Effect of developmental derangements on the proteolytic and protease-inhibitory activities in Galleria mellonella (Insecta)

Comp. Biochem. Physiol. Vol. 79B, No. 2, pp 255-261, 1984 Printed in Great Britain

0305-0491/84$3.00 + 0.00 © 1984Pergamon Press Ltd

EFFECT OF DEVELOPMENTAL DERANGEMENTS ON THE PROTEOLYTIC AND PROTEASE-INHIBITORY ACTIVITIES IN GALLERIA MELLONELLA (INSECTA) M. KUt~ERA, F. SEHNAL and J. MAL.~ Institute of Entomology, Czechoslovak Academy of Sciences, Flemingovo n. 2, 166 09 Prague 6, Czechoslovakia (Received 8 February 1984)

Abstract--1. Protease inhibitory activity in the whole body homogenates of Galleria mellonella larvae exhibits maxima at the beginning of the last larval and pupal instars. Injury, chilling, immobilization, and ligations of larvae cause an increase of inhibition. 2. The inhibitory activity is high in the haemolymph but low in midgut and faty body. By contrast, the proteolytic activity is low in haemolymph and high in both midgut and fat body. 3. Starvation and ligations cause a dramatic fall of the proteolytic activity and increase of the inhibitory activity in examined organs.

INTRODUCTION

The importance of limited proteolysis, which is regulated in part by interaction of proteolytic enzymes with their natural inhibitors, in biological control mechanisms has been recognized only during the last decade. Protease inhibitors are known to control the enzymes involved in blood coagulation, fibrinolysis, inflammation and immunodefense (Heimburger, 1975); in intracellular zymogen activation, fertilization and some morphogenetic processes (Reich et al., 1975). The inhibitors also seem to play a role as defense mechanisms against undesirable proteolytic activities released in vivo after tissue damage, and even against proteolytic enzymes of invader organisms. Studies of the former mechanism in human medicine have proven the suitability of protease inhibitors for therapeutic use (Baugh and Schnebli, 1980); and examinations of the latter mechanism in plants led to a new biochemical model of pestinduced natural plant protection (Ryan, 1978). The role of proteolytic enzymes and especially of their inhibitors in insect development and defense have been studied only to a small extent. A comprehensive review of insect proteases concerns mostly the digestive enzymes and cocoonases and barely touches the role of proteases in development (Law et al., 1977). Attention has been paid in recent years to resolution, isolation and characterization of proteases whereas their physiological and developmental significances have been examined by only a few authors (Lockshin et al., 1980; Mandal et al., 1981; Eguchi et al., 1982; Terra and Ferreira, 1981). Although only a small number of studies has been devoted to naturally-occurring inhibitors of insect proteolytic enzymes, the inhibition has been demonstrated both in the eggs (Kang and Fuchs, 1973; Ku~era and Turner, 1981) and in the postembryonic stages (Kang and Fuchs, 1980). So far the protease inhibitors have been localized in the haemolymph (Hanschke and Hanschke, 1975; Eguchi and Kanbe, 255

1982) and midgut (Houseman, 1980). Some of the identified inhibitors have been partially purified. Engelmann and Geraerts (1980) studied correlations between proteolytic and inhibitory activities in the midgut of the cockroach Leuophaea maderae. The system protease-protease inhibitor apparently acts in the insects as a regulatory mechanism which responds to parasitism (Ku~era and MadziaraBorusiewicz, 1982) and which changes in the course of metamorphosis (Eguchi and Kanbe, 1982). It may also be implicated in other developmental and defense situations but experimental data are lacking. The present report describes fluctuations in protease and protease inhibitory activities in Galleria mellonella larvae and pupae during their normal development and after various adverse treatments. MATERIALS AND METHODS

Larvae and pupae of age known within +6 hr after preceding ecdysis were take from the standard culture of Galleria mellonella (Lepidoptera, Pyralidae) which was maintained as described by Sehnal (1966). Last instar larvae were subjected to treatments listed in Table 1 either at the time of intensive feeding (36 hr, i.e., 2rid day of the VIIth instar, marked as VII/2) or when they finished feeding and were about to initiate cocoon spinning (108-120 hr, VII/6). Before most treatments the larvae were anaesthetized by being submerged in water for 15--30rain. Only starvation, which was combined with isolation of individual larvae in glass tubes (10 mm in diameter, 5 cm in length), was applied without anaesthesia. Chilling was accomplished by placing the larvae directly on ice for about 20 min. In the case of immobilization, the larvae were attached to a glass slide by means of Scotch tape (Edwards, 1967). The attached larvae did not feed and the restraint inhibited pupation. Surgical treatments included removal of a metathoracic wing disc, brain extirpation (decerebration), decapitation by means of a ligation applied between head and thorax, and thoracic ligation at which head, prothorax and mesothorax were cut off. The wing discs were removed through a V-shaped cut in the adjacent integument, the other operations were performed as described previously (Malfi et al., 1977).

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Tissues for each biochemical determination were dissected from 15-20 insects. The haemolymph was collected into a glass capillary, which was rinsed with a concentrated solution ofphenylthiourea, from a wound inflicted by cutting off a thoracic leg. Each larva was then cross-cut at the level of the metathorax and the viscera were pressed out into ice-cold Ephrussi-Beadle saline; the remaining body wall was classified as the integument. Silk glands, fat body and midgut were separated from the other viscera, washed in saline (mJdgut was first cut longitudinally and its contents removed), and collected on glass slides where the excess of saline was drained off. All organs were stored in glass tubes at - 2 0 C until used. The data are means of two parallel experiments. For the determination of proteolytic activity the organs were homogenized (t :2 w/v) in 0.6°, NaCI solution and the homogenates were centrifuged for 10 min at 5000g. Protein content in the supernates was measured according to the method of Warburg and Christian (1957). The 0.2ml of each supernate was added to 0.5 ml of 1'!,~,azocasein (Charney and Tomarelli, 1947) and the mixture was incubated for 4 hr at 30C. Incubation was terminated with 1 ml of 80,, trichloroacetic acid and after 15rain the mixture was filtered. The filtrate was mixed with equal volume of 0.5 M NaOH and the absorbance at 440 nm was measured. One protease unit (PU) was defined as such amount of proteolytic enzymes that caused the same A440 as 1 ~tg trypsin (EC 3.4.4.4) from Serva. In all organs the protease activities were established at optimal pH; haemolymph, pH 8.5, integument and silk glands, pH 9.0; midgut and fat body, pH 10. The protease inhibitory activity was measured after homogenization in 6°Jo perchloric acid (organs/acid w/v, 1:2) (Kurera, 1984). The hornogenates were kept for 3 min at 6 0 C to destroy the remainder of the native proteases; then their pH was adjusted to 6.0 with 5 M K2CO 3 and they were centrifuged for 10min at 5000g (Fritz et al., 1974). A mixture containing 0.2 ml of this perchloric acid extract and 0.2 ml of trypsin solution (10 #g/ml) was preincubated for 10 min at 2ff~C before the addition of 0.5 ml of l",g azocasein at pH 7.8. Incubation and spectrophotometric measurements were the same as described above. Protease inhibitory activity was assessed from the decrease of azocasein hydrolysis by trypsin. One inhibitory unit (IU) was defined as such amounts of inhibitors that suppressed proteolysis caused by I/~g of standard trypsin. Our study

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Fig. 2. Changes in protease inhibitory activity in the whole body homogenates after adverse treatments applied on the 2nd or 6th day of the last larval instar. The differences from initial values established on the day of treatment are given. Treatments: immobilization ( ~ ) , decerebration ( - - - ) , isolation of the abdomen (. . . . ), decapitation (. . . . . ). starvation (..-), chilling ( - - ), and wing extirpation ( I. (not shown) revealed that insect inhibitors impede the activity of trypsin to a similar extent as that of insect proteases. RESULTS

Protease inhibitory activity during development and after adverse treatments Figure 1 d e m o n s t r a t e s that the overall inhibitory activity, as established in the whole homogenates, fluctuated d u r i n g development. A m o d e r a t e activity o f a b o u t 30 I U / g body weight in the penultimate larval instar increased to nearly 50 I U / g at the beginning of the last instar, fell gain to ca. 30 I U / g towards the t e r m i n a t i o n of feeding, a n d during larval-pupal m o u l t it rose to a second peak. Subsequently the inhibitory activity d r o p p e d to a b o u t 15 I U / g by the middle of the p u p a l instar (the last p o i n t measured). These d a t a indicated that each ontogenetic stage was associated with a different level of protease inhibition. The larvae exposed to adverse conditions showed a n increase o f the inhibitory potential (Fig. 2) even t h o u g h their developmental fate was different (Table 1). The treatments applied at VII/2

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Table 1. Effectof adverse treatments on the development of Galleria larvae Larval s t a g e a t t r e a t m e n t

Treatment

VII/2

VII/6 Pupal ec~ysis i n 2 days

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Pupal ecdysia i n 6-7 days

Wing d i s c extirpation

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Starvation and isolation in tubea

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Pupation in 5-I0 days

Chilling

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Inhibition o f spinning, delay of pupal ecdyaia (mobile prepupae o c c u r ' o n days d and 5)

I~obilization

Block o f d e v e l o p m e n t

( r a r e l y only a delay)

Block o f development ( r a r e l y a delay, only 1 prepupa was found on day 5)

No d e v e l o p m e n t

NO development

Decerebration Decapitation Thoracic ligatlon

and g r o v t h

Days in this table are counted from the time o f sixth (VII/6) day o f the last larval instar.

Pupal ecdyaia delayed by 3-5 days; larvae are in cocoons 5 d~ya a f t e r the treatment but t h e i r f u r t h e r d e v e l o p m e n t is h a l t e d

treatment, i.e., from the second(VII/2)or the

always caused an increase in inhibition to a maximal value in 3-5 days, whereas treatments at VII/6 induced a peak of inhibition within a single day (Fig.

Relationship between proteolysis and protease in. hibition in selected organs

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Measurements of proteolytic activity and proteolytic inhibition in several individual organs during the last larval instar disclosed important tissuespecific differences (Fig. 3). Haemolymph contained high inhibitory activity throughout the instar, with a maximum of nearly 1.6 IU/mg of protein on day 4 and minima on days 2 and 6 (both about 0.7 IU/mg). The proteolytic activity of haemolymph was relatively low and decreased as the instar progressed, from ca. 0.3 PU/mg to a little more than 0.1 PU/mg. In contrast, the midgut and fat body were characterized by low inhibitory but high proteolytic activities. The inhibitory activity was maintained below 0.1 IU/mg except for a sharp peak (0.4 IU/mg) detected in the midgut of pharate pupae. The proteolytic activity in the midgut and fat body was high throughout the feeding and post-feeding periods,

When compared to fluctuations in normal development, the change in inhibitory activity was insignificant after the extirpation of a wing disc. Relatively small changes were also caused by starvation and chilling, although both these treatments had pronounced effects on development (Table 1). The greatest alteration of the inhibitory activity was brought about by immobilization (Fig. 2). The decrease, which followed after a sharp increase in inhibition in the immobilized insects, could indicate that some of them had partially overcome the restraint and resumed development. In larvae whose development was prevented permanently by the removal of brain (decerebration, decapitation, and thoracic ligation) the increase of inhibitory activity continued until the end of our observation period.

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Fig. 4. Fluctuations in protease inhibitory and proteolytic activities in haemolymph ( ), gut (---), and fat body (...) in larvae starved since the 2nd day of the last instar (experimental day 0). The values of haemolymph inhibitory activity are to be multiplied by 2. with maxima over 0.6 PU/mg on days 1 and 6 of the instar; the profile of these changes during the instar was reciprocal to that of the inhibitory activity in the haemolymph. In both midgut and fat body the proteolytic activity rapidly dropped to less than 0.1 PU/mg between the onset of spinning (day 6) and pharate pupa (day 7). Changes in inhibitory and proteolytic activities shown in Fig. 3 may result from either changes in the feeding regime of larvae or their developmental progress in course of the last larval instar. In order

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to distinguish between these two alternatives, we have examined larvae starved since VII/2 (Fig. 4). During the first three days of starvation these larvae seemed to reach a developmental stage corresponding to the beginning of cocoon spinning but their further development was halted (Table 1). During these three days the protease inhibiting properties of the haemolymph changed in a similar manner as between the 2nd and 5th day of the normal last larval instar (cf. Figs 3 and 4). Fluctuations in the inhibitory activity in the midgut and fat body resembled the profile which was normally found between days 4 and 7 of the instar. The low proteolytic activity in the haemolymph did not much change after starvation. In the midgut and fat body, however, the originally high activity decreased to very low levels which were normally found only in pharate pupae. The ligation of larvae behind the prothorax prevented feeding and removed all major nervous and endocrine regulatory centres. Further development was blocked. A common feature of larvae ligated on day 2 (Fig. 5) as well as those ligated on day 6 (Fig. 6) was the instant drop of proteolytic activity in all examined organs. Within 3 days after ligation the proteolytic activity in haemolymph and midgut fell under 0.05 PU/mg and in the fat body to about 0.1 PU/mg. The decrease continued until the end ot our observation 6 days after ligation. Activities of protease inhibitors in the ligated larvae showed a diversified pattern. In larvae ligated on day 2, the protease inhibitory capacity of all organs rose until it reached the highest levels established in our study (Fig. 5)+ Less uniform changes in protease inhibition were detected in larvae ligated on day 6 (Fig. 6). A dramatic fluctuation occurred only in the haemolymph where the inhibition first increased (somewhat similar to normal development) but then it decreased to a level lower than was ever found in

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Fig. 6. Fluctuations in the protease inhibitory and in the proteolytic activities in haemolymph ( ), gut (---), and fat body (,-') in isolated larval abdomens. Isolation on the 6th day of the last instar (experimental day 0). The values of haemolymph inhibitory activity are to be multiplied by 2. the normal larvae. It is interesting to note that changes in the protease inhibitory capacity of haemolymph in both the starved (Fig. 4) and ligated larvae (Figs 5 and 6) followed a similar course as fluctuations detected in the whole body homogenates of such larvae (Fig. 2).

three isoinhibitors of identical molecular mass (about 13,000 daltons) but of different isoelectric points. All of them are resistant to 6% perchloric acid. It would be interesting to find out if observed fluctuations in the inhibitory activity concerned all three or only some of the isoinhibitors.

DISCUSSION

The proteolytic enzymes of G. mdloneUa It may seem unlikely that only three proteinase inhibitors would interact with proteolytic enzymes in a large number of diverse tissues which may contain tissues-specific proteases. No attempt has been made to characterize the proteases of different tissues of G. mellonella but our experiments indicate that they are similar at least in respect to their pH optima which are in all cases in the neutral or alkaline regions. It is probable that proteolytic enzymes from diverse tissues possess further similarities and are thus susceptible to identical protease inhibitors. The inhibition of native Galleria proteases and also of various other of both serine and sulfhydryl types (Ku~era, 1984) indicates that Galleria inhibitors are little selective. Inhibition of different proteases of the same and even of other species has been demonstrated with protease inhibitors from several other insects (Houseman, 1980; Kang and Fuchs, 1980).

Protease inhibition during normal development The first peak of the total protease inhibitory activity in the last instar (Fig. I) coincides with the period of intensive food utilization and maximal growth rate (Sehnal, 1971). The second peak extends from larval-pupal apolysis until the second day of the pupal instar. This period is characterized by destruction of larval organs such as gut, silk glands and Malphigian tubuli; by reconstruction of tracheae, nervous system, fat body and some other tissues; and by intensive growth of the imaginal primordia of wings, appendages, gonads, etc. (Sehnal, 1968). Occurrence of high inhibitory activity just in the periods when some tissues contain highly active proteases suggests that the inhibitors are used to suppress undesirable proteolytic activities. We propose that the high level of inhibitors which is found in the haemolymph protects the organism against proteases that "leak" into haemoco¢l from various tissues. Alternatively, the blood inhibitors could pass into the tissues and reduce their endogenous proteolytic activities. It is known that even large protein molecules can penetrate in and out from insect tissues (Wigglesworth, 1943; Locke and Collins, 1967). The protease inhibitors of G. mellonella have not been as yet sufficiently characterize~i. A separate study (Ku~era, 1984) demonstrates that the complex of proteolytic inhibitors in this species is composed of

Proteases and protease inhibitors after adverse treatments A characteristic feature of larvae subjected to adverse treatments was an increase in the protease inhibitory activity (Fig. 2). Larvae treated as VII/2 exhibitecl a maximum inhibition 3-5 days after the treatments whereas in the VII/6 larvae the maximal response occurred already on the first day. The most plausible explanation of this difference can be found in the normal levels of protease inhibitors (Fig. 1). In the VII/2 larvae, the inhibitory activity is at a peak

260

M. KU~ERA et al.

so that no rapid increase is needed to cope with the effects of adverse treatments. In the VII/6 larvae, however, the inhibitory activity is normally at an ebb; its immediate increase appears to be an important component of the adjustment to adverse treatments. The increase of the inhibitory activity presumably serves a similar purpose as in normal development, i.e. the inhibitors suppress excessive proteolytic activity. Proteolytic enzymes may be liberated from damaged tissues after the wing disc extirpation or after ligations. In the starved, the immobilized and the ligated larvae (and to a certain extent, probably also after the other treatments) the inhibitors may be required for the change from the originally high metabolic rate to a low level of nitrogen metabolism. Owing to the reduced metabolic rate, the starved (Sehnal, 1966), the immobilized (Edwards, 1967), and particularly the ligated larvae (Malfi et al., 1977) can all survive for many weeks without any further supply of nutrients. The increase of protease inhibitors in the ligated larvae that are deprived of head also reveals that brain plays no important role in the stimulation of inhibitors. The complexity of the control of proteolytic activity is seen from the analysis of individual organs in larvae subjected to adverse treatments (Figs 4~6). In some instances the reduction in proteolytic activity could be attributed to a rise in protease inhibitors within the examined organ. This is the case of haemolymph, midgut and fat body in the VII/2 isolated abdomens (Fig. 5). In some other cases, however, e.g. in the fat body of the starved VII/2 larvae, a decrease was detected in both the inhibitory and the proteolytic activities (Fig. 4). Control of proteolytic activity by the availability of substrates and by specific metabolic stimulators has been demonstrated at least in some tissues (e.g. Thomsen and Moller, 1963). Compartmentalization of the proteolytic activity within cells is another important mechanism of the in t,ivo regulation of proteolysis (Tashiro et al., 1976). Our study indicates that a separate and not negligible role is played by protease inhibitors. We cannot draw unequivocal conclusions but can ask at least two specific and novel questions: W h a t is the interaction of haemolymph inhibitors with tissue proteases? Are changes in the proteolytic and inhibitory activities a shock response or rather an adaptive reaction to adverse treatments? technical assistance of Miss Alena Alegovfi and Mrs Ludmila Senohrfibkovtl is appreciated. Our thanks are also due to Prof. R. B. Turner of New Mexico State University, Las Cruces, for critical reading of the manuscript. Acknowledgements--Skilful

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