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Entomological Research 44 (2014) 58–64

RESEARCH PA P E R

Detection of antimicrobial substances from larvae of the

black soldier fly, Hermetia illucens (Diptera: Stratiomyidae)
Soon-Ik PARK1, Byung Soo CHANG2 and Sung Moon YOE1
1
Department of Biological Sciences, Dankook University, Cheonan, Korea
2
Department of Cosmetology, Hanseo University, Seosan, Korea

Correspondence Abstract
Sung Moon Yoe, Department of Biological
Sciences, Dankook University, Dandae-ro, Maggots have become highly successful in the treatment of non-healing wounds
Dongnam-gu, Cheonan-si, and multidrug-resistant pathogen infections. The main objective of this study was
Choongcheongnamdo, 330-714, Korea. to extract antibacterial substances from larvae of the black soldier fly, Hermetia
Email: smyoe@dankook.ac.kr illucens. To induce immune responses, we septically injured the larvae with a
contaminated needle. Lyophilized H. illucens larvae were homogenized and
Received 3 January 2014;
extracted with acidic methanol. We examined the antifungal and antibacterial
accepted 3 February 2014.
effects of the low molecular weight antimicrobial factors within the larval extract
doi: 10.1111/1748-5967.12050 on the growth of a broad range of microorganisms, including Gram-positive
Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), and
Gram-negative Pseudomonas aeruginosa. Furthermore, we isolated the anti-
MRSA substances from the larval extract using high performance liquid chroma-
tography. These investigations revealed that the larval extract possessed a broad-
spectrum of antibacterial activity, demonstrating that secretions of H. illucens
larvae prove useful in the fight against MRSA and can potentially be a source of
novel antibiotic-like compounds for infection control.

Key words: acidic methanol, black soldier fly (Hermetia illucens), larval extract, MRSA,
novel antibiotics.

(Bexfield et al. 2004). Recently, several studies reporting

Introduction
antimicrobial activity of the hemolymph and maggot extract,
Maggot therapy is highly successful in cleansing infected either in the whole body or in the excreta/secreta, have
and necrotic wounds (Sherman & Hall 2000) and has been provided impetus for the development of alternative antimi-
practiced since the 1930s when William Baer’s observations crobial products into therapeutically valuable anti-infective
confirmed the reduction of bacterial load at the wound site agents (Dang et al. 2006). It is well known that insects have
by the use of maggots (Baer 1931). The main actions of fly a well-developed innate immune system, subdivided into
maggots on wounds may be categorized into three modes: cellular and humoral defense responses, the latter of which
debridement, disinfection and bacterial death, and stimula- involve production of antimicrobial peptides (AMPs) that
tion of tissue granulation and repair (Richardson 2004). are synthesized in the fat body and subsequently secreted
However, the use of maggot therapy declined as antibiotics into the hemolymph (Bulet & Hetru 1999; Hoffmann &
were developed and surgical techniques improved. Never- Reichhart 2002).
theless, new strains of pathogenic fungi and bacteria devel- The larvae of the black soldier fly, Hermetia illucens, are
oped primarily in response to overdose usage of antibiotics scavengers that can live in extremely harsh environments,
and thus were found commonly in hospitals and the wider such as manures and compost, inhabited by bacteria and
community (Kerridge et al. 2005), renewing our interest in fungi. Animal wastes and rotten plants can be decomposed
maggot therapy in the search for effective methods to treat and recycled by larvae. Additionally, the black soldier fly is
non-healing wounds and control the evolution of resistance sometimes found in carrion. These biological characteristics

© 2014 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd
 Antimicrobial activity of H. illucens

suggest that the soldier fly may be rich in generations of with chloroform and ethyl acetate. All the fractions were
AMPs and other substances possessing activity against drug- lyophilized and stored in a refrigerator at −20°C until use.
resistant “superbugs”. Recently, the antibacterial effect of
H. illucens larval extract against Gram-negative bacteria was Inhibition zone assay
evaluated (Choi et al. 2012). However, insects infected with
the microorganism could contain more biologically and Antimicrobial activity of Sep-Pak C18 (Waters) and high
pharmacologically active chemicals and evidence has been performance liquid chromatography (HPLC) eluent compo-
reported that antibacterial activity of the extract from nents were measured by inhibition zone assay as described
induced larvae was much stronger than that of native larvae previously (Park et al. 2013). Thin plates (1 mm) of 1%
(Hou et al. 2007). The aim of the present study is to improve agarose containing 6 × 104 cells/mL were prepared and wells
antimicrobial substances by inducing immunoreaction in of 3 mm diameter were punched out of the plates. Sep-Pak
H. illucens larvae and testing the antifungal and antibacterial C18 and HPLC eluants were lyophilized and dissolved in
activity of its extract against a range of Gram-positive and distilled water at a final concentration of 1 μg/μL and 5 μL
Gram-negative microorganisms, including the clinically of sample was loaded into each well into appropriate wells.
important strains of methicillin-sensitive (MSSA) and After overnight incubation at 37°C, the diameters of clear
methicillin-resistant Staphylococcus aureus (MRSA), and zones were measured.
also to isolate anti-MRSA substances form the larval extract.
Minimal inhibitory concentration
Minimal inhibitory concentrations (MICs) were performed
Materials and methods
by sequential dilution in sterile 96-well cell culture plates.
The microorganisms were diluted in proper medium at a
Larvae
starting OD600 = 0.001 (approximately 5 × 105 CFU/mL) and
Fifth instar larvae of the black soldier fly (Hermetia illucens) 90 μL dispensed to the plates. Serial dilutions of the larval
were obtained from Nuree Inc., Baekgok, Korea, and reared extract dissolved in distilled water were loaded with 10 μL
at 32°C, 62% humidity with 24 h dark cycle. to the plate and incubated for 16 h (M. luteus was incubated
for 42 h) at 37°C (B. subtilis, E. aerogenes, K. rhizophila,
M. luteus were incubated at 30°C). The MIC was determined
Microorganisms at 600 nm using microplate reader (EL-800, Bio-Tek Instru-
The following bacterial species were used: Escherichia coli ments, Winooski, VT, USA). All experiments were per-
(KCCM 11234), Enterobacter aerogenes (KCCM 12177), formed in quintuplicate.
Psuedomonas aeruginosa (KCCM 11328), MRSA
(methicillin-resistant Staphylococcus aureus), Staphylococ- Purification of antimicrobial substances
cus aureus (KCCM 40881), Bacillus subtilis (KCCM
The water-soluble extract was applied to Sep-Pak C18 and
11316), Kocuria rhizophila (KCCM 11236), Micrococcus
washed with distilled water (20 mL) twice and then eluted
luteus (KCCM 11326), Staphylococcus epidermidis (KCCM
with each 20 mL of 10%, 20%, 30%, 50%, and 80%
35494). The following fungus was used: Candida albicans
acetonitrile (ACN). The anti-MRSA fraction (10% ACN
(KCCM 11282). Escherichia coli and Staphylococcus were
eluant) of preparative purification using Sep-Pak C18 was
grown in tryptic soy broth, while other bacteria were grown
further purified by HPLC on a 4.6 × 250 mm Sim-pack
in nutrient broth, and C. albicans in yeast malt broth.
VP-ODS (Shimadzu) connected to Futecs HPLC system
with a simple linear gradient from 0.1% (v/v) trifluoroacetic
Preparation of the extract of the black soldier acid (TFA) to 25% (v/v) ACN (0.1% (v/v) TFA) at a flow
fly larvae rate of 1 mL/min at room temperature. The elution pattern
was monitored at 214 nm and chromatographic fractions
The larvae were dried after washing with water containing were tested exhibiting activity against MRSA by inhibition
disinfectant and rinsing with sterile water. Subsequently, they zone assay.
were individually pricked deeply with a fine needle dipped in
S. aureus (OD600 = 2.4). The lyophylized larvae were thor-
Results
oughly ground and extracted with acidified methanol
(methanol/water/acetic acid; 90/9/1; v/v/v). The extract was
Antimicrobial activities of the larval extracts
centrifuged at 1600 × g for 10 min at 4°C and taken to dryness
in a rotary evaporator under reduced pressure. Proteins and The water-soluble extract had antibacterial activity against
lipids were removed from the sample by sequential extraction all the Gram-positive and Gram-negative bacteria except

Entomological Research 44 (2014) 58–64 59

© 2014 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd
S.-I. Park et al.

S. aureus and S. epidermidis; MRSA (MIC = 25 mg/mL), ity was observed at the fraction eluted with 10% ACN.
S. epidermidis (MIC = 50 mg/mL), K. rhizophila (MIC = While the eluants did not show any antibacterial activity
25 mg/mL), M. luteus (MIC = 25 mg/mL), B. subtilis (MIC against E. coli, they exhibited a strong activity against
= 12.5 mg/mL), E. coli (MIC = 12.5 mg/mL), E. aerogenes B. subtilis, with the strongest inhibitory zone shown in 30%
(MIC = 25 mg/mL), P. aeruginosa (MIC = 12.5 mg/mL). CAN. Wash1 fraction had antibacterial activity against all
The water-soluble extract also had antifungal activity; the tested bacteria, while wash2 fraction did not have the
C. albicans (MIC = 25 mg/mL). The MIC against S. aureus, same activity (Fig. 2). The second step of purification was
however, could not be determined even at 100 mg/mL. MIC performed using HPLC with a Shim-Pack VP-ODS column
values of the ethyl acetate extract were a similar to that of the (Shimadzu, Kyoto, Japan), which revealed multiple peaks,
water-soluble extract, but the MIC against S. aureus 12256 indicating the presence of various small compounds.
and E. coli 11234 could not be determined even at 100 mg/ Furthermore, large inhibitory zones against MRSA were
mL. Meanwhile, the chloroform extract had no activity detected from fractions 48, 61, and 78, while smaller zones
against all the microorganisms (Table 1). The water-soluble were observed from fractions 50–74 including dark-brown
extract showed a broad-spectrum antimicrobial activity zones (Fig. 3).
against a range of Gram-positive and Gram-negative bacte-
ria and yeast in a concentration-dependent growth manner.
The water-soluble extract exhibited dramatic microbial
Discussion
growth at specific concentrations, but the extract exhibited
concentration-dependent bacterial killing on the growth of It is well known that the larvae of black soldier fly, Hermetia
S. aureus and S. epidermdis. Enhanced bacterial growth was illucens, live in extremely harsh environments, suggesting
observed with the water-soluble extract in low concentration that the soldier fly may be rich in generations of AMPs and
(about 3 mg/mL) (Fig. 1). other substances possessing activity against microorgan-
isms, including MRSA. Previously, it has been reported that
a methanol extract of H. illucens larvae indicated antibacte-
Purification of antimicrobial substances
rial effects against Gram-negative bacteria (Choi et al.
To isolate anti-MRSA substances, the water-soluble fraction 2012). However, according to a study of housefly (Musca
of the larval extract was applied to Sep-Pak C18 cartridges, domestica), antibacterial activities of the extract of inocu-
and eluted with each 20 mL of 10%, 20%, 30%, 50%, and lated larvae were two times stronger and broader than that of
80% ACN. Antibacterial activities of the Sep-Pak C18 the native larvae (Hou et al. 2007). Consistent with this, our
eluants were measured by inhibition zone assay against pre-experimental result also showed that the aqueous extract
MRSA, E. coli, and B. subtilis. Significant anti-MRSA activ- of H. illucens larvae with an immune response induced by

Table 1 Minimum inhibitory concentration (MIC) of the larval extract fractions from Hermetia illucens larvae

MIC of the extract fractions (mg/mL) MIC of antibiotics (μg/mL)

Microorganisms Strain Water Ethyl acetate Chloroform Methicillin Ampicillin

Gram-positive bacteria
MRSA† (clinically isolated) 25 NT‡ >100 >80 >80
Staphylococcus aureus KCCM 40881 >100 >100 >100 80 2.5
S. aureus KCCM 12256 100 NT >100 2.5 2
S. epidermidis KCCM 35494 50 25 >100 >20 10
Kocuria rhizophila KCCM 11236 25 NT >100 <0.3125 <0.3125
Micrococcus luteus KCCM 11326 25 NT >100 NT NT
Bacillus subtilis KCCM 11316 12.5 25 >100 0.078125 0.078125
Gram-negative bacteria
Escherichia coli KCCM 11234 12.5 >100 >100 >80 20
Enterobacter aerogenes KCCM 12177 25 NT >100 >20 >20
Pseudomonas aeruginosa KCCM 11328 12.5 25 >100 NT NT
Yeast
Candida albicans KCCM 11282 25 50 >100 >20 >20

†MRSA, methicillin-resistant Staphylococcus aureus.

‡NT, not tested.

60 Entomological Research 44 (2014) 58–64

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 Antimicrobial activity of H. illucens

Figure 1 Antimicrobial activity of water-soluble extract from Hermetia illucens on the growth of microorganisms at different extract concen-
trations. Each value is expressed as mean ± standard error. Microorganisms were incubated in the presence of different water-soluble extract
concentrations (0–100 mg/mL) for 16 h (except for Micrococcus luteus, which was incubated for 42 h) at 37°C (except for Bacillus subtilis,
Enterobacter aerogenes, Kocuria rhizophila, and M. luteus, which were incubated at 30°C).

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S.-I. Park et al.

Figure 2 Inhibition zone assay of preparative purification against methicillin resistant Staphylococcus aureus (MRSA), Escherichia coli, and
Bacillus subtilis. MtOH, aqueous fraction of acidic methanol extract; Wash1, 2, washing fractions of preparative purification using Sep-Pak C18;
10% –80%, 10% –80% acetonitrile (ACN) eluent; unbind, unbinding fraction.

septic needle, demonstrate stronger antibacterial activity was improved and diversified with the induced immune
against MRSA than that of the native larvae (data not response. The soluble fraction of the extract was tested
shown). against 10 strains of Gram-positive and Gram-negative bac-
The major goals of this study were: (i) to improve the teria and a fungus in a concentration-dependent manner,
expression of antimicrobial substances of H. illucens larvae revealing its broad-spectrum antimicrobial activity as shown
inducing immune response; (ii) to extract the immunized in Figure 1. The enhanced bacterial growth observed with
larvae; and (iii) to test and determine their antibacterial larval extract in concentration dependent growth assay using
activities. The acidic methanol extraction method was low concentration may also be due to the high nutritional
employed for the extraction of antimicrobial substances value of the extract (Bexfield et al. 2004).
from the larvae, because the acidic methanol could denature To isolate anti-MRSA substances, we focused on a spe-
and precipitate large proteins and polypeptides, while effi- cific fraction H. illucens extract, eluted at 10% ACN with
ciently extracting small molecules (Meylaers et al. 2002). Sep-Pak C18 cartridges, which showed a significant activ-
The acidic methanol extract of the larvae was sequentially ity against MRSA. The eluants of Sep-Pak C18 did not
extracted with chloroform and ethyl acetate to remove lipids. show antibacterial activities against E. coli, while the
One of the most commonly used broth dilution techniques soluble-extract did. This result revealed that the antibacte-
was employed to determine the minimal inhibitory concen- rial substances exhibiting activity against E. coli passed
tration (MIC) of the extract under defined conditions. All the through the column, meaning that the anti-E. coli sub-
MIC values of the aqueous fraction of the extract were in a stances were strongly hydrophilic in character (Fig. 2). The
similar range from 12.5 to 25 mg/mL, while Staphylococ- second step of purification was performed using HPLC
cus, including S. aureus and S. epidermidis, demonstrated with a Shim-Pack VP-ODS column, which revealed multi-
relatively lower MIC values. In particular, the MIC value ple peaks, indicating the presence of various small com-
of MRSA was stronger than that of S. aureus and pounds. Large inhibitory zones were detected from
S. epidermidis, suggesting that species-specific antibacterial fractions 48, 61, and 78, while smaller zones were
substances may be expressed in H. illucens larvae and observed from fractions 50–74 (Fig. 3). The fractions dis-
extracted in aqueous fraction. The chloroform fraction of the playing strong activity must be pooled for further purifica-
extract did not show antimicrobial activities against all tion and study of structural properties of the pure
tested bacteria with the result that no antimicrobial sub- substance. It should be noted that H. illucens larvae secrete
stances were extracted in this fraction. According to a pre- dark-brown colored substances due to melanization, or
vious report, the methanol extract of native H. illucens biosynthesis of melanin, a phenolic biopolymer involved in
larvae had Gram-negative specific antibacterial activities the insect immunity. The cytotoxic phenols have already
including Klebsiella pneumonia, Neisseria gonorrhoeae, been investigated extensively (Sugumaran 2002) and are
and Shigella sonnei (Choi et al. 2012). However, the extract well established as compounds displaying broad-spectrum
of acidic methanol of immunized larva used in this study antibacterial effect and, therefore, the presence of these
demonstrated broader antimicrobial activities against all the antibacterial compounds in aqueous extract of H. illucens
bacteria and yeast except S. aureus. This appears to provide is possible (fractions 50–74). Our results show that the
strong evidence that expression of antibacterial substances water-soluble fraction of the whole-body extract possessed

62 Entomological Research 44 (2014) 58–64

© 2014 The Entomological Society of Korea and Wiley Publishing Asia Pty Ltd
 Antimicrobial activity of H. illucens

Figure 3 High performance liquid chromatography (HPLC) purification of the 10% acetonitrile (ACN) solid phase fraction, as prepared from
aqueous extract of Hermetia illucens. (A) Chromatogram of HPLC purification using Sim-pack VP-ODS. The fractions exhibiting activity potent
against methicillin resistant Staphylococcus aureus (MRSA) were represented by arrows. (B) Inhibition zone assay analysis of the fractions
against MRSA.

significantly stronger antibacterial activity against MRSA, 573 Da) was isolated from the adult fleshfly, Sarcophaga
suggesting the presence of more than one “antibacterial” peregrine (Leem et al. 1996), and two small substances,
substance acting in synergy to increase the effect. Addi- β-alanyl-tyrosine (252 Da) and 3-hydroxykynurenine (224
tionally, the aqueous extract of H. illucens has proven to be Da), were isolated from larvae of the grey fleshfly,
highly robust, capable of withstanding several freeze–thaw Neobellieria bullata (Meylaers et al. 2003). Other low
cycles and lyophilization, and is stable as a freeze-dried molecular weight antimicrobial compounds reported from
preparation, all of which are important properties in terms insects include p-hydroxycinnamaldehyde (148 Da), iso-
of development of a product for pharmaceutical purposes. lated from induced larvae of the sawfly, Acantholyda parki
It has been recently reported that the presence and produc- (Leem et al. 1999), and 1-lysophosphatidylethanolamine
tion of a new class of small, perhaps semi-peptidergic (451.2 Da) from native larvae of the housefly, Musca
antimicrobial substances may not be restricted to the domestica (Meylaers et al. 2004).
hemolymph or fat body and therefore, when attempting to In conclusion, all these features provide important evi-
isolate those compounds, preparation of whole-body dence that larval secretions of H. illucens are a very rich
extract could be more efficient (Meylaers et al. 2002). A source of substances with novel antimicrobial properties that
number of such small, low molecular weight antimicrobial could be highly useful in the fight against MRSA and control
dipeptides have been purified from Dipteran species. For of other nosocomial infections. Further research on the
example, the inducible antibacterial compound N-b-alanyl- characterization and purification of the extract may contrib-
5-S-glutathionyl-3,4-dihydroxyphenylalanine (5-S-GAD, ute considerably to the development of new antibiotics.

Entomological Research 44 (2014) 58–64 63

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S.-I. Park et al.

Leem JY, Jeong IJ, Park KT, Park HY (1999) Isolation of

Acknowledgments
p-hydroxycinnamaldehyde as an antibacterial substance
The present research was conducted by the research fund of from the saw fly, Acantholyda parki S. FEBS Letters 442:
Dankook University in 2012. 53–56.
Leem JY, Nishimura C, Kurata S, Shimada I, Kbayashi A, Natori
S (1996) Purification and characterisation of N-b-alanyl-5-
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