<0.02), suggesting that this test is useful for detecting alteration by immunosuppressive agents. In contrast, oxidative function was unaffected by dexamethasone treatment, and results from the oxidative-burst assay were generally less consistent than those from the phagocytosis assay. Based on these results, phagocytic activity of peritoneal neutrophils may be a useful endpoint in immunotoxicologic studies to evaluate the impact of environmental contaminants on innate defense mechanisms in urodele amphibians.     

Molecular characterization of major histocompatibility complex class II alleles in wild tiger salamanders (Ambystoma tigrinum)

Major histocompatibility complex (MHC) class II genes are usually among the most polymorphic in vertebrate genomes because of their critical role (antigen presentation) in immune response. Prior to this study, the MHC was poorly characterized in tiger salamanders (Ambystoma tigrinum), but the congeneric axolotl (Ambystoma mexicanum) is thought to have an unusual MHC. Most notably, axolotl class II genes lack allelic variation and possess a splice variant without a full peptide binding region (PBR). The axolotl is considered immunodeficient, but it is unclear how or to what extent MHC genetics and immunodeficiency are interrelated. To study the evolution of MHC genes in urodele amphibians, we describe for the first time an expressed polymorphic class II gene in wild tiger salamanders. We sequenced the PBR of a class II gene from wild A. tigrinum (n=33) and identified nine distinct alleles. Observed heterozygosity was 73%, and there were a total of 46 polymorphic sites, most of which correspond to amino acid positions that bind peptides. Patterns of nucleotide substitutions exhibit the signature of diversifying selection, but no recombination was detected. Not surprisingly, transspecies evolution of tiger salamander and axolotl class II alleles was apparent. We have no direct data on the immunodeficiency of tiger salamanders, but the levels of polymorphism in our study population should suffice to bind a variety of foreign peptides (unlike axolotls). Our tiger salamander data suggest that the monomorphism and immunodeficiencies associated with axolotl class II genes is a relict of their unique historical demography, not their phylogenetic legacy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Pigment pattern formation in larval ambystomatid salamanders: Ambystoma tigrinum tigrinum

We have begun a comparative study of pigment patterns and their mechanisms of formation in ambystomatid salamanders in an attempt to elucidate the evolution of these traits in this family. In Ambystoma t. tigrinum, the migration of the prospective pigment cells was followed by using scanning electron microscopy and light microscopy combined with markers (dopa incubation for detecting melanophores, ammonia-induced pterin fluorescence for detecting xanthophores). The pigment pattern resulting from the cell migration shares features both with the alternating vertical xanthophore and melanophore bars of A. mexicanum and the horizontal stripes of certain salamandrids and ambystomatids. The pigment pattern of A. t. tigrinum is interpreted here as an intermediate evolutionary step between a primitive horizontal stripe pattern and a derived vertical bar pattern. The initiation of pigment pattern formation resembles the situation in A. mexicanum, probably reflecting the close phylogenetic relationship between the two taxa.     

Effect of microhabitat on fitness components of larval tiger salamanders,Ambystoma tigrinum nebulosum

Summary Survivorship and growth of larval tiger salamanders, Ambystoma tigrinum nebulosum, in the White Mountains of east-central Arizona were compared in six microhabitats using field enclosures during summers, 1983–85. Microhabitats were vegetated and nonvegetated shallows, surface, middle, and bottom horizontal layers of limnetic areas, and the vertical limnetic water column. Initial enclosure densities (0.025 larvae per 1) were identical among microhabitats. Three enclosures were placed in each microhabitat in two ponds. Larval survivorship and growth were usually greatest in vegetated shallows in lowest in middle and bottom limnetic enclosures, despite several population dieoffs. Lower fitness, as reflected in survivorship and growth, in these latter enclosures was correlated with lower food levels, temperatures, and oxygen concentrations in deeper limnetic areas. Relative fitness varied greatly between years while food levels, temperatures, and oxygen concentrations within microhabitats remained relatively constant indicating additional factors affected fitness. Disparities in fitness between microhabitats apparently affect habitat use patterns of larvae.     

Molecular evidence for historical and recent population size reductions of tiger salamanders (Ambystoma tigrinum) in Yellowstone National Park

Population declines caused by natural and anthropogenic factors can quickly erode genetic diversity in natural populations. In this study, we examined genetic variation within 10 tiger salamander populations across northern Yellowstone National Park in Wyoming and Montana, USA using eight microsatellite loci. We tested for the genetic signature of population decline using heterozygosity excess, shifts in allele frequencies, and low ratios of allelic number to allelic size range (M-ratios). We found different results among the three tests. All 10 populations had low M-ratios, five had shifts in allele frequencies and only two had significant heterozygosity excesses. These results support theoretical expectations of different temporal signatures among bottleneck tests and suggest that both historical fish stocking, recent, sustained drought, and possibly an emerging amphibian disease have contributed to declines in effective population size.     

The ontogeny of functional design: metamorphosis of feeding behaviour in the tiger salamander (Ambystoma tigrinum)

The ontogeny of feeding behaviour was studied quantitatively in the tiger salamander, Ambystoma tigrinum , to elucidate the relative importance of morphological and environmental changes on form and function. High-speed films of prey capture provided data for a frame-by-frame analysis of seven kinematic parameters of feeding behaviour. By comparing underwater feeding of larval and metamorphosed individuals, the effect of morphological changes occurring at metamorphosis on the feeding kinematic pattern was determined. By comparing metamorphosed animals feeding in the water and on land, changes in feeding kinematics associated with the environmental transition (and thus the differing physical properties of water and air) were determined. Both univariate and multivariate analyses failed to demonstrate any differences between larval and metamorphosed aquatic feedings for seven kinematic variables. However, when individuals feed on land, a radical shift in hyoid kinematics was observed. In addition, multivariate analysis showed that terrestrial feedings differed from aquatic feedings in having longer duration head movements. The lack of a kinematic difference between larval and metamorphosed individuals feeding in the water indicates that the morphological changes occurring at metamorphosis do not impose any obligatory kinematic consequences. Rather, metamorphosed Ambystoma tigrinum acquire the ability to modulate their kinematic pattern depending on the environment.     

Landscape genetics of the blotched tiger salamander (Ambystoma tigrinum melanostictum)

The field of landscape genetics has great potential to identify habitat features that influence population genetic structure. To identify landscape correlates of genetic differentiation in a quantitative fashion, we developed a novel approach using geographical information systems analysis. We present data on blotched tiger salamanders (Ambystoma tigrinum melanostictum) from 10 sites across the northern range of Yellowstone National Park in Montana and Wyoming, USA. We used eight microsatellite loci to analyse population genetic structure. We tested whether landscape variables, including topographical distance, elevation, wetland likelihood, cover type and number of river and stream crossings, were correlated with genetic subdivision (F(ST)). We then compared five hypothetical dispersal routes with a straight-line distance model using two approaches: (i) partial Mantel tests using Akaike's information criterion scores to evaluate model robustness and (ii) the BIOENV procedure, which uses a Spearman rank correlation to determine the combination of environmental variables that best fits the genetic data. Overall, gene flow appears highly restricted among sites, with a global F(ST) of 0.24. While there is a significant isolation-by-distance pattern, incorporating landscape variables substantially improved the fit of the model (from an r2 of 0.3 to 0.8) explaining genetic differentiation. It appears that gene flow follows a straight-line topographic route, with river crossings and open shrub habitat correlated with lower F(ST) and thus, decreased differentiation, while distance and elevation difference appear to increase differentiation. This study demonstrates a general approach that can be used to determine the influence of landscape variables on population genetic structure.     

Morphology and escape performance of tiger salamander larvae (Ambystoma tigrinum mavortium)

The ability of an individual to escape predators is an important component of fitness. Several adaptive explanations of body shape variation in amphibians hypothesize relationships between swimming performance and morphology, but these ideas have rarely been tested. Here we investigate bivariate and multivariate relationships between natural variation in morphology and performance. We used high-speed video to examine fast-starts associated with escape responses in small tiger salamander larvae (Ambystoma tigrinum). Our results indicate that performance is influenced by interactions among aspects of morphology, physiology, and behavior. Relationships between morphometric variables and velocity could be detected with multivariate, but not bivariate statistical analyses. In particular, relationships between morphology and velocity depend on tail beat frequency (potentially a measure of effort or vigor). Relationships between morphology and acceleration were detected with bivariate analyses, but multivariate analysis suggests that acceleration performance, too, depends on interactions between morphology and tail beat frequency. We found a positive relationship between tail area and propulsive performance, which supports adaptive interpretations of variation in larval tail shape within and between amphibian species.     

Allopurinol-induced melanism in the tiger salamander (Ambystoma tigrinum nebulosum).

The enzyme, xanthine dehydrogenase (XDH), has been examined in Ambystoma tigrinum nebulosum with respect to its role in pigmentation. It now seems probable that the melanoid gene (m) either codes directly for XDH or is somehow intimately connected with the normal function of this enzyme. Inhibition of XDH using the drug, allopurinol, results in animals which appear to be phenocopies of melanoid mutants as described for the Mexican axolotl (Ambystoma mexicanum). The effects of allopurinol in terms of specific pigmentary alterations were examined, and a new method for analyzing heterogeneous extracts of skin pigments (e.g., purines and pteridines) is presented. The significance of the link between XDH and melanism is discussed with emphasis on possible mechanisms of pigment induction and general applicability to biological systems.     

Biochemical plasticity in the Arizona tiger salamander (Ambystoma tigrinum nebulosum).

The Arizona tiger salamander, Ambystoma tigrinum nebulosum, is a developmentally polymorphic species. Some individuals become sexually mature while retaining some larval traits (paedomorphs), while other individuals mature as metamorphosed salamanders. In this study, relative enzyme activities of the products of two duplicate loci in each of three enzyme systems (aconitase, malate dehydrogenase, and isocitrate dehydrogenase) were measured in paedomorphs and in paedomorphs forced to metamorphose by treatment with thyroxine. We found that thyroxine and laboratory conditions affect enzyme activities of four of the six enzymes examined and that activities of products of duplicate loci are altered to different degrees.     

Chondrocranial development in larval Rana sylvatica (Anura: Ranidae): morphometric analysis of cranial allometry and ontogenetic shape change

This study provides baseline quantitative data on the morphological development of the chondrocranium in a larval anuran. Both linear and geometric morphometric methods are used to quantitatively analyze size-related shape change in a complete developmental series of larvae of the wood frog, Rana sylvatica. The null hypothesis of isometry was rejected in all geometric morphometric and most linear morphometric analyses. Reduced major axis regressions of 11 linear chondrocranial measurements on size indicate a mixture of allometric and isometric scaling. Measurements in the otic and oral regions tend to scale with negative allometry and those associated with the palatoquadrate and muscular process scale with isometry or positive allometry. Geometric morphometric analyses, based on a set of 11 chondrocranial landmarks, include linear regression of relative warp scores and multivariate regression of partial warp scores and uniform components on log centroid size. Body size explains about one-quarter to one-third of the total shape variation found in the sample. Areas of regional shape transformation (e.g., palatoquadrate, otic region, trabecular horns) are identified by thin-plate spline deformation grids and are concordant with linear morphometric results. Thus, the anuran chondrocranium is not a static structure during premetamorphic stages and allometric patterns generally follow scaling predictions for tetrapod cranial development. Potential implications regarding larval functional morphology, cranial development, and chondrocranial evolution in anurans are discussed.     

Influence of temperature on Ranavirus infection in larval salamanders Ambystoma tigrinum

Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26 degrees C, whereas all died at 18 degrees C and nearly all died at 10 degrees C. Some asymptomatic salamanders that survived 60 d at 10 or 26 degrees C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10 degrees C than at 18 degrees C but little virus, if any, was present in the small number of salamanders that died at 26 degrees C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely.     

Insights into the mating habits of the tiger salamander (Ambystoma tigrinum tigrinum) as revealed by genetic parentage analyses

Among urodeles, ambystomatid salamanders are particularly amenable to genetic parentage analyses because they are explosive aggregate breeders that typically have large progeny arrays. Such analyses can lead to direct inferences about otherwise cryptic aspects of salamander natural history, including the rate of multiple mating, individual reproductive success, and the spatial distribution of clutches. In 2002, we collected eastern tiger salamander (Ambystoma tigrinum tigrinum) egg masses (> 1000 embryos) from a approximately 80 m linear transect in Indiana, USA. Embryos were genotyped at four variable microsatellite loci and the resulting progeny array data were used to reconstruct multilocus genotypes of the parental dams and sires for each egg mass. UPGMA analysis of genetic distances among embryos resolved four instances of egg mass admixture, where two or more females had oviposited at exactly the same site resulting in the mixing of independent cohorts. In total, 41 discrete egg masses were available for parentage analyses. Twenty-three egg masses (56%) consisted exclusively of full-siblings (i.e. were singly sired) and 18 (44%) were multiply sired (mean 2.6 males/clutch). Parentage could be genetically assigned to one of 17 distinct parent pairs involving at least 15 females and 14 different males. Reproductive skew was evident among males who sired multiply sired clutches. Additional evidence of the effects of sexual selection on male reproductive success was apparent via significant positive correlations between male mating and reproductive success. Females frequently partitioned their clutches into multiple discrete egg masses that were separated from one another by as many as 43 m. Collectively, these data provide the first direct evidence for polygynandry in a wild population of tiger salamanders.