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.     

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.     

Nuclear ribonucleoprotein complexes of amphibian liver. II. Changes in the protein moiety during development.

Ribonucleoprotein complexes composed of small molecular weight nuclear RNA (4--9 S) and proteins were isolated from hepatic nuclei of Rana catesbeiana (bullfrog) and the protein moiety of this nuclear ribonucleoprotein complex compared during different stages of development. SDS-polyacrylamide gel analysis of premetamorphic tadpoles and adult frog nuclear ribonucleoprotein complexes revealed that while the protein profiles of these two particles were very similar polypeptides of 47,000, 70,000, and 11,000 molecular weight were present in significantly higher concentrations in the frog ribonucleoprotein complexes. Comparison of the chromatin proteins isolated from these two developmental stages demonstrated that these three polypeptides of frog ribonucleoprotein were not contaminants from chromatin. Since these three polypeptides could not be preferentially extracted from the frog ribonucleoprotein complex by 0.5 M KCl or 1 M urea, it was unlikely that these polypeptides were bound nonspecifically to the ribonucleoprotein particle. Polypeptide analysis of the nuclear ribonucleoprotein complexes isolated from tadpoles immersed in the thyroid hormone L-thyroxine revealed an increase in two polypeptides of 37,000 and 45,000 molecular weight during metamorphosis. The absence of reduced amount of these two polypeptides in either the premetamorphic tadpole or adult frog demonstrated that their presence in Rana catesbeiana nuclear ribonucleoprotein was transient during development and specifically associated with tadpole metamorphosis. We conclude from these experiments that the nuclear ribonucleoprotein complex is a dynamic structure during Rana catesbeiana development and that specific changes in its protein composition are associated with discrete stages of amphibian development.     

Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.     

牛蛙致病变形菌的鉴定及其敏感药物筛选

【目的】分离鉴定引起牛蛙(Rana catesbeiana)皮肤溃疡病症的致病菌,筛选抗菌药物。【方法】采用无菌解剖组织划线分离法分离致病菌,通过人工感染试验、菌体和菌落形态观察、API20E系统鉴定、16S r RNA基因序列分析,确定分离菌的致病性及分类地位,并对该菌进行药物敏感性试验。【结果】从患病濒死牛蛙体内分离细菌NWG20141026,通过形态特征观察、生理生化试验、API 20E系统鉴定和16S r RNA基因序列相似性分析,认为NWG20141026菌株为普通变形菌(Proteus vulgaris)。人工感染试验显示,NWG20141026菌株不仅可以通过皮肤伤口感染引起蛙体表溃疡溃烂病症,也可通过消化道感染引起蛙肠炎病。药敏实验结果表明,氟苯尼考、四环素、多西环素、萘啶酸、磺胺异噁唑、恩诺沙星、红霉素等7种药物对普通变形菌具有较好的抑杀菌作用。【结论】引起牛蛙(R.catesbeiana)皮肤溃疡病症的致病菌NWG20141026为普通变形菌(P.vulgaris),所患疾病命名为牛蛙变形菌病。普通变形菌对健康牛蛙具有较强致病性,氟苯尼考、四环素、多西环素、萘啶酸、磺胺异噁唑等5种药可作为防治牛蛙变形菌病的候选药物。     

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.     

Tail muscle hydrolases during metamorphosis of the anuran tadpole. I. Histochemical studies

Naphthylamidase, beta-glucuronidase and trimetaphosphatase have been studied histochemically in the tail muscles of Rana catesbeiana, Rana japonica and Xenopus laevis tadpoles. During the early stages of growth, tadpole tail muscles showed very low activity of these hydrolases, whereas during the prometamorphic and metamorphic stages, they exhibited a marked increase in enzyme activity. This was localized in the subepidermal and subnotochordal mesenchymous tissue and in the myoseptum, i.e. between the muscle bundles.     

Regulation of c-erbA-alpha messenger RNA species in tadpole erythrocytes by thyroid hormone.

Putative thyroid hormone (TH) nuclear receptors have been detected in several tissues of Rana catesbeiana tadpoles. T3 receptor number (sites per nucleus) in red blood cells (RBCs) and tail increases substantially just before metamorphic climax or in response to exogenous TH; in contrast, receptor number in liver remains relatively constant. TH receptors in mammals and birds are thought to be encoded by a c-erbA gene. In the present study, two c-erbA cDNAs, one prepared from Xenopus laevis oocytes (XenTR alpha 1) and one prepared from Rana catesbeiana tail (RC12), were used to examine the c-erbA-related mRNA species in Rana catesbeiana tissues and determine their role in the TH induction of tadpole RBC receptor number. XenTR alpha 1 encodes a protein with T3-binding properties typical of TH receptors. RC12 is almost 99% homologous with XenTR alpha 1 at the amino acid level and contains all of the putative T3-binding region and most of the DNA-binding region. Using either cDNA as a probe, it was found that two major species of c-erbA-related mRNA species (2.6 and 4.0 kilobases) were clearly evident in tadpole RBCs, tail, and liver. A third, more diffuse band (approximately 5.0 kilobases) was observed in RBC and tail. In RBCs, but not in liver, the combined level of c-erbA-related mRNA species was increased during spontaneous metamorphosis or after administration of TH. Furthermore, the TH-induced increase in both c-erbA-related mRNA species and receptor number in RBCs was prevented if actinomycin-D was administered with TH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the nematode Gyrinicola batrachiensis on development, gut morphology, and fermentation in bullfrog tadpoles (Rana catesbeiana): a novel mutualism

We describe a novel mutualism between bullfrog tadpoles (Rana catesbeiana) and a tadpole-specific gastrointestinal nematode (Gyrinicola batrachiensis). Groups of tadpoles were inoculated with viable or nonviable nematode eggs, and development, morphology, and gut fermentation activity were compared between nematode-infected and uninfected tadpoles. Nematode infection accelerated tadpole development; the mean time to metamorphosis was 16 d shorter and the range of times to metamorphosis was narrower in nematode-infected tadpoles than in uninfected tadpoles. At metamorphosis, infected and uninfected bullfrogs did not differ in body size or condition. Colon width, wet mass of colon contents, and concentrations of most fermentation byproducts (short-chain fatty acids: SCFAs) in the hindgut were greater in infected tadpoles. Furthermore, in vitro fermentation yields for all SCFAs combined were over twice as high in infected tadpoles than in uninfected tadpoles. One explanation for accelerated development in infected tadpoles is the altered hindgut fermentation associated with the nematodes. Energetic contributions of fermentation were estimated to be 20% and 9% of the total daily energy requirement for infected and uninfected tadpoles, respectively. Infection by G. batrachiensis nematodes potentially confers major ecological and evolutionary advantages to R. catesbeiana tadpoles. The mutualism between these species broadens our understanding of the taxonomic diversity and physiological contributions of fermentative gut symbionts and suggests that nematodes inhabiting the gut regions of other ectothermic herbivores might have beneficial effects in those hosts.     

Prevalence of Batrachochytrium dendrobatidis in American bullfrog and southern leopard frog larvae from wetlands on the Savannah River Site, South Carolina

Batrachochytrium dendrobatidis, an aquatic fungus, has been linked to recent amphibian population declines. Few surveys have assessed B. dendrobatidis infections in areas where the disease is suggested to be less virulent and population declines have not been observed, such as southeastern North America. Although adult Rana catesbeiana and Rana sphenocephala from the Savannah River Site, South Carolina collected in 1979 and 1982 were identified as having B. dendrobatidis, it is unknown whether the fungus is currently present at the site or if susceptibility to infection varies among species or wetlands with different histories of environmental contamination. From 15 May through 15 August 2004, we collected R. catesbeiana and R. sphenocephala tadpoles from three wetlands with differing contamination histories on the Savannah River Site, South Carolina. We found B. dendrobatidis in only one of the wetlands we surveyed. Batrachochytrium dendrobatidis infection was identified in 64% of the R. catesbeiana tadpoles sampled and histologically assessed (n=50) from a wetland contaminated with mercury, copper, and zinc. No R. sphenocephala tadpoles from this site (n=50) were infected. In combination with a recently published report, our data suggest that B. dendrobatidis has been present at the Savannah River Site for over 25 yr but has not caused any apparent population declines. This time period is similar to the known presence of 30 yr of B. dendrobatidis in northeastern North America. Our data suggest that R. sphenocephala larvae might be resistant to infection, even when occupying the same wetland as the infected R. catesbeiana. Our survey did not clarify the effects of environmental contamination on infection severity, but our study stresses the importance of additional field surveys to document how this pathogen is affecting amphibians globally.     

牛蛙皮胶原蛋白酸提取条件优化及其对细胞生长和粘附性的影响

应用羟脯氨酸法测定了牛蛙皮胶原蛋白含量,通过正交实验对牛蛙皮胶原蛋白酸法提取条件进行了优化,并结合MTT法测定了胶原蛋白对细胞生长和粘附性的影响。结果显示,牛蛙皮胶原蛋白的含量约为45．1％;温度条件对提取率影响最大,其次依次为酸种、时间和浓度,最优组合为乙酸、1．5mol／L、37℃、24h;在低浓度时,牛蛙皮胶原蛋白对正常人类肝细胞的生长和粘附性无显著影响,当浓度升高到一定值时,对细胞生长和粘附性有显著促进作用。     

Distribution and reproductive strategies of Gyrinicola batrachiensis (Oxyuroidea: Pharyngodonidae) in larvae of eight species of amphibians from Nebraska

In total, 462 tadpoles and salamander larvae of 8 species were examined for the presence of Gyrinicola batrachiensis from 5 locations in Nebraska. Infection by G. batrachiensis occurred in tadpoles of Rana blairi , Rana catesbeiana, Rana pipiens, and Bufo woodhousii. Tadpoles of Hyla chrysoscelis , Spea bombifrons, and Pseudacris maculata and larvae of Ambystoma mavortium were not infected with G. batrachiensis. Population structure, defined as prevalence, mean abundance, and mean intensity of G. batrachiensis, varied among tadpoles of different amphibian species and was determined by collection locality, developmental period of tadpole hosts, amphibian species co-occurrence, and different reproductive strategies of G. batrachiensis , or a combination. Gyrinicola batrachiensis observed in all ranid tadpoles and B. woodhousii tadpoles from where bufonids were the only anuran species present, confirmed to the didelphic haplodiploidy and monodelphic parthenogenetic reproductive strategies, respectively. However, tadpoles of B. woodhousii that co-occurred with tadpoles of R. pipiens at Cedar Creek were inconsistent with these predictions and contained both male and didelphic female nematodes, but at a low mean intensity (1.61 ± 0.70). Didelphic female nematodes from B. woodhousii tadpoles at Cedar Creek only produced thick-shelled eggs, whereas nematodes in R. pipiens tadpoles had a high mean intensity (14.88 ± 23.83) from this location and contained both thick-shelled and thin-shelled eggs in their respective uteri. More importantly, adult female nematodes from tadpoles of R. pipiens and B. woodhousii from Cedar Creek were morphologically more similar to each other than to female nematodes recovered from tadpoles of other anuran species, other locations, or both. These data suggest that when strains of G. batrachiensis are shared by tadpoles of different amphibian species that differ in developmental period, the nematodes have an intermediate reproductive strategy in amphibian species, with tadpoles having short development.     

Alanine aminopeptidase activity and autolysis in the tails of Rana catesbeiana larvae during metamorphosis.

1. Alanine aminopeptidase activity and autolysis increase concomitantly in tail tissue of Rana catesbeiana tadpoles during metamorphosis. 2. significant increases first appear at Taylor and Kollros state XX and coincide with the beginning of tail regression as determined by the tail wt body wt ration. 3. The results suggest a role of alanine aminopeptidase in the mechanism of tail resorption.     

Developmental implications of differential effects of calcium in tail and body skin of Anuran tadpoles

The effects of external Ca(++) on metamorphosis of Rana catesbeiana tadpoles were assessed. Treatment of tadpoles with Ca(++) (0.05 mM) during early prometamorphic stages induced precocious metamorphic events such as tail regression, shortening of the intestine, forelimb emergence, and keratinization of body epidermis within 23 days of treatment compared to control tadpoles still in mid-prometamorphic stages. These effects of Ca(++) are probably mediated by the thyroid gland, as indicated by histological features of the gland at the light and electron microscopic levels. Calcium levels of tail and body skin were measured at various stages of development by atomic absorption spectrophotometry. In control and experimental groups, body skin had significantly higher Ca(++) concentrations than tail skin. There were no statistically significant effects of developmental stage on Ca(++) levels of tail or body skin. Experimental Ca(++) treatment significantly increased Ca(++) concentration in tail but not body skin. Ultrastructure studies and gel electrophoresis indicated that calcium induced keratinization of body skin, but not tail epidermis. Ca(++)-treated tail epidermis showed various autolysing figures in apoptotic cells. In summary, calcium treatment accelerated metamorphosis and induced the following region-dependent cellular events: keratinization of body skin-a characteristic of adult epidermis-and programmed cell death in the tail. Whatever signal elicited by calcium in this experimentally induced accelerated metamorphosis is probably mediated via the thyroid gland.     

Alternative leech vectors for frog and turtle trypanosomes.

Trypanosoma pipientis infections were achieved by exposing laboratory-raised bullfrog tadpoles (Rana catesbeiana) to the leech Desserobdella picta that had fed on infected frogs. Likewise, a laboratory-raised snapping turtle (Chelydra serpentina) was infected with Trypanosoma chrysemydis following exposure to infected Placobdella ornata. Transmission of the trypanosomes by these leeches constitutes new vector records for the parasites. The biology of D. picta and P. ornata suggests that they are more important in transmitting these flagellates than the species of leech previously reported as vectors.     

Seasonal variation in the active transporting ability and in the membrane ATPase activity of the frog intestinal epithelium.

The active sugar and amino acid transport in the small intestine of the American leopard frog (Rana pipiens) and a species of European frog (Rana esculenta) decreases during the winter months. Parallel with this the (Na+, K+)-stimulated ("pump") ATPase activity is markedly depressed. No seasonal changes are observed in the intestine of the tropical bullfrog (Rana catesbeiana). It is assumed that the low pump-ATPase activity is caused by the hibernation of the frogs living in moderate or subtropical areas and is connected to a biological clock. The decreased active transport of non-electrolytes appears to be a consequence of the change of the ATPase activity.     

Cloning and thyroid hormone regulation of albumin mRNA in Rana catesbeiana tadpole liver.

Thyroid hormones are responsible for the specific biochemical and structural changes that occur during amphibian metamorphosis. In this study we screened a series of cDNAs from a library constructed from T4-treated premetamorphic tadpole liver poly(A)+ RNA in order to identify a clone that could be used to study the influence of T3 on liver-specific gene expression during Rana catesbeiana metamorphosis. The cDNA from one clone exhibited a greater degree of hybridization to liver RNA from thyroid hormone-treated tadpoles than untreated tadpoles and no hybridization to RNA from tail fins of tadpoles of either group. On Northern blots, the mRNA to which the cDNA hybridized was 2.3 kilobases in size. The pattern of hybridization to genomic DNA digested by various restriction enzymes was consistent with the presence of a single gene. Using slot blot analysis we found that the mRNA levels first rose above basal levels only after 5 days of immersion of tadpoles in 12.5 micrograms/liter T3. The mRNA levels increased approximately 10-fold after 7 and 9 days of treatment. Frog livers had mRNA levels that were intermediate between those in untreated tadpoles and tadpoles immersed in T3 for 7 days. Sequence analysis revealed a significant degree of homology to serum albumin and alpha-fetoprotein. While it is known that serum albumin levels rise dramatically during metamorphosis in Rana species, presumably playing a critical role in maintaining water and electrolyte balance during the animals' terrestrial phase, the molecular basis of the induction has not been fully explained.     

Changes in the rate of production of superoxide anion in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine-induced metamorphosis

1. The time course of changes in the rate of production of superoxide anion (O2) was compared with that of beta-glucuronidase in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine (T3)-induced metamorphosis. 2. Superoxide production increases in tadpole tail tissue undergoing regression in vivo during spontaneous metamorphosis and in response to T3 treatment. 3. The specific activity of beta-glucuronidase rises three-fold relative to control levels in tadpoles treated with T3 in vivo. 4. The time of onset of the rise in beta-glucuronidase activity precedes the onset of tissue regression and the onset of the increase in superoxide production precedes the rise in beta-glucuronidase activity.     

Biochemical and Immunological Characterization of Collagenase in Tissues of Metamorphosing Bullfrog Tadpoles

Tissue inhibitor of metalloproteinases (TIMP, a specific inhibitor of collagenase) was found to inhibit thyroid hormone-induced tail regression, suggesting the important role of collagenase in this process. Collagenase was purified from culture media of back skin of tadpole of bullfrog, Rana catesbeiana . Anti-tadpole collagenase polyclonal antisera were obtained against the purified enzyme. The antibody inhibited the activity of tadpole collagenase. The antisera reacted to tissues of adult bullfrogs, tadpoles of african clawed frog, Xenopus laevis , and adult newts, Cynopus pyrrhogaster , and also reacted to human fibroblast collagenase. Immunoblot analyses suggested that tadpole collagenase lacks the procollagenase which is generally found in mammalian collagenases. Intense immunological stains were observed for the tissues of thyroid hormone-treated tadpoles as compared to those of untreated animals. Thyroid hormone increased amounts of collagenase not only in epidermal layer but also in mesenchymal tissues including fibroblastic cells.     

Frog virus 3 prevalence in tadpole populations inhabiting cattle-access and non-access wetlands in Tennessee, USA

Ranaviruses have been associated with most of the reported larval anuran die-offs in the United States. It is hypothesized that anthropogenically induced stress may increase pathogen prevalence in amphibian populations by compromising immunity. Cattle use of wetlands may stress resident tadpole populations by reducing water quality. We isolated a Ranavirus from green frog Rana clamitans (n = 80) and American bullfrog R. catesbeiana (n = 104) tadpoles collected at 5 cattle-access and 3 non-access wetlands on the Cumberland Plateau, Tennessee, USA. Sequencing confirmed Frog virus 3 (FV3); therefore, we compared its prevalence between tadpole populations inhabiting cattle-access and non-access wetlands, and among 3 seasons (winter, summer, and autumn) in 2005. We found FV3 in both tadpole species and cattle land-use types; however, prevalence of FV3 was greater in green frog tadpoles residing in cattle-access wetlands compared to those in non-access wetlands. No difference in FV3 prevalence was detected between cattle land uses for American bullfrog tadpoles. A seasonal trend in FV3 prevalence also existed, with prevalence greater in autumn and winter than in summer for both species. In addition, we found that FV3 prevalence decreased significantly as Gosner stage increased in American bullfrog tadpoles. No trend was detected between FV3 prevalence and developmental stage for green frog tadpoles. Our results suggest that cattle use of wetlands may increase prevalence of FV3 in Rana tadpoles, although this effect may depend on species, season, and tadpole developmental stage.