LOCATION AND VARIABILITY OF CATALASE ACTIVITY WITHIN APHIDS

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

ABSTRACT

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).

INTRODUCTION

Catalase (hydrogen peroxide: hydrogen peroxide oxidoreductase, EC 1.11.1.6, 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.

MATERIAL AND METHODS

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.

RESULTS

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.

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).

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).

DISCUSSION

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.

CONCLUSIONS

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.

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Accepted for print: 19.12.2007

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