Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
Contents lists available at ScienceDirect
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 280–315 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 significantly 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 detoxification mechanism in insects, making them more susceptible to insecticides. Ó 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: [email protected] (M.S. Shivakumar). http://dx.doi.org/10.1016/j.jphotobiol.2014.04.008 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
eliminated by a suite of antioxidant enzymes [6]. Ultraviolet-B radiation (UV-B; 280–315 nm) is a small fraction of the solar spectrum received at ground level. In spite of its modest contribution to the total quantum flux, UV-B can be an important modulator of biological processes in terrestrial ecosystems [7]. Due to deleterious effects of ultraviolet-B radiation (UVB; 280–315 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 fluorescent lamps [10–12], 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 filters specific to UV-B are needed. The effects on plant growth are generally small [15], but solar UV-B can have a large influence 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
2
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
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 (280–315 nm), as it causes a variety of harmful effects. Blacklight, which emits electromagnetic radiation near UV-A region at 315–400 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 [26–28]. 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 280–315 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 five groups. Each group consisting of fifteen 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 final 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 modifications. 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 2–3 min at 20 °C and then transferred to a cuvette in the sample cuvette slot of a UV–Vis Spectrophotometer (Systronics). Reaction mixture (3 ml) without enzyme was placed in the reference slot for setting zero. Absorbance at 340 nm was recorded for 10–12 min employing kinetics (time scan) menu. One unit of GST activity was defined 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 UV–Vis spectrophotometer at 430 nm by catalyzing the oxidation in the presence of H2O2 of a substrate. One unit of POX activity was defined 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 defined as the amount that decomposes H2O2 per second per g protein. CAT activity was expressed as U g 1 protein.
3
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
1.5
2.8. Statistical analysis
3. Results
* U. mg -1 protein
Data are presented as the mean ± SD. Student’s t-test was used for comparison of pairs; One way ANOVA was used for different groups followed by Dunnett’s test for determination of significant 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 significant 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
m
in 60
30
C
m
in m
tro
l
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 significantly (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 significant (p < 0.05) decrease was found after UV-B light irradiation for 90 and 120 min (Fig. 3). A significant (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 significantly 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
Significant 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 significant difference between control and UV-B irradiated adults (p < 0.05). One unit of CAT activity was defined 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 significant difference between control and UV-B irradiated adults (p < 0.05).
12
0m
in
in 90
m
in m 60
in m
C on
in 12 0m
in 90 m
in 60 m
in 30 m
C
on
tro
l
tro
0.0
30
l
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 significant difference between control and UV-B irradiated adults (p < 0.05). One unit of POX activity was defined as the amount that catalyses 1 mg substrate per minute per mg protein. POX activity was expressed as Umg 1 protein.
4
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
250
150
U. mg
*
*
-1
protein
200
100 50
in 0m 12
in 90
m
in m 60
in m 30
C
on
tro
l
0
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 significant difference between control and UV-B irradiated adults (p < 0.05). One unit of GST activity was defined 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 inflammatory 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 filters 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 significantly 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 efficiently 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 significantly 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 significant (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 sufficient 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 verified. 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 significantly (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, reflecting 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 significant 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 modification [56]. UV-B irradiation stress to POX was severe in S. litura adults at longer exposure time. A significant 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) 1–6
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 confirmed 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. References [1] G.W. Felton, C.B. Summers, Antioxidant systems in insects, Arch. Insect Biochem. Physiol. 29 (1995) 187–197. [2] H.U. Dahms, J.S. Lee, UV radiation in marine ectotherms: molecular effects and responses, Aquat. Toxicol. 97 (2010) 3–14. [3] Y. Wang, L.W. Oberley, D.W. Murhammer, Antioxidant defence systems of two lepidopteran insect cell lines, Free Radical Biol. Med. 30 (2001) 1254–1262. [4] K.D. Curtin, Z.J. Huang, M. Rosbash, Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock, Neurons 14 (1995) 365–372. [5] G. Lopez-Martinez, M.A. Elnitsky, J.B. Benoit, R.E. Lee Jr, D.L. Denlinger, High resistance to oxidative damage in the Antarctic midge Belgica Antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins, Insect Biochem. Mol. Biol. 38 (2008) 796– 804. [6] R.R. Aucoin, B.J.R. Philogene, J.T. Arnason, Antioxidant enzymes as biochemical defenses against phototoxin-induced oxidative stress in three species of herbivorous Lepidoptera, Arch. Insect Biochem. Physiol. 16 (1991) 139–152. [7] M.M. Caldwell, C.L. Ballaré, J.F. Bornman, S.D. Flint, L.O. Bjorn, A.H. Teramura, G. Kulandaivelu, M. Tevini, Terrestrial ecosystems, increased solar ultraviolet radiation and interactions with other climatic change factors, Photochem. Photobiol. Sci. 2 (2003) 29–38. [8] T. Suzuki, Environmental engineering approaches toward sustainable management of spider mites, Insects 3 (2012) 1126–1142. [9] W. Dermauw, N. Wybouw, S. Rombauts, B. Menten, J. Vontas, M. Grbic, R.M. Clark, R. Feyereisen, T. Van Leeuwen, A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae, Proc. Natl. Acad. Sci. USA 110 (2013) 113–122. [10] M. Fukaya, R. Uesugi, H. Ohashi, Y. Sakai, M. Sudo, A. Kasai, H. Kishimoto, M. Osakabe, Tolerance to solar ultraviolet-B radiation in the citrus red mite, an upper surface user of host plant leaves, Photochem. Photobiol. 89 (2013) 424– 431. [11] Y. Murata, M. Osakabe, The Bunsen-Roscoe reciprocity law in ultravioletBinduced mortality of the two-spotted spider mite Tetranychus urticae, J. Insect Physiol. 59 (2013) 241–247. [12] F. Tachi, M. Osakabe, Vulnerability and behavioral response to ultraviolet radiation in the components of a foliar mite prey-predator system, Naturwissenschaften 99 (2012) 1031–1038. [13] Y. Sakai, M. Osakabe, Spectrum-specific damage and solar ultraviolet radiation avoidance in the two-spotted spider mite, Photochem. Photobiol. 86 (2010) 925–932. [14] C.A. Mazza, M.M. Izaguirre, J. Curiale, C.L. Ballaré, A look into the invisible: ultraviolet-B sensitivity in an insect (Caliothrips phaseoli) revealed through a behavioural action spectrum, Proc. Royal Soc. B Biol. Sci. 277 (2010) 367–373. [15] C.A. Mazza, D. Battista, A. Zima, M. Szwarcberg-Bracchitta, C.V. Giordano, A. Acevedo, A.L. Scopel, C.L. Ballaré, The effects of solar UV-B radiation on the growth and yield of barley are accompanied by increased DNA damage and antioxidant responses, Plant Cell Environ. 22 (1999) 61–67.
5
[16] N.D. Paul, D. Gwynn-Jones, Ecological roles of solar UV radiation: towards an integrated approach, Trends Ecol. Evol. 18 (2003) 48–55. [17] M.C. Rousseaux, R. Julkunen-Tiitto, P.S. Searles, A.L. Scopel, P.J. Aphalo, C.L. Ballaré, Solar UV-B radiation affects leaf quality and insect herbivory in the southern beech tree Nothofagus antarctica, Oecologia 138 (2004) 505–512. [18] J. Zavala, A.L. Scopel, C.L. Ballaré, Effects of solar UV-B radiation on soybean crops: Impact on leaf herbivory by Anticarsia gemmatalis, Plant Ecol. 156 (2001) 121–130. [19] H. Kamata, H. Hirata, Redox regulation of cellular signaling, Cell Signal 11 (1999) 1–14. [20] S. Ahmad, D.L. Duval, L.C. Winhold, R.S. Parlini, Cabbage looped antioxidant enzymes: tissue specificity, J. Insect Biochem. 21 (1991) 563–572. [21] I.M. Dubovskiy, V.V. Martemyanov, Y.L. Vorontsova, M.J. Rantala, E.V. Gryzanova, V.V. Glupov, Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae), Comp. Biochem. Phys.-C 148 (2008) 1–5. [22] M. Auger, Low nutritive quality as plant defines-effects of herbivore-mediated interaction, Evol. Ecol. 9 (1995) 605–616. [23] G.S. Wei, Q.W. Zhang, M.Z. Zhou, W.G. Wu, Studies on the phototaxis of Helicoverpa armigera, Acta Biophys. Sinica 16 (2000) 89–95. [24] Y. Kojima, K. Aoyagi, T. Yasue, Effect of lithium ion addition on afterglow time of green-emitting Ce3+ and Pr3+ codoped CaS phosphor by black light irradiation, J. Lumin. 115 (2005) 13–18. [25] A. Gunn, The determination of larval phase coloration in the African armyworm Spodoptera exempta and its consequences for thermo regulation and protection from UV light, Entomol. Exp. Appl. 86 (1998) 125–133. [26] S.A.H. Mackerness, S.L. Surplus, P. Blake, C.F. John, V. Buchanan-Wollaston, B.R. Jordan, B. Thomas, Ultraviolet-B-induced stress and changes in gene expression in Arabidopsis thaliana: role of signaling pathways controlled by jasmonic acid, ethylene and reactive oxygen species, Plant Cell Environ. 2 (1999) 1413–1423. [27] S.W. Ryter, R.M. Tyrrell, Singlet molecular oxygen ((1) O2): possible effectors of eukaryotic gene expression, Free Radical Biol. Med. 24 (1998) 1520–1534. [28] J.L. Ravanat, T. Douki, J. Cader, Direct and indirect effects of UV radiation on DNA and its components, J. Photochem. Photobiol.-B 63 (2001) 88–102. [29] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [30] S.L. Marklund, G. Marklund, Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase, Eur. J. Biochem. 47 (1974) 469–474. [31] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione-S-transferase: the first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139. [32] K.P. Reddy, S.M. Subhani, P.A. Khan, K.B. Kumar, Effect of light and benzyladenine on dark treated graving rice (Oryza sativa) leaves-changes in peroxidase activity, Plant Cell Physiol. 26 (1995) 987–994. [33] H. Luck, Catalase, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1971, pp. 885–893. [34] F. Urbach, The biological effects of increased ultraviolet radiation: an update, Photochem. Photobiol. 50 (1989) 439–441. [35] M. Schauen, H.T. Hornig-Do, S. Schomberg, G. Herrmann, R.J. Wiesner, Mitochondrial electron transport chain activity is not involved in ultraviolet A (UVA)-induced cell death, Free Radical Biol. Med. 42 (2007) 499–509. [36] A.T. Dinkova-Kostova, Phytochemicals as protectors against ultraviolet radiation: versatility of effects and mechanisms, Planta Med. 74 (13) (2008) 1548–1559. [37] K.A. Williams, K. Kolappaswamy, L.J. Detolla, I. Vucenik, Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice, Comput. Med. 61 (1) (2011) 39–44. [38] W. Chu, S.C.M. Cheung, R.A.W. Lau, I.F.F. Benzie, Herbal Medicine: Biomolecular and Clinical Aspects, in: I.F.F. Benzie, S. Wachtel-Galor (Eds.), second ed., CRC Press, Boca Raton (FL), 2011 (Chapter 4 PMID 22593936). [39] J. Cadet, E. Sage, T. Douki, Ultraviolet radiation-mediated damage to cellular DNA, Mutat. Res. 571 (2005) 3–17. [40] Y.Y. He, D.P. Häder, UV-B-induced formation of reactive oxygen species and oxidative damage of the Cyanobacterium anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine, J. Photochem. Photobiol. B 66 (2002) 115–124. [41] T. Suzuki, M. Watanabe, M. Takeda, UV tolerance in the two-spotted spider mite, Tetranychus urticae, J. Insect Physiol. 55 (2009) 649–654. [42] A. Veerman, Carotenoid metabolism in Tetranychus urticae Koch (Acari: Tetranychidae), Comp. Biochem. Physiol. B 47 (1974) 101–116. [43] W. Miki, Biological functions and activities of animal carotenoids, Pure Appl. Chem. 63 (1991) 141–146. [44] J. Meng, C. Zhang, F. Zhu, X. Wang, C. Lei, Ultraviolet light-induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults, J. Insect Physiol. 55 (2009) 588–592. [45] A. Somogyi, K.P. Rosta, Z. Tulssay, G. Nagy, Antioxidant measurements, Physiol. Meas. 28 (2007) 41–55. [46] C.H. Foyer, P. Descouvieres, K. Kunert, Protection against oxygen radicals: an important defence mechanism studied in transgenic plants, Plant Cell Environ. 17 (1994) 507–523. [47] N. Krishnan, D. Kodrik, Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they enhanced to protect gut tissues during oxidative stress, J. Insect Physiol. 52 (2006) 11–20.
6
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
[48] D.E. Heck, A.M. Vetrano, T.M. Mariano, J.D. Laskin, UV-B light stimulates production of reactive oxygen species: unexpected role for catalase, J. Biol. Chem. 278 (2003) 22432–22436. [49] T. Polte, R.M. Tyrrell, Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression, Free Radical Biol. Med. 36 (2004) 1566–1574. [50] M.P. Lesser, Oxidative stress in marine environments: biochemistry and physiological ecology, Annu. Rev. Physiol. 68 (2006) 253–278. [51] I. Fridovich, The biology of oxygen radicals, Science 201 (1978) 875–879. [52] W.C. Orr, R.S. Sohal, Extension of life-span by over expression of superoxide dismutase and catalase in Drosophila melanogaster, Science 263 (1994) 1128– 1130.
[53] H. Sies, Oxidative stress-from basic research to clinical application, Am. J. Med. 91 (1991) 313–323. [54] Y. Kono, T. Shishido, Distribution of glutathione-S-transferase activity in insect tissues, Appl. Entomol. Zool. 27 (1992) 391–397. [55] M. Clavaron-Mathews, C.B. Summers, G.W. Felton, Ascorbate peroxidase: a novel antioxidant enzyme in insects, Arch. Insect Biochem. Physiol. 34 (1997) 57–68. [56] T. Tabatabaie, R.A. Floyd, Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agents, Arch. Biochem. Biophys. 314 (1994) 112–119.
Contents lists available at ScienceDirect
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 280–315 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 significantly 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 detoxification mechanism in insects, making them more susceptible to insecticides. Ó 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: [email protected] (M.S. Shivakumar). http://dx.doi.org/10.1016/j.jphotobiol.2014.04.008 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
eliminated by a suite of antioxidant enzymes [6]. Ultraviolet-B radiation (UV-B; 280–315 nm) is a small fraction of the solar spectrum received at ground level. In spite of its modest contribution to the total quantum flux, UV-B can be an important modulator of biological processes in terrestrial ecosystems [7]. Due to deleterious effects of ultraviolet-B radiation (UVB; 280–315 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 fluorescent lamps [10–12], 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 filters specific to UV-B are needed. The effects on plant growth are generally small [15], but solar UV-B can have a large influence 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
2
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
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 (280–315 nm), as it causes a variety of harmful effects. Blacklight, which emits electromagnetic radiation near UV-A region at 315–400 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 [26–28]. 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 280–315 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 five groups. Each group consisting of fifteen 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 final 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 modifications. 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 2–3 min at 20 °C and then transferred to a cuvette in the sample cuvette slot of a UV–Vis Spectrophotometer (Systronics). Reaction mixture (3 ml) without enzyme was placed in the reference slot for setting zero. Absorbance at 340 nm was recorded for 10–12 min employing kinetics (time scan) menu. One unit of GST activity was defined 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 UV–Vis spectrophotometer at 430 nm by catalyzing the oxidation in the presence of H2O2 of a substrate. One unit of POX activity was defined 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 defined as the amount that decomposes H2O2 per second per g protein. CAT activity was expressed as U g 1 protein.
3
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
1.5
2.8. Statistical analysis
3. Results
* U. mg -1 protein
Data are presented as the mean ± SD. Student’s t-test was used for comparison of pairs; One way ANOVA was used for different groups followed by Dunnett’s test for determination of significant 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 significant 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
m
in 60
30
C
m
in m
tro
l
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 significantly (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 significant (p < 0.05) decrease was found after UV-B light irradiation for 90 and 120 min (Fig. 3). A significant (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 significantly 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
Significant 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 significant difference between control and UV-B irradiated adults (p < 0.05). One unit of CAT activity was defined 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 significant difference between control and UV-B irradiated adults (p < 0.05).
12
0m
in
in 90
m
in m 60
in m
C on
in 12 0m
in 90 m
in 60 m
in 30 m
C
on
tro
l
tro
0.0
30
l
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 significant difference between control and UV-B irradiated adults (p < 0.05). One unit of POX activity was defined as the amount that catalyses 1 mg substrate per minute per mg protein. POX activity was expressed as Umg 1 protein.
4
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
250
150
U. mg
*
*
-1
protein
200
100 50
in 0m 12
in 90
m
in m 60
in m 30
C
on
tro
l
0
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 significant difference between control and UV-B irradiated adults (p < 0.05). One unit of GST activity was defined 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 inflammatory 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 filters 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 significantly 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 efficiently 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 significantly 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 significant (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 sufficient 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 verified. 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 significantly (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, reflecting 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 significant 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 modification [56]. UV-B irradiation stress to POX was severe in S. litura adults at longer exposure time. A significant 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) 1–6
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 confirmed 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. References [1] G.W. Felton, C.B. Summers, Antioxidant systems in insects, Arch. Insect Biochem. Physiol. 29 (1995) 187–197. [2] H.U. Dahms, J.S. Lee, UV radiation in marine ectotherms: molecular effects and responses, Aquat. Toxicol. 97 (2010) 3–14. [3] Y. Wang, L.W. Oberley, D.W. Murhammer, Antioxidant defence systems of two lepidopteran insect cell lines, Free Radical Biol. Med. 30 (2001) 1254–1262. [4] K.D. Curtin, Z.J. Huang, M. Rosbash, Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock, Neurons 14 (1995) 365–372. [5] G. Lopez-Martinez, M.A. Elnitsky, J.B. Benoit, R.E. Lee Jr, D.L. Denlinger, High resistance to oxidative damage in the Antarctic midge Belgica Antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins, Insect Biochem. Mol. Biol. 38 (2008) 796– 804. [6] R.R. Aucoin, B.J.R. Philogene, J.T. Arnason, Antioxidant enzymes as biochemical defenses against phototoxin-induced oxidative stress in three species of herbivorous Lepidoptera, Arch. Insect Biochem. Physiol. 16 (1991) 139–152. [7] M.M. Caldwell, C.L. Ballaré, J.F. Bornman, S.D. Flint, L.O. Bjorn, A.H. Teramura, G. Kulandaivelu, M. Tevini, Terrestrial ecosystems, increased solar ultraviolet radiation and interactions with other climatic change factors, Photochem. Photobiol. Sci. 2 (2003) 29–38. [8] T. Suzuki, Environmental engineering approaches toward sustainable management of spider mites, Insects 3 (2012) 1126–1142. [9] W. Dermauw, N. Wybouw, S. Rombauts, B. Menten, J. Vontas, M. Grbic, R.M. Clark, R. Feyereisen, T. Van Leeuwen, A link between host plant adaptation and pesticide resistance in the polyphagous spider mite Tetranychus urticae, Proc. Natl. Acad. Sci. USA 110 (2013) 113–122. [10] M. Fukaya, R. Uesugi, H. Ohashi, Y. Sakai, M. Sudo, A. Kasai, H. Kishimoto, M. Osakabe, Tolerance to solar ultraviolet-B radiation in the citrus red mite, an upper surface user of host plant leaves, Photochem. Photobiol. 89 (2013) 424– 431. [11] Y. Murata, M. Osakabe, The Bunsen-Roscoe reciprocity law in ultravioletBinduced mortality of the two-spotted spider mite Tetranychus urticae, J. Insect Physiol. 59 (2013) 241–247. [12] F. Tachi, M. Osakabe, Vulnerability and behavioral response to ultraviolet radiation in the components of a foliar mite prey-predator system, Naturwissenschaften 99 (2012) 1031–1038. [13] Y. Sakai, M. Osakabe, Spectrum-specific damage and solar ultraviolet radiation avoidance in the two-spotted spider mite, Photochem. Photobiol. 86 (2010) 925–932. [14] C.A. Mazza, M.M. Izaguirre, J. Curiale, C.L. Ballaré, A look into the invisible: ultraviolet-B sensitivity in an insect (Caliothrips phaseoli) revealed through a behavioural action spectrum, Proc. Royal Soc. B Biol. Sci. 277 (2010) 367–373. [15] C.A. Mazza, D. Battista, A. Zima, M. Szwarcberg-Bracchitta, C.V. Giordano, A. Acevedo, A.L. Scopel, C.L. Ballaré, The effects of solar UV-B radiation on the growth and yield of barley are accompanied by increased DNA damage and antioxidant responses, Plant Cell Environ. 22 (1999) 61–67.
5
[16] N.D. Paul, D. Gwynn-Jones, Ecological roles of solar UV radiation: towards an integrated approach, Trends Ecol. Evol. 18 (2003) 48–55. [17] M.C. Rousseaux, R. Julkunen-Tiitto, P.S. Searles, A.L. Scopel, P.J. Aphalo, C.L. Ballaré, Solar UV-B radiation affects leaf quality and insect herbivory in the southern beech tree Nothofagus antarctica, Oecologia 138 (2004) 505–512. [18] J. Zavala, A.L. Scopel, C.L. Ballaré, Effects of solar UV-B radiation on soybean crops: Impact on leaf herbivory by Anticarsia gemmatalis, Plant Ecol. 156 (2001) 121–130. [19] H. Kamata, H. Hirata, Redox regulation of cellular signaling, Cell Signal 11 (1999) 1–14. [20] S. Ahmad, D.L. Duval, L.C. Winhold, R.S. Parlini, Cabbage looped antioxidant enzymes: tissue specificity, J. Insect Biochem. 21 (1991) 563–572. [21] I.M. Dubovskiy, V.V. Martemyanov, Y.L. Vorontsova, M.J. Rantala, E.V. Gryzanova, V.V. Glupov, Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera, Pyralidae), Comp. Biochem. Phys.-C 148 (2008) 1–5. [22] M. Auger, Low nutritive quality as plant defines-effects of herbivore-mediated interaction, Evol. Ecol. 9 (1995) 605–616. [23] G.S. Wei, Q.W. Zhang, M.Z. Zhou, W.G. Wu, Studies on the phototaxis of Helicoverpa armigera, Acta Biophys. Sinica 16 (2000) 89–95. [24] Y. Kojima, K. Aoyagi, T. Yasue, Effect of lithium ion addition on afterglow time of green-emitting Ce3+ and Pr3+ codoped CaS phosphor by black light irradiation, J. Lumin. 115 (2005) 13–18. [25] A. Gunn, The determination of larval phase coloration in the African armyworm Spodoptera exempta and its consequences for thermo regulation and protection from UV light, Entomol. Exp. Appl. 86 (1998) 125–133. [26] S.A.H. Mackerness, S.L. Surplus, P. Blake, C.F. John, V. Buchanan-Wollaston, B.R. Jordan, B. Thomas, Ultraviolet-B-induced stress and changes in gene expression in Arabidopsis thaliana: role of signaling pathways controlled by jasmonic acid, ethylene and reactive oxygen species, Plant Cell Environ. 2 (1999) 1413–1423. [27] S.W. Ryter, R.M. Tyrrell, Singlet molecular oxygen ((1) O2): possible effectors of eukaryotic gene expression, Free Radical Biol. Med. 24 (1998) 1520–1534. [28] J.L. Ravanat, T. Douki, J. Cader, Direct and indirect effects of UV radiation on DNA and its components, J. Photochem. Photobiol.-B 63 (2001) 88–102. [29] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [30] S.L. Marklund, G. Marklund, Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase, Eur. J. Biochem. 47 (1974) 469–474. [31] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione-S-transferase: the first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139. [32] K.P. Reddy, S.M. Subhani, P.A. Khan, K.B. Kumar, Effect of light and benzyladenine on dark treated graving rice (Oryza sativa) leaves-changes in peroxidase activity, Plant Cell Physiol. 26 (1995) 987–994. [33] H. Luck, Catalase, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, 1971, pp. 885–893. [34] F. Urbach, The biological effects of increased ultraviolet radiation: an update, Photochem. Photobiol. 50 (1989) 439–441. [35] M. Schauen, H.T. Hornig-Do, S. Schomberg, G. Herrmann, R.J. Wiesner, Mitochondrial electron transport chain activity is not involved in ultraviolet A (UVA)-induced cell death, Free Radical Biol. Med. 42 (2007) 499–509. [36] A.T. Dinkova-Kostova, Phytochemicals as protectors against ultraviolet radiation: versatility of effects and mechanisms, Planta Med. 74 (13) (2008) 1548–1559. [37] K.A. Williams, K. Kolappaswamy, L.J. Detolla, I. Vucenik, Protective effect of inositol hexaphosphate against UVB damage in HaCaT cells and skin carcinogenesis in SKH1 hairless mice, Comput. Med. 61 (1) (2011) 39–44. [38] W. Chu, S.C.M. Cheung, R.A.W. Lau, I.F.F. Benzie, Herbal Medicine: Biomolecular and Clinical Aspects, in: I.F.F. Benzie, S. Wachtel-Galor (Eds.), second ed., CRC Press, Boca Raton (FL), 2011 (Chapter 4 PMID 22593936). [39] J. Cadet, E. Sage, T. Douki, Ultraviolet radiation-mediated damage to cellular DNA, Mutat. Res. 571 (2005) 3–17. [40] Y.Y. He, D.P. Häder, UV-B-induced formation of reactive oxygen species and oxidative damage of the Cyanobacterium anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine, J. Photochem. Photobiol. B 66 (2002) 115–124. [41] T. Suzuki, M. Watanabe, M. Takeda, UV tolerance in the two-spotted spider mite, Tetranychus urticae, J. Insect Physiol. 55 (2009) 649–654. [42] A. Veerman, Carotenoid metabolism in Tetranychus urticae Koch (Acari: Tetranychidae), Comp. Biochem. Physiol. B 47 (1974) 101–116. [43] W. Miki, Biological functions and activities of animal carotenoids, Pure Appl. Chem. 63 (1991) 141–146. [44] J. Meng, C. Zhang, F. Zhu, X. Wang, C. Lei, Ultraviolet light-induced oxidative stress: effects on antioxidant response of Helicoverpa armigera adults, J. Insect Physiol. 55 (2009) 588–592. [45] A. Somogyi, K.P. Rosta, Z. Tulssay, G. Nagy, Antioxidant measurements, Physiol. Meas. 28 (2007) 41–55. [46] C.H. Foyer, P. Descouvieres, K. Kunert, Protection against oxygen radicals: an important defence mechanism studied in transgenic plants, Plant Cell Environ. 17 (1994) 507–523. [47] N. Krishnan, D. Kodrik, Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they enhanced to protect gut tissues during oxidative stress, J. Insect Physiol. 52 (2006) 11–20.
6
S. Karthi et al. / Journal of Photochemistry and Photobiology B: Biology 135 (2014) 1–6
[48] D.E. Heck, A.M. Vetrano, T.M. Mariano, J.D. Laskin, UV-B light stimulates production of reactive oxygen species: unexpected role for catalase, J. Biol. Chem. 278 (2003) 22432–22436. [49] T. Polte, R.M. Tyrrell, Involvement of lipid peroxidation and organic peroxides in UVA-induced matrix metalloproteinase-1 expression, Free Radical Biol. Med. 36 (2004) 1566–1574. [50] M.P. Lesser, Oxidative stress in marine environments: biochemistry and physiological ecology, Annu. Rev. Physiol. 68 (2006) 253–278. [51] I. Fridovich, The biology of oxygen radicals, Science 201 (1978) 875–879. [52] W.C. Orr, R.S. Sohal, Extension of life-span by over expression of superoxide dismutase and catalase in Drosophila melanogaster, Science 263 (1994) 1128– 1130.
[53] H. Sies, Oxidative stress-from basic research to clinical application, Am. J. Med. 91 (1991) 313–323. [54] Y. Kono, T. Shishido, Distribution of glutathione-S-transferase activity in insect tissues, Appl. Entomol. Zool. 27 (1992) 391–397. [55] M. Clavaron-Mathews, C.B. Summers, G.W. Felton, Ascorbate peroxidase: a novel antioxidant enzyme in insects, Arch. Insect Biochem. Physiol. 34 (1997) 57–68. [56] T. Tabatabaie, R.A. Floyd, Susceptibility of glutathione peroxidase and glutathione reductase to oxidative damage and the protective effect of spin trapping agents, Arch. Biochem. Biophys. 314 (1994) 112–119.
