Reliable identification of mycobacterial species by PCR-restriction enzyme analysis (PRA)-hsp65 in a reference laboratory and elaboration of a sequence-based extended algorithm of PRA-hsp65 patterns

Abstract Background Identification of nontuberculous mycobacteria (NTM) based on phenotypic tests is time-consuming, labor-intensive, expensive and often provides erroneous or inconclusive results. In the molecular method referred to as PRA-hsp65, a fragment of the hsp65 gene is amplified by PCR and then analyzed by restriction digest; this rapid approach offers the promise of accurate, cost-effective species identification. The aim of this study was to determine whether species identification of NTM using PRA-hsp65 is sufficiently reliable to serve as the routine methodology in a reference laboratory. Results A total of 434 NTM isolates were obtained from 5019 cultures submitted to the Institute Adolpho Lutz, Sao Paulo Brazil, between January 2000 and January 2001. Species identification was performed for all isolates using conventional phenotypic methods and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing a 441 bp fragment of hsp65. Phenotypic evaluation and PRA-hsp65 were concordant for 321 (74%) isolates. These assignments were presumed to be correct. For the remaining 113 discordant isolates, definitive identification was based on sequencing a 441 bp fragment of hsp65. PRA-hsp65 identified 30 isolates with hsp65 alleles representing 13 previously unreported PRA-hsp65 patterns. Overall, species identification by PRA-hsp65 was significantly more accurate than by phenotype methods (392 (90.3%) vs. 338 (77.9%), respectively; p < .0001, Fisher's test). Among the 333 isolates representing the most common pathogenic species, PRA-hsp65 provided an incorrect result for only 1.2%. Conclusion PRA-hsp65 is a rapid and highly reliable method and deserves consideration by any clinical microbiology laboratory charged with performing species identification of NTM.

Integrated landmark and outline-based morphometric methods efficiently distinguish species of Euglossa (Hymenoptera, Apidae, Euglossini)

Morphometric methods permit identification of insect species and are an aid for taxonomy. Quantitative wing traits were used to identify male euglossine bees. Landmark- and outline-based methods have been primarily used independently. Here, we combine the two methods using five Euglossa. Landmark-based methods correctly classified 84% and outline-based 77%, but an integrated analysis correctly classified 91% of samples. Some species presented significantly high reclassification percentages when only wing cell contour was considered, and correct identification of specimens with damaged wings was also obtained using this methodology.

Nonculture-based identification of bacteria in milk by protein fingerprinting

Traditional methods for bacterial identification include Gram staining, culturing, and biochemical assays for phenotypic characterization of the causative organism. These methods can be time-consuming because they require in vitro cultivation of the microorganisms. Recently, however, it has become possible to obtain chemical profiles for lipids, peptides, and proteins that are present in an intact organism, particularly now that new developments have been made for the efficient ionization of biomolecules. MS has therefore become the state-of-the-art technology for microorganism identification in microbiological clinical diagnosis. Here, we introduce an innovative sample preparation method for nonculture-based identification of bacteria in milk. The technique detects characteristic profiles of intact proteins (mostly ribosomal) with the recently introduced MALDI SepsityperTM Kit followed by MALDI-MS. In combination with a dedicated bioinformatics software tool for databank matching, the method allows for almost real-time and reliable genus and species identification. We demonstrate the sensitivity of this protocol by experimentally contaminating pasteurized and homogenized whole milk samples with bacterial loads of 10(3)-10(8) colony-forming units (cfu) of laboratory strains of Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus. For milk samples contaminated with a lower bacterial load (104 cfu mL-1), bacterial identification could be performed after initial incubation at 37 degrees C for 4 h. The sensitivity of the method may be influenced by the bacterial species and count, and therefore, it must be optimized for the specific application. The proposed use of protein markers for nonculture-based bacterial identification allows for high-throughput detection of pathogens present in milk samples. This method could therefore be useful in the veterinary practice and in the dairy industry, such as for the diagnosis of subclinical mastitis and for the sanitary monitoring of raw and processed milk products.

The Gracilariaceae Germplasm Bank of the University of So Paulo, Brazil-a DNA barcoding approach

The University of So Paulo Gracilariaceae Germplasm Bank has 50 strains collected mostly in Brazil, but also elsewhere in the world. This bank has been used as a source of material for research developed locally and abroad. With over 200 species, some of which have high economic value, the family Gracilariaceae has been extensively studied. Nonetheless, taxonomic problems still persist by the existence of cryptic species, phenotypic plasticity, and broad geographic distribution. In the case of algae kept in culture for long periods of time, the identification is even more problematic as a consequence of considerable morphological modification. Thus, the use of molecular markers has been shown to be an efficient tool to elucidate taxonomic issues in the group. In this work, we sequenced the 5'-end of the cox1 gene for 41 strains and the universal plastid amplicon (UPA) plastid region for 45 strains, covering all 50 strains in the bank. In addition, the rbcL for representatives of the cox1/UPA clusters was sequenced for 14 strains. The original species identification based on morphology was compared with the molecular data obtained in this work, resulting in the identification of 13 different species. Our analyses indicate that cox1 and UPA are suitable markers for the delineation of species of Gracilariales in the germplasm bank. The addition of DNA barcode tags to the samples in the Gracilariaceae germplasm bank and the molecular identification of the species will make this bank even more useful for future research as the species can be easily traced and confirmed.

NEW RECORDS OF ANOPHELES HOMUNCULUS IN CENTRAL AND SERRA DO MAR BIODIVERSITY CORRIDORS OF THE ATLANTIC FOREST, BRAZIL

Two new records of Anopheles homunculus in the eastern part of the Atlantic Forest are reported. This species was found for the first time in Barra do Ouro district, Maquine municipality, Rio Grande do Sul state, located in the southern limit of the Atlantic Forest. The 2nd new record was in the Serra Bonita Reserve, Camacan municipality, southeast Bahia state. These records extend the geographical distribution of An. homunculus, suggesting that the species may be widely distributed in coastal areas of the Atlantic Forest. It is hypothesized that the disjunct distribution of the species may be caused by inadequate sampling, and also difficulties in species identification based only on female external characteristics. Species identification was based on morphological characters of the male, larva, and pupa, and corroborated by DNA sequence analyses, employing data from both 2nd internal transcribed spacer of nuclear ribosomal DNA and of mitochondrial cytochrome c oxidase subunit I.

A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae)

Abstract Background The ability to successfully identify and incriminate pathogen vectors is fundamental to effective pathogen control and management. This task is confounded by the existence of cryptic species complexes. Molecular markers can offer a highly effective means of species identification in such complexes and are routinely employed in the study of medical entomology. Here we evaluate a multi-locus system for the identification of potential malaria vectors in the Anopheles strodei subgroup. Methods Larvae, pupae and adult mosquitoes (n = 61) from the An. strodei subgroup were collected from 21 localities in nine Brazilian states and sequenced for the COI, ITS2 and white gene. A Bayesian phylogenetic approach was used to describe the relationships in the Strodei Subgroup and the utility of COI and ITS2 barcodes was assessed using the neighbor joining tree and “best close match” approaches. Results Bayesian phylogenetic analysis of the COI, ITS2 and white gene found support for seven clades in the An. strodei subgroup. The COI and ITS2 barcodes were individually unsuccessful at resolving and identifying some species in the Subgroup. The COI barcode failed to resolve An. albertoi and An. strodei but successfully identified approximately 92% of all species queries, while the ITS2 barcode failed to resolve An. arthuri and successfully identified approximately 60% of all species queries. A multi-locus COI-ITS2 barcode, however, resolved all species in a neighbor joining tree and successfully identified all species queries using the “best close match” approach. Conclusions Our study corroborates the existence of An. albertoi, An. CP Form and An. strodei in the An. strodei subgroup and identifies four species under An. arthuri informally named A-D herein. The use of a multi-locus barcode is proposed for species identification, which has potentially important utility for vector incrimination. Individuals previously found naturally infected with Plasmodium vivax in the southern Amazon basin and reported as An. strodei are likely to have been from An. arthuri C identified in this study.

Taxonomic notes, new species and identification key to the New World species of Toxophora Meigen (Diptera, Bombyliidae, Toxophorinae)

Two new species of Toxophora Meigen are described and illustrated-T. paulistana sp. nov. (Neotropical) and T. azteca sp. nov. (Nearctic and Neotropical). An identification key to the New World species is also presented. Morphological differences between populations of T. aurea Macquart (1848) are recorded, illustrated and added to the key. The new species are easily recognized by: scape with long, yellow scales laterally; presence of yellow scales on mesonotum margins; posterior margin of mesonotum with a pre-scutellar pair of setae; and yellow scales forming thin bands on posterior margins of abdominal tergites in T. paulistana sp. nov., and scape entirely covered with long dark-brown scales and yellow scales forming a broad, longitudinal stripe on center of abdominal tergites III-VII in T. azteca sp. nov.

Two new species of Jupiaba (Characiformes: Characidae) from the rio Tapajos and rio Madeira drainages, Brazil, with an identification key to species of the genus

Two new species of Jupiaba Zanata are described from Brazil. Jupiaba iasy, new species, is described from rio Teles Pires and rio Jamanxim, tributaries of rio Tapajos, and from rio Aripuana, in the rio Madeira drainage. It is distinguished from its congeners by its color pattern consisting of a single posteriorly displaced dark crescent-shaped humeral blotch, situated over the first 5 to 7 lateral line scales, and an inconspicuous dark spot at the end of caudal peduncle. It also differs from all remaining Jupiaba for the following combination of characters: 34-36 lateral line scales, 19-21 branched anal-fin rays, 8-10 predorsal scales arranged in a regular row, 6 horizontal series of scales above and 4 series below lateral line, body depth 32.3-36.1% of SL, and absence of filamentous rays in the first dorsal and anal-fin rays. Jupiaba paranatinga, new species, is described from rio Teles Pires, tributary of rio Tapajos. It is distinguished by having 34-35 lateral line scales, two vertically elongated humeral blotches, a conspicuous caudal spot at the end of the caudal peduncle, extending over 8-10 median caudal-fin rays, eye diameter 43.7-46.9% of HL, and relatively low body depth (31.3-35.5% of SL). Additionally, comments on the putative relationships of the new species with their congeners and an updated key to the species of the genus are provided.

Key to the soldiers of Angularitermes Emerson with a new species from Brazilian Amazonia (Isoptera: Termitidae: Nasutitermitinae)

An identification key based on characters of the soldier caste is provided for species of Angularitermes. Soldiers of previously described species in the genus, A. clypeatus, A. nasutissimus, A. orestes, A. pinocchio and A. tiguassu, are illustrated along with a new species, Angularitermes coninasus, n. sp., that is described and illustrated from soldier and worker castes. Samples of the new species were collected from epigeal nests at the Brazilian Amazon rainforest. The soldier of A. coninasus, n. sp. is distinguished from its congeners by having a short conical frontal tube, much wider at its base.

Identification of multi-component trail pheromones in the most evolutionarily derived termites, the Nasutitermitinae (Termitidae)

In the present study, trail pheromone blends are identified for the first time in termites. In the phylogenetically complex Nasutitermitinae, trail-following pheromones are composed of dodecatrienol and neocembrene, the proportions of which vary according to species, although neocembrene is always more abundant than dodecatrienol (by 25-250-fold). Depending on species, termites were more sensitive to dodecatrienol or to neocembrene but the association of both components always elicited significantly higher trail following, with a clear synergistic effect in most of the studied species. A third component, trinervitatriene, was identified in the sternal gland secretion of several species, but its function remains unknown. The secretion of trail pheromone blends appears to be an important step in the evolution of chemical communication in termites. The pheromone optimizes foraging, and promotes their ecological success. (C) 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99, 20-27.

A new species of Cnodalomyia Hull, 1962 (Diptera, Asilidae, Asilinae) from Brazil

The robber fly genus Cnodalomyia Hull, 1962 is a monotypic member of the Asilinae. The type species, C. obtusa Hull, is endemic to Itatiaia, State of Rio de Janeiro, Brazil. A new species, Cnodalomyia catarinensis sp. nov. from southern states of Brazil (Santa Catarina, Parana and Sao Paulo) is herein described and illustrated. Both species are restricted to highland areas of the Atlantic Forest. An identification key to the species is also presented.

Revision of the Neotropical robber fly genus Cnodalomyia Hull (Diptera, Asilidae, Asilinae) with description of two new species from Brazil

The genus Cnodalomyia includes only two Brazilian species: C. obtusa Hull, 1962 and C. catarinensis Lamas and Mellinger, 2008. Studying material deposited in the Museu de Zoologia da Universidade de Sao Paulo (MZUSP), the authors found a long series of unidentified specimens of the genus, some of which are specimens of C. obtusa labelled with the same data as the type series as well as other specimens from different localities, representing an enlargement of the known geographic records of the species. This material, together with the types of C. catarinensis, enables this revision. A redescription of the genus and of its type species, C. obtusa, including descriptions and illustrations of its spermathecae and egg, until now never described, the description and illustration of two new species (C. artigasi sp. nov. and C. papaveroi sp. nov.), the diagnosis and egg description of C. catarinensis and an identification key to all included species are presented herein.

Revision of the Neotropical genus Carrerapyrgota Aczel (Diptera, Pyrgotidae) with the description of two new species

The Neotropical genus Carrerapyrgota Aczel is restricted to the South America (Brazil and Argentina). The genus is composed of two previously described species, which are revised herein: C. miliaria Aczel and C. personata (Lutz & Lima). Two new Brazilian species are described: C. aczeli from Sao Paulo and C. bernardii from Bahia. Illustrations of the external morphology of adults and male and female terminalia are also included. An identification key to the species is presented, as well as a brief discussion of the biology and distribution of the genus.

Taxonomic Revision of the Genus Scorpiodoras (Siluriformes: Doradidae) with Resurrection of Scorpiodoras calderonensis and Description of a New Species

Three species of Scorpiodoras are recognized: S. calderonensis, S. heckelii, and S. liophysus; the latter species is described herein. Scorpiodoras calderonensis occurs in the upper Amazon basin, including the Solimoes, Jurua, Japura, and Tefe rivers. Its type locality, originally stated as ""Calderon"", is elucidated as Tabatinga, Brazil. Scorpiodoras heckelii is the most widespread species, occurring in the Orinoco, Branco, Negro, and Amazonas rivers downstream of its confluence with Rio Negro. Scorpiodoras liophysus is only known from the middle Rio Madeira basin and presents a morphological feature unique within the genus: gas bladder without secondary bladder. An osteological description of the genus is provided, as well as redescriptions of S. calderonensis and S. heckelii. Additionally, a key allowing identification of the species is presented, as well as a biogeographic discussion.