Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes) / Avaliação de oito protocolos na extração de DNA genômico de Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes).

The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Evaluation of eight protocols for genomic DNA extraction of Hypostomus commersoni Valenciennes, 1836 (Loricariidae: Siluriformes)

Abstract The principle and the techniques applied in DNA extraction play a pivotal role in the obtention of a purified genetic material. The present study investigates the efficiency of eight protocols in the DNA extraction of Hypostomus commersoni, an essential component of South American freshwater ichthyofauna. The quality of samples was assessed through spectrophotometry, gel electrophoresis, and PCR-RAPD markers amplification. The efficiency of DNA extraction was influenced both by the method applied and the target-tissue of choice. Higher concentrations and yield of DNA were obtained from ocular tissue, with a positive spectrum of incubation in lysis buffer for up to 36 hours after sample collection, using fresh tissues and in the presence of a high concentration of Proteinase K (20 mg.ml-1). In these conditions, samples were successfully amplified. To date, there is no record of description for the parameters analyzed in this work, neither the description of RAPD markers for the species H. commersoni.

Resumo Os princípios e as técnicas aplicadas na extração de DNA desempenham um papel crucial na obtenção de material genético purificado. O presente estudo investiga a eficiência de oito protocolos na extração de DNA de Hypostomus commersoni, um importante componente ictiofaunístico de riachos da América do Sul. A qualidade das amostras foi avaliada por espectrofotometria, eletroforese em gel e amplificação por marcadores de PCR-RAPD. A eficiência da extração de DNA foi influenciada tanto pelo método aplicado quanto pelo tecido-alvo de escolha. Maiores concentrações e rendimento de DNA foram obtidos a partir do tecido ocular, com um espectro positivo de incubação em tampão de lise por até 36 horas após a coleta da amostra, utilizando tecidos frescos e na presença de alta concentração de proteinase K (20 mg.ml-1). Nestas condições, as amostras foram amplificadas com sucesso. Até o momento, não há registro de descrição para os parâmetros analisados neste trabalho, nem a descrição de marcadores RAPD para a espécie H. commersoni.

Medium- and large-sized mammals in a steppic savanna area of the Brazilian Pampa: survey and conservation issues of a poorly known fauna.

The wildlife of the Brazilian Pampa is threatened by large-scale habitat loss, due in particular to the expansion of soybean cultivation and the conversion of grasslands areas into extensive areas of silviculture. It is essential to study how the mammal fauna copes with the highly fragmented, human-influenced, non-protected landscape. Our study presents the results of a survey of the large- and medium-sized mammals of a typical human-influenced steppic savanna area of the Pampa biome. The survey was conducted exclusively with the use of camera traps over a period of 16 months. The relative frequencies of species in the area were evaluated. We recorded 18 species, some of them locally threatened (Tamandua tetradactyla, Alouatta caraya, Leopardus colocolo, Leopardus geoffroyi, Leopardus wiedii, Puma yagouaroundi, Mazama gouazoubira and Cuniculus paca). Several species were found to thrive in the area; however, many species were considered rare, and undoubtedly new species could be recorded if we continued the sampling. Our results contribute to the knowledge of faunal diversity in the Pampa biome and associated habitats, warn about threats and provide support for conservation measures.

Medium- and large-sized mammals in a steppic savanna area of the Brazilian Pampa: survey and conservation issues of a poorly known fauna / Mamíferos de médio e grande porte em uma área de savana estépica do Pampa Brasileiro: levantamento e questões de conservação sobre uma fauna pouco conhecida

Abstract The wildlife of the Brazilian Pampa is threatened by large-scale habitat loss, due in particular to the expansion of soybean cultivation and the conversion of grasslands areas into extensive areas of silviculture. It is essential to study how the mammal fauna copes with the highly fragmented, human-influenced, non-protected landscape. Our study presents the results of a survey of the large- and medium-sized mammals of a typical human-influenced steppic savanna area of the Pampa biome. The survey was conducted exclusively with the use of camera traps over a period of 16 months. The relative frequencies of species in the area were evaluated. We recorded 18 species, some of them locally threatened (Tamandua tetradactyla, Alouatta caraya, Leopardus colocolo, Leopardus geoffroyi, Leopardus wiedii, Puma yagouaroundi, Mazama gouazoubira and Cuniculus paca). Several species were found to thrive in the area; however, many species were considered rare, and undoubtedly new species could be recorded if we continued the sampling. Our results contribute to the knowledge of faunal diversity in the Pampa biome and associated habitats, warn about threats and provide support for conservation measures.

Resumo A fauna do Pampa brasileiro está sendo ameaçada pela grande perda de habitat que vem sofrendo, em especial, devido à expansão do cultivo da soja e da conversão de áreas de pastagens em extensas áreas de silvicultura. É essencial estudar como a fauna de mamíferos lida com a paisagem altamente fragmenta e não protegida, pela influencia humana. Nosso estudo apresenta os resultados de um levantamento dos mamíferos de médio e grande porte em uma área típica do bioma Pampa com influência humana. O levantamento foi realizado exclusivamente com o uso de armadilhas fotográficas ao longo de um período de 16 meses. Foram avaliadas as frequências relativas das espécies na área. Foram registradas 18 espécies, algumas delas ameaçadas localmente (Tamandua tetradactyla, Alouatta caraya, Leopardus colocolo, Leopardus geoffroyi, Leopardus wiedii, Puma yagouaroundi, Mazama gouazoubira e Cuniculus paca). Várias espécies foram encontradas de forma abundante, no entanto, muitas espécies foram consideradas raras, sendo que novas espécies poderiam ser registradas se continuássemos a amostragem. Nossos resultados contribuem para o conhecimento da diversidade de mamíferos do bioma Pampa e habitats associados, alertam sobre ameaças e fornecem suporte para medidas de conservação.

Ontogenetic allometry in the foot size of Oligoryzomys flavescens (Waterhouse, 1837) (Rodentia, Sigmodontinae).

Ontogenetic allometry is the study of how the size or shape of certain structures changes over the course of an animal's development. In this study, using Huxley's formula of allometric growth (1932), we assessed the changes in the rate of growth of the feet size of the sigmodontine rodent Oligoryzomys flavescens during its ontogeny and compared differences between males and females. We find evidence of a change of polarity during the ontogenetic development of the species, with the presence of positive allometry during pregnancy and negative allometry in adulthood. Moreover, we note the presence of sexual dimorphism in the size of the feet, in which males of the species have a higher rate of growth than females. This growth pattern is positively related to escape from predators in childhood in both sexes and, in adulthood, provides a higher encounter rate of females by males, due to the larger displacement of the latter. We suggest that both the forces of natural selection and sexual selection have acted to shape the evolution of foot size in this species.

Comparison between Oligoryzomys nigripes and O. flavescens by RAPD and genetic diversity in O. nigripes (Rodentia, Cricetidae).

The genus of Oligoryzomys includes species of small size, morphologically similar, which may impede taxonomic identification, mainly between O. flavescens (Waterhouse, 1837) and O. nigripes (Olfers, 1818). The main objective of this work was to investigate whether the RAPD markers are capable of genetically differentiating the specimens O. nigripes and O. flavescens, coming from Rio Grande do Sul (RS) and Santa Catarina (SC) states, and also to estimate the genetic variability among populations of O. nigripes, with the Uruguay River as a geographical barrier. For this purpose, samples were collected in fragments of forests situated in the North of RS, at FLONA (Floresta Nacional de Passo Fundo) and in fragments from SC, close to the Uruguay River. The karyotyping of two samples for each species was carried out and compared using the RAPD technique together with non- karyotyped individuals. Samples of O. nigripes presented 2n = 62; NA = 82, with submetacentric arms on the largest chromosomes, while samples of O. flavescens showed 2n = 64; NA = 66, with the largest chromosomes presenting acrocentric morphology, making such a result the main difference between the species. The analysis was able to detect two distinct groups, being the first one with karyotyped O. flavescens and the second with karyotyped O. nigripes. Identification afforded 211 loci, among them 181 (85.78%) polymorphic. The Jaccard similarity coefficient was in the range of 0.45 to 0.87. The UPGMA and Main Coordinate Analysis techniques demonstrated the existence of heterogeneous genetics among populations, but did not separate them completely in terms of geographical standards, and they are not influenced by the Uruguay River, which did not act as an efficient barrier.

Effects of chronic exposure to coal in wild rodents (Ctenomys torquatus) evaluated by multiple methods and tissues.

Rio Grande do Sul (RS) coal is low quality and typically obtained by strip mining. In a recent study concerning 2 years of biomonitoring in coal regions, we demonstrated the genotoxicity of coal and related products on blood cells of native rodents, from RS, Brazil. With the goal of studying the variations in the effects of RS coal on different tissues of the same rodent, we utilized, besides the single cell gel (SCG) and micronucleus (MN) assay on blood, histological analyses and SCG assay of bone marrow, spleen, kidney, liver and lung cells, and MN assay of bone marrow and spleen cells. In addition, to identify agents that can potentially influence the results, concentrations of several heavy metals were analyzed in livers and in soil, and the total concentration of hydrocarbons in the soil was determined. Rodents exposed to coal were captured at two different sites, Butiá and Candiota, in RS. Reference animals were obtained from Pelotas, where there is no coal mining. This report provides chemical and biological data from coal regions, indicating the possible association between Zn, Ni, Pb and hydrocarbons in the induction of DNA damage (e.g. single strand-breaks and alkali-labile sites) determined by the alkaline SCG assay in cells from Ctenomys torquatus. The results of the present SCG study indicate that coal and by-products not only induce DNA damage in blood cells, but also in other tissue cells, mainly liver, kidney and lung. Neither the MN assay nor histopathological observations showed significant differences; these analyses may be useful under circumstances where genotoxicity is higher. In conclusion we believe that the in vivo genotoxicity of coal can be biomonitored by the SCG assay, and our studies suggest that wild rodents, such as C. torquatus are useful for monitoring genotoxic damage by both methods, the SCG assay and the MN test.

Genotoxicity biomonitoring in coal regions using wild rodent Ctenomys torquatus by Comet assay and micronucleus test.

Coal is a mixture of a variety of chemicals, especially hydrocarbons, which may give rise to polycyclic aromatic hydrocarbons (PAH). Many PAH compounds produce mutagenic and carcinogenic effects. The quality of mineral coal in Rio Grande do Sul (RS) is low and it is typically obtained by stripping operations; it represents approximately 87% of the Brazil reserves. This report concerns the application of the Comet assay to Ctenomys torquatus to detect the effects of coal, comparing the results with a micronucleus (MN) assay, both using peripheral blood. This study was performed over a 2-year period in an attempt to evaluate seasonal patterns. The wild rodent is fossorial, and its geographic distribution in RS coincides with the distribution of coal reserves. Three localitions were studied: two coal fields, Butiá (in a strip coal mine region) and Candiota (near a strip coal mine), and one control region, Pelotas (no coal). At the end of 2 years, 240 rodents had been analyzed. Our results showed that coal and derivatives induced DNA and chromosomal lesions in rodent cells that were demonstrated by Comet and MN assays. These tests also demonstrated quantitative differences between field exposures (Candiota > Butiá). The Comet assay was more sensitive and also showed a direct relationship between age and damage, and an inverse relationship between temperature and damage index.