Oxidative stress and neurotoxicity induced by exposure to settleable atmospheric particulate matter in bullfrog tadpoles, Aquarana catesbeiana, (Shaw, 1802).

Bullfrog tadpoles, Aquarana catesbeiana, were exposed to settleable particulate matter (SePM), (1 g L-1, 96 h) and their organs were collected for analysis of metal/metalloid, oxidative stress and neurotoxicity in liver, muscle, kidney and brain. The SePM water of the exposed groups contained 18 of the 28 metals/metalloids detected in ambient particulate matter (APM). Fe56 and Al were those that presented the highest concentrations, Cr, Mn, Pb and Cu increased from 10 to 20 times and Ti, V, Sr, Rb, Cd, Sn and Ni increased from 1 to 3 times compared to the control. Bioaccumulation of metals/metalloids in the exposure water varied significantly between organs, with the muscle and liver showing the highest concentrations of metals, followed by the brain. Lipoperoxidation and malondialdehyde increased only in muscle, while carbonyl proteins increased only in the liver and brain. Regarding nitric oxide synthase, there was an increase in the liver and brain in the group exposed to SePM. Catalase activity decreased in the liver and muscle, while the activity of glutathione peroxidase, increased in the liver and kidney and decreased in muscle. Glutathione S-transferase, which is mainly responsible for detoxification, increased in the liver and decreased in muscle and the kidney. Cholinesterase activity increased only in the muscle. The results indicate oxidative stress, due to oxidation catalyzed by metals, components of SePM. Thus, the results contribute to the understanding that SePM has a deleterious effect on the aquatic environment, negatively affecting bullfrog tadpoles, in different ways and levels in relation to the analyzed organs.

Comparison of Lipid Composition between Quasipaa spinosa Oil and Rana catesbeiana Oil and Its Effect on Lipid Accumulation in Caenorhabditis elegans.

Frog oil has been recognized for its nutritional and medicinal value. However, there is limited research on the role of frog oil in preventing obesity. In this study, we aimed to investigate the lipid composition of Quasipaa spinosa oil (QSO) and Rana catesbeiana oil (RCO) using lipidomics analysis. We compared the lipid accumulation effects of these two kinds of frog oils and soybean oil (SO) in Caenorhabditis elegans (C. elegans). Additionally, we determined the gene expression related to lipid metabolism and used the nhr-49 mutant (RB1716) and sir-2.1 mutant (VC199) for validation experiments. The results showed that the lipid composition of QSO and RCO was significantly different (p < 0.05), and QSO was rich in more polyunsaturated fatty acids (PUFAs). After feeding C. elegans, the lipid accumulation of the QSO group was the lowest among the three dietary oil groups. In addition, compared with RCO and SO, QSO significantly inhibited the production of malondialdehyde (MDA) and increased the activity of superoxide dismutase (SOD). The effects of three kinds of dietary oils on the fatty acid composition of C. elegans were significantly different. Compared with SO and RCO, QSO significantly up-regulated (p < 0.05) the expression of sir-2.1 and ech-1 genes. The results showed that QSO might reduce lipid accumulation through the SIRT1 and nuclear hormone signaling pathways. Such a situation was verified experimentally by the nhr-49 mutant (RB1716) and sir-2.1 mutant (VC199). This study proposed a new functional oil, laying the groundwork for developing functional foods from Quasipaa spinosa.

High prevalence and concomitant infection of Ranavirus and Eustrongylides sp. in the invasive American Bullfrog in Brazil.

American Bullfrogs, Aquarana catesbeiana, are invasive anuran species distributed worldwide. One of the adverse impacts that this species causes in native communities is as a reservoir host for pathogens and parasites. Here, we report the coinfection of two pathogenic organisms in A. catesbeiana: Ranavirus and the nematode Eustrongylides. Bullfrogs were collected in the wild in a pond close to the urban area of São Paulo, Brazil. The prevalence of both pathogens was high: 77% were infected with ranavirus with a mean viral load of 1010.3 viral copies, and 100% of the bullfrogs were infected by Eustrongylides sp. with a mean intensity of infection of 13.4 specimens per host. Four host specimens (31%) presented pathological signs that seemed to be related to the Eustrongylides sp. infection, such as internal organs adhered to each other due to high intensity and large size of the nematodes, ulcers, and raw flesh wounds caused by the nematode. The pathogenic and concomitant infections have potential zoonotic implications and raise concerns about human infection risks for Eustrongylides infection. Moreover, such infections may represent an additional level of threat to native communities through the potential shifts in patterns of parasite and pathogen transmission. Future research involving the native anuran community is essential to ascertain whether invasive bullfrogs are attenuating or exacerbating diseases such as ranavirosis and eustrongylidiosis.

Strobilurin fungicide increases the susceptibility of amphibian larvae to trematode infections.

The global rise in fungal pathogens has driven the increased usage of fungicides, yet our understanding of their ecotoxicity remains largely limited to acute toxicity. While such data is critical for projecting the risk of fungicide exposure to individual species, the contamination of natural systems with fungicides also has the potential to alter species interactions within communities including host-parasite relationships. We examined the effects of the fungicide pyraclostrobin on the susceptibility of larval American bullfrogs (Rana catesbeiana) to trematode (echinostome) infections using a controlled laboratory experiment. Following a 2-wk exposure to 0, 1.0, 5.2, or 8.4 µg/L of pyraclostrobin, tadpoles were then exposed to parasites either in the 1) presence (continued/simultaneous exposure) or 2) absence (fungicide-free water) of pyraclostrobin. We found that when exposed to pyraclostrobin during parasite exposure, meta cercariae counts increased 4 to 8 times compared to control tadpoles. Additionally, parasite loads were approximately 2 times higher in tadpoles with continued fungicide exposures compared to tadpoles that were moved to fresh water following fungicide exposure. This research demonstrates that fungicides at environmentally relevant concentrations can indirectly alter host-parasite interactions, which could elevate disease risk. It also underscores the need for studies that expand beyond traditional toxicity experiments to assess the potential community and ecosystem-level implications of environmental contaminants.

Microplastics and TiO2 nanoparticles mixture as an emerging threat to amphibians: A case study on bullfrog embryos.

Emerging contaminants can act as contributing factors to the decline of amphibian populations worldwide. Recently, scientists have drawn attention to the potential ecotoxicity of microplastics and nanomaterials in amphibians, however, their possible effects on embryonic developmental stages are still absent. Thus, the present study analyzed the developmental toxicity of environmentally relevant concentrations of polyethylene microplastics (PE MPs; 60 mg/L) and titanium dioxide nanoparticles (TiO2 NPs; 10 µg/L), isolated or in combination (Mix group) on bullfrog embryos, Aquarana catesbeiana, adapting the Frog Embryo Teratogenesis Assay (FETAX, 96h). Allied to the FETAX protocol, we also analyzed the heart rate and morphometric data. The exposure reduced the survival and hatching rates in groups exposed to TiO2 NPs, and to a lesser extent, also affected the Mix group. TiO2 NPs possibly interacted with the hatching enzymes of the embryos, preventing hatching, and reducing their survival. The reduced effects in the Mix group are due to the agglomeration of both toxicants, making the NPs less available for the embryos. PE MPs got attached to the gelatinous capsule of the chorion (confirmed by fluorescence microscopy), which protected the embryos from eventual direct effects of the microplastics on the hatching and survival rates. Although there were no cardiotoxic effects nor morphometric alterations, there was a significant increase in abdominal edemas in the hatched embryos of the PE MPs group, which indicates that osmoregulation might have been affected by the attachment of the microplastics on the embryos' gelatinous capsule. This study presents the first evidence of developmental toxicity of environmental mixtures of microplastics and nanoparticles on amphibians and reinforces the need for more studies with other amphibian species, especially neotropical specimens that could present bigger sensibility. Our study also highlighted several features of the FETAX protocol as useful tools to evaluate the embryotoxicity of several pollutants on amphibians.

Ultrastructural characteristics and morphological relationships of cardiomyocytes and telocytes in the myocardium of the bullfrog (Rana catesbeiana).

Telocytes (TCs) are distinctive interstitial cells due to their characteristic structures and heterogeneity. They are suggested to participate in tissue repair/regeneration. TCs have been identified in many organs of various mammals. However, data on TCs in lower animals are still very limited. In this work, TCs were identified in the myocardium of the bullfrog (Rana catesbeiana) by light and transmission electron microscopy (TEM). The structural relationships between TCs and neighbouring cell types were measured using the ImageJ (FiJi) morphometric software. TCs with slender Tps (telepodes) were located around cardiomyocytes (CMC). TEM revealed TCs with long Tps in the stroma between CMC. The homocellular tight junctions were observed between the Tps. The Tps were also very close to the neighbouring CMC. The distance between Tps and CMC was 0.15 ± 0.08 µm. Notably, Tps were observed to adhere to the periphery of the satellite cells. The Tps and the satellite cells established heterocellular structural connections by tight junctions. Additionally, Tps were frequently observed in close proximity to mast cells (MCs). The distance between the Tps and the MCs was 0.19 ± 0.09 µm. These results confirmed that TCs are present in the myocardium of the bullfrog, and that TCs established structural relationships with neighbouring cell types, including satellite cells and MCs. These findings provide the anatomical evidence to support the note that TCs are involved in tissue regeneration.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles.

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a "molecular memory" of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3'-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

Defining components of early thyroid hormone signalling through temperature-mediated activation of molecular memory in cultured Rana [lithobates] catesbeiana tadpole back skin.

Thyroid hormones (THs) are essential signalling molecules for the postembryonic development of all vertebrates. THs are necessary for the metamorphosis from tadpole to froglet and exogenous TH administration precociously induces metamorphosis. In American bullfrog (Rana [Lithobates] catesbeiana) tadpoles, the TH-induced metamorphosis observed at a warm temperature (24 °C) is arrested at a cold temperature (4 °C) even in the presence of exogenous THs. However, when TH-exposed tadpoles are shifted from cold to warm temperatures (4 â†’ 24 °C), they undergo TH-dependent metamorphosis at an accelerated rate even when the initial TH signal is no longer present. Thus, they possess a "molecular memory" of TH exposure that establishes the TH-induced response program at the cold temperature and prompts accelerated metamorphosis after a shift to a warmer temperature. The components of the molecular memory that allow the uncoupling of initiation from the execution of the metamorphic program are not understood. To investigate this, we used cultured tadpole back skin (C-Skin) in a repeated measures experiment under 24 °C only, 4 °C only, and 4 â†’ 24 °C temperature shifted regimes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA-sequencing (RNA-seq) analyses. RNA-seq identified 570, 44, and 890 transcripts, respectively, that were significantly changed by TH treatment. These included transcripts encoding transcription factors and proteins involved in mRNA structure and stability. Notably, transcripts associated with molecular memory do not overlap with those identified previously in cultured tail fin (C-fin) except for TH-induced basic leucine zipper-containing protein (thibz) suggesting that thibz may have a central role in molecular memory that works with tissue-specific factors to establish TH-induced gene expression programs.

Discovery of a Novel Glucagon-like Peptide-1 (GLP-1) Analogue from Bullfrog and Investigation of Its Potential for Designing GLP-1-Based Multiagonists.

In this study, we aimed to discover novel GLP-1 analogues from natural sources. We investigated GLP-1 analogues from fish and amphibians, and bullfrog GLP-1 (bGLP-1) showed the highest potency. Starting with bGLP-1, we explored the structure-activity relationship and performed optimization and long-acting modifications, resulting in a potent analogue called 2f. Notably, 2f exhibited superior effects on food intake, glycemic control, and body weight compared to semaglutide. Furthermore, we explored the usefulness of bGLP-1 in designing GLP-1-based multiagonists. Using the bGLP-1 sequence, we designed novel dual GLP-1/glucagon receptor agonists and triple GLP-1/GIP/glucagon receptor agonists. The selected dual GLP-1/glucagon receptor agonist 3o and triple GLP-1/GIP/glucagon receptor agonist 4b exhibited significant therapeutic effects on lipid regulation, glycemic control, and body weight. Overall, our study highlights the potential of discovering potent GLP-1 receptor agonists from natural sources. Additionally, utilizing natural GLP-1 analogues for designing multiagonists presents a practical approach for developing antiobesity and antidiabetic agents.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process / Atividade enzimática de marcadores ósseos no crescimento de Lithobates catesbeianus (Shaw, 1802) durante o processo de ossificação

Abstract In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

Resumo Para melhor compreender o processo de ossificação em anuros, nosso estudo foi conduzido em girinos e adultos de Lithobates catesbeianus. Nesse sentido, as propriedades cinéticas da fosfatase alcalina com p-nitrofenilfosfato (pNPP) e pirofosfato (PPi) foram caracterizadas, e as atividades enzimáticas das fosfatases ácida e ácida tartarato resistente foram avaliadas. Os extratos enzimáticos foram obtidos de fêmur de girinos e adultos, divididos em epífise e diáfise. Após a homogeneização as amostras foram submetidas à centrifugação diferencial para obter membrana celular e, em seguida, ao tratamento com fosfolipase C (PIPLC), para remover as proteínas de membrana ancoradas por fosfatidilinositol. A média da atividade específica da fosfatase alcalina, liberada pela PIPLC de Bacillus cereus, para a hidrólise de pNPP (pH 10,5) nas diferentes regiões do fêmur e idades dos animais foi de 1.142,57 U.mg-1, enquanto para a hidrólise do PPi (pH 8,0) foi de 1.433,82 U.mg-1. Entre os compostos testados para a atividade enzimática, o de maior influência foi o EDTA, inibindo aproximadamente 67% e 77% das atividades de pNPPase e PPase, respectivamente. Quanto aos parâmetros cinéticos, a enzima apresentou comportamento Michaeliano para a hidrólise dos dois substratos. O valor de Km foi de 0,6 mM para a atividade de pNPPase e variou de 0,01 a 0,11 para a atividade de PPase, indicando uma maior afinidade por esse substrato. O estudo da hidrólise de pNPP e PPi revelou que o pH ótimo aparente de atuação foi de 10,5 para o pNPP e 8,0 para o PPi, próximo ao fisiológico, sendo que esse é considerado o substrato natural da fosfatase alcalina. Os resultados demonstram que, apesar do tipo de ossificação que ocorre, a mesma enzima ou isoenzimas, atuam nos diferentes locais do osso e estágios de vida dos anuros. A similaridade dos estudos com os realizados com outros vertebrados apontam que os anuros podem ser considerados excelentes modelos animais para o estudo da calcificação biológica.

Immune Activation and Inflammatory Response Mediated by the NOD/Toll-like Receptor Signaling Pathway-The Potential Mechanism of Bullfrog (Lithobates catesbeiana) Meningitis Caused by Elizabethkingia miricola.

Elizabethkingia miricola is an emerging opportunistic pathogen that is highly pathogenic in both immunocompromised humans and animals. Once the disease occurs, treatment can be very difficult. Therefore, a deep understanding of the pathological mechanism of Elizabethkingia miricola is the key to the prevention and control of the disease. In this study, we isolated the pathogenic bacteria from bullfrogs with dark skin color, weak limbs, wryneck, and cataracts. Via subsequent morphological observations and a 16S rRNA gene sequence analysis, the pathogen was identified as Elizabethkingia miricola. The histopathological and transmission electron microscopy analysis revealed that the brain was the main target organ. Therefore, brain samples from diseased and healthy bullfrogs were used for the RNA-Seq analysis. The comparative transcriptome analysis revealed that the diseased bullfrog brain was characterized by the immune activation and inflammatory response, which were mediated by the "NOD-like receptor signaling pathway" and the "Toll-like receptor signaling pathway". We also performed qRT-PCR to examine the expression profile of inflammation-related genes, which further verified the reliability of our transcriptome data. Based on the above results, it was concluded that the NOD/Toll-like receptor-related networks that dominate the immune activation and inflammatory response were activated in the brain of Elizabethkingia miricola-infected bullfrogs. This study contributes to the search for therapeutic targets for bullfrog meningitis and provides basic information for establishing effective measures to prevent and control bullfrog meningitis.

Activation of respiratory-related bursting in an isolated medullary section from adult bullfrogs.

Breathing is generated by a rhythmic neural circuit in the brainstem, which contains conserved elements across vertebrate groups. In adult frogs, the 'lung area' located in the reticularis parvocellularis is thought to represent the core rhythm generator for breathing. Although this region is necessary for breathing-related motor output, whether it functions as an endogenous oscillator when isolated from other brainstem centers is not clear. Therefore, we generated thick brainstem sections that encompass the lung area to determine whether it can generate breathing-related motor output in a highly reduced preparation. Brainstem sections did not produce activity. However, subsaturating block of glycine receptors reliably led to the emergence of rhythmic motor output that was further enhanced by blockade of GABAA receptors. Output occurred in singlets and multi-burst episodes resembling the intact network. However, burst frequency was slower and individual bursts had longer durations than those produced by the intact preparation. In addition, burst frequency was reduced by noradrenaline and µ-opioids, and increased by serotonin, as observed in the intact network and in vivo. These results suggest that the lung area can be activated to produce rhythmic respiratory-related motor output in a reduced brainstem section and provide new insights into respiratory rhythm generation in adult amphibians. First, clustering breaths into episodes can occur within the rhythm-generating network without long-range input from structures such as the pons. Second, local inhibition near, or within, the rhythmogenic center may need to be overridden to express the respiratory rhythm.

Sodium bicarbonate and salbutamol facilitate recovery from hyperkalemia-induced electrocardiogram abnormalities in bullfrog hearts.

Hyperkalemia is a common electrolyte abnormality frequently complicated with chronic kidney disease. By injecting potassium chloride (KCl) solutions intravenously into bullfrogs, we reproduced typical electrocardiogram (ECG) abnormalities of hyperkalemia in the frog hearts, such as the peaked T waves and the widening of QRS complexes. Simultaneous recordings of cardiac action potentials showed morphological changes that synchronized with those of ECG. After 100 mM KCl injection, the widened QRS complexes continued for a while and gradually restored to their baseline widths. However, pre-treatment with sodium bicarbonate or salbutamol, which directly or indirectly stimulates Na+/K+-ATPase activity, significantly facilitated the recovery from the widened QRS duration, indicating the transcellular movement of potassium ions from the extracellular fluid into the intracellular stores.

Defining a species-specific K constant in American Bullfrogs (Lithobates catesbeianus) through use of computed tomography.

OBJECTIVE: To use CT measurements to define the body surface area (BSA) formula in American bullfrogs (Lithobates catesbeianus) and calculate the species-specific shape constant (K) to suggest chemotherapeutic doses. ANIMALS: 12 American bullfrogs owned by the North Carolina State College of Veterinary Medicine Department of Laboratory Animal Resources underwent CT scans without anesthesia or sedation in November 2022. METHODS: As part of this prospective study, each American bullfrog underwent a complete physical exam and CT scan. 3-D surface models were created using CT data, and the resulting measurements were used for BSA calculations. Animals were grouped by sex. Nonlinear regression analysis of BSA versus body weight was performed, and a species-specific formula was derived for calculating BSA in American bullfrogs. RESULTS: The mean body weight of the bullfrogs was 354 grams. The mean CT-derived BSA was 414.92 cm2. The calculated K constant was 8.28 for the 12 American bullfrogs, and the CT-derived BSA formula was BSA in cm2 = 8.28 X (body weight in g)2/3. The K constant was 8.07 for females and 8.44 for males and was not significantly different between sexes (P = .5). CLINICAL RELEVANCE: Results indicated that the species-specific K constant for American bullfrogs is 8.28. This is the first calculated K constant that exists for amphibians to our knowledge.

Hypersensitive response to interferon-stimulated gene (ISG)-inducing double-stranded RNA in American bullfrog tadpole fibroblasts.

American bullfrogs are thought to be carriers of ranaviruses and contribute to their global spread via trade. Bullfrog tadpoles succumb to ranaviral infection's more severe and deadly effects than bullfrog adults. Presently, little is known about bullfrog tadpoles' innate antiviral immunity, possible due to the lack of available bullfrog tadpole cell lines. In this study, we describe a novel bullfrog tadpole fibroblast cell line named BullTad-leg. Its general cellular attributes, gene expression and function of class-A scavenger receptors (SR-As), and responses to poly IC (a synthetic dsRNA mimicking viral dsRNAs and a potent inducer of the interferon (IFN)-mediated antiviral responses) are investigated. Its abundant expression of vimentin corroborated with the cells' fibroblast morphology. BullTad-leg cells expressed transcripts of four SR-A members: SR-AI, SCARA3, SCARA4, and SCARA5, but transcripts of MARCO, the fifth SR-A member, were not detected. BullTad-leg cells expressed functional SR-As and could bind AcLDL. BullTad-leg cells exhibited cytotoxicity in response to poly IC treatment via SR-As. Additionally, very low doses of poly IC were able to induce dose-dependent expressions of ISGs including Mx, PKR, ISG20, and IFI35. This research sheds new light on the innate immune response, particularly SR-A biology and dsRNA responsiveness, in bullfrog tadpoles.

Genotypic and phenotypic analysis of Elizabethkingia meningoseptica in bullfrog Rana catesbeiana isolated in Taiwan.

Elizabethkingia meningoseptica is a hazardous bacterium for agriculture production and human health. The present study identified E. meningoseptica from the bullfrog, human and reference strain BCRC 10677 by API 20NE, 50S ribosome protein L27 sequencing and pulse field gel electrophoresis to differentiate isolates of E. meningoseptica from aquatic animals and humans. All isolates from bullfrogs and humans were identified as E. meningoseptica by DNA sequencing with 98.8%-100% sequence identity. E. meningoseptica displayed significant genetic diversity when analysed using pulsed-field gel electrophoresis (PFGE). There were six distinct pulsotypes, including one pulsotype found in bullfrog isolates and five pulsotypes found in human isolates. However, E. meningoseptica from bullfrog exhibited one genotype only by PFGE. Overall, molecular epidemiological analysis of PFGE results indicated that the frog E. meningoseptica outbreaks in Taiwan were produced by genetically identical clones. The bullfrog isolates were not genetically related to other E. meningoseptica from human and reference isolates. This research provided the first comparisons of biochemical characteristics and genetic differences of E. meningoseptica from human and bullfrog isolates.

The Absence of an Na+/Ca2+Exchanger (NCX) in Bullfrog Proximal Tubules and Cellular pH is More Influential Than Cellular Ca2+ on Proximal Na Transport.

BACKGROUND/AIMS: The functional significance of the Na+/Ca2+ exchanger (NCX) in basolateral membranes in the proximal tubule remains controversial. The key factor in crosstalk between the apical and basolateral sides is not known. METHODS: We investigated the basolateral membranes, using double-barreled Ca2+ or pH ion-selective microelectrodes. We used doubly perfused bullfrog kidneys in vivo, and switched the basolateral solution (renal portal vein) to experimental solutions. RESULTS: In the control, cellular pH (pHi) was 7.33 ± 0.032 (mean ± SE, n = 7) and in separate experiments, cellular Ca2+ activity (aCai) was 249.6 ± 35.54 nM (n = 28). Changing to respiratory acidosis, pHi was significantly acidified by 0.123 pH units on average and the change of aCai was +53.1 nM (n = 9 ns). In metabolic acidosis, pHi was reduced by 0.151 while aCai was reduced by 143.4. Using the 30 mM K+ solution, pHi was increased by 0.233 while aCai was reduced by 203.9, with depolarization. Both ionomycin and ouabain caused aCai to increase. In the 0.5 mM Na+ solution (replaced with BIDAC Cl), pHi was reduced by 0.177. No changes in aCai (+49.8 ns) were observed although we recorded depolarization of 15.2 mV. In the 0.5 mM Na+ solution, replaced with raffinose, no changes in aCai (-126.4 ns) were observed with depolarization (6.5 ns). CONCLUSION: Our results suggest that thermodynamic calculations of cellular Na+ concentration led to the conclusion that either a Na+/HCO3- exchanger (NBC) or NCX could be present in the same basolateral membrane. H+ ions are the most plausible key factor in the crosstalk.

Analysis of evolutionary and functional features of the bullfrog SULT1 family.

We identified the bullfrog Rana catesbeiana sulfotransferase 1 (SULT1) family from the BLAST search tool of the public databases based on the SULT1 families of Nanorana parkeri, Xenopus laevis, and Xenopus tropicalis as queries, revealing the characteristics of the anuran SULT1 family. The results showed that the anuran SULT1 family comprises six subfamilies, four of which were related to the mammalian SULT1 subfamily. Additionally, the bullfrog has two SULT1Cc subfamily members that are consistent with the characteristics of the expanded Xenopus SULT1C subfamily. Several members of the bullfrog SULT1 family were suggested to play important roles in sulfation during metamorphosis. Among these, cDNAs encoding SULT1Cc1 and SULT1Y1 were cloned, and the sulfation activity was analyzed using recombinant proteins. The affinity for 2-naphthol and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the enzymatic reaction rate were higher in SULT1Cc1 than in SULT1Y1. Both the enzymes showed inhibitory effect of many thyroid hormones (THs) analogs on the sulfation of 2-naphthol. The potency of sulfation activities of SULT1Cc1 and SULT1Y1 against T4 indicated their possible role in the intracellular T4 clearance during metamorphosis.

Gastrointestinal parasites in bullfrogs (Lithobates catesbeianus) in aquaculture production units in the Mexican central highlands.

In Mexico, intensive production of bullfrogs is one of the most important aquaculture activities, due to growing demand for their meat. Frogs can be hosts for several parasites that negatively affect their development and health. The objective of this study was to identify the presence of intestinal parasites in bullfrogs in aquaculture production units. Eighteen bullfrogs aquaculture production units were selected, and 20 animals (n=360) from each farm. Fecal samples were obtained by mucosal scraping and processed using the concentration method. The overall prevalence of intestinal parasites was 70.5%, and all farms had frogs infected by some species of parasite. Two species of parasites were identified: Eimeria sp. and Strongyloides sp. Significant differences were found regarding parasite prevalence between males and females (73.8% vs 58.8%) and regarding tibia length (5.5 vs 6.1 cm) and weight (168 vs 187 g) between parasitized and non-parasitized frogs. In conclusion, the present study showed a high prevalence of intestinal parasites, and morphometric alterations (weight, snout-cloaca length, radio-ulna length, tibia length and distance between parotid glands) were identified in the parasitized animals. These results provided useful information that will enable establishment of adequate control measures to help minimize the adverse effects of these parasites.

KEEPING THE HEAT ON: WEIGHTED SURVEILLANCE FOR CHYTRID FUNGUS (BATRACHOCHYTRIUM DENDROBATIDIS) IN DIXIE VALLEY TOADS (ANAXYRUS [= BUFO] WILLIAMSI).

Introduced fungal pathogens have caused declines and extinctions of naïve wildlife populations across vertebrate classes. Consequences of introduced pathogens to hosts with small ranges might be especially severe because of limited redundancy to rescue populations and lower abundance that may limit the resilience of populations to perturbations like disease introduction. As a complement to biosecurity measures to prevent the spread of pathogens, surveillance programs may enable early detection of pathogens, when management actions to limit the effects of pathogens on naïve hosts might be most beneficial. We analyzed surveillance data for the endangered and narrowly endemic Dixie Valley toad (Anaxyrus [= Bufo] williamsi) from two time periods (2011-2014 and 2019-2021) to estimate the minimum detectable prevalence of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). We assessed if detection efficiency could be improved by using samples from both Dixie Valley toads and co-occurring introduced American bullfrogs (Lithobates catesbeianus) and literature-derived surveillance weights. We further evaluated a weighted surveillance design to increase the efficiency of surveillance efforts for Bd within the toad's small (<6 km2) range. We found that monitoring adult and larval American bullfrogs would probably detect Bd more efficiently than monitoring Dixie Valley toads alone. Given that no Bd was detected, minimum detectable prevalence of Bd was <3% in 2011-2014, and <5% (Dixie Valley toads only) and <10% (American bullfrogs only) in 2019-2021. Optimal management for Bd depends on the mechanisms underlying its apparent absence from the range of Dixie Valley toads, but a balanced surveillance scheme that includes sampling American bullfrogs to increase the likelihood of detecting Bd, and adult Dixie Valley toads to ensure broad spatial coverage where American bullfrogs do not occur, would probably result in efficient surveillance, which might permit timely management of Bd if it is detected.