Environmental control of cyclical reproduction of tropical freshwater fish: Evidence from comparative experimental data by Frank Kirschbaum (1, 2), Uwe Leyendecker (3), Betty Nyonge (2), Carsten Schulz (2), Hendrik Weitkamp (2), Salif Didhiou (2), Marielle Thomas (4) & Christian Schugardt (1, 2)

AbsTract. - In 3 neotropical gymnotiform fishes gonad maturation and spawning could be obtained by experimental variation of 3 environmental factors, 1) imitation of rain (R), 2) decreasing conductivity (C), and 3) increasing water level (WL), or only 2 factors (WL and C) (4 species) or just (C) (1 species). The basis of the regulation of cyclical reproduction is induction of gonad maturation (yolk vesicle formation, vitellogenesis) and all stages of spermatogenesis. In more than 10 African mormyrid species decrease of C alone was sufficient to induce gonad maturation, spawning could be obtained in 9 species. In 2 species of the family Schilbeidae (Pareutropius buffei, Parailia pellucida) and 2 species of the genus Syno- dontis (S. nigrita, S. nigriventris) (Mochokidae) variation of all 3 factors led to gonad maturation. Detailed experimental analysis in Pareutropius buffei showed that imitation of R was sufficient to induce complete gonad maturation. In the South-East Asian catfish Kryptopterus bicirrhis variation of all 3 environmental factors led to complete gonad maturation. In the two notopterid knifefishes Xenomystus nigri (Africa) and Notopterus notopterus (South-East Asia) and the African Polypterus senegalus no clear correlation between variation of environmental factors and gonad development or sex hor- mone titers could be found. Key words. - Gonad maturation - Fractional spawning - Cyclical reproduction - Freshwater fish - Environmental factors.

Introduction Photoperiod was held constant (LD 12:12) and temperature Field data from South America, Africa, and South-East was in general kept at 26 to 28°C (range 24-31°C). Correla- Asia indicate that cyclical reproduction of tropical freshwa- tion between GSI values and testosterone and estradiol val- ter fishes is a common phenomenon (Benech and Quensiere, ues were tested for significance with the SPERMAN rank 1985). However, the environmental control (proximate fac- correlation. tors) of the cyclical reproduction is poorly investigated. In Results and discussion this paper experimental variation of environmental factors, Most of the experiments with the neotropical gymnoti- imitation of rain, variation of conductivity and water level, form fishes were done with Eigenmannia lineata. In a first imitating the effect of high and low water season on gonad experiment for 62 days R was imitated, C was decreased maturation and regression are presented comprising various from 1500 µS cm-1 to 380 µS cm-1 and WL increased from taxa of tropical freshwater fishes. Some of the results have 20 cm up to 45 cm three times during that period. This exper- already been published. imental variation lead to a significant (p = 0.014, always Material and Methods U-test) increase of the median GSI values in females (n = 4) Most of the fish were wild specimens. Imitation of high after 18 days from 0.83% (day 1; ovaries containing oocytes water condition was performed by imitation of rain (R), of type I) to 2.89% (ovaries containing oocytes of types I, II (recirculation system with a spray bar, 8 hours per day), and III). After 33 days, the median GSI values were 8.2% decrease of conductivity (C) (addition of deionised water to (value significantly (p = 0.008) different from the 0.83% tap water) and increase of water level (WL). Imitation of low value); these ovaries contained oocyte types I to IV and were water conditions was achieved by increase of conductivity mature. After 62 days, neither a change in the GSI values (addition of neutral salts). The reaction of the fish was meas- (6.9%) nor in the histology of the ovaries was found; these ured either by using the gonado-somatic index (GSI), or by fish remained mature. The males (n = 3-4) also had regressed histological characterization of the gonads, and/or in addi- gonads on day 1 (0.22%) containing spermatogonia and tion by spawning or by determination of testosterone and some spermatocytes I. On day 18 the median GSI value had estradiol titers (competitive ELISA; modified after Cuisset significantly (p = 0.028) increased to 0.56%; these testis now et al., 1994). At the beginning of the experiments the fish contained spermatocytes II, spermatides and spermatozoa; had regressed gonads (induced by decreasing conductivity) they were mature. On day 33 and 62, the testis remained containing type I oocytes (terminology see Wallace and unchanged both concerning histology and median GSI val- Selman, 1981) in females and spermatogonia in the testis. ues (0.59 and 0.68%, respectively). From day 62 to day 102

(1) Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Department of Biology and Ecology of Fishes, Müggelseedamm 310, D-12587 Berlin, Germany. [[email protected]] (2) Institute of Animal Sciences, Humboldt University Berlin, Philippstraße 13, Haus16, D-10099 Berlin, Germany. (3) Zoological Institute, University of Cologne, Weyertal 119, D-50931 Köln, Germany. (4) Université Henri Poincaré-Nancy 1, Communauté urbaine du Grand Nancy, Musée de Zoologie-Aquarium Tropical, 34 rue Sainte- Catherine, F-54000 Nancy, France.

Cybium 2008, 32(2) suppl.: 294-296. Ki r s c h b a u m e t a l . Cyclical reproduction of tropical freshwater fish gonad regression was induced by stop of rain imitation and conductivity increase from 380 µS cm-1 to 610 µS cm-1; WL increase was continued. This lead to a significant (p = 0.014) decrease of the median GSI values in females (n = 4) on day 74 (2.58%) and to a further significant (p = 0.005) decrease on day 89 (1.06%). These ovaries now were regressed like the ovaries at the beginning of the experiment. In males, the experimental variation of the environmental factors also lead to a significant decrease of the median GSI values, but more retarded compared to females. On day 74 and 89, the median GSI values (0.64 and 0.58%, respectively) were not different from the 62-day value, but on day 102 the difference (medi- an GSI value 0.39%) was significant (p = 0.008); in these testis spermatozoa had decreased considerably. In the con- trol females (N = 3-4) kept at constant water level of 30 cm, Figure 1. - Induction of gonad maturation and subsequent spawning no imitation of rain, and slightly increasing conductivity (numbers below the bar; grey bar represents length of reproductive (from 1500 µS cm-1 on day 1 up to 1650 µS cm-1 on day period) and gonad regression (O) by experimental variation of con- 102) no change in mean GSI values was observed (0.83% on ductivity alone in Mormyrus rume proboscirostris (after Schugardt day 1; 1.25% on day 62; 1.01 on day 102). In the control and Kirschbaum, 2004, modified). males on day 62 a significant (p = 0.05) increase in the medi- an GSI value 0.59% (compared to 0.22%) was observed; on onstrated that the decrease of conductivity is important for day 102 the GSI value was 0.33%, however only 1 control gonadal recrudescence independent of the absolute values male was left at that time. We performed additional breeding (Kirschbaum, 1987). In P. isidori 8 spawnings occurred; 25 experiments with E. lineata applying the same experimental spawnings could be obtained in M. sp.; 36 in Hippopot- variation of the 3 environmental factors: 9 breeding experi- amyrus pictus; 13 in Campylomormyrus tamandua, 6 in C. ments with different sex ratios (2 f, 3 m; 3 f, 4 m; 3 f, 7 m) sp.; 2 in Petrocephalus soudanensis, and 2 in Marcusenius yielded 196 spawnings; finally a F3 generation was obtained. mento. M. mento might be a total spawner, the remaining 8 E. lineata revealed to be a fractional spawner with short species all are fractional spawners (intervals a few days up intervals between spawnings: between 2 to 5 days in females to several weeks). fed very well (for further reproductive features see Kirsch- 4 species of two African catfish families (Schilbeidae: baum and Schugardt, 2002). With additional gymnotiform Pareutropius buffei, Parailia pellucida; Mochokidae: Syno- fish experiments were performed using different combina- dontis nigrita, S. nigriventris) were investigated. The reac- tions of the 3 environmental factors; spawning and raising of tion of the fish towards the experimental variation of all three larvae was always taken as indication of complete gonad environmental factors was assessed with GSI values and maturation: in Sternopygus macrurus and Apteronotus albi- gonad histology. As an example the experiments with P. buf- frons 2 and 3 spawnings, respectively, could be obtained fei are described: Imitation of R, decrease of C (from 932 µS after variation of all three environmental factors. Variation cm-1 to 337 µS cm-1) and WL increase (from 25 cm up to 50 of C and WL led to spawning in A. leptorhynchus (many cm) for 6 weeks led to a significant increase in median GSI spawnings over years), Rhamphichthys sp. (7 spawnings), values both in males and females; histology demonstrated Brachyhypopomus pinnicaudatus and B. brevirostris (1 these gonad to be mature. In females the median GSI values spawning each). All gymnotiform species investigated increased from 0.8% (n = 4) to 18.25% (n = 7) (p = 0.016, proved to be fractional spawners (intervals between spawn- U-test); control females had 1.23% (n = 7); in males the val- ings a few days up to several weeks). ues increased from 0.07% (n = 8) to 1.8% (p = 0.003, U-test); 9 African mormyrid fishes were investigated. As exem- control males had a median value of 0.08% (n = 5). Addi- plified in Mormyrus rume proboscirostris (Fig. 1) decrease tional experiments with the same principal experimental set of C alone is sufficient to induce gonad maturation and up showed that imitation of R alone (for 49 days) provoked a spawning, increase of WL and imitation of R did not pro- significant increase of the median GSI values both in females voke gonadal recrudescence. In this species regressed ova- and males leading to mature gonads. Increase of WL as sin- ries had median (n = 4) GSI values of 0.12%, ripe ovaries gle factor led to a significant increase in the median GSI val- amounted to 4.30%; regressed testis showed median (n = 4) ues of the females (8.42% (n = 8), p = 0.002, U-test), but not GSI values of 0.63%, ripe testis median values of 0.31%. In in males (0.1%). Imitation of R, decrease of C and increase Pollimyrus adspersus more than 100 spawnings could be of WL led to significant increases of the median GSI values provoked over many years. In this species it could be dem- both in females and males and mature gonads in Parailia

Cybium 2008, 32(2) suppl. 295 Cyclical reproduction of tropical freshwater fish Ki r s c h b a u m e t a l . pellucid, S. nigrita and, S. nigriventris. The significance of late the cyclical reproduction depend on the biology of the single environmental factors for gonad recrudescence was species and therefore represent an interesting example of not tested. convergent evolution. These data could be helpful for aquac- In the African Polypterus senegalus (Polypteridae, ulture of tropical freshwater fishes. Polypteriformes) variation of all three factors (R, C, and WL) led to spawning, however in a less predictable way as Acknowledgements. - Some of the investigations were supported by grants from the German Science Foundation (DFG). We in the species mentioned above. The endogenous control of acknowledge varies technical support during our stay in France gonad maturation seems to prevail in P. senegalus. For dis- (Gif-sur-Yvette) and Germany (Cologne, Berlin). cussion of this aspect see Schugardt and Kirschbaum References (2006). BENECH V. & J. QUENSIERE, 1985. - Strategies de reproduction In the two notopterid knifefishes Xenomystus nigri (Afri- des poissons du Tchad en periode de “Tchad Normal” (1966- ca) and Notopterus notopterus (South-East Asia) no clear 1971). Rev. Hydrobiol. Trop., 18(3): 227-244. correlation between variation of environmental factors (3 CUISSET B, PRADELLES P., KIME D.E., KUEHN E.R., BABIN factors in X. nigri ) (increase of WL and decrease of C in N. P., DAVAIL S. & F. LE MENN, 1994. - Enzyme immunoassay for 11-ketosterone using acetylcholinesterase as label: Applica- notopterus) and gonad development or sex hormone titers tion to the measurement of 11-ketosterone in plasma of Siberi- (testosterone and estradiol) could be found. However, when an sturgeon. Comp. Biochem. Physiol. Pharmacol. Tox. Endo- these fish were put in large tanks in small numbers without crinol., 108C: 229-241. the presence of other fish gonad maturation (X. nigri) and KIRSCHBAUM F., 1979. - Reproduction of the weakly electric spawning did occur (N. notopterus) after several weeks. fish Eigenmannia virescens (Rhamphichthyidae, Teleostei) in captivity. I. Control of gonadal recrudescence and regression In the South-East Asian Kryptopterus bicirrhis (Siluri- by environmental factors. Behav. Ecol. Sociobiol., 4: 331-355. dae) variation of all three environmental factors led to com- KIRSCHBAUM F., 1987. - Reproduction and development of the plete gonad maturation (gonad histology and GSI). The sig- weakly electric fish Pollimyrus isidori (Mormyridae, Teleostei) nificance of single factors was not investigated. in captivity. Env. Biol. Fishes, 20: 11-31. KIRSCHBAUM F. & C. SCHUGARDT, 2002. - Reproductive Conclusion strategies and developmental aspects in mormyrid and gymno- We showed experimentally that cyclical reproduction is tiform fishes. J. Physiol., 96: 557-566. found in several taxa of tropical fishes from South America, SCHUGARDT C. & F. KIRSCHBAUM, 2004. - Control of gonad maturation and regression by experimental variation of envi- Africa, and South-East Asia. The field data and the many ronmental factors in the mormyrid fish, Mormyrus rume pro- ornamental fish with lacking reproduction in captivity indi- boscirostris. Env. Biol. Fish., 70: 227-233. cate that many more tropical freshwater fishes are character- SCHUGARDT C. & F. KIRSCHBAUM, 2006. - Fortpflanzung und ized by cyclical reproduction though experimental evidence Entwicklung von Polypterus senegalus. In: Biologie der Aquar- ienfische (GREVEN H, RIEHL R, eds), pp. 171-182. Tetra Ver- is still lacking. The experimental data of the African fishes lag GmbH. (mormyrids, schilbeid fishes, mochokid fishes, notopterid WALLACE R. A. & K. SELMAN, 1981. - Cellular and dynamic knifefish) studied show that the proximate factors that regu- aspects of oocyte growth in teleosts. Am. Zool., 21: 325-343.

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