Trophic signatures of small-scale fishing gears: implications for conservation and management

We quantified the ecosystem effects of small-scale gears operating in southern European waters (Portugal, Spain, Greece), based on a widely accepted ecosystem measure and indicator, the trophic level (TL). We used data from experimental fishing trials during 1997 to 2000. We studied a wide range of gear types and sizes: (1) gill nets of 8 mesh sizes, ranging from 44 to 80 mm; (2) trammel nets of 9 inner panel mesh sizes, ranging from 40 to 140 mm; and (3) longlines of 8 hook sizes, ranging from Nos. 15 (small) to 5 (large). We used the number of species caught per TL class for constructing trophic signatures (i.e. cumulative TL distributions), and estimated the TL at 25, 50 and 75% cumulative frequency (TL25, TL50, TL75) and the slopes using the logistic function. We also estimated the mean weighted TL of the catches (TLW). Our analyses showed that the TL characteristics of longlines varied much more than those of gill and trammel nets. The longlines of large hooks (Nos. 10, 9, 7, 5) were very TL selective, and their trophic signatures had very steep slopes, the highest mean TL50 values, very narrow mean TL25 to TL75 ranges and mean TLW > 4. In addition, the mean number of TL classes exploited was smaller and the mean TL50 and TLW were larger for the longlines of small hooks (Nos. 15, 13, 12, 11) in Greek than in Portuguese waters. Trammel and gill nets caught more TL classes, and the mean slopes of their trophic signatures were significantly smaller than those of longlines as a group. In addition, the mean number of TL classes exploited, the mean TL50 and the TLW of gill nets were significantly smaller than those of trammel nets. We attribute the differences between longlines of small hooks to bait type, and the differences between all gear types to their characteristic species and size-selectivity patterns. Finally, we showed how the slope and the TL50 Of the trophic signatures can be used to characterise different gears along the ecologically 'unsustainable-sustainable' continuum.

Insights into population structure of Diplodus vulgaris along the SW Portuguese coast from otolith elemental signatures

The common two-banded sea bream (Diplodus vulgaris) is an important fish in the marine ecosystems of the NW Atlantic and Mediterranean. In southern Portugal it is a major fishery resource being targeted mainly by the artisanal fleets. Although there is some knowledge of the age, growth and reproductive biology of the species, information about its population structure is scarce and somewhat limited to the Mediterranean Sea. In this study the otolith elemental signatures of 90 specimens of D. vulgaris of the same age group (2+ years) and cohort collected from the important fishery regions of SW Portugal (Sesimbra, Sagres and Faro) have been analysed by inductively coupled plasma mass spectrometry (ICP-MS). Two different methodologies have been applied: solution based analysis of the whole otoliths; representative of the entire life-history prior to capture, and laser ablation analysis of otolith cores; representative of the larval and early post-settlement phase. Whole otolith comparisons utilised Sr/Ca, Ba/Ca, Mn/Ca, Li/Ca and Ni/Ca to demonstrate regional population structure. Classification accuracy rates from linear discriminant function analyses (LDFA) of whole otolith chemistry data were high for each region; Faro - 93%, Sagres - 90% and Sesimbra - 80%. Comparison of the otolith core chemistry utilised Sr/Ca, Ba/Ca, Mn/Ca and Mg/Ca and Zn/Ca. LDFA for the otolith core chemistry also achieved accurate classification for samples from Sesimbra (73%), but there was high overlap of otolith chemistry between samples from Faro and Sagres (47 and 43% classification accuracy respectively). The whole otolith results suggest that D. vulgaris are resident in the regional fishing areas during the juvenile phase. Both the core and whole otolith chemistry data supported separation of the Sesimbra fishery region from the more southern and closely associated Faro and Sagres regions for management purposes. However, while the whole otolith data indicated that the populations at Faro and Sagres likely remained separated in the juvenile stage, the otolith core chemistry data was inconclusive as to whether recruitment to these two areas was derived, or not, from different spawning areas.

Biogeography of the fire salamander (Salamandra salamandra, L.) in Portugal

Dissertação de mest., Biologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2008

Profiles of ageing in the South of Portugal: education and cognitive functioning in elderly persons living in institutionalized and community contexts

The region of the Algarve shows huge differences between the coastline where population in the urban areas grows, and the inland rural areas, in some cases very isolated, which frequently have high ageing indexes. This general scenario, with an elderly population with very different economic and social conditions, frames the ongoing PhD research designed as a cross-sectional study of an intentional sample of elderly persons. The basic theoretical framework departs from the perspective of developmental psychology of life-span and the model of selection, optimisation and compensation for optimal ageing (Baltes & Baltes, 1990; Freund & Baltes, 2002). The present study is a first step in the analysis of empirical data collected in the PhD sample (N=156; age range 65 to 97 years; M = 80.4 years; SD = 7.2 years). Its purpose is to assess the cognitive functioning of participants, screening for cognitive impairment and examine the relations between the cognitive status of the subjects and a number of selected variables including educational level, age, physical activity and living contexts of the subjects. We accessed the cognitive status of the participants with the Portuguese version of Mini Mental State Examination (MMSE) finding a 10.3% prevalence of positive cases with cognitive impairment. The results also show significant relationships between the cognitive status accessed by the MMSE and educational level, professional qualification, age, living arrangement and activity level of the participants. The relationship verified between educational level and cognitive status of the participants was the largest correlation found in the study with the variability in educational level accounting for 44.8% of the variability in MMSE score. This results points in the same direction of several lines of research that corroborate the strong intercorrelation between education and cognitive functioning in old age.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

In two waters: contemporary evolution of lagoonal and marine white seabream (Diplodus sargus) populations

Brackish water ecosystems are often exposed to wide variations in environmental variables, including temperature and salinity, which may cause strong selective pressures on organisms modifying the genetic patterns of species. The aim of this work was to test whether there is a ‘divergence-with-gene flow’ in coastal lagoon populations of white seabream (Diplodus sargus) (Ria Formosa, S Portugal and Mar Menor, SE Spain) respect to four marine populations, by using partial sequences of cyt b mitochondrial gene and information from nine microsatellite loci. Genetic diversity was highest in both coastal lagoons (Mar Menor and Ria Formosa) considering mitochondrial and nuclear markers. Although some of FST population pairwise comparisons were not significant, analyses of molecular variance (AMOVAs) detected differences between groups (coastal lagoon and marine) close to significance. Also, only two haplotypes (Cytb-17 and Cytb-18) were detected in both coastal lagoon sampling sites and these localities (Mar Menor and Ria Formosa) showed the highest number of singletons, some of them with a high number of mutations, as has been already described for other Mar Menor populations (Pomatochistus marmoratus and Holothuria polii). Also, several tests detected significant positive and balancing selection considering mtDNA and microsatellite data. These data support the hypothesis of selection as one of the drivers of the genetic differences found between coastal lagoon and marine populations. The life strategy adopted by Diplodus sargus in coastal lagoons allows it to decrease its mortality rate and improve the heritability of its genes. Also, the increase time spent in coastal lagoons with different temperatures and salinities favours the fitness selection and the maintenance of exclusive haplotypes and genotypes in coastal lagoon inhabitants favouring the ‘divergence-with-gene-flow’.

Estudo da variabilidade genética de marcadores moleculares ligados ao gene da MGP e sua aplicação para paternidades em Sparus aurata

Dissertação de Mestrado, Biologia Marinha, Especialização em Biotecnologia Marinha, Faculdade de Ciências do Mar e do Ambiente, Universidade do Algarve, 2008

Marine foundation species shifting ranges and genetic baselines

This thesis revealed the most importance factors shaping the distribution, abundance and genetic diversity of four marine foundation species. Environmental conditions, particularly sea temperatures, nutrient availability and ocean waves, played a primary role in shaping the spatial distribution and abundance of populations, acting on scales varying from tens of meters to hundreds of kilometres. Furthermore, the use of Species Distribution Models (SDMs) with biological records of occurrence and high-resolution oceanographic data, allowed predicting species distributions across time. This approach highlighted the role of climate change, particularly when extreme temperatures prevailed during glacial and interglacial periods. These results, when combined with mtDNA and microsatellite genetic variation of populations allowed inferring for the influence of past range dynamics in the genetic diversity and structure of populations. For instance, the Last Glacial Maximum produced important shifts in species ranges, leaving obvious signatures of higher genetic diversities in regions where populations persisted (i.e., refugia). However, it was found that a species’ genetic pool is shaped by regions of persistence, adjacent to others experiencing expansions and contractions. Contradicting expectations, refugia seem to play a minor role on the re(colonization) process of previously eroded populations. In addition, the available habitat area for expanding populations and the inherent mechanisms of species dispersal in occupying available habitats were also found to be fundamental in shaping the distributions of genetic diversity. However, results suggest that the high levels of genetic diversity in some populations do not rule out that they may have experienced strong genetic erosion in the past, a process here named shifting genetic baselines. Furthermore, this thesis predicted an ongoing retraction at the rear edges and extinctions of unique genetic lineages, which will impoverish the global gene pool, strongly shifting the genetic baselines in the future.