General and Comparative Endocrinology 172 (2011) 77–84

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General and Comparative Endocrinology

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

Oxidative stress elicited by insecticides: A role for the adipokinetic hormone

Mirna Velki a,b,c, Dalibor Kodrík a,b,⇑, Josef Vecera a,b, Branimir K. Hackenberger c, Radomír Socha a

a Institute of Entomology, Biology Centre, Academy of Sciences, Branišovská 31, 370 05 Ceské Budejovice, Czech Republicb Faculty of Science, University of South Bohemia, Branišovská 31, 370 05 Ceské Budejovice, Czech Republicc Department of Biology, Josip Juraj Strossmayer University of Osijek, Trg Ljudevita Gaja 6, 31000 Osijek, Croatia

a r t i c l e i n f o a b s t r a c t

Article history:Available online 23 December 2010

Keywords:InsectAdipokinetic hormoneOxidative stressInsecticideCatalaseGlutathione

0016-6480/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ygcen.2010.12.009

⇑ Corresponding author at: Institute of EntomologySciences, Branišovská 31, 370 05 Ceské Budejovice, C

E-mail address: kodrik@entu.cas.cz (D. Kodrík).

Adipokinetic hormones (AKHs) are insect neuropetides responding to stress situations including oxida-tive stress. Two insecticides – endosulfan and malathion – were used to elicit oxidative stress conditionsin the firebug Pyrrhocoris apterus, and the physiological functions of AKHs and their ability to activateprotective antioxidative reactions were studied. The insecticide treatments elicited only a slight increaseof the AKH level in CNS, but more intensive increase in haemolymph, which indicates an immediateinvolvement of AKH in the stress response. The treatment also resulted in a significant increase of cata-lase activity in the bug’s body and depletion of the reduced glutathione pool in the haemolymph, how-ever, co-application of the insecticides with the AKH (80 pmol) reduced the effect. It has also been foundthat co-application of the insecticides with AKH increased significantly the bug mortality compared tothat induced by the insecticides alone. This enhanced effect of the insecticides probably resulted fromthe stimulatory role of AKH on bug metabolism: the carbon dioxide production was increased signifi-cantly after the co-treatment by AKH with insecticides compared to insecticide treatment alone. It washypothesized that the increased metabolic rate could intensify the insecticide action by an acceleratedrate of exchange of metabolites accompanied by faster penetration of insecticides into tissues.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

Oxidative stress occurs when the production of potentiallydestructive reactive oxygen species (ROS) exceeds the bodiesown natural antioxidative defences, resulting in organismal dam-age or even in its death [41]. This imbalance can result from a lackof antioxidative capacity caused by disturbances in production anddistribution of low molecular-weight antioxidants, as well as dis-turbances in antioxidant-enzyme protective machinery, or by anover-abundance of ROS from environmental or behavioural stress-ors [19,20]. If not regulated properly, the excess ROS can damagecell’s lipids, proteins or DNAs, inhibiting their natural functions.ROS are products of normal cellular metabolism. Most of the body’senergy is produced by the enzymatically controlled reaction ofoxygen with hydrogen in oxidative phosphorylation occurringwithin the mitochondria during oxidative metabolism. Duringthose enzymatic cascades, free radicals are formed [49].

The ROS, such as singlet oxygen, superoxide anion, hydroxylradical and hydrogen peroxide, are eliminated by antioxidant en-zymes and several redox systems [6,9,10]. It is also well acceptedthat the triad of glutathione, ascorbic acid, and vitamin E is central

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, Biology Centre, Academy ofzech Republic.

to the antioxidant defence in both mammals and insects [3,38].Each of these compounds is a major ROS reductant with actionsdistinct from ROS scavenging by superoxide dismutase andcatalase.

Activation of the antioxidative machinery is an essentially com-plex process; there are indications that it could be, at least partially,under hormonal control both in vertebrates and in insects. Lu et al.[37] reported that glucagon-mediated signal transduction path-ways lead to a down-regulation of hepatic reduced glutathione(GSH) synthesis while promoting the efflux of glutathione to theblood plasma in rats which clearly indicates the activation of anti-oxidative systems. The presence of peptides with glucagon-likeactivity has been immunochemically documented also in a numberof insect species [43,53]. Recently the presence of such a peptidehas also been proven in the CNS and the gut of the firebug Pyrrhoc-oris apterus, and its role in initiation of the antioxidative reactionshas been suggested [1]. When co-injected with a herbicide paraquat(diquaternary derivative of 4,40 bipyridyl) that is commonly used asan oxidative stressor, the mammalian glucagon partially eliminatedoxidative stress markers elicited by this redox cycling agent. Thereaction was characterized by increasing GSH and decreasing pro-tein carbonyl levels (a biomarker for oxidative stress and damageto proteins) in haemolymph, and decreasing both protein carbonyland protein nitrotyrosine levels in CNS. A similar antioxidativefunction has also been described for adipokinetic hormones (AKHs)

78 M. Velki et al. / General and Comparative Endocrinology 172 (2011) 77–84

[29,50]. The AKHs that form the AKH/RPCH (adipokinetic hormone/red pigment concentrating hormone) peptide family [14] behave astypical insect stress hormones by stimulating catabolic reactionssuch as mobilization of lipids, carbohydrates and/or certain aminoacids, making energy more available, while inhibiting syntheticreactions. They mobilize entire energy reserves to combat theimmediate stress problems and suppress processes that aremomentarily less important and could, if allowed to continue, evendraw on the mobilized energy. These reactions are controlled viaresponsible enzyme cascades. The whole anti-stress reaction canbe accompanied by stimulation of heart beating [42], increasingof muscle tonus [40], stimulation of general locomotion [48],enhancement of immune response [15,16] and/or some other phys-iological actions (reviewed by Kodrík [26]).

The role of AKH in stimulation of antioxidative mechanisms isnot connected with apparent stimulation and ensuing consump-tion of energy. Application of the oxidative stressors (paraquat,Bacillus thuringiensis toxin – Cry 3Aa or Gallanthus nivalis lectin –GNA) caused multiple increases in the titre of AKH in insect body[29,50]. Moreover, similarly as mentioned for glucagon [1], aninjection of exogenous AKH mobilized antioxidative mechanismsthat reduced biomarkers of oxidative damage incurred by the oxi-dative stressors: protein carbonylation, decrease of GSH level andattenuation of total antioxidant activity in haemolymph. Thesefindings indicate that there is a feed-back regulation between anoxidative stressor and AKH actions, and that AKHs are very proba-bly involved in the activation of antioxidant protection mecha-nisms. The possible mode of action is unclear at present, butoxidative stress could be a causative factor accelerating synthesisor release of AKH in/from the endocrine gland – corpora cardiaca.

In this study we used two insecticides – endosulfan and mala-thion – to elicit oxidative stress in the insect body. Those com-pounds were suitable as they have a well-documented ability toelicit oxidative stress [2,5,13,23]. Thereto endosulfan as organo-chlorine cyclodiene pesticide acts as a neurotoxin by inhibitingGABA receptors at synapses, Ca2+ and Mg2+ ATPase, and acetylcho-linesterase, and also works as an endocrine disruptor [11]. On theother hand malathion is an organophosphorous insecticide thatbinds irreversibly to acetylcholinesterase in insect nervous system[12]. We used the firebug P. apterus as a model species in ourexperiments for the following reasons: (1) Two AKHs have beenidentified in this species in our laboratory: Pyrap-AKH [32] andPeram-CAH-II [31], and (2) a considerable amount of physiological,biochemical and endocrinological data about this species is avail-able. The general biological data has been reviewed by Socha[46], the AKH data including a role of this hormone in oxidativestress by Kodrík [26]. The main aim of this paper was to create con-ditions of oxidative stress in the model insect species by applica-tion of insecticides endosulfan and malathion, and to study thephysiological reaction(s) in the body. We focused on the functionof the AKHs and their ability to activate metabolic protective reac-tions including the antioxidative ones to eliminate or at least to re-duce the impact of the oxidative stressors on their physiologicalfunctions.

2. Materials and methods

2.1. Experimental insects

A stock culture of the firebug, P. apterus (L.) (Heteroptera, Insec-ta), established from wild populations collected at Ceské Budejo-vice (Czech Republic, 49�N), was used in the present study.Larvae and adults of a common reproductive brachypterous morphwere kept in 0.5 l glass jars in a mass culture (approximately 40specimens per jar) and reared at constant temperature of

26 ± 1 �C under long-day conditions (18 h light: 6 h dark). Theywere supplied with linden seeds and water ad libitum, which werereplenished twice a week. Freshly ecdysed adults were transferredto small 0.25 l glass jars (females and males separately) and keptunder the same photoperiod, food and temperature regimes inwhich they developed. Ten-day old individuals (for reasons see[47]) were used for the experiments.

2.2. Determination of insecticide doses

The insecticides endosulfan (commercial name Global E-35; ac-tive compound in preparation 350 g/L, Chromos Agro, Zagreb,Croatia) and malathion (commercial name Radotion-E50; activecompound in preparation 500 g/L, Herbos, Sisak, Croatia) wereused in the study. The experimental bugs were treated individuallyby injection into the haemocoel with endosulfan 100–1000 ng(100, 200, 225, 250, 275, 300, 500 and 1000 ng) or malathion275–1000 ng (275, 300, 350, 400, 450, 475, 500 and 1000 ng). Inanother series of experiments the topical application under thewings with endosulfan 250–3000 ng (250, 450, 750, 1100, 1250,1750, 2000, 2500 and 3000 ng) or malathion 250–3000 ng (250,500, 750, 900, 1250, 1750, 2000, 2500 and 3000 ng) were used.All listed doses represent the amounts of the active compoundsonly. The insecticide doses were always applied in 2 ll Ringersaline and the results were evaluated 24 h later. Each dose wasapplied in 3–5 parallel replicates and each replicate contained 20individuals. The LD15 and LD50 were determined by a probit analy-sis from a number of bugs that died within 24 h of insecticidetreatment (see the Section 3). Those doses were used for analysesof the insecticide interaction with the Pyrap-AKH (see below). Thecontrols were sham-treated (injection or topical application) with2 ll Ringer saline only.

2.3. Adipokinetic hormone

The Pyrap-AKH, custom-synthesized by Polypeptide Laborato-ries s.r.o. (Praha, Czech Republic), was dissolved in 20% methanolin Ringer saline. The experimental bugs were either injected withthe hormonal solution (dose 80 pmol in 2 ll of the solution) in asimilar manner as mentioned above for the insecticide treatments,or applied topically (again 80 pmol in 2 ll of the solution) onto thethorax and abdomen under the wings using a pipette (mortalityexperiments only). Similarly, control bugs were sham-treated(injection or topical application) with 2 ll of 20% methanol in Ring-er saline. In the case of insecticide and AKH co-treatment, a 30 mindelay between the treatments was maintained for recovery of thebugs from the stress of the first treatment. Results of mortalityexperiments were evaluated 24 h later, the other ones as men-tioned in the corresponding text.

2.4. Extraction of the AKH from CNS and haemolymph

Methanolic extract (80% methanol) of the brain with corporacardiaca and corpus allatum attached was used for determiningthe AKH content in the CNS by means of a competitive ELISA (seebelow). For determination of the endogenous AKH titre in the hae-molymph by ELISA, some pre-purification steps were essential[17]. Briefly, haemolymph samples were extracted in 80% metha-nol and after centrifugation the supernatants were evaporated todryness. The residues of the evaporation were dissolved in 0.11%TFA (trifluoroacetic acid), applied to a solid phase extraction car-tridge (Sep Pak C18, Waters), and eluted in 60% acetonitrile. Theeluent was subjected to HPLC analysis on a Waters chromatogra-phy system with a fluorescence detector Waters 2475 using aChromolith Performance RP-18e column (Merck). Fractions elutingbetween 6.5–7.5 min were subjected to competitive ELISA.

M. Velki et al. / General and Comparative Endocrinology 172 (2011) 77–84 79

Retention times of the two Pyrrhocoris synthetic adipokinetic pep-tides Pyrap-AKH and Peram-CAH-II were 6.87 and 7.03 min,respectively.

The efficiency of recovery of haemolymph AKH during theextraction procedure was checked by adding 500 fmol of Pyrap-AKH to 20 ll samples of haemolymph before the extraction. Therecovery of AKH (74.8 ± 8.2 %; mean ± SE) was checked using ELISA(see below) and estimated from five separate parallel measure-ments; all respective data were corrected for these losses.

2.5. Quantification of AKH by ELISA

A competitive ELISA was used for determination of total AKHcontent in P. apterus CNS (IgG dilution 1:10,000, 1=4 CNS equiv.per well, detection limit 20 fmol per well) and haemolymph (IgGdilution 1:2000, 20 ll equiv. per well, detection limit 14 fmol perwell – unpublished data) according to our protocol described ear-lier [17].

Briefly, rabbit antibodies were raised commercially againstCys1-Pyrap-AKH (Sigma Genosys, Cambridge, UK) and the resultingantibody recognised well both the Pyrap-AKH and the Peram-CAH-II. A biotinylated probe was prepared from Cys1-Pyrap-AKHusing Biotin Long Arm Maleimide (BLAM, Vector Laboratories,Peterborough, UK). The ELISA comprised pre-coating of the 96-wellmicrotitre plates (high binding Costar, Corning Incorporated, Corn-ing, NY, USA) overnight with the IgG preparation in coating buffer.After blocking (non-fat dried milk), test samples were added tospecific wells, followed by the biotinylated probe, both in an assaybuffer. After the competition for the binding sites on the IgG boundto the plates a streptavidin conjugated with horseradish peroxi-dase solution (Vector Laboratories), diluted 1:500 in PBS-Tweenwas added to each well. All of the above mentioned steps wereterminated by washing. Finally, freshly prepared OPD (orthophe-nylenediamine) reagent was added and than the reaction wasstopped by adding 0.5 M sulphuric acid. The absorbance valueswere determined in a microtitre plate reader at 492 nm. One rowof each plate always contained a dilution series of synthetic Pyr-ap-AKH which allowed the construction of a competition curveand estimation of the AKH content of unknown samples.

2.6. Oxidative stress determination

The effect of insecticides endosulfan and malathion on the oxi-dative stress level was examined by measuring of two biomarkers– catalase activity and GSH level. The experimental bugs were trea-ted with the LD15 and LD50 insecticide doses (for details refer Sec-tion 2.2) and used 3 h later for the analyses.

2.6.1. Catalase measurementCatalase activity was measured spectrophotometrically accord-

ing to Claiborne [8]. Three hours after the treatment by insecticidesand/or co-treatment by insecticides together with Pyrap-AKH(80 pmol), the whole bugs were homogenized in a sodium phos-phate buffer. The homogenates were centrifuged and the superna-tant used for the reaction with hydrogen peroxide. Degradation ofthe hydrogen peroxide was measured at 240 nm during 30 s, andthe activity of catalase was expressed in lmol min�1 using theextinction coefficient of 42.6 L mol�1 cm�1 for the hydrogenperoxide.

2.6.2. Reduced glutathione measurementReduced glutathione (GSH) and its oxidized product GSSG were

quantified according to Griffith [18] with some modifications. Hae-molymph was sonicated, centrifuged and for the total glutathione(GSH + GSSG) measurement, the supernatant was diluted 100times in N2 purged potassium phosphate buffer (60 mM, pH 7.0).

For the GSSG measurement, the supernatant was diluted 30 timesin the same buffer, then 2-vinylpyridine was added and themixture was incubated 1 h at 30 �C. After that the samples werevortexed, centrifuged and the supernatants used for the GSSGdetermination. The samples of both series (GSH + GSSG and GSSG)were mixed in microtitre wells with 0.2 mM NADPH and glutathi-one reductase (0.01 U/ml). The mixture was incubated at 37 �Cfor 5 min with gentle shaking, and after that 6 mM 5,50-dithi-obis(2-nitrobenzoic acid) was added. The change of absorbancewas recorded for 10 min in 20 s intervals at 412 nm in a Spectra-max microplate reader. Total glutathione content was quantifiedagainst a calibration curve of GSH (0–25 lM) using the kineticmethod (slope) and expressed in nmol/ml of haemolymph. Theamount of GSSG was estimated from the calibration curve of theGSSG standard (0–12.5 lM) by the same way as mentioned forthe total glutathione. The amounts of reduced GSH were calculatedby subtracting the GSSG content from that of the total glutathione(GSH + GSSG) one.

2.7. Metabolic rate measurement

A flow-through respirometry system to measure the rate ofcarbon dioxide production by experimental bugs was used as previ-ously described [28]. Briefly, air is pushed through a chamber withthe experimental insects in this system at a flow rate 60 ml min�1

into the Li-7000 CO2/H2O analyser (Li-COR Biosciences, Lincoln,NE, USA), which is interfaced with a computer. The carbon dioxidemeasurement is based on the difference of infrared radiation pass-ing through two gas cells. A reference gas flows continuously withinthe reference cell, and its signal is subtracted from the sample cellone in order to get rid of any variation in the ground signal. The out-put of the analyzer is proportional to the difference in absorptionbetween the two cells. A group of 10 bugs was measured 0, 1, 3, 6and 24 h after the treatment for a period of 20 min. Only living indi-viduals were used for the analysis. Data were analysed by data-acquisition software (Sable System, Las Vegas, Nevada, USA). Thecarbon dioxide production ðVCO2 Þ was calculated from fractionalconcentrations of carbon dioxide going in (FI) and coming out (FE)of the respirometry chamber using an equation according to With-ers [52] and expressed in ll h�1 bug�1 units:

VCO2 ¼ ðFECO2 � FICO2 Þf where f is the flow rate in ll h�1

2.8. Data presentation and statistical analyses

The obtained results were plotted using the graphic programPrism (GraphPad Software, version 5.0, San Diego, CA, USA). Thebar graphs and the symbols in the Fig. 1 represent the mean ofmeasurement ± SD (Fig. 1: n = 3–5 groups – each containing 20bugs, Fig. 2: n = 12–15, Fig. 3: n = 5–7 groups – each containing20 ll haemolymph from about 5 bugs, Fig. 4: n = 8, Fig. 5: n = 5groups – each containing 15 ll haemolymph from about 5 bugs,Fig. 6: n = 4 groups – each contained 20 bugs, Fig. 7: n = 5 groups– each containing 10 bugs). The significance of the results wasevaluated by one-way ANOVA followed by Tukey’s multiple com-parison test (significance of the effects of different treatments ona single parameter) in Figs. 2–5 and 7, and by the commonStudent’s t-test in Figs. 4 and 6, at 5% significance level.

3. Results

In the first series of experiments, doses of insecticides suitablefor P. apterus treatments, have been standardized. The results pre-sented in Fig. 1 show the effect of increasing doses of insecticideson the mortality of bugs. The LD15 and LD50 were determined from

Fig. 1. The effect of increasing doses of endosulfan and malathion on the mortalityof P. apterus 24 h after injection into the haemocoel (A) or after topical application(B).

Fig. 2. The effect of LD15 and LD50 of endosulfan (A) and malathion (B) injected intothe haemocoel of P. apterus on the level of AKHs in CNS 24 h after the treatment.Control bugs were injected with Ringer saline only. Statistics: one-way ANOVA andTukey’s multiple comparison test: significant differences at the 5% level betweencontrol and experimental treatments are labelled by asterisks. Vertical linesindicate SD.

Fig. 3. The effect of LD15 and LD50 of endosulfan (A) and malathion (B) injected intothe haemocoel of P. apterus on the titre of AKHs in haemolymph 24 h after thetreatment. Control bugs were injected with Ringer saline only. Statistics: one-wayANOVA and Tukey’s multiple comparison test: significant differences at the 5% levelbetween control and experimental treatments are labelled by asterisks. Verticallines indicate SD.

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the dose–response curves by a probit analysis from the number ofbugs that died within 24 h after injection (Fig. 1A) or topical applica-tion (Fig. 1B) of the insecticides. The corresponding doses were asfollows (the numbers in brackets represent rounded doses used for

analyses of the insecticide interactions with the Pyrap-AKH): injec-tion – endosulfan LD15 = 186.0 ng (200 ng), LD50 = 237.4 ng(250 ng), malathion LD15 = 279.3 ng (300 ng), LD50 = 452.6 ng(450 ng); topical application – endosulfan LD15 = 474.2 ng(450 ng), LD50 = 1127.3 ng (1100 ng), malathion LD15 = 519.1 ng(500 ng), LD50 = 894.7 ng (900 ng). The application of insecticideby injection was employed for most of the experiments studyingphysiological aspects of the phenomenon, both injection as well astopical application were used for the interactions that resulted in in-crease of bug mortality (see below).

The insecticide treatment definitely causes a severe stress in in-sects [22] including the P. apterus, which should activate corre-sponding biochemical pathways. This prediction was indicated byincreasing of the AKH level in the P. apterus body 24 h after theendosulfan or malathion injections. The competitive ELISA re-vealed just a slight increase of the AKH amount in the CNS(Fig. 2), however, changes in the haemolymph (Fig. 3) were moredramatic. Significant increase of the AKH level in haemolymphwere recorded for LD50 of endosulfan (1.4 times, Fig. 3A), and forboth doses of malathion (1.4 and 1.7 times, respectively, Fig. 3B).

The status of oxidative stress in P. apterus body after the insec-ticide treatment was assessed by measuring catalase activity(Fig. 4). Differences in catalase activity between the control (injec-tion of Ringer saline only) and experimental bugs increased afterboth LD15 and LD50 of endosulfan injection about 14 times as com-pared with the bugs treated with Pyrap-AKH only (Fig. 4A). Thestimulatory effect of endosulfan was significantly reduced (about2.3 times) when this insecticide was co-injected in combinationwith 80 pmol Pyrap-AKH which indicates a lower production ofhydrogen peroxide elicited by oxidative stress action of endosulfanalone. A similar picture was obtained using malathion (Fig. 4B): inthis case the protective effect when Pyrap-AKH was co-injectedwith the insecticide was even more apparent – the reduction ofthe catalase activity was 3.7-fold (for LD15) and 3.2-fold (LD50),

Fig. 4. The effect of LD15 and LD50 of endosulfan – E (A) and malathion – M (B)injected into the haemocoel of P. apterus alone or in combination with the Pyrap-AKH (80 pmol) on the catalase activity 3 h after the treatment. The results areexpressed as a difference in the catalase activity between the control and theexperimental bugs; control bugs were injected with Ringer saline only. Statistics:one-way ANOVA and Tukey’s multiple comparison test: significant differences atthe 5% level between the Pyrap-AKH and insecticide treatments are labelled byasterisks, and between the insecticide treatments and insecticide co-treatmentswith the Pyrap-AKH by @. Vertical lines indicate SD.

Fig. 5. The effect of LD15 of endosulfan (E) and malathion (M) injected into thehaemocoel of P. apterus alone or in combination with the Pyrap-AKH (80 pmol) onthe reduced glutathione (GSH) level in haemolymph 3 h after the treatment. Theresults are expressed in nmol of GSH per ml of haemolymph; control bugs wereinjected with Ringer saline only. Statistics: one-way ANOVA and Tukey’s multiplecomparison test: significant differences at the 5% level between the control and theinsecticide treatments are labelled by @, and between the insecticide treatmentsand insecticide co-treatments with the Pyrap-AKH by asterisks.

Fig. 6. The effect of LD15 and LD50 of endosulfan and malathion injected into thehaemocoel (A) or applied topically (B) on P. apterus body either alone or incombination with the Pyrap-AKH (80 pmol) on the mortality 24 h after thetreatment. Statistics: Student’s t-test: significant differences at the 5% levelbetween the insecticide treatment and insecticide with the Pyrap-AKH co-treatment are labelled by asterisks. Vertical lines indicate SD.

M. Velki et al. / General and Comparative Endocrinology 172 (2011) 77–84 81

respectively. In other words, malathion appears to be a weakergenerator of oxidative stress than endosulfan at least in the path-ways resulting in hydrogen peroxide production and its decompo-sition by catalase, because the same AKH dose reduced the catalaseactivity increased by malathion more effectively than in the case ofendosulfan.

On examining the next oxidative stress marker – the level ofglutathione in haemolymph – only the effect of LD15 of both insec-ticides was measured. The results presented in Fig. 5 are expressedas the reduced glutathione (GSH) only; the share of the GSH on thetotal glutathione content (GSH + GSSG) was in all samples about90–94%. A small fall of the level that was not significant, was re-corded after the injection of endosulfan (1.2 times). The effect ofmalathion was more pronounced, however, and resulted in signif-icant 1.4-fold reduction of GSH level in the bug’s haemolymph.Injection of the Pyrap-AKH only (80 pmol) had no apparent effect.Co-injection of the insecticides with the Pyrap-AKH however sig-nificantly increased the GSH level as compared with the insecticidealone (using endosulfan 1.4 times and using malathion 1.3 times).After these co-treatments GSH levels stabilized at values that didnot significantly differ from the control one.

In experiments dealing with the insecticide and Pyrap-AKHinteractions and their impact on the oxidative stress, it was re-corded that the bugs treated with both agents showed apparentlyhigher mortality than the others. The subsequent series of

Fig. 7. The effect of LD15 of endosulfan (A) and malathion (B) injected into thehaemocoel of the experimental bugs P. apterus alone or in combination with thePyrap-AKH (80 pmol) on the carbon dioxide production 1, 3, 6 and 24 h after thetreatment. Control bugs were injected with Ringer saline only. The carbon dioxideproduction was measured for a period of 20 min (for details see Section 2).Statistics: one-way ANOVA and Tukey’s multiple comparison test: significantdifferences at the 5% level are indicated as follows: (a) control vs. any other group,(b) AKH vs. any other group, (c) insecticide vs. any other group, (d) insecticide withAKH vs. any other group. Vertical lines indicate SD.

82 M. Velki et al. / General and Comparative Endocrinology 172 (2011) 77–84

experiments revealed that co-injection of LD15 of endosulfan with80 pmol Pyrap-AKH resulted in about 3-fold significant increase ofmortality within 24 h than after the injection of endosulfan only(Fig. 6A); the LD50 with the same dose of Pyrap-AKH elicited1.7-fold increase of mortality. A similar effect was recorded whenmalathion was injected: for the LD15 the mortality increased justslightly after co-injection with the Pyrap-AKH, but using the LD50

again the 1.6-fold significant increase was recorded. The mortalityin control (Pyrap-AKH only) groups was negligible. Since insecti-cides are not usually injected, the phenomenon was confirmedby topical application of these agents (Fig. 6B). The results showedthe same trend: 1.8 and 1.7-fold significant increase of mortalitywas recorded for LD15 and LD50 of endosulfan, respectively, afterthe co-application with 80 pmol Pyrap-AKH. For LD50 of malathionthe increase – 1.4-fold – was also significant. Mortality after LD15 ofmalathion treatment was enhanced just slightly by the Pyrap-AKHtopical application. The mortality in control group was negligibleand similar to that in injected bugs. Statistical comparison of themortality results for the LD15 and LD50 of endosulfan and mala-thion co-injected with Pyrap-AKH between themselves (i.e. LD15

of endosulfan with Pyrap-AKH vs. LD15 of malathion with Pyrap-AKH, etc., Student t-test) suggested that for both doses and for bothmanners of application endosulfan was significantly more effective(on 5% level) than malathion.

A question arose about the role of the AKH as a stress hormonein this phenomenon, how the AKH interacts with the insecticidesand what is the mechanism by which AKH increases the mortalityelicited by the insecticides alone. It was hypothesized that intensi-fication of metabolism after the Pyrap-AKH application could bethe reason for the observed effects. As a marker of metabolic activ-ity, carbon dioxide production was monitored in the experimentalbugs (Fig. 7). The results revealed that application of the Pyrap-AKH (80 pmol) increased the carbon dioxide production, however,a significant difference was recorded only 1 and 3 h after the

treatment; 6 and 24 h later on the effect disappeared. Applicationof endosulfan (LD15) elicited no significant effect in the carbondioxide production (Fig. 7A). On the other hand, as expected, co-application of the LD15 of endosulfan with the Pyrap-AKH signifi-cantly increased carbon dioxide production by 2.6, 2.0 and 1.7times 1, 3 and 6 h after the treatment, respectively, compared withapplication of endosulfan alone. No measurements were possible24 h after co-treatment, because mortality of the bugs was almost100% (see Fig. 6A). A similar effect was recorded using LD15 of mal-athion (Fig. 7B). In contrast to endosulfan, malathion alone en-hanced the carbon dioxide production significantly within thewhole duration of the experiment including 24 h (increasing1.4–1.8 times). Co-application of the LD15 of malathion with thePyrap-AKH significantly increased the production 1.3 and 1.2 times1 and 3 h after the treatment, respectively, compared with applica-tion of endosulfan only. Later on the effect disappeared. It isthought that this increase in metabolic rate might be a crucial fac-tor behind the increased mortality after insecticide plus Pyrap-AKHco-treatment documented in Fig. 6.

Results presented in Fig. 7 also support the fact that endosulfanwas more effective than malathion in the mortality test when co-injected with Pyrap-AKH based upon the results of Fig. 6. The ratiosof carbon dioxide production by bugs treated with endosulfan vs.endosulfan with Pyrap-AKH (2.6, 2.1 and 1.7 after 1, 3 and 6 h,respectively) are evidently higher than those of malathion vs. mal-athion with Pyrap-AKH treatments (1.3, 1.2 and 1.1 after 1, 3 and6 h, respectively). This is consistent with the suggestion that theAKH-stimulated increase in metabolic rate might play an impor-tant role in the increase of mortality in co-treatment tests (detailsare discussed in Section 4.2).

4. Discussion

4.1. AKH and oxidative stress

Anti-stress defence mechanisms in insects can be induced byvarious stressors such as injury, infection, starvation, intensivelocomotion, X-ray irradiation and poisoning, including treatmentby insecticides. All of these stressors disrupt homeostasis andevoke similar defence mechanisms. Biochemical and physiologicalprocesses must be mobilised to eliminate or at least to minimizethe stress impact on body functions. Certain stressors elicit oxida-tive stress in the organism, whereby the production of free radicalsis enhanced and corresponding defence mechanisms are activated.It is supposed that hormones could be responsible for the initiationof the processes [37]. Recently it has been proven that AKHs[29,50], ecdysteroids [34], juvenile hormone [25] and glucagon[1] play this role in insects.

Application of the oxidative stressors such as paraquat [21],Gallanthus nivalis agglutinin and Bacillus thuringiensis toxin [29],and insecticides endosulfan and malathion (this study) elevatesthe titre of AKHs in haemolymph and depending on duration ofthe stressor incidence, also in the CNS. All those findings suggestthat AKH might be involved in the activation of an antioxidant pro-tection processes. Despite of the fact that exact mechanism(s) ofstimulation of antioxidative reactions by AKH is far from clear, aputative role of AKH in regulation of oxidative stress is supportedby biochemical data. Previously we have shown [29,50] that AKHis somehow involved in the processes reducing the degree of car-bonylation of proteins, in enhancing GSH level and in increasingof total antioxidant capacity of the haemolymph. The list of thoseAKH activities has now been extended to also include its impacton catalase activity. Catalase is a 24 kD homotetrameric enzymewith a heme–iron active centre, whose main function is todecompose toxic hydrogen peroxide into water and oxygen

M. Velki et al. / General and Comparative Endocrinology 172 (2011) 77–84 83

(2H2O2 ? O2 + 2H2O) [51]. Catalase activity was measured 3 h afterexposure to insecticides and Pyrap-AKH in this study. Both endo-sulfan and malathion caused a significant increase in the activity,but when co-applied with the Pyrap-AKH a significant decreasewas recorded compared with the insecticides alone. This indicatestwo phenomena: first, both endosulfan and malathion causeoxidative stress in P. apterus; and second, Pyrap-AKH reduces thelevel of catalase activity enhanced by oxidative stress. The induc-tion of oxidative stress and increased formation of ROS wasexpected, because both endosulfan and malathion are known topromote formation of free radicals [2,13,23]. Since free radical-scavenging enzyme complexes like superoxide dismutase, catalaseand glutathione peroxidase, are in the first line of cellular defenceagainst oxidative injury [4,39,44], the induction of catalase afterinsecticide application was anticipated. Possibly, AKH reduces theproduction of hydrogen peroxide by an unknown mechanism,and therefore the activity of catalase necessary for reduction ofhydrogen peroxide is lower. For malathion, co-application withPyrap-AKH decreased the catalase activity closer to control level(Pyrap-AKH only) than for endosulfan, which might mean thatendosulfan is more effective in eliciting hydrogen peroxide produc-tion than malathion.

To confirm a positive role of Pyrap-AKH in stimulating antioxi-dative mechanisms documented by the catalase data, and to com-pare the effect of the insecticide oxidative stressors with paraquat[29,50] the GSH level in the bug’s haemolymph was determined.Glutathione is one of the most important antioxidants: it is a lowmolecular weight thiol (Glu-Cys-Gly) found in the cytosol andother aqueous phases of various living systems [33,35]. In the pres-ence of oxygen radicals the reduced form (GSH) is oxidized (GSSG)and can then be recycled back in a NADPH-dependent reaction cat-alyzed by glutathione reductase or by the thioredoxin reductasesystem (in the case of Drosophila [24]). Results of this paper indi-cate that malathion seems to be more effective in lowering of theGSH level in haemolymph than endosulfan. This corresponds withthe data of Büyükgüzel [5], who found that dietary malathion in 1and 10 ppm concentrations significantly reduced the GSH level inbody of the wax moth Galleria mellonella. Although the role of Pyr-ap-AKH in elevating reduced GSH levels is not as pronounced as itseffect on catalase activity, it none the less evokes an antioxidativeresponse by direct or indirect increase of the GSH efflux from thetissues. This is supported by work of Lu et al. [36] who found thatglucagon injected into rats stimulates massive GSH efflux from theliver into the bloodstream. On the other hand, AKH might upregu-late the enzymes involved in regenerative glutathione cycle trans-forming the oxidised form (GSSG) to its reduced one (GSH).

Interestingly, Pyrap-AKH alone did not cause any significantchanges both in catalase activity and in GSH level as comparedwith controls, indicating that stressor action is a prerequisite forthe Pyrap-AKH activation of the antioxidant protective mecha-nisms. Presence of a stressor for activation of the AKH functionsseems to be an important character of those hormones at least incertain AKH activities. For example, positive correlation betweenthe AKH stimulated mobilization of lipids and enhancement oflocomotory activity was recorded only for injection and not fortopical application of the hormone [30]. This suggests that thestressing factor caused by injection plays a role in co-ordinationof the AKH actions; when the injury stressor is absent (such as intopical application) the response is slow and unbalanced, or evenmissing.

4.2. AKH and insecticide action

The use of toxic chemicals or rather insecticides to evoke stressconditions in insect body is not so exceptional in AKH research[22]. There are probably several reasons for it: the insecticides

are potent stressors, often penetrate the cuticle, are easy to handlewhich from the practical aspect of a pest control is also indispens-able. Poisoning of insects to induce modulation of the AKH titrewas firstly used by Singh and Orchard [45], who described the re-lease of AKHs from isolated corpora cardiaca of the locust Locustamigratoria after treatment by insecticides. They supposed that thisrelease was mediated by direct action of insecticides on the neuro-secretory cells. Later, Candy [7] recorded significant release of thehormones from corpora cardiaca and increase of their titre in hae-molymph of Schistocerca gregaria treated with the insecticide del-tamethrin or excessive doses of KCl. Also, short-term application(20 min) of permethrin was responsible for a significant increaseof the AKH titre in haemolymph of P. apterus [27], however, thetime was too short to affect the AKH level in CNS. Nevertheless,extending the time of the permethrin treatment to 24 h signifi-cantly elevated the AKH level also in corpora cardiaca [28]. Asmentioned before, multiple increases in the titre of AKH (both inCNS and haemolymph) occurred also in the Colorado potato beetle,Leptinotarsa decemlineata, fed on genetically modified potatoesexpressing Bacillus thuringiensis toxin or Gallanthus nivalis lectin[29].

An intriguing finding in the present study is that co-applicationof either of the insecticides with Pyrap-AKH significantly increasedthe mortality of the bugs. Recently, the same effect has been re-ported for a pyrethroid permethrin, a neurotoxin, whose effecton mortality is significantly enhanced by co-injection or topicalco-application with Pyrap-AKH [28]. Almost nothing is knownabout the mechanism of the phenomenon, but it has been sug-gested that the AKH stimulation of metabolism could enhancethe insecticide action. This suggestion is supported by the Pyrap-AKH-induced increase of carbon dioxide production [28, this pa-per] – an indicator of metabolic rate in the experimental bugs.The increased metabolic rate could intensify the insecticide actionby a more intensive exchange of metabolites in biochemically ac-tive cells accompanied by faster penetration of the insecticide intotissues.

The present results indicate that the Pyrap-AKH enhanced mor-tality of the bugs by endosulfan is higher than that induced by mal-athion. This fact correlates with the carbon dioxide data (Fig. 7)that demonstrate a more pronounced effect of the Pyrap-AKH onmetabolic rate in the bugs treated with endosulfan than in thosereceiving malathion. Thus the higher potency of endosulfan sup-ports the hypothesis that stimulation of metabolism by AKH is crit-ical for the enhancement of the insecticide effectivity. However, inthe absence of direct biochemical data it continues to be a theorythat should be experimentally proven.

In summary, the present paper demonstrates that the insecti-cides endosulfan and malathion elicit oxidative stress in the modelinsect species P. apterus. The stress enhances the production ofAKH, which might be involved in activation of antioxidative stressmechanisms. Further, AKH increases insecticidal activity probablyby stimulating metabolism, which can account for the greater tox-icity of endosulfan compared with malathion.

Acknowledgments

This study was supported by the Grant No. P501/10/1215 fromthe Czech Science Foundation (DK), and by the Institute ofEntomology Project No. Z50070508 obtained from the Academyof Sciences of the Czech Republic. The stay of MV in the Universityof South Bohemia was supported by the Erasmus programme. Theauthors thank Dr. I. Bártu for her help with the carbon dioxidemeasuring, Mrs. D. Rienesslová for her technical assistance andDr. N. Krishnan (Oregon State University, USA) for critical readingof the MS.

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