Visual sex discrimination in goldfish: seasonal, sexual, and androgenic influences

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1 Hormones and Behavior 46 (2004) Visual sex discrimination in goldfish: seasonal, sexual, and androgenic influences R.R. Thompson*, K. George, J. Dempsey, J.C. Walton Psychology Department/Neuroscience Program, Bowdoin College, Brunswick, ME 04011, United States Received 27 April 2004; revised 24 June 2004; accepted 28 June 2004 Available online 11 September 2004 Abstract The olfactory signals used by goldfish for sexual and aggressive communication have been studied extensively, but little work has addressed the role of other sensory modalities in social communication in this species. We therefore investigated the role that visual stimuli play in sex discrimination and the ability of androgens, which masculinize courtship behavior, to affect behavioral responses toward female visual stimuli. We found that males selectively orient toward female visual stimuli during the breeding season but not outside it, whereas prostaglandin F2-alpha (PGF 2a )-injected females do not differentially approach male and female visual stimuli, even during the breeding season. Implanting adult females with testosterone (T) and 11-ketotestosterone (KT), however, induced orientation responses toward female visual stimuli similar to those observed in males. These results indicate that visual sexual stimuli are likely important for reproductive signaling in goldfish, potentially helping males identify ovulating females from a distance in a shoal of fish, and that androgens can influence mechanisms associated with orientation responses toward such stimuli. D 2004 Elsevier Inc. All rights reserved. Keywords: Teleost; Sex; Discrimination; Visual; Testosterone; Ketotestosterone Introduction Mechanisms that help animals make accurate determinations of the sex and reproductive state of conspecifics allow them to minimize energy expenditure on unfruitful and potentially dangerous mating attempts. Selective pressures on this ability have led to the evolution of sensory systems that are often sensitive to the particular signals that convey such information, and orientation responses towards those stimuli are often modulated by internal factors that are involved in the control of reproductive processes. Sex steroids, in particular, may affect the ability to detect sexual stimuli and/or sensorimotor mechanisms that orient animals toward those stimuli, as well as the motivation to approach them. Influences on any * Corresponding author. Psychology Department, Banister Hall, Bowdoin College, Brunswick, ME Fax: address: rthompso@bowdoin.edu (R.R. Thompson). or all of these processes could underlie hormonal influences on complex behaviors like the formation of sexual partner preferences. Most of the research on the effects of sex-steroids on mechanisms associated with the detection of and/or orientation towards potential sexual partners has focused on hormonal effects on responses to olfactory sexual stimuli, including work in rodents (Bakker, 2003; Kelliher and Baum, 2001, 2002; Woodley and Baum, 2003), amphibians (Thompson and Moore, 2003), and fish (Cardwell and Stacey, 1995; Murphy and Stacey, 2002). Notable exceptions include studies showing that androgens influence the tuning characteristics of electrosensory afferent neurons in stingrays, making fish with high androgens (typically males) more sensitive to electric signals produced by females (Sisneros and Tricas, 2000), and work in leopard frogs showing that estrogens enhance midbrain auditory sensitivity for frequencies at which males call (Yovanof and Feng, 1983). Estrogens have also been shown to influence pairbonding preferences that are not thought to involve X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi: /j.yhbeh

2 R.R. Thompson et al. / Hormones and Behavior 46 (2004) olfactory processes in zebra finches, in which brief exposure to estrogens soon after hatching induces some females to pair with other females when they reach sexual maturity (Adkins-Regan et al., 1997). In goldfish, as in many other vertebrates, pheromones are important for sexual communication, as initially demonstrated by studies showing that males orient towards the water collected from ovulating females in a Y maze (Partridge et al., 1976). Such female-scented water contains at least two major classes of chemical components that act as pheromones. Females release 17,20 betadihydroxy-4-pregnen-3-one (17,20 BP) and sulfonated conjugates the night before they ovulate and prostaglandins at the time of ovulation (Scott and Sorensen, 1994). Both classes of chemical affect male physiology and behavior differently: 17,20 BP is sufficient to induce elevations of testosterone (T) and milt and produces small but longlasting increases in courtship-related behaviors; whereas prostaglandins, primarily prostaglandin F2-alpha (PGF 2a ), do not affect endocrinological parameters in isolated males but do induce rapid and robust increases in courtshiprelated behaviors (Poling et al., 2001; Sorensen et al., 1989). While the importance of olfactory signals for sexual communication in goldfish is thus well established, the potential role that other sensory modalities play in intersexual signaling in this species has not been as extensively studied. Although courtship is greatly reduced in males by cutting the olfactory tract, it is not completely eliminated (Stacey and Kyle, 1983), and such males follow ovulating females more than they do nonovulating females (Partridge et al., 1976). Thus, while pheromones play an important, perhaps dominant role in eliciting male courtship, other nonolfactory cues are also involved. The presence of androgen receptors and/or high levels of aromatase in regions of the brain involved in the detection of (retina) and orientation towards (optic tectum) visual stimuli suggest that androgens might influence visual processes (Callard et al., 1995; Gelinas and Callard, 1993, 1997) and that visual cues might be important for some component of sexual communication in this species. Goldfish typically aggregate in small groups, or shoals (Kavaliers, 1989; Magguran and Pitcher, 1983), and we have observed that males and females will approach the visual stimuli of conspecifics, in and out of the breeding season (unpublished data). However, we do not yet know whether they can make sexual discriminations using only visual cues, and (if so) if they can use such information to orient towards potential mates during the breeding season. We therefore tested whether or not goldfish prefer to approach opposite sexed individuals when only visual stimuli are available, whether such responses are seasonal and/or sexually dimorphic, and whether or not androgens, which masculinize courtship behavior in this species, can stimulate visually guided responses toward females. Methods General Reproductively mature goldfish (Carassius auratus) were purchased from Hunting Creek Fisheries (Maryland). All fish were cm in length and weighed between 25 and 60 g at the time of testing. Fish were separated by sex and housed in 90-gallon aquaria with varying light and temperature conditions according to the specific experiment. The animals were fed commercial goldfish pellets (Wardleys) once daily. Experimental testing tanks had three separate sections, each divided by acrylic, nonfiltering Plexiglas partitions that allow ultraviolet transmission (Polymershapes, Chicago, IL). The middle, experimental section was 70 l and the two side compartments used to hold stimulus fish were 18 l each. The middle compartment contained floating green yarn for spawning substrate on tests when females were added to the middle tank after visual testing was complete (Experiment 1). Animals were able to see between sections, but the Plexiglas partitions were sealed to prevent water and chemical exchange. Dual, full spectrum light bulbs (Reptisun 5.0, Zoomed, CA) were hung approximately 10 cm above the water in each tank and ran their length. All experiments were recorded with a black and white video camera. No observers were in the experimental room during behavioral testing. Experiment 1 Male goldfish were tested at two times of year to see if they make visual sexual discriminations, and if so to determine if such discriminations are seasonally dependent. The first part of this experiment was performed in April, when the goldfish were in reproductive condition, evidenced by the presence of tubercles and expressible milt. Six male goldfish were kept in warm water (208C) on a long light cycle (16:8 h light dark), and stimulus female fish were kept in cold water (168C) until 2 days before each test. In order to stimulate ovulation, stimulus female fish were placed into buckets of warm water 48 h before testing (Stacey and Liley, 1974). Female stimulus fish were also given 10 Al intramuscular injections of Lutalyse (1 Ag/Al PGF 2, Butler Inc., Guilderland, NY) 10 min before testing, a treatment known to induce receptivity (Kobayashi and Stacey, 1993), pheromone release (Sorensen et al., 1988), and potentially visual attractiveness too. At 8:30 a.m., before the lights came on, each male fish was moved from the group holding aquaria into the middle compartment of an individual testing tank. Full spectrum lights were then turned on, and the fish were left alone for a 30-min habituation period. A 30-min pretest followed the habituation period. One female stimulus fish was then placed in one side compartment of each tank, and one male stimulus fish was placed in the other side compart-

3 648 R.R. Thompson et al. / Hormones and Behavior 46 (2004) ment of each tank. The placement of male and female stimulus fish on different sides was counterbalanced. A 30- min test phase took place once male and female stimulus fish were introduced. A single observer who did not know which stimulus fish was male and which was female (secondary sexual characteristics could not be discerned on the videotapes) recorded the time each experimental fish spent with its nose within 1 cm of each divider during the 30-min pretest and then the time it spent in proximity to each divider during the 30 min after the stimulus fish were introduced. The corrected proximity scores were calculated by subtracting the time spent in proximity to the respective dividers during the pretest period from the time spent in proximity to them when the stimulus fish were present. At the end of the visual tests, the stimulus females were placed in the experimental tanks with the males for 30 min to see if courtship (following, nudging, and spawning) occurred. The second part of Experiment 1 was performed in November, when goldfish were not in reproductive condition. Males lacked tubercles and expressible milt and females did not have expressible eggs, as determined by noting whether or not eggs were present in the nets when they were handled or if they could be expressed by gently pressing the abdomen. All fish were kept in cold water (168C) on a 12:12-h light dark cycle to simulate nonmating conditions. A total of eight male fish were tested in the same behavioral paradigm described above, the only difference being that female stimulus fish were not placed into buckets of warm water 48 h before testing to induce ovulation because the fish would not have been vitellogenic at this time of year. Experiment 2 To test if males and females make visual sexual discriminations during the breeding season, male and female goldfish were tested in April in the same behavioral paradigm as described above. Nine males and nine females were tested. The procedures for this experiment differed from those in Experiment 1 in that all fish were injected 16 h before testing with 25 Al Ovaprim, a commercially available GnRH agonist and dopamine antagonist cocktail that induces final seasonal maturation in male and female teleosts (Syndel, Inc., Vancouver, BC). We used this treatment because our tests were conducted early in the breeding season when fish may not have otherwise undergone the final stages of seasonal reproductive maturation. Also, fish in this experiment were only given a 10-min habituation and 15-min pretest and test periods. Stimulus females were injected with the same dose of Ovaprim before being placed in the buckets of warm water 48 h before testing. Experimental females were injected with 10 Al Lutalyse (1 Ag/Al PGF 2a ) immediately before being placed in the tank and thus 25 min before behavioral testing to induce sexual receptivity (Kobayashi and Stacey, 1993). A single observer that did not know the sex of the experimental or stimulus fish recorded the data. Corrected proximity scores were again calculated by subtracting the time in proximity to the dividers during the pretest period from the time spent in proximity to them during the test period when the stimulus fish were present. The presence of tubercles and expressible milt was verified in all experimental and stimulus males in this experiment by gently pressing on the abdomen, as was the presence of expressible eggs in experimental and stimulus females. Eggs were typically present in the net after routine handling or released after only light pressure to the abdomen. No tests of courtship were performed after the visual tests in this study. Experiment 3 To test if androgens can induce male-typical behaviors associated with visual sexual discrimination, female goldfish were implanted with 2 Silastic capsules (1.98 mm ID, 3.18 mm OD), one containing testosterone (T) and one containing 11-ketotestosterone (KT) dissolved in castor oil (androgen group), or with one empty capsule and one filled with castor oil (blank group). For the surgeries, which were performed in May, 11 months before behavioral testing, goldfish were lightly anesthetized by placing them into 0.1% ethyl-3-aminobenzoate methane sulfonate salt (MS- 222; Sigma) buffered with NaHCO 3, and a small incision was made in the abdomen into which the capsules were inserted. T and KT capsules were prepared according to the protocols of Stacey and Kobayashi (1996), who demonstrated that such implants masculinize courtship behavior in adult females tested 4.5 months after implantation and produce levels of circulating T and KT within the physiological range observed in control males. Briefly, T capsules were filled with approximately 10 mg T by tamping the T into the capsule, and KT capsules were filled with 70 Al solution containing approximately 400 Ag KT, made by first dissolving 5 mg KT in 100 Al ethanol, diluting into 900 Al castor oil, and then letting the ethanol evaporate off overnight before sealing the other end. Nine fish were implanted with androgens and nine with blank capsules, but two from each group died before behavioral testing. Behavioral tests were conducted in April and followed the same protocol described in Experiment 2 except that the experimental fish were not injected with Ovaprim. Because of the small sample size, we attempted to reduce variability due to observer error by averaging the proximity scores recorded by two observers, each of whom was blind to the experimental group of the implanted fish and the sex of the stimulus fish on each side. Immediately after the behavioral tests, the fish were anesthetized in 0.1% MS222. They were weighed and blood was drawn for hormone assays (see below), and the fish were then killed and the implants were recovered and visually inspected. The gonads were removed, weighed, and visually inspected. The presence of tubercles and expressible milt was verified in all stimulus

4 R.R. Thompson et al. / Hormones and Behavior 46 (2004) males and the presence of expressible eggs was verified in all stimulus females. Hormone assays Blood was collected from three males and three females in similar condition to the fish used for spring behavioral tests in Experiment 1; that is, samples were taken in early April from fish that had been housed in long daylight conditions (16:8 h light dark) for at least 3 weeks and that had tubercles and expressible milt (males) or eggs (females), though we did not specifically induce ovulation. Blood was also drawn in early April from four males and four females from the same group of fish but which were in the same condition as fish in Experiment 2; that is, they were injected with Ovaprim and placed in warm water (208C) 16 h before sampling. These fish all had tubercles or expressible milt (males) or expressible eggs (females). Blood was also taken from the seven blank and seven androgen-implanted fish used in Experiment 3. To draw blood, fish were anesthetized by immersing them in 0.1% MS222 and then inserting a 25-g heparinized syringe into the caudal vasculature. Approximately 500 Al of blood was withdrawn from each fish. The samples were centrifuged at 5000 g for 15 min and the plasma was removed and stored at 808C. Steroids were extracted with two diethyl ether separations, resuspended in an equal volume of buffer, and T and KT were measured with enzyme-linked immunoassay kits according to the protocols provided with the kits (Cayman Chemical, Michigan) at 1:20 and 1:100 dilutions. The T antibody provided with these kits has a 2.2% cross-reactivity with KT and the KT antibody a 1.1% cross-reactivity with T. Samples were run in triplicate. All animals were treated according to protocols approved by the research oversight committee at Bowdoin College. females than to other males, though the effect was not significant (t = 1.7, df =5,P = 0.15; see Fig. 1A). The corrected proximity time spent near the female stimulus fish were also greater than the corrected proximity time spent near the male stimulus fish, though the effect was marginal (t = 2.53, df =5,P = 0.053; see Fig. 1B). Four of the six males displayed courtship behavior (following, nudging, and spawning) when the stimulus fish were placed in the tank with them. Males actually spent more time near male visual stimuli than near female visual stimuli in tests performed outside of the breeding season, though the difference was not significant (t = 0.39, df =7,P = 0.71), nor were the corrected proximity scores for the two types of stimulus fish different (t = 0.43, df =7,P = 0.68). None of the eight males tested displayed courtship behaviors when the stimulus females were added to the tanks after the visual tests at this time of year. In Experiment 2, reproductively mature male goldfish spent significantly more time in proximity to PGF 2a -injected gravid females (t = 3.93, df = 8, P = 0.004; see Fig. 2A), and the corrected proximity time spent near stimulus females was significantly greater than the corrected proximity time spent near stimulus males (t = 3.28, df =8,P = 0.01; see Fig. 2B). Intact females injected with PGF 2a did not spend significantly more time near males or females during the test (t = 0.5, df =6,P = 0.64), Statistics Within-subjects paired t tests were used to compare the time each experimental fish spent in proximity to the male and female stimulus fish during the test period as well as the corrected proximity times near the male and female stimulus fish. An alpha level P b 0.05, two tailed, was required for statistical significance. Because of extreme differences in variance across the groups, a nonparametric Kruskal Wallis one-way analysis of variance was performed to see if androgen levels differed across the groups, and follow-up, Mann Whitney U tests were used to compare steroid levels between individual groups if overall significance was found. Results Reproductively mature male goldfish in Experiment 1 spent more time in proximity to PGF 2a -injected gravid Fig. 1. Mean F SEM of the time spent by males tested during the breeding season and males tested outside of the breeding season in proximity to the partitions during the pretest, baseline period before stimulus fish were introduced and during the test period when a male stimulus fish was behind one partition and a female stimulus fish behind the other (A); the corrected time spent near each stimulus fish (B), which was the time spent in proximity to each partition during the test period minus the time spent in proximity to the same partition during the pretest.

5 650 R.R. Thompson et al. / Hormones and Behavior 46 (2004) Fig. 2. Mean F SEM of the time spent by males and females in reproductive condition in proximity to the partitions during the pretest, baseline period before stimulus fish were introduced and during the test period when a male stimulus fish was behind one partition and a female stimulus fish behind the other (A); the corrected time spent near each stimulus fish (B), which was the time spent in proximity to each partition during the test period minus the time spent in proximity to the same partition during the pretest (B). *Indicates P b nor were the corrected proximity scores for the stimulus types different (t = 0.12, df =6,P = 0.91). In Experiment 3, the scores recorded by the two observers differed, on average, by 6% of the total range of scores recorded, and the scores recorded by the two observers were highly correlated (R = 0.89, P b 0.001). Androgen-implanted females spent more time in proximity to stimulus females during the test period, though the effect was not significant (t = 2.2, df =6,P = 0.07; see Fig. 3A). However, the corrected time spent in proximity to stimulus females was significantly higher than the corrected time in proximity to stimulus males in the androgen-implanted group (t = 2.47, df =6,P = 0.049; see Fig. 3B). Blankimplanted females did not spend significantly more time near either stimulus type during the test (t = 1.2, df =6,P = 0.27), nor were the corrected proximity scores for the two types of stimulus fish different within this group (t = 1.39, df = 6, P = 0.22). Androgen-implanted females spent significantly more time near the stimulus females than did blank-implanted females (t = 2.31, df = 11, 9, P = 0.04). All androgen-implanted fish, but no blank-implanted fish, had tubercles at the time of sacrifice. Body weight did not differ between blank- and androgen-implanted females (63.5 F 5.2 g, mean F SEM, vs F 3.2, respectively), nor did gonadal weight (1.23 F 0.15 vs F 0.15 g) or the gonadosomatic index (0.02 F vs F 0.002). Five of the seven androgen-implanted females and all of the blank-implanted females had gonads that were clear or pink, though the gonads in the blank group were more often granular in appearance due to the presence of eggs. Gonads in one of the androgen-implanted fish were white, characteristic of testis development, and gonads in another were partially streaked with white. The time spent in proximity to stimulus females by these two fish were the first and sixth highest of the seven fish in the androgen-implanted group. The corrected difference scores (time in proximity to the female minus time in proximity to the male) for both fish were within two standard deviations of the difference scores for all fish in this experiment, indicating that neither was an outlier. Implants were recovered from all fish, and T could still be observed in capsules in all but one fish. KT could not be visualized in any implant because it was dissolved in a clear fluid. Three assays were run for each androgen, with each containing combinations of male and female samples or blank- and androgen-implanted female samples. The intraassay coefficients of variation (CV) for the T assays were 14.4%, 13.5%, and 18.7%, and intraassay CV s for the KT Fig. 3. Mean F SEM of the time spent by females implanted with T and 11- KT capsules and females implanted with blank capsules in proximity to the partitions during the pretest, baseline period before stimulus fish were introduced and during the test period when a male stimulus fish was behind one partition and a female stimulus fish behind the other (A); the corrected proximity time spent near each stimulus fish (B), which was the time spent in proximity to each partition during the test period minus the time spent in proximity to the same partition during the pretest (B). *Indicates P b 0.05.

6 R.R. Thompson et al. / Hormones and Behavior 46 (2004) assays were 14.5%, 9.7%, and 7.4%. The interassay CV for the T assays was 20% and for the KT assays 11%. Levels of T and KT were first compared between males injected with Ovaprim and those not injected and between females injected with Ovaprim and those not injected. No significant differences were found for either androgen in males or females, though it should be noted that Ovapriminjected and noninjected fish were run in different assays and sample sizes were very small for each subset, so it is possible that differences between these groups could have been masked by interassay variation. Data from Ovapriminjected males and noninjected males and from Ovapriminjected females and noninjected females were combined for all subsequent statistical analyses. For T, there was a significant overall effect of group (Kruskal Wallis test statistic = 21.5, P b 0.001; see Fig. 4A). Follow-up tests revealed that males had significantly higher levels of T than intact females (U = 56.0, P = 0.001), and androgenimplanted females had significantly higher levels of T than intact females (U = 8, P = 0.02) and blank-implanted females (U = 47.0, P = 0.004). Levels of T in males and androgen-implanted females did not differ significantly. KT levels were also significantly different across groups (Kruskal Wallis test statistic = 25.7, P b 0.001; see Fig. 4B). KT levels were significantly higher in males than in females (U = 49, P = 0.002), and KT levels were significantly higher in androgen-implanted females than Fig. 4. Mean F SEM levels of androgens in males, females, androgenimplanted females, and blank-implanted females, as determined by EIA. Bars with the same letters above them are not significantly different. in intact females (U = 49, P = 0.002) and blank-implanted females (U = 49, P = 0.001), in which KT levels were not detectable even at the lowest dilution used (each fish therefore received a score of 0.25 ng/ml, which was the limit of detection for the lowest dilution used, for the statistical analysis). KT levels were significantly higher in intact males than in androgen-implanted females (U = 49, P = 0.002). Discussion The present results demonstrate that male goldfish can make sexual discriminations during the breeding season using nonolfactory cues. Although sound production is important for sexual signaling in some teleosts, including at least one cyprinid (Johnston and Johnson, 2000), no vocal signaling systems have been reported in goldfish, and we have been unable to record vocalizations during courtship with a hydrophone. We therefore think it most likely that males used visual stimuli to make sexual discriminations in these experiments and that androgens stimulated approach responses toward female visual stimuli. Visual signals, as well as pheromones, are thus likely involved in sexual signaling in this species. The ability to use vision for sexual discrimination may help males locate potential mates more accurately from a distance, as identifying the source of a waterborne pheromone in a shoal of fish would require very close physical proximity, if not individual sampling after direct contact. We cannot yet say what visual features of females may be attractive to males during the breeding season. It is possible the distension of the abdomen due to the presence of eggs is an important cue, as it is in some amphibians (Verbell, 1986). Future tests will be able to address this possibility by determining if males orient towards ovulating females whose eggs are stripped immediately before the test. Additionally, all of our tests were done in full spectrum lighting with nonfiltering Plexiglas partitions that allowed UV transmission, so it is possible that ovulating females reflect a pattern of ultraviolet radiation that is attractive to males. Goldfish can discriminate UV wavelengths (Bowmaker et al., 1991; Neumeyer and Arnold, 1989), and UV patterns are used for mate assessment in guppies, Poecilia reticulata, another teleost (Kodric-Brown and Johnson, 2002; Smith et al., 2002), though UV assessments are more strongly associated with female mate choice in that species. Similarly, UV patterns are used for mate assessment in birds (Bennett et al., 1997; Pearn et al., 2001) and lizards (LeBas and Marshall, 2000). It is also possible that ovulating females display proceptive behaviors around the time of ovulation that are visually attractive to males, as do female rats in estrus (reviewed in Erskine, 1989). Partridge et al. (1976) did report that ovulating females solicit courtship from males, but the proceptive behaviors they described involved direct contact

7 652 R.R. Thompson et al. / Hormones and Behavior 46 (2004) (nudging), which was not possible in our test paradigm. However, it remains possible that ovulating females display other behavior patterns that males can observe from a distance. It is even possible that ovulating females are simply more active and that males and androgenimplanted females are attracted to movement. Unfortunately, we were unable to quantify the behavioral activity of the stimulus fish from the videotapes and so cannot assess this possibility within the present data set. That males only made sexual discriminations during the breeding season indicates either that males lose the ability to detect and/or the motivation to approach female visual stimuli outside of the breeding season, that the attractive visual features of our stimulus females were not present outside of the breeding season, or both. Female stimulus fish were treated with PGF 2a in tests conducted in November, which should have stimulated courtship if the males were responsive to female sexual stimuli, as PGF 2a administration can induce receptivity in females independently of the ovaries and/or sex steroids (Kobayashi and Stacey, 1993) and induce attractivity by stimulating pheromone release (Sorensen et al., 1988). In fact, males in reproductive condition will court naturally ovulated females and PGF 2a - injected females equally (Stacey, 1981), and yet males did not court PGF 2a females in November when direct contact was allowed after the completion of the visual tests. Thus, males appear unresponsive to female sexual stimuli, at least to those associated with PGF 2a -injected females, outside of the breeding season. This is consistent with previous findings showing that males that are not producing milt are not attracted to female pheromone signals (Partridge et al., 1976). It is possible that males tested in the fall would have responded more strongly to the visual stimulus of a naturally ovulating female than that of a PGF 2a -injected female, as we have observed that PGF 2a does not make spawned-out females visually attractive to males in late summer even though males will still court them when direct contact is allowed (unpublished data), suggesting the presence of eggs may be critical for female visual attractiveness. However, we think it unlikely that visual responsiveness to female sexual stimuli would be maintained at a time of year when responsiveness to sexual stimuli processed in other sensory modalities, most notably pheromones, is not. Females did not exhibit orientation or approach responses towards stimulus males even when injected with PGF 2a, a treatment that induces female receptivity (Kobayashi and Stacey, 1993; Stacey and Liley, 1974). Partridge et al. (1976) have noted that females actively solicit male courtship, primarily by nudging them, though the male stimuli that elicit solicitations have not been identified. Our data suggest that visual stimuli may not play an important role, though it is possible that our test paradigm was inadequate to assess female proceptive behaviors associated with potential visual sex discrimination processes. Originally, we had planned to test the effects of shortterm androgen treatment on visual sexual discrimination responses in the summer immediately following the steroid implantation. However, all of our stimulus females were spawned out by the time we were ready to run the first round of tests, and as discussed above, we observed that males court spawned-out females injected with PGF 2a in late summer but do not orient towards them in our visual sex discrimination paradigm (unpublished data). We therefore waited until the following spring to test the implanted fish so we could use naturally ovulating females as visual stimuli. Though our androgen implants were thus in place for an extended time period, tubercles, an androgendependent secondary sexual characteristic, were present on all androgen-implanted fish, and our steroid hormone assays confirmed that elevated androgen levels were present at the time of testing. Thus, circulating androgens could have induced the behavioral masculinization of approach responses toward female visual stimuli directly. Alternatively, previous studies have shown that long-term androgen treatment can cause testicular development in adult female goldfish (Kobayashi et al., 1991), raising the possibility that the behavioral effects of androgens in our study could have been a secondary result of gonadal masculinization and the subsequent secretion of other hormones or factors. However, despite the exceptionally long period of hormone exposure in our experiment, most gonads in the androgenimplanted fish were clear or pink, as they were in control fish. We only noted two androgen-implanted females with testicular development. Though one of those fish spent the highest amount of time in proximity to a stimulus female relative to a stimulus male, the other spent less time near a stimulus female relative to a stimulus male than all but one other androgen-implanted fish, which argues that such an indirect mechanism was not likely responsible for the effects of the androgen implants on approach responses toward female visual stimuli. Long-term androgen treatments in adult females have already been shown to stimulate male-typical courtship behavior (Stacey and Kobayashi, 1996), thus demonstrating that goldfish, like many other teleosts, retain plasticity as adults in the brain systems that control sexual behavior. However, the mechanism(s) through which androgens produce such masculinizing effects in adult fish is not yet fully understood. Though androgens are likely to produce such effects in goldfish, at least in part, by stimulating responses toward female sex pheromones, as they do in crucian carp, Carassius carassius (Kobayashi and Nakanishi, 1999), the present results also suggest that androgenic influences on behavioral responses to visual sexual stimuli may be involved in the masculinization process, at least to the extent to which visual sex discrimination processes play a role in orienting maletypical courtship behavior. Androgens could influence the ability to detect some visual stimulus feature of females, sensorimotor mechanisms that control orienting responses

8 R.R. Thompson et al. / Hormones and Behavior 46 (2004) toward female visual features, and/or motivational processes involved in the regulation of approach responses toward female visual stimuli. Such effects could be mediated via androgen actions in regions of the goldfish brain that specifically process visual information and where there are androgen receptors and/or aromatase, most notably the retina and optic tectum (Callard et al., 1995; Gelinas and Callard, 1993, 1997), and/or via actions in androgen sensitive brain regions that have been shown to influence male courtship, including the ventral telencephalon and preoptic area (Kyle and Peter, 1982). In our hormone implant experiment, females were implanted with T and KT, so we cannot determine the unique effects of each hormone on orientation responses toward female visual stimuli. T and KT levels were higher in males than in females and higher in androgen-implanted females than in intact and blank-implanted females, so either or both androgens could be important for regulating orientation responses towards female visual stimuli in intact, reproductively mature males and for the masculinizing effects of androgen treatment on this behavior in adult females. Although the levels of KT produced by our implants were below those observed in intact males in reproductive condition (present study, Kobayashi et al., 1986), they were similar to the levels shown to be sufficient to stimulate male-typical courtship in adult females 6 months after KT implantation in a previous study by Stacey and Kobayashi (1996). It is therefore possible that the small elevations of KT observed in androgen-implanted females in the present study likewise masculinized visual sex-discrimination processes. However, it also remains possible that T, alone or in combination with KT, influences approach responses to female visual stimuli, as aromatase, which can convert T but not KT into estrogenic metabolites, is particularly high in visually sensitive brain regions in goldfish, including the retina and optic tectum (Callard et al., 1995; Gelinas and Callard, 1993, 1997). Interestingly, pheromones released by preovulatory females induce a T surge in males that peaks around the time of spawning, but they do not affect levels of circulating KT (Kobayashi et al., 1986). If estrogenic metabolites from T do sensitize visual processing systems involved in the detection of and/or orientation towards females, and if they do so rapidly, then pheromone exposure the night before spawning could enhance the ability of males to visually locate potential mates in the early morning, dim light conditions when spawning normally occurs (Stacey et al., 1979). In conclusion, we have shown that male goldfish can make sexual discriminations using visual cues during the breeding season and that circulating androgens can masculinize behavioral responses toward female visual stimuli. Future work will seek to determine the female stimulus features important for such discriminations, the mechanisms through which androgens masculinize such responses, and whether or not the olfactory and visual systems influence one another in the context of normal mating behavior. Acknowledgments We wish to thank Elizabeth Adkins-Regan and Peter Sorensen for helpful comments on an earlier version of this manuscript. This work was supported by a grant from the National Science Foundation (SGER # , supplement # ). References Adkins-Regan, E., Mansukhani, V., Thompson, R., Yang, S., Organizational actions of sex hormones on sexual partner preference. Brain Res. 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