Thank you for visiting Nature.com. The browser version you are using has limited CSS support. For best results, we recommend using a newer browser (or disabling compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we will display the site without styles and JavaScript.
The black soldier fly (Hermetia illucens, L. 1758) is an omnivorous detritivorous insect with a high potential for utilizing carbohydrate-rich organic by-products. Among carbohydrates, black soldier flies rely on soluble sugars for growth and lipid synthesis. The aim of this study was to evaluate the effects of common soluble sugars on the development, survival, and fatty acid profile of black soldier flies. Supplement chicken feed with monosaccharides and disaccharides separately. Cellulose was used as a control. Larvae fed glucose, fructose, sucrose, and maltose grew faster than control larvae. In contrast, lactose had an antinutritional effect on larvae, slowing growth and reducing final individual body weight. However, all soluble sugars made larvae fatter than those fed the control diet. Notably, the tested sugars shaped the fatty acid profile. Maltose and sucrose increased the saturated fatty acid content compared to cellulose. In contrast, lactose increased the bioaccumulation of dietary unsaturated fatty acids. This study is the first to demonstrate the effect of soluble sugar on the fatty acid composition of black soldier fly larvae. Our results indicate that the tested carbohydrates have a significant effect on the fatty acid composition of black soldier fly larvae and may therefore determine their final application.
The global demand for energy and animal protein continues to increase1. In the context of global warming, it is imperative to find greener alternatives to fossil energy and traditional food production methods while increasing production. Insects are promising candidates to address these issues due to their lower chemical composition and environmental impact compared to traditional livestock farming2. Among insects, an excellent candidate to address these issues is the black soldier fly (BSF), Hermetia illucens (L. 1758), a detritivorous species capable of feeding on a variety of organic substrates3. Therefore, valorizing these substrates through BSF breeding could create a new source of raw materials to meet the needs of various industries.
BSF larvae (BSFL) can feed on agricultural and agro-industrial by-products such as brewers’ grain, vegetable residues, fruit pulp and stale bread, which are particularly suitable for BSFL growth due to their high carbohydrate (CH)4,5,6 content. Large-scale production of BSFL results in the formation of two products: feces, a mixture of substrate residues and feces that can be used as fertilizer for plant cultivation7, and larvae, which are mainly composed of proteins, lipids and chitin. Proteins and lipids are mainly used in livestock farming, biofuel and cosmetics8,9. As for chitin, this biopolymer finds applications in the agri-food sector, biotechnology and health care10.
BSF is an autogenous holometabolous insect, meaning that its metamorphosis and reproduction, particularly the energy-consuming stages of the insect’s life cycle, can be entirely supported by nutrient reserves generated during larval growth11. More specifically, protein and lipid synthesis leads to the development of the fat body, an important storage organ that releases energy during the non-feeding phases of BSF: prepupa (i.e., the final larval stage during which BSF larvae turn black while feeding and searching for an environment suitable for metamorphosis), pupae (i.e., the non-motile stage during which the insect undergoes metamorphosis), and adults12,13. CH is the main energy source in the diet of BSF14. Among these nutrients, fibrous CH such as hemicellulose, cellulose and lignin, unlike disaccharides and polysaccharides (such as starch), cannot be digested by BSFL15,16. Digestion of CH is an important preliminary step for the absorption of carbohydrates, which are ultimately hydrolyzed to simple sugars in the intestine16. The simple sugars can then be absorbed (i.e., through the intestinal peritrophic membrane) and metabolized to produce energy17. As mentioned above, larvae store excess energy as lipids in the fat body12,18. The storage lipids consist of triglycerides (neutral lipids formed from one glycerol molecule and three fatty acids) synthesized by the larvae from dietary simple sugars. These CH provide the acetyl-CoA substrates required for fatty acid (FA) biosynthesis via the fatty acid synthase and thioesterase pathways19. The fatty acid profile of H. illucens lipids is naturally dominated by saturated fatty acids (SFA) with a high proportion of lauric acid (C12:0)19,20. Therefore, the high lipid content and fatty acid composition are rapidly becoming limiting factors for the use of whole larvae in animal feed, especially in aquaculture where polyunsaturated fatty acids (PUFA) are needed21.
Since the discovery of the potential of BSFL to reduce organic waste, studies on the value of various by-products have shown that the composition of BSFL is partly regulated by its diet. Currently, the regulation of the FA profile of H. illucens continues to improve. The ability of BSFL to bioaccumulate PUFA has been demonstrated on PUFA-rich substrates such as algae, fish waste, or meals such as flaxseed, which provides a higher quality FA profile for animal nutrition19,22,23. In contrast, for by-products that are not enriched in PUFA, there is not always a correlation between the dietary FA profiles and the larval FA, indicating the influence of other nutrients24,25. In fact, the effect of digestible CH on FA profiles remains poorly understood and under-researched24,25,26,27.
To the best of our knowledge, although total monosaccharides and disaccharides are abundant in the diet of H. illucens, their nutritional role remains poorly understood in H. illucens nutrition. The aim of this study was to elucidate their effects on BSFL nutrition and lipid composition. We will evaluate the growth, survival, and productivity of larvae under different nutritional conditions. Then, we will describe the lipid content and fatty acid profile of each diet to highlight the effects of CH on BSFL nutritional quality.
We hypothesized that the nature of the tested CH would affect (1) larval growth, (2) total lipid levels, and (3) modulate the FA profile. Monosaccharides can be absorbed directly, whereas disaccharides must be hydrolyzed. Monosaccharides are thus more available as direct energy sources or precursors for lipogenesis via the FA synthase and thioesterase pathways, thereby enhancing H. illucens larval growth and promoting the accumulation of reserve lipids (especially lauric acid).
The tested CH affected the average body weight of larvae during growth (Fig. 1). FRU, GLU, SUC and MAL increased larval body weight similarly to the control diet (CEL). In contrast, LAC and GAL appeared to retard larval development. Notably, LAC had a significant negative effect on larval growth compared to SUC throughout the growth period: 9.16 ± 1.10 mg versus 15.00 ± 1.01 mg on day 3 (F6,21 = 12.77, p < 0.001; Fig. 1), 125.11 ± 4.26 mg and 211.79 ± 14.93 mg, respectively, on day 17 (F6,21 = 38.57, p < 0.001; Fig. 1).
Using different monosaccharides (fructose (FRU), galactose (GAL), glucose (GLU)), disaccharides (lactose (LAC), maltose (MAL), sucrose (SUC)) and cellulose (CEL) as controls. Growth of larvae fed with black soldier fly larvae. Each point on the curve represents the mean individual weight (mg) calculated by weighing 20 randomly selected larvae from a population of 100 larvae (n = 4). Error bars represent SD.
The CEL diet provided excellent larval survival of 95.5 ± 3.8%. Moreover, the survival of H. illucens fed diets containing soluble CH was reduced (GLM: χ = 107.13, df = 21, p < 0.001), which was caused by MAL and SUC (disaccharides) in the studied CH. The mortality was lower than that of GLU, FRU, GAL (monosaccharide), and LAC (EMM: p < 0.001, Figure 2).
Boxplot of survival of black soldier fly larvae treated with various monosaccharides (fructose, galactose, glucose), disaccharides (lactose, maltose, sucrose) and cellulose as controls. Treatments with the same letter are not significantly different from each other (EMM, p > 0.05).
All diets tested allowed larvae to reach the prepupal stage. However, the CHs tested tended to prolong larval development (F6,21=9.60, p<0.001; Table 1). In particular, larvae fed GAL and LAC took longer to reach the prepupal stage compared to larvae reared on CEL (CEL-GAL: p<0.001; CEL-LAC: p<0.001; Table 1).
The tested CH also had different effects on the larval body weight, with the body weight of larvae fed the CEL diet reaching 180.19 ± 11.35 mg (F6,21 = 16.86, p < 0.001; Fig. 3). FRU, GLU, MAL and SUC resulted in an average final larval body weight of more than 200 mg, which was significantly higher than that of CEL (p < 0.05). In contrast, larvae fed GAL and LAC had lower body weights, averaging 177.64 ± 4.23 mg and 156.30 ± 2.59 mg, respectively (p < 0.05). This effect was more pronounced with LAC, where final body weight was lower than with the control diet (CEL-LAC: difference = 23.89 mg; p = 0.03; Figure 3).
Mean final weight of individual larvae expressed as larval spots (mg) and black soldier flies expressed as histogram (g) fed different monosaccharides (fructose, galactose, glucose), disaccharides (lactose, maltose, sucrose) and cellulose (as control). Columnar letters represent groups significantly different in total larval weight (p < 0.001). Letters associated with larval spots represent groups with significantly different individual larval weights (p < 0.001). Error bars represent SD.
Maximum individual weight was independent of the maximum final total larval colony weight. In fact, diets containing FRU, GLU, MAL, and SUC did not increase the total larval weight produced in the tank compared to CEL (Figure 3). However, LAC significantly decreased the total weight (CEL-LAC: difference = 9.14 g; p < 0.001; Figure 3).
Table 1 shows the yield (larvae/day). Interestingly, the optimal yields of CEL, MAL and SUC were similar (Table 1). In contrast, FRU, GAL, GLU and LAC reduced the yield compared to CEL (Table 1). GAL and LAC performed the worst: the yield was halved to only 0.51 ± 0.09 g larvae/day and 0.48 ± 0.06 g larvae/day, respectively (Table 1).
Monosaccharides and disaccharides increased the lipid content of the CF larvae (Table 1). On the CLE diet, larvae with a lipid content of 23.19 ± 0.70% of the DM content were obtained. For comparison, the average lipid content in larvae fed with soluble sugar was more than 30% (Table 1). However, the tested CHs increased their fat content to the same extent.
As expected, the CG subjects affected the FA profile of the larvae to varying degrees (Fig. 4). The SFA content was high in all diets and reached more than 60%. MAL and SUC unbalanced the FA profile, which led to an increase in the SFA content. In the case of MAL, on the one hand, this imbalance led predominantly to a decrease in the content of monounsaturated fatty acids (MUFA) (F6,21 = 7.47; p < 0.001; Fig. 4). On the other hand, for SUC, the decrease was more uniform between MUFA and PUFA. LAC and MAL had opposite effects on the FA spectrum (SFA: F6,21 = 8.74; p < 0.001; MUFA: F6,21 = 7.47; p < 0.001; PUFA: χ2 = 19.60; Df = 6; p < 0.001; Figure 4). The lower proportion of SFA in LAC-fed larvae appears to increase the MUFA content. In particular, MUFA levels were higher in LAC-fed larvae compared to other soluble sugars except for GAL (F6,21 = 7.47; p < 0.001; Figure 4).
Using different monosaccharides (fructose (FRU), galactose (GAL), glucose (GLU)), disaccharides (lactose (LAC), maltose (MAL), sucrose (SUC)) and cellulose (CEL) as controls, box plot of fatty acid composition fed to black soldier fly larvae. Results are expressed as percentage of total FAME. Treatments marked with different letters are significantly different (p < 0.001). (a) Proportion of saturated fatty acids; (b) Monounsaturated fatty acids; (c) Polyunsaturated fatty acids.
Among the identified fatty acids, lauric acid (C12:0) was dominant in all observed spectra (more than 40%). Other present SFAs were palmitic acid (C16:0) (less than 10%), stearic acid (C18:0) (less than 2.5%) and capric acid (C10:0) (less than 1.5%). MUFAs were mainly represented by oleic acid (C18:1n9) (less than 9.5%), while PUFAs were mainly composed of linoleic acid (C18:2n6) (less than 13.0%) (see Supplementary Table S1). In addition, a small proportion of compounds could not be identified, especially in the spectra of CEL larvae, where unidentified compound number 9 (UND9) accounted for an average of 2.46 ± 0.52% (see Supplementary Table S1). GC×GC-FID analysis suggested that it might be a 20-carbon fatty acid with five or six double bonds (see Supplementary Figure S5).
The PERMANOVA analysis revealed three distinct groups based on the fatty acid profiles (F6,21 = 7.79, p < 0.001; Figure 5). Principal component analysis (PCA) of the TBC spectrum illustrates this and is explained by two components (Figure 5). The principal components explained 57.9% of the variance and included, in order of importance, lauric acid (C12:0), oleic acid (C18:1n9), palmitic acid (C16:0), stearic acid (C18:0), and linolenic acid (C18:3n3) (see Figure S4). The second component explained 26.3% of the variance and included, in order of importance, decanoic acid (C10:0) and linoleic acid (C18:2n6 cis) (see Supplementary Figure S4). The profiles of diets containing simple sugars (FRU, GAL and GLU) showed similar characteristics. In contrast, disaccharides yielded different profiles: MAL and SUC on one hand and LAC on the other. In particular, MAL was the only sugar that changed the FA profile compared to CEL. In addition, the MAL profile was significantly different from the FRU and GLU profiles. In particular, the MAL profile showed the highest proportion of C12:0 (54.59 ± 2.17%), making it comparable to the CEL (43.10 ± 5.01%), LAC (43.35 ± 1.31%), FRU (48.90 ± 1.97%) and GLU (48.38 ± 2.17%) profiles (see Supplementary Table S1). The MAL spectrum also showed the lowest C18:1n9 content (9.52 ± 0.50%), which further differentiated it from the LAC (12.86 ± 0.52%) and CEL (12.40 ± 1.31%) spectra. A similar trend was observed for C16:0. In the second component, the LAC spectrum showed the highest C18:2n6 content (17.22 ± 0.46%), while MAL showed the lowest (12.58 ± 0.67%). C18:2n6 also differentiated LAC from the control (CEL), which showed lower levels (13.41 ± 2.48%) (see Supplementary Table S1).
PCA plot of fatty acid profile of black soldier fly larvae with different monosaccharides (fructose, galactose, glucose), disaccharides (lactose, maltose, sucrose) and cellulose as control.
To study the nutritional effects of soluble sugars on H. illucens larvae, cellulose (CEL) in chicken feed was replaced with glucose (GLU), fructose (FRU), galactose (GAL), maltose (MAL), sucrose (SUC), and lactose (LAC). However, monosaccharides and disaccharides had different effects on the development, survival, and composition of HF larvae. For example, GLU, FRU, and their disaccharide forms (MAL and SUC) exerted positive supportive effects on larval growth, allowing them to achieve higher final body weights than CEL. Unlike indigestible CEL, GLU, FRU, and SUC can bypass the intestinal barrier and serve as important nutrient sources in formulated diets16,28. MAL lacks specific animal transporters and is thought to be hydrolyzed to two glucose molecules before assimilation15. These molecules are stored in the insect body as a direct energy source or as lipids18. First, with regard to the latter, some of the observed intramodal differences may be due to small differences in sex ratios. Indeed, in H. illucens, reproduction may be entirely spontaneous: adult females naturally have sufficient egg-laying reserves and are heavier than males29. However, lipid accumulation in BSFL correlates with dietary soluble CH2 intake, as previously observed for GLU and xylose26,30. For example, Li et al.30 observed that when 8% GLU was added to the larval diet, the lipid content of BSF larvae increased by 7.78% compared to controls. Our results are consistent with these observations, showing that fat content in larvae fed the soluble sugar was higher than that of larvae fed the CEL diet, compared with an 8.57% increase with GLU supplementation. Surprisingly, similar results were observed in larvae fed GAL and LAC, despite the adverse effects on larval growth, final body weight, and survival. Larvae fed LAC were significantly smaller than those fed the CEL diet, but their fat content was comparable to larvae fed the other soluble sugars. These results highlight the antinutritional effects of lactose on BSFL. First, the diet contains a large amount of CH. The absorption and hydrolysis systems of monosaccharides and disaccharides, respectively, may reach saturation, causing bottlenecks in the assimilation process. As for hydrolysis, it is carried out by α- and β-glucosidases 31 . These enzymes have preferred substrates depending on their size and the chemical bonds (α or β linkages) between their constituent monosaccharides 15 . Hydrolysis of LAC to GLU and GAL is carried out by β-galactosidase, an enzyme whose activity has been demonstrated in the gut of BSF 32 . However, its expression may be insufficient compared to the amount of LAC consumed by larvae. In contrast, α-glucosidase maltase and sucrase 15, which are known to be abundantly expressed in insects, are able to break down large amounts of MAL and sucrose SUC, thereby limiting this satiating effect. Secondly, antinutritional effects may be due to the reduced stimulation of insect intestinal amylase activity and the slowing of feeding behavior compared to other treatments. Indeed, soluble sugars have been identified as stimulators of enzyme activity important for insect digestion, such as amylase, and as triggers of the feeding response33,34,35. The degree of stimulation varies depending on the molecular structure of the sugar. In fact, disaccharides require hydrolysis before absorption and tend to stimulate amylase more than their constituent monosaccharides34. In contrast, LAC has a milder effect and has been found to be incapable of supporting insect growth in various species33,35. For example, in the pest Spodoptera exigua (Boddie 1850), no hydrolytic activity of LAC was detected in extracts of caterpillar midgut enzymes36.
Regarding the FA spectrum, our results indicate significant modulatory effects of the tested CH. Notably, although lauric acid (C12:0) accounted for less than 1% of the total FA in the diet, it dominated in all profiles (see Supplementary Table S1). This is consistent with previous data that lauric acid is synthesized from dietary CH in H. illucens via a pathway involving acetyl-CoA carboxylase and FA synthase19,27,37. Our results confirm that CEL is largely indigestible and acts as a “bulking agent” in BSF control diets, as discussed in several BSFL studies38,39,40. Replacing CEL with monosaccharides and disaccharides other than LAC increased the C12:0 ratio, indicating increased CH uptake by larvae. Interestingly, the disaccharides MAL and SUC promote lauric acid synthesis more efficiently than their constituent monosaccharides, suggesting that despite the higher degree of polymerization of GLU and FRU, and since Drosophila is the only sucrose transporter that has been identified in animal protein species, disaccharide transporters may not be present in the gut of H. illucens larvae15, the utilization of GLU and FRU is increased. However, although GLU and FRU are theoretically more easily metabolized by BSF, they are also more easily metabolized by substrates and gut microorganisms, which may result in their more rapid degradation and decreased utilization by larvae compared to disaccharides.
At first glance, the lipid content of larvae fed LAC and MAL was comparable, indicating similar bioavailability of these sugars. However, surprisingly, the FA profile of LAC was richer in SFA, especially with lower C12:0 content, compared to MAL. One hypothesis to explain this difference is that LAC may stimulate the bioaccumulation of dietary FA via acetyl-CoA FA synthase. Supporting this hypothesis, LAC larvae had the lowest decanoate (C10:0) ratio (0.77 ± 0.13%) than the CEL diet (1.27 ± 0.16%), indicating reduced FA synthase and thioesterase activities19. Second, dietary fatty acids are considered to be the main factor influencing the SFA composition of H. illucens27. In our experiments, linoleic acid (C18:2n6) accounted for 54.81% of dietary fatty acids, with the proportion in LAC larvae being 17.22 ± 0.46% and in MAL 12.58 ± 0.67%. Oleic acid (cis + trans C18:1n9) (23.22% in the diet) showed a similar trend. The ratio of α-linolenic acid (C18:3n3) also supports the bioaccumulation hypothesis. This fatty acid is known to accumulate in BSFL upon substrate enrichment, such as the addition of flaxseed cake, up to 6-9% of the total fatty acids in larvae19. In enriched diets, C18:3n3 can account for up to 35% of the total dietary fatty acids. However, in our study, C18:3n3 accounted for only 2.51% of the fatty acid profile. Although the proportion found in nature was lower in our larvae, this proportion was higher in LAC larvae (0.87 ± 0.02%) than in MAL (0.49 ± 0.04%) (p < 0.001; see Supplementary Table S1). The CEL diet had an intermediate proportion of 0.72 ± 0.18%. Finally, the palmitic acid (C16:0) ratio in CF larvae reflects the contribution of synthetic pathways and dietary FA19. Hoc et al. 19 observed that C16:0 synthesis was reduced when the diet was enriched with flaxseed meal, which was attributed to a decrease in the availability of the acetyl-CoA substrate due to a decrease in the CH ratio. Surprisingly, although both diets had similar CH content and MAL showed higher bioavailability, MAL larvae showed the lowest C16:0 ratio (10.46 ± 0.77%), whereas LAC showed a higher proportion, accounting for 12.85 ± 0.27% (p < 0.05; see Supplementary Table S1). These results highlight the complex influence of nutrients on BSFL digestion and metabolism. Currently, research on this topic is more thorough in Lepidoptera than in Diptera. In caterpillars, LAC was identified as a weak stimulant of feeding behavior compared to other soluble sugars such as SUC and FRU34,35. In particular, in Spodopteralittoralis (Boisduval 1833), MAL consumption stimulated amylolytic activity in the intestine to a greater extent than LAC34. Similar effects in BSFL may explain the enhanced stimulation of the C12:0 synthetic pathway in MAL larvae, which is associated with increased intestinally absorbed CH, prolonged feeding, and intestinal amylase action. Less stimulation of the feeding rhythm in the presence of LAC may also explain the slower growth of LAC larvae. Moreover, Liu Yanxia et al. 27 noted that the shelf life of lipids in H. illucens substrates was longer than that of CH. Therefore, LAC larvae may rely more on dietary lipids to complete their development, which may increase their final lipid content and modulate their fatty acid profile.
To the best of our knowledge, only a few studies have tested the effects of monosaccharide and disaccharide addition to BSF diets on their FA profiles. First, Li et al. 30 assessed the effects of GLU and xylose and observed lipid levels similar to ours at an 8% addition rate. The FA profile was not detailed and consisted mainly of SFA, but no differences were found between the two sugars or when they were presented simultaneously30. Furthermore, Cohn et al. 41 showed no effect of 20% GLU, SUC, FRU and GAL addition to chicken feed on the respective FA profiles. These spectra were obtained from technical rather than biological replicates, which, as explained by the authors, may limit the statistical analysis. Furthermore, the lack of iso-sugar control (using CEL) limits the interpretation of the results. Recently, two studies by Nugroho RA et al. demonstrated anomalies in the FA spectra42,43. In the first study, Nugroho RA et al. 43 tested the effect of adding FRU to fermented palm kernel meal. The FA profile of the resulting larvae showed abnormally high levels of PUFA, more than 90% of which were derived from the diet containing 10% FRU (similar to our study). Although this diet contained PUFA-rich fish pellets, the reported FA profile values of the larvae on the control diet consisting of 100% fermented PCM were not consistent with any previously reported profile, in particular the abnormal level of C18:3n3 of 17.77 ± 1.67% and 26.08 ± 0.20% for conjugated linoleic acid (C18:2n6t), a rare isomer of linoleic acid. The second study showed similar results including FRU, GLU, MAL and SUC42 in fermented palm kernel meal. These studies, like ours, highlight serious difficulties in comparing results from BSF larval diet trials, such as control choices, interactions with other nutrient sources, and FA analysis methods.
During the experiments, we observed that the colour and odour of the substrate varied depending on the diet used. This suggests that microorganisms may play a role in the results observed in the substrate and the digestive system of the larvae. In fact, monosaccharides and disaccharides are easily metabolised by colonising microorganisms. The rapid consumption of soluble sugars by microorganisms may result in the release of large quantities of microbial metabolic products such as ethanol, lactic acid, short-chain fatty acids (e.g. acetic acid, propionic acid, butyric acid) and carbon dioxide44. Some of these compounds may be responsible for the lethal toxic effects on larvae also observed by Cohn et al.41 under similar developmental conditions. For example, ethanol is harmful to insects45. Large quantities of carbon dioxide emissions may result in its accumulation at the bottom of the tank, which may deprive the atmosphere of oxygen if air circulation does not allow its release. Regarding SCFAs, their effects on insects, especially H. illucens, remain poorly understood, although lactic acid, propionic acid, and butyric acid have been shown to be lethal in Callosobruchus maculatus (Fabricius 1775)46. In Drosophila melanogaster Meigen 1830, these SCFAs are olfactory markers that guide females to oviposition sites, suggesting a beneficial role in larval development47. However, acetic acid is classified as a hazardous substance and can significantly inhibit larval development47. In contrast, microbially derived lactate has recently been found to have a protective effect against invasive gut microbes in Drosophila48. Furthermore, microorganisms in the digestive system also play a role in CH digestion in insects49. Physiological effects of SCFAs on the gut microbiota, such as feeding rate and gene expression, have been described in vertebrates 50 . They may also have a trophic effect on H. illucens larvae and may contribute in part to the regulation of FA profiles. Studies on the nutritional effects of these microbial fermentation products will clarify their effects on H. illucens nutrition and provide a basis for future studies on beneficial or detrimental microorganisms in terms of their development and the value of FA-rich substrates. In this regard, the role of microorganisms in the digestive processes of mass-farmed insects is increasingly being studied. Insects are beginning to be viewed as bioreactors, providing pH and oxygenation conditions that facilitate the development of microorganisms specialized in the degradation or detoxification of nutrients that are difficult for insects to digest 51 . Recently, Xiang et al.52 demonstrated that, for example, inoculation of organic waste with a bacterial mixture allows CF to attract bacteria specialized in lignocellulose degradation, improving its degradation in the substrate compared to substrates without larvae.
Finally, with regard to the beneficial use of organic waste by H. illucens, the CEL and SUC diets produced the highest number of larvae per day. This means that despite the lower final weight of individual individuals, the total larval weight produced on a substrate consisting of indigestible CH is comparable to that obtained on a homosaccharide diet containing monosaccharides and disaccharides. In our study, it is important to note that the levels of other nutrients are sufficient to support the growth of the larval population and that the addition of CEL should be limited. However, the final composition of the larvae differs, highlighting the importance of choosing the right strategy for valorizing the insects. CEL larvae fed with whole feed are more suitable for use as animal feed due to their lower fat content and lower lauric acid levels, whereas larvae fed with SUC or MAL diets require defatting by pressing to increase the value of the oil, especially in the biofuel sector. LAC is found in dairy industry by-products such as whey from cheese production. Recently, its use (3.5% lactose) improved final larval body weight53. However, the control diet in this study contained half the lipid content. Therefore, the antinutritional effects of LAC may have been counteracted by larval bioaccumulation of dietary lipids.
As shown by previous studies, the properties of monosaccharides and disaccharides significantly affect the growth of BSFL and modulate its FA profile. In particular, LAC seems to play an antinutritional role during larval development by limiting the availability of CH for dietary lipid absorption, thereby promoting UFA bioaccumulation. In this context, it would be interesting to conduct bioassays using diets combining PUFA and LAC. Furthermore, the role of microorganisms, especially the role of microbial metabolites (such as SCFAs) derived from sugar fermentation processes, remains a research topic worthy of investigation.
Insects were obtained from the BSF colony of the Laboratory of Functional and Evolutionary Entomology established in 2017 at Agro-Bio Tech, Gembloux, Belgium (for more details on rearing methods, see Hoc et al. 19). For experimental trials, 2.0 g of BSF eggs were randomly collected daily from breeding cages and incubated in 2.0 kg of 70% wet chicken feed (Aveve, Leuven, Belgium). Five days after hatching, larvae were separated from the substrate and counted manually for experimental purposes. The initial weight of each batch was measured. The average individual weight was 7.125 ± 0.41 mg, and the average for each treatment is shown in Supplementary Table S2.
The diet formulation was adapted from the study by Barragan-Fonseca et al. 38 . Briefly, a compromise was found between the same feed quality for larval chickens, similar dry matter (DM) content, high CH (10% based on fresh diet) and texture, since simple sugars and disaccharides have no textural properties. According to the manufacturer’s information (Chicken Feed, AVEVE, Leuven, Belgium), the tested CH (i.e. soluble sugar) was added separately as an autoclaved aqueous solution (15.9%) to a diet consisting of 16.0% protein, 5.0% total lipids, 11.9% ground chicken feed consisting of ash and 4.8% fibre. In each 750 ml jar (17.20 × 11.50 × 6.00 cm, AVA, Tempsee, Belgium), 101.9 g of autoclaved CH solution was mixed with 37.8 g of chicken feed. For each diet, the dry matter content was 37.0%, including homogeneous protein (11.7%), homogeneous lipids (3.7%) and homogeneous sugars (26.9% of added CH). The CH tested were glucose (GLU), fructose (FRU), galactose (GAL), maltose (MAL), sucrose (SUC) and lactose (LAC). The control diet consisted of cellulose (CEL), which is considered indigestible for H. illucens larvae 38 . One hundred 5-day-old larvae were placed in a tray fitted with a lid with a 1 cm diameter hole in the middle and covered with a plastic mosquito net. Each diet was repeated four times.
Larval weights were measured three days after the start of the experiment. For each measurement, 20 larvae were removed from the substrate using sterile warm water and forceps, dried, and weighed (STX223, Ohaus Scout, Parsippany, USA). After weighing, larvae were returned to the center of the substrate. Measurements were taken regularly three times a week until the first prepupa emerged. At this point, collect, count, and weigh all larvae as described previously. Separate stage 6 larvae (i.e., white larvae corresponding to the larval stage preceding the prepupal stage) and prepupae (i.e., the last larval stage during which BSF larvae turn black, stop feeding, and seek an environment suitable for metamorphosis) and store at -18°C for compositional analysis. The yield was calculated as the ratio of the total mass of insects (larvae and prepupae of stage 6) obtained per dish (g) to the development time (d). All mean values in the text are expressed as: mean ± SD.
All subsequent steps using solvents (hexane (Hex), chloroform (CHCl3), methanol (MeOH)) were performed under a fume hood and required wearing nitrile gloves, aprons and safety glasses.
White larvae were dried in a FreeZone6 freeze dryer (Labconco Corp., Kansas City, MO, USA) for 72 h and then ground (IKA A10, Staufen, Germany). Total lipids were extracted from ±1 g of powder using the Folch method 54. The residual moisture content of each lyophilized sample was determined in duplicate using a moisture analyzer (MA 150, Sartorius, Göttiggen, Germany) to correct for total lipids.
Total lipids were transesterified under acidic conditions to obtain fatty acid methyl esters. Briefly, approximately 10 mg lipids/100 µl CHCl3 solution (100 µl) was evaporated with nitrogen in an 8 ml Pyrex© tube (SciLabware – DWK Life Sciences, London, UK). The tube was placed in Hex (0.5 ml) (PESTINORM®SUPRATRACE n-Hexane > 95% for organic trace analysis, VWR Chemicals, Radnor, PA, USA) and Hex/MeOH/BF3 (20/25/55) solution (0.5 ml) in a water bath at 70 °C for 90 min. After cooling, 10% aqueous H2SO4 solution (0.2 ml) and saturated NaCl solution (0.5 ml) were added. Mix the tube and fill the mixture with clean Hex (8.0 mL). A portion of the upper phase was transferred to a vial and analyzed by gas chromatography with a flame ionization detector (GC-FID). Samples were analyzed using a Trace GC Ultra (Thermo Scientific, Waltham, MA, USA) equipped with a split/splitless injector (240 °C) in split mode (split flow: 10 mL/min), a Stabilwax®-DA column (30 m, 0.25 mm i.d., 0.25 μm, Restek Corp., Bellefonte, PA, USA) and an FID (250 °C). The temperature program was set as follows: 50 °C for 1 min, increasing to 150 °C at 30 °C/min, increasing to 240 °C at 4 °C/min and continuing at 240 °C for 5 min. Hex was used as a blank and a reference standard containing 37 fatty acid methyl esters (Supelco 37-component FAMEmix, Sigma-Aldrich, Overijse, Belgium) was used for identification. The identification of unsaturated fatty acids (UFAs) was confirmed by comprehensive two-dimensional GC (GC×GC-FID) and the presence of isomers was accurately determined by a slight adaptation of the method of Ferrara et al. 55. Instrument details can be found in Supplementary Table S3 and the results in Supplementary Figure S5.
The data are presented in Excel spreadsheet format (Microsoft Corporation, Redmond, WA, USA). Statistical analysis was performed using R Studio (version 2023.12.1+402, Boston, USA) 56 . Data on larval weight, development time and productivity were estimated using the linear model (LM) (command “lm”, R package “stats” 56 ) as they fit a Gaussian distribution. Survival rates using binomial model analysis were estimated using the general linear model (GLM) (command “glm”, R package “lme4” 57 ). Normality and homoscedasticity were confirmed using the Shapiro test (command “shapiro.test”, R package “stats” 56 ) and analysis of data variance (command betadisper, R package “vegan” 58 ). After pairwise analysis of significant p-values (p < 0.05) from the LM or GLM test, significant differences between groups were detected using the EMM test (command “emmeans”, R package “emmeans” 59).
The complete FA spectra were compared using multivariate permutation analysis of variance (i.e. permMANOVA; command “adonis2”, R package “vegan” 58) using the Euclidean distance matrix and 999 permutations. This helps to identify fatty acids that are influenced by the nature of dietary carbohydrates. Significant differences in the FA profiles were further analyzed using pairwise comparisons. The data were then visualized using principal component analysis (PCA) (command “PCA”, R package “FactoMineR” 60). The FA responsible for these differences was identified by interpreting the correlation circles. These candidates were confirmed using a one-way analysis of variance (ANOVA) (command “aov”, R package “stats” 56 ) followed by Tukey’s post hoc test (command TukeyHSD, R package “stats” 56 ). Before analysis, normality was assessed using the Shapiro-Wilk test, homoscedasticity was checked using the Bartlett test (command “bartlett.test”, R package “stats” 56), and a nonparametric method was used if neither of the two assumptions was met. Analyses were compared (command “kruskal.test”, R package “stats” 56 ), and then Dunn’s post hoc tests were applied (command dunn.test, R package “dunn.test” 56 ).
The final version of the manuscript was checked using Grammarly Editor as an English proofreader (Grammarly Inc., San Francisco, California, USA) 61 .
The datasets generated and analyzed during the current study are available from the corresponding author upon reasonable request.
Kim, S. W., et al. Meeting the global demand for feed protein: challenges, opportunities, and strategies. Annals of Animal Biosciences 7, 221–243 (2019).
Caparros Megido, R., et al. Review of the status and prospects of world production of edible insects. Entomol. Gen. 44, (2024).
Rehman, K. ur, et al. Black soldier fly (Hermetia illucens) as a potentially innovative and eco-friendly tool for organic waste management: A brief review. Waste Management Research 41, 81–97 (2023).
Skala, A., et al. Rearing substrate influences growth and macronutrient status of industrially produced black soldier fly larvae. Sci. Rep. 10, 19448 (2020).
Shu, M.K., et al. Antimicrobial properties of oil extracts from black soldier fly larvae reared on breadcrumbs. Animal Food Science, 64, (2024).
Schmitt, E. and de Vries, W. (2020). Potential benefits of using black soldier fly manure as a soil amendment for food production and reduced environmental impact. Current opinion. Green Sustain. 25, 100335 (2020).
Franco A. et al. Black soldier fly lipids—an innovative and sustainable source. Sustainable Development, Vol. 13, (2021).
Van Huis, A. Insects as food and feed, an emerging field in agriculture: a review. J. Insect Feed 6, 27–44 (2020).
Kachor, M., Bulak, P., Prots-Petrikha, K., Kirichenko-Babko, M., and Beganovsky, A. Various uses of black soldier fly in industry and agriculture – a review. Biology 12, (2023).
Hock, B., Noel, G., Carpentier, J., Francis, F., and Caparros Megido, R. Optimization of artificial propagation of Hermetia illucens. PLOS ONE 14, (2019).
Post time: Dec-25-2024