Journal of Fish Biology (2006) 68 (Supplement A), 136–143 doi:10.1111/j.1095-8649.2005.00903.x, available online at http://www.blackwell-synergy.com BRIEF COMMUNICATIONS Sexual genotype markers absent from small numbers of male New Zealand Oncorhynchus tshawytscha V. J. M E T C A L F * AND N. J. G E M M E L L School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand (Received 18 February 2005, Accepted 28 June 2005) In New Zealand chinook salmon Oncorhynchus tshawytscha, sexual genotype did not correspond to phenotype in 1
6% of male fish, contrasting starkly with North American chinook populations, where inconsistencies between female genotype and phenotype have been observed # 2006 The Fisheries Society of the British Isles at frequencies up to 84%. Key words: chinook; masculinization; New Zealand; pollution; salmon; sex reversal. Chinook salmon Oncorhynchus tshawytscha (Walbaum) from the Sacramento River catchment were successfully introduced into New Zealand between 1900 and 1906 (McDowall, 1990, 1994; Quinn et al., 1997) and have maintained a wild population here ever since, colonizing many of the east coast rivers of the South Island (McDowall, 1994). Recently, it has been shown that North American populations of chinook salmon have a varied (16–84%) but generally high prevalence of females bearing a male genetic marker (Nagler et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004), indicative of spontaneous sex reversal. Other natural populations of salmonids, including New Zealand chinook salmon, may also possess a proportion of phenotypically sex-reversed fish, resulting in altered sex ratios and contributing to population decline. In salmonids, like humans, sex is determined by an XY chromosomal system (Hunter et al., 1982, 1983; Johnstone & Youngson, 1984; Okada, 1985; Devlin et al., 1994), resulting in gonochoristic fishes. Sex determination in salmonids was thought to be under strict genetic control, although it is now clear that environment can influence sex, with a labile period identified between hatching and first feeding, during which phenotypic sex may change (Baker et al., 1988; Piferrer & Donaldson, 1989; Piferrer et al., 1993). Four sex-linked DNA markers have been identified in salmonids, with two (GH-Y and OtY1) routinely used in polymerase chain reaction (PCR)-based tests *Author to whom correspondence should be addressed: Tel.: þ64 3 3642987 ext. 4848; fax: þ64 3 3642590; email: victoria.metcalf@canterbury.ac.nz 136 # 2006 The Fisheries Society of the British Isles SEX REVERSAL IN NEW ZEALAND CHINOOK SALMON 137 for sex-identification (Devlin et al., 1991, 1994; Du et al., 1993; Forbes et al., 1994; Clifton & Rodriguez, 1997; Devlin et al., 2001; Brunelli & Thorgaard, 2004). A growth hormone (GH) pseudogene sequence, found on the Y chromosome (GH-Y, GH-C or GH-p) is closely associated with the sex-determination region in a number of salmonids (Du et al., 1993; Forbes et al., 1994; Kavsan et al., 1994; Nakayama et al., 1999; Devlin et al., 2001; Zhang et al., 2001). A second DNA fragment, OtY1, has been isolated from the Y chromosome of chinook salmon (Devlin et al., 1991, 1994). Inconsistencies in both the GH-Y and OtY1 sexing tests have been reported in several Oncorhynchus species (Nagler et al., 2001; Zhang et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004, 2005). A study examining GH-Y in the Oncorhynchus masou (Brevoort) complex, identified phenotypic males lacking GH-Y or phenotypic females bearing GH-Y at levels ranging from 3–7% (Zhang et al., 2001). More dramatic levels of phenotypic sex reversal have been reported in chinook salmon from the Columbia River (Nagler et al., 2001; Chowen & Nagler, 2004). Here an initial study found that 84% of phenotypic females possessed the OtY1 marker in the Hanford Reach population, while hatchery broodstock females from Priest Rapids Hatchery used to supplement this population did not (Nagler et al., 2001). A subsequent and more extensive study of three wild and one hatchery population within the same river system, which included the Hanford Reach and Priest Rapids Hatchery populations, documented females bearing OtY1 at frequencies ranging from 33–63% (Chowen & Nagler, 2004). Contemporaneously, an independent study of chinook salmon populations from the Sacramento and San Joaquin River Basin revealed that overall 16% of phenotypic females bore the OtY1 marker, with a higher frequency in stream as opposed to hatchery populations (Williamson & May, 2002). Absolute causality of the high level of phenotypic sex reversal in the North American chinook salmon population has not yet been established. Variation in the genomic position of the GH-Y marker loci used for sex-tests has been observed in some salmonids (Nakayama et al., 1999), and if such variation occurred among chinook populations it could lead to erroneous predictions of sexual genotype. The most probable explanation, however, is that some extrinsic, environmental factor has led to the sex reversal observed (Nagler et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004). The most common extrinsic variable influencing sex in animals is temperature (Bull, 1983) and temperaturedependent sex differentiation during the labile period has been demonstrated in sockeye salmon Oncorhynchus neska (walbaum) (Craig et al., 1996; Azuma et al., 2004). In addition, pH (Rubin, 1985), exogenous hormones (Yamamoto, 1969; Goetz et al., 1979; Donaldson & Hunter, 1982; Hunter & Donaldson, 1983; Hunter et al., 1986) and pollutants (Torblaa & Westman, 1980; Matta et al., 1998) can also have an effect on sex differentiation in fishes. Several of these environmental factors have now been implicated in the phenotypic sex reversal observed in North American chinook salmon, but since the base-line level of sex reversal prior to environmental modification and pollution is unknown, it is difficult to determine whether the sex reversal observed within these populations is a normal feature of chinook salmon biology or a response to environmental change. # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 68 (Supplement A), 136–143 138 V. J. METCALF AND N. J. GEMMELL This study sought to determine if the high level of phenotypic sex reversal noted in North American chinook salmon populations (Nagler et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004) was also present in the New Zealand chinook salmon stock. Given that this stock shares a common genetic origin with U.S. fish, but inhabits a radically different environment (McDowall, 1994), it provides an opportunity to begin to determine if the high levels of phenotypic sex reversal observed in North America are an intrinsic feature of chinook salmon biology, or if environmental factors are the main causal agents. In May 2002, c. 1000 1-year-old chinook salmon were randomly selected from a hatchery-reared broodstock pool generated from semi-wild returns. These fish are the descendants of juvenile fish collected from all of the major chinook salmon-producing rivers and from several isolated land-locked populations found in the central South Island of New Zealand (M. Unwin, pers. comm.). Each year gametes are harvested from wild returns of this stock released into the Kaiapoi River, which are used to generate broodstock at the National Institute of Water and Atmospheric Research (NIWA) Silverstream Hatchery and this broodstock is in turn released back into the Kaiapoi River, as well as other major South Island Rivers. The fish used in this study were surplus fish of this same stock. These fish were individually PIT passive integrator transponder (PIT) tagged for life long identification (Prentice et al., 1987) and the adipose fin removed and stored in 95% ethanol for subsequent genetic analysis. Fish identification numbers and sample numbers were triple checked to reduce sample handling and labelling errors. DNA was extracted from fin clips by a standard Chelex procedure (Walsh et al., 1991). For the GH-Y test, genomic DNA (gDNA) was amplified by PCR using two GH-specific primers (SEX-F1 50 -CCTGGATGACAATGACTCTCA30 and SEX-R1 50 -CTACAGAGTGCAGTTGGCCTC-30 ) as in Du et al., (1993), in a total volume of 25 ml with c. 50 ng gDNA, 2
5 mM MgCl2, 0
2 mM dNTP, 0
3 mM each primer, 0
5 units Taq DNA polymerase, 20 mM Tris and 50 mM KCl. The PCR reaction was adapted from that described in Du et al. (1993) with 2 min at 93 C, 15 s at 61 C, 1 min 20 s at 72 C, 15 s at 93 C for 9 cycles; then 15 s at 61 C, 45 s at 72 C, 15 s at 93 C for 30 cycles, followed by 7 min at 72 C. The PCR products were visualized on a 2% agarose gel. Both female and male controls were always included in the amplification. GH-Yþ individuals had three PCR products, including the diagnostic male 273 bp band; GH-Y- individuals had two PCR products of 782 and 400 bp and were deemed to be genetically female. To eliminate possible errors associated with a reliance on a single marker for sex identification, the OtY1 genetic test was also used on all putative sex reversed fish, as well as a control group of 52 male and 52 female fish. Genomic DNA was amplified by PCR using two OtY1-specific primers (Y1 50 -GATCTGCTG GCTGGATTTGG-30 and Y2 50 -CCAGCGATGGTTTGTTTGAG-30 ) as in Devlin et al. (1994), in a total volume of 25 ml with c. 50 ng gDNA, 1
25 mM MgCl2, 0
2 mM dNTP, 0
3 mM each primer, 0
5 units Taq DNA polymerase, 20 mM Tris and 50 mM KCl. The PCR reaction was loosely based on that described in Devlin et al. (1994) with 2 min at 94 C, 15 s at 94 C, 15 s at 58 C, 20 s at 72 C for 35 cycles, followed by 5 min at 72 C. The PCR products were # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 68 (Supplement A), 136–143 139 SEX REVERSAL IN NEW ZEALAND CHINOOK SALMON visualized on a 2% agarose gel. Both female and male controls were always included in the amplification. Males were identified by the presence of a bright 209 bp band, whereas females had faint bands at 230, 250, 280 and 360 bp. Surviving fish (n ¼ 876) were culled during the Austral summer of 2002–2003 (mainly in October to November 2002 and some in May 2003 ) and phenotypic sex was determined by visual inspection of the gonads. In New Zealand, male and female chinook salmon generally mature and spawn at 2 and 3 years of age, respectively. Consequently, unambiguous identification of ovaries and testes can be undertaken at least a full year earlier than in their northern hemisphere counterparts. All culled females had clear evidence of ovarian maturation. All males culled in the first round of culling had immature testes and those culled in the later round had clear evidence of testes maturation. The fish whose female genotype disagreed with their male phenotype all came from the first round of culling (i.e. had immature testes) and as such the possibility that some may have been sterile females could not be completely eliminated. After culling, any fish noted to be phenotypically different from their genotypically determined sex had four additional and independent DNA extractions performed using their original fin clip to ensure confidence in the results. A new sample was not used because the correlations of phenotypic and genotypic sex were made subsequent to both PIT tag retrieval and burial of the carcasses. Genotypic sex was then confirmed by at least three independent additional PCR reactions for each genetic test on each extraction. A total of 876 fish had their sex genetically determined by the GH-Y test (Table I). The total number of genotypic females was 471, while the total number of genotypic males was 405. This indicated female:male sex ratios of 1
16:1 (Table I), which represents a significant skew towards females in the adult population (w2, d.f. ¼ 1, P < 0
05), possibly due to death of males that matured as parr. When fish were culled and the phenotypic sex determined, aberrations from the expected results were discovered, in that not all sex phenotypes correlated with their previously determined genotypes. Seven of the 471 fish that were genotyped as female according to GH-Y were phenotypic males since they had testes rather than ovaries [Table I and Fig. 1(a), lanes 1–7). The mean  95% CI incidence for phenotypic and genotypic discordance in this study was 0
0169  0
0120. TABLE I. Phenotypic sex and GH-Y occurrence in New Zealand chinook salmon Genotypic sex Phenotypic male Phenotypic female Total Reversion rate (%) GH-Yþ GH-Y Total 405 7 412 0 464 464 405 471 876 0
8 GH-Yþ, individuals were those exhibiting three PCR products and deemed to be genetically male (XY); GH-Y , individuals were those exhibiting two PCR products and deemed to be genetically female (XX). # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 68 (Supplement A), 136–143 140 (a) bp 805 514 448 339 264 V. J. METCALF AND N. J. GEMMELL Mr 1 2 3 4 5 6 7 M F (b) bp Mr 1 2 3 4 5 6 7 M M F F 514 448 339 264 216 FIG. 1. Genetic sex of New Zealand chinook salmon determined by polymerase chain reactions. (a) Polymerase chain reaction assays for GH-Y in New Zealand chinook salmon. Lanes: Mr, marker DNA; 1, fish 4; 2, fish 9; 3, fish 405; 4, fish 630; 5, fish 661; 6, fish 847; 7, fish 952; M, male control; F, female control. Females have bands at 782 and 400 bp, whereas males have an additional band at 273 bp. Genetically female fish with male phenotype are in lanes 1–7. (b) Polymerase chain reaction assays for OtY1 in New Zealand chinook salmon. Lanes: Mr, marker DNA; 1, fish 4; 2, fish 9; 3, fish 405; 4, fish 630; 5, fish 661; 6, fish 847; 7, fish 952; M, male control; F, female control. Males have a clear singular band at 209 bp while females have bands at 230, 250, 280 and 360 bp. Genetically female fish with male phenotype are in lanes 1–7. To eliminate the possibility that these putative sex reversals were simply methodological artefacts, perhaps caused by mutations in the GH-Y primer binding sites, a second sexing test was used to verify the GH-Y results. The OtY1 locus was amplified using DNA samples from the seven putative sexreversed fish as well as from 52 confirmed male genotype and male phenotype fish and 52 female genotype and female phenotype fish. All seven of the fish found to possess testes but lacking GH-Y were also genotyped as females according to OtY1 [Fig. 1(b), lanes 1–7]. Tests were repeated and were 100% concordant a total of four times. This indicated that 0
8% of fish sampled from this New Zealand chinook salmon cohort were phenotypically male but genetically female (XX) in that they appeared to lack the Y-chromosome specific markers, GH-Y and OtY1. This is the first reported finding of phenotypically male chinook salmon lacking Y-chromosome specific markers (GH-Y and OtY1). While each test appeared to be accurate in itself, it is recommended that both tests are used for any investigation of chinook salmon sex, especially in light of the increasing numbers of sex-reversed fish found in wild populations. In stark contrast to other studies reporting a lack of correlation between phenotypic and genotypic sex in salmonids (Nagler et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004), no phenotypic female fish with a male genotype were observed. Both males lacking GH-Y and females with GH-Y were found in a study of the O. masou complex, where consistency between phenotypic and genotypic sex was determined to be 93
1% (masu), 96
7% (Biwa) and 94% (Honmasu) (Zhang et al., 2001). More dramatic findings of phenotypic sex reversal in North American chinook salmon populations have been documented using the OtY1 test (Nagler et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004), although unlike the present study, these studies all found medium to high rates (16–84%) of females bearing the OtY1 marker. It is known that the GH-Y region exhibits lability in its location within the genome of salmonids. For example, GH-Y is Y-linked in masu salmon but not in the very closely related amago salmon Oncorhynchus masou ishikawai (Jordan & # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 68 (Supplement A), 136–143 SEX REVERSAL IN NEW ZEALAND CHINOOK SALMON 141 McGregor) (Nakayama et al., 1999). Most studies, however, have concluded that fishes with aberrant sexual genotype and phenotype result from spontaneous sex reversion (Nagler et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004). In the present study both the GH-Y and OtY1 tests gave the same genotypic sex for fish identified with aberrant phenotypic sex, so it is unlikely that the inconsistency between genotype and phenotype observed is the result of a chromosomal crossing over event. A lack of sex specificity of the GH-Y and OtY1 region in the New Zealand chinook salmon would also have been expected, even if crossing over occurred at a low rate. The seven phenotypically male fish that are genetically female may have arisen due to environmental influence. This could be due to a temperature effect, but there is no evidence of temperature-induced sex determination in chinook salmon (Nagler et al., 2003). It is well known, however, that exposure to oestrogenic steroids during the labile period of embryonic development can alter the phenotype of male salmonids to female fishes (Goetz et al., 1979; Donaldson & Hunter, 1982; Hunter et al., 1986) with oestrogens and xeno-oestrogens postulated as a reason for sex reversal in North American chinook populations (Nagler et al., 2001; Williamson & May, 2002; Chowen & Nagler, 2004). Most effluents primarily seem to have oestrogenic activity, although masculinization by androgens (Piferrer et al., 1993) and PCBs is known to occur (Matta et al., 1998). The fish in the current study were hatchery-reared fish, from Silverstream Hatchery, resident beside the spring-fed Silverstream stream. No data exists on the presence of oestrogenic or androgenic compounds in this waterway. In comparison, with the North American waterways such as the Columbia and Sacramento Rivers, Silverstream and the bore water supplying the hatchery would be considered relatively pristine. Thus, the low level of masculinization present in the New Zealand fish is unlikely to be the result of environmental androgens present in the water. If the high incidence of apparent feminization found in North American populations is the result of environmental factors like pollutants, then the finding may provide a useful baseline for comparison. New Zealand chinook salmon are believed to have originated from fish resident in Battle Creek, a tributary of the Sacramento River (McDowall, 1994). Recently, it was shown that 35% of phenotypic female chinook from Battle Creek possessed a male genotype based on the OtY1 sex test (Williamson & May, 2002). It remains unknown, however, if this observed level of discordance between phenotypic and genotypic sex is abnormal. The present study showed that none of the New Zealand female chinook salmon surveyed had OtY1 or GH-Y indicating absolute concordance to their genotype, but 1
5% of phenotypic males lacked OtY1 and GH-Y. Given the common genetic origin of these two populations of chinook salmon, the very low frequency of sex-reversed fish (0
8%) in New Zealand may reflect the natural level of spontaneous sex reversal within chinook salmon prior to significant environmental perturbation. We are grateful to K. McBride for assistance in extracting DNA from fin clips for use in this study, to L. Pagarigan for assistance with OtY PCRs, to R. Dungan and M. Griffin for assistance with statistical analyses, to Silverstream Salmon Farm (NIWA), especially S. Hawke and M. Unwin, for supplying the facilities and fish and # 2006 The Fisheries Society of the British Isles, Journal of Fish Biology 2006, 68 (Supplement A), 136–143 142 V. J. METCALF AND N. J. GEMMELL N. Boustead (NIWA) for assistance with the tagging procedure as well as K. Williamson and anonymous reviewers for useful comments on the manuscript. References Azuma, T., Takeda, K., Doi, T., Muto, K., Akutsu, M., Sawada, M. & Adachi, S. (2004). The influence of temperature on sex determination in sockeye salmon Oncorhynchus nerka. Aquaculture 234, 461–473. Baker, I. 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