Patrice Bellot
email: patrice.bellot@univ-amu.fr

Véronique Moriceau
email: moriceau@limsi.fr

Josiane Mothe
email: josiane.mothe@irit.fr

Eric Sanjuan
email: eric.sanjuan@univ-avignon.fr

Xavier Tannier
email: xtannier@limsi.fr

Overview of INEX Tweet Contextualization 2013 track

Keywords: Short text contextualization, Tweet understanding, Automatic summarization, Question answering, Focus information retrieval, XML, Natural language processing, Wikipedia, Text readability, Text informativeness

Twitter is increasingly used for on-line client and audience fishing, this motivated the tweet contextualization task at INEX. The objective is to help a user to understand a tweet by providing him with a short summary (500 words). This summary should be built automatically using local resources like the Wikipedia and generated by extracting relevant passages and aggregating them into a coherent summary. The task is evaluated considering informativeness which is computed using a variant of Kullback-Leibler divergence and passage pooling. Meanwhile effective readability in context of summaries is checked using binary questionnaires on small samples of results. Running since 2010, results show that only systems that efficiently combine passage retrieval, sentence segmentation and scoring, named entity recognition, text POS analysis, anaphora detection, diversity content measure as well as sentence reordering are effective.

Motivation

Text contextualization [START_REF] Sanjuan | Overview of the inex 2011 question answering track (qa@inex)[END_REF][START_REF] Sanjuan | Overview of the inex 2012 tweet contextualization track[END_REF] differs from text expansion in that it aims at helping a human to understand a text rather than a system to better perform its task. For example, in the case of query expansion in IR, the idea is to add terms to the initial query that will help the system to better select the documents to be retrieved. Text contextualization on the contrary can be viewed as a way to provide more information on the corresponding text in the objective to make it understandable and to relate this text to information that explains it.

In the context of micro-blogging, which is increasingly used for many purposes such as for on-line client and audience fishing, contextualization is specifically important since 140 characters long messages are rarely self-content. This motivated the proposal in 2011 of a new track at Clef INEX lab of Tweet Contextualization.

The use case is as follows: given a tweet, the user wants to be able to understand the tweet by reading a short textual summary; this summary should be readable on a mobile device without having to scroll too much. In addition, the user should not have to query any system and the system should use a resource freely available. More specifically, the guideline specified the summary should be 500 words long and built from sentences extracted from a dump of Wikipedia. Wikipedia has been chosen both for evaluation purpose and because this is an increasing popular ressource while being generally trustable. In this paper, details the 2013 track set up and results. The use case and the topic selection remained stable since 2011 [START_REF] Sanjuan | Overview of the inex 2011 question answering track (qa@inex)[END_REF], so that 2011 and 2012 topics could be used as a training set. However, In 2013 we considered more diverse types of tweets for this year edition, so that participants could better measure the impact of hashtag processing on their approaches.

The remaining of the paper is organised as follows: In section 2 we describe in detail the 2013 data collection. Section 3 presents the results and Section 4 concludes this paper.

Data collection

This section describes the document collection that is used as the resource for contextualization, as well as the topics selected for the test set which correspond to the tweets to contextualize.

The document collection has been built based on a recent dump of the English Wikipedia from November 2012. Since we target a plain XML corpus for an easy extraction of plain text answers, like in past years, we used the same perl programs released for all participants to remove all notes and bibliographic references that are difficult to handle and keep only non empty Wikipedia pages (pages having at least one section).

Resulting automatically generated documents from Wikipedia dump, consist of a title (title), an abstract (a) and sections (s). Each section has a sub-title (h). Abstract and sections are made of paragraphs (p) and each paragraph can contain entities (t) that refer to other Wikipedia pages.

Over 2012 and 2013 editions, evaluated topics were made of 120 (60 topics each year) tweets manually collected by organizers. These tweets were selected and checked, in order to make sure that:

-They contained "informative content" (in particular, no purely personal messages); Only non-personal accounts were considered (i.e. @CNN, @TennisTweets, @PeopleMag, @science. . . ).

-The document collection from Wikipedia contained related content, so that a contextualization was possible.

From the same set of accounts, more than 1,800 tweets were then collected automatically. These tweets were added to the evaluation set, in order to avoid that fully manual, or not robust enough systems could achieve the task. All tweets were then to be treated by participants, but only the 120 short list was used for evaluation. Participants did not know which topics were selected for evaluation.

These tweets were provided in a text-only format without metadata and in a JSON format with all associated metadata.

Results

This year the entire evaluation process was carried out by organizers.

Tweet contextualization [START_REF] Sanjuan | Overview of the inex 2010 question answering track (qa@inex)[END_REF] is evaluated on both informativeness and readability. Informativeness aims at measuring how well the summary explains the tweet or how well the summary helps a user to understand the tweet content. On the other hand, readability aims at measuring how clear and easy to understand the summary is.

Informativeness measure is based on lexical overlap between a pool of relevant passages (RPs) and participant summaries. Once the pool of RPs is constituted, the process is automatic and can be applied to unofficial runs. The release of these pools is one of the main contributions of Tweet Contextualization tracks at INEX [START_REF] Sanjuan | Overview of the inex 2011 question answering track (qa@inex)[END_REF][START_REF] Sanjuan | Overview of the inex 2012 tweet contextualization track[END_REF].

By contrast, readability is evaluated manually and cannot be reproduced on unofficial runs. In this evaluation the assessor indicates where he misses the point of the answers because of highly incoherent grammatical structures, unsolved anaphora, or redundant passages. Like in 2012, three metrics were used: Relevancy (or Relaxed) metric, counting passages where the T box has not been checked; Syntax, counting passages where the S box was not checked either, and the Structure (or Strict) metric counting passages where no box was checked at all.

Participant runs were ranked according to the average, normalized number of words in valid passages.

In 2013, a total number of 13 teams from 9 countries (Brasil, Canada, France, India, Ireland, Mexico, Russia, Spain, USA) submitted 24 runs to the Tweet Contextualization track in the framework of CLEF INEX lab 2013.

Infomativity results are presented in Table 1 and statistical significance of differencies between scores are indicated in Table 2. Table 1 shows readability scores.

This year, the best participating system (199) used hashtag preprocessing introduced in [START_REF] Deveaud | Lia/lina at the inex 2012 tweet contextualization track[END_REF]. The best run by this participant used all available tweet features including web links which was not allowed by organisers. However his second best run without using linked web pages is ranked first among official runs. This participant also tried to weight hashtags based on 2012 results but this did not improve results. Perhaps because topics evaluated in 2012 were too specific.

Second best participant (182) in informativity and best in readability used state of the art NLP tools. This participant was first in informativity in 2011 [START_REF] Ermakova | Irit at inex[END_REF]. Differences between these two best systems are not statistically significant. Third best participant system (65) was first in 2012 [START_REF] Ganguly | Dcu@inex-2012: Exploring sentence retrieval for tweet contextualization[END_REF], so the same system performs well even on a more diversify set of tweets.

Reference system by organisers (62-276) available online through an API is not more among three best systems. This systems is a robust focused information retrieval system [START_REF] Sanjuan | Overview of the inex 2010 question answering track (qa@inex)[END_REF] that was not smoothed for tweets. This year we also set up a baseline (62 -278) using a state of the art IR system on sentences. Its informativity scores are high but its readability is very low.

Overall, informativity and readability scores are this year strongly correlated (Kendall test: τ > 90%, p < 10 -3 ) which shows that all systems have integrated this constrain. Remenber that since 2012, readability is evaluated in the context of the tweet. Passages not related to the tweet are considered as unreadable.

Conclusions

Like in 2012, almots all participants used language models.

Terminology extraction and reformulation applied to tweets was also used in 2013 like in previous editions [9]. Appropriate stemming and robust parsing of both tweets and wikipedia pages also seems to be an important issue. Most systems having a run among the top ten in informativeness used the Standford Core NLP tool or the TreeTagger.

It also seems that automatic readability evaluation and anaphora detection helps improving readability scores, but also informativeness density in summaries. It is now clear that state of the art summarization methods based on sentence scoring [START_REF] Moreno | Two statistical summarizers at inex 2012 tweet contextualization track[END_REF] proved to be helpful on this task even though they need to be combined with an IR engine.

Best run in 2013 also experimented a tweet hashtag scoring technique introduced in 2012 [START_REF] Deveaud | Lia/lina at the inex 2012 tweet contextualization track[END_REF] while generating the summary.

Finally, this time the state of the art system proposed by organizers since 2010 combining LM indexation, terminology graph extraction and summarization based on shallow parsing was not ranked among the six best runs which shows that participant systems improved on this task over the three editions.


Dominique Marie

Fabienne Rigaut-Jalabert

Daniel Vaulot

An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples

Preservation of cells, choice of fixative, storage and thawing conditions are recurrent issues for the analysis of phytoplankton by flow cytometry. We examined the effects of addition of the surfactant Pluronic F68 to glutaraldehyde-fixed photosynthetic organisms in cultures and natural samples. In particular, we examined cell losses and modifications of side scatter (a proxy of cell size) and fluorescence of natural pigments. We found that different marine phytoplankton species react differently to the action of Pluronic F68. In particular, photosynthetic prokaryotes are less sensitive than eukaryotes. Observed cell losses may result from cell lysis or from cell adhesion to the walls of plastic tubes that are commonly used for flow cytometry analysis. The addition of the surfactant, Pluronic F68, has a positive effect on cells for long term storage. We recommend to modify current protocols for preservation of natural marine planktonic samples, by fixing them with glutaraldehyde 0.25% (final concentration) and adding Pluronic F68 at a final concentration of 0.01% in the samples before preservation. Pluronic F68 also appears effective for preserving samples without fixation for subsequent sorting, e.g. for molecular biology analyses.

INTRODUCTION

Enumeration of bacteria and phytoplankton both in cultures and natural samples has been one of the major applications of flow cytometry in oceanography during the last 3 decades, [START_REF] Kw | DNA distribution in planktonic bacteria stained with TOTO or TO-PRO[END_REF][START_REF] Vaulot | Application of flow cytometry in marine phytoplankton research: current applications and future perspectives[END_REF][START_REF] Gasol | Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities[END_REF]. Photosynthetic plankton is composed by prokaryotes and eukaryotes that exhibit autofluorescence from pigments, in particular phycoerythrin and chlorophyll. Although flow cytometers can be used on cruise vessels and in situ systems have been developed [START_REF] Campbell | First harmful Dinophysis (Dinophyceae, Dinophysiales) bloom in the US is revealed by automated imaging flow cytometry[END_REF][START_REF] Dubelaar | Design and first results of Cytobuoy: A wireless flow cytometer for in situ analysis of marine and fresh waters[END_REF][START_REF] Dubelaar | RR High frequency monitoring reveals phytoplankton dynamics[END_REF][START_REF] Olson | An automated submersible flow cytometer for analyzing pico-and nanophytoplankton: FlowCytobot[END_REF], it is still often necessary to preserve samples for delayed analysis. For other applications, such as microscopy, lugol, formalin (that contains methanol), and alcohols are used for sample preservation. However, these chemicals drastically affect the fluorescence of natural pigments that is required to distinguish between phytoplanktonic and heterotrophic cells; therefore they cannot be used for flow cytometry [START_REF] Marie | Flow cytometric analysis of phytoplankton, bacteria and viruses[END_REF]. Phytoplankton samples are commonly preserved using aldehydes such as glutaraldehyde or formaldehyde, because they are efficient at preserving both cellular structures and pigment autofluorescence (Vaulot et al., 1989;[START_REF] Lepesteur | Comparative study of different preservation methods for phytoplankton cell analysis by flow cytometry[END_REF][START_REF] Sato | Effects of long-term sample preservation on llow cytometric analysis of natural populations of Pico-and Nanophytoplankton[END_REF]. [START_REF] Lepesteur | Comparative study of different preservation methods for phytoplankton cell analysis by flow cytometry[END_REF] reported that there is no universal freezing process and they suggested that for mixed species populations, it is best to preserve aliquots by 3 methods: 1) rapid freezing with no addition of chemical to analyze fluorescence and size of algae, 2) slow freezing with glutaraldehyde fixation for cyanobacteria samples, and 3) same as the previous method but with addition of glycerol for cell counts (although they recorded large cell loss). However, they remarked that this is not always realistic. Vaulot et al. (1989) have proposed the use of glutaraldehyde 1% following by deep-freezing to preserve phytoplanktonic cells, but some authors have preferred the use of formaldehyde because it minimizes cell losses [START_REF] Sieracki | Plankton community response to sequential silicate and nitrate depletion during the 1989 North Atlantic spring bloom[END_REF] especially for long term preservation (Trousselier et al., 1995, Charpy and[START_REF] Charpy | Photosynthetic picoplankton in French Polynesia atoll lagoon: Estimation of taxa contribution to biomass and production by flow cytometry[END_REF]. [START_REF] Sato | Effects of long-term sample preservation on llow cytometric analysis of natural populations of Pico-and Nanophytoplankton[END_REF] suggested that the effects of fixation and preservation on phytoplankton for flow cytometry are different for cultures and for natural samples. Fixation of samples using aldehydes has been reported to result in cell losses (Vaulot et al., 1989;[START_REF] Lepesteur | Comparative study of different preservation methods for phytoplankton cell analysis by flow cytometry[END_REF][START_REF] Marie | DNA, RNA analysis of phytoplankton by flow cytometry[END_REF]. [START_REF] Pan | On-board flow cytometric observation of picoplankton community structure in the East China Sea during the fall of different years[END_REF] reported up to 70% of Prochlorococcus cells loss in preserved samples from the East China Sea and [START_REF] Sato | Effects of long-term sample preservation on llow cytometric analysis of natural populations of Pico-and Nanophytoplankton[END_REF] suggested that cell losses are more critical in natural than in cultured samples, probably due to the different species composition or physiological status.

If the use of aldehydes for preserving microorganisms is commonly accepted [START_REF] Eschbach | A simple and highly efficient fixation method for Chrysochromulina polylepis (Prymnesiophytes) for analytical flow cytometry[END_REF][START_REF] Marie | Flow cytometric analysis of phytoplankton, bacteria and viruses[END_REF], the type of fixative, its final concentration, as well as the storage process have been debated. Because methanol is added to commercial solutions of formaldehyde, it should be prepared from its polymerized form, para-formaldehyde; however such para-formaldehyde solutions are not stable for more than one week. Because glutaraldehyde is less toxic than formaldehyde and because commercial solutions are available and stable over the time, it appears as the best option for fixation.

Pluronic is a non-ionic surfactant used as a culture media additive and as a non toxic cryoprotectant during cell freezing and thawing procedures [START_REF] Lowe | Beneficial effects of Pluronic F-68 and artificial oxygen carriers on the post-thaw recovery of cryopreserved plant cells[END_REF]. It is also used as anti-foaming, wetting, dispersant, thickener or emulsifier agent, and as stabilizer of cell membranes against shearing during batch cultures [START_REF] Papoutsakis | Media additives for protecting freely suspended animal cells against agitation and aeration damage[END_REF]. [START_REF] Hellung-Larsen P Assaad | Effects of Pluronic F-68 on Tetrahymena cells: protection against chemical and physical stress and prolongation of survival under toxic conditions[END_REF] studied the effect of Pluronic F68 (P68) on the ciliate Tetrahymena. They found a marked protection against physical and chemical stresses, especially at low cell concentrations. P68 has also been used to minimize cells losses due to numerous steps of centrifugation required to couple in situ hybridization with flow cytometry [START_REF] Biegala | Quantitative assessment of picoeucaryotes in the natural environment using taxon specific oligonucleotide probes in association with TSA-FISH (Tyramide Signal Amplification -Fluorescent In Situ Hybridization) and flow cytometry[END_REF].

In the present work, we have tested the use of P68 either alone or in combination with glutaraldehyde to preserve cultured and natural populations of microalgae in order to improve the recovery of cells for subsequent analysis by flow cytometry.

Materials and methods

Cultures and samples: Cultures from the Roscoff Culture Collection (RCC, www.roscoffculture-collection.org) have been used for the different experiments (Table 1). They were grown in 12:12 light:dark cycle and were collected in exponential growth phase. For the natural samples, surface seawater from the SOMLIT-Astan site (48°46'18''N, 3°58'6''W) off Roscoff (Brittany, France) was sampled during winter using a Niskin bottle.

Flow cytometry: Analyses of samples were performed using a FACSCanto II flow cytometer equipped with 488 and 633 nm lasers and standard filter setup. The flow rate was determined by measuring the difference of volume of filtered seawater in a plastic tube before and after run for a minimum of 10 minutes as described in [START_REF] Marie | Flow cytometric analysis of phytoplankton, bacteria and viruses[END_REF]. Data acquisition was triggered on the red fluorescence signal and samples were run for two minutes for cultures at medium rate (~50 µL/min) and three minutes in duplicate for natural samples at high rate (~100 µL/min). To allow comparison between samples, 0.95 µm fluorescent beads were added to each sample as internal reference. Listmode files were computed with Cytowin (Vaulot 1989). Cell parameters (scatter, fluorescence) were then normalized by dividing the mean value for each cell population by the mean value for the beads. In the following, we consider that differences in cell numbers below 5% are not significant, since the coefficient of variation (CV) of 2 replicates of the same sample analyzed by FCM commonly ranges between 1 and 3%.

Epifluorescence microscopy: In order to assess whether cells could be trapped on the side walls of the tubes used for analysis, some tubes that contained eukaryotes were emptied and manually broken into pieces. These pieces were observed by epifluorescence microscopy using an Olympus BX 51 microscope.

Experiment 1. Effect of Pluronic concentration: Different concentrations of Pluronic F-68 (P68, Sigma-Aldrich P5556) from 10 -6 to 10 -1 % were tested on a mixture of Prochlorococcus RCC156, Synechococcus RCC263, Micromonas pusilla RCC114, Pleurochrysis carterae RCC190, and 0.95 µm fluorescent beads (Polysciences). After an initial flow cytometric analysis of the fresh sample, samples were fixed with glutaraldehyde (GLU, Sigma G5882, 0.25% final), incubated for 15 min and then flash-frozen in liquid nitrogen and stored at -80°C. After one day, frozen samples were thawed at room temperature just before a new analysis.

Experiment 2. Fixation of culture mixes: Two different mixes of cultures each containing, one Prochloroccocus strain (RCC156, RCC168), one Synechococcus strain (RCC263, RCC1085), one picoeukaryote strain (RCC114, RCC745) and one nanoeukaryote strain (RCC190, RCC400), and 0.95 µm fluorescent polyspheres (Polysciences) were divided into four 1 mL aliquots. Two of them were unfixed and two were fixed with GLU 0.25% (minimum incubation period of 15 minutes at room temperature). Then, P68 at a final concentration of 0.01% was added into one aliquot of each condition (unfixed and fixed). After a first analysis by flow cytometry, aliquots were deep frozen into liquid nitrogen and then stored at -80°C for one month before a new analysis.

Experiment 3. Fixation of individual cultures: Eukaryotic cultures from the RCC in exponential growth phase were used and diluted if necessary with 0.22 µm-filtered seawater to adapt cell concentrations for flow cytometry analysis. The same treatment than for Experiment 2 was performed.

Experiment 4. Natural samples. Four samples of 40 mL were prepared on board, two without fixative and two others fixed with GLU 0.25% final. P68 at a final concentration of 0.01% was added in one aliquot of each condition. Samples were stored in the dark and brought back to the laboratory. They were analyzed by flow cytometry and then aliquoted into 1.5 mL cryovials, deep frozen in liquid nitrogen and stored at -80°C. These aliquots were analyzed after different storage duration (1, 2, 3 etc... months, up to 15 months).

Results

Experiment 1. Effect of Pluronic concentration.

Our first step was to test the effect of addition of P68 at different concentrations on cell counts for 4 cultures (two cyanobacteria, one picoeukaryote, and one nanoeukaryote). For cyanobacteria, there was little effect of P68 concentrations (Figure 1). For eukaryotes, there was a clear decrease in cell concentration after fixation and freezing compared to fresh sample when no P68 was added. Increasing concentration of P68 had a beneficial effect allowing to obtain cell counts on fixed-frozen samples that were quite similar to the original counts (Figure 1). Overall, concentration of P68 ranging from 0.1 to to 0.001% seemed to be optimal for the 4 species tested and therefore 0.01% was chosen for subsequent analysis.

Effects of fixation and P68 addition

Experiment 2. Mix of cultures. First we tested two mixed cultures, each containing strains of Prochlorococcus, Synechococcus, picoeukaryotes and nanoeukaryotes (see Material and Methods).

For the two Prochlorococcus strains tested in the mixes, fixation had very little effect and we did not observe significant differences in cells counts with and without P68 (Figure 2). The Side Scatter (SSC) of Prochlorococcus was reduced 2.5 -3 fold in unfixed samples after freezing, but was higher after fixation by GLU (Supplementary Figure 1). Chlorophyll fluorescence decreased in GLU-fixed samples and this was more marked for RCC168 (Supplementary Figure 2).

Synechococcus cell concentrations decreased after freezing in unfixed samples, while fixation by GLU allowed to recover the original cell numbers (Figure 2). The SSC of Synechococcus increased after fixation by GLU, but was lower in presence of P68 (Supplementary Figure 1).

The phycoerythrin fluorescence of Synechoccocus cells increased up to 9 and 7 fold after freezing in absence of GLU for RCC263 and RCC1085, respectively, and this was strongly minimized on fixed cells with increases of 10 and 25%, respectively (data not shown). The same trend was observed for the chlorophyll fluorescence of Synechococcus that was increased by the fixation by GLU up to 2.5 and 3.7 fold after freezing in unfixed RCC263 and RCC1085, respectively (Supplementary Figure 2).

Freezing always resulted in cell losses for both picoeukaryotes tested with only 30 and 40% recovery in unfixed RCC114 and RCC745, respectively (Figure 2). The addition of P68 clearly minimized these losses. Fixation by GLU preserved well SSC of both Micromonas and Ostreococcus but it was strongly reduced by freezing, especially for Ostreococcus RCC745 (Supplementary Figue 1). Chlorophyll fluorescence per cell was little affected in both picoeukaryotes (Supplementary Figure 2).

For the nanoeukaryotes RCC190 and RCC400, the use of P68 minimized cell losses after freezing, especially in GLU-fixed samples (Figure 2). SSC was not affected by the fixation, but increased in Pleurochrysis RCC190 and decreased in Chrysochromulina RCC400 after freezing (Supplementary Figure 1). For both nanoeukaryotes, the chlorophyll content per cell was well preserved with and without P68 after freezing (Supplementary Figure 2).

Experiment 3. individual eukaryotic cultures:

Based on the results reported in the previous experiment 2, different cultures seem to react differently to fixation. Therefore, we tested the effect of fixation with GLU and P68 addition on 11 strains of the RCC, belonging to a wide taxonomic range (Figure 3). Fixation and freezing could induce important cell losses as demonstrated with RCC174 (21%) or RCC91 (50%). Addition of P68 in the unfixed, unfrozen samples always resulted in higher cell counts that varied from 1 to 21%. The addition of P68 to fixed and frozen samples always allowed recovery of more cells compared to the original cell number obtained for not fixed and not frozen samples. Cell recoveries higher than 100% have been observed for some strains and could be explained by the fact that cells were trapped on the side walls of the tubes used for analysis. In order to determine whether this was the case, tubes that contained unfixed and GLU-fixed Chrysochromulina RCC400 and Scrippsiella RCC91 were completely emptied and manually broken into pieces. These pieces were observed by epifluorescence microscopy. Photosynthetic cells were easily detected on the walls of the tubes for GLU-fixed samples, very few cells were detected for unfixed samples and no cells was detected on the walls of tubes that had contained P68. Experiment 4. Natural samples: Seawater samples collected in winter off Roscoff showed typical flow cytometric signatures of cyanobacteria (Syn) with orange and red fluorescences, Cryptophyceae (Crypto) with strong orange and red fluorescences. Two populations of pico-and nano-eukaryotes with only red fluorescence, are not clearly distinguishable from each other (Figure 4). As previously observed for cultures, P68 had little effect on cyanobacteria counts. Interestingly, cyanobacteria loss in unfixed sample was only 12% in presence of P68 after 12 months storage (Figure 5). The SSC of cyanobacteria was reduced by about 20% in unfixed and GLU-fixed samples after 1 day of preservation, and then it was stable over the time. As observed with cultures, the orange and red fluorescences increased after fixation with GLU.

Cell loss of total eukaryotes was low in unfixed samples in the absence of P68 reaching 14% after 2 months of storage (Figure 5). For the samples fixed with GLU or containing P68 we recorded up to 22% more cells after 15 months compared to the initial unfixed/unfrozen sample. Note that the percentage of cell loss was negligible in the unfixed sample containing P68, even after 12 months storage (Figure 5). For picoeukaryotes, we observed a slight increase in numbers over the time. The SSC of the picoeukaryotes decreased by about 40% immediately after fixation, but remained constant after that. For the nanoeukaryotes, after 5 months of preservation, we recorded 43% more cells in the GLU-fixed than in the original sample, and 23% more in the unfixed one. The SSC of nanoeukaryotes decreased by 20% immediately after fixation, and slowly decreased with time down to 50% after 15 months preservation. The fixation resulted in a decrease of the chlorophyll fluorescence for both pico-(20 to 24%) and nanoeukaryotes (12 to 21%), data are not shown for SSC and pigments analysis.

For the cryptophyte population, we observed 34% more cells in unfrozen GLU-fixed samples in presence of P68. After 1 month of preservation, 93 % of cryptophytes were lost in unfixed samples, and after 4 months storage, they almost disappeared in these samples (Figure 4). In contrast, the number of cryptophytes remained relatively constant in the fixed samples with or without P68 (Figure 5).

Discussion

Our results demonstrate a large variety of responses to fixation that are often species dependant, especially for eukaryotes. [START_REF] Kamiya | Effects of fixation and storage on flow cytometric analysis of marine bacteria[END_REF] reported that fixation is not responsible of bacterial cell loss, that was attributed to flash freezing and storage at -80°C, with a rapid decrease of counts within the first 3 days, followed by a slower decline. We have not observed such tendency for the photosynthetic prokaryotes used in this study and fixed with GLU. Rapid fixation after sampling appears essential for preserving delicate cells such as cryptophytes. We regularly observed a rapid decline of this population, within a few hours, in unfixed samples. Conversely, abundances of Synechococcus and total eukaryotic cells remained relatively stable in unfixed samples. This can be of interest for some flow cytometry sorting applications for which samples cannot be fixed with aldehydes such as molecular biology analyses (barcoding or single cell genomics). Nevertheless care must be taken since certain types of eukaryotes present important cell losses after freezing (Figure 2).

The most interesting aspect of this study concerns the use of the surfactant Pluronic F68. The effect of P68 is not clear for prokaryotes, but clearly minimizes cell losses for eukaryotes. P68 has a short term effect, as demonstrated on eukaryotes from cultures or from natural assemblages. More cells were recovered in tubes containing the surfactant when analyzing unfrozen samples (Figures 2 and5). These differences can be explained by cell lysis or by a tendency of such organisms to attach to the plastic walls of the tubes. Epifluorescence microscopy observations of the cell walls of tubes that had contained RCC400 (Chrysochromulina sp) or RCC91 (Scrippsiella trochoidea), suggest that, in the absence of P68, GLU-fixed cells have a very strong tendency to attach to the tube walls. These interactions are minimized in presence of P68. Electrostatic effects of plastic tubes can also be observed with the 0.95 µm beads added in natural sample from Roscoff, 20 % more beads were obtained in the unfrozen unfixed samples containing P68. In unfixed sample, we observed a regular increase in beads numbers over the time, up to 85% more after 5 months preservation (data not shown).

In conclusion, we strongly recommend either, to run all marine plankton (cultured and natural) with the addition of Pluronic F68 at a final concentration of 0.01%, or alternatively to fix them with glutaraldehyde (0.25% final concentration) in presence of P68 (0.01% final concentration), to freeze the tubes in liquid nitrogen, and to keep them at -80°C for delayed analysis.

Figure legends

Figure 1: Experiment 1. Effect of Pluronic (P68) concentration on the percentage of cell recovery (defined as the ratio of the cell concentration after treatment to that of initial untreated sample), obtained for a mixture of Prochlorococcus RCC156, Synechococcus RCC263, picoeukaryote RCC114 and nanoeukaryote RCC190, not fixed and non frozen (blue), fixed with GLU 0.25% and frozen for one day (red). Dashed line corresponds to 100% of recovery.

Figure 2: Experiment 2. Effect of freezing and fixation by GLU (0.25%) on the percentage of cell recovery obtained for 2 mixtures each containing one Prochloroccocus strain (RCC156, RCC168), one Synechococcus strain (RCC263, RCC1085), one picoeukaryote strain (RCC114, RCC745) and one nanoeukaryote strain (RCC190, RCC400), in presence (P) or absence of P68 at concentration of 0.01% analyzed by FCM, unfrozen (blue), and frozen for 1 month (red). Percentages of recovery of Synechococcus, Cryptophyceae, and total eukaryotic cells per mL obtained for a natural sample taken off Roscoff, not fixed (blue), not fixed but with P68 (dark blue), fixed with GLU 0.25% (red), fixed with GLU with P68 (dark red). Analyses were performed immediately on non frozen samples (T0). They were then frozen and stored at -80°C for 1 to 15 months. 10 -6 0 10 -5 10 -4 10 -3 10 -2 10 -1 Pluronic concentration (%)

Percentage of recovery

Not fixed not frozen GLU-Fixed and frozen

Figure 1 10 -6 0 10 -5 10 -4 10 -3 10 -2 10 -1 10 -6 0 10 -5 10 -4 10 -3 10 -2 10 -1 10 -6 0 10 -5 10 -4 10 -3 10 -2 10 -1

Figure 3 :

 3 Figure3: Experiment 3. Percentages of recovery of eukaryotes from the RCC unfixed and non frozen (blue), unfixed and non frozen with addition of P68 (dark blue), GLU-fixed and non frozen (red), GLU-fixed and frozen with addition of P68 (dark red).

Figure 4 :

 4 Figure 4: Experiment 4. Phycoerythrin vs chlorophyll fluorescence of a natural sample from the English Channel (Station Astan) analyzed after 5 months preservation with no fixative (A and C) or fixed with GLU 0.25% (B and D) in absence (A and B) or in presence (C and D) of P68 (0.01%). Sub-populations are: 0.95 µm beads (Beads), Synechococcus (Syn), Picoeukaryotes (Pico), Nanoeukaryotes (Nano) and Cryptophyceae (Crypto). Typicall

Figure 5 :

 5 Figure5: Experiment 4. Percentages of recovery of Synechococcus, Cryptophyceae, and total eukaryotic cells per mL obtained for a natural sample taken off Roscoff, not fixed (blue), not fixed but with P68 (dark blue), fixed with GLU 0.25% (red), fixed with GLU with P68 (dark red). Analyses were performed immediately on non frozen samples (T0). They were then frozen and stored at -80°C for 1 to 15 months.

  Figure3

Acknowledgements. We thank the captain and crew of the Neomysis for help with sampling. Financial support for this work was provided by the following EU FP7 programs: ASSEMBLE, MACUMBA (grant agreement no 311975).

Supplementary Figures:

Supplementary Figure 1: Side scatter normalized to that of 0.95 µm beads obtained for 2 mixtures each containing one Prochloroccocus strain (RCC156, RCC168), one Synechococcus strain (RCC263, RCC1085), one picoeukaryote strain (RCC114, RCC745) and one nanoeukaryote strain (RCC190, RCC400), in presence (P) or absence of P68 at concentration of 10 -2 analyzed by FCM, unfrozen (blue), and frozen for 1 month (red).

Supplementary Figure 2: Chlorophyll fluorescence normalized to the red fluorescence of 0.95 µm beads obtained for 2 mixtures containing each, one Prochloroccocus strain (RCC156, RCC168), one Synechococcus strain (RCC263, RCC1085), one picoeukaryote strain (RCC114, RCC745) and one nanoeukaryote strain (RCC190, RCC400), in presence (P) or absence of P68 at concentration of 10 -2 analyzed by FCM, unfrozen (blue), and frozen for 1 month (red).