Jorge Reppold Marinho

An alkaline single-cell gel electrophoresis (comet) assay for environmental biomonitoring with native rodents

The main advantages of single-cell gel electrophoresis (SCG) are its applicability to any eukaryotic organism and cell type, it s low cost and the short time required to obtain results. These properties make the SCG assay particularly useful in screening for environ mental genotoxicity. The present study describes a modified version of this technique for use in field work with native rodents and ex amines some factors which influence the outcome of the assay. Wild rodents ( Ctenomys torquatus, “tuco-tuco”) from a region close to a strip coal mine and from a region with no coal mines were used. Animals from the coal mining region had significantly more DNA damage than those from the control area. The use of this SCG technique for direct sampling in the field should facilitate environmental genotoxic ity studies with natural populations, without the need to remove the animals from their habitat or to sacrifice them.

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. Environ. Mol. Mutagen. 35:270–278, 2000

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.

New Karyotypes and Some Considerations about the Chromosomal Diversification of Ctenomys minutus (Rodentia: Ctenomyidae) on the Coastal Plain of the Brazilian State of Rio Grande do Sul

The dominant mammals occupying the subterranean niche in South America are rodents of the genus Ctenomys, which form a large group of 56 species with chromosome numbers ranging from 2n= 10 to 70. In southern Brazil, Ctenomys minutus is the species with the widest geographic distribution, inhabiting sandy fields and dunes extending from Jaguaruna beach in the state of Santa Catarina to the town of São José do Norte in the state of Rio Grande do Sul. Eleven karyotypes (2n= 42; 2n= 46a; 2n= 46b; 2n= 47a; 2n= 47b; 2n= 48a; 2n= 48b; 2n= 49a; 2n= 49b; 2n= 50a and 2n= 50b) were described for this species and zones of hybridization are also known. A sample of 51 C. minutus specimens was collected from five sampling sites about 20 km apart along the coastal plain of Rio Grande do Sul between the municipalities of Tavares (31°23′S 51°09′W) and São José do Norte (31°52′S 51°54′W). We were able to extend the known geographic distribution of C. minutus by 90 km, from Tavares southwards to São José do Norte. During our study we found five karyotypes (2n= 46b, 47b, 48b, 49b and 50b), four of which (2n= 47b, 48b, 49b and 50b) have not previously been described for this species.

Population dynamics of Akodon montensis and Oligoryzomys nigripes in an Araucaria forest of Southern Brazil

Abstract We investigated the demographic parameters and the relationships between population density and extrinsic factors (temperature, rainfall, humidity and insolation) of two sigmodontine rodents (Akodon montensis and Oligoryzomys nigripes) in an Araucaria forest of southern Brazil. We followed the Cormarck-Jolly-Seber (CJS) method to estimate population parameters and density for each trapping session from November 2008 to August 2009. The relationships between density and extrinsic factors for each month were tested by simple linear regressions. Akodon montensis displayed the highest population size in three of the eight samples and showed no associations with any of the extrinsic factors analyzed. This species had higher estimated mortality than recruitment for all the seasons. Oligoryzomys nigripes presented higher population size in four of the eight samples, and density was associated with low temperatures and higher rainfall. The species showed similar estimated mortality and recruitment during the seasons, and the population size increased substantially during winter. The sex ratio was not biased in any of the species. In general, these rodents could be highly variable in time, and the observed abundance variations between seasons are in accordance with patterns described in other studies.

DNA Barcoding of Sigmodontine Rodents: Identifying Wildlife Reservoirs of Zoonoses

Species identification through DNA barcoding is a tool to be added to taxonomic procedures, once it has been validated. Applying barcoding techniques in public health would aid in the identification and correct delimitation of the distribution of rodents from the subfamily Sigmodontinae. These rodents are reservoirs of etiological agents of zoonoses including arenaviruses, hantaviruses, Chagas disease and leishmaniasis. In this study we compared distance-based and probabilistic phylogenetic inference methods to evaluate the performance of cytochrome c oxidase subunit I (COI) in sigmodontine identification. A total of 130 sequences from 21 field-trapped species (13 genera), mainly from southern Brazil, were generated and analyzed, together with 58 GenBank sequences (24 species; 10 genera). Preliminary analysis revealed a 9.5% rate of misidentifications in the field, mainly of juveniles, which were reclassified after examination of external morphological characters and chromosome numbers. Distance and model-based methods of tree reconstruction retrieved similar topologies and monophyly for most species. Kernel density estimation of the distance distribution showed a clear barcoding gap with overlapping of intraspecific and interspecific densities < 1% and 21 species with mean intraspecific distance < 2%. Five species that are reservoirs of hantaviruses could be identified through DNA barcodes. Additionally, we provide information for the description of a putative new species, as well as the first COI sequence of the recently described genus Drymoreomys. The data also indicated an expansion of the distribution of Calomys tener. We emphasize that DNA barcoding should be used in combination with other taxonomic and systematic procedures in an integrative framework and based on properly identified museum collections, to improve identification procedures, especially in epidemiological surveillance and ecological assessments.

Population structure of Ctenomys minutus (Rodentia, Ctenomyidae) on the coastal plain of Rio Grande do Sul, Brazil

Ctenomys minutus Nehring, 1887 inhabits fields and pastures of the coastal plains of Santa Catarina (SC) and Rio Grande do Sul (RS), southern Brazil. This species shows territoriality, low vagility, and sedentary behavior. The sexr atio is unbalanced; there are more females both in the general (1.3∶1) and in the adult population (1.26∶1). The population was sexually dimorphic, with males being significantly larger than females in all measured in dividuals. Analysis indicated allometric form of variation in both sexes.

Genetic structure of sigmodontine rodents (Cricetidae) along an altitudinal gradient of the Atlantic Rain Forest in southern Brazil

The population genetic structure of two sympatric species of sigmodontine rodents (Oligoryzomys nigripes and Euryoryzomys russatus) was examined for mitochondrial DNA (mtDNA) sequence haplotypes of the control region. Samples were taken from three localities in the Atlantic Rain Forest in southern Brazil, along an altitudinal gradient with different types of habitat. In both species there was no genetic structure throughout their distribution, although levels of genetic variability and gene flow were high.

Anura, Bufonidae, Melanophryniscus simplex: Distribution extension.

The genus Melanophryniscus currently includes 20 species distributed in southern Brazil, southern Bolivia, Paraguay, Uruguay, and central and northern Argentina (Di-Bernardo et al. 2006). To date, nine species of Melanophryniscus are known to occur in the state of Rio Grande do Sul, Brazil. Following Caramaschi and Cruz (2002), three species, M. atroluteus, M. dorsalis, and M. montevidensis (see Braun and Braun 1980; Tedros et al. 2001), are included in the Melanophryniscus stelzneri species group; five, M. macrogranulosus, M. cambaraensis, M. tumifrons M. pachyrhynus, and M. devincenzii (see Braun and Braun, 1973; 1979; 1980; Caramaschi and Cruz 2002; Zanella et al. 2007), are in the Melanophryniscus tumifrons group; and M. admirabilis is unassigned to any group (Di-Bernardo et al. 2006).

New record and distribution extension of the rare Atlantic Forest endemic Abrawayaomys ruschii Cunha & Cruz, 1979 (Rodentia, Sigmodontinae)

We recorded three individuals of Abrawayaomys ruschii Cunha & Cruz, 1979 in Chapeco, Santa Catarina state, expanding the known geographic distribution of the species in approximately 370 km west from its nearest locality. It is the second record of the species in this type of physiognomy, characterized by the transition of Seasonal Deciduous and Mixed Ombrophilous forests. Additionally, this is the closest record, about 200 km east, from the recently described Abrawayaomys chebezi Pardinas, Teta & D’Elia, 2009.