Comparative Biochemistry and Physiology, Part A 210 (2017) 1–6

Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A

journal homepage: www.elsevier.com/locate/cbpa

Changes in free amino acid content during naupliar development of the MARK
Calanoid copepod Acartia tonsa
Thomas Allan Raynera,b,⁎, Niels Ole Gerslev Jørgensenc, Guillaume Drilletd,
Benni Winding Hansena
a
Department of Science and Environment, Roskilde University, DK-4000 Roskilde, Denmark
b
Institute of Marine Biology, National Taiwan Ocean University, Keelung 20224, Taiwan
c
Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark
d
DHI Water & Environment (S) Pte Ltd, 1 Cleantech Loop, #03-05 CleanTech One, 637141, Singapore

A R T I C L E I N F O A B S T R A C T

Keywords: Changes in free amino acids (FAA) were investigated in the potentially important live feed and neritic copepod
Free amino acids species Acartia tonsa during naupliar development. Total content of FAA in A. tonsa nauplii was around 17% of
Calanoid nauplii dry weight at first development stage, and declined to 6% for later stages. Relative to body-volume and biomass,
Acartia tonsa the FAA content indicated possible volume-dependent changes. However, changes in FAA with osmolytic
Live feed
activity could not account for this decline in FAA content, but suggests that the decline reflected degradation of
Glutamic acid
Body volume
residual FAAs from the embryonic stage. Glutamic acid revealed the largest change in relative abundance during
naupliar development and declined from 29.0% at first nauplius stage to 7.1% at later stages. The high FAA pool
in early naupliar stages may be necessary for naupliar development due to an absence of feeding at first
development stages. The high FAA content in early nauplii indicates that A. tonsa is a valuable source for
nutritional energy for first-feeding fish larvae and should be further exploited for aquaculture purposes.
Enhancements to FAA abundances in nauplii through manipulation of maternal diets could be of future interest,
as copepod nauplii can contain a substantial pool of FAAs at first development stage.

1. Introduction
Copepods as live feed for first-feeding marine fish larvae have
demonstrated beneficial results in terms of fish larvae survival and
growth in comparison to other more conventional live feed subjects
such as rotifers and Artemia nauplii (Shields et al., 1999; Toledo et al.,
1999; Payne et al., 2001; Wilcox et al., 2006). At first-feeding, marine
fish larvae have a preferred prey size range of which copepod nauplii
typically fall within (Pepin and Penney, 1997; Engell-Sørensen et al.,
2004), and even small-mouthed fish larvae like the red grouper
(Epinephelus coioides) have a stronger preference towards copepod
nauplii than rotifers in mixed copepod/rotifer diet scenarios (Toledo
et al., 1997). Once consumed, the fish larvae will benefit from the
copepod's more favourable biochemical composition, with fatty acids
being highly focused on in particular (Shields et al., 1999; Reitan et al.,
1994; Sargent et al., 1997; McEvoy et al., 1998; Drillet et al., 2006a,
2006b). In addition to fatty acids, and due to the absence of a fully
developed digestive system at first-feeding, fish larvae have a depen-
dency on free amino acids (FAA) as a source of nutrition and energy
(Rønnestad et al., 1999), and a high content of FAAs in the live feed are
likely to be necessary to fulfil the nutritional demand of first-feeding
fish larvae.
The contents of FAA in different copepods vary considerably. A
review study (Ventura, 2006) reports the relative content of FAA in
marine copepods to range from 5 to 19% of dry weight (DW), whereas
another (Båmstedt, 1986) found that the total FAA content in copepods
ranged from around 1 to 8% of DW. This wide span in FAA contents is
assumed to reflect osmolyte properties of FAAs in marine organisms
(Farmer and Reeve, 1978; Burton, 1991; Huxtable, 1992; Lindley et al.,
2011) as well as FAAs being involved in various metabolic processes (Li
et al., 2009). Since FAAs in calanoid copepods consist of both non-
essential (can be synthesized by the organism) and essential amino
acids (must be obtained from the diet) (Claybrook, 1983), amino acid
compositions in calanoid copepods are affected by diet and develop-
mental stage, since nauplii and adults may have different diets and
metabolic needs, as well as other environmental factors like tempera-
ture of which govern metabolic rates (Brucet et al., 2005). Some
copepod nauplii do not feed within their first three nauplii development

⁎
Corresponding author at: Department of Science and Environment, Roskilde University, DK-4000 Roskilde, Denmark.
E-mail address: tar@ruc.dk (T.A. Rayner).

http://dx.doi.org/10.1016/j.cbpa.2017.04.020
Received 21 December 2016; Received in revised form 25 April 2017; Accepted 30 April 2017
Available online 05 May 2017
1095-6433/ © 2017 Elsevier Inc. All rights reserved.
T.A. Rayner et al.
stages (NI-NIII) (Mauchline, 1998), and therefore the needs for specific
FAAs must be covered by allocation from the adult female to the
embryo. This could mean that the pool of FAAs varies substantially
between NI stage relative to the following development stages. Detailed
studies on FAA changes during nauplii development, however, are
scarce, but such studies are relevant when using live feed in aqua-
culture, since nauplii rather than copepodites or adults are usually
applied as first-feed to fish larvae (Pepin and Penney, 1997; Engell-
Sørensen et al., 2004).
The aim of this study was to evaluate the concentration and
composition of FAAs in the calanoid copepod Acartia tonsa throughout
nauplii development. The reason for choosing A. tonsa is due to its
relevance as a live feed subject in aquaculture through its ability to
produce resting-eggs as well as its generally favourable nutritional
characteristics as live feed. The content of FAA was related to nauplii
body-volume (BV) as well as specific biomass (in carbon units, C).
Understanding the availability of FAAs in NI nauplii will highlight the
benefit of applying A. tonsa resting-eggs, hatched and fed out as nauplii,
in aquaculture hatcheries as well as providing further understanding on
how the FAA profile in nauplii changes during development.
2. Methods
2.1. Culturing of nauplii and algae feed
Production of A. tonsa nauplii for BV estimation and FAA analysis
was done at Roskilde University (RUC), Denmark, by acquiring cold-
stored eggs for culturing in the laboratory. The eggs were collected
from 60 L stock cultures of A. tonsa (ID: DFH-ATI) at RUC and cold-
stored at 4 °C in absence of oxygen for two months (Drillet et al.,
2006a). The continuous A. tonsa stock cultures were fed daily with the
cryptophyte algae Rhodomonas salina (ID: SCCAP K-1487). The algae
feed was fed in excess (20 million cells L− 1) as according to the study
Jepsen et al. (2007). The algae were cultured continuously in auto-
claved seawater (32 g L− 1 salinity) added 1 ml f/2 media L− 1 culture
water (Guillard and Ryther, 1962). The algae were grown at 107 μmol
photons m− 2 s− 1 of PAR irradiance. Cell concentrations were mea-
sured using a Beckman Coulter Counter 3 (Miami, FL, USA).
2.2. Estimation of nauplii body-volume and specific biomass
Nauplii BV for A. tonsa were estimated by regressions that were
determined by measuring the dimensions of numerous nauplii (n = 85)
at different body lengths. The measured nauplii were grown from
18,000 cold-stored A. tonsa eggs divided into three 2 L containers. A
10 ml subsample was taken from each every 24 h for individual
nauplius size measurements and 10 ml of dense R. salina culture were
added. The subsamples were placed in petri-dishes and euthanized with
a few drops of 5% neutral buffered formalin. The nauplii were counted
and measured at ×40 magnification in a Nikon (Tokyo, Japan) SMZ18
dissecting microscope, equipped with a digital camera connected to a
PC. Dimensions were measured on images using NIS-Elements Basic
Research software (Nikon, Tokyo, Japan). The geometry of the nauplii
corresponded to that of an ellipsoid (Fig. 1), and the BV was therefore
calculated by the equation for an ellipsoid with an ellipsoid cross
section, where the most extreme points in length, width and height of
the nauplii were measured (described in Fig. 1). Regression formula for
converting body lengths of nauplii to carbon (C) was obtained
(Berggreen et al., 1988) and applied for the same A. tonsa strain.
2.3. Algae and nauplii sample preparation for free amino acid analysis
Algae samples were made by transferring 5 mL of dense culture
algae onto 13 mm Whatman GF/C while applying suction (< 0.2 bar).
Three analytical replicates were produced and the filters were trans-
ferred subsequently into cryo-tubes for storage at −80 °C.
2
Comparative Biochemistry and Physiology, Part A 210 (2017) 1–6
For nauplii samples, ten thousand cold-stored A. tonsa eggs were
selected and added to each planned 2 L culture beakers. A total of
twelve 2 L cultures were produced and R. salina was added in excess on
a daily basis as nauplii feed. The eggs were incubated in a walk-in
climate-room set to 16 °C. The 2 L culture beakers were placed at
random and contained 0.2 μm filtered 32 g L− 1 salinity seawater while
aerated. Over time-intervals of 24 h, three cultures were selected and
screened for nauplii using 70, 100 and 150 μm mesh size screens,
resulting in four different size categories representing three nauplii
cultures each. The last sample was taken on day four. Between two and
four analytical samples were produced for every nauplii culture
depending on the available number of nauplii. The nauplii samples
were subsequently transferred to 0.2 μm filtered and 32 g L− 1 salinity
seawater in 1 L bottles equipped with a kip-automate (NS 29.2/32;
Buch & Holm, Witeg, Germany). The density of nauplii in the bottles
was determined by adding 10 mL of the nauplii sub-sample to petri-
dishes. The nauplii were then euthanized in the petri-dishes with a few
drops of 5% neutral buffered formalin. The number of nauplii in the
10 mL subsamples were subsequently counted (12% average SD, n = 3)
and their body lengths were measured under a dissecting microscope at
40× magnification. After density determination of the nauplii, the
samples with nauplii were filtered for FAA analysis by transferring the
nauplii (around 1500 per subsample) onto 25 mm GF/C filters by kip-
automate while applying suction. The filters were thereafter stored in
cryo-vials and frozen at −80 °C until further analysis.
2.4. Free amino acid extraction and analysis
Extraction and analysis of FAA was done according to procedures in
Drillet et al. (2006a), Drillet et al. (2006b) and Rayner et al. (2015).
Filters with algae and copepods for FAA analysis were placed in 1.5 mL
GC-vials and freeze-dried for 12 h prior to extraction. Each vial was
added 1 mL MilliQ water and heated to 95 °C in an incubator for
15 min. After cooling, the extracts were filtered through 0.2 μm GHP
polypropylene filters and placed into 2 mL Eppendorf tubes for storage
at −20 °C. FAA concentrations were determined by HPLC and fluores-
cence detection after derivatisation with 6-aminoquinolyl-N-hydroxy-
succinimidyl carbamate (AQC), which was purchased by Waters
(Milford Massachusetts, USA) as an AccQFlour kit (www.waters.com).
The analysis was run as recommended by Waters AccQFlour, and the
amino acid derivatives were analysed on a Waters HPLC system
(Alliance 2695 solvent module, 2475 fluorescence detector and a
3.9 × 150 mm AccQTag column). Chromatogram peaks were identified
from retention times and integrated against an external amino acid
standard mixture supplied by Waters.
2.5. Statistical methods
Covariance between relative abundances of FAAs was tested for by
applying principle component analysis (PCA). Relative abundances of
different FAAs for nauplii were tested for normality and equal variance,
and a non-parametric test was assigned (Mann-Whitney U test Conover-
Inman Pairwise Comparison) whenever these criteria were not met.
Otherwise, analysis of variance (One-Way ANOVA, Tukey's test) was
selected. All tests were performed at significance level 0.05. Tests were
run using the PC software Systat 13 developed by Systat Software Inc.
(San Jose California, USA). All mean values are presented with ± 1
standard deviation. Figures were created using SigmaPlot 12.3 software
for Windows.
3. Results
3.1. Acartia tonsa nauplii body-volume
Measurements of body width and height for A. tonsa nauplii, as well
as calculations of BV, revealed a sigmoidal development when plotted
T.A. Rayner et al. Comparative Biochemistry and Physiology, Part A 210 (2017) 1–6

Fig. 1. Example of ellipsoid cross sections fitted to an Acartia tonsa nauplius. A) Cross section from the top, B) cross section from the side. Body volume (BV) was calculated through the
eq. BV = 1.33π((BL × BW × BH)/8). BL: body length; BW: body width; BH: body height.

Fig. 2. A) Naupliar body dimension regression plot, B) Naupliar body volume regression plot. Nauplii dimensions were used for calculating nauplii body volumes (BV), which were used
for creating the sigmoidal regression plot against nauplii body length (B). Regressions are: BW: y = 31.05 + 107.74 / (1 + exp(−(x − 233.41) / 19.27)0.21. BH: y = 51.83 + 83.65 /
(1 + exp(−(x − 230.12) / 15.50)0.27. BV: y = 0.29 × 105 + 28.30 × 105 / (1 + exp(−(x − 249.04) / 18.64))0.37.

against the nauplii body length (Fig. 2). The shortest and longest
nauplii indicated a pattern where nauplii body length continued to
increase while body width and height exhibited little changes. For these
reasons, a sigmoidal pattern revealed itself. The length-width measure-
ments were used to create a regression model for nauplii BV of which
was applied in analysis of FAA content in the nauplii (equations given
in caption to Fig. 2).
3.2. Observations on feeding and free amino acid contents
In nauplius development stage NI, access to R. salina did not cause
colourisation of the gut, indicating that feeding did not occur. Body size
at this development stage has previously been determined to
116 ± 10 μm for A. tonsa (Drillet et al., 2006a, 2006b; Alver et al.,
2011). Feeding on R. salina was observed in the following nauplii stages
after NI (presence of bright red guts) at mean body lengths of
128 ± 4 μm and onwards.
Relative FAA abundances in R. salina were determined and revealed
high abundances for glycine, glutamic acid (Glu), Alanine (Ala) and
proline (Pro) of which are regarded non-essential amino acids. Arginine
(Arg) and Lysine (Lys) were the most abundant of the essential amino
acids (Fig. 5). Contents of FAAs relative to BV (FAA/BV) for A. tonsa
were determined for nauplii at different sizes (Fig. 3A). According to
the regression model, nauplii at NI stage contained around
290 nmol FAA mm− 3 BV and decreased towards 117 nmol FAA mm− 3
BV for later stages, corresponding to a 60% decline in total FAA
concentration. Changes in FAA relative to carbon content (FAA/C)
revealed a similar trend as FAA/BV (Fig. 3B). According to the
regression model, the FAA content of the nauplii at stage NI was
3.0 nmol FAA μg− 1 C, and it decreased towards 1.0 nmol FAA μg− 1 C
for later nauplii stages. The average molecular weight of FAA in the
nauplii at all sizes was 130.3 g mol− 1. From this average molecular
weight and by applying a C to DW conversion factor of 0.447
(Båmstedt, 1986), the FAA content of the nauplii was estimated to
vary from around 17% of DW for NI stage nauplii to 6% of DW for later
nauplii stages. This corresponds to a drop of 66% in FAA concentration
from stage NI to later nauplii stages. When plotting FAA/C versus FAA/
BV by linear regression (Fig. 3C), the regression slope was significantly

3
T.A. Rayner et al.
Fig. 3. Regression plots on total FAA concentrations against nauplii body lengths. A) FAA contents relative to body volume, B) FAA contents relative to carbon biomass, C) FAA content
over naupliar body volume versus FAA contents over naupliar specific biomass. First nauplius development stage is indicated by NI with an average range as according to Alver et al.
(2011). Regressions are: A: y = 1.16 + 332.46exp(− 0.049 ×). B: y = 0.91 + 684.79exp(−0.055 ×). C: y = 0.39 + 0.86 ×, p: 0.0001, t-test α: 0.05.
Fig. 4. Principle component analysis (PCA) factor plot. The plot shows changes in relative
abundances among different FAAs in Acartia tonsa nauplii. According to the analysis, any
FAAs that are opposite to one-another along a principle component (PC) are correlated
and contribute to the variation observed among the FAA profiles of nauplii at different
sizes. x-axis represents loading values for the first principle component (PC1) and y-axis
represents loading values for the second principle component (PC2).
lower than 1.0 and had a positive intercept, indicating that post NI
stage nauplii have comparatively higher concentrations of FAA/BV
versus FAA/C. Relative abundances of the different FAAs in nauplii at
different stages were tested by PCA analysis. Throughout nauplii
4
Comparative Biochemistry and Physiology, Part A 210 (2017) 1–6
development, Glu, Pro, Arg and Lys accounted for the largest changes
in relative abundance in FAAs. Judging by the first principle component
(PC1), Glu accounted for the largest change in abundances and
correlated with Pro, Arg and Lys (Fig. 4). Furthermore, the changing
abundance of Pro correlated with changes in abundance of Arg and Lys,
as indicated by PC2. Overall, the most dominant FAAs in the nauplii
were Pro, Ala, Arg, Lys and Glu, which together constituted > 71% of
the total FAA content for all nauplii sizes (Fig. 5). Glu abundance
exhibited a sharp and significant decrease (p ≤ 0.001) from develop-
ment stage NI towards larger nauplii, decreasing from 29.0% to 7.1% in
mean relative abundance. Low relative abundances of Arg and Lys
occurred in the early stages of A. tonsa, but their relative abundances
increased significantly (p ≤ 0.001 and p ≤ 0.012, respectively) towards
later nauplii stages. Other essential amino acids, such as methionine
(Met) and valine (Val) saw similar significant increases (p ≤ 0.001 in
both cases) during naupliar development; however these increases were
quite minor. Total relative abundances of essential FAAs went from
19.3 ± 3.6% at NI stage to 49.6 ± 2.0% at later development stages.
Pro was by far the most dominant FAA (> 30% in relative abundance)
for all nauplii sizes, with a significantly higher concentration for nauplii
at mean sizes 128 μm (p ≤0.001) and 149 μm (p = 0.004).
4. Discussion
First-feeding fish larvae favour FAAs for nutritional energy
(Rønnestad et al., 1999) and newly hatched A. tonsa nauplii can
potentially sustain the need for amino acids in fish larvae in aqua-
culture. The analysis of FAAs in A. tonsa nauplii demonstrated that the
total FAA concentration was high at NI stage and declined with nauplii
T.A. Rayner et al.
Fig. 5. Relative abundances of FAAs in different sized Acartia tonsa nauplii. A) Relative
abundance of essential free amino acids. B) Relative abundance of non-essential free
amino acids. Error bars represent mean standard deviation.
ontogeny, i.e. increasing body size, and that the change in FAA
composition occurred between different body sizes. The FAA pools
measured in this study for later staged nauplii following NI stage falls
within the range observed previously (Ventura, 2006; Båmstedt, 1986),
whereas development stage NI nauplii revealed substantially higher
concentrations than elsewhere reported (Ventura, 2006; Båmstedt,
1986). The FAA content declined with nauplii development, whether
the FAA concentrations were related to carbon content (FAA/C) or body
volume (FAA/BV); however the concentration declined at a higher rate
relative to the carbon content than body-volume. Supporting this, the
later staged nauplii to NI contained relatively higher concentrations of
FAA per unit BV compared to C. FAAs are potential osmolytes in
copepods, and the declining concentrations of FAA with BV may
suggests a diminishing body water content in A. tonsa nauplii through-
out nauplii development. Changes in BV in A. tonsa have been
demonstrated when challenged with low and high salinities
(Svetlichny and Hubareva, 2014), which suggest that BV can be
affected by changing osmolyte concentrations. It is hereby suggested
that decreasing concentrations of FAA in the nauplii may have affected
overall BV and could indicate that A. tonsa nauplii were counteracting
any imbalance in osmolyte concentration.
Since A. tonsa nauplii do not feed immediately after hatching, the
observed high FAA content in stage NI nauplii was most likely caused
by residual contents from embryonic development. Among FAAs
allocated from the egg yolk, Glu occurred at a high concentration at
early development stage (Fig. 5), which declined during later stages.
Glu is involved in several metabolic processes in fish and is also an
important metabolic energy substrate (Li et al., 2009). A study by
5
Comparative Biochemistry and Physiology, Part A 210 (2017) 1–6
Conceição et al. (2002) on herring larvae demonstrated that they
preferred Glu to Lys as an energy substrate, and thus Glu-rich copepods,
as in this case A. tonsa NI nauplii, could be a favourable source for
nutrition for first feeding fish larvae. High Glu contents in NI nauplii
will also likely act as nutrition for early nauplii development, and
would explain the drastic drop in relative abundance during the period
of inactive feeding. High Glu abundance at NI stage may be a result of
high Glu contents in the algae feed being transferred from female to
embryo, as Glu occurred in relatively high abundances in the feed
(about 17% of total FAA) of which is in accordance with observations in
other studies (Drillet et al., 2006a; Helland et al., 2003). The abundance
of Pro was also moderately high in the algae; giving the A. tonsa females
the means to allocate large pools of Glu and Pro by trophic transfer.
However, judging by the higher abundances of these amino acids in the
nauplii in comparison to what was available in the algae feed, the adult
female may also have provided these FAAs through metabolic pathways
as these FAAs are non-essential (Claybrook, 1983). Manipulation of
FAA contents in copepod embryos, e.g., by changing the maternal diet
for adult copepods, was found to result in changes in amino acid
abundances at the expense of lowered reproductive success (Helland
et al., 2003; Guisande et al., 1999). Whether it is possible to manipulate
the abundance of specific FAAs in newly hatched A. tonsa nauplii
through various maternal diets has seen little attention.
This study demonstrated significant changes in both essential and
non-essential FAAs with development stage. The relative abundances of
Arg, Lys, and to a lesser extent, Met and Val gradually increased as the
nauplii developed. Since these amino acids are regarded as essential,
i.e. not synthesized in the organisms (Claybrook, 1983), changes in the
FAA profile from stage NI to one that more resembles the external diet
is the most likely cause. Since Arg and Lys were the most dominant
essential FAAs in the algae feed, it comes as little surprise that a similar
trend was observed in post NI nauplii although relative abundances
were substantially higher in later stage nauplii. Acartia tonsa nauplii
seem to be very able to accumulate essential amino acids (particularly
Arg and Lys) into their FAA pool from the diet, as the overall relative
abundance of essential FAAs in later stage nauplii had well exceeded
that of the algae feed. Concentrations of the amino acids Ala, Pro and
taurine (Tau) are typically adjusted in occurrence with changing
external salinities in marine invertebrates (Burton, 1991; Huxtable,
1992). Significant changes in relative abundances of Pro and Tau were
observed in the nauplii at different sizes, possibly indicating that
osmoregulation may have occurred. Changes in concentrations of Glu
relative to changes in external salinities have in fact been demonstrated
in copepods elsewhere (Farmer and Reeve, 1978; Lindley et al., 2011).
If nauplii development resulted in changes in osmolyte abundances, due
to osmoregulation of excess FAAs from the embryonic stage, then Ala,
Pro and Tau would most likely be down-regulated. However, changes in
relative abundance of Ala, Pro and Tau were small, and were coupled
with changes of other FAAs, i.e. Glu and the essential FAAs Arg and Lys.
Hereby, the drop in FAA content was not a direct result of osmoregu-
latory processes, but rather due to degradation of residual Glu from the
embryonic stage. Therefore, during a fish larvae feeding scenario, A.
tonsa resting-eggs should be applied 24 h prior to first feeding to ensure
the availability of newly hatched NI nauplii and hereby take advantage
of their inherent large pool of FAAs. Applying R. salina (or other algae)
in conjunction with A. tonsa nauplii would benefit more developed fish
larvae later on. Despite of a smaller relative FAA pool compared to
biomass in larger nauplii, the total content of FAA per individual as well
as the increasing abundance of essential FAAs should be considered by
hatchery managers. Additionally, manipulation of the FAA pool by
feeding post NI nauplii with algae containing a specific profile of FAAs
may help achieve better nutrition for fish larvae.
5. Conclusions
Analysis of FAAs in early staged A. tonsa nauplii revealed a
T.A. Rayner et al.
relatively high abundance (17% of DW) in NI stage nauplii followed by
a rapid decline through nauplii ontogeny. Residual contents of FAA,
especially of Glu, from the embryonic stage could account for the high
post hatching content, since no external feeding occurred at first
development stage. Changes in osmolyte related FAAs did not con-
tribute to the decline in total FAA content during nauplii development,
and changes in the BV relative to biomass indicated conformation to the
nauplius' external environment. The high content of FAAs in the NI
stage nauplii could be favourable in aquaculture for first feeding fish
larvae with high amino acid demand, and A. tonsa resting-eggs should
be applied 24 h prior to first feeding ensuring newly hatched nauplii to
exploit such high FAA contents. Manipulation of the adult copepod's
diet, e.g., by addition of specific amino acids, might increase the
nutritional value of newly hatched nauplii as live feed for first feeding
fish larvae with special biochemical needs.
Acknowledgements
Our gratitude goes to Minh T.T. Vu for her assistance in producing
algae samples for FAA analysis. This work is supported by the Strategic
Research Council of Denmark IMPAQ grant (j. no. 10-093522) to Benni
W. Hansen. The present work was supported by the Danish Ministry for
Independent Research, Ung Elite Forsk Grants 10-093759 and 10-
094773 to Guillaume Drillet
References
Alver, M.O., Storøy, W., Bardal, T., Overrein, I., Onsøyen, M.K., Tennøy, T., Øie, G., 2011.
Automatic measurement of Acartia tonsa nauplii density, and estimation of stage
distribution. Aquaculture 313, 100–106.
Båmstedt, U., 1986. Chemical Composition and Energy Content. In: Corner, E.D.S.,
O'Hara, S.C.M. (Eds.), The Biological Chemistry of Marine Copepods. Clarendon
Press, Oxford, UK, pp. 1–58.
Berggreen, U., Hansen, B., Kiørboe, T., 1988. Food size spectra, ingestion and growth of
the copepod Acartia tonsa during development: implications for determination of
copepod production. Mar. Biol. 99, 341–352.
Brucet, S., Boix, D., López-Flores, R., Badosa, A., Quintana, X.D., 2005. Ontogenic changes
of amino acid composition in planktonic crustacean species. Mar. Biol. 148, 131–139.
Burton, R.S., 1991. Regulation of proline synthesis during osmotic stress in the copepod
Tigriopus californicus. J. Exp. Zool. 259, 166–173.
Claybrook, D.L., 1983. Nitrogen Metabolism, Chap. 5. In: Mantel, L.H. (Ed.), The Biology
of Crustacea. Anatomy and Physiological Regulation Volume 5. Academic Press, New
York, US, pp. 163–213.
Conceição, L.E.C., Rønnestad, I., Tonheim, S.K., 2002. Metabolic budgets for lysine and
glutamate in unfed herring (Clupea harengus) larvae. Aquaculture 206, 305–312.
Drillet, G., Jørgensen, N.O.G., Sørensen, T.F., Ramløv, H., Hansen, B.W., 2006a.
Biochemical and technical observations supporting the use of copepods as live feed
organisms in marine larviculture. Aquac. Res. 37, 756–772.
Drillet, G., Iversen, M.H., Sørensen, T.F., Ramløv, H., Lund, T., Hansen, B.W., 2006b.
Effect of cold storage upon eggs of a Calanoid copepod, Acartia tonsa (Dana) and their
offspring. Aquaculture 254, 714–729.
Comparative Biochemistry and Physiology, Part A 210 (2017) 1–6
Engell-Sørensen, K., Støttrup, J.G., Holmstrup, M., 2004. Rearing of flounder (Platichthys
flesus) juveniles in semiextensive systems. Aquaculture 230, 475–491.
Farmer, L., Reeve, M.R., 1978. Role of the free amino acid pool of the copepod Acartia
tonsa in adjustment to salinity change. Mar. Biol. 48, 311–316.
Guillard, R.R.L., Ryther, J.H., 1962. Studies of marine planktonic diatoms. I. Cyclotella
nana Hustedt and Detonula confervacea Cleve. Can. J. Microbiol. 8, 229–239.
Guisande, C., Maneiro, I., Riveiro, I., 1999. Homeostasis in the essential amino acid
composition of the marine copepods Euterpina acutifrons. Limnol. Oceanogr. 44 (3),
691–696.
Helland, S., Nejstgaard, J.C., Humlen, R., Fyhn, H.J., Båmstedt, U., 2003. Effects of season
and maternal food on Calanus finmarchicus reproduction, with emphasis on free
amino acids. Mar. Biol. 142, 1141–1151.
Huxtable, R.J., 1992. Physiological actions of taurine. Physiol. Rev. 72 (1), 101–163.
Jepsen, P.M., Andersen, N., Holm, T., Jørgensen, A.T., Højgaard, J.K., Hansen, B.W.,
2007. Effect of adult stocking density on egg production and viability in cultures of
the Calanoid copepod Acartia tonsa (Dana). Aquac. Res. 38, 764–772.
Li, P., Mai, K., Trushenski, J., 2009. New developments in fish amino acid nutrition:
towards functional and environmentally oriented aquafeeds. Amino Acids 37, 43–53.
Lindley, L.C., Phelps, R.P., Davis, D.A., Cummins, K.A., 2011. Salinity acclimation and
free amino acid enrichment of copepod nauplii for first-feeding of Larval marine fish.
Aquaculture 318, 402–406.
Mauchline, J., 1998. Gut, Food and Feeding. In: Blaxter, J.H.S., Southward, A.J., Tyler,
P.A. (Eds.), The Biology of Calanoid Copepods. Elsevier Academic Press Vol. 33.
London, UK, pp. 139–175.
McEvoy, L.A., Naess, T., Bell, J.G., Lie, Ø., 1998. Lipid and fatty acid composition of
normal and malpigmented Atlantic halibut (Hippoglossus hippoglossus) fed enriched
Artemia: a comparison with fry fed wild copepods. Aquaculture 163, 237–250.
Payne, M.F., Rippingale, R.J., Cleary, J.J., 2001. Cultured copepods as food for west
Australian Dhufish (Glaucosoma hebraicum) and pink snapper (Pagrus auratus) larvae.
Aquaculture 194, 137–150.
Pepin, P., Penney, R.W., 1997. Patterns of prey size and taxonomic composition in Larval
fish: are there general size-dependent models? J. Fish Biol. 51 (A), 84–100.
Rayner, T.A., Jørgensen, N.O.G., Blanda, E., Wu, C.H., Huang, C.C., Mortensen, J.,
Hwang, J.S., Hansen, B.W., 2015. Biochemical composition of the promising live feed
tropical Calanoid copepod Pseudodiaptomus annandalei (Sewell 1919) cultured in
Taiwanese outdoor aquaculture ponds. Aquaculture 441, 25–34.
Reitan, K.I., Rainuzzo, J.R., Olsen, Y., 1994. Influence of lipid composition of live feed on
growth, survival and pigmentation of turbot larvae. Aquac. Int. 2, 33–48.
Rønnestad, I., Thorsen, A., Finn, R.N., 1999. Fish larval nutrition: a review of recent
advances in the roles of amino acids. Aquaculture 177, 201–216.
Sargent, J.R., McEvoy, L.A., Bell, J.G., 1997. Requirements, presentation and sources of
polyunsaturated fatty acids in marine fish Larval feeds. Aquaculture 155, 117–127.
Shields, R.J., Bell, J.G., Luizi, F.S., Gara, B., Bromage, N.R., Sargent, J.R., 1999. Natural
copepods are superior to enriched Artemia Nauplii as feed for halibut larvae
(Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology:
relation to dietary essential fatty acids. J. Nutr. 129 (6), 1186–1194.
Svetlichny, L., Hubareva, E., 2014. Salinity tolerance of alien copepods Acartia tonsa and
Oithona davisae in the Black Sea. J. Exp. Mar. Biol. Ecol. 461, 201–208.
Toledo, J.D., Golez, S.N., Doi, M., Ohno, A., 1997. Food selection of early grouper,
Epinephelus coioides, larvae reared by the semi-intensive method. Aquac. Sci. 45 (3),
327–337.
Toledo, J.D., Golez, S.N., Doi, M., Ohno, A., 1999. Use of copepod Nauplii during early
feeding stage of grouper Epinephelus coioides. Fish. Sci. 65 (3), 390–397.
Ventura, M., 2006. Linking biochemical and elemental composition in freshwater and
marine crustacean zooplankton. Mar. Ecol. Prog. Ser. 327, 233–246.
Wilcox, J.A., Patrick, L.T., Marcus, N.H., 2006. Improving live feeds: Effect of a mixed
diet of copepod Nauplii (Acartia tonsa) and rotifers on the survival and growth of first-
feeding larvae of the southern flounder, Paralichthys lethostigma. J. World Aquacult.
Soc. 37 (1), 113–120.