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Effect of Fenvalerate-20EC on Sericigenous Insects
Ecotoxicology and Environmental Safety 53, 212d220 (2002) Environmental Research, Section B doi:10.1006/eesa.2002.2229
Effect of Fenvalerate-20EC on Sericigenous Insects II. Digestive Enzymes in the Nutritive Physiology of Silkworm, Bombyx Mori L. N. Vyjayanthi and M. V. V. Subramanyam Department of Studies in Sericulture, Bangalore University, Bangalore 560 056, India Received April 24, 2001; published online July 25, 2002
Changes in the activities of a few digestive enzymes after fenvalerate-20EC treatment were studied during the late stages of multivoltine and bivoltine silkworms, Bombyx mori. Insecticide treatment induced a reduction in the activity of such enzymes as amylase, sucrase, and protease, whereas the trehalase activity was enhanced when the midgut was used as the source of enzyme. The changes recorded in the activity of these enzymes indicated probable adverse e4ects of fenvalerate on silkworm metabolism through interference with the functional abilities of digestive enzymes. 2002 Elsevier Science (USA) Key Words: Bombyx mori; digestive enzymes; toxicity; pyrethroids; altered metabolism.
INTRODUCTION
Silkworm, Bombyx mori, midgut digestive enzymes have been studied in detail (Kanekatsu, 1972, 1978; Eguchi and Iwamoto, 1976; Sumida et al., 1994; Abraham et al., 1992). Rationalization of some of these enzymes is a feature of the silkworm (Kanekatsu et al., 1989). Midgut enzyme activity is also a developmental stage dependent (Kanekatsu et al., 1993; Sumida et al., 1990), and the diapause nature has relevance to enzymatic activities in the midgut of B. mori (Asakawa and Hamano, 1994). These midgut enzymes also show a di!erential activity in relation to voltinism (Eguchi et al., 1982; Sarangi, 1985). The toxic e!ects of biologically active chemicals like insecticides are known to induce many biochemical and physiological changes in insects (Egaas et al., 1992; Silver et al., 1995; Nath et al., 1997; Nath, 2000). Pyrethroids are known to induce an irritation response in insects (Tan, 1981), with increased locomotor activity, rotation behaviour and abdominal tucking, reduction in feeding (Armstrong and Bonner, 1985), reduced body weight, and refusal of food (Kuribayashi, 1988). Armstrong and Bonner (1985) reported To whom correspondence should be addressed. E-mail: asuba@blr. vsnl.net.in.
that in Drosophila melanogaster, the antifeeding e!ects of pyrethroids were observed to occur with very low doses of chemicals and may be an indirect result of an irritation response, rather than any direct in#uence on the feeding mechanism. They also suggested that the nonspeci"c nature of the response indicates that very low levels of pyrethroids possibly a!ect a diverse range of biological functions and not just feeding. Though the primary site of action of pyrethroids in insects is the nervous system (O'Brien, 1967) these agents can cause side e!ects exhibited as various physiological and biochemical changes (Saleem and Shakoori, 1993). Reports from our laboratory have shown that fenvalerate, a pyrethroid insecticide, interferes with silkworm feeding behavior and physiology of digestion, and the magnitude of changes increased with an increase in insecticide concentration (Vyjayanthi and Subramanyam, 2002). Thus we have further attempted to examine the potential role of fenvalerate in digestive physiology. In this regard, we believe that the changes in digestion, assimilation, and conversion of food on insecticide treatment may be due to the changes in various digestive enzyme activities, which take part in active digestive and assimilative process of the silkworm. Thus, the aim of this study was to determine if pyrethroids a!ect di!erent digestive enzymes isolated from midgut. MATERIALS AND METHODS
Insects. Disease-free layings of B. mori (Pure-Mysore and NB D varieties) were obtained from Government Grainage and freshly hatched larvae were reared on the leaves of Morus alba (M variety) with standard rearing techniques and optimum conditions (Krishnaswami, 1978). Insecticide: Preparation of stock solution. A Technicalgrade sample of Sumicidine-20EC (Fenvalerate-20EC: cyano(3-phenoxyphenyl)methyl 4-chloro-x-(1-methyl)-benzene acetate, brown liquid) was obtained from Rallis India Ltd. A stock solution of 1% strength was prepared with
212 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
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EFFECT OF FENVALERATE ON SILKWORM METABOLISM
TABLE 1 E4ect of Fenvalerate on Amylase Activity in the Hemolymph of Fourth-Instar Silkworm, Bombyx mori L. Amylase activity [(mg glucose/min/mg protein$SD);10\] Bivoltine Development (days) 1 2 3 4
Control
LC
0.549$0.021 0.732$0.029 1.11$0.04 1.0$0.05
0.512$0.025 0.701$0.03 0.93$0.04* 0.82$0.032*
Multivoltine LC
0.480$0.018** 0.514$0.020** 0.58$0.027** 0.50$0.031**
Control
LC
LC
1.02$0.024 1.62$0.0382 2.04$0.1 1.78$0.062
0.8$0.015** 1.2$0.04** 1.48$0.05** 1.19$0.08**
0.41$0.0114*** 0.69$0.0121*** 1.07$0.05** 0.723$0.044**
Note. Values are means$SD, Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
acetone and subsequent dilutions were made with distilled water (Burch"eld et al., 1952; Radhakrishna and Delvi, 1987). The concentrations were calculated on the basis of the active ingredient present in the insecticide.
larvae of uniform size and age. To replace the dead and unequal larvae, a separate batch of worms were maintained under identical conditions. The insecticide was applied just after the "rst feeding in both age groups. A single uniform topical spray was applied to test individuals. Topical application of the insecticide was preferred because it is a method considered for toxicity tests (Kuwana et al., 1967; Ochi & Kiuchi, 1998).
Evaluation of lethal and sublethal concentrations. The silkworms maintained for the experiments were divided into batches of 50 worms each and treated with selected concentrations of fenvalerate. Further, the LC and LC of the insecticide, were determined by the probit analysis method (Finney, 1971). Brie#y, the mean percentage mortality and di!erent concentrations of insecticide used were plotted on the log-probit curve for each race and age. LC and LC values for each race and age were calculated from the curve. From the probit and percentage mortality curves the LC was found to be 8.62 and 9.50 ppm for fourth- and "fthinstar multivoltine worms and 8.11 and 7.50 ppm for fourthand "fth-instar bivoltine worms, respectively, and the LC was 4.00 and 3.76 ppm for multivoltine and bivoltine fourthinstar worms and 4.80 and 4.40 ppm for multivoltine and bivoltine "fth-instar respectively.
Analyses: Preparation of tissue extracts for enzyme assays. For the evaluation of digestive enzyme assays, the whole midgut was excised from prefrozen larvae kept at !203C for 12 h and a 10% homogenate was prepared (after separating the malpighian tubules, fat bodies, and other tissue fragments adhering to the gut) in ice-cold bu!er solution. The homogenate was centrifuged at 3000 rpm and the supernatant was used as the enzyme source with appropriate dilution. Enzyme assay. Amylase activity was evaluated by the Bernfeld (1955) method using glucose as standard. The reaction mixture contained 50 mM phosphate bu!er (pH 6.5), 1% starch (freshly prepared), and appropriately diluted enzyme. Sucrase activity was measured according to the method of Ishaaya and Swirski (1970) with glucose as
¹reatment. Immediately after the third and fourth molts, larvae were divided into three batches with six replicates in each batch. Each replicate consisted of 50 healthy
TABLE 2 E4ect of Fenvalerate on Sucrase Activity in the Whole Midgut of Fourth-Instar Silkworm, Bombyx mori L. Sucrase activity [(mg glucose/min/mg protein);10\$SD] Bivoltine Development (days) 1 2 3 4
Control 6.6$0.145 7.26$0.057 8.07$0.092 8.00$012
LC 6.43$0.16 6.62$0.098** 6.75$0.098** 6.51$0.091**
Multivoltine LC
6.02$0.162** 3.92$0.099*** 4.43$0.091*** 4.40$0.087***
Control 7.02$0.162 7.96$0.10 8.66$0.11 8.21$0.15
Note. Values are means$SD. Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
LC 6.21$0.11* 6.82$0.125** 7.45$0.10** 7.14$0.14**
LC
5.13$0.098*** 6.00$0.14** 5.38$0.092*** 5.22$0.12***
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TABLE 3 E4ect of Fenvalerate on Trehalase Activity in the Whole Midgut of Fourth-Instar Silkworm, Bombyx mori L. Trehalase activity [(mg glucose/min/mg protein);10\$SD] Bivoltine Development (days) 1 2 3 4
Control
LC
1.35$0.01 1.92$0.10 2.2$0.12 2.01$0.08
2.05$0.08* 2.80$0.12** 3.33$0.11** 2.98$0.091*
Multivoltine LC
2.92$0.108** 4.7$0.16*** 5.8$0.14*** 5.0$0.11***
Control
LC
2.115$0.121 2.54$0.09 2.96$0.079 2.80$0.125
3.24$0.089* 3.82$0.14* 4.14$0.010** 3.51$0.21*
LC
3.62$0.112* 4.02$0.201** 4.98$0.13** 3.75$0.159*
Note. Values are means$S.D. Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
standard. The incubation mixture contained 50 mM phosphate bu!er (pH 6.5), 3.42 M sucrose, and appropriately diluted enzyme. Trehalase activity was determined by the Dahlman (1971) method with slight modi"cation of pH from 5.6 to 6.0. The assay mixture contained 50 mM phosphate bu!er (pH 6.0), 3.78 M trehalose, and appropriately diluted enzyme. The incubation period and temperature of incubation were 30 min and 24$13C for amylase, and 60 min and 373C for sucrase and trehalase. The amount of glucose liberated was measured at 540 nm after inhibition of the reaction with dinitrosalicylic acid (DNS) reagent in the cases of amylase and sucrase and with concentrated H SO in the trehalase assay. The mixture was boiled over a boiling water bath for 10 min and diluted with distilled water. Activity is expressed as milligrams of glucose liberated per minute milligram of mg protein in all three estimations. The protease enzyme assay was carried out with the method of Eguchi and Iwamoto (1982) with slight modi"cation of the pH of borate bu!er (pH 11.0) as outlined by Sarangi (1985) using tyrosine as standard. The reaction mixture contained 1% casein, 0.1 M borate bu!er (pH 11.0), and appropriately diluted enzyme. The incubation was carried out for 30 min at 303C. The reaction was inhibited by adding 8.2 M trichloroacetic acid (TCA) and centrifuged. The supernatant was
used with 0.5 N NaOH and Folin's reagent to measure the tyrosine liberated at 660 nm. Protein content in all assays was estimated with the Folin phenol reagent method (Lowry et al., 1951) using bovine serum albumin as standard. Statistical analysis. All data collected were statistically analyzed with Student's t test and the results are expressed as means$SD. The level of signi"cance were expressed as P values (Panse and Sukhatme, 1989). RESULTS
Activity levels of the digestive enzymes amylase, sucrase, trehalase, and protease in normal and treated multivoltine and bivoltine silkworms are presented in Tables 1}4 and Figs. 1}8 for the last two instars. In fourth instar, midgut enzymes increased steadily until a day prior to molting, whereas during "fth instar, activity increased similarly up to the fourth to "fth day and declined thereafter. These enzymes were at higher levels in the last instar in both races. Fenvalerate treatment induced a signi"cant reduction in the activity of amylase, sucrase, and protease, but signi"cantly enhanced the activity of trehalase in both age groups and also in both races. Further, the magnitude of the
TABLE 4 E4ect of Fenvalerate on Protease Activity in the Whole Midgut of Fourth-Instar Bivoltine and Multivoltine Silkworm, Bombyx mori L. Protease activity [(mole tyrosine/min/mg protein);10\$SD] Bivoltine Development (days) 1 2 3 4
Control 10.55$0.08 13.02$0.262 16.3$0.33 16.02$0.23
LC 8.19$0.24* 9.29$0.193** 11.4$0.12** 11.0$0.094**
Multivoltine LC
Control
7.77$0.11** 8.5$0.24*** 9.52$0.08*** 8.91$0.095***
8.89$0.21 10.71$0.226 13.0$0.19 12.72$0.148
Note. Values are means$SD. Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
LC 8.94$0.195 9.02$0.121* 10.6$0.07** 10.1$0.11***
LC
6.92$0.112* 7.19$0.09*** 8.8$0.069*** 8.28$0.051**
EFFECT OF FENVALERATE ON SILKWORM METABOLISM
215
FIG. 1. E!ect of fenvalerate-20EC on amylase activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
changes in enzyme activity was found to depend on the concentration of insecticide used. However, age-dependent activity patterns were not in#uenced by pyrethroid application. The fenvalerate-induced changes in various enzyme activities were much more pronounced in bivoltine worms. DISCUSSION
The process of digestion and the activity of enzymes depend on the quantity and quality of food ingested, age and health of the larva, and few physical factors (Swingle, 1930; Kanekatsu et al., 1989, 1993). Toxic chemicals cause
disturbances in the normal biochemical physiological activities of an insect (Khan, 1993; Bhosale et al., 1988). In the present investigation, it was observed that silkworms treated with fenvalerate had few such responses such as food avoidance and rapid movements of the head and thoracic region at low concentrations, but higher concentrations induced a slow death, before which rectal protrusion occurred (Vyjayanthi and Subramanyam, 2002). As poor nutrition and low-nutrient diets have direct e!ects on primary biochemical and physiological systems, and thus may decrease the performance of insects by e!ecting changes in the detoxi"cation system that can alter the susceptibility of the
FIG. 2. E!ect of fenvalerate-20EC on amylase activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
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FIG. 3. E!ect of fenvalerate-20EC on sucrase activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
insect (Lindroth et al., 1991), the poor feeding behavior in our earlier experiments may be correlated with the alteration in digestive enzyme activity on insecticide treatment. Activity of the enzymes amylase, sucrase, and protease was decreased, which may be due to the insu$cient amount of
substrate resulting from food rejection. Sumida et al. (1990) reported that midgut sucrase is activated by sucrose at a higher concentration ('100 mM) derived from the ingested food in the midgut lumen. The rational food consumption by a lepidotpteran larva was correlated directly with
FIG. 4. E!ect of fenvalerate-20EC on sucrase activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
EFFECT OF FENVALERATE ON SILKWORM METABOLISM
217
FIG. 5. E!ect of fenvalerate-20EC on trehalase activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
the activities of amylase and invertase by Christopher and Mathavan (1985), with the larva receiving 100% food found to have the highest amylase and invertase activites, which
declined as the percentage of food o!ered was reduced. Similarly, the poor feeding rate on fenvalerate treatment (Vyjayanthi and Subramanyam, 2002) could have resulted
FIG. 6. E!ect of fenvalerate-20EC on trehalase activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
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FIG. 7. E!ect of fenvalerate-20EC on protease activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8; Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
in the reduced activity of amylase and sucrase in our present study. Proteases are the proteolytic enzymes that aid in digestion of leaf proteins. Late silkworms are generally coarse leaf feeders, and are suppose to have a highly speci"c protease enzyme system that hydrolyzes the "brous protein found in abundance in coarse mulberry leaves (Ito and Arai, 1966). The proteolytic activity of the alimentary canal in relation to feeding or proteins has been studied in many
insects (Dadd, 1956; Hamano and Mukaiyama, 1970). Changes in protein metabolism have been reported in silkworms treated with organophosphate insecticide (Nath et al., 1997). In the present study, protease activity decreased on insecticide treatment, and it is presumed that the insecticide may even have inhibitory action over the enzyme molecules to act on their substrates, or the enzyme molecules may be devoid of a su$cient amount of substrate. The decline in
FIG 8. E!ect of fenvalerate-20EC on protease activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
EFFECT OF FENVALERATE ON SILKWORM METABOLISM
219
protease activity may also be due to starvation, which is a result of the food rejection behavior on fenvalerate treatment. Protease activity is in#uenced by the age, sex and feeding behaviour of silkworms and decreases signi"cantly on starvation during late "fth instar (Jadhav and Kallapur, 1988). The observations of the present investigation can thus be correlated with feeding behavior and the quantity of food ingested by the silkworm for the active participation of these enzymes in the process of digestion. Trehalase activity to the contrary, was enhanced in the midgut of silkworms treated with fenvalerate. Azuma and Yamashita (1985) reported such an increase in midgut trehalase activity serves for the utilization of hemolymph trehalose for metabolic energy to maintain active processes in various situations. The enhanced trehalase activity may also be due to increased hydrolysis of trehalose to release two glucose moieties, which plays an important role in silkworm carbohydrate metabolism (Hasegawa and Yamashita, 1970). The hyperactivity (irritation response) and stress caused by pyrethroid poisoning in silkworms demands energy that might have been supplied in the form of glucose molecules by the hydrolysis of trehalose in the midgut by trehalase. Similar disturbances in carbohydrate metabolism and related enzymes other than trehalase due to organophosphorous insecticide have been reported in B. mori (Nath, 2000). Though this reduction may be due to the lesser availability of nutrients, there may be some in#uence on enzyme structure and function, and the poison may act as an inhibitor for such enzymes with altered activity.
Bernfeld, P. (1955). Enzymes of carbohydrate metabolism: Amylases, and . In Methods in Enzymology (S. P. Colowick and N. O. Kaplan, Eds.), Vol. I, pp. 149}158. Academic Press, New York.
CONCLUSIONS
Hamano, K., and Mukaiyama, F. (1970). Some properties of digestive #uid proteases in the silkworm, Bombyx mori, with reference to the relation between dissociation degree and nutritive value of some proteins. J. Sericult. Sci. Japan. 39, 371}376.
Fenvalerate toxicity in B. mori interferes with major metabolic functions and checks the production and activity of digestive enzymes. The degree of e!ect depends on age, race, and concentration of the insecticide. The toxic e!ects of pyrethroids hinder normal digestive metabolism and thus growth of the insect, which further greatly in#uences silk production by the worms. REFERENCES Abraham, E. G., Nagaraju, J., and Datta, R. K. (1992). Chemical studies of amylases in the silkworm, Bombyx mori L.: Comparative analysis in diapause and nondiapause strains. Insect Biochem. Mol. Biol. 22, 867}873. Armstrong, K. E., and Bonner, A. B. (1985). Investigation of permethrininduced antifeedent e!ect on Drosophila melanogaster, an ethological approach. Pestic. Sci. 16, 641}650. Asakawa, H., and Hamano, K. (1994). Enzymatic properties of digestive amylase isozymes in silkworms, Bombyx mori L. J. Seric. Sci. Japan 63, 13}20. Azuma, M., and Yamashita, O. (1985). Cellular localization and proposed function of midgut trehalase in silkworm larva, Bombyx mori. ¹issue Cell. 17, 539}551.
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Khan, N. A. (1993). Activation of C-dimethoate in lepidopterous larvae. Comp. Biochem. Physiol. C 105, 63}68. Krishnaswami, S. (1978). New ¹echnology of Silkworm Rearing CSR and TI. Bull. No. 2. Dept. of Sericulture, Govt. of Karnataka, Bangalore. Kuribayashi, S. (1988) Damage of silkworms caused by pesticides and preventive measures. JARQ. 21, 274}283. Kuwana, Z., Nakamura, S., and Sugiyama, H. (1967). E!ects of insecticides on the silkworm larvae, Bombyx mori L. Analysis of the insecticide}silkworm larvae relationship by means of subcutaneous injection, topical and oral applications. Bull. Seric. Exp. Sta. 22, 123}180. Lindroth, R. L., Barman, M. A., and Weisbra, A. V. (1991). Nutritient de"ciencies and the gypsy moth, ¸. dispar: E!ects on larval performance and detoxi"cation enzyme activities. J. Insect Physiol. 37, 45}52. Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265}275. Nath, B. S. (2000). Changes in carbohydrate metabolism in haemolymph and fatbody of the silkworm, Bombyx mori L., exposed to organophosphorus insecticides. Pestic. Biochem. Physiol. 68, 127}137. Nath, B. S., Suresh, A., Varma, B. M., and Kumar, R. P. S. (1997). Changes in protein metabolism in hemolymph and fat body of the silkworm, Bombyx mori (lepidoptera: Bombycidae) in response to organophosphorous insecticides toxicity. Ecotoxicol. Environ. Saf. 36, 169}173. O'Brien, R. D. (1967). Insecticide Action and Metabolism. Academic Press, New York. Ochi, H., and Kiuchi, M. (1998). Application methods and toxic e!ects of an imidazole derivative, tri#umizole, in Bombyx mori. J. Seric. Sci. Japan 67, 303}309. Panse, V. G., and Sukhatme, P. V. (1989). Statistical Method for Agricultural =orkers (rev. by P. V. Sukhatme and V. N. Amble). ICAR, New Delhi.
Radhakrishna, P. G., and Delvi, M. R. (1987). E!ect of ration level food utilization in the silkworm, Bombyx mori. L. (Lepidoptera: Bombycidae). Sericologia 27, 347}354. Saleem, M. A., and Shakoori, A. R. (1993). E!ect of cypermethrin on the free aminoacids pool in an organophosphorous-insecticide-resistant and a susceptible strain of ¹ribolium castaneum. Comp. Biochem. Physiol. C 105, 549}553. Sarangi, S. K. (1985). Alkaline protease in the midgut of the silkworm, Bombyx mori L: Changes during metamorphosis. Proc. Indian Acad. Sci (Anim. Sci.) 94, 567}572. Silver, A. R. J., Emden, H. F. V., and Battersby, M. (1995). A biochemical mechanism of resistance to pirimicarb in two glasshouse clones of Aphis gossypii. Pestic. Sci. 43, 21}29. Sumida, M., and Yamashita, O. (1977). Trehalase transformation in silkworm midgut during metamorphosis. J. Comp. Physiol. 115, 241}253. Sumida, M., Yuan, X. L., and Matsubara, F. (1994). Sucrase activity and its kinetic properties in pertrophic membrans, and in membrane-bound and soluble fractions of midgut in silkworm, Bombyx mori. Comp. Biochem. Physiol. A 108, 255}264. Sumida, M., Yuan, X. L., Mari, Y. I. Mori, H., and Matsubara, F. (1990). Changes in kinetic parameters and total activity of midgut sucrase in the silkworm, Bombyx mori during larval pupal}adult development. Comp. Biochem. Physiol. B 96, 605}611. Swingle, M. C. (1930). Physiology of gut: Popillia. Col. J. Agric. Res. 41, 181}196. Tan, K. H. (1981). Antifeeding e!ect of cypermethrin and permethrin at sub-letahl levels against Pieries brassicae larva-1e. Pesticides 12, 619}626. Vyjayanthi, N., and Subramanyam, M. V. V. (2002). E!ect of fenvalerate20EC on sericigenous insects. I. Food utilization in the late-age larva of the silkworm, Bombyx mori L. Ecotoxicol. Ecol. Saf. 53.
Effect of Fenvalerate-20EC on Sericigenous Insects II. Digestive Enzymes in the Nutritive Physiology of Silkworm, Bombyx Mori L. N. Vyjayanthi and M. V. V. Subramanyam Department of Studies in Sericulture, Bangalore University, Bangalore 560 056, India Received April 24, 2001; published online July 25, 2002
Changes in the activities of a few digestive enzymes after fenvalerate-20EC treatment were studied during the late stages of multivoltine and bivoltine silkworms, Bombyx mori. Insecticide treatment induced a reduction in the activity of such enzymes as amylase, sucrase, and protease, whereas the trehalase activity was enhanced when the midgut was used as the source of enzyme. The changes recorded in the activity of these enzymes indicated probable adverse e4ects of fenvalerate on silkworm metabolism through interference with the functional abilities of digestive enzymes. 2002 Elsevier Science (USA) Key Words: Bombyx mori; digestive enzymes; toxicity; pyrethroids; altered metabolism.
INTRODUCTION
Silkworm, Bombyx mori, midgut digestive enzymes have been studied in detail (Kanekatsu, 1972, 1978; Eguchi and Iwamoto, 1976; Sumida et al., 1994; Abraham et al., 1992). Rationalization of some of these enzymes is a feature of the silkworm (Kanekatsu et al., 1989). Midgut enzyme activity is also a developmental stage dependent (Kanekatsu et al., 1993; Sumida et al., 1990), and the diapause nature has relevance to enzymatic activities in the midgut of B. mori (Asakawa and Hamano, 1994). These midgut enzymes also show a di!erential activity in relation to voltinism (Eguchi et al., 1982; Sarangi, 1985). The toxic e!ects of biologically active chemicals like insecticides are known to induce many biochemical and physiological changes in insects (Egaas et al., 1992; Silver et al., 1995; Nath et al., 1997; Nath, 2000). Pyrethroids are known to induce an irritation response in insects (Tan, 1981), with increased locomotor activity, rotation behaviour and abdominal tucking, reduction in feeding (Armstrong and Bonner, 1985), reduced body weight, and refusal of food (Kuribayashi, 1988). Armstrong and Bonner (1985) reported To whom correspondence should be addressed. E-mail: asuba@blr. vsnl.net.in.
that in Drosophila melanogaster, the antifeeding e!ects of pyrethroids were observed to occur with very low doses of chemicals and may be an indirect result of an irritation response, rather than any direct in#uence on the feeding mechanism. They also suggested that the nonspeci"c nature of the response indicates that very low levels of pyrethroids possibly a!ect a diverse range of biological functions and not just feeding. Though the primary site of action of pyrethroids in insects is the nervous system (O'Brien, 1967) these agents can cause side e!ects exhibited as various physiological and biochemical changes (Saleem and Shakoori, 1993). Reports from our laboratory have shown that fenvalerate, a pyrethroid insecticide, interferes with silkworm feeding behavior and physiology of digestion, and the magnitude of changes increased with an increase in insecticide concentration (Vyjayanthi and Subramanyam, 2002). Thus we have further attempted to examine the potential role of fenvalerate in digestive physiology. In this regard, we believe that the changes in digestion, assimilation, and conversion of food on insecticide treatment may be due to the changes in various digestive enzyme activities, which take part in active digestive and assimilative process of the silkworm. Thus, the aim of this study was to determine if pyrethroids a!ect di!erent digestive enzymes isolated from midgut. MATERIALS AND METHODS
Insects. Disease-free layings of B. mori (Pure-Mysore and NB D varieties) were obtained from Government Grainage and freshly hatched larvae were reared on the leaves of Morus alba (M variety) with standard rearing techniques and optimum conditions (Krishnaswami, 1978). Insecticide: Preparation of stock solution. A Technicalgrade sample of Sumicidine-20EC (Fenvalerate-20EC: cyano(3-phenoxyphenyl)methyl 4-chloro-x-(1-methyl)-benzene acetate, brown liquid) was obtained from Rallis India Ltd. A stock solution of 1% strength was prepared with
212 0147-6513/02 $35.00 2002 Elsevier Science (USA) All rights reserved.
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EFFECT OF FENVALERATE ON SILKWORM METABOLISM
TABLE 1 E4ect of Fenvalerate on Amylase Activity in the Hemolymph of Fourth-Instar Silkworm, Bombyx mori L. Amylase activity [(mg glucose/min/mg protein$SD);10\] Bivoltine Development (days) 1 2 3 4
Control
LC
0.549$0.021 0.732$0.029 1.11$0.04 1.0$0.05
0.512$0.025 0.701$0.03 0.93$0.04* 0.82$0.032*
Multivoltine LC
0.480$0.018** 0.514$0.020** 0.58$0.027** 0.50$0.031**
Control
LC
LC
1.02$0.024 1.62$0.0382 2.04$0.1 1.78$0.062
0.8$0.015** 1.2$0.04** 1.48$0.05** 1.19$0.08**
0.41$0.0114*** 0.69$0.0121*** 1.07$0.05** 0.723$0.044**
Note. Values are means$SD, Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
acetone and subsequent dilutions were made with distilled water (Burch"eld et al., 1952; Radhakrishna and Delvi, 1987). The concentrations were calculated on the basis of the active ingredient present in the insecticide.
larvae of uniform size and age. To replace the dead and unequal larvae, a separate batch of worms were maintained under identical conditions. The insecticide was applied just after the "rst feeding in both age groups. A single uniform topical spray was applied to test individuals. Topical application of the insecticide was preferred because it is a method considered for toxicity tests (Kuwana et al., 1967; Ochi & Kiuchi, 1998).
Evaluation of lethal and sublethal concentrations. The silkworms maintained for the experiments were divided into batches of 50 worms each and treated with selected concentrations of fenvalerate. Further, the LC and LC of the insecticide, were determined by the probit analysis method (Finney, 1971). Brie#y, the mean percentage mortality and di!erent concentrations of insecticide used were plotted on the log-probit curve for each race and age. LC and LC values for each race and age were calculated from the curve. From the probit and percentage mortality curves the LC was found to be 8.62 and 9.50 ppm for fourth- and "fthinstar multivoltine worms and 8.11 and 7.50 ppm for fourthand "fth-instar bivoltine worms, respectively, and the LC was 4.00 and 3.76 ppm for multivoltine and bivoltine fourthinstar worms and 4.80 and 4.40 ppm for multivoltine and bivoltine "fth-instar respectively.
Analyses: Preparation of tissue extracts for enzyme assays. For the evaluation of digestive enzyme assays, the whole midgut was excised from prefrozen larvae kept at !203C for 12 h and a 10% homogenate was prepared (after separating the malpighian tubules, fat bodies, and other tissue fragments adhering to the gut) in ice-cold bu!er solution. The homogenate was centrifuged at 3000 rpm and the supernatant was used as the enzyme source with appropriate dilution. Enzyme assay. Amylase activity was evaluated by the Bernfeld (1955) method using glucose as standard. The reaction mixture contained 50 mM phosphate bu!er (pH 6.5), 1% starch (freshly prepared), and appropriately diluted enzyme. Sucrase activity was measured according to the method of Ishaaya and Swirski (1970) with glucose as
¹reatment. Immediately after the third and fourth molts, larvae were divided into three batches with six replicates in each batch. Each replicate consisted of 50 healthy
TABLE 2 E4ect of Fenvalerate on Sucrase Activity in the Whole Midgut of Fourth-Instar Silkworm, Bombyx mori L. Sucrase activity [(mg glucose/min/mg protein);10\$SD] Bivoltine Development (days) 1 2 3 4
Control 6.6$0.145 7.26$0.057 8.07$0.092 8.00$012
LC 6.43$0.16 6.62$0.098** 6.75$0.098** 6.51$0.091**
Multivoltine LC
6.02$0.162** 3.92$0.099*** 4.43$0.091*** 4.40$0.087***
Control 7.02$0.162 7.96$0.10 8.66$0.11 8.21$0.15
Note. Values are means$SD. Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
LC 6.21$0.11* 6.82$0.125** 7.45$0.10** 7.14$0.14**
LC
5.13$0.098*** 6.00$0.14** 5.38$0.092*** 5.22$0.12***
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TABLE 3 E4ect of Fenvalerate on Trehalase Activity in the Whole Midgut of Fourth-Instar Silkworm, Bombyx mori L. Trehalase activity [(mg glucose/min/mg protein);10\$SD] Bivoltine Development (days) 1 2 3 4
Control
LC
1.35$0.01 1.92$0.10 2.2$0.12 2.01$0.08
2.05$0.08* 2.80$0.12** 3.33$0.11** 2.98$0.091*
Multivoltine LC
2.92$0.108** 4.7$0.16*** 5.8$0.14*** 5.0$0.11***
Control
LC
2.115$0.121 2.54$0.09 2.96$0.079 2.80$0.125
3.24$0.089* 3.82$0.14* 4.14$0.010** 3.51$0.21*
LC
3.62$0.112* 4.02$0.201** 4.98$0.13** 3.75$0.159*
Note. Values are means$S.D. Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
standard. The incubation mixture contained 50 mM phosphate bu!er (pH 6.5), 3.42 M sucrose, and appropriately diluted enzyme. Trehalase activity was determined by the Dahlman (1971) method with slight modi"cation of pH from 5.6 to 6.0. The assay mixture contained 50 mM phosphate bu!er (pH 6.0), 3.78 M trehalose, and appropriately diluted enzyme. The incubation period and temperature of incubation were 30 min and 24$13C for amylase, and 60 min and 373C for sucrase and trehalase. The amount of glucose liberated was measured at 540 nm after inhibition of the reaction with dinitrosalicylic acid (DNS) reagent in the cases of amylase and sucrase and with concentrated H SO in the trehalase assay. The mixture was boiled over a boiling water bath for 10 min and diluted with distilled water. Activity is expressed as milligrams of glucose liberated per minute milligram of mg protein in all three estimations. The protease enzyme assay was carried out with the method of Eguchi and Iwamoto (1982) with slight modi"cation of the pH of borate bu!er (pH 11.0) as outlined by Sarangi (1985) using tyrosine as standard. The reaction mixture contained 1% casein, 0.1 M borate bu!er (pH 11.0), and appropriately diluted enzyme. The incubation was carried out for 30 min at 303C. The reaction was inhibited by adding 8.2 M trichloroacetic acid (TCA) and centrifuged. The supernatant was
used with 0.5 N NaOH and Folin's reagent to measure the tyrosine liberated at 660 nm. Protein content in all assays was estimated with the Folin phenol reagent method (Lowry et al., 1951) using bovine serum albumin as standard. Statistical analysis. All data collected were statistically analyzed with Student's t test and the results are expressed as means$SD. The level of signi"cance were expressed as P values (Panse and Sukhatme, 1989). RESULTS
Activity levels of the digestive enzymes amylase, sucrase, trehalase, and protease in normal and treated multivoltine and bivoltine silkworms are presented in Tables 1}4 and Figs. 1}8 for the last two instars. In fourth instar, midgut enzymes increased steadily until a day prior to molting, whereas during "fth instar, activity increased similarly up to the fourth to "fth day and declined thereafter. These enzymes were at higher levels in the last instar in both races. Fenvalerate treatment induced a signi"cant reduction in the activity of amylase, sucrase, and protease, but signi"cantly enhanced the activity of trehalase in both age groups and also in both races. Further, the magnitude of the
TABLE 4 E4ect of Fenvalerate on Protease Activity in the Whole Midgut of Fourth-Instar Bivoltine and Multivoltine Silkworm, Bombyx mori L. Protease activity [(mole tyrosine/min/mg protein);10\$SD] Bivoltine Development (days) 1 2 3 4
Control 10.55$0.08 13.02$0.262 16.3$0.33 16.02$0.23
LC 8.19$0.24* 9.29$0.193** 11.4$0.12** 11.0$0.094**
Multivoltine LC
Control
7.77$0.11** 8.5$0.24*** 9.52$0.08*** 8.91$0.095***
8.89$0.21 10.71$0.226 13.0$0.19 12.72$0.148
Note. Values are means$SD. Signi"cance at the level of *P(0.01, **P(0.001, ***P(0.005.
LC 8.94$0.195 9.02$0.121* 10.6$0.07** 10.1$0.11***
LC
6.92$0.112* 7.19$0.09*** 8.8$0.069*** 8.28$0.051**
EFFECT OF FENVALERATE ON SILKWORM METABOLISM
215
FIG. 1. E!ect of fenvalerate-20EC on amylase activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
changes in enzyme activity was found to depend on the concentration of insecticide used. However, age-dependent activity patterns were not in#uenced by pyrethroid application. The fenvalerate-induced changes in various enzyme activities were much more pronounced in bivoltine worms. DISCUSSION
The process of digestion and the activity of enzymes depend on the quantity and quality of food ingested, age and health of the larva, and few physical factors (Swingle, 1930; Kanekatsu et al., 1989, 1993). Toxic chemicals cause
disturbances in the normal biochemical physiological activities of an insect (Khan, 1993; Bhosale et al., 1988). In the present investigation, it was observed that silkworms treated with fenvalerate had few such responses such as food avoidance and rapid movements of the head and thoracic region at low concentrations, but higher concentrations induced a slow death, before which rectal protrusion occurred (Vyjayanthi and Subramanyam, 2002). As poor nutrition and low-nutrient diets have direct e!ects on primary biochemical and physiological systems, and thus may decrease the performance of insects by e!ecting changes in the detoxi"cation system that can alter the susceptibility of the
FIG. 2. E!ect of fenvalerate-20EC on amylase activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
216
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FIG. 3. E!ect of fenvalerate-20EC on sucrase activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
insect (Lindroth et al., 1991), the poor feeding behavior in our earlier experiments may be correlated with the alteration in digestive enzyme activity on insecticide treatment. Activity of the enzymes amylase, sucrase, and protease was decreased, which may be due to the insu$cient amount of
substrate resulting from food rejection. Sumida et al. (1990) reported that midgut sucrase is activated by sucrose at a higher concentration ('100 mM) derived from the ingested food in the midgut lumen. The rational food consumption by a lepidotpteran larva was correlated directly with
FIG. 4. E!ect of fenvalerate-20EC on sucrase activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
EFFECT OF FENVALERATE ON SILKWORM METABOLISM
217
FIG. 5. E!ect of fenvalerate-20EC on trehalase activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
the activities of amylase and invertase by Christopher and Mathavan (1985), with the larva receiving 100% food found to have the highest amylase and invertase activites, which
declined as the percentage of food o!ered was reduced. Similarly, the poor feeding rate on fenvalerate treatment (Vyjayanthi and Subramanyam, 2002) could have resulted
FIG. 6. E!ect of fenvalerate-20EC on trehalase activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
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FIG. 7. E!ect of fenvalerate-20EC on protease activity in the "fth-instar multivoltine larva of Bombyx mori. Values are means$SD; n"8; Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
in the reduced activity of amylase and sucrase in our present study. Proteases are the proteolytic enzymes that aid in digestion of leaf proteins. Late silkworms are generally coarse leaf feeders, and are suppose to have a highly speci"c protease enzyme system that hydrolyzes the "brous protein found in abundance in coarse mulberry leaves (Ito and Arai, 1966). The proteolytic activity of the alimentary canal in relation to feeding or proteins has been studied in many
insects (Dadd, 1956; Hamano and Mukaiyama, 1970). Changes in protein metabolism have been reported in silkworms treated with organophosphate insecticide (Nath et al., 1997). In the present study, protease activity decreased on insecticide treatment, and it is presumed that the insecticide may even have inhibitory action over the enzyme molecules to act on their substrates, or the enzyme molecules may be devoid of a su$cient amount of substrate. The decline in
FIG 8. E!ect of fenvalerate-20EC on protease activity in the "fth-instar bivoltine larva of Bombyx mori. Values are means$SD; n"8. Values are signi"cant at *P(0.01, **P(0.001, ***P(0.005.
EFFECT OF FENVALERATE ON SILKWORM METABOLISM
219
protease activity may also be due to starvation, which is a result of the food rejection behavior on fenvalerate treatment. Protease activity is in#uenced by the age, sex and feeding behaviour of silkworms and decreases signi"cantly on starvation during late "fth instar (Jadhav and Kallapur, 1988). The observations of the present investigation can thus be correlated with feeding behavior and the quantity of food ingested by the silkworm for the active participation of these enzymes in the process of digestion. Trehalase activity to the contrary, was enhanced in the midgut of silkworms treated with fenvalerate. Azuma and Yamashita (1985) reported such an increase in midgut trehalase activity serves for the utilization of hemolymph trehalose for metabolic energy to maintain active processes in various situations. The enhanced trehalase activity may also be due to increased hydrolysis of trehalose to release two glucose moieties, which plays an important role in silkworm carbohydrate metabolism (Hasegawa and Yamashita, 1970). The hyperactivity (irritation response) and stress caused by pyrethroid poisoning in silkworms demands energy that might have been supplied in the form of glucose molecules by the hydrolysis of trehalose in the midgut by trehalase. Similar disturbances in carbohydrate metabolism and related enzymes other than trehalase due to organophosphorous insecticide have been reported in B. mori (Nath, 2000). Though this reduction may be due to the lesser availability of nutrients, there may be some in#uence on enzyme structure and function, and the poison may act as an inhibitor for such enzymes with altered activity.
Bernfeld, P. (1955). Enzymes of carbohydrate metabolism: Amylases, and . In Methods in Enzymology (S. P. Colowick and N. O. Kaplan, Eds.), Vol. I, pp. 149}158. Academic Press, New York.
CONCLUSIONS
Hamano, K., and Mukaiyama, F. (1970). Some properties of digestive #uid proteases in the silkworm, Bombyx mori, with reference to the relation between dissociation degree and nutritive value of some proteins. J. Sericult. Sci. Japan. 39, 371}376.
Fenvalerate toxicity in B. mori interferes with major metabolic functions and checks the production and activity of digestive enzymes. The degree of e!ect depends on age, race, and concentration of the insecticide. The toxic e!ects of pyrethroids hinder normal digestive metabolism and thus growth of the insect, which further greatly in#uences silk production by the worms. REFERENCES Abraham, E. G., Nagaraju, J., and Datta, R. K. (1992). Chemical studies of amylases in the silkworm, Bombyx mori L.: Comparative analysis in diapause and nondiapause strains. Insect Biochem. Mol. Biol. 22, 867}873. Armstrong, K. E., and Bonner, A. B. (1985). Investigation of permethrininduced antifeedent e!ect on Drosophila melanogaster, an ethological approach. Pestic. Sci. 16, 641}650. Asakawa, H., and Hamano, K. (1994). Enzymatic properties of digestive amylase isozymes in silkworms, Bombyx mori L. J. Seric. Sci. Japan 63, 13}20. Azuma, M., and Yamashita, O. (1985). Cellular localization and proposed function of midgut trehalase in silkworm larva, Bombyx mori. ¹issue Cell. 17, 539}551.
Bhosale, S. H. ,Yadwad, V. B., and Kallapur, V. L. (1988). Residual toxic e!ect of Ekalux EC-25 on the biochemical constituents of the fat body of 5th instar Bombyx mori. Indian J. Seric. 27, 73}77. Burch"eld, H. P., Hilchey, J. D., and Storrs, E. E. (1952). An objective method for insecticide bioassay based on photomigration of mosquito larvae. Contrib. Boyce ¹homson Inst. 17, 60. Christopher, M. S. M., and Mathavan, S. (1985). Regulation of digestive enzyme activity in the larva of Catopsilia crocale (Lepidoptera). J. Insect Physiol. 31, 217}221. Dadd, R. H. (1956). Proteolytic activity of the midgut in relation to feeding in the beetles, ¹enebrio molitor and Ditiscus marinalis L. J. Exp. Biol. 33, 311}324. Dahlman, D. L. (1971). Puri"cation and properties of trehalase from tobacco hornworm larvae. J Insect Physiol. 17, 1677}1687. Egaas, E., Skaare, J. U., Goksoyr, A., Svendsen, N. O., and Kobro, S. (1992). E!ects of the insecticide Thiodan 35 (endosulfan) on xenobiotic metabolizing enzymes from midgut and gut-free tissues of some coleopteran larva. Comp. Biochem. Physiol. C 102, 385}389. Eguchi, M., and Iwamoto, A. (1976). Alkaline protease in the midgut tissue and digestive #uid of silkworm, Bombyx mori. Insect Biochem. 6, 491}496. Eguchi, M. and Iwamoto, A. (1982). Comparison of three alkaline proteases from digestive #uid of the silkworm, Bombyx mori. L. Comp. Biochem. Physiol. B 71, 663}668. Eguchi, M., Iwamoto, A., and Yamauchi, K. (1982). Interrelation of proteases from the midgut lumen, epithelia and pertrophic membrane of the silkworm, Bombyx mori. L., Comp. Biochem. Physiol. A 72, 359}363. Finney, D. J. (1971). Probit Analysis, 3rd ed. Cambridge Univ. Press, Cambridge, London/New York.
Hasegawa, K., and Yamashita, O. (1970). Mode d'action de I'hormone de diapause dans le metabolisme glucidique de ver a& soie, Bombyx mori L. Ann. Endocrinol. 31, 631}636. Ishaaya, and Swirski, E. (1970). Invertase and amylase activity in the armoured scales Chrysomphalus aordun and Aonidiella auranti. J. Insect Physiol. 16, 1599}1606. Ito, T., and Arai, N. (1966). Amino acid requirements in Bombyx mori. J. Insect Physiol. 23, 861}869. Jadhav, G., and Kallapur, V. L. (1988). In#uence of age, sex and feeding on the protease activity of certain tissues of "fth instar silkworm, Bombyx mori. Entomon. 13, 289}293. Kanekatsu, R. (1972). Amylase in the digestive juice of silkworm larvae, Bombyx mori. J. Seric Sci. 41, 445}451. Kanekatsu, R. (1978). Studies on further properties for an alkaline amylase in the digestive juice of silkworm, Bombyx mori. J. Fac. ¹ext. Sci. ¹echnol. 76, 1}21. Kanekatsu, R., Ichimura, H., and Hori, M. (1989). Distribution and developmental changes in midgut sucrase activity of the silkworm, Bombyx mori. J. Seric. Sci. Japan 58, 517}523. Kanekatsu, R., Satoh, M., Kodaira, R., and Miyashita, T. (1993). Midgut sucrase-1 (suc-1) of the silkworm, Bombyx mori: Geneties and changes in the activities during the pupal}adult development. J. Seric. Sci. Japan 62, 13}19.
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