We studied transcriptomic effects of dietary EPA availability in combination with temperature to disentangle responsive gene networks underpinning the beneficial effects of this long chain ω3-PUFA on a physiological level. We further explore these effects by quantifying somatic growth rates as a fitness proxy, together with the animals’ fatty acid composition. This allows us to discriminate gene expression patterns indicative of a complex interplay between resource availability and temperature responses in the aquatic model herbivore Daphnia magna.
Physiological performance and fatty acid composition
As for most animals, the fatty acid composition of Daphnia sp. reflects the composition of their diet [47]. In nature, the occurrence of PUFA-rich phytoplankton in lakes at cooler temperatures in spring matches the nutritional demand of zooplankton at the beginning of this season, providing high proportions of PUFAs for growth and reproduction [48], as well as for membrane remodelling [32]. Seasonal shifts in temperature and food availability should therefore be mirrored in altered transcript expression with signatures that are particularly attributable to these factors.
In our analysis, responses in life history traits in connection with EPA availability at different temperatures — demonstrated by impaired growth when EPA was limiting (Fig. 1) — showed that Daphnia cultivated at 15°C displayed a higher demand for EPA than specimens at 20°C, which is in line with the findings of an earlier study [49]. However, EPA levels of D. magna were higher at 20°C (Fig. 2), contrary to the assumption that more EPA should be required at 15°C for homeoviscous adaptation. Similar results have been found previously for the same temperature regime [49, 50].
The total amount of EPA as a proportion of total body mass in D. magna is higher at the lower temperature. Nevertheless D. magna may have been ultimately limited by EPA availability due to the enhanced PUFA demand at lower temperatures. A higher amount of EPA accumulation in somatic tissue at 20°C than at 15°C is further supported by a recent study [50].
Overall, when we analysed the daphnids’ fatty acid (FA) composition with respect to saturation state (SAFAs, MUFAs and PUFAs; see Fig. 3) almost no temperature-effects were visible within the different food types (except for MUFAs in the CY + EPA treatment). Consequently, it is likely that the applied thermal difference of 5°C was not severe enough to alter the animals’ FA contents.
Gene expression
By assessing gene expression profiles in D. magna under strictly controlled experimental conditions, we were able to attribute particular functional changes specific to temperature and EPA availability. In general, temperature elicits large responses connected to RNA and DNA related processes (“Information storage and processing”, see Figure 4), which are represented by a complex network of genes involved with replication as well as with transcription and translation. This key abiotic factor also provoked the alteration of transcripts that affect signal transduction mechanisms, posttranslational modification, as well as carbohydrate-, amino acid- and lipid transport mechanisms, and inorganic ionic transport processes.
Although the effect size of EPA altered transcripts was lower than the temperature-induced effects, this dietary constituent is nevertheless a major driver of improved growth at the physiological level.
The transcriptomic responses so far analysed in connection with long chain polyunsaturated fatty acids rely on studies of enzymes that are involved in eicosanoid synthesis of the “arachidonic pathway” [51]. These enzymes are known to convert eicosanoids into important signalling molecules, such as prostaglandins or leukotrienes in invertebrates, but also in mammals [40, 52]. In our study, EPA availability provoked various functional changes in translation and transcription, but also in signal transduction mechanisms, changes in intracellular trafficking, as well as altered transcript levels for carbohydrate-, amino acid-, and lipid metabolism that are detailed below.
Information storage and processing
In this cluster, the strongest thermal effects are seen for the categories ‘RNA processing and modification’ (category A); ‘translation, ribosomal structure and biogenesis’ (J), and ‘transcription’ (K). Adjustments in the transcriptome become visible here, as these functions are modulated as a first response to the altered conditions. A high proportion of maintenance costs is attributed to regulation of this gene, which compensates effects of bio-physical reaction norms [53-55]. Generally, higher expression values were observed at the colder temperature, and were more enhanced than in other functional classes, such as ‘cellular processes and signalling’ or ‘metabolism’ (see Supplementary File 1). This effect is known as the compensatory effect, and was previously shown to vary between clones of D. pulex due to local adaptation [56]. Many candidates attributed in this cluster through artNOG annotation showed high transcript levels at both lower temperature and dietary EPA availability. This indicates adjustments of the transcriptome in response to changes in both factors.
Signalling
Numerous G-protein signalling transcripts, as well as serine/threonine kinases and opsins, were found to be thermally sensitive and were elevated with dietary EPA availability. Such candidates are potential mediators of anti-inflammatory processes, and are connected to healing and growth of cells in mammals [57]. Further, RAs and Ran- transcripts (RAs-related nuclear protein) — that are factors involved in G-protein signalling affecting gene expression cascades involved in cell growth, differentiation and survival [58] — were upregulated. It is likely that RAs and Ran transcripts mediate sensing and signalling cascades for growth in Daphnia and other invertebrates. Similarly, the production of resolvins and protectins, molecules derived from EPA as well as from the longer docosahexaenoic acid (DHA, 22:6 ω3), are involved in cytokine and leukotriene signalling via G-proteins [59]. Transcripts of signalling cascades involving stimulators like dopamine or serotonin (products of the aromatic-L-amino-acid decarboxylase) were found to be upregulated in the EPA treatments. It remains to be investigated whether such products do function as neurotransmitters, or if they serve other endocrine functions in invertebrates. The first indication for the utilization of dopamine in Daphnia sp. was found in connection with predatory stress [60].
We also identified transcripts of cytochrome P450 in connection with the combined effects of temperature and EPA availability (Supplementary File 2). This is an important indicator for the biotransformation of EPA. Potential mechanisms are the conversions of EPA into five regioisomeric epoxyeicosatetraenoic acids (EETeTrs) and ω/(ω-1)- hydroxyeicosapentaenoic acids (19- and 20-HEPE) [61], which mediate (at least in mammals) a delicate balance between pro- and anti-inflammatory responses [62].
Numerous gene families of cytochrome P450, as well as pseudogenes, have been identified across the animal kingdom [63]. For Daphnia, 75 functional CYP genes and 3 pseudogenes belonging to 4 clans, 13 families, and 19 subfamilies are so far identified [64]. However, the particular functional implications for many of these genes are still to be determined.
Cellular structure and metabolism
The higher growth rates were paralleled by higher expression of genes for cytoskeletal structures, accompanied by induced growth factor receptors and fibronectin, which were expressed at higher levels at 15°C when EPA was available.
The different profiles of carbohydrate metabolic transcripts (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 demands at both temperatures. Whilst sugars seem to be strongly metabolized at 20°C, glycogen anabolism becomes more effective at 15°C. This may be due to the fact that a faster metabolism is connected to a higher temperature, and is thus accompanied by a higher demand for sugars; this is mirrored by higher growth rates at the physiological level. Carbohydrate metabolism was also differentially regulated when Daphnia sp. were challenged with diets of different qualities in terms of nutrient stoichiometry [65], which may indicate that this is a very general response to food quality alterations. Genes in carbohydrate metabolism involved in inflammatory processes (SAPA), or connected to chitin and moulting (chitotriosidase), were also regulated in a temperature-dependent manner; these do however reflect a higher variation that may mirror the variability of individuals in sample pools.
Despite low transcriptional levels in the GA-diet regime, peptidases like trypsin or chymotrypsin, aminotransferases and metallopepdiases were up-regulated in CY diets, especially at the lower temperature (E). This was also mirrored in the expression of Eip55E (Ecdysteroid-inducible polypeptide 55 subunit E), which is involved in sulphur amino acid metabolic processes like cysteine and glutathione biosynthesis.
The different expression profiles in carbohydrate and amino acid metabolism indicate a recruitment of different enzymes to extract energetic compounds like sugars or amino acids from the different basal diets [66]. Different digestive efforts for CY-diets are indicated through high expression levels of metallopepdidases, trypsins, and aminotransferases, as well as by chaperones like T-complex proteins. Therefore, different basal diets provoke different phenotypes to handle and digest the different food items.
In the ‘lipid metabolism’ category (I), high levels of acyl-CoA dehydrogenases were expressed at 20°C, especially when EPA was available. This indicates the transcription of RNAs related to the degradation of fatty acids. Transcript levels for transporters and intracellular transport structures associated with the transport mechanisms of long chain fatty acids were up-regulated when EPA was absent. This may be a mechanism to cover the higher demand for long chain PUFAs under EPA limitation. The higher expression levels of fatty acid transporters 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 cytochrome P450 305a1, which are involved prostaglandin-mediated signalling (Q, secondary metabolites).
High vitellogenin levels were pronounced in GA+EPA diets, accompanied by the expression of glycerol-3-phosphate acyltransferase and acyl-CoA-binding domain-containing protein 7, with slightly higher (perhaps compensatory) levels at 15°C, this may be involved in the biogenesis of vitellogenin, as previously observed [43]. Further, a secretory phospholipase A2 was induced, indicating increased effort to digest liposomal-supplemented diets.
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 latter transcripts that are further supported by EPA availability. In this context, higher levels of fibronectin and endothelial growth factor receptor indicate a mediation of processes involved in cell division and growth [67].
Expressed candidate genes connected to ‘inorganic ion transport and metabolism’ (P) were up-regulated in animals feeding on cyanobacteria at 20°C. Further gene upregulation occurred at 15 °C, which indicates dynamic adjustments of the osmotic balance at the lower temperature. Ca2+- and serotonin transporters were particularly more strongly expressed at the lower temperature in the GA- diet supplemented with EPA. This matches the observed pattern for G-protein transcripts and conjoined candidates in category T, which may contribute to the same messaging pathway [68]. Similarly, cytochrome P450 305a1 transcript displayed the same pattern in the category Q ‘secondary metabolites…’, which may also indicate a conjoined function in a signalling pathway. Interestingly, other Cytochrome P450-like proteins seem to be highly temperature sensitive, and were expressed with low or high levels in GA+EPA diets at 20°C and 15°C, respectively.
Cytochrome P450 transcripts and subsequent proteins seem to play an important role in the metabolism and potential transformation of EPA into signalling cascades. Potential pathways for a transformation of the long chain polyunsaturated fatty acid EPA into other endocrine signalling molecules were proposed by [40] and [52], these are: the cyclooxygenase (COx) pathway; 2) the lipoxygenase (LOX) pathway; or 3) the cytochrome P450 pathway. Recent expression studies, however, have shown that COx expression is not affected by EPA-availability [43, 51], and so far, no LOX genes have been found in Daphnia species [43]. Overall, our study delivers a profound insight into EPA-connected metabolism, and indicates that a transformation into endocrine signalling may rely on Cytochrome P450-based conversions; these outcomes should be explored in detail by further studies.