Larval development and survival of pond-breeding anurans in an agricultural landscape impacted more by phytoplankton than surrounding habitat.

The destruction of freshwater habitat is a major contributor to biodiversity loss in aquatic ecosystems. However, created or restored wetlands could partially mitigate aquatic biodiversity loss by increasing the amount of available habitat across a landscape. We investigated the impact of surrounding terrestrial habitat and water quality variables on suitability for two species of pond-breeding amphibians (bullfrogs [Lithobates catesbeianus] and Blanchard's cricket frogs [Acris blanchardi]) in created permanent wetlands located on an agricultural landscape. We examined tadpole growth and survival in field enclosures placed in ponds surrounded by agricultural, forested, or grassland habitats. We also evaluated the potential for carryover effects of the aquatic environment on terrestrial growth and overwinter survival of cricket frog metamorphs. We found that habitat adjacent to ponds did not predict tadpole growth or survival. Rather, phytoplankton abundance, which showed high variability among ponds within habitat type, was the only predictor of tadpole growth. Cricket frogs emerged larger and earlier from ponds with higher phytoplankton abundance; bullfrogs were also larger and at a more advanced developmental stage in ponds with higher levels of phytoplankton. Overwinter survival of cricket frogs was explained by size at metamorphosis and there were no apparent carryover effects of land use or pond-of-origin on overwinter growth and survival. Our results demonstrate that created ponds in human-dominated landscapes can provide suitable habitat for some anurans, independent of the adjacent terrestrial habitat.

Sexual dimorphism in external morphology of the American bullfrog Rana (Aquarana) catesbeiana and the possibility of sex determination based on tympanic membrane/eye size ratio.

The American bullfrog Rana (Aquarana) catesbeiana has been reported to show significant sexual dimorphism based on the size ratio between the tympanic membrane and the eye. In males the tympanic membrane is much larger than the eye, but not in females. The ratio has been used as a convenient criterion to discriminate sexes (sexing) in the American bullfrog, though its reliability is unknown. In this study, we examined 86 adult American bullfrogs to clarify whether the tympanic membrane long diameter/eye long diameter (Dtm/De) ratio is a reliable index to discriminate sexes in this species. In addition, we examined the growth of this sexually dimorphic trait. Results indicated that there is a significant difference but there is a small overlap in this ratio Dtm/De between sexes. The allometric comparisons showed the sexual dimorphism of the Dtm/De ratio was increased during growth and the dimorphism is attributable to the difference in the growth rate of the tympanic membrane (Dtm). Therefore, sex determination of American bullfrogs cannot be wholly reliably achieved by the Dtm/De ratio alone; other external morphological features are required in addition.

Propagule pressure and hunting pressure jointly determine genetic evolution in insular populations of a global frog invader.

Islands are often considered to be more susceptible to biological invasions and to suffer greater impacts from invaders than mainland areas, and this difference is generally attributed to differences in species introductions, ecological factors or human activities between islands and mainland areas. Genetic variation, as a good estimate of evolutionary potential, can influence the invasion process and impacts of alien species. However, few studies have compared the genetic diversity of alien species between islands and a corresponding mainland. Here, we examined the genetic variation and differentiation in feral populations (30 sampled individuals/population) of a globally invasive species (the American bullfrog, Lithobates catesbeianus) that was extensively farmed on 14 islands in the Zhoushan Archipelago of China and in three nearby regions on the mainland. We quantified the relative importance of propagule pressure and hunting pressures on the genetic variation of bullfrog populations and found that insular populations have greater genetic variation than their mainland counterparts. Although genetic differentiation between the populations was observed, no evidence of recent bottlenecks or population expansion in any of the tested population was found. Our results suggest that the propagule pressures of bullfrogs escaping from farms, multiple releases and hunting pressure influence the genetic variation among bullfrog populations. These results might have important implications for understanding the establishment and evolution of alien species on islands and for the management of invasive species.

Seasonal acclimatization and thermal acclimation induce global histone epigenetic changes in liver of bullfrog (Lithobates catesbeianus) tadpole.

The American bullfrog (Lithobates catesbeianus) is a eurythermal amphibian that is naturally distributed from subarctic to subtropical areas. The tadpoles of this species overwinter, in water, in cold environments. Therefore, they may have adapted to a wide range of temperatures in an active state. To understand the adaptation mechanisms to cope with low or high temperatures, we investigated global epigenetic modifications, histone variants, transcript levels of related genes, and the cellular acetyl coenzyme A (acetyl-CoA) and free CoA (CoA-SH) levels, in the livers of tadpoles collected in summer and winter and of those acclimated to 4 °C and 21 °C. Among epigenetic marks tested, the levels of acetylated histones and the histone variant H2A.Z were influenced by different temperature conditions. Histone acetylation levels were higher in summer than in winter and increased within 3 days of warm acclimation, whereas histone H2A.Z levels were higher in winter than in summer and decreased within 2 weeks of warm acclimation. Transcript analysis revealed that decreased expression of histone H2A.Z in warm acclimation was regulated at the transcriptional level. Acetyl-CoA levels were not correlated with those of the acetylated histones, indicating that cellular acetyl-CoA levels may not directly influence the state of histone acetylation in the tadpole liver. Such epigenetic and metabolic changes in the tadpole liver may contribute to the maintenance of energy balance during seasonal acclimatization and thermal acclimation.

Developmental Maturation of Functional Coupling Between Ventilatory Oscillators in the American Bullfrog.

Many vital motor behaviors - including locomotion, swallowing, and breathing - appear to be dependent upon the activity of and coordination between multiple endogenously rhythmogenic nuclei, or neural oscillators. Much as the functional development of sensory circuits is shaped during maturation, we hypothesized that coordination of oscillators involved in motor control may likewise be maturation-dependent, i.e., coupling and coordination between oscillators change over development. We tested this hypothesis using the bullfrog isolated brainstem preparation to study the metamorphic transition of ventilatory motor patterns from early rhythmic buccal (water) ventilation in the tadpole to the mature pattern of rhythmic buccal and lung (air) ventilation in the adult. Spatially distinct oscillators drive buccal and lung bursts in the isolated brainstem; we found these oscillators to be active but functionally uncoupled in the tadpole. Over the course of metamorphosis, the rhythms produced by the buccal and lung oscillators become increasingly tightly coordinated. These changes parallel the progression of structural and behavioral changes in the animal, with adult levels of coupling arising by the metamorphic stage (forelimb eruption). These findings suggest that oscillator coupling undergoes a maturation process similar to the refinement of sensory circuits over development.

Transcriptomics investigation of thyroid hormone disruption in the olfactory system of the Rana [Lithobates] catesbeiana tadpole.

Thyroid hormones (THs) regulate vertebrate growth, development, and metabolism. Despite their importance, there is a need for effective detection of TH-disruption by endocrine disrupting chemicals (EDCs). The frog olfactory system substantially remodels during TH-dependent metamorphosis and the objective of the present study is to examine olfactory system gene expression for TH biomarkers that can evaluate the biological effects of complex mixtures such as municipal wastewater. We first examine classic TH-response gene transcripts using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) in the olfactory epithelium (OE) and olfactory bulb (OB) of premetamorphic Rana (Lithobates) catesbeiana tadpoles after 48 h exposure to biologically-relevant concentrations of the THs, 3,5,3'-triiodothyronine (T3) and L-thyroxine (T4), or 17-beta estradiol (E2); a hormone that can crosstalk with THs. As the OE was particularly sensitive to THs, further RNA-seq analysis found >30,000 TH-responsive contigs. In contrast, E2 affected 267 contigs of which only 57 overlapped with THs suggesting that E2 has limited effect on the OE at this developmental phase. Gene ontology enrichment analyses identified sensory perception and nucleoside diphosphate phosphorylation as the top affected terms for THs and E2, respectively. Using classic and additional RNA-seq-derived TH-response gene transcripts, we queried TH-disrupting activity in municipal wastewater effluent from two different treatment systems: anaerobic membrane bioreactor (AnMBR) and membrane enhanced biological phosphorous removal (MEBPR). While we observed physical EDC removal in both systems, some TH disruption activity was retained in the effluents. This work lays an important foundation for linking TH-dependent gene expression with olfactory system function in amphibians.

The rostral medulla of bullfrog tadpoles contains critical lung rhythmogenic and chemosensitive regions across metamorphosis.

The development of amphibian breathing provides insight into vertebrate respiratory control mechanisms. Neural oscillators in the rostral and caudal medulla drive ventilation in amphibians, and previous reports describe ventilatory oscillators and CO2 sensitive regions arise during different stages of amphibian metamorphosis. However, inconsistent findings have been enigmatic, and make comparisons to potential mammalian counterparts challenging. In the current study we assessed amphibian central CO2 responsiveness and respiratory rhythm generation during two different developmental stages. Whole-nerve recordings of respiratory burst activity in cranial and spinal nerves were made from intact or transected brainstems isolated from tadpoles during early or late stages of metamorphosis. Brainstems were transected at the level of the trigeminal nerve, removing rostral structures including the nucleus isthmi, midbrain, and locus coeruleus, or transected at the level of the glossopharyngeal nerve, removing the putative buccal oscillator and caudal medulla. Removal of caudal structures stimulated the frequency of lung ventilatory bursts and revealed a hypercapnic response in normally unresponsive preparations derived from early stage tadpoles. In preparations derived from late stage tadpoles, removal of rostral or caudal structures reduced lung burst frequency, while CO2 responsiveness was retained. Our results illustrate that structures within the rostral medulla are capable of sensing CO2 throughout metamorphic development. Similarly, the region controlling lung ventilation appears to be contained in the rostral medulla throughout metamorphosis. This work offers insight into the consistency of rhythmic respiratory and chemosensitive capacities during metamorphosis.

Metabolomic insights into the effects of thyroid hormone on Rana [Lithobates] catesbeiana metamorphosis using whole-body Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI).

Anuran metamorphosis involves the transformation of an aquatic tadpole into a juvenile frog. This process is completely dependent upon thyroid hormones (THs). Although much research has been focused on changes in gene expression programs during this postembryonic developmental period, transitions in the metabolic profiles are relatively poorly understood. Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) is a technique that generates highly multiplexed mass spectra while retaining spatial location information on a thin tissue section. Reconstructed ion heat maps are correlated with morphology of the tissue section for biological interpretation. The present study is the first to use whole-body MALDI-MSI on tadpoles to gain insights into anuran metamorphosis. Approximately 1000 features were detected in each of five tissues examined (brain, eye, liver, notochord, and tail muscle) from premetamorphic North American bullfrog (Rana [Lithobates] catesbeiana) tadpoles. Of these detected metabolites, 1700 were unique and 136 were significantly affected by exposure to 50 nM thyroxine for 48 h. Of the significantly-affected metabolites, 64 features were tentatively identified using the MassTRIX annotation tool. All tissues revealed changes in lipophilic compounds including phosphatidylcholines, phosphatidylinositols, phosphatidylglycerols, phosphatidylethanolamines, and phosphatidylserines. These lipophilic compounds made up the largest portion of significantly-affected metabolites indicating that lipid signaling is a major target of TH action in frog tadpoles.

Behavioral and molecular analyses of olfaction-mediated avoidance responses of Rana (Lithobates) catesbeiana tadpoles: Sensitivity to thyroid hormones, estrogen, and treated municipal wastewater effluent.

Olfaction is critical for survival, facilitating predator avoidance and food location. The nature of the olfactory system changes during amphibian metamorphosis as the aquatic herbivorous tadpole transitions to a terrestrial, carnivorous frog. Metamorphosis is principally dependent on the action of thyroid hormones (THs), l-thyroxine (T4) and 3,5,3'-triiodothyronine (T3), yet little is known about their influence on olfaction during this phase of postembryonic development. We exposed Taylor Kollros stage I-XIII Rana (Lithobates) catesbeiana tadpoles to physiological concentrations of T4, T3, or 17-beta-estradiol (E2) for 48h and evaluated a predator cue avoidance response. The avoidance response in T3-exposed tadpoles was abolished while T4- or E2-exposed tadpoles were unaffected compared to control tadpoles. qPCR analyses on classic TH-response gene transcripts (thra, thrb, and thibz) in the olfactory epithelium demonstrated that, while both THs produced molecular responses, T3 elicited greater responses than T4. Municipal wastewater feed stock was spiked with a defined pharmaceutical and personal care product (PPCP) cocktail and treated with an anaerobic membrane bioreactor (AnMBR). Despite substantially reduced PPCP levels, exposure to this effluent abolished avoidance behavior relative to AnMBR effluent whose feed stock was spiked with vehicle. Thibz transcript levels increased upon exposure to either effluent indicating TH mimic activity. The present work is the first to demonstrate differential TH responsiveness of the frog tadpole olfactory system with both behavioral and molecular alterations. A systems-based analysis is warranted to further elucidate the mechanism of action on the olfactory epithelium and identify further molecular bioindicators linked to behavioral response disruption.

Tadpoles fed supplemented diet with probiotic bacterium isolated from the intestinal tract of bullfrog Lithobates catesbeianus: Haematology, cell activity and electron microscopy.

The aim of this study is to select and isolate autochthonous bacteria with probiotic potential for use in a supplemented diet for bullfrog tadpoles, Lithobates catesbeianus. A total of 20 strains of lactic acid bacteria were isolated. Nine out of these were used in the following in vitro assays: antagonism against pathogenic bacteria (ANT), antimicrobial activity from extracellular compounds (MIC), tolerance to bile salts (TBS), pH reduction, protease production, sensitivity to antimicrobial tetracycline, cell viability, growth rate and doubling time. Using these data was defined an ideotype (ideal strain) based on the best results. Distances were estimated with the Mahalanobis (D2) test, and the best candidates, presenting the shortest ideotype distances, were considered to be used. The best strain was found to be Lactobacillus plantarum because it presented 10.00 ± 0.50 mm of ANT against Aeromonas hydrophila, 3.99 ± 0.01 of MIC independent of pathogenic bacteria, 85.07 ± 0.01 of TBS, 4.20 ± 0.02 of final pH, 17.67 ± 1.15 of protease production, 13.50 ± 2.00 sensitivity to antimicrobial tetracycline, 9.36 ± 0.04 of cell viability, 0.20 ± 0.00 of growth rate and 3.46 ± 0.00 doubling time. Therefore this probiotic candidate was then supplemented (2.045 ± 1.07 × 107 colony forming unities. g-1) into the diets of bullfrog tadpoles for a period of 42 days. At the end of the trial, samples of blood and intestines were collected to verify the haematological alterations and the intestinal morphology using transmission and scanning electron microscopy. Tadpoles fed the supplemented diet showed successful lactic acid bacterium colonisation, an increased number of circulating thrombocytes, monocytes, eosinophil and LG-PAS+ and also an increase in the length and density of intestinal microvilli. This study shows the feasibility of using probiotics isolated from farmed bullfrogs as a supplement in the diets of tadpoles, providing a promising alternative for modulating the health of these animals.

Temperature effects on the cardiorespiratory control of American bullfrog tadpoles based on a non-invasive methodology.

Temperature effects on cardiac autonomic tonus in amphibian larval stages have never been investigated. Therefore, we evaluated the effect of different temperatures (15, 25 and 30°C) on the cardiorespiratory rates and cardiac autonomic tonus of premetamorphic tadpoles of the bullfrog, Lithobates catesbeianus To this end, a non-invasive method was developed to permit measurements of electrocardiogram (ECG) and buccal movements (fB; surface electromyography of the buccal floor). For evaluation of autonomic regulation, intraperitoneal injections of Ringer solution (control), atropine (cholinergic muscarinic antagonist) and sotalol (ß-adrenergic antagonist) were performed. Ringer solution injections did not affect heart rate (fH) or fB across temperatures. Cardiorespiratory parameters were significantly augmented by temperature (fH: 24.5±1.0, 54.5±2.0 and 75.8±2.8 beats min-1 at 15, 25 and 30°C, respectively; fB: 30.3±1.1, 73.1±4.0 and 100.6±3.7 movements min-1 at 15, 25 and 30°C, respectively). A predominant vagal tone was observed at 15°C (32.0±3.2%) and 25°C (27.2±6.7%) relative to the adrenergic tone. At 30°C, the adrenergic tone increased relative to the lower temperature. In conclusion, the cholinergic and adrenergic tones seem to be independent of temperature for colder thermal intervals (15-25°C), while exposure to a hotter ambient temperature (30°C) seems to be followed by a significant increase in adrenergic tone and may reflect cardiovascular adjustments made to match oxygen delivery to demand. Furthermore, while excluding the use of implantable electrodes or cannulae, this study provides a suitable non-invasive method for investigating cardiorespiratory function (cardiac and respiratory rates) in water-breathing animals such as the tadpole.

Impact of an environmental relevant concentration of 17α-ethinylestradiol on the cardiac function of bullfrog tadpoles.

This study evaluated if a concentration of 17α-ethinylestradiol (EE2 - 10 ng L(-1) for 96 h) normally found in Brazilian surface waters exerts any impact on cardiac function of bullfrog tadpoles (25 Gosner stage), Lithobates catesbeianus. During exposure, the animals' activity level (AL -% of active individuals) was monitored twice a day. Then, the in loco heart rate (f(H) - bpm) was determined, as well as the relative ventricular mass (RVM - % of body mass). Afterwards, cardiac ventricles were mounted for isometric force recordings (CS - mN mm(-2)), and determination of the cardiac pumping capacity (CPC - mN mm(-2) min(-1)). EE2 did not affect tadpoles' AL, although it resulted in a tachycardia in animals exposed to EE2 (f(H) = 66 bpm) when compared to controls (f(H) = 52 bpm), suggesting that EE2 acts directly on the cardiac muscle of tadpoles, rather than being a result of an increased cardiac demand due to a higher activity level (i.e., avoidance response). Additionally, EE2 exerted a positive inotropic response, which resulted in a higher CPC, which occurred independently of an increase in the number of myofibrils of EE2-exposed animals, since RVM remained similar between experimental groups. Thus, the increase on cardiac demand induced by the exposure to EE2 elevates considerably the animal energy expenditure, diverting a large amount of energy that tadpoles could use for their growth and development. These alterations can make amphibians more susceptible to predators and reduce the likelihood to reach reproductive stage.

Effects of the organophosphorus pesticide Folisuper 600 (methyl parathion) on the heart function of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802).

The aim of this work was to evaluate whether the heart function of bullfrog tadpoles (25 Gosner stage) is affected by their acute exposure (48 h) to a sub-lethal concentration (10 µg.L-1) of the active principle of the organophosphorus pesticide Folisuper 600R (methyl parathion - MP). Our results demonstrated that MP causes not only a reduction in tadpoles' cardiac ventricular mass, resulting in a marked reduction in their cardiac twitch force, but also impairs their swimming performance, irrespective of increasing their heart rate. Together, these findings indicate that low and realistic concentration of MP have a negative impact on tadpoles' performance, jeopardizing their survival.

Effects of the organophosphorus pesticide Folisuper 600 (methyl parathion) on the heart function of bullfrog tadpoles, Lithobates catesbeianus (Shaw, 1802) / Efeitos do pesticida organofosforado Folisuper 600 (metil paration) sobre a função cardíaca de girinos de rã-touro, Lithobates catesbeianus (Shaw, 1802)

Abstract The aim of this work was to evaluate whether the heart function of bullfrog tadpoles (25 Gosner stage) is affected by their acute exposure (48 h) to a sub-lethal concentration (10 µg.L–1) of the active principle of the organophosphorus pesticide Folisuper 600R (methyl parathion - MP). Our results demonstrated that MP causes not only a reduction in tadpoles’ cardiac ventricular mass, resulting in a marked reduction in their cardiac twitch force, but also impairs their swimming performance, irrespective of increasing their heart rate. Together, these findings indicate that low and realistic concentration of MP have a negative impact on tadpoles’ performance, jeopardizing their survival.

Resumo O objetivo do presente estudo foi avaliar se a função cardíaca de girinos de rãs-touro (estágio 25 de Gosner) é afetada pela exposição aguda (48h) a uma concentração sub-letal (10 µg.L–1) do princípio ativo do pesticida organofosforado Folisuper 600R (metil paration – MP). Nossos resultados demonstraram que o MP ocasionou não apenas uma redução na massa ventricular cardíaca dos girinos, como também provocou uma redução na sua força de contração cardíaca e de seu desempenho natatório, a despeito de ter sido observado um aumento de sua freqüência cardíaca. Conjuntamente, os achados aqui obtidos indicam que uma baixa e realística concentração de MP exerce um impacto negativo sobre o desempenho dos girinos, ameaçando sua sobrevivência.

Comparison of thyroid hormone-dependent gene responses in vivo and in organ culture of the American bullfrog (Rana (Lithobates) catesbeiana) lung.

Postembryonic frog development requires a thyroid hormone (TH)-dependent metamorphic transition from an aquatic larva to a terrestrial frog. Such change in environment involves lung maturation in preparation for breathing air. However, little is known regarding the underlying molecular events and the role of THs in this process. Using quantitative real-time polymerase chain reaction, we evaluated Rana (Lithobates) catesbeiana lung mRNA transcripts representing key elements of TH and oxidative stress signaling pathways during natural and TH-induced precocious metamorphosis. TH induction was evaluated in two ways: 1) in vivo through interperitoneal injection of 10pmol/g body weight of 3,3', 5-triiodothyronine (T3) into premetamorphic tadpoles and analysis after 48h, and 2) in serum-free organ culture in the presence of 10nM T3 after 48h. Abundance of transcripts encoding the transcriptional regulators TH receptors α and ß, TH-induced bZip protein, and CCAAT/enhancer binding protein 1 was increased during postembryonic development and following administration of exogenous THs to premetamorphic tadpoles in vivo and culture. In contrast, mRNA representing Krüppel-like factor 9 and cold-inducible RNA binding protein revealed differential effects between natural and precocious metamorphosis. Elevated levels of catalase and Cu/Zn superoxide dismutase mRNA were observed at the end of metamorphosis with transcript levels displaying minimal TH-dependency. No change in stress-responsive heat shock protein 30 mRNA abundance was noted. The results support a role for TH-dependent reprogramming of the lung transcriptome during frog development and reveal a requirement for increased antioxidant capacity following anuran metamorphosis.

Community Structure and Function of Amphibian Skin Microbes: An Experiment with Bullfrogs Exposed to a Chytrid Fungus.

The vertebrate microbiome contributes to disease resistance, but few experiments have examined the link between microbiome community structure and disease resistance functions. Chytridiomycosis, a major cause of amphibian population declines, is a skin disease caused by the fungus, Batrachochytrium dendrobatidis (Bd). In a factorial experiment, bullfrog skin microbiota was reduced with antibiotics, augmented with an anti-Bd bacterial isolate (Janthinobacterium lividum), or unmanipulated, and individuals were then either exposed or not exposed to Bd. We found that the microbial community structure of individual frogs prior to Bd exposure influenced Bd infection intensity one week following exposure, which, in turn, was negatively correlated with proportional growth during the experiment. Microbial community structure and function differed among unmanipulated, antibiotic-treated, and augmented frogs only when frogs were exposed to Bd. Bd is a selective force on microbial community structure and function, and beneficial states of microbial community structure may serve to limit the impacts of infection.

Median lethal concentration of formaldehyde and its genotoxic potential in bullfrog tadpoles (Lithobates catesbeianus).

In order to avoid that contaminated frog farms animals escaping in the environment and become potential vector of emergent diseases, studies with disinfection protocol are strictly necessary. The formaldehyde is one of the compounds tested in fungal disinfection protocols and also used in aquaculture. This study aimed to determine the median lethal concentration (LC50-96h) of formaldehyde in bullfrog tadpoles and to evaluate the possible genotoxic effects in acute exposition. Accordingly, the animals were exposed to formaldehyde in the concentrations of 6, 9, 12, 15, and 18 mg L(-1), and after 96 h blood samples were drawn for the micronucleus (MN) test. The LC50-96h was 10.53 mg L(-1), and the MN frequency increased in proportion to the formaldehyde concentrations, with an estimated frequency in the negative control being 1.35 MN/individual. We concluded that formaldehyde is genotoxic to tadpoles of bullfrogs in the tested concentrations, and the choice of this chemical should be contemplated before its use in animals in captivity.

Larval Environment Alters Amphibian Immune Defenses Differentially across Life Stages and Populations.

Recent global declines, extirpations and extinctions of wildlife caused by newly emergent diseases highlight the need to improve our knowledge of common environmental factors that affect the strength of immune defense traits. To achieve this goal, we examined the influence of acidification and shading of the larval environment on amphibian skin-associated innate immune defense traits, pre and post-metamorphosis, across two populations of American Bullfrogs (Rana catesbeiana), a species known for its wide-ranging environmental tolerance and introduced global distribution. We assessed treatment effects on 1) skin-associated microbial communities and 2) post-metamorphic antimicrobial peptide (AMP) production and 3) AMP bioactivity against the fungal pathogen Batrachochytrium dendrobatidis (Bd). While habitat acidification did not affect survival, time to metamorphosis or juvenile mass, we found that a change in average pH from 7 to 6 caused a significant shift in the larval skin microbial community, an effect which disappeared after metamorphosis. Additionally, we found shifts in skin-associated microbial communities across life stages suggesting they are affected by the physiological or ecological changes associated with amphibian metamorphosis. Moreover, we found that post-metamorphic AMP production and bioactivity were significantly affected by the interactions between pH and shade treatments and interactive effects differed across populations. In contrast, there were no significant interactions between treatments on post-metamorphic microbial community structure suggesting that variation in AMPs did not affect microbial community structure within our study. Our findings indicate that commonly encountered variation in the larval environment (i.e. pond pH and degree of shading) can have both immediate and long-term effects on the amphibian innate immune defense traits. Our work suggests that the susceptibility of amphibians to emerging diseases could be related to variability in the larval environment and calls for research into the relative influence of potentially less benign anthropogenic environmental changes on innate immune defense traits.

Structure and composition of arytenoid cartilage of the bullfrog (Lithobates catesbeianus) during maturation and aging.

The aging process induces progressive and irreversible changes in the structural and functional organization of animals. The objective of this study was to evaluate the effects of aging on the structure and composition of the extracellular matrix of the arytenoid cartilage found in the larynx of male bullfrogs (Lithobates catesbeianus) kept in captivity for commercial purposes. Animals at 7, 180 and 1080 days post-metamorphosis (n=10/age) were euthanized and the cartilage was removed and processed for structural and biochemical analysis. For the structural analyses, cartilage sections were stained with picrosirius, toluidine blue, Weigert's resorcin-fuchsin and Von Kossa stain. The sections were also submitted to immunohistochemistry for detection of collagen types I and II. Other samples were processed for the ultrastructural and cytochemical analysis of proteoglycans. Histological sections were used to chondrocyte count. The number of positive stainings for proteoglycans was quantified by ultrastructural analysis. For quantification and analysis of glycosaminoglycans were used the dimethyl methylene blue and agarose gel electrophoresis methods. The chloramine T method was used for hydroxyproline quantification. At 7 days, basophilia was observed in the pericellular and territorial matrix, which decreased in the latter over the period studied. Collagen fibers were arranged perpendicular to the major axis of the cartilaginous plate and were thicker in older animals. Few calcification areas were observed at the periphery of the cartilage specimens in 1080-day-old animals. Type II collagen was present throughout the stroma at the different ages. Elastic fibers were found in the stroma and perichondrium and increased with age in the two regions. Proteoglycan staining significantly increased from 7 to 180 days and reduced at 1080 days. The amount of total glycosaminoglycans was higher in 180-day-old animals compared to the other ages, with marked presence of chondroitin- and dermatan-sulfate especially in this age. The content of hydroxyproline, which infers the total collagen concentration, was higher in 1080-day-old animals compared to the other ages. The results demonstrated the elastic nature of the arytenoid cartilage of L. catesbeianus and the occurrence of age-related changes in the structural organization and composition of the extracellular matrix. These changes may contribute to alter the function of the larynx in the animal during aging.

Representation of particle motion in the auditory midbrain of a developing anuran.

In bullfrog tadpoles, a "deaf period" of lessened responsiveness to the pressure component of sounds, evident during the end of the late larval period, has been identified in the auditory midbrain. But coding of underwater particle motion in the vestibular medulla remains stable over all of larval development, with no evidence of a "deaf period." Neural coding of particle motion in the auditory midbrain was assessed to determine if a "deaf period" for this mode of stimulation exists in this brain area in spite of its absence from the vestibular medulla. Recording sites throughout the developing laminar and medial principal nuclei show relatively stable thresholds to z-axis particle motion, up until the "deaf period." Thresholds then begin to increase from this point up through the rest of metamorphic climax, and significantly fewer responsive sites can be located. The representation of particle motion in the auditory midbrain is less robust during later compared to earlier larval stages, overlapping with but also extending beyond the restricted "deaf period" for pressure stimulation. The decreased functional representation of particle motion in the auditory midbrain throughout metamorphic climax may reflect ongoing neural reorganization required to mediate the transition from underwater to amphibious life.