ENTOLAB-loppuseminaari: hyönteiskasvatuksen edistäminen Etelä-Pohjanmaalla 4.12.18 Seinäjoki

1. Black soldier fly meal in feed for Atlantic salmon
- nutritional qualities and safety considerations -
Erik-Jan Lock, Irene Biancarosa, Ikram Belghit

2. Institute of Marine Research
• One of the largest Marine Research Institutes in Europe (~1000
employees)
• Main activities: research, advisory work and monitoring

4. Institute of Marine Research
• One of the largest Marine Research Institutes in Europe (~1000
employees)
• Main activities: research, advisory work and monitoring
• Leading supplier of knowledge relating to the sustainable management of
the resources in our marine ecosystems and the whole food chain from the
sea to the table

5. 35-45% Crude protein
20-35% Lipid
5-15% Ash

6. Feed ingredients in Norwegian salmon diets
65.4
24.0
9.6
1990
18.4
36.7
19.2
11.2
10.9
2013
100%
Fish meal
Fish oil
Starch
Plant protein
Plant oil
1.0
Microingredients
Past Present Future ?
3.7
Adapted from Ytrestøyl et al. 2015
?
Yeast, bacteria, microalgae
Seaweeds
Animal non-ruminant by-products
Blue mussel
Insects

7. Natural converters
Feed (% d.w.) Insect (% d.w.)
Protein 4-10 35-45
Lipid 2-5 20-35
Carbohydrates 50-80 8-9
Chitin 0 6
Ash 4-15 5-15

8. Insect as feed ingredients
Black soldier fly (BSF) larvae
0
10
20
30
40
50
60
CP Arg His Ile Leu Lys Met Thr Val
Partially defatted BSF larvae
Atlantic salmon requirements (NRC, 2011)
Most of the indispensable amino acids are close to the requirements in Atlantic salmon
Amino acids composition= % of crude protein
Atlantic salmon requirements

9. Nutrients, contaminants, pathogens
Nutrients
Contaminants
Fillet quality
Fish health and welfare
Sustainability (social, economical)
2014 2015 2016-2017 2018

10. Insect as natural feed ingredients for sustainable salmon
?
Evaluation of seaweed as a feed ingredients
for BSF larvae
Belghit I, Liland NS, Waagbø R, Biancarosa I, Pelusio N, Li Y, Krogdahl Å, Lock EJ. 2018. Potential of insect-based diets for
Atlantic salmon (Salmo salar). Aquaculture 491: 72-81

11. Increased EPA (retention around 35%)
Increased iodine
Increased tocopherols
N.S. Liland, I. Biancarosa, P. Araujo, D. Biemans, C.G. Bruckner, R. Waagbø, B.E. Torstensen, E.-J. Lock, Modulation of nutrient
composition of black soldier fly (Hermetia illucens) larvae by feeding seaweed-enriched media, PLOS ONE, 12 (2017)
I. Biancarosa, N.S. Liland, D. Biemans, P. Araujo, C.G. Bruckner, R. Waagbø, B.E. Torstensen, E.-J. Lock, H. Amlund, Uptake of heavy
metals and arsenic in black soldier fly (Hermetia illucens) larvae grown on seaweed-enriched media, J. Sci. Food Agric.
Black soldier fly larvae experimental trial

12. Objective
Allowed limits for complete animal feed

13. Insect as natural feed ingredients for sustainable salmon
?
Evaluation of insect meal (IM) as a feed
ingredients for Atlantic salmon
Belghit I, Liland NS, Waagbø R, Biancarosa I, Pelusio N, Li Y, Krogdahl Å, Lock EJ. 2018. Potential of insect-based diets for
Atlantic salmon (Salmo salar). Aquaculture 491: 72-81

14. Insect meal (IM)BSF larvae
FM/VP
Control diet (IM-0%)
66%IM
/VP
IM-66%
100%IM
/VP
IM-100%
Cargill
Replacement
60:40
Feed ingredients and experimental diets
Belghit et al
IM-33%
33%IM
/VP
Feeding period:16 weeks
WP2
Experiment 2. Grow-out trial

15. IM -0 IM -6 6IM -1 0 0 IM -0 IM -6 6IM -1 0 0 IM -0 IM -6 6IM -1 0 0
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
Grwoth(g)
Main results
• 3 fold increase in body weight
• No significant effects on growth
between groups
• No effects on nutrient digestibility
• Whole body FA composition was
affected by IM dietary inclusion
0 33 66 100 0 33 66 100 0 33 66 100
Belghit et al., submitted
WP2
Experiment 2. Grow-out trial

16. Head Kidney (HK) Isolated leukocytes
+ LPS
+ Poly I:C
+ Control (non treatment)
BSF insect meal (IM)
IM-0%
IM-66%
IM-100%
Gene expression
3rd October ≈2500 g
54 days
7th August ≈1200 g Isolated for 24h and added
treatment for additional 24h
2 fish/ cages = 6 fish/diet
IM -0 IM -6 6IM -1 0 0 IM -0 IM -6 6IM -1 0 0 IM -0 IM -6 6IM -1 0 0
0
1 0 0 0
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0
Grwoth(g)
0 33 66 100 0 33 66 100 0 33 66 100
WP2
Experiment 2. Grow-out trial
 Inflammatory related gene expression
 Eicosanoid related gene expression
 Stress related gene expression

17. Main results
WP2
Experiment 2. Grow-out trial
• Replacement of fishmeal with IM in the diets of Atlantic
salmon had no effect on the transcription of pro-
inflammatory genes in the head kidney cells.
• Fish fed with IM had down regulated transcription of
antioxidant and stress related genes.
IM - 0 IM - 66 IM - 100
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
S O D
¤
z
y
y
IM - 0 IM - 66 IM - 100
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
5 -LO X
z
y y
In vitro bacterial and viral response in head kidney leukocytes of Atlantic
salmon (Salmo salar) fed dietary insects meal

18. Sensory testing
• Softer texture of the raw fillets in fish
fed 100% IM than fish fed 33 % IM
• Higher rancidity odor of baked fillet
from fish fed 100% IM than the
control group
• No differences in color scores of raw
fish
Belghit et al., submitted
WP2
Experiment 2. Grow-out trial

19. Insect as feed ingredients for Atlantic salmon
Christopher Haatuft

20. -> Must comply with the associated legal constraints to
guarantee their safe use in fish feed ingredients
+ Large variety in origin and quality of insect products
Objective
Analytical approaches are urgently required for a proper detection
and identification of insect species-containing feed ingredients
ENTOFÔR project - From waste to resource

21. Insect meal (IM)
- 18 commercial IM samples
- Different companies - Different processing:
whole larvae meal or defatted insect meal
8 Black soldier fly (Hermetia illucens) (BSF)
5 Yellow mealworm (Tenebrio molitor) (YM)
3 Lesser mealworm (Alphitobius diaperinus) (LM)
2 House cricket (Acheta domesticus) (HC)
Materials

22. The protein content of the 18 IM samples
BSF YM LM HC
True protein content was determined as sum of amino acid residue
%oftotalAAAmino acid composition, protein content, and nitrogen-to-protein conversion factors of 21 seaweed species from Norwegian waters,
Biancarosa et al., 2016
0
2 0
4 0
6 0
8 0
1 0 0
0
2 0
4 0
6 0
8 0
1 0 0
Crude protein from overall nitrogen content, using the traditional
nitrogen-to-protein conversion factor of 6.25
BSF YM LM HC
Crudeprotein

23. Principal component analysis and hierchical clustering of the amino acids calculated
from 18 insect meal samples
BSF
HC
LM
YM
His
Glu
Met
Asp
Phe
Lys
Thr
Tau
Arg
Tyr
Iso
Ser
Pro
Ala
Val
Gly
Leu
BSF
YMLM
HC
BSF

24. Peptidomic profiling of different (commercially)
available insect species

25. PeptidesProteins extraction
Peptidomic profiling of different (commercially) available insect species
LC-MS/MS Spectral
Species Protein Peptides
BSF 1
21 53
BSF 2
43 196
BSF 3
21 103
BSF 4
81 275
BSF 5
18 91
BSF 6
58 303
BSF 7
56 156
BSF 8
55 220
YW 9
127 765
YW 10
138 689
YW 11
133 697
YW 12
54 235
YW 13
130 664
LW 14
94 509
LW 15
90 477
LW 16
94 389
HC 17
77 605
HC 18
75 454
Name Common names
Number of entries
(UNIPROT)
Number of proteins
(NCBI)
ACHETA DOMESTICUS House cricket 159 168
HERMETIA ILLUCENS Black soldier fly, BSF 56 348
TENEBRIO MOLITOR Yellow mealworm, beetles 527 607
ALPHITOBIUS
DIAPERINUS
Lesser mealworms,
buffalo worms, litter
beetles
27 63
ZOPHOBAS MORIO or
ZOPHOBAS ATRATUS
or ZOPHOBAS
RUGIPES
Morio worms, super
worm, king worm
19 38

26. PeptidesProteins extraction
Peptidomic profiling of different (commercially) available insect species
LC-MS/MS Spectral
All tandem mass spectra ≈ 4000
Pairwise comparaison CompareMS2
Records the similarity of the best matching spectrum in the corresponding range
Creating a distance matrix
Phylogenetic tree of the samples

27. Hierarchical Clustering of insect meal Proteomes
LM
LM
LM
YM
YM
YM
YM
YM
HC
HC
BSF
BSF
BSF
BSF
BSF
BSF
BSF
BSF
The phylogenetic analysis distinguished and successfully arranged the 18 different IM samples
species in groups of closely related families based on the comparison of 4000 tandem mass spectra+ increase -> spectral library data base

28. Soil
Crop
production
Biowaste
Feed
Animal production
Food
Insect production
Feed
Insect as feed ingredients and food
Insect species have the capacity to efficiently
convert various organic waste into body mass
Next:
Quantification of specific waste products or
insect species in a feed ingredient mixture
ENTOFÔR project - From waste to resource

29. Quantification of specific waste products or insect species in a feed ingredient mixture

30. Developing peptide markers for the quantitative species specific
differentiation of insect meal
By using the gel-free shotgun proteomics approach in combination
with direct spectral comparison we were able to differentiate the
insect meal samples according to the taxonomic classification of the
insect species
Conclusion

31. Thank you for your attention!