Marc Metian
email: metian@hawaii.edu

Michel Warnau

Jean-Louis Teyssié

Paco Bustamante
email: paco.bustamante@univ-lr.fr

Prof Paco Bustamante

Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways

Keywords: bivalve, scallop, radionuclide, bioaccumulation, uptake, depuration

teaching and research institutions in France or abroad, or from public or private research centers.   Characterization of 241Am and 134Cs bioaccumulation in the king scallop Pecten maximus: investigation via three exposure pathways

Introduction

During the last sixty years, human activities have resulted in various degrees of contamination of the world seas and oceans with anthropogenic radionuclides (Frielander et al. 2005).

Although this contamination tends to decrease (e.g. [START_REF] Toshimichi | Temporal and spatial variations of anthropogenic radionuclides in Japan Sea waters[END_REF], it is still a major concern in coastal areas receiving radioactive inputs mainly from industries, nuclear accidents and fallout from nuclear weapon testing and use. Consequently, monitoring programs were established worldwide to monitor the levels of those radionuclides in the marine environment [START_REF] Nielsen | Environmental radionuclide monitoring programme[END_REF]. Generally, surveys are based on the analysis of seawater, sediments and biological samples (e.g., [START_REF] Fegan | Radioactivity monitoring of the Irish environment[END_REF]. Overall, biomonitoring programs present the advantages 1) to reveal the bioavailability of the considered contaminants and 2) to magnify their levels above the analytical detection limits.

The use of marine organisms for monitoring radionuclide contamination is well established [START_REF] Valette-Silver | Radionuclide concentrations in bivalves collected along the coastal United States[END_REF][START_REF] Burger | Radionuclide levels in benthic invertebrates from Amchitka and Kiska Islands in the Aleutian Chain, Alaska[END_REF][START_REF] Thébault | 137 Cs Baseline levels in the Mediterranean and Black Sea: a cross-basin survey of the CIESM Mediterranean Mussel Watch Programme[END_REF]. In order to understand field measurements, the characterization of bioaccumulation parameters and/or the relative importance of the different exposure pathways has been carried out for several radionuclides [START_REF] Wang | Modeling radiocesium bioaccumulation in a marine food chain[END_REF][START_REF] Wang | Modeling radiocesium bioaccumulation in a marine food chain[END_REF][START_REF] Baines | Influence of temperature on dietary metal uptake in Arctic and temperate mussels[END_REF][START_REF] Borretzen | Bioavailability of sediment-associated and low-molecularmass species of radionuclides/trace metals to the mussel Mytilus edulis[END_REF]. Beside mussels, other bivalve species are used to a lesser extent in biomonitoring programs among which scallops appear of great interest as they highly accumulate trace elements from their environment [START_REF] Bryan | The occurence and seasonal variation of trace metals in the scallops Pecten maximus (L.) and Chlamys opercularis (L.)[END_REF][START_REF] Bustamante | Subcellular and body distributions of 17 trace elements in the variegated scallop Chlamys varia from the Charente-Maritime coast (Bay of Biscay, France)[END_REF], Metian et al. 2008a, 2009a[START_REF] Pan | Validation of biokinetic model of metals in the scallop Chlamys nobilis in complex field environments[END_REF]. Scallops have also been reported to efficiently concentrate natural and anthropogenic radionuclides such as 241 Am, 137 Cs, 210 Po, 238 Pu, 239 Pu and 90 Sr in their tissues [START_REF] Miramand | Histo-autoradiographic localization of americium in the tissues of the scallop Pecten maximus labelled from sea water: preliminary study on the transfer mechanisms[END_REF][START_REF] Nonnis Marzano | Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance[END_REF][START_REF] Bustamante | Concentration and distribution of 210 Po in the tissues of the scallop Chlamys varia and the mussel Mytilus edulis from the coasts of Charente-Maritime (France)[END_REF]. As for some metals [START_REF] Brooks | The biogeochemistry of trace element uptake by some New Zealand bivalves[END_REF][START_REF] Chouvelon | Hg concentrations and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF][START_REF] Bustamante | Interspecific and geographical variations of trace element concentrations in Pectinidae from European waters[END_REF], scallops sometimes display higher bioaccumulation capacity for 137 Cs than other filter-feeders such as oysters and mussels occurring in the same areas (JCAC 2002). Within the Pectinid family, the current knowledge on concentrations of anthropogenic radionuclides is limited to field measurements [START_REF] Bustamante | Concentration and distribution of 210 Po in the tissues of the scallop Chlamys varia and the mussel Mytilus edulis from the coasts of Charente-Maritime (France)[END_REF][START_REF] Nonnis Marzano | Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance[END_REF][START_REF] Miramand | Experimental and in situ data (Goury, North Cotentin, France) on the concentration and distribution of Americium ( 241 Am) in the tissues of scallop Pecten maximus[END_REF], and to waterborne exposure experiment to Am (Miramand andGermain 1986, Miramand et al. 1991). The importance of other contamination pathways in the bioaccumulation process of Am and Cs in scallops is not known although sediment and food have been considered as possible source of Am and Cs following the analyses carried out on scallops from the field or after laboratory studies (Miramand andGermain 1986, Nonnis Marzano et al. 2000) and by histo-autoradiography approach in laboratory [START_REF] Miramand | Histo-autoradiographic localization of americium in the tissues of the scallop Pecten maximus labelled from sea water: preliminary study on the transfer mechanisms[END_REF]. Overall, sediment is considered as a major vector of transuranic elements, among which Am, to biota [START_REF] Miramand | Uptake of americium and plutonium from contaminated sédiments by three benthic species: Arenicola marina, Corophium volutator and Scrobicularia plana[END_REF][START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF][START_REF] Ryan | Transuranic biokinetic parameters for marine invertebrates-a review[END_REF]. In comparison, Cs transfer from sediments appears relatively limited [START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF][START_REF] Borretzen | Bioavailability of sediment-associated and low-molecularmass species of radionuclides/trace metals to the mussel Mytilus edulis[END_REF]. Recently, food has been demonstrated as a major pathway for metal bioaccumulation in scallops (Metian et al. 2009ab). Therefore, it appears necessary to determine experimentally the bioaccumulation of radionuclides in scallops via seawater, food and sediments in order to better understand the relative contribution of these three pathways of exposure.

The aim of this study was thus to determine the kinetics of uptake and depuration of 241 Am and 134 Cs in a typical pectinid from European waters, the king scallop Pecten maximus, following its exposure to radiolabelled seawater, food or sediment. The radionuclides were selected for their contrasting characteristics in seawater (particle-reactive Am and soluble Cs).

There is a particular interest to study this transuranian and the radiocesium in P. maximus since the geographic distribution of this species (European North-Atlantic coasts) coincides with the area subject to direct inputs from the nuclear retreatment plants of Dounreay (Scotland -facility closed in 1996), La Hague (France) and Sellafield (England), which affect the Norway where Pecten maximus is cultured [START_REF] Bergh | Great scallop, Pecten maximus, research and culture strategies in Norway: a review[END_REF].

Materials and Methods

Sampling

In spring 2004 and 2005, seventy king scallops P. maximus were collected on the French Atlantic coast (Pertuis Breton, Charente-Maritime) by SCUBA diving. They were carefully transported to IAEA-MEL premises in Monaco and were acclimated to laboratory conditions for 4 weeks (constantly aerated, open-circuit 800l aquarium; flux: 50 l h -1 ; salinity: 36 p.s.u.; temperature: 17 ± 0.5 °C; pH: 8.0 ± 0.1; light/dark cycle: 12 h/12 h) prior to experimentations.

During this period, scallops were fed daily an algal mixed diet (Isochrysis galbana, Skeletonema costatum).

Radiotracer and counting

Uptake and depuration kinetics of 241 Am and 134 Cs in scallop were determined using highspecific activity radiotracers purchased from Isotope Product Lab ( 241 Am nitrate -0.1 N-, T ½ = 433 years; 134 Cs chloride -0.1 N-, T ½ = 2 years). Tracers were counted using a high-resolution γ-spectrometer system composed of four Germanium (N-or P-type) detectors (EGNC 33-195-R, Canberra® and Eurysis ® ) connected to a multi-channel analyzer and a computer equipped with a spectra analysis software (Interwinner ® 6). The radioactivity was determined by comparison with standards of known activity and of appropriate geometry. Measurements were corrected for counting efficiency and physical radioactive decay. The counting time was adjusted to obtain a propagated counting error less than 5% (Rodriguez y Baena et al. 2006).

Seawater exposure

Twenty five P. maximus (average weight ± SD: 208 ± 46 g) were placed in a 70-l glass aquarium (constantly aerated, closed-circuit aquarium; salinity: 36 p.s.u.; temperature: 17 ± 0.5 °C; pH: 8.0 ± 0.1; light/dark cycle: 12 h/12 h) and simultaneously exposed for 7d to 241 Am and to 134 Cs dissolved in 0.45 µm filtrated seawater (0.3 and 1.4 kBq l -1 , respectively). No change in pH was detectable after the tracer addition. Spiked seawater was renewed twice a day during the first two days and then daily in order to keep radioactivity in seawater constant. Activity of the 241 Am and 134 Cs in seawater was checked before and after each spike renewal, yielding time-integrated activities of 0.13 ± 0.09 kBq l -1 and 1.23 ± 0.03 kBq l -1 ( Nine tag-identified scallops were collected at different time intervals and were whole-body radioanalyzed alive (same identified individual each time). At the end of the 7d exposure period, 5 scallops among the 25 were sacrificed and dissected. Shell, digestive gland, kidneys, gills, gonad, mantle, intestine, adductor muscle and the remaining soft tissues were separated and radioanalyzed in order to assess the radionuclide body distribution. The remaining scallops were then placed in non contaminating conditions (constantly aerated, open-circuit aquarium; flux: 50 l h -1 ; salinity: 36 p.s.u.; temperature: 17 ± 0.5 °C; pH: 8.0 ± 0.1; light/dark cycle: 12 h/12 h) for 36d and the nine tag-identified individuals were regularly radioanalyzed alive in order to follow the depuration of 241 Am and 134 Cs from the scallops. Three nonexposed individuals were introduced in the aquarium in order to control possible tracer recycling from the contaminated scallops. During the 36d depuration period, scallops were fed daily with Skeletonema costatum and Isochrisis galbana (5.10 4 cells ml -1 ). At the end of the depuration period, four contaminated scallops were collected and dissected into several body compartments as previously described.

Food exposure

The Bacillariophyceae Skeletonema costatum was used to study the transfer of 241 Am and 134 Cs to scallops through their diet. Phytoplankton cells were exposed to 4.5 kBq 241 Am l -1 and 7 kBq 134 Cs l -1 during their exponential growing phase (10d). After that period, phytoplankton medium was filtrated (1 µm-mesh size; Osmonic filters), and the phytoplankton cells resuspended in a 70-l aquarium (constantly aerated, closed-circuit aquarium; salinity: 36 p.s.u.; temperature: 17 ± 0.5 °C; pH: 8.0 ± 0.1; light/dark cycle: 12 h/12 h) at a cell concentration of 5 10 4 cell ml -1 to avoid pseudofeces production by the scallops. Nine P. maximus (average weight ± SD: 199 ± 32 g) had been placed in the aquarium for one week before the feeding experiment. Scallops were then allowed to feed on radiolabelled S. costatum for 2h. After the feeding period, all scallops were γ-counted and flowing seawater conditions (50 l h -1 ) were restored in the aquarium. Individuals were then whole-body γcounted alive at different time intervals to follow the depuration kinetics of both elements.

Three non-exposed individuals were introduced in the aquarium in order to control possible tracer recycling from the contaminated scallops. During the 21d depuration period, scallops were fed daily with Skeletonema costatum and Isochrisis galbana (5.10 4 cells ml -1 ). Four contaminated individuals were randomly collected after 21d and dissected to determine the radionuclide distribution among the different body compartments (shell, digestive gland, kidneys, gills, gonad, mantle, intestine, adductor muscle and the remaining soft tissues).

Radiolabelled S. costatum did not allow contaminating significantly the scallops with 134 Cs. Therefore, another phytoplankton species was used to study the trophic transfer of this radionuclide. To this end, the Haptophyceae Isochrisis galbana was used following the same method as previously described for S. costatum except that phytoplankton cells were exposed to 134 Cs during 7d (growing phase of I. galbana). Six P. maximus (average weight ± SD: 127 ± 14 g) were exposed and whole-body γ-counted alive at different time of the depuration experiment (16d). Four individuals were dissected at the end of the depuration period to determine the 134 Cs distribution among the different body compartments (same as described above).

Sediment exposure

Since P. maximus lives buried into the sediment, 241 Am and 134 Cs exposure through sediment was assayed. Sediment was collected in Wimereux (North-Atlantic coast of France).

Sediment grain size distribution was measured on a Mastersizer micro and the evaluation of the dry/wet weight ratio was calculated after freeze drying in a LABCONCO Freezone18. Aerated sediment (9 kg) was placed in a plastic container, spiked with 241 Am (8 kBq) and 134 Cs (13 kBq) for 6d with constant agitation, then used to form a homogeneous sediment layer of 4 cm height in a 20-l aquarium. Weakly bound radioisotopes were allowed to leach overnight under flowing seawater (50 l h -1 ). Ten P. maximus (average weight ± SD: 118 ± 5 g) were then placed for 13d in the aquarium (open circuit; parameters as previously described), and six tag-identified individuals were regularly whole-body radioanalyzed alive.

Sediment samples were also regularly collected and γ-counted to verify that the radiotracer activities in sediment remained constant. Activity of 241 Am and 134 Cs in sediment was constant all along the exposure period (8.2 ± 0.8 and 13.1 ± 3.0 Bq g -1 wet wt, respectively).

At the end of the exposure period, four scallops were collected, dissected (shell, digestive gland, kidneys, gills, gonad, mantle, intestine, adductor muscle and the remaining soft tissues), weighed and radioanalyzed to determine the radionuclide body distribution. The remaining six scallops were placed in non-contaminating depuration conditions for 31d (new 20l glass aquarium with clean sediment under flowing seawater, 50 l h -1 , daily feeding on Skeletonema costatum and Isochrysis galbana at 5 10 4 cells ml -1 ), and regularly γ-counted.

The radioactivity in sediment was regularly checked in order to ensure that no tracer recycling occurred in the sediment. Although no radioactivity was detected, the whole sediment was renewed after 1 week. After 31d of depuration, four scallops were collected and dissected as described above to determine body distribution of 241 Am and 134 Cs.

Data analysis

Uptake of the radioisotope was expressed in term of concentration factors (CF: ratio between the radioisotope activity in scallops -Bq g -1 wet wt-and time integrated activity in the seawater -Bq g -1 -) over time for the seawater exposure and in term of transfer factors (TF: ratio between the radioisotopes activity in scallops -Bq g -1 wet wt-and time-integrated activity in the sediment -Bq g -1 wet wt-) over time for the sediment exposure of P. maximus. Uptake kinetics of 241 Am and 134 Cs in whole-body scallops were fitted (Statistica® 6) using a simple exponential kinetic model (Eq. ( 1)) or using a linear model (Eq. ( 2)): CF t = CF ss (1 -e -k e t ) (Eq. 1)

CF t = k u t (Eq. 2)
where CF t and CF ss (CF ss = k u /k e ) are the concentration factors at time t (d) and at steady state, respectively; k u and k e are the uptake and depuration rate constants (d -1 ), respectively [START_REF] Whicker | Radioecology: Nuclear Energy and the Environment[END_REF].

Depuration of 241 Am and 134 Cs (seawater, food and sediment experiments) was expressed in terms of percentage of remaining radioactivity (radioactivity at time t divided by initial radioactivity measured in scallops at the beginning of the decontamination period*100). The percentages of remaining activity were plotted against time and depuration kinetics were described by a double-component exponential model (Eq. ( 3)):

A t = A 0s e -k es t + A 0l e -k el t (Eq. 3) where A t and A 0 are the remaining activities (%) at time t (d) and 0, respectively; k e is the depuration rate constant (d -1 ); 's' and 'l' are the subscripts for the short-lived' and 'long-lived' components. For each exponential component (s and l), a biological half-life can be calculated (T b½s and T b½l ) from the corresponding depuration rate constant (k es and k el , respectively) according to the relation T b½ = ln2/k e [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: sea water and food exposures[END_REF]. Regarding feeding experiments, the 'long-lived' exponential term describes the fraction of the radiotracer ingested with food that is actually absorbed by the organism [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: sea water and food exposures[END_REF]. The corresponding A 0l represents the assimilation efficiency (AE) of the considered radiotracer. The best fitting regression models were selected according to highest determination coefficient and examination of residuals. The level of significance for statistical analysis was always set at α = 0.05.

Results

No mortality of scallops was recorded neither during acclimatation period nor during the different experiments.

Seawater exposure

Uptake of 241 Am in whole-body P. maximus displayed linear kinetics (R 2 = 0.80; Fig. 1A,1B and Table 1) whereas the uptake of 134 Cs displayed exponential kinetics reaching a steady state (R 2 = 0.79). The values estimated for the kinetic parameters and their associated statistics are shown in Table 1. The concentration factors measured at the end of the exposure period (CF 7d ) of 241 Am and 134 Cs were 63 ± 18 and 1.0 ± 0.2 in whole body scallops, respectively. In the case of 134 Cs, the estimated steady-state CF calculated by the model (CF SS ) reached 0.94 ± 0.05 (Fig. 1B and Table 1).

Calculated CF 7d for the different compartments and organs are shown in Table 2. 241 Am is systematically more concentrated than 134 Cs when considering the same compartment of the scallops (up to 2 orders of magnitude). The shells of the scallops displayed higher capacities of 241 Am bioconcentration than their whole soft parts (CFs: 130 vs. 30) whereas the opposite was observed for 134 Cs (CFs: 1 vs. 3). Among the soft tissues, the digestive gland and the kidneys presented the highest CF of 241 Am and 134 Cs, respectively (Table 2).

At the end of the uptake experiment, the highest 241 Am load was in the shell (more than 90% of the total body load) and that of 134 Cs was in whole soft parts (more than 70% of the total load; Table 2). Among soft tissues, 241 Am was mainly contained in the mantle, digestive gland and gills (34, 23 and 18% of total body load, respectively; Table 2) whereas 134 Cs was mainly present in the adductor muscle and the mantle (41 and 24% of total body load, respectively; Table 2).

When non-contaminating conditions were restored, the whole-body depuration kinetics of both 241 Am and 134 Cs were best described by a two-component exponential model (Fig. 1C and Table 1). The major part of the bioaccumulated 241 Am was efficiently absorbed (A 0l : 78%) whereas only 45% of the bioaccumulated 134 Cs was absorbed in P. maximus. The estimated depuration rate constant of the long-lived components (k el ) for 134 Cs was 0.031 ± 0.004 d -1 and, consequently, the derived biological half-life reached 22 ± 3d (Table 1). In the case of 241 Am, the depuration rate constant was not significantly different from 0 (p<0.05), thus the corresponding T b½l may thus be considered as infinite. However, an estimation of T b½l based on the mean value of k el is shown in Table 1 (892d).

After 36d of depuration, the distribution of both radionuclides between the shell and the whole soft parts remained similar to the one observed at the end of the exposure period:

241 Am was mainly found in the shell and 134 Cs in the soft tissues (Table 2). Within soft tissues, radionuclide distribution displayed a different pattern than the one observed at the end of the exposure period (Table 2). Indeed, the digestive gland contained the major part of total 241 Am load (43%) while the adductor muscle was the main storing organ for 134 Cs (76%).

Nevertheless, the activities of both radionuclides in all the compartments of P. maximus decreased along the depuration phase (data not shown).

Dietary exposure

The depuration kinetics of the radionuclides ingested with food in whole body P. maximus were best fitted by a double exponential model (Fig. 2A and Table 1). 241 Am and 134 Cs displayed similar assimilation efficiencies (20%<AE<30%) and close depuration rate constants, k el , respectively 0.08 ± 0.02 and 0.05 ± 0.02, which give close T b½l (9 ± 2d and 14 ± 7d, respectively). At the end of the depuration period, 241 Am load was essentially in the shell (88% of the total body load) whereas most of the Cs was associated to the soft tissues; Table 2). Among the soft tissues, the digestive gland contained the main part of 241 Am (46%) whereas 134 Cs was mainly distributed between the digestive gland, the kidney, the mantle, the adductor muscle and the remaining tissues, with the digestive gland presenting the higher average load, i.e., 27% (Table 2).

Sediment exposure

Bioaccumulation of sediment-bound radionuclides was measured in P. maximus. However, their whole-body uptake kinetics could not be fitted by a model having a biological meaning (Fig. 2B and Table 3). At the end of the exposure period, the highest transfer factor (TF) measured in for whole scallops was 0.011 ± 0.003 for 241 Am and 0.005 ± 0.002 for 134 Cs.

Overall, the different body compartments displayed low TF 13d with elevated standard deviations (Table 3). Consequently, none of the tissues could be identified as the main bioaccumulation organ using TF. However, in terms of body distribution, the shell displayed the main part of both radionuclides (> 75%). Among soft tissues, the digestive gland and the adductor muscle contained the major part of 241 Am (47 ± 17%) and 134 Cs (41 ± 13%), respectively (Table 3).

The whole-body depuration kinetics of both 241 Am and 134 Cs after exposure to spiked sediment were best described by a two-compartment exponential equation (Fig. 2C and Table 1). The results indicated that 78% of 241 Am and 68% of 134 Cs previously bioaccumulated were efficiently retained, with biological half-lives of 79 ± 42d and 74 ± 49d, respectively. At the end of the 31d depuration period, both radionuclides were mainly associated to the shell (Table 3). The distribution of 241 Am showed that in soft tissues, the major part of the radionuclide was retained in the digestive gland with 49 ± 7% of the total 241 Am load.

Discussion

Biomonitoring programs using scallops (Class Bivalvia, family Pectinidae) already exist [START_REF] Fegan | Radioactivity monitoring of the Irish environment[END_REF], JCAC 2002). Interestingly, scallops show a high accumulation capacity for radionuclides [START_REF] Miramand | Histo-autoradiographic localization of americium in the tissues of the scallop Pecten maximus labelled from sea water: preliminary study on the transfer mechanisms[END_REF][START_REF] Nonnis Marzano | Anthropogenic radionuclides bioaccumulation in Antarctic marine fauna and its ecological relevance[END_REF][START_REF] Bustamante | Concentration and distribution of 210 Po in the tissues of the scallop Chlamys varia and the mussel Mytilus edulis from the coasts of Charente-Maritime (France)[END_REF] that can be accumulated to higher degrees than in other bivalve species such as mussels (JCAC 2002). However, little is known about the behavior of radionuclides in scallops and their mode of uptake in comparison to other bivalve families such as Ostreidae or Mytilidae [START_REF] Ryan | Transuranic biokinetic parameters for marine invertebrates-a review[END_REF]. To the best of our knowledge, experimental investigations were limited to the description of the uptake of waterborne 241 Am in P. maximus and to the localization of this transuranic element in the digestive gland cells (Miramand andGermain 1986, Miramand et al. 1991). Therefore, there is a lack of information on the uptake and retention of Am by scallops following other natural exposure pathways (i.e., food and sediment) although food and/or sediment were shown to constitute the main pathway of accumulation for Ag, Cd, Co, Pb, Zn [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF](Metian et al. , 2008b, 2009abc), 2009abc). Concerning Cs, no information on its bioaccumulation by scallops is currently available in the literature.

Using realistic activities of dissolved 241 Am (i.e., within the range of environmental levels), our study confirmed that 241 Am was efficiently accumulated in hard and soft tissues of the scallops, reaching a whole-body CF of 63 after 7d of exposure. This CF value is relatively high for bivalves. For example, CF were 10-30 after 5d of exposure in Mytilus edulis (Bjeregaard et al. 1985), 230 after 28d of exposure in Cerastoderma edule and 140 after 31d of exposure in Scrobicularia plana [START_REF] Miramand | Uptake of curium ( 244 Cm) by five benthic marine species (Arenicola marina, Cerastoderma edule, Corophium olatator, Nereis diversicolor and Scrobicularia plana): comparison with americium and plutonium[END_REF]. This CF was higher than in other marine invertebrates such as the sea urchin Paracentrotus lividus for comparable exposure time (i.e., approx. 30; [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: sea water and food exposures[END_REF]. For P. maximus, [START_REF] Miramand | Experimental and in situ data (Goury, North Cotentin, France) on the concentration and distribution of Americium ( 241 Am) in the tissues of scallop Pecten maximus[END_REF] showed a CF of 80 but after a longer exposure period to radiolabelled seawater (i.e., 38d).

Based on the linear model describing the uptake kinetics, the scallops from the present experiment should accumulate 241 Am up to a CF of 360 after 38d. This difference could be due to the size/weight difference of the studied organisms since the scallops of [START_REF] Miramand | Experimental and in situ data (Goury, North Cotentin, France) on the concentration and distribution of Americium ( 241 Am) in the tissues of scallop Pecten maximus[END_REF] were half-lighter than our organisms. Indeed, previous works have shown translocated from soft tissues (e.g. the digestive gland) to the shell. It is important to note that the relative affinity of 241 Am with the shell of P. maximus has been previously observed in the field [START_REF] Miramand | Experimental and in situ data (Goury, North Cotentin, France) on the concentration and distribution of Americium ( 241 Am) in the tissues of scallop Pecten maximus[END_REF]. A good perspective for the temporal record of 241 Am in shell would be the use of ICP-MS coupled to laser ablation that has been already used in scallop for chronological survey of other elements [START_REF] Thébault | Barium and molybdenum records in bivalve shells: Geochemical proxies for phytoplankton dynamics in coastal environments?[END_REF].

When scallops were fed radiolabelled phytoplankton, dietary 241 Am and 134 Cs were relatively poorly assimilated (AE: 28 and 20%, respectively; Table 1). Furthermore, both radionuclides were rapidly depurated, resulting in relatively short biological half-lives (between 1 and 2 weeks). These AEs are much lower than in echinoderms and cephalopods for which they vary from 33 to 90% and from 30 to 60%, respectively [START_REF] Ryan | Transuranic biokinetic parameters for marine invertebrates-a review[END_REF][START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF]). However, our results for 241 Am are consistent with data reported for other bivalves, which displayed low 241 Am assimilation. For example, AE of 241 Am ranged between 2 and 13% in mussels of the genus Mytilus [START_REF] Baines | Influence of temperature on dietary metal uptake in Arctic and temperate mussels[END_REF]. Such low AE by mussels could be related to the radionuclide association with mineral fractions of phytoplankton (e.g. diatom shells) or on algal cell surfaces (e.g., Chlorophyceae such as Dunaliella tertiolecta) due to the particlereactive properties of 241 Am [START_REF] Fisher | Interactions of marine plankton with transuranic elements. 1. Biokinetics of neptunium, plutonium, americium, and californium in phytoplankton[END_REF][START_REF] Fisher | Influence of food composition on the biokinetics and tissue distribution of zinc and americium in mussels[END_REF].

The scarcity of data on 134 Cs assimilation in marine invertebrates is conspicuous, especially compared to 241 Am data on the subject. Nevertheless, 134 Cs is not well bioaccumulated by phytoplankton. [START_REF] Heldal | Bioaccumulation of 137 Cs and 57 Co by five marine phytoplankton species[END_REF] working on 134 Cs uptake of five species (three prymnesiophytes and two diatoms) have shown that phytoplankton is unlikely to influence the Cs build-up in marine food webs and Cs flux to deep waters. Bivalves generally showed low AE for Cs with values ranging between 0.4 and 10% in the green mussel Perna viridis [START_REF] Wang | Modeling radiocesium bioaccumulation in a marine food chain[END_REF]. In predators, reported AEs for Cs are higher, ranging between 44 and 58% in the gastropod Babylonia formosae habei and between 23 and 29% in the cuttlefish Sepia officinalis [START_REF] Wang | Modeling radiocesium bioaccumulation in a marine food chain[END_REF][START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF].

Regarding their way of living and their nutrition, scallops are in direct contact with bottom sediments and predispose scallop to filter and ingest contaminated particles. For a species filtering large quantities of sediment particles, the TF obtained at the end of the exposure were quite low (TF 13d of 241 Am and 134 Cs < 0.011 with a maximum TF < 0.05 over the whole exposure period) but consistent with other sediment exposure studies [START_REF] Miramand | Uptake of americium and plutonium from contaminated sédiments by three benthic species: Arenicola marina, Corophium volutator and Scrobicularia plana[END_REF][START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF][START_REF] Borretzen | Bioavailability of sediment-associated and low-molecularmass species of radionuclides/trace metals to the mussel Mytilus edulis[END_REF].

In scallops from the field, radionuclide activity provides an integrated value of the bioaccumulation process. However, experimental approaches are compulsory to quantify the different physiological parameters of element bioaccumulation and to determine the relative importance of the different exposure pathways [START_REF] Warnau | Radiotracer Techniques: A unique tool in marine ecotoxicological studies[END_REF]. According to the bioaccumulation kinetics of the radionuclides, Am seems to be mainly accumulated from the dissolved phase since exposure to particule-associated Am (food and sediment) resulted in quite poor absorption and retention, compared to 241 Am-dissolved bioaccumulation (efficient uptake and strong retention). In the case of the Cs, the kinetic analyses did not clearly revealed a trend on the major uptake pathway, even though the low amount of Cs bioaccumulated through sediment exposure (whole body TF < 0.01) was highly absorbed (A 0l of 68 ± 7%) and strongly retained (T b½ = 74d). The use of a bioaccumulation model would allow to further explore the importance of each exposure pathways sensu [START_REF] Thomann | Steady-state model of biota sediment accumulation factor for metals in two marine bivalves[END_REF] and their use have been already developed and applied on scallops in previous studies [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF](Metian et al. , 2008b(Metian et al. , 2009b)). However, variability of the kinetic parameters obtained during the sediment experiment was high because the exposure was relatively short. Thus the bioaccumulation model with 3 exposure pathways could not be run in the present study.

Experiments with longer exposure periods will be necessary to better specify these parameters.

Conclusion

The present study provided new information about the different bioaccumulation pathways of 241 Am and 134 Cs that scallops are facing in the field. In this context, our data showed that the shell and the adductor muscle appeared to be the best scallop compartments for monitoring respectively 241 Am and 134 Cs in the marine environment.

Table 1. Whole-body uptake and depuration kinetic parameters of 241 Am and 134 Cs in Pecten maximus following different exposure experiments:

1) exposed for 7d to waterborne radionuclides (n = 9) followed by 36d of depuration (n = 9); 2) after a 2h feeding on radiolabelled Skeletonema costatum for 241 Am followed by 21d of depuration (n = 9) and Isochrysis galbana for 134 Cs followed by 16d of depuration (n = 6)

3) exposed for 13d via the radiolabelled sediments (n = 6) and then maintained for 31d in clean sediment and running seawater (n = 6) Uptake parameters: CF ss : concentration factors at steady state; k u : uptake rate constant (d -1 ).

Depuration parameters: A 0s and A 0l : remaining activity (%) according to the short-and the long-lived exponential component, respectively; T b½ : biological half-life (d). ASE: asymptotic standard error; r 2 : determination coefficient of the uptake or depuration kinetics.

Probability of the model adjustment: a p < 0.05. b p < 0.01. c p < 0.001. d p < 0.0001 n.a.: information not available.

Figures captions:

Fig. 1. Uptake kinetics of (A) 241 Am and (B) 134 Cs in Pecten maximus exposed for 7d to dissolved radiotracers (n=9) and their following 36d of depuration kinetics (C) (n=9).

All values are mean ± SD.

Fig. 2 .

 2 Fig.2. Kinetics of 241 Am and 134 Cs in Pecten maximus (A) Depuration after a 2h feeding on radiolabelled Skeletonema costatum for 241 Am followed by 21d of depuration (n = 9) and Isochrysis galbana for 134 Cs followed by 16d of depuration (n = 6); Uptake and depuration kinetics of 241 Am and 134 Cs in Pecten maximus (B) exposed for 13d to the radiolabelled

Figure 2

 2 Figure 2

Acknowledgments

Authors are grateful to F. Oberhansli (IAEA-MEL) for skilful technical assistance and to M. Burlot (University of La Rochelle) for his help in scallop collection. MW is an Honorary Senior Research Associate of the National Fund for Scientific Research (NFSR, Belgium). This work was supported by the IAEA, the Conseil Général de la Charente-Maritime and the GIP Seine-Aval Programme. The IAEA is grateful for the support provided to its Marine Environment Laboratories by the Government of the Principality of Monaco. The experiments described herein comply with the current laws of the Principality of Monaco and France.

that this factor affects metal bioaccumulation [START_REF] Boyden | Trace elements contents and body size in molluscs[END_REF][START_REF] Boyden | Effect of size upon metal content of shellfish[END_REF][START_REF] Warnau | Allometry of heavy metal bioconcentration in the echinoid Paracentrotus lividus[END_REF], Hédouin et al. 2007).

Following waterborne exposure, 241 Am was more efficiently concentrated than 134 Cs in Pecten maximus (~20 times higher, Table 1). This difference is often found when marine organisms are exposed to these two radionuclides through seawater [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: sea water and food exposures[END_REF][START_REF] Bustamante | Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle[END_REF]). It could be related to physico-chemicals properties of each radionuclide: as a transuranic radionuclide, americium (III) is strongly particulate reactive [START_REF] Ryan | Transuranic biokinetic parameters for marine invertebrates-a review[END_REF] while Cs is soluble and not reacting with particles. Such reactive properties of Am would lead to direct adsorption onto shells. It is therefore not surprising that ca. 95% of the accumulated 241 Am was found on the shell of the scallops (Table 2). After they have been accumulated from seawater, the radionuclides were depurated with very different rates with a much shorter biological half-life for Cs (22d) than for Am (892d; Table 1). Such very long biological half-life for Am might result from its retention on the shell. 134 Cs was mainly present in the soft tissues, with 76% in the adductor muscle and the mantle (Table 2). This specific accumulation pattern in muscular tissues is related to the analogous behavior of Cs + for K + (Ke et al. 2000, Smith et al. 2002, Lacoue-Labarthe et al. 2010). The predominant distribution of 241 Am in the calcitic skeleton/endoskeleton have been shown by several authors [START_REF] Grillo | Comparative studies on transuranium nuclide bio-kinetics in sediment-dwelling invertebrates[END_REF][START_REF] Guary | Routes of plutonium uptake and their relation to biomagnification in starfish[END_REF][START_REF] Fowler | Americium biokinetics in benthic organisms as a function of feeding mode[END_REF]. [START_REF] Fowler | Americium biokinetics in benthic organisms as a function of feeding mode[END_REF] demonstrated a positive correlation between the 241 Am CF in different echinoderm species and the proportion of calcitic endoskeleton in the body wall of those species. Recently, [START_REF] Zuykov | Autoradiographic study on the distribution of 241 Am in the shell of the freshwater zebra mussel Dreissena polymorpha[END_REF] showed a preferential accumulation of 241 Am in the organic periostracum of bivalve's shell. In P. maximus, it is noteworthy that the shell contained most of 241 Am (up to 95%) whatever the exposure pathway was (seawater, food or sediment, Tables 2 and3). According to our results of dietary exposure, it clearly appears that, beside a direct adsorption of dissolved 241 Am on the shell, the radionuclide is also Table 2. Concentration Factors (mean CF ± SD) and body distribution (mean % ± SD) of 241 Am and 134 Cs in Pecten maximus during seawater (after 7d of exposure and after 36d of depuration) and feeding experiments (21d after feeding with Skeletonema costatum for 241 Am and 16d after feeding with Isochrysis galbana for 134 Cs).

n.a.: information not available.

Radionuclides

Seawater


Xiyan He

Gilles Mourot

Didier Maquin

José Ragot

Pierre Beauseroy

André Smolarz

One-Class SVM in Multi-Task Learning

has become an active research topic in recent years. While most machine learning methods focus on the learning of tasks independently, multi-task learning aims to improve the generalization performance by training multiple related tasks simultaneously. This paper presents a new approach to multi-task learning based on one-class Support Vector Machine (one-class SVM). In the proposed approach, we first make the assumption that the model parameter values of different tasks are close to a certain mean value. Then, a number of one-class SVMs, one for each task, are learned simultaneously. Our multi-task approach is easy to implement since it only requires a simple modification of the optimization problem in the single one-class SVM. Experimental results demonstrate the effectiveness of the proposed approach.

INTRODUCTION

Classical machine learning technologies have achieved much success in the learning of a single task at a time. However, in many practical applications we may need to learn a number of related tasks or to rebuild the model from new data, for example, in the problem of fault detection and diagnosis of a system that contains a set of equipments a priori identical but working under different conditions.

Here "an equipment" may be a simple machine (a pump, a motor, ...), a system (a car, an airplane, ...), or even an industrial plant (nuclear power plant, ...). This may be the case for a car hire system where we have a fleet of vehicles to serve a set of customers. In industry, it is common to encounter a number of a priori identical plants, such as in the building or maintenance of a fleet of nuclear power plants or of a fleet of their components. In such cases, the learning of the behavior of each equipment can be considered as a single task, and it would be nice to transfer or leverage the useful information between related tasks [START_REF] Pan | A survey on transfer learning[END_REF]. Therefore, Multi-Task Learning (MTL) has become an active research topic in recent years [START_REF] Bi | An improved multi-task learning approach with applications in medical diagnosis[END_REF][START_REF] Zheng | Transferring multi-device localization models using latent multi-task learning[END_REF][START_REF] Gu | Learning the shared subspace for multi-task clustering and transductive transfer classification[END_REF][START_REF] Birlutiu | Multi-task preference learning with an application to hearing aid personalization[END_REF].

While most machine learning methods focus on the learning of tasks independently, multi-task learning aims to improve the generalization performance by training multiple related tasks simultaneously. The main idea is to share what is learned from different tasks (e.g., a common representation space or some model parameters that are close to each other), while tasks are trained in parallel [START_REF] Caruana | Multitask learning[END_REF]. Previous works have shown empirically as well as theoretically that the multi-task learning framework can lead to more intelligent learning models with a better performance [START_REF] Caruana | Multitask learning[END_REF][START_REF] Heskes | Empirical bayes for learning to learn[END_REF][START_REF] Ben-David | Exploiting task relatedness for mulitple task learning[END_REF][START_REF] Evgeniou | Learning multiple tasks with kernel methods[END_REF][START_REF] Ben-David | A notion of task relatedness yielding provable multiple-task learning guarantees[END_REF].

In recent years, Support Vector Machines (SVM) [START_REF] Boser | A training algorithm for optimal margin classifiers[END_REF][START_REF] Vapnik | The Nature of Statistical Learning Theory[END_REF] have been successfully used for multi-task learning [START_REF] Evgeniou | Regularized multi-task learning[END_REF][START_REF] Jebara | Multi-task feature and kernel selection for svms[END_REF][START_REF] Evgeniou | Learning multiple tasks with kernel methods[END_REF][START_REF] Widmer | Inferring latent task structure for multitask learning by multiple kernel learning[END_REF], Yang et al. 2010). The SVM method was initially developped for the classification of data from two different classes by a hyperplane that has the largest distance to the nearest training data points of any class (maximum margin). When the datasets are not linearly separable, the "kernel trick" is used. The basic idea is to map the original data to a higher dimensional feature space and then solve a linear problem in that space. The good properties of kernel functions make support vector machines well-suited for multi-task learning.

In this paper, we present a new approach to multitask learning based on one-class Support Vector Machines (one-class SVM). The one-class SVM pro-posed by Schölkopf et al. (2001) is a typical method for the problem of novelty or outlier detection, also known as the one-class classification problem due to the fact that we do not have sufficient knowledge about the outlier class. For example, in the application of fault detection and diagnosis, it is very difficult to collect samples corresponding to all the abnormal behaviors of the system. The main advantage of one-class SVM over other one-class classification methods [START_REF] Tarassenko | Novelty detection for the identification of masses in mammograms[END_REF][START_REF] Ritter | Outliers in statistical pattern recognition and an application to automatic chromosome classification[END_REF][START_REF] Eskin | Anomaly detection over noisy data using learned probability distributions[END_REF][START_REF] Singh | An approach to novelty detection applied to the classification of image regions[END_REF] is that it focuses only on the estimation of a bounded area for samples from the target class rather than on the estimation of the probability density. The bounded area estimation is achieved by separating the target samples (in a higher-dimensional feature space for non-linearly separable cases) from the origin by a maximum-margin hyperplane which is as far away from the origin as possible.

Recently, [START_REF] Yang | Multi-task learning for one-class classification[END_REF] proposed to take the advantages of multi-task learning when conducting one-class classification. The basic idea is to constrain the solutions of related tasks close to each other. However, they solve the problem via conic programming [START_REF] Kemp | Learning and using relational theories[END_REF], which is complicated. In this paper, inspired by the work of [START_REF] Evgeniou | Regularized multi-task learning[END_REF], we introduce a very simple multi-task learning framework based on the one-class SVM method, a widely used tool for single task learning. In the proposed method, we first make the same assumption as in [START_REF] Evgeniou | Regularized multi-task learning[END_REF], that is, the model parameter values of different tasks are close to a certain mean value. This assumption is reasonable due to the observation that when the tasks are similar to each other, usually their model parameters are close enough. Then, a number of one-class SVMs, one for each task, are learned simultaneously. Our multi-task approach is easy to implement since it only requires a simple modification of the optimization problem in the single one-class SVM. Experimental results demonstrate the effectiveness of the proposed approach.

This paper is organized as follows. In Section 2, a brief description of the formulation of the one-class SVM algorithm and the properties of kernel functions is first discussed. The proposed multi-task learning method based on one-class SVM is then outlined in Section 3. Section 4 presents the experimental results. In Section 5, we conclude this paper with some final remarks and future work propositions.

ONE-CLASS SVM AND PROPERTIES OF KERNELS

One-class SVM

The

A m = {x i }, i = 1, . . . , m is a set of m training samples of a single class. x i is a sample in the space X ⊆ R d of dimension d.
Also suppose that φ is a non-linear transformation. The one-class SVM is predicated on the assumption that the origin in the transformed feature space belongs to the negative or outlier class. The training stage consists in first projecting the training samples to a higher dimensional feature space and then separating most of the samples from the origin by a maximum-margin hyperplane which is as far away from the origin as possible. In order to determine the maximum-margin hyperplane, we need to deduce its normal vector w and a threshold ρ by solving the following optimization problem:

min w,ξ,ρ 1 2 w 2 + 1 νm m i=1 ξ i -ρ subject to: w, φ(x i ) ≥ ρ -ξ i , ξ i ≥ 0 (1)
ξ i are called slack variables, and they are introduced to relax the constraints in some cases for certain training sample sets. Indeed, the optimization algorithm aims at finding the best trade-off between the maximization of the margin and the minimization of the average of the slack variables. The parameter ν ∈ (0, 1] is a special parameter for one-class SVM. It is the upper-bound of the ratio of outliers among all the training samples as well as the lower-bound of the ratio of support vectors among all the samples. Due to the high dimensionality of the normal vector w, the primal problem is solved by its Lagrange dual problem :

min α 1 2 m i,j=1 α i α j φ(x i ), φ(x j ) subject to: 0 ≤ α i ≤ 1 νm , m i=1 α i = 1 (2)
where α i are the Lagrange multipliers. It is worth noting that all the mappings φ occur in the form of inner products. We need not to calculate the non-linear mapping explicitely by defining a simple kernel function that fulfills Mercer's conditions [START_REF] Vapnik | The Nature of Statistical Learning Theory[END_REF]:

φ(x i ), φ(x j ) = k(x i , x j ).
(3)

As an example, the Gaussian kernel

k σ (x i , x j ) = e -x i -x j 2 2σ 2
is a largely used kernel among the community. By solving the dual problem with this kernel trick, the final decision is given by:

f (x) = sign m i=1 α i k(x i , x) -ρ (4)

Properties of kernels

In order to exploit the kernel trick, we need to construct valid kernel functions. A necessary and sufficient condition for a function to be a valid kernel is defined as follows (Schölkopf and Smola 2001): Definition 2.1 Let X be a nonempty set. A function k on X × X which for all m ∈ N and all x 1 , . . . , x m ∈ X gives rise to a positive definite Gram matrix K, with elements:

K ij := k(x i , x j ) (5)
is called a positive definite kernel.

One popular way to construct new kernels is to build them based on simpler kernels. In this section, we briefly gather some results of the properties of the set of admissible kernels that are useful for designing new kernels. For a detailed description concerning the design of kernel functions, interested readers are referred to (Schölkopf and Smola 2001).

Proposition 2.1 If k 1 and k 2 are kernels, and

α 1 , α 2 ≥ 0, then α 1 k 1 + α 2 k 2 is a kernel.
Proposition 2.2 If k 1 and k 2 are kernels defined respectively on X 1 × X 1 and X 2 × X 2 , then their tensor product

k 1 ⊗ k 2 (x 1 , x 2 , x ′ 1 , x ′ 2 ) = k 1 (x 1 , x ′ 1 )k 2 (x 2 , x ′ 2 ) is a kernel on (X 1 × X 2 ) × (X 1 × X 2 ). Here x 1 , x ′ 1 ∈ X 1 and x 2 , x ′ 2 ∈ X 2 .
With these properties, we can now construct more complex kernel functions that are appropriate to our specific applications in multi-task learning.

THE PROPOSED METHOD

In this section, we introduce the one-class SVM method for the purpose of multi-task learning. In the context of multi-task learning, we have T learning tasks on the same space X , with X ⊆ R d . For each task we have m samples {x 1t , x 2t , . . . , x mt }. The objective is to learn a decision function (a hyperplane) f t (x) = sign( w t , φ(x)ρ t ) for each task t. Inspired by the method proposed by [START_REF] Evgeniou | Regularized multi-task learning[END_REF], we make the assumption that when the tasks are related to each other, the normal vector w t can be represented by the sum of a mean vector w 0 and a specific vector v t corresponding to each task:

w t = w 0 + v t (6)

Primal problem

Following the above assumption, we can generalize the one-class SVM method to the problem of multitask learning. The primal optimization problem can be written as follows:

min w0,vt,ξit,ρt 1 2 T t=1 v t 2 + µ 2 w 0 2 + T t=1 1 ν t m m i=1 ξ it - T t=1 ρ t (7)
for all i ∈ {1, 2, . . . , m} et t ∈ {1, 2, . . . , T }, subject to:

(w 0 + v t ), φ(x it ) ≥ ρ t -ξ it (8) ξ it ≥ 0 (9)
where ξ it are the slack variables associated to each sample and ν t ∈ (0, 1] is the special parameter of oneclass SVM for each task. In order to control the similarity between tasks, we introduce a positive regularization parameter µ into the primal optimisation problem. In particular, a big value of µ tends to enforce the system to learn the T tasks independently whereas a small value of µ will lead the system to learn a common model for all tasks. As in the earlier case of a single one-class SVM, the Lagrangien is formed as:

L(w 0 , v t , ξ it , ρ t , α it , β it ) = 1 2 T t=1 v t 2 + µ 2 w 0 2 + T t=1 1 ν t m m i=1 ξ it - T t=1 ρ t - T t=1 m i=1 α it [ (w 0 + v t ), φ(x it ) -ρ t + ξ it ] - T t=1 m i=1 β it ξ it (10)
where α it , β it ≥ 0 are the Lagrange multipliers. We set the partial derivatives of the Lagrangian to zero and obtain the following equations:

(a) w 0 = 1 µ T t=1 m i=1 α it φ(x it ) (b) v t = m i=1 α it φ(x it ) (c) α it = 1 νtm -β it (d) m i=1 α it = 1 (11)
By combining the equations ( 6), (11)(a) and (11)(b), we have:

w 0 = 1 µ T t=1 v t ( 12
)
w 0 = 1 µ + T T t=1 w t ( 13
)
With these relationships, we may replace the vectors v t and w 0 by w t in the primal optimisation function (7), which leads to an equivalent function:

min wt,ξit,ρt λ 1 2 T t=1 w t 2 + λ 2 2 T t=1 w t - 1 T T r=1 w r 2 + T t=1 1 ν t m m i=1 ξ it - T t=1 ρ t (14)
with

λ 1 = µ µ + T and λ 2 = T µ + T (15)
We can see that the objective of the primal optimisation problem (7) in the framework of multi-task learning is thus to find a trade-off between the maximisation of the margin for each one-class SVM model and the closeness of each one-class SVM model to the average model.

Dual problem

The primal optimisation problem (7) can be solved through its Lagrangian dual problem expressed by:

max αit - 1 2 T t=1 T r=1 m i=1 m j=1 α it α jr 1 µ + δ rt φ(x it ), φ(x jr ) (16)
contrainted to :

0 ≤ α it ≤ 1 ν t m , m i=1 α it = 1 (17)
where δ rt is the Kronecker delta kernel:

δ rt = 1, if r = t 0, if r = t (18)
We can see that the main difference between this dual problem ( 16) and that in a single one-class SVM learning (2) is the introduced term 1 µ + δ rt in the multi-task learning framework. Suppose that we define a kernel function as in equation (3):

k(x it , x jr ) = φ(x it ), φ(x jr ) (19)
where r and t are the task index associated to each sample. Taking advantage of the kernel properties presented in section 2.2, we know that the product of two kernels δ rt k(x it , x jr ) is a valid kernel (Proposition 2.2). Further, the following function:

G rt (x it , x jr ) = ( 1 µ + δ rt )k(x it , x jr ) (20) = 1 µ k(x it , x jr ) + δ rt k(x it , x jr )
is a linear combination of two valid kernels with positive coefficients ( 1 µ and 1), and therefore is also a valid kernel (Proposition 2.1). We can thus solve the multitask learning optimisation problem (7) through a single one-class SVM problem by using the new kernel function G rt (x it , x jr ). The decision function for each task is given by:

f t (x) = sign T r=1 m i=1 α ir G rt (x ir , x) -ρ t (21)

EXPERIMENTAL RESULTS

This section presents the experimental results obtained in our analysis. In order to evaluate the effectiveness of the proposed multi-task learning framework, we compare our one-class SVM based multitask learning method (denoted by MTL-OSVM) with two other methods: the traditional learning method that learns the T tasks independently each with a oneclass SVM (denoted by T -OSVM) and the method that uses 1 one-class SVM for all tasks under the assumption that all the related tasks can be considered as one big task (denoted by 1-OSVM).

In our experiments, the kernel of the one-class SVM used for T -OSVM and 1-OSVM is a Gaus-

sian kernel k σ (x it , x jr ) = e -x it -x jr 2 2σ 2
. For the proposed multi-task learning method MTL-OSVM, the new kernel is thus constructed based on the Gaussian kernel as presented in equation ( 20). The optimum values for the two parameters ν and σ of the one-class SVM are determined through cross validation. For the sake of simplicity, we have used a common combination of their values (ν, σ) for all related tasks. In order to ensure the reliability of the performance evaluation, all the results have been averaged over 20 trials each with random draws of training set. As the approaches and comparison are all one-class classification methods, the statistics of both false positive and false negative error rates are reported.

Experiment on nonlinear toy data

We have firstly tested the poposed method on four (T = 4) related simple nonlinear classification tasks. The datasets are created according to the following steps. For the first task, each sample is composed of d = 4 variables of which the first three are uniformly distributed variables. The fourth variable is set by the relation:

x (4) = x (1) + 2x (2) + (x (3) ) 2
The datasets for the other three tasks are then created by adding Gaussian white noises with different amplitudes on the dataset of the first task. The noises are classified respectively as low noise (for Task 2, with an amplitude of about an order of 1% of the first dataset amplitude value), medium noise (for Task 3, with an amplitude of about an order of 8% of the first dataset amplitude value) and high noise (for Task 4, with an amplitude of about an order of 15% of the first dataset amplitude value). In order to evaluate the false positive error rates, we have generated a set of negative samples that are composed of d = 4 uniformly distributed variables. Therefore, the training set of each task contains only positive samples (m = 200) whereas in the test procedure we use the test set of size 400 that contains both positive and negative samples (200 samples for each class). The obtained optimum parameter values of one-class SVM are (ν, σ) = (0.01, 0.5) for this experiment.

Figure 1 illustrates the variation of the average false positive, false negative and total error rates of our multi-task learning method MTL-OSVM for each task along with the value of the regularization parameter µ. The error rates of T -OSVM and 1-OSVM are also presented. We can see that for a very small value of µ, the performance of MTL-OSVM coincides with that of 1-OSVM as if all the tasks were considered as the same task. When the value of µ is very large, the performance of MTL-OSVM is in accordance with that of the traditional independant learning method T -OSVM. With the increase of the value of µ, the behaviors of the first three tasks are similar. The false positive error rate of the MTL-OSVM method tends to decrease whereas its false negative error rate increases. However, for the fourth task, the false positive (false negative) error rate first increases (decreases) and then decreases (increases) after it reaches the maximum (minimum) value. This behavior may be due to the very high noise that we added to the original dataset. In all, with a good choice of µ, the multi-task framework achieves a better performance in terms of the total error rate when compared to the traditional learning methods.

Experiment on textured image data

We have tested the proposed method on several textured gray-scale images that contain artificial textures generated by using Markov chain models [START_REF] Smolarz | Etude qualitative du modèle auto-binomial appliqué à la synthèse de texture[END_REF]. According to the nature of a texture, we first suppose that the useful information for texture caracterization is included in an isotropic neighbourhood of each pixel. In our experiments we use then the gray levels of a local d = 5 × 5 squared window centered to each pixel as its feature vector. Similar to the previous experiment in Section 4.1, four related tasks are created. The dataset for Task 1 contains samples of size d = 5 × 5 = 25 that are selected randomly from the original single texture source image. The samples for the other three tasks are selected from textured images of the same source as Task 1, but contaminated respectively by low noise (Task 2), medium noise (Task 3) and high noise (Task 4). Negative samples used in the test set are generated by using a different single texture source image. Figure 2 Table 1 shows the statistics of the obtained error rates. The corresponding optimum value of µ for MTL-OSVM, which minimises the total error rate, is also presented. According to this table, we can see that the individual learning method T -OSVM has the lowest false positive but a higher false negative. On the contrary, the learning of a single one-class SVM for all tasks (1-OSVM) achieves the lowest false negative at the expense of a higher false positive. The proposed multi-task learning method MTL-OSVM can reach an overall better performance by finding a trade- off between the false positive error rate and the false negative error rate.

The average results of this experiment are depicted in Figure 3. As in the previous experiment on the nonlinear toy data, we can observe the same behaviors of the error rates variations along with the value of the regularization parameter µ. The proposed method MTL-OSVM outperforms the other two methods (T -OSVM and 1-OSVM) for all the tasks. It is worth noting that the optimum value of µ is different for different task. The setting of this parameter is thus very important in order to ensure a good performance. In our experiment we use a validation set to find the optimum value of µ.

Discussion

It is worth noting that in this section only academic experiments on nonlinear toy data with low dimenstional feature space and textured image data with high dimentional feature space are presented. We address the problem of modeling the normal data with the help of one-class SVM method, which is usually considered as an essential step for fault dectection and diagnosis. In each experiment, related tasks are created in order to mimic the application of modeling the normal process behavior of a fleet of plants that are a priori identical but working under different conditions (thus having different noises on the measurements). Here we consider the modeling of the behavior of each plant as a single task and the classification of normal data and anomalies is then performed by the constructed model. The proposed methodologie shows that learning multiple related tasks simultaneously can be beneficial to improve the performance of each constructed model.

One straightforward work is to use the proposed show that learning multiple related tasks simultaneously can achieve a better performance than learning each task independantly.

In our method we have used a common setting of both one-class SVM parameter and kernel parameter values. One future work is thus to use different parameter values for different tasks. The properties of kernels open a wide range of further developpements on constructing new kernels for multi-task learning.

Figure 1 :

 1 Figure 1: The variation of the average (a) false positive, (b) false negative and (c) total error rates for each task (nonlinear toy data) along with the value of the regularization parameter µ.

  illustrates the single texture source images used for generating the datasets. In each trial, the training set of each task contains m = 200 positive samples and the test set is composed of 200 positive and 200 negative samples. The common parameter values of one-class SVM used in this experiment are (ν, σ) = (0.01, 300).

Figure 2 :

 2 Figure 2: Single texture source images used for generating the datasets. (a) Original texture image for Task 1. (b) Texture image of (a) with low noise, for Task 2. (c) Texture image of (a) with medium noise, for Task 3. (d) Texture image of (a) with high noise, for Task 4. (e) Original texture image for generating negative samples.

Figure 3 :

 3 Figure 3: The variation of the average (a) false positive, (b) false negative and (c) total error rates for each task (texture data) along with the value of the regularization parameter µ.

Table 1 :

 1 Error rates (%) of the different methods for all tasks on texture data. FP: false positive error rates, FN: false negative error rates, Total: total error rates.

			Task 1
		FP	FN	Total	µ
	T -OSVM	3.62 ± 1.18 27.0 ± 3.0 15.3 ± 1.4 -
	1-OSVM	27.1 ± 2.6 2.70 ± 1.59 14.9 ± 1.5 -
	MTL-OSVM 14.4 ± 2.2 9.52 ± 3.19 12.0 ± 1.8 0.1
			Task 2
		FP	FN	Total	µ
	T -OSVM	4.27 ± 1.11 28.2 ± 3.6 16.3 ± 1.6 -
	1-OSVM	27.1 ± 2.6 3.27 ± 2.21 15.2 ± 1.8 -
	MTL-OSVM 14.6 ± 2.2	10.3 ± 3.7 12.4 ± 2.0 0.1
			Task 3
		FP	FN	Total	µ
	T -OSVM	7.05 ± 1.41 28.6 ± 3.9 17.8 ± 2.0 -
	1-OSVM	27.1 ± 2.6 6.62 ± 3.73 16.8 ± 2.2 -
	MTL-OSVM 15.7 ± 2.3	14.4 ± 4.1 15.0 ± 2.2 0.1
			Task 4
		FP	FN	Total	µ
	T -OSVM	27.4 ± 3.0	34.4 ± 8.7 30.9 ± 4.5 -
	1-OSVM	27.1 ± 2.6	34.0 ± 8.7 30.5 ± 4.5 -
	MTL-OSVM 29.4 ± 2.6	29.0 ± 9.2 29.2 ± 4.5 0.5

ACKNOWLEDGEMENT

The authors would like to thank the financial support from GIS 3SGS.


Ricardo Cardoso Neves
email: ricardon.6@gmail.com

Marina Ribeiro Da Cunha

Peter Funch

Andreas Wanninger

Reinhardt Møbjerg Kristensen

External morphology of the cycliophoran dwarf male: a comparative study of Symbion pandora and S. americanus

Keywords: sexual reproduction, life cycle, scanning electron microscopy, Spiralia, Lophotrochozoa

Cycliophora is a recently described phylum to which only two species have been assigned so far, Symbion pandora and S. americanus. The cycliophoran life cycle is complex and alternates between asexual and sexual stages. Although not recognized as an entirely independent free-swimming stage when the phylum was first described, the dwarf male has a remarkably complex bodyplan albeit its very small size (approx. 30-40 m in length). Aiming to increase the knowledge on the gross morphology of the cycliophoran dwarf male, specimens from S. pandora and S. americanus were analyzed by scanning electron microscopy. In both species, anterior and ventral ciliated fields, as well as paired lateral sensorial organs were identified, thus confirming previous observations. However, new details are described herein such as the penial pouch that encloses the penis. We compare our findings on both Symbion species with the data currently available and other metazoan dwarf males.

Introduction

Cycliophora is a metazoan phylum recently erected to accommodate minute marine invertebrates that live commensally on clawed lobsters. So far, only two species have been described, Symbion pandora Funch and Kristensen, 1995 and Symbion americanus [START_REF] Obst | A new species of Cycliophora from the mouthparts of the American lobster, Homarus americanus (Nephropidae, Decapoda)[END_REF], which live on the mouthparts of the Norwegian lobster Nephrops norvegicus Linnaeus, 1758, and the American lobster, Homarus americanus H. Milne-Edwards, 1837, respectively. However, cryptic speciation has been suggested for S. americanus by recent molecular studies [START_REF] Obst | Hidden diversity and host specificity in cycliophorans: a phylogeographic analysis along the North Atlantic and Mediterranean Sea[END_REF]Baker & Giribet, 2007;Baker et al., 2007).

Cycliophorans are characterized by a very complex life cycle that alternates between a sexual and an asexual phase, involving various stages. The most prominent stage is the sessile feeding stage, which lives attached to the setae of the host lobster and filters small food particles from the water [START_REF] Funch | Symbiotic relations and feeding biology of Symbion pandora (Cycliophora) and Triticella flava (Bryozoa)[END_REF]. This stage generates several other life cycle stages by internal budding (Funch & Kristensen, 1995, 1997;[START_REF] Kristensen | An introduction to Loricifera, Cycliophora, and Micrognathozoa[END_REF]. Feeding individuals are capable of producing Pandora larvae, Prometheus larvae, or females, one at a time, inside a brood chamber. These stages are later released to commence a free-swimming life style. In the asexual phase of the life cycle the Pandora larva settles close to the maternal feeding stage and develops asexually into a new feeding stage. In the sexual phase of the life cycle a free male stage, the Prometheus larva, settles on the trunk of a feeding stage, probably the maternal individual. The Prometheus larva then produces 1-3 dwarf males inside its body. The dwarf male is regarded as a free stage and is morphologically characterized by a heavy ciliated body and a ventral-posterior penis, among other features [START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF]. The fertilized female is thought to settle on the mouthparts of the same host where it encysts (Funch, 1996). A dispersal stage, the chordoid larva, hatches from this cyst, settles on a new host individual, and develops into a new feeding stage.

The phylogenetic position of Cycliophora has been unclear ever since its original description, in which a relationship to Entoprocta and Ectoprocta had been hypothesized (cf. Funch & Kristensen, 1995). More recently, several studies have suggested a close relationship to either Entoprocta (e.g., [START_REF] Zrzavý | Phylogeny of the Metazoa based on morphological and 18S ribosomal DNA evidence[END_REF][START_REF] Sørensen | On the phylogeny of the Metazoa in the light of Cycliophora and Micrognathozoa[END_REF][START_REF] Obst | Cycliophoran relationships revisited[END_REF][START_REF] Funch | On the phylogenetic position of Rotiferahave we come any further?[END_REF][START_REF] Passamanek | Lophotrochozoan phylogeny assessed with LSU and SSU data: Evidence of lophophorate polyphyly[END_REF][START_REF] Paps | Lophotrochozoa internal phylogeny: new insights from an up-to-date analysis of nuclear ribosomal genes[END_REF][START_REF] Hejnol | Assessing the root of bilaterian animals with scalable phylogenomic methods[END_REF] or gnathiferan taxa such as Rotifera (e.g., [START_REF] Winnepenninckx | Relations of the new phylum Cycliophora[END_REF][START_REF] Giribet | Triploblastic relationships with emphasis on the acoelomates and the position of Gnathostomulida, Cycliophora, Plathelminthes, and Chaetognatha: a combined approach of 18S rDNA sequences and morphology[END_REF][START_REF] Peterson | Animal phylogeny and the ancestry of bilaterians: inferences from morphology and 18S rDNA gene sequences[END_REF][START_REF] Giribet | Investigations into the phylogenetic position of Micrognathozoa using four molecular loci[END_REF][START_REF] Glenner | Bayesian Inference of the Metazoan Phylogeny: A Combined Molecular and Morphological Approach[END_REF].

Because of the incongruent data from gene sequence analyses, further details on the morphology of all life cycle stages are needed for a better understanding of the cycliophoran phylogenetic interrelationships.

The cycliophoran dwarf male has already been described using light and electron microscopy, and at the cytochemical level using confocal laserscanning microscopy (Funch and Kristensen, 1997;[START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF]Neves et al., 2009aNeves et al., , 2009b[START_REF] Neves | Comparative myoanatomy of cycliophoran life cycle stages[END_REF].

However, only one male of Symbion pandora has so far been described by scanning electron microscopy, and its external morphology was apparently altered during the freeze-drying technique used to prepare specimens (cf. [START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF]. Also the high loss of specimens during preparation, due to the minute size of the males, limited the success of this approach. In the study presented herein, we compare the external morphology of the dwarf males of S. pandora and S. americanus.

Materials and Methods

Feeding stages with attached Prometheus larvae of the cycliophoran species Symbion pandora were obtained from mouth parts of Norway lobsters, Nephrops norvegicus, collected off the coast of Sagres, Portugal (January 23 rd , 2008) by local fishermen.

Cycliophorans of the species S. americanus were obtained from mouth parts of the American lobster, Hommarus americanus, collected from Maine (USA; between October 1 st and 15 th , 2006). After fixation for 1h in 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PBS), specimens were washed 3×15 min in 0.1M PBS containing 0.1% sodium azide and stored at 4ºC. PFA-fixed males of Symbion americanus were washed in distilled water and concentrated on a Millipore filter (12 mm pore size) using a Swinnex filter (Millipore, Massachusetts, USA) holder to minimize the loss of specimens. The specimens were then post-fixed in 1% OsO 4 for 1h at room temperature, and subsequently washed with distilled water. Afterwards, specimens were dehydrated in an ethanol and an acetone series and critical point dried (CPD) using carbon dioxide. Finally, the membrane with the attached animals was mounted on an aluminium stub with sticky carbon pads, and sputter-coated with platinum/palladium alloy. Alternatively, dwarf males of S. americanus and S. pandora were attached to circular coverslides coated with poly-Llysine (solution 0.01%, molecular weight 150,000-300,000; Sigma-Aldrich, Brøndby, Denmark). After post-fixation with OsO 4 as described above, the specimens were dehydrated in an ethanol series and immersed in hexamethyldisilazane (HMDS) (Sigma-Aldrich, Brøndby, Denmark) for about 12 hours in a desiccator. Finally, the coverslides with the dwarf males were allowed to dry inside the desiccator for approx. 15 minutes, mounted on an aluminium stub, and sputter-coated as described above. All specimens were analysed and digitally photographed using a JEOL JSM-6335F field emission scanning electron microscope (Tokyo, Japan).

Results and discussion

The external morphology of 10 specimens of Symbion pandora and 16 specimens of S. americanus investigated herein showed the typical features that characterize the cycliophoran dwarf male (Figs. 1,2), including the anterior and ventral ciliated fields, the paired lateral sensory organ, and the ventral-posterior penis (see [START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF][START_REF] Obst | A new species of Cycliophora from the mouthparts of the American lobster, Homarus americanus (Nephropidae, Decapoda)[END_REF]. The penis is located inside a pouch-like structure, which is laterally constricted and forms an elongated slit at the cuticle level (Figs. 1a,b,2a,b).

Only the tip of the penis is protruding from the penial pouch. The body region at the base of the penis, in the posterior-most end of the slit, is the only difference between males from the two cycliophoran species. In S. pandora the cuticle at the base of the penis shows some ridges (Fig. 1b), whereas the same structure in S. americanus is smooth (Fig. 2b). This fact was observed for all specimens investigated and is consistent even for the different drying processes used in this study. No differences were recognized in any other structure due to the different drying processes.

In both cycliophoran species, a ventral ciliated field starts in the very anterior end of the body of the dwarf male and extends posteriorly until the beginning of the penial pouch (Figs. 1a,2a). No clearly distinguishable longitudinal rows of cilia are found in the somewhat horseshoe-shaped ventral ciliated field. An additional distinct ciliated field likewise emerges in anterior position. This is a frontal ciliated field that spans dorsally through the most anterior third of the male body length (Figs. 1c,d,2c,d) and is clearly separated from the ventral ciliated field by a depression. Sensory cilia located laterally as an extension from the frontal ciliated field are found to be very long (10-15 m) in S. pandora and S. americanus (Figs. 1a,c,d,2a,c,d) [START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF]. The non-ciliated parts of the male integument do not exhibit the typical polygonal sculpture observed in other cycliophoran life cycle stages (e.g., the feeding stage). Different shapes of the body of the dwarf male were found for both species. Some specimens have a roundish, oval body (Figs. 1a,2a), whereas other specimens present a more elongated, rectangular shape (Fig. 1c,2c).

Our results corroborate the view of the dwarf male as a totally independent stage in the life cycle of both hitherto described cycliophoran species. The external phenotype of dwarf males of the two species, Symbion pandora and S. americanus, is very similar.

The main difference noticed is related to the cuticle at the base of the penis, which shows some ridges in S. pandora but not in S. americanus. Interestingly, the presence of those ridges in S. pandora is confirmed by transmission electron microscopy (cf. [START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF]. However, there are no data available on the ultrastructure of males of the species S. americanus, which makes further comparisons between both species impossible. Since the ridges were neither observed in HMDS-nor in CPD-dried specimens of S. americanus, this finding is apparently not caused by the different drying processes used for each species.

Earlier descriptions of the dwarf male of Symbion pandora and S. americanus revealed details of sensory elements other than the lateral sensory cilia [START_REF] Obst | Cycliophoran relationships revisited[END_REF]Funch 2003, Obst et al. 2006). In S. pandora a frontal sensorial organ was identified by TEM but not by SEM, and several palps and a tactile papilla (Fig. 3) were described only for one specimen using SEM. Frontal palps were also described for S. americanus [START_REF] Obst | A new species of Cycliophora from the mouthparts of the American lobster, Homarus americanus (Nephropidae, Decapoda)[END_REF]). These sensorial structures could not be identified in any of the males of S. pandora or S. americanus in our study. However, our results do not argue against the existence of such structures since the frontal ciliated field is very dense and the structures mentioned are possibly covered by cilia.

The various shapes found for the male body were already observed in previous studies under light microscopy (RCN pers. obs.). Since the external morphology is not distinct among males with different shapes, all specimens should be fully formed, i.e., at the same developmental phase. Thus, the differences found in the shape of the male body are not indicative of any physiological constraints. It is possible that those differences are related to the number of males developed at the same time and the exact position they adopt inside the attached Prometheus larva.

External morphology and functional implications

In the original description of Cycliophora, the extremely reduced dwarf male had not been recognized as a free swimming, independent stage. Instead, an intermediate stage, now termed the Prometheus larva, was originally proposed as the free mature male (cf. Funch andKristensen 1995, 1997). The cycliophoran dwarf male is now known to have a sophisticated bodyplan that includes a highly complex musculature, a relatively large brain, a pair of ventral longitudinal nerve cords, fully developed gonads and mating structures, sensory organs, and various types of glands (Fig. 3). Accordingly, the details on the external morphology as presented herein strengthen the view of the cycliophoran dwarf male as a free swimming stage with an important role during sexual reproduction.

So far, fertilization in cycliophorans has never been observed and questions such as when, where, and how this process occurs remain unanswered. The presence in the male of extensive locomotory ciliated fields together with the elongated sensory cilia and an elaborated muscle architecture are indicative of its swimming and crawling capabilities (Fig. 3). Therefore, these locomotory and sensory structures might be necessary for the male to carry out its role in the reproductive process, i.e., find the female and copulate (Funch andKristensen 1999, Obst and[START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF]. It is possible that the transfer of sperm cells takes place during the encounter of the male with the female while free swimming stages. The mechanisms involved in the transfer of the sperm cells to the oocytes, however, remains unknown.

The penis of the dwarf male of both species is a cuticular hollow tube without any aperture at the very distal end (Fig. 3; see also [START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF]. The intriguing question is how the sperm cells exit the male body through the penial structure. The fact that the tip of the penis is less than one third of the diameter of the sperm cells raises the possibility that this organ serves as an anchoring device that facilitates copulation rather than being a true copulatory organ [START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF].

The anchoring cirrus organ could then be used by the male to momentarily pierce the female and somehow liberate the sperm cells from the inside of its body. Both hypotheses are supported by the fact that a complex set of muscles are present in the posterior body region in connection with the base of the penial (or anchoring?) structure (Funch and Kristensen 1997[START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF], Neves et al. 2009b).

The penial pouch in which the penis lies is a newly described feature for the cycliophoran dwarf male. This structure may be very important during the release of sperm cells by the cycliophoran dwarf male. If, however, the penis would be shown to be an anchoring organ, the penial pouch may act as a receptaculum where the sperm cells accumulate before being transferred to the female's body. This assumption is obviously speculative but based on the aforementioned morphological details of the penial structure.

The cycliophoran male compared to other metazoan dwarf males Dwarf males are known from several metazoan lineages such as echiurans, polychaetes, siboglinids, monogonont rotifers, barnacles, and spiders [START_REF] Windoffer | The nervous system of the male Dinophilus gyrociliatus (Annelida: Polychaeta). I. Number, types and distribution pattern of sensory cells[END_REF][START_REF] Ricci | Dwarf males in monogonont rotifers[END_REF][START_REF] Rouse | Osedax: bone-eating marine worms with dwarf males[END_REF], for reviews see [START_REF] Gilbert | Sexual dimorphism in zooplankton (Copepoda, Cladocera, and Rotifera)[END_REF]Williamson 1983, Volrath 1998). Frequently in textbooks (e.g., [START_REF] Ruppert | Invertebrate Zoology: A Functional Evolutionary Approach[END_REF]), dwarf males are described as rudimentary sexual intervening forms that are only equipped with the strictly necessary structures to seek a female and copulate. The cycliophoran dwarf male, however, has a very complex body architecture (cf. Funch and Kristensen 1997[START_REF] Obst | Dwarf male of Symbion pandora (Cycliophora)[END_REF], Neves et al. 2009b). The location of the penis inside a pouch is to our knowledge a new feature among metazoan dwarf males.

In some cases, the penial structure is found inside the body (e.g., in polychaetes or monogonont rotifers; see [START_REF] Windoffer | The nervous system of the male Dinophilus gyrociliatus (Annelida: Polychaeta). I. Number, types and distribution pattern of sensory cells[END_REF]Westheide 1988, Clément andWurdak, 1991) or may be entirely absent (e.g., in echiurans or the siboglinid Osedax; [START_REF] Schuchert | Ultrastructural observations on the dwarf male of Bonellia viridis (Echiura)[END_REF][START_REF] Rouse | Osedax: bone-eating marine worms with dwarf males[END_REF][START_REF] Worsaae | The simplicity of males: dwarf males of four species of Osedax (Siboglinidae; Annelida) investigated by Confocal Laser Scanning Microscopy[END_REF]. Therefore, more data are necessary to clarify the male-female interaction and the true function of the penial structure and the penial pouch.

In the future, males of both Symbion species should be compared at the ultrastructural level. A better understanding of, e.g., spermatogenesis and sperm morphology will permit further comparisons with other lophotrochozoan taxa, which could result in further insights into the phylogenetic placement and evolution of 3 Line drawing of the cycliophoran dwarf male (reprinted with permission from Neves et al., 2009b). Lateral view, anterior faces to the left. Scale bar represents 10 μm. cg, cerebral glands; dm, dorsal muscle; dp, dorsal papilla; dvm, dorsoventral muscles; fc, frontal ciliated field; fp, frontal palp; fs, frontal sensoria; ga, cerebral ganglion; hm, horseshoe-shaped muscles; ls, lateral sensoria; mg, medial glands; pe, penial structure;

Figure Legends Fig. 1 .

 Legends1 Figure Legends

Fig. 2

 2 Fig. 2 Symbion americanus, free male. Anterior faces to the left in all aspects. a Ventral

Fig.

  Fig. 3 Line drawing of the cycliophoran dwarf male (reprinted with permission from

Acknowledgments

We are indebted to Lisbeth Haukrogh (Copenhagen) for technical assistance. RCN greatly appreciates the generous support from the staff of the Department of Biology at the University of Aveiro, the Invertebrate Department at Zoological Museum