α-ENaC in bullfrog embryo: expression in cement gland, gills and skin.

The epithelial sodium channel (ENaC) is involved in Na(+) responses such as Na(+) absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.

Microcystin-leucine arginine exposure induced intestinal lipid accumulation and MC-LR efflux disorder in Lithobates catesbeianus tadpoles.

Few studies exist on the toxic effects of chronic exposure to microcystins (MCs) on amphibian intestines, and the toxicity mechanisms are unclear. Here, we evaluated the impact of subchronic exposure (30 days) to environmentally realistic microcystin-leucine arginine (MC-LR) concentrations (0 µg/L, 0.5 µg/L and 2 µg/L) on tadpole (Lithobates catesbeianus) intestines by analyzing the histopathological and subcellular microstructural damage, the antioxidative and oxidative enzyme activities, and the transcriptome levels. Histopathological results showed severe damage accompanied by inflammation to the intestinal tissues as the MC-LR exposure concentration increased from 0.5 µg/L to 2 µg/L. RNA-sequencing analysis identified 634 and 1,147 differentially expressed genes (DEGs) after exposure to 0.5 µg/L and 2 µg/L MC-LR, respectively, compared with those of the control group (0 µg/L). Biosynthesis of unsaturated fatty acids and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were upregulated in the intestinal tissues of the exposed groups, with many lipid droplets being observed on transmission electron microscopy, implying that MC-LR may induce lipid accumulation in frog intestines. Moreover, 2 µg/L of MC-LR exposure inhibited the xenobiotic and toxicant biodegradation related to detoxification, implying that the tadpoles' intestinal detoxification ability was weakened after exposure to 2 µg/L MC-LR, which may aggravate intestinal toxicity. Lipid accumulation and toxin efflux disorder may be caused by MC-LR-induced endoplasmic reticular stress. This study presents new evidence that MC-LR harms amphibians by impairing intestinal lipid metabolism and toxin efflux, providing a theoretical basis for evaluating the health risks of MC-LR to amphibians.

C-fin: a cultured frog tadpole tail fin biopsy approach for detection of thyroid hormone-disrupting chemicals.

There is a need for the development of a rapid method for identifying chemicals that disrupt thyroid hormone (TH) action while maintaining complex tissue structure and biological variation. Moreover, no assay to date allows a simultaneous screen of an individual's response to multiple chemicals. A cultured tail fin biopsy or C-fin assay was developed using Rana catesbeiana tadpoles. Multiple tail fin biopsies were taken per tadpole, cultured in serum-free medium, and then each biopsy was exposed to a different treatment condition. The effects of known disruptors of TH action were evaluated in the C-fin assay. Chemical exposure was performed +/- 10 nM 3,3',5-triiodothyronine and real-time quantitative polymerase chain reaction (QPCR) of two TH-responsive transcripts, TH receptor beta (TRbeta) and the Rana larval keratin type I (RLKI), was performed. Within 48 h of exposure to Triac (1-100 nM), roscovitine (0.6-60 microM), or genistein (1-100 microM), perturbations in TH signaling were detected. Tetrabromobisphenol A (TBBPA) (10-1,000 nM) showed no effect. Acetochlor (1-100 nM) elicited a modest effect on the TH-dependent induction of TRbeta transcript. These data reveal that a direct tissue effect may not be critical for TBBPA and acetochlor to disrupt TH action previously observed in intact tadpoles.

Harmful Algal Bloom Toxicity in Lithobates catesbeiana Tadpoles.

Harmful algal blooms (HAB) have become a major health concern worldwide, not just to humans that consume and recreate on contaminated waters, but also to the fauna that inhabit the environments surrounding affected areas. HABs contain heterotrophic bacteria, cyanobacterial lipopolysaccharide, and cyanobacterial toxins such as microcystins, that can cause severe toxicity in many aquatic species as well as bioaccumulation within various organs. Thus, the possibility of trophic transference of this toxin through the food chain has potentially important health implications for other organisms in the related food web. While some species have developed adaptions to attenuate the toxic effects of HAB toxins, there are still numerous species that remain vulnerable, including Lithobates catesbeiana (American bullfrog) tadpoles. In the current study we demonstrate that acute, short-term exposure of tadpoles to HAB toxins containing 1 µg/L (1 nmol/L) of total microcystins for only 7 days results in significant liver and intestinal toxicity within tadpoles. Exposed tadpoles had increased intestinal diameter, decreased intestinal fold heights, and a constant number of intestinal folds, indicating pathological intestinal distension, similar to what is seen in various disease processes, such as toxic megacolon. HAB-toxin-exposed tadpoles also demonstrated hepatocyte hypertrophy with increased hepatocyte binucleation consistent with carcinogenic and oxidative processes within the liver. Both livers and intestines of HAB-toxin-exposed tadpoles demonstrated significant increases in protein carbonylation consistent with oxidative stress and damage. These findings demonstrate that short-term exposure to HAB toxins, including microcystins, can have significant adverse effects in amphibian populations. This acute, short-term toxicity highlights the need to evaluate the influence HAB toxins may have on other vulnerable species within the food web and how those may ultimately also impact human health.

Acute and chronic effects of perfluoroalkyl substance mixtures on larval American bullfrogs (Rana catesbeiana).

Discovery of elevated concentrations of perfluoroalkyl substances (PFAS) in ground and surface waters globally has heightened concern over their potential adverse health effects. The effects of PFAS are known largely from acute toxicity studies of single PFAS compounds in model organisms, while little is understood concerning effects of mixtures on wildlife. To address this gap, we examined the acute and chronic effects of two of the most common PFAS (perfluorooctanesulfonic acid [PFOS] and perfluorooctanoic acid [PFOA]) and their mixtures on survival, growth, and development of American bullfrog (Rana catesbeiana) tadpoles. In 96 h acute toxicity tests, PFOS was 10X more toxic than PFOA and effects of the two chemicals in combination appeared additive. The effects of PFOS, PFOA, and their interaction varied by the sublethal endpoint under consideration in a 72 d exposure. Effects of PFAS on tadpole mass and developmental stage were largely driven by PFOS and there was no evidence of interactions suggesting deviations from additivity. However, for snout-vent length, reductions in length in mixture treatments were greater than expected based on the effects of the two chemicals independently (i.e. non-additivity). Further, effects on snout-vent length in single chemical exposures were only observed with PFOA. Our results highlight the importance of assessing combined effects of PFAS co-occurring in the environment and suggest caution in extrapolating the effects of acute toxicity studies to more environmentally relevant exposures. Future studies examining effects of environmentally relevant mixtures on wildlife will be essential for effective environmental risk assessment and management.

Genotoxic effects of 4-nonylphenol and Cyproterone Acetate on Rana catesbeiana (anura) tadpoles and juveniles.

Genotoxic analyses are commonly used in ecotoxicological studies as early biomarkers to investigate the potential effects of environmental contaminants on biological models. Several pollutants can induce DNA damage and, therefore, counting micronuclei and other nuclear abnormalities are efficient tools to evaluate genotoxicity. Some pollutants such as 4-nonylphenol (NP), a detergent used mainly in industries, and Cyproterone Acetate (CPA), an antiandrogenic medicine, have already shown genotoxic effects on some vertebrates. However, although amphibians are considered bioindicators of environmental quality and their populations are declining worldwide, the effects of these compounds on anurans are not yet known and, therefore, we believe that it is important to investigate such effects on anurans. Since water contamination is one of the ultimate causes of amphibian decline, ecotoxicological studies are important to discuss the appropriate solutions to avoid species extinction. Thus, this study investigates the genotoxic effects on Rana catesbeiana tadpoles and juveniles after being exposed to 1, 10 and 100 µg/L NP and 0.025, 0.25 and 2.5 ng/L CPA, by counting the nuclear abnormalities after exposure. The laboratory experiments lasted 28 days. The experimental conditions were the same except for the water volume since tadpoles and juveniles exhibit different habits at different developmental stages. Compared to juveniles, tadpoles were more susceptible to both compounds as indicated by the increased nuclear abnormalities observed in the highest NP concentration and all tested CPA concentrations. The juveniles, on the other hand, responded only to the two highest CPA concentrations. We concluded that CPA, even at very low concentrations, is extremely harmful to both anuran developmental stages and, particularly, to tadpoles. The significant effects observed on tadpoles is an important outcome of this study since 100 µg/L or higher NP concentrations are frequently detected in the environment.

Rana catesbeiana virus Z (RCV-Z): a novel pathogenic ranavirus.

A virus, designated Rana catesbeiana virus Z (RCV-Z), was isolated from the visceral tissue of moribund tadpoles of the North American bullfrog Rana catesbeiana. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis of viral proteins and sequence analysis of the amino terminal end of the major capsid protein showed that RCV-Z was similar to frog virus 3 (FV3) and other ranaviruses isolated from anurans and fish. However, analysis of restriction fragment profiles following digestion of viral genomic DNA with XbaI and BamHI indicated that RCV-Z was markedly different from FV3. Moreover, in contrast to FV3, RCV-Z contained a full-length copy of the viral homolog of eukaryotic initiation factor 2 alpha (eIF-2alpha). Experimental infection of bullfrog tadpoles with FV3 and RCV-Z demonstrated that RCV-Z was much more pathogenic than FV3, and that prior infection with FV3 protected them from subsequent RCV-Z induced mortality. Collectively, these results suggest that RCV-Z may represent a novel species of ranavirus capable of infecting frogs and that possession of a viral eIF-2alpha homolog (vIF-2alpha) correlates with enhanced virulence.

Aldosterone, corticosterone, and thyroid hormone and their influence on respiratory control development in Lithobates catesbeianus: An in vitro study.

The emergence of air breathing during Lithobates catesbeianus development requires significant changes to the brainstem circuits that generate and regulate breathing; however, the mechanisms responsible for initiating this transformation remain largely unknown. Because amphibian metamorphosis is regulated by hormones such as aldosterone, corticosterone, and thyroid hormone (T3), we tested the hypothesis that exposing the brainstem to these hormones augments the fictive air breathing frequency in pre-metamorphic tadpoles. Brainstems were isolated and were placed either in the recording chamber (acute; 1h+1h recovery) or in a bottle (chronic exposure; 24h) for treatment. Brainstems were exposed to artificial cerebrospinal fluid (aCSF; sham treatment) or one of the following hormones: aldosterone (100nM), corticosterone (100nM), or T3 (100nM). While acute exposure had limited effects on respiratory motor output, chronic incubation with any hormone significantly increased fictive air breathing; the burst frequencies observed following treatment were similar to those observed in adult bullfrogs. We conclude that through their long term effects, hormones regulating metamorphosis can initiate the maturation of the neural circuits that generate and regulate breathing in this species.

Hemoglobin transition in early development stages of the tadpole, Rana catesbeiana.

Five hemoglobin components (a, I, II, III and IV) were isolated from the hemolysates of the tadpole, Rana catesbeiana. Component a was monomeric molecule and the other four were tetrameric molecules. Component I predominating in younger tadpoles was replaced by component II during tadpole development. Electrophoretic and chemical analyses on the constituent globin chains revealed that component a was very similar to the alpha-chains of components I and II, ad that components I and II differed from one another in their beta chains.

Glycoconjugate localization in larval and adult skin of the bullfrog, Rana catesbeiana: a lectin histochemical study.

This study investigates whether or not the distribution of specific glycoconjugates within the skin is related to the regulation of water balance in the aquatic larvae and semiaquatic adults of the bullfrog, Rana catesbeiana. A lectin histochemical study was carried out on paraffin sections of dorsal and ventral skin from tadpoles in representative stages as well as from adult frogs. Sections were stained with the following horseradish peroxidase (HRP)-conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates: UEA 1 for alpha-L-fucose, SBA for N-acetyl-D-galactosamine, WGA for N-acetyl-B-D-glucosamine, and PNA for beta-galactose. Results indicate that lectins serve as markers for specific skin components (e.g., a second ground substance layer within the dermis was revealed by positive UEA 1 staining). Moreover, each lectin has a specific binding pattern that is similar in dorsal and ventral skin; the larval patterns change as the skin undergoes extensive histological and physiological remodeling during metamorphic climax. These findings enhance our understanding of glycoconjugates and their relationship to skin structure and function-in particular, to the regulation of water balance in R. catesbeiana.

Functional morphology of feeding and gill irrigation in the anuran tadpole: electromyography and muscle function in larval Rana catesbeiana.

This study provides the first data on muscle activity patterns during active feeding in a larval anuran. Data regarding muscle function during gill irrigation and hyperexpiration are also provided. Electromyographic and kinematic data were recorded from six mandibular and hyoid muscles in unanesthetized, unrestrained larvae of Rana catesbeiana. Only three (hyoangularis, orbitohyoideus, anterior interhyoideus) of the six muscles examined are active during gill irrigation. Feeding cycles are characterized by the recruitment of three additional muscles: intermandibularis, suspensorioangularis, and levator mandibulae longus superficialis. The latter two contribute, respectively, to wide opening and forceful closing of the mouth during feeding. Hyperexpiration is characterized by a reversal of water flow anteriorly out of the mouth. This hydrodynamic change occurs due to modulation of the timing of firing of the anterior interhyoideus, as well as recruitment of the posterior interhyoideus, which is only active during hyperexpiration. Both regions of the interhyoideus, which are responsible for evacuation of the buccal cavity, are active during the opening phase of hyperexpiration. Kinematically, transitioning from gill irrigation to feeding involves both an overall shortening of the gape cycle and a shift in the relative length of opening phase vs. closing phase. Our results corroborate many of the findings of Gradwell ([1972] Can J Zool 50:501-521) regarding muscle function during gill irrigation and hyperexpiration. Furthermore, we demonstrate that in larval anurans the transition from gill irrigation to feeding involves modulation of gape cycle kinematics, changes in the level of activity of muscles, and recruitment of muscles that are not active during irrigation. In light of new data presented here, a review of muscle function in tadpoles is also provided.

Early development of descending pathways from the brain stem to the spinal cord in Xenopus laevis.

The early development of descending pathways from the brain stem to the spinal cord has been studied in Xenopus laevis tadpoles. The relatively protracted development of this permanently aquatic amphibian as well as its transparency during development make this animal particularly attractive for experimental studies. Between the 5th and 10th myotome the spinal cord was crushed with a thin needle and dry horseradish peroxidase (HRP) crystals were applied. After a survival time of one day the tadpoles were fixed and the brain and spinal cord were stained as a whole according to a modification of the heavy metal intensification of the DAB-reaction, cleared in cedarwood oil and examined as wholemounts. At stage 28 (the neural tube has just closed) the first brain stem neurons projecting to the spinal cord were found in what appear to be the nucleus reticularis inferior and -medius. At this stage of development the first, uncoordinated swimming movements can be observed. At stage 30/31 (the tailbud is visible) both Mauthner cells project to the spinal cord as well as the interstitial nucleus of the fasciculus longitudinalis medialis situated in the mesencephalon. Towards stage 35/36 (the tail is now clearly visible), a more extensive reticulospinal innervation of the spinal cord appears, now including cells of the nucleus reticularis superior. At this stage also the first vestibulospinal and raphespinal projections were found. At stage 43/44 (the tadpoles have now a well-developed tail) the pattern of reticulospinal projections appears to be completed with the presence of labeled neurons in the nucleus reticularis isthmi. From stage 43/44 on, the number of HRP-positive cells is steadily increasing. At stage 47/48, when the hindlimb buds appear, the descending projections to the spinal cord are comparable with the adult situation except for the absence of a rubrospinal and a hypothalamospinal projection. The observations demonstrate that already very early in development reticulospinal fibers and, somewhat later, Mauthner cell axons and vestibulospinal fibers innervate the spinal cord. Furthermore, a caudorostral gradient appears to exist with regard to the development of descending projections to the spinal cord. However, the interstitial nucleus of the fasciculus longitudinalis medialis forms an exception to this rule.

Integrating individual-based indices of contaminant effects. How multiple sublethal effects may ultimately reduce amphibian recruitment from a contaminated breeding site.

Habitat contamination can alter numerous biological processes in individual organisms. Examining multiple individual-level responses in an integrative fashion is necessary to understand how individual health or fitness reflects environmental contamination. Here we provide an example of such an integrated perspective based upon recent studies of an amphibian (the bullfrog, Rana catesbeiana ) that experiences several, disparate changes when larval development occurs in a trace element-contaminated habitat. First, we present an overview of studies focused on specific responses of individuals collected from, or transplanted into, a habitat contaminated by coal combustion residues (CCR). These studies have reported morphological, behavioral, and physiological modifications to individuals chronically interacting with sediments in the CCR-contaminated site. Morphological abnormalities in the oral and tail regions in contaminant-exposed individuals influenced other properties such as grazing, growth, and swimming performance. Behavioral changes in swimming activities and responses to stimuli appear to influence predation risk in the contaminant-exposed population. Significant changes in bioenergetics in the contaminated habitat, evident as abnormally high energetic expenditures for survival (maintenance) costs, may ultimately influence production pathways (growth, energy storage) in individuals. We then present a conceptual model to examine how interactions among the affected systems (morphological, behavioral, physiological) may ultimately bring about more severe effects than would be predicted if the responses were considered in isolation. A complex interplay among simultaneously occurring biological changes emerges in which multiple, sublethal effects ultimately can translate into reductions in larval or juvenile survival, and thus reduced recruitment of juveniles into the population. In systems where individuals are exposed to low concentrations of contaminants for long periods of time, research focused on one or few sublethal responses could substantially underestimate overall effects on individuals. We suggest that investigators adopt a more integrated perspective on contaminant-induced biological changes so that studies of individual-based effects can be better integrated into analyses of mechanisms of population change.

Estudio morfológico, morfométrico e histoquímico de los epitelios de revestimiento y glandular de la lengua de la rana toro, rana catesbeiana / Morphological, morphometrical and histochemical study of the lining and glandular epithelia of the tongue of the tullfrog rana catesbeiana

La superficie dorsal de la lengua de la rana toro, Rana catesbeiana, presenta un epitelio simple cilíndrico, constituido por células caliciformes y raras células ciliadas. El dorso de la lengua posee numerosas papilas filiformes y algunas fungiformes. Las primeras poseen un epitelio simple cilíndrico, con células secretoras, mientras que las segundas poseen en la región apical, un disco sensorial con epitelio estratificado cilíndrico, con células basales, periféricas, glandulares y receptoras. A lo largo del dorso de la lengua existen numerosas glándulas tubulares, que penetran en profundidad, entremezclándose con las fibras musculares. El epitelio glandular es simple cilíndrico, con células secretoras y de sostén. Las primeras son las únicas en la base de la glándula y las segundas solo se encuentran en número escaso en el tercio superior. La superficie ventral de la lengua posee un epitelio estratificado, con células caliciformes y, entre éstas, células ciliadas. La morfometría de las glándulas mostró que son más cortas en la región anterior de la lengua (330 um) que en la región posterior (450 um). Las células secretoras de las glándulas linguales anteriores son menores (1457,7 um3) que en las posteriores (2645,9 um3). Lo mismo ocurre con los núcleos celulares: 130,0 um3 en las glándulas anteriores y 202,3 um3 en las posteriores. Las células secretoras de las glándulas linguales sintetizan producto rico en proteínas y mucopolisacáridos neutros, pudiendo caracterizarse como seromucoso. Las células caliciformes de las superficies dorsal y ventral secretan proteínas y mucopolisacáridos neutros, clasificándose como del tipo G1, mientras que las células de sostén de las glándulas superficiales de las papilas fungiformes secretan moco rico en mucopolisacáridos neutros, sulfomucinas y sialomucinas.

The dorsal surface of the tongue of the bullfrog, Rana catesbeiana, has simple columnar epithelium with a few ciliated cells and goblet cells. The entire surface is covered with numerous filiform papillae and few fungiform. Filiform papillae have a simple columnar epithelium with secretory cells, while the fungiform have a sensory disc on their upper surface the lined by a stratified columnar epithelium with basal, peripheral, glandular and receptor cells. Over the dorsal lingual surface there are numerous winding tubular glands, which penetrate deeply into the muscle of the tongue, mingling with the fibers. The gland epithelium is cylindrical with secretory and supporting cells. The first are absolute on the basis of the gland and the latter are rare in the upper third. The ventral surface of the tongue is lined by a stratified epithelium, with the presence of goblet cells, with ciliated cells among them. Morphometrically, lingual glands varies in length, according to their location: shorter in the anterior region of the tongue (330 um) than in the posterior region (450 um). Secretory cells of the anterior lingual glands are smaller (1457.7 mm3) than the posterior ones (2645.9 um3). The same can be said of the cell nuclei, 130.0 um3 for the anterior glands and 202.3 um3 for the posterior ones. Secretory cells of the lingual glands contain substances rich in protein and neutral mucopolysaccharides, which characterize the seromucous type. Goblet cells of the dorsal and ventral surface epithelia secrete neutral mucopolysaccharides and proteins, and can be characterized as type G1 cells, and the supporting cells of the superficial glands of the fungiform papillae secrete a mucus rich in neutral mucopolysaccharides, sulfomucins and sialomucins.

Nitric oxide changes its role as a modulator of respiratory motor activity during development in the bullfrog (Rana catesbeiana).

Nitric oxide (NO) is a unique chemical messenger that has been shown to play a role in the modulation of breathing in amphibians and other vertebrates. In the post-metamorphic tadpole and adult amphibian brainstem, NO, acting via the neuronal isoform of nitric oxide synthase (nNOS), is excitatory to the generation of lung burst activity. In this study, we examine the modulation of breathing by NO during development of the amphibian brainstem. Isolated brainstem preparations from pre-metamorphic and late-stage post-metamorphic tadpoles (Rana catesbeiana) were used to determine the role of NO in modulating central respiratory neural activity. Respiratory neural activity was monitored with suction electrodes recording extracellular activity of cranial nerve rootlets that innervate respiratory musculature. Brainstems were superfused with an artificial cerebrospinal fluid (aCSF) at 20-22 degrees C containing l-nitroarginine (l-NA; 1-10 mM), a non-selective NOS inhibitor. In pre-metamorphic tadpoles, l-NA increased fictive gill ventilation frequency and amplitude, and increased lung burst frequency. By contrast, l-NA applied to the post-metamorphic tadpole brainstem had little effect on fictive buccal activity, but significantly decreased lung burst frequency and the frequency of lung burst episodes. These data indicate that early in development, NO provides a tonic inhibitory input to gill and lung burst activity, but as development progresses, NO provides an excitatory input to lung ventilation. This changing role for NO coincides with the shift in importance in the different respiratory modes during development in amphibians; that is, pre-metamorphic tadpoles rely predominantly on gill ventilation whereas post-metamorphic tadpoles have lost the gills and are obligate air-breathers primarily using lungs for gas exchange. We hypothesize that NO provides a tonic input to the respiratory CPG during development and this changing role reflects the modulatory influence of NO on inhibitory or excitatory modulators or neurotransmitters involved in the generation of respiratory rhythm.

An analysis of the influence of thyroid hormone on the synthesis of proteins in the tail fin of bullfrog tadpoles.

By incubation of explants of tail fin from tadpoles of Rana catesbeiana in a solution of 35S-methionine for 4 h, newly synthesized proteins were labeled isotopically. After separation by two-dimensional polyacrylamide gel electrophoresis, those proteins were visualized by fluorography. Exposure of explants to culture medium containing thyroxine (T4) (150 nM) increased the incorporation of 35S-methionine into several proteins with 48 h. Effects of T4 on the relative abundance of two of these newly synthesized proteins were detected after 8 h of hormonal treatment. Very similar patterns of newly synthesized proteins were observed when proteins from explants of tail fin removed from tadpoles at metamorphic climax and immediately incubated with 35S-methionine were compared with proteins produced in fin derived from premetamorphic animals. These results are interpreted to indicate that both treatment of explants with T4 and elevation of endogenous levels of thyroid hormones during spontaneous metamorphosis increased the relative rates of synthesis of several identical proteins. The potential involvement of those proteins in early phases of metamorphic action which eventually lead to cell death and resorption is discussed.

Elevated maintenance costs in an anuran (Rana catesbeiana) exposed to a mixture of trace elements during the embryonic and early larval periods.

We investigated the relationship between maintenance costs (standard metabolic rates, measured as O2 consumption at rest) in tadpoles of the bullfrog, Rana catesbeiana, and exposure to contaminants in a coal ash-polluted habitat (characterized by a variety of trace elements). We compared metabolic rates of tadpoles collected from the polluted site with those from an unpolluted reference pond. Tadpoles collected in the polluted site had 40%-97% higher standard metabolic rates than those collected from the reference pond. We also reciprocally transplanted eggs of the bullfrog between the polluted site and another reference pond and compared standard metabolic rates of tadpoles at 25 and 80 d posthatching. Metabolic rates of tadpoles raised in the polluted site were from 39% to 175% higher than those raised in a reference pond, depending on tadpole age and temperature at which metabolic rates were measured. There were no effects of site of origin of the eggs (polluted or unpolluted) on metabolic rates. Survival to hatching did not differ between sites, although survival to the end of the experiment (80 d posthatching) was lower in the polluted area than in the reference site. Surviving tadpoles were larger in wet body mass in the polluted site than in the reference pond, possibly due to lower survival in the former, but there was no relationship between survival and metabolic rate. It is clear that some feature of the polluted habitat was responsible for causing substantial elevation of standard metabolic rates of tadpoles. We hypothesize that the mixture of trace elements present in sediment and water in the polluted site was responsible for the observed physiological differences.

Embryonic and larval hemoglobins during the early development of the bullfrog, Rana catesbeiana.

The main hemoglobin (Hb) found in Shumway (embryonic) stage 25 bullfrogs is that which we have designated Td-4. The other major tadpole Hbs (Td-1, 2, and 3) predominate during Taylor and Kollros (larval) stages I-XVIII. We propose that Td-4 is an embryonic Hb, whereas Td-1, 2, and 3 are larval (fetal-like) Hbs. Embryonic Hb Td-4 continues to be synthesized during the larval stages. During the larval period, the average peripheral blood Hb profile changes very little with morphological stage or general growth. However, there is great heterogeneity in the embryonic:larval Hb ratio among individual tadpoles of a given stage or weight, apparently due to differential Hb and red cell production by the two active erythropoietic sites, mesonephric kidneys (Td-4), and liver (Td-1, 2, 3).