Corap. Biochem. Physiol., 1973, Vol. 4413, pp. 577 to 585. Pergamon Press. Printed in Great Britain

CHANGES IN ACTIVITY OF PROTEASES DURING EMBRYOGENESIS OF A N A G A S T A KUEHNIELLA

(INSECTA)*

M I L O S K U C E R A 1 and RALPH B. T U R N E R *

1Institute of Entomology, Czechoslovak Academy of Sciences, Prague, Czechoslovakia; and aAgricultural Experiment Station, New Mexico State University, Las Cruces, New

Mexico 88001 U.S.A.

(Received 13 June 1972)

Abstrac t - -1 . Cleavage of hemoglobin by homogenates of eggs of Anagasta kuehniella (Zeller) has been investigated. Three distinct pH optima (4-5, 6"9 and 9"5) of activity are manifested during embryological development. Several characteristics suggest the two activities with lower pH optima are of the cathepsin type while activity at the high pH may be of the trypsin type.

2. The activity having optimum at pH 6"9 fluctuates in a diurnal manner during the 4-day development period. Such diurnal fluctuations in activity persisted even in eggs in which embryonic development had been arrested by exposure to 57°C for 15 min within 6 hr after oviposition.

3. The protein complement undergoes virtually complete alteration during embryogenesis. Loss of soluble proteins from the egg near the end of the developmental period seems to correlate with increases in proteolytic activity.

INTRODUCTION DESPITE considerable interest in the metabolism of nitrogenous compounds during insect embryogenesis, there have been very few reports of work on the activity of proteolytic enzymes. Several reports have indicated that the amounts of free amino acids and peptides increase, as well as vary qualitatively, during embryo- genesis in eggs of several different insect species (Drilhon &Busnel, 1950; Fu, 1957; Colombo et al., 1962; Chen & Briegel, 1965). Proteolytic enzyme activity is most certainly directly involved in such changes and is probably responsible for degradation of yolk proteins for utilization by the developing embryo (Colombo et al., 1962).

Proteolytic activity has been reported to be present in eggs of several different insect species (Lichtenstein, 1947; Lichtenstein et al., 1949; Bodenheimer & Shulov, 1951; Kuk-Meiri et al., 1954; Shulov et al., 1957; Urbani & Rossi, 1959; Kuk-Meiri et al., 1966) and the most recent ones (1957, 1959, 1966) have taken cognizance of changes in specific proteolytic activity during the course of embryo- genesis.

*This research was supported in part by the Czechoslovak and the U.S. National Academy of Sciences. New Mexico Agricultural Experiment Station Journal Series No. 423.

577

578 MILOS KUCERA AND RALPH B. TURNER

T h e present repor t describes some quali tat ive and quant i ta t ive changes in hemoglobin-c leavage activity du r ing embryogenes is of the Med i t e r r anean flour

moth , Anagasta (= Ephestia) kuehniella (Zeller).

MATERIALS AND M E T H O D S

The colony of A. kuehniella was maintained on food prepared as in Table 1. Food was prepared just before use, or stored under refrigeration (4°C) to inhibit microbial growth. Eggs (about 0"2 g) were gently mixed with 1 kg of food into a gallon jar, and a 2"5-cm wide corrugated cardboard roll was placed on top of the inoculated medium. Each jar was covered with a filter paper cap and held at 27°C and 65% relative humidity (r.h.) in a 14 hr light-10 hr dark photoperiod until the adults emerged.

TABLE 1.

Component Parts (by volume)

Lay crumbles 15 Broiler crumbles 30 Rolled barley 30 Raisins 10 Glycerin 8

For oviposition, 200-300 adults were anaesthetized with CO~ and transferred into pint jars fitted with 40-mesh screen caps. Each jar was then inverted over a funnel-shaped device and eggs were collected as they fell into a lower jar.

Thirty-mg portions of eggs which had been deposited during a 6-hr period were washed free of feces and frass with two washings of ice-cold 0"1 M NaC1 solution. Then the eggs were homogenized in a Potter-Elvehjem homogenizer for 3 min with 0"2 ml of 0'1 M NaC1 solution in an ice-water bath. The supernatant liquid was decanted and the residue was washed once with cold 0"1 M NaC1 solution and homogenized again for 3 min in an ice- water bath. The volume of the combined supernates was then adjusted to 1"0 ml with 0"1 M NaC1 solution.

"Heat-killed" eggs were prepared according to the method of Quednau (1957). A 30-mg portion of freshly collected eggs was placed in a small homogenizer tube previously moistened inside with a stream of moist air directed at a point 1 cm above the bottom while immersed in the ice-bath. The tube was shaken so that the eggs formed a single layer adhering to the tube, and then placed in a constant temperature (57°C) water-bath for 15 rain. In our experience this treatment was 100 per cent effective in preventing larval hatch but the specific effect of this heat treatment on embryogenesis is not known.

Determination of protease titer The modified speetrophotometric method of Anson described in Colowick & Kaplan

(1955), in which hemoglobin served as substrate, was used. One unit of proteolytic activity (P.U.) is expressed as 1 x 10 -4 m-equiv, of tyrosine released per hr at 37°C.

Denatured 1% hemoglobin (Miles Laboratories) was brought to appropriate pH values (4"5, 6"9 and 9"5) by the addition of boric, phosphoric or acetic acid, respectively. Each assay tube contained 2"5 ml of 1% hemoglobin solution, 0-1 ml of egg homogenate (except 0"4 ml was used when measuring the low level of protease activity at pH optimum 6"9) and one drop of toluene (as preservative during the 16-hr incubation). Tubes were stoppered, mixed and then incubated at 37°C. After 16 hr 2"5 ml of 10% trichloroacetic acid were

PROTEASES D U R I N G EMBRYOGENESIS OF ANAGASTA KUEHNIELLA 579

mixed into each tube, and the contents allowed to stand for 30 rain before being filtered. Absorbancy of the filtrate was then measured at 280 nm with a Beckman D U speetrophoto- meter. As a control, hemoglobin solution and egg homogenate were incubated for 16 hr in separate tubes and then mixed with trichloroacetic acid and treated as above. All chemicals were of reagent grade or better.

Acrylamide gel electrophoresis The technique described by Davis (1964) was employed. Eggs (30 mg) were homo-

genized for 3 min in 100/zl of Tris-HC1 buffer, 0"15 M at pH 7"0 in 40% sucrose, in an ice-water bath, and centrifuged briefly (ca 10 sec at 5000 g). A 25-/zl portion of the super- natant liquid was applied to the top of each gel and subjected to 3 mA/tube for 80 min. The system having a 5"5 per cent lower gel, pH 8"9, with 3 per cent spacer gel was used. After electrophoresis, the gels were stained with amido black 10B. Materials for the pre- paration of acrylamide gel were purchased from Bio-Rad Laboratories.

RESULTS

Three proteolytic activities with widely differing pH optima are present at different times during embryogenesis.

The first activity designated P1, is apparent as early as 6 hr after oviposition, displays maxima of activity at about 12 and 70 hr (Fig. 1), has a pH optimum of 4.5 and 15 min exposure to 57°C causes a 50 per cent loss of activity.

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FIG. 1. Proteolytic activity, P1, of homogenates made from A. kuehniella eggs as effected by age, by pH and by temperature. P.U., proteolytic unit. One P.U. is defined as that amount of homogenate which liberates 1 x 1074 m-equiv, of tyrosine

per hr at 37°C.

580 MILOS KUCERA AND RALPH B. TURNER

As shown in Fig. 2, a second type of proteolytic activity, P2, is present during early stages of embryogenesis, but its activity is only about 15 per cent that of P1 and its pH optimum is 6.9. It differs also in its sensitivity to heat; 50 per cent activity is lost after 15 min exposure to 62°C. This activity manifests a cyclic

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Fro. 2. Proteolytic activity, P2, of homogenates made from A. kuehniella eggs as effected by age, by pH and by temperature. P.U., proteolytic unit. One P.U. is defined as that amount of homogenate which liberates 1 × 10-4 m-equiv, of tyrosine

per hr at 37°C.

diurnal fluctuation during the 4 days of embryogenesis (Fig. 3). It was most surprising to find that this cyclic diurnal fluctuation of activity actually persisted in eggs which had been exposed to 57°C for 15 min within 6 hr after oviposition to prevent normal development (Fig. 3). The data of Fig. 3 are means of four experiments and the significance of the differences is apparent from the standard deviations drawn as vertical lines.

Figure 4 shows a third protease activity, P3, which becomes apparent in homo- genates of eggs during very late stages of development, has a pH optimum of 9.5 and loses 50 per cent of its activity after 15 min exposure to 63°C.

Changes in protein composition during embryogenesis are revealed by the pattern of protein bands on polyacrylamide gel disc electrophoresis (Fig. 5). Several new protein bands appeared during embryogenesis; bands 3 and 4 at 14 hr and bands 10 and 11 at. 57 hr. As might be expected, virtually all of the original yolk proteins are gone by the end of the 96 hr larval development.

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582 MILOS KUCERA AND RALPH B. TURNER

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Disc electrophoretic patterns of proteins during development of eggs of A. kuehniella.

The protease activities also differ in their sensitivity to metal ions, p CMB and E D T A * (Table 2).

TABLE 2--PERCENTAGE OF PROTEASE ACTIVITY REMAINING AFTER CHEMICAL TREATMENT

Chemical Protease P1 Protease P2 Protease P3

Cu *+ 50"6 7"1 100 Ag + 56 16"2 79"0 Cd ~+ 100 55 100 Hg 2+ 100 6"4 83-8 Co S+ 100 56"6 100 Zn z+ 100 23"2 92'6 Pb 2 + 100 82"8 100 pCMB 40-6 16"2 35'8 EDTA 100 42"5 100

* All metal ions were tested at 10 -3 M. t-pCMB and EDTA were tested at 10 -z M.

DISCUSSION

Our findings indicate that in eggs of A. kuehniella, as in locust eggs (Lichtenstein et M., 1949; Kuk-Meir i et al., 1954; Shulov et al., 1957) there is a definite correla- tion between the stage of embryogenesis and proteolytic activity. In addition our results show cyclic diurnal fluctuation of one protease activity, P2, and changes in activity of two other proteolytic activities, P1 and P3.

Classification of proteolytic enzymes is confusing. Much of the confusion arises from the diversity of assay methods and the variety of substrates employed. Th e term "cathepsin" is generally applied to proteinase activity having acidic pH optima

* pCMB, p-chloromercuribenzoate ; EDTA, ethylenediaminetetracetate.

PROTEASES DURING EMBRYOGENESIS OF ANAGASTA KUEHNIELLA 583

and found in aqueous extracts of tissues. This is differentiated from the better known proteolytic enzymes, pepsin and trypsin, which are secreted into the mammalian alimentary tract (Fruton, 1960). Cathepsin activity has been detected in extracts of numerous organs of various mammals and in extracts of tissues of other vertebrates and invertebrates (Lundblad & Gohl, 1966). Cathepsin activity has also been reported in eggs of the sea urchin (Lundblad et al., 1966) and of the locust (Kuk-Meiri et al., 1966).

On the basis of the above criteria, protease activity P1 and P2 appear to be cathepsin-like in that the pH optima are rather low and activities occur in tissues during early embryogenesis. This classification is consistent with that of Kuk- Meiri et al. (1966), which describes "cathepsin type" activity in locust eggs by the same assay with the same protein substrate that we used.

Classification of P1 and P2 as two distinct protease activities is indicated by their different pH optima; sensitivity to heat; and response to metal ions, pCMB and EDTA (Table 2).

The occurrence of proteolytic activity P1 early in development suggests it is responsible for digestion of egg yolk proteins to provide the embryo with an initial supply of peptides and amino acids as needed at the onset of embryogenesis. It has been suggested (Urbani, 1965) that such acidic protease activity in embryogenesis is of the same type as that in regeneration of tissues. Augmentation of the activity of acidic proteinase and dipeptidase occurs in regions of high RNA content and high respiratory metabolism (Urbani, 1965).

The significance of the circadian fluctuation of P2 is not clear but suggests that a temporal separation of certain nitrogenous metabolites may be necessary at certain times during embryogenesis. At this stage of our investigation the influence of photoperiod has not been established. Nevertheless, it appears that within 6 hr after oviposition a program directing cyclic levels of protease activity at pH 6.9 is established which is not destroyed by a heat treatment sufficient to effectively block larval hatch. The molecular basis to explain the operation and maintenance of such a program in "heat-killed" eggs is not readily apparent, but such cyclic changes may result from fluctuating de novo synthesis and degradation of enzymes or possibly from some other regulatory mechanism involving activation, inhibition or inactivation.

In efforts to detect the presence of activators or inhibitors we mixed portions of low activity preparations with those of high activity. The resulting specific activity was directly proportional to the amount of each particular preparation present. This suggests that the degree of activity is not a result of the presence of any un- bound inhibitor or activator. Additional experiments designed to detect possible bound inhibitors or activators are underway. Other experiments are directed toward determining whether de novo protein synthesis is responsible for the fluctuation. Such an explanation might also require cyclic fluctuation of several additional enzymes, e.g. RNA polymerase, etc.

The absence of proteolytic activity P3 from the egg during early stages of embryogenesis and its increase in activity during development are consistent with

584 MILOS KUCERA AND RALPH B. TURNER

the report by Lichtenstein (1947) that proteolytic activity around pH 8 was not detected in B. mori eggs until a few days before hatching. Activity P3 increases sharply 72 hr after oviposition, and just before hatching it reaches a level of activity which is triple that of the highest activity of P1. This protease activity has a pH optimum similar to that found in newly-hatched larvae and larval feces (Kucera & Turner, unpublished data). This suggests that P3 may be a trypsin-like activity of the larval midgut. In a study of embryonic development and its relation to specific proteolytic activity in the locust, Locusta migratoria migratorioides (R. & F.), Shulov et al. (1957) found that the initiation of proteolytic activity at pH 7.8 coincided with the beginning of the organization of the midgut. This proteolytic activity was attributed to the appearance of a trypsin-like enzyme known to occur in the midgut of the larvae and of adult locust. Such increased activity might also facilitate mobilization of vitelline proteins in response to increased demands of the embryo during this phase of development. The observation that disappearance of several bands from the protein pattern of disc electrophoresis (Fig. 5) corresponds with the increase in activity of enzyme activities, seems to support this suggestion. Gaeta & Zappanico (1959) detected a similar increase in activity of another digestive enzyme, lipase, and suggested the activity was needed to mobilize the vitelline lipids for energy production in late embryogenesis of the silkworm.

The protein pattern of polyacrylamide gel disc electrophoresis reflects the changing pattern of proteins during embryogenesis and demonstrates some general correlations with the fluctuating proteolytic enzyme activity (Fig. 5). As might be expected, almost all of the original yolk proteins are utilized during the 96 hr of egg development (Fig. 5). An interesting decrease in protein bands occurs after 72 hr that coincides generally with the time of the second peak of P1 activity and subsequently with the increased P3 activity. There are several proteins which appear to be synthesized de novo during the course of egg development, labeled as 3 and 4 (at 14 hr) and 10 and 11 (at 57 hr).

The fact that the eggs heated at 57°C for 15 rain do not develop but can still be parasitized by Trichogramma sp. (Quednau, 1957) indicates that the changes which stop the development of the egg are not so drastic as to preclude normal develop- ment of the egg parasite, Trichogramma sp. Such heat treatment which completely inhibits development of the Anagasta embryo is enough to reduce proteolytic activity P1 by only 50 per cent in the egg homogenate (Fig. 1). In the case of the more heat-resistant activities, P2 and P3, about 25 per cent is lost. The significance of the heat sensitivity of these enzymes to the blockage of larval emergence is questionable in consideration of the fact that enzymes are generally more resistant to heat in the protective environs of the intact cell and these heat-sensitivity evalua- tions represent in vitro treatments of egg homogenates.

REFERENCES BODENHEIMER F. S. & SHULOV A. (1951) Egg-development and diapause in the Moroccan

locust. Bull. Res. Court. Israel 1, 59-75.

PROTEASES D U R I N G EMBRYOGENF~IS OF A N A G A S T A K U E H N I E L L A 585

CHEN P. S. & BRIEGEL H. (1965) Studies on the protein metabolism of Culex pipiens L. - -V . Changes in free amino acids and peptides during embryonic development. Comp. Biochem. Physiol. 14, 463-473.

COLOMBO G., BENASSI C. A., ALLEGRI G. & LONGO E. (1962) Free amino acids in eggs of Schistocerca gregaria during development. Comp. Biochem. Physiol. 5, 83-93.

COLOWICK S. P. & KAPL~a'q N. O. (Editors) (1955) Methods in Enzymology, Vol. II , p. 34. Academic Press, New York.

DAVIS B. J. (1964) Disc electrophoresis--II . Method and application to human serum proteins. Ann. N .Y . Acad. Sci. 121, 404-427.

DRILrtON A. & BUSNEL R. G. (1950) Identification of free amino acids in the egg of Bombyx mori. Compt. rend. 230, 1114-1116.

FRUTON J. S. (1960) Cathepsins. In The Enzymes (Edited by BO'C~R P. D., LARDY H. & MYaBACK K.), 2nd edn., 4, 233-241. Academic Press, New York.

Fu Y-Y. (1957) Changes in distribution of sulfur-containing amino acids in developing grasshopper egg (Melanoplus differentialis). Physiol. Zo6l. 30, 1-12.

GAETA I. & ZAPPANICO A. (1959) Lipases in the embryonic development (post-diapause) of Bombyx mori. Ricerca sci. 29, 788-791.

KuK-MEIRI S., LICHTENSTEIN N., SHULOV A. & PENER M. P. (1966) Cathepsin-type proteo- lytic activity in the developing eggs of the African migratory locust (Locusta migratoria migratorioides, R. & F.). Comp. Biochem. Physiol. 18, 783-795.

KuK-MEmI S., SHULOV A. & LICHTENSTEIN N. (1954) Proteases of the eggs of the desert locust. Bull. Res. Coun. Israel 4, 66~8 .

LICHTENSTEIN N. (1947) Proteolytic enzymes of insects--I . Proteases of the silkworm (Bombyx mori L.). Enzymologia 12, 156-165.

LICHTENSTEIN N., BODENHEIM~R F. S. & SHULOV A. (1949) Proteolytic enzymes of insects-- II . Proteases of the eggs of the Moroccan locust (Doeiostaurus maroccanus Thnbg.). Enzymologia 13, 276-280.

LUNDBLAD G. & GOrIL B. (1966) A study of cathepsins in extracts of Brissopsis ovaries. Ark. Kemi 26, 79-86.

LUNDBLAD G., LUNDBLAD M., IMMERS J. & SHILLING W. (1966) Chromatographic analysis of the proteolytic enzymes of the sea urchin egg- - I I . Gel filtration of extracts from unfertilized and fertilized eggs and identification of the catheptic activity. Ark. Kemi 25, 395-409.

QUEDNAU W. (1957) Uber den Einfluss von Temperatur und Luftfeuchtigkeit auf den Eiparasiten Trichogramma cacoeciae Marchal (eine Biometrischen Studie). Mitteil. Biol. Bundesanst. Berlin-Dahlem 90, 63.

SHULOV A., PENER M. P., KuK-MEIRI S. & LICHTENSTEIN N. (1957) Proteolytic enzymes in various embryonic stages of the eggs of Locusta migratoria migratorioides (R. & F.). J . Insect Physiol. 1, 279-285.

URBANI E. (1965) Proteolytic enzymes in regeneration. Regeneration Amin. Related Probl., Int. Symp., Athens, 1964, pp. 39-55.

URRANI E. & ROSSI M. (1959) Dipeptidases in the embryo development of Bombyx mori. Atti accad, nazl. Lincei, Rend., Classe sci. fis., mat. e nat. 26, 54-58.

Key Word Index--Insecta ; protease; egg; diurnal fluctuation; Anagasta kuehnieUa.