Histological Assessment of Organs in Sexually Mature and Post-spawning Steelhead Trout and Insights into Iteroparity

Zachary L. Penney1 and Christine M. Moffitt1,2 *

1Department of Fish and Wildlife Sciences, University of Idaho, Moscow, Idaho, USA;

penn4282@vandals.uidaho.edu; 208-885-7139 (phone)

2 US Geologic Survey, Idaho Cooperative Fish and Wildlife Research Unit, Department of Fish

and Wildlife Sciences, University of Idaho, Moscow, Idaho, USA, cmoffitt@uidaho.edu; 208-

885-7047 (phone); 208-885-9080 (fax)

* Corresponding author, Christine M. Moffitt

For Reviews in Fish Biology and Fisheries

Special issue: Teaming up Atlantic and Pacific Salmonid Biologists to Enhance Recovery of

Endangered Salmon in North America

This draft manuscript is distributed solely for purposes of scientific peer review. Its content is deliberative and predecisional, so it must not be disclosed or released by reviewers. Because the manuscript has not yet been approved for publication by the U.S. Geological Survey (USGS), it does not represent any official USGS finding or policy."

1

23

4

5

6

7

8

9

10

11

12

13

14

15

16

1718

19

20

21

Abstract

Steelhead (Oncorhynchus mykiss) are anadromous and iteroparous, but repeat-spawning rates are

generally low. Like other anadromous salmonids, steelhead fast during freshwater spawning

migrations, but little is known about the changes that occur in vital organs and tissues.

Intuitively, it would seem that any fish capable of repeat spawning would not undergo the same

irreversible cellular degeneration as occurs in semelparous salmon and begin replacing energy

soon after spawning. Using Snake River steelhead as a model we examined the gastrointestinal

tract of post-spawn steelhead (kelts) to document if freshwater feeding was occurring, and used

histological analysis to assess the cellular architecture in the pyloric stomach, ovary, liver, and

spleen in sexually mature and kelt steelhead. We found 38% of the kelts contained food or fecal

material in the gastrointestinal tract and that feeding was related to external condition. We found

a significant renewal of villi in the pyloric stomachs of kelts was associated with feeding. No

vitellogenic oocytes were observed in sections of kelt ovaries, but perinucleolar and early/late

stage cortical alveolus stages were present suggesting iteroparity was possible. We documented a

negative correlation between the quantity of perinucleolar oocytes in ovarian tissues and fork

length suggesting that larger steelhead may invest more into a single spawning event. Liver and

spleen tissues of kelts were found to show minimal cellular deterioration. Together, these

findings indicate that the physiological processes causing rapid senescence and death in

semelparous salmon are not the same in steelhead, and recovery begins in freshwater.

Keywords: Iteroparity, Histology, Steelhead, Fasting

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

1

Introduction

Within the salmonidae family two reproductive life history strategies exist: semelparity and

iteroparity. Semelparity is exclusive to the Pacific salmon (Oncorhynchus sp) that spawn once

and die, although exceptions to this have occasionally been documented (Tsiger 1994; Unwin et

al. 1999). All other salmonids are iteroparous, but the degree of inter and intra-specific post-

spawning survival is highly variable (Crespi and Teo 2002; Quinn and Meyers 2004). Steelhead

O. mykiss, the anadromous form of rainbow trout, and Atlantic salmon Salmo salar are

considered iteroparous, but show complex polytypic life history strategies that involve

residualism (Viola and Schuck 1995, Christie et al. 2011), anadromy (Schaffer and Elson 1975;

McDowall 1987; Pascual et al. 2001), and combinations of life history strategies (Docker and

Heath 2003; Thrower et al. 2004; Pearse et al. 2009; Null et al. 2013). Iteroparity can increase

genetic diversity of stocks and individual lifetime fitness (Seamon and Quinn 2010), as well as

provide a safeguard against complete brood year failures (Narum et al. 2008). Consequently,

anadromy can complicate many of the benefits afforded by iteroparity. Foremost, the energetic

and physiological costs of migrations between seawater and freshwater can be high and repeat

spawning rates among steelhead and Atlantic salmon are generally low (<10%) (Fleming and

Reynolds 2004).

Within steelhead, a range of life history strategies can exist, including stream-maturing

(summer/fall run) and ocean-maturing (winter/spring run) reproductive strategies (Behnke 1992).

Stream-maturing steelhead return to freshwater early (June-November) and undergo the final

stages of gonadal maturation while in freshwater, whereas ocean-maturing steelhead return to

freshwater (December-April) much closer to the time of spawning. As a spring spawner, low

rates of iteroparity in stream-maturing steelhead have been commonly attributed to energy

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

2

limitations due to longer migration distances and the extended time in freshwater compared to

ocean-maturing steelhead (Burgner et al. 1992). However, high post-spawning mortalities in

ocean-maturing steelhead (Busby et al. 1996) has not been well explained. Considering the

differences in migration distance and time spent in freshwater between the two ecotypes, it

appears that factors other than simple energetic depletion may contribute to post-spawning

survival.

A defining characteristic expressed by both semelparous and iteroparous anadromous

salmonids, including steelhead, is the prolonged fasting that accompanies freshwater re-entry to

spawn. Most anadromous salmonids enter a period of voluntary anorexia during spawning

migrations and rely on lipids and protein stored in somatic and visceral tissues. It has been

theorized that by discontinuing active consumption in freshwater during spawning, Pacific

salmonids and Atlantic salmon can curtail the energy required for digestion, which can account

for as much as 40% of basal metabolism in feeding fish (Wang et al. 2006). Through fasting,

anadromous salmonids may able to conserve energy by significantly lowering the energetic

demands of their basal metabolism, thus allocating the bulk of their stored energy to support

upstream migration, completion of gonadal maturation, the physical exertion of spawning, and in

the case of iteroparous species, emigration back to the ocean.

Post-spawn steelhead and Atlantic salmon, known as kelts (Allan and Ritter 1976), are

known to re-initiate feeding activity in freshwater following spawning (Quinn and Myers 2004),

but it is presumed that majority of somatic energy is replaced in the ocean. However, the low

proportion of repeat-spawners in steelhead and Atlantic salmon populations suggests that most

kelts do not survive despite attempts at feeding. In some cases, such as stream-maturing

Columbia River steelhead the rates of repeat-spawning are so low that the stocks have often been

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

3

regarded, as functionally semelparous (Burgner et al. 1992). Little is known about the effects that

prolonged fasting and catabolism have on organ systems in steelhead or Atlantic salmon,

especially those involved in digestion, energy storage, and vitellogensis. Studies with other non-

mammalian vertebrates has shown that there is a gradual reduction of intestinal epithelium

(atrophy) and nutrient transport capacities of the gastrointestinal tract during fasting, but that

digestive processes are rapidly restored at the onset of feeding (Wang et al. 2006; Secor et al.

2002). It therefore seems apparent that for steelhead or Atlantic salmon kelts to recover

energetically, their digestive functions must also be restored. Additionally, little is known about

how other organ systems involved in energy storage, blood filtration, waste removal, and red

blood cell production respond in recovering kelts.

In the following study, Snake River steelhead were used as a model to qualitatively and

quantitatively evaluate the histological architecture of liver, spleen, gastrointestinal, and ovary

tissues to provide insight on the physiological effects of prolonged fasting in an iteroparous

salmonid. Histological assessments provide information about tissue function at the cellular level

and can reveal physiological information that may not be apparent via external condition and

non-lethal measures of nutrition (i.e. blood chemistry, fat-meter). The primary objectives of this

study were to (1) determine of Snake River kelts were attempting to replace energy via during

freshwater emigration, and (2) describe the gross changes in cellular architecture in pre-

spawning and post-spawning fish and evaluate the capacity for repeat-spawning based on the

functionality of tissues.

Methods

Sample locations and procedures

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

4

Sections of the stomach, ovarian, liver, and spleen tissues of Snake River steelhead were

sampled in 2009 and 2010 at two phase of the reproductive cycle, first at or near sexual maturity

(mature) and secondly in migrating kelts (Table 1). Samples collected at maturity were obtained

from hatchery stocks returning to Dworshak National Fish Hatchery (DNFH) (46°30’N, -

116°19’W) or intercepted at Nez Perce Tribal Hatchery on the Clearwater River, ID (46°30’N, -

116°39’W ). Samples from kelts were collected from mixed stocks of Snake River steelhead at

the Lower Granite Dam juvenile bypass facility (46°39’N, -117°26’W), WA (Fig 1). We

sampled to select representative proportions of female and male steelhead at both phases for

comparison, but few male kelts were ever captured (Table 1).

Before sampling, steelhead were euthanized with a lethal dose (200mg/L) of MS-222

(Finquel, Argent Laboratories, Redmond, WA) buffered with NaHCO3 or in some cases CO2 was

used at DNFH. Fish were measured for fork length (nearest 0.5 cm). All mature steelhead were

considered to be in good external condition, whereas the condition of kelts was rated as good,

fair, or poor. Kelts in good condition exhibited no or only minor injuries, minimal fungal

infection, minor fin deterioration, silver or bright coloration, firm tissue texture, and were active.

Kelts in fair condition exhibited moderate injuries (non-life threatening), fungal infections over

1-10% of the body, moderate fin deterioration, pale coloration, and moderate activity. Kelts in

poor condition exhibited severe injuries (life threatening), fungal infections greater than 10% of

the body, severe fin deterioration, pale coloration, spongy tissue texture, and were listless. The

gastrointestinal tract of kelts was examined at the time of necropsy for the presence of

identifiable food or evidence of digestion via the presence of fecal material. Sections of the

stomach, liver, spleen, pyloric stomach, and ovarian tissues (kelts only) were removed using a

scalpel and transferred to jars containing 10% buffered neutral formalin (pH 6.8) at a 10:1 ratio

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

5

of formalin for tissue fixation. After fixation the stomach was sagittally bisected to provide a

cross section of the pyloric stomach. Fixed tissues were shipped to Colorado Histo-Prep (Fort

Collins, Colorado) for processing, where tissues were dehydrated, embedded in paraffin,

sectioned at ~ 4 to 6 μm, mounted on glass microscope slides, and stained with hematoxylin and

eosin (H&E) (Luna 1968).

Histological analysis

Histological analyses and scoring were accomplished with a compound light microscope

(Leitz Laborlux) fitted with a Leica EC3 camera and photographic software (LAS EZ 1.8.0;

Leica Microsystems, Cambridge, Ltd). Evaluations were made at magnifications of 4X, 10X, and

40X, which provided fields of view at 9.6 mm2, 1.5 mm2, and 0.1 mm2, respectively. We

evaluated tissue architecture with a categorical scoring system (Table 2). All scoring was

blinded.

Pyloric stomach

We evaluated and scored cross sections of the pyloric stomach using six metrics. We

quantified the detachment of the submucosa from the surrounding muscularis and stratum

compactum as severe, moderate, and minor to no detachment. The density of villi, which are

comprised of columnar epithelial cells and goblet cells lining the stomach lumen was scored as

low, moderate, and high. The extent of villiar invagination was estimated based on depth or

distance of the tip of villi (lamina epithelialis) to the stratum compactum. Villi invagination was

then scored as <1/4 the distance to the stratum compactum, 1/4 to 1/2 the distance to the stratum

compactum, or > 1/2 the distance to the stratum compactum. The cellular integrity of columnar

epithelial cells comprising the villi was evaluated and ranked by observing the severity of

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

6

deterioration of the cell membrane, cytoplasm and nucleus. The results were scored as severe,

moderate, and minor to no (absent) deterioration. The length to width ratio of columnar epithelial

cells was assessed to examine changes in overall cell size. As the name suggests, it was assumed

that columnar epithelial cells would exhibit length to width ratios similar to a rectangle (length >

width) rather than square (length = width). The length to width ratio of columnar epithelial cells

was ranked either as < 1/2 length to width ratio or > 1/2 length to width ratio. These measures

were validated via micrometer-calibrated measurements of photographs. Finally, we evaluated

the lamina epithelialis for the presence or absence of goblet or mucous cells to provide an

indication of mucous secretion. All scores for the pyloric stomach were assessed over the entire

tissue, regardless of magnification.

Ovary

We enumerated the quantity of perinucleolar oocytes (pre-lipid deposition), the quantity of

early/late cortical alveolus stage oocytes (lipid deposition present), and the quantity of

vitellogenic oocytes ( lipid and yolk deposition) in three to four fields for each ovary section.

Only oocytes sectioned through the nucleus or exhibiting clear lipid or yolk deposition were

enumerated in each field and it was assumed that all oocytes were randomly distributed within

the ovary. To validate this assumption, we compared the frequency of oocyte counts by category

and field. We found 92% of the ovarian tissues (N = 51) showed no significant variations across

fields and concluded pre and post-lipid oocyte distribution was random and could be averaged

across all fields by dividing the total sum of pre and post-lipid oocytes by the total number of

fields examined.

Liver

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

7

Five metrics were assessed (Table 2): The density of melanomacrophage aggregates

dispersed in the parenchyma was scored as none visible, low (< 5 aggregates), and high (> 5

aggregates). The separation of hepatocytes within their respective cords was evaluated for the

amount of detachment and scored as light, moderate, and minor or none. The overall separation

between heptatocyte cords (sinusoid spaces) was estimated and classified as less than 10μm or

greater than 10μm. The proportion of vacuolization within parenchyma was observed and ranked

as absent, between 1-10% in the field of view, and greater than 10% in the field of view.

Hepatocyte cellular structure was scored to consider integrity of the cell membrane, cytoplasm,

and nucleus and ranked as severe, moderate, or minor to no (absent) cellular deterioration. The

density of melanomacrophage aggregates, the apparent hepatocyte detachment, and sinusoid

spacing were scored over the entire liver tissue area at 10X. Scores for the degree of

vacuolization and hepatocyte integrity from each microscopic field assessed at 40X were

averaged and rounded up.

Spleen

Spleen tissues were evaluated to consider the distribution of white pulp nodules within the

red pulp as discrete, or diffuse wherein no clear separation of the white and red pulp was visible.

The percent of fields occupied by red pulp was estimated and categorized as 0-39%, 40-60%, or

61-100%. We evaluated the presence or absence of maturing erythrocytes and leukocytes by

noting the presence of differentiating cells or cell types. All scores for the spleen were derived

from assessments of the entire tissue, regardless of the magnification used.

Statistical analysis

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

8

Statistical comparisons of the frequency of scoring metrics by category within each tissue

were made to explore the differences within and between both mature and kelt phases. Within

mature steelhead we evaluated differences between sexes. Within the sample of female kelts we

evaluated metrics by condition, and also to evaluated differences between kelts with and without

presence of food at necropsy. Because sample sizes for male kelts were small (poor = 3, fair = 5,

and good = 5) we did not make statistical comparisons between males and female kelts. We

compared metrics between mature and kelt steelhead for good condition females only.

Statistical comparisons of the frequency of scores by category were made using exact Chi-

square analyses with Monte Carlo estimates derived from 20,000 permutations. These estimates

were used to account for occasional low frequencies (< 5 per cell) in which case the use of an

asymptotic Chi-Square test was not valid. Comparisons of oocyte counts in kelts by condition

were tested using Wilcoxon rank-sum tests. The relationship of fish length to several scoring

metrics transformed to progressive numerical scores was examined using Spearman correlations.

All statistical tests were conducted using SAS 9.3 (SAS Institute, Carey, North Carolina), and

statistical significance against a null hypotheses was reported as a P value.

Results

Evidence of feeding

Of the 65 migrating kelts examined, 25 had food or fecal material in the gastrointestinal tract.

Food items included salmon smolts, other small unidentified fish, fish eggs, and various

terrestrial and aquatic invertebrates. The proportion of feeding kelts significantly decreased with

decreasing condition (58% of good condition versus 7% of poor condition; P < 0.01, Table 3). In

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

9

male kelts, we detected no significant relationship between the proportion of feeding fish and

fish condition.

Pyloric stomach

We found no significant variation between male and female mature steelhead in any of the

metrics scored in the pyloric stomach, and all fish were considered in good condition. However,

we found significant differences attributed to fish condition in female kelt scores of submucosa

integrity, villi density, villi invagination, and columnar epithelial cell integrity (P < 0.02; Fig 2).

We found 23 of the 38 female kelts in good or fair condition had minor to no detachment of the

submucosa from the muscularis of the pyloric stomach, whereas the majority of poor condition

kelts (12/14) exhibited severe and moderate detachment of the submucosa. The villi density was

highest in good condition female kelts (Figs 3 and 4). The invagination of villi to the stratum

compactum was highly variable between good, fair, and poor condition kelts. Invaginations in

good condition kelts were all > 1/4 the distance to the stratum compactum and 60% of those

evaluated were > 1/2 the distance. Fair condition kelts exhibited a wide range of villi

invagination, and nearly all poor condition kelts showed villi invagination < 1/2 the distance to

the stratum compactum (Fig 2). Regardless of condition, the majority of kelts showed minor to

no signs of columnar epithelial cell deterioration.

Comparisons between stomach tissues of mature and kelt steelhead showed several

differences in structural patterns likely related to feeding recovery. Over 60% of mature

steelhead had low densities of villi but over 70% of kelts had significantly higher villi density (P

< 0.0001; Fig 5). We found significant differences between the extent of invagination of villi

between mature and kelt steelhead (exact P < 0.0001). The depth of villi invagination in mature

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

10

steelhead was very shallow, and nearly 80% of villi measured were <1/4 the distance to the

stratum compactum. In kelts, villi invagination was more pronounced and all were > 1/4 the

distance to the stratum compactum. No significant variations in submucosa integrity, columnar

epithelial cell integrity, length to width ratio of columnar epithelial cells, or presence of goblet

cells were found between mature or kelt steelhead.

We found a significant negative correlation of submucosa integrity with fish size in mature

steelhead (r = -0.4654; P <0.007; N = 33). However, compared to kelts, the range in length of

mature fish sampled (72 to 82 cm) was different from that of kelts (52 to 82 cm) sampled. We

found no such relationship with fork length in kelts. No mature steelhead exhibited severe

submucosa deterioration and very few (< 5) showed moderate deterioration, and the lack of

correlation could be influenced by sample sizes in each scoring category.

Ovary

No vitellogenic oocytes were observed in any of the ovarian tissues from kelts. However,

perinucleolar and early/late stage cortical alveolus stage oocytes were observed indicating that

Snake River steelhead are capable of repeat-spawning. Oocyte atresia was infrequent. We found

no variations in perinucleolar and early/late stage cortical alveolus oocytes by fish condition (Fig

6). However, we detected a negative correlation between fish fork length and perinucleolar

oocytes (r = -0.606 ; P < 0.002; N = 24). There was no significant correlation with length and

counts of early/late stage cortical alveolus oocytes.

Liver

200

201

202

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

11

We detected a significantly higher proportion of vacuolization in liver parenchyma of mature

steelhead males compared to that observed in mature females (P = 0.043; Fig 7). Mature female

steelhead had significantly lower proportions of cellular deterioration in hepatocytes than

proportions observed in males (P <0.001). We found no significant differences in the density of

melanomacrophage aggregates, the amount of hepatocyte detachment, or sinusoid spacing

between male and female mature steelhead.

In kelts, we observed that hepatocyte integrity varied among good, fair, and poor condition

females. Over 60% of good condition (N=24) and poor condition (N=14) kelts had minor to no

hepatocyte deterioration, whereas approximately 80% of fair condition kelts (N=13) showed

moderate deterioration (Fig 9). In samples with deterioration, we noted disintegration or loss of

the cellular membrane and nucleation within hepatocytes. Hypertrophy of hepatocytes was

occasionally observed and some exhibited vacuolization within the cytoplasm (Fig 9). We found

no significant variation in the proportion of melanomacrophage aggregates, attachment of

hepatocytes, spacing of sinusoids, or vacuolization among good, fair, and poor female kelts.

Our comparisons of liver tissues between good condition mature and kelt steelhead

determined that the proportion of mature females with minor to no hepatocyte cellular

deterioration was significantly higher than that of kelts (P = 0.006; Figs 8 and 9). Good condition

female kelts exhibited moderate hepatocyte detachment within cords (P = 0.002), in contrast

with samples from mature fish that had little to none. Variations in the degree of vacuolization in

livers between mature and kelt steelhead were noted (P = 0.05). We observed approximately

60% of livers from mature steelhead with vacuolization scores between 1 to 10%, and 20% with

scores > 10%, while nearly 50% of kelts showed a complete absence of liver vacuolization.

Small areas of melanomacrophage aggregates were present in all mature and kelt livers and

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

12

clustered near bile ducts and veins. Sinusoid spacing was consistent between phases and rarely

exceeded 10 μm between hepatocyte cords.

Spleen

We found significant differences in the proportion of red pulp in male versus female mature

steelhead (P = 0.001; Fig 10). Proportionally, 70% of male steelhead exhibited red pulp ratios

greater than 61%. In migrating female kelts, differences in the proportions of cellular

differentiation in the spleen were associated with fish condition. We observed some evidence of

red blood cell differentiation as well as leukocyte production or infiltration in 75% of kelt

spleens. Only fair and poor condition kelt spleens were observed without cell production or

monocyte activity (Fig 11). No other metrics were associated with fish condition. Regardless of

reproductive phase, we observed that spleens with higher proportions of white pulp had greater

amounts of cell differentiation in the white pulp. In spleens with elevated proportions of red

pulp, mature erythrocytes were observed as the dominant cell type.

We found a statistically significant relationship between fish length and the proportion of red

pulp in kelts (r = 0.4347; P = 0.034; N = 24). Although this trend suggested that

larger kelts had higher proportions of red pulp in spleens, these relationships were driven by two

large kelts (73 and 75 cm) with red pulp ratios > 61%.

Discussion

The energetic and physiological deterioration of semelparous Pacific salmon during

spawning has been documented extensively (Green 1916; Robertson and Wexler 1960;

Robertson et al. 1961; Hane et al. 1959; Brett 1995; McPhee and Quinn 1998; Barry et al. 2010;

Morbey et al. 2005), but has not been well described in anadromous iteroparous salmonids

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

13

(Belding 1934). Intuitively, it would seem that any fish capable of repeat spawning would not

undergo the same irreversible cellular degeneration as occurs in semelparous salmon. We found

little evidence of extensive cellular deterioration in the pyloric stomach, ovary, liver or spleen in

mature or kelt steelhead suggesting that the physiological processes causing rapid senescence

and death in semelparous salmon are likely not the same in steelhead. Furthermore, the

observation of active feeding in migrating kelts indicates that these fish are attempting to replace

energy even while still in freshwater.

Gastrointestinal response

Steelhead are not believed to feed actively during freshwater migrations prior to spawning,

but occasionally freshwater feeding has been documented in mature Pacific (Garner et al. 2009)

and Atlantic salmon (Johansen 2001). Reductions or loss of villi and microvilli in the

gastrointestinal tract is a common characteristic found in fasting or starving poikilothermic

organisms (i.e. fish, snakes) (Ehrlich et al. 1976; Secor et. al. 2002; Krogdahl and Bakke-

McKellep 2005; Wang et al. 2006). Considering that the majority of mature steelhead at DNFH

were held on site for 2-3 months and not provided any opportunities to feed prior to sampling,

we are confident these fish were in a fasting state. The pyloric stomach of mature steelhead had

low densities of villi and almost little villiar invagination. These observation were similar to the

histological assessments of post-spawn sockeye O. nerka and Chinook O. tshawytscha salmon

(Robertson and Wexler 1960). The reduction of villi in fasting fish has also been documented in

non-salmonid species. Zeng et al. (2012) showed that the gastrointestinal tract and liver in food

deprived juvenile catfish (Siluris meridionalis) were down-regulated as a physiological response

to endure unfavorable environmental conditions. However, it is important to note that cellular

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

14

deterioration of the columnar epithelial cells rarely occurred in mature steelhead suggesting that

an irreversible degeneration was not occurring.

From an evolutionary perspective, the volitional anorexia in anadromous semelparous and

some iteroparous salmonids is likely the result of returning to a less productive environment

(freshwater) at a generally much larger size (McDowell 1987; Gross 1987; Fleming 1998). Wang

et al. (2006) hypothesized that reductions in organ size and enzymatic activity, specifically in the

gastrointestinal tract, could provide considerable energy savings to fasting or starving organisms

via lowering basal metabolic demands. This hypothesis is especially relevant to fasting

anadromous salmonids preparing to spawn, which primarily rely on somatic energy stores for

migration and spawning. Bioenergetically, it is conceivable that anadromous salmon returning to

spawn in a less productive environment at a larger size would save energy by fasting for three

primary reasons: (1) they would not actively pursue food with little energetic gain, (2) lower

basal metabolic costs by not operating the gastrointestinal tract, and (3) the use of stored somatic

energy forgoes the costs of specific dynamic action (loss of energy during digestion). Therefore

we agree with Wang et al (2006) hypothesis and further hypothesize that the gastrointestinal tract

of iteroparous salminds enters a cellular stasis or hibernation during reproduction, which is

supported by the observed renewal of villi and invagination found in kelts.

It is unclear if steelhead kelts must first ingest food to begin gastrointestinal recovery. Secor

et al. (2002) found that following prolonged periods of fasting in pythons that intestinal mass and

microvilli density rapidly increased following the ingestion of specific amino acids and peptides.

The columnar epithelial cells and goblet cells comprising the villi are important in the secretion

of enzymes, mucous, as well as the uptake of nutrients in the gastrointestinal tract (Mader 2001).

Presumably, the observed increases in villi density and invagination provide more surface area

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

15

for the digestion and absorption of food. Significant variations in villi density and invagination

did occur between good, fair, and poor kelts, which is possibly due to the finding that good

condition kelts were more likely to feed than fair or poor condition kelts. However, comparisons

between feeding and non-feeding good condition female kelts found no significant variations in

any of the pyloric scoring metrics. In reconditioning experiments with Atlantic salmon kelts,

Johnston et al. (1987) found that many kelts began feeding soon after spawning, but that many

had difficulties in swallowing feed and only after swallowing food several times did the rejection

of feed become less prevalent. This suggests that gastrointestinal regeneration may not occur at

the same rate or may be more difficult for some kelts than others. One possibility is that non-

feeding kelts had simply evacuated food from the gastrointestinal tract prior to sampling.

Carnivorous species like steelhead have short gastrointestinal tracts and the residence period of

food in the gut is relatively short (Arrington et al. 2002), thus it is possible feeding was

undetected in some kelts. Additionally, it has been shown that acute stress can alter the cellular

composition and reduce the permeability of the gastrointestinal tract (Olsen et al. 2005; Olsen et

al. 2008). Therefore, it is possible that stresses not detected via external examination could also

potentially explain the variations in cellular microstructure between feeding and non-feeding

kelts.

In addition to feeding, the gastrointestinal tract is also important in osmoregulation in

migrating kelts. As in smolts, the migration of kelts from a hypo- to hyper-tonic environment

requires a change in the osmoregulation of ions from the gills to the gastrointestinal tract. The

process of the re-acclimation to seawater has been shown to be greatly accelerated in kelts.

Talbot et al. (1992) found that Atlantic salmon kelts were able to re-adapt to seawater within 48

hours, thus indicating that the gut and all organs responsible for the salt and water balance had

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

16

recovered some or all function 4 to 6 weeks after spawning. This quick acclimation is likely

beneficial to kelts on two levels. Not only does it reduce the stress and time required to re-

acclimate in the estuary allowing for quicker access to high energy food in the ocean, but it likely

aids in the recovery from many freshwater-specific external infections such as (Saprolegnia sp.)

and parasitic copepods (Salmincola sp).

Ovarian composition

We observed perinucleolar (pre-lipid) and early/late stages of cortical alveolus oocytes (post-

lipid) in Snake River kelts, suggesting that these populations are capable of re-maturation. Like

their freshwater resident conspecifics, steelhead exhibit group synchronous ovarian development

characterized by the development of one primary group of oocytes at a time, which are

accompanied by numerous “resting” previtellogenic oocyte stages (Blazer 2002). This type of

ovarian development pattern has also been defined as determinant fecundity, where the

recruitment of oocytes in relation to secondary growth is arrested prior to spawning (Rideout and

Tomkiewicz 2011). Interestingly, Robertson and Wexler (1960) found small nucleated oocytes

present in the ovaries in spawning sockeye salmon, but not in the ovaries of Chinook salmon.

Exceptions to repeat spawning have been found for some salmonids that were previously

considered to follow strict semelparous life histories. Tsiger et al. (1994) found that male masu

salmon O. masou maturing and spawning in freshwater could later adopt anadromous life

histories and return to spawn again. Unwin et al. (1999) found that precocious male Chinook

salmon could be spawn and remature to spawn again under experimental conditions.

All oocytes found in Snake River steelhead ovaries were in previtellogenic stages indicating

that vitellogenesis was not occurring. This finding is not surprising considering that these fish

had recently ovulated the primary group of oocytes during spawning (<1 – 2 months earlier).

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

17

Oogenesis is an energetically expensive process for all fish species (Rideout and Tomkiewicz

2011), thus immediate gonadal recrudescence would not be expected to occur in a post-spawning

fish with depleted somatic energy. Johnson et al. (1987) found oogenesis and vitellogenesis

proceeded in Altantic salmon kelts only after restoration of body energy reserves. In our studies,

the exact date and location of spawning were unknown for kelts, but it is unlikely that any of

these fish had adequate time to restore their total body energy to re-initiate oogenesis and

vitellogenesis, even with the re-initiation of feeding.

Recent studies by Null et al. (2013) on acoustically tagged steelhead kelts in the Sacramento

documented that 10% of kelts rematured or residualized in freshwater. Although residualized

kelts grew less than those that returned to the ocean, their survival was higher. Steelhead that re-

mature can remain in the ocean a year or more before return migration (Burgner et al.1992).

Variations in spawning periodicity, known as skip-spawning, are common and can occur in

mature fish due to low somatic energy, poor physical health, or poor environmental conditions

(Rideout et al. 2005; Rideout and Tomkiewicz 2011). In anadromous species, migrating long

distances, skip spawning is common. We found no female kelts in our study that had retained

ripe eggs, and some follicular atresia was observed. The absence of vitellogenisis in Snake River

kelt ovaries in our studies suggests that the pre-vitellogenic oocytes were in a resting stage,

presumably until re-feeding was initiated and somatic energy reserves restored.

With the extended migration distance to return to the ocean it is likely that Snake River

steelhead would exhibit skip-spawning to re-build energy stores. Jonsson et al. (1991) found

smaller Atlantic salmon kelts (< 60cm) were more likely to exhibit consecutive spawning,

whereas larger kelts were more likely to exhibit skip-spawning (> 90 cm), and smaller kelts had

higher survival. The residualized and anadromous steelhead kelts tagged by Null et al. (2013)

356

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

18

were observed as consecutive spawners, and were <60 cm. It has been shown that larger

iteroparous salmonids generally exhibit lower post-spawning survival over smaller salmonids

likely due to difficulty in replacing energy stores (Fleming 1998).

We found the number of pre-lipid oocytes was negatively correlated with fork length. Our

finding is of particular interest as stocks in the Snake River basin that contain two steelhead run-

types, known as A and B-run (Brannon et al. 2004). The A-run steelhead return to freshwater

earlier (July to August), have shorter marine residences (1-2 years), are smaller in size (<70 cm),

and generally spawn earlier (March-April) than B-run fish. The B-run steelhead return later

(August to October), have longer marine residence (2-3 years), are generally larger (>70 cm),

and spawn later (April-June). During our sampling of kelts in 2009 and 2010, we observed lower

proportions of large kelts in the juvenile bypass system at the Lower Granite Dam (unpublished

data).

We hypothesize that the lower number of perinucleolar oocytes in larger B-run kelts may

relate to greater energetic investments in size to increase fecundity, egg size, and juvenile

survival, thus exhibit a life history similar to semelparity (Fleming 1998; Crespi and Teo 2002;

Fleming and Reynolds 2004). Observations of acoustically tagged Snake River steelhead by

Jones (2013) do not support a higher mortality of large B-run kelts following spawning. Of the

30 B-run kelts tagged at Fish Creek on the Lochsa River, ID, 29 were detected at the Lower

Granite Dam forebay indicating they successfully migrated from the spawning system.

Unfortunately, estimates of the quantity of B-runs steelhead passing the Lower Granite Dam via

routes other than the bypass facility are unavailable. Regardless, the successful migration of

large B-run kelts does not suggest that size is as important as restoring energy.

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

19

Liver and spleen responses

Our assessments of mature and kelt steelhead livers showed little evidence of severe liver

deterioration or loss of function at the cellular level. Of the two phases examined, tissues in kelts

had the most hepatocyte detachment and deterioration, although samples from < 10% of kelts

showed severe hepatocyte deterioration. Robertson and Wexler (1960) reported pronounced

hepatocyte degeneration, that included diminishments in cytoplasm and occasionally a complete

loss of nucleation in spawning semelparous salmon.

Compared to white muscle tissue the liver is not a primary energy storage tissue in

anadromous salmonids (Brett 1995), but it does serve as a readily accessible energy source

during fasting or starvation. In semelparous pink salmon O. gorbuscha, Phleger (1971) found

that the liver lost the ability to synthesize triglycerides following freshwater re-entry and

spawning. Mommsen et al. (1980) examined the enzymatic activity of Fraser River sockeye

salmon O. nerka livers during spawning migrations and found a gradual decrease in metabolic

enzymes and protein content as fish progressed toward spawning. In salmonids, carbohydrates

comprise only a very small component of white muscle tissue composition and are a not a

significant source of energy (Brett 1995), however the liver does store glycogen as a small

source of energy for immediate use when needed. French et al. (1983) found that that the liver of

post-spawn sockeye salmon increased in its capacity to synthesize glucose following complete

exhaustion of somatic lipids, perhaps providing a final energy source for nest guarding. Thus, it

appears that a complete loss of liver function is rare even among spawning semelparous species

and may explain the overall lack of hepatocyte deterioration found in Snake River steelhead

livers.

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

20

The effects of starvation and prolonged fasting on the cellular architecture of the liver have

been documented for a variety of fish species (Hochachka 1961; Connor et al. 1964; Vijayan and

Moon 1992; Hur et al. 2006; Rios et al. 2007; Kullgren et al. 2010). Among the noticeable

histological changes in the livers of starving fish are losses of glycogen or lipid vacuolization

(Wolf and Wolfe 2005; Hur et al. 2006). In our study, the only significant variation in liver

vacuolization was observed between male and female pre-spawn steelhead, where males had a

higher proportion of vacuolization. French et al. (1983) found that liver glycogen reserves in

Fraser River sockeye were highest in females just before spawning, but did not examine males.

As livers are important to vitellogenin synthesis, it is possible that glycogen reserves in female

were used to complete oogenesis before spawning. This difference between sexes may also

partially explain the variations in hepatocyte integrity between male and females. Wolf and

Wolfe (2005) note that it is not uncommon to observe hepatocye hypertrophy in the livers of

sexually maturing females due increases in estrogen vitellogenin. Therefore, it is possible that

the variations in vacuolization and hepatocyte integrity between mature male and female

steelhead reflected these differences in energy allocation for oogenesis.

Regardless of condition, sex, or phase all steelhead livers had > 5 melanomacrophage

aggregates. Agius and Roberts (2003) noted the melanomacrophage aggregates are generally

prolific in the kidney, spleens, and livers in starving fish. We did not observe large numbers of

aggregates, but perhaps a more refined method of scoring aggregates would have resolved

potential variation among factors of condition, sex, and mature and kelt phases.

We found little evidence of cellular deterioration or loss of spleen function in either mature

or kelt steelhead. Only fair and poor condition kelts showed a complete loss of cellular activity in

the spleen suggesting other potential pathological problems. Robertson and Wexler (1960)

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

21

detected a reduction in the quantity or complete disappearance of lymphoid cells and increase in

fibrous tissue in the spleen of spawning semelparous Chinook and sockeye salmon, but found

only 1 of 16 spleens examined with cellular necrosis.

We found a higher proportion of red pulp in males over females in comparisons between

mature steelhead. Rebok et al. (2011) found that white pulp increased in the spleens of female

Ohrid trout Salmo letnica during spawning, but did not examine males. Rebok et al. (2011)

postulated that gonadal maturation likely reduced the proportion of circulating lymphocytes

thereby potentially increasing the white pulp in the spleen of females. Variations in red pulp can

be caused by the use of erythrocytes for exercise and activity. Strenuous exercise influences the

proportion of erythrocytes stored in the spleens of O. mykiss, and other teleosts (Kita and Itazawa

1989; Franklin et al.1993). It is possible that the spleens of mature male steelhead in our study

contained higher proportions of erythrocytes in red pulp in preparation for competition on the

spawning grounds.

We found no variations in the arrangement of white pulp nodes by sex, condition, or phase in

our study, but red and white pulp nodes in teleosts are reported to be more diffuse than in

mammals (Mumford et al. 2012). Although not scored, melanomacrophage aggregates were

present in all steelhead spleens. Like the liver and kidney, prolonged fasting and starvation

generally increases the number of melanomacrophage aggregates (Agius and Roberts 1981,

Mizuno et al. 2002), thus future evaluations of spleen in response to prolonged fasting during

spawning may be warranted.

Summary

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

466

22

This study was the first to examine in detail the histological architecture of organs of

steelhead at maturity and as migrating kelts. Although many changes can be detected between

these two stages, the majority of steelhead showed little cellular atrophy that would be indicative

of loss of cellular function. Moreover, our measures of tissue histology have been paired with

assessment of plasma blood profiles (unpublished data) that indicate kelts are in homeostasis.

The evidence of developing oocytes, changes in the epithelial structures of the pyloric stomach

between maturity and kelt migration, and observations of feeding, all provide strong support for

potential iteroparity.

467

468

469

470

471

472

473

474

475

476

23

Acknowledgments

Funding for this study was provided by the Columbia Inter-Tribal Fish Commission, Doug

Hatch, project manager. We thank the staff at Dworshak National Fish hatchery for their

cooperation with sampling. The US Army Corps of Engineers, and Nez Perce Tribe provided

assistance with logistics of sampling at Lower Granite Dam juvenile fish bypass facility. We

thank Dr. Rajan Bawa and staff at Colorado Histo-prep for processing tissues samples for

microscopic analysis. Ann Norton, Boling Sun, James Nagler, Chris Williams, Andrew Pierce,

Victor Cajas of the University of Idaho provided invaluable assistance in interpretation of

samples, laboratory and field assistance. We thank Amy Long and Vicki Blazer for their reviews

of earlier drafts. Additional sampling support was provided by Aaron Penney, Jessica Buelow,

Kala Hamilton, Andrew Paper, William Schrader, Martin Perales, Veatasha Dorsey, and Bryan

Jones. This study was performed under the auspices of University of Idaho protocol # 2009-10.

Any use of trade, firm, or product names is for descriptive purposes only and does not imply

endorsement by the U.S. Government.

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

24

References

Agius C, Roberts RJ (2003) Melano-macrophage centres and their role in fish pathology.

Journal of Fish Diseases 26: 499-509

Allan I.R.H, Ritter J.A. (1976) Salmonid terminology. Journal Du Conseil International

37(3): 293-299

Arrington DA, Winemiller KO, Loftus WF, Akin S ( 2002) How often do fishes “run on

empty”. Ecology 83(8):2145-2151.

Barry TP, Marwag A, Nunez S (2010) Inhibition of cortisol metabolism by 17α,20β-P:

Mechanism mediating semelparity in salmon? General and Comparative

Endocrinology 165:53-59

Behnke RJ (1992) Native trout of western North America. American Fisheries Society,

Bethesda, Maryland

Belding DL, (1934) The cause of the high mortality in the Atlantic salmon after

spawning. Transactions of the American Fisheries Society 64(1): 219-224

Blazer VS (2002) Histopathological assessment of gonadal tissue in wild fishes. Fish

Physiology and Biochemistry 26: 85-101

Brannon EL, Powell MS, Quinn TP,Talbot A (2004) Population structure of Columbia

River basin Chinook salmon and steelhead trout. Reviews in Fisheries Science

12:99-232

Brett JR (1995) Energetics. In: Groot C, Margolis L, Clarke WC (ed) Physiological

ecology of Pacific salmon. UBC Press, Vancouver British Columbia, pp 3-68

Burgner RL, Light JT, Margolis L, Okazaki T, Tautz A, Ito S (1992) Distribution and

origins of steelhead trout (Oncorhynchus mykiss) in offshore waters of the North

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

25

Pacific. International North Pacific Fisheries Commission. Bulletin Number 51:

1-92

Busby PJ, Wainwright TC, Bryant GJ, Lierheimer LJ, Waples RS, Waknitz FW,

Lagomarsino IV (1996) Status review of west coast steelhead from Washington,

Idaho, Oregon, and California. NOAA Technical Memorandum NMFS-NWFSC-

27. Northwest Fisheries Science Center, Seattle. Available:

http://www.nwr.noaa.gov. (September 2004)

Celius T, Walther BT, (1998) Oogenesis in Atlantic salmon (Salmo salar L.) occurs by

zonagenesis preceding vitellogenesis in vivo and in vitro. Journal of

Endocrinology 158:259-266

Christie MR, Marine ML, Blouin MS, (2011) Who are the missing parents?

Grandparentage analysis identifies multiple sources of gene flow into a wild

population. Molecular Ecology: 1-14

Crespi BJ, Teo R, (2002) Comparative phylogenetic analysis of the evolution of

semelparity and life history in salmonids fishes. Evolution 56(5): 1008-1020.

Docker MF, Heath DD, (2003) Genetic comparison between sympatric anadromous

steelhead and freshwater resident rainbow trout in British Columbia, Canada.

Conservation Genetics 4: 227-231

Dyck JC van, Pieterse GM, van Vuren JHJ (2007) Histological changes in the liver of

Oreochromis mossambicus (Cichlidae) after exposure to cadmium and zinc.

Ecotoxicology and Environmental Safety 66:432-440

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

26

Ehrich K., Blaxter JHS, Pemberton R, (1976) Morphological and histological changes

during the growth and starvation of herring and plaice larvae. Marine Biology 35:

105-118

Fleming IA, (1998) Pattern and variability in the breeding system of Atlantic salmon

(Salmo salar), with comparisons to other salmonids. Canadian Journal of

Fisheries and Aquatic Sciences 55 (Supplement 1): 59-76

Fleming IA, Reynolds JD, (2004) Salmon breeding systems. In: Hendry AP, Stearns SC

(ed) Evolution illuminated salmon and their relatives. Oxford University Press,

USA, pp 264-294

Franklin CE, Davison W, Mckenzie JC, (1993) The role of the spleen during exercise in

the Antarctic teleost, Pagothenia Borchgrevinki. Journal of Experimental Biology

174: 381-386

French, CJ, Hochachka PW, Mommsen TP, (1983) Metabolic organization of liver

during spawning migration sockeye salmon. American Journal of Physiology

245(6):R827-830

Garner, SR, Heath JW, Neff BD, (2009) Egg consumption in mature Pacific salmon

(Oncorhynchus sp.). Canadian Journal of Fisheries and Aquatic Sciences 66:

1546-1553

Gauthier D, Desjardins L, Robitaille JA,Vigneault Y (1989) River spawning of

artificially reconditioned Atlantic salmon (Salmo salar). Canadian Journal of

Fisheries and Aquatic Sciences 46: 824-826

537

538

539

540

541

542

543

544

545

546

547

548

549

550

551

552

553

554

555

556

557

27

Green C W (1916) On Some Quantitative Physiological Changes in the Pacific Salmon

during the Run to the Spawning Grounds. Transactions of the American Fisheries

Society, 45:1, 5-12

Gross MR, (1987) Evolution of diadromy in fishes. American Fisheries Society

Symposium 1: 14-25

Hane S, Robertson OH (1959) Changes in plasma 17-hydoxycorticosteroids

accompanying sexual maturation and spawning of the Pacific salmon (Oncorhynchus

tschawtscha) and rainbow trout (Salmo gairdnerii). Proceedings of the National

Academy of Sciences of the United States of America 45(6):886-893

Hatch D, Anders P, Evans A, Blodgett J, Bosch B, Fast D, Newsome T, (2002) Kelt

Reconditioning: A Research Project to Enhance Iteroparity in Columbia Basin

Steelhead (Oncorhynchus mykiss)". 2001 Annual Report. Project No. 200001700, 89

electronic pages (BPA Report DOE/BP-00004185-2) Bonneville

Hochachka PW (1961) Liver glycogen reserves of interacting resident and introduced

trout populations. Journal of Fisheries Research Board of Canada 18(1): 125-135

Hur JW, Jo JH, Park IS (2006) Effects of long-term starvation on hepatocyte

ultrastructure of olive flounder Paralichthys olivaceus. Ichthyological Reseach 53:

306-310

Johansen MJ (2001) Evidence of freshwater feeding by adult salmon in the Tana River,

northern Norway. Journal of Fish Biology 59: 1405-1407

Johnston CE, Hambrook MJ, Gray RW, Davidson KG, (1992) Manipulation of

reproductive function in Atlantic salmon (Salmo salar) kelts with controlled

558

559

560

561

562

563

564

565

566

567

568

569

570

571

572

573

574

575

576

577

578

579

28

photoperiod and temperature. Canadian Journal of Fisheries and Aquatic Sciences 49:

2055-2061

Johnston CE, Gray RW, McLennan A, Paterson A, (1987) Effects of photoperiod,

temperature, and diet on the reconditioning response, blood chemistry, and gonad

maturation of Atlantic salmon (Salmo salar) held in freshwater. Canadian Journal of

Fisheries and Aquatic Sciences 44:702-711

Jones B, (2013) Migratory and physiological characteristics of steelhead kelts from the

Clearwater River, Idaho, and Lower Granite Dam, Washington. Master’s thesis,

University of Idaho

Jonsson N, Hansen LP, Jonsson B, (1991) Variation in age, size, and repeat spawning of

adult Atlantic salmon in relation to river discharge. Journal of Animal Ecology 60(3):

937-947

Kita J, Itazawa Y, (1989) Release of erythrocytes from the spleen during exercise and

splenic constriction by adrenaline infusion in the rainbow trout. Japanese Journal of

Ichthyology 36: 48-52

Krogdahl Å, Bakke-McKellep AM (2005) Fasting and refeeding cause rapid changes in

intestinal mass and digestive enzyme capacities of Atlantic salmon (Salmo salar L.).

Comparative Biochemistry and Physiology, Part A 141: 450-460

Kullgren A, Samuelsson LM, Joakim Larsson DG, Gjornsson BT, Bergman EJ (2010) A

metabolic approach to elucidate effects of food deprivation in juvenile rainbow trout

(Oncorhynchus mykiss). American Journal Physiology-Regulatory, Integrative, and

Comparative Physiology 299:R1440-R1448

580

581

582

583

584

585

586

587

588

589

590

591

592

593

594

595

596

597

598

599

600

601

29

Luna LG, (1968) Manual of histological staining methods of the Armed Forces Institute

of Pathology, 3rd edition. McGraw-Hill Book Company, New York

Mader SS, (2001) Biology, 7th edition. McGraw-Hill Inc. New York, NY. pp 786-789

McDowall RM, (1987) The occurrence and distribution of diadromy among fishes.

American Fisheries Society Symposium 1: 1-13

McDowall RM, (1997) The evolution of diadromy in fishes (revisited) and its place in

phylogenetic analysis. Reviews in Fish Biology and Fisheries 7: 443-462

McPhee MV, Quinn TP, (1998) Factors affecting the duration of nest defense and

reproductive lifespan of female sockeye salmon, Oncorhynchus nerka.

Environmental Biology of Fishes 52: 369-375

Mizuno S, Misaka N, Miyakoshi Y, Takeuchi K, Kasahara N, (2002) Effects of starvation

on melano-macrophages in the kidney of masu salmon (Oncorhynchus masou).

Aquaculture 209: 247-255

Mommsen TP, French CJ, Hochachka PW (1980) Sites and patterns of protein and amino

acid utilization during the spawning migration of salmon. Canadian Journal of

Zoology 58: 1785-1799

Morbey YE, Brassil CE, Hendry AP (2005) Rapid senescence in Pacific salmon. The

American Naturalist 166(5):556-568

Mumford S, Heidel J,Smith C, Morrison J, MacConnell B, Blazer V (2007) Fish

Histology and Histopathology. United States Fish and Wildlife Service Manual.

http://training.fws.gov/EC/Resources/Fish_Histology/Fish_Histology_Manual_v4.pdf

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

30

Narum S R, Hatch D, Talbot A, Moran P, Powell M, (2008) Iteroparity in complex

mating systems of steelhead Oncorhynchus mykiss (Walbaum). Journal of Fish

Biology 72:45-60

Null R E, Niemela KS, Hamelberg SF, (2013) Post-spawn migrations of hatchery-origin

Oncorhynchus mykiss kelts in the Central Valley of California. Environmental

Biology of Fishes 96:341-353

Olsen RE, Sundell K, Mayhew TM, Myklebust R, Ringǿ E (2005) Acute stress alters

intestinal function of rainbow trout Oncorhynchus mykiss (Walbaum).

Aquaculture 250: 480-495

Olsen, R.E., K. Sundell, E. Ringǿ, R. Myklebust, G. Hemre, T. Hansen, Karlsen Ø (2008)

The acute stress response in fed and food deprived Atlantic cod, Gadus morhua L.

Aquaculture 280: 232-241

Pascual M, Bentzen P, Rossi CR, Mackey G, Kinnison MT, Walker R (2001) First

documented case of anadromy in a population of introduced rainbow trout in

Patagonia, Argentina. Transactions of the American Fisheries Society. 130: 53-67

Phleger CF, (1971) Liver triglyceride failure in postspawning salmon, Lipids 6: 347-349

Pearse DE, Hayes SA, Bond MH, Hanson CV, Anderson EC, MacFarlane RB, Garza JC

(2009) Over the Falls? Rapid evolution of ecotypic differentiation in

steelhead/rainbow trout (Oncorhynchus mykiss). Journal of Heredity 100(5): 515-

525

QuinnTP, Myers KW (2004) Anadromy and the marine migrations of Pacific salmon and

trout: Rounsefell revisited. Reviews in Fish Biology and Fisheries 14:421-442

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

31

Rebok K, Joranova M, Tavciovska-Vasileva I (2011) Spleen histology in the female

Ohrid trout, Salmo letnica (Kar.) (Teleostei, Salmonidae) during the reproductive

cycle. Archives of Biological Science 63 4:1023-1030

Rideout RM, Rose GA, Burton MPM, (2005). Skipped spawning in female iteroparous

fishes. Fish and Fisheries 6:50-72

Rideout RM, Tomkiewicz J (2011) Skipped spawning in fishes: More common than you

think. Marine and Coastal Fisheries 3(1): 176-189

Rios FS, Donatti L, Fernandes MN, Kalinin AL, Rantin FT (2007) Liver histopathology

and accumulation of melano-macrophage centres in Hoplias malabaricus after long-

term food deprivation and re-feeding. Journal of Fish Biology 71:1393-1406

Robertson OH, Wexler BC (1960) Histological changes in the organs and tissues of

migrating and spawning Pacific salmon (Genus Oncorhynchus). Endocrinology

66(2): 222-239

Robertson OH, Wexler BC, Miller BF (1961) Degenerative changes in the cardiovascular

system of spawning Pacific salmon (Oncorhynchus tshawtscha). Circulation Research

9: 826-834

Schaffer WM, Elson PF, (1975) The adaptive significance of variations in life history

among local populations of Atlantic salmon in North America. Ecology 56(3): 577-

590

Secor SM, Lane JS, Whang EE, Ashley SW, Diamond J, (2002) Luminal nutrient signals

for intestinal adaptation in pythons. American Journal of Gastrointestinal Physiology

283: G1298-G1309

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

32

Talbot C, Stagg RM, Eddy FB (1992) Renal, respiratory and ionic regulation in Atlantic

salmon (Salmo salar L.) kelts following transfer from freshwater to seawater. Journal

of Comparative Physiology 162(4): 358-364.

Thrower,FP, Hard JJ, Joyce JE (2004) Genetic architecture of growth and early life-

history transitions in anadromous and derived freshwater populations of steelhead.

Journal of Fish Biology 65 (Supplement A): 286-307

Tsiger VV, Skirin VI, Krupyanko NI, Kashlin KA, Semenchenko AY (1994) Life history

form of male masu salmon (Oncorhynchus masou) in South Primore, Russia.

Canadian Journal of Fisheries and Aquatic Sciences 51: 197-208.

Unwin MJ, Kinnison MT,Quinn, TP (1999) Exceptions to semelparity: Postmaturation

survival, morphology, and energetic of male Chinook salmon (Oncorhynchus

tshawytscha). Canadian Journal of Fisheries and Aquatic Sciences 56: 1172-1181/

Vijayan MM, Moon TW (1992) Acute handling stress alters hepatic glycogen

metabolism in food-deprived rainbow trout (Oncorhynchus mykiss). Canadian Journal

of Fisheries and Aquatic Sciences 49: 2260-2266

Viola A E, Schuck ML, (1995) A method to reduce the abundance of residual hatchery

steelhead in rivers. North American Journal of Fisheries Management 15:488–493

Wang T, Hung CY, Randall DJ (2006) The comparative physiology of food deprivation:

From feast to famine. Annual Review of Physiology 68:223-251

Wingfield B, (1976) Holding summer steelhead adults over to spawn a second year.

Northwest Fish Culture Conference 27: 63-64

Wolf JC, Wolfe MJ (2005) A brief overview of nonneoplastic hepatic toxicity in fish.

Toxilogical Pathology 33: 75-85

667

668

669

670

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

33

Zeng LQ, Li FJ, Li XM, Cao AD, Fu SJ,Zhang YG (2012) The effects of starvation on

digestive tract function and structure in juvenile southern catfish (Siluris meridionalis

Chen). Comparative Biochemistry and Physiology, Part A 162: 200-211

690

691

692

34

Table 1. Number of fish sampled and tissues examined, separated by spawning year, and

reproductive stage. All but four sexually mature males and one female in 2010 were sampled at

Dworshak National Fish Hatchery. All kelts were sampled at Lower Granite Dam.

Phase Sex 2009 2010 Total

Liver, spleen and pyloric stomach

Sexually mature F 16 17 33

M 0 14 14

Kelt F 24* 29* 0

M 3 10 13

Ovary

Sexually mature F 0 11 11

Kelt F 24 29 53

* Indicates one sample was missing from analysis of pyloric stomach and liver.

693

694

695

696697

698

699

700

701

702

703

704

705

706

707

708

35

Table 2. Summary of histological evaluations by tissue type and magnification in pyloric

stomach, liver, spleen, and ovarian tissues. Numerical scores and criteria are presented for each

metric.

Tissue Metric (magnification) Scoring criteria

Pyloric

stomach

Submucosa integrity (4X) 1 = Severe detachment from

muscularis ; 2 = Moderate

detachment from muscularis; 3

= Light or no detachment from

muscularis

Villi density (4X) 1 = Low ; 2 = Moderate; 3 =

High

Villi invagination (10X) 1 = <1/4 distance to stratum

compactum; 2 = 1/4 ≤ 1/2

distance to stratum compactum;

3 = 1/2 distance to stratum

compactum

Columnar epithelial cell integrity

(40X)

1 = Severe cell deterioration,

poor nucleation; 2 = Moderate

cell deterioration, nucleation

present ; 3 = Light to no cell

deterioration, nucleus present

L:W ratio of columnar epithelial cells

(40X)

1 = ≤1/2 length to width ratio; 2

= >1/2 length to width ratio;

Presence of goblet cells (40X) 1 = Absent; 2 = Present

709

710

711

712

36

Table 2, continued.

Tissue Metric (magnification) Scoring criteriaLiver Melanomacrophage aggregates (10X) 1 = Absent; 2 = < 5; 3 = > 5

Hepatocyte dettachment from rosette

(10X)

1 = Severe detachment

2 = Moderate;

3 = Light to no detachment

Hepatocyte rosette spacing (10X) 1 = <10μm; 2 = >10μm

Vacuolization in field of view (40X) 1 = absent; 2 = 1 to 10%

3 = > 10%

Hepatocyte cell integrity (40X) 1 = Severe cell deterioration, poor

nucleation ; 2 = Moderate cell

deterioration, nucleation present;

3 = Light to no cell deterioration,

nucleation present

Spleen Distribution of white pulp 1 = Discrete; 2 = Diffuse

Percentage of red pulp coverage (10X) 1 = 0-39%; 2 = 40-60% ; 3 = 61-

100%

Cell production/differentiation (40X) 1 = Absent ; 2 = Present

713

37

Tissue Metric (magnification) Scoring criteria

Ovary Quantity of perinucleolar oocytes (4X) Directly quantified and averaged

in 3 to 4 separate zones

Quantity of early/late cortical alveolus oocytes

(4X)

Directly quantified and averaged

in 3 to 4 separate zones

 

Quantity of vitellogenic oocytes (4X) Directly quantified and averaged

in 3 to 4 separate zones

714

38

Table 3. Summary of proportion of kelts with food or evidence of feeding at time of necropsy at

Lower Granite Dam, by sex and fish condition. Monte Carlo permutation exact P values are

provided for comparisons by sex and condition.

Sex Condition N Food (%) PFemal

e Good 24 14 (58)0.0061

Fair 14 6 (43)

Poor 14 1 (7)

Male Good 5 2 (40)

0.5809Fair 5 2 (40)

Poor 3 0

  Total 65 25 (38)  

715

716

717

718

719

720

721

722

39

Figure Captions

Fig 1 Map of Columbia/Snake River sub-basin with sampling sites indicated.

Fig 2 Significant scoring metrics for the pyloric stomach among good, fair, and poor condition

kelts for scores of A) submucosa integrity, B) villi density, C) villi invagination, and D)

columnar epithelial cell integrity.

Fig 3 Photomicrograph (4X) of pyloric stomach architecture in (a) mature steelhead (good

submucosa intergrity, low villi density, <1/4 invagination), (b) poor condition kelt (poor

submucosa integrity, low villi density, <1/4 invagination), (c) fair condition kelt (fair submucosa

integrity, moderate villi density, and <1/2 invagination), and (d) good condition kelt (good

submucosa integrity, high villi density, >1/2 invagination).

Fig 4 Photomicrograph (40X) of columnar epithelial cell integrity in the pyloric stomach villi of

(a) poor condition kelt (severe to moderate cellular deterioration), (b) fair condition kelt

(moderate to minor cellular deterioration), and (c) good condition kelt (minor to no cellular

deterioration).

Fig 5 Significant scoring metrics for pyloric stomach histology between good condition female

sexually mature and kelt steelhead: A) villi density and B) villi invagination.

Fig 6 Photomicrograph (4X) of kelt ovaries (a) kelt ovary with high quantity of perinucleolar

(pre-lipid) and some early and late stage cortical alveolus (lipid deposition present) oocytes and

(b) ovary from a large kelt (> 70cm) with only early and late stage cortical alveolus oocytes.

723

724

725

726

727

728

729

730

731

732

733

734

735

736

737

738

739

740

741

742

743

744

745

746

40

Fig 7: Significant scoring metrics for the liver between male and female pre-spawn steelhead: A)

vacuolization and B) hepatocyte integrity.

Fig 8: Significant scoring metrics for liver histology between good condition female sexually

mature and kelt steelhead: A) hepatocyte detachment, B) vacuolization, and C) hepatocyte

integrity.

Fig 9. Photomicrograph (10X) of liver cellular architecture in (a) mature steelhead (high

vacuolization, minor to no hepatocyte detachment, minor to no hepatocyte deterioration), (b)

poor condition kelt (no vacuolization, severe hepatocyte detachment, severecellular

deterioration), (c) fair condition kelt (no vacuolization, minor to no hepatocyte deterioration),

and (d) good condition kelt (vacuolization present, minor to no hepatocyte attachment, minor to

no hepatocyte deterioration).

Fig 10 Comparison of red pulp coverage between the spleen of male and female pre-spawn

steelhead.

Fig 11 Photomicrograph (10X) of spleen cellular architecture in (a) mature steelhead (40-60%

red pulp, diffuse white pulp, and cellular activity present) (b) poor condition kelt (40-60% red

pulp, diffuse white pulp, loss of cellular activity), (c) fair condition kelt (61-100% red pulp,

white pulp discrete), cellular activity present), and (d) good condition kelt (0-39% red pulp,

white pulp diffuse, cellular activity present).

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

41

Fig 1.

771

772

773

774

775

776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

42

Fig 2.

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

810

811

43

Fig 3.

812

813

814

815

816

817

818

819

820

821

822

823

824

825

826

827

828

44

Fig 4.

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

45

Fig 5.

845

846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

865

866

867

46

Fig 6.

868

869

870

871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

886

47

Fig 7.

887

888

889

890

48

Fig 8.

891

892

893

49

Fig 9.

894

895

896

897

898

899

900

901

902

50

Fig 10.

903

904

905

906

907

908

909

910

911

912

913

914

915

916

917

918

919

920

921

51

Fig 11.

922

923

924

925

926

927

928

929

930

931

932

933

934

935