www.nature.com/scientificreports OPEN About lipid metabolism in Hermetia illucens (L. 1758): on the origin of fatty acids in prepupae B. Hoc1, M. Genva2, M.‑L. Fauconnier2, G. Lognay1, F. Francis1 & R. Caparros Megido1* Although increasingly targeted in animal nutrition, black soldier fy larvae or prepupae (BSF, Hermetia illucens L. 1758) require the characterization and modulation of their fatty acid profle to become fully integrated within the feed sector. This improvement will only be possible by the understanding of underlaying biochemical pathways of fatty acid synthesis in BSF. In this study, we hypothesized a labelling of de novo synthesized fatty acids in BSF by the incorporation of deuterated water (D2O) in their feed. Three batches of ffty larvae were reared on two diets with diferent polyunsaturated fatty acid profles moistened with 40% of H2O or D2O: chicken feed or 40% of chicken feed and 60% of fax cake. Although the occurrence of D2O in insect feed increased the larval development time and decreased prepupal weight, it was possible to track the biosynthesis of fatty acids through deuterium labelling. Some fatty acids (decanoic, lauric or myristic acid) were exclusively present in their deuterated form while others (palmitic, palmitoleic or oleic acid) were found in two forms (deuterated or not) indicating that BSF can partially produce these fatty acids via biosynthesis pathways and not only by bioaccumulation from the diet. These results suggest the importance of carbohydrates as a source of acetyl‑CoA in the constitution of the BSF fatty acid profle but also the potential importance of specifc enzymes (e.g. thioesterase II or Δ12 fat2 desaturase) in BSF fatty acid metabolism. Finally, nearly no deuterated polyunsaturated fatty acids were found in BSF fed with deuterium confrming that BSF is not able to produce these types of fatty acids. Despite the high levels of linolenic acid in fax‑enriched diets, BSF will simply bioaccumulate around 13% of this fatty acid and will metabolize approximately two‑thirds of it into saturated fatty acids as lauric or myristic acid. Insects are increasingly identifed as an environmentally sustainable source of proteins and lipids for food and feed but also for pharmaceutical or biodiesel applications1–3. While yellow mealworm larvae (Tenebrio molitor L. 1758) appears to be the preferred model in human nutrition for environmental, nutritional and accept- ability considerations4–6, larvae or prepupae of black soldier fy (BSF, Hermetia illucens L. 1758) emerges by their ability to develop rapidly and extensively on a wide range of decomposing material for the feed sector 7–9. Although possessing unquestionable nutritional qualities9,10, the incorporation of BSF into animal feed is still limited by several factors including their high lipid content (till approximately 40%) and their unbalanced fatty acid profle10,11. As BSF fatty acid profle is known to be related with the one of their diet, several experiments have tried to manipulate their fatty acid profle with enriched feed formulation8,12–14. By analysing the fatty acid profles in BSF fed with diferent diets, it appeared that BSF larvae contain a high level of saturated fatty acids (SFAs, lauric acid C12:0 or myristic acid C14:0) not found in their diet. Moreover, when BSF diets were rich in mono- and polyunsaturated fatty acids (MUFAs, PUFAs), it seemed that BSF larvae bioaccumulate some of them (i.e. oleic acid (Z)-C18:1n9; linoleic acid (Z,Z)-C18:2n6 and α-linolenic acid (Z,Z,Z)-C18:3n3) in order to metabolize them principally into C12:08,12–15. Tese results suggest that the understanding of biochemical pathways for fatty acid synthesis by BSF will be one of the next challenges for their use as a sustainable source of lipids. Nevertheless, beside the study of Giannetto et al. and Zhu et al. on the expression of key genes involved in BSF lipid metabolism 15,16, no other studies were performed in order to understand the lipid metabolism in this species of great interest for multiple sectors. 1Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech − University of Liège, TERRA , Passage des Déportés 2, 5030 Gembloux, Belgium. 2Chemistry of Natural Molecules, Gembloux Agro-Bio Tech − University of Liège, TERRA , Passage des Déportés 2, 5030 Gembloux, Belgium. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:11916 | https://doi.org/10.1038/s41598-020-68784-8 1 Vol.:(0123456789) www.nature.com/scientificreports/ To give insights into BSF fatty acid metabolism, we hypothesized that a complete replacement of water by deuterated water (D 2O) in diets will allow us to identify which fatty acid is completely/partially biosynthesized by BSF and which one is simply bioaccumulated from their feed. When the only water source for an organism is D2O, NADPH, a key coenzyme from the pentose-phosphate cycle, will be produced in deuterated form and lead to deuterium atom incorporation during fatty acid biosynthesis 17–19. Moreover, in elongation stages, it was 20,21 shown that deuterium from D2O is incorporated into fatty acids by introducing CD2 instead of a CH2 . To test this hypothesis, batches of BSF larvae were reared until prepupal stage on PUFAs-diferentiated diets (chicken feed or CF and chicken feed with a 60% substitution by fax cake or FL) with water (BSF-CF and BSF-FL) or with deuterated water (BSF-CFD and BSF-FLD with D for deuterated). Te fatty acid profle of these larvae was subsequently analysed by gas chromatography coupled with mass spectrometry (GC–MS) in order to identify biosynthesised or bioaccumulated fatty acids. Results and discussion First of all, despite survival rates above 80% on each diet, the presence of D 2O in insect feed had a negative impact on life history traits of BSF larvae. Te individual prepupal weight was signifcantly higher on standard diets (respectively 251.16 mg ± 4.47a and 200.04 mg ± 6.17b for BSF-CF and BSF-FL; diferent letters indicate signifcant diferences) than on deuterated diets (respectively 113.56 ± 5.89c and 118.01 ± 6.31c for BSF-CFD and BSF-FLD; F3,8 = 134.49; p < 0.001) and the larval development was longer on deuterated diets (from 24 days) than on standard diets (from 9 days). By being more stable than H 2O, D2O is efectively known to decrease some biochemical reactions by a special efect called the secondary isotope efect: the more deuterated bonds being 22 found in a compound, the lower will be the compound overall reactivity . Consequently, D2O have shown to decrease respiratory cycles or to slow down the heart beat in other Arthropods or Diptera species 22,23 and may explain growth delays in BSF larvae. Te fatty acid profle of BSF-CF showed high level of SFAs (C12:0, C14:0 and C16:0) followed by MUFAs ((Z)-C16:1n9 and (Z)-C18:1n9) and about ffeen percent of PUFAs (principally (Z,Z)-C18:2n6) in line with other studies8,12–15 (Fig. 1a, Table S1 for details). For BSF-CFD, the global fatty acid profle followed the same pattern as BSF-CF excepted for the appearance of deuterated fatty acids. As expected, decanoic acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) were almost entirely found in deuterated form as they were not found or in very low proportion (i.e. 0.00% for C10:0, 1.27 ± 0.33% for C12:0 and 0.76 ± 0.18% for C14:0) in the chicken feed. Tese results confrmed our hypothesis since fatty acids supposed to be fully produced by insects were only present in their deuterated form 17,18. SFAs from C12:0 to stearic acid (C18:0) are known to be de novo biosynthesized by several insect species from carbohydrates from their diet (Fig. 2). Tis results was expected for BSF larvae as two genes coding for acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) were recently characterized15,24–26. BSF larvae also produced some proportions of palmitic acid (C16:0) and C18:0. As natural and deuterated C16:0 were found, the present results showed that, despite a high level of this fatty acid in chicken feed (28.78 ± 0.34%), larvae biosynthesized approximately 50% of the C16:0 found in their whole body instead of a simple accumulation. High level in biosynthesized C16:0 could be expected as the fatty acid synthase complex was known to synthetize direct and overwhelming quantity of C16:0 from acetyl-CoA through malonyl-CoA synthesis27–29 due to the very high specifcity of the terminal thioesterase (which releases the biosynthesized fatty acid) for C16:028,30. Production of C16:0D (or other unexpected deuterated fatty acids) could also result from stress conditions experienced by insects in the presence of D 2O. Although this hypothesis seems weak based on the expected and obtained results for C:10D and C:12D, combined genomic and proteomic studies, such as the study of Li et al., are essential to improve our knowledge of fatty acid production in BSF 31. Following this synthesis, C16:0 will be elongated to form longer-chain fatty acids (as C18:0D via the fatty acid elongase 6 /ELOVL 632), desaturated to generate unsaturated fatty acids or used for the synthesis of storage lipids27. Te occurrence of C10 to C14 fatty acids can be explained by the action of an additional cytosolic thioesterase (e.g. thioesterase II) or by retroconversion by partial peroxisomal β-oxidation28,33,34. Finally, SFAs’ levels were lower in BSF-CFD than in BSF-CF excepted for the bioaccumulated C18:0. Tis result can be justifed by the decrease in overall reactivity caused by the occurrence of D2O in metabolic pathways which reduces the typical production of these fatty acids 22; the replacement of an hydrogen atom by deuterium has an infuence on enzyme activities by reducing their specifcities and/or kinetic parameters 20.