Characteristics of Lipid Fractions of Larvae of The Black Soldier Fly

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ISSN 16076729, Doklady Biochemistry and Biophysics, 2016, Vol. 468, pp. 209–212. © Pleiades Publishing, Ltd., 2016.

Original Russian Text © N.A. Ushakova, E.S. Brodskii, A.A. Kovalenko, A.I. Bastrakov, A.A. Kozlova, D.S. Pavlov, 2016, published in Doklady Akademii Nauk, 2016, Vol. 468, No. 4,
pp. 462–465.

BIOCHEMISTRY, BIOPHYSICS
AND MOLECULAR BIOLOGY

Characteristics of Lipid Fractions of Larvae


of the Black Soldier Fly Hermetia illucens
N. A. Ushakova, E. S. Brodskii, A. A. Kovalenko, A. I. Bastrakov,
A. A. Kozlova, and Academician D. S. Pavlov
Received February 2, 2016

Abstract—The lipid fraction of larvae of the black soldier fly Hermetia illucens was shown to contain lauric
acid (38.43 wt %) and its esters, azelaic and sebacic acids, and azelaic acid dibutyl ester. The dominant com
pound in the group of identified glycerides was lauric acid monoglyceride (0.70 wt %). Glycerides were also
represented by triglycerides and diglycerides of lauric acid. Sterols were represented primarily by phytosterols
(over 75%), the major of which was alphasitosterol (45%). The identified lipid complex composition is appar
ently determined by the biological characteristics of the fly Hermetia illucens and ensures antibacterial defence
of larvae and stability of lipids at changing ambient temperature.

DOI: 10.1134/S1607672916030145

The black soldier fly (Hermetia illucens) is consid with a DB5ms capillary column (25 m × 0.25 mm ×
ered one of the most promising species for artificial 0.25 µm) and a Finnigan Polaris Q massspectromet
breeding [1]. The main attention is given to the possi ric detector. The sample was injected into the injector
bility of using insect protein in animal feed [2, 3]. of the chromatograph at a temperature of 240°C in the
Among the biomass of black soldier fly larvae, lipids 1/12 flow dividing mode. The sample was heated
account for up to 45% [4], which allows this biological under the following conditions: initial heating at 60°C
object to be considered as a natural source of biologi for 2 min, subsequent heating to 300°C at a rate of
cally active substances of hydrophobic nature. 6°C/min, and incubation at this temperature for
The aim of this work was to study the characteris 6 min. Helium was used as a carrier gas, the flow rate
tics of the composition of the lipid fraction of Herme was 1 mL/min. The interface temperature was 250°C,
tia illucens larvae fed on wheat grain. the temperature of the source of ions in the mass spec
trometer was 220°C. Mass spectra were recorded in
This study was performed with the Hermetia the mass range of 40–550 atomic mass units (amu).
illucens larvae kept in the laboratory of the Severtsov Concentrations were evaluated by the areas of chro
Institute of Ecology and Evolution, Russian Academy matographic peaks on the chromatogram by the total
of Sciences. Larvae were separated from the food sub ion current (TIC) using the internal normalization.
strate when they reached the maximum linear sizes—
length of 20–25 mm, diameter of 6–7 mm, and weight The mass chromatogram of the silylated extract for
of up to 0.3 g. Lipids were extracted from the larvae TIC is shown in Fig. 1.
that were frozen at –25°C and then ground [5] in The extract contained primarily fatty acids (Table 1),
order to maximize the yield of steroids. the major of which was lauric acid (38.4 wt %). Other
An aliquot (1.075 g) of the homogenate obtained dominant components were myristic, palmitic, and
from the biomass of larvae was extracted twice with stearic saturated acids as well as hexadecenoic and
2 mL of dichloromethane in a glass vial with a screw octadecenoic unsaturated acids. Branched acids were
cap. The extract was silylated with N,Obis(trimethyl present in lesser amounts, and polyunsaturated acids
silyl)trifluoroacetamide containing 1% trimethylchlo were almost absent. The extract also contained dibasic
rosilane (BSTFA, Tokyo Chemical Industry Co., Ltd., azelaic and sebacic acids. The minor components
Japan) and analyzed in a Finnigan Trace GC Ultra gas included decanoic (capric) acid, a fragrant compound
chromatograph (Thermo Scientific, United States) with citrus odor, which is found in certain essential oils
(lemon, lime, and anise). This acid determines the
pleasant smell of lipids of the black soldier fly.
Severtsov Institute of Ecology and Evolution, The extract also contained various glycerides
Russian Academy of Sciences, Leninskii pr. 33, Moscow, (Table 2). This group was dominated by the dode
119071 Russia canoic (lauric) acid monoglyceride (0.70 wt %).
email: naushakova@gmail.com A number of mono and diglycerides as well as lauric

209
210 USHAKOVA et al.

Relative intensity, % 25.92


100
95
90
85
80
75
70
65 35.88
60 31.04
55
50
39.59
45
40
35
30
25
35.19
20 40.26
15
10 19.99 24.55
29.77 39.19
5 7.72 8.21 13.26 24.05 27.13 34.89
13.84 16.98 37.42 43.03 43.21 49.01
0
10 15 20 25 30 35 40 45 50
Time, min

Mass chromatogram silylated dichloromethane extract larvae Hermetia illucens.

acid triglyceride were also identified. Mono and dig low temperatures [7]. Since insects are poikilothermic
lycerides included compounds in which acids were animals, their body temperature depends on the ambi
attached both at the terminal and central hydroxyl ent temperature. Therefore, normal functioning of the
group of glycerol as well as glycerides with two differ organism in a wide range of temperatures (both low
ent acid residues. and high) should be ensured in a biological system
with a high fat content. The presence of large amounts
The extract also contained a number of other com of saturated lauric acid provides solid consistency of
pounds (Table 3), including sterols. However, com larvae, which is not subject to rapid oxidation of fat
pounds of this group were also dominated by lauric (melting point of lauric acid is 43.2°C), which allows
acid methyl ester (0.48 wt %). the larvae to survive in rotting vegetable substrates at
The lipid fraction of Hermetia illucens larvae is a temperatures above 40°C. Azelaic acid can provide the
complex set of substances. However, the main lipid protection of the lipid complex of the insect from infec
component of this insect is the lauric acid and its tions, because it can inhibit the reproduction of lipo
esters. Among the identified glycerides, lauric acid philic microorganisms in skin sebaceous glands [8].
monoglyceride, which is known to exhibit a significant A characteristic feature of the composition of ste
biological activity, was present in highest quantities. In rols is the presence of phytosterols, which constitute
animals and humans, lauric acid is converted into more than 75% of all compounds of this class. This
monolaurin, which is an antiviral, antibacterial, and group was dominated by alphasitosterol (45% of total
antiprotozoal glyceride [6]. Lipids of black soldier lar sterols). It can be assumed that the larvae feeding on
vae also contained lauric acid diglycerides and triglyc plant substrate do not transform plant sterols and use
erides. Chemically, diglycerides of lauric acid and these substances as part of their own lipid complex.
other fatty acids are emulsifiers and stabilizers of dis
perse systems. Lipids of Hermetia illucens larvae also The discovered structural organization of the lipid
included azelaic and sebacic acids, whose esters, complex is apparently associated with the biological
including the identified azelaic acid dibutyl ether, may peculiarities of the fly Hermetia illucens, which can
ensure the plasticity of the lipid complex of larvae at feed and store lipids only at the larval stage during the

DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 468 2016


CHARACTERISTICS OF LIPID FRACTIONS OF LARVAE 211

Table 1. The content of carboxylic acids in the extract of Her Table 2. The composition of glycerides in the extract of Her
metia illucens larvae metia illucens larvae
Acid Content, wt % Compound Content, wt %
Hydroxypropanoic 0.22 Dodecanoic acid 2monoglyceride 0.15
Hexanoic 0.56 Dodecanoic acid 1monoglyceride 0.70
Glycerol 0.36 Tetradecanoic acid 1monoglyceride 0.11
Octanoic 0.16 Tetradecanoic acid 2monoglyceride 0.08
Hydroxyheptanoic 0.07 Hexadecenoic acid 1monoglyceride 0.04
Nonanoic 0.13 Hexadecanoic acid 1 monoglyceride 0.07
Decanoic (capric) 1.22 Hexadecenoic acid 2 monoglyceride 0.04
Dodecanoic (lauric), nC12 : 0 38.43 Hexadecanoic acid 2monoglyceride 0.01
Nonadecenoic acid 1monoglyceride 0.01
Tetradecanoic, isoC14 : 0 0.11
9,12,15Octadecatrienoic acid 1monoglyc 0.01
Tetradecenoic, nC14 : 1 0.19 eride
Tetradecanoic (myristic), nC14 : 0 12.33 Octadecanoic acid 1monoglyceride 0.01
Pentadecanoic, isoC15 : 0 0.13 Octadecenoic acid 1monoglyceride 0.02
Pentadecanoic, isoC15 : 0 0.22 Tetradecanoic and dodecanoic acid 1,2dig 0.09
Pentadecanoic, nC15 : 0 0.15 lyceride
Hexadecanoic, isoC16 : 0 0.37 Dodecanoic and tetradecenoic acid 1,3dig 0.004
lyceride
Hexadecenoic, isoC16 : 1 0.10
Dodecanoic acid 1,3diglyceride 0.05
Hexadecenoic, isoC16 : 1 2.95
Dodecanoic and hexadecanoic acid 1,3dig 0.02
Hexadecanoic (palmitic), nC16 : 1 15.71 lycerides
Heptadecanoic, isoC17 : 0 0.04 Dodecanoic acid triglyceride 0.04
Heptadecanoic, isoC17 : 0 0.22 C12 : 0, C12 : 0, and C14 : 0 triglyceride 0.02
Heptadecenoic, nC17 : 0 0.08
Heptadecanoic, nC17 : 0 0.13 Table 3. Other components of the extract of Hermetia illucens
larvae
Octadecadienoic, isoC18 : 2 0.23
Octadecenoic (oleic), isoC18 : 1 8.81 Compound Content, wt %

Octadecenoic, nC18 : 1 0.16 Ethylamine 0.02


Octadecanoic (stearic), nC18 : 0 2.95 Nonanal 0.01
1Methylheptanol 0.16
Eicosanoic, C20 : 0 0.09
2Nonenal 0.01
Octadicarboxylic 0.11
Glycerol 0.07
Nonanedicarboxylic (azelaic) 0.83 Nonanedicarboxylic acid dibutyl ester 0.27
Decanedicarboxylic (sebacic) 0.09 Dodecanoic acid ethyl ester 0.48
1,4Benzenedicarboxylic 0.08 9Hexadecenoic acid ethyl ester 0.04
Ethyl oleate 0.25
3,11Diacetoxypregnan20ol 0.01
development in rotting organic matter [9, 10]. This Cholesterol 0.01
feature determines the necessity to maintain the sta
bility of lipids at elevated ambient temperatures and Unidentified sterol 0.04
ensure their antibacterial protection for providing nor Stigmasterol 0.02
mal life activity of larvae. αSitosterol 0.08
The properties of the lipid fraction of Hermetia Unidentified sterol 0.01
illucens larvae, found in this study, are important for Stigmasta3,5dien7one 0.01
the understanding of insect biology and assessment of
the prospects of using the larval biomass of the black Unidentified sterol 0.01
soldier fly in artificial breeding. Unidentified sterol 0.01

DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 468 2016


212 USHAKOVA et al.

ACKNOWLEDGMENTS 5. Kates, M., Techniques of Lipidology. Isolation, Analysis


and Identification of Lipids, Laboratory Techn. Bio
The work was supported by the program of the chem. Mol. Biol., New York: Elsevier, 1986, vol. 3,
Department of Biological Sciences of the Russian pt. 2, p. 103.
Academy of Sciences “Sustainable Use of Biological 6. Goncharov, A.T. and Khamidullin, T.N., Ptitsa Ptit
Resources of Russia: The Fundamental Bases of Man seprodukty, 2012, no. 3, pp. 30–34.
agement.”
7. chemistry_ru_en.academic.ru
8. Mashkovskii, M.D., Lekarstvennye sredstva (Drugs),
REFERENCES Moscow: Novaya volna, 2014.
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Silvicult. Mendelianae Brunensis, 2013, vol. LXI, no. 1, soldier fly Hermetia illucens Linnaeus (Insecta: Diptera:
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DOKLADY BIOCHEMISTRY AND BIOPHYSICS Vol. 468 2016

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