Multiple aquatic invertebrates have the necessary enzymatic machinery for de novo biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) including the so-called “omega-3” EPA (20:5n-3) and DHA (22:6n-3) . Two distinct types of fatty acyl desaturases, namely methyl-end (ω des) and front-end desaturases (Fed), are known to be involved in animals’ LC-PUFA biosynthesis . Iron (Fe) has a prominent role in LC-PUFA biosynthesis since it is a cofactor of desaturases, and is present in the active di-iron centres of all aerobic desaturases . Since expression of desaturases involved in LC-PUFA biosynthesis can be increased by feeding a low LC-PUFA diet, an innovative strategy to enhance the endogenous production of LC-PUFA in aquatic organisms consists of dietary Fe supplementation to guarantee adequate supply under conditions resulting in desaturase activation . Supplementation of Fe has been shown to positively influence LC-PUFA biosynthesis in salmonids  but, to the best of our knowledge, has not been yet investigated in invertebrates. The present study aimed to assess dietary Fe supplementation as an enhancer of LC-PUFA biosynthesis in the nereid polychaete Hediste diversicolor, a commercially important species with great interest for aquaculture.
Materials and Methods
First, an in vitro trial was carried out by growing t ransgenic yeast expressing the H. diversicolor desaturases (two ω des and two Fed) in the presence of specific fatty acid (FA) substrates. For each desaturase , the following treatments were tested: no Fe supplementation (control), supplementation with FeSO4 (inorganic Fe), supplementation with ProPath® Fe (organic Fe) , and supplementation with an Fe chelating agent (chelator) . The desaturase activity of transgenic yeast was estimated by calculating the conversion of the FA substrate into the FA product. Second, an in vivo trial with H. diversicolor juveniles was carried out. Briefly, 20 worms (25-50 mg ww) were randomly distributed in nine experimental units (3 units x 3 diets) . The worms were fed for 7 weeks on an experimental diet with low LC-PUFA (control) , which was supplemented with either FeSO4 (inorganic Fe ) or ProPath® Fe ( organic Fe). Worms were fed to 4% of the biomass 5 d per week. Survival and specific growth rate ( SGR) were recorded. After 7 weeks, the animals were starved for 24h prior sampling for lipid analysis. Total lipids were extracted and quantified gravimetrically, with an aliquot being transmethylated to fatty acid methyl esters (FAME), and analysed using gas chromatography. The results were processed using principal component analysis (PCA). FA analyses from yeast (in vitro assay) were carried out as described above for worm samples .
Results and Discussion
In the in vitro trial all the H. diversicolor desaturases converted the specific FA substrate into the corresponding product with i norganic and organic Fe treatment exhibiting higher enzyme activity than control . The Fe c helating agent reduced the activity of al desaturases (Figure 1a). These results suggest that Fe can effectively enhance desaturase activity as previously reported in yeast . At the end of the experiment, no significant differences (p>0.05) in SGR (average of 0.084) nor s urvival (96.1±4.2 %) were found among treatments. Moreover, t he results from the in vivo trial did not show any clear effect of organic and in organic Fe supplementation on the FA composition of H. diversicolor whereas a clear segregation of the day 0 samples was found (Figure 1b). High levels of 18:1n-9 and 18:2n-6 (23.6 and 19.3%, respectively) on the FA profile of all treatments were found , suggesting a strong dietary effect (34.4 and 15.4%, respectively). Besides, in all treatments, the FA composition of polychaetes showed high levels of PUFA (33.4%) such as 20:4n-6 (ARA), EPA and DHA, indicating bioconversion and trophic upgrading. The reasons underlying the apparent discrepancy between the enhanced desaturase activity observed in vitro and the lack of increased LC-PUFA biosynthesis in worms fed on Fe supplemented diets (in vivo trial) remain unclear. However, it is reasonable to speculate that the Fe enhancing effect on LC-PUFA biosynthesis could not be detected in the present study due to an insufficient capacity of experimental diet to increase the expression of fatty acyl desaturases in vivo. Further analyses on gene expression, as well as FA composition of the polar and neutral lipid fractions, will contribute to clarify the role that dietary Fe supplementation may play as enhancer of LC-PUFA biosynthesis in H. diversicolor.
This study forms part of the ThinkInAzul programme and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Generalitat Valenciana (THINKINAZUL/2021/26). Additional funding was received from the project "SPACE” (TED2021-129647B-I00), fund ed by MCIN/AEI /10.13039/501100011033 and by European Union NextGenerationEU/ PRTR. We thank Zinpro Corporation for supplying ProPath ® Fe for the experimental diets .