Introduction
Many aquatic invertebrates, including mollusks, crustaceans, cnidarians and annelids, may serve as exceptional sources of the essential nutrients, including the long-chain (≥C20) polyunsaturated fatty acids (LC-PUFAs), namely arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3), and docosahexaenoic acid (DHA, 22:6n-3). While some of the LC-PUFAs contained in invertebrate body lipids derive from dietary input, some aquatic invertebrates have the capacity to inherently synthesize LC-PUFAs from adequate short-chain precursors thanks to the presence of complementary set of enzymes called elongases (Elovl), front-end desaturases (Fed) and methyl-end desaturases (��x). Polychaetes are arguably the most studied group within the Annelida phylum regarding their LC-PUFA biosynthetic capacity; however, other groups, such as oligochaetes, have been largely overlooked. Focusing on the terrestrial oligochaete Enchytraeus albidus, an organism often used as live feed and functional feed ingredient in fish larviculture , the present study aims to determine the capacity of E. albidus to synthesize LC-PUFAs through the identification and characterization of its enzymatic repertoire of fatty acid elongases and desaturases.
Materials and Methods
To identify the potential elongases (Elovl) and desaturases (Fed and ��x), tblastn searches were conducted against publicly available Transcriptome Shotgun Assembly (TSA) databases from E. albidus, using as query known sequences of desaturases and elongases from marine polychaetes. After sequence validation, the putative elongase and desaturase sequences retrieved from E. albidus were used as a reference to design primers enabling the amplification of the complete open reading frames (ORFs) by PCR. The PCR template was cDNA prepared from total RNA from fresh worms (pools of three individuals). The PCR products corresponding to the ORFs of the putative elongases and desaturases from E. albidus were digested and cloned into pYES2 for functional assays in yeast (see below). Correctness of the ORF inserts in pYES2 was confirmed by sequencing. For the molecular characterization of the encoded enzymes, we compared the deduced protein sequences with other annelid elongases and desaturases, identified conserved domains, and performed a Maximum likelihood phylogenetic tree along with known sequences from multiple invertebrates. For functional characterization, transgenic yeast transformed with the pYES2 constructs containing the E. albidus ORF of desaturases and elongases were supplemented with potential fatty acid substrates selected based on currently established LC-PUFA biosynthetic pathways (Monroig et al., 2022). After 48 h, yeast cultures were harvested, and total lipids were extracted for further derivatization into fatty acid methyl esters (FAME). FAMEs were analyzed through gas chromatography and mass spectrometry detection (GC-MS), and conversions were calculated using the formula [product area / (product area +substrate area)] × 100.
Results and Discussion
Three Elovl, two Fed and two ��x with putative roles in the LC-PUFA biosynthesis were identified in E. albidus. Both the elongases and desaturases contained histidine boxes and conserved domains expected for their respective protein family . The phylogenetic analyses of the three E. albidus Elovl sequences clustered closely with well-described elongases from other invertebrates, including annelids, allowing us to classify them as Elovl2/5, Elovl4 and Elovl8. The two E. albidus Fed sequences, namely Fed1 and Fed2, were clustered in the so-called "Group A" and "Group B", respectively. The E. albidus ��x desaturases, termed hereafter as ��x1 and ��x2, clustered closely to other methyl-end desaturases from Annelida, within Clade 3 as referred to by Concerning the functional assays, yeast expressing the three E. albidus elongases showed activity towards C18 and C20 fatty acid precursors. Regarding desaturases, the E. albidus Fed1 exhibited ��5 desaturation activity, being able to produce LC-PUFAs like ARA and EPA, while Fed2 showed a combination of ��6 and ��8 activities. These newfound enzymes displayed similar functions to their homologs found in polychaetes such as Hediste diversicolor . The assays on the E. albidus methyl-end desaturases showed complementary functions. Yeast expressing the E. albidus ��x1 was able to convert oleic acid (18:1n-9) into linoleic acid (18:2n-6), confirming this enzyme is a ��12 desaturase. The ��x2 showed ��15, ��17 and ��19 activities, converting a variety of omega-6 substrates into omega-3 LC-PUFAs (e.g., EPA), and exhibiting analogous activities to other methyl-end desaturases of polychaetes like H. diversicolor . Collectively, the present investigation demonstrates that E. albidus has the complete enzymatic repertoire needed for the de novo biosynthesis of LC-PUFAs including ARA and EPA, thus allowing the production of live prey with high nutritional value and eliminating the requirement for exogenous enrichment prior to use as larval feed.
Acknowledgment
The POLYPUFA grant PID2022-136234OB-C21 was funded by MICIU/AEI /10.13039/501100011033 and by FEDER, UE. Additional funding was received from Independent Research Fund Denmark for the NOVOPUFA project to MH (grant no. 3164-00138B).
References
Grantland, D., Holmstrup, M., Overgaard, J., Holmstrup, M. E., Slotsbo, S. (2025). Tiny worms, big potential: Enchytraeus albidus (Annelida: Clitellata) as starter feed for rainbow trout. Aquaculture Research 2025, 339644. https://doi.org/10.1155/are/7339644
Hashimoto, K., Yoshizawa, A. C., Okuda, S., Kuma, K., Goto, S., Kanehisa, M. (2008). The repertoire of desaturases and elongases reveals fatty acid variations in 56 eukaryotic genomes. Journal of Lipid Research 49 (1), 183–191. https://doi.org/10.1194/jlr.M700377-JLR200
Kabeya, N., Fonseca, M. M., Ferrier, D. E. K., Navarro, J. C., Bay, L. K., Francis, D. S., Tocher, D. R., Filipe, L., Castro, C., Monroig, ��. (2018). Genes for de novo biosynthesis of omega-3 polyunsaturated fatty acids are widespread in animals. Science Advances 4 (5), aar6849. https://doi.org/10.1126/sciadv.aar6849
Kabeya, N., G��r, I., Oboh, A., Evjemo, J. O., Malzahn, A. M., Hontoria, F., Navarro, J. C., Monroig, ��. (2020). Unique fatty acid desaturase capacities uncovered in Hediste diversicolor illustrate the roles of aquatic invertebrates in trophic upgrading. Philosophical Transactions of the Royal Society B: Biological Sciences 375 (1804), 20190654. https://doi.org/10.1098/rstb.2019.0654
Monroig, ��., Shu-Chien, A. C., Kabeya, N., Tocher, D. R., Castro, L. F. C. (2022). Desaturases and elongases involved in long-chain polyunsaturated fatty acid biosynthesis in aquatic animals: From genes to functions. Progress in Lipid Research 86, 101157. https://doi.org/10.1016/j.plipres.2022.101157
Villena-Rodr��guez, A., Navarro, J. C., Hontoria, F., Castro, L. F. C., Malzahn, A. M., Hagemann, A., Monroig, ��. (2025). Biosynthesis of long-chain polyunsaturated fatty acids in the nereid polychaete Hediste diversicolor: Molecular cloning and functional characterisation of three fatty acyl elongases and two front-end desaturases. Aquaculture 595, 741497. https://doi.org/10.1016/j.aquaculture.2024.741497