General and Comparative Endocrinology 196 (2014) 112–122

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Growth and endocrine effect of growth hormone transgene dosage in diploid and triploid coho salmon Robert H. Devlin a,⇑, Dionne Sakhrani a, Carlo A. Biagi a, Jack L. Smith a, Takafumi Fujimoto b, Brian Beckman c a b c

Fisheries and Oceans Canada, 4160 Marine Drive, West Vancouver, BC V7K 1N6, Canada Faculty and Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato, Hakodate, Hokkaido 041-8611, Japan Northwest Fisheries Science Center, 2725 Montlake Boulevard East, Seattle, WA 98112, USA

a r t i c l e

i n f o

Article history: Received 21 August 2013 Revised 19 November 2013 Accepted 26 November 2013 Available online 7 December 2013 Keywords: Transgenic Dosage Triploid GH Salmon Growth

a b s t r a c t Growth-hormone transgene dosage, polyploidy, and parental effects on growth and endocrine responses have been assessed in coho salmon. Diploid fry with one or two transgene doses grew equally, whereas later-stage juvenile homozygotes grew faster than hemizygotes. In contrast, homozygotes and hemizygotes grew equally after smoltification, both in sea water and fresh water. Triploid transgenic salmon showed impaired growth which could not be fully overcome with additional transgene copies. Levels of muscle GH mRNA were elevated in two vs. one transgene dose diploids, but in triploids, a dosage effect was observed in muscle but not for animals carrying three transgene doses. IGF-I mRNA levels were elevated in transgenic vs. non-transgenic animals, but a dosage effect was not observed. Diploids and triploids with two transgenes had higher plasma GH levels than one-dose animals, but three-dose triploids showed no further elevation. Circulating IGF-I levels also showed a dosage effect in diploids, but not among any transgene doses in triploids. The present study reveals complex interactions among transgene dosage, maternal effects, developmental stage, and ploidy on growth and endocrine parameters in GH transgenic coho salmon. Specifically, GH transgenes do not always express nor have effects on growth that are directly correlated with the number of transgenes. Further, the reduced growth rate seen in triploid transgenic animals could not be fully overcome by increasing transgene dosage. The findings have relevance for understanding growth physiology, transgene function, and for environmental risk assessments that require understanding phenotypes of hemizygous vs. homozygous transgenic animals in populations. Crown Copyright Ó 2013 Published by Elsevier Inc. All rights reserved.

1. Introduction Growth regulation is critical for animals to respond effectively to seasonal variations in environmental conditions, food availabilities, and energy expenditures, and is regulated in part by the neuroendocrine control of growth hormone (GH) and insulin-like growth factor-1 (IGF-I) production (Beckman, 2011; Bjornsson et al., 2002). Control is exerted via feed back regulation of growth hormone (GH) production in vertebrates, mediated by IGF-I and GH interacting with their respective receptors in the hypothalamus and pituitary gland, coupled with information from the brain which integrates environmental conditions. These control systems can be overridden by overexpression of GH from transgenes in genetically modified animals, causing GH to be expressed in tissues other than the pituitary gland, and from promoters lacking capacity to respond to regulatory feedback signals (Du et al., 1992;

⇑ Corresponding author. Fax: +1 604 666 3497. E-mail address: [email protected] (R.H. Devlin).

Palmiter et al., 1982). For GH transgenic coho salmon (Devlin et al., 2004), elevated circulating GH causes very strong feedback inhibition of host GH gene expression in the pituitary gland (Mori and Devlin, 1999), rendering such animals essentially unregulated with regard to environmental controls influencing GH production. Extra-pituitary expression of GH at high levels causes continuous rapid growth rate which is largely uncoupled from normal seasonal cues that adjust growth rates in accordance with environmental circumstances (e.g., low growth during winter when food supplies and water temperatures are low). The growth rate of specific strains of GH transgenic fish differ widely (Devlin et al., 1995a; Leggatt et al., 2012; Nam et al., 2001; Rahman and Maclean, 1999; Razak et al., 1999), likely due to insert site position effects and also to a degree transgene copy number. In some cases, transgenic fish may be over stimulated by GH exposure such that morphological abnormalities (analogous to acromegaly), reduced viability, and sub-maximal growth can arise (Devlin et al., 1995a, 2001; Figueiredo et al., 2007; Nam et al., 2001; Ostenfeld et al., 1998; Rosa et al., 2008). However, strains showing very highly stimulated growth rates but with

0016-6480/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.11.023

R.H. Devlin et al. / General and Comparative Endocrinology 196 (2014) 112–122

few morphological abnormalities can also be generated, suggesting an optimal level of overexpression may exist for achieving maximal growth stimulation. For one GH transgenic coho salmon strain, further treatment with GH protein did not promote additional growth (Raven et al., 2012), whereas slower-growing wild-type salmon responded significantly. These data suggested that, for this strain, expression of the transgene was causing maximal growth possible by modification of the GH regulatory pathway. Different non-transgenic strains also may respond distinctly to GH treatment. For example, fast-growing strains of catfish (Silverstein et al., 2000), rainbow trout (Devlin et al., 2001), and Atlantic salmon (Neregard et al., 2008) respond less to GH treatment than do slow-growing strains. Molecular analysis has shown that GH transgenic and domesticated strains (both fast growing relative to wild type) show elevated GH and IGF-I levels and have similar changes in mRNA profiles (Devlin et al., 2009; Fleming et al., 2002; Tymchuk et al., 2009). Circulating GH levels in GH transgenic salmon arise primarily from constitutive expression of the transgene, and the level of GH may be expected to correlate positively with the number of copies of the transgenic locus (gene dosage). Such dosage effects are seen for many gene products when their locus is duplicated (Devlin et al., 1982; Kahlem et al., 2004; Rawls and Lucchesi, 1974). Alternatively, GH levels may not be correlated with transgene dosage if regulatory mechanisms exist to limit GH production, for example via gene regulatory networks or post-nuclear cellular or organismal-level endocrine processes (Malone et al., 2012). Transmission of GH signaling may also be buffered, affecting downstream endocrine responses and growth. Indeed, a relatively poor correlation between circulating GH levels and growth rate is often found in diploid non-transgenic fish (Beckman, 2011; Bjornsson et al., 2002) indicating other complex regulatory processes are presumably functioning which could also impact the effects of GH transgene dosage. Triploidy causes sterility in many fishes, and hence is an approach proposed for transgenic technology to reduce genetic interaction with wild stocks and to protect their proprietary genetic resources (Devlin and Donaldson, 1992). However, triploid coho salmon possessing a GH transgene in one set of chromosomes have been observed to grow slower than diploid salmon with one transgene dose (Devlin et al., 2004; Leggatt et al., 2012). The reasons for this effect are unclear, but could be due to several factors. Nontransgenic triploid coho salmon have been found to grow slower than their diploid counterparts in some (Devlin et al., 2004; Withler et al., 1995, 1998) but not all cases (Johnson et al., 1986), suggesting there could be an inherent growth-retarding effect of the triploidy condition per se in this species (e.g., arising from disrupted gene regulation and physiology). Alternatively, triploid animals have 50% larger cells, but only two-thirds of the number of cells compared to diploids of the same body size (Benfey, 1999), and hence animals with one transgene copy in one set of chromosomes (hemizygotes) will, overall, possess one-third less transgenes expressing GH. It is also possible that the triploid condition could affect expression of the transgene at the cellular level, for example if transcription factors do not operate in a concentration-dependant fashion within the larger cell volume (e.g., triploids could have higher absolute amounts of specific suppressive transcription factors per cell). Investigating the effects of triploidy on GH transgene expression is important to understand basic regulatory controls acting on transgenes, for strain assessments for aquaculture, and for environmental risk assessments of potential effects of animals with different transgene dosages in populations (e.g., one-dose hemizygotes vs. two-dose homozygotes), or in cases where the dosage of the transgene may change due to unequal recombination exchanges between flanking repetitive sequences at transgenic loci

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(Uh et al., 2006; Yaskowiak et al., 2006). A specific objective of the present study is to understand whether reduced growth seen in transgenic triploids is due to effects on GH transgene expression, and further whether such effects can be overcome by increased transgene dosage. Thus, to assess the influence of transgene dosage and polyploidy, we have examined the early growth rate of diploid coho salmon with 0, 1, or 2 doses, and triploids with 0, 1, 2, or 3 doses. Full life cycle growth rates have also been examined for one and two dose diploid salmon raised in fresh water and in sea water. Plasma GH and IGF-I levels, and amounts of liver and muscle GH, IGF-I, and GH receptor (GHR) mRNA have also been determined to assess their involvement in the observed growth responses. 2. Materials and methods 2.1. Fish strains and culture conditions All animals were reared according to the guidelines from the Canadian Council for Animal Care under institutional permission from the Fisheries and Oceans Canada Pacific Regional Animal Care Committee. Transgenic coho salmon (Oncorhynchus kisutch) were developed as described by Devlin et al. (1994, 2004). The strain utilized (M77) is fast-growing and contains the OnMTGH1 gene construct located at a single genetic locus (Uh et al., 2006) and is maintained in a wild genetic background by backcrosses at each generation to non-transgenic salmon obtained from nature (Chehalis River, BC, Canada). Crosses were performed by in vitro fertilization of stripped gametes, and embryonic development occurred in standard Heath incubation trays supplied with aerated well water (10 °C). Fish were reared under natural photoperiods in densities less than 10 kg/m3 to minimize tank effects, Well water was supplied at a rates of 1 L/(kg body weight) until smoltification, at which time they were either maintained in fresh water or were transferred to sea water (8–14 °C). Food was provided to groups initially 5–7 times/day, reducing to 2 feedings/ day near sexual maturation. All groups were fed to excess throughout these experiments with commercial Pacific salmon diets (Skretting Canada). 2.2. Production of homozygous and hemizygous GH transgenic coho salmon Inter se crosses between hemizygous (transgene present in one set of chromosomes only) individuals from one strain (M77) were performed to generate homozygous individuals with two transgene copies. Six F1 transgenic salmon (of unknown zygosity) of each sex were raised to sexual maturity and individually: (1) test crossed with non-transgenic individuals to determine (from transgene frequency in the progeny) whether each parent was homozygous or hemizygous for the transgene insert, and (2) incrossed to each other in all possible combinations (36 crosses). These crosses were reared until the eyed stage, at which time DNA was isolated from 25 individuals from each family and analyzed by PCR (Devlin et al., 2004) for the presence or absence of the transgene. Parents producing 100% transgenic offspring were bred together to establish an homozygous F2 line of transgenic salmon for strain M77. Fish from this strain were subsequently genotyped to confirm they were homozygous using PCR tests specific to the transgene and to the genomic site of integration (Uh et al., 2006). Groups of salmon were retained from the family crosses involving the homozygous parents (i.e., homozygous  homozygous, and reciprocal homozygous  wild type). Families (n = 168–188 fish per family) were reared separately in 200 L tanks until the smolt

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stage (210 days post fertilization). Fish were PIT tagged and half of each family transferred into one of two 20 m3 tanks where growth and survival were subsequently followed in both freshwater or marine conditions. Non-transgenic fish were grown only in freshwater conditions as they required an additional year to acquire smolt status (Devlin et al., 2004).

statements in the text referring to group differences (e.g., smaller, larger) were only made when this significance level was met.

3. Results 3.1. Production of homozygous GH transgenic salmon

2.3. Production of diploid and triploid salmon with different transgene dosages Diploids and triploids with different transgene doses were generated on Feb 4, 2005 as follows: (1) Diploids and triploids without transgenes were generated by crosses between wild-type salmon, and triploids were induced in half the progeny in of each family by pressure shocking eggs (Devlin et al., 2010); (2) Diploids and triploids with single transgene doses were produced as in (1), but using sperm from homozygous transgenic fathers as described above; (3) Diploids with two transgene doses were produced from crosses between homozygous fathers and mothers; (4) Triploids with two transgene doses were generated from crosses between homozygous transgenic mothers and wild-type fathers, followed by pressure shocking; (5) Triploids with three transgene doses were produced as in (4) but using sperm from homozygous transgenic fathers. To detect maternal effects on growth rate between eggs produced from mothers reared in laboratory conditions vs. nature, crosses were performed both with parents grown in the laboratory through their full life history (such rearing is necessary for transgenic but not wild-type fish) and with non-transgenic parents obtained from nature. Fry were initially transferred from Heath incubation trays to tanks on April 16, 2005. Weight and length determinations were made for group on April 21 (5 days post first feeding, dpff), June 18 (63 dpff), August 19 (125 dpff), and November 3 (201 dpff). During the growth trial, some families of the same cross type were combined (see cross IDs in Table S1) at sampling dates due to limitations in culture facilities to retain all groups separately as fish grew, or if families had few progeny. These pooled groups are indicated by family names separated by /. 2.4. Molecular analyses

Hemizygous GH transgenic salmon (strain M77) were incrossed and F1 progeny reared until sexual maturity. At 319 days post fertilization, a bimodal size distribution which contained 889 large and 257 small fish (ratio 3.46:1) was observed (Fig. 1). This ratio for growth-enhanced to wild growth rate F1 progeny does not differ from 3:1 (P = 0.141, Chi Square), and suggests the transgene insert is homozygous viable (a ratio of 2:1 would expected for a homozygous lethal transgene insert). The bimodal size distribution is consistent both with homozygotes and hemizygotes both surviving but growing at the same rate relative to slow-growing non-transgenic salmon (rather than trimodal as anticipated if hemizygous and homozygous salmon were growing at different rates). These progeny were reared to maturity, and twelve were crossed inter se, and also test crossed with wild-type salmon. Homozygous GH transgenic salmon for strain M77 were found to be viable (test crosses with wild-type salmon yielded 100% transgenic offspring), and progeny from incrossed homozygous transgenic salmon allowed establishment of a stable strain of homozygous transgenic salmon. Southern blots of genomic DNA from homozygous and hemizygous parents were also probed with GH sequences (Devlin et al., 2004) to examine the autoradiogram densities of the transgene bands relative to those from host GH genes. The GH transgene bands appeared darker in homozygotes compared to hemizygotes when compared to endogenous host GH sequences (example autoradiogram shown in Fig. 1 inset). On average, the ratio of transgene/GH2 band densities was consistent with a two-fold gene dosage difference (autoradiogram scan densities were 1.19 ± 0.31 and 0.66 ± 0.06 in homozygotes and hemizygotes, respectively (n = 10)). Two homozygous GH transgenic males were also separately crossed to a larger number of eggs from wildtype coho salmon females, and each produced 100% transgenic progeny (n = 1855 and 964, respectively). Of these progeny, 7 fish (0.14%) failed to show growth enhancement, but all tested positive for the OnMTGH1 transgene in blood.

Genotyping transgenic progeny to distinguish homozygotes from hemizygotes was performed using specific PCR tests (Uh et al., 2006) that can distinguish between the insert-site locus with and without the transgene present. Quantitative PCR methods for determination of mRNA levels for GH, IGF-I, IGF-II, thyroid hormone receptor-b (TRB), and GH receptor were performed as described (Raven et al., 2008). Southern blots were performed using GH exon sequences as a probe (Devlin et al., 2001). Radioimmunoassays for salmon growth hormone (GH) were performed as described by Swanson (1994), and for insulin-like growth factor I (IGF-I) were developed using antibodies and antigens supplied from GroPep, Adelaide, Australia (www.gropep. com). IGF-I antigen was iodinated with 125I, purified by chromatography, and used in radioimmunoassays as described by the manufacturer. 2.5. Statistical analysis Group comparisons were performed (SigmaStat) using ANOVA followed by Student–Newman–Keuls post-hoc test, or ANOVA on Ranks followed by Dunn’s post-hoc test for non-normal data. For liver GH mRNA levels between 1-dose and 2-dose diploids or triploids, Student’s t-test was used to make paired comparisons. Comparisons among groups were deemed different if P < 0.05, and all

Fig. 1. Frequency distribution of weights at age 319 days post fertilization of F1 progeny derived from an incross of male and female hemizygous GH transgenic salmon (strain M77). n = 1146. Inset: Southern blot analysis of genomic DNA from hemizygous and homozygous GH transgenic salmon, digested with Bam HI and Xba I and probed with salmon GH exon sequences. From left to right: heterozygous GH transgenic male, homozygous GH transgenic male, heterozygous GH transgenic female, homozygous GH transgenic female.

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3.2. Effect of transgene dosage on growth From the crosses described above, homozygous and hemizygous GH transgenic, as well as wild-type, coho salmon families were reared to determine whether the effect of the transgene was dominant or co-dominant in its genetic effect on growth rate. Fig. 2 shows frequency distributions for weight of progeny from each of these groups at age 159 days post fertilization. All three transgenic groups had a mean size significantly greater than that of non-transgenic controls (Fig. 2a vs. b–d,). Homozygous and hemizygous fish derived from the same egg clutch Fig. 2b vs. d) indicated these two genotypes were growing at the same rate at this early stage. However, hemizygotes generated from wildcaught mothers (Fig. 2c) grew significantly faster than the reciprocal cross (Fig. 2b), indicating the presence of growth-retarding maternal effect from transgenic mothers in these crosses. It is important to note that, despite being transgenic, a small proportion of individuals in these crosses did not grow at a rapid rate, particularly those derived from homozygous transgenic mothers. The quality of eggs in these cases appeared inferior (fewer and more dispersed lipid droplets) which may have caused some progeny to not thrive following first feed offering. Growth of the homozygous and hemizygous families was followed for two years until sexual maturity. Growth trials were performed in both fresh and sea water (Fig. 3a and b, respectively). During the presmolt interval (159–210 days post fertilization) the growth rate (Fig. 3c and d) of salmon with two doses of the transgene exceeded that of those with one dose (see more detailed experiment below examining gene dosage at early fresh water stages). From 210 to 398 days post fertilization, in both sea and fresh water, homozygous fish had lower growth rates than hemizygotes, whereas in the final interval, this was the case only in fresh water. Thus, following smoltification (210 days), hemizygotes either outgrew or had the same growth as homozygotes in both fresh water and sea water conditions. The fastest growing salmon in the trial were those that were hemizygous for the transgene and derived from eggs from non-transgenic wild females. Homozygotes sexually matured during the same season (January and February) as hemizygotes, one to two full years earlier than non-transgenic control salmon grown in culture conditions. 3.3. Evaluation of transgene dosage and maternal effects on growth in triploid and diploid salmon To examine the effect of transgene dosage in more detail, multiple crosses were performed to generate diploids with either 0, 1, or 2 doses of the transgene, and triploids with 0, 1, 2, or 3 doses (Supplemental Table S1). The parents of these families were either nontransgenic cultured or wild salmon, or cultured salmon hemizygous or homozygous for the GH transgene. At the fry stage (April), it was apparent that factors other than transgene dose or ploidy were influencing growth rate (Table S1), as seen above in the previous generation (Fig. 2). For example, a significant (P < 0.05) maternal effect causing reduced growth from cultured vs. wild mothers was detected both in diploid non-transgenic progeny (compare families 108 and 115 vs. 128, 135, 129, and 136) and in hemizygous transgenic progeny (families 112, 119, 143, 149 vs. 133 and 140). The same effects were observed in triploids (compare families 102, 109, and 116 vs. 130 and 137 for non-transgenic salmon, and families 106, 113, and 120 vs. 134 and 141 for hemizygous salmon). However, these effects became inconsistent by June with only one family pair differing in both non-transgenic and transgenic diploid progeny. A similar response was seen for triploids in June, except that for transgenic progeny only, both

Fig. 2. Size distributions of fry derived from crosses among homozygous transgenic, hemizygous transgenic, and control coho salmon (159 days post fertilization). Cross types are indicated above each figure. Mean weights ± SE and sample size (n) are also shown in panels for each group.

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Fig. 3. Growth of post-smolt transgenic salmon to maturation (a, b) and specific growth rates for weight (c, d), of homozygous and hemizygous transgenic salmon (strain M77) and controls, in sea water (a, c) and fresh water (b, d). Crosses involving different sexes (M and F) and different genotypes (Hom, homozygous transgenic; Het, hemizygous transgenic; Wild, wild nontransgenic) are shown in legends for each graph. Values are group means ± SE. In (b) and (d) SGRs for hemizygous transgenic salmon derived from a hemizygous male by wild female are not shown in the first interval because they were not yet size differentiated from co-cultured non-transgenic siblings. Error estimates also cannot be provided for this interval as fish were not yet tagged to allow tracking individual growth, as was done for later ages. Different letters above symbols or bars within a figure panel represent statistical differences among groups. Sample sizes: Fresh water start n = 23–84 group, final n = 9–54; sea water start n = 65–93, final n = 28–42.

families derived from cultured mothers remained significantly smaller. No consistent paternal effect derived from sperm from cultured vs. wild fathers was detected in diploid non-transgenic progeny: in April, all such families (128, 135, 129, 136) differed in weight from each other, with one family from a cultured father (136) particularly slow growing; in June, the families from cultured fathers (129 vs. 136) differed from each other, but only one (136) was significantly smaller than both families from wild fathers. These effects had reversed by August, with the pooled families from cultured fathers (129/136 vs. 128/135) being larger than from wild fathers. Thus, when assessing the effects of transgene dosage at young ages among a complex series of crosses involving both cultured and wild eggs, it is important to account for influences arising from egg quality. Hence comparisons below are made among families with the same maternal influence (either wild or cultured) unless otherwise indicated. In April, the weight of newly emerged fry was not elevated by the presence of any dose of transgene in diploids and triploids, in either a cultured or wild maternal background (Table S1). However, by June, a clear effect of the transgene was apparent in diploids (for diploid progeny from cultured mother, compare families 108 and 115 vs. 112, 119,143, and149 hemizygotes vs. 146 and 152 homozygotes; for diploid progeny from wild mothers, compare families 128, 135, 129, and136 vs. 133 and 140). However, in neither ploidy was a dosage effect apparent. In triploids from wild mothers, hemizygous fry were significantly larger than

non-transgenic fry (compare 130 and 137 vs. 134 and 141), whereas from cultured mothers, relationships between weight and transgene dose were less consistent (compare families 102/ 109 and 116 vs. 106/113 and 120 vs. 144 and 150 vs.147 and 153). By August, transgenic progeny (all dosages) were more than 8-fold (for diploids) and 5-fold (for triploids) heavier than their non-transgenic counterparts, and while no maternal influence was observed in diploid hemizygotes (compare families 112/119, 143/ 149, and 133/140), triploid hemizygotes from cultured mothers were smaller than hemizygotes from wild mothers (compare families 106/113/120 vs. 134/141). For progeny from cultured mothers in August, salmon with one or two transgene doses for diploids, or one, two, or three transgene doses for triploids, did not differ in weight (compare families 112/119, 143/149, and 146/152 for diploids, and 106/113/120, 144/150 and 147/153 for triploids). In November, a non-linear dosage effect on body size was seen (Fig. 4) between hemizygous and homozygous diploid salmon. Similarly, two-dose triploids were larger than one-dose triploids (dosage effect), but the former were still smaller than two-dose diploids. Triploids possessing 3 doses showed no further increase in growth beyond triploids with 2 doses. Thus, in all cases, triploids grew slower than diploids with equivalent transgene dose, such that adding additional transgene doses in triploids could not compensate for their general growth retardation. Crosses involving hemizygous parents (145, 151, 165, 169, 166, 170, 167, and 171) also allowed assessment of growth rates among

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Transgene Dose Fig. 4. Effects of transgene dosage on body size in diploid and triploid coho salmon (November time point). Data shown for progeny from cultured mothers only (Diploids: non-transgenic, group 108/115 (n = 40); one-dose, average of groups 112/119 and 143/149 (n = 24. 43); two-dose, group 146/152 (n = 17). Triploids: non-transgenic, group102/109/116 (n = 25); one-dose,:106/113/120 (n = 19); twodose, 144/150 (n = 27); three-dose, group 147/153 (n = 26). Different letters above symbols represent statistical differences among groups across all ploidies and transgene dosages.

genotypes with different transgene doses, but within rather than among families by application of transgene and insert-site specific PCR tests. In June, hemizygous and homozygous progeny did not differ in weight among six families generating hemizygous and homozygous progeny, whereas by August one of three family groups possessed within-family homozygotes that were larger than hemizygotes (Table S1). The effect of the origin of the transgene (maternal vs. paternal), and maternal transgene dosage, were examined in diploid and triploid non-transgenic and hemizygous transgenic salmon derived from cultured mothers (Table S1). The use of hemizygous mothers produces both transgenic and non-transgenic progeny and allows examination of whether the maternal presence of a transgene can influence growth of non-transgenic progeny compared with progeny from non-transgenic mothers. Among all possible comparisons (for families 108, 115, 165, and 169), non-transgenic salmon from hemizygous transgenic mothers (165) were heavier than one family from a cultured non-transgenic mother (115), and the other family from hemizygous transgenic mothers (169) did not differ from family 108 from a cultured non-transgenic mother. For hemizygous transgenic progeny in April, both families derived from cultured mothers carrying two transgene doses had average weights between families derived from non-transgenic mothers (i.e., reciprocal crosses, compare families 112 and 119 vs. 143 and 149), and all interfamily comparisons were significantly different except for one. From reciprocal crosses of homozygous transgenic  wild parents, by June, and in August, no interfamily effects were significant for their hemizygous transgenic progeny. Thus, evidence for a maternal effect caused by the presence of a transgene in mothers is weak, or at least is influenced by independent family effects of similar strength. 3.4. mRNA levels in liver and muscle in triploid and diploid salmon with different transgene dosages In August, levels of GH mRNA (Fig. 5) in non-transgenic liver RNA samples were not detectable as expected, but did show a dosage effect in diploid salmon with one vs. two transgene doses in a paired (t-test; P < 0.01) comparison, but not when all groups were analyzed together. Triploids showed more erratic levels that did not differ with transgene dosage, although some indication seems apparent between one and two dose salmon (Fig. 5). GH mRNA was

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lower in single transgene dose triploids compared to diploid hemizygotes, however diploid and triploid two-dose genotypes did not differ. IGF-I mRNA was elevated in transgenic salmon diploids relative to their respective non-transgenic controls, but a dosage effect was not observed. A trend towards a dosage effect was seen in triploids, except for those with 3 doses, but differences were not significant. Triploids with 3 doses had levels that did not differ from non-transgenic salmon. GH receptor mRNA was elevated in diploid transgenic salmon relative to non-transgenic diploids, but a dosage effect was not observed for these groups, nor in triploids. Levels of IGF-II and TRB showed no consistent trend with transgene dosage. In muscle (Fig. 6), GH mRNA was detectable in non-transgenic animals as previously described for coho salmon (Devlin et al., 2009; Mori and Devlin, 1999). GH transgenic salmon with one dose did not have significantly elevated mRNA from non-transgenic fish, diploids with two doses did. A similar response was seen in triploids, with elevated levels seen in 2 and 3 dose animals relative to 0 and 1 dose genotypes. One vs. two dose salmon were significantly different in paired comparisons within diploid and triploid groups, but were non-significant (shown) when all groups were analyzed together (as seen for liver GH mRNA levels). All transgenic salmon had IGF-I mRNA levels greater than non-transgenic salmon, but a significant dosage effect was not detected among any other transgenic or ploidy genotypes. 3.5. Plasma GH and IGF-I levels Levels of plasma GH and IGF-I were determined in hemizygous and homozygous GH transgenic coho salmon from marine conditions with average fish sizes of 2706 ± 104 and 2040 ± 167 g, respectively. GH levels were significantly greater in homozygotes than hemizygotes (1.54-fold higher) indicating a partial dosage effect on the production of this hormone (Fig. 7a). However, IGF-I levels did not differ significantly between the groups. Plasma levels of GH and IGF-I were also examined (August samples) in the diploid and triploid salmon series possessing different transgene dosages. GH levels showed a clear dosage effect in onedose vs. two-dose animals in both diploids and triploids (Fig. 7b; age-matched non-transgenic (0-dose) animals did not provide enough plasma to allow GH determinations). Levels of GH in threedose triploids were intermediate between one a two-dose triploids, but significantly lower than two-dose diploids. In contrast, IGF-I showed significant dosage effects among fish with 0, 1, and 2 doses in diploid fish (Fig. 7c). Triploid transgenic salmon possessed elevated IGF-I relative to non-transgenic fish, whereas a dosage effect among transgenic genotypes was not statistically significant. For both 1 and 2 dose salmon, triploid animals possessed lower IGF-I than their diploid counterparts with the same transgene dose. 4. Discussion The insertion of foreign DNA in the GH transgenic coho salmon strain (M77) studied is not disrupting a critically vital function in the host genome which renders homozygotes lethal (Uh et al., 2006). Nevertheless, the present study has found that GH transgene dosage, ploidy, and maternal rearing conditions can affect growth rate, gene product levels in the GH/IGF-I pathway. Maternal effects were derived largely from fish being reared in culture conditions, which is known to reduce egg quality, and were largely dissipated after early developmental stages (Bessey et al., 2004; Campbell et al., 1994; McGinnity et al., 1997). The presence of a maternal transgene was not found to influence growth in nontransgenic nor transgenic progeny. Transgene dosage effects were found to act in a developmental-stage-dependant and

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Fig. 5. Levels of mRNA (mean ± SE) for specific genes in liver RNA as determined by Q-PCR. X axis labels (e.g., 2N-0) indicate ploidy (2N or 3N) and transgene dosage (0, 1, 2, 3). n = 8–11 samples per group. Different letters above bars within a panel represent statistical differences among groups. GH mRNA was undetectable in non-transgenic livers and was therefore not included in the statistical analysis.

ploidy-dependant fashions. At the newly emerged fry stage, no effect of transgene dose was detected in diploids, whereas at a juvenile fresh-water stage, homozygous transgenic salmon grew somewhat faster than hemizygotes. In the marine phase, no difference in growth was detected between hemizygotes and homozygotes. In other species, no effect of transgene dose on growth rate was detected between hemizygotes and homozygotes tilapia in one case (Rahman et al., 1998), whereas within a different tilapia strain, a dosage effect on growth was observed (Martinez et al., 1999). In loach, both elevated growth, and elevated and reduced growth at different developmental stages, have been observed among strains (Nam et al., 2002). For a strain of GH transgenic zebrafish, negative effects of transgene dosage have been observed for

growth (Figueiredo et al., 2007; Rosa et al., 2008). Interestingly, GH transgenic amago salmon using the same gene construct as used in the present study do show a transgene dosage effect, for both growth and lipid metabolism markers (Sugiyama et al., 2012). In GH transgenic mice, homozygotes have been observed to grow faster than hemizygotes in some (Yun et al., 1990) but not all (Eisen and Murray, 2001) cases. Together these data show that phenotypic responses to GH expression via alterations in transgene dosage (at the same locus) are likely strain and perhaps species specific, dependent on the specific level of GH produced in the line and the physiological reception of that signal in the species. It is possible that the lack of further growth enhancement seen in two vs. one-dose transgenics is due to pathological effects. Indeed,

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in some transgenic strains, a single dose of a GH transgene locus may be able to stimulate endocrine growth pathways maximally, whereas two doses may induce abnormalities (Devlin et al., 1995b; Jhingan et al., 2003; Ostenfeld et al., 1998) that counter potential growth stimulating effects. In many cases, when genes are duplicated in diploid organisms, their gene product levels increase, and when present in one copy are reduced (Devlin et al., 1988; Rawls and Lucchesi, 1974). However, the phenotypic consequences of two-fold differences in gene products can appear small for many loci (e.g., most null mutations are recessive), which seems paradoxical and wasteful for organisms to produce such an excess of gene product. However, it has been suggested (Kacser and Burns, 1981; Muller, 1948) that fully diploid levels of gene product are actually necessary to develop a completely wild-type phenotype that affords normal fitness under natural selection conditions (hence the evolution of various dosage compensating mechanisms (Lucchesi, 1978). Such a situation may not be always expected for anthropogenically generated (transgenic) loci as examined here, where natural selection has not acted to adjust expression levels to a fitness optimum. Further, inverse effects of duplicated chromosomal regions are known to suppress expression of other loci in a trans fashion that can lead to the lack of a dosage effect (Birchler, 2013; Devlin et al., 1988; Rawls and Lucchesi, 1974). Indeed, in transgenic salmon, levels of both liver

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Transgene Dosage Fig. 7. Plasma hormone levels in GH transgenic coho salmon (strain M77). (a) Levels of GH and IGF-I in plasma from diploid 21-month-old hemizygous (n = 13) and homozygous (n = 31 for GH, n = 32 for IGF-I) salmon. (b) Plasma GH levels in August (125 days post fertilization) fish 2n and 3n salmon possessing different transgene dosages. Insufficient plasma was obtained for determination of GH levels from non-transgenic fish (i.e., 0 dose) due to their size. n = 10 for each group. (c) Plasma IGF-I levels in August sample of 2n and 3n salmon possessing different transgene dosages. n = 8–12 for each group. Different letters above bars or symbols within a panel represent statistical differences among groups.

and muscle GH mRNA, and plasma GH, were elevated in two-dose vs. one-dose fish for both diploids and triploids. However, no further increase was seen in three dose triploids. IGF-I mRNA levels in liver and muscle were elevated above non-transgenic salmon, but did not show a dosage effect in either ploidy. In contrast, circulating plasma IGF-I levels were elevated in young homozygous vs. hemizygous salmon in diploids, but not in near-mature fish and not in triploids. For both mRNA and plasma hormone levels, triploid animals had lower levels than their diploid counterparts with the same transgene dose. Together these data indicate that the GH

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transgene is being expressed in a largely unregulated way, but that influences exist which can, with elevated transgene dose, dampen expression and signaling within the GH/IGF-I axis to influence IGF-I production and consequent phenotypic effects (growth). Lower growth was also observed for triploids compared to diploids with the same transgene dose, correlating with observed hormone and mRNA levels between ploidies. In previous studies, GH transgenic coho salmon carrying a single dose of the transgene were found to grow slower as triploids than diploids, paralleling reduced growth of triploids seen in non-transgenic coho and closely related salmon species (Devlin et al., 2004; Shrimpton et al., 2012; Withler et al., 1995, 1998). Reduced growth of triploid vs. diploid GH transgenic fish has also been shown for loach (Nam et al., 2001) and tilapia (Razak et al., 1999). Reduced growth in triploids in both one-dose and two-dose transgenic salmon, and in non-transgenic salmon, suggests that the triploid condition itself could be inducing a degree of genetic and physiological change that disrupts phenotypes from those highly evolved in diploids, with consequent reduction in vitality and growth (Benfey, 1999; Jhingan et al., 2003). In Chinook salmon (Oncorhynchus tshawytscha), IGF-I levels were reduced in non-transgenic triploids relative to diploids, and other physiological parameters showed dosage effects between ploidies (Shrimpton et al., 2012). In Drosophila, diploids and triploids have the same level of gene products at the organism level, and whereas this reflects a 50% increase in gene product per cell due to elevated cell volumes of triploids, it also indicates that the expression state of the gene has not been altered (Ching et al., 2010; Guo et al., 1996; Lucchesi, 1973). Nevertheless, gene regulatory systems have likely evolved to function best in a diploid condition, and if placed within the context of a larger cell volume with 1.5 times as many loci to control, may not function with perfect fidelity in some cases (Guo et al., 1996). Such effects could arise if cellular processes act in a non-concentration-dependant fashion (e.g., if transcription factors act by amounts per cell basis rather than amounts per cellular volume). It should also be noted that triploidy in some fish species is associated with more rapid growth. For example, triploid crucian carp grow faster than diploids and have elevated levels of GH, GHR, and IGF-I mRNAs (Zhong et al., 2012). The mechanisms of incomplete dosage effects and influences of ploidy could be acting at multiple levels. Lower levels of transgene GH mRNA and protein in triploids compared to diploids suggests a cellular level of control. Although triploid fish have larger cells (Benfey, 1999), the organism compensates for this by possessing fewer cells, thus retaining similar developmental trajectories, overall morphology, and body size at age. The consequence of this cellular compensation in the present context is that GH transgenes will be present in one-third fewer number in the entire animal compared to diploids with the same transgene dose. In this case, an overall reduction in GH production and growth may be expected in triploid vs. diploid animals carrying the same transgene dosage. This hypothesis that reduced growth in triploid GH transgenic salmon arises from lower levels of circulating GH is supported by the observation that adding a second transgene dose to triploids does further elevate their growth, albeit not to the level seen for homozygous diploids. Whereas such effects could influence the utility of sterilized genetically distinct strains for application, it should be noted that any negative effects of triploidy on growth are much less than the growth-stimulating capabilities of GH transgenes seen for multiple fish species (Devlin et al., 2004; Nam et al., 2001; Razak et al., 1999). Previous research has shown that challenges to physiological pathways can modify transcript abundance between diploid and triploid salmon (Ching et al., 2010). Analogously, for downstream gene products regulated by GH, a lack of dosage effects might arise from saturation of a signaling pathway. For example, IGF-I mRNA

levels are elevated in hemizygous transgenic salmon relative to non-transgenic fish, but no further increase is seen with additional transgene dosages in diploids or triploids. Similarly, treatment of hemizygous GH transgenic salmon with additional GH protein does not further elevate IGF-I (Raven et al., 2012). In diploid GH transgenic zebrafish, hemizygotes have elevated levels of growth and IGF-I, whereas homozygotes possess GH levels and growth rates the same as wild type (Figueiredo et al., 2007). It is possible that the amount of GH produced from one transgene in hemizygotes is able to saturate the GH receptor signaling pathway, such that higher levels of GH have no effect on IGF-I mRNA production and growth. For GH receptor, mRNA levels were found to be elevated in transgenic salmon relative to controls, but no clear dosage effect was apparent. Thus, GH signaling may be stimulated by both elevated GH and GHR, but since the latter does not increase with transgene dosage, stimulation of the system may be limited to a degree (depending on the degree of receptor saturation). It is also known that nutritional status of vertebrates has a strong capacity to limit IGF-I mRNA production under different GH signaling conditions (Beckman, 2011; Duan, 1998; Duan and Plisetskaya, 1993; Raven et al., 2008), a mechanism that could prevent direct dosage effect relationships between GH levels and IGF-I mRNA production depending on feed intake levels among genotypes (which could be influenced by the degree of endocrine disruption and pathology they experience from elevated GH levels). Incongruently, both IGF-I protein levels and growth both do show a dosage effect in diploids and triploids despite mRNA levels not showing this response, suggesting post-transcriptional regulation could also be operating. For example, IGF-I mRNA stability may be inversely linked to translation rates such that higher levels of protein production cause a consequent reduction in mRNA levels, resulting in similar steady-state IGF-I mRNA levels among genotypes with different IGF-I transcription rates. It is also possible that different levels of IGF-I binding proteins (IGFBP) could cause differences between IGF-I protein and mRNA levels, since IGFBPs are known to influence the stability of circulating IGF-I (Baxter, 1994). IGFBP1 mRNA is influenced by GH transgenesis and by nutritional status in coho salmon, with reduced growth rates correlated with elevated IGFBP1 levels (Overturf et al., 2010; Shimizu et al., 2009). In the present experiments, IGFBPs may be present at higher levels in some genotypes, which could alter the stability of circulating IGF-I and hence divergence between steady-state levels of IGF-I protein and mRNA. In these scenarios, growth rates would be expected to correlate better with plasma IGF-I than IGF-I mRNA levels (see similarity of curves for both diploids and triploids in Figs. 4 and 7c in the present experiment). Indeed, circulating IGF-I levels do correlate closely with growth rate within many conditions (Beckman, 2011). It should be noted that negative feedback regulation of GH production arising from elevated GH and IGF-I in transgenic fish is not anticipated to play a major role in modulating transgene dosage effects, since constitutive ectopic expression of GH from the transgene in salmon and other species causes a strong reduction in pituitary GH mRNA production (i.e., the pituitary is no longer the only major regulator of the GH/IGF-I axis in GH transgenic fish) (Eppler et al., 2007; Mori and Devlin, 1999). Further, steady-state IGF-I levels may not correlate well with GH levels in transgenic animals due to changes in hormone turnover and dilution effects arising from differential growth (Devlin et al., 2000). The mode of action of GH may also be altered significantly in transgenic animals expressing GH in all cells, potentially acting locally in a paracrine fashion (Eppler et al., 2007; Raven et al., 2008), or intracellulalry. The present study has shown that GH transgenes do not always act in a dosage-dependent manner with respect to gene product levels, endocrine responses, and influences on growth rate. The data reveal complex interactions among transgene dosage,

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maternal effects, developmental stage, and ploidy on growth and endocrine parameters in GH transgenic coho salmon. Specifically, reduced growth seen in triploid transgenic coho salmon appears to arise from the triploid condition itself, and cannot be fully overcome by addition of additional transgene dosage. A further important purpose for the present data is to provide understanding of transgene dosage effects for environmental risk assessments. Should GH transgenic fish enter nature, the persistence of the transgene will depend to a large degree on fitness effects caused by the transgene (Ahrens and Devlin, 2010; Kapuscinski et al., 2007; Maclean and Laight, 2000; Muir and Howard, 2002). If different effects on phenotype and fitness exist between homozygous vs. hemizygous transgenic fish, or from changes in transgene copy number at the transgenic locus, then these should be taken into account when modeling the introgression and subsequent potential ecological consequences of a transgene to ecosystem components. Acknowledgments The authors acknowledge funding support from the Canadian Regulatory System for Biotechnology to R.H.D. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ygcen.2013. 11.023. References Ahrens, R.N.M., Devlin, R.H., 2010. Standing genetic variation and compensatory evolution in transgenic organisms: a growth-enhanced salmon simulation. Transgenic Res. 20, 583–597. Baxter, R., 1994. Insulin-like growth factor binding proteins in the human circulation: a review. Horm. Res. 42, 140–144. Beckman, B., 2011. Perspectives on concordant and discordant relations between insulin-like growth factor 1 (IGF1) and growth in fishes. Gen. Comp. Endocrinol. 170, 233–252. Benfey, T.J., 1999. The physiology and behavior of triploid fishes. Rev. Fish. Sci. 7, 39–67. Bessey, C., Devlin, R.H., Liley, N.R., Biagi, C.A., 2004. Reproductive performance of growth-enhanced transgenic coho salmon (Oncorhynchus kisutch). Trans. Am. Fish. Soc. 133, 1205–1220. Birchler, J., 2013. Aneuploidy in plants and flies: the origin of studies of genomic imbalance. Semin. Cell Dev. Biol. 24, 315–319. Bjornsson, B.T., Johansson, V., Benedet, S., Einarsdottir, I.E., Hildahl, J., Agustsson, T., et al., 2002. Growth hormone endocrinology of salmonids: regulatory mechanisms and mode of action. Fish Physiol. Biochem. 27, 227–242. Campbell, P.M., Pottinger, T.G., Sumpter, J.P., 1994. Preliminary evidence that chronic confinement stress reduces the quality of gametes produced by brown and rainbow trout. Aquaculture 120, 1–2. Ching, B., Jamieson, S., Heath, J.W., Heath, D.D., Hubberstey, A., 2010. Transcriptional differences between triploid and diploid Chinook salmon (Oncorhynchus tshawytscha) during live Vibrio anguillarum challenge. Heredity 104, 224–234. Devlin, R.H., Biagi, C.A., Smailus, D.E., 2001. Genetic mapping of Y-chromosomal DNA markers in Pacific salmon. Genetica 111, 43–58. Devlin, R.H., Biagi, C.A., Yesaki, T.Y., 2004. Growth, viability and genetic characteristics of GH transgenic coho salmon strains. Aquaculture 236, 607– 632. Devlin, R.H., Biagi, C.A., Yesaki, T.Y., Smailus, D.E., Byatt, J.C., 2001. Growth of domesticated transgenic fish. Nature 409, 781–782. Devlin, R.H., Donaldson, E.M., 1992. Containment of genetically altered fish with emphasis on salmonids. In: Hew, C.L., Fletcher, G.L. (Eds.), Transgenic Fish. World Scientific Press, Singapore, pp. 229–265. Devlin, R., Holm, D., Grigliatti, T.A., 1982. Autosomal dosage compensation in Drosophila melanogaster strains trisomic for the left arm of chromosome 2. Proc. Natl. Acad. Sci. USA 79, 1200–1204. Devlin, R.H., Holm, D.G., Grigliatti, T.A., 1988. The influence of whole-arm trisomy on gene expression in Drosophila. Genetics 118, 87–101. Devlin, R.H., Sakhrani, D., Biagi, C.A., Eom, K.-W., 2010. Occurrence of incomplete paternal-chromosome retention in GH-transgenic coho salmon being assessed for reproductive containment by pressure-shock-induced triploidy. Aquaculture 304, 66–78. Devlin, R.H., Sakhrani, D., Tymchuk, W.E., Rise, M.L., Goh, B., 2009. Domestication and growth hormone transgenesis cause similar changes in gene expression in coho salmon (Oncorhynchus kisutch). Proc. Natl. Acad. Sci. USA 106, 3047–3052.

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