Journal of Phytopathology and Pest Management 7(1): 14-30, 2020
pISSN:2356-8577 eISSN: 2356-6507
Journal homepage: http://ppmj.net/
∗
Corresponding author:
Olalekan J. Soyelu,
E-mail: jlekan2001@yahoo.co.uk
14
Copyright © 2020
Insecticidal effect of lemongrass oil on behavioural
responses and biochemical changes in cowpea
weevil,
Callosobruchus maculatus
(Fabricius)
Segun E. Omotoso
1
, Bolajoko A. Akinpelu
1
, Olalekan J. Soyelu
2*
1
Department of Biochemistry and Molecular Biology, Faculty of Science, Obafemi Awolowo
University, 220005 Ile-Ife, Osun State, Nigeria
2
Department of Crop Production and Protection, Faculty of Agriculture, Obafemi Awolowo
University, 220005 Ile-Ife, Osun State, Nigeria
Abstract
Keywords: bioassay, Cymbopogon citratus, Callosobruchcus maculatus, enzymes, essential oil, insecticides.
The cowpea weevil, Callosobruchus maculatus (Fabricius), is the most important postharvest
insect pest of cowpeas throughout the tropics causing about 70% infestation within 6 months
of storage. Control interventions have often been by way of synthetic chemical application
but the negative impact of these chemicals on biotic and abiotic elements of the environment
continues to motivate search for alternative control measures. The present study was
conducted to evaluate insecticidal effect of essential oil (EO) from lemongrass, Cymbopogon
citratus (DC.) Stapf, against the weevil. Air-dried lemongrass leaves were milled into powder,
the EO was extracted by hydro-distillation using a Clevenger-type apparatus and the extract
was dried over anhydrous sodium sulphate. Acute toxicity was determined by inhalation over
a period of 24 h using five serial concentrations (0.62, 1.28, 2.50, 5.00 and 10.00 mg/ml) of the
EO on filter papers. The median lethal concentration (LC
50
) was determined from mortality
data using probit analysis and three sublethal concentrations (1.41, 2.83 and 5.66 mg/ml)
were used in subsequent antioviposition test and biochemical assays. Repellency against the
weevils was tested using 2 ml 10 mg/ml EO concentration applied evenly on ½ disc of filter
paper. Weevil homogenates were assayed for acetylcholinesterase (AChE), glutathione-S-
transferase (GST), reduced glutathione (GSH), superoxide dismutase (SOD), lactate
dehydrogenase (LDH), alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) activities. Changes in the concentrations of total protein (TPR) and glycogen (GLY) as
influenced by dosage and exposure time to the EO were also determined. The results showed
that the EO had effective insecticidal properties against the pest. Weevil mortality was
concentration- and time-dependent and 80% death rate was attained within 24 h of inhaling
10 mg/ml EO with a LC
50
value of 7.07 mg/ml. The EO repelled a significantly higher number
of weevils (66%) and fewer eggs were laid on cowpea seeds treated with the EO. The values
of AChE (P ≤ 0.01), GLY (P ≤ 0.001), SOD (P ≤ 0.001) and TPR (P ≤ 0.001) reduced
significantly with increasing EO concentrations giving an evidence of induced physiological
stress in exposed weevils. Kinetics of the assayed enzymes and physiological implications for
the storage pest were highlighted. In addition, potential of lemongrass EO as an alternative to
synthetic chemicals was discussed.
Omotoso et al., 2020
15
1. Introduction
Plant-derived insecticides are a group of
naturally occurring protective agents that
are usually safer to humans and with
minimal residual effects in the
environment compared to synthetic
pesticides. Biopesticides contain
alkaloids, rotenoids and pyrethrins and
other groups of natural chemicals that
could act as insecticides, deterrents or
repellents (Adeyemi, 2010). These
secondary metabolites protect plants
against herbivores by acting as toxicants
and interfering with reproductive system
and other physiological processes in
insect pests (Rattan, 2010). Essential oils
(EOs) are natural volatile and strongly
odorous compounds produced as
secondary metabolites by aromatic plants.
They were formed by complex volatile
mixtures of chemical compounds, with
predominance of terpene associated with
aldehyde, alcohols and ketone deposited
in various plant structures (Linares et al
.,
2005). Naturally, EOs protect plants by
fighting against bacteria, viruses and
fungi. They also act as insecticides by
functioning as feeding deterrents,
attractants or repellents (Bakkali et al
.,
2008). Some EOs have been reported to
possess ovicidal and larvicidal properties
against various insect pests (Cetin et al
.,
2004; Isman, 2000). Studies by Ayvaz et
al. (2010) showed that EOs from oregano
and savory were highly effective against
Plodia interpunctella
(Hübner) and
Ephestia kuehniella
Zeller, with 100%
mortality obtained after 24 h at 9 and 25
µl/l. Kabera et al. (2011) also reported
100% mortality in maize weevil,
Sitophilus zeamais
(Motschulsky), treated
with EOs of
Pelargonium graveolens
L’Hér. and
Cymbopogon citratus
(DC.)
Stapf.
Cymbopogon citratus
commonly
known as citronella grass or lemongrass,
is a bunch of perennial grass growing to a
height of 1 m with numerous stiff leafy
stems arising from short rhizomatous
roots with an economic lifespan of about
5 years (Carianne, 2005). Many
ethnobotanical and medicinal uses of the
plant have been documented in literature
(Tripathi et al., 2009). The EO in
lemongrass is biosynthesized in the
rapidly growing leaves and stored in
specific oil cells in the parenchymal
tissues (Santoro et al
.,
2007; Luthra et al
.,
1999). The major constituent of
lemongrass oil is citral (a mixture of
geranial and neral) and other unusual
active components include limonene,
citronellal, ß-myrcene and geraniol
(Schaneberg & Khan, 2002). Studies
have shown that citral reduces attractivity
of sex pheromone in the codling moth,
Cydia pomonella
(L.), thus acting as a
mating disruptor (Hapke et al
.,
2001).
Stored product insects constitute pest
problems all over the world causing
significant economic losses. The cowpea
weevil,
Callosobruchus maculatus
(Fabricius), is a cosmopolitan field-to-
store insect pest ranked as the principal
post-harvest pest of cowpea in the tropics
(Caswel, 1981). It causes substantial
quantitative and qualitative losses
manifested by seed perforation and
reductions in weight, market value and
germination ability of seeds (Oluwafemi,
2012). The use of synthetic insecticides
to control pest infestation has impacted
drastically on biotic and abiotic elements
of the environment through excessive or
indiscriminate use (Al-Zaidi et al
.,
2011).
Some of the negative impacts are
contamination of food and water sources,
toxicity against beneficial insects and
pesticide resistance in target insects
(Kumar et al
.,
2008). A large number of
synthetic pesticides exhibit potential for
biomagnification, thereby, affecting food
chains adversely (Gavrilescu, 2005;
Linde, 1994). Detoxification is the
metabolic process by which toxins or
contaminated resources are changed into
Omotoso et al., 2020
16
less toxic or more readily excretable
substances. Several defensive
mechanisms and biochemical reactions
are involved in detoxification processes
against chemical intruders. These
mechanisms predominantly involve either
metabolic detoxification of the insecticide
before it reaches its target site or the
sensitivity of the target site changes so
that it is no longer responsive to the
active ingredient (Sun, 1992; Hama et al.,
1987). A number of enzymes such as
esterase, glutathione-S-transferase (GST),
superoxide dismutase (SOD), reduced
glutathione (GSH) and lactate
dehydrogenase (LDH) participate in
various defensive mechanisms (Nedal &
Hassan, 2009). The present study
investigated repellency and
antioviposition activity of lemongrass oil
against the cowpea weevil. It also
assessed the effects on oxidative stress
enzymes (SOD and GSH), xenobiotic
detoxifying enzyme (GST),
neurochemical enzyme
(acetylcholinesterase: AChE) and energy
metabolism biomolecules (glycogen and
protein) in the insect pest with a view to
establishing the insecticidal efficacy of
the EO.
2. Materials and methods
2.1 Insect rearing
Callosobruchus maculatus
was reared on
cowpea seeds inside the Insect
Physiology Laboratory, Faculty of
Agriculture, Obafemi Awolowo
University, Ile-Ife at 28 ± 2°C and 75 ±
5% RH. Cowpea seeds were bought from
the local market and cleaned by
handpicking. The pack of seeds was then
kept in a deep freezer (sub-zero) for at
least a week to eliminate infesting
insects, mites or disease-causing
microorganisms. At the end of the cold
treatment, cowpea seeds were poured
into 2 L glass jars, cowpea weevils were
introduced and the containers were
covered with muslin cloth for ventilation.
The insects were reared for about three
generations before newly-emerged adult
weevils were selected for different
experiments.
2.2 Extraction of essential oil from
lemongrass
Fresh leaves of
C. citratus
were collected
from Mokuro Road, Ile-Ife and
authenticated at Ife Herbarium,
Department of Botany, Obafemi
Awolowo University. The materials were
air-dried until crispy and milled to
powder using a mill with rotatory knives
for proper extraction. The essential oil
was extracted from milled leaves
following the procedure of hydro-
distillation described by Papachristos and
Stamopoulos (2004). Powdered
C.
citratus
(500 g) was mixed in distilled
water (3000 ml) and hydro-distilled for 6
h in a Clevenger apparatus. The oil was
dried over anhydrous sodium sulphate
(Na
2
SO
4
) to remove water molecules and
refrigerated at 4°C for subsequent
experiments.
2.3 Toxicity of lemongrass oil against
adult
Callosobruchus maculatus
The acute toxicity of
C. citratus
oil
against
C. maculatus
was investigated by
inhalation according to the method
described by Ogunsina et al
.
(2011).
Separate Whatman No. 1 filter papers
were treated with different
concentrations (0.62, 1.28, 2.50, 5.00 and
10.00 mg/ml) of
C. citratus
oil while
Omotoso et al., 2020
17
other filter papers were treated separately
with ethanol and distilled water for
comparison of results. The filter papers
were air-dried for ethanol to evaporate
after which each of them was placed
inside 4.5 cm diameter Petri dishes. Ten
newly emerged
C. maculatus
were
transferred into each Petri dish which was
then covered and its side sealed using a
paper tape to prevent escape of vapour.
Each treatment was replicated three times
and insects were monitored for signs of
toxicity and mortality at 1, 6, 12, 18 and
24 h after exposure. The number of
insects that were dead at each observation
hour was recorded per treatment.
2.4 Repellency test
A slight modification of the still-air
olfactometer described by Weeks et al
.
(2011) was used to evaluate the action of
C. Citratus
EO against
C. maculatus.
A
Whatman’s No. 1 filter paper was
divided into two halves; 2 ml 10 mg/ml
essential oil was applied evenly on one
while 2 ml 95% ethanol was applied on
the other half using a micropipette. The
two halves were allowed to air-dry for 5-
10 min before they were attached
lengthwise and placed inside the Petri
dish. Twenty newly emerged adult
C.
maculatus
were released in the middle of
the Petri dish and proportion (%) of
weevils that settled on each half of the
filter paper disc was determined after 1 h
as
If in a trial 30% of introduced weevils
were found in the EO portion of the disc,
it means that percent repellency by the oil
was 70%. A total of 20 trials was carried
out and percent repellency due to the
essential oil and ethanol was recorded
accordingly.
2.5 Anti-oviposition activity of
lemongrass oil
The method described by Parugrug and
Roxas (2008) was followed with little
modification. Cowpea grains (50 g) were
treated with three sublethal
concentrations (1.41, 2.83 and 5.66
mg/ml) of the EO in triplicated 1.5 L
glass jars. Jars containing grains for
positive and negative control
experiments were treated with distilled
water and 95% ethanol, respectively. The
jars were left opened long enough for the
solvent to evaporate after which an
aspirator was used to introduce 30 newly
emerged C.
maculatus
into each of them.
The jars were then covered with net
material and left for 7 days. At the
expiration of this period of oviposition,
the weevils were removed and each grain
per glass jar was examined carefully
under a dissecting microscope to count
the number of eggs laid on the surface.
Eggs (about 0.75 mm long) are glued on
the surface of seeds and they appear as
whitish specks.
2.6 Preparation of insect homogenate
The weevils were reared on cowpea
grains that had been treated with the
three sublethal concentrations listed in
subsection 2.5 and insect homogenate
was prepared using the method of
Upadhyay (2011). The insects (200 mg)
were homogenized in 1.5 ml freshly
prepared phosphate buffer (100 mM, pH
6.8) and the homogenate was centrifuged
at 12,000 rpm for 20 min using Ice-cold
Omotoso et al., 2020
18
Bench Centrifuge (Searchtech 90-2). The
supernatant was carefully collected in a
clean vial and stored in a freezer until it
was needed for biochemical assays.
2.7 Determination of total protein
concentration in insect homogenate
Protein estimation was carried out using
the method of Lowry et al
.
(1951). Insect
homogenate (0.2 ml) was added to 2.1 ml
alkaline copper reagent which was
freshly prepared by mixing 2% Na
2
CO
3
in 0.1 M NaOH, 1% CuSO
4
.5H
2
O and
1% Na-K tartrate.4H
2
O (98:1:1 v/v/v).
The mixture was vortexed and allowed to
stand for 10 min followed by the addition
of 0.2 ml Folin-Ciocalteu colour reagent.
The resulting reaction mixture was
vortexed and allowed to stand at room
temperature in the dark for an hour after
which absorbance of the solution was
read at 550 nm against a reagent blank.
The blank was made of 0.2 ml distilled
water and appropriate volume of the
diluents and colour reagent. The protein
concentration of the homogenate was
estimated from a standard curve obtained
using Bovine Serum Albumin (BSA).
2.8 Estimation of reduced glutathione
Reduced glutathione (GSH) was
measured according to the method of
Beutler et al
.
(1963). One millimetre (1
ml) of the supernatant was added to 0.5
ml 10 mM Ellman’s reagent (5,5'-dithio-
bis-[2-nitrobenzoic acid]) and 2 ml
phosphate buffer (0.2 M, pH 8.0) was
added. The yellow colour developed was
read at 412 nm with a blank containing
3.5 ml phosphate buffer. A series of
standards were also treated in similar
manner and the amount of GSH was
expressed in mg/200 mg homogenate.
2.9 Glutathione-S-transferase activity
Activity of Glutathione-S-transferase
(GST) was determined as described by
Habig et al
.
(1974). Into a clean, dry test
tube was pipetted 2.7 ml sodium
phosphate buffer (0.1 M; pH 6.5), 0.1 ml
30 mM reduced glutathione and 0.1 ml
30 mM 1-chloro-2,4-dinitrobenzene
(CDNB). To the resulting mixture, 0.2
ml of insect hemolysate was added. The
absorbance of the reacting mixture was
measured at 340 nm at 15 s intervals for
3 min. Distilled water (0.2 ml) was used
in lieu
of hemolysate for the blank. The
GST activity was calculated as below:
Where
= extinction coefficient of
CDNB (9.6 mM
-1
cm
-1
). ΔA/min =
change in absorbance per minute. V =
reaction volume. DF = dilution factor. v
= sample volume.
2.10 Estimation of Superoxide
dismutase
Superoxide dismutase (SOD) was
estimated as described by McCord and
Fridovich (1969). To 50 μL lysate,
75 mM Tris-HCl buffer (pH 8.2), 30 mM
EDTA and 2 mM pyrogallol were added
and thoroughly mixed. An increase in
absorbance was recorded at 420 nm for
3 min. One unit of enzyme activity
represented 50% inhibition of the rate of
autooxidation of pyrogallol as
determined by change in absorbance/min
at 420 nm. The activity of SOD was
measured in unit/mg protein as expressed
below:
Omotoso et al., 2020
19
Where A
0
= absorbance after 30 s. A
3
=
absorbance after 150 s.
(ΔA/min)
Blank
= Increase in absorbance
per minute for blank. (ΔA/min)
Test
=
Increase in absorbance per minute for test
sample. 1 unit of SOD activity was given
as the amount of SOD necessary to cause
50% inhibition of the oxidation of
pyrogallol.
2.11 Lactate dehydrogenase activity
The activity of Lactate dehydrogenase
(LDH) in insect homogenate was assayed
using Randox Diagnostic Kit (Randox
Laboratories Ltd, Antrim, UK) as
described by Weisshaar et al. (1975).
Insect homogenate (0.02 ml) was pipetted
into 1 cm cuvette at room temperature
(25°C) and the reaction was initiated by
adding 1.0 ml substrate. The initial
absorbance was taken after 12 s at 340
nm and repeated first, second and third
minute consecutively so as to obtain
change in absorbance/min. The LDH
activity was calculated as expressed
below:
LDH activity (U/L) = 4127 × ∆A 340 nm/min
Where ∆A = change in absorbance.
2.12 Alanine aminotransferase activity
Alanine aminotransferase activity (ALT)
was estimated according to the method of
Reitman and Frankel (1957) using
Randox Diagnostic Kit. The homogenate
(0.1 ml) was pipetted into clean test tubes
in triplicates and 0.5 ml of the buffer was
added and mixed gently. The reaction
mixture was incubated at 37°C for 30
min in a water bath. The reaction mixture
was cooled and 0.5 ml 2.0 mM 2, 4-
dinitrophenlhydrazine was added. The
mixture was mixed properly and allowed
to stand at room temperature for
additional 20 min after which 5 ml 0.4 M
NaOH solution was added and mixed
thoroughly. The absorbance of the
reaction mixture was taken at 546 nm
against a blank prepared with distilled
water (0.1 ml) in place of the sample.
The enzyme activity (expressed in IU/L
protein) was extrapolated from a
calibration curve obtained from an
absorbance-enzyme activity table of
values provided by the kit manufacturer.
2.13 Aspartate aminotransferase
activity
The procedure was similar to that
described for aminotransferase (AST)
except that the buffered substrate
consisted of 100 mM phosphate buffer
(pH 7.4), 100 mM L-aspartate and 2 mM
α-ketoglutarate.
2.14 Acetylcholinesterase activity
Acetylcholinesterase (AChE) activity
was determined according to the method
of Ellman et al. (1961). Into microplate
well was added, in triplicate, 240 µl
buffer (0.1 M phosphate buffer, pH 8.0)
and 20 µl insect homogenate. The
reaction mixture was incubated in a dry
bath incubator for 30 min at 37°C after
which 20 µl 25 mM acetylthiocholine
iodide and 20 µl 10 mM 5,5'-dithio-bis-
[2-nitrobenzoic acid] were added.
Change in absorbance at 30 s intervals
Omotoso et al., 2020
20
was measured spectrophotometrically for
4 min at 412 nm. The enzyme activity
was calculated as follows:
Where ∆A/min = change in absorbance
per minute. V = total volume of reaction
mixture. v = volume of test sample in
reaction mixture.
= extinction
coefficient of DTNB (1.36 x 10
4
mM
-1
cm
-1
). d = light path length (1cm).
2.15 Estimation of Glycogen
concentration
The insect glycogen was isolated and
estimated according to the method of
Oyedapo and Araba (2001). Insects (200
mg) were homogenized and transferred
into separate test tubes containing 10 ml
30% (w/v) KOH. The tubes were heated
at 70ºC until the tissues were fully
digested. Distilled water was added to the
resulting burgundy red suspension to
make the volume 20 ml followed by the
addition of 18 ml 95% (v/v) cold ethanol.
The mixture was mixed by inversion and
chilled with ice. A white flocculent
precipitate of impure glycogen was
formed and this was collected by
centrifugation at 3,000 rpm for 15 min.
The precipitate was dissolved in 15 ml
5% (w/v) cold trichloroacetic acid (TCA)
and stirred vigorously after which it was
heated at 85ºC for 5 min. The suspension
was centrifuged at 5,000 rpm for 10 min
and 25 ml cold 95% (v/v) ethanol was
added to the supernatant, mixed by
inversion and allowed to precipitate the
glycogen. The precipitate was collected
by centrifugation at 3,000 rpm for 10
min. The precipitated glycogen was
dissolved in 5 ml distilled water and
shaken thoroughly until fully dissolved.
To the solution was added 12 ml 95%
(v/v) ethanol, while mixing with stirring
rod and a white flocculent precipitate of
pure glycogen was formed. The
precipitate was centrifuged at 2,000 rpm
for 10 min and the residue obtained was
kept in a deep freezer for analysis. The
standard glycogen calibration curve was
prepared by pipetting 0, 0.2, 0.4, 0.6, 0.8
and 1.0 ml of the working solution (250
µg/ml), in triplicates, into clean dry test
tubes and volumes were adjusted to 1.0
ml with distilled water. To each of the
tubes was added 0.5 ml concentrated
HCl, followed by 0.5 ml 88% (v/v)
formic acid and 2.0 ml Anthrone reagent
(0.02 g in 68% (v/v) sulphuric acid). The
tubes were transferred into boiling water
for 10 min after which they were cooled
under running water. The absorbance
was read at 630 nm against the blank and
the insect glycogen concentration was
extrapolated from the standard
calibration curve.
2.16 Statistical analysis
Percent mortality data were subjected to
square root transformation before
analysis of variance (ANOVA) was
carried out and mean values were
separated using Tukey’s HSD test.
Percent repellency data were also
transformed and analyzed in a similar
manner but mean values were separated
using Fisher’s LSD at 0.05 level of
probability. On the other hand, count
data obtained from anti-oviposition
experiment were subjected to natural log
transformation before ANOVA and mean
values were separated using Tukey’s
HSD test. The median lethal
concentration (LC
50
) was determined
Omotoso et al., 2020
21
from mortality data using probit analysis
in Microsoft Excel. Bioassay data were
subjected to ANOVA procedure and
mean values were separated using
Tukey’s HSD test.
3. Results and Discussion
Generally, insect mortality was dose- and
observation time-dependent (Table 1).
Mortality was reported among insects
exposed to 10 mg/ml EO within 1 h of
application and it increased to an
excellent level of 80% within 24 h. The
maximum efficacies attained by other
concentrations were 25% (2.5 mg/ml and
5.0 mg/ml), 10% (1.28 mg/ml) and 15%
(0.62 mg/ml). Ketoh et al. (2000) in an
earlier study recorded > 90% mortality in
C. maculatus
within 24 h of exposure to
EOs from two
Cymbopogon
spp. This
report and the results obtained in the
current study indicated that members of
the genus
Cymbopogon
may possess
good insecticidal properties against the
cowpea weevil. The 24 h LC
50
obtained
for the EO against cowpea weevil was
7.07 mg/ml. This is an indication that
toxicity of the EO against
C. maculatus
is
superior to those of
Cordia millenii
Baker
(Manjack) (LC
50
= 36.3 mg/ml)
, Zingiber
officinale
Roscoe (Ginger) (LC
50
= 37.5
mg/ml),
Xylopia aethiopica
(Dunal)
(Negro pepper) (LC
50
= 43.8 mg/ml),
Monodora myristica
(Gaertn.) (Nutmeg)
(LC
50
= 47.5 mg/ml) and
Allium sativum
L. (Garlic) (LC
50
= 55.0 mg/ml) even at
96 h post-treatment (Edwin & Jacob,
2017). The lemongrass EO exhibited
significant repelling and oviposition
deterrence abilities against the cowpea
weevil (Table 2). Approximately ⅔ of
tested weevils was repelled by the oil and
this effectiveness corroborates an earlier
report (Jayasingha et al., 1999)
establishing repelling efficacy of the
essential oil against
Dacus dorsalis
(Diptera: Tephritidae). The anti-
oviposition effect was dose-dependent
with an average of 16 eggs laid on
cowpea grains treated with 1.44 mg/ml
EO. This was not significantly different
from the number of eggs laid on grains
without plant extract while significantly
fewer eggs were laid when higher
concentrations of EO were applied. This
report is in agreement with Ketoh et al
.
(2000) where EOs of
C. citratus
and
C.
nardus
(L.) Rendle elicited significant
dose-dependent oviposition deterrence in
C. maculatus
. Plant EOs have also been
reported to have ovicidal effect on eggs
of
C. chinensis
(Dwivedi & Kumari,
2000; Pathak et al
.,
1997).
Table 1: Percent mortality of cowpea weevils exposed to serial concentrations of lemongrass oil over a period of 24 hours.
Time (h)
Concentration of essential oil (mg/ml)
Ethanol
Blank
10.00
5.00
2.50
1.28
0.62
1
15cA
0bB
0cB
0aB
0bB
0aB
0aB
6
35bA
15aAB
5bcBC
0aC
0bC
0aC
0aC
12
45bA
15aAB
10abcAB
10aAB
5abAB
5aAB
0aB
18
75aA
20aB
20abB
10aBC
5abBC
5aBC
0aC
24
80aA
25aAB
25aAB
10aB
15aB
5aB
5aB
Values with similar capital letters in the same row and similar small letters in the same column are not significantly different at 0.05
level of probability.
Omotoso et al., 2020
22
Table 2: The repelling efficacy (A) and antioviposition effect (B) of lemongrass oil against the cowpea weevil.
A
Proportion of cowpea weevils repelled (%)
Lemongrass oil (10 mg/ml)
Ethanol (extraction solvent)
LSD
0.05
65.83 ± 0.32
34.17 ± 0.19
15.45
B
Average number of eggs laid on cowpea seeds
Concentration of lemongrass oil (mg/ml)
Ethanol
Distilled water
5.66
2.83
1.41
0.33c
4.00b
16.00a
17.33a
22.67a
Values with similar letters in the same row are not significantly different at 0.05 level of probability.
This has been attributed to ability of the
oils to penetrate egg chorion, thereby,
creating the deleterious hypercarbic
condition (Don-Pedro, 1989). The mean
square value and contribution of each
source of variation to activity of each
biochemical entity that was quantified in
the present study is presented in Table
(3). Time post-exposure and
concentration of essential oil
had
significant effect (
P
≤ 0.001) on activity
of superoxide dismutase and total protein.
They also affected activity of
acetylcholinesterase, glycogen and lactate
dehydrogenase significantly. However,
these two experimental sources of
variation did not have any significant
effect on activity of alanine
aminotransferase. Time post-exposure
accounted for most of the variation in the
activity of acetylcholinesterase (35%),
aspartate aminotransferase (20%),
alanine aminotransferase (11%), reduced
glutathione (35%), glutathione-S-
transferase (21%) and superoxide
dismutase (47%) while concentration of
essential oil accounted for a larger
percentage in the case of glycogen
depletion (75%), superoxide dismutase
(28%) and total protein (65%).
Table 3: Mean square values from analysis of variance and percent contribution of each source of variation to
biochemical reactions in cowpea weevils exposed to lemongrass oil.
Source of variation
df
Mean square value
AChE
AST
ALT
GLY
GSH
GST
LDH
SOD
TPR
Time post-exposure
3
0.001064***
(35.36%)
0.006382*
(20.11%)
0.000709
(10.91%)
76.683**
(9.17%)
0.002849***
(34.79%)
4.75 × 10
-7
*
(21.18%)
6.068*
(20.45%)
0.175***
(46.97%)
2.360***
(17.53%)
Concentration of
essential oil
4
0.000497**
(2.20%)
0.001368
(5.75%)
0.000280
(5.74%)
469.846***
(74.88%)
0.000605
(9.85%)
1.28 × 10
-7
(7.62%)
5.426*
(24.39%)
0.077***
(27.66%)
6.535***
(64.75%)
Replication
1
0.000004
(0.04%)
0.003054
(3.21%)
0.000281
(1.44%)
24.613
(0.98%)
0.001172
(4.77%)
2.60 × 10
-14
(0.00%)
0.000
(0.00%)
0.008
(0.72%)
0.000
(0.00%)
Error
39
0.000124
(42.58%)
0.002179
(70.94%)
0.000515
(81.91%)
12.124
(14.97%)
0.000401
(50.59%)
1.54 × 10
-7
(71.20%)
1.583
(55.16%)
0.009
(24.65%)
0.231
(17.72%)
R
2
0.574
0.291
0.181
0.850
0.494
0.288
0.448
0.753
0.823
CV
46.60
13.21
10.41
19.96
27.83
49.10
20.69
34.19
16.16
*,**,*** significance at 0.05, 0.01 and 0.001 levels of probability, respectively. Percent contribution of each source of variation to
changes in biochemical activity within exposed insects is presented in parentheses. AChE: Acetylcholinesterase, AST: Aspartate
aminotransferase, ALT: Alanine aminotransferase, GLY: Glycogen, GSH: Reduced glutathione, GST: Glutathione-S-transferase,
LDH: Lactate dehydrogenase, SOD: Superoxide dismutase, TPR: Total protein
This is an indication that the last three
biochemical parameters were more
sensitive to the active fraction of the
EO. The
in vitro
biochemical activity of
tested weevils, in both dose- and time-
dependent situations, is presented in
Tables (4) and (5), respectively.
Activity of alanine aminotransferase
and reduced glutathione did not vary
with concentration of EO whereas
reduction in acetylcholinesterase,
aspartate aminotransferase and
glutathione-S-transferase activity, and
depletion of glycogen and total protein
Omotoso et al., 2020
23
were more pronounced at highest EO
concentration of 5.66 mg/ml. The
highest lactate dehydrogenase activity
was recorded when 5.66 mg/ml EO was
applied and the oil, irrespective of its
concentration, clearly reduced the
activity of superoxide dismutase
.
Activity of alanine aminotransferase
remained comparable between the 6
th
and 24
th
hour of exposure whereas the
suppression of glutathione-S-transferase
by the EO waned with time post-
treatment. Acetylcholinesterase and
aspartate aminotransferase activity
increased significantly after 18 h of
inhalation while a similar trend was
observed in superoxide dismutase
activity after 12 h. Glycogen depletion
increased with time post-exposure to the
EO and protein depletion was more
evident at the 6
th
hour of observation.
Acetylcholinesterase is a serine protease
found at cholinergic synapses where it
terminates synaptic transmission by
hydrolysis of the neurotransmitter
acetylcholine. Some insecticidal agents
have been reported to inhibit
acetylcholinesterase activity, thereby,
producing a deleterious effect on the
insect as it causes excessive
accumulation of the neurotransmitter.
This results in hyperactivity, paralysis
and eventual death of insect (Fournier
& Mutero, 1994). During normal
function of AChE, a serine-histidine-
glutamate triad, located in the active
site of the enzyme, catalyzes the
hydrolysis of acetylcholine in a step-
wise manner releasing choline and
acetic acid (Quinn, 1987). Usually,
acetylcholinesterase inhibitors attack
the serine hydroxyl group in the
enzyme active site and form a covalent
bond (Mercey et al., 2012). The AChE
inhibition observed in this study could
be due to the formation of a complex at
the enzyme active site but the inhibition
was not irreversible as AChE activity
increased significantly after 18 h of
exposure to the EO.
Table 4: Activity of biochemical parameters determined in cowpea weevils exposed to serial concentrations of
lemongrass oil.
Concentration
of EO (mg/ml)
Biochemical activity
AChE
AST
ALT
GLY
GSH
GST
LDH
SOD
TPR
0
0.035a
0.380a
0.215a
26.049a
0.076a
0.0010a
6.294ab
0.450a
3.884a
1.41
0.027ab
0.363b
0.224a
17.426b
0.074a
0.0009a
5.877bc
0.213b
2.802b
2.83
0.024abc
0.347c
0.219a
12.486c
0.064a
0.0008a
6.157abc
0.248b
2.572b
5.66
0.014c
0.338d
0.223a
7.576d
0.062a
0.0005b
7.183a
0.227b
1.757c
n-hexane
0.019bc
0.378a
0.210a
23.683a
0.083a
0.0010a
4.895c
0.242b
3.850a
Values with similar letters in the same column are not significantly different at 0.05 level of probability. AChE:
Acetylcholinesterase, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase, GLY: Glycogen, GSH: Reduced
glutathione, GST: Glutathione-S-transferase, LDH: Lactate dehydrogenase, SOD: Superoxide dismutase, TPR: Total protein.
Table 5: Activity of biochemical parameters determined at 6-hour intervals in cowpea weevils exposed to serial
concentrations of lemongrass oil.
Time post-
exposure (h)
Biochemical activity
AChE
AST
ALT
GLY
GSH
GST
LDH
SOD
TPR
6
0.016b
0.344b
0.224a
20.433a
0.057c
0.0006c
6.531ab
0.180b
2.394c
12
0.018b
0.354ab
0.210a
19.072a
0.075b
0.0007bc
5.204c
0.150b
2.762b
18
0.022b
0.328b
0.227a
14.520b
0.094a
0.0010a
6.910a
0.425a
3.277a
24
0.039a
0.388a
0.211a
15.750b
0.062bc
0.0010a
5.680bc
0.348a
3.459a
Values with similar letters in the same column are not significantly different at 0.05 level of probability. AChE:
Acetylcholinesterase, AST: Aspartate aminotransferase, ALT: Alanine aminotransferase, GLY: Glycogen, GSH: Reduced
glutathione, GST: Glutathione-S-transferase, LDH: Lactate dehydrogenase, SOD: Superoxide dismutase, TPR: Total protein.
Omotoso et al., 2020
24
The reduced glutathione (GSH), a free
radical scavenger, is known to be an
important cellular protectant against
reactive oxygen metabolites in many
insect cells that are concerned with
pesticide detoxification (Büyükgüzel,
2009). The enhanced GSH level
observed in this study within 18 h of
inhaling fumes from the EO could be an
adaptive mechanism which allows the
insects to scavenge the free radical
damaging systems thereby maintaining
the integrity of the cell membrane and
other biomolecules like proteins,
polysaccharides and DNA. Glutathione-
S-transferase (GST) which catalyzes the
conjugation of GSH with a variety of
electrophilic metabolites participates in
defense against oxidative stress. It does
this by detoxifying endogenous harmful
compounds like hydroxyl alkenal and
base propenal or DNA hydroperoxides
and electrophilic xenobiotic, and is
known to provide protection against
oxidative/nitrosative stress by GSH-
mediated process of reactive products of
lipid peroxidation (Kim et al
.,
2003).
Similar to GSH, the GST activity was
induced and this might indicate an
adaptive response to the oxidative stress
triggered by the applied EO. This
probably suggests involvement and
activation of GST-dependent xenobiotic
metabolism (Kolawole et al
.,
2011). The
induction of GST is considered
beneficial to handle environmental stress
(van der Oost et al
.,
2003) while
overexpression of GST could be an
important means of cell protection during
physiological stress. The GST
stimulation in treated insects might be
due to citral that is in the EO. It is a
constituent of
C. Citratus
oil-made of
mixture of geranial and neral
stereoisomers. Citral was reported to
possess a significant ability to suppress
oxidative stress probably through
induction of the endogenous antioxidant
glutathione system (Nakamura et al
.,
2003). Superoxide dismutases (SODs)
are metalloproteins with sufficient
activity for dismutation of superoxide
anions that are produced as a result of
oxidative stress (Beedham et al
.,
1995)
thereby protecting cells against damage.
The SOD reacts with superoxide radicals
and converts them to H
2
O
2
, catalyzed by
catalase or GSH peroxidase (Kakkar et
al
.,
1984). It could be inferred that the
time-dependent increase in SOD activity
in treated bean weevils was a possible
survival mechanism in order to reduce
possible oxidative (or toxic) stress posed
by the applied essential oil (Al-Omar et
al
.,
2004). Aspartate aminotransferase
(AST) and alanine aminotransferase
(ALT) are key enzymes that catalyze the
synthesis of glutamic acid from aspartic
acid and alanine, respectively. These
important enzymatic processes usually
occur in the final stage of development
in insects (Gowda & Ramaiah, 1976)
and changes in transaminase levels are
consistent with the sum total of
anabolism and catabolism of protein.
The time-dependent increase in AST
activity observed in this study was a
form of physiological response aimed at
initiating amino acid synthesis with a
view to overcoming damage caused by
the EO-induced stress (Gowda &
Ramaiah, 1976). Lactate dehydrogenase
(LDH) is an important glycolytic
enzyme that is present in almost all
Omotoso et al., 2020
25
animal tissues (Kaplan & Pesce, 1996).
The enzyme helps in energy generation
especially when a considerable amount
of additional energy is required
immediately (Nathan et al
.,
2006). A
marginal reduction in LDH activity was
observed when lower concentrations
(1.41 and 2.83 mg/ml) of the EO were
applied. The decrease denotes reduction
in energy metabolism of the insect
probably because of the effect of toxic
phytoconstituents on membrane
permeability, especially of the gut
epithelium (Nathan et al
.,
2006).
Reduction in LDH activity could also be
due to a physiological disturbance at the
molecular level as a result of depression
or mutation of genes responsible for
biosynthesis of the polypeptide chains
that build LDH (Hassanien et al
.,
1996).
However, a stress-induced marginal
increase in LDH activity resulted when a
higher concentration (5.66 mg/ml) of the
oil was applied, apparently as a means of
compensating for depleted protein. The
depletion of glycogen in stressed
organisms is associated with an increase
in the utilization of food reserve (Sancho
et al
.,
1998). The consistent
concentration-dependent reduction in
glycogen level reported in the current
study is an evidence that treated weevils
were under stress and they needed
additional energy to match the increase
in demand as a result of the physiological
stress induced by the EO. Inhalation of
the EO caused significant protein
depletion in bean weevils and this was
more evident within the first 12 h of
exposure. Protein depletion correlates
with break-down of proteins into amino
acids, and their entrance into the Krebs
cycle as keto acid. Thus, protein
depletion in tissues plays a role in
compensatory mechanisms under
insecticidal stress to provide
intermediates in the Krebs cycle (Shekari
et al
.,
2008; Zibaee et al
.,
2008). Protein
depletion in a stressed organism could
also be attributed to inability of protein-
synthesizing mechanisms to function
properly. The subsequent increase in
total protein level beyond 12
th
hour of
inhalation could be due to ability of the
insects to counter damaging effects of
the EO through specific physiological
mechanisms. The lemongrass oil
exhibited effective insecticidal action
against the cowpea weevils and could,
therefore, be considered as an alternative
agent for protecting cowpea seeds in
storage, especially, by rural farmers in
tropical and subtropical regions.
Insecticides are costly and not
sustainable in the long run due to
environmental contamination whereas
EOs contain a range of selective
bioactive chemicals with insignificant or
no harmful effect on non-target
organisms and the environment
(Vinayaka et al., 2010). The use of plant
oil would be cost effective and
sustainable, especially considering that
this plant is easy to grow. In addition,
the grass is safe to users as evidenced by
the fact that it is used as culinary spices
and herbs.
Author Contributions
BA conceptualised this work as part of
SE’s postgraduate Degree program. The
research was co-designed and executed
by BA, SE and OJ; BA and SE carried
Omotoso et al., 2020
26
out the biochemical assays while OJ and
SE investigated the behavioural
responses. SE, BA and OJ analysed
obtained data and prepared the
manuscript for publication.
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