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Different course of proteolytic inhibitory activity and proteolytic activity in Galleria mellonella larvae infected by Nosema algerae and Vairimorpha heterosporum
JOURNAL
OF INVERTEBRATE
PATHOLOGY
45, 41-46 (1985)
Different Course of Proteolytic Inhibitory Activity and Proteolytic Activity in Galleria me/lone//a Larvae Infected by Nosema algerae and Vairimorpha heterosporum M. KUCERA Department
of Insect
Pathology,
AND J. WEISER
Institute of Entomology, Prague. Czechoslovakia
Czechoslovak
Academy
of Sciences,
Received April 26, 1984: accepted July 9, 1984 The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th.instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia. 0 1985 Academic Press. Inc. KEY WORDS: Galleria mellonella, larvae of: Nosema algerae; Vairimorpha heterosporrrm: proteolytic inhibitors; proteases.
fected by two species of microsporidia vading different larval organs.
In diseased plants and animals inhibitors of proteolytic activity can act as a defense mechanism against undesired proteolytic enzymes released from the damaged tissue of the host, as well as against the proteases released from the pathogen (Hanschke and Hanschke, 1975; Ryan, 1978). We found enhanced proteolytic inhibitory activity in diseased larvae of Cephalcia falleni (Kucera and Madziara-Borusiewicz, 1982). In the greater wax moth, Galleria mellonella, the presence of a potent proteolytic inhibitor which inhibits the toxic protease of the entomopathogenous fungus, Metarhizium anisopliae, was established. The inhibitor was separated into three isoinhibitors, and the fluctuation of inhibitory activity in the organs during the larval development was described (Kucera 1984; KuEera et al., 1984). In this report we describe differences in protease inhibitory activity and in protease activity in the hemolymph, gut, and fat body in the larvae of G. mellonella in-
MATERIALS
Two-day-old
in-
AND METHODS
larvae of the 7th instar of G.
mellonella were used for all experiments.
This insect has been maintained in the insectary of the Department of Insect Pathology for several years on a diet described by Haydak (1936). The larvae were divided into three groups. The first group was infected with a suspension of spores of Nosema algerae perorally, with 30,000 spores per loop; the second group was injected with spores of Vairimorpha heterosporum, 25,000 spores per inoculum, into the body cavity; and the third group was used as control. All experiments were performed in duplicate. The hemolymph was collected into a glass capillary, after cross cutting of larvae, and the fat body and midgut were separated and kept in glass tubes at -20°C and used
41 0022-2011/85 $1.50 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
42
KUtERA
AND
for analysis within 48 hr. Ten to fifteen animals were used for preparation of both perchloric acid and saline homogenates. Determination of inhibitory activity in perchloric acid homogenates of the organs (6% perchloric acid, 1:2 w/v) was performed by the method based on hydrolysis of azocasein described previously (Kucera et al., 1984). One inhibitory unit (IU) is defined as the activity which inhibits the proteolysis caused by 1 kg of standard trypsin (EC 3.4.4.4) from Serva. Inhibitory activity is expressed in IU per milligram of protein. Determination of proteolytic activity in saline homogenates of the organs (0.6% NaCl, 1:2 w/v) was performed with 1% azocasein as substrate after the method of Charney and Tomarelli (1947). One unit of protease activity (PU) is defined as the activity of 1 )-Lgof standard trypsin. Proteolytic activity is expressed in PU per milligram of protein. In all organs the protease activities were measured at optimal pH (KuCera et al., 1984). Protein content of the supernates from saline homogenates were measured according to the method of Warburg and Christian (1957). RESULTS Larvae of G. mellonella infected with the microsporidia N. algerae or V. heterosporum did not pupate at the usual time for normal larval development (8th day of 7th instar), but remain in the developmental stadium with the infection until Day 12 (N. algerae infection), or Day 22 (V. heterosporum infection). Inhibitory and proteolytic activities followed the same pattern in both normal and diseased larvae up to Day 8 of the 7th instar (= 6th day after infection), which corresponds with our previous finding in normal G. mellonella larvae (Kucera et al., 1984). After this time, however, when normal larvae have pupated, proteolytic and inhibitory activities responded in different ways to the infection with N. algerae and V. heterosporum, as can be seen from Figures I-3. Figure 1 demon-
WEISER
strates that the inhibitory activity, which is normally high in hemolymph compared to other organs, increases in N. algeraeinfected larvae on the 6th day, from about 0.4 to 1.5 IU (statistically significant at the P = 0.05 level), and maintains this level up to the 12th day, when some of the larvae (60%) pupate. In V. heterosporum-infected larvae, the elevation of inhibitory activity in hemolymph occurred later, on the 8th to 9th days, and the maximum activity was somewhat lower (1.3 IU) than with N. algerae infection (P = 0.05 against the 6th day of infection). Up to the 20th day there was a slow decrease of inhibitory activity to the value of 0.9 IU. Proteolytic activity in the hemolymph remained low with both microsporidia, at about the same level as found in normal larvae. With V. heterosporum, at the end of the experiment the activity increased to 0.3 PU (P = 0.1 against the 13th day of infection), in accordance with the slowly decreasing level of inhibitors. Inhibitory activity increased in hemolymph, gut, and fat body following infection by either N. algerae or V. heterosporum. The specific inhibitory activity in hemolymph increased to the same extent when the wax moth was infected with either microsporidium, but increased more rapidly in response to N. algeare than to V. heterosporum (Fig. 1). Similarly, inhibitory activity increased sooner in the gut in response to infection with N. algerae, and the maximum specific inhibitory activity was almost double that attained in response to infection with V. heterosporum (Fig. 2) (P = 0.05 if values obtained at the peak plus two following days are considered). There was a concomitant reduction in specific proteolytic activity, being more pronounced after infection with N. algerae than with V. heterosporum. In contrast, there was a reverse situation in fat body (Fig. 3). The inhibitory activity was increased much less in larvae infected with N. algerae, reaching its peak at 0.2 IU. than in those infected with V. hetero-
PROTEOLYTIC
INHIBITORS
b
1.6
‘4
‘O
DAYS
AND PROTEASES
OF IllfECllON
FIG. 1. Proteolytic inhibitory activity and proteolytic activity in the hemolymph of Galleria me/lonella larvae infected by Nosema algerae (a) and Vairimorpha heterosporum (b). Vertical lines represent standard errors. Arrow indicates pupation of normal larvae.
up to the end of the experiment. With V. heterosporum-infected larvae, the activity increased from the 8th day, with a maximum of 0.6 IU on the 14th day of infection (P = 0.05 for both maxima). The proteolytic activity in N. algerae-infected larvae, after a drop to ca. 0.3 PU (6th day sporum
after infection), remained constant, while in V. heterosporum, after a temporary drop and elevation, the activity remained below 0.4 PU and then rapidly decreased to less than 0.1 PU, again inverse to the increasing inhibitory activity. At the end of the experiment, in moribund larvae, an increase of
I
0.0 -
b
0.6 -
. T
DAYS
ii.
. Yo'
.
. '1;.
. .
OF IWECTIOM
FIG. 2. Proteolytic inhibitory activity and proteolytic activity in the gut of Caller& mellonellu larvae infected by Nosema algerae (a) and Vuirimorpha heterosporum (b). Vertical lines represent standard errors. Arrow indicates pupation of normal larvae.
44
KUCERA
proteolytic curred.
AND
activity to more than 0.5 PU oc-
WEISER
sporidia do not pupate represents the first visible change as compared with normal larvae which pupate on the 8th day of the last instar. The absence of pupation may be connected with the release of some hormone-like compounds which were first studied by Fisher and Sanborn (1962). Besides this, there are other possible factors influencing pupation. After infection by microsporidia, changes in enzyme activities can be found: alanine aminotransferase increases on the 5th day after infection (KuCera and Weiser, 1973a) and, in our experiments, enhanced protease inhibitor levels have been found on the 6th and 9th days, depending on the microsporidia used. The high activity of proteolytic inhibitors in the hemolymph compared with other organs coincides with the appearance of vegetative stages in the gut (N. algerae infection) and the fat body (V. heterosporum infection). The hemolymph can serve as a pool for proteolytic inhibitory activity, which is subsequently transported to the infected organs to inactivate released proteolytic enzymes from the cells. At the same time hemolymph may serve also as reservoir where the inactivation of proteases
DISCUSSION
N. algerae infects the Malphigian tubules and the anterior part of the midgut muscular layer. Spores appear first in the Malphigian tubules in 5-6 days. At that time only vegetative stages are seen in the midgut. After S- 10 days, small irregular centers of developing microsporidia spread over all the midgut, and the Malphigian cells in the infected areas are inflated and do not function. Not all tubules are infected. The molts and pupation are postponed to the 12th or 13th days of infection, but not to an extent which may produce mummies. V. heterosporurn concentrates only in the fat body of injected caterpillars. Here the vegetative stages concentrate, with evidence of the infection, after 8 days. First spores appear, and they accumulate subsequently and fill all cells of the fat body. The larvae then changes into a mummy. The average final spore count in N. algerae was 2 x lo* per animal and in V. heterosporum IO9 per animal. The fact that larvae infected with micro-
I 0.D .
0.0 .
. 0.0
a
‘4
IS
DAYS
OF IIFECTIOI
I
- 0.1
b
.
FIG. 3. Proteolytic inhibitory activity and proteolytic activity in the fat body of Galleria me/lone//u larvae infected by Nosema algerae (a) and Vairimorpha heterosporum (b). Vertical lines represent standard errors. Arrow indicates pupation of normal larvae.
PROTEOLYTIC
INHIBITORS
leaking from infected organs takes place. We are aware that these explanations are only speculations at the present time, and further work must be done to substantiate these suggestions. The course of inhibitory activity in the gut and in the fat body differs with each of the microsporidia, reflecting the specificity of the organs for the disease. V. heterosporum also evokes, in the gut, some limited reaction, whereas N. algerae, which develops and attacks only the tissue of the gut, does not evoke any elevation of inhibitory activity in the fat body. Proteolytic activity in the organs declines quickly to very low levels as a response to the increased inhibitory activity. Only in the fat body of N. algerae-infected larvae was there no elevation of inhibitory activity and decrease of proteolytic activity. The inverse course of both the activities points to a direct regulation of proteolytic enzymes by their natural inhibitors in the course of microsporidian diseases. The increase of inhibitory activity and corresponding decrease of proteolytic activity in the gut and fat body reduces the level of nitrogen metabolism in the particular organs, and thus enables the larvae to survive with the infection for a long period of time until the microsporidian finishes its development. Thus, the stabilized level of inhibitory activity in the fat body between 12th and 17th days of V. heterosporum infection fits this “surviving phase” of the larvae, whereas the onset of elevation of inhibitory activity between the 9th and 12th days is connected with the beginning of sporogony. Only at the end of V. heterosporum infection, i.e., on the 18th to 20th days, does the proteolytic activity increase again in moribund larvae, which may indicate that the capacity of the inhibitor available is exhausted and cannot compensate for the proteolytic activity released from the organs and produced by the microflora penetrating from the gut. The longlasting control of proteases by inhibitors of proteolytic activity during the disease further supports our interpretation of the re-
AND PROTEASES
45
sults of experiments with Nosema plodiaeinfected larvae of Barathra brassicae (KuEera and Weiser, 1973b), in which the elevation of proteolytic activity has been found also only at the very end of the disease. In our recent work (Kucera et al., 1984) we found enhanced inhibitory activity under several adverse treatments. With microsporidia, the elevation of inhibitory activity lasts much longer, especially with V. heterosporum infection where it persists for at least 9 days or almost until the larvae die. This long persistence is apparently a reflection of the long-lasting interaction between the host and the pathogen. Data explaining the function of proteolytic inhibitors remain fragmentary. Their fluctuations during insect development, their enhanced activity in insect diseases, and their relatively broad spectrum of activity against proteases of different origins point to their importance in physiopathology. ACKNOWLEDGMENTS The skillful technical assistance of Miss Alena AleSova is highly appreciated. We are also indebted to Professor R. B. Turner from New Mexico State University, Las Cruces, for critical reading of the manuscript.
REFERENCES CHARNEY, J., AND TOMARELLI, R. M. 1947. A colorimetric method for the determination of the proteolytic activity of duodenal juice. J. Biol. Chem., 171, 501-505. FISHER, F. M., AND SANBORN, R. C. 1962. Production of insect juvenile hormone by the microsporidian parasite Nosema. Nature (London), 194, 1193. HANSCHKE, R., AND HANSCHKE, M. 1975. Untersuchungen zum Vorkommen und zur Funktion eines Proteinaseinhibitors in der Hamolymphe von Insekten. Acta E&l. Med. Germ. 34, 531-537. HAYDAK, M. H. 1936. A food for rearing laboratory insects. J. Econ. Entomol., 29, 1026. K&ERA, M. 1984. Partial purification and properties of Galleria mellonella larvae proteolytic inhibitors acting on Metarhizium anisopliae toxic protease. J. Znvertebr. Pathol., 43, 190- 196. K&ERA, M., AND MADZIARA-BORUSIEWICZ, K. 1982. Proteolytic inhibitors, proteases and hemocyte changes in diapausing and diseased larvae of Ce-
KUCERA
46 phalcia falleni (Hymenoptera, Em. Bohemoslors.. 79, 401-405.
Pamphiliidae).
KUCERA, M., SEHNAL. F., AND MALL of developmental derangements on and protease inhibitory activities in nella. Comp. Biochem. Physiol., in KUCERA, M., AND WEISER, J. 1973a. transferase in the three last larval athra brassicue infected by Nosemu vertebr.
Pathol.,
AND WEISER
Actu
J. 1984. Effect the proteolytic G~IIeria me/lopress. Alanine aminoinstars of Burplodiae.
.I. In-
21, 287-292.
KUC‘ERA, M., AND WEISER, J. 1973b. Alanine amino-
transferase. alkaline phosphatase. and protease activity in Burathru brassicae during microsporidian infection. 1. Inlaertebr. Puthol., 21, I?1 - 122. RYAN, G. A. 1978. Proteinase inhibitors in plant leaves: A biochemical model for pest-induced natural plant protection. Trerlds Biochem. Sci., 7, 148150.
WARBURG, 0.. AND CHRISTIAN. W. 1957. Protein estimation by ultraviolet absorption. III “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan. eds.). Vol. 3, p. 451, Academic Press, New York.
OF INVERTEBRATE
PATHOLOGY
45, 41-46 (1985)
Different Course of Proteolytic Inhibitory Activity and Proteolytic Activity in Galleria me/lone//a Larvae Infected by Nosema algerae and Vairimorpha heterosporum M. KUCERA Department
of Insect
Pathology,
AND J. WEISER
Institute of Entomology, Prague. Czechoslovakia
Czechoslovak
Academy
of Sciences,
Received April 26, 1984: accepted July 9, 1984 The activity of protease inhibitors and proteases was studied in the hemolymph, gut, and fat body of 7th.instar larvae of Galleria mellonella infected by two microsporidia, Nosema algerae and Vairimorpha heterosporum. The increase in inhibitory activity in the hemolymph was substantial, and coincided with the development of the disease. The increase in inhibitory activity in the gut was almost doubled by N. algerae as compared with V. heterosporum, whereas the increase in inhibitory activity in fat body was found only in V. heterosporum-infected larvae. The course of proteolytic activity followed an inverse pattern to the elevated activity of inhibitors in the gut and the fat body, and rose only in moribund larvae at the end of the course of V. heterosporum infection. The differences in the pattern of proteases and inhibitors reflect the organ specificity of each of the microsporidia. 0 1985 Academic Press. Inc. KEY WORDS: Galleria mellonella, larvae of: Nosema algerae; Vairimorpha heterosporrrm: proteolytic inhibitors; proteases.
fected by two species of microsporidia vading different larval organs.
In diseased plants and animals inhibitors of proteolytic activity can act as a defense mechanism against undesired proteolytic enzymes released from the damaged tissue of the host, as well as against the proteases released from the pathogen (Hanschke and Hanschke, 1975; Ryan, 1978). We found enhanced proteolytic inhibitory activity in diseased larvae of Cephalcia falleni (Kucera and Madziara-Borusiewicz, 1982). In the greater wax moth, Galleria mellonella, the presence of a potent proteolytic inhibitor which inhibits the toxic protease of the entomopathogenous fungus, Metarhizium anisopliae, was established. The inhibitor was separated into three isoinhibitors, and the fluctuation of inhibitory activity in the organs during the larval development was described (Kucera 1984; KuEera et al., 1984). In this report we describe differences in protease inhibitory activity and in protease activity in the hemolymph, gut, and fat body in the larvae of G. mellonella in-
MATERIALS
Two-day-old
in-
AND METHODS
larvae of the 7th instar of G.
mellonella were used for all experiments.
This insect has been maintained in the insectary of the Department of Insect Pathology for several years on a diet described by Haydak (1936). The larvae were divided into three groups. The first group was infected with a suspension of spores of Nosema algerae perorally, with 30,000 spores per loop; the second group was injected with spores of Vairimorpha heterosporum, 25,000 spores per inoculum, into the body cavity; and the third group was used as control. All experiments were performed in duplicate. The hemolymph was collected into a glass capillary, after cross cutting of larvae, and the fat body and midgut were separated and kept in glass tubes at -20°C and used
41 0022-2011/85 $1.50 Copyright All rights
0 1985 by Academic Press, Inc. of reproduction in any form reserved.
42
KUtERA
AND
for analysis within 48 hr. Ten to fifteen animals were used for preparation of both perchloric acid and saline homogenates. Determination of inhibitory activity in perchloric acid homogenates of the organs (6% perchloric acid, 1:2 w/v) was performed by the method based on hydrolysis of azocasein described previously (Kucera et al., 1984). One inhibitory unit (IU) is defined as the activity which inhibits the proteolysis caused by 1 kg of standard trypsin (EC 3.4.4.4) from Serva. Inhibitory activity is expressed in IU per milligram of protein. Determination of proteolytic activity in saline homogenates of the organs (0.6% NaCl, 1:2 w/v) was performed with 1% azocasein as substrate after the method of Charney and Tomarelli (1947). One unit of protease activity (PU) is defined as the activity of 1 )-Lgof standard trypsin. Proteolytic activity is expressed in PU per milligram of protein. In all organs the protease activities were measured at optimal pH (KuCera et al., 1984). Protein content of the supernates from saline homogenates were measured according to the method of Warburg and Christian (1957). RESULTS Larvae of G. mellonella infected with the microsporidia N. algerae or V. heterosporum did not pupate at the usual time for normal larval development (8th day of 7th instar), but remain in the developmental stadium with the infection until Day 12 (N. algerae infection), or Day 22 (V. heterosporum infection). Inhibitory and proteolytic activities followed the same pattern in both normal and diseased larvae up to Day 8 of the 7th instar (= 6th day after infection), which corresponds with our previous finding in normal G. mellonella larvae (Kucera et al., 1984). After this time, however, when normal larvae have pupated, proteolytic and inhibitory activities responded in different ways to the infection with N. algerae and V. heterosporum, as can be seen from Figures I-3. Figure 1 demon-
WEISER
strates that the inhibitory activity, which is normally high in hemolymph compared to other organs, increases in N. algeraeinfected larvae on the 6th day, from about 0.4 to 1.5 IU (statistically significant at the P = 0.05 level), and maintains this level up to the 12th day, when some of the larvae (60%) pupate. In V. heterosporum-infected larvae, the elevation of inhibitory activity in hemolymph occurred later, on the 8th to 9th days, and the maximum activity was somewhat lower (1.3 IU) than with N. algerae infection (P = 0.05 against the 6th day of infection). Up to the 20th day there was a slow decrease of inhibitory activity to the value of 0.9 IU. Proteolytic activity in the hemolymph remained low with both microsporidia, at about the same level as found in normal larvae. With V. heterosporum, at the end of the experiment the activity increased to 0.3 PU (P = 0.1 against the 13th day of infection), in accordance with the slowly decreasing level of inhibitors. Inhibitory activity increased in hemolymph, gut, and fat body following infection by either N. algerae or V. heterosporum. The specific inhibitory activity in hemolymph increased to the same extent when the wax moth was infected with either microsporidium, but increased more rapidly in response to N. algeare than to V. heterosporum (Fig. 1). Similarly, inhibitory activity increased sooner in the gut in response to infection with N. algerae, and the maximum specific inhibitory activity was almost double that attained in response to infection with V. heterosporum (Fig. 2) (P = 0.05 if values obtained at the peak plus two following days are considered). There was a concomitant reduction in specific proteolytic activity, being more pronounced after infection with N. algerae than with V. heterosporum. In contrast, there was a reverse situation in fat body (Fig. 3). The inhibitory activity was increased much less in larvae infected with N. algerae, reaching its peak at 0.2 IU. than in those infected with V. hetero-
PROTEOLYTIC
INHIBITORS
b
1.6
‘4
‘O
DAYS
AND PROTEASES
OF IllfECllON
FIG. 1. Proteolytic inhibitory activity and proteolytic activity in the hemolymph of Galleria me/lonella larvae infected by Nosema algerae (a) and Vairimorpha heterosporum (b). Vertical lines represent standard errors. Arrow indicates pupation of normal larvae.
up to the end of the experiment. With V. heterosporum-infected larvae, the activity increased from the 8th day, with a maximum of 0.6 IU on the 14th day of infection (P = 0.05 for both maxima). The proteolytic activity in N. algerae-infected larvae, after a drop to ca. 0.3 PU (6th day sporum
after infection), remained constant, while in V. heterosporum, after a temporary drop and elevation, the activity remained below 0.4 PU and then rapidly decreased to less than 0.1 PU, again inverse to the increasing inhibitory activity. At the end of the experiment, in moribund larvae, an increase of
I
0.0 -
b
0.6 -
. T
DAYS
ii.
. Yo'
.
. '1;.
. .
OF IWECTIOM
FIG. 2. Proteolytic inhibitory activity and proteolytic activity in the gut of Caller& mellonellu larvae infected by Nosema algerae (a) and Vuirimorpha heterosporum (b). Vertical lines represent standard errors. Arrow indicates pupation of normal larvae.
44
KUCERA
proteolytic curred.
AND
activity to more than 0.5 PU oc-
WEISER
sporidia do not pupate represents the first visible change as compared with normal larvae which pupate on the 8th day of the last instar. The absence of pupation may be connected with the release of some hormone-like compounds which were first studied by Fisher and Sanborn (1962). Besides this, there are other possible factors influencing pupation. After infection by microsporidia, changes in enzyme activities can be found: alanine aminotransferase increases on the 5th day after infection (KuCera and Weiser, 1973a) and, in our experiments, enhanced protease inhibitor levels have been found on the 6th and 9th days, depending on the microsporidia used. The high activity of proteolytic inhibitors in the hemolymph compared with other organs coincides with the appearance of vegetative stages in the gut (N. algerae infection) and the fat body (V. heterosporum infection). The hemolymph can serve as a pool for proteolytic inhibitory activity, which is subsequently transported to the infected organs to inactivate released proteolytic enzymes from the cells. At the same time hemolymph may serve also as reservoir where the inactivation of proteases
DISCUSSION
N. algerae infects the Malphigian tubules and the anterior part of the midgut muscular layer. Spores appear first in the Malphigian tubules in 5-6 days. At that time only vegetative stages are seen in the midgut. After S- 10 days, small irregular centers of developing microsporidia spread over all the midgut, and the Malphigian cells in the infected areas are inflated and do not function. Not all tubules are infected. The molts and pupation are postponed to the 12th or 13th days of infection, but not to an extent which may produce mummies. V. heterosporurn concentrates only in the fat body of injected caterpillars. Here the vegetative stages concentrate, with evidence of the infection, after 8 days. First spores appear, and they accumulate subsequently and fill all cells of the fat body. The larvae then changes into a mummy. The average final spore count in N. algerae was 2 x lo* per animal and in V. heterosporum IO9 per animal. The fact that larvae infected with micro-
I 0.D .
0.0 .
. 0.0
a
‘4
IS
DAYS
OF IIFECTIOI
I
- 0.1
b
.
FIG. 3. Proteolytic inhibitory activity and proteolytic activity in the fat body of Galleria me/lone//u larvae infected by Nosema algerae (a) and Vairimorpha heterosporum (b). Vertical lines represent standard errors. Arrow indicates pupation of normal larvae.
PROTEOLYTIC
INHIBITORS
leaking from infected organs takes place. We are aware that these explanations are only speculations at the present time, and further work must be done to substantiate these suggestions. The course of inhibitory activity in the gut and in the fat body differs with each of the microsporidia, reflecting the specificity of the organs for the disease. V. heterosporum also evokes, in the gut, some limited reaction, whereas N. algerae, which develops and attacks only the tissue of the gut, does not evoke any elevation of inhibitory activity in the fat body. Proteolytic activity in the organs declines quickly to very low levels as a response to the increased inhibitory activity. Only in the fat body of N. algerae-infected larvae was there no elevation of inhibitory activity and decrease of proteolytic activity. The inverse course of both the activities points to a direct regulation of proteolytic enzymes by their natural inhibitors in the course of microsporidian diseases. The increase of inhibitory activity and corresponding decrease of proteolytic activity in the gut and fat body reduces the level of nitrogen metabolism in the particular organs, and thus enables the larvae to survive with the infection for a long period of time until the microsporidian finishes its development. Thus, the stabilized level of inhibitory activity in the fat body between 12th and 17th days of V. heterosporum infection fits this “surviving phase” of the larvae, whereas the onset of elevation of inhibitory activity between the 9th and 12th days is connected with the beginning of sporogony. Only at the end of V. heterosporum infection, i.e., on the 18th to 20th days, does the proteolytic activity increase again in moribund larvae, which may indicate that the capacity of the inhibitor available is exhausted and cannot compensate for the proteolytic activity released from the organs and produced by the microflora penetrating from the gut. The longlasting control of proteases by inhibitors of proteolytic activity during the disease further supports our interpretation of the re-
AND PROTEASES
45
sults of experiments with Nosema plodiaeinfected larvae of Barathra brassicae (KuEera and Weiser, 1973b), in which the elevation of proteolytic activity has been found also only at the very end of the disease. In our recent work (Kucera et al., 1984) we found enhanced inhibitory activity under several adverse treatments. With microsporidia, the elevation of inhibitory activity lasts much longer, especially with V. heterosporum infection where it persists for at least 9 days or almost until the larvae die. This long persistence is apparently a reflection of the long-lasting interaction between the host and the pathogen. Data explaining the function of proteolytic inhibitors remain fragmentary. Their fluctuations during insect development, their enhanced activity in insect diseases, and their relatively broad spectrum of activity against proteases of different origins point to their importance in physiopathology. ACKNOWLEDGMENTS The skillful technical assistance of Miss Alena AleSova is highly appreciated. We are also indebted to Professor R. B. Turner from New Mexico State University, Las Cruces, for critical reading of the manuscript.
REFERENCES CHARNEY, J., AND TOMARELLI, R. M. 1947. A colorimetric method for the determination of the proteolytic activity of duodenal juice. J. Biol. Chem., 171, 501-505. FISHER, F. M., AND SANBORN, R. C. 1962. Production of insect juvenile hormone by the microsporidian parasite Nosema. Nature (London), 194, 1193. HANSCHKE, R., AND HANSCHKE, M. 1975. Untersuchungen zum Vorkommen und zur Funktion eines Proteinaseinhibitors in der Hamolymphe von Insekten. Acta E&l. Med. Germ. 34, 531-537. HAYDAK, M. H. 1936. A food for rearing laboratory insects. J. Econ. Entomol., 29, 1026. K&ERA, M. 1984. Partial purification and properties of Galleria mellonella larvae proteolytic inhibitors acting on Metarhizium anisopliae toxic protease. J. Znvertebr. Pathol., 43, 190- 196. K&ERA, M., AND MADZIARA-BORUSIEWICZ, K. 1982. Proteolytic inhibitors, proteases and hemocyte changes in diapausing and diseased larvae of Ce-
KUCERA
46 phalcia falleni (Hymenoptera, Em. Bohemoslors.. 79, 401-405.
Pamphiliidae).
KUCERA, M., SEHNAL. F., AND MALL of developmental derangements on and protease inhibitory activities in nella. Comp. Biochem. Physiol., in KUCERA, M., AND WEISER, J. 1973a. transferase in the three last larval athra brassicue infected by Nosemu vertebr.
Pathol.,
AND WEISER
Actu
J. 1984. Effect the proteolytic G~IIeria me/lopress. Alanine aminoinstars of Burplodiae.
.I. In-
21, 287-292.
KUC‘ERA, M., AND WEISER, J. 1973b. Alanine amino-
transferase. alkaline phosphatase. and protease activity in Burathru brassicae during microsporidian infection. 1. Inlaertebr. Puthol., 21, I?1 - 122. RYAN, G. A. 1978. Proteinase inhibitors in plant leaves: A biochemical model for pest-induced natural plant protection. Trerlds Biochem. Sci., 7, 148150.
WARBURG, 0.. AND CHRISTIAN. W. 1957. Protein estimation by ultraviolet absorption. III “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan. eds.). Vol. 3, p. 451, Academic Press, New York.