The distinctive location along the continuum between dry land and completely submerged land, between continental fresh water and sea water, gives these environments specific ecological characteristics and an intrinsic heterogeneity, represented both by the intra-habitat variations of the chemical and physical parameters (e.g. salinity, nutrients and hydro-dynamics), and by the variability between habitats, characterized by extremely different “earth-water” interfaces.

Moreover, transitional and marine-coastal ecosystems receive the products of the metabolism of roughly 60% of the total population of the biosphere, since this is how much of the population lives along the coasts, and they are thus exposed to high risks of perturbation and deterioration of their ecological state.

These distinctive characteristics of aquatic transitional and marine-coastal ecosystems have highlighted the need to develop effective plans for monitoring their environmental state.

Recent national and EU regulations [Legislative Decree 152/99 and subsequent modifications, Water Framework Directive (WFD, 2000/60/EC)] place great importance on the provision of guidelines for the establishment of monitoring plans that entail the classification of the ecological state of such ecosystems.

The accuracy of the monitoring activities is fundamentally linked to the choice of the descriptors and the correspondence between the spatial and temporal scales in which the information is gathered and the dynamics of the descriptors themselves.

The aforementioned national and EU regulations see phytoplankton as one of the most significant biological elements for the evaluation of the state of health of aquatic transitional and marine-coastal ecosystems. However, these regulations provide only general indications on the relative descriptors, which represent the measurable variables of such elements, to be included in the monitoring plans.

The selection of suitable descriptors is a critical part of the creation of a monitoring programme. For this reason the scientific community has been working for many years on the development, both conceptual and methodological, of descriptors that are effective, reliable, and simple to apply [2].

As for the phytoplankton, there is currently much debate among scientists over the suitability of descriptors and indices based on the taxonomic recognition of the species (for which the regulations also provide classification criteria), rather than on aspects relating to functional relationships or size fractions. The latter group of descriptors are generally referred to as non-taxonomic descriptors.

Non-taxonomic descriptors have a number of advantages on the operational and conceptual level, but currently lack an adequate experimental basis, sufficient data, tested evidence and methodologies comparable to those used for taxonomic study, for which, in contrast, there exist standardized methodological procedures. For many years, the ecology group of the University of Salento has been involved in the development of non-taxonomic descriptors of the state of health of aquatic transitional and marine-coastal ecosystems based on the determination of the bodily dimensions of phytoplankton cells.

These descriptors are linked to aspects relating to the morphological and dimensional characteristics of the phytoplankton organisms and may be defined both on an individual level (as bio-volume, surface area or ratio of surface area to volume [26]), and on the corporation level, either as the distributions of the bodily dimensions of the organisms in relation to their abundance (body size-abundance spectra [42]), or as the contribution to phytoplankton biomass of the size classes, consisting of pico-, nano- and micro-phytoplankton, following Sieburth (1979) [34]).

On a conceptual level, these descriptors have a strong theoretical basis, which is associated with body-size theory [23][20] [44][24] and metabolic theory [3], since the whole of the individual metabolism, a large part of the biological cycle [4][7] and even the interactive relationships [1][38][14][15] that the individuals have with individuals of the same and different species, all depend on the bodily dimensions of the individuals themselves.

On these themes the ecology group of the University of Salento has produced important scientific papers that have been published in the most prestigious periodicals of this sector [1][2][32][33].

• they are easier to determine;
• they are easier to inter-calibrate;
• they eliminate the difficulties linked to the taxonomic classification of the species;
• they allow for an easier comparison between ecosystems, since they make it possible to resolve the difficulties linked to the heterogeneity of the taxonomic structure in transitional and marine-coastal ecosystems;
• they are quicker and the costs in terms of personnel can be lower.

Finally, there is much experimental evidence showing the sensitivity of dimensional structures to environmental forcing and human-generated pressure. Indeed, much recent research, conducted above all in marine/coastal settings, has demonstrated the existence of significant variations in the size fractions [12][37][43] as a function of the trophic state of the ecosystems – as this increases, so does the largest dimensional class (micro-phytoplankton). Other studies have shown how the size-abundance distribution of the phytoplankton corporations varies significantly in relation to human-generated or environmental forcing [5][6][9][8][11][17][19][25][30][28][27][29][40][46]. On these aspects the University of Salento has published papers [32][33][41] and cooperated with the National Agency for the Environment and Technical Services, which in Italy has the task of drawing up plans and defining national criteria for monitoring the waters, for which it has also set up a training course in the innovative methodological tools for monitoring for all the Italian Environmental Protection Agencies (ARPA). On the procedural and methodological level the development of descriptors linked to bodily dimensions depends on the capacity to support biometric and ecological knowledge with a technologically adequate detection tool. One of the starting points in the determination of descriptors of the bodily dimensions of the phytoplankton concerns the way in which the bio-volume and the cellular surface of the algal cells is estimated [10][13][35][36][39]. Specifically, the accurate determination of the bio-volume is fundamental in that it constitutes the starting point for establishing the size-abundance spectra, on which the scientific community has been focusing its attention in view of its potential use as a descriptor of the ecosystem’s state of health. Bio-volume can be estimated by various automatic or semiautomatic methods. The automatic systems include methods based on the electronic counting of particles (using the Coulter principle), those based on flow cytometry and those based on automatic image analysis systems [13][36]. The use of these systems, however, is affected by a series of problems that prevent them from being applied in routine analysis. For example, both methods based on the use of electronic particle counters and methods that use flow cytometry yield a low taxonomic resolution, limited to the level of the groups that are discernable with such methods; with the former, this means size classes, with no distinction between living and non-living parts, while in the latter case we are dealing with groups of algae with a different pigmentary structure. These methods therefore do not allow for estimates of bio-volume for species or genera, necessary in many studies of phytoplankton ecology. Finally, regarding methods based on the use of automatic image analysis systems, this technique has been very popular in the fields of cytology and bacteriology, where the homogeneity of the particles that constitute the object of study (tissue cells, bacteria etc) has facilitated the development of techniques of automatic detection both for counting and for the determination of the structural (dimensional) characteristics of the particles analysed. However, the application of such techniques for counting and determining the morphometric characteristics of phytoplankton cells has proved to be more problematic, since they show much broader morphological variability than tissue or bacteria cells. For this reason, the most common method today among the scientific community for estimating bio-volume and cellular surfaces is manual or semiautomatic, in which the algal cells are assimilated to the geometric solids that are most similar to the cellular shape, of which the linear dimensions necessary for the calculation are measured by optical microscope. The accuracy of the method depends on the set of geometrical forms selected, which must be as similar as possible to the morphological structure of the species. Concerning this aspect, one of the problems most frequently discussed by the scientific community is whether such selection should aim to enhance practicality or accuracy. In the first case geometrically simple forms are selected which, despite being in some cases rather inaccurate, do allow for considerable savings in terms of the costs and the time required for each determination [10]. In the second case, more complex geometrical forms are selected, which require the measurement of a larger number of dimensions and consequently also more time and higher costs, especially in terms of personnel [36]. Another problematic aspect of the application of this method is a consequence of the application of different sets of geometrical forms identified in different research groups, which very often results in the production of data that are not comparable between ecosystems. The different sets of geometrical forms proposed are characterised by their local application, limited to the eco-regional level, the ecosystem level (freshwater or marine ecosystems) and the corporation level (phytoplankton or phytobenthos) [13]. Some of these sets of geometrical forms furthermore are strongly influenced by the type of dominant species, and some are published in periodicals that are not widely available, meaning that they are difficult to access for the majority of the scientific community in this sector. In the last few years some sets of geometrical forms have begun to be used more widely. However they need to be expanded to include the phytoplankton of transitional waters, for which specific sets of geometrical forms have never been published. Furthermore, besides these two aspects (selection of the set of forms and their availability), it is necessary to take another purely methodological aspect into account, which has until now posed a further obstacle to the widespread adoption of the method; in many laboratories the determination of bio-volume is based almost entirely on manual measurement, using a micrometer associated with an eyepiece, of the cellular dimensions necessary for estimating the bio-volume and the cellular surface of each algal cell. This manual method requires long analysis times and high costs in terms of personnel. The introduction of image analysis systems applied to the field of microscopy has opened up new perspectives for the determination of the morphometric characteristics of phytoplankton cells. Indeed, image analysis systems enable the acquisition of images in the eyepiece field and subsequently make it possible to highlight the objects (phytoplankton cells) and measure some of their dimensional characteristics (direct and derived). For phytoplankton however, although image analysis systems open up broader perspectives than manual techniques, their application to estimating morphometric characteristics cannot yet be completely automated, as has been done in the biological fields mentioned above (i.e. cytology, bacteriology). The impossibility of using a completely automated procedure is linked to the heterogeneity of phytoplankton, characterized by cellular forms that are highly diverse both in terms of structure and of dimensions, as well as the lack of a clear contrast between cellular structure and the bottom of sedimentation chambers (some phytoplankton cells are completely transparent), the presence of colonial forms and the presence of inert material (detritus or dead cells), whose colouring and dimensional characteristics are often similar to the algal cells. Therefore, the application of image analysis systems currently makes it possible to determine the dimensional characteristics of the phytoplankton cells only semi-automatically, i.e. it is the operator who selects the counting unit on the basis of which the morphometric characteristics are subsequently automatically determined, using specific image processing programmes. However none of the image processing programmes currently on the market are specifically designed for phytoplankton corporations, for which their output is generally limited and non-specific. Another limitation of the most widely used image analysis systems is that they acquire the image and process it only in 2D, which means that the information relative to the thickness of the algal cells is completely lost. A series of suggestions for overcoming this problem are currently available [21][22], but they make it possible to provide only estimates of the third dimension of the algal cells. The new technologies in the field of microscopy, supported by systems of analysis, are developing new systems that will enable a correct visualization of the algal cells in 3D. The department of engineering of innovation has been active in the 3D imaging sector for years. Its specialization in the field of high performance processing has had interesting results for the treatment of images and volumetric data in terms of their interactive 3D visualization, possible from remote client workstations [47], and in terms of their utility in data mining, using traditional or innovative techniques linked to evolutionary computing (genetic algorithms and neural networks).

The other special characteristic of the department of engineering of innovation is its outstanding research record in the field of Grid Computing, which has interesting implications for the management of scientific data distributed over a number of sites. This helps to optimize the use of available hardware, [48] which can be managed by the user efficiently and transparently, ensuring maximum usability by the scientific community.

Concerning the current state of knowledge in the field of microscopy supported by image analysis systems, the project intends to move in two directions; on the one hand it will develop a specific software for the determination of cellular bio-volume and the surface area of the algal cells, starting from 2D images, which takes account of the most recent scientific developments concerning both the sets of geometric models proposed and the proposed and tested suggestions for estimating the third dimension.

The software will be easy to use and available on the market and usable in routine analysis and together with instrumentation commonly available in the laboratory for the analysis of phytoplankton. Moreover the project will experiment with the new technologies in the field of 3D image analysis, with the aim of achieving a more accurate and faster estimate of the bio-volume and surface area of the algal cells. The project also aims to create the software for classification of health status based on estimated values of non-taxonomic descriptors of phytoplankton guild, the latter being identified in the descriptors of the size-abundance distributions.

The way in which these descriptors will be estimated will take account of scientific evidence that will study the statistical parameters of the size-abundance distributions (Mean, Mode, median, Kurtosis and Skewness, and line slope and intercept in the case of normalized distributions) with a view to evaluating their potential as descriptors [16][20][32][33][27][28][29][42][45]. The model for classifying the ecological status of marine and transitional ecosystems criteri will take in account of recent laws and regulation of “resource water” (D.L. 152/99; WFD 2000/60/CE).

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