Combined effects of dietary polyunsaturated fatty acids and parasite exposure on eicosanoid-related gene expression in an invertebrate model

Abstract

Eicosanoids derive from essential polyunsaturated fatty acids (PUFA) and play crucial roles in immunity, development, and reproduction. However, potential links between dietary PUFA supply and eicosanoid biosynthesis are poorly understood, especially in invertebrates. Using Daphnia magna and its bacterial parasite Pasteuria ramosa as model system, we studied the expression of genes coding for key enzymes in eicosanoid biosynthesis and of genes related to oogenesis in response to dietary arachidonic acid and eicosapentaenoic acid in parasite-exposed and non-exposed animals. Gene expression related to cyclooxygenase activity was especially responsive to the dietary PUFA supply and parasite challenge, indicating a role for prostanoid eicosanoids in immunity and reproduction. Vitellogenin gene expression was induced upon parasite exposure in all food treatments, suggesting infection-related interference with the host's reproductive system. Our findings highlight the potential of dietary PUFA to modulate the expression of key enzymes involved in eicosanoid biosynthesis and reproduction and thus underpin the idea that the dietary PUFA supply can influence invertebrate immune functions and host-parasite interactions.

Introduction

Polyunsaturated fatty acids (PUFA) are essential lipids required for animal growth and reproduction. Dietary C20 PUFA, such as arachidonic acid (ARA) and eicosapentaenoic acid (EPA), serve as precursors of eicosanoids which are known to modulate reproduction and immunity of vertebrates (Calder, 1998, de Pablo and de Cienfuegos, 2000, Fritsche, 2006, Wathes et al., 2007). In invertebrates, however, eicosanoid biosynthesis has been scarcely linked to the dietary PUFA supply (Schlotz et al., 2012). This is surprising considering the severe physiological and ecological consequences that are associated with an inadequate dietary PUFA provisioning (Fraenkel and Blewett, 1947, Martin-Creuzburg et al., 2012, Müller-Navarra et al., 2000).

In various invertebrate species, the occurrence of eicosanoids has been thoroughly documented (Rowley et al., 2005, Stanley, 2000). Reproductive processes (Machado et al., 2007, Tootle and Spradling, 2008, Wimuttisuk et al., 2013) as well as immune responses have been shown to depend on eicosanoid action (Merchant et al., 2008, Shrestha and Kim, 2010, Stanley-Samuelson et al., 1991). In contrast to eicosanoid biosynthesis in mammals, invertebrate eicosanoid biosynthesis has been suggested to split into two instead of three pathways; an epoxygenase pathway seems to be absent (Heckmann et al., 2008b, Stanley, 2000). The presence of the leukotriene branch has also been doubted as no leukotrienes or orthologs of LOX could be found in a number of invertebrates, including Daphnia pulex (Morgan et al., 2005, Yuan et al., 2014). In the biosynthesis of prostanoid eicosanoids (Fig. 1), phospholipases A₂ (PLA2) represent the first step of a chain of consecutive reactions. PLA2 are responsible for the hydrolysis of the sn-2 ester of phospholipids resulting in the release of free PUFA. Both secretory and cytosolic forms of PLA2 have been shown to be potent in mobilizing eicosanoid precursors and to play a role in host defense against microbial pathogens (Balsinde and Dennis, 1997, Boyanovsky and Webb, 2009, Park et al., 2005, Shrestha et al., 2010). Once in their free form, ARA and EPA compete for the same enzymes for further metabolism (Lands, 1992) and are converted by a cyclooxygenase (COX or PXT) to prostanoids. The following steps of eicosanoid biosynthesis are performed by various enzymes which form the functional eicosanoids (Fig. 1). Eicosanoids are suspected to play a regulatory role in reproductive processes and thus reproduction-related genes constitute possible downstream targets of eicosanoid action.

Species of the genus Daphnia have become important model organisms in ecology, ecotoxicology and evolutionary biology (Lampert, 2011). Owing to the long history of ecological research, our knowledge of e.g. life history traits or phenotypic plasticity is vast compared to many other model organisms. With the fully sequenced genome of Daphnia pulex another major advantage was given to Daphnia as a model (Colbourne et al., 2011, Ebert, 2011). However, potential links between eicosanoids, reproduction, and immune defense have been poorly studied in Daphnia although the enzymatic machinery for eicosanoid biosynthesis is present and seems to have undergone substantial restructuring (Colbourne et al., 2011, Heckmann et al., 2008b, Yuan et al., 2014). It is well established that the fecundity of Daphnia can be increased by supplementing the eicosanoid precursors ARA and EPA, implying that eicosanoid biosynthesis is linked to reproduction (Martin-Creuzburg et al., 2010, Schlotz et al., 2013). Moreover, it has been shown recently that the dietary PUFA supply can improve the performance of Daphnia magna under pathogen challenge, suggesting a link between eicosanoid biosynthesis and immunity (Schlotz et al., 2013, Schlotz et al., 2014). These latter studies also proposed that the presence of eicosanoid precursors (i.e. EPA or ARA) in the diet affects the resource allocation trade-off between reproduction and immune function. Thus, revealing the physiological and genetic mechanisms responsible for these interactions may provide crucial insights into the role of PUFA in modulating host-parasite interactions.

The objective of the present study was to explore whether genes coding for key enzymes of the eicosanoid biosynthesis machinery as well as reproduction-related genes are differentially expressed in female D. magna in response to dietary PUFA supply and parasite challenge. Target genes were chosen to cover several steps of biosynthesis starting from substrate release via central conversion to final formation of bioactive eicosanoids. In addition, three genes related to oogenesis were investigated as potential targets of eicosanoid action.

Experimental animals were raised on food sources naturally differing in their C20 PUFA content and composition. In addition, to specifically test for the potential of single PUFA to modulate gene expression, the C20 PUFA-deficient diet was experimentally enriched with either ARA or EPA. To challenge the immune system, half of the animals of each group were exposed to endospores of the parasitic bacterium Pasteuria ramosa (Ebert et al., 1996, Ebert et al., 2016); subsamples for gene expression analyses were taken 12 and 24  h after parasite exposure.