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Behavioral Ecology

Original Article

Exploring simultaneous allocation to mating effort, sperm production, and body growth in male guppiesAlessandro Devigili,a Victoria Doldán-Martelli,a,b and Andrea Pilastroa

aDepartment of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy and bUniversidad Autónoma de Madrid, Facultad de Ciencias, Madrid, 28049 Cantoblanco, SpainReceived 10 February 2015; revised 28 April 2015; accepted 30 April 2015; Advance Access publication 3 June 2015.

In several species, males increase their mating and sperm investment in the presence of unfamiliar females, the so-called Coolidge effect. Such an elevated reproductive effort is expected to be associated with a decreased investment in other costly traits, such as somatic growth and maintenance. How precopulatory, postcopulatory, and somatic investments interact one with each other, however, has been rarely evaluated simultaneously. We used the guppy (Poecilia reticulata), a polyandrous livebearing fish with alternative male mating tactics, to compare these 3 investments between males whose encounter rate with unfamiliar females was experimentally maintained at either high (high mate encounter rate [HER]) or low rate (low mate encounter rate [LER]) for 4 months. At the end of this period, HER males showed an increased sperm production as compared with their LER counterparts. This increment was accompa-nied with a reduction in the time spent following the female and by a shift from costly courtship displays to gonopodial thrusting, a less expensive coercive mating tactic. This effect may indicate a trade-off between these 2 components of male reproductive invest-ment or may reflect a change in male optimal reproductive strategy associated with different mate encounter rate. In contrast, body growth was positively correlated with the increase in sperm production, and did not differ between experimental groups. Collectively, these results suggest that males did not vary their overall reproductive investment in response to their encounter rate with unfamiliar females, but differed significantly in the amount of resources available individually.

Key Words: Coolidge effect, phenotypic flexibility, Poecilia reticulata, sexual investment, social context, somatic investment, sperm competition, trade-offs.

INTRODUCTIONIn polyandrous species, in which males compete both to acquire mates and to fertilize their eggs (Parker 1970, 1984), selection is expected to favor males investing in traits associated with both pre-copulatory and postcopulatory components of the reproductive success. Within a set amount of resources allocated to reproduc-tion, trade-offs are expected to arise between mate acquisition/monopolization and fertilization traits (Parker 1998). Comparative evidence of a trade-off between precopulatory and postcopulatory traits has accumulated (see Kvarnemo and Simmons 2013 for a recent review), although the sign of the covariation between these traits depends on male capability to monopolize females and do not, if negative, demonstrate a trade-off per se (Lüpold et al. 2014). Similarly, trade-offs have also been evidenced at the intraspecific level, for example, by revealing the occurrence of negative genetic

covariation between precopulatory and postcopulatory traits (e.g., Evans 2010).

Within species, trade-offs between costly traits can also be revealed by experimentally manipulating the investment in 1 trait and observing the change in the others during the ontogeny (Reznick et al. 2000; Simmons and Buzatto 2013 for a recent exam-ple). After sexual maturation, however, the allocation to precopula-tory and postcopulatory traits can be experimentally manipulated only in those species in which these traits are plastic throughout the lifespan (sensu Piersma and Drent 2003). Postcopulatory traits such as sperm number and performance (for example swimming velocity, viability, and number) are typically plastic and can show very rapid changes. For example, in domestic fowl males, Gallus gallus domesticus, sperm quality rapidly drops in the winner of the contest, while it remains constant in the looser (Pizzari et al. 2007). In the cockroach Nauphoeta cinerea, frequent competitive interac-tions between males are associated with a decrease in spermato-phore size and sperm numbers (Montrose et  al. 2008). Although these results are likely to be the consequence of a trade-off between Address correspondence to A. Devigili. E-mail: alessandro.devigili@unipd.it.

Behavioral Ecology (2015), 26(4), 1203–1211. doi:10.1093/beheco/arv067

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Behavioral Ecology

precopulatory and postcopulatory investment, there is an alterna-tive explanation: dominant/guarding males usually face a lower level of sperm competition compared with subordinate/sneaker males and their optimal postcopulatory investment is therefore lower than that expected for subordinate/sneakers (Parker 1990; Parker et  al. 2013). When an increased precopulatory investment is associated with a reduction of sperm competition risk, a nega-tive correlation between the 2 types of traits is expected (Lüpold et  al. 2014). Indeed, there is ample evidence that males respond to a perceived change in the risk of sperm competition by strate-gically adjusting their sperm allocation (Kelly and Jennions 2011). Therefore, the experimental design adopted to manipulate male investment in precopulatory or postcopulatory traits should ideally maintain constant the perceived level of sperm competition across treatments.

There is another complication associated with studies on the trade-off between precopulatory and postcopulatory traits: this trade-off is expected if the overall resources allocated to reproduc-tion are constant. Thus, an experimental manipulation of a male’s precopulatory investment may, for example, be obtained by real-locating resources from somatic maintenance, without affecting his postcopulatory allocation. Indeed, reproduction and survival are commonly traded off, and evidence has been found in both females (e.g., Dijkstra et  al. 1990) and males (e.g., Van Voorhies 1992; Miller and Brooks 2005; Sentinella et al. 2013). Additionally, nega-tive genetic covariance between male ornaments and survival has also been found (Brooks 2000; Hunt et al. 2004). Trade-offs among reproductive traits may therefore be masked if males adjust their resource allocation from maintenance to reproduction (Parker et al. 2013). Although there is evidence that both elevated precopulatory and postcopulatory investment have a negative effect on body con-dition (e.g., Mappes et al. 1996) and maintenance (McNamara et al. 2013), studies simultaneously investigating the intercorrelated allo-cation to precopulatory and postcopulatory traits and to somatic investment are scarce. The only study we are aware of indeed found evidence that, in horned beetles, males prevented from devel-oping horns attained a larger body size and invested relatively more in testes than males which were allowed to grow horns (Simmons and Emlen 2006).

We investigated these potential 3-way trade-offs in the guppy (Poecilia reticulata), a live-bearing fish with internal fertilization char-acterized by alternative male mating behaviors: individual males can interchangeably adopt either a cooperative or a coercive mating tactic (Liley 1966). In the first case, males actively court the female with a stereotypical behavior: sigmoid displays (SDs), whereas in the second case males attempt to forcibly inseminate the female (“gonopodial thrust”, [GT]); Houde 1997). Among the numerous male characteristics that influence female mate choice, the most important are body size, size and brightness of color spots, and courtship rate (Houde 1997). When sexually receptive, females mate cooperatively, often with several males (Magurran 2005). As a consequence, guppies exhibit one of the highest levels of multiple paternity (and hence sperm competition) reported for vertebrates (e.g., Hain and Neff 2007). Sexual selection is therefore expected to be strong on both precopulatory and postcopulatory male traits (Evans and Pilastro 2011; Rios-Cardenas and Morris 2011).

As proposed for species where female monopolization is low (Lüpold et  al. 2014), in guppies, precopulatory and postcopula-tory male sexual traits positively covary phenotypically (Pitcher and Evans 2001; Locatello et al. 2006), although not in all populations (Evans 2010; Gasparini and Evans 2013). This positive correlation

may be due to a variation in the allocation between reproduction and survival and/or to a variation in the total resources available to males (van Noordwijk and de Jong 1986). Our experimental manipulation of male reproductive effort is aimed at exploring the pattern of male resource allocation between precopulatory and postcopulatory traits, on the 1 hand, and somatic investment (body growth), on the other hand.

Male sexual behavior in guppies is flexible and can be adjusted to the actual (e.g., Jirotkul 1999a; Evans et al. 2002) and previously experienced social conditions (Jordan and Brooks 2012; Barrett et al. 2014). In particular, males prefer to court postpartum (=sexu-ally receptive) over gravid (=unreceptive) females (Ojanguren and Magurran 2004), large over small females (Herdman et  al. 2004), and unfamiliar over familiar females (Kelley et  al. 1999; Mariette et al. 2010). When they encounter an unfamiliar female, male gup-pies tend to maintain a sustained sexual effort for about 5  days, when the rate of courtship display and gonopodial thrusting start to decrease as familiarity arises (Jordan and Brooks 2010). Males show a restoration of their mating behavior when the familiar female is replaced with a novel female, the so-called Coolidge effect (Wilson et al. 1963; Dewsbury 1981).

Male sexual effort is also influenced by the likelihood of encoun-tering mates, which induces a shift from costly courtship to a sneaky mating tactic (Jordan and Brooks 2012). Similarly, males can adaptively tune their sperm investment and respond to the pres-ence of available females by increasing the number (Bozynski and Liley 2003) and the velocity of their “ready” sperm (Gasparini et al. 2009). The increased sexual effort triggered by the presence of unfamiliar females is costly in guppies, as it reduces male post-maturation growth (Jordan and Brooks 2010). In fact, male body growth, although strongly reduced, continues after sexual maturity (i.e., when males are approximately 3 months old; Yamanaka et al. 1995; Nakajima and Taniguchi 2002; Miller and Brooks 2005).

We took advantage of adult male guppies’ flexible reproduc-tive allocation and their continued, although slow, body growth after sexual maturity to simultaneously investigate the relationship between precopulatory, postcopulatory, and somatic investment in 2 groups of males in relation with their encounter rate with unfa-miliar females. In particular, we presented 2 groups of adult male guppies with unfamiliar females every 2 (high mate encounter rate [HER]) or 10 days (low mate encounter rate [LER]) for 4 consecu-tive months. Body size, relative area of color spots, and ejaculate characteristics were measured both before and after the 4-month treatment, whereas male mating behavior was measured at the end of the 4-month treatment in a standard condition. For the reasons explained above, we predicted that HER males should 1) shift their precopulatory investment from costly courtship displays to the less costly gonopodial thrusting; 2)  show an elevated postcopulatory investment (sperm number and velocity); and 3) show reduced body growth as a consequence of a greater overall reproductive invest-ment when compared with their LER counterparts.

MATERIALS AND METHODSFish maintenance

Guppies used in the experiment were descendants of fish caught in the Tacarigua river, Trinidad, in 2002 (national grid reference PS 787 804) and were maintained in several large stock tanks (130 L, approximately 0.70 fish/L) with a balanced sex ratio. Fish were maintained as a large outbred population by periodically moving individuals through different stocks. The bottom of the tanks was

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Devigili et al. • Courtship, sperm and growth in male guppies

covered with mixed color gravel and the tanks were provided with aquatic plants. Water temperature was maintained between 25 and 27 degrees and illumination was set on a 12:12 light:dark cycle (Philips TLD 36W fluorescent lamps). All fish were fed ad libitum twice a day a mixed diet of brine shrimp nauplii and commer-cially prepared flake food (see Pilastro et al. 2007 for details of fish maintenance).

Experimental design

We randomly assigned 80 sexually mature (6 ± 1 months old) males to 2 groups exposed to a higher or lower encounter rate with unfa-miliar females (HER, N  =  40, LER, N  =  40). Before the experi-ment, males were physically isolated but kept in visual contact with females for 3  days to ensure a full replenishment of sperm reserves, according to previous studies on guppies (Bozynski and Liley 2003; see also O’Dea et al. 2014 for similar results on another poeciliid species). At the end of the 3 days, males were anaesthe-tized for measurements and sperm collection. Fish anesthesia was conducted by an expert operator (A.D.) according to standard pro-cedures (Gasparini et  al. 2010) and was usually completed in less than 2 min. Anesthetization was obtained with a solution of tric-aine methanesulfonate (MS222) at a concentration of 150 mg/L. This concentration allows a short induction and recovery time and reduces the risk of mortality (Chambel et al. 2013). All individuals recovered fully from anesthesia and, at the end of the experiment, were returned to postexperimental tanks where mortality rate was similar to that observed in our other stock tanks. We used the lowest number of individuals necessary to achieve the aims of the experi-ment. This experiment was conducted according to the Italian legal requirements and was approved by the Ethics committee of the University of Padova (permit n. 36/2011 to AP).

Subdued males were photographed for morphological analysis (body size and coloration) and stripped for ejaculate character-ization (sperm number at rest and sperm velocity, see below for a description of the methods). Subsequently, males were revived in conditioned water and transferred to individual tanks, where they were allowed to recover for 2 days before being randomly assigned to 60-L experimental tanks. Each tank contained 4 experimental males and 6 females, with 10 tanks per experimental group. Males were allowed to freely interact with the females for approximately 16 weeks (115 ± 5 days).

Previous experiments have shown that male sexual interest drops on average after 5 days after being housed with females that were initially unfamiliar (Jordan and Brooks 2010). To manipulate sexual investment, every 2  days we replaced the 6 females in each tank with other 6 unfamiliar females in the HER group, whereas in the LER group they were replaced every 10 ± 1 days. To equalize treatments for fish handling, females in the LER treatment were captured and released back in the same tank every 2 days. A total of 198 nonvirgin females were used. Females originated from stock aquaria where the sex ratio was approximately 1:1, and were of mixed age and size. Females were switched between tanks and treatment according to a schedule that ensured that each female was used in both treatments, but never more often than once per month in the same tank.

Digital photographs taken before the beginning of the experi-ment were used to identify individual males within each tank according to their color pattern. Fourteen males (8 HER and 6 LER) died during the treatment and were replaced with other males from stock tanks to maintain a constant sex ratio and fish density, but these males were excluded from the subsequent analyses. Two

experimental tanks were invaded by unicellular green algae approx-imately 2 months after the beginning of the experiment. Although this did not affect the health of the fish, the water in the tank was very turbid and the operation of exchanging females became so dif-ficult that these 2 replicates (1 HER and 1 LER) had to be removed from the study. Final sample size was therefore represented by 58 males (HER, N  =  28; LER, N  =  30). At the end of the experi-ment, males were isolated in individual 1-L tanks for 3 days during which time they were in visual contact with 2 unfamiliar females. Subsequently, male sexual behavior was measured in a large tank (see below). After the behavioral assay, males were anaesthetized for ejaculate collection and morphological analysis. Male body size, color, and ejaculate were therefore measured before and after the 4-month treatment, whereas the standardized sexual behavior assays were conducted only at the end of the 4-months treatment.

Body size and coloration

To measure body size and coloration (see Pilastro et  al. 2008 for details), males were anaesthetized with MS-222 (see above) and put on a plastic reference ruler. Excess water was removed using blot-ting paper. A digital photograph of the left side of each male was taken using a Canon EOS 450D camera mounting a Canon EF-S 60-mm Macro USM lens. Body area and standard length (from the snout to the base of the tail fin, SL), total area of carotenoid and pteridine spots (hereafter “orange”), iridescent (from violet to green-and-white structural spots), and black (melanistic spots) were measured using ImageTool 3.0 (http://compdent.uthscsa.edu/dig/itdesc.html). Color spot area was subsequently standardized to body area (%).

Repeatability of male SL measure was estimated by taking a digital photo of the same 10 random males twice 2 days apart. The same operator (A.D.) took all photos and all the body measurements in the experiment. The repeatability assay was conducted blind of male identity. Repeatability was then calculated following Lessells and Boag (1987). SL repeatability (R) was 0.98 ± 0.013 SE and cor-responded to a mean error of 0.33 mm, significantly smaller than the mean SL increment observed during the experiment (1.05 mm ± 0.15 95% CI).

In this population, males with larger relative orange spots are preferred by females (Evans et  al. 2004) and produce faster and more viable sperm (Locatello et al. 2006). We thus controlled that, at the beginning of the experiment, males in the 2 treatments did not differ in body size (SL, Student’s t-test: T78 = 0.925, P = 0.358) or relative area of orange color spots (Student’s t-test: T78 = 0.142, P  =  0.887). The area of the other 2 components of male color pattern, iridescent and black spots, did not differ between groups (Student’s t-tests: T78 = 0.202, P = 0.841; T78 = 0.162, P = 0.827, respectively).

Ejaculate assays

After anaesthetization (see above), sperm were stripped for base-line sperm counts and sperm velocity assay (Gasparini et al. 2010). Each male was placed on a slide under a dissection microscope with 0.5 mL of physiological solution (0.9% NaCl). A  gentle pressure was applied to the side of the abdomen to eject all sperm (packaged in bundles). Six sperm bundles were collected with a Drummond Micropipette and immediately used for computer-assisted sperm analysis (Hamilton-Thorne CEROS). To this end, the sperm bun-dles were placed on a multiwell slide and activated with 150-mM KCl and 4-mg mL−1 BSA solution (Gasparini and Pilastro 2011)

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and the velocity of the sperm moving away from the opening bun-dle was registered (the threshold values defining static cells were predetermined at 25 µm/s). We considered the average path veloc-ity (VAP), as this measure of sperm velocity is that commonly used in guppies (e.g., Evans 2011; Gasparini et  al. 2012; Barrett et  al. 2014; Fitzpatrick et  al. 2014). All other bundles were collected in a known volume of saline solution for sperm count. Sperm num-ber was estimated using an improved Neubauer haemocytometer under a 400× magnification (see Devigili et al. 2013 for details).

Males of the 2 experimental groups did not differ at the beginning of the experiment in the number of sperm produced (Student’s t-test: T78  =  1.052, P  =  0.296) nor in the sperm veloc-ity (VAP, Student’s t-test: T78 = 0.987, P = 0.327). After the treat-ment (i.e., during the second measurement) 1 male (LER group) produced no bundles and was thus excluded from the subsequent analysis. For another male (LER group), the vial containing the bundles for sperm counting was lost and for this male we have esti-mated the sperm velocity only.

Male sexual behavior assay

At the end of the treatment, sexual behavior of individual males was measured in a standardized assay. Briefly, 1 unfamiliar, gravid (i.e., sexually nonreceptive, Houde 1997) female was allowed to settle overnight in a 20-L tank. The following morning, a male was introduced into the tank containing the female, allowed to acclima-tize for 5 min (normal behavior is usually resumed within 3 min of introduction), and his sexual behavior was recorded for 5 min. The use of sexually nonreceptive females excluded possible confound-ing effects due to female preference. Furthermore, we wanted to avoid the reduced risk of copulation during the test, as we wanted an estimation of their intact sperm reserves. Moreover, males show a refractory period after copulation (Jirotkul 1999b; Pilastro and Bisazza 1999), and we were interested in obtaining a continuous sexual effort by males during the test. In particular, we recorded the number of SDs, the number of GTs and the time spent following (min), chasing or courting the female (hereafter following; see Head and Brooks 2006). After a 10-min interval (during which the male remained with the female but his behavior was not recorded), male behavior was recorded again for a further 5 min in order to evalu-ate male persistence (Hayes et al. 2013). Male sexual behavior was then compared between HER and LER groups and between the two 5-min observation periods (hereafter: first period and second period).

Statistical analysis

If not otherwise stated, male traits were analyzed using linear mixed models (restricted maximum likelihood [REML]), or gen-eralized linear mixed models (GLMM), in which tank and fish identity were entered as random factors to control for nonindepen-dence of observations, and treatment (HER vs. LER), period (for the behavioral assay), and time (before and after the treatment, for male size, sperm number, and sperm velocity) as fixed factors. In all the models with a continuous predictor term, interactions between this predictor and fixed factors were tested. If not significant, the interaction was excluded from the final model. Only final models are shown. Iridescent and black coloration variables were not nor-mally distributed, even after appropriate transformation, due to 2 outlier individuals whose percentage of body covered by iridescent and black coloration was particularly high (both from LER group). However, because results were not affected by the inclusion or the

exclusion of these individuals, we present the results for the com-plete dataset. The number of SDs and GTs followed a Poisson distribution and were therefore analyzed using the appropriate GLMM. Finally, to determine the relative use of 1 tactic over the other, we used a GLMM with binomial distribution in which the number of GT was the dependent variable and the total number of GT and SD the binomial total. Means and their standard errors are given. Statistical analyses were performed using Genstat 16.0 and SPSS 18.0.

RESULTSBody growth, color pattern, and sperm investment

We observed a significant increase in body size during the 4-month treatment. Mean SL increase was 1.05 mm ± 0.074 and did not differ between HER (1.07 ± 0.109 mm) and LER (1.03 ± 0.101 mm) males (REML, fixed factor: treatment, F1,16.6 = 1.214, P = 0.286; time, F1,55  =  192.66, P  <  0.001; treatment × time, F1,55  =  0.188, P  =  0.666; random factors: tank, male identity). The relative size of male color spots did not change during the experiment nor differ between experimental groups (orange: REML, fixed fac-tor: treatment, F1,17.7  =  0.980, P  =  0.333; time, F1,55  =  3.15, P = 0.081; treatment × time, F1,55 = 0.288, P = 0.594; iridescent: REML, fixed factor: treatment, F1,16.5  =  0.001, P  =  0.999; time, F1,55 = 2.36, P = 0.139; treatment × time, F1,55 = 0.001, P = 0.960; black: REML, fixed factor: treatment, F1,17.1  =  0.312, P  =  0.583; time, F1,55  =  0.375, P  =  0.583; treatment × time, F1,55  =  2.580, P = 0.114; random factors: male identity, tank). Using absolute spot area and body area as covariate gave substantially identical results (data not shown). In contrast, sperm number at rest and sperm velocity increased significantly during the experiment (Figure  1). Although the increase in ejaculate investment did not differ signifi-cantly between HER and LER (interaction between treatment and time), HER males showed a tendency to produce on average more sperm (Table 1).

The increase in sperm number (after–before, ∆Sperm) and the increase in sperm velocity (after–before, ∆VAP) were posi-tively correlated, indicating a phenotypic integration between these 2 ejaculate traits (GLMM. Dependent variable: ∆VAP; pre-dictors: ∆Sperm, F1,46  =  4.46, P  =  0.04; treatment, F1,46  =  1.15, P  =  0.286; random factor: tank). Moreover, body growth (∆SL: SL after–SL before), ∆Sperm, and ∆VAP were also positively cor-related (GLMM. Dependent variable: ∆Sperm; predictors: ∆SL, F1,36.7 = 5.33, P = 0.027; treatment, F1,13.1 = 0.35, P = 0.567; ran-dom factor: tank. Dependent variable: ∆VAP; predictors: ∆SL, F1,47 = 5.19, P = 0.027; treatment, F1,47 = 2.07, P = 0.157; random factor: tank; Figure 2).

Sexual behavior

Eight males (4 in each group) did not perform any SD or GT during the behavior assay (although 5 of them actively followed the female) and were therefore excluded from the analysis. Sexual behavior did not differ between HER and LER males during the initial 5 min of observation (Figure  3). Once compared with the first observa-tion period, the time spent by HER males following the female decreased in the second observation period, whereas the opposite trend was observed in LER males (Table 2). Similarly, HER males decreased their SD rate whereas LER increased it (Table 2). This effect was particularly evident when the proportion of SD over the total number of SD and GT was considered (Table 2).

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Association between sperm number, body growth, and sexual behavior

We evaluated if there was an association between the postcopula-tory investment (change in sperm number) and the somatic invest-ment (change in SL) during the 4  months treatment with sexual behavior at the end of the treatment. To this end, we entered ∆Sperm and ∆SL (representing the postcopulatory and somatic investment during the previous 4 months) as continuous predictors in the respective GLMM (Table 2). We found negative not statisti-cally significant covariation between ∆Sperm and both SD and fol-lowing. ∆SL did not covary with any of the behaviors measured (Table 2). We did not find any significant association between male colors and any of the above traits (data not shown).

DISCUSSIONOur initial predictions were only partly confirmed by the results of our experiment. Males from both experimental groups increased their postcopulatory investment (sperm number and velocity) dur-ing the experiment. As predicted, we found that HER males’ post-copulatory investment (sperm number) was more pronounced than

that of their LER counterparts, although the difference was mar-ginally significant. In contrast, male coloration appeared to be fixed, and changed neither within males nor between groups. In the stan-dardized behavioral assay conducted at the end of the treatment, sexual behavior toward an unfamiliar female did not differ between male groups during the initial 5 min of the trial. In contrast, during the second 5-min observation period (which was conducted 15 min after the initial encounter with the female), HER males reduced their time following the female and their rate of courtship display, and preferentially used the less costly gonopodial thrusting strat-egy over the more costly sigmoid courtship display, as compared with their LER counterparts. Overall, a higher encounter rate with unfamiliar females was associated with increased investment in postcopulatory traits (sperm number) and decreased invest-ment in precopulatory traits (time following and courtship display). A negative correlation between the increase in sperm number and the frequency of courtship display (Table  2), although only close to statistical significance, was also found. In contrast, we did not find any evidence of a trade-off between somatic and reproductive investment. First, the increase in body length during the 4-months treatment did not differ between HER and LER males; secondly, a

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Table 1Effect of treatment (HER and LER of unfamiliar females) and time (before and after the 4 months of treatment) on sperm number and velocity

Estimatea SE F df P

Sperm numberb Treatment −0.13 0.08 6.16 1,11.5 0.030Time 0.42 0.09 48.65 1,54.0 <0.001Time × Treatment 0.03 0.12 0.08 1,54.0 0.785

Sperm velocity (VAP) Treatment 3.78 2.92 0.73 1,14.9 0.406Time 8.31 2.50 13.66 1,55.0 <0.001Time × Treatment −3.58 3.51 1.04 1,55.0 0.312

Significant effects are in bold.Linear mixed model. Fixed factors: treatment and time (before and after treatment); random factors: male identity and experimental tank.aEstimated effect relative to HER and to measure before the treatment.bSperm number was log transformed.

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male’s body growth during the experiment was positively correlated with his sperm investment, contrary to what would be expected if somatic and reproductive investments were traded off. The aver-age observed increase in body size largely exceeded the measure-ment error (see Methods), indicating that this result was robust and suggesting that the variation in the total resources available to males exceeded the variation in allocation between reproductive and somatic investment (Reznick et  al. 2000). Overall, our results therefore suggest a trade-off between the 2 components (precopu-latory and postcopulatory) of reproductive investment, but at the same time revealed that some male were able to increase both their sperm and somatic investment during the 4-months of the experi-ment. We will discuss each of these results in more detail below.

The increase in sperm number observed in both groups during the 4-month experiment was expected as sperm number increases with age in this population (Gasparini et  al. 2010). For 2 reasons, however, we think that the increased postcopulatory investment we observed during the 4  months experiment was, at least partly, influenced by the high encounter rate with unfamiliar females and its associated Coolidge effect. First, in our experiment the mean increase in sperm number was twice the increase of that reported in the study by Gasparini et  al. (2010). Second, although sperm velocity declines with age (Gasparini et  al. 2010), it increased in our experiment, in agreement with the observation that male gup-pies respond to an increased mating opportunities by increasing the number (Bozynski and Liley 2003) and the velocity of their “ready”

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Figure 3Sexual behavior after the treatment. Mean values and standard errors are given for each behavior. The bars on the left of each panel (“first”) show behavior measured during the first 5-min observation period and those on the right (“second”) show behavior after a 10-min interval. The HER group is represented by gray bars and the LER group by white bars. Males that performed neither thrusts (GT) nor displays (SD) were excluded from the graph.

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Figure 2Relationship between male body growth (SL after–SL before, ∆SL) and the change in sperm number at rest (sperm number after–sperm number before, ∆Sperm) and in sperm velocity (sperm velocity after–sperm velocity before, ∆VAP) during the 4 months treatment. Gray dots and solid line represent HER males. Open dots and dashed line represent LER males.

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Devigili et al. • Courtship, sperm and growth in male guppies

sperm (Gasparini et  al. 2009). In agreement with these studies, we found a significant positive correlation between the increase in sperm number and in sperm velocity, also at the individual level, confirming that these 2 ejaculate traits are phenotypically inte-grated (Locatello et al. 2006; Gasparini et al. 2013). This covaria-tion may ultimately depend on the fact that both sperm number and sperm velocity are important determinants of male competi-tive fertilization success (Boschetto et  al. 2011) and therefore we might expect that a male’s postcopulatory investment is allocated to both aspects of ejaculate quality.

Producing numerous (Gasparini et al. 2013) and fast swimming sperm (Rahman et al. 2013) is costly, and an increased investment in these ejaculate traits is expected to result in a reduced invest-ment in other costly traits such as those associated with mate acqui-sition and somatic growth (Parker et  al. 2013). Indeed, after the 4-month treatment, male guppies of the 2 experimental groups showed a divergent pattern of sexual behavior. When presented with 1 unfamiliar female, males from the 2 groups showed a similar sexual behavior during the first 5 min. However, after 10 min, LER males were able to afford an increased courtship rate (a costly mat-ing tactic, Kolluru and Grether 2005; Head et  al. 2010; Devigili et  al. 2013; Rahman et  al. 2013), whereas HER males reduced activity and switched to gonopodial thrusting, a less-expensive mat-ing tactic (Houde 1997). Similar trade-offs between precopulatory and postcopulatory traits has been found in a number of other

species (e.g., Rudolfsen et al. 2006; Pizzari et al. 2007; Kelly 2008; Montrose et al. 2008; Kvarnemo et al. 2010). An alternative expla-nation may be that, in the presence of sexually unreceptive females, HER males have switched to a coercive mating tactic in order to save their energy for successive, possibly receptive females, given that new, unfamiliar females were provided every other day in this group and that recent social history has been shown to influence male guppy behavior (Jordan and Brooks 2012). Moreover, despite the number of known competitors was taken constant within and across groups, the different turnover of familiar females may have influenced a male perception of sperm competition risk (although it has to be noted that the perception of sperm competition appar-ently does not influence male ejaculate allocation in guppies, Evans 2009).

Contrary to our prediction, the increase in sperm number and velocity covaried positively with the rate of body growth during the experiment. As typical of this fish family males strongly decrease their growth after sexual maturity (Magurran 2005) as a conse-quence of their intense sexual activity (Magurran and Seghers 1994; Jordan and Brooks 2010). The observed differences in post-maturation body growth and sperm investment suggest that there was a large variation in the total resources allocated by males to these 2 components between 6 and 10 months of age, possibly due to the coexistence of different life-history strategies in the popula-tion (e.g., early vs. late sexual and somatic investment, Hunt et al.

Table 2Male sexual behavior at the end of the 4-months period during which males were maintained at a higher or lower encounter rate with unfamiliar females (Model A) and its covariation with the change in sperm production and body size during the previous 4 months (Model B)

Estimatea SE F df P

Model ATime following the femaleb Treatment 0.06 0.86 2.42 1,13.1 0.144

Observation period −1.36 0.67 0.4 1,46.0 0.530Treatment × observation period 2.13 0.94 5.08 1,46.0 0.029

SDc Treatment 0.21 0.43 1.84 1,12.3 0.199Observation period −0.28 0.22 0.51 1,44.2 0.477Treatment × observation period 0.63 0.28 4.92 1,44.2 0.032

Proportion of SDs over total mating attemptsd Treatment 0.42 0.69 4.06 1,40.6 0.050Observation period −1.32 0.36 4.34 1,39.5 0.044Treatment × observation period 1.53 0.50 9.31 1,39.6 0.004

Model BTime following the femaleb Treatment −0.05 0.85 2.51 1,12.3 0.138

Observation period −1.37 0.67 0.4 1,46.0 0.530Treatment × observation period 2.13 0.95 5.08 1,46.0 0.029∆Sperm −0.09 0.05 3.38 1,31.1 0.073∆SL 0.04 0.08 0.31 1,43.0 0.582

SDc Treatment 0.19 0.42 1.81 1,11.6 0.205Observation period −0.28 0.22 0.52 1,45.0 0.473Treatment × observation period 0.63 0.28 5.02 1,45.0 0.030∆Sperm −0.05 0.03 3.24 1,54.9 0.077∆SL −0.06 0.38 0.49 1,29.1 0.490

Proportion of SDs over total mating attemptsd Treatment 0.38 0.69 3.92 1,39.1 0.055Observation period −1.35 0.36 4.33 1,40.3 0.044Treatment × observation period 1.59 0.50 9.35 1,40.2 0.004∆Sperm −0.08 0.05 2.73 1,58.8 0.104∆SL −0.43 0.66 1.35 1,37.7 0.253

Significant effects are in bold.GLMMs. Fixed factors: treatment and period; random factors: male identity, experimental tank.aEstimated effect relative to HER and to the first observation period.bNormal error distribution.cPoisson error distribution.dBinomial error distribution (binomial total: GTs + SDs). ∆Sperm (number of sperm after—number of sperm before the treatment) and ∆SL (SL after–SL before the treatment) were entered as continuous predictors in Model B). All continuous predictors × treatment and observation period interactions were not significant and therefore excluded from the final model.

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Behavioral Ecology

2004). Although the variation in female life-history strategies has been extensively studied in females (e.g., Torres-Dowdall et  al. 2012), much less is known about males and further studies will be necessary to investigate this possibility.

Finally, we do not observe any change, between or within groups, in male coloration. Male coloration, and in particular orange spot size, is known to be condition dependent in this species (e.g., van Oosterhout et al. 2003) and to respond to diet restriction, although to a lesser extent as compared with male sexual behavior (Devigili et al. 2013) and sperm traits (Rahman et al. 2013). Our results con-firm that male coloration is less plastic than other sexual traits, rein-forcing previous evidence that social environment do not influence color spot characteristics in this species (Miller and Brooks 2005).

In conclusion, we demonstrated that the frequency at which males encounter unfamiliar females affects their allocation between precopulatory and postcopulatory traits, suggesting a trade-off between the investment in mate acquisition (courtship rate) and the postcopulatory investment (sperm number, see also Fitzpatrick et  al. 2014). However, the alternative explanation that HER and LER males may have different precopulatory and postcopula-tory allocation optima cannot be ruled out. We also found that the within-male increase in sperm number and in sperm velocity were positively correlated. Whether this phenotypic integration we observed is adaptive or is the result of constraints in the physiologi-cal or genetic machinery responsible for plasticity in sperm produc-tion remains to be understood. Finally, we revealed an unexpected positive correlation between a male’s increase in sperm number and velocity during the 4-months treatment and his body growth. This latter result suggests that there may be a large phenotypic variation in the life-history trajectories among males, whose genetic basis warrants further investigation.

FUNDINGThis work was supported by the Fondazione CARIPARO (Progetto di Eccellenza 2007), the Ministero Istruzione Università e Ricerca (PRIN 2008 no. 2008Z8ACTN), and the University of Padova (Progetto PRAT 2012 no. CPDA 120105).

We thank 2 anonymous referees, J.  Fitzpatrick, C.  Gasparini, and M. Jennions for their comments on previous versions of the manuscript.

Handling editor: John Fitzpatrick

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