The protease activity in the midgut of bivoltine and muitivoltine races of the silkworm,Bombyx mori, was studied. The enzyme activity increased during fifth instar reached a peak, and decreased significantly through pre-pupal stage to the lowest level at the pupal stagTouring pharate adult period, the protease activity increased to reach another peak just before emergence of the moth, and decreased thereafter. The enzyme activity m bivoltmes was about 2–3 times higher than multivoltines at the peak levels. Bivoltines showed a sex difference in midgut protease activity while no significant difference was observed in multivoltines. Larvae and pharate adults showed a difference in the pH optima for the enzyme activity. From the results the possible role of midgut protease during the process of metamorphosis is discussed

Sarangi, S.K.

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Proc. Indian Acad. Sci. (Anim. Sci.), Vol. 94, 1'1/1). 5, October 1985, pp. 567-572.© Printed in India.Alkaline protease in the midgut of the silkworm Bombyx mori L:changes during metamorphosisS K SARANGIR&D Programme in Sericulture, Bangalore University, Bangalore 560009, IndiaMS received 13 February 1985; revised 22 April 1985Abstract. The protease activity in the midgut of bivoltine and multivoltine races of thesilkworm, Bombyx mori, was studied. The enzyme activity increased during fifth instar, reacheda peak, and decreased significantly through pre-pupal stage to the lowest level at the pupalstage. During pharate adult period, the protease activity increased to reach another peak justbefore emergence of the moth, and decreased thereafter. The enzyme activity in bivoltines wasabout 2-3 times higher than multivoltines at the peak levels.Bivoltines showed a sex differencein midgut protease activity while no significant difference was observed in multivoltines.Larvae and pharate adults showed a difference in the pH optima for the enzyme activity. Fromthe results the possible role of midgut protease during the process of metamorphosis isdiscussed.Keywords. Bombyx mori; bivoltine; multivoltine; midgut protease; metamorphosis.1. IntroductionThe most common proteolytic enzymes in the alimentary canal of insects have longbeen recognised as trypsin-like enzymes (Day and Waterhouse 1953;House 1974;Lawet al 1977; Jany et al 1978). Eguchi and Iwamoto (1982) have also established thatmidgut protease, the activity of which has been earlier demonstrated in the alimentarycanal of the larva of the silkworm, Bombyx mori (Horie et at 1963; Eguchi andYoshitake 1967; Hamano and Mukaiyama 1970; Eguchi and Iwamoto 1976) is atrypsin-like enzyme possessing certain properties (Eguchi and Arai 1983). However,most of the studies on the midgut protease ofsilkworms are restricted to the larval stagemainly because of its important role in protein digestion. Eventhough Eguchi et al(1972)have studied the proteolytic enzymes in pharate adults, very little information isavailable on the pattern of change in total protease activity during larval-pupal-adulttransformations in silkworms. It has already been reported that the trypsin-like enzymein mosquitoes changes significantly during metamorphosis (Chen 1978).The presentcommunication deals with the pattern ofchange in total protease activity ofthe midgut,a comparison of the enzyme activity in different races and sexesand the pH optima formidgut protease at different stages of metamorphosis.2. Materials and methods2.1 AnimalsBivoltine (NB18 and NB4D2) and multivoltine (Pure Mysore) silkworm races weremaintained under standard laboratory conditions at 25-28°C and a relative humidity567568 S K Saranqiof 70-90 %on mulberry leaves (M, variety). Silkworms were used from fifth larvalinstar upto the adult stage for the present experiment. The midgut was excised alongwith its contents after freezing the animals at - 200 e for about 12 hr, to avoid any lossof protease activity as most of it is detected in the digestive juice. A 10% (WIV)homogenate of the tissue was prepared in ice-cold borate buffer, pH 9·0and pH 11·0 foradults and larvae respectively, with glass homogenizer. The homogenate was centri-fuged in an IEC refrigerated centrifuge at 3000 RPM for 15 minutes and the supernatantwas used as the enzyme source. Four to five individuals were taken at each time pointfor independent determination and only the mean value of four independentdeterminations is presented in the graph. Further, student's t test was conducted toassess the level of significance.2.2 Enzyme assayProtease activity was assayed according to Eguchi and Iwamoto (1982) with slightmodifications. The pH optima were 11 and 9 respectively for the larval and pharateadult stages. The time of incubation was 30 min for enzyme from the larva and 60 minfor enzyme from the pharate adult. Half ml of 1%casein and 2 m1 of 0-1 M boratebuffer were incubated with 0-5 ml of appropriately diluted enzyme solution at 30°C.The reaction was stopped by adding 2 m1 of 10% TCA and centrifuged. Theconcentration of digested protein in the supernatant was determined colorimetricallywith Beckman DU2 spectrophotometer using Folin's reagent at 660 nm. Proteinconcentration was determined according to the method of Lowry et al (1951). Thespecificactivity of the enzyme was expressed as pmol of tyrosine formed per minute permg of protein.3. ResultsIn bivoltines, males and females showed a significant difference (P = < 0-(01) inmidgut protease,activity during development except the pupal stage (figure 1).In males,the peak enzyme activity was observed around the third day which significantlydecreased(P = < 0-(01) thereafter during the fifth instar development. While infemales, the enzyme activity increased significantly (P = < 0-(01) to reach a peakaround the sixth day and decreased thereafter. In general, midgut protease activity washigh in females compared to males during the larval stage. In both males and females,there was about 50-70 fold decrease in total protease activity from larval to pre-pupalstage which decreased further to attain the lowest level during pupal stage. Duringpharate adult stage, protease activity increased gradually to attain another peak justbefore emergence of the moth and decreased thereafter during adult period. Theenzyme activity in males during pharate adult stage was about 2 times higher than thatof females.In Mysore race, there was no significant difference in midgut protease activitybetween male and female throughout the development except the pharate adult stage(P = < 0-025) (figure 2).Peak activity ofthe enzyme was observed around the sixth dayof fifth instar development in both the sexes. There was about 75-90 fold decrease intotal protease activity from larval to pre-pupal stage which reached its minimum valueMidgut protease in silkworms100,----------------------,569'"10 10xc.0;'0L.o,e-~cE"-.,..0E::L• MALEo FEMALEV3 V6 V9 PP P P4 P8 E AFigure 1. Changes in midgut protease activity during development of bivoltines (V) fifthinstar in days (PP) pre-pupa (P) pupa in days (E) before emergence (A) adult .N'0xc~a.Cl'E<,cE<,.,..~::L10• MALEo FEMALEV3 V6 V9 PP P P4 P8 E AFigure 2. Changes in midgut protease activityof pure Mysore during development (V) fifthinstar in days (PP) pre-pupa (P) pupa in days (E) before emergence (A) adult.during the pupal stage in both males and females. During pharate adult stage, theenzyme activity increased slowly' to reach another peak just before emergence anddecreased rapidly (P = < 0'(01) in both the sexes during adult stage.Figure 3 shows the effectofpH on the total protease activity from larval and pharateadult stages of both bivoltines and multivoltine (Pure Mysore). During larval stage,only one peak was observed at pH 11·0 in both the races and the enzyme activity in570 S K Saranqi40 I LARVA I'"'0- 20xc:~'-G-O'~c:~IIIc:.;;;e~II>III'sE:l0.4/.-~ BIVOLTINE•0-0",O-~ 0 PURE MYSOREI PHARATE ADULT I..--~'"• BIVOLTINE0·2 /~""o...........0 PURE MYSORE8 10pH12Figure 3. Effect of pH on the alkaline protease of the midgut.bivoltines was about 2·5 times higher than that of multivoltine. During pharate adultstage, the bivoltines interestingly showed two Peaks ofenzyme activity at pH 9 and pH11.But in case ofpure Mysore only one peak was observed at pH 9.The enzyme activityin bivoltines was about 2·5 times higher than multivoltines during pharate adult stage.4. DiscussionMidgut enzymes in insects are synthesized in the midgut epithelium and secreted intothe gut lumen (Engelmann 1969;Engelmann and Geraerts 1980;Eguchi and Arai 1983).Thus changes in total protease activity of the midgut reflect changes in the overallproduction of the enzyme during .metamorphosis. A significantly high activity ofmidgut protease during fifth instar larval development of silkworms shows a higherrate of enzyme synthesis corresponding to enhanced food intake (Waldbauer 1968).This might facilitate a greater utilization of proteins for larval growth and silkMidgut protease in silkworms 571production as well. Higher protease activity in bivoltines might result in betterconversion of exogenous proteins which ultimately lead to production of more silkcompared to multivoltines (Tanaka 1964). The sharp decrease in midgut proteaseactivity from larval to pupal stage shows the difference between active feeding and non-feeding stages during metamorphosis. Thus it can be presumed that the rate ofsynthesisof midgut protease is dependent upon the rate of food intake. Furthermore, traces ofprotease activity during pupal stage might be the residual larval enzymes. In pharateadults, protease activity increases and reaches another peakjust before emergence. Thisis in accordance with earlier reports (Eguchi et al 1972) and supports their view thatmidgut protease in pharate adult might contribute either in part or full towards theformation of cocoonase.The present study shows that the optimum pH for total protease activity of themidgut is around pH 11'0 in the larval stage. Eguchi and Iwamoto (1976) have shownthat the pH optima for protease from midgut tissue and digestive fluid are the same.Interestingly a shift in pH optima for protease activity is observed during meta-morphosis from pH 11·0 at larval stage to pH 9·0 at pharate adult stage. It cannot beconfirmed at present whether the larval protease is different from adult protease orwhether they are two isoenzymes of protease active at different stages of metamor-phosis. The presence of two peaks, each at pH 9·0 and pH 11,0, in midgut proteaseactivity of bivoltine pharate adult, likewise, again provides a basis to speculate theexistence of two isoenzymes of protease having different pH optima characteristics.However, the single peak at pH 9'0, in the enzyme activity ofmultivoltine pharate adultrequires further studies to draw any conclusion.AcknowledgementGrateful acknowledgements are due to the World Bank for financial assistance and tothe co-ordinator, R&D Programme in Sericulture, Bangalore University for facilities.ReferencesChen P S 1978 Regulation of proteolytic enzymes In: Biochemistry ofinsects (ed.) M Rockstein (New York:Academic Press) pp. 162-168Day M F and Waterhouse D F 1953 The mechanism of digestion. In: Insect physiology (ed.) K D Roeder(London: Chapmann & Hall) pp. 311-330Eguchi M and Arai M 1983 Relationship between alkaline proteases from the midgut lumen and epithelia ofthe silkworm; solubilization and activation of epithelial protease (6B3); Comp. Biochem: Physiol., B7S589--593Eguchi M and Iwamoto A 1976 Alkaline proteases in the midgut tissue and digestive fluid of the silkworm,Bombyx mari; Insect Biochem. 6491-496Eguchi M and Iwamoto A 1982 Comparison of three alkaline proteases from digestive fluid of the silkworm,Bombyx mari, L.; Compo Biochem. Physiol~ B71 663-668Eguchi M and Yoshitake N 1967 Electrophoretic variation of Proteinase in the digestive juice of thesilkworm, Bombyx mari, L.; Nature (London) 214 843-844.Eguchi M, Furukawa S and Iwamoto A 1972 Proteolytic enzyme in the midgut of the pharate adult of thesilkworm Bombyx mori; J. Insect. Physiol., 182457-2467Eguchi M, Iwamoto A and Yamauchi K 1982 Interrelation ofproteases from the midgut lumen, epithelia andperitrophic membrane of the silkworm, Bombyx mori, L.; Compo Biochem. Physiol. A72 359--363572 S K SarangiEngelmann F 1969Food-stimulated synthesis of intestinal proteolytic enzymes in the cockroach Leucophaeamoderae; J. Insect Physiol. 15217-235Engelmann F and Geraerts WPM 1980 The proteases and the protease inhibitor in the midgut ofLeucophaea moderae; J. Insect Physiol. 26703-710Hamano K and Mukaiyama F 1970 Some properties of digestive fluid proteinase in the silkworm Bombyxmarl with references to the relation between dissociation degree and nutritive value of some proteins;J. Sericult. Sci. Jpn 39 371-376Horie Y, Tenaka M and Ito T 1963 Proteolytic enzymes of digestive juice and midgut of the silkworm,Bombyx marl; J. Sericult. Sci. Jpn 32 8-15House H L 1974Digestion In:The physiologycfinsecta(ed.) M Rockstein (New York: Academic Press) Vol. 5,pp.63-117Jany K D, Haug Hand Ishay J 1978 Trypsin-like endopeptidases from the midguts of the larvae from thehornets of Vespa orientalis and Vespa crabro; Insect Biochem. 8221-230Law J H, Dunn P F and Kramer K J 1977 Insect proteases and peptidases; Adv. Enzymol. 45 389-425Lowry 0 H, Rosebrough N J, Fare A L and Randall R J 1951 Protein measurement with the Folin phenolreagent; J. Bioi.Chem. 193265-275Tanaka Y 1964 Silkworm Races. In Sericoloqy (Bombay: Central Silk Board) pp. 99-104Waldbauer G P 1968 The consumption and utilization of food by insects In: Advances in insect physiology(eds) J W L Beament, J E Treherme and V B Wigglesworth (London: Academic Press) Vol. 5, pp. 229--288

Alkaline protease in the midgut of the silkwormBombyx mori L: changes during metamorphosis

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Alkaline protease in the midgut of the silkwormBombyx mori L: changes during metamorphosis

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