Journal of Fish Biology (2013) 83, 1381–1386 doi:10.1111/jfb.12210, available online at

BRIEF COMMUNICATIONS Are zebrafish Danio rerio males better swimmers than females? I. Leris*†‡, D. G. Sfakianakis* and M. Kentouri* *Biology Department, University of Crete, P.O. Box 2208, Heraklion, 71409, Crete, Greece and †Biology Department and Helmholtz Institute, Utrecht University, P.O. Box 80086, Utrecht, 3508TB, The Netherlands (Received 16 January 2013, Accepted 2 July 2013) Swimming performance of zebrafish Danio rerio males and females initially reared in four different temperatures was assessed by measuring their relative critical swimming speed after acclimation to a common temperature. Males reared at 31◦ C achieved higher swimming speeds than females reared at the same temperature. Morphometric measurements indicate differences in body shape between sexes that could account for the difference in swimming speed. © 2013 The Fisheries Society of the British Isles

Key words: body shape; developmental temperature; locomotion; morphometric character; plasticity; U crit .

Swimming performance is critical for fish survival as it can strongly affect food acquisition, reproduction and escape from predators (Dr¨ucker, 1996). Fish swimming performance can be influenced by several abiotic factors such as water temperature (Koumoundouros et al., 2002), oxygen concentration (Widmer et al., 2006), light and photoperiod (Beamish, 1978). Although differences between males and females are quite common in several aspects of fish biology, the effect of sex on swimming performance has received little attention (Hinch & Rand, 1998; James & Johnston, 1998; Plaut, 2002; Ghalambor et al., 2004). Interestingly, most of the published studies on zebrafish Danio rerio (Hamilton 1822) swimming performance (Plaut & Gordon, 1994; Plaut, 2000; Widmer et al., 2006) do not report the sex of their experimental subjects, with only Sfakianakis et al. (2011a) specifying the use of males. Therefore, the aim of this study was to examine the effect of sex on the swimming performance of D. rerio. Moreover, as developmental temperature can also influence the swimming performance of D. rerio (Sfakianakis et al., 2011a), the effect of sex was studied in respect to different rearing temperatures. Danio rerio eggs were collected from stock tanks and reared in duplicate tanks (indicated as A and B) at four temperatures (22, 25, 28 and 31◦ C) according to ‡Author to whom correspondence should be addressed. Tel.: +31 302 535 412; email: [email protected]

1381 © 2013 The Fisheries Society of the British Isles



Westerfield (1995) (for detailed information on rearing conditions, see Sfakianakis et al., 2011a, b). At c. 100 days post hatching, all fish were acclimatized to a new common temperature, which was also the water temperature during swimming trials. The chosen temperature, 26·5◦ C, was the intermediate of all four and well inside the temperature range preferred by D. rerio in the wild (24–30◦ C; Lawrence, 2007). This acclimation period lasted 45 days. Males and females were separated 3 days before the swimming trials to avoid spawning, which could affect performance (James & Johnston, 1998). All females chosen were in advanced reproductive state since they had already started to spawn. All fish were deprived of food for 24 h before use in experiments, and were examined prior to the trials for visible morphoanatomical malformations. Swimming performance was assessed as the relative critical swimming speed (U critR ) using Brett’s method (1964). Initially, critical swimming speed (U crit ) was calculated and then it was transformed to U critR according to Beamish (1978), so that comparisons were independent from individual total length (LT ). A custom-designed apparatus with a swimming tunnel, described in Koumoundouros et al. (2002), was used to test the fish. Each subject was randomly taken from its holding area with the use of a net and was placed in the swimming tank for a 5 min acclimatization. Later, it was placed inside the swimming tunnel and forced to swim against a current of two body lengths (LT ) per second with water velocity then increased by 1 LT s−1 every 10 min (Koumoundouros et al., 2002). Although this 10 min time interval between velocity increments is an intermediate of those previously reported in published D. rerio studies (5 min, Plaut, 2000; Scott & Johnston, 2012 and 30 min, Plaut & Gordon, 1994), it is at the lower end of the range of time increments typically used in U crit tests (e.g. 60 min, Brett, 1964). Therefore, the measured swimming speeds are possibly slightly higher than the actual U crit of the species (Farrell, 2008). When the fish was unable to hold its position against the current or react to stimulation (e.g. the observer poking the side or the rear of the tunnel), it was considered fatigued and the trial concluded (for detailed information see Sfakianakis et al., 2011a). At the end of the trial, fish were anaesthetized (ethylene glycol monophenyl ether, 0·2–0·3 ml l−1 , Merck;, individually photographed using a digital camera (Olympus, Camedia C3030 Zoom;, weighed (M , 0. 001 g) and the maximum body width (W B ) measured under a stereoscope (0·01 mm). Digital photographs and image processing software (tpsDig2, Rohlf, version; were used for accurate measurement of LT , maximum body depth (D Bmax ), body depth at the anus (D B ) and caudal peduncle depth (D Cp ). Moreover, selective double staining of cartilage and bone tissue (Park & Kim, 1984) was performed on all specimens, to examine for skeletal deformities under a stereoscope. Abnormal fish were discarded from the analysis. Data were analysed (SPSS, IBM, 20; spss/products/statistics) by means of two-way ANOVA with sex and rearing temperature as factors. Normality and homoscedasticity were tested using Shapiro-Wilk’s and Levene’s tests, between-group comparison was conducted using Tukey HSD post hoc test. Duplicates did not differ statistically and were therefore pooled for male and female comparisons. All tests were performed at a minimum level of significance of 0·05.

© 2013 The Fisheries Society of the British Isles, Journal of Fish Biology 2013, 83, 1381–1386



Table I. Relative critical swimming speed (U critR ) measurements of male (M) and female (F) Danio rerio reared at four different temperatures T R (◦ C) M

22 25 28 31


22 25 28 31

Duplicate (n) A B A B A B A B A B A B A B A B

(6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6) (6)

U critR (LT s−1 )


7·44 7·84 8·82 8·53 8·67 8·56 9·29 9·35 7·89 7·67 8·06 8·35 8·14 7·98 8·02 8·26

0·68 1·61 1·09 1·09 0·83 0·49 1·11 1·26 0·67 0·77 0·21 0·40 0·91 0·56 0·66 0·98

Average U critR (LT s−1 ) 7·64a 8·67a,b 8·62a,b 9·32b, * 7·78a 8·2a 8·06a 8·14a

T R , rearing temperature; n, sample size; LT , total length; s.d., standard deviation. *Statistical differences within each temperature group; different superscript lower case letters indicate statistical differences within each sex group (P < 0·05; Tukey HSD post hoc test).

According to the analysis, fish sex had a significant effect on swimming performance (two-way ANOVA, F 1,88 = 8·536, P < 0·01), with males achieving higher U critR than females when reared at 31◦ C (Tukey HSD post hoc test, P < 0·05, Table I). Rearing temperature also had a significant effect on swimming performance (two-way ANOVA, F 3,88 = 5·901, P < 0·001) as expected, with males reared at 31◦ C being better swimmers than those reared at 22◦ C (Tukey HSD post hoc test, P < 0·001, Table I). This is in accordance with previous reports (Sfakianakis et al., 2011a). Females, on the other hand, did not present any significant difference in swimming performance with temperature (Table I). The observed difference between males reared at 22 and 31◦ C could be attributed to temperature-dependent differences in: (1) muscle ontogeny and fibre type composition (Johnston et al., 2009; Scott & Johnston, 2012), (2) body shape and meristic count (Sfakianakis et al., 2011b), (3) muscle lactate decomposition rate (Sfakianakis et al., 2012) or (4) a combination of the above. The majority of morphometric characters of D. rerio were significantly affected both by rearing temperature and sex (Table II). The effect of sex was statistically significant for LT , M LT −1 , W B LT −1 , D Bmax LT −1 and D B LT −1 ratios (two-way ANOVA, F 1,88 > 71·096, P < 0·001). Specifically, females were longer and had higher M LT −1 , W B LT −1 and D Bmax LT −1 ratios than males at all rearing temperatures (Table II). D B LT −1 differed significantly between males and females reared at 22, 25 and 28◦ C, whereas D Cp LT −1 did not differ between the two sexes (Table II). Plaut (2002) suggested that the poorer swimming performance of pregnant mosquitofish Gambusia affinis (Baird & Girard 1853) was probably a result of aerobic constraints due to high oxygen demands of the embryos. On the other

© 2013 The Fisheries Society of the British Isles, Journal of Fish Biology 2013, 83, 1381–1386



Table II. Sampling information (duplicates pooled) and morphometric measurements (per total length, LT ) of male (M) and female (F) Danio rerio reared at different temperatures TR ( C) 22 25 28 31 22 25 28 31




Mean ± s.d. LT (mm)

M LT −1 (g mm−1 )

WB LT −1

D Bmax LT −1

DB LT −1

D Cp LT −1

12 12 12 12 12 12 12 12

32·62 ± 1·44 32·4 ± 2·71 32·57 ± 2·00 32·49 ± 1·66 37·02 ± 2·20 35·4 ± 2·21 36·05 ± 2·29 36·48 ± 1·48

0·0109*,a 0·0104*,a 0·0104*,a 0·0104*,a 0·0197a 0·0164b 0·0159b 0·0162b

0·1452*,a 0·1484*,a 0·1497*,a 0·1432*,a 0·1977a 0·1874a,c 0·1700b 0·1788b,c

0·2058*,a 0·2048*,a 0·2078*,a 0·2010*,a 0·2630a 0·2531a,b 0·2417b 0·2422b

0·1713*,a 0·1718*,a 0·1747*,a 0·1712a 0·1923a 0·1858a,b 0·1857a,b 0·1789b

0·0912 0·0919 0·0900 0·0872 0·0934 0·0939 0·0924 0·0868

T R , rearing temperature; n, sample size; M , mass; W B , body width; D Bmax , maximum body depth; D B , body depth at the anus; D Cp , caudal peduncle depth. *Statistical differences within each temperature group; different superscript upper case letters indicate statistical differences within each sex group (P < 0·05; Tukey HSD post hoc test). In the case of LT and D Cp LT −1 no post hoc comparisons were conducted since there was no significant interaction between factors sex and rearing temperature in the two-way ANOVA.

hand, James & Johnston (1998) attributed decreased performance of gravid shorthorn sculpin Myoxocephalus scorpius (L. 1758) to the additional mass of eggs, the larger girth and the associated changes in the contractile properties of the muscles. The results of this study indicate that, at this developmental stage, males are generally lighter, thinner and more slender than females (Table II). Fusiform or streamlined fish body shapes have often been associated with higher swimming efficiency, by comparison to deep-bodied and less streamlined shapes (Crossin et al., 2004). The sex-related differences in body shape in this study can, therefore, explain the differences in swimming capacity. Rearing temperature significantly affected most of the morphometric characters, i.e. M LT −1 , W B LT −1 , D Bmax LT −1 , D B LT −1 and D Cp LT −1 (two-way ANOVA, F 3,88 > 3·248, P < 0·05) and although the post hoc comparison tests did not discriminate the 31◦ C male fish from the rest of the males (so as to account for the observed difference in U critR ), there was a tendency for lower values of D Cp LT −1 ratios in fish reared at 31◦ C (Tukey HSD post hoc test between temperature groups, P < 0·01). Since D Cp is known to play an important role in swimming performance, with thinner peduncles associated with higher sustained swimming speeds (Webb, 1984), it may have contributed to the differences in swimming capacity in this study. The observed differentiation in swimming performance of males and females occurs only at the 31◦ C temperature group. This is particularly important, as this is the rearing temperature that has been found to produce the fastest swimmers in the case of males (Sfakianakis et al., 2011a; present results). In addition, Scott & Johnston (2012) reported that developmental temperatures in the higher end of D. rerio’s optimum thermal range (32◦ C in their study) are associated with improved thermal acclimation later in life. It could be therefore argued that underlying male and female differences in swimming performance might also exist at other temperature groups, but they emerge only at the thermal regime where fish have the potential to reach higher swimming capacities.

© 2013 The Fisheries Society of the British Isles, Journal of Fish Biology 2013, 83, 1381–1386



These results also show that females coming from different temperature groups do not present any differences in their swimming ability as opposed to males. A possible explanation for this could be the fact that physiological constrains associated with gravidity (i.e. increased mass and volume, body stiffness or different energy demands) mask any propensity of 31◦ C reared females to swim at higher performance (as dictated by their developmental temperature). Although females appear to be worse swimmers than males in this study, the fact that they exhibited stable swimming performance at all rearing temperatures, even the theoretically marginal temperature of 22◦ C, demonstrates the species’ ability to acclimate to a broad thermal range. All protocols were approved by the Departmental Animal Care Committee, in accordance to Greek (N. 2015/1992) and EE (Directive 63/2010) legislation on the care and use of experimental animals. This study was financed by the European Social Fund and National Resources (EPEAEK II–PYTHAGORAS I) to M.K. We thank the two anonymous reviewers and the editor for their helpful comments on the manuscript.

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Are zebrafish Danio rerio males better swimmers than females?

Swimming performance of zebrafish Danio rerio males and females initially reared in four different temperatures was assessed by measuring their relati...
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