ISSN 0013-8738, Entomological Review, 2012, Vol. 92, No. 1, pp.??????. Pleiades Publishing, Inc., 2012. Original Russian Text I.M. Dubovskiy, N.D. Slyamova, V.Yu. Kryukov, O.N. Yaroslavtseva, M.V. Levchenko, A.B. Belgibaeva, A. Adilkhankyzy, V.V. Glupov, 2011, published in Zoologicheskii Zhurnal, 2011, Vol. 90, No. 12, pp. 1360 1364. The Activity of Nonspecific Esterases and Glutathione-S- Transferase in Locusta migratoria Larvae Infected with the Fungus Metarhizium anisopliae (Ascomycota, Hypocreales) I. M. Dubovskiy a, N. D. Slyamova b, V. Yu. Kryukov a, O. N. Yaroslavtseva a, M. V. Levchenko c, A. B. Belgibaeva b, A. Adilkhankyzy b, and V. V. Glupov a a Institute of Animal Systematics and Ecology, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630091 Russia b Research Institute of Plant Protection, Rakhat, Karasai District, Almaty, 040924 Kazakhstan c All-Russian Institute of Plant Protection, Russian Academy of Agricultural Sciences, Pushkin, St. Petersburg, 196608 Russia e-mail: dubovskiy2000@yahoo.com Received January 26, 2011 Abstract The activity of nonspecific esterases and glutathione-s-transferase in whole body homogenates, hemolymph plasma, and fat body of the larvae of the locust Locusta migratoria was analyzed during development of infection with the fungus Metarhizium anisopliae. The lethal dose of the fungus (LC 80 ) was found to enhance the activity of detoxifying enzymes in the whole body homogenate of the larvae on the 3rd day after infection. The activity of nonspecific esterases and glutathione-s-transferase in the plasma and fat body of the infected larvae increased on the 3rd day but dropped to the control levels by the 6th day, during the acute period of infection. The detoxifying enzymes may participate in defense reactions at the early stage of the acute fungal infection. DOI: 10.1134/S0013873807090199 The process of infection of insects with entomopathogenic fungi starts with adhesion and germination of conidia on the cuticle surface, the thickness, structure, and chemical composition of the cuticle being of great importance for the progress of mycosis and development of resistance in insects (Leger et al., 1988; Glupov, 2001). Besides the cuticular barrier, the mechanisms of insect resistance to entomopathogenic fungi include some systems aimed at pathogen elimination and destruction of the toxic products of fungal metabolism (Glupov, 2001; Serebrov et al., 2001). In studying resistance of insects to entomopathogenic fungi, a considerable role of mechanisms directed at detoxication of the fungal metabolites during mycoses was demonstrated (Serebrov et al., 2001, 2003, 2006). Activation of detoxifying systems in infected insects may be related to the fact that entomopathogenic fungi possess a wide range of metabolites participating in the infection process, intoxication of the host organism being typical of mycoses (Hajek and Leger, 1994; James et al., 1994; Vilcinskas et al., 1999; Charnley, 2003). The main enzyme systems in insects participating in detoxication of various xenobiotics are monooxygenases, esterases, and glutathione-s-transferase (GST) (Li et al., 2007). Nonspecific esterases perform important functions in the insect organism: they perform catabolism of esters of higher fatty acids that proceeds actively in the flight muscles and enables insects to fly, mobilization of lipids, including those of the fat body (Roslavtseva et al., 1993), and degradation of inert metabolic esters, including various xenobiotics (Terriere, 1984). A wide substrate specificity of esterases testifies to their exceptional role in the degradation of toxins of different origins. The researchers interest in insect GST is first of all associated with participation of these enzymes in insecticide degradation. GST activity has been found to increase in insects resistant to insecticides (Papadopoulos et al., 2000). Besides degradation of xenobiotics, GST takes part in metabolite removal and protection of tissues from damage by free radicals (Kolesnichenko and Kulinskii, 1969; Bakanova et al., 1992).???
2 DUBOVSKIY et al. Nonspecific esterases and GST were found to participate in metabolism and detoxication of organophosphates, pyrethroids, carbamates, and juvenoids (Small and Hemingway, 2000; Pasteur et al., 2001). An increased expression of genes of detoxifying enzymes responsible for resistance to different xenobiotics was shown in insects of various orders, such as Hemiptera: the tarnished plant bug Lygus lineolaris Pal. De Beauv. and the peach aphid Myzodes persicae Sulz., Hymenoptera: the parasitoid wasp Habrobracon hebetor Say., Lepidoptera: the striped stem borer Chilo suppressalis Walker, and Diptera: the horn fly Haematobia irritans L. and the northern house mosquito Culex pipiens L. (Field and Devonshire, 1998; Hemingway et al., 1998; Field, 2000; Hawkes and Hemingway, 2002). The function of degradation of toxic molecules with esterases and GST during the progress of infection may play one of the key roles in protecting insects from pathogens. Induction of new isoforms of nonspecific esterases and a change in their activity in different organs were demonstrated in larvae of the greater wax moth Galleria mellonella and the silkworm Bombyx mori L. infected with microsporidia, bacteria, or fungi (Shiotsuki and Kato, 1999; Serebrov et al., 2001; Vorontsova et al., 2006). Use of synthetic inhibitors of the detoxifying system reduced resistance of G. mellonella to entomopathogenic fungi (Serebrov et al., 2003, 2006). Practically no studies of detoxifying enzymes of grasshoppers during mycosis were made (Xia et al., 2000). The goal of the present work is the study of activity of nonspecific esterases and GST in whole body homogenates, hemolymph plasma, and fat body of the larvae of the migratory locust Locusta migratoria during the progress of infection with the fungus Metarhizium anisopliae. MATERIALS AND METHODS Larvae of the migratory locust Locusta migratoria were collected in the nature in rice paddies near Bakanas (Kazakhstan) and kept in the laboratory at 12 h of light per day, on a diet of the common reed grass Phragmites communis Trin. The experiments were carried out with young (II III instar) and old (IV V instar) larvae. After infection with the fungus, activity of nonspecific esterases and GST was assessed in the whole body homogenate of young instar larvae and in the hemolymph plasma and fat body of old instar larvae. The entomopathogenic fungus Metarhizium anisopliae (Metsch.) Sorokin, strain P-72, was used for experimental infestation. The insects were infested by single submerging into water suspension of the conidia (titer 1 10 7 ). The whole body and the fat body homogenates were prepared in 0.1 M Na-phosphate buffer (PB) with ph of 7.2. One replication included 5 larvae. Insects and extracted organs were ground in a glass homogenizer containing cold PB (0.06 g of tissues per 1 ml PB). Then the homogenates were centrifuged at 4 C for 15 min at 10 000 RCF. The supernatant was used to determine the enzyme activity and protein concentration. The hemolymph was sampled with a glass capillary through an incision in the cuticle and placed in cooled tubes, to which 4 mg/ml phenylthiourea was added to prevent melanization. The hemolymph was centrifuged at 4 C for 5 min at 500 RCF, and the cell-free plasma fraction was used to determine the enzyme activity and protein concentration. Esterase activity in the samples was determined spectrophotometrically by Asperen s method (1962) with minor modifications. The incubation mixture contained 1 ml of 0.54 mm 1-naphthylacetate in PB and 20 μl of the sample. The concentration of 1- naphthyl produced during the reaction was measured spectrophotometrically at the wave length of 550 nm. GST activity was determined with respect to 2-nitro-5-chlorobenzoic acid (DNCB) by Habig s method (Habig et al., 1974). Incubation was carried out at 25 C for 5 min in 0.1 M Na-phosphate buffer (ph 6.5) containing 1 mm glutathione, 1 mm DNCB, and 20 μl of the sample. The reaction was initiated by adding DNCB solution in acetone. Concentration of 5-(2,4-dinitrophenyl) glutathione produced during the reaction was measured spectrophotometrically at the wave length of 340 nm. The specific activity of nonspecific esterases and GST was expressed in terms of change in the optical density (ΔA) of the incubation mixture per 1 min and 1 mg of protein. Pprotein concentration in the samples was determined by Bradford s method (1976), with bovine serum albumin used to build a calibration curve. The data obtained are presented as the mean and standard error (SE). The normality of the data was assessed using the Shapiro-Wilk W test. The statistical significance of the differences was determined by Student s t test within the STATISTICA 6.0 software.
THE ACTIVITY OF NONSPECIFIC ESTERASES 3 Fig. 1. The mortality dynamics of young and old instar larvae of the migratory locust Locusta migratoria infected with the entomopathogenic fungus M. anisopliae: K1, intact young instar larvae; Ma1, infected young instar larvae; K2, intact old instar larvae; Ma2, infected old instar larvae (* p < 0.05 as compared to the control). Fig. 2. Activity of nonspecific esterases (Est) and glutathione-stransferase (GST) in the whole body homogenates of intact young instar larvae of the migratory locust Locusta migratoria (K) and young instar larvae infected with the entomopathogenic fungus M. anisopliae (Ma) (n = 10, ** p < 0.001 as compared with the control). Fig. 3. Activity of glutathione-3-transferase (GST) in the hemolymph plasma and the fat body of old instar larvae of the migratory locust Locusta migratoria at different stages of fungal infection: K, intact larvae; Ma, larvae infected with the fungus M. anisopliae (n = 20, ** p < 0.001 as compared with the control). RESULTS AND DISCUSSION Infestation of larvae of the migratory locust with the fungus M. anisopliae resulted in an infection process with the overall mortality of 81.5 ± 2.7% of young instar larvae and 87.1 ± 8.3% of old instar larvae by the 6 7th day (Fig. 1). By the 3rd day of the progress of the disease, mortality in both groups of infested insects was 10 15% and did not significantly differ from the control (Fig. 1). The first 3 days may therefore be regarded as the initial period of infection. The general dynamics of mortality indicated acute fungal pathogenesis. The action of the fungus was found to be accompanied by activation of detoxicating enzymes on the 3rd day of the progress of infection. Analysis of nonspecific esterase and GST activity in the whole body homogenates of young instar larvae revealed a significant (p 0.001) two-fold increase in esterase activity on the 3rd day after infestation (Fig. 2). A 2.5-fold (p 0.001) increase in GST activity was also noted (Fig. 2). Old instar larvae infected with M. anisopliae showed a significant increase in nonspecific esterase and GST activity in the hemolymph plasma (by 1.7 and 2 times, respectively; p 0.001), whereas the nonspecific esterase activity in the fat body grew 1.3-fold, and that of GST, 1.4-fold (p 0.001) (Figs. 3, 4). Activation of the components of the detoxifying system at the initial stage of acute fungal infection (LC 80 ) may testify to the participation of nonspecific esterases and GST in defense reactions of insects directed at destruction of the toxins produced by entomopathogenic fungi. Similar results were obtained earlier from the study of the role of nonspecific esterases during mycoses in larvae of the greater wax moth Galleria mellonella. In particular, infestation of insects with entomopathogenic fungi was found to be accompanied by an abrupt increase in activity of nonspecific esterases and GST in the hemolymph plasma. The activity of nonspecific esterases increases due to induction of additional isoenzymes (Serebrov et al., 2001). Also, in studying the activity of acid phosphatases during mycosis in the desert locust Schistocerca gregaria Forsk., an increase in enzyme activity in the hemolymph was recorded (Xia et al., 2000). The main factor of increasing activity of detoxifying enzymes during mycoses may be mechanical damage to the insect cuticle by the hyphae as they penetrate into the host organism and the action of fungal toxins re-
4 DUBOVSKIY et al. leased into the hemocoel (Xia et al., 2000; Serebrov et al., 2001, 2006). Thus, the observed increase in activity of nonspecific esterases and GST suggests that the activity of detoxicating enzymes of the migratory locust larvae may be directed at elimination of fungal metabolites and toxic substances formed in the course of penetration of the entomopathogenic fungus into the hemocoel. By the 6th day of mycosis, during the acute period, activity of the enzymes was observed to fall to the control values; in particular, reduction in nonspecific esterase and GST activity was recorded in the whole body homogenates of young instar larvae (Fig. 2). Analysis of enzyme activity in old instar larvae showed reduction in GST activity down to the control values in the plasma and the fat body (Fig. 3), and also that of esterases in the fat body on the 6th day after infestation (Fig. 4). A significant (p 0.001) 1.6-fold decrease in esterase activity was observed in the plasma of infected insects (Fig. 4). Reduction in esterase and GST activity during the acute period of mycosis may be associated with effective inhibition of the host defense systems by entomopathogenic fungi. This suggestion is supported by studies of defense reactions of the desert locusts during the progress of M. anisopliae mycosis. In particular, an abrupt reduction in activity of phenoloxidases, antibacterial activity, and the total number of hemocytes was recorded in infected insects (Gillespie et al., 2000). Based on the data obtained, it can be suggested that the detoxicating system of grasshoppers participates in the defense reactions against entomopathogenic fungi. One of the modern biotechnological methods is search for ways of blocking or suppressing activity of insect defense systems, which would increase their susceptibility to entomopathogens used for biocontrol purposes. Our results indicate that at the initial stages of mycosis, nonspecific esterase and GST activity in larvae of L. migratoria may be directed at detoxication of metabolites and toxins of entomopathogenic fungi. The use of mechanisms affecting the detoxicating system of the insect (secondary plant metabolites, synthetic inhibitors, etc.) may reduce resistance of grasshoppers to entomopathogens, in particular, to fungi. ACKNOWLEDGMENTS The work was financially supported by the Russian Foundation for Basic Research (grant no. 09-04- Fig. 4. Activity of nonspecific esterases (Est) in the hemolymph plasma and the fat body of old instar larvae of the migratory locust Locusta migratoria at different stages of fungal infection: K, intact larvae; Ma, larvae infected with the fungus M. anisopliae (n = 20, ** p < 0.001 as compared with the control). 00380), the Integration grant from the Siberian Branch of the Russian Academy of Sciences, the Russian Presidential grant, and the grant from Scientific Council of Ministry for Agriculture of the Republic of Kazakhstan. REFERENCES 1. Asperen, K., Van, A Study of Housefly Esterase by Means of a Sensitive Colorimetric Method, J. Insect Physiol. 8, 401 416 (1962). 2. Bakanova, E.I., Eremina, O.Yu., and Roslavtseva, S.A., The Properties and Functions of Glutathione-S- Transferase in Arthropods, Izv. RAN Ser. Biol., No. 4, 537 545 (1992). 3. Bradford, M.M., A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem. 72, 248 254 (1976). 4. Charnley, A.K., Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins, Adv. Botan. Res. 40, 241 321 (2003). 5. Field, L.M., Methylation and Expression of Amplified Esterase Genes in the Aphid Myzus persicae (Sulzer), Biochem. J. 349, 863 868 (2000). 6. Field, L.M. and Devonshire, A.L., Evidence that the E4 and FE4 Esterase Genes Responsible for Insecticide Resistance in the Aphid Myzus persicae (Sulzer) are Part of a Gene Family, Biochem. J. 330, 169 173 (1998). 7. Gillespie, J.P., Burnett, C., and Charnley, A.K., The Immune Response of the Desert Locust Schistocerca gregaria during Mycosis of the Entomopathogenic Fungus Metarhizium anisopliae var acridum, J. Insect Physiol. 46, 429 437 (2000). 8. Glupov, V.V., Insect Pathogens: the Structural and Functional Aspects (Kruglyi God, Moscow, 2001) [in Russian].
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