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Prostaglandin induction of spawning behavior in Cichlasoma bimaculatum (Pisces Cichlidae)
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235-248 (1984)
Prostaglandin induction of Spawning Behavior in Cichlasoma bimaculatum (Pisces Cichlidae) KATHLEEN
S. COLE’ AND NORMAN E. STACEY
Department of Zoology, University of Alberta, Edmonton, Alberta TtjC; 2El, Canada Prostaglandin (PG) stimulates female spawning behavior in goldfish and in some other teleosts in which female reproductive behaviors consist of postovulatory oviposition acts. This study examined the effects of PG on female sexual behavior in a teleost fish, Cichlasoma bimaculatum, in which female reproductive behaviors involve both preovulatory courtship and substrate cleaning behaviors, and postovulatory oviposition behavior. In females of established pairs, PGF2 a injection (5 pg, im) at any stage of the spawning cycle, or in the parental phase, rapidly induced substrate cleaning which soon merged into oviposition behavior (without egg release). These results support a role for PG in oviposition behavior of Cichlasomu. However, indomethacin (1 mg, ip), a PG synthesis inhibitor, did not block oviposition in ovulated females which had begun to spawn. lndomethacin may not have lowered PG levels sufficiently. Alternatively, as shown by J. J. Polder (1971, Neth. J. Zoo/. 21, 265), oviposition behavior may be induced or maintained by other factors associated with the spawning situation.
Prostaglandins (PGs) have been implicated in the female sexual behavior of mammals (Rodriguez-Sierra and Komisaruk, 1978; Buntin and Lisk, 1979; Mart-one, Rodriguez-Sierra, and Feder, 1979), amphibians (Diakow and Nemiroff, 1981), and fish (Stacey, 1976, 1981; Stacey and Goetz, 1982; Villars and Burdick, 1982). In both mammals and teleosts, PGs apparently act on the brain, and may be effective within minutes (RodriguezSierra and Komisaruk, 1978; Stacey and Peter, 1979). In the goldfish, Carassius auratus, female spawning or oviposition behavior normally occurs only after ovulation but can be induced in nonovulated females by the intraovarian injection of ovulated eggs (Stacey and Liley, 1974), and inhibited in egg-injected females by indomethacin (Id), an inhibitor of PG synthesis (Stacey, 1976). PG (PGFZII.especially) injection induces apparently normal female spawning behavior in egginjected, Id-treated females, in nonovulated females which are otherwise untreated, and also in males (Stacey, 1976; 1981). In support of these ’ Present address: Department of Biology. University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada. 235 CQ18-506X/84$1.50 Copyright CJ 1984 by Academic Press. Inc. All rigbrs of reproduction in any iorm rcxrved
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behavioral studies, PGF has been shown to increase in the ovaries of several teleost species around the time of ovulation (Ogata, Nomura, and Hata, 1979; Cetta and Goetz, 1982) and also in the blood of both ovulated goldfish (Bouffard, 1979) and brook trout, Salvelinus fontin& (Cetta and Goetz, 1982). Together, these findings in externally fertilizing fish have been interpreted as indicating that ovarian PG, released into the bloodstream and acting within the brain, stimulates female sexual behaviors during the time in which ovulated eggs arc ready for release (Stacey, 1981; Stacey and Goetz, 1982; Liley and Stacey, 1983). This study examined the effects of PG on female spawning behavior in another externally fertilizing teleost, Cichlusoma bimaculatum, (Pisces: Cichlidae). Unlike goldfish, C. bimacufutum demonstrate long-term pair bonding, preparation of and joint defense of a spawning site, and parental care. Three questions were asked: (1) does PGFzoelicit behavior associated with oviposition in nonparental and parental female C. bimuculutum? (2) does PGF2, elicit similar behavior in males? and (3) does indomethacin block normal spawning in females, or affect female response to exogenous PG? METHODS AND MATERIALS
Maintenance Twenty spawning pairs of C. bimucuiutum, of 25-35 g body wt, were established from a single spawn of 8-month-old individuals. Pairs were placed in separate aquaria (67 or 102 liters) and maintained at 24-28°C under a 16-hr light: 8-hr dark photoperiod. Each aquarium had gravel substrate to a depth of 5 cm, two small clay pots, and a piece of slate. All pairs were allowed to spawn undisturbed at least once prior to experimental use. Experiments were carried out from June 9 to August 30, 1982. Description of Normul Spawning, and Behaviors Recorded Normal spawning of C. bimuculatum in our laboratory fish consisted of three phases: (1) an initial heightened defense of a nest area and an increase in mate-directed behaviors, (2) the subsequent onset of substratedirected behaviors, and (3) the synchronized release of eggs and sperm. Each of the three phases will be briefly described below. Ttio or three days prior to oviposition, both the female and male developed a darkened body color. Adults are normally pallid, with two dark, prominant spots on each side, one located midlaterally and the other at the base of the caudal fin. A barred pattern consisting of one longitudinal bar running from the eye to the caudal peduncle, and 5-6 vertical bars, may or may not be visible. In prespawning individuals, however, the background became dark grey, and the barred pattern became dusky grey to black, often obscuring the black spots completely.
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The fins, particularly the pelvics, also developed a dark grey or black appearance. Coincident with a color change, both the female and male became overtly aggressive to apparent intrusions into the area of the tank near to the spawning site. Movements outside the tank caused one or both members of the pair to attack the glass closest to the movement. Introduced food was also attacked, but not eaten. Interactive behaviors between the male and female during this phase also increased. “Rushing” by the female consisted of a rapid and directed approach toward the male, terminating in an abrupt halt in front of or parallel to him, and a brief extension of the dorsal, anal, caudal, and pelvic fins. This was usually followed by brief bursts of accelerated swimming by the female alongside or directly in front of the male. “Anal nudge,” a behavior performed only by males, consisted of the male placing his mouth close to the female’s anal region and gently butting the female’s anal or adjacent flank region with a closed or slightly open mouth. In the 24-hr period immediately preceding oviposition, the male and female started to direct increased attention toward a suitable spawning surface (usually a slate placed on the gravel substrate of the tank) and the immediately surrounding substrate. The two activities most frequently observed were associated with removing debris from the slate, and with displacing gravel adjacent to the slate. “Gravel-digging” consisted of the displacement of gravel, either by vigorous lateral undulations of the body. or by pushing movements with an open mouth, resulting in depressions in the gravel substrate near the spawning slate. This was usually done by the male. “Surface cleaning” consisted of the removal of detritus, gravel, and algae from a selected spawning surface (usually a piece of slate) by either member of the pair. This was done by assuming a verticai head-down position and biting off or picking up particles with the mouth and expelling them away from the cleaned surface. In the 4-6 hr directly preceding oviposition, the genital papilla of both adults, which are normally retracted and not visible outside of spawning and immediate postspawning phases, became fully distented. The female frequently made rushing movements toward the male, then approached the slate. Often, the male followed the female to the slate, then moved a short distance away. Shortly before oviposition (30-90 min) the female, upon approaching the slate, hovered over it in a slightly head-down position. This soon developed into jerky head-down movements over the slate surface so the gular region of the female grazed the slate as she moved forward. As the female swam forward. the tail dropped until the pelvics touched the substrate, at which point the female would halt, then briefly move away from the slate. Chin movements gradually became less jerky, and merged into smooth forward movements as the posterior portion of the female’s body became lower and the genital papilla came into direct contact with the spawning
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surface. This activity was soon followed by continuous movements over the slate, the female turning only at the edge of the slate and approaching again on a slightly different trajectory. After several such passes over the slate, the female commenced ovipositon. At the onset of female passing movements over the slate, the male approached the slate, assumed a head-down position and appeared to inspect the slate surface, shifting his orientation slightly and directing first one eye, then the other, towards the slate. As the female moved by, the male often placed his mouth close to the slate, sometimes touching it or nibbling the surface. At the onset of egg release, the male immediately adopted a horizontal posture close enough to the slate to allow contact with his genital papilla, and followed the female’s path after she had made a complete pass over the slate surface, to release sperm. Oviposition was generally complete after 15-20 min, after which both adults initiated egg-tending behaviors. Female behaviors in C. bimaculutum appear similar to the three progressive phases of spawning described by Greenberg, Zijlstra, and Baerends, (1965) and Polder (1971) for Aequidens portalegrensis.’ Initial slate-directed movements by female A. portalegrensis that consisted of short, forward movements from above the substrate and coming to a halt when the pelvic fins and abdomen came into contact with the substrate were termed “skimming.” Similar head-down movements over the slate by female C. bimuculutum were sometimes interspersed with bouts of surface cleaning, and by rushing movements directed toward the male. In A. portulegrensis, skimming soon merged into “pseudospawning,” which consisted of sliding, propulsive movements forward over the substrate, with the pelvic fins and abdomen in direct contact with the substrate, and with the anterior portion of the dorsal fin slightly spread. With the appearance of eggs, Polder distinguished the onset of “true spawning,” which differed from pseudospawning by the compression of the anterior portion of the dorsal fin towards the body, and by the presence of contractions occurring in the belly region, In C. bimucuhtum in our laboratory, differences in the position of the dorsal fin during normal spawning were not always clear-cut, but in other respects appeared identical to the behaviors of A. portulegrensis. ’ Cichlasoma bimac~tlatum is frequently marketed as Aequidens porralegrensis in both North America and Europe (see Baerends and Baerends-Van Roon, 1956,p. 2). We initially selected what we believed to be a A. porralegrensis stock for this study, because of the detailed descriptions of the reproductive behavior available on this species (Polder, 1971; Greenberg er al., 1%5). We subsequently discovered that our fish were not A. portalegrensis, but rather C. bimaculatunz (identity confirmed by D. Stewart, Field Museum of Natural History, Chicago, III.; specimen catalog Nos. 534-94748,534-94749). Nevertheless, Polder and Greenberg’s descriptions still accurately described the reproductive behaviors of our fish.
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For the purposes of recording the gross effects of PGF, on nonovulated pairs of C. bimuculatum, we did not attempt to record all the action patterns found in the spawning sequence. Instead, we selected frequently occurring behaviors that were typical of each phase, that had clear-cut onset and termination points, and that consisted of several motor patterns that usually occurred together. These behaviors were “rushing,” “anal nudge, ” “surface cleaning,” “gravel-digging,” and “spawning.” We defined “spawning” as the slow movement of a fish across a cleaned surface, with the genital papilla in close or direct contact with the surface, the tail lifted slightly, and with the anterior portion of the dorsal fin either partially or totally compressed against the body. These behaviors were recorded on a 20 channel Esterline Angus event recorder during experimental observations. Experiment
I: Female Response to Prostaglandin
(a) Nonparental females. Forty-eight hours after spawning, the eggs were removed from a pair, and 24 hr later the female was given an intramuscular injection of either 10~1 buffered saline or 5 pg PGFz, tromethamine salt (Sigma) in buffered saline. The 72-hr delay ensured that the female was not ovulating at the time of the injection (there was a minimum 5day interval between subsequent spawnings in any one female in our laboratory fish). The behavior of both the male and female were then recorded for l-hr postinjection. The durations of the most commonly observed of these behaviors, for both the male and female, were summed over successive 5-min intervals. In addition, the time from the injection to the onset of sustained surface cleaning and spawning behavior (latent period) was recorded for each test fish. Each treatment (saline or PG) was carried out on the female of seven pairs of fish. (b) Parental females. Six females from parental pairs of fish were injected with 5 pg PGFk, and subsequent behaviors were recorded for a I-hr period. In each case, young had hatched within the last 7 days. Experiment
2: Female and Male Response to Prostaglandin
One member of a pair was given an intramuscular injection of 5 pg PGF&. Two to three days later, the other member of the pair was given a similar injection. Two to three days following this, both members of the pair were reinjected with PG. For 1 hr following each injection treatment, the behaviors described in Experiment 1 were recorded for both the male and female, using an event recorder, and the latent period also was noted. Durations of the most commonly observed behaviors for both the male and female were summed over the 60-min observation period. This was repeated with eight pairs of fish.
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Experiment 3: Response to Indomethacin (a) Effect of Id on normal oviposition. Just after the onset of natural spawning, the ovipositing female was removed, given an intraperitoneal injection of either 0.15 ml saline (0.6% NaCl) or 0.9 mg Id (Sigma) in saline, and held in isolation, with no appropriate spawning surface, for 3 hr. The male and the spawning slate with attached eggs were also removed from the aquarium and placed together in isolation. Before returning the male and spawning slate to the resident aquarium, the eggs were removed. Five minutes after the male’s reintroduction, the female was returned, and the time to resumption of ovipositing was recorded. If spawning was not resumed within 24 hr, spawning behavior was recorded as blocked (i.e., no latency). Seven spawning females were Id-treated, and seven were saline-treated. (6) Effect of Id on Female Response to PGF,,. To determine whether inhibition of normal female spawning behavior following Id treatment might be attributable to interference with the female’s response to endogenous PG, rather than to an inhibition of PG synthesis, nonparental females were injected intraperitoneally with either Id (0.9 mg; n = 6) or saline (n = 7). This was followed 3 hr later by an intramuscular injection of 5 pugPGFZLl. Following the PG treatment, the latent period and the females’ behavioral responses to the PG were recorded. If spawning behavior did not occur within 4 hr, it was recorded as a nonresponse, and no latency value was assigned. RESULTS
Experiment I: Female Response to PGF,, All seven nonparenting females injected with PG subsequently showed behavior normally associated with oviposition. The two behaviors most frequently observed were surface cleaning and spawning. In six trials, females responded with sustained surface cleaning, followed almost immediately by spawning behavior 6-26 min after the injection. In the seventh pair, the male initially attacked the female when she attempted to surface clean. Sustained surface cleaning started 9 min after the injection, but sustained spawning behavior did not start until 85 min after the administration of PGFza. The mean latency for a sustained response to PG was 11.9 & 6.7 min (range = 6.8-26.1 min). Among the six females that did respond shortly after the PG injection, similar behaviors were seen. After the initial fright response to handling had passed, each PG-injected female exhibited both increased respiration rate and increased motor activity. The body color darkened in a manner similar to that of normally spawning fish, and the genital papilla became distended. Increased aggression directed toward apparent intrusions into the tank, surface-cleaning behaviors, rushing motions directed toward
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the male, and spawning movements over the slate occurred in a manner identical to normal female spawning behaviors, with the exception that egg release did not occur, and that all PG-induced behavioral elements were induced very soon after the time of injection. None of the behaviors associated with normal spawning was observed in PG-treated females immediately prior to the time of the injection. Spawning movements over the slate substrate persisted for 3-5 hr following PC injection. The proportion of time spent spawning by nonovulated, nonparental females showed an increasing trend over the 60min observation period, but the time spent surface cleaning, after initial high levels, decreased, and was maintained at a low level until the end of the observation period (Fig. 1A). Untreated males showed variable responses to the PG-induced spawning behavior of their partners. Some males showed no response at all, or retreated to a pot or aquarium corner; others sometimes developed a dark coloration typical of naturally spawning males, joined the female in surface cleaning, and initiated gravel digging. Unlike females, however, males were easily distracted, and their behaviors were often brief and incomplete. Female rushing behavior sometimes elicited anal nudging of the female by a responsive paired male; anal nudging was not observed in the male prior to the administration of PGF2, to the female. At the onset of female spawning behavior, some responsive males adopted a head-down position. and following the female, put the mouth in contact with the spawning surface directly behind the female, where thin mucous tracks were sometimes evident. However, male spawning behavior in synchrony with female spawning was uncommon, and usually sporadic. All females injected with teleost saline demonstrated a fright response typified by shelter seeking, immobility, and pale coloration. In control pairs, no reproductive behaviors were observed in either adult. All parental females, as with nonparental females, responded to PGFz,, by demonstrating spawning behavior, with a mean latency of 14.1 -C 7.8 min (range = 4.9-25.6 min). Initially, the time spent surface cleaning increased, but then declined and remained low, while the time spent in spawning behavior progressively increased (Fig. 1B). Parental females spent less time surface cleaning than did nonparental females (c’ = 2, P = 0.002), but the two experimental groups did not differ in the amounts of time spent spawning (V = 20, P = 0.473) or in their latency to response (V = 17, P = 0.314, Mann-Whitney U test) (Siegel, 1956). Experiment 2: Male-Female Response to PGF,, Of the eight experimental pairs used in Experiment 2, two spawned before the completion of all three treatments, and were omitted from the data analysis. The remaining six females responded to PG treatment regardless of the interval (which varied from 2 days to 2 weeks) between
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FIG. 1. Mean duration per 5-min observation interval of behavioral responses by (A) nonparental and (B) parental females for 1 hr following PG injection. Vertical brackets indicate 1 SE above and below the mean.
the time of the injection and the time of the last ovulation, as indicated by normal spawning. When the behavioral responses of females under all conditions were compared, the levels of female surface cleaning and spawning behavior were very low when only the male was injected, but were greater when either the female alone, or both the female and male were injected (Table la).
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TABLE 1 Total Duration (set) of Spawning and Surface Cleaning Responses of (a) Female and (b) Male Fish under Different Experimental Conditions
Fish pair -___-._____ 1 2 3 4 5 6
(a) Duration of female response Surface cleaning Spawning __--_--_-.-..-. -.-.-.-.--. F F-M F-M M F .- -.-.-.-_._--__1316 1505 1 43 30 1537 758 0 123 432 933 1283 0 122 204 1697 1173 0 27 222 24 1474 0 63 91 1651 745 0 289 72 X,2 = 9.0, P = 0.008 X,, = 6.5. P = 0.05
1 2 3 4 5 6
0 2 0 37 0 0
M -37 0 0 0 0 0
(b) Duration of male response 7 0 233 246 0 225 130 71 0 0 253 0 0 129 80 0 0 0 105 0 0 0 50 0 X,z = 4.2, P = 0.18 X+ = 9.8, P = 0.006 123 34 38 20 93 175
Note. F indicates treatment where only the female of each pair was PG-injected; F-M indicates both the female and male were injected; M indicates only the male of each pair was injected. X,? values are based on Friedman two-way ANOVA tests.
The behavior of responsive PG-injected males consisted primarily of surface cleaning and spawning. However, unlike PG-treated females, males were easily distracted, causing their motor patterns to be discontinuous and of short duration. When both the male and female were injected, the male spawning response was significantly greater than when only one partner was injected. However, the male surface cleaning response was not affected by different injection conditions (Table lb). Experiment
3
(a) Effect of Id on normal oviposition. Of seven ovipositing females injected with saline, all continued to spawn, with a mean time to resumption of spawning of 76.1 ? 52.6 min (range = 36-180 min) after their return to the aquarium. Of seven ovipositing females treated with Id, two individuals did not resume spawning within 24 hr; the mean time for resumption of spawning of the remaining five females was 127.0 +- 63.8 min (range = 30-200 min). The control and experimental groups did not differ significantly in their latency periods (U = 11, P = 0.172, MannWhitney U test) or in the degree of blocking of normal spawning by Id or saline 0, = 0.23, Fisher exact test) (Siegel, 1956).
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(b) Effect ofld on jkmale response to PGFzU. When six females were treated first with Id, then with PGFzp 3 hr later, five females showed surface cleaning and spawning behavior similar to that of PGFz,,-treated females in Experiments 1 and 2; the sixth female showed no reproductive behavior within the next 4 hr. The mean latent period of the five responsive females was 17.1 + 8.3 min (range = 4-26 min). Of the seven females injected first with saline, followed by PGFzOafter 3 hr, three showed no reproductive behavior typical of a PGF2, response. The four remaining females responded in a manner similar to Id-PGF2,treated females, with a mean latent period of 35.0 + 26.5 min (range = 6-65 min). There were no significant differences between the groups, either in the proportion of females responding after Id or saline treatments (P = 0.29, Fisher exact test), or in response latency (U = 5, P = 0.14, Mann-Whitney U test). DISCUSSION
The results of this study demonstrate clearly that PG injection in female rapidly induces several behavioral components normally associated with oviposition. These behaviors include aggressive defense of the spawning site, certain behaviors directed toward the male, cleaning of potential spawning substrates, and spawning (oviposition without egg release) behavior. The findings indicate that PG may be the endogenous signal synchronizing oviposition behavior with ovulation in this species, as had been suggested for goldfish (Stacey, 1981). Normal spawning behavior in ovulated C. bimaculatum involves egg release. However, other behaviors similar to skimming and pseudospawning, as described by Greenberg et ul. (1965) and Polder (1971) for A. portulegrensis, precede the onset of oviposition by one or more hours. Polder (1971) found that during the intense skimming phase, ovulation (follicular rupture) had occurred in only a few follicles in A. portalegrensis. However, with the appearance of pseudospawning, ovulation was completed, and shortly thereafter true spawning with egg release commenced. Except for the difference in dorsal fin position and the absence of egg release, pseudospawning in A. portulegrensis is virtually identical to true spawning. Possibly, the latency between the onset of pseudospawning and the onset of true spawning represents the time required for ovulated eggs to travel through the oviduct to the genital papilla. Close temporal synchrony between ovulation and the onset of spawning also has been noted in the cichlid Sarotherodon melunotheron (= Tilapiu macroceph&) (Aronson and Holz-Tucker, 1949) and likely is typical of many teleosts (see Liley and Stacey, 1983). It is not clear whether skimming behavior in C. bimuculutum, which likely begins near the time of ovulation, also is stimulated by cndogenous PG. However, ovarian PG is involved in follicular rupture in teleosts C. bimaculutum
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(Stacey and Goetz, 1982), and it is possible that follicular PG synthesis prior to ovulation is sufficient to stimulate skimming behavior. This possibility is supported by the finding that plasma PGF levels increase in brook trout coincident with oocyte final maturation, prior to ovulation (Cetta and Goetz, 1982). Both the effects of PG injection, and the temporal relationship between ovulation and spawning, support a role for endogenous PG in female C. himaculutum spawning behavior. However, Polder (1971) has shown that skimming and pseudospawning can also be induced within minutes in nonovulated female A. porta1egrensi.s simply by exposing the female to a clutch of eggs from another pair; similar responses were observed in males. This effect of eggs, which likely is not mediated by endogenous PG, indicates that in normally spawning pairs, once spawning has commenced, it may be maintained by visual or chemical cues from the nest site. lndomethacin did not block the resumption of normal spawning in ovulated C. bimacufutum, or reduce female responsiveness to PG. These findings are in contrast to the complete blockage of spawning behavior in egg-injected (Stacey, 1976) or ovulated goldfish (Stacey and Goetz. 1982) after Id treatment. One explanation for this discrepancy is that indomethacin may not be a potent inhibitor of PG synthesis in C. himuculutum. Another possibility is that PG may not be rapidly metabolized in Id-treated females that have already ovulated. PG has been found in the ovarian fluid of ovulated female goldfish (Bouffard, 1979), and the coelomic fluid of ovulated brook trout (Cetta and Goetz, 1982). If PG is present in the ovarian fluid of ovulated female C. himuculutum, it may be sufficient to sustain spawning behavior even after subsequent PG synthesis has been inhibited by Id. The responsiveness of female C. bimuculutum to experimental levels of exogenous PG did not vary with the reproductive cycle. Whether they were tested several days after spawning, at a time of rapid oocyte growth (Polder, 1971), or more than a week after spawning, at a time when oocyte growth has been completed (Polder, 1971), females exhibited equivalent levels of PG-induced spawning behavior. Furthermore. responsiveness was not affected by the presence of recently hatched young. As all tests involved a similar high PG dose (approximately 150 ng.g-’ body wt), the possibility remains that the minimum dosage required to elicit spawning may vary with ,ovarian condition. However, the present results suggest that, as in goldfish (Stacey, 1981; Stacey and Goetz, 1982). responsiveness to PG in female C. bimuculutum is not obviously influenced by reproductive condition. This situation is in marked contrast to that in the paradise fish, Mucropodus operculuris, in which female spawning responsiveness to exogenous PGF declines dramatically several days after spawning (Villars and Burdick. 1982).
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Unlike female C. bimacufatum, which spawned readily following PG treatment, males responded consistantly to PG only if their female partner also was PG-treated. In contrast, PG treatment of goldfish of either sex induces equivalent levels of female spawning behavior (Stacey, 1981; Stacey and Kyle, 1983). The low responsiveness to PG exhibited by male C. bimaculatum is difficult to interpret because their behavioral responses were too brief and incomplete to clearly indicate whether male or female behavior was being displayed. In natural spawning, there are some qualitative differences in the spawning behavior of males and females. Males displace gravel to form pits in the substrate, direct anal nudging towards females, and before and during egg release by the female, repeatedly approach the slate in a head down position and appear to inspect the slate for the presence of eggs. None of these behaviors is typical of females, and none was seen in the repertoire of PG-injected males. If PG-treated males are indeed performing female spawning behavior, two factors could contribute to their low responsiveness. First, male C bimaculatum may be behaviorally defeminized through developmental processes, as has been well documented in mammals and birds [see reviews by Feder (1981) and Adkins-Regan (1981)]. This is in contrast to goldfish, where PG-treated males readily show female behavior (Stacey, 1981; Stacey and Kyle, 1983). Second, male C. bimaculatum may be physiologically capable of female levels of responsiveness to PG, but may require specific stimuli from a partner showing male behavior in order to express this female potential. In goldfish, PG-treated males require the presence of a responsive male in order to display female spawning behavior. This is easy to provide, since untreated males readily court and spawn with PG-treated males (Stacey, 1981). In contrast to goldfish, however, male-to-male aggression in C. bimaculatum would likely preclude the use of homosexual male pairs to examine this possibility. Nonetheless, the dramatic increase in male spawning responses to PG when the female partner also was PG-treated indicates that the behavior of the female partner can influence male responsiveness to PG in this species. Initial reports of the effects of PG on female sexual behavior in goldfish (Stacey, 1976, 1981; Stacey and Goetz, 1982), a teleost with relatively simple reproductive behavior involving no pair formation and no territorial or parental activities, demonstrated that PG-treatment of nonovulated females induces normal spawning interactions between the sexes. The present study demonstrates that, even in a teleost where complex and protracted interactions both precede and follow oviposition, PG treatment induces in nonovulated females a set of behaviors associated with normal spawning. The use of PG treatment as a technique for investigating sexual interactions may become an important tool in future studies of the reproductive behavior of cichlids and other teleostean fishes.
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ACKNOWLEDGMENTS We thank D. L. G. Noakes, R. J. F. Smith, and two anonymous reviewers for critical comments on the manuscript, and J. R. Baylis for bringing to our attention the problem of species identification for Aequidens portalegrensis and Cichlasoma himaculatum. This study was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) University Research Fellowship (UOOO7)to N.E.S.
REFERENCES Adkins-Regan, E. (1981). Early organizational effects of hormones: An evolutionary perspective. In N. T. Adler (Ed.), Neuroendocrinology of Reproduction: Physiology and Behavior, pp. 159-228. Plenum, New York. .4ronson, L. R., and Holz-Tucker, A. M. (1949). Ovulation in the mouthbreeding cichlid fish, Tilapia macrocephala. Anar. Rec. 105, 568-569. Baerends, G. P., and Baerends-Van Roon, J. M. (1950). An introduction to the study of the ethology of cichlid fishes. Behaviour Suppl. 1, l-243. Bouffard, R. E. (1979). Role of Prostaglandins during Sexual Maturation, Ovulation, and Spermiation in the Goldfish, Carassius auratus. M.Sc. thesis, University of British Columbia. Buntin, J. D., and Lisk, R. D. (1979). Prostaglandin E,-induced lordosis in estrogen-primed female hamsters: Relationship to progesterone action. Physiol. Behav. 23, 569-575. Cetta, F., and Goetz, F. W. (1982). Ovarian and plasma prostaglandin E and F levels in brook trout (Salvelinus fantinalis) during pituitary-induced ovulation. Biol. Reprod. 27, 1216-1221. Diakow, C., and Nemiroff, A. (1981). Vasotocin, prostaglandin, and female reproductive behavior in the frog, Rana pipiens. Horm. Behav. 15, 86-93. Feder, H. H. (1981). Perinatal hormones and their role in the development of sexually dimorphic behaviors. In N. T. Adler (Ed.), Neuroendocrinology of Reproduction: Physiology and Behavior, pp. 127-157. Plenum, New York. Greenberg, B., Zijlstra. J. J., and Baerends, G. P. (1%5). A quantitative description of the behavior changes during the reproductive cycle of the cichlid fish Aequidens portalegrensis Hensel. Proc. K. Ned. Akad. Wet. Ser. C, 68, 135-149. Liley, N. R., and Stacey, N. E. (1983). Hormones, pheromones and reproductive behavior in fish. In W. S. Hoar, D. J. Randall, and E. M. Donaldson (Eds.), Fish Physiology, Vol. IXB, Behavior and Fertility Control. Academic Press, New York. Marrone, B. L., Rodriguez-Sierra, J. F., and Feder, H. H. (1979). Differential effects of prostaglandins on lordosis behaviour in female guinea pigs and rats. Biol. Repro. 20, 853-861. Ogata, H., Nomura, T., and Hata, M. (1979). Prostaglandin F2 alpha changes induced by ovulatory stimuli in the pond loach, Misgurnus anguillicaudatus. Bull. Japan Sot. Sci. Fish. 45, 929-931. Polder, J. J. (1971). On gonads and reproductive behaviour in the cichlid fish Aequidens portalegrensis (Hensel). Neth. J. Zool. 21, 265-365. Rodriguez-Sierra, J. F., and Komisaruk, B. R. (1978). Lordosis induction in the rat by prostaglandin E, systemically or intracranially in the absence of ovarian hormones. Prostaglandins
15, 513-524.
Siegel, S. (1956). Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill, New York. Stacey, N. E. (1976). Effects of indomethacin and prostaglandins on spawning behavior of female goldfish. Prostaglandins 12, 113-126. Stacey, N. E. (1981). Hormonal regulation of female reproductive behavior in fish. Amer. Zool. 21, 305-316.
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Stacey, N. E., Cook, E. F., and Peter, R. E. (1979). Ovulatory surge of gonadotropin in the goldfish, Carassius auralus. Gen, Comp. Endocrinol. 37, 246-249. Stacey, N. E., and Goetz, F. W. (1982). Role of prostaglandins in tish reproduction. Cunad. J. Fish. Aquat.
Sci. 39, 92-98.
Stacey, N. E., and Kyle, A. L. (1983). Effects of olfactory tract lesions on sexual and feeding behavior in the goldfish. Physiol. Behav. 30, 621-628. Stacey, N. E., and Liley, N. R. (1974). Regulation of spawning behavior in the female goldfish. Nature (London) 247, 71-72. Stacey, N. E., and Peter, R. E. (1979). Central action of prostaglandins in spawning behavior of female goldfish. Physiol. Behav. 22, 1191-l 196. Villars, T. A., and Burdick, M. (1982). Rapid decline in the behavioral response of female paradise fish to prostaglandin treatment. Amer. Zool. 22, 948.
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18,
235-248 (1984)
Prostaglandin induction of Spawning Behavior in Cichlasoma bimaculatum (Pisces Cichlidae) KATHLEEN
S. COLE’ AND NORMAN E. STACEY
Department of Zoology, University of Alberta, Edmonton, Alberta TtjC; 2El, Canada Prostaglandin (PG) stimulates female spawning behavior in goldfish and in some other teleosts in which female reproductive behaviors consist of postovulatory oviposition acts. This study examined the effects of PG on female sexual behavior in a teleost fish, Cichlasoma bimaculatum, in which female reproductive behaviors involve both preovulatory courtship and substrate cleaning behaviors, and postovulatory oviposition behavior. In females of established pairs, PGF2 a injection (5 pg, im) at any stage of the spawning cycle, or in the parental phase, rapidly induced substrate cleaning which soon merged into oviposition behavior (without egg release). These results support a role for PG in oviposition behavior of Cichlasomu. However, indomethacin (1 mg, ip), a PG synthesis inhibitor, did not block oviposition in ovulated females which had begun to spawn. lndomethacin may not have lowered PG levels sufficiently. Alternatively, as shown by J. J. Polder (1971, Neth. J. Zoo/. 21, 265), oviposition behavior may be induced or maintained by other factors associated with the spawning situation.
Prostaglandins (PGs) have been implicated in the female sexual behavior of mammals (Rodriguez-Sierra and Komisaruk, 1978; Buntin and Lisk, 1979; Mart-one, Rodriguez-Sierra, and Feder, 1979), amphibians (Diakow and Nemiroff, 1981), and fish (Stacey, 1976, 1981; Stacey and Goetz, 1982; Villars and Burdick, 1982). In both mammals and teleosts, PGs apparently act on the brain, and may be effective within minutes (RodriguezSierra and Komisaruk, 1978; Stacey and Peter, 1979). In the goldfish, Carassius auratus, female spawning or oviposition behavior normally occurs only after ovulation but can be induced in nonovulated females by the intraovarian injection of ovulated eggs (Stacey and Liley, 1974), and inhibited in egg-injected females by indomethacin (Id), an inhibitor of PG synthesis (Stacey, 1976). PG (PGFZII.especially) injection induces apparently normal female spawning behavior in egginjected, Id-treated females, in nonovulated females which are otherwise untreated, and also in males (Stacey, 1976; 1981). In support of these ’ Present address: Department of Biology. University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO, Canada. 235 CQ18-506X/84$1.50 Copyright CJ 1984 by Academic Press. Inc. All rigbrs of reproduction in any iorm rcxrved
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behavioral studies, PGF has been shown to increase in the ovaries of several teleost species around the time of ovulation (Ogata, Nomura, and Hata, 1979; Cetta and Goetz, 1982) and also in the blood of both ovulated goldfish (Bouffard, 1979) and brook trout, Salvelinus fontin& (Cetta and Goetz, 1982). Together, these findings in externally fertilizing fish have been interpreted as indicating that ovarian PG, released into the bloodstream and acting within the brain, stimulates female sexual behaviors during the time in which ovulated eggs arc ready for release (Stacey, 1981; Stacey and Goetz, 1982; Liley and Stacey, 1983). This study examined the effects of PG on female spawning behavior in another externally fertilizing teleost, Cichlusoma bimaculatum, (Pisces: Cichlidae). Unlike goldfish, C. bimacufutum demonstrate long-term pair bonding, preparation of and joint defense of a spawning site, and parental care. Three questions were asked: (1) does PGFzoelicit behavior associated with oviposition in nonparental and parental female C. bimuculutum? (2) does PGF2, elicit similar behavior in males? and (3) does indomethacin block normal spawning in females, or affect female response to exogenous PG? METHODS AND MATERIALS
Maintenance Twenty spawning pairs of C. bimucuiutum, of 25-35 g body wt, were established from a single spawn of 8-month-old individuals. Pairs were placed in separate aquaria (67 or 102 liters) and maintained at 24-28°C under a 16-hr light: 8-hr dark photoperiod. Each aquarium had gravel substrate to a depth of 5 cm, two small clay pots, and a piece of slate. All pairs were allowed to spawn undisturbed at least once prior to experimental use. Experiments were carried out from June 9 to August 30, 1982. Description of Normul Spawning, and Behaviors Recorded Normal spawning of C. bimuculatum in our laboratory fish consisted of three phases: (1) an initial heightened defense of a nest area and an increase in mate-directed behaviors, (2) the subsequent onset of substratedirected behaviors, and (3) the synchronized release of eggs and sperm. Each of the three phases will be briefly described below. Ttio or three days prior to oviposition, both the female and male developed a darkened body color. Adults are normally pallid, with two dark, prominant spots on each side, one located midlaterally and the other at the base of the caudal fin. A barred pattern consisting of one longitudinal bar running from the eye to the caudal peduncle, and 5-6 vertical bars, may or may not be visible. In prespawning individuals, however, the background became dark grey, and the barred pattern became dusky grey to black, often obscuring the black spots completely.
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The fins, particularly the pelvics, also developed a dark grey or black appearance. Coincident with a color change, both the female and male became overtly aggressive to apparent intrusions into the area of the tank near to the spawning site. Movements outside the tank caused one or both members of the pair to attack the glass closest to the movement. Introduced food was also attacked, but not eaten. Interactive behaviors between the male and female during this phase also increased. “Rushing” by the female consisted of a rapid and directed approach toward the male, terminating in an abrupt halt in front of or parallel to him, and a brief extension of the dorsal, anal, caudal, and pelvic fins. This was usually followed by brief bursts of accelerated swimming by the female alongside or directly in front of the male. “Anal nudge,” a behavior performed only by males, consisted of the male placing his mouth close to the female’s anal region and gently butting the female’s anal or adjacent flank region with a closed or slightly open mouth. In the 24-hr period immediately preceding oviposition, the male and female started to direct increased attention toward a suitable spawning surface (usually a slate placed on the gravel substrate of the tank) and the immediately surrounding substrate. The two activities most frequently observed were associated with removing debris from the slate, and with displacing gravel adjacent to the slate. “Gravel-digging” consisted of the displacement of gravel, either by vigorous lateral undulations of the body. or by pushing movements with an open mouth, resulting in depressions in the gravel substrate near the spawning slate. This was usually done by the male. “Surface cleaning” consisted of the removal of detritus, gravel, and algae from a selected spawning surface (usually a piece of slate) by either member of the pair. This was done by assuming a verticai head-down position and biting off or picking up particles with the mouth and expelling them away from the cleaned surface. In the 4-6 hr directly preceding oviposition, the genital papilla of both adults, which are normally retracted and not visible outside of spawning and immediate postspawning phases, became fully distented. The female frequently made rushing movements toward the male, then approached the slate. Often, the male followed the female to the slate, then moved a short distance away. Shortly before oviposition (30-90 min) the female, upon approaching the slate, hovered over it in a slightly head-down position. This soon developed into jerky head-down movements over the slate surface so the gular region of the female grazed the slate as she moved forward. As the female swam forward. the tail dropped until the pelvics touched the substrate, at which point the female would halt, then briefly move away from the slate. Chin movements gradually became less jerky, and merged into smooth forward movements as the posterior portion of the female’s body became lower and the genital papilla came into direct contact with the spawning
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surface. This activity was soon followed by continuous movements over the slate, the female turning only at the edge of the slate and approaching again on a slightly different trajectory. After several such passes over the slate, the female commenced ovipositon. At the onset of female passing movements over the slate, the male approached the slate, assumed a head-down position and appeared to inspect the slate surface, shifting his orientation slightly and directing first one eye, then the other, towards the slate. As the female moved by, the male often placed his mouth close to the slate, sometimes touching it or nibbling the surface. At the onset of egg release, the male immediately adopted a horizontal posture close enough to the slate to allow contact with his genital papilla, and followed the female’s path after she had made a complete pass over the slate surface, to release sperm. Oviposition was generally complete after 15-20 min, after which both adults initiated egg-tending behaviors. Female behaviors in C. bimaculutum appear similar to the three progressive phases of spawning described by Greenberg, Zijlstra, and Baerends, (1965) and Polder (1971) for Aequidens portalegrensis.’ Initial slate-directed movements by female A. portalegrensis that consisted of short, forward movements from above the substrate and coming to a halt when the pelvic fins and abdomen came into contact with the substrate were termed “skimming.” Similar head-down movements over the slate by female C. bimuculutum were sometimes interspersed with bouts of surface cleaning, and by rushing movements directed toward the male. In A. portulegrensis, skimming soon merged into “pseudospawning,” which consisted of sliding, propulsive movements forward over the substrate, with the pelvic fins and abdomen in direct contact with the substrate, and with the anterior portion of the dorsal fin slightly spread. With the appearance of eggs, Polder distinguished the onset of “true spawning,” which differed from pseudospawning by the compression of the anterior portion of the dorsal fin towards the body, and by the presence of contractions occurring in the belly region, In C. bimucuhtum in our laboratory, differences in the position of the dorsal fin during normal spawning were not always clear-cut, but in other respects appeared identical to the behaviors of A. portulegrensis. ’ Cichlasoma bimac~tlatum is frequently marketed as Aequidens porralegrensis in both North America and Europe (see Baerends and Baerends-Van Roon, 1956,p. 2). We initially selected what we believed to be a A. porralegrensis stock for this study, because of the detailed descriptions of the reproductive behavior available on this species (Polder, 1971; Greenberg er al., 1%5). We subsequently discovered that our fish were not A. portalegrensis, but rather C. bimaculatunz (identity confirmed by D. Stewart, Field Museum of Natural History, Chicago, III.; specimen catalog Nos. 534-94748,534-94749). Nevertheless, Polder and Greenberg’s descriptions still accurately described the reproductive behaviors of our fish.
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For the purposes of recording the gross effects of PGF, on nonovulated pairs of C. bimuculatum, we did not attempt to record all the action patterns found in the spawning sequence. Instead, we selected frequently occurring behaviors that were typical of each phase, that had clear-cut onset and termination points, and that consisted of several motor patterns that usually occurred together. These behaviors were “rushing,” “anal nudge, ” “surface cleaning,” “gravel-digging,” and “spawning.” We defined “spawning” as the slow movement of a fish across a cleaned surface, with the genital papilla in close or direct contact with the surface, the tail lifted slightly, and with the anterior portion of the dorsal fin either partially or totally compressed against the body. These behaviors were recorded on a 20 channel Esterline Angus event recorder during experimental observations. Experiment
I: Female Response to Prostaglandin
(a) Nonparental females. Forty-eight hours after spawning, the eggs were removed from a pair, and 24 hr later the female was given an intramuscular injection of either 10~1 buffered saline or 5 pg PGFz, tromethamine salt (Sigma) in buffered saline. The 72-hr delay ensured that the female was not ovulating at the time of the injection (there was a minimum 5day interval between subsequent spawnings in any one female in our laboratory fish). The behavior of both the male and female were then recorded for l-hr postinjection. The durations of the most commonly observed of these behaviors, for both the male and female, were summed over successive 5-min intervals. In addition, the time from the injection to the onset of sustained surface cleaning and spawning behavior (latent period) was recorded for each test fish. Each treatment (saline or PG) was carried out on the female of seven pairs of fish. (b) Parental females. Six females from parental pairs of fish were injected with 5 pg PGFk, and subsequent behaviors were recorded for a I-hr period. In each case, young had hatched within the last 7 days. Experiment
2: Female and Male Response to Prostaglandin
One member of a pair was given an intramuscular injection of 5 pg PGF&. Two to three days later, the other member of the pair was given a similar injection. Two to three days following this, both members of the pair were reinjected with PG. For 1 hr following each injection treatment, the behaviors described in Experiment 1 were recorded for both the male and female, using an event recorder, and the latent period also was noted. Durations of the most commonly observed behaviors for both the male and female were summed over the 60-min observation period. This was repeated with eight pairs of fish.
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Experiment 3: Response to Indomethacin (a) Effect of Id on normal oviposition. Just after the onset of natural spawning, the ovipositing female was removed, given an intraperitoneal injection of either 0.15 ml saline (0.6% NaCl) or 0.9 mg Id (Sigma) in saline, and held in isolation, with no appropriate spawning surface, for 3 hr. The male and the spawning slate with attached eggs were also removed from the aquarium and placed together in isolation. Before returning the male and spawning slate to the resident aquarium, the eggs were removed. Five minutes after the male’s reintroduction, the female was returned, and the time to resumption of ovipositing was recorded. If spawning was not resumed within 24 hr, spawning behavior was recorded as blocked (i.e., no latency). Seven spawning females were Id-treated, and seven were saline-treated. (6) Effect of Id on Female Response to PGF,,. To determine whether inhibition of normal female spawning behavior following Id treatment might be attributable to interference with the female’s response to endogenous PG, rather than to an inhibition of PG synthesis, nonparental females were injected intraperitoneally with either Id (0.9 mg; n = 6) or saline (n = 7). This was followed 3 hr later by an intramuscular injection of 5 pugPGFZLl. Following the PG treatment, the latent period and the females’ behavioral responses to the PG were recorded. If spawning behavior did not occur within 4 hr, it was recorded as a nonresponse, and no latency value was assigned. RESULTS
Experiment I: Female Response to PGF,, All seven nonparenting females injected with PG subsequently showed behavior normally associated with oviposition. The two behaviors most frequently observed were surface cleaning and spawning. In six trials, females responded with sustained surface cleaning, followed almost immediately by spawning behavior 6-26 min after the injection. In the seventh pair, the male initially attacked the female when she attempted to surface clean. Sustained surface cleaning started 9 min after the injection, but sustained spawning behavior did not start until 85 min after the administration of PGFza. The mean latency for a sustained response to PG was 11.9 & 6.7 min (range = 6.8-26.1 min). Among the six females that did respond shortly after the PG injection, similar behaviors were seen. After the initial fright response to handling had passed, each PG-injected female exhibited both increased respiration rate and increased motor activity. The body color darkened in a manner similar to that of normally spawning fish, and the genital papilla became distended. Increased aggression directed toward apparent intrusions into the tank, surface-cleaning behaviors, rushing motions directed toward
PG INDUCTION
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BEHAVIOR
241
the male, and spawning movements over the slate occurred in a manner identical to normal female spawning behaviors, with the exception that egg release did not occur, and that all PG-induced behavioral elements were induced very soon after the time of injection. None of the behaviors associated with normal spawning was observed in PG-treated females immediately prior to the time of the injection. Spawning movements over the slate substrate persisted for 3-5 hr following PC injection. The proportion of time spent spawning by nonovulated, nonparental females showed an increasing trend over the 60min observation period, but the time spent surface cleaning, after initial high levels, decreased, and was maintained at a low level until the end of the observation period (Fig. 1A). Untreated males showed variable responses to the PG-induced spawning behavior of their partners. Some males showed no response at all, or retreated to a pot or aquarium corner; others sometimes developed a dark coloration typical of naturally spawning males, joined the female in surface cleaning, and initiated gravel digging. Unlike females, however, males were easily distracted, and their behaviors were often brief and incomplete. Female rushing behavior sometimes elicited anal nudging of the female by a responsive paired male; anal nudging was not observed in the male prior to the administration of PGF2, to the female. At the onset of female spawning behavior, some responsive males adopted a head-down position. and following the female, put the mouth in contact with the spawning surface directly behind the female, where thin mucous tracks were sometimes evident. However, male spawning behavior in synchrony with female spawning was uncommon, and usually sporadic. All females injected with teleost saline demonstrated a fright response typified by shelter seeking, immobility, and pale coloration. In control pairs, no reproductive behaviors were observed in either adult. All parental females, as with nonparental females, responded to PGFz,, by demonstrating spawning behavior, with a mean latency of 14.1 -C 7.8 min (range = 4.9-25.6 min). Initially, the time spent surface cleaning increased, but then declined and remained low, while the time spent in spawning behavior progressively increased (Fig. 1B). Parental females spent less time surface cleaning than did nonparental females (c’ = 2, P = 0.002), but the two experimental groups did not differ in the amounts of time spent spawning (V = 20, P = 0.473) or in their latency to response (V = 17, P = 0.314, Mann-Whitney U test) (Siegel, 1956). Experiment 2: Male-Female Response to PGF,, Of the eight experimental pairs used in Experiment 2, two spawned before the completion of all three treatments, and were omitted from the data analysis. The remaining six females responded to PG treatment regardless of the interval (which varied from 2 days to 2 weeks) between
242
COLE AND STACEY 200 160 160
“, z
140 120
z 0 100
40 20
PG 140
-I
TIME
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’ SPAWNING
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- loo v w UJ 60 z 0 la L 3 0
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TIME
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60
SURFACE CLEANING
(MINI
FIG. 1. Mean duration per 5-min observation interval of behavioral responses by (A) nonparental and (B) parental females for 1 hr following PG injection. Vertical brackets indicate 1 SE above and below the mean.
the time of the injection and the time of the last ovulation, as indicated by normal spawning. When the behavioral responses of females under all conditions were compared, the levels of female surface cleaning and spawning behavior were very low when only the male was injected, but were greater when either the female alone, or both the female and male were injected (Table la).
PG INDUCTION
OF SPAWNING BEHAVIOR
243
TABLE 1 Total Duration (set) of Spawning and Surface Cleaning Responses of (a) Female and (b) Male Fish under Different Experimental Conditions
Fish pair -___-._____ 1 2 3 4 5 6
(a) Duration of female response Surface cleaning Spawning __--_--_-.-..-. -.-.-.-.--. F F-M F-M M F .- -.-.-.-_._--__1316 1505 1 43 30 1537 758 0 123 432 933 1283 0 122 204 1697 1173 0 27 222 24 1474 0 63 91 1651 745 0 289 72 X,2 = 9.0, P = 0.008 X,, = 6.5. P = 0.05
1 2 3 4 5 6
0 2 0 37 0 0
M -37 0 0 0 0 0
(b) Duration of male response 7 0 233 246 0 225 130 71 0 0 253 0 0 129 80 0 0 0 105 0 0 0 50 0 X,z = 4.2, P = 0.18 X+ = 9.8, P = 0.006 123 34 38 20 93 175
Note. F indicates treatment where only the female of each pair was PG-injected; F-M indicates both the female and male were injected; M indicates only the male of each pair was injected. X,? values are based on Friedman two-way ANOVA tests.
The behavior of responsive PG-injected males consisted primarily of surface cleaning and spawning. However, unlike PG-treated females, males were easily distracted, causing their motor patterns to be discontinuous and of short duration. When both the male and female were injected, the male spawning response was significantly greater than when only one partner was injected. However, the male surface cleaning response was not affected by different injection conditions (Table lb). Experiment
3
(a) Effect of Id on normal oviposition. Of seven ovipositing females injected with saline, all continued to spawn, with a mean time to resumption of spawning of 76.1 ? 52.6 min (range = 36-180 min) after their return to the aquarium. Of seven ovipositing females treated with Id, two individuals did not resume spawning within 24 hr; the mean time for resumption of spawning of the remaining five females was 127.0 +- 63.8 min (range = 30-200 min). The control and experimental groups did not differ significantly in their latency periods (U = 11, P = 0.172, MannWhitney U test) or in the degree of blocking of normal spawning by Id or saline 0, = 0.23, Fisher exact test) (Siegel, 1956).
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(b) Effect ofld on jkmale response to PGFzU. When six females were treated first with Id, then with PGFzp 3 hr later, five females showed surface cleaning and spawning behavior similar to that of PGFz,,-treated females in Experiments 1 and 2; the sixth female showed no reproductive behavior within the next 4 hr. The mean latent period of the five responsive females was 17.1 + 8.3 min (range = 4-26 min). Of the seven females injected first with saline, followed by PGFzOafter 3 hr, three showed no reproductive behavior typical of a PGF2, response. The four remaining females responded in a manner similar to Id-PGF2,treated females, with a mean latent period of 35.0 + 26.5 min (range = 6-65 min). There were no significant differences between the groups, either in the proportion of females responding after Id or saline treatments (P = 0.29, Fisher exact test), or in response latency (U = 5, P = 0.14, Mann-Whitney U test). DISCUSSION
The results of this study demonstrate clearly that PG injection in female rapidly induces several behavioral components normally associated with oviposition. These behaviors include aggressive defense of the spawning site, certain behaviors directed toward the male, cleaning of potential spawning substrates, and spawning (oviposition without egg release) behavior. The findings indicate that PG may be the endogenous signal synchronizing oviposition behavior with ovulation in this species, as had been suggested for goldfish (Stacey, 1981). Normal spawning behavior in ovulated C. bimaculatum involves egg release. However, other behaviors similar to skimming and pseudospawning, as described by Greenberg et ul. (1965) and Polder (1971) for A. portulegrensis, precede the onset of oviposition by one or more hours. Polder (1971) found that during the intense skimming phase, ovulation (follicular rupture) had occurred in only a few follicles in A. portalegrensis. However, with the appearance of pseudospawning, ovulation was completed, and shortly thereafter true spawning with egg release commenced. Except for the difference in dorsal fin position and the absence of egg release, pseudospawning in A. portulegrensis is virtually identical to true spawning. Possibly, the latency between the onset of pseudospawning and the onset of true spawning represents the time required for ovulated eggs to travel through the oviduct to the genital papilla. Close temporal synchrony between ovulation and the onset of spawning also has been noted in the cichlid Sarotherodon melunotheron (= Tilapiu macroceph&) (Aronson and Holz-Tucker, 1949) and likely is typical of many teleosts (see Liley and Stacey, 1983). It is not clear whether skimming behavior in C. bimuculutum, which likely begins near the time of ovulation, also is stimulated by cndogenous PG. However, ovarian PG is involved in follicular rupture in teleosts C. bimaculutum
PG INDUCTION
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(Stacey and Goetz, 1982), and it is possible that follicular PG synthesis prior to ovulation is sufficient to stimulate skimming behavior. This possibility is supported by the finding that plasma PGF levels increase in brook trout coincident with oocyte final maturation, prior to ovulation (Cetta and Goetz, 1982). Both the effects of PG injection, and the temporal relationship between ovulation and spawning, support a role for endogenous PG in female C. himaculutum spawning behavior. However, Polder (1971) has shown that skimming and pseudospawning can also be induced within minutes in nonovulated female A. porta1egrensi.s simply by exposing the female to a clutch of eggs from another pair; similar responses were observed in males. This effect of eggs, which likely is not mediated by endogenous PG, indicates that in normally spawning pairs, once spawning has commenced, it may be maintained by visual or chemical cues from the nest site. lndomethacin did not block the resumption of normal spawning in ovulated C. bimacufutum, or reduce female responsiveness to PG. These findings are in contrast to the complete blockage of spawning behavior in egg-injected (Stacey, 1976) or ovulated goldfish (Stacey and Goetz. 1982) after Id treatment. One explanation for this discrepancy is that indomethacin may not be a potent inhibitor of PG synthesis in C. himuculutum. Another possibility is that PG may not be rapidly metabolized in Id-treated females that have already ovulated. PG has been found in the ovarian fluid of ovulated female goldfish (Bouffard, 1979), and the coelomic fluid of ovulated brook trout (Cetta and Goetz, 1982). If PG is present in the ovarian fluid of ovulated female C. himuculutum, it may be sufficient to sustain spawning behavior even after subsequent PG synthesis has been inhibited by Id. The responsiveness of female C. bimuculutum to experimental levels of exogenous PG did not vary with the reproductive cycle. Whether they were tested several days after spawning, at a time of rapid oocyte growth (Polder, 1971), or more than a week after spawning, at a time when oocyte growth has been completed (Polder, 1971), females exhibited equivalent levels of PG-induced spawning behavior. Furthermore. responsiveness was not affected by the presence of recently hatched young. As all tests involved a similar high PG dose (approximately 150 ng.g-’ body wt), the possibility remains that the minimum dosage required to elicit spawning may vary with ,ovarian condition. However, the present results suggest that, as in goldfish (Stacey, 1981; Stacey and Goetz, 1982). responsiveness to PG in female C. bimuculutum is not obviously influenced by reproductive condition. This situation is in marked contrast to that in the paradise fish, Mucropodus operculuris, in which female spawning responsiveness to exogenous PGF declines dramatically several days after spawning (Villars and Burdick. 1982).
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Unlike female C. bimacufatum, which spawned readily following PG treatment, males responded consistantly to PG only if their female partner also was PG-treated. In contrast, PG treatment of goldfish of either sex induces equivalent levels of female spawning behavior (Stacey, 1981; Stacey and Kyle, 1983). The low responsiveness to PG exhibited by male C. bimaculatum is difficult to interpret because their behavioral responses were too brief and incomplete to clearly indicate whether male or female behavior was being displayed. In natural spawning, there are some qualitative differences in the spawning behavior of males and females. Males displace gravel to form pits in the substrate, direct anal nudging towards females, and before and during egg release by the female, repeatedly approach the slate in a head down position and appear to inspect the slate for the presence of eggs. None of these behaviors is typical of females, and none was seen in the repertoire of PG-injected males. If PG-treated males are indeed performing female spawning behavior, two factors could contribute to their low responsiveness. First, male C bimaculatum may be behaviorally defeminized through developmental processes, as has been well documented in mammals and birds [see reviews by Feder (1981) and Adkins-Regan (1981)]. This is in contrast to goldfish, where PG-treated males readily show female behavior (Stacey, 1981; Stacey and Kyle, 1983). Second, male C. bimaculatum may be physiologically capable of female levels of responsiveness to PG, but may require specific stimuli from a partner showing male behavior in order to express this female potential. In goldfish, PG-treated males require the presence of a responsive male in order to display female spawning behavior. This is easy to provide, since untreated males readily court and spawn with PG-treated males (Stacey, 1981). In contrast to goldfish, however, male-to-male aggression in C. bimaculatum would likely preclude the use of homosexual male pairs to examine this possibility. Nonetheless, the dramatic increase in male spawning responses to PG when the female partner also was PG-treated indicates that the behavior of the female partner can influence male responsiveness to PG in this species. Initial reports of the effects of PG on female sexual behavior in goldfish (Stacey, 1976, 1981; Stacey and Goetz, 1982), a teleost with relatively simple reproductive behavior involving no pair formation and no territorial or parental activities, demonstrated that PG-treatment of nonovulated females induces normal spawning interactions between the sexes. The present study demonstrates that, even in a teleost where complex and protracted interactions both precede and follow oviposition, PG treatment induces in nonovulated females a set of behaviors associated with normal spawning. The use of PG treatment as a technique for investigating sexual interactions may become an important tool in future studies of the reproductive behavior of cichlids and other teleostean fishes.
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ACKNOWLEDGMENTS We thank D. L. G. Noakes, R. J. F. Smith, and two anonymous reviewers for critical comments on the manuscript, and J. R. Baylis for bringing to our attention the problem of species identification for Aequidens portalegrensis and Cichlasoma himaculatum. This study was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) University Research Fellowship (UOOO7)to N.E.S.
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