Microplastics and TiO2 nanoparticles mixture as an emerging threat to amphibians: A case study on bullfrog embryos.

Emerging contaminants can act as contributing factors to the decline of amphibian populations worldwide. Recently, scientists have drawn attention to the potential ecotoxicity of microplastics and nanomaterials in amphibians, however, their possible effects on embryonic developmental stages are still absent. Thus, the present study analyzed the developmental toxicity of environmentally relevant concentrations of polyethylene microplastics (PE MPs; 60 mg/L) and titanium dioxide nanoparticles (TiO2 NPs; 10 µg/L), isolated or in combination (Mix group) on bullfrog embryos, Aquarana catesbeiana, adapting the Frog Embryo Teratogenesis Assay (FETAX, 96h). Allied to the FETAX protocol, we also analyzed the heart rate and morphometric data. The exposure reduced the survival and hatching rates in groups exposed to TiO2 NPs, and to a lesser extent, also affected the Mix group. TiO2 NPs possibly interacted with the hatching enzymes of the embryos, preventing hatching, and reducing their survival. The reduced effects in the Mix group are due to the agglomeration of both toxicants, making the NPs less available for the embryos. PE MPs got attached to the gelatinous capsule of the chorion (confirmed by fluorescence microscopy), which protected the embryos from eventual direct effects of the microplastics on the hatching and survival rates. Although there were no cardiotoxic effects nor morphometric alterations, there was a significant increase in abdominal edemas in the hatched embryos of the PE MPs group, which indicates that osmoregulation might have been affected by the attachment of the microplastics on the embryos' gelatinous capsule. This study presents the first evidence of developmental toxicity of environmental mixtures of microplastics and nanoparticles on amphibians and reinforces the need for more studies with other amphibian species, especially neotropical specimens that could present bigger sensibility. Our study also highlighted several features of the FETAX protocol as useful tools to evaluate the embryotoxicity of several pollutants on amphibians.

Mutagenicity, hepatotoxicity, and neurotoxicity of glyphosate and fipronil commercial formulations in Amazon turtles neonates (Podocnemis expansa).

Pesticides are considered one of the main causes of the population decline of reptiles worldwide, with freshwater turtles being particularly susceptible to aquatic contamination. In this context, we investigated the potential mutagenic, hepatotoxic, and neurotoxic effects in neonates of Podocnemis expansa exposed to substrate contaminated with different concentrations of glyphosate and/or fipronil during embryonic development. Eggs collected from the natural environment were artificially incubated in sand moistened with pure water, water added with glyphosate Atar 48® at concentrations of 65 and 6500 µg/L (groups G1 and G2, respectively), water added with fipronil Regent® 800WG at 4 and 400 µg/L (groups F1 and F2, respectively) and, water added with the combination of 65 µg/L glyphosate and 4 µg/L fipronil or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). For mutagenicity analysis, we evaluated the frequency of micronuclei (MN) and other erythrocyte nuclear abnormalities (ENAs), while for evaluation of hepatotoxicity and neurotoxicity, livers and encephalon were analyzed for histopathological alterations. Exposure to pesticides, alone or in combination, increased the frequency of erythrocyte nuclear abnormalities, particularly blebbed nuclei, moved nuclei, and notched nuclei. Individuals exposed to fipronil exhibited congestion and inflammatory infiltrate in their liver tissue, while, in the encephalon, congestion, and necrosis were present. Our study confirms that the incubation of eggs in substrate polluted with glyphosate and fipronil causes histopathological damage and mutagenic alteration in P. expansa, highlighting the importance of using different biomarkers to evaluate the ecotoxicological effects of these pesticides, especially in oviparous animals.

Eggshell composition of Amazon turtle (Podocnemis expansa) is altered after incubation in substrates containing glyphosate and fipronil formulations.

Besides its crucial role during embryo development, eggshells are an essential bioindicator of environmental contaminants. However, the effects of contaminant exposure during incubation on the eggshell composition remain poorly known for freshwater turtles. Accordingly, we tested the effects of incubating the eggs of Podocnemis expansa in substrates containing glyphosate and fipronil formulations on the eggshell's level of mineral and dry matter, crude protein, nitrogen, and ethereal extract. Eggs were incubated in sand moistened with water contaminated with glyphosate Atar 48 at concentrations of 65 or 6500 µg/L, fipronil Regent 800 WG at concentrations of 4 or 400 µg/L, or the combination of 65 µg/L glyphosate and 4 µg/L fipronil and 6500 µg/L glyphosate with 400 µg/L fipronil. Exposure to the tested pesticides, alone or in association, altered the chemical composition of the eggshell of P. expansa, reducing the eggshell's moisture and crude protein content and increasing levels of ethereal extract. These changes may cause significant deficiencies in the mobilization of water and nutrients to the embryo, affecting the development and reproductive success of P. expansa.

Plastic microfibers as a risk factor for the health of aquatic organisms: A bibliometric and systematic review of plastic pandemic.

Plastic microfibers (PMFs) are emerging pollutants widely distributed in the environment. In the early 2020s, the need for personal protection due to the COVID-19 pandemic led to increased consumption of plastic materials (e.g., facemasks and gloves) and ultimately to increased plastic pollution, especially by PMFs. The PMFs present in the environment may be released in this form (primary particles) or in larger materials, that will release them as a result of environmental conditions. Although a considerable number of studies have been addressing the effects of microplastics, most of them studied round particles, with fewer studies focusing on PMFs. Thus, the current study aimed to summarize and critically discuss the available data concerning the ecotoxicological impact of PMFs on aquatic organisms. Aquatic organisms exposed to PMFs showed accumulation, mainly in the digestive tract, and several toxic effects, such as DNA damage, physiological alterations, digestive damage and even mortality, suggesting that PMFs can pose a risk for the health of aquatic organisms. The PMFs induced toxicity to aquatic invertebrate and vertebrate organisms depends on size, shape, chemical association and composition of fibers. Regarding other size range (nm) of plastic fibers, the literature review highlighted a knowledge gap in terms of the effects of plastic nanofibers on aquatic organisms. There is a knowledge gap in terms of the interaction and modes of action of PMFs associated with other pollutants. In addition, studies addressing effects at different trophic levels as well as the use of other biological models should be considered. Overall, research gaps and recommendations for future research and trends considering the environmental impact of the COVID-19 pandemic are presented.

Assessment of multiple biomarkers in Lithobates catesbeianus (Anura: Ranidae) tadpoles exposed to zinc oxide nanoparticles and zinc chloride: integrating morphological and behavioral approaches to ecotoxicology.

The ecotoxicological risk to vertebrates posed by zinc oxide nanoparticles (ZnO NPs) is still poorly understood, especially in animals with a biphasic life cycle, which have aquatic and terrestrial phases, such as amphibians. In the present study, we investigated whether acute exposure (7 days) to ZnO NPs and zinc chloride (ZnCl2) at three environmentally relevant concentrations (0.1, 1.0, and 10 mg L-1) induces changes in the morphology, chondrocranium, and behavior of the tadpoles of Lithobates catesbeianus (Anura: Ranidae). Tadpoles exposed to both forms of Zn did not undergo any morphological or behavioral changes at the lowest concentrations (0.1 and 1.0 mg L-1). However, the animals exposed to the highest concentration (10 mg L-1) lacked oral disc structures, were smaller in size, had a longer tail, and presented changes in the position and coiling of the intestine and malformations of the chondrocranium in comparison with the control group. This indicates that ZnO NPs and ZnCl2 altered the development of the tadpoles, causing delays in their metamorphosis and even reducing individual fitness. The tadpoles exposed to both forms of Zn at 10 mg L-1 also had reduced mobility, especially in the presence of conspecifics. Based on these findings, we emphasize the importance of studying morphological, skeletal, and behavioral biomarkers to evaluate the toxic effects of metal-based nanoparticles in amphibians.

Prenatal effects of alprazolam treatment on the immature cerebellum of rats.

The use of benzodiazepines (BZDs) during pregnancy, especially alprazolam, is common and its impact on the fetal neural tissue is not known. In this sense, the present study aimed to investigate the effects of prenatal treatment with alprazolam on the cerebellum of Wistar rat pups. Thirty animals (24 females and six males, CEUA protocol 014/17) were separated into pairs for copulation. Females were divided into three groups: Control (CT), treatment 1 (T1, 1.25 mg per animal), and treatment 2, which is an overdose (T2, 30 mg per animal). Alprazolam was administered 10 days before copulation and throughout pregnancy. We evaluated the number and weight of pups and the macroscopic changes in the brain. Eight neonates (n = 8) from each group were used in the following analyses: Cellular and chromatin density, gliosis, synaptic density, inflammation, and oxidative stress. The results showed no significant differences regarding the number of pups, body weight, and macroscopic changes. The morphological study focused on the external granular layer (EGL) that is presented only in the immature cerebellum. Here, we detected more cells after alprazolam treatment; the T2 group showed large nuclei and some pyknotic nuclei; also, both treated groups presented an increase in the euchromatin density compared with the control. The molecular and biochemical analyses used the total protein extract of the entire cerebellum and showed an increased expression of Iba-1 and NF-κBp65 but without indication of inflammation or degeneration in the T1 group. Overdose of alprazolam presented an increased level of oxidative degradation of lipids. The treatment with alprazolam during pregnancy involved cellular and molecular changes in the immature cerebellum.

The exposure in ovo of embryos belonging to Amazonian turtle species Podocnemis expansa (Testudines) to commercial glyphosate and fipronil formulations impairs their growth and changes their skeletal development.

Pesticides are widely used in agricultural production; moreover, they can have direct and indirect effect on both flora and fauna. Aquatic organisms, among other animals, including reptiles, are mainly susceptible to contamination effects. Accordingly, the aim of the present study is to test the hypothesis that the incubation of Podocnemis expansa eggs in substrate added with glyphosate and fipronil formulations changes their viability, interferes with their growth and induces bone alterations. Eggs collected in natural environment were artificially incubated in sand moistened with water added with glyphosate Atar 48, at concentrations of 65 or 6500 µg/L (groups G1 and G2, respectively), and with fipronil Regent 800 WG at 4 or 400 µg/L (groups F1 and F2, respectively) or, yet, with the combination of 65 µg/L glyphosate and 4 µg/L fipronil, or with 6500 µg/L glyphosate and 400 µg/L fipronil (groups GF1 and GF2, respectively). The level of exposure to the herein assessed pesticides was quantified at the end of the incubation period; it was done by dosing its concentration in eggshells. Eggs exposed to the tested pesticides did not have their viability affected by it; however, all embryos exposed to the tested pesticides showed lowered body mass at hatch, as well as impaired development. In addition, bone malformation in the scleral ossicular ring was observed in individuals in groups F1, F2 and GF1. Pesticides accumulated in eggshells at concentrations related to exposure level. Thus, the recorded results have evidenced some remarkably relevant, and previously unknown, impacts associated with the exposure of a species listed as lower risk/conservation dependent, which spends most of its life in the water, to two widely used pesticides, at a very sensitive stage of its life, namely: egg incubation on land.

Comparative developmental toxicity of iron oxide nanoparticles and ferric chloride to zebrafish (Danio rerio) after static and semi-static exposure.

Iron oxide nanoparticles (IONPs) are used in several medical and environmental applications, but their mechanism of action and hazardous effects to early developmental stages of fish remain unknown. Thus, the present study aimed to assess the developmental toxicity of citrate-functionalized IONPs (γ-Fe2O3 NPs), in comparison with its dissolved counterpart, in zebrafish (Danio rerio) after static and semi-static exposure. Embryos were exposed to environmental concentrations of both iron forms (0.3, 0.6, 1.25, 2.5, 5 and 10 mg L-1) during 144 h, jointly with negative control group. The interaction and distribution of both Fe forms on the external chorion and larvae surface were measured, following by multiple biomarker assessment (mortality, hatching rate, neurotoxicity, cardiotoxicity, morphological alterations and 12 morphometrics parameters). Results showed that IONPs were mainly accumulated on the zebrafish chorion, and in the digestive system and liver of the larvae. Although the IONPs induced low embryotoxicity compared to iron ions in both exposure conditions, these nanomaterials induced sublethal effects, mainly cardiotoxic effects (reduced heartbeat, blood accumulation in the heart and pericardial edema). The semi-static exposure to both iron forms induced high embryotoxicity compared to static exposure, indicating that the nanotoxicity to early developmental stages of fish depends on the exposure system. This is the first study concerning the role of the exposure condition on the developmental toxicity of IONPs on fish species.

Mutagenic, genotoxic and morphotoxic potential of different pesticides in the erythrocytes of Podocnemis expansa neonates.

Despite the damaging effects of pesticides glyphosate (Gly), atrazine (Atra) and fipronil (Fip) on different organisms, the mutagenic, genotoxic and morphotoxic potential of testudine erythrocytes in freshwater remains unknown. Thus, the aim of the present study is to assess the toxicological potential of these compounds in Podocnemis expansa (Amazonian turtles) neonates from eggs artificially incubated in substrate at different concentrations of herbicides Gly and Atra and insecticide Fip. Micronucleus test and other nuclear abnormalities, as well as comet assay and morphometric measurements taken of models' circulating erythrocytes were used as toxicity biomarkers. Pups exposed to Gly (groups Gly-65 ppb and Gly-6500 ppb) were the ones recording the largest amount of nuclear abnormalities; erythrocytes with multilobulated, notched and displaced nucleus were mostly frequent in groups Atra-2 ppb and Gly -65 ppb. All treatments (Gly-6500 ppb, Atra-2 ppb, Atra-200 ppb, Fip-4 ppb and Fip-400 ppb), except for group Gly-65 ppb, led to decreased erythrocyte area, increased "nuclear area: erythrocyte area" ratio, as well as to decreased erythrocyte and erythrocyte nuclei circularity, which highlights the clear effect on the size and shape of these cells. On the other hand, the comet assay did not evidence any genotoxic effect caused by the assessed pesticides. This is a pioneer study on the mutagenic and morphotoxic potential of pesticides in P. expansa eclodides exposed in ovo to Gly, Atra and Fip; therefore, it is an insight on how these compounds can affect the health of these animals.

Effects of egg exposure to atrazine and/or glyphosate on bone development in Podocnemis unifilis (Testudines, Podocnemididae).

This study was designed to investigate skeletal changes in Podocnemis unifilis embryos derived from artificially incubated eggs exposed to different concentrations of atrazine, glyphosate or atrazine and glyphosate mixture. Forty-two eggs were randomly allocated to one of seven trays containing vermiculite treated distilled water (control group) or the following solutions: 2 or 200 µg L-1 of atrazine (groups A1 and A2 respectively); 65 or 6500 µg L-1 of glyphosate (groups G1 and G2 respectively); 2 µg L-1 and 65 µg L-1 or 200 µg L-1 and 6500 µg L-1 of atrazine and glyphosate mixture (groups AG1 and AG2 respectively). Three eggs per tray were randomly collected on days 30 and 50 of the incubation period. Embryos were submitted to soft tissue diaphanization and stained with Alizarin red S or Alcian blue for morphological analysis of bone and cartilage tissues; histological analysis was performed to confirm ossification changes. Findings were compared between groups. Morphological changes were limited to sclerotic ring features and number of ribs. Malformations rates differed significantly (p < 0.05) between embryos in the control and treated groups A2, AG1 and AG2. Concurrent exposure to atrazine and glyphosate did not affect the presence or severity of embryonic malformations and was not associated with appendicular skeleton changes in P. unifilis embryos. However, further studies focusing on the axial skeleton with particular emphasis on rib abnormalities are warranted.

Ontogeny of the Postcranial Axial Skeleton of Melanosuchus niger (Crocodylia, Alligatoridae).

This study proposes the description of the development of the postcranial axial skeleton, including vertebrae, gastralium, ribs, sternum, and interclavicle, in Melanosuchus niger. Six nests were marked and two eggs removed from each nest at 24-hr intervals until hatching. For posthatching evaluation, 30 hatchlings were kept in captivity and one exemplar was euthanized at three-day intervals. Samples were diaphanized using potassium hydroxide (KOH), alizarin red S, and Alcian blue. A routinely generally used method was applied for histological evaluation. It was difficult to define in which vertebrae the development of cartilaginous centers began, but it was possible to observe that this condensation advanced in the craniocaudal direction. The condensation started in the vertebral arches and was visibly stronger in the cervical and dorsal regions, advancing to the lumbar, sacral and, last, to the caudal region. The atlas showed a highly different morphology compared with the other cervical vertebrae, with a short intercenter, two neural arches, and a proatlas. The ossification process began in the body of cervical vertebrae III to VIII and alizarin retention decreased in the last vertebrae, indicating a craniocaudal direction in bone development, similar to cartilage formation. In the histological sections of gastralium and interclavicles of M. niger at several development stages, it was possible to observe that these elements showed intramembranous development. Anat Rec, 301:607-623, 2018. © 2017 Wiley Periodicals, Inc.

Ontogeny of the Appendicular Skeleton in Melanosuchus niger (Crocodylia: Alligatoridae).

The objective of the present study was to analyze chondrogenesis and the ossification pattern of the limbs of Melanosuchus niger in order to contribute with possible discussions on homology and the fusion pattern of autopodial elements and phylogeny. In the Reserva Extrativista do Lago Cuniã, Rondônia, Brazil, six nests were marked and two eggs removed from each nest at 24-hour intervals until hatching. Embryos were cleared using KOH; bone tissue was stained with alizarin red S and cartilage with Alcian blue. Routine staining with HE was also performed. In the pectoral girdle, the scapula showed ossification centers before the coracoid process. In the pelvic girdle, the ilium and the ischium were condensed as a single cartilage, although ossification took place through two separate centers, forming distinct elements in the adult. The pubis developed from an independent cartilaginous center with free end, which reflects its function in breathing. In the initial stages, the stylopodium and the zeugopodium developed from the condensation of a Y-shaped cartilage in the limbs, and differentiation of the primary axis and digital arch were observed. The greatest changes were observed in the mesopodia. In their evolution, Crocodylia underwent a vast reduction in the number of autopodial elements as a consequence of fusions and ossification of some elements. This study shows that the chondrogenesis and ossification sequences are dissociated. Moreover, the differences between M. niger and other species show clear variation in the patterns for these events in Alligatoridae.

Chemical characteristics and thickness of Podocnemis expansa post-hatching eggshells (Testudines, Podocnemididae) / Aspectos químicos e espessura da casca de ovos de Podocnemis expansa pós-eclosão (Testudines, Podocnemididae)

Knowledge on chemical components of the post -hatching eggshell of reptiles may provide indicators of the quality of the diet offered to females kept in captivity. Therefore, the objective of this study was to investigate the chemical characteristics of the calcareous layer, as well as the thickness of Podocnemis expansa post-hatching eggshells. Eggshell thickness was 183±1.405 µm. This value is similar to that of the eggs of other Testudines with flexible eggshells. As for the chemical composition, the following percentages were observed: nitrogen 7.983 ± 0.054; crude protein 49.91 ± 0.324; crude fat 0.068 ± 0.002; mineral matter 20.302 ± 0.807; calcium 13.374 ± 0.647; and phosphorus 0.176 ± 0.003. Knowledge on chemical composition of the eggshell may aid the nutrition of P. expansa raised in commercial facilities, once this species is an alternative and promising source of exotic meat.

O conhecimento sobre os constituintes químicos da casca de ovos pós-eclosão de répteis pode ser um referencial de qualidade da dieta fornecida para as fêmeas que vivem em cativeiro. Assim, este trabalho visa investigar os aspectos químicos que compõem a camada calcária, bem como a espessura da casca do ovo de Podocnemis expansa no período pós-eclosão. A espessura média da casca dos ovos foi de 183 ± 1,405 µm. Esta espessura é condizente com ovos de outros Testudines com casca flexível. Para os constituintes químicos, foram encontrados os seguintes valores médios percentuais: nitrogênio 7,983 ± 0,054, proteína bruta 49,91 ± 0,324, gordura bruta 0,068 ± 0,002, matéria mineral 20,302 ± 0,807, cálcio 13,374 ± 0,647 e fósforo 0,176 ± 0,003. Este conhecimento sobre a composição química da casca poderá auxiliar na criação em escala comercial dessa espécie, uma vez que a P. expansa tem se apresentado como fonte alternativa e promissora de carne exótica.

Effects of the exposure to atrazine on bone development of Podocnemis expansa (Testudines, Podocnemididae).

The use of pesticides is a widely spread practice in Brazilian agriculture, and dispersion of these substances is an important factor for the fauna and flora. Atrazine is an endocrine disruptor in the xenoestrogen class that is used worldwide in agricultural practices. In Brazil, its use is permitted in several crops. Podocnemis expansa is a representative of the Testudines order that is the largest freshwater reptile of South America. Its distribution enables it to get in contact with molecules that are commonly used as pesticides, which may cause deleterious effects in target populations. In order to evaluate the possible effects of the exposure to atrazine on bone ontogeny of this species, eggs were artificially incubated in sand moistened with water contaminated with atrazine at concentrations equal to 0, 2, 20 or 200 µg/L. Embryos were collected throughout incubation and submitted to diaphanization of soft tissues with potassium hydroxide (KOH); bones were stained with Alizarin red S and cartilages by Alcian blue. Embryos were evaluated for the presence of abnormalities during the different stages of pre-natal development of skeletal elements. No effect of atrazine was observed on bone development during the embryonic phase in P. expansa individuals, in the conditions of this study.

Effects of Exposure to Methyl Parathion on Egg Hatchability and Eggshell Chemical Composition in Podocnemis expansa (Testudines, Podocnemididae).

Eggshell evaluation may serve as an indicator of the effect of substances released in the environment, which may change eggshell shape, size, structure, and/or chemical composition. Additionally, exposure may interfere with hatching rates in contaminated eggs. The objective of this study was to better understand how exposure to the insecticide methyl parathion interferes with chemical changes in eggshells of Podocnemis expansa throughout their artificial incubation, as well as with egg hatchability. A total of 343 P. expansa eggs were collected in a natural reproduction area for the species. These eggs were transferred to and artificially incubated in the Wild Animal Teaching and Research Laboratory at Universidade Federal de Uberlândia. On the first day of artificial incubation, 0, 35, 350, and 3500 ppb of methyl parathion were incorporated to the substrate. Eggs were collected every three days for chemical analysis of eggshells. Hatchability was evaluated as the number of hatchlings in each treatment, for the eggs that were not used in the chemical analysis. Student's T-test was used for data on eggshell chemical composition, and the Binomial Test for Two Proportions was used in the hatchability analysis, at a 5% significance level. It was observed that the incorporation of methyl parathion to the substrate on the first day of artificial incubation of P. expansa eggs reduced the levels of total fat in the shells throughout their incubation, besides reducing egg hatchability.

Morphological aspects of the liver of the Podocnemis expansa(Testudines, Podocnemididae)

The liver of P. expansa was characterized morphohistologically. To this end, twenty livers from clinicallyhealthy male and female Podocnemis expansa, weighing from 2.0 to 4,5 kg, supplied by the commercial breederFazenda Moenda da Serra, in Araguapaz, state of Goiás, Brazil, were analyzed macro - and microscopically.The coelomatic cavity was opened and the topography of the fresh organs was examined visually. After thehistological preparation, the slides were stained with Hematoxylin and Eosin (HE), Periodic Acid-Schiff(PAS), Gomori Trichrome, Reticulin and Picrosirius. The liver of P. expansa is a voluminous organ with anapproximately rectangular shape and brown coloration, varying from light to dark shades, and is divided into aright lobe, left lobe, and a central portion. The right lobe is the largest of the three portions. The gall bladderis located in a depression in the caudal portion of the right lobe, where the gall duct begins and emptiesinto the duodenum. Histologically, the hepatocytes are arranged in the form of double cords surroundedby winding sinusoidal capillaries. In cross section, they resemble acini containing approximately two to fivehepatocytes surrounding a probable central biliary canaliculus. The hepatocytes are polyhedral or pyramidalin shape, of uniform size, with a few central nuclei and others displaced peripherally, and the cytoplasm islittle eosinophilic when analyzed by the HE staining technique. The parenchyma is supported by delicatereticular fibers surrounding hepatocytes and sinusoids. The parenchyma and perisinusoidal spaces containlarge quantities of melanomacrophages, mainly close to the portal spaces.

Esqueletogênese dos ossos da coluna vertebral, costelas e esqueleto abdominal em embriões de Caiman Yacare (Daudin, 1802) (Crocodylia: Alligatoridae) / Skeletogenesis of the vertebral column bones, ribs and inscriptional ribs in embryos of Caiman yacare (Crocodylia: Alligaroridae)

Dentre os répteis, os lagartos e os testudines são o foco de inúmeras investigações que contemplam o estudo de sua ontogenia, contudo, descrições do desenvolvimento e da sequência de ossificação em crocodilianos são escassas. Assim, objetivou-se investigar o padrão de formação e a sequência de ossificação das vértebras e costelas em Caiman yacare (DAUDIN, 1802). Embriões foram coletados em intervalos regulares e submetidos ao protocolo de diafanização e coloração por Aliarina red S. O processo de ossificação da coluna vertebral ocorre no sentido crânio-caudal a partir dos 33 dias. As vértebras se formam a partir de centros de ossificação distintos para os centros, o arco e as apófises. O pró-atlas se forma a partir de dois centros de ossificação distintos que se fundem até o fim do período de incubação. A fusão dorsal dos processos transversos ocorre parcialmente aos 51 dias, dando origem ao diminuto esboço do processo espinhoso. No embrião de 57 dias a articulação entre as costelas, o sacral e o ílio não estava evidente. A morfologia e tamanho das vértebras caudais variam consideravelmente no sentido crânio-caudal. Todas apresentam os processos hemais e iniciam sua ossificação até os 48 dias. C. yacare apresenta um número variado de costelas abdominais, que se ossificam ainda durante o período embrionário. O padrão da esqueletogênese de C. yacare é congruente com de outros répteis, embora existam algumas variações, com particular ênfase na remodelação de estruturas, o que possivelmente reflete as variações do período de incubação entre os diversos répteis relatados.

Among reptiles, lizards and Testudines are the focus of numerous investigations that include the study of its ontogeny, however, descriptions of development and ossification sequence in crocodilians are scarce. Thus, it was aimed to investigate the pattern of training and sequence of ossification of the vertebrae and ribs in Caiman yacare (Daudin, 1802). Embryos were collected at regular intervals and submitted to the protocol of clearing and staining Aliarina red S. The process of ossification of the vertebral column occurs in the craniocaudal direction from the 33 days. The vertebrae are formed from separate ossification centers for the centers, the arch and the transverse processes. The proatlas is formed from two separate ossification centers that fuse to the end of the incubation period. The fusion of dorsal transverse process is partly to 51 days, giving rise to a small sketch of the spinous process. In the embryo of 57 days the link between the ribs, the sacrum and the ilium was not evident. The morphology and size of caudal vertebrae vary considerably in the craniocaudal direction. All have the hemal processes and initiate its ossification up to 48 days. C. yacare has a varied number of abdominal ribs, which ossify even during the embryonic period. The pattern of skeletogenesis C. yacare is consistent with other reptiles, although some variations, with particular emphasis on the remodeling of structures, which possibly reflects the variations in incubation period among the various reptiles reported.

Ontogeny of the shell bones of embryos of Podocnemis unifilis (Troschel, 1848) (Testudines, Podocnemididae).

The aim of the present study was to investigate the sequence of shell bone formation in the embryos of the Pleurodira, Podocnemis unifilis. Their bones and cartilage were collected and cleared before staining. The shell was also examined by obtaining a series of histological slices. All the bony elements of the plastron have independent ossification centers, which subsequently join together and retain two fontanelles until the period of hatching. This turtle has a mesoplastra, which is characteristic of the Podocnemididae. The carapace begins to form concurrently with the ossification of the ribs at the beginning of stage 20. All the plates, except the suprapygal, initiate ossification during the embryonic period. The main purpose of the histological investigation was to highlight the relationship between the formation of the carapace and ribs with that of the neural plates. The costal and neural plates were found not to independent ossification centers, but to be closely related to components of the endoskeleton, originating as expansions of the perichondral collar of the ribs and the neural arches, respectively. Considering the ribs as an endoskeletal element of the carapace, the carapace and plastron begin ossification at the same stage in P. unifilis. This pattern reveals similarities with other Pleurodira, as well as evident variations, such as the presence of the seven neural bones and the presence of only one ossification center in the nuchal plate.

Anatomical and topographical description of the digestive system of caiman crocodilus (Linnaeus 1758), melanosuchus niger (Spix 1825) and paleosuchus palpebrosus (Cuvier 1807) / Descripción anatómica y topográfica del sistema digestivo del caimam crocodillus (Linnaeus 1758), melanosuchus niger (Spix 1825) y paleosuchus palpebrosus (Cuvier 1807)

This paper describes the digestive system of Caiman crocodilus, Melanosuchus niger and Paleosuchus palpebrosus based on anatomical and topographical inferences. The study involves two digestive systems of C. crocodilus, one of M. niger and one of P. palpebrosus, already fixed in 10 percent formaldehyde, belonging to the collection of the Wild Animal Research Laboratory (LAPAS) of the Federal University of Uberlândia. The work begins with a description of the digestive system of the aforementioned crocodilians, followed by topographical associations, aided by photographs taken with a SONY® DSC-H20 camera, X-rays of the gastrointestinal tract and a photograph of the digestive system of C. crocodilus prior to formaldehyding, which also belong to the LAPAS collection. The results indicate that the digestive system of crocodilians consists of a wide mouth, short pharynx, long straight esophagus, dilated stomach in relation to the rest of the tract, pancreas lodged between the first two ventral duodenal loops, coiled small intestine, large intestine with diameter larger than the preceding segments, and cloaca as the terminal portion of the digestive, urinary and reproductive systems. The anatomical and topographical description of the digestive system of C. crocodilus (Linnaeus, 1758) (Crocodylia: Alligatoridae), M. niger (Spix, 1825) (Crocodylia: Alligatoridae) and P. palpebrosus (Cuvier, 1807) (Crocodylia: Alligatoridae) can be extended to the other crocodilians due to interspecies and intraspecific behavioral similarities.

Mediante comparaciones anatómicas y topográficas describimos el sistema digestivo de C. crocodillus, M. nigeri y P. palpebrosus. Se utilizaron dos sistemas digestivos de C. crocodillus, uno de M. nigeri y uno de P. palpebrosus, fijados en formalina al 10 por ciento y pertenecientes al laboratorio de enseñanza e investigación de fauna silvestre (LAPAS) de la Universidade Federal de Uberlândia. En primera instancia se realizó la descripción del sistema digestivo de las mencionadas especies, seguido de comparaciones topográficas. Luego, fueron tomados registros fotográficos con cámara DSC H20 SONY. Adicional a esto, nos apoyamos con radiografías del tracto gastrointestinal y fotografías del sistema digestivo de C. crocodillus antes de ser fijados en formalina. Los resultados demuestraron que el sistema digestivo de estas tres especies de caimanes está constituido por una boca grande, faringe corta, esófago alargado y rectilíneo, estómago dilatado con relación a las otras partes del tracto digestivo, páncreas alojado entre las dos primeras curvaturas duodenales ventrales, intestino delgado plegado, intestino grueso con un diámetro mayor en relación a los segmentos anteriores y una cloaca donde también terminan los sistemas urinarios y reproductor. La descripción anatómica y topográfica del sistema digestivo de C. crocodillus, M. niger y P. palpebrosus puede ser extrapolada para otros cocodrilos por la semejanza comportamental interespecies e intraespecíficas.

Chondrogenesis of the limbs and mesopodial ossification of Podocnemis expansa Schweigger, 1812 (Testudines: Podocnemidae).

We address the chondrogenic formation of the limbs and the mesopodial ossification pattern of the Pleurodira Podocnemis expansa, to resolve the homology of these elements as well as the pattern of connection of the autopodial elements and the origin of the digital arch. Embryos and juveniles of P. expansa were cleared and stained for cartilage and bone. The fore- and hind-limbs were also studied histologically. We describe the development of the stylopodium and zeugopodium originating from a Y-shaped cartilaginous condensation, and the differentiation of the primary axis and the digital arch in the initial stages of limb development. The most pronounced changes were observed in the chondrogenic pattern and ossification of the mesopodium, although development of the digits is similar and we found no ontogenetic reduction such as that described for other Testudines. In this study, as in previous research involving several groups of reptilian sauropsids, we found an inconsistent pattern between the chondrogenic formation and mesopodial ossification of the limbs, indicating that these developmental events are dissociated. In summary, the chondrogenic and ossification sequences of these elements do not follow the same pattern. In addition, the differences found between P. expansa and other species to which it was compared clearly indicate that these events follow more than one pattern in Testudines.