Hindawi

Aquaculture Nutrition
Volume 2022, Article ID 4717014, 16 pages
https://doi.org/10.1155/2022/4717014

Research Article
Apparent Digestibility Coefficients of Black Soldier Fly (Hermetia
illucens), Yellow Mealworm (Tenebrio molitor), and Blue Bottle
Fly (Calliphora vicina) Insects for Juvenile African Catfish
Hybrids (Clarias gariepinus×Heterobranchus longifilis)

Zsuzsanna J. Sándor ,1 Vojislav Banjac ,2 Strahinja Vidosavljević ,2 Jenő Káldy,1

Robert Egessa,3,4 Éva Lengyel-Kónya ,5 Rita Tömösközi-Farkas ,5 Zsolt Zalán ,5
Nóra Adányi ,5 Balázs Libisch ,6 and Janka Biró 1
1
Research Centre for Aquaculture and Fisheries (HAKI), Hungarian University of Agriculture and Life Sciences, Anna liget u. 35,
Szarvas, Hungary
2
University of Novi Sad, Institute of Food Technology, Bulevar cara Lazara br. 1, Novi Sad, Serbia
3
Doctoral School of Animal Husbandry Science, Hungarian University of Agriculture and Life Sciences, Gödöllő, Hungary
4
National Agricultural Research Organisation (NARO), Jinja, Uganda
5
Research Group of Food Science, Institute of Food Science and Technology, Hungarian University of Agriculture and Life Sciences,
Herman Ottó u. 15, Budapest, Hungary
6
Institute of Genetics and Biotechnology, Hungarian University of Agriculture and Life Sciences, Szent-Györgyi Albert u. 4,
Gödöllő, Hungary

Correspondence should be addressed to Zsuzsanna J. Sándor; jakabne.sandor.zsuzsanna@uni-mate.hu

Received 21 March 2022; Revised 12 August 2022; Accepted 22 August 2022; Published 18 October 2022

Academic Editor: Mahmoud Dawood

Copyright © 2022 Zsuzsanna J. Sándor et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

A digestibility trial was conducted with African catfish hybrid juveniles in order to determine the apparent digestibility coefficients
(ADCs) of different nutrients. The experimental diets contained defatted black soldier fly (BSL), yellow mealworm (MW), or fully
fat blue bottle fly (BBF) meals, in a 70 : 30 ratio between the control diet and the tested insect meals. The indirect method for the
digestibility study was performed using 0.1% yttrium oxide as an inert marker. Fish juveniles of 217:4 ± 9:5 g initial weight were
distributed in 1 m3 tanks (75 fish/tank) of a recirculating aquaculture system (RAS), in triplicates, and fed until satiation for 18
days. The average final weight of the fish was 346 ± 35:8 g. The ADCs of the dry matter, protein, lipid, chitin, ash, phosphorus,
amino acids, fatty acids, and gross energy for the test ingredients and diets were calculated. A six-month storage test was
carried out to evaluate the shelf life of the experimental diets, while the peroxidation and microbiological status of the diets
were also assessed. The ADC values of the test diets differed significantly (p < 0:001) compared to those of the control for most
of the nutrients. Altogether, the BSL diet was significantly more digestible for protein, fat, ash, and phosphorus than the
control diet but less digestible for essential amino acids. Significant differences were found between the ADCs of the different
insect meals evaluated (p < 0:001) for practically all nutritional fractions analyzed. The African catfish hybrids were able to
digest BSL and BBF more efficiently than MW, and the calculated ADC values agreed with those of other fish species. The
lower ADCs of the tested MW meal correlated (p < 0:05) with the markedly higher acid detergent fiber (ADF) levels present in
the MW meal and MW diet. Microbiological evaluation of the feeds revealed that mesophilic aerobic bacteria in the BSL feed
were 2–3 orders of magnitude more abundant than those in the other diets and their numbers significantly increased during
storage. Overall, BSL and BBF proved to be potential feed ingredients for African catfish juveniles and the shelf life of the
produced diets with 30% inclusion of insect meal retained the required quality during a six-month period of storage.
2 Aquaculture Nutrition

1. Introduction The digestibility of feeds containing different propor-

tions of insect meals has been examined, and the concerned
Feed manufacturers show an increasing interest in insect- studies were reviewed by Gasco et al. [22]. The most fre-
derived raw materials because of their potential as fish meal quently investigated insects in these studies were the black
substituents. The ecological footprint of insect cultivation is soldier fly and mealworm, and finally, the optimal inclusion
much lower than that of field crops, which make up the vast level for nutrition was suggested. Only a few studies esti-
majority of animal feeds. The development and reproduc- mated the digestibility of insect meals by different fish spe-
tion cycle of insects requires a short period of time. They cies. Among them, apparent digestibility coefficients
can be grown on biowaste, and being ectotherm organisms, (ADCs) of housefly maggot meal were determined for Nile
they have high feed utilization rates [1]. The need for valu- tilapia (Oreochromis niloticus) and common carp (Cyprinus
able proteins in fish diets is increasing due to the scarcity carpio) [23] and ADCs of several Coleoptera, Orthoptera,
and limited availability of marine raw material resources and Blattodea species in Nile tilapia fingerlings [24].
and the limited suitability of some terrestrial plants as ingre- Mohamad-Zulkifli et al. [25] presented ADC values of a
dients of aquafeeds. Various feeding experiments with processed black soldier fly (BSL) meal fed to hybrid grouper
insects have been carried out using many aquaculture spe- (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂),
cies in the last ten years, and the results so far are also and ADC results for BSL are also available for Atlantic
encouraging for their industrial production [2–6]. Since the salmon (Salmo salar) [26].
authorization of insect farming in the European Union Black soldier fly and yellow mealworm (MW) are the
(EU Regulation no. 2017/893), more than 5000 tons of insect most frequently utilized insects in fish nutrition. BSL
protein have been commercialized by European insect pro- belongs to the Diptera, Stratiomyidae family and is present
ducers [7]. According to the recommendation of the Euro- on all major continents, predominantly in temperate and
pean Food Safety Authority, the following species are tropic regions. It can successfully develop on vertebrate
eligible for farming purposes: black soldier fly (Hermetia remains, kitchen waste, fruits and vegetables, raw liver, fish
illucens), common housefly (Musca domestica), yellow meal- offal, municipal and human waste, and dairy cattle manure.
worm (Tenebrio molitor), lesser mealworm (Alphitobius dia- MW belongs to the order Coleoptera, and it has been pro-
perinus), house cricket (Acheta domesticus), banded cricket duced in large quantities since the 1950s, initially for fishing
(Gryllodes sigillatus), and field cricket (Gryllus assimilis). bait and later for pet food and for the songbird feed market.
Nevertheless, in non-EU countries, regulations are different Nowadays, thousands of tons of dry mealworm are pro-
and other insects are also considered useful for fish nutrition duced and sold worldwide [27–29]. The MW can grow and
[8, 9]. reproduce when fed exclusively on wheat bran or additional
The chemical composition and nutritional value of food supplements [30, 31]. Blue bottle fly (BBF) (Calliphora
insect larvae are variable and depend on many factors. The vicina, Calliphoridae, and Diptera), commonly referred to as
most important factors are the rearing substrate and pro- “meat fly,” is used as bait or boilies ingredient for fishing or
cessing methodology ([2, 10]. Insect meals are high in pro- angling. However, its utilization in fish feeds is not yet
tein and have an immunostimulatory effect due to their authorized in EU countries (EU Regulation no. 2017/893)
chitin content [9, 11–13]. Insect proteins have the advantage but it is present in several fishing products available in
of high levels of the essential amino acid lysine (LYS), methi- Europe, too.
onine (MET), and leucine (LEU), which are usually limiting The microbiological composition of the fish feed has a
in plants [14]. Another product made from insects is insect high impact on the weight gain and fish health condition
fat, which is favorable for fish mainly due to the presence [32]. A wide range of microbes occurs naturally in feeds or
of lauric acid, a medium-chain fatty acid considered to have as contaminants of feeds. These microbes could be non-
antibacterial effect on fish [15]. Besides the available nutri- pathogenic bacteria, but often, these are molds or also harm-
ents in the feedstuffs, knowledge of the digestibility of the ful bacteria such as Salmonella, Listeria, and E. coli [33]. The
various feed ingredients is also required. Together with insects may contain several insect-specific pathogenic
chemical analysis, determination of digestibility concerning microorganisms that have to be considered when the feed
the nutrients and energy may allow a more thorough estima- safety aspect is assessed. The risk of transmission from a
tion of the nutritive value of a particular protein source in a rearing substrate to the insect can be reduced by hygienic
complete feed for fish. culturing. Fortunately, the risk posed by pathogenic micro-
Although insects have the potential as good protein and organisms is mitigated during the insect meal production
fat sources, less information is known about their utilization process or later in the feed extrusion step. Autochthonous
as feed ingredients for intensively reared African catfish microbiota of the insects including bacteria, fungi, and
(Clarias gariepinus), an important and dominant aquacul- viruses have been explored by different authors [34, 35].
ture species in Hungary. Meals from insects such as shea However, in case of most pathogens, no active growth occurs
caterpillar (Cirina butyrospermi), housefly, variegated grass- in the intestinal tract of insects [36].
hopper (Zonocerus variegatus L.), and black solider fly have Taking into consideration the abovementioned gaps in
previously been included in the diets of African catfish as our knowledge, we aimed to determine the apparent digest-
alternative protein sources [16–21]. However, no data are ibility coefficients (ADCs) of African catfish hybrid juveniles
available on the digestibility of insect meals as ingredients for BSL, MW, and BBF meals. Accordingly, the present
for this fish species. study demonstrates a short-term digestibility trial conducted
Aquaculture Nutrition 3

in a recirculating aquaculture system (RAS) using three then extruded. The control feed was prepared to be a high
experimental diets containing different insect meals. In addi- fish meal diet in order to be easily digestible for fish.
tion, the stability and shelf life of these feeds were followed The dry ingredients were mixed in a double-shaft paddle
up during a six-month storage period. mixer (model SLHSJ0.2A, Muyang, Yangzhou, China) for
120 s according to Table 2 and the experimental setup.
2. Materials and Methods Yttrium-oxide, as a marker for digestibility assessment, was
added to each diet at a 0.1% level. Dry mixtures were proc-
2.1. Description of the Insect Meals. The defatted black sol- essed using a twin-screw extruder (Bühler BTSK-30, Bühler,
dier fly meal was supplied by Agroloop Ltd., Netherlands; Uzwil, Switzerland) and then subsequently dried in the con-
defatted yellow mealworm meal was imported from Berg tinuous vibro dryer, model FB 500 × 2000 (Amandus Kahl
and Schmidt Pte. Ltd., Singapore; while the fully fat blue bot- GmbH & Co., KG, Germany) at 80°C for approximately 10
tle fly meal was produced by Csali Hungary Ltd. (Kiskunha- minutes. The final pellets were 4.5 mm in diameter and
las, Hungary). The composition (% DM) and gross energy semifloating. Proximate, fatty acid, amino acid composition,
value (MJ·kg−1) of insect meals are summarized in Table 1. and gross energy of diets are shown in Table 2.
There was high variability in chemical composition of the
ingredients. The protein content was high in BSL and MW, 2.3. Fish Feeding and Faeces Collection. The research was
while in the case of BBF, the protein level was lower due to carried out with African catfish hybrid (Clarias gariepi-
the high amount of fat in it. Crude ash, Ca, P, and chitin nus × Heterobranchus longifilis) juveniles. The experiments
content were the highest in BSL compared to the others, were conducted according to the European Union Directive
while the acid detergent fiber (ADF) content was the highest 2010/63/EU regarding the protection of animals for scien-
in MW. The chitin content ranged between 5.8 and 9.6%. tific purposes. The animal experiments and related sam-
The ash-free ADF of BBF was the lowest (15%), while for plings were approved by the Ethical Committee of HAKI
BSL and MW, 22% and 27% were determined, respectively. (license no. BE/25/4302-3/2017), which was established
The gross energy content of feed ingredients ranged from according to the Hungarian State law 9/1999 (I. 27.), and it
20.7 MJ·kg−1 (for MW) to 25.64 MJ·kg−1 (for BBF). The is operated according to the relevant Hungarian legislation
essential amino acid content differed significantly in HIS concerning animal experiments, transportation of animals,
between the meals. The highest level of LYS and MET and and their welfare (40/2013. II. 14).
the lowest level of LEU have been observed in the BBF meal. Nine hundred African catfish juveniles (average weight
Finally, the sum of essential amino acids (EAA) was the low- of 217:4 ± 9:5 g) originating from the institutional hatchery
est in MW. The fatty acid profile of the meals differed in facility of HAKI were distributed in a RAS equipped with
some cases. The lauric acid (12 : 0) content was the highest twelve 1 m3 fiberglass tanks (75 fish per tank). Three exper-
in BSL (43.12%) although the sample was defatted, while imental groups and one control group were set up and ran-
in MW and BBF, its level was very low (0.12–0.25% or domly distributed in tank triplicates. The water flow was
4.01 mg/100 g D.M., 0.02 mg/100 g D.M., and 0.04 mg/100 g adjusted to an average of 4.5 L/min per tank, the dissolved
D.M., respectively). In BBF and MW, the oleic acid oxygen level was kept above 80% saturation, ammonia-N
(18 : 1n − 9) and linoleic acid (18 : 2n − 6) levels were about was below 0.1 mL/L, and pH varied between 7.8 and 8.4.
two times higher than those in the BSL sample. Conse- The water temperature was set to 23 ± 1° C. During three
quently, total monounsaturated (MUFA) and polyunsatu- days of acclimatization, the fish were fed with a commercial
rated (PUFA) fatty acid levels were also high in the MW diet and thereafter switched to experimental diets. Fish were
and BBF samples. Regarding the long chain n-3 polyunsatu- hand fed till apparent satiation with the experimental diets 3
rated FAs (Lc-PUFAs), the insects do not contain them, thus times per day for 18 consecutive days. On the last day of
depending on the rearing substrate, they can be detected feeding, 15 individuals from the fish stock per tank were
only at trace levels in insects. sampled in order to collect faeces from the intestine [45].
The average final weight of fish was 346 ± 35:8 g. Before har-
2.2. Diet Preparation for the Digestibility Trial. The nutrient vesting, fish were anesthetized with norcaicum-/tonogen-
content of the tested insect meals was examined in detail in (50 mL/100 L) based anesthesia [46]. The whole intestines
order to satisfy the needs of omnivorous fish species since were removed and the solid part of the faeces was collected
information on the nutrient requirements of African catfish as pooled samples per treatment. The fecal samples were
[38] is scarcely available. The amino acid balance of these refrigerated, freeze dried, and stored in exicator until analy-
insect meals was comparable to that of fish meal [39] and sis. The evaluation of growth parameters was not considered
was sufficient to meet the dietary requirement for catfish. in this trial.
However, the quantity of MET and LYS should be increased
in feed formulations. Four different feeds for African catfish 2.4. Analytical Methods. The chemical composition of test
were produced at the Institute of Food Technology (Univer- ingredients, feeds, and faeces was analyzed by standard
sity of Novi Sad, Novi Sad, Serbia) for digestibility trials of methods of the AOAC [47]. Crude protein (CP) was deter-
three different insect meals. For this purpose, a control feed mined by the Kjeldahl method [47] using digestion block
(reference) was formulated, which was then mixed individu- (KJELDATHERM, Gerhardt, Germany) via a distillation
ally with the test ingredients at a 70 : 30 ratio (control: test, as procedure (VAPODEST 450, Gerhardt, Germany). 0.5 g
is basis) to produce the experimental diets [40–43] and were dry samples were digested with 10 mL of cc H2SO4 and
4 Aquaculture Nutrition

Table 1: Composition of the tested insect meals (dry weight, %).

Ingredients BSL MW BBF

Dry matter 93:19 ± 0:61 91:96 ± 0:30 95:15 ± 0:16
Crude protein∗ 52:46 ± 0:42 56:53 ± 0:14 42:54 ± 0:25
Crude fat 9:29 ± 0:40 6:20 ± 0:17 29:41 ± 0:04
Crude ash 7:80 ± 0:25 7:01 ± 0:05 4:75 ± 0:12
Crude fiber 4:81 ± 1:01 3:43 ± 0:13 5:30 ± 0:25
Phosphorus 1.01 0.54 0.78
Calcium 3.64 0.65 0.26
Gross energy (MJ·kg−1) 22:76 ± 0:12 20:72 ± 0:03 25:64 ± 0:07
Acid detergent fiber (ADF) 22:10 ± 0:92 27:69 ± 0:12 15:11 ± 0:33
Chitin 9:62 ± 2:01 5:81 ± 2:08 8:05 ± 1:50

Amino acids (%)

Arginine (ARG) 3.30 3.54 3.05
Histidine (HIS) 1.46 0.91 1.99
Isoleucine (ILE) 3.03 2.18 2.35
Leucine (LEU) 5.02 5.22 3.84
Lysine (LYS) 3.89 3.81 4.43
Methionine (MET) 1.26 1.19 1.46
Threonine (THR) 2.78 2.37 2.66
Phenylalanine (PHE) 3.17 2.38 4.13
Tryptophan (TRP) 0.58 0.42 0.35
Valine (VAL) 3.96 3.35 3.23
ΣEAA 32.09 27.62 31.23
ΣAA 55.76 52.61 54.62
Fatty acids (FA) (%)
12 : 0 43:12 ± 1:53 0:25 ± 0:01 0:12 ± 0:04
14 : 0 8:05 ± 0:45 1:09 ± 0:02 1:73 ± 0:00
16 : 0 11:65 ± 0:20 18:14 ± 0:05 18:12 ± 0:13
16 : 1n − 9 0:29 ± 0:00 0:28 ± 0:01 5:12 ± 0:14
16 : 1n − 7 3:22 ± 0:09 1:96 ± 0:03 10:79 ± 0:00
18 : 0 1:69 ± 0:16 7:52 ± 0:06 2:64 ± 0:16
18 : 1n − 9 15:52 ± 1:03 33:83 ± 0:10 30:36 ± 0:14
18 : 2n − 6 12:38 ± 0:52 27:44 ± 0:28 24:60 ± 0:34
18 : 3n − 3 0:57 ± 0:01 1:17 ± 0:17 0:65 ± 0:04
20 : 4n − 6 0:07 ± 0:00 0:27 ± 0:02 1:68 ± 0:00
20 : 5n − 3 (EPA) 0:00 ± 0:00 0:00 ± 0:00 0:20 ± 0:01
22 : 6n − 3 (DHA) 0:00 ± 0:00 0:69 ± 0:01 0:00 ± 0:00
SFA 65:68 ± 1:63 28:85 ± 0:09 22:91 ± 0:06
MUFA 19:67 ± 1:14 39:65 ± 0:12 47:54 ± 0:26
PUFA 14:58 ± 0:48 29:86 ± 0:10 28:73 ± 0:20
Total lipid (mg·g−1) 59:27 ± 2:81 15:77 ± 0:89 192:75 ± 1:78
∗
Protein was calculated by applying a nitrogen to protein conversion factor of Kp = 4:76 [37].

10 mL of 30% H2O2. Afterwards, the generated ammonium the 5 g dry sample according to the AOAC 945.16 Soxhlet
sulphate was distilled off by using 2% H3BO3. The CP was method using an automatic system (SOXTHERM® Unit
calculated as N × 4:75 in the case of insects and N × 6:25 SOX416, Gerhardt, Germany) and diethyl ether (boiling
for diets and faeces. The crude fat was determined from point, 40–60°C) as a solvent. The crude ash content was
Aquaculture Nutrition 5

Table 2: Formulation (g·kg−1), proximate composition (%, wet weight), gross energy (MJ·kg−1, wet weight) amino acid and fatty acid profile
(w %) of the control, and experimental diets used in the digestibility experiment.

Ingredients CONTR diet BSL diet MW diet BBF diet

1
Fish meal 399 280 280 280
Winter wheat2 330 230 230 230
Soybean protein concentrate3 130 91 91 91
Corn gluten4 110 77 77 77
Vitamin/mineral premix5 30 21 21 21
Insect meal6 0 300 300 300
Yttrium-oxide7 1 1 1 1
Proximate composition % (mean ± SD)
Dry matter 95:69 ± 0:02 95:30 ± 0:06 96:87 ± 0:06 96:05 ± 0:01
Crude protein∗ 47:13 ± 0:14 50:10 ± 1:00 53:14 ± 0:75 48:96 ± 0:60
Crude fat 5:80 ± 0:12 8:50 ± 0:05 5:90 ± 0:45 12:10 ± 0:79
Crude fiber 1:43 ± 0:07 4:05 ± 0:03 1:70 ± 0:01 3:55 ± 0:17
Crude ash 9:01 ± 0:04 8:33 ± 0:04 8:34 ± 0:04 7:66 ± 0:01
Phosphorus 1:03 ± 0:01 0:89 ± 0:01 0:85 ± 0:02 0:94 ± 0:03
Calcium 1:79 ± 0:01 1:98 ± 0:00 1:35 ± 0:00 1:40 ± 0:07
Gross energy (MJ·kg−1) 18:63 ± 0:06 19:92 ± 0:04 19:23 ± 0:02 20:66 ± 0:04
Acid detergent fiber (ADF) 2:13 ± 0:33 6:11 ± 0:76 10:06 ± 0:47 6:34 ± 0:12
Chitin8 0:13 ± 0:05 4:91 ± 0:54 2:94 ± 0:56 3:06 ± 0:37
Essential amino acid (EAA) (%)
Arginine (ARG) 2.59 2.80 3.01 2.79
Histidine (HIS) 0.97 1.12 1.01 1.28
Isoleucine (ILE) 1.82 2.01 1.83 1.96
Leucine (LEU) 4.01 3.98 4.22 3.91
Lysine (LYS) 4.35 4.61 4.45 5.29
Methionine (MET) 0.89 0.94 0.93 1.01
Threonine (THR) 1.88 2.05 2.02 2.09
Phenylalanine (PHE) 2.21 2.36 2.42 2.82
Tryptophan (TRP) 3.07 3.07 3.64 3.38
Valine (VAL) 2.19 2.70 2.46 2.46
ΣEAA 23.99 25.63 25.99 27.00
ΣAA 42.88 45.49 46.16 47.22
Fatty acids (FA) w % (mean ± SD)
12 : 0 0:08 ± 0:00 20:79 ± 0:25 0:09 ± 0:00 0:29 ± 0:02
14 : 0 4:33 ± 0:02 6:22 ± 0:02 3:87 ± 0:03 2:44 ± 0:00
16 : 0 18:20 ± 0:66 14:91 ± 0:07 18:15 ± 0:03 18:26 ± 0:04
16 : 1n − 9 0:29 ± 0:01 0:31 ± 0:01 0:32 ± 0:01 3:47 ± 0:00
16 : 1n − 7 4:51 ± 0:00 3:81 ± 0:02 4:12 ± 0:03 8:76 ± 0:04
18 : 0 2:83 ± 0:01 2:25 ± 0:03 3:61 ± 0:01 2:79 ± 0:03
18 : 1n − 9 15:54 ± 0:04 15:45 ± 0:10 18:57 ± 0:04 26:35 ± 0:12
18 : 2n − 6 12:04 ± 0:07 12:34 ± 0:12 14:65 ± 0:10 20:59 ± 0:12
18 : 3n − 3 1:67 ± 0:06 1:12 ± 0:01 1:56 ± 0:02 0:96 ± 0:02
20 : 4n − 6 0:48 ± 0:01 0:26 ± 0:00 0:46 ± 0:00 1:29 ± 0:00
20 : 5n − 3 (EPA) 7:08 ± 0:01 3:75 ± 0:05 5:89 ± 0:03 2:07 ± 0:05
22 : 6n − 3 (DHA) 12:43 ± 0:03 6:27 ± 0:12 10:24 ± 0:12 3:33 ± 0:04
6 Aquaculture Nutrition

Table 2: Continued.

Ingredients CONTR diet BSL diet MW diet BBF diet

SFA 26:86 ± 0:03 45:47 ± 0:16 27:24 ± 0:07 24:47 ± 0:04
MUFA 31:34 ± 0:02 25:34 ± 0:16 32:69 ± 0:09 42:63 ± 0:19
PUFA 38:08 ± 0:02 26:51 ± 0:11 36:71 ± 0:01 29:88 ± 0:03
EPA + DHA 19:51 ± 0:02 10:02 ± 0:07 16:14 ± 0:09 5:40 ± 0:10
−1
Total lipid (mg·g ) 37:15 ± 0:80 47:79 ± 0:33 30:57 ± 0:07 90:10 ± 1:03
1
999 LT Fish meal, TripleNine Fish Protein A/S, Esbjerg, Denmark. 2Supplied from a local feed mill, Novi Sad, Serbia. 3Tradkon SPC500-P, Sojaprotein, Bečej,
Serbia. 4Starch Industry, Jabuka DOO, Pančevo, Serbia. 5Ravago Chemicals (Feketić, Serbia). 6BSL: black soldier fly larvae supplied by Agroloop Ltd.; MW:
yellow mealworm from Berg and Schmidt Pte. Ltd., Singapore; BBF: blue bottle fly produced by Csali Hungary Ltd. 7Alfa Aesar, Thermo Fisher (Kandel)
GmbH, Karlsruhe, Germany. 8 Chitin% = ash free ADF% − ADF protein% following the method presented by Marono [44]. ∗ Protein was calculated by
applying a nitrogen to protein conversion factor of Kp = 6:25.

estimated according to the AOAC 942.05 method. Two mate (AQC) reagent. The analysis was performed with AccQ
grams of each sample were weighed, placed in a furnace, UPLC BEH C18 2:1 × 100 mm, 1.7 μm column (Waters),
and heated at 550°C for 4 h. The amount of remaining ash and AccQ Tag Ultra eluents A, B, and water in the gradient
was recorded. The crude fiber content was determined from mode, the flow rate being 0.7 mL/min. The chromatograms
defatted samples [47]. The sample amount was 1.5–2.0 were evaluated at 260 nm, using amino acid standards. Acid
grams, and the digestion procedure was carried out using hydrolysis was carried out for amino acid analysis. Twenty-
0.13 M H2SO4 and 0.313 M NaOH in a GERHARDT five milligrams of the samples were hydrolyzed by 6 N HCl
Fibretherm FT12 apparatus (Königswinter, Germany). The containing 1% of phenol in a Milestone Ethos One Micro-
acid detergent fiber (ADF) was determined with the same wave digestion system. Hydrolysates were completed to
equipment by using ADF solution prepared from N-cetyl- 5 mL by 1 M borate buffer (pH 8.51).
trimethyl-ammonium bromide dissolved in 0.5 M H2SO4 Yttrium, calcium, and phosphorus contents were ana-
(100 g/5 L) and a few drops of antifoaming agent. The chitin lyzed by the ICP method. The digestion of samples was car-
content was determined as the difference between ash-free ried out with mixtures of acids, including nitric acid (R.G.
ADF and protein linked to ADF (ADIP) 65%) and hydrogen peroxide (R.G. 30%). The extraction
(chitin% = ADF% − ADIP%) according to Finke [48] and was realized by using the microwave digestion technique
Marono [44]. The gross energy was determined by a Parr under high pressure and a Milestone Ethos Plus (Sorisole,
Instruments 6400 calorimeter bomb (Moline, Illinois, USA) Italy) microwave apparatus. The concentrations of elements
calibrated with benzoic acid. were measured by Thermo Scientific 6500 ICP-OES (Massa-
The fatty acid composition of different samples was ana- chusetts, USA) equipment.
lyzed by the capillary gas chromatographic method. Lipids
were extracted from the samples with a 2 : 1 mixture of chlo- 2.5. Calculations and Statistical Analyses. The apparent
roform and methanol. The extracts were purified according digestibility coefficients (ADCs) of dry matter, protein, lipid,
to the method by Folch et al. [49]. Aliquots of total lipid fiber, chitin, ash, phosphorus, amino acids, fatty acids, and
samples were trans-esterified using a methanolic solution gross energy for the test ingredients and diets were calcu-
of HCl [50]. Fatty acid methyl esters (FAMEs) were sepa- lated as follows [40, 53].
rated on fused silica capillary columns (DB-225; Agilent)     
in an Agilent (HP) gas chromatograph system (AGILENT Y diet Dfaeces
ADCdiet = 1 – × × 100, ð1Þ
6890N, California, USA) equipped with a flame ionization Y faeces Ddiet
detector (FID) and a mass spectrometer detector (MSD)
(Agilent, B5973N). The FAMEs were identified using where Y diet is the dietary yttrium level, Y faeces is the faeces
authentic primary (Supelco, Bellefonte, NJ, USA) or second- yttrium level, Ddiet is the dietary nutrient level, and Dfaeces
ary (e.g., linseed oil and cod liver oil) standards and by is the faeces nutrient level.
means of the relationship between the logarithms of relative The apparent digestibility coefficients of the test ingredi-
retention times and the carbon number (Cn) of fatty acids. ent (BSL, MW, and BBF) were calculated according to
Fatty acid concentrations were expressed as a weight per- Bureau et al. [53] as follows:
centage of the FA sample, as assessed by the relative
" !#
response factor (RRF) and molar concentration of FAME 0:7 × Dcontr
[51, 52]. Total lipids were calculated by summing the milli- ADCingredient = ADCtest diet + ðADCtest diet – ADCcontr diet Þ x ,
0:3 Dingredient
gram per gram values of the present fatty acids in the
samples. ð2Þ
The amino acid content of samples was analyzed using
the UPLC-DAD method (Waters Acquity UPLC H-Class, where Dcontr is the % nutrient (or kJ g−1) of control diet (dry
Milford, USA) after acid hydrolysis and precolumn derivati- matter basis) and Dingr is the % nutrient (or kJ g−1) of insect
zation with 6-aminoquinolyl-N-hydroxysuccinimidyl carba- ingredient (dry matter basis).
Aquaculture Nutrition 7

All data are presented as means ± SD and subjected to (ADCFA), phosphorus (ADCP), crude ash (ADCA), and
one-way analysis of variance (ANOVA) to determine chitin (ADCCh) of the diets were estimated based on the
whether significant differences occurred among treatments. digestibility trial and sampling of faeces. The results of dif-
If a significant difference was identified, differences among ferent test diets and the control diet are shown in Table 3.
means were compared with Tukey’s post hoc test and two- The ADCDM, ADCPr, and ADCGE of BSL and BBF diets
sample t-test. All statistical analyses, including Pearson cor- did not differ significantly from the control diet, while
relations between dietary nutrient levels in insects and ADC digestibility coefficients for other nutrients, such as ADCF
values, were performed by the SPSS 22 (SPSS Inc., Chicago, and ADCP, significantly differed. The ADC of each nutrient
IL, USA) software package. determined in the MW diet was significantly lower com-
pared to the control diet except for phosphorus and differed
2.6. Shelf-Life Tests of the Experimental Feeds. The peroxida- from the BSL and BBF diets as well. The ADCAs were typi-
tion and microbiological status of the diets were evaluated cally low and ranged between 37.01 and 61.92%, with signif-
during a six-month period of storage. The experimental icant differences in the case of BSL and MW compared to
feeds were stored in the storage room of the fish rearing the control diet. The ADCEAA values ranged between
facility. Samples were collected at appropriate times and 79.34 and 93.65% for the BSL diet, except ILE and THR
stored at −80°C until analysis. The peroxide value (POV) is (73.81% and 73.00%, respectively), and differed significantly
defined as the reactive oxygen content expressed in terms from the control except ARG. The ADCEAA values
of milliequivalents (meq) of free iodine per kilogram of fat. obtained for the MW diet differed from the control diet in
It is determined by titrating iodine liberated from potassium all parameters and from other testing diets as well. Signifi-
iodide with sodium thiosulphate solution. Briefly, 50 g of cantly lower ADCEAAs were calculated for BBF in most of
each feed sample was extracted with petrol ether (30–40°C) the cases compared to the reference diet except HIS and
and evaporated at 40–45°C. The etheric fat solution (approx. LYS. The ADCFAs were the highest among the tested nutri-
0.3 g fat) was vacuum evaporated and mixed with 0.5 g of KI ents in the range of 96.36–99.45%, except for lauric acid in
(powdered) and 10 mL of an acetic acid and chloroform the MW diet (77.81%) and control diet (82.23%). The ADCs
(3 : 2) solvent mixture. Titration was carried out with 0.1 M of lauric acid in the BBF and BSL diets were significantly
Na2S2O3 solution using a 1% starch indicator [47]. higher compared to the control and MW diets. Moreover,
The mesophilic aerobic microbial cell count, the mold, in most of the cases, ADCFAs were significantly higher for
Enterobacteriaceae, E. coli, Salmonella, and Clostridium per- BBF compared to the control diet (except for DHA).
fringens cell numbers were measured to assess the microbial
contamination of feeds. Samples were taken on weeks 1, 4, 8, 3.2. Apparent Digestibility Coefficient of the Ingredients. Fol-
16, and 21 of the storage period. An aliquot of each sample lowing the calculated ADC values of the diets, the ADC of
(1.0 g) was weighed under sterile conditions into 9 mL pep- the test ingredients could be determined using the equations
tone salt solution for the determination of mesophilic aero- presented in Section 2.5. These data are presented in Table 4.
bic microbes and mold or into an enrichment broth in the The ADCPr for insects from the order Diptera (BSL and
case of Enterobacteriaceae, E. coli (EE-Mossel broth), Salmo- BBF) ranged between 76.04% and 83.93%, while 49.28%
nella (Rappaport Vassiliadis (R.V.S.) broth), and Clostridium was found for MW (order Coleoptera). Similarly, the ADCFs
(DRCM broth). The samples were vortexed intensively for were 93.90% and 96.41% for these species, respectively, but
30 s and allowed to dissolve, and from the salt solution, serial only 61.86% was obtained for MW. The same tendency
decimal dilutions were prepared and 0.1 mL of each dilution was found for some other parameters, with a much lower
was spread onto dishes containing Dichloran Rose-Bengal value for MW. The availability of P was remarkable in the
Chloramphenicol (DRBC) agar for mold and Plate Count Diptera meals (ADCP between 80.61% and 94.16%), while
Agar (PCA) for mesophilic aerobic microbes and incubated for MW, the ADCP was about 64.16%. As for gross energy,
at 25°C for 2–3 days. The enrichment broths were incubated ADCGEs for BSL and BBF were significantly (p < 0:001)
at 35°C for 24–48 hours. For the identification of Enterobac- higher than those of MW. The chitin digestibility was rela-
teriaceae, E. coli, Salmonella, and Clostridium Violet Red Bile tively high for BSL (96.05%) and significantly differed from
(VRB) agar, FluoroBio® VRBL agar, Harlequin™ Salmonella other meals. In respect of the digestibility of several micro-
ABC agar, and Tryptose Sulfite Cycloserine (TSC) selective nutrients of the insect meals, the ADC values are summa-
agar were used, respectively. If any of the investigated bacte- rized in Table 4. The best digestible AAs were ARG, MET,
ria were identified from the enrichment broth, the quantita- and LYS in the range of 79.15–83.91% for BSL, 75.68–
tive analysis was carried out from the original sample by 90.45% for BBF, and 33.84–60.45% for MW. In the case of
using the selective media. All experiments were performed MW, some data obtained had negative digestibility values
in triplicates. after using the mathematical digestibility equations and
these data were excluded. The ADC of fatty acids was gener-
3. Results ally high in all insect meals.
Pearson correlation analyses were performed between
3.1. Apparent Digestibility Coefficient of the Diets. The appar- the nutrient contents of the insect meals and experimental
ent digestibility coefficients of dry matter (ADCDM), crude diets and between the ADC values calculated for the insect
protein (ADCPr), crude fat (ADCF) and gross energy meal ingredients. While the chitin contents of the three
(ADCGE), essential amino acids (ADCEAAs), fatty acids tested insect meals did not differ significantly (p > 0:05),
8 Aquaculture Nutrition

Table 3: Apparent digestibility coefficients (ADC) of the diets prepared by using different insects as test ingredients.

ADC (%) Control diet BSL diet MW diet BBF diet p value
Dry matter 79:18 ± 0:37ab 79:60 ± 0:25a 69:17 ± 0:58c 78:76 ± 1:20b <0.001
ab a c b
Crude protein 83:28 ± 0:55 83:47 ± 0:06 72:07 ± 0:77 81:32 ± 0:76 <0.001
c b d a
Crude fat 89:70 ± 0:24 91:86 ± 0:10 81:24 ± 0:35 94:23 ± 0:32 <0.001
b a c b
Crude ash 55:27 ± 1:65 61:92 ± 0:10 37:01 ± 0:15 55:16 ± 2:25 <0.001
c a c b
Phosphorus 65:07 ± 1:32 73:39 ± 1:57 64:91 ± 1:13 68:74 ± 1:42 <0.001
a a b a
Gross energy 81:82 ± 0:53 82:61 ± 0:09 72:21 ± 0:63 81:59 ± 1:01 <0.001
Essential amino acids (EAA)
Arginine 89:27 ± 0:23a 87:43 ± 0:21a 69:35 ± 0:80c 84:85 ± 0:33b <0.001
a b c a
Histidine 84:96 ± 0:32 79:34 ± 0:35 61:87 ± 0:99 83:24 ± 0:37 <0.001
a c d b
Isoleucine 82:83 ± 0:36 73:81 ± 0:44 49:51 ± 1:31 77:24 ± 0:50 <0.001
a b c b
Leucine 87:89 ± 0:26 79:57 ± 0:34 59:67 ± 1:05 81:13 ± 0:41 <0.001
a b c a
Lysine 90:73 ± 0:20 87:62 ± 0:21 80:18 ± 0:51 90:65 ± 0:21 <0.001
a b c b
Methionine 90:93 ± 0:19 87:86 ± 0:20 80:14 ± 0:52 89:01 ± 0:24 <0.001
a b c b
Threonine 81:06 ± 0:28 73:00 ± 0:45 45:20 ± 1:42 74:95 ± 0:55 <0.001
a c d b
Phenylalanine 91:55 ± 0:18 86:71 ± 0:22 80:22 ± 0:51 89:09 ± 0:24 <0.001
a b c d
Tryptophan 95:17 ± 0:10 93:65 ± 0:11 90:57 ± 0:24 90:37 ± 0:12 <0.001
a b c b
Valine 86:15 ± 0:29 78:38 ± 0:36 54:20 ± 0:36 82:82 ± 2:37 <0.001
Fatty acids (FA)
12 : 0 82:23 ± 2:27b 99:97 ± 0:00a 77:81 ± 2:32b 98:02 ± 0:19a <0.001
b a c a
14 : 0 98:63 ± 0:05 99:22 ± 0:09 98:06 ± 0:14 99:03 ± 0:01 <0.001
c b c a
16 : 0 97:66 ± 0:23 98:29 ± 0:04 97:69 ± 0:03 98:96 ± 0:03 <0.001
16 : 1n − 7 98:12 ± 0:05 b
98:08 ± 0:07 b
98:60 ± 0:02 b 98:78 ± 0:04 a
<0.001
b bc a ac
18 : 0 97:07 ± 0:56 97:64 ± 0:09 96:74 ± 0:05 98:65 ± 0:05 0.009
18 : 1n − 9 97:52 ± 0:75 b
98:48 ± 0:13 ab
98:00 ± 0:01 ab 99:32 ± 0:05 a
0.036
18 : 2n − 6 96:36 ± 0:68 b 98:43 ± 0:13 a
97:85 ± 0:06 a
99:18 ± 0:04 a
0.006
18 : 3n − 3 (LNA) 97:30 ± 0:76 98:28 ± 0:19 98:00 ± 0:20 98:38 ± 0:13 0.169
a a a
22 : 6n − 3 (DHA) 99:36 ± 0:05 99:20 ± 0:01 99:30 ± 0:01 98:90 ± 0:07 b
0.003
a a a b
EPA + DHA 99:45 ± 0:05 99:37 ± 0:00 99:36 ± 0:03 99:20 ± 0:05 0.091
The statistical IDs marked with different letters within the same row translate into a deviation on a significance level of p < 0:05.

the acid detergent fiber (ADF) levels in the MW meal and used in the fish trial was monitored during a six-month stor-
also in its experimental diet (27.69% and 10.6%, respec- age period. Significant differences were found in peroxide
tively) were significantly higher (p < 0:01) compared to the values (POVs) between the control diet and the experimen-
other tested insect meals and diets, respectively (see tal diets after four weeks of storage, where the highest value
Tables 1 and 2). As shown in Table 5, Pearson correlation was detected in the control diet (3:98 ± 0:16 meq/kg fat).
analyses revealed significant negative correlations between Moreover, the POV of the BBF diet was significantly lower
the ADC values calculated for protein, fat, phosphorus, gross than those of the BSL and MW diets (Figure 1). The highest
energy, LYS, MET, and saturated fatty acids as insect meal POV was measured in the control diet after 12 and 16 weeks
ingredients and between the ADF levels of the experimental of storage as well. The BBF-containing feed showed the low-
diets and in some cases also between the ADF levels of insect est oxidation during storage, having only 0:66 ± 0:06 meq/kg
meals (for ADCLYS, ADCMET, and ADC18 : 0). fat as the highest value.
During the microbiological evaluation of the feeds, the
3.3. Shelf-Life Tests of the Diets. The microbiological and total number of mesophilic aerobic bacteria was examined
peroxidation status of the insect meal supplemented feeds by repeated sampling for 21 weeks (Figure 2). The average
Aquaculture Nutrition 9

Table 4: Apparent digestibility coefficients of nutrients, gross energy, and chitin of the tested insect meals.

ADC (%) BSL MW BBF p value

Dry matter 80:59 ± 0:83a 44:86 ± 1:93c 77:76 ± 4:02b <0.001
a c b
Crude protein 83:93 ± 0:19 49:28 ± 2:39 76:04 ± 2:82 <0.001
b c a
Crude fat 93:90 ± 0:19 61:86 ± 1:51 96:41 ± 0:48 <0.001
a b
Crude ash 80:66 ± 0:40 N/A 54:66 ± 9:67 <0.001
a c b
Phosphorus 94:16 ± 5:49 64:16 ± 6:44 80:61 ± 6:02 <0.001
a b a
Gross energy 84:10 ± 0:26 52:05 ± 1:96 81:20 ± 2:71 <0.001
a b b
Chitin 96:05 ± 0:37 72:84 ± 7:57 68:18 ± 5:69 <0.001
Essential amino acids (EAA)
Arginine 83:91 ± 0:61a 33:84 ± 2:21c 75:68 ± 1:02b <0.001
b a
Histidine 69:80 ± 0:93 N/A 81:10 ± 0:83 <0.001
b a
Isoleucine 60:61 ± 1:08 N/A 66:66 ± 1:45 <0.001
Leucine 63:39 ± 1:01 N/A 63:92 ± 1:47 <0.511
b c a
Lysine 79:15 ± 0:77 50:85 ± 0:56 90:45 ± 0:70 <0.001
b c a
Methionine 82:56 ± 0:56 60:45 ± 1:46 86:16 ± 0:60 <0.001
b a
Threonine 59:85 ± 1:20 N/A 64:55 ± 1:02 <0.001
b c a
Phenylalanine 78:48 ± 0:60 54:58 ± 1:68 85:87 ± 0:55 <0.001
b a
Tryptophan 74:03 ± 1:49 N/A 77:53 ± 2:76 <0.001
b a
Valine 67:89 ± 0:85 N/A 69:30 ± 1:18 <0.031
Fatty acids (FA)
10 : 0 99:26 ± 0:22a 62:86 ± 2:77b N/A <0.001
b c a
12 : 0 100:01 ± 0:01 70:61 ± 6:22 102:51 ± 1:82 <0.001
a b a
14 : 0 99:64 ± 0:11 84:66 ± 1:28 99:59 ± 0:06 <0.001
a b a
16 : 0 99:61 ± 0:17 97:85 ± 0:18 99:50 ± 0:03 <0.001
b c a
18 : 0 98:91 ± 0:09 96:07 ± 0:17 99:35 ± 0:10 <0.001
18 : 1n − 9 99:75 ± 0:28 99:16 ± 0:07 99:70 ± 0:06 <0.068
18 : 1n − 7 99:78 ± 3:64b 102:47 ± 0:32a 99:43 ± 0:07b <0.001
18 : 2n − 6 101:09 ± 0:32a 101:29 ± 0:37a 99:75 ± 0:05b <0.001
18 : 3n − 3 102:11 ± 1:00 a
103:18 ± 1:13 a
99:54 ± 0:33b <0.001
22 : 6n − 3 N/A 90:16 ± 0:22 N/A —
b a
EPA + DHA N/A 84:94 ± 3:07 89:08 ± 1:41 <0.001
N/A: not applicable. N/A values assigned when nutrient levels in the ingredient were traced, resulting in a negative digestibility value using the mathematical
digestibility equation. The statistical IDs marked with different letters translate into a significant difference at the level of p < 0:05.

Table 5: Pearson correlation coefficients between ADCs of insect meal ingredients and ADF levels in the tested insect meals and diets.

ADCPr ADCF ADCP ADCGE ADCLYS ADCMET ADC12:0 ADC16:0 ADC18:0

ADF in IMa −0.575 −0.806 −0.274 −0.686 −0.915b −0.845b −0.804 −0.739 −0.847b
ADF in diet −0.964c −0.979c −0.84b −0.983c −0.931c −0.971c −0.976c −0.969c −0.945c
IM: insect meal. bCorrelations significant at the p < 0:05 level. cCorrelations significant at the p < 0:01 level.
a

colony forming unit (CFU) of the feeds was initially 102–103, orders of magnitude of more microorganisms than the other
except for the BSL feed, which was outstanding in terms of samples. Nevertheless, there was a significant difference
the number of mesophilic aerobic bacteria, containing 2–3 between the samples (p < 0:05) except for MW and CONTR.
10 Aquaculture Nutrition

4.5
a
4.0

3.5

Peroxide value (meq/kg fat)

3.0

2.5
a
b
2.0
a
a
1.5 b a a
1.0 a a
b b b
c a
0.5 b b b b
b b c
b c b c
0.0 b b
0 4 8 12 16 20 24
Number of the weeks

Contr BBF
BSL MW

Figure 1: The peroxide value of the diets (meq/kg fat) during a 6-month storage period. The statistical IDs marked with lowercase letters
translate into a difference between treatments at the same time at the significance level of p < 0:05.

1.E + 07

1.E + 06

1.E + 05

1.E + 04
CFU (g)

1.E + 03

1.E + 02

1.E + 01

1.E + 00
0 5 10 15 20 25
Number of weeks
Contr BBF
BSL MW

Figure 2: Mesophilic aerobic microbial cell count changes over time.

During the 21-week storage period, the total mesophilic aer- experiment. Based on these results, it can be concluded that
obic bacterial cell counts did not change much from a the number of molds did not change during the study and
microbiological point of view, as only one order of magni- they were in the undetectable range or just reached the 102
tude increase or decrease was observed, but for BBF and mold/g level, while members of Enterobacteriaceae appeared
BSL, the cell numbers at the end of the storage were statisti- in BBF feed in small numbers. Salmonella spp. (<25 CFU/g),
cally significantly higher (p < 0:05) compared to the first E. coli, and Clostridium perfringens were not found (<
week. The cell numbers in the MW and CONTR samples 102 CFU/g) in the samples during the storage period.
did not change significantly during the storage, although a
slight decrease was observed in the MW sample (Table 6).
In a more detailed examination, the number of molds 4. Discussion
and the Enterobacteriaceae, Salmonella spp., E. coli, and 4.1. Digestibility of the Insect Meals. The digestibility of the
Clostridium perfringens appearance was investigated from feed ingredients and the availability of nutrients are the most
samples taken at the beginning and at the end of the storage important factors in fish nutrition. In the current study, the
Aquaculture Nutrition 11

Table 6: Microbiological evaluation of the diets during the storage period (CFU·g−1 feed).

Feeds Mesophilic aerobic microbes Enterobacteriaceae

Tolerance threshold 106 CFU·g−1 103 CFU·g−1
Week 1st 21st 1st 21st
MW 700 ± 480cC 50 ± 33cC 0 0
bB bA
BBF 7600 ± 4400 21000 ± 11000 <100 <100
BSL 140000 ± 410000aB 1100000 ± 300000aA 0 0
CONTR 100 ± 52cC 550 ± 220cC 0 0
Tolerance threshold according to the 65/2012. (VII. 4.) Hungarian Ministry of Rural Development regulation. Different lowercase letters within a column
indicate a significant difference between the samples according to the two-sample t-test (p < 0:05). Different uppercase letters within a row indicate a
significant difference between the storage time within the same sample according to the two-sample t-test (p < 0:05).

ADCs of three possible insect protein sources were assessed the BSL was still highly digestible for Atlantic salmon. The
in a digestibility trial for African catfish juveniles. ADC of arginine was the highest among the EAAs in our
BSL is one of the most frequently studied insects in fish study, demonstrating its high bioavailability in BSL meal.
nutrition. In the current study, the ADCs of dry matter, This observation is in line with results presented for Atlantic
crude protein, and gross energy in the BSL diet were not sig- salmon [61]. Higher ADCAA was reported for the BSL
nificantly different from the reference control feed, while ingredient in rainbow trout [59] or channel catfish (Ictalurus
others such as ADCF and ADCP were significantly higher punctatus) compared to our results obtained for African cat-
compared to the control (Table 3). The ADCPr of the test fish. However, investigated insects are still more digestible
BSL diet (83.47%) in our study was lower than those than several plants such as maize in the case of striped cat-
reported for BSL meal in European sea bass (Dicentrarchus fish (Pangasianodon hypophthalmus) [62] and sunflower
labrax) (91–93%) ([54], rainbow trout (Oncorhynchus meal in the case of African catfish [43]. Regarding the
mykiss) (87–91%) [55], and Atlantic salmon (90%) [26], ADCFAs, Belghit et al. [15, 61] demonstrated highly avail-
but it was similar to turbot (Psetta maximus) (81.1%) [56] able digestible FA in the BSL-based diets for Atlantic
and close to the 86% found for Siberian sturgeon (Acipenser salmon. In our study, the ADCFAs in the test diet in most
baerii) [5]. African catfish also showed similar digestibility of the cases were significantly higher compared to the refer-
regarding crude protein (81.2%), lipid (89.8%), and dry mat- ence control diet, indicating that the BSL contains well
ter (74%) [57] when cricket meal (Gryllus bimaculatus) was digestible FAs. The level of Lc-PUFA was below the limit
fed. In terms of ADC of the ingredients, the protein digest- of detection in BSL meal, making the calculation of ADC
ibility of the BSL is in line with data presented for hybrid meaningless.
grouper (81–88%) [25], higher than for turbot (63.1%) [56] All ADC values determined for the MW diet were the
and lower than for African catfish fingerlings (85–91%, lowest among the tested diets in this study and differed sig-
depending on feeding regime) [43], and Atlantic salmon nificantly from the control diet in respect of all investigated
(89%) [26]. macro- and micronutrients (Table 3). Based on our findings,
The lipid ADC was higher than the protein digestibility it seems that the tested MW is less suitable for African cat-
value. Lipids are a preferable energy source to carbohydrates fish juveniles; however, a study by Ng et al. [63] demon-
and are almost completely digestible by fish. The high ADCF strated that MW used as insect meal is a potential
indicates a strong ability of African catfish to utilize the lipid protein source for this fish species. It was found to be
components of insects. Comparable high ADCFs of the diets highly palatable and could replace up to 40% of the fish
and ingredients have been reported for Atlantic salmon, meal component in diets for African catfish without any
rainbow trout, turbot, and hybrid grouper [25, 26, 55, 56]. significant reduction in growth performance and feed effi-
The ADC of gross energy (ADCGE) in the diet with BSL ciency ratio. In the case of meagre (Argyrosomus regius),
inclusion agrees with the results of the abovementioned pub- a limited capacity to utilize MW was found, with a 10%
lications except for rainbow trout where only 60–65% was dietary inclusion already resulting in significant impair-
reported [55]. Considering BSL as ingredient, the ADCGE ment of fish digestive capacity and growth performance
in African catfish was generally higher than that in turbot [64]. There are some studies where ADC data were inves-
(54.5%) [56] or maggot in carp and tilapia (74.9% and tigated. For example, Chemello et al. [65] reported coeffi-
58.1%) [23]. This suggests that BSL is a promising ingredient cients of total tract apparent digestibility (CTTAD) of
in relation to energy utilization in African catfish juveniles. MW supplemented diets for rainbow trout, where ADCPr
The ADCs of amino acids in the diet were comparable to was between 97 and 98%, while for gilthead sea bream
those reported for Atlantic salmon [58], rainbow trout [59], (Sparus aurata), 79–87% was determined by Piccolo et al.
and European seabass [54] with different inclusion levels of [66]. These values are higher compared to our findings
BSL. The ADCAA decreased with BSL inclusion in the diet probably due to the different methodologies applied. Also,
of Atlantic salmon in the study by Belghit et al. [60], but this a higher (93%) ADCPr value was obtained by Rema
reduction did not affect the growth performance of the fish et al. [67] for rainbow trout compared to ours. In terms
or feed conversion ratio, and finally, it was concluded that of MW as ingredient, the ADC values for protein and
12 Aquaculture Nutrition

crude fat (Table 4) were much lower compared to tilapia activity was detected in some fish species, but chitinolytic
(85.4% and 90.6%) reported by Fontes et al. [24]. action seems to be limited or completely absent for most fish
The ADC data calculated for the BBF diet were not sig- [56, 74–76]. Chitinolytic activity was measured in the intes-
nificantly different from the control diet in respect of dry tine and stomach of African catfish juveniles fed on mopane
matter, crude protein, crude ash, and gross energy. At the worm (Imbrasia belina) meal; however, the results showed
same time, a significant increase was found for ADCF and no discernible trend with increased mopane worm inclu-
ADCP. Generally, ADCs of BBF meal were significantly sion [77].
lower than those of BSL, but higher than those of MW. Whole insects contain variable but significant amounts
Compared with other feed ingredients, the ADCPr data indi- of fiber as measured by ADF, although the components that
cates that BBF meal is better digested than plant feedstuffs. make up the ADF fraction have not yet been fully character-
Such ADC data were reported for several catfish species like ized [44]. Finke [48] reported that the fiber content of insects
striped surubim (Pseudoplatystoma reticulatum) [68] or measured as ADF consists chitin with significant amounts of
striped catfish [62] except for soybean meal which has the associated cuticular proteins. The acid detergent fiber (ADF)
highest value among the plant products. Among the tested level of MW diet in our study was high (10.1% as fed) com-
insects, BBF has the highest ADCAA except for arginine pared to the control feed (2.13% as fed, p < 0:01). The ADF
and leucine. Although BSL contained an appropriate level content of MW meal was much higher (27.7% d. m.) than
of lauric acid, a similar level was not detected in BBF even the 7–11% and 7.2% reported by Marono et al. [44] and Pic-
though both belong to the order Diptera. In contrast, the colo et al. [66], respectively.
investigated BBF meal contained the highest Lc-PUFA level Many studies have shown that as ADF increases, digest-
compared to BSL and MW. To the best of our knowledge, ibility and nutrient availability decreases [78]. Crude protein
this is the first report on the ADC of BBF meal in fish and digestibility was negatively correlated (p < 0:05) to the ADF
our ADC data are well comparable to other examined insects content in an in vitro digestibility study of T. molitor and
in fish nutrition. Considering our study, BBF could be a rel- H. illucens insect meals [44]. Pearson correlation analyses
evant protein and oil source for diet formulation. showed that the ADF level of insect meals was associated
The digestibility of nutrients may depend on other com- with a lower in vitro digestibility of organic matter
ponents also present in insects. One such compound is chi- (R = −0:59; p < 0:05) and lower in vitro digestibility of crude
tin, a nondigestible fiber, that is, a polymer of N-acetyl- protein (R = −0:68; p < 0:01) in experiments using crude
glucosamine with β-(1/4) linkages. Chitin is known to inter- enzyme extracts from digestive tracts of meat-type ducks
fere with protein use [69]. Based on the analytical results, the [79]. Similar observations were made in the current study
amount of chitin in the tested insect meals in our study where calculated ADC values negatively correlated with the
agrees with other insects’ data. Piccolo et al. [66] found ADF level of the insect meals and experimental diets
4.6% chitin (as fed) for MW, but 12% (dry matter) is (Table 5), indicating that their ADF fraction could inhibit
reported by Fontes et al. [24] and 13.7% (dry matter) by the digestion process thereby contributing to the limited
Finke [48]. In this study, the estimated range of the chitin MW digestibility. Likewise, high ADF levels in mopane
level in the several insect meals was between 1.16% and worm meal have previously been proposed to reduce insect
13.72%. meal digestibility in a feeding experiment of African catfish
The role of chitin in feed digestion may be influenced by (Clarias gariepinus) juveniles [77].
several factors, considering that chitin has an immunostimu- Another factor that may impair insect protein digestibil-
latory effect to the intestine [13, 70] and chitin was also ity is the release of insects’ proteases and phenoloxidases
shown to stimulate bile acid excretion resulting in an during the grinding of whole insects [72]. Phenoloxidases
increased fecal loss of bile acids [71]. Compared to BSL, are responsible for the formation of crosslinked structures
MW presents a more complex chitin-protein matrix [44] between o-quinone and AA, which may negatively affect
and lower trypsin susceptibility [72]. The chitin-bound protein digestibility and digestive enzyme activities. This
nitrogen in mealworms is about 5–6% of total nitrogen suggests that besides the insects’ fiber content, other insect
[73]. Even though this is only a relatively small amount, it components, especially at the enzymatic level, may also
would still be translated into a slight decrease of available influence the overall insect digestibility [64].
dietary protein. Nevertheless, the chitin levels of the three
insect meals applied in the current study were not signifi- 4.2. Shelf-Life Tests. The intestinal tract of insects harbors
cantly different (p > 0:05) from each other, suggesting that high numbers of microorganisms, which play an important
the chitin level was not the primary factor influencing the role in the insects’ life activity, mainly in the digestion of
markedly lower ADCs obtained for the MW meal. feed [80, 81]. The average total microbial cell counts of
The digestibility of chitin was determined in the current insects are generally high, including total mesophilic aerobes
study, together with other nutrients. The ADCs for chitin (3.6–9.4 logCFU/g), Enterobacteriaceae (4.2–7.8 logCFU/g),
(Table 4) show that African catfish can digest chitin from bacterial endospores or spore-forming bacteria (0.5–
the investigated insect meals in different ratios. Moreover, 5.8 logCFU/g), lactic acid bacteria (LAB) (5.2–9.1 logCFU/
these results are comparable with ADCCh values obtained g), psychrotrophic aerobes (4.5–7.2 logCFU/g), and yeasts
in tilapia [24]. Although the chitinase activity was not mea- and molds (3.4–7.2 logCFU/g) [82]. However, between dis-
sured in our study, many fish species, including carnivorous tinct insect types, there can be a great difference in the size
ones, are assumed to be unable to digest chitin [9]. Chitinase and composition of the microbial community. Moreover,
Aquaculture Nutrition 13

the microbiota of insects is greatly influenced by the feed this type of diet, regarding its origin, production, and post-
supply, rearing process, and practices [80, 81]. De Smet harvest processes.
et al. [83] reported that microorganisms occurring in the
feed can also be present in the microbial community of the Data Availability
insects and their diversity was found to be linked to nutri-
tional complexity. Besides, there is a unique “core” compo- All data generated or analyzed during this study are included
nent of the gut microbiota for every species but it may also in this published article.
vary with location and with feed type [83–85]. After the
postharvest treatments, the processed insects generally show Conflicts of Interest
a lower microbial count than the fresh ones [82]. In a two-
year study where fish feeds of different origin were investi- The authors declare that they have no conflict of interest.
gated, Petreska et al. [32] found high numbers of total bacte-
ria, followed by yeast and molds and E. coli to a lesser extent, Authors’ Contributions
which is similar to our results. The observed differences
Conceptualization was done by ZJS and VB; methodology
between the microbial characteristics of the investigated
was done by ZJS, AN, SV, BJ, and KJ; analysis was done by
samples might be explained by the distinct insect types, geo-
ZZ, TFR, RE, LKÉ, and BL; data evaluation was done by
graphic locations, and different rearing and postharvest pro-
ZJS, TFR, RTK, and RE; writing—original draft prepara-
cesses. During storage, a fraction of the initially present
tion—was done by ZJS, BJ, and AN; writing—review—was
microbial species will become dominant [80]. Vandeweyer
done by BJ, VB, LKÉ, RE, and BL, and editing was done by
et al. [86] have found that after a postharvest heat treatment,
ZJS. All authors have read and agreed to the published ver-
the microbial numbers of crickets remained constant over a
sion of the manuscript.
6-month storage experiment even at ambient temperature,
which is similar to our results.
The insect microbiota is complex and contains a great Acknowledgments
variety of different microorganisms due to the abovemen- The authors are thankful to the technical staff of HAKI for
tioned effects. These and the postharvest processes of feed their support in fish husbandry (Csaba Weber) and labora-
production could play an important role in the distinct tory sample analyses (Nándor Kugyela, Judit Molnár). This
results obtained for feeds [87]. However, it should also be work was supported by the European Regional and Develop-
mentioned that in the MW feed, the total microbial colony ment Fund and the Government of Hungary through the
forming units were by one order of magnitude lower com- project TKP2020-NKA-24 and by the RRF-2.3.1-21-2022-
pared to the control feed. 00007 NKFIH project of the National Research, Develop-
The oxidation of fat in the experimental diets was low ment and Innovation Office (NRDI Office), Hungary.
during the storage period compared to rancid oils [88],
despite that the fat content of the diets was different from
each other. None of these results indicated excessive fat oxi-
References
dation and deterioration of the diets after a six-month [1] J. A. Cortes Ortiz, A. T. Ruiz, J. A. Morales-Ramos et al.M. G.
period of storage. Rojas et al., “Insect mass production technologies-chapter 6,”
in Insects as Sustainable Food Ingredients, A. T. Dossey and J.
5. Conclusions A. Morales-Ramos, Eds., pp. 153–201, Academic Press, 2016.
[2] K. B. Barragan-Fonseca, M. Dicke, and J. J. A. van Loon,
The apparent digestibility coefficients determined for the “Nutritional value of the black soldier fly (Hermetia illucensL.)
BSL meal in African catfish hybrid juveniles agreed with and its suitability as animal feed – a review,” Journal of Insects
those reported earlier for other fish species. Based on our as Food and Feed, vol. 3, no. 2, pp. 105–120, 2017.
digestibility experiments, the BBF meal also seems to be well [3] P. Ferrer Llagostera, Z. Kallas, L. Reig, and D. Amores de Gea,
digestible for African catfish, similarly to BSL. However, sig- “The use of insect meal as a sustainable feeding alternative in
nificantly lower digestibility coefficients were obtained for aquaculture: current situation, Spanish consumers’ percep-
the MW meal in the current study. An interaction effect tions and willingness to pay,” Journal of Cleaner Production,
vol. 229, pp. 10–21, 2019.
between the digestibility of various insect meal ingredients
and between the ADF levels was indicated by correlation [4] S. N. Musyoka, D. M. Liti, E. Ogello, and H. Waidbacher, “Uti-
lization of the earthworm, Eisenia fetida (Savigny, 1826) as an
analyses. Overall, our data suggest that replacing fish meal
alternative protein source in fish feeds processing: a review,”
with up to 30% BSL or BBF meal in the African catfish diet Aquaculture Research, vol. 50, no. 9, pp. 2301–2315, 2019.
would not cause difficulties in the digestibility and utilization [5] C. Caimi, M. Renna, C. Lussiana et al., “First insights on black
of nutrients. On the other hand, the tested MW meal proved soldier fly (Hermetia illucens L.) larvae meal dietary adminis-
less suitable for African catfish, partly due to its high ADF tration in Siberian sturgeon (Acipenser baerii Brandt) juve-
content and possibly also due to certain enzymes that may niles,” Aquaculture, vol. 515, p. 734539, 2020.
occur among its ingredients. From the feed safety aspects, [6] L. Gasco, G. Acuti, P. Bani et al., “Insect and fish by-products
the BSL containing diet showed a potential for increasing as sustainable alternatives to conventional animal proteins in
numbers of mesophilic aerobic bacteria during storage that animal nutrition,” Italian Journal of Animal Science, vol. 19,
would require further microbiological characterization of no. 1, pp. 360–372, 2020.
14 Aquaculture Nutrition

[7] International Platform of Insects for Food and Feed parameters,” Journal of the World Aquaculture Society,
(IPIFF)https://ipiff.org/wp-content/uploads/2019/12/ vol. 51, no. 4, pp. 1024–1033, 2020.
2019IPIFF_VisionPaper_updated.pdf. [22] L. Gasco, I. Biasato, S. Dabbou, A. Schiavone, and F. Gai, “Ani-
[8] F. G. Barroso, C. de Haro, M. J. Sánchez-Muros, E. Venegas, mals fed insect-based diets: state-of-the-art on digestibility,
A. Martínez-Sánchez, and C. Pérez-Bañón, “The potential of performance and product quality,” Animals, vol. 9, no. 4,
various insect species for use as food for fish,” Aquaculture, p. 170, 2019.
vol. 422-423, pp. 193–201, 2014. [23] J. O. Ogunji, T. Pagel, C. Schulz, and W. Kloas, “Apparent
[9] M. Henry, L. Gasco, G. Piccolo, and E. Fountoulaki, “Review digestibility coefficient of housefly maggot meal (magmeal)
on the use of insects in the diet of farmed fish: past and future,” for Nile tilapia (Oreochromis niloticus L.) and carp (Cyprinus
Animal Feed Science and Technology, vol. 203, no. 1, pp. 1–22, carpio),” Asian Fisheries Science, vol. 22, pp. 1095–1105, 2009.
2015. [24] T. V. Fontes, K. R. B. . de Oliveira, I. L. Gomes Almeida et al.,
[10] T. Spranghers, M. Ottoboni, C. Klootwijk et al., “Nutritional “Digestibility of insect meals for Nile tilapia fingerlings,” Ani-
composition of black soldier fly (Hermetia illucens) prepupae mals, vol. 9, no. 4, p. 181, 2019.
reared on different organic waste substrates,” Journal of the [25] N. F. N. Mohamad-Zulkifli, A. S. K. Yong, G. Kawamura et al.,
Science of Food and Agriculture, vol. 97, no. 8, pp. 2594– “Apparent digestibility coefficient of black soldier fly (Herme-
2600, 2017. tia illucens) larvae in formulated diets for hybrid grouper (Epi-
[11] M. A. Esteban, A. Cuesta, J. Ortuño, and J. Meseguer, “Immu- nephelus fuscoguttatus ♀ x epinephelus lanceolatus ♂),” AACL
nomodulatory effects of dietary intake of chitin on gilthead Bioflux, vol. 12, no. 2, pp. 513–522, 2019.
seabream (Sparus aurata L.) innate immune system,” Fish & [26] H. J. Fisher, S. A. Collins, C. Hanson, B. Mason, S. M.
Shellfish Immunology, vol. 11, no. 4, pp. 303–315, 2001. Colombo, and D. M. Anderson, “Black soldier fly larvae meal
[12] M. A. Henry, F. Gai, P. Enes, A. Peréz-Jiménez, and L. Gasco, as a protein source in low fish meal diets for Atlantic salmon
“Effect of partial dietary replacement of fishmeal by yellow (Salmo salar),” Aquaculture, vol. 521, p. 734978, 2020.
mealworm (Tenebrio molitor) larvae meal on the innate [27] International Platform of Insects for Food and Feed
immune response and intestinal antioxidant enzymes of rain- (IPIFF)https://ipiff.org/wp-content/uploads/2021/09/Draft-
bow trout (Oncorhynchus mykiss),” Fish & Shellfish Immunol- IPIFF-position-paper-EU-stds-for-whole-insects-as-feed-for-
ogy, vol. 83, pp. 308–313, 2018. food-producing-animals-.pdf.
[13] R. Kumar, N. Kaur, and D. Kamilya, “Chitin modulates immu- [28] A. Bordiean, M. Krzyżaniak, M. J. Stolarski, S. Czachorowski,
nity and resistance of Labeo rohita (Hamilton, 1822) against and D. Peni, “Will yellow mealworm become a source of safe
gill monogeneans,” Aquaculture, vol. 498, pp. 522–527, 2019. proteins for Europe?,” Agriculture, vol. 10, no. 6, p. 233, 2020.
[14] S. Nogales-Mérida, P. Gobbi, D. Józefiak et al., “Insect meals in [29] A. Bordiean, M. Krzyżaniak, M. J. Stolarski, and D. Peni,
fish nutrition,” Reviews in Aquaculture, vol. 11, no. 4, “Growth potential of yellow mealworm reared on industrial
pp. 1080–1103, 2019. residues,” Agriculture, vol. 10, no. 12, p. 599, 2020.
[15] I. Belghit, R. Waagbø, E. J. Lock, and N. S. Liland, “Insect- [30] D. G. A. B. Oonincx, S. Van Broekhoven, A. Van Huis, and J. J.
based diets high in lauric acid reduce liver lipids in freshwater A. Van Loon, “Feed conversion, survival and development,
Atlantic salmon,” Aquaculture Nutrition, vol. 25, no. 2, and composition of four insect species on diets composed of
pp. 343–357, 2019. food by-products,” PLoS One, vol. 10, no. 12, 2015.
[16] E. A. Fasakin, A. M. Balogun, and O. O. Ajayi, “Evaluation of [31] S. van Broekhoven, D. G. A. B. Oonincx, A. Van Huis, and J. J.
full-fat and defatted maggot meals in the feeding of clariid cat- A. Van Loon, “Growth performance and feed conversion effi-
fish Clarias gariepinus fingerlings,” Aquaculture Research, ciency of three edible mealworm species (Coleoptera: Teneb-
vol. 34, no. 9, pp. 733–738, 2003. rionidae) on diets composed of organic by-products,” Journal
of Insect Physiology, vol. 73, pp. 1–10, 2015.
[17] A. O. Aniebo, E. S. Erondu, and O. J. Owen, “Replacement of
fish meal with maggot meal in African catfish (Clarias gariepi- [32] M. Petreska, J. Ziberoski, and M. Zekiri, “Fish feed microbio-
nus) diets,” Revista Cientifica UDO Agricola, vol. 9, no. 3, logical status,” Journal of Hygienic Engineering and Design,
pp. 666–671, 2009. vol. 4, pp. 16–19, 2013.
[18] W. O. Alegbeleye, S. O. Obasa, O. O. Olude, K. Otubu, and [33] J. P. F. D’Mello, “Microbiology of animal feeds,” in FAO ani-
W. Jimoh, “Preliminary evaluation of the nutritive value of mal production and health 160, assessing quality and safety of
the variegated grasshopper (Zonocerus variegatus L.) for Afri- animal feeds, FAO, Rome, 2004.
can catfish Clarias gariepinus (Burchell. 1822) fingerlings,” [34] H. C. Klunder, J. Wolkers-Rooijackers, J. M. Korpeal, and M. J.
Aquaculture Research, vol. 43, no. 3, pp. 412–420, 2012. R. Nout, “Microbiological aspects of processing and storage of
edible insects,” Food Control, vol. 26, no. 2, pp. 628–631, 2012.
[19] I. G. Olaleye, “Effects of grasshopper meal in the diet of Clarias
Gariepinus fingerlings,” Journal of Aquaculture Research & [35] O. Schlüter, B. Rumpold, T. Holzhauser et al., “Safety aspects
Development, vol. 6, no. 4, pp. 6–8, 2015. of the production of foods and food ingredients from insects,”
Molecular Nutrition & Food Research, vol. 61, no. 6, 2017.
[20] M. P. M. Anvo, B. R. D. Aboua, I. Compaoré et al., “Fish meal
replacement by Cirina butyrospermi caterpillar’s meal in prac- [36] W. C. Hazeleger, N. M. Bolder, R. R. Beumer, and W. F. Jacobs
tical diets for Clarias gariepinus fingerlings,” Aquaculture Reitsma, “Darkling beetles (Alphitobius diaperinus) and their
Research, vol. 48, no. 10, pp. 5243–5250, 2017. larvae as potential vectors for the transfer of Campylobacter
jejuniand Salmonella enterica Serovar Paratyphi B variant Java
[21] A. A. Adeoye, Y. Akegbejo-Samsons, F. J. Fawole, and S. J.
between successive broiler flocks,” Applied and Environmental
Davies, “Preliminary assessment of black soldier fly (Hermetia
Microbiology, vol. 74, no. 22, pp. 6887–6891, 2008.
illucens) larval meal in the diet of African catfish (Clarias gar-
iepinus): impact on growth, body index, and hematological [37] R. H. Janssen, J. P. Vincken, L. A. M. Van Den Broek,
V. Fogliano, and C. M. M. Lakemond, “Nitrogen-to-protein
Aquaculture Nutrition 15

conversion factors for three edible insects: Tenebrio molitor, of fatty acids and esters,” Journal of Chromatography, vol. 16,
Alphitobius diaperinus, and Hermetia illucens,” Journal of pp. 298–305, 1964.
Agricultural and Food Chemistry, vol. 65, no. 11, pp. 2275– [53] D. P. Bureau, A. M. Harris, and C. Y. Cho, “Apparent digest-
2278, 2017. ibility of rendered animal protein ingredients for rainbow
[38] National Research Council (NRC), Nutrient Requirements of trout (Oncorhynchus mykiss),” Aquaculture, vol. 180, no. 3-4,
Fish and Shrimp, The National Academic Press, Washington, pp. 345–358, 1999.
DC, 2011. [54] R. Magalhães, A. Sánchez-López, R. S. Leal, S. Martínez-Llo-
[39] “Feedipedia - Animal Feed Resources Information System, rens, A. Oliva-Teles, and H. Peres, “Black soldier fly (Hermetia
INRAE CIRAD AFZ and FAO © 2012-2022,” http:// illucens) pre-pupae meal as a fish meal replacement in diets for
feedipedia.org. European seabass (Dicentrarchus labrax),” Aquaculture,
[40] C. Y. Cho, S. J. Slinger, and H. S. Bayley, “Bioenergetics of sal- vol. 476, no. March, pp. 79–85, 2017.
monid fishes: energy intake, expenditure and productivity,” [55] M. Renna, A. Schiavone, F. Gai et al., “Evaluation of the suit-
Comparative Biochemistry and Physiology, vol. 73, no. 1, ability of a partially defatted black soldier fly (Hermetia illu-
pp. 25–41, 1982. cens L.) larvae meal as ingredient for rainbow trout
[41] N. Révész, S. Kumar, A. S. Bogevik et al., “Effect of temperature (Oncorhynchus mykiss Walbaum) diets,” Journal of Animal
on digestibility, growth performance and nutrient utilization Science and Biotechnology, vol. 8, no. 1, pp. 57–70, 2017.
of corn distiller’s dried grains with soluble (DDGS) in com- [56] S. Kroeckel, A. G. E. Harjes, I. Roth et al., “When a turbot
mon carp juveniles,” Aquaculture Research, vol. 51, no. 2, catches a fly: evaluation of a pre-pupae meal of the black sol-
pp. 828–835, 2020. dier fly (Hermetia illucens) as fish meal substitute - growth per-
[42] Z. J. Sándor, N. Révész, K. K. Lefler, R. Čolović, V. Banjac, and formance and chitin degradation in juvenile turbot (Psetta
S. Kumar, “Potential of corn distiller’s dried grains with solu- maxima),” Aquaculture, vol. 364-365, pp. 345–352, 2012.
bles (DDGS) in the diet of European catfish (Silurus glanis),” [57] N. M. Taufek, H. Muin, A. A. Raji, S. A. Razak, H. M. Yusof,
Aquaculture Reports, vol. 20, p. 100653, 2021. and Z. Alias, “Apparent digestibility coefficients and amino
[43] F. E. Elesho, S. Kröckel, D. A. H. Sutter et al., “Effect of feeding acid availability of cricket meal, Gryllus bimaculatus, and fish-
level on the digestibility of alternative protein-rich ingredients meal in African catfish, Clarias gariepinus, diet,” Journal of the
for African catfish (Clarias gariepinus),” Aquaculture, vol. 544, World Aquaculture Society, vol. 47, no. 6, pp. 798–805, 2016.
p. 737108, 2021. [58] E. R. Lock, T. Arsiwalla, and R. Waagbø, “Insect larvae meal as
[44] S. Marono, G. Piccolo, R. Loponte et al., “In vitro crude protein an alternative source of nutrients in the diet of Atlantic salmon
digestibility of Tenebrio molitor and Hermetia illucens insect (Salmo salar) postsmolt,” Aquaculture Nutrition, vol. 22, no. 6,
meals and its correlation with chemical composition traits,” pp. 1202–1213, 2016.
Italian Journal of Animal Science, vol. 14, no. 3, pp. 338–343, [59] A. Dumas, T. Raggi, J. Barkhouse, E. Lewis, and E. Weltzien,
2015. “The oil fraction and partially defatted meal of black soldier
[45] E. Austreng, “Digestibility determination in fish using chromic fly larvae (Hermetia illucens) affect differently growth perfor-
oxide marking and analysis of contents from different seg- mance, feed efficiency, nutrient deposition, blood glucose
ments of the gastrointestinal tract,” Aquaculture, vol. 13, and lipid digestibility of rainbow trout (Oncorhynchus
no. 3, pp. 265–272, 1978. mykiss),” Aquaculture, vol. 492, pp. 24–34, 2018.
[46] K. Matuk and T. Gulyás, “New possibilities in fish anaesthesia [60] I. Belghit, N. S. Liland, R. Waagbø et al., “Potential of insect-
process [A halak altatásának újabb lehetőségei],” Halászat, based diets for Atlantic salmon (Salmo salar),” Aquaculture,
vol. 33, pp. 11–13, 1987. vol. 491, pp. 72–81, 2018.
[47] AOAC, 2000, Official methods of analysis. 17th edition, the [61] I. Belghit, N. S. Liland, P. Gjesdal et al., “Black soldier fly larvae
Association of Official Analytical Chemists, Gaithersburg, meal can replace fish meal in diets of sea-water phase Atlantic
MD, USA. AOAC 928.08 – nitrogen in meat; Kjeldhal method, salmon (Salmo salar),” Aquaculture, vol. 503, pp. 609–619,
AOAC 945.16 – oil in cereal adjuncts, AOAC 962.09 – fiber 2019.
(crude) in animal feed and pet food; AOAC 942.05 – ash of [62] C. T. Da, T. Lundh, and J. E. Lindberg, “Digestibility of dietary
animal feed; AOAC 965.33 – peroxide value of oil and fats. components and amino acids in plant protein feed ingredients
[48] M. D. Finke, “Estimate of chitin in raw whole insects,” Zoo in striped catfish (Pangasianodon hypophthalmus) finger-
Biology, vol. 26, no. 2, pp. 105–115, 2007. lings,” Aquaculture Nutrition, vol. 19, no. 4, pp. 619–628, 2013.
[49] J. Folch, M. Lees, and G. H. S. Stanley, “A simple method for [63] W. K. Ng, F. L. Liew, L. P. Ang, and K. W. Wong, “Potential of
the isolation and purification of total lipides from animal tis- mealworm (Tenebrio molitor) as an alternative protein source
sues,” The Journal of Biological Chemistry, vol. 226, no. 1, in practical diets for African catfish,Clarias gariepinus,” Aqua-
pp. 497–509, 1957. culture Research, vol. 32, SUPPL. 1, pp. 273–280, 2001.
[50] W. Stoffel, F. Chu, and E. H. Ahrens, “Analysis of long-chain [64] F. Coutinho, C. Castro, I. Guerreiro et al., “Mealworm larvae
fatty acids by gas-liquid chromatography,” Analytical Chemis- meal in diets for meagre juveniles: growth, nutrient digestibil-
try, vol. 31, no. 2, pp. 307-308, 1959. ity and digestive enzymes activity,” Aquaculture, vol. 535,
[51] R. G. Ackman and J. C. Sipos, “Application of specific response p. 736362, 2021.
factors in the gas chromatographic analysis of methyl esters of [65] G. Chemello, M. Renna, C. Caimi et al., “Partially defatted
fatty acids with flame ionization detectors,” Journal of the tenebrio molitor larva meal in diets for grow-out rainbow
American Oil Chemists’ Society, vol. 41, no. 5, pp. 377-378, trout, Oncorhynchus mykiss (Walbaum): effects on growth
1964. performance, diet digestibility and metabolic responses,” Ani-
[52] R. G. Ackman and J. C. Sipos, “Flame ionization detector mals, vol. 10, no. 2, p. 229, 2020.
response for the carbonyl carbon atom in the carboxyl group
16 Aquaculture Nutrition

[66] G. Piccolo, V. Iaconisi, S. Marono et al., “Effect of Tenebrio [80] J. Stoops, S. Crauwels, M. Waud, J. Claes, B. Lievens, and
molitor larvae meal on growth performance, in vivo nutrients L. Van Campenhout, “Microbial community assessment of
digestibility, somatic and marketable indexes of gilthead sea mealworm larvae (Tenebrio molitor) and grasshoppers
bream (Sparus aurata),” Animal Feed Science and Technology, (Locusta migratoria migratorioides) sold for human consump-
vol. 226, pp. 12–20, 2017. tion,” Food Microbiology, vol. 53, no. Part B, pp. 122–127,
[67] P. Rema, S. Saravanan, B. Armenjon, C. Motte, and J. Dias, 2016.
“Graded incorporation of defatted yellow mealworm (Teneb- [81] D. Vandeweyer, S. Crauwels, B. Lievens, and L. Van Campenh-
rio molitor) in rainbow trout (Oncorhynchus mykiss) diet out, “Microbial counts of mealworm larvae (Tenebrio molitor)
improves growth performance and nutrient retention,” Ani- and crickets (Acheta domesticus and Gryllodes sigillatus) from
mals, vol. 9, no. 4, p. 187, 2019. different rearing companies and different production batches,”
[68] T. S. C. Silva, G. V. Moro, T. B. A. Silva, J. K. Dairiki, and J. E. International Journal of Food Microbiology, vol. 242, pp. 13–
P. Cyrino, “Digestibility of feed ingredients for the striped sur- 18, 2017.
ubimPseudoplatystoma reticulatum,” Aquaculture Nutrition, [82] C. Garofalo, V. Milanović, F. Cardinali, L. Aquilanti,
vol. 19, no. 4, pp. 491–498, 2013. F. Clementi, and A. Osimani, “Current knowledge on the
[69] M. Reyes, M. Rodríguez, J. Montes et al., “Nutritional and microbiota of edible insects intended for human consumption:
growth effect of insect meal inclusion on seabass (Dicentrarch- a state-of-the-art review,” Food Research International,
uss labrax) feeds,” Fishes, vol. 5, no. 2, p. 16, 2020. vol. 125, p. 108527, 2019.
[70] S. Errico, A. Spagnoletta, A. Verardi, S. Moliterni, S. Dimatteo, [83] J. De Smet, E. Wynants, P. Cos, and L. Van Campenhout,
and P. Sangiorgio, “Tenebrio molitor as a source of interesting “Microbial community dynamics during rearing of black sol-
natural compounds, their recovery processes, biological effects, dier fly larvae (Hermetia illucens) and impact on exploitation
and safety aspects,” Comprehensive Reviews in Food Science potential,” Applied and Environmental Microbiology, vol. 84,
and Food Safety, vol. 21, no. 1, pp. 148–197, 2022. no. 9, 2018.
[71] S. Meyer, D. K. Gessner, G. Maheshwari et al., “Tenebrio moli- [84] M. Shelomi, M. K. Wu, S. M. Chen, J. J. Huang, and C. G.
torlarvae meal affects the cecal microbiota of growing pigs,” Burke, “Microbes associated with black soldier fly (Diptera:
Animals, vol. 10, no. 7, pp. 1–17, 2020. Stratiomiidae) degradation of food waste,” Environmental
[72] R. H. Janssen, J. P. Vincken, N. J. G. Arts, V. Fogliano, and Entomology, vol. 49, no. 2, pp. 405–411, 2020.
C. M. M. Lakemond, “Effect of endogenous phenoloxidase [85] E. Gorrens, L. Van Moll, L. Frooninckx, J. De Smet, and L. Van
on protein solubility and digestibility after processing of Campenhout, “Isolation and identification of dominant bacte-
Tenebrio molitor, Alphitobius diaperinus and Hermetia illu- ria from black soldier fly larvae (Hermetia illucens) envisaging
cens,” Foodservice Research International, vol. 121, no. 8, practical applications,” Frontiers in Microbiology, vol. 12, 2021.
pp. 684–690, 2019.
[86] D. Vandeweyer, E. Wynants, S. Crauwels et al., “Microbial
[73] D. Barker, M. P. Fitzpatrick, and E. S. Dierenfeld, “Nutrient dynamics during industrial rearing, processing, and storage
composition of selected whole invertebrates,” Zoo Biology, of tropical house crickets (Gryllodes sigillatus) for human con-
vol. 17, no. 2, pp. 123–134, 1998. sumption,” Applied and Environmental Microbiology, vol. 84,
[74] G. J. H. Lindsay, M. J. Walton, J. W. Adron, T. C. Fletcher, no. 12, article e00255, 2018.
C. Y. Cho, and C. B. Cowey, “The growth of rainbow trout
[87] N. T. Grabowski and G. Klein, “Bacteria encountered in raw
(Salmo gairdneri) given diets containing chitin and its rela-
insect, spider, scorpion, and centipede taxa including edible
tionship to chitinolytic enzymes and chitin digestibility,”
species, and their significance from the food hygiene point of
Aquaculture, vol. 37, no. 4, pp. 315–334, 1984.
view,” Trends in Food Science and Technology, vol. 63,
[75] R. Abro, K. Sundell, E. Sandblom et al., “Evaluation of chitino- pp. 80–90, 2017.
lytic activities and membrane integrity in gut tissues of Arctic
[88] M. H. Gordon, “Measuring antioxidant activity,” In Antioxi-
charr (Salvelinus alpinus) fed fish meal and zygomycete bio-
dants in food, pp. 71–84, 2001.
mass,” Biochemistry and Physiology - B Biochemistry and
Molecular Biology, vol. 175, pp. 1–8, 2014.
[76] I. Guerreiro, C. R. Serra, F. Coutinho et al., “Digestive enzyme
activity and nutrient digestibility in meagre (Argyrosomus
regius) fed increasing levels of black soldier fly meal (Hermetia
illucens),” Aquaculture Nutrition, vol. 27, no. 1, pp. 142–152,
2021.
[77] B. Glencross, N. Rutherford, and N. Burne, “The influence of
various starch and non-starch polysaccharides on the digest-
ibility of diets fed to rainbow trout (Oncorhynchus mykiss),”
Aquaculture, vol. 356-357, no. 367, pp. 141–146, 2012.
[78] A. Kovitvadhi, P. Chundang, K. Thongprajukaew,
C. Tirawattanawanich, S. Srikachar, and B. Chotimanothum,
“Potential of insect meals as protein sources for meat-type
ducks based on in vitro digestibility,” Animals, vol. 9, no. 4,
p. 155, 2019.
[79] M. M. Rapatsa and N. A. G. Moyo, “Enzyme activity and his-
tological analysis of Clarias gariepinus fed on Imbrasia belina
meal used for partial replacement of fishmeal,” Fish Physiology
and Biochemistry, vol. 45, no. 4, pp. 1309–1320, 2019.