Report on the technical and analytical improvements of innovative techniques and recommendations on their use

This is a summary of the activities and results of JERICO-NEXT Work Package 3 Innovations in Technology and Methodology, Task 3.1 Automated platform for the observation of phytoplankton diversity in relation to ecosystem services. The aim is to provide an advanced report on the last developments dedicated to the observation of the phytoplankton diversity by applying novel techniques on automated platforms. The work was carried out in close connection with task 4.1 Biodiversity of plankton, harmful algal blooms and eutrophication. The partners involved in these developments are CNRS, SYKE, SMHI, HZG, RWS, VLIZ CEFAS and Ifremer. Subcontractors in WP4, task 4.1 are WHOI, Scanfjord AB, Tomas Rutten b.v., CytoBuoy b.v. and UGent - PAE.
The work was carried out mainly in the field with activities in the Baltic Sea, the Kattegat-Skagerrak, the Celtic seas-English Channel-North Sea Area, the Western Mediterranean, as well as in shared studies with other WP3.4 and WP4.4 in the Bay of Biscay and, out of Europe, in the Benguela Current. Instrument platforms included continuous recording (Ferrybox or assimilated) systems on research vessels, on ships of opportunity, instrumented oceanographic buoys/fixed platforms and land-based systems.
Common implementations were carried out in some observatories, allowing inter comparison of sensors and some techniques. In addition, work on developing and testing new algorithms have been carried out in offices and laboratories. Three international workshops have been successfully arranged, one in Wimereux, France (June 2016 – organised by CNRS-LOG), one in Gothenburg, Sweden (September 2016, organised by SMHI) and one in Marseille (March 2019 – organised by CNRS MIO) connected to a EuroMarine workshop. Partners presented, discussed, were able to inter compare the sensors and techniques to be implemented in the field (Goetborg) and were trained to different automated analytical procedures and tools (for automated flow cytometry, Marseille).
The work was divided into three sections but there is substantial overlap and cooperation. One example is that reference samples analysed in the microscope were used for completing and/or evaluating the quality of some of the automated methods.
Imaging in flow and in situ imaging of plankton (led by SMHI)
The work included evaluating instruments and developing algorithms for automated identification of phytoplankton automated image acquisition (in flow or in situ). Three different commercial instruments and one instrument prototype were used. On the Swedish west coast (Skagerrak coast) a study of harmful algae and other phytoplankton was carried out near a mussel farm. The Imaging Flow Cytobot was deployed in situ and collected samples at six different depths for approximately two months. In the English Channel the old generation of FlowCAM and a prototype system, the FastCam, were used to analyse samples on research vessels or in the laborator. A colour version of FlowCAM was used both during a cruise in the Baltic Sea-Kattegat-Skattegat (July 2017) as well as in coastal monitoring in the Baltic and the Channel). In addition, the CytoSense and CytoSub were used to collect images in flow.
The in situ video system UVP5 was implemented during a cruise in the Baltic Sea-Skagerrak-Kattegat area, together with a new generation of FlowCAM of faster acquisition and providing colour images and CytoSense.
A major task was to develop and evaluate plankton identification algorithms. This included the use of a subset of images of organisms for training the software tools. Existing software were improved (as the PhytoZooImage) and an image data system/platform named EcoTaxa was described and is currently available for storing and cooperative analysis/discrimination of plankton images.
Single-cell optical characterization (led by CEFAS)
Automated flow cytometers (FCM, CytoSense/CytoSub, Cytobuoy b.v.) were implemented on a Ferry line, on research vessels and a fixed platform to investigate functional groups of phytoplankton. In the Western Mediterranean the main targets were the picoplankton and the nanoplankton while in the other areas pico-, nano- and microplankton were in focus. Several cruises were carried out in the Channel and North Sea to follow combined diatoms and Phaeocystis bloom development. A cruise covering the Baltic Sea and Skagerrak-Kattegat area had a main focus on cyanobacteria and dinoflagellates. Moreover, inter comparisons of machines and on clustering analysis methods were performed. Finally, a combination of FCM and multi-spectral fluorometer continuous recording was coupled with physical and hydrological continuous measurements in the southern Bay of Biscay.

Bio-optical Instrumentation (led by SYKE)
Novel multi-wavelength fluorometers for detecting phycoerythrin indicative e.g. of certain cyanobacteria and of cryptophytes were evaluated in the Baltic Sea. Multi wavelength fluorometers were also used in the Benguela current, during the Gothenburg workshop, as well as on a variety of field cruises from the southern Bay of Biscay to the E. Channel and North Sea, in order to discriminate amongst main phytoplankton pigmentary groups. The manufacturers’ algorithms were found to be partly inaccurate for detecting algal groups based on photosynthetic pigment composition. New dedicated fingerprints were used in field work to improve discrimination amongst phytoplankton groups. A principle component analyses approach was also evaluated.
Single wavelength fluorometers were evaluated in several sea areas. Sun induced photoquenching had a strong effect on fluorescence yield. In the North Sea and the Norwegian Sea multi spectral absorption was used to detect chlorophyll and phytoplankton groups based on pigment content.
Variable fluorometers were implemented on both samples, continuous recording and profiles in the E. Channel and North Sea, as well as in the Baltic and Skagerrak-Kattegat, for studying photosynthetic parameters and potential primary productivity. Recommendations are made on the strategy and type of measurements to carry out.
Recent work (since mid 2017) in task 3.1
Some field work was continued, especially at the Utö observatory in the Baltic Sea. The new data collected was processed together with old data and used for improving the discrimination of phytoplankton taxa or functional groups by inter-comparison of techniques and continued algorithm development which are described in the present deliverable 3.2. Scientific publication of results is in progress. A special issue in the open access journal Diversity (MPI) is being discussed. Some results and strategy were presented during a symposium in Hannover, in October 2017 and during the FerryBox workshop on board the ship Colour Fantasy later in October 2017 and in FerryBox meetings in 2018 and 2019. Results were also presented during the third JERICO-NEXT workshop on automated plankton observation in Marseille in March 2018 and some analytical tools for flow cytometry were presented and further directions as well as connections with modelling and remote sensing were discussed during the EuroMarine meeting that followed. A special session was held during the International Conference on Harmful Algae (ICHA) in Nantes in October 2018, and more presentations were carried out in 2019 meetings (IMBER, OceanObs, etc.).
Main conclusions
1. The methods used are reliable for automated observation of phytoplankton biodiversity (functional groups, size classes, taxa when possible) and biomass, complementing manual methods for sampling and microscope analyses.
2. Operating the equipment and interpreting the results still need a lot of knowledge and time. Even though some operational procedures can be established, the standardization of analytical and data processing as well as data management need more development. The degree of automation varies depending on the method considered.
3. Imaging in flow and in situ imaging provide means for identifying and counting phytoplankton at the genus or species level. Also, biomass based on cell volume of individual cells can be estimated. Development of classifiers for automated identification of organisms is time consuming and requires specific skills on signal analysis and on taxonomy.
4. Flow cytometry has proven to be a useful tool for counting phytoplankton and for describing the phytoplankton community as size-based classes and functional groups. There was an agreement to report the phytoplankton count in four groups for inter comparison purposes: Synechococcus (pico-cyanobacteria), pico-eukaryotic organisms, nanoplankton and microplankton.
5. Single and multi-wavelength fluorometry makes it possible to estimate phytoplankton biomass (at a chlorophyll-a basis) and to differentiate phytoplankton based on photosynthetic pigments. Sunlight induced photo-quenching is a problem for estimating chlorophyll a from fluorescence. For instruments mounted buoys or vessels, night time data can be used to minimize the problem.
6. Multi-wavelength absorption is a useful tool for estimating chlorophyll a and is also useful for discriminating between phytoplankton groups based on pigment content.
7. Variable (active) fluorescence is available for addressing phytoplankton physiology, photosynthetic parameters and we could estimate primary productivity on both continuous sub-surface recording and water column profiles, mediating careful coupling with other optical and also biogeochemical analysis.

How to cite
Artigas Felipe, Creach Veronique, Houliez Emilie, Karlson Bengt, Lizon Fabrice, Seppala Jukka, Wacquet Guillaume (2019). Report on the technical and analytical improvements of innovative techniques and recommendations on their use. Ref. ERICO-NEXT-WP3-D3.2-120819-V5.

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