THE EFFECTS OF XANTHOTOXIN
ON THE BIOLOGY AND
BIOCHEMISTRY OF
Galleria
mellonella
L. (LEPIDOPTERA:
PYRALIDAE)
Meltem Erdem
Ahmet Erdo˘gan Vocational School of Health Services, B¨ulent Ecevit University, Zonguldak, Turkey
Ender B ¨uy ¨ukg ¨uzel
Department of Molecular Biology and Genetics, Faculty of Arts and Science, B¨ulent Ecevit University, Zonguldak, Turkey
The effects of a dietary plant allelochemical, xanthotoxin (XA), on survivorship, development, male and female adult longevity, fecundity, and hatchability of the greater wax moth Galleria mellonella L. were investigated. Oxidative stress indicators, the lipid peroxidation product, malondialdehyde (MDA), and protein oxidation products, protein carbonyl (PCO) contents, and activities of a detoxification enzyme glutathione S-transferase (GST) activity were determined in wax moth adults. The insect was reared from first-instar larvae on an artificial diets containing XA at 0.001, 0.005, or 0.1% to adult stage in laboratory conditions. Relative to the controls, the diets containing XA concentrations led to decreased survivorship in seventh instar, pupal, and adult stages. Compared to control diet (77.7%), the highest dietary XA concentration decreased survivorship to adulthood to 11.0%. The highest XA
concentration (0.1%) reduced female longevity from 10.4 to 5.7 days and decreased egg numbers from 95.0 to 33.5 and hatchability from 82.7 to 35.6%. The lowest XA concentration (0.001%) led to about a sixfold increase in MDA content. XA at high concentrations (0.005 and 0.1%) increased MDA (by threefold) and protein carbonyl (by twofold) contents
Grant sponsor: B ¨ulent Ecevit University.
Correspondence to: Ahmet Erdo˘gan, Vocational School of Health Services, B ¨ulent Ecevit University, 68080 Zonguldak, Turkey. E-mail: meltemerdem1927@hotmail.com
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 89, No. 4, 193–203 (2015)
Published online in Wiley Online Library (wileyonlinelibrary.com). C
decreased GST activity. The highest dietary XA concentration decreased GST activity from 0.28± 0.025 to 0.16 ± 0.005 μmol/mg protein/min. We infer from these findings that XA-induced oxidative stress led to decreased biological fitness. C 2015 Wiley Periodicals, Inc.
Keywords: Galleria mellonella; oxidative stress; xanthotoxin; GST; survivorship; fecundity; protein carbonyl
INTRODUCTION
Herbivores are challenged by a variety of prooxidant allelochemicals that produce re-active oxygen species (ROS; Barbehenn et al., 2008). Allelochemicals include pheno-lic compounds that range from small acids to complex polymers, such as tannins and lignins (Harborne, 1997); and furanocoumarin compounds, such as xanthotoxin (XA), isopimpinellin, and angelicin (Brown et al., 2005), exert antifeedant or toxic activities, deterring insect herbivores (Duffey and Stout, 1996). Apiaceous and rutaceous plants produce XA, potentially for their protection. XA is a strong prooxidant that selectively inhibits DNA synthesis by binding with guanine and cytosine bases of DNA and cross-links opposing strands, effectively preventing transcription (Scott et al., 1976). XA is oxidized to produce ROS, such as superoxide and hydroxyl radicals, and singlet oxygen (Pritsos et al., 1990; Pardini, 1995). When the rate of ROS generation exceeds capacity of the antioxidant and detoxification systems, oxidative stress damages lipids, proteins, and nu-cleic acids (Stadtman, 1992; Halliwell and Gutteridge, 2007). Malondialdehyde (MDA) and carbonyls contents are used as markers of oxidative stress. MDA is the principal and most studied product of polyunsaturated fatty acid peroxidation as a commonly used marker of oxidative stress and antioxidant status. Protein carbonylation is an indicator of protein oxidation (B ¨uy ¨ukg ¨uzel et al., 2010).
Insects have biochemical defense mechanisms, including antioxidant or detoxifica-tion enzyme systems, to detoxify host plant allelochemicals (Harrison et al., 2001).The pri-mary antioxidative enzymatic defense system includes glutathione-S-transferases (GSTs; Krishnan and Kodrik, 2006). GSTs comprise a group of multifunctional detoxification enzymes that neutralize the toxic effects of electrophilic substrates or xenobiotics by con-jugating reduced glutathione (GSH) to these compounds. This process reduces toxic substrates in invertebrates and vertebrates (Grant and Matsumura, 1989; Vontas et al., 2001).
Dietary XA decreased the survivorship, delayed development, and reduced the body sizes in tested insect species (Chen, 2008; Lampert et al., 2008). Increased life expectancy attended elevated P450 enzyme activity in larvae of Heliothis zea exposed toα-cypermethrin (Li et al., 2000a,b). The growth and reproduction of the moth Spodoptera littoralis was re-duced following transfer of caterpillars from a semiartificial diet to potato plants (Hussein et al., 2006). This switch led to increased oxidative radicals and antioxidant enzyme ac-tivities in the digestive tract (Krishnan and Kodr´ık, 2006; Krishnan et al., 2007). Some studies examined possible roles of other allelochemicals, mainly the alkaloidα-solanine in the induction and management of oxidative stress in midgut and fat body of Galle-ria mellonella larvae (B ¨uy ¨ukg ¨uzel et al., 2013; Adamski et al., 2014) and in the gut of S. littoralis caterpillar (Krishnan and Sehnal, 2006). Our study showed that XA induced lipid and protein oxidation in hemolymph (B ¨uy ¨ukg ¨uzel et al., 2010). It may be surmised that prooxidant chemicals in natural diets lead to substantial fitness costs in herbivorous
insects; however, more details on the influence of the prooxidants on specific physiolog-ical systems will shed light on the mechanisms of prooxidative challenge in plant/insect interactions. We addressed this issue by posing the hypothesis that oxidative stress in-terferes with the developmental physiology and reproductive biology of wax moths, G. mellonella.
MATERIALS AND METHODS
Insect Culture
Greater wax moth larvae and pupae were collected from infected hives in apicultural areas around Zonguldak, Turkey, and the newly emerged adults were used to maintain the stock culture. The insects were reared in 1000 ml glass jars with an artificial diet (Bronskill, 1961), at 30 ± 1°C, 65 ± 5% relative humidity in constant darkness. The standard diet was composed of 420 g of bran, 150 ml of filtered honey, 150 ml of glycerol, 20 g of ground old dark honeycomb, and 30 ml of distilled water. Newly emerged adult females were placed in the jars and provided a piece of old honeycomb on the diet for egg deposition and feeding of newly hatched larvae. For continuing insect culture, these newly hatched larvae were reared through seventh instar at the same artificial diets. And then last larval instars were transferred new jars to obtain pupal stage and adult stage of insect B ¨uy ¨ukg ¨uzel et al. (2010).
Chemicals
All reagents were analytical grade. XA, phenylmethylsulphonyl fluoride (PMSF), dithiotre-itol (DTT), dipotassium hydrogen phosphate (K2HPO4), potassium chloride (KCl), ethylendiaminetetraecetic acid (EDTA), butylated hydroxytoluene (BHT), thiobabituric acid (TBA), trichloroacetic acid (TCA), bovine serum albumin (BSA), Folin–Ciolcalteu reagent, guanidine hydrochloride, 2,4 dinitrophenylhydrazine (DNPH), streptomycin sulfate, GSH, 1-chloro-2,4-dinitrobenzene (CDNB), glycerol, ethanol, sodium chloride (NaCl), and phenylthiourea (PTU) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Experimental Designs
XA was directly incorporated into diets at concentrations of 0.001, 0.005, or 0.1%. Our preliminary experiments showed that these concentrations enable larvae to complete their adult development with gradually increasing mortality. Larvae reared on diets with-out XA were used as controls in all treatments. The XA concentrations used in this study were taken from Timmermann et al. (1999) and B ¨uy ¨ukg ¨uzel et al. (2010). Using standard laboratory rearing conditions, two series of experiments were carried out. In the first series, the effects of XA on survivorship, development, adult longevity, egg production (fecundity), and egg hatchability (fertility) were determined. Newly emerged adults were used in the biochemical analyses because our unpublished observations demonstrated that some plant allelochemicals in the larval diet exerted their effects thorough adult stage (B ¨uy ¨ukg ¨uzel et al., 2010). In the second series, we determined the influence of dietary XA on lipid peroxidation and protein oxidation levels, and on GST activities in whole bodies of newly emerged adults.
Survivorship and Development
First instars were reared through adult emergence on the artificial diets amended with given concentrations of XA. Time to reach the seventh instar, pupal stage, adult stage, and survivorship to those stages were recorded. Control larvae were reared on diet with-out XA. Seventh instars were transferred into another jar lined with a filter paper for pupation and adult emergence. The filter paper was used to provide a dry surface for pupation. Numbers of pupae and adults were recorded, and their developmental times were calculated for each replicate. Each experiment included three XA concentrations and a control. We performed four independent biological replicates with 20 larvae per replicate.
Longevity, Fecundity, and Hatchability
Newly emerged adults from the experimental diets were transferred into 30 ml plastic cups (35× 55 mm, ORLAB Ltd., Ankara, Turkey) covered with a screened lids. To determine average adult longevity, the number of dead adults in each of the treatment group was counted and recorded daily until all adults died. Fecundity was determined by recording the number of eggs laid and hatch rates. Neonate larvae were reared to the adult stage on the diets containing XA.
Females reared on XA were placed in 30 ml plastic cups with screened lids. Our previous experience showed that newly emerged females laid most of their eggs within the first 2 days. Experimental females were allowed to oviposit in cups for 2 days, then adults were removed from the cups, and eggs were transferred into Petri dishes using a fine brush. Total numbers of eggs laid per female per day were recorded. Egg counts were performed in the Petri dish on a black background to make the eggs more visible. Eggs also were kept for hatching. Numbers of eggs hatched per female per day and their percentages were recorded. Females were not provided with food during the oviposition period. Experiments were carried out under standard laboratory conditions. Numbers of hatched larvae in each Petri dish were recorded daily. Egg production and larval hatch were monitored continuously from the first oviposition day until experiments were stopped. Egg production was calculated as eggs laid per female per day and, termed fecundity; percentage of larval hatch is termed fertility.
Biochemical Assays
First instar larvae were reared through adult emergence on an artificial diet amended with given concentrations of XA. Newly emerged adults were used for determining the content of the lipid peroxidation product MDA, protein oxidation product, protein carbonyl (PCO), and detoxification enzyme GST activity. All adults were of the same age.
Adults were chilled on ice for 5 min and surface sterilized in 95% ethanol. Adults were collected into a chilled Eppendorf tube charged with cold homogenization buffer
(w/v 1.15% KCl, 25 mM K2HPO4, 5 mM EDTA, 2mM PMSF, 2mM DTT, pH 7.4) and
stored at−80°C. A few crystals of PTU were added to each sample to prevent melaniza-tion. The cryotubes were kept at room temperature until the tissue began to thaw before using. Adults were at 4°C by an ultrasonic homogenizer (Bandelin Sonoplus, HD2070, Berlin, Germany) at 50 W, 40–50 sec in homogenization buffer, and subsequent cen-trifugation at 10,000× g for 10 min. The resulting cell-free preparations were collected for biochemical analysis. Protein concentrations were determined according to Lowry
et al. (1951) by using BSA as a quantitative standard. A dual beam spectrophotometer (Shimadzu 1700, UV/VIS Spectrophotometer, Kyoto, Japan) was used for all absorbance measurements.
Determination of MDA, PCO Content, and GST Activity
MDA content was assayed according to Jain and Levine (1995). MDA reacts with TBA to form a colored complex. MDA contents were determined after incubation at 95°C with TBA (1% w/v). Absorbances were measured at 532 nm to determine MDA content. MDA content was expressed as nanomole per milligram protein by using 1.56× 105/M/cm for extinction coefficient.
Protein carbonyl was assayed according to Levin et al. (1990) with some modifications (Krishnan and Kodr´ık, 2006). Carbonyl content was quantified spectrophotometrically after reaction of carbonyl groups with DNPH, leading to the formation of a stable 2,4-dinitrophenylhydrazone. Absorbances were measured at 370 nm. Results are expressed as nanomole per milligram protein by using 22.000/M/cm as the extinction coefficient. Protein concentrations in guanidine solutions were measured spectrophotometrically (280 nm) and quantified with a BSA standard curve. Protein carbonyls values were cor-rected for interfering substances by subtracting the A370/mg protein measured without DNPH.
GST (EC 2.5.1.18) activity was assayed by measuring the formation of the GSH and CDNB conjugate (Habig et al., 1974). The increase in absorbance was recorded at 340 nm for 3 min. The specific activity of GST was expressed as nanomole GSH–CDNB conjugate formed per minute per milligram protein using an extinction coefficient 9.6 mM−1cm−1. All assays were corrected for nonenzymatic reactions using corresponding substrate in phosphate buffer (50 mM, pH 7.0).
Statistical Analysis
Data on the development, longevity, egg production, hatchability, and adult whole body MDA, protein carbonyl content, and GST activity were analyzed by one-way analysis of variance (ANOVA). To determine significant differences between means least significant difference (LSD) test (SPSS, 1997) was used. Data on survivorship were compared by a chi-squared test (Snedecor and Cochran, 1989). Each experiment was replicated four times. When the F andχ2estimate exceeded the probability of 0.05, the differences were considered significant.
RESULTS
Influence of Dietary XA on Survival and Development
Dietary XA did not influence development rates, recorded as days to seventh instar (approximately 35–39 days), pupa (about 41–47 days), or adult (circa 48 to 59 days; data not shown). Insects reared on diets containing XA suffered decreased survivorship at seventh-instar, pupal and adult stages. Figure 1 shows the influence of XA on survivorship was expressed in a dose-dependent manner. Adult developmental time was significantly prolonged by 10 days (Fig. 2).
Figure 1. Effects of dietary XA (at the indicated proportions of diet) on survival of the Galleria mellonella. Each histogram bar represents the mean of four replicates in each treatment group, 20 larvae were used per replicate, and the error bars represent 1 SEM. Histogram bars annotated with the same letter are not significantly different within each developmental stage, P< 0.05 (χ2test, LSD test). Controls were reared on standard media without
XA.
Influence of Dietary XA on Longevity, Fecundity, and Hatchability
Dietary XA did not influence male adult longevity (about 6–8 days). But the highest XA significantly shorted female longevity from 10 to 6 days (Fig. 3) and XA-laced media led to decreased egg numbers (95–33) and hatchability (83–36%), again in a dose-dependent manner (Fig. 4). Female longevity (R= 0.876, P < 0.05), number of eggs laid (R = 0.732, P < 0.05), and hatchability (R = 0.844, P < 0.05) were negatively correlated with XA concentrations.
The Effects of XA on MDA and PCO Contents and on GST Activity
Dietary XA treatments influenced three parameters of oxidative biology. XA exposure led to increased whole body MDA levels, by about sixfold at 0.001% and threefold at 0.005 and 0.1%, increased PCO levels by about twofold and decreased GST activity, albeit with no indication of a dose–response relationship (Table 1).
DISCUSSION
The results of this study support our hypothesis that oxidative stress interferes with the developmental physiology and reproductive biology of wax moths, G. mellonella. The mech-anism appears to be related to the effects of XA-induced oxidative stress on fundamental
Figure 2. Dietary XA (at the indicated proportions of diet) did not extend Galleria mellonella developmental time, except for time to adulthood. Each histogram bar represents the mean of four replicates in each of treatment groups, 20 larvae per replicate. Histogram bars annotated with the same letter are not significantly within each developmental stage, P< 0.05 (χ2test, LSD test). Controls were reared on standard media without
XA.
Figure 3. Effects of XA on male and female longevity of Galleria mellonella. Each point represents mean adult
longevity (four replicates, 10 adults/replicate) and the error bars represent 1 SEM. Points annotated with the same letter are not significant, P< 0.05 (χ2test, LSD test). Controls were reared on standard media without
Figur e 4 . The influence of dietar y X A o n fecundity (panel l A ) a nd fertility (panel 1 B) of Galleria m ellonella . Each d iamond point represents mean numbers o f e ggs laid and e ach square p oint represents the m ean h atchability (%) and the error b ars represent 1 S EM. P oints annotated w ith the same letter are not significant ly different, P < 0.05 (χ 2test, L SD test). Controls were reared o n standard d iets without X A.
Table 1. Effect of XA on MDA and Protein Carbonyl Content, and GST Activity of the Galleria mellonella Adults
XA (%)
MDA (nmol/mg
protein; Mean± SE)*
Protein carbonyl (nmol/mg
protein; Mean± SE)*
GST (μmol/mg
protein/min; mean± SE)*
0.00† 0.01± 0.0005a 1.38± 0.082a 0.28± 0.025a
0.001 0.03± 0.001b 1.05± 0.099a 0.31± 0.035a
0.005 0.02± 0.001c 2.48± 0.137b 0.19± 0.005b
0.1 0.02± 0.0005c 2.23± 0.105b 0.16± 0.005b
*Four replicates with five adults per replicate. Values followed by the same letter are significantly different from each other, P< 0.05 (LSD test).
†Control (without XA).
physiological processes. First, we recorded decreased survival to the last larval stage, to pupae, and to adulthood. Second, the XA-treated females laid fewer eggs and the de-posited eggs suffered reduced hatchability. Third, dietary XA led to increased whole-body levels of oxidative products, MDA, and PCO. Finally, XA exposure resulted in reduced GST activity, again at the whole-body level. These points construct a firm argument that exposure to dietary XA leads to increased oxidative effects on cellular lipids (↑ MDA) and proteins (↑ PCO) and to decreased antioxidant enzyme activity (↓ GST activity). Taken with literature reporting similar results (Barbehenn et al., 2005a; Krishnan and Sehnal, 2006), we infer that XA acts as a powerful allelochemical on G. mellonella and possibly other insect herbivores.
Wax moth survivorship, adult longevity, and reproduction parameters (fecundity and fertility) were adversely affected by dietary XA exposure. The influence of dietary XA was expressed in dose-dependent manners, from which we infer that XA toxicity operates in physiological, rather than toxic, mechanisms. In the lepidopteran S. littoralis, feeding onα-solanine-containing potato plants resulted in toxic or nutritionally unsuitable diets, recorded as decreased growth and reproduction (Hussein et al., 2006). Alterations in GST activity have been reported in many insects following severe toxicity of plant allelo-chemicals. For example, Francis et al. (2005) reported that aphids, Myzus persicae, reared on white mustard, Sinapis alba, accumulate glucosinolates, which led to increased GST activity. Allelochemicals in plants influence the relationship of oxidative and antioxida-tive actions (Barbehenn et al., 2005a,b; Krishnan and Kodrik, 2006). Survival is increased with increasing detoxication enzyme activities (Adamski et al., 2003; B ¨uy ¨ukg ¨uzel, 2006). However, low levels of GST activity in adults may not be sufficient to reduce the oxidative stress elicited by allelochemicals, such as XA, which may help understand the influence of dietary XA on G. mellonella. Yang et al. (2013) reported that exogenous application of methyl jasmonate to cotton leaves led to marked reductions in several parameters, in-cluding carboxylesterase and GST activities, and prolonged larval development of cotton bollworm Helicoverpa armigera pupae.
XA and many other compounds are collectively known as plant secondary com-pounds, so called because they are not essential to the plants. In our view, detailed understanding of plant biosynthesis of these chemicals and their actions within insects and other herbivores has very broad significance in several otherwise disconnected ar-eas, including the biology of plant/insect interactions, medical pharmacology, illicit drug economics, and conservation of plant-rich areas. Our goal is to advance understanding of the chemicals to the molecular level.
ACKNOWLEDGMENTS
The authors are grateful to Prof. Dr. Kemal B ¨UY ¨UKG ¨UZEL (Department of Biology, Faculty of Arts and Science, Zonguldak, B ¨ulent Ecevit University, Turkey) for reviewing and providing useful comments on earlier draft of this article. This study was supported by B ¨ulent Ecevit University, Research Fund (project no.: 2011-10-06-08).
LITERATURE CITED
Adamski Z, Ziemnicki K, Fila K, ˇZiki´c R, ˇStajn A. 2003. Effects of long-term exposure to fenitrothion on Spodoptera exigua and Tenebrio molitor larval development and antioxidant enzyme activity. Biol Lett 40:43–52.
Adamski Z, Marciniak P, Ziemnicki K, B ¨uy ¨ukg ¨uzel E, Erdem M, B ¨uy ¨ukg ¨uzel K, Ventrella E, Falabella P, Cristallo M, Salvia R, Scrano L, Bufo SA. 2014. Potato leaf extract and its component, α-solanine, exert similar impacts on development and oxidative stress in Galleria mellonella L. Arch Insect Biochem Physiol 87(1):26–39.
Barbehenn RV, Cheek S, Gasperut A, Lister E, Maben R. 2005a. Phenolic compounds in red oak and sugar maple leaves have prooxidant activities in the midguts of Malacosoma disstria and
Orgyia leucostigma caterpillars. J Chem Ecol 31(5):969–988.
Barbehenn RV, Dodick T, Poopat U, Spencer B. 2005b. Fenton-type reactions and iron concentra-tions in the midgut fluids of tree-feeding caterpillars. Arch Insect Biochem Physiol 60:32–43. Barbehenn RV, Maben RE, Knoester JJ. 2008. Linking phenolic oxidation in the midgut lumen
with oxidative stress in the midgut tissues of a tree-feeding caterpillar Malacosoma disstria (Lep-idoptera: Lasiocampidae). Env Entomol 37:1113–1118.
Bronskill J. 1961. A cage to simplify the rearing of the greater waxmoth, Galleria mellonella (Pyralidae). J Lepid Soc 15:102–104.
Brown RP, McDonnell CM, Berenbaum MR, Schuler MA. 2005. Regulation of an insect cytochrome P450 monooxygenase gene (CYP6B1) by aryl hydrocarbon and xanthotoxin response cascade. Gene 26:39–52.
B ¨uy ¨ukg ¨uzel E, Hyrsl P, B ¨uy ¨ukg ¨uzel K. 2010. Eicosanoids mediate hemolymph oxidative and antiox-idative response in larvae of Galleria mellonella L. Comp Biochem Physiol 156(2):176–183. B ¨uy ¨ukg ¨uzel E, Adamski Z, B ¨uy ¨ukg ¨uzel K, Erdem M, Marciniak P, Ziemnicki K, Ventrella E, Scrano
L, Aurelio Bufo S. 2013. The influence of dietaryα-solanine on the waxmoth Galleria mellonella L. Arch Insect Biochem 83:15–24.
B ¨uy ¨ukg ¨uzel K. 2006. Malathion-induced oxidative stress in a parasitoid wasp: effect on adult emer-gence, longevity, fecundity, oxidative and antioxidative response of the Pimpla turionellae. J Econ Entomol 99:1225–1234.
Chen MS. 2008. Inducible direct plant defence against insect herbivores: a review. Insect Sci 15(2):101-114.
Duffey SS, Stout MJ. 1996. Antinutritive and toxic components of plant defense against insects. Arch Insect Biochem Physiol 32:3–37.
Francis F, Vanhaelen N, Haubruge E. 2005. Glutahthione-S-transferases in the adaptation to plant secondary metabolites in the Myzus persicae Aphid. Arch Insect Biochem Physiol 58:166–174. Grant DF, Matsumura F. 1989. Glutathione S-transferase 1 and 2 in susceptible and resistant
insec-ticide resistant Aedes aegypti. Pestic Biochem Physiol 33:132–143.
Habig WH, Pabst MJ, Jakoby WB. 1974. Glutathione-S-transferases: the first enzymatic step in mer-capturic acid formation. J Biol Chem 249:7130–7139.
Halliwell B, Gutteridge JMC. 2007. Free radicals in biology and medicine (4th ed.). Oxford: Press Biosciences.
Harborne JB. 1997. Biochemical plant ecology. In: Dey, PM, Harborne, JB, editors. Plant Biochem-istry. London: Academic Press. p 503–515.
Harrison TL, Zangerl AR, Schuler MA, Berenbaum MR. 2001. Developmental variation in cy-tochrome P450 expression in Papilio polyxenes in response to xanthotoxin, a hostplant allelo-chemical. Arch Insect Biochem Physiol 48:179–189.
Hussein HM, Habuˇstov´a O, Turanli F, Sehnal F. 2006. Potato expressing beetle-specific Bacillus
thuringiensis Cry3Aa toxin reduces performance of a moth. J Chem Ecol 32:1–13.
Jain SK, Levine SN. 1995. Elevated lipid peroxidation and vitamin E-quinone levels in heart ventricles of streptozotocin-treated diabetic rats. Free Radic Biol Med 18:337–341.
Krishnan N, Kodrik D. 2006. Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they en-hanced to protect gut tissues during oxidative stress? J Insect Physiol 52: 11–20.
Krishnan N, Sehnal F. 2006. Compartmentalization of oxidative stress and antioxidant defense in the larval gut of Spodoptera littoralis. Arch Insect Biochem Physiol 63:1–10.
Krishnan N, Kodr´ıc D, Turanli F, Sehnal F. 2007. Stage- specific distribution of oxidative radicals and antioxidant enzymes in the midgut of Leptinotarsa decemlineata. J Insect Physiol 53:67–74. Lampert EC, Zangerl AR, Berenbaum MR, Ode PJ. 2008. Tritrophic effects of xanthotoxin on
the polyembryonic parasitoid Copidosoma sosares (Hymenoptera: Encyrtidae). J Chem Ecol 34(6):783–790.
Levin RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG. 1990. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 186:464–478.
Li X, Berenbaum MR, Schuler MA. 2000a. Molecular cloning and expression of CYP6P8: a xan-thotoxin inducible cytochrome P450 cDNA from Helicoverpa zea. Insect Biochem Mol Biol 30:75–84.
Li X, Zangerl AR, Schuler MA, Berenbaum MR. 2000b. Cross-resistance toα-cypermethrin after Xanthotoxin ingestion in Helicoverpa zea (Lepidoptera: Noctuidae). J Econ Entomol 93(1):18– 25.
Lowry OH, Rosebrough NL, Farr AL, Randall RJ. 1951. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275.
Pardini RS. 1995. Toxicity of oxygen from naturally occurring redox-active pro-oxidants. Arch Insect Biochem Physiol 29:101–118.
Pritsos CA, Ahmad S, Elliott AJ, Pardini RS. 1990. Antioxidant enzyme level response to prooxidant allelochemicals in larvae of the southern armyworm moth, Spodoptera eridania. Free Radic Res Commun 9(2):127–133.
Scott BR, Pathak MA, Mohn GR. 1976. Molecular and genetic basis of furocoumorin reactions. Mutat Res 39:29–74.
Snedecor GS, Cochran WG. 1989. Statistical methods (8th ed.). Ames, IA: Iowa State University Press.
SPSS Inc. 1997. User’s manual, version 10. Chicago, IL: SPSS Inc. Stadtman ER. 1992. Protein oxidation and aging. Science 257:1220–1224.
Timmermann SE, Zangerl AR, Berenbaum MR. 1999. Ascorbic and uric acid responses to xan-thotoxin ingestion in a generalist and a specialist caterpillar. Arch Insect Biochem Physiol 42:26–36.
Vontas JG, Small GJ, Hemingway J. 2001. Glutathione-S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens. J Biochem 357:65–72.
Yang S, Wu H, Xie J, Rantala MJ. 2013. Depressed performance and detoxification enzyme activities of Helicoverpa armigera fed with conventional cotton foliage subjected to methyl jasmonate exposure. Entomol Exp App 147:186–195.
