Journal of Photochemistry and Photobiology B: Biology 135 (2014) 16

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

Ultraviolet-B light induced oxidative stress: Effects on antioxidant

response of Spodoptera litura
Sengodan Karthi, R. Sankari, Muthugounder S. Shivakumar
Molecular Entomology Lab, Department of Biotechnology, Periyar University, Salem 11, Tamil Nadu, India

a r t i c l e

i n f o

Article history:
Received 18 January 2014
Received in revised form 18 March 2014
Accepted 7 April 2014
Available online 19 April 2014
Keywords:
Ultraviolet light
Oxidative stress
Antioxidant enzymes
Spodoptera litura
Reactive oxygen species

a b s t r a c t
Ultraviolet light (UV-B), which emits radiation in the range of 280315 nm, has been used worldwide in
light trapping of insect pests. In this article, we test the hypothesis that one of the duration of UV-B exposure has a differential impact on oxidative stress marker enzymes in Spodoptera litura. Effect of UV-B
exposure on total protein and antioxidant activities of superoxide dismutase (SOD), catalase (CAT),
peroxidases (POX) and glutathione-S-transferase (GST) were investigated in S. litura. The adults were
exposed to UV-B light for various time periods (0, 30, 60, 90 and 120 min). We found that exposure to
UV-B light for 30 and 60 min resulted in increased activities of POX. When the exposure time lasted
for 60 and 90 min, the activities of SOD remained signicantly higher than the control. However, the
POX, CAT and GST activity decreased to control levels at 90 and 120 min. whereas relatively long duration
exposure activates the xenobiotics detoxifying enzymes like GST and POX and CAT enzymes. Longer UV-B
exposure may interfere with pesticide detoxication mechanism in insects, making them more susceptible to insecticides.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Insects are exposed to various abiotic and biotic stress throughout their life time, which can induce excessive reactive oxygen
species (ROS). To protect against the damage of ROS, insects have
evolved a complex network of enzymatic antioxidant systems,
which include enzymatic and non-enzymatic components [1]. UV
radiation can cause oxidative stress in insects [2] and may disturb
the functional activity of protein [3]. Organisms that live in surface
region of the earth have evolved many mechanisms to reduce UV
irradiation damage including behavioral avoidance of UV exposure,
acquisition of sunscreens, repair of macromolecules such as proteins and DNA or elimination of ROS and toxic compounds created
by UV exposure [4]. Reactive oxygen species (ROS), including superoxide anion, hydrogen peroxide and hydroxyl radical, are thought
to be generated during normal oxidative processes in all aerobic
organisms. It is believed that low levels of ROS are not harmful to
cells and play an important role in cell signaling and the induction
of host defense genes [2,5]. However, under environmental stress,
UV radiation, ROS level may increase dramatically and result in oxidative stress [3]. Only a small portion of ROS is scavenged by dietary
antioxidants, such as ascorbate and carotenoids, whereas most are

Corresponding author. Mobile: +91 9942044479; fax: +91 0427 2345124.

E-mail address: skentomol@gmail.com (M.S. Shivakumar).
http://dx.doi.org/10.1016/j.jphotobiol.2014.04.008
1011-1344/ 2014 Elsevier B.V. All rights reserved.

eliminated by a suite of antioxidant enzymes [6]. Ultraviolet-B radiation (UV-B; 280315 nm) is a small fraction of the solar spectrum
received at ground level. In spite of its modest contribution to the
total quantum ux, UV-B can be an important modulator of biological processes in terrestrial ecosystems [7]. Due to deleterious
effects of ultraviolet-B radiation (UVB; 280315 nm), its application
has been considered in pest control, particularly for organisms such
as insects and mites [8] are one of the agricultural pests that are
hardest to control because of their rapid development of pesticide
resistance [9]. Therefore, investigation of their susceptibility to
UV-B is important to determine whether this light offers an alternative measure for controlling insects using various light sources such
as uorescent lamps [1012], xenon lamps [13], halogen lamps [14]
or sunlight [10]. However, such long exposures of UV-B irradiation
systems occupy a large space and take some time for the irradiance
to stabilize. In addition, spectral apparatuses such as diffraction
gratings or lters specic to UV-B are needed. The effects on plant
growth are generally small [15], but solar UV-B can have a large
inuence on the interactions between plants and phytophagous
insects [16]. The most common effect of exposing plants to UV-B
is a decrease in the intensity of insect herbivory [17,18]. The mechanisms responsible for the reduced herbivory in UV-B-exposed
plants, compared to plants grown under attenuated UV-B, are not
well understood. To counteract the toxicity of ROS, insects have
developed a suite of antioxidant enzymes like other eukaryotes to

S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 16

cope with oxidative stress. Various antioxidant enzymes may

decrease the level of lipid peroxidation as well as protein and
DNA damage [19]. Major components of the antioxidant enzyme
system of insects include superoxide dismutase (SOD), catalase
(CAT) and peroxidases (POX) [2,20]. In the enzymatic ROS-scavenging pathways, SOD breaks down superoxide into oxygen and hydrogen peroxide. Hydrogen peroxide is then scavenged by CAT and a
variety of POX into oxygen and water. In addition to SOD, CAT
and POX, another major component of the enzymatic defense is glutathione-S transferase (GST) which can remove the products of lipid
peroxidation or hydroperoxides from cells [1,20]. It has been demonstrated that the activities of antioxidant enzymes in insects can
be induced by environmental stress [1,3,20].
Spodoptera litura (Lepidoptera: Noctuidae) is a polyphagous
pest, attacking many important crops worldwide [21]. Direct
effects of UV upon insect behavior, developmental physiology
and biochemistry have been reported, with a particular focus on
UV-B (280315 nm), as it causes a variety of harmful effects. Blacklight, which emits electromagnetic radiation near UV-A region at
315400 nm, has been widely used in Integrated Pest Management
(IPM) to control lepidopteran pests [22]. Direct effects of UV insect
behavior and developmental physiology [23] and biochemistry
[24] have been reported. UV irradiation can cause oxidative stress
through the generation of ROS such as singlet oxygen, superoxide
anion, hydrogen peroxide, and others [25], which in turn leads to
damage to membrane lipids and proteins [2628]. However, information about oxidative stress induced by UV-B radiation in insects
is scanty except a recent report that shows that UV-B radiation
from direct Antarctic sunlight has the potential to generate high
levels of oxidative stress in the Antarctic midge Belgica antarctica
[3]. Moreover, with regard to whether UV-B light radiation can
increase oxidative stress in phototactic insects and the responses
of antioxidant enzymes in phototactic insects under UV-B
radiation.
The purpose of the present study was to test whether UV-B
exposure affects the balance between ROS production and their
inhibition by some antioxidant enzymes in the moths of S. litura.
To test the hypothesis we studied the antioxidant response by
determining the activities of Total protein, SOD, CAT, POX and
GST in S. litura adults.
2. Materials and methods
2.1. Insects
S. litura was obtained from the National Bureau of Agricultural
important insects (NBAII), Bangalore. Insects were fed on castor
leaf and maintained at 28 1 C, 70 10% Relative Humidity (RH)
under a photoperiod of 12L:12D. Males and females were seggregated based on the morphology of the abdominal terminal segments of the pupa. Virgin males and females were separated into
different plastic cups (20  20  30 cm). Adults 3 days after the
emergence were used for the experiments. The adults used for
the experiments were held in 100 ml plastic containers and provided with a 10% honey solution.
2.2. UV irradiation
UV-B light (Philips, Holland) which emits UV-B in the range
280315 nm was used as the source to irradiate S. litura adults.
The irradiance was 300 lW/cm2. Prior to use in our experiments,
the adults were divided into ve groups. Each group consisting of
fteen adults were exposed to UV-B light 2 h after the start of
scotophase. Fifteen adults per treatment were randomly selected
at the start of the experiment control (0 min) and at 30, 60, 90

and 120 min after UV light treatment commenced. Samples were

immediately frozen in liquid nitrogen and stored at 80 C for subsequent analysis.

2.3. Sample preparation

Before homogenization of whole moth, the wings were
removed and the remaining moth was weighed. The moth (without its wings) was homogenized in ice-cold buffer (0.1 M phosphate buffer, 1 mM EDTA, 1 mM DTT, 1 mM PTU, 1 mM PMSF
and 20% glycerol, pH 7.2) in a proportion of 0.1 g of body weight
to 1 ml of buffer. The homogenates were centrifuged at 10,000g
for 15 min at 4 C and the supernatant was used for subsequent
analysis. Protein concentrations were determined according to
[29].

2.4. Superoxide dismutase assay

SOD activity was assayed using the method described by [30].
Reaction mixtures were prepared in 3-ml glass spectrophotometer
cuvettes by adding 2.8 ml of Tris-EDTA (50 mM Tris and 10 mM
EDTA, pH 8.2) buffer and 50 ll of enzyme supernatant. The content
was mixed and the nal volume was adjusted to 2.9 ml with TrisEDTA buffer. Reaction in the cuvette was started with the addition
of 100 ll of pyrogallol (15 mM). The rates of autoxidation were followed at 440 nm in the UV-Vis spectrophotometer (Systronics),
and absorbance was measured for 3 min. One unit total SOD activity was calculated as the amount of protein per milligram causing
50% inhibition of pyrogallol autoxidation. SOD activity was
expressed as U mg 1 protein.

2.5. Glutathione-S-transferase assay

GST activity assay as per the mentioned protocol [31] with
minor modications. 50 ll of 50 mM 1-chloro-2, 4-dinitrobenzene
(CDNB) and 150 ll of 50 mM reduced glutathione (GSH) were
added to 2.78 ml of sodium phosphate buffer (100 mM, pH 6.5).
Twenty ll of enzyme stock was then added. The reaction was carried out in duplicate. The contents were shaken gently, incubated
23 min at 20 C and then transferred to a cuvette in the sample
cuvette slot of a UVVis Spectrophotometer (Systronics). Reaction
mixture (3 ml) without enzyme was placed in the reference slot for
setting zero. Absorbance at 340 nm was recorded for 1012 min
employing kinetics (time scan) menu. One unit of GST activity
was dened as the amount that catalyses the conjugation of
1 lmol/L GSH with CDNB per minute per mg protein. GST activity
was expressed as U mg 1 protein.

2.6. Peroxidase assay

POX activity was determined by [32] using UVVis spectrophotometer at 430 nm by catalyzing the oxidation in the presence of
H2O2 of a substrate. One unit of POX activity was dened as the
amount that catalyses 1 mg substrate per minute per mg protein.
POX activity was expressed as U mg 1 protein.

2.7. Catalase assay

CAT activity was spectrophotometrically measured by the rate
of decomposition of H2O2 by catalase [33]. One unit of CAT activity
was dened as the amount that decomposes H2O2 per second per g
protein. CAT activity was expressed as U g 1 protein.

S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 16

1.5

2.8. Statistical analysis

3. Results

*
U. mg -1 protein

Data are presented as the mean SD. Students t-test was used
for comparison of pairs; One way ANOVA was used for different
groups followed by Dunnetts test for determination of signicant
differences (p < 0.05). Statistical analysis was performed with SPSS
11.5 software.

1.0

0.5

3.1. Total protein

12

0m
in

m
in
90

30

60

m
in

l
tro
C
on

Fig. 2. Effects of UV-B light on SOD activity of Spodoptera litura adults for different
lengths of time. Values are mean S.D. (n = 15). Asterisk designates statistically
signicant difference between control and UV-B irradiated adults (p < 0.05). One
unit total SOD activity was calculated as the amount of protein per milligram
causing 50% inhibition of pyrogallol autoxidation. SOD activity was expressed as
U mg 1 protein.

200

150

100

50

12

0m

in

in
90

in
60

30

in
m

tro

0
on

A marked elevation of SOD activity (p < 0.05) in S. litura adults

was recorded when insects were exposed to UV-B light for 60
and 90 min. However, at 30 and 120 min exposure the SOD activity
decreased to the control level (Fig. 2).
CAT activity was signicantly (p < 0.05) enhanced in S. litura
adults following exposure to UV-B light for 60 min in comparison
with the control. However, exposure to UV-B light for 90 and
120 min resulted in a decline in the enzyme activity in comparison
with the control adults, and a signicant (p < 0.05) decrease was
found after UV-B light irradiation for 90 and 120 min (Fig. 3).
A signicant (p < 0.05) increase of POX activity in S. litura adults
was recorded when insects were exposed to UV-B light for 30 and
60 min. However exposure to UV-B light for 90 and 120 min
resulted in a decline in the enzyme activity in comparison with
the control adults (Fig. 4).
We found signicantly decreased GST activity (p < 0.05) in S.
litura adults exposed to UV-B light for 90 and 120 min in comparison with control, 30 and 60 min exposure (Fig. 5).

Exposure time

U. g -1 protein

3.2. Antioxidant enzymes

m
in

0.0

Signicant increases in total protein level (p < 0.05) were

observed in 120 min UV-B treatment groups. However there are
no changes between control and 30, 60 and 90 min of exposure
(Fig. 1).

Exposure time

4. Discussion
UV irradiation is generally considered to be a common and
strong environmental stress factor for animals [34,35]. UV light
used as light sources in light traps might be regarded as an environmental stress factor to insects. UV-B radiations are harmful to

Fig. 3. Effects of UV-B light on CAT activity of Spodoptera litura adults for different
lengths of time. Values are mean S.D. (n = 15). Asterisk designates statistically
signicant difference between control and UV-B irradiated adults (p < 0.05). One
unit of CAT activity was dened as the amount that decomposes H2O2 per second
per g protein. CAT activity was expressed as U g 1 protein.

0.10

*
protein
U. mg

mg/ g

0.08

*
0.06

-1

0.2

-1

protein

0.3

0.1

0.04
0.02

Exposure time
Fig. 1. Effects of UV-B light on total protein of Spodoptera litura adults for different
lengths of time. Values are mean S.D. (n = 15). Asterisk designates statistically
signicant difference between control and UV-B irradiated adults (p < 0.05).

12

0m

in

in
90

in
m
60

in
m

C
on

in
12
0m

in
90
m

in
60
m

in
30
m

on

tro

tro

0.0

30

0.00

Exposure time
Fig. 4. Effects of UV-B light on POX activity of Spodoptera litura adults for different
lengths of time. Values are mean S.D. (n = 15). Asterisk designates statistically
signicant difference between control and UV-B irradiated adults (p < 0.05). One
unit of POX activity was dened as the amount that catalyses 1 mg substrate per
minute per mg protein. POX activity was expressed as Umg 1 protein.

S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 16

250

150

U. mg

-1

protein

200

100
50

in
0m
12

in
90

in
m
60

in
m
30

on

tro

Exposure time
Fig. 5. Effects of UV-B light on GST activity of Spodoptera litura adults for different
lengths of time. Values are mean S.D. (n = 15). Asterisk designates statistically
signicant difference between control and UV-B irradiated adults (p < 0.05). One
unit of GST activity was dened as the amount that catalyses the conjugation of
1 lmol/L GSH with CDNB per minute per mg protein. GST activity was expressed as
U mg 1 protein.

living organisms in a variety of ways. They cause photo determination [36], DNA damage directly or indirectly via exposure production of ROS [37]. UV-B can also activate inammatory pathways,
through the transcription and release of cytokines and chemokines
from skin keratinocytes, leading to skin damage [38]. Total antioxidant capacity is a resultant measure of the ability of all antioxidants present in an organism to counteract the oxidation of an
indicator by an oxidant, or to reduce an indicator substance [35].
Conventional UV-B irradiation systems often occupy a large
space because of the large distance from the light source that is
required to achieve the desired irradiation level or the need for
neutral-density lters to control the irradiance [39]. They are
therefore awkward to use in the laboratory and create the risk of
a health hazard to researchers due to exposure to UV-B, but not
in experiments on animals, for which the effect of irradiation has
only been examined for UV-B in the absence of other wavelengths.
The mechanism of UV-B tolerance in T. okinawanus eggs might
be explained by an antioxidative function in addition to photoreactivation. Besides causing direct DNA damage, UV-B produces reactive oxygen species (ROS), including superoxide radicals, hydroxyl
radicals, hydrogen peroxide and singlet oxygen as a result of electron or energy transfer to oxygen, and these substances cause lipid
peroxidation and DNA damage [40]. [41] showed that diapausing
adult females of T. urticae became more tolerant of UV-B than
non-diapausing females. The diapausing females showed a bright
orange coloration because of the accumulation of hydroxy-ketocarotenoids such as astaxanthin [42], which has a strong quenching effect against singlet oxygen and a strong scavenging effect
against hydroxyl radicals [43].
A study on Helicoverpa armigera has shown that UV-A exposure
for shorter duration of (30 min) has no effect on the ROS formation.
However prolonged exposure for 60 and 90 min results in oxidative stress [44]. This shows that persistent stress conditions are
detrimental to antioxidant systems [45]. Induction of oxidative
stress in S. litura adults exposed to UV-B is observed by the
substantial increase in protein content, superoxide dismutase, catalase, peroxidase and glutathione-S-transferase activity to counteract oxidative stress generated by high concentration of ROS
inside the cell [46].
SOD plays an important role as an antioxidant protein by reducing the high level of intracellular superoxide radical induced by
extracellular stimuli such as UV irradiation. Similar study reported

that the change in SOD activity suggested that UV-A light irradiation induces superoxide radical in H. armigera adults [44]. SOD
activity signicantly increased when the insects were exposed to
UV-B light for 60 and 90 min, suggesting that SOD was stimulated
by superoxide radical to protect the adults from UV-B stress. It has
been reported that an increase in SOD activity is probably a
response towards increased ROS generation [47]. However, exposure to UV-B light for longer time (120 min) resulted in inhibition
of SOD activity in comparison with the adults at 30 min exposure
group and the activity decreased to the control level. This is consistent with previous reports showing that high doses of UV irradiation suppress the activity of protective enzymes, such as SOD in
normal cells [48]. We suspect that SOD cannot efciently withdraw
superoxide radicals that accumulated in cells of S. litura adults at
longer duration of UV-B exposure.
Catalase is an unusual antioxidant enzyme which is sensitive to
light [49]. It is perfectly suited for reducing the high amount of
H2O2 and directly regulated by the concentration of H2O2 [50]. It
is known that SOD and CAT together take part in stepwise oxygen
reduction [51]. Since SOD activity was enhanced when the adults
S. litura were exposed to UV-B light for 60 min, we assume that this
increased SOD activity would result in an increased H2O2 concentration and consequently in a further increase in CAT activity. In
our study CAT activity in S. litura adults the time of exposure UVB at 60 min will be signicantly increased as compared to the control (p < 0.05). However, exposure to UV-B light for 90 and 120 min
resulted in a decline in the enzyme activity in comparison with the
control adults, and a signicant (p < 0.05) decrease was found after
UV-B light irradiation for 90 and120 min (Fig. 3). Although UV-B
generated an oxidative stress, the CAT activity was sufcient to
cope with excess H2O2. High level of CAT activity was observed
in response to UV-B exposure, suggesting a possible increase of
ROS production. CAT is a vital component of antioxidant system
in insects. Studies have shown that expression of CAT gene is
correlated with longevity in Drosophila melanogaster. A disrupted
CAT gene results in early death of adult D. melanogaster [52]. We
thus suspect that the high levels of CAT in the irradiated adults
serve as a protective mechanism against DNA damage. However
this needs to be veried.
GST in insects can be considered as a primary antioxidant
enzyme, which is effective in metabolizing lipid peroxides
[53,54]. Similar study reported that GST activity in the UV-A
radiated adults was quite high. With increased levels of GST,
H. armigera adults exposed to UV light appeared exceedingly capable of removing the lipid peroxidation products generated during
UV irradiation stress, and were protected from potential cellular
damage. In our study GST activity in the UV-B irradiated adults
was signicantly (p < 0.05) decreased at 90 min and 120 min as
compared with control insects, showing that a prolonged UV-B
exposure results in disruption of GST activity.
The results obtained showed that CAT was present at lower
levels than SOD, reecting that alternate enzymes, such as POX,
may be associated with the scavenging of H2O2 [55], and an
increase in POX activity is related to increase in stress tolerance.
When S. litura adults were exposed to UV-B light for 30 and
60 min, a signicant increase in POX activity functions to keep
the balance of H2O2 components. However, exposure to UV-B light
for longer time (90 and 120 min) resulted in a decrease in enzyme
activity (Fig. 4). Previous studies have shown that enzyme activity
can be decreased by negative feedback from an excess of substrate
or damage by oxidative modication [56]. UV-B irradiation stress
to POX was severe in S. litura adults at longer exposure time. A signicant increase in CAT activity in response to UV light irradiation
at longer exposure time and a simultaneous decrease in POX activity suggested that CAT may have a more important role in scavenging H2O2 than POX at longer exposure times.

S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 16

5. Conclusion
In conclusion, UV-B exposure of adults in S. litura for various time
exposure of UV-B radiation has the potential to generate oxidative
stress. We have conrmed that UV-B light may disturb the functional activity of protein and intensify the activity of protein oxidation processes. The induction of antioxidant enzymes as a result of
UV-B irradiation may also indicate the over-production of ROS.
The strong and constant enhanced activities of SOD, CAT and POX
activity of S. litura adults were exposed to UV-B irradiation at
60 min exposure and the rapid enhanced activities of GST in
response to UV-B light are a likely defense against oxidative damage
due to the accumulation of ROS. These processes may be mirrored in
insect physiological adaptations. However, prolonged exposure to
UV-B light resulted in decreased activities of CAT, POX and GST,
accompanied by impaired antioxidant capacity and high levels of
oxidative stress, suggesting that oxidative stress marker enzymes
are down regulated in response to continuous UV-B exposure.
Declaration of interest
We thank the Department of Biotechnology, Periyar University;
Salem for providing funding (URF) and UGC-MRP No: 42-201/2013
(SR) and infrastructure facilities for carrying out this research work
and for providing giving instrument sterilization facilities. We
thank our lab research scholars for help during these experiments.
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