Professional Documents
Culture Documents
doi:10.1111/j.1365-2109.2007.01730.x
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.
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
Aquaculture Research, 2007, 38, 764^772 Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al.
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
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.
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
Cultivation parameters
The temperature, oxygen and algal concentration
were tested during experimental time. The mean
Aquaculture Research, 2007, 38, 764^772 Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al.
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).
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
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
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
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
768
600
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.
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
60%
40%
20%
0%
Initial
100
200
300
Density
Copepodite
Male
400
600
Female
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
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.
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Eect of stocking density of the calanoid copepod Acartia tonsa P M Jepsen et al. Aquaculture Research, 2007, 38, 764^772
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