Accepted Manuscript

Title: Insects as food: Enrichment of larvae of Hermetia

illucens with omega 3 fatty acids by means of dietary
modifications

Authors: Fernando G. Barroso, Marı́a-José Sánchez-Muros,

Macarena Segura, Elvira Morote, Alejandro Torres, Rebeca
Ramos, José-Luis Guil

PII: S0889-1575(17)30099-6
DOI: http://dx.doi.org/doi:10.1016/j.jfca.2017.04.008
Reference: YJFCA 2879

To appear in:

Received date: 24-1-2017

Revised date: 18-3-2017
Accepted date: 12-4-2017

Please cite this article as: Barroso, Fernando G., Sánchez-Muros, Marı́a-José., Segura,
Macarena., Morote, Elvira., Torres, Alejandro., Ramos, Rebeca., & Guil, José-Luis.,
Insects as food: Enrichment of larvae of Hermetia illucens with omega 3 fatty
acids by means of dietary modifications.Journal of Food Composition and Analysis
http://dx.doi.org/10.1016/j.jfca.2017.04.008

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 Original Research Article
Insects as food: Enrichment of larvae of Hermetia illucens with omega 3
fatty acids by means of dietary modifications
Fernando G. Barroso a,*, María-José Sánchez-Muros a; Macarena Segura, Elvira Morote a,
Alejandro Torres b, Rebeca Ramos c, José-Luis Guil c
a
Department of Biology and Geology, University of Almería, Almería, Spain.
b
ENTOMOTECH. S.L., P.Y.T.A., Almería, Spain
c
Department of Agronomy, University of Almería, Almería, Spain.
* Corresponding author. Tel.: +34 950 015918; fax: +34 950 015476; e-mail address:
fbarroso@ual.es

1
Highlights

 Insects might become a key element of sustainable foods in the future.

 The modification of the fatty acid composition of Hermetia illucens is proposed
 The amounts of EPA and DHA in insect meal increased by dietary manipulation.
 These results could have a strong impact on the mass rearing of insects

Abstract
Insects can become a key element of sustainable foods in the future. However, terrestrial insects
have high levels of saturated fatty acids (SFA) and are very low in very long change
polyunsaturated fatty acids (VLCPUFA), which could limit their use as food. In this experiment,
we studied the modification of the FA composition of Hermetia illucens larvae by varying the
composition of the larval feed. The effects of diets enriched in n-3 VLCPUFA on FA
compositions of insect larvae were studied. Eight experimental groups of larvae were fed with an
experimental diet for different durations before slaughter. The addition of n-3 to the larval diets
resulted in an insect meal with three times more n-3 and therefore a lower n-6:n-3 ratio than those
of the control insect meal. The amounts of n-3 VLCPUFA in Hermetia larvae could be altered by
dietary manipulation in a short period of time.
Keywords : Hermetia illucens; food; insect meal; enrichment nutritive; EPA; DHA
1. Introduction
With a growing world population and increasingly demanding consumers, the production of
sufficient protein from livestock, poultry, and fish represents a serious challenge for the future
(van Huis, 2013). According to recent FAO (2014) estimates, approximately 805 million people
were chronically undernourished in 2012–14.
Although insects are considered an extravagant and unappealing food source for a large part of
the population (mainly in developed countries), the FAO claims that insects could mitigate this
lack of nutritive resources. For thousands of years, insects have been a common food in the diet
of a large population. Although 2,000 documented food insects are currently consumed, this
number seems very low, considering that there are approximately one million described species
of insects in the world.
Compared with conventional livestock, “minilivestock,” i.e. insects, have several advantages
(Raubenheimer and Rothman, 2013, van Huis, 2013): (1) they have higher reproduction rates and
food conversion efficiencies than those of poultry, swine, and beef; (2) they require less water;
(3) they emit low levels of greenhouse gases; and (4) owing to their lack of similarity to humans,
they have a lower risk of producing pathogens that are threatening to human health.
Insects and other terrestrial arthropods are nutritious food (Wang et al., 2007). These insects can
provide ample bio-available proteins, fats, vitamins, minerals, and fibre (Looy et al., 2014).
Nevertheless, low levels of polyunsaturated fatty acids (n-3 PUFA) have been observed in
analyses of insect meals (Akinnawo and Ketiku, 2000, Barroso et al., 2014, Ekpo and Onigbinde,

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2007, Finke, 2002, Katayama et al., 2008). If we want to use insects as food, their lack of EPA
and DHA could be a major limitation (Barroso et al., 2014).
Consumption of the n-3 fatty acid (FA) EPA (20:5 n-3) and DHA (22:6 n-3) have been shown to
have numerous health benefits (Ruxton et al., 2004, Watters et al., 2012). PUFA appear to be an
integral part of membrane phospholipids for optimal biological function, particularly in
specialized cells and tissues, such as the brain, retina, testes, heart, liver and kidneys (Tinoco,
1982). Many epidemiological studies and clinical trials have shown a relationship between the
intake of n-3 FA and beneficial effects in various diseases: cardiovascular disorders (McDaniel
et al., 2013), different cancers, asthma, inflammatory bowel disease, rheumatoid arthritis and
osteoporosis, among others me (Gómez et al., 2011). On the other hand, increasing evidence
for competitive interactions between PUFA of the n-6 and n-3 FA families (Lands, 1986).
Several researchers have indicated that a low n-6:n-3 ratio results in relatively low inflammation
and potential improvements in or the prevention of cardiovascular heart disease (Fernandes,
2002, Kafatos and Codrington, 1999, McDaniel et al., 2013).
The black soldier fly, Hermetia illucens (Linnaeus), is often around organic matter waste.
Although, at their last larval stage, these flies only weigh 220 mg and measure 27 mm, this insect
has an advantage with respect to rapid food intake, ranging from 25 to 500 mg of fresh
matter/larva/day, on a wide range of decaying organic materials, such as rotting fruits and
vegetables, coffee bean pulp, distiller grains, fish offal and particularly animal manure and human
excreta (Diener et al., 2009, Hardouin and Mahoux, 2003, Van Huis, 2013). Larvae pass through
six instars and require approximately 14 days to complete development (Hall and Gerhardt 2002).
As adults they do not need to feed and rely on the fats stored from the larval stage (Newton et al.
2005).
Fish are not able to synthesize PUFA, they get it from their diet. Marine phytoplankton contains
a high concentration of omega 3. The lipid content of insects is largely dependent on their diets
and stage of development (Stanley-Samuelson and Dadd, 1983). Therefore, we hypothesize that,
as monogastric animals, the composition of FA in Hermetia larvae is influenced by the
composition of FA in their diet. We therefore consider that the larvae of Hermetia could similarly
accumulate PUFA, thereby improving their dietary value. Accordingly, the aim of the present
work was to determine if it is possible to enrich Hermetia larvae with n-3 FA via the exposure to
feed containing PUFA and to determine the time required to modify their FA profile.
2. Materials and Methods
2.1. Larvae of Hermetia illucens
This study was conducted using the black soldier fly because the system for the mass production
of this insect is highly developed, and the species has been studied extensively as food and feed

3
(De Marco et al., 2015; Maurer et al, 2015; Kroeckel et al., 2012, among others). Larvae of H.
illucens were rearing in ENTOMOTECH S.L. Company.
2.2. Experimental design
The experiment was carried out in a climate control chamber of 550 l at 26±1ºC and 65±5%. All
the H. illucens larvae were raised during 10 days feeding on control diet, based on a commercial
composition of laying hen feed (NANTA®, Spain) 30 % and distilled water 70% w/v. After the
10th growing day, the larvae were divided in groups of 5000 larvae per treatment, and those larvae
were fed on control diet until switched to the experimental diet with a 40% w/w substitution of
the laying hen feed by fish meal (SUYSEGALA, Spain). 40% fish meal inclusion is the maximum
without apparent detrimental in the larva growth Cortes (ENTOMOTECH S.L personal
communication). Fish meal is rich in n-3 FA; accordingly, the experimental diet was enriched
with a high percentage of PUFA compared to the level in the control diet (Table 2).
To establish the optimal time required to increase levels of n-3 in H. illucens, 8 treatments were
used: 30 m, 60 m, 3 h, 6 h, 12 h, 24 h, 48 h and 4d, representing the times that the larvae were
eating the experimental diet (enriched with fish meal) until slaughter.
2.3. Samples
Larvae were harvested before they reached the prepupal stage (at day 14 th). The larvae were
subsequently killed (by freezing) and stored at -20 °C. All samples, larvae and diets were
lyophilized prior to the analysis (Cryodos, Ima-Telstar, Terrassa, Spain) and were later ground
and kept frozen until analysis.
2.4. Oil extraction and transesterification
Simultaneous oil extraction and transesterification was done according to previous works (Lepage
and Roy, 1984). Ground seeds were weighed in 10 ml-test tubes and then 1 ml of the methylation
mixture (methanol:acetyl chloride 20:1 v/v) and 1 ml of n-hexane were added. Tubes were capped
and later on heated at 100 ºC for 30 min. Afterwards, the tubes were cooled to room temperature,
1 ml of distilled water was added, and after centrifugation (3500 rpm, 3 min), the upper hexane
layer was removed for GLC analysis.
2.5. FA analyses
FA methyl esters (FAMEs) were analyzed using a Focus GC (Thermo Electron, Cambridge, UK)
equipped with a flame ionization detector (FID) and an Omegawax 250 capillary column (30 m
x 0.25 mm i.d. x 0.25 µm film thickness; Supelco, Bellefonte, USA). The temperature program
was: 1 min at 90 ºC, heating until 220 ºC at a rate of 10 ºC/min, constant temperature at 220 ºC
(2 min), heating until 250 ºC at a rate of 10 ºC/min and constant temperature at 250 ºC (1 min).
The injector temperature was 250 ºC with a split ratio of 50:1. The injection volume was 4 µl.
The detector temperature was 260 ºC. Nitrogen was used as carrier gas (1 ml/min).
Peaks were identified by retention times obtained for known FAME standards (PUFA No. 1,
47033; methyl γ-linolenate 98.5% purity, L6503; and methyl stearidonate 97% purity, 43959

4
FLUKA) from Sigma, (St. Louis, USA), and FA contents were estimated by using methyl
pentadecanoate (15:0; 99.5% purity; 76560 Fluka) from Sigma as internal standard.
2.6. Quality control
The repeatability of the direct methylation was checked by analyzing replicates of the same
sample daily. The intermediate precision was evaluated by measuring samples on different days
throughout the study. Also, blank samples were analyzed every time when the methylations were
performed. Control oil samples were analyzed prior and after running samples. Canola oil (46961
SUPELCO, from SIGMA) was used for the control tests. As quality control of GLC, a blank
sample (hexane) was run together with the samples in every batch to check the GLC performance.
Experiments for all samples were conducted in triplicate. Results are expressed as mean value ±
S.D in Table 2.
2.7. Proximal composition
Content of crude protein (CP) was determined by Kjeldahl (AOAC, 2005; #954.01) (Nx6.25),
and total lipid (EE) was determined by ethyl ether extraction (Soxhlet technique) (AOAC, 2005;
#920.39). Ash were determined gravimetrically after drying at 105±0.5ºC (AOAC, 2005;
#934.01) and after combustion at 500ºC in a mufla oven (AOAC, 2005; #942.05), respectively,
to constant weight. All analyses were performed in triplicate
2.8. Statistical analysis
Experimental results were expressed as means ± SD for three different determinations. The
Kruskal-Wallis test and comparisons among pairs using Tukey–Kramer HSD tests (JMP 9.0.0 for
Macintosh) were performed for comparison of the results of proximate composition and FA data
obtained from the larvae meal.
3. Results
The proximal composition of the experimental diets and insects is summarised in Table 1.
Statistical differences were found between different time of sampling. The CP seem to increase
along the time while nitrogen-free extract (NFE) decrease. The EE and Ash varies among the
sampling time.
The FA composition of the diets and Hermetia larvae fed with the experimental diet for various
durations before slaughter are shown in Table 2. It is remarkable that the control diet had higher
proportion of linoleic acid (18:2n-6) (26.0%) and lower DHA (0.0%) than the experimental diet
(18.1% and 6.4% respectively). The larvae fed on the experimental diet reflect these differences
with regards to these FA. Control diet also has higher level of SFA (32.5%) and lower PUFA
(28.25%) than the experimental diet (27.4% and 32.2%, respectively). Although, the larvae fed
on the experimental diet showed lower proportion of SFA in agreement with the previous results,
on the contrary this tendency was not observed in PUFA.

5
The values of the most relevant FA (EPA, DHA, n-3, and n-6/n-3) are summarized in Figure 1.
Overall, the administration of the experimental diet (enriched with PUFA) modified the lipid
profile in a few hours, increasing the amount of PUFA and decreasing the n-6/n-3 ratio.
4. Discussion
As detailed in the table 1, the proximate composition of larvae varies over time without a clear
tendency. The larval development stage might influence both the noted increasing of CP and the
decreasing of NFE.
In addition to use insects as protein-rich food, their lipids might constitute a source of energy and
essential FA; therefore, they could be used to combat malnutrition in developing countries (Smit
et al., 2004).
As shown in Figure 1, the larvae fed the experimental diet enriched with n-3 had a significantly
greater amount of PUFA than control larvae, and this fact agrees with previous ones described
St-Hilaire et al. (2007). Barroso et al. (2014) found evidences that the FA profiles of insects reflect
the FA composition of their food; that is, plants constitute a rich source of α-linolenic acid (ALA,
18:3 n-3), and wild orthopterans consuming plants in the field showed higher amounts of ALA
than other ones bred in captivity and fed only with flour and bran cereals.
Regarding n-3 PUFA, the maximum increase in the larval body was obtained at 3 hours, reaching
almost triple the amount observed in the control. It was noted that larvae can select the FA that
they incorporate. EPA seems to accumulate over time; i.e., high levels of EPA were observed in
larvae fed for more than 2 days; however, although the levels of DHA were significantly higher
in the experimental diet, this FA did not accumulate in larvae over time. At 30 min, there was a
significant increase of DHA, and the maximum concentration was observed in larvae fed for 3 h.
However, although the diet was provided for several days before slaughter, differences of DHA
concentration between larvae fed n-3 PUFA-rich diet and control gradually decreased..
In insects, PUFA is mainly incorporated into phospholipids rather than triacylglycerols, as seen
in mammals (Stanley-Samuelson et al., 1985). The selective accumulation of EPA by H. illucens
larvae detected in this work indicates that EPA could be an essential FA for larval-pupal
development of this species, as it has been cited in the malarial vector mosquito, Anopheles
stephensi (Moribayashi et al., 2004). Therefore, the enzymes involved in the synthetic pathways
for phospholipids and triacylglycerol synthesis might exert a selective action for the incorporation
of EPA against DHA in the larvae lipids.
The bioaccumulation of EPA and DHA noted in insect larvae has nutritional relevance for
humans. DHA is a key nutritional PUFA and needs to be supplied by the human diet (Cardoso et
al., 2016), while EPA is a precursor in the synthesis of DHA and also has undoubtedly beneficial
actions per se. For instance, PPARγ1 is a molecular target of EPA in human colon cancer (HT-
29) cells, by which EPA suppress tumour cell growth (Allred et al., 2008). The eicosanoids
produced from EPA frequently have properties that are different from those that are produced

6
from arachidonic acid. Moreover, EPA and DHA are also substrates for production of resolvins
and protectins, which seem to be biologically extremely potent (Carlder, 2012).
At this point in the development of research, the effect of changing the proportion of n-3 FAs in
diets to feed insects has yet to be studied. However, the fact of reaching the top of the
concentration of n-3 VLCPUFA in the larvae after few hours of the initiation of the feeding trial,
indicates that larvae actively select these PUFAs from the diet to be incorporated into their tissues,
mainly into membrane phospholipids, as previously indicated. And the evolution of the
concentrations of EPA and DHA over time suggests that an increasing of n-3 VLCPUFA
supplementation might have no effects in the concentration of such compounds in the larvae, as
well as the total n-3.
Three hours after start the feeding trial, the fat percentage reaches 22.9 g per 100 g of dry matter
(Table 1). At such time, EPA + DHA reached 2.7% of total FAs (Table 2), thus 5.2 g EPA +
DHA per 100 g of dry larvae are expected. Given that the recommended daily intake of EPA +
DHA is 500 mg daily (Vannice and, Rasmussen, 2014), 9.6 g of dried larvae would be enough to
meet such needs. This value is lower than other ones referred to usually consumed fish, such as
sardine or hake, when used for the same purpose. Such fish contains ~4300 and ~4800 mg of
EPA+DHA by 100 g dry matter (Domingo et al., 2007). Accordingly, 12 g of dry sardine and
10.5 g of dry hake would be also enough to meet EPA + DHA daily needs.
Another important point is that the n-6:n-3 ratio of larvae fed with the experimental diet is reduced
by up to 60% compared with that of the control (Figure 1). Several researchers have indicated
that a low n-6:n-3 ratio results in relatively low inflammation and potential improvements in or
the prevention of cardiovascular heart disease (Fernandes, 2002, Kafatos and Codrington, 1999,
McDaniel et al., 2013). The potential dietary benefits attributed to this low ratio appear to be
directly related to the competitive interactions between PUFA of the n-6 and n-3 FA series for
the biosynthesis and action of eicosanoids, thromboxanes, prostacyclins, prostaglandins, and
leukotrienes (Lands, 1986).
Of course, rearing insect larvae on fish meal for human consumption is not directly applicable. In
this experiment, this substrate, which is rich in n-3 VLCPUFA, was used owing to its ease of
handling and mixing in the laboratory; working with fish meal was easier than working with fish
by-products. The basic aim of this study was not to characterize the recycling of these by-
products, but to determine the dynamics of n-3 PUFA accumulation in Hermetia. However, the
effective implementation of these results, as St-Hilaire et al. (2007) suggest, might involve
feeding black soldier fly larvae a diet composed of waste products containing desirable n-3
VLCPUFA (e.g. fish by-products), thereby enriching the final insect meal.
An important finding of this study is that the significant increasing of n-3 VLCPUFA in larvae,
obtained by applying this food management strategy, lies with the reduction in time necessary to
provide fish waste to not too many hours before the slaughter of the larvae. Logically, reducing

7
the feeding time of larvae with fish by-products also reduce the complexity of the process;
therefore, this method could be viewed as a finisher diet, similar to that used in other livestock
sectors.
Future investigations could be focused to know the bioaccessibility of the n-3 VLCPUFA present
in the larvae; however, since these are composed by water, protein and fat as major components,
and lack fiber as opposed to algae and vegetables, such bioaccessibility is expected to be
reasonably high. Another line of research could be oriented to know the degree of accumulation
of n-3 VLCPUFA by larvae using other feeding sources, such as macro and microalgae, for using
larvae as bioaccumulators, since these are more digestible than algae.
5. Conclusions
The FA profiles of Hermetia meal could be easily modified by dietary manipulation of larvae, to
improve the percentages of the healthy EPA and DHA. Such findings could have a strong impact
on research related to the mass rearing of insects because short times of feeding with enriched
diet are needed to obtain a EPA+DHA-rich food. Specially, between 30 min and 3 h before
slaughter is enough to obtain significant improvements.
Acknowledgments
The authors are grateful to the Consejería de Innovacion y Ciencia (project AGR5273), European
Regional Development Funds (FEDER funds), Campus de Excelencia Internacional
Agroalimentaria (CEI3) Campus de Excelencia Internacional del Mar (CEImar), Ministerio de
Educación (Spain) for financial support.
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Aquaculture: The Importance of Omega-3 Fatty Acids in Fish Development and Human
Health. College of Tropical Agriculture and Human Resources: Food and Nutrition July 2012
FN-11.

11
CP - Crude protein, EE - Crude fat and NFE: Nitrogen-free extract. Values are means ± SD of
triplicate determinations. Values within a column followed by the same superscript letter are not
significantly different (P < 0.05) by the Tukey's range test.

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1 Table 2. Fatty acid content (expressed as percentage of total fatty acids) of control and experimental diets and Hermetia illucens larvae.
2 Larvae were fed on diets throughout different experimental periods.

Fatty Contro Exp. H.illuc H.illuc H.illuc H.illuc H.illuc H.illuc H.illuc H.illuc H.illuc
l diet Diet ens 0 ens 30 ens 60 ens 3 ens 6 ens 12 ens 24 ens 2 ens 4
acid (40% min. min. min. hours hours hours hours days days
FM)

8:0 1.1 ± 0.7 0.3 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.1 ±
0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2

10:0 0.6 ± 0.2 ± 0.6 ± 0.4 ± 0.5 ± 0.4 ± 0.8 ± 0.7 ± 0.4 ± 0.3 ± 0.9 ±
0.4 0.3 0.8 0.6 0.7 0.5 1.1 1.0 0.6 0.5 0.1

12:0 0.4 ± 0.6 ± 39.0 ± 31.0 ± 33.8 ± 33.6 ± 33.1 ± 32.9 ± 33.0 ± 32.9 ± 22.6 ±
0.1 0.0 6.1 0.2 1.4 6.5 6.8 3.7 0.8 1.3 0.5

14:0 0.7 ± 2.1 ± 8.0± 8.1 ± 7.8 ± 7.8 ± 7.2 ± 7.1 ± 7.8 ± 6.8 ± 5.3 ±
0.1 0.3 1.1 0.7 0.2 0.5 0.2 0.0 0.8 0.0 0.2

15:0 1.6 ± 0.5 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ±
2.2 0.7 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

16:0 19.6 ± 17.7 ± 12.7 ± 14.6 ± 14.3 ± 13.5 ± 13.2 ± 11.1 ± 15.0 ± 15.0 ± 17.9 ±
1.5 2.4 2.1 1.5 1.3 3.7 2.1 4.6 0.7 0.9 1.0

16:1n- 0.7 ± 2.3 ± 1.3 ± 4.0 ± 3.9 ± 1.5 ± 2.7 ± 4.8 ± 4.0 ± 4.6 ± 1.9 ±
7 0.4 0.6 1.9 1.6 1.6 2.2 0.1 3.1 1.4 1.3 1.3

17:0 0.0 ± 0.2 ± 0.2 ± 0.1 ± 0.1 ± 0.1 ± 0.2 ± 0.1 ± 0.2 ± 0.2 ± 0.3 ±
0.0 0.2 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.4

18:0 6.5 ± 5.2 ± 3.1 ± 2.8 ± 2.8 ± 3.4 ± 4.8 ± 3.7 ± 3.0 ± 1.7 ± 4.6 ±
0.0 1.6 1.0 1.9 1.5 0.9 1.6 0.1 1.6 2.4 0.3

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18:1n- 35.3 ± 34.3 ± 15.1 ± 17.7 ± 16.9 ± 16.7 ± 16.8 ± 17.7 ± 17.5 ± 18.8 ± 22.9 ±
9 2.3 6.3 0.6 1.8 1.9 0.9 4.0 3.8 0.7 2.6 0.1

18:1n- 0.8 ± 0.8 ± 0.2 ± 0.3 ± 0.3 ± 0.5 ± 0.5 ± 0.3 ± 0.4 ± 0.3 ± 1.0 ±
7 0.0 1.2 0.3 0.4 0.4 0.7 0.6 0.4 0.5 0.4 0.2

18:2n- 26.0 ± 18.1 ± 16.1 ± 15.4 ± 13.7 ± 14.2 ± 13.7 ± 15.5 ± 13.2 ± 11.8 ± 15.0 ±
6 2.9 0.0 0.8 0.0 0.5 0.5 1.7 1.1 0.0 0.2 0.7

18:3n- 1.4 ± 1.5 ± 0.8 ± 0.8 ± 0.8 ± 1.0 ± 0.9 ± 1.0 ± 0.8 ± 0.8 ± 0.9 ±
3 0.1 0.3 0.0 0.0 0.1 0.2 0.3 0.3 0.1 0.1 0.1

18:4n- 0.0 ± 0.7 ± 1.0 ± 0.9 ± 0.8 ± 0.9 ± 0.9 ± 1.0 ± 1.4 ± 2.0 ± 0.9 ±
3 0.0 0.4 0.2 0.1 0.0 0.0 0.2 0.1 0.1 0.0 0.0

20:0 0.8 ± 0.2 ± 0.0 ± 0.1 ± 0.0 ± 0.1 ± 0.0 ± 0.1 ± 0.1 ± 0.1 ± 0.0 ±
0.0 0.4 0.0 0.2 0.0 0.2 0.0 0.1 0.1 0.1 0.0

20:1n- 0.3 ± 0.9 ± 0.0 ± 0.3 ± 0.6 ± 0.6 ± 0.7 ± 0.4 ± 0.3 ± 0.5 ± 0.4 ±
9 0.4 0.5 0.1 0.1 0.3 0.2 0.3 0.0 0.0 0.4 0.1

20:4n- 0.3 ± 0.9 ± 0.1 ± 0.2 ± 0.4 ± 0.2 ± 0.4 ± 0.3 ± 0.1 ± 0.3 ± 0.2 ±
6 0.4 0.3 0.0 0.2 0.5 0.3 0.5 0.4 0.1 0.1 0.1

20:4n- 0.0 ± 0.3 ± 0.0 ± 0.1 ± 0.0 ± 0.0 ± 0.1 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ±
3 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0

20:5n- 0.0 ± 2.8 ± 0.0 ± 0.5 ± 0.5 ± 0.8 ± 0.9 ± 0.8 ± 1.1 ± 1.8 ± 1.6 ±
3 0.0 0.7 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1

22:0 1.1 ± 0.3 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ± 0.0 ±
0.2 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

22:1n- 0.0 ± 0.6 ± 0.1 ± 0.2 ± 0.2 ± 0.1 ± 0.3 ± 0.2 ± 0.0 ± 0.0 ± 0.0 ±
11 0.0 0.2 0.1 0.0 0.1 0.2 0.4 0.2 0.0 0.0 0.0

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 22:4n- 0.0 ± 0.3 ± 0.3 ± 0.2 ± 0.3 ± 0.0 ± 0.3 ± 0.4 ± 0.2 ± 0.1 ± 0.0 ±
6 0.0 0.2 0.4 0.3 0.4 0.0 0.4 0.5 0.3 0.1 0.0

22:5n- 0.0 ± 0.2 ± 0.1 ± 0.0 ± 0.4 ± 0.6 ± 0.7± 0.4 ± 0.1 ± 0.2 ± 0.0 ±
6 0.0 0.3 0.1 0.0 0.5 0.8 1.0 0.6 0.1 0.3 0.0

22:5n- 0.6 ± 1.1 ± 0.0 ± 0.5 ± 0.1 ± 0.6 ± 0.2 ± 0.2 ± 0.1 ± 0.1 ± 0.5 ±
3 0.1 0.1 0.0 0.4 0.1 0.4 0.0 0.1 0.1 0.1 0.4

22:6n- 0.0 ± 6.4 ± 0.1 ± 1.3 ± 1.3 ± 1.9 ± 1.6 ± 1.3 ± 0.8 ± 0.8 ± 0.7 ±
3 0.0 0.7 0.0 0.0 0.2 0.4 0.3 0.1 0.2 0.1 0.2

Others 2.6 ± 1.5 ± 1.3 ± 0.6 ± 0.5 ± 1.4 ± 0.3 ± 0.3 ± 0.5 ± 0.9 ± 2.1 ±
1.7 1.3 0.9 0.1 0.5 1.3 0.0 0.3 0.1 1.3 1.0

32.5 ± 27.4 ± 63.6 ± 57.1 ± 59.4 ± 58.9 ± 59.2 ± 55.7 ± 59.6 ± 57.0 ± 51.7 ±
SFA
4.7 6.2 3.0 3.7 4.9 1.6 8.8 9.5 1.6 2.9 0.3

37.1 ± 39.0 ± 16.7 ± 22.5 ± 21.9 ± 19.5 ± 20.8 ± 23.3 ± 22.2 ± 24.2 ± 26.2 ±
MFA
3.1 5.1 1.6 2.9 3.4 1.9 4.2 6.8 1.6 3.8 1.0

28.2 ± 32.2 ± 18.4 ± 19.9 ± 18.2 ± 20.2 ± 19.6 ± 20.7 ± 17.8 ± 17.8 ± 19.7 ±
PUFA
2.7 2.3 0.6 0.8 2.0 1.7 4.6 3.0 0.2 0.4 0.8

3
4 SFA - Saturated fatty acid; MFA– Monounsaturated fatty acids; monounsaturated; PUFA - Polyunsaturated fatty acids

15
Figure caption

Figure 1. Changes in fatty acids composition of Hermetia illucens larvae according to the period
consuming the experimental diet (enriched with PUFA) before slaughtering. FA sharing the same
letter indicate no significant differences (P < 0.05, Tukey–Kramer’s test).

16
17
Figr-1

Table 1. Proximate analysis (% dry matter) of control and experimental diets and Hermetia
illucens larvae.

CP % EE % ASH % NFE %

Control diet 15.2 ± 0.6 8.5 ± 0.4 17.0 ± 0.2 59.4 ± 0.3
Exp. diet (40% FM) 32.8 ± 3.9 10.2 ± 0.5 18.1 ± 0.4 38.9 ± 3.9
H.illucens 0 min. 36.7 ± 4.2 c 15.8 ± 0.8 b 11.8 ± 0.3 bc 35.6 ± 3.2 a
H.illucens 30 min. 44.8 ± 5.5 abc 21.2 ± 0.8 ab 11.7 ± 0.3 bc 22.3 ± 3.3 bcd
H.illucens 60 min. 37.5 ± 1.8 bc 22.6 ± 0.7 a 11.6 ± 0.2 bc 28.2 ± 0.9 b
H.illucens 3 hours 40.6 ± 2.0 bc 22.9 ± 3.1 a 12.4 ± 0.2 a 24.0 ± 1.4 bc
H.illucens 6 hours 43.2 ± 0.2 bc 20.6 ± 2.4 ab 12.7 ± 0.1 a 23.5 ± 2.5 bc
H.illucens 12 hours 46.0 ± 0.2 abc 19.0 ± 2.1 ab 12.2 ± 0.2 ab 22.8 ± 1.8 bcd
H.illucens 24 hours 55.2 ± 3.8 a 17.2 ± 2.7 b 11.4 ± 0.2 c 16.3 ± 2.5 d
H.illucens 2 days 47.8 ± 1.1 ab 21.0 ± 0.3 ab 10.5 ± 0.1 d 20.7 ± 0.7 cd
H.illucens 4 days 53.8 ± 3.4 a 15.8 ± 0.2 b 11.7 ± 0.1 bc 18.7 ± 3.4 cd

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