Vascular resistance and vasoactivity of gills and pulmonary artery of the salamander,Ambystoma tigrinum

Summary In order to understand the blood flow patterns and their regulation in the gills and pulmonary artery ofAmbystoma tigrinum, the vascular resistance and vasoactivity of the two major branchial perfusion pathways and a vascular plexus in the pulmonary artery were investigated using an isolated-tissue perfusion method. Acetylcholine and epinephrine were both pressor agents in all three vascular segments. Angiotensin II also constricted the branchial respiratory vasculature. Norephinephrine was primarily a vasodilator in the branchial respiratory vasculature, however, it had no effect on the shunt vessels of the gill or the pulmonary arterial plexus. Both gill circulations were insensitive to alterations in CO₂ and pH. Anoxia produced a slight vasodilation of the branchial respiratory vessels but had no effect on the shunt vasculature. Mild hypoxia had no effect on either branchial circulations. The results suggest that: (1) blood flow through the respiratory section of the gill may vary between 8 and 47% of total gill flow, (2) the major perfusion pathway to the lung is probably from the efferent artery of the third gill through the ductus arteriosus and then into the pulmonary artery, (3) O₂, CO₂ and pH exert no local control of branchial perfusion, (4) both cholinergic and adrenergic regulation of branchial and proximal pulmonary arterial vascular resistance is possible, (5) a rise in circulating norepinephrine should increase blood flow to the respiratory section of the gill.Abbreviations AII angiotensin II - ACh acetylcholine - EPi epinephrine - NE norepinephrine     

Evidence for multiple Pleistocene refugia in the postglacial expansion of the eastern tiger salamander,Ambystoma tigrinum tigrinum

Abstract Pleistocene glaciations were important determinants of historical migration and, hence, current levels of genetic diversity within and among populations. In many cases, these historical migrations led to the existence of disjunct populations of plants and animals. However, the origin and timing of arrival of these disjunct populations is often debated. In the current study, we identify potential refugia and estimate the timing of vicariance events of the eastern tiger salamander, Ambystoma tigrinum tigrinum , using mitochondrial sequence data. The results suggest a vicariant event 0.75–2 million years ago, separating the tiger salamanders to the east and west of the Apalachicola River Basin. East of the Appalachians, there appear to be multiple independent refugia with little migration among the remaining populations. In particular, populations along the Atlantic Coastal Plain were likely isolated in a coastal plain refugium in the Carolinas. Migrants from this refugium were the likely source of colonists for populations occupying previously glaciated areas along the northeastern Atlantic Coast. A second potential refugium occurs in the Blue Ridge Mountains of western Virginia. This refugium contains a disjunct population of the eastern tiger salamander, as well as a community of nearly 70 other disjunct plant and animal species. The tiger salamanders here have been isolated from other populations for 200,000–500,000 years. These results suggest that disjunct mountain populations of Coastal Plain species may have existed in situ throughout the Pleistocene in Appalachian refugia. Therefore, these disjunct populations are not of recent origin, but rather exist as relicts of a warmer, more widespread fauna and flora that is now restricted to the Coastal Plain.     

The composition and function of the pulmonary surfactant system during metamorphosis in the tiger salamander Ambystoma tigrinum

Mammalian lungs secrete a mixture of surface-active lipids (surfactant), which greatly reduces the surface tension of the fluid coating the inner lung surface, thereby reducing the risk of collapse upon deflation and increasing compliance upon inflation. During foetal lung maturation, these lipids become enriched in the primary and active ingredient, a disaturated phopholipid. However, disaturated phospholipids exist in their inactive gellike form at temperatures below 37°C and thus are inappropriate for controlling surface tension in the lungs of many ectotherms. We examined the development of the composition and function of the surfactant system of the tiger salamander (Ambystoma tigrinum) during metamorphosis from the fully aquatic larva (termed stage I) through an intermediate air-breathing larval form (stage IV) to the terrestrial adult (stage VII). Biochemical analysis of lung washings from these three life stages revealed a decrease in the percentage of disaturated phospholipid per total phospholipid (23.03 versus 15.92%) with lung maturity. The relative cholesterol content remained constant. The increased level of phospholipid saturation in the fully aquatic larvae may reflect their generally higher body temperature and the higher external hydrostatic compression forces exerted on the lungs, compared to the terrestrial adults. Opening pressure (pressure required for initial lung opening) prior to lavage decreased from larval to adult salamanders (7.96 versus 4.69 cm H₂O), indicating a decrease in resistance to opening with lung development. Opening pressure increased after lavage in older aquatic (stage IV) larvae (5.36 versus 9.80 cm H₂O) and in the adults (4.69 versus 7.65 cm H₂O), indicating that the surfactant system in salamanders may have an antiglue function which prevents apposing epithelial surfaces from adhering together.Abbreviations bm body mass - Chol cholesterol - DSP disaturated phospholipid - PC phosphatidylcholine - PL phospholipid - postlav postlavage - prelav prelavage - P-V pressure-volume - RH relative humidity - T_b body temperature - USP unsaturated phospholipid - WL wet lung mass     

Antimicrobial peptide defenses in the salamander, Ambystoma tigrinum, against emerging amphibian pathogens

Skin peptides were collected from living Ambystoma tigrinum larvae and adults and tested against two emerging pathogens, Batrachochytrium dendrobatidis and the Ambystoma tigrinum virus (ATV), as well as bacteria isolated from A. tigrinum. Natural mixtures of skin peptides were found to inhibit growth of B. dendrobatidis, Staphylococcus aureus, and Klebsiella sp., but activity against ATV was unpredictable. Skin peptides collected from salamanders held at three environmentally relevant temperatures differed in activity against B. dendrobatidis. Activity of the A. tigrinum skin peptides was found to be strongly influenced by pH.     

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.     

Growth and foraging consequences of facultative paedomorphosis in the tiger salamander,Ambystoma tigrinum nebulosum

Summary Facultative paedomorphosis in salamanders occurs when larvae respond to varying environmental conditions by either metamorphosing into terrestrial metamorphic adults or retaining their larval morphology to become sexually mature paedomorphic adults. Several hypotheses have been proposed for the evolutionary maintenance of this environmentally induced dimorphism, but few data are available to assess them adequately. We studied a montane population of the tiger salamander,Ambystoma tigrinum nebulosum, and measured the adult growth rate and body condition across three growing seasons to assess the relative costs and benefits of each morph. Metamorphic adults grew more than paedomorphic adults in terms of snout—vent length across years and in weight within years. Dietary analyses and foraging experiments revealed some of the proximate factors that may underlie these differential growth patterns. Across all prey, metamorphs had significantly higher biomass and calories per stomach sample than paedomorphs. Metamorphic diets primarily consisted of the fairy shrimpBranchinecta coloradensis, whereas paedomorphic diets contained a variety of benthic and terrestrial invertebrates. Foraging experiments revealed that both morphs are more successful at capturing fairy shrimp relative to other prey types and both show high electivity toward this prey. However, fairy shrimp occurred only in non-permanent ponds and thus are inaccessible to paedomorphs, which can survive only in permanent ponds. Paedomorphs also experience higher levels of intraspecific competition with large larvae in permanent ponds than metamorphs do in non-permanent ponds. Thus, metamorphs obtain a growth advantage over paedomorphs by foraging in non-permanent ponds that contain fairy shrimp and have reduced intraspecific competition. These results suggest that paedomorphs should have decreased fitness relative to metamorphs, primarily because metamorphs can move into the best habitats for growth. The net fitness effect of morph-specific differences in dispersal depend on whether there are trade-offs with other life history traits. Nonetheless, because the relative benefit of metamorph dispersal will change with environmental conditions in permanent ponds and the surrounding habitat, the relative fitness payoff to each morph should track changes in the environment. Thus, facultative paedomorphosis may be maintained in part by variable, environmentally-specific fitness payoffs to each morph.     

Effects of metamorphosis on water permeability of skin in the salamander, Ambystoma tigrinum

Developmentally associated changes in the pressure driven water permeability of the skin of the salamander Ambystoma tigrinum were measured at 20 degrees C in neotenic (gilled), transitional, and fully transformed adults. Mean values for the hydraulic conductivity of the skin (Lp, X 10(-5) cm.sec-1.ATM-1) were, respectively, 1.54, 0.54 and 0.13. This nearly 12-fold decrease in the H2O permeability coincides with the transition from aquatic to terrestrial life and may be related to the changing role of the skin in water conservation. The increase in hydraulic conductivity is opposite to the decrease in H2O diffusion rates reported by others. We suggest a theoretical basis for the apparently conflicting results.     

Peripheral projections of nervus terminalis LHRH-containing neurons in the tiger salamander,Ambystoma tigrinum

The peripheral projections of the nervus terminalis (NT) have been difficult to examine due to the weak immunoreactivity of the processes to various antibodies. We performed two experimental manipulations in the tiger salamander in an attempt to increase the luteinizing hormone-releasing hormone-immunoreactive (LHRH-ir) labelling in the peripheral processes of the NT: 1) the NT was sectioned centrally, or 2) a 100 mg melatonin pellet was embedded subcutaneously for 3 days prior to sacriffice. Following these manipulations, animals were sacrifficed and tissue was processed with standard immunocytochemical techniques for the analysis of the distribution of LHRH-ir processes. In the nasal cavity, LHRH-ir fibers were observed projecting 1) into the rostral olfactory epithelium, 2) to Bowman's glands in the lamina propria of the rostromedial olfactory mucosa and ventrolateral mucosa between the main nasal cavity and Jacobson's organ, 3) into the naris constrictor muscle, and 4) along the palatine nerves and ganglia. These lesion and hormone manipulations have enabled the detection of peripheral projections of the NT not observed previously with immunocytochemical procedures alone. The wide distribution of LHRH-ir NT processes in the nasal cavity and cranium suggests that this nerve may influence many different cranial structures during appropriate pheromonal or neuroendocrine events.     

Basis of selective predation by the aquatic larvae of the salamander, Ambystoma tigrinum

SUMMARY. 1. The effect of several factors on feeding selectivity by three size classes of larvae of Ambystoma tigrinum (Green) was examined. Five types of invertebrate prey were used: Chaoborus, Daphnia , damselflies, Hyalella and water boatmen.
2. Five variables were examined in relation to the electivity indices associated with each prey type: ease of prey consumption, ease of prey capture, relative prey size, prey activity, and microhabitat overlap. No single variable explained the observed electivity.
3. For small larvae (snout-vent length, SVL, <2.5cm) relative prey size was correlated with electivity. For large larvae (SVL >6.5cm), ease of prey consumption, prey activity, and relative prey size predicted electivity values. However, none of the measured variables was related to electivity values found for medium sized larvae (SVL >3.5 and <5.5cm).     

Active transport of Calcium ions in the skin of the salamander Ambystoma tigrinum

We measured Ca²⁺ exchanges across the skin of larval and adult Ambystoma tigrinum using the radio-isotope influx method. We found that the skin of both morphs takes up Ca²⁺ in a manner that is proportional to external [Ca²⁺], saturable and oriented against the electrochemical gradient for Ca²⁺. We conclude that this uptake occurs by active transport. Kinetic analysis yields affinities for calcium ions that are similar to the affinities for both Ca²⁺ and Na⁺ in the skin of other amphibians. The capacity for calcium is similar to Ca²⁺ capacity in other amphibians. The capacity for Ca²⁺ is lower than the capacity for Na⁺. Cutaneous Ca²⁺ deposits are lower in this urodele than found in anurans. Adults tend to have higher levels of Ca in their skin than do larvae. Accepted: 25 June 1999     

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.     

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.     

Adenohypophysial-thyroid activity of the tiger salamander, Ambystoma tigrinum, as a function of metamorphosis and captivity

The aim of this study was to determine the timing of adenohypophysial activation during metamorphosis of the tiger salamander, Ambystoma tigrinum. It consisted of two parts: 1) determination of plasma thyroid hormone concentrations and analysis of thyroid gland histology as a function of metamorphic stage and 2) analysis of the time-course of uptake of 125I by the thyroids during metamorphosis as an indicator of endogenous thyrotropin (TSH) levels. Significant increases in both triiodothyronine (T3) and thyroxine (T4) first were evident at the onset of metamorphic climax (stage II). Maximum levels of both hormones were not observed, however, until the completion of gill resorption (stage VII). No changes in thyroid histology were observed that could be unambiguously related to metamorphic transformation. The thyroids accumulated 125I in a slow but linear fashion in premetamorphic larvae (stage I). However, uptake exhibited a rapid peak during early climax (stage II), before maximum concentrations of thyroid hormones were observed. In addition, uptake was maintained above premetamorphic levels at stage VII, in conjunction with maximum levels of T4 and T3. Captivity alone produced a small but significant increase in plasma concentrations of T3. It produced no significant effect on either thyroid histology or uptake of 125I. These results indicate that adenohypophysial activation occurs rapidly and is maximal at the onset of metamorphic climax.     

The effects of amiloride on electrolyte transport and acid-base balance in the larval salamander,Ambystoma tigrinum

Summary The responses of net and unidirectional fluxes of Na⁺ and acid-base balance to the drug amiloride were assessed during normocapnia and hypercapnia in larval salamanders, Ambystoma tigrinum. Isotope flux measurements demonstrated that 10^-4 M amiloride in the external medium inhibits Na⁺ influx during normocapnia and reverses the usual increase in influx of this ion during hypercapnia, causing a significant decrease instead. Measurements of blood-gas/acid-base balance conditions of artcrially cannulated salamanders demonstrated a significant metabolic acidosis in amiloridetreated animals that did not occur in untreated animals over the same period. the same concentration of amiloride also blocked the normal compensatory increase in [HCO _- ³ ] that follows a respiratory acidosis produced by a hypercapnic environment.Abbreviations IU international nnits - J _in influx - J _net net flux - PCO ₂ parial pressure of carbon dioxide     

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.     

Population structure and genetic diversity of metamorphic and paedomorphic populations of the tiger salamander,Ambystoma tigrinum

Genetic relationships, population subdivision and genetic diversity were estimated from mtDNA and allozyme data for two subspecies of tiger salamander, one of which is obligately metamorphic and the other polymorphic for paedomorphosis (larval reproduction). Far greater genetic differentiation exists between subspecies than within subspecies, suggesting that the subspecies have evolved in allopatry. Values of F_st calculated from both mtDNA and allozymes were greater than 0.400 for each subspecies. Significant population subdivision was detected even on a microgeographic scale. This extensive population subdivision indicates that populations can respond to extremely localized selection pressures. In the case of paedomorphosis, populations in permanent water should evolve paedomorphosis as long as the appropriate genes exist. For both mtDNA and allozymes, comparisons of population structure within the polymorphic subspecies and between polymorphic and metamorphic subspecies reveal no discernible effects of paedomorphosis. However, a comparison of paedomorphic and metamorphic populations of the polymorphic subspecies showed significantly higher mtDNA diversity in paedomorphic populations. The discrepancy between the allozyme and mtDNA results may be due to the lower effective population size of mtDNA compared to autosomal genes.     

Experimental analysis of character coupling across a complex life cycle: Pigment pattern metamorphosis in the tiger salamander,Ambystoma tigrinum tigrinum

Developmental relationships among characters are expected to bias patterns of morphological variation at the population level. Studies of character development thus can provide insights into processes of adaptation and the evolutionary diversification of morphologies. Here I use experimental manipulations to test whether larval and adult pigment patterns are coupled across metamorphosis in the tiger salamander, Ambystoma tigrinum tigrinum (Ambystomatidae). Previous investigations showed that the early larval pigment pattern depends on interactions between pigment cells and the lateral line sensory system. In contrast, the results of this study demonstrate that the major features of the adult pigment pattern develop largely independently of both the early larval pattern and the lateral lines. These results suggest that ontogenetic changes that occur across metamorphosis decouple larval and adult pigment patterns and could thereby facilitate independent evolutionary modifications to the patterns during different stages of the life cycle. J. Morphol. 237:53–67, 1998. © 1998 Wiley-Liss, Inc.