Reproductive Biology of Amphiprion Nigripes (Regan, 1908) at Lakshadweep Archipelago: Implications for Specific Reef Fish Conservation in Asia Swagat Ghosh*, T.T

Ghosh et al., 1:11 http://dx.doi.org/10.4172/scientificreports.511 Open Access Open Access Scientific Reports Scientific Reports Research Article OpenOpen Access Access Reproductive Biology of Amphiprion nigripes (Regan, 1908) at Lakshadweep Archipelago: Implications for Specific Reef Fish Conservation in Asia Swagat Ghosh*, T.T. Ajith Kumar, S. Gunasundari and T. Balasubramanian Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608 502, Tamil Nadu, India

Abstract Maldives clown (Amphiprion nigripes) is a commercially important species that contributes to marine ornamental fisheries in Asian countries. In the present study, the reproductive biology of A. nigripes in Lakshadweep, India is determined for the first time. Six maturity stages were described using ovarian and testicular histology throughout the annual cycle. Spawning was estimated to be 2 years, with a few exceptions of 1 year in well developed pairs. Ovaries that contained both tertiary yolk oocytes and postovulatory follicles occurred within 15 days, indicating that A. nigripes is a multiple spawner. The size frequency distribution of oocytes provided evidence for its multiple spawning and accuracy of the fecundity estimates. The fecundity of this species was estimated at between 200- 1,500 eggs per individual, increasing with age between two and six years. The present findings on the reproductive strategy of A. nigripes in Lakshadweep are generally consistent with those in reef populations, but quite different from those of tropical population where parental care, and egg maturation occurs seasonal or long term.

Keywords: Amphiprion nigripes; Reproduction; Conservation; Size- presents the first detailed information on the reproductive biology ofA. frequency; Maturation nigripes. This study aims to further stimulate investigation and stronger protection of A. nigripes, as well as other reef fishes whose habitats are Introduction being irrevocably damaged by global worming in Asian countries. The Union Territory of Lakshadweep consists of 36 islands covering Materials and Methods an area of 32 sq km. The islands are being oceanic in nature; the continental shelf is small extending to an area of about 4336 sq km, Sampling and biological data with the total lagoon area of about 4200 sq km, territorial water area A. nigripes were collected monthly over a year from 24 June 2008 of 20,000 sq km and an exclusive economic zone of 400,000 sq km [1]. to 23 July 2010 in the lagoon area of Lakshadweep islands (8º and 12º Anemone fishes are typical components of tropical and subtropical 30´ N Latitude and 71º-74º E Longitude). All of the fish were selectively shallow reefs, where they constitute a common target species and sampled by SCUBA diving and gill nets at Lakshadweep. A total of 60 Amphiprion nigripes [2] is of particular interest in this context. The A. specimens were examined for the reproductive study. Body weight of nigripes is a Pomacentridae fish of high ornamental value because it can each individual was recorded to avoid the unstable factors caused by be found only in the Indian Ocean specially, Maldives, Sri Lanka [3] gonads at different maturity stages for males (Figure 1) and females and Lakshadweep in Indian waters [4]. This implies that the fish might (Figure 2). The gonad of each specimen was also weighed to the have varying life strategies and population structure when environment accuracy of 0.01 g (GW), while the sex was determined by examination changes occur. Although A. nigripes is commercially important and, has of the gonads either with the naked eye or with the aid of a binocular consequently suffered from the hands of human pressure, there are no microscope, if necessary. The gonads were then preserved in 10% studies of its reproductive biology to develop its effective management. buffered formaldehyde for subsequent histological analysis. Recent developments in the anemonefish market due to the openings of an export market increased the concerns of researchers and breeders [5]. Histological examination There have been several papers published on clownfish and maturation and spawning of the populations in captivity and reefs. Most of these, Following standard histological manual [10], a histology study on however, are limited due to short-period data set, bias on female, and gonads was conducted on 20 females, 20 males and 20 sub-adults. less detailed recent information on reproductive biology of this species. The fishes were sacrificed in MS-222 and placed in Bouin’s fixative Within a social setting, all anemonefishes are born as male [6,7] solution for future analysis such as histological sections. The fishes were and the dominant ‘member’ of the group reverses its sex to become the female. The second dominant male subsequently becomes the male. In *Corresponding author: Swagat Ghosh, Centre of Advanced Study in Marine any circumstances, when the female dies, the dominant male reverses Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608 sex and all other subordinate males move up in the hierarchical ladder 502, Tamil Nadu, India, E-mail: [email protected] like other fishes [7-9]. If a social surrounding lacks a female, the sensory Received July 20, 2012; Published October 29, 2012 nerves of an adolescent anemonefish stimulates its hypothalamus to Citation: Ghosh S, Ajith Kumar TT, Gunasundari S, Balasubramanian T (2012) start pumping out hormones that cause the fish to grow, become more Reproductive Biology of Amphiprion nigripes (Regan, 1908) at Lakshadweep aggressive and develop into a functioning female. Once an anemonefish Archipelago: Implications for Specific Reef Fish Conservation in Asia. 1:511. becomes female, it cannot return to adolescent again [7]. Thus, it will be doi:10.4172/scientificreports.511 highly valuable to elucidate the maturation and spawning of the species. Copyright: © 2012 Ghosh S, et al. This is an open-access article distributed under Lacking of the specific sensory stimuli to mature sexually adolescent the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and anemonefish remains small and reasonably submissive. This study source are credited.

Volume 1 • Issue 11 • 2012 Citation: Ghosh S, Ajith Kumar TT, Gunasundari S, Balasubramanian T (2012) Reproductive Biology of Amphiprion nigripes (Regan, 1908) at Lakshadweep Archipelago: Implications for Specific Reef Fish Conservation in Asia. 1:511. doi:10.4172/scientificreports.511

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measured to the nearest 0.01 mm in diameter under a profile projector. Total weight For each individual, at least 100 oocytes were measured. 20 Gonad weight Fecundity 18 All ovaries used to estimate fecundity were histologically screened 16 to check for the occurrence of postovulatory follicles. Ovaries with

14 recent postovulatory follicles were eliminated for fecundity calculations as their presence indicated part of the batch which ovulated. 12 Approximately 1.00 g of subsamples were extracted from the anterior,

10 middle, and posterior parts of the left ovary of 20 females, and weighed to the nearest 0.01 g. The fecundity was estimated by scaling the average 8 Weight (g) Weight number of maturing or ripe oocytes in subsamples to the weight of 6 whole ovary.

4 Results and Discussions 2 Histological observations 0 Microscopic histology of ovarian tissue: The six stages of sexual 2 4 6 8 10 12 14 16 18 20 maturity for females are defined as follows: Number of samples (Male) Virgin/Immature: The ovary consists of oocytes in the early to Figure 1: Relation between total body weight and gonad weight in male A. nigripes. late peri-nucleolus stage (Figure 3A). This is determined by a large and bright nucleus containing peripheral nucleoli surrounded by basophilic cytoplasm.

30 Total weight Developing: The most advanced oocytes are at the cortical alveoli Gonad weight stage or primary yolk stage (Figure 3B). At this phase, yolk granules accumulation is initiated in cytoplasm. 25 Late developing: Oocytes in several developmental stages may

20 exist together in an ovary. The most advanced oocyte group may have already reached the secondary yolk stage and the other developmental

15 Weight (g) Weight

0 0 2 4 6 8 10 12 14 16 18 20 Number of samples (Female) Figure 2: Relation between total body weight and gonad weight in female A. nigripes. dissected and gonads were removed and embedded in 2-hydroxyethyl methacrylate (GMA). Serial cross sections (6-8 mm thick) of the gonads were stained with toluidine blue (pH 4.4) and with basic fuchsin-methylene blue. The female histology was studied following the methods proposed by [11], west while those for the males were adapted from [12] Scott and Pankhurst. The histological staging was done based on the most advanced oocytes and the occurrence of postovulatory follicles in ovaries [13] and the predominant cell types in testes [14] respectively. Maturity index and reproductive mode Gonado somatic index (GSI) of each fish was calculated as Figure 3: Different maturity stages of A. nigripes (testes) (120 × µ) A - 2 Immature, B - developing, C - mature, D – gravid, E – Spawning and F - spent GSI=(GW/BW)×10 . The spawning season was determined based on (where T.T. - Testicular Tissue, O.L. - Ovarian Lumen, P.O. - Perinucleolus the monthly changes of GSI indices and proportions of each maturity oocytes, C.S. - Spent Crypt, S.C. - Spermatocysts, D.T.T. - Degenerating stage. In order to establish the size–frequency distribution of oocytes Testicular Tissue, C.R. – Spermatozo - Crypt, S.P. - Spermatozoa, S.T. - Spermatid, R.O. - Resting oocytes, I.O. - Infested oocytes, P.V. - Periviteline in A. nigripes, female individuals with ovaries representing maturing or oocytes). more advanced stages were sub-sampled. The sizes of the oocytes were

Page 3 of 5 oocyte groups may be at the primary yolk stage or the cortical alveoli lumen. Cysts of spermatocytes and spermatids are observed from the stage (Figure 3C). periphery of each lobule (Figure 4D). Maturing: The most advanced oocytes are at the tertiary yolk stage Ripe/Spawning: The lobule lumens are full of spermatozoa with (Figure 3D). sparse peripheral spermatogonia and spermatocytes (Figure 4E). Ripe/Spawning: Most oocytes are at the tertiary yolk or nucleus Spent: Diffused residual spermatozoa combined with migration stage. Postovulatory follicles may coexist with oocytes spermatogonia or spermatocytes are present in the empty space in the (Figure 3E). lobule lumen (Figure 4F). Spent/Regressing: Postovulatory follicles and atretic oocytes that Reproduction failed to spawn are frequently observed. No oocyte develops beyond the cortical alveoli stage (Figure 3F). Size variation in maturity stages: According to the size percentage composition of ovarian maturity stages (Figure 5), females at stage I was Microscopic histology of testicular tissue observed from 50 to 60 mm. The females at stage II occurred in 60 to Spermatogenesis can be divided into six stages depending on the 70 mm, and then developed gradually to the more advanced stages III, degree of testis development. IV and V from the beginning of 70 to 80 mm. The female fish at stage VI began to appear in the size of 85 mm and thereafter. The annual Immature: The testis consists of a large number of spermatogonia testicular development of A. nigripes in Lakshadweep during the period with a few spermatocytes (Figure 4A). of the study is as follows: from below 30 to 40 mm testes of most males Developing: The most predominant spermatogenic cells are were at stage I, while some had already developed into stage II; in 41 spermatocytes (Figure 4B). to 50 mm size, 100% of the male individuals had testes at stage II, and Late developing: The testis contains spermatogonia, spermatocytes some the testes had converted to stage III in after 49 mm. Testes at stage and spermatids. Spermatozoa may initially appear at this stage (Figure IV and stage V appeared in 50 to 60 mm. Testes were observed at stage 4C). VI 60 to 70 mm and thereafter. Males were observed to develop fast than females, while all females were previously functionally male. However, Maturing: Spermatozoa proliferate moderately within the lobule both spawning females and males (stage V) were synchronously found in separate pair form with the sea anemone. GSI index: The mean values of GSI ranged from 14% to 19.8% in females, and 7% to 15.3% in males. The GSI remained very low thereafter until the following a non-specified period during the year. The variation of GSI is very fast and it consistent with the percentage composition of maturity stages in gonads (Figure 6), suggesting that the spawning throughout the year. Minimum size at maturity Minimal size at first gonadal maturity in females was 55 mm (TL) and 35 mm (TL) in males. The average size of collected fishes were viz., sub adult 20 to 40 mm, male 40 to 70 mm and female 50 to above 120 mm ( Figure 7). The combined information on histological observation

8 Male Female 7 Sub-adult 6

Figure 4: Different maturity stages of A. nigripes (overy) (120 × µ) maturity Individuals 4 stages, A - Immature, B - developing, C - mature, D - gravid, IX - Spawning and X - Spent (where T.T. - Testicular Tissue, O.L. - Ovarian Lumen, P.O. 3 - Perinucleolus oocytes, C.S. - Spent crypt, S.C. - Spermatocysts, D.T.T. - Degenerating testicular tissue, C.R. - Spermatozoa - Crypt, S.P. - Spermato- 2 zoa, S.T. - Spermatid, O.L.L. - Ovarian lamella, A.R. - Artesia, I.O. - Infested 1 oocytes, C.A. - Cortical alveolus oocytes, M.V. - Mid vitelogenic oocytes, V.O. - Vitelogenic oocytes.where T.T. - Testicular tissue, O.L. - Ovarian lumen, P.O. 0 - Perinucleolus oocytes, C.S. - Spent crypt, S.C. - Spermatocysts , D.T.T. >20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 <100 Degenerating testicular tissue, C.R. - Spermatozo - crypt, S.P. - Spermato- zoa., S.T. - Spermatid, O.L.L. - Ovarian lamella, A.R. - Artesia, I.O. - Infested Size (mm) oocytes, C.A. - Cortical alveolus oocytes). Figure 5: Different stages of male, female and sub adult.

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0.24 75 A 60 0.22 45 30 0.20 15 0.18 40 B 0.16 30 0.14 20

0.12 10

0.10 25 C 20 0.08 15 Gonado Somatic Index Somatic Gonado 10 0.06 (%) Frequency 5 Male GSI 0.04 Female GSI 20 D 0.02 15 Figure 6: Relation between male and female Gonado Somatic Index (G.S.I.) 10 in A. nigripes. 5

40 E 32 24 Pair 1 16 Pair 2 8 Pair 3 Pair 4 0 1200 Pair 5 Pair 6 600 800 1000 1200 1400 1600 1100 Pair 7 1000 Pair 8 Ova diameter (µ) Pair 9 900 Pair 10 Figure 8: Ova diameter (µ) and frequency (%) of different maturity stage. 800 700 600 of oocytes. Thus, the total number of oocytes beyond the tertiary yolk Fecundity 500 stage, defined as the oocytes with a diameter ≥ 0.3 mm by histological 400 300 observation, was estimated. As a result, the fecundity ranged from 200 200-1500 eggs per individual and increased with size throughout the 100 year. The relationship between fecundity (F) and length (TL) and body 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec weight (BW) also described by the following equations, respectively: -2 2 Months F=1:56×10 FL 2:6857 (r =0:7536; n=15) Figure 7: Monthly changes in the fecundity of female A. nigripes at F=4427+172:76 BW (r2=0:8800; n=15) Lakshadweep. Fish of the Pomacentridae genus Amphiprion comprise 28 species. They are mainly distributed in the all tropical and sub tropical coral and age shows that the female and most males mature within 2 year, reefs. In the present study, five lines of evidence have been examined while few males mature within a year after hatching. to determine the reproductive biology of A. nigripes in Lakshadweep, Size–frequency distribution of oocytes India. Based on the above-mentioned information, it can be concluded that A. nigripes spawns throughout the year. The fish are iteroparous Five types of size–frequency distribution of oocytes were and reach their first maturity at 55 mm in size for female and 35 mm determined (Figure 8A-E) by examining oocyte diameters from 20 of size in well-developed males. In the present study the mean sex ratio individuals. In type A, the ovary had oocytes within the tertiary yolk also varies wide range (Table 1) and ovaries contained both tertiary stage. The oocyte diameters were all less than 0.6-0.7 mm. The spawning yolk oocytes and the postovulatory follicles occurred from size 50 batch began to separate around 0.5 mm in diameter from the adjacent mm, implying that some well-developed oocytes will have also been group of smaller oocytes at the secondary yolk stage. In type B, a well- recruited for ovulating during before spawning. Thus, A. nigripes developed size-hiatus was established at the maturing stage, forming releases eggs throughout the year. Moreover, individuals which reached two groups of distinct diameter distributions. One ranged from 0.5 to the spawning ground had oocytes that were mainly at the maturing 0.7 mm in diameter, most of which were at the tertiary yolk stage. The stage (stage IV). The present findings on spawning, parental care, other ranged from 0.8 to 1.0 mm in diameter, dominantly containing GSI and fecundity of A. nigripes under captive condition were well oocytes at the nucleus migration stage. In type C, the oocytes had documented. The mean sex ratio in both male and female showed that been hydrated, and showed a remarkable increase in diameter of 1.1- the gonadal maturation of anemonefishes was very fast and they bred 1.3 mm; in type D, one group still remained after a recent ovulation, throughout the year (Table 2). which mainly consisted of tertiary yolk stage oocytes. Finally, types E, In case of male, the immature testis consists of large number of again ovulation were started, which mainly indicated that the species is spermatogonia with few spermatocytes but all are in inactive stage a prolific breeder. when it start developing, spermatogenic cells and spermatocytes getting Fecundity maturity and in late developing stage the testis contains spermatogonia, spermatocytes and spermatids including spermatozoa may initially The fecundity calculated ofA. nigripes of size-frequency distribution appear at this stage. The spermatozoa proliferate moderately within

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Ratio Chi- Available information on the reproductive biology of genus Maturity Fe- Males Females Males% males/ square P-value stages males% Amphiprion is quite limited because only few species had been reported females value [16]. Although the present study reveals the reproductive characteristics 1 35 23 70.01 30.16 1.52 3.248 P<0.05 of A. nigripes illustrate that many gaps need to be filled in terms of the 2 28 45 35.46 65.27 0.62 3.124 P<0.05 reproduction of this species. Lakshadweep, in India 2008 the authorities 3 75 60 55.28 45.96 1.25 1.256 P<0.01 4 56 42 62.48 67.62 1.33 1.159 P<0.01 of Annamalai University and the Centre for Marine Living Resources 5 30 28 52.36 47.64 1.08 3.245 P<0.05 and Ecology, Ministry of Earth Sciences, Govt. of India established marine ornamental fish hatchery and one of the major target species Table 1: Mean sex ratio of different stages. for conservation is A. nigripes which is running successfully. Further Numbers Chi- investigation needs to be done at the Maldives and SriLankan reefs Length Number of Males Females of male / square P-value group specimen to establish effective conservation and management strategies for this females value vulnerable species in the forth future. >30 0 0 0 0 - - 31-40 0 0 0 0 - - Acknowledgments 41-50 15 0 0 0 - - The authors are facilitating by Annamalai University and funding by the Centre 51-60 45 45 0 0 - - for Marine Living Resources and Ecology, Ministry of Earth Sciences, Govt. of 61-70 60 60 0 0 - - India. 71-80 90 75 15 5.00 6.298 P<0.05 References 81-90 42 12 30 2.56 4.236 P<0.05 1. Kumar TTA, Balasubramanian T (2009) Broodstock development, spawning 91-100 40 0 40 0 - - and larval rearing of the false clownfish, Amphiprion ocellaris in captivity using 101-110 60 0 60 0 - - estuarine water. Current Science 97: 1483-1486. 111-120 15 0 15 0 - - 2. Allen GR, Mowka EJ (1980) The anemonefishes of the world: Species, <120 6 0 6 0 - - care and breeding: handbook for aquarists, divers, and scientists. Aquarium Systems 104. Table 2: Sex ratio of different size groups. 3. Blaber SJM, Staunton-Smith J, Milton DA, Fry G, Van der Velde T, et al. (1998) the lobule lumen at the stage of maturing and ripe the lobule lumens The biology and life-history strategies of Ilisha (Teleostei: Pristigasteridae) in the coastal waters and estuaries of Sarawak. Estuarine, coastal and Shelf are full of spermatozoa with sparse peripheral spermatogonia and Science 47: 499-511. spermatocytes. Diffused residual spermatozoa combined with spermatocytes are present in the empty space in the lobule lumen in 4. Elliott J, Mariscal R (1996) Ontogenetic and interspecific variation in the protection of anemonefishes from sea anemones. J Exp Mar Bio Ecol 208: spent stage. In female, ovary consists of oocytes in the early to late 57-72. perinucleolus stage and developing oocytes are at the cortical alveoli stage and yolk granules accumulation was started in cytoplasm. In 5. Fautin DG, Allen GR (1992) Field guide to anemone fishes and their host sea late developing stage, oocytes consists several developmental stages anemones. Western Australian Museum 3-10. may exist together in an ovary and the most advanced oocytes are at 6. Grier HJ (1981) Cellular organization of the testis and spermatogenesis in the tertiary yolk at the stage of maturing and then in ripe stage, most fishes. Amer Zool 21: 345-357. oocytes are at the tertiary yolk or nucleus migration stage. At least, in 7. Hinton DE (1990) Histological techniques. In: Schreck CB, Moyle PB (Eds.), regressing time postovulatory follicles and atretic oocytes that failed to Methods for Fish Biology AFS, Bethesda, MD, 191-211. spawn are frequently observed. To reveal the reproductive features of A. 8. Limburg KE, Waldman JR (2003) Biodiversity Status and Conservation of the nigripes, the present findings on spawning, range of GSI, fecundity and World’s Shads. American Fisheries Society. size at first maturity are generally consistent with total reproduction. 9. Murty SV (2002) Marine Ornamental Fish Resources of Lakshadweep. CMFRI The mean values of GSI in both males and females remained nearly 72: 1-134. same throughout the year as a certain time it’s went down for a resting stage. Moreover, individuals which reached the spawning activities had 10. Regan CT, Gardiner JS (1908) Report on the marine fishes collected by Mr. J. oocytes that were mainly at the maturing stage, (stage IV) while those Stanley Gardiner in the Indian Ocean. Trans Linn Soc. still making cultch and continuously ejaculating their body. 11. Rosenberg S, Cruz G (1988) The anemonefishes of the Indo-Pacific. Sea Frontiers 34: 16-21. Geographic differences in reproductive modes are believed linked 12. Satheesh JM (2002) Biology of the clownfish,Amphiprion sebae (Bleeker) from to varying environmental conditions, and this type of fine tuning is Gulf of Mannar (South east coast of India) 1-140. considered strong evidence of intraspecific life history evolution in marine fish [15]. There are no meaningful reports at reproductive levels 13. Scott SG, Pankhurst NW (1992) Inter annual variation in the reproductive cycle of the New Zealand snapper Pagrus auratus (Bloch and Schneider) (Sparidae). in clownfish species. But in A. sebae with different reproductive modes Journal of Fish Biology 41: 685-696. were thought to have nearly equivalent lifetime reproductive output due to reciprocal latitudinal trends in fecundity and degree of repeat 14. Smith BB, Walker KF (2004) Spawning dynamics of common carp in the River Murray, South Australia, shown by macroscopic and histological staging of spawning [16]. In contrast, a estuarine fish temperate and subtropical gonads. Journal of Fish Biology 64: 336-354. populations, tropical Ilisha elongata have relatively larger body sizes at age 1 and age 2 years, first maturation at less than 1 year (2 years in 15. Wallace RA, Selman K (1981) Cellular and dynamic aspects of oocyte growth compared populations), and shorter life spans with a maximum age of in teleosts. Amer Zool 21: 325-343. less than 3 years [17,18]. Furthermore, A. nigripes has the most selective 16. West G (1990) Methods of assessing ovarian development in fishes: a review. distribution one of the other clown species in reefs. The fish provides Australian Journal of Marine and Freshwater Research 41: 199–222. an opportunity to test the assertions for its Indo-Pacific distribution 17. Wellington GM, Victor BC (1989) Planktonic larval duration of one hundred and various life history parameters that had been clarified between the species of Pacific and Atlantic damselfishes (Pomacentridae). Marine Biology temperate and tropical populations. The percentage mean composition 101: 557-567. of sub adults, males and females in different size and maturity were also 18. Yamamoto K, Yamazaki F (1961) Rhythm of development in the oocyte of the analysed in the present findings. gold-fish. Bulletin of the Faculty of Fisheries. 12: 93-110.

Volume 1 • Issue 11 • 2012