Erred directly into dichloromethanemethanol for subsequent fatty acid extraction (as described
Erred straight into dichloromethanemethanol for subsequent fatty acid extraction (as described under). At least 3 Daphnia have been applied to collect a minimum of 25 eggs per sample. All eggs sampled have been within the first egg stage and did not show any morphological differentiation.Parasite handlingThe experiments had been performed having a clone of Daphnia magna (clone HO2, originating from Hungary). StockFor the infection of your host a clone of your Gram good bacterium Pasteuria ramosa (C19, PKD3 custom synthesis derived from a D. magna population from Garzerfeld, Germany and characterized in Luijckx [52] was employed. Stocks of P. ramosa endospores had been stored at -20 within the infected host. Before use, the stock was thawed and also the infected animal squashed inside a small volume of ADaM. Endospore concentrations within these suspensionsSchlotz et al. BMC Ecology 2013, 13:41 http:biomedcentral1472-678513Page eight ofwere determined beneath a microscope employing a counting chamber (Neubauer improved).Biochemical analyses Elemental compositionLife history experimentsA two generation life history experiment was conducted to assess food quality effects on healthy and P. ramosa-challenged D. magna. Inside the 1st generation experiment animals (third-clutch neonates born inside 12 h) had been kept individually in 80 mL of ADaM at 20C in addition to a 16:eight h light:dark cycle. They were randomly assigned to one of the following food regimes: S. obliquus (Scen), S. obliquus supplemented with handle liposomes ( lipo), S. obliquus supplemented with ARAor EPA-ULK2 list containing liposomes ( ARA, EPA), N. limnetica (Nanno), or Cryptomonas sp. (Crypto). For the second generation experiment, mothers from the first generation had been placed into fresh medium with out algae shortly before the expected release of their second clutch neonates. These neonates had been collected and placed individually in jars exclusively containing S. obliquus, irrespective from the food conditions below which they were produced. The mothers have been place back into their previous meals therapies. Culturing conditions corresponded to these of the initially generation. All animals have been transferred to fresh medium and received freshly prepared food suspensions corresponding to a total of 2 mg C L-1 every other day. 18 animals of every treatment have been not exposed to parasite spores, 30 animals were subjected towards the parasite. For infection, all animals have been placed individually in 20 mL of medium at day 3 of your experiment and have been exposed on three consecutive days to a total of ca. 12,000 P. ramosa spores per person (four,000 spores every day) in the initially generation experiment and to a total of ca. six,000 spores per person (2,000 spores each day) inside the second generation experiment. This was performed due to high infections rates within the initial generation. Manage animals in both experiments were treated as described for the spore-exposed animals; rather than infectious spores a suspension of uninfected, macerated D. magna was added (mockexposure). Subsequently, animals have been transferred to new, spore-free jars containing 80 mL of ADaM. Both experiments were terminated following 30 days as a result of expected high death prices of infected animals after about 40 days [53]. For the duration of this time period reproduction (viable offspring) and infection status were recorded. On day 30, all infected people have been stored at -20 for subsequent determination from the spore load per animal. Subsamples of infected animals of every therapy have been dried for 24 h and their dry mass deter.