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Effects of aflatoxin B1, aflatoxin B2, aflatoxin G1, and sterigmatocystin on viability, rates of development, and body length in two strains of Drosophila melanogaster (Diptera)
JOURNAL
OF
INVERTEBRATE
PATHOLOGY
39, 388-394 (1982)
Effects of Aflatoxin B,, Aflatoxin B2, Aflatoxin G,, and Sterigmatocystin on Viability, Rates of Development, and Body Length in Two Strains of Drosophila melanogaster (Diptera) KATHLEEN Department
GUNST, of Biology,
JOSEPH P. CHINNICI, Virginia
Commonwealth
AND GERALD University,
C. LLEWELLYN
Richmond,
Virginia
23284
Received March 5, 1981; accepted May 15, 1981 Two wild-type laboratory populations of Drosophila melanogaster, Florida-9 (sensitive to aflatoxin (AF) B,-induced toxicity) and Lausanne-S (resistant to AFB,-induced toxicity) were tested to determine relative degrees of sensitivity to growth from the egg stage on media containing 0.2, 0.6, 2.0, and 4.0 ppm AFB,, AFG,, AFB2, or sterigmatocystin (ST). Data indicate that strain Florida-9 is quite sensitive to AFG, toxicity at both the egg-pupa and egg-adult stages of development while Lausanne-S is quite resistant to such toxic effects. For Lausanne-S, AFB, > AFG, in relative toxicity, while for Florida-9, AFG, > AFB,. The latter is noteworthy since vertebrate studies consistently show that AFB, is a significantly stronger carcinogen and mutagen than AFG,. Possible explanations are discussed. Neither strain tested displayed toxic responses to the presence of AFBZ or ST in the culture media; however, the 4.0-ppm Lausanne-S treatment displayed a significantly lower adult mortality rate than the control, indicating that Lausanne-S flies may benefit from the presence of ST in the culture medium. KEY WORDS: atlatoxin B,, aflatoxin B,, aflatoxin G,, sterigmatocystin, mycotoxins, Drosophila. Aspergillus
jlavus.
effects of aflatoxins on viability, fertility, or mutagenesis involve aflatoxin B, (AFB,). The aflatoxins are a group of at least 12 Various wild-type laboratory strains of the fruit fly Drosophila melanogaster differ highly substituted polycyclic compounds produced by several species of fungi, espe- significantly in their levels of sensitivity to cially Aspergillus flaws and A. parasiticus the toxic effects of AFB, in the culture (Diener and Davis, 1969; Wogan 1975). medium (Llewellyn and Chinnici, 1978; Discovered after several incidences of Chinnici and Llewellyn, 1979). Toxicity is demonstrated by high levels of larval and poultry kills due to eating contaminated pupal lethality, significantly reduced pupa feed (Blount, 1961; Asplin and Carnaghan, 1961), the aflatoxins are highly potent ver- case and adult body lengths, and signifitimes induced tebrate hepatocarcinogens (Newberne and cantly longer development Butler, 1969; Kocylawski et al., 1973; by AFB, in the growth medium. Sensitive Reddy et al., 1976; Wray and Hayes, 1980) strains show marked toxic effects at low of AFBl while resistant and effective mutagens in a wide variety of concentrations strains do not show toxic effects unless organisms (Ong, 1975; Wong and Hsieh, 1976; Fahmy et al., 1978; Thomson and subjected to concentrations two- to threeEvans, 1979). In addition, aflatoxins dis- fold higher than the effective doses for sensitive strains (Llewellyn and Chinnici, play insecticidal, larvicidal, and chemosterilizing properties against a wide variety 1978). Using a resistant strain, continuous egg-adult exposure to 0.67 ppm AFBl in of insect species (Lalor et al., 1976; Moore et al., 1978; Chinnici et al., 1979; Nguyen et the culture medium caused a significant reduction in the fertility of surviving females al., 1979; Gaston and Llewellyn, 1980). In insects, the only detailed studies of the but did not affect the rate of meiotic INTRODUCTION
388 0022-201 l/82/030388-07$01.00/O Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
EFFECT
OF
MYCOTOXINS
IN
Drosophila
389
crossing-over in these females (Chinnici et MATERIALS AND METHODS al., 1976). Chinnici et al. (1979) have shown that Insects. Two standard wild-type laborasensitive and resistant strains differ in the tory strains of D. melunoguster were used stages of larval development sensitive to in these studies: Lausanne-S and Florida-9. AFB,-induced toxicity and in the degree of Complete descriptions of these strains are such sensitivity at each stage. Also, D. found in Lindsley and Grell (1968). Flies were obtained from the Mid-America mefanogaster with DNA repair-deficient mutations are hypersensitive to the lethal Drosophila Center, Bowling Green, Ohio, effects of growing larvae in media containand were maintained by mass culture. A ing 0.001 mM AFB, (Nguyen et al., 1979). previous study (Chinnici and Llewellyn, AFB, also will induce recessive lethal sec- 1979) has shown that Florida-9 is quite sensitive to the toxic effects of lifetime expoond chromosome mutations in D. melasure (egg-adult) to AFB, while Lausannenoguster (Lamb and Lilly, 1971). When males are injected with AFB1, X chromoS is relatively resistant to the treatments. some recessive lethals and X chromosome Media. Five types of media were prorecessive visible mutations are induced duced: control medium containing no afwith both whole body and mosaic mutants latoxin, medium containing AFB, (Grade seen (Fahmy et al., 1978). A, stock 121741, lot 387032), media conAlthough considerable data exist on the taining AFB, (Grade A, stock 121743, lot effects of AFB1, nothing is known con- 500003), medium containing AFG, (Grade cerning the toxic effects of other aflatoxins B, stock 121748, lot 300644), and medium and mycotoxins related to AFB, in D. containing ST (Grade B, stock 568611, lot mefanoguster. This paper reports the toxic 800779). All mycotoxins were obtained effects of the mycotoxins aflatoxin G, from Calbiochem-Behring, La Jolla, (AFG,), aflatoxin B, (AFB,), and sterig- California. matocystin (ST) on two strains of D. The various types of media were premelunoguster previously tested for degree pared according to Chinnici et al. (1976) exof sensitivity to AFB,. It is of interest to cept that in this case each toxic stock was determine if a correlation exists between diluted to yield measureable concentrations sensitivity or resistance to AFB, and the of 0.2, 0.6, 2.0, and 4.0 ppm of AFB1, effect of AFG,, which has a similar mechaAFB2, AFG1, and ST. After partial cooling, nism of toxic activation in vertebrate sys- each type of medium was poured into a tems (Gut-too et al., 1978; Gamer et al., series of 8-dram glass shell vials, stoppered 1979). Also, the insecticidal properties of with foam plastic plugs, and refrigerated AFBz and ST are of interest since neither is until used. Samples of control medium and a strong toxigen or mutagen in vertebrate each type of experimental medium were systems (Carnaghan et al., 1963; Ong, analyzed quantitatively and qualitatively 1975). The responses of different strains of for mycotoxins using a thin-layer chromaD. melunoguster to these various mycotoxtography system (Horwitz et al., 1975). ins may determine whether this species is Experimental procedures. All flies were a good assay system for detecting the pres- maintained at 25” -t 2°C in a constant temence of such mycotoxins in the environment. perature incubator. Initially, flies were Finally, the demonstration of differential placed in half-pint plastic bottles containing sensitivities to these mycotoxins can lead to control media and allowed to lay eggs on an analysis of the genetic differences af- the surface of the media for 12 hr. Eggs then fecting sensitivity in D. melunoguster, as is were collected and placed onto small pieces currently being done with AFB, (Chinnici, of blotting paper, 25 eggs per piece. Imme1980). diately thereafter, one piece of paper with
390
GUNST,
CHINNICI,
eggs was placed in each vial containing experimental or control media. For each of the two strains of D. melanogaster used, six vials for each treatment were set up (control; 0.2, 0.6, 2.0, 4.0 ppm AFB1, AFB*, AFGI, and ST; 102 vials per strain). Each vial was checked daily and data collected for pupation and eclosion times. Pupae were counted as observed. However, some larvae pupated under or within the blotting paper squares lying on the food surface or slightly below the food surface. This made it impossible to count the total number of pupal cases actually present. For this reason, in some cases the number of pupae is less than the number of adults in Tables 1 and 2. Adults were counted, sexed, and measured by placing them onto millimeter graph paper and measuring to the nearest 0.5 mm. Data were analyzed using orthogonal linear contrast statistics. Differences referred to as being significant in the sections below have probabilities of 0.05 or lower. RESULTS
Data for the AFB,-sensitive Florida-9 strain are given in Table 1 and data for the AFB,-resistant Lausanne-S strain are given in Table 2. Number ofpupae. In the Florida-9 strain, toxic sensitivity is seen for the AFB, and AFG, treatments. A significant reduction in number of pupae is seen at the 0.6-ppm AFB, and the 0.2- and O.dppm AFG, concentrations. No pupae are produced above 0.6 ppm on either AFB, or AFG,. However, Florida-9 pupae numbers are not reduced significantly by growth on media with up to 4.0 ppm of either AFB2 or ST, indicating lack of sensitivity to these mycotoxins at the concentrations tested. In the Lausanne-S strain, toxic resistance (relative to Florida-9) is seen for the AFBr and AFG, treatments. A significant decrease in number of pupae is seen only at the 2.0- and 4.0-ppm-AFB, treatments, whereas pupal number is not affected significantly by any AFG, or AFB2 treatments
AND
LLEWELLYN
reported. Interestingly, the only ST treatment significantly different is 4.0 ppm which produced more pupae than the control. Egg-pupal development time. In the Florida-9 strain, significant increases in egg-pupal development time is seen for the 0.6-ppm-AFB, and 0.6-ppm-AFG, treatments, illustrating the sensitive nature of Florida-9 to these toxins. Growth on AFB, and ST do not affect development times in Florida-9. In the Lausanne-S strain, significant increases in development times occur for the 2.0- and 4.0-ppm-AFB, and the 4.0ppm-AFG, treatments, indicating sensitivity only to relatively high doses of these toxins. Interestingly, Lausanne-S pupae developed significantly faster than the controls when treated with 0.6, 2.0, or 4.0 ppm ST. Number ofadults. The Florida-9 strain is quite sensitive to egg-adult mortality due to the presence of AFB, or AFG, in the media. Significant decreases in adult number are seen in the 0.2- and 0.6-ppmAFB, treatments (no adults at all at 2.0 and 4.0 ppm), and the 0.2-ppm-AFG, treatment (no adults at 0.6, 2.0, and 4.0 ppm). The number of Florida-9 adults produced is not affected by any of the AFB, or ST treatments reported. The Lausanne-S strain displays much higher resistance levels than does Florida-9 to egg-adult mortality induced by either AFBl or AFG1, since Lausanne-S adult mortality increases significantly only in the 2.0- and 4.0-ppm-AFB, treatments and in the 4.0-ppm-AFG, treatment. None of the AFB, or ST treatments results in a significant increase in adult mortality, although the 2.0- and 4.0-ppmAFB2 and the 4.0-ppm-ST treatments produce significantly more adults than does the control. Egg-adult development times. In the Florida-9 strain, no significant differences in egg-adult development times are observed for the AFBl or AFG, treatments, but significant decreases in development time occur in all the AFBz treatments and in
EFFECT
OF
MYCOTOXINS
IN
TABLE TOXICITY
DATA
FOR FLORIDA-9
391
Drosophila
1
STRAIN
Pupae *
OF Drosophila
melanogaster
Adults’
Number
Time
Number
Time
Body length of females (mm)
11.50 2 0.89
6.65 2 0.14
15.67 2 1.17
11.02 2 0.08
2.82 ? 0.05
AFB, 0.2 0.6 2.0 4.0
9.67 k 0.95 6.17 k 0.54 0 0
6.57 +- 0.08 9.23 AT0.37 -
8.50 + 1.02 0.17 k 0.17 0 0
10.62 2 0.06 4f;OO
2.42 f 0.07 2.50
-
-
AFG, 0.2 0.6 2.0 4.0
4.17 k 0.60 0.83 k 0.54 0 0
7.80 +- 0.56 8.35 k 0.35 -
6.00 f 1.29 0 0 0
11.30 k 0.24 -
2.32 t 0.06 -
-
-
Toxin”
Control
AFB, 0.2 0.6 2.0 4.0
11.33 14.33 12.00 11.00
rt t 2 t
1.23 1.20 0.97 1.24
6.57 6.87 6.28 6.35
f t ” t
0.08 0.12 0.07 0.02
15.17 15.00 16.67 15.33
t k 2 *
1.56 1.93 1.20 0.71
10.65 10.53 10.17 10.08
f ? f f
0.11 0.07 0.06 0.03
2.70 2.55 2.62 2.67
t t e t
0.04 0.05 0.05 0.05
ST 0.2 0.6 2.0 4.0
13.33 11.17 11.50 9.50
2 2 2 t
0.67 0.40 1.12 0.85
6.38 7.72 5.23 5.55
k 0.05 2 0.11 -c 0.10 2 0.12
16.50 16.83 16.33 16.67
t 2 f r
1.06 0.83 1.15 0.95
10.55 k 10.95 t 9.23 k 9.42 k
0.05 0.10 0.15 0.09
2.88 2.78 2.67 2.65
k 2 k f
0.06 0.05 0.03 0.06
R Parts per million (ppm) atlatoxin B1 (AFB,), aflatoxin G, (AFG,), atlatoxin B, (AFB&, or sterigmatocystin (ST) in the culture media. b Number of pupal cases observed and egg-pupa development time in days, means and standard errors of the mean for six replications, 25 eggs per replication. CNumber of adults eclosing and egg-adult development times in days, means and standard error of the means.
the 0.2-, 2.0-, and 4.0-ppm-ST treatments. For Lausanne-S, no significant differences in development time are seen for the AFB, treatments, but the 0.2- and O.dppm-AFG, treatments display significantly faster development times and the 4.0-ppm-AFG, treatment displays significantly slower development. Some decrease is seen in the AFB, treatments and significant decreases are found for all ST treatments. Adult female body lengths. In the Florida-9 strain, significant decreases in female adult body lengths are seen in the O.ZppmAFB, and 0.2-ppm-AFG1 treatments as well as in all AFBz and the 2.0- and 4.0ppm-ST treatments. For Lausanne-S length
occur for the 2.0- and 4.0-ppm-AFG1 treatments, whereas a significant increase in body length occurs for the 2.0-ppm-AFB, treatment. Other treatments do not show significant differences from the control values. DISCUSSION
In vertebrate species, the most toxic and carcinogenic aflatoxin is AFBl (Wogan, 1973). The other aflatoxins and related mycotoxins such as ST are much less mutagenic and carcinogenic than AFB, (Wong and Hsieh, 1976; Ong, 1975; Gurtoo et al., 1978). Carnaghan et al. (1963) measured the relative potency of several afla-
392
GUNST,
TOXICITY
DATA
CHINNICI,
AND
TABLE 2 FOR LAUSANNE-S STRAIN
Pupae b Toxin”
Number
LLEWELLYN
OF Drosophila
melanogaster
Adults’ Time
Number
Body Time
length of females (mm)
6.48 k 0.31
17.83 + 1.82
11.62 k 0.39
2.68 * 0.03
6.73 k 0.09 6.65 + 0.06 9.72 ‘- 0.11 11.00
22.67 2 0.49 16.00 + 0.58 0.33 k 0.33 0
10.95 2 0.07 10.63 f 0.06 15.00 -
2.63 + 0.04 2.60 k 0.04 2.00
k 0.05 + 0.05 -+- 0.12 + 0.20
22.00 20.83 19.17 2.50
T ” 2 k
0.82 0.98 0.70 0.96
9.53 10.10 11.43 14.38
+ 2 ? 2
0.08 0.05 0.10 0.39
2.62 2.58 2.40 2.10
k 2 + k
0.04 0.02 0.07 0.10
6.70 6.95 6.95 6.78
2 f -c 2
0.09 0.10 0.14 0.14
16.33 15.33 22.00 21.00
k 2 + +
1.65 1.28 0.26 0.68
10.51 11.03 10.92 10.73
f t ? f
0.11 0.10 0.05 0.12
2.73 2.70 2.82 2.73
+ ? 5 +
0.07 0.04 0.04 0.03
7.15 5.27 5.68 6.02
” k + 2
0.06 0.08 0.13 0.16
15.17 15.50 15.50 21.33
+ ? t f
2.88 0.67 1.36 0.88
10.62 9.33 9.53 9.83
t 2 4 2
0.22 0.15 0.04 0.08
2.80 2.80 2.65 2.63
k + 2 2
0.05 0.07 0.04 0.03
Control
13.33 2 2.28
AFB( 0.2 0.6 2.0 4.0
14.83 11.67 5.50 0.17
2 2 k +
1.05 0.80 1.34 0.17
AFG, 0.2 0.6 2.0 4.0
19.83 17.63 11.83 10.00
2 + t 2
1.83 0.65 2.75 1.73
5.62 5.97 7.00 9.55
AFB, 0.2 0.6 2.0 4.0
15.67 16.67 16.50 16.17
k f k 2
1.23 1.20 1.06 1.62
12.50 15.50 15.17 17.67
k k + +
2.13 0.76 1.19 1.05
ST 0.2 0.6 2.0 4.0
~MX See footnotes
to Table
1.
toxins in ducklings and showed the LD,, values to be 18.2 pg for AFB,, 84.8 ,ug for AFB2, and 39.2 pg for AFG, per 50 g body wt. In the rat, ST is one-sixth as toxic to the liver as AFB,. It is 200 times less carcinogenic than AFB, when administered subcutaneously and 10 times less carcinogenic when given orally (Purchase and Watt, 1968, 1969; Englebrecht, 1971). The carcinogenic effects of AFB, and AFGl are due to their possession of an exposed epoxide double bond in the 2,3 position which allows conversion, upon microsomal activation, to an active epoxide intermediate which binds to DNA covalently (Gurtoo et al., 1978; Garner et al., 1979). AFBz lacks the 2,3 double bond but may be converted to AFB, in limited quantities. Data presented in this report indicate that strain Florida-9 is quite sensitive to the toxic effects on growth from the egg stage
in media containing small doses of either AFB, or AFG, but is not sensitive to the toxic effects of AFB, or ST in the growth media. In contrast, strain Lausanne-S is significantly more resistant to both AFB,and AFG,- induced toxicity while also not being sensitive to the effects of AFB, or ST. The differential response to AFG, is most striking, since Florida-9 flies are more sensitive to AFG, than to AFB, while Lausanne-S flies are more resistant to AFG, than to AFB,. Florida-9 produces no adults (100% lethality) at 0.6 ppm and higher concentrations of AFG, while Lausanne-S produces adults on 4.0 ppm AFG,. It may not be surprising that Florida-9, which is sensitive to AFB,-induced toxicity, is also sensitive to the toxic effects of AFG, possibly due to the similarities of their structures. But the extreme sensitivity of Florida-9 to AFG, is noteworthy since
EFFECT
OF
MYCOTOXINS
vertebrate studies consistently show that AFGl is not as potent a toxigen as AFB,. The reverse is true, however, in strain Florida-9. Drosophila possesses a microsomal system capable of activating mutagens and carcinogens, especially polycyclic hydrocarbons (Baars et al., 1980), and it has been suggested that this system may show strain and species differences in drosophila. Such differences may be present in the Lausanne-S and Florida-9 strains of D. melanogaster. Studies to determine this are now underway. Neither strain displayed any toxic responses to the presence of either AFB, or ST in the media. In fact, Lausanne-S flies actually seem to benefit from the presence of 4.0 ppm ST in the food, since a significant increase in pupal and adult numbers occurs for this treatment, indicating a decrease in egg-pupa and egg-adult mortalities. ACKNOWLEDGMENTS We wish to thank Ms. Almedia Bell for her technical assistance in collecting data. Mr. Thomas Eadie of the Virginia Division of Consolidated Laboratory Service, Mycotoxin Laboratory, Richmond, Virginia kindly performed the mycotoxin analyses.
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J. P., AND LLEWELLYN, G. C. 1979. Reduced atlatoxin toxicity in hybrid crosses of aflatoxin B1 sensitive and resistant strains ofDrosophila melanogaster (Diptera). J. Invertebr. Pathol., 33, 81-85. DIENER, V. L., AND DAVIS, N. D. 1969. Aflatoxin formation by Aspergillus j7avus. In “AflatoxinScientific Background, Control and Implications” (L. A. Goldblatt, ed.), pp. 13-57. Academic Press, New York. ENGELBRECHT, J. C. 1971. The effects of aflatoxin B1 and sterigmatocystin on two different types of cell cultures. In “Symposium on Mycotoxins in Human Health” (I. F. H. Purchase, ed.), pp. 215-222. Macmillan, New York. FAHMY, M. J., FAHMY, 0. G., AND SWENSON, D. H. 1978. Aflatoxin B,-2, 3-dichloride as a model of the active metabolite of aflatoxin B, in mutagenesis and carcinogenesis. Cancer Res., 38, 2608-2616. GARNER, R. C., MARTIN, C. N., SMITH, J. R. L.. COLES, B. F., AND TOLSON, M. R. 1979. Comparison of aflatoxin B, and aflatoxin G, binding to cellular macromolecules in vitro, in vivo and after peracid oxidation; characterization of the major nucleic acid adducts. Chern-Biol. Interact., 26, 57-73. GASTON, J. Q., AND LLEWELLYN, G. C. 1980. Sexdetermined differential mortality of milkweed bugs, Oncopeltus fasciatus (Hemiptera), to aflatoxins. J. Invertebr. Pathol., 35, 195-199. GURTOO, H. L., DAHMS, R. P., AND PAIGEN. B. 1978. Metabolic activation of aflatoxins related to their mutagenicity. Biochem. Biophys. Res. Cotnmun., 81,965-972. HORWITZ, W., SENZEL, A., AND REYNOLDS, H. 1975. Natural poisons. In “Official Methods of Analysis,” pp. 465-473. Association of Official Analytical Chemists, Washington, D. C. CHINNICI,
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OF
INVERTEBRATE
PATHOLOGY
39, 388-394 (1982)
Effects of Aflatoxin B,, Aflatoxin B2, Aflatoxin G,, and Sterigmatocystin on Viability, Rates of Development, and Body Length in Two Strains of Drosophila melanogaster (Diptera) KATHLEEN Department
GUNST, of Biology,
JOSEPH P. CHINNICI, Virginia
Commonwealth
AND GERALD University,
C. LLEWELLYN
Richmond,
Virginia
23284
Received March 5, 1981; accepted May 15, 1981 Two wild-type laboratory populations of Drosophila melanogaster, Florida-9 (sensitive to aflatoxin (AF) B,-induced toxicity) and Lausanne-S (resistant to AFB,-induced toxicity) were tested to determine relative degrees of sensitivity to growth from the egg stage on media containing 0.2, 0.6, 2.0, and 4.0 ppm AFB,, AFG,, AFB2, or sterigmatocystin (ST). Data indicate that strain Florida-9 is quite sensitive to AFG, toxicity at both the egg-pupa and egg-adult stages of development while Lausanne-S is quite resistant to such toxic effects. For Lausanne-S, AFB, > AFG, in relative toxicity, while for Florida-9, AFG, > AFB,. The latter is noteworthy since vertebrate studies consistently show that AFB, is a significantly stronger carcinogen and mutagen than AFG,. Possible explanations are discussed. Neither strain tested displayed toxic responses to the presence of AFBZ or ST in the culture media; however, the 4.0-ppm Lausanne-S treatment displayed a significantly lower adult mortality rate than the control, indicating that Lausanne-S flies may benefit from the presence of ST in the culture medium. KEY WORDS: atlatoxin B,, aflatoxin B,, aflatoxin G,, sterigmatocystin, mycotoxins, Drosophila. Aspergillus
jlavus.
effects of aflatoxins on viability, fertility, or mutagenesis involve aflatoxin B, (AFB,). The aflatoxins are a group of at least 12 Various wild-type laboratory strains of the fruit fly Drosophila melanogaster differ highly substituted polycyclic compounds produced by several species of fungi, espe- significantly in their levels of sensitivity to cially Aspergillus flaws and A. parasiticus the toxic effects of AFB, in the culture (Diener and Davis, 1969; Wogan 1975). medium (Llewellyn and Chinnici, 1978; Discovered after several incidences of Chinnici and Llewellyn, 1979). Toxicity is demonstrated by high levels of larval and poultry kills due to eating contaminated pupal lethality, significantly reduced pupa feed (Blount, 1961; Asplin and Carnaghan, 1961), the aflatoxins are highly potent ver- case and adult body lengths, and signifitimes induced tebrate hepatocarcinogens (Newberne and cantly longer development Butler, 1969; Kocylawski et al., 1973; by AFB, in the growth medium. Sensitive Reddy et al., 1976; Wray and Hayes, 1980) strains show marked toxic effects at low of AFBl while resistant and effective mutagens in a wide variety of concentrations strains do not show toxic effects unless organisms (Ong, 1975; Wong and Hsieh, 1976; Fahmy et al., 1978; Thomson and subjected to concentrations two- to threeEvans, 1979). In addition, aflatoxins dis- fold higher than the effective doses for sensitive strains (Llewellyn and Chinnici, play insecticidal, larvicidal, and chemosterilizing properties against a wide variety 1978). Using a resistant strain, continuous egg-adult exposure to 0.67 ppm AFBl in of insect species (Lalor et al., 1976; Moore et al., 1978; Chinnici et al., 1979; Nguyen et the culture medium caused a significant reduction in the fertility of surviving females al., 1979; Gaston and Llewellyn, 1980). In insects, the only detailed studies of the but did not affect the rate of meiotic INTRODUCTION
388 0022-201 l/82/030388-07$01.00/O Copyright All rights
@ 1982 by Academic Press, Inc. of reproduction in any form reserved.
EFFECT
OF
MYCOTOXINS
IN
Drosophila
389
crossing-over in these females (Chinnici et MATERIALS AND METHODS al., 1976). Chinnici et al. (1979) have shown that Insects. Two standard wild-type laborasensitive and resistant strains differ in the tory strains of D. melunoguster were used stages of larval development sensitive to in these studies: Lausanne-S and Florida-9. AFB,-induced toxicity and in the degree of Complete descriptions of these strains are such sensitivity at each stage. Also, D. found in Lindsley and Grell (1968). Flies were obtained from the Mid-America mefanogaster with DNA repair-deficient mutations are hypersensitive to the lethal Drosophila Center, Bowling Green, Ohio, effects of growing larvae in media containand were maintained by mass culture. A ing 0.001 mM AFB, (Nguyen et al., 1979). previous study (Chinnici and Llewellyn, AFB, also will induce recessive lethal sec- 1979) has shown that Florida-9 is quite sensitive to the toxic effects of lifetime expoond chromosome mutations in D. melasure (egg-adult) to AFB, while Lausannenoguster (Lamb and Lilly, 1971). When males are injected with AFB1, X chromoS is relatively resistant to the treatments. some recessive lethals and X chromosome Media. Five types of media were prorecessive visible mutations are induced duced: control medium containing no afwith both whole body and mosaic mutants latoxin, medium containing AFB, (Grade seen (Fahmy et al., 1978). A, stock 121741, lot 387032), media conAlthough considerable data exist on the taining AFB, (Grade A, stock 121743, lot effects of AFB1, nothing is known con- 500003), medium containing AFG, (Grade cerning the toxic effects of other aflatoxins B, stock 121748, lot 300644), and medium and mycotoxins related to AFB, in D. containing ST (Grade B, stock 568611, lot mefanoguster. This paper reports the toxic 800779). All mycotoxins were obtained effects of the mycotoxins aflatoxin G, from Calbiochem-Behring, La Jolla, (AFG,), aflatoxin B, (AFB,), and sterig- California. matocystin (ST) on two strains of D. The various types of media were premelunoguster previously tested for degree pared according to Chinnici et al. (1976) exof sensitivity to AFB,. It is of interest to cept that in this case each toxic stock was determine if a correlation exists between diluted to yield measureable concentrations sensitivity or resistance to AFB, and the of 0.2, 0.6, 2.0, and 4.0 ppm of AFB1, effect of AFG,, which has a similar mechaAFB2, AFG1, and ST. After partial cooling, nism of toxic activation in vertebrate sys- each type of medium was poured into a tems (Gut-too et al., 1978; Gamer et al., series of 8-dram glass shell vials, stoppered 1979). Also, the insecticidal properties of with foam plastic plugs, and refrigerated AFBz and ST are of interest since neither is until used. Samples of control medium and a strong toxigen or mutagen in vertebrate each type of experimental medium were systems (Carnaghan et al., 1963; Ong, analyzed quantitatively and qualitatively 1975). The responses of different strains of for mycotoxins using a thin-layer chromaD. melunoguster to these various mycotoxtography system (Horwitz et al., 1975). ins may determine whether this species is Experimental procedures. All flies were a good assay system for detecting the pres- maintained at 25” -t 2°C in a constant temence of such mycotoxins in the environment. perature incubator. Initially, flies were Finally, the demonstration of differential placed in half-pint plastic bottles containing sensitivities to these mycotoxins can lead to control media and allowed to lay eggs on an analysis of the genetic differences af- the surface of the media for 12 hr. Eggs then fecting sensitivity in D. melunoguster, as is were collected and placed onto small pieces currently being done with AFB, (Chinnici, of blotting paper, 25 eggs per piece. Imme1980). diately thereafter, one piece of paper with
390
GUNST,
CHINNICI,
eggs was placed in each vial containing experimental or control media. For each of the two strains of D. melanogaster used, six vials for each treatment were set up (control; 0.2, 0.6, 2.0, 4.0 ppm AFB1, AFB*, AFGI, and ST; 102 vials per strain). Each vial was checked daily and data collected for pupation and eclosion times. Pupae were counted as observed. However, some larvae pupated under or within the blotting paper squares lying on the food surface or slightly below the food surface. This made it impossible to count the total number of pupal cases actually present. For this reason, in some cases the number of pupae is less than the number of adults in Tables 1 and 2. Adults were counted, sexed, and measured by placing them onto millimeter graph paper and measuring to the nearest 0.5 mm. Data were analyzed using orthogonal linear contrast statistics. Differences referred to as being significant in the sections below have probabilities of 0.05 or lower. RESULTS
Data for the AFB,-sensitive Florida-9 strain are given in Table 1 and data for the AFB,-resistant Lausanne-S strain are given in Table 2. Number ofpupae. In the Florida-9 strain, toxic sensitivity is seen for the AFB, and AFG, treatments. A significant reduction in number of pupae is seen at the 0.6-ppm AFB, and the 0.2- and O.dppm AFG, concentrations. No pupae are produced above 0.6 ppm on either AFB, or AFG,. However, Florida-9 pupae numbers are not reduced significantly by growth on media with up to 4.0 ppm of either AFB2 or ST, indicating lack of sensitivity to these mycotoxins at the concentrations tested. In the Lausanne-S strain, toxic resistance (relative to Florida-9) is seen for the AFBr and AFG, treatments. A significant decrease in number of pupae is seen only at the 2.0- and 4.0-ppm-AFB, treatments, whereas pupal number is not affected significantly by any AFG, or AFB2 treatments
AND
LLEWELLYN
reported. Interestingly, the only ST treatment significantly different is 4.0 ppm which produced more pupae than the control. Egg-pupal development time. In the Florida-9 strain, significant increases in egg-pupal development time is seen for the 0.6-ppm-AFB, and 0.6-ppm-AFG, treatments, illustrating the sensitive nature of Florida-9 to these toxins. Growth on AFB, and ST do not affect development times in Florida-9. In the Lausanne-S strain, significant increases in development times occur for the 2.0- and 4.0-ppm-AFB, and the 4.0ppm-AFG, treatments, indicating sensitivity only to relatively high doses of these toxins. Interestingly, Lausanne-S pupae developed significantly faster than the controls when treated with 0.6, 2.0, or 4.0 ppm ST. Number ofadults. The Florida-9 strain is quite sensitive to egg-adult mortality due to the presence of AFB, or AFG, in the media. Significant decreases in adult number are seen in the 0.2- and 0.6-ppmAFB, treatments (no adults at all at 2.0 and 4.0 ppm), and the 0.2-ppm-AFG, treatment (no adults at 0.6, 2.0, and 4.0 ppm). The number of Florida-9 adults produced is not affected by any of the AFB, or ST treatments reported. The Lausanne-S strain displays much higher resistance levels than does Florida-9 to egg-adult mortality induced by either AFBl or AFG1, since Lausanne-S adult mortality increases significantly only in the 2.0- and 4.0-ppm-AFB, treatments and in the 4.0-ppm-AFG, treatment. None of the AFB, or ST treatments results in a significant increase in adult mortality, although the 2.0- and 4.0-ppmAFB2 and the 4.0-ppm-ST treatments produce significantly more adults than does the control. Egg-adult development times. In the Florida-9 strain, no significant differences in egg-adult development times are observed for the AFBl or AFG, treatments, but significant decreases in development time occur in all the AFBz treatments and in
EFFECT
OF
MYCOTOXINS
IN
TABLE TOXICITY
DATA
FOR FLORIDA-9
391
Drosophila
1
STRAIN
Pupae *
OF Drosophila
melanogaster
Adults’
Number
Time
Number
Time
Body length of females (mm)
11.50 2 0.89
6.65 2 0.14
15.67 2 1.17
11.02 2 0.08
2.82 ? 0.05
AFB, 0.2 0.6 2.0 4.0
9.67 k 0.95 6.17 k 0.54 0 0
6.57 +- 0.08 9.23 AT0.37 -
8.50 + 1.02 0.17 k 0.17 0 0
10.62 2 0.06 4f;OO
2.42 f 0.07 2.50
-
-
AFG, 0.2 0.6 2.0 4.0
4.17 k 0.60 0.83 k 0.54 0 0
7.80 +- 0.56 8.35 k 0.35 -
6.00 f 1.29 0 0 0
11.30 k 0.24 -
2.32 t 0.06 -
-
-
Toxin”
Control
AFB, 0.2 0.6 2.0 4.0
11.33 14.33 12.00 11.00
rt t 2 t
1.23 1.20 0.97 1.24
6.57 6.87 6.28 6.35
f t ” t
0.08 0.12 0.07 0.02
15.17 15.00 16.67 15.33
t k 2 *
1.56 1.93 1.20 0.71
10.65 10.53 10.17 10.08
f ? f f
0.11 0.07 0.06 0.03
2.70 2.55 2.62 2.67
t t e t
0.04 0.05 0.05 0.05
ST 0.2 0.6 2.0 4.0
13.33 11.17 11.50 9.50
2 2 2 t
0.67 0.40 1.12 0.85
6.38 7.72 5.23 5.55
k 0.05 2 0.11 -c 0.10 2 0.12
16.50 16.83 16.33 16.67
t 2 f r
1.06 0.83 1.15 0.95
10.55 k 10.95 t 9.23 k 9.42 k
0.05 0.10 0.15 0.09
2.88 2.78 2.67 2.65
k 2 k f
0.06 0.05 0.03 0.06
R Parts per million (ppm) atlatoxin B1 (AFB,), aflatoxin G, (AFG,), atlatoxin B, (AFB&, or sterigmatocystin (ST) in the culture media. b Number of pupal cases observed and egg-pupa development time in days, means and standard errors of the mean for six replications, 25 eggs per replication. CNumber of adults eclosing and egg-adult development times in days, means and standard error of the means.
the 0.2-, 2.0-, and 4.0-ppm-ST treatments. For Lausanne-S, no significant differences in development time are seen for the AFB, treatments, but the 0.2- and O.dppm-AFG, treatments display significantly faster development times and the 4.0-ppm-AFG, treatment displays significantly slower development. Some decrease is seen in the AFB, treatments and significant decreases are found for all ST treatments. Adult female body lengths. In the Florida-9 strain, significant decreases in female adult body lengths are seen in the O.ZppmAFB, and 0.2-ppm-AFG1 treatments as well as in all AFBz and the 2.0- and 4.0ppm-ST treatments. For Lausanne-S length
occur for the 2.0- and 4.0-ppm-AFG1 treatments, whereas a significant increase in body length occurs for the 2.0-ppm-AFB, treatment. Other treatments do not show significant differences from the control values. DISCUSSION
In vertebrate species, the most toxic and carcinogenic aflatoxin is AFBl (Wogan, 1973). The other aflatoxins and related mycotoxins such as ST are much less mutagenic and carcinogenic than AFB, (Wong and Hsieh, 1976; Ong, 1975; Gurtoo et al., 1978). Carnaghan et al. (1963) measured the relative potency of several afla-
392
GUNST,
TOXICITY
DATA
CHINNICI,
AND
TABLE 2 FOR LAUSANNE-S STRAIN
Pupae b Toxin”
Number
LLEWELLYN
OF Drosophila
melanogaster
Adults’ Time
Number
Body Time
length of females (mm)
6.48 k 0.31
17.83 + 1.82
11.62 k 0.39
2.68 * 0.03
6.73 k 0.09 6.65 + 0.06 9.72 ‘- 0.11 11.00
22.67 2 0.49 16.00 + 0.58 0.33 k 0.33 0
10.95 2 0.07 10.63 f 0.06 15.00 -
2.63 + 0.04 2.60 k 0.04 2.00
k 0.05 + 0.05 -+- 0.12 + 0.20
22.00 20.83 19.17 2.50
T ” 2 k
0.82 0.98 0.70 0.96
9.53 10.10 11.43 14.38
+ 2 ? 2
0.08 0.05 0.10 0.39
2.62 2.58 2.40 2.10
k 2 + k
0.04 0.02 0.07 0.10
6.70 6.95 6.95 6.78
2 f -c 2
0.09 0.10 0.14 0.14
16.33 15.33 22.00 21.00
k 2 + +
1.65 1.28 0.26 0.68
10.51 11.03 10.92 10.73
f t ? f
0.11 0.10 0.05 0.12
2.73 2.70 2.82 2.73
+ ? 5 +
0.07 0.04 0.04 0.03
7.15 5.27 5.68 6.02
” k + 2
0.06 0.08 0.13 0.16
15.17 15.50 15.50 21.33
+ ? t f
2.88 0.67 1.36 0.88
10.62 9.33 9.53 9.83
t 2 4 2
0.22 0.15 0.04 0.08
2.80 2.80 2.65 2.63
k + 2 2
0.05 0.07 0.04 0.03
Control
13.33 2 2.28
AFB( 0.2 0.6 2.0 4.0
14.83 11.67 5.50 0.17
2 2 k +
1.05 0.80 1.34 0.17
AFG, 0.2 0.6 2.0 4.0
19.83 17.63 11.83 10.00
2 + t 2
1.83 0.65 2.75 1.73
5.62 5.97 7.00 9.55
AFB, 0.2 0.6 2.0 4.0
15.67 16.67 16.50 16.17
k f k 2
1.23 1.20 1.06 1.62
12.50 15.50 15.17 17.67
k k + +
2.13 0.76 1.19 1.05
ST 0.2 0.6 2.0 4.0
~MX See footnotes
to Table
1.
toxins in ducklings and showed the LD,, values to be 18.2 pg for AFB,, 84.8 ,ug for AFB2, and 39.2 pg for AFG, per 50 g body wt. In the rat, ST is one-sixth as toxic to the liver as AFB,. It is 200 times less carcinogenic than AFB, when administered subcutaneously and 10 times less carcinogenic when given orally (Purchase and Watt, 1968, 1969; Englebrecht, 1971). The carcinogenic effects of AFB, and AFGl are due to their possession of an exposed epoxide double bond in the 2,3 position which allows conversion, upon microsomal activation, to an active epoxide intermediate which binds to DNA covalently (Gurtoo et al., 1978; Garner et al., 1979). AFBz lacks the 2,3 double bond but may be converted to AFB, in limited quantities. Data presented in this report indicate that strain Florida-9 is quite sensitive to the toxic effects on growth from the egg stage
in media containing small doses of either AFB, or AFG, but is not sensitive to the toxic effects of AFB, or ST in the growth media. In contrast, strain Lausanne-S is significantly more resistant to both AFB,and AFG,- induced toxicity while also not being sensitive to the effects of AFB, or ST. The differential response to AFG, is most striking, since Florida-9 flies are more sensitive to AFG, than to AFB, while Lausanne-S flies are more resistant to AFG, than to AFB,. Florida-9 produces no adults (100% lethality) at 0.6 ppm and higher concentrations of AFG, while Lausanne-S produces adults on 4.0 ppm AFG,. It may not be surprising that Florida-9, which is sensitive to AFB,-induced toxicity, is also sensitive to the toxic effects of AFG, possibly due to the similarities of their structures. But the extreme sensitivity of Florida-9 to AFG, is noteworthy since
EFFECT
OF
MYCOTOXINS
vertebrate studies consistently show that AFGl is not as potent a toxigen as AFB,. The reverse is true, however, in strain Florida-9. Drosophila possesses a microsomal system capable of activating mutagens and carcinogens, especially polycyclic hydrocarbons (Baars et al., 1980), and it has been suggested that this system may show strain and species differences in drosophila. Such differences may be present in the Lausanne-S and Florida-9 strains of D. melanogaster. Studies to determine this are now underway. Neither strain displayed any toxic responses to the presence of either AFB, or ST in the media. In fact, Lausanne-S flies actually seem to benefit from the presence of 4.0 ppm ST in the food, since a significant increase in pupal and adult numbers occurs for this treatment, indicating a decrease in egg-pupa and egg-adult mortalities. ACKNOWLEDGMENTS We wish to thank Ms. Almedia Bell for her technical assistance in collecting data. Mr. Thomas Eadie of the Virginia Division of Consolidated Laboratory Service, Mycotoxin Laboratory, Richmond, Virginia kindly performed the mycotoxin analyses.
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WRAY, B. B., AND HAYES, A. W. 1980. Aflatoxin B, in the serum of a patient with primary hepatic carcinoma. Environ Res., 22, 400-403.