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Glucocorticoid action on the growth and development of insects
Life Sciences, Vol. 40, pp. 1725-1730 Printed in the U.S.A.
Pergamon Journals
GLUCOCORTICOID ACTION ON THE GROWTH AND DEVELOPMENT OF INSECTS Anthony M. Gawienowski, Lawrence J. Kegsler, Barrie S. Tan I and Chih-Ming YinDepartments of Biochemistry, Chemistry 1 and Entomology 2, University of Massachusetts, Amherst, Massachusetts 01003 (Received in final form February 9, 1987) Summary Cortisol increased growth and differentiation in the large milkweed insect (Oncopeltus fasciatus). The glucocorticoid significantly increased the growth of the insect as analyzed by wet and dry weights. Cortisol also stimulated the development of the insects over that of the controls during the six day bioassay. The presence and activity of various mammalian hormones in non-mammalian life systems have been demonstrated in many cases from sterols in AzoCobacCer chroococcum and Escherichia coli (I); to, in our own laboratory, estrone and cholesterol in plants (2,3); to testosterone in invertebrates (4). Androgens and estrogens have been found in dytiscid beetles (5) and dipterans (6,7). Insulin, glucagon, somatostatin, gastrin, cholecystokinin, vasopressin, neurophysin, enkephalin, and endorphin-like peptides have been localized in a wide variety of species (8). Thus, a considerable overlap may exist in the endocrine mechanisms of insects and m~mmals despite the evolutionary separation between them. Studies have indicated that both prokaryotes and eukaryotes possess some similar biochemical mechanisms for the synthesis and/or catabolism of steroids (4). In recent articles (9,10), the physiological effects of several corticosteroids on mung bean seedlings (Phaseolus aureus Roxb.) were demonstrated to enhance the plant growth. This effect on the plant metabolism prompted this examination of the possible effects of a number of glucocorticoids and mineralocorticoids on the large milkweed bug Oncopeltus fasc~a~us (Dallas). Methods Steroids, obtained from Sigma Co., St. Louis, M0, were dissolved in pesticide grade acetone to obtain the respective concentrations for administration to the insect. 0nly acetone was applied to the control insects. In each experimental run, every group began with ten randomly selected third instars of comparable weights and age. All weights were measured on a Mettler H-20 (± 0.01 mg) balance. Average and individual weights for each group were measured and recorded. To,facilitate treatment, each group of insects was ventilated with CO 2 for i to 2 minutes to induce immobility. While immobile, the insects were administered with I #I of the treatment solution on their lower abdominal surface. This i ~i was measured with an Instrumentation Co. microapplicator and a Thomas Scientific apparatus D-10 250 #i syringe. After treatment, the groups were housed separately in containers provided with distilled water and cracked-raw unsalted sunflower seeds, enough for more than 6 days. These 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1726
Glucocorticoid Action
containers were kept in a Percival cycle at 28°C (±2°C).
Vol. 40, No. 17, 1987
incubator under a 16 hour light: dark
After the six day period each group was exposed to a lethal dose of hydrogen cyanide and then individual weights were obtained and recorded. Subsequently, the insects were placed in an oven at 76°C for 2 days of drying. After the drying period, the insects were again individually weighed. All weights were compared using a single analysis of variance (II). The cortisol trial was conducted with five dilutions, 0.8, 0.5, 0.I, 0.01 and 0.001 #g/~l which were compared to a standard control. The six-day assay was utilized and the results of the trial are reported in Table i. Earlier trials of 30 days showed no significant differences between controls and treated bugs since they apparently all were at the growth plateau. This earlier work led us to the 6-day trials which were at the rapid growth sta~e. Results The slopes of each growth curve (Figure i) indicate that most of the treated bugs molted to the fourth {nstar (steepest curve) between the third and fourth days. The duration of the third stadium for the controls was approximately six day s and this was confirmed in the literature to be 6.1 days (12). In order to evalute any action of the cortisol on the development of the insects, they were observed in this experiment for seven days after treatment (Figure i). All the control and cortisol treated insects (groups of I0) were still in the third instar on day 4. Therefore the changes in development from day 4 to day 7 are given in Figure I. As can be noted only 8 of the control bugs were in the fourth instar by day 7, which contained the least development among the groups. In the cortisol groups treated with 0.5 ~g/~l or 0.01 #g/#l, three bugs in the former and two in the latter group were already at the fifth instar on day 7. A comparable effect was noted for the ll-dehydrocorticosterone (II-DHC) treatment. All relative measurements from experimental trials are cited in Tables i, 2 and 3. Weights, both individual wet and dry, of cortisol-treated insects and statistical analysis are presented in Table I. A similar growth pattern to cortisol was noted in a trial with varying concentrations of IIDHC. In this respect, the largest rate differences appeared to be between the 0.I #g/ #I treatment and the controls. Statistical analysis of individual weights is reported in Table 2. The results in Table 3 illustrates the hormone actions which are similar to the past cortisol-mung bean seedling studies (9,10). In addition to an effect on development, cortisol treatments increased significantly the body weight of recipients at certain doses. Table i shows that cortisol treatments at 0.8, 0.5, 0.01, and 0.001 #g/insect caused significant increases in body wet and dry weights when compared to controls. Interestingly, only the cortisol treatment at 0.I #g/insect did not cause a significant increase in either wet or dry weight. In each treatment group, wet or dry weights followed by the same letter are not significantly different from each other at p <0.05 (analysis of variance). In contrast to cortisol treatments, II-DHC caused no significant increases in weight at 0.5, 0.01, and 0.001 #g/insect. However, at 0.i ~g/insect, a significant increase was recorded for II-DHC (table 2), also a similar dose effect was noted in a preliminary trial.
Vol. 40, No. 17, 1987
Glucocorticoid Action
1727
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DAYS Figure i.
Action of cortisol on the development of
0"4eopel#us fascians
1728
Glucocorticoid Action
Vol. 40, No. 17, 1987
Topical application of four different corticoids (Table 3) demonstrated no significant differences between the treatment and control insects. A significant weight difference was found between the insects treated with 58pregnane-3~, 21-diol-20-one and deoxycorticosterone acetate. Table I Effect of Cortisol on Weight Gain of Oncopelrus fasciarus
Treatment ~g/~I/insect 0.8 0.5 0.i 0.01 0.001 Control
No.
6 17 16 18 19 19
Final Weight (mg) Wet Dry Mean i S.E.* Mean i S.E.* 40.91 41.37 31.19 38.37 37.87 25.11
± ± ± ± ± ±
1.78 a 2.08~ 2.30 b'c 2.99 a'c 1.77~ 'c 1 83 b
18.39 20.07 13.75 16.44 16.33 9,73
± ± ± ± ± ±
1.50 a 1.15 a 1.31 b c 1.66 a'c 1.01 a'c 1.01 b
*For each experiment, data from different treatments were analyzed with ANOVA. Means followed by different letters are significantly different from each other at p < 0.05 Table 2 Action of II-DHC on Weight Gain of Oncopelcus fasciarus
Treatment #g/#i/insect 0.5 0.i 0.01 0.001 Control
No.
Final Weight (mg) Wet Dry Mean i S.E.* Mean i S.E.*
9 9 9 i0 i0
27.06 43.09 28.17 27.50 23.69
± ± ± ± ±
1.85~ 2.45 ° 2.12 a 3.22 a 1.20 a
9.45 19 97 11.14 10.33 8.87
± ± ± ± ±
0.91~ 1.37 b 0.93 a 1.69 a 0.47 a
*See Table I Discussion This research demonstrated, for the first time, the stimulatory effects of cortisol on the growth and rate of development of the large milkweed bug OncopelCus fascia~us in its change from third to fourth instars. It is very interesting to note that the growth patterns of these treated insects (Table i) were very similar to those reported in the mung bean seedling (9) and later confirmed by our laboratory (I0). The significance of these findings may indicate a wider potential use of glucocorticoids and mineralocorticoids outside of mammalian systems, specifically in the insects. As noted earlier in the introduction, so-called mammalian hormones are found in plants (2). Therefore, the treatment with cortisol is not that unusual as insects and their relatives are phylogenetically closer to mammals than are plants. In fact, cortisol therapy has been shown to increase the mite (D. brevis) population in human skin (13).
Vol. 40, No. 17, 1987
Glucocorticoid Action
1729
Cortisol and II-DHC both had stimulatory effects in the present study, and in past plant studies (9,10). However, the 5~-compound used in this study demonstrated virtually no effect on growth, while Geuns (9) noted that 5~-compounds generally induced growth in plants. Although the plant and insect did show common responses to some pharmacological agents, the differences indicate a potential use of adrenal cortical metabolites for additional biochemical studies and possible future use for insect control. Table 3 Effects of: i) 5~-Pregnane-3,21-diol-20-one 2) Aldosterone 3) ll-Deoxycortisol 4) Deoxycorticosterone acetate on Weight Gain
Treatment ~g/~I/insect i) 0.I 2) 0.i 3) 0.i A) 0.i Control
No.
Final Weight (mg) Wet Dry Mean ± S.E.* Mean ± S.E.*
8 i0 9 8 9
23.51 31.95 23.15 37.48 26.86
+ ++ + +
2.60 a 2.96 a'c 1.44 a 1.83 b'c'd 3.43 a'd
9.73 13.26 9.65 16.78 11.82
+ + + +_ +
1.32 a 1.78 a'c 0.54 a 1.18 D'c'd 1.98 a'd
*See Table i The statistical significant data substantiated the importance of our findings, but it would be worthwhile to add refinements to the procedures. It would benefit future researchers to use individual insects and house them separately as opposed to groups. This type of experiment would enable the measurement of other parameters as each individual insect could be labeled and traced over a growth period. The six-day assay with which we achieved the present results in growth patterns from the application of cortisol, an endogenous mammalian hormone, may lead to the development of bioassays using the insects as opposed to laboratory m=mm, ls. This switch would effectively lead to less expensive experiments and produce rapid bioassays that do not require elaborate preparation. In this sense, Oncopeltus fasciatus has the potential of becoming a popular research animal as suggested by Feir (12). References and Notes i. 2. 3. 4.
5. 6. 7. 8.
9.
N. G. CARR and I. W.CRAIG, Phytochemical Phylogeny, J. B. Hardborne, pp. 119-143, Academic Press, New York (1970).. A. M. GAWIENOWSKI and C. C. GIBBS, Phytochem. 8, 685-686 (1969). A. M. GAWIENOWSKI and C.C. GIBBS, Phytochem. 8, 2317-2319 (1969). T. SANDOR, S. SONEA and A. Z. MEHDI, Trends in Comparative Enzymology (Amer. Zool. 15 suppl.), E. J. W. Barrington, pp. 227-253 T. J. Griffiths Sons, New York (1975). H. SCHILDKNECHT, Agnew. Chem. Int. Ed. 9, 1-9 (1970). R. MECHOULAM, R. W. BRUEGGEMEIER and D. L. DENLINGER, Experentia AO, 942-944 (1984). D. DeCLERCK, H. DIEDERIK, and A. DeLOOF, Insect Bioohem. IA, 199-208 (1984). K. J. KRAMER, Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 7, G. A. Kerkut and L. I. Gilbert, pp. 511-536, Pergamon Press, Oxford, England (1985). J. M. C. GEUNS, Aspects and Prospects of Plant Growth Regulators, B. Jeffcoat, pp. 209-217, Vantage Press, Oxfordshire, England (1980).
1730
i0. ii.
12. 13. 14.
Glucocorticoid Action
Vol. 40, No. 17, 1987
A. M. GAWIENOWSKI, K. M. CSERNUS, J. E. SIMON and L. E. CRAKER, Steroids, 46, 727-733 (1986). R. R. SOKAL and F. J. ROHLE, Biometry, The Principles and Practice of Statistics in Biological Research, W. H. Freeman and Co., San Francisco (1969). D. Feir, Ann. Rev. Entomology 19, 81-96 (1974). Y. SATO, H. HIGUCHI and U. SAITO, Jap. J.Dermatology 75, 331 (1965). This study was supported in part from Mass. Agric. Exp. Sta. Project Hatch 570 (C.-M. Y.), and listed as publication no. 2767.
Pergamon Journals
GLUCOCORTICOID ACTION ON THE GROWTH AND DEVELOPMENT OF INSECTS Anthony M. Gawienowski, Lawrence J. Kegsler, Barrie S. Tan I and Chih-Ming YinDepartments of Biochemistry, Chemistry 1 and Entomology 2, University of Massachusetts, Amherst, Massachusetts 01003 (Received in final form February 9, 1987) Summary Cortisol increased growth and differentiation in the large milkweed insect (Oncopeltus fasciatus). The glucocorticoid significantly increased the growth of the insect as analyzed by wet and dry weights. Cortisol also stimulated the development of the insects over that of the controls during the six day bioassay. The presence and activity of various mammalian hormones in non-mammalian life systems have been demonstrated in many cases from sterols in AzoCobacCer chroococcum and Escherichia coli (I); to, in our own laboratory, estrone and cholesterol in plants (2,3); to testosterone in invertebrates (4). Androgens and estrogens have been found in dytiscid beetles (5) and dipterans (6,7). Insulin, glucagon, somatostatin, gastrin, cholecystokinin, vasopressin, neurophysin, enkephalin, and endorphin-like peptides have been localized in a wide variety of species (8). Thus, a considerable overlap may exist in the endocrine mechanisms of insects and m~mmals despite the evolutionary separation between them. Studies have indicated that both prokaryotes and eukaryotes possess some similar biochemical mechanisms for the synthesis and/or catabolism of steroids (4). In recent articles (9,10), the physiological effects of several corticosteroids on mung bean seedlings (Phaseolus aureus Roxb.) were demonstrated to enhance the plant growth. This effect on the plant metabolism prompted this examination of the possible effects of a number of glucocorticoids and mineralocorticoids on the large milkweed bug Oncopeltus fasc~a~us (Dallas). Methods Steroids, obtained from Sigma Co., St. Louis, M0, were dissolved in pesticide grade acetone to obtain the respective concentrations for administration to the insect. 0nly acetone was applied to the control insects. In each experimental run, every group began with ten randomly selected third instars of comparable weights and age. All weights were measured on a Mettler H-20 (± 0.01 mg) balance. Average and individual weights for each group were measured and recorded. To,facilitate treatment, each group of insects was ventilated with CO 2 for i to 2 minutes to induce immobility. While immobile, the insects were administered with I #I of the treatment solution on their lower abdominal surface. This i ~i was measured with an Instrumentation Co. microapplicator and a Thomas Scientific apparatus D-10 250 #i syringe. After treatment, the groups were housed separately in containers provided with distilled water and cracked-raw unsalted sunflower seeds, enough for more than 6 days. These 0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
1726
Glucocorticoid Action
containers were kept in a Percival cycle at 28°C (±2°C).
Vol. 40, No. 17, 1987
incubator under a 16 hour light: dark
After the six day period each group was exposed to a lethal dose of hydrogen cyanide and then individual weights were obtained and recorded. Subsequently, the insects were placed in an oven at 76°C for 2 days of drying. After the drying period, the insects were again individually weighed. All weights were compared using a single analysis of variance (II). The cortisol trial was conducted with five dilutions, 0.8, 0.5, 0.I, 0.01 and 0.001 #g/~l which were compared to a standard control. The six-day assay was utilized and the results of the trial are reported in Table i. Earlier trials of 30 days showed no significant differences between controls and treated bugs since they apparently all were at the growth plateau. This earlier work led us to the 6-day trials which were at the rapid growth sta~e. Results The slopes of each growth curve (Figure i) indicate that most of the treated bugs molted to the fourth {nstar (steepest curve) between the third and fourth days. The duration of the third stadium for the controls was approximately six day s and this was confirmed in the literature to be 6.1 days (12). In order to evalute any action of the cortisol on the development of the insects, they were observed in this experiment for seven days after treatment (Figure i). All the control and cortisol treated insects (groups of I0) were still in the third instar on day 4. Therefore the changes in development from day 4 to day 7 are given in Figure I. As can be noted only 8 of the control bugs were in the fourth instar by day 7, which contained the least development among the groups. In the cortisol groups treated with 0.5 ~g/~l or 0.01 #g/#l, three bugs in the former and two in the latter group were already at the fifth instar on day 7. A comparable effect was noted for the ll-dehydrocorticosterone (II-DHC) treatment. All relative measurements from experimental trials are cited in Tables i, 2 and 3. Weights, both individual wet and dry, of cortisol-treated insects and statistical analysis are presented in Table I. A similar growth pattern to cortisol was noted in a trial with varying concentrations of IIDHC. In this respect, the largest rate differences appeared to be between the 0.I #g/ #I treatment and the controls. Statistical analysis of individual weights is reported in Table 2. The results in Table 3 illustrates the hormone actions which are similar to the past cortisol-mung bean seedling studies (9,10). In addition to an effect on development, cortisol treatments increased significantly the body weight of recipients at certain doses. Table i shows that cortisol treatments at 0.8, 0.5, 0.01, and 0.001 #g/insect caused significant increases in body wet and dry weights when compared to controls. Interestingly, only the cortisol treatment at 0.I #g/insect did not cause a significant increase in either wet or dry weight. In each treatment group, wet or dry weights followed by the same letter are not significantly different from each other at p <0.05 (analysis of variance). In contrast to cortisol treatments, II-DHC caused no significant increases in weight at 0.5, 0.01, and 0.001 #g/insect. However, at 0.i ~g/insect, a significant increase was recorded for II-DHC (table 2), also a similar dose effect was noted in a preliminary trial.
Vol. 40, No. 17, 1987
Glucocorticoid Action
1727
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°
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i 4
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DAYS Figure i.
Action of cortisol on the development of
0"4eopel#us fascians
1728
Glucocorticoid Action
Vol. 40, No. 17, 1987
Topical application of four different corticoids (Table 3) demonstrated no significant differences between the treatment and control insects. A significant weight difference was found between the insects treated with 58pregnane-3~, 21-diol-20-one and deoxycorticosterone acetate. Table I Effect of Cortisol on Weight Gain of Oncopelrus fasciarus
Treatment ~g/~I/insect 0.8 0.5 0.i 0.01 0.001 Control
No.
6 17 16 18 19 19
Final Weight (mg) Wet Dry Mean i S.E.* Mean i S.E.* 40.91 41.37 31.19 38.37 37.87 25.11
± ± ± ± ± ±
1.78 a 2.08~ 2.30 b'c 2.99 a'c 1.77~ 'c 1 83 b
18.39 20.07 13.75 16.44 16.33 9,73
± ± ± ± ± ±
1.50 a 1.15 a 1.31 b c 1.66 a'c 1.01 a'c 1.01 b
*For each experiment, data from different treatments were analyzed with ANOVA. Means followed by different letters are significantly different from each other at p < 0.05 Table 2 Action of II-DHC on Weight Gain of Oncopelcus fasciarus
Treatment #g/#i/insect 0.5 0.i 0.01 0.001 Control
No.
Final Weight (mg) Wet Dry Mean i S.E.* Mean i S.E.*
9 9 9 i0 i0
27.06 43.09 28.17 27.50 23.69
± ± ± ± ±
1.85~ 2.45 ° 2.12 a 3.22 a 1.20 a
9.45 19 97 11.14 10.33 8.87
± ± ± ± ±
0.91~ 1.37 b 0.93 a 1.69 a 0.47 a
*See Table I Discussion This research demonstrated, for the first time, the stimulatory effects of cortisol on the growth and rate of development of the large milkweed bug OncopelCus fascia~us in its change from third to fourth instars. It is very interesting to note that the growth patterns of these treated insects (Table i) were very similar to those reported in the mung bean seedling (9) and later confirmed by our laboratory (I0). The significance of these findings may indicate a wider potential use of glucocorticoids and mineralocorticoids outside of mammalian systems, specifically in the insects. As noted earlier in the introduction, so-called mammalian hormones are found in plants (2). Therefore, the treatment with cortisol is not that unusual as insects and their relatives are phylogenetically closer to mammals than are plants. In fact, cortisol therapy has been shown to increase the mite (D. brevis) population in human skin (13).
Vol. 40, No. 17, 1987
Glucocorticoid Action
1729
Cortisol and II-DHC both had stimulatory effects in the present study, and in past plant studies (9,10). However, the 5~-compound used in this study demonstrated virtually no effect on growth, while Geuns (9) noted that 5~-compounds generally induced growth in plants. Although the plant and insect did show common responses to some pharmacological agents, the differences indicate a potential use of adrenal cortical metabolites for additional biochemical studies and possible future use for insect control. Table 3 Effects of: i) 5~-Pregnane-3,21-diol-20-one 2) Aldosterone 3) ll-Deoxycortisol 4) Deoxycorticosterone acetate on Weight Gain
Treatment ~g/~I/insect i) 0.I 2) 0.i 3) 0.i A) 0.i Control
No.
Final Weight (mg) Wet Dry Mean ± S.E.* Mean ± S.E.*
8 i0 9 8 9
23.51 31.95 23.15 37.48 26.86
+ ++ + +
2.60 a 2.96 a'c 1.44 a 1.83 b'c'd 3.43 a'd
9.73 13.26 9.65 16.78 11.82
+ + + +_ +
1.32 a 1.78 a'c 0.54 a 1.18 D'c'd 1.98 a'd
*See Table i The statistical significant data substantiated the importance of our findings, but it would be worthwhile to add refinements to the procedures. It would benefit future researchers to use individual insects and house them separately as opposed to groups. This type of experiment would enable the measurement of other parameters as each individual insect could be labeled and traced over a growth period. The six-day assay with which we achieved the present results in growth patterns from the application of cortisol, an endogenous mammalian hormone, may lead to the development of bioassays using the insects as opposed to laboratory m=mm, ls. This switch would effectively lead to less expensive experiments and produce rapid bioassays that do not require elaborate preparation. In this sense, Oncopeltus fasciatus has the potential of becoming a popular research animal as suggested by Feir (12). References and Notes i. 2. 3. 4.
5. 6. 7. 8.
9.
N. G. CARR and I. W.CRAIG, Phytochemical Phylogeny, J. B. Hardborne, pp. 119-143, Academic Press, New York (1970).. A. M. GAWIENOWSKI and C. C. GIBBS, Phytochem. 8, 685-686 (1969). A. M. GAWIENOWSKI and C.C. GIBBS, Phytochem. 8, 2317-2319 (1969). T. SANDOR, S. SONEA and A. Z. MEHDI, Trends in Comparative Enzymology (Amer. Zool. 15 suppl.), E. J. W. Barrington, pp. 227-253 T. J. Griffiths Sons, New York (1975). H. SCHILDKNECHT, Agnew. Chem. Int. Ed. 9, 1-9 (1970). R. MECHOULAM, R. W. BRUEGGEMEIER and D. L. DENLINGER, Experentia AO, 942-944 (1984). D. DeCLERCK, H. DIEDERIK, and A. DeLOOF, Insect Bioohem. IA, 199-208 (1984). K. J. KRAMER, Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 7, G. A. Kerkut and L. I. Gilbert, pp. 511-536, Pergamon Press, Oxford, England (1985). J. M. C. GEUNS, Aspects and Prospects of Plant Growth Regulators, B. Jeffcoat, pp. 209-217, Vantage Press, Oxfordshire, England (1980).
1730
i0. ii.
12. 13. 14.
Glucocorticoid Action
Vol. 40, No. 17, 1987
A. M. GAWIENOWSKI, K. M. CSERNUS, J. E. SIMON and L. E. CRAKER, Steroids, 46, 727-733 (1986). R. R. SOKAL and F. J. ROHLE, Biometry, The Principles and Practice of Statistics in Biological Research, W. H. Freeman and Co., San Francisco (1969). D. Feir, Ann. Rev. Entomology 19, 81-96 (1974). Y. SATO, H. HIGUCHI and U. SAITO, Jap. J.Dermatology 75, 331 (1965). This study was supported in part from Mass. Agric. Exp. Sta. Project Hatch 570 (C.-M. Y.), and listed as publication no. 2767.