Apoptosis in the digestive tract of herbivorous Rana pipiens larvae and carnivorous Ceratophrys ornata larvae: An immunohistochemical study

The lifespan of herbivorous Rana pipiens larvae is ～3 months, while that of carnivorous Ceratophrys ornata larvae is only about 2 weeks. During metamorphic climax, the larval gut shortens dramatically, especially in R. pipiens, and its luminal epithelium is replaced by adult‐type epithelium. To determine when programmed cell death occurs during the metamorphic restructuring of the gut, we prepared cross‐sections of the stomach, small intestine, and large intestine from representative larval stages and from juvenile frogs of both species. The sections were incubated with monoclonal antibody against active caspase‐3, one of the key enzymes in the apoptotic cascade. We observed apoptosis in some luminal epithelial cells in each of the three regions of the larval gastrointestinal tract of both species. However, apoptotic cells appeared earlier in larval stages of R. pipiens than C. ornata and few were seen in juvenile frogs of either species. The results demonstrate the occurrence of apoptosis in the metamorphic remodeling of the gut of both R. pipiens larvae and C. ornata larvae. J. Morphol., 2011. © 2011 Wiley Periodicals, Inc.     

Apoptosis in larval and frog skin of Rana pipiens,R. catesbeiana,and Ceratophrys ornata

Apoptosis (programmed cell death) occurs during normal development of anurans in organs such as gills, gut, and tail. For example, apoptotic cells have been reported in the luminal epithelium along the length of the digestive tract of both larvae and frogs; however, timing of the peak number of such cells varies in different species. The purpose of the present study was to ascertain whether apoptosis also varies by species during metamorphic restructuring of the skin (as larval epithelium is replaced by adult epidermis). To determine this, cross‐sections of dorsal skin from representative larval stages and frogs of Rana pipiens, R. catesbeiana, and Ceratophrys ornata were incubated with monoclonal antibody against active caspase‐3, one of the main enzymes in the apoptotic cascade. We observed apoptotic cells in the epidermis of the skin of the three species and found that such cells were more numerous in larval stages than in frogs and more abundant in the two ranid species than in C. ornata. These results contribute to our understanding of metamorphic changes in anuran skin. J. Morphol. 275:51–56, 2014. © 2013 Wiley Periodicals, Inc.     

Surface ultrastructure of the cornea and adjacent epidermis during metamorphosis of Rana pipiens A scanning electron microscopic study

The external surface of the cornea and adjacent epidermis of larvae in representative developmental stages and of adult frogs, Rana pipiens, was studied by scanning electron microscopy. Surface cells are polygonal, usually hexagonal, in outline and covered with microprojections. During larval development prior to metamorphic stages, neither eyelids nor Harderian glands have developed; microprojections on the corneal surface are high and branched, and cell boundaries are elevated. On the anterior portion of the cornea and on the epidermis near the eye, the surface pattern is less dense, and ciliated cells are present. During metamorphic stages, corneal cell boundaries become less prominent and the pattern of microprojections more variable and markedly different from that of larvae of earlier stages. Corneal cells have a spongy appearance, are covered by a coating material, or are characterized as light or dark based on their brightness and surface texture. As eyelids develop in metamorphic stages XX–XXI, the numbers of ciliated cells increase dramatically, both on the corneal surface and on the edges of the developing lids. In later metamorphic stages XXII–XXV, lids and Harderian glands become well-developed, and cilia are no longer observed. The adjacent epidermal surface becomes devoid of cilia but perforated by openings of cutaneous glands. Its spongy appearance is similar to that of both the cornea and neighboring epidermis of the mature frog. Changes in corneal surface features are probably metamorphic events associated with development of lids and Harderian glands and a shift from an aqueous to an air environment.     

The genetic basis of altitudinal variation in the wood frog Rana sylvatica II. An experimental analysis of larval development

Summary The variation in larval developmental patterns in the wood frog, Rana sylvatica, along an elevation gradient of 1,000 m was experimentally studied. Larval populations at high elevation ponds had lower growth rates, developmental rates and were larger at all stages (including metamorphic climax) than larval populations developing in low elevation ponds. There was considerable variation among ponds within each elevation in both the length of the larval period and size at metamorphic climax. Reciprocal transplant experiments and controlled laboratory experiments revealed that most of the observed variation between high and low elevation populations could be explained by the effects of temperature induction during ontogeny. Significant genetic differences in growth rates and non-genetic maternal effects on developmental rates between larvae of mountain origin and lowland origin were also demonstrated. Selection in both environments has acted to minimize the prevailing environmental effect of pond temperature on developmental rates, but has accentuated the prevailing environmental effects on larval body size. As a consequence mountain larvae were capable of completing metamorphosis sooner and at a larger size in all environments than lowland larvae.     

DNA SYNTHESIS AND CELL TURNOVER IN RANA PIPIENS TADPOLE LIVER DURING SPONTANEOUS METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of ³H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented.     

Metamorphic changes in localization of sugars in skin of the leopard frog,Rana pipiens

A lectin histochemical study was carried out to determine the distribution of specific sugars in glycoconjugates within an important osmoregulatory organ, amphibian skin. Paraffin sections were made of Rana pipiens skin from dorsal and ventral regions of aquatic larvae in representative developmental stages as well as from several body regions of semiaquatic adult frogs. Sections were incubated with horseradish peroxidase (HRP)‐conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates. Such sites were visualized by DAB‐H₂O₂. The following HRP‐lectins were used: UEA‐1 for α‐L ‐fucose, SBA for N‐acetyl‐D ‐galactosamine, WGA for N‐acetyl‐β‐D ‐glucosamine, PNA for β‐galactose, and Con A for α‐mannose. We found that lectin binding patterns in larvae change during metamorphic climax as the skin undergoes extensive histological remodeling; this results in adult skin with staining patterns that are specific for each lectin and are similar in all body regions. Such findings in R. pipiens provide additional insight into the localization of molecules involved in osmoregulation in amphibian skin. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Histology and lectin-binding patterns in the digestive tract of the carnivorous larvae of the anuran, Ceratophrys ornata

Larvae of Ceratophrys ornata are carnivorous, have relatively short digestive tracts and continue to feed during metamorphic climax, in contrast to those of more typical herbivorous anuran larvae. The present study describes both histological and histochemical changes in the stomach, small intestine, and large intestine of C. ornata prior to and during metamorphic climax. Modifications in these organs were found to be similar to but less dramatic than those in herbivorous larvae. Luminal epithelial cells in the three regions develop vacuoles, suggesting degeneration, but sloughing of this epithelium, as occurs in herbivorous larvae, was not observed in C. ornata. Multicellular tubular glands develop gradually in the gastric area during the larval stages, gastric pits appear during metamorphic climax, and mucous neck cells are first visible in the juvenile. Goblet cells in the small and large intestine increase in number during larval life, as do the number of folds in the intestinal wall. Increase in diameter and thickness of the wall occurs in the stomach as well as in the small and large intestine. Such changes result in an adult-type digestive tract characteristic of frogs in general. Staining with two horseradish peroxidase conjugated lectins, soybean agglutinin (SBA) and Ulex europaeus agglutinin I (UEA I), demonstrated specific sites along the digestive tract of glycoconjugates with terminal sugars N-acetylgalactosamine and alpha-fucose, respectively. As metamorphic climax approaches, staining intensities decrease--thus providing evidence for metamorphic changes in the sugar moieties of glycoconjugates present in the digestive tract of carnivorous larvae.     

Energetics of metamorphic climax in the pickerel frog (Lithobates palustris)

Anuran metamorphosis, the transition from aquatic larvae to terrestrial juveniles, is accompanied by significant morphological, physiological, and behavioral changes. Timing of metamorphosis and final size, which can influence adult fitness, may depend on sufficient energy accumulated during the larval period to support metamorphosis. However, only two species of anurans have been examined for energetic costs of metamorphosis, Rana tigrina and Anaxyrus terrestris. Based on these species, it has been hypothesized that differences in energy expenditure are related to duration of metamorphosis. To compare energetic costs of metamorphosis among species and examine this hypothesis, we quantified the total energy required for metamorphosis of Lithobates palustris tadpoles by measuring oxygen consumption rates over the duration of metamorphic climax using closed-circuit respirometry. Total energy costs for L. palustris were positively related to tadpole mass and duration of metamorphic climax. However, larger tadpoles completed metamorphosis more efficiently because they used proportionally less total energy for metamorphic climax than smaller counterparts. Costs were intermediate to R. tigrina, a larger species with similar metamorphic duration, and A. terrestris, a smaller species with shorter metamorphic climax. The results supported the hypothesis that amphibian species with more slowly developing tadpoles, such as ranids, require more absolute energy for metamorphosis in comparison to more rapidly developing species like bufonids.     

Changes in Structure and Function of Ventral Epidermis in Hyla arborea savignyi Aud. (Anura; Hylidae) Throughout Metamorphosis

The changes in epidermal ultrastructure during the metamorphic cycle of Hyla arborea are described. The number of cell layers increased from two to four in the late tadpole stages. The cell layers flatten and the process of stratification reaches its peak after the completion of metamorphosis. The mitochondria-rich cell appears early in the tadpole stages. Numerous flask cells are noticeable in the post-metamorphic stages. K+–p-NPPase activity was localized cytochemically in the epidermis of H. arborea during its metamorphic cycle. In the epidermis of the legless tadpole, evidence for K+–p-NPPase activity was confined intracellularly. During the later tadpole stages, preceding metamorphic climax, the main ATPase activity shifted to the baso-lateral cell membranes bordering with the intercellular spaces under the surface and later the stratum corneum. This continued after metamorphic climax in the juvenile toadlets, diminishing later in the adult stage.     

The role of the thyroid in the development of Mauthner's neuron

Summary Although the normal postmetamorphic atrophy of the giant neurons of Mauthner in anuran amphibians has been attributed to the direct or indirect action of thyroid hormone, the administration of exogenous thyroxin to Rana pipiens larvae appears to facilitate growth of the neuron rather than inhibit it. In the present investigation, Rana pipiens embryos were surgically thyroidectomized. In the absence of the thyroid, metamorphosis never occurred, and hind limb development was arrested at larval stages VI–VIII. However, these animals continued their growth and often exceed their controls in overall size. Mauthner cells of thyroidectomized larvae have nuclei of significantly smaller size than their controls. The observations are consonant with the view that thyroid hormones play an important positive role in the growth and maintenance of Mauthner's cell. It is further indicated that the growth of Mauthner's neuron is more dependent upon the titer of thyroid hormone in the blood than it is upon age, brain size, tail motor influences, or the state of the lateral line system.Dedicated to Professor Berta Scharrer on her 60th birthday. This study was supported by Grants NB-04555, IGS-98 and 5 T 1-GM-102 from the U.S.P.H.S.I am pleased to acknowledge the excellent technical assistance of Mrs. Celia Grubman.     

Past and Present Risk: Exposure to Predator Chemical Cues Before and after Metamorphosis Influences Juvenile Wood Frog Behavior

Animals are exposed to different predators over their lifespan. This raises the question of whether exposure to predation risk in an early life stage affects the response to predators in subsequent life stages. In this study, we used wood frogs (Rana sylvatica) to test whether exposure to cues indicating predation risk from dragonfly larvae during the wood frog larval stage affected post‐metamorphic activity level and avoidance of garter snake chemical cues. Dragonfly larvae prey upon wood frogs only during the larval stage, whereas garter snakes prey upon wood frogs during both the larval stage and the post‐metamorphic stage. Exposure to predation risk from dragonflies during the larval stage caused post‐metamorphic wood frog juveniles to have greater terrestrial activity than juvenile wood frogs that were not exposed to larval‐stage predation risk from dragonflies. However, exposure to predation risk as larvae did not affect juvenile wood frog responses to chemical cues from garter snakes. Wood frogs exposed as larvae to predation risk from dragonfly larvae avoided garter snake chemical cues to the same extent as wood frog larvae not exposed to predation risk from dragonfly larvae. Our results demonstrate that while some general behaviors exhibit carry‐over effects from earlier life stages, behavioral responses to predators may remain independent of conditions experienced in earlier life stages.     

The fat body of bullfrog (Lithobates catesbeianus) tadpoles during metamorphosis: changes in mass, histology, and melatonin content and effect of food deprivation

The fat body of Lithobates catesbeianus (formerly Rana catesbeiana) tadpoles was studied during metamorphosis and after food deprivation in order to detect changes in its weight, adipocyte size, histology, and melatonin content. Bullfrog tadpoles have large fat bodies throughout their long larval life. Fat bodies increase in absolute weight, and weight relative to body mass, during late stages of prometamorphosis, peaking just before climax, and then decreasing, especially during the latter stages of transformation into the froglet. The climax decrease is accompanied by a reduction in size of adipocytes and a change in histology of the fat body such that interstitial tissue becomes more prominent. Food deprivation for a month during early prometamorphosis significantly decreased fat body weight and adipocyte size but did not affect the rate of development. However, food restriction just before climax retarded development, suggesting that the increased nutrient storage in the fat body before climax is necessary for metamorphic progress. Melatonin, which might be involved in the regulation of seasonal changes in fat stores, stayed approximately at the same level during most of larval life, but increased sharply in the fat body during the late stages of climax. The findings show that the rate of development of these tadpoles is not affected by starvation during larval life as long as they can utilize fat body stores for nourishment. They also suggest that the build up of fat body stores just before climax is necessary for progress during the climax period when feeding stops.     

Developmental changes in cell proliferation in the auditory midbrain of the bullfrog, Rana catesbeiana

We examined patterns of cell proliferation in the auditory midbrain (torus semicircularis) of the bullfrog, Rana catesbeiana, over larval and early postmetamorphic development, by visualizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) in cycling cells. At all developmental stages, BrdU-labeled cells were concentrated around the optic ventricle. BrdU-labeled cells also appeared within the torus semicircularis itself, in a stage-specific manner. The mitotic index, quantified as the percent of BrdU-positive cells outside the ventricular zone per total cells available for label, varied over larval development. Mitotic index was low in hatchling, early larval, and late larval stages, and increased significantly in deaf period, metamorphic climax, and froglet stages. Cell proliferation was higher in metamorphic climax than at other stages, suggesting increased cell proliferation in preparation for the transition from an aquatic to an amphibious existence. The change in mitotic index over development did not parallel the change in the total numbers of cells available for label. BrdU incorporation was additionally quantified by dot-blot assay, showing that BrdU is available for label up to 72 h postinjection. The pattern of change in cell proliferation in the torus semicircularis differs from that in the auditory medulla (dorsal medullary nucleus and superior olivary nucleus), suggesting that cell proliferation in these distinct auditory nuclei is mediated by different underlying mechanisms.     

Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867)

Jungblut, L.D., Pozzi, A.G. and Paz, D.A. 2010. Larval development and metamorphosis of the olfactory and vomeronasal organs in the toad Rhinella (Bufo) arenarum (Hensel, 1867). — Acta Zoologica (Stockholm) 92 : 305–315. The olfactory and the vomeronasal system are the two major chemosensory systems found in terrestrial vertebrates. Among tetrapods, amphibians are unique in having an aquatic larval stage, followed by metamorphosis to a terrestrial adult. In the present work, we studied the histological development of the olfactory and vomeronasal organ and associated multicellular glands of the toad Rhinella (Bufo) arenarum, from early poshatching larva to postmetamorphic toadlets. As in other bufonids, the olfactory epithelium of R. arenarum in larvae is divided into dorsal and ventral branches in the rostral and mid‐nasal regions. At metamorphic climax, the larval pattern changes drastically and the adult olfactory configuration develops. Bowman’s glands appear in the olfactory epithelium of R. arenarum at the onset of metamorphic climax. The vomeronasal epithelium develops early in larval development in R. arenarum, around the time of operculum development. Interestingly, a novel sensory epithelium develops in the floor of the principal chamber of R. arenarum at metamorphic climax. This novel sensory epithelium resembles larval sensory epithelium lacking Bowman’s glands, and suggests that these animals would be able to sense not only air‐borne, but also water‐borne odors during their adult terrestrial life.     

Degeneration of the tail notochord of Rana temporaria at metamorphic climax

Summary An electron microscopical study has been made of the notochordal cells of the tail of the larval Rana temporaria, before and after they commence to degenerate at metamorphic climax.Degeneration of the tail begins at the tip; the necrotic area proceeds to extend proximally and becomes more widespread. Simultaneously during climax the tail shortens and finally disappears as the animal acquires the froglet form.Notochordal cells autolyse independently and at random, when there is disruption and disorganisation of the surrounding notochordal sheath. The release of lysosomal enzymes intracellularly elicits organelle necrosis.Evidence is provided to support the view that necrotic notochordal tissue is phagocytosed by invading mesenchymal macrophages.     

Immunocytochemical distribution of corticotropin-releasing factor in the brain and hypophysis of larval Bufo arenarum; effect of KClO4 during early development

The maturation of the corticotropin-releasing factor (CRF) neuronal system was evaluated by immunocytochemistry and morphometry in Bufo arenarum, during spontaneous metamorphosis and in tadpoles with inhibited thryroid function. The first appearance of CRF immunoreactive fibers was at the end of premetamorphosis (stage VIII). These fibers were found in small numbers and weakly stained in the median eminence and infundibular stalk. With the advance of larval development, CRF-like material stained intensely and tended to aggregate in the external zone of the median eminence. At climax stages, immunoreactive fibers and perikarya (weakly stained) were identified in the interpeduncular nucleus and in the dorsal infundibular nucleus. Morphometric and immunocytochemical results indicate that the maturation of the CRF neuronal system in Bufo arenarum occurs just before metamorphic climax, coinciding with high levels of thyroid and steroid hormones. We have also found that in larvae with inhibited thyroid function, the CRF neuronal system is able to develop, and that thyroid hormone could exert a negative feedback control on the synthesis of CRF.     

Experimental analysis of hematopoietic cell development in the liver of larval Rana pipiens.

Hematopoietic cell development in the liver of Rana pipiens throughout the larval period was examined. Differential counts of Wright/Giemsa-stained cell suspensions demonstrated that hepatic hematopoiesis includes lymphoid, myeloid, and erythroid cell lineages. Hematopoietic activity appeared to be initiated near the end of embryonic development (Shumway stages 24–25) and reached a substantial level by 30 days of development (Taylor and Kollros stage IV). Although lymphopoiesis became highly variable during later larval stages, granulopoiesis remained stable and erythropoiesis increased throughout the larval period. Erythropoiesis is the predominant hematopoietic activity in the larval liver. Histological examination of plastic embedded tissue demonstrated that hematopoietic activity is localized in discrete foci within the endothelial-lined sinusoids. These hematopoietic foci are found in both the subcapsular region and deeper portions of the organ. Microdensitometric analysis of experimental embryos which received chromosomally labeled presumptive ventral blood island transplants at Shumway stage 15 indicated that the derivatives of this embryonic region are limited to the circulating erythrocyte population of the early larvae. The hematopoietic cell populations of the liver appear to be derived from an extrinsic stem cell source which may be located in the dorsal region of the early embryo.     

The epidermis and its degeneration in the larval tail and adult body of Rana temporaria and Xenopus laevis (Amphibia: Anura)

The tail epidermis of the larva and the body epidermis of adults of Rana temporaria and Xenopus laevis are described in terms of electron microscopy.
The activity of lysosomes (determined by the localization of acid phosphatase) in relation to autolysis and the process of cellular cornification, is considered during the periods of climactic disappearance of the larval tail and skin sloughing of adults. The results obtained generally correspond for both genera.
Larval tail epidermal cells completely disappear at metamorphic climax; those of the adult, which are shed, are replaced throughout life after each periodic sloughing. Nevertheless the mechanisms of their epidermal cell loss are comparable, though the level of lysosomal activity in larval tail epidermal cells is higher than in the adult body epidermis. This higher activity of lysosomal enzymes may facilitate the heavy necrosis which ensues in the larval tail at metamorphic climax.     

Morphological evolution in Ceratophryinae frogs (Anura,Neobatrachia): the effects of heterochronic changes during larval development and metamorphosis

Heterochrony produces morphological change with effects in shape, size, and/or timing of developmental events of a trait related to an ancestral ontogeny. This paper analyzes heterochrony during the ontogeny of Ceratophryinae (Ceratophrys, Chacophrys, and Lepidobatrachus), a monophyletic group of South American frogs with larval development, and uses different approaches to explore their morphological evolution: (1) inferences of ancestral ontogenies and heterochronic variation from a cladistic analysis based on 102 morphological larval and adult characters recorded in ten anuran taxa; (2) comparisons of size, morphological variation, and timing (age) of developmental events based on a study of ontogenetic series of ceratophryines, Telmatobius atacamensis, and Pseudis platensis. We found Chacophrys as the basal taxon. Ceratophrys and Lepidobatrachus share most derived larval features resulting from heterochrony. Ceratophryines share high rates of larval development, but differ in rates of postmetamorphic growth. The ontogeny of Lepidobatrachus exhibits peramorphic traits produced by the early onset of metamorphic transformations that are integrated in an unusual larval morphology. This study represents an integrative examination of shape, size, and age variation, and discusses evolutionary patterns of metamorphosis. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154 , 752–780.     

Cell proliferation in the Rana catesbeiana auditory medulla over metamorphic development

During metamorphic development, bullfrogs (Rana catesbeiana) undergo substantial morphological, anatomical, and physiological changes as the animals prepare for the transition from a fully-aquatic to a semi-terrestrial existence. Using BrdU incorporation and immunohistochemistry, we quantify changes in cell proliferation in two key auditory brainstem nuclei, the dorsolateral nucleus and the superior olivary nucleus, over the course of larval and early postmetamorphic development. From hatchling through early larval stages, numbers of proliferating cells increase in both nuclei, paralleling the overall increase in total numbers of cells available for labeling. Numbers of proliferating cells in the superior olivary nucleus decrease during the late larval and deaf periods, and significantly increase during metamorphic climax. Proliferating cells in the dorsolateral nucleus increase in number from hatchling to late larval stages, decrease during the deaf period, and increase during climax. In both nuclei, numbers of proliferating cells decrease during the postmetamorphic froglet stage, despite increases in the number of cells available for label. Newly generated cells express either glial- or neural-specific phenotypes beginning between 1 week and 1 month post-BrdU injection, respectively, while some new cells express gamma-aminobutyric acid within 2 days of mitosis. Our data show that these auditory nuclei dramatically up-regulate mitosis immediately prior to establishment of a transduction system based on atmospheric hearing.