Frequency and periodicity of spawning in the clownfish Amphiprion akallopisos under aquarium conditions ANDREW K. GORDON and ANDRE W. BOK Oceanographic Research Institute, P.O. Box 10712, Durban, South Africa Received 7 July 1999; accepted 19 August 2000 Key words: Amphiprion akallopisos, spawning, lunar cycle, clownfish ABSTRACT Amphiprion akallopisos kept under aquarium conditions in South Africa spawned on average 2.2± 0.8 (standard deviation) times per month throughout the year. There was no significant correlation between percentage of A. akallopisos pairs spawning and phase of the moon or tidal patterns. The increase in the number of spawnings during some spring and summer months appeared to correlate with changing photoperiod rather than changing water temperature.

INTRODUCTION

In wild populations of clownfishes, spawning frequency and spawning periodicity appear to vary with latitude. Clownfish species occurring in tropical waters spawn throughout the year (Allen, 1972; Ross, 1978), whereas in more temperate regions spawning occurs only during the warmer summer months (Bell, 1976; Moyer, 1980; Ochi, 1985; Richardson et al., 1997). Spawning periodicity in tropically occurring clownfishes has also been strongly correlated with lunar cycles (Allen, 1972; Ross, 1978). However, in temperate clownfish species, the correlation between spawning periodicity and moon phase becomes much weaker (Richardson et al., 1997). In aquaria, the environmental cues that control reproductive cycles in wild fishes are very often absent or weak. As a result, fish become entrained by the simulated temperatures and lighting regimes imposed by the aquarist, and reproductive activity changes accordingly (Kohler et al., 1994). Clownfish, in aquaria, appear to breed throughout the year (Alava and Gomes, 1989; Hoff, 1996), with an increase in the number of spawnings during the spring and summer months (Hoff, 1996). There appears to be no correlation between spawning periodicity and lunar cycle (Alava and Gomes, 1989). This paper presents both spawning frequency and periodicity data for Amphiprion akallopisos, a tropical and warm temperate Indian Ocean clownfish, investigated under hatchery conditions in South Africa.

MATERIALS AND METHODS

Five pairs of A. akallopisos were collected from Sodwana Bay (27◦ 24.9 S; 32◦ 43.6 E), South Africa and transported to the ornamental fish hatchery at the Aquarium Sciences and Conservation 3: 307–313, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Oceanographic Research Institute (ORI) in Durban. Pairs of clownfish were each placed in 188 L glass aquaria and connected to a 4000 L partially recirculating system. Each pair were assigned a code number (AK1 to AK5). During the course of the study pairs were fed once daily, to satiety, on female brown mussel (Perna perna) gonad. Pairs were observed twice daily, in the morning and late afternoon, for evidence of spawning. Natural seawater exchanged with system water at a rate of 48 L h−1 . The natural seawater was drawn directly from a well point situated 4 m below the sand in the intertidal zone of the shore adjacent to the Institute. The system incorporated three biological trickle-tower filters (length = 137 cm, diameter = 16 cm) filled with shadecloth. Aquarium floors were covered with sand and spawning décor was provided in the form of pieces of rock and 50 mm PVC T-pieces. Throughout the study period water quality parameters remained within acceptable limits with pH ranging from 8.08 to 8.12, and specific gravity from 1.020 to 1.025. Aquarium test kits (Red Sea Fish Pharm Ltd.) measured NH4+ /NH3 and NO−2 values at 0 mg L−1 and NO−3 levels below 6 mg L−1 . Water temperature was maintained by means of solar heat and forced hot air heaters. Water temperature data were collected during 1999 from the broodstock system, and ranged from 25◦ C in winter to 31◦ C in summer (Figure 1). Ambient light entered the hatchery through glass windows in the walls. Supplementary artificial lighting was provided by cool white flourescent tubes situated 30 cm above each tank and set on a 12 h light : 12 h dark cycle. The resultant photoperiod in the hatchery ranged from 12 h light : 12 h dark in winter to approximately 14 h light : 10 h dark in summer. Light intensity 5 cm below the water surface of aquaria was measured with a Licor photometer model LI-185B and ranged from 63 to 75 Lux.

Figure 1. Average number of A. akallopisos spawnings over a three-year period (1997–1999) and monthly water temperatures (±standard deviation) for 1999 in the Oceanographic Research Institute marine hatchery.

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RESULTS AND CONCLUSIONS

Data on spawning were collected from November 1996 until November 1999. However, not all the five pairs provided data for the duration of the study as pairs began to breed at different times and an individual from each of three pairs died before the end of the study. One individual died from a suspected internal infection, while the other two died after jumping out of their aquariums. Consequently, all data were analyzed as percentages of the number of pairs reproductively active at that time. At the beginning of June 1997 there was an outbreak of disease, and the broodstock were temporarily removed to a different system while the original system was treated with freshwater flushes. Consequently, none of the pairs spawned during June or July 1997, thus these two months were excluded from the statistical analyses. Data were statistically analyzed using analysis of variance (ANOVA) and correlation analysis (Zar, 1974). A. akallopisos in this study spawned on average 2.2 ± 0.8 (SD) times per month throughout the year (Table 1). Table 2 presents spawning data for various wild and captive populations of clownfish species. Year-round spawning for captive Amphiprionae at Instant Ocean Hatcheries (IOH) in Florida is reported (Hoff, 1996), with A. akallopisos spawning an average of 2.4 times per month (Table 2). Year-round spawning is also recorded for wild populations of tropically occurring A. chrysopterus, A. perideraion (Allen, 1972) and A. melanopus (Allen, 1972; Ross, 1978). Year-round spawning appears to be an indication of the stability of environmental conditions both in captivity (hatcheries) and in the wild within tropical latitudes. In contrast, the low water temperatures and more unstable environmental conditions of temperate regions result in spawning only occuring during the summer months (Richardson et al., 1997). Overall, there is a higher spawning frequency for captive clownfish as compared with wild populations (Table 2), although this increased spawning frequency in captivity did not appear to have had any deleterious effect on the health of the A. akallopisos at ORI over the three-year study period. Wild populations of A. clarkii from Miyake-jima and Shiko-ku island spawned 7.0 and 5.5 times per Table 1. Monthly spawning records for A. akallopisos at the Oceanographic Research Institute marine hatchery Pair code

AK1 AK2 AK3 AK4 AK5 Total

Number of nests produced over study period 15 70 8 14 73 180

Number of months of study

Average spawnings per month (±SD)

7 34 4 7 31 83

2.1 ± 0.7 2.1 ± 0.7 2.0 ± 0.0 2.0 ± 0.8 2.4 ± 1.0 2.2 ± 0.8

Wild: tropical conditions A. melanopus A. chrysopterus A. perideraion Wild: warm temperate conditions A. clarkii A. clarkii A. akindynos A. latezonatus Aquarium/hatchery conditions A. percula P. biaculeatus A. clarkii A. ephippium A. frenatus A. melanopus A. ocellaris A. akallopisos A. akallopisos

Species

Miyake-jima (34◦ N) Shiko-ku Is. (33◦ N) Julian Rocks (27◦ S) Julian Rocks (27◦ S)

Guam (13◦ N) Eniwetok (11◦ N) Eniwetok (11◦ N)

Location

0.6 0.5 0.6 0.7 1.2 1.7 2.9 2.2 2.3 1.8 2.1 2.4 2.2

14.0 20.7 34.6 25.9 27.2 21.3 24.8 29.0 26.0

1.6 0.7 0.7

Nests per month

7.0 5.5 6.7 8.0

19.8 8.5 8.5

Annual number of spawnings

67–649 146–986 435–981 225–869 309–551 172–339 168–313 212–392 –

1000–2500 1600–5400 700–5025 800–3870

200–400 ∼400 300–700

Eggs per nest

804–7788 1752–11 832 5220–11 772 2700–10 428 3708–6612 2064–4068 2016–3756 2544-4704 –

8000–17 500 11 000–15 000 2810–26 890 10 470–33 140

7200 3000–5000 2000–4000

Annual number of eggs per pair

Hoff (1996) Hoff (1996) Hoff (1996) Hoff (1996) Hoff (1996) Hoff (1996) Hoff (1996) Hoff (1996) Present study

Bell (1976) Ochi (1985, 1989) Richardson et al. (1997) Richardson et al. (1997)

Ross (1978) Allen (1972) Allen (1972)

Reference

Table 2. Fecundity and spawning frequency for captive, and wild populations of clownfishes in tropical and warm/temperate regions

310 A.K. GORDON AND A.W. BOK

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year, respectively (Bell, 1976; Ochi, 1985, 1989), while A. clarkii in captivity spawned 34.6 times per year (Hoff, 1996). Conversely, the clutch size and annual fecundity of A. clarkii from Miyake-jima (Bell, 1976) was significantly higher than tropical wild (Allen, 1972; Ross, 1982) and captive (Hoff, 1996) clownfish. The greater fecundity of A. clarkii at Miyake-jima is most probably a selective response to the wider range of environmental pressures that characterize temperate environments (Moyer, 1980). Further, the spawning patterns of A. akindynos and A. latezonatus (Richardson et al., 1997) found on a high latitude reef on the east coast of Australia are more similar to A. clarkii (Bell, 1976; Moyer, 1980; Ochi, 1985) in temperate Japan, in terms of spawning frequency, periodicity and fecundity, than to congeners in tropical regions (Table 2). This suggests that the environmental factors associated with high latitude environments may modify the reproductive behavior and biology of the anemonefish (Moyer, 1980; Richardson et al., 1997). A. akallopisos in this study exhibited increased spawning frequency during the spring months of 1997 and the spring and summer months of 1998, although these increases were not significant (ANOVA, p > 0.05) (Figure 1). Hoff (1996) reported increased spawning frequency during spring and summer months in captive Amphiprionae at IOH. Broodstock at IOH, like the ORI hatchery, were exposed to ambient light in addition to artificial light, and dependant to a degree on solar heat to maintain water temperatures. Consequently, photoperiod and water temperatures fluctuated with the seasons. Increased spawning frequency in Amphiprionae has been attributed to the shorter time taken for the incubation of embryos at higher water temperatures (Bell, 1976; Hoff, 1996; Richardson et al., 1997). It has also been suggested that larval and juvenile fitness may be enhanced at warm water temperatures due to higher growth rates and a reduction in temperature related stress (Moyer, 1980). For A. akallopisos in this study it is not possible to discern whether water temperature or photoperiod were factors influencing spawning frequency. Fluctuations in water temperature in the ORI hatchery had no correlation with spawning frequency of clownfish (correlation analysis = −0.12). Perhaps temperatures in the hatchery did not drop to the level where embryo incubation time is increased, and consequently spawning frequency reduced. It is, therefore, postulated that the increase in photoperiod or light intensity associated with spring may play more of a role in increasing the number of spawnings at this time. This postulation is supported by Moyer (1980), but not by Hoff (1996). The influence of photoperiod on wild clownfish spawning was observed by Moyer (1980) in A. clarkii at Miyake-jima (34◦ N). He suggested that diminishing light and not water temperature restricted their breeding season. However, Hoff (1996), believed light intensity and photoperiod were not major factors influencing spawning frequency at IOH. Data showed that clownfish at IOH consistently spawned more times during December than the other autumn and winter months, even though December experienced the shortest day in terms of photoperiod and lowest light intensity.

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The highly seasonal water temperatures and unpredictable environment in higher latitude regions result in embryo incubation times for A. clarkii varying from 6 to 12 days (Bell, 1976). It has been suggested that this would cause the time of hatching to be unpredictable and, as a result, not synchronized with the phase of the moon or associated tidal patterns (Ochi, 1985; Richardson et al., 1997). In contrast, spawning periodicity in tropically occurring clownfishes has been strongly correlated with lunar cycles (Allen, 1972; Ross, 1978). In A. melanopus spawning peaks coincide approximately with the first and third quarters of the moon (Ross, 1978). In clownfishes from Eniwetok Atoll, Allen (1972) reported significantly more spawnings in the period 6 days before or after the full moon. Both these situations result in eggs hatching at a time when the highest tides were encountered on the reef (Allen, 1972; Ross, 1978). It has been suggested that this occurs because the stronger currents associated with spring tides at new and full moon aid larval dispersal (Allen, 1972). In this study there was an increased percentage of A. akallopisos pairs spawning over new and full moon. However, neither the correlation between the percentage spawning and phase of the moon (Table 3), nor tidal patterns (Table 4) was statistically significant (ANOVA, p > 0.05). A. clarkii and A. percula spawned in aquaria in the Philippines also showed no significant correlation with lunar cycle (Alava and Gomes, 1989), however it is not stated whether these fish were exposed to ambient light and whether they would thus be aware of the different phases of Table 3. Percentage of A. akallopisos spawnings in the Oceanographic Research Institute marine hatchery at different phases of the moon Pair code AK1 AK2 AK3 AK4 AK5 Average ± SD

Moon phase New moon

1st quarter

Full moon

3rd quarter

13.3 30.0 37.5 28.6 28.8 27.6 ± 8.8

26.7 27.1 12.5 21.4 23.3 22.2 ± 5.9

26.7 27.1 50.0 42.9 23.3 34.0 ± 11.7

33.3 15.7 0 7.1 24.7 16.2 ± 13.3

Table 4. Percentage of A. akallopisos spawnings in the Oceanographic Research Institute marine hatchery at spring and neap tides Pair code

Spring tide

Neap tide

AK1 AK2 AK3 AK4 AK5 Average ± SD

40 57.1 88 71 52 61.6 ± 18.3

60 42.9 12 29 48 38.4 ± 18.3

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the moon. In the ORI hatchery clownfish are exposed to ambient light, but it is likely that the surrounding city lights override any effect moon phase could have as a reproductive cue. This could account for the lack in significantly different spawning frequencies at different moon phases. Although this study was a small-scale pilot study, it is hoped that this paper will contribute to the understanding of interactions between environmental factors and spawning frequency and periodicity in Amphiprionae maintained under aquarium conditions. Further trials should be conducted using larger numbers of broodstock, and focus specifically on the manipulation of selected environmental factors such as photoperiod and water temperature to try and elucidate the influencing environmental factors in clownfish spawning. ACKNOWLEDGMENTS

Funding was provided by the South African Association for Marine Biological Research (SAAMBR), with additional financial assistance from NBS and Spawnrite. Thanks to R. Van der Elst and L. Beckley for reviewing earlier drafts, and to the staff of SAAMBR for assistance in running the hatchery and collecting broodstock. REFERENCES Alava, V.R. and Gomes, L.A.O. (1989) Breeding marine aquarium animals: the anemonefish. Naga, ICLARM Quarterly 12, 12–13. Allen, G.R. (1972) Anemonefishes. Neptune City: T.F.H. Publications, 288 pp. Bell, L.J. (1976) Notes on the nesting success and fecundity of anemonefish Amphiprion clarkii at Miyake-Jima, Japan. Japanese Journal of Ichthyology 22, 207–211. Hoff, F.H. (1996) Conditioning, Spawning and Rearing of Fish with Emphasis on Marine Clownfish. Dade City: Aquaculture Consultants, 212 pp. Kohler, C.C., Sheehan, R.J., Habiche, C., Malison, J.A. and Kayes, T.B. (1994) Habituation to captivity and controlled spawning of white bass. Transactions of the American Fisheries Society 123, 964–974. Moyer, J.T. (1980) Influence of temperate waters on the behavior of the tropical anemonefish Amphiprion clarkii at Miyake-Jima, Japan. Japanese Journal of Ichthyology 23, 23–32. Ochi, H. (1985) Temporal patterns of breeding and larval settlement in a temperate population of the tropical anemonefish, Amphiprion clarkii. Japanese Journal of Ichthyology 32, 248–257. Ochi, H. (1989) Mating behavior and sex change of the anemonefish, Amphiprion clarkii, in the temperate waters of Japan. Environmental Biology of Fishes 26, 257–275. Richardson, D.L., Harrison, P.L. and Harriott, V.J. (1997) Timing of spawning and fecundity of a tropical and subtropical anemonefish (Pomacentridae: Amphiprion) on a high latitude reef on the east coast of Australia. Marine Ecology Progress Series 156, 175–181. Ross, R.M. (1978) Reproductive behavior of anemonefish Amphiprion melanopus on Guam. Copeia 1978, 103–107. Zar, J.H. (1974) Biostatistical Analysis. Englewood Cliffs: Prentice-Hall, 620 pp. Address for correspondence: A.K. Gordon, Oceanographic Research Institute, P.O. Box 10712, Durban, South Africa Phone: +27 31 3373536; Fax: +27 31 3372132; E-mail: [email protected]