A TEMPERATURE SHIFT DURING EMBRYOGENESIS IMPACTS PREVALENCE OF DEFORMITY IN DIPLOID AND TRIPLOID ATLANTIC SALMON ( Salmo salar L.)

35 The study investigated effects of a temperature shift during embryogenesis on diploid and 36 triploid Atlantic salmon ( Salmo salar L.) embryo development and juvenile skeletal 37 deformities. From fertilisation, sibling populations were incubated under one of three 38 temperatures (6, 8 or 11 °C) until 400 °days when all fish were then reared under a common 39 temperature until smolt. Survival was negatively impacted by increasing temperatures 40 irrespective of ploidy. There was no effect of incubation temperatures on growth in diploids, 41 but triploids incubated at 6 °C had improved growth rates (thermal growth coefficient; TGC 6 42 °C: 1.05, 8 °C: 0.94, 11 °C: 0.48). Fish from 11 °C in both ploidies showed increased jaw and 43 vertebral deformity prevalence. In response to the temperature change at 400 °days post- 44 fertilisation, upregulation of bmp2 , bmp4 , col2a1 , mmp13 , opn , sparc , and downregulation of 45 ocn further suggest that bone and cartilage formation is compromised after experiencing a 46 thermal shift. The data show that temperature profile during embryogenesis strongly influences 47 future growth and deformity prevalence. Triploids appear to require a lower incubation 48 temperature than the current industry standard of 8 °C to promote better overall performance, 49 however, a thermal shift during embryogenesis was shown to impact expression of important 50 developmental genes. 51 52 53 54 55 56


Introduction
water bath at 8 °C prior to triploid induction. Triploidy was induced in one group (655 bar of 171 hydrostatic pressure for 6.25 mins. at 8 °C, 37 mins. post-fertilisation) according to Smedley 172 et al. (2016), while the others experienced the handling but did not receive a hydrostatic shock 173 and were maintained as diploids. After water hardening, eggs were further divided into three 174 incubation temperatures (5.8 ± 0.7, 8.3 ± 0.1 or 10.8 ± 0.1 °C, referred to as 6, 8 and 11) and 175 incubated in triplicate in aluminium egg trays (total = 54; 3 trays -1 family -1 temperature -1 ploidy -176 1 ) within temperature-specific trough systems (15 mL sec -1 flow) in darkness. Temperature was 177 monitored in 30 min intervals with temperature data loggers (HOBO 64K Pendant®, Onset 178 Computer Corporation) submerged in the middle of the incubation trays, central in each trough 179 system. At 400 °days post-fertilisation, ~800 eggs from the two best performing families (400 180 eggs family -1 ) were pooled from each treatment (2 ploidies x 3 temperatures) and transferred 181 to single 6 x 0.3 m 3 recirculation system (RAS) tanks and incubated at a constant temperature 182 of 8 °C until first feeding (Fig. 1). One of the three families was removed from the study due 183 to a high mortality rate in both ploidies suspected to be due to poor egg quality. Embryos in all 184 treatments were kept in constant darkness until first feeding at which point fry were given 185 constant light (LL). Three treatments per ploidy (a history of 6, 8 and 11 °C) were created, with 186 eggs incubated at 8 °C referred to as the "control treatment" as routine in commercial practice. 187 Whilst in RAS, pH measurements were regularly checked and maintained (pH 6.8 -7.2), with 188 the addition of sodium bicarbonate (NaHCO3) as necessary. At 1 g, all fish were transferred to and subsequent impact on developmental speed, the number of feeding days (d) from first 193 feeding until the end of the trial differed between treatments (6, 364d; 8, 386d; 11, 395d). 194 Diploids were fed a standard commercial diet (BioMar INICIO Plus, 4.9 g kg -1 available P) 195 and triploids were fed a triploid-specific diet (BioMar INICIO TRI-X, 7.7 g kg -1 available P), 196 with the only difference in the formulation being P supplementation in TriX, and thus in 197 accordance with previously published triploid specific P requirements (Fjelldal et   To confirm ploidy status, blood smears were prepared from samples taken from the caudal 203 peduncle of euthanised fish (20 fish treatment -1 ploidy -1 ; 49.6 ± 25.8 g). Air dried slides were 204 fixed in 100 % methanol and then placed into Giemsa stain for 10 mins. Slides were digitised 205 using a slide scanner at 20x magnification (Axio Scan Z1, Zeiss) and erythrocyte length and 206 diameter was determined by Fiji software (ImageJ). A total of 30 randomly chosen nuclei per 207 slide were measured to the nearest 0.01 μm and a mean taken for presumed diploid and triploid 208 fish. Diploid control groups had significantly smaller erythrocyte nuclear lengths, with no 209 overlaps with the triploid groups (2N, 7.2 -8.6 μm; 3N, 9.2 -11.1 μm) confirming that all 210 sampled fish that were subjected to hydrostatic pressure shock were likely to be triploids. frozen flat at -20 °C for later radiological deformity analysis (50 individuals treatment -1 ploidy -218 1 ) and to assess whole carcass mineral composition (3 pools; 3 individuals pool -1 treatment -1 219 ploidy -1 ). Additional smolts were selected using the same method and sacrificed (Tricaine,220 Pharmaq; 1000 ppm) for myogenic morphology assessment (6 individuals treatment -1 ploidy -221 1 ). A 6 mm thick cross section was excised from the trunk immediately anterior to the dorsal 222 fin. Cross sections were then mounted onto cork using optimal cutting temperature (O.C.T.) 223 compound and frozen in isopentane cooled to -170 °C in liquid nitrogen and subsequently 224 stored at -70 °C until processing.

225
Samples for expression of genes associated with lipid metabolism, muscle and bone 226 formation were collected as whole individuals at eyeing and at first feeding (6 individuals 227 treatment -1 ploidy -1 ). No further samples were collected to analyse lipid metabolism as 228 subsequent samples were tissue specific for remaining genes. At smolt, muscle-related genes 229 were analysed in dissected muscle from the Norwegian Quality Cut (NQC) region and the 230 vertebral column under the dorsal fin (2 cm, ~10 vertebrae) was sampled for bone associated 231 genes (6 individuals treatment -1 ploidy -1 ). All samples for gene expression analysis were 232 collected into 'RNA Later', stored at 4 °C for 24 hours and then frozen at -20 °C until 233 processing.

283
Final cDNA 10 μL reactions were diluted 1:10 in nuclease-free water to a total volume of 100 284 μL and 2.5 μL was used for each 10 μL (2.5 ng µL -1 ) qPCR reaction.   Master Mix (Applied Biosystems, UK) in a total reaction volume of 10 μL. Amplification was 308 achieved in 384-well plates and conducted in a thermo cycling program consisting of a pre-309 incubation of 95 °C for 10 mins. followed by 40 cycles of; 95 °C for 15 secs., TA °C for 30 310 secs., and 72 °C for 30 secs. This was followed by a temperature ramp from 60 to 90 °C for 311 melt-curve analysis to verify that no primer-dimer artefacts were present and only one product 312 was generated from each qPCR assay. Quantification was achieved by a parallel set of reactions 313 containing standardised plasmids described above.

314
Results from cDNAs were normalised by relating expression data to geometric mean 315 of two reference genes with stable gene expression levels across samples; β-actin and elf-α. 316 Gene activity was expressed as a fold change from 8 °C (current industry incubation    For mortality and gross deformity, 95% confidence intervals were estimated according to the 333 central limit theorem (CLT;Feller, 1968Feller, , 1971) and overlapping standard deviations between 334 treatments were considered not significantly different. Use of this approach is referred to as 335 "Analysis of CLT" throughout. All significance was accepted at p < 0.05.  Table 2). Irrespective of ploidy, there was an increase in cumulative mortality in 341 response to increasing embryo incubation temperatures. Diploids appeared to have a linear 342 increase in cumulative mortality (~10 % between each temperature), whilst triploids had a more 343 prominent increase from 6 °C (49.3%) to 8 and 11 °C (68.7 and 72.9%, respectively).

344
Triploid embryos had a consistently higher mortality compared to diploids under all 345 temperature regimes between fertilisation to 400 °days, however no significant differences 346 were observed due to the large variation between families (Table 2).

347
No ploidy difference was also shown in mortality between 400 °days to hatch in 6 or 348 11 °C treatments, however triploids had a significantly higher mortality than diploids when 349 incubated at 8 °C (Table 2). In diploids, mortality was comparable in 6 and 8 °C treatments, 350 however a significant increase was shown in embryos incubated at 11 °C. In triploids, a 351 significant increase in mortality was found in both 8 and 11 °C compared to 6 °C.

352
Between hatch and first feeding, no ploidy differences were observed in any 353 temperature treatment (Table 2). Within diploids, there was a significant increase in mortality 354 from 6 to 8 °C, however mortality in 11 °C showed comparable rates to both 6 and 8 °C.

355
Triploids showed a similar trend with mortality significantly increasing between 6 to 8 °C. 356 However, mortality in 11 °C was comparable to 8 °C, but not 6 °C.

357
During the final window (first feeding to smolt), triploids had a higher mortality than 358 diploids in all treatments (Table 2). Both ploidies showed the same trend of increasing mortality 359 between 6 and 8 °C, and comparable mortality between 8 and 11 °C. An overall effect of ploidy (p < 0.001, 2N > 3N) but no overall temperature effect was observed 363 (p = 0.234) on first feeding weight (BWi). No difference between ploidies was observed for 364 BWi at 6 °C, however, diploids had a significantly higher BWi than triploids in 8 and 11 °C 365 treatments ( Fig. 2a). Within each ploidy, no differences were found in BWi between 366 temperature treatments.

367
There was a significant effect of both ploidy (p < 0.001, 2N > 3N) and temperature (p 368 < 0.001, 8 > 6 > 11 °C) on final smolt weight (BWf) and a significant interaction between 369 ploidy and temperature (p < 0.001). BWf was comparable between ploidies in fish at 6 °C, 370 however, diploids had significantly higher BWf than triploids in 8 and 11 °C treatments ( Fig.   371 2b). Within diploids, there was an increase in fish BWf between 6 and 8 °C treatments, and then comparable thereafter. Conversely, BWf of triploids was comparable between 6 and 8 °C 373 treatments, but lower in fish at 11 °C.

374
Although statistical differences could not be determined, TGC of both diploid and 375 triploid fish at 6 °C appeared to be similar (Fig. 2c). However, triploids appeared to have a 376 lower TGC at 8 and 11 °C compared to diploids. Diploids had an overall higher final fibre number (FFN) compared to triploids, however within 380 temperature treatments, differences were significant only in fish from the 6 °C treatment with 381 a higher (+23 %) FFN in diploids than triploids (Table 3). An overall temperature effect was 382 observed with FFN in fish from 8 °C being significantly greater than 11 °C, but both 383 comparable to FFN in fish at 6 °C. In diploids, there was no significant difference in FFN 384 between any temperature treatments, while in triploids, fish at 8 °C had significantly more 385 fibres per mm 2 compared to 6 °C, but both were comparable to 11 °C.

386
Triploids had an overall higher muscle fibre cross-sectional surface area than diploids, 387 however within temperature treatments, differences were significant only in fish from the 6 °C 388 treatment with a higher (+23 %) area in triploids than diploids. There was a significant 389 interaction between ploidy and temperature on muscle fibre cross-sectional surface area 390 whereby no differences were found within diploids, while triploids had significantly higher 391 area at 6 °C compared to 8 °C, but both were comparable to 11 °C.  (Table 4a). Within 396 diploids, visible external deformity prevalence was significantly greater in 11 °C compared to 397 8 °C but not different from 6 °C. Triploids also had increased deformity prevalence in 11 °C 398 compared to 8 °C, but also significantly higher when compared to 6 °C. These deformities were 399 mainly comprised of jaw malformation as externally visible vertebral deformities were 400 negligible. Triploids had higher jaw deformity prevalence in 8 °C and 11 °C compared to 401 diploids but no difference between ploidy was evident at 6 °C. In diploids, jaw deformity  (Table 4b). 409 Triploids had a significantly higher deformity prevalence than diploids in 8 °C (72 vs. 410 24 %), and 11 °C (88.2 vs. 40 %) treatments, but prevalence was comparable between ploidies 411 at 6 °C (Table 4b). Within diploids, there was no significant difference in the prevalence of 412 radiologically deformed individuals with increasing incubation temperatures. Within triploids 413 a comparable deformity prevalence was exhibited between 8 and 11 °C, however those from 6 414 °C had a significantly lower prevalence.  The most common location for these deformed vertebrae was in the tail fin (R4) 425 irrespective of ploidy or temperature (Fig. 4a, b, c). In triploids, there is a common peak at v54 426 in all temperatures, however, in diploids there was a shift in the peak with increasing 427 temperatures (6, v54-55 < 8, v54-56 < 11 °C, v56-57). The prevalence in this region appeared 428 to be consistent in diploids in each temperature treatment (~10 %), however an increase was 429 observed in triploids in response to increasing temperature treatments (6, ~20 % < 8, ~30 % < 430 11 °C, ~50 %). Both diploids and triploids showed a similarly increased deformity prevalence 431 in the caudal trunk (R2) when incubated at 11 °C, however triploids had a much larger increase 432 in the cranial trunk (R1) compared to diploids (35 vs. 10 %).  utilisation at the eyeing stage in both ploidy (Fig. 5a, b). 444 Conversely, at first feeding (after the temperature shift), an upregulation of fas was 445 found in diploids from 6 and 11 °C treatments (~2 fold), and an upregulation of srebp1 was 446 found in both ploidy from 6 and 11 °C (4 -8 fold) compared to those in 8 °C (Fig. 5c, d) At the eyeing stage, there was an upregulation in igfbprp found in ova from 11 °C in both 453 diploids (~4.5 fold) and triploids (~3 fold) compared to 8 °C but were only significantly greater 454 than expression at 6 °C within respective ploidies (Fig. 6a, b). No other differences were found 455 between temperature treatments in diploids, however there was a significant downregulation 456 of igf1 and myod in triploid ova from 6 °C compared to 8 °C.

457
At first feeding, there was a significant upregulation in igf1 (~3.5 fold), igf2 (~6.5 fold) 458 and igfbrp (~8.5 fold) found in fish from both 6 and 11 °C treatment compared to 8 °C in 459 diploids (Fig. 6c). Conversely, igf1r showed lower expression in diploids from 6 and 11 °C 460 treatments compared to 8 °C. In triploids, igf1 had significantly greater expression in fish from 461 6 °C (~4 fold) and 11 °C (~8 fold) compared to 8 °C (Fig. 6d). Further, upregulation in fish 462 from 11 °C was significantly higher than in 6 °C. Like in diploids, igf1r expression was also  At smolt, there were also no differences found in expression of any of the genes 468 involved with muscle development in diploids (Fig. 6e). In triploids, a significant difference 469 between temperatures was only observed in igf2, with fish from 6 °C showing an upregulation 470 (~2 fold) compared to those from 8 °C (Fig. 6f). 471 472 3.6.3. Genes associated with bone formation and mineralisation compared to 8 and 6 °C (Fig. 7a). Both col1a1 and col2a1 expression was significantly higher 475 in diploids of 11 °C compared to 6 °C, although were comparable to 8 °C. In triploids, there 476 was a significant downregulation of alp, col2a1, opn and sparc in 6 °C compared to both 8 and 477 11 °C (Fig. 7b). No other differences were observed.

478
At first feeding in diploids, no difference in alp expression was observed (Fig. 7c), 479 however a significant upregulation in first feeding triploids from 11 °C (~2 fold) compared to 480 8 °C was found (Fig. 7d). A significant upregulation of bmp2, bmp4, col2a1, mmp13, opn and 481 sparc occurred in first feeding fry from the 6 and 11 °C treatments, irrespective of ploidy ( Fig.   482 6c, d). An upregulation was also found in col1a1 expression in diploids from 11 °C compared 483 to 8 °C (~2 fold), however no differences were found between any of the temperature 484 treatments in triploids. ocn was the only gene associated with bone formation to show a 485 significant downregulation in 6 and 11 °C compared to 8 °C, and this occurred in both ploidies.

486
At smolt, there were no differences found in expression of any of the genes associated 487 with bone formation within each ploidy (Fig. 7e, f).

490
The present study showed that incubation temperature during embryonic development had an 491 evident effect on both diploid and triploid Atlantic salmon survival, growth and skeletal health.

492
Results confirmed that triploids have a lower optimal thermal range than diploids, and they 493 showed an improved survival when incubated at ~6 °C during embryogenesis pre-hatch. 494 Moreover, growth rate was comparable between ploidies incubated at 6 °C, but triploids had 495 poorer growth compared to diploids at 8 and 11 °C. This study intends to build on results from with incubation history, with those exposed to a higher temperature during incubation having 510 the highest mortality rates. This was true for both ploidies, highlighting the need for lower 511 temperatures during this developmental stage. for all temperature treatments in this study were higher in diploids than in triploids, and 519 significantly so in 8 and 11 °C. This was supported by the lack of differences in ploidy-specific 520 expression of myf5 and myod between treatments during embryogenesis, both genes being 521 involved in the initial stages of muscle development. Moreover, there was an altered expression 522 in genes associated with the somatotropic axis in both ploidies, whereby igf1, igf2, and igfbprp 523 all showed increased expression in fish from 6 and 11 °C treatments relative to the 8 °C control and it is well established that temperature has a direct impact on enzyme activity and the 538 ionisation of a solution, subsequently altering the pH. Therefore, it is highly likely that 539 temperature not only affects somatic metabolism, but more fundamentally, the utilisation of 540 yolk during embryogenesis.
In the present study, we observed no differential expression in genes associated with lipid utilisation between incubation temperatures at the eyeing stage (~250 °days) in both 543 ploidies. However, the ~2 °C temperature change at 400 °days clearly influenced the ability to 544 utilise the yolk later during embryonic development as individuals from both the 6 and 11 °C 545 treatments showed upregulation of fas in diploids and srebp1 in both ploidies at first feeding 546 (~900 °days). The expression of these genes, involved with fatty acid biosynthesis and 547 subsequent storage, appears to be significantly increased as a result of the temperature shift, 548 suggesting that acute changes in temperature has a direct influence on the energy status of a 549 developing fish. Although the gene expression pattern appeared to be the same in fish incubated 550 at 6 and 11 °C, the resulting growth was very different. Furthermore, a downregulation of lxr 551 was found in triploid first feeding fry previously incubated at 6 °C, whist no differences were

562
This initial difference in first feeding weight would certainly impact the subsequent 563 growth trajectory of the cohorts. Due to fertilisation of all treatments occurring on the same 564 day (to control for parental effect) and each treatment experiencing different thermal regimes, 565 the first feeding dates ultimately differed. As a result, treatments experienced a different 566 number of feeding days up to the point of smoltification. This differential grow out duration in 567 part resulted in different final body weights (BWf) at smolt between temperature treatments 568 that could not be compared directly. However, when comparing ploidies, the growth rate, and 569 therefore BWf, was reduced in triploids from the 8 and 11 °C treatments relative to diploids.

570
This agrees with the theory that triploids have a lower thermal tolerance than diploids and 571 concurs with a recent study where metabolic rate and aerobic scope in triploids suffered at 10.5 572 °C but was comparable to diploids at 3 °C (Riseth, Fraser, Sambraus, Stien and Hvas, 2020).

573
Nevertheless, triploids have also been reported to have better growth rates than diploids under 574 different experimental incubation temperatures (Taylor et al., 2011;Fraser et al., 2015). In the present study, both ploidies had comparable growth rates when incubated at 6 °C, suggesting 576 that triploids held for this short duration at the lower temperature was enough to match the 577 growth performance of diploids.

578
In order to standardise smolt weights for comparison, final weights were predicted 579 using TGC in each treatment to adjust to the same number of developmental grow out days 580 (Fig. 8). Diploid smolts were predicted to be in the range of 72 -82 g with no clear effect of 581 incubation temperature. Triploids at 6 °C were predicted to match their diploid siblings (71.7 582 and 72.6 g, respectively), however, they were predicted to be much smaller at 8 °C (53.5 g) temperatures, albeit using a commercial diet with no P supplementation from first feeding.

627
Ca and P are both key minerals for bone mineralisation (Fjelldal, Nordgarden and 628 Hansen, 2007) and thus it appears that incubation temperature (or the temperature shift) had no 629 impact on mineralisation within a given ploidy. There was an overall ploidy effect in whole 630 body Ca, P and Ca:P ratio, with triploids having significantly higher concentrations, however, 631 this most likely reflected the ploidy-specific diets used to ensure optimal nutritional 632 requirements were met. Nonetheless, whole body P and Ca levels in both ploidy and treatments 633 were within normal accepted ranges generally accepted to be reflective of good bone contrast, in the present study, a significant upregulation of col2a1 was observed at first feeding in fish that experienced a temperature switch at 400 °days compared to the constant 8 °C 644 control. Furthermore, there was an increase in prevalence of jaw malformation in fish exposed   The data that support the findings of this study are available from the corresponding author 724 upon reasonable request.  Table 2. Mortality (%) during specific developmental windows in diploid and triploid Atlantic salmon incubated as embryos under different temperature treatments. Where there was replication (between fertilisation and 400 °days), data are expressed as means ± SD (p < 0.05, Two-Way ANOVA). Where there were single groups thereafter, data are expressed as tank value ± SD (p < 0.05; Analysis of CLT). Superscripts denote significant differences between treatments and ploidy within specific developmental stages.  2. Growth summary of diploid (black) and triploid (grey) Atlantic salmon incubated under different temperature treatments including (a) body weight in first feeding alevins; BWi (g), (b) final body weight in smolts; BWf (g), and (c) thermal growth coefficient (TGC) between first feeding and smolt. Data are expressed as mean ± SD (n = 34 -50 fish treatment -1 ploidy -1 ). Superscripts denote significant differences between treatments (p < 0.05, Two-Way ANOVA).     Temperature treatment ( C)