Fish Lateral System Is Required for Accurate Control of Shoaling

Home , Hemigrammus bleheri, Lateral line, Shoaling and schooling

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Animal Behaviour xxx (2010) 1–9

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Animal Behaviour

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55 1 56 2 Fish lateral system is required for accurate control of shoaling behaviour 57 3 58 4 59 a,* a b b a 5 Karine Faucher , Eric Parmentier , Christophe Becco , Nicolas Vandewalle , Pierre Vandewalle 60 6 a Laboratoire de Morphologie Fonctionnelle et Evolutive, Universite´ de Lie`ge 61 7 b De´partement de Physique, Universite´ de Lie`ge 62 8 63 9 64 10 article info 65 11 In teleost fishes, the lateral system is assumed to contribute, among other roles, to maintaining schooling 66 behaviour. Sight is also assumed to play a role in schooling, as fish with a cut lateral line do not stop 12 Article history: 67 Received 8 July 2009 schooling unless they are also blinded. This conclusion, however, is based on experiments where only 13 68 Initial acceptance 8 September 2009 the trunk lateral line was inactivated, leaving the head lateral system intact. We investigated how 14 69 Final acceptance 9 December 2009 inactivation of the whole lateral system affects fish shoaling behaviour. Groups of firehead tetras, 70 15 Available online xxx Hemigrammus bleheri, were videorecorded before and after inactivation of their whole lateral system 16 MS. number: 09-00464R with aminoglycoside antibiotics (and also in sham-treated specimens). Shoaling behaviour was 71 17 characterized by nearest distance to thePROOF first, second and third neighbours, shoal radius, shoal order 72 18 Keywords: parameter and the number of collisions between individuals. Scanning electron microscope observations 73 firehead tetra 19 showed damage to most superficial neuromasts as a result of antibiotic treatment. Importantly, the 74 fish antibiotic-treated fish proved unable to maintain a shoal. After the end of the treatment, however, they 75 20 hair cell bundle 21 Hemigrammus bleheri recovered both a normal tissue morphology and normal shoaling behaviour within about a month. The 76 22 lateral system lateral system is thus more crucial to shoaling behaviour than previously believed. 77 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. 23 neuromast 78 shoaling behaviour 24 79 25 80 26 81 27 82 28 In fish, the lateral system is involved in many behaviours, migration, spawning and predator avoidance (Partridge et al. 1980; 83 29 such as predator and prey detection (Hoekstra & Janssen 1986; Pitcher & Parrish 1993), and may reduce energy costs owing to 84 30 Montgomery 1989; Janssen et al. 1999), rheotaxis (Montgomery hydrodynamic interactions (Weihs 1973; Svendsen et al. 2003). It 85 31 et al. 1997; Northcutt 1997), obstacle avoidance (Blaxter & Batty seems that the primary function of school structure is to position 86 32 1985) and intraspecific interactions (Partridge & Pitcher 1980). The individuals so that they can most quickly respond to their neigh- 87 33 functional units of the lateral system are mechanoreceptors, the bours (Partridge et al. 1980). For each schooling fish species, there is 88 34 neuromasts, distributed over the head, trunk and tail of the fish a specific preferred distance to the nearest neighbour, which is 89 35 (Coombs et al. 1988). usually about one body length (Partridge 1982). Until now, it has 90 36 The lateral system is notably believed to play a role in the been commonly accepted that both the lateral line and vision are 91 37 cohesive swimming behaviours, called shoaling or schooling, involved in collective swimming behaviours (Pitcher et al. 1976; 92 38 displayed by most of the 24 000 species of teleost fish at some Partridge 1982; Pitcher 1983, 1998; Pitcher & Parrish 1993). On the 93 39 period in their lives (Pitcher 1998). The difference between shoals one hand, blinded saithe, Pollachius virens, with an intact lateral line 94 40 and schools has been defined in previous studies (Pitcher 1983). are able to maintain their positions in a school, but with greater 95 41 Shoaling applies to fish swimming together in an unstructured distances between neighbours (Pitcher et al. 1976; Partridge 1982). 96 42 manner, whereas schooling implies synchronized and polarized On the other hand, saithe with normal vision but with the lateral 97 43 swimming. There is no clear dichotomy between schooling and line cut at the level of the operculum can also maintain the shoal, 98 44 shoaling, since all schools are shoals by definition (Pitcher 1983). but with closer distances between neighbours (Partridge & Pitcher 99 45 The tendency to form shoals or schools varies both between species 1980; Partridge 1982). After trunk lateral line section, however, 100 46 (according to the ecological niche) and within species (according to disruption of the saithe school by deliberate startling leads to 101 47 the motivational state). These collectiveUNCORRECTED swimming behaviours may collisions with sufficient force to stun the fish (Partridge & Pitcher 102 48 facilitate many ecological functions, such as foraging (Breder 1959), 1980). These results have led to the conclusion that altering their 103 49 sensory capabilities by lateral line inactivation or temporary 104 50 blindfolding merely results in the fish taking up different positions 105 * Correspondence: K. Faucher, Laboratoire de Morphologie Fonctionnelle et with respect to their neighbours, without stopping schooling 106 51 Evolutive, Universite´ de Lie`ge, Institut de Chimie – B6C, Sart Tilman, 4000 Lie`ge, 52 Belgium. behaviour. Schooling behaviour stops only when the fish are both 107 53 E-mail address: [email protected] (K. Faucher). blinded and have their lateral lines cut (Pitcher et al. 1976; Partridge 108 54 109 0003-3472/$38.00 2009 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. 110 doi:10.1016/j.anbehav.2009.12.020

Please cite this article in press as: Faucher, K., et al., Fish lateral system is required for accurate control of shoaling behaviour, Animal Behaviour (2010), doi:10.1016/j.anbehav.2009.12.020 ARTICLE IN PRESS YANBE18556_proof 2 January 2010 2/9

2 K. Faucher et al. / Animal Behaviour xxx (2010) 1–9

111 1982). A potential pitfall of all these studies is that they concluded not the inner ear when administered by immersion (Matsuura et al. 176 112 that inactivation of the trunk lateral line equated to inactivation of 1971). Gentamicin sulphate and streptomycin sulphate were used to 177 113 the whole lateral system. Indeed, in these studies only the trunk inactivate selectively canal and superficial neuromasts, respectively 178 114 lateral system was inactivated (Pitcher et al. 1976; Partridge & (Blaxter & Fuiman 1989; Song et al. 1995; Coombs et al. 2001). This 179 115 Pitcher 1980; Partridge 1982), as each lateral line was cut just method affected the whole fish lateral system. The water was 180 116 behind the operculum. It seems that the cephalic part of the lateral completely changed each day and fresh aminoglycoside antibiotics 181 117 system, well developed in schooling fish (Blaxter et al. 1983; were added. At the end of each treatment day, the fish were care- 182 118 Janssen et al. 1995; Pitcher 2001; Diaz et al. 2003), was left intact. fully rinsed before being placed in their maintenance tanks. At the 183 119 Nevertheless, it has been shown that the entire lateral system is end of the treatment, two specimens were collected, anaesthetized 184 120 involved in the determination of the distance and direction of with 75 mg/litre MS 222, and killed for SEM observation of the 185 121 a stimulus (Janssen & Corcoran 1998), which is useful for a fish to lateral system. Sham fish were subjected to the same handling for 4 186 122 maintain its position relative to its neighbours in a school. days, but without any antibiotic. After the 4 days, two specimens 187 123 Our aim in the present study was to assess the involvement of were collected, anaesthetized with 75 mg/litre MS 222, and killed 188 124 the lateral system in the shoaling behaviour of firehead tetra, for SEM observation of the lateral system, to check the effect of 189 125 Hemigrammus bleheri (Characidae) when their whole lateral system frequent handling on the neuromasts. 190 126 was inactivated by treatment with aminoglycoside antibiotics, as After lateral-system inactivation, we randomly collected 10 fish 191 127 checked by scanning electron microscopy (SEM). from each group (sham or treated) and recorded their swimming 192 128 behaviour three times a week (same days for both groups) until 193 129 METHODS restoration of normal shoaling behaviour in the treated fish. At the 194 130 end of each week after the start of treatment, two treated fish were 195 131 Animals collected, anaesthetized and killed to observe their lateral line 196 132 system by SEM. At the end of the experiment, two sham fish were 197 133 Hemigrammus bleheri was chosen because: (1) it shoals also killed for SEM observations. 198 134 frequently, (2) it is easy to obtain, and (3) it is easy to maintain in 199 135 the laboratory. Sixty specimens (average length ¼ 3.4 0.2 cm) SEM Lateral Line System Observations 200 136 provided by the breeder Bassleer (Westmeerbeek, Belgium) were PROOF 201 137 housed for 1 week before the experiment in two 120-litre tanks To check the efficiency of sensory inactivation by aminoglyco- 202 138 with oxygenated freshwater in a recycled system. Water tempera- side antibiotics, we examined by SEM superficial neuromasts 203 139 ture was maintained at 26 C. The fish were fed three times a week from the trunk lateral line system in controls and both sham- and 204 140 with frozen artemias. The photoperiod was 9:15 h light:dark. antibiotic-treated fish. As the scales were fragile, the canal roofs of 205 141 the modified scales composing the trunk lateral line system could 206 142 Swimming Behaviour Recording not be removed without tissue deterioration, so very few canal 207 143 neuromasts were observed. The difficulty of observing these 208 144 We recorded the swimming behaviour of control fish with neuromasts under good conditions led us to exclude them from our 209 145 a numerical video camera (25 frames/s) three times a week for 3 observations and to assume that the damage to canal neuromasts 210 146 weeks (2 min per video session). For each video session, we mirrored the state of the superficial neuromasts following antibi- 211 147 randomly chose 10 of the 60 fish. They were placed in a Plexiglas otic or sham treatment. Both trunk lateral lines were sampled, 212 148 circular tank (diameter 67 cm). A small round cylinder (diameter immediately fixed in 4% glutaraldehyde (Fisher Scientific Labosi, 213 149 33 cm) placed in the middle of the tank and associated with a slight Elancourt, France) in sodium cacodylate buffer (0.4 M, pH 7.2), and 214 150 current produced by a water pump (100 litres/h) was used to incite then dehydrated in a graded ethanol series and critical point dried 215 151 shoaling behaviour. The water height was limited to 3 cm to restrict with liquid CO2. They were then mounted on brass supports and 216 152 fish shoaling to two dimensions. The experimental tank was lit with sputter-coated with platinum (SCD 030 Balzers Union, FL 9496, 217 153 neon tubes, but the surface of the tank was covered with paper to Balzers, Lichtenstein). Blind observation was ensured by numbering 218 154 avoid light reflexion at the water surface. A mirror placed under the the various samples, so that the viewer would not know if they 219 155 tank enabled the video camera to record pictures from underneath. were from control, sham or treated fish. Observations were 220 156 The fish were allowed to settle for about 5 min before recording. performed with a JEOL JSM 840A scanning electron microscope. 221 157 At the end of each week, two fish were killed by over- 222 158 anaesthetizing with 75 mg/litre MS 222 (3-aminobenzoic acid Data Analysis 223 159 ethyl, SIGMA A-5040). To ensure brain death, we left fish in the 224 160 anaesthetic for about an extra 10 min after they stopped reacting We analysed the videos collected as picture sequences with Java 225 161 visibly to physical stimuli. Their trunk lateral line system was then ImageJ version 1.39u software (http://rsb.info.nih.gov/ij). For this, 226 162 observed by SEM. the picture background was eliminated by (iteratively) subtracting 227 163 two randomly chosen pictures, subtracting the whole picture 228 164 Lateral-System Inactivation sequence from the background, and inverting black and white. Each 229 165 UNCORRECTEDfish was thus represented by a black trace on a white background. 230 166 After 3 weeks of recording under control conditions, we divided Fish trajectories were calculated by the plugin MultiTraFish (Java 231 167 the fish into two groups: (1) lateral-system-inactivated fish (N ¼ 32) language program written by C. Becco) of ImageJ (Becco et al. 2006). 232 168 and (2) sham-inactivated (sham) fish (N ¼ 22). The whole lateral Each fish was identified by a number that remained the same from 233 169 system of the first fish group was inactivated by immersion of the one picture to another, so that the position of each fish was 234 170 animals in small tanks containing water from their maintenance obtained according to the time. Speeds were then determined from 235 171 tank supplemented with aminoglycoside antibiotics: 0.02 g/litre differences between consecutive pictures. Despite the low water 236 172 gentamicin sulphate (SIGMA G-3632; Song et al.1995; Coombs et al. height, some fish were able to cross each other’s paths, but the 237 173 2001) and 0.5 g/litre streptomycin sulphate (SIGMA S-9137; Blaxter algorithm was able to dissociate fish identity from fish speed and 238 174 & Fuiman 1989) for 8 h per day over a 4-day period. Aminoglycoside surface variation. After using this algorithm, we calculated several 239 175 antibiotics are known to damage specifically the lateral system but parameters. 240

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241 (1) Interindividual distances: the distances of each fish to its 0° 306 242 first (NND1), second (NND2) and third (NND3) nearest neighbours 307 243 were calculated and expressed in fish body length units (BLU). 308 244 309 These distances were averaged for each video session and − 45° 45° 245 compared between groups (control, sham and treated fish) by 310 246 means of Kruskal–Wallis and two-tailed Mann–Whitney tests. For 311 247 the latter, the U statistic value corresponded to the larger value 312 248 between U1 (sum of the ranks for sample 1) and U2 (sum of the 313 249 ranks for sample 2). 314 250 (2) Shoal radius r: the gyration radius, that is, the average 315 251 distance of all fish to the shoal mass centre (D ), was first esti- 316 max − 252 mated. Assuming a homogeneous distribution of fish inside the 90° 90° 317 253 shoal and a circular shoal, the shoal radius was calculated as: 318 254 319 255 D 3 Figure 1. The shoal order parameter was estimated from fish body orientations on 100 320 r ¼ max (1) 256 2 pictures selected from each video session. Fish swimming in the same main direction 321 257 (from 90 to þ90 , indicated with a tick) were numbered whereas those swimming 322 The r values obtained were averaged for each video session and in different directions (indicated with a cross) were not considered. 258 323 compared between groups with the Mann–Whitney test. 259 324 (3) Degree of distribution: to describe the pattern of distribution 260 325 exhibited by the fish population (random, regular or aggregative) in software (Addinsoft, Paris, France). The significance level was set at 261 326 the experimental tank, the average distance from each individual to P 0.05. 262 327 its nearest neighbour (NND1) was used according to the method of 263 328 Clark & Evans (1954). The fish density (r) was calculated. The 264 Ethical Note 329 average distance to the nearest neighbour that would be expected if 265 330 the individuals were randomly distributed (NND1T) was also 266 ThroughoutPROOF the experiment, mortality was low and did not 331 calculated: 267 exceed the average rate that we have observed in the species in 332 captivity. Twenty-two fish (six controls, four sham and 12 treated 268 1 333 NND ¼ pffiffiffi (2) fish) out of 60 were killed to observe the tissue state of their lateral 269 1T 2 r 334 270 line system in SEM. The number of fish killed in the treated group 335 271 The ratio R of the observed average distance to the expected was higher than in other groups, to examine the changes in their 336 272 average distance was calculated and used as a measure of departure lateral line system tissue state with time. Two treated fish were 337 273 from randomness (Clark & Evans 1954): killed at the end of each week to enable a comparison with sham 338 274 fish over time. 339 275 The aminoglycoside antibiotic treatment by intramuscular or 340 NND1 276 R ¼ (3) intravenous injection is well known to involve some health disor- 341 NND 277 1T ders (loss of appetite, erratic swimming behaviour, bloating of the 342 abdomen, nephrotoxicity, haemorrhages) leading to the death of 278 In a random distribution, R ¼ 1. When R is between 0 and 1, the 343 the animals (Yan et al. 1991; Lombarte et al. 1993; Faucher et al. 279 distribution is aggregative and when R is between 1 and 2.1491, the 344 2008a). However, administering the same antibiotic treatment by 280 individuals present a regular distribution (Clark & Evans 1954). 345 immersion did not present any systemic side-effects, as has 281 Shapiro–Wilk normality tests performed on the R values confirmed 346 previously been described (Faucher et al. 2008b). For example, no 282 the normal distribution of data in the control (P ¼ 0.242), sham 347 mortality from this antibiotic treatment was recorded, and no fish 283 (P ¼ 0.928) and treated fish (P ¼ 0.496). A Student’s t test was used 348 were observed swimming on their sides, either in the sham or 284 to test the significance of the departure of R values from 1. 349 treatment groups. Indeed, the only differences in swimming 285 (4) Shoal order parameter: to estimate the quality of the 350 behaviour observed are those presented in the Results. 286 shoaling behaviour, we analysed fish body orientations. For this, 351 The experiments were carried out under the approval of the 287 100 pictures out of 3000 from each video session were sequentially 352 Animal Care Committee of the University of Lie`ge. After the study, 288 selected (1/30) and examined. The number of fish (out of 10) 353 fish were retained in captivity to be used in other studies. 289 swimming in the same gross direction (from 90 to þ90; Fig. 1) 354 290 was calculated and recorded as the average percentage SD of 355 291 shoaling fish. Chi-square tests were used to compare average RESULTS 356 292 percentages of shoaling fish for control, sham and treated fish. 357 293 (5) Number of collisions: in all groups and for each video Interindividual Distances 358 294 session, fish collisions were counted and expressed per min. The 359 295 data obtained were compared betweenUNCORRECTED groups. Figure 2 shows the changes over time in average distances to the 360 296 (6) The average number of superficial neuromasts damaged was first (NND1, Fig. 2a), second (NND2, Fig. 2b) and third closest 361 297 calculated and expressed as a percentage. Chi-square tests were neighbour (NND3, Fig. 2c) in each group. In sham fish, the average 362 298 then used to compare the percentages recorded for the three NND1 (1.45 0.22 BLU, N ¼ 15) did not differ significantly from that 363 299 different groups. Intact neuromasts showed sensory maculae determined for the 3 weeks of recording under control conditions 364 300 provided with well-developed hair bundles from subjacent sensory (1.36 0.13 BLU, N ¼ 9; U ¼ 51.00, N1 ¼ 15, N2 ¼ 9, P ¼ 0.325; 365 301 hair cells. Neuromasts were considered damaged when their hair Fig. 2a). In contrast, the average NND1 measured in treated fish 366 302 cell bundles were sparse, shortened or not visible. (2.49 0.46 BLU, N ¼ 5) was significantly higher than in sham fish 367 303 Quantitative data are expressed as the mean SD followed (1.23 0.11 BLU, N ¼ 5) up to day 14 (U ¼ 25.00, N1 ¼ N2 ¼ 5, 368 304 by the number of data used to calculate the mean. All statistical P ¼ 0.01), but not from day 16 onwards (U ¼ 26.00, N1 ¼ N2 ¼ 10, 369 305 tests were conducted with the Xlstat-Pro 6.0 statistical analysis P ¼ 0.070). 370

4 K. Faucher et al. / Animal Behaviour xxx (2010) 1–9

371 12 436 372 (a) Control 437 373 10 Sham 438 374 Treated 439 375 440 8 ** NS

376 (BLU) 441 1 377 442 378 6 443 379 444 380 4 445 381 446 Average NND 382 2 447 383 448 384 0 449 − − − − − − − − − 385 21 19 17 14 12 10 7 5 30 4 7 9 11 14 16 18 21 23 25 27 29 31 34 36 450 386 12 451 Control 387 (b) 452 388 10 Sham 453 389 Treated 454 NS 390 8 * 455

391 (BLU) 456 2 392 457 6 393 458 394 459 395 4 460

396 Average NND PROOF 461 397 2 462 398 463 399 0 464 −21 −19 −17 −14 −12 −10 −7 −5 −3 0 4 7 9 111416182123252729313436 400 465 401 12 466 Control 402 (c) 467 Sham 403 10 * NS 468 404 Treated 469 405 8 470 (BLU)

406 3 471 407 6 472 408 473 409 474 4 410 475

411 Average NND 476 412 2 477 413 478 414 0 479 −21 −19 −17 −14 −12 −10 −7 −5 −3 0 4 7 9 111416182123252729313436 415 480 Time (days) 416 481 417 Figure 2. Changes in interindividual distances (body length units, BLU) over time in the three groups of ﬁsh (control, sham and treated). Day 0 is the ﬁrst day of the 4-day sham or 482

418 antibiotic treatment. (a) Average distance to the first nearest neighbour (NND1). (b) Average distance to the second nearest neighbour (NND2). (c) Average distance to the third 483 419 nearest neighbour (NND3). Vertical bars represent SDs. The solid line indicates the start of the treatment. The dashed vertical line corresponds to the time at which the difference 484 420 between treated and sham fish ceased to be significant. *P < 0.05; **P 0.01. 485 421 486 422 487

423 The average NND2 was also comparable (U ¼ 46.00, N1 ¼ 9, than for the sham fish (2.38 0.05 BLU, N ¼ 3) up to day 9 if 488 424 N2 ¼ 15, P ¼ 0.200; Fig. 2b) in control (1.99 0.10 BLU, N ¼ 9) and a significance level of 10% is exceptionally considered in this case. 489 425 sham fish (2.14 0.11 BLU, N ¼ 15)UNCORRECTED and significantly higher in Thereafter, the NND3 decreased in the treated fish, so that no 490 426 antibiotic-treated fish (4.37 0.78 BLU, N ¼ 4) than in sham fish significant difference was observed between treated and sham fish 491 427 (1.82 0.08 BLU, N ¼ 4; U ¼ 16.00, N1 ¼ N2 ¼ 4, P ¼ 0.05) up to day from day 11 onwards (U ¼ 41.00, N1 ¼ N2 ¼ 12, P ¼ 0.073). 492 428 11. Like the average NND1, the average NND2 gradually decreased in 493 429 treated fish from day 4 to the end of the experiment, the difference Shoal Radius 494 430 between these fish and sham fish becoming nonsignificant from 495 431 day 14 onwards (U ¼ 31.00, N1 ¼ N2 ¼ 11, P ¼ 0.053). The average radius of the shoal was about 15.27 2.86 cm 496 432 The NND3 values were similar (U ¼ 47.00, N1 ¼ 9, N2 ¼ 15, (N ¼ 9) in control fish and 15.10 3.70 cm (N ¼ 15) in sham fish, 497 433 P ¼ 0.222; Fig. 2c) for control (2.63 0.10 BLU, N ¼ 9) and sham fish the difference between these groups being nonsignificant 498 434 (2.89 0.16 BLU, N ¼ 15) but higher (U ¼ 9.00, N1 ¼ N2 ¼ 3, (U ¼ 70.00, N1 ¼ 9, N2 ¼ 15, P ¼ 0.881; Fig. 3a). Antibiotic-treated 499 435 P ¼ 0.10) for the antibiotic-treated fish (6.02 0.60 BLU, N ¼ 3) fish showed a significantly higher average shoal radius 500

K. Faucher et al. / Animal Behaviour xxx (2010) 1–9 5

501 40 566 502 (a) Control 567 * NS 503 35 Sham 568

504 , cm) Treated 569 r 30 505 570 506 25 571 507 572 20 508 573 509 15 574 510 575 10 511 576

512 Average shoal radius ( 5 577 513 578 0 514 −25 −20 −15 −10 −5 0 5 10 15 20 25 30 35 40 579 515 580 516 581 517 (b) Control 582 2 518 Sham 583 ) Regular 519 R Treated 584 520 Expected R 585 521 1.5 586 522 ** NS 587 523 588 524 1 Random 589 525 590 526 PROOF 591 527 0.5 592 Degree of distribution ( 528 593 529 Aggregative 594 530 0 595 − − − − − 531 25 20 15 10 5 0 5 10152025303540 596 532 Time in days 597 533 598 Figure 3. (a) Average shoaling radius in control, sham and treated fish. Day 0 is the first day of the 4-day sham or antibiotic treatment. Vertical bars represent SDs. (b) Fish 534 599 distribution inside the experimental tank. The horizontal dotted line represents the value 1, corresponding to a random distribution. The solid line indicates the start of the 535 treatment. The vertical dashed line corresponds to the time at which the difference between treated and sham fish ceased to be significant. *P 0.05; **P 0.01. 600 536 601 537 602 538 (25.97 4.27 cm, N ¼ 4; U ¼ 16.00, N1 ¼ N2 ¼ 4, P ¼ 0.05) than (97.54 1.97%, N ¼ 1500 pictures) among the sham fish than in the 603 2 539 sham fish (11.90 1.82 cm, N ¼ 4) up to day 11. From day 14 control group (c1 ¼ 148.865, P < 0.0001). Just after antibiotic 604 540 onwards, this parameter decreased in the group of treated fish until treatment, in contrast, only 63.30% of the treated fish (N ¼ 100 605 541 no significant difference from sham fish was observed (U ¼ 41.00, pictures) swam in the same direction, this being a significantly 606 542 N1 ¼ N2 ¼ 11, P ¼ 0.20). lower percentage than in the sham group on the same day (96.20%, 607 2 543 N ¼ 100 pictures; c1 ¼ 335.124, P < 0.0001). After day 4, the 608 544 Degree of Random Distribution percentage of fish swimming together increased daily in the treated 609 545 group (Fig. 4a), so that the difference between antibiotic-treated 610 2 546 The average degree of random distribution was 0.58 0.05 and sham fish was no longer significant (c1 ¼ 0.922, P ¼ 0.337) 611 547 (N ¼ 9) for controls and 0.62 0.09 (N ¼ 15) for sham fish (Fig. 3b). from day 29 onwards (98.75 1.21%, N ¼ 400 pictures versus 612 548 These values below 1 are indicative of an aggregative distribution. 98.50 1.75%, N ¼ 400 pictures). 613 549 From day 4 to day 18, the average R value calculated for the anti- 614 550 biotic-treated fish (0.88 0.20, N ¼ 7) was significantly higher than Number of Collisions per Min 615 551 that calculated for sham fish (0.58 0.10, N ¼ 7; t6 ¼4.630, 616 552 P ¼ 0.002). From day to day, the treated fish displayed a distribution In controls and in sham fish, no collision was observed at any 617 553 changing from regular (from day 4 to day 7) to random (day 9) and time during the experiment. In contrast, treated fish showed about 618 554 then aggregative (from day 11 to day 36). The R value difference 3 collisions/min from day 4 to day 9, sometimes colliding with 619 555 between treated and sham fish ceasedUNCORRECTED to be significant on day sufficient force to stun themselves. After day 9, this swimming 620 556 21 (0.74 0.11, N ¼ 8 versus 0.66 0.06, N ¼ 8; t7 ¼1.381, behaviour disorder gradually decreased, disappearing totally by 621 557 P ¼ 0.210), by which time both populations displayed an aggrega- day 25. 622 558 tive distribution. 623 559 Observation by SEM of the Lateral Line System 624 560 Shoal Order Parameter 625 561 The lateral line system was distributed over the whole body. At 626 562 Over the 3 weeks of recording under control conditions, the level of the head, canals were observed with pores allowing 627 563 94.51 3.06% (N ¼ 900 pictures) of the fish were found to swim in communication with the external medium. Some superficial neu- 628 564 the same direction (Fig. 4a); the remainder tended to disperse. Over romasts were also seen clustered in patches in the supraorbital, 629 565 the next 3 weeks, the percentage of shoaling fish was higher nasal, mandibular and preopercular areas (Fig. 5a). In all 630

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631 696 632 100 (a) 697 633 698 634 699 635 80 700 636 701 637 702 60 638 *** NS 703 639 704 640 705 641 40 706 % Fish shoaling 642 707 643 Control 708 20 644 Sham 709 645 710 Treated 646 711 0 647 −25 −20 −15 −10 −5 0 5 10152025303540 712 648 713 Time (days) 649 714 650 715 651 716 (b) N = 2 652 717 n = 90 653 718 N = 2 654 100 *** 719 n = 100 655 720 *** 656 80 N = 2PROOF 721 657 n = 102 722 658 723 60 *** 659 724 660 N = 2 725

% SN damaged n = 100 661 40 726 662 *** N = 2 N = 2 727 N = 6 N = 4 663 20 n =102 n = 100 728 664 n = 102 n = 102 NS NS 729 665 730 0 666 Control Sham D3 D11 D18 D25 D31 D36 731 667 732 668 Days post-treatment 733 669 Fish 734 670 735 Figure 4. (a) Shoal order parameter expressed as the percentage of fish shoaling together in the same gross direction (from 90 to þ90) in the three groups (control, sham and 671 736 treated fish). Day 0 is the first day of the 4-day sham or antibiotic treatment. The solid line indicates the start of the treatment. The vertical dashed line corresponds to the time at 672 which the difference between treated and sham fish ceased to be significant. (b) Percentage of damaged superficial neuromasts (SN) in the three groups of fish (control, sham and 737 673 treated fish). N and n correspond to the number of fish and the number of superficial neuromasts observed, respectively. Vertical bars represent SDs. ***P < 0.0001. 738 674 739 675 740 676 specimens, the trunk lateral line was restricted to a single row of neuromasts represented a much higher percentage of the 741 677 mid-flank modified scales, with a canal located in the anterior part observed superficial neuromasts (96.7%, N ¼ 2 fish; Fig. 4b) than in 742 2 678 of each flank (Fig. 5a). The canal appeared open to the external sham fish (c1 ¼ 62.579, N ¼ 6 fish, P < 0.0001). After day 3, the 743 679 medium through a series of pores (see inserts, Fig. 5a). The few percentage of damaged superficial neuromasts gradually 744 680 canal neuromasts that could be observed were located in the first decreased. The difference between treated and sham fish 745 681 third of the canal on each modified scale. The trunk lateral system remained significant on days 11, 18 and 25, when the percentage of 746 682 additionally comprised round superficial neuromasts along the damaged neuromasts was, respectively, 78.0% (N ¼ 2 fish), 53.9% 747 683 whole body of the fish. They were numerous (two to five) on the (N ¼ 2 fish), and 28.0% (N ¼ 2 fish; significance tests yielding, 748 2 2 684 row of modified scales but were also observed, in lesser number respectively, c1 ¼ 51.881, N ¼ 6 fish, P < 0.0001; c1 ¼ 35.531, N ¼ 6 749 2 685 (zero to two), in the rows of scalesUNCORRECTED located beneath and above this fish, P < 0.0001; c1 ¼ 16.094, N ¼ 6 fish, P < 0.0001). On day 18 750 686 line. In both controls (Fig. 5b) and sham fish (Fig. 5c), most (Fig. 4b), the hair cell bundles appeared sparse and shortened, and 751 687 superficial neuromasts appeared intact, possessing well-developed some cuticular plates could be seen (Fig. 5e). By day 25, they were 752 688 hair cell bundles. Damaged neuromasts displaying sparse or less sparse but still slightly shortened (Fig. 5f). The percentages of 753 689 shortened hair cell bundles represented only 2.0% (N ¼ 6 fish) of damaged neuromasts observed on days 31 and 36 (respectively, 754 690 the superficial neuromasts in controls and 3.9% (N ¼ 4 fish) in 5.9%, N ¼ 2 fish and 4.0%, N ¼ 2 fish; Fig. 4b) no longer differed 755 691 sham fish (Fig. 4b). These percentages were not significantly significantly from those observed in sham fish (statistical tests 756 2 2 2 692 different (c1 ¼ 0.648, N ¼ 10 fish, P ¼ 0.421). In contrast, at the end yielding, respectively, c1 ¼ 0.381, N ¼ 6 fish, P ¼ 0.537; c1 ¼ 0.001, 757 693 of treatment (day 3) the aminoglycoside-treated fish had many N ¼ 6 fish, P ¼ 0.978). The hair cell bundles appeared intact, that is, 758 694 greatly damaged superficial neuromasts, whose hair cell bundles similar to those of control and sham-treated fish, from day 31 759 695 were shortened or even no longer visible (Fig. 5d). These onwards (Fig. 5g). 760

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761 826 762 827 763 828 764 829 765 830 766 831 767 832 768 833 769 834 770 835 771 836 772 837 773 838 774 839 775 840 776 841 777 842 778 843 779 844 780 845 781 846 782 847 783 848 784 849 785 850 786 PROOF 851 787 852 788 853 789 854 790 855 791 856 792 857 793 858 794 859 795 860 796 861 797 862 798 863 799 864 800 865 801 866 802 867 803 868 804 869 805 870 806 871 807 872 808 873 809 874 810 875 811 876 812 877 813 878 814 879 815 UNCORRECTED 880 816 881 817 882 818 883 819 884 Figure 5. SEM pictures of the firehead tetra lateral line system. (a) Lateral system showing the cephalic pores (white arrowheads) and the trunk canal (*) running along the mid- 820 885 section of each flank and located only on the anterior part of the fish’s body. The three inserts located underneath show high magnifications of two types of scales: (1) a normal scale 821 showing superficial neuromasts (white arrowheads) in large numbers, (2) a modified scale showing the pore of the canal, also provided with some superficial neuromasts (white 886 822 arrowheads) and (3) a cephalic superficial neuromast. (b) Well-developed superficial neuromast hair cell bundles of a control fish. (c) Intact superficial neuromast of a sham fish. (d) 887 823 Greatly damaged superficial neuromast, with very few hair cell bundles, of an antibiotic-treated fish just after treatment. (e) Superficial neuromast of treated fish on day 18 showing 888 824 some signs of regeneration: shortened hair cell bundles were observed. (f) Superficial neuromast on day 25, appearing less damaged but with shorter hair cell bundles than in sham 889 fish. (g) Superficial neuromast on day 36, similar to those of sham and control fish. 825 890

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891 DISCUSSION species that subsequent completion of the buccal lateral lines 956 892 coincides with the appearance of a well-established schooling 957 893 In this study on the characin H. bleheri, we have sought to avoid behaviour (Masuda & Tsukamoto 1996). 958 894 a potentially misleading pitfall of studies aiming to assess the role In conclusion, this is the first study to demonstrate that the 959 895 of the lateral system in the cohesive swimming behaviours of whole lateral system is crucial to shoaling behaviour. Further 960 896 teleosts. Such studies have relied on inactivating only the trunk studies are required to establish the precise role of the anterior 961 897 lateral line, leaving the head lateral system intact. They have led to lateral system in fish shoaling/schooling. 962 898 the commonly accepted view that disruption of the fish lateral 963 899 system does not cause shoals or schools to disband, but leads to 964 Acknowledgments 900 closer distances between neighbours (Pitcher et al. 1976; Pitcher 965 901 1979; Partridge & Pitcher 1980; Burgess & Shaw 1981; Partridge 966 We thank N. Decloux for preparing lateral line samples for 902 1982, 2002). Furthermore, because cohesive swimming did not 967 critical point drying and platinum sputter-coating. We also thank 903 stop unless both sight and the lateral system were inactivated, 968 the CATm (Cellule d’Appui Technologique en Microscopie, 904 it was proposed that sight may be of prime importance in 969 Universite´ de Lie`ge) for allowing the use of a scanning electron 905 maintaining a particular position and angle with respect to shoal/ 970 microscope. This study was funded thanks to a grant from the 906 schoolmates, whereas the lateral line may be important in 971 Fonds de la Recherche Fondamentale Collective (FNRS/FRFC 907 monitoring their swimming speeds and directions (Pitcher 1979; 972 2.4.569.06.F). 908 Partridge & Pitcher 1980). 973 909 We used gentamicin and streptomycin to inactivate the whole 974 910 lateral system in H. bleheri. Our SEM data are indicative of References 975 911 successful inactivation, as 97% of the superficial neuromasts 976 912 Becco, C., Vandewalle, N., Delcourt, J. & Poncin, P. 2006. Experimental evidences 977 appeared damaged. Although we observed only the superficial of a structural and dynamical transition in fish school. Physica A: Statistical 913 neuromasts by SEM, exactly the same treatment has been shown to Mechanics and its Applications, 367, 487–493. 978 914 damage both the superficial and the canal neuromasts in various Blaxter, J. H. S. & Batty, R. S. 1985. Herring behaviour in the dark: responses to 979 915 fish species, inactivating the whole lateral system (Blaxter & stationary and continuously vibrating obstacles. Journal of the Marine Biological 980 Association U.K., 65, 1031–1049. 916 PROOF 981 Fuiman 1989; Song et al. 1995; Coombs et al. 2001). Blaxter, J. H. S. & Fuiman, L. A. 1989. Function of the free neuromasts of marine 917 To make sure that any effects observed were not due to the teleost larvae. In: The Mechanosensory Lateral Line (Ed. by S. Coombs, P. Go¨rner & 982 918 handling and stress associated with treatment, we included sham- H. Mu¨ nz), pp. 481–499. New York: Springer Verlag. 983 Blaxter, J. H. S., Gray, J. A. B. & Best, A. C. G. 1983. Structure and development of the 919 treated fish in our study. These fish showed only slight tissue free neuromasts and lateral line system of the herring. Journal of the Marine 984 920 damage and, throughout the study, the control and sham fish Biological Association U.K., 63, 247–260. 985 921 behaved similarly, maintaining their distances to their neighbours Breder, C. M. Jr. 1959. Studies on social groupings in fishes. Bulletin of the American 986 Museum of Natural History, 117, 393–482. 922 987 (NND1, NND2 and NND3) and always showing an aggregative Burgess, J. W. & Shaw, E. 1981. Effects of acoustico-lateralis denervation in 923 distribution with a low and stable shoaling radius (about 15 cm), a facultative schooling fish: a nearest-neighbor matrix analyses. Behavioral and 988 924 many fish swimming in the same direction (between 95 and 98%), Neural Biology, 33, 488–497. 989 Clark, P. J. & Evans, F. C. 1954. Distance to nearest neighbor as a measure of spatial 925 990 and no collision between individuals. Their shoaling behaviour was relationships in populations. Ecology, 35, 445–453. 926 thus constant and reproducible. Coombs, S., Janssen, J. & Webb, J. F. 1988. Diversity of lateral systems: evolutionary 991 927 Aminoglycoside antibiotic treatment had striking effects on fish and functional considerations. In: Sensory Biology of Aquatic Animals (Ed. by 992 928 J. Atema, R. R. Fay, A. N. Popper & W. N. Tavolga), pp. 553–593. New York: 993 behaviour: (1) significantly increased NNDs, (2) an approximately Springer Verlag. 929 25% expansion of the shoal radius, (3) random fish distribution Coombs, S., Braun, C. B. & Donovan, B. 2001. The orienting response of 994 930 inside the tank, (4) fewer fish swimming in the same direction Lake Michigan mottled sculpin is mediated by canal neuromasts. Journal of 995 931 (only 63%) and (5) collisions between neighbours. After the anti- Experimental Biology, 204, 337–348. 996 Diaz, J. P., Prie´-Granie´, M., Kentouri, M., Varsamos, S. & Connes, R. 2003. 932 biotic treatment, the fish recovered gradually, according to a similar Development of the lateral line system in the sea bass. Journal of Fish Biology, 997 933 time frame, both at tissue level and behaviourally. Shoaling 62, 24–40. 998 934 behaviour reappeared between days 11 and 16, with restoration of Faucher, K., Aas-Hansen, O., Damsgard, B. & Stenklev, N. C. 2008a. Effects 999 of systemic versus local gentamicin on the inner ear in the Atlantic cod, 935 normal interindividual distances. By day 21 the fish showed an Gadus morhua (L.), relevance for fish hearing investigations. Hearing Research, 1000 936 aggregative distribution and from day 25 onwards, no collision was 240, 12–21. 1001 937 recorded. The shoal order parameter became similar to those of Faucher, K., Fichet, D., Miramand, P. & Lagarde`re, J. P. 2008b. Impact of chronic 1002 cadmium exposure at environmental dose on escape behaviour in sea 938 1003 sham fish by day 29. On day 31, the lateral line mechanoreceptors bass (Dicentrarchus labrax L.; Teleostei, Moronidae. Environmental Pollution, 151, 939 appeared intact. Complete recovery of both the sensory tissue and 148–157. 1004 940 swimming behaviour thus took about a month. Hoekstra, D. & Janssen, J. 1986. Lateral line receptivity in the mottled sculpin 1005 (Cottus bairdi). Copeia, 1, 91–96. 941 1006 Our results thus clearly demonstrate, in contrast to previous Janssen, J. & Corcoran, J. 1998. Distance determination via the lateral line in the 942 studies, that fish totally deprived of the lateral system (both trunk mottled sculpin. Copeia, 3, 657–662. 1007 943 and head) cannot maintain a shoal, and that they move apart Janssen, J., Jones, W. R., Whang, A. & Oshel, P. E. 1995. Use of the lateral line in 1008 944 particulate feeding in the dark by juvenile alewife (Alosa pseudoharengus). 1009 instead of moving closer. We thus show for the first time that the Canadian Journal of Fisheries and Aquatic Sciences, 52, 358–363. 945 lateral system is more crucial toUNCORRECTED shoaling than is commonly Janssen, J., Sideleva, V. & Biga, H. 1999. Use of the lateral line for feeding in two 1010 946 accepted. The closer swimming observed previously after inacti- Lake Baikal sculpins. Journal of Fish Biology, 54, 404–416. 1011 947 vation restricted to the trunk lateral line may mean that fish with Lombarte, A., Yan, H. Y., Popper, A. N., Chang, J. S. & Platt, C. 1993. Damage and 1012 regeneration of hair cell ciliary bundles in a fish ear following treatment with 948 few mechanoreceptors (and only on the head) need to be closer to gentamicin. Hearing Research, 64, 166–174. 1013 949 detect their neighbours. Furthermore, the head lateral line system Masuda, R. & Tsukamoto, K. 1996. Morphological development in relations to 1014 950 alone appears insufficient for maintaining the shoal in blinded fish. phototaxis and rheotaxis in the striped jack, Pseudocaranx dentex. Marine and 1015 Freshwater Behaviour and Physiology, 28, 75–90. 951 The view that the anterior lateral-system organ is important is Masuda, R. & Tsukamoto, K. 1999. School formation and concurrent develop- 1016 952 supported by the observation that cohesive swimming behaviours mental changes in carangid fish with reference to dietary conditions. Environ- 1017 953 (mutual attraction, association) appear in the striped jack, Pseu- mental Biology of Fish, 56, 243–252. 1018 Matsuura, S., Ikeda, K. & Furukawa, T. 1971. Effects of streptomycin, kanamycin, 954 1019 docaranx dentex, just after the cephalic lateral-system canals begin quinine, and other drugs on the microphonic potentials of goldfish sacculus. 955 to form (Masuda & Tsukamoto 1999). It has also been shown in this Japanese Journal of Physiology, 21, 579–590. 1020

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Please cite this article in press as: Faucher, K., et al., Fish lateral system is required for accurate control of shoaling behaviour, Animal Behaviour (2010), doi:10.1016/j.anbehav.2009.12.020