Larval Tiger Salamanders Research Articles

Age- and dose-dependent susceptibility of axolotls (Ambystoma mexicanum) by bath exposure to Ambystoma tigrinum virus (ATV)

Ranaviruses are large, dsDNA viruses that have significant ecological and economic impact on cold-blooded vertebrates. However, our understanding of the viral proteins and subsequent host immune response(s) that impact susceptibility to infection and disease is not clear. The ranavirus Ambystoma tigrinum virus (ATV), originally isolated from the Sonoran tiger salamander (Ambystoma mavortium stebbinsi), is highly pathogenic at low doses of ATV at all tiger salamander life stages and this model has been used to explore the host-pathogen interactions of ATV infection. However, inconsistencies in the availability of laboratory reared larval tiger salamanders required us to look at the well characterized axolotl (A. mexicanum) as a model for ATV infection. Data obtained from five infection experiments over different developmental timepoints suggest that axolotls are susceptible to ATV in an age- and dose-dependent manner. These data support the use of the ATV-axolotl model to further explore the host-pathogen interactions of ranavirus infections.

Testing whether adrenal steroids mediate phenotypic and physiologic effects of elevated salinity on larval tiger salamanders.

Salinity (sodium chloride, NaCl) from anthropogenic sources is a persistent contaminant that negatively affects freshwater taxa. Amphibians can be susceptible to salinity, but some species are innately or adaptively tolerant. Physiological mechanisms mediating tolerance to salinity are still unclear, but changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO) are prime candidates. We exposed larval barred tiger salamanders (Ambystoma mavortium) to environmentally relevant NaCl treatments (<32-4000 mg·L-1 ) for 24 days to test effects on growth, survival, and waterborne CORT responses. Of those sampled, we also quantified waterborne ALDO from a subset. Using a glucocorticoid antagonist (RU486), we also experimentally suppressed CORT signaling of some larvae to determine if CORT mediates effects of salinity. There were no strong differences in survival among salinity treatments, but salinity reduced dry mass, snout-vent length, and body condition while increasing water content of larvae. High survival and sublethal effects demonstrated that salamanders were physiologically challenged but could tolerate the experimental concentrations. CORT signaling did not attenuate sublethal effects of salinity. Baseline and stress-induced (after an acute stressor, shaking) CORT were not influenced by salinity. ALDO was correlated with baseline CORT, suggesting it could be difficult to decouple the roles of CORT and ALDO. Future studies comparing ALDO and CORT responses of adaptively tolerant and previously unexposed populations could be beneficial to understand the roles of these hormones in tolerance to salinity. Nevertheless, our study enhances our understanding of the roles of corticosteroid hormones in mediating effects of a prominent anthropogenic stressor.

User-friendly Taqman probe coupled-insulated isothermal PCR (iiPCR) for rapid detection of emerging Ambystoma tigrinum virus (ATV) in western tiger salamanders (Ambystoma mavortium) on a compact, portable instrument

Portable user-friendly diagnostic tests can benefit detection and surveillance of wildlife diseases. Here, the performance of a compact POCKIT™ Nucleic Acid Analyzer for detection of Ambystoma tigrinum virus (ATV), an emerging Iridovirus that is associated with high host mortality in the western tiger salamander (Ambystoma mavortium) in North America was assessed. Tissue samples from 188 larval tiger salamanders collected from sites in Alberta, Canada were tested by both iiPCR and by conventional PCR. Results of the two assays showed 96.3% agreement. All 176 samples that tested positive by conventional PCR were also positive by iiPCR, while 12 of the samples that were negative by conventional PCR were positive by iiPCR. Comparison of the limits of detection of the two assays shows that the iiPCR assay was more sensitive than conventional PCR and had a LOD95 of 20 copies per reaction. The instrument automatically analyzes and displays results within 40min following nucleic acid extraction. The novel technology could enhance detection of, and response to, wildlife pathogens, particularly those that occur sporadically, cause rapid outbreaks, and/or occur within isolated geographical regions.

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4. A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording. Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

Comparative transcriptomics and gene expression in larval tiger salamander ( Ambystoma tigrinum) gill and lung tissues as revealed by pyrosequencing

Biologists are beginning to unravel the complexities of gene expression in model organisms by studying the transcriptome, the complement of genes that are transcribed in a given tissue. It is unclear, however, if findings from model systems apply to non-model organisms because of environmental effects on gene expression. Furthermore, there have been few efforts to quantify how transcriptome or gene expression varies across individuals and across tissues in natural environments. Herein, we describe transcriptomic profiling of gene expression in lung and gill tissue of three larval tiger salamanders. We do so with a hierarchical experimental design that captures variation in expression among genes, among tissues, and among individuals. Using 454 pyrosequencing, we produced high-quality sequence data of 59 megabases and assembled ~ 200,000 reads into 19,501 contigs. These contigs BLASTed to 3,599 transcripts, of which 721 were expressed in both tissues, 1,668 were unique to gill, and 1,210 unique to lung. Our data showed tissue-specific patterns in gene expression level with variation among transcripts and individuals. We identified genes and gene ontology terms related to respiration and compared their relative expression levels between gill and lung tissues. We also found evidence of exogenous genes associated with larval salamanders, and we identified ~ 1400 potential molecular markers (microsatellites and single nucleotide polymorphisms) that are associated with expressed genes. Given the tissue-specific differences we observed in transcriptomes, these data reinforce the idea that changes in gene expression serve as a primary mechanism underlying phenotypic plasticity.

Amphibian malformations and inbreeding.

Inbreeding may lead to morphological malformations in a wide variety of taxa. We used genetic markers to evaluate whether malformed urodeles were more inbred and/or had less genetic diversity than normal salamanders. We captured 687 adult and 1,259 larval tiger salamanders (Ambystoma tigrinum tigrinum), assessed each individual for gross malformations, and surveyed genetic variation among malformed and normal individuals using both cytoplasmic and nuclear markers. The most common malformations in both adults and larvae were brachydactyly, ectrodactyly and polyphalangy. The overall frequency of adults with malformations was 0.078 compared to 0.081 in larval samples. Genetic diversity was high in both normal and malformed salamanders, and there were no significant difference in measures of inbreeding (f and F), allele frequencies, mean individual heterozygosity or mean internal relatedness. Environmental contaminants or other extrinsic factors may lead to genome alternations that ultimately cause malformations, but our data indicate that inbreeding is not a causal mechanism.

Hybrid vigor between native and introduced salamanders raises new challenges for conservation

Hybridization between differentiated lineages can have many different consequences depending on fitness variation among hybrid offspring. When introduced organisms hybridize with natives, the ensuing evolutionary dynamics may substantially complicate conservation decisions. Understanding the fitness consequences of hybridization is an important first step in predicting its evolutionary outcome and conservation impact. Here, we measured natural selection caused by differential viability of hybrid larvae in wild populations where native California Tiger Salamanders (Ambystoma californiense) and introduced Barred Tiger Salamanders (Ambystoma tigrinum mavortium) have been hybridizing for 50-60 years. We found strong evidence of hybrid vigor; mixed-ancestry genotypes had higher survival rates than genotypes containing mostly native or mostly introduced alleles. Hybrid vigor may be caused by heterozygote advantage (overdominance) or recombinant hybrid vigor (due to epistasis or complementation). These genetic mechanisms are not mutually exclusive, and we find statistical support for both overdominant and recombinant contributions to hybrid vigor in larval tiger salamanders. Because recombinant homozygous genotypes can breed true, a single highly fit genotype with a mosaic of native and introduced alleles may eventually replace the historically pure California Tiger Salamander (listed as Threatened under the U.S. Endangered Species Act). The management implications of this outcome are complex: Genetically pure populations may not persist into the future, but average fitness and population viability of admixed California Tiger Salamanders may be enhanced. The ecological consequences for other native species are unknown.

Phenotypically Plastic Responses of Larval Tiger Salamanders, Ambystoma tigrinum, to Different Predators

Abstract Studies of prey responses to different predators are needed to investigate costs and benefits of particular antipredator responses and to unravel community-level effects on phenotypic plasticity. We reared laboratory-bred larvae of Arizona Tiger Salamanders, Ambystoma tigrinum nebulosum with either of two common predators, diving beetle larvae (Dytiscus sp.) or dragonfly naiads (Anax junius). Relative to controls, salamander larvae in both predator treatments had shorter snout–vent lengths and deeper tails; these differences may be related to increased swimming ability. In addition, larvae reared with dragonfly naiads had shorter tails than those reared with diving beetle larvae, possibly in response to different predator foraging strategies or differences in strength of selection imposed by each. Salamander larvae from predator treatments weighed less than controls, with salamanders reared with dragonflies weighing the least. This suggests that salamanders respond more strongly to dragonfly naia...

Heterospecific Prey and Trophic Polyphenism in Larval Tiger Salamanders

Polyphenisms (environmentally cued polymorphisms) are ubiquitous, yet the specific proximate mechanisms producing alternative morphs are generally not well known. We tested hypotheses for the role of large heterospecific prey in the cannibalistic polyphenism within larval tiger salamanders, Ambystoma tigrinum nebulosum, to determine whether heterospecific prey directly or indirectly influence the production of cannibal morphs. Field surveys suggested, and laboratory experiments confirmed, that macroinvertebrate prey induce cannibals via an increase in body size variation within larval salamander populations. Dietary data and laboratory foraging experiments revealed that cannibals preferred conspecifics even when their capture success was greater on macroinvertebrates. Typical morphs, in contrast, consumed only macroinvertebrate and other prey and never successfully cannibalized conspecifics. Our results support the indirect hypothesis that cannibals are induced via increased body size variation within a population of larvae, as a result of differential consumption of large heterospecific prey, and do not rely on consumption of heterospecific prey once they are cannibals. The cannibalistic polyphenism is one example of phenotypic plasticity in which the functional significance and the proximate mechanisms producing the two morphs are becoming clearer, allowing further study of the molecular and physiological basis of the alternative phenotypes.

Effects of fathead minnows and restoration on prairie wetland ecosystems

SUMMARY 1. Research has shown that fish influence the structure and processes of aquatic ecosystems, but replicated studies at the ecosystem level are rare as are those involving wetlands. Some wetlands of the Prairie Pothole Region (PPR) of North America support fish communities dominated by fathead minnows (Pimephales promelas) while others are fishless, providing an opportunity to assess the influence of these fish on wetland ecosystems. Additionally, many wetlands have previously been drained and subsequently restored, but the success of these efforts is poorly known and restoration may be impeded by the presence of fish.2. We assessed the effects of fathead minnows and drainage by studying 20 semipermanent, prairie wetlands in Minnesota from 1996 to 1999. We used a 2 × 2 factorial design to examine the effects of presence and absence of minnows and drainage history (restored/never drained) on the abundance of aquatic invertebrates and amphibians, as well as on the concentrations of chlorophyll a, total phosphorus, total nitrogen and turbidity in the water column.3. Results showed that fathead minnows are an important determinant of many biotic and abiotic characteristics of wetlands in the eastern PPR. Wetlands with fathead minnows had fewer aquatic insects, large‐ and small‐bodied cladocerans, calanoid copepods, ostracods and larval tiger salamanders, as well as a higher abundance of corixids and greater turbidity and chlorophyll a. A higher concentration of phosphorus in restored basins was the only consistent effect of past management.4. Fathead minnows usually dominate fish communities in eastern PPR wetlands where fish are present, and can have several strong ecosystem effects. While abiotic variables are important determinants of ecosystem structure in prairie wetlands, they can be strongly influenced by biotic factors.

Patterns of habitat and invertebrate diet overlap between tiger salamanders and ducks in prairie potholes

During the breeding season, migratory waterfowl are attracted to wetlands characterized by high macroinvertebrate availability. Many of these prairie potholes are fishless and this apparent void is filled, at least partially, by tiger salamanders. Based on gut contents from 98 tiger salamanders and published diet data from over 1500 ducks, we show that there is general overlap in diet between both larval and adult tiger salamanders and 10 duck species. Furthermore, when the ducks were split into foraging guilds and compared with tiger salamanders, prey type overlap was 1.7 times higher and prey size was 1.8 times higher with dabbling ducks than diving ducks. Field surveys show that tiger salamander density is more highly correlated with diving duck density across potholes than dabbling duck density. Tiger salamanders have higher diet overlap with dabbling ducks than diving ducks whereas tiger salamanders have higher spatial overlap with diving ducks than dabbling ducks suggesting that these consumers coarsely partition diet and habitat resources. It has been reported that tiger salamanders have specialized diets that are associated with foraging preferences for benthic habitats. This view is too narrow: in southwestern Manitoba, Canada, tiger salamanders are more general consumers with diets more like dabbling ducks that forage mostly in planktonic and littoral habitats. Our results suggest that dabbling and diving ducks are, to different extents, liable to the effects of indirect interactions, specifically competition for common prey, with tiger salamanders.

Development of size structure in tiger salamanders: the role of intraspecific interference.

Cannibalism affects patterns of density-dependent mortality and may regulate population size. In many cases, rates of cannibalism depend on size structure, the frequency distribution of body sizes in the population, because cannibals can often only capture and consume smaller individuals. Size differences within single-age groups can be caused by a variety of factors. In this research we tested the hypothesis that size variation among larval tiger salamanders is due, in part, to interference interactions among individuals of different sizes. We found that size variation was greater when we raised larvae in groups rather than in isolation. This increase in size variation was due more to a relative deceleration of growth among smaller individuals rather than acceleration among larger individuals. We also found that smaller larvae had lower feeding rates than larger larvae when in groups, but not when isolated. Including spatial structure to limit physical interactions did not affect the size specificity of feeding rate, although it reduced feeding rates overall. We argue that these results are consistent with the hypothesis that larger larvae interfere, probably indirectly, with the feeding behavior of small individuals and this contributes to increases in size variation over time. We hypothesize that this indirect interference is caused by a behavioral response of smaller larvae to the risk of predation (cannibalism) by larger individuals.

Pathology, isolation, and preliminary molecular characterization of a novel iridovirus from tiger salamanders in Saskatchewan.

All iridovirus was confirmed to be the cause of an epizootic in larval and adult tiger salamanders (Ambystoma tigrinum diaboli) from four separate ponds in southern Saskatchewan (Canada) during the summer of 1997. This organism also is suspected, based on electron microscopic findings, to be the cause of mortality of larval tiger salamanders in a pond over 200 km to the north during the same year. Salamanders developed a generalized viremia which resulted in various lesions including: necrotizing, vesicular and ulcerative dermatitis; gastrointestinal ulceration; and necrosis of hepatic, splenic, renal, lymphoid, and hematopoietic tissues. In cells associated with these lesions, large lightly basophilic cytoplasmic inclusions and vacuolated nuclei with marginated chromatin were consistently found. Virus was isolated from tissue homogenates of infected salamanders following inoculation of epithelioma papilloma cyprini (EPC) cells. The virus, provisionally designated Regina ranavirus (RRV), was initially identified as an iridovirus by electron microscopy. Subsequent molecular characterization, including partial sequence analysis of the major capsid protein (MCP) gene, confirmed this assignment and established that RRV was a ranavirus distinct from frog virus 3 (FV3) and other members of the genus Ranavirus. Intraperitoneal inoculation of 5 x 10(6.23) TCID50 of the field isolate caused mortality in inoculated salamanders at 13 days post infection. Field, clinical, and molecular studies jointly suggest that the etiological agent of recent salamander mortalities is a highly infectious novel ranavirus.

PROXIMATE CAUSES OF CANNIBALISTIC POLYPHENISM IN LARVAL TIGER SALAMANDERS

Larval tiger salamanders, Ambystoma tigrinum, exist as small-headed “typical” morphs that feed mainly on aquatic invertebrates and as large-headed “cannibal” morphs that often prey on conspecifics. Previous experiments have demonstrated that cannibals are induced facultatively when crowded with conspecific larvae. We asked: (1) What sensory cues trigger expression of cannibals? (2) Does larval age influence expression of cannibals? (3) Are cannibals also induced by crowding with heterospecifics? (4) Do cannibals suppress others from developing into cannibals? We found that tactile cues from other salamander larvae were necessary to elicit cannibals. Cannibals were produced when tiger salamander larvae were crowded with congeneric larvae, and these heterospecific-induced cannibals were expressed at an earlier age than those induced by conspecifics. Furthermore, younger larvae were more likely to develop into cannibals. Finally, visual and chemical cues from cannibals did not suppress other larvae from deve...

Mechanics of Lung Ventilation in a Larval Salamander Ambystoma Tigrinum

The larval stage of the tiger salamander Ambystoma tigrinum is entirely aquatic, but the larvae rely on their lungs for a large proportion of their oxygen uptake. X-ray video and pressure measurements from the buccal and body cavities demonstrate that the larvae inspire using a two-stroke buccal pump and exhale actively by contracting the hypaxial musculature to increase body pressure. Larvae begin a breath by expanding the buccal cavity to draw in air through the mouth, while simultaneously exhaling air from the lungs to mix with the fresh air in the buccal cavity. The mouth then closes, and the buccal cavity compresses to pump a portion of the mixture into the lungs. The remaining air in the buccal cavity is then released as bubbles from the mouth and gill slits. Ventilatory volumes estimated from X-ray video records indicate that approximately 80% of the air pumped into the lungs is fresh air and 20% is previously expired air. Exhalation in larval tiger salamanders is active, powered by contraction of all four layers of lateral hypaxial musculature. Electromyography indicates that the transverse abdominis (TA) muscle is active for the longest duration and shows the highest-amplitude activity, but the external oblique superficialis, the external oblique profundus and the internal oblique also show consistent, low-level activity. The finding that the TA muscle is active during exhalation in larval tiger salamanders contributes to a growing body of evidence that the use of the TA for exhalation is a primitive character for tetrapods.

Group size structure affects patterns of aggression in larval salamanders

The potential importance of intrapopulation phenotypic variability to population-level ecology has been demonstrated in both theoretical and field studies. One way to connect individuals to the dynamics of populations they compose is to study behavioral response (an individual-level characteristic) to variability in conspecific phenotypes (a population-level characteristic). We examined effects of variation in size of individuals on patterns of aggression in larval tiger salamanders (Ambystoma tigrmum nebulosum) by observing aggressive behavior in groups of three larvae in a laboratory experiment. We assessed effects of variability in size of conspecdfics independently of mean larval size and larval density. Overall levels of aggression were generally higher in groups in which all individuals were similarly sized than in groups of variably sized individuals. Medium-sized individuals exhibited significantly higher levels of aggression and were attacked significantly more often when in groups consisting only of similarly sized larvae as compared to groups composed of larvae representing a wider range of body sizes. Activity levels of larvae were also generally lower when all individuals were the same size, resulting in a negative correlation between activity and levels of aggression. These results suggest that groups of similarly sized individuals experience a more aggressive social environment than groups of variably sized individuals, and they suggest a promising avenue of research for connecting individual behavioral and physiological responses to size structure (phenotypic variability) with population dynamics.

Metamorphosis of Freshwater Mussel Glochidia (Bivalvia: Unionidae) on Amphibians and Exotic Fishes

This study determined that inexpensive and easily maintained amphibians and exotic fishes could act as hosts for two species of native North American unionid mussels, and bypass the need to identify native hosts when the object is to culture mussels. Two mussel species, Lampsilis cardium and Utterbackia imbecillis, were used to parasitize 42 exotic fishes and seven potential nonpiscine host species. Nonpiscine hosts included amphibians and decapod crustaceans. Lampsilis cardium successfully metamorphosed on six species of exotic fishes, as well as on larval tiger salamanders. Utterbackia imbecillis successfully metamorphosed on 30 species of exotic fishes and all four amphibian species tested. No glochidia metamorphosed on crustaceans. Successful metamorphosis on amphibians indicates that mussel zoogeography may be more complicated than previously thought. Using surrogate hosts may be a valuable alternative to natural hosts in laboratory culture of mussels.

Effects of the herbicide atrazine on Ambystoma tigrinum metamorphosis: duration, larval growth, and hormonal response.

We exposed larval tiger salamanders (Ambystoma tigrinum) reared in the laboratory from eggs collected from a prairie wetland in North Dakota to three concentrations of atrazine (0, 75, and 250 micrograms/L) in a static renewal test to determine the pesticide's effect on (1) plasma corticosterone and thyroxine concentrations, (2) larval size, and (3) days-to-stage at stages 2 and 4 of metamorphic climax. We found significant effects of atrazine on each of these response variables. Plasma thyroxine was elevated in both atrazine-exposed groups compared with the control group; plasma corticosterone was depressed in the 75 micrograms/L treatment compared with both the control and 250 micrograms/L treatment. Larvae exposed to 75 micrograms/L atrazine reached stage 4 later but at a size and weight comparable to the control group. By contrast, larvae in the 250 micrograms/L treatment progressed to stage 4 at the same time but at a smaller size and lower weight than larvae in the control group. These results indicate that the herbicide has the potential to influence tiger salamander life history. We present a model consistent with our results, whereby corticosterone and thyroxine interact to regulate metamorphosis of tiger salamanders based on nutrient assimilation and adult fitness.

Interactions of Androgens and Estradiol on Sex Accessory Ducts of Larval Tiger Salamanders,Ambystoma tigrinum

Immature tiger salamander larvae were treated with 12.5 or 25 μg of estradiol, testosterone, or dihydrotestosterone (DHT), or 12.5 μg of estradiol combined with 12.5 μg of either testosterone or DHT. Müllerian duct epithelium was more stimulated by combined steroid treatment than by any steroid alone. Estradiol antagonized the action of DHT in the Wolffian duct. Both of the androgens and estradiol when administered alone at the higher dose stimulated enlargement of connective tissue surrounding the ducts, but the combined 12.5 μg androgen/12.5 μg estrogen treatment was more effective even though the total steroid administered was the same. The effectiveness of DHT on Müllerian cells of this species is evidence against a required aromatization of androgens to explain paradoxical steroid effects and suggests that fundamental differences may exist in steroid receptors of Müllerian ducts, connective tissue, and Wolffian ducts. A possible role for the urodele duct system for assessing estrogenic activity of environmental contaminants is discussed.