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.