1 Bulletin of Insectology 64 (2): , 2011 ISSN Effect of host plants on antioxidant system of pea aphid Acyrthosiphon pisum Iwona ŁUKASIK 1, Sylwia GOŁAWSKA 1, Agnieszka WÓJCICKA 1, Artur GOŁAWSKI 2 1 Department of Biochemistry and Molecular Biology, University of Natural Sciences and Humanities, Siedlce, Poland 2 Department of Zoology, University of Natural Sciences and Humanities, Siedlce, Poland Abstract The effect of some species of family Fabaceae plants on the non-enzymatic and enzymatic antioxidants of the pea aphid, Acyrthosiphon pisum (Harris) has been studied. The highest concentration of ascorbate was noted for aphids fed on Pisum sativum L.. The content of another non-enzymatic antioxidant, GSH, was the highest within the tissues of the morphs fed on Vicia faba L.. The aphids reared on this host plant had 3-fold higher activity of the antioxidant enzymes than the ones fed on Pisum sativum L. and Vicia sativa L.. The influences of the host plants on antioxidant defence mechanisms within the pea aphid species are discussed. Key words: Acyrtosiphon pisum, Fabaceae, aphids, reactive oxygen species, oxidative stress, antioxidants. Introduction In the natural environment, plants develop complex direct and indirect defence strategies against herbivores (Cory and Hoover, 2006). The basal defences are based on cell wall modification, antixenotic or antibiotic compounds and plant volatiles that repel aphids or attract their natural enemies (Goggin, 2007). Aphid feeding may trigger plant signalling pathways driven by jasmonic acid (JA), salicylic acid (SA), ethylene (ET), abscisic acid (ABA), gibberelic acid (GA), reactive oxygen species (ROS) or nitric oxide (NO) that induce the production of chemical defences (Smith and Boyko, 2007; Goggin, 2007; Walling, 2008). Aphids developed deceptive strategies to avoid or deter many plant defences. During feeding aphids introduce salivary effectors into the plant tissues to affect wound healing, defence-signalling pathways and volatile production (Walling, 2008). One of the most rapid plant defence reactions to biotic stress is oxygen burst which constitutes the production of reactive oxygen species (ROS) including superoxide radical anion (O 2 ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical ( OH) (Kuźniak and Urbanek, 2000; Mittler, 2002). Among the generated ROS, a central role in plant defence responses is played by hydrogen peroxide. H 2 O 2 exhibits direct toxicity toward herbivores, contributes to cell wall strengthening processes, triggers the hypersensitive responses (HR) and acting as signal molecule for the induction of defence genes (Kuźniak and Urbanek, 2000). Moloi and van der Westhuizen (2006) found that during infestation of wheat by Russian wheat aphid ROS, produced as a result of NADPH oxidase activation, act as signal for activation of defence enzymes intercellular peroxidase and β-1,3-glucanase. Additionally, some of the plant pro-oxidant compounds upon photochemical or metabolic activation may also generate ROS (Barbehenn et al., 2003; Krishnan et al., 2007). In response to oxidative stress arising from host plants, phytophagous insects have evolved specific defence mechanisms to deal with ROS. Herbivores posses system of antioxidant enzymes composed of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), Se-independent glutathione peroxidase (GSTPX) and glutathione reductase (GR), as well as low-molecular-weight antioxidants such as glutathione (GSH) and ascorbic acid (ASA). Among them, two enzyme-catalyzed pathways for destroying toxic H 2 O 2 : catalase and antioxidant system consisting of ascorbate peroxidase, dehydroascorbate reductase, ascorbic acid and glutathione play an important role (Barbehenn et al., 2001; Summers and Felton, 1993). CAT: 2H 2 O 2 2H 2 O + O 2 APX: ASA + H 2 O 2 DHA + 2H 2 O DHAR: DHA + 2GSH AA + 2H 2 O The pea aphid, Acyrthosiphon pisum (Harris) (Homoptera Aphididae) is becoming an increasingly important phytophagous on legumes due to the rising economic importance of these crops. The species is frequently used as a model piercing-sucking insect and recently its entire genome sequence has been published (The International Aphid Genomics Consortium, 2010). A. pisum is an oligophagous aphid species, one of the most serious pests of the Fabaceae plants: peas, alfalfas, vetchs, and red clovers (Cuperus et al., 1982; Lane and Walters, 1991). A. pisum is a vector of more than 30 viral diseases, including bean yellow mosaic virus, red clover vein mosaic virus and pea streak virus (Barnett and Diachun, 1986; Jones and Proudlove, 1991). Several reports have been published on the effect of different plant species and plant morphology on pea aphid development (Soroka and Mackay, 1990, 1991; Kaakeh and Dutcher, 1993; Sandström, 1994; Sandström and Pettersson, 1994) but there is a lack of studies relating to the role of oxidative stress in the process of adaptation of A. pisum to various host plants. Thus, the aim of the present paper was to investigate the effect of some Fabaceae plant species on antioxidant system removing hydrogen peroxide in A. pisum.
2 Materials and methods Aphids The insects came from the aphid stock cultures kept at the University of Natural Sciences and Humanities at Siedlce. The aphids were reared on pea seedlings, in an environmental chamber (21 C, L16:D8 photoperiod, and 70% RH). Experiments were conducted on wingless females (apterae) and nymphs of the pea aphid. Plants Three species of Fabaceae were used in the experiments: Pisum sativum L. var. Tulipan, Vicia faba L. var Start and Vicia sativa L. var. Jaga. Seed samples were bought in Horticultural Plant Breeding Seed Production and Nursery in Ożarów Mazowiecki (administrative Warsaw, Poland). Seed samples were germinated in a climate chamber, which was kept at 21 ± 1 C, L16:D8 photoperiod, and 70% RH. The seedlings were grown in plastic pots (10 x 10 cm, 5 seedlings per pot) filled with medium nutrient fine structure compost with sand. The soil was bought in ZPHU Umex from Łochów (administrative Warsaw, Poland). Nine-day-old seedlings of Fabaceae were infested each 20 aphids. After the aphids had fed on seedlings for seven days, they were collected and taken for assays of antioxidants. The experiment was conducted in four replicates. Ascorbic acid assay ASA concentration in aphid tissues was determined by adapting the procedure of (Omaye et al., 1979). 200 collected aphids were homogenized in a 67 mm potassium phosphate buffer (ph 6.5) containing 0.2 M EDTA and centrifuged at 3000 g for 15 min. Trichloroacetic acid (TCA) (1 ml of a 5% solution) was added to 0.5 ml of the supernatant, and the 1.5-ml volume was centrifuged at g for 20 min. ASA content was quantified after mixing 0.27 ml of the supernatant containing the aphid extract, 0.08 ml of 85% H 3 PO 4, 1.37 ml of 0.5% α,α' dipirydyl, and 0.28 ml of 1% FeCl 3. The reaction mixtures were incubated at 42 C for 40 min, and absorbance at 525 nm was measured against a control containing 0.27 ml of 67 mm K-phosphate buffer (ph 6.5) instead of the aphid homogenate. ASA content was calculated from a calibration curve prepared with standards and was expressed in nmol per mg of protein. Glutathione assay Total glutathione concentration in aphid tissues was determined with an enzymatic recycling assay based on glutathione reductase (Griffith, 1980). Briefly, 200 collected aphids were homogenized in 50 mm phosphate buffer ph = 7.8 containing 0.2 M EDTA and centrifuged at 3000 g for 15 min. The obtained supernatants were recentrifuged after addition of 50% TCA. Content of glutathione was determined after mixing 0.5 ml of the aphid extracts, 0.3 ml of 0.3 mm NADPH, 0.1 ml of 6 mm DTNB [5,5 -dithiobis-(2-nitrobenzoic acid)] and 0.1 ml of GR (50 units/ml). The absorbance at 412 nm was recorded after 5 min at room temperature. The GSH content within the aphid homogenates was determined by comparing the rate of absorbance change to that of glutathione standards of known concentrations and was expressed as nmol per mg of protein. Catalase assay CAT activity was measured as described by Aebi (1984). 20 collected aphids were homogenized in 67 mm potassium phosphate buffer (ph 7) for 5 min at 0 C. The homogenates were filtered through two layers of cheesecloth and centrifuged at 3000 g for 15 min. 0.5 ml of aphid extracts was added to 0.5 ml 30 mm H 2 O 2 and disappearance of hydrogen peroxide was measured at 240 nm during 3 min at 30 s intervals. Activity of the catalase was expressed as µmol decomposed H 2 O 2 per minute per mg protein. Ascorbate peroxidase assay APX activity was determined according to Asada (1984). 100 collected aphids were homogenized in 67 mm potassium phosphate buffer (ph 7) for 5 min at 0 C. The homogenates were filtered through two layers of cheesecloth and centrifuged at 3000 g for 15 min. The reaction mixture consisted of 0.75 ml of crude homogenate of aphids and 0.25 ml of 67 mm potassium phosphate buffer (ph 7) containing 2.5 mm ascorbic acid and 0.2 ml of 30 mm H 2 O 2. The decrease in absorbance at 290 nm was monitored for 5 min using a spectrophotometer. Boiled samples served as controls. APX activity was expressed as µmol ascorbate oxidized/min/mg protein, using an extinction coefficient of 2.8 mm -1 cm -1. Dehydroascorbate reductase assay DHAR activity was determined by adapting the procedure of Asada (1984). 100 collected aphids were homogenized in 67 mm potassium phosphate buffer (ph 7) for 5 min at 0 C. The homogenates were filtered through two layers of cheesecloth and centrifuged at 3000 g for 15 min. The reaction mixture consisted of 0.6 ml of crude homogenate of aphids, 0.1 ml of 5 mm GSH, 0.1 ml of 0.1 mm EDTA and 0.2 ml of 0.06 mm DHA in 67 mm potassium phosphate buffer ph 7. The enzyme activity was determined by the formation of ASA at 265 nm. The increase in absorbance at 265 nm was monitored for 6 min against the blank contained boiled homogenates. DHAR activity was calculated as nmol ASA/min/mg protein. Protein assay The protein content in the studied aphid supernatants was determined using the method given by Bradford (1976). Statistics All data are reported as means ± SD, n = 4, where each replication represents one independent aphid homogenate. Data were subjected to a one-way analysis of variance (ANOVA) followed by the Tukey s multiplerange test. 154
3 Results and discussion Removing toxic hydrogen peroxide is very important for herbivorous insects because they lack a Sedependent glutathione peroxidase, which is a main antioxidant enzyme for neutralizing hydrogen peroxide in mammalian tissues (Ahmad et al., 1987). The genome of A. pisum lacks the genes encoded selenocysteine (The International Aphid Genomics Consortium, 2010). However, it may be compensated for the presence of the specific peroxidase activity of glutathione transferase (GSTPX), which catalyzes the reduction of wide range of hydroperoxides (Łukasik and Goławska, 2007). The pea aphid have a least 18 genes encoding glutathione-stransferases (GST) that may detoxify stress agents, including ROS (Gerardo et al., 2010; Ramsey et al., 2010). A comparison of Myzus persicae (Sulzer) cdna and A. pisum genomic sequences showed no differences in the number of glutathione-s-transferase genes but cytochrome P-450 gene family was 40% larger in M. persicae than in A. pisum. It is partially consistent with hypothesis that the different host ranges of the two aphids caused that generalist (M. persicae) requires a greater number of detoxification enzymes than a specialist (A. pisum) (Ramsey et al., 2010). Since catalase is inefficient at removing low concentrations of hydrogen peroxide, alternative mechanism in the form of an ascorbate-recycling system (ASA, APX, GSH and DHAR) seems to be very important in protection of herbivores against oxidative stress. Hitherto antioxidant system for removing hydrogen peroxide has been detected in Lepidoptera larvae and other leafchewing insects (Barbehenn et al., 2001; Mathews et al., 1997; Wang et al., 2001; Krishnan and Kodrik, 2006) as well as in sucking-piercing insects, including aphids (Figueroa et al., 1999; Loayza-Muro et al., 2000; Lukasik, 2007). The obtained results showed the pea aphid had less efficient antioxidant mechanisms than the cereal aphids with regard to lower level of CAT and APX (Lukasik, 2007; Łukasik et al., 2009). It may be associated with the presence of specific compounds in the cereals (e.g. hydroxamic acids) that affected antioxidant system (Figueroa et al., 1999). The results of this study demonstrate that Fabaceae plants clearly affected the content of non-enzymatic antioxidants and antioxidant enzymes in the pea aphid. The concentration of GSH and ASA within studied aphid morphs varied depending on the host plant on which they were reared (table 1). The highest GSH level was recorded for morphs that fed on a broad bean (ANOVA: F 1.5 = , p < 0.001). The aphids reared on this host plant had 2-fold higher concentration of GSH in comparison with insects fed on vetch. In contrast, the content of another non-enzymatic antioxidant, ASA, was the highest in tissues of morphs reared on pea (ANOVA: F 1.5 = , p < 0.001). The wingless females that fed on vetch and broad bean had similar ASA concentration whereas the nymphs showed 2-fold higher level of this antioxidant during feeding on vetch. Because the level of low-molecular-weight antioxidants is one of the biochemical markers of oxidative stress, differences in ASA and GSH content within aphid tissues point to diversity of defence mechanisms of host plants against the aphids. Petrić-Mataruga et al. (1997) suggest that regulation of the level of GSH in herbivores may be one of the rapid forms of adaptive responses against nutritive and oxidative stress. Previously, it has been reported that the alteration of the diet caused changes in the GSH amount in the midgut of Lymantria dispar (L.) (Petrić-Mataruga et al., 1997). The concentration of ASA within gut tissues of Trichoplusia ni (Hubner) and Depressaria pastinacella (Duponchel) varied in proportion to levels of ASA in the diets (Timmermann et al., 1999). Our previous studies demonstrated the depletion of GSH and ASA in the cereal aphids upon treatment of plant o-dihydroxyphenols (Łukasik, 2006; Łukasik et al., 2009). Thus pro-oxidants status of host plants and their qualitative composition may affect level of nonenzymatic antioxidants within aphid tissues. Among the studied morphs, GSH concentrations were higher for apterae females in comparison to nymphs fed on all studied host plants. A different pattern was recorded for ascorbate, where the studied morphs reared on pea and broad bean had similar ASA content whereas nymphs fed on vetch contained significantly higher level of ASA than apterae. Krishnan et al. (2007, 2009) noted a lower level of ROS and a higher antioxidant potential in adults of Leptinotarsa decemlineata (Say) than in the larval stage. Different results were obtained for morphs of cereal aphids, in which the ASA and GSH concentrations were higher within nymph tissues than apterae ones (Łukasik, 2006; Łukasik et al., 2009). Goławska (unpublished data) proved that the most suitable host for A. pisum is pea plant. The aphids fed on pea showed higher fecundity and survival than those feeding on broad bean, alfalfa or clover. This may be conditioned by a very high level of ASA within tissues of aphids fed on the pea. ASA is an essential nutrient for insects that is Table 1. Content of the GSH and ASA (nmol/mg protein) within tissues of the pea aphid fed on Fabaceae plants. Host plant Aphid morphs GSH ASA Pea apterae ± 0.51 b 880 ± 27 a nymph ± 0.36 c 850 ± 30 a Broad bean apterae ± 0.42 a 280 ± 12 c nymph ± 0.39 b 240 ± 19 c Vetch apterae ± 0.43 c 280 ± 17 c nymph 8.11 ± 0.19 d 440 ± 21 b Data are presented as mean ± SD; n = 4. Values in columns not followed by the same letter are significantly different (ANOVA: p < 0.001). 155
4 Table 2. Activity of the CAT (µmol H 2 O 2 /min/mg protein), APOX (µmol ASA oxidized/min/mg protein) and DHAR (nmol ASA/min/mg protein) within tissues of the pea aphid fed on Fabaceae plants. Host plant Aphid morphs CAT APOX DHAR Pea apterae 7.61 ± 0.30 e 0.08 ± 0.02 c ± 2.63 d nymph 8.50 ± 0.25 d 0.11 ± 0.03 c ± 5.33 c Broad bean apterae ± 0.21 b 0.22 ± 0.05 ab ± 3.28 c nymph ± 0.40 a 0.29 ± 0.07 a ± a Vetch apterae 8.59 ± 0.16 d 0.12 ± 0.03 c ± 2.29 d nymph ± 0.35 c 0.16 ± 0.02 bc ± 6.31 b Data are presented as mean ± SD; n = 4. Values in columns not followed by the same letter are significantly different (ANOVA: p < 0.001). involved in many metabolic processes and required for normal growth, reproduction and development. The highest level of studied antioxidant enzymes was noted in morphs reared on broad bean (ANOVA: F 1.5 = , p < for CAT; F 1.5 = 16.26, p < for APX; F 1.5 = , p < for DHAR). In comparison to broad bean, vetch and pea reduced aphid enzyme activities. The aphids fed on these host plants had 2- or 3- fold lower levels of the studied enzymes than insects originating from broad bean. Comparing the other host plants, the induction of enzyme activities was shown to be greater in aphids fed on vetch than in those fed on pea (table 2). Krishnan and Kodrik (2006) observed a significant up-regulation of CAT and APX activity in Spodoptera littoralis (Boisduval) fed on plant diet in comparison to semi-artificial diet. Thus insects are able to adapt quickly to increased oxidative stress arising from plant pro-oxidant allelochemicals by regulation of antioxidant enzyme activities (Ahmad and Pardini, 1990; Krishnan and Kodrik, 2006; Lukasik, 2007). Petrić-Mataruga et al. (1997) observed induction or inhibition of antioxidant enzymes when the effect of host plant of different population origins on the antioxidant defence of L. dispar was studied. A comparison of antioxidant status of three species of Lepidoptera differing in their preference of host plant T. ni, Spodoptera eridania (Stoll), Papilio polyxenes F. pointed towards a correlation of antioxidant defence with natural feeding habits of these insects and their susceptibility to pro-oxidant plant compounds in the diet (Ahmad and Pardini, 1990). The high antioxidant enzyme activities within tissues of aphids fed on broad bean suggest that this host plant may possess allelochemical compounds acting as inducers of antioxidant system. Figueroa et al. (1999) reported the twofold increase of CAT activity in the grain aphid Sitobion avenae (F.) fed on DIM- BOA-containing artificial diets with respect to control diets without DIMBOA. This contrasts with the results obtained by Loayza-Muro et al. (2000) that indicate no significant effect of wheat cultivars differing in the hydroxamic acid concentrations on CAT activity within the grain aphid. Łukasik (2009) noted the induction of CAT activity in migrants of Rhopalosiphum padi (L.) after transfer from primary (bird cherry) to secondary (triticales) host plants, but the level of activity was closely associated with feeding duration on the secondary host. Krishnan and Sehnal (2006) noted that the ingestion of tannic acid significantly increased the activity of CAT and APOX in the midgut of S. littoralis larvae. These results are in accordance with our studies in which exposure of cereal aphids to plant pro-oxidants clearly induced the activity of APOX (Łukasik et al., 2009). This increase of APOX activity within cereal aphids under the influence of o-dihydroxyphenols may compensate for the inhibition of catalase by these compounds (Lukasik, 2007). However, studies of the ascorbate-recycling system in the midgut of caterpillars Malacosoma disstria (Hubner) and Orgyia leucostigma (Smith) showed that APOX and CAT activities were reduced by ingestion of tannic acid (Barbehenn et al., 2001). Thus the induction/inhibition of antioxidant system might depend on insect species and on biochemical properties of enzymes. For example, quercetin, a pro-oxidant flavonoid, caused an increase of superoxide dismutase (SOD) activity within cereal aphid tissues, but significantly suppressed the CAT and the glutathione reductase (GR) activity (Lukasik, 2007; Łukasik and Goławska, 2007). Among the studied aphid morphs the highest activities for CAT and DHAR were found for nymphs. In case of the DHAR, nymphs had nearly 3-fold higher activity than wingless apterae fed on the studied host plants. Different results were obtained for the APX, where apterae as well as nymphs reared on all tested Fabaceae plants had comparable enzyme activities (table 2). These results may point out more effective antioxidant mechanisms that protect nymphs from damages caused by oxidative stress. It was evidenced by results of our earlier studies related to antioxidant mechanisms in cereal aphids in which nymphs had higher levels of enzymes targeting H 2 O 2 than apterae adults (Lukasik, 2007; Łukasik et al., 2009). Conclusions It was evidenced by results presented here that host plants clearly affected the antioxidant defence mechanisms in the pea aphid tissues. The pea seems to be the most suitable host to A. pisum since aphids reared on this plant had the lowest level of antioxidant enzymes and the highest content of the main non-enzymatic antioxidant - ascorbate. This is probably conditioned by a lower level of specific plant metabolites that may promote the oxidative stress within herbivores tissues. The larval stages of A. pisum seem to posses more effective antioxidant mechanisms than wingless females with regard to higher activity of enzymes removing H 2 O
5 References AEBI H., Catalase in vitro.- Methods in Enzymology, 105: AHMAD S., PARDINI R. S., Mechanisms for regulating oxygen toxicity in phytophagous insects.- Free Radicals in Biology and Medicine, 8: AHMAD S., PRITSOS C. A., BOWEN S. M., KIRKLAND K. E., BLOMQUIST G. J., PARDINI R. S., Activities of enzymes that detoxify superoxide anion and related oxyradicals in Trichoplusia ni.- Archives of Insect Biochemistry and Physiology, 7: ASADA K., Chloroplasts: formation of active oxygen species and its scavenging.- Methods in Enzymology, 105: BARBEHENN R. V., BUMGARNER S. L., ROOSEN E. F., MARTIN M. M., Antioxidant defenses in caterpillars: role of the ascorbate-recycling system in the midgut lumen.- Journal of Insect Physiology, 47: BARBEHENN R. V., POOPAT U., SPENCER B., Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry.- Insect Biochemistry and Molecular Biology, 33: BARNETT O. W., DIACHUN S., Virus diseases of clovers: etiology and crop losses, pp In: Viruses infecting forage legumes (EDWARDSON J. R.,CHRISTIE G., Eds).- Florida Agricultural Experiment Station, Gainesville, USA. BRADFORD M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.- Analytical Biochemistry, 72: CORY J. S., HOOVER K., Plant-mediated effects in insect-pathogen interactions.- Trends in Ecology and Evolution, 21 (5): CUPERUS C. W., RADCLIFFE E. B., BARNES D. K., MARTEN G. C., Economic injury levels and economic thresholds for pea aphid, Acyrthosiphon pisum (Harris) on alfalfa.- Crop Science, 1: FIGUEROA C. C., KOENIG C., ARAYA C., SANTOS M. J., NIE- MEYER H. M., Effect of DIMBOA, a hydroxamic acid from cereals, on peroxisomal and mitochondrial enzymes from aphids: evidence fort he presence of peroxisomes in aphids.- Journal of Chemical Ecology, 25: GERARDO N. M., ALTINCICEK B., ANSELME C., ATAMIAN H., BARRIBEAU S. M., DE VOS M., DUNCAN E. J., EVANS J. D., GABALDÓN T., GHANIM M., HEDDI A., KALOSHIAN I., LA- TORRE A., MOYA A., NAKABACHI A., PARKER B.J., PÉREZ- BROCAL V., PIGNATELLI M., RAHBÉ Y., RAMSEY J. S., SPRAGG C. J., TAMAMES J., TAMARIT D., TAMBORINDEGUY C., VINCENT-MONEGAT C., VILCINSKAS A., Immunity and other defenses in pea aphid, Acyrtosiphon pisum.- Genome Biology, 11: R21. GOGGIN F. L., Plant-aphid interactions: molecular and ecological perspectives.- Current Opinion in Plant Biology, 10: GRIFFITH O. W., Determination of glutathione and glutathione disulfide using glutathione reductase and 2- vinylpyridine.- Analytical Biochemistry, 106: JONES R. A. C., PROUDLOVE W., Further studies on cucumber mosaic virus infection of narrow-leafed lupin (Lupinus angustifolius): seed-borne infection, aphid transmission, spread and effects on grain yield.- Annals of Applied Bioloy, 118: KAAKEH W., DUTCHER J. D., Rates of increase and probing behaviour of Acyrthosiphon pisum (Homoptera: Aphididae) on preferred and nonpreferred host cover crops.- Environmental Entomology, 22: KRISHNAN N., KODRIK D., Antioxidant enzymes in Spodoptera littoralis (Boisduval): are they enhanced to protect gut tissues during oxidative stress?- Journal of Insect Physiology, 52: KRISHNAN N., SEHNAL F., Compartmentalization of oxidative stress and antioxidant defense in the larval gut of Spodoptera littoralis.- Archives of Insect Biochemistry and Physiology, 63: KRISHNAN N., KODRIK D., TURANLI F., SEHNAL F., Stagespecific distribution of oxidative radicals and antioxidant enzymes in the midgut of Leptinotarsa decemlineata.- Journal of Insect Physiology, 53: KRISHNAN N., KODRIK D., KŁUDKIEWICZ B., SEHNAL F., Glutathione-ascorbic acid redox cycle and thioredoxin reductase activity in the digestive tract of Leptinotarsa decemlineata (Say).- Insect Biochemistry and Molecular Biology, 39: KUŹNIAK E., URBANEK H., The involvement of hydrogen peroxide in plant responses to stresses.- Acta Physiologiae Plantarum, 22: LANE A., WALTERS K. F. A., Effect of pea aphid (Acyrthosiphon pisum) on the yield of combining peas.- Aspects of Applied Biology, 27: LOAYZA-MURO R., FIGUEROA C. C., NIEMEYER H. M., Effect of two wheat cultivars differing in hydroxamic acid concentration on detoxification metabolism in the aphid Sitobion avenae.- Journal of Chemical Ecology, 26: ŁUKASIK I., Effect of o-dihydroxyphenols on antioxidant defence mechanisms of cereal aphids associated with glutathione.- Pestycydy/Pesticides, 3-4: LUKASIK I., Changes in activity of superoxide dismutase and catalase within cereal aphids in response to plant o- dihydroxyphenols.- Journal of Applied Entomology, 131: ŁUKASIK I., Effect of host alternation on the activity of adaptive enzymes of the bird cherry-oat aphid Rhopalosiphum padi (L.).- Journal of Pest Science, 82: ŁUKASIK I., GOŁAWSKA S., Activity of Se-independant glutathione peroxidase and glutathione reductase within cereal aphid tissues.- Biological Letters, 4: ŁUKASIK I., GOŁAWSKA S., WÓJCICKA A., Antioxidant defense mechanisms of cereal aphids based on ascorbate and ascorbate peroxidase.- Biologia, 64: MATHEWS M. C., SUMMERS C. B., FELTON G. W., Ascorbate peroxidase: a novel antioxidant enzyme in insects.- Archives of Insect Biochemistry and Physiology, 34: MITTLER R., Oxidative stress, antioxidants and stress tolerance.- Trends in Plant Science, 7: MOLOI M. J., VAN DER WESTHUIZEN A. J., The reactive oxygen species are involved in resistance responses of wheat to the Russian wheat aphid.- Journal of Plant Physiology, 163: OMAYE S. T., TURNBULL J. D., SAUBERLICH H. E., Selected methods for the determination of ascorbic acid in animal cells, tissues and fluids.- Methods in Enzymology, 62: PETRIĆ-MATARUGA W., BLAGOJEVIĆ D., SPASIĆ M.B., IVANO- VIĆ J., JANKOVIĆ-HLADNI M., Effect of host plant on the antioxidative defence in the midgut of Lymantria dispar L. caterpillars of different population origins.- Journal of Insect Physiology, 43: RAMSEY J. S., RIDER D. S., WALSH T. K., DE VOS M., GORDON K. H. J., PONNALA L., MACMIL S. L., ROE B. A., JANDER G., Comparative analysis of detoxification enzymes in Acyrtosiphon pisum and Myzus persicae.- Insect Molecular Biology, 19:
6 SANDSTRÖM J., High variation in host adaptation among clones of the pea aphid, Acyrthosiphon pisum on peas, Pisum sativum.- Entomologia Experimentalis at Applicata, 71: SANDSTRÖM J., PETTERSSON J., Amino acid composition of phloem sap and the relation to intraspecific variation in pea aphid (Acyrthosiphon pisum) performance.- Journal of Insect Physiology, 40: SMITH C. M., BOYKO E. V., The molecular bases of plant resistance and defense responses to aphid feeding: current status.- Entomologia Experimentalis et Applicata, 122: SOROKA J. J., MACKAY P. A., Growth of pea aphid, Acyrthosiphon pisum (Homoptera: Aphididae), populations on caged plants of six cultivars of field peas and the effects of pea aphids on harvest components of caged field peas.- The Canadian Entomologist, 122: SOROKA J. J., MACKAY P. A., Antibiosis and antixenosis to pea aphid (Homoptera: Aphididae) in cultivars of field peas.- Journal of Economic Entomology, 84: SUMMERS C. B., FELTON G. W., Antioxidant role of dehydroascorbic acid reductase in insects.- Biochimica at Biophysica Acta, 1156: THE INTERNATIONAL APHID GENOMICS CONSORTIUM, Genome sequence of the pea aphid Acyrtosiphon pisum.- PloS Biology, 8: e TIMMERMANN S. E., ZANGERL A. R., BERENBAUM M. R., Ascorbic and uric acid responses to xanthotoxin ingestion in a generalist and a specialist caterpillar.- Archives of Insect Biochemistry and Physiology, 42: WALLING L. L., Avoiding effective defenses: strategies employed by phloem-feeding insects.- Plant Physiology, 146: WANG Y., OBERLEY L. W., MURHAMMER D. W., Evidence of oxidative stress following the viral infection of two lepidopteran cell lines.- Free Radicals in Biology and Medicine, 31: Authors addresses: Iwona ŁUKASIK (corresponding author, Sylwia GOŁAWSKA, Agnieszka WÓJCICKA, Department of Biochemistry and Molecular Biology, University of Natural Sciences and Humanities, ul. B. Prusa 12, Siedlce, Poland; Artur GOŁAWSKI, Department of Zoology, University of Natural Sciences and Humanities, ul. B. Prusa 12, Siedlce, Poland. Received December 12, Accepted June 6,
Reactive oxygen species in leaves and how to catch these Éva Hideg Molecular Stress- & Photobiology Group, BRC Szeged EPPN Summer School 2013 Outline How to prove that ROS are involved? The central role
International Journal of PharmTech Research CODEN( USA): IJPRIF ISSN : 0974-4304 Vol.1, No.3, pp 870-874, July-Sept 2009 CLINICAL STUDY OF MARKETED AYURVEDIC PREPARATION IN THYROID INDUCED STRESS A. A.
Laboratory 5: Properties of Enzymes Technical Objectives 1. Accurately measure and transfer solutions with pipettes 2. Use a Spectrophotometer to study enzyme action. 3. Properly graph a set of data. Knowledge
The Physiology of Hyperbaric Oxygen Therapy Free Radicals and Reactive Oxygen Species I. Introduction Definition, Source, function and Purpose A. Definition of free radicals and reactive oxygen species
INTRODUCTION Factors Affecting Enzyme Activity The chemical reactions occurring in living things are controlled by enzymes. An enzyme is a protein in the cell which lowers the activation energy of a catalyzed
Ann.wellhouse@TouchStoneScience.net 1 Enzyme Function National Science Standards Science as Inquiry: Content Standard A: As a result of activities in grades 9-12, all students should develop: Abilities
LAB 5 PLANT NUTRITION I. General Introduction All living organisms require certain elements for their survival. Plants are known to require carbon (C), hydrogen (H), oxygen (O), nitrogen (N), phosphorus
Rubisco; easy Purification and Immunochemical Determination Ulrich Groß Justus-Liebig-Universität Gießen, Institute of Plant Nutrition, Department of Tissue Culture, Südanlage 6, D-35390 Giessen e-mail:
= Fields of Application / Industry: Chemistry / Polymer Industry Clinical Chemistry / Medicine / Hygiene / Health Care Cosmetics Electronics Energy Environment / Water / Waste Food / Agriculture Geology
Plant Genetic Resources Green sources of plant protein Gert Poulsen Department of Plant and Environmental Science Faculty of Science NORDGEN IS AN INSTITUTE UNDER THE NORDIC COUNCIL OF MINISTERS Protein
Antioxidant Products A comprehensive range of ALB BIL FERR GR Ransel Ransod antioxidant TAS testing TIBC kits TF BIL FERR GR Ransel Ransod TAS TIBC TF UA A FERR GR Ransel Ransod TAS TIBC TF UA ALB GR Ransel
Enzyme Action: Testing Catalase Activity (Method 1 O 2 Gas Sensor) Computer 2 Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically. Enzymes are globular proteins, responsible for most
Hydrogen Peroxide Cell-Based Assay Kit Item No. 600050 www.caymanchem.com Customer Service 800.364.9897 Technical Support 888.526.5351 1180 E. Ellsworth Rd Ann Arbor, MI USA TABLE OF CONTENTS GENERAL INFORMATION
Enzyme Action: Testing Catalase Activity DataQuest 12 Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities
Catalase Assay Kit Catalog Number CAT100 Storage Temperature 2 8 C TECHNICAL BULLETIN Product Description Catalase is an antioxidant enzyme ubiquitously present in mammalian and non-mammalian aerobic cells
Understanding the Soil Test Report Page 1 of 7 Crops absorb the nutrients required from soil in order to grow, so ensuring that your soil is meeting the crops needs is critical. Having the proper level
148 Bishop s University 2015/2016 Biochemistry The Biochemistry program at Bishop s is coordinated through an interdisciplinary committee of chemists, biochemists and biologists, providing students with
et al., 1981a,c) and predators were free to leave a host plant at any time. The interactions between predator and prey were quantified and the fate of the aphids in the colony was noted. MATERIAL AND METHODS
17 th INTERNATIONAL BIOLOGY OLYMPIAD 9-16 JULY 2006 Río Cuarto República Argentina PRACTICAL TEST 3 Biochemistry Student code: 17 th INTERNATIONAL BIOLOGY OLYMPIAD 9-16 JULY 2006 Río Cuarto República Argentina
Name Section Lab 5 Photosynthesis, Respiration and Fermentation Plants are photosynthetic, which means that they produce their own food from atmospheric CO 2 using light energy from the sun. This process
Effect Of Amino Acids On Plants Agriculture production is a very intensive business and is related to better quality and better yield leading to better profitability Every farmer s dreams to achieve this
CHAPTER 10 BACTERIAL GROWTH Eye of Science / Science Photo Library WHY IS THIS IMPORTANT? Increase in numbers is one of the requirements for infection. This increase is dependent upon bacterial growth.
Tech Prep Articulation Agriculture & Natural Resources Tech Prep Education: Tech Prep education in Missouri is an articulated two-year secondary and two or more year post-secondary education program which:
DOE Office of Biological & Environmental Research: Biofuels Strategic Plan I. Current Situation The vast majority of liquid transportation fuel used in the United States is derived from fossil fuels. In
Experiment 7 (Lab Period 8) Quantitative Determination of Phosphatase Activity Phosphatases are enzymes that catalyze the hydrolysis of organic-phosphate compounds, releasing inorganic phosphate from the
1. The diagram below represents a biological process 5. The chart below indicates the elements contained in four different molecules and the number of atoms of each element in those molecules. Which set
Enzyme Action: Testing Catalase Activity Experiment 6A Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities
Enzyme Action: Testing Catalase Activity Experiment 6A Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically. Enzymes are globular proteins, responsible for most of the chemical activities
Exercise 6 - Enzyme Activity Introduction Enzymes are biological catalysts that regulate the rate of chemical reactions. Their 3-dimensional conformation and therefore their function can be affected by
Product Manual Phosphate Assay Kit (Colorimetric) Catalog Number STA-685 1000 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures Introduction Phosphorus exists as a soluble free phosphate
TECHNICAL REPORT STUDY CHEMICAL PROPERTIES OF RATTAN SHOOT FROM PLANTATION IN THAILAND by Assistant Professor Dr. Noojaree Prasitpan Chemist Analyzer of the ITTO Project on PD 24/00 Rev.1 (I): Promotion
GENE CLONING AND RECOMBINANT DNA TECHNOLOGY What is recombinant DNA? DNA from 2 different sources (often from 2 different species) are combined together in vitro. Recombinant DNA forms the basis of cloning.
1) Complete the table: MEDCHEM 562 Fall 2013 Vitamin Problem Set 1: Vitamin Function / Uses Deficiency States Toxicities Daily Value Upper Limit A D E K 2) Complete the metabolic pathway shown below: Retinyl
HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for
Enzymes: Amylase Activity in Starch-degrading Soil Isolates Introduction This week you will continue our theme of industrial microbiologist by characterizing the enzyme activity we selected for (starch
Names: LabQuest Enzyme Action: Testing Catalase Activity 50 Points 6A Many organisms can decompose hydrogen peroxide (H 2 O 2 ) enzymatically. Enzymes are globular proteins, responsible for most of the
Glutathione and Oxidative Stress - Part I By: James L. Holly, MD Oxidative Stress refers to effects from endogenous (produced in the body) toxins (free radicals) produced in the body by normal metabolism
77:222 Spring 2003 Free Radicals in Biology and Medicine Page 0 This student paper was written as an assignment in the graduate course Free Radicals in Biology and Medicine (77:222, Spring 2003) offered
Curriculum Policy of the Graduate School of Agricultural Science, Graduate Program Agricultural Science plans to conserve natural and artificial ecosystems and its ideal of "Sustainable coexistence science"
Lab 10: Bacterial Transformation, part 2, DNA plasmid preps, Determining DNA Concentration and Purity Today you analyze the results of your bacterial transformation from last week and determine the efficiency
Unit 2 Metabolism and Survival Summary 1 Metabolism pathways and their control (a) Introduction to metabolic pathways This involves integrated and controlled pathways of enzymecatalysed reactions within
Is Exercise the Best Antioxidant Supplement? Len Kravitz, Ph.D. As an unexpected consequence of the metabolic steps that convert food into energy, the body produces molecules commonly called free radicals.
AP Biology Unit I: Ecological Interactions Essential knowledge 1.C.1: Speciation and extinction have occurred throughout the Earth s history. Species extinction rates are rapid at times of ecological stress.
Cautions: 6 M hydrochloric acid is corrosive. Purpose: To colorimetrically determine the mass of iron present in commercial vitamin tablets using a prepared calibration curve. Introduction: Iron is considered
D. A. Lewis Anti-Inflammatory Drugs from Plant and Marine Sources 1989 BirkhauserVerlag Basel Boston Berlin TABLE OF CONTENTS Page No. Preface. Acknowledgements..12 SECTION I : INFLAMMATION AND INFLAMMATORY
EXPERIMENT 3 Potato Polyphenol Oxidase (I) 1 BACKGROUND Polyphenol oxidase, also known as tyrosinase (EC 18.104.22.168) is an enzyme responsible for one of the steps in the conversion of tyrosine into melanine,
Cattle and Horse Nutrition Dona Goede Livestock Specialist Introduction Many health, reproductive and production problems can be prevented with good nutrition. Poor nutrition results in: Poor conception
Free radicals - Enemies of health, beauty and youth Free radicals (FR) are natural molecules produced in the body following biochemical reactions related to the behaviour of singlet oxygen. The human body
Creatine Kinase Activity Colorimetric Assay Kit ABE5487 100 assays; Store at -20 C I. Introduction: Creatine Kinase (CK) also known as creatine phosphokinase (CPK) and ATP: creatine N- phosphotransferase
Vol. 9, pp. 50-58 (2009) Journal of Agricultural Physics http://www.agrophysics.in Germination Characteristics of Seeds of Maize (Zea mays L.) Exposed to Magnetic Fields under Accelerated Ageing Condition
Free Energy and Enzymes (Chapter 6) Outline Growing Old With Molecular Mayhem A. Free radicals are molecules with extra electrons. 1. The "extra" electrons have been stripped from other atoms in the cell.
A Degree in Science is: Intellectually stimulating Can lead to a variety of careers: Scientific Research (with opportunities for working with animals) Teaching Science-related jobs Advisory Sales Patents..
1 Name: SARA CUARESMA Lesson Title: WHAT IS AN ENZYME? Lesson Length (2 CLASS PERIODS) Grade Level(s): 9 TH GRADE BIOLOGY SSI Topic: SSI Topic: SC.912.L.18.11 Explain The role of enzymes as catalysts that
Induction of Enzyme Activity in Bacteria:The Lac Operon Preparation for Laboratory: Web Tutorial - Lac Operon - submit questions I. Background: For the last week you explored the functioning of the enzyme
Chapter 2 Integrated Pest Management In This Chapter Keywords After learning the information in this chapter, you will be able to: 1. Define Integrated Pest Management (IPM). 2. List and describe the 5
Viruses and Prokaryotes Cellular Basis of Life Q: Are all microbes that make us sick made of living cells? 20.1 What is a virus? WHAT I KNOW SAMPLE ANSWER: A virus is a tiny particle that can make people
Mammalian digestive tracts Mouth: mastication, some digestive enzymes Esophagus: simple transport tube Stomach: most initial digestion, some physical processing Small intestine: digestion continues, some
Investigation 2-13 ENZYME ACTIVITY How do abiotic or biotic factors influence the rates of enzymatic reactions? BACKGROUND Enzymes are the catalysts of biological systems. They speed up chemical reactions
Catalytic Activity of Enzymes Introduction Enzymes are biological molecules that catalyze (speed up) chemical reactions. You could call enzymes the Builders and Do-ers in the cell; without them, life could
Introduction to Integrated Pest Management John C. Wise, Ph.D. Michigan State University MSU Trevor Nichols Research Complex What is Integrated Pest Management? Integrated Pest Management (IPM) New concept;
Academic Offerings This section contains descriptions of programs, majors, minors, areas of concentration, fields of specialization, and courses. Semesters following course titles indicate when each course
Bio 210A Bacterial Transformation-Gene Cloning Purpose To understand the relevance of gene cloning to the biotechnology industry. To understand the definition of transformation, clone, and cloning vector.
DNA Biotechnology and Enzymes 35 Background Unit 2~ Lesson 1 The Biotechnology Industry Biotechnology is a process (or a technology) that is used to create products like medicines by using micro-organisms,
Inhibition of dopamine conversion to norepinephrine by Clostridia metabolites appears to be a (the) major cause of autism, schizophrenia, and other neuropsychiatric disorders. All these factors can now
r r r rr EDUCATION RESEARCH EXTENSION The Texas A&M University System Soil & Crop Sciences Managing of Annual Winter Forages in Southwest Texas Mr Charles Stichler Assocaite Professor and Extension Agronomist
Fighting the Battles: Conducting a Clinical Assay 6 Vocabulary: In Vitro: studies in biology that are conducted using components of an organism that have been isolated from their usual biological surroundings
Name Lab 3: ENZYMES In this lab, you ll investigate some of the properties of enzymes. So what are enzymes? Enzymes are large protein molecules (macromolecules) They catalyze or speed up chemical reactions
Chapter 7 Lecture Notes: Control of Microbes by Physical and Chemical Agents I. Definitions A. Antimicrobial agent = general terms for an agent that kills microbes or inhibits their growth 1. prefix designates
THE KILL DATE AS A MANAGEMENT TOOL TO INCREASE COVER CROPS BENEFITS IN WATER QUALITY & NITROGEN RECYCLING María ALONSO-AYUSO José Luis GABRIEL Miguel QUEMADA Technical University of Madrid (Spain) INDEX
Human serum albumin (HSA) nanoparticles stabilized with intermolecular disulfide bonds Wentan Wang, Yanbin Huang*, Shufang Zhao, Ting Shao and Yi Cheng* Department of Chemical Engineering, Tsinghua University,
The Huntington Library, Art Collections, and Botanical Gardens How Sweet It Is: Enzyme Action in Seed Germination Overview This experiment is intended to familiarize students with the macromolecule starch,
Keystone Biology Exam Information: Module A: Cell and Cell Processes Basic Biological Principles- Day 1 Describe the characteristics of life shared by prokaryotic and eukaryotic organisms. Compare cellular
Doctor of Philosophy in Biochemistry FACULTY OF MEDICAL SCIENCE Naresuan University 73 Doctor of Philosophy in Biochemistry The Biochemistry Department at Naresuan University is a leader in lower northern
Bachelor s Programme in Agricultural and Environmental Management Study line: Environmental Management Total Study description 2011-2013 Table of contents: 1. The Programme... 3 2. Total study overview...
Dana Hitchcock Dr. Cummings - Prd. 1 September 9, 1996 Sample Lab Report Effect of the Hormone Gibberellic Acid on the Growth of Dwarf Peas Objective In this experiment students will grow genetically dwarf
7 Answers to end-of-chapter questions Multiple choice questions 1 B 2 B 3 A 4 B 5 A 6 D 7 C 8 C 9 B 10 B Structured questions 11 a i Maintenance of a constant internal environment within set limits i Concentration
Enzyme Lab DEFINITIONS: 1. Enzyme: 2. Catalase: 3. Catalyze: 4. Hydrolysis: 5. Monomer: 6. Digestion: BACKGROUND INFORMATION Many living tissues contain the enzyme catalase. This enzyme breaks down hydrogen
5 Training Objectives:! Knowledge of the most important function of nutrients! Description of both, mechanism and function of gluconeogenesis! Knowledge of the difference between essential and conditionally
Running protein gels and detection of proteins 1. Protein concentration determination using the BIO RAD reagent This assay uses a colour change reaction to give a direct measurement of protein concentration.
The Nature of Science Science is an ongoing, human, cultural activity which involves the efforts of many people to describe the world in mutually acceptable terms. Scientific knowledge is the cumulative