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.

Histology and lectin-binding patterns in the skin of the terrestrial horned frog Ceratophrys ornata.

The terrestrial horned frog, Ceratophrys ornata, lives on a wet substratum and absorbs water through the ventral epidermis; water is lost by evaporation from the dorsal skin. Thus, this species may be useful as a model for determining whether or not skin histology and lectin-binding patterns, indicative of glycoconjugates, are related to skin functions such as osmoregulation and water balance. With this in mind, a histological and lectin-histochemical study was carried out on dorsal and ventral skin of aquatic tadpoles and of a young terrestrial frog of C. ornata. Sections of skin were stained with various dyes to demonstrate general histological features and with two horseradish peroxidase (HRP)-conjugated lectins, Ulex europaeus agglutinin (UEA 1) and soybean agglutinin (SBA) which bind to specific terminal sugar residues of glycoconjugates, namely L-fucose and N-acetyl-D-galactosamine or D-galactose, respectively. In early stage tadpoles both lectin-binding patterns were similar in the bilaminar epidermis of dorsal and ventral skin (i.e., each lectin stained the apical cell layer). However, metamorphic changes resulted in a young frog with typical adult-type skin composed of a stratified squamous epidermis and three distinct types of glands containing glycoconjugates in their secretions. Strikingly different lectin-binding patterns were evident in the epidermis from dorsal and ventral regions of the body. The epidermis from the dorsal region was stained by both lectins; in contrast, that from the ventral region, although stained strongly by HRP-SBA, did not react with HRP-UEA 1, indicating that few, if any, fucose residues were present in the ventral epidermis.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of xenopsin and xenopsin precursor fragment immunoreactivities in the skin and gastrointestinal tract of Xenopus laevis.

Xenopsin (Xp) and xenopsin precursor fragment (XPF) are bioactive peptides derived from a single precursor molecule; both were isolated previously from extracts of Xenopus laevis skin. The present immunohistochemical study was undertaken to determine the specific cellular localization of these two peptides in the skin and also in the gastrointestinal tract of adult Xenopus. We report here that Xp-like and XPF-like immunoreactivities co-exist in the granular glands of the skin and specific granular cells in the lower esophagus and stomach. However, only Xp-like immunoreactivity, not XPF-like immunoreactivity, was detected in tall, thin cells of the duodenum and in club-shaped cells of the large intestine. The immunochemical co-localization of the two peptides in specific cells of the skin, lower esophagus and stomach suggests that the same gene is expressed in each of these cells, and that the precursor molecule undergoes similar post-translational processing. In contrast, the observation that certain cells of the duodenum and large intestine display only one peptide immunoreactivity suggests an alternative phenomenon, possibly involving selective peptide accumulation or expression of a different gene.

Thyrotropin-releasing hormone (TRH): immunohistochemical distribution in tadpole and frog brain.

The anatomical localization of immunoreactive TRH (IR-TRH) was demonstrated by the peroxidase-antiperoxidase technique in the brain and pituitary gland of larval and adult Rana catesbeiana. In the adult frog main sites of IR-TRH are perikarya and neuronal fibers in the preoptic and infundibular nuclei of the hypothalamus and in the amygdala and diagonal band of Broca of the telencephalon. In addition, TRH-positive neuronal fibers and endings were found in the septum, pallium, and brain stem as well as in the preoptico-hypophyseal tract, the external zone of the median eminence (which matures during late larval stages), and the pars nervosa; fibers were less extensive in the pars intermedia, and were absent from the pars distalis. In early larval stages, the magnocellular nucleus of the posterior preoptic area is the main site of immunoreactive perikarya. During late stages the extensive adult pattern of distribution of IR-TRH becomes established. The study represents the first immunohistochemical demonstration of IR-TRH in larval anurans, and serves as a basis for clarification of the neuroendocrine regulation of metamorphosis.

Immunofluorescent detection and localization of thyroxine in blood of adult amphibians.

Indirect immunofluorescent staining was used to detect and localize thyroxine (T4) in blood smears from different species of adult amphibians, namely, Rana pipiens, Rana catesbeiana, Bufo marinus, Xenopus laevis, and Notopthalmus viridescens. Fluorescence, indicative of T4, was observed in both plasma and erythrocytes (RBC) from all individuals of the five species studied. It was weak and diffuse in the plasma and in the cytoplasm of the RBC but was intense in the nuclei (especially the nuclear perimeter) of these cells. The finding of intracellular T4 suggests that thyroid hormone may be of some physiological importance in adult amphibians.

Immunofluorescent detection and localization of thyroxine in blood of Rana catesbeiana from early larval through metamorphic stages.

Indirect immunofluorescent staining was used to detect and localize thyroxine (T4) in blood smears from individual Rana catesbeiana tadpoles in almost every stage of larval development. Earlier radioimmunoassays revealed a surge of T4 in the blood plasma during metamorphic stages, but plasma T4 concentrations in earlier stages were either very low or below the minimal detectable limits of the assays. With the present immunofluorescent method, T4 was found in plasma of tadpoles throughout the entire larval period from early limb bud stages to the end of metamorphosis. Moreover, T4 was also found in association with cytoplasm and nuclei of red blood cells, particularly nuclei of the new population of adult red cells differentiating during metamorphic stages. In conclusion, thyroid hormone is present in both blood plasma and erythrocytes of R. catesbeiana from early through late stages of larval development.

Tadpole erythrocytes: luminescent properties with dark field microscopy.

To characterize and classify erythrocytes of ranid tadpoles, alcohol-fixed blood smears were studied with dark field illumination. All preclimax stage (limb bud-foot) Rana castesbeiana tadpoles from ponds in Massachusetts had red blood cells that were polymorphic. The majority of cells (88%) showed a bright, granular luminescence varying from white to blue-grey, whereas, cytoplasm of the other cells was smooth, black, and nonluminescent. On the other hand, tadpoles in similar stages from other species (Rana clamitans, Mass. and Rana pipens, Vermont) and from R. catesbeiana tadpoles from other locations (Wisconsin and North Carolina) had no observable cytoplasmic luminescence in any of their red blood cells. Moreover, as Mass. R. catesbeiana underwent metamorphic climax their luminescent cells disappeared and were replaced by small, round, dark, nonluminescent cells, precursors of the oval, nonluminescent erythrocytes characteristic of adult frogs. Cells with black nonluminescent cytoplasm generally contained nuclei which were luminescent. In conclusion, two main types of red blood cells-those with and those without cytoplasmic luminescence-are distinguishable with dark field microscopy. Luminescence of the cells varies with species, geographic location, and developmental stage of the tadpoles.

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.