Multiple morphophysiological responses of a tropical frog to urbanization conform to the pace-of-life syndrome.

The Pace-of-Life syndrome proposes that behavioural, physiological and immune characteristics vary along a slow-fast gradient. Urbanization poses several physiological challenges to organisms. However, little is known about how the health status of frogs is affected by urbanization in the Tropics, which have a faster and more recent urbanization than the northern hemisphere. Here, we analysed a suite of physiological variables that reflect whole organism health, reproduction, metabolic and circulatory physiology and leukocyte responses in Leptodactylus podicipinus. Specifically, we tested how leukocyte profile, erythrocyte morphometrics and germ cell density, as well as somatic indices and erythrocyte nuclear abnormalities differ throughout the adult life span between urban and rural populations. We used Phenotypic Trajectory Analysis to test the effect of age and site on each of the multivariate data sets; and a Generalised Linear Model to test the effect of site and age on nuclear abnormalities. Somatic indices, erythrocyte nuclear abnormalities, erythrocyte morphometrics and leukocyte profile differed between populations, but less so for germ cell density. We found a large effect of site on nuclear abnormalities, with urban frogs having twice as many abnormalities as rural frogs. Our results suggest that urban frogs have a faster pace of life, but the response of phenotypic compartments is not fully concerted.

The amphibian's spleen as a source of biomarkers for ecotoxicity assessment: Historical review and trends.

Amphibians are very sensitive to many environmental changes, so these animals are considered good bioindicator models for ecotoxicology. Given the importance of the amphibian spleen for hematopoietic and immune responses, this can be a key organ for the evaluation of biomarkers to monitor the health of individuals in nature or in captivity. In this systematic review, we searched databases and summarized the main findings concerning the amphibian spleen as a source of possible biomarkers applied in different scientific fields. The searches resulted in 83 articles published from 1923 to 2022, which applied the use of splenic samples to evaluate the effects of distinct stressors on amphibians. Articles were distributed in more than twenty countries, with USA, Europe, and Brazil, standing out among them. Publications focused mainly on anatomical and histomorphological characterization of the spleen, its physiology, and development. Recently, the use of splenic biomarkers in pathology and ecotoxicology began to grow but many gaps still need to be addressed in herpetological research. About 85 % of the splenic biomarkers showed responses to various stressors, which indicates that the spleen can provide numerous biomarkers to be used in many study fields. The limited amount of information on morphological description and splenic anatomy in amphibians may be a contributing factor to the underestimated use of splenic biomarkers in herpetological research around the world. We hope that this unprecedented review can instigate researchers to refine herpetological experimentation, using the spleen as a versatile and alternative source for biomarkers in ecotoxicology.

Ecotoxicological Methodologies to Evaluate Biomarkers at Different Scales in Neotropical Anurans.

The new questions in ecotoxicology highlight the importance of applying a battery of biomarkers, as this results in ecotoxicological predictions that improve not only the interpretation of the effects of environmental stressors on organisms but also the determination of their possible impact. It is well known that the use of ecotoxicological biomarkers at different levels of organization allows for the prediction of the biological responses of organisms to environmental stressors, which is useful in environmental risk assessment. Nevertheless, it is necessary to consider the optimization of basic procedures, to generate historical data in control groups, and to employ specific bioassays to evaluate responses in organs and tissues in order to elucidate the nature and variation of the effects observed. Therefore, the present work aims to describe several ecotoxicological methodologies employed in all stages of neotropical anurans at different ecological levels and to validate them as useful biomarkers to be used both in wildlife and in laboratory conditions. In this work, these biomarkers were applied at the individual/organismic level (body condition index), histological/physiological level (histopathology, histometric, and pigmentary analyses), biochemical level (oxidative stress enzymes), and genetic level (direct and oxidative damage in DNA by comet assay). Although these methodologies have small variations or modifications depending on the species, these techniques provide effective biomarkers for evaluating the effect of xenobiotics on anurans, which possess certain characteristics that make them useful indicator species of aquatic and terrestrial ecosystems. In conclusion, the battery of biomarkers employed in the present study has proven to be adequate for estimating toxic responses in Neotropical anurans and can be further recommended as bioindicators for identifying the impact of pollutants on the aquatic ecosystems of the region. Finally, it is recommended to achieve the standardization of these important biomarkers for anurans in specific regions as well as to possibly include them in risk assessments and decision-making.

Short-term effects of α-melanocyte-stimulating hormone in three distinct melanin-pigmented cell types of Anura.

Ectothermic animals present melanin-containing cells in their integument and viscera. Besides cutaneous melanophores, amphibians have melanomacrophages in the hepatic parenchyma and melanocytes in the viscera, which are also present in their testicular stroma. The native melanocyte stimulating hormone (α-MSH) is the main hormone that modulates the color change in melanophores. However, we still know too little about how the α-MSH acts in vivo on visceral melanin-containing cells. In this study, we collected 30 adult males of Physalaemus nattereri (Anura, Leptodactylidae) to evaluate the short-term effects of α-MSH on melanophores, melanocytes and melanomacrophages under light microscopy. For this, we injected 0.05 ml of a single intraperitoneal dose containing 2.5x10-7 mmol/10g of α-MSH, diluted in ringer solution, in five experimental groups with five individuals each one. The different groups were analyzed after 1, 3, 6, 12 and 24h. The control group with five other individuals received only 0.05 ml of ringer solution. The skin pigmentation increased quickly after animals received the hormone α-MSH with the consequent darkening of the body (body darkness). Melanophores, melanocytes and melanomacrophages responded similarly to the test, with an increase in the area containing melanin. However, melanophores and melanomacrophages reached their darkest pigmentation in a shorter period of time in comparison to the testicular melanocytes, probably due to specific metabolic characteristics of each organ. Thus, we verified that the three types of cells, although present in different organs, are responsive to the native hormone α-MSH, which enables us to treat them as a pigmentary system.

Intestinal morphometry and cell density features in tropical farmed fish: A methodological approach.

The digestive tract of fish has many morphological adaptations related to habitat and nutrition. Intestinal biometry may reflect these adaptations. Here, we aimed to describe histometric patterns in farmed fish and their relationship with feeding by using a standardized protocol considering cell density by tissue area. Five juvenile specimens of each species (Pseudoplatystoma corruscans, Piaractus mesopotamicus, and Oreochromis niloticus) were used. O. niloticus possessed higher intestinal weight and length besides higher intestinal quotient and intestinal somatic index than the other species. The general histological composition was similar between species. However, P. corruscans showed differences in thickness between the anterior and posterior segments. O. niloticus had thinner serosa and muscularis layers than the other species. The cell density was distinct in both species and segments. Comparing the intestinal segments, O. niloticus displayed the lowest count of granulocytes. Goblet cell density was lower in P. mesopotamicus in all segments. However, the volume of these cells was higher in the anterior and middle anterior segments. Our data demonstrated that intestinal structural plasticity is associated with the difference in feeding habits. Here, we used quantitative standardized histometric criteria to understand the morphophysiological diversity of the fish digestive tract, and this technique can be applied in future studies to evaluate changes in the digestive tracts of vertebrates.

Changes in blood parameters and metabolism in bullfrog tadpoles, Lithobates catesbeianus, (Shaw, 1802) after exposure to the Sorocaba River (São Paulo, Brazil) water.

This study evaluated the genetic damage, oxidative stress, neurotoxicity, and energy metabolism in bullfrog tadpoles (Lithobates catesbeianus) exposed to water from two sites of the Sorocaba River, Ibiúna (PI), and Itupararanga reservoir (PIR), in summer and winter. After 96-h exposure, the erythrocyte number decreased in PI and increase in PIR in summer. Bullfrogs show oxidative unbalance (liver, kidney, and muscle), with alterations in the nitric oxide synthase and glucose 6-phosphate dehydrogenase. Cholinesterase increased in the brain in PI and PIR in the summer and decreased in PI in the winter. It also increased in the muscle in both PI and PIR in the winter. Tadpoles show alterations in the activity of the metabolic enzymes (liver, kidney, and muscle), such as phosphofructokinase, pyruvatokinase, malate dehydrogenase, and lactate dehydrogenase; and in the amount of glucose and triglycerides metabolites. Exposure to the Sorocaba River reflected a stressful situation for L. catesbeianus as the changes caused to their metabolism associated with oxidative stress and neurotoxicity may have effects on the development of tadpoles.

Hematological parameters of a Neotropical wild frog population, with a phylogenetic perspective on blood cell composition in Anura.

Hematological parameters can provide key information to an animal health status. However, this information is usually hard to obtain. Here, we described the hematological parameters of Leptodactylus podicipinus in the Brazilian Pantanal. We measured red blood cell morphometrics, erythrogram, and leukogram. We also tested for phylogenetic signal in the erythrogram and leukogram of 48 frog species from 15 families, testing if body size explains their variation. Lymphocytes were the most abundant leukocytes (>60%) in L. podicipinus, followed by neutrophils (∼10%). Given that L. podicipinus is an abundant and widely distributed species in central Brazil, knowing its hematological pattern can help establish a baseline and improve its use as a bioindicator of environmental degradation. Mean corpuscular hemoglobin and value contributed more to the phylomorphospace of erythrogram, in which Leptodactylus spp. and Hypsiboas raniceps had lower values of these variables, whereas Bufotes viridis and Hyla arborea had high values. The phylogenetic signal was spread throughout the dimensions of the leukogram phylomorphospace. The variables that most contributed to it were total leukocytes counts, lymphocytes, and neutrophils. We also found a moderate phylogenetic signal for both the erythrogram and leukogram. Accordingly, body size accounted for a low proportion of variation in both the leukogram (4.7%) and erythrogram (0.57%). By applying phylogenetic comparative methods to hematological parameters, our results add a new perspective on the evolution of blood cell physiology in frogs.

Sperm morphology and testicular histology of the polyandric species Leptodactylus podicipinus (Anura: Leptodactylidae) from an urban environment.

Anurans have a high reproductive diversity, which is closely associated with testicular dynamics and sperm production. This variety in reproduction is modulated by the sexual characteristics of reproductive strategies, such as polyandry. Leptodactylus podicipinus has high reproductive plasticity, wide geographical distribution, and polyandric behavior. Although aspects of the testes and sperm of this species are known, knowledge about the relationship between these aspects and reproductive investment is scarce. The present study evaluated the morphological characteristics of the testes and sperm in an urban environment. We used 11 sexually mature males from the city of Campo Grande, Mato Grosso do Sul, Brazil. The specimens were obtained from night excursions between April and September, the months that constitute the post-rainy season. The biometric data (snout-vent length, testes, and body mass) were associated with sperm parameters (length of the spermatozoon head and tail). Stereological analyses of sperm morphology and the testes were performed. The results showed correlations between testis mass and spermatozoon length, which were positively related to head length and negatively related to tail length. The locular area and tail length were also negatively correlated. The percentage of normal spermatozoa was 97%; however, some sperm heads and tails exhibited different morphologies from the pattern described for the species. The germinative structure comprised more than 50% of the locular area composed of spermatozoa. The results of this study contribute to the knowledge of aspects related to the reproductive biology of L. podicipinus obtained from an urban environment.

Response of hepatic biomarkers in Physalaemus nattereri (Anura) to different benzo(α)pyrene exposure routes.

For over 40 years, anurans have been used as a study model to assess the adverse effects of benzo(α)pyrene (BαP), which include genotoxic, hepatotoxic, and immunotoxic effects. In these studies, BαP is administered cutaneously or by injection, with no comparison between two or more routes. The purpose of this study is to assess whether the effect of BαP is influenced by its route of administration, using the response of hepatic biomarkers of Physalaemus nattereri. Specimens (n = 108) were collected and divided into three experimental treatments (cutaneous, injection, and oral) and three experimental times (one, three, and seven days). Specimens received 0.02 ml of pure mineral oil (control) or mineral oil containing 2 mg/kg of BαP. The BαP causes changes in morphological (melanin, hemosiderin, lipofuscin, and mast cells) and biochemical (superoxide dismutase and glutathione S-transferase) hepatic biomarkers. Compared to biochemical, morphological biomarkers underwent a greater number of significant changes due to the treatment with BαP. The route of exposure alters the effects of BαP, mainly seen in morphological biomarkers, especially the pigments melanin, hemosiderin, and lipofuscin. In these pigments, the effect of the exposure pathway changes according to the analyzed biomarker, and the exposure time modulates the exposure pathway effect. These results are unprecedented for anurans and contribute to the field of herpetology and ecotoxicology.

Nonylphenol and cyproterone acetate effects in the liver and gonads of Lithobates catesbeianus (Anura) tadpoles and juveniles.

Environmental pollution plays an important role in amphibian population decline. Contamination with endocrine disrupting chemicals (EDCs) is particularly worrying due to their capacity to adversely affect organisms at low doses. We hypothesized that exposure to EDCs such as 4-nonylphenol (NP) and cyproterone acetate (CPA) could trigger responses in the liver and gonads, due to toxic and endocrine disrupting effects. Growth rate may also be impaired by contamination. We investigated sublethal effects of a 28-day exposure to three different concentrations of NP and CPA on liver pigmentation, gonadal morphology, body mass, and length of tadpoles and juveniles Lithobates catesbeianus. Liver pigmentation and the gonadal morphologies of treated tadpoles did not differ from control, but growth rate was impaired by both pollutants. Juveniles treated with 10 µg/L NP and 0.025 and 0.25 ng/L CPA displayed increased liver melanin pigmentation, but gonadal morphologies, sex ratios, and body mass were not affected after treatments. The increase in liver pigmentation may be related to defensive, cytoprotective role of melanomacrophages. The decreased growth rate in tadpoles indicates toxic effects of NP and CPA. Thus, contamination with NP and CPA remains a concern and sublethal effects of different dosages of the compounds on native species should be determined.

Habitat differences affect the nuclear morphology of the erythrocytes and the hepatic melanin in Leptodactylus fuscus (Anura) in the Brazilian Cerrado savanna.

The sensitivity of anuran to the effects of habitat destruction and contamination has led to a preoccupying global decline in their populations. Morphological biomarkers such as micronuclei and other erythrocyte nuclear abnormalities (ENAs), as well as the occurrence of hepatic melanin, can be used to evaluate the effects of habitat impacts. In the present study, these two parameters were combined for the in situ assessment of the effects of soybean cultivation on the grassfrog, Leptodactylus fuscus. Specimens were also collected from a protected area to provide a reference site (non-agricultural environment). The frequency of some of the nuclear abnormalities in the animals from the soybean plantation was much higher than that recorded at the reference site, in particular micronuclei, which were 3.6 times more frequent in the plantation, lobulated nuclei (3.4 times more frequent), and reniform nuclei, which were four times more common than at the reference site. The combined analysis of all the ENAs together also revealed a frequency approximately 1.4 times higher in the animals from the soybean plantation, in comparison with the protected area. Smaller areas of hepatic melanin were observed in the specimens from the soybean plantation. These results provide further evidence of the sensitivity of anurans to habitat impacts and indicate that animals found in soybean plantations are susceptible to systematic alterations of their cells.

The evolution of red blood cell shape in fishes.

The size and shape of red blood cells (RBCs) provide key information on life-history strategies in vertebrates. However, little is known about how RBC shape evolved in response to environmental factors, body size and the role of evolutionary rate. Here, we analysed RBC morphometrics in a set of Teleostei (bony fishes) and Elasmobranchii (sharks and rays) species testing the hypothesis that phylogenetic relationship explains species occupation of morphospace. We collected data on cell and nucleus area and volume, nucleus:cytoplasm ratio and shape factor for 65 species belonging to 28 orders. Then, we built phylomorphospaces separately for bony fish and sharks and rays. To test whether phylogenetic relationships predicted phenotypic similarity, we calculated multivariate phylogenetic signal. We also estimated the evolutionary rate of RBC shape for each node and tip using ridge regression. Finally, we tested whether habitat and body size influenced RBC shape using a PGLS. We found a significant phylogenetic signal in RBC shape for bony fish, but not sharks and rays. Saltwater teleost species were more clustered than freshwater ones in the phylomorphospace, suggesting clade disparity. Accordingly, the rate of evolution was highly heterogeneous, with significant decrease in Acanthopterygii. Neither habitat nor body size influenced RBC shape. In conclusion, RBC shape seems to have evolved in fishes in response to multiple selective pressures independent of life-history characters.

Genotoxic and melanic alterations in Lithobates catesbeianus (anura) tadpoles exposed to fipronil insecticide.

The aim of this study was to evaluate the genotoxic and morphological systemic effects of both an acute and a chronic exposure of bullfrog tadpoles to fipronil. Lithobates catesbeianus tadpoles had morphological biomarkers (skin, liver, and blood) analyzed at Gosner stages 36-38, when exposed to four different concentrations of Regent® 800 WG (80% fipronil): 0.00 (control), 0.04, 0.08, 0.4 mg/L, and four experimental times: 4, 8, 12, and 16 days. Body darkness responded directly to the treatment and exposure time. There was a treatment-dependent decrease in darkness of heads and tails. In relation to the biometric analysis, fipronil induced a decrease in the individual weight and liver mass at the end of the experiments, whereas the hepatosomatic index did not vary according to the treatment. For the exposed animals and for the control group, the area of hepatic melanin increased as exposure time increased. Fipronil has genotoxic effects on L. catesbeianus tadpoles even after short exposure times (e.g., 4 and 8 days), and the main nuclear abnormality is in the anucleate cells. A relevant correlation was observed between genotoxic biomarkers and cutaneous and internal melanin. The frequency of nuclear abnormalities is inversely correlated both with the hepatic melanin area and with the cutaneous melanin of animals. Fipronil has distinct systemic effects on tadpoles based on its concentration, as well as on its exposure time. Such alterations (pigmentation level and rate of erythrocyte abnormality) result in morphological and physiological effects, which may compromise the behavior and survival of the anurans.

Transaminase profile and hepatic histopathological traits in Piaractus mesopotamicus exposed to insecticide Diflubenzuron.

Diflubenzuron (DFB) is a widely used insecticide to control ectoparasites in fish farming. Although therapeutic concentrations (i.e., 50 to 100 mg/L) are safe as they fail to induce mortality, they can promote tissue changes. In Brazil, Pacu (Piaractus mesopotamicus) is a native species used for commercial production, and it remains crucial to determine underlying mechanisms to mitigate the potential effects of pathogens on productivity. The aim of this study was to analyze the transaminase profile and histopathological changes in the liver of P. mesopotamicus exposed to a DFB bath. Hence, the fish were exposed to an immersion bath containing a 70 mg/L nominal concentration of Difluchem 240 SC® (24% (m/m) DFB) for 30 (n = 10), 60 (n = 10), and 120 min (n = 10), every 24 h for 3 days. Following exposure, plasma transaminases and liver histology were analyzed. In DFB-exposed fish, levels of aspartate transaminase (AST) and alanine transaminase (ALT) were elevated when compared with the control at 30 and 60 min. Furthermore, liver morphology was altered based on exposure times. Compared with controls, the degree of reversible damage (degree of tissue change (DTC)) demonstrated high scores for all exposure times, with no difference between individual groups. Irreversible changes were increased in the 60 and 120-min baths. These findings highlight the impact of the therapeutic DFB concentration (i.e., 70 mg/L), revealing that 60-min and 120-min bathing induces irreversible and progressive hepatic changes.

Testicular morphometric changes in neotropical anurans from agroecosystems.

One of the causes of the global decline of amphibians is agricultural activity, responsible for causing habitat fragmentation and bringing a range of agrochemicals and fertilizers in the environment, compounds with a potential disrupting effect on non-target organisms, such as frogs. Exposure to these compounds has numerous harmful effects on the testes of these animals, which can compromise reproduction and, consequently, the maintenance of their communities. In this context, we compared the morphology and morphometry of the testes of three species of neotropical anurans (Physalaemus cuvieri, Dendropsophus minutus, and Boana albopunctata) from an agricultural area and a conservation unit. Histologically, the testicular morphology of the species was similar for both environments; however, morphometrically, there was a difference in the measured testicular parameters (locular area and area of spermatogenic cysts). Physalaemus cuvieri presented higher averages of locular and spermatogonial area in the agricultural environment, whereas the area occupied by the spermatozoa was smaller. Additionally, the testicular pigmentation, which is only present in this species, was greater in animal from the agricultural area. In D. minutus, the locular, spermatogonial, and sperm areas showed lower values in the agricultural area, whereas in B. albopunctata, the opposite pattern was found, with the area of the locule, spermatocytes, and spermatozoids being higher. Agricultural activities influence the testicular metric parameters in different species, and our results suggest that D. minutus is most sensitive to anthropic pressures. The least sensitive species is B. albopunctata. We highlight the importance of evaluating different species, since each species responds differently to agricultural activities.

Comparative liver morphology associated with the hepatosomatic index in five Neotropical anuran species.

The liver is an important metabolic organ in vertebrates. In anurans, the hepatosomatic index (HSI) reflects differences in energy storage and reproductive activities between males and females. The objective of this study was to describe the histological and histometric parameters of the livers of five species of Neotropical anurans, taking sex-related differences into account. We also tested how the relationship between quantitative histometric variables and HSI varied between males and females in different species. Five males and five females of Elachistocleis matogrosso, Leptodactylus podicipinus, Lysapsus limellum, Pseudis platensis, and Trachycephalus typhonius were captured in central Brazil during the rainy season. HSI did not vary according to sex, but it varied among species. Elachistocleis matogrosso had the highest HSI due to the large hepatocyte size. The percentage of melanomacrophage centers (MMCs) was higher in P. platensis and L. limellum. In T. thyphonius, hepatocyte area was negatively associated with HSI, while the MMC percentages were positively associated with HSI. The liver plays a key role in reproductive activities, especially for species with explosive reproduction. Additionally, histometric patterns and volumetric structural density varied between males and females due to energy utilization for reproduction. Not only are these results important for future studies on hepatic morphophysiology but they also provide tools for evolutionary and phylogenetic studies.

Hepatotoxicity of the anionic surfactant linear alkylbenzene sulphonate (LAS) in bullfrog tadpoles.

The liver of anurans play an important role in metabolism, including detoxification, the biotransformation of molecules, and the storage of metabolites. Surfactants are part of domestic and industrial effluents. The effects of linear alkylbenzene sulfonate (LAS) on anuran liver remain unknown, however, some studies have evaluated the effects of LAS on the skin, gills, heart, testes, and liver of fishes. Here, we tested the hypothesis that LAS is hepatotoxic, promoting morphometric alterations in hepatocytes along with inflammation in the tissue, altering hepatic catabolism. We evaluated the effects of a LAS concentration that is considered environmentally safe in Brazilian inland waters on the liver of Lithobates catesbeianus tadpoles, including studies on morphology, morphometry, immunology, and metabolism. LAS exposure promoted enlargement of liver sinusoids and vacuolization of hepatocytes. Exposure to LAS also increased the area of mast cells and melanomacrophages (MMs). Additionally, LAS exposure increased hemosiderin inside MMs, suggesting alterations in the catabolism and storage of iron. Hepatocyte size increased after exposure to LAS, suggesting cytotoxic effects. Integrative analyses (i.e., morphometric, metabolic, and immunological) demonstrated hepatotoxic effects of LAS. These types of studies are key to understanding the negative effects of these substances on tadpole health, as these liver alterations impair anuran homeostasis.

Age-associated hemogram and ultrastructural leukocyte morphology in Pygocentrus nattereri (Characiformes: Serrasalmidae) from the Brazilian Pantanal

Pygocentrus nattereri is a carnivorous fish widely distributed in the Brazilian wetland and occupies different river strata. Based on the sexual maturity, the standard length (SL) has been used to categorize age and physiological attributes. However, hematological parameters and their relationship with age and growth features are unknow. Here, the aim was to evaluate the hematological profile and leukocyte data of juvenile and adult specimens, associating it with SL. Specimens of both sexes were weighted, and SL were measured for classify as juveniles (SL ≤16.0 cm) and adults (SL ≥16.1 cm). Blood was collected to perform the hemogram, for leukocyte quantification and ultrastructural descriptions. In general, hemogram values and leukocyte, lymphocyte, and monocyte counts were high in juveniles. Hemogram variables were negatively correlated with SL. Thrombocyte and neutrophil counts were higher in adults than in juveniles. SL was correlated with leukogram variables. Ultrastructural analysis showed that granular leukocyte count in P. nattereri was similar another fishes. PAS-positive granular leukocytes are slightly smaller than neutrophils and are characterized by dense elongated and semi-lunar shapes in the cytoplasm. Our results suggest that age-based SL may be useful for the analysis of the relationship between P. nattereri health status and wetland aquatic environments.(AU)

Pygocentrus nattereri é um peixe carnívoro amplamente distribuído no pantanal brasileiro e ocupa diferentes estratos de rios. Com base na maturidade sexual, o comprimento padrão (CP) tem sido usado para categorizar a idade e os atributos fisiológicos. No entanto, os parâmetros hematológicos e sua relação com a idade e características de crescimento são desconhecidos. O objetivo foi avaliar o perfil hematológico e leucocitário de espécimes juvenis e adultos, associando-o ao CP. Espécimes de ambos os sexos foram pesados e os CPs foram medidos para classificação em juvenis (CP ≤16,0 cm) e adultos (CP ≥16,1 cm). Foi coletado sangue para realização do hemograma, perfil e morfologia dos leucócitos. Em geral, os valores do hemograma e as contagens de leucócitos, linfócitos e monócitos foram maiores nos jovens. A contagem de trombócitos e neutrófilos foi maior em adultos do que em jovens. A análise ultraestrutural mostrou que a contagem de leucócitos granulares em P. nattereri foi semelhante a outros peixes. Os leucócitos granulares PAS-positivos são menores do que os neutrófilos, sendo caracterizados por formas alongadas e semilunares densas no citoplasma. Nossos resultados sugerem que CP é útil para a análise da relação entre o estado de saúde de P. nattereri em ambientes aquáticos alagados.(AU)

Differential liver histopathological responses to amphibian chytrid infection.

Amphibians have been facing a pandemic caused by the deadly fungus Batrachochytrium dendrobatidis (Bd). Although studies have elucidated cutaneous and homeostatic disturbances, it is still unknown if the hepatic function can be affected or if hepatic effects differ among host species. Thus, we evaluated the effects of an experimental Bd infection on the liver (histopathology and the hepatosomatic index) of 2 anuran species (Xenopus laevis and Physalaemus albonotatus) with different susceptibilities to Bd infection and compared them to uninfected controls. Bd infection increased the melanomacrophage cell area and induced leukocyte infiltration in both species. The effects were more pronounced in the sensitive species, P. albonotatus, which showed severe reduction in glycogen stores and liver atrophy, due to energetic imbalance. Hepatocytes of P. albonotatus also showed ballooning degeneration (vacuolization), which could lead to cell death and liver failure. Our results provide evidence that although the sensitive species showed more severe effects, the tolerant species also had hepatic responses to the infection. These findings indicate that hepatic function can play an important role in detoxification and in immune responses to chytridiomycosis, and that it may be used as a new biomarker of health status in chytrid infections.

Idiosyncratic liver pigment alterations of five frog species in response to contrasting land use patterns in the Brazilian Cerrado.

BACKGROUND: Changes in land use trigger environmental changes that can lead to decreased biodiversity and species loss. The liver is an essential detoxification organ that reflects systemic physiological responses to environmental changes. Here, we tested whether contrasting land use patterns influence the amount of substances from the hepatic cellular catabolism and melanomacrophages (MMs) of five anuran species in the Brazilian Cerrado. METHODS: We collected the same five species of pond-dwelling frogs in one protected area and in an area with intense agricultural activity. We used routine histological and histochemical techniques to quantify the area occupied by lipofuscin, melanin, and hemosiderin in the liver of two frogs Leptodactylus fuscus, Physalaemus cuvieri, and three tree-frogs Dendropsophus minutus, Scinax fuscomarginatus, and Boana albopunctata. We classified land use types in a buffer around each pond based on satellite images. We then used a double-constrained Correspondence Analysis, a recently developed ecological method to relate functional traits to environmental variables, to test the effect of each land use type on the area of each liver pigment. RESULTS: There was an increase in the amount of melanin in environments with high proportion of agriculture, as well as variation in the amount of lipofuscin and hemosiderin. Liver pigments of P. cuvieri and B. albopunctata varied more strongly in response to land use types, suggesting they could be good indicator species. Therefore, the area of MMs in the liver and the metabolic products in their cytoplasm can be used as biomarkers of environmental changes in regions with intense agricultural activities. Our results add a new perspective to the influence of land use patterns on environmental health by highlighting the effect of environmental changes on internal morphological aspects of animals.