Effect of rearing substrate on growth performance, waste reduction

efficiency and chemical composition of black soldier fly (Hermetia illucens)

larvae†

RUNNING TITLE: Rearing substrate effects on performance and nutritional

composition of black soldier fly

Accepted Article
Marco Meneguz,a Achille Schiavone,bc Francesco Gai,c Andrea Dama,a Carola

Lussiana,a Manuela Renna,a* Laura Gascoac

a
Department of Agricultural, Forest and Food Sciences, University of Torino, Largo

P. Braccini 2, 10095 Grugliasco (TO), Italy

b
Department of Veterinary Science, University of Torino, Largo P. Braccini 2, 10095

Grugliasco (TO), Italy

c
Institute of the Science of Food Production, National Research Council, Largo P.

Braccini 2, 10095 Grugliasco (TO), Italy

* Corresponding author: manuela.renna@unito.it

† The paper was partly given at the 68th Annual Meeting of the European

Federation of Animal Science (Tallin, Estonia, 28/08-01/09/2017)

ABSTRACT

BACKGROUND: Wastes can be used as rearing substrate by black soldier fly (BSF)

larvae, the latter being exploitable as protein source in animal feed. This research

aimed to assess the influence of four rearing substrates [Trial 1 (organic wastes): a

mixture of vegetable and fruit (VEGFRU) vs a mixture of fruits only (FRU); Trial 2

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which
may lead to differences between this version and the Version of Record. Please cite this
article as doi: 10.1002/jsfa.9127

This article is protected by copyright. All rights reserved.

 (agro-industrial by-products): brewery (BRE) vs winery (WIN) by-products] on BSF

larvae development, waste reduction efficiency, and nutritional composition.

RESULTS: If respectively compared to FRU and WIN, VEGFRU and BRE larvae

needed less time to reach the prepupae stage (22.0, 22.2, 20.2 and 8.0 days of
Accepted Article
trial, respectively) and had higher protein content (229.7, 257.3, 312.9 and 395.7

g kg-1 DM). The waste reduction index ranged from 2.4 (WIN) to 5.3 g d-1 (BRE).

BRE larvae showed the lowest saturated and the highest polyunsaturated fatty

acids proportions (612.4 and 260.1 g kg-1 total fatty acids, respectively).

CONCLUSION: Vegetable and fruit wastes and winery by-products can be used as

rearing substrates for BSF larvae mass production. Brewery by-products led to very

promising larvae performances and nutritional composition. However, given BRE

limited availability, low BRE dietary inclusion levels could be used with the purpose

of increasing larvae performances.

Keywords: organic waste, agro-industrial by-product, Hermetia illucens, animal

feed, crude protein, fatty acid profile

This article is protected by copyright. All rights reserved.

 INTRODUCTION

The world population is estimated around 7.3 billion, with a growth rate of about 83

million per year. This increase will generate an increment of food demand with a

consequent rise in waste and by-products production.1 Urgent and innovative

Accepted Article
solutions are needed for the management of the waste streams (WS) that

nowadays are estimated around 1.3 billion and 100 million tons per year in the

world and in the European Union, respectively.1,2 Furthermore, the EC Directive No

2008/98 unequivocally establishes the order of priority in the choice of WS

treatment, the first being their reuse and the last their landfill disposal.

Some WS could be valorized through the recovery of the residual bio-elements they

contain, with a cost reduction both for the industry (disposal cost) and the

environment (pollution).3 The use of insects in the bioconversion of WS constitutes

a new approach and an interesting example of sustainable circular economy. This

bioconversion can generate new elements such as proteins and lipids for animal

feeds,4,5,6,7 biodiesel,8 high value products as chitin9 or anti-microbial peptides.10

Processed proteins from seven insect species have recently been approved for

aquafeed by the EC Regulation No 2017/893, which also lists the licensed rearing

substrates. Among authorized species, black soldier fly (BSF; Diptera:

Stratiomydae) is one of the most promising and researches recently aimed to

increase knowledge on optimal rearing substrates for larvae and prepupae. In this

respect, BSF has shown great flexibility as it can be used to reduce volume and add

value to various wastes.8,11,12 The available literature has highlighted that BSF life

cycle and nutritional composition are noticeably influenced by the rearing

substrate,13,14 with the crude protein (CP) content of the larvae ranging from about

317 to 630 g kg-1 dry matter (DM).7,15,16

In 2014, around 90 million tons of slaughter and vegetable WS were produced in

Europe

(EUROSTAT(http://appsso.eurostat.ec.europa.eu/nui/show.do?dataset=env_wasge

n&lang=en)). Considering the Italian context, 54% of the total production of waste

This article is protected by copyright. All rights reserved.

 and agro-industrial by-products is generated by the manufacturing of vegetable

products.17 About 1.5 million tons of winery by-products and 406 tons of brewery

by-products are produced every year

(EUROSTAT(http://ec.europa.eu/eurostat/tgm/table.do?tab=table&init=1&language
Accepted Article
=en&pcode=tag00034&plugin=1; EU Report

(https://www.brewersofeurope.org/site/media-

centre/index.php?doc_id=905&class_id=31&detail=true)).18

The aim of this research was to evaluate the effects of organic wastes (vegetables

and fruits) and agro-industrial by-products (winery and brewery) generated by the

Italian food sector as rearing substrates for BSF larvae on their development, waste

reduction efficiency and chemical composition.

MATERIAL AND METHODS

Two trials were carried out at the Experimental Facility of the Department of

Agricultural, Forest and Food Sciences (DISAFA; University of Torino, Torino, Italy).

Rearing substrates

In Trial 1, two organic wastes were compared:

- Vegetable-fruit waste (VEGFRU) obtained from a street market (Torino, Italy) and

containing a mixture of vegetables and fruits (celery 43.4%, oranges 28.9% and

peppers 27.7%);

- Fruit waste (FRU) obtained from a fruit market (Torino, Italy) and containing fruits

only (apples 47.8%, oranges 15.5%, apple leftovers 13.8%, strawberries 7.1%,

mandarins 4.8%, pears 4.1%, kiwis 3.4%, bananas 1.9% and lemons 1.6%).

In Trial 2, two agro-industrial by-products were used:

- Winery by-product (WIN) obtained during the wine making process, before the

alcohol extraction, from a private distillery (Distilleria Santa Teresa dei Fratelli

Marolo S.R.L., Alba (CN), Italy) and containing grape seeds, pulp, skins, stems and

leaves;

This article is protected by copyright. All rights reserved.

 - Brewery by-product (BRE) obtained during beer production (IFN 5-00-517 Barley

brewers grains wet) from a private brewery (“Birrificio dei Santi”, Castelnuovo Don

Bosco (AT), Italy).

Each substrate was ground with a 3 mm die meat mincer (FTS136; Fama Industrie
Accepted Article
S.r.l., Rimini, Italy) and carefully mixed.

A sample of each substrate was freeze-dried and frozen at -80°C for further

chemical analysis, while the remaining was stored at -20°C until it was fed to the

larvae.

BSF eggs

BSF eggs laid on corrugated cardboards for less than 24 h, were purchased from a

private company (CIMI S.r.l., Cervasca (CN), Italy). The cardboards with the eggs

were immediately transported to the DISAFA Experimental Facility. The cardboards

were put onto plastic boxes (25cm × 33cm × 12cm) which contained whole rye

thoroughly mixed with water (60% moisture) as rearing substrate for the newborn

larvae. The plastic boxes were placed into climatic chambers under controlled

environmental conditions (T: 27±0.5°C; RH: 70±5%; 24:0 L:D photoperiod). The

eggs hatched approximately three days after oviposition.

Experimental design and calculations

Larvae development and waste reduction efficiency

Six-day-old larvae were used in both trials. In each trial, for the evaluation of

larvae development (weight and length) and waste reduction efficiency, six

replicates of 100 larvae were weighed (KERN PLE-N v. 2.2; KERN & Sohn GmbH,

Balingen-Frommern, Germany; d: 0.001) and assigned to each rearing substrate.

The method reported by Harnden and Tomberlin19 was used to count the larvae. For

each replicate, the larvae were placed into plastic containers (10cm × 17.5cm ×

7cm), directly on the rearing substrate (100 g per replicate). The containers were

covered with a perforated cap with a black nylon grid and placed in a climatic

This article is protected by copyright. All rights reserved.

 chamber under controlled environmental conditions (T: 27±0.5°C; RH: 70±5%;

24:0 L:D photoperiod).

Each replicate was monitored daily to control the quantity of available feed. If

needed, as reported by Harnden and Tomberlin,19 50 g of substrate per replicate

Accepted Article
was added in all replicates at the same time.

To avoid the effect of handling on the considered dependent variables,13 weight and

length data were collected every four days until the appearance of the first

prepupae, thereafter every day for the relative substrate. Thirty larvae were

randomly sampled for three consecutive times from each container to measure

weight and length. As measurement was not destructive, the larvae were re-

introduced into the containers between two consecutive samplings. The sampled

larvae were individually cleaned, dried with a paper towel and weighed, and

photographed orthogonally (Lumix G1; Panasonic Corporation, Kadoma, Osaka,

Japan) with a metric scale (mm). The images were analyzed with ImageJ software

package (v. 1.50b) to record larvae length (i.e., from mouthpart to the bottom of

the last abdominal segment).

For each container, weight and length data collection ended when 30% of the

larvae reached the prepupae stage. The prepupae were removed from the

containers. The remaining 70% of the larvae were hand-counted, washed, dried

with a paper towel and individually weighed and photographed. The total final

biomass (larvae + prepupae) and the residual rearing substrate were also weighed.

The following parameters were then calculated:

– larvae mortality (LM)

LM = [initial number of larvae - (final number of larvae + number of prepupae)] /

initial number of larvae * 100;

- growth rate (GR),20 readapted for this research substituting prepupa body weight

(g) with larva body weight (g)

GR = (larva average final body weight (g) - larva initial body weight (g)) / days of

trial (d);

This article is protected by copyright. All rights reserved.

 - substrate reduction (SR)21

SR = [(distributed substrate (g) - residual substrate (g)) / distributed substrate

(g)] * 100;

- waste reduction index (WRI)20

Accepted Article
WRI = [(W – R) / W] / days of trial (d) * 100

where W = total amount of rearing substrate distributed during the trial (g); R =

residue substrate (g);

- efficiency of conversion of digested food (ECD)20

ECD = total final biomass (g) / (total feed distributed (g) - residual substrate (g))

where total final biomass = larvae + prepupae; residual substrate = undigested

food + excretory products.

Parameters related to waste reduction efficiency (SR, WRI and ECD) were

calculated on a fresh matter basis.

Larvae nutritional composition

For each trial, a second set of six replicates per rearing substrate was

simultaneously prepared with the aim to rear a sufficient amount of larvae to be

analyzed for their proximate composition and fatty acid - FA - profile. Five hundred

hand-counted 6-day-old larvae were placed into plastic containers of bigger size

(25cm × 33cm × 15cm) than those used for the larvae development and waste

reduction efficiency test, following the same relationships between (i) number of

larvae / container size surface, and (ii) amount of administered feed / larvae

density. The larvae were not handled until the appearance of the first prepupa.

Then, each container was checked daily and the identified prepupae were removed.

The trial ended when the 30% of the larvae reached the prepupae stage. The

remaining larvae were then manually separated from the residual rearing substrate,

washed, slightly dried with paper towel, weighed and frozen at -80°C until being

freeze-dried.

This article is protected by copyright. All rights reserved.

 Chemical analyses of rearing substrates and larvae

Samples of freeze-dried rearing substrates and larvae were ground using a cutting

mill (MLI 204; Bühler AG, Uzwil, Switzerland). They were analyzed for DM, ash, CP

and EE following AOAC International methods as detailed in Gasco et al.5 For the
Accepted Article
determination of the CP of whole BSF larvae, in addition to the conventional

nitrogen-to-protein (N-factor) conversion factor of 6.25, the more accurate N-factor

of 4.67 suggested by Janssen et al.22 was used. Neutral detergent fiber (NDF) was

analyzed according to Van Soest et al.23 Acid detergent fiber and acid detergent

lignin (ADF and ADL) were determined according to method no. 973·18 of AOAC

International.24 The residual nitrogen in ADF (ADFN) was determined according to

method no. 984.13 of AOAC International.24 Chitin (CHI, g kg-1 DM) was estimated

as: ash free ADF (g kg-1) – ADFN * N-factor (g kg-1).9 Gross energy (GE) was

determined using an adiabatic calorimetric bomb (C7000; IKA, Staufen, Germany).

The FA composition of substrates and larvae was assessed.25 The results were

expressed as g kg-1 of total detected fatty acids (TFA) (Table 2).

Statistical analyses

The statistical analysis of data was performed using IBM SPSS Statistics v. 20.0 for

Windows. The two trials were considered separately. Larvae weights and lengths

were subjected to a Two-Way Mixed ANOVA. The Shapiro-Wilk test was used to

verify if the dependent variables were normally distributed for each combination of

the groups of within- (test day, considered as a repeated measure) and between-

(rearing substrate) subjects factors. The Levene’s test was used to verify the

homogeneity of variances for each combination of the groups of within- and

between-subjects factors. The Mauchly's test was used to verify the assumption of

sphericity; if such an assumption was violated, the Greenhouse-Geisser or the

Huynh-Feldt correction (in cases of estimates of sphericity lower or higher than

0.75, respectively) was applied to correct the degrees of freedom of the F-

distribution. Final larvae weights and lengths (average weight and length of the

This article is protected by copyright. All rights reserved.

 leftover 70% larvae after removing the 30% of prepupae) were further subjected to

independent samples Student’s t-tests to assess differences between rearing

substrates.

Differences in terms of larvae growth performances, waste reduction efficiency,

Accepted Article
proximate composition and FA profile between substrates were also assessed using

independent-samples Student’s t-tests.

The Kruskal-Wallis test was used to compare the time needed by the larvae to

reach the prepupae stage.

Significance was declared at P<0.05.

RESULTS

Growth performances and waste reduction efficiency of the BSF larvae

The effect of the rearing substrate on the development of BSF larvae over time is

reported in Figure 1 (Trial 1) and Figure 2 (Trial 2).

In both trials, results from the Two-Way Mixed ANOVA showed that, for larvae

development (weight and length), rearing substrate, test day and their interaction

were highly significant (P<0.001), VEGFRU and BRE performing better than FRU

and WIN, respectively.

In Trial 1, no differences were observed for larvae weight (mean ± SD:

0.004±0.0002 g) at the beginning of the trial (day 0; 6 days-old larvae) (Figure

1A). Differences appeared after 4 days of trial, with a higher weight in the VEGFRU

larvae (0.055±0.0084 g) compared to the FRU larvae (0.037±0.0058 g). Such

trend was maintained at each test day until day 16 (VEGFRU: 0.148±0.0103 g;

FRU: 0.120±0.0094 g) when VEGFRU larvae started to enter in the prepupae stage.

The final weight of the larvae did not show differences between the two rearing

substrates. At the beginning of Trial 1, VEGFRU and FRU larvae showed length

values of 5.7±1.32 mm and 5.3±1.17 mm, respectively (P<0.05; Figure 1B).

VEGFRU larvae continued showing higher length values than FRU larvae until the

This article is protected by copyright. All rights reserved.

 last statistical assessment (day 16). VEGFRU and FRU larvae achieved a final length

of 17.7±0.46 mm and 17.8±0.51 mm, respectively (P>0.05).

At the beginning of Trial 2, no differences were observed between WIN and BRE for

larvae weight (0.007±0.0011 g) (Figure 2A). Remarkable differences were reported

Accepted Article
after 4 days of trial, with a higher weight in the BRE larvae compared to the WIN

larvae (0.092±0.0063 and 0.017±0.0018 g, respectively). The final weight of the

larvae (reached after 8 and 26 days of trial for BRE and WIN, respectively) did not

show differences between treatments. The mean length of 6-day-old larvae (day 0)

was 6.5±1.36 and 6.4±1.24 mm for BRE and WIN, respectively (P>0.05; Figure

2B). After 4 days of trial, differences in larvae length were highlighted, with

recorded values of 15.1±1.84 mm (BRE) and 8.7±1.23 mm (WIN).

Dynamic of growth and waste reduction efficiency parameters are reported in Table

1. In Trial 1, VEGFRU larvae showed lower LM and time needed to reach the

prepupae stage, as well as higher ECD than FRU larvae. In Trial 2, BRE larvae

showed lower LM, time needed to reach the prepupae stage and SR, and

contemporarily higher total final biomass, GR, WRI and ECD than WIN larvae.

Proximate and fatty acid compositions of the rearing substrates

The proximate and FA compositions of the rearing substrates are reported in Table

2 and Table 3, respectively.

In Trial 1, VEGFRU showed lower values of DM and NSC and higher contents of ash,

CP, NDF and ADF than FRU, while comparable EE and ADL contents were found. In

Trial 2, WIN showed higher DM, ash, NDF, ADF, and ADL contents and lower CP and

NSC contents than BRE. VEGFRU and FRU showed similar GE values which were

lower than those obtained in the second trial for WIN and BRE.

Total FA ranged from 10.04 (FRU) to 82.47 g kg-1 DM (BRE). VEGFRU showed

higher total polyunsaturated fatty acids (PUFA) and lower total monounsaturated

fatty acids (MUFA) than FRU. WIN had higher MUFA and lower SFA when compared

to BRE. Linoleic acid (C18:2 n6) was the most abundant FA in all substrates.

This article is protected by copyright. All rights reserved.

 Proximate and fatty acid compositions of the BSF larvae

The proximate and FA compositions of the BSF larvae are reported in Table 4 and

Table 5, respectively.
Accepted Article
Concerning Trial 1, ash, CP and ADF values in the VEGFRU larvae were higher than

those in FRU larvae. Conversely, the FRU larvae showed higher DM, EE and NDF

contents than the VEGFRU larvae. In Trial 2, the WIN larvae showed lower DM and

CP contents when compared to the BRE larvae, while all the other parameters

showed an opposite trend.

Considering the FA composition of the larvae, FRU larvae showed higher TFA than

VEGFRU larvae. On the contrary, in Trial 2 similar TFA contents were observed for

BRE and WIN larvae. Significant differences between treatments were observed in

both trials for almost all considered FA groups and individual FA. PUFA were higher

in VEGFRU and BRE larvae when compared to FRU and WIN larvae, respectively,

while an opposite trend was observed for SFA. The most represented individual FA

in BSF larvae from all treatments was C12:0, which showed higher amounts in FRU

and WIN when compared to VEGFRU and BRE, respectively. C18:1 c9 and C18:2 n6

were the most represented unsaturated FA in all treatments.

DISCUSSION AND CONCLUSIONS

Our study investigated, through 2 trials, the effects of different rearing substrates

on development, waste reduction efficiency, and nutritional composition of BSF

larvae.

VEGFRU and BRE larvae showed higher weights after 4 days from the beginning of

the trial, had lower mortality and needed less time to reach the prepupae stage

than FRU and WIN larvae, respectively. Such results were obtained in spite of

comparable GE values found in Trial 1 for VEGFRU and FRU and in Trial 2 for WIN

and BRE substrates, and can be at least partly ascribed to the higher CP and

This article is protected by copyright. All rights reserved.

 moisture contents of VEGFRU and BRE, confirming the results obtained by other

authors.26,27

The need for high dietary moisture content could be ascribed to the morphology of

the mouthparts of BSF larvae, which resembles the characteristics of scavenger

Accepted Article
insects.28,29 This kind of macerating mouth apparatus allows BSF larvae to scrape

off the food from the feeding surface. By softening the feed solids, increased

dietary moisture content makes easier for the larvae to feed.30

The results obtained in our trials could be also reflective of possible differences

between rearing substrates in terms of the content of nutrients other than CP (e.g.,

ether extract, structural and non-structural carbohydrates, amino acids) and/or in

terms of nutrient digestibility. In both trials, the EE content of substrates (<90 g

kg-1 DM) was far below the 200-260 g kg-1 found by Nguyen et al.12,13 to have

detrimental effects for the survival of BSF larvae and adults. BRE larvae showed

very good performances despite the high structural carbohydrates content of the

relative rearing substrate (NDF: 447 g kg-1 DM; ADF: 225 g kg-1 DM). Such a result

clearly demonstrates that BSF larvae are also able to efficiently bioconvert wastes

and by-products characterized by high fiber content, thanks to the presence, in the

digestive tract of the insect, of intestinal bacteria able to degrade cellulose.31 The

amino acid composition of the rearing substrates was not analyzed in our trials, and

little literature is available concerning the effects of dietary amino acids on

development and nutritional composition of BSF larvae.32,33 Studying the nutritional

composition of BSF prepupae reared on different organic waste substrates,

Spranghers et al.32 showed that the amino acid content of the prepupae had narrow

ranges, particularly when compared to the noticeable differences found in the

amino acid composition of the rearing substrates. Concerning nutrient digestibility,

to the best of our knowledge no studies are currently available. Further studies are

necessary to deepen these aspects for the optimization of BSF feeding and

nutrition.

This article is protected by copyright. All rights reserved.

 In Trial 2 the differences in larvae growth performances between treatments were

very pronounced. We may speculate that the GE of the WIN substrate was not fully

available for the larvae. The methodology used to grind the WIN substrate could

have influenced the availability of the oil present inside the grape seeds. Indeed, a
Accepted Article
3-mm grinder was used, and this size could not have completely milled the seeds.

Moreover, the WIN substrate could have contained substances unsuitable for the

BSF larvae development. Indeed, winery by-products usually contain high levels of

polyphenols.34 It is known that plants use polyphenolic compounds to protect

themselves from herbivore insect attacks.35 Some studies also showed how the

grape seeds can accumulate high doses of pesticides and insecticides used in wine

grapevines management.35,36

Hard et al.37 reported that larvae rearing density affects competition for food, low

densities usually leading to highest larvae weights. This was also reflected in our

trial, as no (Trial 1) or slight (Trial 2) differences were observed for the total final

biomass despite the differences found in LM between treatments. The observed LM

for VEGFRU was lower than that reported by Nguyen et al.13 using a vegetable and

fruit rearing substrate.

In both trials, the differences highlighted in terms of LM and ECD were closely

connected, and treatments leading to lower mortality allowed obtaining the best

performances in terms of ECD. BRE larvae reduced a lesser quantity of substrate

compared to WIN larvae; nevertheless, the WRI was higher in the BRE larvae as

they took less time to reach the prepupae stage, which is also confirmed by the

higher GR results. The SR was particularly high (above 65%) in Trial 1, showing the

great potential of BSF larvae in the degradation of vegetable and fruit wastes.12,13

Overall, the BRE larvae showed the best ECD combined with the absolute highest

total final biomass production and the shortest developmental period.

The time needed by the larvae to reach the prepupae stage seemed to influence

their chitin content. Such results agree with the findings of Diener et al.11 who

This article is protected by copyright. All rights reserved.

 reported how small larvae grown in 42 days showed a chitin level higher than

heavy larvae grown in 16 days.

In both trials, substrates containing the highest CP and moisture contents (VEGFRU

and BRE) allowed obtaining BSF larvae with the highest CP level, which is
Accepted Article
consistent with the results obtained by other authors.26,27 Consistently with the

findings of Janssen et al.,22 the use of the conventional N-factor of 6.25 led to a CP

overestimation of about 25%.

Despite comparable EE values of the rearing substrates, FRU larvae showed higher

EE content than VEGFRU larvae, probably as a consequence of the higher NSC level

of FRU.38 Insects have the ability to convert carbohydrates into lipids.32,39 Insects

store lipids for two reasons. Firstly, as energy reserve for the adult stage.14

Secondly because, as insect body presents an open blood system and a high

surface compared to volume and the combination of these two factors could be a

problem for the loss of water and the drying out process, lipids allow them to avoid

transpiration and store non-imbibed water.40 However, the influence of the NSC

content of substrates on the EE content of BSF larvae should be further

investigated as higher NSC in BRE substrate did not lead to higher EE content in

BSF larvae in Trial 2.

The FA composition of the rearing substrates did not directly affect the larvae FA

composition, which was also influenced by carbohydrates (starch and sugars),

confirming other researches.26,32 Being of vegetable origin, all rearing substrates

had PUFA as the most abundant FA group. Notwithstanding, as typically observed

for Diptera, the BSF larvae FA profile was dominated by SFA, mainly C12:0 (which

showed the absolute highest values among individual detected FA), C14:0, C16:0

and C18:0.32,41 The high presence of SFA in insects is connected with cold-

adaptation.42 Indeed, larvae from some species showed a SFA decrease from

summer to autumn while PUFA increased highlighting a correlation between the

change in FA composition and the temperatures due to seasonal change.42,43,44 BSF

is a sub-tropical species growing with high temperatures (27-32°C) and the difficult

This article is protected by copyright. All rights reserved.

 adaptation to low temperatures was demonstrated by the lowest BSF survival rate

at about 16°C.45 We can argue that the high SFA presence could be ascribed to BSF

adaptation to the sub-tropical climate. In particular the high content of lauric acid

(melting point: 43.2°C) could preserve BSF larvae from lipid oxidation and allow
Accepted Article
them to survive at temperatures above 40°C.41 Consistent with other findings,7,15,32

C18:1 c9 was the main represented MUFA in the larvae, while C18:2 n6 and C18:3

n3 were the main represented PUFA n6 and PUFA n3, respectively. The low quantity

of recovered n3 PUFA in the larvae could represent a problem if insect meals are

intended to be used for animal feed. Indeed, researches highlighted a decrease in

nutritional product quality with the inclusion of insect meals in animal diets

especially when full-fat meals are used.5,6,46 Nevertheless, BSF larvae can be

enriched in n3-PUFA through the substrate.33,47 Authors 10,48

reported that C12:0 is

a good inhibitor of bacteria strains and could be of great interest in the reduction of

the use of antibiotics in animal feeding.10,49,50 In this context, BSF larvae reared on

organic wastes resulted very interesting with up to 574 g kg-1 TFA of lauric acid.

Especially in Southern Europe, the large availability of vegetable and fruit wastes

(mainly from markets and supermarkets) may allow the development of a BSF

larvae mass production, enabling as well to obtain economic and environmental

benefits from the sustainable management of organic wastes. Regarding the

considered agro-industrial by-products, the use of winery by-products as rearing

substrate for BSF larvae could be conditioned, both from a technological and

economical point of view, by the need of preliminarily processing to remove or

reduce polyphenols, pesticides and insecticides contents, which could exert a

negative influence on the growth performance of the larvae. Remarkable positive

results were obtained in terms of overall development time, growth performances

and nutritional composition for the larvae reared on brewery by-products, which

should therefore be considered promising rearing substrates. However, as brewery

by-products are characterized by a more limited availability than vegetables, fruits

This article is protected by copyright. All rights reserved.

 or winery by-products, it could be advisable to use BRE at low dietary inclusion

levels with the purpose of increasing BSF larvae performances.

Overall, our results show that the performance and chemical composition of BSF

larvae are largely affected by the chemical composition of the provided substrate.
Accepted Article
This clearly demonstrates that insects, like farm animals, have nutritional

requirements which have to be met for optimal performance.

The performances obtained in our bench top trials may vary when transferred to an

industrial scale. For instance, the large volumes of waste used as well as the high

larvae concentration could result in environmental oxygen depletion and heat

production.51 Attention should then be placed to the airflow to guarantee

appropriate rearing conditions. In addition, to optimize land use, insect breeding

should exploit the verticality of the breeding structure. However, this can lead to

the stratification of temperature and particular attention must be given to an

adequate air circulation to guarantee a homogeneous temperature in all parts of

the building. At industrial level, the production system would also require a

constant supply of substrates, possibly with a fairly constant chemical composition,

as to obtain BSF larvae with relatively constant nutrient profile.

Future studies should be designed to assess the nutritional requirements of BSF

larvae and to evaluate other agro-industrial by-products, as well as the effect of

mixing different organic wastes and agro-industrial by-products, to obtain optimal

BSF larvae performances in terms of development, waste reduction efficiency and

nutritional composition.

ACKNOWLEDGEMENTS

The research was partially supported by GERV_CRT_17_01 grant.

REFERENCES

1. van Huis A, Van Itterbeeck J, Klunder H, Mertens E, Halloran A, Muir G,

Vantomme P, Edible Insects – Future Prospects for Food and Feed Security. FAO

This article is protected by copyright. All rights reserved.

 Forestry Paper 171. Food and Agriculture Organization of the United Nations,

Roma, pp. 59-66 (2013).

2. Salomone R, Saija G, Mondello G, Giannetto A, Fasulo S, Savastano D,

Environmental impact of food waste bioconversion by insects : Application of Life

Accepted Article
Cycle Assessment to process using Hermetia illucens. J Clean Prod 140:890–905

(2017).

3. Arancon RAD, Lin CSK, Chan KM, Kwan TH, Luque R, Advances on waste

valorization: new horizons for a more sustainable society. Energy Science &

Engineering 1:53–71 (2013).

4. Biasato I, De Marco M, Rotolo L, Renna M, Lussiana C, Dabbou S, Capucchio MT,

Biasibetti E, Costa P, Gai F, Pozzo L, Dezzutto D, Bergagna S, Martínez S,

Tarantola M, Gasco L, Schiavone A, Effects of dietary Tenebrio molitor meal

inclusion in free-range chickens. J Anim Physiol Anim Nutr 100:1104–1112

(2016).

5. Gasco L, Henry M, Piccolo G, Marono S, Gai F, Renna M, Lussiana C,

Antonopoulou E, Mola P, Chatzifotis S, Tenebrio molitor meal in diets for

European sea bass (Dicentrarchus labrax L.) juveniles: Growth performance,

whole body composition and in vivo apparent digestibility. Anim Feed Sci Technol

220:34–45 (2016).

6. Renna M, Schiavone A, Gai F, Dabbou S, Lussiana C, Malfatto V, Prearo M,

Capucchio MT, Biasato I, Biasibetti E, De Marco M, Zoccarato I, Gasco L,

Evaluation of the suitability of a partially defatted black soldier fly (Hermetia

illucens L.) larvae meal as ingredient for rainbow trout (Oncorhynchus mykiss

Walbaum) diets. J Anim Sci Biotechnol 8:57, 1–13 (2017).

7. Barragan-Fonseca KB, Dicke M, van Loon JJA, Nutritional value of the black

soldier fly (Hermetia illucens L.) and its suitability as animal feed – a review. J

Insects Food Feed 3(2):105-120 (2017).

8. Surendra KC, Olivier R, Tomberlin JK, Jha R, Kumar S, Bioconversion of organic

wastes into biodiesel and animal feed via insect farming. Renew Energy 98:197–

This article is protected by copyright. All rights reserved.

 202 (2016).

9. Finke MD, Estimate of chitin in raw whole insects. Zoo Biol 26:105–115 (2007).

10. Spranghers T, Joris M, Vrancx J, Ovyn A, Eeckhout M, De Clercq P, De Smet S,

Gut antimicrobial effects and nutritional value of black soldier fly (Hermetia
Accepted Article
illucens L.) prepupae for weaned piglets Anim Feed Sci Technol 235:33-42

(2018).

11. Diener S, Zurbrügg C, Tockner K, Conversion of organic material by black

soldier fly larvae: establishing optimal feeding rates. Waste Manag Res 27:603–

610 (2009).

12. Nguyen TTX, Tomberlin JK, Vanlaerhoven S, Ability of Black Soldier Fly

(Diptera: Stratiomyidae) larvae to recycle food waste. Environ Entomol 44:406–

410 (2015).

13. Nguyen TTX, Tomberlin JK, Vanlaerhoven S, Influence of resources on Hermetia

illucens (Diptera: Stratiomyidae) larval development. J Med Entomol 50:898–

906 (2013).

14. Tomberlin JK, Sheppard DC, Joyce JA, Selected life-history traits of Black

Soldier Flies (Diptera: Stratiomyidae) reared on three artificial diets. Ann

Entomol Soc Am 95:379–386 (2002).

15. Barroso FG, de Haro C, Sánchez-Muros MJ, Venegas E, Martínez-Sánchez A,

Pérez-Bañón C, The potential of various insect species for use as food for fish.

Aquaculture 422–423:193–201 (2014).

16. Wang YS, Shelomi M, Review of Black Soldier Fly (Hermetia illucens) as animal

feed and human food. Foods 6:91 (2017).

17. Segrè A, Falasconi L, Il libro nero dello spreco in Italia: il cibo. Edizioni

Ambiente, Milano, pp 1-124 (2011).

18. Briggs DE, Boulton CA, Brookes PA, Steven R, Brewing: science and practice.

Woodhead Publishing Limited and CRC Press, Boca Raton, pp. 1-863 (2004).

19. Harnden LM, Tomberlin JK, Effects of temperature and diet on black soldier fly,

Hermetia illucens (L.) (Diptera: Stratiomyidae), development. Forensic Sci Int

This article is protected by copyright. All rights reserved.

 266:109–116 (2016).

20. Leong SY, Kutty SRM, Malakahmad A, Tan CK, Feasibility study of biodiesel

production using lipids of Hermetia illucens larva fed with organic waste. Waste

Manage 47:84–90 (2016).

Accepted Article
21. Tschirner M, Simon A, Influence of different growing substrates and processing

on the nutrient composition of black soldier fly larvae destined for animal feed. J

Insects Food Feed 1:1–12 (2015).

22. Janssen RH, Vincken JP, van den Broek LAM, Fogliano V, Lakemond CMM,

Nitrogen-to-protein conversion factors for three edible insects: Tenebrio molitor,

Alphitobius diaperinus, and Hermetia illucens. J Agric Food Chem 65:2275–2278

(2017).

23. Van Soest PJ, Robertson JB, Lewis BA, Methods for dietary fiber, neutral

detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J

Dairy Sci 74:3583–3597 (1991).

24. AOAC International, Official methods of analysis of AOAC International. 16th ed.

Gaithersburg, MD: Association of Official Analytical Chemists (2000).

25. Renna M, Gasmi-Boubaker A, Lussiana C, Battaglini LM, Belfayez K, Fortina R,

Fatty acid composition of the seed oils of selected Vicia L. taxa from Tunisia. Ital

J Anim Sci 13:308–316 (2014).

26. Oonincx DGAB, van Broekhoven S, van Huis A, van Loon JJA, Feed conversion,

survival and development, and composition of four insect species on diets

composed of food by-products. PLoS ONE 10:1–20 (2015).

27. Cammack JA, Tomberlin JK, The impact of diet protein and carbohydrate on

select life-history traits of the Black Soldier Fly Hermetia illucens (L.) (Diptera:

Stratiomyidae). Insects 8,56:1-14 (2017).

28. Purkayastha D, Sarkar S, Roy P, Kazmi AA, Isolation and morphological study

of ecologically-important insect “Hermetia illucens” collected from Roorkee

compost plant. Pollution 3:453–459 (2017).

29. Kim W, Bae S, Park H, Park K, Lee S, Choi Y, Han S, Koh Y, The larval age and

This article is protected by copyright. All rights reserved.

 mouth morphology of the black soldier fly, Hermetia illucens (Diptera:

Stratiomyidae). Int J Indust Entomol 21:185–187 (2010).

30. Banks IJ, To assess the impact of black soldier fly (Hermetia illucens) larvae on

faecal reduction in pit latrines. Doctoral Thesis, London School of Hygiene

Accepted Article
Tropical Medicine (2014). Available at:

http://researchonline.lshtm.ac.uk/1917781/ (last accessed: 20 April 2018).

31. Kim E, Park J, Lee S, Kim Y, Identification and physiological characters of

intestinal bacteria of the Black Soldier Fly, Hermetia illucens. Korean J Appl

Entomol 53:15–26 (2014).

32. Spranghers T, Ottoboni M, Klootwijk C, Ovyn A, Deboosere S, De Meulenaer B,

Michiels J, Eeckhout M, De Clercq P, De Smet S, Nutritional composition of black

soldier fly (Hermetia illucens) prepupae reared on different organic waste

substrates. J Sci Food Agric 97:2594–2600 (2017).

33. Liland NS, Biancarosa I, Araujo P, Biemans D, Bruckner CG, Waagbø R,

Torstensen BE, Lock EJ, Modulation of nutrient composition of black soldier fly

(Hermetia illucens) larvae by feeding seaweed-enriched media. PLoS ONE

12(8): e0183188 (2017).

34. Garavaglia J, Markoski MM, Oliveira A, Marcadenti A, Grape seed oil

compounds : biological and chemical actions for health. Nutr Metab Insights

9:59–64 (2016).

35. Lattanzio V, Lattanzio VM, Cardinali A, Role of phenolics in the resistance

mechanisms of plants against fungal pathogens and insects, in Phytochemistry:

Advances in Research, ed. by Imperato F. Research Signpost, Trivandrum, pp.

23–67 (2006).

36. Rose G, Lane S, Jordan R, The fate of fungicide and insecticide residues in

Australian wine grape by-products following field application. Food Chem

117:634–640 (2009).

37. Hard JJ, Bradshaw WE, Malarkey DJ, Resource- and density-dependent

development in tree-hole mosquitoes. Oikos 54:137 –144 (1989).

This article is protected by copyright. All rights reserved.

 38. Arrese EL, Soulages JL, Insect fat body: energy, metabolism, and regulation.

Annu Rev Entomol 55:207–225 (2010).

39. Inagaki S and Yamashita O, Metabolic shift from lipogenesis to glycogenesis in

the last instar larval fat body of the silkworm, Bombyx mori. Insect Biochem
Accepted Article
16:327–331 (1986).

40. Downer RGH, Matthews JR, Patterns of lipid distribution and utilisation in

insects. Am Zool 16:733–745 (1976).

41. Ushakova NA, Brodskii ES, Kovalenko AA, Bastrakov AI, Kozlova AA, Pavlov

ADS, Characteristics of lipid fractions of larvae of the black soldier fly Hermetia

illucens. Dokl Biochem Biophys 468:209–212 (2016).

42. Bennett VA, Lee RE, Modeling seasonal changes in intracellular freeze-tolerance

of fat body cells of the gall fly Eurosta solidaginis (Diptera, Tephritidae). J Exp

Biol 200:185–192 (1997).

43. Atapour M, Moharramipour S, Barzegar M, Seasonal changes of fatty acid

compositions in overwintering larvae of rice stem borer, Chilo suppressalis

(Lepidoptera: Pyralidae). J Asia-Pacific Entomol 10:33–38 (2007).

44. Kalyoncu L, Çetin H, Effects of low temperature on the fatty acid compositions

of adult of Acanthoscelides obtectus (Say) (Coleoptera: Chrysomelidae). Selçuk

Tarım ve Gıda Bilimleri Dergisi 31:11–15 (2017).

45. Holmes LA, VanLaerhoven SL, Tomberlin JK, Lower temperature threshold of

black soldier fly (Diptera: Stratiomyidae) development. J Insects Food Feed

2:255–262 (2016).

46. Bovera F, Loponte R, Marono S, Piccolo G, Parisi G, Iaconisi V, Gasco L, Nizza A,

Use of Tenebrio molitor larvae meal as protein source in broiler diet: effect on

growth performance, nutrient digestibility, carcass and meat traits. J Anim Sci

94:639-647 (2016).

47. Barroso FG, Sánchez-Muros MJ, Segura M, Morote E, Torres A, Ramos R, Guil

JL, Insects as food: Enrichment of larvae of Hermetia illucens with omega 3 fatty

acids by means of dietary modifications. J Food Compos Anal 62:8-13 (2017).

This article is protected by copyright. All rights reserved.

 48. Skrivanova E, Marounek M, Benda V, Brezina P, Susceptibility of Escherichia

coli, Salmonella sp. and Clostridium perfringens to organic acids and monolaurin.

Vet Med (Praha) 51:81–88 (2006).

49. Schiavone A, Cullere M, De Marco M, Meneguz M, Biasato I, Bergagna S,

Accepted Article
Dezzutto D, Gai F, Dabbou S, Dalle Zotte A, Partial or total replacement of

soybean oil by black soldier fly larvae (Hermetia illucens L.) fat in broiler diets:

effect on growth performances, feed-choice, blood traits, carcass characteristics

and meat quality. Ital J Anim Sci 16:93–100 (2017).

50. Schiavone A, Dabbou S, De Marco M, Cullere M, Biasato I, Biasibetti E,

Capucchio MT, Bergagna S, Dezzutto D, Meneguz M, Gai F, Dalle Zotte A, Gasco

L, Black soldier fly (Hermetia illucens L.) larva fat inclusion in finisher broiler

chicken diet as an alternative fat source. Animal. Accepted on December 13

2017 (2018).

51. Cheng JYK, Chiu SLH, Lo IMC, Effects of moisture content of food waste on

residue separation, larval growth and larval survival in black soldier fly

bioconversion. Waste Manage 67:315–323 (2017).

This article is protected by copyright. All rights reserved.

Accepted Article
Table 1: Dynamic of growth and waste reduction efficiency (on a fresh matter basis) of black soldier fly larvae reared on

organic wastes (vegetables and fruits) and agro-industrial by-products (winery and brewery) generated by the Italian food

sector (mean ± SD; n = 6).

Trial 1 Trial 2
(organic wastes) (agro-industrial by-products)
VEGFRU FRU P WIN BRE P
Larvae mortality (%) 11.2±4.35 19.3±5.24 0.015 24.8±10.53 9.5±5.68 0.011
Total final biomass (g) 10.42±0.648 10.92±2.057 0.584 9.90±0.785 11.32±0.864 0.014
Time needed to reach prepupae stage (days of trial) 20.2±1.33 22.0±0.89 0.031 22.2±0.98 8.0±0.01 0.003
-1
Growth rate (g d ) 0.006±0.0018 0.007±0.0007 0.451 0.006±0.0009 0.014±0.0009 0.000
Substrate reduction (%) 65.2±5.54 70.8±8.39 0.129 53.0±5.28 42.5±8.41 0.027
-1
Waste reduction index (g d ) 3.2±0.26 3.2±0.41 0.952 2.4±0.32 5.3±1.05 0.000
Efficiency of conversion of digested food 0.07±0.009 0.05±0.011 0.004 0.06±0.002 0.14±0.034 0.000
VEGFRU: 70% vegetable and 30% fruit waste; FRU: 100% fruit waste; WIN: winery by-product; BRE: brewery by-product.

This article is protected by copyright. All rights reserved.

Accepted Article
Table 2: Proximate composition (g kg-1 dry matter, unless otherwise stated) of organic wastes (vegetables and fruits) and

agro-industrial by-products (winery and brewery) generated by the Italian food sector and used as rearing substrates by

black soldier fly larvae.

Trial 1 Trial 2
(organic wastes) (agro-industrial by-products)
VEGFRU FRU WIN BRE
Dry matter (g kg-1) 82.7 131.9 358.3 232.1
Ash 91.1 30.4 103.0 39.8
Crude protein 119.9 46.0 117.4 200.5
Ether extract 26.0 27.8 79.0 86.7
Neutral detergent fiber 178.0 139.3 566.4 447.1
Acid detergent fiber 110.5 91.1 462.4 225.3
Acid detergent lignin 12.9 13.1 323.5 62.1
Non-structural carbohydrates* 585.0 756.5 134.2 225.9
-1
Gross energy (MJ kg DM) 15.1 15.6 19.5 19.4
VEGFRU: 70% vegetable and 30% fruit waste; FRU: 100% fruit waste; WIN: winery by-product; BRE: brewery by-product.

*Calculated as: 1000 – (crude protein + ether extract + ash + neutral detergent fiber).

This article is protected by copyright. All rights reserved.

Accepted Article
Table 3: Fatty acid composition (g kg-1 total fatty acids, unless otherwise stated) of organic wastes (vegetables and fruits)

and agro-industrial by-products (winery and brewery) generated by the Italian food sector and used as rearing substrates by

black soldier fly larvae.

Trial 1 Trial 2
(organic wastes) (agro-industrial by-products)
VEGFRU FRU WIN BRE
-1
Total fatty acids (g kg dry matter) 20.91 10.04 73.57 82.47
C12:0 0.73 3.52 0.93 0.80
C14:0 8.85 8.44 1.99 3.22
C16:0 184.90 192.71 100.49 252.48
C16:1 c9 5.89 5.96 4.43 1.75
C18:0 26.14 43.36 50.24 15.42
C18:1 c9 65.91 208.61 185.09 103.27
C18:1 c11 20.85 29.62 8.51 7.46
C18:2 n6 575.23 333.38 630.32 554.90
C18:3 n3 111.50 174.40 18.00 60.70
Saturated fatty acids 220.62 248.03 153.65 271.92
Monounsaturated fatty acids 92.65 244.19 198.03 112.48
Polyunsaturated fatty acids 686.73 507.78 648.32 615.60
VEGFRU: 70% vegetable and 30% fruit waste; FRU: 100% fruit waste; WIN: winery by-product; BRE: brewery by-product.

This article is protected by copyright. All rights reserved.

Accepted Article
Table 4: Proximate composition (g kg-1 dry matter, unless otherwise stated) of black soldier fly larvae reared on organic

wastes (vegetables and fruits) and agro-industrial by-products (winery and brewery) generated by the Italian food sector

(mean ± SD; n = 6).

Trial 1 Trial 2
(organic wastes) (agro-industrial by-products)
VEGFRU FRU P WIN BRE P
-1
Dry matter (g kg ) 219.6±10.22 282.9±6.57 0.000 265.4±5.93 290.8±6.96 0.000
Ash 129.8±6.50 72.2±2.22 0.000 145.7±6.67 73.0±1.89 0.000
Crude protein1 418.8±13.24 307.5±10.29 0.000 344.3±7.63 529.6±5.27 0.000
2
Crude protein 312.9±9.89 229.7±7.69 0.000 257.3±5.70 395.7±3.94 0.000
Ether extract 262.8±18.01 407.0±18.83 0.000 322.2±19.60 298.7±6.49 0.031
Neutral detergent fiber 170.9±16.49 197.9±13.48 0.011 177.3±13.08 87.0±9.89 0.000
Acid detergent fiber 113.1±20.09 93.4±3.55 0.014 98.5±10.16 64.8±9.17 0.000
Acid detergent lignin 14.9±7.75 8.9±2.47 0.104 44.8±17.80 8.3±9.35 0.001
3
Chitin 62.4±19.63 56.0±3.96 0.453 52.9±9.25 14.2±6.06 0.000
Chitin4 75.2±19.7 65.5±3.53 0.283 64.5±9.48 27.0±6.59 0.000
VEGFRU: 70% vegetable and 30% fruit waste; FRU: 100% fruit waste; WIN: winery by-product; BRE: brewery by-product.
1
Obtained using the nitrogen-to-protein conversion factor of 6.25.

2
Obtained using the nitrogen-to-protein conversion factor of 4.67.
3
Calculated using the nitrogen-to-protein conversion factor of 6.25.

4
Obtained using the nitrogen-to-protein conversion factor of 4.67.

This article is protected by copyright. All rights reserved.

Accepted Article
Table 5: Fatty acid composition (g kg-1 total fatty acids, unless otherwise stated) of black soldier fly larvae reared on organic

wastes (vegetables and fruits) and agro-industrial by-products (winery and brewery) generated by the Italian food sector

(mean ± SD; n = 6).

Trial 1 Trial 2
(organic wastes) (agro-industrial by-products)
VEGFRU FRU P WIN BRE P
-1
TFA (g kg dry matter) 253.02±18.512 398.40±18.547 0.000 287.41±16.973 282.93±6.936 0.563
C12:0 520.61±17.505 574.32±11.060 0.000 346.91±16.840 323.73±9.277 0.014
C14:0 103.55±3.303 96.39±3.471 0.004 65.54±4.283 66.49±2.687 0.654
C16:0 138.95±7.338 130.57±3.846 0.040 189.36±7.434 204.15±5.772 0.003
C16:1 c9 33.57±3.606 37.45±0.956 0.046 60.63±4.718 29.45±2.639 0.000
C18:0 25.90±1.693 17.51±0.539 0.000 28.32±2.139 18.07±0.599 0.000
C18:1 c9 85.37±4.075 93.19±2.086 0.002 124.59±4.280 92.23±2.414 0.004
C18:1 c11 4.31±0.381 2.79±0.157 0.000 4.46±0.261 5.75±1.155 0.040
C18:2 n6 70.41±7.408 40.70±1.534 0.000 175.76±14.935 235.47±6.593 0.000
C18:3 n3 17.31±1.370 7.06±0.729 0.000 4.44±0.392 24.65±0.504 0.000
SFA 789.02±10.854 818.81±4.632 0.000 630.13±16.745 612.45±8.784 0.045
MUFA 123.26±6.829 133.44±2.773 0.013 189.68±6.220 127.43±5.354 0.000
PUFA 87.72±7.333 47.75±2.083 0.000 180.19±15.244 260.12±6.843 0.000
VEGFRU: 70% vegetable and 30% fruit waste; FRU: 100% fruit waste; WIN: winery by-product; BRE: brewery by-product.

This article is protected by copyright. All rights reserved.

Accepted Article
Fo
rP
ee
rR
ev
iew

Figure 1. Trial 1: Development (A: weight; B: length) of black soldier fly larvae reared on organic wastes
(VEGFRU: 70% vegetable and 30% fruit waste; FRU: 100% fruit waste) generated by the Italian food
sector. P-value: *P<0.05, **P<0.01, ***P<0.001. Error bars represent the standard error of the mean.

This article is protected by copyright. All rights reserved.

Accepted Article
Fo
rP
ee
rR
ev
iew

Figure 2. Trial 2: Development (A: weight; B: length) of black soldier fly larvae reared on agro-industrial by-
products (WIN: winery by-product; BRE: brewery by-product) generated by the Italian food sector. P-value:
*P<0.05, **P<0.01, ***P<0.001. Error bars represent the standard error of the mean.

This article is protected by copyright. All rights reserved.