Data and code from: The temperature-size rule in Daphnia magna across different genetic lines and ontogenetic stages: multiple patterns and mechanisms

2 MATERIALS AND METHODS 2.1 Collection and maintenance of animals Daphnia magna is a small cladoceran crustacean that inhabits freshwater habitats. Its short life cycle and abundance have made this species a popular species for studies on life-history and energy budgets (Martínez-Jerónimo, Villaseñor, Rios, & Espinosa, 1994). Resting eggs of Daphnia magna were collected from a small lake in Hilversum, The Netherlands, in spring 2014. These resting eggs are produced after sexual reproduction with hatchlings being genetically different from each other (Robinson, Wares, & Drake, 2013). Resting eggs were kept at continuous darkness and 4°C for at least 3 months before they were hatched in the laboratory to give rise to different genetic lines of which two were used in this study. These two genetic lines were designated lines D and E. Newly produced clonal neonates from these lines were separated until 5–10 vials per genetic line contained offspring of that line. A third genetic line was obtained from a laboratory at Wageningen University, The Netherlands, where it had been kept at room temperature for 15 years since collection from Lake Zwemlust, The Netherlands (Lürling & Tolman, 2010). This laboratory line was designated line C. The stock culture of D. magna in our laboratory was kept at a constant temperature of 10°C and 16:8 hr light:dark. Fourteen days before the onset of the experiment (May 2015), five juveniles of each of the three genetic lines were separated into 80-ml glass vials with Dutch standard water (DSW, 200 mg/L CaCl2.2H2O, 180 mg/L MgSO4.7H2O, 100 mg/L NaHCO3, 20 mg/L KHCO3; NEN 1980) and kept at 20°C. Offspring from these mothers’ second clutch onward were used for the actual experiment in accordance with OECD guidelines (Dufresne & Hebert, 1998; OECD/OCDE 2012) and individually and randomly assigned to one of the eight temperature treatments (10, 15, 18, 20, 23, 26, 28, 30°C). At least five individuals from each of the three genetic lines were placed in each temperature, and individuals that died within the first seven days were replaced resulting in a total of 216 individuals in the experiment. Survival, growth, development, and reproduction were monitored three times per week for each individual from birth to death. The medium consisted of 1.6 × 105 cells/ml (McKee & Ebert, 1996; Algal diet 1800) suspended in DSW and was replaced at each measurement to ensure ad libitum food conditions. 2.2 Measurement of physiological rates Length of each individual D. magna was measured to the nearest 0.04 mm three times per week using a stereo microscope with 25 times magnification. The frequency of measurement caused an uncertainty in age estimates of approximately 1 day. Length was measured in live animals from the middle of the eye to the base of the caudal spine following Chopelet, Blier, and Dufresne (2008). Movement of the eye and contraction of the body were sometimes observed to cause a small (maximum 0.12 mm) inaccuracy in the length measurement. Width and thickness of D. magna were measured for a representative set of animals to derive an equation for converting length into volume (Equation 1). We assumed a constant weight-to-volume ratio for D. magna, so that volume could be used as a proxy for weight. The unit volume was used for statistical analyses and for fitting growth curves. The term body size in this study always refers to (calculated) body volume. Individual Daphnia that lived at least 20 days at 10°C or 15°C, or 15 days at 18, 20, 23, 26, 28 or 30°C, were used for characterization of individual growth trajectories (105 in total) and also for fitting the resource allocation model. To estimate asymptotic size, a modified von Bertalanffy growth function for body size (Equation 2) was fitted to data based on individual Daphnia using the nonlinear least square function nls() in R. Of the four parameters in Equation 2, Vmax and D were estimated and V0 and K were constants. (1) (2) where length is in mm, V0 is the volume at age zero, and Vmax is the estimated asymptotic volume. We assumed a 10% increase in length on the first day, which results in a volume at birth of 0.7 × volume at first measurement. K is the von Bertalanffy growth parameter (which is not actual growth in the sense of mm/d, but the rate of change in the slope). We fixed (instead of estimated) K at a value of 0.046 (see Appendix S1) because of its correlation with Vmax (Pauly, 1979). D = 3 × (1 − d) and can be interpreted as a parameter to correct for nonisometric growth of gill surface (Pauly, 1981), where d is the mass scaling exponent for catabolism (von Bertalanffy, 1934). Time is age of the individual in days, with the first measurement at day 1, which was 0–72 hr after release from the mother's brood chamber. The maximum slope of the estimated individual growth curve was used as maximum growth rate (g/d). The first day when eggs or neonates were observed was taken as the moment of maturity. Animals were considered juvenile before this point and adult beyond this point. Length and age were noted as above. When neonates were observed before eggs were observed (embryonic development occurring between two measurements), age at maturity was approximated by subtracting 2 days from the age at first neonates. Development rate is the inverse of time taken to reach maturity and thus has unit day−1. Rates of growth and development were standardized by expressing them as a percentage of the maximum growth rate (0.56 mm3/d) and maximum development rate (0.20 d−1) observed in this study, and subsequently compared by calculating the ratio between these standardized rates. Free-living neonates were counted at the first measurement day after each release, and lengths of three neonates per clutch were measured. Young were then removed from the experimental unit.