Growth hormone transgenesis and polyploidy increase metabolic … · 27 28 To whom correspondence should be addressed: ... 37 ontogenic effects of GH-transgenesis on the respiratory

copy 2013 Published by The Company of Biologists Ltd

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Growth hormone transgenesis and polyploidy increase metabolic rate alter 1

the cardiorespiratory response and influence HSP expression to acute 2

hypoxia in Atlantic salmon (Salmo salar) yolk-sac alevins 3

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Polymeropoulos E T 1 Plouffe D 2 LeBlanc S3 Elliott NG1 Currie S3 Frappell PB4 7

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9 1CSIRO National Food Futures Flagship CSIRO Marine and Atmospheric Research GPO Box 1538 Hobart Tasmania 10

7001 Australia 2AquaBounty Farms Souris Prince Edward Island C0A 2B0 Canada3Department of Biology Mount 11

Allison University Sackville NB E4L 1G7 Canada 4University of Tasmania 7001 Hobart Australia 12

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To whom correspondence should be addressed Elias Polymeropoulos CSIRO National Food Futures Flagship CSIRO 28

Marine and Atmospheric Research Marine Laboratories Castray Esplanade 7000 Hobart Australia Email 29

eliaspolymeropouloscsiroau Phone +61-3-6232-5208 30

httpjebbiologistsorglookupdoi101242jeb098913Access the most recent version at J Exp Biol Advance Online Articles First posted online on 27 March 2014 as doi101242jeb098913httpjebbiologistsorglookupdoi101242jeb098913Access the most recent version at

First posted online on 27 March 2014 as 101242jeb098913

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Summary 31

Growth hormone (GH) transgenic Atlantic salmon display accelerated growth rates compared 32

to non-transgenics GH-transgenic fish also display cardiorespiratory and metabolic 33

modifications that accompany the increased growth rate An elevated routine metabolic rate 34

has been described for pre- and post-smolt GH-transgenic salmon that also display 35

improvements in oxygen delivery to support the increased aerobic demand The early 36

ontogenic effects of GH-transgenesis on the respiratory and cellular physiology of fish 37

especially during adverse environmental conditions and the effect of polyploidy are unclear 38

Here we investigated the effects of GH-transgenesis and polyploidy on metabolic heart and 39

ventilation rates and heat shock protein (HSP) levels after exposure to acute hypoxia in post-40

hatch Atlantic salmon yolk-sac alevins Metabolic rate decreased with decreasing partial 41

pressures of oxygen in all genotypes In normoxia triploid transgenics displayed the highest 42

mass specific metabolic rates in comparison to diploid transgenics and non-transgenic 43

triploids which in contrast had higher rates than diploid non-transgenics In hypoxia we 44

observed a lower mass-specific metabolic rate in diploid non-transgenics compared to all 45

other genotypes However no evidence for improved O2 uptake through heart or ventilation 46

rate was found Heart rate decreased in diploid non-transgenics while ventilation rate 47

decreased in both diploid non-transgenics and triploid transgenics in severe hypoxia 48

Regardless of genotype or treatment inducible HSP70 was not expressed in alevins 49

Following hypoxia the constitutive isoform of HSP70 HSC70 decreased in transgenics and 50

HSP90 expression decreased in all genotypes These data suggest that physiological changes 51

through GH-transgenesis and polyploidy are manifested during early ontogeny in Atlantic 52

salmon 53

54

55

56

57

58

59

60

Key words Salmo salar hypoxia growth hormone transgenesis polyploidy metabolic rate 61

oxygen transport heat shock proteins 62

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Abbreviations 63

2n = diploid 64

3n = triploid 65

bpm = beats per minute 66

βwO2 = solubility of oxygen in water 67

fH = heart rate 68

fV = ventilation rate 69

GH = growth hormone 70

HSP = heat shock protein 71

kPa = kilopascal 72

M O2 = metabolic rate 73

M O2g = mass specific metabolic rate 74

PO2 = partial pressure of oxygen 75

Ta = ambient temperature 76

Tx = transgenic 77

Nt = non transgenic 78

79

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Introduction 80

Growth hormone (GH) transgenic fish have gained increasing attention in the aquaculture 81

industry as a result of accelerated growth rates (~two to tenfold) when compared to non-82

transgenic conspecifics (Devlin et al 1994 Martinez et al 1999 Shao Jun et al 1992 83

Stokstad 2002) Physiologically faster growth entails a higher demand for O2 through an 84

increase in metabolism In salmon from the parr stage onwards an increase in resting 85

routine metabolic rate has been verified in GH-transgenic fish (Cook et al 2000 Lee et al 86

2003 Stevens et al 1998) In association with an increased O2 demand modifications to the 87

cardiorespiratory physiology in GH-transgenic Atlantic salmon (Salmo salar) have been 88

documented that include an increase in heart size cardiac output hemoglobin concentration 89

muscle aerobic enzyme activity (Deitch et al 2006) gill surface area (Stevens and Sutterlin 90

1999) and modifications to cardiorespiratory function (Blier et al 2002 Cogswell et al 91

2002) 92

93

Due to the concerns of non-native transgenes impacting wild fish stocks in the event of an 94

escape from commercial aquaculture it has been suggested that transgenic fish species 95

should be reproductively sterile In some fish species including salmonids polyploidy 96

sporadically occurs as a natural evolutionary trait (Allendorf and Thorgaard 1984 Ferris 97

1984 Schultz 1980) and is often artificially induced to produce commercial triploid 98

reproductively sterile progeny The biological effects associated with triploidy in fish have in 99

part been investigated and are mainly attributed to heterozygosity cell size or gonadal 100

development (Benfey 1999 Maxime 2008) As a consequence of the additional complement 101

of chromosomes in triploid organisms nuclear size is increased in the cells of most organs 102

and tissues which in turn causes an increase in cell volume with overall body size being 103

maintained through hypoplasia in triploids (Benfey 1999) In theory sterile triploid 104

organisms have the potential for greater growth due to the abundance of genetic material as 105

well as the diversion of energy from gonadal growth to somatic growth during sexual 106

maturation In practice the evidence supporting increased growth rates in triploid organisms 107

is inconclusive (Tiwary et al 2004) since both slower (Galbreath et al 1994) as well as 108

equivalent growth rates have been observed in triploid populations relative to that of diploid 109

conspecifics (McGeachy et al 1995 Sacobie et al 2012) In the past poor performance of 110

triploid fish in comparison to diploids especially under conditions of high O2 demand has 111

been suggested to be related to changes to their respiratory physiology and therefore hypoxia 112

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tolerance (Benfey 1999) However poor growth performance and increased prevalence of 113

deformities in triploids has been refuted in more recent studies (OFlynn et al 1997 Oppedal 114

et al 2003 Sadler et al 2001 Taylor et al 2011) suggesting that triploid salmon can 115

perform as well as diploid siblings Differences in hematological parameters associated with 116

triploidy as well as a reduction in gill surface area and metabolism have been reported for 117

salmonid fish (Benfey 1999) but the physiological effect of triploidy remains poorly 118

understood Triploid Atlantic salmon have been documented to display a reduction in O2 119

carrying capacity (Graham et al 1985) whereas triploid rainbow trout show higher rates of 120

O2 consumption (Oliva-Teles and Kaushik 1987) and early collapse of the cardiovascular 121

systems ability to deliver O2 to the body during warming (Verhille et al 2013) Triploid 122

brook trout have been shown to have lower rates of O2 consumption in comparison to diploid 123

counterparts (Stillwell and Benfey 1996) 124

125

Respiration in Atlantic salmon yolk-sac alevins at hatching is primarily cutaneous under 126

normoxic conditions (Rombough 1988 Wells and Pinder 1996a Wells and Pinder 1996b 127

Pelster 1999) and at this stage the larvae are exclusively dependent on the energy supply 128

contained within the yolk-sac Hence both O2 supply and demand may be under different 129

regulatory control compared to later stages of development or adulthood and may be 130

differentially affected by ploidal level or GH-transgenesis 131

132

Collectively these findings suggest that in salmonids GH-transgenesis and triploidy have 133

significant impacts on the oxygen cascade (ie the series of diffusive and conductive steps in 134

the pathway of O2 from the environment to the mitochondria) Further such impacts might be 135

exacerbated under adverse environmental conditions In order to cope with the potential 136

damaging and even lethal effects of environmental stress fish respond with behavioural (eg 137

evasion) physiological (eg release of stress hormones) and cellular (eg induction of heat 138

shock proteins [HSPs]) strategies (Barton 2002 Currie 2011) The cellular stress response 139

characterised by an increase in the synthesis of HSPs is generally exhibited by all organisms 140

(Schlesinger 1990 Feder and Hofmann 1999) More recently the temporal expression of 141

HSPs throughout ontogeny has gained increased attention and it has been suggested that 142

HSPs play a key role during early embryonic development in fish (Deane and Woo 2011) 143

Expression of genes encoding HSPs show spatial and temporal specificity during early 144

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development of zebrafish and in the case of HSP70-4 and HSP90α they appear to be 145

required for normal tissue development (Krone et al 2003) 146

147

Given that GH-transgenic Atlantic salmon show phenotypic differences in metabolism and 148

cardiorespiratory function and that hypoxiaanoxia elicits a physiological stress response and 149

alters HSP expression (Kregel 2002) it is likely that genotypic differences resulting in 150

changes to respiration will affect the heat shock response under conditions of low O2 151

availability The cellular stress response to low environmental O2 levels is variable in fish and 152

high levels of tissue cell and temporal specificity in terms of the levels of HSP expression 153

have been reported (Airaksinen et al 1998 Currie and Tufts 1997 Currie and Boutilier 154

2001 Currie et al 2010) Furthermore a decrease in HSP70 mRNA production in hepatic 155

tissue of sparids (family Sparidae) has been reported after exogenous administration of 156

growth hormone (Deane et al 1999) Despite this body of research we know very little 157

regarding the effects of ploidy andor transgenesis on the cellular stress response in fish 158

159

In the present study we tested for differences in metabolic rate cardiorespiratory function 160

and the expression of HSPs (HSP70 HSC70 and HSP90) between diploid and triploid 161

transgenic and non-transgenic newly hatched yolk-sac alevins We further investigated how 162

these parameters are altered under hypoxic conditions It was hypothesised that GH-163

transgenesis will lead to accelerated growth rates that are reflected in an increase in oxygen 164

uptake at this early developmental stage This increase may be supported by improved O2 165

delivery through cardiorespiratory modifications especially when exposed to acute hypoxia 166

Despite the apparent susceptibility of triploid Atlantic salmon (Benfey 1999 and citations 167

within) to hypoxia there is no clear evidence that this is the case in the present study 168

Therefore it was hypothesized that the O2 demand of triploids is unlikely to differ from that 169

of diploids A disturbance to cellular homeostasis by hypoxic exposure will elicit a cellular 170

stress response that may be augmented by increased anabolic processes or an increased 171

metabolism associated with GH-transgenesis 172

173

174

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Results 175

176

Treatments and Measurements 177

Measurements of metabolic rate (M O2) were performed at 8degC on 2n and 3n transgenic 178

(2nTx 3nTx) as well as 2n and 3n non-transgenic (2nNt 3nNt) Atlantic salmon yolk-sac 179

alevins (Series I as described in Polymeropoulos et al 2013) Subsequent measurements 180

were conducted of MO2

heart rate (fH) as well as ventilation rate (fV see Series II) Mass-181

specific M O2 was calculated on the basis of yolk-free wet body mass 182

183

Additionally we tested the cellular stress response of alevins (see Series III) of the different 184

genotypes (2nNt 2nTx 3nNt 3nTx) to a similar hypoxic stress as in Series I Measurements 185

of M O2 as described in Series I were repeated in 2nNt 2nTx 3nNt 3nTx (n = 5 respectively) 186

alevins at 8degC Here the alevins were successively exposed to decreasing levels of O2 187

(hypoxic stress) as they consumed the O2 in closed system respirometry chambers 188

Experiments were performed until a PO2 of 5 kPa was reached After this procedure 189

experimental animals were transferred to normoxic water for 24 h of recovery In parallel 10 190

alevins per genotype were maintained under indentical experimental conditions in normoxia 191

(control group) For this purpose the alevins were placed in multiwell plates (similar to that 192

described in Polymeropoulos et al 2013) submerged in well aerated water with only a thin 193

mesh separating the alevins from escaping into the surrounding water The control group was 194

maintained within the same multiwell plate for a duration that matched that of the 195

experimental animals (measurement + 24 h) this included a quick removal from the wells to 196

mimic the handling stress to which the experimental animals were exposed After the 197

required interval specimens were taken from the chambers kept in ice water measured for 198

length and weight then decapitated and kept at -80degC until HSP immunodetection could be 199

performed 200

201

Body mass and morphometry 202

Total body and yolk mass was lower in 3nTx yolk-sac alevins in comparison to all other 203

genotypes (Table 1) Yolk-free body mass was highest in 2nNt and differed from 3nNt and 204

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3nTx alevins The number of caudal fin rays as a measure of developmental stage and body 205

length did not differ between genotypes Yolk-sac alevins that were used in Series II or III 206

did not differ statistically in any of the parameters measured in animals used in Series I 207

208

Series I Mass-specific metabolic rate 209

Generally M O2g decreased with increasing hypoxia the decrease displaying a triphasic 210

pattern Within a PO2 range of 15 to 11 kPa values for M O2g within the groups remained 211

constant (two-way-RM-ANOVA P = 01) Therefore the data were separated into three 212

sections (Fig 2 dashed lines) and values for M O2g were pooled within a range of 3 kPa 213

(PO2) resembling normoxic (21-19 kPa) intermediate hypoxic (14-12 kPa) and severely 214

hypoxic (7-5 kPa) conditions M O2g in 3nTx alevins was higher (~8) than in 2nTx and 215

3nNt alevins while the latter two groups in turn had elevated metabolic rates (~8) in 216

comparison to 2nNt (Fig 2 inset One-way ANOVA) Within intermediate and severe 217

hypoxia treatments 2nNt alevins showed the lowest values for M O2g while 2nTx 3nNt and 218

3nTx alevins were not statistically different from each other (Fig 2 inset) 219

220

Series II Simultaneous measurement of mass-specific metabolic rate heart- and 221

ventilation rate 222

The cardiorespiratory function in the two metabolically most distinct genotypes from Series I 223

(2nNt and 3nTx) was subsequently studied Measurements of M O2g heart rate (fH) and 224

ventilation rate (fV) were made on individual 2nNt and 3nTx (n = 9 per group) alevins in 225

normoxia and the levels of intermediate and severe hypoxia as determined in Series I As 226

previously described in Series I (Fig 2) here M O2g in both groups decreased with declining 227

levels of PO2 compared to normoxic values (Fig 3a d) Using this experimental design the 228

overall M O2g in normoxia measured in Series II was ~175 times higher in comparison to 229

Series I As such 3nTx alevins displayed elevated M O2g of ~26 in normoxia compared to 230

16 in Series I and as in Series I remained elevated at all levels of PO2 in comparison to 231

2nNt alevins 232

233

3nTx and 2nNt alevins had a similar fH in normoxia (Fig 3b e) Despite a small reduction in 234

fH in 2nNt alevins in intermediate hypoxia it was not statistically different compared to 235

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normoxic values However in severe hypoxia fH in 2nNt alevins was reduced (~10) while 236

in 3nTx fH remained similar to measurements in normoxia In contrast to these findings both 237

3nTx and 2nNt yolk-sac alevins reduced their fV under severe hypoxia (Fig 3c f) In 238

normoxic and intermediate hypoxic conditions no difference in fV was detected between 239

genotypes 240

241

Series III HSP levels 242

The M O2g pattern in Series III was similar to that observed in Series I and was therefore 243

incorporated within Series I results Western blot analysis of whole animal samples revealed 244

that HSP70 (inducible isoform) was undetectable in the control or hypoxia treated alevins In 245

contrast HSC70 (constitutive isoform) and HSP90 were detectable but no differences in 246

relative expression of HSC70 between genotypes were observed in the control samples kept 247

in normoxic conditions (Fig 4a) However HSC70 levels in acute hypoxia-treated GH-248

transgenic alevins were significantly lower than in non-transgenics (P = 0031) Likewise 249

relative HSP90 expression in control groups were similar but significantly lower in all 250

hypoxia treated alevins (Fig 4b P lt 0001) 251

252

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Discussion 253

In the present study we show that exposure to acute hypoxia generally leads to a decrease in 254

metabolic rate in Atlantic salmon yolk-sac alevins when measured at 8degC In addition we 255

present evidence that GH-transgenesis and triploidy result in elevated mass-specific 256

metabolic rates in normoxia as well as acute hypoxia and the combination of GH-257

transgenesis and triploidy appear to have additive effects Furthermore we confirmed higher 258

metabolic rates of triploid transgenic alevins compared to diploid non-transgenic alevins in a 259

separate methodogical approach while simultaneously measuring heart rate and ventilation 260

rate Neither of these latter measures correlated with metabolic rate at this ontogenic stage 261

Hence the increase in metabolic rate through GH-transgenesis and triploidy is not reflected 262

in improved O2 uptake through heart or ventilation rate and therefore must relate to other 263

physiological mechanisms to ensure O2 delivery These might include morphological changes 264

such as gill structure or cutaneous thickness haematological parameters such as hemoglobin 265

concentration or changes in cardiac stroke volume The difference in metabolic rate among 266

groups was also not reflected in altered expression of HSPs (HSP70 HSC70 and HSP90) 267

under control conditions and hypoxia did not appear to elicit a cellular stress response 268

However hypoxia resulted in differential decreased HSP levels among groups with 269

transgenics showing reduced levels of HSC70 while HSP90 expression was significantly 270

decreased under hypoxic conditions across all genotypes 271

272

Series I and Series II 273

The M O2 curves in alevins that were measured in Series I using the closed system approach 274

generally showed a triphasic pattern Here an initial decline in M O2 with decreasing PO2 (21-275

14 kPa) is followed by a period where M O2 is decreasing at a slower rate (14-9 kPa) in 276

intermediate hypoxia before decreasing at a faster rate with more severe hypoxia (9-5 kPa) 277

This pattern was unexpected as it does not fully comply with either classic oxygen 278

conformity or regulation curves for M O2 (see Richards 2009) 279

280

It is possible that the initial steep decrease in M O2 is related to an increased activity of the 281

unconstrained alevin at the beginning of the measurement where the alevins have not fully 282

settled or reached resting M O2 after handling However once the animals were put in the 283

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respirometry chambers they settled on the mesh within a few minutes and did not move 284

(personal observation) In addition when M O2 was measured in the open flow respirometry 285

system and heart rate and ventilation rates where measured simultaneously it was verified 286

that resting heart and ventilation rates were established at a minimum usually within ~30 287

min The period of the initial steep decline in the closed system is significantly longer (~90-288

120 min) and therefore unlikely to be related to activity 289

290

The switch to a period of relative oxyregulation (slower decline in M O2) is difficult to 291

explain In section II the alevins show a greater ability to maintain M O2 which could be 292

indicative of a switch to a more ldquoO2 efficientrdquo metabolic pathway which is triggered at a 293

specific PO2 This pathway might involve the reallocation of ATP to different ATP 294

consuming pathways (ie protein synthesis degradation ion transport) that are more 295

critically involved in the hypoxic metabolic response During the exposure to a gradually 296

decreasing PO2 the alevins might have also adjusted to hypoxia through other pathways of 297

ATP production that support the maintenance of M O2 Even though the alevins appear to 298

settle relatively quickly in the respirometers a measure of activity (eg frequency of fin 299

movement) during an experiment is likely to add further insight into the M O2 pattern 300

observed In addition the analysis of changes in substrate utilisation or anaerobic enzyme 301

activity (eg lactate dehydrogenase pyruvate kinase creatine phosphokinase) might add 302

further insight into changes in metabolic pathways Another simple explanation for the 303

triphasic pattern could be that if section I corresponds to an elevated M O2 due to an unsettled 304

state of the animal section II might then correspond to the true settled state The decrease in 305

M O2 in section III might then be analogous to the Pcrit where cellular changes are made to 306

further reduce metabolic rate as part of ldquometabolic depressionrdquo 307

In adult salmonids the physiological response to acute hypoxia is constituted by metabolic 308

depression cholinergic bradycardia as well as an increase in ventilation (Burggren and 309

Pinder 1991 Holeton and Randall 1967 Randall 1982) to counteract the reduced 310

availability of O2 and high aerobic demand typical for salmonids In salmonid and other 311

lower order vertebrate larvae however this cardiorespiratory response appears to be absent 312

(Holeton 1971 McDonald and McMahon 1977) At this early developmental stage 313

diffusion of O2 across the skin is the primary avenue for O2 uptake and the reliance on gills as 314

the main respiratory organ occurs at the end of the larval stage (Wells and Pinder 1996a 315

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Wells and Pinder 1996b) This precondition might provide an explanation for the difference 316

in respiratory response to hypoxia in comparison to adult fish but also supports the notion of 317

an uncoupling of heart rate as well as ventilation from metabolic rate as it has been observed 318

in other vertebrates such as zebrafish larvae (Barrionuevo and Burggren 1999) and the 319

chicken embryo (Mortola et al 2009) 320

321

The uncoupling of heart and ventilation rate from metabolic rate questions the relevance of 322

convective O2 transport in the O2 cascade and the importance of gill ventilation at this early 323

ontogenic stage It has been suggested that the embryonic heart beat might primarily serve 324

alternative functions such as nutrient transport and angiogenesis (Burggren 2004) whereas 325

the gills serve in ionoregulation (Fu et al 2010) Alternatively the absence of a 326

cardiorespiratory response to hypoxia could be explained by a lack of chemosensory 327

feedback at this life stage In a recent study on the ontogeny of rainbow trout cardiac control 328

this hypothesis was refuted as adrenergic and cholinergic control of the heart is present 329

shortly after hatching and the latter can be delayed by chronic hypoxic exposure (Miller et 330

al 2011) 331

332

In the present study we observed bradycardia and a decrease in ventilation in response to 333

severe hypoxia in diploid Atlantic salmon alevins In contrast 3nTx alevins did not display 334

this bradycardic response but a decrease in fV at a low PO2 was observed in both genotypes 335

This result implies that although there was no general coupling of fH or fV to M O2 the 336

chemosensory precondition for a cardiorespiratory response to hypoxia are met but an 337

increase in metabolism did not lead to increased O2 uptake through heart or ventilation rate 338

339

The differences in M O2g in comparison between Series I and Series II are most likely due to 340

the different experimental designs where the relatively greater constraint of the animal in 341

Series II might have led to an elevation of M O2g Even though the overall reduction in M O2 342

with hypoxia is similar between genotypes in Series II (unlike in Series I) we observed a 343

more severe hypoxic cardiorespiratory response in 2nNt through a decrease in heart rate and a 344

trend towards lower ventilation rate The lack of a bradycardic response in 3nTx could be 345

indicative of a higher tolerance to severe hypoxia despite elevated M O2 and smaller relative 346

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reduction in M O2 in severe hypoxia compared to normoxic values (60 in 3nTx vs 80 in 347

2nNt) 348

349

If O2 uptake was purely limited by physical constraints and diffusive distances were similar 350

between genotypes of similar body mass the same response in fH and fV could be expected 351

As this is not the case the animal must be using physiological strategies to maximize O2 352

uptake These may include improved O2 extraction capabilities via an increase in cardiac 353

output (increase in cardiac stroke volume) hyperventilation through an increase in ventilation 354

volume or an increased hemoglobin concentration Equally changes to the diffusive 355

properties of the skin (eg diffusion distance) could affect O2 uptake and therefore the 356

cardiorespiratory response These possibilities require further experimentation 357

358

It is generally accepted that GH-transgenesis leading to accelerated growth rates is 359

associated with an increase in metabolism in juvenile and adult salmon (Deitch 2006) In this 360

study it was demonstrated that in normoxia GH-transgenesis and triploidy leads to an 361

increase in metabolism in alevins which appears to be additive when GH-transgenesis and 362

ploidy are combined This raises the question about the interactive effects of GH-transgenesis 363

and triploidy on metabolic physiology which so far has not been investigated Whereas the 364

increased metabolism as a consequence of GH-transgenesis is physiologically conclusive in 365

the light of a greater energy requirement for developmental and anabolic processes 366

alterations to respiratory mechanisms due to triploidy remain elusive It has been 367

hypothesised that the larger cell size of most tissues and the distinct morphology 368

characteristic of triploids (Benfey 1999) would have an impact on O2 transport to the cell and 369

therefore on overall metabolism (Maxime 2008) According to Fickrsquos law of diffusion 370

(Dejours 1981) the increase in cell volume of triploid organisms should increase the 371

distance for O2 diffusion to reach the centre of the cell A greater O2 gradient must then be 372

established to meet the O2 demand which in turn increases O2 uptake Despite numerous 373

accounts of alterations to hematological parameters in the literature in particular the size and 374

shape of triploid erythrocytes the effects of triploidy on metabolic rate in fish remains 375

inconclusive and does not appear to follow a consistent pattern (Benfey 1999 Maxime 376

2008) If triploidy affects the hematology of fish it stands to reason that further investigation 377

of O2 binding properties of embryonic larval hemoglobin in triploid fish should help answer 378

this question 379

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380

Series III 381

On a cellular level we observed decreases in predominantly constitutive HSPs after exposure 382

to acute progressive hypoxia and for HSC70 this decrease was genotype dependent 383

(transgenic polyploid) Expression of the stress inducible HSP70 was not observed thus 384

hypoxia does not appear to induce HSPs at least at this early developmental stage The effects 385

of hypoxia on HSP induction in fish are variable Our finding is consistent with Zarate and 386

Bradley (2003) who show that HSP30 HSP70 and HSP90 mRNA were not induced with 387

hypoxia in Atlantic salmon Similarly anoxia did not induce HSP70 in the red blood cells of 388

rainbow trout (Currie and Tufts 1997) On the other hand HSPs were induced with hypoxia 389

in rainbow trout gill epithelial cells (Airaksinen et al 1998) Likewise the air breathing 390

mudminnow Umbra limi responded to hypoxia and 6 h of air exposure with significant 391

increases in HSP70 and HSP90 in liver tissue (Currie et al 2010) In the anoxia tolerant 392

western painted turtle long-term but not short-term anoxia lead to an induction of HSPs 393

(constitutive HSP73 inducible HSP72 and HSP90) in several tissues (Ramaglia and Buck 394

2004) Hypoxic submergence also resulted in elevated levels of HSP70 in the hearts of the 395

hypoxia-tolerant common frog Rana temporaria (Currie and Boutilier 2001) These latter 396

studies suggest that the induction of HSPs may be at least part of the strategy of low oxygen 397

tolerance protecting proteins from damage associated with low oxygen andor the sudden 398

return of oxygen to tissues These studies also illustrate the complexity of the HSP response 399

to hypoxiaanoxia and highlight the necessity to improve our understanding of its temporal 400

spatial and species-specific regulation 401

402

Atlantic salmon are considered hypoxia intolerant and thus it may not be surprising that HSP 403

induction was not observed The aerobic thermal limit and thus the susceptibility to hypoxia 404

is thought to be greater during early life stages when the oxygen supply shifts from cutaneous 405

to ventilatory and circulatory systems It is possible that the lack of a heat shock response 406

(eg HSP70) with hypoxia observed here contributes to the oxygen sensitivity at the alevin 407

stage It is also possible that the decrease in constitutive HSPs with hypoxia might be 408

indicative of a reduced ability for protein synthesis due to the lack of sufficient energy 409

obtained via aerobic pathways during hypoxic hypometabolism That said the red blood cells 410

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of adult rainbow trout are able to mount an inducible heat shock response under severe 411

hypoxia (Currie and Tufts 1997) and during pharmacological inhibition of both oxidative 412

phosphorylation and glycolysis (Currie et al 1999) suggesting that the cells of adult 413

salmonids at least are not energetically limited in terms of their heat shock response Protein 414

synthesis in earlier developmental stages however may be more susceptible to these 415

energetic constraints 416

417

Interestingly the decrease in HSC70 observed during hypoxia appears to be restricted to GH-418

transgenic alevins As 3nNt larvae display a similar increase in metabolic rate as 2nTx this 419

finding can not be explained by differences in metabolism Our knowledge of the cellular and 420

molecular consequences resulting from GH-transgenesis are limited It is possible that protein 421

synthesis is preferentially directed towards muscle growth in transgenic animals over 422

constitutive HSPs particularly under harsh environmental conditions (eg hypoxia) 423

Interestingly the differences in metabolic rate between groups was not reflected in 424

differences in HSP levels Differences in HSPHSC levels with hypoxia may have been 425

anticipated due to the higher demand for O2 in eg 3nTx over all other groups It appears 426

however that generally the HSP levels in normoxia or hypoxia are not influenced by the 427

initial metabolic rate Future studies should examine the effects of long-term exposure to 428

hypoxia on the cellular stress response throughout development in transgenic animals to 429

improve our understanding of the involvement of HSPs in normal growth and development as 430

well as in hypoxia tolerance 431

432

Conclusions and perspectives 433

This is the first study examining the metabolic cardiorespiratory and cellular stress response 434

to hypoxia of a GH-transgenic Atlantic salmon under consideration of the effects of 435

polyploidy We conclude that GH-transgenesis or artificially induced triploidy increase 436

metabolism and together alter cardiorespiratory function under oxygen limiting conditions in 437

Atlantic salmon during early development Hypoxia did not induce a cellular stress response 438

which may have a negative effect on the ability of the animal to deal with harsh 439

environments We see an important role in further investigating respiratory and cellular 440

responses to environmental stress in triploid and GH-transgenic vertebrates during ontogeny 441

and comparing them to juvenile and adult life stages It is likely that physiological differences 442

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during early life stages are carried on to adulthood and might affect robustness or 443

susceptibility to environmental stress in an aquaculture environment 444

Materials and Methods 445

Experimental animals 446

Atlantic salmon eggs used in this study were all fertilized on the same day and hatched at 447

~400 degree days post-fertilisation (50 days at 8degC post-fertilisation) Yolk-sac alevins were 448

reared under standard hatchery conditions (8degC in darkness) at AquaBounty Canada 449

(Fortune Prince Edward Island Canada) in heath-stack incubators fed with flow-through 450

UV-treated ground water Four groups of alevins were used diploid (2nNt) triploid (3nNt) 451

diploid transgenic (2nTx) and triploid transgenic (3nTx) Transgenic Atlantic salmon carry a 452

single copy of the α-form of a salmon growth hormone transgene (opAFP-GHc2) that is 453

stably integrated at the α-locus in the EO-1α lineage and exhibit a rapid growth phenotype 454

(AquaBounty Technologies 2010) Triploidy in Atlantic salmon was induced by a 455

hydrostatic pressure shock for 5 minutes at a constant 9500 psi initiated at 300degC minutes 456

after fertilisation which prevents the extrusion of the second polar body during meiotic 457

development and renders the animals sterile with an effectiveness of better than 99 458

459

Yolk-sac alevin mass and morphometry 460

Yolk-sac alevin (alevin) dimensions were measured using a horizontal dissecting microscope 461

(Leica EZ4D) with an integrated ocular micrometer Alevins were killed immediately 462

following each experiment The total wet weight of each alevin was measured in grams to the 463

nearest 0001g prior to yolk-sac removal The wet yolk-free body and wet yolk mass of 464

each alevin was measured in grams following the separation of the the yolk-sac from the 465

alevin body using micro-operating scissors Total body length (mm) and number of caudal fin 466

rays (n as a proxy for developmental stage Gorodilov 1996) were measured under a 467

dissecting microscope 468

469

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Series I Measurement of metabolic rate in alevins 470

The rate of oxygen consumption (cong metabolic rate = M O2) in alevins was measured using 471

fluorescence-based closed system respirometry Each alevin was placed in an individual well 472

in a polystyrene multiwell plate (24 wells x 33 ml Oxodishreg PreSens Regensburg 473

Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure 474

of O2 PO2 = 21 kPa) Each well contained an optode to monitor O2 levels and was fitted with 475

a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact 476

the optode (as described in Polymeropoulos et al 2013) Four wells remained empty of 477

animals for calibration purposes two were filled with normoxic water from the rearing trays 478

and two with a zero solution (1 sodium sulphite solution which acted as an O2 scavenger) 479

Each well was sealed with a glass cover slip using vacuum grease care being taken to 480

exclude all air bubbles The multiwell plate was placed on a SensorDishReader (SDR 481

PreSens) on an orbital mixer inside a temperature controlled cabinet The orbital mixer was 482

set to the lowest speed (70 rpm) required to move a small (2mm Oslash) stainless steel ball around 483

the perimeter at the bottom of each well ensuring the water in each well was gently mixed 484

the alevins remained motionless on the mesh The PO2 in each well was measured at 2 min 485

intervals starting from 21 kPa conditions until the PO2 reached 5 kPa Movement response to 486

physical disturbance confirmed that all alevins were alive at the end of each experiment 487

Before every measurement optodes were calibrated by alternatively filling the wells with 1 488

sodium sulphite solution containing 10-4 M cobalt chloride (PO2 = 0 kPa) and air-equilibrated 489

water (PO2 = 21 kPa) 490

491

The M O2 (μmol O2 min-1) for each egg or alevin was calculated from the decline in PO2 over 492

time in each well after taking into account the diffusive flux of O2 that occurs through the 493

polystyrene Oxodishreg well plate (see Polymeropoulos et al 2013) 494

495

Mass-specific M O2 was adjusted for yolk-free wet body mass (g) to account for differences in 496

body mass among individuals The volume of the chamber was corrected for the volume 497

displaced by each individual according to its total wet mass assuming a density of 1gml the 498

stainless steel ball bearing and mesh 499

500

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Series II Simultaneous measurement of metabolic rate heart rate and ventilation rate in 501

individual alevins 502

Simultaneous measurements of metabolic rate (M O2) heart rate (fH) and ventilation rate (fV) 503

were performed on individual alevins using a combined optophysiological and respirometry 504

system (Fig 1) Individual alevins were placed in a sealed flow-through respirometry 505

chamber (15 ml) under the exclusion of air the chamber being partially submerged in a 506

temperature controlled waterbath (Polyscience accuracy plusmn 0025degC) at the respective 507

temperature Water temperature in the chamber was measured with a thermocouple (accuracy 508

plusmn 01degC traceable to a National Standard) Normoxic water (PO2 = 21 kPa) was pumped 509

through the chamber at a constant flow rate of 15ml min-1 using a peristaltic pump (Living 510

Systems Instrumentation Burlington Vermont) the water first passing through a heat 511

exchanger in the water bath to maintain the required temperature 512

513

514

To minimise handling stress alevins were acclimated within the chamber under constant 515

flow of normoxic water for 1 h before measurement The chamber was then sealed for 516

approximately 30 min prior to flushing with water at the original incoming PO2 until the PO2 517

within the chamber had returned to the original PO2 (approx 8 min) The process of sealing 518

and flushing was repeated for each experiment where the desired incoming PO2 (kPa) was 519

achieved by bubbling water with the required mixture of O2 and N2 (high precision low-flow 520

gas blender V10040A Bird USA) and was confirmed with an optochemical O2 sensor 521

(Oxygen meter HQ10 Hach USA) It was assumed that the constant movement of the 522

pectoral fins and opercular pumping during periods where the chamber was sealed allowed 523

sufficient mixing of the water and prevented the formation of hypoxic boundary layers 524

surrounding the alevin 525

526

For the measurement of M O2 an optochemical O2 sensor in a flow-through cell (FTC-PSt3 527

PreSens) in combination with an O2 analyser (Fibox PreSens) was used This was connected 528

in series downstream to the respirometry chamber and maintained at the same ambient 529

temperature (Fig 1) In the closed but intermittently flushed respirometry system the rates of 530

O2 consumption (M O2 μmol min-1) were calculated as previously described (Clark et al 531

2005) Following a period where the chamber was sealed it was then flushed with fresh 532

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water and the M O2 was determined from the time integral of the resulting PO2 curve ( PO2 533

kPasdotmin-1) multiplied by the oxygen capacitance (βwO2 μmolsdotL-1sdotkPa-1) the flow of water (V 534

mlsdotmin-1) used to flush the chamber and the reciprocal of the time (t min) during which the 535

compartment was sealed (equation 1) 536

537

M O2= βwO2middotPO2middotV

t (1) 538

539

Mass-specific M O2 was calculated by adjusting for wet yolk-free body mass (g) 540

541

The validation of the intermittantly sealed flow-through respirometry system is described in 542

detail in the Appendix 543

544

During periods when the chamber was sealed heart rate (fH) and ventilation rate (fV) were 545

measured using optophysiological methods For this purpose a stereomicroscope (Stemi 546

SV6 Zeiss Germany) was equipped with a CCD-camera (SSC-DC50P Sony Japan) which 547

was situated above the transparent respirometry chamber to visually monitor the fish (Fig 1) 548

Video information was processed by a video dimension analyser (V94 Living Systems 549

Instrumentation Burlington Vermont) and displayed on a computer via a video capture USB 550

card (Roxio 315-E) In principle the instrument operates on the relative optical density 551

changes of border structures (ie border of beating heart or moving operculum) and generates 552

a linear voltage signal that can be read by a digital interface (PowerLab 8sp 553

ADInstruments) Separate measurements of fH and fV were performed for each level of PO2 554

within the first 5 min of the chamber being sealed Within this timeframe the PO2 within the 555

sealed chamber did not drop lower than 125 kPa from the initial value This was verified by 556

measuring the PO2 within the chamber using a microoptode Therefore the measurements for 557

fH and fV are representative of the desired PO2 A minimum of 30 consecutive heart beats and 558

operculum movements were recorded using LabChartreg7 Pro (ADInstruments) Movement 559

response to physical disturbance confirmed that all alevins were alive at the end of each 560

experiment 561

562

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Series III Heat shock protein (HSP) immunodetection 563

Whole alevins were ground in liquid nitrogen and soluble protein was extracted as in LeBlanc 564

et al (2012) Briefly 25-35 mg of ground sample was homogenized in tissue homogenization 565

buffer (50 mM Tris Sigma-Aldrich 2 SDS Bio-Rad 01 mM protease inhibitor cocktail 566

Sigma-Aldrich) using a PowerGen 125 polytron (Fisher Scientific) Samples were then 567

pushed through a 27G needle several times and spun at 14000 g for 10 min in a Sorvall 568

Legend RT microcentrifuge (Mandel) The supernatant containing the soluble protein was 569

removed and stored at -80oC The soluble protein concentration of each sample was measured 570

using the DC protein assay kit (Bio-Rad) based on the Lowry method (Lowry et al 1951) 571

572

To determine HSP levels immunodetection via western blot was performed using the Novex 573

Midi Gel System (Invitrogen) Briefly 15 μg of soluble protein per sample were loaded onto 574

4-12 Bis-Tris gels electrophoresed and transferred to PVDF membranes using the Iblot 575

system (Invitrogen) according to the manufacturerrsquos instructions Commercial standards 576

(HSC70 SPP-751 HSP90 SPP-770 Assay Designs) or a red blood cell sample from a heat 577

shocked rainbow trout (HSP70) were loaded onto each gel to allow direct comparison 578

between gels For immunodetection rabbit salmonid-specific primary antibodies against 579

HSP70 (AS05061) HSC70 (AS05062) and HSP90 (AS05063) were used at a final 580

concentration of 150 000 (Agrisera) The HSP70 antibody detects only the inducible form of 581

HSP70 and does not cross-react with the constitutively expressed isoform (HSC70) while the 582

HSP90 antibody does not distinguish between the HSP 90α HSP 90βa and HSP 90βb 583

isoforms of the protein (Rendell et al 2006) A goat anti-rabbit antibody (SAB-300 Enzo 584

Life Sciences) was used at a concentration of 150 000 as secondary antibody 585

586

Protein bands were visualized using the ECL Advance Western Blotting Detection Kit (GE 587

Healthcare) and a Versadoc Imaging System (Bio-Rad) Bands were then quantified using 588

ImageLab software (Bio-Rad) and the band density of each sample was divided by the band 589

density of the appropriate standard to obtain the relative band density After each round of 590

immunoblotting antibodies were stripped from the membranes using acid stripping buffer 591

(04 M glycine Sigma-Aldrich 007 M SDS Bio-Rad 0018 M Tween 20 Sigma-Aldrich 592

pH 22) for 2 X 30 min followed by 3 X 5 min washes in PBS (137 M NaCl 27 mM KCl 593

The

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43 mM Na2HPO47H2O 14 mM KH2PO4 Sigma-Aldrich) and membranes were re-probed 594

using a different antibody 595

596

Statistical analysis 597

Morphometrical parameters were compared between genotypes using one-way ANOVA 598

Comparisons of M O2 in Series I and M O2 fH and fV in Series II between and within groups 599

were performed using two-way repeated measures ANOVA with PO2 and genotype (2nNt 600

2nTx 3nNt 3nTx) as factors and the level of significance set to P lt 005 For HSC70 and 601

HSP90 three-way ANOVAs with treatment (hypoxia or control) ploidy (2n or 3n) and 602

transgenesis (transgenic or non transgenic) as factors were performed All pairwise multiple 603

comparisons procedures were performed using adjusted Bonferroni post-hoc modified t-tests 604

605

606

Acknowledgements 607

Funding (SC) Natural Sciences and Engineering Research Council (NSERC) Engage and 608

Discovery Grants Harold Crabtree Foundation 609

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oxygen-affinity of intact lamprey erythrocytes J Exp Biol 198 2393-2396 612

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Blier P U Lemieux H and Devlin R H (2002) Is the growth rate of fish set by 634

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Breau C Cunjak R A and Bremset G (2007) Age-specific aggregation of wild 638

juvenile Atlantic salmon Salmo salar at cool water sources during high temperature events 639

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Cook J T Sutterlin A M and McNiven M A (2000) Effect of food deprivation on 656

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Currie S and Boutilier R G (2001) Strategies of hypoxia and anoxia tolerance in 665

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Currie S and Tufts B L (1997) Synthesis of stress protein 70 (HSP70) in rainbow trout 669

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Deane E E Kelly S P Lo C K M and Woo N Y S (1999) Effects of GH prolactin 672

and cortisol on hepatic heat shock protein 70 expression in a marine teleost Sparus sarba J 673

Endocrinol 161 413-421 674

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Deitch E J Fletcher G L Petersen L H Costa I A S F Shears M A Driedzic 676

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Polymeropoulos ET Elliott NG Wotherspoon SJ and Frappell PB (2013) 759

Respirometry Correcting for diffusion and validating the use of plastic multiwell plates with 760

integrated optodes Physiol Biochem Zool 86(5) 588-592 761

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804

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Jour

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2028-2035 807

808

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61-68 818

819

Tiwary B K Kirubagaran R and Ray A K (2004) The biology of triploid fish Rev 820

Fish Biol Fish 14 391-402 821

822

Verhille C Anttila K and Farrell AP (2013) A heart to heart on temperature Impaired 823

temperature tolerance of triploid rainbow trout (Oncorhynchus mykiss) due to early onset of 824

cardiac arrhythmia Comp Biochem Physiol A Mol Integr Physiol 164 653657 825

826

Wells P R and Pinder A W (1996a) The respiratory development of Atlantic salmon I 827

Morphometry of gills yolk sac and body surface J Exp Biol 199 2725-2736 828

829

Wells P R and Pinder A W (1996b) The respiratory development of Atlantic salmon II 830

Partitioning of oxygen uptake among gills yolk sac and body surfaces J Exp Biol199 831

2737-2744 832

833

Zarate J and Bradley T M (2003) Heat shock proteins are not sensitive indicators of 834

hatchery stress in salmon Aquaculture 223 175-187 835

836

837

838

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Table 1 Comparisons of morphometric parameters of newly hatched Atlantic salmon (Salmo 839

salar) yolk-sac alevins of different genotypes diploid (2nNt) triploid (3nNt) diploid 840

transgenic (2nTx) and triploid transgenic (3nTx) reared at 8degC Data show mean plusmn SE 841

Different letters between groups indicate significant differences at P lt 005 while their 842

absence indicate no statistical difference 843

844

2nNt (n=28) 2nTx (n=30) 3nNt (n=30) 3nTx (n=29)

total body mass (g) 0132 plusmn 0002 0124 plusmn 0002 0123 plusmn 0002 a0112 plusmn 0002

yolk-free body mass (g) a0064 plusmn 0001 0060 plusmn 0001 b0058 plusmn 0001 b0057 plusmn 0001

yolk mass (g) 0068 plusmn 0002 0064 plusmn 0002 0065 plusmn 0002 a 0055 plusmn 0002

caudal fin rays (n) 172 plusmn 17 169 plusmn 17 170 plusmn 16 166 plusmn 16

total length (mm) 208 plusmn 17 203 plusmn 09 205 plusmn 07 205 plusmn 08

845

846

Figure 1 Respirometry setup Depicted is a diagram of the respirometry setup in 847

combination with the optophysiological system to simultaneously measure metabolic rate 848

(M O2) heart rate (fH) and ventilation rate (fV) in individual Atlantic salmon (Salmo salar) 849

alevins at different levels of PO2 In principle the respirometry chamber containing the alevin 850

was sealed for a period of ~30 min while fH and fV were recorded each recorded at a separate 851

time M O2 was calculated from the time integral of the gas concentration curve derived from 852

the PO2 recording after flushing the chamber 853

854

Figure 2 Changes in M O2 in response to progressive acute hypoxia (21 5 kPa) 855

Responses of diploid (2nNt) triploid (3nNt) diploid transgenic (2nTx) and triploid 856

transgenic (3nTx) are shown Atlantic salmon (Salmo salar) yolk-sac alevins measured at 857

8degC Dashed lines separate sections where M O2 decreases (I III) or remains constant (II) as 858

determined by two-way repeated measures ANOVA Inset panel shows M O2 data for 859

normoxia intermediate and severe hypoxia (data enclosed in boxes) and statistical 860

differences determined by two-way ANOVA (P lt 005) are indicated using different letters 861

Data shown are means plusmn SE for the PO2 ranges specified 862

863

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Figure 3 Changes in M O2 (a) heart rate fH (b) and ventilation rate fV (c) Responses of 864

diploid (2nNt) and triploid transgenic (3nTx) Atlantic salmon (Salmo Salar) yolk-sac alevins 865

in response to a stepwise (PO2 of 21 13 and 5 kPa) challenge of hypoxia are shown Right 866

panels show relative changes in M O2 (d) heart rate fH (e) and ventilation rate fV (c) at 13 and 867

5 kPa with respect to values measured in normoxia (21 kPa) The asterisks indicate a 868

significant difference (P lt 005) in respective parameters compared to normoxic (control) 869

conditions All values are expressed as mean plusmn SE 870

871

Figure 4 HSP levels Relative quantification of a) HSC70 and b) HSP90 in diploid (2nNt) 872

triploid (3nNt) diploid transgenic (2nTx) and triploid transgenic (3nTx) Atlantic salmon 873

(Salmo Salar) yolk-sac alevin whole body homogenates in normoxia (control n = 10 per 874

group) and after acute progressive hypoxia exposure (hypoxia n = 5 per group) The bar 875

graphs depict protein band density obtained from densitometric scans of immunoblots The 876

asterisks indicate a significant difference (P lt 005) from respective control conditions within 877

each genotype All values are expressed as mean plusmn SE 878

879

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Summary 31

Growth hormone (GH) transgenic Atlantic salmon display accelerated growth rates compared 32

to non-transgenics GH-transgenic fish also display cardiorespiratory and metabolic 33

modifications that accompany the increased growth rate An elevated routine metabolic rate 34

has been described for pre- and post-smolt GH-transgenic salmon that also display 35

improvements in oxygen delivery to support the increased aerobic demand The early 36

ontogenic effects of GH-transgenesis on the respiratory and cellular physiology of fish 37

especially during adverse environmental conditions and the effect of polyploidy are unclear 38

Here we investigated the effects of GH-transgenesis and polyploidy on metabolic heart and 39

ventilation rates and heat shock protein (HSP) levels after exposure to acute hypoxia in post-40

hatch Atlantic salmon yolk-sac alevins Metabolic rate decreased with decreasing partial 41

pressures of oxygen in all genotypes In normoxia triploid transgenics displayed the highest 42

mass specific metabolic rates in comparison to diploid transgenics and non-transgenic 43

triploids which in contrast had higher rates than diploid non-transgenics In hypoxia we 44

observed a lower mass-specific metabolic rate in diploid non-transgenics compared to all 45

other genotypes However no evidence for improved O2 uptake through heart or ventilation 46

rate was found Heart rate decreased in diploid non-transgenics while ventilation rate 47

decreased in both diploid non-transgenics and triploid transgenics in severe hypoxia 48

Regardless of genotype or treatment inducible HSP70 was not expressed in alevins 49

Following hypoxia the constitutive isoform of HSP70 HSC70 decreased in transgenics and 50

HSP90 expression decreased in all genotypes These data suggest that physiological changes 51

through GH-transgenesis and polyploidy are manifested during early ontogeny in Atlantic 52

salmon 53

54

55

56

57

58

59

60

Key words Salmo salar hypoxia growth hormone transgenesis polyploidy metabolic rate 61

oxygen transport heat shock proteins 62

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Abbreviations 63

2n = diploid 64

3n = triploid 65



fH = heart rate 68




kPa = kilopascal 72





Tx = transgenic 77


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Introduction 80












2002) 92

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125







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147












159














173

174

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Results 175

176








183

















performed 200

201




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208












220












2nNt alevins 232

233



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genotypes 240

241











252

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Discussion 253



















272








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290


























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321










al 2011) 331

332







339








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2nNt) 348

349









358





















this question 379

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380

Series III 381





















402









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417















432











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459










469

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491




495





500

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513

514












526







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537


t (1) 538

539


541



544

















experiment 561

562

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572














586








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596









605

606




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References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

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644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

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675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



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708




712




716



719





724



727



1467 730

731



333-341 734

735




739

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743



Sci 54 1160-1165 746

747




751




755



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765



768




772

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775




779



369-386 782

783




787


789



792




796





801



804

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2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

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844







845

846








854









863

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871








879

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3

Abbreviations 63

2n = diploid 64

3n = triploid 65



fH = heart rate 68




kPa = kilopascal 72





Tx = transgenic 77


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Introduction 80












2002) 92

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5













125







132













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147












159














173

174

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Results 175

176








183

















performed 200

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208












220












2nNt alevins 232

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9





genotypes 240

241











252

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Discussion 253



















272








280




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290


























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321










al 2011) 331

332







339








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2nNt) 348

349









358





















this question 379

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380

Series III 381





















402









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15







417















432











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16

















459










469

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17






















491




495





500

The

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513

514












526







The

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537


t (1) 538

539


541



544

















experiment 561

562

The

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20










572














586








The

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596









605

606




The

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NU

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22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

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CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

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f Exp

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CC

EPTE

D A

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OR

MA

NU

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24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

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EPTE

D A

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NU

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IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

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26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

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27



775




779



369-386 782

783




787


789



792




796





801



804

The

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2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

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844







845

846








854









863

The

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871








879

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The

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The

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4

Introduction 80












2002) 92

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125







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UTH

OR

MA

NU

SCR

IPT

6



147












159














173

174

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

7

Results 175

176








183

















performed 200

201




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

8




208












220












2nNt alevins 232

233



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

9





genotypes 240

241











252

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

10

Discussion 253



















272








280




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

11







290


























The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

5













125







132













The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

6



147












159














173

174

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

7

Results 175

176








183

















performed 200

201




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

8




208












220












2nNt alevins 232

233



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

9





genotypes 240

241











252

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

10

Discussion 253



















272








280




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

11







290


























The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

6



147












159














173

174

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

7

Results 175

176








183

















performed 200

201




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

8




208












220












2nNt alevins 232

233



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

9





genotypes 240

241











252

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

10

Discussion 253



















272








280




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

11







290


























The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

7

Results 175

176








183

















performed 200

201




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

8




208












220












2nNt alevins 232

233



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

9





genotypes 240

241











252

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

10

Discussion 253



















272








280




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

11







290


























The

Jour

nal o

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ndash A

CC

EPTE

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12






321










al 2011) 331

332







339








The

Jour

nal o

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logy

ndash A

CC

EPTE

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UTH

OR

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NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

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erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

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enta

l Bio

logy

ndash A

CC

EPTE

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UTH

OR

MA

NU

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IPT

15







417















432











The

Jour

nal o

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l Bio

logy

ndash A

CC

EPTE

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UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

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UTH

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18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

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IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

8




208












220












2nNt alevins 232

233



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

9





genotypes 240

241











252

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

10

Discussion 253



















272








280




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

11







290


























The

Jour

nal o

f Exp

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enta

l Bio

logy

ndash A

CC

EPTE

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UTH

OR

MA

NU

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IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

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UTH

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IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

9





genotypes 240

241











252

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

10

Discussion 253



















272








280




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

11







290


























The

Jour

nal o

f Exp

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enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

10

Discussion 253



















272








280




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

11







290


























The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

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enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

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EPTE

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UTH

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MA

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

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EPTE

D A

UTH

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MA

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IPT

The

Jour

nal o

f Exp

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enta

l Bio

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ndash A

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D A

UTH

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MA

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SCR

IPT

The

Jour

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enta

l Bio

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ndash A

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UTH

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MA

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IPT

11







290


























The

Jour

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ndash A

CC

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IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

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UTH

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MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

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UTH

OR

MA

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IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

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UTH

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MA

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18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

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enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

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l Bio

logy

ndash A

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D A

UTH

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MA

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The

Jour

nal o

f Exp

erim

enta

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logy

ndash A

CC

EPTE

D A

UTH

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MA

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IPT

The

Jour

nal o

f Exp

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enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

12






321










al 2011) 331

332







339








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

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IPT

15







417















432











The

Jour

nal o

f Exp

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enta

l Bio

logy

ndash A

CC

EPTE

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UTH

OR

MA

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IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

17






















491




495





500

The

Jour

nal o

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enta

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logy

ndash A

CC

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UTH

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18













513

514












526







The

Jour

nal o

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enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

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NU

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IPT

20










572














586








The

Jour

nal o

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enta

l Bio

logy

ndash A

CC

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UTH

OR

MA

NU

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IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

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enta

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logy

ndash A

CC

EPTE

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UTH

OR

MA

NU

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IPT

The

Jour

nal o

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enta

l Bio

logy

ndash A

CC

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D A

UTH

OR

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IPT

The

Jour

nal o

f Exp

erim

enta

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logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

13


2nNt) 348

349









358





















this question 379

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

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UTH

OR

MA

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IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

14

380

Series III 381





















402









The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

15







417















432











The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

16

















459










469

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

17






















491




495





500

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

18













513

514












526







The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

19





537


t (1) 538

539


541



544

















experiment 561

562

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

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logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

20










572














586








The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

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logy

ndash A

CC

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D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

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erim

enta

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logy

ndash A

CC

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D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

21



596









605

606




The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

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enta

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logy

ndash A

CC

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D A

UTH

OR

MA

NU

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IPT

The

Jour

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ndash A

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The

Jour

nal o

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logy

ndash A

CC

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D A

UTH

OR

MA

NU

SCR

IPT

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

22

References 610



613



616



619




623



626



Gene 295 173-183 629

630


67 632

633




637



JFish Biol 71 1179-1191 640

641

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

27



775




779



369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

29






844







845

846








854









863

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

30








871








879

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

nal o

f Exp

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enta

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logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

The

Jour

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logy

ndash A

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The

Jour

nal o

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logy

ndash A

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UTH

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NU

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The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

23



644



647




78 347-355 651

652



655




659





664



Zool 74 420-428 667

668



671



Endocrinol 161 413-421 674

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

27



775




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369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

SCR

IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

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NU

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IPT

29






844







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854









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The

Jour

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ndash A

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30








871








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The

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The

Jour

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The

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ndash A

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The

Jour

nal o

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enta

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logy

ndash A

CC

EPTE

D A

UTH

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24

675




679



682



685



688



691




695



FASEB J 14(4) A586 698

699



702



705



The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

25

708




712




716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

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enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

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NU

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IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

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NU

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IPT

27



775




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369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

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enta

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logy

ndash A

CC

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D A

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IPT

29






844







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ndash A

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30








871








879

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Jour

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ndash A

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IPT

25

708




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716



719





724



727



1467 730

731



333-341 734

735




739

The

Jour

nal o

f Exp

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enta

l Bio

logy

ndash A

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D A

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IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

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enta

l Bio

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ndash A

CC

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MA

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IPT

27



775




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369-386 782

783




787


789



792




796





801



804

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

UTH

OR

MA

NU

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IPT

28



2028-2035 807

808




812


Science 297 1797-1799 814

815



61-68 818

819



822




826



829



2737-2744 832

833



836

837

838

The

Jour

nal o

f Exp

erim

enta

l Bio

logy

ndash A

CC

EPTE

D A

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NU

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IPT

29






844







845

846








854









863

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Jour

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ndash A

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30








871








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enta

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D A

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IPT

26




743



Sci 54 1160-1165 746

747




751




755



758




762



765



768




772

The

Jour

nal o

f Exp

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enta

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logy

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CC

EPTE

D A

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MA

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