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SUPPORTING INFORMATION
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Appendix S1. Sealing of microplate wells
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For sealing of microplate wells we used transparent adhesive PCR film (Thermo Scientific, USA).
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The characteristics of this film are ideal for our application, as they allow inspection of wells
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following sealing, and the glue used does not influence the animals negatively and does not stick to
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the wells upon removal of the film (own observations). However, animals may get stuck to the film
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if they come into contact with the glue during the experiment. Thus, to avoid this we made
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transparent plastic discs with a diameter equal to that of the inner walls of the wells, washed these
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with ethanol, and distributed them on the film at locations corresponding to the locations of the
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wells on the plate (Fig. S1). This was done by placing the film inverted on a photocopy of the plate,
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which showed the exact positions of the wells and thus allowed accurate placements. For sealing of
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the wells, the prepared film with plastic discs was applied to the plate while making sure that the
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discs fit into their respective wells. Then, to ensure efficient and consistent sealing, pressure was
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applied sequentially on top of each well by the use of a glass tube with the same diameter as the
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wells. Using this method prevents effects of pressure on oxygen readings due to the flexibility of
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the film, but allows some diffusion into wells during experiments. We estimated this diffusion and
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accounted for it when calculating Daphnia oxygen consumption rates (Appendix S3).
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Appendix S2. Selection of measurement interval
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Based on pilot experiments we evaluated oxygen consumption estimates when using different
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measurement intervals. The quality of such estimates may be compromised by a change in activity
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of the organisms during the period. This may occur for example if the animals show an initial stress
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response, or if oxygen concentrations are allowed to become sufficiently low toward the end of the
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period to cause a reduced (or increased) metabolism. Thus, one of the selection criteria should be to
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what extent the decline in oxygen concentrations over time deviates from linearity. Furthermore, the
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interval should be of sufficient length to produce low uncertainty in slope estimates, and this can be
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evaluated by the standard error of the estimates. For our set-up the period 20 – 80 minutes
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performed best with respect to these criteria. To evaluate the degree of curvilinearity in the decrease
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of oxygen over time within this interval for the final data we compared the AIC value of linear vs.
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quadratic models for all wells in all runs. For all of these, linear models outperformed (i.e. had
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lower AIC values than) curvilinear ones, and the smallest AIC difference for any well was 18.9.
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Furthermore, the uncertainties in slope estimates for the linear models were small over this interval,
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with standard errors being on average 5.3% of the estimates. Fig. S2a shows an example of declines
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in oxygen content of wells from one of the experimental runs obtained from the SDR v38 Software
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(PreSens, Germany), and Fig. S2b the control adjusted declines during the chosen measurement
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interval. To further evaluate whether oxygen consumption rate was influenced by changes in
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oxygen concentration during the chosen 60 minute period we used the data from the experiment
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testing for effects of density during respirometry (where total biomass, and hence declines in
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oxygen concentration was highest). We calculated consumption for each replicate well during the
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first and last 30 minutes of the chosen period and compared these using a paired samples t-test.
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Mean ± SD consumption during the first and second interval was 0.201 ± 0.145 and 0.216 ± 0.143
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µg h-1, respectively, and were not significantly different (t = 1.92, df = 173, P = 0.057).
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Furthermore, the change in consumption from the first to the second 30 minute interval (i.e.
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consumption first interval – consumption second interval) was independent of the number of
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individuals in a well (F3, 170 = 0.20, P = 0.894) and total body mass (F1, 172 = 0.41, P = 0.524),
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which would also be expected if oxygen consumption depended on oxygen concentrations. Thus,
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declines in oxygen concentration during experiments did not influence consumption.
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Appendix S3. Estimating and correcting for oxygen diffusion into wells during experiment
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To estimate oxygen diffusion into wells we filled wells with undersaturated (by boiling) water that
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had been brought to 17°C. The wells were then sealed and readings were taken inside the incubator
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at 17°C with 5 min intervals for 48 hours, by which time the undersaturated water had regained
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100% oxygen saturation. For each reading for each well we calculated the deviation from final
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values that was due to undersaturation, ΔO2 (mg). According to Fick’s first law, diffusion rate into
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wells will be proportional to this undersaturation. Thus, we fitted the model D = kΔO2, where D (mg
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s-1) is the rate of change in O2 content due to diffusion, and k (s-1) is an empirically obtained
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diffusion constant that is specific to the experimental conditions (e.g. temperature, type of wells and
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sealing used). Assuming that oxygen consumption by the animal is constant (see Appendix S2), the
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deviation in oxygen content due to undersaturation during an experiment can be expressed as ΔO2 =
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bt, where t is time elapsed since the sealing of the well (s), and b is the observed slope of the
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function describing how oxygen content changes through time during an experiment due to
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consumption (mg s-1). The rate of diffusion can then be written as D = kΔO2 = kbt. To calculate the
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total amount of oxygen diffusion during the measurement period this function is integrated over the
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given period from t1 to t2:
t2
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 kbt  2  t
kb
2
2
 t12 
t1
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The diffusion will lead to an underestimation of oxygen consumption if not corrected for, and
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should therefore be added to the apparent consumption measured as b(t2-t1). However, given that
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the diffusion constant k and measurement interval is constant across experimental treatments, the
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magnitude of the underestimation (i.e. the proportional relationship between diffusion and true
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consumption) is constant and independent of the slope b:
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kb 2
k 2
t2  t12 

 t2  t12 
Diffusion
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2


kb
k
Consumption
b  t2  t1    t2 2  t12   t2  t1    t2 2  t12 
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2
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It is important to note from this however that increasing the duration of the interval will increase the
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proportional contribution of diffusion (as the interval length goes towards infinity the above
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expression approaches 1). Thus, if diffusion in such experiments occur but is not accounted for, the
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true consumption will be underestimated more for longer measurement intervals. For our set-up the
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estimate of the diffusion constant k was 0.00004143 s-1, and with the given constant measurement
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interval this would cause the apparent consumption (ignoring diffusion) to be 11% lower than the
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diffusion-corrected consumption.
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Figure S1. Sealing of microplate wells with PCR film. A. Plastic discs of the same diameter as
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inner walls of the wells are placed on the film according to the photocopy of the plate. B. After
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removing the plastic cover of the film, discs are pushed into the glue of the film with a blunt
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instrument to make sure that there is no air between the disc and the film (such bubbles are difficult
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to distinguish from any bubbles appearing inside the well). The film cover may be replaced back for
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storage if the film is not used immediately. C. Film is applied on the wells overfilled with ADaM.
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D. Pressure is applied sequentially on top of each well using a glass tube. E. Side view scheme of
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the sealed well with Daphnia. F. Top view of the sealed well with Daphnia. Wells can be easily
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inspected for air bubbles and Daphnia activity. G. Schematic drawing of the 24-wells plate
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equipped with planar oxygen sensor spots and sealed with PCR film, plastic discs positioned on top
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of each well. GT – glass tube, PF – PCR film, S – sensor spot, L – plastic disc.
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Figure S2a: Screen shot of the output from the SDR SensorDish® Reader (PreSens, Germany)
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showing decline in oxygen concentration over time in 24 wells. Wells A1, B1, B4, C4 and D6 were
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control wells (without animals), and the remaining wells each contained a single individual D.
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magna. The higher rate of decline during the initial period is caused by temperature changes (being
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slightly raised above the measurement temperature of 17°C during preparation of the plate).
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Because this effect is common to both the control wells and wells containing animals it can be
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accounted for when calculating consumption.
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Figure S2b: Declines in oxygen concentrations of the same wells as those shown in Fig. S2a over
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the measurement period used for calculations of consumption (20 – 80 minutes after sealing wells).
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Data shown here are calculated while accounting for the mean change observed in control wells.
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