Male African Clawed Frogs Research Articles

Cardiac and Cerebellar Histomorphology and Inositol 1,4,5-Trisphosphate (IP3R) Perturbations in Adult Xenopus laevis Following Atrazine Exposure

Despite several reports on the endocrine-disrupting ability of atrazine in amphibian models, few studies have investigated atrazine toxicity in the heart and cerebellum. This study investigated the effect of atrazine on the unique Ca2+ channel-dependent receptor (Inositol 1,4,5-trisphosphate; IP3R) in the heart and the cerebellum of adult male Xenopus laevis and documented the associated histomorphology changes implicated in cardiac and cerebellar function. Sixty adult male African clawed frogs (Xenopus laevis) were exposed to atrazine (0 µg/L (control), 0.01 µg/L, 200 µg/L, and 500 µg/L) for 90 days. Thereafter, heart and cerebellar sections were processed with routine histological stains (heart) or Cresyl violet (brain), and IP3R histochemical localization was carried out on both organs. The histomorphology measurements revealed a significant decrease in the mean percentage area fraction of atrial (0.01 µg/L and 200 µg/L) and ventricular myocytes (200 µg/L) with an increased area fraction of interstitial space, while a significant decrease in Purkinje cells was observed in all atrazine groups (p < 0.008, 0.001, and 0.0001). Cardiac IP3R was successfully localized, and its mean expression was significantly increased (atrium) or decreased (cerebellum) in all atrazine-exposed groups, suggesting that atrazine may adversely impair cerebellar plasticity and optimal functioning of the heart due to possible disturbances of calcium release, and may also induce several associated cardiac and neural pathophysiologies in all atrazine concentrations, especially at 500 µg/L.

Cardiotoxicity in African clawed frog (Xenopus laevis) sub-chronically exposed to environmentally relevant atrazine concentrations: Implications for species survival

The toxic effects of different atrazine concentrations on tadpoles and adult male African clawed frogs (Xenopus laevis) were assessed in a controlled laboratory environment following 90 days’ exposure. The aim was to elucidate the danger of atrazine exposure on the cardiac tissue relative to its critical function of rhythmic contractility, fundamental for optimal blood circulation and homeostasis.Tadpoles and adult frogs were exposed to 0 μg/L (control), 0.01 μg L−1, 200 μg L−1 and 500 μg L−1 concentrations of atrazine for 90 days. Mortality was concenration-dependent and significantly increased in juvenile group (77%, 43%, 23% and 0 respectively for 500 μg L−1, 200 μg L−1, 0.01 μg L−1, and control group). While the mean juvenile heart area decreased concentration-dependently, adult frog mean heart area significantly increased in the 200 μg L−1 group only and mean heart weight change was variable across all exposure levels.Light microscopy of hematoxylin and eosin (H&E) and Mallory-Heidenhain rapid one-step staining techniques on cardiac tissue sections of the juvenile and adult frogs revealed shrinkage of cardiac muscle cells into thin wavy myocytes. Additionally, disorganized branching of muscle fibres with reduced striations were observed in 0.01 μg L−1 and 200 μg L−1 but hypertrophied myocytes, thickened intensely staining myofibrils in the 500 μg L−1 group in juvenile and adult frogs. Significant increase in the mean percentage area of connective tissue in all the treated groups (p < 0.036) were also recorded. Immunohistochemistry analysis showed decreased eNOS localization in cardiac tissue in 200 μg L−1 and 500 μg L−1 of both juvenile and adult group, suggestive of decreased cardiac contractility due to atrazine exposure.The results indicate that environmentally relevant atrazine concentrations cause significant mortality in tadpoles while concentrations ≥200 μg L−1 adversely affect cardiac muscle morphology and may induce functional perturbations in cardiac tissue contractility and consequent dysfunction which generally may have an adverse impact on their survival and longevity.

Condition dependence of advertisement calls in male African clawed frogs

Variations in the costs or benefits of signaling may result in variations in signaling strategies. The adverse conditions hypothesis suggests that reliable signaling can be improved under stress or adverse environmental conditions, but the empirical cases are limited. In the present study, we manipulated food availability in male Xenopus laevis in order to determine whether certain characteristics of advertisement call in X. laevis are condition-dependent and how food depletion modulates the association between individual condition and signaling performance. The results showed that although frogs tend to decrease their within-bout call rate compared to other call characteristics (calling effort and dominant frequency) under food constraint, none of them was statistically significantly affected by food restriction. However, within-bout call rate was positively coupled with adaptive immunocompetence in starved frogs, and the correlation was decoupled in those with free access to food. These results support that within-bout call rate is a condition-dependent characteristic of advertisement call and even mild adverse conditions may enhance reliable signaling in X. laevis, probably due to enlarging the relative fitness difference between high- and low-condition individuals.

Premotor Neuron Divergence Reflects Vocal Evolution.

To identify mechanisms of behavioral evolution, we investigated the hindbrain circuit that generates distinct vocal patterns in two closely related frog species. Male Xenopus laevis and Xenopus petersii produce courtship calls that include a fast trill: trains of ∼60 Hz sound pulses. Although fast trill rates are similar, X. laevis fast trills have a longer duration and period than those of X. petersii To pinpoint the neural basis of these differences, we used whole-cell patch-clamp recordings in a key premotor hindbrain nucleus (the Xenopus parabrachial area, PBX) in ex vivo brains that produce fictive vocalizations, vocal nerve activity corresponding to advertisement call patterns. We found two populations of PBX neurons with distinct properties: fast trill neurons (FTNs) and early vocal neurons (EVNs). FTNs, but not EVNs, appear to be intrinsically tuned to produce each species' call patterns because: (1) X. laevis FTNs generate longer and slower depolarizations than X. petersii FTNs during their respective fictive vocalizations, (2) current steps in FTNs induce burst durations that are significantly longer in X. laevis than X. petersii, and (3) synaptically isolated FTNs oscillate in response to NMDA in a species-specific manner: longer and slower in X. laevis than in X. petersii Therefore, divergence of premotor neuron membrane properties is a strong candidate for generating vocal differences between species.SIGNIFICANCE STATEMENT The vertebrate hindbrain includes multiple neural circuits that generate rhythmic behaviors including vocalizations. Male African clawed frogs produce courtship calls that are unique to each species and differ in temporal patterns. Here, we identified two functional subtypes of neurons located in the parabrachial nucleus: a hindbrain region implicated in vocal and respiratory control across vertebrates. One of these neuronal subtypes exhibits distinct properties across species that can account for the evolutionary divergence of song patterns. Our results suggest that changes to this group of neurons during evolution may have had a major role in establishing novel behaviors in closely related species.

Effects of environmentally relevant sub-chronic atrazine concentrations on African clawed frog (Xenopus laevis) survival, growth and male gonad development

Sub-chronic toxicity of environmentally relevant atrazine concentrations on exposed tadpoles and adult male African clawed frogs (Xenopus laevis) was evaluated in a quality controlled laboratory for 90 days. The aim of this study was to determine the effects of atrazine on the survival, growth and gonad development of African clawed frogs. After exposure of tadpoles to atrazine concentrations of 0 (control), 0.01, 200 and 500 μg L−1 in water, mortality rates of 0, 0, 3.3 and 70% respectively were recorded for the 90 day exposure period. Morphometry showed significantly reduced tadpole mass in the 500 μg L−1 atrazine exposed tadpoles (p < 0.05). Light microscopy on testes of adult frogs exposed to the same atrazine concentrations using hematoxylin and eosin (H&E) and Van Gieson staining techniques revealed gonadal atrophy, disruption of germ cell lines, seminiferous tubule structure damage and formation of extensive connective tissue around seminiferous tubules of frogs exposed to 200 μg L−1 and 500 μg L−1 atrazine concentrations. Ultrastructural analysis of the cellular organelles using transmission electron microscopy (TEM) revealed significant amounts of damaged mitochondria in testosterone producing Leydig cells as well as Sertoli cells. Biochemical analysis revealed reduced serum testosterone levels in adult frogs at all exposure levels as well as presence of six atrazine metabolites in frog serum and liver. The results indicate that atrazine concentrations greater than the calculated LC50 of 343.7 μg L−1 cause significant mortality in tadpoles, while concentrations ≥200 μg L−1 adversely affect reproductive health of adult frogs and development of tadpoles sub-chronically exposed to atrazine.

Rhythm generation, coordination, and initiation in the vocal pathways of male African clawed frogs

Central pattern generators (CPGs) are considered to underlie vocalizations in many vertebrate species, but the detailed mechanisms underlying their functions remain unclear. We addressed this question using an isolated brain preparation of African clawed frogs. We discovered that two vocal phases are mediated by anatomically distinct CPGs, that there are a pair of CPGs contained in the left and right half of the brain stem, and that mechanisms underlying initiation of the two vocal phases are distinct.

Tebuconazole disrupts steroidogenesis in Xenopus laevis

A 27-day controlled exposure study of adult male African clawed frogs (Xenopus laevis) was conducted to examine the mechanism by which tebuconazole may disrupt steroidogenesis. The fungicide was measured by LC–MS/MS in tank water and in target tissues (adipose, kidney, liver, and brain), and we observed tissue-specific bioconcentration with BCF up to 238. Up to 10 different steroid hormones were quantified in gonads using LC–MS/MS and in plasma using GC–MS/MS and a radioimmunoassay was performed for further measurement of androgens. In order to assess whether effects increased with exposure or animals adapted to the xenobiotic, blood samples were collected 12 days into the study and at termination (day 27). After 12 days of exposure to 100 and 500μgL−1 tebuconazole, plasma levels of testosterone (T) and dihydrotestosterone (DHT) were increased, while plasma 17β-estradiol (E2) concentrations were greatly reduced. Exposure to 0.1μgL−1, on the other hand, resulted in decreased levels of T and DHT, with no effects observed for E2. After 27 days of exposure, effects were no longer observed in circulating androgen levels while the suppressive effect on E2 persisted in the two high-exposure groups (100 and 500μgL−1). Furthermore, tebuconazole increased gonadal concentrations of T and DHT as well as expression of the enzyme CYP17 (500μgL−1, 27 days). These results suggest that tebuconazole exposure may supress the action of CYP17 at the lowest exposure (0.1μgL−1), while CYP19 suppression dominates at higher exposure concentrations (increased androgens and decreased E2). Increased androgen levels in plasma half-way into the study and in gonads at termination may thus be explained by compensatory mechanisms, mediated through increased enzymatic expression, as prolonged exposure had no effect on circulating androgen levels.

Species-specific loss of sexual dimorphism in vocal effectors accompanies vocal simplification in African clawed frogs (Xenopus).

Phylogenetic studies can reveal patterns of evolutionary change, including the gain or loss of elaborate courtship traits in males. Male African clawed frogs generally produce complex and rapid courtship vocalizations, whereas female calls are simple and slow. In a few species, however, male vocalizations are also simple and slow, suggesting loss of male-typical traits. Here, we explore features of the male vocal organ that could contribute to loss in two species with simple, slow male calls. In Xenopus boumbaensis, laryngeal morphology is more robust in males than in females. Larynges are larger, have a more complex cartilaginous morphology and contain more muscle fibers. Laryngeal muscle fibers are exclusively fast-twitch in males but are both fast- and slow-twitch in females. The laryngeal electromyogram, a measure of neuromuscular synaptic strength, shows greater potentiation in males than in females. Male-specific physiological features are shared with X. laevis, as well as with a species of the sister clade, Silurana tropicalis, and thus are likely ancestral. In X. borealis, certain aspects of laryngeal morphology and physiology are sexually monomorphic rather than dimorphic. In both sexes, laryngeal muscle fibers are of mixed-twitch type, which limits the production of muscle contractions at rapid intervals. Muscle activity potentiation and discrete tension transients resemble female rather than male X. boumbaensis. The de-masculinization of these laryngeal features suggests an alteration in sensitivity to the gonadal hormones that are known to control the sexual differentiation of the larynx in other Xenopus and Silurana species.

Distinct neural and neuromuscular strategies underlie independent evolution of simplified advertisement calls

Independent or convergent evolution can underlie phenotypic similarity of derived behavioural characters. Determining the underlying neural and neuromuscular mechanisms sheds light on how these characters arose. One example of evolutionarily derived characters is a temporally simple advertisement call of male African clawed frogs (Xenopus) that arose at least twice independently from a more complex ancestral pattern. How did simplification occur in the vocal circuit? To distinguish shared from divergent mechanisms, we examined activity from the calling brain and vocal organ (larynx) in two species that independently evolved simplified calls. We find that each species uses distinct neural and neuromuscular strategies to produce the simplified calls. Isolated Xenopus borealis brains produce fictive vocal patterns that match temporal patterns of actual male calls; the larynx converts nerve activity faithfully into muscle contractions and single clicks. In contrast, fictive patterns from isolated Xenopus boumbaensis brains are short bursts of nerve activity; the isolated larynx requires stimulus bursts to produce a single click of sound. Thus, unlike X. borealis, the output of the X. boumbaensis hindbrain vocal pattern generator is an ancestral burst-type pattern, transformed by the larynx into single clicks. Temporally simple advertisement calls in genetically distant species of Xenopus have thus arisen independently via reconfigurations of central and peripheral vocal neuroeffectors.

Atrazine induces complete feminization and chemical castration in male African clawed frogs (Xenopus laevis)

The herbicide atrazine is one of the most commonly applied pesticides in the world. As a result, atrazine is the most commonly detected pesticide contaminant of ground, surface, and drinking water. Atrazine is also a potent endocrine disruptor that is active at low, ecologically relevant concentrations. Previous studies showed that atrazine adversely affects amphibian larval development. The present study demonstrates the reproductive consequences of atrazine exposure in adult amphibians. Atrazine-exposed males were both demasculinized (chemically castrated) and completely feminized as adults. Ten percent of the exposed genetic males developed into functional females that copulated with unexposed males and produced viable eggs. Atrazine-exposed males suffered from depressed testosterone, decreased breeding gland size, demasculinized/feminized laryngeal development, suppressed mating behavior, reduced spermatogenesis, and decreased fertility. These data are consistent with effects of atrazine observed in other vertebrate classes. The present findings exemplify the role that atrazine and other endocrine-disrupting pesticides likely play in global amphibian declines.

Pesticide mixtures, endocrine disruption, and amphibian declines: are we underestimating the impact?

Amphibian populations are declining globally at an alarming rate. Pesticides are among a number of proposed causes for these declines. Although a sizable database examining effects of pesticides on amphibians exists, the vast majority of these studies focus on toxicological effects (lethality, external malformations, etc.) at relatively high doses (parts per million). Very few studies focus on effects such as endocrine disruption at low concentrations. Further, most studies examine exposures to single chemicals only. The present study examined nine pesticides (four herbicides, two fungicides, and three insecticides) used on cornfields in the midwestern United States. Effects of each pesticide alone (0.1 ppb) or in combination were examined. In addition, we also examined atrazine and S-metolachlor combined (0.1 or 10 ppb each) and the commercial formulation Bicep II Magnum, which contains both of these herbicides. These two pesticides were examined in combination because they are persistent throughout the year in the wild. We examined larval growth and development, sex differentiation, and immune function in leopard frogs (Rana pipiens). In a follow-up study, we also examined the effects of the nine-compound mixture on plasma corticosterone levels in male African clawed frogs (Xenopus laevis). Although some of the pesticides individually inhibited larval growth and development, the pesticide mixtures had much greater effects. Larval growth and development were retarded, but most significantly, pesticide mixtures negated or reversed the typically positive correlation between time to metamorphosis and size at metamorphosis observed in controls: exposed larvae that took longer to metamorphose were smaller than their counterparts that metamorphosed earlier. The nine-pesticide mixture also induced damage to the thymus, resulting in immunosuppression and contraction of flavobacterial meningitis. The study in X. laevis revealed that these adverse effects may be due to an increase in plasma levels of the stress hormone corticosterone. Although it cannot be determined whether all the pesticides in the mixture contribute to these adverse effects or whether some pesticides are effectors, some are enhancers, and some are neutral, the present study revealed that estimating ecological risk and the impact of pesticides on amphibians using studies that examine only single pesticides at high concentrations may lead to gross underestimations of the role of pesticides in amphibian declines.

Plasma concentrations of estradiol and testosterone, gonadal aromatase activity and ultrastructure of the testis in Xenopus laevis exposed to estradiol or atrazine

The ultrastructure of testicular cells of adult male African clawed frogs ( Xenopus laevis) exposed to either estradiol (0.1 μg/L) or 2-chloro-4-ethylamino-6-isopropyl-amino- s-triazine (atrazine; 10 or 100 μg/L) was examined by electron microscopy and compared to plasma concentrations of the steroid hormones, testosterone (T) and estradiol (E2), testicular aromatase activity and gonad growth expressed as the gonado-somatic index (GSI). Exposure to E2 caused significant changes both at the sub-cellular and biochemical levels. Exposure to E2 resulted in significantly fewer sperm cells, inhibition of meiotic division of germ cells, more lipid droplets that are storage compartments for the sex steroid hormone precursor cholesterol, and lesser plasma T concentrations. Although not statistically significant, frogs exposed to E2 had slightly smaller GSI values. These results may be indicative of an inhibition of gonad growth and disrupted germ cell development by E2. Concentrations of E2 in plasma were greater in frogs exposed to E2 in water. Exposure to neither concentration of atrazine caused effects on germ cell development, testicular aromatase activity or plasma hormone concentrations. These results suggest that atrazine does not affect testicular function. In contrast, exposure of male X. laevis to E2 led to sub-cellular events that are indicative of disruption of testicular development, and demasculinization processes (decrease of androgen hormone titers). These results indicate that atrazine does not cause responses that are similar to those caused by exposure to E2.

Changes in expression and honesty of sexual signalling over the reproductive lifetime of sticklebacks.

Fitness costs of signalling are essential in order for reliable sexual signalling to prevail when the interests of the sexes conflict. This means that signalling can be subjected to a life history trade-off between present and future signalling effort. Here, I show that three-spined stickleback males (Gasterosteus aculeatus), who have a single breeding season during which they breed repeatedly, change their red nuptial coloration over the season depending on their body size at the start of breeding. Large males that completed several breeding cycles increased their red coloration over the season, whereas small males, who completed only a few cycles, did not. The increase in coloration was accompanied by an increase in parental success when males were energy constrained, but not when they had access to an unlimited food supply. Red coloration was thus an honest signal of male parental ability despite changes in signal expression when both signalling and parental care were costly and the investments in them changed simultaneously over the reproductive lifetime. However, the honesty of the signal varied over a lifetime. At the penultimate cycle, bright males cannibalized some of their eggs, probably to increase survival to the last cycle, whereas males cared for their offspring independent of coloration at the ultimate cycle.