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Male zebrafish (Danio rerio) odorants attract females and induce spawning
Aquaculture and Fisheries 3 (2018) 139e144
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Aquaculture and Fisheries journal homepage: www.keaipublishing.com/en/journals/ a q u a c u l t u r e - a n d - fi s h e r i e s /
Male zebrafish (Danio rerio) odorants attract females and induce spawning rio a, b Jie Li a, Peter C. Hubbard b, *, Adelino V.M. Cana a b
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China Centro de Ci^ encias do Mar, Universidade do Algarve, Campus de Gambelas, 8005-139, Faro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2018 Received in revised form 8 June 2018 Accepted 23 June 2018 Available online 11 July 2018
In zebrafish, the chemical identity and biological roles of reproductive pheromones have yet to be clarified. The current study assessed the role(s) of male-released odorants in a reproductive context, and evaluated the possible involvement of steroids and their metabolites. Females were placed in chemical and/or visual contact with males. In chemical contact, females ovulated as frequently whether they could see the males or not. Conversely, females with visual contact alone with males ovulated as frequently as isolated females. Male-released odorants attracted females; visual contact with males attracted females, but to a lesser extent; the two effects were additive. The olfactory potency of solid-phase extraction of male- and female-conditioned water was assessed by the electro-olfactogram; males released more potent odorants than females, but the most potent extract came from water in which males and females interacted freely. HPLC fractionation of these extracts revealed that, while in all samples some fractions contained odorants, male-specific activity was contained in the first four fractions, suggesting that the compounds involved are relatively hydrophilic. Finally, the olfactory potency of some steroids, previously suggested to have a pheromonal role, was assessed. However, it is unlikely that these steroids contribute significantly to the odor of fish-conditioned water. © 2018 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Pheromone Zebrafish Reproduction Behavior Olfaction Spawning
1. Introduction Chemical communication is an important facet of reproduction in teleost fishes. The zebrafish (Danio rerio) is an important model species for developmental, genetic and behavioral studies. Yet the chemical identities of its reproductive pheromones remain unknown. Pioneer work in the Netherlands suggested that steroid glucuronates, produced by the testes, are capable of attracting females (Van Den Hurk & Resink, 1992; Lambert, Van Den Hurk, Schoonen, Resink, & Van Oordt, 1986; Van Den Hurk, Schoonen, Van Zoelen, & Lambert, 1987); however, these were never tested individually, or assessed for olfactory potency. On the other hand, 4pregnen-17,20b-diol-3-one 20-sulfate (17,20b-P-sulfate), one of the female-released steroid pheromones identified in the goldfish (Carassius auratus; like the zebrafish, a cyprinid) (Dulka, Stacey, Sorensen, & Van Der Kraak, 1987; Poling, Fraser, & Sorensen, 2001; Sorensen, Scott, Stacey, & Bowdin, 1995) is a potent odorant for the zebrafish
^ncias do Mar, Universidade do Algarve, * Corresponding author. Centro de Cie Campus de Gambelas, 8005-139, Faro, Portugal. E-mail address: [email protected] (P.C. Hubbard).
(Belanger, Pachkowski, & Stacey, 2010; Friedrich & Korsching, 1998). Another female-released goldfish reproductive pheromone, prostaglandin F2a is detected with high sensitivity by zebrafish and mutant male zebrafish deficient in its receptor have impaired courtship towards females (Yabuki et al., 2016). Here we tested the hypothesis that male zebrafish release odorants that attract females and promote spawning, using a combination of behavioral experiments, electrophysiology and high performance liquid chromatography (HPLC). Firstly, we tested the relative importance of visual and chemical cues from the males on female ovulation and behavior. Secondly, we subjected maleand female-conditioned water to solid-phase extraction and HPLC. We then the assessed olfactory potency of these fractions by the electro-olfactogram (EOG) and compared these responses to those evoked by a range of steroids that may form part of the male pheromone. We conclude that males release powerful compounds to the water that attract females and induce them to ovulate but, unlike previous suggestions, sex steroids and their conjugates do not appear to be a major component of the male-released odorants. Thus, more work is needed to identify the male reproductive pheromone in zebrafish.
https://doi.org/10.1016/j.aaf.2018.06.004 2468-550X/© 2018 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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2. Materials and methods 2.1. Fish and housing conditions Wild-type zebrafish of the AB strain three to six months old (3e4 cm standard length, 2.0e3.0 g) and sexually mature were used in this study. Fish were kept at 26e28 C on a 14:10 light:dark photoperiod and fed twice daily at 11:00 and 18:00 with dried brine shrimp (Artemia salina). As soon as their sex could be determined (three months onwards), males were separated from females and kept in single-sex groups. Seven days before the experiments (the ovulatory cycle is five to seven days) single males and females were mated to ensure that they were reproductively active. Fish that spawned were placed again in tanks of single-sex groups to be used in all experiments described below. Fish used for electro-olfactogram (EOG) recording were taken from a separate stock; all female and similar age and size as those used in the behavior experiments.
Fig. 1. Schematic front view of tanks used in the behavioral experiments. Each tank was divided by a partition separating the male from the female side. The partition could be either transparent without holes (vision only), transparent with a 2 mm diameter perforation per cm2 (vision and olfaction), opaque without holes (neither vision nor olfaction), or opaque with a 2 mm diameter perforation every cm2 (olfaction only). The 3 cm wide rectangular region on the female side (broken line) next to the partition was defined as the "attraction zone".
2.2. Ovulation experiment Twenty females were placed in one side of a 24 cm 10 cm 10 cm Perspex tanks separated into two by a division, each division with 1 l of water. The division could be transparent or opaque without or with 1 mm in diameter perforations per cm2. For the control group ‘no vision, no olfaction’, 20 females were placed in one side of the tank and 20 males in the other separated by an opaque division. For the 'vision only' group, 20 females were placed in one side of the tank and 20 males in the other separated by a transparent division without perforations. For the 'olfaction only' group 20 females were placed in one side of the tank and 20 males in the other separated by an opaque division with perforations. Finally, for 'vision and olfaction' group, 20 females were placed in one side of the tank and 20 males in the other separated by a transparent division with perforations. The 20 males and females were placed in their respective compartments at 18:00 the previous day. The next morning, 30 min after 'lights on' (10:00 a.m.) the females were checked for ovulation by applying gentle pressure to the abdomen (without anesthesia, but under water); those which released eggs were removed from the experimental tank. The procedure was repeated three times at 30 min intervals. The experiment was repeated six times independently with different fish (i.e, six times with 20 fish in each treatment group). The data were compared by one-way ANOVA followed by Tukey's post hoc test (data were normally distributed with equal variance).
the female spent in the attraction zone was measured. The data were arc-sign transformed and compared by One-Way ANOVA followed by Tukey's post hoc test (data were normally distributed with equal variance). 2.4. Fish-conditioned water To extract and, eventually, identify odorants - putative pheromones - released by zebrafish, fish-conditioned water was prepared by using a similar experimental approach outlined above for the ovulation experiment with 500 mL water on each side of the division: i) five males and five males that could not see each-other but with chemical contact; ii) five males and five females with both visual and chemical contact. The fish were placed in the tanks at 17:00 of one day until 11:00 the following morning (18 h) to allow plenty of time for odorants to be released. The fish were then removed and the water extracted through a 500 mg C18 solidphase extraction column (Cronus, SMI-LabHut Ltd., Gloucester, UK) using methanol to prime the cartridges and to elute the retained compounds. The methanol was evaporated to dryness (under nitrogen) and the pellet re-dissolved in 100 mL HPLC grade methanol. 2.5. High performance liquid chromatography (HPLC) of water extracts
2.3. Attraction experiment The attraction experiment was carried out in 24 cm 5 cm 10 cm Perspex tanks with divisions as in the ovulation experiment, each half containing 0.5 l water. A 3 cm wide region on the female side, contiguous to the middle division, was defined as the attraction zone (Fig. 1). One female and one male zebrafish were placed separately either each side of the division, opaque or transparent, with or without holes as above; the control group "no vision, no olfaction" had an opaque division with no holes, the "vision only" group had a transparent division, the "olfaction only" group had an opaque division with holes, the "olfaction and vision" group had a transparent division with holes. Experiments started at 30 min after lights on (peak time of day for spawning activity) and the fish was filmed for 30 min by a digital camera (Sony Handycam FDRAXP55). Each treatment was repeated six times (independently with different fish each replicate). The videos were analyzed by Ethovision XT software (version 11.5, Noldus Information Technology, Wageningen, the Netherlands). The proportion of time that
20 mL of the solid-phase extract of fish-conditioned water (described in the previous section) was injected onto a HPLC system (Smartline KNAUER, Berlin, Germany) with a C18 column (3.9 mm 300 mm; 4 mm particle size; Nova-Pak, Waters). HPLC conditions were as follows: mobile phase was pure water (MilliQ) and methanol (HPLC-grade), both containing 0.001% FA; 0e4 min isocratic at 15% methanol, 5e91 min linear gradient from 15% to 100% methanol, 91e96 min isocratic at 100% methanol; flow-rate: 0.7 mL/min, the same conditions used to isolate the steroid glucoronate pheromone from urine of male tilapia (Keller-Costa et al., 2014). The column eluate was first routed to a diode array UV detector (Smartline 2600, KNAUER, Germany), then split off and half routed to an evaporative light scattering detector (Varian 380-LC ELSD, Polymer Laboratories) and the other half collected by an Advantec CHF100SA fraction collector into 32 fractions, each 3 min (i.e. 2.1 mL). ELSD conditions were as follows: nitrogen carrier gas (98% purity, 1.6 SLM), 65 C nebulization temperature, 110 C evaporation temperature.
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2.6. Recording the electro-olfactogram (EOG) The EOG is a robust and sensitive method for assessing olfactory potency of a given compound or biological sample. In the current study, it was used to screen the HPLC fractions and steroids (and some conjugates) for olfactory activity. The EOG was recorded from female zebrafish as previously described for goldfish (Hubbard, rio, 2002) with the following modifications. ZebraBarata, & Cana fish were anesthetized by immersion in water containing 250 mg/L 3-aminobenzoic acid ethyl ester (MS222; Sigma-Aldrich) but not given any neuro-muscular blocker. They were maintained alive by aerated, anesthetic-containing water flowing over the gills (by gravity) at a flow of 2e3 mL/min, and odorant-containing water was delivered to the olfactory epithelium via a thin glass tube (under gravity) at a flow of around 1 mL/min; no surgery was necessary to expose the olfactory rosette. The EOG was recorded using borosilicate glass micropipettes filled with 1 mol/L NaCl in 2% agar bridged to the D.C. amplifier with Ag/AgCl pellets in 3 mol/L KCl. Pulses (4 s) of odorant-containing solution were introduced into the flow of water over the olfactory rosette via a computeroperated solenoid valve. The optimal position for the recording electrode was found using 105 M L-serine as a stimulus; this was also used to normalize the response amplitude and check the stability of the preparation throughout the recording period (approximately every 12 min), usually evoking a response between 1 and 2 mV (1.46 ± 1.02 mV, N ¼ 11). HPLC fractions were diluted 1:100 in water (i.e. odorants would be at approximately the same concentration as in the original water sample). Pure steroids were all given at 0.5 mg/L (approximately 106 M). Data are shown as arithmetic mean ± standard deviation (SD). 3. Results 3.1. Spawning When female zebrafish could neither see nor smell the males, few of them ovulated (Fig. 2). Similarly, when they could not smell the males, but could see them, few females ovulated. However, when the females could smell the males, about half of them ovulated, whether they could see the males or not. This suggests that olfaction of male-released odorants is much more important for female ovulation than visual contact with the males. In all cases, with or without visual and chemical contact, the females ovulated almost entirely within the first hour of the experiment, mostly within the first 30 min (data not shown). 3.2. Attraction When female zebrafish could neither see nor smell the males, they spent less than 10% of their time in the attraction zone (Fig. 3). However, if they had chemical contact with the males but could not see them, they spent significantly more time in the attraction area (approximately 45%) than when in visual contact but without chemical contact (approximately 25%). With both visual and chemical contact with the males, females spent significantly more time (approximately 70%) in the attraction area. Thus, olfaction is more important than vision in this behavior, but both play a role; their effects are additive. 3.3. EOG responses to HPLC fractions The EOG responses to HPLC fractions proved to be rather variable (only female zebrafish were used for EOG recording), so only qualitative comparisons were made. Nevertheless, several features became clear. Firstly, males released more potent odorants than
Fig. 2. Effect of visual and/or chemical contact (olfaction) with males on spawning in female zebrafish. Numbers of females (out of a total of twenty) that spawned within two hours in the four different treatments (with or without visual and/or chemical contact). Groups with different letters are significantly different from each-other (P < 0.05; One-Way ANOVA followed by Tukey's test, F3,23 ¼ 55.556, N ¼ 6 per treatment group).
females (Fig. 4). A large part of these male-released odorants exited the column in the first four fractions (0e12 min); the activity in these fractions was much greater when males and females were in chemical contact, suggesting that males may be stimulated to release more putative pheromones when in the presence of females (it should be noted that spawning occurred in this tank). Similarly, responses to fractions 15 and 20 were greater in water with males and females together than in that of males or females alone. In this case, however, it is less clear which sex released these odorants. Finally, fraction 7 evoked similar amplitude EOGs in water with males and females, and water with males alone; this fraction was less potent in water from females alone. 3.4. EOG responses to steroids and metabolites Olfactory responses to the steroids (all at 0.5 mg/L; around 106 mol/L) were also variable, but rarely exceeded the response to 105 mol/L L-serine (Fig. 5). The most potent androgens (C19 steroids) were 4-androstenedione, a steroid previously shown to have a pheromonal role in the goldfish (Sorensen, Pinillos, & Scott, 2005), and the structurally related 4-androstene-11b,17b-diol-3one (11b-hydroxytestosterone) and 11b-hydroxyandrostenedione. 5a-androstane-3a,17b-diol was also among the most potent; however, its glucuronated metabolite was without olfactory potency. Among the progestogens (C21 steroids), 5a-pregnane-3a, 17,
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Fig. 3. Effect of visual and/or chemical contact (olfaction) with males on attraction in female zebrafish. A. Percentage of time spent by the female in the predefined ‘attraction area’ adjacent to the male's half of the tank. Groups with different letters (with or without visual and/or chemical contact) are significantly different from eachother (P < 0.05, One-Way ANOVA (arc-sign transformed data) followed by Tukey's test, F3,23 ¼ 50.146, N ¼ 6 per treatment group).
20b-triol, 5a-pregnane-3a, 17-diol-20-one and 4-pregnane-17, 20a-diol-3 one were the most potent; more potent than 4pregnene-17, 20b-diol-3-one, the well characterized maturationinducing steroid (Nagahama, 1997) and goldfish pre-ovulatory pheromone (Dulka et al., 1987). 4. Discussion The current study has shown that female zebrafish are attracted to odorants released by males, consistent with earlier studies (Bloom & Perlmutter, 1977). Furthermore, it shows that the attraction to odorants is greater than that to the sight of the males, and that these two effects are additive. Male odorants also induce ovulation in females, again consistent with earlier studies (Chen & Martinich, 1975; Van Den Hurk & Resink, 1992; Van Den Hurk et al., 1987). Furthermore, olfaction is of prime importance; visual contact alone with males does not appear to have any effect, as few females ovulated in visual contact alone as with neither visual nor chemical contact. Thus, putative male zebrafish pheromones have both ‘primer’ and ‘releaser’ effects on females. The goldfish (like the zebrafish, a cyprinid) female pre-ovulatory pheromone has been identified as a mixture of the maturationinducing steroid 17,20b-dihydroxy-4-pregnen-3-one and its glucuronate and sulfate metabolites (Dulka et al., 1987; Scott & Sorensen, 1994; Sorensen et al., 1995; Van Der Kraak, Sorensen, Stacey, & Dulka, 1989). It was proposed that steroid glucuronates
Fig. 4. Olfactory responses to fractions of male and female holding water. A. HPLC fractions of male holding water (in visual but not chemical contact with each-other). B. HPLC fractions of female holding water (in visual but not chemical contact with eachother). C. HPLC fractions of holding water with males and females in visual and chemical contact. Data are shown as mean þ SD (N ¼ 3 or 4).
also comprise the male pheromone in zebrafish and 7 steroid glucoronates in testis and 5a-androstan-3a,17b-diol and cholesterol glucoronates in male holding water have been identified (Lambert & Resink, 1991; Lambert et al., 1986; Van Den Hurk et al., 1987); however, in the present study, the olfactory potency of 5a-
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Fig. 5. Olfactory responses to steroids in zebrafish. Normalized EOG amplitudes (mean þ SD, N ¼ 3 or 4) to a single concentration (0.5 mg/L; approximately 106 mol/L) of steroids, and some glucoronate and sulfate metabolites, suspected to play pheromonal roles in the zebrafish.
androstan-3a,17b-diol was much higher than its glucoronate. In fact, none of the steroid sulfates or glucuronates tested showed sufficient olfactory potency to justify further investigation into their possible pheromonal roles; the concentrations used (around 106 M) would be extremely high for a pheromone. Nevertheless, that males are releasing potent odorants is clear from the EOG responses to the HPLC fractions. Firstly, only those odorants retained by the solid-phase extraction (SPE) cartridges can be responsible for the EOG responses seen to the HPLC fractions. SPE is commonly used to extract steroids and their metabolites for measurement (Scott et al., 2008) or identification (Keller-Costa et al., 2014). It is likely, therefore, that any steroids and/or conjugates released by zebrafish would be present in the solid-phase extract. However, it seems unlikely that any of the tested steroids contribute to the olfactory responses to the HPLC fractions of male-conditioned water, as they are insufficiently
potent odorants. Nevertheless, we cannot exclude steroids per se; it is necessary to identify the compounds present in the more olfactory potent fractions. Secondly, it is clear that male zebrafish release more potent odorants than females; at least, those that are retained on the solidphase extraction cartridges. Although females are thought to release ovarian steroids that play a pheromonal role on males (Van Den Hurk & Lambert, 1983), the primacy of male released odorants in reproductive chemical communication in zebrafish is suggested by both physiology and behavior in the current study. That a significant proportion of the odorants are found in the first four fractions (first 12 min) means that they have relatively high polarity and, perhaps, are non-steroidal in nature. Future work needs to clarify this, although odorants eluting later from the HPLC column (e.g., fractions 15, 19 and 20) may well be steroidal compounds. Thirdly, the larger EOG responses to fractions from water
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containing both females and males suggests that, when in chemical communication with each-other, one sex is stimulated by the other or that they are mutually stimulated to release odorants. Since water was collected soon after lights on some females ovulated and spawned, prostaglandin F2a, which influences attraction and spawning behavior (Yabuki et al., 2016), could be the female chemical responsible for the activation of the male pheromone release. The large responses to the first four fractions are also seen in the male-alone conditioned water, so it is likely that these odorants are male-released. On the other hand, the odorants in fraction 15 could come from either sex; this odorant does not appear to be present in either female-alone or male-alone conditioned water. Nevertheless, it seems that the smell of the opposite sex can induce odorant - putative pheromone - release in either or both sexes. In conclusion, the current study supports the role of pheromones in zebrafish reproduction, and indicates the primacy of male-released pheromones - as yet unidentified - on both the physiology and behavior of females. However, the contribution of steroids and/or their metabolites, at least those tested, must be augmented by other male-released odorants; zebrafish pheromones are likely to be complex mixtures. Conflicts of interest The authors have no conflicts of interest to declare. Ethics statement The animal experimentation procedures used in this study followed the institutional guidelines for the use of animals in experimentation and were approved by the Ethics Committee of Shanghai Ocean University (behavior experiments) and of the Centre of Marine Sciences (electrophysiology experiments) and by the National Veterinary Authority (DGAV, Portugal; group 1 license). Acknowledgements Li Jie was supported by a grant from Shanghai Aquatic Products (grant no. A1-2035-150048C1). This study received national funds ~o para a Cie ^ncia e a Tecnologia (FCT; Portuguese from Fundaça Foundation for Science and Technology) through project UID/Multi/ 04326/2013. References Belanger, R. M., Pachkowski, M. D., & Stacey, N. E. (2010). Methyltestosteroneinduced changes in electro-olfactogram responses and courtship behaviors of cyprinids. Chemical Senses, 35, 65e74. https://doi.org/10.1093/chemse/bjp085. Bloom, H. D., & Perlmutter, A. (1977). A sexual aggregating pheromone system in
the zebrafish, Brachydanio Rerio (Hamilton-Buchanan). Journal of Experimental Zoology, 199, 215e226. https://doi.org/10.1002/jez.1401990206. Chen, L.-C., & Martinich, R. L. (1975). Pheromonal stimulation and metabolite inhibition of ovulation in zebrafish, Brachydanio Rerio. Fishery Bulletin, 73, 889e894. Dulka, J. G., Stacey, N. E., Sorensen, P. W., & Van Der Kraak, G. J. (1987). A steroid sex pheromone synchronizes male-female spawning readiness in goldfish. Nature, 325, 251e253. Friedrich, R. W., & Korsching, S. I. (1998). Chemotopic, combinatorial, and noncombinatorial odorant representations in the olfactory bulb revealed using a voltage- sensitive axon tracer. Journal of Neuroscience, 18, 9977e9988. Hubbard, P. C., Barata, E. N., & Can ario, A. V. M. (2002). Possible disruption of pheromonal communication by humic acid in the goldfish, Carassius auratus. Aquatic Toxicology, 60, 169e183. Keller-Costa, T., Hubbard, P. C., Paetz, C., Nakamura, Y., Da Silva, J. P., Rato, A., et al. (2014). Identity of a tilapia pheromone released by dominant males that primes females for reproduction. Current Biology, 24, 2130e2135. https://doi.org/ 10.1016/j.cub.2014.07.049. Lambert, J. G. D., & Resink, J. W. (1991). Steroid glucuronides as male pheromones in the reproduction of the African catfish Clarias gariepinus - a brief review. The Journal of Steroid Biochemistry and Molecular Biology, 40, 549e556. Lambert, J. G. D., Van Den Hurk, R., Schoonen, W. G. E. J., Resink, J. W., & Van Oordt, P. G. W. J. (1986). Gonadal steroidogenesis and the possible role of steroid glucoronides as sex pheromones in two species of teleosts. Fish Physiology and Biochemistry, 2, 101e107. https://doi.org/10.1007/BF02264077. Nagahama, Y. (1997). 17 a,20b-dihydroxy-4-pregnen-3-one, a maturation-inducing hormone in fish oocytes: Mechanisms of synthesis and action. Steroids, 62, 190e196. Poling, K. R., Fraser, E. J., & Sorensen, P. W. (2001). The three steroidal components of the goldfish preovulatory pheromone signal evoke different behaviors in males. Comparative Biochemistry and Physiology B, 129, 645e651. Scott, A. P., Hirschenhauser, K., Bender, N., Oliveira, R. F., Earley, R. L., Sebire, M., et al. (2008). Non-invasive measurement of steroids in fish holding water: Important considerations when applying the procedure to behaviour studies. Behaviour, 145, 1307e1328. Scott, A. P., & Sorensen, P. W. (1994). Time course of release of pheromonally active gonadal steroids and their conjugates by ovulatory goldfish. General and Comparative Endocrinology, 96, 309e323. Sorensen, P. W., Pinillos, M., & Scott, A. P. (2005). Sexually mature male goldfish release large quantities of androstenedione into the water where it functions as a pheromone. General and Comparative Endocrinology, 140, 164e175. https:// doi.org/10.1016/j.ygcen.2004.11.006. Sorensen, P. W., Scott, A. P., Stacey, N. E., & Bowdin, L. (1995). Sulfated 17,20bdihydroxy-4-pregnen-3-one functions as a potent and specific olfactory stimulant with pheromonal actions in the goldfish. General and Comparative Endocrinology, 100, 128e142. Van Den Hurk, R., & Lambert, J. G. D. (1983). Ovarian steroid glucuronides function as sex pheromones for male zebrafish, Brachydanio Rerio. Canadian Journal of Zoology-revue Canadienne De Zoologie, 61, 2381e2387. Van Den Hurk, R., & Resink, J. W. (1992). Male reproductive system as sex pheromone producer in teleost fish. Journal of Experimental Zoology, 261, 204e213. https://doi.org/10.1002/jez.1402610211. Van Den Hurk, R., Schoonen, W. G. E. J., Van Zoelen, G. A., & Lambert, J. G. D. (1987). The biosynthesis of steroid glucuronides in the testis of the zebrafish, Brachydanio rerio, and their pheromonal function as ovulation inducers. General and Comparative Endocrinology, 68, 179e188. Van Der Kraak, G., Sorensen, P. W., Stacey, N. E., & Dulka, J. G. (1989). Periovulatory female goldfish release three potential pheromones: 17a,20b-dihydroxyprogesterone, 17a,20b-dihydroxyprogesterone glucuronide, and 17a-hydroxyprogesterone. General and Comparative Endocrinology, 73, 452e457. https:// doi.org/10.1016/0016-6480(89)90202-5. Yabuki, Y., Koide, T., Miyasaka, N., Wakisaka, N., Masuda, M., Ohkura, M., et al. (2016). Olfactory receptor for prostaglandin F2a mediates male fish courtship behavior. Nature Neuroscience, 19, 897e904. https://doi.org/10.1038/nn.4314.
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Aquaculture and Fisheries journal homepage: www.keaipublishing.com/en/journals/ a q u a c u l t u r e - a n d - fi s h e r i e s /
Male zebrafish (Danio rerio) odorants attract females and induce spawning rio a, b Jie Li a, Peter C. Hubbard b, *, Adelino V.M. Cana a b
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China Centro de Ci^ encias do Mar, Universidade do Algarve, Campus de Gambelas, 8005-139, Faro, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2018 Received in revised form 8 June 2018 Accepted 23 June 2018 Available online 11 July 2018
In zebrafish, the chemical identity and biological roles of reproductive pheromones have yet to be clarified. The current study assessed the role(s) of male-released odorants in a reproductive context, and evaluated the possible involvement of steroids and their metabolites. Females were placed in chemical and/or visual contact with males. In chemical contact, females ovulated as frequently whether they could see the males or not. Conversely, females with visual contact alone with males ovulated as frequently as isolated females. Male-released odorants attracted females; visual contact with males attracted females, but to a lesser extent; the two effects were additive. The olfactory potency of solid-phase extraction of male- and female-conditioned water was assessed by the electro-olfactogram; males released more potent odorants than females, but the most potent extract came from water in which males and females interacted freely. HPLC fractionation of these extracts revealed that, while in all samples some fractions contained odorants, male-specific activity was contained in the first four fractions, suggesting that the compounds involved are relatively hydrophilic. Finally, the olfactory potency of some steroids, previously suggested to have a pheromonal role, was assessed. However, it is unlikely that these steroids contribute significantly to the odor of fish-conditioned water. © 2018 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Keywords: Pheromone Zebrafish Reproduction Behavior Olfaction Spawning
1. Introduction Chemical communication is an important facet of reproduction in teleost fishes. The zebrafish (Danio rerio) is an important model species for developmental, genetic and behavioral studies. Yet the chemical identities of its reproductive pheromones remain unknown. Pioneer work in the Netherlands suggested that steroid glucuronates, produced by the testes, are capable of attracting females (Van Den Hurk & Resink, 1992; Lambert, Van Den Hurk, Schoonen, Resink, & Van Oordt, 1986; Van Den Hurk, Schoonen, Van Zoelen, & Lambert, 1987); however, these were never tested individually, or assessed for olfactory potency. On the other hand, 4pregnen-17,20b-diol-3-one 20-sulfate (17,20b-P-sulfate), one of the female-released steroid pheromones identified in the goldfish (Carassius auratus; like the zebrafish, a cyprinid) (Dulka, Stacey, Sorensen, & Van Der Kraak, 1987; Poling, Fraser, & Sorensen, 2001; Sorensen, Scott, Stacey, & Bowdin, 1995) is a potent odorant for the zebrafish
^ncias do Mar, Universidade do Algarve, * Corresponding author. Centro de Cie Campus de Gambelas, 8005-139, Faro, Portugal. E-mail address: [email protected] (P.C. Hubbard).
(Belanger, Pachkowski, & Stacey, 2010; Friedrich & Korsching, 1998). Another female-released goldfish reproductive pheromone, prostaglandin F2a is detected with high sensitivity by zebrafish and mutant male zebrafish deficient in its receptor have impaired courtship towards females (Yabuki et al., 2016). Here we tested the hypothesis that male zebrafish release odorants that attract females and promote spawning, using a combination of behavioral experiments, electrophysiology and high performance liquid chromatography (HPLC). Firstly, we tested the relative importance of visual and chemical cues from the males on female ovulation and behavior. Secondly, we subjected maleand female-conditioned water to solid-phase extraction and HPLC. We then the assessed olfactory potency of these fractions by the electro-olfactogram (EOG) and compared these responses to those evoked by a range of steroids that may form part of the male pheromone. We conclude that males release powerful compounds to the water that attract females and induce them to ovulate but, unlike previous suggestions, sex steroids and their conjugates do not appear to be a major component of the male-released odorants. Thus, more work is needed to identify the male reproductive pheromone in zebrafish.
https://doi.org/10.1016/j.aaf.2018.06.004 2468-550X/© 2018 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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2. Materials and methods 2.1. Fish and housing conditions Wild-type zebrafish of the AB strain three to six months old (3e4 cm standard length, 2.0e3.0 g) and sexually mature were used in this study. Fish were kept at 26e28 C on a 14:10 light:dark photoperiod and fed twice daily at 11:00 and 18:00 with dried brine shrimp (Artemia salina). As soon as their sex could be determined (three months onwards), males were separated from females and kept in single-sex groups. Seven days before the experiments (the ovulatory cycle is five to seven days) single males and females were mated to ensure that they were reproductively active. Fish that spawned were placed again in tanks of single-sex groups to be used in all experiments described below. Fish used for electro-olfactogram (EOG) recording were taken from a separate stock; all female and similar age and size as those used in the behavior experiments.
Fig. 1. Schematic front view of tanks used in the behavioral experiments. Each tank was divided by a partition separating the male from the female side. The partition could be either transparent without holes (vision only), transparent with a 2 mm diameter perforation per cm2 (vision and olfaction), opaque without holes (neither vision nor olfaction), or opaque with a 2 mm diameter perforation every cm2 (olfaction only). The 3 cm wide rectangular region on the female side (broken line) next to the partition was defined as the "attraction zone".
2.2. Ovulation experiment Twenty females were placed in one side of a 24 cm 10 cm 10 cm Perspex tanks separated into two by a division, each division with 1 l of water. The division could be transparent or opaque without or with 1 mm in diameter perforations per cm2. For the control group ‘no vision, no olfaction’, 20 females were placed in one side of the tank and 20 males in the other separated by an opaque division. For the 'vision only' group, 20 females were placed in one side of the tank and 20 males in the other separated by a transparent division without perforations. For the 'olfaction only' group 20 females were placed in one side of the tank and 20 males in the other separated by an opaque division with perforations. Finally, for 'vision and olfaction' group, 20 females were placed in one side of the tank and 20 males in the other separated by a transparent division with perforations. The 20 males and females were placed in their respective compartments at 18:00 the previous day. The next morning, 30 min after 'lights on' (10:00 a.m.) the females were checked for ovulation by applying gentle pressure to the abdomen (without anesthesia, but under water); those which released eggs were removed from the experimental tank. The procedure was repeated three times at 30 min intervals. The experiment was repeated six times independently with different fish (i.e, six times with 20 fish in each treatment group). The data were compared by one-way ANOVA followed by Tukey's post hoc test (data were normally distributed with equal variance).
the female spent in the attraction zone was measured. The data were arc-sign transformed and compared by One-Way ANOVA followed by Tukey's post hoc test (data were normally distributed with equal variance). 2.4. Fish-conditioned water To extract and, eventually, identify odorants - putative pheromones - released by zebrafish, fish-conditioned water was prepared by using a similar experimental approach outlined above for the ovulation experiment with 500 mL water on each side of the division: i) five males and five males that could not see each-other but with chemical contact; ii) five males and five females with both visual and chemical contact. The fish were placed in the tanks at 17:00 of one day until 11:00 the following morning (18 h) to allow plenty of time for odorants to be released. The fish were then removed and the water extracted through a 500 mg C18 solidphase extraction column (Cronus, SMI-LabHut Ltd., Gloucester, UK) using methanol to prime the cartridges and to elute the retained compounds. The methanol was evaporated to dryness (under nitrogen) and the pellet re-dissolved in 100 mL HPLC grade methanol. 2.5. High performance liquid chromatography (HPLC) of water extracts
2.3. Attraction experiment The attraction experiment was carried out in 24 cm 5 cm 10 cm Perspex tanks with divisions as in the ovulation experiment, each half containing 0.5 l water. A 3 cm wide region on the female side, contiguous to the middle division, was defined as the attraction zone (Fig. 1). One female and one male zebrafish were placed separately either each side of the division, opaque or transparent, with or without holes as above; the control group "no vision, no olfaction" had an opaque division with no holes, the "vision only" group had a transparent division, the "olfaction only" group had an opaque division with holes, the "olfaction and vision" group had a transparent division with holes. Experiments started at 30 min after lights on (peak time of day for spawning activity) and the fish was filmed for 30 min by a digital camera (Sony Handycam FDRAXP55). Each treatment was repeated six times (independently with different fish each replicate). The videos were analyzed by Ethovision XT software (version 11.5, Noldus Information Technology, Wageningen, the Netherlands). The proportion of time that
20 mL of the solid-phase extract of fish-conditioned water (described in the previous section) was injected onto a HPLC system (Smartline KNAUER, Berlin, Germany) with a C18 column (3.9 mm 300 mm; 4 mm particle size; Nova-Pak, Waters). HPLC conditions were as follows: mobile phase was pure water (MilliQ) and methanol (HPLC-grade), both containing 0.001% FA; 0e4 min isocratic at 15% methanol, 5e91 min linear gradient from 15% to 100% methanol, 91e96 min isocratic at 100% methanol; flow-rate: 0.7 mL/min, the same conditions used to isolate the steroid glucoronate pheromone from urine of male tilapia (Keller-Costa et al., 2014). The column eluate was first routed to a diode array UV detector (Smartline 2600, KNAUER, Germany), then split off and half routed to an evaporative light scattering detector (Varian 380-LC ELSD, Polymer Laboratories) and the other half collected by an Advantec CHF100SA fraction collector into 32 fractions, each 3 min (i.e. 2.1 mL). ELSD conditions were as follows: nitrogen carrier gas (98% purity, 1.6 SLM), 65 C nebulization temperature, 110 C evaporation temperature.
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2.6. Recording the electro-olfactogram (EOG) The EOG is a robust and sensitive method for assessing olfactory potency of a given compound or biological sample. In the current study, it was used to screen the HPLC fractions and steroids (and some conjugates) for olfactory activity. The EOG was recorded from female zebrafish as previously described for goldfish (Hubbard, rio, 2002) with the following modifications. ZebraBarata, & Cana fish were anesthetized by immersion in water containing 250 mg/L 3-aminobenzoic acid ethyl ester (MS222; Sigma-Aldrich) but not given any neuro-muscular blocker. They were maintained alive by aerated, anesthetic-containing water flowing over the gills (by gravity) at a flow of 2e3 mL/min, and odorant-containing water was delivered to the olfactory epithelium via a thin glass tube (under gravity) at a flow of around 1 mL/min; no surgery was necessary to expose the olfactory rosette. The EOG was recorded using borosilicate glass micropipettes filled with 1 mol/L NaCl in 2% agar bridged to the D.C. amplifier with Ag/AgCl pellets in 3 mol/L KCl. Pulses (4 s) of odorant-containing solution were introduced into the flow of water over the olfactory rosette via a computeroperated solenoid valve. The optimal position for the recording electrode was found using 105 M L-serine as a stimulus; this was also used to normalize the response amplitude and check the stability of the preparation throughout the recording period (approximately every 12 min), usually evoking a response between 1 and 2 mV (1.46 ± 1.02 mV, N ¼ 11). HPLC fractions were diluted 1:100 in water (i.e. odorants would be at approximately the same concentration as in the original water sample). Pure steroids were all given at 0.5 mg/L (approximately 106 M). Data are shown as arithmetic mean ± standard deviation (SD). 3. Results 3.1. Spawning When female zebrafish could neither see nor smell the males, few of them ovulated (Fig. 2). Similarly, when they could not smell the males, but could see them, few females ovulated. However, when the females could smell the males, about half of them ovulated, whether they could see the males or not. This suggests that olfaction of male-released odorants is much more important for female ovulation than visual contact with the males. In all cases, with or without visual and chemical contact, the females ovulated almost entirely within the first hour of the experiment, mostly within the first 30 min (data not shown). 3.2. Attraction When female zebrafish could neither see nor smell the males, they spent less than 10% of their time in the attraction zone (Fig. 3). However, if they had chemical contact with the males but could not see them, they spent significantly more time in the attraction area (approximately 45%) than when in visual contact but without chemical contact (approximately 25%). With both visual and chemical contact with the males, females spent significantly more time (approximately 70%) in the attraction area. Thus, olfaction is more important than vision in this behavior, but both play a role; their effects are additive. 3.3. EOG responses to HPLC fractions The EOG responses to HPLC fractions proved to be rather variable (only female zebrafish were used for EOG recording), so only qualitative comparisons were made. Nevertheless, several features became clear. Firstly, males released more potent odorants than
Fig. 2. Effect of visual and/or chemical contact (olfaction) with males on spawning in female zebrafish. Numbers of females (out of a total of twenty) that spawned within two hours in the four different treatments (with or without visual and/or chemical contact). Groups with different letters are significantly different from each-other (P < 0.05; One-Way ANOVA followed by Tukey's test, F3,23 ¼ 55.556, N ¼ 6 per treatment group).
females (Fig. 4). A large part of these male-released odorants exited the column in the first four fractions (0e12 min); the activity in these fractions was much greater when males and females were in chemical contact, suggesting that males may be stimulated to release more putative pheromones when in the presence of females (it should be noted that spawning occurred in this tank). Similarly, responses to fractions 15 and 20 were greater in water with males and females together than in that of males or females alone. In this case, however, it is less clear which sex released these odorants. Finally, fraction 7 evoked similar amplitude EOGs in water with males and females, and water with males alone; this fraction was less potent in water from females alone. 3.4. EOG responses to steroids and metabolites Olfactory responses to the steroids (all at 0.5 mg/L; around 106 mol/L) were also variable, but rarely exceeded the response to 105 mol/L L-serine (Fig. 5). The most potent androgens (C19 steroids) were 4-androstenedione, a steroid previously shown to have a pheromonal role in the goldfish (Sorensen, Pinillos, & Scott, 2005), and the structurally related 4-androstene-11b,17b-diol-3one (11b-hydroxytestosterone) and 11b-hydroxyandrostenedione. 5a-androstane-3a,17b-diol was also among the most potent; however, its glucuronated metabolite was without olfactory potency. Among the progestogens (C21 steroids), 5a-pregnane-3a, 17,
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Fig. 3. Effect of visual and/or chemical contact (olfaction) with males on attraction in female zebrafish. A. Percentage of time spent by the female in the predefined ‘attraction area’ adjacent to the male's half of the tank. Groups with different letters (with or without visual and/or chemical contact) are significantly different from eachother (P < 0.05, One-Way ANOVA (arc-sign transformed data) followed by Tukey's test, F3,23 ¼ 50.146, N ¼ 6 per treatment group).
20b-triol, 5a-pregnane-3a, 17-diol-20-one and 4-pregnane-17, 20a-diol-3 one were the most potent; more potent than 4pregnene-17, 20b-diol-3-one, the well characterized maturationinducing steroid (Nagahama, 1997) and goldfish pre-ovulatory pheromone (Dulka et al., 1987). 4. Discussion The current study has shown that female zebrafish are attracted to odorants released by males, consistent with earlier studies (Bloom & Perlmutter, 1977). Furthermore, it shows that the attraction to odorants is greater than that to the sight of the males, and that these two effects are additive. Male odorants also induce ovulation in females, again consistent with earlier studies (Chen & Martinich, 1975; Van Den Hurk & Resink, 1992; Van Den Hurk et al., 1987). Furthermore, olfaction is of prime importance; visual contact alone with males does not appear to have any effect, as few females ovulated in visual contact alone as with neither visual nor chemical contact. Thus, putative male zebrafish pheromones have both ‘primer’ and ‘releaser’ effects on females. The goldfish (like the zebrafish, a cyprinid) female pre-ovulatory pheromone has been identified as a mixture of the maturationinducing steroid 17,20b-dihydroxy-4-pregnen-3-one and its glucuronate and sulfate metabolites (Dulka et al., 1987; Scott & Sorensen, 1994; Sorensen et al., 1995; Van Der Kraak, Sorensen, Stacey, & Dulka, 1989). It was proposed that steroid glucuronates
Fig. 4. Olfactory responses to fractions of male and female holding water. A. HPLC fractions of male holding water (in visual but not chemical contact with each-other). B. HPLC fractions of female holding water (in visual but not chemical contact with eachother). C. HPLC fractions of holding water with males and females in visual and chemical contact. Data are shown as mean þ SD (N ¼ 3 or 4).
also comprise the male pheromone in zebrafish and 7 steroid glucoronates in testis and 5a-androstan-3a,17b-diol and cholesterol glucoronates in male holding water have been identified (Lambert & Resink, 1991; Lambert et al., 1986; Van Den Hurk et al., 1987); however, in the present study, the olfactory potency of 5a-
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Fig. 5. Olfactory responses to steroids in zebrafish. Normalized EOG amplitudes (mean þ SD, N ¼ 3 or 4) to a single concentration (0.5 mg/L; approximately 106 mol/L) of steroids, and some glucoronate and sulfate metabolites, suspected to play pheromonal roles in the zebrafish.
androstan-3a,17b-diol was much higher than its glucoronate. In fact, none of the steroid sulfates or glucuronates tested showed sufficient olfactory potency to justify further investigation into their possible pheromonal roles; the concentrations used (around 106 M) would be extremely high for a pheromone. Nevertheless, that males are releasing potent odorants is clear from the EOG responses to the HPLC fractions. Firstly, only those odorants retained by the solid-phase extraction (SPE) cartridges can be responsible for the EOG responses seen to the HPLC fractions. SPE is commonly used to extract steroids and their metabolites for measurement (Scott et al., 2008) or identification (Keller-Costa et al., 2014). It is likely, therefore, that any steroids and/or conjugates released by zebrafish would be present in the solid-phase extract. However, it seems unlikely that any of the tested steroids contribute to the olfactory responses to the HPLC fractions of male-conditioned water, as they are insufficiently
potent odorants. Nevertheless, we cannot exclude steroids per se; it is necessary to identify the compounds present in the more olfactory potent fractions. Secondly, it is clear that male zebrafish release more potent odorants than females; at least, those that are retained on the solidphase extraction cartridges. Although females are thought to release ovarian steroids that play a pheromonal role on males (Van Den Hurk & Lambert, 1983), the primacy of male released odorants in reproductive chemical communication in zebrafish is suggested by both physiology and behavior in the current study. That a significant proportion of the odorants are found in the first four fractions (first 12 min) means that they have relatively high polarity and, perhaps, are non-steroidal in nature. Future work needs to clarify this, although odorants eluting later from the HPLC column (e.g., fractions 15, 19 and 20) may well be steroidal compounds. Thirdly, the larger EOG responses to fractions from water
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containing both females and males suggests that, when in chemical communication with each-other, one sex is stimulated by the other or that they are mutually stimulated to release odorants. Since water was collected soon after lights on some females ovulated and spawned, prostaglandin F2a, which influences attraction and spawning behavior (Yabuki et al., 2016), could be the female chemical responsible for the activation of the male pheromone release. The large responses to the first four fractions are also seen in the male-alone conditioned water, so it is likely that these odorants are male-released. On the other hand, the odorants in fraction 15 could come from either sex; this odorant does not appear to be present in either female-alone or male-alone conditioned water. Nevertheless, it seems that the smell of the opposite sex can induce odorant - putative pheromone - release in either or both sexes. In conclusion, the current study supports the role of pheromones in zebrafish reproduction, and indicates the primacy of male-released pheromones - as yet unidentified - on both the physiology and behavior of females. However, the contribution of steroids and/or their metabolites, at least those tested, must be augmented by other male-released odorants; zebrafish pheromones are likely to be complex mixtures. Conflicts of interest The authors have no conflicts of interest to declare. Ethics statement The animal experimentation procedures used in this study followed the institutional guidelines for the use of animals in experimentation and were approved by the Ethics Committee of Shanghai Ocean University (behavior experiments) and of the Centre of Marine Sciences (electrophysiology experiments) and by the National Veterinary Authority (DGAV, Portugal; group 1 license). Acknowledgements Li Jie was supported by a grant from Shanghai Aquatic Products (grant no. A1-2035-150048C1). This study received national funds ~o para a Cie ^ncia e a Tecnologia (FCT; Portuguese from Fundaça Foundation for Science and Technology) through project UID/Multi/ 04326/2013. References Belanger, R. M., Pachkowski, M. D., & Stacey, N. E. (2010). Methyltestosteroneinduced changes in electro-olfactogram responses and courtship behaviors of cyprinids. Chemical Senses, 35, 65e74. https://doi.org/10.1093/chemse/bjp085. Bloom, H. D., & Perlmutter, A. (1977). A sexual aggregating pheromone system in
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