- Email: [email protected]
Chemosterilant and insecticidal activity of mixed aflatoxins against Anthonomus grandis (Coleoptera)
JOURNAL
OF INVERTEBRATE
PATHOLOGY
31, 365-367
(1978)
Chemosterilant and Insecticidal Activity of Mixed Aflatoxins against Anthonomus grandis (Coleoptera) JACK H. MOORE,~ABNER Depurtmrnt
M. HAMMOND,AND
GERALD C.LLEWELLYN
of Biology, Uni\,rrsity oj North Alabama, Florence, Alabama 35630; Department oj’Entomology, Loaisiana State University. Baton Rouge. Louisiana 70803: and Department ofBiology. Virginia Common~~~ealth University, Richmond. Virginiu 23284 Received
August
20, 1977
A mixture boll weevils, KEY
of the four major aflatoxins at 0.06 ppm in food Anthonomus grandis, was an effective chemosterilant. WORDS: Anthonomus grandis, adult; aflatoxin: aziridine: chemosterilant
INTRODUCTION
all correspondence
should
to
adult
effects.
ing aflatoxin with a known chemosterilant (aziridine) to evaluate aflatoxin as a chemosterilant and insecticide.
Certain strains of the fungi Aspergillus flc~rus and A. parasitic-us produce a group of mycotoxic metabolites commonly known as the aflatoxins (Diener and Davis, 1969). The aflatoxins have been studied extensively since they were found first in peanuts, stored grains, and other agricultural products (Golumbic and Kulick, 1969). Aflatoxin B, (AFB,) is a known hepatocarcinogen and hepatotoxic agent (Wogan, 1968). Additional aflatoxins include B, (AFB,). G, (AFG,), and G, (AFGJ. Aflatoxin has insecticidal effects upon Heliotlzis I-irescens (Gudauskas et al.. 1967), Drosuplzila melanogaster (Lalor et al., 1976), Musca domestica (Beard and Walton, 1971), Epilachna \,igintiopunctatcze (Krishnamoorthy and Naidu, 1971). and Trombidium gigas (Sannasi and Amirthavalli, 1970). Chemosterilant effects were produced by aflatoxin in Aedes aegypti, M. domestica, and D. melanogaster (Matsumara and Knight, 1967); Sitophilus oryzae (Srinath et al., 1973); and T. gigas (Sannasi and Amirthavalli, 1970). The present study was undertaken on the boll weevil, Anthonomus grandis, compar’ To whom
supplied
MATERIALS
AND METHODS
Aflatoxic corn meal containing 17 ppm of AFB,, 4.3 ppm of AFB,, 30 ppm of AFG,, and 9 ppm of AFG, was mixed into an agar-liquid weevil food (Vanderzant and Davich, 1961). Three concentrations of aflatoxic corn meal were prepared: 1.OOO, 0.100 and 0.010%. The corn meal was substituted for a portion of the sucrose (equal weight basis) in the food. Food containing 0.005% aziridine, a known chemosterilant, and a food containing 1.000% toxin-free corn meal as a control were prepared. They were mixed for 3 min at 60°C. Each mixture was poured into glass tubing, 1 cm in diameter, and allowed to cool and solidify. The solidified food was removed from the tube and cut into lengths of 1 cm, each weighing 1.5 g. These were dipped into melted wax to retard desiccation and were stored at 5°C. Twenty-five marked weevils of each sex (2.5-30 hr old) were placed together into pint containers. They were maintained at 30°C and 50% RH. One piece of food per five weevils was provided and changed twice daily. Daily observations were made for mortality. The test diets
be addressed. 365
0022-201
l/78/0313-0365$01.00/O
Copyright 0 1978 by Academic Press. Inc. All rights of reproduction m any form reserved
366
MOORE.
HAMMOND.
AND
were provided to the weevils for 3 days and, on the fourth day, were replaced with control food for 4 additional days; on the seventh day, the eggs were harvested by dissection and screen washing (Vanderzant and Davich, 1961). Microbial contamination was controlled by suspending the eggs in a solution of 40% formalin diluted 5O:l. Fifty eggs were placed into small depressions in a pour plate of larval diet, one egg per depression. The plates were maintained at 30°C and 50% RH, and the eggs were observed twice daily for 7 days to determine hatch. RESULTS
AND DISCUSSION
The data in Table 1 show that the highest concentration of the aflatoxins studied (0.603 ppm) prevented egg production and 36% of the animals died within 7 days. The toxin was fed for only 3 of the 7 days and approached the LD,,, level for the weevils. The second highest concentration (0.060 ppm) was an effective chemosterilant. The lowest concentration (0.006 ppm) had no chemosterilant effect. This group had a hatch value of 113.73 as compared to 100.00 for the control. The middle concentration of the aflatoxins produced a hatch value of 1.96 and azaridine produced a value of 0.00. McHaffey et al. (1972) consider a treatment effective as a chemosterilant if the hatch value is 4.00 or less. TABLE CHEMOSTERILANT
Treatment Aflatoxin Aflatoxin Aflatoxin Apholate (aziridine) Control (1 Treated/Control
AND INSECTICIDAL
Concentration in food
ACTIVITY
Total number of animals
0.603
LLEWELLYN
Lalor et al (1976) found that 1.5 pgiml of AFB, reduced the hatching percentage for a specific strain of D. melanogaster (wild type Oregon R) to 35%. No hatching occurred in concentrations above 2.8 pgiml. Chinnici et al. (1976) found that AFB,-treated D. melanogaster (wild type, Oregon R) males were more sensitive than females during their development from eggs to adults. It is not possible to attribute the chemosterilization observed in this study to any one of the four afatoxins since they were fed as a mixture. Neither can the sterilization be attributed to a specific sex since both males and females had access to the food. This potent hepatocarcinogen should be studied more thoroughly. One consideration should be the possibility of bioconcentration and biotransfer of such an agent (Nevins and Grant, 1971). Further studies are planned to identify sex-related responses of the boll weevil to each of the four major aflatoxins. ACKNOWLEDGMENTS The boll weevils, aziridine. and laboratory space were provided by Southern Research Institute. Birmingham. Alabama. The corn meal containing the aflatoxins and the analysis were supplied by Dr. Urban Diener and Dr. N. D. Davis Auburn University. Auburn. Alabama. Mrs. Kathryn Collendra. Virginia Commonwealth University, Richmond. Virginia, assisted with data analysis. technical, clerical. and editorial aspects of the work. I
OF MIXED
AFLATOXINS
Mortality after 7 days (S:)
AGAINST Number eggs plated
ANTHONOMUSGRANDIS
of Hatch (So)
Hatch value”
ppm 0.060 ppm 0.006 ppm
50
36
0
50 50
4 8
100 100
I 58
1.96 113.73
0.005% 0.000
50 50
8 2
97 100
0 51
0.00 100.00
x 100 = hatch
value.
-
CHEMOSTERILANT
ACTIVITY
REFERENCES BEARD, R. L.. AND secticidal mycotoxins Jla~,us var. columnaris. No. 125. l-26.
WALTON.
produced Bull. Corm.
G.
S. 1971. Inby Aspergillus Agr. Exp. Sta..
J. P., BOOKER, M. A.. AND LLEWELLYN. G. C. 1976. Effect of Aflatoxin B, on viability, growth, fertility, and crossing over in Drosophila melanogaster (Diptera). .I. Inwrtebr. Pathol.. 21, 255-258. DIENER, U. L.. AND DAVIS, N. D. 1969. Aflatoxin formation by Aspergillus jarus. IN “AflatoxinScientific Background, Control and Implications” (L. A. Goldblatt. ed.). pp. 13-54. Academic Press. New York. GOLUMBIC. C.. AND KULIK, M. 1969. Fungal spoilage on stored crops and its control. In “AflatoxinScientific Background, Control and Implications” (L. A. Goldblatt. ed.), pp. 307-327. Academic Press, New York. GUDAUSKAS, R. T.. DAVIS, N. D.. AND DIENER, U. L. 1967. Sensitivity of Heliothis virescens larvae to aflatoxin in ad libitum feeding. J. Invertebr. Pathol., 9, 132-133. KRISHNAMOORTHY, K.. AND NAIDLJ. M. S. 1971. Aspergillus Jklvus Link-A fungal parasite of Epilachna vigintiopunctatae F. Curr. SC;.. 40, 666-667. CHINNICI,
OF AFLATOXINS
367
LALOR, J. H., CHINNICI, J. P., AND LL.EWELLYN G. C. 1976. Effects of a fungal metabolite. aflatoxin B, on larval viability and gross morphology in Drosophila melanogastrr. Develop. Ind. Micro&~/.. 11. 443-449. M4TSUMARA. F., AND KNIGHT, S. G. 1967. Toxicity and chemosterilizing activity of aflatoxin against insects. ./. Econ. Entomol.. 60, 871-872. MCHAFFEY, D. G.. FLINT. H. M.. H.~YF~~s. J. W.. KLASSEN, W.. MITI.IN. N.. AND DALICH. T. B. 1972. Sterility induced in boll weevils by alkylating agents administered in an adult diet. .1. Econ. Entomol.. 6.5, 13- 19. NEVINS, M. P., AND GRANT, D. W. 1971. BIOconcentration and biotransfer of aflatoxin. Bull. Environ. Contam. Tfkc-01.. 6, 552-558. SANNASI. A., AND AMIRTHAVALLI. S. 1970. Infection of the velvet mite. Trombrdium gigas by Aspc’rgillw j!u~~us. .I. Investebr. Pa~hol.. 16, 54-56. SRINATH, D.. RAGUNAI-HAN, A. N., AND MAJUMDAR. S. K. 1973. Influence of some Asprrgillus species on the population ofSitophi/lts u~:cre L. (Coleptera: Curculionidae). Curr. Sci.. 42, 683-684. VANDERZANT. E. S.. AND DAVICH, T. B. 1961. Artificial diets for adult boll weevils and techniques for obtaining eggs. J. Econ. En/omol.. 51, 923-938. WOGAN. G. N. 1968. Biochemical responses to aflatoxin. Cancer Res., 28, 2282-2287.
OF INVERTEBRATE
PATHOLOGY
31, 365-367
(1978)
Chemosterilant and Insecticidal Activity of Mixed Aflatoxins against Anthonomus grandis (Coleoptera) JACK H. MOORE,~ABNER Depurtmrnt
M. HAMMOND,AND
GERALD C.LLEWELLYN
of Biology, Uni\,rrsity oj North Alabama, Florence, Alabama 35630; Department oj’Entomology, Loaisiana State University. Baton Rouge. Louisiana 70803: and Department ofBiology. Virginia Common~~~ealth University, Richmond. Virginiu 23284 Received
August
20, 1977
A mixture boll weevils, KEY
of the four major aflatoxins at 0.06 ppm in food Anthonomus grandis, was an effective chemosterilant. WORDS: Anthonomus grandis, adult; aflatoxin: aziridine: chemosterilant
INTRODUCTION
all correspondence
should
to
adult
effects.
ing aflatoxin with a known chemosterilant (aziridine) to evaluate aflatoxin as a chemosterilant and insecticide.
Certain strains of the fungi Aspergillus flc~rus and A. parasitic-us produce a group of mycotoxic metabolites commonly known as the aflatoxins (Diener and Davis, 1969). The aflatoxins have been studied extensively since they were found first in peanuts, stored grains, and other agricultural products (Golumbic and Kulick, 1969). Aflatoxin B, (AFB,) is a known hepatocarcinogen and hepatotoxic agent (Wogan, 1968). Additional aflatoxins include B, (AFB,). G, (AFG,), and G, (AFGJ. Aflatoxin has insecticidal effects upon Heliotlzis I-irescens (Gudauskas et al.. 1967), Drosuplzila melanogaster (Lalor et al., 1976), Musca domestica (Beard and Walton, 1971), Epilachna \,igintiopunctatcze (Krishnamoorthy and Naidu, 1971). and Trombidium gigas (Sannasi and Amirthavalli, 1970). Chemosterilant effects were produced by aflatoxin in Aedes aegypti, M. domestica, and D. melanogaster (Matsumara and Knight, 1967); Sitophilus oryzae (Srinath et al., 1973); and T. gigas (Sannasi and Amirthavalli, 1970). The present study was undertaken on the boll weevil, Anthonomus grandis, compar’ To whom
supplied
MATERIALS
AND METHODS
Aflatoxic corn meal containing 17 ppm of AFB,, 4.3 ppm of AFB,, 30 ppm of AFG,, and 9 ppm of AFG, was mixed into an agar-liquid weevil food (Vanderzant and Davich, 1961). Three concentrations of aflatoxic corn meal were prepared: 1.OOO, 0.100 and 0.010%. The corn meal was substituted for a portion of the sucrose (equal weight basis) in the food. Food containing 0.005% aziridine, a known chemosterilant, and a food containing 1.000% toxin-free corn meal as a control were prepared. They were mixed for 3 min at 60°C. Each mixture was poured into glass tubing, 1 cm in diameter, and allowed to cool and solidify. The solidified food was removed from the tube and cut into lengths of 1 cm, each weighing 1.5 g. These were dipped into melted wax to retard desiccation and were stored at 5°C. Twenty-five marked weevils of each sex (2.5-30 hr old) were placed together into pint containers. They were maintained at 30°C and 50% RH. One piece of food per five weevils was provided and changed twice daily. Daily observations were made for mortality. The test diets
be addressed. 365
0022-201
l/78/0313-0365$01.00/O
Copyright 0 1978 by Academic Press. Inc. All rights of reproduction m any form reserved
366
MOORE.
HAMMOND.
AND
were provided to the weevils for 3 days and, on the fourth day, were replaced with control food for 4 additional days; on the seventh day, the eggs were harvested by dissection and screen washing (Vanderzant and Davich, 1961). Microbial contamination was controlled by suspending the eggs in a solution of 40% formalin diluted 5O:l. Fifty eggs were placed into small depressions in a pour plate of larval diet, one egg per depression. The plates were maintained at 30°C and 50% RH, and the eggs were observed twice daily for 7 days to determine hatch. RESULTS
AND DISCUSSION
The data in Table 1 show that the highest concentration of the aflatoxins studied (0.603 ppm) prevented egg production and 36% of the animals died within 7 days. The toxin was fed for only 3 of the 7 days and approached the LD,,, level for the weevils. The second highest concentration (0.060 ppm) was an effective chemosterilant. The lowest concentration (0.006 ppm) had no chemosterilant effect. This group had a hatch value of 113.73 as compared to 100.00 for the control. The middle concentration of the aflatoxins produced a hatch value of 1.96 and azaridine produced a value of 0.00. McHaffey et al. (1972) consider a treatment effective as a chemosterilant if the hatch value is 4.00 or less. TABLE CHEMOSTERILANT
Treatment Aflatoxin Aflatoxin Aflatoxin Apholate (aziridine) Control (1 Treated/Control
AND INSECTICIDAL
Concentration in food
ACTIVITY
Total number of animals
0.603
LLEWELLYN
Lalor et al (1976) found that 1.5 pgiml of AFB, reduced the hatching percentage for a specific strain of D. melanogaster (wild type Oregon R) to 35%. No hatching occurred in concentrations above 2.8 pgiml. Chinnici et al. (1976) found that AFB,-treated D. melanogaster (wild type, Oregon R) males were more sensitive than females during their development from eggs to adults. It is not possible to attribute the chemosterilization observed in this study to any one of the four afatoxins since they were fed as a mixture. Neither can the sterilization be attributed to a specific sex since both males and females had access to the food. This potent hepatocarcinogen should be studied more thoroughly. One consideration should be the possibility of bioconcentration and biotransfer of such an agent (Nevins and Grant, 1971). Further studies are planned to identify sex-related responses of the boll weevil to each of the four major aflatoxins. ACKNOWLEDGMENTS The boll weevils, aziridine. and laboratory space were provided by Southern Research Institute. Birmingham. Alabama. The corn meal containing the aflatoxins and the analysis were supplied by Dr. Urban Diener and Dr. N. D. Davis Auburn University. Auburn. Alabama. Mrs. Kathryn Collendra. Virginia Commonwealth University, Richmond. Virginia, assisted with data analysis. technical, clerical. and editorial aspects of the work. I
OF MIXED
AFLATOXINS
Mortality after 7 days (S:)
AGAINST Number eggs plated
ANTHONOMUSGRANDIS
of Hatch (So)
Hatch value”
ppm 0.060 ppm 0.006 ppm
50
36
0
50 50
4 8
100 100
I 58
1.96 113.73
0.005% 0.000
50 50
8 2
97 100
0 51
0.00 100.00
x 100 = hatch
value.
-
CHEMOSTERILANT
ACTIVITY
REFERENCES BEARD, R. L.. AND secticidal mycotoxins Jla~,us var. columnaris. No. 125. l-26.
WALTON.
produced Bull. Corm.
G.
S. 1971. Inby Aspergillus Agr. Exp. Sta..
J. P., BOOKER, M. A.. AND LLEWELLYN. G. C. 1976. Effect of Aflatoxin B, on viability, growth, fertility, and crossing over in Drosophila melanogaster (Diptera). .I. Inwrtebr. Pathol.. 21, 255-258. DIENER, U. L.. AND DAVIS, N. D. 1969. Aflatoxin formation by Aspergillus jarus. IN “AflatoxinScientific Background, Control and Implications” (L. A. Goldblatt. ed.). pp. 13-54. Academic Press. New York. GOLUMBIC. C.. AND KULIK, M. 1969. Fungal spoilage on stored crops and its control. In “AflatoxinScientific Background, Control and Implications” (L. A. Goldblatt. ed.), pp. 307-327. Academic Press, New York. GUDAUSKAS, R. T.. DAVIS, N. D.. AND DIENER, U. L. 1967. Sensitivity of Heliothis virescens larvae to aflatoxin in ad libitum feeding. J. Invertebr. Pathol., 9, 132-133. KRISHNAMOORTHY, K.. AND NAIDLJ. M. S. 1971. Aspergillus Jklvus Link-A fungal parasite of Epilachna vigintiopunctatae F. Curr. SC;.. 40, 666-667. CHINNICI,
OF AFLATOXINS
367
LALOR, J. H., CHINNICI, J. P., AND LL.EWELLYN G. C. 1976. Effects of a fungal metabolite. aflatoxin B, on larval viability and gross morphology in Drosophila melanogastrr. Develop. Ind. Micro&~/.. 11. 443-449. M4TSUMARA. F., AND KNIGHT, S. G. 1967. Toxicity and chemosterilizing activity of aflatoxin against insects. ./. Econ. Entomol.. 60, 871-872. MCHAFFEY, D. G.. FLINT. H. M.. H.~YF~~s. J. W.. KLASSEN, W.. MITI.IN. N.. AND DALICH. T. B. 1972. Sterility induced in boll weevils by alkylating agents administered in an adult diet. .1. Econ. Entomol.. 6.5, 13- 19. NEVINS, M. P., AND GRANT, D. W. 1971. BIOconcentration and biotransfer of aflatoxin. Bull. Environ. Contam. Tfkc-01.. 6, 552-558. SANNASI. A., AND AMIRTHAVALLI. S. 1970. Infection of the velvet mite. Trombrdium gigas by Aspc’rgillw j!u~~us. .I. Investebr. Pa~hol.. 16, 54-56. SRINATH, D.. RAGUNAI-HAN, A. N., AND MAJUMDAR. S. K. 1973. Influence of some Asprrgillus species on the population ofSitophi/lts u~:cre L. (Coleptera: Curculionidae). Curr. Sci.. 42, 683-684. VANDERZANT. E. S.. AND DAVICH, T. B. 1961. Artificial diets for adult boll weevils and techniques for obtaining eggs. J. Econ. En/omol.. 51, 923-938. WOGAN. G. N. 1968. Biochemical responses to aflatoxin. Cancer Res., 28, 2282-2287.