Detection of virulent Klebsiella pneumoniae strains causing intestinal and extraintestinal infections during the 80s and 90s in Brazil.

Klebsiella pneumoniae is an important pathogen that causes several human infections, which is currently among the main bacterial species of clinical importance. Given the importance of understanding the characteristics of this pathogen and its evolutionary aspects, in this study, we sought to characterize strains of K. pneumoniae recovered in the 1980s and 1990s in São Paulo, Brazil. Our analyses included 48 strains recovered from diarrheagenic stools and extraintestinal infections. These strains were submitted to screening for virulence and ESßL-encoding genes, antimicrobial susceptibility tests, biofilm formation, and hypermucosity and hemolytic activity tests. Our results revealed that among the studied virulence genes, the most frequent were entB (100%), followed by iutA (100%), mrkD (98%), and ycfM (72%). Phenotypic tests revealed that the strains were non- hemolytic, and two strains were positive for the hypermucoviscosity phenotype but did not have the genetic markers associated with this phenotype. Furthermore, 17% of the isolates proved to be strong biofilm producers. Antimicrobial susceptibility testing demonstrated that most strains were susceptible to the tested antimicrobials, with the exception of five isolates that produced CTX-M-2. Our findings indicate that the collection of strains studied showed variability in virulence factors, as well as biofilm production. Still, a minority of the strains showed clinically significant resistance mechanisms. As far as we know, this is the oldest collection of K. pneumoniae studied in the country.Keywords: Bacterial virulence; Ancient bacterial strains; Enterobacterales; Bacterial infection; Diarrhea.

Comparative anatomy and evolution of the atlantoaxial complex in the fossorial lineage Amphisbaenia (Squamata: Lacertoidea).

The atlas and axis are the first two vertebrae from the cervical series; these two vertebrae are responsible for neck flexion, extension, and rotation movements, while providing insertion points for muscles and tendons. Amphisbaenia is a group of fossorial squamates known for having four distinctive head shapes, which are related to different excavation methods. However, little is known about the relationship between these different digging patterns and the anatomy and evolution of the atlantoaxial complex. In this study, we used computed microtomography data to describe in detail of the atlantoaxial complex for 15 species, belonging to all six current families of Amphisbaenia. Furthermore, we evaluate evolutionary scenarios of selected characters related to the atlantoaxial complex in the most recent phylogeny for Amphisbaenia, using the criteria of parsimony and maximum likelihood. Our results indicate that the evolutionary pattern of the atlantoaxial complex presents a diversification in its morphology that is not always correlated with the shape of the head. This analysis reinforces the hypothesis of remarkable morphological convergences in the evolutionary history of Amphisbaenia. Additionally, some of the characters studied may represent independent evolution through convergence in some cases (e.g., horizontal axis of the neural column) and parallelism in others (e.g., present or absent from the transverse process).

Ciliate diversity in rodrigo de freitas lagoon (Rio de Janeiro, Brazil) from an integrative standpoint.

The Rodrigo de Freitas Lagoon is a highly eutrophic lacustrine system and has one of the longest histories of exploration and anthropic alteration in Brazil. Despite its relevance, limited studies explored the diversity of micro-eukaryotes in the lagoon. Ciliates (Alveolata, Ciliophora) are overlooked in environmental microbiology, especially in tropical and subtropical ecosystems, resulting in limited knowledge about their diversity and functional relevance in South American habitats, particularly in coastal lagoons. To fill this gap, here we investigated the diversity of ciliates in a brackish coastal lagoon in an urban area of Rio de Janeiro, Brazil, applying and comparing the performance of morphological and metabarcoding approaches. The metabarcoding analysis, based on high-throughput sequencing of the hipervariable region V4 of the 18S rRNA genes detected 37 molecular operational taxonomic units (MOTUs) assigned to Ciliophora, representing only about a half (56.9%) of the diversity detected by microscopy, which counted 65 ciliate morphotypes. The most representative classes in both approaches were Spirotrichea and Oligohymenophorea. The metabarcoding analysis revealed that 35.3% of the ciliate MOTUs had less than 97% similarity to available sequences in the NCBI database, indicating that more than one-third of these MOTUs potentially represents still not represented or undescribed ciliate species in current databases. Our findings indicate that metabarcoding techniques can significantly enhance the comprehension of ciliate diversity in tropical environments, but the scarcity of reference sequences of brackish ciliates in molecular databases represents a challenge to the taxonomic assignment of the MOTUs. This study provides new insights into the diversity of ciliates in a threatened coastal lagoon, revealing a vast array of still unknown and rare ciliate taxonomic units in tropical environments.

Evaluation of the Pathogenic Potential of Escherichia coli Strains Isolated from Eye Infections.

While primarily Gram-positive bacteria cause bacterial eye infections, several Gram-negative species also pose eye health risks. Currently, few studies have tried to understand the pathogenic mechanisms involved in E. coli eye infections. Therefore, this study aimed to establish the pathogenic potential of E. coli strains isolated from eye infections. Twenty-two strains isolated between 2005 and 2019 from patients with keratitis or conjunctivitis were included and submitted to traditional polymerase chain reactions (PCR) to define their virulence profile, phylogeny, clonal relationship, and sequence type (ST). Phenotypic assays were employed to determine hemolytic activity, antimicrobial susceptibility, and adhesion to human primary corneal epithelial cells (PCS-700-010). The phylogenetic results indicated that groups B2 and ST131 were the most frequent. Twenty-five virulence genes were found among our strains, with ecp, sitA, fimA, and fyuA being the most prevalent. Two strains presented a hemolytic phenotype, and resistance to ciprofloxacin and ertapenem was found in six strains and one strain, respectively. Regarding adherence, all but one strains adhered in vitro to corneal cells. Our results indicate significant genetic and virulence variation among ocular strains and point to an ocular pathogenic potential related to multiple virulence mechanisms.

Alternative use of phage display: phage M13 can remain viable in the intestines of poultry without causing damage.

Phage display (PD) is a tool for developing new molecules to control pathogens. Peptides selected by PD are commonly synthesised and tested, but the use of phage M13 displaying the selected peptides as a direct biding in the intestinal tract has not yet been tested. This study evaluated whether phage M13 can remain viable in the chicken gastrointestinal tract and whether it causes injury or humoral immune response. We inoculated phage M13 or E. coli ER2738 (ECR) infected with M13 into birds at different ages. We found the virus in faeces at 5 or 13 days after inoculation, just when it infected the ECR. The presence of phage M13 or ECR did not result in gut injuries and had no impacts on weight gain and bird health. Furthermore, the levels of IgY were similar in all treatments, which indicates that the virus can be used in chicken until 42 days without being recognised by the immune system. This work provides a scientific basis for the use of PD as a tool in numerous applications to control different pathogens.

Checklist, diversity and biogeography of ciliates (Ciliophora) from Chile.

There is a significant gap in research and knowledge on the diversity and distribution of Chilean ciliates. To tackle these issues, we used cultures and protargol preparations to describe the ciliates present in poorly explored areas. At these sites, we identified 45 ciliate morphospecies, 35 of which represent unprecedent records to Chile. Then, we brought together our records with literature data to construct a species checklist. This checklist summarises 132 years of data and describes the identity, habitat and distribution of 207 species, including 15 species potentially endemic to Chile. This checklist is far from complete: a diversity estimate suggests that at least two-thirds of the ciliate species occurring in Chile have yet to be described. The checklist is dominated by freshwater taxa because ciliates from marine, brackish and terrestrial environments have rarely been investigated in Chile. Finally, after controlling for sampling artefacts, we found that ciliates exhibit a bell-shaped latitudinal diversity gradient in Chile. This peculiar biogeographical pattern is common in Chile. Plants, animals and testate amoebae also exhibit a bell-shaped latitudinal diversity gradient in Chile. This finding suggests that the historical contingencies that drove the biogeography of the Chilean biota also shaped ciliate biogeography.

Two new species of geckos of the genus Phyllopezus Peters, 1878 (Squamata: Gekkota: Phyllodactylidae) from northeastern Brazil.

We describe two new species of Brazilian geckos of the genus Phyllopezus based on morphological and molecular data. The first species is currently known from a relictual Cerrado enclavecampos rupestres, in the mountains of the Serra do Espinhao in the state of Bahia. The second species is known from northeastern Atlantic Forest and transitional areas with Caatinga biome in the state of Alagoas. The two new species are sister taxa and together are the sister clade to the remaining species in the Phyllopezus pollicaris species complex. These new species can be morphologically distinguished from their congeners by meristic and morphometric characters, in addition to color pattern and genetic differentiation.

Unilateral Versus Bilateral Resistance Exercise in Postoperative Rehabilitation After ACL Reconstruction With Bone-Patellar Tendon-Bone Graft: A Randomized Controlled Trial.

Background: Lower limb muscle strength symmetry has been suggested as an essential criterion for the safe return to sports after anterior cruciate ligament (ACL) reconstruction. Limited evidence is available regarding the most effective intervention to achieve symmetry after reconstruction with contralateral bone-patellar tendon-bone (BPTB) graft. Purpose: To verify whether unilateral isotonic resistance exercise is more effective than bilateral exercise for obtaining postoperative functional and muscular strength symmetry between the donor limb and reconstructed limb for patients who received BPTB graft. Study Design: Randomized controlled trial; Level of evidence, 1. Methods: A total of 88 patients were randomly divided into a control group (n = 44) and an intervention group (n = 44). All participants performed an 8-week exercise program starting at the beginning of the fourth postoperative month and were evaluated before and after the program. The control group performed bilateral exercises, and the intervention group performed unilateral exercises for the donor limb only (the limb with the greatest disability). The primary outcome was muscle strength (peak torque and hamstrings to quadriceps [H:Q] ratio), and the secondary outcomes were range of motion (ROM; goniometry), KT-1000 arthrometer side-to-side difference in anteroposterior knee laxity, and objective (single-leg hop test) and subjective (Lysholm score) functionality. Results: Both groups improved significantly from before to after the exercise program. The improvements were significantly greater in the intervention group regarding peak torque, H:Q ratio, flexion ROM, single-leg hop test, and Lysholm score in the donor limb (P < .001 for all), and the improvements were significantly greater in the control group regarding peak torque and single-leg hop test in the reconstructed limb (P < .001 for both). Comparison between the groups showed significantly increased symmetry regarding peak torque, H:Q ratio, and single-leg hop test in the intervention group compared with the control group (P < .001), with large effect sizes (>0.80) except for the H:Q ratio. Conclusion: Although postoperative, bilateral, isotonic resistance exercise provided better strength gains to the reconstructed limb, unilateral exercise was more effective in obtaining functional and muscle strength between-limb symmetry in patients who underwent ACL reconstruction with contralateral BPTB graft. Registration: Brazilian Registry of Clinical Trials (number RBR-22rnjh).

Inhibitory effect of different probiotic formulations on salmonella heidelberg, avian pathogenic escherichia coli and campylobacter jejuni / Inhibitory effect of different probiotic formulations on salmonella heidelberg, avian pathogenic escherichia coli and campylobacter jejuni

A wide variety of microorganism species have been used as probiotics to improve the intestinal microbial balance, control and prevent the colonization of pathogenic bacteria and promote growth in animal production. Based on the various beneficial factors of probiotic agents, this study aimed to evaluate the effectiveness of three different formulations of probiotics strains through the analysis of antagonistic capacity against common pathogens from chickens gastrointestinal tract. Three formulations composed by Bacillus, Lactobacillus, Saccharomyces, Enterococcus, Bifidobacterium and Pediococcus were tested in vitro by the method of probiotic culture spot in plates seeded with inocula of Escherichia coli, Salmonella heidelberg and Campylobacter jejuni. The inhibition halos were measured and classified according to the degree of pathogenic bacteria inhibition. The formulation containing an association among Bacillus, Lactobacillus and Saccharomyces presented better results when compared to the other formulations.

Uma enorme variedade de espécies de microrganismos tem sido usada como probiótico visando melhorar o equilíbrio microbiano intestinal, controlar e prevenir a colonização de bactérias patogênicas e promover o crescimento na produção animal. Baseado nos diversos fatores benéficos dos agentes de ação probiótica no organismo, objetivou-se avaliar a eficácia das linhagens de três formulações diferentes de probióticos pela análise de capacidade inibitória contra patógenos comuns do trato gastrointestinal de frangos. Três formulações compostas por microrganismos dos gêneros Bacillus, Lactobacillus, Saccharomyces, Enterococcus, Bifidobacterium e Pediococcus foram testadas in vitro pelo método de spot de cultura probiótica em placas para inibição de Escherichia coli, Salmonella Heidelberg e Campylobacter jejuni. Os halos de inibição foram medidos e classificados de acordo com o grau de inibição das bactérias patogenicas. A formulação contendo a associação entre Bacillus, Lactobacillus e Saccharomyces apresentou melhores resultados quando comparado as demais formulações.

Inhibitory effect of different probiotic formulations on Salmonella Heidelberg, avian pathogenic Escherichia coli and Campylobacter jejuni

A wide variety of microorganism species have been used as probiotics to improve the intestinal microbial balance, control and prevent the colonization of pathogenic bacteria and promote growth in animal production. Based on the various beneficial factors of probiotic agents, this study aimed to evaluate the effectiveness of three different formulations of probiotics strains through the analysis of antagonistic capacity against common pathogens from chickens gastrointestinal tract. Three formulations composed by Bacillus, Lactobacillus, Saccharomyces, Enterococcus, Bifidobacterium and Pediococcus were tested in vitro by the method of probiotic culture spot in plates seeded with inocula of Escherichia coli, Salmonella Heidelberg and Campylobacter jejuni. The inhibition halos were measured and classified according to the degree of pathogenic bacteria inhibition. The formulation containing an association among Bacillus, Lactobacillus and Saccharomyces presented better results when compared to the other formulations.

Morphological and phylogenetic investigations shed light on evolutionary relationships of the enigmatic genus Copemetopus (Ciliophora, Alveolata), with the proposal of Copemetopus verae sp. nov.

Copemetopus Villeneuve-Brachon, 1940 is a rare, poorly known sapropelic ciliate genus composed of only two valid nominal species. Over time, Copemetopus was taxonomically assigned to Heterotrichea and Armophorea classes, but its phylogenetic affinities remained unknown. Until the present study, there were no molecular data available for Copemetopus representatives. Here, we present the 18S and 28S-rDNA sequences and the phylogenetic position of Copemetopus verae sp. nov., as well as its detailed morphological description based on live observations, protargol impregnation, and scanning electron microscopy. Transmission electron micrographs of the type species C. subsalsus Villeneuve-Brachon, 1940 reveal new morphological traits and a unique somatic ciliature pattern of Copemetopus, composed by short segments of dikinetids with one or two supplementary kinetosomes. The phylogenetic trees recovered Copemetopus as the sister group of the genus Protocruzia, both constituting early-divergent lineages that split first from a common ancestor of Intramacronucleta. Morphological and molecular evidence suggest that Copemetopus is neither a heterotrichean nor an armophorean ciliate, but a distinct clade related to Protocruzia.

Decreased susceptibility to imipenem and ceftazidime in early virulent Raoultella spp. strains retrieved from human intestinal infections.

The genus Raoultella spp. is comprised of four species, namely, R. electrica, R. ornithinolytica, R. planticola, and R. terrigena, which are rarely reported to cause infections in humans. This study aimed to characterize six strains of Raoultella spp. isolated from stool samples from patients with diarrhea. The strains included in the study were previously identified by biochemical methods as K. pneumoniae, during a surveillance study conducted in 1987. In the present study, the strains were re-identified by MALDI TOF and 16S rRNA sequencing and subsequently subjected to virulence gene screening by PCR, hemolytic activity, biofilm formation, hypermucoviscosity phenotype, capacity to interact with Caco-2 cells, and antimicrobial susceptibility test. Our results revealed that, among the six strains, three were identified as R. ornithinolytica and three as R. planticola. The genes related to iron uptake systems (aero1, aero2, iutA, entB, and ybtS) and adhesin (mrkD) were found in all strains. Furthermore, all strains demonstrated the ability to interact in vitro with Caco-2 cells and form biofilms. In general, the strains studied were sensitive to the antimicrobials tested; however, it was possible to observe high MICs for imipenem compared to ertapenem and meropenem and high minimal inhibitory concentrations (MICs) for ceftazidime, except for one strain. Our results show the occurrence of virulent strains of Raoultella spp. with high MICs for imipenem and ceftazidime causing diarrhea. We hope that our findings can contribute to the understanding of the evolution of this species since, as far as we know, these are the oldest isolates reported so far.

A new species of Oreosaurus (Squamata: Gymnophthalmidae) from the Turimiquire Massif, northeastern Venezuela.

We describe a new species of gymnophthalmid lizard, Oreosaurus bisbali sp. nov., from the Turimiquire Massif in northeastern Venezuela. It is one of three reptile species known to be endemic to the region and the fifth species of Oreosaurus from Venezuela. It can be distinguished from all congeners by a combination of rectangular dorsal scales at midbody, having two scales separating femoral pores medially, and lacking a nasoloreal suture. The new species is likely to be restricted in distribution to the highlands of the Turimiquire Massif, which have been significantly degraded by agriculture.

Nanocomposite of Ag-Doped ZnO and Ag2O nanocrystals in control of Salmonella Heidelberg biofilms formed in commercial egg / Nanocompostos de Ag-Doped ZnO e nanocristais de Ag2O no controle da formação de biofilmes de Salmonella Heidelberg em ovos comerciais

Salmonella spp. is an important causal agent of salmonellosis in humans. Controlling Salmonella spp. in eggs is important as the bacterium passes through the shell to an embryo and remains in the terrain. Disinfection is usually performed by using several sanitizers. However, novel, more efficient ways of controlling this agent have been studied with advances in nanotechnology, including nanoparticles. Preliminary studies of nanoparticles have shown they are successful in controlling such microorganisms. Standardizing the ideal concentration of this nanocomposite is fundamental for optimum efficiency in the control of Salmonella spp. In this study, eggs from commercial laying chickens were purchased from local trade and treated in laboratory with silver and zinc nanoparticles in different concentrations. Biofilm was formed 24 hours after that; then, the eggs were washed for the removal of free bacteria. Conventional microbiology was performed to isolate Salmonella spp., and PCR was performed to identify colonies. The effectiveness of using nanocomposite of silver oxide with silver-doped zinc oxide (ZnO:Ag-AgO) was evaluated in different concentrations to prevent the formation of eggshell biofilms.(AU)

As Salmonellas spp. são importantes agentes causadores de salmonelose em humanos. O controle da Salmonella spp. é importante, pois a bactéria ultrapassa a barreira da casca atingindo o embrião e infecta lotes de aves que podem levar a infecção ao ser humano. A desinfecção costuma ser feita por vários sanitizantes; porém, com os avanços da nanotecnologia, formas novas e mais eficientes de controle desse agente estão sendo estudadas, como as nanopartículas. Estudos preliminares dessas nanopartículas têm mostrado o sucesso de seu uso no controle de microrganismos. A padronização da concentração ideal de uso desse nanocomposto é fundamental para a máxima eficiência no controle de Salmonella spp. Ovos vermelhos oriundos de postura comercial foram comprados no comércio local e tratados em laboratório com as nanopartículas em diferentes concentrações; após 24 horas, formaram o biolfilme. Os ovos foram lavados para a retirada das bactérias livres. Realizaram-se exame microbiológico convencional, para isolamento de Salmonella spp., e PCR, para identificação das colônias. O objetivo deste artigo foi avaliar a eficácia da utilização de nanocompostos de óxido de prata com óxido de zinco dopado com óxido de prata (ZnO: Ag-Ago) em diferentes concentrações na prevenção da formação de biofilmes na casca dos ovos.(AU)

Nanocomposite of Ag-Doped ZnO and Ag2O nanocrystals in control of Salmonella Heidelberg biofilms formed in commercial egg / Nanocompostos de Ag-Doped ZnO e nanocristais de Ag2O no controle da formação de biofilmes de Salmonella Heidelberg em ovos comerciais

Salmonella spp. is an important causal agent of salmonellosis in humans. Controlling Salmonella spp. in eggs is important as the bacterium passes through the shell to an embryo and remains in the terrain. Disinfection is usually performed by using several sanitizers. However, novel, more efficient ways of controlling this agent have been studied with advances in nanotechnology, including nanoparticles. Preliminary studies of nanoparticles have shown they are successful in controlling such microorganisms. Standardizing the ideal concentration of this nanocomposite is fundamental for optimum efficiency in the control of Salmonella spp. In this study, eggs from commercial laying chickens were purchased from local trade and treated in laboratory with silver and zinc nanoparticles in different concentrations. Biofilm was formed 24 hours after that; then, the eggs were washed for the removal of free bacteria. Conventional microbiology was performed to isolate Salmonella spp., and PCR was performed to identify colonies. The effectiveness of using nanocomposite of silver oxide with silver-doped zinc oxide (ZnO:Ag-AgO) was evaluated in different concentrations to prevent the formation of eggshell biofilms.

As Salmonellas spp. são importantes agentes causadores de salmonelose em humanos. O controle da Salmonella spp. é importante, pois a bactéria ultrapassa a barreira da casca atingindo o embrião e infecta lotes de aves que podem levar a infecção ao ser humano. A desinfecção costuma ser feita por vários sanitizantes; porém, com os avanços da nanotecnologia, formas novas e mais eficientes de controle desse agente estão sendo estudadas, como as nanopartículas. Estudos preliminares dessas nanopartículas têm mostrado o sucesso de seu uso no controle de microrganismos. A padronização da concentração ideal de uso desse nanocomposto é fundamental para a máxima eficiência no controle de Salmonella spp. Ovos vermelhos oriundos de postura comercial foram comprados no comércio local e tratados em laboratório com as nanopartículas em diferentes concentrações; após 24 horas, formaram o biolfilme. Os ovos foram lavados para a retirada das bactérias livres. Realizaram-se exame microbiológico convencional, para isolamento de Salmonella spp., e PCR, para identificação das colônias. O objetivo deste artigo foi avaliar a eficácia da utilização de nanocompostos de óxido de prata com óxido de zinco dopado com óxido de prata (ZnO: Ag-Ago) em diferentes concentrações na prevenção da formação de biofilmes na casca dos ovos.

Potential of grazing management to improve beef cattle production and mitigate methane emissions in native grasslands of the Pampa biome.

We tested the hypothesis that improving sward structure through adjustments in forage allowance results in greater forage intake and live weight (LW) gains by beef cattle and lower CH4 emissions per unit LW gain and unit area in a native grassland ecosystem of the Pampa biome. The experiment was carried out during 2012 and 2013 in southern Brazil. The experimental design was a randomized complete block with two replicates. Treatments consisted of five contrasting forage allowances of a native grassland managed under continuous stocking: 4, 8, 8-12, 12, and 16 kg of dry matter (DM) 100 kg LW-1 day-1 (or % LW). The 8-12% LW treatment had a variable forage allowance of 8% LW in spring and 12% LW in summer, autumn, and winter. Forage allowance was controlled by changes in stocking rate (kg LW ha-1). Average daily gain (kg LW day-1) was high for forage allowances of 12 and 16% LW but decreased at 8%, reaching the lowest value at 4% LW treatment (p < 0.001). Live weight gain ha-1 year-1 was the greatest at forage allowance of 8-12% LW (p < 0.001). Forage DM intake peaked at a forage allowance of 12% LW (p = 0.005). Individual CH4 emissions remained constant around 150 g day-1 for the two highest forage allowances and decreased to 118 and 107 g day-1 under forage allowances of 8 and 4% LW, respectively (p = 0.002). Emissions per unit LW gain and unit area were driven by animal productivity changes and decreased with increasing forage allowance (p = 0.001 and p = 0.040, respectively). We propose that the combination of 8% LW forage allowance during spring and 12% LW during the rest of the year should be targeted to best balance animal production and environmental impact in the Pampa biome.

Macronuclear Plasticity in Two South American Populations of Spirostomum (Ciliophora, Heterotrichea) Warns About its Use for Species Classification: Revision and New Insights.

Spirostomum is a widely distributed heterotrichean genus composed of well-known species with described ecology and phylogenetic affinities. The morphological classification of Spirostomum species is mostly based on the body size/shape, number of cortical granule rows and macronuclear characteristics. These features along with molecular phylogenies based on ribosomal genes divide the genus into two phylogroups, one including species with a compact macronucleus, and another including species with a moniliform macronucleus. Here, we present our observations on atypical Spirostomum specimens with unusually two distinct macronuclei and shortened adoral zone of membranelles. These atypical forms appeared in the cultures of S. minus and S. yagiui, sampled at different sites in South America (Chile and Brazil) and associated with unrelated substrate types. Morphological observations of living and stained cells, 18S rRNA gene analyses, and a thorough investigation of the literature suggest that the atypical phenotype may be a result of uncommon pathways during the conjugative process. Thus, we demonstrate that studies of ciliate natural populations and their morphological variations, especially from undersampled biogeographical regions, can reveal the boundaries of widely used morphological characters for Spirostomum taxonomy and species identification.

Long known, brand new, and possibly threatened: a new species of watersnake of the genus Helicops Wagler, 1828 (Serpentes; Xenodontinae) from the Tocantins-Araguaia River Basin, Brazil.

We describe a new species of watersnake of the genus Helicops from the Tocantins-Araguaia River Basin based on characters of coloration and hemipenial morphology, also corroborated by a molecular phylogenetic analysis including 18 terminals of 10 additional congeners. The new species superficially resembles the spotted-patterned He. leopardinus, but differs from it in exhibiting dorsal spots fused into irregular bands, and a unique hemipenial structure with two pairs of distinctly enlarged basal spines. In addition, despite the superficial resemblance of color pattern, our genetic evidence indicates that the new species does not belong to the He. leopardinus radiation, representing the sister group of a component including terminals of this species, He. modestus, and He. infrataeniatus. Finally, we discuss the apparent restriction of the new species to the Tocantins-Araguaia Basin with focus on vicariant processes that may have promoted its differentiation, as well as on the conservation challenges in an area severely affected by anthropic impacts associated with the rapid expansion of hydroelectric and agricultural sectors in the Brazilian Cerrado.