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.

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.

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.

Exposure of Physalaemus cuvieri (Anura) to benzo[a]pyrene and α-naphthoflavone: Morphofunctional effects on hepatic melanomacrophages and erythrocytes abnormalities.

Benzo[a]pyrene (BaP) is a high-risk contaminant of elevated toxicity. Its biotransformation process occurs as the expression of CYP1A1 increases and produces toxic metabolites. In turn, α-naphthoflavone (aNF) represents an inhibitor of CYP1A1, preventing BaP metabolism. Toxicological studies in anurans show alterations in the melanomacrophage (MM) detoxification cell after exposure to xenobiotics. In this study, the production of melanin by MMs was evaluated, as were morphological alterations in the cytoskeleton, phagocytosis and the genotoxicity effects after exposure of an anuran species to BaP and aNF. Physalaemus cuvieri received subcutaneous injections of 2 mg/kg and/or 20 mg/kg aNF. For phagocytosis analyses, animals received an intraperitoneal injection with 0.4% trypan blue. The results revealed that melanin synthesis increased by 503.2% in animals exposed to BaP after 48 h, which was related to the antioxidant action of melanin, whereas the decreased in synthesis of 25.6% with the BaP + aNF interaction resulted in high toxicity to MMs and cell degeneration. The phagocytic activity reduced to 37.6% in animals exposed to BaP, characterizing a functional impairment; however, the BaP + aNF interaction led to the restoration of phagocytosis, reaching 419.23%. The decreased rate or absence of abnormalities may be explained by the fact that only the less damaged erythrocytes remained in the bloodstream, whereas the most damaged cells died. In conclusion, BaP and aNF are toxic to P. cuvieri, bringing risks to herpetofauna.

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.

Enzymatic, morphological, and genotoxic effects of benzo[a]pyrene in rainbow trout (Oncorhynchus mykiss).

Fish have defense systems that are capable of repairing damages caused by xenobiotics like benzo[a]pyrene (BaP), so the aims of this study were to identify BaP toxicity in melanomacrophages (MMs) cytoskeleton, evaluate the melanin area in MMs, and analyze genotoxicity. Rainbow trout juveniles (n = 24) were split in 48h and 7d treatments that received 2 mg/kg of BaP. After the experiment, blood samples were collected and liver was removed, to proceed with the analysis: EROD activity, MMs melanin area quantification, melanosomes movements, and a genotoxicity test. The results revealed increased in EROD activity after 48-h and 7-day BaP exposure. The group 7d displayed a reduction in MMs pigmented area, melanosomes aggregation, in addition to an increased frequency of micronucleus. By means of the EROD assay, it was possible to confirm the activation of BaP biotransformation system. The impairment of the melanosomes' movements possibly by an inactivation of the protein responsible for the pigment dispersion consequently affects the melanin area and thus might negatively impact the MMs detoxification capacity. In addition to this cytotoxicity, the increased frequency of micronucleus might also indicate the genotoxicity of BaP in this important fish species.

Ecotoxicological evaluation of water from the Sorocaba River using an integrated analysis of biochemical and morphological biomarkers in bullfrog tadpoles, Lithobates catesbeianus ().

Lithobates catesbeianus tadpoles were exposed for 96 h to water from two sites of the Sorocaba River (summer and winter), Ibiúna (PI) and Itupararanga reservoir (PIR) that contained metals. In the liver, in PI, the glutathione peroxidase (GPx) decreased, and the glutathione S-transferase (GST) and carbonyl proteins (PCO) increased. In PIR, the glutathione reduced (GSH) increased, while there was a decrease in catalase (CAT), GPx, GST, PCO, and superoxide dismutase (SOD). In winter, GPx and GST increased in both points. Regarding the kidneys, lipoperoxidation (LPO) levels and GST decreased, while GSH increased in the summer. In the winter, LPO increased in PI. In the muscle, in the summer, there was an increase in GSH and GST and change in PCO. In the winter, the levels of PCO increased and CAT decreased in PIR. The area and volume of the hepatocyte and nucleus area increased in the summer and decreased in the winter. Hepatic melanin decreased in the summer after exposure to PIR water. There were the systemic effects of Sorocaba River water exposure at different times of the year with alterations in biomarkers at different levels, in which kidney shows highest Integrated Response of Biomarkers (IBR) value followed by liver and muscle. Biochemical biomarkers were more sensitive than morphological ones. The more sensitive biochemical markers were MT, PCO, GST and LPO. These effects confirm the hypothesis of metabolic alteration in bullfrog tadpoles by the Sorocaba River water.

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.

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.

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.

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.

Gonadal, body color, and genotoxic alterations in Lithobates catesbeianus tadpoles exposed to nonylphenol.

Endocrine disrupting chemicals are one of the most important factors contributing to worldwide amphibian decline. The 4-nonylphenol (NP) is a degradation product of several compounds, such as detergents and pesticides, affecting the aquatic environment. Here, we test whether treatment with NP has an effect on developing ovarian tissue, nuclear abnormalities in erythrocytes, and body darkness in pre-metamorphic tadpoles of the bullfrog Lithobates catesbeianus. Tadpoles were exposed for 14 days to three different concentrations of NP (1, 10, and 100 µg/L) besides the control group, which was maintained only with water. After determining body coloration, animals were euthanized and gonads and blood were collected and processed for histology and genotoxic analysis. Even though most animals were females, intersex tadpoles were observed in control and treated groups and there were no males in any group. The highest concentration of NP showed an increase in atretic oocytes, but the area corresponding to somatic compartment and early and late germ cells were not affected. Furthermore, all treated groups presented higher amount of nuclear abnormalities in erythrocytes and body darkening when compared with the control group. These results suggest that NP causes genetic damage and morphological alterations in L. catesbeianus tadpoles by disrupting oogenesis, inducing genotoxicity and increasing body coloration. Its effects on gonadal development could cause future impairments in reproduction, while its deleterious effects on genotoxicity and body pigmentation could be used as a biomarker of effect to this compound.

Genotoxic Evaluation in Tadpoles Associated with Agriculture in the Central Cerrado, Brazil.

Many agricultural practices cause environmental degradation that affects the cellular integrity of anurans. In the present study, we provided in situ data of Dendropsophus minutus, Physalaemus cuvieri, and Scinax fuscovarius collected in soybean/corn and conservation units in the Brazilian Cerrado. The in situ data showed no significant variation in the micronucleus frequency between the sites, only the reniform cells had a higher rate for the agricultural environment. A combined analysis of all nuclear erythrocyte abnormalities (ENAs = nuclear buds, reniform nuclei, apoptotic cell, binucleated, and anucleated cells) was recorded higher frequencies in farmland. Overall, Scinax fuscovarius was considered the best potential bioindicator for soybean/corn plantations. Finally, we recommend expanding the micronucleus test for in situ studies to expand our understanding of the sensitivity of native anuran species and provide a more systematic assessment of the adverse effects of environmental pollutants on wildlife.

Monitoring the morphological integrity of neotropical anurans.

Amphibians are considered to be excellent bioindicators to their morphophysiological characteristics and life cycle. In this context, the present study investigated the morphological integrity of anuran larvae collected in preserved environments in the Emas National Park, in the municipality of Mineiros (Goiás state, Brazil), and in environments representative of the agricultural matrix of the Rio Verde region, also in Goiás, where there is a long history of the use of agricultural pesticides. Samples of water from temporary ponds, permanent dams, and veredas were analyzed for the presence of pesticides and, especially atrazine (5350 µg/L), found at significantly higher concentrations in the agricultural matrix. We observed a high percentage (approximately 10%) of morphological malformations including alterations of the fins in Boana albopunctatus and Scinax fuscovarius; alteration in oral structures in B. albopunctatus, Dematonotus muelleri, Physalaemus centralis, Physalaemus cuvieri, and Leptodactylus fuscus mainly in the tadpoles collected in the agricultural environment in comparison with those from the protected area (3.5%; P < 0.0001, χ2 31.75). However, changes in the eyes, mouth, intestines, and nostrils, as well as amelia were observed only in the agricultural environment. The vast majority of the observed malformations were associated with the tail and oral disc, which suggests that these anatomical parameters may be used as sensitive morphological biomarkers. Given these findings, we reinforce that areas of agricultural land may have a deleterious effect on the morphological integrity of the tadpoles and consequently, on their development, and that these features may be used as indicators of environmental quality and health.

Biomarkers at different levels of organisation after atrazine formulation (SIPTRAN 500SC®) exposure in Rhinella schineideri (Anura: Bufonidae) Neotropical tadpoles.

Brazil is an important consumer of herbicides. In sugarcane cultivation-the country's most extensive agricultural crop-atrazine-based formulations are the principal form of weed control. Several studies have investigated adverse effects of atrazine or their formulations on anurans, but not specifically on Brazilian species. Our aim was therefore to investigate the lethal and sublethal effects of an atrazine-based herbicide in Rhinella schneideri tadpoles and, in particular, effects on the pigmentation system as a new endpoint in ecotoxicological studies. Rhinella schneideri tadpoles at the Gosner-30 stage were exposed to the atrazine-based herbicide formulation, SIPTRAN 500 SC®, in acute bioassays at concentrations of 1.5-25 mg/L. The lethal and sublethal effects induced were analysed at different ecotoxicological levels: organismal level (alterations in behaviour, growth, development, and body mass; morphologic abnormalities), histological level (liver histopathology), the pigmentation system (melanomacrophages and dermal-melanophores), and cellular level (erythrocyte micronucleus formation and other nuclear-abnormalities). This herbicide induced sublethal effects at the organismal level with alterations in swimming and growth and morphologic abnormalities. These results demonstrated that, in anuran tadpoles, the atrazine-based agrochemical increased the frequency of micronucleus formation and other nuclear-abnormalities in erythrocytes and caused liver damage. In addition, we demonstrated for the first time effects of an atrazine-based formulation on the pigmentation system of anuran tadpoles, specifically an increase in the number of melanomacrophages and dermal melanophores. This study is the first to use several widely differing endpoints at different ecotoxicological levels in a comprehensive manner for assessment of the effects of environmental stressors in order to determine the health status of Neotropical anuran species. In doing so, this study establishes a foundation for future ecological assessments.

How Does Oocyte Uptake Occur? A Macroscopic Study of the Ovarian and Oviductal Modifications for Egg Capture in the Coral-Snake Micrurus corallinus.

The processes of follicular development, ovulation, egg capture, and egg transport vary among vertebrates. Therefore, analysis of the reproductive tract of several lineages is needed for understanding the evolutionary changes of the reproductive system. In turtles, the ovulated eggs are released into the coelomic cavity and taken up by one of the two oviducts, a phenomenon called extrauterine migration of eggs. However, the process of egg uptake in lizards is different. The egg is ovulated directly into the infundibulum, and oocyte uptake by the contralateral oviduct rarely occurs. The same pattern has been hypothesized to occur in snakes. To test this hypothesis, we analyzed the reproductive tract of female Micrurus corallinus preserved in zoological collections. We examined the anatomical characteristics of the infundibulum and ovary and compared the reproductive output between the ovaries to verify the mechanism of egg capture and the anatomical viability of extrauterine migration of eggs. The reproductive output of the right ovary was higher than that of the left ovary, and the higher number of eggs in the right oviduct is due exclusively to the production of the ipsilateral ovary. Several anatomical features prevent extrauterine migration of eggs, including the asymmetry of the reproductive system, the arrangement of the ovarian follicles in a single row, and the formation of a wrapping around the ovary and infundibulum by the visceral pleuroperitoneum membrane (preventing against ectopic eggs). Therefore, the hypothesis of egg capture by the contralateral oviduct is anatomically infeasible in M. corallinus and possibly in other snakes. Anat Rec, 301:1936-1943, 2018. © 2018 Wiley Periodicals, Inc.

Effects of benzo[a]pyrene on the blood and liver of Physalaemus cuvieri and Leptodactylus fuscus (Anura: Leptodactylidae).

Benzo[a]pyrene (BaP) is a bio-accumulative toxic compound found in the atmosphere, water, and soil that may affect the life cycle of amphibians. In this study, a few contamination biomarkers, such as hepatic melanomacrophages (MMs), mast cells, erythrocyte micronuclei (MN) and white blood cells were used to determine how BaP acts in these cells in the anurans Physalaemus cuvieri and Leptodactylus fuscus. Animals of both species were divided into three treatment groups: 1 day, 7 days and 13 days, subcutaneously injected 2 mg/kg BaP diluted in mineral oil and control group with only mineral oil. After 7 days, BaP caused the frequency of MN to increase in both species while reducing melanin area. The micronucleus frequency increased due to the genotoxicity of BaP, while the decreasing melanin area may be related to the inhibition of tyrosinase activity, an enzyme responsible for regulating melanogenesis, decreasing the synthesis of melanin. The mast cell density increased in all groups and in both species as a response to the inflammatory action of BaP. These cells respond to nonspecific inflammatory effects leading, therefore, to this response in all treatments. The percentage of leukocytes remained unchanged probably due to great intraspecific variability. Additionally, the leukocyte profiles of both species were characterized and the differences were attributed to extrinsic factors. In short, BaP can affect the integrity of several organs and tissues, and cell functions leading to the conclusion that this compound is hepatotoxic, genotoxic and immunotoxic for anurans.