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Growth pattern, diet and reproductive biology of the clownfish Amphiprion

ocellaris in waters of Pulau Tioman, Malaysia

Article in The Egyptian Journal of Aquatic Research · July 2018

DOI: 10.1016/j.ejar.2018.07.003

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Full length article

Growth pattern, diet and reproductive biology of the clownfish Amphiprion ocellaris
in waters of Pulau Tioman, Malaysia
Mei Ling Khooa, Simon Kumar Dasa, b, ⁎, Mazlan Abd Ghaffarc

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a
Marine Ecosystem Research Centre (EKOMAR), Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor D.E., Malaysia
b
School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor D.E., Malaysia
c
Institute of Oceanography and Environment, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

ARTICLE INFO ABSTRACT

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Article history: The growth pattern, diet and reproductive biology of the clownfish Amphiprion ocellaris collected from wa-
Received 15 January 2018 ters of Pulau Tioman were investigated. The length-weight relationship showed an isometric growth pattern
Received in revised form 17 July 2018 (b = 3) in A. ocellaris. The stomach contents mainly consisted of zooplankton and algae, which showed that
Accepted 17 July 2018
the fish is omnivorous and was confirmed by trophic level analysis (2.98 ± 0.29). Fecundity of A. ocellaris
Available online xxx
ranged from 23-1518 with mean egg count of 582 ± 478, and has positive relationships with body length, body
weight, eviscerated weight and ovary weight. The gonads were also described and examined histologically.
Keywords: The ovaries showed 4 stages of maturity, displaying different colours for each stage. The different devel-
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Reproductive biology opments of oocytes were also found in each stage of maturity. The males and non-breeders comprised of
Growth pattern both testicular and ovarian tissues. In the males, the testicular tissues were more prominent in the ovotestes,
Stomach content whereas in non-breeders the primary oocytes were more prominent. Absence of testicular tissue in ovaries of
Amphiprion ocellaris females showed that the fish is a protandrous hermaphrodite and sex change may not be reversible. Similar
Protandrous hermaphrodite
observations have been reported in A. ocellaris of other countries and other Amphiprion species indicating
that Amphiprion species show consistency in their reproduction strategy throughout their range.
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© 2018.

Introduction is still low (Abol-Munafi et al., 2011). In Malaysia, Liew et al. (2006)
has successfully bred the clownfish in captivity but failed to develop
Malaysia initially focused on the export of freshwater species, but broodstocks from F1 generations and still needs to collect broodstocks
now is one of the main suppliers of marine ornamental species which pairs from the wild.
are becoming increasingly popular among aquarist (Abol-Munafi et Amphiprion ocellaris are protandrous hermaphrodites; they have
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al., 2011; Cato and Brown, 2003). The false clownfish, Amphiprion the ability to change sex from male to female at maturity (Thresher,
ocellaris, is one of the most commercially exploited species in the 1984; Fautin and Allen, 1992). Sex change occurs in relation to the
aquarium trading as ornamental fishes (Wabnitz et al., 2003). It is social hierarchy, where the two largest individuals (female being the
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unfortunate though that in the present situation with increasing de- largest of the two) forms a strong monogamous breeding pair while
mand for the clownfishes, supplies are mostly dependant on the wild the rest are non-breeders (Allen, 1975; Moyer and Nakazono, 1978;
catches (Abol-Munafi et al., 2011; Cato and Brown, 2003). Studies on Thresher, 1984; Fautin and Allen, 1992; Hirose, 1995; Liew et al.,
Malaysian pomacentrids are still lacking (Sin et al., 1994), and con- 2006). When the female dies (or is removed), the male changes sex to
cerns have been raised about the declining numbers of this species become the dominant breeding female and the second largest member
in Malaysian waters due to the possibility of overfishing and deterio- from the non-breeders becomes the dominant male (Rosenberg and
ration of its natural habitat caused by destructive collection methods Cruz, 1988; Fautin and Allen, 1992). This has made the breeding of
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(Abol-Munafi et al., 2011; Livengood and Chapman, 2007). There- clownfish challenging in captivity; the need for research arose from
fore, it is important to be able to propagate this species in captivity the lack of detailed information concerning the feeding and reproduc-
for both commercial and conservation purposes. Although the clown- tive biology of A. ocellaris in Malaysian waters, which are important
fish Amphiprion ocellaris has been successfully reared in captivity in to facilitate effective management in culturing the species to provide
other countries (Juhl, 1992; Moe, 1992), the production in Malaysia for local and global demands. Therefore, this study was conducted to
measure important biological aspects of A. ocellaris; namely (i) pop-
ulation growth pattern (ii) stomach content analysis (iii) trophic level
⁎ (iv) fecundity estimates, (v) observations of changes in maturity stages
Corresponding author at: School of Environmental and Natural Resource Sciences,
Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, in the gonads and, (vi) histology studies to determine the stages of the
Selangor D.E., Malaysia. reproductive organ.
Email address: [email protected] (S.K. Das)

https://doi.org/10.1016/j.ejar.2018.07.003
1687-4285/ © 2018.
2 The Egyptian Journal of Aquatic Research xxx (2018) xxx-xxx

Material and methods equipped with a camera. Diameter of oocytes of all stages were ran-
domly selected and measured to the nearest 0.001mm.
Fish sampling
Results
A total of 65 fishes were collected from waters of Pulau Tioman
using plastic bags while SCUBA diving. The fishes were then killed Length-weight relationship

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by a sharp blow on the head or by pithing. The specimens were then
measured to the nearest 1 cm (total length TL and standard length SL) Length-weight relationship was derived from 65 individuals of

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and weighed to the nearest 0.1 g (total body weight TW and eviscer- clownfishes. The length weight relationship was a combination for
ated weight EW). Each fish was dissected, the gonads were removed, both sexes and was represented as W = 0.0197L3.06 (Fig. 1). Inter-
weighed and photographed. cept ‘a’ was 0.0197 ± 0.0037 and the value of exponent ‘b’ was
3.0674 ± 0.0917, with coefficient of determination ‘r2’ value of 0.9429
Length-weight relationship (P < 0.001). The value ‘b’ was in close proximity of 3, indicating an
isometric growth pattern where the body changes form in proportion-
Population growth pattern was estimated based on length-weight ate to the body weight (following cube law; volume = L3).
relationship (LWR), expressed by the equation W = aLb where W is

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body weight (g), L is total length (cm), a is the intercept and b is the Stomach content and trophic level
slope. A non-linear regression was used to determine the values of a
and b. Curve fitting was carried out by a non-linear iterative method The stomachs of 60 fishes were examined, most of which were
using Levenberg-Marquardt and simplex logarithm using MicroCal empty or contained few food item. Those food items were identified
OriginTM Version 8.0 programme with a statistical significance of 5%. to mainly consist of various larvae (barnacle, tunicate, crustaceans and
The degree of adjustment of the model studied was assessed by the gastropods), copepods, algae, fish eggs and ctenoid scales (Fig. 2).
coefficient of determination (r2). A student t-test was applied to ver- The estimated trophic level ranged from 2.00 to 4.10 with mean values

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ify if the b value presented a significant difference to 3.0, indicating of 2.98 ± 0.29, and remained at the same level as size increased (Fig. 3a
the type of growth; isometric (b = 3.0), positive allometric (b > 3.0) and and b).
negative allometry (b < 3.0) (Froese 2006; Simon and Mazlan, 2008;
Simon et al. 2009; Froese et al., 2011, 2014; Simon et al. 2014). Fecundity estimates

Stomach content analysis and trophic level Total fecundity ranged from 23-1516 eggs with a mean egg count
of 582±478. An overall positive correlation was obtained with fecun-
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In the laboratory, the digestive tracts were removed and their con- dity and each and every comparison including body length (TL), body
tent analysed under a microscope and identified. Diet composition weight (TW), body eviscerated weight (EW) and ovary weight (Fig. 4)
data were used to estimate the trophic level of the fish. TROPH value with r values of 0.308, 0.223, 0.154, and 0.660 respectively. Fecundity
was calculated from the dataset using TrophLab; an application for showed a strong correlation with ovary weight (r = 0.660) but a weaker
estimating TROPH and its standard error using weight or volumetric correlation with body length (r = 0.308).
contribution and trophic level of each prey species to the diet, where
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individuals with a value of 2.0 or lower were considered herbivores, Gonad development in A. ocellaris
5.0 and above were carnivores and any value in between those values
were omnivores (Pauly et al., 1998, 2001; Simon and Mazlan, 2010). The gonads of A. ocellaris are bi-lobed and asymmetrical (Fig. 5e
and 6e). Ovotestes are found in males and non-breeders. The shape of
the ovotestis is flat compared to the ovary and is translucent white in
Fecundity estimates
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Fecundity was estimated from 30 females (n = 30) as the total num-

ber of oocytes present in the mature gonad. Prepared samples were
fixed in Gilson’s fluid to loosen the oocytes from the lobe. The total
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number of oocytes were then counted and reported as total fecundity.

Relationships between fecundity and total length, total weight, ovary
weight and eviscerated weight were then derived by regression analy-
sis using MicroCal Origin™ software and were analysed using Pear-
son’s correlation coefficient (Simon et al. 2012).

Gonad histology
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A total of 30 gonads from all development stages were prepared

and placed in cassettes, dehydrated (tissue processing) and embed-
ded in paraffin wax following the procedures as practiced by the Na-
tional Fish Health Research Centre of Fisheries Research Institute lab-
oratory. Sections were cut at 5µm thickness and then dewaxed, dried
and stained with hematoxylin and eosin. They were then mounted on
slides before being observed under the microscope (Leica DM1000)
Fig. 1. Length weight relationship of A. ocellaris from Pulau Tioman waters. Red line
represents a non-linear fit of the A. ocellaris, both sexes combined.
 The Egyptian Journal of Aquatic Research xxx (2018) xxx-xxx 3

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Fig. 2. Major food items consumed by A. ocellaris in Pulau Tioman. (a) barnacle larvae (b) tunicate larvae (c) copepod and (d) zoea larvae of crustaceans
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Fig. 3. Trophic level of A. ocellaris. (a) Identification of trophic levels of A. ocellaris individuals. (b) Identification of trophic levels of A. ocellaris according to size. Open circles
represent mean TROPH values/trophic level and solid bars represent variants of prey items.

colour. Histological examinations revealed that the ovotestes in male according to major morphological characteristics of oocytes growth.
and non-breeders consist of both testicular and ovarian cells. Terminology used for categorising oocytes based on the microscopic
Ovotestes of male fishes comprised all stages of spermatogonic cells and macroscopic features of the ovaries were adapted from
(spermatogonia, spermaticytes, spermatids and spermatozoa) and are Abol-Munafi et al. (2011), McMillan (2007), Genten et al. (2009),
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located in the centre region (TR), surrounded by oogonia with Wallace and Selman (1981) and Selman and Wallace (1989).
pre-vitellogenic oocytes at the periphery (OR) (Fig. 5a and b). Stage I ovaries are small in size, colour ranging from white to pale
Ovotestes of non-breeders consist mainly of oogonia and previtel- yellow (Fig. 6e). Primary growth oocytes are predominant (Fig. 6a).
logenic oocytes (OR) , with less area of the testicular region (TR) al- Oogonia are small (54.03±1.2µm) with the presence of one nucleolus
though all stages of spermatogenic cells can be found (Fig. 5c and d). and are highly basophilic. The nuclei may contain many prominent nu-
The heterosexual germinal elements are in direct contact with each cleoli, often at the periphery of the nucleus. Oogonia initiated meiosis
other with no connective tissues in both ovotestes. and entered into chromatin nucleolus stage of development. They are
As for the ovary, testicular tissues are not observed in immature considered oocytes in this stage. Oocytes are surrounded by a single
and mature females, where only mixtures of all stages of oocytes are layer of squamous follicle cells (zona granulosa).
present (Fig. 5e and f). Four developmental stages were recognised
4 The Egyptian Journal of Aquatic Research xxx (2018) xxx-xxx

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Fig. 4. Relationship between fecundity and a) body weight b) total length c) eviscerated weight and d) ovary weight in A.ocellaris. Solid line represents a non linear fit in (a) and
linear fit in (b-d) of A. ocellaris

The colour deepens as ovaries turns from pale to bright yellow Nakamura et al. (2003). A number of stomachs contained fish eggs
in Stage II (Fig. 6e). This is the beginning of secondary growth with and scales, suspected to be the result of egg guarding behaviour of the
cortical alveoli (CA). Vacuoles start to appear and progressively in- clownfish.
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crease in number to form peripheral rows (Fig. 6b). Oocytes are The results showed that A. ocellaris exhibited an isometric growth
167.11±18.52µm in size. This can be found in premature females. pattern where the length increased proportionately to weight, similarly
In Stage III, the ovaries are matured and are bright orange in colour Froese et al. (2014) reported matching value in the estimation of pa-
(Fig. 6e). The appearance of yolk granules (protein) and fat vacuoles rameter ‘b’ for the majority of Amphiprion members; 3.04 for A. per-
in the cytoplasm defines the vitellogenic oocytes (Vtg). The cyto- cula, A. melanopus and A. akallopisos and 2.99 in A. perideraion, A.
plasm begins to fill with yolk spheres, granules or globules (Fig. 6c). polymnus and A. clarkii, indicating that the members of the genus may
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These structures maintained their shape throughout the growth with- all show the same isometric growth strategy.
out merge into a mass of yolk fluid, with an average size of The fecundity estimates of A. ocellaris were similar as reported by
239.23 ± 23.24µm. This stage is predominant in mature female Fautin and Allen (1992) and Gordon and Bok (2001). The increase
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ovaries. in fecundity with body length and weight is consistent with the ob-
Stage IV ovaries range from dark yellow to brownish (Fig. 6e). The servations in other Amphiprion species (Beldade et al., 2012; Hattori,
germinal vesicle begins to breakdown and migrates towards the an- 2012), which makes body length and weight reliable indicators of the
imal pole with the dissolution of the nucleolus membrane (Fig. 6d). capacity of egg production (Bagenal, 1978). The histological results
Rapid uptake of fluid by the oocytes (hydration) causes the oocytes to were alike to those observed in A. ocellaris as reported by many others
swell and ovulation occurs when the follicle ruptures after the com- (Moyer and Nakazono,1978; Madhu et al., 2010; Abol-Munafi et al.,
pletion of oocyte maturation. The oocytes in this stage measured an 2011). The present study showed that the ovaries consisted of oocytes
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average of 280.25 ± 17.52 µm in size. The remnants follicles are called of various stages and were randomly scattered in the gonad, indicat-
postovulatory follicles. ing an asynchronous ovarian development organization. Such organi-
zation in general may be found in iteroparous species with multiple
Discussion spawning seasons that mostly relies on food availability in the envi-
ronment at that time (Hunter and Leong, 1981), which is the case for
In the current study, most of the stomachs were empty due to re- A. ocellaris as they are reported to spawn throughout the year in trop-
gurgitation of the fish during capture process. Therefore, stomach full- ical countries (Fautin and Allen, 1992).
ness analysis was unable to be carried out and thus, the diet com- The presence of both ovarian and testicular tissues in the gonad
position analysis was based on the remaining stomach content. The of male and non-breeders and the absence of testicular tissues in the
contents found were similar to the reports by Myers (1999) and ovary of the females suggested that A. ocellaris undergo a change of
 The Egyptian Journal of Aquatic Research xxx (2018) xxx-xxx 5

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Fig. 5. Histological examination of the gonads of A. ocellaris. (a-b): Male ovotestes which consists of testicular region (TR) at the centre surrounded by ovarian cells (OR) at the
periphery (stage I only), (c-d): ovotestes of non-breeder which consists of stage I and II oocytes (majority)(PO) and all stages of spermatogenic cells, (e-f): Mature ovary with all
stages of oocytes, (PO, primary oocytes; CA, cortical alveoli; Vtg, vitellogenic oocytes).
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sex from male to female (protandry) that may not be reversible. Sim- consisted of ovarian tissues without any connective tissue between the
ilar observations were also reported by Abol-Munafi et al. (2011) and two regions, with the exception of the ovarian and testicular regions
also in gonads of other Amphiprion species (Moyer and Nakazono, that do not intermix as in A. ocellaris. The change of sex from male
1978; Ochi, 1989; Miura et al., 2003; Nakamura et al. 2005; to female in A. ocellaris may not be reversible; however Madhu et al.
Casadevall et al., 2009). This indicates that Amphiprion species (2010) discovered that when reared in the absence of the breeding pair,
showed consistency in their reproduction strategy disregarding of their the largest non-breeder member can become female without passing
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locality. through the male stage and the second largest non-breeder developed
Our results showed that in the male ovotestes, the testicular tis- into the breeding male. Similar results were obtained by Brusle-Sicard
sues were found located at the centre while the ovarian tissues were et al. (1991) and Stahlschmidt-Allner and Reinboth (1991) in A. fre-
located at the periphery. A reverse organization was observed in the natus, A. clarkii (Miura et al., 2003; Nakamura et al., 2005) and A.
male ovotestes of A. polymnus and A. clarkii where testicular tissues akallopisos (Mills et al, 2018). This emphasises the importance of
were peripherally located while the ovarian tissues were centrally lo- pairing and social structure in the sex changing mechanism of clown-
cated (Abol-Munafi et al., 2011; Ratanayuvakorm et al., 2006; Miura fish and suggests that protandry in clownfish could be facultative
et al., 2003). The ovotestes in the non-breeders of A. polymnus and (Madhu et al., 2010).
A. clarkii were also similar to that A .ocellaris, where they primarily
6 The Egyptian Journal of Aquatic Research xxx (2018) xxx-xxx

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Fig. 6. Stages of oogonia; (a) stage I (Primary growth): N, nucleus; PN: peripheral nucleoli; PO, primary oocyte (b) stage II (cortical alveoli): YV, yolk vesicle; ZR, zona radiata (c)
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Stage III (vitellogenesis): Vtg, vitellogenic oocyte; VE, vitelline envelope; PO, primary oocyte, (d) Stage IV (oocyte maturation): YG, yolk globule and (e) The change in colours in
the ovary of A.ocellaris in different maturity stage.

Conclusion Acknowledgement

This study has provided a more detailed understanding of the ba- Heartiest thanks to the National Fish Health Research Centre of
sic diet and reproduction biology of A. ocellaris in Malaysian waters. Fisheries Research Institute, for the assistance provided regarding the
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A .ocellaris is omnivorous; stomach content consisted of variety of histological works and permission to use their equipment and Dr. Kua
larva and algae. Results were further supported by trophic level analy- Beng Chu for the helpful advices. Thanks to UKM for the financial
sis of 2.98 ± 0.29 with no shift in diet with increasing size. Population support through the research grant GUP-2017-023.
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growth pattern for clownfish estimated from a length-weight relation-

ship (W = aLb) exhibited an isometric growth (b = 3.0). An estimated References
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