Bullfrog Research Articles

Systematic evaluation of the influence of Taq enzyme choice and amplification conditions on targeted environmental DNA assay performance and detection in field samples

AbstractTargeted environmental DNA (eDNA) studies mainly rely on quantitative real‐time polymerase chain reaction (qPCR) to amplify extremely low concentrations of DNA present in environmental samples. Understanding factors that influence targeted eDNA assay performance and detection in field samples, such as Taq DNA polymerase enzyme type and thermocycle protocol, is critical for the interpretation of qPCR results. We completed a systematic performance evaluation of five distinct targeted eDNA assays (eANFI6, eFISH1, eANBO5, eGLIN1, and eLICA3 targeting sablefish, general fish, Boreal toad, wood turtle, and the American bullfrog) by subjecting the same samples to analysis by five different Taq enzyme reagent mixes (Immolase, Environmental Master Mix (EMM), Amplitaq, QIAcuity, and QuantiNova) and the commonly used 2‐step (95°C, 60°C) and 3‐step (95°C, 64°C, 72°C) thermocycle protocols. We evaluated assay performance using a standardized dilution series of synthetic dsDNA target sequences and calculated limits of detection (LOD) and quantification (LOQ) and 95% confidence intervals for each combination of Taq enzyme and thermocycle protocol. All assays performed within acceptable performance criteria as defined by the Canadian national standard for targeted eDNA assays. Based on data generated by synthetic dsDNA fragments, the eDNA assays performed comparably regardless of the enzyme reagent mix and protocol used, except for eANFI6 and eGLIN1 using EMM and the 3‐step protocol, where no amplification was observed. On freshwater field samples, EMM and Immolase performed best. On marine field samples, Immolase, EMM, Qiacuity, and QuantiNova performed equally well, although EMM failed to amplify some samples. The work reveals that an enzyme reaction mix or thermocycle protocol can affect the result of an eDNA assay, but the appropriate choice also depends on the nature of the field sample. It is therefore imperative that these are considered when selecting appropriate reaction conditions and that they are clearly reported.

Transcriptomic profiling of Rana [Lithobates] catesbeiana back skin during natural and thyroid hormone-induced metamorphosis under different temperature regimes with particular emphasis on innate immune system components

As amphibians undergo thyroid hormone (TH)-dependent metamorphosis from an aquatic tadpole to the terrestrial frog, their innate immune system must adapt to the new environment. Skin is a primary line of defense, yet this organ undergoes extensive remodelling during metamorphosis and how it responds to TH is poorly understood. Temperature modulation, which regulates metamorphic timing, is a unique way to uncover early TH-induced transcriptomic events. Metamorphosis of premetamorphic tadpoles is induced by exogenous TH administration at 24 °C but is paused at 5 °C. However, at 5 °C a “molecular memory” of TH exposure is retained that results in an accelerated metamorphosis upon shifting to 24 °C. We used RNA-sequencing to identify changes in Rana (Lithobates) catesbeiana back skin gene expression during natural and TH-induced metamorphosis. During natural metamorphosis, significant differential expression (DE) was observed in >6500 transcripts including classic TH-responsive transcripts (thrb and thibz), heat shock proteins, and innate immune system components: keratins, mucins, and antimicrobial peptides (AMPs). Premetamorphic tadpoles maintained at 5 °C showed 83 DE transcripts within 48 h after TH administration, including thibz which has previously been identified as a molecular memory component in other tissues. Over 3600 DE transcripts were detected in TH-treated tadpoles at 24 °C or when tadpoles held at 5 °C were shifted to 24 °C. Gene ontology (GO) terms related to transcription, RNA metabolic processes, and translation were enriched in both datasets and immune related GO terms were observed in the temperature-modulated experiment. Our findings have implications on survival as climate change affects amphibia worldwide.

Dietary phosphorus requirement of bullfrog Aquaranacatesbeiana

This experiment investigated the dietary phosphorus (P) requirement of bullfrogs Aquaranacatesbeiana. Six diets containing available P (AP) levels of 0.29% (the control group), 0.47%, 0.65%, 0.88%, 1.06% and 1.24% were formulated and fed to bullfrogs for 56 days. As dietary AP level increased from 0.29% to 1.24%, weight gain rate increased and then decreased, reaching the highest at the AP level of 0.88% (P < 0.05). Compared to the control group, content of crude protein in the whole-body was significantly increased in bullfrogs fed the 0.88% AP diet (P < 0.05). Content of crude lipid in the whole-body was significantly decreased in bullfrogs fed the 0.65%, 0.88%, 1.06% and 1.24% AP diets compared to those fed the 0.29% and 0.47% AP diets (P < 0.05). Bullfrogs fed the 0.65%, 0.88%, 1.06%, and 1.24% AP diets had higher content of serum P than those fed the 0.29% and 0.47% AP diets (P < 0.05). Activity of glutamic pyruvic transaminase in the serum was significantly decreased in bullfrogs fed the 0.88%, 1.06% and 1.24% AP diets compared to those fed the 0.29% and 0.47% AP diets (P < 0.05). Bullfrogs fed the 0.65%, 0.88%, 1.06% and 1.24% AP diets had lower contents of total cholesterol in the liver and triglycerides in the serum than the control group (P < 0.05). Meanwhile, bullfrogs fed the 0.47%, 0.65%, 0.88% and 1.06% AP diets had higher total antioxidant capacity in the serum than the control group (P < 0.05). Compared to the control group, contents of P and calcium in the whole-body were significantly increased in bullfrogs fed the 0.65%, 0.88%, 1.06% and 1.24% AP diets (P < 0.05). Compared to the control group, bullfrogs fed the 0.88%, 1.06% and 1.24% AP diets had lower expression of sodium-dependent phosphate transporter in the intestine and kidney, and higher expression of hormone-sensitive triglyceride lipase in the liver (P < 0.05). Bullfrogs fed the 0.88% and 1.06% AP diets had higher the mucosal fold height than those fed the 0.29% and 1.24% AP diets (P < 0.05). Compared to those fed the 0.29% and 1.24% AP diets, the abundance of potentially pathogenic bacteria Serratia and Acinetobacter in the intestine was decreased in bullfrogs fed the 0.88% AP diet. Using the weight gain rate, feed efficiency, whole-body and serum P content as evaluation indicators, the AP requirements of bullfrogs was 0.82% to 0.89%.

Evaluation of sex-based differences in central control of breathing in American bullfrogs

The neural control of breathing exhibits sex differences. There is now a large effort to account for biological sex in mammalian research, but the degree to which ectothermic vertebrates exhibit sex differences in the central control of breathing is not well-established. Therefore, we compared respiratory-related neural activity in brainstem-spinal cord preparations from female and male bullfrogs to determine if important aspects of the central control of breathing vary with sex. We found that the breathing pattern was similar across males and females, but baseline frequency of the respiratory network was faster in females. The magnitude of the central response to hypercapnia was similar across sexes, but the time to reach maximum burst rate occurred more slowly in females. These results suggest that sex differences may account for variation in traits associated with the control of breathing and that future work should carefully account for sex of the animal in analysis.

Rearing American bullfrog tadpoles in different tank colors: Effect on metamorphosis, performance, and biochemistry responses

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.

Variations in Genetic Diversity of Invasive Species Lithobates catesbeianus in China.

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.

Immune-pineal-ocular axis in amphibians: unveiling a novel connection?

Melatonin is a hormone known as an endogenous temporal marker signaling the dark phase of the day. Although the eyes seem to be the main site of melatonin production in amphibians, little information is available about the natural variation in the ocular melatonin levels and its modulation following immune stimulation. We investigated the daily variation of plasma and ocular melatonin levels in bullfrogs (Lithobates catesbeianus) and their modulation following an immune stimulation with lipopolysaccharide (LPS) in yellow cururu toads (Rhinella icterica). For the daily variation, bullfrogs were bled and then euthanized for eye collection every 3h over 24h to determine plasma and ocular melatonin levels. We found a positive correlation between ocular and plasma melatonin levels, with maximum values at night (22h) for both plasma and the eyes. For immune stimulation, yellow cururu toads received an intraperitoneal injection of LPS or saline solution during the day (10h) or at night (22h). Two hours after injection, toads were bled and euthanized for eye collection to obtain plasma and ocular melatonin levels. In addition, the liver and bone marrow were collected to investigate local melatonin modulation. Our results demonstrate that retina light-controlled rhythmic melatonin production is suppressed while liver and bone marrow melatonin levels increase during the inflammatory assemblage in anurans. Interestingly, the LPS injection decreased only ocular melatonin levels, reinforcing the central role of the eyes (i.e., retina) as an essential organ of melatonin production, and a similar role to the pineal gland during the inflammatory response in amphibians. Together, these results point to a possible immune-pineal-ocular axis in amphibians, yet to be fully described in this group.

Biochemical analysis of bullfrog liver and blood: insights into growth and development

The aim of this study was to evaluate the productive and physiological performance of bullfrogs (Lithobates catesbeianus) after metamorphosis, through zootechnical growth parameters and the quantification of proteins and enzymes present in the liver. In addition we observed the behavior of circulating amino acids in the blood. The experiment was conducted at the Experimental Station of the Aquaculture, APTA Regional – SAA, in São Paulo, Brazil. We used 400 L. catesbeianus froglets weighing 5.76±1.32g, which were distributed in four fattening tanks in the wet system, installed under an agricultural greenhouse at density of 51 frogs/m². The animals were fed with 5% of the biomass of each tank every day, with extruded feed for carnivorous fish with 40% Crude Protein. Every 28 days, they were weighed to calculate weight gain and apparent feed conversion ratio. At the same time, two animals from each tank were chosen randomly to have their blood and liver collected for biochemical analysis to quantify amino acids, proteins, phenols, peroxidase and polyphenol oxidase. The results obtained indicate that the absence of screening every 15 days affected the development and uniformity of the animals, favoring cannibalism. The average apparent feed conversion was 1.84. There was an increasing and proportional formation of proteins with low degradation as the animals gained weight. As for amino acids, we detected arginine, isoleucine, alanine and tryptophan in the first month, with trypsin appearing after 60 days. These results lead us to state that total or partial screening is essential during the first month after metamorphosis, and that the feed provided, although not specific to the species, fulfilled its role in animal metabolism, with little formation of phenolic compounds and free radicals.

Defenses of whirligig beetles against native and invasive frogs.

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.

Effects of dietary lipid and protein levels on metamorphosis, growth, metabolism and gut microbiota of tadpole (Lithobates catesbeianus)

Tadpole cultivation is a crucial step in the artificial production of bullfrogs (Lithobates catesbeiana), but its feed nutrition is poorly understood. This study examined how dietary lipid and protein levels affect metamorphosis, growth and intestinal health of tadpole. Six groups of tadpoles were fed six diets with two lipid levels (7% and 10%) and three protein levels (36%, 40% and 44%) for 70 days. The results showed that at 7% lipid level, increasing protein enhanced metamorphosis rate and mean weight of tadpoles, reaching the optimal value at 40% protein (P < 0.05). At 10% lipid level, increasing protein also improved mean weight, peaking at 40% protein. Dietary nutrition also influenced liver amino acid and lipid metabolism directly. At 7% lipid level, increasing protein significantly increased the activities of hepatic enzymes GLS, PDH, ALT, AST, etc. (P < 0.05), and up-regulated the expression of hepatic genes fas, accb, fadd6 and cpt1a involved in lipid metabolism (P < 0.05). At 10% lipid level, increasing protein also up-regulated fadd6 and cpt1a genes (P < 0.05), but down-regulated acadl gene. Meanwhile, increasing dietary lipid level elevated TG and T-CHO levels in liver. Increasing dietary protein enhanced gut microbiota richness, while increasing lipid reduced it. Moreover, different gut microbiota groups had distinct biomarkers, such as Rikenellaceae, Desulfovibrionaceae, Burkholderiales and Bacteroidales, which were related to amino acid or lipid metabolism. In summary, under the test conditions, 7% lipid and 40% protein in diet are suitable levels, which can promote metamorphosis, growth and metabolism of tadpoles, and are more economical than high-lipid and high-protein diets.

Food preference for native and invasive prey in the naive lancehead pitviper

AbstractThe presence of invasive species in the environment can be highly detrimental. The success of these species depends on their interaction with native ones. The prey–predator relationship between invasive and native species can result in biodiversity loss or the introduction of new food items for local predators. Due to its tropical and extensive nature, Brazil is vulnerable to the colonization of invasive species. Snakes are a group of animals that can act as both predators and prey for invasive species. The pitviper Bothrops jararaca is an abundant and widely distributed native species on the Brazilian coast. Based on this, we conducted a study on the feeding preferences of naive B. jararaca juveniles regarding invasive and native prey. We found that this species exhibits a greater preference for native anurans compared to Rana catesbeiana, an invasive amphibian associated with significant environmental impacts. Additionally, the gecko Hemidactylus mabouia, a prey that triggered intense predatory responses from the pitviper, reveals that this species, even with a short period of coexistence with B. jararaca, has established an intense prey–predator relationship. The negative correlation between prey size and feeding interest, guided solely by olfactory cues, adds a layer of complexity to understanding the feeding choices of B. jararaca, providing valuable insights for conservation and environmental management strategies.Abstract in Portuguese is available with online material.

Immunohistochemical Characterization of Langerhans Cells in the Skin of Three Amphibian Species.

The amphibian taxon includes three orders that present different morphological characteristics: Anura, Caudata, and Apoda. Their skin has a crucial role: it acts as an immune organ constituting a physical, chemical, immunological, and microbiological barrier to pathogen insult and conducts essential physiological processes. Amphibians have developed specialized features to protect the vulnerable skin barrier, including a glandular network beneath the skin surface that can produce antimicrobial and toxic substances, thus contributing to the defense against pathogens and predators. This study aims to characterize Langerhans cells in the skin of Lithobates catesbeianus (order: Anura; Shaw, 1802), Amphiuma means (order: Caudata; Garden, 1821), and Typhlonectes natans (order: Apoda; Fischer, 1880) with the following antibodies: Langerin/CD207 (c-type lectin), Major Histocompatibility Complex (MHC)II, and Toll-like receptor (TLR)2 (expressed by different types of DCs). Our results showed Langerhans cells positive for Langerin CD/207 in the epidermis of the three species; moreover, some antigen-presenting cells (APCs) in the connective tissue expressed TLR2 and MHCII. The distribution of the Langerhans cells is very similar in the three amphibians examined, despite their different habitats. A greater knowledge of the amphibian immune system could be useful to better understand the phylogeny of vertebrates and to safeguard amphibians from population declines. Furthermore, the similarities between amphibians' and human skin concerning immunological features may be useful in both biology and translational medicine.

Ranavirus and helminth parasite co-infection in invasive American bullfrogs in the Atlantic forest, Brazil

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.

Multivariate data analysis for semen quality in two frog species

Despite the growing body of research on frog reproduction and semen quality, studies employing statistical methods and analyses are scarce. The present study aimed to examine the use of exploratory multivariate data analysis for evaluating the seminal quality of frogs, determining a possible hierarchy among frog species based on semen characteristics, and identifying the most effective clustering strategy. Data on the seminal quality of the butter frog (Leptodactylus ocellatus, native specie) were collected from seven samples, and for the bullfrog (Lithobates catesbeianus, exotic specie), five samples were used, all derived from a literature review. Four distinct clustering strategies were tested: Single Linkage, Unweighted Pair Group Method with Arithmetic Mean (UPGMA), Complete Linkage, and Ward’s method. Exploratory multivariate data analysis could effectively evaluate frog seminal quality, allowing the classification of frog species (both native and exotic) based on semen. Among the tested strategies, UPGMA emerged as the most suitable grouping method.

Large-landscape connectivity models for pond-dwelling species: methods and application to two invasive amphibians of global concern

ContextFunctional 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.ObjectivesWe 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.MethodsAll 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.ResultsThe 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.ConclusionOur 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.

Heritability Estimates for Growth Traits and Correlation Analysis between Weight and Metamorphosis Rate in the Bullfrog Rana (Aquarana) catesbeiana

Metamorphosis is a crucial process in the life cycle of Rana (Aquarana) catesbeiana. R. catesbeiana tadpoles, in their short larval period, possess a high survival rate and also a highly competitive ability in the amphibious stage. In actual seed production, the economic traits of larval period and metamorphosis rate are used as quantifiable indicators of quality for individuals and populations, respectively. However, studies of these economic traits in larval cultivation and production are still lacking. In this study, we constructed 40 full-sib families of R. catesbeiana and measured the weight and metamorphosis rate of tadpoles at different developmental stages. Subsequently, we calculated the phenotypic and genetic association between weight and metamorphosis rate in tadpoles and assessed the heritability of these two traits. The heritabilities of weight at three developmental stages were all higher than 0.40 and decreased with advancement of the developmental stage; the heritability of the metamorphosis rate was 0.18 ± 0.20, a moderate level. Correlation analysis of weight and metamorphosis rate at each developmental stage in each tadpole family showed that weight at stages 25-I, 25-II, and 25-III was significantly correlated at the phenotypic level but non-significantly at the genetic level. The metamorphosis rate was only moderately associated with stage 25-III weight (0.38, p &lt; 0.05). The results of this study confirm the importance and transportability of tadpole weight in actual seed production and provide basic data and a potentially optimized direction for the selective breeding of high-metamorphosis-rate bullfrogs.

Using acoustic energy of vocalizations to monitor population size and phenology of anurans

As a species that lives at the land/water interface, the American bullfrog (Rana catesbeianus) serve as a bioindicator in many habitats, yet also invasive in many locations. Due to challenges with traditional monitoring approaches, there is a lack of fine-scale population and phenological data for bullfrogs. Passive acoustic monitoring (PAM) can provide a low-cost alternative with high-resolution data for monitoring vocal animals. Sexually mature male bullfrogs attract mates by calling from exclusive territories. These vocalizations can be used to explore bullfrog behavior, population size, and phenology. We describe the analysis framework and initial results from an project monitoring the vocal behavior of frogs in 25 ponds in southeastern New Hampshire during the reproductive season using acoustic arrays. By using an acoustic energy index (RMS amplitude), we can estimate numbers of frogs in ponds, determine timing of reproduction, and even document anthropogenic disturbance. Our results can lead to future uses of PAM to monitor population size and phenology and develop reliable long-term management and conservation strategies.

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

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

Strobilurin fungicide increases the susceptibility of amphibian larvae to trematode infections

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

Evaluation of sensory modalities involved in finding food and foraging behaviour in light/dark conditions in tadpoles of the invasive species Lithobates catesbeianus

AbstractThe ability of anuran larvae to efficiently locate food is essential for their growth and development, but little is known about the sensory modalities they use to find food. We experimentally assessed the role of visual and chemical cues in finding food in tadpoles of the invasive species Lithobates catesbeianus. In the two‐choice experiment, using rectangular containers that offer visual and/or chemical food cues on opposite ends, tadpoles chose the side where the chemical cues of the food were present consistently. The visual cues alone showed an initial transient attraction of tadpoles, which disappeared after a few minutes, suggesting that tadpoles are potentially capable of seeing contrasting objects, at least, at a close range and in clear waters and daylight conditions. We also evaluated the foraging behaviour and the capability of tadpoles to find food in an open field experiment under two different lighting conditions: natural daylight and darkness. Results showed that tadpoles find food faster in dark conditions, confirming that chemical cues alone are sufficient for tadpoles of this species to find food. Moreover, the time spent feeding and the number of tadpoles simultaneously exploiting the food patch were greater in darkness suggesting that environments with low visibility conditions could favour the establishment of L. catesbeianus. Understanding the sensory modalities used by tadpoles of this invasive species to find food could help to understand the potential abilities to expand and establish in novel environments.