These data are associated with a study that tests the hypothesis of thermal plasticity in the rigid setae of the second limb of Daphnia. The data are body size and length measurements (mm) of three limb setae (limb 2; endite 2). The second seta is often called the "rigid" seta because it is heavily chitinized. In all, 300 specimens of Daphnia (including Daphnia umbra and Daphnia galeata) were dissected from four sets of clonal thermal transfers and seasonal variants from nature. Specimens of D. galeata were cultured under identical conditions save temperature (10 and 20◦C). Clonal neonates were reciprocally transferred across temperature regimes to determine the degree of postnatal plasticity.

Midsummer and Midwinter samples of Daphnia galeata were collected by a zooplankton throw net (30 cm dia., 200-250 um mesh size) from Guelph Lake, a eutrophic reservoir in Guelph Ontario, Canada, and Lake Ontario near Rochester, New York. Midwinter specimens were collected when the lake was covered with ice. We used ethanol preserved specimens of Daphnia umbra from a pond on the northern Melville Peninsula, Nunavut, Canada in August.

To quantify the seasonal differences in seta lengths, midsummer and midwinter specimens (i.e., the coldest and warmest lake temperatures) were collected and mature females (ovigerous females) were photographed in lateral view and then dissected to remove and mount the second thoracic limbs. To test for phenotypic plasticity of the rigid setae, ovigerous specimens of D. galeata from cold water conditions (winter; 4–5◦C) were transferred to individual glass vials at 20◦C and 24-h light photoperiod until they released their embryos. The first mature adults were then preserved for measurement and dissection (Zuykova and Bochkarev, 2010). The clonal offspring were raised in vials at a constant 20◦C until maturity. One mature clonal offspring per vial was used for morphological measurements. Selenastrum capricornutum algae were provided daily (1.9 104 cells per single Daphnia) to the vials. The third set of experiments tested the hypothesis that temperature change alone (in both directions) is sufficient to induce phenotypic plasticity. The hypothesis of postembryonic induction of phenotypic plasticity was also tested in this experiment. Clones from Lake Ontario (collected in midsummer) were cultured individually in 7-dram borosilicate glass vials (Kimble Glass Inc.) and 20mL of culturing solution in 20◦C experimental chambers. Broods were split and clonal juveniles haphazardly assigned to either 20◦C or 10◦C vials with otherwise identical growth conditions (24-h light photoperiod and 100 μL algal food +50 μL homogenized fish food per vial every 3 days). Vials from both treatments were examined for mature females (with eggs)—when detected, such females were removed and preserved in 50% at −20◦C for dissection. A similar experiment was run in the other thermal direction with the seed specimens here grown at 10◦C for more than one generation.

The culturing solution was made by mixing 10-mL synthetic freshwater (96mgNaHCO3, 60mgCaSO4 2H2O, 60 mg MgSO4, and 4 mg KCl in 1 L of double distilled water) (EPA, 2002) with 100 μL algal (S. capricornutum) solution (4.8 106 cells per mL) and 50 μL homogenized fish food. Homogenized fish food was made by blending 10 g of Tetrafin™ Goldfish flakes in 1 L of double distilled water (Baldwin and LeBlanc, 1994; Olmstead and LeBlanc, 2000). Solids were settled out overnight and the resulting supernatant was used as homogenized fish food. The dry weight of 1 mL of homogenized fish food is 4 mg.

Experimental animals were killed in 50% ethanol and stored at −20◦C. Microphotographs were taken using a Leica MZ16 motor-focus microscope equipped with a Leica DC480 camera. The initial pictures of the entire specimen were used for measurements of body length. Specimens were then transferred into a drop of distilled water and dissected to separate the second thoracic limbs from the body. Minuten needles (from WARDS, item # 14-0185) were used for the dissection. Pictures of one thoracic limb II were then taken for measurement (Fig. 1). Body length was measured as the distance (mm) between the anterior margin of the eye and the inflection point of the curve formed by the posteroventral margin of the valves at the base of the tail-spine in mature females (Dodson, 1988). The rigid seta of Endopodite 4 (seta 2) thoracic limb II has adjacent setae (seta 1and seta 3). Each seta was measured in ImageJ by tracing the arc of the curved seta from base to tip.

Table of measurements from experiments to test the hypothesis of thermal plasticity of the rigid seta of the second endite, second limb, in Daphnia. Daphnia galeata was collected from Glen Haven Park, Irondequoit Bay, Lake Ontario, Rochester, NY and Guelph Lake, Ontario, Canada. The columns are: body (body length from lateral view of adult females in mm), seta 1 length (mm), seta 2 length (rigid seta), seta 3 length (mm), group number in the manuscript, temp (growth temp warm >20C or cold <10C), source (wild type from nature, F1 or multigenerational culture), and transfer (life stage of transfer to temperature treatment).