Sperm characteristics associated with different male reproductive tactics in bluegills (Lepomis macrochirus)

Transcription

1 Behav Ecol Sociobiol (2000) 49:31 37 Springer-Verlag 2000 ORIGINAL ARTICLE Brenda Leach Robert Montgomerie Sperm characteristics associated with different male reproductive tactics in bluegills (Lepomis macrochirus) Received: 18 February 2000 / Revised: 4 September 2000 / Accepted: 14 March 2000 Abstract We examined the availability and motility of sperm from parental and sneaker male bluegills (Lepomis macrochirus), a colonially nesting sunfish (Family Centrarchidae) with male parental care and a high incidence of cuckoldry by both sneaker and satellite males. We found no differences between sneakers and parentals in length and swimming speed of sperm, or percent and duration of sperm activity. In sneaker milt, however, sperm was almost 50% more concentrated than in parental milt ( vs sperm/µl of milt, respectively). Despite this difference in sperm concentration, stripped ejaculates from sneakers contained almost 400 million fewer sperm (only 32% as many sperm) than those from parentals due to their much smaller stripped ejaculate volumes (only about 19% that of parentals). Thus unless sneakers can compensate by releasing more sperm or gaining closer proximity to eggs at the time of spawning, they may be at a disadvantage with respect to sperm competition. We discuss these results in relation to models for the evolution of alternative reproductive behaviours in this species and suggest that the cuckolders may be making the best of a bad situation. Keywords Sperm competition Sperm Fish Alternative reproductive strategies Introduction Sexually mature male bluegills (Lepomis macrochirus) employ one of three different mating tactics, representing two distinct life histories. Parental males delay reproduction until age 7 years when they are approximately 17 cm long (Gross 1982). When sexually mature, they Communicated by J. Reynolds B. Leach R. Montgomerie ( ) Department of Biology, Queen s University, Kingston, Ontario, K7L 3N6, Canada montgome@biology.queensu.ca Tel.: , Fax: build nests, court females and defend their brood. Sneaker and satellite males, on the other hand, mature younger (usually age 2 years) and at a smaller size (7.3 cm and 9.5 cm, respectively), and adopt cuckolder reproductive tactics. Cuckolder males do not defend nests but instead parasitize the nests of parental males by attempting to fertilize the eggs of females during spawning, either darting into the nest from nearby cover (sneakers) or mimicking female behaviour and appearance (satellites) and following females into nests to avoid detection by parental males (Gross 1982). Such alternative mating tactics are common, particularly in fish species with external fertilization (Taborsky 1994, 1998), and the physical and behavioural differences between the males adopting the different tactics can be striking. Early maturing or precocious males are usually at a disadvantage with respect to size, female preference (Jones 1959; Gage et al. 1995), and conspecific aggression (Sandercock 1991), but appear to obtain some reproductive success by parasitizing the reproductive effort of their larger counterparts. Many studies have compared the mating success of alternative male tactics, but determining both the lifetime fitness of individuals and the underlying genetic mechanisms maintaining these alternate tactics is difficult. Both of these details are required to ascertain whether the different phenotypes are (1) alternative strategies with equal fitnesses resulting from a genetic polymorphism, (2) alternative tactics with equal fitnesses (within a mixed strategy) resulting from a genetic monomorphism or (3) alternative tactics with unequal fitnesses (within a conditional strategy) resulting from a genetic monomorphism (Gross 1996). The demonstration of genetic polymorphism for mating tactics is extremely rare, suggesting that alternative tactics are usually maintained within mixed or conditional strategies (for a review see Gross 1996). Concomitant with the physical and behavioural differences between male tactics, one might expect differences at the gametic level, particularly if cuckolder males are to compensate for their size disadvantage. Gametic differences are expected because sperm competition be-

2 32 tween males with different phenotypes is often intense, particularly in species with external fertilization where females have limited control of paternity (Parker 1990; Ball and Parker 1996; Parker et al. 1996). Interspecific comparative studies have shown that both sperm length and sperm production are positively correlated with the intensity of sperm competition in internal fertilizers such as mammals (Harcourt et al. 1981; Møller 1988a; Gomendio and Roldan 1991), birds (Møller 1988b; Briskie and Montgomerie 1992), and butterflies (Gage 1994). Recently, however, Stockley et al. (1997) found a negative relationship between sperm length and the intensity of sperm competition across fish species, though both sperm numbers and relative gonad size did increase when competition was more intense. At the gametic level, there are four main ways by which cuckolder males could offset their apparent mating disadvantage and thereby increase their reproductive success. First, they could enhance reproductive success by increasing sperm production (Parker et al. 1996). In a study comparing precocious (parr or sneaker) and anadromous (parental) male Atlantic salmon (Salmo salar), Gage et al. (1995) found that parr had significantly larger testes relative to body mass (often called the gonadosomatic index or GSI), and larger stripped volumes of ejaculate relative to body mass. GSI is assumed to reflect relative investment in sperm production by males (Gage et al. 1995) and is commonly found to be high in cuckolder (or parasitic) males (Taborsky 1994). Although no studies have compared sperm production by precocious and adult males, there is a positive relationship between GSI and the intensity of sperm competition across fish species (Stockley et al. 1997). Increased total production or rate of production by precocious males could increase their fitness by allowing them to participate in more spawnings per unit time or expel more sperm per ejaculate. Second, by increasing sperm concentration, cuckolders could compensate for their usually smaller body and testis size. If sneaker reproductive success is limited by their production or storage of milt, increasing the concentration of sperm in their milt may be a way of maximizing the number of sperm expelled in a small volume. Data in Kazarov (1981), however, suggest that milt from parr (sneaker) males is in fact less concentrated than that from anadromous (parental) male Atlantic salmon. Third, if all eggs shed by the female are not fertilized immediately, sperm that stay active longer will have more opportunity for fertilization (Ball and Parker 1996). Thus, the longevity of active sperm may be a better predictor of fertilization success than the total number of sperm, especially if it is difficult to time ejaculation to the shedding of eggs. In the rose bitterling (Rhodeus ocellatus), for example, sneakers often ejaculate before the female lays eggs. Sneaker sperm stays active and is maximally capable of fertilizing eggs for at least 60 s and can fertilize some eggs for as long as 180 s (Kanoh 1996). Moreover, parr (sneaker) Atlantic salmon sperm have a higher percent motility and longer duration of motility than sperm from anadromous (parental) males (Gage et al. 1995), suggesting that selection is capable of altering these sperm traits within species. Fourth, faster-swimming sperm could have an advantage over other sperm spawned simultaneously from the same distance, or could counter the disadvantage of being ejaculated later or further from the eggs. Although various measures of motility have been used, few studies have estimated actual swimming speeds of individual fish sperm (Trippel and Neilson 1992; Lahnsteiner et al. 1996), and no studies have compared the swimming speed of sperm from parental and cuckolder males. In this study we examined sneaker and parental male bluegills for differences in stripped ejaculate volume, milt concentration, sperm swimming speed, and the percent and duration of sperm motility. Previous work on alternative reproductive tactics in this species has assumed that sperm competition between parentals and cuckolders is probably not an important factor influencing their reproductive success (Gross 1991) and that the reproductive success of males adopting different tactics could be estimated simply by determining mating (ejaculation) frequency (Gross 1982). A successful cuckolder intrusion has also been assumed to result in most of the eggs released by the female being fertilized by that cuckolder (Gross 1982). Although we cannot test these assumptions directly with our data, our results suggest that they may be in error. Methods Parental and sneaker male bluegills were taken from active colonies in Lake Opinicon, Ontario, Canada, between 27 June and 6 July Satellite males were not included in this study because it was difficult to distinguish them from females during spawning. We used a dip net to capture males near nests during the short spawning period at each colony and transported them to the laboratory where they were kept in a tank maintained at lake temperature (14 18 C). Between 12 and 18 h after capture, we measured total length and body mass of each male, and stripped them of milt by applying gentle pressure to their abdomen. We collected milt from these stripped ejaculates in 40-µl microcapillary tubes, recorded total volume collected, and used subsamples for immediate analysis of motility and preparation of slides for light microscopy. Measuring motility A sample of µl of freshly collected milt was activated by mixing it in 1 2 ml distilled water (i.e. 100 the volume of milt) at 18 C. Sperm swimming speed can vary with water temperature, so we used 18 C water to standardize across measurements since actual water temperature in the lake varied considerably during the course of our study. Each activated sample was stirred vigorously for 10 s, and then a drop was added to an Improved Neubauer haemocytometer. Activity was recorded on videotape using a COHU High-Performance CCD camera mounted on a Leitz DMRB light microscope using a 40 objective. A few samples that did not have 100% motility when first recorded (about 30 s after activation) were assumed to have been activated by urine during sampling and were, therefore, discarded. We played back videotapes frame by frame into version 1.60 of the public domain NIH Image program (developed at the U.S.

3 33 National Institutes of Health and available on the Internet at running on a Macintosh computer. Using this program, we marked the position of a focal sperm every 0.5 s over a 2-s interval and recorded the total distance travelled. The total distance travelled was divided by the 2-s swimming time to give an average speed. This procedure was repeated for ten haphazardly chosen sperm from each sample at intervals of 45, 60, 90, 120, 150, 180 and 240 s after activation. On the computer monitor, the sperm were approximately 25 mm long, or about 500 times their actual size. The percentage of sperm active (showing propulsive motility) at each of these times was also measured by estimating the number of sperm in the field of view (range 300 1,000) and the number of those that were still motile (the percentage was calculated to the nearest 5%). Even though the haemocytometer chamber was deep enough to allow sperm to swim in and out of focus, all movement in the field of view was readily detectable. After all forward motility had ceased, we determined the concentration of sperm in the milt by counting the number of sperm in five haemocytometer cells and multiplying by the appropriate dilution factor. In a separate study of bluegills in 1999, using the same methods developed here, we determined that the repeatabilities of our estimates of both swimming speed and motility were both statistically significant (R. Montgomerie, unpublished data). To estimate repeatability, we took up to four sperm samples from each of nine parental males and measured the motility of each sample after 45 s and the speeds of ten haphazardly chosen sperm. Repeatability of swimming speed was 0.64 and that of motility was 0.72 Measuring sperm tail lengths To prepare a slide for light microscopy, a drop of milt was placed on a clean glass slide, and a drop of distilled water added. We then spread the diluted sperm sample thinly across the slide and allowed it to air dry. For each male, ten haphazardly chosen sperm that could be clearly seen were videotape recorded and digitized using the NIH Image program. Tail lengths of sperm in the digitized images were measured to the nearest 0.01 µm (calibrated using the grid scale on the haemocytometer under the same magnification). Statistical analysis All statistical analysis was performed with JMP software (ver , SAS Institute). To help normalize residuals and provide a more linear fit in correlation analyses, we transformed data on swimming speed (log) and the percentage of sperm showing forward motility (angular) before statistical analysis. Residuals from the regressions of body mass on total length (for sneaker and parental males separately) were calculated for each male and used as a measure of condition. When more than one analysis was applied to a set of data to search for significant relationships, we applied the sequential Bonferroni correction when testing for statistical significance (Rice 1989). Results Milt production The total volume of milt in a single stripped ejaculate was greater in parental (mean=63.4 µl) than sneaker males (11.90 µl; t-test, t=3.40, P=0.003, n=12, 10; Fig. 1a). In fact, the largest stripped ejaculate from a sneaker male was 30 µl, smaller than that of most ejaculates (75%) from parental males (Fig. 1a). Per gram body mass, sneakers did have a significantly greater volume of milt (2.29 vs 0.45 µl/g body mass; t=2.13, P=0.046, n=12, 10; Fig. 1b) and significantly greater number of sperm ( vs sperm/g body mass; t=2.10, P=0.049, n=11, 10) per stripped ejaculate than parentals. Mean sperm concentration in the milt of sneakers (16.5 million sperm/µl) was significantly and about 50% greater than in parentals (11.5 million sperm/µl; t=2.53, P=0.02, n =11, 10; Fig. 1c). Despite having a higher sperm concentration in their milt, the total number of sperm in a stripped ejaculate was still significantly smaller in sneakers ( ) than in parentals ( ; t=3.28, P=0.004, n=11, 10; Fig. 1d). Volume of milt and total number of sperm in a stripped ejaculate were not significantly related to body mass, total length or condition for either parentals or sneakers (Table 1). Sample sizes for these analyses were small, however, so statistical power was low. Fig. 1 Box plots comparing stripped ejaculates of parental and sneaker male bluegills with respect to volume of milt (a), ejaculate volumes per gram fish body mass (b), concentration of sperm in the milt (c) and total number of sperm per ejaculate (d). Box plots show 10th, 25th, 50th, 75th and 90th percentiles with horizontal lines, and all data points outside this range, with sample sizes at the top of each box

4 34 Table 1 Correlations between stripped ejaculate characteristics (volume and number of sperm) and the body mass, total length and condition index of parental and sneaker male bluegills. None of the correlations is significant (all P>0.13) Mass Length Condition Parentals Ejaculate volume (n=12) Number of sperm (n=11) Sneakers Ejaculate volume (n=10) Number of sperm (n=11) Table 2 Results of repeated-measures ANOVAs examining the effects of time since activation on motility (angular-transformed) and swimming speed (log-transformed) of bluegill sperm. Motility is measured as the percentage of sperm active at each time interval since activation; swimming speed is the average speed of about ten sperm sampled haphazardly from the same ejaculate of each male at each time interval. Note that time is a fixed effect in these models Source of variation df MS F P Motility Fish < Tactic Time 5 17, < Tactic time Error Table 3 Correlations between mean sperm length per male and other male and ejaculate characteristics of parental (n=12) and sneaker (n=10) bluegills. After Bonferroni correction only *P 0.01 is significant Parentals Sneakers r P r P Body length (cm) * Body mass (g) Condition (g/cm) Number of sperm/ejaculate Ejaculate volume (µl) Swimming speed Fish < Tactic Time 5 12, < Tactic time Error Activity and swimming speed The percentage of sperm showing forward motility decreased significantly over time (Table 2), with all samples having virtually all sperm motile at 45 s and practically all activity ceasing within 4 min after activation (Fig. 2). The motility of sperm over time did not differ significantly between parental and sneaker males (Table 2). Average swimming speeds also decreased significantly over time for both types of male (Table 2, Fig. 3), but there was a significant interaction between time (since activation) and tactic (parental or sneaker). This interaction suggests that the average sperm swimming speeds of parentals and sneakers declined at different rates. Indeed, contrast analysis shows that parental sperm swam significantly faster than sneaker sperm 45 s after activation (t=5.32, P<0.0001, df=68; Fig. 3). The mean swimming speed for parental sperm was faster than that for sneakers at all other times, though the differences were not significant. Sperm morphometry Average sperm lengths in parental and sneaker males were not significantly different (repeated-measures ANOVA, F=0.82, P=0.37, df=1,198; Fig. 4), but there were significant differences among males within tactics Fig. 2 Percentage of sperm showing forward motility at different intervals since activation for parental (n=8) and sneaker (n=8) male bluegills. Dot size is relative to the number of coincident data points; lines connect mean values (F=2.74, P=0.0002, df=21,198). Most of the variation in sperm length across both tactics occurred within (85.4%) rather than between males (14.6%). Moreover, average sperm length per male was not significantly related to average sperm swimming speed at 45 s after ac-

5 35 Fig. 3 Mean swimming speeds of sperm (n=5 10 sperm per male) from parental (n=8) and sneaker (n=7) male bluegills at different intervals since activation. Dot size is relative to the number of coincident data; the positions of some dots have been shifted slightly on the abscissa to improve clarity; lines connect mean values. The dashed line on the sneakers graph is the parentals curve Fig. 5 Relationship between mean sperm length (SPL; n=5 sperm per male) and body length (BL) for parental (n=12) and sneaker (n=10) male bluegills. The model II regression line is shown for sneakers, SPL= BL (r= 0.76, P=0.01) Discussion Fig. 4 Mean lengths of sperm (n=5 sperm per male) from parental (n=12) and sneaker (n=10) male bluegills. Box plots as in Fig. 1 tivation for either parentals (r=0.15, P=0.66, n=12) or sneakers (r=0.40, P=0.32, n=10). Sperm length of parental males was not significantly correlated with any male trait (Table 3) though the positive correlation with condition is suggestive, especially given the low statistical power of this analysis. In sneakers, however, sperm length was significantly, but negatively, related to total length (Fig. 5, Table 3). Thus larger sneakers had shorter sperm and again the positive relationship between sperm length and condition is interesting though far from significant. The sperm characteristics (morphology, motility) of sneaker male bluegills do not appear to be much different from those of parentals. Based on previous empirical studies, we expected a difference in sperm length to have evolved due to sperm competition, though whether sneakers or parentals would be expected to have the longer sperm is not clear (contrast Gomendio and Roldan 1991; Briskie and Montgomerie 1992; Gage 1994 with Stockley et al. 1997). Nonetheless, the sperm lengths of sneaker and parental male bluegills did not differ. Theory initially suggested that sperm number rather than size would most likely respond to sperm competition (Parker 1993). More recently, however, Ball and Parker (1996) have shown that sperm size should increase with the intensity of sperm competition when sperm longevity (i.e. duration of forward motility) decreases with size but should decrease under more intense competition when sperm longevity increases with size. Though studies so far indicate that longevity decreases with size across fish species (Stockley et al. 1997), there is no information on this relationship within species, in part because relatively little intraspecific variation has been detected so far. Therefore, other factors possibly constrain the evolution of sperm morphology and behaviour within species (Parker et al. 1996).

6 36 Interestingly, there was a negative relationship between sperm length and body length in both sneakers and parentals (though significant only for sneakers). The (non-significant) tendency for both parental and sneaker males in better condition to have larger ejaculates and longer sperm is also interesting and deserves further study. Neither of these patterns is easily explained nor have they been found previously or predicted from theory. If there is a positive relationship between sperm length and swimming speed, as has been suggested for mammals (Gomendio and Roldan 1991), males in better condition would be at a competitive advantage in sperm competition. Recent evidence, however, suggests that there is no interspecific relationship between sperm length and swimming speed in birds (T.R. Birkhead, personal communication) or Pacific salmon (Leach 1997). Moreover, why selection would favour shorter sperm in larger fish, within species, is not clear. In our samples there were also no clear differences between parentals and sneakers in sperm motility or swimming speed, except that sneaker sperm swam significantly and considerably slower (73% as fast) at the first interval we sampled (45 s) after activation (Fig. 3). Thus sneaker sperm do not have any of the expected adaptations to increase fertilization success under intense sperm competition and, if anything, would appear to be at a disadvantage in sperm competition during the first minute after ejaculation. Further study is required to determine whether this pattern holds from the instant of activation and what mechanism might be responsible for variation in swimming speed among sperm of the same size and general morphology. We assume that the earliest swimming speeds are most important, but exactly how long after ejaculation (and sperm activation) the majority of fertilization takes place in external spawners is not known. In addition, the volume of milt obtained from a single stripped ejaculate was far smaller in sneaker than parental males. Even though the milt of sneakers was almost 50% more concentrated with sperm than that of parentals, the average stripped ejaculate from a sneaker was so much smaller in volume that it contained 400 million fewer sperm than that from a parental male. This pattern of higher sperm concentration in sneakers has also been found in Atlantic salmon (Kazakov 1981; Gage et al. 1995), but its significance is unknown. Milt production and storage seem to be limited in some species (Foerster 1968; Gharrett and Shirley 1985; Pitnick and Markow 1994; Shapiro and Giraldeau 1996), and increasing concentration may enable sneakers to maximize sperm numbers in an ejaculate when volume is constrained (see Taborsky 1994 for a discussion of such trade-offs). Is there any disadvantage to producing a highly concentrated milt? This has not been examined directly but levels of citric acid (a sperm motility inhibitor) are known to increase with sperm concentration in the milt (Piironen and Hyvärinen 1983). Glucose, fructose and glycerol levels are similarly variable in milt (Piironen and Hyvärinen 1983) but the effect of these substances on sperm motility or fertilization success is unknown. Given the potential advantages of higher sperm concentration in the milt, a cost also seems likely, otherwise the lower concentration of parental sperm is difficult to explain. Sneakers do produce more gonad per unit body mass than parentals, but this obviously does not translate into more sperm in a stripped ejaculate. Sneaker male bluegills clearly suffer more intense sperm competition than parentals since they always attempt to fertilize eggs in the presence of parentals whereas the reverse is not true (Gross 1982). Thus a higher relative investment in gonads and sperm production in sneakers is expected from theory (Parker 1990; Gage et al. 1995) and has been found in all relevant studies of fish published to date (e.g. Kazakov 1981; Gross 1982; Gage et al. 1995; Leach 1997; Stockley et al. 1997; Taborsky 1998). The relationship between stripped and natural ejaculate volumes is not known for this (or any) fish species, nor is the relationship between relative gonad size (measured as GSI) and the rate of milt production. Both of these relationships will need to be determined before betweentactic differences in GSI or stripped ejaculate volume can be accurately interpreted. Faster-swimming sperm may be advantageous if there is active competition among sperm at the site of fertilization (Gage et al. 1995). While it seems logical that there would be a correlation between sperm swimming speed and fertilization success, this idea has not been tested. Sperm swimming behaviour is also believed to change when sperm are near the micropyle of the egg (Stoss 1983; Iwamatsu et al. 1993). In our study, swimming speeds were calculated for sperm that were not in the presence of eggs, so further testing is needed to determine if parental male sperm also swim faster in the vicinity of a micropyle. One possibility is that there is a chemical substance or guiding factor on the surface of the micropyle (Iwamatsu et al. 1993) and this could differentially affect parental and sneaker sperm. Since the sperm of parental and sneaker male bluegills are not different in length, swimming speed must be influenced by some other factor such as energy (ATP) stores (Billard et al. 1995). How do these results help to address the assumptions of existing models for the evolution of alternative reproductive tactics in this species (Gross 1982, 1991)? Our results show that sneakers do not appear to have evolved any sperm traits (morphology, concentration or swimming behaviour) that would provide them with an advantage over parental males at the level of sperm competition. That is not to say that sneakers could not still gain some advantage by timing their ejaculations carefully (e.g. Kanoh 1996), by positioning themselves closer than parental males to the unfertilized eggs when ejaculating, or by having larger ejaculates (we measured only stripped ejaculates). So far we have no quantitative data on these details of bluegill spawnings. Indeed, predictions of lifetime fitness in this species have assumed a lack of sperm competition in general (Gross 1982), and

7 attributed fertilization success as random when parental and sneaker males ejaculated simultaneously and complete fertilization success by sneakers when they were the first to ejaculate (Gross 1991). We have shown, however, that parental males could gain a clear advantage in sperm competition via their superior (threefold) sperm numbers and their faster-swimming sperm shortly after activation. Thus, our results suggest that sneaker male bluegills may have lower fertilization success than parentals during fertilization contests at a given spawning event and as a result, assuming the other aspects of Gross (1982, 1991) models are correct, they could have lower lifetime fitness than parentals. If this is so, the sneaker tactic would not be evolutionarily stable but simply making the best of a bad situation in a conditional strategy. 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