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.

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.

Evidence for multi-scale power amplification in skeletal muscle.

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.

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

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

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

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

Bullfrogs (Lithobates catesbeianus) as a Potential Source of Foodborne Disease.

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.

Influence of temperature on the biomarker responses of bullfrog tadpoles (Lithobates catesbeianus) to 2-hydroxyatrazine exposure.

The influence of temperature (25 and 32 °C) on the biomarker responses of bullfrog tadpoles (Lithobates catesbeianus) to different concentrations of the atrazine metabolite 2-hydroxyatrazine (2-HA, 0, 10, 50 and 200 ng.L-1, 16 days), was evaluated. Temperature affected the activities of superoxide dismutase, glutathione S-transferase and acetylcholinesterase. The activities of catalase, glutathione peroxidase, glucose-6-phosphate dehydrogenase and carboxylesterase presented no alterations. Frequencies of micronuclei and nuclear abnormalities were also not altered. 2-HA decreased SOD activity at 25 °C and caused histopathological changes in the liver and the kidney at both temperatures, with the kidney being more affected by the combination of higher temperature and 2-HA exposure, presenting glomerular shrinkage and an increase in Bowman's space. Our results indicate that at environmentally relevant concentrations, 2-HA can cause changes in biomarker responses as well as in the morphology of liver and kidney in L. catesbeianus tadpoles. Temperature has an important influence on biomarker response and histopathological alterations.

Environmental DNA Assay for the Detection of the American Bullfrog (Lithobates catesbeianus) in the Early Stages of the Invasion in the Ebre Delta.

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.

Microcystin-leucine arginine induces skin barrier damage and reduces resistance to pathogenic bacteria in Lithobates catesbeianus tadpoles.

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.

Microcystin-leucine arginine exposure induced intestinal lipid accumulation and MC-LR efflux disorder in Lithobates catesbeianus tadpoles.

Few studies exist on the toxic effects of chronic exposure to microcystins (MCs) on amphibian intestines, and the toxicity mechanisms are unclear. Here, we evaluated the impact of subchronic exposure (30 days) to environmentally realistic microcystin-leucine arginine (MC-LR) concentrations (0 µg/L, 0.5 µg/L and 2 µg/L) on tadpole (Lithobates catesbeianus) intestines by analyzing the histopathological and subcellular microstructural damage, the antioxidative and oxidative enzyme activities, and the transcriptome levels. Histopathological results showed severe damage accompanied by inflammation to the intestinal tissues as the MC-LR exposure concentration increased from 0.5 µg/L to 2 µg/L. RNA-sequencing analysis identified 634 and 1,147 differentially expressed genes (DEGs) after exposure to 0.5 µg/L and 2 µg/L MC-LR, respectively, compared with those of the control group (0 µg/L). Biosynthesis of unsaturated fatty acids and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were upregulated in the intestinal tissues of the exposed groups, with many lipid droplets being observed on transmission electron microscopy, implying that MC-LR may induce lipid accumulation in frog intestines. Moreover, 2 µg/L of MC-LR exposure inhibited the xenobiotic and toxicant biodegradation related to detoxification, implying that the tadpoles' intestinal detoxification ability was weakened after exposure to 2 µg/L MC-LR, which may aggravate intestinal toxicity. Lipid accumulation and toxin efflux disorder may be caused by MC-LR-induced endoplasmic reticular stress. This study presents new evidence that MC-LR harms amphibians by impairing intestinal lipid metabolism and toxin efflux, providing a theoretical basis for evaluating the health risks of MC-LR to amphibians.

Evaluation of nutritional composition and technological functionality of whole American Bullfrog (Lithobates catesbeianus), its skin, and its legs as potential food ingredients.

Frog farming systems do not take advantage of their byproducts, which represents health risks and environmental pollution. The present study aimed to evaluate the proximate composition, amino acid, and fatty acid profile of American Bullfrog byproducts (whole frogs (WF), legs (LF), and skin (SF)) and their technological functionality. Results showed that WF, LF, and SF protein content was 47.6, 88.4, and 91.1% dry base (d.b.), correspondingly. Fat content resulted in 34.6, 3.2, and 4.2% (d.b.), respectively. Moreover, byproducts contain all the essential amino acids (23.8-46.6%), and the unsaturated fatty acids predominated the saturated fats. Samples showed water and oil absorption capacities of 1.8-2.6% and 1.8-4.0%, respectively, while oil and water emulsion capacities were 76.7-98.3% and 36.1-85.6%, correspondingly. Additionally, SF presented a gelling capacity in a 5% concentration. These results showed that frogs' byproducts have adequate nutritional and functional capacities, compared to other vegetable and animal flours used in the industry.

Resilience of native amphibian communities following catastrophic drought: Evidence from a decade of regional-scale monitoring.

The increasing frequency and severity of drought may exacerbate ongoing global amphibian declines. However, interactions between drought and coincident stressors, coupled with high interannual variability in amphibian abundances, can mask the extent and underlying mechanisms of drought impacts. We synthesized a decade (2009 - 2019) of regional-scale amphibian monitoring data (2273 surveys, 233 ponds, and seven species) from across California's Bay Area and used dynamic occupancy modeling to estimate trends and drivers of species occupancy. An extreme drought during the study period resulted in substantial habitat loss, with 51% of ponds drying in the worst year of drought, compared to <20% in pre-drought years. Nearly every species exhibited reduced breeding activity during the drought, with the occupancy of some species (American bullfrogs and California newts) declining by >25%. Invasive fishes and bullfrogs were also associated with reduced amphibian occupancy, and these taxa were locally extirpated from numerous sites during drought, without subsequent recovery-suggesting that drought may present an opportunity to remove invaders. Despite a historic, multi-year drought, native amphibians rebounded quickly to pre-drought occupancy levels, demonstrating evidence of resilience. Permanent waterbodies supported higher persistence of native species during drought years than did temporary waterbodies, and we therefore highlight the value of hydroperiod diversity in promoting amphibian stability.

Effects of Supplementing Intestinal Autochthonous Bacteria in Plant-Based Diets on Growth, Nutrient Digestibility, and Gut Health of Bullfrogs (Lithobates catesbeianus).

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.

Vitamin C dietary supplementation influence tadpoles of bullfrog Lithobates catesbeianus reared in low water temperature / A suplementação da dieta com vitamina C influencia os girinos de rã-touro Lithobates catesbeianus criados em baixa temperatura da água

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.(AU)

A suplementação de vitamina C é importante para o crescimento e desenvolvimento de girinos de rã-touro sob condições ótimas de temperatura da água. Desta forma, foi realizado um experimento para avaliar o efeito da suplementação de vitamina C sobre a dieta de girinos de rã-touro em baixa temperatura. O experimento foi realizado com 480 girinos com peso médio de 0,078g, distribuídos em 12 aquários com 40L de água em sistema fechado de recirculação de água. O delineamento experimental foi inteiramente casualizado, com quatro tratamentos (0, 150, 300 e 600 mg kg-1 de monofosfato de ácido L-ascórbico) e três repetições. O desempenho produtivo foi avaliado por meio de ganho de peso, conversão alimentar, consumo de ração, eficiência proteica, rendimento de carcaça, índice hepatossomático, índice viscerossomático, índice de gordura visceral, matéria seca e extrato etéreo de carcaça. A temperatura da água durante o período experimental foi de 21,74 ± 0,43 °C. A suplementação com vitamina C não influenciou o rendimento de carcaça e o índice viscerossomático. No entanto, houve efeito quadrático da suplementação com vitamina C sobre o ganho de peso, a conversão alimentar aparente, a eficiência protéica, o índice de gordura visceral, o índice hepatossomático e o extrato etéreo de carcaça. Com base nos resultados do presente estudo, os girinos de rã-touro devem ser suplementados com 600 mg vitamina C kg-1 da dieta quando submetidos em temperaturas médias de 22 °C.(AU)

Complete mitochondrial genome of the American bullfrog in Korea, Lithobates catesbeianus (Anura: Ranidae).

The complete mitochondrial (mt) genome of Lithobates catesbeianus was sequenced and characterized. The circular mt genome was constituted of of 37 genes (13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs) and a non-coding region (NCR). Phylogenetic analysis based on the full mt genome sequences confirmed that among the genus Lithobates, L. catesbeianus Korea is included in a monophyletic group with L. catesbeianus China, but not with either L. catesbeianus Japan or L. catesbeianus Canada. This is the first completed mt genome from L. catesbeianus Korea, which provide data for further study of phylogeny in Lithobates spp. that have been introduced into a number of different countries originally from North America.

Gut microbiota of invasive bullfrog tadpoles responds more rapidly to temperature than a noninvasive congener.

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.

Experimental Frog Virus 3 infection using Brazilian strain: amphibians susceptibility / Infecção experimental utilizando estirpe brasileira de Frog Virus 3: suceptibilidade de anfíbios

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.(AU)

Um número alarmante de notificações globais sobre surtos de mortalidade de anfíbios tem sido realizado nos últimos anos. As doenças emergentes destacam-se como as principais causas potenciais. O ranavírus é uma doença altamente infecciosa disseminada em todo o mundo, capaz de afetar até outros animais ectotérmicos como peixes e répteis. Uma questão importante em relação a essa patologia é a falta de sinais clínicos antes de levar à morte. Com o objetivo de compreender melhor a suscetibilidade dos anuros, o presente trabalho analisou a taxa de sobrevivência de rãs-touro (Lithobates catesbeianus), desafiadas com três doses de uma estirpe brasileira do Frog virus 3 (FV3). A análise de qPCR indicou baixa taxa de infectividade nesses animais, tanto como larvas quanto como adultos. Procurando esclarecer os resultados, foram formuladas as seguintes hipóteses: 1) A quantidade de inóculo aplicada nas rãs foi insuficiente para desencadear uma infecção; 2) Para que o FV3 dê sinais clínicos nesta espécie, é necessário um cofator; 3) Os animais sofreram infecção por FV3, mas se recuperaram no decorrer do experimento, e 4) O inóculo utilizado pode ter sido de baixa virulência. Finalmente, foi discutida a presença de sinais clínicos reais de ranavírus e levantada a hipótese mais provável(AU)

Presence of ascites in bullfrog breeders reared and kept on a frog farm / Presença de ascite em reprodutores de rã-touro criados e mantidos em ranário

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.(AU)

A ascite é uma condição patológica que se caracteriza pelo extravasamento do líquido dos vasos sanguíneos e por seu acúmulo na cavidade abdominal provocado por uma somatória de fatores. É considerada uma síndrome de caráter multifatorial e, uma vez desencadeado o processo ascítico, esse se torna, na maioria dos casos, irreversível. O objetivo deste trabalho foi relatar a ocorrência dessa síndrome em machos e fêmeas reprodutores de rã-touro (Lithobates catesbeianus), alimentados com ração comercial para peixes, com 40% de proteína bruta, durante três anos e já sendo observada má formação de fígado, por falta de deposição proteica, em animais adultos da mesma desova. A cultura do líquido ascítico demonstrou ausência de crescimento bacteriano. Na histopatologia, observou-se, para ambos os sexos, nos rins, grande número de estruturas tubulares com material fortemente eosinofílico e hialino, sugerindo coloide, tanto na região cortical como na medular. Visualizaram-se também granulomas em vários estágios de desenvolvimento, muitos apresentando material necrótico central. Ainda se pôde observar que os aparelhos renais, os glomérulos, em sua maioria, estavam hipoplásicos e com aumento do espaço de Bowman, e muitos hialinizados ou hemorrágicos; além de calcificação distrófica no parênquima e nos dos túbulos, havia material fibrinoide no seu interior. No fígado, pôde-se ver grande quantidade de melanomacrófagos no parênquima e focos de hepatite monolinfocitária e acentuada rarefação citoplasmática, assim como vários hepatócitos com núcleo em picnose e células em necrose e dissociação das trabéculas hepáticas. A causa da ascite pode ser devido às lesões observadas nesses órgãos.(AU)

Differential expression of RNA exosome subunits in the amphibian Lithobates catesbeianus during reproductive and non-reproductive periods.

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.

Histopathological Evaluation of the Exposure by Cyanobacteria Cultive Containing [d-Leu¹]Microcystin-LR on Lithobates catesbeianus Tadpoles.

This study evaluated the effects of [d-Leu¹]Microcystin-LR variant by the exposure of Lithobates catesbeianus tadpole to unialgal culture Microcystis aeruginosa NPLJ-4 strain. The Tadpole was placed in aquariums and exposed to Microcystis aeruginosa culture or disrupted cells. For 16 days, 5 individuals were removed every 2 days, and tissue samples of liver, skeletal muscle, and intestinal tract were collected for histopathology and bioaccumulation analyses. After exposure, those surviving tadpoles were placed in clean water for 15 days to evaluate their recovery. A control without algae and toxins was maintained in the same conditions and exhibited normal histology and no tissue damage. In exposed tadpoles, samples were characterized by serious damages that similarly affected the different organs, such as loss of adhesion between cells, nucleus fragmentation, necrosis, and hemorrhage. Samples showed signs of recovery but severe damages were still observed. Neither HPLC-PDA nor mass spectrometry analysis showed any evidence of free Microcystins bioaccumulation.