Journal of Fish Biology (2003) 63, 1615–1620 doi:10.1046/j.1095-8649.2003.00260.x, available online at http://www.blackwell-synergy.com Androgen levels of reproductive competitors in a co-operatively breeding cichlid R. F. O L I V E I R A *†, K. H I R S C H E N H A U S E R *, A. V. M. C A N Á R I O ‡ A N D M. T A B O R S K Y § *Unidade de Investigação em Eco-Etologia, Instituto Superior de Psicologia Aplicada, Rua Jardim do Tabaco 34, 1149–041 Lisboa, Portugal, ‡Centro de Cieˆncias do Mar, Universidade do Algarve, Campus de Gambelas, 800–117 Faro, Portugal and §Department of Behavioural Ecology, Zoological Institute, University of Bern, Wohlenstrasse 50a, CH-3032 Hinterkappelen, Switzerland (Received 7 February 2003, Accepted 10 September 2003) Androgen levels of family groups of Neolamprologus pulcher were assessed using non-invasive methods. There were no significant differences in the excretion rates neither of testosterone nor of 11-ketotestosterone between territorial and helper males. # 2003 The Fisheries Society of the British Isles Co-operative breeding has been described in many birds and mammal species, in which young individuals, usually genetically related to the breeding pair, help to raise the offspring produced in a group (Emlen, 1991). In teleosts co-operative breeding has been described only for a few species (Taborsky & Limberger, 1981; Taborsky, 1984, 1994, 2001). It can take the form of associations between a bourgeois and a satellite male that trade help in defending the territory, courting females and building nests for easier access to parasitic fertilizations (Taborsky et al., 1987; Taborsky, 1994, 1999, 2001; Martin & Taborsky, 1997; Oliveira et al., 2002). Co-operative brood care has also been documented in teleosts and an extreme specialization of this pattern is the occurrence of extended families in which younger individuals stay with the breeding pair and actively participate in parental care (Taborsky & Limberger, 1981; Taborsky, 1984, 1994; Balshine-Earn et al., 1998, 2001). Helpers of co-operatively breeding birds and mammals generally have lower androgen levels than breeding males, which have been interpreted as a psychological castration of helpers by the breeding dominant male of the family group (Reyer et al., 1986; Schmidt et al., 1991; Schoech et al., 1991; Wingfield et al., 1991; Poiani & Fletcher, 1994; Vleck & Brown, 1999; Peters et al., 2001). This pattern, however, has not been found in species in which helpers have substantial mating opportunities and may share paternity of the offspring †Author to whom correspondence should be addressed. Tel.: þ351 21 8811708; fax: þ351 21 8860954; email: ruiol@ispa.pt 1615 # 2003 The Fisheries Society of the British Isles 1616 R. F. OLIVEIRA ET AL. (e.g. beta males in co-operatively breeding packs of African wild dogs Lycaon pictus; Creel et al., 1997). Neolamprologus pulcher (Trewavas & Poll) is a teleost species with extended families, among which males compete for reproduction (Taborsky & Limberger, 1981; Taborsky, 1984, 1985), and male helpers are able to share in paternity (Dierkes et al., 1999). Thus differences in androgen levels between breeding and helper males are not expected in this species. In the present study the androgen levels (testosterone, T; 11-ketotestosterone, KT) of the different members of family groups of N. pulcher were investigated (i.e. breeding male, breeding female, male helpers and female helpers). The dominant male breeder, the dominant female breeder, the largest male helper and the largest female helper of each family group were sampled in a controlled laboratory situation. Only families were sampled in which the standard length (LS) of the largest male helper was >3
5 cm since this is considered the size at maturity for male helpers (Taborsky, 1984). The composition of each family group is summarized in Table I. Sampling occurred always between 1700 and 1900 hours to avoid effects of the daily variations in androgen levels that are known to occur in cichlids (Oliveira et al., 2001a). Since individuals were too small to allow blood sampling without sacrificing the fish androgen excretion was assayed. Steroid excretion rates of goldfish Carassius auratus (L.) measured in holding water have been shown (Scott & Sorensen, 1994) to match well blood plasma levels in fish receiving similar treatments (Moriwaki et al., 1991). The injection of males of Oreochromis mossambicus L. with luteinizing hormone-releasing hormone (LHRH) leads to subsequent increase in excretion of KT and T measured in holding water (Hirschenhauser et al., 2002). Individual N. pulcher were caught with a hand net and placed in a small aquarium (beaker diameter ¼ 15 cm), which contained TABLE I. Group composition of the studied families of Neolamprologus pulcher Family Number of helpers in the group Presence of fry or eggs Mass of territorial male (g) Mass of breeding female (g) Mass of male helper (g) Mass of female helper (g) 15 3 4 5 3 5 18 9 3 11 4 11 13 No Yes Yes Yes No Yes Yes No Yes Yes Yes Yes Yes 19
7 11
2 11
5 7
5 25
9 16
0 18
3 25
3 14
5 15
2 17
7 13
2 20
1 15
1 8
7 11
5 10
5 12
6 9
6 16
8 16
7 8
7 17
2 13
4 10
4 7
6 n.a. 8
6 5
4 2
5 9
3 n.a. 2
0 5
3 4
3 1
3 7
4 3
8 3
4 n.a. n.a. n.a. 1
7 n.a. 2
5 10
4 8
9 n.a. 2
2 8
3 3
9 7
9 A B C D E F G H I J K L M n.a., not available in this family group. # 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1615–1620 ANDROGENS IN A CO-OPERATIVELY BREEDING CICHLID 1617 500 ml of water and were left for 1 h. Free, sulphated and glucuronidated steroids were solid phase extracted (Sep Pak RP-18, Merck) following procedures previously described (Scott & Sorensen, 1994; Greenwood et al., 2001) and stored at 20 C until the radioimmunoassays (RIA) for T (Scott et al., 1984) and KT (Kime & Manning, 1982) were performed. A pool of water extracts were separated on thin-layer chromatography and fractions assayed to confirm the specificity of the antisera for the samples. Only one immunoreactive peak was detected for each of the RIAs matching the elution position of the respective androgens (R.F. Oliveira, K. Hirschenhauser, T. Oliveira & A.V.M. Canário, unpubl. data). Since excretion rates are expected to be related to body mass and body mass varied significantly among the four types of individuals (Kruskal–Wallis ANOVA, d.f. ¼ 3 and 52, P ¼ 0
01), androgen values were related to individual body mass (ng g1 body mass). Since only one breeding female and three female helpers had androgen levels above the limit of reliable measurements of the assays used (0
4 ng sample1), females were not included in the statistical analysis (androgens levels in the breeding female: T excretion rate ¼ 0
40 ng h1 g1 body mass, KT excretion rate ¼ 0
29 ng h1 g1 body mass; female helpers: average T excretion rate ¼ 0
57 ng h1 g1 body mass, average KT excretion rate ¼ 0
60 ng h1 g1 body mass). Non-parametric statistics were applied using the statistical package Statistica # V.5.0A ( Statsoft Inc., U.S.A.). There were no significant differences in the excretion rates of T or KT between territorial (n ¼ 9) and helper males (n ¼ 11) (Mann–Whitney U test: KT, P ¼ 0
34 and T, P ¼ 0
24; Fig. 1). There were no effects of family group size on androgen levels (either KT or T) neither among territorial males (n ¼ 9; Spearman rank correlation, KT, P ¼ 0
80 and T, P ¼ 0
87) nor among male helpers (n ¼ 11; Spearman rank correlation, KT, P ¼ 0
27 and T, P ¼ 0
54). Again, with the sample size used in this study, the variation in male androgen levels was not explained by the presence of eggs and fry (0/1) in the family group, neither among territorial males (nwithout fry ¼ 5, nwith fry ¼ 4; Mann– Whitney U test, KT, P ¼ 0
22 and T, P ¼ 0
22) nor among male helpers (nwithout fry ¼ 6, nwith fry ¼ 5; Mann–Whitney U test, KT, P ¼ 0
36 and T, P ¼ 0
27). Neolamprologus pulcher male helpers also share paternity and their helping behaviour is therefore part of an alternative reproductive tactic. In all teleost species with alternative reproductive tactics for which androgen levels are known, the bourgeois courting morph has higher KT levels than the parasitic non-courting morph (Brantley et al., 1993; Oliveira et al., 2001b). Therefore, the present results may be seen as the first exception to this rule. In N. pulcher, however, there is no qualitative differentiation of helpers as an alternative morphotype. Helpers are subordinate to breeders but they display all reproductive behavioural patterns present in breeders, including territorial defence and brood care (Taborsky & Limberger, 1981; Limberger, 1983; Taborsky, 1984). Moreover, in this species helper males do not have higher gonado-somatic indices (IG) than territorial males (F. Neat, M. Taborsky & S. Balshine, unpubl. data), which do not conform to the pattern found in teleost species with alternative reproductive tactics, in which parasitic spawners (e.g. sneakers) generally have higher IG values than bourgeois males (Taborsky, 1994, 1999, 2001). # 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1615–1620 1618 11-Ketotestosterone excretion (ng h–1 g–1 body mass) R. F. OLIVEIRA ET AL. 2 1 0 Territorial Helper Male type Testosterone excretion (ng h–1 g–1 body mass) 2 1 0 Territorial Helper Male type FIG. 1. Androgen levels in territorial males (n ¼ 9) and male helpers (n ¼ 11); bar ¼ median; box ¼ quartiles; whiskers ¼ min–max values; open circles ¼ outliers. In conclusion, the results of this study confirm for the first time in a teleost the prediction derived from observations in higher vertebrates that in co-operatively breeding species in which helpers’ reproduction is not suppressed, helpers and breeding males have similar androgen levels. The authors acknowledge E. Couto’s technical assistance in the steroid measurements and the comments provided by A. Ros and the late L. Carneiro that contributed to improve the manuscript. This study is part of a larger research project of RFO funded by Fundação para a Ciência e a Tecnologia (FCT, Praxis XXI/P/BIA/10251/1998). RFO research is supported by a Plurianual Research Grant (FCT R&D research unit 331/94) and MT by a SFN Grant (31-64396.01). # 2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 1615–1620 ANDROGENS IN A CO-OPERATIVELY BREEDING CICHLID 1619 References Balshine-Earn, S., Neat, F. C., Reid, H. & Taborsky, M. (1998). Paying to stay or paying to breed? Field evidence for direct benefits of helping behaviour in a cooperative breeding cichlid fish, Neolamprologus pulcher. Behavioral Ecology 9, 432–438. Balshine-Earn, S., Leach, B., Neat, F., Reid, H., Taborsky, M. & Werner, N. (2001). 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