The purpose is to create a data-base of the phytoplankton species present in transitional and marine-coastal environments. The data-base will be constructed on the basis of pre-existing data and will include the phytoplankton species observed in the transitional and marine-coastal environments of various eco-regions. For the transitional and marine-coastal environments of the Mediterranean eco-region, reference will be made to scientific papers (Vadrucci et al, in press) or to lists of species already identified, including those identified during previous projects (INTERREG IIIB- TWReferencenetnet, Marine Coastal Monitoring, Puglia Region) conducted by the local experts belonging to RU1. For the other eco-regions, the foreign experts participating in RU1 will provide a data base of the phytoplankton species present in their specific eco-regions. These include the lagoon systems of the southern Pacific area (Polynesia and New Caledonia), the coastal lagoons of Brazil, the lagoon and marine-coastal systems typical of the Baltic eco-region, the North Sea, the northern Atlantic both on the western shore (Sea lochs and estuaries) and the eastern shore (estuaries and coastal lagoons), and finally the lagoon and marine coastal systems of the China Sea and the northern Pacific.

The professional figures involved will be the local personnel of RU1, the foreign experts working with the post-doc research fellows, and other staff involved in the Research unit. - Duration of activity: 12 months Start: 1 month

This activity can be carried out together with the previous one and entails the attribution of the most suitable geometric models to each species identified in the data-base. The geometric shape will then be associated with the genus, taking account of all the deviations that may occur on the species level. The set of geometric models to be associated with the phytoplankton taxa will then be defined. This set will be constructed taking account of the suggestions and the sets of geometrical forms available in the literature (Edler, 1979; Rott, 1981, Hillebrand et al., 1989, Sun and Liu, 2003, Manual for Marine monitoring in the HELCOM Combine programme, 2000), as well as the suggestions of the international experts involved in the project. Specifically, the set of geometric forms will be identified by associating with each genus the most similar geometric form. Since the bio-volume is calculated by measuring the linear dimensions of the associated geometric solid under an optical microscope and applying the relative calculation model, the geometric models will be selected in such a way that they are able to provide a good estimate of the biovolume in reasonable analysis times, the latter being evaluated in terms of the number and type of linear dimension to be measured under the microscope. For this reason, the Ecology laboratory has already carried out research (Vadrucci et al in press) with the aim of identifying a set of geometrical forms to be applied in marine coastal environments of the Mediterranean eco-region. An example of the type of results that will be achieved in this activity is shown in the following table.

The professional figures involved are the local personnel of the research unit and the foreign experts involved in the project - Duration of activity: 24 months; Start: 1 month

The need to implement inter-calibration procedures between experts in this sector emerges in much of the literature (Hillebrand et al., 1989, Sun and Liu 2003), in such a way as to obtain a set of geometric models that are suitable, shared, and applicable in diverse types of aquatic environment and in diverse eco-regions. Concerning the suitability of the models, the selection of the geometric models is always made on the basis of human-eye examination under the microscope, which has led in some cases to errors of evaluation that are often considerable (Kononen et al, 1984, Vadrucci et al in press). For this project, the models will be validated using state-of-the-art instrumentation, especially motorized microscopes and image analysis systems able to reconstruct the structure of the algal cell in 3D. These systems will make it possible to conduct the selection of the geometric models objectively. Concerning the wide applicability, it will be guaranteed by substantial amount of data on which the selection of geometric shapes was based The activity will be carried out both on samples gathered in the field and on isolated algal lines.

The professional figures involved are the local personnel of RU1 and the foreign experts involved in the project - Duration of activity : 36 months Start: 1 month

The activity entails the creation of a gallery of images of the genera and the geometric forms associated with them. The gallery of images will be constructed using images of the algal genera observed in a bright field or in phase contrast. In this way the image visible in the gallery will correspond to the one that the operator commonly observes in the sample during routine analysis. For the geometric models, the the model to be associated with the genus, the number and the type of linear dimensions to be measured and the formula used are all shown. For species that require an estimate of the thickness, a series of indications will be provided for measuring this, extrapolated from the literature of the sector (Menden-Deur, 2000; Verity et al., 1992). However, the final objective is to arrive at an objective and real evaluation of this dimension by means of the research discussed in point T5, which will be used both to validate the models (T3).

The professional figures involved are the local personnel of RU1 and the foreign experts involved in the project - Duration of activity: 30 Start: 1 month

For the execution of these activities there will be a series of experiments in the laboratory, designed to evaluate the effectiveness of using instrumentation that enables the visualization of the algal cells in 3D e for validation of geometic models (T3). This entails the use of a completely motorized inverted microscope, with a reading resolution in the Z axis of 0.05 μm. This microscope is particularly useful for capturing images in 3D, even for species with a cellular thickness of a few μm (for example certain Diatoms). In addition, procedures will be sought that resolve the problems of contrast between the algal cells (linked to the transparency of some forms) and the bottom of the chamber, which can create problems for the capture of images during routine analysis.

The professional figures involved are the local personnel of RU1 and RU2. - Duration of activity: 36 months; Start: 1 month

The activity aims to identify the hardware to be used. The possible interfacing will be analysed, and the modules that enable the analysis software to acquire the images provided by the hardware and to control the hardware regarding the automatic mechanisms that will manage the motorized focusing and the servo-controlled plane of acquisition in the x-y axes will be implemented.

The professional figures involved are the local personnel of RU1 and that of Nikon Instruments S.p.A. - Duration of activity : 6 months Start: 1 month

The activity is aimed at the creation of the core of the software. The basic imaging functions (zoom, movement, luminosity and contrast, balancing, windowing, visualization by channel etc) will be identified and these will be implemented together with the options for saving the images in the most common graphic formats (bmp, tiff, gif, jpg, raw). A proprietary saving format will be defined that will make it possible to save, as well as the image, the optical calibration data with which the image was acquired. Furthermore, once the analysis software that will enable us to identify the structures of interest has been created, it will be possible to view the three-dimensional structures on active apparatus and stereo protections as well as through passive filters and anaglyphic visualizations.

The professional figures involved are the local personnel of RU1 and that of Nikon Instruments S.p.A. - Duration of activity : 6 months; Start: 7 month

Commercial and non-commercial software will be analysed with a view to identifying the most suitable analysis functions (segmentation based on thresholds, isoclines, manual polygonal and continuous selection, segmentation based on region-growing etc). The functions identified that use the calibration data to carry out the necessary measurements (lengths, perimeters, areas, volumes, shape factors) will be implemented.

The professional figures involved are the local personnel of RU1 and that of Nikon Instruments S.p.A. - Duration of activity: 12 months; Start: 5th month

Commercial and non-commercial software will be analysed and the state of the art in the fields of imaging, data mining and machine learning will be evaluated with a view to identifying the most suitable algorithms for the recognition and analysis of known structures that can be used as training sets for processes of automatic classification. The functions identified will be implemented by analysing the complexity of the algorithms. For particularly complex evolutionary algorithms the possibility of parallel or distributed implementations will be considered; the latter can be used by the software by means of remote calls or web services.

The professional figures involved are the local personnel of RU1 - Duration of activity : 24 months Start: 12 month

The most practical way of determining the useful information will be analysed and an appropriate context for integration of the data and cooperation between the users of the system will be established, with a view to identifying the functionalities to be implemented; the objective is to create a collaborative environment that enables the users to exchange data and cooperate in the phase of studying the image and identifying the information. This context will be useful both to promote and facilitate scientific cooperation at a distance, and as an instrument for teaching, by creating an environment in which the teacher shares the instrument (the available views and analysis tools) with students located in whatever part of the world.

The professional figures involved are the local personnel of RU1 and that of IPA s.r.l. - Duration of activity: 12 months; Start: 24 month

In accordance with the indications of the recent Water Framework Directive, WFD 60/2000/EC, the classification of the ecological status of the aquatic ecosystems is based on a comparison between the conditions of each single ecosystem and the so-called "ecosystems of reference", which have never previously been exposed to pressures as a result of human activity or have been exposed to negligible pressure.

The search for ecosystems of reference is thus a key point for the definition of any classification system. In this task, the project aims to analyse the characteristics of the phytoplankton corporations in transitional ecosystems of reference that are present in diverse Eco-Regions, in the search for common models of distribution that make it possible to define quality standards (i.e. optimal conditions) that can also be applied to the Mediterranean area and the North Atlantic, in which it is not possible to observe ecosystems of reference but for which the conditions of reference can be assumed to be similar to those of ecosystems with low human impact.

This activity will be developed by means of a series of pilot studies designed to analyse the characteristics of the size/abundance distributions among the phytoplankton corporations of marine-coastal and transitional environments present in eco-regions other than the Mediterranean; for each eco-region, “reference sites”, i.e. ecosystems in which the impact of human activity is low or absent, will be identified. Specifically, these studies will be carried out in lagoon ecosystems that may be considered of reference, and that is practically immune to significant human pressure, in the following Eco-Regions:

1. the southern Pacific (i.e. Polynesia and New Caledonia),
2. marine-coastal transitional environments along the coasts of Brazil,
3. transitional environments typical of the North Sea and Atlantic eco-region, as well as, for comparison,
4. ecosystems with different levels of human impact in Puglia, including both transitional ecosystems and marine-coastal ecosystems.

This activity is closely linked to the second task and aims to identify the general models of variation of the size-abundance distributions linked exclusively to the climatic variability, geomorphology and hydrodynamics of the sites under study. The experimental activity will be conducted in at least two types of transitional and marine-coastal ecosystem for each eco-region included in the project, with at least three ecosystems and three experimental stations per ecosystem in each type.

The ecosystems identified are classified in relation to their type, according to Roff and Taylor (2000). In accordance with this approach, the transitional ecosystems are classifiable into n° types, identified on the basis of the granulometry of the sediments and the presence and type of vegetation. For each ecosystem considered, a maximum of three categories of habitat are selected and in each of them a sampling point is specified. Since the project intends to provide a range of values of the descriptors on a spatial scale, a single sampling will be carried out. The water samples gathered will be analysed to determine the taxonomic parameters and non-taxonomic parameters.

The professional figures involved are the local personnel of RU1 and the foreign experts involved in the project - Duration of activity: 12 months Start: 7 month

The samples of phytoplankton gathered will be analysed in the laboratory for their taxonomic structure and size structure and the following characteristics of the phytoplankton corporations will be measured: wealth in species, taxonomic diversity, and distinctiveness (as taxonomic indices); total biomass and cellular density (as indices of abundance); biomass by fraction, measurements of central tendency, shape and breadth of the size-abundance distributions (as non-taxonomic indices of size). For all the phytoplankton characteristics, the models of variation between ecosystems, habitats, and types of ecosystem within the same eco-region and between Eco-regions will be analysed. The objective of task 2 is to find common models, invariant for habitat, type of ecosystem and bio-geographical region, of the characteristics of phytoplankton corporations, in which the patterns of variation in response to environmental pressures are clearly distinguishable from variations between different conditions of reference. The set of data gathered in this way will enrich the data we already have and will serve to establish the classification criteria of the non-taxonomic descriptors.

The professional figures involved: local personnel of RU1 - Duration of activity: 24 months Start: 7 month

For each of the characteristics of the phytoplankton corporations a methodological standardization will be carried out in order to evaluate:

1. the dimension of the sample (n° of cells to be counted and measured) necessary for the accurate analytical determination of the values of the characteristic (coefficient of variation of the average ≤ 10%);
2. the relations existing between the dimension of the sample and density of the phytoplankton corporations;
3. the number and optimal position of the sampling stations for each ecosystem as a function of [3a] the surface area of the ecosystem, [3b] water circulation; [3c] the types of habitat in the sediments.

The aims of task 3 are thus to evaluate:

• The suitability of the diverse characteristics for describing perturbation pressure;
• The accuracy of the size-abundance distribution descriptors as descriptors of the ecological status of transitional ecosystems;
• The optimal model of classification for each of the descriptors selected (linear vs. type 3 functional response)
• The distribution of the normalized values into classes of ecological status, in accordance with the different national and international regulations in force (in Europe in accordance with the Water Framework Directive)

The professional figures involved are the local personnel of RU1 and the foreign experts involved in the project - Duration of activity : 36 months Start: 1 month

The models of classification determined on the basis of the data gathered in the ecosystems of reference and in the ecosystems in Puglia with different levels of perturbation will be validated with data already available at the Laboratory of Ecology at the University of Salento and compared to previous analysis in the Mediterranean and Black Sea Eco-regions. Specifically, conditions will be analysed in which pronounced gradients of perturbation have already been recognised; the perturbation we are dealing with here is saline [Margherita di Savoia Saltpans (IT), Narta lagoons (AL) Kalloni lagoon (GR)]; thermal [Piallassa Baiona (IT)]; chemical [the wetlands in Brindisi and Grado Marano (IT), Varna Lagoon (BG)]. The task is centred in particular on the validation of the descriptors of phytoplankton size-abundance distributions.

The professional figures involved: local personnel of RU1 - Duration of activity: 30 months Start: 7 month

The aim of this task is to obtain a certification of quality of the descriptors. The task thus entails a precise methodological validation and a calibration of all the methodological phases of the procedure for analysing a sample that lead to the calculation of the descriptors.This experimental activity will be carried out by the local personnel of the Research Unit and the foreign institutions

The professional figures involved: local personnel of RU1 - Duration of activity: 30 months Start: 7 month

The development of this task entails three phases of implementation. In the first phase a thorough bibliographical survey will be performed of the international scientific literature and the literature on the international and national regulations governing the ecological status of aquatic ecosystems, the existing biological descriptors and the relevant guidance that the regulations provide. The resulting database will be analysed with reference to the following criteria: the scientific robustness, efficacy and accuracy of the descriptors, the potential for generalizing their use, the simplicity of obtaining and interpreting the data and the existence of arbitrary assumptions (e.g. allocation of the taxa to classes of sensitivity). In the second phase, on the basis of the results obtained in WP2, the characteristics of the phytoplankton corporations suitable to be used as descriptors will be selected, taking account of the requirements of the regulations. The descriptors will then be normalized in such a way as to render them suitable for a classification. In the third phase, the phytoplankton descriptors will be selected that are most appropriate for the classification of the ecological status of the aquatic ecosystems from which the original samples were taken. From these, by means of image analysis or other techniques, analytical data will be obtained regarding the taxonomic structure and the size-abundance distribution of the individuals in the samples.

The professional figures involved are the local personnel of RU1 and IPA s.r.l. - Duration of activity: 6 months; Start: 25 month

The task is based on the products of T1 and is developed in two phases of implementation. The first phase entails designing the data structure and the calculation models of the software. This phase includes: i. the identification of the input file structure, whose design will be parallel to that of the output files of the image analysis system; ii. the definition and the optimisation of the calculation models for the descriptors; iii. the extension of the calculation models to the zooplankton, macro-zoobenthos and ichthyic fauna. The second phase of implementation entails: i. the construction of a statistical and graphical software for the analysis of the databases obtained as the final product of WP1; ii. the specification of parameters for each descriptor for classifying the ecological status of the ecosystem from which each individual water sample was taken.

The professional figures involved are the local personnel of RU1,RU2 and IPA s.r.l. - Duration of activity: 6 months; Start: 30 month

The project includes the activation of a training plan relevant to the scientific issues tackled in the project.

For this project the following training plan is proposed:

Title of the training plan: Methodological Aspects linked to the use of morphometric descriptors of phytoplankton corporations in the evaluation of the state of health of transitional and marine-coastal ecosystems.

Location of the training plan: Laboratory of Ecology, Department of Biological and Environmental Science and Technology, Centro Ecotekne, 73100 LECCE (Italy)

Duration of the training plan: 2 weeks + 1 week

Proposed Start Date: 7th month (2-week module) 33rd month (1-week module)

Description of the training plan: The training plan has the aim of training and increasing the knowledge of personnel who are already qualified for the identification and the classification of phytoplankton species and the use of programming tools. Specifically, the course will follow three parallel training programs.

The first is the “ecological” training program, intended for students with skills in the field of phytoplankton biology; it will be designed to improve knowledge of the descriptors of the ecological status of aquatic ecosystems, in order to apply the technological product to monitoring programmes;

The second is the "ICT" training program, intended for students with computer skills in the sectors of image analysis and parallel and distributed programming; it will aim at enhancing skills in the use of tools for analysing biometric data and the dimensional structures of phytoplankton; specifically it will look at themes relating to image analysis, tools for evolutionary computation (Genetic Algorithms and Neural Networks), Parallel Computing and Grid Computing, Data Mining and the application of these concepts to the analysis of the state of health of the ecosystems.

The third is the “PMI and software for the environment” training program, intended for potential stakeholders such as representative of national and international environmental protection agencies and administrations involved in the monitoring plans for the protection of transitional and coastal marine ecosystems. They could be the potential users for acquisition of software. The training program will aim to presentation of software for the environment and at their use in the monitoring plans.

The training plan related to the ecological and ICT training programs will be organised into 10 training modules, which will be taught in the course of the first two weeks of the training plan. Some of the modules are common to both programs, others are specific, plus one training module (PMI and Software for the Environment) which will be developed in the 33rd month of the project which will entail practical activities aimed at the application and the validation of ICT tools for the evaluation of the state of quality of the aquatic ecosystems.

The implementation of the training plan involves both theoretical lessons in the classroom and practical activities in biology or computing labs, for which there will also be e-learning activities. The common training, for the first two training programs plans, will be designed: • to develop issues related to ecological principles of monitoring the state of quality of aquatic transitional and marine coastal ecosystems and in particular the use of phytoplankton as an indicator of the ecological status of these environments; • to develop aspects related to the new methodological approaches to classifying the state of health of aquatic environments based on non-taxonomic descriptors (i.e. size-abundance distributions) of phytoplankton corporations. The “ecological” training program will have the following objectives:

1. to provide theoretical aspects that take account the most recent literature concerning morphology, taxonomy, classification and phylogenesis of phytoplankton corporations,
2. to teach theoretical and methodological aspects of phytoplankton as bio-indicators of the ecological quality of the aquatic transitional and marine-coastal ecosystems
3. to provide indications regarding the new methodological approaches to classifying the state of quality of the aquatic transitional and marine-coastal ecosystems based on the use of non-taxonomic descriptors, measured with optical microscopy techniques supported by image analysis systems.
4. to teach, by means of practical activities, methods and criteria for the correct identification of species
5. to teach, by means of practical activities, ways of recording phytoplankton descriptors, with particular reference to non-taxonomic descriptors, linked to the dimensional characteristics of the individuals, measured with optical microscopy techniques, and to train students in the use of specific software
6. to provide indications for the evaluation of the criteria for the classification of the state of health by means of non-taxonomic descriptors and to train students in the use of specific software.

This objective will be fulfilled in the last week of the training plan

The "ICT" training program will have the following objectives:

1. to provide general principles concerning image analysis systems
2. to teach the general principles of evolutionary computation (genetic algorithms and neural networks)
3. to teach the general principles of parallel and distributed programming and grid computing
4. to teach the general principles of treatment of data and data mining in particular

The professional figures involved: local personnel of RU1 and RU2 ¬- Duration of activity: 1 month Start: 18 month

The teachers will be selected on the basis of the training plan established and will include both teachers belonging to RU1 and RU2 and university and non-university teachers, who may also belong to foreign institutions.

The professional figures involved: local personnel of RU1 - Duration of activity: 1 month Start: 18 month

Persons eligible for the training plan:

The course will be open to about 20 people resident in the EU.

The criteria for the selection of the participants are as follows:

1. the student must hold a degree in Biological or Environmental Science or in Information and Communication Technology.
2. almost the 50% of the student must be compatible with the figure of young researcher, i.e. he/she must be less than 35 years of age and have at least 4 years of experience in the specific sector of reference.
3. the student must have previous experience in the field of the taxonomy of phytoplankton or in the field of software programming for image analysis systems.
4. the student must be involved, in his/her institution of origin, in research specific to the sector.

The professional figures involved: local personnel of RU1 Duration of activity: 1 month Start: 18 month