Appendix A. Validation of the comparability of visual and isotope methods for estimating gut passage time.
Methods
We conducted an experiment to compare the visual and isotope methods for estimating gut passage time (GPT) using surface water samples collected between early July and early August from eight lakes in southwest Michigan that varied widely in seston concentration (0.39 to 1.75 mg C/L). Methods for collecting the samples and measuring particulate organic carbon content followed DeMott and Tessier (2002). Measurements using the two methods were conducted on a single day with the same water sample for each lake. Each trial was conducted with a cohort of 4-day-old Daphnia pulex that had been acclimated to experimental conditions for at least 1 h.
The visual method used carmine red particles as a tracer as in this study. Estimates are the means of individual trials for 20 animals for seven lakes and 10 animals for the eighth lake.
The isotope method is based on the inflection point in the uptake of radioactively labeled food over time (Geller 1975). Methods for labeling seston with 32P-phosphate and measuring radioactivity in animals and their food followed DeMott and Tessier (2002). To start each trial, a negligible volume of radioactively labeled seston was added to a beaker with about 30 animals feeding in 500 mL of seston. At periodic intervals three animals were removed from the beaker to measure net uptake of the radioactive isotope. The time interval ranged from 2–5 min during the first 20 min and 5–20 min thereafter, for a total of 30–80 min for 7–10 samples. Since GPT increases with decreasing food concentration, longer time intervals and total time were used for resources from lakes with lower food concentrations. An initial experiment with anesthetized animals showed negligible adsorption of radioactive isotope by the nonfeeding animals. A data point for zero uptake at time zero was therefore included in the analysis of each replicate.
The net isotope uptake versus time data were fit to two linear regressions (Geller 1975). The initial, steeper line is a measure of the ingestion rate, whereas the second line, with a lower slope, is a measure of the assimilation rate. The intersection of the two lines marks the beginning of defecation of radioactively labeled food and is therefore an estimate of GPT. Each estimate is the mean for three replicate beakers for a lake.
Results
We found a tight linear relationship between the isotope and visual methods for estimating GPT (Fig. A1; linear regression, R2 = 0.93; P < 0.001; visual estimate = 0.98* isotope estimate + 0.10). Thus, our results show that the two methods give comparable values for the passage of undigested particles through the gut.
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FIG. A1. Comparison of the visual and isotope methods for estimating gut passage time. Each symbol represents an estimate for one of eight lakes. The solid line is a linear regression whereas the dotted line has a slope of 1.0 and an intercept of zero. |
LITERATURE CITED
DeMott, W. R., and A. J. Tessier. 2002. Stoichiometric constraints vs. algal defenses: Testing mechanisms of zooplankton food limitation. Ecology 83:3426–3433.
Geller, W. 1975. Die Nahrungsaufnahme von Daphnia pulex in Abhaengigkeit von der Futterkonzentration, der Temperature, der Korpergrosee und dem Hungerzustand der Tiere. Archiv für Hydrobiologie, Supplement 48:47–107.