Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 10
Issue 4
Available Online: http://www.ejpau.media.pl/volume10/issue4/art-38.html


Anna Urbańska
Department of Biochemistry and Molecular Biology, University of Natural Sciences and Humanities in Siedlce, Poland



This paper concerns with the catalase (CAT) activity in the grain aphid Sitobion avenae (F.), in particular its location/distribution within the body tissues and variability in consequence of the chemical compounds of the aphid diets. There is specified that the CAT activity is ‘focused’ within the midgut, whereas the salivary secretions lack of the CAT. Dietary o-dihydroxyphenolics i.e. caffeic acid, (+) – catechin and dihydroxyphenylalanine, similarly to hydrogen peroxide (H2O2) show inductive effects on the CAT activity. Moreover, ingestion of the chemicals present within wheat foliages also has significant positive effect on the CAT activity of S.avenae. Role of the CAT activity for antioxidant response of S.avenae against the o-diphenolics is argued.

Key words: catalase (CAT), CAT activity, grain aphid, Sitobion avenae, o-dihydroxyphenolic compound, hydrogen peroxide H2O2).


Catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase, EC, CAT) is the classical enzyme of the aerobic organisms which catalyses the decomposition of hydrogen peroxide (H2O2) into water (H2O) and oxygen (O2), according to the following reaction: 2H2O2 → 2H2O + O2. This activity of CAT is defined by biochemists as ‘catalase’ activity or ‘catalitic’ reaction. CAT also acts as a peroxidase, but its ‘peroxidatic’ activity also called ‘peroxidatic’ reaction is relatively slow and noticeable at low H2O2 concentration. The reaction for which CAT is commonly know since 1900, is its ‘catalase’ activity/ ‘catalitic’ reaction; in short, the CAT activity. It proceeds very rapidly, as compared to the ‘peroxidatic’ activity and predominates at higher H2O2 concentration [1,13,18].

The biochemical function/ ‘purpose’ of the CAT activity is ‘scavening’ of toxic H2O2, representing the reactive oxygen species (ROS), having adverse effect towards almost all cell compounds including membrane lipids, DNA and proteins. Participation of the CAT activity in antioxidant defence has been shown for spectrum of organisms, from bacteria to mammals [6,12,13,25,27]. A lot of knowledge is available on CAT activity within the phytophagous insects, mostly amongst Lepidoptera, especially on its protective action against H2O2 realized from the prooxidant plant chemicals [3,4,7,14,15,16,22,23,24,30]. Whereas, the CAT of Aphidoidea seems to be the enzyme with ‘mysteries’ still, both in regard to its occurrence/localization and role for antioxidant response against ROS generated during oxidative metabolism of plant metabolites. Positive results of CAT activity were shown for the whole-body homogenates of Rhopalosiphum padi (L.) and Sitobion avenae (F.) [17,19] On the other hand, no activity was found in the salivary secretions of Therioaphis trifolii (Buckton) and Acyrthosiphon pisum (Harris) [20]. Moreover, there was argued that CAT activity of S.avenae was modified by the cereal chemicals i.e. hydroxamic acids and phenolics [8,19].

This paper aims to investigate three aspects of the CAT within the grain aphid, Sitobion avenae (F.), ‘useful’/essential to ‘assess’ the role of this enzyme for antioxidant defence towards the plant allelochemicals, namely [1] the occurrence within the salivary secretions and midgut; [2] the activity variability as a consequence of the o-dihydroxyphenolics and H2O2 incorporated into the diets; [3] the effect of the feeding within plant tissues on the activity level.


Chemicals. Agarose, 3-amino-1,2,4-triazole, ferric chloride (FeCl3) horseradish peroxidase, hydrogen peroxide (H2O2), potassium cyanide (KCN), potassium ferricyanide (K3Fe(CN)3), sucrose, tetramethylbenzidine (TMBZ) and the phenolics: caffeic acid, (+)-catechin, L-dihydroxyphenylalanine (L-DOPA) were purchased from Sigma Chemical Co., (St. Louis, Mo, USA).

Aphids. The adults of S.avenae used in the experiments were collected from the stock culture reared on seedlings of Polish winter wheat cv Sakva, in insectary chamber at L 18: D 8 photoperiod and 22°C.

Assay of CAT in situ and its localization. Ten aphids brushed into ‘feeding chamber’ done from plastic ring and placed in an environmental cabinet, were allowed to probe for 15 hrs the agarose (1.25% w/v) – sucrose (30% w/v) gels. Aphids readily accepted the gels as diets and fed through parafilm M®. After feeding, visualization of CAT in the gels was accomplished via the methods I and II detailed in Gregory & Fridovich [9] and Sun et al. [26], respectively.

Method I: Here the gels were soaked for 1h in a mixture of 50 mM Na-phosphate buffer (pH 7.0), 1.25 mL TMBZ (0.4%) and 500 μL horseradish peroxidase (0.1%), and next washed with distilled water (d H2O) and immersed in 20 mM H2O2 for 30 min. Achromatic/bleached zones vs a brown background indicate the CAT presence.

Method II: Here, the gels were incubated in 0.003% H2O2 for 10 min, then rinsed with d H2O and stained in a aqual volume of 1% K3Fe(CN)3 and 1% FeCl3 for 10 min. Finally, the gels were washed with d H2O. Appearance of achromatic/ bleaching or yellow colouration vs blue-green background indicates the CAT activity location.

Ten replications were done for each procedure.

Moreover, for detection of CAT activity groups of ten midguts were dissected from adults actively feeding on wheat seedlings. Then the midguts washed in dH2O and immediately followed the methods for CAT visualization [9,26].

For control, gels/midguts were treated with 10mM 3-amino-1,2,4-triazole or 2 mM KCN.

Stained gels/midguts were then examined under a light microscope for CAT activity location.

Dilute saliva collection for CAT test. Hundred aphids were allowed for 15 hrs to probe d H2O enclosed between two parafilm M® membranes streached across Mittler and Dadd dishes [20,21]. For CAT, 500μL of ‘probed-water’ added to 500 μL of 20 mM H2O2 in Na-phosphate buffer, pH 7.0, and analysis was by change in A240 over 3 min [2].

Aphid feeding experiment on liquid diets. Used 0.1, 1.0 and 10.0 mM caffeic acid, 1.0 mM (+) – catechin; 1.0 mM L-DOPA, 2.0, 20.0 and 200.0 mM H2O2, all in 15% w/v aqueous sucrose solution. 500 μl aliquot of each diet enclosed between two parafilm M® membranes stretched across Mittler and Dadd dish [21], placed in a environmental cabinet and a hundred adults were allowed for 15 hrs to feed the diet. Ten replications were done for each amongst the studied diets.

For control, the aphids were allowed to feed 15 % w/v aqueous sucrose.

CAT activity assay. Sources of the enzyme were 1000 g X 15 min supernatants of crude homogenates of 30 actively feeding adults, in ice-cold 50 mM sodium phosphate buffer, pH 7.0. CAT activity was analysed spectrophotometrically following Aebi [2]. To 500μl extracts added 500 μl of the substrate, (20 mM H2O2) in 50 mM Na-phosphate buffer, pH 7.0 and immediately disappearance of H2O2 was measured, at 240 nm, for 3 min at 30s intervals. For control, buffer replaced the H2O2. The CAT activity expressed as IU mg protein. One IU (International Unit) is the enzyme activity that transforms/decomposes 1 mmole of H2O2 in 1 minute. Protein content was determined by the method of Bradford [5] with bovine serum albumin as a standard.

Statistics. The activity of CAT was subjected to an analysis of variance followed by Duncan’s test.


CAT activity localization. Not distinct staining was noted for the stylet sheaths visible against the background after incubation in the CAT medium (Fig. 1A,B1,B2). Moreover, no decrease of absorbance at 240 nm, was registered for the CAT reaction mixture containing the substrate, H2O2 and the dilute saliva (‘fed-water’). In this way, results for the CAT activity were negative with the salivary secretions.

On the contrary, the midguts appeared prominently stained for the CAT reaction
(Fig. 1C1,C2). In short, these figures illustrate the CAT activity in midgut of S.avenae.

Fig. 1. Light micrographs showing results of the CAT staining within the salivary secretions and midgut of S. avenae, by the classical histochemical techniques [9,26].
A – the salivary sheats with negative CAT reaction after incubation in the medium detailed in Sun et al. [26]; CAT is not visualised.
B1 – the salivary sheat incubated for CAT reaction by the Gregory & Fridovich original method [9]; B2 – the control, the salivary sheat after incubation in the CAT medium [9] in the presence of 10.0 mM 3-amino-1,2,4-triazole; the reaction is negative.
C1 – these figures show the midgut incubated in the H2O2 and postfixed by the ferricyanide, according to procedure detailed in Sun et al. [26]; the CAT reaction is exclusively positive; C2: the control, the midgut after incubation in the CAT medium [26] and 2 mM KCN, the reaction is not positive.

Effect of o-dihydroxyphenolics on CAT activity. The results obtained for the CAT activities of the aphid adults feeding on the artificial diets range from approx. 15.0 to 89.0 (Fig.2). The CAT activity of S.avenae fed caffeic acid, (+)-catechin and L-DOPA was significantly higher compared with the control (P<0.05) and was induced 3.5-fold by (+) catechin and 4.2 – fold by L-DOPA, at concentration of 1.0 mM. When the aphids were fed on diets containing caffeic acid at the concentrations of 0.1, 1.0 and 10.0 mM the CAT activity increased 3.2 – fold, 4.5 – fold and 6.0-fold, respectively.

How hydrogen peroxide induces CAT activity. When S.avenae was fed on the 15% sucrose diets containing 2.0, 20.0 and 200.0 mM H2O2 then the CAT activity values were of the increase, and were determined as 51.33, 63.21 and 70.61, respectively. Fig. 2 shows that the CAT activity of S.avenae was significantly induced for all treatments with H2O2 relative to the control treatment (P<0.05).

Fig. 2. CAT activity variation in S. avenae fed on diet with o-phenolics and H2O2

Values not followed by the some letter are significantly different at the 0.05% level (Duncan’s test)

How foliage feeding affects CAT activity. The CAT activity of S.avenae was significantly (2.0-fold) increased by the ingestion of the phloem sap of the wheat foliage as compared to the control (P<0.05). When adults of S.avenae taken from the wheat foliage and starved for 15 hrs then their CAT activity was found to be slightly lower (1.4 – fold) than in the aphids actively feeding on the seedlings, but still significantly higher as compared with the 15% sucrose treatment (Fig.3).

Fig. 3. Values of CAT activity in S. avenae when fed on the ‘natural’ diets as compared with the sucrose/ ‘artificial’ diet. Mean values with different letters are significantly different (p ≤ 0.05).


Results for the CAT activity were negative with the salivary secretions and positive with the midgut of S.avenae. CAT activity of phytophagous insect tissues varies greatly, from rich in CAT to CAT free e.g. for fifth-instar larvae of cabbage looper, Trichoplusia ni no activity was found in the salivary glands and relatively high for the midgut [3,4]. Madhusudhan & Miles [20] did not find CAT in the saliva from the pea aphid and spotted alfalfa aphid. However, CAT was found in salivary glands of the Australian froghopper Eoscarta carnifex [24] and its high levels was detected in the midgut tissues of lepidopteran pest i.e. Heliothis zea, H. virescens, Manduca sexta and Spodoptera exigua [3,4,7,14,15,16,22,23,24,30].

The CAT activity values of S.avenae ranged from 14.86 to 88.89 U mg protein and were similar to those accessed for lepidopterans i.e. H.virescens, M.sexta, S.eridania, S.exigua but distinctly lower in comparison with Paplio polyxenes (231.0 U mg protein), T.ni (361.0 U mg protein) and Depressaria pastinacella (637.8 U mg protein) [8,17].

I present evidence, that the o-dihydroxyphenolics e.g. caffeic acid, (+) – catechin, similarly to dietary H2O2 induce the CAT activity of S.avenae. Moreover, the ingestion of the wheat foliage sap also has significant positive effect on the aphid’s CAT activity. These data seem to be essential for specifying of role of CAT for the aphid – diphenolic interactions. As a matter of fact, the aphid’s CAT via removing of H2O2 ‘appears’ as defensive enzyme against H2O2 generated by ingestion of the plant allelochemicals e.g. o- dihydroxyphenolics. According to research Figuera et al. [8] CAT activity of S.avenae was increased 2-fold when aphids fed with 2 mM DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one). On the other hand, Lukasik [19] observed an opposite tendency; namely, the CAT activity was reduced in S.avenae fed on diet containing caffeic acid.

Phenolic compounds, in particular o-dixydroxyphenolics appear to be bioactivated through oxidative metabolism catalysed by various enzymes within phytophagous insect tissues and the result of this activation is the production of the ROS e.g. H2O2, a substance toxic to living cells [6,25,27,10,11]. I my opinion just in that bioactivation may participate the polyphenol oxidase (PPO), the enzyme which oxidizes spectrum of the o-dihydroxyphenolics both within the salivary secretions and midgut of S.avenae [28,29].

There was suggested important role of the CAT in antioxidant protection from oxidative stress (ROS, H2O2) consequent of dietary pro-oxidants i.e. flavonoids and quinones, for the lepidopterans, e.g. S.eridania and P.polyxenes [10,11,22,23]. However, CAT of P.polyxenes was inhibited by increased consumption of quercetin – common flawonoid in plant tissues, which generates H2O2 [23]. Also CAT activity of T.ni was decreased slightly in response to increasing concentration of dietary quercetin [3]. In contrast with quercetin the prooxidant furanocoumarin – harmine induced the CAT activity of T.ni; although the furanocoumarins did not increased level of the CAT of D.pastinacella [16].

In the case of phytophagous insects, exogenous oxidative stress/generation of ROS e.g. H2O2 is exerted mostly in the midgut – the tissue which is the ‘locus’ of the enzymatic biotransformation/detoxification of the plant pro-oxidant allelochemicals, specifically the oxidative reactions/conversions of the o-dihydroxyphenolics [3,4,7].

The generation of H2O2 in the midgut of Aphididae may result from a variety of enzymes, and in above-mentioned particular PPO [28,29]. This enzyme oxidizes o-diphenolics via quinones till biological inactive melanins and a by-product of this conversion may by superoxide radical, O2. Next, H2O2 may be produced from O2 as a product of reaction: 2O2 + 2H+ → O2 + H2O2, catalysed by superoxide dismutase [27]. The CAT activity located in the midgut of S.avenae is likely to counteract the exogenous oxidative stress ‘amplified’ by the dietary o-diphenolics. Felton & Duffey proposed similar action for the midgut CAT of various Lepidopteran against oxidative defence of their host-plants [7].

The CAT via removal of H2O2, co-substrate of the enzyme – peroxidase (POD) may inhibit its activity. POD is the enzyme which oxidizes o-diphenolics to quninones. This aspect seems to be remarkable here because the POD activity was found in the midgut of S.avenae.

The o-dihydroxyphenolics e.g. caffeic acid and (+) – catechin, implicated in the plant resistance/defence against S.avenae, provide a source of H2O2 [29]. Relatively high values of the aphid’s CAT activity both constitutive and inductive, in response to the dietary o-diphenolics, H2O2 and also wheat foliage sap may offer an efficient elimination of H2O2 and thus ‘suppressing’ the plant o-diphenolic defence/prooxidant response.


Accordingly to the obtained results the CAT of S.avenae appears to be real antioxidant enzyme against H2O2/oxidative stress generated/derived within the midgut tissues during the red-ox metabolism of the plant allelochemicals e.g. caffeic acid and related o-diphenolics and polyphenolics.


  1. Aebi H., 1974. Catalase: Methods of Enzyme Analysis Vol. II (ed. By H.U. Bergmeyer) Verlag Chemie, Weinheim, 673-684.

  2. Aebi H., 1984. Catalase in vitro. Meth. Enzymol. 105, 121-126.

  3. Ahmad S., Duval D.L., Weinhold L.C., Pardini R.S., 1991. Cabbage looper antioxidant enzymes: tissue specificity. Insect Biochem. 21, 563-572.

  4. Ahmad S., Pardini R.S., 1990. Antioxidant defense of the cabbage looper, Trichoplusia ni: Enzymatic responses to the superoxide-generating flavonoid, quercetin, and photodynamic furanocoumarin, xanthotoxin. Phytochemistry and Photobiology 51, 305-311.

  5. Bradford M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248-254.

  6. Cadens E., 1989, Biochemistry of oxygen toxicity. Ann. Rev. Biochem. 58, 79-110.

  7. Felton G.W., Duffey S.S., 1991. Protective action of midgut catalase in lepidopteran larvae against oxidative plant defenses. J. Chem. Ecol. 17, 1715-1732.

  8. Figueroa C.C., Koenig C., Araya C., Santos M.J., Niemeyer H.M., 1999. Effect of Dimboa, A hydroxamic acid from cereals, on peroxisomal and mitochondrial enzymes from aphids: evidence for the presence of peroxisomes in aphids. J.Chem.Ecol. 25, 2465-2475.

  9. Gregory E.M., Fridovich I., 1974. Vizualization of catalase on acrylamide gels. Anal. Biochem. 58, 57-62.

  10. Hassan H.M., Fridovich I., 1979. Interacellular production of superoxide radical and hydrogen peroxide by redox active compounds. Archs. Biochem. Biophys. 196, 385-395.

  11. Hodnik W.F., Kung F.S., Roetter W.J., Bohmont C.W., Pardini R. S., 1986. Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids: a structure – activity study. Biochem. Pharmac. 35, 2345-2357.

  12. Isik K., Kayali H.A., Sahin N., Gündogdu E.Ő., Tarhan L., 2007. Antioxidant response of a novel Streptomyces sp. M3004 isolated from legume rhizosphere to H2O2and paraguat. Process Biochemistry 42, 235-243.

  13. Kirkman H.N., Gaetani G.F., 2007. Mammalian catalase: a venerable enzyme with new mysteries. Trends Biochem. Sci. 32, 44-50.

  14. Lee K., Berenbaum M.R., 1989. Action of antioxidant enzymes and cytochrome P-450 Monooxygenases in the cabbage looper in response to plant phototoxins. Arch. Insect Biochem. Physiol. 10, 151-162.

  15. Lee K., Berenbaum M.R., 1990. Defense of parsnip webworm against phototoxic furanocoumarins: role of antioxidant enzymes. J. Chem. Ecol. 16, 2451-2460.

  16. LEE K., Berenbaum M.R., 1993. Food utilization and antioxidant enzyme activities of black swallowtail in response to plant phototoxins. Arch. Insect Biochem. Physiol. 23, 79-89.

  17. Loayza – Muro R., Figueroa C.C., Niemeyer H.M., 2000. Effect of two wheat cultivars differing in hydroxamic acid concentration on detoxification metabolism in the aphid Sitobion avenae. J.Chem. Ecol. 26, 1725-2736.

  18. Loew O., 1900. A new enzyme of general occurrence in organisms. A preliminary note. Science 11, 701-702.

  19. Lukasik I., 2007. Changes in activity of superoxide dismutase and catalase within cereal aphids in response to plant o-dihydroxyphenols. J.Appl. Entomol. 131, 209-214.

  20. Madhusudhan V.V., Miles P., 1998. Mobility of salivary components as a possible reason for differences in the responses of alfalfa to the spotted alfalfa aphid and pea aphid. Ent Exp. Appl. 86, 25-39.

  21. Mittler T.E., Dadd R.H., 1963. Studies on artificial feeding of the aphid Myzus persicae (Sulzer) – I. Relative uptake of water ad sucrose solutions. J. Ins. Physiol. 9, 623-645.

  22. Pritsos C.A., Ahmad S., Bowen S.M., Elliot A.J., Blomquist G.J., Paradini R.S., 1988. Antioxidant enzymes of the black swallowtail butterfly, Papilio polyxenes and their response to the prooxidant allelochemical quercetin. Arch. Insect Biochem. Physiol. 8, 101-112.

  23. Pritsos C.A., Ahmad S., Bowen S.M., Blomquist G.J., Pardini R.S., 1988. Antioxidant enzyme activities in the southern armyworm, Spodoptera eridania. Comp. Biochem. Physiol. 90 C, 423-427.

  24. Rodman D.H., Miller D.J., 1992. Enzyme activities associated with salivary glands of the froghopper Eoscarta carnifex (F.) (Homoptera: Carcopoidae): Possible role of salivary catalase in phytotoxicity. Aust. J. Zool. 40, 365-370.

  25. Sies H., de Groot H., 1992. Role of reactive oxygen species in cell toxicity. Toxicol. Lett., 64/65, 547-551.

  26. Sun Y., Elwell J.H., Oberley L.W., 1988. A simultaneous visualization of the antioxidant enzymes glutathione peroxidase and catalase on polyacrylamide gels. Free Radic. Res. Commun. 5, 67-75.

  27. Timbrell J., 2000, Principles of Biochemical Toxicology. Taylor & Francis Ltd, London, UK.

  28. Urbanska A., 2007, Some biochemical aspects of plant-aphid interactions. 32nd FEBS Congress, Abstr.7-12 July, Vienna (Austria), 194.

  29. Urbanska A., Leszczynski B., 2007, Oxidation of phenolics within aphids-toxication versus detoxication ? 13th Symposium on Insect-Plant Relationships, Abstr. July 29-August 2, Uppsala (Sweden), 166.

  30. Wang Y., Oberley L.W., Murhammer D.W., 2001, Antioxidant defense systems of two lepidopteran insect cell lines. Free Radical Biology & Medicine 30, 1254-1262.


Accepted for print: 19.12.2007

Anna Urbańska
Department of Biochemistry and Molecular Biology,
University of Natural Sciences and Humanities in Siedlce, Poland
B. Prusa 12, 08-110 Siedlce, Poland
phone: +48 25 643 12 22,
fax: +48 25 644 59 59
email: annau@uph.edu.pl

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