Evaluation of Sediment Particle Size Selection During Feeding by The

1 2 3 1 Evaluation of sediment particle size selection during feeding by the holothurian 4 5 6 2 Parastichopus regalis (Cuvier, 1817) 7 8 3 9 10 4 Montserrat Ramón*, Gonzalo Simarro*, Eve Galimany, Jordi Lleonart 11 12 5 Marine Sciences Institute (ICM-CSIC) 13 14 6 Passeig Marítim de la Barceloneta 37-49, 08003, Barcelona (Spain) 15 16 7 17 18 8 19 20 9 M. Ramón (Corresponding Author) e-mail: [email protected] 21 22 10 G. Simarro e-mail: [email protected] 23 24 25 11 E. Galimany e-mail: [email protected] 26 27 12 J. Lleonart e-mail: [email protected] 28 29 13 (*) The authors contributed equally to this work. 30 31 14 32 33 15 Abstract 34 35 16 Parastichopus regalis is an epibenthic holothurian common in the 36 37 17 Mediterranean Sea and the NE Atlantic, which feeds on the upper layer of the sediment 38 39 18 playing a significant role on soft-bottom dynamics. Whether or not P. regalis is able to 40 41 42 19 select the sediment ingested by size is the question of this study. For this purpose, a 43 44 20 comparison between grain size distributions of the seabed sediments and the digestive 45 46 21 contents of sea cucumbers were carried out. We performed the comparisons among 47 48 22 sediment distributions through the median diameter D 50 and the granulometric 49

50 23 dispersion D 84 /D 16 . The results showed that the size of the sediment within the 51 52 24 holothurians was significantly smaller and more uniform than the ones in the seabed. 53 54 25 Evidence showed that P. regalis select sediment by particle size during feeding, 55 56 57 58 59 60 61 62 26 choosing the smaller particles. This finding reports novel information on the feeding 63 64 65 27 behaviour of this species, a fishery resource of local interest and importance in the 66 67 28 Western Mediterranean region. 68 69 29 70 71 30 Key words: Holothurian; feeding; grain size selection; Mediterranean Sea 72 73 31 74 75 32 1. Introduction 76 77 33 Holothuroidea is a conspicuous and diverse group of invertebrates in the worlds 78 79 34 oceans. Aspidochirotid holothurians provide important ecosystem services enhancing 80 81 35 nutrient cycling and sediment dynamics through their feeding activities and bioturbation 82 83 84 36 (Uthicke, 1999) . Most species live on the sea bottom surface, where they ingest the 85 86 37 upper layer of sediment from which they assimilate a fraction of its organic matter 87 88 38 content. The capture of food is made with the tentacles, provided with contractile and 89 90 39 adhesive elements on its surface. During feeding, the tentacles extend over the 91 92 40 substratum, collect the particles, and contract and fold into the mouth to release the food 93 94 41 (Roberts et al., 2000). 95 96 42 Bottom sediment characteristics, hydrodynamics, and depth are crucial factors 97 98 43 affecting habitat preference of sea cucumbers (Sloan and Von Bodungen, 1980; Slater 99 100 101 44 and Jeffs, 2010; Dissanayake and Stefansson, 2012). Sediment characteristics have a 102 103 45 direct influence in feeding particle selection, which may be based on sediment grain size 104 105 46 or organic-rich content, depending on species and habitats (Roberts, 1979). For 106 107 47 example, no evidences of selectivity by size were found in Parastichopus parvimensis 108 109 48 (Yingst, 1976), Holothuria mexicana, H. arenicola (Hammond, 1982), Leptosynapta 110 111 49 tenuis (Powell, 1977), H. atra (Massin and Doumen, 1986), and Isostichopus 112 113 50 badionotus (Sloan and Von Bodungen, 1980; Hammond, 1982). Coarser grain sizes 114 115 116 117 118 119 120 121 51 tend to be excluded by H. atra , Bohadschia marmorata , and H. leucospilota (Massin 122 123 124 52 and Doumen, 1986; Dar, 2004). In addition, the burrower sea cucumber Molpadia 125 126 53 oolitica preferred smaller grain sizes (Rhoads and Young, 1971). On the contrary, 127 128 54 selection for larger grain sizes have been found in Stichopus tremulus (Hauksson, 1979) 129 130 55 and Holothuria scabra (Baskar, 1994). Grain size selection varies between species and 131 132 56 has been proposed as an important resource to establish niche separation between co- 133 134 57 existing deposit feeders (Massin and Doumen, 1986; Mezali and Soualili, 2013). For 135 136 58 instance, analyses of the digestive contents of the holothurian species H. tubulosa , H. 137 138 59 poli and H. stellate revealed that these ingested coarse and fine sediment, whereas H. 139 140 60 forskali and H. sanctori selected fine and very fine sediment (Mezali and Soualili, 141 142 143 61 2013). The size of the particles ingested influences the time needed for the holothurian 144 145 62 gut to efficiently extract the organic matter (Hudson et al., 2004), with implications in 146 147 63 the amount of sediment reworked by the holothurians and, by extension, in the 148 149 64 evaluation of their role in the ecosystems. 150 151 65 The importance of sea cucumbers in reworking muddy and sandy bottoms has 152 153 66 long been emphasized. Much of the work carried out worldwide on holothurian feeding 154 155 67 has been focused on shallow water species, mainly on tropical reefs, for its easy 156 157 68 accessibility and the high diversity of species (Yingst, 1976; Hauksson, 1979; Navarro 158 159 160 69 et al., 2013) . Knowledge on the feeding of deep-sea holothurians has increased recently 161 162 70 given the improvement of deep-sea exploration methods, highlighting the abundance of 163 164 71 sea cucumbers in such ecosystems (Wigham et al., 2003; Hudson et al., 2005; Navarro 165 166 72 et al., 2013). Nevertheless , information on holothurian species inhabiting the continental 167 168 73 shelf below 50 m depth is scarce. The sea cucumber Parastichopus regalis (Cuvier, 169 170 74 1817) belongs to the family Stichopodidae and is abundant in the Mediterranean Sea 171 172 75 and the NE Atlantic, between 50 and 300 m depth (Ramón et al., 2010). Its five 173 174 175 176 177 178 179 180 76 longitudinal muscle bands are commercialized for human consumption in Catalonia 181 182 183 77 (NW Mediterranean), where it is captured using bottom trawls (Ramón et al., 2010). 184 185 78 Despite its ecological and economic importance, there is little information on its basic 186 187 79 ecology. Understanding habitat use of P. regalis by studying feeding selectivity is a 188 189 80 necessary step towards the knowledge of its ecological role in the Mediterranean 190 191 81 continental shelf. 192 193 82 The aim of this study is to find out whether the sea cucumber P. regalis is able 194 195 83 to select the sediment ingested by size and, if so, to elucidate if selection is affected by 196 197 84 particle size availability. For this purpose, we compared the grain size distribution of the 198 199 85 ambient sediments to that of the gut sediments in situ. The statistical treatment of the 200 201 202 86 sediment grain distributions takes into account the particularity of the data set (each data 203 204 87 is a distribution of the sizes of the sediment). 205 206 88 207 208 89 2. Materials and Methods 209 210 90 2.1. Sampling 211 212 91 Specimens of Parastichopus regalis were collected on board a commercial 213 214 92 trawler from Arenys de Mar (41.5768°N, 2.5593ºE, NW Mediterranean) using a bottom 215 216 93 otter trawl operating around 100 m depth. They were transported to the laboratory in 217 218 219 94 coolers with sea water. On arrival, the sea cucumbers had ejected their internal organs, 220 221 95 making it impossible to perform feeding experiments in tanks therefore forcing a new 222 223 96 experimental approach, an in situ study. Then, sediments were collected on the 224 225 97 fishing ground, at the same time as sea cucumbers, using a stainless steel cylinder 226 227 98 coupled to the fishing gear (Del Rio et al., 2012) . This methodology has been 228 229 99 demonstrated to collect superficial sediment, which is where the sea cucumbers feed on 230 231 100 (Rufino et al., 2018) . Once on board, holothurians were dissected, the gut extracted and 232 233 234 235 236 237 238 239 101 placed individually in plastic bags to be transported to the Institute of Marine Sciences 240 241 242 102 (ICM-CSIC) in coolers with ice. A sample of 50g of sediment from each haul was kept 243 244 103 on board and stored in the fridge after returning to the laboratory. 245 246 104 Five daily surveys were performed in 2013 in the fishing ground of Arenys de 247 248 105 Mar. A total of eleven hauls were carried out at depths between 82 and 129 m. In 249 250 106 addition, a sediment sample from a trawl belonging to the oceanographic cruise 251 252 107 MEDITS_ES_CAL_13 on the Ebro river shelf (40.2983ºN, 01.2682ºE, NW 253 254 108 Mediterranean ) was added in order to analyze a different area. A total of 121 specimens 255 256 109 were analyzed (Table 1). 257 258 110 259 260 261 111 2.2. Sediment analysis 262 263 112 Sediment samples analyzed included 12 samples of seabed (one for each case in 264 265 113 table 1) and 121 of holothurians gut content, which allowed to perform a total of 133 266 267 114 analyses. Sediment was treated for size particle analysis, i. e., t he organic matter was 268 269 115 removed using hydrogen peroxide at 20%, followed with a treatment with 270 271 116 pyrophosphate to disaggregate the sediment particles. Samples were analyzed using a 272 273 117 Horiba LA940V2 Laser scattering particle size distribution analyzer. The instrument 274 275 118 provides results of grain size grouped into 48 size intervals (or channels), from 0.031 276 277 278 119 µm up to 6 mm in a logarithmic scale. The organic matter (OM) content of the ambient 279 280 120 sediment was quantified as weight loss on ignition at 475ºC for 12 h. The limited 281 282 121 amount of sediment inside the guts did not allow performing more analysis than size 283 284 122 particle; thus, organic matter content was not calculated for these samples. 285 286 123 To determine whether the holothurian P. regalis selects by grain size while foraging it 287 288 124 was necessary to perform a comparison of seabed and gut sediment distributions, and 289 290 125 determine whether the differences were statistically significant. In the statistical 291 292 293 294 295 296 297 298 126 treatment of the data, to establish that two populations (i.e. a sample of bed sediment 299 300 301 127 and a sample of gut sediment) were statistically different, the difference between the 302 303 128 populations had to be much larger than the difference within each population, since each 304 305 129 population is not uniform . 306 307 130 In this study, we compared arrays of numbers because each measure has 48 308 309 131 channels. Therefore, at first, a MANOVA should be employed. A first approach to 310 311 132 estimate the variability within samples is to obtain several measurements (or 312 313 133 realizations ) of a same sediment with the laser particle analyzer. The above 314 315 134 repetitions give an indication of the variance corresponding to the instrument, but not of 316 317 135 the variance within the sediment. Hence, to estimate the within variance, several 318 319 320 136 subsamples were obtained from the samples that contained more than 9 g of sediment, 321 322 137 to get 3 subsamples of at least the 3 g required by the particle analyzer. Table 1 includes 323 324 138 the number of specimens subsampled. Out of the 121 specimens analyzed, 23 of them 325 326 139 allowed the subsampling, and correspond to 7 different cases (case 1, 2, 3, 6, 8, 9 and 327 328 140 10, see Table 1). This constraint did not affect the seabed samples, available in high 329 330 141 quantities. 331 332 142 For each of the 30 above mentioned sediment samples (23 gut contents and 7 333 334 143 seabed sediments), 3 subsamples were obtained, as mentioned. For each subsample, 20 335 336 337 144 realizations were taken in order to compute the variability associated of the analyzer 338 339 145 measurement process (Horiba LA940V2 laser). In summary, a total of 60 realizations (3 340 341 146 subsamples x 20 realizations) for each of these 30 sediment samples allowed the 342 343 147 variance-type analysis. 344 345 148 To analyze whether two sediment samples were significantly different, and 346 347 149 having 60 realizations of the size distribution for each sediment sample, a first possible 348 349 150 approach would be to perform a Multivariate ANalysis Of VAriance (MANOVA). It is 350 351 352 353 354 355 356 357 151 Multivariate because results for the samples are not just a given number but 358 359 360 152 distributions with 48 channels (i.e., dimensions). However, each realization is built of 361 362 153 48 numbers that will always satisfy the condition that they sum up to 100, i.e., they are 363 364 154 not independent but compositional data (Aitchison, 1982; Pawlowsky-Glahn and 365 366 155 Egozcue, 2006). This dependency prevents from the use of classical statistical tools to 367 368 156 the raw data. Recent works propose the use of complex compositional data techniques 369 370 157 to transform the raw data so that statistical techniques can be employed (Flood et al., 371 372 158 2015; Martín-Fernández et al., 2015). A simpler approach shows to be useful for this 373 374 159 data set, in particular to circumvent the composition data problem, while guaranteeing 375 376 160 statistical rigor. Here, we consider D for comparison purposes, where D is the 377 50 50 378 379 161 percentile 50% of the grain-sediment distribution: the size so that half of the distribution 380 381 162 (in weight) is above and half below the value. Thus, the whole distribution (48 values 382 383 163 that sum up to 100) is reduced to a single value (D 50 ) which, moreover, is not restricted 384 385 164 to satisfy any condition (contrary to compositional data, which sum up to 100). A 386

387 165 classical one-way ANOVA analyses was used to compare the D 50 of different samples. 388 389 166 This simplification implicitly assumes that two distributions are different (with 390 391 167 statistical significance) if their D are different. However, the contrary is not always 392 50 393 168 true: two distributions not having significantly different D values could we statistically 394 50 395 396 169 different. Given the shape of the distributions in this study, this issue was not a 397 398 170 limitation in our study. Even though it is not strictly necessary, the granulometric 399 400  171 dispersion  = 84 16 was also used to analyze the uniformity of the distributions. 401 402 172 Recall that, by definition, the smaller the value of  , the more uniform is the sediment 403  404 405 173 (Blott and Pye, 2001). 406 407 174 408 409 410 411 412 413 414 415 416 175 3. Results 417 418 419 176 3.1. General description of sediment characteristics 420 421 177 The sediment from P. regalis habitat was mainly composed by a mix of sand 422 423 178 and silt in different proportions, with a small amount of clay, as described in Table 2. 424 425 179 Cases 1, 2, 11 and 12 were classified following (Folk, 1954) as muddy sands; cases 4, 5, 426 427 180 6, 7, 8, 9 and 10 as sandy muds and case 3 as gravelly mud. Gravel was only present in 428 429 181 2 of the 12 samples analyzed (case 3 and 6). 430 431 182 Note that in this section we use data for all 121 + 12 samples (subsampling 432 433 183 analysis is considered below). The data used for calculations is one measurement 434 435 184 (realization) for each of the 133 samples. 436 437 438 185 The size of the ingested particles by P. regalis ranged from 0.103 µm to ~1mm, 439 440 186 while that of the seabed was 0.103 µm to > 3mm. The grain-size sediment distributions, 441 442 187 both for seabed sediments (N = 12) and gut (N = 121 holothurians), had three main 443 444 188 modes, around 10 -0.5 ~ 0.3 µm, 10 0 = 1 µm, and 10 2.5 ~ 300 µm (Figure 1). 445

446 189 As shown in Figure 1, for the largest D 50 values of the seabed sediment (cases 1, 447 448 190 2, 10, 11 and 12), the values of D 50 for the guts were much smaller than the 449 450 191 environmental ones. From Table 3, which shows basic statistics of the samples, the 451 452 192 differences in the D values among the bottom sediments are quite large, ranging from 453 50 454 1.03 2.28 455 193 10 = 10.7 µm for case 5 to 10 = 190.5 µm in case 12, i.e., one order of magnitude. 456 457 194 In contrast, the range of mean values of the gut sediments (mean values computed for 458 459 195 each case using the different specimens) was smaller: from 10 1.00 = 10.0 µm for case 5 460 461 196 to 10 1.59 = 38.9 µm in case 2. 462 463 197 From the above, the sediment within the guts of the holothurians was not much 464 465 198 influenced by the grain-size distribution of the seabed (Figure 1 and Table 3). This is 466 467 199 further emphasized in Figure 2, which shows the relationship between the values of D 50 468 469 470 471 472 473 474 475 200 of the environment (12 samples) and the guts (121 samples) sediment. Working in the 476

477 2 478 201 logarithmic scale, the regression analysis yields R = 0.037, confirming that such a 479 480 202 dependence is very low. Furthermore, there was slightly more variability for the seabed 481 482 203 sediment (log 10 D50 (in µm) = 1.54 ± 0.36, i.e. 15µm ≲ D 50 ≲ 80µm) than that within the 483 484 204 guts (log 10 D50 (in µm) = 1.26 ± 0.20, i.e. 11µm ≲ D 50 ≲ 28µm), as mentioned above. In 485 486 205 addition, the environmental sediment was larger, in average, than the sediment in the 487 488 206 guts. Actually, only a 29.8% of the 121 holothurians had sediment D 50 values larger 489 490 207 than that of their environment, while the individuals with gut contents with smaller D 50 491 492 208 values than the seabed were 70.2% (85 out 121). 493 494 495 209 In regard to using the diameter D 50 (and not another percentile) to discriminate 496 497 210 whether two distributions are similar or not, Figure 3, equivalent to Figure 1 but plotting 498 499 211 the D 75 of the distributions, shows how the trends are similar to those in Figure 1 for 500 501 212 D50 . This is actually a general trend for all percentiles from 25 to 75 (not shown). It is 502

503 213 worth noting that in case 9 the differences appear clearer using D 75 than D 50 . In fact, in 504 505 214 order to show that two distributions are different, it would be enough to find significant 506 507 215 differences for a percentile . 508 509 216 The organic matter content of the seabed ranged between 2.25 and 3.52% (Table 510 511 512 217 2). 513 514 218 515 516 219 3.2. Statistical analysis with subsamples 517 518 220 To determine whether the differences among sediment distributions are 519 520 221 statistically significant or not, an ANOVA test was performed to the 23 subsampled 521 522 222 holothurians (Table 1) and the seabed samples. Figure 4 shows the distribution of the 523 524 223 log 10 D50 both for the seabed sediment and the given holothurian specimen, obtained 525 526 527 528 529 530 531 532 533 534 224 from the 60 realizations for each sample (20 different measures for each of the 3 535 536 537 225 subsamples). 538 539 226 One-way ANOVA results showed that the values of log 10 D50 of the seabed 540 541 227 sediment were significantly different to those for the holothurian gut sediment, except 542 543 228 for 2 individuals (case 03 specimen N01 and case 06 specimen N59). The size of the 544 545 229 sediment within the holothurians is smaller than the ones in the seabed (Figure 4). 546 547 230 Actually, as a general trend, the larger the size of the seabed sediment (cases 01, 02 and 548 549 231 10), the clearer the histograms are differenced. Furthermore, the two cases 03 and 06, 550 551 232 for holothurians whose gut sediment was not significantly different to that of the 552 553 233 seabed, coincided with seabed where the sediment particles were small. Similar results 554 555 556 234 were obtained using D 25 or D 75 instead of D 50 . 557 558 235 The distribution of the granulometric dispersion ( ) for the seabed and the 559 560 236 holothurians gut sediment is shown in Figure 5. One-way ANOVA results showed that 561 562 237  values are statistically different between the seabed sediment and holothurians gut 563 564 238 content in all cases. Unless for very few exceptions (i.e. case 03-specimen N01 and case 565 566 567 239 06specimen N59), the values of  are smaller for the gut sediment than for the 568 569 240 environment, indicating that the gut sediment is more uniform and that holothurians 570 571 241 select the particle size of the sediment during feeding. 572 573 242 Results also show, for each case, significant differences between the specimens 574 575 243 (all them considered as a whole) and the seabed sediment, both in D 50 and  (see last 576 577 244 columns in Figures 4 and 5). Note that the vertical axis is scaled differently than in the 5 578 579 580 245 first columns, given that the number of measurements is increased. The gut sediment, 581 582 246 taken as a whole and thus including the variance created by different specimens, is 583 584 247 significantly different (p < 0.001) from the seabed sediment, with the only exception of 585 586 248  values in case 06. Not surprisingly, the trends are the same as the ones observed for 587 588 589 590 591 592 593 249 each one of the specimens: D is smaller for the gut sediment than for the bottom 594 50 595 596 250 sediment (particularly for the cases with larger particle size in the seabed) and the 597 598 251 sediments within the guts of the animals are more uniform in size than that of the 599 600 252 environment ones. 601 602 253 603 604 254 4. Discussion 605 606 255 Our results showed that Parastichopus regalis selected the sediment by size to 607 608 256 obtain its food and that the use of D 50 , complemented with  , has shown to be, while 609 610 611 257 rigorous, a simple way to compare grain size frequency distributions . The finer particles 612 613 258 were ingested in preference to the larger ones in 85 specimens from a total of 121 614 615 259 analyzed. This pattern of selection was more evident in all the specimens collected in 616 617 260 sediments composed of more than 45% sand . Holothurians collected in five of the seven 618 619 261 sediments with sand content <45% showed the same behavior, with the exception of 620 621 262 case 6 and 7, where gut sediment had higher values of D 50 than the seabed, but selection 622 623 263 of finer particles was the general trend found in most of the cases analyzed. 624 625 264 The role of other sediment characteristics in the particle selection of P. regalis , 626 627 265 628 such as OM content, has not been analyzed in the current study. Nonetheless, our results 629 630 266 showed high selection in case 11, related to sediment with 3.52% OM (the highest in 631 632 267 our study), and also in case 10, corresponding to sediment with 2.31% OM (the lowest 633 634 268 in our study). The OM content values of the seabed sediments here analyzed were quite 635 636 269 similar among them, so OM was not expected to affect the holothurians selection. 637 638 270 The ability of holothurians to select food is believed to vary between species and 639 640 271 habitats (Roberts et al., 2000) with some contradictory results found in the literature 641 642 272 when studying the same species. For example, selectivity by size has been shown in 643 644 273 Stichopus variegatus (Roberts, 1979), Holothuria atra , and H. leucospilota (Massin and 645 646 647 648 649 650 651 652 274 Doumen, 1986). However, no selectivity by size was also reported for these same 653 654 655 275 species by (Levin, 1989) . Information related to feeding rate and sediment reworking on 656 657 276 Mediterranean species is limited to H. tubulosa, H. poli , and H. forskali (Massin and 658 659 277 Jangoux, 1976; Coulon and Jangoux, 1993) . 660 661 278 Holothurians feed on the particles available at the surficial layer of the sediment. 662 663 279 It should be expected that the particles found inside P. regalis were a reflection of the 664 665 280 available particles on the seabed, with potential differences derived from the preferential 666 667 281 selection of a certain range of particles. Our results showed that the correlation between 668 669 282 the values of D of the seabed and the gut sediments was very low. The bigger the 670 50 671 672 283 seabed grain size, the more the animals select the sediment. Accordingly, in seabed 673 674 284 sediments with small grain sizes, the holothurian seem not to select by size as their 675 676 285 preferred sizes are already available. Our results showed that for seabed with larger 677 678 286 sediment sizes, the differences in D 50 values found among the bed and guts sediments 679 680 287 were up to an order of magnitude. The values of  showed that gut sediments were 681 682 288 more uniform than seabed sediments, which confirms particle size selection during 683 684 289 feeding. 685 686 687 290 The particles ingested by P. regalis in the studied area showed a wide size 688 689 291 range; from 0.103 µm up to 1 mm. Khripounoff and Sibuet (1980) suggested that deep- 690 691 292 sea holothurians ingest finer sediment fractions than their shallow water counterparts. 692 693 293 Many abyssal species select particles between 2060 µm (i.e., log 10 D from 1.30 to 1.78) 694 695 294 (Sokolova, 1972; Khripounoff and Sibuet, 1980) whereas some reef dwelling 696 697 295 holothurians (e.g. Holothuria atra ) can select sizes bigger than 500 µm (i.e., log 10 D > 698 699 296 2.70) (Hauksson, 1979; Uthicke, 1999; Uthicke and Karez, 1999) . The influence of 700 701 297 habitat was understood when analyzing the results of two studies carried out with the 702 703 704 298 same species in two different habitats: (Hauksson, 1979)) concluded that shallow-water 705 706 707 708 709 710 711 299 specimens Stichopus tremulus had a preference for fecal pellets (500 µm), whereas 712 713 714 300 Hudson et al. (2004) found that the majority of particles ingested by deep-sea specimens 715 716 301 were <50 µm ( log 10 D 1.70) . It is known that deep-sea sediments generally consist of 717 718 302 finer particles than coastal ones, a fact that can explain these differences in selection 719 720 303 between habitats. 721 722 304 P. regalis is the most abundant species of holothurians found in the continental 723 724 305 shelf between 50-300 m depth in the NW Mediterranean. Thus, the species does not 725 726 306 have to compete for food resources with other deposit feeding holothurians, as usually 727 728 307 occurs in tropical shallow waters (Roberts, 1979) . This would explain its wide range of 729 730 308 preferential particle size as there is no need of partitioning of substrata by different 731 732 733 309 species. 734 735 310 The optimal foraging model assumes that the food abundance of a particle is 736 737 311 proportional to its surface area; i.e., smallest particles are more nutritious (Taghon et al., 738 739 312 1978). Then, the preference for small grains in P. regalis should be expected and it was 740 741 313 confirmed by this study. However, we did not have information on the OM content of 742 743 314 the gut sediment for comparisons with the one from the seabed. 744 745 315 In conclusion, the edible sea cucumber P. regalis has selective feeding toward 746 747 316 fine sediment particles, being this preference more evident in thick-textured than in 748 749 750 317 fine-grained sediments. This conclusion was reached by the use of D 50 , complemented 751 752 318 with , which analysis has shown to be a simple way to compare grain size frequency 753 754 319 distributions. These results are of importance to u nderstand the ecological role of a 755 756 320 valuable seafood resource in the Mediterranean continental shelf. 757 758 321 759 760 761 762 763 764 765 766 767 768 769 770 322 Acknowledgements 771 772 773 323 The authors wish to thank Mariangeles Millán Álvarez and Elena Martínez 774 775 324 Planchart, technical assistants of the sediment analyzer, for their fine work. Also to Juan 776 777 325 José Egozcue and Vera Pawlowsky-Glahn, for their advice, and to the fisherman Jaume 778 779 326 Germà . This research has been supported through the LLONGO project (AGL2011- 780 781 327 25382) financed by the Ministry of Science and Innovation of Spain. 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 328 References 830 831 832 329 Aitchison J. (1982) The statistical analysis of compositional data, Journal of the Royal 833 834 330 Statistical Society: Series B (Methodological) 44 , 13977. 835 836 331 Baskar B.K. (1994) Some observations on the biology of the holothurian Holothuria 837 838 332 scabra , Bull. Cent. Mar. Fish. Res. Inst. 46 , 3943. 839 840 333 Blott S.J., Pye K. (2001) Gradistat: a grain size and statistics package for the analysis of 841 842 334 unconsolidated sediments, Earth Surf. Process. Landf. 26 , 1237-48. 843 844 335 Coulon P., Jangoux M. (1993) Feeding rate and sediment reworking by the holothuroid 845 846 336 Holoturia tubulosa (Echinodermata) in a Mediterranean sea grass bed off Ischia Island, 847 848 337 Italy, Mar. Ecol. Prog. Ser. 92 , 2014. 849 850 851 338 Dar M.A. (2004) Holothurians role in the marine sediments reworking processes, 852 853 339 Sedimentology Egypt 12 , 17383. 854 855 340 Del Rio J.L., Acosta-Yepes J., Cristobo J., Martinez J., Parra-Descalzo S., Tel E., Viñas 856 857 341 L., Muñoz A., Vilela R., Jimenez E.E., Patrocinio T., Ríos P., Almón-Pazos B., Blanco 858 859 342 R., Murillo J., Polonio-Povedano V., Fernández J., Cabanas-López J.M., Gago J., 860 861 343 Gonzalez-Nuevo G., Cabrero-Rodríguez Á.H., Besada V., Schultze F., Franco- 862 863 344 Hernández M.Á. (2012) Estudio de los ecosistemas marinos vulnerables en aguas 864 865 345 internacionales del Atlántico sudoccidental , Madrid (Spain). 866 867 868 346 Dissanayake D.C.T., Stefansson G. (2012) Habitat preference of sea cucumbers: 869 870 347 Holothuria atra and Holothuria edulis in the coastal waters of Sri Lanka, J. Mar. Biol. 871 872 348 Ass. U. K. 92 , 58190. 873 874 349 Flood R.P., Orford J.D., McKinley J.M., Roberson S. (2015) Effective grain size 875 876 350 distribution analysis for interpretation of tidal-deltaic facies: West Bengal Sundarbans, 877 878 351 Sediment. Geol. 318 , 5874. 879 880 881 882 883 884 885 886 887 888 352 Folk R.L. (1954) The distinction between grain size and mineral composition in 889 890 891 353 sedimentary rock nomenclature, J. Geol. 62 , 34459. 892 893 354 García-Arrarás J.E., Greenberg M.J. (2001) Visceral regeneration in holothurians, 894 895 355 Microsc. Res. Tech. 55 , 43851. 896 897 356 Hammond L.S. (1982) Patterns of feeding and activity in deposit-feeding holothurians 898 899 357 and echinoids (Echinodermata) from a shallow back-reef lagoon, Discovery Bay, 900 901 358 Jamaica, Bull. Mar. Sci. 32 , 54971. 902 903 359 Hauksson E. (1979) Feeding biology of Stichopus tremulus, a deposit feeding 904 905 360 holothurian, Sarsia 65 , 15560. 906 907 361 Hudson I.R., Wigham B.D., Solan M., Rosenberg R. (2005) Feeding behaviour of deep- 908 909 910 362 sea dwelling holothurians: Inferences from a laboratory investigation of shallow fjordic 911 912 363 species, J. Mar. Syst. 57 , 20118. 913 914 364 Hudson I.R., Wigham B.D., Tyler P.A. (2004) The feeding behaviour of a deep-sea 915 916 365 holothurian, Stichopus tremulus (Gunnerus) based on in situ observations and 917 918 366 experiments using a Remotely Operated Vehicle, J. Exp. Mar. Biol. Ecol. 301 , 7591. 919 920 367 Khripounoff A., Sibuet M. (1980) La nutrition d'echinodermes abyssaux I. Alimentation 921 922 368 des holothuries, Mar. Biol. 60 , 1726. 923 924 369 Levin V.S. (1989) Trophoecology of holothurians of the nearshore sea zone, P. P. 925 926 927 370 Shirshov Institute of Ocenography, Moscow. 928 929 371 Martín-Fernández J.A., Daunis-i-Estadella J., Mateu-Figueras G. (2015) On the 930 931 372 interpretation of differences between groups for compositional data, SORT 39 , 23152. 932 933 373 Massin C., Doumen C. (1986) Distribution and feeding of epibenthic holothuroids on 934 935 374 the reef flat of Laing Island (Papua New Guinea), Mar. Ecol. Prog. Ser. 31 , 18595. 936 937 938 939 940 941 942 943 944 945 946 947 375 Massin C., Jangoux M. (1976) Observations écologiques sur Holothuria tubulosa , H. 948 949 950 376 poli et H. forskali et comportement alimentaire de H. tubulosa , Cah. Biol. Mar. 17 , 45 951 952 377 59. 953 954 378 Mezali K., Soualili D.L. (2013) The ability of holothurians to select sediment particles 955 956 379 and organic matter, SPC Beche-de-mer Inf. Bull. 33 , 3843. 957 958 380 Navarro P.G., García-Sanz S., Barrio J.M., Tuya F. (2013) Feeding and movement 959 960 381 patterns of the sea cucumber Holothuria sanctori , Mar. Biol. 160 , 295766. 961 962 382 Pawlowsky-Glahn V., Egozcue J.J. (2006) Compositional data and their analysis: an 963 964 383 introduction, in Buccianti A., Mateu-Figueras G., Pawlowsky-Glahn V. (Eds) 965 966 384 Compositional data analysis in the geosciences: from theory to practice , pp. 110. 967 968 969 385 Geological Society Special Publications, London. 970 971 386 Powell E.N. (1977) Particle size selection and sediment reworking in a funnel feeder, 972 973 387 Leptosynapta tenuis (Holothuroidea, Synaptidae), Int. Rev. gesamten Hydrobiol. 62 , 974 975 388 385408. 976 977 389 Ramón M., Lleonart J., Massutí E. (2010) Royal cucumber ( Stichopus regalis ) in the 978 979 390 northwestern Mediterranean: Distribution pattern and fishery, Fish. Res. 105 , 217. 980 981 391 Rhoads D.C., Young D.K. (1971) Animal sediment relations in Cape Cod Bay, 982 983 392 Massachusetts II. Reworking by Molpadja oolitica (Holothuroidea), Mar. Biol. 11 , 255 984 985 986 393 61. 987 988 394 Roberts D. (1979) Deposit feeding mechanisms and resource partitioning in tropical 989 990 395 holothurians, J. Exp. Mar. Biol. Ecol. 15 , 6980. 991 992 396 Roberts D., Gebruk A.V., Levin V., Manship B.A.D. (2000) Feeding and digestive 993 994 397 strategies in deposit-feeding holothurians, Oceanogr. Mar. Biol. Annu. Rev. 38 , 257 995 996 398 310. 997 998 999 1000 1001 1002 1003 1004 1005 1006 399 Rufino M., Baptista P., Pereira F., Gaspar M. (2018) Semi-automatic surface sediment 1007 1008 1009 400 sampling system A prototype to be implemented in bivalve fishing surveys, Cont. 1010 1011 401 Shelf Res. 152 , 715. 1012 1013 402 Slater M.J., Jeffs A.G. (2010) Do benthic sediment characteristics explain the 1014 1015 403 distribution of juveniles of the deposit-feeding sea cucumber Australostichopus mollis ?, 1016 1017 404 J. Sea Res. 64 , 2419. 1018 1019 405 Sloan N.A., Von Bodungen B. (1980) Distribution and feeding of the sea cucumber 1020 1021 406 Isostichopus badionotus in relation to shelter and sediment criteria of the Bermuda 1022 1023 407 platform, Marine Ecology Progress Series 2, 25764. 1024 1025 408 Sokolova M.N. (1972) Trophic structure of the macrobenthos, Mar. Biol. 16 , 1-12. 1026 1027 1028 409 Taghon G.L., Self R.F.L., Jumars P.A. (1978) Predicting particle selection by deposit 1029 1030 410 feeders: a model and its implications, Limnol. Oceanogr. 23 , 7529. 1031 1032 411 Uthicke S. (1999) Sediment bioturbation and impact of feeding activity of Holothuria 1033 1034 412 (Halodeima ) atra and Stichopus chloronotus , two sediment feeding holothuroids, at 1035 1036 413 Lizard Island, Great Barrier Reef, Bull. Mar. Sci. 64 , 12941. 1037 1038 414 Uthicke S., Karez R. (1999) Sediment patch selectivity in tropical sea cucumbers 1039 1040 415 (holothurioidea: aspidochirotida) analysed with multiple choice experiments, J. Exp. 1041 1042 416 Mar. Biol. Ecol. 236 , 69-87. 1043 1044 1045 417 Wigham B.D., Hudson I.R., Billett D.S.M., Wolff G.A. (2003) Is long-term change in 1046 1047 418 the abyssal Northeast Atlantic driven by qualitative changes in export flux? Evidence 1048 1049 419 from selective feeding in deep-sea holothurians, Prog. Oceanogr. 59 , 40941. 1050 1051 420 Yingst J.Y. (1976) The utilisation of organic matter in shallow marine sediments by an 1052 1053 421 epibenthic deposit-feeding holothurian, J. Exp. Mar. Biol. Ecol. 23 , 5569. 1054 1055 422 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 423 Figure 1. Seabed sediment distributions (thick red line) and Parastichopus regalis gut 1066 1067 1068 424 sediment distributions (thin black line). Particle diameters (in µm) at the horizontal axis 1069 1070 425 (in log-10 scale). Vertical lines indicate D 50 value. 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 426 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 427 Figure 2. Diameter D of the environmental sediment versus D of the gut sediment. 1125 50 50 1126 1127 428 Axis in logarithmic scale for D 50 expressed in µm. 1128 1129 429 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 430 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 431 Figure 3. Seabed sediment distributions (thick red line) and Parastichopus regalis gut 1184 1185 1186 432 sediment distributions (thin black line). Particle diameters (in µm) at the horizontal axis, 1187 1188 433 in log-10 scale. Vertical lines indicate D 75 value. 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220 1221 1222 1223 1224 1225 1226 1227 1228 1229 434 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 435 Figure 4. Histograms of D (in µm) corresponding to the 60 realizations for seabed 1243 50 1244 1245 436 (red) and gut (black) sediment for the subsampled samples. The first five columns 1246 1247 437 correspond to a different specimen each (up to 5 for cases 6 and 9), coded as NNo. The 1248 1249 438 sixth column corresponds to the merging of the realizations of all the specimens. The 1250 1251 439 vertical axis is not to scale. Note that the seabed (red) histograms are the same within 1252 1253 440 each case. One-way ANOVA results are displayed inside each graph: significant 1254 1255 441 differences are indicated by (p < 0.05) *, (p < 0.01) **, (p < 0.001) *** and n.s. 1256 1257 442 stands for no significant. 1258 1259 1260 1261 1262 1263 1264 1265 1266 1267 1268 1269 1270 1271 1272 1273 1274 1275 1276 1277 1278 1279 1280 1281 1282 1283 1284 1285 1286 1287 1288 1289 1290 1291 1292 1293 1294 1295 443 1296 1297 1298 1299 1300 1301 444 Figure 5. Histograms of  corresponding to the 60 realizations for seabed (red) and gut 1302  1303 1304 445 (black) sediment for the subsampled samples. The first five columns correspond to a 1305 1306 446 different specimen each (up to 5 for cases 6 and 9), coded as NNo. The sixth column 1307 1308 447 corresponds to the merging of the realizations of all the specimens. The vertical axis is 1309 1310 448 not to scale. Note that the seabed (red) histograms are the same within each case. One- 1311 1312 449 way ANOVA results are displayed inside each graph: significant differences are 1313 1314 450 indicated by (p < 0.05) *, (p < 0.01) **, (p < 0.001) *** and n.s. stands for no 1315 1316 451 significant. 1317 1318 1319 1320 1321 1322 1323 1324 1325 1326 1327 1328 1329 1330 1331 1332 1333 1334 1335 1336 1337 1338 1339 1340 1341 1342 1343 1344 1345 1346 1347 1348 1349 1350 1351 1352 452 1353 1354 453 1355 1356 1357 1358 1359 1360 454 Table 1. Case, number of P. regalis analyzed, sampling depth and number of gut 1361 1362 1363 455 sediment samples that allowed to analyze the within variance. Case 7 correspond to 1364 1365 456 the Ebro river shelf sampling, the rest of samples were collected in Arenys de Mar. 1366 1367 457 1368 1369 Number of 1370 Number of Case Depth (m) subsampled 1371 specimens 1372 specimens 1373 1374 01 4 103 2 1375 02 5 107 4 1376 1377 03 5 92 2 1378 1379 04 10 111 0 1380 05 10 93 0 1381 1382 06 24 99 5 1383 1384 07 11 132 0 1385 08 15 113 2 1386 1387 09 10 108 5 1388 1389 10 9 91 3 1390 1391 11 12 129 0 1392 12 6 82 0 1393 1394 Total 12 Total 121 Total 23 1395 1396 458 1397 1398 1399 1400 1401 1402 1403 1404 1405 1406 1407 1408 1409 1410 1411 1412 1413 1414 1415 1416 1417 1418 1419 459 Table 2. Percentage of each one of the size fractions, textural group (following Folk, 1420 1421 1422 460 1954) classification ) and organic matter content of the sediment where the holothurians 1423 1424 461 P. regalis were captured, with indication of depth, by case. 1425 1426 % sand % silt Textural group 1427 Depth % gravel % clay % organic 1428 Case (2mm- (63µm- 1429 (m) (>2mm) (<4 µm) matter 1430 63µm) 4µm) 1431 1432 1433 01 103 0 52.05 34.52 13.44 Muddy sand 2.78 1434 1435 1436 02 107 0 53.03 34.24 12.73 Muddy sand 2.96 1437 1438 1439 03 92 14.81 37.17 33.93 14.09 Gravelly mud 2.25 1440 1441 1442 04 111 0 16.55 63.97 19.48 Sandy mud 3.1 1443 1444 1445 05 93 0 31.44 52.44 16.12 Sandy mud 3.37 1446 1447 1448 06 99 6.08 32.79 46.36 14.77 Sandy mud 2.90 1449 1450 1451 07 132 0 39.46 41.35 19.19 Sandy mud --- 1452 1453 1454 08 113 0 34.09 54.07 11.84 Sandy mud 2.65 1455 1456 1457 09 108 0 33.87 50.48 15.65 Sandy mud 2.98 1458 1459 1460 10 91 0 46.99 42.81 10.2 Sandy mud 2.31 1461 1462 1463 11 129 0 52.34 35.12 12.55 Muddy sand 3.52 1464 1465 1466 12 82 0 65.48 24.76 9.76 Muddy sand 2.98 1467 1468 1469 462 1470 1471 1472 1473 1474 1475 1476 1477 1478 463 Table 3. D values of the seabed and holothurian guts sediment by case. 1479 50 1480 1481 464 1482 1483 log 10 D50 D50 seabed Mean D 50 1484 log 10 D50 guts Number of 1485 seabed (µm) guts (µm) 1486 case (mean ± standard guts 1487 (µm) 1488 deviation, in µm) analyzed 1489 1490 1491 01 1.85 70.79 1.39 ± 0.05 24.55 4 1492 1493 02 1.89 77.62 1.59 ± 0.13 38.90 5 1494 1495 1496 03 1.52 33.11 1.48 ± 0.25 30.20 5 1497 1498 04 1.25 17.78 1.05 ± 0.13 11.22 10 1499 1500 05 1.03 10.72 1.00 ± 0.05 10.00 10 1501 1502 06 1.24 17.38 1.39 ± 0.17 24.55 24 1503 1504 07 1.25 17.78 1.31 ± 0.15 20.42 11 1505 1506 1507 08 1.31 20.42 1.20 ± 0.07 15.85 15 1508 1509 09 1.29 19.50 1.24 ± 0.08 17.38 10 1510 1511 10 1.70 50.12 1.16 ± 0.10 14.45 9 1512 1513 11 1.87 74.13 1.33 ± 0.20 21.38 12 1514 1515 12 2.28 190.55 1.16 ± 0.13 14.45 6 1516 1517 1518 465 1519 1520 1521 1522 1523 1524 1525 1526 1527 1528 1529 1530 1531 1532 1533 1534