Assessment of sublethal ecotoxicity of solvents on larvae of a model native amphibian (Lithobates pipiens).

Carrier solvents are used frequently in toxicity testing to assist hydrophobic chemicals into solution, but such solvents may have toxic effects on test subjects. Amphibians are model organisms in toxicity studies; however, little is known about the direct effects of solvents on native amphibians. Following modifications to standardized guidelines for native species, we used acute 96-hour exposures to assess the direct effects of three common solvents on survival, differences in morphology and occurrence of abnormalities of northern leopard frog larvae (Lithobates pipiens). The solvents, dimethyl sulfoxide (DMSO), ethanol (ETOH) and acetone (ACE) were used at nominal concentrations ranging from 1 to 100 µL/L. We also conducted a 30-day exposure to assess the direct chronic effects of DMSO at 1 and 5 µL/L, on larval growth, development and sex differentiation, but found no effects. Acute exposure to solvents also had no effect on the survival of larvae, but we found significant abnormalities in tadpoles acutely exposed to 100 µL/L ACE. Acute exposure to DMSO and ETOH had further concentration-dependent effects on larval morphological traits. Our study suggests that DMSO and ETOH at ≤20 µL/L may be used as solvents in amphibian ecotoxicological studies, but ACE should be limited to ≤50 µL/L in ecotoxicity studies and perhaps much less (≤10 µL/L) in studies with other amphibians, based on a review of existing literature. We emphasize pilot studies when using solvents on acute and chronic ecotoxicity tests, using native amphibians.

Role of sediments in modifying the toxicity of two Roundup formulations to six species of larval anurans.

The role of sediment in modifying the toxicity of the original formulation of Roundup® and Roundup WeatherMAX® was examined in aqueous laboratory tests. Six species of anurans (Bufo fowleri, Hyla chrysoscelis, Rana catesbeiana, Rana clamitans, Rana sphenocephala, and Rana pipiens) were exposed at Gosner stage 25 to concentrations of the 2 herbicide formulations in 96-h, static, nonrenewal experiments in the presence and absence of sediment. All species tested had lower median lethal concentration values in water-only exposures of both formulations compared with exposures with sediment. Sediment significantly altered the potency slopes in all tests with the exceptions of H. chrysoscelis and R. clamitans when exposed to the original formulation of Roundup and H. chrysoscelis and R. sphenocephala when exposed to Roundup WeatherMAX. Thresholds were significantly different in all tests, including those in which potency slopes did not differ. Based on water-sediment exposures of the original formulation of Roundup, all 6 species tested had a margin of safety when compared with the predicted environmental concentration of the highest label application rate. Of the 6 species, 5 had a margin of safety when exposed to Roundup WeatherMAX. During incidental exposures in the field, sediments and organic matter present in aquatic systems provide significant sources of environmental ligands. If used according to label instructions, both herbicides should pose minimal risk to anuran amphibians in actual field applications. Environ Toxicol Chem 2014;33:2616-2620. © 2014 SETAC.

Plasma membrane events associated with the meiotic divisions in the amphibian oocyte: insights into the evolution of insulin transduction systems and cell signaling.

BACKGROUND: Insulin and its plasma membrane receptor constitute an ancient response system critical to cell growth and differentiation. Studies using intact Rana pipiens oocytes have shown that insulin can act at receptors on the oocyte surface to initiate resumption of the first meiotic division. We have reexamined the insulin-induced cascade of electrical and ion transport-related plasma membrane events using both oocytes and intact plasma membranes in order to characterize the insulin receptor-steroid response system associated with the meiotic divisions. RESULTS: [(125)I]Insulin binding (K(d) = 54 ± 6 nM) at the oocyte plasma membrane activates membrane serine protease(s), followed by the loss of low affinity ouabain binding sites, with a concomitant 3-4 fold increase in high affinity ouabain binding sites. The changes in protease activity and ouabain binding are associated with increased Na(+)/Ca2(+) exchange, increased endocytosis, decreased Na(+) conductance resulting in membrane hyperpolarization, increased 2-deoxy-D-glucose uptake and a sustained elevation of intracellular pH (pHi). Hyperpolarization is largely due to Na(+)-channel inactivation and is the main driving force for glucose uptake by the oocyte via Na(+)/glucose cotransport. The Na(+) sym- and antiporter systems are driven by the Na(+) free energy gradient generated by Na(+)/K(+)-ATPase. Shifts in α and/or ß Na(+)-pump subunits to caveolar (lipid raft) membrane regions may activate Na/K-ATPase and contribute to the Na(+) free energy gradient and the increase in both Na(+)/glucose co-transport and pHi. CONCLUSIONS: Under physiological conditions, resumption of meiosis results from the concerted action of insulin and progesterone at the cell membrane. Insulin inactivates Na(+) channels and mobilizes fully functional Na(+)-pumps, generating a Na(+) free energy gradient which serves as the energy source for several membrane anti- and symporter systems.

Effects of chronic copper exposure on development and survival in the southern leopard frog (Lithobates [Rana] sphenocephalus).

Exposure to environmental contaminants contributes to the global decline of amphibian populations. The impacts of organic contaminants on amphibians are well documented. However, substantially less is known concerning the potential effects of metals on amphibian populations. Copper (Cu) is an essential element, but it can be toxic at concentrations only slightly higher than the normal physiological range. The present study examines the effects of chronic Cu exposure on embryos and larvae of southern leopard frogs, Lithobates (Rana) sphenocephalus. Groups of eggs from multiple clutches were collected from two wetlands and exposed to a range of Cu concentrations (0-150 µg/L) until they reached the free-swimming stage, and then individual larvae were reared to metamorphosis. Higher Cu concentrations significantly reduced embryo survival to the free-swimming stage but did not further reduce survival to metamorphosis. Larval period was affected by Cu treatment, but the clutch from which larvae originated (i.e., parentage) explained a higher proportion of the variation. Embryo survival to hatching varied significantly among clutches, ranging from 42.9 to 79.2%. Measurable levels of Cu were found in larvae with body burdens up to 595 µg Cu/g dry mass in the 100 µg/L treatment, and larval Cu body burdens were higher than in metamorphs. The present study also demonstrated that higher initial egg density ameliorated embryo mortality at higher Cu levels and should be accounted for in future studies.

Chronic, dietary polybrominated diphenyl ether exposure affects survival, growth, and development of Rana pipiens tadpoles.

Levels of polybrominated diphenyl ethers (PBDEs) in the environment have been increasing rapidly over the past two decades; however, the toxicology of these compounds to aquatic organisms is poorly understood. Because amphibians play a role in both aquatic and terrestrial food webs, and are currently undergoing worldwide population declines, it is of interest to determine how PBDEs may affect amphibian health. This is the first study that reports chronic, dietary effects of environmentally relevant levels (7-277 ng/g wet food) of PBDEs in amphibians throughout larval development. Beginning at the free-swimming stage (Gosner Stage [GS] 25), Rana pipiens tadpoles were orally exposed to a technical pentabromodiphenyl ether mixture (DE-71) through metamorphic climax (GS 42). On exposure day 43, a subset of tadpoles was removed for body residue analysis. Sum PBDEs in whole-body tissue correlated linearly to dietary concentrations with BDE-99 represented as the highest contributing congener in both diet and tissue. Survival among all treatments compared to the control was decreased by DE-71 exposure. Further, growth and development were delayed in all but the highest treatment, perhaps indicating greater PBDE tolerance among those individuals that survived the highest treatment. Time to metamorphic climax was delayed, on average, 22 to 36 d in DE-71-treated tadpoles compared to control tadpoles. Additionally, size at metamorphosis was smaller in the highest treatment, suggesting that individuals that survived and metamorphosed similarly to the controls did so with a trade-off in size. At environmentally relevant levels, PBDEs induced mortality as well as sublethal effects on developing tadpoles through dietary exposure.

Effect of mono-ortho and di-ortho substituted polychlorinated biphenyl (PCB) congeners on leopard frog survival and sexual development.

We tested the effect of mono-ortho and di-ortho PCB congeners on northern leopard frog (Rana pipiens) hatching success, survival and sexual development. Embryos and tadpoles were exposed to two levels (0.5 and 50 microg/l) of two PCBs. PCBs 101 and 70 were selected because they were present in amphibians collected in the Fox River-Green Bay ecosystem and they have the theoretical structural requirements to be able to bind to the estrogen receptor and mediate estrogenic responses. The exposure of leopard frog embryos and tadpoles to PCB 70 and 101 did not significantly affect hatchability, survival, deformities or growth. There were significant departures from the expected 50:50 sex ratio in tadpoles/froglets exposed to PCB 101 and PCB 70. In all the cases of significant departure, the bias was towards higher number of females. Decrease in the proportion of male gonads and increase in the proportion of intersex gonads were observed with increasing PCB tissue concentrations. The effects of PCB congeners on sexual differentiation occur at concentrations higher than observed in frogs in the Fox River/Green Bay ecosystem.

Origin and progress of nuclear transfer in nonmammalian animals.

This chapter traces the origin and progress of nuclear transfer that later became the paradigm for cloning animals. Classic studies in cytology, embryology, or genetics spanning more than five centuries that led to nuclear transfers in unicellular animals and to those in oocytes of insects, fish and amphibians are reviewed. The impetus for the development of successful nuclear transfers in amphibian oocytes in 1952 was to determine whether or not differentiated somatic cell nuclei are developmentally equivalent to zygote nuclei. Experiments in amphibians demonstrated several important results: (1) specialized somatic cell nuclei are extensively multipotent; (2) fertile adult amphibians can be cloned from embryonic and larval nuclei; (3) serial cloning expands the number of clones; (4) transplanting nuclei into oocyte cytoplasm induces reprogramming of their gene function; and (5) amphibian cloning became the model for cloning mammals. Subsequent studies in mice, a more technically favorable species, revealed that specialized cell nuclei are equivalent to zygote nuclei.

Effects of nitrate and atrazine on larval development and sexual differentiation in the northern leopard frog Rana pipiens.

Pollution from agrochemicals may be contributing to the global decline in amphibian populations. Environmentally relevant concentrations of nitrate and/or atrazine on anuran development and gonadal differentiation were tested. Four replicates of 20 tadpoles per tank (80/treatment) were exposed from Taylor-Kollros stage 2 to 3 to stage 23 to 34 to either 10 mg/L nitrate, 10 microg/L atrazine, a combined exposure of 10 mg/L nitrate plus 10 microg/L atrazine, or untreated controls. No treatment-dependent effects on weight, snout-vent or hind limb length, or time to forelimb emergence were observed. The proportions of females increased in all treatments compared to the controls, especially in the combined treatment (chi2 = 17.90, df = 6, p = 0.0065, combined = 66.4% female, control = 41% female). The frequency of intersex was low in all treatments. No treatment-related effects on the total number of spermatogenic cells were observed, but the ratio of cell types differed in that testes from animals in the treated groups exhibited more spermatogonia, fewer spermatocytes, and more spermatids than the control (significantly different, Kruskal-Wallis, p < 0.05). Ovaries from animals treated with nitrate or atrazine exhibited larger immature (previtellogenic) and mature (vitellogenic) follicles, but ovaries from the combined treatment had larger immature follicles only. Testicular oocytes were observed in the nitrate-only and atrazine-only treatments, and the control treatment, but not the combined treatment. Overall, this study has demonstrated changes in sex ratios that are more marked in response to combined nitrate/atrazine exposure than with these chemicals alone. Histological evidence suggests that premature maturation of gonad may occur as a result of nitrate and/or atrazine exposure during larval development.

Relationship of tadpole stage to location of echinostome cercariae encystment and the consequences for tadpole survival.

The effect of echinostome infections on the survival of Rana pipiens tadpoles was examined in relation to developmental stage of tadpoles. Individual tadpoles of Gosner stages 25, 27, 32-33, and 37-39 were exposed to 1 of 4 levels of cercariae (0, 20, 50, or 100). Only tadpoles at stage 25, the earliest stage infected, died within a 5-day experimental period. This stage-specific mortality rate could be explained, in part, by the stage-specific location of encystment of cercariae, which was documented in a separate experiment. In accordance with kidney development, cercariae predominately encysted in the pronephroi during early stages of tadpole development (stages 25 through 31-32) and only in the mesonephroi and associated ducts at later stages (stages 37 through 46). As the mesonephros develops, renal capacity presumably increases. Thus, tadpoles died only when metacercariae concentrated in the functional portion of the kidney with the most limited renal capacity. As tadpoles aged, they also became less susceptible to infections. On average, 69.5% of cercariae that were exposed to stage 25-26 tadpoles successfully encysted. compared with only 8.4% of cercariae exposed to stage 37-38 tadpoles. Exposures of metamorphic frogs (poststage 46) to cercariae revealed that these individuals can become infected with echinostomes. Collectively, our data highlight the host stage-dependent dynamics of tadpole-echinostome interactions.

Effects of atrazine on embryos, larvae, and adults of anuran amphibians.

We examined the effects of atrazine (0-20 mg/L) on embryos, larvae, and adult anuran amphibian species in the laboratory. Atrazine treatments did not affect hatchability of embryos or 96-h posthatch mortality of larvae of Rana pipiens, Rana sylvatica, or Bufo americanus. Furthermore, atrazine had no effect on swimming speed (measured for R. pipiens only). However, there was a dose-dependent increase in deformed larvae of all three species with increasing atrazine concentration. In adult R. pipiens, atrazine increased buccal and thoracic ventilation, indicating respiratory distress. However, because atrazine had no affect on hemoglobin, this respiratory distress was probably not indicative of reduced oxygen-carrying capacity of the blood. Frogs exposed to the highest atrazine concentration stopped eating immediately after treatment began and did not eat during the 14-d experiment. However, no decreases in mass were measured even for frogs that were not eating, probably because of compensatory fluid gain from edema. Atrazine concentrations found to be deleterious to amphibian embryos and adults are considerably higher than concentrations currently found in surface waters in North America. Therefore, direct toxicity of atrazine is probably not a significant factor in recent amphibian declines.

Ontogeny of B cells expressing IgM in embryonic and larval tissues of the American grass frog, Rana pipiens.

Affinity-purified, fluorochrome-tagged F(ab')(2) antibody fragments specific for heavy (mu) chains of Rana pipiens IgM were prepared from hyperimmune rabbit sera. By using two-color immunofluorescent procedures we observed that (1) the first cells expressing IgM, termed pre-B cells, lack detectable quantities of membrane or surface IgM but contain detectable quantities of cytoplasmic IgM (smu(-)/cmu(+)), (2) sIgM(+) B cells were the second type of IgM containing cell to appear in development, and (3) plasma cells, which contain copious quantities of cIgM, were the final phenotype to appear in the development of B cells expressing IgM. These cells were first observed in the pronephros of the developing urogenital system. Shortly after their appearance in the pronephros, cells in B lineages were observed in the liver. These observations (1) are consistent with recent studies of B lymphopoiesis in the aorta-gonad-mesonephros (AGM) region in endothermic vertebrates, including mice, (2) suggest that there are fundamental ontogenetic and phylogenetic similarities between cells and tissues of developing vertebrate immune systems, and (3) evoke questions concerning the possible function(s) of lymphocytes in developing anurans up to metamorphosis and beyond.

Effect of alpha-tomatine and tomatidine on membrane potential of frog embryos and active transport of ions in frog skin.

alpha-Tomatine, a glycoside in which four carbohydrate residues are attached to the 3-OH group of the aglycone tomatidine, occurs naturally in tomatoes (Lycopersicon esculentum). The glycoalkaloid is reported to be involved in host-plant resistance against phytopathogens and to have a variety of pharmacological and toxicological properties in animals and humans. As part of an effort designed to establish the mechanism of action of glycoalkaloids in cells, frog embryos and frog skin were exposed to varying concentrations of alpha-tomatine and tomatidine. alpha-Tomatine increased the fluorescence-measured membrane permeability of frog embryos by about 600% compared with control values; the corresponding value for tomatidine was about 150%. alpha-Tomatine also diminished sodium-active transport in frog skin by about 16% compared with control values, as estimated from the change in the interstitial short-circuit current. Tomatidine had no effect on frog skin. As these findings complement similar results with glycoalkaloids from potatoes and eggplants, the fundamental mechanism governing their action both against fungi, insects and other phytopathogens and in animal and human cells may be disruption of cell membranes and changes in ion fluxes and interstitial currents of the membranes. The described methodologies should make it possible to define the relative potencies of both adverse and beneficial effects of glycoalkaloids and metabolites in cell membranes without the use of animals.

Germ-layer surface tensions and "tissue affinities" in Rana pipiens gastrulae: quantitative measurements.

The morphogenetic properties causing germ-layer spreading and stratification in amphibian gastrulation were called "tissue affinities" by Holtfreter. The differential adhesion hypothesis (DAH) attributes such liquid-like tissue rearrangements to forces generated by intercellular adhesions within and between the migrating cell populations. This theory predicts that, among the primary germ layers, the cohesiveness of deep ectoderm should be the greatest, that of deep mesoderm should be intermediate, and that of deep endoderm should be the least. Also, the cohesiveness of differentiating neural ectoderm should increase after induction, causing it to internalize and segregate from epidermis. The DAH also explains why the cohesiveness of "liquid" tissues, whose cells are free to rearrange, should be measurable as tissue surface tensions. Using a specially designed tissue surface tensiometer, we demonstrate that (i) aggregates of Rana pipiens deep germ layers do possess liquid-like surface tensions, (ii) their surface tension values lie in precisely the sequence necessary to account for germ-layer stratification in vitro and in vivo, and (iii) the surface tension of deep ectoderm just underlain by the archenteron roof is twice that of not-yet-underlain deep ectoderm. These measurements provide direct, quantitative evidence that the "tissue affinities" governing germ-layer flow during early stages of vertebrate morphogenesis are reflected in tissue surface tensions.

Investigation of developing embryonic morphology using optical coherence tomography.

Improved imaging of morphological changes has the potential of offering new insight into the complex process of embryonic development. Optical coherence tomography (OCT) is a new imaging technique for performing in vivo cross-sectional imaging of architectural morphology by measuring backscattered infrared light. This study investigates the application of OCT for imaging developing structure in Rana pipiens, Xenopus laevis, and Brachydanio rerio. Images are compared to conventional histological baselines. Cross-sectional imaging can be performed and structural morphology identified at greater imaging depths than possible with confocal and light microscopy. Repeated OCT imaging may be performed in vivo in order to track structural changes throughout development.

A fate map for the 32-cell stage of Rana pipiens.

A fate map of the progeny derived from all blastomeres of the 32-cell stage embryo of the leopard frog Rana pipiens has been generated. Embryos presenting regular cleavages were injected into two pairs of blastomeres with fluorescein- and Texas red-lysine dextran. By stage 21, embryos were sectioned and the tissue distribution of labeled clones was determined. The results of 93 clones were pooled to give a fate map which represents the derivation of each tissue from the different blastomeres of the 32-cell embryo. The results show that all blastomeres give rise to multiple tissues and all tissues are derived from at least two, and usually more, pairs of blastomeres. Although there is a general tendency for ectoderm to derive from the animal hemisphere, endoderm from the vegetal hemisphere, and mesoderm from the equatorial region, the boundaries between germ layers are not sharply defined at the 32-cell stage but rather appear as a series of overlapping zones. The fate map of R. pipiens is quite similar to that of Xenopus laevis but differs in some details that are discussed. As in all vertebrates, the R. pipiens fate map is not fully deterministic but nevertheless has predictive value in that tissues are populated by the progeny of the same blastomeres in different embryos.

Superficial cells in the early gastrula of Rana pipiens contribute to mesodermal derivatives.

We have studied the fate of presumptive mesodermal cells in the early Rana pipiens gastrula. We labeled superficial cells of the early gastrula with 125I. We also labeled all cells in a gastrula with rhodamine-lysine-dextran cell lineage tracer and superficial cells with 125I and then grafted small pieces of the marginal zone orthotopically into unlabeled host embryos. Labeled progeny were identified in sectioned embryos at the tail bud stage. The use of double-labeled grafts allowed us to study the relative contributions by superficial and deep cells to different derivatives. We found that the notochord and somite regions are both derived from the superficial and deep portions of circumblastoporal regions. In contrast, pronephros, lateral plate, cardiac anlagen, and blood cells only arise from deep cells in circumblastoporal regions. Such observations indicate that the fate map for R. pipiens is different from that of Xenopus laevis, where mesodermal derivatives appear to be restricted to the deep layers, and from that of Pleurodeles waltl, where all mesodermal derivatives are formed both from superficial and deep layers. We also have shown that from the neurula stage there is substantial mixing between cells due to the ingression of cells in the dorsal region so that superficial labeled cells, initially located in the roof of the archenteron, contribute to ventral regions of both the somites and notochord.