Physiological Characterization of Hatchery-Origin Juvenile Steelhead Oncorhynchus mykiss Adopting Divergent Life-History Strategies

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1 Articles Physiological Characterization of Hatchery-Origin Juvenile Steelhead Oncorhynchus mykiss Adopting Divergent Life-History Strategies Kyle C. Hanson*, William L. Gale, William G. Simpson, Benjamen M. Kennedy, Kenneth G. Ostrand U.S. Fish and Wildlife Service, Abernathy Fish Technology Center, 1440 Abernathy Creek Road, Longview, Washington Present address of W.L. Gale: U.S. Fish and Wildlife Service, Mid-Columbia River Fishery Resource Office, 7501 Icicle Road, Leavenworth, Washington Abstract Smoltification by juvenile Pacific salmonids has been described as a developmental conflict whereby individuals face several life-history decisions. Smoltification occurs as a result of interactions between organismal condition and environmental cues, although some fish may forgo ocean migration and remain in freshwater streams for some time (residualize). We compared the physiological profiles of steelhead that were actively migrating to the ocean (migratory fish) and those that remained in fresh water (residuals) for at least a period of between 2 wk and 3 mo after release from a hatchery facility. In addition, we investigated the physiological characterization of residuals that further differentiated into precocial freshwater residents or parr that will either precocially mature in fresh water or migrate to the ocean in the future. Residuals had higher condition factors and gonadosomatic index than migratory fish and were characterized as less prepared for saltwater due to low levels of gill Na +,K + -ATPase activity and Na +,K + -ATPase a1bsubunit expression. Residuals tended to be males with the highest condition factors. Sex-specific differences are probably reflective of male fish adopting an alternative life-history strategy foregoing outmigration as a result of condition at the time of release. Collection of residuals throughout the fall suggested that residual hatchery fish further diversify into precocially mature fish that will presumably attempt to spawn without ever migrating to the ocean or into parr that will precocially mature or migrate in a future year. Keywords: smoltification; physiology; Na +,K + -ATPase activity; Na +,K + -ATPase a-subunit expression; residual salmon; hatchery management Received: September 1, 2010; Accepted: April 12, 2011; Published Online Early: April 2011; Published: June 2011 Citation: Hanson KC, Gale WL, Simpson WG, Kennedy BM, Ostrand KG Physiological characterization of hatcheryorigin juvenile steelhead (Oncorhynchus mykiss) adopting divergent life-history strategies. Journal of Fish and Wildlife Management 2(1):61 71; e x. doi: / jfwm-032 Copyright: All material appearing in the Journal of Fish and Wildlife Management is in the public domain and may be reproduced or copied without permission unless specifically noted with the copyright symbol ß. Citation of the source, as given above, is requested. The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service. * Corresponding author: kyle_hanson@fws.gov Introduction The yearly movement of juvenile Pacific salmonids Oncorhynchus spp. from freshwater natal streams to the ocean where rapid growth and maturation occur is one of the most prevalent forms of migration (Hinch et al. 2006). However, participation in juvenile migration to the ocean is not required to reach maturity and reproduce for all salmonid species. One such species that has evolved a complex set of alternative life-history strategies is the steelhead trout Oncorhynchus mykiss (Thorpe et al. 1998; Quinn 2005). In preparation for migration, juvenile salmonids undergo smoltification, a series of changes to body morphology, coloration, behavior, and physiology that allow for survival in saltwater (Folmar and Dickhoff 1980; McCormick and Saunders 1987; Hoar 1989). Principal in smoltification is the remodeling of the osmoregulatory system in preparation for the hyperosmotic environment of the ocean. This remodeling includes an increased Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 61

2 proliferation of gill chloride cells and the well-documented up-regulation of gill Na +,K + -ATPase (hereafter NKA) activity, an enzyme required to maintain osmotic balance by aiding in the extrusion of NaCl across the gills (Hoar 1989; Evans et al. 2005). In addition, expression of different NKA a-subunit isoforms, particularly the a1aand a1b-subunits, has been linked to seawater tolerance in salmonid fishes (Richards et al. 2003; Bystriansky et al. 2006, 2007). Smoltification has been described in Thorpe (1994) as a developmental conflict whereby juvenile steelhead are faced with three distinct possibilities every year: 1) undergo smoltification, followed by migration to the ocean (individuals are termed smolts, hereafter referred to as migratory fish); 2) begin maturation and attempt to spawn as a resident fish in the following winter (hereafter precocial residuals); and 3) remain in fresh water (natal steams, other tributaries, or the main channel of large rivers such as the Columbia River, etc.) and revisit these options in the following year (hereafter residual parr; precocial residuals and residual parr hereafter referred to a residuals collectively). These possibilities represent a case of developmental plasticity where adoption of one of these three life-history strategies is initiated through the interplay of phenotypic expression with environmental and biological cues (Björnsson 1997; Stefansson et al. 2007) rather than the result of a fixed genotype (Thorpe 1994; Thorpe and Metcalfe 1998; Thorpe et al. 1998). In the face of population declines for many Pacific salmonids, fisheries managers have increasingly relied on artificial propagation of species in hatchery facilities with the common goal of producing juvenile fish that undergo smoltification and rapidly migrate to the ocean upon release (Moring 1986; Lichatowich and McIntyre 1987). The presence of residual salmonids from a production facility may be viewed as either beneficial or detrimental given the potential ecological risks that residual hatchery fish pose to wild salmonids (Kostow 2009). Managers have long noted that hatchery-origin juveniles differ significantly from natural-origin conspecifics in regard to a host of behavioral (Symons 1969; Kostow 2004; Hill et al. 2006) and physiological (Folmar and Dickoff 1980; Shrimpton et al. 1994; Kennedy et al. 2007) traits that may decrease the survivability of hatchery-origin fish to adulthood, thereby reducing the conservation benefit of hatchery propagation (Campton 1995; Brannon et al. 2004; Araki et al. 2008). Because residuals remain in freshwater rearing habitats for extended durations, the potential for competitive interactions with wild conspecifics for limited resources may be exacerbated (McMichael et al. 1997; Hayes et al. 2004; Hill et al. 2006). In addition, precocial residuals may be a source of unaccounted for gene flow between hatchery and wild populations potentially leading to fitness declines in wild stocks due to outbreeding depression (Araki et al. 2008). Conversely, residuals may represent a benefit to the conservation goals of hatchery facilities. Low numbers of hatchery-origin steelhead fish have been documented as migrating to the ocean during the migration period in years after release from facilities on tributaries of the Columbia River (Ward and Slaney 1990; Tipping et al. 1995; Hayes et al. 2007; Sharpe et al. 2007). These fish represent an increase in life-history diversity of fish produced at hatchery facilities. With a greater diversity in life-history strategies, hatchery origin fish are more likely to express traits that are similar to those of wild fish, possibly increasing the odds of survival to adulthood for some individuals (Campton 1995; Brannon et al. 2004; Araki et al. 2008). Several physiological indicators have been linked to migratory tendency and residualism among salmonid species, including sex steroid levels (Larson et al. 2004), growth hormone (Sweeting et al. 1985), gill NKA activity (Gale et al. 2009), plasma [Na + ] or osmolality levels (Kennedy et al. 2007), and thyroid steroid levels and plasma insulin-like growth factor I (McCormick et al. 2002). Residualism of juvenile steelhead leads to individuals adopting multiple life-history strategies that each impose differing physiological demands on the organism (Thorpe 1994; Thorpe et al. 1998). Precocially mature residuals require development of mature gonads at the time of residualism, a physiological process that can be measured in a manner similar to that for other salmonids (Schmidt and House 1979). Residual parr do not undergo the same maturation process, making it impossible to enumerate these fish by using measures of sex hormones required for maturation. However, all residuals require an osmoregulatory system adapted to freshwater residence. For these reasons, a combination of physiological variables indicative of osmoregulatory status, condition factor (a proxy for overall animal condition and health and an indirect indicator of energy reserves; Hanson et al. 2010), and maturation are required to distinguish between the two residual life-history strategies in steelhead. The goal of this study was to characterize the differences in physiological status among migratory fish, precocially mature residual fish, and residual parr after release from a hatchery to determine whether physiological parameters can be used to predict life-history strategies. We predicted that all residuals would have higher condition factors and lower NKA activity (indicative of no osmoregulatory preparedness for saltwater) and show higher mrna expression of the NKA a1a-subunit and decreased mrna expression of the NKA a1b-subunit than migratory fish. We predicted that residuals would further differentiate into either precocious residuals or residual parr (Thorpe 1994; Thorpe et al. 1998). Finally, we predicted that males would be more likely to precocially mature in fresh water than females (Hanson et al. 2008). Methods Study site and fish collection This study took place on Abernathy Creek, a small, thirdorder tributary to the Columbia River (located 87 rkm from the Pacific Ocean), in Longview, Washington, USA, with a drainage area of approximately 110 km 2 (Figure 1). The creek supports several native salmonids: steelhead trout, coho salmon Oncorhynchus kisutch, cutthroat trout Oncorhynchus clarki clarkii, chum salmon Oncorhynchus keta, and an introduced run of fall Chinook salmon Oncorhynchus tshawytscha. In addition to wild propaga- Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 62

3 Figure 1. Migratory (migrating out of Abernathy Creek within 2 wk of hatchery release) and residual (remaining in freshwater for more than 2 wk after hatchery release) steelhead Oncorhynchus mykiss were collected in Abernathy Creek, Longview, Washington, USA (87 rkm from the ocean; denoted by the star in the inset), at a rotary screwtrap (300 m upstream from the confluence with the Columbia River) or by electrofishing within the creek after release of hatchery fish from Abernathy Fish Technology Center ([AFTC]; 3.4 km upstream from the confluence with the Columbia River). tion, the U.S. Fish and Wildlife Service maintains a research hatchery on the stream (3.4 km from the confluence of Abernathy Creek with the Columbia River) that produces steelhead smolts from wild brood stock as part of a larger study (Ostrand et al. 2007). Initial hatchery broodstock was produced by raising a large number of wild juvenile steelhead to adulthood. Subsequent generations of broodstock are crossed with wild fish to maintain genetic variability within the hatchery population. This hatchery method maintains genetic variability of individuals in an effort to retain genetic similarity between hatchery and wild fish (Hill et al. 2006; Ostrand et al. 2007). A series of passive integrated transponder (PIT) antennae located at both Abernathy Fish Technology Center and approximately 300 m upstream from the Abernathy Creek confluence with the Columbia River allowed for the movements of individual fish to be tracked. Fish capture Hatchery-origin steelhead were released in three time periods: mid-april (,7,000 fish released), early-may (,7,000 fish released), and mid-may (,7,000 fish released), Before release, all hatchery-origin fish were marked with an adipose clip and coded wire tag, and a subset of fish (approximately 1,000 spread evenly across release groups) were implanted with a PIT tag following the methods described in Kennedy et al. (2007). Migrating fish were captured at a screw trap (operated by the Washington Department of Fish and Wildlife from April to June) located 300 m upstream from the Abernathy Creek confluence with the Columbia River and approximately 3.1 km downstream from Abernathy Fish Technology Center. Upon capture at the screw trap, fish origin was determined by scanning for the presence of a PIT tag or visual identification of an adipose fin clip; Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 63

4 Table 1. Migration timing of hatchery-origin steelhead Oncorhynchus mykiss internally tagged with passive integrated transponder (PIT) tags and later captured in a screwtrap located 300 m upstream from the mouth of Abernathy Creek, Longview, Washington, USA, after release from Abernathy Fish Technology Center (located 3.4 km upstream from the mouth of Abernathy Creek) in the study year (2007) and two preceding years ( ). Year No. of fish captured at screw trap,13 days from release (% of total) No. of fish captured at screw trap.13 days from release (% of total) (87.9) 13 (12.1) (86.5) 31 (13.5) (84.2) 19 (15.8) Total 393 (86.2) 63 (13.8) hatchery-origin fish (n = 73) were retained for sampling as detailed below. Single-pass electrofishing surveys were used on a single day 2 wk after each hatchery release (but preceding the next release), spanning 2.8 km of Abernathy Creek adjacent and immediately upstream and downstream of the hatchery facility to assess the physiological status of hatchery-origin residuals (n = 76). Hatchery-origin fish captured during electrofishing were considered to be residuals; PIT tag detections from the PIT antenna array at the mouth of the creek indicate that the majority of migratory fish leave the system within 13 days after release from the facility (Table 1). This was further supported by physiological sampling of the osmoregulatory capacity of fish captured at the screw trap (migratory) and those captured via electrofishing near the release facility (residuals; Table 2). Residuals also were captured by electrofishing a 2.8-km section of Abernathy Creek immediately upstream and downstream of the hatchery facility on June 21 and July 26, 2007, to determine whether variable life-history strategies were being adopted by residuals. Physiological sampling At the time of capture, fish were euthanized by overdose in anesthetic (buffered MS-222, 180 mg/l) and then weighed (grams) and measured for fork length ([FL]; millimeters). A small gill biopsy also was taken for physiological assessment of NKA activity (McCormick 1993); biopsies were placed into ice-cold SEI buffer (250 mm sucrose, 10 mm Na 2 -EDTA, and 50 mm imidazole, ph 7.3), frozen on dry ice, and stored at 280uC. In addition, whole gill arches were removed and rapidly frozen in liquid N 2 for later analysis of NKA isoform a1a and a1b mrna expression. The FL and weight (W) measurements were used to calculate condition factor (K) for each individual fish by the following equation (Anderson and Gutreuter 1983): K~ W FL 3 100,000 Finally, the sex of each fish was determined by visual inspection of the gonads, and gonadosomatic index (GSI) Table 2. Physiological variables (mean 6 SD) measured in migratory (migrating out of Abernathy Creek, Longview, Washington, USA, within 2 wk of hatchery release) and residual (remaining in freshwater for.2 wk after hatchery release) hatchery-origin steelhead Oncorhynchus mykiss by sex after release into Abernathy Creek between April and May, Minimum and maximum values are presented in parentheses for each group. Male Female Variable Migratory (n = 37) Residual (n = 40) Migratory (n = 36) Residual (n = 36) Fork length (mm) ( ) ( ) ( ) ( ) Condition factor ([K]: g/mm ) ( ) ( ) ( ) ( ) Gonadosomatic index ( ) ( ) ( ) ( ) (GSI) a Na +,K + -ATPase activity (mmol-adp 6mg protein 21 6 h 21 ) Na +,K + -ATPase a1a expression Na +,K + -ATPase a1b expression ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) a GSI was determined as the relationship of gonad wet weight to total body wet weight. Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 64

5 was determined as the relationship of gonad wet weight to total body wet weight. Lab analyses Both NKA activity and NKA isoform expression assays were performed on all samples taken from residuals captured 2 wk after release and on migratory fish captured at the screw trap. Samples from residuals collected in June and July, 2007, were not analyzed in this manner because we believe there would be little change in osmoregulatory function given these fish never migrated to the ocean. Gill biopsies were assayed for NKA activity by using the method developed by McCormick (1993). Measurement of NKA isoform a1a and a1b mrna expression followed the methods developed by Bystriansky et al. (2006). Total RNA was extracted from the gills by using Tripure isolation reagent (Roche Applied Science, Indianapolis, IN), following the manufacturer s guidelines. Extracted RNA purity and concentration were determined by measuring absorbance at 260/280 nm, and the integrity of extracted RNA was assessed by agarose gel electrophoresis. First strand cdna was produced by reverse transcription-polymerase chain reaction using Reverse-iT first strand synthesis kits (Thermo Scientific, Epsom, UK). The quantitative polymerase chain reaction reactions (25 ml/well) consisted of cdna (2 ml/well), forward and reverse primers (70 nm), Rox dye (50 nm), and SYBR Green master mix (12.5 ml; Applied Biosystems, Foster City, CA). Primers (obtained from Integrated DNA Technologies, Coralville, IA) were used as described by Bystriansky et al. (2006) for the NKA a1a- and a1b-subunits and for the control gene, elongation factor-1a (Richards et al., 2003). Primer sequences were as follows: a1a forward, 59-GGC CGG CGA GTC CAA T-39 and a1a reverse, 59-GAG CAG CTG TCC AGG ATC CT-39; a1b forward, 59-CTG CTA CAT CTC AAC CAA CAA CAT T-39 and a1b reverse, 59-CAC CAT CAC AGT GTT CAT TGG AT-39; and EF1a forward, 59-GAG ACC CAT TGA AAA GTT CGA GAA G-39 and EF1a reverse, 59-GCA CCC AGG CAT ACT TGA AAG-39. The quantitative polymerase chain reaction reactions consisted of 15 min at 95uC, 40 cycles of 95uC for 15 s, and 60uC for 1 min. The presence of a single product was confirmed using melt curve analysis. The presence of nontarget genomic DNA was assessed using negative control reactions with extracted RNA, and in all cases genomic DNA contamination was negligible. A pool of RNA collected from anadromous steelhead parr (n = 6 fish collected March 6, 2008) was used as a calibrator. The relative quantities of the target genes were expressed using the comparative C T method (Schmittgen and Livak 2008). Quantities were normalized using the endogenous reference EF1a and are expressed relative to the calibrator pool. Statistical analyses Analyses were performed in the statistical package JMP version 7.0 (SAS Institute, Cary, NC) and significance was assessed at a = In addition, all values are presented in this study as means 6 SD unless otherwise noted. Before statistical analysis, variables that did not meet the criterion of univariate normality were log 10 (condition factor, NKA activity, and both NKA isoform a1a and a1b mrna expression) or square root (GSI)- transformed (Zar 1999). Differences in physiological status between fish exhibiting each life history were assessed by two-way analysis of variance with physiological measures (FL, condition, GSI, NKA activity, and NKA isoform a1a and a1b mrna expression) as dependent variables and sex, migratory behavior (migratory or residual), and the interaction of sex and migratory behavior as independent variables (Zar 1999). Tukey s post hoc tests were used to determine differences between group means (Zar 1999). Finally, we used simple linear regressions to compare gill NKA activity to NKA isoform a1a and a1b mrna expression to determine whether changes in gill NKA activity were related to expression of a specific a-isoform (Zar 1999). Results Residuals differed physiologically from migratory fish even though fish in both groups were of similar size at the time of capture (Tables 2, 3, and S1; org/ / jfwm-032.s1). Residuals also had higher condition factors (Tables 2 and 3) and GSI (Tables 2 and 3), lower overall gill NKA activity (Tables 2 and 3), and lower expression of mrna for the a1bisoform of NKA (Tables 2 and 3). Sex ratios differed between migratory (a female-to-male ratio of 1.09:1) and residual (a female-to-male ratio of 0.49:1) fish. In addition, sex specific differences in GSI and gill NKA activity were noted among the groups. Specifically, male residuals had higher GSI compared with all other groups (Tables 2 and 3), and gill NKA activity was the lowest in male residuals (Tables 2 and 3). Male steelhead GSI was below 0.5 for all individuals captured in the screwtrap (Figure 2A). The GSI of male residuals temporally varied in the months (April July) after release (Figures 2B 2D; Table S2; / JFWM-032.S2). Among residuals captured by electrofishing after release, a pattern emerged whereby male GSI increased (.0.5) in a subset of individuals (suggestive of precocial maturation), whereas GSI remained comparable with that of migratory fish in the majority of individuals (suggestive of adoption of a residual parr life-history strategy) (Figures 2B 2D). This was especially evident 2 mo after release (Figure 2D). Finally, our results suggest that a1a isoform expression was not related to changes in NKA activity (df = 145, F = 1.47, P = 0.23; Figure 3A). Conversely, a1b-isoform expression was positively related to overall gill NKA activity (r 2 = 0.27, df = 145, F = 53.9, P, 0.001; Figure 3B). Discussion In the current study, hatchery-origin fish adopted three alternative life-history strategies (migratory, precocious residuals, or residual parr) and exhibited several physiological differences probably as a result of interaction between rearing practices and plastic phenotypic expression. Similar to previous studies (e.g., McCormick and Bjornsson 1994; Shrimpton et al. 1994), fish that residualized did not up-regulate NKA activity, thereby Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 65

6 Table 3. Results from two-way analysis of variance comparing physiological variables between migratory types (migratory fish vs. residuals) of hatchery-origin steelhead Oncorhynchus mykiss after release into Abernathy Creek, Longview, Washington, USA, in April and May, Variable Source df F P Fork length (mm) Model 3, Migratory type Sex Migratory type 6 sex Condition factor ([K]: g/mm ) a Model 3, ,0.001 Migratory type 53.21,0.001 Sex Migratory type 6 sex Gonadosomatic index (GSI) b,c Model 3, ,0.001 Migratory type 27.34,0.001 Sex Migratory type 6 sex 17.08,0.001 Na +,K + -ATPase activity a Model 3, ,0.001 Migratory type ,0.001 (mmol-adp 6 mg protein 21 h 21 ) Sex Migratory type 6 sex Na +,K + -ATPase a1a expression a Model 3, Migratory type Sex Migratory type 6 sex Na +,K + -ATPase a1b expression a Model 3, Migratory type Sex Migratory Type 6 sex a Denotes log 10 -transformed variables. b Denotes square root-transformed variable. c GSI was determined as the relationship of gonad wet weight to total body wet weight. making them physiologically unprepared for the saltwater environment. A long line of research has investigated the relationship between gill NKA activity and saltwater preparedness, noting that smolts with low NKA activity are less able to survive in saltwater environments (McInerney 1964; Harache et al. 1980; Mesa et al. 1994; Price and Schreck 2003), because gill NKA has a primary role in extrusion of salt in the marine environment (Evans et al. 2005). Expression patterns of the a-subunit isoforms of NKA measured in this study also followed similar patterns to previous laboratory studies, indicating decreased preparedness for saltwater among residuals. Specifically, residuals tended to have lower expression of mrna for the a1b-isoform of NKA. Reciprocal switching between expression of the a-subunit isoforms of NKA has been correlated with seawater tolerance and survival in several salmonid species (Bystriansky et al. 2006; Nilsen et al. 2007; Madsen et al. 2009). Previous studies have shown that up-regulation of a1b is required for seawater survival (Bystriansky et al. 2006, 2007; Nilsen et al. 2007), and it has been speculated that the a1a-isoform primarily plays a role in ionic regulation in the freshwater environment (Bystriansky et al. 2006; Madsen et al. 2009). Our data conform to these ideas, given the lack of change in a1a expression while migratory fish inhabit freshwater streams (Bystriansky et al. 2006; Nilsen et al. 2007; Madsen et al. 2009). In the current study, migratory fish undergoing smoltification were characterized by increased a1b expression, whereas a1a expression remained similar to that in residuals. Any differences in overall gill NKA activity between the two groups would reflect only the up-regulation of the a1b-isoform in migratory individuals. This finding corresponds with previous work that attributed the increases in NKA a- subunit mrna expression commonly seen during seawater acclimation (D Cotta et al. 2000; Seidelin et al. 2000; Singer et al. 2002; Tipsmark et al. 2002; Madsen et al. 2009) to up-regulation of the a1b-isoform (Bystriansky et al. 2006). Residual hatchery steelhead further diverged into two groups in the summer after release from the facility. In the first group, increases in GSI of some males showed that they were starting to precocially mature in the months after release. Increases in GSI are related to sexual maturation in seasonally spawning fish wherein the gonads begin to enlarge before development of mature gametes (Snyder 1983). Male residuals characterized by high GSI are more likely to spawn at a future date without ever migrating to saltwater. Because the hatchery environment provides ample forage and low predation, Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 66

7 Figure 2. Frequency histograms of male steelhead Oncorhynchus mykiss gonadosomatic index (GSI), measured as the relationship of gonad wet weight to total body wet weight, of (A) migratory (n = 49), (B) residual hatchery fish 2 wk after release (n = 75), (C) residual hatchery fish 1 mo after hatchery release (n = 32), and (D) residual hatchery fish 2 mo after hatchery release (n = 24). juvenile steelhead are generally able to devote the majority of energy resources to growth and attain comparably higher condition factors than wild fish (McCormick and Björnsson 1994; Hill et al. 2006). Smoltification has been described as a negative developmental decision wherein only individuals that have not attained a certain body condition will emigrate to the ocean (Thorpe 1994; Thorpe et al. 1998); thus, hatchery rearing conditions may facilitate residualism. Sexual predetermination of migration tendency is most likely rooted in differences in fitness between the sexes as adults. Because male fitness is largely constrained by the number of females that the individual spawns with, males may suffer fewer fitness consequences by forgoing ocean migration to remain in freshwater and spawn at a smaller size (Trivers 1972; Gross 1984). Conversely, as female fitness is dictated through egg production that is positively correlated to body size (Sargent et al. 1987), failure to secure the growth benefit from migration to the ocean would result in severe reductions in female fitness. Sex-specific differences in physiological status may be indicative of a sexual predetermination for migration tendency. As predicted, male fish were more likely to residualize as evidenced by a skewed sex ratio compared with migratory fish, indicating that female fish did not residualize as often. Precocious residual males were characterized by the highest GSI and lowest NKA activity, indicating a higher likelihood of foregoing emigration and starting the maturation process in freshwater. In practice, these precocious residual males represent an unaccounted for source of gene flow between hatchery and wild populations, which may potentially lead to deleterious genetic effects within wild stocks (Araki et al. 2008). However, to date, the extent to which precocial residuals are reproductively successful in the wild is unknown, and research is required to determine the full impact of these individuals on the genetic makeup of local stocks. A second group of residuals was characterized by low GSI, suggestive of individuals remaining in natal watersheds as parr to revisit the options of either maturing as freshwater residents or migrating to saltwater in the next year (Thorpe 1994). Previous studies have documented hatchery steelhead emigrating from systems in years after release (Ward and Slaney 1990; Tipping et al. 1995; Hayes et al. 2004; Sharpe et al. 2007). The successful migration of a hatchery-origin residual parr out of a freshwater system in a year after release may represent an increase in life-history diversity within the hatchery population. The presence of hatchery-origin juveniles in a system for an extended duration after release may be deleterious to wild juveniles due to competitive interactions (McMichael et al. 1997; Hayes et al. 2004; Hill et al. 2006). However, the increased number of life-history strategies among hatchery-origin juveniles may prove beneficial to maintaining phenotypic diversity within the hatchery population. Further research is required to determine whether residual parr that eventually migrate to the ocean display differences in behavior and survival relative to both wild and hatchery-origin conspecifics. In addition, future research should focus on determining when the onset of changes in the molecular mechanisms Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 67

8 Figure 3. Gill Na +,K + -ATPase activity (micromoles-adp/milligrams of protein/hour) relative to (A) Na +,K + -ATPase a1a expression (relative to an endogenous reference, elongation factor-1a [EF-1a]) and (B) Na +,K + -ATPase a1b expression (relative to an endogenous reference, EF-1a) in 147 hatchery-origin steelhead Oncorhynchus mykiss at Abernathy Creek, Longview, Washington, USA. (NKA a1a- and a1b-subunits) responsible for the rise in gill NKA activity occurs. Changes in NKA a1a- and a1bsubunit activity may potentially be used as a measure to predict steelhead residualism rates before release, allowing for alteration of rearing and release protocols to modify residualism rates if desired. Efforts are ongoing to modify hatchery practices to preserve genetic diversity within the hatchery populations and avoid potentially deleterious interactions between hatchery and natural populations (Hindar et al. 1991; Waples 1991; Goodman 2005; Araki et al. 2008). Data from this study and others (e.g., Sharpe et al. 2007) indicate that the hatchery environment can interact with plastic phenotypic expression. This interaction, in turn, can lead to the propagation of fish that do not follow the commonly desired life-history strategy of smoltification and migration to the ocean and are maladapted to survival in the marine environment. Further assessment of the physiological status of juveniles and modification of hatchery practices may produce a higher proportion of fish exhibiting desired life histories to support conservation or hatchery production goals. Modification Journal of Fish and Wildlife Management June 2011 Volume 2 Issue 1 68

9 of hatchery techniques should focus on the physiological as well as genetic effects of rearing practices to promote adoption of the desired life-history strategy by juveniles. In particular, manipulation of condition factor through modification of rearing practices (e.g., Brockmark et al. 2007) may lead to a cascade of changes in many of the physiological variables measured in this study, potentially inducing an increased rate of smoltification and a reduction in the number of residuals produced. Supplemental Material Please note: The Journal of Fish and Wildlife Management is not responsible for the content or functionality of any supplemental material. Queries should be directed to the corresponding author for the article. Table S1. Physiological variables measured in migratory (migrating out of Abernathy Creek within 2 wk of hatchery release) and residual (remaining in freshwater for more than 2 wk after hatchery release) hatcheryorigin steelhead Oncorhynchus mykiss after release into Abernathy Creek, Longview, Washington, USA, between April and May, 2007, where TYPE represents migratory type (migratory or residual), NKA represents Na +,K + - ATPase activity (micromoles-adp/milligrams of protein/ hour), NKA 1A represents Na +,K + -ATPase a1a expression (relative to an endogenous reference, elongation factor-1 a [EF-1a]), NKA 1B represents Na +,K + -ATPase a1b expression (relative to an endogenous reference, EF- 1a), GSI represents gonadasomatic index (the relationship of gonad wet weight to total body wet weight), K represents condition factor (gram per cubid millimeter ), and FL represents fork length (millimeters). Found at DOI: JFWM-032.S1 (19 KB XLSX). Table S2. Temporal trends in gonadasomatic index (GSI), measured as the relationship of gonad wet weight to total body wet weight, among residual hatchery-origin steelhead Oncorhynchus mykiss in Abernathy Creek, Longview, Washington, USA, 2007, where Date represents sampling date, Type represents residual fish sampling period, and sample_loc represents sampling location. Found at DOI: JFWM-032.S2 (12 KB XLSX). Acknowledgments Data collection and reporting for 2007 were funded through the Bonneville Power Administration. We thank Jeff Poole and John Holmes for spawning, handling, and maintaining hatchery brood stock and their progeny. We thank the Washington Department of Fish and Wildlife for maintaining the screwtrap on Abernathy Creek for collection of migrating smolts. We also thank Patty Crandell and four anonymous reviewers for commenting on an earlier version of this manuscript. 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