Open Access Scientific Reports

Your Research - Your Rights

Reproductive Biology of Amphiprion nigripes (Regan, 1908) at Lakshadweep Archipelago: Implications for Specific Reef Fish Conservation in Asia

Research Article Open Access
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
*Corresponding authors: Swagat Ghosh
Centre of Advanced Study in Marine Biology
Faculty of Marine Sciences
Annamalai University, Parangipettai - 608 502
Tamil Nadu, India
E-mail: swagotor@gmail.com
Â
Received July 20, 2012; Published October 29, 2012
Â
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
Â
Copyright: © 2012 Ghosh S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Â
Abstract
Â
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.
Â
Keywords
Â
Amphiprion nigripes; Reproduction; Conservation; Sizefrequency; Maturation
Â
Introduction
Â
The Union Territory of Lakshadweep consists of 36 islands covering 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, with the total lagoon area of about 4200 sq km, territorial water area of 20,000 sq km and an exclusive economic zone of 400,000 sq km [1]. Anemone fishes are typical components of tropical and subtropical shallow reefs, where they constitute a common target species and Amphiprion nigripes [2] is of particular interest in this context. The A. nigripes is a Pomacentridae fish of high ornamental value because it can be found only in the Indian Ocean specially, Maldives, Sri Lanka [3] and Lakshadweep in Indian waters [4]. This implies that the fish might have varying life strategies and population structure when environment changes occur. Although A. nigripes is commercially important and, has consequently suffered from the hands of human pressure, there are no studies of its reproductive biology to develop its effective management. Recent developments in the anemonefish market due to the openings of an export market increased the concerns of researchers and breeders [5]. There have been several papers published on clownfish and maturation and spawning of the populations in captivity and reefs. Most of these, however, are limited due to short-period data set, bias on female, and less detailed recent information on reproductive biology of this species.
Â
Within a social setting, all anemonefishes are born as male [6,7] and the dominant ‘member’ of the group reverses its sex to become the female. The second dominant male subsequently becomes the male. In any circumstances, when the female dies, the dominant male reverses sex and all other subordinate males move up in the hierarchical ladder like other fishes [7-9]. If a social surrounding lacks a female, the sensory nerves of an adolescent anemonefish stimulates its hypothalamus to start pumping out hormones that cause the fish to grow, become more aggressive and develop into a functioning female. Once an anemonefish becomes female, it cannot return to adolescent again [7]. Thus, it will be highly valuable to elucidate the maturation and spawning of the species. Lacking of the specific sensory stimuli to mature sexually adolescent anemonefish remains small and reasonably submissive. This study presents the first detailed information on the reproductive biology of A. nigripes. This study aims to further stimulate investigation and stronger protection of A. nigripes, as well as other reef fishes whose habitats are being irrevocably damaged by global worming in Asian countries.
Â
Materials and Methods
Â
Sampling and biological data
Â
A. nigripes were collected monthly over a year from 24 June 2008 to 23 July 2010 in the lagoon area of Lakshadweep islands (8º and 12º 30´ N Latitude and 71º-74º E Longitude). All of the fish were selectively sampled by SCUBA diving and gill nets at Lakshadweep. A total of 60 specimens were examined for the reproductive study. Body weight of each individual was recorded to avoid the unstable factors caused by gonads at different maturity stages for males (Figure 1) and females (Figure 2). The gonad of each specimen was also weighed to the accuracy of 0.01 g (GW), while the sex was determined by examination of the gonads either with the naked eye or with the aid of a binocular microscope, if necessary. The gonads were then preserved in 10% buffered formaldehyde for subsequent histological analysis.
Â
Figure 1: Relation between total body weight and gonad weight in male A. nigripes.
Â
Figure 2: Relation between total body weight and gonad weight in female A. nigripes.
Â
Histological examination
Â
Following standard histological manual [10], a histology study on gonads was conducted on 20 females, 20 males and 20 sub-adults.
Â
The fishes were sacrificed in MS-222 and placed in Bouin’s fixative solution for future analysis such as histological sections. The fishes were 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.
Â
Maturity index and reproductive mode
Â
Gonado somatic index (GSI) of each fish was calculated as GSI=(GW/BW)×102. The spawning season was determined based on the monthly changes of GSI indices and proportions of each maturity stage. In order to establish the size–frequency distribution of oocytes in A. nigripes, female individuals with ovaries representing maturing or more advanced stages were sub-sampled. The sizes of the oocytes were measured to the nearest 0.01 mm in diameter under a profile projector. For each individual, at least 100 oocytes were measured.
Â
Fecundity
Â
All ovaries used to estimate fecundity were histologically screened to check for the occurrence of postovulatory follicles. Ovaries with recent postovulatory follicles were eliminated for fecundity calculations as their presence indicated part of the batch which ovulated. Approximately 1.00 g of subsamples were extracted from the anterior, 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 number of maturing or ripe oocytes in subsamples to the weight of whole ovary.
Â
Results and Discussions
Â
Histological observations
Â
Microscopic histology of ovarian tissue: The six stages of sexual maturity for females are defined as follows:
Â
Figure 3: Different maturity stages of A. nigripes (testes) (120 × μ) A - Immature, B - developing, C - mature, D – gravid, E – Spawning and F - spent (where T.T. - Testicular Tissue, O.L. - Ovarian Lumen, P.O. - Perinucleolus oocytes, C.S. - Spent Crypt, S.C. - Spermatocysts, D.T.T. - Degenerating Testicular Tissue, C.R. – Spermatozo - Crypt, S.P. - Spermatozoa, S.T. - Spermatid, R.O. - Resting oocytes, I.O. - Infested oocytes, P.V. - Periviteline oocytes).
Â
Virgin/Immature: The ovary consists of oocytes in the early to late peri-nucleolus stage (Figure 3A). This is determined by a large and bright nucleus containing peripheral nucleoli surrounded by basophilic cytoplasm.
Â
Developing: The most advanced oocytes are at the cortical alveoli stage or primary yolk stage (Figure 3B). At this phase, yolk granules accumulation is initiated in cytoplasm.
 
Â
Late developing: Oocytes in several developmental stages may exist together in an ovary. The most advanced oocyte group may have already reached the secondary yolk stage and the other developmental oocyte groups may be at the primary yolk stage or the cortical alveoli stage (Figure 3C).
Â
Maturing: The most advanced oocytes are at the tertiary yolk stage (Figure 3D).
Â
Ripe/Spawning: Most oocytes are at the tertiary yolk or nucleus migration stage. Postovulatory follicles may coexist with oocytes (Figure 3E).
Â
Spent/Regressing: Postovulatory follicles and atretic oocytes that failed to spawn are frequently observed. No oocyte develops beyond the cortical alveoli stage (Figure 3F).
Â
Microscopic histology of testicular tissue
Â
Spermatogenesis can be divided into six stages depending on the degree of testis development.
Â
Figure 4: Different maturity stages of A. nigripes (overy) (120 × μ) maturity 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. - Perinucleolus oocytes, C.S. - Spent crypt, S.C. - Spermatocysts, D.T.T. - Degenerating testicular tissue, C.R. - Spermatozoa - Crypt, S.P. - Spermatozoa, S.T. - Spermatid, O.L.L. - Ovarian lamella, A.R. - Artesia, I.O. - Infested 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. - Perinucleolus oocytes, C.S. - Spent crypt, S.C. - Spermatocysts , D.T.T. Degenerating testicular tissue, C.R. - Spermatozo - crypt, S.P. - Spermatozoa., S.T. - Spermatid, O.L.L. - Ovarian lamella, A.R. - Artesia, I.O. - Infested oocytes, C.A. - Cortical alveolus oocytes).
Â
Immature: The testis consists of a large number of spermatogonia with a few spermatocytes (Figure 4A).
Â
Developing: The most predominant spermatogenic cells are spermatocytes (Figure 4B).
Â
Late developing: The testis contains spermatogonia, spermatocytes and spermatids. Spermatozoa may initially appear at this stage (Figure 4C).
Â
Maturing: Spermatozoa proliferate moderately within the lobule lumen. Cysts of spermatocytes and spermatids are observed from the periphery of each lobule (Figure 4D).
Â
Ripe/Spawning: The lobule lumens are full of spermatozoa with sparse peripheral spermatogonia and spermatocytes (Figure 4E).
Â
Spent: Diffused residual spermatozoa combined with spermatogonia or spermatocytes are present in the empty space in the lobule lumen (Figure 4F).
Â
Reproduction
Â
Size variation in maturity stages: According to the size percentage composition of ovarian maturity stages (Figure 5), females at stage I was observed from 50 to 60 mm. The females at stage II occurred in 60 to 70 mm, and then developed gradually to the more advanced stages III, 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 testicular development of A. nigripes in Lakshadweep during the period of the study is as follows: from below 30 to 40 mm testes of most males were at stage I, while some had already developed into stage II; in 41 to 50 mm size, 100% of the male individuals had testes at stage II, and some the testes had converted to stage III in after 49 mm. Testes at stage IV and stage V appeared in 50 to 60 mm. Testes were observed at stage VI 60 to 70 mm and thereafter. Males were observed to develop fast than females, while all females were previously functionally male. However, both spawning females and males (stage V) were synchronously found in separate pair form with the sea anemone.
Â
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.
Â
Figure 5: Different stages of male, female and sub adult.
Â
Figure 6: Relation between male and female Gonado Somatic Index (G.S.I.) in A. nigripes
Â
Minimum size at maturity
Â
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 and age shows that the female and most males mature within 2 year, while few males mature within a year after hatching.
Â
Figure 7: Monthly changes in the fecundity of female A. nigripes at Lakshadweep.
Â
Size–frequency distribution of oocytes
Â
Five types of size–frequency distribution of oocytes were determined (Figure 8A-E) by examining oocyte diameters from 20 individuals. In type A, the ovary had oocytes within the tertiary yolk stage. The oocyte diameters were all less than 0.6-0.7 mm. The spawning batch began to separate around 0.5 mm in diameter from the adjacent group of smaller oocytes at the secondary yolk stage. In type B, a well-developed size-hiatus was established at the maturing stage, forming two groups of distinct diameter distributions. One ranged from 0.5 to 0.7 mm in diameter, most of which were at the tertiary yolk stage. The other ranged from 0.8 to 1.0 mm in diameter, dominantly containing oocytes at the nucleus migration stage. In type C, the oocytes had been hydrated, and showed a remarkable increase in diameter of 1.1-1.3 mm; in type D, one group still remained after a recent ovulation, which mainly consisted of tertiary yolk stage oocytes. Finally, types E, again ovulation were started, which mainly indicated that the species is a prolific breeder.
Â
Figure 8: Ova diameter (μ) and frequency (%) of different maturity stage.
Â
Fecundity
Â
The fecundity calculated of A. nigripes of size-frequency distribution of oocytes. Thus, the total number of oocytes beyond the tertiary yolk stage, defined as the oocytes with a diameter ≠mm by histological observation, was estimated. As a result, the fecundity ranged from 200-1500 eggs per individual and increased with size throughout the year. The relationship between fecundity (F) and length (TL) and body weight (BW) also described by the following equations, respectively: F=1:56×10-2 FL 2:6857 (r2=0:7536; n=15)
Â
F=4427+172:76 BW (r2=0:8800; n=15)
Â
Fish of the Pomacentridae genus Amphiprion comprise 28 species. They are mainly distributed in the all tropical and sub tropical coral reefs. In the present study, five lines of evidence have been examined to determine the reproductive biology of A. nigripes in Lakshadweep, India. Based on the above-mentioned information, it can be concluded that A. nigripes spawns throughout the year. The fish are iteroparous and reach their first maturity at 55 mm in size for female and 35 mm of size in well-developed males. In the present study the mean sex ratio also varies wide range (Table 1) and ovaries contained both tertiary yolk oocytes and the postovulatory follicles occurred from size 50 mm, implying that some well-developed oocytes will have also been recruited for ovulating during before spawning. Thus, A. nigripes releases eggs throughout the year. Moreover, individuals which reached the spawning ground had oocytes that were mainly at the maturing stage (stage IV). The present findings on spawning, parental care, GSI and fecundity of A. nigripes under captive condition were well documented. The mean sex ratio in both male and female showed that the gonadal maturation of anemonefishes was very fast and they bred throughout the year (Table 2).
Â
Table 1: Mean sex ratio of different stages.
Â
Table 2: Sex ratio of different size groups.
Â
In case of male, the immature testis consists of large number of spermatogonia with few spermatocytes but all are in inactive stage when it start developing, spermatogenic cells and spermatocytes getting maturity and in late developing stage the testis contains spermatogonia, spermatocytes and spermatids including spermatozoa may initially appear at this stage. The spermatozoa proliferate moderately within the lobule lumen at the stage of maturing and ripe the lobule lumens are full of spermatozoa with sparse peripheral spermatogonia and spermatocytes. Diffused residual spermatozoa combined with spermatocytes are present in the empty space in the lobule lumen in spent stage. In female, ovary consists of oocytes in the early to late perinucleolus stage and developing oocytes are at the cortical alveoli stage and yolk granules accumulation was started in cytoplasm. In late developing stage, oocytes consists several developmental stages may exist together in an ovary and the most advanced oocytes are at the tertiary yolk at the stage of maturing and then in ripe stage, most oocytes are at the tertiary yolk or nucleus migration stage. At least, in regressing time postovulatory follicles and atretic oocytes that failed to spawn are frequently observed. To reveal the reproductive features of A. nigripes, the present findings on spawning, range of GSI, fecundity and size at first maturity are generally consistent with total reproduction. The mean values of GSI in both males and females remained nearly same throughout the year as a certain time it’s went down for a resting stage. Moreover, individuals which reached the spawning activities had oocytes that were mainly at the maturing stage, (stage IV) while those still making cultch and continuously ejaculating their body.
Â
Geographic differences in reproductive modes are believed linked to varying environmental conditions, and this type of fine tuning is considered strong evidence of intraspecific life history evolution in marine fish [15]. There are no meaningful reports at reproductive levels in clownfish species. But in A. sebae with different reproductive modes were thought to have nearly equivalent lifetime reproductive output due to reciprocal latitudinal trends in fecundity and degree of repeat spawning [16]. In contrast, a estuarine fish temperate and subtropical 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 compared populations), and shorter life spans with a maximum age of less than 3 years [17,18]. Furthermore, A. nigripes has the most selective distribution one of the other clown species in reefs. The fish provides an opportunity to test the assertions for its Indo-Pacific distribution and various life history parameters that had been clarified between the temperate and tropical populations. The percentage mean composition of sub adults, males and females in different size and maturity were also analysed in the present findings.
Â
Available information on the reproductive biology of genus Amphiprion is quite limited because only few species had been reported [16]. Although the present study reveals the reproductive characteristics of A. nigripes illustrate that many gaps need to be filled in terms of the reproduction of this species. Lakshadweep, in India 2008 the authorities of Annamalai University and the Centre for Marine Living Resources and Ecology, Ministry of Earth Sciences, Govt. of India established marine ornamental fish hatchery and one of the major target species for conservation is A. nigripes which is running successfully. Further investigation needs to be done at the Maldives and SriLankan reefs to establish effective conservation and management strategies for this vulnerable species in the forth future.
Â
Acknowledgments
Â
The authors are facilitating by Annamalai University and funding by the Centre for Marine Living Resources and Ecology, Ministry of Earth Sciences, Govt. of India.
Â
 
References
Â

















Â
Â