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.

Avaliação do efeito do corte horizontal da copa, com máquina de podar de discos, em pomares de pera “Rocha

Current pear pruning making use of pneumatic shears still is a very labour intensive operation. The Proder project “Avaliação da poda mecânica em pomares de pera” was designed to contribute to solutions that would reduce the present dependence in labour and therefore to promote a reduction in pruning costs. This paper shows the results of a trial made to evaluate the influence of mechanical topping in manual pruning complement field work and pear yield. Topping was performed using a Reynolds 6DT 3.0m cutting bar with six hydraulic-driven circular disc-saws mounted in the three point tractor linkage system. The field trial was performed in a commercial orchard with 20 years, planted in an array of 4m x 2m with tree lines oriented in North-South direction. Trees were trained as the central leader system. In this trial, in a randomised complete block design with four replications, two treatments are being compared leading to 8 plots with one line of 14 trees per plot. The treatments tests were: T1 - manual pruning performed by workers using pneumatic shears, in each year; T2 - Topping the canopy parallel to the ground, using a discs-saw pruning machine mounted in a front loader of an agricultural tractor, followed by manual pruning complement performed by workers with pneumatic shears. Tree height and width was measured, before and after pruning. Work was timed and pear yields evaluated. Mechanical topping seems to be effective in the control of tree height, which can contribute to increase 14% of work rates on manual pruning complement. No significant differences in pear yield were found between treatments.