Albert Calbet
email: acalbet@icm.csic.es

Enric Saiz

Rodrigo Almeda

Juan Ignacio Movilla

Miguel Alcaraz

Miquel Alcaraz

Low microzooplankton grazing rates in the Arctic Ocean during

Keywords: Arctic Ocean, Microzooplankton, Dilution technique, Food web Arctic Ocean, Microzooplankton, Phaeocystis pouchetii, Dilution technique, Food web

come

Low microzooplankton grazing rates in the Arctic Ocean during a Phaeocystis pouchetii bloom (Summer 2007): fact or artifact of the dilution technique?

Introduction

The fast melting of Arctic ice, a consequence of global warming [START_REF] Cavalieri | 30-Year satellite record reveals contrasting Arctic and Antarctic decadal sea ice variability[END_REF]Johannessen et al., 2004), has profound ecological consequences on Arctic wildlife, including polar bears, walruses, and ringed seals, which are threatened by the reduction of sea-ice cover. Not so evident are the changes that the inhabitants of the waters underneath and surrounding the Polar ice cap will suffer under progressive melting conditions. One of the key groups in marine food webs, which are potentially sensitive to these Arctic scenario changes, are the microzooplankton.

Microzooplankton are of paramount importance in controlling primary production [START_REF] Calbet | Phytoplankton growth, microzooplankton grazing, and carbon cycling in marine systems[END_REF]. Regrettably, their role in the high Arctic still remains uncertain and needs to be determined to fully understand and predict the consequences of the changes this ecosystem will experience in the future. For instance, the relevance of the trophic impact of this group in the Arctic Ocean food web has mostly been studied either in bays or in relatively low latitude sites [START_REF] Paranjape | Grazing by microzooplankton in the eastern Canadian Arctic in summer 1983[END_REF][START_REF] Gifford | Grazing by microzooplankton and mesozooplankton in the high-latitude North Atlantic Ocean: Spring versus summer dynamics[END_REF][START_REF] Olson | Phytoplankton growth, microzooplankton herbivory and community structure in the southeast Bering Sea: insight into the formation and temporal persistence of an Emiliania huxleyi bloom[END_REF][START_REF] Verity | Grazing of phytoplankton by microzooplankton in the Barents Sea during early summer[END_REF][START_REF] Strom | Intense stratification leads to phytoplankton nutrient limitation and reduced microzooplankton grazing in the southeastern Bering Sea[END_REF], or it has been derived indirectly [START_REF] Levinsen | Plankton community structure and carbon cycling on the western coast of Greenland during the stratified summer situation. II. Heterotrophic dinoflagellates and ciliates[END_REF][START_REF] Rysgaard | Seasonal variation in nutrients, pelagic primary production and grazing in a high-Arctic coastal marine ecosystem, Young Sound, Northeast Greenland[END_REF][START_REF] Levinsen | The trophic role of marine pelagic ciliates and heterotrophic dinoflagellates in arctic and temperate coastal ecosystems: A crosslatitude comparison[END_REF]. Most of these studies indicate a strong control of primary production by microzooplankton grazing. However, recent work in high Arctic waters by Sherr et al. [START_REF] Sherr | Microzooplankton grazing impact in the Western Arctic Ocean[END_REF] questions such strong control, likely due, according to the authors, to a strong top-down impact of copepods on microzooplankton [START_REF] Levinsen | The trophic role of marine pelagic ciliates and heterotrophic dinoflagellates in arctic and temperate coastal ecosystems: A crosslatitude comparison[END_REF][START_REF] Campbell | Mesozooplankton prey preference and grazing impact in the Western Arctic Ocean[END_REF].

For this reason, we undertook a study of the microbial interactions in Arctic waters during the melting season. Our research coincided with a bloom of P.

pouchetii (Lasternas and Agustí, in press), which will add further value to our results because, despite being a successful species in Arctic waters [START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF], the very few data dealing with the impact of microzooplankton feeding on these algae have resulted in contradictory conclusions. [START_REF] Weisse | Growth and grazing loss rates in single-celled Phaeocystis sp. (Prymnesiophyceae)[END_REF]) measured microzooplankton grazing in a P. cf. pouchetii bloom in the North Sea using the dilution technique [START_REF] Landry | Estimating the grazing impact of marine microzooplankton[END_REF] F o r P e e r R e v i e w 4 finding grazing loss rates from 0.037 to 0.174 h -1 , grazing rates increasing in the course of the bloom and exceeding phytoplankton growth rates at the end. Gifford et al. [START_REF] Gifford | Grazing by microzooplankton and mesozooplankton in the high-latitude North Atlantic Ocean: Spring versus summer dynamics[END_REF], also using the dilution technique, did not detect any grazing on phytoplankton in the high-latitude North Atlantic Ocean during a P. pouchetii bloom, but they obtained increased microzooplankton grazing as the bloom declined.

Archer et al. [START_REF] Archer | Impact of microzooplankton on the progression and fate of the spring bloom in fjords of northern Norway[END_REF] measured microzooplankton grazing impact under relatively low abundance of P. pouchetii in 3 fjords of the northern Norway, and obtained significant grazing rates on total phytoplankton and on fluorescently labeled algae of similar size to P. pouchetii. On the other hand, [START_REF] Wolfe | DMSP and DMS dynamics and microzooplankton grazing in the Labrador Sea: application of the dilution technique[END_REF] only found significant grazing on chlorophyll and DMSP when P. pouchetii cells were in poor condition in the Labrador Sea. In this regard, the ability of microzooplankton to ingest Phaeocystis spp. has been widely discussed in the literature (see reviews by [START_REF] Whipple | Life cycle of the marine alga Phaeocystis: a conceptual model to summarize literature and guide research[END_REF][START_REF] Nejstgaard | Zooplankton grazing on Phaeocystis: a quantitative review and future challenges[END_REF]. It is usually accepted that microzooplankton exerts their pressure mostly on single cells; however, there is evidence that Noctiluca scintillans [START_REF] Weisse | The trophic significance of Phaeocystis blooms[END_REF]Jakobsen and Tang, 2002) and Gyrodinium cf. spirale [START_REF] Stelfox-Widdicombe | Microzoo-plankton grazing in Phaeocystis and diatom-dominated waters in the southern North Sea in spring[END_REF] are able to ingest small colonies.

Method

This study was part of a multidisciplinary project (ATOS: POL2006-00550/CTM), and took place on board the research vessel BIO Hespérides from July 1 to 24, 2007.

The cruise departed Reykjavik (Iceland), and sampling started northward across the Greenland Sea (Table 1, Fig. 1). In Arctic waters, we sampled a series of stations in the vicinity of the ice-edge, alternating between several stations free of ice and two coastal stations near the Svalbard Islands. During the study, we reached a historical minimum of Arctic ice cover [START_REF] Zhang | What drove the dramatic retreat of Arctic sea ice during summer 2007?[END_REF], allowing samples to be taken from areas up to 80º 50' N. The stations lasted 24 h, starting with profiles for the At each station we conducted standard grazing dilution experiments [START_REF] Landry | Estimating the grazing impact of marine microzooplankton[END_REF] to assess the microzooplankton grazing impact on primary producers and on other components of the food web (see below). This technique consists of the sequential dilution of natural water with filtered seawater to obtain a gradient of net grazing impact on phytoplankton. The water for these experiments was collected at the fluorescence maximum (Table 1) using a rosette equipped with 12-L Niskin bottles, according to the fluorescence profile. Once on deck, the water was gravity-filtered through a Pall Acropak 0.8/0.2 500 capsule (previously flushed, including tubing, with diluted HCl and thoroughly rinsed with deionised water), and then 2 replicate bottles (2.3-L acid washed polycarbonate) for each of the dilution treatments were filled with the corresponding required amount of filtered seawater.

Afterwards, we added natural non-filtered seawater from the selected depth to the bottles to generate experimental water percentages of 13, 27, 50, 73, and 100%. At some stations, the presence of a dense bloom of the haptophyte Phaeocystis pouchetii made it impossible to efficiently filter the water through the Acropak capsules; therefore, filtered seawater originated from below the bloom depth.

To guarantee the homogeneity of the natural water poured from different Niskin bottles filled at the same depth, we used a 20-L intermediary carboy in which the water was gently mixed by its own flow. All the process was carried out under dim light conditions to avoid cell light-damage. To promote constant and saturated phytoplankton growth in the dilution series, each bottle received added nutrients (10 µM NH 4 Cl and 0.7 µM Na 2 HPO 4 ). In addition, four 100% (i.e., not diluted) bottles were prepared without nutrients to assess the natural growth of the algae. Two of these latter bottles were sacrificed for initial samples. Because we did not screen the water used for the dilution series to avoid damaging delicate microzooplankton, the experimental suspension may have contained some mesozooplankton. We examined the bottles by eye to observe the presence of large copepods, and the very few times this occurred did not result in inconsistent results in the dilution series. open-circuit water running from a 5-m depth at a temperature about the same as in situ. The natural sunlight was dimmed with appropriate dark plastic mesh to mimic the light intensity at the fluorescence maximum. We gently mixed the bottles by repeatedly turning them upside down and moving them around the incubator at least three times per day. After 27-32 h, we finished the incubations and took samples for quantification of total and > 5-µm Chl a concentration. To get further insight into the actual trophic interactions during dilution experiments and to detect possible artifacts [START_REF] Dolan | Dilution effects on microzooplankton in dilution grazing experiments[END_REF][START_REF] Agis | A cautionary note: Examples of possible microbial community dynamics in dilution grazing experiments[END_REF][START_REF] Modigh | Changes in phytoplankton and microzooplankton populations during grazing experiments at a Mediterranean coastal site[END_REF], we additionally took samples for the determination of nano-and microplankton from the 2 initials and in one of the replicates per dilution level, and preserved them with Acidic Lugol's solution (2% final concentration). To avoid damaging the delicate cells we first added the fixative and then gently siphoned the water sample directly into the sample bottle For total Chl a, we filtered 50-250 ml of water (depending on station and dilution level) under low vacuum pressure (< 100 mm Hg) through Whatmann glass fiber filters (GF/F, 25 mm diameter). For the > 5-µm fraction, we filtered 100-300-ml samples through 5-µm pore-size polycarbonate Osmonics Inc. filters (25 mm diameter). After filtration, the filters were stored frozen at -20°C until fluorometric analysis of acetone extracts, with and without acidification [START_REF] Parsons | A manual of chemical and biological methods for sea water analysisedn[END_REF] on a Turner Designs Fluorometer.

Lugol-preserved samples were processed in the laboratory. We concentrated the most diluted treatments (13%, 25% and 50%) by first settling the entire bottle for 72 h and gently siphoning off 50-75% of the supernatant water without re-suspending the sample. Then, for all the samples, 100 ml of the concentrate was settled in Utermöhl chambers for at least 48 h prior and counted under the microscope. The whole chamber, or a fraction of it for the smallest and more abundant organisms, was counted under an inverted microscope (XSB-1A) at 100, 250, and 400X magnification, depending on the group. Fifty to one-hundred cells per group were sized, adjusted to their closest geometric shape, and converted into carbon using the equations of Menden-Deuer and [START_REF] Menden-Deuer | Carbon to volume relationships for dinoflagellates, diatoms, and other protist plankton[END_REF]. We did not use any correction factor to compensate for ciliate losses due to fixation, as previously suggested by [START_REF] Broglio | Trophic impact and prey selection by crustacean zooplankton on the microbial communities of an oligotrophic coastal area (NW Mediterranean Sea)[END_REF], and Calbet and Saiz [START_REF] Calbet | The ciliate-copepod link in marine ecosystems[END_REF], because recent research has revealed that such corrections should apply to many other planktonic groups, not only ciliates [START_REF] Zarauz | Effects of Lugol's fixation on the size structure of natural nano-microplankton samples, analyzed by means of an automatic counting method[END_REF], and universal factors have not been developed yet. Because plankton were preserved with acidic Lugol's solution, no distinction between strict heterotrophs and auto/mixotrophs was made for flagellates, ciliates, and some dinoflagellates.

However, the groups identified to species level were classified trophically according to the literature.

Instantaneous growth rates in dilution grazing experiments were derived from net growth in the un-amended bottles (no nutrients added) adding the mortality by microzooplankton grazing from dilution experiments when the latter was significant.

We should note that in the cases where the grazing mortality was not significant the instantaneous growth rate of prey would be equivalent to the net growth rate in the un-amended bottles. Therefore, we may be underestimating the actual instantaneous growth rate of the prey because some mortality may still occur in the bottles, even if not measured by the dilution method. All statistical tests were conducted with JMP 7.0 statistical software.

Results

Plankton biomass and distribution

We present a summary of the different fractions of Chl a in Table 1 and the contribution of micro-and nanoplankton to total plankton community biomass at the sampled stations in Table 2 (a detailed summary of the species composition is presented in the Supplementary Material online). The main feature of the data for the Arctic stations is the almost ubiquitous presence of the haptophyte Phaeocystis pouchetii, which formed dense blooms (Table 2, Fig. 2) and whose distribution was directly correlated with total Chl a (r=0.76, p<0.01), and inversely correlated with temperature (r = 0.77; p < 0.05). Following P. pouchetii, diatoms (mostly chain forming species > 20 µm in total length of the genera Chaetoceros, Fragilariopsis, Nitzschia, Rhizosolenia, Thalassiothrix, and Thalassiosira) were the second biomass grouping within the phytoplankton, being dominant in the Greenland Sea and at warmer stations. Regarding micrograzers, the important contribution of large ciliates 2). Actually, ciliates were the overall major contributors to microzooplankton during the study and were substantial components of the total plankton biomass (ca. 25%), whereas heterotrophic dinoflagellates (considering 50% of unidentified dinoflagellates as heterotrophic; [START_REF] Lessard | Species-specific grazing rates of heterotrophic dinoflagellates in oceanic waters, measured with a dual radioisotope technique[END_REF][START_REF] Sherr | Heterotrophic dinoflagellates: a significant component of microzooplankton biomass and major grazers of diatoms in the sea[END_REF] represented, on average, 12% of the total carbon biomass (Table 2). In relation to the relevance of heterotrophs, it is interesting to note that the quotient between heterotrophic and autotrophic carbon (indicative of the trophic characteristics of the system) was > 1 in the Greenland Sea and Svalbard Islands coastal stations (Fig. 3).

Despite the high heterotrophic biomass, we did not find significant correlations between any of the size fractions of Chl a, large heterotrophic dinoflagellates (e.g., Gyrodinium spp., Protoperidinium spp., and Katodinium spp.), and ciliates. However, large mixotrophic dinoflagellates (Dinophysis spp., Ceratium spp., Gonyaulax spp., and Amphidinium spp.) were positively correlated with diatoms (r = 0.85; p < 0.05). Correlations between groups do not necessarily mean causality, but they can indicate that an association exists. Likewise, unidentified > 20-µm dinoflagellates were also positively correlated with P. pouchetti biomass (r = 0.72; p < 0.05). However, when excluded from the analysis station 43 (the station with the maximum biomass of P. pouchetii) this relationship is strongly affected and it becomes non-significant.

We calculated the C:Chl a ratios using the Chl a data and the autotrophic biomass (in carbon), obtained by cell counting and conversion to carbon using literature equations (Table 2). For unidentified dinoflagellates we assumed 50% of the organisms were phototrophs. Because our microscope counting technique, based on Lugol-preserved samples, does not allow for good resolution of the lower sizefractions, we can better estimate the C:Chl a ratio using only > 5-µm Chl a. The values averaged 18 for the entire data-set, and ranged from 4 to 47. Table 3 shows the outcome of the dilution grazing experiments based on the different size-fractions of Chl a. Unanticipated results from these experiments include the following: i) the little, or even at times negative, phytoplankton instantaneous growth (range: -0.24 to 0.14; average -0.04 for total Chl a) and ii) the low microzooplankton grazing impact on primary producers. Significant microzooplankton grazing (i.e., significant negative slopes in the dilution experiments) were found only in 6 out of 16 experiments for total and <5-µm Chl a, and no significant mortality was detected in the >5-µm fraction at any of the stations. Overall, microzooplankton grazing cannot explain the observed phytoplankton negative net growth rates (especially in the >5µm size-fractions) and must be attributed to other causes (see discussion). However, it is interesting to note the inverse relationship between the phytoplankton net growth rates and Chl a concentration (Fig. 4) in the Arctic Ocean stations. This points towards a naturally-occurring, density-dependent mortality effect, probably linked to the presence of Phaeocystis pouchetii, although not significantly related to the occurrence of either this or any other planktonic group. Likewise, and related to net phytoplankton growth rates, there was no clear evidence of nutrient limitation at most of the stations (Fig. 5).

Dilution grazing experiments

The phytoplankton and microzooplankton composition analysis in the initial and final dilution experiments revealed a very complex food web scenario, with frequent negative growth rates both for autotrophs and heterotrophs, frequent positive slopes, and very few cases of significant microzooplankton grazing impact (Table 4).

Overall, it is difficult to extract any clear interpretation or global pattern out of the dilution data for the different plankton groups. Nevertheless, we decided to present these data because negative dilution grazing results in the literature have seldom been discussed [START_REF] Dolan | The reliability of grazing rate estimates from dilution experiments: Have we over-estimated rates of organic carbon consumption by microzooplankton?[END_REF]. As example, we show in Fig. 6 the dilution experiment plots for stations 4 and 33. It is interesting to note in these plots the positive slopes for some prey and the negative slopes (theoretically indicative of grazing) for some potential grazers of phytoplankton. It should be noted that the mortality rates of top predators (large ciliates and heterotrophic dinoflagellates) are not necessarily estimated well by the dilution method. However, we think the information about the changes of these groups in the incubation bottles is relevant and should be also presented in the results.

Discussion

Community composition and microzooplankton grazing on phytoplankton

A main characteristic that distinguishes our study from previous ones in Arctic waters is the peculiarity of the composition of the heterotrophic microbial community. Past works stressed the relevance of heterotrophic dinoflagellates in Arctic waters, especially when diatoms dominated the autotrophic community [START_REF] Levinsen | Plankton community structure and carbon cycling on the western coast of Greenland during the stratified summer situation. II. Heterotrophic dinoflagellates and ciliates[END_REF][START_REF] Rysgaard | Seasonal variation in nutrients, pelagic primary production and grazing in a high-Arctic coastal marine ecosystem, Young Sound, Northeast Greenland[END_REF][START_REF] Sherr | Heterotrophic protists in the Central Arctic Ocean[END_REF][START_REF] Sherr | Heterotrophic dinoflagellates: a significant component of microzooplankton biomass and major grazers of diatoms in the sea[END_REF][START_REF] Sherr | Microzooplankton grazing impact in the Western Arctic Ocean[END_REF]. We found a population rich in ciliates, which accounted for ca. 2/3 of the total microzooplankton biomass (assuming 50% of the unidentified dinoflagellates were heterotrophs). This high contribution was especially evident in the Greenland Sea, where they completely dominated the microplankton. We suspect that in these waters we were facing a very unusual situation, likely the result of temporal imbalances in the structure of the community (e.g., the end of a phytoplankton bloom), as the biomass of producers seems unable to maintain such a high abundance of grazers.

Despite the relatively high biomass of microzooplankton, the grazing rates on phytoplankton (Chl a) were low. Certainly, trophic relationships between predators and prey do not necessarily have to be directly related to biomass, especially in areas where annual blooms are intense. If a phytoplankton bloom is senescent, grazers, even if abundant, may not graze phytoplankton cells in poor health (the end of the bloom situation). Similar inconsistencies between grazing impacts and grazer biomass have also been reported for Antarctic waters [START_REF] Caron | Microzooplankton herbivory in the Ross Sea, Antarctica[END_REF]. Regarding the Arctic Ocean, we find contrasting results for microzooplankton grazing impact in different areas. For instance, during the summer along the western coast of Greenland, Levinsen et al. [START_REF] Levinsen | Plankton community structure and carbon cycling on the western coast of Greenland during the stratified summer situation. II. Heterotrophic dinoflagellates and ciliates[END_REF] studied the microzooplankton grazing impact by indirect methods and concluded that if this group had a purely autotrophic diet they could remove 362% of primary daily production. Nevertheless, the authors pointed out that cannibalism could likely reduce the actual impact of this group on phytoplankton. Also by indirect measurements, Rysgaard et al. [START_REF] Rysgaard | Seasonal variation in nutrients, pelagic primary production and grazing in a high-Arctic coastal marine ecosystem, Young Sound, Northeast Greenland[END_REF] estimated that the combined grazing activity of ciliates and dinoflagellates in Young Sound (NE Greenland) would potentially remove only 14% of the annual Levinsen and Nielsen [START_REF] Levinsen | The trophic role of marine pelagic ciliates and heterotrophic dinoflagellates in arctic and temperate coastal ecosystems: A crosslatitude comparison[END_REF] found that potential microzooplankton grazing could account for 32-55% of the primary production in Disko Bay. These data, as well as the 40-114% primary production daily grazed in Jones Sound [START_REF] Paranjape | Grazing by microzooplankton in the eastern Canadian Arctic in summer 1983[END_REF], the 37-88% in Baffin Bay [START_REF] Paranjape | Grazing by microzooplankton in the eastern Canadian Arctic in summer 1983[END_REF], the 64-97% in the Barents Sea [START_REF] Verity | Grazing of phytoplankton by microzooplankton in the Barents Sea during early summer[END_REF], and the 2-293% (average 110 and 81% of phytoplankton growth rates for > 10 µm and < 10 µm phytoplankton, respectively) grazed in the southeast Bering Sea [START_REF] Olson | Phytoplankton growth, microzooplankton herbivory and community structure in the southeast Bering Sea: insight into the formation and temporal persistence of an Emiliania huxleyi bloom[END_REF] are much higher than the values observed in our study. Recent research in the high Western Arctic Ocean [START_REF] Sherr | Microzooplankton grazing impact in the Western Arctic Ocean[END_REF], however, advocates a lower control of microzooplankton grazing on primary producers (average 22±26%). The study by [START_REF] Sherr | Microzooplankton grazing impact in the Western Arctic Ocean[END_REF], even if in a different area, with lower average temperatures, and with a community of phytoplankton not dominated by Phaeocystis, but by diatoms, is the study that a priori seems more appropriate to contrast with ours; both were located in open waters near the ice-edge zone, and both were conducted at high latitudes using the same methodology. Our data agree with the results of the Sherr et al. [START_REF] Sherr | Microzooplankton grazing impact in the Western Arctic Ocean[END_REF] study, which does not show significant grazing in about half of the experiments at the fluorescence maximum and contains total average grazing rates of < 0.1 d -1 .

Similarly, in the Sherr et al. [START_REF] Sherr | Microzooplankton grazing impact in the Western Arctic Ocean[END_REF] study they found low and even negative values for phytoplankton growth rates during summer. They attributed this natural mortality to low light levels (samples were collected at the base of the euphotic zone) and to post-bloom conditions (protist grazing rates were low because the diatom blooms were senescent).

Despite the overall low grazing rates, some associations between the distributions of several organisms seem to be evident. For instance, mixotrophic dinoflagellates were positively correlated with diatoms. Mixotrophy is widespread among dinoflagellates, and it is not uncommon that these organisms contribute significantly to the community grazing on phytoplankton [START_REF] Stoecker | Mixotrophy among dinoflagellates[END_REF][START_REF] Stoecker | Acquired phototrophy in aquatic protists[END_REF], and particularly on diatoms [START_REF] Yoo | Feeding by phototrophic red-tide dinoflagellates on the ubiquitous marine diatom Skeletonema costatum[END_REF]. Moreover, the role of mixotrophic dinoflagellates in the fate of primary producers is likely to have been underestimated for several reasons. First, the presence of their own chloroplasts may mask the detection of prey inside the organisms, and second, the different feeding mechanisms displayed by this group (direct engulfment, tube-feeding, and pallium-F o r P e e r R e v i e w feeding; [START_REF] Hansen | Phagotrophic mechanisms and prey selection in free-living dinoflagellates[END_REF]) make it quite difficult to correctly assess their contribution to total community grazing. Because pallium-feeding and tube-feeding do not leave evident remains of the preyed cell inside the predator and because the pallium and peduncles are not persistent structures, they are not easily quantified when microscopically observing preserved samples. Therefore, we are of the opinion that a predator-prey association between armoured mixotrophic dinoflagellates and diatoms is meaningful. Certainly, other microbial grazers, such as ciliates among others, can impact on diatoms [START_REF] Aberle | Spring bloom succession, grazing impact and herbivore selectivity of ciliate communities in response to winter warming[END_REF], but we did not obtain proof of this behaviour in our study. On the other hand, the diatom-dinoflagellate relationship has been widely suggested in the literature (e.g., [START_REF] Sherr | Heterotrophic dinoflagellates: a significant component of microzooplankton biomass and major grazers of diatoms in the sea[END_REF][START_REF] Saito | Role of heterotrophic dinoflagellate Gyrodinium sp. in the fate of an iron induced diatom bloom[END_REF][START_REF] Calbet | The trophic roles of microzooplankton in marine systems[END_REF], although seldom quantified in natural communities [START_REF] Archer | Microbial dynamics in coastal waters of East Antarctica: herbivory by heterotrophic dinoflagellates[END_REF].

Finding explanations for the low microzooplankton grazing impact, but the high net mortality rates of phytoplankton:

It is difficult to ascertain whether the low grazing found in our study is a general characteristic describing the system or if it was the result of some particular conditions. It is surprising, however, that we found many negative net phytoplankton growth rates (based on Chl a changes) not associated with microzooplankton grazing. The simplest explanation for this would be that the incubation light-level was not appropriate, the cells adjusting their Chl a contents to the new conditions. This could actually be the cause at some stations; however, the same pattern persists in cell counts in many others. Therefore, we have to find alternative hypotheses, which may be nonexclusive:

-The natural mortality hypothesis Parallel to our study, Lasternas and Agustí (Lasternas and Agustí, in press) used a membrane permeability probe [START_REF] Agustí | Cell viability in natural phytoplankton communities quantified by a membrane permeability probe[END_REF] to estimate the natural mortality of the P. pouchetii community, and they observed that higher percentages of dead P. pouchetii cells (up to ca. 90% at some stations) were associated to both cold and less saline waters across the area studied. The mechanisms behind this mortality, not related to grazing rates, can be several. It could well be that we were facing a 13 senescent community at the end of the bloom. On the other hand, we cannot disregard viruses as playing a role in controlling the population of these and other protists and responsible for the frequent negative growth rates [START_REF] Baudoux | Virally induced mortality of Phaeocystis globosa during a spring bloom in temperate coastal waters[END_REF][START_REF] Jacobsen | Susceptibility of colonies and colonial cells of Phaeocystis pouchetii (Haptophyta) to viral infection[END_REF]. Unfortunately, our experimental set up was not adequate for virus-related mortality quantification.

Given the contribution of P. pouchetii to the total phytoplankton biomass, the natural mortality of these algae could be driving the growth rates observed on the basis of Chl a. Corroborating this, we observed an inverse relationship between phytoplankton instantaneous growth rates and Chl a distribution. Although other groups of plankton could also have shown natural mortality rates, we do not have solid evidence of this.

-The Phaeocystis pouchetii hypothesis

As previously mentioned, our cruise coincided with high abundances of Phaeocystis pouchetii, which commonly blooms in these waters in July [START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF].

The peculiarities of the food web dominated by these algae make it difficult to extract general conclusions. We believe that our low grazing estimates (not significant at most of the stations) could be partially a consequence of the presence of P. pouchetii .

We observed the presence of many colonies in the samples, although we have not been able to quantify the percentage of free cells and colonies in the Lugol preserved samples. However, it is quite likely this species either introduced variability into the samples (colonies can be heterogeneously distributed in the samples) and precluded the establishment of significant regressions based on Chl a, evaded grazing when in colonial form [START_REF] Hansen | On the trophic fate of Phaeocystis pouchetti (Harriot). 5. Trophic relationships between Phaeocystis and zooplankton: An assessment of methods and size dependence[END_REF][START_REF] Weisse | The trophic significance of Phaeocystis blooms[END_REF][START_REF] Tang | Grazing and colony size development in Phaeocystis globosa (Prymnesiophyceae): the role of a chemical signal[END_REF], or chemically deterred grazers [START_REF] Barnard | Dimethylsulde and Phaeocystis poucheti in the Southeastern Bering Sea[END_REF][START_REF] Nejstgaard | Zooplankton grazing on Phaeocystis: a quantitative review and future challenges[END_REF][START_REF] Van Rijssel | Haemolytic activity of living Phaeocystis pouchetii during mesocosm blooms[END_REF]. Certainly, P. pouchetii seems the most obvious candidate when seeking chemical deterrence of grazing. Similar to macroalgae and other phytoplankters, Phaeocystis spp. exude chemicals that can interfere with grazing activity (see review by [START_REF] Van Rijssel | Haemolytic activity of living Phaeocystis pouchetii during mesocosm blooms[END_REF]. Although the chemicals involved in this process have not yet been identified, it has been suggested that grazing-activated DMSP cleavage by P. pouchetii contributes to grazing deterrence [START_REF] Wolfe | DMSP and DMS dynamics and microzooplankton grazing in the Labrador Sea: application of the dilution technique[END_REF]. Allelopathic interactions could also be responsible for the negative growth rates of a phytoplankton (and other protists) community, which apparently was not limited by nutrients. Overall, we believe that the net transfer of energy to higher trophic levels in this ecosystem would be greatly diminished if the autotrophic community was dominated by Phaeocystis, provided that other groups not considered here (e.g., copepods) do not exert a strong impact on this alga. However, the low tolerance of P. pouchetii to relatively high temperatures [START_REF] Schoemann | Phaeocystis blooms in the global ocean and their controlling mechanisms: a review[END_REF], as shown by the reduced presence at St. 27 where temperatures reached 7.5ºC, indicates a limited relevance of this species under global warming scenarios. Certainly, this does not mean that other Phaeocystis species, such as P. globosa, which is adapted to warmer waters, cannot replace P. pouchetii, further diminishing the trophic efficiency of the system because P. globosa is seldom consumed by zooplankton (see review by [START_REF] Nejstgaard | Zooplankton grazing on Phaeocystis: a quantitative review and future challenges[END_REF].

-The dilution grazing artifact hypothesis

Chl a is a rough proxy for phytoplankton because it does not capture the complexity of this group, and its use in dilution experiments has been questioned because chlorophyll content per cell may change during the incubation [START_REF] Mcmanus | Phytoplankton abundance and pigment changes during simulated in situ dilution experiments in estuarine waters: possible artifacts caused by algal light adaptation[END_REF].

Moreover, the need for a close examination of the microzooplankton community during dilution experiments to detect and correct possible artifacts has also been discussed [START_REF] Dolan | Dilution effects on microzooplankton in dilution grazing experiments[END_REF][START_REF] Agis | A cautionary note: Examples of possible microbial community dynamics in dilution grazing experiments[END_REF][START_REF] Modigh | Changes in phytoplankton and microzooplankton populations during grazing experiments at a Mediterranean coastal site[END_REF]. To shed light on this point and to deepen our understanding of the food web interactions in Arctic waters, we further examined changes in the entire microbial community during the dilution experiments. We did not make any attempt to compare the rates obtained based on Chl a, with those derived from cell counts and their corresponding conversion to carbon because the uncertainties associated when depicting a trophic role (autotrophy vs heterotrophy) to unidentified dinoflagellates and nanoflagellates.

Besides, the contribution of mixotrophic species at some stations (e.g. the mixotrophic ciliate Laboea sp. represented most of the planktonic biomass at station St. 3; Supplementary Material online) precluded any comparison.

When opening the planktonic black box in the dilution experimental bottles we faced unanticipated results suggesting a complex and intricate food web, in which choosing the major microbial grazers of phytoplankton was not so straightforward. A clear example of a puzzling response was the occurrence of positive regression slopes between the net growth rates of certain groups against the dilution factor. Positive slopes for heterotrophs and mixotrophs, even if sometimes the result of complicated 15 ecological interactions, can be easily interpreted as growth enhancement due to increased feeding in the more concentrated treatments. However, positive slopes of phytoplankton occur when the organism considered is adversely affected by the dilution treatment. The explanation for these particular responses is not easy because they can have different non-exclusive causes. For instance, they may either be the result of strong trophic cascade effects during the incubations [START_REF] Calbet | Impacts of micro-and nanograzers on phytoplankton assessed by standard and size-fractionated dilutions[END_REF], chemical grazing deterrence by the algae or other organisms, toxic effects of the filtered seawater [START_REF] Landry | Estimating rates of growth and grazing mortality of phytoplankton by the dilution method[END_REF], mixotrophs being important contributors of phytoplankton biomass (then, favoured in less diluted conditions), or of complex cycling of nutrients between internal and external pools [START_REF] Landry | Estimating rates of growth and grazing mortality of phytoplankton by the dilution method[END_REF], because nutrients would be taken up by smaller algae more efficiently and would become limiting for larger phytoplankton. We do not believe that the last three hypotheses apply to our experiments for the following reasons: i) any toxic effect would likely be persistently evident at all the groups and stations, ii) some of the groups of phytoplankton showing positive slopes, as far as we know, were not mixotrophic (e.g., diatoms), and iii) nutrients were supplied in excess. Therefore, either (or both) trophic cascades or grazing deterrence seem to be the most reasonable explanation. If P. pouchetii was responsible for the positive slopes found for diatoms and other groups, its effects would not be apparent at the stations where the haptophyte was not present (stations 2 and 3). While this was the case and thus supporting the feeding deterrence hypothesis, it did not fully demonstrate the hypothesis because the response was not directly related to the P. pouchetii concentration.

We, therefore, contemplated the trophic cascade explanation for the positive slopes found in our (and others) study. It has been argued that changes of grazer abundance during dilution grazing incubation may result in results that are artifacts [START_REF] Dolan | Dilution effects on microzooplankton in dilution grazing experiments[END_REF][START_REF] Agis | A cautionary note: Examples of possible microbial community dynamics in dilution grazing experiments[END_REF][START_REF] Modigh | Changes in phytoplankton and microzooplankton populations during grazing experiments at a Mediterranean coastal site[END_REF]. These changes usually involve a decrease in abundance in the most diluted treatments, the result of starvation. However, our experiments showed on many occasions the opposite, the grazers diminishing in the most concentrated treatments. This can be a consequence of predation from other microzooplankers, either protozoans (intraguild predation) or metazoans (e.g., copepod nauplii; not included in our sampling), during the incubations. It is relatively easy to mathematically simulate a dilution grazing experiment involving a grazer that reduces their abundance inversely related to the dilution level during the incubation. We can actually base our example on data from one of our experiments. For instance, we can use as an example the response of < 20 µm ciliates at Station 4, and the positive slope for nanoflagellates, one of their likely prey (Fig. 6). We assume nanoflagellates doubled their abundance in 24h, and that < 20 µm ciliates were the only group grazing on them. If ciliate feeding rates were linearly related to food concentration we will most likely obtain, after the incubation period, a negative slope for nanoflagellates when plotting net growth rates as function of dilution level, as predicted by dilutions (although it would not be a true estimate of the natural grazing rate on this group because grazers varied their concentrations in the experimental bottles). However, if feeding was saturated, we could easily mimic the results found in the experiments using a constant feeding rate of only 76 nanoflagellates consumed per ciliate per day (Fig. 7). This happens because the grazing pressure is in this case only proportional to the abundance of grazers, and we have a higher net growth rate of grazers (< 10 µm ciliates) in the more concentrated treatments. Moreover, varying the concentration of grazers, prey and the growth and grazing rates we can also obtain non significant from zero grazing estimates, which are not true rates, but artifacts of the method in very special situations. This mathematical exercise was not intended to correct our grazing rates, as suggested by [START_REF] Modigh | Changes in phytoplankton and microzooplankton populations during grazing experiments at a Mediterranean coastal site[END_REF], because given the complexity of the food web we cannot anticipate the microzoplankton group responsible for most of the phytoplankton grazing impact. However, we can use our reasoning to prove that positive slopes (and non-significant slopes) are easily the result of a combination of trophic cascades during the incubations (the main grazers decreasing their abundance in the most concentrated treatments) with a saturated feeding responses. The picture complicates further if microzooplankton feeding behaviour changes with food concentration, as described by Teixeira and Figueras (Teixeira and Figueras, 2009), and if there is nutrient limitation during the incubations. Actually, and regarding this latter artifact, severe nutrient limitation during the dilution experiments inversely proportional to the dilution level will most likely favour fake negative slopes, exaggerating the grazing activity of microzooplankton because phytoplankton instantaneous growth rates will be higher in the most diluted treatments, where more nutrients per cell are available. In summary, the data presented here depict a planktonic Arctic community dominated by P. pouchetii and rich in microzooplankton, which at first sight did not seem to be exerting a strong control on a phytoplankton community in decline. However, several natural and causes as well as artifacts may have been playing important roles in some of our experiments, precluding clear grazing estimates in this very complex food web. Even though, it may seem disappointing to conclude that our rates might not be actual estimates, but bound for the lower grazing impact of the microzooplankton in the area, they point out the need for presenting negative results, when these are not consequence of evident mistakes or artifacts [START_REF] Dolan | The reliability of grazing rate estimates from dilution experiments: Have we over-estimated rates of organic carbon consumption by microzooplankton?[END_REF]. Only with a whole picture of the existing data we will be able to extract solid conclusions on the dynamics of marine systems. Maybe in the future someone will find the way of extracting information from such results. Specifically regarding dilution grazing experiments, this study calls for special caution when applying the technique, originally developed for oligotrophic areas, to rich environments were saturated feeding responses may be common. In any case, as previously suggested [START_REF] Dolan | Dilution effects on microzooplankton in dilution grazing experiments[END_REF][START_REF] Agis | A cautionary note: Examples of possible microbial community dynamics in dilution grazing experiments[END_REF][START_REF] Modigh | Changes in phytoplankton and microzooplankton populations during grazing experiments at a Mediterranean coastal site[END_REF], it is evident we need a detailed examination of the grazer and prey dynamics during the incubations if we want to present trustable microzooplankton grazing estimates. By presenting data on counts-based rates we will enhance our resolution and avoid artifacts associated with chlorophyll analysis. However, these sorts of data involve a considerable amount of time and are highly dependent on the taxonomic skills of the researcher. Table 3. Dilution grazing experiments. Phytoplankton instantaneous growth rates without nutrients (µ±SE; d -1 ) and mortality rates (m±SE; d -1 ) for total and the 2 fractions of chlorophyll a at the different stations. %SS correspond to the calculated impact on the phytoplankton standing stock. Determination coefficient for the regression analysis is also provided. SE error for µ was obtained using the equation (SE k 2 +SE m 2 ) 1/2 were SE k is the SE of the average of the 100% un-amended bottles and SE m is the standard error associated to the slope of the regression equation (when significant).

Station

  , temperature, and fluorescence during the early morning using a Seabird CTD911, followed by collection of water samples for the determination of chlorophyll a concentration (hereafter Chl a) with 12 l Niskin bottles fitted to a rosette during the ascending CTD casts.

6

  All bottles were incubated on deck in a large (600 L), dark incubator with

  , Strombidium spp., and the mixotroph Laboea spp.) was noticeable in the Greenland Sea and Svalbard Islands coastal station (Table

  a value that contrasts strongly with the previous estimate.

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Phaeocystis pouchetii biomass as related to latitude.

Figure 3 .

 3 Figure 3. The quotient heterotrophic carbon / autotrophic carbon as a function of latitude. Greenland Sea and Coastal stations are indicated, the rest of stations correspond to Arctic Ocean open waters.

Figure 4 .

 4 Figure 4. Relationship between instantaneous phytoplankton growth rates in the unamended (without added nutrients) bottles(d -1) and chlorophyll a concentration (µg Chl a l -1 ).

Figure 5 .

 5 Figure 5. Comparison of instantaneous phytoplankton growth rates (from total Chl a analysis) in bottles amended (with added nutrients) and un-amended. The discontinuous line represents the 1:1.

Figure 6 .

 6 Figure 6. Example of grazing dilution plots for the microbial components of the planktonic community of stations 4 (left) and 33 (right).

Figure 7 .

 7 Figure 7. Simulated outcome of a dilution grazing experiment using the abundance and growth rates of < 20 µm ciliates in St. 4 as grazers and the abundance of nanoflagellates as prey. See text for further details.

  Fig.2

  Fig.3

  Fig.4

  Fig.5

  Fig.6

  Fig.7

F

TABLE 1 .

 1 Summary of the dates of sampling (July 2007), geographic position and area, depth of sampling (m) and in situ chlorophyll a concentration (µg l -1 ± SE) and temperature for the different stations sampled.

	Station	Date	Latitude	Longitude	Area	Sampling depth	Total Chl a > 5 µm Chl a	Temp. (ºC)
	2	2	70º 43.19 N	17º 07.70 W	Greenland Sea	20	0.48 ± 0.02	0.15 ± 0.00	-1.2
	3	3	72º 57.21 N	12º 39.19 W	Greenland Sea	25	1.31 ± 0.21	0.79 ± 0.00	0
	4	4	74º 53.89 N	7º 24.50 W	Greenland Sea	32	1.41 ± 0.02	0.49 ± 0.01	0.8
	5	5	77º 23.29 N	1º 40.57 W	Arctic Ocean	15	6.67 ± 0.42	2.90 ± 0.13	4
	6	6	78º 00.44 N	2º 29.94 E	Arctic Ocean	23	1.75 ± 0.03	1.09 ± 0.03	4
	9	7	78º 43.72 N	2º 58.51 E	Arctic Ocean	15	2.97 ± 0.07	1.48 ± 0.03	5
	12	8	79º 30.83 N	7º 29.74 E	Arctic Ocean	20	5.21 ± 0.33	2.57 ± 0.07	5
	15	9	80º 08.39 N	11º 19.54 E	Svalbard Coast	20	3.30 ± 0.13	1.81 ± 0.36	5
	18	10	80º 26.90 N	15º 35.38 E	Svalbard Coast	35	2.67 ± 0.10	0.81 ± 0.08	5
	20	12	80º 13.98 N	10º 10.97 E	Arctic Ocean	24	4.81 ± 0.20	2.31 ± 0.07	5
	23	13	79º 22.16 N	6º 49.39 E	Arctic Ocean	17	8.97 ± 0.63	5.01 ± 0.08	5
	27	15	79º 52.71 N	8º 36.44 E	Arctic Ocean	30	1.81 ± 0.09	1.38 ± 0.07	7.5
	33	17	80º 23.46 N	12º 25.98 E	Arctic Ocean	25	1.97 ± 0.01	0.66 ± 0.00	5
	39	19	80º 49.96 N	13º 12.82 E	Arctic Ocean	39	0.77 ± 0.02	0.28 ± 0.03	5
	43	22	80º 25.29 N	7º 57.57 E	Arctic Ocean	20	8.97 ± 0.21	2.28 ± 0.12	-1
	46	23	79º 59.15 N	3º 39.63 E	Arctic Ocean	16	4.94 ± 0.07	0.73 ± 0.06	-1.5

Table 2 .

 2 In situ biomass in µgC L -1 of the different protist groups considered at the stations sampled. Numbers between parentheses are SE of 2 replicates. The quotient "total carbon / > 5 µm Chl a" are also shown.

	Station Diatoms Nanofl P. pouchetii < 20 µm dinofl. > 20 µm dinofl. Mixo. dinofl. Het. dinofl. < 20 µm ciliat > 20 µm ciliat Total biomass C / > 5 µm Chl a
	2	1.25	0.36	0.00	0.51	0.24	0.24	0.28	4.48	34.91	42.26	14.03
		(0.07)	(0.06)	(0.00)	(0.08)	(0.03)	(0.02)	(0.07)	(0.46)	(4.88)
	3	1.55	3.12	0.00	0.84	0.22	0.12	0.08	4.74	50.53	61.18	4.75
		(0.21)	(0.05)	(0.00)	(0.02)	(0.03)	(0.01)	(0.00)	(0.33)	(4.00)
	4	1.26	0.60	0.83	0.77	0.57	0.72	0.43	12.2	20.90	38.31	7.73
		(0.15)	(0.01)	(0.12)	(0.12)	(0.01)	(0.08)	(0.08)	(0.00)	(2.31)
	5	1.77	0.51	2.81	14.1	0.26	1.76	0.47	0.66	11.49	33.83	4.75
		(0.47)	(0.12)	(0.32)	(3.40)	(0.06)	(0.28)	(0.13)	(0.19)	(3.03)
	6	1.53	3.78	12.2	10.3	0.87	1.20	0.69	3.70	6.08	40.42	20.64
		(0.05)	(0.81)	(2.30)	(4.38)	(0.23)	(0.27)	(0.13)	(0.11)	(1.66)
	9	1.79	1.02	1.59	1.93	0.34	0.72	0.15	0.40	0.83	8.77	3.88
		(0.11)	(0.09)	(0.10)	(0.32)	(0.03)	(0.06)	(0.02)	(0.11)	(0.06)
	12	0.81	1.81	5.90	1.49	0.75	0.11	0.17	1.91	0.94	13.88	3.44
		(0.03)	(0.05)	(0.82)	(0.04)	(0.11)	(0.04)	(0.01)	(0.15)	(0.27)
	15	2.97	3.82	13.0	5.56	3.09	0.99	1.53	2.45	15.09	48.50	12.80
		(0.48)	(0.12)	(1.27)	(0.14)	(0.74)	(0.05)	(0.24)	(0.33)	(6.67)
	18	0.18	4.01	1.19	6.14	5.24	0.27	3.33	2.12	59.9	82.38	11.46
		(0.02)	(1.87)	(0.71)	(0.72)	(0.28)	(0.05)	(0.67)	(0.15)	(7.61)
	20	4.24	3.43	14.8	3.99	3.89	0.98	0.57	6.59	4.52	43.02	10.39
		(0.36)	(0.22)	(2.40)	(0.07)	(0.73)	(0.08)	(0.17)	(0.42)	(0.50)
	23	67.8	3.10	24.8	5.00	1.79	3.65	0.65	5.73	7.12	119.58	20.18
		(5.19)	(0.39)	(2.63)	(0.28)	(0.53)	(1.20)	(0.11)	(0.21)	(0.81)
	27	25.1	1.36	0.09	5.49	1.91	2.55	0.51	0.81	1.74	39.59	23.22
		(1.21)	(0.10)	(0.01)	(0.34)	(0.10)	(0.23)	(0.04)	(0.24)	(0.38)
	33	6.47	2.33	7.89	3.36	2.70	1.05	0.70	4.56	5.60	34.67	29.68
		(0.38)	(0.28)	(4.22)	(0.47)	(0.53)	(0.17)	(0.05)	(0.13)	(0.63)
	39	6.73	1.61	1.79	2.10	2.50	0.25	0.34	1.45	1.02	17.78	42.16

Table 4 .

 4 Dilution grazing experiments. Instantaneous growth rates without nutrients (µ; d -1 ) and mortality rates (m; d -1 ) for the different planktonic groups considered.Determination coefficient for the regression analysis is also provided. Positive m values indicate positive slope.

		µ (TOTAL)	m (TOTAL) r 2 (TOTAL)	µ (< 5 µm)	m (< 5 µm)	r 2 (< 5 µm)	µ (> 5 µm)	m (> 5 µm)	%SS
	2	0.12±0.029	ns	-	0.10±0.035	ns		-	0.17±0.14	ns	0.0
	3	0.07±0.11	ns	-	-0.05±0.019	ns		-	0.13±0.18	ns	0.0
	4	-0.23±0.11	-0.31±0.11	0.50	-0.39±0.25	-0.44±0.19		0.45	-0.22±0.13	ns	23.9
	5	-0.11±0.077	-0.13±0.039	0.59	0.00±0.21	-0.34±0.056		0.84	-0.15±0.069	ns	11.6
	6	-0.05±0.010	ns	-	0.29±0.11	-0.30±0.086**	0.67	-0.03±0.050	ns	0.0
	9	-0.11±0.046	ns	-	-0.33±0.026	ns		-	0.07±0.090	ns	0.0
	12	0.15±0.113	-0.31±0.11*	0.73	-0.25±0.026	ns		-	-0.08±0.022	ns	28.6
	15	0.09±0.030	ns	-	-0.16±0.24	ns		-	0.23±0.079	ns	0.0
	18	-0.06±0.059	ns	-	-0.11±0.00	ns		-	0.19±0.036	ns	0.0
	20	-0.24±0.015	ns	-	-0.29±0.031	ns		-	-0.19±0.060	ns	0.0
	23	-0.24±0.00	ns	-	-0.48±0.15	ns		-	-0.09±0.07	ns	0.0
	27	-0.04±0.043	ns	-	-0.04±0.16	ns		-	-0.04±0.007	ns	0.0
	33	0.14±0.081	-0.24±0.073	0.57	0.00±0.11	-0.30±0.11		0.48	-0.06±0.041	ns	22.9
	39	-0.04±0.022	ns	-	-0.05±0.002	ns		-	-0.03±0.061	ns	0.0
	43	0.01±0.152	-0.35±0.15	0.41	-0.05±0.19	-0.46±0.19**		0.45	0.17±0.069	ns	29.7
	46	0.01±0.030	-0.16±0.027	0.83	-0.02±0.43	-0.25±0.058		0.70	0.34±0.70	ns	14.9
	ns: not significant (p > 0.05); * Grazing saturation: 3-point method used (Gallegos 1989); ** One outlier removed

  Web Annex 1. Species composition and abundance of the plankton at the different stations sampled. SE c to the Standard Error of 2 replicate samples. Sizes correspond to equivalent spherical diameter.

	stations sampled. SE corresponde to the Standard Error of 2 replicate samples. Sizes correspond to equivalent spheric
	Station	4 9 18 27 43		5 12 20 33 46	2 12 5 20 33 46	2 15 6 23 39	3 15 6 23 39	3 18 9 27 43	4
	SE SE SE SE SE	Cells L -1 Cells L -1 Cells L -1 Cells L -1 Cells L -1	Cells L -1 SE SE SE SE SE	SE Cells L -1 Cells L -1 Cells L -1 Cells L -1 Average cell size (ESD; µm) Cells L -1 SE SE SE SE	SE Cells L -1 Cells L -1 Cells L -1 Cells L -1	Cells L -1
	Diatoms
	Chaetoceros spp. 180 30 5 7434.5 260		1630 3115 9960 2610 350	1224 530 805 180 190 250	526 2875 1845 149858 2345	80959 465 325 4990 675 21.2	11497 50 5 27281.5 1350	1770
	Corethron hystrix 0 0 0 150 0		0 55 440 220 0	0 40 0 5 40 0	0 110 0 1600 75	0 60 0 280 15 44.2	0 810 0 0 0	0
	Eucampia zodiacus 0 450 0 5 0		35 10 25 5 0	0 15 10 25 5 0	0 30 0 60 15	20 30 0 40 5 16	20 3600 0 25 0	0
	Fragilariopsis spp. 130 760 15 50 430	10545 370 1280 450 240	280 1110 50 540 110 240	20 1330 1080 5148.5 300	60 10 260 901.5 20 8.6	60 1820 135 1030 890	130
	Nitzschia spp. 225 2475 90 72075 2250	41850 9450 57825 25650 5175	0 15750 450 4275 2250 675	0 15300 27000 380556 9675	20 900 5850 46128 225 4.2	20 28575 170 533355 9000	2025
	Rhizosolenia spp. 0 0 0 10 0		0 0 0 25 0	10 15 0 0 0 0	10 10 0 0 0	0 10 0 0 0 36	0 10 0 0 0	0
	Skeletonema spp. 0 0 0 0 0		0 0 1575 0 0	0 225 0 0 0 0	0 1575 0 2475 0	0 675 0 225 0 12.4	0	0 0 0 0	0
	Thalassiosira spp. 90 195 315.5 1195 1035	10405 1770 10530 11000 11475	955 1325 410 1310 1750 225	445 6930 15935 79785 4760	290 550 675 2585 340 18	20 10195 849.5 12135 15885	750
	Other centric diatoms 0 5 5 225 65	30 10 10 1660 725	220 10 10 10 250 205	20 125 242 21190 2410	80 232 5 2240 470 24.24	10 8365 5 5 255	10
	Pennated diatoms 5 1350 68.5 0 0	12375 900 4725 4275 450	0 3825 450 1125 225 0	0 2025 3150 6300 2475	0 1125 900 2250 225 9.6	0 9450 479.5 450 0	5
	o r 35 0 0 F o r 1138785 Phaeocystis pouchetii Flagellates Choanoflagellidea Dictyocha sp. 30758 129735 8649 0 0 20 75 25 F o r 23064 1326180 184512 0 0 0 25 10 0 F o r 158565 2485146 403620 0 0 0 5 15 5 F o r 2883 1588533 850485 0 0 0 0 60 20 F 1210860 4050615 1484745 o 900 1350 1350 r 65 630 110	0 0 5 4947228 0 35 3992955 49011 5 5420040 0 10 360375 0 20	0 371907 10 934092 0 5 389205 14415 5 576600 0 10 8649 0 10 3.8 4 24.4	0 8649 10 357492 0 35 268119 0 35 20181 0 0 8014740 900 285	212940 100905 50
	Other nanoflagellates 2366 262353 14415 194553 282534 804186 11532 194553 34821 284559 Dinoflagellates Amphidinium spp. Ceratium spp. Dinophysis spp. Gyrodinium spp. 10 15 10 0 5 10 130 155 10 60 0 0 5 0 10 145 110 230 0 0 0 0 167 195 20 140 0 0 0 10 50 215 20 300 5 0 5 15 20 370	P e e r 29250 4500 P 60543 227757 P 4641 224874 P 50586 323880 P 24189 154599 5991 P 65 35 10 0 0 0 85 15 e e r 5 240 0 5 10 5 55 705 e e r 20 120 0 0 0 15 15 790 e e r 10 140 0 0 0 0 65 445 e e r 20 65 0 0 10 0 25 180 20 e 0 e 5 r 30	42588 49011 23064 41262 13965 4.04 35 0 0 45 80 5 5 95 20 0 5 170 20 0 0 35 15 0 0 30 13.8 52.2 32.8 32.4	2366 170097 605430 149916 207801 5 0 0 15 10 0 5 50 390 0 0 935 80 0 0 210 90 5 35 150	227136 40 10 5 520
	Katodinium glaucum Micracanthodinium claytonii Prorocentrum balticum 5 20 10 30 35 7425 0 120 5 20 225 110 274 220 0 10 68.5 2475 10 410 20 25 900 2250 60 370 25 260 225 3825	70 20 70 10 20 1125 0 10 50 80 10 225 30 15 450 10 20 675	R 30 20 10 R 370 20 4050 R 120 80 3825 R 460 10 14175 R 110 20 45 R	15 95 0 130 0 900 20 20 225 100 10 4725 10 0 5 15 20 19.4	5 15 0 15 0 2925 685 10 479.5 190 30 9900 320 75 3825	75 100 165
	Protoperidinium bipes Protoperidinium spp. Torodinium robustum Dinoflagellates < 20 µm Dinoflagellates > 20 µm 5 888 5 25 25 35 388.5 45675 20 550 30 40 5 5 5 15 1800 8325 45 435 68.5 100 0 60 5 120 11532 24750 117 1065 20 70 5 65 0 160 1575 16200 20 1745 20 50 5 35 15 40 1575 12375 680 1510	85 25 50 1644 479.5 222 5 15 11025 110 10 5 5 225 75 40 10 40 450 165 50 5 20 2250 345 20 5 10 2925 200	e v i e 15 20 5 10 10 10 274 2719.5 68.5 490 e v i e 20 10 40 10 55 5 33525 14175 1835 455 e v i e 105 25 70 40 10 10 18000 450 1695 405 e v i e 70 30 35 5 120 20 20025 1125 990 290 e v i e 10 0 25 15 15 5 16875 675 1040 150 18.8 e 36.6 27.6 v i e	10 10 10 55.5 60 230 5 5 10800 335 890.5 10 5 98022 2429 140 45 60 25425 950 120 55 45 15075 3340	25 5 265 2497.5 1270
	Ciliates Didinium sp. 5 5 0 0 10		20 5 0 0 45	80	0 5 0 0 5	0 15 10 10 0	w 15 w 5 w 0 w 0 w 0 w 28.8	5 10 5 20 30	5
	Laboea sp. 50 0 130 5 20		0 0 0 40 20	545	0 0 0 0 0	65 45 0 15 0	2220 71 0 5 5 0	180 1400 0 5 110	310
	Mesodinium spp. 0 0 0 0 20		0 0 0 0 20	55 20 0 0 0 0	35	0 0 0 0	5 21 0 0 0 0	5 120 0 0 0	0
	Strombidium spp. 304.5 0 25 25 95		65 65 180 310 440	505 15 15 0 80 100	65 95 145 120 155	680 35 5 30 35 33	120 35 0 65 225	2004.5
	Tintinnids	10 0 5 0 0		0 0 0 15 5	0 15 0 0 0 5	0	0 5 0 0	5 57.2 0 5 0 0	5	0 5 0 0	20
	< 20 µm ciliates 301 55 132 120 55	686.5 1040 4650 1905 2735	3699 203.5 70 310 145 195	548 6555 2555 2636.5 725	1494.5 215 325 72.5 115	24.5 205 1465 400 1055	4131
	> 20 µm ciliates 100.5 10 140 50 40		530 140 720 520 400	420 140 40 80 80 20	70 330 810 1000 150	240 90 370 120 30	20 130 760 250 280	1120.5

e

http://mc.manuscriptcentral.com/jplankt Journal of Plankton Research

Acknowledgements

This research was supported by the Spanish grants POL2006-0550/CTM to C. Duarte, CTM2009-08783 to A.C., CTM2006-12344-C02-01 to M. A, CTM2007-60052 to E.S., and grant 2005SGR-00219 from the Generalitat de Catalunya to the research group, and a PhD fellowship, BES-2005-7491 to RA. The authors wished to thank the crew of the BIO Hesperides and the special help of the technical staff of the UTM (D. Afonso, R. Ametller, J. Llinàs J. Pozo, J. Sanz, and X. Vidal) for their invaluable logistic help. Plankton composition was analyzed by Dr. M. Delgado. Five anonymous reviewers highly contributed to improve earlier versions of the ms.


Inhee Choi

Young In Yang

Hyeon Don Song

Seon Lee

Taewook Kang

Jung-Joon Sung

Jongheop Yi

Lipid molecules induce the cytotoxic aggregation of Cu/Zn superoxide dismutase with structurally disordered regions

Keywords: Amyotrophic lateral sclerosis, aggregation, Cu/Zn-superoxide dismutase, cytotoxicity, lipid molecules, Lou Gehrig's disease

Cu/Zn-superoxide dismutase (SOD1) is present in the cytosol, nucleus, peroxisomes and mitochondrial intermembrane space of human cells. More than 114 variants of human SOD1 have been linked to familial amyotrophic lateral sclerosis (ALS), which is also known as Lou Gehrig's disease. Although the ultimate mechanisms underlying SOD1-mediated cytotoxicity are largely unknown, SOD1 aggregates have been strongly implicated as a common feature in ALS. This study examined the mechanism for the formation of SOD1 aggregates in vitro as well as the nature of its cytotoxicity. The aggregation propensity of SOD1 species was investigated using techniques ranging from circular dichroism spectroscopy to fluorescence dye binding methods, as well as electron microscopic imaging. The aggregation of SOD1 appears to be related to its structural instability. The demetallated (apo)-SOD1 and aggregated SOD1 species, with structurally disordered regions, readily undergo aggregation in the presence of lipid molecules, whereas metallated (holo)-SOD1 does not.

The majority of aggregated SOD1s that are induced by lipid molecules have an amorphous morphology and exhibit significant cytotoxicity. The lipid binding propensity of SOD1 was found to be closely related to the changes in surface hydrophobicity of the proteins, even at very low levels, which induced further binding and assembly with lipid molecules. These findings suggest that lipid molecules induce SOD1 aggregation under physiological conditions and exert cytotoxicity, and might provide a possible mechanism for the pathogenesis of ALS.

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Introduction

Cu/Zn superoxide dismutase (SOD1) [START_REF] Valentine | Copper-Zinc superoxide dismutase and amyotrophic lateral sclerosis[END_REF], a 32 kDa homodimeric protein comprised of two identical subunits arranged in eight β-strands connected by seven loops, is found in the cytosol, nucleus, peroxisomes, lysosomes, and mitochondrial intermembrane space of eukaryotic cells. It functions primarily as an antioxidant enzyme to reduce the steady-state concentration of superoxide anions. However, structural disorders in SOD1, such as mutations and aggregation, have been linked

to the development of familial amyotrophic lateral sclerosis (ALS). ALS is a progressive, fatal neurodegenerative disease that mainly affects the motor neurons in the brain stem and spinal cord.

Recent studies have shown that SOD1 aggregates are present in the tissues from ALS patients [START_REF] Bruijn | Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1[END_REF][START_REF] Koide | Formation of granular cytoplasmic aggregates in COS7 cells expressing mutant Cu/Zn superoxide dismutase associated with familial amyotrophic lateral sclerosis[END_REF],

ALS-SOD1 transgenic mice [START_REF] Higgins | ALS-associated mutant SOD1 G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes[END_REF][START_REF] Liu | Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria[END_REF], as well as in cell culture models, which indicates that SOD1associated familial ALS is a protein conformational disorder that is similar to other neurodegenerative diseases in which protein aggregates play a role [START_REF] Dobson | Protein folding and misfolding[END_REF]. Although the ultimate mechanisms underlying SOD1-mediated cytotoxicity are unknown, SOD1 aggregates have been strongly implicated as a causative factor in the disease. Many experimental studies have attempted to define the critical factors leading to SOD1 aggregation because the aggregation of SOD1 is a complex multistep process consisting of dimer dissociation, loss of metals, conformational changes, nucleation and accretion, and involves a variety of cellular components [START_REF] Zhang | Intracellular conformational alterations of mutant SOD1 and the implications for fALSassociated SOD1 mutant induced motor neuron cell death[END_REF][START_REF] Khare | The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis[END_REF]. In this regard, it is generally accepted that altering the physiological conditions, such as increasing the temperature, lowering the pH, adding a metal chelator, reduction of disulfide linkages, and oxidative modification, can affect the formation of SOD1 aggregates [START_REF] Khare | The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis[END_REF][START_REF] Banci | Metal-free superoxide dismutase forms soluble oligomers under physiological[END_REF][10][START_REF] Gaggelli | Copper homeostasis and neurodegenerative disorders (Alzheimer's, Prion, and Parkinson's diseases and amyotrophic lateral sclerosis)[END_REF]. Recently, the formation of SOD1 aggregates as a result of interactions with other cellular components was also suggested [START_REF] Jiang | DNA is a template for accelerating the aggregation of copper, zinc superoxide dismutase[END_REF][START_REF] Kim | Unsaturated fatty acids induce cytotoxic aggregate formation of amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutants[END_REF]. Considering the ubiquitous distribution of SOD1 in a cell (i.e. cytosol, nucleus, peroxisomes, lysosomes, and mitochondrial intermembrane space), it would not be surprising that aberrant SOD1 species can interact with other cellular components, which may eventually be involved in the formation of heterogeneous aggregates without perturbing physiological conditions.

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In this study, we examined the in vitro SOD1 aggregation in the presence of lipid molecules as a possible aggregation model system for ALS. Lipid molecules serve as mediators for signaling and inflammation during the progression of neurodegeneration. Although SOD1 is frequently in contact with lipids, the interactions with SOD1 have not been investigated extensively compared to other disease linked proteins [START_REF] Chi | Ganglioside GM1-mediated amyloid-beta fibrillogenesis and membrane disruption[END_REF][START_REF] Madine | The aggregation and membrane-binding properties of an α-synuclein peptide fragment[END_REF][START_REF] Kazlauskaite | Structural changes of the Prion protein in lipid membranes leading to aggregation and fibrillization[END_REF]. The lipid molecule-associated behavior of proteins can be generally derived from interactions between the lipid molecules and proteins, and the extent of these interactions would be expected to promote the lipid molecule mediated abnormal assembly of SOD1.

These findings suggest that lipid molecules promote the abnormal assembly and cytotoxic aggregation of SOD1. This aggregation was found to be strongly related to the structural instability and affinity of SOD1 for binding to lipid molecules. These results show that lipid molecules, acting as a promoter, lead to a significant acceleration of SOD1 aggregation, as evidenced by fluorescence dye binding and the negatively-stained electron micrographs. The cytotoxicity of the resulting aggregates was also evaluated through cellular viability measurements.

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Materials and methods

Chemicals

Bovine serum albumin (BSA), DPPC (1,2-dipalmitoyl-sn-glycero-3-Phosphocholine), POPC (1palmitoyl-2-oleoyl-phosphatidylcholine), cholesterol, Thioflavin-T (ThT) and 8-Anilino-1naphthalene-sulfonic acid (ANS) were purchased from Sigma Chemical Co. 2,2,2-trifluoroethanaol (TFE) was supplied by Aldrich Chemical Co. All chemicals were used as received without further purification. All water was purified using a Milli-Q water system (18 MΩ, Millipore).

Purification of SOD1

The proteins were prepared using the purification protocol reported elsewhere [START_REF] Kang | Reversible pH-driven conformational switching of tethered superoxide dismutase with gold nanoparticle enhanced surface plasmon resonance spectroscopy[END_REF][START_REF] Hong | Sensitive and colorimetric detection of the structural evolution of superoxide dismutase (SOD1) with gold nanoparticles[END_REF][START_REF] Lee | Highly selective detection of Cu 2+ , utilizing specific binding between Cu-demetallated superoxide dismutase 1 and the Cu 2+ ion via surface plasmon resonance spectroscopy[END_REF][START_REF] Mccord | Superoxide dismutase: An enzymic function for erythrocuprein (hemocuprein)[END_REF]. Human SOD1 genes encoding the wild type enzyme were cloned into the pET23b(+) (Novagen) vector and the proteins were expressed in E. coli. The cultures were induced by the addition of 0. For the pre-formation of SOD1 aggregates, purified WT apo SOD1 molecules were dissolved in acidic phosphate buffered saline (PBS) solution (pH 5.4) at a concentration of 0.1 mg/ml, followed by the previously reported protocol with a solution containing 20 % (v/v) TFE [START_REF] Stathopulos | Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro[END_REF].

Preparation of lipid vesicles

To prepare all lipid vesicles, a 40 mg of lipids was dissolved in 4 mL chloroform in a small vial, agitated and exposed to three freeze-thaw cycles using liquid nitrogen. The organic solvent was

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evaporated with a N 2 stream, leaving a thin lipid layer on the inner wall of the vial. The dried lipid films were resuspended in a 25 mM PBS solution (pH 7.0) at a concentration of 0.1 mg/mL. The suspension was sonicated at low amplitude in an ice bath for 30 min. Sonication was carried out at 5 minute intervals to avoid an excessive increase in suspension temperature. The resulting vesicle solution was stored at 4 °C for 1 hr prior to use.

Incubation of proteins with lipid vesicles

To determine the effect of the interaction of lipid molecules with SOD1 on aggregation, SOD1 was incubated with the DPPC (a neutral, saturated lipid) vesicles in PBS at 37.5 °C. The proteins, apo SOD1, holo SOD1, SOD1 aggregates and BSA, were dissolved in 25 mM PBS at a concentration of 0.1 mg/ml and added to the vesicle solution to yield incubation mixtures with a SOD1 to lipid molar ratio of 1:45. As controls, proteins without DPPC vesicles were also incubated under the same conditions at 37.5 °C. All samples were incubated without agitation. At each time point, an aliquot of each sample was taken and the presence of aggregates was determined using the analytical methods described below. To further understand the relevance of lipid molecule-promoted SOD1 aggregation, the above mentioned procedures were also performed with POPC (a neutral, unsaturated lipid molecules) vesicles and lipid vesicles containing cholesterols.

Circular Dichroism spectroscopy

The secondary structures of the proteins were examined by circular dichroism (CD) spectroscopy (Jasco J-715 spectropolarimeter, USA) equipped with a Naslab temperature controller. A 0.1-cm quartz cell was used for the measurements and the CD spectra were recorded from 190 to 250 nm. All CD measurements were carried out using the following parameters: 1 nm bandwidth, 50 nm/min run speed, 1 nm step size, 8.3 s response time and an average of three runs.

Fluorescence spectroscopy

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To monitor the formation of SOD1 aggregates, the ThT fluorescence of the incubated samples was measured by fluorescence spectroscopy (LS 55, PerkinElmer, UK). ThT is associated rapidly with aggregated structures, giving rise to a new excitation maximum at approximately 450 nm and enhanced emission at around 482 nm. Therefore, previous studies employed the ThT binding characteristics to identify the aggregation propensity of SOD1 [START_REF] Banci | Metal-free superoxide dismutase forms soluble oligomers under physiological[END_REF][START_REF] Stathopulos | Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro[END_REF][START_REF] Banci | SOD1 and amyotrophic lateral sclerosis: Mutations and oligomerization[END_REF]. At each time point, 150 μL of each sample was mixed with 15 mL of 10 μM ThT in PBS, and the fluorescence emission intensity was determined at least 3 times for each sample.

ANS fluorescence [START_REF] Jiang | DNA is a template for accelerating the aggregation of copper, zinc superoxide dismutase[END_REF][START_REF] Tiwari | Aberrantly increased hydrophobicity shared by mutants of Cu,Zn-superoxide dismutase in familial amyotrophic lateral sclerosis[END_REF] was used to determine the surface hydrophobicity of the protein. The ANS fluorophore (final concentration 20 μM) was incubated at 37 °C with the proteins in 25 mM PBS. After excitation at 360 nm, the emission spectra were recorded between 400 and 700 nm.

Transmission Electron Microscopy

Transmission electron microscopy (TEM) was used to characterize the morphology of the resulting SOD1 aggregates. The samples (150 μL aliquots) were incubated overnight on 400-mesh carboncoated formvar copper grids. The grids were air-dried and stained with a 1 % (wt/vol) solution of uranyl acetate. Specimens were viewed using a LIBRA energy-filtering transmission electron microscope at an accelerating voltage of 120 kV. The images were digitized using a Gatan slow-scan CCD camera and analyzed using a Gatan Pigtal micrograph.

Cell culture and Toxicity Assay

The cytotoxicity of the prepared protein samples was measured using a simple cell viability assay.

The PC-12 cell line, which was derived from Rattus norvegicus pheochromocytoma (KCLB No, 21721), was obtained from the Korean Cell Line Bank (http://cellbank.snu.ac.kr). The PC-12 cells were grown in RPMI-1640 media supplemented with 10 % fetal bovine serum and 1 % antibiotics in a humidified atmosphere containing 5 % CO 2 at 37 o C. The cells were cultured on poly-L-Lysine coated plates and slides.

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The cell viability was determined by a MTT assay for the mitochondrial function, which is based on the conversion of a tetrazolium salt into an insoluble formazan product by various dehydrogenases in the mitochondria. The cells were plated at approximately 10 6 cells/ml, grown to 80 % confluence, and exposed to the prepared protein samples for 24 hr. At the end of incubation, the cells were treated with the MTT solution for 30 min at 37 °C in a cell culture incubator. The resulting blue-colored formazan product was dissolved in 1 ml of acidified isopropanol, and the absorbance was measured at 570 nm using a VersaMax tunable Microplate reader (Molecular Devices Corporation, USA).

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Results

Structural instability of metal-free SOD1

The propensity of a native protein to undergo aggregation is a complex property that involves a range of biophysical and physicochemical parameters, including the net charge, surface hydrophobicity, and intrinsic secondary structure [START_REF] Shaw | How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?[END_REF]. Therefore, aggregated SOD1 species would be expected to have different structural and physicochemical properties from proteins in the native state.

Before measuring the interactions between the SOD1 and lipid molecules, the structural and superficial properties of SOD1s were examined under different conditions by CD spectroscopy and ANS fluorescence spectroscopy.

Fig. 1A shows the CD pattern for fully metallated (holo) SOD1 (①), which shows the characteristic peak for an abundant β-sheet structure. Note that the secondary structure of holo SOD1 is comprised mainly of 60 % β-sheets and 30 % random coils with the remainder being α-helixes [START_REF] Nagami | Liposome-assisted activity of superoxide dismutase under oxidative stress[END_REF].

On the other hand, the spectrum of apo SOD1 (②) shows a broad characteristic peak due to the structural changes caused by the absence of Cu 2+ and Zn 2+ , which play a role in the catalytic and structural cores of SOD1, respectively. The structure of SOD1 becomes ordered as a result of Cu 2+ and Zn 2+ coordination [START_REF] Valentine | Copper-Zinc superoxide dismutase and amyotrophic lateral sclerosis[END_REF]. Importantly, these disordered regions as a result of the metal-free state of SOD1

have been shown to be involved in initiating protein aggregation, and in some cases, are believed to become integrated into the aggregate core [START_REF] Khare | The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis[END_REF][START_REF] Shaw | How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?[END_REF][START_REF] Lynch | Kinetic stability of Cu/Zn superoxide dismutase is dependent on its metal ligands: Implications for ALS[END_REF]. This is also supported by observations of apo SOD1 species that had been incubated for 2 months at room temperature. In this case, the characteristic CD patterns (③) were broadened compared to the patterns of the freshly prepared apo SOD1. The characteristic pattern of SOD1 totally disappeared (④) under more severe conditions, such as treatment with TFE (an aggregation-enhancing agent) [START_REF] Hong | Sensitive and colorimetric detection of the structural evolution of superoxide dismutase (SOD1) with gold nanoparticles[END_REF][START_REF] Stathopulos | Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro[END_REF]. Apo SOD1 species readily assemble into largely aggregated structures under destabilizing conditions [START_REF] Khare | The rate and equilibrium constants for a multistep reaction sequence for the aggregation of superoxide dismutase in amyotrophic lateral sclerosis[END_REF][START_REF] Banci | Metal-free superoxide dismutase forms soluble oligomers under physiological[END_REF][START_REF] Kim | Unsaturated fatty acids induce cytotoxic aggregate formation of amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutants[END_REF][START_REF] Stathopulos | Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro[END_REF]. The CD spectra clearly show the structural changes in SOD1 during the aggregation progress. In a previous study [START_REF] Hong | Sensitive and colorimetric detection of the structural evolution of superoxide dismutase (SOD1) with gold nanoparticles[END_REF], the unique secondary structures of native state SOD1 were lost within 2 days under the TFE treatment conditions. These conformational changes in the case of the metal-deficient state and/or This can be attributed to a conformational change due to the removal of metal ions from its structure, and is in good agreement with data that appeared in a previous report [START_REF] Tiwari | Metal deficiency increases aberrant hydrophobicity of mutant[END_REF]. Moreover, the ANS fluorescence enhancements were more prominent in the case of lengthy incubation (2 months) and a TFE treatment of apo SOD1. Obviously, this enhancement is caused by conformational changes in SOD1, resulting in the exposure of more hydrophobic residues at the surfaces during the aggregation process.

Time-resolved Analysis of Lipid Molecule-Induced SOD1 Aggregation

Three types of SOD1 variants, apo SOD1, holo SOD1 and as-prepared SOD1 aggregate, were incubated with DPPC lipid vesicles to examine the lipid molecule-induced aggregation propensity of SOD1. As a control protein, BSA, which contains abundant α-helixes unlike SOD1, was also examined. DPPCs are neutral, saturated lipid molecules that are relatively unreactive compared to other types of lipid molecules (i.e. cationic, anionic and/or unsaturated lipid molecules). For example, the electrostatic interactions between proteins and lipid molecules are also involved in protein aggregation because the net charge of the protein can be altered by conformational changes [START_REF] Shaw | How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?[END_REF]28].

However, the charge of lipid molecules is not a shared property for all types of lipid molecules.

Although many other parameters can affect interactions between proteins and lipid molecules [28,[START_REF] Pinheiro | The role of rafts in the fibrillization and aggregation of prions[END_REF], in this study, we focused on the examining whether lipid molecule-induced protein aggregation can be induced by the general nature of lipid molecules, i.e., amphiphilicity. After incubation with DPPC lipid vesicles, the aggregated SOD1 was sampled and then subjected to ThT fluorescence

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measurements. ThT [START_REF] Banci | Metal-free superoxide dismutase forms soluble oligomers under physiological[END_REF][START_REF] Stathopulos | Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro[END_REF][START_REF] Banci | SOD1 and amyotrophic lateral sclerosis: Mutations and oligomerization[END_REF] is a fluorescence probe used to detect the presence of aggregates and the amount of aggregated proteins, compared to the initial state of each sample (Supplement, Fig. S1).

Fig. 2A shows the results for time-resolved analysis of the process of SOD1 aggregation. Apo SOD1 and an as-prepared SOD1 aggregate, when incubated with lipid molecules, showed a higher propensity for aggregation than holo SOD1 or BSA depending on the incubation time. Native holo SOD1 and BSA, which are the stable forms, did not show any significant changes in fluorescence intensity. This suggests that they interact minimally or not at all with lipid molecules. In the case of apo SOD1, which is the unstable form due to the absence of Zn 2+ as a structural core, an increase in fluorescence intensity was observed during incubation. Similarly, a significant increase in fluorescence intensity was also observed for an as prepared SOD1 aggregate. Based on control measurements, lipid molecules also have a propensity to bind to ThT; however, no significant change in the time-resolved fluorescent intensities was observed (Supplement, Fig. S2). Therefore, the addition of lipid molecules to all protein samples generally induced an increase in ThT fluorescent intensities in the initial state (0 day). However, the magnitude of the increase was not the same for all samples. Thus, the initial large increase for an aggregated SOD1 can be attributed to a rapid association between aggregated SOD1 and lipid molecules. These results suggest that the conformational changes in SOD1s, which are observed in apo SOD1 and its aggregate, can promote further aggregation by assembling with lipid molecules. The propensity of lipid molecule-induced aggregation was readily confirmed by the enhanced ThT fluorescence intensity after 5 days incubation (Fig. 2B).

Changes in Hydrophobicity during Lipid Molecule-Induced SOD1 Aggregation

The hydrophobicity of SOD1 aggregates assembled with lipid molecules might be altered to some degree owing to their amphiphilicity, which is a unique property of lipid molecules. The hydrophobicity of the prepared samples was measured to obtain clear evidence of an assembly with lipid molecules. After incubation for 5 days, changes in ANS fluorescence intensities were observed

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before and after incubation with lipid molecules, as shown in Fig. 3. A plot for a control sample shows the intrinsic fluorescence of ANS dye (without lipid) and ANS fluorescence of lipid molecules (with lipid), respectively. This result shows that lipid molecules induce an enhancement in ANS fluorescence. This can be attributed to the adsorption of ANS dyes to the hydrocarbon chains in lipid molecules [START_REF] Conti | Fluorescence signals in ANS-stained lipid bilayers under applied potentials[END_REF][START_REF] Jendrasiak | The interaction of 1-anilino-8-naphthalenesulfonate with thyroid lipids and membranes: A nuclear magnetic resonance study[END_REF][START_REF] Merkle | Dynamic fluorescence quenching studies on lipid mobilities in phosphatidylcholine-cholesterol membranes[END_REF]. Interestingly, apo SOD1s with lower ANS intensities, compared to lipid molecules, showed enhanced ANS fluorescence, and that of the SOD1 aggregate with higher ANS intensities showed a decrease in intensity after incubation with the lipid molecules. These slight discrepancies are not due simply to the co-existence of two components remaining unassembled in the mixtures considering the protein to lipid molar ratio of the incubation mixtures, 1:45. Specifically, it is due to changes in the surface hydrophobicity of the newly formed structures formed from the assembly between lipids and proteins. Native holo SOD1 and BSA, which are the stable forms, did not show any significant changes in ANS fluorescence intensity. Therefore, these changes in ANS fluorescence intensities can be interpreted as changes in the hydrophobicity of the proteins caused by further binding and the formation of assemblies with lipid molecules.

Morphological Analysis of Lipid Molecule-Induced SOD1 Aggregates

Negatively stained samples were observed by TEM to visualize the protein structures and examine the morphology of the resulting structures. In the case of apo SOD1, which had been incubated in PBS buffer for 2 weeks, no visible macrostructures, except for a few small structures, were observed (Fig. 4A). In the case of SOD1 aggregate (Fig. 4C) prepared by the TFE treatment, some individual aggregates ranging in size from several hundreds of nanometers were observed frequently. In particular, apo SOD1, which had been incubated with lipid molecules, formed large, amorphous network structures (Fig. 4B). Dramatic growth of these structures (Fig. 4D) was also observed in the case of the SOD1 aggregate that had been incubated with lipid molecules. On the other hand, no visible protein aggregates were observed in the BSA, BSA-lipid, lipid only samples (Supplement, Fig. S3). The ThT binding propensities for the resulting aggregated structures were visualized by

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fluorescence imaging (insets of each TEM image). The fluorescence images are completely consistent with the morphological properties revealed from the TEM images. In particular, large fluorescence spots, observed in the SOD1 aggregate samples that had been incubated with lipid vesicles, support the existence of enormous aggregates formed from the further assembly with lipid molecules.

From the results, it was concluded that favorable interactions between lipid molecules and denaturated proteins (i.e. apo SOD1 and as prepared SOD1 aggregate) promotes the lipid moleculemediated abnormal assembly of denaturated SOD1s. As a control, all data (Fig. 2 and Supplement, Fig. S4) obtained from structurally ordered holo SOD1 strongly support this finding. After incubation of holo-SOD1 with or without lipid vesicles, their structural and superficial properties did not change, unlike those of apo SOD1 (metal-deficient).

Cellular toxicity of Lipid Molecule-Induced SOD1 Aggregates

Having established that SOD1 variants with structurally disordered regions (i.e. apo SOD1 and as prepared SOD1 aggregate) have stronger affinity to lipid molecules than the native-state-stable protein forms (i.e., holo SOD1 and BSA) and consequently form larger protein aggregates assembled with lipid molecules, their cellular toxicity was examined using a cell viability assay. Aliquots of the samples that had been incubated in the presence or absence of lipid molecules were diluted with the culture medium, and added directly to the cultures of PC-12 cells. PC-12 cells are a cell line originating from the pheochromocytoma in rats, and are commonly used as a research model for examining neurodegenerative diseases and spinal cord injuries [START_REF] Seth | Induced expression of early response genes/oxidative injury in rat pheochromocytoma (PC12) cell line by 6-hydroxydopamine: implication for Parkinson's disease[END_REF][START_REF] Harris | Cellular biology of prion diseases[END_REF][START_REF] Kim | Potentiation of amyotrophic lateral sclerosis (ALS)-associated TDP-43 aggregation by the proteasome-targeting factor, ubiquilin 1[END_REF]. After incubation, the toxicity was evaluated using a MTT assay. As shown in Fig. 5, the SOD1 aggregates incubated with lipid molecules as well as the SOD1 aggregate themselves are quite toxic and clearly reduced the cell viability (*). In contrast, no significant decrease in viability was detected when the cells were exposed to most of the incubated samples containing only proteins and/or only lipids. They showed a cell viability of more than 80 %, as shown in control samples. However, the aggregates formed from the

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incubation of apo SOD1 with lipid significantly reduced the cell viability by up to 55 % (*). This suggests that lipid-induced SOD1 aggregates are quite toxic to cells.

Relevance of Lipid Molecule-Induced SOD1 Aggregates

To identify the relevance of this lipid molecule-promoted SOD1 aggregation, apo SOD1 was also incubated with POPC, a neutral, unsaturated lipid molecule. After incubating apo SOD1 with different lipid vesicles, the amorphousness of the resulting structures was determined from CD spectra (Fig. 6A). Similarly, the negatively stained electron micrographs also revealed the presence of large amorphous networked structures of SOD1 (Fig. 6B). Moreover, we performed additional analyses for apo SOD1s that have been incubated with different lipid vesicle compositions, which are prepared by inserting cholesterol molecules to lipid vesicles, in an attempt to mimic the raft domains of real cell membranes. Analogous results, structural variations (Fig. 7A) and resulting amorphous morphologies (Fig. 7B), were also obtained for these types of lipid vesicles. This strongly suggests that lipid molecules can the promote aggregation of SOD1s with abundant hydrophobic parts exposed by structural disordering, due to the amphiphilic property as a general feature of lipid molecules. Fig. 8 shows a schematic description of the molecular mechanism for the lipid molecule-associated SOD1 aggregation. Based on our findings, it appears that the structurally disordered SOD1 variants (apo SOD1 and aggregated SOD1) with abundant hydrophobic regions can be readily inserted to the inner space of lipid vesicles, disrupt them, and consequently become assembled with the hydrophobic tails of lipid molecules.

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Discussion

This study examined a molecular mechanism involving lipid molecules in the formation of SOD1 aggregates, as well as the cytotoxicity of these aggregates. In vitro biophysical analyses showed that apo SOD1 and aggregated SOD1 species, which undergo conformational changes from their native structure, aggregated readily in the presence of lipid molecules, even under physiological conditions.

Conformational changes in SOD1s result in an increase in surface hydrophobicity, which promotes binding to lipid molecules with amphiphilic properties. This is consistent with results observed for structurally ordered proteins (i.e., holo SOD1 and BSA), showing no significant formation of lipidassociated aggregates. That is, the formation of lipid-associated aggregates can be attributed to differential interactions between lipid molecules and denaturated (or native) proteins. Therefore, the extent of these interactions promotes the lipid molecule-mediated abnormal assembly of denaturated SOD1s.

It is important to note that interactions between proteins and lipid molecules can be controlled by many factors. G. P. Gorbenko, et al. reported that these interactions are controlled by not only the general physicochemical characteristics of a membrane (i.e., its phase state, bilayer curvature and elasticity, surface charge, and degree of hydration), but also the exact chemical nature of membrane lipids (i.e., the extent of acyl chain unsaturation, conformation and dynamics of lipid head groups and acyl chains, and protein-lipid selectivity arising from factors such as the hydrophobic matching) [28].

In addition, the role of lipid rafts in the protein aggregation has been also emphasized in some proteins [START_REF] Pinheiro | The role of rafts in the fibrillization and aggregation of prions[END_REF].

In this regard, we focused on the aggregation induced by the general nature of lipid molecules, which is shared between all of lipids, that is "amphiphilicity". We concluded that other parameters, as mentioned above, can also accelerate or decelerate aggregation to some extent, compared with our present observation results. To address this, first of all, DPPC (a neutral, saturated lipid molecule), that is relatively inactive compared to other types of lipid molecules, was selected as a component of model membrane system. Furthermore, we performed additional analyses with other different types of aggregation. Similar results (Fig. 6 and Fig. 7), structural variations and resulting amorphous morphologies, were also observed for these types of lipid vesicles. These findings suggest that lipid molecules can promote the aggregation of SOD1s with abundant hydrophobic parts exposed by structural disordering, due to their amphiphilic properties as a general nature of lipid molecules.

Since lipid molecules are the main components of the cellular membrane, the proposed molecular mechanism of SOD1 aggregation can be directly associated with the membrane-related behavior of SOD1s, such as membrane binding and membrane disruption. In particular, membrane linked SOD1 aggregates can be formed by the direct binding of SOD1 species to membranes, and/or the assembly of SOD1 with detached lipid molecules. Consequently, these assembled macrostructures would be expected to have an effect on cell viability, for example, homeostasis and permeability [START_REF] Chi | Ganglioside GM1-mediated amyloid-beta fibrillogenesis and membrane disruption[END_REF][START_REF] Engel | Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane[END_REF][START_REF] Friedman | Amyloid aggregation on lipid bilayers and its impact on membrane permeability[END_REF]. It has been extensively reported that interactions between proteins and lipid molecules may play a crucial role in determining the membrane-associated cytotoxicity of many other proteins that have been implicated in conformational diseases [START_REF] Chi | Ganglioside GM1-mediated amyloid-beta fibrillogenesis and membrane disruption[END_REF][START_REF] Madine | The aggregation and membrane-binding properties of an α-synuclein peptide fragment[END_REF][START_REF] Kazlauskaite | Structural changes of the Prion protein in lipid membranes leading to aggregation and fibrillization[END_REF][START_REF] Engel | Membrane damage by human islet amyloid polypeptide through fibril growth at the membrane[END_REF][START_REF] Friedman | Amyloid aggregation on lipid bilayers and its impact on membrane permeability[END_REF].

Recently, the hypothesis that SOD1 variants might aggregate on mitochondrial membrane surfaces has received support by the demonstration that SOD1 aggregates are attached to the cytoplasmic face of the mitochondrial membrane in transgenic ALS mice [START_REF] Higgins | ALS-associated mutant SOD1 G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes[END_REF][START_REF] Liu | Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria[END_REF][START_REF] Shaw | How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?[END_REF][START_REF] Zhai | Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord[END_REF]. Interestingly, it has been reported that SOD1 may form pore structures in membranes [START_REF] Chung | Cu/Zn superoxide dismutase can form pore-like structures[END_REF][START_REF] Ray | An intersubunit disulfide bond prevents in vitro aggregation of a superoxide dismutase-1 mutant linked to familial amytrophic lateral sclerosis[END_REF]. Furthermore, we observed abnormal ion permeability in real cell membranes induced by aggregated SOD1 (Choi. I et al., unpublished data).

The findings reported herein suggest that an aggregate with abundant hydrophobic parts, induced by conformational changes, can be readily inserted into the inner space of a membrane and assembled with the hydrophobic tails of lipid molecules, in a similar manner to that described in Fig. 8. That is, defects within a membrane can be formed by the detachment of lipid molecules that are co-assembled with structurally disordered SOD1species. Such behaviors of lipid membranes provide a plausible explanation for the abnormal aggregation and resulting cytotoxicity of disordered SOD1 variants. We

  5 mM isoprophyl b-D-thiogalactopyranoside for 3 to 6 hr at 30 o C. The cells were then lysed by sonication in a buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF). The proteins were eluted with a linear gradient of ammonium sulfate (0.75-0 M) in 50 mM sodium phosphate (pH 7.0), 150 mM NaCl, 0.1 mM EDTA and 0.25 mM DTT. Wild type (WT) SOD1 was released with high specificity from the column between 1.3 and 0.8 M ammonium sulfate. Apo type SOD1 was produced by the demetallation of Zn and Cu.

  A C C E P T E D M A N U S C R I P TACCEPTED MANUSCRIPTother destabilizing conditions are generally accompanied by changes in the superficial properties of the proteins, e.g. hydrophobicity. The ANS fluorescence results (Fig.1B) are consistent with the hydrophobic residues being exposed as the result of conformational changes because the ANS dye is a probe for identifying the extent of exposed hydrophobic surfaces. As shown in Fig.1B, holo SOD1 did not show any significant ANS fluorescence enhancement from the base line for a control sample (ANS dye only), whereas substantial ANS fluorescence enhancement was observed for apo SOD1.

A

  C C E P T E D M A N U S C R I P TACCEPTED MANUSCRIPTlipid vesicles including unsaturated lipids (POPC) and lipid rafts (lipid vesicles containing cholesterol), in an attempt to identify the relevance of this lipid molecule-promoted SOD1

AFigure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .

 12345678 Figure 1. Structural and superficial properties of the proteins. (A) Secondary structures of the proteins

Acknowledgements

This study was supported by a grant (no. R01-2006-000-10239-0) from the Basic Research Program of the Korea Science & Engineering and a grant (101-082-032) from the Ministry of Environment as "The Eco-technopia 21 project". This research was also supported by the WCU (World Class University) program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-10013).