Interannual dynamics of putative parasites (Syndiniales Group II) in a coastal ecosystem.

Temporal dynamics of Syndiniales Group II were investigated combining 18S rDNA amplicon sequencing and direct microscopy counts (fluorescence in situ hybridization-tyramide signal amplification [FISH-TSA]) during 5 years. The study was undertaken in meso-eutrophic coastal ecosystem, dominated by diatoms, the haptophyte Phaeocystis globosa and exhibiting relatively low dinoflagellate abundance (max. 18.6 × 103 cells L-1 ). Consistent temporal patterns of Syndiniales Group II were observed over consecutive years highlighting the existence of local populations. According to sequencing data, Syndiniales Group II showed increasing abundance and richness in summer and autumn. Dinospores counted by microscopy, were present at low abundances and were punctuated by transient peaks. In summer dinospore highest abundance (559 × 103 L-1 ) and prevalence (38.5%) coincided with the peak abundance of the dinoflagellate Prorocentrum minimum (13 × 103 L-1 ) while in autumn Syndiniales Group II likely had more diversified hosts. Although, several peaks of dinospore and read abundances coincided, there was no consistent relation between them. Ecological assembly processes at a seasonal scale revealed that stochastic processes were the main drivers (80%) of the Group II community assembly, though deterministic processes were noticeable (20%) in June and July. This latter observation may reflect the specific Syndiniales-dinoflagellate interactions in summer.

Taxonomic and functional dynamics during chytrid epidemics in an aquatic ecosystem.

Fungal parasitism is common in plankton communities and plays a crucial role in the ecosystem by balancing nutrient cycling in the food web. Previous studies of aquatic ecosystems revealed that zoosporic chytrid epidemics represent an important driving factor in phytoplankton seasonal successions. In this study, host-parasite dynamics in Lake Pavin (France) were investigated during the spring diatom bloom while following chytrid epidemics using next generation sequencing (NGS). Metabarcoding analyses were applied to study changes in the eukaryotic microbial community throughout diatom bloom-chytrid epidemics. Relative read abundances of metabarcoding data revealed potential "beneficiaries" and "victims" during the studied period. Subsequently, metatranscriptomic analyses on samples before and during the chytrid epidemic unveiled the active part of the community and functional/metabolic dynamics in association with the progress of chytrid infection. Diatom functions involving lipases, transporters, histones, vacuolar systems, the proteasome, proteases and DNA/RNA polymerases were more abundant during the diatom bloom. Chytrid functions related to a parasitic lifestyle including invasion, colonization and stress tolerance were up-regulated during the chytrid epidemic. In addition, functions related to the degradation/metabolism of proteins, lipids and chitin were in higher proportion in the community during the epidemic event. Results of NGS and bioinformatics analyses offered a panorama of dynamic biodiversity and biological functioning of the community.

Winter-summer succession of unicellular eukaryotes in a meso-eutrophic coastal system.

The objective of this study was to explore the succession of planktonic unicellular eukaryotes by means of 18S rRNA gene tag pyrosequencing in the eastern English Channel (EEC) during the winter to summer transition. The 59 most representative (>0.1%, representing altogether 95% of total reads), unique operational taxonomic units (OTUs) from all samples belonged to 18 known high-level taxonomic groups and 1 unaffiliated clade. The five most abundant OTUs (69.2% of total reads) belonged to Dinophyceae, Cercozoa, Haptophyceae, marine alveolate group I, and Fungi. Cluster and network analysis between samples distinguished the winter, the pre-bloom, the Phaeocystis globosa bloom and the post-bloom early summer conditions. The OTUs-based network revealed that P. globosa showed a relatively low number of connections-most of them negative-with all other OTUs. Fungi were linked to all major taxonomic groups, except Dinophyceae. Cercozoa mostly co-occurred with the Fungi, the Bacillariophyceae and several of the miscellaneous OTUs. This study provided a more detailed exploration into the planktonic succession pattern of the EEC due to its increased depth of taxonomic sampling over previous efforts based on classical monitoring observations. Data analysis implied that the food web concept in a coastal system based on predator-prey (e.g. grazer-phytoplankton) relationships is just a part of the ecological picture; and those organisms exploiting a variety of strategies, such as saprotrophy and parasitism, are persistent and abundant members of the community.

Effect of natural iron fertilization on carbon sequestration in the Southern Ocean.

The availability of iron limits primary productivity and the associated uptake of carbon over large areas of the ocean. Iron thus plays an important role in the carbon cycle, and changes in its supply to the surface ocean may have had a significant effect on atmospheric carbon dioxide concentrations over glacial-interglacial cycles. To date, the role of iron in carbon cycling has largely been assessed using short-term iron-addition experiments. It is difficult, however, to reliably assess the magnitude of carbon export to the ocean interior using such methods, and the short observational periods preclude extrapolation of the results to longer timescales. Here we report observations of a phytoplankton bloom induced by natural iron fertilization--an approach that offers the opportunity to overcome some of the limitations of short-term experiments. We found that a large phytoplankton bloom over the Kerguelen plateau in the Southern Ocean was sustained by the supply of iron and major nutrients to surface waters from iron-rich deep water below. The efficiency of fertilization, defined as the ratio of the carbon export to the amount of iron supplied, was at least ten times higher than previous estimates from short-term blooms induced by iron-addition experiments. This result sheds new light on the effect of long-term fertilization by iron and macronutrients on carbon sequestration, suggesting that changes in iron supply from below--as invoked in some palaeoclimatic and future climate change scenarios--may have a more significant effect on atmospheric carbon dioxide concentrations than previously thought.

On the conditions promoting Pseudo-nitzschia spp. blooms in the eastern English Channel and southern North Sea.

This study investigated the drivers of the blooms of Pseudo-nitzschia seriata and Pseudo-nitzschia delicatissima complexes in the eastern English Channel and southern North Sea. Phytoplankton data series acquired from 1992 to 2020 were analyzed with a multivariate statistical approach based on Hutchinson's niche concept. P. seriata and P. delicatissima complexes were found to be typically present year round, but they bloomed at different periods because they occupied different realized ecological niches. P. delicatissima complex occupied a more marginal niche and was less tolerant than P. seriata complex. P. delicatissima complex typically bloomed in April-May at the same time as Phaeocystis globosa while P. seriata complex blooms were more frequently observed in June during the decline of low intensity P. globosa blooms. P. delicatissima and P. seriata complexes were both favored by low-silicate environments and relatively low turbulence but they responded differently to water temperature, light, ammonium, phosphate and nitrite + nitrate conditions. Niche shifts and biotic interactions played important roles in the control of the blooms of P. delicatissima and P. seriata complexes. The two complexes occupied different sub-niches during their respective low abundance and bloom periods. The phytoplankton community structure and the number of other taxa presenting a niche overlapping the niches of P. delicatissima and P. seriata complexes also differed between these periods. P. globosa was the taxa contributing the most to the dissimilarity in community structure. P. globosa interacted positively with P. delicatissima complex and negatively with P. seriata complex.

Stochastic and Deterministic Processes Regulate Phytoplankton Assemblages in a Temperate Coastal Ecosystem.

Assessing the relative contributions of the interacting deterministic and stochastic ecological processes for phytoplankton community assembly is crucial in understanding and predicting community organization and succession at different temporal and spatial scales. In this study, we hypothesized that deterministic and stochastic ecological processes regulating phytoplankton, present seasonal and repeating patterns. This hypothesis was explored during a 5-year survey (287 samples) conducted at a small spatial scale (~15km) in a temperate coastal ecosystem (eastern English Channel). Microscopy and flow cytometry quantified phytoplankton abundance and biomass, while metabarcoding data allowed an extended evaluation of diversity and the exploration of the ecological processes regulating phytoplankton using null model analysis. Alpha diversity of phytoplankton was governed by the effect of environmental conditions (environmental filtering). Temporal community turnover (beta diversity) evidenced a consistent interannual pattern that determined the phytoplankton seasonal structure. In winter and early spring (from January to March), determinism (homogeneous selection) was the major process in the phytoplankton community assembly. The overall mean in the year was 38%. Stochastic processes (ecological drift) prevailed during the rest of the year from April to December, where the overall mean for the year was 55%. The maximum values were recorded in late spring and summer, which often presented recurrent and transient monospecific phytoplankton peaks. Overall, the prevalence of stochastic processes rendered less predictable seasonal dynamics of phytoplankton communities to future environmental change. IMPORTANCE While ecological deterministic processes are conducive to modeling, stochastic ones are far less predictable. Understanding the overall assembly processes of phytoplankton is critical in tracking and predicting future changes. The novelty of this study was that it addressed a long-posed question, on a pluriannual scale. Was seasonal phytoplankton succession influenced by deterministic processes (e.g., abiotic environment) or by stochastic ones (e.g., dispersal, or ecological drift)? Our results provided strong support for a seasonal and repeating pattern with stochastic processes (drift) prevailing during most of the year and periods with monospecific phytoplankton peaks.

The effect of inorganic particle concentration on bacteria-virus-nanoflagellate dynamics.

The effect of inorganic particle concentrations on bacteria-virus-nanoflagellate dynamics in an oligotrophic coastal system was investigated using a model aluminosilicate, kaolinite, with a modal size of 2.1 µm. Virus-only, bacteria-only and bacteria-virus-nanoflagellate incubations were carried out at increasing kaolinite concentrations to elucidate the microbial response. The sorption of bacteria and viruses to kaolinite particles was negligible over a concentration range of 1-50 mg l(-1). In contrast, the abundance of heterotrophic nanoflagellates was negatively correlated with kaolinite concentrations following both 48 and 96 h incubations. Calculated nanoflagellate bacterial ingestion rates were reduced by 5-35% depending on kaolinite particle concentration. In the bacteria-virus-nanoflagellate incubations viral production increased by 56 × 10(3) to 104 × 10(3) VLPs ml(-1) h(-1) as a function of kaolinite particle concentration. Our results demonstrate for the first time that the interaction of microbial populations with inorganic particles can shift the balance between protist and virally mediated mortality of marine heterotrophic prokaryotes.

Exploring and quantifying fungal diversity in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing.

Water samples were collected along transects from the shore to the centre of two French lakes: the deep, volcanic, oligomesotrophic and low allochthonic-impacted Lake Pavin, and the productive and higher allochthonic-impacted Lake Aydat. The biodiversity was analysed using two approaches: the classical approach consisting of cloning/sequencing of the 18S, ITS1, 5.8S, ITS2 and partial 28S region using primers designed for fungus sequences, and the pyrosequencing of 18S rRNA hypervariable V2, V3 and V5 regions using two primer sets (one universal for eukaryotes and one for fungi). The classical approach yielded 146 (Lake Pavin) and 143 (Lake Aydat) sequences, corresponding to 46 and 63 operational taxonomic units (OTUs) respectively. Fungi represented half of the OTUs identified in Lake Pavin and 30% in Lake Aydat, and were dominated by sequences from Chytridiomycota found throughout Lake Pavin but mostly in the central pelagic zone of Lake Aydat. The pyrosequencing approach yielded 42,064 (Pavin) and 61,371 (Aydat) reads, of which 12-15% and 9-19% reads were assigned to fungi in Lakes Pavin and Aydat respectively. Chytridiomycota members were also dominant among these reads, with OTUs displaying up to > 33-fold overrepresentation in the centre compared with the riparian areas of Lake Aydat. Besides fungi, both approaches revealed other major eukaryote groups, with the highest diversity in the central areas of lakes. One of the major findings of our study was that the two lakes displayed contrasting spatial distributions, homogenous for Lake Pavin and heterogeneous for Lake Aydat, which may be related to their peculiarities. This study represents the first unveiling of microbial eukaryote and fungus diversity assessed with two complementary molecular methods, and is considered a major milestone towards understanding the dynamics and ecology of fungi in freshwater lake ecosystems, which are directly link to the abundance and distribution of taxa.

Small phytoplankton contribute greatly to CO2-fixation after the diatom bloom in the Southern Ocean.

Phytoplankton is composed of a broad-sized spectrum of phylogenetically diverse microorganisms. Assessing CO2-fixation intra- and inter-group variability is crucial in understanding how the carbon pump functions, as each group of phytoplankton may be characterized by diverse efficiencies in carbon fixation and export to the deep ocean. We measured the CO2-fixation of different groups of phytoplankton at the single-cell level around the naturally iron-fertilized Kerguelen plateau (Southern Ocean), known for intense diatoms blooms suspected to enhance CO2 sequestration. After the bloom, small cells (<20 µm) composed of phylogenetically distant taxa (prymnesiophytes, prasinophytes, and small diatoms) were growing faster (0.37 ± 0.13 and 0.22 ± 0.09 division d-1 on- and off-plateau, respectively) than larger diatoms (0.11 ± 0.14 and 0.09 ± 0.11 division d-1 on- and off-plateau, respectively), which showed heterogeneous growth and a large proportion of inactive cells (19 ± 13%). As a result, small phytoplankton contributed to a large proportion of the CO2 fixation (41-70%). The analysis of pigment vertical distribution indicated that grazing may be an important pathway of small phytoplankton export. Overall, this study highlights the need to further explore the role of small cells in CO2-fixation and export in the Southern Ocean.

Protist Interactions and Community Structure During Early Autumn in the Kerguelen Region (Southern Ocean).

This study investigated protist community composition and biotic interactions focusing on microplankton at four distinct sites around the Kerguelen Islands (Southern Ocean) after the summer phytoplankton bloom. Protist diversity in different size fractions, sampled with Niskin bottles and plankton nets, was assessed by sequencing of the V4 18S rDNA region. Combining different approaches, i.e. sequencing of different plankton size fractions, and isolation and sequencing of single cells, provided new insights into microbial interactions in protist communities. The communities displayed high variability, including short-term fluctuations in relative abundance of large protists (>35µm) highlighted by the plankton net samples. Size fractionation of protist communities showed high concentrations of free Syndiniales spores but relatively few Syndiniales associated with microplankton, suggesting low parasitic infection in early autumn. Co-variance network analyses and sequencing of individually isolated single cells highlighted the important role of Rhizaria as consumers of a wide range of different diatom taxa. The data also raised the hypothesis that different Syndiniales clades might be directly or indirectly associated with some diatom genera, thus suggesting a potentially wider host range of these parasites than has been previously reported. These associations and the potential impact on carbon fluxes are discussed.

Cosmopolitan heterotrophic microeukaryotes are active bacterial grazers in experimental oil-polluted systems.

We investigated the population dynamics and prevailing 18S rDNA phylotypes of microeukaryotes (

Diversity and potential activity patterns of planktonic eukaryotic microbes in a mesoeutrophic coastal area (eastern English Channel).

The diversity of planktonic eukaryotic microbes was studied at a coastal station of the eastern English Channel (EEC) from March 2011 to July 2015 (77 samples) using high throughput sequencing (454-pyrosequencing and Illumina) of the V2-V3 hypervariable region of the 18S SSU rDNA gene. Similar estimations of OTU relative abundance and taxonomic distribution for the dominant higher taxonomic groups (contributing >1% of the total number of OTUs) were observed with the two methods (Kolmogorov-Smirnov p-value = 0.22). Eight super-groups were identified throughout all samples: Alveolata, Stramenopiles, Opisthokonta, Hacrobia, Archeaplastida, Apusozoa, Rhizaria, and Amoebozoa (ordered by decreasing OTU richness). To gain further insight into microbial activity in the EEC, ribosomal RNA was extracted for samples from 2013-2015 (30 samples). Analysis of 18S rDNA and rRNA sequences led to the detection of 696 and 700 OTUs, respectively. Cluster analysis based on OTUs' abundance indicated three major seasonal groups that were associated to spring, winter/autumn, and summer conditions. The clusters inferred from rRNA data showed a clearer seasonal representation of the community succession than the one based on rDNA. The rRNA/rDNA ratio was used as a proxy for relative cell activity. When all OTUs were considered, the average rRNA:rDNA ratio showed a linear trend around the 1:1 line, suggesting a linear relation between OTU abundance (rDNA) and activity (rRNA). However, this ratio was highly variable over time when considering individual OTUs. Interestingly, the OTU affiliated with P. globosa displayed rRNA:rDNA ratio that allowed to delimit high vs low abundance and high vs low activity periods. It unveiled quite well the Phaeocystis bloom dynamic regarding cell proliferation and activity, and could even be used as early indicator of an upcoming bloom.

Development of a qPCR Method for the Identification and Quantification of Two Closely Related Tuna Species, Bigeye Tuna (Thunnus obesus) and Yellowfin Tuna (Thunnus albacares), in Canned Tuna.

Bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) are among the most widely used tuna species for canning purposes. Not only substitution but also mixing of tuna species is prohibited by the European regulation for canned tuna products. However, as juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional substitutions may occur during the canning process. In this study, two mitochondrial markers from NADH dehydrogenase subunit 2 and cytochrome c oxidase subunit II genes were used to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR methodology. Two different qPCR-based methods were developed to quantify the percentage of flesh of each species used for can processing. The first one was based on absolute quantification using standard curves realized with these two markers; the second one was founded on relative quantification with the universal 12S rRNA gene as the endogenous gene. On the basis of our results, we conclude that our methodology could be applied to authenticate these two closely related tuna species when used in a binary mix in tuna cans.

Shifts in bacterial community composition associated with increased carbon cycling in a mosaic of phytoplankton blooms.

Marine microbes have a pivotal role in the marine biogeochemical cycle of carbon, because they regulate the turnover of dissolved organic matter (DOM), one of the largest carbon reservoirs on Earth. Microbial communities and DOM are both highly diverse components of the ocean system, yet the role of microbial diversity for carbon processing remains thus far poorly understood. We report here results from an exploration of a mosaic of phytoplankton blooms induced by large-scale natural iron fertilization in the Southern Ocean. We show that in this unique ecosystem where concentrations of DOM are lowest in the global ocean, a patchwork of blooms is associated with diverse and distinct bacterial communities. By using on-board continuous cultures, we identify preferences in the degradation of DOM of different reactivity for taxa associated with contrasting blooms. We used the spatial and temporal variability provided by this natural laboratory to demonstrate that the magnitude of bacterial production is linked to the extent of compositional changes. Our results suggest that partitioning of the DOM resource could be a mechanism that structures bacterial communities with a positive feedback on carbon cycling. Our study, focused on bacterial carbon processing, highlights the potential role of diversity as a driving force for the cycling of biogeochemical elements.

Mussel biofiltration effects on attached bacteria and unicellular eukaryotes in fish-rearing seawater.

Mussel biofiltration is a widely used approach for the mitigation of aquaculture water. In this study, we investigated the effect of mussel biofiltration on the communities of particle-associated bacteria and unicellular eukaryotes in a sea bass aquaculture in southern North Sea. We assessed the planktonic community changes before and after biofiltration based on the diversity of the 16S and 18S rRNA genes by using next generation sequencing technologies. Although there was no overall reduction in the operational taxonomic units (OTU) numbers between the control (no mussels) and the test (with mussels) tanks, a clear reduction in the relative abundance of the top three most dominant OTUs in every sampling time was observed, ranging between 2-28% and 16-82% for Bacteria and Eukarya, respectively. The bacterial community was dominated by OTUs related to phytoplankton blooms and/or high concentrations of detritus. Among the eukaryotes, several fungal and parasitic groups were found. Their relative abundance in most cases was also reduced from the control to the test tanks; a similar decreasing pattern was also observed for both major higher taxa and functional (trophic) groups. Overall, this study showed the effectiveness of mussel biofiltration on the decrease of microbiota abundance and diversity in seawater fueling fish farms.

Composition of diatom communities and their contribution to plankton biomass in the naturally iron-fertilized region of Kerguelen in the Southern Ocean.

In the naturally iron-fertilized surface waters of the northern Kerguelen Plateau region, the early spring diatom community composition and contribution to plankton carbon biomass were investigated and compared with the high nutrient, low chlorophyll (HNLC) surrounding waters. The large iron-induced blooms were dominated by small diatom species belonging to the genera Chaetoceros (Hyalochaete) and Thalassiosira, which rapidly responded to the onset of favorable light-conditions in the meander of the Polar Front. In comparison, the iron-limited HNLC area was typically characterized by autotrophic nanoeukaryote-dominated communities and by larger and more heavily silicified diatom species (e.g. Fragilariopsis spp.). Our results support the hypothesis that diatoms are valuable vectors of carbon export to depth in naturally iron-fertilized systems of the Southern Ocean. Furthermore, our results corroborate observations of the exported diatom assemblage from a sediment trap deployed in the iron-fertilized area, whereby the dominant Chaetoceros (Hyalochaete) cells were less efficiently exported than the less abundant, yet heavily silicified, cells of Thalassionema nitzschioides and Fragilariopsis kerguelensis Our observations emphasize the strong influence of species-specific diatom cell properties combined with trophic interactions on matter export efficiency, and illustrate the tight link between the specific composition of phytoplankton communities and the biogeochemical properties characterizing the study area.

Microzooplankton community associated with phytoplankton blooms in the naturally iron-fertilized Kerguelen area (Southern Ocean).

The spatial and temporal community composition of microzooplankton (dinoflagellates and ciliates) was assessed in the Kerguelen area (Southern Ocean) during the KEOPS2 cruise in early spring (October-November) 2011. This naturally iron-fertilized region was characterized by a complex mesoscale circulation resulting in a patchy distribution of phytoplankton blooms. Collectively, 97 morphospecies of dinoflagellates and ciliates belonging to 41 genera were identified by microscopy, and 202 Alveolata-related OTUs (operational taxonomical units) were retrieved with tag-pyrosequencing. Microscopy and pyrosequencing data were in accordance, in that diatom-consuming dinoflagellates were the most enhanced taxa in the blooms. Dinoflagellates also showed significant positive relationships with phytoplankton pigments, while no major differences were found in the ciliate abundances inside and outside the blooms. Cluster analysis showed clear differences in the phytoplankton and microzooplankton community structures between the iron-fertilized and HNLC (high nutrient low chlorophyll) waters, and between the blooms, concerning their location and the fertilization mechanisms. These results were combined with the rates of primary production and mesozooplankton consumption determined for the study area. The potential role of dinoflagellates and ciliates as phytoplankton consumers and as prey for mesozooplankton was then evaluated. Overall, heterotrophic dinoflagellates were probably the most important group of phytoplankton grazers, and a potential food source for copepods.

Seasonal variations of marine protist community structure based on taxon-specific traits using the eastern English Channel as a model coastal system.

Previous microscopy-based studies in the eastern English Channel have revealed it to be a productive meso-eutrophic coastal ecosystem, characterized by strong repeating patterns in microplankton succession. The present study examines the seasonal structure of the entire protistan community from March 2011 to July 2013, using tag pyrosequencing of the V2-V3 hypervariable region of the 18S rRNA gene. A total of 1242 OTUs and 28 high-level taxonomic groups, which included previously undetected taxa in the area, were identified. The detected OTUs were considered according to taxon-specific traits, which included their trophic role, abundance and specialization level. Taxa differentiation based on specialization level rather than abundance was more informative in describing community organization. While generalists were always abundant, numerous specialists that were either rare or absent in most samples, increased in abundance for short periods, appearing to be overall abundant. Statistical and network analyses showed that the protistan seasonal organization was influenced by environmental parameters. It also highlighted that in addition to grazers, fungi and parasites played potentially significant roles during phytoplankton blooms. Overall, while the protistan succession was mainly shaped by environmental variations, biotic interactions among co-occurring taxa were the main structural drivers of the temporal assemblages.

Size-fractionated diversity of eukaryotic microbial communities in the Eastern Tropical North Pacific oxygen minimum zone.

Oxygen minimum zones (OMZs) caused by water column stratification appear to expand in parts of the world's ocean, with consequences for marine biogeochemical cycles. OMZ formation is often fueled by high surface primary production, and sinking organic particles can be hotspots of interactions and activity within microbial communities. This study investigated the diversity of OMZ protist communities in two biomass size fractions (>30 and 30-1.6 µm filters) from the world's largest permanent OMZ in the Eastern Tropical North Pacific. Diversity was quantified via Illumina MiSeq sequencing of V4 region of 18S SSU rRNA genes in samples spanning oxygen gradients at two stations. Alveolata and Rhizaria dominated the two size fractions at both sites along the oxygen gradient. Community composition at finer taxonomic levels was partially shaped by oxygen concentration, as communities associated with versus anoxic waters shared only ∼32% of operational taxonomic unit (OTU) (97% sequence identity) composition. Overall, only 9.7% of total OTUs were recovered at both stations and under all oxygen conditions sampled, implying structuring of the eukaryotic community in this area. Size-fractionated communities exhibited different taxonomical features (e.g. Syndiniales Group I in the 1.6-30 µm fraction) that could be explained by the microniches created on the surface-originated sinking particles.

Evaluation of double formalin--Lugol's fixation in assessing number and biomass of ciliates: an example of estimations at mesoscale in NE Atlantic.

Ciliated protozoa are potential grazers of primary and bacterial production and act as intermediaries between picoplankton and copepods and other large suspension feeders. Accurate determination of ciliate abundance and feeding mode is crucial in oceanic carbon budget estimations. However, the impact of different fixatives on the abundance and cell volume of ciliates has been investigated in only a few studies using either laboratory cultures or natural populations. Lugol's solution and formalin are the most commonly used fixatives for the preservation of ciliates samples. In the present study, the aim was to compare 0.4% Lugol's solution and 2% borated-formalin fixation and evaluate the need of counting duplicate samples each using a different fixative. For this, a large number of samples (n = 110) from the NE Atlantic was analyzed in the frame of POMME program (Multidisciplinary Mesoscale Ocean Program). We established a statistically significant relationship (p < 0.0001) between Lugol's and formalin fixed samples for both abundance (r2 = 0.50) and biomass (r2 = 0.76) of aloricate ciliates which showed that counts were higher in Lugol's solution by a factor of 2 and a non-taxon specific cell-loss in formalin. However, loricate ciliate abundance in our samples which were represented primarily by Tintinnus spp. did not show any difference between the two treatments. Abundance and biomass of mixotrophic ciliates (chloroplast-bearing cells) were for various reasons underestimated in both treatments. Our results show that unique fixation by formalin may severely underestimate ciliates abundance and biomass although their population may not alter. For this reason, Lugol's solution is best for the estimation of their abundance and biomass. However, for counts of mixotrophs and the evaluation of the ecological role of ciliates in carbon flux, double fixation is essential. Compromises regarding the fixatives have lead to severe underestimations of mixotrophs in studies conducted by now.