Molecular tools in the diagnostic and epidemiology of infections caused by members of Mycobacterium avium Complex

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

Tree species diversity in a seasonally-dry forest: the case of the Pinkaití site, in the Kayapó Indigenous Area, Southeastern limits of the Amazon

This study investigates patterns of forest structure and tree species diversity in an anthropogenic palm grove and undisturbed areas at the seasonally-dry Pinkaití research station, in the Kayapó Indigenous Area. This site, managed by the Conservation International do Brasil, is the most southeastern site floristically surveyed in the Amazon until now. The secondary and a nearby undisturbed forest were sampled in a group of 52 floristic plots of 0.0625-ha (25x25-m) where all trees with DBH > 10 cm were measured and identified. The analyses were complemented with other two floristic plots of 1-ha (10x1000-m). The present study has shown that the Pinkaití, like other seasonally-dry forests, have great heterogeneity in forest structure and composition, associated with biotic characteristics of the most important tree species, natural disturbance and history of land-use. The palm grove, moderately dominated by the arborescent palm Attalea maripa (Aubl.) Mart., presented high tree species diversity and was floristically similar to undisturbed forests at the study site. It is discussed the importance of large arborescent palms for the seasonally-dry Amazon forests regeneration.

Geographic pattern of genetic diversity in natural populations of Rosewood (Aniba rosaeodora), in the Central Amazonia

Rosewood (Aniba rosaeodora Ducke, Lauraceae) is an Amazonian evergreen tree and a source of the purest linalool, the main component of its essential oil, which is very valuable in the international perfumery market. After decades of over-exploitation it is currently considered as threatened. We evaluated the genetic diversity and its distribution in four populations in Central Amazonia. Thirty-five reliable RAPD markers were generated, of which 32 were polymorphic (91.4%). Variation was higher within the populations (76.5%; p < 0.0001) and geographic distribution contributed to population differentiation (23.4%; p < 0.0001). The Amazon River had a small influence on gene flow (3.3%; p < 0.0001), but we identified evidence of gene flow across the river. There were significant differences in marker frequencies (p < 0.05), in agreement with the low gene flow (Nm = 2.02). The correlation between genetic distance and gene flow was - 0.95 (p = 0.06) and between geographic distance and gene flow was -0.78 (p = 0.12). There was a geographic cline of variability across an East-West axis, influenced as well by the Amazon River, suggesting the river could be a barrier to gene flow. Although threatened, these Rosewood populations retain high diversity, with the highest levels in the Manaus population, which has been protected for over 42 years in a Reserve.

The trading relationship between the EU and Mercosur: the mediatory role of Brazil

Dissertação de mestrado em European and Transglobal Business Law

Structural and successional variability of periphytic algal community in a Amazonian lake during the dry and rainy season (Rio Branco, Acre)

The colonization process and successional patterns of a periphytic algal community were evaluated in a Amazonian Viveiro Lake (Rio Branco, Acre, Brazil). Sampling was performed over a period of 35 days; at four-day intervals for 20 days, and then at five-day intervals. Water sampling for physical, chemical and biological analyses was done during the dry and rainy season. Glass slides were used as artificial substrates for periphyton colonization. The structural community was evaluated through population density, algae class, diversity indices and descriptive species. Species richness, diversity and evenness increased as succession progressed. While density of Bacillariophyceae, Euglenophyceae and Zygnemaphyceae increased with succession, Cyanobacteria remained dominant. Synechocystis aquatilis, Synechocystis diplococcus and Navicula pseudolanceolata were the main descriptive species in both the dry and rainy season. Cymbela tumida, Frustulia rhomboides, Trachelomonas lacustris and Closterium acicularis was correlated with an increase in hydrologic level during the rainy season. Conversely, the density of Chlamydomonas sp., Chroomonas nordstedtii, Trachelomonas volvocinopsis, Trachelomonas volvocina and Synechococcus linearis was correlated with an increase in water transparency during the dry season. In general, the periphytic algal community showed high diversity and species richness independent of season. Season also had little influence on representation of algae class and main descriptive species. However, successional patterns varied by season, and changes in hydrologic levels acted directly on the succession path of periphytic algae. More research on periphyton dynamics is needed to improve our understanding of tropical lake ecosystems, especially in Amazonian.

Tree communities of white-sand and terra-firme forests of the upper Rio Negro

The high tree diversity and vast extent of Amazonian forests challenge our understanding of how tree species abundance and composition varies across this region. Information about these parameters, usually obtained from tree inventories plots, is essential for revealing patterns of tree diversity. Numerous tree inventories plots have been established in Amazonia, yet, tree species composition and diversity of white-sand and terra-firme forests of the upper Rio Negro still remain poorly understood. Here, we present data from eight new one-hectare tree inventories plots established in the upper Rio Negro; four of which were located in white-sand forests and four in terra-firme forests. Overall, we registered 4703 trees > 10 cm of diameter at breast height. These trees belong to 49 families, 215 genera, and 603 species. We found that tree communities of terra-firme and white-sand forests in the upper Rio Negro significantly differ from each other in their species composition. Tree communities of white-sand forests show a higher floristic similarity and lower diversity than those of terra-firme forests. We argue that mechanisms driving differences between tree communities of white-sand and terra-firme forests are related to habitat size, which ultimately influences large-scale and long-term evolutionary processes.

Composition and structure of the fig wasp community in Amazonia

An understanding of the complex ecological interaction between fig wasps and their host plants in Amazonia requires previous knowledge of their distribution and diversity. The objective of this study was to describe the composition and structure of the wasp community associated with four species of Ficus in the municipal area of Manaus, Amazonas, Brazil. A total of 600 syconia from four species were collected. The study species were: Ficus obtusifolia Kunth; Ficus citrifolia Mill; F. americana subspecies guianensis Desv. form mathewsii; and F. americana subspecies guianensis Desv. form parkeriana. Statistical analyses were used to examine the relationship between fig wasp diversity and syconium diameter, and the effect of non-pollinating wasps on numbers of pollinators and seeds. Forty three species of fig wasp were identified, distributed across seven genera (Pegoscapus, Idarnes, Aepocerus, Physothorax, Anidarnes, Heterandrium , Eurytoma). Idarnes (carme group) was the wasps genus non-pollinator with greatest number of individuals with the greatest number of infested syconia (7409 wasps in 376 syconia). Analysing non-pollinating wasp diversity in relation to fig diameter, a significant difference was observed between the four fig species. Ficus citrifolia and F. americana subspecies guianensis form mathewsii had the smallest diameter but the greatest diversity of fig wasp. Ficus obtusifolia was the only species in which the non-pollinating wasps had a significant negative effect on the number of Pegoscapus sp. and on seed production.

Comparative dietary analysis of Eurycheilichthys pantherinus and Pareiorhaphis hystrix: two Loricariidae species (Ostariophysi, Siluriformes) from Campos Sulinos biome, southern Brazil

This work was carried out in Marco river, São José dos Ausentes municipality, state of Rio Grande do Sul, southern Brazil. This region is located within the Campos Sulinos biome, an area of great biological importance due to its high diversity and endemism of fish. The feeding habits and food overlap between Eurycheilichthys pantherinus (Reis & Schaefer, 1992) (n=108) and Pareiorhaphis hystrix Pereira & Reis, 2002 (n=60) are described. Monthly samples were obtained between September 2000 and July 2001 with dipnets using the kick sampling technique. Stomach contents were analyzed based on frequency of occurrence, volumetric frequency and index of alimentary importance. The level of dietary specialization and food overlap were determined by the Levins measure and the Morisita index, respectively. Eurycheilichthys pantherinus fed mainly on immature aquatic insects, such as Dipterans and Ephemeropterans, and was classified as insectivore. Pareiorhaphis hystrix fed mainly on detritus associated with small amounts of aquatic insects, and was classified as detritivore. Species are clearly segregated by the use of food resources in Marco River. Their diets did not overlap and the differences observed in their feeding habits probably contribute for their coexistence.

Temporal and spatial variation of rocky shores intertidal benthic communities in Southeast Brazil

The relationships between environmental factors and temporal and spatial variations of benthic communities of three rocky shores of the state of Espírito Santo, Southeast Brazil, were studied. Sampling was conducted every three months, from August 2006 to May 2007, using intersection points. Chthamalus bisinuatus (Pilsbry, 1916) (Crustacea) and Brachidontes spp. (Mollusca) were the most abundant taxa, occupying the upper level of the intertidal zone of the rocky shore. The species richness was higher at the lower levels. The invasive species Isognomon bicolor (C. B. Adams, 1845) (Mollusca) occurred at low densities in the studied areas. The clustering analysis dendrogram indicated a separation of communities based on exposed and sheltered areas. According to the variance analyses, the communities were significantly different among the studied areas and seasons. The extent of wave exposure and shore slope influenced the species variability. The Setibão site showed the highest diversity and richness, most likely due to greater wave exposure. The communities showed greater variation in the lower levels where environmental conditions were less severe, relative to the other levels.

Microbial spoilage and quality of marinated dark, firm, dry meat

Estudi elaborat a partir d’una estada al Royal Veterinary and Agricultural University of Denmark entre els mesos de Març a Juny del 2006. S’ha investigat l’efecte dels envasats amb atmosferes modificades (MAP), així com la marinació amb vi tint, sobre l’evolució de la contaminació bacteriològica de carns fosques, dures i seques (DFD). Les carns DFD es troben a les canals d’animals que, abans del sacrifici, han estat exposades a activitats musculars prolongades o estrès. Les carns DFD impliquen importants pèrdues econòmiques degut a la contaminació bacteriològica i als problemes tecnològics relacionats amb la alta capacitat de retenció d’aigua. A més a més, és crític per la indústria investigar la diversitat de la contaminació bacteriana, identificar les espècies bacterianes i controlar-les. Però és difícil degut a la inhabilitat per detectar algunes bactèries en medis coneguts, les interaccions entre elles, la complexitat dels tipus de contaminació com són aigua, terra, femtes i l’ambient. La Polymerasa chain reaction- Denaturating Electrophoresis Gel (PCR-DGEE ) pot sobrepassar aquests problemes reflectint la diversitat microbial i les espècies bacterianes. Els resultants han indicat que la varietat bacteriana de la carn incrementava amb els dies d’envasat independentment del mètode d’envasat, però decreixia significativament amb el tractament de marinació amb vi tint. La DGEE ha mostrat diferències en les espècies trobades, indicant canvis en la contaminació bacteriana i les seves característiques en la carn DFD sota els diferents tractaments. Tot i que la marinació és una bona alternativa i solució a la comercialització de carn DFD , estudis de seqüenciació són necessaris per identificar les diferents tipus de bactèries.

Discordances between phylogenetic and morphological patterns in alpine leaf beetles attest to an intricate biogeographic history of lineages in postglacial Europe.

Pleistocene glacial and interglacial periods have moulded the evolutionary history of European cold-adapted organisms. The role of the different mountain massifs has, however, not been accurately investigated in the case of high-altitude insect species. Here, we focus on three closely related species of non-flying leaf beetles of the genus Oreina (Coleoptera, Chrysomelidae), which are often found in sympatry within the mountain ranges of Europe. After showing that the species concept as currently applied does not match barcoding results, we show, based on more than 700 sequences from one nuclear and three mitochondrial genes, the role of biogeography in shaping the phylogenetic hypothesis. Dating the phylogeny using an insect molecular clock, we show that the earliest lineages diverged more than 1 Mya and that the main shift in diversification rate occurred between 0.36 and 0.18 Mya. By using a probabilistic approach on the parsimony-based dispersal/vicariance framework (MP-DIVA) as well as a direct likelihood method of state change optimization, we show that the Alps acted as a cross-roads with multiple events of dispersal to and reinvasion from neighbouring mountains. However, the relative importance of vicariance vs. dispersal events on the process of rapid diversification remains difficult to evaluate because of a bias towards overestimation of vicariance in the DIVA algorithm. Parallels are drawn with recent studies of cold-adapted species, although our study reveals novel patterns in diversity and genetic links between European mountains, and highlights the importance of neglected regions, such as the Jura and the Balkanic range.

The Argentine ant (Linepithema humile) invasion effects on Mediterranean forests’ trophic web: blue tit (Parus caeruleus) reproduction and development

Les invasions biològiques representen una greu amenaça per al funcionament dels ecosistemes i per a la preservació de la biodiversitat.. La formiga argentina (Linepithema humile) està considerada com una de les 100 espècies invasores més nocives. Prospera en extenses àrees de clima mediterrani de regions temperades i subtropicals de tots els continents amb l’excepció de l’Antàrtida. És una formiga dominant i una competidora agressiva que mitjançant múltiples mecanismes, des de predació directe a competència, produeix efectes negatius en una amplia varietat de taxons, principalment formigues i altres artròpodes, però també vertebrats. S’ha investigat, per primera vegada, els efectes de la formiga invasiva sobre les comunitats d’artròpodes de fullatge i com aquestes pertorbacions es transmeten en la xarxa tròfica del bosc esclerofil•le mediterrani. En les suredes estudiades la invasió de formiga argentina és causa directe de la extinció local de la gran majoria de poblacions de formigues natives. En el període mostrejat s’han constatat també impactes negatius en la diversitat i en l’abundància d’artròpodes natius en les capçades dels arbres, particularment d’erugues. Una avaluació preliminar basada únicament amb dades del 2005 indica que, reduint la disponibilitat d’erugues, la formiga argentina empobreix l’hàbitat reproductiu de la mallerenga blava (Parus caeruleus). La mallerenga blava basa la dieta insectívora estricte de la seva pollada fonamentalment en les erugues. No hem detectat impactes en l’èxit reproductiu de les mallerengues blaves en zones envaïdes. Els polls crescuts en àrees envaïdes assoleixen una condició física similar als de les zones no envaïdes, però la reducció en la disponibilitat d’erugues associada a la invasió de formiga argentina es tradueix en un creixement descompassat i en una menor mida estructural del polls volanders. Així, les pertorbacions en la comunitat d’artròpodes associades a la invasió de la formiga argentina promouen efectes bottom-up que acaben perjudicant el desenvolupament dels polls de mallerenga blava.

Shifts in species richness, herbivore specialization, and plant resistance along elevation gradients.

Environmental gradients have been postulated to generate patterns of diversity and diet specialization, in which more stable environments, such as tropical regions, should promote higher diversity and specialization. Using field sampling and phylogenetic analyses of butterfly fauna over an entire alpine region, we show that butterfly specialization (measured as the mean phylogenetic distance between utilized host plants) decreases at higher elevations, alongside a decreasing gradient of plant diversity. Consistent with current hypotheses on the relationship between biodiversity and the strength of species interactions, we experimentally show that a higher level of generalization at high elevations is associated with lower levels of plant resistance: across 16 pairs of plant species, low-elevation plants were more resistant vis-à-vis their congeneric alpine relatives. Thus, the links between diversity, herbivore diet specialization, and plant resistance along an elevation gradient suggest a causal relationship analogous to that hypothesized along latitudinal gradients.

The effect of host social system on parasite population genetic structure: comparative population genetics of two ectoparasitic mites and their bat hosts.

BACKGROUND: The population genetic structure of a parasite, and consequently its ability to adapt to a given host, is strongly linked to its own life history as well as the life history of its host. While the effects of parasite life history on their population genetic structure have received some attention, the effect of host social system has remained largely unstudied. In this study, we investigated the population genetic structure of two closely related parasitic mite species (Spinturnix myoti and Spinturnix bechsteini) with very similar life histories. Their respective hosts, the greater mouse-eared bat (Myotis myotis) and the Bechstein's bat (Myotis bechsteinii) have social systems that differ in several substantial features, such as group size, mating system and dispersal patterns. RESULTS: We found that the two mite species have strongly differing population genetic structures. In S. myoti we found high levels of genetic diversity and very little pairwise differentiation, whereas in S. bechsteini we observed much less diversity, strongly differentiated populations and strong temporal turnover. These differences are likely to be the result of the differences in genetic drift and dispersal opportunities afforded to the two parasites by the different social systems of their hosts. CONCLUSIONS: Our results suggest that host social system can strongly influence parasite population structure. As a result, the evolutionary potential of these two parasites with very similar life histories also differs, thereby affecting the risk and evolutionary pressure exerted by each parasite on its host.

Spatial predictions of phylogenetic diversity in conservation decision making.

Considering genetic relatedness among species has long been argued as an important step toward measuring biological diversity more accurately, rather than relying solely on species richness. Some researchers have correlated measures of phylogenetic diversity and species richness across a series of sites and suggest that values of phylogenetic diversity do not differ enough from those of species richness to justify their inclusion in conservation planning. We compared predictions of species richness and 10 measures of phylogenetic diversity by creating distribution models for 168 individual species of a species-rich plant family, the Cape Proteaceae. When we used average amounts of land set aside for conservation to compare areas selected on the basis of species richness with areas selected on the basis of phylogenetic diversity, correlations between species richness and different measures of phylogenetic diversity varied considerably. Correlations between species richness and measures that were based on the length of phylogenetic tree branches and tree shape were weaker than those that were based on tree shape alone. Elevation explained up to 31% of the segregation of species rich versus phylogenetically rich areas. Given these results, the increased availability of molecular data, and the known ecological effect of phylogenetically rich communities, consideration of phylogenetic diversity in conservation decision making may be feasible and informative.