Aquaculture 293 (2009) 57–61

Contents lists available at ScienceDirect

Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Enrichment potential of HUFA-rich thraustochytrid Schizochytrium mangrovei for the

rotifer Brachionus plicatilis
Chona Estudillo-del Castillo ⁎, Rolando S. Gapasin, Eduardo M. Leaño
Southeast Asian Fisheries Development Center, Aquaculture Department (SEAFDEC/AQD), 5021 Tigbauan, Iloilo, Philippines

a r t i c l e i n f o a b s t r a c t

Article history: An enrichment experiment was performed to evaluate the changes in lipid and essential fatty acid contents
Received 25 August 2008 of the rotifer Brachionus plicatilis fed with freeze-dried cells of tropical thraustochytrid Schizochytrium
Received in revised form 12 April 2009 mangrovei (Isolate IAo-1). Rotifers starved for 24 h were fed with S. mangrovei cells at 200, 300, 400, 500, 600
Accepted 13 April 2009
and 700 mg L− 1. Enrichment was carried out at two periods (Short-term = 5 h; Long-term = 10 h) to
determine the optimum time needed for the maximum enrichment of the rotifers. There was an overall
Keywords:
Thraustochytrid
significant increase in the total lipid, arachidonic acid (AA) and docosahexaenoic acid (DHA) contents of
Rotifer rotifers after feeding with freeze-dried S. mangrovei indicating the successful uptake of these nutrients in the
Lipid rotifer's biochemical composition. On the other hand, docosapentaenoic acid (DPA) did not change
DHA significantly in enriched rotifers. Results of the present study indicate that both factors, feeding
AA concentrations and enrichment periods, significantly affected the lipid, AA and DHA contents of rotifers.
Enrichment Uptakes of lipid, AA and DHA significantly increased with increasing feeding concentrations except for those
Feeding fed the highest feeding concentration of 700 mg L− 1 for 10 h. Moreover, lipid and AA contents of enriched
rotifers were significantly higher during the short-term enrichment period while DHA contents were
significantly higher during the long-term enrichment period. Therefore, it is concluded that the feeding
concentration of 700 mg L− 1 at an enrichment period of 5 h is optimum in the AA and DHA enrichment of
rotifers. The strategic scheme of combining the proper amount of enrichment product and the duration of
enrichment in boosting the DHA contents of rotifers will effectively ensure a reliable production of
nutritionally superior rotifers at a minimal cost. This will ultimately contribute to the success of rearing
marine fish larvae in the hatchery.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction known as stramenopiles, thraustochytrids are reported to produce a

Larvae of many marine fish require a certain amount of long-chain
highly unsaturated fatty acids (HUFAs) of the n-3 series such as
arachidonic acid (AA, 20:4), docosapentaenoic acid (DPA, 22:5),
eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA,
22:6) for optimum survival and growth. Common sources of these
essential fatty acids include: traditional commercial fish oils (by-
products of pelagic fisheries); oils rich in DHA/EPA; and, single-celled
eukaryotes such as heterotrophic dinoflagellates and phototrophic
prymnesiophyceans (Sargent et al., 1997; Leaño and Liao, 2004; Leaño
and Liao, 2007). Originally thought to be primitive fungi, the
heterotrophic thraustochytrids (e.g. Schizochytrium spp.) are now
classified under Kingdom Chromista and Phylum Stramenopila, which
is closely related to heterokont microalgae (e.g. brown algae and
diatoms) (Cavalier-Smith et al., 1994; Parfrey et al., 2006). Commonly
⁎ Corresponding author. Present address: University of Asia and the Pacific, Pearl
Drive, Ortigas Center, Pasig, Metro Manila, Philippines.
E-mail address: mcdelcastillo@uap.edu.ph (C. Estudillo-del Castillo).
high amount of fatty acids, particularly DHA (Barclay and Zeller, 1996;
Li and Ward, 1994; Yaguchi et al., 1997; Lewis et al., 1999; Fan et al.,
2002; Leaño et al., 2003; Leaño and Liao, 2004), which is superior to
EPA as feeds for fish larvae.
Fish do not synthesize long-chain HUFAs in significant quantities.
They acquire HUFAs through their diet, such as eating zooplankton
(e.g. rotifers, crustaceans), which are enriched with these nutrients.
Increasing the HUFA content of zooplankton prior to feeding larval fish
and shrimp is a common practice in the aquaculture industry
(reviewed by Apt and Behrens, 1999). Due to the high cost of
producing sufficient and reliable live HUFA-rich microalgae, spray-
dried heterotrophic microalgae have been tested as aquaculture feeds.
However, the strains tested were selected primarily for their
heterotrophic growth potential with only a secondary concern for
their nutritional value, particularly on their HUFA content resulting in
poor larval feed performance (Laing and Verdugo, 1991). The
heterotrophic Schizochytrium mangrovei was chosen as the test
organism for the enrichment of rotifers in this study because of its
high long-chain HUFA contents of the n-3 series, particularly DHA
(Leaño et al., 2003). Thraustochytrids offer advantages over other

0044-8486/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2009.04.008
58 C. Estudillo-del Castillo et al. / Aquaculture 293 (2009) 57–61
sources of HUFA for aquaculture (Leaño and Liao, 2004). Many fish
larval aquaculture species require proportionally more DHA than EPA
(Furuita et al., 1996; Furuita et al., 1998; Pousao-Ferreira et al., 2001).
The HUFA profile of S. mangrovei fits this criterion, while most oils
from the fish-meal industry contain more EPA than DHA. The cells of S.
mangrovei are small in size and have excellent suspension character-
istics in seawater which facilitate easy ingestion. The physical
conditions applied in the drying process can also facilitate easy
ingestion by rotifers (Laing and Verdugo, 1991). Freeze-dried
S. mangrovei may have fragile cell walls as a result of the drying
process; this may make the diet more easily digested and physically
broken down in the gut of the rotifer, leading to efficient assimilation
and utilization. Schizochytrium sp. has been shown to enrich and boost
the fatty acid and DHA content in rotifers Brachionus plicatilis and
Artemia sp. (Barclay and Zeller, 1996). Schizochytrium-based products
have also been available in the market for enriching rotifers and Ar-
temia sp. in the U.S.A. (www.aquafauna.com). The manufacturer
suggests an 8 h enrichment of rotifers prior to feeding to target larval
fish. On the other hand, Kraul (2006) reported that a 2 h enrichment of
rotifers prior to feeding greatly increases the health and growth rate of
marine fish larvae. Unfortunately, the rotifer's fatty acid composition
after enrichment for 2 h was not evaluated to support his observation.
This study evaluated the potential of locally isolated tropical
thaustochytrid S. mangrovei (Isolate IAo-1) as live food enrichment
for the rotifers B. plicatilis. It addressed the need to identify the
optimum feeding concentration and period for the enrichment of
rotifers B. plicatilis using locally isolated tropical Schizochytrium
species. The changes in the lipid, AA, DPA and DHA contents were
analyzed in rotifers enriched with freeze-dried S. mangrovei cells at
different feeding concentrations during short-term and long-term
feeding.
2. Methodology
2.1. Mass production of S. mangrovei
Axenic small-scale heterotrophic mass production of S. mangrovei
(Isolate IAo-1) in the laboratory was conducted on peptone-yeast-
extract high glucose medium (PYG medium) following the procedure
of Leaño et al. (2003) with slight modifications. Cultures were grown
in a 2 L flask filled with 700 mL culture medium under 150 rpm
shaking condition (Orbit Shaker, Labline Instrument Incorporated) at
25 °C for 3–4°d (days). Each culture flask was plugged with sterile
cotton to avoid contamination and wrapped with black plastic to
eliminate light. These cultures were later used as inoculum for the
medium-scale mass production of S. mangrovei in a 30 L fermentor
carried out at the Biotechnology Laboratory, University of the
Philippines, Los Baños, Laguna, Philippines. At late-exponential
phase, S. magrovei cultures were scaled up by inoculating into sterile
15 L PYG medium in 30 L fermentors as described previously in the
small-scale mass production, except that the live cell inoculum was
increased from 5% to 10% of the total volume of the culture.
Fermentor-cultured S. mangrovei was harvested through centrifuga-
tion, freeze-dried and stored inside sealed containers at −70 °C until
fatty acid analysis or until use in the rotifer enrichment experiments.
2.2. Rotifer source
The rotifers B. plicatilis were obtained from a routine culture at the
finfish hatchery of the Southeast Asian Fisheries Development Center.
They were grown semi-continously on a “green water” system as
described by Duray et al. (1996) using live microalgal diet Nanno-
chloropsis sp. Prior to the enrichment experiment, rotifers were
harvested at the late exponential phase, washed with 0.2 µm-filtered
seawater, stocked in 10 L plastic containers at a density of 5000–
10,000 individuals mL− 1, and provided with adequate aeration. The
rotifers were starved for 24 h (hours) to ensure that the gut content
was clear of the microalga Nannochloropsis sp. with which the rotifers
were originally fed.
2.3. Enrichment/feeding experiment
Prior to the experiment, samples from the starved rotifers were
taken to determine the initial (time = 0 h) lipid and fatty acid
contents. This was designated as time = 0 in the enrichment period.
The remaining starved rotifer suspension was stocked into each of
the cone-shaped plastic containers (5 L) filled with 3.5 L filtered
seawater. Each rotifer culture was provided with adequate aeration and
maintained at a density of 1500 individuals mL− 1. Rotifers were then fed
with dried S. mangrovei freeze-dried cells at different feeding concen-
trations of 200, 300, 400, 500, 600 and 700 mg L− 1 of rotifer culture
(expressed as mg L− 1). Short (time = 5 h) and long (time= 10 h)
enrichment periods were tested for all feeding concentrations. Experi-
mental treatments were replicated three times which were randomly
assigned. Enrichment experiments were conducted at a temperature
(26–27 °C) controlled laboratory.
At the end of each enrichment period, rotifers (120–200 µm in
size) were collected from each treatment using a plankton net (mesh
size = 37 μm) to separate them from uneaten S. mangrovei cells with
cell sizes of 8–14 µm (Fan et al., 2002). Rotifer samples were then
washed with 0.5 M ammonium formate to remove salts, freeze-dried
and stored desiccated at −70 °C until further analysis.
2.4. Lipid and fatty acid methyl ester (FAME) extraction/analysis
Lipid extraction and FAME analysis of starved (initial, time = 0)
and enriched rotifers, and sample of freeze-dried S. mangrovei cells
were done following Lepage and Roy (1984) and Metcalfe and Schmitz
(1961) with minor modifications. Lipids were trans-esterified with an
acetylchloride/methanol mixture. For quantitative purposes, an
internal standard heneicosanoic acid methyl ester (21:0, SIGMA,
MO, U.S.A.) was added equivalent to 3.5% of the total weight FAMEs.
FAME extracts were evaporated under vacuum, dried under a stream
of nitrogen, weighed and resuspended in isooctane (LiChroSolv,
Merck, Germany) prior to analysis with a GC-17A gas chromatograph
(Shimadzu, Kyoto, Japan). Helium was the carrier gas. Injector and
detector temperatures were both at 250 °C. Individual FAME was
identified by comparing the relative retention times of the fatty acids
with that of the known reference standard and quantified by a
Chromatopac C-R7 integrator. FAMEs were quantified using the
internal standard method and expressed as percent of the total fatty
acids (TFA).
2.5. Statistical analysis
All values on biochemical composition (lipid and fatty acids) are
presented on a dry weight basis. Lipid and fatty acid compositions of
rotifers after the feeding experiments were analyzed using a two-way
ANOVA to compare the effects of different feeding concentrations on
the changes in the chemical composition of starved against the
enriched rotifers at two enrichment periods. Data showing significant
differences at (P b 0.05) across treatments were further analyzed by
Duncan multiple range test (DMRT). All statistical analysis was
performed using the statistical program SAS (Statistical Analysis
System, Version 8, Windows 2000).
3. Results
3.1. Lipid and fatty acid profiles of S. mangrovei
The lipid and fatty acid profiles of the DHA-rich traustochytrid
S. mangrovei used as enrichment feed for rotifers B. plicatilis are
 C. Estudillo-del Castillo et al. / Aquaculture 293 (2009) 57–61 59

Table 1 and 400 mg L− l. Lipid contents in rotifers fed with freeze-dried

Lipid (expressed as % of the dry weight, % DW) and fatty acid composition (expressed as
% of the total fatty acid, % TFA) of Schizochytrium mangrovei (enrichment organism).
Lipid/FA composition
Lipid
Fatty acid composition
Saturated fatty acid (SFA)
14:0
unk1
unk2
16:0
18:0
Polyunsaturated fatty acid (PUFA)
18:2
18:3
20:4 n-3
21:0
unk3
unk4
22:5 n-3 (DPA)
22:6 n-3 (DHA)
Each value in the fatty acid composition is the mean of two replicate samples.
presented in Table 1. The major fatty acid component was palmitic
acid (16:0), a saturated fatty acid (SFA), comprising up to 34.91% TFA
and the n-3 polyunsaturated fatty acid (PUFA) docosahexaenoic fatty
acid (DHA, 22:6), comprising up to 31.53% TFA. There was no
monounsaturated fatty acids (MUFA) found in S. mangrovei.
3.2. Changes in lipid and fatty acid compositions of enriched rotifers
Table 2 shows the total lipid, arachidonic acid (AA, 20:4),
docosapentaenoic acid (DPA, 22:5) and the docosahexaenoic acid
(DHA, 22:6) contents of starved rotifers and changes in these lipid
compositions after the feeding experiments. The lipid content of
starved rotifers (initial) was 17.43% dry weight (DW). Starved rotifers
contained only two HUFA, AA (4.19% TFA) and DPA (2.29% TFA), while
DHA was not detected. The results showed no significant interaction
between the feeding rates and the enrichment periods tested
(P N 0.05). On the other hand, two-way ANOVA showed significant
effects of different feeding concentrations on the changes in the
chemical composition of starved against enriched rotifers at two
enrichment periods.
A significant increase (P b 0.05) in the lipid contents of the rotifers
fed with freeze-dried S. mangrovei cells was observed, ranging from
23.17 to 42.81% DW compared to the initial level of 17.43%. In both
enrichment periods, lipid contents increased significantly (P b 0.05) at
feeding concentrations of 600 and 700 mg L− l while lower lipid
contents were observed at lower feeding concentrations of 200, 300
S. mangrovei cells for a short period (5 h) were consistently higher
compared to those fed for a longer period (10 h). Two-way ANOVA
Percent (%) indicated that both factors, feeding concentrations and enrichment
33.2–43.9 periods, significantly affected the lipid contents in enriched rotifers.
AA contents of rotifers in both enrichment periods tested increased
consistently (P b 0.05) with increasing feeding concentrations except
4.73 for those fed at 700 mg L− 1 for 10 h. At the 5 h enrichment period, the
2.22 highest AA level in rotifers was attained at the feeding concentration
8.15 of 700 mg L− l, while at 10 h, the highest AA level was obtained from
34.91 rotifers fed with 600 mg L− l for 10 h. The AA content did not differ
1.30
significantly between these two enrichment periods tested indicating
that only one factor (feeding concentrations) affected the uptake of
1.78 AA in rotifers.
4.32 Rotifers enriched with S. mangrovei also contain DPA, but did not
2.46
differ significantly compared from the initial contents of the starved
1.56
3.29 rotifers in both enrichment periods and feeding concentrations.
3.70 There was a significant uptake of DHA by rotifers fed with freeze-
31.53 dried S. mangrovei. From the undetected levels in the initial starved
rotifers, DHA increased significantly (P b 0.05) to a range of 4.52–
33.65% TFA. As with AA, DHA levels increased with increasing feeding
concentrations in both enrichment periods tested, except for those fed
at 700 mg L− l for 10 h. Highest DHA levels were attained in rotifers fed
at 700 mg L− 1 for 5 h (27.37% TFA) and at 600 mg L− 1 for 10 h (33.65%
TFA). DHA uptake was lowest at 200 mg L− 1 in both enrichment
periods tested (ST = 4.52% TFA and LT = 5.2% TFA). Two-way ANOVA
indicated that at higher feeding concentrations of 500–700 mg L− 1,
both factors, enrichment periods and feeding concentrations, sig-
nificantly affected the uptake of DHA in starved rotifers enriched with
freeze-dried S. mangrovei cells. Rotifers fed for a longer period of 10 h
have significantly (P b 0.05) higher DHA contents compared to those
fed for a shorter period of 5 h.
4. Discussion
The rearing of marine fish larvae requires the use of microalgae as
live food for the zooplankton rotifers (Brachionus spp.), which in turn
are consumed by the first-feeding larvae. The critical factor in the
dietary value of rotifers is their highly unsaturated fatty acid (HUFA)
content, which may be improved by feeding them with HUFA-rich
products such as microalgae and fish oil emulsions. It has been shown
that the fatty acid composition of rotifers is closely related to that of
the diets used, and short-term feeding of rotifers with HUFA-rich
microalgae/algal-like microorganisms shifts the fatty acid composi-
tion towards that of the species they were fed on (Barclay and Zeller,
1996; Lewis et al., 1998; Loung-Van et al., 1999; Estudillo, 2001).
The results of the present study showed that feeding rotifers
B. plicatilis with freeze-dried S. mangrovei cells can effectively enrich

Table 2
Mean total lipid (expressed as % of the dry weight, % DW), AA, DPA and DHA contents (expressed as % of the total fatty acid, % TFA) of starved (initial) and Schizochytrium mangrovei-
enriched rotifers Brachionus plicatilis at different feeding concentrations (expressed as mg L− 1 of rotifer culture) at two enrichment periods (Short-term = 5 h; Long-term = 10 h).

Total lipids AA (20:4) DPA (20:5) DHA (22:6)

Short-term Long-term Short-term Long-term Short-term Long-term Short-term Long-term
Starved rotifers (time = 0) 17.43 ± 1.98 4.19 ± 1.51 2.29 ± 0.20 Not detected

Feeding rates
200 31.66 ± 2.52c 23.39 ± 3.69c 4.48 ± 0.65c 3.63 ± 0.44e 1.30 ± 0.22b 1.99 ± 0.20a 4.52 ± 2.27e 5.20 ± 0.95e
300 28.44 ± 3.48d 22.37 ± 0.87c 4.61 ± 0.35c 4.26 ± 0.25d 1.67 ± 0.14b 2.07 ± 0.13a 5.27 ± 0.30d 7.57 ± 0.25d
400 23.17 ± 1.34e 21.62 ± 0.90c 6.58 ± 0.76b 3.42 ± 0.07e 2.71 ± 1.17a 1.29 ± 0.30a 15.20 ± 0.98c 9.31 ± 0.68c
500 30.90 ± 0.49c 27.01 ± 2.76b 6.79 ± 0.25b 5.72 ± 0.72c Traces Traces 14.45 ± 0.77c 28.34 ± 3.26b
600 35.16 ± 1.71b 34.61 ± 0.05a 6.65 ± 0.09b 7.52 ± 0.51a Traces Traces 22.90 ± 0.33b 33.65 ± 1.43a
700 42.81 ± 0.85a 32.72 ± 0.74a 7.25 ± 0.17a 6.16 ± 0.64b Traces Traces 27.37 ± 0.56a 27.27 ± 2.87b

Each value is the mean of two replicate samples.

Treatment means (± SEM) in the same row followed by the same superscripts are not significantly different (P N 0.05). ANOVA.
Traces (trace amount = less than 0.1% of the total fatty acids).
60 C. Estudillo-del Castillo et al. / Aquaculture 293 (2009) 57–61
and boost their essential fatty acid contents such as AA and DHA.
Significant uptake of DHA by starved rotifers (which initially does not
contain DHA) was evident after feeding them with freeze-dried
S. mangrovei. The successful uptake of DHA occurred even at the
lowest feeding concentration of 200 mg L− 1 and at a shorter
enrichment period of 5 h. The DHA contents were significantly
highest (33.65% TFA) for rotifers fed at a rate of 600 mg L− 1 for a
longer enrichment period of 10 h. Similarly, the highest uptake of AA
by the rotifers was also attained at the same feeding concentration
(600 mg L− 1) and enrichment period (10 h).
Barclay and Zeller (1996) reported that DHA contents of rotifers
fed with Schizochytrium sp. cells for 8 h increased significantly to
18.3% TFA. No DHA was detected in rotifers fed with brewer's yeasts
(control group). The feeding concentration used in their study was
slightly higher (140 mg 600 mL− 1; rotifer density = 400 individuals
mL− 1) compared to the highest feeding concentration (700 mg L− 1;
rotifer density = 1500 individuals mL− 1) used in the present study.
DHA levels obtained by enriched rotifers in this study appeared to be
higher compared to Barclay and Zeller (1996). This could be attributed
to the differences on the strain/species of Schizochytrium and
enrichment period used in this study. Feeding different strains of
Schizochytrium has different effects on the final HUFA contents of
B. plicatilis. Marked variations in the final HUFA composition of
enriched rotifers were achieved by changing the strains of Schizo-
chytrium with which the rotifers are enriched (Lewis et al., 1998).
Aside from the feeding concentration and the strains, the length of
time of feeding is also an important consideration in the successful
enrichment of rotifers. Feeding Schizochytrium sp. for a longer period
of 24 h (16 h longer compared to the standard feeding period of 8 h)
did not exhibit a further increase in the DHA contents of rotifers
(Barclay and Zeller, 1996). In the present study, feeding the rotifers
with freeze-dried S. mangrovei for a period of 10 h and at a rate of
600 mg L− 1 produced the best enrichment results with regard to
essential fatty acids such as AA and DHA. Consequently, the DHA as
well as the lipid and AA contents started to decrease significantly in
rotifers fed at the highest feeding concentration of 700 mg L− 1 for
10 h. In a similar study, the DHA content of B. plicatilis fed with
S. limacinum (OUC88) reduced sharply if the enrichment time was
longer than 12 h (Song et al., 2007). These results could indicate an
unfavorable environmental condition such as deterioration of the
water quality due to a high concentration of freeze-dried thrausto-
chytrid cells for the long enrichment period used. In this study, a foul
smell in the rotifer cultures was already observed during the
enrichment period of 10 h. Unfortunately, water quality parameters
were not measured in this study. Further studies on water quality are
needed to support this observation.
The present study demonstrated the importance of combining the
proper timing and amount of Schizochytrium cells to be used to obtain
the desired enrichment levels of lipid, AA and DHA for rotifers. Rotifers
enriched with high levels of lipids, especially HUFAs, can in turn be
used for improved and more successful larval rearing operations in
marine fish hatcheries. The reason behind the significant decrease in
the DPA contents at higher feeding concentrations of 500, 600 and
700 mg L− 1 is unknown and difficult to explain. It is, therefore,
recommended that studies be conducted for further investigation.
5. Summary and conclusion
The use of HUFA-rich microalgae/algal-like organisms to improve
the nutritional value of zooplankton such as rotifers in the first-
feeding process of many marine fish larvae may enhance their rearing
success in the hatcheries. The results of this study indicate that the
tropical thraustochytrid S. mangrovei (Isolate IAo-1) can be used as a
feed to effectively increase the HUFA contents of rotifers prior to
feeding to fish larvae in the hatchery. The strains/species of Schizo-
chytrium, feeding concentrations and the length of feeding are the
important considerations in the success of the enrichment of rotifers
with HUFA at a minimal cost. It is concluded that the feeding
concentration of 700 mg L− 1 at an enrichment period of 5 h is
optimum in the successful AA and DHA enrichment of rotifers,
although maximum uptake of these essential fatty acids in rotifers is
attained at feeding of 600 mg L− 1 after a 10 h enrichment period.
Feeding freeze-dried cells of S. mangrovei at the highest rate of 700 mg
L− l did not exhibit a further increase in the uptake of lipid, AA and
DHA in rotifers. The strategic combination of knowing the proper
amount of enrichment product and the length of the enrichment
period to use to boost the DHA contents of rotifers will effectively
ensure a reliable and cost-effective production of nutritionally
superior rotifers which will ultimately contribute to the success of
rearing marine fish larvae in the hatchery.
Acknowledgements
The authors thank Dr. Josephine Nocillado for her critical review of
the manuscript. This study is funded by SEAFDEC/AQD under the
Study Code: NR-05-F99T.
References
Apt, K.E., Behrens, P.W., 1999. Commercial developments in microalgal biotechnology.
J. Phycol. 35, 215–226.
Barclay, W.R., Zeller, S., 1996. Nutritional enhancement of n-3 and n-6 fatty acids in
rotifers and Artemia nauplii by feeding spray-dried Schizochytrium sp. J. World
Aquac. Soc. 27, 314–322.
Cavalier-Smith, T., Allsopp, M.T.E.P., Chao, E., 1994. Thraustochytrids are chromists, not
fungi: 18s rRNA signatures of Heterokonta. Philos. Trans. Lond. Biol. Sci. 346,
387–397.
Duray, M.N., Estudillo, C.B., Alpasan, L.G., 1996. The effect of background color and
rotifer density on rotifer intake, growth and survival of the grouper (Epinephelus
suillus) larvae. Aquaculture 146, 217–224.
Estudillo, C.B., 2001. Production of rotifer Brachionus rotundiformis for use in
aquaculture. Thesis. Master of Agricultural Science. University of Queensland,
School of Land and Food. QLD. Australia. 155 pp.
Fan, K.W., Vrijmoed, L.L.P., Jones, E.B.G., 2002. Physiological studies of subtropical
mangrove thraustochytrids. Bot. Mar. 45, 50–57.
Furuita, H., Takeuchi, T., Watanabe, T., Fujimoto, H., Sekiya, S., Imaizumi, K., 1996.
Requirements of larval yellowtail for eicosapentaenoic acid, docosahexaenoic acid,
and n-3 highly unsaturated fatty acid. Fish. Sci. 62, 372–379.
Furuita, H., Takeuchi, T., Umatso, K., 1998. Effects of eicosapentaenoic and docosahex-
aenoic acids on growth, survival and brain development of larval Japanese flounder
(Paralichthys olivaceus). Aquaculture 161, 269–279.
Kraul, S., 2006. Live food for marine fish larvae. In: Suarez, E.C., Marie, D.R., Salazar, M.T.,
Nieto Lopez, M.G., Villareal Cavazos, D.A., Puello Cruz, A.C., Ortega, A.G. (Eds.),
Advances en Nutricion Acuicola VIII, VIII Symposium Internacional de Nutricion
Acuicola. Universidad Autonoma de Nuevo leon, Monterrey, Nuevo Leon, Mexico.
15–17 Noviembre, 55–60 pp.
Laing, I., Verdugo, C.G., 1991. Nutritional value of spray-dried Tetraselmis suecica for
juvenile bivalves. Aquaculture 92, 207–218.
Leaño, E.M., Liao, I.C., 2004. Thraustochytrids: potential DHA source for marine fish
nutrition. Glob. Aquac. Advocate 2004, 87–88 (February).
Leaño, E.M., Liao, I.C., 2007. Thraustochytrids: ecological role and potential commercial
use. In: Reichardt, W. (Ed.), DAAD Conference Proceedings: Challenges of Applied
and Environmental Microbiology in Marine Science. Marine Science Institute,
University of the Philippines, Diliman, Quezon City, Philippines, pp. 39–42.
Leaño, E.M., Gapasin, R.S.J., Polohan, B., Vrijmoed, L.L.P., 2003. Growth and fatty acid
production of thraustochytrids from Panay mangroves, Philippines. Fungal Divers.
12, 111–122.
Lepage, G., Roy, C.C., 1984. Improved recovery of fatty acid through direct transester-
ification without prior extraction or purification. J. Lipid Res. 28, 1391–1396.
Lewis, T.E., Nichols, P.D., Hart, P.R., Nichols, D.S., McMeekin, T.A., 1998. Enrichment of
rotifers with eicosapentaenoic acid and docosahexaenoic acid produced by
bacteria. J. World Aquac. Soc. 29, 313–318.
Lewis, T.E., Nicholas, P.D., McMeekin, T.A., 1999. The biotechnological potential of
thraustochytrids. Mar. Biotechnol. 1, 580–587.
Li, Z.Y., Ward, O.P., 1994. Production of docosahexaenoic acid by Thraustochytrium
roseum. J. Ind. Microbiol. 13, 138–141.
Loung-Van, T., Renaud, S.M., Parry, D.L., 1999. Evaluation of recently isolated Australian
tropical microalgae for the enrichment of the dietary value of brine shrimp, Artemia
nauplii. Aquaculture 170, 161–173.
Metcalfe, L.D., Schmitz, A.A., 1961. The rapid preparation of fatty acid methyl esters for
gas chromatography analysis. Anal. Chem. 33, 363–364.
Parfrey, L.W., Barbero, E., Lasser, E., Dunthorn, M., Bhattacharya, D., Patterson, D.J., Katz,
L.A., 2006. Evaluating support for the current classification of eukaryotic diversity.
PLOS Genetics 2, e220.
 C. Estudillo-del Castillo et al. / Aquaculture 293 (2009) 57–61 61

Pousao-Ferreira, P., Dores, E., Morais, S., Narciso, L., 2001. Lipid requirements of the
white seabream (Diplodus sargus Linnaeus, 1758) larvae. In: Henry, C.I., Van
Stappen, G., Wille, M., Sorgeloos, P. (Eds.), Larvi '01 — Fish and Shellfish Larviculture
Symposium. European Aquaculture Society, Special Publication, vol. 30, pp.
482–485.
Sargent, J.R., McEvoy, L.A., Bell, L.J., 1997. Requirements, presentation and sources of
polyunsaturated fatty acids in marine fish larval feeds. Aquaculture 155, 117–127.
Song, X., Zhang, X., Guo, N., Zhu, L., Kuang, C., 2007. Assessment of marine
thraustochytrid Schizochytrium limacinum OUC88 for mariculture by enriched
feeds. Fish. Sci. 73, 565–573.
Yaguchi, T., Tanaka, S., Yokochi, T., Nakahara, T., Higashuhara, T., 1997. Production of high
yields of docosahexaenoic acid by Schizochytrium sp. JAOCS 74, 1431–1434.