Control Frogs Research Articles

Inhibition of antipredator behavior caused by methylmercury in newly metamorphosed Physalaemus ephippifer (Anura, Leptodactylidae)

The aim of our study was to evaluate how methylmercury (MeHg) at sublethal concentrations may affect the anti-predator behavioral performance of newly metamorphosed Physalaemus ephippifer. Accordingly, P. ephippifer larvae were exposed to different concentrations of MeHg: 0 (control), 0.0007 and 0.007 μg/ml, and the animals were then evaluated post-metamorphosis. Newly metamorphosed individuals were submitted to the behavior assay of chemical perception of injured conspecifics. Control frogs showed normal behavior, that is a reduction in the level of activity (immobility). However, exposure to MeHg promoted inactivation of the anti-predator behavior, which was more evident in the treatment with the higher concentration, with increased movement, indicating behavioral hyperactivity. Therefore, it is concluded that the model species is sensitive to MeHg, where sublethal concentrations cause behavioral damage, namely behavioral hyperactivity.

Compatibility of eugenol anesthesia with classroom physiology experiments on nerve and muscle of frogs

Frogs serve as valuable model organisms for studying physiological responses of nerve, skeletal muscle and the heart in undergraduate biology labs. Heretofore, induced hypothermia has been the preferred technique for pain and stress reduction prior to euthanasia by pithing. However, AVMA guidelines discourage hypothermia for restraint or euthanasia of amphibians and advocate anesthetic overdose as preferable to pithing alone. Unfortunately, the primary anesthetic used in fish and other aquatic invertebrates, MS-222, blocks normal nerve and muscle function in leopard frogs and renders them useless for classroom physiology experiments, as demonstrated by Medler (2019). Eugenol is a safe and affordable anesthetic that is effective in amphibians. We sought to test the hypothesis that eugenol anesthesia is compatible with classroom nerve and muscle experiments on frogs. Bullfrogs and leopard frogs were immersed in a bath of eugenol suspended in tap water at 275 to 350 mg/L. At various time points, a four step reflex test was performed including tests of righting response, superficial pain reflex, deep pain reflex, and corneal blink reflex. Once all reflexes were absent, the frog was euthanized by pithing and the gastrocnemius muscle was prepared for force recordings. The sciatic nerve was stimulated with a bipolar stimulating electrode while gastrocnemius force was measured using an isometric force transducer. The threshold voltage eliciting a muscle contraction and the peak isometric twitch force were recorded at 15 minute intervals for 2 hours. Control frogs were sedated by immersion in ice water for 20 to 30 minutes, pithed when reflexes were substantially weakened, and then subjected to the same stimulation protocol and force measurements. At these doses, eugenol reliably induces loss of reflexes in 20 to 35 minutes and neuromuscular preparations maintain the ability to respond to electrical stimulation. Preliminary results suggest that eugenol may produce a modest elevation of threshold voltage and a modest reduction in maximum twitch force compared to control frogs euthanized without chemical anesthesia. Eugenol may be an effective anesthetic to prepare bullfrogs and leopard frogs for classroom experiments on nerve and muscle. Funding was provided by the Bill and Linda Frost Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pseudacris regilla metamorphs acquire resistance to Batrachochytrium dendrobatidis after exposure to the killed fungus.

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) is associated with drastic global amphibian declines. Prophylactic exposure to killed zoospores and the soluble chemicals they produce (Bd metabolites) can induce acquired resistance to Bd in adult Cuban treefrogs Osteopilus septentrionalis. Here, we exposed metamorphic frogs of a second species, the Pacific chorus frog Pseudacris regilla, to one of 2 prophylactic treatments prior to live Bd exposures: killed Bd zoospores with metabolites, killed zoospores alone, or a water control. Prior exposure to killed Bd zoospores with metabolites reduced Bd infection intensity in metamorphic Pacific chorus frogs by 60.4% compared to control frogs. Interestingly, Bd intensity in metamorphs previously exposed to killed zoospores alone did not differ in magnitude relative to the control metamorphs, nor to those treated with killed zoospores plus metabolites. Previous work indicated that Bd metabolites alone can induce acquired resistance in tadpoles, and so these findings together indicate that it is possible that the soluble Bd metabolites may contain immunomodulatory components that drive this resistance phenotype. Our results expand the generality of this prophylaxis work by identifying a second amphibian species (Pacific chorus frog) and an additional amphibian life stage (metamorphic frog) that can acquire resistance to Bd after metabolite exposure. This work increases hopes that a Bd-metabolite prophylaxis might be widely effective across amphibian species and life stages.

When Defenses Fail: Atelopus zeteki Skin Secretions Increase Growth of the Pathogen Batrachochytrium dendrobatidis.

To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.

Changes in the gut microbiota diversity of brown frogs (Rana dybowskii) after an antibiotic bath

BackgroundCaptive amphibians frequently receive antibiotic baths to control bacterial diseases. The potential collateral effect of these antibiotics on the microbiota of frogs is largely unknown. To date, studies have mainly relied on oral administration to examine the effects of antibiotics on the gut microbiota; in contrast, little is known regarding the effects of bath-applied antibiotics on the gut microbiota. The gut microbiota compositions of the gentamicin, recovery, and control groups were compared by Illumina high-throughput sequencing, and the functional profiles were analysed using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt). Furthermore, the relationship between the structure and predicted functional composition of the gut microbiota was determined.ResultsThe alpha diversity indices were significantly reduced by the gentamicin bath, illustrating that this treatment significantly changed the composition of the gut microbiota. After 7 days, the gut microbiota of the recovery group was not significantly different from that of the gentamicin group. Forty-four indicator taxa were selected at the genus level, comprising 42 indicators representing the control group and 2 indicators representing the gentamicin and recovery groups. Potential pathogenic bacteria of the genera Aeromonas, Citrobacter, and Chryseobacterium were significantly depleted after the gentamicin bath. There was no significant positive association between the community composition and functional composition of the gut microbiota in the gentamicin or control frogs, indicating that the functional redundancy of the gut bacterial community was high.ConclusionsGentamicin significantly changed the structure of the gut microbiota of R. dybowskii, and the gut microbiota exhibited weak resilience. However, the gentamicin bath did not change the functional composition of the gut microbiota of R. dybowskii, and there was no significant correlation between the structural composition and the functional composition of the gut microbiota.

Pharmacokinetics of ceftazidime in Northern leopard frogs (Lithobates pipiens) at two different doses and administration routes.

To determine an optimal ceftazidime dosing strategy in Northern leopard frogs (Lithobates pipiens) by evaluation of 2 different doses administered SC and 1 dose administered transcutaneously. 44 Northern leopard frogs (including 10 that were replaced). Ceftazidime was administered to frogs SC in a forelimb at 20 mg/kg (n = 10; SC20 group) and 40 mg/kg (10; SC40 group) or transcutaneously on the cranial dorsum at 20 mg/kg (10; TC20 group). Two frogs in each ceftazidime group were euthanized 12, 24, 48, 72, and 96 hours after drug administration. Plasma, renal, and skin concentrations of ceftazidime were measured by means of reversed-phase high-performance liquid chromatography. Four control frogs were used for assay validation. Mean plasma half-life of ceftazidime in the SC20, SC40, and TC20 groups was 9.01 hours, 14.49 hours, and too low to determine, respectively. Mean maximum plasma ceftazidime concentration was 92.9, 96.0, and 1.3 μg/mL, respectively. For 24 hours after drug administration in the SC20 and SC40 groups, plasma ceftazidime concentration exceeded 8 μg/mL. Renal and skin concentrations were detectable at both doses and routes of administration; however, skin concentrations were significantly lower than renal and plasma concentrations. Findings indicated that ceftazidime administration to Northern leopard frogs at 20 mg/kg, SC, every 24 hours would achieve a plasma concentration exceeding the value considered effective against common amphibian pathogens. Transcutaneous administration of the injectable ceftazidime formulation at 20 mg/kg warrants further investigation but is not currently recommended because of a potential lack of efficacy.

Polybrominated Diphenylether (DE-71) Exposure Skews Phenotypic Sex Ratio, and Alters Steroid Hormone Levels and Steroidogenic Enzyme Activities in Juvenile Silurana tropicalis.

The impact of the brominated flame-retardant mixture, DE-71, on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined. A partial lifecycle study exposing S. tropicalis to varying concentrations of DE-71 (0.0, 0.65, 1.3, 2.5, and 5.0 μg/l [nominal]) was conducted from early gastrula-stage embryo to 150 days postmetamorphosis (dpm). Exposure of S. tropicalis to DE-71 induced liver necrosis and induced abnormal ovary development characterized by previtellogenic oocyte necrosis and arrested development of vitellogenic oocytes in females in a concentration-dependent manner. Decreased mean plasma dihydrotestosterone (DHT) and T, gonad T, and increased mean plasma E2 levels were found in 150 dpm DE-71-treated male S. tropicalis compared to controls. Plasma E2 levels in females were not significantly altered compared to control S. tropicalis, although lower plasma and gonad T were detected. Mean gonadal CYP 19 aromatase activity in both male and female S. tropicalis exposed to DE-71 was not appreciably affected. Decreased mean male 5α-reductase and CYP17 activities in both male and females were observed compared to control frogs. Overall, these studies suggested that PBDE exposure induced liver necrosis and abnormal ovary development; and reduced circulating and gonadal androgens resulting in a phenotypic skew in sex ratio toward the female sex in S. tropicalis.

Effect of perfluorooctanesulfonate exposure on steroid hormone levels and steroidogenic enzyme activities in juvenile Silurana tropicalis.

The impact of the perfluoro-chemical, perfluorooctanesulfonate (PFOS), on gonadal steroidogenesis during sexual differentiation in Silurana tropicalis was examined because of its ubiquity in the environment, bioaccumulative nature and potential to disturb endocrine activity. A partial life cycle study exposing S. tropicalis to varying concentrations of PFOS 0.06, 0.13, 0.25, 0.50 and 1.0mg PFOS/L [nominal]) was conducted. Gonad and plasma samples were collected from juvenile control specimens and organisms exposed to PFOS from early embryo through 150days post-metamorphosis. Gonad CYP17, aromatase and 5α-reductase activities were measured. Plasma estradiol, testosterone, dihydrotestosterone (DHT) and gonadal testosterone were measured in both males and females. Increased plasma DHT and gonadal testosterone were found in PFOS-treated juvenile male S. tropicalis compared to controls. Decreased plasma estradiol, but not testosterone, was detected in PFOS-treated female S. tropicalis compared to controls. Plasma DHT was not detected and an increase in gonadal testosterone was detected in PFOS-treated female frogs. Female S. tropicalis exposed to PFOS exhibited a concentration-related decrease in the mean aromatase activity, but not 5α-reductase. PFOS exposure in male frogs induced a concentration-related increase in 5α-reductase activity, but did not alter aromatase activity compared to control frogs. A concentration-related increase in CYP 17,20-lyase activity, but not 17-hydroxylase activity, was found in both female and male S. tropicalis exposed to PFOS.

Evaluation of tree frog tracking methods using Phyllomedusa trinitatis (Anura: Phyllomedusidae)

Evaluation of tree frog tracking methods using Phyllomedusa trinitatis (Anura: Phyllomedusidae). Investigating the behaviors of small, inconspicuous and cryptic animals can be helped by tracking their movements. The effectiveness of different tracking methods can be very dependent on behavior and ecology; radio-telemetry and thread bobbins have been widely used over a range of environments and taxa, but each presents problems. Phyllomedusa trinitatis is a tree frog found in Trinidad and Venezuela and has mostly been studied for its nest building and breeding behavior, but little is known about its behavior away from breeding ponds. This study aimed to identify the strengths and weaknesses, including impacts on the welfare of these frogs, of different tracking methods, thread bobbins and radio-telemetry, when used to track them in a dense rainforest environment. A pilot study found that fuorescent dyes were unsuitable for this species. Individuals were tested in laboratory conditions to determine the application time for each tracker and to test on this species the tracker 10% weight rule. The rule was found to be too restrictive for this frog; trackers up to 15% of body weight were used with no signifcant impacts on distances travelled. Frogs became lethargic when bearing trackers longer than two days, so we limited tracking in the feld to one overnight period. Of the 26 frogs tracked in the feld (nine radio-tags, 17 bobbins), 16 were successful (six radio-tags, 10 bobbins) and six untracked frogs were found in the feld during the day as controls. Bobbins were cheaper and allowed visualization of the detailed path taken, including substrates used, but caused more bruising due to entanglement, and individuals tracked with this method were less likely to return on following nights to the breeding ponds. Radio-tags had no threat of entanglement but were much more expensive and the signal was interrupted by the dense vegetation preventing some individuals from being found. There were no signifcant differences in the distances travelled by tracked or control frogs, from which we infer that these tracking methods did not impact signifcantly on movement. It appears that neither of these tracking methods work perfectly for Phyllomedusa in a densely vegetated environment, and that both incur welfare problems. Our study emphasizes the need to test out tracking methods on each species in each habitat.

Effects of neonicotinoids on putative escape behavior of juvenile wood frogs (Lithobates sylvaticus) chronically exposed as tadpoles.

Neonicotinoids are water-soluble neurotoxic insecticides widely used in agriculture that are being detected in nontarget aquatic environments. Nontarget aquatic wildlife, such as amphibians, may be at risk of exposure. Studies using larval stages suggest neonicotinoids are a minor concern to amphibians; however, behavioral effects manifesting later in life are not often considered. Behavioral endpoints could further our understanding of potential sublethal neurotoxic effects after exposure has ended. Using juvenile wood frogs (Lithobates sylvaticus), we investigated the effects of chronic larval exposure to 3 concentrations (1, 10, and 100 μg/L) of formulations containing imidacloprid or thiamethoxam on the putative escape response to a simulated heron attack. We found that control frogs actively responded (i.e., moved or jumped) to the simulated predator attack but frogs exposed to imidacloprid at 10 and 100 μg/L were less likely to respond. The exposed frogs, specifically from the imidacloprid treatment at 10 μg/L (tendency at 100 μg/L) were less likely to leave the attack area compared with controls. However, frogs used refuge similarly among all treatments. Finally, there were no differences in locomotor performance, as measured by total number of jumps and distance traveled during a trial among treatments. In conclusion, our study suggests that exposure to neonicotinoids during amphibian larval development may affect a juvenile frog's ability to perceive or respond to a predator, potentially increasing their vulnerability to predation. Future studies should validate and explore this potential effect further. Environ Toxicol Chem 2018;37:3115-3123. © 2018 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

Environment friendly management of mosquito: a short review

Despite the large scale use of insecticides, capacity building, municipality, community and metropolis awareness, and preventive measures to counter vector borne diseases which are mounting day-by-day, new tools are now been introducing to prevent the spread of mosquito transmitted diseases. The low efficacy status of chemical pesticides have lead to the interest of researchers in search of fresh and even more practicable vector control methodologies to be applied. In this regards, multiple alternatives have been monitoring to develop control practice measures for the eradication, observation and control of mosquitoes at larval level by the use of a sustainable biological monitoring and control by an ordinary constructive predator, to exercise monitoring and practical control measures over parasites at larval stages in environmental and eco-friendly techniques. In particular, bio-control measures to monitor and control practical practices, context predatory larvivorous fish, dragonfly nymph, frogs, copepods, turtle, Entomopathogenic bacillus, Bacillus sphaericus and Bacillus thuringiensis israelensis are beings tried in different regions of world. The available research on the subject recommends that there exist multiple direct and indirect growth factors that could play a dynamic role in prey and predator’s survival. Species controphic that have an impact on concerned eco-relation reflect significant effect. In addition to this, certain eco-relations represent positive stimuli for the control of vector borne viral diseases. As a bio-control achieving feasible agent for vector monitoring, pointing, management and control predatory larvivorous fish, dragonfly nymph, frogs, copepods, turtle, Entomopathogenic bacillus, Bacillus sphaericus and Bacillus thuringiensis israelensis are not only considered as a liberated intervention for disease vector control of practices and mechanical control cost deterrents as well. Further research has been suggested on the subject so as to find out even more practicable and effective mosquito monitoring and practical control practices.Bangladesh J. Sci. Ind. Res.53(3), 169-178, 2018

Cytotoxicity in the frog (Fejervarya limnocharis) after acute cadmium exposure in vivo

Chromosome aberrations (CA) in frogs (Fejervarya limnocharis) exposed to 5, 10 and 20 mg/L of cadmium chloride (CdCl2) for 24, 48 and 72 h were invetegated. Treated frogs were compared to a control group. Cadmium (Cd) was not detected in the water or control frogs. The highest Cd concentrations in water and frog samples were found at 20 mg/L exposed for 72 h. The water samples indicated that exposure to 10 mg/L of Cd for 24 h was significantly different from the control (p < 0.05). Cd concentrations in frogs differed significantly between the control and experimental groups (p < 0.05). The cytotoxicity assessment revealed ten types of CA in the frogs, including single chromatid gap (SCG), isochromatid gap, single chromatid break, isochromatid break, iso-arm fragmentation, single chromatid decomposition, centric fragmentation, centromere gap, deletion and fragmentation. The most common CA in the study was SCG. The statistical analysis indicated significant differences in the percentage of cells with CA for exposures of 20 mg/L (24 h), 10 and 20 mg/L (48 h) and 5, 10 and 20 mg/L (72 h) compared to the control (p < 0.05). The results of this study show that high Cd concentrations and long duration exposure can cause CA in frogs.

Role of the Nrf2-ARE pathway in perfluorooctanoic acid (PFOA)-induced hepatotoxicity in Rana nigromaculata

Perfluorooctanoic acid (PFOA) is widely distributed in various environmental media and is toxic to organisms. This study demonstrated that PFOA induces hepatotoxicity in the frog and evaluated the role of CYP3A and the Nrf2-ARE signaling pathway in regulating responses to PFOA-induced hepatotoxicity. Rana nigromaculata were exposed to 0, 0.01, 0.1, 0.5, or 1 mg/L PFOA solutions in a static-renewal system for 14 days. Liver tissue samples were collected 24 h after the last treatment. Hepatic histology was observed by HE staining and transmission electron microscopy. The oxidative stress levels in the liver were measured. The expression levels of CYP3A, Nrf2, NQO1, and HO-1 mRNA were measured by quantitative reverse transcription–polymerase chain reaction. PFOA-treated frog liver tissue exhibited diffuse cell borders, cytoplasmic vacuolization, broken nuclei, nuclear chromatin margination, and swollen mitochondria. In addition, the livers of PFOA-treated frogs showed a significantly elevated content of reactive oxygen species, malondialdehyde, glutathione and glutathione S-transferase activity compared to the livers of control frogs. However, the glutathione peroxidase activities concomitantly decreased in PFOA-treated frogs compared to those in the control group. Furthermore, compared with control frogs, the expression levels of CYP3A, Nrf2, and NQO1 mRNA significantly increased in PFOA-treated frogs. HO-1 mRNA expression remarkably increased only in groups treated with 0.5 or 1 mg/L PFOA. Our results indicate that PFOA induces hepatotoxicity in a dose-dependent manner. Furthermore, the results of the comparison analysis between different gender groups illustrated that PFOA is more toxic to female frogs than male frogs. Our results demonstrated that PFOA causes liver damage and that CYP3A enhances PFOA-induced female frogs hepatotoxicity are more virulent than male through biotransformation, and the activation of the Nrf2-ARE pathway is induced to protect against hepatotoxicity in Rana nigromaculata, all of which provide the scientific basis for the protection of amphibians against environmental contaminants.

Increased transcript levels and kinetic function of pyruvate kinase during severe dehydration in aestivating African clawed frogs, Xenopus laevis.

The African clawed frog, Xenopus laevis, can withstand extremely arid conditions through aestivation, resulting in dehydration and urea accumulation. Aestivating X. laevis reduce their metabolic rate, and rely on anaerobic glycolysis to meet reduced ATP demands. The present study investigated how severe dehydration affected the transcript levels, kinetic profile, and phosphorylation state of the key glycolytic enzyme pyruvate kinase (PK) in the liver and skeletal muscle of X. laevis. Compared to control frogs, severely dehydrated frogs showed an increase in the transcript abundance of both liver and muscle isoforms of PK. While the kinetics of muscle PK did not differ between dehydrated and control frogs, PK from the liver of dehydrated frogs had a lower Km for phosphoenolpyruvate (PEP) (38%), a lower Ka for fructose-1,6-bisphosphate (F1,6P2) (32%), and a greater activation of PK via F1,6P2 (1.56-fold). PK from dehydrated frogs also had a lower phosphorylation-state (25%) in comparison to the enzyme from control frogs in the liver. Experimental manipulation of the phosphorylation-state of liver PK taken from control frogs by endogenous protein phosphatases resulted in decreased phosphorylation, and a similar kinetic profile as seen in dehydrated frogs. The physiological consequence of dehydration-induced PK modification appears to adjust PK function to remain active during a metabolically depressed state. This study provides evidence for the maintenance of PK activity through elevated mRNA levels and a dephosphorylation event which activates frog liver PK in the dehydrated state in order to facilitate the production of ATP via anaerobic glycolysis.

Reduced innate immunity of Cuban Treefrogs at leading edge of range expansion.

During geographic range expansion, populations of non-indigenous species at the invasion front may benefit from directing resources away from immune defense. To test this hypothesis, we investigated the strength of two innate immune components in populations of invasive Cuban Treefrogs (Osteopilus septentrionalis) in a long-colonized area (core region) and at the invasion front (leading-edge region). First, we compared the region-specific metabolic response of frogs injected with an endotoxin that induces systemic inflammation (lipopolysaccharide, LPS) to sham-injected control frogs pooled from both regions. Males and females were analyzed independently because we detected a sex-related difference in mass-independent metabolism of control frogs, with males exhibiting a significantly higher metabolic rate (F1, 21 =29.02, P<0.001) than females. We observed a significantly higher metabolic rate in LPS-injected core frogs compared with control frogs for both males (P = 0.041) and females (P = 0.007). Conversely, in leading-edge populations, there was no significant difference in the metabolic rate of LPS-injected and control frogs (males, P=0.195; females, P=0.132). Second, we directly compared bacterial killing ability of frog blood plasma between regions. Bactericidal ability of plasma was significantly greater in frogs from the core region in comparison with those at the leading edge (F1, 26 =28.67, P<0.001). For both immune components that we examined, populations from the core exhibited stronger immune responses. Our findings support hypotheses predicting an inverse relationship between immunity and range expansion.

The regulation of heat shock proteins in response to dehydration in Xenopus laevis.

African clawed frogs (Xenopus laevis) endure bouts of severe drought in their natural habitats and survive the loss of approximately 30% of total body water due to dehydration. To investigate molecular mechanisms employed by X. laevis during periods of dehydration, the heat shock protein response, a vital component of the cytoprotective stress response, was characterized. Using western immunoblotting and multiplex technology, the protein levels of HSP27, HSP40, HSP60, HSP70, HSC70, and HSP90 were quantified in the liver, skeletal muscle, kidney, lung, and testes from control frogs and those that underwent medium or high dehydration (~16 or ~30% loss of total body water). Dehydration increased HSP27 (1.45-1.65-fold) in the kidneys and lungs, and HSP40 (1.39-2.50-fold) in the liver, testes, and skeletal muscle. HSP60 decreased in response to dehydration (0.43-0.64 of control) in the kidneys and lungs. HSP70 increased in the liver, lungs, and testes (1.39-1.70-fold) during dehydration, but had a dynamic response in the kidneys (levels increased 1.57-fold with medium dehydration, but decreased to 0.56 of control during high dehydration). HSC70 increased in the liver and kidneys (1.20-1.36-fold), but decreased in skeletal muscle (0.27-0.55 of control) during dehydration. Lastly, HSP90 was reduced in the kidney, lung, and skeletal muscle (0.39-0.69 of control) in response to dehydration, but rose in the testes (1.30-fold). Overall, the results suggest a dynamic tissue-specific heat shock protein response to whole body dehydration in X. laevis.

Fear of the human 'super predator' reduces feeding time in large carnivores.

Large carnivores' fear of the human 'super predator' has the potential to alter their feeding behaviour and result in human-induced trophic cascades. However, it has yet to be experimentally tested if large carnivores perceive humans as predators and react strongly enough to have cascading effects on their prey. We conducted a predator playback experiment exposing pumas to predator (human) and non-predator control (frog) sounds at puma feeding sites to measure immediate fear responses to humans and the subsequent impacts on feeding. We found that pumas fled more frequently, took longer to return, and reduced their overall feeding time by more than half in response to hearing the human 'super predator'. Combined with our previous work showing higher kill rates of deer in more urbanized landscapes, this study reveals that fear is the mechanism driving an ecological cascade from humans to increased puma predation on deer. By demonstrating that the fear of humans can cause a strong reduction in feeding by pumas, our results support that non-consumptive forms of human disturbance may alter the ecological role of large carnivores.

Reduced immune function predicts disease susceptibility in frogs infected with a deadly fungal pathogen.

The relationship between amphibian immune function and disease susceptibility is of primary concern given current worldwide declines linked to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). We experimentally infected lowland leopard frogs (Lithobates yavapaiensis) with Bd to test the hypothesis that infection causes physiological stress and stimulates humoral and cell-mediated immune function in the blood. We measured body mass, the ratio of circulating neutrophils to lymphocytes (a known indicator of physiological stress) and plasma bacterial killing ability (BKA; a measure of innate immune function). In early exposure (1-15 days post-infection), stress was elevated in Bd-positive vs. Bd-negative frogs, whereas other metrics were similar between the groups. At later stages (29-55 days post-infection), stress was increased in Bd-positive frogs with signs of chytridiomycosis compared with both Bd-positive frogs without disease signs and uninfected control frogs, which were similar to each other. Infection decreased growth during the same period, demonstrating that sustained resistance to Bd is energetically costly. Importantly, BKA was lower in Bd-positive frogs with disease than in those without signs of chytridiomycosis. However, neither group differed from Bd-negative control frogs. The low BKA values in dying frogs compared with infected individuals without disease signs suggests that complement activity might signify different immunogenetic backgrounds or gene-by-environment interactions between the host, Bd and abiotic factors. We conclude that protein complement activity might be a useful predictor of Bd susceptibility and might help to explain differential disease outcomes in natural amphibian populations.

A hydrogen peroxide safety valve: The reversible phosphorylation of catalase from the freeze-tolerant North American wood frog, Rana sylvatica

BackgroundThe North American wood frog, Rana sylvatica, endures whole body freezing while wintering on land and has developed multiple biochemical adaptations to elude cell/tissue damage and optimize its freeze tolerance. Blood flow is halted in the frozen state, imparting both ischemic and oxidative stress on cells. A potential build-up of H2O2 may occur due to increased superoxide dismutase activity previously discovered. The effect of freezing on catalase (CAT), which catalyzes the breakdown of H2O2 into molecular oxygen and water, was investigated as a result. MethodsThe present study investigated the purification and kinetic profile of CAT in relation to the phosphorylation state of CAT from the skeletal muscle of control and frozen R. sylvatica. ResultsCatalase from skeletal muscle of frozen wood frogs showed a significantly higher Vmax (1.48 fold) and significantly lower Km for H2O2 (0.64 fold) in comparison to CAT from control frogs (5°C acclimated). CAT from frozen frogs also showed higher overall phosphorylation (1.73 fold) and significantly higher levels of phosphoserine (1.60 fold) and phosphotyrosine (1.27 fold) compared to control animals. Phosphorylation via protein kinase A or the AMP-activated protein kinase significantly decreased the Km for H2O2 of CAT, whereas protein phosphatase 2B or 2C action significantly increased the Km. ConclusionThe physiological consequence of freeze-induced CAT phosphorylation appears to improve CAT function to alleviate H2O2 build-up in freezing frogs. General significanceAugmented CAT activity via reversible phosphorylation may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.

English

The natural pyrethroids are not so much effective against agricultural pests, thus artificial synthesis of synthetic pyrethroids like fenvalerate was carried out by scientists. Fenvalerate is a insecticide has been proved to be highly toxic to non-target species like amphibians. In our study, Healthy frogs, Rana tigrina weighing 50 ± 3 gms were collected from the pond, acclimated to the laboratory conditions in large glass aquaria with water. The frogs were divided into groups of ten animals. They were exposed to different pesticide concentrations of fenvalerate, both commercial and technical grade, according to biomass ratio as suggested by Doundroff et al., (1951). The Total carbohydrate content in the present study Control Muscle tissue has shown higher amount of carbohydrates than compare with Brain and Intestine tissue. Under fenvalerate intoxication, the frog tissues showed consistent decrement in all the periods and it may be due to rapid utilization to withstand pesticide of stress. Total protein contents in control frog tissues the Muscle showed higher concentration of proteins than Intestine and Brain. The protein content in experimental frog tissues showed depleted levels when compared to controls. The significant decrease in protein and it may be due rapid utilization to withstand pesticide of stress. Total free amino acid (FAA) showed enhanced Intestine FAA levels, the levels in fenvalerate exposed frog tissues were increased significantly when compared to control. These elevated levels may be due to improved protease activity or enhanced transamination. From the above observations fenvalerate seems to be hazards to the aquatic life, causing severe biochemical changes and permanent architectural changes investigated in the present investigation.