Assessment of different minute zooplankton in
the larval rearing of rusty angelfish Centropyge
ferrugata1

Stenly Wullur, 2Shigeru Kumagai, 3Yoshitaka Sakakura, 3Atsushi Hagiwara

1

Faculty of Fisheries and Marine Science, Sam Ratulangi University, Manado, Indonesia;
Faculty of Science, Okayama University of Science, Okayama, Japan; 3 Graduate Schoolof Fisheries Sciensce and Environmental Studies, Nagasaki University, Nagasaki, Japan.
Corresponding author: S. Wullur, stenlywullur@unsrat.ac.id2

Abstract. Angelfishes of the genus Centropyge are commercially valuable aquarium fish. Although
success in domesticating the broodstock of angelfishes is already reported, larval rearing remains a
failure because their larvae require very small live food. Recognizing this demand, we assess theacceptability of four minute zooplankton including rotifers Proales similis, Keratella sinensis, Brachionus
rotundiformis (SS-type), and nauplii of copepod Paracyclopina nana as feed for rusty angelfish
Centropyge ferrugata larvae. The average size of the four zooplankton we used ranged from 80 to 160
m. Results showed that feeding incidence on P. similis on the first day of feeding (3 days after hatching,
3 DAH) was 40%, and as high as 60% on day 6 after hatching (6 DAH), while it was 0 to 20% on otherzooplankton tested. Larvae in the control group (without feeding) did not survive on 5 DAH. Standard
length of the larvae in the two trials was not significantly different among treatments, but survival was
significantly higher in P. similis (19 to 38%) than other zooplanktons (0 to10%). Results of this study
indicate the usefulness of P. similis as starter food for C. ferrugata larvae.
Key Words: Proales similis, Brachionus rotundiformis, rotifer, live food, copepod.

Introduction. Angelfishes of the genus Centropyge are among the highly prized andoften sought coral reef fish because of their prominent coloration (Olivotto et al 2006). At
present, the main source of angelfishes for aquarium rearing is from the wild (Wood2001), especially the coral reef ecosystem where these fishes reside. However, collection
from the wild is often unsustainable and stresses the coral reef ecosystems (Rubec 1988;
Jones & Steven 1997; Jones et al 1999). Furthermore, the declining condition of coral
reefs has caused increasing regulation on any activities that impact the reef environment
including the restriction or even banning of ornamental fish collection activities which
includes angelfishes (Rubec 1988; Wood 2001). These problems have created acompelling need to develop an alternative mariculture technology to wild collection inorder to meet the market demand and possibly restore degraded wild population (Laidley
et al 2008). Despite much success in captive maturation and spawning of angelfishes in
the last three decades (Suzuki et al 1979; Arai 1994; Hioki et al 1990; Olivotto et al
2006; Leu et al 2009), massive mortality related to poor initial feeding of the larvae
remain a bottleneck for their successful captive production (Olivotto et al 2006; Leu et al2009).
Larvae of angelfishes require small food due to their small mouth size (Olivotto et
al 2006; Laidley et al 2008; Leu et al 2009). SS-type rotifer Brachionus rotundiformis(lorica length 90 to 150 m) which is a common starter food for rearing marine fish withsmall mouth size (Lubzens et al 1989; Hagiwara et al 2001; Ogello et al 2018) isineffective in rearing angelfish larvae (Olivotto et al 2006; Laidley et al 2008; Leu et al2009). Efforts had been geared to identify appropriate starter food organism for angelfish
larvae including the use of copepod nauplii (mean body length <100 m, Olivotto et al
2006; Laidley et al 2008), dinoflagellate (Gonyaulax sp., 65 m in length and 38 m inwidth, Leu et al 2009), sea urchin eggs, ciliates or oyster trochopores (mean diameter
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<70 m, Laidley et al 2008). The use of copepods showed promising results on growthand survival of angelfish larvae (Olivotto et al 2006; Laidley et al 2008), but since mostof the tested copepod species are difficult to culture at high density (Schipp et al 1999;
McKinnon et al 2003; Shields et al 2005), their use as starter food is still limited toexperimental scale only. The use of other food organisms including dinoflagellates, sea
urchin, ciliates or oyster trochopores were reported to have modest success (Laidley et al2008) or no beneficial effect on the larval growth and survival (Leu et al 2009). Although
their minute size could fit the mouth of the larvae, they are nutritionally inferior andcould not meet the requirement of the larvae for growth and survival. Moreover,nutritional enrichment of these food organisms is difficult because at the stage where
their size fits the mouth of the angelfish larvae, the organisms are not yet on theirfeeding stage (Hoff & Snell 1987).
Recognizing the demand of smaller starter food, we conducted two feeding trials
on rusty angelfish (Centropyge ferrugata) using four-minute zooplankton including
rotifers Proales similis, Keratella sinensis, SS-type Brachionus rotundiformis, and nauplii
of a copepod Paracyclopina nana. These zooplanktons were selected mainly because of
their small size and easiness of culture.
Material and Method
Zooplankton food. Zooplanktons used in the present study were collected from various
habitats around the world. The rotifer P. similis was collected in Ishigaki island, Okinawa,
Japan (Wullur et al 2009, 2011, 2013), B. rotundiformis in Bali island, Indonesia(Hagiwara et al 1995; Wullur et al 2013), K. sinensis in South-Korea (J.C. Park,
Kangnung National University, South-Korea; Wullur et al 2013) and copepod P. nana in
Hwajinpo salt lake, Gangwondo, South-Korea (Lee et al 2006; Wullur et al 2013).
All zooplanktons were cultured in 2-5 L culture containers using diluted seawater(25 ppt) at 25oC, adapting the semi-continuous culture method and feed with super fresh
Chlorella vulgaris V-12® (Chlorella Industry Co. Ltd., Fukuoka, Japan). Our previous
study showed that by feeding super fresh C. vulgaris V-12 to P. similis, nutritional value
greatly increased (Wullur et al 2011), and can be used to feed directly to the fish larvae.
The population growth of the cultured zooplanktons was monitored daily by taking aliquot
samples from each culture tank and counting under a stereomicroscope. The density wasmaintained at 100 to 300 ind/mL for P. similis, B. rotundiformis and K. sinensis, and at 5
to 10 ind/mL for P. nana by harvesting certain culture volume of the culture andreplenishing with diluted seawater depending on their density. The zooplanktons were
harvested using plankton net (mesh size = 10 to 45 m mesh size depending on bodysize of the zooplankton). Nauplii of P. nana were obtained by separating them from themother and copepodids using a 100 m net.
Feeding experiment. Feeding experiment was conducted in an aquarium filled with 2.5 L
filtered natural seawater (32 ppt) at a temperature-controlled (25oC) and well lighted(500-600 lux) room. The room was lighted all day (24L:0D) and the aquarium wasprovided with slow (5 mL/min) aeration. Two feeding trials were conducted.
In both trials, fertilized eggs of naturally spawn C. ferrugata were obtained from a
private hatchery in Kagawa, Japan. In the first trial, newly hatched larvae were stocked
at 200 larvae/aquarium and the water temperature was maintained at 25 0C, while in thesecond trial, the larvae were stocked at 50 larvae/aquarium and the temperature wasmaintained at 280C.
In our preliminary experiments, we knew that first feeding of C. ferrugata
occurred on day 3 after hatching (3 DAH). Thus, during the experiment, zooplankton
foods were added in each aquarium on 3 DAH. P. similis, K. sinensis, and B.
rotundiformis were added in each aquarium at 20 ind/mL, while P. nana at 10 ind/mL.
We prepared another aquarium stocked with 50 larvae, but no food was added, as ourcontrol. Super fresh C. vulgaris V-12® was added into the culture water as backgroundalgae.

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Starting from 3 DAH, five larvae were sampled from each aquarium, anesthetizedwith 0.01% MS 222 (Tricaine; Sigma Chemical Co., St Louis, MO, USA), fixed with 5%
formaldehyde, and measured under a digital microscope (VH-6300, Keyence Corp.,
Japan). Feeding incidence (percent of larvae with rotifers inside the gut) and feeding
amount (number of rotifers in larval gut) were investigated by dissecting the larval gut
under a stereomicroscope. Feeding amount was determined by counting the trophi ofdigested rotifers as well as number of undigested body of rotifers or copepod (Akazawa
et al 2008). Percent survival of the larvae in all treatments was estimated by counting
the number of surviving larvae at the end of the experiment.
Results. The body length and width of the four zooplankton used in the present study
are shown in Table 1. Among the zooplankton, mean body size of P. similis (90.910.9
m in length and 44.46.8 m in width) was the smallest, while B. rotundiformis was thelargest (137.515.7 m in length and 106.915.6 m in width). Mouth size of C.
ferrugata larvae on 3 DAH (when the mouth opens) was 16016 m.
Table 1
Body length and width (meanSD) of the zooplankton used in present study (each n=50)
Zooplankton species
Proales similis
Keratella sinensis
Brachionus rotundiformis
Paracyclopina nana

Body dimension (m)LengthWidth90.910.9
44.46.8
107.310.2
62.37.5
137.515.7
106.915.6
113.546.9
61.811.8

Feeding incidence of the larvae in the first trial occurred from 4 DAH, with 60% feeding
incidence (food intake, 1.6 ind/larvae) on P. similis while it was 0 to 20% (food intake, 0
to 0.2 ind/larvae) for those fed with other zooplankton. The larvae kept ingesting P.
similis (feeding incidence 40%, feeding amount 2.2 ind/larvae) and K. sinensis (feeding
incidence 20%, feeding amount 0 to 0.2 ind/larvae) on the next day (5 DAH) while noingestion by the larvae was observed for B. rotundiformis and P. nana (Figure 1).

Figure 1. Feeding incidence (mean%SD, n=3) (I) and feeding amount (meanSD, n=3)(II) of Centropyge ferrugata larvae in first trial fed with Proales similis (□), Brachionus
rotundiformis (■), Keratella sinensis ( ) and nauplii or the copepod Paracyclopina nana( ).
In the second trial, we observed the first feeding on 3 DAH (feeding incidence = 40%,
feeding amount = 0.8 ind/mL) in P. similis-fed treatment and none in other treatments.
From 4 DAH to 6 DAH, 20 to 60% feeding incidence was observed on P. similis while it
was 0 to 20% on other zooplankton (Figure 2).
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Figure 2. Feeding incidence (mean%SD, n=3) (I) and feeding amount (meanSD, n=3)(II) of Centropyge ferrugata larvae in second trial fed with Proales similis ( □), Brachionus
rotundiformis (■), Keratella sinensis () and nauplii or the copepod Paracyclopina nana( ).
Standard length of larvae fed with P. similis (2.2 0.0 m), K. sinensis (2.10.1 m), B.
rotundiformis (2.00.2 m), P. nana (2.10.1 m) was not significantly different among
treatments, but survival was significantly higher on P. similis (18.5%) than otherzooplankton (6 to 11.5%) (Tukey-Kramer test, p<0.05) (Figure 3-I). Larvae in thecontrol group (no feeding) did not survive on 5 DAH.
No significant difference was observed in the standard length of larvae in alltreatments. The standard length of larvae fed with P. similis was 2.20.1 m, 2.10.1 m
for those fed with K. sinensis and 2.00.1 m with B. rotundiformis. Survival was
significantly higher on P. similis (38%) than other zooplankton (0 to10%) (Tukey-Kramer
test, p<0.05) (Figure 3-II). No larvae survived in the control group (no feeding) on 6
DAH.

Figure 3. Survival (mean%SD, n=1) of rusty angelfish Centropyge ferrugata larvae in
the first (I) and second (II) trial fed with Proales similis, Brachionus rotundiformis,
Keratella sinensis and nauplii of copepod Paracyclopina nana. Different alphabeticalletters indicates significant differences, Tukey Kramer test, p<0.05, a>b>c.

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Discussion. The first characteristic to consider in choosing food organism for smallmouth fish larvae is the body size of their prey (Shirota 1970; Fernandez-Diaz et al
1994; Cunha & Planas 1999; Olivotto et al 2006; Yufera & Darias 2007). The mouth sizeof C. ferrugata larvae in the present study was 16016 m at first opening. It has been
known that most fish larvae could prey on food 20 to 70% smaller than their mouth size
(Shirota 1970; Fernandez-Diaz et al 1994; Cunha & Planas 1999; Yufera & Darias 2007).
Considering that C. ferrugata used in this study has a mouth size of 16016 m, thissuggests that they require food with size from 32 to 112 m. P. similis (meanSD;
90.910.9 m in length and 44.46.8 m in width) and K. sinensis (107.310.2 m inlength and 62.311.5 m in width) are within the required size, but those of B.
rotundiformis (137.515.7 m in length and 106.915.6 m in width) and nauplii of P.
nana (113.546.9 m and 61.811.8 m) slightly exceed the requirement.
At first feeding (3 DAH) we found that C. ferrugata larvae could ingest only P.
similis (Figure 1) and occurred only when the larvae were reared at higher temperature(28oC, first run), and not at 25oC. We hypothesized that in addition to the size of food,environmental factors such as temperature also affect the initial feeding of C. ferrugata
larvae. Furthermore, larvae on 4, 5 and 6 DAH showed 20 to 60% feeding incidence on P.
similis, while it was less than 20% for those larvae in other treatments indicating thepotential of P. similis to be used as starter food for angelfish larvae. Less feeding
incidence by the larvae in the treatments of Keratella sp., B. rotundiformis and copepod P.
nana nauplii could be due to the less proportion of food with suitable size for larvae
available in the water column affecting the encounter rate with the larvae and the prey.
Past feeding trials on angelfish larvae used bigger tanks of 150 L or more (Olivotto
et al 2006; Leu et al 2009), while we used small (2.5 L) aquaria and with high stocking
density (50 to 200 larvae), and also high food density (10 to 20 ind/mL). With theserearing conditions, water quality is difficult to maintain for an extended time which was
the reason why we conducted the experiment for a short period by counting the survival
on 5 or 6 DAH where the larvae stocked without feeding in two trials showed zerosurvival. On these days, growth of the larvae in the two trials was not significantly
different among treatments but survival was significantly higher on larvae fed P. similis(18.5 to 38%) than those fed with other zooplankton (0 to 11.5%). As survival during
the early larval stage depends on various factors including feeding, environmental
requirements or age at which the survival were recorded (Yufera & Darias 2007), it issuggested that angelfish larvae to be reared at suitable environmental condition using P.
similis as food source so that survival of the larvae could be enhanced and prolonged.
Higher survival of angelfish larvae in the treatment of P. similis correlated to the feedingof the larvae in this treatment during the experiment.
The result of the present study could serve as a starting point for successful larvalrearing of angelfish and other fish larvae with small mouth size including humphead
wrasse (Cheilinus undulatus) by providing P. similis as starter food. Besides their small
size, more advantages of P. similis is that it can be mass propagated and nutritionallyenriched (Wullur et al 2009, 2011; Hirai et al 2012; Hagiwara et al 2014). These features
are in contrast with those food organisms that have been previously evaluated for theirpotentials as starter food for angelfish larvae including copepod naupliar (Olivotto et al
2006; Laidley et al 2008), dinoflagellates (Leu et al 2009), sea urchin eggs, ciliates oroyster trochophores (Laidley et al 2008), which are difficult to mass culture (Schipp et al
1999; McKinnon et al 2003; Shields et al 2005) or nutritionally enrich (Hoff & Snell 1987).
Conclusions. Our results demonstrate the usefulness of P. similis as a starter food for C.
ferrugata larvae during the onset of first feeding. The larvae effectively consumed P.
similis and were able to use nutritional benefits of the rotifer to support their survival.
The present results can be a starting point for successful larval rearing of marine fish
species with small mouth size at first feeding by providing P. similis as starter food.
Acknowledgements. The authors express thanks to Hellen Marcial for reviewing themanuscript. This research was partially supported by The Ministries of Research and

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Higher
Education
Republicof
Indonesia,
PPUPT-grant,
No:
SP.DIPA042.06.1.401516/2018, and Jasso Follow-up Research Fellowship Fiscal 2016 to SW.
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Received: 22 June 2018. Accepted: 08 October 2018. Published online: 15 October 2018.
Authors:
Stenly Wullur, Sam Ratulangi University, Faculty of Fisheries and Marine Science, Indonesia, Jl. Kampus Unsrat
Manado 95-115, e-mail: stenlywullur@unsrat.ac.id
Shigeru Kumagai, Okayama University of Science, Faculty of Science, Japan, Okayama 700-005, Kita, 1-1
Ridaicho, e-mail: budidaya_ikan@live.jp
Yoshitaka Sakakura, Nagasaki University, Graduate School of Fisheries Sciences and Environmental Studies,Japan, Nagasaki 852-8521, 1-14 Bunkyomachi, e-mail: sakakura@nagasaki-u.ac.jp
Atsushi Hagiwara, Nagasaki University, Graduate School of Fisheries Sciences and Environmental Studies,Japan, Nagasaki 852-8521, e-mail: hagiwara@nagasaki-u.ac.jp
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.
How to cite this article:
Wullur S., Kumagai S., Sakakura Y., Hagiwara A., 2018 Assessment of different minute zooplankton in thelarval rearing of rusty angelfish Centropyge ferrugata. AACL Bioflux 11(5):1495-1501.
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