- Background: Nutritional quality of phytoplankton is a major determinant of the trophic transfer efficiency at the plant-herbivore interface in freshwater food webs. - In particular, the phytoplankton ’ s content of the essential polyunsaturated omega-3 fatty acid eicosapentaenoic acid (EPA) has been repeatedly shown to limit secondary production in the major zooplankton herbivore genus Daphnia. - Searching for orthologous genes (COG-analysis) yielded a functional overview of the altered transcriptomes. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Full list of author information is available at the end of the article. - In aquatic environments, the photosynthetic base of the food web consists of small unicellular phytoplankton that is consumed by herbivor- ous zooplankton. - In freshwater ecosystems, crustacean zooplankton of the genus Daphnia are the major pelagic herbivores, and form a crucial link between primary producers and con- sumers [12]. - The genomes of several Daphnia species have been se- quenced, and it is therefore one of the few animal genera for which extensive ecological and genomic information is available [17, 18]. - Beyond their role in growth and reproduction, PUFAs are well recognised critical components of the so-called. - In particular, the availability of the highly unsaturated ω3-PUFA eicosapentaenoic acid (EPA, C-20:5 ω3) was repeatedly shown to be crucial for Daphnia growth and reproduction, via controlled PUFA supplementation ex- periments [5, 6, 36]. - Despite the growing body of evidence underscoring the importance of dietary PUFAs in general — and of EPA in particular — our understanding of the molecular physiology underlying the PUFA/EPA metabolism, and the gene networks responsive to the availability of this critical dietary compound remain very limited. - [40] conducted an in-silico analysis of the genome of Daphnia pulex, which produced the first in- sights on potential mechanisms that are affected by ω6 – eicosanoids. - We discuss these results in con- nection with the animals’ respective growth performance and fatty acid composition. - Due to the high level of control of the ex- perimental factors, the results illuminate the genetic basis underlying EPA (and more generally ω3 PUFA)- dependent metabolism in this keystone herbivore. - In general, growth rates were much lower at 15 °C, reaching only of the performance at 20 °C.. - EPA incorporation and fatty acid composition. - The supplementation of EPA and the natural differences in fatty acid composition in basal diets were considered as main drivers for the observed growth performances. - The two different basal diets resulted in different tis- sue fatty acid compositions in D. - 3), with re- spect to the proportions of different fatty acid species (state of saturation). - However, monounsaturated fatty acid (MUFA) proportions dif- fered significantly between diets. - We did not detect dif- ferences in the total expression output among treat- ments or temperatures, thus sequencing depth of the samples was comparable. - 3 Fatty acid composition of D. - magna in the experiment. - magna in the respective treatments. - The total amount of the respective transcripts was then functionally annotated by the ArtNOG categorisation (given in % of the total response). - These alterations were paral- leled by changes in the ‘Transcription machinery’ (K), as well as alterations in ‘Translational-’ (J) and ‘RNA processing’ (A) transcripts, which were strongly affected by the factor temperature.. - To provide a more detailed overview of the large set of re- sponsive genes depicted in Fig. - in the respective categories to extract common responses in connection with the factors: temperature, EPA availabil- ity, and combined effects of both factors. - The strongest altered gene expression was detected in the cluster of. - Most of the genes were up-regulated at 15 °C when compared to 20 °C. - An exception to this are the transcripts of the carboxylic ester hydrolase and the aromatic-L-amino- acid decarboxylase, which were expressed at the highest levels in animals on GA diets supplemented with EPA.. - At the higher temperature, this enzyme was upregulated in the CY + EPA diet, and at the lower temperature in the GA + EPA regime. - We further explore these effects by quantifying somatic growth rates as a fitness proxy, together with the animals’ fatty acid composition. - temperature responses in the aquatic model herbivore Daphnia magna.. - Physiological performance and fatty acid composition As for most animals, the fatty acid composition of Daph- nia sp. - Overall, when we analysed the daphnids’ fatty acid (FA) composition with respect to saturation state (SAFAs, MUFAs and PUFAs. - Gene expression. - The transcriptomic responses so far analysed in con- nection with long chain polyunsaturated fatty acids rely on studies of enzymes that are involved in eicosanoid synthesis of the “arachidonic pathway” [52]. - This indicates adjustments of the transcrip- tome in response to changes in both factors.. - The different profiles of carbohydrate metabolic tran- scripts (G, but also in E) maltase, amylase and alpha- glucan branching enzymes indicate a different quality of the basal food sources, as well as different energetic de- mands at both temperatures. - In the ‘lipid metabolism’ category (I), high levels of acyl-CoA dehydrogenases were expressed at 20 °C, espe- cially when EPA was available. - The higher expression levels of fatty acid trans- porters was accompanied by the expression of a transporter in the category ‘inorganic ion transport and metabolism’ (P), as well as by ABC transport proteins (ATP-Binding Cassette sub-family C/ member 4) and cyto- chrome P450 305a1, which are involved prostaglandin- mediated signalling (Q, secondary metabolites).. - High levels of dynein, myosin, and tubulin (Z) indicate a remodelling of the cytoskeleton at lower temperatures.. - As the solubility and viscosity of the cytosol seems to be affected, a structural remodelling is indicated by the lat- ter transcripts that are further supported by EPA avail- ability. - Further gene up- regulation occurred at 15 °C, which indicates dynamic adjustments of the osmotic balance at the lower temperature. - and serotonin transporters were par- ticularly more strongly expressed at the lower temperature in the GA- diet supplemented with EPA.. - Similarly, cytochrome P450 305a1 transcript displayed the same pattern in the category Q ‘secondary metabo- lites. - Potential pathways for a transformation of the long chain polyunsaturated fatty acid EPA into other endo- crine signalling molecules were proposed by [40, 53], these are: the cyclooxygenase (COx) pathway. - The expression patterns in differ- ent phenotypes were highly dependent on the dietary availability of the ω3-polyunsaturated fatty acid EPA.. - study thus reveals some of the molecular mechanisms underlying the positive effects of a particular dietary omega-3 fatty acid, and constitutes an important re- source of transcriptional patterns, even in genes with lit- tle or no annotation. - The diets were chosen to monitor effects of the fatty acid EPA via supplementation, as they do naturally not contain long chain (>. - Individuals were sampled when they reached maturity, indicated by the deposition of the first clutch of eggs into their brood pouch. - magna requires a minimum of PUFASs and sterols to produce offspring [8], cyanobacterial diets were further supplemented with at alpha linoleic acid (ALA, C-18:3 ω3) and cholesterol to ensure the reach of the sampling end-points. - As a reference we used the mean dry weight of 40 neonates at the beginning of the respect- ive experimental temperature. - number of days until reaching maturity, which was indi- cated by formation of the first clutch of offspring in their brood chamber [75].. - Fatty acid composition. - The analysis of fatty acid composition was done by means of fatty acid methyl esters (FAME), which were subsequently quantified by gas chromatography (GC).. - Internal standards with uneven length of the fatty acid chain, which do not naturally occur in Daphnia or Alga, were added to the extraction as a mass reference: 10 μg heptadecanoic acid methyl ester (C-17:0 ME) and 5 μg tricosanoic acid methyl ester (C-23:0 ME). - Extraction of FAMES was car- ried out by adding ~ 2 ml isohexane, vigorously vortexing for a minute and collection of the upper liquid phase. - After solvent evaporation, FAMEs were collected from the glass vial by pipetting 3 × 100 μl, rinsing the walls of the vial. - Single fatty acid contents were. - related to the carbon content of the body tissue using the previously determined carbon to dry mass conversion fac- tor for body tissue, 0.41 μg carbon (μg dry mass. - The RNA-extract was analyzed on a Bioanalyzer (Agilent Technologies, Santa Clara/USA) to determine quality and quantity of the RNA. - The experiments were carried out at 20 °C and daphnids were fed with either a green alga (Scenedes- mus obliquus, now Acutudesmus obliquus), or a crypto- monad (Cryptomonas spec.) until the generation of the third clutch of offspring. - The transcript for juvenile hormone was another tar- get in the study of Schlotz et al. - Fatty acid binding protein 3 was another candidate gene derived from the study of Schlotz et al. - Respective transcript levels in our RNA-seq data (product accessions KZS15286.1, KZS06031.1, KZS02020.1, KZS01487.1, KZS01268.1, KZS98609.1, KZR98522.1) also displayed only minor variation among treatments, thus corroborating the house-keeping char- acter of the target gene.. - A respective COG-annotation of single transcripts was included by colour of the data points. - FA: Fatty acid. - FAME: Fatty acid methyl esters. - MUFA: Monounsaturated fatty acid. - PUFA: Polyunsaturated fatty acid. - SAFA: Saturated fatty acid. - Z/4: Name of the medium used for Acutodesmus obliquus culture. - ω 3: Fatty acid species with. - ω 6: Fatty acid species with unsaturation (carbon double bond) at the n-6 position. - We also thank Amy MacLeod for language editing of the manuscript.. - HW conducted the experiments and analysed physiological, fatty acid and transcriptomic data and wrote the first draft of the manuscript. - The project was funded by the Institute of Zoology and Cell Biology of the Heinrich-Heine-University Düsseldorf and by project FI1548/6 – 1 within the Priority Programme SPP 1704 “ DynaTrait ” funded by the Deutsche For- schungsgemeinschaft to PF. - The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - A detailed description and overview of the respective sampling material is available under the BioProject accession number PRJNA391248. - The results of the RNA-seq experiment are also accessible under the GEO entry GSE130674.. - Specimen of the clone were kindly provided to the group “ Aquatic chemical Ecology ” headed by Prof. - No permissions for the experiments were necessary, as animal experiments on invertebrates (except cephalopods and higher crustaceans, which does not apply here) do not require regulatory approval according to § 8 TierSchG of the national regulations of Germany.. - The molecular basis of essential fatty acid limitation in Daphnia magna: a transcriptomic approach. - Dietary Essential Amino Acids Affect the Reproduction of the Keystone Herbivore Daphnia pulex. - De novo transcriptome assembly and sex-biased gene expression in the cyclical parthenogenetic Daphnia galeata. - A test of the role of polyunsaturated fatty acids in phytoplankton food quality for Daphnia using liposome supplementation. - Chapter 7 Fatty acid desaturation and chain elongation in eukaryotes. - Predator evasion in zooplankton is suppressed by polyunsaturated fatty acid limitation. - Climate warming is predicted to reduce omega-3, long- chain, polyunsaturated fatty acid production in phytoplankton. - Outlining eicosanoid biosynthesis in the crustacean Daphnia. - The essential omega-3 fatty acid EPA affects expression of genes involved in the metabolism of omega-6-derived eicosanoids in Daphnia magna. - Daphnia fatty acid composition reflects that of their diet. - Temperature affects the limitation of Daphnia magna by eicosapentaenoic acid, and the fatty acid composition of body tissue and eggs. - Coping with cold: an integrative, multitissue analysis of the transcriptome of a poikilothermic vertebrate. - Lipid Mediators in the Resolution of Inflammation. - Dopamine is a key regulator in the signalling pathway underlying predator-induced defences in Daphnia.. - Invasion of quality: high amounts of essential fatty acids in the invasive Ponto-Caspian mysid Limnomysis benedeni. - Effects of temperature and dietary sterol availability on growth and cholesterol allocation of the aquatic keystone species Daphnia.
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