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Genetic Resistance to DDT in the Japanese Quail Coturnix Coturnix Japonica1,2
MAREK'S DISEASE VACCINE Neuritis-epizootie bij kippen te Barneveld in 1921. Tijdschr. voor verg. Geneesk. 10: 34-50. Witter, R. L., G. H. Burgoyne and J. J. Solomon, 1969. Evidence for a herpesvirus as an etiologic agent of Marek's disease. Avian Dis. 13:171-184. Witter, R. L., J. I. Moulthrop, Jr., G. H. Burgoyne and H. C. Cornell, 1970. Studies on the epidemiol-
841
ogy of Marek's disease herpesvirus in broiler flocks. Avian Dis. 14: 255-267. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 3 1 : 525-538.
Genetic Resistance to D D T in the Japanese Quail Coturnix Coturnix Japonic^'2
(Received for publication May 25, 1972) ABSTRACT Selection studies of resistance to D D T included eight generations of quail consisting of two selected lines and one control line. One selected line differed from the other in that, in addition to mass selection, birds were chosen on the basis of family merit. Selected lines were fed a diet containing 200 p.p.m. D D T during the first 30 days of life. In order to test for resistance, part of the control lines were also given DDT diets, and the mortality of the control was compared to the mortality of the selected lines. Development of resistance started to appear after the third generation of selection. This was evidenced by the lower mortality among the selected lines than in the control line fed DDT. If the selected lines had stored DDT in the body, cold exposure or partial starvation (release of energy from the fat) would presumably increase the D D T levels in the blood and other sensitive tissue, causing mortality due to DDT toxicity. This was put to test by exposing DDT-fed resistant fines and the DDT-fed control group to partial starvation and cooler temperatures. Under these conditions, the selected lines were lower in mortality than the control. Among the resistant lines, the females were less resistant than males. These lines showed some cross-resistance to another hydrochlorinated pesticide lindane. A conclusion drawn from this study is that it is possible to develop a strain of quail resistant to D D T out of the less resistant population by selection. POULTRY SCIENCE 52: 841-846, 1973
INTRODUCTION
A
RTHROPODS maydevelop resistance L toward insecticides used extensively for many generations for their control (Babers and Pratt, 1951; Babers, 1953). Experimentation has shown that resistance can be developed as a consequence of selection pressure exerted by the presence of a toxicant in the environment (Crow, 1957). House flies, Musca domes1
Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin. 1 Paper Number 1550 from the Laboratory of Genetics.
tica, resistant to DDT were first reported in 1947 (Barber and Schmitt, 1948; Lindquist and Wilson, 1948). Many strains of cattle tick, Boophilus microplus, have been found to be resistant to organophosphorus compounds (Shaw and Malcolm, 1964; Shaw, 1965; Roulston et ah, 1968). Oshima (1964) reported that occurrence of resistance to insecticides in wild population of Drosophila. The first report of pesticide resistance in a natural population involved DDTresistant mosquito fish from an intensively sprayed cotton producing area (Ferguson, 1963) and in the cricket frogs, Acris sp.
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K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN Departments of Poultry Science and Genetics, University of Wisconsin, Madison, Wisconsin 53706
842
K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN
MATERIALS AND METHODS
Quail chicks from an untreated (no DDT in ration) population were reared on a 28 percent protein ration and assigned at random to "Artificial Selection" (A), to "Natural Selection" (N) and to "Control" (C) groups. A and N lines were fed 100 p.p.m. DDT in the ration for the first 30 days after hatching. In succeeding generations the amount of DDT in the above mentioned ration was changed to 200 p.p.m. in an attempt to increase mortality to 50 percent during 30 days in the A and N Groups. The control was not fed DDT. Daily mortality was recorded. After 30 days of age the quail were fed a 23 percent protein ration without DDT. One male and one female were assigned to a cage in generation 1. In succeeding generations two females were paired with each male. Eggs were collected from the cages daily and were stored for no longer than 13 days in a cooler. Two hatches were used. The first hatch was used for selecting parents for the next generation in the
A, N and C lines. The second hatch was not used for breeding, but served as a test for resistance by providing a comparison between the A, N and C groups challenged with DDT in the feed. The test was not carried out in generations in which mortality of the birds had reached 50 percent by 30 days of age. The A-line differed from the N-line in that, in addition to mass selection, birds were chosen on the basis of family merit. The index (Lush, 1948) on which selection was based is: (G - G) =
nrh 2 1 + r(n - l)h 2
(x - *)
(G — (?) = estimated breeding worth of an individual n = number of survivors in the family x = percent survivors in a particular family x = average percent surviving in the population r = relationship within family = .5 (assumed h2 = 1) 2 h = portion of the observed variance for which difference in heredity is responsible In the first experiment, sixth-generation progeny of the three lines (A, N and C) were given 200 p.p.m. DDT diet from one day to 23 days of age and then were exposed to cold (18.3 to 20.0°C.) from 15 to 23 days of age. In experiment two, seven-day old progeny of the seventh-generation from A, N and C lines were fed 100, 200 and 300 p.p.m. DDT in the diet for four days. They were then starved for five different lengths of time—8, 14, 16, 18 and 20 hours per day for a period of 30 days. The birds surviving in each group were exposed to the next starvation time. Experiment three used seventh-genera-
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(Boyd et al., 1963). Boyd and Ferguson (1964) demonstrated that as much as 300fold resistance persisted among the first few generations of descendants of these resistant fishes. Wally et al. (1966) made observations on survival of grackles, redwinged blackbirds and starlings in heavily DDT treated areas, while Gill and Verts (1970) investigated the tolerance of a population of ring-necked pheasants occupying an area to which DDT was applied for eight years. The object of this investigation was to determine if resistance to DDT could be developed in the Japanese quail, Coturnix coturnix japonica. Also the experimental design facilitated determining the effects that environmental stress had on the resistance of birds selected for resistance to DDT.
843
GENETIC RESISTANCE TO DDT
RESULTS
The percent mortality of the first and the third to sixth generations is presented in Table 1. In the second generation testing for resistance was not done because the selected lines had 50 percent mortality before the birds were 30 days old. The C-line fed DDT had significantly higher mortality than the two selected lines (A and N) in the later generations. Mortalities of birds exposed to cold as well as those kept at normal room temperatures are compared (Table 2). In all three lines, mortality was higher in the groups exposed to cold (18.3 to 20.0°C.) than in the groups kept at normal room temperatures. At normal room temperature (24°C), the N-line had significantly higher mortality than the A-line, whereas, TABLE 1.—Average percent mortality of the four treatments in each generation for the second hatch
1 2
A
N
C-DDT<
C
46.4 44.0 67.1 36.5 32.2
46.4 49.3 56.9 40.3 47.1
36.4 48.2 75.6 48.4 61.6
11.5 22.3 30.4 31.0 17.6
Groups come from Hatch One. Groups fed 200 p.p.m. DDT in the diet. ' Groups ted 300 p.p.m. DDT in the diet. Controls challenged with DDT. 1
TABLE 2.—Accumulated percent mortality of ZOO P.P.M. DDT fed young growing quail of the sixth generation when exposed to cold and normal room temperatures Normal Room Temp. (24°C.)
Days of Ex- • posure
A
N
C
A
N
C
2 3 4 6 8
13.9 27.8 33.3 36.1 38.9
5.6 22.2 25.0 25.0 27.8
30.6 44.4 61.1 63.9 69.4
2.8 5.6 5.6 5.6 5.6
5.6 13.9 13.9 16.7 16.7
16.7 22.2 25.0 25.0 25.0
Cold (18.3-20.0°C.)
in the groups exposed to cold, the A-line had higher mortality than the N-line. At both temperatures, the control line had significantly higher mortality than the A and N lines. Mortality within the control line exposed to cold was almost twice that of the one not exposed to the cold. Mortality of the three lines at three different levels of DDT is summarized in Table 3. On the average, there was a significant difference in the mortality between the three different levels. The average mortality at the 300 p.p.m. level was approximately twice that at the 200 p.p.m. level and about four times that at the 100 p.p.m. level. Mortality of the N-line was significantly lower than the A-line at the 200 p.p.m. level. The opposite was true at the 300 p.p.m. level. Mortality of the C-line was significantly higher than that of the selected lines at all three levels of DDT. Percent mortality of the adult quail fed 500 p.p.m. DDT in the ration was determined and is presented in Table 4. On the average, the mortalities among all three lines (A, N and C) were significantly different from each other. Average mortality of the control line was significantly higher than that of the selected lines. Females were significantly (P < .01) more susceptible to DDT toxicity than males. Accumulated mortality (in Probits) of adult quail of the eight generation fed a diet containing lindane was also determined and is illustrated in Figure 1. All
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tion adult quail of the three lines (A, N and C) and each line was fed a diet containing 500 p.p.m. DDT throughout the experiment. This experiment was terminated after a period of 18 days. A fourth experiment group containing part of the eighth-generation of adult quail was fed 400 p.p.m. of lindane in the diet for the first four days, 500 p.p.m. for the next 12 days and 1000 p.p.m. for the rest of the period and partially starved each day. Daily mortality was recorded. The mortality percentages were transformed to arcsins (Steel and Torrie, 1960). The transformed data were treated by analysis of variance.
844
K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN TABLE 3.—Percent mortality of growing quail of the seventh generation given DDT-diet and partially starved (8 to 20 hours/day) for 26 days Days of Exposure 1
A
100 P.P.M. N
C
A
200 P.P.M N
C
A
6 9 12 14 19 21 23 24 25 26
0 0 0 0 0 5.3 10.5 10.5 21.1 52.6
0 0 0 0 0 5.6 16.8 16.8 27.8 44.4
0 0 0 0 13.3 20.0 26.7 26.7 73.3 93.3
0 0 11.1 11.1 38.9 38.9 38.9 44.4 66.7 88.9
0 0 5.3 5.3 10.5 15.8 15.8 21.1 31.6 57.9
0 6.7 26.7 26.7 40.0 46.7 46.7 53.3 80.0 100.0
5.9 5.9 17.6 23.5 35.3 52.9 82.4 94.1 100.0
300 P.P.M N
—
0 0 27.8 55.6 61.1 66.7 94.4 100.0
— —
C 10.5 26.3 42.1 63.2 73.7 84.2 89.5 94.7 100.0
—
• See Materials and Methods.
DISCUSSION
Quail were selected for resistance to DDT for a period of eight generations. Several tests were made to determine the presence of resistance in each generation. They were challenged with a higher level of DDT than the level used for selection purposes to see how much resistance had been developed. Resistance to DDT started to appear after the third generation (Table 1). This development of resistance by selection had been shown to oc-
TABLE 4.—Percent mortality of adult quail of the seventh generation given a diet containing 500 P.P.M. of DDT for IS days 500 P.P.M. Full-fed
5 7 8 9 10 11 12 13 14 18
Female
Male
Days of Exposure
Both Sexes Combined
A
N
C
A
N
c
A
N
C
0 0 0 9.1 9.1 18.2 18.2 27.3 27.3 54.6
0 0 0 0 20.0 20.0 30.0 40.0 50.0 50.0
0 0 0 18.2 27.3 36.4 45.5 63.6 63.6 72.7
11.1 11.1 22.2 22.2 33.3 33.3 55.6 66.7 77.8 88.9
0 0 0 0 22.2 44.5 44.5 66.7 77.8 100.0
7.7 30.8 38.5 46.2 61.5 61.5 69.2 92.3 92.3 92.3
5.0 5.0 10.0 15.0 20.0 25.0 35.0 45.0 50.0 70.0
0 0 0 0 21.1 31.6 36.9 52.6 63.2 73.7
4.2 16.7 20.8 33.3 45.8 50.0 58.3 79.2 79.2 83.3
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cur in the house fly (Barber and Schmitt, 1948; Lindquist and Wilson, 1948), and in the natural populations of fishes found in watersheds with a history of intense spraying with pesticides (Ferguson, 1963). The development of DDT resistance was also demonstrated in the laboratory in Drosophila by Weiner and Crow (1951), Tsukamoto and Ogaki (1953) and Oshima and Heroygoshi (1955). Ozburn and Morrison (1962) produced mice that developed DDT tolerance after eight generations of selection. Fat soluble chlorinated hydrocarbon insecticides were shown to accumulate in various fatty tissues of the body (Woodard et al., 1945; Moore et al, 1964; and
three lines (A, N and C) were shown to be significantly different from each other. Selected lines had lower mortality than the control line.
GENETIC RESISTANCE TO
845
5.5i
7 ' 9
11 13 15 ' 17 " 19 21 23 25 Days of Treatment
FIG. 1. Eight generations of accumulated mortality of adult quail fed a diet containing lindane plotted against days.
found that the selected lines had two to three times less mortality than the DDTfed control line (Table 2). Menn et al. (1957) showed that susceptible house flies exposed to DDT at 15°C. were killed rapidly. A much longer time was needed to influence the final mortality of resistant flies. Results obtained from the DDT-fed quail during exposure to partial starvation also demonstrated that the selected lines had lower mortality than the control line fed DDT. This is a confirmation of the results showing that resistance had been developed in the selected lines. Further testing for resistance was done by feeding adult quail higher levels of DDT in the diet. There was some mortality among the selected lines. Males were more resistant than females (Table 4). Normally, females are heavier than males. They have a tendency to have more fatty tissues in their body and hence a greater opportunity to store DDT and residues. This could have caused higher mortality in females than males. There are several reports of one insecticide-resistant strain of insects being resistant to several other related or non-
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Hickey et al., 1966). Thus, animals might have become tolerant to chlorinated hydrocarbon pesticides when they were fed at low levels and the animals were kept at normal room temperature and not starved. When such animals were exposed to either starvation or to lower temperatures, the insecticide residues stored in the body fat were thought to be released into various other tissues such as liver, brain, kidney, etc., causing toxicity (Dale et al., 1962; Bernard, 1963; and Fitzhugh and Nelson, 1947). Experiments summarized in Table 2 were designed to determine whether the resistance in quail was mainly due to tolerance (accumulation of DDT residue in the fat depots) or to the presence of some sort of resistance. The development of resistance may have been by a degradative mechanism which breaks down DDT to simpler non-toxic compounds with rapid elimination or lower rate of absorption from the gastrointestinal tracts or to low nerve sensitivity to DDT. By exposing the DDT-fed selected lines of quail to cold environment, the hypothesis that they developed a resistance mechanism, other than tolerance which prevented them from dying of DDT toxicity, was supported (Table 2). If the selected lines had stored DDT in the body, it might have been released from the body fat during cold exposure (release of energy from the fat) and there would have been increased DDT levels in the blood and other tissues. This would be likely to cause mortality due to DDT toxicity. On the other hand, the low level of DDT consumed by the selected lines could have been metabolized and rapidly eliminated from the system. Normally an animal can store DDT in the body fat in much larger quantities than the level fed (Draper et al., 1952). In the current experiments, in which a diet containing DDT was fed and the birds exposed to cold stress, it was
DDT
846
K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN
related insecticides (Barber et al., 1948; Weiner and Crow, 1951; and Tsukamoto, 1957). Selected lines of quail were shown to have less mortality than the control line when they were fed lindane and then partially starved. This result is consistent with the result of Ozburn and Morrison (1962), in which DDT-tolerant mice showed cross-resistance to lindane and to dieldrin. REFERENCES
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Babers, F. H., 1953. Chemical control and resistance to insecticides by agricultural pests. J. Econ. Entomol. 46: 869-873. Babers, F. H., and J. J. Pratt, Jr., 1951. Development of insect resistance to insecticide II. U.S. Dept. Agri., Bur. Entomol. Plant Quarant. No. E818: 44. Barber, G. W., and J. B. Schmitt, 1948. House flies resistant to D D T residual sprays. New Jersey Expt. Sta. Bull. 742: 1-8. Bernard, R. F., 1963. Studies on the effects of D D T on birds. Michigan State University, Misc. Publ., Biol. Ser. 2: 159-192. Boyd, C. E., and D. E. Ferguson, 1964. Spectrum of cross-resistance to insecticides in the mosquito fish, Gambusia qffinis. Mosquito News, 24: 19-21. Boyd, C. E., S. B. Vinson and D. E. Ferguson, 1963. Possible DDT resistance to two species of frogs. Copeia, 1963; 426-429. Crow, J. F., 1957. Genetics of insecticide resistance to chemicals. Ann. Rev. Entomol. 2: 227-246. Dale, W. E., T. B. Gaines and W. J. Hayes, Jr., 1962. Storage and excretion of D D T in starved rats. Toxicol. Appl. Pharmacol. 4: 89-106. Draper, C. I., J. R. Harris, D. A. Greenwood and C. H. Biddulph, 1952. The transfer of D D T from the feed to eggs and body tissues of White Leghorn hens. Poultry Sci. 31: 388-393. Ferguson, D. E., 1963. Developing resistance to pesticides. Agric. Chem. 18: 32-34. Fitzhugh, O. G., and A. A. Nelson, 1947. The chronic oral toxicity of DDT. J. Pharmacol. Exptl. Therap. 89: 18-30. Gill, J. A., and B. J. Verts, 1970. Tolerances of two populations of ring-necked pheasants to DDT. J. Wildl. Mgmt. 34: 630-636. Hickey, J. J., J. A. Keith and F. B. Coon, 1966. An exploration of pesticides in a Lake Michigan
ecosystem. J. Appl. Ecol. 3 (Suppl.): 141-154. Lindquist, A. W., and H. G. Wilson, 1948. Development of a strain of houseflies resistant to DDT. Science, 107: 276. Lush, J. L., 1948. The Genetics of Populations. 364 pp. Mimeographed notes. Iowa State University, Ames, Iowa. Menn, J. J., E. Benjamini and W. M. Hoskins, 1957. Effects of temperature and stage of life cycle upon the toxicity and metabolism of D D T in the house fly. J. Econ. Entomol. 50: 67-74. Moore, N. W., and C. H. Walker, 1964. Organic chlorine insecticide residues in wild birds. Nature, 201: 1072-1073. Oshima, C , 1964. Genetics of resistance to insecticides in Drosophila. Proc. Interm. Cong. Entomol. 12. 243. Ozburn, G. W., and F. O. Morrison, 1962. Development of a DDT-tolerant strain of laboratory mice. Nature, 196: 1009-1010. Roulston, W. J., B. F. Stone, J. T. Wilson and L. I. White, 1968. Chemical control of an organophosphorus and carbamate resistant strain of cattle tick from the Rockhampton area of Queensland. Bull. Ent. Res. 58: 379-392. Shaw, R. D., 1965. Culture of an organophosphorusresistant strain of Boopilus microplus and an assessment of its resistance spectrum. Bull. Ent. Res. 56: 389-405. Shaw, R. D., and H. A. Malcolm, 1964. Resistance to cattle tick to organophosphorus insecticides. Vet. Rec. 76:210-211. Steel, R. G. D., and J. H. Torrie, 1960. Principles and Procedures of Statistics. 481 pp. McGrawHill Book Company, Inc., New York. Tsukamoto, M., 1957. Cross-resistance to insecticides in Drosophila melanogaster. Drosophila Information Service, 31: 168. Tsukamoto, M., and M. Ogaki, 1953. Inheritance of resistance to D D T in Drosophila melanogaster. Botyu-Kagaku, 18: 39-44. Wally, W. W., D. E. Ferguson and D. D. Culley, 1966. The toxicity, metabolism and fate of D D T in certain icterid birds. Mississippi Acad. Sci. 12: 281-300. Weiner, R., and J. F. Crow, 1951. The resistance of DDT-resistant Drosophila to other insecticides. Science, 113:403-404. Woodard, G., R. R. Ofner and C. M. Montgomery, 1945. Accumulation of DDT in the body fat and its appearance in the milk of dogs. Science, 102: 177-178.
841
ogy of Marek's disease herpesvirus in broiler flocks. Avian Dis. 14: 255-267. Witter, R. L., K. Nazerian, H. G. Purchase and G. H. Burgoyne, 1970. Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. Am. J. Vet. Res. 3 1 : 525-538.
Genetic Resistance to D D T in the Japanese Quail Coturnix Coturnix Japonic^'2
(Received for publication May 25, 1972) ABSTRACT Selection studies of resistance to D D T included eight generations of quail consisting of two selected lines and one control line. One selected line differed from the other in that, in addition to mass selection, birds were chosen on the basis of family merit. Selected lines were fed a diet containing 200 p.p.m. D D T during the first 30 days of life. In order to test for resistance, part of the control lines were also given DDT diets, and the mortality of the control was compared to the mortality of the selected lines. Development of resistance started to appear after the third generation of selection. This was evidenced by the lower mortality among the selected lines than in the control line fed DDT. If the selected lines had stored DDT in the body, cold exposure or partial starvation (release of energy from the fat) would presumably increase the D D T levels in the blood and other sensitive tissue, causing mortality due to DDT toxicity. This was put to test by exposing DDT-fed resistant fines and the DDT-fed control group to partial starvation and cooler temperatures. Under these conditions, the selected lines were lower in mortality than the control. Among the resistant lines, the females were less resistant than males. These lines showed some cross-resistance to another hydrochlorinated pesticide lindane. A conclusion drawn from this study is that it is possible to develop a strain of quail resistant to D D T out of the less resistant population by selection. POULTRY SCIENCE 52: 841-846, 1973
INTRODUCTION
A
RTHROPODS maydevelop resistance L toward insecticides used extensively for many generations for their control (Babers and Pratt, 1951; Babers, 1953). Experimentation has shown that resistance can be developed as a consequence of selection pressure exerted by the presence of a toxicant in the environment (Crow, 1957). House flies, Musca domes1
Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin. 1 Paper Number 1550 from the Laboratory of Genetics.
tica, resistant to DDT were first reported in 1947 (Barber and Schmitt, 1948; Lindquist and Wilson, 1948). Many strains of cattle tick, Boophilus microplus, have been found to be resistant to organophosphorus compounds (Shaw and Malcolm, 1964; Shaw, 1965; Roulston et ah, 1968). Oshima (1964) reported that occurrence of resistance to insecticides in wild population of Drosophila. The first report of pesticide resistance in a natural population involved DDTresistant mosquito fish from an intensively sprayed cotton producing area (Ferguson, 1963) and in the cricket frogs, Acris sp.
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K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN Departments of Poultry Science and Genetics, University of Wisconsin, Madison, Wisconsin 53706
842
K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN
MATERIALS AND METHODS
Quail chicks from an untreated (no DDT in ration) population were reared on a 28 percent protein ration and assigned at random to "Artificial Selection" (A), to "Natural Selection" (N) and to "Control" (C) groups. A and N lines were fed 100 p.p.m. DDT in the ration for the first 30 days after hatching. In succeeding generations the amount of DDT in the above mentioned ration was changed to 200 p.p.m. in an attempt to increase mortality to 50 percent during 30 days in the A and N Groups. The control was not fed DDT. Daily mortality was recorded. After 30 days of age the quail were fed a 23 percent protein ration without DDT. One male and one female were assigned to a cage in generation 1. In succeeding generations two females were paired with each male. Eggs were collected from the cages daily and were stored for no longer than 13 days in a cooler. Two hatches were used. The first hatch was used for selecting parents for the next generation in the
A, N and C lines. The second hatch was not used for breeding, but served as a test for resistance by providing a comparison between the A, N and C groups challenged with DDT in the feed. The test was not carried out in generations in which mortality of the birds had reached 50 percent by 30 days of age. The A-line differed from the N-line in that, in addition to mass selection, birds were chosen on the basis of family merit. The index (Lush, 1948) on which selection was based is: (G - G) =
nrh 2 1 + r(n - l)h 2
(x - *)
(G — (?) = estimated breeding worth of an individual n = number of survivors in the family x = percent survivors in a particular family x = average percent surviving in the population r = relationship within family = .5 (assumed h2 = 1) 2 h = portion of the observed variance for which difference in heredity is responsible In the first experiment, sixth-generation progeny of the three lines (A, N and C) were given 200 p.p.m. DDT diet from one day to 23 days of age and then were exposed to cold (18.3 to 20.0°C.) from 15 to 23 days of age. In experiment two, seven-day old progeny of the seventh-generation from A, N and C lines were fed 100, 200 and 300 p.p.m. DDT in the diet for four days. They were then starved for five different lengths of time—8, 14, 16, 18 and 20 hours per day for a period of 30 days. The birds surviving in each group were exposed to the next starvation time. Experiment three used seventh-genera-
Downloaded from http://ps.oxfordjournals.org/ by guest on January 13, 2015
(Boyd et al., 1963). Boyd and Ferguson (1964) demonstrated that as much as 300fold resistance persisted among the first few generations of descendants of these resistant fishes. Wally et al. (1966) made observations on survival of grackles, redwinged blackbirds and starlings in heavily DDT treated areas, while Gill and Verts (1970) investigated the tolerance of a population of ring-necked pheasants occupying an area to which DDT was applied for eight years. The object of this investigation was to determine if resistance to DDT could be developed in the Japanese quail, Coturnix coturnix japonica. Also the experimental design facilitated determining the effects that environmental stress had on the resistance of birds selected for resistance to DDT.
843
GENETIC RESISTANCE TO DDT
RESULTS
The percent mortality of the first and the third to sixth generations is presented in Table 1. In the second generation testing for resistance was not done because the selected lines had 50 percent mortality before the birds were 30 days old. The C-line fed DDT had significantly higher mortality than the two selected lines (A and N) in the later generations. Mortalities of birds exposed to cold as well as those kept at normal room temperatures are compared (Table 2). In all three lines, mortality was higher in the groups exposed to cold (18.3 to 20.0°C.) than in the groups kept at normal room temperatures. At normal room temperature (24°C), the N-line had significantly higher mortality than the A-line, whereas, TABLE 1.—Average percent mortality of the four treatments in each generation for the second hatch
1 2
A
N
C-DDT<
C
46.4 44.0 67.1 36.5 32.2
46.4 49.3 56.9 40.3 47.1
36.4 48.2 75.6 48.4 61.6
11.5 22.3 30.4 31.0 17.6
Groups come from Hatch One. Groups fed 200 p.p.m. DDT in the diet. ' Groups ted 300 p.p.m. DDT in the diet. Controls challenged with DDT. 1
TABLE 2.—Accumulated percent mortality of ZOO P.P.M. DDT fed young growing quail of the sixth generation when exposed to cold and normal room temperatures Normal Room Temp. (24°C.)
Days of Ex- • posure
A
N
C
A
N
C
2 3 4 6 8
13.9 27.8 33.3 36.1 38.9
5.6 22.2 25.0 25.0 27.8
30.6 44.4 61.1 63.9 69.4
2.8 5.6 5.6 5.6 5.6
5.6 13.9 13.9 16.7 16.7
16.7 22.2 25.0 25.0 25.0
Cold (18.3-20.0°C.)
in the groups exposed to cold, the A-line had higher mortality than the N-line. At both temperatures, the control line had significantly higher mortality than the A and N lines. Mortality within the control line exposed to cold was almost twice that of the one not exposed to the cold. Mortality of the three lines at three different levels of DDT is summarized in Table 3. On the average, there was a significant difference in the mortality between the three different levels. The average mortality at the 300 p.p.m. level was approximately twice that at the 200 p.p.m. level and about four times that at the 100 p.p.m. level. Mortality of the N-line was significantly lower than the A-line at the 200 p.p.m. level. The opposite was true at the 300 p.p.m. level. Mortality of the C-line was significantly higher than that of the selected lines at all three levels of DDT. Percent mortality of the adult quail fed 500 p.p.m. DDT in the ration was determined and is presented in Table 4. On the average, the mortalities among all three lines (A, N and C) were significantly different from each other. Average mortality of the control line was significantly higher than that of the selected lines. Females were significantly (P < .01) more susceptible to DDT toxicity than males. Accumulated mortality (in Probits) of adult quail of the eight generation fed a diet containing lindane was also determined and is illustrated in Figure 1. All
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tion adult quail of the three lines (A, N and C) and each line was fed a diet containing 500 p.p.m. DDT throughout the experiment. This experiment was terminated after a period of 18 days. A fourth experiment group containing part of the eighth-generation of adult quail was fed 400 p.p.m. of lindane in the diet for the first four days, 500 p.p.m. for the next 12 days and 1000 p.p.m. for the rest of the period and partially starved each day. Daily mortality was recorded. The mortality percentages were transformed to arcsins (Steel and Torrie, 1960). The transformed data were treated by analysis of variance.
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K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN TABLE 3.—Percent mortality of growing quail of the seventh generation given DDT-diet and partially starved (8 to 20 hours/day) for 26 days Days of Exposure 1
A
100 P.P.M. N
C
A
200 P.P.M N
C
A
6 9 12 14 19 21 23 24 25 26
0 0 0 0 0 5.3 10.5 10.5 21.1 52.6
0 0 0 0 0 5.6 16.8 16.8 27.8 44.4
0 0 0 0 13.3 20.0 26.7 26.7 73.3 93.3
0 0 11.1 11.1 38.9 38.9 38.9 44.4 66.7 88.9
0 0 5.3 5.3 10.5 15.8 15.8 21.1 31.6 57.9
0 6.7 26.7 26.7 40.0 46.7 46.7 53.3 80.0 100.0
5.9 5.9 17.6 23.5 35.3 52.9 82.4 94.1 100.0
300 P.P.M N
—
0 0 27.8 55.6 61.1 66.7 94.4 100.0
— —
C 10.5 26.3 42.1 63.2 73.7 84.2 89.5 94.7 100.0
—
• See Materials and Methods.
DISCUSSION
Quail were selected for resistance to DDT for a period of eight generations. Several tests were made to determine the presence of resistance in each generation. They were challenged with a higher level of DDT than the level used for selection purposes to see how much resistance had been developed. Resistance to DDT started to appear after the third generation (Table 1). This development of resistance by selection had been shown to oc-
TABLE 4.—Percent mortality of adult quail of the seventh generation given a diet containing 500 P.P.M. of DDT for IS days 500 P.P.M. Full-fed
5 7 8 9 10 11 12 13 14 18
Female
Male
Days of Exposure
Both Sexes Combined
A
N
C
A
N
c
A
N
C
0 0 0 9.1 9.1 18.2 18.2 27.3 27.3 54.6
0 0 0 0 20.0 20.0 30.0 40.0 50.0 50.0
0 0 0 18.2 27.3 36.4 45.5 63.6 63.6 72.7
11.1 11.1 22.2 22.2 33.3 33.3 55.6 66.7 77.8 88.9
0 0 0 0 22.2 44.5 44.5 66.7 77.8 100.0
7.7 30.8 38.5 46.2 61.5 61.5 69.2 92.3 92.3 92.3
5.0 5.0 10.0 15.0 20.0 25.0 35.0 45.0 50.0 70.0
0 0 0 0 21.1 31.6 36.9 52.6 63.2 73.7
4.2 16.7 20.8 33.3 45.8 50.0 58.3 79.2 79.2 83.3
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cur in the house fly (Barber and Schmitt, 1948; Lindquist and Wilson, 1948), and in the natural populations of fishes found in watersheds with a history of intense spraying with pesticides (Ferguson, 1963). The development of DDT resistance was also demonstrated in the laboratory in Drosophila by Weiner and Crow (1951), Tsukamoto and Ogaki (1953) and Oshima and Heroygoshi (1955). Ozburn and Morrison (1962) produced mice that developed DDT tolerance after eight generations of selection. Fat soluble chlorinated hydrocarbon insecticides were shown to accumulate in various fatty tissues of the body (Woodard et al., 1945; Moore et al, 1964; and
three lines (A, N and C) were shown to be significantly different from each other. Selected lines had lower mortality than the control line.
GENETIC RESISTANCE TO
845
5.5i
7 ' 9
11 13 15 ' 17 " 19 21 23 25 Days of Treatment
FIG. 1. Eight generations of accumulated mortality of adult quail fed a diet containing lindane plotted against days.
found that the selected lines had two to three times less mortality than the DDTfed control line (Table 2). Menn et al. (1957) showed that susceptible house flies exposed to DDT at 15°C. were killed rapidly. A much longer time was needed to influence the final mortality of resistant flies. Results obtained from the DDT-fed quail during exposure to partial starvation also demonstrated that the selected lines had lower mortality than the control line fed DDT. This is a confirmation of the results showing that resistance had been developed in the selected lines. Further testing for resistance was done by feeding adult quail higher levels of DDT in the diet. There was some mortality among the selected lines. Males were more resistant than females (Table 4). Normally, females are heavier than males. They have a tendency to have more fatty tissues in their body and hence a greater opportunity to store DDT and residues. This could have caused higher mortality in females than males. There are several reports of one insecticide-resistant strain of insects being resistant to several other related or non-
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Hickey et al., 1966). Thus, animals might have become tolerant to chlorinated hydrocarbon pesticides when they were fed at low levels and the animals were kept at normal room temperature and not starved. When such animals were exposed to either starvation or to lower temperatures, the insecticide residues stored in the body fat were thought to be released into various other tissues such as liver, brain, kidney, etc., causing toxicity (Dale et al., 1962; Bernard, 1963; and Fitzhugh and Nelson, 1947). Experiments summarized in Table 2 were designed to determine whether the resistance in quail was mainly due to tolerance (accumulation of DDT residue in the fat depots) or to the presence of some sort of resistance. The development of resistance may have been by a degradative mechanism which breaks down DDT to simpler non-toxic compounds with rapid elimination or lower rate of absorption from the gastrointestinal tracts or to low nerve sensitivity to DDT. By exposing the DDT-fed selected lines of quail to cold environment, the hypothesis that they developed a resistance mechanism, other than tolerance which prevented them from dying of DDT toxicity, was supported (Table 2). If the selected lines had stored DDT in the body, it might have been released from the body fat during cold exposure (release of energy from the fat) and there would have been increased DDT levels in the blood and other tissues. This would be likely to cause mortality due to DDT toxicity. On the other hand, the low level of DDT consumed by the selected lines could have been metabolized and rapidly eliminated from the system. Normally an animal can store DDT in the body fat in much larger quantities than the level fed (Draper et al., 1952). In the current experiments, in which a diet containing DDT was fed and the birds exposed to cold stress, it was
DDT
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K. B. POONACHA, B. C. WENTWORTH AND A. B. CHAPMAN
related insecticides (Barber et al., 1948; Weiner and Crow, 1951; and Tsukamoto, 1957). Selected lines of quail were shown to have less mortality than the control line when they were fed lindane and then partially starved. This result is consistent with the result of Ozburn and Morrison (1962), in which DDT-tolerant mice showed cross-resistance to lindane and to dieldrin. REFERENCES
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Babers, F. H., 1953. Chemical control and resistance to insecticides by agricultural pests. J. Econ. Entomol. 46: 869-873. Babers, F. H., and J. J. Pratt, Jr., 1951. Development of insect resistance to insecticide II. U.S. Dept. Agri., Bur. Entomol. Plant Quarant. No. E818: 44. Barber, G. W., and J. B. Schmitt, 1948. House flies resistant to D D T residual sprays. New Jersey Expt. Sta. Bull. 742: 1-8. Bernard, R. F., 1963. Studies on the effects of D D T on birds. Michigan State University, Misc. Publ., Biol. Ser. 2: 159-192. Boyd, C. E., and D. E. Ferguson, 1964. Spectrum of cross-resistance to insecticides in the mosquito fish, Gambusia qffinis. Mosquito News, 24: 19-21. Boyd, C. E., S. B. Vinson and D. E. Ferguson, 1963. Possible DDT resistance to two species of frogs. Copeia, 1963; 426-429. Crow, J. F., 1957. Genetics of insecticide resistance to chemicals. Ann. Rev. Entomol. 2: 227-246. Dale, W. E., T. B. Gaines and W. J. Hayes, Jr., 1962. Storage and excretion of D D T in starved rats. Toxicol. Appl. Pharmacol. 4: 89-106. Draper, C. I., J. R. Harris, D. A. Greenwood and C. H. Biddulph, 1952. The transfer of D D T from the feed to eggs and body tissues of White Leghorn hens. Poultry Sci. 31: 388-393. Ferguson, D. E., 1963. Developing resistance to pesticides. Agric. Chem. 18: 32-34. Fitzhugh, O. G., and A. A. Nelson, 1947. The chronic oral toxicity of DDT. J. Pharmacol. Exptl. Therap. 89: 18-30. Gill, J. A., and B. J. Verts, 1970. Tolerances of two populations of ring-necked pheasants to DDT. J. Wildl. Mgmt. 34: 630-636. Hickey, J. J., J. A. Keith and F. B. Coon, 1966. An exploration of pesticides in a Lake Michigan
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