The molecular basis of essential fatty acid limitation in Daphnia magna: A transcriptomic approach

It is widely accepted that in many food webs, the trophic transfer efficiency among primary producers and herbivores is determined by the nutritional value of primary producers. In pelagic freshwater and marine ecosystems, secondary production by herbivorous crustacean zooplankton is often limited by the seston's content of essential ω3 polyunsaturated fatty acids (ω3 PUFAs). However, little is known about the genetic network behind the positive relationship between phytoplankton ω3 PUFA content and zooplankton growth and reproduction. In our experimental study, we analysed gene expression changes of the freshwater cladoceran Daphnia magna under different food regimes differing in their ω3 PUFA composition. To disentangle ω3 PUFA effects from other factors, we fed D. magna with different pure phytoplankton cultures (i.e., algal and cyanobacterial diets) with or without supplementing the essential ω3 PUFA eicosapentaenoic acid (EPA). As hypothesized, we observed enhanced growth on diets supplemented with EPA. We applied an Illumina RNA-seq approach to D. magna from different diet treatments to find and monitor genes that are regulated dependent on EPA availability. Of 26,646 potential protein products (mapped to the D. magna genome), we identified transcriptomic signatures driven by the different food sources. Further analyses revealed specific candidate genes involved in EPA metabolism, irrespective of the basal food source. This allows a first functional annotation of previously uncharacterized genes involved in the EPA-specific response of D. magna and may finally provide a link to molecular processes connected to ω3 PUFA metabolism and conversion and thus trophic transfer efficiency in pelagic food webs. We conducted a feeding experiment in Daphnia magna with strictly controlled diets to address EPA-dependent expression profiles. Two different EPA-free basis food sources were used in the experiment: the green alga (GA) Acutodesmus obliquus and the Cyanobacterium (CY) Synechococcus elongatus. The PUFA EPA was added via liposomes to half of the experimental groups fed with GA or CY to provide a comparable design for monitoring effects of EPA availability. In addition, we also monitored Daphnia magna fed with Cryptomonas ssp. (CRY), serving as a natural positive control (contains naturally EPA). We analysed animals that were exposed to the defined food conditions from hatch until they reached maturity. In total, 5 treatment groups were prepared for sequencing with 3 replicates each. Replicate sample pools consisted of 5 mature specimens to ensure appropriate amounts for RNA extraction and comparability. Using the Illumina HiSeq 4000 platform in paired-end mode we gathered ~ 52.9 million reads per single replicate for transcriptomic analyses.