Journal of Phytopathology and Pest Management 5(3): 43-54, 2018
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Eman M. Hassan,
E-mail: emanhassan_67@yahoo.com
43
Copyright © 2018
Influence of some natural products of
Moringa
oleifera
(L.) on some biochemical and economical
characters of infected mulberry silkworm,
Bombyx mori
L. (Lepidoptera: Bombycidae)
Eman M. Hassan
1*,
Ghada M. A. Morsy
2
,
Mohamed E. Hashish
3
1
Sericulture Research Department, Plant Protection Research Institute, Agricultural Research
Center, Dokki, Giza, Egypt.
2
Horticulture Insects Department, Plant Protection Research
Institute, Agricultural Research Center, Dokki, Giza, Egypt.
3
Bee Research Department, Plant
Protection Research Institute, Agricultural Research Center, Dokki, Giza, Egypt
Abstract
Keywords: Bacterial diseases, botanical extracts, Moringa oleifera, Bombyx mori.
44
Introduction
The mulberry silkworm,
Bombyx mori
L.,
(Lepidoptera: Bombycidae) is one of the
most economic important insects. This
importance developed from the ability of
silkworm to secrete the natural silk
filament from its silk gland. Silk
production is only about 0.2 % of the
total textile fiber production in the world
which represents a slow production
increase. Recently a considerable
attention has been given to improve
rearing technology and consequently
increasing natural raw silk production.
However, this production may be
suddenly falls due to many technical and
non-technical problems. Silkworm
diseases are considered as one of the
major technical problem; Bacterial
diseases (Flacherie) are usually only
secondary to virus diseases. Several
bacteria cause Septicaemia and toxaemia
(
Bacillus
,
Streptococcus
,
Staphylococcus
and
Escherichia coli
) cause softening and
putrefaction of the dead worms.
Tolerance to all the agents is another
theory to decreasing the incidence rate of
those diseases. Finding natural and eco-
friendly plant products that prevent or
treat these diseases could be an
alternative treatment process (Kumar et
al., 2009; Mesbah et al., 2000; Mahesha
et al., 1999; Parra, 1991). Use of herbal
plants in medicine which having anti-
microbial property, non-toxic,
biodegradable and non pollutant for
controlling diseases of silkworm rearing.
Plant extracts contain variety of
components that can either inhibit the
growth of the microorganisms or
eradicate them were used by many
authors (Abalaka et al., 2009; Nigam,
1982).
Moringa oleifera
L
.
belongs to
family Moringaceae. It originally located
in Asia then spread in many parts of
Africa. This family contains around 13
species relocated from tropical to
subtropical regions and ranging in size
from little herbs to huge trees and
recently grows successfully in Egypt.
The importance of this plant is due to its
multiple uses and benefits to agriculture
and industry and the all parts of
Moringa
plant are used for medicinal and other
purposes (Barakat & Ghazal, 2016;
Janick & Paull, 2008; Anwar et al., 2007;
Price, 2000).
M. oleifera
contains many
essential nutrients, vitamins, minerals,
amino acids, beta-carotene, omega 3 and
6 fatty acids, also it consists of
antioxidants, anti-inflammatory, anti-
spasmotic, anti-hypertensive, anti-
tumour, anti-pyretic, anti-ulcer,
cholesterol lowering and anti-diabetic
nutrients (Sharma et al., 2012; Paliwal et
al., 2011; Kasolo et al., 2010; Hsu et al.,
2006; Fahey, 2005). Physicochemical
properties of
Moringa
seeds oil proved
that this oil bodes rewarding potential
application in nutrition aspects. The
higher content of unsaturated fatty acids
may present a healthy influence of
Moringa
seeds oil in terms of nutrition
(Barakat & Ghazal, 2016). It has good
quantity of oleic acid (57%) and omega 3
(13.28%) and rich in natural antioxidants
that are scavenging of free radicals in the
body due to the presence of tocopherols,
phenolics and carotenoids (Khattab &
Shakak, 2012). Leaves of
Moringa
species are rich in various phytochemical
components like carotenoids, amino
acids, sterols, glycosides, alkaloids,
flavonoids, moringine, moringinine,
phytoestrogens caffeoylquinic acids and
phenolics, and it works as an effective
source of natural antioxidants due to the
presence of flavonoids, ascorbic acid,
carotenoids, and phenolics (Anwar et al.,
2007; Siddhuraju & Becker, 2003;
Dillard & German, 2000). The present
study aims to evaluate the role of some
natural products of
Moringa
plant (seed
oil, leaves extract, root powder and
Moringa
honey bee) with different
45
concentrations in treatment of bacterial
infected mulberry silkworm,
B. mori.
Also, to select the effective natural part
product through studying their effects on
the activity of some protein enzymes,
cocoon and filament characters.
Materials and methods
The present study was carried out in the
laboratory of Sericulture Research
Department of Plant Protection Research
Institute, Agricultural Research Center,
Cairo, Egypt. Mulberry silkworm,
B.
mori
eggs (G
2
× V
2
× H
8
× KK hybrid)
were obtained from the Sericulture
Research Department of Plant Protection
Research Institute, Agricultural Research
Center, Giza, Egypt.
Silkworm rearing technique:
Rearing
of silkworm was carried out in laboratory
under the hygro-thermic conditions 28 ±
1°C and 75 ± 5% RH, according to the
technique of Krishnaswami (1978). The
larval bed was cleaned daily. Cleaning
net was used for removing the remained
dried food and feces. The newly hatched
larvae were fed on fresh clean mulberry
leaves until the beginning of the 5
th
instar. The 5
th
instar larvae were used in
the present study. The source of
Moringa
seed oil, leaves extract and root powder
were kindly prepared and obtained from
Egyptian Scientific Society of
Moringa
(ESSM), National Research Center,
Cairo, Egypt.
Moringa
bee honey was
obtained from Bee Research Department,
Plant Protection Research Institute,
Agricultural Research Center, Giza,
Egypt. The collected plant parts (seeds,
leaves and roots) of
Moringa
were
washed under tap water followed by
distilled water and dried under shade.
Dried samples were powdered in
mixer/grinder. Powder of leaves and
roots was mixed individually with
distilled water in a ratio of 1:1 (w/v) and
left overnight to allow the constituents to
get dissolved in water, then filtered
through muslin cloth and 100% plant
extract solution was prepared according
to El–Mohamedy and Abdalla (2014).
Extraction of seeds oil was prepared
according to Harvey and John (1898)
protocol. Each was used with three
concentrations. 5 ml of
Moringa
oil
dissolved in 1 L. distilled water to
prepare a concentration of 0.5% by
adding Tween 80 to disperse the oil in
the solution and 10 ml of
Moringa
oil to
prepare a concentration of 1%; and so the
remaining concentration to 1.5%. By the
same way the other concentrations of
Moringa
leaves extract (0.5%, 1% &
1.5%),
Moringa
root extract (0.5%, 1%
& 2%) and
Moringa
honey (1%, 2% &
4%) were prepared without tween 80.
Schedule of application:
Larvae were
divided into four groups; each group was
fed on mulberry leaves treated with
(
Moringa
seeds oil,
Moringa
leaves
extract,
Moringa
honey and
Moringa
root powder). Each group was divided
into three subgroups representing three
concentrations with 3 replicates each
(100 larvae for each replicate). Mulberry
leaves were washed and let to dry. Fresh
mulberry leaves were dipped in each
concentration for 5 minutes and left to
dry then offered to larvae (three
diets/day, five times during the larval
instar) to obtain the full growth. Fifth
group (control) fed on leaves was treated
only with distilled water. Chicken egg
cartons plates were used as montages for
cocoon spinning as described by
46
(Zannoon & Shadia, 1994).
Infecting of silkworm larvae with
bacteria:
Bacterial pathogens were
collected and isolated from diseased
larvae (Aneja, 2003). After bacterial
culture prepared using Luria agar
medium (Suparna et al., 2011),
B. mori
artificially infected by spraying mulberry
leaves with the concentration (15 ppm) of
bacterial flacherrie (
Streptococcus
pneumoniae
) then fed to larvae one time
on the 2
nd
day of the 5
th
instar larvae.
The biochemical characters:
Samples
of haemolymph were collected at the 7
th
day of the 5
th
larval instar made by
removing one of the thoracic legs of the
larvae and bending the body to expose
the sternum at the position of the
removed leg. This ensured proper
drainage of the haemolymph, and
avoided any risk of internal organs to be
destructed. The haemolymph of each
treatment was collected in eppendorf
tubes 1.5 ml with small crystal of phenyl
thiourea (PTU) to prevent melanization
of sample and prepared for biochemical
assays according to Mahmoud (1988)
protocol. The supernatant was
immediately assayed colorimetrically to
determine Aspartate transaminase (AST),
Alanine transaminase (ALT) activities
according to the method of Reitman and
Frankel (1957). The proteolytic enzyme
activity was determined by the casein
digestion method described by (Ishaaya
et al., 1971).
The economical characters:
The
resulted cocoons were collected after 7
days from the beginning of spinning for
studying the economical characters. Half
number of the resulted cocoons of each
replicate was used for determining the
cocoon characters (cocoon weight, shell
weight). The other half was dried
at
80
ºC in oven and used to study the filament
characters (filament length, weight and
size).
Statistical Analysis:
Obtained data was
subjected to analysis of variance
(ANOVA) as one way to find the
differences among different treatments.
Statistical analysis was conducted using
Proc ANOVA in SAS (SAS Institute,
1988).
Means separation was conducted
using Duncan Multiple Range Test in the
same program. Later factorial analysis
was conducted to elucidate the
significance among different materials
and tested concentrations using the same
procedures.
Results and Discussion
Biochemical aspects:
The biochemical
aspects represent one of the most
important aspects in silk production.
Proteins constitute the major working
force for all forms of biological work.
Alanine transaminase (ALT):
As
represented in Table (1a), data exhibited
a significant increase in ALT means of
larvae fed on mulberry leaves treated
with different concentrations of
Moringa
products. The root extract concentration
(1 and 2%) showed the highest means
(82.18, 87.72 μg pyruvate/min/ml)
respectively, followed by the
concentration of seed oil 1.5% (63.71μg
pyruvate/min/ml). Also the concentration
of
Moringa
honey 4% recorded the
higher ALT value (61.87μg
pyruvate/min/ml), comparing to the
control group (27.70 μg pyruvate
47
/min/ml). Data represented in Table (2a)
indicated that,
Moringa
root extract
exhibited significantly the best result
among
Moringa
products (73.56 μg
pyruvate/min/ml), followed by seed oil
(52.94 μg pyruvate/min/ml).
Aspartate transaminase (AST):
Table
(1a) showed that a significant increase in
AST values with different concentrations
compared with control (70.09 μg
oxaloacetate/min/ml), especially the root
extract (2%) exhibited (148.59μg
oxaloacetate/min/ml) followed by the 2
nd
concentration (1%) of the same treatment
(132.70 μg oxaloacetate/min/ml), then
the 3
rd
concentration of seed oil (1.5%)
followed by the 3
rd
concentration (4%) of
Moringa
honey (122.42, 104.66 μg
oxaloacetate/min/ml) respectively. As
shown in Table (2a), a significant
increase in AST values of diseased larvae
with mulberry leaves supplemented with
Moringa
root extract (125.54 μg
oxaloacetate/min/ml), followed by seed
oil of
Moringa
treatment (106.22 μg
oxaloacetate/min/ml). At the same time,
AST means increased significantly with
concentrations increasing. According to
the obtained results, the significant
increase in protein enzymes activities
may be attributed to the useful effect of
constituents of botanical extracts on the
haemolymph protein content of infected
silkworm,
B. mori
. These findings are
close to those of Yousef (2014), revealed
that, the improvement of silk production
depending on amines transfer mechanism
involved in the uptake of used
compounds constituted in mulberry
leaves by the body tissues and silk
glands, resulted in the subsequent
promotion of silk protein synthesis. By
the same way, Arora et al.
(2013) stated
that the various extracts of Moringa’s
morphological parts such as seeds, steam
park, leaves, root bark possess
antimicrobial potentiality. Fahey (2005)
stated that, the chemical compounds
which isolated from the plant
M. oleifera
contain numerous antibacterial
compounds such as, glucosinolates,
rhamanose, pterygospermin, and
isothiocyanates. Gaceres et al. (1992)
and Ezeamuzie et al. (1996) reported
that, the root extracts of
Moringa oleifera
possess anti-inflammatory activity, so, it
has been used for treatment of a diseases
number. Similarly, Khattab and Shakak
(2012) and Barakat and Ghazal (2016)
showed that, the protein content of
Moringa
seed oil was 41.13% and the
amino acids content demonstrated a high
potential application of seed extracts in
nutrition being the highly nutrition value
of its protein.
Protease:
According to the results
obtained in Table (1a), the 3
rd
concentration (2%) of root extract
followed by the 2
nd
concentration (1%)
exhibited significantly the highest means
of protease enzyme activity (194.71,
177.18 O.D. units x10
3
/min/ml),
respectively compared to the control
group (99.72 O.D. units x10
3
/min/ml). At
the same way, the 3
rd
concentration
(1.5%) of seed oil and the 3
rd
concentration (4%) of honey also showed
significant increase of protease values
(152.64 and 149.66 O.D. units x10
3
/min/ml), respectively. On the other
hand, as shown in Table (2a), a highly
significant difference was noticed with
Moringa
root extract treatment (168.40
O.D. units x10
3
/min/ml), followed by the
seed oil treatment (138.65 O.D. units
x10
3
/min/ml). These results are
48
confirmed by Prajapati et al. (2003)
finding. Who described the presence of
the essential amino acids - arginine,
histidine, lysine, tryptophan,
phenylalanine, methionine, threonine,
leucine, isoleucine, valine among the
biochemical constituents of
Moringa
oleifera
which also consists of Carotene,
nicotinic acid, ascorbic acid, ascorbic
acid oxidase sulphur, a prolamin. As the
same trend, roots have been found to
possess several distinct pharmacological
properties (Goyal et al., 2007). Similarly,
Haristory et al. (2005) reported that, the
isothiocyanate structure and its
predecessor, glucosinolate, as primary
constituents from
M. oleifera
seed
extracts are grand antibacterial agents.
Also, protease regulates the fate,
localization, and activity of many
proteins, modulate protein-protein
interactions and create new bioactive
molecules and influence immunity and
apoptosis (Turk, 2006), it represents an
important tool of the biotechnological
industry because of its usefulness as
biochemical reagent or in the
manufacture of numerous products
(Saeki et al., 2007). Generally, the
significant increases of the biochemical
aspects (protein transaminases and
protease enzyme) of the present study are
in good agreement with previous work
demonstrating that, the natural
disinfectants may stimulate the enzymes
activities which influence the
biochemical contents of the haemolymph
of the silkworm,
B. mori
;
also, increased
the amount of haemolymph protein of
larval instars as suggested by El-Sayed
et
al.
(1990).
Table 1a: Effect of different treatment concentrations of Moringa on the biochemical characters of
diseased mulberry silkworm, B. mori.
Treatments
Conc. (%)
ALT
AST
Protease
Moringa seed oil
0.5
42.48
cde
95.32
cde
123.99
bc
1
52.63
cde
100.93
cde
139.32
bc
1.5
63.71
abc
122.42
abc
152.64
abc
Moringa leaves extract
0.5
60.94
bcd
102.80
bcde
149.44
abc
1
34.17
e
74.76
de
106.72
c
1.5
37.04
de
81.30
de
114.38
c
Moringa honey
1
32.32
e
74.76
de
104.52
c
2
34.17
e
78.50
de
111.78
c
4
61.87
bcd
104.66
bcd
149.66
abc
Moringa root extract
0.5
50.79
cde
95.32
cde
133.32
bc
1
82.18
ab
132.70
ab
177.18
ab
2
87.72
a
148.59
a
194.71
a
Control
27.70
e
70.09
e
99.72
c
P value
0.0001
0.0001
0.01
L.S.D.
22.91
28.37
49.17
Means in the same column not followed by the same letter are significantly different (P≤ 0.05).
49
Table 1b: Effect of different treatment concentrations of Moringa on the economical characters of diseased mulberry
silkworm, B. mori.
Conc.
(%)
Cocoon
weight(g)
Shell
weight(g)
Filament
length(m)
Filament
weight(g)
Filament
size(dn)
0.5
0.70
abc
0.11
ab
385.00
bcd
0.11
bcd
2.53
bc
1
0.71
abc
0.09
ab
452.00
abcd
0.12
abc
2.33
bcd
1.5
0.79
ab
0.13
ab
494.67
ab
0.13
abc
2.39
bcd
0.5
0.73
abc
0.07
b
462.67
abcd
0.11
bcd
2.14
cd
1
0.52
bc
0.09
ab
287.67
cde
0.07
de
2.07
cd
1.5
0.69
abc
0.13
ab
382.33
bcd
0.10
cd
2.35
bcd
1
0.52
bc
0.08
ab
276.00
de
0.10
cd
3.17
a
2
0.65
abc
0.10
ab
351.00
bcde
0.07
de
1.70
d
4
0.76
abc
0.12
ab
469.00
abc
0.12
abc
2.27
bcd
0.5
0.70
abc
0.13
ab
431.00
bcd
0.11
bcd
2.46
bc
1
0.82
a
0.14
a
506.00
ab
0.15
ab
2.62
ab
2
0.92
a
0.13
ab
626.00
a
0.16
a
2.31
bcd
Control
0.49
c
0.11
ab
193.00
e
0.05
e
2.17
cd
P value
0.0328
0.2504
0.0013
0.0002
0.0001
L.S.D.
0.2367
-
163.8
0.03986
0.626
Means in the same column not followed by the same letter are significantly different (P≤ 0.05).
Economic aspects (Cocoon indices):
Cocoon indices represented the most
important economic parameters in silk
industry. Fresh cocoon weight, cocoon
shell weight and silk ratio were recorded
in Tables (2a & 2b). Fresh cocoon weight
(g): Results in Table (1b) indicated that
all different concentrations of used
treatments recorded significant increases
in cocoon weights, compared with
control group (0.49 g). Among different
concentrations, (1% & 2%) of
Moringa
root extract exhibited the highest weights
(0.82& 0.92 g) followed by the
concentration 1.5% of
Moringa
oil (0.79
g). By the same way, data tabulated in
Table (2b) cleared that, a significant
increase in fresh cocoon weights was
noticed among all treatments; root extract
recorded the highest value (0.82 g)
followed by seed oil (0.73g). Cocoon
shell weight (g): Data represented in
Tables (1b & 2b) showed that, no
significant differences in shell weights
were noticed among investigated
treatments and with their different
concentrations. Increasing of cocoon
parameters in the present study are due to
the stimulation after using different
Moringa
extracts for treating the 5
th
instar larvae. These findings are in
confirmation with those of Rajeswari and
Isaiarasu (2004) who found that, the
dietary supplementation of leaf, flower
and pod extracts of
M. oleifera
(1% w/v)
trigger varied responses in the 5
th
instar
larvae of
Bombyx mori;
as a result of this
supplementation. The means of larval
weight and the weight and size of cocoon
were increased significantly. Patil et al.
(2005) reported that
Parthenium
root
extract motivated silkworms to feed
more, resulting in the increase of larval,
cocoon and pupal weight. Furthermore,
Mbikay (2012) noticed that, studies with
the
Moringa
plant have recommended its
efficacy in treating inflammation and
these properties of its phytochemicals
due to the presence of flavonols, and
phenolic acids which related to the anti-
inflammatory, anti-oxidants and anti-
bacterial activities. Karagiorgou et al.
(2016) cleared that, the polyphenolic
composition of
M. oleifera
roots remains
50
largely unexamined and the results
indicated that water extracts were the
richest in total polyphenols, exhibiting
strong antioxidant activity.
Table 2a: Effect of different treatments and different concentrations of Moringa on the biochemical
characters of diseased mulberry silkworm, B. mori.
Factor
Level
ALT
AST
Protease
Treatments
M. seed oil
52.94
b
106.22
ab
138.65
ab
M. leave extract
44.05
b
86.29
b
123.51
b
M. honey
42.79
b
85.97
b
121.99
b
M. root extract
73.56
a
125.54
a
168.40
a
P value
0.0024
0.0006
0.0173
L.S.D.
16.73
19.66
31.29
Concentrations
C1
46.63
b
92.05
b
127.82
a
C2
50.79
ab
96.72
b
133.75
a
C3
62.59
a
114.24
a
152.85
a
P value
0.0819
0.0303
0.1608
L.S.D.
-
17.03
-
Means in the same column not followed by the same letter are significantly different (P≤ 0.05).
Table 2b: Effect of different treatments and different concentrations of Moringa on the economical characters of diseased
mulberry silkworm, B. mori.
Factor
Level
Cocoon
weight (g)
Shell weight
(g)
Filament
length (m)
Filament
weight (g)
Filament size
(dn)
Treatments
M. seed oil
0.73
ab
0.11
a
443.89
ab
0.12
b
2.42
ab
M. leave extract
0.65
b
0.10
b
377.56
b
0.09
c
2.19
b
M. honey
0.64
b
0.10
a
365.33
b
0.10
c
2.38
b
M. root extract
0.82
a
0.13
a
521.00
a
0.14
a
2.46
a
P value
0.0396
0.1021
0.0168
0.0004
0.0026
L.S.D.
0.1342
-
103.6
0.0229
0.425
Concentrations
C1
0.66
b
0.10
a
388.67
b
0.11
b
2.58
a
C2
0.68
ab
0.11
a
399.17
b
0.10
b
2.18
b
C3
0.79
a
0.13
a
493.00
a
0.13
a
2.34
b
P value
0.0607
0.0734
0.0458
0.0042
0.0007
L.S.D.
-
-
89.68
0.199
0.368
Means in the same column not followed by the same letter are significantly different (P≤ 0.05).
Reelable filament characters:
Silk
filament characters are economically
very important. A significant increase
was recorded in silk filament characters
as a result of feeding diseased silkworm
during rearing
on mulberry leaves
supplemented with different
concentrations of natural
Moringa
extracts. Filament length (m): According
to the results obtained in Table (1b), a
significant increase in filament lengths
for all treatments with different
concentrations was exhibited; the 3
rd
concentration (2%) of root extract
showed the highest filament length (626
m), followed by the 2
nd
concentration of
it (1%) and the concentration (1.5%) of
seed oil (506 & 494.67 m) respectively,
followed by the 3
rd
concentration (4%)
of honey (469 m), while the control
group recorded the lowest length (193
m). By the same way, data in Table (2b)
revealed that
Moringa
root extract
exhibited significantly the highest silk
filament lengths (521 m) among other
treatments, followed by
Moringa
oil
51
(443.89 m) with increased
concentrations. Filament weight (g):
means of silk filament weights
represented in Table (1b), showed that
the highest weights significantly
recorded by 2% followed by 1% of
Moringa
root extract (0.16 & 0.15g)
respectively, followed by the
concentrations 1%, 1.5% of seed oil and
4% of
Moringa
honey (0.12, 0.13 and
0.12g) respectively. While the control
recorded (0.05g). Data in table (2b),
revealed that root extract treatment
significantly exhibited the highest means
values in filament weight (0.14g)
followed by seed oil treatment (0.12g)
with increased concentrations. Filament
size (dn): The results as shown in Table
(2b) indicated that, root extract treatment
was effective significantly (2.46 dn)
followed by seed oil treatment (2.42 dn).
It might be due to bioactive compounds
which have growth promoting and
nutritive nature of this plant. These
results are in line with Murugan et al.
(1998), noticed a strong correlation
between the growth of silkworm and the
silk production after the treatment of
plant extracts. And with Karthikairaj et
al. (2014), proved that aqueous and
alcoholic extracts of Ocimum, Acalypha,
and Leucas can be exploited to control
the microbial pathogens during silkworm
rearing resulting improved silk yield.
This has been further confirmed by
Fakurazi et al. (2012) and Anwar et al.
(2007) reported that, the various parts
(roots, leaves, gum, flowers and seed
infusion) of
Moringa oleifera
contain
nitrile, mustard oil glycosides and
thiocarbamate glycosides which
represent important bioactive
constituents and have wide medicinal
applicability. Conclusively, Plant
derived medicines have been part of our
conventional health care in most parts of
the world and now there is an increasing
interest in using plants as the sources of
agents to fight microbial diseases
(Sandhya et al., 2006).
So, the findings
of this study proved that
Moringa
oliefera
extracts (seeds, leaves, honey
and roots) has a protective and
therapeutic role in body cells of
silkworm
B. mori
against bacterial
infections; especially, the root and seed
oil extracts with increasing
concentrations compared with the
infected control group.
Acknowledgments
We are thankful to the Egyptian
Scientific Society of
Moringa
(ESSM),
National Research Center, Cairo, Egypt
and Plant Protection Research Institute,
Agricultural Research Center, Giza,
Egypt for supplementation test materials.
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