Eva M Mateo

F M Valle-Algarra

R Mateo-Castro

M Jiménez

Impact of non-selective fungicides on growth and production of 1 ochratoxin A by Aspergillus ochraceus and A. carbonarius in barley 2 -based medium 3 4

Keywords: fungicides, barley, mancozeb, copper oxychloride, sulfur, Aspergillus, ochratoxin

The aim of this study was to assess the influence of the non-selective fungicides mancozeb, 18 copper oxychloride and sulfur on the growth and capability for producing ochratoxin A (OTA) 19 of ochratoxigenic isolates of Aspergillus carbonarius and A. ochraceus in barley-based 20 medium. Lag phases and growth rates were determined for each fungicide at different doses, 21 at 15 ºC and 25 ºC and at 0.97 a w . Mancozeb at 40 mg L -1 inhibited fungal growth and 22 provided lag phases > 24 days at 10-20 mg L -1 and 15 ºC. OTA was observed only at 25 ºC 23 and doses <10 mg L -1 . At 15 ºC, copper oxychloride proved inhibitory at 800 mg L -1 while at 24 25 ºC growth was not delayed and only high doses decreased OTA levels. Sulfur was 25 inhibitory or provided large lag phases at 5-8 g L -1 (at 15 ºC) while at 25 ºC growth took place 26 even at 8 g L -1 although OTA levels were low or undetectable. The antifungal activity 27 decreased in the order mancozeb > copper oxychloride > sulfur and was lower at 25 ºC than 28 at 15ºC. OTA accumulation was affected by the type of fungicide, dose, temperature and 29 time. The efficacy of these fungicides on the growth of A. carbonarius and A. ochraceus and 30 OTA production in barley-based medium is assessed for the first time.

 2009a, 2009b;Belli et al. 2004;Kapetanakou et al. 2009;Romero et al. 2007). Culture 79 nutrients affect OTA production by Aspergillus spp. [START_REF] Medina | Influence of 557 nitrogen and carbon sources on the production of ochratoxin A by ochratoxigenic isolates 558 of Aspergillus spp. isolated from grapes[END_REF] and OTA 80 biosynthetic genes in A. ochraceus are differentially regulated by pH and nutritional stimuli 81 (O'Callaghan et al. 2006). In many cases, usually during pre-harvest, the use of fungicides is 82 the only efficient, cost-effective and often successful way to prevent mould growth 83 (Munimbaz et al. 1997) thus being an attractive strategy to prevent mycotoxin production 84 (FAO/WHO/UNEP 1999). However, fungicides must be applied carefully since some of them, 85 such as carbendazim, can reduce fungal growth but can also stimulate OTA production Mycelial extension rates were measured over time. Lag phase was considered as the 170 time (days) to reach a colony five mm diameter [START_REF] Bellí | Influence of water activity and temperature on 458 growth of isolates of Aspergillus section Nigri obtained from grapes[END_REF]Medina et al. 2007a; 171 Astoreca et al. 2009aAstoreca et al. , 2009b;;Samapundo et al. 2007). For each treatment, 15 plates were 172 prepared and two right-angled diameters of the colonies were randomly chosen and 173 measured every day until the colony filled the dish or the cultures were used for OTA 174 determination. The sum of two diameters was divided by four to calculate the mean radius.

175

These measurements were then averaged over the number of measured dishes. The slope 176 of the line obtained by linear regression of colony radius against time (days elapsed from the 177 lag time) was used to determine growth rates (mm day -1 ). For OTA determination, 20 g of culture (substrate plus fungal biomass) was cut into small 196 pieces, homogenised in a stomacher, extracted with methanol (50 mL) and analyzed using As there were 15 replicates per treatment, the remaining three ones were carried out for 203 safety.

204

The LC system was a Waters 600E system controller, a Millipore Waters 717 plus 205 autosampler and a Waters 470 scanning fluorescence detector (Waters, Milford, MA, USA).

206

Excitation and emission wavelengths were 330 and 460 nm, respectively. The samples were 207 separated using a C18 Phenomenex Gemini ® column (150 × 4.6 mm, 5 µm particle size)

208

(Phenomenex, Macclesfield, UK), with a guard column of the same material. Run time for 209 samples was 20 min with OTA being detected at about 12 min. The flow rate of the mobile 210 phase (acetonitrile/water/acetic acid, 44:55:1, v/v/v) was 1 mL min -1 . Measurements were 211 processed using the Millennium ® 4.0 software (Waters).

212

The limit of detection was 0.5 ng OTA g -1 culture, based on a signal-to-noise ratio of 3:1.

213

The average recovery rate for OTA from the agar-based medium was 89 %. were about 3 days at 1-10 mg L -1 , about 6 days at 20 mg L -1 and about 9 days at 30 mg L -1 ; 231 however, at 15 ºC, lag phases lasted 25 -40 days at 10 -20 mg L -1 (Figure 1A). The lag 232 phase of A. carbonarius also increased with mancozeb dose but they were higher than lag 233 phases observed in A. ochraceus cultures under the same conditions. Sulfur also increased the lag phase more at 15 ºC than at 25 ºC (Figure 1E). At 25 ºC, 297 sulfur had no ability to significantly delay fungal development up to a dose of 8 g L -1 . At 15 298 ºC, a dose of 3 g L -1 resulted in lag phases of about 21 days for both isolates, which were 299 much longer than lag phases attained at doses ≤ 1 g L -1 , while 5 g L -1 prevented fungal 300 growth. Temperature, dose and cross interactions, but not the fungi, significantly influenced 301 the lag-times. Tukey-HSD test found six homogeneous groups concerning dose (P < 0.05).

302

Growth rates 303 Growth rates were higher at 25 ºC than at 15 ºC at the same dose (Figure 1F). At 25 ºC, 304 the growth rate of A. ochraceus at 8 g L -1 was lower than rates observed at 3 -5 g L -1 . At this 305 temperature, in the case of A. carbonarius, there were not significant differences concerning 306 growth rate between controls and cultures at 3 g L -1 . At 15 ºC, however, growth rates 307 decreased by 25%. The ANOVA revealed that temperature, fungi and dose and their 308 interactions, except the interaction fungi × temperature, were significant (P < 0.05). Tukey-309 HSD test found six homogeneous groups related to sulfur dose (P < 0.05).

310

OTA levels 311

At 25 ºC, OTA was not detectable in cultures of A. ochraceus at <1 g L -1 or at 8 g L -1 312 (Table 1). However, OTA accumulation was high at 3 g L -1 . At 15 ºC, OTA levels were 313 significantly higher than those found in controls in cultures containing 0.2 -3 g sulfur L -1 314 (Table 2).

315

At 25 ºC OTA level in cultures of A. carbonarius was low at intermediate/high sulfur 316 doses. The highest toxin level was found in control and at the dose of 10 mg L -1 (Table 1) 317 while at 15 ºC, OTA was not detected (Table 2) and was not assayed when growth was 318 negligible. The influence of temperature, dose and time on OTA accumulation was significant In the present study, two ochratoxigenic isolates, one of A. carbonarius and another of A. 323 ochraceus, were selected to investigate the effect of three antifungal agents on fungal growth 324 and OTA production when the fungi were cultured on barley-based medium at 15 ºC and 25 325 ºC and at 0.97 a w .

326

The doses of the fungicides were chosen on the basis of preliminary experiments 327 performed in our laboratory, which had shown that growth of these isolates in BMEA was 328 inhibited at doses above those used in the present study (data not shown). Sub-inhibitory 329 doses were assayed because mycotoxin production may be stimulated when stressing 330 environmental conditions and low fungicide doses are maintained during growth of 331 mycotoxin-producing fungi (D' Mello et al. 1998;Magan et al. 2002;Medina et al. 2007b;332 Nesci et al. 2003). This hypothesis is supported by the results obtained in this work.

333

The lowest assayed temperature (15 ºC) was less favorable than 25ºC to the growth of production of OTA but also to minimize the toxic effects of ETU in humans and animals.

376

Any of the assayed doses of copper oxychloride proved inhibitory at 25 ºC. However, at 377 15 ºC fungal growth was inhibited at 0.8 g L -1 . This fungicide produced shorter lag phases 378 and slower growth rates than mancozeb at the same doses, especially in the case of A. 379 carbonarius. At 25ºC, OTA levels were undetectable or unquantifiable at doses higher than 380 500 mg L -1 in cultures of A. ochraceus and higher than 10 mg L -1 in cultures of A. 387 of this antifungal agent [START_REF] Gharieb | Transformation of copper oxychloride fungicide into 503 copper oxalate by tolerant fungi and the effect of nitrogen source on tolerance[END_REF] or that 8 g L -1 are needed to completely inhibit 388 the growth of A. flavus [START_REF] Mishra | Evaluation of some essential oils for their toxicity against fungi 560 causing deterioration of stored food commodities[END_REF]. But up to now its effectiveness for 389 controlling the production of OTA in ochratoxigenic species from barley has not been 390 assessed.

391

Sulfur was unable to produce inhibition of the assayed fungi and its capacity to delay 392 growth was irrelevant at 25 ºC. At 15 ºC, doses equal or higher than 5 g sulfur L -1 were 393 needed to inhibit mycelial extension of both isolates. Therefore, sulfur does not appear 394 suitable to control these fungi. In addition, the results shown in the present report indicate 395 that sulfur can increase OTA production by the two species at sub-inhibitory doses, mainly in 396 cultures of A. ochraceus. It has been reported that sulfur can inhibit the growth of A. flavus at 397 7 g L -1 and 75% inhibition took place at 5 g L -1 (Mishra and Dubey 1994) but little is known 398 [START_REF] Bellí | Impact of fungicides on Aspergillus carbonarius 464 growth and ochratoxin A production on synthetic grape-like medium and on grapes[END_REF] about its effect on mycotoxin production by mycotoxigenic fungi at sub-399 inhibitory doses. Thus, this antifungal agent should be applied with caution.

400

The assayed fungi showed some differences concerning lag phase, growth rate and OTA 401 production with regard to the fungicide and its dose. Differences in lag phases are higher at the lowest temperature in cultures of the isolates of the two assayed species. In cultures 403 containing any of the three fungicides, at 15 ºC, A. ochraceus showed shorter lag phases and 404 higher growth rates than A. carbonarius with the three antifungal agents; at 25 ºC, the two 405 isolates showed similar lag phases, although growth rates were higher in cultures of A.

406

ochraceus, like at 15 ºC. Concerning OTA production, the isolate of A. ochraceus produced 407 more toxin that the isolate of A. carbonarius in controls, under the same conditions and, 408 generally, this behavior was observed in fungicide-containing cultures at 25 ºC. At 15 ºC, the 409 stress due to temperature significantly affected to OTA production in controls, mainly in those 410 of A. carbonarius. However, it is remarkable that at this temperature, less favorable for fungal 411 growth and toxin production, some doses of copper oxychloride and, especially sulfur, have a 412 positive impact on OTA production by A. ochraceus. This effect is also observed in cultures 413 at 25 ºC supplemented with certain doses of these two fungicides. Unfortunately, scarcity of 414 previous reports about these fungi and fungicides difficult the discussion of the results, 415 although it is generally known that interspecific and intraspecific differences in fungicide 416 susceptibility are frequent in fungi [START_REF] Antachopoulos | Use of high inoculum for 443 early metabolic signalling and rapid susceptibility testing of Aspergillus species[END_REF]Pell et al. 2001).

417

The effects of mancozeb, copper oxychloride and sulfur in isolates of A. carbonarius and 418 A. ochraceus from barley grown in Spain and cultured on BMEA have been studied for the 419 first time. On the basis of the results obtained in this study it seems that risk of barley 420 contamination with OTA might be higher on spring crops than on winter crops treated with 421 these fungicides at sub-inhibitory levels. Low fungicide doses combined con mild

  , the mean temperatures recorded in Spanish crop areas (25 ºC and 15 ºC during 166 spring and winter crops, respectively), and previous studies on the growth of A. ochraceus in 167 fungicide-free barley medium.

  OTA standard (2-1000 ng mL -1 ) were used to obtain a 180 calibration line by linear regression after injecting 100 µl into the liquid chromatography (LC) 181 system. These solutions were obtained by dissolution of the standard (Sigma-Aldrich, 182 Alcobendas, Spain) in chloroform and further dilution in dimethylsulfoxide (DMSO)/water 183 (60:40, v/v). 184 Recovery studies were performed by adding variable amounts of OTA standard, 185 dissolved in chloroform, at 45 ºC to Erlenmeyer flasks containing 20 g of autoclaved (115 ºC, 186 30 min) BMEA. The concentration range for recovery studies was 5-300 ng g -1 . After 187 homogenisation, the medium was poured in a Petri dish and let to cool at room temperature. 188 Twenty grams of solid medium was cut into small pieces and homogenised in a stomacher. A 189 fraction of about 1.5 g of the homogenate was taken and extracted with five milliliters of 190 methanol for one h using a rotating plate stirrer (model F-205, FALC Instruments S.R.L., 191Treviglio, Italy)[START_REF] Bragulat | An easy screening method for fungi producing 467 ochratoxin A in pure culture[END_REF]. Four mL of extract was filtered through filter paper 192 . 4) containing 5-10 g of Celite 545 (Sigma-Aldrich). The filtrate was dried 193 under a N 2 stream and re-dissolved in 0.50 mL ofDMSO/water (60:40, v/v). Finally, 100 µL of 194 solution was injected into the LC system.

  195

197

  the procedure indicated for recovery studies. Once the colony diameter reached 5 mm, the 198 OTA level in the cultures was determined. Samples were taken on days 5, 10, 15 and 20 199 when incubation temperature was 25 ºC and on days 20, 30, 40 and 60 when incubation 200 temperature was 15 ºC because previous experiments had shown that growth was favoured 201 at 25 ºC. This protocol was performed each time with three dishes from the same treatment.

  202

  Figure 1A. Lag phases were always longer at 15 ºC than at 25ºC under all the assayed

  319for both isolates. Concerning dose and time Tukey-HSD test found three and two

334

  the studied isolates. The impact of temperature on growth parameters agrees well with 335 studies performed with other strains of A. carbonarius(Barberis et al. 2009; Medina et al. 336 2007b; Romero et al. 2007) and A. ochraceus (Pardo et al. 2005; Ramos et al. 1998), even 337 though in all these studies the culture media, a w -values, presence of fungicides and hosts 338 were different.339OTA accumulation in BMEA was affected by temperature both in fungicide-containing 340 media and controls. Toxin levels were generally higher at 25 ºC than at 15 ºC at a given 341 fungicide dose. These results agree with those ofKapetanakou et al. (2009), who report that 342 OTA production by A. carbonarius and A. ochraceus in malt extract agar was higher at 25 ºC 343 than at 15, 20 or 30 ºC, although fungicides were not used and a w conditions were different 344 from those employed in the present study. However, other authors(Barberis et al. 2009) 345 have found that temperature is the factor of minor influence on OTA production by isolates in They can be very useful to control and to prevent the growth of Aspergilli and 375

381carbonarius.

  At 15ºC OTA production was inhibited at doses higher than 50 mg L -1 in cultures 382 of A. ochraceus and at all the assayed doses in cultures of A. carbonarius. This antifungal 383 agent, though not very effective to control the growth of the assayed species, plays a 384 relevant role in the inhibition of OTA production. Other authors have found that this fungicide 385 was scarcely effective to control Fusarium oxysporum f.sp. cubense(Nel et al. 2007), that A. 386 niger and Penicillium chrysogenum were able to grow in a medium containing up to 0.5 g kg -1

Figure 1 .

 1 Figure caption

Figure 1 .

 1 Figure 1. Effect of three fungicides on lag phase and radial growth rate of isolates of A. ochraceus and A. carbonarius cultured on BMEA at 0.97 aw. Mancozeb (A and B); Copper oxychloride (C and D); Sulfur (E and F). Line key: A. ochraceus: dotted line with white squares (15 ºC) or white rhombi (25 ºC); A. carbonarius: solid line with black circles (15 ºC) or black triangles (25 ºC). 234x303mm (600 x 600 DPI)

Table 1 .

 1 Mean concentration ± standard deviation of OTA (ng g -1 ) (N = 3) found in cultures of isolates of A. ochraceus and A. carbonarius carried out at 25 ºC and 0.97 a w on BMEA treated with three fungicides. No fungicide was added; ND, not detected; NQ, detected but not quantified (< 1.5 ng g

	Incubation time (days)

*

Table 2 .

 2 Mean concentration ± standard deviation of OTA (ng g -1 ) (N = 3) found in cultures of isolates of A. ochraceus and A. carbonarius carried out at 15 ºC and 0.97a w on BMEA treated with three fungicides.

	Incubation time (days)

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgements 430

The authors acknowledge financial support from FEDER, the Spanish Ministry of Science 431 and Innovation (Project AGL2007-66416-C05-01/ALI and a research grant) and from the


Stamatina ; Isari
email: misari@icm.csic.es

Feeding performance of the copepod Clausocalanus lividus (Frost and Fleminger 1968)

Keywords: copepod, Clausocalanus lividus, feeding, functional response, size selection copepod, Clausocalanus lividus, feeding, functional response, size selection

Clausocalanus is a small-sized copepod genus that is widespread in the world ocean, but it has been poorly investigated from a biological and ecophysiological point of view. In this study we targeted the species Clausocalanus lividus, one of the less studied circumglobally occurring congeners of the genus, to explore a number of aspects of its feeding. We examined the functional feeding response of adult wild females to seven monospecific diets with prey items differing in terms of size (ESD: 4-28 µm) and motility. C. lividus did not significantly clear the small flagellate, Isochrysis sp., while for the larger-sized food items offered (ESD>5 µm), it exhibited a type II functional feeding response. Its maximum daily carbon intake ranged from 54 to 137 % of its body carbon and was generally higher on prey with ESD>15 µm, being up to 17 µg C cop -1 day -1 for the diatom Thalassiosira weissflogii. The clearance maxima (10 to 308 mL cop -1 day -1 ) were generally associated with prey size (increasing with the ESD). The estimated feeding rates of C. lividus are discussed in relation to the concentration and size of food and compared to findings for other congeners.

INTRODUCTION

One major paradigm in copepod ecology and biological oceanography in recent years has been the overwhelming numerical dominance of small-sized copepods in planktonic communities (e.g., [START_REF] Calbet | Annual zooplankton succession in coastal NW Mediterranean waters: the importance of the smaller size fractions[END_REF][START_REF] Gallienne | Is Oithona the most important copepod in the world's ocean?[END_REF][START_REF] Turner | The importance of small planktonic copepods and their roles in pelagic marine food webs[END_REF]Zervoudaki et al., 2006). This perception seems to challenge the view that we have had for the last 50 years about the ecological role of planktonic copepods in marine ecosystems (e.g., [START_REF] Mauchline | The biology of calanoid copepods[END_REF], because most of the scientific effort directed toward elucidating the biogeochemical impact of this group has been focused on a very small number of copepod species: both large-sized copepods, (i.e. Calanus) and medium-sized representatives of shelf and coastal waters [START_REF] Saiz | Scaling of feeding in marine calanoid copepods[END_REF]. Due to the bias in the body size structure of copepod communities and the disproportionate importance given to some particular species, there is an obvious need to extend research relevant to future estimations of copepod-mediated fluxes and to consider the functional diversity encompassed within the "neglected" smaller copepod taxa. This is particularly the case in relation to future scenarios of climate forcing, in which phenological and distributional changes in zooplankton communities may involve shifts to smaller-sized copepods (e.g., [START_REF] Batten | Changes in oceanic zooplankton populations in the north-east Pacific associated with the possible climatic regime shift of 1998/1999[END_REF].

A typical representative of this widespread copepod size fraction is the calanoid genus Clausocalanus (♀ total length: 0.66-1.90 mm but usually <1 mm) [START_REF] Frost | A revision of the genus Clausocalanus (Copepoda: Calanoida) with remarks on distributional patterns in diagnostic characters[END_REF], which has been regarded as a worldwide genus with a high ecological success. It is distributed circumglobally with 13 congeners [START_REF] Frost | A revision of the genus Clausocalanus (Copepoda: Calanoida) with remarks on distributional patterns in diagnostic characters[END_REF]), forming an important numerical component of epipelagic mesozooplankton communities both in terms of abundance and biomass in different trophic environments, from low-to high-latitude ecosystems (e.g., Webber and Roff, 1995: up to 32% of copepod biomass in tropical oceans; [START_REF] Calbet | Latitudinal changes of copepod egg production rates in Atlantic waters: temperature and food availability as the main driving factors[END_REF] up to 30% of copepod abundance in the Atlantic; [START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF] up to 55% and 73% in the eastern and western Mediterranean respectively, up to 57% of copepod abundance in the Atlantic).

Existing information on fundamental ecological aspects of the genus, such as species-specific distribution patterns (e.g., [START_REF] Fragopoulu | Patterns of vertical distribution of Pseudocalanidae and Paracalanidae (Copepoda) in pelagic waters (0 to 300 m) of the Eastern Mediterranean Sea[END_REF][START_REF] Paffenhöfer | Vertical distribution of subtropical epiplanktonic copepods[END_REF][START_REF] Peralba | Vertical and seasonal distribution of eight Clausocalanus species (Copepoda: Calanoida) in oligotrophic waters[END_REF], seasonal cycles (e.g., Mazzocchi and Ribera d' Alcala, 1995;[START_REF] Siokou-Frangou | Zooplankton annual cycle in a Mediterranean coastal area[END_REF][START_REF] Peralba | Vertical and seasonal distribution of eight Clausocalanus species (Copepoda: Calanoida) in oligotrophic waters[END_REF] and reproductive parameters [START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF], has considerably broadened our insight into the niche characterisation of several Clausocalanus species [START_REF] Peralba | Vertical and seasonal distribution of eight Clausocalanus species (Copepoda: Calanoida) in oligotrophic waters[END_REF][START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF]. Nonetheless, our knowledge regarding many behavioural and physiological mechanisms of the genus remains scarce. Substantial difficulties related to the taxonomic distinction among its congeners [START_REF] Blanco-Bercial | RFLP procedure to discriminate between Clausocalanus Giesbrecht, 1888 (Copepoda, Calanoida) species in the Central Cantabrian Sea[END_REF] and the cultivation of these species [START_REF] Mazzocchi | First observations on the biology of Clausocalanus furcatus (Copepoda, Calanoida)[END_REF] have made this particular genus less During the last three decades some assessments of Clausocalanus vital rates (i.e., egg production, respiration, grazing, gut evacuation, growth) have been sporadically included in published studies estimating a number of biological processes of field copepod assemblages (e.g., [START_REF] Sazhina | Fecundity and growth rate of copepods in different dynamic zones of equatorial counter current of the Indian Ocean[END_REF][START_REF] Landry | Mesozooplankton grazing in the Southern California Bight. II. Grazing impact and particulate flux[END_REF]Webber and Roff et al., 1995;[START_REF] Hopcroft | Zooplankton growth rates: the influence of female size and resources on egg production of tropical marine copepods[END_REF]Saiz et al., 1999;[START_REF] Mayzaud | Feeding, respiration and egg production rates of copepods during austral spring in the Indian sector of the Antarctic Ocean: role of the zooplankton community in carbon transformation[END_REF]. However, the first laboratory research purely focused on the biological mechanisms of the genus Clausocalanus was conducted by Mazzocchi andPaffenhöfer (1998, 1999). These authors, targeting the species Clausocalanus furcatus, provided preliminary experimental data on the feeding and swimming behaviour of the genus, along with some information on its reproductive and developmental processes. Through a detailed observation of the swimming trajectories of the species [START_REF] Mazzocchi | Swimming and feeding behaviour of the planktonic copepod Clausocalanus furcatus[END_REF]Uttieri et al., 2008a), a unique motion strategy was discovered for small planktonic copepods, which is not only displayed by C. furcatus, but presumably by the other congeners as well. This strategy involves a rapid (ca. 10 body lengths s -1 ), continuous movement in convoluted small loops and the capture of dinoflagellates while swimming at high speed, with no evidence of feeding current generation. This foraging tactic also implies that food is perceived within a restricted horizon by direct encounter (intercept feeding), which is a feature that has been argued to be an efficient adaptation of the species in the oligotrophic areas (Uttieri et al., 2008a).

Although in recent years there has been an increasing interest in the variables regulating population dynamics of Clausocalanus species (e.g., [START_REF] Buttino | A novel method to dectec embryo viability in the egg-carrying copepod Clausocalanus furcatus[END_REF][START_REF] Bi | Egg production rates and stage-specific development times of Clausocalanus furcatus (Copepoda, Calanoida) in the northern Gulf of Mexico[END_REF]Cornils et al., 2007a;[START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF], no thorough studies describing the feeding performance and prey size spectrum of this copepod genus have yet been conducted. To the best of our knowledge, since the pioneering works by Mazzocchi andPaffenhöfer (1998, 1999), dietary studies on Clausocalanus congeners have involved mainly in situ trials with the natural particle composition and concentration [START_REF] Batten | The contribution of microzooplankton to the diet of mesozooplankton in an upwelling filament off the north west coast of Spain[END_REF]Broglio et al., 2004: on Clausocalanus spp;Paffenhöfer et al., 2006: on C. furcatus;Cornils et al., 2007b: on C. farrani and C. furcatus).

In this paper, we deal with the first experimental data on the functional feeding response of this genus, targeting one of the larger sized congeners, C. lividus. This species is characterised by an antitropical distribution in the Atlantic (Williams and Wallace, 1975;[START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF] and Pacific Ocean [START_REF] Frost | A revision of the genus Clausocalanus (Copepoda: Calanoida) with remarks on distributional patterns in diagnostic characters[END_REF]). In the Mediterranean, it occurs at relatively low abundances compared with smaller congeners, though a more significant presence has been reported during spring in the less oligotrophic western part of the basin [START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF]. Thus far, C. lividus has attracted attention due to its different spawning behaviour (free spawner) compared to the rest of its small and medium sized congeners (sac spawners) (Saiz and Calbet, 1999;[START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF]. However, there is no information about the biological mechanisms (e.g., feeding behaviour) that define the success of this species and shape its distribution patterns.

With the aim of shedding some light on this topic, we exploited the spring-early summer population of C. lividus off the Barcelona coast (NW Mediterranean) to explore the single-resource feeding functional response of wild females on prey differing in size and motility. Feeding rates were estimated as a function of the food concentration and the relevance of prey-specific characteristics in the maximum feeding capacity of C. lividus were evaluated. We consider the information provided in this paper to be a prerequisite that will allow future comparisons of the feeding behaviour and requirements among congeners and will facilitate our understanding of the ecological interactions of this widespread genus and the functional role of its species.

METHOD

Sample collection

Live zooplankton were collected ca. five nautical miles from the coast of Barcelona, Spain (41 o 20΄Ν, 002 o 15΄Ε; ca. 100 m depth) on five dates from April to June 2009 (Table I). Oblique slow-speed tows of a 100-µm mesh plankton net were deployed for five minutes at an approximate depth of 40 m order to collect live material. The end of the net was equipped with a non-filtering cod-end (plastic bag) that, upon retrieval, was transferred to an isothermal container filled with surface seawater, and tied with a string to avoid the presence of an air interface to which Clausocalanus might stick on. In the laboratory, zooplankton were concentrated and adult females of C. lividus were sorted with a wide-mouth Pasteur pipette under a dissecting microscope.

Feeding experiments

The feeding functional response of C. lividus was explored on seven monospecific diets (Table I). The prey selected ranged in size (equivalent spherical diameter, ESD) from 4 µm to 28 µm and were kept in batch cultures growing exponentially at 20ºC under a 12:12 h light:dark cycle.

Autotrophic prey were grown in f/2 medium with silicon added for the maintenance of the diatom Thalassiosira weissflogii. Heterotrophic dinoflagellates (i.e. Gyrodinium sp. and Oxyrrhis marina) were grown on the cryptophyte Rhodomonas salina, whereas the ciliate Strombidium sulcatum was fed on the small-sized (ca. 2 µm ESD) autotrophic flagellates Micromonas sp.

Food suspensions in all of the experiments were prepared in 0.2 µm filtered sea water (FSW) using stock cultures; in some cases, prior to their use the cultures were screened through a larger mesh to remove any cell aggregates. In the case of T. weissflogii, three days before the initiation of the experiments cultures were aerated to induce the removal of cell spines because these structures 6 have been reported to influence copepod feeding behaviour [START_REF] Gifford | Spines on diatoms: Do copepods care?[END_REF]. Heterotrophic prey stocks were not fed on the day preceding the experiment to ensure that food algae were grazed down, and hence, our results would not be associated with a potential presence of algae. The concentrations of the stock cultures and the cell volume were determined using a Beckman Coulter Multisizer III electronic particle counter. For each food item, the adjustment of the initial suspensions to the desired concentrations was achieved through successive dilutions. Finally all suspensions (autotrophic, heterotrophic) were enriched with 5 mL of f/2 medium per litre of suspension to (a) avoid a difference in algal growth among the treatments with or without copepods due to nutrient excretion by the copepods and (b) assure the same conditions in experiments with autotrophs or heterotrophs. While preparing the ciliate suspension, EDTA (ethylenediaminetetraacetic acid 10 µM) was added to the FSW before the inclusion of the prey to ensure their survival [START_REF] Jonsson | Feeding-behavior, prey detection and capture efficiency of the copepod Acartia tonsa feeding on planktonic ciliates[END_REF] Each experiment consisted of six consecutively doubled food levels (Table I). A total of five replicate 625-mL Pyrex screw-cap bottles were prepared per food level. Once the bottles were filled near the neck with the appropriate algal suspensions, copepods (4-12, the number was inversely dependent on the food concentration and according to copepod size, which was found to vary significantly among the different experiments) were added to only two of the bottles (experimental: 2 x Exp), while the other three served as initial (1 x I) and control bottles (2 x C). The copepods used in the experiments had been acclimated for the preceding 24 h at the experimental temperature and light conditions and exposed to a prey concentration equal to the corresponding mean value of the range of concentrations to be used during the experiment. After the addition of copepods, all the bottles were filled to the top and prior to the tightening of the screw-cap, a plastic film was placed over their mouth to prevent bubble formation. The initial bottles corresponding to each experimental concentration were sampled as described below, whereas both the experimental and control bottles were incubated on a slowly rotating plankton wheel (0.2 rpm) for 24 h at 16 o C with a light: dark cycle of 12h:12h. At the end of each experiment, the content of all bottles were carefully poured through a 200-µm mesh, and the actual number and condition of copepods was checked.

The mortality of females was negligible in our experiments, with one or two dead animals occasionally being observed (not considered for calculations). After mixing the contents of each bottle well, a sample of 50 mL (or of a higher volume in the case of ciliates) was preserved with 2%

Lugol solution. The abundance of S. sulcatum in the experiment was corrected for 25% fixation losses according to [START_REF] Broglio | Effect of heterotrophic versus autotrophic food on feeding and reproduction of the calanoid copepod Acartia tonsa: relationship with prey fatty acid composition[END_REF]. Cell numbers were counted visually by inverted microscopy, except for the smallest prey item, Isochrysis sp for which the food concentrations at the termination of grazing experiments were determined with a Beckman Coulter Multisizer III fitted with a 70-µm aperture tube. The clearance and ingestion rates as well as average food concentrations, were computed for each food level according to the equation of [START_REF] Frost | Effects of size and concentration of food particles on the feeding behaviour of the marine planlktonic copepod Calanus pacificus[END_REF]. Carbon-specific ingestion rates for each food were also estimated taking into account the average copepod carbon content in each experiment and the cell carbon content of the prey. For copepods as well as the algae Heterocapsa sp. and Isochrysis sp. the carbon content was determined with a CHN analyser. For the other prey items the carbon content was obtained by the conversion of the cell volume (as obtained using the Multisizer III) with different equations found in the literature (Table I).

To assess the functional response to food concentration, an exponential decay equation (F=F max *e -bC ) was fitted to the weighted clearance rates (using the inverse of the standard error as weight), where F max is the maximum clearance rate (mL cop -1 d -1 ); b is a prey-specific constant;

and C is the food concentration (cells mL -1 ). Regarding ingestion rates, asymptotic maxima were estimated by fitting the data (after weighting with the inverse of the standard error) to the model described by [START_REF] Ivlev | Experimental ecology of feeding fishes[END_REF]: I=I max *(1-exp -aC ), where I max is the asymptotic maximum ingestion rate, a is a constant defining the rate at which ingestion (I) approaches the maximum ingestion (I max ); and

C is the food concentration (cells mL -1 or µg C L -1 ).

The maximum clearance rates of C. lividus were plotted against the ESD of the prey to examine the effect of prey size in the feeding behaviour of the females of the species. Additionally, to compare the capability of the copepods to approach saturation (i.e., the shape of the curve) under the different diets, we computed the parameter C Imax/2 (Fig. 2C), which is the concentration at which ingestion equals half of the maximum rate (C Imax/2 = ln(0.5)/-α); equivalent to K m in the Michaelis-Menten equation).

RESULTS

The C. lividus cleared the other offered food resources (ESD>5 µm) at different rates, ranging from 3 mL cop -1 day -1 (for R. salina at an average concentration of ca. 2000 cells mL -1 ) to 323 mL cop -1 d -1 (for S. sulcatum at a mean concentration of ca. 1 cell mL -1 ) (Fig. 1). The clearance rates (F, mL cop -1 d -1 ) of C. lividus typically declined with increasing food concentration (Fig. 1). The parameters of the fitted exponential decay equations for each prey are shown in Table II. The maximum clearance rates ranged from 10 to 308 mL cop -1 day -1 and increased with food particle size. Among the offered prey, the lowest clearance rates were found for the autotrophic flagellate R.

salina and the highest for the ciliate, whereas dinoflagellates ranging from 13 to 17 µm in ESD were cleared at rates that increased with the size (i.e., Heterocapsa<Gyrodinium sp.<Oxyrrhis marina) (Fig. 2A). The diatom T. weissflogii was cleared more efficiently than expected on the basis of its size, with an F max ca. 166 mL cop -1 day -1 .

With respect to ingestion, the estimated rates of C. lividus on prey larger than 5 µm generally increased with prey concentration up to a feeding plateau, resembling an asymptotic Type II functional response [START_REF] Gentleman | Functional responses for zooplankton feeding on multiple resources: A review of assumptions and biological dynamics[END_REF]. The tendency towards saturation was more evident for the species Rhodomonas salina, Heterocapsa sp. and Oxyrrhis marina (Fig. 1), while in the rest of the cases the satiating conditions seemed to exceed our highest food levels. The observed ingestion rates varied considerably among food types and concentrations, ranging from 215 to 166,004 cells cop -1 mL -1 and between 0.36 and 15.25 µg C cop -1 day -1 in terms of cells and carbon respectively (Fig. 1,3A). Incubations with the ciliate Strombidium sulcatum at an average concentration of ca. 1 cell mL -1 (corresponding to 1 µg C L -1 ) resulted in the lowest ingestion records during our study.

The highest rates in cell units were exhibited when feeding on the flagellate Rhodomonas salina (when offered at an average concentration 59,096 cell mL -1 ), whereas when carbon units were considered the diatom T. weissflogii resulted in the highest ingestion rates. The estimated parameters of the Ivlev models fitted to the ingestion rate functional responses are provided in Table II. Maximum ingestion rates ranged from 8,112 to 169,960 cells cop -1 day -1 and were lower when feeding on S. sulcatum and higher in the case of R. salina. The estimated maxima appeared to be quite similar for the three dinoflagellates, whereas the diatom T. weissfloggi was unexpectedly ingested at rates more than two times higher compared to dinoflagellates (Table II). When expressed in terms of carbon, the maximum intake per individual was higher overall on prey with an ESD>15 µm,; however a maximum value of 17 µg C cop -1 day -1 was found for the diatom species tested (Table II, Fig. 3A). Daily ration curves (% body C day -1 ) are shown in Fig. 3B. The carbon ingestion in relation to body carbon ranged from 2-33 % in the lowest food concentrations and between 49.2 to 112.5 % in the highest (Fig. 3B), with maximum ingestion ranging from 54 to 137 % body C day -1 (Table II). When the maximum carbon daily rations were plotted against prey size (Fig. 2B), no clear trend was shown, though C. lividus displayed the highest ingestion rates when offered T. weissflogii and Gyrodinum sp.

When we compared the capability of the copepod to approach saturation under the different diets tested (Fig. 2C), we also found no clear pattern in relation to size and dinoflagellates and ciliates resulted in lower C Imax/2 values. 2007b) or cultured monospecific diets [START_REF] Mazzocchi | First observations on the biology of Clausocalanus furcatus (Copepoda, Calanoida)[END_REF][START_REF] Montresor | Viability of dinoflagellate cysts after the passage through the copepod gut[END_REF].

The food resources utilised in all previous studies involved only a small variation in the food density, which was always within the ranges of oligotrophy (Table III). Hence, there is no comprehensive information regarding the ability of this genus to perform in the richer environments that they can also inhabit (e.g. [START_REF] Valdés | A decade of sampling in the Bay of Biscay: What are the zooplankton time series telling us?[END_REF]. In addition, even in the most trophically poor environments, the ephemeral patchiness of resources at small spatial scales within the water column is likely to be encountered by copepods [START_REF] Tiselius | Behavior of Acartia tonsa in patchy food environments[END_REF][START_REF] Saiz | Experimental records of the effects of food patchiness and predation on egg production of Acartia tonsa[END_REF]. In this regard, Uttieri et al. (2008a, b), taking into consideration the peculiar swimming behaviour of C. furcatus and the sensory structure of the A1 and A2 antennae, hypothesised that Clausocalanus might be especially suited to profit from food patches. Thus, it would be also of particular ecological interest to consider how extreme feeding conditions (e.g., towards satiation) would be perceived by this presumably "intercept feeder" and test its physiological response.

Except for Isochrysis, which was not ingested, C. lividus typically exhibited an asymptotic, density-dependent feeding pattern (Type II, [START_REF] Gentleman | Functional responses for zooplankton feeding on multiple resources: A review of assumptions and biological dynamics[END_REF]: ingestion rates increased up to satiation with increasing food concentration, whereas clearance rates showed the opposite trend.

The ranges of prey concentrations chosen for conducting the experiments did not allow full satiation in some cases. This was, in part, a consequence of the restrictions in the stock cell densities that we were able to reach for the slow-growing heterotrophic prey (S. sulcatum, Gyrodinium sp.). For this reason, in these cases, the maximum feeding rates of C. lividus obtained by the fit of Ivlev's model must be taken as a proxy for the prey-specific maximum potential of the species. (2003), the estimated feeding rates compared well to those reported in these previous studies. Our data also agree with those for Clausocalanus spp. [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], C.

furcatus [START_REF] Paffenhöfer | Living on the edge: Feeding of subtropical open ocean copepods[END_REF]Cornils et al., 2007b) and Clausocalanus farrani (Cornils et al., 2007b) under natural particle composition and concentration (Table III).

Food limitation is considered to be the main factor that limits the feeding rates of copepods in the field with respect to their potential maxima, with this effect being more pronounced in the case of more oligotrophic environments and for the larger-sized species [START_REF] Saiz | Scaling of feeding in marine calanoid copepods[END_REF]. The capability of Clausocalanus lividus to exploit food resources (C Imax/2 ) seems to be inversely dependent on prey size overall, reaching satiation at lower food concentrations when prey is larger (Fig. 3C), as observed in other copepod species [START_REF] Frost | Effects of size and concentration of food particles on the feeding behaviour of the marine planlktonic copepod Calanus pacificus[END_REF][START_REF] Uye | Predatory feeding of the planktonic copepod Tortanus forcipatus on three different prey[END_REF][START_REF] Støttrup | Influence of algal diet on feeding and egg-production of the calanoid copepod Acartia tonsa Dana[END_REF]. One can tentatively estimate the metabolic needs of C. lividus to assess the critical food concentration at which their metabolic demands can be satisfied. Using the equation of [START_REF] Ikeda | Metabolic rates of epipelagic marine copepods as a function of body mass and temperature[END_REF] to estimate oxygen consumption as a function of body mass and temperature, and assuming a respiratory quotient of 0.97 [START_REF] Omori | Methods in Marine Zooplankton Ecology[END_REF], C. lividus metabolic demands are on the order of 11.5 % of body carbon respired per day (for an average body mass of 14.9 µg C ind - 1 at 16 ºC). However, this figure is probably in the lower range because is based on 24h incubations in filtered seawater and there is evidence that under feeding conditions, respiration rates can increase up to 2.3 times [START_REF] Alcaraz | Metabolic rates and energy budget of the early developmental stages of the marine cyclopoid copepod Oithona davisae[END_REF]. For the sake of the comparison, Fig. 3B shows the ingestion rates (assuming 90% assimilation efficiency) required to satisfy the metabolic demands of C. lividus both under starved conditions and under feeding conditions (assuming a 2.3-fold increase). The critical concentrations obtained are relatively high (ca. 14-70 µg C L -1 for prey with an ESD>15 µm; >49 µg C L -1 for prey with an ESD<15 µm) compared to what is expected in oligotrophic natural environments. The above calculations may also provide a reasonable explanation for the rarity of C. lividus in the more oligotrophic eastern part of the Mediterranean [START_REF] Fragopoulu | Patterns of vertical distribution of Pseudocalanidae and Paracalanidae (Copepoda) in pelagic waters (0 to 300 m) of the Eastern Mediterranean Sea[END_REF][START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF]. Interestingly, within the boundaries of the oligotrophic Ionian waters (Eastern Mediterranean), where C. lividus is usually recorded only in low numbers, [START_REF] Peralba | Niche separation of Clausocalanus species in the Mediterranean Sea and in the Atlantic Ocean[END_REF] demonstrated the association of high fluorescence waters offshore the Calabrian coast ("the Calabrian bloom" in the NW Ionian Sea) with a distinct Clausocalanus assemblage, mainly comprising of C. lividus and copepodites of the genus. This appears to contrast with what has been assumed for the smaller sized C. furcatus, the basal metabolism of which may be satisfied even under the particularly limited food resources of the subtropical open ocean [START_REF] Paffenhöfer | Living on the edge: Feeding of subtropical open ocean copepods[END_REF]. Hence, one can assume that under low food conditions, the energetic demands of C. lividus may not be satisfied to the same extent as those of its lower body weight congeners. In contrast to the low daily rations of the species observed when prey availability was low, at saturation levels the ingestion rates increased up to 17.12 µg C cop -1 day -1 , representing a daily food ration equal to 137% of body carbon. The satiating feeding rates of C. lividus estimated during our study (Table II) compare well with those reported for other calanoid copepods with a body mass in the range of C. lividus (daily ration: average=102% body C d -1 , range=53 to 189% body C d -1 , computed from Appendix A1.2 in [START_REF] Saiz | Scaling of feeding in marine calanoid copepods[END_REF].

Feeding rates of C. lividus: the effect of food size

Feeding rates of planktonic copepods have been considered to be the outcome of a complex multi-factorial influences, associated with prey characteristics (e.g., cell size, motility, palatability, food quality) and structural properties of the food assemblage (i.e., species and size distribution), as well as the predator itself (i.e., previous feeding history) (as reviewed by [START_REF] Paffenhöfer | Feeding rates and behavior of zooplankton[END_REF][START_REF] Mauchline | The biology of calanoid copepods[END_REF]. Among these parameters, the role of cell size in prey selection has been the central subject of debate over the last 40 years, often prompting laboratory estimations of the foodparticle size spectra in different species (e.g., [START_REF] Frost | Effects of size and concentration of food particles on the feeding behaviour of the marine planlktonic copepod Calanus pacificus[END_REF][START_REF] Frost | Feeding behaviour of Calanus pacificus in mixtures of food particles[END_REF][START_REF] O' Connors | Particle-Size-dependent maximum grazing rates for Temora longicornis fed natural particle assemblages[END_REF][START_REF] Bartram | Experimental development of a model for the feeding of neritic copepods of phytoplankton[END_REF][START_REF] Vanderploeg | Feeding rate of Diaptomus sicilis and its relation to selectivity and effective food concentration in algal mixtures and in Lake Michigan[END_REF][START_REF] Berggreen | Food size spectra, ingestion and growth of the copepod Acartia tonsa during development: Implications for determination of copepod production[END_REF]. The study of feeding as a function of particle size has provided the chance to make comparisons among species regarding important aspects of ecology (i.e., the lower, optimum and higher limit in food size).

The prey items selected for the present study had a 4-28 µm ESD size range. Although this is rather restricted when compared to other studies (e.g., Vanderploeg et al., 1984: natural assemblage;Berggreen et al., 1998: ESD= 1.90 to 247 µm), this choice actually encompasses the majority of food items abundant in oligotrophic environments. For instance, the particle range 10-20 µm is considered to represent a major component of the material ingested by C. furcatus in the subtropical open ocean due to a combination of high clearance rates and particle concentration in this size range [START_REF] Paffenhöfer | Living on the edge: Feeding of subtropical open ocean copepods[END_REF]. In our study, the flagellates Isochrysis sp., located at the lower end (ca. 4 µm) of the food size spectrum offered, were not significantly consumed and represent the lower threshold in the particle capture efficiency of C. lividus. Copepods are not passive filter feeders [START_REF] Koehl | Copepod feeding currents: food capture at low Reynolds numbers[END_REF][START_REF] Paffenhöfer | Suspension-feeding by herbivorous calanoid copepods: A cinematographic study[END_REF][START_REF] Price | Modes of cell capture in calanoid copepods[END_REF], as their feeding appendages work like paddles but also as leaky sieves under certain circumstances (see [START_REF] Koehl | Biomechanics of microscopic appendages: Functional shifts caused by changes in speed[END_REF]. In this regard our finding fits well with the 15% retention of particles ≤5 µm calculated by [START_REF] Boyd | Selection of particles by filter-feeding copepods: a plea for reason[END_REF] for C. arcuicornis based on the intersetular distance measurements made by [START_REF] Nival | Efficacite de filtration des copkpodes planctoniques[END_REF].

Unfortunately the in situ feeding measurements on Clausocalanus species (i.e., C. furcatus, C. farrani) did not provide any information on this topic, because inefficient feeding on particles < 5 or 6 µm had been considered a priori [START_REF] Paffenhöfer | Living on the edge: Feeding of subtropical open ocean copepods[END_REF]Cornils et al., 2007b). Similar or even lower values of the lower size limit have been reported for adult females of other copepod species e.g., ca. 2 µm for Acartia tonsa (Berggreen et al., 1998;[START_REF] Bartram | Experimental development of a model for the feeding of neritic copepods of phytoplankton[END_REF], 1.5 µm for Paracalanus parvus [START_REF] Bartram | Experimental development of a model for the feeding of neritic copepods of phytoplankton[END_REF], and 3 to 4 µm for Diaptomus sicilis females [START_REF] Vanderploeg | Feeding rate of Diaptomus sicilis and its relation to selectivity and effective food concentration in algal mixtures and in Lake Michigan[END_REF]. In this regard, it is quite puzzling to understand how Clausocalanus, which is reportedly unable to create feeding currents and behaves as a fast-swimming intercept feeder [START_REF] Mazzocchi | Swimming and feeding behaviour of the planktonic copepod Clausocalanus furcatus[END_REF]Uttieri et al., 2008a), can manage to capture relatively small prey (<15 µm ESD: R. salina, T. weissflogii, Heterocapsa sp.) at rates comparable to those exhibited by feeding-current copepods (see review in [START_REF] Saiz | Scaling of feeding in marine calanoid copepods[END_REF]. Further studies using high-speed video will be required to provide better insights into the particular feeding mechanisms of Clausocalanus.

In contrast to the small cell Isochrysis sp., all of the larger prey items offered (ESD>5 µm)

were consumed by C. lividus and cleared at rates proportional to the prey size. Hence, the maximum clearance was lower on Rhodomonas salina (ESD= 7 µm), increased successively with prey size and was found to be highest for the larger-sized cell, the ciliate S. sulcatum (ESD= 28 µm). The prey size spectra for the other copepod species, resembling either increasing [START_REF] Frost | Feeding behaviour of Calanus pacificus in mixtures of food particles[END_REF][START_REF] Bartram | Experimental development of a model for the feeding of neritic copepods of phytoplankton[END_REF] or bell-shaped curves [START_REF] Vanderploeg | Feeding rate of Diaptomus sicilis and its relation to selectivity and effective food concentration in algal mixtures and in Lake Michigan[END_REF]Berggreen et al., 1998), showed similar size-dependent clearance patterns, which can be explained by an increase in realised encounter rate due to a higher percentage of individual cell detections with size [START_REF] Price | Perception of food availability by calanoid copepods[END_REF] and to a prey size-dependent capture efficiency.

Nevertheless, a discrepancy to the above-mentioned pattern was also observed in our study due to the feeding performance of C. lividus on the diatom T. weissflogii. The clearance rates on this food species showed a three-fold increase compared to the similarly sized autotrophic dinoflagellate Heterocapsa sp. Additionally, C. lividus cleared the diatoms at rates 2 and 1.3 times higher than the larger dinoflagellates Gyrodinium sp. and O. marina respectively. Although the estimated clearance values on this particular prey are realistic in the overall context of copepod feeding rates, this finding has to be treated cautiously because this particular observation seems to challenge not only the "anticipated" influence of size on copepod feeding but also an anticipated positive stimulus of prey motility on the feeding procedure [START_REF] Paffenhöfer | Feeding rates and behavior of zooplankton[END_REF]. A similar deviation from what is expected on the basis of size was observed for females of A. tonsa, which cleared the dinoflagellate Scrippsiella faröense less efficiently than expected (Berggreen et al., 1998). This result was attributed to copepod "taste preferences", implying a rejective behaviour to chemical substances excreted by dinoflagellates (e.g., [START_REF] Huntley | Chemically mediated rejection of dinoflagellate prey by the copepods Calanus pacificus and Paracalanus parvus: mechanisms, occurrence and significance[END_REF]. Similarly, the nutritional characteristics or chemical properties of the diatom may make it more "preferable" prey item, but this speculation deserves further study.

Although the series of experiments described here allows conclusions related to the lower prey size limit of C. lividus, as well as the relationship between F max vs. prey size, unfortunately the (Cornils et al., 2007b). These authors concluded that both species mainly feed on small-sized food items between 5 and 20 µm in terms of abundance, whereas the main carbon uptake was observed in the size classes larger than 20 µm.

We must also take into account that apart from the cell size, the food items we utilised varied in terms of nutritional mode (autotrophs vs. heterotrophs), motility (motile vs. immotile) and phylogeny (flagellates, dinoflagellates, diatoms, ciliates), and it is not only cell dimensions that may have influenced the feeding performance of C. lividus in our experiments. For instance, we cannot exclude the possibility that the high clearance rates on the ciliate S. sulcatum could be a combined function of a size and taxon selectivity of C. lividus. Ciliates form an important component in the diet of copepods [START_REF] Calbet | The ciliate-copepod link in marine ecosystems[END_REF] and represent a selected prey for a variety of copepod species [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]. Taxon-related selectivity in the feeding of the genus Clausocalanus was examined during in situ feeding studies of congeners (C. furcatus and C. farrani in Cornils et al., 2007b;Clausocalanus spp. in Broglio et al., 2004), but the responses were rather contradictory.

Incubation experiments coupled with fatty acid and stable isotope analysis, were employed by Cornils et al. (2007b) to study this phenomenon, and both methods revealed no apparent selectivity for the major taxa (e.g. diatoms, ciliates, dinoflagellates) comprising the natural particle assemblage. This overall finding was interpreted as a reflection of the strategy adopted by the species in the very oligotrophic waters of the Red Sea (Cornils et al., 2007b). However, [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] observed a clear selection for ciliates by individuals of the genus in the NW Mediterranean, in accordance with what has been shown for many other copepod species in their and other studies (e.g., Atkinson, 1996;[START_REF] Batten | The contribution of microzooplankton to the diet of mesozooplankton in an upwelling filament off the north west coast of Spain[END_REF][START_REF] Calbet | The ciliate-copepod link in marine ecosystems[END_REF]. Similarly, gut pigment analysis in Clausocalanus sp. also revealed high carotenoid concentrations of animal and dinoflagellate origin [START_REF] Kleppel | Natural diets of zooplankton off southern California[END_REF].

Although the present study provided preliminary information with respect to the feeding processes of C. lividus, an in-depth understanding of these processes would be advanced by the Information on body mass (B. M.), temperature (Temp), food type (size fraction of natural assemblage or species in the single diets) and food concentration (F. C.) have also been provided. rates (mL cop -1 day -1 ) and ingestion rates (cells cop -1 day -1 ) are presented as a function of the average food concentration (cells mL -1 ). Parameters of the fitted models are provided in Table II.

Error bars indicate the standard errors of the mean values. day -1 (A) and carbon-specific ingestion rates (% of body carbon day -1 ) (B) are presented in relation to the mean food concentration (µg C L -1 ). The curves correspond to the fitting of Ivlev's model to the data (estimated parameters for each prey item are provided in Table II). Horizontal dashed lines correspond to the required ingestion rates to satisfy the metabolic demands of C. lividus (assuming 90% assimilation efficiency), both under starved (12.8%) and feeding conditions (29.4%).

F o r P e e r R e v i e w

Table II. Maximum ingestion (I max ) and clearance (F max ) rates estimated by the Ivlev model [I=I max *(1-exp -αC )] and the exponential decay equation [F=F max *e bC ], respectively. Parameters a and b of the equations are also given.

Clearance rates

Ingestion rates mL -1 cop -1 day -1 cells cop -1 day -1 µg C cop [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] 2 : NW Spain [START_REF] Batten | The contribution of microzooplankton to the diet of mesozooplankton in an upwelling filament off the north west coast of Spain[END_REF] 3 : Gulf of Aqaba: averaged data of four and seven experiments with C. furcatus and C. farrani respectively (Cornils et al., 2007) 4 : subtropical Atlantic Ocean: averaged data of six experiments in subtropical [START_REF] Paffenhöfer | Living on the edge: Feeding of subtropical open ocean copepods[END_REF] 5 : S.E. USA [START_REF] Mazzocchi | First observations on the biology of Clausocalanus furcatus (Copepoda, Calanoida)[END_REF] 6 : Gulf of Naples: values correspond to vegetative forms that have been approximately (≈) extracted from their Fig. 1 [START_REF] Montresor | Viability of dinoflagellate cysts after the passage through the copepod gut[END_REF] 7 : NW Mediterranean (present study; * : range of body carbon in our experiments; n.s.: not significantly Comparative presentation of the carbon intake of C. lividus (♀) on prey of equivalent spherical diameter (ESD) smaller and larger than 15 µm. Ingestion rates expressed as µg C cop -1 day -1 (A) and carbon-specific ingestion rates (% of body carbon day -1 ) (B) are presented in relation to the mean food concentration (µg C L -1 ). The curves correspond to the fitting of Ivlev's model to the data (estimated parameters for each prey item are provided in Table II). Horizontal dashed lines correspond to the required ingestion rates to satisfy the metabolic demands of C. lividus (assuming 90% assimilation efficiency), both under starved (12.8%) and feeding conditions (29.4%). 192x175mm (600 x 600 DPI)

  experimentation and have discouraged in-depth, species-specific ecophysiological studies.

  feeding response of C. lividus on the different food particles as a function of resource density is presented in Fig. 1. Two-way ANOVA tests showed significant consumption in the experimental bottles for all of the tested prey (P<0.01), except for the autotrophic flagellates Isochrysis sp. (ESD<5 µm), the smallest item offered. For this reason we only computed clearance and ingestion rates for the other prey items tested.

  C. lividus: effect of resource densityThe available feeding rates reported for the genus in the literature, which were based on few observations and primarily focused on C. furcatus, were estimated mainly via bottle incubation experiments (but also see[START_REF] Mazzocchi | Swimming and feeding behaviour of the planktonic copepod Clausocalanus furcatus[END_REF] using either the natural prey assemblage of oligotrophic environments (NW Spain:[START_REF] Batten | The contribution of microzooplankton to the diet of mesozooplankton in an upwelling filament off the north west coast of Spain[END_REF] NW Mediterranean: Broglio et al., 2004; subtropical ocean: Paffenhöfer et al., 2006; Gulf of Aqaba: Cornils et al.,

Clausocalanus

  feeding rates on single nutritional resources have previously been quantified by supplying dinoflagellates or flagellates at concentrations resembling oligotrophic field conditions: (a) Gymnodinium nelsoni and Rhodomonas sp. to individuals of C. furcatus and copepodites of Clausocalanus (Mazzocchi and Paffenhöfer, 1998) and (b) Scrippsiella trochoideaand Scrippsiella ramonii to C. lividus[START_REF] Montresor | Viability of dinoflagellate cysts after the passage through the copepod gut[END_REF]. In the current study, when C. lividus was offered three dinoflagellate species (Heterocapsa sp., Gyrodinium sp. and Oxyrrhis marina) at carbon concentrations within the range employed by[START_REF] Mazzocchi | First observations on the biology of Clausocalanus furcatus (Copepoda, Calanoida)[END_REF]

  tested (28 µm ESD) did not allow us to estimate the upper limit of the prey size range. Reviewing their own data on Acartia tonsa and published studies on Calanus pacificus and Diaptomus sicilis,Berggreen et al. (1998) suggested an optimum particle size for copepods equal to ca. 2-5% their body length. Taking into account the average prosome length of C. lividus found in our experiments (1.16 mm), the optimum particle range in our case would be 23-58 µm. Thus, among the prey examined, only the ciliate S. sulcatum (ESD 28 µm) might fall within the optimum particle range. Regarding the other Clausocalanus congeners, the feeding selectivity in terms of size has been recently investigated for females of the species C. furcatus and C. farrani in the very oligotrophic waters of Red Sea

Fig. 1 :

 1 Fig. 1: Functional feeding response of C. lividus (♀) on six different-sized food items. Clearance

Fig. 2 :

 2 Fig. 2: Maximum clearance rates (A) and daily ration (B) of C. lividus, as well as the C Imax/2 (concentration at which ingestion equals half the maximum rate), plotted against the equivalent spherical diameter (ESD) of the different prey items (Iso: Isochrysis sp., Rho: Rhodomonas salina, Hete: Heterocapsa sp., Thala: Thalassiosira weissflogii, Gyro: Gyrodinium sp., Oxy: Oxyrrhis marina, Stro: Strombidium sulcatum).

Fig. 3 :

 3 Fig. 3: Comparative presentation of the carbon intake of C. lividus (♀) on prey of equivalent spherical diameter (ESD) smaller and larger than 15 µm. Ingestion rates expressed as µg C cop -1

  Functional feeding response of C. lividus (♀) on six different-sized food items. Clearance rates (mL cop -1 day -1 ) and ingestion rates (cells cop -1 day -1 ) are presented as a function of the average food concentration (cells mL -1 ). Parameters of the fitted models are provided in TableII. Error bars indicate the standard errors of the mean values. 213x249mm (600 x 600 DPI) Maximum clearance rates (A) and daily ration (B) of C. lividus, as well as the CImax/2 (concentration at which ingestion equals half the maximum rate), plotted against the equivalent spherical diameter (ESD) of the different prey items (Iso: Isochrysis sp., Rho: Rhodomonas salina, Hete: Heterocapsa sp., Thala: Thalassiosira weissflogii , Gyro: Gyrodinium sp., Oxy: Oxyrrhis marina , Stro: Strombidium sulcatum ). 93x219mm (600 x 600 DPI)

  observation of free-swimming animals in different trophic regimes in terms of prey size and concentration. Future studies should include the performance of selective feeding experiments in Webber, M. K. and Roff J. C. (1995) Annual biomass and production of the oceanic copepod community off Discovery Bay, Jamaica. Mar. Biol., 123, 481--495. Williams, R. and Wallace M. A. (1975) Continuous Plankton Records: a plankton Atlas of the North Atlantic and North Sea: supplement 1 -The genus Clausocalanus (Crustacea:

	which certain food types at varying concentrations are offered concurrently, as well as observations
	of the behavioural responses of both predator and prey.
	Copepoda, Calanoida) in 1965. Bull. Mar. Ecol., 8, 167--184.
	Zervoudaki, S., Christou, E. D., Nielsen, T. G., Siokou-Frangou, I., Assimakopoulou, G.,
	Giannakourou, A., Maar, M., Pagou, K., Krasakopoulou, E., Christaki, U. and Moraitou-
	Apostolopoulou, M. (2007) The importance of small-sized copepods in a frontal area of the
	Aegean Sea. J. Plankton Res., 29, 317--338.
	F o r F o r F o r F o r F o r F o r
	P P P P P P
	e e e e e e
	e r e r e r e r e r e r
	R R R R R R
	e e e e e e
	v i e v i e v i e v i e v i e v i e
	w w w w w w

TABLE AND FIGURE LEGENDS Table I :

 ANDLEGENDSI Overview of the experiments conducted. Equivalent spherical diameter (ESD) was estimated in live cells with a Beckman Coulter Multisizer III. Prey carbon contents were estimated by (a) application of conversion factors found in the literature, (b) through CHN analysis carried out either within the present study or during previous experimental studies using the same strains and growing conditions. Date Samp : sampling date, Date Exp : date of experiment, T: sea surface temperature.

Table II :

 II Maximum ingestion (I max ) and clearance (F max ) rates estimated by the Ivlev model [I=I max *(1-exp -aC ) ] and the exponential decay equation (F=F max *e -bC ), respectively. Parameters a and b of the equations are also given.

Table III :

 III Feeding rates of the genus Clausocalanus on natural field assemblages or monospecific

diets compiled from the literature (C:ciliate; D: dinoflagellate; Dia: diatom; F: flagellate).

Table III .

 III Feeding rates of the genus Clausocalanus on natural field assemblages or monospecific diets compiled from the literature (CIL: ciliate; DIN: dinoflagellate; DIA: diatom; F: flagellate; HNF: heterotrophic nanoflagellates). Information on copepod body mass (B.M.), experimental temperature (Temp), food type (size fraction of natural assemblage or species in the single diets) and food concentration (F. C.) have also been provided.

	Taxon	B.M.	Temp	Food type	F. C.	Clearance rate	Ingestion rate	%BC d -1
		(µg C cop -1 )	( o C)		(µg C L -1 )	(mL -1 cop -1 day -1 )	(µg C cop -1 day -1 )
				Natural assemblage
	Clausocalanus spp. 1	2.26; 1.40 21.8; 23.1 total particles (autotrophic + ciliates)	10.00; 38.00	-		0.12; 0.48	5.18; 34.64
	Clausocalanus spp. 2	-	18 CIL: 16-20 µm; 21-30 µm	0.92 (16-30 µm)	36; 74.4		-	-
				DIN: 16-20 µm; 21-30 µm; 31-45 µm HNF: 2-5 µm; 5-20 µm	0.32(Din+HNF)	49.92; 38.16; 35.76 -		-≈5.38; 1.89	--
	♀ C. farrani 3 Isochrysis sp. Food type Rhodomonas salina Heterocapsa sp. Thalassiosira weissflogii 4.20 Gyrodinium sp. Oxyrrhis marina * Strombidium sulcatum ♀ C. furcatus 3 3.69 ♀ C. furcatus 4 2.74 Clausocalanus spp. 5 -" " ♀ C. furcatus 5 3.60 ♀ C. lividus 6 -" " ♀ C. lividus 7 9.7-19.9 * " " " " " " " " " " " " 1 : NW Mediterranean	-1 day -1 134.00 I max a (x10 -3 ) No model was fitted 9.35 1.16 9.29 5.49 17.12 1.19 12.59 4.43 14.15 7.64 13.78 13.58 50.00 56; 71; 114; 218 9.60 29.04; 31.68 29.00; 47.00 --48.27 80.78 115.50 n.s. consumed 10.40 128.81 323.22 F o r P e e r R e v i e w 21-25.5 particles >5 µm 1.58 F max b (x10 -3 ) r 2 I max a (x10 -3 ) r 2 No model was fitted No model was fitted 10.49 0.02 0.86 169960 0.06 0.99 52.55 0.77 0.99 32298 1.58 0.99 166.41 1.72 0.99 77627 0.26 0.77 77.197 1.48 0.88 30255 1.84 0.99 120.43 1.74 0.97 29412 3.67 0.84 307.84 24.39 0.77 8112 23.08 0.99 21-24 particles >5 µm 1.41 23 6-8 µm; 8-10 µm; 10-20 µm; 20-40 µm 1.07; 0.93; 3.11; 1.18 Monospecific diets 20 F Rhodomonas sp. 29.67 " DIN Gymnodinium nelsoni 17.42; 29.03 20 " " 12.90; 25.80 20 " Scrippsiella trochoidea 55.95 " " Scrippsiella ramonii 56.65 16 " Heterocapsa sp. 42.58 " " Gyrodinium sp. 15.82 " " Oxyrrhis marina 19.76 " F Isochrysis sp. 48.04 " " Rhodomonas salina 93.06 " DIA Thalassiosira weissflogii 35.50 " CIL Strombidium sulcatum 2.72	0.48 % body carbon day -1 13.00 I max a (x10 -3 ) No model was fitted 54 1.16 54 5.49 137 1.19 125 4.43 71 7.64 76 13.58 0.01 1.94 --0.28 -0.51; 0.92 -0.37; 1.21 10.30; 33.60 ≈2.93 -≈1.58 -1.75 10.08 0.90 8.99 3.28 16.43 n.s. consumed r 2 0.99 0.99 0.77 0.99 0.84 0.99 n.s. consumed 1.13 6.50 4.15 33.35 0.36 2.01	r 2 0.99 0.99 0.77 0.99 0.84 0.99

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ACKNOWLEDGMENTS

We acknowledge Dr. Miguel Alcaraz for assistance with the CHN analysis, Meritxell Antó, Patricia Jiménez, Isaac Cereijo, Jordi Felipe, Sara Zamora and Dr. Albert Calbet for assistance and discussion and the captain Jordi Carol for assistance at sea. This work was supported by the projects OITHONA (CTM2007-60052) and PERFIL (CTM2006-12344-C02-01) from the Spanish Ministry of Education and Science and by the postdoctoral grant "Modalidad B: Estancias de jóvenes doctores extranjeros en centros de investigación españoles" to S.I (grant no. SB2009-0020). Thanks also go to a Leonardo da Vinci Training fellowship that partially funded the work of S.I. and to the two anonymous reviewers for their useful comments on the manuscript.