Radioresistance in natural populations of Drosophila nebulosa from a brazilian area of high background radiation

Mutation Research, 27 (1975) 347-355 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

347

R A D I O R E S I S T A N C E IN N A T U R A L P O P U L A T I O N S OF D R O S O P H I L A

N E B U L O S A FROM A B R A Z I L I A N AREA OF H I G H BACKGROUND RADIATION*

FERNANDO

LUIZ KRATZ

Departamento de Biologia Germ do Instituto de Cigncias Bioldgicas, Universidade Federal de Golds, 74ooo-Goidnia-GO (Brazil) (Received J u n e 24th, 1974) (Revision received N o v e m b e r 5th, 1974)

SUMMARY

D. nebulosa, collected in two woods of a high background radiation area (both in Iron Hills, State of Minas GerMs, Brazil), were compared to and found to be more resistant than flies collected as controls in two other woods of an adjacent area. This was the second time that average differences in radioresistance between natural populations were established. Previous experiments were carried out with D. willistoni, in the same area and in comparable fashion. In spite of their higher radiation resistance the flies from the radiation area carried a higher expressed load than the controls. The following tests were performed to estimate the differences: (a) survival, after whole body exposure to 90000 R o t 6°Co-gamma-rays on 12o strains set up from single inseminated females and (b) reproductive performance, in 24 ° duplicate crosses, measured in terms of the difference between irradiated series (males received 3000 R of "°Co-gamma-rays) and their unirradiated counterparts. The data are based on an offspring of 293784 individuals. Furthermore, two diallel crosses between sensitive and resistant strains have shown that the differences probably are due mainly to additive genes.

INTRODUCTION

Although many papers dealing with radiobiological effects state considerable differences in radiosensitivity where different species or strains are concerned (suggesting that the individual genetic constitution influences the response to irradiation (rets. 8, 14, 26, 28), the genetics ot radioresistance is fairly well known only in microorganismsl°,ll,15,~4,a°; little is known of higher organisms in this respect. It was found that radioresistance in Mus depends on both nonadditive and additive genetic factors, but the detected nonadditive seems to be primarily a heterotic expression 9. In Bom* T h i s p a p e r is p a r t of a P h . D . T h e s i s for t h e P o s t g r a d u a t e Genetics Course of t h e U n i v e r s i d a d e F e d e r a l do Rio G r a n d e do Sul. A b b r e v i a t i o n s : CW, control woods; R W , r a d i o a c t i v e woods.

348

r . L . KRATZ

b y x it was shown that the radiosensitivity of embryos is controlled by at least two major genes is. OGAKI AND NAKASHIMA-TANAKA2°, working with wild-type and m u t a n t strains of Drosophila mdanogaster selected for heat tolerance, found that radioresistance is dominant over susceptibility. More recently PARSONS et al. 22, studying 18 strains derived from single inseminated females oi D. melanogaster from a wild population, concluded that the differences in radioresistance among the strains were due mainly to additive genes. Little is also known about the genetic effects of low level radiation, especially in natural populations which have been exposed to radiation for m a n y generations. Populations living in an area of natural high background radioactivity are certainly the best material for genetic and ecological studies involving long-term exposure to low doses of radiation. The Brazilian regions of Iron Hills (Minas Gerais), which shows an atmospheric radioactivity, at 50 cm from the soil, of up to 3.2 m R / h 2., is suitable ior such studies. Two major types of radiation effects are expected at a high population level: first, an increase in mutation frequencies with a consequent increase in genetic variability; and, second, selection pressures caused mainly by the effects at the chromosomal level 25, as well as by lethal, semi-lethal and detrimental gene actions. The conditions for such selection were demonstrated by CORDEIRO et al. ~ concerning the frequencies of lethals, semi-lethals and detrimentals in Drosophila, as well as by TAKAHASHI2~ in cytogenetic studies of the scorpion Tityus at the chromosomal level. Then again, several papers dealing with irradiated experimental populations in Drosophila presented evidence not only of an increase in the rate of adaptation to a new environment but even of the development oi radioresistance as well1, ~,4,5,19,~9. We reier especially to D. nebulosa, a common species in Iron Hills, which was studied by MARQUES16,17 who demonstrated the development of radioresistance in experimental populations. This was one of the reasons why D. nebulosa was chosen for our experiments. Furthermore, average differences in radioresistance were demonstrated on a previous occasion by CORDEIRO et al. 7 in the same region of Iron Hills. This situation thus enabled us to work with D. nebulosa for the purpose of: (a) testing and extending the findings already obtained in D. willistoni 7, to determine whether these low levels of chronic radiation are enough to change a population's average radioresistance; (b) studying the genetic nature of this natural radioresistance to increase our knowledge about the polymorphism of natural populations for genes controlling radioresistance in Drosophila. MATERIAL AND METHODS

The experiments involved strains set up from single inseminated females collected in J a n u a r y 1972 in two isolated woods in the "hot region" (Iron Hills); controls were collected from two other nearby isolated woods in the adjacent areas. The "radiative woods" were named RW I and RW I I ; the control, CW I and CW II. As we pointed out, the "hot region" is one of the highest and largest radioactive regions in the world. One area of more than IOOOOm 2 shows levels higher than 1.5 m R / h ; another covering more than 30000 m 2 has levels higher than I.O m R / h ~7. Inseminated females were placed singly in culture vials and allowed to oviposit. The irradiations were performed with several doses of gamma-rays at an intensity of about 230 R/min from a 6°Co source.

R A D I O R E S I S T A N C E IN D R O S O P H I L A

349

To estimate the extent of the polymorphism as well as the differences in radioresistance between the populations with different backgrounds of radiation the following tests were performed: (a) survival within 3 days after whole body exposure to 90000 R of S°Co-gamma-rays on 12o strains (30 strains from each wood) set up from single inseminated females; (b) reproductive performance between homologous series. Because of the large amount of material, differences in reproductive performance were tested in two separate experiments that did not differ in procedure and experimental design. The experimental unit consisted of two groups of 3 pairs, obtained by crossing 6 full-sib males from one strain with 6 full-sib females from another. The males of the first group had been exposed to 3000 R; the males of the second group had not. The data were obtained by the difference in the number of descendants between the duplicates with irradiated and unirradiated males. Each experiment involved 12o different units, i.e., 30 from each of the 4 woods of origin (CW I, CW II, R W I and R W II). 4-day-old flies were prepared and similarly handled, except that the males had not been irradiated. Reproductivity was defined as the total number of offspring from 3 pairs in culture vials. The pairs were allowed to cross and to oviposit for a period of 21 days in 7 vials (3 days of oviposition in each vial under standardized conditions). Counts were made 16 days after the oviposition period had ended in each vial. Observation of the progenies of irradiated males was then to make clear the action of dominant, semi-dominant, lethal and detrimental genes. To infer the genetic nature of differences in radioresistance two diallel crosses were performed to test additive and dominance effects TM. RESULTS

Survival experiment with 9oooo R This experiment was designed to detect the influence of sex and the varying response (different levels of background radioactivity) to whole-body exposure to 90000 R of °°Co-gamma-rays on the part of F1 strains obtained from single females inseminated in nature, with 30 strains collected from each wood. Survivors were counted 72 h after irradiation. Each experimental unit was composed of 25 individuals aged 5 ~ I days. Table I gives the most important results. A goodness of fit test was also performed (CW I and CW II, pooled, and R W I and R W I I pooled) and the data proved not to differ from a normal distribution. As we can see, the survival test shows that the populations from the radioactive woods (RW I and R W II) survived better than both controls (CW I and CW II). Nevertheless, CW I I suffered more from irradiation than CW I. In general, the females were more resistant than the males.

Reproductive per]brmance As indicated, the decrease in reproductive pelformance resulting from exposure to irradiation was tested in two paired experiments. In both experiments the males within one series received 3000 R of e°Co-gamma-rays before mating, while the other (homologous) series remained as controls, without irradiation. Sensitivity to irradiation on the part of the male% measured in terms of the difference between irradiated and unirradiated duplicates is presented in Table I I for the first experiment (the results are based on a total of 181142 offspring). Table I I I summarizes the results of the second experiment, based on a total of 112 642 offspring.

350

F.L.

TABLE

KRATZ

I

PERCENTAGE SURVIVAL 72 h AFTER 9 0 0 0 0 R IRRADIATION WITH e°Co (APPLYING ANGULAR TRANSFORMATION) AND ANALYSIS OF VARIANCE

Overall mean survival Data

CW I

C W II

RW I

RW II

%

42.4

52.5

65.8

66.0

M e a n survival by sex Data

Male

Female

%

51.1

62.3

F values Source of variation d f

F

Woods high × low between low between high Sexes Wood × sex Error

3

1 3

232

Total a p

<

I6.233 a 42.483 a 6.212 b o . o o 3 ns I5.27oa 2 . I I 3 ns

i i i

239 o.ooi.

b p < 0.05 . ns N o t s i g n i f i c a n t .

TABLE

II

Average decrease IN THE REPRODUCTIVE PERFORMANCE OF D. nebulosa AFTER IRRADIATION OF MALES ( 3 0 0 0 R ) , BETWEEN 3 0 DUPLICATED CROSSES (IRRADIATED AND UNIRRADIATED) BY WOOD OF ORIGIN First experiment.

Origin

Replication I st 2rid

3rd

4th

5th

6th

7th

Total

CW I

84.30

61-5o (145.8o)

45.90 (191.7o)

44.23 (235.93)

58.67 (294.6o)

58.8o (348.4 ° )

51.1o (399.5 ° )

399.5o

CW II

95-97

64-63 (16o.6o)

19.33 (179.93)

31.27 (211.2o)

71.oo (282.2o)

58.23 (340.43)

46.7 ° (387.13)

387.13

RWI

24.77

--2.6 (22-17)

11.2o (33.37)

39.43 (72.80)

46.o0 (118.8o)

54.97 (173.77)

31.9 ° (205.67)

205.67

RWII

15.57

17.8o (33.37)

8.47 (41.84)

42.13 (83.97)

66.80 (15o.77)

42.87 (193.64)

8.47 (2o2.1o)

2o2.1o

F

24.87a

I4.9Ia

MSD 2 MSD 3 MSD 4

22.93 24.16 24.98

24.02 25.31 26.17

5.43 b 20.52 21.62 22.33

o . 6 o ns

1 . 9 3 ns

o.58ns

-

-

-

< o.ooi. b p < o.oi. ns N o t s i g n i f i c a n t . MSD X, Minimal significant difference between X ordered means. ( ), A c c u m u l a t e d m e a n d i f f e r e n c e s . a p

5.57 b 22.79 24.Ol 24.83

i4.76a 79.80 84.o8 86.93

351

R A D I O R E S I S T A N C E IN D R O S O P H I L A

These reproductive performance tests confirmed the results obtained in the survival tests. The productivity decrease is less in the strains obtained from the "hot regions" than in their controls.

Influence of the background level on the reproductive performance The reproductive capacity of the unirradiated duplicates of the two experiments described was used to estimate and compare the population expressed loads according to their radiation backgrounds. Table IV presents the results of this analysis for the first and second experiments, respectively. TABLE

III

Average decrease IN THE REPRODUCTIVE PERFORMANCE OF O. nebulosa AFTER IRRADIATION OF MALES (3OOO R), BETWEEN 3 ° DUPLICATED CROSSES (IRRADIATED AND UNIRRADIATED) BY WOOl) OF ORIGIN Second experiment.

Origin

Replication I St 2nd

3rd

4th

5th

6th

7th

Total

CW I

20.30

8.60 (28.90)

39.77 (68.67)

68-17 (136"84)

97.43 (234"27)

67.93 (3 ° 2 . 2 0 )

35-33 (337.53)

337.53

CW It

14.97

29-47 (44-44)

39-73 (84.17)

4°"17 (124.34)

72"4 ° (196.74)

75-07 (271.78)

34-97 (306.77)

3o6.77

RW I

8.13

8"71 (16.84)

14.26 (31-1o)

26-13 (57.23)

35.93 (93.16)

25.58

1-58 (12o.32)

129.63

(118.74)

18.1o (31-73)

18.63 (50.36 )

29.00 (79.36 )

59.13 (137.49)

46.90 (184.39)

15.93 (2oo.33)

200.33

RW II

13.63

F

o . 5 5 ns

1 . 2 o ns

1 . 9 ons

MSD 2 MSD 3 MSD 4

-

-

-

a p

<

6.56a

7.3ia

21.o3 22.15 22.91

26.91 28.35 19-31

5.46b

26.99 28.44 19.4 °

7.24 a 17.14 18.o5 18.67

7.32a 95.00 lOO.99 lO3.49

o.ooi.

b p < o.oi. ns N o t s i g n i f i c a n t . MSD X, Minimal significant differences between X ordered means. ( ), A c c u m u l a t e d m e a n d i f f e r e n c e s . TABLE

IV

BACKGROUND INFLUENCE ON REPRODUCTIVITY

Level

Wood

Ist experiment Total Productivity offspring (• of D pairs)

2rid experiment Total Productivity offspring ( X of 3 pairs)

Control

CW I CW II

28o44 28517

934.8o 950.57

22185 18548

739.50 618.27

"Hot"

RW I RW II

24329 27529

81o.97 917.63

13693 16292

441.71 543.07

F

3.o93 b

7.349 a

MSD 2 MSD 3 MSD 4

lOO.52 lO5.9o lO9.49

123.93 13o.57 134.99

~ o.oi. b p ~ 0.05 . MSD X, Minimal significant difference between X ordered means. a p

F. L. KRATZ

352

I t is interesting that the D. nebulosa populations of RW I and RW II, which were shown to be radioresistant, exhibited greater genetic expressed load. This allows us to suppose that radioresistance did not prevent an increase in the load. Diallel crosses

After preliminary tests to determine the radioresistance characteristics of the strains used, a diallel cross was developed with two radioresistant natural strains (RW2o4 and RW2oo) and two radiosensitive natural strains (CWI28 and CW97). These strains have an inbreeding coefficient of 0.5. The offspring received a 90000 R whole body exposure. The survival data, obtained 72 h a i t e r irradiation, are presented in Table V. Each entry in this table is based on the observation oi 50 flies (25 males and 25 females) and is transformed to arcsines to remove the dependence of the variance on the mean TM. The second diallel experiment was developed with the following strains: E85 (an inbred radioresistant induced strain developed by MARQUESle'I~); RW2oo (a resistant natural strain) and CW97 (a sensitive natural strain); the latter two were also used in the first diallel experiment but now had an inbreeding coefficient of 0.594. The data are presented in Table VI (each entry is based on the observation of 15o flies). TABLE V FIRST DIALL]~L EXPERIMENT A, Survival percentage 72 h following 90000 R irradiation w i t h e°Co in the 4 × 4 diallele cross f r o m two n a t u r a l resistant strains and t w o n a t u r a l sensitive ones. D a t a w i t h angular t r a n s f o r m a tion. E a c h e n t r y in the table is based on observation of 50 flies. B, analysis of variance. A Males

1RW RW CW CW

2o 4 2o0 128 97

Total

Females R W 204

R W 200

C W i28

C W 97

Total

80.02 66.42 74.66 73.57

73-57 68.87 69.73 64.90

64.90 59.34 62-o3 62.03

7o.63 50.77 60.00 52"24

289.12 245.4 ° 266.42 252.74

294.67

277.07

248.3o

233.64

I o53.68

B Source of variation

df

F

(a) General combining ability (bl) Dominance (b2) A s y m m e t r y of frequence (b3) Dominance and others

3 i 3 2

3 6.°°a o.oo3 ns 3.4 °ns 3.56ns

(b) Specific combining ability

6

2.88 ns

(c) Average m a t e r n a l effect of each strain (d) Variation in the reciprocal not a t t r i b u t a b l e to (c) Total a P

<

3 3 15

o.oi.

b p < 0.05 . ns N o t significant.

12.32b

353

RADIORESISTANCE IN DROSOPHILA T A B L E VI

SECOND DIALLEL EXPERIMENT A, S u r v i v a l p e r c e n t a g e 72 h a f t e r 9 0 0 0 0 R i r r a d i a t i o n w i t h e°Co in t h e following s t r a i n s : E85 (a h i g h l y i n b r e d i n d u c e d strain) ; R W 2 o o (a r e s i s t a n t n a t u r a l strain) ; a n d CW97 (a s e n s i t i v e n a t u r a l strain). D a t a w i t h a n g u l a r t r a n s f o r m a t i o n . E a c h e n t r y in t h e t a b l e is b a s e d on o b s e r v a t i o n of 15o flies. B, A n a l y s i s of variance. A Males

E 85 R W 20o C W 97 Total

Females E 85

R W 200

C W 97

Total

81.87 70.63 67.21

74.66 73.57 62.72

71.56 67.21 54.33

228.09 211.41 184.26

219.71

21o.95

193.1o

623.76

B Source of variation

df

F

(a) General c o m b i n i n g a b i l i t y (b) Specific c o m b i n i n g ability (c) M a t e r n a l effects of each s t r a i n (d) V a r i a t i o n in t h e reciprocal n o t a t t r i b u t a b l e to (c)

2 3 2

I7.55 a

Total

8

4.27 ns

i

a p < 0.05 . ns N o t significant.

The diallel analysis showed that the differences found have significant genetic components and that these differences were due mainly to additive genes. The first experiment suggested the presence of maternal or cytoplasmic effects but these were not confirmed by the second experiment. DISCUSSION

This was the second time, as far as we know, that an average difference in radioresistance between two natural populations was established. The first occasion also involved Iron Hills 7. It is interesting that the levels of radiation which induced the population differences al e, in absolute value, low if compared with other experimental chronic irradiation levels. Nevertheless, we must consider that terrestrial radioactivity contributes both to external and internal radiation, and that high natural radiation together with special food chain concentrating mechanisms pioduce internal radiation many times greater than background radioactivity. Since tests to estimate the isolation of D. nebulosa were not performed in the woods studied, we cannot affirm whether or not there is any migration of flies in the hot region and in the surrounding areas. Migration is likely but its effects seem to be more limited than those of natural selection. The region is covered with grassland and with scattered and insulated ciliary woods. The distance between the hot and the control woods is several kilometer. The genetic nature of differences in radioresistance was studied by diallel analysis under standardized conditions. It is interesting that the strains used had never before received experimental irradiation ; they were chosen from the strains ob-

354

v.L. KRATZ

tained in nature (except E85), and their radioresistance parameters were well established by additional tests. The detection of additive effects confirms and extends the results in Mus", in Drosophila melanogaster ~ and in Bombyx 18. OGAKI AND NAKASHIMA-TANAKA ~°, working with wild and mutant strains of D. melanogaster selected for heat tolerance, found that radioresistance was dominant over susceptibility. However, this may be due to a correspondence between the genetic architecture of a characteristic and the kind of selection acting on it, as proposed by KEARSEY AND KOJIMA13 and BREESE AND MATHER3. It is postulated that continuous directional selection tends to produce directional dominance. Thus if there is a positive correlation between radioresistance and heat tolerance (and this seems to be the case 21) this might help to explain the dominance observed by OGAKI AND NAKASHIMATANAKA. Given the significance of genetic components with respect to radioresistance, as shown by diallele analysis, the variability among the founder females implies that the population studied is polymorphic for these additive genes. Moreover, if we allow the hypothesis that the genetic architecture reflects the kind of acting selection, we may infer that the populations studied are mainly under stabilizing selection for the trait studied. Finally, we wish to emphasize that the radioresistant populations also showed a higher expressed load, suggesting that the acquired radioresistance could not completely avoid an increase in genetic load. ACKNOWLEDGEMENTS

I am grateful to my adviser Prof. A. R. CORDEIROfor the guidance given to me in the course of this work, and for bis invaluable help in the collecting, isolating and culturing of the females of D. nebulosa in the field laboratory during the expedition to Minas GerMs; to my colleagues A. VEIGA-NETO and V. ZANETTE and my student A. FURLANETOwho made possible the simultaneous collecting in four isolated woods. To Prof. E. K. MARQUESgo my thanks for his helpful discussions and for making available the radioresistant laboratory strains. I am grateful to Prof. L. E. MAGELH~,ES,N. FREIRE-MAIA and W. E. KERR for their comments on my thesis. This work was partially supported by the Comiss~o Nacional de Energia Nuclear (CNEN), Conselho Nacional de Pesquisas (CNPq), Coordenaq~o de Aperfei~oamento do Pessoal de Nfvel Superior (CAPES) and CAmara Especial de Pos-gradua~Ao e Pesquisa da Universidade Federal do Rio Grande do Sul and was developed at the Department of Genetics of the Universidade Federal do Rio Grande do Sul. REFERENCES V. J., Evolution of fitness, I. Improvement in the productivity and size of irradiated populations of Drosophila serrata and Drosophila birchii, Genetics, 53 (1966) 883-895. 2 BONNIER, G., U. n. JONSSON AND C. RAMEL, Selection pressure on irradiated populations of Drosophila melanogaster, Genetics, 39 (1958) 77-88. 3 BREESE,E . , AND K. MATHER,The organization of polygenic activity within a chromosome in Drosophila, II. Viability, Heredity, 14 (196o) 375-399. 4 CARFAGNA, M., Selezioni per la radioresistenza in popolazioni artificiali di Drosophila melanogaster, I. Dati popolazionistici, Atti. Mccad. Naz. Lincei Rend. Classe Sci. Fiz. Mat. Nat., 34 (1963) 681-684. I AYALA,

RADIORESISTANCE IN DROSOPHILA

355

5 CARSON, H. L., T r a n s i t o r y increase in genetic load in irradiated l a b o r a t o r y p o p u l a t i o n s of Drosophila melanogaster, in S. J. GEERTS (Ed.), Proc. Intern. Congr. Genet. IIth, The Hague, ±963, Vol. I, Pergamon, 1965, p. 74. 6 CORDEIRO, A. R., A. KALISZ, N. B. MORALES AND R. P. BARROS, Genetic analysis of D. willistoni n a t u r a l p o p u l a t i o n s inhabiting high n a t u r a l radiation environment, in Radiochemical and Radioecological Studies on Brazilian Areas of High Natural Radiation, U.S. Atomic E n e r g y Comission, NYo3273- 7, 9 (1966) 1-6. 7 CORDEIRO, A. R., E. K. MARQUES AND A. VEIGA-NETO, Radioresistance of a n a t u r a l p o p u l a t i o n of Drosophila willistoni living in a radioactive e n v i r o n m e n t , Mutation Res., 19 (1973) 325 329 • 8 EHLING, U. H., Strain variation in reproductive capacity and radiation response of female mice, Radiation Res., 23 (1964) 6o3-61o9 GRAHN, D., Acute radiation response of mice from a cross b e t w e e n radiosensitive and radioresistant strains, Genetics, 43 (1958 ) 835-843. IO GREENBERG, J., A locus for radiation resistance in Escherichia coli, Genetics, 49 (1964) 771-778. I I GREENBERG, J., A locus for radiation sensitivity in Escherichia coli Bs-2 Genetics, 5 ° (I964) 639 648. I2 HAYMAN, B. I., The analysis of variance of diallel tables, Biometrics, io (1954) 235-244. 13 KEARSEY, M. Y., AND K. KOJIMA, The genetic architecture of b o d y weight and egg h a t c h a b i l i t y in Drosophila melanogaster, Genetics, 56 (1967) 23-37. 14 KOHN, H. I., AND R. F. KALMAN, The influence of strains on acute X - r a y lethality in mouse, I. LDs0 and d e a t h rate studies, Radiation Res., 15 (1956) 3o9-312. 15 LEE, B. T. O., AND B. W. HOLLO'vVAY,The genetic control of radiosensitivity in Pseudomonas aeruginosa, Radiation Res., 25 (1965) 68-77. 16 MARQUES, E. K., Efeito das radia~6es no valor a d a p t a t i v o e o desenvolvimento de radioresistSncia em popula~6es de Drosophila, Bol. I.C.N.-U.F.R.G.S. Porto Alegre, 27 (1968) i i i o . 17 MARQUES, E. K., The d e v e l o p m e n t of radioresistance in irradiated Drosophila nebulosa populations, Mutation Res., 17 (1973) 59-72. I8 MURAKAMI, A., AND Y. TAZIMA, Studies on strain differences in radiosensitivity in silkworm, I. Screening of sensitive and resistant strains to embryonic radiation killing, Ann. Rept. Natl. Inst. Genet. (Japan), 17 (1966) 98-1oo. 19 N6THEL, H., I n v e s t i g a t i o n s on radiosensitive and radioresistant populations of Drosophila melanogaster, I. Decreased radiosensitivity in stage- 7 oocytes of the irradiated p o p u l a t i o n R() 1, Mutation Res., IO (197 o) 463-474 . 20 OGAKI, I. I., AND E. N. TANAKA, I n h e r i t a n c e of radioresistance in Drosophila, I. Mutation Res., 3 (1966) 438 443. 21 PARSONS, P. A., A correlation between the ability to w i t h s t a n d high t e m p e r a t u r e s and radioresistance in Drosophila melanogaster, Experientia, 25 (1969) IOOO. 22 PARSON'S, P. A., I. T. MACBEAN AND B. T. O. LEE, P o l y m o r p h i s m in n a t u r a l p o p u l a t i o n s for genes controlling radioresistance in Drosophila, Genetics, 61 (1969) 211-218. 23 PENNA FRANCA, E., Y. C. ALMEIDA, Y. BECKER, M. EMMERICH, V. X. ROSER, G. HEGEL, L. HAINSBERGER, T. L. CULLEN, H. PETROW, R. T. DREW AND M. EISENBUND, S t a t u s of investigation in the Brazilian areas of high n a t u r a l radioactivity, Hea~th Phys., I I (1965) 699-712. 24 RESNICK, M. A., Genetic control of radiation sensitivity in Saccharomyces cerevisae, Genetics, 62 (1968) 519-531 . 25 SOBELS, E. H., Recent advances in radiation genetics with emphasis on repair p h e n o m e n a , Proc. Intern. Congr. Genet. I2th., 3 (1969) 205 223. 26 STROMAES, O., Stock differences in X - r a y m u t a t i o n a l sensitivity p a t t e r n of Drosophila melanogaster, Hereditas, 45 (1959) 221-229. 27 TAKAHASHI, C. S., E s t u d o s sobre a influ~ncia de elevada r a d i a ~ o n a t u r a l no Morro do Ferro, P o l o s de Caldas, MG, em alguns organismos que h a b i t a m a ~rea, P h . D . Thesis, F.F.C.L.Ribeir~o Preto-USP, 1972. 28 TAZIMA, Y., Differences in sensitivity of g e r m cells and c h r o m o s o m e s to radiation a m o n g sonle m u t a n t strains of the silkworm, Cytologia, Suppl. (I957) 280-286. 29 WALLACE, B., The estimation of a d a p t a t i v e values of experimental populations, Evolution, 6 (1952) 333-341 . 3 ° WITKIN, E. M., Genetics of resistance in Escherichia coli, Genetics, 32 (1947) 221-248-