Theoretical and Applied Gcnetics 42, 208--214 (t 972) 9 b y Springer-Verlag t 972
Ether Resistance in Drosophila melanogaster BARBARA J. DEERY a n d P. A . PARSONS D e p a r t m e n t of G e n e t i c s a n d H u m a n V a r i a t i o n , L a T r o b e U n i v e r s i t y , B u n d o o r a , V i c t o r i a (Australia) Summary. Strains set up from single inseminated females of D. melanogaster from the wild differ in their resistance to the anaesthetics, ether and chloroform. The main differences between four selected extreme strains could be explained b y additive genes, which in the case of ether resistance were located to regions of chromosomes 2 and 3. The lack of correspondence between ether and chloroform resistance between strains indicates t h a t although the t y p e of genetic architecture controlling the traits is similar, the actual genes differ, which is reasonable in view of their differing chemical structures. Quite high heritabilities were found for resistance to ether based on five inbred strains. I~o significant associations between resistance to ether and body weight, developmental rate or longevity were found. I t is clear t h a t resistance to both anaesthetics would be amenable to more detailed genetic analyses. I t is pointed out t h a t the general conclusions reached from such studies will have implications with respect to the effect of chemicals such as insecticides, not naturally present in nature.
Introduction A l t h o u g h e t h e r h a s long b e e n u s e d as an a n a e s t h e t i c a g e n t , l i t t l e is k n o w n a b o u t v a r i a t i o n s b e t w e e n org a n i s m s in t h e i r r e s i s t a n c e to it. W h e r e v a r i a t i o n s h a v e b e e n found, few g e n e t i c a n a l y s e s h a v e been c a r r i e d out. S t e r n , Schaeffer a n d S p e n c e r (1944) f o u n d t h a t D. virilis was s u b s t a n t i a l l y m o r e r e s i s t a n t to e t h e r t h a n D. americana. I t was shown t h a t t h e difference was c h r o m o s o m a l l y c o n t r o l l e d , a n d Crow (1957) r e p o r t e d t h e t r a i t to be polygenic. R a s m u s o n (1955) f o u n d t h a t a w i l d - t y p e c h a e t a selection line of D. melanogaster was e x t r e m e l y s e n s i t i v e to ether. T h e sensit i v i t y d e p e n d e d on b o t h c y t o p l a s m i c f a c t o r s a n d c h r o m o s o m a l genes, t h e gene d e p e n d e n t s e n s i t i v i t y being polygenic. When the sensitive and normal s t r a i n s were crossed, it was f o u n d t h a t a s t i m u l a t i n g a g e n t was t r a n s m i t t e d v i a t h e s p e r m of t h e s e n s i t i v e s t r a i n w h i c h c h a n g e d t h e r e a c t i v e s y s t e m p r e s e n t in t h e c y t o p l a s m of t h e n o r m a l s t r a i n . Tile s t i m u l a t i n g a g e n t in t h e m a l e was o n l y p r o d u c e d if t h e g e n o t y p e a n d c y t o p l a s m were sensitive. On t h e o t h e r h a n d Ogaki, N a k a s h i m a - T a n a k a a n d M u r a k a m i (1967) f o u n d no e v i d e n c e for c y t o p l a s m i c f a c t o r s in D. melanogaster b u t t h e y d e v e l o p e d an e t h e r r e s i s t a n t s t r a i n in w h i c h t h e t h i r d c h r o m o s o m e was m a i n l y r e s p o n sible, w i t h m a j o r g e n e t i c a c t i v i t y a t 61, a n d m i n o r a c t i v i t y on t h e X a n d f o u r t h c h r o m o s o m e s . In this paper, investigations into ether resistance in w i l d p o p u l a t i o n s will be d e s c l i b e d , w i t h s o m e g e n e t i c a n a l y s e s of e t h e r r e s i s t a n t a n d s e n s i t i v e s t r a i n s . T h e p o s s i b i l i t y of c o r r e l a t i o n s w i t h c e r t a i n o t h e r t r a i t s will be discussed. Method The strains used consisted of fifteen from Leslie Manor (LM) near Camperdown, Victoria collected in December 1965, and seventeen from E l t h a m (E) near Melbourne
collected in J a n u a r y t968. They were set up from single inseminated founder females from the wild populations. Five strains, inbred b y sib-mating for at least 300 generations were also used in some of the experiments. The method of etherization was similar to t h a t developed by Ogaki, Nakashima-Tanaka and Murakami (1967). Etherizations were carried out in a corked 50 ml. vial at 25 ~ A flat headed screw was inserted into the cork and around the exposed end, lgm. of absorbent cotton wool was wrapped and then covered with a piece of gauze. In order to anaesthetize the flies, 2.8 ml. of diethyl ether was pipetted onto the cotton wool plug and the vial immediately stoppered. After leaving the vial for four minutes, 30 virgin male or female flies were rapidly introduced, after which the vial was again corked and left for a further two minutes (unless otherwise specified). At the conclusion of the two minutes, the flies were transferred to a fresh vial containing medium. Mortalities were assessed 24 hours later. Flies were etherized at 24 hours of age, because the effect of ether was found to be age-dependent, in t h a t percentage m o r t a l i t y was found to increase over the period 4--120 hours (Figure 1). In particular at four 100
I ., Holes o Femotes
80 >. 5o
~ 4o g_
0
20
40
60 80 10{3 120 A0eIin h0ursl Fig. 1. Percentage mortality after etherization plotted against age, for males (o) and females (o)
E t h e r Resistance in Drosophila melanogaster
]Zol. 42, No. 5
hours, the percentage m o r t a l i t y was low and b y t 20 hours very high. Twenty-four hours was chosen rather t h a n t20 hours, because at t20 hours it would have been necessary to reduce the time of exposure quite considerably, and it was thought t h a t this might decrease the accuracy of the testing time. I t will also be noted from Figure t t h a t males are in general more sensitive than ~emales, in agreement with Ogaki, Nakashima-Tanaka and Murak a m i (1967). All analyses of variance based on percentage mortalities were carried out after applying the angular transformation to avoid a dependence of the variance on the mean.
Table 2. a. Mean percentage mortalities o/30 flies 24 hours after etherization for the x7 E strains (Note: each entry represents the mean of two replicates for each o/ the two generations tested)
Ether resistance in natural populations Tile LM s t r a i n s were t e s t e d f o u r t i m e s o v e r a p e r i o d of e i g h t g e n e r a t i o n s ; t w o r e p l i c a t e s p e r sex b e i n g t e s t e d e a c h t i m e ; a n d tile m e a n p e r c e n t a g e m o r t a l i t i e s are g i v e n for t h e 15 s t r a i n s in T a b l e I t o g e t h e r w i t h a n a n a l y s i s of v a r i a n c e . T h e m a i n effects were all h i g h l y s i g n i f i c a n t . T h e sexes effect reflects g r e a t e r m a l e s u s c e p t i b i l i t y , a n d t h e c o u n t s effect m i n o r e n v i r o n m e n t a l differences b e t w e e n g e n e r a t i o n s . Of p a r t i c u l a r i n t e r e s t is t h e h i g h l y s i g n i f i c a n t s t r a i n s effect. This shows g e n e t i c effects p r e s u m a b l y a r i s i n g f r o m differences b e t w e e n t h e f o u n d e r females. T h u s t h e Leslie M a n o r p o p u l a t i o n f r o m w h i c h t h e f o u n d e r f e m a l e s were d e r i v e d is p o l y m o r p h i c for genes cont r o l l i n g r e s i s t a n c e a n d s e n s i t i v i t y t o ether. This agrees w i t h w o r k on a n u m b e r of o t h e r p h y s i o l o g i c a l stresses s u c h as h i g h t e m p e r a t u r e s ( H o s g o o d a n d P a r s o n s , 1968; P a r s o n s , t969), Co6~ r a y s (Parsons, Table t. a. Mean percentage mortalities o/3o flies 24 hours after etherization for the 15 L M strains (Note : each entry represents the mean o/ two replicates for each o/ the four generations tested) Strain
Females Males
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
26.27 26.30 55.63 24.99 t8.84 11.45 53.50 19.87 36.91 t8.59 53.64 32.65 12.03 31.52 t8.38
53.85 53.96 72.18 76.19 53.48 43.70 86.32 54.48 52.96 54.79 86.82 40.91 51.89 52.69 46.41
b. Analysis o/variance Sources of variation
d.f.
M.S.
F
Strains Sexes Counts Strains • sexes Strains • counts Sexes • counts Error
14 1 3 t4 42 3 42
639.62 10962.83 1492.53 96.14 99.07 53.26 95.18
6.72*** 115.t9"** 15.68"** t.01 t.04 0.56
*** P ~ 0.00t
209
Strain
Females Males
t 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
21.96 15.45 25.40 13.29 37.t2 20.46 2.50 15.83 18.41 11.92 3.12 17.59 t6.04 6.83 6.71 7.51 30.13
47.46 69.46 76.43 40.73 58.43 45.64 t7.24 41.71 20.24 42.89 t3.80 31.33 42.46 25.90 20.26 26.84 40.37
b. Analysis of variance Source of variation
d.f.
M.S.
F
Strains Sexes Counts Strains • sexes Strains • counts Sexes • counts Error
16 1 1 16 16 t t6
3t0.25 4371.54 2.92 62.09 69.53 26.09 26.49
11.71"** 165.03"** 0.11 2.34 2.62* 0.99
* -P ]0.4971 for d.f. significance at P ~ 0.05.) Hence the genetic control Source of v a r i a t i o n Females Males of the two anaesthetics differs at least partly in General combining ability 3 38.O1 * * * 68.51 * * * natural populations. Specific combining ability 6 2.48 11.54" * * The investigation of the genetic basis of chloroform R e c i p r o c a l e f f e c t 6 5.99" * 2.85 * resistance was taken one step further by setting up Error 16 a 4 • 4 diallel cross between two chloroform resistant * P
Ether Resistance in Drosophila melanogaster BARBARA J. DEERY a n d P. A . PARSONS D e p a r t m e n t of G e n e t i c s a n d H u m a n V a r i a t i o n , L a T r o b e U n i v e r s i t y , B u n d o o r a , V i c t o r i a (Australia) Summary. Strains set up from single inseminated females of D. melanogaster from the wild differ in their resistance to the anaesthetics, ether and chloroform. The main differences between four selected extreme strains could be explained b y additive genes, which in the case of ether resistance were located to regions of chromosomes 2 and 3. The lack of correspondence between ether and chloroform resistance between strains indicates t h a t although the t y p e of genetic architecture controlling the traits is similar, the actual genes differ, which is reasonable in view of their differing chemical structures. Quite high heritabilities were found for resistance to ether based on five inbred strains. I~o significant associations between resistance to ether and body weight, developmental rate or longevity were found. I t is clear t h a t resistance to both anaesthetics would be amenable to more detailed genetic analyses. I t is pointed out t h a t the general conclusions reached from such studies will have implications with respect to the effect of chemicals such as insecticides, not naturally present in nature.
Introduction A l t h o u g h e t h e r h a s long b e e n u s e d as an a n a e s t h e t i c a g e n t , l i t t l e is k n o w n a b o u t v a r i a t i o n s b e t w e e n org a n i s m s in t h e i r r e s i s t a n c e to it. W h e r e v a r i a t i o n s h a v e b e e n found, few g e n e t i c a n a l y s e s h a v e been c a r r i e d out. S t e r n , Schaeffer a n d S p e n c e r (1944) f o u n d t h a t D. virilis was s u b s t a n t i a l l y m o r e r e s i s t a n t to e t h e r t h a n D. americana. I t was shown t h a t t h e difference was c h r o m o s o m a l l y c o n t r o l l e d , a n d Crow (1957) r e p o r t e d t h e t r a i t to be polygenic. R a s m u s o n (1955) f o u n d t h a t a w i l d - t y p e c h a e t a selection line of D. melanogaster was e x t r e m e l y s e n s i t i v e to ether. T h e sensit i v i t y d e p e n d e d on b o t h c y t o p l a s m i c f a c t o r s a n d c h r o m o s o m a l genes, t h e gene d e p e n d e n t s e n s i t i v i t y being polygenic. When the sensitive and normal s t r a i n s were crossed, it was f o u n d t h a t a s t i m u l a t i n g a g e n t was t r a n s m i t t e d v i a t h e s p e r m of t h e s e n s i t i v e s t r a i n w h i c h c h a n g e d t h e r e a c t i v e s y s t e m p r e s e n t in t h e c y t o p l a s m of t h e n o r m a l s t r a i n . Tile s t i m u l a t i n g a g e n t in t h e m a l e was o n l y p r o d u c e d if t h e g e n o t y p e a n d c y t o p l a s m were sensitive. On t h e o t h e r h a n d Ogaki, N a k a s h i m a - T a n a k a a n d M u r a k a m i (1967) f o u n d no e v i d e n c e for c y t o p l a s m i c f a c t o r s in D. melanogaster b u t t h e y d e v e l o p e d an e t h e r r e s i s t a n t s t r a i n in w h i c h t h e t h i r d c h r o m o s o m e was m a i n l y r e s p o n sible, w i t h m a j o r g e n e t i c a c t i v i t y a t 61, a n d m i n o r a c t i v i t y on t h e X a n d f o u r t h c h r o m o s o m e s . In this paper, investigations into ether resistance in w i l d p o p u l a t i o n s will be d e s c l i b e d , w i t h s o m e g e n e t i c a n a l y s e s of e t h e r r e s i s t a n t a n d s e n s i t i v e s t r a i n s . T h e p o s s i b i l i t y of c o r r e l a t i o n s w i t h c e r t a i n o t h e r t r a i t s will be discussed. Method The strains used consisted of fifteen from Leslie Manor (LM) near Camperdown, Victoria collected in December 1965, and seventeen from E l t h a m (E) near Melbourne
collected in J a n u a r y t968. They were set up from single inseminated founder females from the wild populations. Five strains, inbred b y sib-mating for at least 300 generations were also used in some of the experiments. The method of etherization was similar to t h a t developed by Ogaki, Nakashima-Tanaka and Murakami (1967). Etherizations were carried out in a corked 50 ml. vial at 25 ~ A flat headed screw was inserted into the cork and around the exposed end, lgm. of absorbent cotton wool was wrapped and then covered with a piece of gauze. In order to anaesthetize the flies, 2.8 ml. of diethyl ether was pipetted onto the cotton wool plug and the vial immediately stoppered. After leaving the vial for four minutes, 30 virgin male or female flies were rapidly introduced, after which the vial was again corked and left for a further two minutes (unless otherwise specified). At the conclusion of the two minutes, the flies were transferred to a fresh vial containing medium. Mortalities were assessed 24 hours later. Flies were etherized at 24 hours of age, because the effect of ether was found to be age-dependent, in t h a t percentage m o r t a l i t y was found to increase over the period 4--120 hours (Figure 1). In particular at four 100
I ., Holes o Femotes
80 >. 5o
~ 4o g_
0
20
40
60 80 10{3 120 A0eIin h0ursl Fig. 1. Percentage mortality after etherization plotted against age, for males (o) and females (o)
E t h e r Resistance in Drosophila melanogaster
]Zol. 42, No. 5
hours, the percentage m o r t a l i t y was low and b y t 20 hours very high. Twenty-four hours was chosen rather t h a n t20 hours, because at t20 hours it would have been necessary to reduce the time of exposure quite considerably, and it was thought t h a t this might decrease the accuracy of the testing time. I t will also be noted from Figure t t h a t males are in general more sensitive than ~emales, in agreement with Ogaki, Nakashima-Tanaka and Murak a m i (1967). All analyses of variance based on percentage mortalities were carried out after applying the angular transformation to avoid a dependence of the variance on the mean.
Table 2. a. Mean percentage mortalities o/30 flies 24 hours after etherization for the x7 E strains (Note: each entry represents the mean of two replicates for each o/ the two generations tested)
Ether resistance in natural populations Tile LM s t r a i n s were t e s t e d f o u r t i m e s o v e r a p e r i o d of e i g h t g e n e r a t i o n s ; t w o r e p l i c a t e s p e r sex b e i n g t e s t e d e a c h t i m e ; a n d tile m e a n p e r c e n t a g e m o r t a l i t i e s are g i v e n for t h e 15 s t r a i n s in T a b l e I t o g e t h e r w i t h a n a n a l y s i s of v a r i a n c e . T h e m a i n effects were all h i g h l y s i g n i f i c a n t . T h e sexes effect reflects g r e a t e r m a l e s u s c e p t i b i l i t y , a n d t h e c o u n t s effect m i n o r e n v i r o n m e n t a l differences b e t w e e n g e n e r a t i o n s . Of p a r t i c u l a r i n t e r e s t is t h e h i g h l y s i g n i f i c a n t s t r a i n s effect. This shows g e n e t i c effects p r e s u m a b l y a r i s i n g f r o m differences b e t w e e n t h e f o u n d e r females. T h u s t h e Leslie M a n o r p o p u l a t i o n f r o m w h i c h t h e f o u n d e r f e m a l e s were d e r i v e d is p o l y m o r p h i c for genes cont r o l l i n g r e s i s t a n c e a n d s e n s i t i v i t y t o ether. This agrees w i t h w o r k on a n u m b e r of o t h e r p h y s i o l o g i c a l stresses s u c h as h i g h t e m p e r a t u r e s ( H o s g o o d a n d P a r s o n s , 1968; P a r s o n s , t969), Co6~ r a y s (Parsons, Table t. a. Mean percentage mortalities o/3o flies 24 hours after etherization for the 15 L M strains (Note : each entry represents the mean o/ two replicates for each o/ the four generations tested) Strain
Females Males
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
26.27 26.30 55.63 24.99 t8.84 11.45 53.50 19.87 36.91 t8.59 53.64 32.65 12.03 31.52 t8.38
53.85 53.96 72.18 76.19 53.48 43.70 86.32 54.48 52.96 54.79 86.82 40.91 51.89 52.69 46.41
b. Analysis o/variance Sources of variation
d.f.
M.S.
F
Strains Sexes Counts Strains • sexes Strains • counts Sexes • counts Error
14 1 3 t4 42 3 42
639.62 10962.83 1492.53 96.14 99.07 53.26 95.18
6.72*** 115.t9"** 15.68"** t.01 t.04 0.56
*** P ~ 0.00t
209
Strain
Females Males
t 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
21.96 15.45 25.40 13.29 37.t2 20.46 2.50 15.83 18.41 11.92 3.12 17.59 t6.04 6.83 6.71 7.51 30.13
47.46 69.46 76.43 40.73 58.43 45.64 t7.24 41.71 20.24 42.89 t3.80 31.33 42.46 25.90 20.26 26.84 40.37
b. Analysis of variance Source of variation
d.f.
M.S.
F
Strains Sexes Counts Strains • sexes Strains • counts Sexes • counts Error
16 1 1 16 16 t t6
3t0.25 4371.54 2.92 62.09 69.53 26.09 26.49
11.71"** 165.03"** 0.11 2.34 2.62* 0.99
* -P ]0.4971 for d.f. significance at P ~ 0.05.) Hence the genetic control Source of v a r i a t i o n Females Males of the two anaesthetics differs at least partly in General combining ability 3 38.O1 * * * 68.51 * * * natural populations. Specific combining ability 6 2.48 11.54" * * The investigation of the genetic basis of chloroform R e c i p r o c a l e f f e c t 6 5.99" * 2.85 * resistance was taken one step further by setting up Error 16 a 4 • 4 diallel cross between two chloroform resistant * P
