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Variation in environmental chemistry affects animal health and demographics. For instance, acidification of aquatic ecosystems (e.g., due to acid rain) can have consequences for organismal fitness. Elevated atmospheric concentrations of CO2, which reacts with water to produce carbonic acid, may affect aquatic animal physiology in multiple ways, including via reductions in pH. Such impacts have been assessed to a large extent in marine systems. Similar effects are also likely occurring in freshwater ecosystems but are relatively understudied. The goal of this project was to determine how changes in pH and CO2 concentrations influence traits and survival of animals in freshwater systems, using larval American Bullfrogs (Lithobates catesbeianus (also known as Rana catesbeiana) [Shaw, 1802]) as a model. We hypothesized that elevated CO2 concentrations and reduced pH negatively affect larval frog immune function, growth, and survival, which we tested in a laboratory aquarium experiment. Over the 27-d experiment, tadpoles were exposed to 4 treatments: 1) near-neutral pH and no gas addition (mean ± SE pH = 7.17 ± 0.05), 2) CO2 gas sparged into aquariums (pH = 5.32 ± 0.04), 3) acidified water without gas addition (pH = 6.29 ± 0.08), and 4) ambient air sparged into aquariums (control for effects of gas addition) at near-neutral pH (pH = 7.46 ± 0.03). We measured tadpole innate immune response, growth, and survival. Results from the experiment partially supported our hypothesis, with lower immune function in tadpoles exposed to acidified water or elevated CO2 compared with the control treatments and lower final mass and survival under elevated CO2. These findings motivate additional research on pH and CO2 effects on freshwater animals and suggest that increased environmental acidity and elevated CO2 can lead to negative impacts, which could contribute to reductions in wildlife health and population declines in light of ongoing environmental changes. [ABSTRACT FROM AUTHOR]

Simple Summary: This study investigates the genetic structure and population dynamics of invasive bullfrogs in China employing cutting-edge genomic technologies. By analyzing microsatellite loci and single nucleotide polymorphisms (SNPs), the study identifies three distinct genetic subpopulations of bullfrogs and explores their origins and dispersal pathways. Findings reveal the varying degrees of gene flow between the subpopulations and a severe bottleneck effect followed by a rapid population expansion. Comparison with American bullfrog populations highlights the differences in genetic diversity and suggests potential multiple introductions. The introduction and subsequent range expansion of the American bullfrog (Lithobates catesbeianus) is part of a rising trend of troublesome biological invasions happening in China. This detrimental amphibious invasive species has strong adaptability. After its introduction and spread, it established its own ecological niche in many provinces of China, and its range has continued to expand to more areas. Previous studies recorded the introduction time of bullfrogs and calculated the changes in their genetic diversity in China using mitochondria, but the specific introduction route in China is still unknown. Expanding upon previous research, we employed whole-genome scans (utilizing 2b-RAD genomic sequencing) to examine single nucleotide polymorphisms (SNPs) and microsatellites within Lithobates catesbeianus to screen the genomes of these invasive amphibian species from eight Chinese provinces and two U.S. states, including Kansas, where bullfrogs originate. A total of 1,336,475 single nucleotide polymorphic loci and 17 microsatellite loci were used to calculate the genetic diversity of bullfrogs and their migration pathways. Our results suggest that the population in Hunan was the first to be introduced and to spread, and there may have been multiple introductions of subpopulations. Additionally, the genetic diversity of both the SNP and microsatellite loci in the Chinese bullfrog population was lower than that of the US population due to bottleneck effects, but the bullfrogs can adapt and spread rapidly. This study will offer crucial insights for preventing and controlling future introductions into the natural habitats in China. Additionally, it will assist in devising more precise strategies to manage the existing populations and curtail their continued expansion, as well as aim to improve clarity and originality while mitigating plagiarism risk. [ABSTRACT FROM AUTHOR]

Many native insects have evolved defenses against native predators. However, their defenses may not protect them from non-native predators due to a limited shared history. The American bullfrog, Aquarana catesbeiana (Anura: Ranidae), which has been intentionally introduced to many countries, is believed to impact native aquatic animals through direct predation. Adults of whirligig beetles (Coleoptera: Gyrinidae), known for swimming and foraging on the water surface of ponds and streams, reportedly possess chemical defenses against aquatic predators, such as fish. Although whirligig beetles potentially encounter both bullfrogs and other frogs in ponds and lakes, the effectiveness of their defenses against frogs has been rarely studied. To assess whether whirligig beetles can defend against native and non-native frogs, we observed the behavioral responses of the native pond frog, Pelophylax nigromaculatus (Anura: Ranidae), and the invasive non-native bullfrog, A. catesbeiana, to native whirligig beetles, Gyrinus japonicus and Dineutus orientalis, in Japan. Adults of whirligig beetles were provided to frogs under laboratory conditions. Forty percent of G. japonicus and D.orientalis were rejected by P. nigromaculatus, while all whirligig beetles were easily consumed by A. catesbeiana. Chemical and other secondary defenses ofG. japonicus and D. orientalis were effective for some individuals of P. nigromaculatus but not for any individuals of A. catesbeiana. These results suggest that native whirligig beetles suffer predation by invasive non-native bullfrogs in local ponds and lakes in Japan. [ABSTRACT FROM AUTHOR]

Context: Functional connectivity models are essential in identifying major dispersal pathways and developing effective management strategies for expanding populations of invasive alien species. However, the extrapolation of models parameterized within current invasive ranges may not be applicable even to neighbouring areas, if the models are not based on the expected responses of individuals to landscape structure. Objectives: We have developed a high-resolution connectivity model for both terrestrial and aquatic habitats using solely potential sources. The model is used here for the invasive, principally-aquatic, African clawed frog Xenopus laevis, which is a species of global concern. Methods: All ponds were considered as suitable habitats for the African clawed frog. Resistance costs of lotic aquatic and terrestrial landscape features were determined through a combination of remote sensing and laboratory trials. Maximum cumulative resistance values were obtained via capture-mark-recapture surveys, and validation was performed using independently collected presence data. We applied this approach to an invasive population of the American bullfrog, Lithobates catesbeianus, in France to assess its transferability to other pond-dwelling species. Results: The model revealed areas of high and low functional connectivity. It primarily identified river networks as major dispersal pathways and pinpointed areas where local connectivity could be disrupted for management purposes. Conclusion: Our model predicts how the dispersal of individuals connect suitable lentic habitats, through river networks and different land use types. The approach can be applied to species of conservation concern or interest in pond ecosystems and other wetlands, including aquatic insects, birds and mammals, for which distribution data are limited or challenging to collect. It serves as a valuable tool for forecasting colonization pathways in expanding populations of both native and invasive alien species and for identifying regions suitable for preventive or adaptive control measures. [ABSTRACT FROM AUTHOR]

Toll‐like receptors (TLRs) are an important family of pattern‐recognition receptors that recognize a series of pathogen‐associated molecular patterns to trigger the host immune response. In the present study, we identified three TLR13 genes (i.e., LcLTR13a, LcTLR13b, and LcTLR13c) in American bullfrogs, Lithobates catesbeianus, and explored their constitutive and pathogen‐induced expression profile in different tissues. The open reading frames of LcLTR13a, LcTLR13b and LcTLR13c are 2811, 2844, and 2814 bp long, encoding 955, 947, and 933 amino acids, respectively. Phylogenetic analysis showed that three LcTLR13 genes grouping in different subclades indicate the gene duplication of bullfrog TLR13 as in teleost. The three LcTLR13 genes were expressed in all the tissues examined, with the liver and spleen expressing the higher levels. Moreover, the expression levels of LcLTR13a, LcTLR13b, and LcTLR13c were significantly induced in the liver and spleen after bullfrogs were challenged with bacterial (i.e., Citrobacter freundii, Aeromonas hydrophila, and Edwardsiella tarda) pathogens. These results indicate that LcTLR13 genes play an essential role in the bullfrog antibacterial innate immune response, providing new insights into immune mechanisms of bullfrogs. [ABSTRACT FROM AUTHOR]

Biological invasions pose significant threats to biodiversity and ecosystem functioning. Removal of introduced species is most successful when detected early. We evaluate the effectiveness of passive acoustics combined with automated recognition in detecting the invasive American bullfrog (Lithobates catesbeianus). We applied this technique to two real-world monitoring programs aimed at determining the optimal time of day for monitoring the species in Europe, for which we recorded the species in Belgium and Italy; and for evaluating the effectiveness of BirdNET (a free and user-friendly automated recognizer) in analyzing a large dataset collected in Spain. BirdNET was highly effective in automatically detecting the bullfrog presence, with a detection rate (compared to visual inspection of sonograms) of 89.5% using default settings (85 of 95 recordings with known presence), and 95.8% with user-specific settings (91 of 95 recordings detected). The system showed remarkable precision, correctly identifying 99.7% (612 out of 614) of the verified predictions, and with only one mislabelled recording (predicted to be present when it was absent). The species' vocal activity in Belgium and Italy was higher during the night compared to crepuscular periods. Recording analyses and output verification of the dataset collected in Spain was carried out in 3.8% of the recorded time, and resulted in significantly reduced effort compared to visual inspection. Our study highlights the effectiveness of this technique for remotely surveying the American bullfrog, making it a significantly potential tool for informing management decisions, particularly for the early detection of the species' arrival in new areas. [ABSTRACT FROM AUTHOR]

Abstract In recent years, bullfrog (Lithobates catesbeianus) has become one of the most commercially important amphibians for aquaculture in China, and its annual production has grown rapidly. However, bacterial diseases caused by Citrobacter freundii infection have resulted in enormous economic losses. The present study used RNA sequencing technology to analyze the transcriptomic profile and differentially expressed genes (DEGs) in the bullfrog liver and spleen post C. freundii infection. De novo assembly resulted in the generation of 77,556 L. catesbeianus transcripts, and 49,421 of these genes were successfully annotated to the databases. Overall, 1487 DEGs were identified in the liver group, including 788 upregulated and 709 downregulated DEGs. A total of 799 DEGs, including 311 upregulated and 488 downregulated genes, were detected in the spleen groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed significantly enriched pathways, including pathways involved in metabolism and immune responses. The processes in which the DEGs were enriched were those related to metabolic activities, including the lipid metabolism pathway, amino acid pathway, immune‐related pathway, and inflammatory responses. The results of this study provide insight into the response of bullfrogs against C. freundii at the transcriptome level and reveal the mechanism of the immune response against C. freundii.

The American bullfrog Lithobates catesbeianus is a well-known invasive alien species recorded in over 40 countries and islands. It is necessary to elucidate their seasonal emergence and habitat water body characteristics for implementing efficient eradication activities. Sado Island, Japan, is home to several endemic and rare species, including the endangered Sado wrinkled frogs (Glandirana susurra) that are concerned to be negatively impacted by invasive American bullfrogs. Here, we surveyed 41 ponds for invasive American bullfrogs over the period preceding emergence to the start of oviposition to determine their seasonal emergence and habitat pond use. As in previous studies, we observed initiations of their post-hibernation activities such as emergence, mating call, and oviposition in that order as air temperature increased, and those activities were thought to begin as late as late-April, mid-May, and mid-July, respectively on Sado Island. For their habitat preferences, we confirmed that they mainly inhabit relatively deep ponds surrounded by rice paddy fields. Our results were broadly consistent with those of previous studies, with respect to the seasonal emergence and habitat pond characteristics of American bullfrogs on Sado Island. Since our results suggest an overlap in habitat and pond usage season between American bullfrogs and Sado wrinkled frogs, we highly recommend implementing bullfrog eradication activities to conserve the Sado wrinkled frog population. This study provided the basic ecological information for selecting appropriate seasons and ponds for implementing bullfrog eradication activities. [ABSTRACT FROM AUTHOR]

In recent years, bullfrog (Lithobates catesbeianus) has become one of the most commercially important amphibians for aquaculture in China, and its annual production has grown rapidly. However, bacterial diseases caused by Citrobacter freundii infection have resulted in enormous economic losses. The present study used RNA sequencing technology to analyze the transcriptomic profile and differentially expressed genes (DEGs) in the bullfrog liver and spleen post C. freundii infection. De novo assembly resulted in the generation of 77,556 L. catesbeianus transcripts, and 49,421 of these genes were successfully annotated to the databases. Overall, 1487 DEGs were identified in the liver group, including 788 upregulated and 709 downregulated DEGs. A total of 799 DEGs, including 311 upregulated and 488 downregulated genes, were detected in the spleen groups. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed significantly enriched pathways, including pathways involved in metabolism and immune responses. The processes in which the DEGs were enriched were those related to metabolic activities, including the lipid metabolism pathway, amino acid pathway, immune‐related pathway, and inflammatory responses. The results of this study provide insight into the response of bullfrogs against C. freundii at the transcriptome level and reveal the mechanism of the immune response against C. freundii. [ABSTRACT FROM AUTHOR]

Abstract Toll‐like receptors (TLRs) are an important family of pattern‐recognition receptors that recognize a series of pathogen‐associated molecular patterns to trigger the host immune response. In the present study, we identified three TLR13 genes (i.e., LcLTR13a, LcTLR13b, and LcTLR13c) in American bullfrogs, Lithobates catesbeianus, and explored their constitutive and pathogen‐induced expression profile in different tissues. The open reading frames of LcLTR13a, LcTLR13b and LcTLR13c are 2811, 2844, and 2814 bp long, encoding 955, 947, and 933 amino acids, respectively. Phylogenetic analysis showed that three LcTLR13 genes grouping in different subclades indicate the gene duplication of bullfrog TLR13 as in teleost. The three LcTLR13 genes were expressed in all the tissues examined, with the liver and spleen expressing the higher levels. Moreover, the expression levels of LcLTR13a, LcTLR13b, and LcTLR13c were significantly induced in the liver and spleen after bullfrogs were challenged with bacterial (i.e., Citrobacter freundii, Aeromonas hydrophila, and Edwardsiella tarda) pathogens. These results indicate that LcTLR13 genes play an essential role in the bullfrog antibacterial innate immune response, providing new insights into immune mechanisms of bullfrogs.

Emerging infectious diseases threaten amphibian species across the globe. In Brazil, the American bullfrog (Aquarana catesbeiana) is a highly invasive species that can potentially transmit parasites and pathogens to native amphibians. This is the first assessment of co-infection of Ranavirus and helminth macroparasites in invasive populations of bullfrogs in South America. We collected, measured, and euthanized 65 specimens of A. catesbeiana sampled from 9 sites across three states of Brazil in the Atlantic Forest biome. We collected and identified helminth macroparasites and sampled host liver tissue to test for the presence and load of Ranavirus with quantitative PCR. We documented patterns of prevalence, parasite load, and co-infection with generalized linear mixed models, generalized logistic regressions, and randomization tests. Most individual bullfrogs did not exhibit clinical signs of infection, but the overall Ranavirus prevalence was 27% (95% confidence interval, [CI 17–38]). Bullfrogs were infected with helminth macroparasites from 5 taxa. Co-infection of helminth macroparasites and Ranavirus was also common (21% CI [12–31]). Bullfrog size was positively correlated with total macroparasite abundance and richness, and the best-fitting model included a significant interaction between bullfrog size and Ranavirus infection status. We observed a negative correlation between Ranavirus viral load and nematode abundance (slope = −0.22, P = 0.03). Invasive bullfrogs (A. catesbeiana) in Brazil were frequently infected with both Ranavirus and helminth macroparasites, so adult bullfrogs could serve as reservoir hosts for both pathogens and parasites. However, many macroparasites collected were encysted and not developing. Coinfection patterns suggest a potential interaction between Ranavirus and macroparasites because helminth abundance increased with bullfrog size but was lower in Ranavirus infected individuals. Future studies of bullfrogs in the Atlantic Forest should investigate their potential role in pathogen and parasite transmission to native anurans.

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 speciesspecific 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Amphibians are declining globally, and the fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been the culprit in many of these declines. In the Mediterranean region of Baja California, Mexico—a biodiversity hotspot—native amphibians are also in decline, and Bd is present. To determine which factors best predict Bd infection prevalence and intensity in Baja California anurans, we conducted Bd sampling at three disparate sites at varying elevations. We observed an overall Bd prevalence of 68%, and found that species was an important predictor of pathogen prevalence and burden (load) both across and within sites. Pathogen prevalence and load increased positively with elevation. The highest Bd load we detected was 139,188 zoospore equivalents (ZE) in a western toad ( Anaxyrus boreas)—more than 10 times the pathogen burden at which chytridiomycosis is lethal in some species. We observed high Bd prevalence (84 – 99%) and Bd loads (> 44,000 ZE) in the California red-legged frog ( Rana draytonii)—a cause for concern in this threatened anuran, especially at La Grulla—the highest elevation (2050 m) site for this species across its range. We sampled museum specimens to determine how long Bd has been present in the region, and detected the pathogen as early as 1932. Contemporary Bd prevalence and load in Baja California are considerably higher than would be expected given similar habitats and the same community composition as in southern California, USA. We therefore highlight the importance of historical context and comparative analyses in examining contemporary Bd dynamics, as well as adhering to strict biosecurity measures when implementing reintroductions and translocations.

The introduction and subsequent range expansion of the American bullfrog (Lithobates catesbeianus) is part of a rising trend of troublesome biological invasions happening in China. This detrimental amphibious invasive species has strong adaptability. After its introduction and spread, it established its own ecological niche in many provinces of China, and its range has continued to expand to more areas. Previous studies recorded the introduction time of bullfrogs and calculated the changes in their genetic diversity in China using mitochondria, but the specific introduction route in China is still unknown. Expanding upon previous research, we employed whole-genome scans (utilizing 2b-RAD genomic sequencing) to examine single nucleotide polymorphisms (SNPs) and microsatellites within Lithobates catesbeianus to screen the genomes of these invasive amphibian species from eight Chinese provinces and two U.S. states, including Kansas, where bullfrogs originate. A total of 1,336,475 single nucleotide polymorphic loci and 17 microsatellite loci were used to calculate the genetic diversity of bullfrogs and their migration pathways. Our results suggest that the population in Hunan was the first to be introduced and to spread, and there may have been multiple introductions of subpopulations. Additionally, the genetic diversity of both the SNP and microsatellite loci in the Chinese bullfrog population was lower than that of the US population due to bottleneck effects, but the bullfrogs can adapt and spread rapidly. This study will offer crucial insights for preventing and controlling future introductions into the natural habitats in China. Additionally, it will assist in devising more precise strategies to manage the existing populations and curtail their continued expansion, as well as aim to improve clarity and originality while mitigating plagiarism risk.

The aim was to evaluate the recovery of bullfrog homeostasis after biometry, a routine management. An experiment in a completely randomized design was conducted with six treatments and 10 repetitions: the bullfrog being the experimental unit. Five treatments consisted of evaluating the bullfrog recovery times after biometry (0 - immediately after biometry, 6, 12, 24 and 48 h) and a control (animals in the pen before biometry). Sixty bullfrogs (285.33 ± 10.00 g) were subjected to a 12-h fasting. Subsequently, 50 animals underwent biometry and 10 were used for blood collection before biometry (control). A significant increase was observed in all variables analyzed for animals subjected to density stress, except for total proteins, globulin, and hemoglobin. There was a significant increase in blood glucose and erythrocyte numbers in the subjects immediately after biometry. Plasma proteins and globulin had no significant difference in any of the groups that underwent biometry. Lactate, albumin, and triglycerides levels were significantly elevated in animals shortly after biometry and remained elevated until 12 h after management. The results showed that 24 h after biometry stress, all the analyzed variables were already at similar levels as to the levels of control group animals. [ABSTRACT FROM AUTHOR]

Establishing historical species distributions can assist conservation translocations for threatened species, and yet, ecological changes necessitate developing restoration targets that are not analogous to historical baselines. Despite its recent conservation translocation to Yosemite Valley in Yosemite National Park, Sierra Nevada Mountains, USA, the historical distribution of the federally threatened California Red-legged Frog (Rana draytonii) in the valley remains unclear. Using archival records, interviews, and museum specimens, we examined the historical evidence for California Red-legged Frogs and sympatric amphibian species in the Yosemite region. We found a paucity of reliable amphibian records for Yosemite Valley since the 19th century, one of the most-visited sites in the US National Park System, and conclude that this is the result of historically low collecting and survey effort prior to the introduction of invasive American Bullfrogs (Lithobates catesbeianus; also Rana catesbeiana after Yuan and others 2016) in concert with a bird and mammal study bias from largely diurnal collecting that occurred when California Red-legged Frogs were extant regionally. We found previously undocumented records for individuals of the genus Rana for Yosemite Valley, consistent with a dominant historical hydrology more compatible for Foothill Yellow-legged Frogs (Rana boylii), though none could be definitively identified as California Red-legged Frogs. We conclude that extensive anthropogenic impacts, including acute ecosystem alteration and American Bullfrog introduction, contributed to the failure to detect California Red-legged Frogs in many places regionally once amphibians became a research priority in the latter 20th century. The conservation translocation of California Red-legged Frogs to Yosemite Valley illustrates the integration of historical baselines with contemporary realities, allowing for the complexities of change over time rather than focusing on restoration to an imagined, ideal environment in the past. [ABSTRACT FROM AUTHOR]

Simple Summary: The introduction of alien species is one of the major causes of biodiversity loss. The American bullfrog (Lithobates catesbeianus) is considered to be one of the most harmful invasive species which overall threatens native amphibians. The detection of an invasive species in the first stage of its arrival is critical to control its colonization and to avoid its establishment. This early detection requires tools with high sensitivity. Methods based on the analysis of free environmental DNA (eDNA) are promising. The present article develops an eDNA assay to monitor the early process of invasion of the American bullfrog in the Ebre Delta (Spain), in a scenario where the presence of bullfrog specimens is really low and scarcely detected by traditional methods. In 2018, the first bullfrog tadpoles were found for the first time in the Ebre Delta. Two years after, despite the species not being well established, our eDNA assay detected the presence of bullfrogs in several locations. This methodology proved to yield a higher sensitivity with a lower sampling effort than traditional methods. Based on our experience, we also provided solutions to face challenges associated with the use of eDNA. Developing a rapid and low-cost-effective protocol to use in the early stages of an invasion (as occurred with the American bullfrog in the Iberian Peninsula) is essential to facilitate the detection, control, and eradication of an invasive species in the early stages of the invasion process. The American bullfrog (Lithobates catesbeianus) is considered to be one of the most harmful invasive species. In the Iberian Peninsula, this species had been cited occasionally until the year 2018, when L. catesbeianus appeared in the Ebre Delta, and, for the first time, it started breeding in a territory of the Peninsula. Using environmental DNA (eDNA) analysis and visual surveys, the American bullfrog invasion in the Ebre Delta was monitored across two consecutive years (2019–2020). No specimens were observed in 2019, and results for the eDNA survey also failed to detect this species in the Delta. In 2020, two individuals were captured and, under the most conservative criteria to constrain the number of positive detections, eDNA analyses detected the presence of the American bullfrog in at least five locations. Performing an eDNA assay yielded a higher sensitivity with a lower sampling effort than traditional methods. Although the American bullfrog does not appear to still be well-established in the Ebre Delta, only a few bullfrog individuals could be enough for their establishment in suitable habitats. In this context, eDNA assays are essential tools to facilitate the detection, control, and eradication of this species in the first stage of the invasion process. [ABSTRACT FROM AUTHOR]

Abstract 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.

In Mexico, bullfrogs (Lithobates catesbeianus) are produced as gourmet food. However, bullfrogs can be carriers of pathogens because the frogs' preferred living conditions occur in stagnant water. The present study aimed to identify bacteria that cause foodborne diseases or are associated with human diseases. For molecular identification, based on the sequential analysis by 16S rRNA or rpoD was conducted on all isolates obtained from bullfrog. A total of 91 bacterial isolates were obtained from bullfrogs; 14 genera and 23 species were identified, including Acinetobacter johnsonii 16.5%; Aeromonas media 14.3%; Aeromonas veronii 13.2%; Providencia rettgeri 7.7%; Citrobacter freundii 6.6%; Aeromonas caviae 4.4%; Aeromonas hydrophila and Elizabethkingia ursingii 3.3%; Pseudomonas stutzeri, Raoultella ornithinolytica, and Shewanella putrefaciens 2.2%; Acinetobacter guillouiae, Acinetobacter pseudolwoffii, Citrobacter portucalensis, Citrobacter werkmanii, Edwardsiella anguillarum, Klebsiella michiganensis, Kluyvera intermedia, Kocuria rosea, Myroides odoratimimus, Myroides odoratus, Proteus sp., and Proteus hauseri 1.1%. In this study, 49.4% of the isolates obtained cause foodborne disease, 19.8% are bacteria that play an important role in the spoilage of food, 5.5% of isolates have nosocomial significance, 13.2% of bacteria are considered to be pollutants of the ecosystem, and in the case of A. salmonicida and Edwardsiella anguillarum (12.1%) to have a negative impact on aquaculture. Acinetobacter pseudolwoffii and Citrobacter portucalensis have not been reported to cause disease. Lastly of these isolates, 97.8% (89/91) can cause disease by food consumption or by direct contact for immunocompromised persons. The presence of these bacteria in bullfrogs represents a significant problem for human health. There is evidence that these microorganisms are pathogenic and frogs may also be reservoirs.

Invasive alien species are worldwide one of the main reasons for the decline of species in native communities and hence biodiversity. Control or eradication programs are often fragmented leading to cost-inefficient and inadequate results. This paper addresses a state-of-the-art framework of an integrated management approach based on the case study on the control of the American bullfrog in the Flemish region in Belgium. This approach is based on the close cooperation of scientists, policymakers, field managers and conservationists to create a science-based policy in the management of invasive alien species. Innovative applied research poses an important foundation in the steering process to establish efficient control measures. These are integrated in the management process that is coincided with raising awareness and dissemination of information throughout the whole plan. The results are based on 10 years of experience and cooperation in the management of the American bullfrog in Flanders, Belgium. The several stages of which this framework is built are crucial in the strategy to tackle the invasion in the most integrated way and can be transposed to control other invasive aquatic alien animals.

This study examined the influence of tank color on the performance, time to metamorphosis, skin pigmentation, and biochemistry parameters of American bullfrog tadpoles and froglets. Six hundred tadpoles (0.94 ± 0.11 g) were selected (Gosner stage – 25) and distributed in a completely randomized design in 15 tanks of 40 L (1 tadpole L−1) in a recirculating aquaculture system, five repetitions of each color (blue, black, and white). Biometrics were performed every 14 days in all experimental units, and when the tadpoles at metamorphic climax and following complete transformation. Animals were weighed, measured, and photographed to evaluate the growth performance, uniformity, and skin pigmentation analyses. At the conclusion of metamorphosis, twenty-four animals randomly selected per treatment were euthanized. Twelve of them were macerated to performed of the whole-body biochemistry analysis, and the other twelve were dissected to obtain the biological indexes. The tadpoles raised in blue, and black tanks reached the metamorphosis climax and transitioned into froglets more rapidly (P < 0.05) than animals held in white tanks. However, tadpoles at metamorphic climax reared in black tanks had prolonged time to complete the metamorphosis (P < 0.05). The tadpoles raised in blue background had the higher values for final body weight throughout the experiment (P < 0.05). The tadpoles raised at blue and black tanks had the higher values for body weight gain, body length, body length gain, protein efficiency ratio, specific growth rate and lower feed conversion ratio throughout the experiment (P < 0.05). The pre- and pro-metamorphic uniformity of tadpoles was affected by the color of the tank, with the white treatment having lower uniformity at the 14, 42, 56, 70 and 98 days of the experiment (P < 0.05). In addition, tadpoles on metamorphic climax and froglets raised on black, and blue tanks had darker color of skin compared with the animals raised in white tanks, with animals raised in black tanks showing darker skin tones (P < 0.05). However, neither the growth, and uniformity of tadpoles on metamorphic climax and froglets, nor the biological indexes and whole-body biochemistry of froglets were affected by tank color (P > 0.05). Therefore, considering the homogeneity in growth, and time to metamorphosis, blue colored tanks represent best choice for reared of American bullfrog tadpoles. • Bullfrog tadpoles raised at blue tanks concludes the metamorphosis on shorter time. • Bullfrog tadpoles raised in black tanks complete metamorphic climax more slowly. • The bullfrog tadpoles raised at white tanks present worse uniformity. • Bullfrog tadpoles raised at blue tanks showed better performance. • The color of the tank did not change the biochemistry parameters of the froglets. [ABSTRACT FROM AUTHOR]

Identifying the ecological factors that determine the spread of invasive species is key to adequately managing endangered species in freshwater ecosystems. Invasive species are a main threat to turtles, which are targets of major conservation efforts worldwide.In freshwater ecosystems of the south‐western USA, invasive bullfrog (Lithobates catesbeianus) and crayfish species (Faxonius virilis and Procambarus clarkii) represent a major risk to the desert mud turtle (Kinosternon sonoriense sonoriense), state‐listed as a Species of Greatest Conservation Need in Arizona. As a species in the early stages of population decline, the desert mud turtle is a top candidate for the development of management plans to decrease extinction risk.An invasion risk assessment tool was built from available occurrence data for K. s. sonoriense and the invasive bullfrog and crayfish species in Arizona using 5,886 de‐duplicated records from public databases and reports from the Arizona Game and Fish Department. The occurrence density of K. s. sonoriense was calculated state‐wide to define populations in which the level of invasion by bullfrog and crayfish was assessed. The environmental factors associated with the abundance of invasive species in populations of K. s. sonoriense were then analysed.A higher prevalence of crayfish and bullfrog was detected in turtle populations located in perennial streams. Invasive abundance was significantly higher in turtle populations at higher elevation and closer to the main river trunk for both invasive taxa. Higher bullfrog abundance was detected near human settlements, whereas crayfish were more abundant further from human settlements.These results will inform which populations of K. s. sonoriense require intensive surveying and control of invasive species to maintain the health of native desert mud turtle populations. This study provides valuable information regarding the environmental conditions associated with the abundance of invasive species threatening turtle populations, helping to develop science‐based management of freshwater ecosystems. [ABSTRACT FROM AUTHOR]

Despite the importance of the skin mucosal barrier and commensal microbiota for the health of amphibians, the potential of environmental contaminants to disrupt the skin mucosal barrier and microbiota have rarely been studied in toxicology. In this study, tadpoles (Lithobates catesbeianus) were exposed to 0, 0.5, and 2 μg/L of microcystin-leucine arginine (MC-LR) for 30 days to explore the impacts of environmentally realistic MC-LR concentrations on the physical skin barrier, immune barrier, commensal microbiota, and skin resistance to pathogenic bacterial invasion. MC-LR exposure significantly reduced the collagen fibrils in the dermis of skin tissues and down-regulated tight junction and stratum corneum-related gene transcriptions, suggesting the damage caused by MC-LR to the physical barrier of the skin. Increased skin eosinophils and upregulated transcriptions of inflammation-related genes in the exposed tadpoles underline the development of skin inflammation resulting from MC-LR exposure even at environmentally realistic concentrations. Comparative transcriptome and immunobiochemical analyses found that antimicrobial peptides (Brevinin-1PLc, Brevinin-2GHc, and Ranatuerin-2PLa) and lysozyme were down-regulated in the exposed groups, while complement, pattern recognition receptor, and specific immune processes were up-regulated. However, the content of endotoxin lipopolysaccharide produced by bacteria increased in a dose-dependent pattern. The disc diffusion test showed a reduced ability of skin supernatant to inhibit pathogenic bacteria in the exposed groups. Analysis of microbial 16 S rRNA gene by high-throughput sequencing revealed that MC-LR interfered with the abundance, composition, and diversity of the skin commensal microbiota, which favored the growth of pathogen-containing genera Rhodococcus, Acinetobacter, and Gordonibacter. In summary, the current study provides the first clues about the impact of MC-LR on the integrity and function of skin barrier of amphibians. These new toxicological evidences can facilitate a more comprehensive evaluation of the ecological risk of MC-LR to amphibians.

As a common aquaculture pathogen, Citrobacter freundii has resulted in inconceivable economic losses in aquaculture. In this study, we investigated the spleen immune response of Lithobates catesbeianus to C. freundii infection at the protein level. A comparative proteomic analysis of spleens from C. freundii-infected L. catesbeianus was performed by using isobaric tags for relative and absolute quantitation (iTRAQ) approaches. A total of 2400 proteins were identified by LC-MS/MS spectra and sequencing data. In addition, 316 differentially expressed proteins (DEPs) were identified, of which 154 were significantly upregulated and 162 were significantly downregulated. Seven DEPs that are immune related including Kelch-like protein 22 (KLHL 22), saxiphilin (Sax), integrin beta-3 (ITGB3), retinoic acid-inducible gene I (RIG-I), laboratory of genetics and physiology 2 (LGP2), E3 ubiquitin-protein ligase TRIM39-like (TRIM39L), and phospholipid scramblase 1 (PLSCR1) were selected for further study. The expression profile of these genes in the spleen at 48 h post-C. freundii-infection showed a similar trend as the iTRAQ data. Altogether, the current study is the first to report the spleen proteome of bullfrogs (L. catesbeianus), providing insight into the immune response to C. freundii infection.

Emerging infectious diseases threaten amphibian species across the globe. In Brazil, the American bullfrog (Aquarana catesbeiana) is a highly invasive species that can potentially transmit parasites and pathogens to native amphibians. This is the first assessment of co-infection of Ranavirus and helminth macroparasites in invasive populations of bullfrogs in South America. We collected, measured, and euthanized 65 specimens of A. catesbeiana sampled from 9 sites across three states of Brazil in the Atlantic Forest biome. We collected and identified helminth macroparasites and sampled host liver tissue to test for the presence and load of Ranavirus with quantitative PCR. We documented patterns of prevalence, parasite load, and co-infection with generalized linear mixed models, generalized logistic regressions, and randomization tests. Most individual bullfrogs did not exhibit clinical signs of infection, but the overall Ranavirus prevalence was 27% (95% confidence interval, [CI 17–38]). Bullfrogs were infected with helminth macroparasites from 5 taxa. Co-infection of helminth macroparasites and Ranavirus was also common (21% CI [12–31]). Bullfrog size was positively correlated with total macroparasite abundance and richness, and the best-fitting model included a significant interaction between bullfrog size and Ranavirus infection status. We observed a negative correlation between Ranavirus viral load and nematode abundance (slope = −0.22, P = 0.03). Invasive bullfrogs (A. catesbeiana) in Brazil were frequently infected with both Ranavirus and helminth macroparasites, so adult bullfrogs could serve as reservoir hosts for both pathogens and parasites. However, many macroparasites collected were encysted and not developing. Coinfection patterns suggest a potential interaction between Ranavirus and macroparasites because helminth abundance increased with bullfrog size but was lower in Ranavirus infected individuals. Future studies of bullfrogs in the Atlantic Forest should investigate their potential role in pathogen and parasite transmission to native anurans. [Display omitted] • First documented evidence of widespread Ranavirus infection in invasive adult bullfrogs (Aquarana catesbeiana) in Brazil. • Invasive adult bullfrogs may act as reservoirs of both viral and macroparasitic infections, potentially impacting native anuran communities. • Patterns of co-infection indicate a potential interaction between Ranavirus and helminth macroparasites. [ABSTRACT FROM AUTHOR]

The American bullfrog (Lithobates catesbeianus) is considered to be one of the most harmful invasive species. In the Iberian Peninsula, this species had been cited occasionally until the year 2018, when L. catesbeianus appeared in the Ebre Delta, and, for the first time, it started breeding in a territory of the Peninsula. Using environmental DNA (eDNA) analysis and visual surveys, the American bullfrog invasion in the Ebre Delta was monitored across two consecutive years (2019–2020). No specimens were observed in 2019, and results for the eDNA survey also failed to detect this species in the Delta. In 2020, two individuals were captured and, under the most conservative criteria to constrain the number of positive detections, eDNA analyses detected the presence of the American bullfrog in at least five locations. Performing an eDNA assay yielded a higher sensitivity with a lower sampling effort than traditional methods. Although the American bullfrog does not appear to still be well-established in the Ebre Delta, only a few bullfrog individuals could be enough for their establishment in suitable habitats. In this context, eDNA assays are essential tools to facilitate the detection, control, and eradication of this species in the first stage of the invasion process.

Amphibians are declining globally, and the fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been the culprit in many of these declines. In the Mediterranean region of Baja California, Mexico—a biodiversity hotspot—native amphibians are also in decline, and Bd is present. To determine which factors best predict Bd infection prevalence and intensity in Baja California anurans, we conducted Bd sampling at three disparate sites at varying elevations. We observed an overall Bd prevalence of 68%, and found that species was an important predictor of pathogen prevalence and burden (load) both across and within sites. Pathogen prevalence and load increased positively with elevation. The highest Bd load we detected was 139,188 zoospore equivalents (ZE) in a western toad (Anaxyrus boreas)—more than 10 times the pathogen burden at which chytridiomycosis is lethal in some species. We observed high Bd prevalence (84 – 99%) and Bd loads (> 44,000 ZE) in the California red-legged frog (Rana draytonii)—a cause for concern in this threatened anuran, especially at La Grulla—the highest elevation (2050 m) site for this species across its range. We sampled museum specimens to determine how long Bd has been present in the region, and detected the pathogen as early as 1932. Contemporary Bd prevalence and load in Baja California are considerably higher than would be expected given similar habitats and the same community composition as in southern California, USA. We therefore highlight the importance of historical context and comparative analyses in examining contemporary Bd dynamics, as well as adhering to strict biosecurity measures when implementing reintroductions and translocations.

Poor utilization efficiency of plant protein diets always leads to intestinal barrier dysfunction and growth inhibition in animals. Probiotics have shown promise in improving growth performance and gut health of the host. However, obtaining the host-beneficial probiotic from thousands of bacterial phylotypes is challenging. Here, four intestinal autochthonous bacteria were isolated from fast-growing bullfrog after a 60-day feeding on a soybean meal (SM)-based diet. Another feeding trial was conducted to evaluate the effects of supplementing these strains in an SM-based diet on growth, nutrient digestibility, immunity, and gut health of bullfrog. A high-SM basal diet was used as a non-supplemented control group (NC), and four other diets were prepared by supplementing the basal diet with 1 × 107 CFU/g of Bacillus siamensis , Bacillus tequilensis (BT), Bacillus velezensis , and Lactococcus lactis (LL). Results showed that weight gain, feed efficiency, nitrogen retention, and apparent digestibility coefficients of dry matter and protein were significantly higher in the LL group compared with the NC group (p < 0.05). Furthermore, compared with the NC group, both BT and LL groups showed markedly higher jejunal protease and amylase activities, serum complement 4 and immunoglobulin M levels, jejunal muscularis thickness (p < 0.05), and up-regulated expression of il-10 and zo-1 genes (p < 0.05). High-throughput sequencing revealed higher abundances of Bacillus and Cetobacterium in BT and LL groups, respectively, accompanied with decreased abundances of Enterobacter and Escherichia–Shigella. Besides, KEGG pathways related to metabolisms were significantly enhanced by the LL diet relative to the NC diet (p < 0.05). Overall, the beneficial effects of two frog-derived probiotics were determined: supplementation of L. lactis in SM-based diet promoted growth and nutrient digestibility; both B. tequilensis and L. lactis supplementation improved immune response and intestinal barrier function of bullfrogs. [ABSTRACT FROM AUTHOR]

ABSTRACT Ascites is a pathology characterized by the extravasation of fluid from blood vessels and its accumulation in the abdominal cavity, caused by several associated factors. In this paper, we report the occurrence of this syndrome in breeding male and female Bullfrogs fed a commercial fish feed with 40% crude protein. Liver malformation was already observed due to lack of protein deposition in adult animals from the same spawning. The culture of ascitic fluid showed absence of bacterial growth. Kidney histopathology showed, in both sexes, a large number of tubular structures with strongly eosinophilic hyaline material suggesting colloid growth in both the cortical region and spinal cord. The analysis also revealed granulomas in various stages of development, many showing central necrotic material. The kidneys, glomerular, were mostly hypoplastic, with enlarged Bowman space, and many were hyalinized or hemorrhagic; the parenchyma showed dystrophic calcification, and many tubules containing fibrinoid material. The liver displayed a large amount of melanomacrophages in the parenchyma and foci of mono-lymphocytic hepatitis and marked cytoplasmic rarefaction, as well as several hepatocytes with pyknotic nuclei and necrotic cells and dissociation of the hepatic trabeculae. Ascites may be caused by the lesions observed in these organs.

Poor utilization efficiency of plant protein diets always leads to intestinal barrier dysfunction and growth inhibition in animals. Probiotics have shown promise in improving growth performance and gut health of the host. However, obtaining the host-beneficial probiotic from thousands of bacterial phylotypes is challenging. Here, four intestinal autochthonous bacteria were isolated from fast-growing bullfrog after a 60-day feeding on a soybean meal (SM)-based diet. Another feeding trial was conducted to evaluate the effects of supplementing these strains in an SM-based diet on growth, nutrient digestibility, immunity, and gut health of bullfrog. A high-SM basal diet was used as a non-supplemented control group (NC), and four other diets were prepared by supplementing the basal diet with 1 × 107 CFU/g of Bacillus siamensis, Bacillus tequilensis (BT), Bacillus velezensis, and Lactococcus lactis (LL). Results showed that weight gain, feed efficiency, nitrogen retention, and apparent digestibility coefficients of dry matter and protein were significantly higher in the LL group compared with the NC group (p < 0.05). Furthermore, compared with the NC group, both BT and LL groups showed markedly higher jejunal protease and amylase activities, serum complement 4 and immunoglobulin M levels, jejunal muscularis thickness (p < 0.05), and up-regulated expression of il-10 and zo-1 genes (p < 0.05). High-throughput sequencing revealed higher abundances of Bacillus and Cetobacterium in BT and LL groups, respectively, accompanied with decreased abundances of Enterobacter and Escherichia–Shigella. Besides, KEGG pathways related to metabolisms were significantly enhanced by the LL diet relative to the NC diet (p < 0.05). Overall, the beneficial effects of two frog-derived probiotics were determined: supplementation of L. lactis in SM-based diet promoted growth and nutrient digestibility; both B. tequilensis and L. lactis supplementation improved immune response and intestinal barrier function of bullfrogs.

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease among the main causes of amphibian declines worldwide. However, Bd studies on Neotropical amphibians from temperate areas are scarce. We present a comprehensive survey of Bd in Uruguay, in temperate central eastern South America, carried out between 2006 and 2014. Skin swabs of 535 specimens of 21 native and exotic frogs were tested by PCR. We used individual-level data to examine the relationship between infection, climatic variables, and their effects on body condition and the number of prey items found in stomach contents. Infection was widespread in free-ranging anurans with an overall prevalence of 41.9%, detected in 15 native species, wild American bullfrogs Aquarana catesbeiana, and captive specimens of Ceratophrys ornata and Xenopus laevis. Three haplotypes of the Bd ITS region were identified in native amphibians, all belonging to the global panzootic lineage (BdGPL), of which only one was present in exotic hosts. Despite high infection frequencies in different anurans, we found no evidence of morbidity or mortality attributable to chytridiomycosis, and we observed no discernible impact on body condition or consumed prey. Climatic conditions at the time of our surveys suggested that the chance of infection is associated with monthly mean temperature, mean humidity, and total precipitation. Temperatures below 21°C combined with moderate humidity and pronounced rainfall may increase the likelihood of infection. Multiple haplotypes of BdGPL combined with high frequencies of infection suggest an enzootic pattern in native species, underscoring the need for continued monitoring.

Many native insects have evolved defenses against native predators. However, their defenses may not protect them from non-native predators due to a limited shared history. The American bullfrog, Aquarana catesbeiana (Anura: Ranidae), which has been intentionally introduced to many countries, is believed to impact native aquatic animals through direct predation. Adults of whirligig beetles (Coleoptera: Gyrinidae), known for swimming and foraging on the water surface of ponds and streams, reportedly possess chemical defenses against aquatic predators, such as fish. Although whirligig beetles potentially encounter both bullfrogs and other frogs in ponds and lakes, the effectiveness of their defenses against frogs has been rarely studied. To assess whether whirligig beetles can defend against native and non-native frogs, we observed the behavioral responses of the native pond frog, Pelophylax nigromaculatus (Anura: Ranidae), and the invasive non-native bullfrog, A . catesbeiana , to native whirligig beetles, Gyrinus japonicus and Dineutus orientalis , in Japan. Adults of whirligig beetles were provided to frogs under laboratory conditions. Forty percent of G . japonicus and D . orientalis were rejected by P . nigromaculatus , while all whirligig beetles were easily consumed by A . catesbeiana . Chemical and other secondary defenses of G . japonicus and D . orientalis were effective for some individuals of P . nigromaculatus but not for any individuals of A . catesbeiana . These results suggest that native whirligig beetles suffer predation by invasive non-native bullfrogs in local ponds and lakes in Japan., Competing Interests: The authors declare there are no competing interests., (©2024 Sugiura and Hayashi.)

Vitamin C supplementation is important for the growth and development of bullfrog tadpoles under optimum water temperature conditions. Therefore, an experiment was carried out to evaluate the effects of vitamin C supplementation on the diet of bullfrog tadpoles at a low temperature. A total of 480 tadpoles with a mean weight of 0.078 g were distributed in 12 aquariums each containing 40 L of water in a closed water recirculation system. The experimental design was entirely randomized with four treatments (0, 150, 300, and 600 mg kg-1 of L-ascorbic acid monophosphate) and three replicates. The productive performance was measured by the weight gain, feed conversion, diet consumption, protein efficiency, carcass yield, hepatosomatic index, viscerosomatic index, visceral fat index, dry matter, and ethereal carcass extract. The water temperature during the experimental period was 21.74 ± 0.43 °C. Vitamin C supplementation did not influence carcass yield and viscerosomatic index. However, there was a quadratic effect of vitamin C supplementation on the weight gain, apparent feed conversion, protein efficiency, visceral fat index, hepatosomatic index, and ethereal carcass extract. Based on these results, bullfrog tadpoles should be supplemented with 600 mg vitamin C kg-1 of the diet when subjected to water temperatures of around 22 °C.

Abstract Objective The RNA exosome is an evolutionarily conserved 3′–5′ exoribonucleolytic protein complex involved in processing and degradation of different classes of nuclear and cytoplasmic RNAs, and, therefore, important for the posttranscriptional control of gene expression. Despite the extensive in vivo functional studies and the structural data on the RNA exosome, few studies have been performed on the localization and expression of exosome subunits during gametogenesis, process during which gene expression is largely controlled at the posttranscriptional level. Results We report the identification of exosome subunits in Lithobates catesbeianus and analysis of the differential subcellular localization of RNA exosome core and catalytic subunits in testis cells. In addition, we show seasonal differences in the expression levels of four exosome subunits in different organs. In addition to being part of the RNA exosome complex, its subunits might participate independently of the complex in the control of gene expression during seasonal variation in bullfrog tissues. These results may be relevant for other eukaryotic species.

This work aims to evaluate the effect of transport stress on the hematological and biochemical responses in males and females of bullfrogs (Lithobates catesbeianus). Frog conditions were evaluated before and at five times after transport (0, 6, 12, 24, and 48 h) for both genders. Glycemia increased in the frogs evaluated at time 0 h and recovered 24 h after transport. Lactate increased in the animals evaluated at time 0 h, recovered at time 6 h and reduced 48 h after transport. Aspartate-aminotransferase (AST) increased in animals analysed 0, 6, and 12 h and recovered 24 h after transport. Alanine-aminotransferase (ALT) increased in the frogs analysed at time 0 h, recovered at time 6 h and decrease 48 h after transport. Total proteins, albumin and globulins decreased 48 h after transport. Triglycerides and total cholesterol decrease in the animals evaluated 24 and 48 h after transport. Males had high levels of total cholesterol. Number of erythrocytes increased in the animals evaluated 0 and 6 h after transport and recovered 12 h after transport. Mean corpuscular volume (MCV) of the frogs evaluated 0 and 6 h after transport were lower and recovered 12 h after transport. Hematocrit and hemoglobin decrease 48 h after transport. Mean corpuscular hemoglobin (MCH) decreased in the animals evaluated 0, 6, and 12 h and recovered 24 h after transport. Frogs demonstrated rapid recovery of homeostasis after transport, between 6 and 24 h. Between 24 and 48 h after transport, a decrease in energy reserves and red blood series is observed, caused by prolonged fasting.

Conboy Lake National Wildlife Refuge (CLNWR) in Washington State supports one of the largest remaining populations of federally listed Oregon Spotted Frogs (OSF; Rana pretiosa). Invasive American Bullfrogs (Lithobates catesbeianus [Rana catesbeiana]), implicated in amphibian declines, are also abundant at CLNWR. Owing to the likely negative effect of this aquatic invader on the highly aquatic OSF, bullfrog control strategies are needed. One method is to limit habitat used by bullfrogs while retaining or enhancing OSF habitat, but for such an approach to be implemented, the 2 species must partition habitat sufficiently. We used radiotelemetry to characterize OSF and bullfrog active-season and pre-overwintering habitat in the human-engineered ditch system at CLNWR. We tracked 12 OSF and 10 bullfrogs from mid-to-late summer, recorded data on frog location and habitat, and analyzed movement patterns, niche breadth, and spatial differences between species. Based on minimum convex polygons, bullfrogs overlapped extensively with OSF habitat space in the ditch system. During the active season, niche analyses also showed extensive overlap in the species' vegetation associations, with selected exceptions at the microhabitat scale. However, each species used distinct pre-overwintering habitats. The small differences in active-season microhabitats and pre-overwintering habitats, and our relatively narrow study conditions, create uncertainty about these species' habitat use in a broader context. Nonetheless, these results underscore the challenge of managing an invasive species that is both a habitat generalist and congener and expose the need for further research to guide management approaches to favor OSFs at CLNWR. [ABSTRACT FROM AUTHOR]

The bullfrog, Lithobates catesbeianus, is one of the most important invasive anurans, affecting especially native anurans due to their similar habits. Here we used a hierarchical co-occurrence model fit in a Bayesian framework to investigate the effects of the bullfrog on two native frog species from southern Brazil, testing the hypothesis that bullfrog presence changes the activity of native species and their relationship with habitat. We found that both occupancy and detection probabilities of native species were similar with bullfrog presence or absence at a site. However, we observed changes in activity and microhabitat use preferences of both native species when the bullfrog was present, suggesting that the presence of the invasive species altered the behavior of the native species. Changes induced by invasive species can result in severe long-term consequences for native species since niche differentiation may not mediate the ability of species to persist together indefinitely. [ABSTRACT FROM AUTHOR]

In Mexico, bullfrog (Lithobates catesbeianus) production is carried out in only 35 animal production units, and improvements in its commercialization are necessary to increase demand. Faced with these challenges, innovative technological options such as Biofloc are emerging, which improve production efficiency in favor of environmental, economic and social sustainability. Thus, this study measured the effect of using reused water from a Biofloc system for tilapia (Oreochromis niloticus) culture in the intensive production of bullfrogs (Lithobates catesbeianus). With a biological material of 4,000 organisms (Lithobates catesbeianus), treatments were evaluated: T1 (drinking water), T2 (30% Biofloc reuse water), T3 (60% Biofloc reuse water) and T4 (90% Biofloc reuse water); the variables Weight Gain (WG), Specific Growth Rate (SGR), Survival Percentage (%S), Survival Rate (SR) and Feed Conversion (FC) were measured. The results were analyzed in SPSS Statistic version 27.0.0 with an ANOVA and Mauchly's test of sphericity. The results showed that intensive production of bullfrogs (Lithobates catesbeianus) with reused water from a Biofloc system of tilapia (Oreochromis niloticus) culture is feasible. [ABSTRACT FROM AUTHOR]

An alarming number of global warnings concerning amphibian mortality outbreaks have been released in recent years. Emerging diseases stand out as the main potential causes. Ranavirus is a worldwide-spread highly infectious disease capable of affecting even other ectothermic animals such as fish and reptiles. One major issue regarding this pathology is the lack of clinical signs before it leads up to death. Aiming at having a better understanding of anurans susceptibility, this study analyzed bullfrog (Lithobates catesbeianus) survival rate, when challenged with three doses of a Brazilian strain of Frog Virus 3 (FV3). The qPCR analysis indicated a low infectivity rate in these animals both as larvae and as adults. To elucidate the results, the following hypothesis was performed: 1) The amount of inoculum used on the frogs was insufficient to trigger an infection; 2) For the FV3 to produce clinical signs in this species, there is the need for a cofactor; 3) The animals did undergo FV3 infection but recovered in the course of the experiment, and 4) The inoculum utilized might have been low-virulence. Finally, the presence of actual clinical signs of ranavirus is discussed, with the more likely hypothesis.

Native to the eastern United States, American bullfrogs have been introduced throughout the western U.S. and to several other countries and islands around the world. Bullfrogs are well adapted for many of the permanent water sources that occur within the U.S., and once introduced, they typically become dominant. Because of their large size and voracious appetite, bullfrogs outcompete and prey upon many indigenous species. They are hypothesized to be cause significant negative impacts, which may contribute to the endangerment and extinction of some sensitive species. There are few, if any, effective and efficient control methods to manage invasive bullfrogs. Current methods such as hand or net capture, shooting, and gigging can be labor intensive and often fail to reduce bullfrog numbers. Draining wetland habitats and broadcasting toxicants have severe negative effects on non-target species. New management options, such as locally-sprayed toxicants and multiple-capture traps, could be useful for reducing populations of invasive bullfrogs. However, researchers should make certain that non-target species are not affected by these management techniques.

Environmental temperature can alter the composition, diversity, and function of ectothermic vertebrate gut microbial communities, which may result in negative consequences for host physiology, or conversely, increase phenotypic plasticity and persistence in harsh conditions. The magnitude of either of these effects will depend on the length of time animals are exposed to extreme temperatures, and how quickly the composition and function of the gut microbiota can respond to temperature change. However, the temporal effects of temperature on gut microbiota are currently unknown. Here, we investigated the length of time required for increased temperature to alter the composition of gut bacterial communities in tadpoles of two frog species, the green frog, Lithobates clamitans, and its congener, the globally invasive American bullfrog, L. catesbeianus. We also explored the potential functional consequences of these changes by comparing predicted metagenomic profiles across temperature treatments at the last experimental time point. Bullfrog‐associated microbial communities were more plastic than those of the green frog. Specifically, bullfrog communities were altered by increased temperature within hours, while green frog communities took multiple days to exhibit significant changes. Further, over ten times more bullfrog bacterial functional pathways were temperature‐dependent compared to the green frog. These results support our hypothesis that bullfrog gut microbial communities would respond more rapidly to temperature change, potentially bolstering their ability to exploit novel environments. More broadly, we have revealed that even short‐term increases in environmental temperature, expected to occur frequently under global climate change, can alter the gut microbiota of ectothermic vertebrates. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effect of the probiotic Bacillus subtillis and beta-glucan from the fungus Agaricus blazei on survival, growth and immunological capacity in bullfrogs (Lithobates catesbeianus) cultured in low and high stocking densities. Animals weighing 24.3 ± 2.38 g were randomly distributed into four treatments with four simultaneous replicates: D100: 100 frogs/m2 (control); D236: 236 frogs/m2; D236 + Prob.: 236 frogs/m2 supplemented with probiotic; and D236 + BG: 236 frogs/m2 supplemented with beta-glucan. The parameters evaluated were weight gain, survival, plasma corticosterone (CORT), phagocytic capacity (PC) and phagocytic index (PI), at 24 h and 15 and 30 days. There is significant interaction between treatments and time for CORT levels. At 30 days, these values were very close for the D100 (control) and D236 + BG groups. Meanwhile, no statistical differences were observed between treatments for PC and PI. These results indicate that beta-glucan reduced the effects of stress caused by high density in bullfrogs, but the probiotic did not reduce these effects. Both compounds are not efficient at increasing survival rates, weight gain and neither immune response of animals. Thus, the use of commercial food additives may not have the favorable impact desired by the farmer. Their use in aquaculture should be further studied in experiments involving a longer trial period and taking into account the cost of their use.

Many animals use a combination of skeletal muscle and elastic structures to amplify power output for fast motions. Among vertebrates, tendons in series with skeletal muscle are often implicated as the primary power-amplifying spring, but muscles contain elastic structures at all levels of organization, from the muscle tendon to the extracellular matrix to elastic proteins within sarcomeres. The present study used ex vivo muscle preparations in combination with high-speed video to quantify power output, as the product of force and velocity, at several levels of muscle organization to determine where power amplification occurs. Dynamic ramp-shortening contractions in isolated frog flexor digitorum superficialis brevis were compared with isotonic power output to identify power amplification within muscle fibers, the muscle belly, free tendon and elements external to the muscle tendon. Energy accounting revealed that artifacts from compliant structures outside of the muscle-tendon unit contributed significant peak instantaneous power. This compliance included deflection of clamped bone that stored and released energy contributing 195.22±33.19 W kg-1 (mean±s.e.m.) to the peak power output. In addition, we found that power detected from within the muscle fascicles for dynamic shortening ramps was 338.78±16.03 W kg-1, or approximately 1.75 times the maximum isotonic power output of 195.23±8.82 W kg-1. Measurements of muscle belly and muscle-tendon unit also demonstrated significant power amplification. These data suggest that intramuscular tissues, as well as bone, have the capacity to store and release energy to amplify whole-muscle power output., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

We identified the stomach contents of 65 American Bullfrogs collected in two counties in Virginia. Both vertebrate and invertebrate prey were consumed, including a worm, mollusks, crustaceans, millipedes, centipedes, spiders, insects, a fish, amphibians, and a mammal. In an analysis of 40 of the stomachs containing food, 91 prey items were identified, a third of which were beetles. Crayfish and millipedes were other major prey, representing 27% and 15% of the total prey items, respectively. Several highly poisonous animals, including the cyanide-producing millipede, Apheloria tigana, and the tetrodotoxin-producing salamander, Notophthalmus viridescens, were found as prey. [ABSTRACT FROM AUTHOR]

The potential mutagenic and histopathological effects of the hexavalent chromium were investigated in Lithobates catesbeianus tadpoles. These larvae (GS 25-31) were exposed to three nominal concentrations of potassium dichromate (4, 12, and 36 mg L −1 ) and 5 mg L −1 of Cyclophosphamide as a positive control (PC), for 24 h. A negative control (NC) was also added to the experiment. Our results showed that, in general, the micronuclei (MN) were less frequent than the erythrocyte nuclear abnormalities (ENA); there was a significant difference in the frequency of MN between the NC and all treated groups (p < 0.05) in a concentration-dependent curve, in addition the PC did not differ from the chromium treatments. Also, only PC and the group treated with potassium dichromate at 36 mg L −1 showed significantly higher frequencies of ENA than NC (p < 0.05). Chromium treatments promoted cell retention in the Sub-G1 phase and a decrease of cells in the S and G2/M phases indicating inhibition of the cell cycle. All treatments with chromium led to liver and kidney histopathological lesions, especially with 36 mg L −1 (greater number of lesions). In conclusion, hexavalent chromium was mutagenic to L. catesbeianus tadpoles and its toxic effects also resulted in anti-mitotic activity, besides inducing histopathological alterations in liver and kidney. Amphibians have been proven to be useful bioindicators, and we suggest that tadpoles of different species can be used to represent the environmental impacts in aquatic ecosystems. [ABSTRACT FROM AUTHOR]

Resumo: Para verificação da ocorrência de alterações morfológicas no fígado, foram coletados dez animais de cada dieta, no 30º dia (1º período experimental) e no 60º dia (2º período experimental), sendo esse, o último dia do período experimental e início do clímax da metamorfose. Os girinos foram distribuídos em delineamento experimental inteiramente casualizado (DIC) com três tratamentos: dieta experimental com 32,68% de proteína bruta e duas dietas comerciais com 37,92% e 57,53% de proteína bruta. Os órgãos coletados foram fixados em solução de Bouin e depois submetidos às práticas da rotina histotecnológica e coradas com hematoxilina - Eosina (HE). Na avaliação microscópica do fígado foram encontradas diversas alterações morfológicas como: Desorganização e vacuolização de hepatócitos, infiltrado inflamatório mononuclear periportal, multifocal e difuso, infiltrado inflamatório eosinofílico, congestão, hemorragia, hemólise e necrose. As alterações hepáticas encontradas neste estudo sugerem que as dietas utilizadas para os girinos de rã-touro, não atenderam suas necessidades dietéticas, afetando a homeostasia dos mesmos, comprometendo assim, sua sanidade.

Xenopus laevis oocytes are a valuable tool for investigating the function of membrane proteins. However, regulations around the world, specifically in Brazil, render the import of Xenopus laevis frogs impractical, and, in some cases, impossible. Here, as an alternative, we evaluate the usefulness of the North American aquatic bullfrog Lithobates catesebeianus, which is commercially available in Brazil, for the heterologous expression of aquaporin (AQP) proteins. We have developed a method that combines a brief collagenase treatment and mechanical defolliculation for isolating individual oocytes from Lithobates ovaries. We find that they have a similar size, shape, and appearance to Xenopus oocytes and can tolerate and survive following injections with cRNA or water. Furthermore, surface biotinylation, western blot analysis, and measurements of osmotic water permeability (Pf) show that Lithobates oocytes can express AQPs to the plasma membrane and significantly increase the Pf of the oocytes. In fact, the Pf values are similar to historical values gathered from Xenopus oocytes. Due to the presence of a mercury sensitive cysteine (Cys or C) in the throat of the water channel, the Pf of oocytes expressing human (h) AQP1, hAQP1FLAG [FLAG, short protein tag (DYKDDDDK) added to the N-terminus of AQP1], hAQP8, and rat (r) AQP9 was inhibited with the mercurial compound p-chloromercuribenzene sulfonate (pCMBS), whereas AQPs lacking this Cys – hAQP1C189S mutant [residue Cys 189 was replaced by a serine (Ser or S)] and hAQP7 – were mercury insensitive. Contrary to previous studies with Xenopus oocytes, rAQP3 was also found to be insensitive to mercury, which is consistent with the mercury-sensitive Cys (Cys 11) being located intracellularly. Thus, we consider Lithobates oocytes to be a readily accessible system for the functional expression and study of membrane proteins for international researchers who do not currently have access to Xenopus oocytes.