Journal of Ethnopharmacology 111 (2007) 227–231

Antibacterial activity and in vitro anti-tumor activity of the extract of the

larvae of the housefly (Musca domestica)
Lixia Hou, Yonghui Shi, Pei Zhai, Guowei Le ∗
Key Laboratory of Food Science and Safety, Ministry of Education, School of Food Science and Technology,
Southern Yangtze University, Wuxi 214036, PR China
Received 16 December 2005; received in revised form 30 October 2006; accepted 18 November 2006
Available online 28 November 2006

Abstract
One of the significant characteristics of traditional Chinese medicine is the use of insects, other terrestrial arthropods and their products as
drugs. In the present work, the extract of housefly (Musca domestica) larvae was studied by an agar well diffusion assay and minimum bactericidal
concentration (MBC) determination for detection of antimicrobial activity and by MTT assay method to test its in vitro anti-tumor activity. In our
studies, the extract inhibited six tested bacterial pathogens and Escherichia coli Jm109, a gene-modified strain resistant to ampicillin with the MBCs
ranging from 200 to 1600 ␮g/ml, while Gram-positive bacteria were more sensitive than Gram-negative bacteria. The extract showed high in vitro
anti-tumor activity against human colon cancer cell line CT26 with the IRs ranging from 62 to 89%. The extract of housefly larvae inoculated with
Shigella dysenteriae 51302 exhibited higher antibacterial activity and in vitro anti-tumor activity than that of native larvae, which may indicate the
same principle involved in the use of Bombyx mori larvae infected with silk moth fungus Beauveria bassiana and Hepialis larvae infected with
Cordiceps fungus in the traditional Chinese medicine. Both extracts did not show any coagulation activity and haemolysis activity against rabbit
erythrocytes at 500 and 1000 ␮g/ml. The results support its use in the traditional Chinese medicine, and warrant the further identification of the
active molecule in the housefly larvae.
© 2006 Elsevier Ireland Ltd. All rights reserved.

Keywords: Housefly (Musca domestica); Antibacterial activity; In vitro anti-tumor activity

1. Introduction
A lot of laboratory work on the bioactivity and use of
plants and their components in folk medicine has been reported
(Dobner et al., 2003; Rojas et al., 2003; Tadeg et al., 2005),
on the other hand, there are few reports on the ethnopharma-
cological use of insects. Insects and arthropods “are a large,
unexplored and unexploited source of potentially useful com-
pounds for modern medicine” (Pemberton, 1999). One of the
significant characteristics of traditional Chinese medicine is the
use of insects, other terrestrial arthropods and their products as
drugs (Jiang, 1999). The housefly (Musca domestica) belongs
to the Diptera. The housefly larvae have been used clinically to
cure malnutritional stagnation, decubital necrosis, osteomyeli-
tis, ecthyma and lip boil since the Ming/Qing Danysty (1368
Anno Domini) up to now in China, and also used to treat coma
∗ Corresponding author. Tel.: +86 510 85869236; fax: +86 510 85869236.
E-mail address: lgw@sytu.edu.cn (G. Le).
and gastric cancer when combined with other drugs (Jiang,
1999).
Despite the popular use of the housefly larvae in traditional
Chinese medicine and the fact that they are commonly assumed
to be safe for medical use, to our knowledge, no laboratory
reports on its bioactivity are available. The aim of the present
study is to test the antimicrobial activity, in vitro anti-tumor
activity, and blood coagulation activity and haemolysis activity
of the extract of housefly larvae.
2. Materials and methods
2.1. Preparation of the extract of the housefly larvae
The third instar larvae of the housefly (Musca domestica)
were obtained from the Wuxi Kunlong nursery, Wuxi, China.
The larvae were divided into two groups. For the first group,
preparation of the extract was performed according to Zhang
(1999) but slightly modified. The native insect bodies were

0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2006.11.015
228 L. Hou et al. / Journal of Ethnopharmacology 111 (2007) 227–231
washed with sterile water, frozen with liquid nitrogen and
homogenized thoroughly with a tissue blender in three-fold vol-
ume ammonium acetate (50 mM, pH 5.0) containing 35 ␮g/ml
phenylmethylsulfonyl fluoride (PMSF), 0.2 mg/l ethylenedi-
amine tetraacetic acid (EDTA) and 2‰ 2-mercaptoethonal
(5 mM). The homogenate was centrifuged at 4800 × g for 30 min
in a centrifuge. The supernatant was heat-treated at 100 ◦ C for
5 min with continuous agitation, then centrifuged at 12,000 × g
for 30 min at 4 ◦ C and lyophilized to dry.
For the second group, the preparation of the extract was the
same except that we pricked the insect larvae evenly with a self-
made iron brush dipped into the overnight culture of Shigella
dysenteriae 51302 (2.5 × 108 CFU/ml) at 37 ◦ C, the insect bod-
ies were collected 24 h after inoculation, and then frozen with
liquid nitrogen.
2.2. Antibacterial activtity test of the extract
2.2.1. Microorganisms and media
Six pathogenic microorganisms used for the antimicrobial
assay were kindly provided by Wuxi disease prevention and con-
trol center, Wuxi, China. They are Escherichia coli ATCC25922,
Bacterium pyocyaneum ATCC27553, Salmonella typhimurium
50013, Shigella dysenteriae 51302, Staphylococcus aureus 6538
and Bacillus subtilis 9372. Other microorganisms used are
preserved in our laboratory, they are Escherichia coli Jm109
(ampicillin-resistant) and a Penicillium sp. and Aspergillus
niger. The bacteria were grown in beef-extract peptone medium,
Penicillium sp. and Aspergillus niger were grown in Czapek’s
medium.
2.2.2. Agar well diffusion assay
Overnight culture of the respective test bacteria and 24 h cul-
ture of fungi were adjusted to 2–5 × 108 colony forming units
per ml (CFU/ml), 1ml of such a culture was added to 15 ml
respective medium, evenly mixed and poured into Petri dishes
(9 cm in diameter). The lyophilized extract was dissolved in
10mM sodium phosphate buffer, pH 7.4 (NAPB) to reach final
500 ␮g/ml and 1000 ␮g/ml as the test solutions. The discs (Ø
6 mm) were then applied, 10 ␮l of the test solutions and con-
trol were added to each disc, respectively. Petri dishes were
incubated at 37 ◦ C for 16–24 h (bacteria) and 28 ◦ C for 72–96 h
(fungi). The average diameters of the inhibition zone surround-
ing the discs were measured visually, and did not include the
diameter of the paper disc. NAPB was used as negative control,
Norfloxacin (10 ␮g/disk) was used as antibacterial positive con-
trol and ketoconazole (10 ␮g/disk) as antifungal positive control.
The experiments were carried out in triplicate.
2.2.3. Minimum bactericidal concentration (MBC)
Stock solution of the extract was diluted two-fold serially in
NAPB. The 1ml of the test solutions was mixed with 100 ␮l of
culture medium containing bacteria at 2–4 × 107 CFU/ml. After
24 h of incubation with the extract at 37 ◦ C with shaking, cultures
were diluted 10-fold serially. Three 20 ␮l drops were applied
onto the appropriate agar plates. Cell counts were determined
by counting the colonies after plates were incubated at 37 ◦ C for
16–24 h. The MBC were recorded as the lowest concentration of
the extract that did not permit any visible bacteria colony growth
on agar plate after the period of incubation. The experiments
were carried out in triplicate.
2.3. In vitro anti-tumor activity test
The in vitro anti-tumor activity test was performed according
to Kang et al. (2004) but slightly modified. Human colon cancer
cell line CT26 was purchased from Shanghai cell institute, Chi-
nese Academy of Sciences, Shanghai, China. The experimental
cells were grown in RPMI 1640 medium with supplemented
with 10% (v/v) fetal bovine serum (FBS), 100 units/ml peni-
cillin and 100 ␮g/ml streptomycin in a 5% CO2 atmosphere at
37 ◦ C in a humidified incubator.
The experimental cells were counted in a hemacytometer
using the inverted microscope (Olympus Co., Japan). The cells
were plated on 40-well flat-bottomed plates, with each well
at a density of 5 × 105 cells/well, and incubated for 24 h at
37 ◦ C in the CO2 incubator. After the cells were attached on
40-well flat-bottomed plates, 100 ␮l of the test solutions and
control (NAPB) were directly added to the wells containing
100 ␮l cell suspension, respectively. The cells were incubated
with the test solutions for 48 h in 5% CO2 atmosphere at 37 ◦ C.
After incubation, the supernatant was removed and cells were
washed with PBS (pH 7.4). Cells were then incubated with
100 ␮l of the MTT solution (0.5 mg/ml) in RPMI 1640 medium
without FBS for 4 h at 37 ◦ C, then the supernatant after cen-
trifuging at 1000 × g for 5 min was removed. The 100 ␮l of
dimethyl sulfoxide (DMSO) was then added in order to dis-
solve the formazan crystals formed. After solubilizing, the
optical density (OD) was measured with the ELISA microplate
reader (Thermo Molecular Devices Co., USA) at a wave-
length of 492 nm. The inhibition rate (IR%) was calculated as
follows: IR% = (ODcontrol − ODsample )/ODcontrol × 100%. All
experiments were performed in triplicate.
2.4. Blood coagulation activity and haemolysis activity of
the extract
Fresh rabbit blood was washed three times with physiological
saline PBS (pH 7.4) by centrifuging at 1000 × g for 10 min, and
resuspended in PBS with concentration of 105 erythrocytes/ml.
The 50 ␮l extract solution (500 and 1000 ␮g/ml) was added to
200 ␮l PBS containing erythrocytes respectively. Haemolysis
activity and coagulation activity of the extract was determined
by observing under the microscope at 0, 10, 20, 30, and 60 min
of incubation, wheat germ agglutinin was used as coagulation
positive control.
3. Results
3.1. Antibacterial activity of the extract
The 2.4 g solid extract (lyophilized) could be obtained from
100 g fresh larvae for both groups. The antimicrobial activity of
the extract was determined by two different methods (Table 1).
 L. Hou et al. / Journal of Ethnopharmacology 111 (2007) 227–231 229

Table 1
Antibacterial activity of the extract of the larvae of the housefly (Musca domestica)
Microbials Mean zone of inhibition (mm)a MBC (␮g/ml)
Extract of native larvae Extract of inoculated larvae Antibiotic controlb NAPB Extract of Extract of
controlc native larvae inoculated larvae
500 ␮g/ml 1000 ␮g/ml 500 ␮g/ml 1000 ␮g/ml

Gram-negatives
Escherichia coli 1 ± 0.22 1 ± 0.13 1 ± 0.37 2 ± 0.23 6 ± 0.55 0 600 200
ATCC25922
Shigella dysenteriae 0 2 ± 0.23 2 ± 0.26 4 ± 0.37 4 ± 0.34 0 600 200
51302
Salmonella 0 0 2 ± 0.32 2 ± 0.21 2 ± 0.36 0 1000 400
typhimurium 50013
Bacterium pyocyaneum 0 0. 1 ± 0.48 1 ± 0.16 2 ± 0.44 0 1600 400
ATCC27553
Escherichia coli Jm109 0 0 0 0 2 ± 0.38 0 1200 1200
Gram-positives
Staphylococcus aureus 1 ± 0.24 2 ± 0.46 3 ± 0.23 4 ± 0.38 3 ± 0.12 0 600 200
6538
Bacillus subtilis 9372 0 2 ± 0.25 0 4 ± 0.26 2 ± 0.23 0 600 600
Fungi
Penicillium sp. 0 0 0 0 4 ± 0.47 0 – –
Aspergillus niger 0 0 0 0 4 ± 0.59 0 – –
a Mean of three assays; (±) standard deviation.
b Antibiotic control, norfloxacin (10 ␮g/disk) was used as antibacterial positive control, ketoconazole (10 ␮g/disk) as antifungal positive control.
c NAPB (10 mM sodium phosphate buffer, pH 7.4) was used as negative control.

In the agar diffusion assay, the negative control (NAPB)
did not show any inhibition, while antibiotic control (Nor-
floxacin was used as antibacterial positive control, ketoconazole
as antifungal positive control) showed mean zones of inhibition
ranging from 2 to 6 mm. The mean zones of inhibition against
bacteria was ranging from 1 to 4 mm, except that the extract of
native larvae showed no zone of inhibition against Salmonella
typhimurium 50013, Bacterium pyocyaneum ATCC27553 and
Escherichia coli Jm109, a gene-modified strain resistant to
ampicillin, while the extract of inoculated larvae indicated no
inhibition against Escherichia coli Jm109. Two test fungi were
not inhibited by the extract under the test concentrations (500
and 1000 ␮g/ml).
The results of the MBC showed that both extract were active
against all six aerobic bacterial pathogens and non-pathogenic
gene-modified strain resistant to ampicillin, Escherichia coli
Jm109. The MBCs of the extracts of native larvae ranged from
600 to 1600 ␮g/ml. For Gram-positive strains, Staphylococcus
aureus 6538 showed the same sensitivity as Bacillus subtilis
9372 to the extract of native larvae. Escherichia coli ATCC25922
and Shigella dysenteriae 51302 were most sensitive among the
Gram-negative strains with the same MBC of 600 ␮g/ml, fol-
lowed by Salmonella typhimurium 50013 and Escherichia coli
Jm109 with MBC of 1000 and 1200 ␮g/ml, respectively. Bac-
terium pyocyaneum ATCC27553 was most resistant.
When the extract of inoculated larvae was tested to determine
the MBC, for Gram-positive strains, Staphylococcus aureus
6538 was more sensitive than Bacillus subtilis 9372. Escherichia
coli ATCC25922 and Shigella dysenteriae 51302 was most sen-
sitive among the Gram-negative strains with the same MBC
of 200 ␮g/ml, followed by Salmonella typhimurium 50013 and
Bacterium pyocyaneum ATCC27553 with MBC of 400 ␮g/ml,
and Escherichia coli Jm109 was most resistant.
In general, both extracts of housefly larvae showed higher
activity against Gram-positive bacteria than Gram-negative bac-
teria, and did not show any antifungal activity. The antibacterial
activity of extract of inoculated larvae was higher than that of
extract of native larvae.

Table 2
Dose-Dependent Inhibition of human colon cancer cell line CT26 by the extract of the larvae of the housefly (Musca domestica)

Test solution Concentration (␮g/ml) ODa 492nm IR (%)b P-value

Extract of native larvae 500 0.361 ± 0.023 62 <0.01

1000 0.228 ± 0.026 76 <0.01
Extract of innoculated larvae 500 0.257 ± 0.031 73 <0.01
1000 0.102 ± 0.021 89 <0.01
Control (NAPB) 0.950 ± 0.039 – –
a Mean of three assays; (±) standard deviation.
b IR% = (ODcontrol − ODsample )/ODcontrol × 100%.
230 L. Hou et al. / Journal of Ethnopharmacology 111 (2007) 227–231
3.2. In vitro anti-tumor activity of the extract
The MTT assay is an indirect measure of cell density or num-
ber of living cells attached to the culture plate by formation of
colored formazan crystals. OD492nm of control was 0.950 after
48 h, that of extract of native larvae at 500 and 1000 ␮g/ml was
0.361 and 0.228, with 62 and 76% inhibition rate (IR), respec-
tively. OD492nm of the extract of innoculated larvae at 500 and
1000 ␮g/ml was 0.257 and 0.102, with 73 and 89% inhibition
rate (IR), respectively (Table 2.). The experiments were per-
formed in triplicate, and the differences between OD492 nm of
control and that of the different extract dose treatment were
significant (P < 0.01).
3.3. Blood coagulation activity and haemolysis activity of
the extract
Both extracts did not show any coagulation activity and
haemolysis activity when observed under the microscope,
treated rabbit erythrocytes had normal appearance and were
evenly distributed, on the contrary wheat germ agglutinin
induced the rabbit erythrocytes to aggregate to clusters.
4. Discussion
Traditional medicines such as traditional Chinese medicine
use a great array of insects, terrestrial arthropods and their
products as drugs, but little literature is available, insects
and arthropods “are a large, unexplored and unexploited
source of potentially useful compounds for modern medicine”
(Pemberton, 1999).
The present study tested the antimicrobial activity, in vitro
anti-tumor activity, blood coagulation activity and haemolysis
activity of the extract of housefly larvae. The prepared extracts
included two groups: the extract of native larvae and extract of
innoculated larvae, the latter showed higher antibacterial activity
and in vitro anti-tumor activity than the extract of native larvae,
which may indicated the same principle involved in the use of
Bombyx mori larvae infected with silk moth fungus Beauveria
bassiana and Hepialis larvae infected with Cordiceps fungus in
traditional Chinese medicine. When infected with microorgan-
isms, the insects activate three tightly interconnected reactions:
proteolytic cascades, cellular defense reactions, which consist
predominantly of phagocytosis and/or encapsulation of invad-
ing microorganisms and the rapid and transient synthesis of an
array of antimicrobial peptides by the fat body (Hoffmann et
al., 1996). Therefore, infected insects may contain more bio-
logically and pharmacologically active chemicals. Traditional
medicines are generally effective, but they also have shortcom-
ings such as unknown active molecules and uncertain clinical
efficacy.
In the present work, the extracts of both groups showed higher
antibacterial activity against Gram-positive bacteria than against
Gram-negative bacteria, which is similar with the essential oils
of Thymus vulgaris L. (Marino et al., 1999), Syzygium jam-
bolanum seeds (Chandrasekaran and Venkatesalu, 2004), the
extract of sumac (Rhus coriaria L.) (Nasar-Abbas and Halkman,
2004), the extracts of 25 medicinal plants of the island Soqo-
tra (Mothana and Lindequist, 2005) and the extracts of six
ethnobotanically selected medicinal plants (Anredera cordi-
folia, Elaeodendron transvaalense, Elephantorrhiza burkei,
Senna petersiana, Terminalia sericea and Rauvolfia caffra)
(Tshikalange et al., 2005). The difference in sensitivity between
Gram-positive and Gram-negative bacteria might be attributed
to the differences in morphological constitutions between these
microorganisms. Gram-negative bacteria have an outer phospho-
lipidic membrane containing lipopolysaccharide components,
on the other hand, the Gram-positive bacteria only have an outer
peptidoglycan layer which is not as an effective permeability
barrier as the former (Nostro et al., 2000). Wiart et al. (2004)
reported the similar sensitivity of Staphylococcus aureus and
Shigella sonnei to ethyl acetate fraction of Acalypha siamensis
Oliv. ex Gage.
In our studies, the extract of housefly larvae at 1000 ␮g/ml
showed higher zone of inhibition than at 500 ␮g/ml concen-
tration, as the disc dose increased, the inhibitory effect also
increased. The extract showed dose-dependent antibacterial
activity.
The MTT assay is an assay of metabolic competence based
upon assessment of mitochondrial performance by colorimetri-
cally measuring the conversion of yellow tetrazolium bromide
(MTT) to the purple formazan derivative by mitochondrial suc-
cinate dehydrogenase in viable cells (Mosmann, 1983). In our
present work, the wells incubated with control (NAPB) showed
deep purple, while those incubated with the extract was light
yellow, IR% of the extract ranged from 62 to 89%. Most strik-
ing in our experiment, the extract showed much higher in vitro
anti-tumor activity than peptidoglycan (data not shown). The
combination of radiation therapy and chemotherapy is com-
monly used in the treatment of cancer, but majority of the
chemotherapy drugs are cytotoxic to both normal cells and tumor
cells, leading to the increased cytotoxicity when the curative
effects increase. In the present work, the extract of housefly
larvae specifically inhibited the proliferation of tumor cells in a
dose-dependent fashion, and our results indicated that the extract
was not cytotoxic to rabbit erythrocytes, and did not show any
blood coagulation activity and haemolysis activity, which may
provide useful information for research in the area of anti-tumor
and for the development of anti-tumor drugs.
The chemical compostions of housefly larvae (dry weight/dry
starting material weight, w/w) are protein (54.47%), carbohy-
drate (12.04%), fat (11.60%), crude fibre (5.70%) and ashes
(11.43%), indicating a high content of protein (Li et al., 1997).
In our experiment, specific activity of the extract increased by
3.4 folds after heating at 100 ◦ C for 5 min with continuous agi-
tation (data not shown). In summary, we suppose that the active
molecules in housefly larvae may be some heat-stable peptides.
Chernysh et al. (2002) isolated two anti-viral and anti-tumor
peptides from the blow fly Calliphora vicina (Diptera), with
the following amino acid sequences: HGVSGHGQHGVHG
(alloferon 1) and GVSGHGQHGVHG (alloferon 2).
In conclusion, the extract of housefly larvae possesses broad
antibacterial activity against both Gram-negative bacteria and
Gram-positive bacteria and has in vitro anti-tumor activity, but
 L. Hou et al. / Journal of Ethnopharmacology 111 (2007) 227–231
does not show any coagulation activity and haemolysis activity
against rabbit erythrocytes, which support its use in traditional
Chinese medicines.
Acknowledgements
This research was supported in part by a grant (No.
2001DEA20022) from the Special Foundation of Basic
Research of Science and Technology for the Central Research
Institute of China. We thank Dr. Li Yin for kind assistance in
English revising of this manuscript.
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