Aquaculture 308 (2010) 124–131

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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

Preserved copepods as a new technology for the marine ornamental fish aquaculture:
A feeding study
I. Olivotto a,⁎,1, N.E. Tokle b, V. Nozzi a, L. Cossignani c, O. Carnevali a,1
a
Dipartimento di Scienze del Mare, Università Politecnica delle Marche, via Brecce Bianche, 60131 Ancona, Italy
b
Planktonic AS, 8850 Herøy, Norway
c
Università degli Studi di Perugia, Dip. Scienze Economico-Estimative e degli Alimenti, Sez. Chimica Bromatologica, Biochimica, Fisiologia e Nutrizione, Perugia, Italy

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

Article history: The aim of this study was to evaluate the potential use of preserved copepod as prey in Amphiprion clarkii
Received 27 April 2010 larviculture. After hatching, A. clarkii larvae were divided in three experimental groups for feeding studies as
Received in revised form 25 August 2010 follows: group A (control group) fed rotifers (Brachionus plicatilis) followed by Artemia nauplii; group B fed a
Accepted 31 August 2010
mixed diet of rotifers-Artemia salina nauplii and preserved copepods and group C fed preserved copepods
solely. In this study we observed a positive effect of feeding preserved copepods in A. clarkii larviculture as a
Keywords:
Marine ornamentals
supplement food to the traditional diet based on rotifers and Artemia nauplii. In group B larvae, fed a
Copepods combination of rotifers/Artemia and copepods, a significant increase of insulin like growth factor I and II,
Rotifers peroxisome proliferator activated receptor α−β and thyroid receptor α and β gene expression together with
Artemia a significant decrease of myostatin gene expression was evidenced by real time PCR compared to the other
Amphiprion clarkii experimental groups. In this same group we also observed the best results in terms of growth (total length
and weight) and survival. These preserved copepods may be considered a suitable food for marine fish larvae
larviculture when used as a supplement to the traditional diet based on rotifers and Artemia nauplii.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
The main critical bottleneck that scientists have to face in rearing
fish larvae is the transition from an endogenous to an exogenous
feeding by the larvae. Most of the commercial species are reared
using rotifers (B. plicatilis, B. rotundifornis) and Artemia nauplii
since they can be cultured in large quantities at high densities.
Unfortunately, using rotifers and Artemia during this early period in
life history does not always promote optimal larval growth since
these live prey may contain an inadequate fatty acid profile and, in
some cases, be of an inappropriate size (Kahan, 1981; Sargent et al.,
1999; Holt, 2003; Olivotto et al., 2003; Faulk and Holt, 2005). Because
of this, there is a need for identification of alternative food sources
that do not have these inadequacies and can promote satisfactory
growth (Sun and Fleeger, 1995). It is well established that copepods,
copepodites and nauplii are feeding items preferred by fish larvae
and when used as live prey (solely or in combination with rotifers
and Artemia nauplii), they usually dominate the gut content of the
⁎ Corresponding author. Oce.AN, via Brecce Bianche, 60131 Ancona, Italy. Tel.: + 39
0712204643; fax: + 39 071 2204650.
E-mail address: i.olivotto@univpm.it (I. Olivotto).
1
Oce.AN, Dipartimento di Scienze del Mare, via Brecce Bianche, 60131 Ancona, Italy.
larvae (Holt, 2003). Copepods show a wide range of body size
between nauplii and adults, typical movement that stimulate the
predatory activity of the larvae, and they also show a high content of
highly unsaturated fatty acids (HUFAs) (Delbare et al., 1996). These
fatty acids, in particular eicosapentaenoic acid (EPA, 20:5n-3) and
docosahexaenoic acid (DHA, 22:6n-3), are extremely important for
larval fish survival and growth and several studies have demonstrat-
ed that they are essential in larval diets (Sargent et al., 1999).
Deficiencies in these fatty acids can cause a general decrease of larval
health, poor growth, low feed efficiency, anaemia and high mortality
(Sargent et al., 1999; Bell et al., 2003; Olivotto et al., 2003, 2005,
2006b; Faulk and Holt, 2005;).Moreover, the retention time of
copepods in the gut of fish is much higher than for the Artemia which
passes through the digestive system very fast. This quality of
copepods ensures that they are better digested and therefore offer
more potential for nutrient uptake to the fish than Artemia (Pedersen
et al., 1989).
For these reasons and with the increasing worldwide interest in
aquaculture, copepods may be considered as a valid and alternative food
source for the culture of many larval fish. The use of cultured copepods
in intensive fish larviculture (Pedersen et al., 1989; Van der Meeren
and Naas, 1997; Papandroulakis et al., 2005) has involved calanoids such
as Acartia spp. (Schipp et al., 1999), Eurytemora spp. (Shields et al.,
1999), Parvocalanus spp. (Olivotto et al., 2006a), Centropages typicus

0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.08.033
 I. Olivotto et al. / Aquaculture 308 (2010) 124–131
(Olivotto et al., 2008c, 2009) and harpacticoid copepods such as
Euterpina acutifrons , Tisbe spp. (Kahan et al., 1982; Støttrup and Norsker,
1997; Olivotto et al., 2008a,b) and Tigriopus japonicus (Fukusho, 1980).
The best results have been obtained using calanoid copepods which
have a higher content of HUFAs, are entirely pelagic and thus more
available as prey for marine fish larvae, and usually have very small
naupliar stages which are more readily captured by fish larvae with
small gapes at first feeding (Payne and Rippingale, 2001; Olivotto et al.,
2008a,b; Olivotto et al., 2009). Unfortunately, there are several
difficulties in culturing calanoid copepods on a continuous basis, since
they are usually cultured at very low densities, in large tanks, and need
to be fed different algal combinations (Holt, 2003).
Alternatives may be represented by harvesting live copepods from
natural habitats: these productions are highly dependent on natural
meteorological cycles which limit the annual production of fish in
addition to be potentially affected by parasite that use copepods as an
intermediate host. Prey selection in fish larvae is affected by a great
number of factors related to the characteristics of both the larvae and
the prey. These factors include visual acuity (Neave, 1984), visual
threshold and spectral sensitivity (Drost, 1987), prey contrast and size
(Dendrinos et al., 1984), shape, mobility (Holmes and Gibson, 1986),
and concentration and by the presence of chemical stimulants
(Knutsen, 1992).
The aim of the present study was to test the efficiency of preserved
copepods in Amphiprion clarkii larviculture as a possible alternative
live prey to rotifers Artemia nauplii or alive copepods. Different
feeding combinations and different sizes of preserved copepods have
been tested in order to verify the efficiency of this innovative larval
food on A. clarkii larviculture.
In this study, results obtained from morphometric analysis and live
prey fatty acid composition are supported by a molecular approach. In
particular, genes related to growth such as insulin like growth factor I
and II and myostatin-(IGFI, II, MSTN), to lipid metabolism such as
peroxisome activated proliferator receptors (PPAR-α, -β) and to
metamorphosis (thyroid hormone receptor-α, -β (TR α, β) have been
analyzed through real time PCR. Our results showed that preserved
copepods maintained their nutritional characteristics and may be
easily stored for a long time at −80 °C to be used upon request, and
may improve larval fish survival and growth.
2. Material and methods
2.1. Animals and rearing system
Sexually mature fish (A. clarkii), measuring approximately 5–
10 cm, were bought from a pet shop (Fauna Esotica, Civitanova
Marche, Italy) in February 2005. The pairs were held in 200 L cubic
breeding tanks in a closed recirculating system equipped with
biological, UV and mechanical filtration (Panaque srl, Capranica,
Italy).
The temperature in the breeding tanks was maintained at 28±
0.5 °C, salinity 30‰, pH 8–8.2 and NO2 and NH3 b 0.03 mg/L. A
photoperiod of 14 h light/10 h dark was provided by two 30 W
incandescent lights suspended 20 cm above the water surface. Burned
clay flowerpots (20 cm diameter) were placed in the tank as a substrate
where the fish could spawn. The fish were fed twice a day using frozen
adult Artemia, frozen plankton and chopped fish and shrimp.
Under these conditions, the fish started spawning 1 year after
they were put in the broodstock tanks. Spawning occurred every
12 days and the egg clutches, each with about 350–400 embryos,
were left to parental care for embryo development. Hatching took
place at 28 °C, 144 h post fertilization. An hour before hatching, the
flowerpot with the egg clutch was transferred to 20 L larval rearing
tanks with the same chemical–physical characteristics of the
breeding tanks. An air stone was gently put into the pot and
the egg clutch was left in darkness for about 50 min. After this
125
period synchronous hatching took place and the average percent
hatch for the different clutches considered for the feeding studies
was 96 ± 3%.
The water in the 20 L larval tank was gently replaced 10 times a
day by a dripping system. The sides of the tank were covered with
black panels to reduce light reflection, while the phytoplankton
Nannochloropsis oculata was used (50,000 cells/mL) to condition the
tanks from the first day after hatching.
2.2. Zooplankton culturing
Rotifers (Brachionus plicatilis) characterized by an average size of
239 μm were cultured on N. oculata (50,000 cells/mL) in 100 L tanks
(salinity 30‰, pH 8.2, NO2 and NH3 b 0.03 mg/L) and subjected to
constant light. Each day, the necessary amount of rotifers was gently
transferred to 50 L cone-shaped tanks for enrichment. Enrichment
was performed with Algamac 3000 (Aquafauna Bio-Marine, Inc.,
Hawthorne, CA USA) using 0.5 g/million rotifers. Algamac 3000 was
homogenized in 500 mL salt water for 1 min and then distributed to
the rotifers for enrichment. As recommended by the company,
enrichment lasted for 8 h.
AF 430 Artemia salina cysts (Inve Technologies, Belgium) were
incubated and hatched following INVE instructions. The cysts were
hatched in cone-shaped tanks, in artificial seawater (Prodac Int.,
Cittadella, Italy) (salinity 30‰, pH 8.2, NO2 and NH3 b 0.03 mg/L) at a
density of 1–1.5 g cysts/L of water. The temperature in the hatching
thanks was maintained at 25 °C and the photoperiod consisted of
constant light at 2000 lux. The aeration was vigorous to keep all cysts in
suspension throughout the hatching period and dissolved oxygen was
maintained greater than 5 mg/L. Under these conditions, the cysts
hatched in 24 h. After hatching, the nauplii were separated from the
cysts by siphoning and enrichment was performed with Algamac 3000
(0.2 g/100,000 Artemia nauplii) in 10 L buckets filled with filtered
seawater at a concentration of 200 nauplii mL−1 for 8 h at 25 °C under
continuous aeration and illumination.
Before feeding to the larvae, rotifers and Artemia nauplii were
concentrated on a 30 μm mesh and rinsed 10 times with clean seawater
(salinity 30‰) in order to remove the remaining enrichment.
A mixture of Copepodites (CI–CIII) and nauplii (NIII–NVI) of the
copepods Temora longicornis (76%), Acartia clausi (12%), Centropages
hamatus (7%) and others (5%; Pseudocalanus spp., Microcalanus spp.
and Oithona similis) were harvested outside Herøy in Northern
Norway during spring 2009. The zooplankton were harvested using
horizontally towed plankton net (10 m2 mouth opening, 150 μm
mesh size) at 1 knot speed for 6 h at 1 m depth. After landing of the
catch, the zooplankton was screened on a 400 μm plankton net to
remove large copepods. The size fraction 150–400 μm was thereafter
further separated into size fractions 2–300 and 3–400 by sieving using
a nylon filter mesh. 2–300 and 3–400 μm size fractions represents the
width of the copepodites and nauplii, and their average length were
approximately twice the width. The size fractions consisting of
copepodites and nauplii were processed using a patent pending
method, and thereafter packed in 20 ml plastic vials (polyethylene)
and immediately frozen using liquid N2. Number of copepodites/
nauplii in the 2–300 and 3–400 μm size fractions were approximately
600 000 and 200 000 per gram, respectively. The preserved
copepodites/nauplia were stored in a −80 °C freezer. Analysis showed
a percentage dry weight (DW) of 10.5 ± 0.2 (SD) of the frozen
copepodites/nauplii. Immediately after thawing of the copepodites/
nauplii, the leaking of water soluble proteins were 1.5 ± 0.9 (mg/g wet
weight). Leaking of water soluble proteins increased to 4.3 ± 0.1 (mg/g
wet weight) when copepodites/nauplia were kept in sea water (12 °C)
for 4 h.
The right amount of preserved copepodites/nauplii was defrozen
before feeding the larvae in order to obtain a final concentration in
the larval tank of 5 ind/mL.
126 I. Olivotto et al. / Aquaculture 308 (2010) 124–131
2.3. Experimental design
Immediately after hatching, A. clarkii larvae were divided into 3
experimental groups (200 ± 5 larvae per group, with three replicates
each) as follows:
Group A (control): fed Algamac 3000 enriched rotifers (Brachionus
plicatilis) (10 ind/mL) from day 1 to day 7 post hatching (ph) followed
by Algamac 3000 enriched AF430 Artemia nauplii (6 ind/mL)
introduced from day 7 ph till the end of the experiment.
Group B: fed Algamac 3000 enriched rotifers (5 ind/mL) and
preserved copepodites/nauplii (5 ind/mL) (200–300 μm) from day 1
to day 7 ph, followed by Algamac 3000 enriched AF430 Artemia
nauplii (3 ind/mL) and 300–400 μm preserved copepodites/nauplii
(3 ind/mL) introduced from day 7 ph to metamorphosis.
Group C: preserved copepodites/nauplii (10 ind/mL) (200–
300 μm) from day 1 to day 7 ph, followed by 300–400 μm preserved
copepodites/nauplii (6 ind/mL) introduced from day 7 ph to
metamorphosis.
The feeding experiments were conducted in 20 L cubic larval tanks
(Olivotto et al., 2003, 2005, 2008a) connected to the broodstock tank
system and the phytoplankton N. oculata (introduced at 08.00, 12.00,
16.00, 20.00 h) was used (50,000 cells/mL) to condition the tanks
from day 1 to day 7 ph (Olivotto et al., 2003).
Each day at 08.00 and 18.00 h, dead A. clarkii larvae were siphoned
and counted in order to estimate the survival in the different
experimental groups.
2.4. Sampling of larvae
Samples of A. clarkii larvae (20 ± 1, in three replicates), for each
experimental group, were collected 6 and 10 days ph. The experiment
terminated on day 15 ph since A. clarkii undergo metamorphosis
around day 12–13 ph. All samplings were performed at 09.00 h, prior
to feeding the larvae. Ten larvae in triplicate were used for
morphological measurements (total length and wet weight) using a
Stemi 2000 micrometric microscope and a microbalance (OHAUS
Explorer E11140 accurate to 0.1 mg); the remaining larvae were
stored at −80 °C for molecular analysis.
2.5. Fatty acid analysis
Total lipids of rotifers, Artemia salina and the 200–300 μm / 300–
400 μm preserved copepods were extracted with chloroform/meth-
anol (2:1 v/v). Methanolic transesterification of lipids was carried out
using 2 N methanolic KOH and hexane; the fatty acid methyl esters
(FAME) obtained were analyzed by high resolution gas chromatog-
raphy (HRGC). A DANI 1000DPC gas-chromatograph (Norwalk, CT,
USA), equipped with a split–splitless injector and a flame ionization
detector (FID), was used. The separation was obtained using a fused
silica WCOT capillary column CP-Select CB for FAME (50 m x 0.25 mm
i.d., 0.25 μm f.t.; Varian, Superchrom, Milan, Italy). The chromato-
grams were acquired and processed using Clarity integration software
(DataApex Ltd., Prague, Czech Republic). The oven temperature was
held at 180 °C for 6 min, then was programmed at 3 °C/min to 225 °C
and held for 10 min, while the injector and the FID temperatures were
set at 250 °C. Carrier gas (He) flow rate was 1 ml/min. FAs were
identified by comparing the retention times of their methyl esters
with standard FAME mixtures. Repeated injections of standard
solutions were carried out to test the analytical precision. The relative
standard deviations were less than 5% for all the FA, both considering
the intradie precision, calculated on six repeated injections, and the
interdie precision, evaluated over 6 days. All solvents and reagents
were of analytical grade and were purchased from Carlo Erba
Reagents (Milan, Italy). FAME standards were obtained from Supelco
(Bellefonte, PA).
2.6. Molecular analysis
2.6.1. RNA extraction and cDNA synthesis
Total RNA was extracted from the larvae using a Minikit RNAeasy®
Qiagen extraction kit following the manufacturer's protocol. Final RNA
concentrations were determined by optical density measurement at
260 nm, and the RNA integrity was verified by ethidium bromide
staining of 28 S and 18 S ribosomal RNA bands on 1% agarose gel. Total
RNA was treated with DNAse (10 UI at 37 °C for 10 min, MBI
Fermentas); a total amount of 1 mg of RNA was used for cDNA
synthesis, employing iScript cDNA Synthesis Kit (Bio-Rad).
2.6.2. Primer design
The following primers were used at final concentration of
10 pmol/μL:
β-ACT: For: 5′-TTCCTCGATATGGAGTCCT-3′; Rev: 5′- TGGGGCAAT
GATCTTGATCTT-3′.
IGF I: For: 5′-AGTGCGATGTGCTGTATC-3′; Rev: 5′- CAGCTCA
CAGCTTTGGAAGCA-3′.
IGF II: For: 5′- CGGCAGAAACGCTATGTGGA-3′; Rev: 5′TGCTG
GTTGGCCTACTGAAA -3′.
MSTN: For: 5′-TTTTGAGCAAACTGCGAATG-3′; Rev: 5′- CACGTCGT
ACTGGTCGAGAA-3′.
PPARα: For: 5′-TTC AGC GAC ATG ATG GAG CC-3′; Rev: 5′-CAG TTT
CTG CAG CAG ATT GG-3′
PPARβ: For: 5′-AGG AGA TAG GGG TAC ACG TG -3′; Rev: 5′-CAG
GAA CTC CCG GGT CAC AA -3’.
TRα: For: 5′-GGAAACAGAAGCGCAAGTTC-3′; Rev: 5′-TCTTCA
CAAGGCAGCTCTGA-3′.
TRβ: For: 5′-TGCATGGAAGATCATGTCG-3′; Rev: 5′-AGGCC
TGGCTTCTTAAGCTG-3′.
2.6.3. Real time PCR
Triplicate PCRs were carried out for each sample analysed. After
real time conditions optimization (primers annealing temperatures
and cDNA dilutions) the PCRs were performed with the SYBR green
method in a Chromo 4 Real-Time system (M J Research). The reactions
were set up in a 96-well plate by mixing, for each sample, 1 μL of
diluted (1/20) cDNA, 5 μL of 2× concentrated SYBR Green PCR Master
Mix (Finnzymes) containing SYBR Green as fluorescent intercalating
agent, 0.3 μM forward primer and 0.3 μM of reverse primer. The
thermal profile for all reactions was 15 min 95 °C and then 45 cycles of
20 s at 95 °C, and 20 s at 55 °C and 20 s at 72 °C except for PPARα
which thermal profile was 15 min 95 °C and then 45 cycles of 20 s at
95 °C, and 20 s at 51 °C and 20 s at 72 °C and for PPARβ which thermal
profile was 15 min 95 °C and then 45 cycles of 20 s at 95 °C, and 20 s at
49 °C and 20 s at 72 °C. Fluorescence monitoring occurred at the end
of each cycle. Additional dissociation curve analysis was performed
and showed, in all cases, one single peak. A relative quantification of
cDNA was made using β-ACT as a reference gene (internal standard).
The data obtained were analyzed using the iQ5 optical system
software version 2.0 (Bio-rad). Modification of gene expression is
represented with respect to the control.
2.7. Data analysis
The real-time data were analyzed using the iQ5 optical system
software version 2.0 (Bio-Rad). Modifications of gene expression are
represented compared to a control sampled at the same time of the
treatment, which is assumed to have the value of 1.
Morphological, survival data and fatty acid analysis are expressed
as means ± SD and were analyzed by two-way ANOVA followed by
Tukey's test. The level for accepted statistical significance was P b 0.05.
 I. Olivotto et al. / Aquaculture 308 (2010) 124–131 127

3. Results Table 1
Percentage of some fatty acids in rotifers, Artemia salina nauplii enriched with Algamac
3000® and in 200–300/400–300 mm fractions preserved copepods.
3.1. Survival percent
% relative total Rotifers + Artemia nauplii + Copepods Copepods
On day 15 ph, group A larvae, fed rotifers followed by Artemia free fatty acid Algamac 3000 Algamac3000 200–300 μm 300–400 μm

nauplii, had a 41 ± 5% survival, while group B larvae, fed a combination EPA 6.90 ± 1.6 7.4 ± 0.7 22.4 ± 1.3 13.7 ± 0.3
of rotifers/copepodites/nauplii and Artemia nauplii/copepodites/nauplii, DHA 12.8 ± 0.8 9.2 ± 0.9 19.3 ± 1.3 27.3 ± 1.8
%ω3 19.6 ± 1.7 18.7 ± 1.1 43.9 ± 1.1 46.0 ± 1.2
showed a 62 ± 2% survival (Fig. 1). Group C larvae, fed solely on
copepodites/nauplii, did not survive past day 6 ph (Fig. 1). Significant Fatty acid content was based on HRGC data and expressed as relative area percentage
differences in survival between these groups were evident from day on total fatty acid area measured by peak integration. The best fatty acid profiles are
preserved copepods respect to rotifers and Artemia salina.
3 ph. A combined diet was able to accelerate development in A. clarkii
larvae, since in group B, clownfish larvae started metamorphosis 3 days
(day 9 vs. day 12 in controls) before the larvae from the control group.
3.4. Molecular analysis

3.2. Morphometric results
On day 6 ph significant differences (P b 0.05), either in terms of TL or
body weight, were observed among groups A, B and C (4.61 ± 0.2 mm;
5.25 ± 0.20 mm; 4.35 ± 0.40 mm; 1.11 ± 0.2 mg, 1.72 ± 0.3 mg, 0.64 ±
0.25 mg, respectively). On day 10 ph, TL and body weight (7.88 ±
0.5 mm, 7.91 ± 0.8 mg) of group B larvae, fed on rotifers and preserved
copepodites/nauplii, were significantly higher (P b 0.05) than group A
larvae fed a standard rotifers–Artemia diet (6.1 ± 0.3 mm; 3.88 ±
0.6 mg).
Group C larvae were not considered at this stage since all the
larvae died on 7 day ph.
The expressions of the different genes herein analyzed, were
evaluated by real time PCR and a partial nucleotide sequence was
obtained for each of them and deposited in GeneBank. [PPAR-α
(FJ713638); PPAR-β (FJ713639); β-ACT (FJ713637) Tr-α
(HM041220), Tr-β (HM041221)].
On day 6 and 10 ph, a significant increase (p b 0.05) due to a
positive effect of copepods feeding on IGFI and II, PPAR α−β and Tr α
and β gene expression (compared to control group A) was evidenced
in group B larvae fed a mixed standard/preserved copepodites/nauplii
diet (group B) (Figs. 2A, B; 3A, B; 4A, B; 5A, B; 6A, B; 7A, B;). About
group C larvae (6 days ph), fed on preserved copepodites/nauplii
solely, only a significant increase in PPAR α−β gene expression was

3.3. Lipid analysis

Table 1 reports the percentage DHA and EPA in the different diets,
based on HRGC data and expressed as the relative area percentage on
total fatty acids. In rotifers and Artemia nauplii, both enriched with
Algamac 3000®, the relative percentage of EPA and DHA ranged
between 6.9% and 12.8% with respect to the other fatty acids, the
percentage of total ω3 was less than 20% (Table 1). On the contrary, in
preserved copepods, the DHA content was 19.3% in the 200–300 μm
fraction and 27.3% in the 300–400 μm one. EPA was 22.4% in the 200–
300 μm fraction and 13.7 in the 300–400 μm one. Furthermore, in
preserved copepods the percentage of ω3 fatty acids was very high
(ranging from 43.9 ± 1.1 to 46.0 ± 1.2) with respect to the one
observed in rotifers and Artemia nauplii (19.6 ± 1.7 and 18.7 ± 1.1,
respectively) (Table 1).

Fig. 2. (A and B) IGF I gene expression in A. clarkii larvae fed different live prey at 6 and
Fig. 1. A. clarkii survival % in groups A, B, and C fed the different diets. 10 days ph. Values with different letters indicate statistical significance (P b 0.05).
128 I. Olivotto et al. / Aquaculture 308 (2010) 124–131
Fig. 3. (A and B) IGF II gene expression in A. clarkii larvae fed different live prey at 6 and
10 days ph. Values with different letters indicate statistical significance (P b 0.05).
observed compared to control (group A) (Figs. 4A, 5A). About MSTN
significant (P b 0.05) differences between group A and B larvae were
evident only 10 days ph with a significant decrease in group B larvae
respect to control (Fig. 8A, B) while group C larvae showed a
significant increase of its gene expression during the 6 days ph
sampling (Fig. 8A). Group C larvae were not considered during the
10 days ph sampling since this experimental group did not survived
past day 7 ph.
4. Discussion
A wide range of factors are acknowledged to influence larval prey
selection including prey characteristics such as size, density and
motion (O'Brien, 1979); larval characteristics such as sensory
capabilities, prior experience, motor ability, mouth dimensions
(mouth gape) and body size (Godin, 1978, Cox and Pankhurst,
2000); and environmental factors such as light intensity and
spectrum, water turbidity and temperature (Furnass, 1979, Cobcroft
et al., 2001). Much literature concentrates on the relationship
between prey size and mouth size as the primary determinant of
prey selection (Cunha and Planas, 1999), with prey width rather than
length being the prey dimension that appears to limit the size of prey
ingested by larvae ( Fernandez-Diaz et al., 1994) but other factors
such as colour, smell and movement are important attributes in prey
selection as well. An understanding of the factors (besides prey size)
which are involved in live prey selection and consumption is essential
Fig. 4. (A and B) PPAR α gene expression in A. clarkii larvae fed different live prey at 6
and 10 days ph. Values with different letters indicate statistical significance (P b 0.05).
for effective feed management in intensive aquaculture of marine fish
larvae; an industry still largely based upon live feed rather than
preserved/artificial diets ( Planas and Cunha, 1999).
In the present study we tested the efficiency of a new technology
able to produce preserved copepods that maintain, through the time,
their lipid composition on A. clarkii larval survival and growth. In
particular, analyzing the morphometric results, we demonstrated that
live prey motion is an essential attribute in stimulating larval
predatory activity and the best results, in terms of survival and
growth, were achieved when the preserved copepods were admin-
istered together with live preys (rotifers and Artemia). In fact group C
larvae, fed on copepods solely, did not survive longer than day 7 ph.
This is probably due to the fact that even if there was a slight
turbulence in the larval tank, most of the copepod nauplii sunk to the
tank bottom in a short time. In this way, live preys were not more
available for larval predation and larvae were not able to eat enough
copepods to reach metamorphosis. A similar result was recently
obtained in a study using Tisbe spp. nauplii (Olivotto et al., 2008a,b).
Since the naupliar phase of this species was predominantly found on
the tank walls rather than in the water column, these live preys were
less available to the fish larvae for predation than the pelagic rotifers,
causing lower survival rates (Olivotto et al., 2008a,b). The present
study demonstrated that the best results in terms of growth and
survival were achieved when a combined diet is used. Larvae fed this
diet were able to eat a part of the preserved copepods (they were
found in the gut contents) before that they sunk and were able to feed
on rotifers, after that copepods were no more available. In this way,
group B larvae were able to take advantage of copepod's biochemical
 I. Olivotto et al. / Aquaculture 308 (2010) 124–131
Fig. 5. (A and B) PPAR β gene expression in A. clarkii larvae fed different live prey at 6
and 10 days ph. Values with different letters indicate statistical significance (P b 0.05).
composition that is known to match the nutritional requirements of
fish larvae (Bell et al., 2003; Delbare et al., 1996; Olivotto et al., 2006a,
2006b, 2009). Copepods (as demonstrated from the fatty acid analysis
performed in this study) are particularly rich in HUFAs with these
molecules being essential in larval diets (Sargent et al., 1999) since
deficiencies may result in a general decrease of animal health, causing
anomalies not only in the visual and central nervous system (CNS)
development (Sargent et al., 1999) but also in growth (Furuita et al.,
1999; Copeman et al., 2002), in stress tolerance (Vagelli, 2004), in
pigmentation (McEvoy et al., 1998; Estévez et al., 1999; Copeman et
al., 2002; Bell et al., 2003) in swimming and feeding activity (Benìtez-
Santana et al., 2007) and in the immune system (Izquierdo, 1996).
In the present study, morphometric data were fully supported by
the molecular ones as well. It is well known that fish growth is
controlled by the complex processes of myogenesis which are
regulated by several extrinsic regulators such as the growth factors
insulin-like growth factor-I and II and myostatin. IGFs are considered
positive signals in the complex processes of myogenesis, while MSTN,
a member of the transforming growth factor-β (TGF- β) family, is
considered as a negative factor that inhibits myoblast proliferation
(McPherron et al., 1997). Final growth is thus determined by the
interplay of these positive and negative signals. In this study a positive
effect of feeding preserved copepods was evidenced since the best
results in terms of IGF I and IGF II gene expression improvement and
MSTN decrease were detected in group B larvae fed a combined diet of
rotifers/Artemia/copepods. This may be explained through the role of
copepods as a supplemental source of fatty acids for marine fish
larvae. At this regard, recent studies showed a positive role of HUFAs
129
Fig. 6. (A and B) Tr α gene expression in A. clarkii larvae fed different live prey at 6 and
10 days ph. Values with different letters indicate statistical significance (P b 0.05).
administration on larval fish growth (Avella et al., 2007; Olivotto et al.,
2008a,b; 2009). HUFAs are able to act directly on the genome, via
specific nuclear receptors, the peroxisome proliferator activated
receptors (PPAR-α, -β, and γ), transcription factors belonging to the
ligand-activated nuclear hormone receptor super-family. These
nuclear receptors are involved in several biological processes such
as skeletal development during ontogenesis, adipogenesis, lipid
homeostasis, lipid metabolism regulation, lipid transport, lipid and
glucose oxidation, peroxisomal biogenesis, immune functions, cell
proliferation and epithelial cell differentiation (Burdick et al. 2006)
and are thus considered good fatty acid sensors (Grimaldi, 2007). The
PPAR-α, -β up-regulation observed in group B larvae may thus been
explained through the higher HUFAs content detected in the copepod
samples with respect to rotifers and Artemia ones. Finally, the time to
metamorphosis was also significantly reduced by copepod adminis-
tration. The importance of HUFAs for a correct development and
functionality of the central nervous system (CNS) is well established
(Vaidyanathan et al., 1994, Sargent et al., 1997) and we can assume
that the improvement of the larval diet with a supplemental amount
of HUFAs derived from copepods may result in an adequate
development of the CNS and hence, in a fully functional hypothala-
mus–pituitary–thyroid axis which has a key role in metamorphosis
regulation. This was supported by the higher TR α and β gene
expression in group B with respect to group A and B larvae and by the
earlier metamorphosis observed in these larvae with respect to
control. This hypothesis is supported by the fact that TRs mRNA levels
are directly modulated by thyroid hormone concentrations as already
demonstrated in several fish species where significantly higher levels
130 I. Olivotto et al. / Aquaculture 308 (2010) 124–131
Fig. 7. (A and B) Tr β gene expression in A. clarkii larvae fed different live prey at 6 and
10 days ph. Values with different letters indicate statistical significance (P b 0.05).
of TRs have been evaluated during the metamorphosis event, with the
highest levels simultaneously occurring with metamorphosis climax
(Galay-Burgos et al., 2008).
5. Conclusion
Optimal prey size during development is of great value for
cultured species, particularly at the very early stages when the
availability of adequate prey determines survival and growth of
cultivated larvae. In this study we demonstrated that during these
early periods, a crucial role is played by live prey motion and their
biochemical characteristics as well. In fact, static live preys represent a
limiting factor in larval rearing that in turn negatively affects growth
and survival. On the other hand, we demonstrated that the new
technology here presented is able to preserve copepods maintaining
their precious fatty acid characteristics. For this important reason they
can be considered as a valid source of HUFAs in aquaculture improving
the expression of important genes involved in larval growth, lipid
metabolism and metamorphosis. As a consequence, larval growth can
be faster, survival better, and larval phase shorter.
Acknowledgements
Special thanks to Beatrice Migliarini, Gioacchini Giorgia, Chiara
Piccinetti for their help at various stages of this study. Funding for this
study was provided by the “Fondi di Ateneo 2009” to Ike Olivotto and
Oliana Carnevali, and Oce.AN soc.coop. Finally, thanks to our
government for not supporting scientific research.
Fig. 8. (A and B) MSTN gene expression in A. clarkii larvae fed different live prey at 6 and
10 days ph. Values with different letters indicate statistical significance (P b 0.05).
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