ARTICLE IN PRESS

Journal of Insect Physiology 53 (2007) 67–74

0022-1910/$ - se

doi:10.1016/j.jin

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kodrik@entu.ca

sehnal@entu.ca1Present addr

Plant Protectio

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Stage-specific distribution of oxidative radicals and antioxidant enzymesin the midgut of Leptinotarsa decemlineata

Natraj Krishnan, Dalibor Kodrık, Ferit Turanli1, Frantisek Sehnal�

Institute of Entomology, Biology Centre of the Academy of Sciences, and the Faculty of Biological Sciences, University of South Bohemia; Branisovska 31,

Ceske Budejovice 37005, Czech Republic

Received 17 June 2006; received in revised form 29 September 2006; accepted 3 October 2006

Abstract

The titers of reactive oxygen species (ROS) represented by superoxide anion and general peroxides, and the activities of antioxidant

enzymes superoxide dismutase (SOD) and catalase (CAT), are regulated in the midgut of the Colorado potato beetle (CPB) relative to

the gut compartment, developmental stage, and food intake. ROS concentration is low in the potato leaves but it is very high in their

digest in insect’s anterior midgut. It is proposed that intensive ROS production in this gut region is linked to the processing of

allelochemicals. SOD and CAT activities, low oxygen tension, and unidentified redox systems that maintain a slightly reducing milieu in

the midgut lumen (pe+pH ¼ 6.95 declining to 5.36), obviously contribute to the decrease of ROS concentration along the gut length to a

minimum in the wall of posterior midgut region. SOD and CAT activities are higher in the potato leaves than in the midgut tissues but

the role of plant enzymes in ROS elimination within the gut lumen remains to be shown. A lower level of ROS and a higher antioxidant

potential in the adult than in the larval midgut indicate stage specificity in the management of oxidative stress. The antioxidant defense is

high in the diapausing adults that contain no detectable superoxide and about ten times less peroxides than the reproducing adults.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Catalase; Insect digestion; Peroxides; Superoxide anion; Superoxide dismutase

1. Introduction

All aerobic organisms must eliminate mutually conver-tible reactive oxygen species (ROS) singlet oxygen, super-oxide anion, hydroxyl anion, and hydrogen peroxide,which are generated by oxidative metabolism. Herbivorousinsects are also challenged by exogenous ROS that are partof the plant defense against them (Orozco-Cardenas andRyan, 1999) and the pathogens (Doke et al., 1996). Plantdefense further includes a variety of allelochemicals, mostoften phenolic compounds, that range from small acids tocomplex polymers such as tannins and lignins (Harborne,1997). Ingested phenolics may undergo oxidation (Appel,1993) to products that curb digestion by cross-linking

e front matter r 2006 Elsevier Ltd. All rights reserved.

sphys.2006.10.001

ing author. Tel.: +420 387775220; fax: +420 385310354.

esses: krish@entu.cas.cz (N. Krishnan),

s.cz (D. Kodrık), ferit.turanli@ege.edu.tr (F. Turanli),

s.cz (F. Sehnal).

ess: Ege University, Faculty of Agriculture, Department of

n, 35100 Bornova, Izmir, Turkey.

ingested amino acids and proteins (Harborne, 2001) andenhance the load of ROS in the digestive tract (Barbehennet al., 2005; Krishnan and Sehnal, 2006). High ROSconcentration impairs the absorption of ingested nutrientsand can cause oxidative damage to the midgut cells (Bi andFelton, 1995). Without a counter-defense, ROS couldrender the digestive tract non-functional. A portion ofROS is scavenged by dietary antioxidants such as ascorbateand carotenoids but most are eliminated by a suite ofantioxidant enzymes (Aucoin et al., 1991). For example,superoxide dismutases (SODs) convert the superoxideanion Od�

2 to oxygen and hydrogen peroxide at rateslimited only by diffusion (ca. 2� 109M�1 s�1), and thecatalase (CAT) reduces hydrogen peroxide to water andoxygen (Fridovich, 1978). CAT is a low-affinity but highcapacity enzyme, perfectly suited for scavenging highamounts of H2O2 that occur in the mitochondria andperoxisomes. Both SOD and CAT are principally intracel-lular enzymes but their activities, which reflect the grossrate of ROS turnover, were also recorded in insect gut

ARTICLE IN PRESSN. Krishnan et al. / Journal of Insect Physiology 53 (2007) 67–7468

lumen (Felton and Summers, 1995; Krishnan and Kodrık,2006). The insects also possess ascorbate peroxidase andthe glutathione S-transferase with peroxidase-like activity(Ahmad, 1992; Claravon-Mathews et al., 1997; Krishnanand Kodrık, 2006).

The Colorado potato beetle (CPB), Leptinotarsa decem-

lineata, consumes potato leaves that harbor up to 3.2%tannins (Duke and Atchley, 1986) and contain additionalphenolic and steroid compounds typical for the Solanaceaeplant family. An abundant biphenol, the chlorogenic acid,accumulates in the potato leaves to 0.2% fresh weight andthe concentration of steroid glycoalkaloids reaches asimilar level (Friedman, 2004). The content of endogenousROS has not been measured in potato leaves but it isprobably similar as in other plants (Kuzniak and Urbanek,2000). One aim of the present study was to assess thecontribution of plant ROS to the oxidative environment ininsect gut. We chose to measure the concentrations ofsuperoxide anion and total peroxides (including hydrogenperoxide) that provide an adequate indication of the ROSlevel. SOD and CAT activities were selected as antioxidantenzymes that may also be derived from the ingested leaves.Our second aim was to compare the efficiency of ROSmanagement in the larval gut and in the gut of active anddiapausing adults. We distinguished anterior and posteriormidgut regions and in each of them examined separatelythe gut wall and the gut contents.

2. Materials and methods

2.1. Insects and chemicals

L. decemlineata (Say) (CPB) larvae 2–3 days after the lastlarval ecdysis and the non-diapausing adults 5–6 days afterthe emergence were analyzed. The insects were reared onthe potato cultivar Desiree grown in the greenhouse at25 1C under long day conditions (16 h light: 8 h darkness).A few measurements were done on the non-feedingdiapausing adults resting in the soil for about 3 months.Diapause was induced by keeping the insects under a shortday photoperiod (light:darkness 12:12 h) at 15–20 1C sincethe last larval ecdysis. For the food passage studies, newlyecdysed last instar larvae and adults were fed ad libidumfor 2 and 5 days, respectively, then starved for 24–32 h, andallowed to feed a gain. The passage of food through thedigestive tract was tracked in insects dissected at specifictimes after the feeding resumption. All chemicals werepurchased from Sigma Chemical Company, St. Louis, MO,USA. To remove oxygen that could interfere with theassays, the buffers (50–100ml) were bubbled through withN2 (1ml/min) for 10min.

2.2. Gut pH, redox potential, and O2 content

The measurements of pH and the redox calculationswere done as described by Krishnan and Kodrık (2006).Selected insect specimen was immobilized on ice and the

gut exposed through a dorsal incision. Microelectrodeneedles with beveled tips were inserted through the gut wallthat tightened around the insertion and restricted air accessinto the gut lumen. Measurements were done in theforegut, anterior midgut, medial midgut, and posteriormidgut lumen, respectively. Microneedle pH electrode MI408B (Microelectrodes Inc., Bedford, NH, USA), oxida-tion–reduction potential microelectrode MI 800-408B, andthe silver–silver chloride reference electrode MI 401 wereconnected to the Inolab Level 1 pH and Millivolt Meter(WTW GmbH, Weilheim, Germany). Redox potentialswere read in millivolts (Ehobs) and converted to standardredox potentials (Eh) by adding 200mV. Electron avail-ability was calculated with the Nernst equation (pe ¼ Eh/59.2). Oxidative properties of the system were eventuallyexpressed as the redox parameter pe+pH. Dissolvedoxygen was measured in the anterior and posterior midgutregions at 25 1C and atmospheric pressure 728mmHg usingthe oxygen electrode DO-166MT-1 (Lazar Research LabsInc., Los Angeles, CA, USA). The probe was calibratedagainst 0.2% sodium metabisulfite solution (zero oxygenconcentration).

2.3. Sample preparation for biochemical assays

Potato leaves (40mg) were minced mechanically with arazor blade and the mash of ca. 80 mm pieces was quicklysubmerged in 100 ml ice-cold 50mM sodium phosphatebuffer (pH 6.4, corresponding to the gut pH) purgedwith nitrogen. Aliquots (50 ml) of the suspension wereeither analyzed immediately or incubated first for 15 or30min at 25 1C and under gentle shaking in tubes flushedwith N2.CPB chosen for analysis were immobilized at �20 1C for

10min and the digestive tract between oesophagus and theattachment of Malpighian tubes was dissected on ice. Thetract was cleaned of the fat body and Malpighian tubes,blotted briefly on a filter paper and separated into ananterior and a posterior part. The anterior part includedthe foregut and a broad proximal midgut section; since theforegut is negligible in comparison to the midgut, the wholepreparation is referred to as anterior midgut. The posteriorpart consisted of the narrower but longer posterior midgut.The anterior midgut content (AC), anterior midgut wall(AW), posterior midgut content (PC), and posteriormidgut wall (PW) were collected separately in pre-weighedmicrocentrifuge tubes.Weighed samples (leaves or midgut compartments) were

mixed with the N2-saturated buffer appropriate for theintended analysis in tubes flushed with N2. Minced leavesand gut walls were crushed for 1min in a motor-drivenglass homogenizer and sonicated (Ultrasonic homogenizer,4710 series, Cole–Parmer Instrument Co., Chicago, IL,USA) at 40–50MHz for 20–30 s, while samples of the gutcontents were subjected only to sonication. Furtherprocessing is described in individual assays. The contentof soluble proteins was measured in aliquots centrifuged at

ARTICLE IN PRESSN. Krishnan et al. / Journal of Insect Physiology 53 (2007) 67–74 69

15,000g for 10min. Proteins were precipitated from thesupernatant with 5% trichloroacetic acid, partially hydro-lyzed with 1N NaOH for 15min on boiling water bath,and quantified with the Coomasie Blue binding method ofBradford (1976). Bovine serum albumin was used asstandard. Spectrophotometric measurements were donewith the Spectramax 340PC (Molecular Devices, Sunny-vale, CA, USA) microtitre plate reader unless indicatedotherwise.

2.4. Determination of the superoxide content

The content of O2 was assessed from the change ofaconitase activity after its activation (Hausladen andFridovich, 1996). The samples were homogenized (2% w/v) in 500 ml ice-cold 0.2mM sodium citrate in 50mMTris–HCl buffer, pH 6.4 (purged with N2), centrifuged at800g for 10min, and the supernatants were spun again at20,000g for 10min. The pellets were re-suspended in cold0.2mM sodium citrate and stored for up to a week at�80 1C in tubes filled with N2. Aconitase activity wasassayed in a reaction mixture containing 50mM Tris–HCl(pH 6.4), 30mM sodium citrate, 0.6mM MnCl2, 0.2mMNADP+, and 1 unitml�1 isocitrate dehydrogenase.Changes in absorbance at 340 nm were read at 25 1C for5min. One milliunit of aconitase activity corresponded to1 nmol of NADPH formed per minute (Rose andO’Connell, 1967). To determine how much of the enzymewas inactivated by Od�

2 present in the sample (Kennedyet al., 1983), aconitase was reactivated in the presence of0.5M 1,4-dithio-DL-threitol (DTT), 20mM ferrous am-monium sulfate, and 20mM N2S. The rate constants forinactivation [kinact. ¼ 2� 107mol�1 s�1] and reactivation[kreact ¼ 0.004� 107mol�1 s�1] of purified aconitase weredetermined and Od�

2 amounts in pmolmg�1 sample freshweight were calculated with the equation [Od�

2 ] ¼([aconitaseinact]/[aconitaseact])(2� 10�10M).

2.5. Determination of total peroxide content

The samples were extracted (2% w/v) with 50mM sodiumphosphate buffer, pH 6.4, containing 5mM sodium azideand purged with N2. The supernatants obtained bycentrifugation (14,000g, 10min, 4 1C) were used for themodified FOX2 (ferrous oxidation—xylenol orange) assaybased on the oxidation of ferrous ions (Fe2+) to ferric ions(Fe3+) by the peroxides (Hermes-Lima et al., 1995; Deianaet al., 1999). The stable complex of Fe3+ with xylenolorange formed under acidic conditions was quantified at595nm and the amount of total peroxides was assessedagainst hydrogen peroxide standards. Samples chosen forthe quantification of endogenous hydrogen peroxide wereprepared as above but without sodium azide. CAT (1.3EU/ml in N2-saturated water) was used to selectively destroyhydrogen peroxide in sample aliquots that were subse-quently taken for the quantification of lipid peroxidesagainst the tert-butyl hydroperoxide standard. The content

of hydrogen peroxide was then calculated by subtracting theperoxide amount in the CAT-containing from that in theCAT-free samples. The samples were assayed simulta-neously with a microplate reader. The total peroxide content(H2O2+lipid peroxides) was expressed in nmol/mg samplefresh weight.

2.6. Superoxide dismutase (SOD)

Samples sonicated in 50 mM N2-saturated sodiumphosphate buffer (pH 6.4) were centrifuged (12,000g,10min, 4 1C) and the supernatants transferred to –80 1C.Samples boiled for 5min were run in parallel to subtractthe non-enzymatic superoxide dismutation. Frozen super-natants were thawed within a week and filtered through a10,000 Da cut-off membrane (Ultrafree MC, Millipore,Billerica, MA, USA). Reconstituted retentates in phos-phate buffer were used for the analysis. Preliminaryexperiments were conducted to differentiate Mn–SODfrom the Cu–Zn SOD using 5mM cyanide. Cu–Zn SODclearly dominated and was targeted with our assay. SODreaction mixture consisted of 15 ml sample extract in bufferwith 0.125mM EDTA (pH 6.4) and 1.3 EU/ml CAT;epinephrine in 100mM HCl was added to a 13.6mMconcentration to start the reaction. The rate of absorbancechange at 490 nm was measured with a microtiter platereader for 10min. Kinetic measurements were made usingsix different dilutions of each sample extract. One unit ofSOD activity was defined as the mass of protein (mg)corresponding to a 50% inhibition in the rate ofepinephrine oxidation (Misra and Fridovich, 1972;Munkres, 1990). SOD activity was expressed in enzymeunitsmg�1 protein.

2.7. Catalase (CAT)

Supernatants (8000 g, 5min, 4 1C) of protein extracts in50mM N2-purged sodium phosphate buffer (pH 6.4) weretaken for the assay with 3% H2O2 (Aebi, 1984). Boiledsamples served as controls. Decrease in absorbance at240 nm was measured for 3min in 30 s intervals with theUV 1601 spectrophotometer (Shimadzu Corporation,Kyoto, Japan). Extinction coefficient 39.4mM�1 cm�1

was used to calculate the H2O2 content. CAT activitywas expressed in enzyme unitsmg�1 protein.

2.8. Statistical analysis

All parameters were measured in 8–10 individualsamples that were each analyzed in three or four aliquots.Data were expressed as mean7SEM and two-wayANOVA followed by Bonferroni’s post-test was used forstatistical testing of significance. Graphs were generatedusing GraphPad Prism 4 (GraphPad software, San Diego,CA, USA).

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Fig. 2. Progress of food bolus through the digestive tract of larvae (A) and

adults (B) re-fed after starvation. The tract includes foregut (FG), midgut

terminating at the attachment of Malpighian tubuli (MT), and hindgut

(HG). Solid bars represent progress of the semi-solid, and gray bars of the

liquid food digest in min after the resumption of feeding. Oxidative

conditions were analyzed separately in the wall and content of the anterior

(AMG, includes the foregut) and posterior (PMG) midgut sections.

Table 1

Means7S.D. (n ¼ 8) of the pH and pe+pH (redox parameter) values and

N. Krishnan et al. / Journal of Insect Physiology 53 (2007) 67–7470

3. Results

3.1. The rate of food passage through the alimentary tract

Average weight of the fully-grown CPB larvae selectedfor the study was 149714mg, similar to the adult weight14678mg. The two stages possessed digestive tract ofsimilar morphology and size with a very short and thin-walled foregut, voluminous midgut, and a long hindgut. Ashort but broad anterior and narrow but long posteriormidgut regions were distinguished. The anterior region wasfully extended with food in both larvae and feeding adultsbut the posterior region only in the larvae. This is reflectedin a significantly (po0.01) higher weight of the PC inlarvae, whereas weights of the AC and both anterior andposterior midgut walls (AW and PW, respectively) weresimilar as in the adults (Fig. 1). Since, the difference inmidgut load could be caused by longer food retention inthe larval gut, the rate of food passage through the gut wasexamined. After a 24–32 h starvation the gut of both larvaeand adults was empty except for brownish residues at thevery end of the midgut. The progress of food bolus afterthe feeding resumption is shown in Fig. 2. Larvae excretedliquid gut contents faster but the move of solid digest wasslower than in the adults. Ingested food passed withinminutes through the foregut and in 20min filled theanterior midgut in both larvae and adults. The fluidpreceding the advance of solid digest appeared in thehindgut in 30min and was partly excreted in 40min in thelarvae, while in the adults it occurred in the distal hindgutregion and in the excreta only in 60min. The diapausingadults contained in the midgut small amount of a brownishfluid without any leaf residues.

3.2. pH, redox potential, and dissolved oxygen in the gut

lumen

CPB gut lumen proved to be acidic, with a pH decreasingin larvae from 6.99 in the foregut to 5.37 in the posterior

Fig. 1. Fresh weights of anterior midgut content (AC) and wall (AW) and

posterior midgut content (PC) and wall (PW) in CPB last instar larvae and

adults. Values are means7S.E. (n ¼ 5). Asterisk indicates that larval PC

was significantly (po0.05) heavier than the adult PC.

the range of ppm concentrations of dissolved O2 in the lumen of selected

gut regions in the last instar larvae and feeding adults

Gut region pH (pe+pH) O2 content

Last instar larva

Foregut 6.9970.18 7.6670.01 Not measured

Anterior midgut 6.3070.05 6.9570.03 0.11–0.15

Medial midgut 5.7070.07 6.1170.12 Not measured

Posterior midgut 5.3870.15 5.9370.13 0.08–0.12

Adult

Foregut 6.4570.13 7.2470.02 Not measured

Anterior midgut 6.3170.05 6.3370.12 0.10–0.13

Medial midgut 5.5870.11 6.1270.05 Not measured

Posterior midgut 5.3770.10 5.3670.13 0.08–0.11

midgut (Table 1). A similar decrease from 6.45 to 5.37was found in the adult gut. The redox parameter de-creased along the gut length from mildly oxidizing to

ARTICLE IN PRESS

Fig. 3. The contents of superoxide anion (A) and total peroxides (B) in the

minced potato leaves incubated for 0–15min and 30min, respectively, and

in the anterior midgut content (AC), anterior midgut wall (AW), posterior

midgut content (PC), and posterior midgut wall (PW) of the last instar

larvae and adults. Data are means7S.E. of 10 individuals measured

independently. Values significantly lower in adults than in the larvae are

marked with symbols: Cpo0.001, *po0.01, and **po0.05.

N. Krishnan et al. / Journal of Insect Physiology 53 (2007) 67–74 71

reducing in both larvae (pe+pH ¼ 7.66–5.93) and adults(pe+pH ¼ 7.24–6.33). The anterior section of the gut(foregut+anterior midgut) had more oxidizing conditionsthan the posterior midgut section. Dissolved oxygencontent was similar in the larval and adult gut and variedbetween 0.1 and 0.15 ppm in the anterior and between 0.08and 0.12 ppm in the posterior midgut sections. Due to thesmall size, oxygen content could not be measured in theforegut that often contained air bubbles and the amount ofdissolved oxygen was probably high.

3.3. ROS and antioxidant enzymes in minced potato leaves

Johnson and Felton (2001) working with tobacco plantsexpressing different amounts of chlorogenic acid noted thatantioxidant properties of crushed leaves prevail over theirROS content. This was also true for the potato leaves. Thesuperoxide was hardly detectable in the fresh leaves andremained low even after its 40-fold increase (po0.01)during incubation of the minced leaves (Table 2). Totalperoxide was also low in spite of its 1.6–2.2-fold, (po0.01)elevation in course of the incubation. The low level of ROSwas consistent with relatively high leaf activities of SODand CAT that increased slightly after the incubation. Theactivity rise between 0 and 15–30min was significant in caseof SOD (po0.01) but not CAT.

The ROS and antioxidant enzymes present in the leavesapparently influenced oxidative conditions in the digestivetract. Changes occurring in the minced leaves indicatedthat mechanical leaf chopping during feeding had aconsiderable influence on the oxidative food properties.Considering the rate of food passage through the tract(Fig. 2), we regard values measured in the minced leaves at0 and 15min as a token of the plant contribution tooxidative conditions in the anterior midgut. Valuesestablished at 30min indicate to which extent the oxidativeconditions in posterior midgut were independent of insecttissues.

3.4. ROS in the digestive tract

CPB larvae contained very high superoxide and peroxideamounts in the AC compartment (Fig. 3). Substantially

Table 2

Mean7S.E. (n ¼ 10) values of the superoxide anion and total peroxides

contents (pmolmg�1 fresh weight) and of the SOD and CAT activities

(enzyme units per mg protein) in the minced potato leaves incubated in

buffer

Incubation (min) Superoxide Peroxides SOD CAT

0 0.0370.01a 87715a 0.6170.1a 123.2726.3a

15 0.8770.2b 141724b 0.9570.08b 154.8718.0a

30 1.2170.2b 192746b 0.9570.09b 164.8711.0a

Values marked with different superscripts within a column are signifi-

cantly different from one another at po0.01.

lower ROS levels occurred in the AW and still lower inboth posterior midgut compartments (except for similarperoxide levels in AW and PC). ROS distribution in theadult gut was similar but absolute amounts were lowerthan in the larvae and the concentration in AC was onlyslightly higher than in the other compartments. Similar tothe larvae, Od�

2 was low in both the content and the wall ofposterior midgut whereas peroxides were relatively high inPC. The difference between larvae and adults in the Od�

2

content was highly significant (po0.01) for all compart-ments. The peroxide content was similar in the PW but inall other compartments, it was significantly lower (po0.05)in the larvae than adults.A comparison of ROS levels in the minced leaves and the

gut showed that the food digest might contribute to theOd�

2 level in the midgut lumen. Minced leaves maceratedfor 0–15min contained 0.45 pmolmg�1 and those incu-bated for 30min nearly 1.2 pmolmg�1 Od�

2 (Fig. 3A).Superoxide levels in the AC of larvae and adults reached5.8 and 1.8 pmolmg�1, and in the PC 2.6 and0.2 pmolmg�1 fresh weight, respectively. By contrast, totalperoxide content in the leaves (0.08–0.19 nmolmg�1) wasnegligible compared to 73.5 and 11.6 nmolmg�1 in the AC,and to 23.8 and 5.58 nmolmg�1 in the PC of the larvae andadults, respectively (Fig. 3B).

ARTICLE IN PRESSN. Krishnan et al. / Journal of Insect Physiology 53 (2007) 67–7472

3.5. Antioxidant enzyme activities in the digestive tract

SOD activity in the analyzed gut compartments de-creased in the sequence AC, AW, PC, and PW (Fig. 4A).The activity in adult gut was significantly (l.5–2.5-fold,po0.05) higher than in the corresponding compartments ofthe larval gut, and the activity in the gut content was higherthan in the gut wall in both larvae and adults. Aconsiderable portion of SOD activity in the content couldbe derived from the ingested leaves that harbored 0.61–0.95enzyme unitmg�1 fresh weight. Maximal value measured inthe insects was 1.91 unitmg�1 in the AC of adults.

CAT activity was also higher in adults than in the larvae(Fig. 4B) and the difference was highly significant for bothAW and PW (po0.001 and po0.01, respectively). Incontrast to SOD, most of the CAT activity occurred in thegut wall. The epithelia of both anterior and posteriormidgut regions of adults exhibited nearly twice higher CATactivity than the gut contents. In the larvae, AW containedonly slightly more CAT activity than the AC and nodifference was found between PC and PW. CAT activity inany gut compartment of either larvae or adults was lowerthan in the minced leaves, indicating that a part of CATpresent in the digest could be derived from the ingestedleaves.

Fig. 4. Superoxide dismutase, SOD (A) and catalase, CAT (B) activities in

minced potato leaves incubated for 0–15min and 30min, respectively, and

in the anterior midgut content (AC), anterior midgut wall (AW), posterior

midgut content (PC), and posterior midgut wall (PW) of the last instar

larvae and adults. Data are means7S.E. of 6–8 individuals measured

independently. Values significantly higher in adults than larvae are marked

with symbols: Cpo0.001, *po0.01, and **po0.05.

3.6. Oxidative conditions in the digestive tract of diapausing

adults

The ROS titer and to lesser extent also the activities ofantioxidant enzymes were low in the diapausing adults(Table 3). No inactive aconitase could be detected;suggesting that the Od�

2 level was extremely low. The totalperoxide content ranged in different gut compartmentsfrom 0.19 to 0.75 nmolmg�1 fresh weight, in contrast to thenon-diapausing adults that harbored 2.5–11.6 nmolmg�1

(Fig. 3B). In the diapausing adults, AC had significantly(po0.001) less peroxides than AW; still lower level wasfound in the PC, and a minimal level in the PWcompartment.SOD activity reached in the diapausing adults 0.58 uni-

tmg�1protein in both compartments of the anteriormidgut, was about three times lower in PC and droppedto 0.085 unitmg�1 in the PW. By comparison, the non-diapausing adults possessed 1.9 and 1.06 SODunitmg�1 inthe AC and AW, respectively, and 0.7 unitmg�1 in the PW(Fig. 4A). Diapause was associated with CAT activitybelow 20 unitsmg�1protein in the anterior midgut com-partments (Table 3), in contrast to 68.1 and 109.6 uni-tsmg�1 in the AC and AW, respectively, of the activeadults (Fig. 4B). CAT activity in the posterior midgut wasunaffected by the diapause phenomenon, being 28.4 and57.8 unitsmg�1 protein in the PC and PW of the active, and43 and 35 unitsmg�1 of the diapausing adults, respectively.In the active adults, CAT activity was higher in the midguttissue than in the lumen contents but the ratio was reversedin the diapausing adults.

4. Discussion

ROS are a common component of the plant protectionagainst herbivores and pathogens (Orozco-Cardenas andRyan, 1999; Kuzniak and Urbanek, 2000). One of themechanisms promoting ROS generation in plants is theactivation of polyphenol oxidases following insect attack(Felton et al., 1989). The importance of plant ROS isaccentuated by the observation that the resistance ofcertain potato genotypes to CPB is correlated with a high

Table 3

Means7S.E. (n ¼ 8) of the superoxide anion and total peroxides contents

(pmol per mg fresh weight, n.d. ¼ not detectable) and of the SOD and

CAT activities (enzyme units per mg protein) measured in the anterior

midgut content (AC), anterior midgut wall (AW), posterior midgut

content (PC), and posterior midgut wall (PW) of the diapausing adults

Region Superoxide Peroxide SOD CAT

AC n.d. 0.42670.024a 0.58070.03a 17.872.8a

AW n.d. 0.75370.014b 0.58070.07a 11.273.2b

PC n.d. 0.21670.021c 0.18470.002b 43.177.6c

PW n.d. 0.19570.018c 0.08570.001c 34.775.4c

Values with different superscripts within a column differ from one another

at po0.001 probability level.

ARTICLE IN PRESSN. Krishnan et al. / Journal of Insect Physiology 53 (2007) 67–74 73

level of polyphenol oxidases (Castanera et al., 1996). SinceROS affect plant palatability and interfere with insectdigestion (Bi and Felton, 1995), we expected their rapidelimination in the gut of CPB, with possible contribution ofthe plant-derived ROS scavengers such as the ascorbateand carotenoids and plant antioxidant enzymes such asSOD and CAT. We found, however, that the contents ofsuperoxide and total peroxides were dramatically enhancedin the leaf digest in the anterior midgut section. Mincingand incubation of the leaves, a procedure mimickingmechanical leaf disruption during insect feeding and thedigest retention in the gut, did not elevate ROS content tothe level found in the midgut. This indicates that aparticular environment of the digestive tract is crucial forthe ROS increase in the digest passing through the lumenof anterior midgut.

We did not identify the source of ROS in anteriormidgut. ROS generation as a byproduct of tissue respira-tion is rendered unlikely by the lower ROS titer in theposterior midgut that is likely to have a similar level ofrespiration as the anterior midgut. Also, the size of anteriormidgut is similar in the larvae and adults but ROSconcentration is several times higher in the larvae. Themost plausible explanation of these differences is that aconsiderable portion of ROS is not derived from respira-tion but from a non-respiratory oxidation of allelochem-icals (Ahmad, 1992; Bi and Felton, 1995; Summers andFelton 1994). We suppose that oxygenases linked to thecytochrome P-450 system play a role in the ROSgeneration in CPB but this possibility remains to beexplored. A direct dependence of ROS on the level of plantphenolics was demonstrated in the caterpillars of Spodop-

tera littoralis that responded to 5% tannic acid in their dietby a 70-fold increase of the superoxide content in theforegut (Krishnan and Sehnal, 2006). Phenolic compounds,which represent a major line of the chemical plant defenseagainst herbivores (Harborne, 2001), may contribute to theROS level by undergoing spontaneous oxidation tosemiquinones (Barbehenn et al., 2001, 2005). This processis promoted by the highly alkaline environment in the gutof caterpillars but probably curbed in the mildly acidic andreducing CPB midgut. On the other hand, the reducingmidgut milieu probably limits protein cross-linking bypromoting the non-reactive, protonated state in thealkylable functional groups of amino acids (�NH2, �SH)(Felton et al., 1992).

Our investigations on S. littoralis revealed that plantphenolics cause a burst of ROS in the foregut and elicitantioxidant defense in the midgut (Krishnan and Kodrık,2006; Krishnan and Sehnal, 2006). In CPB with tinyforegut, a ROS gradient was found along the length of themidgut, with a maximum in the lumen of its anterior and aminimum in the wall of its posterior sections. Most of theROS produced in anterior midgut probably move with thefood bolus to the distal region where they are damped bythe increasingly reducing conditions and eliminated by theantioxidant enzymes and other mechanisms. The manage-

ment of ROS in the midgut indicates that CPB is welladapted to the consumption of plants with a high contentof allelochemicals. Adults seem to possess a more efficientcontrol of oxidative burst as indicated by the lowercontents of ROS and higher activities of the antioxidantenzymes.The superoxide was not detectable and the total

peroxides were much reduced in the midgut of thediapausing adults. The low peroxide concentration prob-ably reflected an ebb in the tissue respiration and thevirtual lack of Od�

2 was probably due to the absence offeeding and food processing. The low levels of ROScontrasted with the relatively high activities of SOD andCAT. The enzymes obviously dispose off ROS producedby midgut respiration and are ready to eliminate additionalROS after the resumption of feeding.

Acknowledgments

This work was supported by Grant 522/06/1591 andGrant 522/05/0151 from the Grant Agency of the CzechRepublic and Project Z50070508 of the Institute ofEntomology (Biology Center, Academy of Sciences of theCzech Republic).

References

Ahmad, S., 1992. Biochemical defense of pro-oxidant plant allelochem-

icals by herbivorous insects. Biochemical Systematics and Ecology 20,

269–296.

Aebi, H., 1984. Catalase in vitro. In: Packer, L. (Ed.), Methods in

Enzymology, vol. 105. Academic Press, London, pp. 121–126.

Appel, H.M., 1993. Phenolics in ecological interactions: the importance of

oxidation. Journal of Chemical Ecology 19, 1521–1552.

Aucoin, R.R., Philogene, B.J.R., Arnason, J.T., 1991. Antioxidant

enzymes as biochemical defenses against phototoxin induced oxidative

stress in three species of herbivorous Lepidoptera. Archives of Insect

Biochemistry and Physiology 16, 139–152.

Barbehenn, R.V., Bumgarner, S.L., Roosen, E., Martin, M.M., 2001.

Antioxidant defenses in caterpillars: role of the ascorbate recycling

system in the midgut lumen. Journal of Insect Physiology 47, 349–357.

Barbehenn, R., Cheek, S., Gasperut, A., Lister, E., Maben, R., 2005.

Phenolic compounds in red oak and sugar maple leaves have

prooxidant activities in the midgut fluids of Malacosoma disstria and

Orgyia leucostigma caterpillars. Journal of Chemical Ecology 31,

969–988.

Bi, J.L., Felton, G.W., 1995. Foliar oxidative stress and insect herbivory:

primary compounds, secondary metabolites and reactive oxygen

species as components of induced resistance. Journal of Chemical

Ecology 21, 1511–1530.

Bradford, M.M., 1976. A rapid and sensitive method for the quantitation

of microgram quantities of protein utilizing the principle of protein-

dye binding. Analytical Biochemistry 72, 248–254.

Castanera, P., Steffens, J.C., Tingey, W.M., 1996. Biological performance

of Colorado potato beetle larvae on potato genotypes with differing

levels of polyphenol oxidase. Journal of Chemical Ecology 22, 91–101.

Claravon-Mathews, M., Summers, C.B., Felton, G.W., 1997. Ascorbate

peroxidase: a novel antioxidant enzyme in insects. Archives of Insect

Biochemistry and Physiology 34, 57–68.

Deiana, L., Carru, C., PWs, G., 1999. Spectrophotometric measurement

of hydroperoxides at increased sensitivity by oxidation of Fe2+ in the

presence of xylenol orange. Free Radical Research 31, 237–244.

ARTICLE IN PRESSN. Krishnan et al. / Journal of Insect Physiology 53 (2007) 67–7474

Doke, N., Miura, Y., Sanchez, L.M., Park, H.-J., Noritake, T., Yoshioka,

H., Kawakita, K., 1996. The oxidative burst protects plants against

pathogen attack: mechanism and role as an emergency signal for plant

bio-defence. Gene 179, 45–51.

Duke, J.A., Atchley, A.A., 1986. Handbook of proximate analysis tables

of higher plants. CRC Press, Boca Raton, FL, pp. 389.

Felton, G.W., Donato, K., Del Vecchio, R.J., Duffey, S.S., 1989.

Activation of plant foliar oxidases by insect feeding reduces nutritive

quality of foliage for noctuid herbivores. Journal of Chemical Ecology

12, 2667–2694.

Felton, G.W., Summers, C.B., 1995. Antioxidant systems in insects.

Archives of Insect Biochemistry and Physiology 29, 187–197.

Felton, G.W., Workman, J., Duffey, S.S., 1992. Avoidance of antinu-

tritive plant defense: role of midgut pH in Colorado potato beetle.

Journal of Chemical Ecology 18, 571–583.

Friedman, M., 2004. Analysis of biologically active compounds in

potatoes (Solanum tuberosum), tomatoes (Lycopersicon esculentum)

and jimson weed (Datura stramonium) seeds. Journal of Chromato-

graphy A 1054, 143–155.

Fridovich, I., 1978. The biology of oxygen radicals. Science 201, 875–879.

Harborne, J.B., 1997. Biochemical plant ecology. In: Dey, P.M.,

Harborne, J.B. (Eds.), Plant Biochemistry. Academic Press, London,

pp. 503–515.

Harborne, J.B., 2001. Twenty-five years of chemical ecology. Natural

Product Reports 18, 361–379.

Hausladen, A., Fridovich, I., 1996. Measuring nitric oxide and superoxide:

rate constants of aconitase reactivity. In: Packer, L. (Ed.), Methods in

Enzymology, 269. Academic Press, San Diego CA, pp. 37–41.

Hermes-Lima, M., Willmore, W.G., Storey, K.B., 1995. Quantification of

lipid peroxidation in tissue extracts based on Fe(III)–xylenol orange

complex formation. Free Radicals in Biology and Medicine 19,

271–280.

Johnson, K.S., Felton, G.W., 2001. Plant phenolics as dietary antioxidants

for herbivorous insects: A test with genetically modified tobacco.

Journal of Chemical Ecology 27, 2579–2597.

Kennedy, M.C., Emptage, M.H., Dreyer, J.L., Beinert, H., 1983. The role

of iron in the activation-inactivation of aconitase. Journal of

Biological Chemistry 258, 11098–11105.

Krishnan, N., Kodrık, D., 2006. Antioxidant enzymes in Spodoptera

littoralis (Boisduval): are they enhanced to protect gut tissues during

oxidative stress? Journal of Insect Physiology 52, 11–20.

Krishnan, N., Sehnal, F., 2006. Compartmentalization of oxidative stress

and antioxidant defense in the larval gut of Spodoptera littoralis.

Archives of Insect Physiology and Biochemistry 63, 1–10.

Kuzniak, E., Urbanek, H., 2000. The involvement of hydrogen peroxide in

plant responses to stress. Acta Physiologiae Plantarum 22, 195–203.

Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the

autoxidation of epinephrine and a simple assay for superoxide

dismutase. Journal of Biological Chemistry 247, 3170–3175.

Munkres, K.D., 1990. Purification of exocellular superoxide dismutases.

In: Abelson, J.N., Simon, M.I. (Eds.), Methods in Enzymology, 186.

Academic Press, New York, pp. 249–259.

Orozco-Cardenas, M., Ryan, C.A., 1999. Hydrogen peroxide is generated

systemically in plant leaves by wounding and systemin via the

octadecanoic pathway. Proceedings of National Academy of Science

USA 96, 6553–6557.

Rose, I.A., O’Connell, E.L., 1967. Mechanism of Aconitase action I: the

hydrogen transfer reaction. Journal of Biological Chemistry 242,

1870–1879.

Summers, C.B., Felton, G.W., 1994. Prooxidant effects of phenolic acids

on the generalist herbivore Helicoverpa zea (Lepidoptera: Noctuidae):

potential mode of action for phenolic compounds in plant anti-

herbivore chemistry. Insect Biochemistry and Molecular Biology 24,

943–953.