KEY WORDS: adaptation, Artemia salina, brine shrimp, effect of temperature, filter-feeding, ... ies, or used in aquaculture hatcheries and by aquarium hob-.
J OURNAL OF C RUSTACEAN B IOLOGY, 35(5), 650-658, 2015
ADAPTATION OF THE BRINE SHRIMP ARTEMIA SALINA (BRANCHIOPODA: ANOSTRACA) TO FILTER-FEEDING: EFFECTS OF BODY SIZE AND TEMPERATURE ON FILTRATION AND RESPIRATION RATES Hans Ulrik Riisgård 1,∗ , David Zalacáin 1 , Nathanaël Jeune 1 , Jan Brandt Wiersma 1 , Florian Lüskow 1 , and Daniel Pleissner 2 1 Marine
Biological Research Centre, University of Southern Denmark, Hindsholmvej 11, 5300 Kerteminde, Denmark of Bioengineering, Leibniz-Institute for Agricultural Engineering Potsdam-Bornim e. V., Max-Eyth-Allee 100, 14469 Potsdam, Germany
2 Department
ABSTRACT In spite of wide use of the brine shrimp, Artemia salina (Linnaeus, 1758), as feed and model organism in evolutionary, ecological, physiological, and ecotoxicological investigations, only a few studies have attempted to quantify filtration and respiration rates in order to characterize A. salina as a filter-feeder. Herein, we measured that the maximum filtration rate (F , ml/h) as a function of body length (L, mm) can be expressed by means of two equations, one that applies for small ( 10 l of water filtered per ml of O2 consumed (Jørgensen, 1975; Riisgård and Larsen, 2000). The aim of the present study was to measure the maximum filtration rate as a function of body size of A. salina, to measure the effects of temperature on filtration rate and respiration rate, and further to lay down the relationship between body size and starvation respiration rate. Finally, it has been a goal to characterize A. salina as a filter-feeder by means of the F /R-ratio.
where V = volume of seawater, n = number of A. salina, and b = slope of regression line in a semi-ln plot for the reduction in algal concentration with time in the beaker. In the present work it has been assumed that the about 6 μm diameter R. salina-cells are 100% efficiently retained by the Artemiafilter-setae where the intersetular distance has been measured to be about 1.5 μm in the present work (see also Mertens et al., 1991; Makridis and Vadstein, 1999; Williams, 2007). The clearance method is quick, simple and reliable; a prerequisite for its use is full mixing and a constant water volume, while the disadvantage is that the concentration of particles is declining during the experiment. These drawbacks can be eliminated by making new algal additions to keep the concentration within a certain range, and by transferring all water samples back to the beaker after measurement (which removes only about 1.5 ml water from the sample). All experiments were conducted at 21.4 ± 1.7°C.
M ATERIALS AND M ETHODS
Cultivation of Algae
Experimental Animals
Rhodomonas salina (about 6 μm in diameter) was grown phototrophically in repeated batch cultures at 20°C in 10-l bottles containing 9 l of sea water (20 psu) enriched with f/2-medium. Cultures were continuously illuminated by fluorescent light tubes. Aeration and mixing were carried out by injection of compressed air. Every day, 3 l of algal suspension was withdrawn and replaced by fresh medium.
Because Artemia is important for aquaculture, the trade name “Artemia salina” is being widely used for any population in this genus, although the study of Asem et al. (2010) indicates the existence of 7 species, 3 living in the Americas, one in Europe, and 3 in Asia. Due to the taxonomic confusion, we have used the trade name in the present study. Artemia salina used in the present experiments were cultivated from commercially manufactured brine shrimp eggs (cysts) harvested from salt lakes of Siberia (‘Artemia Koral,’ Biotrade, Barnaul, Russia). The eggs hatched after 1 to 2 days when added to a bucket with bio-filtered seawater (20 psu), and algal cells (Rhodomonas salina) were subsequently added in relatively high concentrations (>100,000 cells/ml) every day. The cultivation bucket was mixed by means of air-stones, and the water was changed every week to ensure good water quality. The mean ± SD body length of the experimental animals (n = 10) was measured using a stereo-microscope (Leica MZ 125) every day during the growth period of about 3 weeks. Under optimal conditions the growth was exponential during the first 2 to 3 weeks after which the adult stage was obtained (Fig. 1, see also Mason, 1963). During the growth period individuals from the cohort, with about the same body length, were transferred to glass beakers for measurement of the filtration rate. Measurement of Filtration Rate The filtration rate of A. salina was measured using the clearance method, where the filtration rate is measured as the volume of water cleared of suspended particles that are 100% efficiently retained by the filter per unit of time. The reduction in the number of particles as a function of time was followed by taking water samples at fixed time intervals (every 5 or 10 minutes) from a glass beaker with well-mixed 500 ml seawater (20 psu) with a known number of A. salina of same size, i.e., same age, added algal cells (R. salina) and measuring the particle concentration with an electronic particle counter (Elzone 5380). The filtration rate (F ) was determined from the exponential decrease in algal concentration (verified as a straight line in a semi-ln plot) as a function of time using the formula (Riisgård et al., 2011): F = V × b/n,
(1)
Filtration Rate Experiments For performing a filtration rate experiment, a sample of water with A. salina of same age (and size) was taken from the cultivation flask and transferred to a glass beaker and supplemented with a known volume of bio-filtered seawater which was mixed by means of an air stone. The total number of A. salina in the beaker was exactly counted by making series of water drops with animals from the culture and subsequently counting the number of individuals in each drop, before the drops were washed into the experimental beaker. The number of individuals used in an experiment was adjusted so that a decreasing number of larger individuals (with increasing filtration rate) were added to the same volume of seawater (usually 500 ml) in order to ensure that the algal concentration decreased to about 50% within 30 to 60 minutes. Then algal cells were added to the seawater to make up the desired initial algal concentration in the experiment, and subsequently the reduction in algal concentration was followed in order to measure the filtration rate using Eq. (1). In most filtration rate experiments, 2 or 3 repeated algal additions were made (see below). Effect of Temperature on Filtration Rate An experimental beaker with 150 ml seawater and A. salina, and a control beaker without animals, both with gentle air mixing, were placed in a temperature-controlled 5 l water-bath. In the first series of experiments (Series 1), starving brine shrimps were acclimated for 16 hours to 15°C previous to measurement of the filtration rate at 15, 20, 25, and 30°C, and then backwards from 30 to 25, 20, and 15°C, respectively, and the new temperature was established within 10 minutes by adjusting the temperature of the water-bath. In the second series (Series 2) of experiments, starving brine shrimps were acclimated for 16 hours to 30°C previous to measurement of the filtration rate at 25, 20, and 15°C, and then backwards from 15 to 20, 25, and 30°C. In this way, an experimental series with one size group of A. salina lasted about 380 minutes. The volume of water was initially adjusted so that the half-life of added R. salina was about 30 minutes. At each new temperature, algal cells were added to establish an initial concentration of about 2000 cells/ml in the experimental beaker. The algal concentration of samples taken from the experimental and control beakers were counted with the electronic particle counter. At each temperature, 5 samples were taken with 10 minutes intervals in order to determine the filtration rate according to Eq. (1). The volume of water removed from the beakers, i.e., sucked in by the particle counter, was approximately 4 ml per sample and therefore, after each specifictemperature experiment of 40 minutes, the initial volume of water in the experimental beaker (150 ml) was re-established by adding new seawater. Respiration Rate
Fig. 1. Artemia salina. Mean body length (± SD) a function of time of animals fed algal cells (Rhodomonas salina) in a growth experiment where eggs were added to growth tank with sea water on Day 0. The newly hatched and juvenile animals growth exponentially during the first 16 days and then succeeded by stagnation in the adult phase of the animal’s life cycle.
The respiration rate of A. salina was measured as the consumption of oxygen using a fibre-optic oxygen meter (FireSting O2 , Pyro Science, Aachen, Germany) for measurement of the dissolved oxygen (DO) concentration in a closed respiration chamber. The fibre-optic oxygen meter, using REDFLASH technology, was connected via a USB-cable to a computer with
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 35, NO. 5, 2015
Pyro Oxygen Logger© software installed. Red light (λ = 620 nm) excited REDFLASH indicators show luminescence in the near infrared (NIR), which decreases with increasing oxygen concentration. The analogous signal is transported via a fibre-optical cable and shown digitally in the software as DO concentration. For oxygen sensing 3 channels were used while an external temperature sensor was running. Prior to respiration measurements a two-point calibration (100% oxygen saturation and full oxygen depletion to 0% by adding Na2 S2 O2 ) was conducted. The optical fibre was placed at the wall of the respiration chamber (V = 4 ml) on top of a REDFLASH spot and the DO concentration (mg O2 /l) was recorded in the Pyro Oxygen Logger© software every 10 seconds. Preliminary experiments prior to the respiration measurements showed that mechanical stirring to supplement the mixing caused by swimming A. salina was not needed for obtaining a homogenous DO concentration. A control with bio-filtered seawater without A. salina was conducted parallel to the respiration measurements. Depending on the size of A. salina, the number of individuals in the respiration chamber was adjusted. All measurements were conducted with respiration chambers submersed in a temperature-controlled water bath to minimise temperature variations. Salinity in respiration chambers was the same as in A. salina cultures. Fully oxygenated bio-filtered seawater (20 psu) was used in respiration chambers. The DO concentration was plotted as a function of time, and expressed by a linear regression line. The starvation respiration rate (μg O2 /day) was calculated as: R = b × V /n,
(2)
where b = slope of the regression line, V = water volume (4 ml) in respiration chamber, n = number of A. salina. The oxygen consumption in the control chamber was subtracted from R to obtain the true respiration rate of the A. salina which were starved for 16 hours before the measurements. The respiration rate was measured in individuals from 5 size classes of A. salina cultivated during several weeks on a diet of R. salina. After the respiration measurements, the body length of 10 A. salina was measured under a stereo-microscope (Leica MZ 125) to the nearest 0.1 mm.
Fig. 3. Artemia salina. Decreasing filtration rates at high algal concentrations. Semi-ln plot of algal concentration (C, cells/ml) as a function of time in a series of clearance experiments with repeated algal additions (indicated by different symbols). The linear regression lines and their equations for the different series are shown. See also Table 2. 0.5499. The regression line in Fig. 4 for larger animals was separated into 3 regression lines with following slopes: 1.3339, 1.7761 and 2.1324. In both experiments (Figs. 3 and 4), the t-test was applied in order to investigate the statistical difference between groups. Statistical difference was accepted for P < 0.05. Analysis of Covariance (ANCOVA) was carried out in Systat and used for investigation of slopes of associations of body length and respiration rate (Table 5, Fig. 8). Hypothesis of significant difference of slopes was accepted for P > 0.05.
One-way analysis of variance (OWANOVA) and t-test were carried out in SigmaPlot and used to investigate if slopes of regression lines are statistically different or not (Figs. 2, 3, 4, 5). Slopes in Fig. 2 were separated in 3 groups, group 1: −0.019 and −0.0186, group 2: −0.0214 and −0.0217, group 3: −0.0172 and −0.021. Slopes in Fig. 5 were separated according to the temperatures used in experiments. In both cases, the difference of slopes was investigated using OWANOVA. Statistical difference was accepted for P < 0.05. In order to investigate the statistical difference of slopes in Fig. 3, slopes were separated in two groups, group 1: −0.0066, −0.0048, −0.0058 and −0.0056 (Series 1 to 4, Table 2), and group 2: −0.0032, −0.0031 and −0.0031 (Series 5 to 7, Table 2). The regression line in Fig. 4 for animals with a body length < 2.5 mm was separated into two regression lines with following slopes: 0.8629 and
R ESULTS Filtration Rate Experiments Figure 2 shows a typical clearance experiment with A. salina where the algal concentration was measured as a function of time in 6 series of measurements with repeated algal additions and plotted in a semi-ln plot of algal concentration versus time. The linear regression lines and their equations for the different series are shown along with a control experiment without animals. The mean estimated individual filtration rate of the 2.32 ± 0.39 mm long A. salina was found to be 1.17 ± 0.12 ml/h at a mean algal concentration of about 3500 cells/ml (Table 1).
Fig. 2. Artemia salina. High and constant filtrations rates at low algal concentrations. Semi-ln plot of algal concentration (C, cells/ml) as a function of time in a series of clearance experiments with repeated algal additions (indicated by different symbols). The linear regression lines and their equations for the different series are shown. See also Table 1.
Fig. 4. Artemia salina. Filtration rate (F ) as a function of body length (L). Two regression lines have been shown, for small ( 0.05). However, in other clearance experiments where the initial algal concentration was increased above about 5000 cells/ml, a reduction in the filtration rate could be noticed after some time. Thus, Fig. 3 shows the effect of increasing the repeated initial algal concentration to about 10,000 cells/ml during a 7.5-hourlong feeding experiment. After about 4 hours the filtration rate was reduced by 50%, from 0.21 to 0.10 ml/h (Table 2). The t-test revealed that slopes of Series 1 to 4 and Series 5 to 7 (Table 2) are statistically different (P < 0.05). Therefore, in order to measure the maximum filtration rate of A. salina as a function of size the algal concentrations were always kept below 5000 cells/ml. Figure 4, based on data in Table 3, shows that the maximum filtration rate (F , ml/h) at 21.4 ± 1.7°C as a function of body length (L, mm) can be expressed by means of two regression lines, one that applies for small ( 0.05).
Table 5 shows the estimated F /R-ratio which expresses the litres of water filtered per ml of oxygen consumed (i.e., l H2 O/ml O2 ). The filtration rates at 25 and 15°C have been estimated from equations for 22°C shown in Fig. 4 and subsequently corrected for deviating higher or lower temperature using that F25°C /F15°C = 1.53 ± 0.19 (Table 4), or a factor of ((1 + 3) × 0.53/10) = 1.159 and ((1 − 7) × 0.53)/10) = 0.629 for 3 and 7°C higher and lower temperatures, respectively. It is seen that the F /R-ratio is 7.4 ± 3.3 and 6.6 ± 2.6 l H2 O/ml O2 at 25 and 15°C, respectively. D ISCUSSION Filtration Rates It is interesting to note that the filtration rate as a function of body length of A. salina has to be expressed by means of two regression lines, one that applies to small (
