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Polylactic acid microplastics (PLA-MPs), biobased plastics made from renewable resources, are considered to be a potential solution for alleviating the pollution pressure of plastics; however, the potential environmental risks of PLA-MPs must be further evaluated. In this study, the effects of PLA-MPs on the tadpoles of Pelophylax nigromaculatus were investigated by designing different PLA-MP exposure experiments. We found that PLA-MPs negatively affected the survival, growth and development of tadpoles. In addition, in open field tests, PLA-MPs also reduced tadpole locomotion while resulting in more repetitive searching behavior within a restricted area. This effect was more pronounced at higher concentrations of PLA-MPs (20 mg/mL) and in combination with the heavy metal Cd 2+ . In the tank tests, PLA-MPs increased tadpole aggregation, and their combined effect with Cd 2+ resulted in a tendency for tadpole aggregation to increase and then decrease, with the distribution tending to favor aggregation in edge regions. PLA-MPs also strongly inhibited the spatiotemporal exploratory activities of tadpoles in the tanks. This study provides a more detailed investigation of the behavioral effects of PLA-MPs on tadpoles and provides a theoretical basis for subsequent ecotoxicological studies of PLA-MPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Vertebrate freeze tolerance requires multiple adaptations underpinned by specialized biochemistry. Freezing of extracellular water leads to intracellular dehydration as pure water is incorporated into growing ice crystals and also results in the cessation of blood supply to tissues, creating an anoxic cellular environment. Hence, the freeze tolerant wood frog, Rana sylvatica, must endure both dehydration and anoxia stresses in addition to freezing. The metabolic responses to freezing, dehydration and anoxia involve both protein/enzyme adaptations and the production of metabolites with metabolic or osmotic functions, particularly glucose and urea. The present study uses a phosphoproteome analysis to examine the differential phosphorylation of metabolic enzymes involved in the production of these two metabolites in liver in response to freezing, anoxia, or dehydration exposures. Our results show stress-specific responses in the abundance of phosphopeptides retrieved from nine glycolytic enzymes and three urea cycle enzymes in liver of wood frogs exposed to 24 h freezing, 24 h anoxia, or dehydration to 40% of total body water loss, as compared with 5 °C acclimated controls. Data show changes in the abundance of phosphopeptides belonging to glycogen phosphorylase (GP) and phosphofructokinase 2 (PFK2) that were consistent with differential phosphorylation control of glycogenolysis and a metabolic block at PFK1 that can facilitate glucose synthesis as the cryoprotectant during freezing. Anoxia-exposed animals showed similar changes in GP phosphorylation but no changes to PFK2; changes that would facilitate mobilization of glycogen as a fermentative fuel for anaerobic glycolysis. Urea is commonly produced as a compatible osmolyte in response to amphibian dehydration. Selected urea cycle enzymes showed small changes in phosphopeptide abundance in response to dehydration, but during freezing differential phosphorylation occurred that may facilitate this ATP expensive process when energy resources are sparse. These results add to the growing body of literature demonstrating the importance and efficiency of reversible protein phosphorylation as a regulatory mechanism allowing animals to rapidly respond to environmental stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Predictions of the International Panel for Climate Changes on sea level rise foresee that the number of coastal regions impacted with salinization will increase in a near future. The present work intended to evaluate the sensitivity to salinization of two freshwater vertebrate species (the frog Pelophylax perezi and the fish Lepomis gibbosus) and their ability to acclimate to this stressor. For this, three specific objectives were targeted: (i) to assess if NaCl may be used as a safe surrogate for risk assessment of seawater (SW) intrusion for freshwater vertebrates; (ii) to evaluate the sensitivity of two freshwater vertebrate models to increased salinity (both due to NaCl or SW); (iii) to determine the capacity of the studied species to acclimate to low levels of salinization. To assess specific objectives (i) and (ii), organisms were exposed to serial concentrations of NaCl or SW dilutions. To assess the capacity of acclimation of both species to salinization, organisms were exposed to low serial concentrations of NaCl during the embryonic development or for a period of two months, respectively, and their sensitivity to NaCl was re-evaluated after this period. Results showed that fish juveniles were more tolerant (96-h LC 50 of 21.3 mS cm -1 for NaCl and 23.6 mS cm -1 for SW) than frog embryos (96-h LC 50 of 10.7 mS cm -1 for NaCl and 10.7 mS cm -1 for SW) and tadpoles (96-h LC 50 of 19.4 mS cm -1 for NaCl and 8.72 mS cm -1 for SW). The fish was able to cope with conductivities of almost one third of SW conductivity, while effect conductivities computed for the amphibian were much lower than SW conductivity (≈ 52 mS cm -1 ). The two-fold difference between the sensitivity of the two tested species reinforces the idea that ecological risk assessment for amphibians based on fish toxicity data may underestimate the risk to the former. Acclimation to low levels of salinity caused an increase in tolerance to salinization in P. perezi tadpoles but not in fish., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The amphibian temporins, amongst the smallest antimicrobial peptides (AMPs), are α-helical, amphipathic, hydrophobic and cationic and are active mainly against Gram-positive bacteria but inactive or weakly active against Gram-negative bacteria. Here, we report two novel members of the temporin family, named temporin-1Ee (FLPVIAGVLSKLFamide) and temporin-1Re (FLPGLLAGLLamide), whose biosynthetic precursor structures were deduced from clones obtained from skin secretion-derived cDNA libraries of the European edible frog, Pelophylax kl. esculentus, by 'shotgun' cloning. Deduction of the molecular masses of each mature processed peptide from respective cloned cDNAs was used to locate respective molecules in reverse-phase HPLC fractions of secretion. Temporin-1Ee (MIC = 10 μM) and temporin-1Re (MIC = 60 μM) were both found to be active against Gram-positive Staphylococcus aureus, but retaining a weak haemolytic activity. To our knowledge, Single-site substitutions can dramatically change the spectrum of activity of a given temporin. Compared with temporine-1Ec, just one chemically-conservative substitution (Val8 instead of Leu8), temporin-1Ee bearing a net charge of +2 displays broad-spectrum activity with particularly high potency on the clinically relevant Gram-negative strains, Escherichia coli (MIC = 40 μM). These factors bode well for translating temporins to be potential drug candidates for the design of new and valuable anti-infective agents., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The present study examined the adverse effects of fluoride exposure on embryos and larvae of Rana chensinensis. Survival, morphological abnormalities, growth and development, time to metamorphosis and size at metamorphic climax of R. chensinensis were examined. Our results showed that embryos malformation occurred in all fluoride treatments. Morphological abnormalities of embryos are characterized by axial flexures, the extrusion of fin axis, edema, and ruffled dorsal and ventral fin. Additionally, 4.1mg F(-)/L and above could significantly inhibit embryos growth and development. On day 15, total length and weight of tadpole were significantly lower in 19.6 and 42.4 mg F(-)/L treatments compared to control. However, significant reductions in total length and weight were observed only at 42.4 mg F(-)/L on day 30. Moreover, significant metamorphic delay and decrease in the size at metamorphic climax were found in larvae exposed to 42.4 mg F(-)/L. Taken together, embryos of R. chensinensis are more vulnerable to fluoride exposure than their tadpoles. Our results suggested that the presence of high concentrations fluoride might increase mortality risk and a reduction in juvenile recruitment in the field by increasing embryos malformation, delaying metamorphosis and decreasing size at metamorphosis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Acute lethal and sublethal toxicity of the imidazolinone imazethapyr (IMZT)-based commercial formulation herbicide Pivot H® (10.59% IMZT) was evaluated on Hypsiboas pulchellus tadpoles. Whereas mortality was used as the end point for lethality, frequency of micronuclei (MNs) and other nuclear abnormalities as well as DNA single-strand breaks evaluated by the single cell gel electrophoresis assay were employed to test genotoxicity. Behavioral, growth, developmental, and morphological abnormalities were also employed as sublethal end points. Mortality studies revealed equivalent LC50 (96h) values of 1.49mg/L (confidence limit, 1.09-1.63) and 1.55mg/L (confidence limit, 1.51-1.60) IMZT for Gosner stage (GS) 25 and GS36, respectively. Behavioral changes, i.e., irregular swimming and immobility, as well as a decreased frequency of keratodonts were observed. The herbicide increased the frequency of MNs in circulating erythrocytes of tadpoles exposed for 48h to the highest concentration assayed (1.17mg/L). However, regardless of the concentration of the herbicide assayed, an enhanced frequency of MNs was observed in tadpoles exposed for 96h. The herbicide was able to induce other nuclear abnormalities, i.e., blebbed and notched nuclei, only when tadpoles were exposed for 96h. In addition, we observed that exposure to IMZT within the 0.39-1.17mg/L range increased the genetic damage index in treatments lasting for both 48 and 96h. This study represents the first evidence of acute lethal and sublethal effects exerted by IMZT on amphibians. Finally, our findings highlight the properties of this herbicide that jeopardize nontarget living species exposed to IMZT., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Lead (Pb) is a common heavy metal in the natural environment, but its concentration has been increasing alongside widespread industrial and agricultural development in China. The dark-spotted frog Pelophylax (formerly Rana) nigromaculata (Anura: Ranidae) is distributed across East Asia and inhabits anthropogenic habitats such as farmland. Here, P. nigromaculata tadpoles (Gosner stage 19-46) were exposed to Pb at different concentrations (0, 40, 80, 160, 320, 640 and 1280µg/L) and Pb-induced survival, metamorphosis time, development, malformations, mobility and gonad structure were monitored. The results showed that above the threshold concentration of Pb, adverse effects were obvious. As the concentration of Pb increased, the adverse effects on different traits followed different patterns: the effects on hindlimb length, survival rate, metamorphosis rate, total malformation rate, swimming speed and jumping speed largely exhibited a linear pattern; the effects on snout-vent length, body mass and forelimb length largely exhibited a bimodal pattern. Sex ratio and gonadal histology were not affected by Pb, suggesting that Pb is not strongly estrogenic in P. nigromaculata., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The majority of studies on the toxicity of glyphosate-based herbicides to amphibians have focused on larval life stages exposed in aqueous media. However, adult and juvenile amphibians may also be exposed directly or indirectly to herbicides. The potential for such exposures is of particular interest in the littoral zone surrounding wetlands as this is preferred habitat for many amphibian species. Moreover, it may be argued that potential herbicide effects on juvenile or adult amphibians could have comparatively greater influence on overall recruitment, reproductive potential and thus stability of local populations than effects on larvae. In this experiment, juvenile green frogs (Lithobates clamitans) were exposed to two concentrations (2.16 and 4.27 kg a.e./ha) of a glyphosate-based herbicide formulation (VisionMax®), which were based on typical application scenarios in Canadian forestry. The experimental design employed frogs inhabiting in situ enclosures established at the edge of small naturalized wetlands that were split in half using an impermeable plastic barrier. When analyzed using nominal target application rates, exposure to the glyphosate-based herbicide had no significant effect on survival, body condition, liver somatic index or the observed rate of Batrachochytrium dendrobatidis infection. However, there were marginal trends in both ANOVA analysis and post-hoc regressions regarding B. dendrobatidis infection rates and liver somatic index in relation to measured exposure estimates. Results from this study highlight the importance of field research and the need to include multiple endpoints when examining potential effects of a contaminant on non-target organisms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Despite numerous mechanistic studies on physiological responses supporting freeze tolerance in anurans, few have addressed the evolutionary significance of this trait. We thus investigated the phylogenetic relationships among anuran species whose freeze tolerance has been assessed and in combination with new data on freezing tolerance of two closely related species of the European brown frogs (Rana temporaria and Rana dalmatina). The species we studied exhibited short survival times in frozen state (around 8 h for both species). Phylogenetic analysis suggests that freeze tolerance evolved at least two times among Ranidae and one or two times among Hylidae and never in Bufonidae. Furthermore, in order to assess the timing of divergence of this character we used a relaxed molecular clock created, and found that the most recent separation between a freeze tolerant species and a freeze intolerant species dates from 15.9 +/- 7.6 Myr (Rana arvalis and R. temporaria). The comparison between these two species thus represents the best current model to understand freeze tolerance evolution. Addressing the evolution of this trait with such large-scale approaches will not only improve our understanding of cold hardiness strategies, but might also create a framework guiding future comparative studies.

Survival in the frozen state depends on biochemical adaptations that deal with multiple stresses on cells including long-term ischaemia and tissue dehydration. We investigated whether the AMP-activated protein kinase (AMPK) could play a regulatory role in the metabolic re-sculpting that occurs during freezing. AMPK activity and the phosphorylation state of translation factors were measured in liver and skeletal muscle of wood frogs (Rana sylvatica) subjected to anoxia, dehydration, freezing, and thawing after freezing. AMPK activity was increased 2-fold in livers of frozen frogs compared with the controls whereas in skeletal muscle, AMPK activity increased 2.5-, 4.5- and 3-fold in dehydrated, frozen and frozen/thawed animals, respectively. Immunoblotting with phospho-specific antibodies revealed an increase in the phosphorylation state of eukaryotic elongation factor-2 at the inactivating Thr56 site in livers from frozen frogs and in skeletal muscles of anoxic frogs. No change in phosphorylation state of eukaryotic initiation factor-2alpha at the inactivating Ser51 site was seen in the tissues under any of the stress conditions. Surprisingly, ribosomal protein S6 phosphorylation was increased 2-fold in livers from frozen frogs and 10-fold in skeletal muscle from frozen/thawed animals. However, no change in translation capacity was detected in cell-free translation assays with skeletal muscle extracts under any of the experimental conditions. The changes in phosphorylation state of translation factors are discussed in relation to the control of protein synthesis and stress-induced AMPK activation.

Anuran tadpoles (Rana pirica) are induced to develop a higher tail and a bulgy body as predator-specific morphological responses when they are exposed to predatory larval salamanders. Subtractive hybridization was performed using induced tadpole body skin and normal tadpoles' body skin. A total of 196 clones showed higher expression, and 104 clones showed lower expression, when they formed bulgy bodies. In the subtraction, carboxypeptidase B, trypsinogen, elastase I, fibrinogen, elastase II, triacyl-glycerol lipase, and alpha1-antitrypsin genes showed lower expression. In contrast, RT-like protein, bullous pemphigoid antigen, phosphoserine aminotransferase, uromodulin, tetranectin, chaperonin-like protein, zinc finger protein, osteonectin, aldehyde dehydrogenase, Sec 23A protein, and ribosomal protein showed higher gene expression. Microarray analysis was also performed using this subtracted cDNA (nine replicates). Results of the microarray data essentially corresponded with those of the subtraction data, and the degree of the suppressed genes was much stronger than that of the expressed genes. Carboxypeptidase B showed the strongest suppression, and its inhibition range was from 1/100 to 3/100 compared with that of control body skin. Strong suppression was also observed with trypsinogen, elastase I, fibrinogen, and elastase II as well. These results can be interpreted as increases of fibrinolysis by strong depression of both carboxypeptidase B and other genes simultaneously, resulting in the retention of blood vessels and facilitating the circulation of blood. Expression was observed in phosphoserine aminotransferase, aldehyde dehydrogenase, RT-related protein, chaperonin-like protein, tetranectin, bullous pemphigoid antigen, uromodulin, and Sec 23A protein. They were significantly (p<0.05) increased and were at least 1.5 times greater compared with the control. From the appearance, it seems that the bulgy shaped body is highly connecting to the bullous pemphigoid (BP) antigen that causes the skin blistering disorder, and tetranectin and uromodulin may be related to the extracell matrix through myogenesis, protein secretion, and ion transport, respectively. Since the RT-related protein gene derived from retrotransposon (L1) is known to disrupt mammalian transcriptomes, retrotransposon may be involved with phenotypic plasticity for morphological defense by Rana prica against predator threat.

Cypermethrin is widely used as an insecticide on animals and in agriculture, the home, and the garden. The effect of inhaled cypermethrin on the cardiac mechanics, electrophysiology, and ultrastructure in frogs was investigated in this study. Four groups received 100 microL of cypermethrin via inhalation for different exposure times, and one group was used as a control. Electrical and mechanical activities of the heart were recorded, and heart samples were examined at light and transmission electron microscopic levels for all groups. The atrial and ventricular contractile forces on the mechanogram, the amplitude of the P wave and the QRS complex on electrocardiogram, and the heart rate were significantly decreased in cypermethrin-inhalated frogs. The total duration of contraction was prolonged in the study groups. Ultrastructurally, dilatation in smooth endoplasmic reticulum cisterns, a decrease in the number of mitochondria, disorganization in the myofibrils of myocytes, and necrotic changes in endothelial cells were observed. These results suggest that cypermethrin has cardiotoxic effects that increase with exposure time.

To ascertain whether styrene monomer and trimer induce an estrogen-like effect, all-male tadpoles of Rana rugosa were exposed to dilute solutions of styrene monomer and trimer (2,4,6-tripfenyl-1-hexene) at concentrations of 0.1, 1, or 10 microM during days 19-23 after fertilization, which is the critical period of gonadal sex differentiation. As positive and vehicle controls, tadpoles were exposed to dilute solutions of estradiol-17beta at concentrations of 0.01, 0.1, or 1 microM and 0.01% ethanol, respectively, during the same period. The influence was estimated by examining the inner structure of the gonads of 40-day-old tadpoles. All the gonads of tadpoles in the vehicle and untreated controls showed histological characteristics of testes, whereas all those of tadpoles treated with 1 microM estradiol-17beta showed a structure typical for ovaries, having an ovarian sac and many meiotic germ cells. Of the genetically male tadpoles treated with styrene monomer and trimer, 97 and 96%, respectively, showed normal testicular structure of the gonads. In contrast, the remaining tadpoles had gonads showing coexistence of testicular and ovarian structure, irrespective of the strength of styrene concentration. These findings suggest that the styrene monomer and trimer induced a weak estrogen-like effect on the pathways of testicular differentiation in genetically male tadpoles.

Comparison was made of abiotic conditions, amphibian reproductive outputs, and the survival of embryonic and larval amphibians in wastewater effluent-irrigated and natural temporary ponds in an area in Centre County, Pennsylvania that has been spray-irrigated with secondarily treated, chlorinated wastewater effluent from The Pennsylvania State University for approximately 14 years. Three species of temporary pond-breeding amphibians were studied: wood frogs (Rana sylvatica LeConte), Jefferson salamanders (Ambystoma jeffersonianum Green), and spotted salamanders (A. maculatum Gravenhorst). Comparisons of physico-chemical parameters in 10 wastewater-irrigated and 10 natural temporary ponds over 19 weeks in 1997 indicated that wastewater -irrigated ponds had significantly higher median conductance, pH, ¿Na, ¿K, ¿Ca, ¿Mg, and ¿N-NO(3) and lower ¿dissolved oxygen. Many of the wastewater-irrigated ponds supported large mats of duckweed (Lemna spp.) that completely blanketed the pond's surface by mid-May. There were significantly fewer egg masses of all three species in wastewater-irrigated ponds than in natural ponds in both 1997 and 1998. In situ egg hatching success and larval survival (over a 6-day period) of all species was lower in wastewater-irrigated ponds than in natural ponds. Cumulatively, these studies suggest that wastewater effluent irrigation may impact amphibian populations by reducing the survival of amphibian eggs and larvae., (Copyright 2000 Academic Press.)

Mammalian cloning technologies originally developed as methods of testing hypotheses about the mechanisms of cell differentiation. Embryo splitting procedures demonstrated that each of the cells in the early embryo are capable of developing into a complete new individual. Nuclear transplantation technologies have shown that loss of genetic sequences or even irreversible repression of gene function are also not mechanisms of cell differentiation. Therefore, both of these methods can be used for producing genetically identical animals. Nuclear transplantation has the advantage of being able to produce unlimited numbers of identical offspring. Highly efficient procedures have been developed for nuclear transplantation in mammals and several important characteristics of donor cells have been described. Unfortunately, the efficiency of producing cloned offspring is still low and many factors affecting the development of nuclear transfer embryos to term remain to be investigated. The tremendous potential of the technology for use in agriculture and medicine, however, will ensure that these problems are addressed and solved.

The ocular lenses of the diurno-nocturnal gecko Lepidodactylus lugubris contain a monomeric 38-kDa protein at a level of 20 to 22% of the total water-soluble protein. Amino acid sequences of peptides from this protein are most similar--up to 72% identity--to mammalian aldose reductase, an NADPH-dependent reductase which normally occurs at house-keeping levels in the eye lens, and which is involved in the development of diabetic cataract. The sequences show 56% identity with rho-crystallin from lenses of the frog genus Rana. It is concluded that different genes from the same superfamily of NADPH-dependent reductases have been recruited to become highly expressed as lens proteins in at least two different evolutionary lineages. To reflect the relationship with frog rho-crystallin, the gecko lens protein is designated as rho B-crystallin. As for frog rho-crystallin, no enzymatic activity could be established for rho B-crystallin in the gecko lens. Up to now, rho B-crystallin has not been detected in lenses of other reptiles or amphibians.

Ice-nucleating active (INA) bacteria were isolated from the gut of field-collected freeze-tolerant wood frogs (Rana sylvatica) collected in winter. Thirteen strains of Pseudomonas fluorescens, four strains of Pseudomonas putida, and two strains of Enterobacter agglomerans had ice-nucleating activity. Each of the INA pseudomonad strains was psychrophilic. P. putida strains were differentiated from P. fluorescens strains by gelatinase, lecithinase, and lipase production. The maximum nucleation temperatures (Tmax) of aqueous suspensions (10(9) bacteria/ml) of the four INA P. putida strains ranged from -1.6 to -3.0 degrees C, which places this INA species among the most potent known biological nucleators. Ingestion of INA P. putida isolated from R. sylvatica by another freeze-tolerant frog. Pseudacris crucifer, decreased the capacity of this frog to supercool and remain unfrozen at -2 degrees C. This is the first report of INA bacteria isolated from a vertebrate, and suggests that, as part of the gut flora in some posthibernation freeze-tolerant wood frogs, these bacteria may play a role in enhancing winter survival by promoting ice nucleation at high subzero temperatures (ca. -2 degrees C).

Hydrin 2 (vasotocinyl-Gly) is an intermediate in pro-vasotocin processing found, along with vasotocin, only in the neurohypophysis of anuran amphibians. It increases cutaneous water permeability in the frog and is likely involved in neuroendocrine control of osmoregulation. The relative amounts of vasotocin and hydrin 2 stored in neurohypophysis have been measured on the one hand in amphibian species known not to adapt in dry areas, on the other hand in two species, Bufo regularis (Africa) and Bufo viridis (Near-East) able to survive in an arid environment. In the first group, the proportions of the two peptides are approximately equal whereas in the two toads the molar ratio hydrin 2 to vasotocin reaches 2. The ratio does not appear to vary significantly when these toads are either submitted to dehydration or placed in saline solutions. Predominance of hydrin 2 suggests an adaptive decrease of the activity of the alpha-amidating enzymatic system involved in the conversion of vasotocinyl-Gly into mature amidated vasotocin.

The lungs of the tree frog Hyla arborea and the toad Bufo bufo were fixed in glutaraldehyde with the addition of picric acid or tannic acid to visualize tubular myelin (TM), one of the forms of pulmonary surfactant. The lungs of both Hyla and Bufo are complex in structure and possess one type of pneumocytes containing numerous lamellar bodies (LBs). The alveolar lining layer in the lung of Hyla includes numerous LBs at different stages of transformation into a monolayer film, but the lattice is sparse, delicate and unstable. The structure of smooth membranes in the alveolar layer is not clearly visible and their thickness varies from 4.0 to 7.2 nm. In the lung of Bufo TM occurs commonly in the alveolar lining layer. Membranes of LBs are smooth particle-free bilayers of 8.1 to 12.0 nm thick. During transformation into TM, the membranes become covered by fuzzy coat and irregularly spaced rod-like particles of 8 to 13 nm in diameter. I suppose that these particles facilitate the maintenance of TM structures.

A static bioassay is performed for measuring the short-term (96 h) toxicity of endosulfan to juvenile catfish Clarias batrachus, insect nymph Enallagma sp. and frog tadpoles of Rana tigrina. Median Tolerance Limit, Slope Function, Confidence Limit, and Presumable Harmless Concentration were computed. The results showed that frog tadpoles are more susceptible to endosulfan than insect nymph and catfish. Morphological changes and behavioural alterations were evaluated as symptoms of endosulfan toxicity.

The cells of the frog pars intermedia synthesize a 36 000 (36K) protein called proopiomelanocortin (POMC). After [3H]glucosamine incorporation, separation of newly synthesized products by SDS-polyacrylamide gel electrophoresis showed that this 36K protein was glycosylated. Tryptic mapping revealed only one site of glycosylation and showed that the carbohydrate side-chain was located in the N-terminal region of POMC. The 36K protein was not released by the melanotrophs, but it generated, through specific intracellular proteolytic cleavage, a number of smaller peptides which were subsequently released. These peptides were identified by various methods including selective amino-acid incorporation, HPLC purification, acid-urea gel electrophoresis, tryptic and chymotryptic mapping, assay of melanotropic activity, radioimmunoassays and immunoprecipitations. Some of the newly synthesized N-terminal (18K) fragment of the POMC was secreted intact while a portion of it was further processed, via an intermediate peptide, to give mature gamma-MSH. All three of these peptides were glycosylated. In addition, the mature peptide (gamma-MSH) exhibited a low but significant melanotropic activity. The C-terminal portion of the prohormone was very rapidly processed to give des N alpha-acetyl alpha-MSH, corticotropin-like-intermediate lobe peptide (CLIP) and beta-endorphin. Authentic alpha-MSH was always absent in cellular extracts: acetylation to give rise to alpha-MSH was a late enzymatic process strictly linked to hormonal release. Since acetylation of alpha-MSH is required for full biological activity of this peptide, it is possible to conceive that this later step could be under neuroendocrine control. Using the perifusion technique we have been able to show the complexity of the control mechanisms regulating amphibian melanotrophs. It is generally accepted that the aminergic innervation of the intermediate lobe of the pituitary is involved in the hypothalamic control of melanotropin release. We have demonstrated that, in amphibians, dopamine inhibits alpha-MSH secretion through D2-type dopaminergic receptors whereas norepinephrine and (or) epinephrine stimulate alpha-MSH secretion via beta-adrenergic receptors. The existence of peptidergic fibers within parenchymal cells of the pars intermedia has been demonstrated. Evidence for TRH-containing fibers has been obtained by immunohistochemistry. Using a specific radioimmunoassay for TRH, we have confirmed the presence of TRH in the neurointermediate lobe of the frog. We have shown that TRH is a powerful MSH-releasing factor in these animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Seven species of amphibian larvae were exposed to toxaphene and endrin in a continuous-flow dosing system to determine differences in sensitivity to the two compounds, EC50 and LC50 estimates varied from those for Rana sphenocephala by no more than one order of magnitude when calculated on the basis of intended concentrations. Removal of pesticides from water by the test animals was significant and it makes interpretation of results difficult. Continuous-flow toxicity tests conflict with the adaptations of amphibian larvae for static water; use of such tests for amphibians requires further evaluation.