Contribution to the study of prognostic factors in canine oral squamous cell carcinoma

Tese de Doutoramento em Ciências Veterinárias, na Especialidade de Clínica

Characterization of 10 new tetranucleotide microsatellite markers for the European eagle owl, Bubo bubo: Useful tools for conservation strategies

Bubo bubo is the largest owl in the world, showing a wide geographical distribution throughout the Palaearctic region. It underwent a demographic decline in many European countries during the last century and was considered “vulnerable” (Annex II of the CITES). Nowadays, it is classified as “Least Concern” according to IUCN. Despite its ecological importance and conservation status, few polymorphic molecular markers are available to study its diversity and population genetics. We report on the isolation and development of 10 new microsatellites for the Eagle owl, B. bubo. All loci (10 tetra-nucleotide) are characterized by high polymorphism levels. Number of alleles ranged from 5 to 13 and expected heterozygosity varied from 0.733 to 0.840. These microsatellites would be very useful to assess the genetic diversity, connectivity patterns and parentage of B. bubo. This information will allow to establish new conservation strategies and improve the management of the species.

DNA extraction and RAPD markers to assess the genetic similarity among Gelidium sesquipedale (Rhodophyta) populations

A simple method developed for genomic DNA isolation from fungus was tested on the red alga, Gelidium sesquipedale (Clem.) Born et Thur., which is commercially exploited for its high sulfated polysaccharide (agar) content. This method is faster, cheaper, and less toxic than conventional phenol/chloroform methods. Random amplified polymorphic DNA (RAPD) amplifications were performed successfully without the necessity of purifying the DNA. RAPD markers were used to investigate the genetic similarity among three natural populations of G. sesquipedale from southern Portugal. Bulked-genomic DNA samples of 15 different individuals were made in each population. These can be conceived of as a sample of the population DNA. Of the 62 primers screened, 41 produced bands and 22 revealed polymorphisms. Genetic similarities among populations were high. Populations that are further away from each other have the lowest similarity coefficients, whereas the intermediate Ingrina population, located on the south coast, showed higher genetic similarity with the Odeceixe population located on the southwest coast, than with the Sao Rafael southern population. This suggests a higher genetic flow between Odeceixe and Ingrina or the result may be a founder effect in the sense that the species has propagated from the east coast to the south coast of Portugal. We conclude that the use of this isolation method with RAPD analysis is appropriate to characterize the genetic variability of this commercial species along its geographical distribution. Large sample sizes can be screened at a relatively low cost. Finding genetic markers for commercial populations of C. sesquipedale may be of industrial interest.

Dieta do ouriço-branco, Tripneustes ventricosus (Lamarck, 1816), no Arquipélago de Fernando de Noronha, PE

Sea urchins are benthic macroinvertebrates that inhabit shallow coastal waters in tropical and temperate zones. Urchins are usually classified as generalists or omnivores as they can adjust their diet according to the food resources available in the environment. Due to the strong grazing pressure they may exert, urchins have an important role in marine ecosystems, occupying different trophic levels and stimulating the intensification of the dynamics of communities where they occur. In 2004, a monitoring program focused on the population dynamics of the white sea urchin, Tripneustes ventricosus, has been initiated in the Fernando de Noronha Archipelago. At the same time, a surprisingly lack of information on the species biology has been noted, despite their wide geographical distribution and economic importance in many parts of its range. Hence, this work was developed to provide information on the feeding habits of T. ventricosus in the archipelago. Ten specimens were collected between December 2006 and July 2007 at two sites of the archipelago, Air France and Sueste Bay for biometrics and analysis of gut contents. Test diameters ranged from 9.19 cm (± 1.1) to 10.08 cm (± 0.58). Calculated stomach repletion index (IRE) was higher (p <0.05) in the Air France site and also during January and July. The IRE was not correlated to the gonad index. Fifteen different species of algae were detected in a total of 120 stomachs examined: 4 Chlorophytas, 4 Phaeophytas and 6 Rhodophytas. Food diversity (p <0.05) was higher in December 2006 and January 2007. Although several items had a high frequency of occurrence, they were low represented in terms of weight, and consequently, had a low level of relative importance. The brown algae Dictyopteris spp and Dictyota spp, followed by the green algae Caulerpa verticillata accounted for the greatest importance in T. ventricosus diet, comprising about 90% of the consumed items

Distribuição e abundância da malacofauna epibentônica no Parracho de Maracajaú, RN, Brasil

The mollusks present a great taxon variety and life habits in coral reefs being good environmental indicators. It is important to know the distribution pattern of the mollusks and the processes that influence it, so that disturbances in sea ecosystems could be monitored. The present study aims to accomplish the inventory and distribution of epibenthic mollusks in the Parracho of Maracajaú. 23 sites in different habitats of the Parracho were settled: 11 in the reef habitat, 3 in the sandy bottom and 9 in the seagrass bed. Qualitative and quantitative samplings have been done through snorkeling and scuba diving. Three band transects (10m²) were sampled in each site and the data were obtained to each m² of the transect, where the species were counted and the environmental variables (rugosity and recovery of the substratum) were valued. The data were submitted to multivariate analyses in order to find possible distribution patterns that could be associated to the substratum variables. The diversity indexes were calculated for each reef sites and compared with each other. A number of 46 species were registered. The reef habit at should to be the richnest area while the sandy bottom was poorest one. In the reef habitat, the mollusks were associated to rugosity and recovering of frondose algae and zoanthids, while for the seagrass bed, the animals exhibited a richness variation associated to the muddy and sandy sediment. There were found 3 species economically explored, what requires an appropriate management for the maintenance and conservation of the area resources in a sustainable way

Estrutura da população de Coleodactylus natalensis Freire, 1999 (Squamata: Sphaerodactylidae) no Parque Estadual Dunas de Natal, Rio Grande do Norte, Brasil

Coleodactylus natalensis Freire, 1999, an endemic species of Atlantic Forest fragments around the Natal municipality, Rio Grande do Norte State, Brazil, has as type locality the Parque Estadual Dunas de Natal (05º48 S to 05º53 S and 35º09 W to 35º12 W), one of the largest restinga (herb and shrub association on sand dunes along the Brazilian coastline) associate fragment, surrounded by urban zone, placed on setentrional Atlantic Forest limits. We made estimates on populational density, spatial distribution, habitat and microhabitat preferences and feeding ecological aspects like sazonal and sexual variations on diet, prey electivities and niche breadth. We randomly sampled ninety-six 50m2 quadrants in each of the four habitats identified in the study area. Were collected 49 specimens and their stomach contents were analyzed; prey items found were correlated with leaf-litter invertebrates from habitat samples. We found a 98,5 ± 75,5 individuals/ha density, in grouped distribution pattern on densest habitats and random distribution on others habitats. This species lives mostly on leaf-litter in forest habitats, in higher humidity points, with lower temperatures, deeper leaf litter and lower sea level elevations than the randomly chosen points in the study area. Isopoda and Aranae were the most important prey categories in numeric, frequency and volumetric terms. Niche breadth has an intermediate value and was variable in sexual and in habitat terms. There was no correlation between morfometric measures and prey size on diet. The C. natalensis population studied seems to be diet opportunist, although selects larger prey items. The Parque Estadual das Dunas do Natal has several indications of anthropic pressure from the surrounding urban area that may affects the local C. natalensis population. Thus, the fragility of this species calls for urgent conservation efforts

Biologia reprodutiva da Tainha, Mugil curema valenciennes, 1836 (osteichthyes: mugilidade) nas águas costeiras do Rio Grande do Norte

The marine fish white mullet, Mugil curema Valenciennes, 1836 (Osteichthyes: Mugilidae) exhibits a wide geographical distribution, being common in the Brazilian coast and is an important component of the artisanal fisheries. The objective of this study was to investigate the reproductive biology of M. curema in the coastal waters of Rio Grande do Norte. Fish samples were captured on a monthly basis during August, 2008 to July, 2009. The fish specimens were numbered, weighed, measured, dissected and their gonads were removed, weighed, their sex and gonadal development were identified. The length-weight relationship was determined for males and females. The sex ratio, the size at first gonadal maturation and gonadosomatic index (GSI) were calculated and ovarian development was investigated using macroscopic and histological techniques. The fecundity, spawning type and the reproductive period of the species were determined. A total of 366 specimens (186 males and 180 females) were captured. The sex ratio was 1:1 and the females were heavier than males. The estimated values of the angular coefficient for both sexes suggest that the species has isometric growth. The size at which 50% of the population began the process of maturation was 25.9 cm of total length for grouped sex. The macroscopic characteristics of the ovaries showed four stages of development: immature, maturing, mature and spent. However, the microscopic characteristics of the ovaries showed five stages of development: immature, early maturing, late maturing, mature and spent. The development of oocytes indicated five phases: Chromatin-nucleolus (phase I), initial perinucleolar (phase II), final perinuclear (phase II), formation of vitelline vesicle or yolk (phase III), vitellogenic (phase IV) and complete vitellogenesis (phase V). The species has a prolonged spawning period, with two peaks coinciding with the rainy season.

Splitting of a prevalent Mycobacterium bovis spoligotype by variable-number tandem-repeat typing reveals high heterogeneity in an evolving clonal group

Mycobacterium bovis populations in countries with persistent bovine tuberculosis usually show a prevalent spoligotype with a wide geographical distribution. This study applied mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing to a random panel of 115 M. bovis isolates that are representative of the most frequent spoligotype in the Iberian Peninsula, SB0121. VNTR typing targeted nine loci: ETR-A (alias VNTR2165), ETR-B (VNTR2461), ETR-D (MIRU4, VNTR580), ETR-E (MIRU31, VNTR3192), MIRU26 (VNTR2996), QUB11a (VNTR2163a), QUB11b (VNTR2163b), QUB26 (VNTR4052), and QUB3232 (VNTR3232). We found a high degree of diversity among the studied isolates (discriminatory index [D] = 0.9856), which were split into 65 different MIRU-VNTR types. An alternative short-format MIRU-VNTR typing targeting only the four loci with the highest variability values was found to offer an equivalent discriminatory index. Minimum spanning trees using the MIRU-VNTR data showed the hypothetical evolution of an apparent clonal group. MIRU-VNTR analysis was also applied to the isolates of 176 animals from 15 farms infected by M. bovis SB0121; in 10 farms, the analysis revealed the coexistence of two to five different MIRU types differing in one to six loci, which highlights the frequency of undetected heterogeneity.

First record of Ocinebrina nicolai (Mollusca: Gastropoda: Muricidae: Ocenebrinae) in North-Eastern Atlantic waters

Ocinebrina nicolai Monterosato, 1884 a marine mollusc belonging to the Muricidae family is reported from Algarve, south coast of Portugal for the first time and is a new record for the biodiversity of the Portuguese malacological fauna and northeastern Atlantic waters. This species with a medium-size shell for the genus (14–16 mm) was initially sampled during a baseline project that studied marine biotopes in the central Algarve region. This short note presents a brief diagnosis of the species, provides local information on geographical distribution, habitat, and compares it with other congeneric species found in Portugal: Ocinebrina aciculata (Lamarck, 1822) and Ocinebrina edwardsii (Payraudeau, 1826).

First record of Calcinus tubularis on the southern coast of Portugal (Crustacea: Decapoda: Anomura: Diogenidae)

Twenty-five specimens of the hermit crab Calcinus tubularis were observed during SCUBA dives for fauna and flora assessment in the shallow rocky reefs of the southern coast of Portugal between 2003 and 2008. Of the specimens observed, only one specimen used a tube gallery, while the others occupied unidentified gastropod shells with strong bio-cover. Calcinus tubularis is here recorded from the mainland Portuguese coast for the first time. This is the first record of the species in European coastal waters outside the Mediterranean Sea (excluding the Atlantic Islands) and extends the known geographical distribution of the species further north in the north-eastern Atlantic.

Identification of a bitter-taste receptor gene repertoire in different Lagomorphs species

The repertoires of bitter-taste receptor (T2R) gene have been described for several animal species, but these data are still scarce for Lagomorphs. The aim of the present work is to identify potential repertoires of T2R in several Lagomorph species, covering a wide geographical distribution. We studied these genes in Lepus timidus, L. europaeus, Oryctolagus cuniculus algirus, Romerolagus diazi, and Sylvilagus floridanus, using O. cuniculus cuniculus as control species for PCR and DNA sequencing. We studied the identities of the DNA sequences and built the corresponding phylogenetic tree. Sequencing was successful for both subspecies of O. cuniculus for all T2R genes studied, for five genes in Lepus, and for three genes in R. diazi and S. floridanus. We describe for the first time the partial repertoires of T2R genes for Lagomorphs species, other than the common rabbit. Our phylogenetic analyses indicate that sequence proximity levels follow the established taxonomic classification.

An update on the genera Longidorus , Paralongidorus and Xiphinema (Family Longidoridae) in Portugal

The tribe Longidorini within the subfamily Longidorinae (Longidorus spp. and Paralongidorus spp.) and the subfamily Xiphineminae (Xiphinema spp.) are two large nematode groups with about 260 and 230 known species, respectively. They are globally two important groups of ectoparasitic nematodes considered to be major pests because of their activity as vectors of important plant nepovirus, with some species included in the list of quarantine pathogenic organisms in many European countries. Knowledge of the biodiversity and occurrence of this nematode group is a prerequisite for the establishment of sound management strategies and control measures. According to data collected from the databases (such as EPPO, FSTA, and Web of Science) and published in specialised literature, a total of 15 Longidorus, 1 Paralongidorus and 40 Xiphinema species have been recorded as present in Portugal. However, the taxonomic status of some species is controversial, and thus needs to be clarified. A comprehensive review for unravelling the biodiversity and occurrence of nematode species of the genus Longidorus, Paralongidorus and Xiphinema in Portugal is herein provided. This review includes an updated checklist of species with information on the localities, host plants and geographical distribution. Additionally, maps on the species distributions of Longidorinae and Xiphineminae nematodes present in Continental Portugal and the Portuguese Macaronesian islands are provided, as well as unpublished data on authors and comments on the current taxonomic status. Finally, new insights and directions for future research on Longidoridae in Portugal are presented.

Molecules, wing pattern and distribution: an approach to species delimitation in the "loxurina group" (Lepidoptera: Lycaenidae: Penaincisalia)

The wide range of morphological variations in the “loxurina group” makes taxa identification difficult, and despite several reviews, serious taxonomical confusion remains. We make use of DNA data in conjunction with morphological appearance and available information on species distribution to delimit the boundaries of the “loxurina” group species previously established based on morphology. A fragment of 635 base pairs within the mtDNA gene cytochrome oxidase I (COI) was analysed for seven species of the “loxurina group”. Phylogenetic relationships among the included taxa were inferred using maximum parsimony and maximum likelihood methods. Penaincisalia sigsiga (Bálint et al), P. cillutincarae (Draudt), P. atymna (Hewitson) and P. loxurina (C. Felder & R. Felder) were easily delimited as the morphological, geographic and molecular data were congruent. Penaincisalia ludovica (Bálint & Wojtusiak) and P. loxurina astillero (Johnson) represent the same entity and constitute a sub-species of P. loxurina. However, incongruence among morphological, genetic, and geographic data is shown in P. chachapoya (Bálint & Wojtusiak) and P. tegulina (Bálint et al). Our results highlight that an integrative approach is needed to clarify the taxonomy of these neotropical taxa, but more genetic and geographical studies are still required.

Impacts of climatic factors on evolution of molecular diversity and the natural distribution of wild stocks of the giant freshwater prawn (Macrobrachium rosenbergii)

Natural distributions of most freshwater taxa are restricted geographically, a pattern that reflects dispersal limitation. Macrobrachium rosenbergii is unusual because it occurs naturally in rivers from near Pakistan in the west, across India and Bangladesh to the Malay Peninsula, and across the Sunda Shelf and Indonesian archipelago to western Java. Individuals cannot tolerate full marine conditions, so dispersal between river drainage basins must occur at limited geographical scales when ecological or climatic factors are favorable. We examined molecular diversity in wild populations of M. rosenbergii across its complete natural range to document patterns of diversity and to relate them to factors that have driven evolution of diversity in this species. We found 3 clades in the mitochondrial deoxyribonucleic acid (mtDNA) data set that corresponded geographically with eastern, central, and western sets of haplotypes that last shared a common ancestor 1 × 106 y ago. The eastern clade was closest to the common ancestor of all 3 clades and to the common ancestor with its congener, Macrobrachium spinipes, distributed east of Huxley's Line. Macrobrachium rosenbergii could have evolved in the western Indonesian archipelago and spread westward during the early to mid-Pleistocene to India and Sri Lanka. Additional groups identified in the nuclear DNA data set in the central and western clades probably indicate secondary contact via dispersal between regions and modern introductions that have mixed nuclear and mtDNA genes. Pleistocene sea-level fluctuations can explain dispersal across the Indonesian archipelago and parts of mainland southeastern Asia via changing river drainage connections in shallow seas on wide continental shelves. At the western end of the modern distribution where continental shelves are smaller, intermittent freshwater plumes from large rivers probably permitted larval dispersal across inshore areas of lowered salinity.

The geographical co-distribution and socio-ecological drivers of childhood pneumonia and diarrhoea in Queensland, Australia

This study aimed to explore the spatiotemporal patterns, geographic co-distribution, and socio-ecological drivers of childhood pneumonia and diarrhea in Queensland. A Bayesian conditional autoregressive model was used to quantify the impacts of socio-ecological factors on both childhood pneumonia and diarrhea at a postal area level. A distinct seasonality of childhood pneumonia and diarrhea was found. Childhood pneumonia and diarrhea mainly distributed in northwest of Queensland. Mount Isa was the high-risk cluster where childhood pneumonia and diarrhea co-distributed. Emergency department visits (EDVs) for pneumonia increased by 3% per 10-mm increase in monthly average rainfall, in wet seasons. In comparison, a 10-mm increase in monthly average rainfall may increase 4% of EDVs for diarrhea. Monthly average temperature was negatively associated with EDVs for childhood diarrhea, in wet seasons. Low socioeconomic index for areas (SEIFA) was associated with high EDVs for childhood pneumonia. Future pneumonia and diarrhea prevention and control measures in Queensland should focus more on Mount Isa.