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 12
Issue 4
Available Online: http://www.ejpau.media.pl/volume12/issue4/art-27.html


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



This paper concerns with the production of the hydrogen peroxide (H2O2), representative of the reactive oxygen species (ROS), in aphids Rhopalosiphum padi L. and Sitobion avenae F. Three aspects of the subject have been investigated: (I) 'centre' of H2O2 occurrence; (II) variability of H2O2 production in aphids, in response to dietary chemicals; (III) 'molecular source' of H2O2, amongst free radiacals and oxidases.  It is known that the H2O2 production focuses in the midgut and in the salivary secretions. Compounds among o-dihydroxyphenolics e.g. caffeic acid, (+) – catechin, L-dihydoxyphenylalanine, increase rate of H2O2  production. Whereas, monophenolics e.g. p-coumaric acid, p-hydroxybenzolic acid, ferulic acid are rather non-prooxidant. Presence of superoxide anion (O2·–) in the midgut – major 'compartment' of H2O2 generation – evidences that O2·– participates in 'chemical route' of H2O2 generation. Polyphenol oxidase (EC, primary biocatalyst of Aphididae for the oxidative metabolism of the phenolic allelochemicals, seems to provide O2·–, precursor of H2O2. The aphid H2O2 generated in the midgut and additionally also in the saliva appears as toxic by-product of the oxidation of the o-dihydroxyphenolic allelochemicals. Visualization of H2O2 is accomplished via the ferricyanide-ferrichloride staining method. 'Amount' of H2O2 is determined via UV-spectrophotometry, A240. O2·–  is examined with the conventional Nitro blue tetrazolium (NBT) microscopic assay. The results indicate that each amongst used assay is simple, sensitive and specific for determined molecule and that all they can be used to visualize/measure of H2O2 and O2·– in animal and plant cells and tissues. I recommend these methods for  education in biochemistry.

Key words: hydrogen peroxide, H2O2, superoxide anion, O2·– aphid, Rhopalosiphum padi, Sitobion avenae, phenolic compound, polyphenol oxidase, aphid – plant interaction.


The major part of molecular oxygen (O2) consumed by aerobic cell in consequence of four electron reduction (O2 + 4ē + 4H+ → 2H2O) is converted into water. This reaction is catalysed by the cytochrome c oxidase – terminal biocatalyst of the mitochondrial respiratory chain. A much smaller part of O2 is converted into hydrogen peroxide (H2O2) via several' metabolic pathways'. Basic level of H2O2 is generated by the electron transport processes i.e. photosynthesis and respiration. Moreover, H2O2 is by-product of the biocatalysis of a variety of oxidases i.e. amino acid oxidase, amine oxidase, glucose oxidase, urate oxidase, xanthine oxidase, NAD(P)H oxidase etc. [3,4,8,9,20,35].

H2O2 is synthesized from O2 directly, O2 + 2ē + 2H+ → H2O2. This way of H2O2  formation is connected with the flavin adenine dinucleotide (FAD) oxidases (e.g. amino acid oxidase)  and reduced FAD oxidases are oxidized by O2 and yield H2O2. Moreover, H2O2 is produced via initial step of one electron reduction of O2 into the superoxide anion, (O2·–), followed by dismutation/protonation to H2O2, 2O2·– + 2H+ → H2O2 + O2. The biocatalyst, superoxide dismutase (SOD; EC participates in dismutation of O2·– to H2O2, although some O2·– spontaneously becomes H2O2 [3,9,22].

It is assessed that 1–2% of O2 utilized by cell is converted to O2·– [35]. Production of O2·– is a 'mistake' in cell biochemistry, and an 'introduction' to cytotoxic oxidative stress. Both the mitochondrial chain and numerous oxidases e.g. xathine oxidase and NAD(P)H oxidase provide O2·–. In  absence of oxidative stress the concentration of H2O2 in vivo is as low as 5 x 10–11 M [13]. H2O2 present in such low concentration is normal metabolite of cell biochemistry. H2O2 is destructive towards fundamental bio-chemicals at relatively high concentration. For example, at concentrations higher than 10–5 M, H2O2 has adverse effect on chemicals of cell membrane [13,35]. H2O2 is not free radical per se and itself is not particularly reactive yet, it has oxidizing effect that contributes to oxidative stress by 'disturbance' of oxidant balance in cell. H2O2 diffuses through cell membranes easly and interacts with a variety of bio-molecules. Namely, H2O2 impairs biological functions of proteins, enzymes and glutathione (GSH), due to oxidation of their thiol (-SH) groups and decarboxylation of amino acids [3,4,8,9,20,35].

H2O2 is precursor of the hydroxyl radical (OH), the most strong oxidant known, and extremely destructive. In vivo, in the presence of Fe and Cu ions H2O2 is reduced to OH via the reactions: (I) O2·– + H2O2 + H+OH + O2 + H2O; (II) Fe+2/Cu+ + H2O2 + H+ → Fe+3/Cu+2 + OH + H2O, well known to biochemists as Fenton – type and Haber-Weiss reactions [3,9,22]. H2O2, O2·– and OH are the reactive oxygen species (ROS), constantly formed and removed from cells. Removal of H2O2 is catalyzed by catalase (CAT, EC, GSH peroxidase (GPOD, EC, ascorbate peroxidase (APOD, EC and peroxidase (POD, EC CAT converts H2O2 to O2 and H2O. GPOD and APOD release H2O. Whereas POD in conjunction with H2O2 oxidise phenols to toxic quinones. CAT removes H2O2 when present in high concentration, whereas GPOD removes rather low concentration of H2O2 [3,4,11,13,25,35].

H2O2 is representative of ROS well known to have wide range effects in biological systems. H2O2 is assigned role in plant protecting  from infection, and in particular the role of direct anti-fungal molecule [12]. Gracia-Olmedo et al. [7] reported on antibiotic activity of H2O2 in plant defence to relevant pathogens, and in vitro studies showed strong anti-microbial activity of H2O2. In plants, H2O2 is studied in details as signaling molecule and it is proved that exposure of plants to various abiotic and biotic stresses e.g. pathogens, induce 'burst' of H2O2 [5]. A lot of research is done on H2O2 in the context of the free radical theory of aging [14,33]. The majority of research suggests that H2O2 provokes aging and death  and aging cells generate and accumulate H2O2 [34]. Concentration of H2O2 and other ROS are known to increase also during plant senescence [25]. Increased H2O2/ROS level may be a result of disease pathology. Extremely, when ROS exceed 'antioxidant defenses' they can destroy and damage cell biochemistry and subcellular structures till cell death by apoptosis or necrosis [34,35].

The respiratory chain generates more than 90% of intracellular H2O2/ROS, and 1-5% of O2 consumed by mitochondria is converted to ROS [33,35].

A variety of xenobiotics enhance generation of H2O2. Classical pro-oxidant phyto-chemicals include furanocoumarins (e.g. xanthotoxin, bergapten, benzofurans, chromenes), flavonoids (quercetin), and quinones. Some of them e.g. flavonoids are involved in plant defence against herbivores [2,6,15,16,23,24]. There is controversy concerning phenolics as regards their pro-oxidant and anti-oxidant capacity. Namely, quercetin is considered as antioxidant compound for mammals and as pro-oxidant for phytophagous insects [6,14,15,22,23,24].

Reference on H2O2/ROS production in phytophagous insects i.e. Aphididae is not numerous [17,18,29]. Madhusudhan and Miles [17] suggested that H2O2 is produced when phenolics were oxidized by saliva of Acyrthosiphum pisum (Harris) and Therioaphis trifolii maculate (Buckton). As yet, there is no progress is this subject, although it is important for recognition of molecular toxicity of the phenolic allelochemicals towards aphids.

This paper provides some data on identification and production of H2O2 in Rhopalosiphum padi L. and Sitobion avenae F., in particular: (1) occurrence of H2O2 synthesis in digestive system and salivary secretions; (2) 'induction' of H2O2 production by a variety of phenolics present in diets; (3) O2·– as a 'chemical precursor' of H2O2. Moreover, the polyphenol oxidase, PPO (EC is considered as a 'enzymatic source' of H2O2.


Aphids. The adults of R. padi and 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 L18: D8 photoperiod at 22°C.

Reagents. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

A qualitative assay of H2O2. The salivary secretions and midguts were used for this assay. Ten aphids set in plastic ring were allowed to puncture through stretched Parafilm M® membrane the agarose (1.25% w/v) – sucrose (30% w/v), (a-s g) incorporating one amongst chemicals i.e. L-dihydroxyphenylalanine (L-DOPA), (+)-catechin and caffeic acid, all at concentration 1.0mM. For H2O2 visualization the gels were immersed in freshly prepared mixture of a equal volume of 0.1% potassium ferricyanide (KFe(CN)6) and 0.1% ferric chloride (FeCl3), for 10 min. The blue-green staining (Prussian blue) demonstrated H2O2 occurrence [26]. Ten replications were done for each version of these investigations. Ten digestive systems were dissected from actively feeding adults. Then, they were washed in distilled H2O (dH2O) and immediately followed the procedure of H2O2 visualization by soaking in a freshly prepared the K Fe(CN)3-FeCl3 reagent, for 10 min. Finally, the midguts washed again with dH2O and blue-green colouration indicating the H2O2 production was examined under a light microscope.

Control for H2O2 detection. For control experiments, the PPO inhibitor, 0.005 phenylthiourea (PTU) was added into the a-s gels.

A qualitative O2·– assay. The conventional microscopic nitroblue tetrazolium (NBT) assay was used to determine production of  O2·– in aphid digestive system [10]. Yellow – coloured nitroblue tetrazolium, Y-NBT (NBT +2) was reduced  to water-insoluble deep blue farmazon particles/deposits (NBT+) by O2·–. Ten freshly dissected midguts were treated with 100 µl aqueous solution of Y-NBT (5.0mM), for 20 min. NBT positive 'regions' (deep blue) of digestive system were observed microscopically.

Aphid feeding experiment on liquid diets with phenolics. Each diet incorporated one chemical amongst diphenolics (DP), monophenolics (MP) and polyphenolics (PP); as follows, caffeic acid (0.1, 1.0 and 10.0mM), (+) – catechin (0.01, 1.0mM, and 10mM), chlorogenic acid (1.0mM), p-coumaric acid (1.0mM), ferulic acid (1.0mM), gallic acid (1.0mM), hydroquinone (1.0mM), p-hydroxybenzoic acid (1.0mM) and L-DOPA, (1mM). All diets were dissolved in 15% w/v aqueous sucrose solution. 500 µl aliquot of each diet has been enclosed between two parafilm M® membranes stretched across plastic ring and, placed in a environmental cabinet. A hundred adults were allowed to feed the diet. Ten replications were done. For the control diet 15% w/v sucrose have been used.

A quantitative assay for determining H2O2 production in liquid diets and in whole aphids. The UV-measurement, at 240 nm was accomplished [1]. Samples for H2O2 assay were the 500 µl of liquid diets probed by aphids and 1000 µl of 1000g x 15min supernatant of crude homogenate of 10 aphids – unstarved that actively fed the 'artificial liquid' diets and wheat foliage and also aphids-starved during 24 hrs. H2O2 was assessed based on the standard curvae, obtained for 5, 10, 15, 20, 25 and 30 µM H2O2. Rate of H2O2 generation in the liquid diets and in the aphids was expressed as: (I) A240/10 or 100 aphids/15 hrs; (II) as µM/100 aphids/15hrs. The autooxidation of MP DP, PP in the diets-without aphids was measured by the same method.

Statistics. The H2O2 production was subjected to an analysis of variance followed by Duncan's test. Results with p-value ≤ 0.05 were considered as significant.


Generation of O2·– and H2O2 in aphid. The results (Fig 1, 2, 3) illustrate that the midgut is a 'major locus' of O2·– and H2O2 occurrence in R. padi and S. avenae. H2O2 is easly detected in the salivary secretions when aphids probe a-s g incorporating o-diphenolics e.g. caffeic acid, (+) – catechin and L-DOPA, and also in the liquid diets containing quoted phenolic chemicals.

Fig. 1. Exemplary light micrographs for H2O2 visualization by the KFe (CN)6 – FeCl3 reagent.
(A1, A2, A3) these figures illustrate H2O2 staining/occurrence (dark blue-green colour)  in the 'saliva deposit' of S.avenae within a-s g incorporating caffeic acid,  (+) catechin and L-DOPA, respectively.
(A') lack of H2O2 staining in the salivary secretions in presence of 0.005 M PTU, inhibitor of PPO activity.
(B1,B2) the blue-green colouring indicated by arrows observed on surface of digestive system showing 'centre' of H2O2 production in the midgut of aphid fed wheat foliage. Results for S.avenae and R.padi, respectively

Fig. 2. Detection of O2·– production using the microscopic NBT (5mM) assay;
in S. avenae and R. padi midguts, respectively. The arrows indicate deep blue/violet colouring corresponding with 'regions' of O2·– detection

Fig. 3. H2O2 production in the liquid diets incorporating the phenolic compounds and probed by S. avenae
A240/100 aphids/15 hrs;    µM/100 aphids/15 hrs

How dietary phenolics effect on H2O2 generation in aphid. Not all tested phenolics show the same capacity for H2O2 generation. In particular, DP and PP e.g. L-DOPA, caffeic acid, catechin, gallic acid and chlorogenic acid induce H2O2 production. Moreover, H2O2 production is associated with increased DP (e.g.caffeic acid) and PP (e.g. (+)–catechin concentration (Fig 3, 4). However, according to the results of the quantitative assay of H2O2, rather none inductive effect is showed for such chemicals as p-coumaric acid, p-hydroxybenzoic acid, ferulic acid and hydroquinone.

Fig. 4. H2O2 production in R. padi (A) and S. avenae (B) during enzymatic oxidation of the dietary
o-diphenolics incorporated in to the liquid diets. Values not followed by the same letter are significantly different  at the 0.05 level (Duncan's test)

How foliage feeding affects H2O2 generation. When R. padi and S. avenae ingest phloem sap from wheat foliage then their H2O2 production significantly increases, as compared to aphids ingest 15% w/v sucrose or starving during 24 hrs (Fig. 5). Then, the 'plant diet' similarlly to DP and PP present in the artificial diets, amplifies the H2O2 generation in the aphids.

Fig. 5. Mean rates of H2O2 production in R. padi and S.avenae fed the 'artificial' diet, plant foliage and starving, respectively. Values with different letters are significantly different (P ≤ 0.05)


The results show the H2O2 occurrence in two Aphididae species, R. padi and S. avenae. The H2O2 production in single aphid both within the midgut and salivary secretions is easly visualized by the classical histochemical procedure.

Presence of H2O2 in the aphid is not a novelty, because H2O2 is constant chemical of the  aerobics due to their obligatory oxidative metabolism. In aerobic cell H2O2 is released by the mitochondrial respiratory chain. Moreover, H2O2 may be provide via a variety of metabolic pathways catalysed by oxidases [3,4,8,9,33,35].

The real new aspect of this study is the recognition of H2O2 occurrence in the aphid, namely in the midgut and in the salivary secretions. Then, the aphid during feeding produces H2O2 both in vivo and in vitro – which is to say within artificial diets or plant foliage.

As yet, Madhusudhun and Miles [17] reported that H2O2 was produced in water incorporating (+) – catechin and probed by T.trifolii maculate and A. pisum.

The evidence that the salivary metabolism of the aphids produces H2O2 is significant for understanding of the fact that H2O2 detected in plant tissues after aphid feeding de facto may be aphid's H2O2.

H2O2 detected in plant tissues is very broadly demonstrated as direct antibiotic 'agent' e.g. anti-microbial, anti-fungal and anti-insect. Now, H2O2 of plants is the subject of many biochemical studies, just in these aspects [5,7].

The finding of the H2O2 synthesis in vivo has meaning as regards oxidative stress in the aphids. Namely, H2O2, O2·– and OH are representatives of ROS and are responsible for cytotoxic oxidative stress. They cause damage of DNA, proteins, phospholipids followed by cell death [3,8,20]. The H2O2 present in aphids at relatively high level, more than 10-5M, may adversely affect 'cell biochemistry' and subcellular structures and thereby may inhibit aphid growth and survival.

In general, the results show that H2O2 in the aphids is provided by oxidation of a variety of phenolics incorporated into the diets.  Yet, not every phenolic group has the same capacity for production of H2O2 during oxidation. DP and PP ‘appear’ as the primary compounds in this aspect. Contrary to dietary DP and PP for MP incorporated into diets, generation of H2O2 is zero, for both aphid species.

A variety of chemicals amongst DP and PP is involved in defence/resistance of Poaceae against R. padi and S.avenae[18,31,32]. Yet, biochemical mechanisms of DP and PP toxicity, in particular toxic implications of their oxidative metabolism, both within the diet and the aphid midgut remain rather debatable theme, still.

The data on the H2O2 production in the aphid body in response to the dietary DP and PP point out their prooxidant impact towards the aphids. Numerous compounds involved in defence against herbivores (e.g. furanocoumarins, flavonoids and quinones) 'provide' H2O2. Flavonoids and quinones seem to be activated by insect enzymes for production of ROS/H2O2 e.g. flavonoid, quercetin generates O2·–, H2O2 and OH [2,6,14,15,17,23,24,28].

DP i.e. caffeic acid and chlorogenic acid autooxidize at basic conditions to form H2O2. The pH of the midgut, for R.padi and S.avenae is acid and ranges 4.6–6.2. Then, the pH value of the aphid midgut excludes autooxidation of DP and PP.

Reduction of the NBT (showed in this study) indicates generation of  O2·– in the midgut of R. padi and S.avenae. This shows that O2·– mediates H2O2 synthesis in the aphid midgut. Thus, it is important to establish the enzymatic source of O2·– which in consequence of protonation is converted into H2O2.

In a general way, oxidases 'consume' O2 for their own reactions. The increase of the  H2O2 synthesis in R.padi and S.avenae as consequence of DP and PP intake suggests that just PPO reaction is potential source of O2·– and H2O2, in sequence.

PPO (EC o-diphenol: O2 oxidoreductase, well known as o-diphenol oxidase, catechol oxidase, tyrosinase and phenolase) is the enzyme that catalyses the conversion of o-diphenols via quinones till a brown or black pigments, melanins. Munoz et al. [19] reported the H2O2 generation in the melanin biosynthesis pathway, in series of chemical reactions coupled with the enzymatic formation of quinones by PPO acting on o-diphenols.

According to the earlier studies [21,31,32] PPO in Aphididae, i.e. Macrosiphum rosae, R.padi and S.avenae is dominant biocatalyst amongst the oxidoreductases of the midguts and the salivary secretions. The aphid PPO oxidizes spectrum of DP and PP - exactly the same that induce the H2O2 production. Then, the results presented here clarify the role of PPO. This oxidase can be regarded as an essential enzyme for the H2O2 production, via O2·–.

It is known that (+) catechin and other PP and DP during the oxidation by PPO provide aryloxyradicals [12,18,29]. As the result these free radicals may reduce O2 into O2·– that may dismutate directly to H2O2.

Jiang and Miles [12] showed that 'mushroom tyrosinase' and 'potato phenolase' produced H2O2 by oxididation of (+)- catechin and some other phenolics – probably via O2·– , and released H2O2 that could then be used as an electron acceptor for POD reaction.

POD is the bio-catalyst responsible for the oxidation of DP, PP and MP into quinones that en route eliminates H2O2. In R.padi and S.avenae POD reaction is located within the midgut and salivary secretions [28,30]. The oxidation of DP, PP and MP catalysed by POD may generate H2O2. This generation is achieved by formation of DP, PP and MP free radicals and hence O2·– followed by dismutation till H2O2.

R.padi and S.avenae possess biocatalyst of H2O2 breakdown in their midguts, CAT [27,30]. Action of the CAT is inducible by dietary DP, PP and H2O2. I argue that 'biochemical function' of catalytic activity of the aphid POD and CAT was the elimination of H2O2, and in consequence reduction of oxidative stress within the aphid organism.

Finally, I emphasize that key finding of this study is identification of H2O2 in the aphids, and recognition of the H2O2 as  by-product of the oxidative metabolism of the dietary DP and PP.

Moreover, I specify a direct link between H2O2 status and action of the aphid oxidoreductases i.e PPO, POD and CAT. It is illustrated by simplified scheme (Fig. 6).

Fig. 6. Summarizing scheme of the plant phenolic prooxidants and the aphid oxidoreductases  involved in the H2O2 production and decomposition [27,28,29]


  1. H2O2 is produced in the midgut and salivary secretions of R.padi and S.avenae.

  2. The phenolic chemicals with prooxidant capacity include mostly DP and PP i.e. caffeic and gallic acids, L-DOPA and also (+) – catechin. Instead, MP i.e. p-coumaric, p-hydroxybenzoic and ferulic acids are not specified as source of H2O2 production in the aphids.

  3. H2O2 is produced in the aphids via superoxide anion, O2·– due to the oxidation of DP and PP catalysed by PPO.


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Accepted for print: 10.12.2009

Anna Urbañska
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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