Source of Organisms
This experiment used P. ramosa spores and D. magnaephippia from natural pond sediments, and D. magna genotypes collected earlier from the field site and propagated clonally in the laboratory. Sediment from the Aegelsee was collected in September 2021 and stored in darkness at 10 oC. Sediment was filtered using distilled water and 80-μm mesh to remove large particles,Daphnia ephippia and rotifer resting eggs from the sediment, but not the much smaller (about 5-6 µm diameter) P. ramosa spores, and was mixed homogeneously. D. magna ephippia were collected under a dissecting microscope from a sediment sample collected in 2019 and stored at 4 oC in a refrigerator. This prolonged resting period increases hatching success and synchrony (Stross 1966). For the live Daphnia experiment, D. magna clones were isolated from the Aegelsee from previous field seasons (2019 and 2014) and maintained in isogenic lines under laboratory conditions (ADaM media (Klüttgen et al. 1994) at 20 oC, 16 hour light:8 hour dark cycle, and 80 % humidity and fed with a suspension ofTetradesmus obliquus ). Prior to this experiment, each clone was assessed for its resistance/susceptibility to a panel ofP. ramosa isolates maintained in the lab, two of which are present in the Aegelsee, using an fluorescence based “attachment test” (Duneau et al. 2011). For the current experiment, genotypes that were susceptible to all P. ramosa isolates of the test-panel (type SSSSS, hereafter referred to as “susceptible”), and clones that were resistant to most tested isolates (type RRSRS hereafter referred to as “resistant”) (clones from (Ameline et al. 2020)) were propagated by transferring and multiplying cultures twice weekly. These two resistotypes are very common in the Aegelsee population (Ameline et al. 2020), but are not “susceptible” or “resistant” to all P. ramosa isolates present in the study pond. Clones were kept in population cultures in 360-mL jars.
Once population sizes were large enough, adult females were transferred to isolated jars in an incubator at 15 oC to acclimate them to a temperature in the centre of the experimental treatment range (10 to 20 oC). To reduce maternal effects of temperature on resistance to P. ramosa (Garbutt et al. 2014), these females were bred two generations under these conditions by removing adult females once they had given birth to offspring, which were then isolated upon reaching maturity. Third- or subsequent-brood juveniles from the second generation were used for the actual experiment, to account for the fact that the first two broods are often smaller in numbers and size of offspring than subsequent broods (Lampert 1993). Prior to the start of the experiment, these juveniles were moved to experimental incubators and allowed to acclimate for 48 hours to their experimental temperatures.
We aimed to run the experiment at each temperature for the same length of biological time, since D. magna physiology and life cycles are accelerated by higher temperature. A pilot experiment was conducted to calibrate the biological scale for D. magna and estimate the hatching rate and time to reach maturity (first eggs) at 10 and 20oC. This scale was used to estimate how long it would be necessary to run the experiment for all temperatures to achieve an approximately equal physiological age.
Experimental Design
This study featured a 5x3 factorial design of temperature and host availability to assess the role of temperature on the start of seasonal epidemics. Five incubators at 10, 12.5, 15, 17.5 and 20oC were used for temperature treatments, into which 360-mL jars with media and a thin layer of filtered sediment were placed. These temperatures were chosen to determine infection dynamics in a range around 15 oC, the temperature at which infections are first observed in the pond (Ameline et al. 2020). Infections in the pond are typically observed when D. magna of different age classes are already present (Ameline et al. 2020), but further hatchlings from resting eggs may still arise. Therefore, three treatments of host availability were used: broadly susceptible or broadly resistant juvenile D. magna from asexually reproducing cultures, and ephippia from which genetically diverse hatchlings could arise. We placed either 15 juvenile D. magna of a broadly susceptible or broadly resistant clone, or 10 ephippia (each containing two eggs, but with an average hatch rate of ~30-50 % as determined in a pilot experiment) into each jar. These treatments were chosen to disentangle the effects of the presence of susceptible hosts on temperature, as behaviour or physiology of hatched ephippia may differ from asexual juveniles. To increase hatching rate, ephippia were treated with a 50:50 solution of commercial bleach (4 %) and distilled water for three minutes (Catur and Ebert 2016), before being rinsed and added to jars. A total of 20 replicates of the full experimental design were performed, with 20 jars of ephippia, 20 jars of broadly susceptible juveniles and 20 jars of broadly resistant juveniles each at each temperature. Within the live D. magna treatments, five different clones were used for each resistotype, with four jars of each clone. All treatments and replicates were run concurrently. Within incubators, trays of 12 jars were arranged randomly and rotated each day to reduce potential position effects. All jars were also covered with a transparent lid to reduce temperature fluctuations due to evaporation. Live D. magna jars were fed with T . obliquus at a rate adjusted to account for differences in D. magnametabolic/filtering rates at different temperatures (Burns 1969) to avoid build-up of algae, and to avoid interactive effects of food availability with temperature on D. magna development (McKee and Ebert 1996, Giebelhausen and Lampert 2001). Specifically, live D. magna jars were fed three times per week with 100x106cells at 20 oC, 70x106cells at 17.5oC, 50x106 cells at 15oC, 20x106 cells at 12.5oC and 10x106 cells 10oC based on our pilot. Ephippia treatment jars were fed at these same rates once hatchlings were observed.