Use of RNA-seq to identify genes encoding cytokines and chemokines activated following uptake and processing a candidate peptide vaccine developed against Mycobacterium avium subsp. paratuberculosis.

Analysis of the primary and recall responses to a membrane molecule (MMP), encoded by MAP2121c demonstrated that tri-directional signaling between the antigen-presenting cell (APC), CD4 and CD8 is essential for eliciting a CD8 cytotoxic T cell (CTL) response against Mycobacterium avium subsp. paratuberculosis. As reported here, RNA-sequencing was used to initiate the characterization of the signaling pathways involved in eliciting the development of CD8 CTL, starting with the characterization of the activation status of genes in monocyte-derived macrophages (MoMΦ) following uptake and processing MMP for the presentation of antigenic epitopes to CD4 and CD8 T cells. Activation status was compared with the uptake and processing of LPS, a nonspecific stimulator of macrophages. 1609 genes were identified that were upregulated, and 1277 were downregulated three hours after uptake and processing MMP. No significant difference was observed in the cytokine genes selected for analysis of the signaling that must occur between APC, CD4, and CD8 for the development of CTL. The initial observations indicate screening of the transcriptome should include genes involved in signaling between APC and CD4, and CD8 regardless of their activation status. Four genes of interest in this study, IL12A, IL12B, IL15, and IL23A, were not significantly different from control values. The initial studies also indicate MoMΦ can be included with dendritic cells and monocyte-derived dendritic cells for further analysis of the tri-directional signaling required for the development of CTL.

A análise das respostas primárias e de recall a uma molécula de membrana (MMP), codificada por MAP2121c demonstrou que a sinalização tridirecional entre a célula apresentadora de antígeno (APC), CD4 e CD8 é essencial para provocar uma resposta de células T citotóxicas CD8 (CTL) contra Mycobacterium avium subsp. paratuberculose. Conforme relatado aqui, o sequenciamento de RNA foi usado para iniciar a caracterização das vias de sinalização envolvidas na indução do desenvolvimento de CTL CD8, começando com a caracterização do status de ativação de genes em macrófagos derivados de monócitos (MoMΦ) após captação e processamento de MMP para a apresentação de epítopos antigênicos às células T CD4 e CD8. O status de ativação foi comparado com a captação e processamento de LPS, um estimulador inespecífico de macrófagos. Foram identificados 1.609 genes que foram regulados positivamente e 1.277 foram regulados negativamente três horas após a captação e processamento de MMP. Nenhuma diferença significativa foi observada nos genes de citocinas selecionados para análise da sinalização que deve ocorrer entre APC, CD4 e CD8 para o desenvolvimento de CTL. As observações iniciais indicam que o rastreio do transcriptoma deve incluir genes envolvidos na sinalização entre APC e CD4 e CD8, independentemente do seu estado de activação. Quatro genes de interesse neste estudo, IL12A, IL12B, IL15 e IL23A, não foram significativamente diferentes dos valores de controle. Os estudos iniciais também indicam que o MoMΦ pode ser incluído com células dendríticas e células dendríticas derivadas de monócitos para análise adicional da sinalização tridirecional necessária para o desenvolvimento de CTL.

Phylogeography, genetic diversity, and intraspecific genetic structure of the black-horned capuchin (Sapajus nigritus).

Recent molecular studies have clarified the overarching taxonomy of capuchin monkeys, but intraspecific genetic diversity remains unexplored for most capuchin species. One example is Sapajus nigritus, the southernmost capuchin monkey, found in Brazil and Argentina; its phenotypic diversity has been recognized as two geographic subspecies, but the intraspecific genetic structure of this taxon is poorly known. Here, we sampled across most of this species' geographic distribution, producing a newly sequenced data set for genetic analyses that included 78 individuals from 14 populations. We investigated the intraspecific diversity, genetic structure, and evolutionary history using three mitochondrial markers. Our results indicated that S. nigritus populations exhibited high levels of genetic structure. We found strong support for two monophyletic clades within this species with a deep phylogenetic split, and clear separation from other related taxa. Vicariance events seem to have played a prevalent role in shaping S. nigritus genetic differentiation. The Paraíba do Sul River may have driven the deep divergence between southern and northern clades, whereas the Tietê River may have had a weaker, more recent effect on the divergence of populations within the southern clade.

Aplicación de PCR a partir de cultivo en la identificación de subespecies del complejo Mycobacterium Tuberculosis / The use of culture-based PCR for the identification of subspecies of the mycobacterium tuberculosis complex

Los métodos diagnósticos clásicos de tuberculosis (TB) se basan en la utilización de baciloscopía y cultivo. La identificación del agente etiológico desde la positivización del cultivo requiere entre 10 y 15 días, mientras que el empleo de la reacción en cadena de la polimerasa (PCR) disminuye el tiempo a 24 h, lo que permite no solo identificar las subespecies del complejo Mycobacterium tuberculosis (CMTB) sino también diferenciarlas de otras especies ambientales clínicamente importantes (MOTT) facilitando el diagnóstico y tratamiento. El objetivo del presente trabajo fue determinar la utilidad de la PCR en la identificación temprana de las micobacterias pertenecientes al CMTB, a partir de cultivos positivos, de pacientes con sospecha de TB, atendidos en un hospital pediátrico de alta complejidad, durante un período de cuatro años. A cada muestra, se le realizó baciloscopía y cultivo en medio líquido. A los cultivos positivos, una inmunocromatografía lateral (TBIDR) y luego PCR. El 4,6% del total de muestras (510/11.162) pertenecientes a 198 pacientes presentó cultivos positivos. Cuatrocientos veintiseis (84%) correspondieron a muestras respiratorias. El rendimiento de la baciloscopía directa fue del 41% (194/470). Cuatrocientos treinta y ocho (86%) resultaron M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG y 44 (9%) MOTT. La utilización de medios de cultivos líquidos junto con el empleo de PCR favorecen una rápida orientación microbiológica y constituye una estrategia útil para optimizar el manejo clínico de estas infecciones, desde el punto de vista terapéutico y epidemiológico, especialmente en pediatría (AU)

Classical diagnostic methods for tuberculosis (TB) are based on the use of smear microscopy and culture. The identification of the etiological agent from positive culture requires 10 to 15 days, while the use of the polymerase chain reaction (PCR) reduces the time to 24 h, which allows not only to identify the subspecies of the Mycobacterium tuberculosis complex (MTC) but also to differentiate them from clinically important environmental mycobacteria other than tuberculosis (MOTT), facilitating diagnosis and treatment. The aim of this study was to determine the usefulness of PCR in the early identification of mycobacteria belonging to the MTC, from positive cultures of patients with suspected TB seen in a pediatric tertiary hospital over a 4-year period. For each sample, smear microscopy and culture in liquid medium was performed. Positive cultures were subjected to lateral immunochromatography (TBIDR) and then PCR. Of the total number of samples (510/11,162) belonging to 198 patients, 4.6% showed positive cultures; 426 (84%) were respiratory samples. The direct smear microscopy yield was 41% (194/470). Overall, 438 (86%) were found to be M. tuberculosis, 21 (4%) Mycobacterium bovis, 7 (1%), M. bovis-BCG, and 44 (9%) MOTT. The use of liquid culture media together with the use of PCR favors a rapid microbiological orientation and is a useful strategy to optimize the clinical management of these infections, from a therapeutic and epidemiological point of view, especially in children (AU)

Eastern Colombian Páramo Liodessus Guignot, 1939 diving beetles are genetically structured, but show signs of hybridization, with description of new species and subspecies (Coleoptera, Dytiscidae).

We studied Liodessus diving beetles from six eastern Colombian Páramo areas, as well as from the Altiplano. We discovered a highly characteristic new species, based on male genital morphology, Liodessussantarositasp. nov., in the Páramo de Guantiva-Rusia. Specimens from the Altiplano around Bogotá, and the Páramos of Almorzadero, Chingaza, Matarredonda, Rabanal y Rio Bogotá and Sumapaz form one clade of genetically similar populations based on mitochondrial Cox1 sequence data. The individuals of this clade are sub-structured according to their geographic distribution. The populations differ from each other mainly in terms of body size and coloration and, at most, subtly in their genital morphology. In two cases, we find putative hybrid populations between Altiplano and Páramo areas. We suggest that the different Páramo populations are in an early phase of speciation, and perhaps already genetically isolated in some cases. They are here assigned subspecies status to highlight these ongoing processes pending more comprehensive geographic sampling and use of genomic data. We refer to this clade as the Liodessusbogotensis complex, containing Liodessusb.bogotensis Guignot, 1953; Liodessusb.almorzaderossp. nov.; Liodessusb.chingazassp. nov.; Liodessusb.lacunaviridis Balke et al., 2021, stat. nov.; Liodessusb.matarredondassp. nov., and Liodessusb.sumapazssp. nov.

Revising the diversity within the Dwarf Dog-faced Bat, Molossopstemminckii (Chiroptera, Molossidae), with the revalidation of the endangered Molossopsgriseiventer.

The genus Molossops includes two monotypic species of insectivore bats distributed in South America: Molossopsneglectus and Molossopstemminckii. Both can be differentiated, based on sizes, M.temminckii being smaller (forearm less than 33 mm). Despite being monotypic, at least two additional subspecies have been described for M.temminckii, of which M.temminckiigriseiventer from the inter-Andean Valley of the Magdalena River in Colombia might represent a valid taxon. To test the taxonomic status of M.t.griseiventer, we reviewed specimens of M.temminckii from cis- and trans-Andean localities in Colombia. We used Cytochrome-b and Cytochrome Oxidase I comparisons to test the phylogenetic position of cis- and trans-Andean samples and compared qualitative morphology, morphometric and bioacoustics. Our results show that M.t.griseiventer is differentiated from cis-Andean specimens, providing further evidence of its validity at the species level. Furthermore, M.temminckii (sensu stricto) is also distributed in Colombia, but both M.griseiventer and M.temminckii are allopatric, with the Andes acting as a barrier. The specific identity of the specimens from the Caribbean Region of Colombia needs a new evaluation, but our results clearly show that the diversity of Molossops is underestimated.

Presence of antibodies against Mycobacterium avium subspecies paratuberculosis in Brazilian high-producing dairy herds.

This study aimed to determine the presence of antibodies against Mycobacterium avium subspecies paratuberculosis (MAP) in high-producing dairy cows, the presence of the pathogen in the feces, and the risk factors associated with the disease. Blood and fecal samples were collected from 708 dairy cows over 2 years from 54 herds located in five municipalities of Paraná, Brazil. The serum samples were evaluated for the presence of antibodies against MAP using enzyme-linked immunosorbent assay (ELISA). Fecal samples from 100 cows (69 seropositive and 31 seronegative) were assessed using real-time PCR (qPCR) for IS900 of MAP. The herd prevalence of antibodies against MAP was 61.1% (33/54; 95% CI 46.88-74.08), ranging from 12.5 to 80% across the municipalities, and the prevalence in the animals was 9.8% (69/708; 95% CI 7.77-12.15); it ranged from 0 to 87.5% per herd. Only one of the 69 (1.45%) fecal samples from the seropositive cows was positive for the qPCR. The factors associated with the occurrence of paratuberculosis in herds were the use of compost barn system and the type of bed, whereas only the type of bed was associated with the infection of cows. The only risk factor (OR = 2.45; 95% CI 1.03-5.85) associated with the occurrence of paratuberculosis was the introduction of animals purchased from other dairy farms. The prevalence of active infection was low; however, our results demonstrate the presence of MAP in high-producing dairy herds in Paraná state, Brazil.

Notes on Peruvian Ribes cuneifolium and Ribes ovalifolium (Grossulariaceae)

The genus Ribes (Grossulariaceae) is widespread across the northern hemisphere, but also species-rich in the tropical Andes. In the Peruvian Andes the genus is mostly found in at least seasonally moist cloud and scrub forests, subparamo habitats and hedges. However, some taxa are from more extreme habitats in semi-arid habitats of the western slope of the Andes (Andean scrub, Ribes ovalifolium) respectively high Andean puna and paramo habitats at elevations of up to 5100 m asl (Ribes cuneifolium and some doubtful segregates). These species share small, weakly divided leaves, making them quite atypical for the genus, usually with large, deeply threeto five-lobed leaves. Both the geographical ranges and the species delimitation for both taxa are poorly understood. We here propose the recognition of only two, well-differentiated species. Ribes ovalifolium can be shown to be wide-ranging from northern Ancash to Tacna, covering nearly the entire western flank of the Peruvian Andes. Similarly, Ribes cuneifolium can be shown to represent a single, wide-ranging species from high elevations of San Martín/La Libertad to Cuzco. There is considerable diversity on details of indument, flower color and leaf shape, but no clear dividing lines permitting the recognition of segregates such as Ribes incertum J.F.Macbr. The only exception are cloud-forest populations of Ribes cuneifolium in Pasco, which we propose to segregate as a new subspecies Ribes cuneifolium subsp. pascoense based on their considerably larger leaves and inflorescences.

El género Ribes (Grossulariaceae) es principalmente distribuido en el hemisferio norte, pero también presente con muchas especies en los Andes tropicales. En los Andes del Perú el género principalmente se encuentra en bosque nublado, el subpáramo, cercos vivos y matorrales de zonas por lo menos estacionalmente húmedos. Sin embargo, algunas especies son presentes en hábitats más extremos, así como matorrales del flanco occidental de los Andes (matorral Andino, Ribes ovalifolium) respectivamente la puna y el páramo altoandino hasta los 5100 m de altitud. (Ribes cuneifolium y algunos segregados dudosos). Estas especies tienen hojas pequeñas, poco divisas, muy atípicas para el género, normalmente provisto de hojas largas, con tres ó cinco lobos profundos. Tanto la distribución como la delimitación de las especies son poco entendidas. El presente estudio presenta una revisión taxonómica de las especies, proponiendo el reconocimiento de solamente dos especies bien diferenciadas. Ribes ovalifolium tiene un rango amplio desde el Norte de Ancash hasta Tacna a lo largo del flanco occidental de los Andes del Perú. Igualmente, demostramos que Ribes cuneifolium representa una sola especie de amplia distribución de grandes alturas desde San Martín/La Libertad hasta Cuzco. Ribes cuneifolium demuestra una diversidad morfológica considerable en detalles del indumento, color de las flores y morfología foliar, pero no encontramos morfotipos claramente delineados justificando la segregación de especies adicionales, como el Ribes incertum J.F.Macbr. Las únicas excepciones son las poblaciones de Ribes cuneifolium del bosque nublado de Pasco. Proponemos el reconocimiento de este material como subespecie Ribes cuneifolium subsp. pascoense basado en sus hojas e inflorescencias mucho más grandes.

Antibodies from healthy or paratuberculosis infected cows have different effects on Mycobacterium avium subspecies paratuberculosis invasion in a calf ileal loop model.

In this work, we used a calf ileal loop model to evaluate whether the preincubation of Mycobacterium avium subspecies paratuberculosis (MAP) with antibodies from healthy, MAP-positive or Lipoarabinomannan (LAM) immunized cows could affect the results of infection after 3.5 h. Bacterial load in tissue was assessed by Ziehl-Neelsen and by culture for each loop. MAP was detectable in all infected loops after 3.5 h.p.i.; although the presence of antibodies from MAP-positive cows significantly reduced bacterial load in loops as compared with antibodies from healthy donors (by Ziehl-Neelsen and culture, p-value < 0.003 and 0.0203, respectively). A possible direct effect of antibodies on MAP viability was shown to be not significant. Severity of histopathologic changes induced by MAP infection also varied according to the pretreatment: MAP induced less changes when inoculated in the presence of antibodies from MAP-positive cows as compared with antibodies from healthy donors. Overall, our results show that the presence of antibodies from MAP-positive cows reduced MAP invasion and consequent early histological changes in this ileal short-term loop model. These results may suggest a protective role of antibodies in the response against MAP at the portal of entry in cattle.

Association between the severity of histopathological lesions and Mycobacterium avium subspecies paratuberculosis (MAP) molecular diversity in cattle in southern Chile.

The objective was to evaluate the association between the severity of histopathological lesions caused by Mycobacterium avium subspecies paratuberculosis (MAP) infection and the molecular diversity of this pathogen. Blood, ileum, and mesenteric lymph node samples were collected at slaughter, from 1,352 adult cattle [source population 1 (SP1)]. In addition, 42 dairy herds (n = 4,963 cows) were followed for 2 years, and samples from compatible paratuberculosis clinical cases [source population 2 (SP2)] were collected. MAP infection was confirmed using an ELISA test, liquid media culture, and PCR. Isolates were genotyped using five MIRU-VNTR markers. Tissues from confirmed samples were subjected to a histopathological examination. A histopathological severity score (HSS) system was developed and used to grade (0 to 5) the magnitude of lesions caused by MAP. In general, the HSS system assesses the number of foci and degree of macrophage infiltration, together with the presence of multinucleated giant cells (MGCs) and acid-fast bacilli (AFB), in addition to the fusion of the intestinal villi and hyperplasia of the crypts. Despite the large sampling effort, only 79 MAP isolates were successfully genotyped, where 19 different haplotypes were described. A mixed-effect Poisson regression model was used to assess the relationship between haplotypes and HSS values. The model was controlled by animal age, and the farm was used as a random effect. Haplotypes were grouped based on their relative frequency: the most frequent haplotype (group i, 49.4%), the second most frequent haplotype (group ii, 12.7%), and all other haplotypes (group iii, 37.9%). Model outputs indicated that group i had significantly higher HSS values than group iii. In addition, group i was also associated with higher optical density (OD) values of the ELISA test. These results support the existence of differences in pathogenicity between MAP haplotypes. However, results were based on a relatively small sample size; thus, these should be taken with caution. Despite this, study findings suggest that haplotypes would be associated with differences in disease progression, where the dominant haplotype tends to generate more severe lesions, which could be linked to a greater shed of MAP cells than non-dominant haplotypes, increasing their chances of transmission.

Performance of bovine genital campylobacteriosis diagnostic tests in bulls from Uruguay: a Bayesian latent class model approach.

The sensitivity (Se) and specificity (Sp) of three diagnostic tests for the detection of Campylobacter fetus venerealis (Cfv) using field samples were estimated using a Bayesian latent class model (BLCM), accounting for the absence of a gold standard. The tests included in this study were direct immunofluorescence antibody test (IFAT), polymerase chain reaction (PCR), and real-time PCR (RT-PCR). Twelve farms from two different populations were selected and bull prepuce samples were collected. The IFAT was performed according to the OIE Manual. The conventional PCR was performed as multiplex, targeting the gene nahE for C. fetus species identification and insertion element ISCfe1 for Cfv identification. The RT-PCR was performed as uniplex: one targeting the gene nahE for C. fetus and the other targeting the insertion ISCfe1 (ISC2) for Cfv. Results from the BLCM showed a median Se of 11.7% (Bayesian credibility interval (BCI): 1.93-29.79%), 53.7% (BCI: 23.1-95.0%), and 36.1% (BCI: 14.5-71.7%) for IFAT, PCR, and RT-PCR respectively. The Sp were 94.5% (BCI: 90.1-97.9%), 97.0% (BCI: 92.9-99.3%), and 98.4% (BCI: 95.3-99.7%) for IFAT, PCR, and RT-PCR respectively. The correlation between PCR and RT-PCR was positive and low in samples from both sampled population (0.63% vs 8.47%). These results suggest that diagnostic sensitivity of the studied tests is lower using field samples than using pure Cfv strains.

Morphological and genetic diversification of Russula floriformis, sp. nov., along the Isthmus of Panama.

Species of Russula are ubiquitous members of ectomycorrhizal fungal communities in tropical ecosystems. However, an important part of the total tropical diversity of this genus and its biogeographic patterns is unknown due to the lack of studies on Russula in tropical ecosystems. We combined molecular, morphological, ecological, and biogeographic data to elaborate concepts for two new subspecies of R. floriformis (subsection Substriatinae). Russula floriformis subsp. floriformis and R. floriformis subsp. symphoniae are described as new from montane forest dominated by Quercus and/or Oreomunnea (Fagales) from Colombia and Panama, respectively. Phylogenies were constructed using nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), D1-D2 domains of nuc 28S rDNA (28S), and partial regions of the second largest subunit of RNA polymerase II (rpb2) and translation elongation factor 1-alpha (tef1). Similar environmental conditions, similar morphology, and an ITS sequence similarity higher than 99% with only three different positions indicate that these two subspecies are closely related. Detailed observations of microscopic structures and analyses of further DNA loci, however, revealed morphological and molecular characteristics that allow distinguishing the two subspecies of R. floriformis. Spatial distribution and phylogenetic proximity of the two Russula subspecies and their ectomycorrhizal hosts, i.e., species of Quercus, suggest that their diversification is a result of comigration, adaptation, and geographic isolation along the Isthmus of Panama during the Pliocene and Pleistocene.

Secretome characterization of clinical isolates from the Mycobacterium abscessus complex provides insight into antigenic differences.

BACKGROUND: Mycobacterium abscessus (MAB) is a widely disseminated pathogenic non-tuberculous mycobacterium (NTM). Like with the M. tuberculosis complex (MTBC), excreted / secreted (ES) proteins play an essential role for its virulence and survival inside the host. Here, we used a robust bioinformatics pipeline to predict the secretome of the M. abscessus ATCC 19977 reference strain and 15 clinical isolates belonging to all three MAB subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. RESULTS: We found that ~ 18% of the proteins encoded in the MAB genomes were predicted as secreted and that the three MAB subspecies shared > 85% of the predicted secretomes. MAB isolates with a rough (R) colony morphotype showed larger predicted secretomes than isolates with a smooth (S) morphotype. Additionally, proteins exclusive to the secretomes of MAB R variants had higher antigenic densities than those exclusive to S variants, independent of the subspecies. For all investigated isolates, ES proteins had a significantly higher antigenic density than non-ES proteins. We identified 337 MAB ES proteins with homologues in previously investigated M. tuberculosis secretomes. Among these, 222 have previous experimental support of secretion, and some proteins showed homology with protein drug targets reported in the DrugBank database. The predicted MAB secretomes showed a higher abundance of proteins related to quorum-sensing and Mce domains as compared to MTBC indicating the importance of these pathways for MAB pathogenicity and virulence. Comparison of the predicted secretome of M. abscessus ATCC 19977 with the list of essential genes revealed that 99 secreted proteins corresponded to essential proteins required for in vitro growth. CONCLUSIONS: This study represents the first systematic prediction and in silico characterization of the MAB secretome. Our study demonstrates that bioinformatics strategies can help to broadly explore mycobacterial secretomes including those of clinical isolates and to tailor subsequent, complex and time-consuming experimental approaches accordingly. This approach can support systematic investigation exploring candidate proteins for new vaccines and diagnostic markers to distinguish between colonization and infection. All predicted secretomes were deposited in the Secret-AAR web-server ( http://microbiomics.ibt.unam.mx/tools/aar/index.php ).

Phenotypic Diversity and Productivity of Medicago sativa Subspecies from Drought-Prone Environments in Mediterranean Type Climates.

The phenotypic diversity and productivity of a diverse alfalfa (M. sativa subspp.) panel of cultivars, landraces and wild relatives with putative drought tolerance were evaluated in two Mediterranean environments (central Chile and Southern Australia). In Chile, 70 accessions were evaluated in rainfed conditions and in Australia 30 accessions under rainfed and irrigated conditions, during three growing seasons. Large phenotypic variation was observed among and within subspecies for NDVI, stem length, intercepted PAR and forage yield. Principal component analysis indicated that the first two principal components (PC) accounted for 84.2% of total variance; fall dormancy, taxa, and breeding status were closely related to the agronomical performance of alfalfa accessions. Forage yield varied largely among accessions across years and locations. A linear relationship was found between annual forage yield and annual water added to the experiments (R2 = 0.60, p < 0.001). The GxE analysis for forage yield allowed the detection of the highest yielding accessions for each of the two mega-environments identified. The accessions CTA002 and CTA003 showed greater forage yield in both Chile and Australia environments. It is concluded that new breeding lines derived from crosses between cultivated alfalfa (M. sativa subsp. sativa) and wild relatives belonging to the primary (M. sativa subsp. falcata) and tertiary (M. arborea) gene pool, achieve outstanding agronomical performance in drought-prone environments.

On the validity and taxonomic status of emCrypturellus/em emobsoletus/em emgriseiventris/em (Salvadori, 1895) and emC. o. hypochraceus/em (Miranda-Ribeiro, 1938) (Aves, Tinamidae).

The nine currently recognized subspecies in the Brown Tinamou (Crypturellus obsoletus) complex are disjunctly widespread in South America, and at least three of them occur in Brazil. Morphological diagnosis of most of these taxa is imprecise, in contrast with consistent vocal differences described in the literature. We conducted a taxonomic review of two Amazonian taxa, C. o. griseiventris and C. o. hypochraceus, using morphological, morphometric, and vocal characters. Our results indicate that C. o. hypochraceus (Miranda-Ribeiro, 1938) is a junior synonym of C. o. griseiventris (Salvadori, 1895), and that Crypturellus griseiventris (Salvadori, 1895) must be treated as a full species, based on unique and fully diagnosable plumage and vocal patterns.

Scanning Electron Microscopy of Sand Flies of the Chagasi Series, Psychodopygus (Diptera: Psychodidae) Genus, Focusing on the Genitalia.

Males of cryptic or closely related species present great morphological variation in their genitalia, whereas females, such as those of the Chagasi Series of the Psychodopygus Mangabeira, 1941 genus, are more similar. Therefore, our aim was to study the fine structure of the male genitalia of five species of the Chagasi Series to better understand the variation in their morphology and its influence on the copulatory process. The sand fly species were captured in the following Brazilian states: Psychodopygus chagasi (Costa Lima, 1941) (Rondônia), Psychodopygus complexus (Mangabeira, 1941) (Tocantins), Psychodopygus squamiventris maripaensis (Floch & Abonnenc, 1946) (Amapá), Psychodopygus squamiventris squamiventris (Lutz & Neiva, 1912) (Amazonas), and Psychodopygus wellcomei Fraiha, Shaw & Lainson, 1971 (Pará and Ceará). Insects were stored in ethanol 70% (then dehydrated) and dry after they were sputtered with gold. The samples were observed under a scanning electron microscope. Microtrichiae, two types of trichoid sensilla, coeloconic and chaetic sensillae, were observed on the antenna of all species, with no difference between them. Only on the anepimeron of P. squamiventris squamiventris a modified 'racket'-like scale was observed. As for the male genitalia, the setae and structures of each species were fully described, such as the small setae on the paramere apex of the P. squamiventris subspecies, and the grooves present in this region and on the paramere lobe of P. complexus and P. wellcomei, which are impossible to observe with optic microscopy. New information is thus provided on the male genitalia, which can contribute to future bionomic studies of these species.

Phylogeography and Population Genetics of Vicugna vicugna: Evolution in the Arid Andean High Plateau.

The vicuña (Vicugna vicugna) is the most representative wild ungulate of the high Andes of South America with two recognized morphological subspecies, V. v. mensalis in the north and V. v. vicugna in the south of its distribution. Current vicuña population size (460,000-520,000 animals) is the result of population recovery programs established in response to 500 years of overexploitation. Despite the vicuña's ecosystemic, economic and social importance, studies about their genetic variation and history are limited and geographically restricted. Here, we present a comprehensive assessment of the genetic diversity of vicuña based on samples collected throughout its distribution range corresponding to eleven localities in Peru and five in Chile representing V. v. mensalis, plus four localities each in Argentina and Chile representing V. v. vicugna. Analysis of mitochondrial DNA and microsatellite markers show contrasting results regarding differentiation between the two vicuña types with mitochondrial haplotypes supporting subspecies differentiation, albeit with only a few mutational steps separating the two subspecies. In contrast, microsatellite markers show that vicuña genetic variation is best explained as an isolation by distance pattern where populations on opposite ends of the distribution present different allelic compositions, but the intermediate populations present a variety of alleles shared by both extreme forms. Demographic characterization of the species evidenced a simultaneous and strong reduction in the effective population size in all localities supporting the existence of a unique, large ancestral population (effective size ∼50,000 individuals) as recently as the mid-Holocene. Furthermore, the genetic variation observed across all localities is better explained by a model of gene flow interconnecting them rather than only by genetic drift. Consequently, we propose space "continuous" Management Units for vicuña as populations exhibit differentiation by distance and spatial autocorrelation linked to sex biased dispersal instead of population fragmentation or geographical barriers across the distribution.

Fate of Mycobacterium avium subsp. paratuberculosis and changes in bacterial diversity populations in dairy slurry after chemical treatments.

AIMS: A major drawback of using dairy slurry as fertilizer is that it may contains pathogens such as Mycobacterium avium subsp. paratuberculosis (MAP), and it could represent a risk to animal and public health. Thus, the aim of this study was to evaluate the fate of MAP and bacterial communities in dairy slurry after chemical treatments. METHODS AND RESULTS: Cattle slurry, naturally contaminated with MAP, was collected from a dairy herd and divided into 32 glass bottles which were assigned to eight different treatments (control, 3·0% CaO, 0·5% NaOH; 0·087%, 0·11% and 0·14% H2 SO4 ; and 1·0 and 2·5% KMnO4 ). Treated dairy slurry samples were evaluated at 0, 1, 3, 7, 15, 30 and 60-days following treatment application for viable MAP and dairy slurry pH, and in addition temperature in this material was monitored continuously. Bacterial counts were estimated at each sampling time. A Bayesian zero-inflated Poisson mixed model was fitted to assess the effect of each treatment on the count of MAP cells. Model results indicated that only the 3·0% CaO treatment had a statistically important negative effect on MAP counts during the study period. For most treatments, MAP was undetectable immediately after chemical treatment but re-appeared over time, in some replicates at low concentrations. However, in those cases MAP counts were not statistically different than the control treatment. Regarding the fate of the other bacterial populations, the Firmicutes phylum was the dominant population in the un-treated slurry while Clostridia class members were among the most prevalent bacteria after the application of most chemical treatments. CONCLUSION: Only 3% CaO treatment had a statistically important negative effect on MAP viability in cattle slurry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence of MAP partial control in dairy slurry. This information should be considered as a best management practice to reduce MAP and other pathogens for slurry management on dairy farms.

Unveiling patterns of genetic variation in parasite-host associations: an example with pinworms and Neotropical primates.

Patterns of genetic variation among populations can reveal the evolutionary history of species. Pinworm parasites are highly host specific and form strong co-evolutionary associations with their primate hosts. Here, we describe the genetic variation observed in four Trypanoxyuris species infecting different howler and spider monkey subspecies in Central America to determine if historical dispersal processes and speciation in the host could explain the genetic patterns observed in the parasites. Mitochondrial (cox1) and ribosomal (28S) DNA were analysed to assess genetic divergence and phylogenetic history of these parasites. Sequences of the 28S gene were identical within pinworms species regardless of host subspecies. However, phylogenetic analyses, haplotype relationships and genetic divergence with cox1 showed differentiation between pinworm populations according to host subspecies in three of the four Trypanoxyuris species analysed. Haplotype separation between host subspecies was not observed in Trypanoxyuris minutus, nor in Trypanoxyuris atelis from Ateles geoffoyi vellerosus and Ateles geoffoyi yucatanensis. Levels of genetic diversity and divergence in these parasites relate with such estimates reported for their hosts. This study shows how genetic patterns uncovered in parasitic organisms can reflect the host phylogenetic and biogeographic histories.

Molecular identification and physiological characterization of Zymomonas mobilis strains from fuel-ethanol production plants in north-east Brazil.

Zymomonas mobilis has long attracted attention owing to its capacity to ferment hexose to ethanol. From a taxonomic viewpoint, Z. mobilis is a unique species of the genus Zymomonas, separated into three subspecies, Z. mobilis subsp. mobilis, Z. mobilis subsp. pomaceae and Z. mobilis subsp. francensis on the basis of physiological tests, which are often unreliable owing to the genetic proximity among these species. Currently, the use of molecular techniques is more appropriate for identification of these bacterial subspecies. In this study, the 32 strains of Z. mobilis present in the UFPEDA bacterial collection were characterized using molecular techniques, such as sequencing of the 16S rDNA gene and its theoretical restriction profile, classifying them as members of the subspecies, Z. mobilis subsp. mobilis. In addition, anaerobic cultivations were performed, which showed the biological diversity of the strains in terms of growth, sugar consumption and ethanol production. From these results, it was possible to identify the strain Z-2-80 as a promising bacterium for use in the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Zymomonas mobilis is a bacterium of great relevance to biotechnology, owing to its capacity to ferment hexose to ethanol. On a molecular basis, 32 isolates were identified as Z. mobilis subsp. mobilis. However, intraspecific diversity was identified when these were grown under strictly anaerobic conditions. The results obtained from this study suggest a strain of Z. mobilis as an alternative for use in the fermentation process.

Combining morphology and molecular data to improve Drosophila paulistorum (Diptera, Drosophilidae) taxonomic status.

The willistoni species subgroup has been the subject of several studies since the latter half of the past century and is considered a Neotropical model for evolutionary studies, given the many levels of reproductive isolation and different evolutionary stages occurring within them. Here we present for the first time a phylogenetic reconstruction combining morphological characters and molecular data obtained from 8 gene fragments (COI, COII, Cytb, Adh, Ddc, Hb, kl-3 and per). Some relationships were incongruent when comparing morphological and molecular data. Also, morphological data presented some unresolved polytomies, which could reflect the very recent divergence of the subgroup. The total evidence phylogenetic reconstruction presented well-supported relationships and summarized the results of all analyses. The diversification of the willistoni subgroup began about 7.3 Ma with the split of D. insularis while D.paulistorum complex has a much more recent diversification history, which began about 2.1 Ma and apparently has not completed the speciation process, since the average time to sister species separation is one million years, and some entities of the D. paulistorum complex diverge between 0.3 and 1 Ma. Based on the obtained data, we propose the categorization of the former "semispecies" of D. paulistorum as a subspecies and describe the subspecies D. paulistorum amazonian, D. paulistorum andeanbrazilian, D. paulistorum centroamerican, D. paulistorum interior, D. paulistorum orinocan and D. paulistorum transitional.