THE EFFECT OF MALE FAMILIARITY ON PROXIMITY TIME IN FEMALE EASTERN MOSQUITOFISH (Gambusia holbrookl)

The Psychological Record, 2001, 51, 237-250 THE EFFECT OF MALE FAMILIARITY ON PROXIMITY TIME IN FEMALE EASTERN MOSQUITOFISH (Gambusia holbrookl) MARY E. MCLAUGHLIN and KATHERINE E. BRUCE University of North Carolina at Wilmington Among live-bearing fish, females often prefer larger or more colorful males, or males engaging in ritualized courtship. However, in eastern mosquitofish, Gambusia holbrooki, the factors affecting females' choice of males are unclear. We tested whether female Gambusia prefer to spend time near unfamiliar males over males with whom they have prior experience, expecting that females would prefer unfamiliar males. We examined both visual/olfactory and full exposure experience with males. As a control, we tested females with pairs of unfamiliar males. Control females showed no preferences between males. Females in the visual/olfactory experiment spent more time with males in general following 30- minute exposure, compared to 24-hour exposure. In the f.ull exposure experiment, females spent more time with unfamiliar males only after 24 hours of previous exposure to the familiar male. We discuss the data in the context of mate choice, familiarity, female deprivation, and sexual satiation. In female mate choice, females prefer characteristics of certain males over others and males who exhibit a preferred characteristic will have a reproductive advantage over those who do not. Because female live-bearing fish show considerable parental investment, intersexual selection probably influences their mate selection. In fact, intersexual selection has been documented in a variety of Poeciliid fish, including mosquitofish, Gambusia spp.; guppies, Poecilia reticulata; swordtails, Xiphophorus spp.; and mollies, p. shenops. Typically such research is conducted using indirect measures of mate choice-that is, measuring the time females spend in proximity to males with particular characteristics (see Houde, 1997, for discussion of methods for testing mate choice). This is done primarily so that female choice can be distinguished from the male's behavior. Choice tests mayor may not be followed by actual mating tests, but typically correlate with male reproductive success when it can be directly measured. This research was conducted in partial fulfillment of the requirements for the M.A. degree at UNC-Wilmington by Mary E. McLaughlin. We appreciate the assistance of Brandy, Burros, Christy Jordan, and Shana Wadsworth with data collection. We also appreciate helpful comments from D. Kim Sawrey and William Overman on an earlier version of the manuscript. Please address all correspondence concerning this manuscript to Katherine Bruce, Department of Psychology, UNC-Wilmington, Wilmington, NC 28403. (E-mail may be sent to bruce@uncwil.edu).

238 MCLAUGHLIN AND BRUCE A series of studies by Houde and colleagues (Houde, 1988; Long & Houde, 1989) has confirmed that the degree of orange pigment in male guppies influences female choice. In addition, Bischoff, Gould, and Rubenstein (1985) found that female guppies, when presented largetailed and short-tailed males, spent more time with large-tailed males. Basolo (1990) reported that female swordtails, X. helleri, spent a greater amount of time with males who had long swords than those with short swords, and that the time increased with the length of the sword. The Poeciliid fish used in the present study were Gambusia holbrooki, or eastern mosquitofish. They are found primarily in the southeastern United States, but have been introduced all over the world (Courtenay, 1984). Adult females average 30 mm in length and can be identified by an egg spot on the lower abdomen that darkens and swells when the female is pregnant. Adult males average 25 mm in length and can be identified by a gonopodium, a modified anal fin that acts as an intromittive organ. Mating occurs when the male swims up to the female from behind, swings his gonopodium down and onto the female's egg spot, and delivers sperm. Both sexes are grayish in color and neither seems to have any striking coloration or ornamentation. An exception is the presence of melanism in a small percentage «3%) of the male Gambusia population which is dark and mottled in appearance and easily distinguished from normally pigmented males (Martin, 1986). Intersexual selection has been studied in Gambusia, including whether females prefer males based on size, ornamentation, or courtship display. Bisazza and Marin (1991) found no female preference for large or small males in groups of adult females that had been (a) housed with males, (b) male deprived for 30 days, (c) male deprived for 4 months, or (d) were virgin. Across female groups, females showed no preference for spending time with either large or small males during dichotomous choice tests when males differed in size by at least 5 mm. Contrary to these findings, McPeek (1992) noted that large females and large males tended to associate in the field and that females appeared to prefer larger males in laboratory tests. Melanism has been studied in relation to intersexual selection and the results have been somewhat ambiguous. Martin (1986) found no female preference for melanistic males, and Taylor, Burt, Hammond, and Relyea (1996) found a preference for normally pigmented over melanistic males. This indicates that melanism is maintained in the Gambusia population through some mechanism other than intersexual selection, possibly dominance via larger size, given that melanistic males tend to be larger than normally pigmented males (Martin, 1986). Eastern mosquitofish, unlike western mosquitofish G. affinis, do not display courtship behavior (Bisazza, 1993). Mating is accomplished solely by a gonopodial thrust, in which the male approaches the female, extends his gonopodium, and delivers sperm. No female cooperation is required for this type of mating. Thus, it does not appear that courtship can influence intersexual selection in eastern mosquitofish.

MALE FAMILIARITY AND PROXIMITY TIME 239 The present study was designed to extend the research on female mate choice in Gambusia to determine if novelty or unfamiliarity with males will influence female mate choice. The relative rarity or novelty of a male may be preferred by females, as in frequency-dependent selection (Dewsbury, 1978). In addition, pregnant female rodents may respond to the presence (or odor alone) of an unfamiliar adult male by reabsorbing fetal material and going into estrus (Bruce, 1966). Further, although the Coolidge Effect (resumption of sexual behavior by a previously sexually satiated animal who is presented with a novel partner) is most often noted for males, females may show changes in receptive behavior when presented with an unfamiliar or unmated male (see Dewsbury, 1981 for a review). Studies of female guppies' sexual behavior or mating preferences in the presence of other fish provide evidence that females can distinguish males based on past interactions. Farr (1977) established small groups of guppies (Le., 1 male and 2 females, or 2 males and 3 females) for 24 to 72 hr. He then introduced a new male into each group and recorded the sexual behavior of the group members. Females increased their sexual responses to the new male, compared to resident males, and the new male was more sucqessful at achieving copulation than resident males. Using a different design, Bruce and White (1995) found dissimilar results. When a new male was introduced into a mixed-sex group of guppies that had acclimated for 7 to 10 days, the aggression aimed towards the new male by resident males may have suppressed the new male's sexual displays and chance at copulation. The authors did find, however, that females showed less aggressive behavior towards new males, as measured by nipping, than to resident males, indicating the ability to distinguish new from resident males. These studies indicate that prior tactile, social, or sexual exposure to the opposite sex may influence sexual selection in fish. We tested whether such prior exposure influences the preferences of female Gambusia for familiar or unfamiliar males. We planned to study three types of familiarity: visual exposure alone, visual and olfactory exposure, and full exposure (visual, olfactory, and tactile). Olfactory cues were investigated because they may be important to Poeciliids when choosing mates (Crapon de Caprona & Ryan, 1990; Crow & Liley, 1978). Crapon de Caprona and Ryan found that female swordtails spent more time on the side of the test tank filled with water that had been inhabited by a male swordtail than on the side filled with uncontaminated water, indicating that the female recognized and preferred olfactory cues given off by the male. Further, Crow and Liley (1978) found that male guppies spent more time in water that had held intact females over water that had held ovariectomized females, males, or control water. Crow and Liley suggested that females may excrete some type of chemical substance, under ovarian control, that the males prefer. It seems reasonable that, if female swordtails can distinguish the olfactory cues of other fish and exhibit mating preferences based on this distinction, and male guppies can distinguish odor cues of female guppies, female Gambusia may

240 MCLAUGHLIN AND BRUCE make a similar distinction and may exhibit a preference for males they are chemically familiar or unfamiliar with. If unfamiliarity or novelty of mates confers a reproductive advantage to females, we expected female Gambusia to spend more time with males with whom they have had no previous contact. We also expected that females would not show a preference for two similar-looking, unfamiliar males. For all experiments, main effects of familiarity were expected, such that females would prefer unfamiliar males over familiar males. No main effects for exposure time (e.g., 30 min, 24 hr) were expected. Interactions between familiarity and exposure time were expected, such that the female preference for unfamiliar males would depend on how long the females were exposed to the familiar male. We expected that longer familiar male exposure times would generate longer times spent in proximity to the unfamiliar male compared to shorter male exposure times. We also expected females would spend different amounts of time with males with whom they had visual and olfactory experience only compared to males with whom they had full exposure experience. As the type of exposure increases, for example, from visual and olfactory to full exposure, the number of cues available to the female for discrimination increases. In addition, during full exposure, the chance of prior mating experience increases, and such experience may influence female choice. Method Subjects Subjects for this study were 233 sexually mature female Gambusia ho/brooki at various stages of gestation. We chose to randomize rather than control for gestation, for example, use only virgin females, because females may be more or less receptive to sexual encounters at different stages of pregnancy and using females at different stages more closely mirrors naturally occurring choice conditions (Bisazza & Marin, 1991). Fish were collected every 2 to 4 weeks from small freshwater pools found in Wilmington and Carolina Beach in southeastern North Carolina. Females were naive to experimentation prior to testing. Twenty-one pairs of males were collected from similar sources at the NC State Agricultural Research Station and on the campus of UNC-Wilmington, with no stimulus male collected from the same pool of water as a test female. Methods of subject collection and treatment were approved by the university's Institutional Animal Care and Use Committee. Apparatus Females were initially housed with males in a 55-gallon community tank for at least 1 week. At least 24 hr prior to testing, females were moved to a 10-gallon all-female tank. All tanks contained gravel, a heater, thermometer, and aeration filters. The community tank included artificial plants. A 10-gallon exposure tank was used to familiarize females with

MALE FAMILIARITY AND PROXIMITY TIME 241 males. In the visual/olfactory experiment, this exposure tank was divided into two halves, separated by a perforated Plexiglas partition to allow the flow of water between sides of the tank. In the full exposure experiment, no partition was used and females swam freely with males. (Because of the results of the visual/olfactory experiment, a visual alone experiment was not conducted.) During testing, fish were restricted to the front half of a 10-gallon aquarium using a Plexiglas partition. The front half of the tank was visually divided into thirds to distinguish right, center, and left sides. Three-hundred-ml glass beakers (8 cm diameter, water level to 9 cm) were used to hold the males during testing. To minimize outside distractions, black plastic was placed on the outside of each side of exposure and test tanks except the side used for observation. Both exposure and test tanks remained covered and a fluorescent light illuminated the tanks during exposure and testing. All aquaria were maintained at 34-38 C with a ph of 6.6-7.0, and lights were set on a 12:12-hr photoperiod (light onset at 6 a.m. EST). Fish were fed commercial flake food (Tetramin ) in the community, all-female, and 24- and 18-hr exposure tanks only. A lap-top computer with an event recorder program was used to record the time female fish spent in proximity. Procedure Each week, females were removed from the community tank at least 24 hr prior to testing and placed in an all-female tank. Two males of similar size, defined as no more than 3 mm smaller or larger than one another, were removed from the stimulus tank and isolated in a separate, partitioned tank. The males appeared similar in coloration, body mass, and health when visually inspected. The control tests were run to determine whether or not female fish would treat two similar-looking males differently. The visual/olfactory experiment was conducted first. After at least 24 hr of male deprivation, 3 of the females were placed in the front of the exposure tank for another 24 hr. A stimulus male was placed in the other side of the exposure tank, in full view of the resident females, but behind the partition. In Condition 1, females were exposed for 30 min (± 5 min) of the 24 hr, immediately prior to the choice test. After exposure, the now ''familiar'' male was removed from the exposure tank and placed in a beaker. One female was moved to the test tank and allowed to habituate for 5 min. During habituation, empty stimulus jars were placed on either side of the tank where the males were later placed, and the lighted hood was in place. The second male of similar size was placed in a second beaker and was designated as the "unfamiliar" male. Following habituation, the beaker containing the familiar male was clipped in place on one side of the test tank and the beaker containing the unfamiliar male was clipped in place on the other. The female was then given a choice test for 10 min (600 s) and the amount of time the female spent in proximity to either male, defined as within one fish length of the beaker,

242 MCLAUGHLIN AND BRUCE was recorded as the dependent variable. Reliability tests conducted on all pairs of observers indicated that there was greater than 90% agreement in scoring this dependent variable. A test was considered valid if the test female swam within proximity to both males at least one time. Data from 9 females that did not swim in proximity to 1 or both males were excluded from data analysis. Data from 2 females that appeared to have ick spots that were noticed during the test were also excluded. After the choice test, the female was removed, measured, and placed in a holding tank. The beakers holding the males were removed and replaced with empty beakers. A 2nd female was moved from the exposure tank to the test tank and allowed to habituate. The side position of the familiar male was alternated throughout the experiment to avoid a side preference confound. The 3rd female was tested as above. At the end of the testing day, males were returned to their home tank, 3 new test females were moved from the isolation tank to the exposure tank, and all fish were fed. The next test day, 3 more females were familiarized with a male and then given a choice test. The identity of the male, that is, whether he was familiar or unfamiliar, was altemated each test day. When 12 females had been tested in the 30-min condition, we ran a replication with new females and a new pair of males. The same 2 males were used for all females in a replicate. Two replicates were then tested at a 24-hr (± 30 min) exposure time. Data from replicates of the same condition were combined for data analysis. In the full exposure experiment, the same procedure was followed, but no partition was used in the exposure tank. Females were exposed to males for 30 min, 3 hr, 18 hr, or 24-hr. All testing was done during the light period. The fish in the 18-hr exposure condition were tested in the morning (9:30-11 :30 a.m.) for logistical reasons. All other fish were tested in the afternoon (between 1 and 5 p.m.). After these studies were completed, we investigated the possibility that the female fish were unable to distinguish males because they lacked sufficient cues during the choice test. We designed square perforated clear Plexiglas containers to hold the stimulus animals (8 x 8 x 11 cm 3, filled to 9 cm). These containers replaced the glass containers used; all other procedures were similar to those described above. Two mixed factorial analyses were used for the visual/olfactory and full exposure experiments, 2 x 2 and 2 x 4 designs, respectively. Familiarity (unfamiliar vs. familiar) was the within-subjects independent variable and exposure time (e.g., 30 min, 24 hr) was the betweensubjects independent variable. Dependent t tests were used to analyze the control condition in which females were tested with a pair of unfamiliar males. Dependent t tests were also used to analyze all data from the testing apparatus modification study. Two-tailed tests were always used.

MALE FAMILIARITY AND PROXIMITY TIME 243 Results Control Experiment Mean times spent in proximty to two similar, unfamiliar males are presented in Figure 1. As expected, no difference in proximity times was found between unfamiliar males [t (23) = 1.27, P =.21]. This was true for both replicates even when the identities of the males (i.e., unfamiliar male --- CJ rj'.l 120... e --... 160 E-4 80 s. -40 Q =.c o Unfamiliar 1 Unfamiliar 2 Identity of Stimulus Male Figure 1. Mean times and standard errors (in seconds) spent in proximity to males during control experiment. 1 and unfamiliar male 2) were switched for analysis [23) =.875, P =.39]. The results confirmed that males chosen for similarity by our visual inspection did not have any discernible physical characteristics that alone would attract females, and that our procedure for selecting visually similar males was effective. ' Visual/Olfactory Exposure Experiment Figure 2 shows the mean times that females spent in proximity to familiar and unfamiliar males following visual and olfactory exposure. Females' time in proximity with males depended on the length of exposure [F(1, 46) = 11.42, P =.002]. Females spent greater amounts of time with males following the 30-min exposure compared to the 24-hr exposure. Unexpectedly, neither a significant main effect for familiarity [F (1, 46) =.063, P =.80] nor an interaction between length of exposure and familiarity [F(1, 46) =.644, P =.43] was found to affect female proximity time. As a check for consistency in responding, the replicates were analyzed separately, with both showing the same pattern of results as seen when replicates were combined.

244 MCLAUGHLIN AND BRUCE..- f:i.l -- 120.9... 160 80 s. = 40 - Familiar D Unfamiliar 0 0.5 24 Hours of Exposure Figure 2. Mean times and standard errors (in seconds) spent in proximity to familiar and unfamiliar males during visual/olfactory experiment. Because an effect for familiarity was not found, it appeared that female fish might require a greater number or intensity of cues to distinguish familiar from unfamiliar males. Therefore, the visual alone experiment was not conducted and the next experiment involving full exposure was conducted. Full Exposure Experiment Figure 3 shows the mean time females spent in proximity to familiar and unfamiliar males following full exposure to a male for 30 min, 3 hr, 18 hr, and 24 hr. In addition, Table 1 shows the mean total amounts of time (out of 600 seconds) females spent in proximity to both males, and the percentage of that time spent near either the familiar or unfamiliar male. Females appeared to prefer unfamiliar males as the length of prior Table 1 Mean Times (in sec) and Standard Errors (SE) Spent in Proximity to Both Males and Percentage of Time Spent in Proximity to Familiar (FAM) and Unfamiliar (UNF) Males during Full Exposure Experiment Exposure Total Time SE % Time % Time Condition near Males' near FAM near UNF 30 min 3 hr 18 hr 24 hr 213.31 190.72 192.35 163.91 Choice tests lasted 600 sec. 24.25 15.86 15.98 17.45 57 55 45 41 43 45 55 59

MALE FAMILIARITY AND PROXIMITY TIME 245 160,.-.. CJ 120... 5 80 1 40 Familiar ---0-- Unfamiliar O+--'--'--r'--- o 3 6 9 12 15 18 21 24 Hours of Exposure Figure 3. Mean times and standard errors (in seconds) spent in proximity to familiar and unfamiliar males during full exposure experiment. exposure increased. Females did not appear to spend time with both males depending on the length of exposure or to prefer unfamiliar males in all conditions. There were no significant main effects for type of exposure [F(1, 92) = 1.18. P =.32] or length of exposure [F(3, 92) =.037, p =.85] on female proximity time. However, the effect of familiarity on the time a female spent in proximity to a male depended upon the length of prior exposure to the familiar male [F(3, 92) = 2.81, P =.044]. Simple main effects analysis, using dependent t tests, was used to analyze the source of this significant interaction. Females spent significantly more time with unfamiliar males compared to males to whom they had been exposed for 24 hr (p <.05). However, there were no significpnt differences in female preference for familiar or unfamiliar males in the 30-min (p>.05), 3-hr (p >.05), or 18-hr exposure conditions (p >.05). A simple main effects analysis, using one-way between subjects ANOVA, was also conducted to see if the amount of time spent with either male (familiar vs. unfamiliar) significantly increased or decreased across time exposures. The amount of time females spent with the familiar male significantly decreased from 30 min to 24 hr (p <.05); however, there were no differences across time exposures for the unfamiliar male (all ps >.05). Statistical analyses of replicates in the 30-min, 3-hr, and 18-hr exposure conditions were consistent, indicating stability in subject responding. However, in the 24-hr condition, the first replicate did not show an effect of familiarity [1(10) =.91, P =.38), but the second replicate did [1(12) = 2.93, P =.007]. Sign tests indicated, however, that the direction of change in favor of preference for the unfamiliar male was

246 MCLAUGHLIN AND BRUCE consistent across the replicates. In the first replicate of the 24-hr condition, 8 of the 11 fish preferred the unfamiliar male (p =.11). Similarly, in the second replicate, 11 of 13 females preferred the unfamiliar male (p =.01). Differences in responding by 1 or 2 fish may have masked the effect in the first replicate. Testing Apparatus Modification Study It was possible that female fish were unable to distinguish males because they lacked sufficient cues during the choice test. After exposure to a male through a perforated partition, the females could only visually inspect the males in glass containers during the choice test. Thus, we used perforated containers to hold the males to allow both visual and olfactory cues during testing. Using this modification, we conducted two replicates of the control condition, two replicates of the 24-hr visual/olfactory exposure, one replicate of the 3-hr full exposure, and three replicates of the 18-hr full exposure conditions. Control condition. Females were still unable to distinguish two visually similar males when the males were presented in perforated containers in the preference test [M1 = 121.82s, SE = 14.24; M2 = 81.11s, SE= 12.63; 1(10)=1.7, p=.11]. Visual/olfactory exposure. FemaleS still did not show a preference for either the familiar or unfamiliar male during a choice test using perforated containers after 24 hr of visual and olfactory exposure to the familiar male [Familiar M = 97.13s, SE = 14.15; Unfamiliar M = 111.76s, SE = 14.12; 1(23) =.80, p =.43]. This finding was consistent for both replicates analyzed separately. Full exposure. USing the perforated containers for male presentation, females showed no clear preference for either the familiar or unfamiliar male during a choice test after 3 hr of full exposure [Familiar M = 101.57s, SE= 19.4; Unfamiliar M= 63.78s, SE= 7.89; 1(11) = 1.819, P =.096]. After 18 hr of full exposure, females still showed no consistent preference for either male when testing used perforated stimulus containers [Familiar M = 97.42s, SE = 13.95; Unfamiliar M = 80.36s, SE = 8.35; 1(30) =.913, p =.37]. We tested three replicates in this condition because there was some inconsistency in the results. In the first replicate, there was a justsignificant unexpected preference for the familiar male (p =.053, n = 11). However in two additional replicates, no preference for males was found (p =.71, n = 12; and p =.55, n = 8). Thus, we are convinced that females are not making a consistent preference for males based on this amount of familiarity; of the 31 females tested, 16 females preferred the unfamiliar male and 15 females preferred the familiar male (sign test, NS). Discussion The results of this study indicated that female Gambusia did not distinguish between two visually similar unfamiliar males, or between an unfamiliar male and a familiar male with whom they have limited exposure

MALE FAMILIARITY AND PROXIMITY TIME 247 (i.e., visual and olfactory cues alone). Females could distinguish between an unfamiliar male and a familiar male with whom they have had full exposure, given that such exposure lasted at least 24 hr before they were given a choice test. Then the female preference was for the unfamiliar male. We also found that females spent less time with the familiar male as prior exposure to him increased, from 30 min to 24 hr. Females did not show a preference for unfamiliar males during the visual/olfactory exposure experiment, regardless of the amount of time spent with the familiar male. Females did spend more time with males in general when the length of previous exposure to the familiar male was 30 min and not 24 hr. Female deprivation before exposure may account for such a finding. All females were housed in a female-only tank for at least 24 hr before exposure to a stimulus male. Females in the 24-hr exposure condition were then exposed to 1 male, blocked by a perforated partition, for 24 hr. However, females in the 30-min condition waited in the exposure tank without a male for an additional 23.5 hr before exposure to a male; they were exposed to a male only for the final 30 min of a 48-hr period. The females in the 24-hr condition had been exposed to a male for the final 24 hr of a 48- hr period. The females exposed for 30 min may have not received enough visual and/or chemical cues from the male during exposure to assess the characteristics that would affect their choice of him as a mate. The choice test may have served as additional time to make such a determination during which time the females inspected the cues of the unfamiliar male as well. Deprivation has been reported to influence female sexual behavior. Hughes (1985) reported that female western mosquitofish (G. affinis) who were deprived of males for 30 days showed greater receptivity towards males, compared to the receptivity of nondeprived females. Thus, a deprivation theory may explain why females did not distinguish between males during the visual/olfactory experiment, but instead spent more time with both males when prior exposure to a male was limited. We ruled out the possibility that female fish were unable to distinguish males during the visual/olfactory experiment because they lacked sufficient cues during the choice test. The results of the testing apparatus modification study still showed that females had no preference for either male during a choice test using perforated containers after 24 hr of visual and olfactory exposure. Although further study is needed to fully test this idea, the current data support the conclusion that prior full, free swimming exposure of at least 24 hr is required to affect female preference for familiar and unfamiliar males. Because females did distinguish between males after full exposure when the length of prior exposure was sufficiently long (24 hr), it may be that the type of interactions during the exposure influenced the ability of females to distinguish between males. Thus, we are currently examining the behavior of the fish during the exposure period. During full exposure, mating opportunities and aggression probably occur. Prior mating experience may be a key factor in determining future preference in female fish. Aggression by the male may also playa role in influencing female preference. Females

248 MCLAUGHLIN AND BRUCE may have avoided males who showed aggression towards them during full exposure, appearing to prefer a male with whom they have had no prior aggressive experience. Bisazza and Marin (1991) suggested that forced inseminations by aggressive Gambusia males may damage the genital tissue of females and females may seek to reduce further mating by swimming away from males. The authors also suggest that constant attempts at mating by males may interrupt the foraging efforts of females or increase their risk of predation. This type of "harassment by males" (Bisazza & Marin, 1991, p. 734) may influence females to avoid mating attempts by males. In addition, Taylor et al. (1996) pointed out that aggression from familiar melanistic males may have been a factor contributing to female Gambusia's preference for normal over melanistic males. Bruce and White (1995) also suggested that male-female aggression may playa role in determining female proximity to males. The present data further support such hypotheses in G. holbrooki. Our data suggest that at least 24 hr of exposure is required for Gambusia females to accumulate enough information from visual cues, olfactory cues, and physical interactions, to distinguish males. As noted above, the direction of female "preference" may have been influenced by habituation to (or avoidance of) the familiar male. The time that females spent with the familiar male decreased across time exposures, with females spending less time with the familiar male as the length of prior exposure increased (see Table 1). After 30 min of exposure to a familiar male, females spent 57% of their time near the familiar male. After 24 hr of exposure to a familiar male, females spent only 41 % of their time near the familiar male. The time spent with the unfamiliar male across exposure times also increased, from 43% after 30 min of exposure to 59% after 24 hr of exposure, although because of individual variation in the dependent measure, this change was not statistically significant. These data offer evidence that the females may not prefer the unfamiliar male as much as they seek to avoid the familiar male. As the time a female is exposed to a male increases, her receptivity to that male may decrease. Farr's (1977) data on guppies are consistent with this idea. Farr allowed male-female groups of guppies to acclimatize for 24-72 hr before introducing a new male. This procedure elicited sexual behavior from females towards the new male regardless of females' gestational state. Thus, it may be that female receptivity or that male attractiveness decreases over a 24-hr time period. This may be what occurred to the female Gambusia in the present study. In addition, the unfamiliar males in the present study may have behaved differently because they had been isolated from females during the familiar males' exposure period. In subsequent studies, we will control for possible changes in male proceptivity or attractivity by allowing the unfamiliar male to have access to (untested) females during the familiar males' exposure period. The factors influencing female mate choice in Poeciliid fish are varied. Females sometimes prefer males who exhibit.certain

MALE FAMILIARITY AND PROXIMITY TIME 249 morphological or behavioral traits, such as larger size, greater ornamentation, and higher rates of courtship behavior. It is apparent that the prior experience with a male may influence female mate choice as well. Future research quantifying the specific characteristics of that prior experience, such as the rate and type of aggressive and sexual interactions that occur during exposure, will help shed further light on female mate choice in Poeciliid fish. References BASOlO, A. L. (1990). Female preference for male sword length in the green swordtail, Xiphophorus helleri (Pisces: Poeciliidae). Animal Behaviour, 40, 332-338. BISAZZA, A (1993). Male competition, female mate choice and sexual size dimorphism in Poecillid fishes. Mar. Behavior Physiology, 23, 257-286. BISAZZA, A, & MARIN, G. (1991). Male size and female mate choice in the eastern mosquitofish (Gambusia affinis holbrooki: Poeciliidae). Copeia, 3, 730-735. BISCHOFF, R. J., GOULD, J. L., & RUBENSTEIN, D. I. (1985). Tail size and female choice in the guppy (Poecilia reticulata). Behavioral Ecology and Sociobiology, 17, 253-255. BRUCE, H. M. (1966). Smell as an exteroceptive factor. Journal of Animal Science, Suppl. 25, 83-89. BRUCE, K. E., & WHITE, W. (1995). Agonistic relationships and sexual behaviour patterns in male guppies, Poecilia reticulata. Animal Behaviour, 50, 1009-1021. COURTENAY, W. (1984). Distribution, biology, and management of exotic fishes. Baltimore: John Hopkins University Press. CRAPON DE CAPRONA, M., & RYAN, M. (1990). Conspecific mate recognition in swordtails, Xiphophorus nigrensis and X. pygmaeus (Poeciliidea): Olfactory and visual cues. Animal Behaviour, 39, 290-296. CROW, R. T., & LILEY, N. R. (1978). A sexual pheromone in the guppy, Poecilia reticulata (Peters). Canadian Journal of Zoology, 57, 184-188. DEWSBURY, D. A. (1978). Comparative animal behavior. New York: McGraw-HilI. DEWSBURY, D. A (1981). Effects of novelty on copulatory behavior: The Coolidge effect and related phenomena. Psychological Bulletin, 89, 464-482. FARR, J. A. (1977). Male rarity or novelty, female choice behavior, and sexual selection in the guppy, Poecilia reticulata. Evolution, 31, 162-168. HOUDE, A. (1988). Genetic difference in female choice between two guppy populations. Animal Behavior, 36, 510-516. HOUDE, A (1997). Sex, color, and mate choice in guppies. Princeton, NJ: Princeton University Press. HUGHES, A L. (1985). Male size, mating success, and mating strategy in the mosquito fish Gambusia affinis (Poeciliidae). Behavioral Ecology and Sociobiology, 17, 271-278. long, K. D., & HOUDE, A E. (1989). Orange spots as a visual cue for female mate choice in the guppy (Poecilia reticulata). Ethology, 82, 316-324.

250 MCLAUGHLIN AND BRUCE MARTIN, R. G. (1986). Behavioral response of female mosquitofish, Gambusia affinis holbrooki, to normal versus melanistic male mosquitofish. Journal of the Elisha Mitchell Scientific Society, 102, 129-136. MCPEEK, M. A. (1992). Mechanisms of sexual selection operating on body size in the mosquitofish (Gambusia holbrookl). Behavioral Ecology, 3, 1-12. TAYLOR, S. A., BURT, E., HAMMOND, G., & RELYEA, K. (1996). Female mosquitofish (Gambusia affinis holbrooki) prefer normally pigmented over melanistic males. Journal of Comparative Psychology, 110, 260-266.