Definition of a saxitoxin (STX) binding code enables discovery and characterization of the anuran saxiphilin family.

American bullfrog (Rana castesbeiana) saxiphilin (RcSxph) is a high-affinity "toxin sponge" protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (NaVs). How specific RcSxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX "recognition code" enabled engineering of RcSxph to improve its ability to rescue NaVs from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by ∼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.

Phenological mismatches mitigate the ecological impact of a biological invader on amphibian communities.

Horizon scans have emerged as a valuable tool to anticipate the incoming invasive alien species (IAS) by judging species on their potential impacts. However, little research has been conducted on quantifying actual impacts and assessing causes of species-specific vulnerabilities to particular IAS due to persistent methodological challenges. The underlying interspecific mechanisms driving species-specific vulnerabilities therefore remain poorly understood, even though they can substantially improve the accuracy of risk assessments. Given that interspecific interactions underlying ecological impacts of IAS are often shaped by phenological synchrony, we tested the hypothesis that temporal mismatches in breeding phenology between native species and IAS can mitigate their ecological impacts. Focusing on the invasive American bullfrog (Lithobates catesbeianus), we combined an environmental DNA (eDNA) quantitative barcoding and metabarcoding survey in Belgium with a global meta-analysis, and integrated citizen-science data on breeding phenology. We examined whether the presence of native amphibian species was negatively related to the presence or abundance of invasive bullfrogs and whether this relationship was affected by their phenological mismatches. The field study revealed a significant negative effect of increasing bullfrog eDNA concentrations on native amphibian species richness and community structure. These observations were shaped by species-specific vulnerabilities to invasive bullfrogs, with late spring- and summer-breeding species being strongly affected, while winter-breeding species remained unaffected. This trend was confirmed by the global meta-analysis. A significant negative relationship was observed between phenological mismatch and the impact of bullfrogs. Specifically, native amphibian species with breeding phenology differing by 6 weeks or less from invasive bullfrogs were more likely to be absent in the presence of bullfrogs than species whose phenology differed by more than 6 weeks with that of bullfrogs. Taken together, we present a novel method based on the combination of aqueous eDNA quantitative barcoding and metabarcoding to quantify the ecological impacts of biological invaders at the community level. We show that phenological mismatches between native and invasive species can be a strong predictor of invasion impact regardless of ecological or methodological context. Therefore, we advocate for the integration of temporal alignment between native and IAS's phenologies into invasion impact frameworks.

Rosmarinic acid mitigates intestinal inflammation and oxidative stress in bullfrogs (Lithobates catesbeiana) fed high soybean meal diets.

High proportions of soybean meal in aquafeed have been confirmed to induce various intestinal pathologies. This study aims to investigate the regulatory effects of rosmarinic acid (RA), an antioxidant with anti-inflammatory and antimicrobial properties, when added to high soybean meal feeds in different doses, (0, 0.5, 1, and 4 g/kg). During the 56-day feeding trial, results indicated that, compared to the control group without RA (0 g/kg), the 1 g/kg and 4 g/kg RA groups increased bullfrog survival rates and total weight gain while reducing feed coefficient. Additionally, these doses markedly suppressed the expression of key intestinal inflammatory markers (tlr5, myd88, tnfα, il1ß, cxcl8, cxcl12) and the activity and content of intestinal antioxidants (CAT, MDA, GSH, GPX). Concurrently, RA significantly downregulated the transcription levels of antioxidant-related genes (cat, gpx5, cyba, cybb, mgst, gclc, gsta, gstp), suggesting RA's potential to alleviate intestinal inflammation and oxidative stress induced by high soybean meal and to help downregulate and restore normal expression of antioxidant enzyme genes. However, the 0.5 g/kg RA group did not show a significant improvement in survival rates; instead, it upregulated the transcription of some antioxidant genes (cat, gpx5, cyba, cybb), revealing the complexity and dose-dependency of RA's antioxidant action. Furthermore, RA supplementation significantly reshaped the intestinal microbial community structure and relative abundance in bullfrogs, particularly affecting the genera Hafnia, Phascolarctobacterium, and Lactococcus. Notably, high doses of RA (1 g/kg, 4 g/kg) were able to downregulate pathways associated with the enrichment of gut microbiota in diseases such as Parkinson's, Staphylococcus aureus infection, and Systemic lupus erythematosus, suggesting its potential in anti-inflammatory action and health maintenance to prevent potential diseases.

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.

Recovery of the baroreflex and autonomic modulation after anesthesia with MS-222 in bullfrogs.

The time course for recovery after anesthesia is poorly described for tricaine methanesulfonate (MS-222). We suggest that the baroreflex and the heart rate variability (HRV) could be used to index the recovery of the autonomic modulation after anesthesia. We analyzed the recovery profile of behavioral and physiological parameters over time to analyze the progression of recovery after anesthesia of American bullfrogs with MS-222. Mean heart rate stabilized after 17 h, whereas the baroreflex efficiency index took 23 h and the baroreflex operating gain, 29 h. Mean arterial pressure recovered after 26 h. Power spectral density peaked at 23 h and again after 40 h. Baroreflex was a relevant component of the first phase of HRV, while autonomic modulation for resting may take longer than 40 h. We suggest that physiological recovery is a complex phenomenon with multiple progressive phases, and the baroreflex may be a useful tool to observe the first substantial recovery of post-instrumentation capacity for autonomic modulation.

Morphological and molecular data establishes Clinostomum dolichorchum n. sp. (Digenea: Clinostomidae) in the great blue heron Ardea herodias L. and American bullfrog Rana catesbeiana Shaw.

Clinostomum is a cosmopolitan genus of trematodes that infect piscivorous birds, freshwater molluscs, freshwater fish and amphibians. Herein, a novel species of Clinostomum is described based on morphological and molecular data from an adult in the oral cavity of the great blue heron Ardea herodias and metacercariae collected from the gills and skin of American bullfrog tadpoles Rana catesbeiana. The novel species shares similar qualitative and quantitative morphological features with a congener, Clinostomum marginatum, which has overlap in host and geographic distribution. The most notable morphological difference when compared to C. marginatum is the greater posterior testis length of the novel species. Molecular data resolved similarities with morphological comparisons to nominal species and supports the establishment of a novel species. Molecular data include partial small ribosomal subunit (18S rRNA gene), ribosomal internal transcribed spacer regions (ITS1, 5.8S rRNA gene, and ITS2), partial large ribosomal subunit (28S rRNA gene), cytochrome c oxidase subunit 1 gene (cox1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene (nad1) sequences. Phylogenetic analyses place the novel species in a sister clade to C. marginatum. Morphological and molecular data, combined with phylogenetic analyses support the establishment of Clinostomum dolichorchum n. sp.

Behavioral and biochemical consequences after chronic exposition to the herbicide atrazine in tadpoles.

Atrazine (ATZ) is the third most sold herbicide in Brazil, occupying the seventh position between most widely used pesticides. Due to its easy outflow, low reactivity and solubility, moderate adsorption to organic matter and clay, and long soil persistence, residual herbicide can be identified after long periods following application, and its usage has been prohibited in diverse countries. Amphibians are important bioindicators to assess impact of pesticide like atrazine, due to having a partial aquatic life cycle. This study had as objective to assess the response of bullfrog (Lithobates catesbeianus) tadpoles when exposed to this herbicide. Animals were exposed for a total of 168h to following concentrations: negative control, 40 µg/L, 200 µg/L, 2000 µg/L, 20000 µg/L of ATZ. Analysis of swimming activity was performed, and biochemical profile was assessed by analysis of blood and plasma glucose levels, urea, creatinine, cholesterol, HDL, triglycerides, glutamic pyruvic transaminase (GPT), alkaline phosphatase (AP), calcium, total proteins, phenol, peroxidase and polyphenol oxidase activity. Results exhibited malnutrition, anemia, likely muscle mass loss, and hepatic damage, indicating that ATZ can lead to an increase in energy to maintain homeostasis for animal survival.

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.

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.

Empirical evidence for effects of invasive American Bullfrogs on occurrence of native amphibians and emerging pathogens.

Invasive species and emerging infectious diseases are two of the greatest threats to biodiversity. American Bullfrogs (Rana [Lithobates] catesbeiana), which have been introduced to many parts of the world, are often linked with declines in native amphibians via predation and the spread of emerging pathogens such as amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]) and ranaviruses. Although many studies have investigated the potential role of bullfrogs in the decline of native amphibians, analyses that account for shared habitat affinities and imperfect detection have found limited support for clear effects. Similarly, the role of bullfrogs in shaping the patch-level distribution of pathogens is unclear. We used eDNA methods to sample 233 sites in the southwestern USA and Sonora, Mexico (2016-2018) to estimate how the presence of bullfrogs affects the occurrence of four native amphibians, Bd, and ranaviruses. Based on two-species, dominant-subordinate occupancy models fitted in a Bayesian context, federally threatened Chiricahua Leopard Frogs (Rana chiricahuensis) and Western Tiger Salamanders (Ambystoma mavortium) were eight times (32% vs. 4%) and two times (36% vs. 18%), respectively, less likely to occur at sites where bullfrogs occurred. Evidence for the negative effects of bullfrogs on Lowland Leopard Frogs (Rana yavapaiensis) and Northern Leopard Frogs (Rana pipiens) was less clear, possibly because of smaller numbers of sites where these native species still occurred and because bullfrogs often occur at lower densities in streams, the primary habitat for Lowland Leopard Frogs. At the community level, Bd was most likely to occur where bullfrogs co-occurred with native amphibians, which could increase the risk to native species. Ranaviruses were estimated to occur at 33% of bullfrog-only sites, 10% of sites where bullfrogs and native amphibians co-occurred, and only 3% of sites where only native amphibians occurred. Of the 85 sites where we did not detect any of the five target amphibian species, we also did not detect Bd or ranaviruses; this suggests other hosts do not drive the distribution of these pathogens in our study area. Our results provide landscape-scale evidence that bullfrogs reduce the occurrence of native amphibians and increase the occurrence of pathogens, information that can clarify risks and aid the prioritization of conservation actions.

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.

First evidence of ranavirus in native and invasive amphibians in Colombia.

Ranaviruses can cause mass mortality events in amphibians, thereby becoming a threat to populations that are already facing dramatic declines. Ranaviruses affect all life stages and persist in multiple amphibian hosts. The detrimental effects of ranavirus infections to amphibian populations have already been observed in the UK and in North America. In Central and South America, the virus has been reported in several countries, but the presence of the genus Ranavirus (Rv) in Colombia is unknown. To help fill this knowledge gap, we surveyed for Rv in 60 species of frogs (including one invasive species) in Colombia. We also tested for co-infection with Batrachochytrium dendrobatidis (Bd) in a subset of individuals. For Rv, we sampled 274 vouchered liver tissue samples collected between 2014 and 2019 from 41 localities covering lowlands to mountaintop páramo habitat across the country. Using quantitative polymerase chain reaction (qPCR) and end-point PCR, we detected Rv in 14 individuals from 8 localities, representing 6 species, including 5 native frogs of the genera Osornophryne, Pristimantis and Leptodactylus, and the invasive American bullfrog Rana catesbeiana. Bd was detected in 7 of 140 individuals, with 1 co-infection of Rv and Bd in an R. catesbeiana specimen collected in 2018. This constitutes the first report of ranavirus in Colombia and should set off alarms about this new emerging threat to amphibian populations in the country. Our findings provide some preliminary clues about how and when Rv may have spread and contribute to understanding how the pathogen is distributed globally.

Internal fluid pressure influences muscle contractile force.

Fluid fills intracellular, extracellular, and capillary spaces within muscle. During normal physiological activity, intramuscular fluid pressures develop as muscle exerts a portion of its developed force internally. These pressures, typically ranging between 10 and 250 mmHg, are rarely considered in mechanical models of muscle but have the potential to affect performance by influencing force and work produced during contraction. Here, we test a model of muscle structure in which intramuscular pressure directly influences contractile force. Using a pneumatic cuff, we pressurize muscle midcontraction at 260 mmHg and report the effect on isometric force. Pressurization reduced isometric force at short muscle lengths (e.g., -11.87% of P0 at 0.9 L0), increased force at long lengths (e.g., +3.08% of P0 at 1.25 L0), but had no effect at intermediate muscle lengths ∼1.1-1.15 L0 This variable response to pressurization was qualitatively mimicked by simple physical models of muscle morphology that displayed negative, positive, or neutral responses to pressurization depending on the orientation of reinforcing fibers representing extracellular matrix collagen. These findings show that pressurization can have immediate, significant effects on muscle contractile force and suggest that forces transmitted to the extracellular matrix via pressurized fluid may be important, but largely unacknowledged, determinants of muscle performance in vivo.

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.

Persistent augmentation of fictive air breathing by hypoxia: An in vitro study of the role of GABAB signaling in pre-metamorphic tadpoles.

Tadpole development is influenced by environmental cues and hypoxia can favor the emergence of the neural networks driving air breathing. Exposing isolated brainstems from pre-metamorphic tadpoles to acute hypoxia (∼0% O2; 15 min) leads to a progressive increase in fictive air breaths (∼3 fold) in the hours that follow stimulation. Here, we first determined whether this effect persists over longer periods (<18 h); we then evaluated maturity of the motor output by comparing the breathing pattern of hypoxia-exposed brainstems to that of preparations from adult bullfrogs under basal conditions. Because progressive withdrawal of GABAB-mediated inhibition contributes to the developmental increase in fictive lung ventilation, we then hypothesised that hypoxia reduces respiratory sensitivity to baclofen (selective GABAB-agonist). Experiments were performed on isolated brainstem preparations from pre-metamorphic tadpoles (TK stages IV to XIV); respiratory-related neural activity was recorded from cranial nerves V/VII and X before and 18 h after exposure to hypoxia (0% O2 + 2% CO2; 25 min). Time-control experiments (no hypoxia) were performed. Exposing pre-metamorphic tadpoles to hypoxia did not affect gill burst frequency, but augmented the frequency of fictive lung bursts and the incidence of episodic breathing levels intermediate between pre-metamorphic and adult preparations. Addition of baclofen to the aCSF (0,2 µM - 20 min) reduced lung burst frequency, but the response of hypoxia-exposed brainstems was greater than controls. We conclude that acute hypoxia facilitates development and maturation of the motor command driving air breathing. We propose that a greater number of active rhythmogenic neurons expressing GABAb receptors contributes to this effect.

Molecular profiling of CO2/pH-sensitive neurons in the locus coeruleus of bullfrogs reveals overlapping noradrenergic and glutamatergic cell identity.

Locus coeruleus (LC) neurons regulate breathing by sensing CO2/pH. Neurons within the vertebrate LC are the main source of norepinephrine within the brain. However, they also use glutamate and GABA for fast neurotransmission. Although the amphibian LC is recognized as a site involved in central chemoreception for the control of breathing, the neurotransmitter phenotype of these neurons is unknown. To address this question, we combined electrophysiology and single-cell quantitative PCR to detect mRNA transcripts that define norepinephrinergic, glutamatergic, and GABAergic phenotypes in LC neurons activated by hypercapnic acidosis (HA) in American bullfrogs. Most LC neurons activated by HA had overlapping expression of noradrenergic and glutamatergic markers but did not show strong support for GABAergic transmission. Genes that encode the pH-sensitive K+ channel, TASK2, and acid-sensing cation channel, ASIC2, were most abundant, while Kir5.1 was present in 1/3 of LC neurons. The abundance of transcripts related to norepinephrine biosynthesis linearly correlated with those involved in pH sensing. These results suggest that noradrenergic neurons in the amphibian LC also use glutamate as a neurotransmitter and that CO2/pH sensitivity may be linkedto the noradrenergic cell identity.

Atrazine contaminated sites and bullfrog tadpoles: evasive trends and biochemical consequences.

Atrazine (ATZ) is one of the most used active principles in agricultural systems. This pesticide has the ability to easily accumulate in terrestrial and aquatic environments, causing impacts with chronic adverse effects. Avoidance tests are tests that seek to assess the concentration from which a given organism escapes, that is, migrates to another habitat. They are being used as a modality of innovative and minimally invasive ecotoxicological tests. Our objective was to evaluate the sensitivity and possible toxic effects of ATZ in bullfrog tadpoles (Lithobates catesbeianus), through avoidance tests and oxidative stress analyses. We performed the behavioral avoidance test lasting 12 h, with observations every 60 min in a linear multi-compartment system with seven compartments. Each compartment corresponded to a concentration: negative control, 1, 2, 20, 200, 2000, 20,000 µg L-1. After the selection of habitat, organisms were forcedly maintained in the chosen concentrations for 48 h and then, metabolic effects were measured assessing the blood plasma amino acid profile and liver protein degradation. We also determined the effective concentrations of ATZ tested at 0 h and 48 h. The results showed that there was an effect of the treatment on the distribution of tadpoles, but not on the hours or on the combined effect (interaction). The biochemical analyses also showed a concentration-dependent relationship which caused significant toxic effects even in a short period of time. In conclusion, these frogs were able to avoid places with high concentrations of ATZ in the first hours of exposure, which suggests that in the natural environment these animals can migrate or avoid areas contaminated by this herbicide; however, depending on the selected concentration, serious biochemical consequences can occur.

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.

Identification and pathogenicity of multidrug-resistant Elizabethkingia miricola isolated from farmed American bullfrogs Rana catesbeiana in China with in vitro screening of herbal antimicrobial agents.

OBJECTIVE: In 2021, an outbreak of an infectious disease characterized by torticollis, cataracts, and neurological disorders caused massive mortality in farmed American bullfrogs Rana catesbeiana in Hubei province, China. We identified the causal agent in this outbreak, characterized its pathogenicity, and screened candidate antimicrobial agents for future disease control. METHODS: Bacterium was isolated from the diseased American bullfrogs and identified based on biochemical tests, sequence analyses (16S ribosomal RNA; DNA gyrase subunit B), and experimental challenge. Furthermore, antibiotic sensitivity of the isolated strain was detected with Kirby-Bauer paper diffusion method, and the antibacterial activity of 60 traditional Chinese herbal extracts against the isolated strain was evaluated by agar disc diffusion and broth dilution assays. RESULT: We identified Elizabathkingia miricola strain FB210601 as the causative agent of this disease. The isolated E. miricola strain FB210601 exhibited extensive antibiotic resistance to all tested quinolones, ß-lactam antibiotics, and aminoglycosides. Eight herbal extracts exhibited excellent antimicrobial activity against E. miricola FB210601, especially Caesalpinia sappan and Rhus chinensis, with minimal inhibitory concentrations less than 0.2 mg/mL. Additionally, the combined effects of two-component herbal mixtures containing C. sappan or R. chinensis were greater than those of the individual extracts. CONCLUSION: Our results provide a reference for understanding the pathogenesis of Elizabethkingia infection in frogs. Furthermore, this study will aid in the application of herbal extracts for protection against infections caused by multidrug-resistant Elizabathkingia in the future.

Phase-Locking Requires Efficient Ca2+ Extrusion at the Auditory Hair Cell Ribbon Synapse.

Proper perception of sounds in the environment requires auditory signals to be encoded with extraordinary temporal precision up to tens of microseconds, but how it originates from the hearing organs in the periphery is poorly understood. In particular, sound-evoked spikes in auditory afferent fibers in vivo are phase-locked to sound frequencies up to 5 kHz, but it is not clear how hair cells can handle intracellular Ca2+ changes with such high speed and efficiency. In this study, we combined patch-clamp recording and two-photon Ca2+ imaging to examine Ca2+ dynamics in hair cell ribbon synapses in the bullfrog amphibian papilla of both sexes. We found that Ca2+ clearance from single synaptic ribbons followed a double exponential function, and the weight of the fast component, but not the two time constants, was significantly reduced for prolonged stimulation, and during inhibition of the plasma membrane Ca2+ ATPase (PMCA), the mitochondrial Ca2+ uptake (MCU), or the sarcolemma/endoplasmic reticulum Ca2+ ATPase (SERCA), but not the Na+/Ca2+ exchanger (NCX). Furthermore, we found that both the basal Ca2+ level and the Ca2+ rise during sinusoidal stimulation were significantly increased by inhibition of PMCA, MCU, or SERCA. Consistently, phase-locking of synaptic vesicle releases from hair cells was also significantly reduced by blocking PMCA, MCU, or SERCA, but not NCX. We conclude that, in addition to fast diffusion mediated by mobile Ca2+ buffer, multiple Ca2+ extrusion pumps are required for phase-locking at the auditory hair cell ribbon synapse.SIGNIFICANCE STATEMENT Hair cell synapses can transmit sound-driven signals precisely in the kHz range. However, previous studies of Ca2+ handling in auditory hair cells have often been conducted in immature hair cells, with elevated extracellular Ca2+ concentration, or through steady-state stimulation that may not be physiologically relevant. Here we examine Ca2+ clearance from hair cell synaptic ribbons in a fully mature preparation at physiological concentration of external Ca2+ and at physiological temperature. By stimulating hair cells with sinusoidal voltage commands that mimic pure sound tones, we recapitulated the phase-locking of hair cell exocytosis with an in vitro approach. This allowed us to reveal the Ca2+ extrusion mechanisms that are required for phase-locking at auditory hair cell ribbon synapses.