Biochemical Stress Indicators of Greater Wax Moth Exposure ... - BioOne

ECOTOXICOLOGY

Biochemical Stress Indicators of Greater Wax Moth Exposure to Organophosphorus Insecticides ¨ YU ¨ KGU ¨ ZEL,1, 3 ENDER I˙ÇEN,1 FERAH ARMUTÇU,2 KEMAL BU

AND

¨ REL2 AHMET GU

J. Econ. Entomol. 98(2): 358Ð366 (2005)

ABSTRACT Although acetylcholinesterase (AChE) is the primary target of organophosphorus insecticides (OPs), increasing evidence regarding their secondary effects suggests that OPs disturb homeostasis of insects by generating free radical intermediates that trigger lipid peroxidation. We therefore investigated alterations in lipid peroxidation product, malondialdehyde (MDA) content, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, in conjunction with AChE activity as biochemical stress indicators in greater wax moth, Galleria mellonella (L.) larvae for OPs methyl parathion (MP) and ethyl parathion (EP). The effects of MP and EP were Þrst investigated by rearing the young larvae on an artiÞcial diet containing 0.01, 0.1, 1, 10, and 100 ppm of each insecticide. Second, the mature larvae were injected with 0.05, 0.5, 5, 50, and 500 ng of insecticides for determining the changes in biochemical stress responses. The diet with lowest level of MP signiÞcantly decreased the activities of all measured enzymes, whereas it increased MDA content. However ALT and AST were signiÞcantly higher in the larvae reared with the diet with high levels of MP than in control larvae. All tested levels of MP resulted in a decrease in AChE activity. The lowest level of EP in diet (0.01 ppm) signiÞcantly increased ALT activity, whereas it reduced that of AChE. This insecticide at 0.1 ppm resulted in reduced AST activity, but 1 ppm in diet elevated AST activity and MDA content. EP at 0.1 ppm and higher levels in the diet reduced ALT activity. All dietary EP levels signiÞcantly decreased AChE activity. ALT, AST, and AChE were lower in larvae fed with the diet containing 100 ppm ethyl parathion compared with larvae on control diet. MP at 50 ng per larva increased ALT and AST activities from 35.42 ⫾ 0.74 and 26.34 ⫾ 0.83 to 203.57 ⫾ 1.09, and 122.90 ⫾ 1.21 U/g, respectively, when the mature larvae were injected. All injected doses of EP dramatically reduced both ALT and AST activities, but only the lowest and highest levels of this insecticide decreased AChE activity. The lowest level of this insecticide also signiÞcantly increased MDA content in larvae. High levels of both insecticides increased MDA content. We observed a signiÞcant higher increase in MDA content in the larvae reared with 10 ppm EP (102.16 ⫾ 1.57 nmol/g protein) than the control group (30.28 ⫾ 1.42 nmol/g protein). These results suggest that OPs caused the metabolic and synaptic dysfunctions in greater wax moth and alter its biochemical physiology in response to oxidative stress. KEY WORDS Galleria mellonella, organophosphorus insecticides, malondialdehyde, synthetic diet, nutrition

CHEMICAL PESTICIDES ARE ONE of the major sources of environmental pollution. Of these chemicals, insecticides used in agricultural Þelds for pest management programs pose a threat to nontarget organisms and the environment (He et al. 2002). These compounds, including organophosphorus insecticides (OPs), at sublethal levels effectively induce biochemical stress responses in invertebrates (Saravana Bhavan and Geraldine 2001). OPs produce their main toxicity through irreversible inhibition of acetylcholinesterase (AChE) by phosphorylating a serine hydroxyl group within the enzyme active site, leading to hyperexcit1 Department of Biology, University of Karaelmas, Faculty of Science, Zonguldak, Turkey. 2 Department of Biochemistry, University of Karaelmas, Faculty of Medicine, Zonguldak, Turkey. 3 Corresponding author, e-mail: [email protected].

ability at peripheral and central cholinergic synapses. AChE is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine in nervous system (Howard and Pope 2002). OPs also generate free radicals, mainly reactive oxygen species, probably because of alteration in the homeostasis of the body resulting in oxidative stress (Felton 1995). The most important targets for free radical attack are polyunsaturated fatty acids in tissue and membrane lipids, which are oxidized to lipid hydroperoxides. Lipid peroxidation produces a variety of products, including aldehydes, the most important of which is the reactive carbonyl compound malondialdehyde (MDA). These products may impair cellular functions, including nucleotide and protein synthesis and enzyme activity. Increased MDA levels, after ex-

0022-0493/05/0358Ð0366$04.00/0 䉷 2005 Entomological Society of America

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I˙ Ç EN ET AL.: BIOCHEMICAL STRESS INDICATORS OF G. mellonella

posure to OPs, have been associated with a variety of tissue damage and cell membrane distribution in animals (Suwalsky et al. 2001). There is some information on effects of various environmental pollutants on lipid peroxidation in insects (Cervera et al. 2003). However, there is no study in the literature on the effects of OPs on this oxidative stress reaction of insects. Methyl parathion (MP) and ethyl parathion (EP) are important broad-spectrum organophosphorus insecticides, which are used in agriculture and public health. MP is safer to nontargets than its ethyl analog parathion, whereas both have similar toxicity to insects (He et al. 2002). These insecticides are also highly used in our country to control pest insects on certain agricultural crops not only in the Þeld but also in stored products because of their easy availability and accessibility. Recently, they have received attention as a consequence of their illegal use in most areas. MP and EP exert their effects by inhibiting esterases, especially AChE that is essential enzyme for life (Belden and Lydy 2001). Greater wax moth, Galleria mellonella (L.) (Lepidoptera: Pyralidae), is a serious honey bee, Apis mellifera L., pest that feeds on combs, wax, and honey in beehives (Charriere and Imdorf 1997). We use the pupae of this moth as factitious host for propagation of ichneumonid parasitoids for the purpose of nutritional studies under laboratory conditions (Bu¨ yu¨ kgu¨ zel 2001a, b). The larvae and pupae of this insect, collected from such hives in apicultural area, were used to start of G. mellonella culture in our laboratory. The insects might have been indirectly exposed to the OPs in their natural environment (Wilson et al. 1988). There is evidence of accumulation of the some environmental pollutants in G. mellonella and of transmission of these pollutants to their parasitoids (Ortel 1995). Biochemical and nutritional state of this host are important in determining its acceptance and use by parasitoids (Sandlan 1982, Harvey et al. 1995). Because OPs are potent cholinesterase inhibitors, most previous work has focused on AChE activity as a robust and well established speciÞc biomarker in insects (Nath and Kumar 1999). Evidence has been growing to suggest that OP toxicity is not only the result of inhibition of AChE but also of a number of pathophysiological alterations in nervous and other tissues of animals. Generation of excess free radicals by OPs, in addition to triggering lipid peroxidation, leads to the release of some stress proteins, necrosis factors, and activates membrane bound and cytosolic enzymes (Baba et al. 1981). It has been reported that transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities are highly present in ganglia of various insect species (Sugden and Newsholme 1975). Furthermore, these transaminases were used as a bioindicator of pesticide contamination in some invertebrates under laboratory conditions (Mosleh et al. 2003). It is generally suggested that an increase in activities of these enzymes reßects metabolic disruption in a few insects (Verma and Rahman 1984, Eid et al. 1989, Theophilis

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1997) contaminated by various pesticides. Based on these results, we hypothesized that alterations in the transaminase activities in relation to OP toxicity would be associated with lipid peroxidation in tissues of insects. With exception of nontarget organisms (Popova and Chavdar 2002, Patil et al. 2003), changes in the transaminase activities, in conjunction with lipid peroxidation levels and their relationships to synaptic transmission, have not been studied in insects as biochemical stress indicators for OP toxicity. For assessing of new biomarkers as nonacethylcholinesterase targets to understand the secondary effects of OPs impairing homeostasis of a laboratory-reared host insect, we measured lipid peroxidation product MDA content as indicator of oxidative stress, and transaminase enzymes ALT and AST activities associated with the amino acid metabolism, in addition to speciÞc target enzyme AChE participated in neurotransmission, in G. melonella larvae exposed to MP and EP. This study shows that sublethal doses of the insecticides are still neurotoxic and that they may very well spark metabolic dysfunction in G. mellonella and its parasitoids emerged from such hosts. Materials and Methods Insect Stock Culture Greater wax moth larvae and pupae were collected from infected hives in apicultural area of Zonguldak, Turkey, and the newly emerged adults were used to maintain the stock culture. The adults were placed in 1000-ml glass jars and provided with diet to lay eggs. Neonate larvae were reared in glass jars (1000 ml) Þlled to one-third with an artiÞcial diet, at 30 ⫾ 1⬚C in constant darkness, in an incubator (Nu¨ ve ES500, Nu¨ ve Co., Ankara, Turkey) as a stock culture. The synthetic diet described by Bronskill (1961) was used for rearing G. mellonella larvae. The diet contained 420 g of bran, 150 ml of Þltered honey, 150 ml of glycerol (Merck, Darmstadt, Germany), 20 g of ground old dark honeycomb, and 30 ml of distilled water. The methods used to prepare and dispense of diets into containers, to obtain eggs and larvae, and to inoculate them onto diets described previously (Laing and Hagen 1970). Insecticides Technical grade samples of methyl [(Penncap, O,O-dimethyl O-(4-nitrophenyl) phosphorothioate] and ethyl parathion [(Pestanal, O,O-diethyl O-(4-nitrophenyl) phosphorothioate] (PESTANAL, liquid form, 100 ng/␮l ⫾ 5%) were obtained from SigmaAldrich GmbH (Seelze, Germany). Test solutions were prepared by serially diluting these technical grade stock solutions with distilled water. Commercial-grade insecticides that are in a liquid form were Þrst diluted in 1 ml of ethanol and completed with distilled water to prepare solutions of the required concentrations. Dilution of the stock solutions was made immediately before injection. Because ethyl al-

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cohol is less toxic and stimulates food consumption of insects (Norris and Baker 1969), it was used as a solvent. The insecticides were tested for their individual sublethal effects on the activities of enzymes ALT (E.C.2.6.1.2, L-alanine 2-oxoglutarate aminotransferase), AST (E.C.2.6.1.1, L-aspartate 2-oxoglutarate aminotransferase), AChE (E.C.3.1.1.7, acetylcholine acetylhidrolase), and lipid peroxidation product MDA in the whole body of the insect. Two groups of larvae in different developmental stages were used in the experimental series. In the Þrst series of experiments, fourth instars were reared until the seventh instar (last instar) on the artiÞcial diets containing various levels of methyl and ethyl parathion. Before starting the feeding experiments dealing with the effects of methyl and ethyl parathion on the activities of enzymes and MDA content of G. mellonella larvae, some preliminary experiments were carried out to determine the effects of these organophosphorus insecticides on survival and development of the insect on artiÞcial diet. The Þrst instars (newly hatched larvae) were inoculated on the artiÞcial diet with graded levels of the organophosphorus insecticides. On all diets containing each level of methyl and ethyl parathion, none of the Þrst instars survived to subsequent stage and died within 24 h. By contrast, all fourth instars on the diets with low levels of the insecticides (0.01, 0.1, 1, and 10 ppm) were able to complete their development to the seventh instar with the exception of the diet containing 100 ppm, which killed most fourth instars in 1 h. Based on these preliminary experiments, fourth instars were used for determining biochemical stress responses of the G. mellonella to organophosphorus insecticides. In the second series of experiments, effects of the methyl and ethyl parathion were tested by injecting the insecticides into seventh instars of G. mellonella. Seventh instars were used in injection experiments because they are most suitable for physiological experiments (Jarosz 1989). Feeding Experiments Five artiÞcial diets containing 0.01, 0.1, 1, 10, and 100 ppm of each insecticides were compared with control diet (without insecticides). Larvae were reared in 250-ml glass jars (12 by 6 cm) containing 100 g of diet treated with desired concentrations of each insecticide. One-day-old fourth instars were obtained from rearing container of standard stock culture of G. mellonella regardless of their weight because of small variation in their body volume. These larvae were placed on the diets contaminated with insecticides and reared until the seventh instar for 10 d. We choose 10-d exposures because a shorter period could result in starvation for avoiding stress. Control larvae were reared on the normal diet treated with an equal volume of distilled water. After 10 d the mature larvae were weighed and kept at ⫺30⬚C until biochemical analysis. Each experiment including Þve concentrations and one control was replicated four times with 10 larvae. Rearing was conducted under the same

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laboratory conditions as mentioned for stock insect culture. Injection Experiments One-day-old seventh instars were selected and ice chilled for 5 min to reduce their mobility before injection. The larvae were injected with 5 ␮l of the required concentrations of methyl and ethyl parathion solutions containing 0.05, 0.5, 5, 50, and 500 ng per larva. Injections were performed dorsolaterally in the intersegmental region between last two abdominal segments into hemocoel of the larva with 25-␮l Hamilton microvolume syringe. Control insects were injected with 5 ␮l of distilled water. Treated larvae were placed in 30-ml plastic cups (35 by 55 mm, OrLab Ltd., Ankara, Turkey) lined with paper towel and covered with a screen lid. The larvae were allowed to stand for 48 h under laboratory conditions as mentioned for the stock culture. The 48-h period was chosen because this is the maximum time required to reach the prepupal stage. Larvae were considered dead if unable to walk in a coordinated way in Þrst 24 h after injection, and these larvae were excluded from the experiments. Larvae were taken from each replicate of each injection. They were weighed and kept at Ð30⬚C until biochemical analysis. Each injection experiment for the insecticides tested was done with four replicates. Ten larvae were tested in each replication. Biochemical Analysis Enzyme Extraction and Determination. The larvae were homogenized in phosphate buffer (0.05 M, pH 8) in a tissue grinder with homogenizator (IKA ULTRATURRAX, IKA-WERKE GMBH Co., Staufen, Germany) at 24,000 rpm. Whole body homogenates were centrifuged at 10,000 ⫻ g for 10 min at 4⬚C to remove debris. Clear supernatant was used to estimate enzyme activities. The results are reported in SI units per gram of larval wet weight. Enzyme extraction in whole body of insect was made according to methods of Azmi et al. (2002). The content of enzymes was determined spectrophotometrically (Jenway 3600, Essex, England) at 340 nm for ALT and AST and at 405 nm for AChE. For the determination of enzyme content, Sigma (St. Louis, MO) diagnostic kits (ALT [kit no. 505], AST [kit no. 505], and AChE [kit no. 420]) were used. L-Aspartate and 2-oxoglutarate for AST, L-alanine and 2-oxoglutarate for ALT, and acetylthiocholine chloride for AChE determination were provided as substrates. The methods used to determine the activities of transaminase enzymes (ALT and AST) and AChE were the same as those described previously (Reitman and Frankel 1957, Frankel 1970). Determination of Malondialdehyde. MDA content was assayed using the method described by Ohkawa et al. (1979). The whole larvae were homogenized in 0.8 ml of phosphate buffer (0.05 M, pH 8.0). Homogenate (0.4 ml) was mixed with 0.025 ml of butylated hydroxytoluene and 0.5 ml of 30% tricholoroacetic acid. After 2-h incubation at ⫺20⬚C, the mixture was cen-

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Table 1. Activities (mean ⴞ SD) of enzymes and MDA content of G. mellonella larvae reared on an artificial diets containing different concentrations of organophosphorus insecticides Concn insecticide in diet (ppm) Methyl parathion Control 0.01 0.1 1 10 100 (4) Ethyl parathion Control 0.01 0.1 1 10 100 (3)

ALT (U/g)

AST (U/g)

AChE (U/g)

MDA (nmol/g protein)

40.13 ⫾ 1.3a 12.25 ⫾ 0.9b 47.82 ⫾ 2.3c 52.41 ⫾ 2.5c 78.89 ⫾ 3.1d 82.47a

34.28 ⫾ 1.72a 12.21 ⫾ 1.02b 57.56 ⫾ 2.12c 49.82 ⫾ 1.30d 63.93 ⫾ 2.80e 76.52a

2.98 ⫾ 0.72a 1.92 ⫾ 0.25b 1.72 ⫾ 0.57b 1.90 ⫾ 0.83b 1.02 ⫾ 0.95b 0.97a

43.48 ⫾ 1.21a 55.21 ⫾ 0.87b 12.36 ⫾ 1.49c 15.39 ⫾ 1.46c 75.34 ⫾ 1.45d 70.61a

38.42 ⫾ 2.12a 45.22 ⫾ 2.84b 32.95 ⫾ 0.96c 33.73 ⫾ 1.25c 16.54 ⫾ 0.75d 7.43a

31.43 ⫾ 1.91a 28.31 ⫾ 1.25a 18.27 ⫾ 0.73b 42.29 ⫾ 2.50c 32.48 ⫾ 1.48a 12.84a

3.25 ⫾ 0.28a 2.01 ⫾ 0.10b 0.89 ⫾ 0.10c 0.75 ⫾ 0.07c 0.80 ⫾ 0.11c 0.71a

30.28 ⫾ 1.42a 27.33 ⫾ 1.46a 25.37 ⫾ 1.57a 45.46 ⫾ 2.01b 102.16 ⫾ 1.57c 58.31a

Data are the average of four replicates, with 10 larvae per replicate. Values followed by the same letter are not signiÞcantly different from each other, P ⬎ 0.05. Value in parentheses is number of the larvae analyzed. a Average of three replicates.

trifuged (2,500 ⫻ g) for 15 min. Aliquots of supernatant (1 ml) were added to each tube and then 0.075 ml of 0.1 M EDTA and 0.25 ml of 1% thiobarbituric acid were added. These tubes with Teßon-lined screw caps were incubated at 100⬚C in a water bath for 15 min and cooled to room temperature. Afterwards, 1.5 ml of butanol was added to samples and mixed vigorously. The absorbance of sample was measured at 532 nm in a spectrophotometer (Jenway 3600). 1,1,3,3-Tetrametoxypropane was used as standard. Results are expressed as nanomoles of MDA formed per gram of protein Protein concentrations in the supernatants were determined by the procedure of Lowry et al. (1951) by using bovine serum albumin (Sigma) as standard. Statistical Analysis The effects of insecticides at different levels on the biochemical parameters in whole body were measured by determining the average activities of the AST, ALT, AChE enzymes, and MDA content of G. mellonella larvae. Data on the activities of enzymes and MDA content in the insect were evaluated by analysis of variance (ANOVA) (Snedecor and Cochran 1967). To determine signiÞcant differences between means, DuncanÕs multiple range test (Duncan 1955) was used. When F exceeded the 0.05 value, the differences were considered signiÞcant. All data are expressed as means ⫾ SD. Results All tested diet concentrations of methyl and ethyl parathion showed lethal effects on Þrst instars. However, the fourth instars had wide tolerance against lower levels of the insecticides than the highest level of 100 ppm per os. However, most larvae on the diet with the highest level could not complete their development to last instar. Most mature larvae injected with 500 ng per larva died within 24 h posttreatment.

Data on the activities of the enzymes AST, ALT, and AChE and MDA content in the whole body of G. mellonella exposed to sublethal and lethal levels of MP and EP are shown in Tables 1 and 2. The diet with lowest MP level (0.01 ppm) signiÞcantly decreased the activities of all enzymes and increased MDA content. Compared with controls, ALT and AST were signiÞcantly higher in the larvae reared with the diet containing other tested levels of MP. This insecticide, at 0.1 and 1 ppm, resulted in signiÞcantly decreased MDA content. All tested levels of MP in the diet decreased the AChE activity. ALT and AST activities were higher by approximately twofold in the larvae reared on the diet with 100 ppm MP (Table 1). Contrary to MP, the lowest dietary level of EP (0.01 ppm) resulted in signiÞcantly increased ALT activity and decreased AChE activity, whereas it had no signiÞcant effect on AST activity and MDA content. EP at 0.1 ppm resulted in declined ALT and AST activity, but 1 ppm EP resulted in elevated AST activity and MDA content. EP at 0.1 ppm and higher resulted in reduced ALT activity. MDA content was signiÞcantly increased from 30.28 ⫾ 1.42 nmol in the control group to 102.16 ⫾ 1.57 nmol in the diet containing 10 ppm EP. The activities of ALT, AST, and AChE were lower in the fourth instar on the diet with 100 ppm EP than in control diet. All levels of EP resulted in signiÞcantly decreased AChE activity (Table 1). MP at a dose of 50 ng caused a signiÞcant increase in the activities of ALT, AST, and MDA content but decreased the AChE activity when the mature larvae were injected. ALT and AST activities were increased from 35.42 ⫾ 0.74 and 26.34 ⫾ 0.83 U/g to 203.57 ⫾ 1.09 and 122.90 ⫾ 1.21 U/g, respectively. MP at 5 ng and lower had no signiÞcant effect on the AChE activity. However, 5 ng of this insecticide per larva caused a signiÞcant increase in MDA content. Compared with control, injection of this insecticide at 500 ng decreased the activities of all enzymes in mature larvae (Table 2). All tested doses of EP dramatically declined both ALT and AST activities, but only its

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Table 2. Activities (mean ⴞ SD) of enzymes and MDA content of G. mellonella larvae injected with different doses of organophosphorus insecticides Doses of insecticide (ng/larva) Methyl parathion Control 0.05 0.5 5 50 500 (4) Ethyl parathion Control 0.05 0.5 5 50 500 (3)

ALT (U/g)

AST (U/g)

AchE (U/g)

MDA (nmol/g protein)

35.42 ⫾ 0.74a 39.35 ⫾ 1.50a 46.37 ⫾ 0.73b 65.78 ⫾ 1.11c 203.57 ⫾ 1.09d 14.69a

26.34 ⫾ 0.83a 26.10 ⫾ 0.91a 19.85 ⫾ 1.14b 24.26 ⫾ 0.68ab 122.90 ⫾ 1.21c 17.83a

3.51 ⫾ 0.92a 3.12 ⫾ 0.85a 2.97 ⫾ 0.62a 2.67 ⫾ 0.45a 1.06 ⫾ 0.43b 0.85a

27.81 ⫾ 0.70a 33.02 ⫾ 1.13a 22.06 ⫾ 1.09a 59.92 ⫾ 0.95b 98.70 ⫾ 1.48c 60.47a

39.25 ⫾ 1.80a 20.46 ⫾ 1.72b 5.12 ⫾ 0.53c 13.25 ⫾ 2.12d 23.27 ⫾ 2.81b 11.89a

28.82 ⫾ 1.21a 17.57 ⫾ 2.17bc 12.93 ⫾ 0.93b 21.74 ⫾ 1.50c 16.41 ⫾ 1.02b 9.97a

3.58 ⫾ 0.71a 2.42 ⫾ 0.62b 3.24 ⫾ 0.70a 2.98 ⫾ 0.56a 2.02 ⫾ 0.54b 0.94a

47.11 ⫾ 1.02a 64.30 ⫾ 1.53b 41.16 ⫾ 1.52a 38.38 ⫾ 1.02a 83.85 ⫾ 1.67c 65.47a

Data are the average of four replicates, with 10 larvae per replicate. Values followed by the same letter are not signiÞcantly different from each other, P ⬎ 0.05. The value in parentheses is number of larvae analyzed. a Average of three replicates.

lowest and highest (0.05 and 500 ng) doses signiÞcantly decreased AChE activity when mature larvae were injected. Injection of the lowest dose (0.05 ng per larva) resulted in signiÞcantly increased MDA content (Table 2). Discussion Although much has been published regarding effects of OPs on transaminase activities in relation to lipid peroxidation as biochemical stress indicators in vertebrate models (Patil et al. 2003), not much attention has been given to related studies in insects. This information is an attempt to relate new possible biomarkers as nonacetylcholinesterase targets for assessing secondary effects of OPs on G. mellonella. The data show that MP and EP induced biochemical stress responses in the larvae and registered changes in enzyme activities and the content of lipid peroxidation product. Such biochemical responses have been reported for a lepidopteran (Biddinger and Hull 1999) and various other insect species (Verma and Rahman 1984, Wakgari and Giliomee 2001) exposed to some pesticides. Effects of the organophosphorus insecticides on G. mellonella larvae varied with insecticide used. Most of the fourth instars fed on the diet with highest levels (100 ppm) of MP and EP showed uncoordinated movements, leading to incapacity of food intake and they eventually died within 1 h. Similar results were obtained by injection of the insecticides at highest doses to mature larvae. Mortality may be due to lethal effects of the highest levels of the insecticides. These alterations also might be attributed to differences in physiology of each larval stage of G. mellonella (Jegorov et al. 1992). Therefore, the effects of sublethal levels of such insecticides would be toxicologically important to evaluate their physiological impairment of insects. Our results provide support for the hypothesis that an increase in lipid peroxidation leads to an increase

in activity of transaminases ALT and AST when G. mellonella larvae were fed or injected with high levels of MP. However, decreased ALT and AST activities with increased lipid peroxidation were observed in the larvae treated with the same levels of EP in both exposure routes. The effects on transaminases and lipid peroxidation vary with insecticide used and exposure route. These data suggest that EP may cause severe metabolic dysfunction, leading to necrotic cell death in tissue of the insect, probably generation of free radicals initiating lipid peroxidation. Increased free radicals mediated by OPs lead to the release of tumor necrosis factors, heat shock proteins, and various cellular enzymes (Baba et al. 1981). A signiÞcant increase in activities of the transaminase enzymes at some levels of OPs might be a result of an adaptive mechanism of the larvae due to oxidative stress. ALT and AST activities serve as biomarkers for assessment of tissue injury of vertebrates (Patil et al. 2003). These transaminases are widely distributed in most tissues and catalyze the interconversions of the amino acids by transfer of amino groups. It is reasonable to suggest that increased ALT and AST activities might be attributed to an impairment in amino acid metabolism and eventually increased metabolic activity of G. mellonella larvae exposed to MP and EP. This was in accordance with the Þndings of Eid et al. (1989) dealing with the increased activities of the transaminases as biochemical response in silk glands of Philosamia ricini (Boisd) treated with some antibiotic insecticides. Furthermore, there is evidence for a relationship between OPs and amino acid metabolism in which alanine content was decreased in organophosphorus insecticide-susceptible strains of some insects (Saleem and Shakoori 1993). The discrepant effects of MP and EP at certain levels on transaminase activity may be a result of differences in their metabolism and chemical structure, although they seem structurally very similar. Methyl parathion may be rapidly transformed to methyl paraoxon, which is an oxidatively activated

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intermediate responsible for its toxicity but through similar pathways to those of ethyl parathion (Blasiak and Kowalik 1999). However, EP is rapidly distributed through tissues and may be stored in lipids because of their high liposolubility, whereas MP does not accumulate in the tissues of animals (Cremlyn 1974). The fat body, which is responsible for detoxiÞcation and nitrogen metabolism, may be one of the other target tissues for these insecticides or their oxidized metabolites. The role of the fat body in insects is similar to the function of liver in vertebrates. Therefore, these OPs may have affected fat body and cellular lipid by leading peroxidation, resulting in tissue injury in G. mellonella. A similar suggestion was made for other lepidopterans Spodoptera exigua (Hu¨ bner) (Adamski et al. 2003) and Bombyx mori (L.) (Nath 2000) exposed to liposoluble insecticide fenitrothion, altering various metabolic activities in fat body. Evidence exists that the toxicity of OPs is connected with their lipophilicity, which enables them to alter cellular lipid proÞle and thus to initiate physicochemical changes leading to metabolic disruption in insects (Cunningham et al. 2002) Alterations in activities of the enzyme systems and lipid peroxidation level also might be attributed to nutritional impairment of the insecticides in the diet. This may be reasonable suggestion because previous studies (Bu¨ yu¨ kgu¨ zel 2001a, b) in which most of the responses of an insect to nonnutritive dietary supplements occur within a nutritional context. The activities of ALT and AST and MDA content of G. mellonella larvae do not show regular correlation with graded levels of the insecticides. As previously suggested by Bu¨ yu¨ kgu¨ zel and I´ ç en (2004), for some antimicrobial insecticides, the signiÞcance of our Þnding is that the irregular effects of the OPs might be dependent on their dietary interactions. In artiÞcial rearing, the diets are not only food source but also they are an environment for the larvae (Grenier et al. 1986). Availability and quality of larval food affect the physiological process of some pyralid larvae, which are very sensitive to changes in microhabitats such as temperature, humidity, and nutritional value of artiÞcial diets (Bell 1975). MP and EP that are given orally decreased AChE activity in G. mellonella larvae. AChE is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine in the nervous system. OPs inhibit AChE by phosphorylating a serine hydroxyl group within the enzyme active site. These insecticides may lead to disruption of synaptic transmission by inhibiting the enzyme. The decline in AChE activity of the insect may have led to hyperexcitability of synaptic transmission, ultimately affecting olfactory functions of the insect. Such a suggestion has been advanced to explain these synaptic depression occurring in lepidopterous pests such as B. mori (Nath and Kumar 1999); tobacco budworm, Heliothis virescens (F.); and corn earworm, Helicoverpa zea (Boddie) (Hamadain and Chambers 2001); and some other pest insects (Theophilis 1997, Barata et al. 2001, Belden and Lydy 2001) on exposure to MP and EP. Tox-

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icity of MP and EP might be attributed to their enhanced oxidative activation by oxygenase enzymes into their intermediate metabolites with increased anticholinesterase activity, as suggested for toxicity of some organophosphate insecticides in a dipteran (Anderson and Zhu 2004). Alterations in AChE activity after exposure of sublethal doses of these insecticides indicate that potential use of this enzyme in the greater wax moth as biomarker for evaluating organophosphorus contamination. Continuous feeding of neonate larvae on diets with MP or EP until last larval stage for a 10-d exposure caused dramatic decrease in AChE activity compared with the insecticides injected singly to mature larvae. Moreover, EP at all dietary levels caused greater decrease in AChE activity than the same levels of MP in the diets. Dietary exposure of both insecticides caused dose-dependent inhibition of AChE activity. It also seems that effects of the insecticides vary with insecticide used and exposure route. The dramatic inhibition of AChE by dietary levels of EP might be due to differences in their intermediate metabolism. Barata et al. (2001) demonstrated that ethyl parathion, which is highly toxic insecticide, produces potent cholinesterase-inhibiting intermediate products such as aminoparaoxon and aminoparathion. AChE activity was not signiÞcantly inhibited after intraheamocoelic injection of low MP levels. This may reßect reactivation and suggests a rapid clearance of the insecticides or their active intermediate metabolites. The enzyme activity may have been recovered within 48 h after single injection of the insecticides to mature larvae. This suggestion is mostly supported with results of a study that AChE activity may be recovered after single injection of MP at low doses compared with repeated exposure (Zhu et al. 2001). The present work suggests that MP and EP caused lipid peroxidation, leading to oxidative stress in the larvae. Lipid peroxidation propagates free radicals and leads to the release of a variety of toxic products, mainly MDA (Felton 1995). The aldehydic product MDA reacts with amino groups on proteins and other biomolecules, impairing cellular functions including nucleotide, protein synthesis, and enzyme activity. Increased MDA level has been identiÞed as an important indicator of oxidative reaction in a lepidopteran pests (Ahmad et al. 1995) and various insect species (Singh et al. 2001, Cervera et al. 2003) exposed to some insecticides. The considerably higher content of MDA in larvae of G. mellonella exposed to high levels of MP and EP also may be a result of insufÞciency of antioxidative protection. Antioxidant activity decreased with increasing level of lipid peroxidation in some vertebrates (Koç ak-Toker et al. 1993). Decreased MDA content in the larvae exposed to low dietary levels of MP and EP might be a result of its transformation into various ßuorescent biomolecules, such as lipofuscin, regarded as an indicator of tolerance to long-term oxidative stress. This suggestion also was made in the case of decreasing MDA content in some pest insects exposed with various environmental pollutants (Sheldahl and Tappel 1974).

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Free radicals generated by xenobiotics also may be important intermediates in enzymatic peroxidation of polyunsaturated fatty acids, especially arachidonic acid (Alric et al. 2000). Enzimatic oxidation of this fatty acid produces various free radical intermediates and its hydroperoxide metabolites. These free radicals may provide biosynthesis of prostaglandins from arachidonic acid released from membranes by means of phospholipase activation as inßammatory responses. Some oxygenated metabolites, prostaglandins, are known to be biosynthesized from arachidonic acid in different stages of various lepidopterans (Bu¨ yu¨ kgu¨ zel et al. 2002). Peroxidation of arachidonic acid induced by OP toxicity in the larvae may have produced excess MDA and free radicals affecting a number of cellular enzymes. Microsomal and cytosolic ALT and AST activities are present in nervous tissue from invertebrates, including various insect species, to vertebrates (Sugden and Newsholme 1975). The fact that some inhibitor of arachidonic acid metabolism caused signiÞcant decrease in content of MDA and ALT and AST activities by reducing the incidence of tissue lesions (Wea-Lung et al. 2000) supports that a relation exist between peroxidation of polyunsaturated fatty acids and tissue damage. It is reasonable to suggest that MP and EP or their oxidized metabolites might act as a pro-oxidant in G. mellonella, leading to generation free radicals that damage cellular polyunsaturated fatty acids, and possibly other structural and functional proteins. Biochemical responses of G. mellonella larvae to OPs vary with insecticide used and exposure route. Alterations in the activities of the enzymes ALT, AST, and AChE and the lipid peroxidation product MDA content in the larvae of G. mellonella provided strong evidence for the involvement of pesticidal contamination in the biochemical changes in insects. Sublethal levels of the organophosphorus insecticides impair some enzyme systems and induce lipid peroxidation, suggesting that these levels are enough to produce oxidative stress in the insect. It is possible for the nutritional quality of the host insect to be affected by these impairments. This was mostly supported by suggestion of Thompson and Lee (1993) in which alterations in metabolism are of nutritional signiÞcance in lepidopteran host larvae parasitized by parasitoids. Investigations are in progress to determine biochemical responses of parasitoids emerged from G. mellonella pupae contaminated by these organophosphorus insecticides.

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