IMIS - Marine Onderzoeksgroepen

The experiment employed a full-factorial design with seven Asterionella formosa (diatom) genotypes (S24, S26, S37, S38, S43, S49 and S53) exposed to one Zygorhizidium planktonicum (chytrid parasite) genotype (F12) at five different temperatures (1°C, 6°C, 11°C, 16°C and 21°C ) in five replicates. This resulted in 35 experimental combinations and 175 experimental units. To compare the performance of Asterionella in parasite exposed and non-exposed populations, non-exposed controls of the seven Asterionella genotypes were grown at the same experimental temperatures resulting in 35 control units (one per temperature-host strain combination).For temperature acclimation, parasite exposed and non-exposed stock cultures of each host genotype were split into five subcultures and stepwise acclimated in semi-continuous batch cultures in temperature-controlled water baths for at least five generations prior to the experiment. The light was set to 160610µmol quanta m2 s-1 provided by cool-white fluorescent lamps (TL-D 30W/830) at a 14: 10 h light: dark cycle. To start the experiment, each experimental and control unit was inoculated from the corresponding non-exposed subculture to a starting concentration of ca. 15 000 uninfected host cells mL-1 into a total volume of 60 mL CHU-10 medium. Parasite exposure was achieved by inoculating ca. 5 000 live infection carrying host cells mL-1 from a nearly 100% infected, temperature- and host genotype-matching exposed subculture. Based on results of pre-experiment trials, the host inoculum of ca 15 000 cells mL-1 was small enough to ensure that the culture medium could support several generations of unlimited host growth before light or nutrient availability could become limiting in the batch set up, but also large enough to support the infection. Similarly, the parasite inoculum of ca 5 000 live infection carrying cells mL-1 (i.e. a starting prevalence of ca 25%) was large enough to follow both increase and decrease in prevalence over time. All experimental and control units were started on the same day. Each unit was shaken manually twice and their position within the water bath was randomized once each day. Samples for microscopy enumeration were taken every second day for the temperatures 6°C to 21°C and every fifth day for temperature 1°C. Counting protocol A minimum of 200 Asterionella cells or 20 fields of view were counted in a 1 mL sample under an inverted microscope (Leica, DMI 4000B, Wetzlar, Germany) according to the thermal settling method. Each sample was counted for abundance of: (i) living uninfected host cells mL-1 (uninf); (ii) infected host cells carrying one or more living infection(s) mL-1 (inf); (iii) infected host cells carrying only dehisced / dead infection(s) mL-1; (iv) sporangia mL-1 , and (v) resting spores mL-1. Infection prevalence, i.e. proportion of cells carrying live infections in the live host population, was calculated as inf/(uninf+inf).Asterionella cells carrying empty or dead sporangia, i.e. class (iii), were excluded from the calculation of infection prevalence as they did not contribute further to population growth of the host or the parasite.