Sep 1, 2015 - hemolymph, midgut and fat body of Galleria mellonella larvae was investigated. Larvae were exposed to LD50 values of pyriproxyfen and B.
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Volume 25 – No. 5/ 2016, pages 1660-1665
Fresenius Environmental Bulletin
COMBINED EFFECTS OF PYRIPROXYFEN AND Bacillus thuringiensis ON ANTIOXIDANT ACTIVITY OF HEMOLYMPH, MIDGUT AND FAT BODY OF Galleria mellonella LARVAE Benay Sezer Tuncsoy, Pinar Ozalp Çukurova University, Faculty of Science and Letters, Department of Biology 01330, Balcali, Adana/ Turkey
ABSTRACT
generation of insect populations resistant to cry toxins. The increasing resistance in the insects can be due to the increasing metabolic capability of detoxification systems and/or reducing xenobiotic target site sensitivity [3]. Insect growth regulators (IGRs) are produced naturally by insects to regulate the processes of molting and development from the egg to the adult stage [4]. Pyriproxyfen is a pyridine-based juvenile hormone analogue that competes for juvenile hormone binding site receptors in insects, mimicking the action of juvenile hormone and thus maintaining an immature state [5]. This compound has a relatively low toxicity for mammals. Uses of pyriproxyfen cause severe morphological disorders [6-7] and many physiological and biochemical changes in metabolic pathway caused by this compound [8-9]. In our previous study, we showed that the solitary use of pyriproxyfen caused necrotic results in hemocytes of G. mellonella [10]. Resistance and cross-resistance problems are increasing and new products have to meet the rising standards of environmental and toxicological safety [11]. Insecticides were used in mixture due to the limitation of new type of insecticides to prevent the progress of resistance. Some insecticides that used in mixture have synergism effects and this causes the decline of expenditure. The use of the low doses of this insecticides benefits differently ecologically, biologically and economically. The aim of this study is to find the possible changes in the biochemical compounds of hemolymph, fat body and midgut and their correlation with each other when pyriproxyfen and B. thuringiensis were applied singly and mixture. Pyriproxyfen act only on target species and effect immune system with its usage together with B. thuringiensis should give rapid and reliable results in controlling G. mellonella. Also, it is expected that by the use of Bt products with pyriproxyfen will be useful for controlling the pests in a short time. Understanding of this biochemical effects may definitely help to prove a physiological and safe procedure to control the population of G. mellonella larvae in the hives.
Insect growth regulators and microbial insecticides are mostly used in pest management since they are nontoxic to other organisms and have short half-life in the environment. Effects of juvenile hormone analogue pyriproxyfen and Bacillus thuringiensis on antioxidant enzyme (catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities of hemolymph, midgut and fat body of Galleria mellonella larvae was investigated. Larvae were exposed to LD50 values of pyriproxyfen and B. thuringiensis (LD of B. thuringiensis: 359 mg/L; 50
LD
50
of pyriproxyfen: 2385 mg/L), then enzyme
activities were measured spectrophotometrically after 72 hours from treatment. When LD 50 value of pyriproxyfen and B. thuringiensis were applied to G. mellonella larvae singly and in mixture, the activities of SOD, CAT and GPx were significantly increased in hemolymph and fat body of G. mellonella larvae, whereas the enzyme activities were significantly decreased in midgut of larvae compared with the control group. As a result of this study, we observed that pyriproxyfen and B. thuringiensis effect the antioxidant defence system of G. mellonella larvae even if at low doses. If this kind of insecticides were applied at sublethal doses, economical, biological and ecological benefits were achieved as well. KEYWORDS: Antioxidant enzymes, Fat Body, Galleria mellonella, Hemolymph, Midgut
INTRODUCTION B. thuringiensis (Bt) is a naturally occurring spore-forming, soil bacteria and different preparations containing Bt are widely used as a microbial pesticide in agriculture [1]. Its bio preparations act by disintegrating intestinal membranes. Bt has been effectively used in control programs against lepidopteran larvae in Europe and North America [2]. One of the major concerns regarding the use of B. thuringiensis is the
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Volume 25 – No. 5/ 2016, pages 1660-1665
centrifuged at 500 × g for 15 min (+4 ◦C) and supernatants recentrifuged at 12,000 × g for 45 min (4 ◦C). Fat body tissues were centrifugated at 50 W, 40-50 s. with ultrasonic homogeniser (Bandelin Sonoplus. HD 2070, Berlin, Germany). Cytosolic fraction volumes were measured reserving a 100 µl aliquot of each sample for total protein quantification and purified applying Sephadex® G-25 gel columns to remove low molecular weight proteins [13]. The samples for biochemical assays were frozen – 80 °C until use.
MATERIALS AND METHODS Insects. G. mellonella larvae were reared at 30±1 °C, 65±5 % RH on a diet composed of bran, honey, glycerol, honeycomb and distilled water [12]. The continuity of the stock culture was supplied by mating the female and male adult insects and hatching the eggs. Chemicals. Juvenile hormone analogue (10% EC), Pyriproxyfen (Admiral) was tested in the present study. Its chemical formula is (2-[1-methyl2-(4-phenoxyphenoxy) ethoxy] pyridine). A larvicide based on B. thuringiensis var. kurstaki (Delfin WG, consisting of 32,000 IU mg-1 spores) was used for the bio tests.
Antioxidant enzymes. Antioxidant enzymatic activities were measured in the haemolymph, fat body and midgut cytosolic fraction of ten larvae from control groups and exposed to LD50 of pyriproxyfen and B. thuringiensis singly and mixture. To determine SOD activity, the reduction of cytochrome c by the system xanthine oxidase/ hypoxanthine was measured at 550 nm [14]. CAT activity was determined by the decrease in absorbance at 240 nm due to H2O2 consumption, with a molar extinction coefficient of 40 M-1 cm-1 [15]. GPx activity was measured through NADPH oxidation in the presence of excess glutathione reductase, reduced glutathione and hydroperoxide as substrate, at 340 nm [16].
Biochemical Assays. The lethal effects of various concentrations of B. thuringiensis and pyriproxyfen were determined. These data were used for probit analysis of the lethal activity of these insecticides to fifth instar larvae of G. mellonella. Eight groups of larvae were exposed to B. thuringiensis and pyriproxyfen in the following concentrations: control, 1500, 1750, 2000, 2400, 2800, 3000 and 3500 µg/mL. After the treatment, number of dead larvae were calculated and LD50 values were determined using SPSS 21.00, Probit Analysis programme. Fifth instar larvae were reared for 72 hours on honeycomb with LD concentrations of B.
Total protein concentration. Total protein content was determined in the cytosolic fraction of haemolymph, fat body and midgut of unexposed and pyriproxyfen and B. thuringiensis exposed larvae of G. mellonella according to Bradford method [17] using bovine serum albumin as a standard.
50
thuringiensis and
Pyriproxyfen (LD
thuringiensis: 359 µg/mL; LD
50
50
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of B.
of pyriproxyfen:
2385 µg/mL). After 72 hours, hemolymph, midgut and fat body were dissected from larvae into a chilled Eppendorf tubes with cold homogenization buffer (20 mM Tris buffer pH 7.6; 1 mM of EDTA+ 0.5 M of saccharose + 0.15 M of KCl + 1 mM of DTT). A few crystals of phenylthiourea (PTU) were added to each sample to prevent melanisation. Resulting homogenates were
Statistical Analysis. The statistical analyses of the data were carried out by a series of Analysis of Variance and Student-Newman Keul’s (SNK) test using SPSS 21.00 pocket program. The differences between the means were considered significant at the 0.05 probability level.
TABLE 1 Toxicity results of the pyriproxyfen bioassay on G. mellonella larvae. Points of Lethal Concentration LD 10.00 LD 15.00 LD 20.00 LD 30.00 LD 40.00 LD 50.00 LD 80.00 LD 90.00 LD 95.00 LD 99.00
Concentration (µg/ml) 1044 1223 1386 1701 2026 2385 4104 5450 6888 10688
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% 95 Confidences limits (µg/ml) 142-1506 243-1665 371-1809 728-2098 1247-2476 1845-3228 3098-17045 3713-44502 4287-98868 5583-166257
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Volume 25 – No. 5/ 2016, pages 1660-1665
Fresenius Environmental Bulletin
TABLE 2 Toxicity results of the B. thuringiensis bioassay on G. mellonella larvae. Points of Lethal Concentration LD 10.00 LD 15.00 LD 20.00 LD 30.00 LD 40.00 LD 50.00 LD 80.00 LD 90.00 LD 95.00 LD 99.00
Concentration (µg/ml) 14 26 43 95 189 359 2997 9090 12719 22667
RESULTS
% 95 Confidences limits (µg/ml) 0,002-84 0,018-132 0,096-197 1,341-424 10-1037 45-3460 633-6246 1452-12347 2696-27567 8025-83912
antioxidant enzymes of fifth instar larvae of G. mellonella was investigated and the results are given in Table 2. Significant increases in SOD, CAT and GPx activities were observed in hemolymph of larvae exposed to LD50 values of pyriproxyfen, B. thuringiensis and mixture (1:1) for 72h compared with the control group (p
