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.