The mechanics of air breathing in gray tree frog tadpoles, Hyla versicolor (Anura: Hylidae).

We describe air-breathing mechanics in gray tree frog tadpoles (Hyla versicolor). We found that H. versicolor tadpoles breathe by 'bubble-sucking', a breathing mode typically employed by tadpoles too small to break the water's surface tension, in which a bubble is drawn into the buccal cavity and compressed into the lungs. In most tadpoles, bubble-sucking is replaced by breach breathing (breaking the surface to access air) at larger body sizes. In contrast, H. versicolor tadpoles bubble-suck throughout the larval period, despite reaching body sizes at which breaching is possible. Hyla versicolor tadpoles exhibit two bubble-sucking behaviors: 'single bubble-sucking', previously described in other tadpole species, is characterized by a single suction event followed by a compression phase to fill the lungs; 'double bubble-sucking' is a novel, apparently derived form of bubble-sucking that adds a second suction event. Hyla versicolor tadpoles transition from single bubble-sucking to double bubble-sucking at approximately 5.7 mm snout-vent length (SVL), which corresponds to a period of rapid lung maturation when they transition from low to high vascularization (6.0 mm SVL). Functional, behavioral and morphological evidence suggests that double bubble-sucking increases the efficiency of pulmonary gas exchange by separating expired, deoxygenated air from freshly inspired air to prevent mixing. Hyla versicolor, and possibly other hylid tadpoles, may have specialized for bubble-sucking in order to take advantage of this increased efficiency. Single and double bubble-sucking represent two- and four-stroke ventilation systems, which we discuss in the context of other anamniote air-breathing mechanisms.

Tadpole nutritional ecology and digestive physiology: Implications for captive rearing of larval anurans.

The adaptive tadpole stage allows anurans to exploit food resources in two vastly different environments, and the transition from aquatic larvae to terrestrial carnivores is both dramatic and complex. As seen in many other members of the freshwater aquatic community, the nutritional requirements and characteristic feeding strategies of anuran larvae (tadpoles) are extremely diverse, ranging from herbivory to carnivory and including predation and cannibalism, oophagy, coprophagy, filter-feeding, and hindgut microbial fermentation. Whereas tadpoles as a group are commonly considered herbivorous or omnivorous, many are specialists; understanding species-specific dietary habits is critical for captive rearing projects in zoos and amphibian habitat conservation efforts. Practical applications of this review also encompass studies of amphibian declines, herpetoculture, ecology and evolution, and comparative gastrointestinal morphology and physiology.

Comparative assessment of individual and mixture chronic toxicity of glyphosate and glufosinate ammonium on amphibian tadpoles: A multibiomarker approach.

The aim of the present study was to assess the ecotoxicity of glyphosate and glufosinate ammonium mixtures on amphibian tadpoles and the potential impact of mixture in aquatic ecosystems health. The bonding properties of the mixture based on computational chemistry and an experimental bioassay on morphology, DNA damage and biochemical biomarkers on tadpoles of the common toad Rhinella arenarum were studied. The results of the density functional theory analysis showed trends of the pesticides clustering to form exothermic mixtures, suggesting the likelihood of hot-spots of pesticides in real aquatic systems. In addition, biological effects of individual pesticides and the mixture were studied on tadpoles over 45 days-chronic bioassay. The bioassay consisted of four treatments: a negative control (CO), 2.5 mg L-1 of a glyphosate-based herbicide (GBH), 2.5 mg L-1 of a glufosinate ammonium-based herbicide (GABH) and their 50:50 (% v/v) mixture (GBH-GABH). Morphological abnormality rates were significantly higher in all herbicide treatments with respect to CO at 48 h of exposure. Abdominal edema was the most frequent type of abnormality recorded at 48 h, 10 and 45 days of exposure. DNA damage was recorded in all herbicides treatments. Thyroxin increased only in GABH treatment. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) significantly increased in GBH treatment, indicating a GBH-neurotoxic effect. Glutathione S-transferase decreased in GABH and GBH-GABH treatments, while catalase decreased in individual GBH and GABH treatments. Overall, teratogenicity, DNA damage, hormonal disruption (T4), and oxidative stress were greater in GABH-treated tadpoles than GBH-treated tadpoles. This study also highlights the robust chemical interaction between the active ingredients of both herbicides, which is reflected on antagonisms in most of analyzed biomarkers, as well as potentiation and additivity in others. Based on our results, the GABH had a higher toxicity than GBH for amphibian tadpoles.

Influence of substrate orientation on tadpoles' feeding efficiency.

In nature, tadpoles encounter food on substrates oriented at different angles (e.g. vertically along stems, horizontally on the bottom of the pond). We manipulated the orientation of food-covered surfaces to test how different orientations of surfaces affect tadpoles' feeding efficiency. We studied taxa that differed in the oral morphology of their larvae and position in the water column. We hypothesized that species would differ in their ability to graze upon surfaces at different orientations and that differences in the tadpoles' feeding ability would result in different growth rates. The orientation of food-covered surfaces did not affect the growth rate of bottom-dwelling tadpoles (whose growth rate varied only between species). Among midwater tadpoles, some species appear to have a generalist strategy and experienced a high relative growth rate on numerous substrate orientations, whereas others achieved high growth rates only on flat substrates (i.e. at 0° and 180°). We conclude that oral morphology constrains tadpoles' ability to feed at different substrate orientations, and this could lead to niche partitioning in structurally complex aquatic environments. Because physical parameters of the environment can affect tadpoles' growth rate, characterizing these features might help us better understand how competition structures tadpole assemblages.

Effects of glyphosate and its commercial formulation, Roundup® Ultramax, on liver histology of tadpoles of the neotropical frog, Leptodactylus latrans (amphibia: Anura).

In the last years, the agricultural expansion has led to an increased use of pesticides, with glyphosate as the most widely used worldwide. This is also the situation in Argentina, where glyphosate formulations are the most commercialized herbicides. It is known that glyphosate formulations are much more toxic than the active ingredient, and this difference in toxicity can be attributed to the adjuvants present in the formula. In this context, the aim of the present study was to evaluate and compare sub-lethal histological effects of the glyphosate formulation Roundup Ultramax and glyphosate active ingredient on Leptodactylus latrans tadpoles at Gosner-stage 36. Semi-static bioassays were performed using 96 h of exposure with Roundup Ultramax formulation (RU; 0.37-5.25 mg a.e./L), glyphosate (GLY; 3-300 mg/L), and a control group. RU exposure showed an increment in the melanomacrophagic cells (MMc) and melanomacrophagic centers (MMCs) from 0.37 mg a.e./L. GLY exposure showed a significant increment in the number of MMc from 15 mg/L, and of MMCs from 3 mg/L. Also, histopathological lesions were observed in the liver of tadpoles exposed to both, GLY and RU. These lesions included: lipidosis and hepatic congestion, but only RU showed significant differences respect to control, with a LOEC value of 2.22 mg a.e./L for both effects. In sum, this study represents the first evidence of adverse effects of glyphosate and RU formulation on the liver of anuran larvae at concentrations frequently found in the environment.

High frequency of larval abnormalities of the toad Rhinella arenarum (Anura: Bufonidae) in an Argentinian agroecosystem

Introduction: The frequent use of pesticides is currently considered a cause of environmental pollution due to the high rate of entry of these substances into agroecosystems. This constitutes a risk for the species that inhabit these ecosystems, in particular anurans whose characteristics make them prone to exposure to and interaction with environmental pollutants. Objective: To report the occurrence of abnormalities in larvae of the common toad Rhinella arenarum inhabiting ponds surrounded by agroecosystems. Methods: In two consecutive springs (2015 and 2016), reproductive events of common toads were monitored in temporary pond systems in agricultural and non-agricultural areas, located near the city of La Plata (Buenos Aires, Argentina). The physicochemical parameters of the ponds were measured, and the stage of each reproductive event was recorded, such as the numbers of adult toads, amplexus and clutches. In the laboratory, the larvae were measured and photographed, their stage of development was recorded, and their morphology was examined under a stereomicroscope. Representative samples (normal and abnormal) from each pond studied were processed for histopathological analysis. Results: In the field studies carried out on a population of R. arenarum collected in an agroecosystem, a lower number of reproductive adults and clutches were observed in relation to the population of a non-agricultural pond. A total of 1 910 larvae were collected: 529 and 1 381 larvae from ponds located in non-agricultural and agricultural areas, respectively. Larvae from the agroecosystem showed two types of abnormalities: severe tail flexure and abdominal bloating. In addition, five degrees of severity could be determined in relation to abdominal bloating. Conclusions: This work reports the high frequency and severity of abnormalities observed in the early stages of R. arenarum larvae living within an agroecosystem, providing evidence of the negative impact that agricultural activities cause on aquatic ecosystems surrounded by farming areas.

Introducción: El uso frecuente de plaguicidas es considerado actualmente una causa de contaminación ambiental debido a las altas tasas de ingreso de estas sustancias a los agroecosistemas. Esta situación es un riesgo para las especies que habitan en estos ecosistemas, en particular los anuros cuyas características los hacen propensos a la exposición e interacción con contaminantes ambientales. Objetivo: Informar la presencia de anormalidades en larvas del sapo común Rhinella arenarum que habitan en estanques rodeados por un agroecosistema. Métodos: En dos primaveras consecutivas (2015 y 2016), se monitorearon los eventos reproductivos del sapo común proveniente de sistemas de estanques temporales ubicados en zonas agrícolas y no agrícolas, cerca de la ciudad de La Plata (Buenos Aires, Argentina). Se midieron los parámetros fisicoquímicos de los estanques y se registraron las etapas de cada evento reproductivo como el número de sapos adultos, amplexos y nidadas. En el laboratorio, las larvas fueron medidas y fotografiadas, se registró su estado de desarrollo y se examinó la morfología de cada una bajo microscopio estereoscópico. Se procesaron muestras representativas (normales y anormales) de cada estanque estudiado para análisis histopatológico. Resultados: En la población de R. arenarum que vive dentro de un agroecosistema, se observó un menor número de adultos reproductores y puestas en relación con la del estanque en la zona no agrícola. Se recolectaron un total de 1 910 larvas: 529 y 1 381 larvas de estanques ubicados en zonas no agrícolas y agrícolas, respectivamente. Las larvas del agroecosistema mostraron dos tipos de anormalidades: severa flexión de la cola y distensión abdominal. Además, se pudo determinar cinco grados de gravedad en relación con la distensión abdominal. Conclusiones: Una alta frecuencia y severidad de anormalidades en los estadios tempranos de larvas de R. arenarum que viven dentro de un agroecosistema proporciona evidencia del impacto negativo que las actividades agrícolas causan en los ecosistemas acuáticos rodeados por áreas de cultivo.

Effect on the growth and development and induction of abnormalities by a glyphosate commercial formulation and its active ingredient during two developmental stages of the South-American Creole frog, Leptodactylus latrans.

We evaluated the acute lethal and sublethal effects of technical-grade glyphosate (GLY) and the GLY-based commercial formulation Roundup ULTRA MAX® (RU) on two Gosner stages (Gss) 25 and 36 of the South-American Creole frog, Leptodactylus latrans. Bioassays were performed following standardized methods within a wide range of concentrations (0.0007-9.62 mg of acid equivalents per liter-a.e./L-of RU and 3-300 mg/L of GLY). The endpoints evaluated were mortality, swimming activity, growth, development, and the presence of morphologic abnormalities, especially in the mouthparts. No lethal effects were observed on larvae exposed to GLY during either Gs-25 or Gs-36. The concentrations inducing 50 % lethality in RU-exposed larvae at different exposure times and Gss ranged from 3.26 to 9.61 mg a.e./L. Swimming activity was affected by only RU. Effects on growth and development and the induction of morphologic abnormalities-like oral abnormalities and edema-were observed after exposure to either GLY or RU. Gs-25 was the most sensitive stage to both forms of the herbicide. The commercial formulation was much more toxic than the active ingredient on all the endpoints assessed. Effects on growth, development, and the induction of morphologic abnormalities observed in the range of environmental concentrations reported for agroecosystems of Argentina constitute an alert to the potential detrimental effects of the herbicide that could be affecting the fitness and survival of anurans in agroecosystems.

Attraction to conspecifcs in Rhinella icterica and R. ornata tadpoles (Anura: Bufonidae) / Atração por coespecíficos em girinos de Rhinella icterica e R. ornata (Anura: Bufonidae)

Abstract: Tadpoles are able to perceive and discriminate signals from environment and they may use this ability in behaviors and ecological processes. Recognition mechanisms may be involved in schooling by means of attraction among individuals, characterizing a social process. By means of laboratory experiments the present study investigated the presence or absence of attraction to conspecifics in tadpoles of Rhinella icterica and R. ornata, two sympatric species from the Atlantic Forest of southeastern Brazil. We collected eggs of these two species in the field and reared them in laboratory according to two different methods (isolated or in groups of siblings). Tadpoles were submitted to experiments of choice between conspecifics, heterospecifics, and an empty compartment. Rhinella icterica tadpoles preferred to associate with conspecifics rather than R. ornata tadpoles, and we verified this is an innate behavior. Rhinella ornata tadpoles failed to discriminate between conspecifics and R. icterica tadpoles. When submitted to choice between a group of tadpoles of the other species and an empty compartment, R. icterica tadpoles presented random distribution, while R. ornata tadpoles preferred to associate with heterospecifics. Our results indicate R. icterica tadpoles have preference to associate with conspecifics, while R. ornata tadpoles may school indiscriminately. This study contributes for a better understanding of larval anuran social behavior.

Resumo: Os girinos são capazes de perceber e discriminar sinais do ambiente e podem usar essa capacidade em comportamentos e processos ecológicos. Mecanismos de reconhecimento podem estar envolvidos na agregação por meio da atração entre indivíduos, caracterizando um processo social. O presente estudo investigou, por meio de experimentos de laboratório, a presença ou ausência de atração por coespecíficos em girinos de Rhinella icterica e R. ornata, duas espécies simpátricas, da Mata Atlântica do sudeste do Brasil. Coletamos ovos dessas duas espécies no campo e os cultivamos em laboratório de acordo com dois métodos diferentes (isolados ou em grupo de irmãos). Os girinos foram submetidos a experimentos de escolha entre coespecíficos, heteroespecíficos e compartimento vazio. Girinos de Rhinella icterica preferiram associar-se a coespecíficos do que com girinos de R. ornata, e verificamos que este é um comportamento inato. Os girinos de Rhinella ornata não conseguiram discriminar entre coespecíficos e girinos de R. icterica. Quando submetidos à escolha entre um grupo de girinos da outra espécie e um compartimento vazio, os girinos de R. icterica apresentaram distribuição aleatória, enquanto os girinos de R. ornata preferiram associar-se a girinos heteroespecíficos. Nossos resultados indicam que girinos de R. icterica preferem associar-se a coespecíficos, enquanto girinos de R. ornata podem agregar-se indiscriminadamente. Este estudo contribui para uma melhor compreensão do comportamento social de larvas de anuros.

Assessment of water pollution in the Brazilian Pampa biome by means of stress biomarkers in tadpoles of the leaf frog Phyllomedusa iheringii (Anura: Hylidae).

The Brazilian Pampa biome is currently under constant threat due to increase of agriculture and improper management of urban effluents. Studies with a focus on the assessment of impacts caused by human activities in this biome are scarce. In the present study, we measured stress-related biomarkers in tadpoles of the leaf frog Phyllomedusa iheringii, an endemic species to the Pampa biome, and tested its suitability as a bioindicator for the assessment of potential aquatic contamination in selected ponds (S1 and S2) nearby agricultural areas in comparison to a reference site. A significant decrease in acetylcholinesterase activity was observed in S2 when compared to S1 and reference. The levels of total-hydroperoxides were increased in S2 site. In parallel, increased activity of the antioxidant enzymes catalase, superoxide dismutase and glutathione S-transferase were observed in S2 when compared to S1 and reference. Further studies are necessary in order to correlate the changes observed here with different chemical stressors in water, as well as to elucidate mechanisms of toxicity induced by pesticides in amphibian species endemic to the Pampa biome. Nevertheless, our study validates Phyllomedusa iheringii as a valuable bioindicator in environmental studies.

Naturally occurring variation in tadpole morphology and performance linked to predator regime.

Divergent natural selection drives a considerable amount of the phenotypic and genetic variation observed in natural populations. For example, variation in the predator community can generate conflicting selection on behavioral, life-history, morphological, and performance traits. Differences in predator regime can subsequently increase phenotypic and genetic variations in the population and result in the evolution of reproductive barriers (ecological speciation) or phenotypic plasticity. We evaluated morphology and swimming performance in field collected Bronze Frog larvae (Lithobates clamitans) in ponds dominated by predatory fish and those dominated by invertebrate predators. Based on previous experimental findings, we hypothesized that tadpoles from fish-dominated ponds would have small bodies, long tails, and large tail muscles and that these features would facilitate fast-start speed. We also expected to see increased tail fin depth (i.e., the tail-lure morphology) in tadpoles from invertebrate-dominated ponds. Our results support our expectations with respect to morphology in affecting swimming performance of tadpoles in fish-dominated ponds. Furthermore, it is likely that divergent natural selection is playing a role in the diversification on morphology and locomotor performance in this system.

Spawn predation and larval growth inhibition as mechanisms for niche separation in anurans.

We have investigated interspecific interactions between larvae of the 3 native British anurans, Rana temporaria, Bufo bufo and B. calamita in the field and in the laboratory. Over a 3 year period, predation of B. calamita spawn by the two competitively superior species accounted for only 0.3-3.0% of the total deposited in the dune pools of the study site. However, in ponds with marked niche overlap destruction of natterjack spawn was often 100%. Furthermore, growth rates of B. calamita larvae surviving to hatch were grossly inhibited when large numbers (>0.1 per l) of common frog or toad tadpoles were present. Overall survival to metamorphosis by the competitively inferior species was always negligible in these situations. These results were confirmed by experiments using caged larvae, and by movement of R. temporaria spawn between pools. Growth could be reduced to <25% of uninhibited controls, and time taken to achieve metamorphosis extended by at least 40% in the presence of larger larvae. Laboratory experiments confirmed that inhibitory effects of both R. temporaria and B. bufo larvae on growth by B. calamita tadpoles was demonstrable at tadpole densities comparable with those found in the field.

Suspension feeding of anuran larvae at low concentrations of Chlorella algae (Amphibia, Anura).

Ingestion and filtering rates in larval Xenopus laevis, Bufo calamita, Rana temporia and Bufo bufo fed suspensions of Chlorella fusca were investigated. Concentrations were measured with a Coulter Counter. (1) For all species, filtration occurred at concentrations far below those reported by other authors for Rana sylvatica feeding on Chlorella pyrenoidosa. For Bufo bufo, only larvae near metamorphosis showed ingestion at low particle concentrations. Since buccopharyngeal ventilation continues even in the absence of food particles, this threshold feeding behaviour in the younger larvae must be due to different mechanisms to those found in Daphnia and Calanus studied by other authors: probably reduction of the buccal pumping rate and the mucus production of the filter apparatus. (2) For B. calamita, R. temporaria and X. laevis the highest suspension feeding efficiency was at early tadpole stages, corresponding with the high growth rate of these stages. (3) The life histories of the species provide the basis for understanding their different retention efficiencies and functional responses.

Copper-driven avoidance and mortality in temperate and tropical tadpoles.

Amphibians have experienced an accentuated population decline in the whole world due to many factors, one of them being anthropogenic contamination. The present study aimed to assess the potential effect of copper, as a worldwide and reference contaminant, on the immediate decline of exposed population due to avoidance and mortality responses in tadpoles of three species of amphibians across climatic zones: a South American species, Leptodactylus latrans, a North American species, Lithobates catesbeianus, and a European species, Pelophylax perezi. A non-forced exposure system with a copper gradient along seven compartments through which organisms could freely move was used to assess the ability of tadpoles to detect and avoid copper contamination. All species were able to avoid copper at a concentration as low as 100 µg L(-1). At the lowest (sublethal) concentrations (up to 200 µg L(-1)) avoidance played an exclusive role for the population decline, whereas at the highest concentrations (> 450 µg L(-1)) mortality was the response determining population decline. The median concentrations causing exposed population immediate decline were 93, 106 and 180 µg L(-1) for Le. latrans, Li. catesbeianus and P. perezi, respectively. Contaminants might, therefore, act as environmental disruptors both by generating low-quality habitats and by triggering avoidance of tadpoles, which could be an important response contributing to dispersion patterns, susceptibility to future stressors and decline of amphibian populations (together with mortality).

Girinos de três espécies de Aplastodiscus Lutz, 1950 (Anura - Hylidae) ocorrentes no Estado da Bahia, Brasil / Tadpoles of three species of Aplastodiscus Lutz, 1950 (Anura - Hylidae) occurring in the State of Bahia, Brazil

Nós estudamos a morfologia dos girinos de Aplastodiscus sibilatus (Cruz, Pimenta, & Silvano, 2003); Aplastodiscus cavicola (Cruz & Peixoto, 1985) Aplastodiscus ibirapitanga (Cruz, Pimenta, & Silvano, 2003) e a comparamos com os girinos de outras espécies do grupo Aplastodiscus albosignatus disponíveis na literatura. Os resultados reforçam um dos dois agrupamentos do complexo de A. albosignatus propostos na literatura, que inclui A. cavicola, A. ibirapitanga e A. leucopygius. Os girinos das espécies deste grupo apresentam "gap" no lábio anterior, três reentrâncias no lábio posterior, fórmula dentária 2(2)/4(1) e distância internasal maior do que as distâncias entre as bordas internas das órbitas dos olhos. Para outro grupo, no qual A. sibilatus está incluído, não observamos nenhuma característica em comum. Girinos de A. sibilatus se diferenciaram dos girinos das demais espécies de Aplastodicus pela fórmula dentária variável 4(1,4)/7(1), 4(1,4)/6(1), 4(1,2,4)/6(1) e 3(1,3)/5(1).

We studied the morphology of tadpoles of Aplastodiscus sibilatus (Cruz, Pimenta, & Silvano, 2003); Aplastodiscus cavicola (Cruz & Peixoto, 1985) and Aplastodiscus ibirapitanga (Cruz, Pimenta, & Silvano, 2003) and compared it with tadpoles of other species of Aplastodiscus albosignatus group available in literature. The results reinforce one of the two species groups of A. albosignatus complex, which includes A. cavicola, A. ibirapitanga and A. leucopygius. Tadpoles of species of this group have anterior lip gap, three indentations in the posterior lip, labial tooth row formula 2(2)/4(1) and internarial distance greater than the distance between inner margins of the eyes orbits. For another group, in which A. sibilatus is included, we did not observe any characteristic in common. Tadpoles of A. sibilatus differ from those of other Aplastodiscus species by the dental formula variable 4(1,4)/7(1), 4(1,4)/6(1), 4(1,2,4)/6(1) e 3(1,3)/5(1).