Bullfrog Research Articles

Hibernation reduces GABA signaling in the brainstem to enhance motor activity of breathing at cool temperatures

BackgroundNeural circuits produce reliable activity patterns despite disturbances in the environment. For this to occur, neurons elicit synaptic plasticity during perturbations. However, recent work suggests that plasticity not only regulates circuit activity during disturbances, but these modifications may also linger to stabilize circuits during future perturbations. The implementation of such a regulation scheme for real-life environmental challenges of animals remains unclear. Amphibians provide insight into this problem in a rather extreme way, as circuits that generate breathing are inactive for several months during underwater hibernation and use compensatory plasticity to promote ventilation upon emergence.ResultsUsing ex vivo brainstem preparations and electrophysiology, we find that hibernation in American bullfrogs reduces GABAA receptor (GABAAR) inhibition in respiratory rhythm generating circuits and motor neurons, consistent with a compensatory response to chronic inactivity. Although GABAARs are normally critical for breathing, baseline network output at warm temperatures was not affected. However, when assessed across a range of temperatures, hibernators with reduced GABAAR signaling had greater activity at cooler temperatures, enhancing respiratory motor output under conditions that otherwise strongly depress breathing.ConclusionsHibernation reduces GABAAR signaling to promote robust respiratory output only at cooler temperatures. Although frogs do not ventilate lungs during underwater hibernation, we suggest this would be beneficial for stabilizing breathing when the animal passes through a large temperature range during emergence in the spring. More broadly, these results demonstrate that compensatory synaptic plasticity can increase the operating range of circuits in harsh environments, thereby promoting adaptive behavior in conditions that suppress activity.

The overlooked shadow killer: Elizabethkingia anophelis as a potent pathogen in American bullfrogs (Aquarana catesbeiana)

The overlooked shadow killer: Elizabethkingia anophelis as a potent pathogen in American bullfrogs (Aquarana catesbeiana)

Combining graph theory and spatially-explicit, individual-based models to improve invasive species control strategies at a regional scale

ContextInvasive species cause widespread species extinction and economic loss. There is an increasing need to identify ways to efficiently target control efforts from local to regional scales.ObjectivesOur goal was to test whether prioritizing managed habitat using different treatments based on spatial measures of connectivity, including graph-theoretic measures, can improve management of invasive species and whether the level of control effort affects treatment performance. We also explored how uncertainty in biological variables, such as dispersal ability, affects measures performance.MethodsWe used a spatially-explicit, individual-based model (sIBM) based on the American bullfrog (Lithobates catesbeianus), a globally pervasive invasive species. Simulations were informed by geographic data from part of the American bullfrog’s non-native range in southeastern Arizona, USA where they are known to pose a threat to native species.ResultsWe found that total bullfrog populations and occupancy declined in response to all treatments regardless of effort level or patch prioritization methods. The most effective spatial prioritization was effort-dependent and varied depending on spatial context, but frequently a buffer strategy was most effective. Treatments were also sensitive to dispersal ability. Performance of treatments prioritizing habitat patches using betweenness centrality improved with increasing dispersal ability, while performance of eigenvalue centrality improved as dispersal ability decreased.ConclusionsWith the careful application of connectivity measures to prioritize control efforts, similar reductions in invasive species population size and occupancy could be achieved with less than half the effort of sub-optimal connectivity measures at higher effort rates. More work is needed to determine if trait-based generalities may define appropriate connectivity measures for specific suites of dispersal abilities, demographic traits, and population dynamics.

Dynamics of Amphibian Pathogen Detection Using Extended Museum Specimens.

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection.

Hibernation reduces GABA signaling in the brainstem to enhance motor activity of breathing at cool temperatures.

Neural circuits produce reliable activity patterns despite disturbances in the environment. For this to occur, neurons elicit synaptic plasticity during perturbations. However, recent work suggests that plasticity not only regulates circuit activity during disturbances, but these modifications may also linger to stabilize circuits during future perturbations. The implementation of such a regulation scheme for real-life environmental challenges of animals remains unclear. Amphibians provide insight into this problem in a rather extreme way, as circuits that generate breathing are inactive for several months during underwater hibernation and use compensatory plasticity to promote ventilation upon emergence. Using ex vivo brainstem preparations and electrophysiology, we find that hibernation in American bullfrogs reduces GABA A receptor (GABA A R) inhibition in respiratory rhythm generating circuits and motor neurons, consistent with a compensatory response to chronic inactivity. Although GABA A Rs are normally critical for breathing, baseline network output at warm temperatures was not affected. However, when assessed across a range of temperatures, hibernators with reduced GABA A R signaling had greater activity at cooler temperatures, enhancing respiratory motor output under conditions that otherwise strongly depress breathing. Hibernation reduces GABA A R signaling to promote robust respiratory output only at cooler temperatures. Although animals do not ventilate lungs during hibernation, we suggest this would be beneficial for stabilizing breathing when the animal passes through a large temperature range during emergence in the spring. More broadly, these results demonstrate that compensatory synaptic plasticity can increase the operating range of circuits in harsh environments, thereby promoting adaptive behavior in conditions that suppress activity.

The first report of prion protein gene sequences in Dybowski’s frog and the American bullfrog: high amyloid propensity of the frog prion protein

Prion diseases are fatal infectious neurodegenerative diseases caused by the proteinase K-sensitive form of prion protein (PrPSc). The exact origin of prion seeding and the transition factor of PrPSc has not been elucidated. The main hosts of prion diseases are herbivores, so the feces and corpses of Amphibians can seed PrPSc through ecosystems. The frog is an excellent candidate for transmission studies for this reason, but genetic analyses of the prion protein gene (PRNP) in the context of prion-related characteristics of frog species are lacking. We amplified frog PRNP gene sequences in Dybowski’s frog and the American bullfrog by polymerase chain reaction (PCR) and amplicon sequencing. In addition, we carried out multiple sequencing alignments and annotated major PrP components including signal peptide, tandem repeat domain, and PrPC-PrPSc interaction region of frog PrPs by bioinformatics tools. We predicted secondary and tertiary structures and amyloid propensities of frog PrPs using AlphaFold2 and AMYCO, respectively. We obtained DNA sequences of the PRNP gene in Dybowski’s frog and the American bullfrog, as well as a partially conserved palindromic sequence (PrPC-PrPSc interaction region) and absence of tandem repeat region of PrP in seven frog species. We analyzed protein structure of among these frog species and found that the high Himalaya frog has high aggregation propensity and the western clawed frog does not have the N-terminal signal peptide. To the best of our knowledge, this was the first comparative genetic study regarding prion-related features of frog species.

Increased Reaction Vessel Surface Area Decreases the Overall Mortality Rate of Rana catesbeiana Larvae during Chemically Induced Metamorphosis

Stimulating precocious metamorphosis in anuran larvae is an important pedagogical tool for understanding vertebrate development. However, historically, artificially provoking metamorphosis by immersing tadpoles in exogenous inducing agents (e.g., thyroxine, and iodine) compromises the longevity of the experimental animals, resulting in up to 100% mortality within a week. In our undergraduate teaching lab, we house our experimental tadpoles in circular glass dishes having a surface area of 182 cm2. Over the past four academic years this lab was performed, we observed 100% mortality of experimental animals within 10, 12, or 15 days when treated with 10-5 M, 10-6 M, or 10-7 M thyroxine, respectively. Here, we investigated whether increasing the surface area to 413 cm2 using square glass dishes would reduce the mortality of the treated animals. Omnibus Kaplan-Meier survival analysis demonstrates a statistically significant decrease in mortality in tadpoles reared in the larger square dishes compared to those housed in the smaller round dishes (P < 0.05). However, increasing the surface area of our reaction vessels could not rescue survivability of those tadpoles immersed in thyroxine, but did increase survivability of control tadpoles maintained in pond water (P < 0.01), tadpoles subjected to iodine (P < 0.05) or treated with actinomycin D (P < 0.05). These data demonstrate that increasing available reaction vessel surface area reduces overall tadpole mortality during chemically modified metamorphosis in an undergraduate teaching lab setting.

Anuran occupancy varies with stream characteristics and flow across Arizona wilderness areas

Abstract Riparian ecosystems comprise less than 2% of the landscape in the arid western United States, yet they provide habitat and resources to over half of arid‐land wildlife species, including a broad diversity of anurans (frogs and toads). Despite this, relatively few studies describe riparian herpetofaunal communities in the Southwest, and remote wilderness areas are particularly undersurveyed. We employed passive acoustic monitoring to capture anuran advertisement calls in wilderness area tributaries of the Verde River, Arizona, USA. In the spring and summer of 2021 and 2022, 13–29 autonomous recording units (ARUs) were deployed along perennial, intermittent and ephemeral tributary stream reaches. We characterized sites based on the per cent of pool, riffle, run and side channel habitat within 100 m of each ARU and quantified substrate, wetted channel width and canopy cover. To relate anuran occupancy to environmental and hydrological variables, we evaluated acoustic detection data using single‐species, single‐season occupancy models. Four species were detected in this study: canyon treefrog (Hyla arenicolor), red‐spotted toad (Anaxyrus punctatus), Woodhouse's toad (Anaxyrus woodhousii) and non‐native American bullfrog (Lithobates catesbeianus), with canyon treefrog being the most ubiquitous species observed. Vocal activity of canyon treefrog was higher at perennial and intermittent sites compared to ephemeral sites, and the presence of pools was most strongly associated with canyon treefrog occupancy. Persistent, slow‐moving water best predicted the presence of breeding canyon treefrogs. Notably, this study did not detect several species with historical records in the middle Verde River catchment, including Arizona toad (Anaxyrus microscaphus) and northern leopard frog (Lithobates pipiens). Given climate change‐related flow declines and intensifying demands for water in arid lands globally, maintaining stream flows that provide consistent and suitable hydroregimes for anuran breeding and larval development is of increasing importance. Determining habitat use and flow regimes necessary to support anuran populations can aid in prioritization of conservation actions related to water management and predict how changes in water availability may impact stream‐breeding anurans.

Transport of American Bullfrogs with Moistened Foam and without Foam: Plasma Biochemistry and Erythrogram Responses

This study aimed to evaluate two transportation methods (with moistened foam and without foam) for 10 h on blood parameters of bullfrogs 0, 6, 12, 24, and 48 h after transportation. There was no mortality. The glucose increased at 0 and 12 h after transportation and returned to baseline at 24 h in both transportations. Triglycerides increased at 0 and 6 h in both transportations and were restored 12 h after transport with foam and 24 h in transport without foam. Plasma proteins and globulins increased at 0 h after transportation under both transportations. After 48 h, there was a reduction in transport without foam. Globulins decreased 48 h under both transportations. Albumin increased at 12, 24, and 48 h after both transportations. Transport with foam had high albumin. The albumin/globulin ratio increased 24 and 48 h after both transportations. The number of erythrocytes increased at 0 h and recovered after 6 h in transport with foam and 12 h in transport without foam. Hematocrit and hemoglobin increased at 0 h and recovered at 6 h in both transportations. MCV increased 48 h after transportation with foam. MCHC decreased 12, 24, and 48 h after both transportations. MCH was lower in the transport carried out with foam.

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.

DESCRIPTIONS AND PHYLOGENETIC AFFINITIES OF TWO NEW SPECIES OF RHABDIAS STILES AND HASSALL, 1905 (NEMATODA: RHABDIASIDAE) FROM NORTH AMERICAN FROGS: ARE WE STILL ONLY SCRATCHING THE SURFACE?

Two new species of lung-dwelling nematodes are described from North American frogs: Rhabdias aurorae n. sp. from Rana aurora and Rhabdias conni n. sp. from Rana clamitans and Rana catesbeiana from Arkansas; the latter species was also found in Oklahoma and Georgia. Rhabdias aurorae n. sp. differs from other Nearctic congeners in the combination of the following characteristics: buccal capsule 22-25 μm wide, elongated tail covered with inflated cuticle, esophagus with prominent dilatation in anterior part and 6 small circumoral lips. Rhabdias conni n. sp. is morphologically closest to Rhabdias ranae Walton, 1929 and Rhabdias joaquinensisIngles, 1936; it differs from them in the shape of lateral pseudolabia, the dimensions of the body, and the egg size. Both new species were found to be significantly different from the Nearctic congeners in the nucleotide sequences of nuclear ribosomal DNA (18S-ITS-28S region), 12S, and CO1 mitochondrial genes. The 2 new species differ from other currently sequenced Nearctic congeners by 1.1-2.7% of nucleotide positions in the nuclear rDNA region, 1.3-3.4% in the 12S gene, and 3.4-9.4% in CO1 gene. Molecular phylogenetic analysis based on nuclear ribosomal DNA sequences placed both new species into the clade consisting of Nearctic and Neotropical Rhabdias spp. The position of Rh. aurorae n. sp. within the clade is uncertain because of a polytomy, but Rh. conni n. sp. is nested within the "Rh. joaquinensis complex" related to Rh. ranae and Rhabdias tarichaeKuzmin, Tkach, and Snyder, 2003. The phylogenetic analysis based on nuclear ribosomal DNA sequences has revealed 3 evolutionary host-switching events from anuran to caudatan hosts among Rhabdias spp. that occurred in the Nearctic and Palearctic. The molecular phylogeny also suggests that Rhabdias may have originally evolved in what is now Africa.

Chicken Meal as a Fishmeal Substitute: Effects on Growth, Antioxidants, and Digestive Enzymes in Lithobates catesbeianus.

In pursuit of sustainable aquaculture, this study was performed to evaluate chicken meal as a substitute for fishmeal in bullfrog diets. Three experimental groups were established: a control group (FM) with 20% fishmeal, a CM50 group with 50% replacement (10% fishmeal), and a CM100 group with 100% replacement (0 fishmeal). Bullfrogs were fed for 56 days. The CM50 group exhibited significant increases in total weight gain and survival rate and a notable decrease in feed coefficient (p < 0.05). However, the CM100 group showed contrary effects. Increasing chicken meal substitution correlated with decreased amino acid content in muscle. Notably, the CM50 group demonstrated enhanced activities of antioxidant enzymes (CAT, T-AOC) and elevated gene expression levels (cat, sod, gst, etc.) in muscle and the intestine (p < 0.05), improved intestinal morphology, enhanced digestive enzyme activities (amylase, lipase), and reduced expression of inflammatory factors (il-1β, il-8, il-17, etc.). Conversely, the CM100 group's indicators regressed to levels similar to or worse than those of the FM group. Therefore, a 50% substitution of fishmeal with chicken meal effectively promoted bullfrog survival, protected the intestines, and enhanced antioxidant capacity, supporting its potential as a fishmeal alternative. However, the adverse outcomes of the CM100 strategy, including growth retardation and reduced amino acid content in muscle, indicate that complete replacement is unsuitable.

Technological and Bioactive Properties of Collagen Extracts Obtained from Bullfrog (Lithobates catesbeianus) Skin

ABSTRACT The objective was to extract type I collagen and determine its ideal extraction conditions, technological parameters, and antioxidant properties. A 2 × 3factorial design was used, with variations in the type of enzyme and hydrolysis time. The treatments that presented the highest yield based on protein content (70.46% and 64.57%) and collagen content (34.65 and 49.05 g/100 g) were the samples subjected to acid hydrolysis-enzymatic for 12 h (CP12 and CB12). CB12 demonstrated greater water absorption capacity (WA), while CP12 demonstrated greater water solubility (WS) and antioxidant activity. CP12 is the most promising collagen extract for use as a bioactive ingredient in foods.

Horizontal plasmid transfer promotes antibiotic resistance in selected bacteria in Chinese frog farms

The emergence and dissemination of antibiotic resistance genes (ARGs) in the ecosystem are global public health concerns. One Health emphasizes the interconnectivity between different habitats and seeks to optimize animal, human, and environmental health. However, information on the dissemination of antibiotic resistance genes (ARGs) within complex microbiomes in natural habitats is scarce. We investigated the prevalence of antibiotic resistant bacteria (ARB) and the spread of ARGs in intensive bullfrog (Rana catesbeiana) farms in the Shantou area of China. Antibiotic susceptibilities of 361 strains, combined with microbiome analyses, revealed Escherichia coli, Edwardsiella tarda, Citrobacter and Klebsiella sp. as prevalent multidrug resistant bacteria on these farms. Whole genome sequencing of 95 ARB identified 250 large plasmids that harbored a wide range of ARGs. Plasmid sequences and sediment metagenomes revealed an abundance of tetA, sul1, and aph(3″)-Ib ARGs. Notably, antibiotic resistance (against 15 antibiotics) highly correlated with plasmid-borne rather than chromosome-borne ARGs. Based on sequence similarities, most plasmids (62%) fell into 32 distinct groups, indicating a potential for horizontal plasmid transfer (HPT) within the frog farm microbiome. HPT was confirmed in inter- and intra-species conjugation experiments. Furthermore, identical mobile ARGs, flanked by mobile genetic elements (MGEs), were found in different locations on the same plasmid, or on different plasmids residing in the same or different hosts. Our results suggest a synergy between MGEs and HPT to facilitate ARGs dissemination in frog farms. Mining public databases retrieved similar plasmids from different bacterial species found in other environmental niches globally. Our findings underscore the importance of HPT in mediating the spread of ARGs in frog farms and other microbiomes of the ecosystem.

Green algae-produced multimer cathelicidin-RC1 disrupts membrane integrity for inhibiting bacterial growth

Cathelicidin-RC1, extracted from the bullfrog Rana catesbeiana, has been found to effectively combat gram-negative bacteria but has limited inhibitory effects on gram-positive bacteria. Additionally, the specific mechanism of cathelicidin-RC1’s interaction with bacteria remains unclear. In this research, we present the expression analysis of a chimeric peptide composed of three repetitive units of cathelicidin-RC1 (3×cathelicidin-RC1) with a hemagglutinin (HA) and a 6×His tag attached at its C-terminus in Chlamydomonas reinhardtii, leading to the production of a chimeric peptide with a molecular mass of ∼16.2 kDa. The recombinant peptide, 3×cathelicidin-RC1-HA-6×His, demonstrates a wide range of antimicrobial activity, with minimum inhibitory concentration (MIC) values of approximately 20–30 µg/ml and 10–60 µg/ml against gram-positive and negative bacteria, respectively. The minimum bactericidal concentration (MBC) values of 3×cathelicidin-RC1-HA-6×His varies from 1× to 3×MIC. Furthermore, 3×cathelicidin-RC1-HA-6×His demonstrates excellent stability, pH stability, and resistance to digestion by certain proteases. It also exhibits low cytotoxicity and negligible hemolytic activity against rat erythrocytes. Scanning electron microscopy and propidium iodide analysis indicate that 3×cathelicidin-RC1-HA-6×His disrupts bacterial cell membranes, leading to inhibition of bacterial growth. Overall, our findings demonstrate that 3×cathelicidin-RC1-HA-6×His, produced in the heterologous expression host C. reinhardtii, effectively inhibits bacterial growth and holds great promise for potential application.

Comparison of Two Methods to Detect the Northwestern Pond Turtle (Actinemys marmorata) and the Invasive American Bullfrog (Lithobates catesbeianus) in Interior Northern California

Comparison of Two Methods to Detect the Northwestern Pond Turtle (Actinemys marmorata) and the Invasive American Bullfrog (Lithobates catesbeianus) in Interior Northern California

Desenvolvimento de biscoitos com vísceras de rã-touro (Lithobates catesbeianus) para cães adultos

The study aimed to develop dog biscuits using bullfrog (Lithobates catesbeianus) viscera. After sterilizing the viscera, a pâté was produced and combined with lard, wheat flour, chemical yeast, and water to make biscuits. Microbiological safety was ensured by analyzing total mesophilic aerobes, molds, and yeasts through colony counts. The chemical composition of the biscuits (moisture, crude protein, ether extract, crude fiber, and ash) was determined. A sensory analysis, known as a preference test, was conducted where dogs were offered both the bullfrog biscuits and a well-accepted commercial brand to evaluate their preferences based on First Choice, First Consumption, and Reason for Ingestion. The commercial biscuits also underwent microbiological analysis. Results showed that both the commercial and bullfrog biscuits met international standards for mesophilic aerobes, molds, and yeasts. The proximate composition of the bullfrog biscuits was: moisture (4.52%), crude protein (14.6%), ether extract (10%), carbohydrates (68.26%), crude fiber (0.01%), and ash (1.55%), with a caloric value of 421.66 kcal per 100g. Sensory analysis revealed no significant difference in First Choice and Intake Ratio. However, dogs preferred the bullfrog biscuits for First Consumption (64.92%) compared to the commercial biscuits (35.02%). Despite the notable preference for First Consumption, no significant differences were observed for other sensory variables, suggesting that the bullfrog biscuits were well accepted by the dogs and had the potential to be a marketable product.

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.

Assessment of American Bullfrog (Lithobates catesbeianus) spreading in the Republic of Korea using rule learning of elementary cellular automata

The spread of American Bullfrog has a significant impact on the surrounding ecosystem. It is important to study the mechanisms of their spreading so that proper mitigation can be applied when needed. This study analyzes data from national surveys on bullfrog distribution. We divided the data into 25 regional clusters. To assess the spread within each cluster, we constructed temporal sequences of spatial distribution using the agglomerative clustering method. We employed Elementary Cellular Automata (ECA) to identify rules governing the changes in spatial patterns. Each cell in the ECA grid represents either the presence or absence of bullfrogs based on observations. For each cluster, we counted the number of presence location in the sequence to quantify spreading intensity. We used a Convolutional Neural Network (CNN) to learn the ECA rules and predict future spreading intensity by estimating the expected number of presence locations over 400 simulated generations. We incorporated environmental factors by obtaining habitat suitability maps using Maxent. We multiplied spreading intensity by habitat suitability to create an overall assessment of bullfrog invasion risk. We estimated the relative spreading assessment and classified it into four categories: rapidly spreading, slowly spreading, stable populations, and declining populations.

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.