2.1 Rotifer culture and life table experimental conditions
Lab-reared, asexually reproducing clones of the heat-tolerant B. calyciflorus s.s. (clone IGB; CTmax =43.18oC) and the heat-sensitive B. fernandoi (clone A10; CTmax =38.49 oC) were selected for our experiments. Both clones originate from Northern Germany and were reared under laboratory conditions for more than 10 years. Species identity was previously confirmed by amplifying a portion of the ITS1 genetic marker (Paraskevopoulou et al., 2018). Stock cultures were maintained as batch cultures in glass flasks containing WC medium (Guillard & Lorenzen, 1972) at 20 °C under a 16:8 light:dark photoperiod. A food combination of two algae species,Monoraphidium minutum (Culture collection Göttingen, strain SAG-243-1) and Cryptomonas sp. (Culture collection Göttingen, strain SAG-26-80), was provided weekly.
Before starting the life table experiments, cultures were exposed to a period of gradual acclimatization by increasing the temperature 2 °C every 2 days until reaching the experimental temperature. Because of this, the acclimation period varied among cultures, with the longest adaptation period (2 weeks) for the highest temperature (32 °C). After reaching the experimental temperature, we maintained the rotifer cultures for one week (at least two to four generations) before starting the experiment to allow for acclimation, and to reduce potential maternal effects. Food was supplied ad libitum daily. ForB. calyciflorus s.s., experiments were conducted at four temperatures (20 °C, 23 °C, 26 °C, 32 °C), while for B. fernandoithree temperature assays (20 °C, 23 °C, 26 °C) were used. We tried several times to acclimatize B. fernandoi also to 32 °C, but high mortality always led to culture collapse before the initiation of the experiment.
The experiments basically followed the procedure from Weithoff (2004, 2007): single females bearing a subitaneous, asexual egg were isolated from the culture, placed in a microtitre well, and inspected thoroughly for hatched neonates. Life-table experiments were started by introducing one neonate into a new well and adding 1ml of algal suspension composed of Monoraphidium minutum (5 x 105 cells/ml) and Cryptomonas sp. (2 x 104 cells/ml), to avoid food limitation. For each temperature and species, at least 24 individuals were recorded. Survival and reproduction were recorded every 12 h and any newly hatched neonates were removed. Every 24h the maternal individuals were transferred into a new well with fresh food suspension. The experiment was conducted in the dark and continued until all individuals of each cohort died.