Molecular phylogeny of advanced snakes (Serpentes, Caenophidia) with an emphasis on South American Xenodontines: a revised classification and descriptions of new taxa

We present a molecular phylogenetic analysis of caenophidian (advanced) snakes using sequences from two mitochondrial genes (12S and 16S rRNA) and one nuclear (c-mos) gene (1681 total base pairs), and with 131 terminal taxa sampled from throughout all major caenophidian lineages but focussing on Neotropical xenodontines. Direct optimization parsimony analysis resulted in a well-resolved phylogenetic tree, which corroborates some clades identified in previous analyses and suggests new hypotheses for the composition and relationships of others. The major salient points of our analysis are: (1) placement of Acrochordus, Xenodermatids, and Pareatids as successive outgroups to all remaining caenophidians (including viperids, elapids, atractaspidids, and all other "colubrid" groups); (2) within the latter group, viperids and homalopsids are sucessive sister clades to all remaining snakes; (3) the following monophyletic clades within crown group caenophidians: Afro-Asian psammophiids (including Mimophis from Madagascar), Elapidae (including hydrophiines but excluding Homoroselaps), Pseudoxyrhophiinae, Colubrinae, Natricinae, Dipsadinae, and Xenodontinae. Homoroselaps is associated with atractaspidids. Our analysis suggests some taxonomic changes within xenodontines, including new taxonomy for Alsophis elegans, Liophis amarali, and further taxonomic changes within Xenodontini and the West Indian radiation of xenodontines. Based on our molecular analysis, we present a revised classification for caenophidians and provide morphological diagnoses for many of the included clades; we also highlight groups where much more work is needed. We name as new two higher taxonomic clades within Caenophidia, one new subfamily within Dipsadidae, and, within Xenodontinae five new tribes, six new genera and two resurrected genera. We synonymize Xenoxybelis and Pseudablabes with Philodryas; Erythrolamprus with Liophis; and Lystrophis and Waglerophis with Xenodon.

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA3) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO3 staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.

Análise das relações taxonômicas e sistemáticas entre espécies de triatomíneos (Hemiptera, Reduviidae) de colônias mantidas pelo Serviço Especial de Saúde de Araraquara, inferida de seqüências do 16S rDNA mitocondrial

Foram analisadas seqüências de nucleotídeos do gene 16S do rDNA mitocondrial em 14 populações de triatomíneos mantidos em colônias no insetário SESA de Araraquara- SP, comparando-as com seqüências do mesmo gene disponíveis no GenBank. Os fragmentos variaram de 311 a 317 pb com baixa variação intra-específica entre as distâncias genéticas (0% a 0,6%), exceto para os relacionamentos entre espécimes de Triatoma sordida (1%) e espécimes de T. brasiliensis (1,3%) atribuídos a populações geográficas diferentes. A parafilia de Rhodniini e do gênero Panstrongylus foi evidenciada pelas analises, confirmando resultados anteriores entre estes e os estreitos relacionamentos de R. prolixus com R. robustus e de T. infestans e T. platensis. O relacionamento entre T. maculata e T. pseudomaculata não foi solucionado, uma vez que, esses táxons apareceram tanto em monofilia quanto em parafilia: T. pseudomaculata (SESA) está agrupado com T. maculata (seqüência do GenBank) e associados a T . brasiliensis (SESA), enquanto T. maculata (SESA) aparece agrupado com T. pseudomaculata do SESA e do GenBank. Os resultados evidenciam a utilidade do gene 16S como marcador de espécies de triatomíneos e sua importância em questões de sistemática e taxonomia. Há necessidade de novos estudos envolvendo outros marcadores associados a caracteres sistemáticos clássicos de morfologia, ecologia e comportamento para decisões sistemáticas adequadas uma vez, que teriam impacto não apenas sistemático mas, para as estratégias de controle.

Anopheles (Nyssorhynchus) atacamensis (Diptera: Culicidae), a new species from Northern Chile

Anopheles (Nyssorhynchus) atacamensis, a new species in the subgenus Nyssorhynchus, is described and validated using morphological characters of the male and female adult, male genitalia and immature stages. Molecular characterization employing sequences of the ITS2 rDNA and COI mtDNA are provided. The new taxon is compared with Anopheles (Nyssorhynchus) pictipennis (Philippi) from central Chile based on morphological features of the adults, male genitalia and larva. Illustrations of the diagnostic characteristics of the male genitalia, fourth-instar larva and pupa are provided.

Imagem por ressonância magnética na investigação da cabeça de cães

A imagem por ressonância magnética (IRM) é o método de diagnóstico por imagem não invasivo mais sensível para avaliar as partes moles, particularmente o encéfalo, porém trata-se de uma técnica onerosa. O método fundamenta-se no fenômeno da ressonância magnética nuclear que ocorre quando núcleos atômicos com propriedades magnéticas presentes no corpo são submetidos a um campo magnético intenso, sendo posteriormente excitados por energia de radiofrequência e gerando, por sua vez, um sinal de onda de radiofrequência capaz de ser captado por uma antena receptora, passando por um processo matemático, chamado Transformada de Fourier, para posterior formação da imagem. Esse estudo objetivou realizar 10 exames completos da cabeça em cadáveres de cães normais à IRM e confeccionar um Atlas com as estruturas identificadas. As imagens foram adquiridas em um aparelho de ressonância magnética Gyroscan S15/HP Philips com campo magnético de 1,5Tesla. Os cadáveres foram posicionados com a cabeça no interior de uma bobina de cabeça humana e foram submetidos a cortes iniciais sagitais a partir de onde se planejou os cortes transversais e dorsais nas sequências de pulso spin-eco T1, T2 e DP. Em T1 utilizou-se TR=400ms e TE=30ms, T2 utilizou-se TR=2000ms e TE=80ms e na DP utilizou-se TR=2000ms e TE=30ms. A espessura do corte foi de 4mm, o número de médias foi igual a 2, a matriz foi de 256x256, o fator foi igual a 1,0 e o campo de visão foi de 14cm. A duração do exame completo da cabeça foi de 74,5minutos. As imagens obtidas com as sequências utilizadas e com a bobina de cabeça humana foram de boa qualidade. Em T1 a gordura tornou-se hiperintensa e o líquido hipointenso. Em T2 a gordura ficou menos hiperintensa e o líquido hiperintenso. A cortical óssea e o ar foram hipointensos em todas as sequências utilizadas devido a baixa densidade de prótons. A sequência DP mostrou o melhor contraste entre a substância branca e cinzenta quando comparada a T2 e a T1. T2 evidenciou o líquido cefalorraquidiano tornando possível a distinção dos sulcos e giros cerebrais. Através do exame de IRM foi possível, pelo contraste, identificar as estruturas ósseas componentes da arquitetura da região, músculos, grandes vasos venosos e arteriais e estruturas do sistema nervoso central, além de elementos do sistema digestório, respiratório e estruturas dos olhos entre outras. Nesse estudo as IRM adquiridas nas sequências T1, DP e T2 foram complementares para o estudo dos aspectos anatômicos da cabeça de cães demonstrando-os com riqueza de detalhes. O tempo requerido para o exame completo da cabeça é compátivel para uso em animais vivos desde que devidamente anestesiados e controlados. Os resultados obtidos por esse trabalho abrem caminho em nosso meio, para o estudo de animais vivos e para o início da investigação de doenças, principalmente as de origem neurológica, visto ser esta técnica excelente para a visibilização do encéfalo.

Occurrence and molecular characterization of Cryptosporidium spp. isolated from domestic animals in a rural area surrounding Atlantic dry forest fragments in Teodoro Sampaio municipality, State of São Paulo, Brazil

The aim of this study was to assess the occurrence of Cryptosporidium in domestic animals in rural properties surrounding rain forest fragments within the municipality of Teodoro Sampaio, southeastern Brazil. Conventional sucrose flotation method followed by molecular characterization of the parasites by sequencing PCR products amplified from SSU rRNA gene were used. Stool samples were collected from domestic animals raised as pets and livestock in all rural properties surrounding three forest fragments. Samples from cattle (197), equine (63), pigs (25), sheep (11), and dogs (28) were collected from 98 rural properties. The frequency of occurrence of Cryptosporidium within each animal species was 3.0% (6/197) among cattle and 10.7% (3/28) among dogs. Cryptosporidium was not detected in stool samples from equine, sheep, and pigs. All sequences obtained from the six samples of calves showed molecular identity with Cryptosporidium andersoni while all sequences from dog samples were similar to C. canis. The frequency of occurrence of Cryptosporidium in these domestic animal species was low. The absence of C. parvum in the present study suggests that the zoonotic cycle of cryptosporidiosis may not be relevant in the region studied. The presence of Cryptosporidium species seldom described in humans may be, otherwise, important for the wild fauna as these animals are a source of infection and dissemination of this protozoan to other animal species. The impact and magnitude of infection by C. andersoni in wild ruminants and C. canis in wild canids have to be assessed in future studies to better understand the actual importance of these species in this region.

Rapid detection of bovine coronavirus by a semi-nested RT-PCR

Bovine coronavirus (BCoV) is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae) and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested) with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256) in DEPC treated ultra-pure water, in fetal bovine serum (FBS) and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694). The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.

Caracterização molecular do vírus da raiva isolado de Desmodus rotundus capturados no Estado do Rio de Janeiro

Caracterizou-se filogeneticamente o vírus da raiva, isolado de morcegos hematógafos (Demodus rotundus). Cento e noventa e nove D. rotundus foram capturados em cinco abrigos, no Norte e Noroeste do Estado do Rio de Janeiro e sul do Espírito Santo. Sete deles foram positivos para a raiva. Amostras desses vírus foram sequenciadas e comparadas com sequências provenientes de diversos estados brasileiros. As sequências de vírus da raiva isoladas, na região norte do Estado do Rio de Janeiro, mostraram características que as distinguem de amostras de vírus isoladas em outras regiões do país, no entanto foram idênticas às isoladas de bovinos no noroeste do Rio de Janeiro.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data

Redescription of Anopheles (Nyssorhynchus) antunesi Galvão & Amaral and description of a new species of the Myzorhynchella Section (Diptera: Culicidae) from Serra da Mantiqueira, Brazil

Anopheles (Nyssorhynchus) pristinus Nagaki & Sallum, n. sp. of the Myzorhynchella Section is described based on morphological characters of adult females, males, fourth-instar larvae, pupae and male genitalia. Anopheles (Nyssorhynchus) antunesi Galvão & Amaral is characterized to fix its identity and distinguish it from An. pristinus. The eggs of An. antunesi are described for the first time. Molecular characterization employing sequences of the COI mitochondrial gene and the ITS2 region of ribosomal DNA are provided for each species. An. antunesi and An. pristinus are compared with morphologically similar species of the Myzorhynchella Section. The results of the present study suggest that the new species has been misidentified as both An. antunesi and Anopheles lutzii Cruz. An. antunesi and An. pristinus are sympatric, occurring at high altitudes in Serra da Mantiqueira, southeastern Brazil

Phylogenetic placement of the enigmatic parasite, Polypodium hydriforme, within the Phylum Cnidaria

Background: Polypodium hydriforme is a parasite with an unusual life cycle and peculiar morphology, both of which have made its systematic position uncertain. Polypodium has traditionally been considered a cnidarian because it possesses nematocysts, the stinging structures characteristic of this phylum. However, recent molecular phylogenetic studies using 18S rDNA sequence data have challenged this interpretation, and have shown that Polypodium is a close relative to myxozoans and together they share a closer affinity to bilaterians than cnidarians. Due to the variable rates of 18S rDNA sequences, these results have been suggested to be an artifact of long-branch attraction ( LBA). A recent study, using multiple protein coding markers, shows that the myxozoan Buddenbrockia, is nested within cnidarians. Polypodium was not included in this study. To further investigate the phylogenetic placement of Polypodium, we have performed phylogenetic analyses of metazoans with 18S and partial 28S rDNA sequences in a large dataset that includes Polypodium and a comprehensive sampling of cnidarian taxa. Results: Analyses of a combined dataset of 18S and partial 28S sequences, and partial 28S alone, support the placement of Polypodium within Cnidaria. Removal of the long-branched myxozoans from the 18S dataset also results in Polypodium being nested within Cnidaria. These results suggest that previous reports showing that Polypodium and Myxozoa form a sister group to Bilateria were an artifact of long-branch attraction. Conclusion: By including 28S rDNA sequences and a comprehensive sampling of cnidarian taxa, we demonstrate that previously conflicting hypotheses concerning the phylogenetic placement of Polypodium can be reconciled. Specifically, the data presented provide evidence that Polypodium is indeed a cnidarian and is either the sister taxon to Hydrozoa, or part of the hydrozoan clade, Leptothecata. The former hypothesis is consistent with the traditional view that Polypodium should be placed in its own cnidarian class, Polypodiozoa.

The expression of genes coding for distinct types of glycine-rich proteins varies according to the biology of three metastriate ticks, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus and Amblyomma cajennense

Background: Ticks secrete a cement cone composed of many salivary proteins, some of which are rich in the amino acid glycine in order to attach to their hosts' skin. Glycine-rich proteins (GRPs) are a large family of heterogeneous proteins that have different functions and features; noteworthy are their adhesive and tensile characteristics. These properties may be essential for successful attachment of the metastriate ticks to the host and the prolonged feeding necessary for engorgement. In this work, we analyzed Expressed Sequence Tags (ESTs) similar to GRPs from cDNA libraries constructed from salivary glands of adult female ticks representing three hard, metastriate species in order to verify if their expression correlated with biological differences such as the numbers of hosts ticks feed on during their parasitic life cycle, whether one (monoxenous parasite) or two or more (heteroxenous parasite), and the anatomy of their mouthparts, whether short (Brevirostrata) or long (Longirostrata). These ticks were the monoxenous Brevirostrata tick, Rhipicephalus (Boophilus) microplus, a heteroxenous Brevirostrata tick, Rhipicephalus sanguineus, and a heteroxenous Longirostrata tick, Amblyomma cajennense. To further investigate this relationship, we conducted phylogenetic analyses using sequences of GRPs from these ticks as well as from other species of Brevirostrata and Longirostrata ticks. Results: cDNA libraries from salivary glands of the monoxenous tick, R. microplus, contained more contigs of glycine-rich proteins than the two representatives of heteroxenous ticks, R. sanguineus and A. cajennense (33 versus, respectively, 16 and 11). Transcripts of ESTs encoding GRPs were significantly more numerous in the salivary glands of the two Brevirostrata species when compared to the number of transcripts in the Longirostrata tick. The salivary gland libraries from Brevirostrata ticks contained numerous contigs significantly similar to silks of true spiders (17 and 8 in, respectively, R. microplus and R. sanguineus), whereas the Longirostrata tick contained only 4 contigs. The phylogenetic analyses of GRPs from various species of ticks showed that distinct clades encoding proteins with different biochemical properties are represented among species according to their biology. Conclusions: We found that different species of ticks rely on different types and amounts of GRPs in order to attach and feed on their hosts. Metastriate ticks with short mouthparts express more transcripts of GRPs than a tick with long mouthparts and the tick that feeds on a single host during its life cycle contain a greater variety of these proteins than ticks that feed on several hosts.

Tool wear aspects when applying high-speed tapping on grey cast iron

Tool wear is a very important subject affecting the economics of machining, especially in tapping, since it is one of the last operations to be performed within most operation sequences. In the present study, some aspects of tapping such as the mechanisms and types of wear were investigated in taps working at conventional and high-speed cutting (HSC). Additionally, different types of coatings and cooling /lubrication conditions were used. The tapping operation (M8 x 1.25) was performed in through holes with two cutting speeds (30 and 60 m/min) in grey cast iron GG25. Lubrication conditions tested were dry and with minimal quantity of lubricant. Tap materials were manufactured by powder metallurgy and coated with (TiAl)N and with TiCN. A go-non-go gauge criterion was used to assess tool life. The wear and surface aspects of the tools and workpiece were evaluated by scanning electron microscopy and energy dissipation spectroscopy. Torque signals were also measured during the tests. The main wear mechanism observed was adhesion, although some abrasion and diffusion may also have occurred, and the main type of wear was flank wear. The adhesion of workpiece material on the tool was the main and decisive factor ending tool life. Tool coatings proved to be an efficient way to minimize adhesion. Torque signals followed the same pattern as the flank wear and no significant change was observed when the cutting speed was increased.

In silico analysis of Simple Sequence Repeats from chloroplast genomes of Solanaceae species

The availaibilty of chloroplast genome (cpDNA) sequences of Atropa belladonna, Nicotiana sylvestris, N tabacum, N tomentosiformis, Solanum bulbocastanum, S lycopersicum and S tuberosum, which are Solanaceae species, allowed us to analyze the organization of cpSSRs in their genic and intergenic regions In general, the number of cpSSRs in cpDNA ranged from 161 in S tuberosum to 226 in N tabacum, and the number of intergenic cpSSRs was higher than genic cpSSRs The mononucleotide repeats were the most frequent in studied species, but we also identified di-, tri-, tetra-, penta- and hexanucleotide repeats Multiple alignments of all cpSSRs sequence from Solanaceae species made the identification of nucleotide variability possible and the phylogeny was estimated by maximum parsimony Our study showed that the plastome database can be exploited for phylogenetic analyses and biotechnological approaches

Systematic review of genus Cerradomys Weksler, percequillo and Voss, 2006 (Rodentia : Cricetidae : Sigmodontinae : Oryzomyini), with description of two new species from eastern Brazil

Cerradomy's is a monophyletic genus that includes four known species, Cerradomys subflavus, C maracajuensis, C. marinhus, and C. scotti, distributed throughout the open vegetation belt across South America, from northeastern Brazil to southeastern Bolivia, and from eastern to northwestern Paraguay. We revised the status of the species currently assigned to this genus by analyzing skins, skulls, karyotypes, and cytochrome b DNA sequences. We also described two novel species, one distributed in the Brazilian states of Minas Gerais, Bahia, and Sergipe, and the other in the states of Paraiba, Pernambuco, Piaui, Ceara, and Maranhao. Molecular analysis suggested the following phylogenetics arrangement: (((C. subflavus-C. sp.n.2) C. sp.n.1) C scotti)(C. marinhus-C. maracajuensis)). Apparently, both novel species inhabit the Caatinga domain and penetrated the coastal Atlantic rainforest, differing from the remaining congeneric species that are typical open-area inhabitants.