Protease and amylase activity in larvae of the Egyptian cotton worm, Spodoptera littoralis

J. hwct Physiol.,1971, Vol. 17, pp. 945 to 953. Pcrgamon Press. Printed in Great Britain

PROTEASE AND AMYLASE ACTIVITY IN LARVAE OF THE EGYPTIAN COTTON WORM, SPODOPTERA

LITTORALIS I. ISHMYA, The

I. MOORE, and D. JOSEPH

Volcani Institute of Agricultural Research, Dagan, Israel (Received 16 April

Division

of Entomology,

Ret

1970)

Abstract-ln

the larvae of the Egyptian cotton worm, Spodopt.era l&oral& the optimum for protease activity was found to be a reaction mixture of pH 11.0 plus 0.75% caaein at 37°C for 60 min. For amykae activity the optimum was a reaction mixture of pH 9.5 plus 0.25% starch at 37% for 30 min. Roth protease and amykse are adapted to high temperaturas in vitro. Proteolytic enzymes lose their activity above SS”C and amykae above 65°C. An artificial diet of high protein content (8 per cent instead of * 2.5 per cent in clover) strongly stimulates the proteolytic activity, eapeciahy in the midgut arPIl,whenit~fourtofivetimes~~inknnenondaP1clovsr. Gntheotherhar&onlya&ghtincmase of 10 to 15 per cent in amykae activity wasfoundinkrvaeraaradonthear&cialdiet. lncmaaingtheproteinkvel from 3.6 to 7.6 per cent in an artificial diet, stimuhted both proteolytic activity and larval weight. hnyltme activity rose four- to fivefold when the protein content increased from 3.6 to 5.6 per cent, but kvelkd off at 7.6 per cent. Enzymatic activity increased when the temperature rose from 10 to 32°C. At 10°C (i.e. below the threshold of larval development), both proteolytic and amylolytic activity in the midgut wall were less than 10 per cent of that obtained at 32°C.

INTRODUCTION THE EGYPTINV cotton. worm, Spodoptera ZittorahkBoisd. (previously known as Rodttka okra F.), is one of the moat voracious pests of the Mediterranean region, heavily attacking field crops, vegetables, ornamental plants, and fruit trees. The larval life span and percentage pupation or emergence differ widely according to the food. On foliage of various apple varieties, larvae develop in 26 to 39 days, and on some of these varieties do not pupate; on Anumnthus retro+u.s, the larvae develop relatively well, whereas they fare poorly on Amaranth gnzmkm (N . Plaut, personal communication). In general, soluble carbohydrates and proteins are very efkiently utilized by insects and moat species derive the largest share of their nouriahment from these nutrients. while the utilization of these nutrients depends on digestive enzymes,

* Contribution from the Volcani Institute of A&ultural Raearch, Ret Dagan, Israel, 1970 Series, No. 1707-E. This work was financed in part by Grant No. loo0822 from the Israel Ministry of Agriculture. 945

I. ISHMYA,I. Mooq

946

.UD D. JOSEPH

very few data are available concerning the effect of the plant host or its constituents on these enzymes. According to HORI (1%9), certain plant compounds can stimulate or inhibit digestive enzymes; it can be assumed that the latter, in turn, affect digestion and food utilization. Since proteaae and amylase activity are of great importance in the digestive system, this project was conducted to determine their optimum activity in S. littoralis, their relationship with the diet, and with the temperature at which the larvae are held. Reming method

MATERIALS AND METHODS

As soon as possible after hatching, the larvae were bred singly until needed for the various tests. Throughout this period they were kept at 25 + l”C, with the exception of the last 3 days for those larvae to be used in the tests on the effect of temperature on amyIolytic and proteolytic activity. All larvae were fed every 2 days. For the teats on enzymatic activity the larvae were fed various modifications of a calcium-alginate medium previously described (MOOREand NAVON,1969). The composition of these diets is shown in Table 1. TABLE~--COMPOSITION

OF ARTIFICIAL

DlFPSFBDIN Tsgig ONTHBxPPPcrsOFPROTEIN LxvEL ONBNZYMATIC ACTIVITIES Calcuhted protein level (%)

Ingredients Full-fat coy meal* Todyeaett CaSeinS Sodium al&ate 8 Methyl-p-hydroxybete cakium carbonate Akorbic acid Triethanolamine Glacial acetic acid Distilled water Ether extract of soya Carbohydratelj Alpha-cellulose Total (B)

7.6

II

527.0 183.0 230.0 110.0 7.0 6.0 33.5 28.0 67.0 5500*0 60.0 6764.5

5.6 357.0 183.0 156.0 110.0 7.0 6.0 33.5 28.0 67.0 5500.0 34.9 22.: 6764.5

3.6 187.0 183.0 81.7 110.0 7.0 6.0 33.5 28-O 67.0 5500*0 69.9 93.7 384.7 6764.5

1.6 17.0 183-O 7.4 110.0 7.0 6.0 33.5 28.0 67.0 5500*0 104.9 140.6 547.1 6764.5

* Protein, 35.9 per cent; fat 20.6 per cent; carbohydrate minus fibre, 27.6 per cent (HAWK et al., 1954). t Type N.F. XI, protein SO-54 per cent, Lake Statsa Yeast and Chemical Divieion of St. Regie Paper Co., Wisconsin, U.S.A. $ Vitamin-free, Nutritional Biochemical Corp., Cleveland, Ohio, U.S.A. # Protanol S.F., Protan, Drammen, Norway. IIMostly (NaPO&, B.D.H. 7 See text.

PROTEASE AND AMYLME ACMVITY IN LARVAE OF EGYPTIAN COTTON WORM

947

As shown in Table 1, while the yeast content was the same in the various diets, the amounts of soya and casein were varied to obtain di&rcnt protein levels. However, the soya : cash ratio remained constant so that protein quality would not be altered. To maintain an equal fat content, appropriate amounts of ether extract, from the same soya that was used in the diet, were added. As regards carbohydrate, each quantity supplied in compensation consisted of equal amounts of sucrose and maize starch. Total weight was kept constant by adding alphacellulose where needed. The larvae used in the determination of optimum enzymatic activity as well as the effect of temperature on amylase and protease, .were fed an artiticial diet similar to the 7.6% protein diet described in Table 1 but without cellulose and, all others factors being equal, containing 450 g full-fat soy meal, 350 g Torula yeast, and 200 g caaein. The calculated protein content of this diet was about 8-O per cent.

Rtpara&m

of larval enzyme solution

The larval enzyme solution was prepared according to Applebaum et al. (1964). Midguts of the 8fth instar larvae (200-250 mg) were collected by exposing the alimentary canal and sectioning it first slightly posterior to the cardiac sphincter and again slightly anterior to the pyloric sphimtcr. A light pressure at one end of the excised midgut reaulted in the extrusion of the midgut contents. Both midgut contents and washed walls were c&c-ted and separately homogenized in water or buff&r solution in a chilled glass-&ion tissue grinder. Brei of the midgut wall or contents representing 1 g larval weight was suspended in 10 ml HsO or buffer solution, filtered or centrifuged in the cold at 10,OOOg for 15 mm, and the supernatant was used as enzyme solution. The homogenate of the midgut wall was used for determining optimum conditions for enzymatic activity, while that of the contents was used for comparison. Determination of proteolytic activity This was determined according to BIRK et al. (1962) by the casein digestion method at an absorbancy of 280 w (KUNITZ, 1947). The reaction mixture consisted of 0.2 ml buffer, 04 ml 1.5% casein solution, and O-2 ml enzyme solution. Optimum pH, enzyme, and substrate concentration were determined in preliminary experiments. Phosphate or glycine-NaOH buffer (0.075 M) was used. Enzymatic activity was terminated after 60 min incubation at 37°C by adding l-2 ml of 5% trichloroacetic acid solution. The reaction mixture was lihered or centrifug6d at 13,000 g for 15 min and the supematant was taken for enzymatic activity evaluation. The proteolytic activity was determined as O.D. units x 108 at an absorbancy of 280 w with a Gilford spectrophotometer (Type 240). Deteknation

of amyhse activity

This determination was baaed on the method of NOELTINC and BBRNFELD (1948), using a modified 3,5-dinitro-salicylic acid reagent. The reaction mixture

I. ISHMYA,I. MOORE,ANDD. JOSKPH

948

consisted of 100~ buffer, 50 /JJ 1% starch solution (B.D.H.) and 50 ~1 enzyme solution. After 30 min incubation at 37”C, ezqmatic activity was termhat by adding 04 ml of the above reagent. The reaction mixture was heated for 5 min at 100°C followed by dilution with 04 ml H,O. The amyloiytic activity was determined according to ISEUAYAand SWIRSI (1970), as O.D. units x lo” at 550 m/h. RESULTS

AND DISCUSSION

Optimum conditiona for reaction, i.e. pH, initial velocity, enzyme, and substrate concentrations, were 6rat evaluated in a series of preliminaq experiment& Thereafter, optimum conditions were determined for each factor separately, all other factors being at optimum. (a) Substrate concaaoa’on. Proteolytic activity wan determined on cakn solution8 of 0*l-l*5°h concentrations in the reaction mixture; 0*75% casein was found n&factory for maximum eqmatic activity. (b) @b PH. The optimum pH for enzymatic activity m found to be 11.0 (Fig. 1). Most of the proteolytic activity occurs above pH 9*0. At pH 8*0, 85 per cent of the potential proteolytic activity was suppressed, and at pH 6.0, 95 per cent. Below pH 60 this activity was not tested, since it is far from the pH

7cQ-

600-

500 -

400-

3aI--

2QO-

100 -0

6.0

7.0

8.0 PH

9.0

10.0

11.0

12.0

FIG. 1. pH-Proteolytic activity curve of S. Iittomh larvae. Enzymatic activity in the 64) to 8-OpH range carried out in phoaphte bu&r (0) ; in the 8-O to 12-O pH range, in glycin*NaOH buffer (0).

PROTEASE AND AMYLASS

ACTIVITY

IN IARVAE

OF EGYPTIAN

COWN

WORM

949

range existing in the larval midgut.

The optimum pH for proteolytic activity was found to be higher than the pH of the midgut contents, which is 9-Z to 9-7 (average = 95), measured with a Radiometer pH meter. Similar values were recorded in the larvae of other Lepidoptera (WMZREIOUSE, 1952; HEIMPEL, 1955). In S. Zittordis, some 40 per cent of the potential protease activity is suppressed at the pH prevailing in the midgut. (c) Initial docity. The initial velocity curve of proteolytic activity is shown in Fig. 2. A digestion period of 60 min was still on the linear part of the enzymatic activity curve.

FIG. 2. Time-prom&tic

activity curve of S. litto*& larvae.

(d) meet of temperature. Enzymatic activity was determined at 20 to 65°C (Fig. 3). The linear phase was found to be between 30 and 40°C. From 40 to 55°C and below 30°C the linearity tapers off and enzymatic activity is destroyed only above 55°C. These results indicate that the proteases of S. littoralis are adapted to high temperatures. A temperature of 37”C, which is on the linear part of the enzymatic activity curve and at which a high proteolytic activity still could be performed, was chosen for the in &TO enzymatic activity.

Optimum conditkms fm amylase activity A reaction mixture of 100 ~1 O-05 M glycine-NaOH buffer at pH 95, 50 ~1 1% starch solution, and 50 4 enzyme solution, incubated for 30 min at 37”C, was most suitable for enzymatic activity. The pH for maximum larval amylase activity, pH 9-5 (Fig. 4), is half a pH unit lower than that obtained by A~PLEBAUM et al. (1964). This may be due to genetic or nutritional factors. Similar to the

950

I. ISHMYA, I. MOORE, ANDD. JOSWH

midgut proteolytic enzymes, amylase also is adapted to high temperatures; its activity only above 65°C. $12001

I

FIG. 3. Temperature-proteolytic

ii

I

g

01

3

I

activity curve of S. littomEs larvae.

0

1

L.0

it loses

-6.0

I

0.0

10.0

/

,

12.0

PH

FIG. 4. pH-Amylase activity curve of S. littoralislarvae. Jhqmatic activity in the 4-O to 6-O pH range carried out in acetate buffer (A) ; in the 6-O to 8-O pH range in phosphate buffer (0); in the 8-O to 11-O pH range in glycine-NaOH buffer (0). E#ect of food cm enzymatic activity Digestibility differs widely with the kind of food given to the polyphagous Proahiu en&n&a Cramer (Soo Hoe and FRAENKEL,1966) and to the oligophagous Protopce sextu Johan (WALDBAUER,1%2). In some protein quality was found to affect proteolysis (BIRK et al., 1962) or to counteract enzyme inhibitors (ISHAAYA and BIRK, 1965). Our results indicate that larvae of S. littoralis reared on an

PROTEASJi

AND

AMYUSE

ACTIVITY

IN

LARVAE

OF EcYPTlAN

COTTON

WORM

951

artificial diet exhibit a much higher proteolytic activity than larvae reared on clover (Fig. 5). About a fivefold increase in enzymatic activity was found in the homogenate of the midgut wall and an increase of about 30 per cent activity in that Similar results were obtained when a population of of the midgut contents. neonates-progeny from insects reared on artificial diet-was divided between artificial diet and clover. On the other hand, only a slight increase of about 10 to

FIG. 5. Effect of food on the proteolytic activity of the larval midgut. Midgut wall (m, 0) and midgut contents (A, A) homogenates of S. Iittoralis larvae reared onfmartifkialdiet( -) or on clover (- - - -).

15 per cent in amylase activity was recorded in larvae reared on an artificial diet as opposed to clover. These results indicate that food type can afkt the larval digestive enzymes in tiwo. The high increase in proteolytic activity in the midgut wall homogenate of larvae reared on an artificial diet, may indicate a high secretion of proteolytic enzymes resulting from physiologically active compounds present in the food. Since the artificial diet contains a high level of protein (8 per cent) compared to clover (u 2-S), protein seems to be a critical factor affecting digestive enzymes. Both enzymatic activities and larval weight were affected by the protein level in the diet. Reducing the protein content from 76 to 3.6 per cent resulted in a decrease of about 75 per cent in the proteolytic and amylolytic activity of the midgut wall, with a simii decrease being observed in the larval weight. Protease activity of the midgut contents was less afkctexl than that of amylase. This seems due, at least in part, to micro-organisms present in the midgut lumen. Our results indicate that protein afkts digestive enzymes in general and not only the enzyme specifically acting on the protein substrate, i.e. protease.

952

I.

hHAAYA,

I.

MOORR,ANDD. JO~BPH

On clover-which contains about 2.5 per cent protein-amylase activity was much higher than expected from a similar level of protein in the artificial diet (Table 2). This may be due to non-protein factors present in clover, such as a high level of carbohydrates and/or other active compounds which may stimulate amylase (APPLEBAUMet al., 1964; HORI, 1969). 2-EFFECTOF PROTEINUWELINTHEDIET,ONLARVALDWELOPMENT

TABLE

ANDON DIGESTIVE

ENZYMES

Protease activity Protein Larval weight 11 days in diet (%) after emergence (me) 1.6 3.6 5.6 7.6

0.6 37.8 91.3 137.2

Amylaae activity

Midgut wall

Midgut contents

110 397 531

911 1258 1446

Midgut Widl

Midgut contcntd

-

208 887 843

51 236 211

Proteaae activity expressed as O.D. units x 10’ at an abeorbancy of 280 rnp; amylaae activity expreaaed aa O.D. units x 10’ at an abaorbancy of 550 mp.

Our data concur with the concept that certain protein fractions (FISK and SHAMBAUGH,1952, 1964) or other active compounds present in the food (HORI, 1%9), can stimulate digestive enzymes, probably through a hormonal mechanism (DADD, 1961; WIGGLESWORTH,1965). Eflect of enviromnentaZtemperature on the amylase andpotease Temperature is a factor of paramount life processes. It was therefore of interest on amylase and protease activity (Table homogenate indicate that at lO”C, which is

activity in larvae

importance in the regulation of insect to determine the effect of this factor 3). The results for the midgut wall below the threshold for larval develop-

AMYLASANDPROTE.4SlI NMENTAL TEMPRRATXmS ONTHRLARVAL TABLX3-EPPBCT OF ENVIRO ACTIVITY

Proteaae activity Temperature (“C) 10 19 26 32

Amylaae activity

Midgut wall

Midgut contents

Midgut wall

Midgut contents

3;

1281 1241 1485 1847

26 168 287 349

144 754 890 1022

557 833

Thee-day exposure prior to sacrificing; enxymatic activities expressed aa O.D. unita aa described in Table 1.

PmTmsEANDAMYLAsEAcMvrrY

INLARVAEOFWYPTIAN

COlTON

WORM

953

ment (BICHARA,1934), these enzymes lose about 90 per cent of their activity as compared to 32°C. As regards the midgut contents, proteases seem to be afkted by temperature less than amylase, similarly to the efkt obtained with various levels of protein (Table 2). Increasing the temperature from 10 to 32°C enhanced both amylase and protease activity. REFERENCES S. W., HARPM I., and BONDI A. (1964) Amylase wxetion in the larvae of PYO&& litwo F. (Inaecta). Camp. B&hem. PhysioZ. 13, 107-l 11. BICHARAI. (1934) The cotton worm, Prodenh Iituru F., in Egypt. Bull. Sot. R. ent. Egypt. l&288-420. BIRKY., HARPAZI., ISHMYA I., and BONDIA. (1962) Studies on the proteolytic activity of the beetle Tewbrio and Tribolium. J. Insect Physiol. 8,417429. DADD R. H. (1961) Evidence for hormonal of digestive secretion in the beetle, Tenebrio molitor. J. esp. Biol. 38, 259-266. FISK F. W. and SHAMFJAUGH G. F. (1952) Protease activity in adult A&s aqypti mesquites as related to feed& OtioJ. Sei. 52,80-88. FISKF. W. and SHNUEAUGH G. F. (1954) Invertase activity in adult A&es aegyptimosquitoes. OhioJ. Sci. 54, 237-239. HAWKP. B., Oag~ B. L., and SUMMBRBON W. H. (Eds.) (1954) Practical PhysiologicaI Chemistry, 3rd ed. McGraw-Hi& New York. HXMPEL A. M. (1955) The pH in the gut and blood of the larch sawfly, Peristiphora erichrti (HTG), and other insects with reference to the patho8enicity of BucciUus cereus Fr. and Fr. Ccm.J. 2002.33,99-106. HORIK. (1969) E&ct of vahus activators on the salivary amylaw of the bug Lygus dispok. J. Insect Physiol. 15,2305-2317. ISHAAYA I. and BIIU Y. (l%S) The effect of proteina on the inhibitory activity of soybean naponh on certain enzymes. J. Fd Sci. 30,118-120. hAAYA I. and S-1 E. (1970) Invertrae and amyhe activity in the armoured scales Chtysowt$hah aonidumand Aonidisllrr aurantii. J. Insect Physiol. 16,1599-1606. KUNITZM. (1947) CrywaUine soybam trypsin inhibitor-II. General properties. J. gen. Physiol. 30,291-310. MOOREI. and NAVON A. (1969) Calcium al&we: A new approach in the arhhial culturing of insects, appliedto Spodopter~l&or& (Boisduval). Exphanh U, 221-222. NOELTING G. and BPRNWLDP. (1948) sur Aeaenzymes amylolytiques-III. La j9-amylalu: Dosage d’activiti et corm&e de I’abunce d’u-amyhe. H&I. chim. Acta 31, 286-290. SooHooC.F.andFm G. (1966) The consumption, d@tion, and utilization of food plants by a polyphagous inaect, P~odeha cridrmio(Cramer). J. insect Physiol. 12,711730. WALDBAUER G. P. (1%2) The growth end reproduction of maxiliectomhd tobacco homworms feeding on normally rejected non-solanaceous plants. Entomologia exp. a@. 5, 147-158. WA~OUSE D. F. (1952) Studies on the digestion of wool by insects-VI. The pH and oxidation-reduction potential of the alimentary canal of the clothes moth larvae (TeneoZu bisseliiella Humm.). Aust.9. Sci. Res. (B) 5,178-188. WIGGL~SWORTX V. B. (l%S) The Princ@s of Insect Physiology, 6th ed. Methuen, London.

A~PLEBNJM