Studies of the trehalase activity in eggs of the egyptian cotton worm, Spodoptera littoralis

Insect Biochem., 1978, Vol. 8, pp. 81 to 85. Pergamon Press. Printed in Great Britain

STUDIES OF THE TREHALASE ACTIVITY IN EGGS OF THE EGYPTIAN COTTON WORM, SPODOPTERA L I T T O R A L I S * ISAAC ISHAAYAand SARA YABLONSKI Division of Entomology, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel (Received 28 January 1977; revised 18 February 1977) Abstract--ln eggs of Spodoptera littoralis, the optimum conditions for trehalase activity involve a reaction mixture of 0.05 M acetate buffer (pH 3.5) and 1.5% trehalose at 37°C for 60 min. A catalytic period of up to 90 min was found to be linear, the gra value was 0.03 M, and the enzyme activity reached its maximum at 55°C. In the presence of sodium deoxycholate, the trehalase activity was strongly enhanced, indicating the presence of an active system of membrane-bound trehalase. A decrease of about 20% in the activity of the membrane enzyme was observed in the first instar larvae shortly before hatching, with no appreciable change in the activity of the soluble enzyme. Urea and divalent cations were found to suppress considerably the enzyme activity, HgCI2 being the most effective compound.

INTRODUCTION

Trehalase activity in the black scale (Saissetia oleae) is affected by the quality of food which the insect ingests, and may serve as a parameter for assessing the adaptability of the scale to its host plant (ISnAAYA and SWIRSKI,1976). In spite of the fact that ample information is available concerning trehalase activity in the larval and adult insect, very little is known about the r61e of this enzyme during embryonic and pharate first instar larval development. Consequently, this study was conducted to (a) isolate egg trehalase from Spodoptera littoralis, (b) determine its optimum condition for reaction, and (c) bring to light some characteristic properties of its activation and inhibition.

TREHALOSE is generally present in large amounts in the haemolymph of most insects (CHEra3Rr,A, 1965; WYATT, 1967). It is metabolised by a thoracic trehalase and decreases dramatically during continuous flight (CLEGG and EVANS, 1961; FORD and CANDY, 1972). Despite the apparent importance of the trehalose-trehalase system for supporting flight, not enough information is available on the relative importance of this system as metabolic fuel for various physiological processes, significant during embryonic and larval development. During molting cycles, this system is activated to generate production of glucose needed, probably, for chitin build-up in the newly synthesized cuticle (CANDY and KILBY, 1962). Numerous publications have appeared in the last MATERIALS AND METHODS decade concerning the isolation, purification and characterization of trehalase activity from larvae and Enzyme preparation One hundred to 200 mg 2- to 3-day-old eggs of Spodopadults of various insects (LEFEBVREand HUBER, 1970; tera littoralis (collected from a laboratory standardized colDAHLMAN, 1971 ; YANAGAWA,1971; DUVE, 1972; TALony reared on alfalfa at 25°C) were homogenized in 40-fold BOT and HUBER, 1975). The enzyme was identified in 0.3% sodium deoxycholate solution (for extracting free and the digestive walls (YANAGAWA, 1971; TALBOT and membrane-bound trehalase); using a chilled glass Teflon HUBER, 1975), the haemolymph (FRIEDMAN, 1961; tissue grinder. In some cases, distilled water was used for MATTHEWSet al., 1976), the thoracic muscles (GussrN homogenization instead of the sodium c]eoxycholate,soluand WYATT,1965; REED and SACKTOR,1971), and the tion for soluble trehalase determination. The homogenate was centrifuged for 15 min at 12,000g at 2°C, the supernaBombyx mori silk glands (SHIMADA, 1976). Two distinct types of trehalase have been identified: tant fraction being used for the enzyme assays. Using one is water soluble and located in the intestinal tis- bovine serum albumin for reference, the protein level sues, and the other is a membrane-bound enzyme (LowRY et al., 1951) in the enzyme solution prepared with sodium-deoxycholate was 5.5 mg/ml, and that prepared generally found in muscles (GuSSlN and WYATT,1965; with water was 1.6 mg/ml. YANAGAWA, 1971). The latter could be released or activated by using detergents or altemating freezing Determination of enzyme activity and thawing (GILBY et al., 1967; CANDY, 1974). Trehalase was determined as described previously (IsH~YA and SwmsKI, 1976), using the 3,5-dinitrosalicylic acid reagent for determining the free aldehyde groups of glucose formed after trehalose digestion. This reaction is * Contribution from the Agricultural Research Organi- based on the reduction of dinitrosalicylic acid by the aldezation, The Volcani Center, Bet Dagan, Israel. 1977 Series, hyde groups of glucose units in basic medium. The optiNo..106-E. This research was supported in part by a grant mum enzyme reaction (see Results) consisted of 0.1 ml 6% from the United States--Israel Binational Science Founda- trehalose, 0.1 ml 0.2M acetate buffer (pH 3.5), and 0.2ml tion (BSF), Jerusalem, Israel. en2=yme solution. After 60 min incubation at 37°C, 0.8 ml 81

82

ISAAC ISHAAYAAND SARA YABLONSK1

3,5-dinitrosalicylic acid reagenl~was added, the mixture was heated for 5 rain at 100°C, and then immediately cooled in an ice bath. The absorbency at 550 nm was determined in extinction units (E) with a Gilford type 240 spectrophotometer. Under conditions similar to those for the enzyme assay, direct reaction of glucose with dinitrosalicylic acid reagent gives 1 E unit = 0.44 mg glucose. For standardization, the activity was expressed in #g glucose/enzyme reaction. The dinitrosalicylic acid reagent was prepared by ~l procedure similar to that of NOEL~NG and BERNFELD(1948). One gram of 3,5 dinitrosalicylic acid was dissolved in 20 ml of 2 N NaOH and 50 ml of water with the aid of a magnetic stirrer. Potassium sodium tartarate (30 g) was added, and magnetic stirring was continued until a clear solution was obtained. Distilled water was then added to bring the final volume to 100 ml. The reagent, when stored in the dark, is usable for at least 3 months.

300

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250

~0200

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150

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Inhibition of enzyme activity Various monovalent and divalent cations or other indicated compounds were added to the 0.2 ml enzyme solution and incubated for 60 win at 37°C before the initiation of the enzyme reaction. Enzyme activity is expressed as a percentage of that of the appropriate control. The in vitro inhibition studies are reported as the average of two or more experiments or as a typical experiment confirmed in principle by two or more separate determinations. RESULTS AND DISCUSSION

Optimum conditions for trehalase activity Optimum conditions for reaction, i.e., pH, initial velocity, enzyme and substrate concentration, and temperature were evaluated in a series of preliminary experiments. Thereafter, optimum conditions were determined for each factor separately, all other factors being at the optimum. The optimum enzyme assay involves a reaction of 0.4 ml containing 1.5~o trehalose, 0.05 M acetate buffer (pH 3.5), and an enzyme level (prepared with sodium deoxycholate) being equilibrated to 1.0mg protein. The trehalase activity curves for the optima of pH, initial velocity, reaction temperature, and the Lineweaver-Burk plot for K m determination are shown in Figs. l, 2, and 3. The optimum pH for trehalase reaction (3.5) found in our assays with either acetate buffer or citrate-phosphate buffer (Fig. 1) was markedly lower than the usual optimum pH (5.0-6.0) found for trehalases of various insects such as Drosophila melanogaster (HUBER and LEFEBVRE, 1971), Calliphora erythrocephala (DuvE, 1972), Manduca sexta (DAHLMAN, 1971), Apis melliphera (TALBOT and HUBER, 1975), and Saissetia oleae (ISHAAYAand Swmsrd, 1976), but resemble the optimum pH obtained from the haemolymph of the American cockroach, Periplaneta americana (MA'rrHEWS et al., 1976). A catalytic period of up to 90 rain at 37°C was found to be linear (Fig. 2), indicating the stability of this enzyme at the described reaction conditions. The enzymatic activity increased with the reaction temperature, reaching its maximum at 55°C, above which the enzyme was gradually inactivated, its activity dropping at 65°C to about 30% of its maximum. The temperature curve of this enzyme resembled those found with other trehalases obtained from various sources of insect species (HUB~R and L~'EaVRE, 1971; DUVE, 1972; TALBOT and HUBm, 1975). Various concentrations of substrate (trehalose) produced a typical Michaelis-Menten relationship,

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Fig. 1. pH optimum curve for trehalase activity of S. littoralis eggs. Enzyme activity was carried out with acetate buffer (It), phosphate buffer (O), and citrate-phosphate buffer (A). and a plot of the reciprocal values, l/v and 1/s (Fig. 3), produced a Km of 0.03 M. Sodium deoxycholate, a protein-solubilizing agent, was used for extracting membrane-bound trehalase. Various concentrations of sodium deoxycholate used in the homogenization and the enzyme preparation process, resulted in a consistent increase in trehalase activity, reaching more than two-fold at 0.3~o compared with that of the water-soluble enzyme (Fig. 4). At higher sodium deoxycholate concentrations the enzyme lost its activity, reaching less than 10~ of its maximum activity at 1.6~. These results indicate that trehalase enzyme is present in S. littoralis eggs in both free and membrane-bound form, and that the activity of the enzyme-bound form at optimum ranges between 50 and 60~ of the total activity.

Effect of age and environmental temperature on egg trehalase activity The egg of S. littoralis is laid in the blastula stage. The outline of the larva appears on the 2nd day and the pharate first instar larva reaches maturity on the 3rd day. The mature pharate 1st instar larva can be recognized by its black head and fully formed body; it can hatch 12 to 24hr later. It is clear from this observation that development of S. littoralis continues after the egg is laid. The soluble and membranous trehalase were tested in S. littoralis eggs kept for various periods at 25°C (Table 1) for determining their activities during development. There was no appreciable difference in water-soluble trehalase activity during 3 days of development; however, a marked decrease of about 20~ in trehalase activity was obtained at 2 to 3 days (at the stage of pharate first instar larva) in sodium deoxycholate enzyme preparation, compared with earlier stages. These results indicate a decrease in membrane-bound treha-

Trehalase in S. ltttoralis eggs I 400

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Fig. 2. Rate and temperature curves of trehalase activity of S. littoralis eggs. lase in embryo at or near m~urity and may reflect determined in an attempt to elucidate some charactera depiction of the level of trehalose at this stage. istic properties of this enzyme. A typical biphasic acEggs, 2 to 3 days old, were incubated for 60 min tivity curve was obtained with the increase in urea at various temperatures, homogenized in water or in concentration. Concentrations of up to 0.2 M urea in 0.3% sodium deoxycholate solution, and tested for the pretest enzyme solution, resulted in a linear their free and membrane-bound trehalase (Table 2). enzyme inhibitory effect reaching 42~o. Higher urea No appreciable, difference was found in the activity concentrations had almost no further effect on the of the various preparations of sodium deoxycholate- enzyme activity, indicating enzyme saturation by soluble enzyme, indicating that the activity of the urea. Urea was reported to inhibit trehalase activity membrane-bound enzyme was not altered when eggs of Apis mellifera abdomen, but not that of Apis melliwere kept at temperatures ranging between 3 and fera thorax (TALBOT and Huen~, 1975). 35°C. On the other hand, the activity of the waterDivalent cations (but not monovalent cations) were soluble trehalasc increased with the temperature, found to inhibit considerably the enzyme activity reaching at 25°C about 1307/o of that at 3°C, with I I T { no further increase at 35°C. The decreased activify level of the water-soluble trehalase at low temperature 300 may reflect a reduced glucose level needed for energy I supply for slow development. _o pEffect of urea and various di- and monovalent cations on enzyme activity The effect of urea (Fig. 5) and various selected cations (Table 3) on egg trehalase of S. littoralis was

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Fig. 4. Effect of sodium deoxycholate on trchalase activity of S. littoralis eggs. Eggs were homogenized in various concentrations of sodium deox~cholate and the enzyme preparations were tested for their trehalase activity.

84

ISAAC ISHAAYA AND SARA YABLONSKI

Table 1. Effect of egg age on trehalase activity

Egg age* (days)

Trehalase activity, expressed in/ig glucose/reaction Water-soluble Sodium deoxycholateenzyme soluble enzyme

0-1 1-2 2-3

114 ± 11 126 ± 14 110 ± 12

330 ± 34 334 ± 37 261 ± 27t

(Table 3). HgCI 2 was the most effective compound, resulting--at a concentration of 10- 2 a n d 10- a M in the pretest e n z y m e - i n h i b i t o r incubation m i x t u r e - - i n 100 a n d 90% inhibition, respectively. CuSO4 had a n intermediate effect, resulting in 100 a n d 43% inhibition at a concentration of 10 -1 a n d 10 - z M, respectively. MgC12, CaC12, Z n S O 4 , a n d COC12 h a d the Table 2. Effect of environmental temperature on egg trehalase activity

(°c)

Trehalase activity, expressed in/zg glucose/reaction Water-soluble Sodium deoxycholateenzyme soluble enzyme

3 15 25 35

77 91 102 99

• 1001

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CuSO4

MgC12

CaC12

ZnSO4 COC12 MnCI2 KCI NaC1

Concentration in the pre-test enzyme solution (M)

Enzyme activity, as % of control

-10- 5 10 -4 10 -3 10- 2 10 -4 10- 3 10 -2 10 -1 10 -4 10- 3 10 -2 10-1 10 4 10 -3 10 -2, 10-1 10- 3 10 2 10-1 10- 3 10- 2 10-1 10 3 10 -2 10 -1 10-1 10-1

100.0 116.1 93.1 6.6 0.0 104.0 86.2 57.2 0.0 99.8 96.4 82.4 47.2 95.3 88.1 75.2 65.2 104.5 89.6 56.4 109.5 99.2 76.9 127.6 141.7 113.0 104.6 95.2

Various concentrations of each compound were incubated with the enzyme before the start of the reaction (see Methods). The sulphate form of the copper and the zinc was used instead of the chloride form, because of better solubility in the enzyme system reaction.

238 248 246 244

* Eggs 2 to 3 days old. were incubated at various temperatures for 60 min and were assayed for their soluble (water-soluble enzyme) and membrane-bound (sodium deoxycholate-soluble enzyme) trehalase. ;....

Compound None HgC12

* Eggs were kept at 28°C for various periods, homogenized in H 2 0 (for soluble trehalase) or in 0.3% sodium deoxycholate (for soluble and membrane-bound trehalase), and assayed for enzyme activity. Data are the average of three replicates followed by their S.E. values. ~"Significantly different from the other data of the same row at 5% level (according to Duncan's new multiple-range test).

Incubation temperature*

Table 3. Effect of di- and monovalent cations on the trehalase activity obtained from eggs of S. littoralis

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least effect, resulting in a 50% or less inhibition at a concentration of 10 -1 M. O n the other hand, MnCI2 enhanced enzyme activity to a b o u t 40% at a concentration of 10 -2 M. KC1 a n d NaCI h a d no appreciable effect at a concentration as high as 10-1 M. O u r results agree with previous reports, indicating that divalent but not m o n o v a l e n t cations inhibit trehalase activity in the lepidopterous larvae Manduca sexta (DAHLMAN, 1971) a n d Bombyx mori (SmMADA, 1976), and that HgCIE is the most effective inhibitor (DAHLMAN,1971).

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Acknowledgement--The authors thank the Department of Toxicology at the Volcani Center for the supply of Spodoptera littoralis eggs.

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REFERENCES

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Fig. 5. Effect of urea on from eggs of S. littoralis. were incubated with the reaction

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the trehalase activity obtained Various concentrations of urea enzyme before the start of the (see Methods).

CANDY D. J. (1974) The control of muscle trehalase activity during locust flight. Biochem. Soc. Trans. 2, 1107-1109. CANDY D. J. and KILBY B. A. (1962) Studies on chitin synthesis in the desert locust. J. exp. Biol 39, 129-140. CHEFURKA W. (1965) Some comparative aspects of the metabolism of carbohydrates in insects. A. Rev. Ent. 10, 345-382. CLEGG J. S. and EVANS D. R. (1961) The physiology of blood trehalose and its function during flight in the blowfly. J. exp. Biol. 38, 771-792.

Trehalase in S. littoralis eggs DAHLMAND. L. (1971) Purification and properties of trehalaNe from tobacco hornworm larvae. J. Insect Physiol. 17, 1677-1687. Drdv'E H. (1972) Purification and properties of trehalase isolated from the blowfly Calliphora erythrocephala. Insect Biochem. 2, 445-450. FORD W. C. L. and CANDY D. J. 0972) The regulation of glycolysis in perfused locust flight muscle. Biochem. J. 130, ll01-1112. FRIEDMANS. (1961) Inhibition of trehalase activity in the haemolymph of Phormia regina Meig. Arch. Biochem. Biophys. 93, 550-554. GILBY A. R., WYATTS. S., and WYATTG, R. (1967) TrehalaNes from the cockroach, Blaberus discoidalis: Activation, solubilization, and properties of the muscle enzyme and some properties of the intestinal enzyme. Acta Biochim. polon. 14, 83-100. GussrN A. E. S. and WYATT. G. R. (1965) Membranebound trehalase from Cercopia silkmoth muscle. Arfh. Biochem. Biophys. 112, 626-634. HUBER R. E. and LEFEBVREY. A. (1971) The purification and some properties of soluble trehalase and sucrase from Drosophila melanogaster. Can. J. Biochem. 49, 1155-!164. ISHAAYAI. and SWmSKI E. (1976) Trehalase, invertase, and amylase activities in the black scale, Saissetia oleae, and their relation to host adaptability. J. Insect Physiol. 22, 1025-1029. L~EBVRE Y. A. and HtmEg R. E. (1970) Solubilization, purification, and some properties of trehalase from honey bee (Apis mellifera). Arch. Biochem. Biophys. 140, 514-518. LOWRY O. H., ROSESROUGHN. J., FARR A. L., and RAN-

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DALL R. L. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. MATrHEWS J. R., DOWNER R. G. H., and MORRISON P. E. (1976) ~-Glucosidase activity in haemolymph of the American cockroach, Periplaneta americana. J. Insect Physiol. 22, 157-163. NOELXrNG G. and BVatN~LD P. (1948) Sur les enzymes amylolytiques--III. La fl-amylase: Dosage d'activit6 et contr61e de l'absence d'~-amylase. Helv. chim. Acta 31, 289-290. REED W. D. and SACKTORB. (1971) Localization of trehalaNe in flight muscle of the blowfly Phormia regina. Arch. Biochem. Biophys. 145, 392-401. SHIMADAS. (1976) Transition of properties in the silk gland trehalase during metamorphosis of the silkworm, Bombyx mori. Comp. Biochem. Physiol. 54B, 47-50. TALBOTB. G. and HUBER R~ E. (1975) Partial purification, stabilization and characterization of adult honey bee midgut trehalase and a new trehalase specific disc gel stain method. Insect Biochem. 5, 337-347. WYAYr G. R. (1967) The biochemistry of sugars and polysaccharides in insects. Adv. Insect Physiol. 4, 287-360. YANAGAWAH. A. (1971) Purification and properties of trehalases from larval muscle and midgut of the silkworm, Bombyx mori. Insect Biochem. 1, 102-112. Key Word Index: trehalase activity, pH ; trehalase activity, initial velocity; trehalase activity, K,,; trehalase activity, environmental temperature effect on; trehalase activity, egg development effect on; trehalase activity, divalent cations effect on; trehalase activity, monovalent cations effect on; trehalase activity, urea effect on