Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Assessment of the Availability of Forest Biomass for Bioenergy Production in Southwest Portugal.

In 2014, Portugal was the seventh largest pellets producer in the World. Since the shortage of raw material is one of the major obstacles that the Portuguese sellets market faces, the need for a good assessment of biomass availability for energy purposes at both country and regional levels is reinforced. This work uses a Geographical Information System environment and remote sensing data to assess the availability and sustainability of forest biomass residues in a management unit with around 940 ha of maritime pine forest. The period considered goes from 2004 to 2015. The study area is located in Southwestern Portugal, close to a pellets factory; therefore the potential Contribution of the residual biomass generated in the management unit to the production of pellets is evaluated. An allometric function is used for the estimation of maritime pine above ground biomass. With this estimate, and considering several forest operations, the residual biomass available was assessed, according to stand composition and structure. This study shows that, when maritime pine forests are managed to produce wood, the amount of residues available for energy production is small (an average of 0.37 t ha -1  year -1 were generated in the study area between 2004 and 2015). As a contribution to the sustainability of the Portuguese pellets industries, new management models for maritime pine forests may be developed. The effect of the pinewood nematode on the availability of residual biomass can be clearly seen in this study. In the management unit considered, cuts were made to prevent dissemination of the disease. This contributes to a higher availability of forest residues in a specific period of time, but, in the medium term, they lead to a decrease in the amount of residues that can be used for energy purposes.

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Contributo para a caracterização das raças serpentinas e charnequeira

Esta dissertação retoma um estudo com 49 cabras e 45 cabritos da raça Serpentina, 24 cabras e 28 cabritos da raça Charnequeira, exploradas em regime extensivo. Pretende ser um contributo para a caracterização destas raças. Com este objectivo calculou-se a fertilidade que foi de 92% nas duas raças e a prolificidade foi de 160% (62,5% partos duplos) na Serpentina e de 141% (57,1% partos duplos) na Charnequeira. A mortalidade dos cabritos, do nascimento até ao desmame, foi de 15,7%, na Serpentina e de 6,6%, na Chamequeira. Os pesos das cobrições ao parto, na Serpentina aumentaram 17% e 13,6% na Chamequeira, apresentando variações significativas na raça, mas não entre raças. Os cabritos de partos simples obtiveram maiores ganhos médios diários do nascimento ao desmame. Não houve variações significativas nos pesos entre machos e fêmeas. Analisaram-se 24 carcaças de cabritos, machos inteiros. O Rendimento Corrigido foi de 52% para as duas e a relação Músculo/Osso de 2,13 (Charnequeira) e 2,12 (Serpentina). O Índice Compacidade para a Charnequeira foi de 11,78 e 11,29 para a Serpentina, evidenciando esta melhor conformação. Definiram-se os seguintes cortes nas carcaças: I-Pá; II-Perna; III- Costela+Sela; IV-Aba; V-Pescoço. Analisou-se o músculo quanto à gordura, proteína, cálcio e fósforo. ABSTRACT: This dissertation retrieves a study involving 49 goats and 45 kids of Serpentina breed, 24 goats and 28 kids of Chamequeira breed exploited in an extensive management. It is intended as a contribution to the characterization of these breeds. To this end, various reproductive parameters were calculated. The fertility stood at 92% in both breeds and the prolificacy in the breed Serpentina was 160% (62.5% in twin births) and in the breed of Chamequeira was 141% (57.1% in twin births). We noted a mortality rate in kids from birth to weaning higher in the Serpentina breed (15.7%), while the Chamequeira breed recorded 6.6%. The evolution of the weights during the mating period to childbirth, increasing 17% in the Serpentina breed and 13.6% in Chamequeira breed. We noted significant variations in each breed, but not between each other. The kids resulting of simple birth had higher average daily weight gain from birth to weaning. We did not note significant variations of weight between males and females. We analysed the carcasses of 24 male kids. The corrected yield was 52% for both breeds and the muscle-bone ratio was 2,13 (Chamequeira) and 2,12 (Serpentina). The compactness index for the Chamequeira breed was 11.78 and 11.29 for the Serpentina breed, showing Serpentina breed had better conformation. We proposed the following cuts in the carcass: I-Shovel, II Leg, III- Rib +Sela, IV-Aba and V-Neck. We also analysed the muscle according to their fat, protein, calcium and phosphorus, aiming to show the dietary interest of this muscle.