You are on page 1of 9

Aquaculture Research, 2007, 38, 764^772

doi:10.1111/j.1365-2109.2007.01730.x

Effects of adult stocking density on egg production and


viability in cultures of the calanoid copepod Acartia

tonsa (Dana)
Per M Jepsen, Nikolaj Andersen,Thue Holm, Anders T Jrgensen, Jonas K Hjgaard &
Benni W Hansen
Department of Environment, Social and Spatial Change, Roskilde University, Roskilde, Denmark
Correspondence: B W Hansen, Department of Environment, Social and Spatial Change, Roskilde University, Universitetsvej 1, PO Box
260, DK-4000 Roskilde, Denmark. E-mail: bhansen@ruc.dk

Abstract
The eect of stocking density of the calanoid copepod
Acartia tonsa was evaluated in a 96 h rearing experiment. Possible density-dependent egg production and
egg viability were analysed at stocking densities of
100, 200, 300, 400 and 600 adults L 1. Temperature,
oxygen saturation and algal concentration were kept
optimal. A non-density-dependent mortality rate of
15^19% day 1 was documented. A non-signicant
density-dependent egg production was observed between 100 and 600 adults L 1. The average egg production was 22.5  8.8 egg female 1 day 1 in all
densities. The average egg hatching success was
84.7  4.8% and was never observed below 76.1%,
with no signicant dierences across the stocking
densities. Conclusively, as a practical recommendation for the aquaculture industry, copepod cultures
with densities ranging from 100 to 600 adults L 1
and presumably even more dense cultures are possible with the studied species obtaining a steady egg
production and still high egg viability.

Keywords: Acartia tonsa, aquaculture, copepod


density, egg production, egg viability, live feed

Introduction
Historically, the most-used live feed sources for marine sh farms have been rotifers (Brachionus genus)
and brine shrimp (Artemia salina). However, recent
research has shown that sh larvae used in aquaculture obtain a higher nutritional value from calanoid
copepods leading to higher growth and survival
rates, stronger pigmentation and higher rates of suc-

764

cessful metamorphosis than when fed traditional live


feed (Holmefjord, Bolla & Raitan 1989; Nss, Germain-Herry & Naas 1995; Nss & Lie 1998; Shields,
Bell, Bromage, Gara, Luizi & Sargent 1999). Wilcox,
Patrick and Marcus (2006) has showed interesting
result using a mixed diet of Acartia tonsa nauplii and
enriched rotifers (1:1) as a feed source for rst-feeding
larvae of the Southern Flounder (Paralichthys lethostigma). Although recent development has taken the
use of copepods to a higher level of production, the
use of copepods are still by far smaller than the super
intensive production of the traditional food items
(Lubzens, Minko, Barr & Zmora 1997; Payne & Rippingdale 2001). This is most likely due to lack of
knowledge regarding rearing techniques. To optimize production of A. tonsa, Medina and Barata
(2004) achieved stocking densities of 500, 1000 and
2000 individuals L 1, Stttrup, Richardson, Kirkegaard and Pihl (1986) reported stocking densities of
100 adults L 1, and Peck and Holste (2006) of 65,
166 and 425 adult L 1. However, only few researchers address the question of declining egg production
as a function of the stocking density (Ogle 1979;
Stttrup et al. 1986; Medina & Barata 2004; Peck &
Holste 2006). The individual as well as the total egg
production are crucial for an eventual mass production. Additionally, the subitaneous eggs are recently
reported to be able to tolerate cold storage for up to 1
year at 2^3 1C without a severe loss of amino acid,
but with a 50% decrease in fatty acids and with a
hatching success of 40% (Drillet, Iversen, Srensen,
Ramlv, Lund & Hansen 2006). This supports the fact
that calanoid copepod eggs are potentially an interesting live prey in parallel toArtemia for the aquaculture industry.

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd

Aquaculture Research, 2007, 38, 764^772 Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al.

Acartia tonsa cultured in high densities could be


negatively inuenced by density-provoked stress factors, eventually adversely inuencing survival and
egg production. According to Medina and Barata
(2004), the mortality rate when keeping A. tonsa at
high stocking densities is about 8% day 1. Stttrup
et al. (1986) reported a daily mortality rate of 5%.
Concerning egg production, Medina and Barata
(2004) concluded a signicant decline from cultures
with densities of 500^2000 individuals L 1. Peck
and Holste (2006) observed essentially the same tendency where the lowest stocking density
(65 adultsL 1) produced most eggs per female. However, the same study indicates that the accumulated
egg production was increasing as a function of increased copepod densities (Peck & Holste 2006). We
initiated a study on our culture copepod wherein we
obtained quite promising results with copepod
stocking densities of up to 1200 L 1; however, we
consider that as a pilot phase, which is why we here
report a thorough study with the appropriate experimental design. The above-mentioned perspectives
raise the important need of the identication of the
optimal operational stocking density where the
trade-o between individual and total egg harvest
from a given copepod culture is determined.
This knowledge is crucial for continuous protable
mass cultivation.

Materials and methods


The experiments were conducted on the calanoid copepod A. tonsa, supplied by the Danish Institute for
Fisheries and Marine Research, Charlottenlund. The
copepods originated from individuals isolated from
net samples in 1981 in resund, Denmark, and assigned the identication code DFH-ATI (Stttrup
et al. 1986). Separate cultures were raised in several
generations in a continuous culture at Roskilde University, Denmark. The adult A. tonsa used in the present experiments originated from this culture kept in
60 L dark tanks in 0.22 mm ltered seawater (30 psu)
at 17 1C. The culture was fed in excess with monocultures of the phytoplankton Rhodomonas salina. The
phytoplankton cultures were grown in logarithmic
phase under constant light within the PAR spectrum
of 150  5 mmol photons m 2 sek 1 (Licor-198 with
Li-192 SR. Nr. UWQ 4294, Li-Cor, Lincoln, NE, USA) in
round-bottom glass asks and fertilized with the culture media B1 (Hansen 1989). All experiments were
conducted under the above-described conditions.

Experimental design
The experiment was designed as a randomized block
design, ensuring independence from potential preexisting gradients or demonic interactions (Hurlbert 1984).
The experimental equipment consisted of ve polyethylene dark tanks with a volume of 60 L, each tank a replicate of each other. In each tank were eight replicate
jars. Each jar consists of a spawning chamber holding
the adult copepods and an egg chamber. The spawning
chambers were equipped with false bottoms, which
consist of a 200 mm lter and contains a volume of
250 mL (Fig. 1). The false bottoms of the egg chambers
were 45 mm lter ensuring retaining of the produced
eggs and simultaneously a free ow of seawater around
the eggs. The 200 mm lter ensured that the copepods
were retained in the spawning chamber while still allowing the eggs to pass the 200 mm lter into the compartment conserved by the 45 and 200 mm lters. The
jars were placed radial symmetrical in a circular acrylglass plate with eight holes (F 586 mm) (Fig. 1c). The
bottom of the acryl-glass plate was covered with foam,
keeping the experimental device oating, ensuring a
constant volume in the spawning chambers. In the centre of the acryl-glass plate, an air stone inside a glass
tube dragged air and algal-enriched seawater to the
top acryl-glass plate (Fig. 1a). The top acryl-glass plate
had eight cone-shaped holes (F 51mm), matching
each spawning chamber (Fig. 1b). The transported
water slowly dripped through the cone-shaped holes
and thereby ensured a constant ow of food-enriched
and aerated seawater through the spawning and egg
chambers creating a recirculated system.
Density experiment
The density experiment was carried out in the abovedescribed experimental design. Five replicates were
stocked with densities of 100, 200, 300, 400 and
600 adult copepods L 1, corresponding to 25, 50, 75,
100 and150 adult copepods per 250 mL 1. A batch of
adult copepods was isolated from the renewal culture
and used in the experiment. Furthermore, the sex ratio was determined on187 randomly selected adults in
order to obtain the initial sex ratio. The adults were
s
gently sorted out in 250 mL Nalgene (Sybron Co.,
Rochester, NY, USA) bottles in their respective densities. When all the copepod densities were sorted out,
the replicates were randomly added to each dark tank.
Before the copepods were added, factors that could potentially inuence on the result were controlled (temperature, oxygen level, algal concentration). These factors were measured twice a day during experimental

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

765

Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al. Aquaculture Research, 2007, 38, 764^772

Figure 1 Acartia tonsa. Experimental design.1, Experimental design, showing the direction of the water ow indicated by
arrows; 2, spawning chamber with a 200 mm lter (false bottom) and egg chamber with a 45 mm lter (false bottom).
Experimental design. A, Glass tube with an air stone for dragging air and algal-enriched seawater to the top acrylic-glass
plate; B, acrylic-glass plate with a water barrier and cylindrical holes matching each spawning chamber; c, bottom acrylicglass plate covered in foam, stabilizing and keeping the experimental design buoyant and ensuring a constant water
volume in the chambers.

time. The temperature and oxygen concentration were


measured with OxyGuard Handy Polaris Portable DO
Meter (OxyGuard International A/S, Birkevd, Denmark), the algal concentrations were measured
with a Coulter Counter Z2 from Beckman Coulter
(Miami, FL, USA). Algal concentrations were kept
at  20 000 cells mL 1, corresponding to  950 mg
cells L 1, during the experiment and considered in
excess (Kirboe, Mhlenberg & Hamburger 1985;
Berggreen, Hansen & Kirboe 1988). Algae were automatically fed every 6 h by a peristaltic pump and the
algae samples were randomly picked twice a day in
two dierent spawning chambers. This tested if the
suspected elevated grazing levels in the high-density
chambers were kept at fed in excess levels. Experimental tanks were kept under total darkness, under the assumption that adult A. tonsa exhibit optimal food
uptake at night (Sterns, Tester & Walker 1989). Before
the experiment, the copepods were acclimated to experimental densities in 3 days based on guidelines in
Kirboe et al. (1985). The experimental time was 96 h.
The egg chambers were emptied every 12 h in order to
avoid egg hatching during the experiment, and the
collected eggs were further tested for hatching.

Results

Hatching experiment
The eggs used in the following hatching experiment
were left for hatching in 20 mL Petri dishes. When
emptying the egg chambers, the following procedure

766

was followed: both the spawning chamber and the


egg chamber were removed from the experimental
tank. The spawning chamber was gently ushed
with 0.22 mm ltered seawater (30 psu), ushing any
remaining eggs down in the egg chamber. Later, a
new egg chamber was placed under the spawning
chamber and the chambers were randomly assigned
in the experiment. The entire procedure lasted
o1min ensuring a minimum of stress to the adult
copepods. The total content of the egg chamber was
ushed down in a 20 mL Petri dish where the eggs
were counted and left for hatching in 17 1C. After
48 h, the nauplii and the remaining eggs were xed
with a 1% nal concentration of acid Lugols solution
and counted by using a dissection microscope (Olympus SZ 40, Olympus Optical (Europe) GmBH, Hamburg, Germany). The hatching success was
examined in the experiment to test the viability of
the eggs. The hatching experiment tested if the viability of the eggs potentially responded adversely to
stress induced by the adult stocking densities. When
terminating the experiment, the sex of all adult copepods used in the experiments was determined.

Cultivation parameters
The temperature, oxygen and algal concentration
were tested during experimental time. The mean

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

Aquaculture Research, 2007, 38, 764^772 Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al.

temperature in the ve tanks were 15.9  0.1 1C


with no statistical signicant temperature dierences between the tanks (t-test, Po0.012, n 5 30).
The mean oxygen concentrations in the ve
tanks were 4.88  0.20 mg L 1 with no statistical
signicant oxygen dierences between the tanks
(t-test, Po0.037, n 5 30). The mean concentration
of R. salina present in the spawning chambers was
always above the intended 20 000 cells mL 1. Mean
algal concentration was 22623  1837 cells mL 1.
There was no signicant variance (F-test, Po0.2091,
n 5 60), which allowed the use of Levene
test of homogeneity of variances (P 5 0.6527,
n 5 60), which showed no dierence over time and
among tanks.

Copepod survival
Mortality was calculated by knowing the initial
and terminal number of animals, considering
mortality as a linear development (Table 1).
There were no signicant mortality dierences between any of the copepod densities. Hence, no
density-dependent mortality was detected in
stocking densities of relevance for mass cultivation
(Keul^Newman test, Po0.05, n 510). There was no
necessity in testing the mortality rate, because it
was calculated from the mortality and thereby shows
the same result. The egg production per female and
the volume-specic egg production in the tanks are
calculated from the same observations of egg production during the experimental time and are therefore
statistically treated as the same (see Density-dependent egg production). The egg productions were calculated by counting the eggs harvested every 12 h.
There was no statistical dierence in hatching
success among tanks or among copepod densities
in the experiment (Keul^Newmann test, Po0.05,
n 5 240).

Egg production
Density-dependent egg production
The egg production across the dierent stocking
densities was tested for each day.
There were only signicant dierences in egg
production between all stocking densities (Keul^
Newmann test, Po0.05, n 5 60) (Fig. 2).

Time-dependent egg production


The egg production over time was tested against the
adult stocking densities. There were tendencies to
higher egg production along the experiment with
the highest egg productions at day 4 (Spearmans
rank order correlation P-values within the range
o0.0000001^0.00013, n 5 20 for each of the stocking densities).
The egg productions during the experiment were
dierent over days in density 100. The egg production
at day 1 (24 h) was dierent from the egg production
at day 3 (72 h) and day 4 (96 h), the egg production
also diered between day 3 (72 h) and day 4 (96 h)
(Keul^Newmann test, Po0.05, n 5 20).
In density 200, there was no dierence in the egg
production at day 1 (24 h) and day 2 (48 h). Day 3
(72 h) and day 4 (96 h) were dierent from all other
days including each other (Keul^Newmann test,
Po0.05, n 5 20).
An increasing trend in egg production in density
300 was observed during all days except between
day 2 (48 h) and day 3 (72 h) (Keul^Newmann test,
Po0.05, n 5 20).
A dierence in egg production in density 400 was
observed between day 1 (24 h), day 3 and day 4 (72
and 96 h), no other dierences were documented
(Keul^Newmann test, Po0.05, n 5 20).
There was a signicant dierence in the egg production between day 1 (24 h) and the last 2 days (72
and 96 h) in density 600. The same coherence in egg

Table 1 Acartia tonsa: stocking density, mortality, mortality rate, egg production and hatching success. Means  SD densitydependent mortality, egg production and egg viability during a 96 h laboratory experiment (n 5 40).
Culture experiment

100 adults L

Stocking density (adults L 1)


Mortality (%)
Mortality rate (% day 1)
Egg production (female 1 day
Egg production (L 1 d 1)
Hatching success (%)

63.2
36.8
15.8
37.3
1121
90.7

 13.1
 13.1
 13.9
 367
 4.7

200 adults L
120.8
39.6
15.1
31.3
1847
86.7

 18.2
 18.2
 11.4
 551
 9.4

300 adults L
202.0
32.4
16.8
26.4
2855
84.2

 31.1
 31.1
 10.3
 989
 7.3

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

400 adults L
300.8
24.80
18.8
30.7
4148
86.3

 43.7
 43.67
 9.4
 1184
 5.3

600 adults L
448.0
25.3
18.7
22.5
5575
84.1

 30.5
 30.5
 8.8
 2123
 8.6

767

Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al. Aquaculture Research, 2007, 38, 764^772

1.5

100

200

300

400

600

100

200

300

400

600

12 hours

24 hours

36 hours

48 hours

60 hours

72 hours

84 hours

96 hours

1.0

0.5

0.0

Eggs female1 hour1

1.0

0.5

0.0

1.0

0.5

0.0

1.0

0.5

0.0

100

200

300

400

600

100

200

300

400

Adult stocking densities (Adult L1)

production was observed between day 2 and the


last 2 days in density 600 (72 and 96 h) (Keul^
Newmann test, Po0.05, n 5 20). The accumulated
egg production reects the total egg production
for each stocking density during the experiment
(Fig. 3). There were neither density nor time-dependent dierences between the egg productions at time
12 h. All other egg production measurements are
characterized by being both dierent from each other
when tested over time and across copepod stocking
densities (Keul^Newmann test, Po0.05, n 5 320).
This gives a steady increase in accumulated egg
production when the adult copepod stocking density
increased.

768

600

Figure 2 Acartia tonsa. Number of eggs produced (egg per


female per hour) for tanks having mean adult stocking densities of 100, 200, 300, 400,
600 adults L 1. Box values
are mean  SE. Error bars are
SD (n 5 5).

Sex ratio
The sex ratio in the ve dierent stocking densities
were tested against each other and the initial value
obtained by subsampling in the renewal culture before the experiment (Fig. 4). The test showed no dierence between the ve densities 100^600 adults L 1,
but the sex ratio in all densities were dierent from
the initial sex ratio (Keul^Newmann test, Po0.05,
n 5 43). There was a tendency to an elevated female
ratio between the termination of the experiment and
the initial ratio (Spearmans rank order correlation
Po0.000002, n 5 43). Thereby, an elevated mortality
of the males during the experiment is indicated.

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

Aquaculture Research, 2007, 38, 764^772 Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al.

6000

5000

100
200

Eggs egg-chamber1

300

4000

400
600

3000

2000

1000

0
0

20

Discussion
The experiment illustrates the relationship among
adult copepod stocking densities, mortality and egg
production. Mortality reported by Fryd, Haslund and
Wolgemuth (1991) for a Centropages hamatus culture
was 16% over the entire experimental period of
400 h, and Berggreen et al. (1988) reported a daily
mortality of 5% for the same culture as used in the
present experiment. In the present experiment, an
average mortality rate of 17.04  1.67% day 1 was
documented. This relatively high mortality rate is
most likely an eect of the handling of the adults
every 12 h during the experiment. Simply avoiding
mechanical manipulation with the copepods during
cultivation can reduce this. During daily care sorting,
water shift, feeding, etc. can be easily optimized with
minimum contact with the animals, thereby fullling the prerequisites for reducing the loss of copepods. This is supported by Drillet et al. (2006) with
handling every second day and a following low mortality.
Adult stocking densities had no eect on the egg
production, in the entire range of 100^600 adult
copepods L 1 during the experiment (Fig. 2). Additionally, the stocking densities had no signicant effect on the egg-hatching success. Overall, the
hatching success of 84.7  4.8% corresponds to the
ranges reported in the literature (Holste & Peck
2005; Drillet et al. 2006).

40

60

80

100

Hours
100%

80%
Percent age of sex

Figure 3 Acartia tonsa.


Accumulated total egg
production across adult
stocking densities over
time (mean  SD; n 5 5).

60%

40%

20%

0%

Initial

100

200
300
Density

Copepodite

Male

400

600

Female

Figure 4 Acartia tonsa. Fraction of copepodites, males


and females of initial density 100, 200, 300, 400 and
600 adults L 1 after 96 h (n 51721).

The observed egg production of 23^37 eggs


female 1 day 1 when densities were100^600 adults
L 1 corresponds to the range reported by Stttrup
et al. (1986) with an average egg production of
25 eggs female 1 day 1 with densities between 50
and 100 copepods L 1. Medina and Barata (2004)
showed a maximum egg production of 32 eggs

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

769

Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al. Aquaculture Research, 2007, 38, 764^772

female 1 day 1 at a nominal density of 500 individuals L 1. However, scrutinizing the study by
Medina and Barata (2004) reveals that at day 12,
where the egg production was identied, a loss of copepods had lead to that only 191  19 individuals L 1 being left, leading to a signicant
discrepancy between nominal and actual copepod
densities. Peck and Holste (2006) reported an egg
production of 40 eggs female 1 day 1 at a stocking
density of 65 adults L 1 and 16 eggs female 1day 1
when 166 adults L 1, but with a decrease to
10 eggs female 1 day 1 at a stocking density of
425 adults L 1. In contradiction, a negative densitydependent egg production was not observed in this
study. Between 100 and 600 adults L 1 there was a
stable and equal egg production. Furthermore, the
accumulated egg production developed with a coherent increase in total egg production as a function of
the increased adult stocking density (Fig. 3). This
leads to the conclusion that in an aquaculture mass
production situation, there is no prevention in operating with a stocking density at 600 adult L 1, still
resulting in a high total egg production with a high
viability. In a pilot study before the present experiment, we stocked our experimental set-up with adult
densities up to 1200 L 1 with the same promising experience as in the present one (data not shown). Drillet et al. (2006) stocked their 2 L incubation bottles to
densities around 3000 adults and with as many as
2000 copepodites of A. tonsa per litre with a low mortality of 0.035^0.13 day 1. They changed the water by
inverse ltration every second day not touching the
copepods. Hence, our recommendation is that future
studies could further challenge the density dependency on egg production by experimenting with
stocking densities of 600 adults L 1 and even higher;
e.g. 41000 adults L 1, to determine the expected
trade-o level.
In the literature, there is no consistent evidence of
the duration of the acclimation period of A. tonsa exposed to sudden changes in dierent vital factors.
Some experiments are simply conducted without
any acclimation period at all (e.g. Andersen, HallingSrensen & Kusk1999; Medina & Barata 2004), while
Berggreen et al. (1988) acclimated the copepods for 2^
3 h before grazing experiments. Thor (2003) acclimated the copepods for 48 h when monitoring oxygen consumption. However, none of the cited studies
report the data showing that the copepods had in fact
reached a steady state. Kirboe et al. (1985) showed
an acclimation period of 3 days where the response
factor was egg production related to time after a shift

770

in food concentration. In this study, the 3-day criteria


for an acclimation period was followed, and after that
measurements were initiated. However, the observed
egg productions still tend to increase as a function of
time (Fig. 2). This supports an extension of the acclimation period concerning the adults respond to the
stocking densities. In an aquaculture situation, the
total realized egg production is the only crucial factor. Hence, an eventual increase in egg production
over time is not considered a practical problem as
long as the applied food is used optimally. The acclimation period could be an area for further exploration, where the synergy between various stress
factors such as shift in food concentration, temperature, salinity, etc. would be investigated in order to
document the time frame for the acclimation period.
An important factor inuencing egg production is
the food, as algae are costly to produce and therefore
important to optimize. In this study, A. tonsa were fed
in excess on a monoculture of R. salina as originally
used in the continuous DFH-ATI A. tonsa culture
(Stttrup et al. 1986) based on inspiration from Klein
Breteler, Schogt and Gonzalez (1990) who was the
founder of this concept. However, Klein Breteler
et al. (1990) always fed with a mixture of Rhodomonas
sp. and the larger heterotrophic agellate Oxhyrrhis
marina. Naturally, food limitation of A. tonsa is a
factor limiting the egg production. The relationship
between egg production and food quantity measured
as algal cells per millilitre follows a saturation curve
(e.g. Berggreen et al. 1988). However, numerous
authors suggest that egg production is limited not
only by food quantity but also by food quality, including possible eects due to particle size, phytoplankton physiological state and biochemical composition
(e.g. Berggreen et al. 1988; Kleppel & Burkart 1995).
Hence, it is necessary to ensure a high-quality feed
in an aquaculture mass production of suspensionfeeding copepods. This is eventually solved with several feed items covering dierent size ranges for nauplii and for copepodites and adult copepods in order
to ensure an optimal somatic growth and nally egg
production. As algal feed is a cost-expensive part of
an intensive production of calanoid copepods elements such as space consumption, electricity for
light sources and the need for man-hours have to be
optimized like for the existing systems used for cultivating the traditional live feed items.
The copepod sex ratio was documented before and
at termination of the experiment in order to determine an eventual gender-specic mortality. However,
no statistical dierences in the sex ratios were

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

Aquaculture Research, 2007, 38, 764^772 Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al.

detected between the densities during the experimental period. Nevertheless, there was a tendency to
a negative correlation between the prior male stock
from the continuous culture and to the terminal
male stock in all stocking densities (Fig. 4). This possible elevated gender-specic mortality is probably
due to an unintended human factor where one might
tend to pick a female before a male when manually
sorting. This is most likely due to their bigger body
size and the easiness by which the female sex can be
determined. Furthermore, the true sex ratio in a
culture can be dicult to determine only by subsampling. The sex dierentiation along the present
experiment is for sure not likely to be caused by genetic drift, due to the relative short experimental period, but in long-term cultivation of A. tonsa genetic
drift probably occurs (Klein Breteler et al.1990; Drillet
et al. 2006). In an aquaculture situation, the optimal
sex ratio is basically least males and most females,
still obtaining a viable egg production. Thereby, the
observed gender-specic mortality has no inuence
in an aquaculture situation as long as there are enough males for mating and remating. The females
are the important organisms that produce eggs while
the males only function is fertilization. As such,
males are besides that just tolerated acting as a feed
expense. The present experiment had a terminate sex
ratio 3:1 (female:male). Holste and Peck (2005) used a
sex ratio of 5:1in their experiment and yielded a hightemperature-specic egg production. Their copepod
sex ratio can probably be applied to density-related
experiments, also obtaining a satisfying egg production and viability. Further experiments should determine the optimal sex ratio leading to high viable
egg production, followed by an eort to manipulate
the sex ratio, which nally could be applied to mass
cultures in the aquaculture industry.

Conclusion
The present experiment showed no decrease in egg
production at adult A. tonsa densities within the
range 100^600 L 1 besides a steady increase in accumulated egg production with adult densities.
Furthermore, the viability of the eggs produced at
the various adult densities was similar. A recommendation for mass cultivation of A. tonsa and possibly
other small calanoid copepods is therefore that densities of up to 600 adults L 1 and presumably even
more dense cultures are promising. We propose that
the next step for reaching a protable rearing techni-

que of calanoid copepods for live feed are (1) optimizing the use of a mixture of phytoplankton algae by
feeding ontogenetically advanced copepod stages
with successively larger food items, (2) manipulating
the sex ratio.

Acknowledgments
Thanks are due to Jack Melby, Danish Institute for
Fisheries Research, Department of Ecology and Aquaculture for delivery of copepod eggs. We also thank
Guillaume Drillet,Thomas F. Srensen and Anne Faarborg for technical assistance with the copepod eggs.
The study was supported by the Danish National Research Council (grant no. 21-01-0549) to B.W. H.

References
Andersen H.R., Halling-Srensen B. & Kusk K.O. (1999) A
parameter for detecting estrogenic exposure in the copepod Acartia tonsa. Ecotoxicology and Environmental Safety
44, 56^61.
Berggreen U., Hansen B. & Kirboe T. (1988) Food size
spectra, ingestion and growth of the copepod Acartia tonsa during development: implication for determination of
copepod production. Marine Biology 99, 341^352.
Drillet G., Iversen M.H., Srensen T.F., Ramlv H., Lund T. &
Hansen B.W. (2006) Eect of cold storage upon eggs of a
calanoid copepod, Acartia tonsa (Dana) and their ospring. Aquaculture 254,714^729.
Fryd M., Haslund O.H. & Wolgemuth O. (1991) Development,
growth and egg production of the two copepod species
Centropages hamatus and Centropages typicus in the
laboratory. Journal of Plankton Research 13, 683^689.
Hansen P.J. (1989) The red tide dinoagellate Alexandrium
tamarense: eects on behaviour and growth of a tintinnid
ciliate. Marine Ecology Progress Series 53, 105^116.
Holmefjord I., Bolla S. & Raitan K.I. (1989) Start feeding of
Atlantic halibut (Hippoglossus hippoglossus L.) on enriched
rotifers and Artemia compared with collected plankton.
In: The Early Life History of Fish. The Third ICES Symposium, Bergen, 3^5 October 1988 (ed. by J.H.S. Blaxter, J.C.
Gamble & H.VonWesternhagen) Rapp. P-V. Reun. Ciem.
Holste L. & Peck M.A. (2005) The eects of temperature and
salinity on egg production and hatching success of Baltic
Acartia tonsa (Copepoda: Calanoida): a laboratory investigation. Marine Biology 148,1061^1070.
Hurlbert S.H. (1984) Psedoreplication and the design of ecological eld experiments. Ecological Monographs 54,187^211.
KirboeT., Mhlenberg F. & Hamburger K. (1985) Bioenerginetics of the planctonic copepod Acartia tonsa: relation
between feeding, egg production and respiration and
composition of the specic dynamic action. Marine Ecology Progress Series 26, 85^97.

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

771

Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al. Aquaculture Research, 2007, 38, 764^772

Klein Breteler W.C.M., Schogt N. & Gonzalez S.R. (1990) On


the role of food quality in grazing and development of life
stages, and genetic change of body size during cultivation
of pelagic copepods. Journal of Experimental Marine Biology and Ecology 135, 177^189.
Kleppel G.S. & Burkart C.A. (1995) Egg production and the
nutritional environment of Acartia tonsa: the role of food
quality in copepod nutrition. ICES Journal of Marine
Science 52, 297^304.
Lubzens E., Minko G., Barr Y. & Zmora O. (1997) Mariculture in Israel ^ past achievements and future directions
in raising rotifers as food for marine sh larvae. Hydrobiologia 358,13^20.
Medina M. & Barata C. (2004) Static-renewal culture of Acartia tonsa (Copepoda: Calanoida) for ecotoxicological testing. Aquaculture 229, 203^213.
Nss T., Germain-Henry M. & Naas K.E. (1995) First feeding
of Atlantic halibut (Hippoglossus hippoglossus) using different combination of Artemia and wild zooplankton.
Aquaculture 130, 235^250.
Nss T. & Lie . (1998) A sensitive period during rst feeding for the determination of pigmentation pattern in halibut, Hippoglossus hippoglossus L., juveniles: the role of
diet. Aquaculture Research 29, 925^934.
Ogle J. (1979) Adaptations of a brown water culture technique to the mass culture of the copepod Acartia tonsa. Gulf
Research Reports 6, 291^292.
Payne M.F. & Rippingale R.J. (2001) Eects of salinity, cold
storage and enrichment on the calanoid copepod Gladioferens imparipes. Aquaculture 201, 251^262.

772

Peck M.A. & Holste L. (2006) Eect of salinity, photoperiod


and adult stocking density on egg production and egg
hatching success in Acartia tonsa (Calanoida: Copepoda):
optimizing intensive cultures. Aquaculture 255,
341^350.
Shields R.J., Bell J.G., Bromage N.R., Gara B., Luizi F.S. & Sargent J.R. (1999) Natural copepods are superior to enriched
Artemia nauplii as fed for larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids. Journal
of Nutrition 129, 1186^1194.
Sterns D.E.,Tester P.A. & Walker R.L. (1989) Diel changes in
the egg production rate of Acartia tonsa (Copepoda, Calanoida) and related environmental factors in two estuaries.
Marine Ecology Progress Series 52,7^16.
Stttrup J.G., Richardson K., Kirkegaard E. & Pihl N.J.
(1986) The cultivation of Acartia tonsa Dana for use as a
live food source for marine sh larvae. Aquaculture 52,
87^96.
Thor P. (2003) Elevated respiration rates of the neritic
copepod Acartia tonsa during recovery from starvation.
Journal of Experimental Marine Biology and Ecology 283,
133^143.
Wilcox J.A., Patrick L.T. & Marcus N.H. (2006) Improving live
feeds: eect of a mixed diet of copepod nauplii (Acartia
tonsa) and rotifers on the survival and growth of rstfeeding larvae of the southern Flounder, Paralichthys
lethostigma. Journal of the World Aquaculture Society 37,
113^120.

r 2007 The Authors


Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 764^772

You might also like