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.

Toxicological assessment of novel green biocides for cultural heritage.

The damaging of buildings and monuments by biological contamination is a cause of serious concern. Biocides based on chemical toxic compounds have been used to mitigate this problem. However, in the past decade many of the most effective biocides have been banned due to their environmental and health hazards. Therefore, proper remediation actions for microbiologically contaminated historic materials based on environmentally safe solution is of vital importance. Bacillus species are emerging as a promising alternative for built heritage treatment. They produce a great diversity of secondary metabolites with biological activity, well known to possess antagonistic activities against many fungal pathogens. In order to evaluate the antifungal activity of the novel biocides produced in our laboratory by cultures of selected bacterial strains, liquid interaction assays using four biodeteriogenic fungi were achieved, revealing a nearly 100% of inhibitory capacity to fungal proliferation. To confirm their effective safe toxicological properties, in vivo tests using two different biological models were performed. The lyophilized supernatant of the Bacillus culture broth showed no lethality against brine shrimp and also no toxicological effects in Swiss mice through administration of acute dose of 5000 mg/kg by oral gavage. In fact, the bioactive compounds were no lethal at the tested dose unlike Preventol® (commercial biocide) that induced acute toxicity with 10 times minor concentration dose administrated in the same conditions. Therefore, the new bioactive compounds that suppress growth of biodeteriogenic fungi on historical artworks, presenting at the same time no toxicity against other living organisms, constituting an efficient and green safe solution for biodegradation/biodeterioration treatment of Cultural Heritage.

Transferability of environmental favourability models in geographic space: The case of the Iberian desman (Galemys pyrenaicus) in Portugal and Spain

Transferring distribution models between different geographical areas may be problematic, as the performance of models outside their original scope is hard to predict. A modelling procedure is needed that gets the gist of the environmental descriptors of a distribution area, without either overfitting to the training data or overestimating the species’ distribution potential.We tested the transferability power of the favourability function, a generalized linear model, on the distribution of the Iberian desman (Galemys pyrenaicus) in the Iberian territories of Portugal and Spain.We also tested the effects of two of the main potential constraints on model transferability: the analysed ranges of the predictor variables, and the completeness of the species distribution data. We modelled 10 km×10km presence/absence data from Portugal and Spain separately, extrapolated each model to the other country, and compared predictions with observations. The Spanish model, despite arguably containing more false absences, showed good predictive ability in Portugal. The Portuguese model, whose predictors ranged between only a subset of the values observed in Spain, overestimated desman distribution when transferred.We discuss possible reasons for this differential model behaviour, and highlight the importance of this kind of models for prediction and conservation applications

ASSESSMENT OF ENERGY CONSUMPTION IN GREENHOUSE PRODUCTION IN THE WEST REGION OF PORTUGAL

Greenhouse production is a very important activity in the West region of Portugal, with an area of approximately 800 ha where the regular production consists in two crops per year, one in winter-spring and the other in summer-autumn. Many growers are now prepared to better exploit market opportunities, since they know that the big export window opportunity is from June to September, when the production is difficult in other regions of south due to high temperatures. Grower’s use new and more productive varieties, either in soil or hydroponic systems, mostly in unheated greenhouses, naturally ventilated, and equipped with modern fertigation systems. Greenhouse production causes some environmental impacts due to the high use of inputs. Several improvements in technologies and crop practices may contribute to increase the use efficiency of resources, decreasing the negative environmental impacts. Greenhouse vegetable production in Northern EU countries is based on the supply of heating and differs significantly from the production system in the Southern EU countries. In the Northern countries, direct energy inputs, mostly for heating, are predominant while in the South the indirect energy input is also important, mainly associated with fertilizers, plastic cover materials and other auxiliary materials. The main objective of this work was to characterise the greenhouse production systems in the West region of Portugal, in order to evaluate the energetic consumptions (direct and indirect), the GHH emissions, the production costs and the farmer’s income. With this work the mostly important inputs were identified, allowing proposing alternative measures to improve efficiency and sustainability. All the data was obtained by surveys performed directly with growers, previously selected to be representative of the crop practices and greenhouse type of the region. However, more research should be performed in order to develop and to test technologies capable to improve resources use efficiency in greenhouse production.

Assessment of the spatial variability in Tall wheatgrass forage using LANDSAT 8 satellite imagery to delineate potential management zones

Little information is available on the degree of within-field variability of potential production of Tall wheatgrass (Thinopyrum ponticum) forage under unirrigated conditions. The aim of this study was to characterize the spatial variability of the accumulated biomass (AB) without nutritional limitations through vegetation indexes, and then use this information to determine potential management zones. A 27-×-27-m grid cell size was chosen and 84 biomass sampling areas (BSA), each 2 m(2) in size, were georeferenced. Nitrogen and phosphorus fertilizers were applied after an initial cut at 3 cm height. At 500 °C day, the AB from each sampling area, was collected and evaluated. The spatial variability of AB was estimated more accurately using the Normalized Difference Vegetation Index (NDVI), calculated from LANDSAT 8 images obtained on 24 November 2014 (NDVInov) and 10 December 2014 (NDVIdec) because the potential AB was highly associated with NDVInov and NDVIdec (r (2) = 0.85 and 0.83, respectively). These models between the potential AB data and NDVI were evaluated by root mean squared error (RMSE) and relative root mean squared error (RRMSE). This last coefficient was 12 and 15 % for NDVInov and NDVIdec, respectively. Potential AB and NDVI spatial correlation were quantified with semivariograms. The spatial dependence of AB was low. Six classes of NDVI were analyzed for comparison, and two management zones (MZ) were established with them. In order to evaluate if the NDVI method allows us to delimit MZ with different attainable yields, the AB estimated for these MZ were compared through an ANOVA test. The potential AB had significant differences among MZ. Based on these findings, it can be concluded that NDVI obtained from LANDSAT 8 images can be reliably used for creating MZ in soils under permanent pastures dominated by Tall wheatgrass.

Assessment of environment, land management, and spatial variables on recent changes in montado land cover in southern Portugal

Montado decline has been reported since the end of the nineteenth century in southern Portugal and increased markedly during the 1980s. Consensual reports in the literature suggest that this decline is due to a number of factors, such as environmental constraints, forest diseases, inappropriate management, and socioeconomic issues. An assessment on the pattern of montado distribution was conducted to reveal how the extent of land management, environmental variables, and spatial factors contributed to montado area loss in southern Portugal from 1990 to 2006. A total of 14 independent variables, presumably related to montado loss, were grouped into three sets: environmental variables, land management variables, and spatial variables. From 1990 to 2006, approximately 90,054 ha disappeared in the montado area, with an estimated annual regression rate of 0.14 % year-1. Variation partitioning showed that the land management model accounted for the highest percentage of explained variance (51.8 %), followed by spatial factors (44.6 %) and environmental factors (35.5 %). These results indicate that most variance in the large-scale distribution of recent montado loss is due to land management, either alone or in combination with environmental and spatial factors. The full GAM model showed that different livestock grazing is one of the most important variables affecting montado loss. This suggests that optimum carrying capacity should decrease to 0.18–0.60 LU ha-1 for livestock grazing in montado under current ecological conditions in southern Portugal.