Quantifying the effects of post-surgery recovery time on the migration dynamics and survival rates in the wild of acoustically tagged Atlantic Salmon Salmo salar smolts


 
 The experimental effects of surgically implanting fish with acoustic transmitters are likely to have negative effects on survival and behaviour. Measuring the extent of these negative effects is important if we wish to extrapolate inferences from tagged animals to un-manipulated animals. In this study, we examine the effect of surgery and post-tagging recovery time on the survival and migration rate of acoustically tagged wild Atlantic Salmon (Salmo salar) smolts through freshwater, estuarine and ocean phases of migration. Four treatment groups were used: pre-smolt captured in the fall that overwintered in a hatchery and were tagged either 75 days prior to release (winter hatchery) or within 24 h prior to release (spring hatchery) and smolt captured during the spring smolt run, tagged 24 h prior to release and released during the day (day-released) or night (night-released).
 
 
 The spring hatchery treatment group served as a reference treatment group such that recovery time (comparison to winter hatchery treatment) and hatchery effects (comparison to day-released and night-released treatments) could both be discerned. The hatchery effect increased migration rate, whereas short recovery times and captivity in a hatchery negatively affected survival. These effects were most pronounced within the first 5 days and/or 48 km downstream post-release, however, the residual recovery time effects appeared to persist during the transition from the estuary into salt water.
 
 
 Even with smolts originating from the wild and spending relatively little time within the hatchery environment, post-release survival was still negatively affected. Migration speed was faster for hatchery smolts, but is likely only due to their larger size. Recovery time effects were most prominent during the initial migration period in freshwater and again in the transition from the estuary to saltwater which may be due to added stress during these transitional zones. As surgery-related bias will likely never be completely removed from telemetry studies, it is important to quantify and account for these effects in situ when making inferences on the un-manipulated component of the population.



Introduction 31
Acoustic telemetry has become a broadly applied technology for studying the migration 32 dynamics and for estimating survival rates of many fish species, including juvenile stages of wild 33 Atlantic salmon in both fresh and salt water [1][2][3][4][5]. The use of acoustic technologies generally 34 involves the surgical implantation (hereafter "tagging") of acoustic transmitters (hereafter "tags") 35 inside the abdominal cavity of fish which are released back to the wild and remotely detected 36 when in range of receivers placed in the study area. The primary purpose of these studies is to 37 draw inferences on the behaviour and survival of wild untagged animals which are otherwise 38 difficult to observe and monitor.  Table 1), a trip which took 123 approximately 45 minutes. Upon arrival at the release site, river water was gradually added to the 124 transport containers for approximately 30 minutes for purposes of temperature and water 125 chemistry acclimation. River water temperatures at the time of transfer ranged from 8.5 to 9.5°C. 126 The winter tagged captive smolts were released as a group immediately following acclimation. 127 The remaining 34 untagged captive smolts were transported to the release site where 29 were 128 tagged for the second treatment group (spring hatchery), held for a minimum of one hour to 129 allow restoration of equilibrium of the fish, then released (

Collection of wild Atlantic Salmon smolts for day and night releases 133
A total of 61 wild salmon smolts were selected during May 17 -19 (2016) from catches in a 134 rotary screw trap set at Trout Brook in the Northwest Miramichi ( Figure 1; Table 1). Only smolts 135 with a fork length ≥ 13 cm were selected for the experiment to accommodate the size of the 136 acoustic tag. All smolts selected for tagging were held in in-stream tanks for 20 to 24 hours prior 137 to tagging to allow for digestion of stomach contents. Smolts were then transported by truck in 138 oxygenated tanks to the Miners Bridge release site, a trip which took approximately 15 minutes, 139 where surgery and tagging were conducted. After surgery, smolts were held in plastic flow 140 through stream side containers (length: 80 cm; depth: 35 cm; width: 50 cm) for a minimum of 141 one hour after equilibrium had been restored before being released (Table 1). Collected smolts 142 were divided daily into two treatment groups. The first treatment group, consisting of a total of 143 8 consisting of 29 smolts, was tagged and released in the late evening at 2200 hours, 145 approximately one-hour post-sunset (night-released). Wild tagged smolts from these treatment 146 groups were released on May 18, 2016 (22 for day release), May 19, 2016 (9 and 19 for day and  147 night released, respectively) and May 20, 2016 (one and 10 for day and night released, 148 respectively; Table 1). The combination of both day-released and night-released treatments are 149 referred to as a spring wild treatment. 150

Tagging procedure 151
The tagging procedure was the same for all treatment groups and as previously described by 152 Daniels et al.
[10] and Chaput et al. [5]. Fish were anaesthetised using clove oil (40 mg/L 153 concentration) until loss of equilibrium and very little operculum movement was observed 154 (generally 3 -5 minutes). All surgical tools and tags were disinfected in anhydrous ethyl alcohol 155 and rinsed in distilled water. Wet weight (g) and fork length (mm) were taken before the fish was 156 placed ventral side up on a v-shaped operating board lined with a chamois leather. An ≈11 mm 157 incision was made along the mid-ventral line about 10 mm anterior to the pelvic girdle. Acoustic 158 tags (Innovasea Marine Systems Canada, Inc., Halifax, NS; model V8; diameter=8 mm, 159 length=20.5 mm, weight in air=2.0 g; output = 144 dB re 1uPa@1m, transmission cycle = 160 random 25-55 s) were inserted into the body cavity of the fish via the incision. The fish's gills 161 and body were continuously irrigated with anaesthetic or water during the surgery while 162 avoiding the incision area. One suture per five mm of incision (typically two sutures) was used to 163 close the incision and the fish was placed in a recovery bath, with aerated water, for observation. 164 Two surgeons tagged the same number of smolts in each treatment. The acoustic tags for the 165 winter tagged group were programmed for a delayed start on May 14, 2016. 9 Fish from all treatment groups were only weighed and measured at time of tagging. 167 Winter tagged hatchery fish were weighed and measured at the time of surgery (March 8) but not 168 at the time of release (May 19) as we did not want to introduce anaesthetic and handling effects 169 immediately before release. The captive treatment group tagged in the winter was of intermediate 170 size at time of tagging (median fork length 149 mm) to captive fish tagged in the spring (median 171 FL = 172 mm) and to wild smolts captured and tagged in the spring (median FL = 138 mm; 172 Note that the fish in the reference treatment have |1 = 0 and |2 = 0 hence logit survival 227 of the reference group in zone j = . We set the spring hatchery tagging group as the reference 228 treatment because we were interested in estimating the expected survival rate of wild captured 229 smolts with minimal tagging effect (i.e. long recovery time). However, to do so, we also had to 230 account for the effect of captive holding of fish for several months. Spring wild (Treatment 1) 231 relative to spring hatchery represents the hatchery holding effect ( ), whereas Winter hatchery 232 (Treatment 2) relative to the spring hatchery represents the tagging effect ( ). From equation 3, the adjusted logit survival rate of wild captured smolts with a long recovery time is the sum of 234 the posterior distributions of , and . The odds ratio of survival among treatment groups is 235 used to make inference on the effect of captivity in the hatchery (wild spring tagged relative to 236 spring hatchery tagged) and the effect of recovery period (captive winter tagged relative to 237 captive spring tagged and to wild spring tagged). CJS models were run with and without total unique tags inferred to introduce predation induced 280 bias to explore potential interactions between predation induced bias and tagging induced bias. It 281 should be noted, however, that an increased rate of predation and therefore, the potential rate of 282 predation induced bias, could be an indirect result of tagging effects. 283 284

Smolts size 286
Overall, there was a significant difference in fork length (F=43.24; df=3; p<0.0001) between 287 treatment groups. Post-hoc treatment comparisons showed only spring hatchery smolts to be 288 significantly longer than all other treatments (Table 1; Figure 2). Tag burden also differed 289 significantly between treatment groups (F=59.73; df=3; p<0.0001). Post-hoc comparisons 290 showed that spring hatchery smolts tag burdens were smaller than all other treatment groups 291 while winter hatchery smolts had smaller tag burdens compared to day-and night-released wild 292 smolts (Table 1). Day-released and night-released wild spring smolt groups had similar tag 293 burdens. 294

Day-release vs night-release 295
There were no significant differences in the apparent survival and migration rate to arrays of 296 day-released compared to night-released wild smolts (Table 2). Therefore, day-released and 297 night-released treatments were combined for comparisons to the captive treatments. 298

Observed detections 299
Observed detections of tagged smolts varied between zones and treatments. Of the 61, 29, and 29 300 tagged smolts released for the spring wild, winter hatchery, and spring hatchery treatments, 50 301 (82%), 25 (86%), and 13 (45%) smolts, respectively, were subsequently detected at downstream receivers. Only 13 of 29 tags (45%) of the spring hatchery treatment were detected at the HoT 303 array, much lower than the 79% of tags from the winter hatchery treatment and the spring wild 304 treatment detected at the HoT array ( Figure 3). The relative reduction in detections between the 305 HoT and the outer bay array was similar for the spring wild treatment and the spring hatchery 306 treatment (-61%) in contrast to the relatively lower reduction in tags detected for the hatchery 307 winter treatment (-52%). There was no evidence of size selection in survival between receiver 308 arrays and within treatments. 309

Predation induced bias 310
Based on the interpretation of detection patterns, 8 of 61 tags from spring wild tagging, 2 of 29 311 tags from spring hatchery tagging, and 4 of 29 tags from winter hatchery tagging were inferred to 312 have been predated by Striped Bass and introduce predation bias (Table 1). This represents 13% 313 of tags of the spring wild release group (95% Confidence Interval range, 5% -20%), 7% (95% 314 C.I. 1% -17%) of the spring hatchery release group, and 14% (95% C.I. 3% -26%) of the winter 315 hatchery release group. Based on tags detected post-release, the number of tags with detections 316 consistent with Striped Bass movement and inducing predation bias was similar among 317 treatments; 17% (95% C.I. 6% -24%) of the spring wild group, 15% (95% C.I. 2% -34%) of the 318 spring hatchery group, and 17% (95% C.I. 4% -30%) for the winter hatchery group. As there 319 was no evidence of differential rates of predation induced bias between treatments, CJS model 320 results including total unique tags are presented and discussed. 321

Smolt survival and probability of detection 322
The mean probabilities of detection at the arrays within the river and estuary were generally 323 high, ranging from 0.914 to 0.984. The outer bay and SoBI arrays, however, were estimated to have lower detection probabilities, with mean values of 0.731 and 0.702, respectively (Figure 4), 325 consistent with previous analyses [5]. 326 Evidence of statistically significant negative hatchery and tagging effects were present 327 from release to the HoT array ( Figure 5). There was no evidence of hatchery or tagging effects 328 present through the estuary (HoT to inner bay). A potentially significant (p = 0.06) negative 329 tagging effect was noted between the inner and outer Miramichi bay whereas there was no 330 evidence of a hatchery effect in this zone ( Figure 5). 331 Results suggest that the winter hatchery treatment group may have had slightly improved 332 survival through the bay in comparison to the other treatments, however, small sample sizes 333 preclude firm conclusions (Figure 4). Throughout all regions survival odds generally favored the 334 winter hatchery treatment (Figure 4; Table 3). An extended recovery period prior to release 335 improved the odds of survival post-release (winter hatchery treatment) with survival odds of just 336 under 2.53; this is due to the poor survival immediately post release of the spring hatchery 337 treatment ( Figure 5; Table 3). The survival odds of a long recovery period were also improved 338 relative to wild spring tagged smolts, with a median value of 1.38 favouring the survival of the 339 long recovery treatment. After the initial mortalities in the freshwater portion of the river, above 340 the HoT, the survival odds still favoured the long recovery period treatment, 1.34 to 1.47 relative 341 to the short recovery treatments, however the statistical evidence for this was weaker (p-values 342 0.11 to 0.14) because of small sample sizes. A hatchery effect was noted for the initial survival 343 odds, favouring by a factor of 1.83 the survival of spring wild smolts relative to the spring 344 hatchery treatment (Table 3). After the initial triage of compromised fish from the spring  We report on a field experiment to assess the effects of handling/tagging on migrating Atlantic 373 Salmon smolts and on inferences of survival and migration rates using acoustic technologies. In 374 particular, we assess the extent to which recovery time post-surgery can affect those common 375 metrics and the inferences which could be made from such studies relative to migration and 376 survival of untagged smolts. The results suggest that there is a detectable negative effect on 377 survival and migration rates when fish are only allowed short recovery times post-surgery before 378 release. We found that in this first experiment, capturing juveniles in the fall, holding them in 379 captivity over the winter and releasing back to the river in the spring immediately after a tagging 380 intervention imparted a very strong compromise on survival immediately post release. Although 381 we might suspect the manipulation, transport, and short period of acclimation prior to release 382 would be stressful to all fish, immediate short-term survival was not compromised in the winter 383 hatchery treatment subjected to the same handling procedures, absent the tagging. 384 In comparison to the other treatment groups, it is unclear why the spring hatchery smolts 385 experienced such low short-term survival post release. Both hatchery treatments underwent the 386 same transportation to and release protocols at the release site. As well, the wild spring smolts 387 were also manipulated, transported for a shorter distance and time, and held in stream-side 388 containers post tagging. Surgery and handling protocols were identical between the spring wild 389 and spring hatchery treatments, but these groups had very different estimated survivals to HoT 390 from the release site. Therefore, it seems unlikely that solely transportation or surgery and 391 handling protocols resulted in the high early mortality observed for the spring hatchery 392 treatment. The most likely explanation for the high rate of early mortality of spring hatchery smolts is the result of cumulative stress from transportation and acclimation immediately 394 followed by surgery. There is evidence that cumulative stress results in higher than expected 395 mortality. For example, Handeland et al. [29] found that the interaction between osmotic stress 396 and predator induced stress resulted in higher rates of predation than the combined effects of 397 predation and osmotic stress solely. Similarly, Dietrich et al. [30] present evidence which 398 suggests sublethal doses of organophosphates increase mortality rate of Chinook salmon in the 399 presence of thermal stress. We suggest that the unexpectedly high mortality may have been due 400 to the cumulative impacts of hatchery transport, surgery, and release to the wild following a short 401 recovery period. 402

Tagging effects on migration speed and survival 403
While there was no statistical difference in migration rates between the winter and spring 404 hatchery treatments, there was a slightly faster mean migration speed for winter hatchery smolts 405 between release and HoT. This was unexpected as the size of the spring hatchery treatment 406 appeared to be much larger based on visual inspection pre-release and larger fish tend to have 407 faster swimming speeds [6,8]. While small sample size prohibits any definitive conclusions, the 408 slight difference could be indicative of a tagging effect and short recovery period on the 409 migration speed. 410 There was a noticeable tagging effect on apparent survival from release to HoT. 411 Although, tag burden is most often viewed as the leading cause for reduced survival the results 412 from this study imply surgery may be the larger effect. Compared to the spring hatchery 413 treatment, the winter hatchery group had higher tag burdens but assumingly without the tagging 414 effects and they presented a higher survival rate. It is important to note, however, that we did not 415 measure size of the winter hatchery group at time of release and their tag burdens were likely to 416 have been exaggerated in relation to what would have been experienced at the time of release. 417 Other studies have also drawn similar conclusions that surgery has a larger effect than tag 418 burden. Studies have found no difference in mortality rates between tagged smolts and sham 419 treatments (ie. received surgery, yet no tag was implanted) also suggesting that surgery related 420 mortality may be more important than the size of the tag [9,12]. 421 Determining when and where mortality occurs is a key question however, experimental 422 manipulations such as those considered here can affect the conclusions from the interpretation of 423 acoustic telemetry data [31]. For example, a study presented by Chaput et al. [5] involved a 424 fourteen-year multi-stock inference on the survival and migration of Atlantic Salmon smolts and 425 post-smolts which suggested that differences between stocks migrating through a shared 426 environment may be indicative of post-surgery acclimation factors. They also suggest that 427 mortality associated with migration duration could be enhanced as a result of direct tagging 428 effects and/or vulnerability to predation that occurred within the first 8 to 12 days of migration. 429 While field-based studies and analysis of telemetry detections provide some information on 430 survival and behaviour of tagged fish, they generally are inadequate to ascribe causal factors of 431 mortality. Laboratory-based studies report similar time frames of surgery related mortality as 432 found in this study. Brunsdon et al. [12] found that the majority of the mortality of tagged 433 Atlantic Salmon juveniles occurred within the first 10 days after surgery using the same tags 434 used in this study (Vemco V8) and did not find any difference in survival rates between tagged 435 smolts and a sham treatment. Ammann et al.
[9] also found no difference in mortality rates 436 between acoustically tagged individuals and a sham treatment. These results suggest that for 437 smolts of the size used in these laboratory studies and in this experiment, mortality may be more 438 likely a consequence of surgery related effects as opposed to effects related to tag burden. 439

Hatchery effects on migration speed and survival 440
The juvenile salmon held in the hatchery were collected from the wild and held in captivity for a 441 period of less than six months. Despite this relatively short-term holding period we did note an 442 effect on migration rates associated with captivity through the freshwater zone of migration. The 443 larger size of the hatchery held smolts in comparison to spring wild smolts may partially explain 444 the faster migration speed observed. Many studies have found a positive relationship between 445 fork length and speed of Atlantic salmon [22,32,33] and fish with higher tag burdens tend to 446 have slower swimming speeds [6,8]. However, the difference in migration speed was only noted 447 for survivors from release to HoT (<5 days). This suggests that migration rate in freshwater to 448 the estuary may be related to fork length, whereas, migration rate through the estuary is 449 conditioned by other factors such as variation in smoltification stage and acclimation to saltwater 450 In general, wild smolt survival rates tend to be higher than for hatchery-reared smolts, 452 interpreted to be a consequence of poorer foraging and predator avoidance abilities of hatchery 453 reared smolts [35][36][37][38]. The effect of the hatchery holding (i.e. the spring wild relative to the 454 spring hatchery treatment) appeared to negatively impact survival within the same zone of the 455 river in which migration speed appeared to have been affected, between release and HoT. 456 Jokikokko et al. [39] reported that survival of wild smolt and hatchery reared Atlantic Salmon 457 released as parr was twice that of hatchery reared Atlantic Salmon released as smolts. Thorstad et 458 al. [40] found that hatchery released smolts suffered approximately 50 percent mortality near the 459 transition from fresh to salt water and imply that hatchery rearing of these fish may have resulted 460 in poor predator avoidance as the vast majority of mortality in their study was likely due to 461 piscine predators. 462 Most of the comparisons of wild versus hatchery survivals and behaviour are from 463 studies in which the hatchery reared smolts originated from spawning, hatching and rearing in 464 the hatchery environment. We found no information regarding the impacts of short-term captive 465 holding of wild fish on their subsequent behavior and survival once released back into their natal 466 river. There is however evidence from this study that some effects of captivity may manifest 467 themselves even for relatively short periods of captivity. 468

Expected survival 469
The effect of the tagging procedure on survival after controlling for a hatchery effect is 470 quite apparent in the very early stages of the migration, between release and HoT and to a lesser 471 and more uncertain degree between the inner and outer bay zone. Our results suggest that smolt 472 survival could have been greater than 95% and nearly 90% through the freshwater and bay 473 regions of the migration, respectively. The uncertainty in these estimates primarily resulting from 474 higher than expected spring hatchery mortality limits our ability to suggest these effects are 475 short-lived, however, we have presented information from both field and laboratory settings 476 which suggests that direct and indirect tagging related mortality is temporally short-lived or a 477 result of increased stress when transitioning through different environments. 478 The effect of the tag burden on the survival and behavior of smolts was not quantified in 479 this study. Tag burden has been extensively investigated and results have shown mortality 480 increases with tag burden while swimming speed and predator avoidance decrease [6][7][8]41]. 481 Studies have also reported that larger tagged smolts have greater survival during the early stages 482 of migration compared to smaller counterparts [5]. However, this study found the larger spring 483 hatchery smolts had lower survival compared to the smaller spring wild and winter hatchery 484 treatments but hatchery and tagging effects, respectively, likely influenced results. Furthermore, there was also no evidence that larger smolts within each treatment had higher survival rates (see 486 Supplementary materials). Nevertheless, the much higher mortality of the spring hatchery 487 treatment was surprising as larger smolts were expected to have greater survival due to a reduced 488 vulnerability to predators and a reduced tag burden [5,36,42]. 489 Vollset et al. [18] reported that acoustically tagged wild Atlantic Salmon smolts released 490 in the evening had higher estimated survival rates in freshwater and in a fjord than tagged smolts 491 released in the morning. We examined and did not find any evidence of differences in inferred 492 survival rates of smolts released during the day or the late evening. The objective of most acoustic telemetry studies is to quantify survival and behavior of tagged 501 individuals and to use these to infer survival and behaviour of untagged and un-manipulated 502 animals [22]. In this experiment, we examined the effects of the length of post-recovery period 503 on the migratory behaviour and inferred survivals of tagged animals post-release. We found that 504 allowing for a long post-surgery recovery period can improve the survival rates of tagged smolts 505 relative to smolts with only a short recovery time post-surgery. There is evidence of an 506 immediate short-term improvement in survival as well as a later benefit as smolts moved from 507 brackish water into a more saline environment, a stressful environmental transitional zone. In order to have a treatment group with a long recovery period, we captured juveniles from the wild 509 and held them in captivity for a relatively short overwinter period. This may have introduced 510 unintended and undesired effects on behaviour and survival of individuals post release. However, 511 the results from this one-year study suggest that the effects of captivity if present are minor 512 compared to the effects related to surgery and short recovery times. An alternate experimental 513 design that would provide a long recovery period without captivity would involve capturing, 514 tagging and releasing in the late fall salmon juveniles of an appropriate size to accommodate the 515 acoustic tags and that would be indicative of a high likelihood of becoming a smolt the following 516 spring. The long recovery treatment group would then consist of those fall-tagged juveniles 517 which survived and became smolts and were detected at a key receiver array in freshwater. This 518 treatment group could be monitored in concert with the reference treatment involving capture, 519 tagging and releasing actively migrating smolts but with a short recovery period. This type of 520 study was conducted and reported by Gregory et al. [44] on salmon juveniles which were tagged 521 with small passively induced transponder tags in the fall prior to smolt emigration and tracked 522 using instream antennas and loggers as they migrated in the spring and returned as adult salmon 523 one and two years later. Furthermore, different sized tags could also be incorporated into the 524 proposed study designs to examine the effects of tag burden in addition to the tagging (i.e. 525 surgical) effects. 526 Acoustic telemetry studies of wild fish will always introduce a certain degree of bias as 527 they involve the manipulation of animals. Provided the bias is consistent over time, which 528 requires establishing and respecting standardized experimental procedures and protocols, long-529 term multi-stock telemetry studies such as that presented by Chaput et al. [5] Table 1. Experimental details and observed detections of smolts per receiver array. For the detections, the values in parentheses are 748 the number of tags detected at each array inferred to be of a smolt tag ingested by a predator which induced predation bias rather 749 than of a free-swimming smolt relative to the total number of unique tags which were inferred to have been ingested by a predator 750 and induced predation bias over the whole migration period.