OCCURRENCE AND SOURCE OF HYDROGEN PEROXIDE IN APHIDS

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

ABSTRACT

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 1.10.3.1), 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.

INTRODUCTION

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 1.15.1.1) 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 1.11.1.6), GSH peroxidase (GPOD, EC 1.11.1.9), ascorbate peroxidase (APOD, EC 1.11.1.11) and peroxidase (POD, EC 1.11.1.7). 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 1.10.3.1) is considered as a 'enzymatic source' of H₂O₂.

MATERIAL AND METHODS

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.

RESULTS

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.

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.

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.

DISCUSSION

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 1.10.3.1 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).

CONCLUSIONS

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

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