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The effect of staphylococcal enterotoxin B on pheromone production in fat bodies isolated from male boll weevils
JOURNAL
OF INVERTEBRATE
PATHOLOGY
47, 116- 119 (1986)
The Effect of Staphylococcal Enterotoxin B on Pheromone Production in Fat Bodies Isolated from Male Boll Weevils GLENN U.S.
Department
and Department
WIYGUL
AND P. P. SIKOROWSKI
of Agriculture, Agricultural Research Service, Boll Weevil Research of Entomology, Mississippi Agricultural and Forestry Experiment Mississippi State University, Mississippi State, Mississippi 39762
Laboratory, Station,
Received May 17, 1985; accepted August 22, 1985 In this study we treated fat bodies excised from 1- to lo-day-old male boll weevils with staphylococcal enterotoxin B. After the compound was incubated with fat bodies for 2 hr, the incubating medium was extracted and analyzed for boll weevil sex pheromone production by gas chromatographic analysis. Of the four compounds that make up the boll weevil male sex pheromone, differences were found between the control (no enterotoxin) and test groups in production of the two aldehydes. We concluded from this that the staphylococcal enterotoxin does affect male pheromone production in boll weevils. 0 1986 Academic press. IX. KEY WORDS: staphylococcal enterotoxin B; sex pheromone; boll weevils; Anthonomus grandis.
weevil fat bodies incubated in vitro by the method Of wiygul et a1* (1982)*
INTRODUCTION
A number of bacteria have been found to contaminate cotton boll weevils, Anthonomus grandis, under mass-rearing conditions (Sikorowski and Thompson, 1980). Such contamination has been shown to reduce production of boll weevil sex pheromone (Gueldner et al., 1977), amino acids (Thompson and Sikorowski, 1978), fatty acids (Thompson et al., 1977), and oxygen uptake (Wiygul and Sikorowski, 1981). In that earlier work the authors studied oxygen uptake and weight gain and loss in lto IO-day-old boll weevils contaminated with Staphylococcus aureus. There was a significant positive relationship between the number of bacteria present in the insect and the weight of the insect. There was no significant relationship between oxygen uptake and the number of bacteria present. These studies all indicated a direct effect by bacteria on the metabolism of boll weevils. Bacteria are known to produce various toxins that exert metabolic effects (Stephen and Pietrowski, 1981). The present study was conducted to determine the effects, if any, of staphylococcal enterotoxin B on pheromone production by male boll
METHODS
Newly emerged boll weevils used in this study were reared by the methods of Roberson and Wright (1984). Males were selected and were placed in 2 x 10 x 19-cm cages and were kept in an incubator at 27°C 50% RH, and a 163 LD cycle. The weevils were held 100 to the cage and were fed fresh cotton squares (buds) daily. Fat bodies were excised from insects and incubated in saline solution + ATP (Wiygul and Sikorowski, 1985) (controls) or saline solution + ATP + staphylococcal enterotoxin B (Sigma) (test) for 2 hr at 22°C. Fat bodies from 20 insects were used to make one replicate and the study was replicated four times for each day of adult age (days l- 10) so that fat bodies from 800 insects were used for the entire experiment. The fat bodies were incubated for 2 hr in 5 ml of the test or control solutions. One microgram of enterotoxin was added to the test solutions so that the final concentration was 200 &ml. At the end of the 2-hr incubation periods, pheromone was ex-
116 0022-2011186 $1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND MATERIALS
EFFECT OF ENTEROTOXIN
z E -. 2 g $ i; g K e P
80 60 40 20 600 80 60 40 20 500 8C 60 40 2c 4oc 8C , 6C 4c 1 2c I 3oc , 8CI 6C, 4i ) 2C/ I 2oc , 8C1 6i ) 4c ) 2c ,lO( )8C) 6( )4( )2( ).
_ ---
ON PHEROMONE
Control Test
PRODUCTION
117
60 40 20 300 80 P 60
cmtrd ---Ted
$ 40 20 @200 g 80 f 60
*
I
2
3
4
5
6
7
8
9
10
FIG. 2. Production of compound 1 (mean ng 2 SE. *significant difference (P < 0.05) by r test) in fat bodies from l- to IO-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
FIG. 1. Total pheromone production (mean ng t SE, *significant difference (P < 0.05) by r test) in fat bodies from I- to lo-day-old male boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
tracted and analyzed by gas chromatography (GLC) (Wiygul and Sikorowski, 1985). Results were calculated on a nanogram of pheromone per insect basis. The four compounds that comprise the boll weevil pheromone are: (compound 1) cis2-isopropenyl1 -methylcyclobutaneethanol, (compound 22 cis-3,3-dimethyl-Al, pcyclohexaneethanol, (compound 3) cis-3, 3-dimethyl-A’,o-cyclohexaneacetaldehyde, and (compound 4) truns-3,3-dimethyl-A2,cxcyclohexaneacetaldehyde. The results were analyzed using standard error of the mean and t test (Steele and Torrie, 1960). RESULTS
AND DISCUSSION
Figure 1 shows the results for total pheromone production (results for the two al-
cohols and aldehydes summed) from excised fat bodies of l- through IO-day male boll weevils. There were significant differences (P < 0.05) by f test on day 1 between the control and test groups. There was also a significant difference between the two groups when the l- through lo-day results were pooled. The means were 223.06 and 113.09 ng pheromone/insect/2-hr incubation period for the control and test groups, respectively, for the whole IO-day test period. The results for compound 1 (Fig. 2) show that there were significant differences between the test and control groups on days 1,4, and 5. As with the summed pheromone production, there were significant differences between the two groups when the Ito IO-day results for compound 1 were pooled. The means for these two groups were 74.12 and 36.66 r&insect/incubation period for the control and test groups, respectively. There were significant differences between the control and test groups on day I for compound 2 (Fig. 3). As with the other results, when the I- to IO-day compound 2 results were pooled there was a significant difference between the two groups with the control group having a mean of 106.74 and the test group having a mean of 54.51 ng of
118 420 400 360 360 340 320 300.
WIYGUL
AND SIKOROWSKI 60 . 40 20 300 60 . - 60. H
. . . .
-CUllld ---Test
\ -
-CUM --Ted
40 .
1 200 60 . 20. t 60 . m 40. t * 100
20-
I
I
2
3
4 5 6 7 Age of Adult Weevils Chys)
6
9
IO
FIG. 3. Production of compound 2 (mean ng f SE, *significant difference (P < 0.05) by t test) in fat bodies from l- to lo-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
compound l/insect/incubation period, respectively. There were no differences between the control and test groups for compound 3 (Fig. 4) for any of the 10 days of the experiment, nor did the pooled I- to IO-day results present any differences. There was a difference in the control and test results for compound 4 (Fig. 5) on day 2. In this case there was a greater quantity of this compound produced in the test group than in the control group (51.9 ng vs 33.3 ng). There was no difference in the l- to IO-day pooled results for this compound. In this study we have found that an enterotoxin produced by S. aureus decreases pheromone production in fat bodies isolated from l- to lo-day-old male boll weevils. The differences were greatest in the results from compounds 1 and 2. The production of compounds 3 and 4 (aldehydes) was not affected by the enterotoxin and in one case (compound 4, day 2) more of the compound was produced in the enterotoxin-treated group than in the controls. We have no explanation for this anomaly. The differences in the effect of the enterotoxin
2
3
4 5 6 7 Pg of &At Weevils (Days)
6
9
IO
FIG. 4. Production of compound 3 (mean ng 2 SE, *significant difference (P < 0.05) by t test) in fat bodies from l- to lo-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
on the production of the alcohols (compounds 1 and 2) and aldehydes (compounds 3 and 4) is very likely a reflection on the mode of action of the enterotoxin and differences in the metabolic pathways involved in the production of the components of the pheromone. The weight gain observed in the earlier study as an effect of S. aureus contamination could be the result of increased uric 60 40 20 300 I 60 . 60 i 40. B 200. 20 . P 2 5 s
---
control Test
so60. 40.
20.
IOOr
12345676910
FIG. 5. Production of compound 4 (mean ng k SE, *significant difference (P =Z0.05) by t test) in fat bodies from l- to lo-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
EFFECT
OF
ENTEROTOXIN
acid concentration in the infected insects (Thompson and Sikorowski, 1982). The increased weight gain observed in the earlier study after S. aureu~ infection occurred on day 5 of the adult stage as did a decrease in production of compound 1 after treatment of the fat bodies with enterotoxin; however, there is no evidence that there is a relationship between weight gain and pheromone production. There is evidence that reduced concentrations of pheromone components 1 and 2 will reduce female response (Hardee et al., 1974). REFERENCES GUELDNER, R. C., SIKOROWSKI, P. P., AND WYATT, J. M. 1977. Bacterial load and pheromone production in the boll weevil, Anthonomus grandis. J. Znverrebr.
Parhol.,
29, 397-398.
HARDEE, D. D., MCKIBBEN. G. H., RUMMEL, D. R., HUDDLESTON, P. M., AND COPPEDGE,J. R. 1974. Response of boll weevils to component ratios and doses of the pheromone. Grandlure. Environ. Entomol.,
3, 135-138.
ROBERSON,J.. AND WRIGHT, J. E. 1984. Production of boll weevils. In “Advances and Challenges in Insect Rearing” (E. G. King and N. C. Leppla, eds.). Science and Education Administration, U.S. Department of Agriculture, Washington, D.C. SIKOROWSKI, P. P, AND THOMPSON, A. C. 1980. “Effects of Bacterial Contaminants on Mass Reared Boll Weevils (Anfhonomus grandis Boheman).”
ON
PHEROMONE
PRODUCTION
119
Tech. Bull. No. 103, Mississippi Agriculture and Forestry Experiment Station, l-6. STEEL, R. G. D., AND TORRIE. J. H. 1960. “Principle5 and Procedures of Statistics.” McGraw-Hill. New York. STEPHEN, J.. AND PIETROWSKI, R. A. 1981. Bacterial toxins. In “Aspects of Microbiology” (J. A. Cole. C. J. Knowles, and D. Schlessinger, eds.). Am. Sot. Microbial., Washington, D.C. THOMPSON. A. C., AND SIKOROWSKI, P. P. 1978. The effect of bacterial load on amino acids in the boll weevil, Anthonomus grandis. J. Invertebr. Potho/.. 32, 388-389.
THOMPSON, A. C., AND SIKOROWSKI. P. P. 1982. Hemolymph analysis of irradiated and Dimilin-treated boll weevils, Anthonomus grandis. J. Invertebr. Pathol.. 39, 158-163. THOMPSON, A. C., SIKOROWSKI, P. P., AND WYATT. J. M. 1977. The effect of bacterial load on fatty acids in the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae). .I. Invevtebr. Pathol., 30, 274-275. WIYGUL. G., MACGOWN, M. W., SIKOROWSKI, P. P., AND WRIGHT. J. E. 1982. Localization of pheromone in male boll weevils Anrhonomus grandis Boheman (Coleoptera: Curculionidae). Entomol. Exp. Appl.. 31, 330-331.
WIYGUL, G. AND SIKOROWSKI,P. P. 1981. The effect of various levels of contamination by two species of bacteria (Sraph$ococcus aureus and StreptococcLrs sp.) on oxygen uptake of the boll weevil. Con~p. Biochem.
Physiol.
A, 68, 527-530.
WIYGUL, G., AND SIKOROWSKI, P. P. 1985. The effect of glucose and ATP on sex pheromone production in fat bodies from male boll wevils Anfhonomus grandis Boheman (Coleoptera: Curculionidae). Comp.
Biochem.
Physiol.
B. 81,
1073-1075.
OF INVERTEBRATE
PATHOLOGY
47, 116- 119 (1986)
The Effect of Staphylococcal Enterotoxin B on Pheromone Production in Fat Bodies Isolated from Male Boll Weevils GLENN U.S.
Department
and Department
WIYGUL
AND P. P. SIKOROWSKI
of Agriculture, Agricultural Research Service, Boll Weevil Research of Entomology, Mississippi Agricultural and Forestry Experiment Mississippi State University, Mississippi State, Mississippi 39762
Laboratory, Station,
Received May 17, 1985; accepted August 22, 1985 In this study we treated fat bodies excised from 1- to lo-day-old male boll weevils with staphylococcal enterotoxin B. After the compound was incubated with fat bodies for 2 hr, the incubating medium was extracted and analyzed for boll weevil sex pheromone production by gas chromatographic analysis. Of the four compounds that make up the boll weevil male sex pheromone, differences were found between the control (no enterotoxin) and test groups in production of the two aldehydes. We concluded from this that the staphylococcal enterotoxin does affect male pheromone production in boll weevils. 0 1986 Academic press. IX. KEY WORDS: staphylococcal enterotoxin B; sex pheromone; boll weevils; Anthonomus grandis.
weevil fat bodies incubated in vitro by the method Of wiygul et a1* (1982)*
INTRODUCTION
A number of bacteria have been found to contaminate cotton boll weevils, Anthonomus grandis, under mass-rearing conditions (Sikorowski and Thompson, 1980). Such contamination has been shown to reduce production of boll weevil sex pheromone (Gueldner et al., 1977), amino acids (Thompson and Sikorowski, 1978), fatty acids (Thompson et al., 1977), and oxygen uptake (Wiygul and Sikorowski, 1981). In that earlier work the authors studied oxygen uptake and weight gain and loss in lto IO-day-old boll weevils contaminated with Staphylococcus aureus. There was a significant positive relationship between the number of bacteria present in the insect and the weight of the insect. There was no significant relationship between oxygen uptake and the number of bacteria present. These studies all indicated a direct effect by bacteria on the metabolism of boll weevils. Bacteria are known to produce various toxins that exert metabolic effects (Stephen and Pietrowski, 1981). The present study was conducted to determine the effects, if any, of staphylococcal enterotoxin B on pheromone production by male boll
METHODS
Newly emerged boll weevils used in this study were reared by the methods of Roberson and Wright (1984). Males were selected and were placed in 2 x 10 x 19-cm cages and were kept in an incubator at 27°C 50% RH, and a 163 LD cycle. The weevils were held 100 to the cage and were fed fresh cotton squares (buds) daily. Fat bodies were excised from insects and incubated in saline solution + ATP (Wiygul and Sikorowski, 1985) (controls) or saline solution + ATP + staphylococcal enterotoxin B (Sigma) (test) for 2 hr at 22°C. Fat bodies from 20 insects were used to make one replicate and the study was replicated four times for each day of adult age (days l- 10) so that fat bodies from 800 insects were used for the entire experiment. The fat bodies were incubated for 2 hr in 5 ml of the test or control solutions. One microgram of enterotoxin was added to the test solutions so that the final concentration was 200 &ml. At the end of the 2-hr incubation periods, pheromone was ex-
116 0022-2011186 $1.50 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND MATERIALS
EFFECT OF ENTEROTOXIN
z E -. 2 g $ i; g K e P
80 60 40 20 600 80 60 40 20 500 8C 60 40 2c 4oc 8C , 6C 4c 1 2c I 3oc , 8CI 6C, 4i ) 2C/ I 2oc , 8C1 6i ) 4c ) 2c ,lO( )8C) 6( )4( )2( ).
_ ---
ON PHEROMONE
Control Test
PRODUCTION
117
60 40 20 300 80 P 60
cmtrd ---Ted
$ 40 20 @200 g 80 f 60
*
I
2
3
4
5
6
7
8
9
10
FIG. 2. Production of compound 1 (mean ng 2 SE. *significant difference (P < 0.05) by r test) in fat bodies from l- to IO-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
FIG. 1. Total pheromone production (mean ng t SE, *significant difference (P < 0.05) by r test) in fat bodies from I- to lo-day-old male boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
tracted and analyzed by gas chromatography (GLC) (Wiygul and Sikorowski, 1985). Results were calculated on a nanogram of pheromone per insect basis. The four compounds that comprise the boll weevil pheromone are: (compound 1) cis2-isopropenyl1 -methylcyclobutaneethanol, (compound 22 cis-3,3-dimethyl-Al, pcyclohexaneethanol, (compound 3) cis-3, 3-dimethyl-A’,o-cyclohexaneacetaldehyde, and (compound 4) truns-3,3-dimethyl-A2,cxcyclohexaneacetaldehyde. The results were analyzed using standard error of the mean and t test (Steele and Torrie, 1960). RESULTS
AND DISCUSSION
Figure 1 shows the results for total pheromone production (results for the two al-
cohols and aldehydes summed) from excised fat bodies of l- through IO-day male boll weevils. There were significant differences (P < 0.05) by f test on day 1 between the control and test groups. There was also a significant difference between the two groups when the l- through lo-day results were pooled. The means were 223.06 and 113.09 ng pheromone/insect/2-hr incubation period for the control and test groups, respectively, for the whole IO-day test period. The results for compound 1 (Fig. 2) show that there were significant differences between the test and control groups on days 1,4, and 5. As with the summed pheromone production, there were significant differences between the two groups when the Ito IO-day results for compound 1 were pooled. The means for these two groups were 74.12 and 36.66 r&insect/incubation period for the control and test groups, respectively. There were significant differences between the control and test groups on day I for compound 2 (Fig. 3). As with the other results, when the I- to IO-day compound 2 results were pooled there was a significant difference between the two groups with the control group having a mean of 106.74 and the test group having a mean of 54.51 ng of
118 420 400 360 360 340 320 300.
WIYGUL
AND SIKOROWSKI 60 . 40 20 300 60 . - 60. H
. . . .
-CUllld ---Test
\ -
-CUM --Ted
40 .
1 200 60 . 20. t 60 . m 40. t * 100
20-
I
I
2
3
4 5 6 7 Age of Adult Weevils Chys)
6
9
IO
FIG. 3. Production of compound 2 (mean ng f SE, *significant difference (P < 0.05) by t test) in fat bodies from l- to lo-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
compound l/insect/incubation period, respectively. There were no differences between the control and test groups for compound 3 (Fig. 4) for any of the 10 days of the experiment, nor did the pooled I- to IO-day results present any differences. There was a difference in the control and test results for compound 4 (Fig. 5) on day 2. In this case there was a greater quantity of this compound produced in the test group than in the control group (51.9 ng vs 33.3 ng). There was no difference in the l- to IO-day pooled results for this compound. In this study we have found that an enterotoxin produced by S. aureus decreases pheromone production in fat bodies isolated from l- to lo-day-old male boll weevils. The differences were greatest in the results from compounds 1 and 2. The production of compounds 3 and 4 (aldehydes) was not affected by the enterotoxin and in one case (compound 4, day 2) more of the compound was produced in the enterotoxin-treated group than in the controls. We have no explanation for this anomaly. The differences in the effect of the enterotoxin
2
3
4 5 6 7 Pg of &At Weevils (Days)
6
9
IO
FIG. 4. Production of compound 3 (mean ng 2 SE, *significant difference (P < 0.05) by t test) in fat bodies from l- to lo-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
on the production of the alcohols (compounds 1 and 2) and aldehydes (compounds 3 and 4) is very likely a reflection on the mode of action of the enterotoxin and differences in the metabolic pathways involved in the production of the components of the pheromone. The weight gain observed in the earlier study as an effect of S. aureus contamination could be the result of increased uric 60 40 20 300 I 60 . 60 i 40. B 200. 20 . P 2 5 s
---
control Test
so60. 40.
20.
IOOr
12345676910
FIG. 5. Production of compound 4 (mean ng k SE, *significant difference (P =Z0.05) by t test) in fat bodies from l- to lo-day-old boll weevils. Fat bodies were incubated in saline solution + ATP or in saline solution + ATP + staphylococcal enterotoxin.
EFFECT
OF
ENTEROTOXIN
acid concentration in the infected insects (Thompson and Sikorowski, 1982). The increased weight gain observed in the earlier study after S. aureu~ infection occurred on day 5 of the adult stage as did a decrease in production of compound 1 after treatment of the fat bodies with enterotoxin; however, there is no evidence that there is a relationship between weight gain and pheromone production. There is evidence that reduced concentrations of pheromone components 1 and 2 will reduce female response (Hardee et al., 1974). REFERENCES GUELDNER, R. C., SIKOROWSKI, P. P., AND WYATT, J. M. 1977. Bacterial load and pheromone production in the boll weevil, Anthonomus grandis. J. Znverrebr.
Parhol.,
29, 397-398.
HARDEE, D. D., MCKIBBEN. G. H., RUMMEL, D. R., HUDDLESTON, P. M., AND COPPEDGE,J. R. 1974. Response of boll weevils to component ratios and doses of the pheromone. Grandlure. Environ. Entomol.,
3, 135-138.
ROBERSON,J.. AND WRIGHT, J. E. 1984. Production of boll weevils. In “Advances and Challenges in Insect Rearing” (E. G. King and N. C. Leppla, eds.). Science and Education Administration, U.S. Department of Agriculture, Washington, D.C. SIKOROWSKI, P. P, AND THOMPSON, A. C. 1980. “Effects of Bacterial Contaminants on Mass Reared Boll Weevils (Anfhonomus grandis Boheman).”
ON
PHEROMONE
PRODUCTION
119
Tech. Bull. No. 103, Mississippi Agriculture and Forestry Experiment Station, l-6. STEEL, R. G. D., AND TORRIE. J. H. 1960. “Principle5 and Procedures of Statistics.” McGraw-Hill. New York. STEPHEN, J.. AND PIETROWSKI, R. A. 1981. Bacterial toxins. In “Aspects of Microbiology” (J. A. Cole. C. J. Knowles, and D. Schlessinger, eds.). Am. Sot. Microbial., Washington, D.C. THOMPSON. A. C., AND SIKOROWSKI, P. P. 1978. The effect of bacterial load on amino acids in the boll weevil, Anthonomus grandis. J. Invertebr. Potho/.. 32, 388-389.
THOMPSON, A. C., AND SIKOROWSKI. P. P. 1982. Hemolymph analysis of irradiated and Dimilin-treated boll weevils, Anthonomus grandis. J. Invertebr. Pathol.. 39, 158-163. THOMPSON, A. C., SIKOROWSKI, P. P., AND WYATT. J. M. 1977. The effect of bacterial load on fatty acids in the boll weevil, Anthonomus grandis (Coleoptera: Curculionidae). .I. Invevtebr. Pathol., 30, 274-275. WIYGUL. G., MACGOWN, M. W., SIKOROWSKI, P. P., AND WRIGHT. J. E. 1982. Localization of pheromone in male boll weevils Anrhonomus grandis Boheman (Coleoptera: Curculionidae). Entomol. Exp. Appl.. 31, 330-331.
WIYGUL, G. AND SIKOROWSKI,P. P. 1981. The effect of various levels of contamination by two species of bacteria (Sraph$ococcus aureus and StreptococcLrs sp.) on oxygen uptake of the boll weevil. Con~p. Biochem.
Physiol.
A, 68, 527-530.
WIYGUL, G., AND SIKOROWSKI, P. P. 1985. The effect of glucose and ATP on sex pheromone production in fat bodies from male boll wevils Anfhonomus grandis Boheman (Coleoptera: Curculionidae). Comp.
Biochem.
Physiol.
B. 81,
1073-1075.