An experimental study of environmental factors in selection and population

AN EXPERIMENTAL STUDY O F ENVIRONMENTAL FACTORS IN SELECTION AND POPULATION

MORRIS HENRY HARNLY

FOUR FIQUREs

THE PROBLEM1

The following experiments with Drosophila melanogaster were planned to study the effects of four environmental factors on a mixed population. Varying the environmental conditions under which the larvae developed would be expected to show to what'extent these factors affect the total yield and also the ratio of the types to each other.

METHODS

All experiments were carried out in incubators at approximately 25°C. The food was the customary banana-agar mixture in the proportion of 500 grams of banana, 500 cc. of water, and 10 grams of agar-agar. The bananas were always overripe (the skins dark brown or black), but not decayed. The amount of yeast used varied with the a rea -one drop of a yeast suspension (one cake of fresh yeast mixed with 100 cc. of water) was used in vials, two drops in 4-ounce bottles, four drops in half-pint and pint milk bottles, and six drops in 250-cc. Erlenmeyer flasks.

Virgin wild females of a particular inbred stock (wild- type flies from the Star Dichaete stock) were crossed to vestigial males. Virgin F, females were backcrossed to vestigial males. The use of the backcross assures equal numbers of two kinds of eggs being deposited in each culture. A

'The author wiehea to express to Prof. T. H. Morgan and Dr..C. B. Bridges his gratitude for suggestions in connection with this work.

141

THE JOURNAL OF EXPERIMENTAL ZO6LOOY, VOL. 53, NO. 2

142 MORRIS HENRY HARNLY

mixed population should be produced, with equal numbers of vestigial and of wild-type flies, unless this ratio is modified by differential mortality. One mated female was put into each vial, bottle, or flask and allowed to lay eggs during a ten-day period. The counting period for the emerging off- spring was usually eighteen days, which was possible without mixing generations, since. the emerging flies were removed daily from the cultures.

GENERAL RESULTS

It was found that an increase in the depth, area, or hardness of the food or in the amount of paper in the surface of the food, up to an optimum, carried with it an increase in total yield or productivity. The theoretical ratio of one wild to one vestigial was not obtained even under the optimum conditions of the experiments, the average ratio of wild to vestigial being about 1: 0.8. Increase in depth of food had little differential effect on viability. Decrease in area had a marked effect, changing the ratio from 1: 0.8 to as little as 1 wild to 0.46 vestigial. Increase in the hardness of the food (amount of agar-agar) or in the amount of paper in the surface improved the ratio from 1: 0.8 to 1: 0.9. The mutant gene vestigial was found to have a definite retarding effect on the rate of development during the larval period.

EFFECTS O F ENVIRONMENTAL FACTORS ON THE TOTAL POPULATION

A . Experiments on depth

In these experiments half-pint bottles were used throughout, keeping the area constant at 24 sq.cm. The quantities of food used were 5, 10, 15, 20, 30, 40, 50, 60, 70, and 80 cc. The depths corresponding to these volumes have been de- termined by measuring from the bottom to the top of the food at the wall of the bottle. The conversion scale is as follows : 5 cc. = 4 mm. ; 10 cc. = 7 mm. ; 15 cc. = 9 mm. ; 20 cc. = 11 mm. ; 30 cc. = 14 mm. ; 40 cc. = 18 mm. ; 50 cc. = 22

SELECTION AND POPULATION 143

mm.; 60 cc.=26 mm.; 70 cc.=30 mm.; 80 cc.=34 mm. The depths will be referred to throughout in terms of this conversion scale of millimeters.

In table 1 and in one of the curves of figure 1 is shown the average number of flies per pair obtained for the various

TABLE 1

Effect of depth of food'

4 I 7 ' 9 ' 11 14 I 18 '1SA

Cultures 23 1 53 ~ 30 1 47 1 266 1 2"9 ~ 34

Depth Trials 2 , 9 , 3 l 9 2

_____ !

7

S

9

10

18

1 4

16

18

__ Viability

- 116 73

169 145 241 169 277 249 187 159 121 95

122 76 55 32 83 38 88 65 19 12 6 7 3 -

- 856 566 697 585 754 673 502 459 452 407 320 316 252 209 237 176 241 180 260 205 130 92 97 67

120 60

110 86

- i89 134 362 297 392 311 230 206 246 225 192 187 179 136 135 122 129 132 184 141 230 204 260 152 262 208 254 149

.77€

.-

-

- 940 602 649 592 624 529 446 397 446 352 361 327 315 267 262 235 247 220 264 234 414 319 463 361 391 352 152 123

.821 -

- 513 262 485 333 421 302 263 199 266 195 243 174 202 127 179 135 160 113 161 104 310 209 257 206 209 165 112 103

.695 -

-

- 622 310 614 459 417 396 365 287 324 272 245 227 172 180 178 160 165 129 156 108 346 251 246 228 199 169 142 93

.78( _ _

-

- 266 113 077 888 989 944 592 600 555 484 341 287 294 313 318 296 317 264 307 262 689 574 631 549 933 932 37E 397

A95 _ _

-

18B ' 22 1 6

15 ~ 22

126 52

457 368 481 449 401 375 266 234 240 213 278 252 189 147 165 139 218 167 294 289 158 123

.R58 __

- 697 334 465 353 338 302' 236 198 186 157 192 137 121 103 121 78

128 95

13 1 79

280 204 303 245 175 181 141 128

.73E __ -

__ 451 248 335 228 297 250 187 178 173 133 152 180 122 109 118 94

122 94 96 65

292 202 287 231 183 159 131 115

.776 __

- 483 205 430 332 296 243 177 147 142 94

148 102 113 73

134 97

164 108 134 78

268 207 250 156 135 109 79 62

.685 _ _

-

__ 366 205 421 307 366 265 206 138 230 143 176 134 119 115 145 93

157 90

121 66

310 163 258 174 156 127 98 75

.671 __

Depth of food in millimeters 1 per cent agar-agar, except 18A = 2.5 per cent and 1SB = 5 per cent agar.


depths. It is apparent from the curve (smoothed by eye) that there was a sharp and striking increase in yield through the first three depths (4 to 9 mm.) followed by a gradual increase in the number of flies obtained until a depth of 22 mm. was reached. Beyond 22 mm. a larger reserve supply of food seemed to have no effect upoii the yield. Doubling the food supply in the cultures, even in the case of the

smallest amount, did not double the yields. Thus, with 5 cc. of food the yield' averaged 113 flies per bottle, while 10 cc. of food produced only 172 flies, instead of the 226 that would have been produced by that amount of food in two cultures. Similarly, bottles with 10 cc. of food averaged 172 flies, or at the rate of 344 flies for 20 cc. of food, but 20 cc. of food in one bottle produced only 232 flies. Bottles containing 30 cc. of food yielded an average of 246 flies; but this amount


of food divided equally into two bottles produced an ag- gregate average of 444 flies. It is evident that there must have been a reserve supply of food in these bottles not used by the larvae, or used and lost due to the death of the larvae. Increases in the food supply followed the law of diminishing returns-marked increases in the yield at first, gradually stepped down until further additions had no effect.

The number of flies obtained daily from the 4-mm. bottles became negligible after the twelfth day, although an occa- sional fly emerged to the sixteenth day.. The larger number of flies obtained from the second depth (7 mm.) was due to a slight increase of the daily yield plus a few flies obtained from the eleventh to the eighteenth day of the count. The higher yield in the bottles that contained food 9 mm. deep was due to a general increase during the entire eighteen- day period of counting, and was especially marked during the last eight days. Beyond this depth (9 mm.) the increased yield was due to a slight daily increase throughout the entire eighteen-day period. It was evident that, with increasing depth, the major increase in the total yield per bottle was due to the added reserve supply of food which carried through development those larvae hatching from the eggs produced during the latter part of the ten-day period of egg laying. The minor increase was due to a slightly larger number of flies obtained in the earlier counts.

It is improbable that, with a constant surface area, there was any significant difference in the number of eggs laid by the females on the various depths. It was evident from the conditions of the bottles that actual starvation was the con- trolling factor when only 5 cc. of food was supplied to the larvae in the half-pint bottle, for the food was entirely consumed in a few days in these bottles.

B. Experiments on area

The depth of the food was kept constant at 1 inch (25 mm.) throughout these experiments. Five areas were tried-


vials, 4-ounce bottles, half-pint and pint milk bottles, and 250-cc. Erlenmeyer flasks. The areas were: 4.4, 11, 24, 40, and 52 sq.cm.

The effect, on the average total yield, of increasing the surface area available to the larvae is seen in table 2 and in the lower curve of figure 1. The units of area are too far apart to give as smooth and characteristic a curve as the

TABLE 2

Effec t of surface crowding, depth 26 mm., 1 per cent agar-agar

Area Triale Cultures

Vials 8

256

4-oqnce bottles 8

104

Pints 2 10

Flasks 2 8

DAY or COUNT 1

1

2

3

4

5

6

7

8

9

10

12

14

16

18

370 108

1975 793

2120 1240 1640 1083 1444 917

1237 835

1132 714 759 554 585 453 439 314 944 707 908 662 934 637 721 492

343 46

1924 729

1666 739

1184 502

1140 496 832 453 696 400 524 313 418 245 371 244 907 454 875 405 889 488 799 593

162 118 124 99

192 169 86 58 63 47 69 25 81 61

183 131 206 157 124 113

Viability 1 .625 1 .486 1 .758

51 28 48 30

192 143 53 59 51 56 35 53 28 21 97 57

165 129 135 110

3 0 2


points selected for depth; but they indicate that properly selected increments of area would give a similar curve, with a sharp rise flattening off to a plateau. As in the case of depth, an increase of area carried with it an increase in the yield up to the optimum. There was a marked increase in the 4-ounce bottles over the vials, and in the half-pint milk bottles over the 4-ounce bottles. The pint milk bottle had a somewhat larger yield than the half-pint, but the 250-c~. flask was about the same as the half-pint. With regard to productivity, the optimum area for single pair matings seemed to be approached with the half-pint bottle. The half- pint bottle was the largest area that could be handled con- veniently and rapidly. Areas larger than this required care- ful handling and much more time to manipulate in removing the flies from the bottle, since the food cake tended to break loose from the bottom-of the culture bottle. The half-pint bottle with the area of 24 sq.cm. is the same as the half-pint bottle used in the experiment on depth, and the amount of food used, 25 mm., corresponded closely to the 26-mm. depth in that series. But in the area experiment the counting was continued for only ten days, and accordingly the absolute values obtained in this experiment are fa r below those obtained in the eighteen-day counts of the corresponding cultures in the depth experiment.

C . Experiments on consistency Half-pint milk bottles were used, and the food was poured

to a depth of 26 mm. in each bottle. Eight different amounts of agar-agar were tried, namely, 0.7 per cent, 1.0 per cent, 1.48 per cent, 1.96 per cent, 2.44 per cent, 2.91 per cent, 3.85 per cent, and 4.76 per cent agar-agar media. Three trials were run for these amounts of agar-agar. In a given trial enough bananas were mashed and thoroughly mixed to make the food for one set of ten bottles of every degree, and 350 grams were weighed out for each percentage of agar. The bottles for all percentages of agar in each trial were sterilized together and kept in the same incubator. The females were


forty-eight hours old and had been mated for twenty-four hours.before they entered the bottles in which they laid eggs for nine days. Eighteen-day counts were made.

There was a marked difference in the consistency of the food. The 0.7 per cent was noticeably softer than the usual 1.0 per cent mixture. The higher percentages of agar were quite hard, 1.5 per cent being noticeably firmer than 1.0 per cent, and from 2 per cent upward there was a marked increase in stiffness. In the higher percentages of agar the food tended to shrink away from the walls of the bottle as the count progressed. The shrinkage took place after the egg-laying period was completed and varied in amount from a partial movement of the food from one side of the bottle to a complete freeing of the food from the walls of the bottle. This would change the free area exposed to the larvae from 24 sq.cm. by varied increments with a maximum area of 56 sq.cm. This might introduce an error in the upper range of the experiment.

At and above 2.44 per cent there was noticeable in all three trials, late in the count, a very fine powdery green mold not produced at all in the lower percentages of agar-agar. Gas- sing, the pushing up of the food in the bottle by thc C 0 2 gas produced during fermentation, was a serious problem for all percentages of agar in all three trials. It was necessary to jar the food down twice daily during the egg-laying period. Many eggs, as is usual in cultures, were laid on the wall of the bottle at the edge of the food. When the cake was shaken down the eggs were left far from the food. Some just-hatched larvae probably never found their way down to the food and died. In previous trials on 2.44 per cent and 4.76 per cent agar (table 1, MA, 18B) an average of fifty more flies were obtained than in these trials. It may be that the disturbance due to the excessive gassing in these trials explains the difference in the total yield. It has been the practice of many workers to make a channel in the food at the wall of the bottle to allow the escape of gas and remove this hazard. Since surface area was to be kept constant in


this experiment, it was not thought feasible to take this precaution against gassing.

In the study of depth it was found that a 1 per cent medium with a depth of 26 mm. produced an average of 275 flies, the average for the four greater depths being 270 flies. In the experiment on consistency, a 1 per cent medium averaged 267 flies for the same depth and area--a value in close ac- cordance with the previous one.

The results of the test of the effect of increasing hardness (table 3 and figure 1) were striking; the cultures with a hardness above the standard 1 per cent produced a progressively higher output until about 3 per cent was reached. With food at the depth and area that the previous experiments had shown to be approximately optimum, an increase of 46 per cent in the yield followed an increase in hardness corresponding to 3 per cent agar. Hardness above that of the 3 per cent cultures did not produce further increase in yield, or even brought a slight decline.

The production of a population corresponding to the maximum output of eggs for a given female is thus seen to depend upon the consistency of the food as well as upon the amount and surface area of that food. Experiments (Clausen, '24), in which females were transferred to fresh food frequently, have shown that the maximum yields here reported for the optimum conditions of area (about 40 sq.cm.), of depth (about 22 mm.), or for consistency are in each case far below the population that might be produced if all the eggs resulted in flies. It seems hardly probable that the differences observed in the series on depth, on area, and on hardness repre- sent differences in egg laying primarily, but rather differences in the effectiveness of the environment in permitting a higher proportion of those eggs to survive through larval and pupal development and to emerge as imagoes. Increases in depth of food, in area, and in hardness allowed greater survival until an optimum was reached beyond which further additions did not better the conditions of survival. It was anticipated that the lower degrees of hardness would give

150 MORRIS H E N R Y HARNLY

106 36

lower yields, because a higher percentage of eggs, larvae, and pupae would be smothered by the working over of the softer

TABLE 3

E r e c t of consistency, onc-half-pint bottle, depth $6 mm.

160 46

Agar (per cent) Trials Cultures

360 137 658 513 651

0; I 1;" 1 1.48 3

28 1 27 28

363 107 830 631 621

l i 6 I 2 1

26

209 124 262 242 208 219 375 324 351 312 295 264

2.91 3 26

183 181 211 192 178 157 340 271 327 247 208 155

3*."3" 24 ' DAY OF COUNT I

1

2

3

4

5

6

7

8

9

10

12

14

16

18

36 15

599 286 486 411 451 383 325 285 305 214 229 196 156 122 177 128 167 141 358 274 400 298 240 218 99

137

Viability 1 .771 .816 1 .794

147 46

752 488 710 647 378 426 188 206 153 126 159 114 256 189 306 329 244 239 467 433 411 411 119 165 100

1 A90 I

137 44

675 444 515 530 257 330 158 192 174 142 206 187 207 162 236 200 170 149 356 309 247 223 174 150 104 94

2 7 5

239 66

885 687 686 665 367 449 196 197 246 206 311 256 269 272 328 265 293 261 503 420 382 354 208 245 130 120

.885 .849 1 A17

medium. But inspection of the cultures suggested that, at 1.5 per cent or certainly at 2 per cent of agar, the hardness of the medium was sufficient to prevent much loss from drowning. However, the increase in productivity did not


cease there, but continued until 3 or more per cent was reached.

The daily yields for the six percentages of agar-agar showed a marked increase in production in successive percentages of agar on the second and third days, with a general increase throughout the entire period of counting, with the exception of the fourth and fifth days, when it was nearly the same for all consistencies, and the seventeenth and eighteenth days, when the cultures ran out. Something had operated to cause a tremendous drop in the yield on the fourth day of the count. This drop was followed by low production for about two days. Subsequent to this there was a minor rise that was maintained as a rough plateau until the counts began to run out. These sudden changes were probably due to some environmental factor or factors, for no results corresponding to them have been obtained when females were transferred to fresh cultures from day to day. Pearl and Penniman ('26) have recorded the pH in the banana medium in which larvae were developing. Their observations are as follows: 1st day, 5.3; 2nd day, 5.15; 3rd day, 4.85; 4th day, 4.7; 5th day, 4.6; 6th day, 4.8; 7th day, 4.7; 8th day, 4.8; 9th day, 4.8. There was a slow drop in the pH through the first four days, reaching a low point on the fifth day. A slight rise followed this, reaching an apparently more or less stable condition slightly above the lowest point. It may be that the similarity in the two curves is merely a coincidence or it may be that the environmental factor, pH, had an effect on the yield.

D. Experiments with paper

Half-pint milk bottles were used, the food was poured to a depth of 26 mm., and the media was the standard mixture of 1 per cent agar. The variable was the amount of paper placed on the surface and worked into the surface of the food by the larvae. No paper was used in the previously described experiments.


0.25 3 24

4719 4089 8808

0.866 ~ _ _ . _ _ ~

‘Scott tissue toweling,’ cut in strips 4 inches long and g inch wide, was weighed out in various amounts and placed in the bottles. Control bottles containing no paper and units of 0.25 gram, 0.5 gram, 0.75 gram, and 1.0 gram of paper were used in the experiment. The strips of paper, loosely wadded, were placed in the bottles and pressed onto the surface of the food. This paper, taking up moisture from the food, collapsed on the surface and was worked into the surface of the food by the larvae. The results are shown in figure 1 derived from table 4.

The average total yield for the controls without paper was 278 flies, which corresponds closely to 275 obtained in the 26-mm. grade in the depth experiment and to 267 for the 1

0.5 3

23 4968 4371 9339

0.879

TABLE 4

Effect of paper

Paper (grams) Trials Cultures + Vt3

Total

Viability

0.0 3 20

3073 2488 5561

0.809

0.75 3 25

5,877 5,285

11,162

0.899 ~ -

1.0 3 23

5,246 4,774

10,020

0.908

per cent agar in the experiment on consistency, the conditions being the same in all three experiments for the grades just compared.

The paper was found to have a marked effect on the size of the population that could be supported by the same amount of food and on the same area. The maximum increase in productivity in these experiments was 60 per cent from the control grade with no paper to the grade with 0.75 gram of paper per bottle. Here, again, a mechanical or physical factor other than food supply or crowding of the larvae mas operating. With no change in the food supply, or relief of crowding, a given area and amount of food were able to sup- port a population 60 per cent larger. The larvae in their burrowing carried or worked the paper fibers down into the


food to the depth to which they operated. The food to this depth appeared to be porous and quite firm. The paper may have served as reinforcement, holding the food in place where the larvae had burrowed through and so increasing the ef- fective sutface and insuring a more certain supply of air to the larvae. Furthermore, there was less danger of pupae or eggs being buried and suffocated in the working over of the medium by the larvae.

SELECTIVE EFFECTS O F ENVIRONMENTAL FACTORS ON A MIXED POPULATION

The experiments just described were so planned that eggs of two genetic compositions, heterozygous wild-vestigial and homozygous vestigial, were present in equal numbers in the cultures. If no selective mortality occurs, the adult population should consist of two classes in equal numbers, heterozygous wild-type flies and homozygous vestigial flies. The environmental factors in the experiments were such that their selective action, if any, would operate on the larvae. I f any or all of these factors had a selective action, it would show in the ratio of the two classes in this mixed population. The index of viability of vestigial is the quotient obtained by dividing the number of vestigial flies by the number of wild flies.

A . Depth The quotient or viability curve for the various depths is

shown in figure 2 derived from table 1. The curve shows that the proportion ranged from 1 wild to 0.8 vestigial (100: 82) to 1 wild to 0.7 vestigial (100 : 67). An increase in the available food might seem to be advantageous to the weaker vestigial larvae and result in a nearer approach to equality in the two kinds of flies obtained, but it is evident that the deeper cultures gave poorer results on the average than the shallower. In the greater depths there was a tendency for the gas produced by fermentation to collect under the food and drive it toward the top of the bottle. The disturbance resulting from this and the necessary jarring down of the

154 MORRIS H E N R Y H A R N L Y

food (it would frequently go out of the top if not shaken down), which, of course, disturbed the surface of the food in which the larvae were working, might tend to pack the soft surface food and injure, submerge, and perhaps drown the weaker larvae.

It is evident from figure 2 that with an exposed food area as extensive as that of the half-pint bottle, reduction of the food supply, even to the point where it formed only a film of food and larvae were eliminated by starvation in large numbers, did not exercise selection adverse to the vestigial type.

Figure 2

The first larvae that hatched consumed the food and pupated; larvae hatching later perhaps never reached a size permitting pupation. Larger supplies of food with the same surface area might increase the yield by 143 per cent, yet have no apparent effect upon the quotient; in fact, other unknown factors have a slightly detrimental effect in the greater depths tried.

B. Selective e f f ec t o f crowdirng

Figure 2 shows the quotients obtained from the various areas, based on the ten-day counts of table 2. The quotients


obtained for the half-pint and pint bottles and 250-cc. Erlen- meyer flasks were practically identical with those derived from the shallower and middle depths of the half-pint milk bottles and the egg-transfer work (to be described later). The increase in area beyond the half-pint bottle did not affect the proportion of wild to vestigial. On the other hand, the vials and 4-ounce bottles exercised a marked selective effect against the vestigial type. The food, as stated before, was 1 inch (25 mm.) deep-a volume large enough in proportion to the exposed area to supply moisture throughout the entire experiment. That is, the effect was very evidently not due to a lack of moisture.

TABLE 5

Effec t of length of egg-laying pet-iod

Cultures Wild-type Vestigial

I 2: 1 33 ~ 34 1 25 ~ 37 I 38 ~ 20 1 55 1 116 1102 1087 1153 2189 1419 1651 1107 570 712

857 1028 942 1352 1432 815 385 525

Viability 1 .56 1 .79 1 .89 ~ .43 I .95 [ .87 1 .74 1 .68 1 .74 C. Duration of egg-laying period

If , under the conditions of these experiments, the difference in the quotients obtained was due to surface crowding of the larvae, then a shorter period of egg laying on the smaller areas, placing a more nearly equivalent number of larvae on each unit of area, should result in approximately the same quotient for the smaller and the larger areas. This experiment was performed and its results are given in table 5 and figure 3. Pairs were placed in vials and 4-ounce bottles for twenty-four, forty-eight, and ninety-six hours (one, two, and four days). Pairs were also placed in half-pint bottles for three, six, and twenty-four hours. The results were striking. The vials and 4-ounce bottles gave for the shorter egg-laying periods a much higher quotient than the half-pint bottles for any period whatever. The quotient for a two-day period of


egg laying in the vials was slightly better than that of the half-pint bottles, while that of the 4-ounce bottles was much the best of all the conditions tested. The quotients for a four-day period for the vials and 4-ounce bottles were practically equivalent to their quotients for a ten-day period of egg laying, and were fa r from the optimum.

Clausen ('24) found a low survival value for vestigial in experiments conducted by his two-day vial method, which he

- .q5 - .YO - .85 -.a0

- 65 -.60 -35 -.50 - .Y5 -.YO I 3 , 6 k 1 2 h EGG LAYING PERIOD /!DAYS

I I I

I I b

Figure 3

attributed to unfavorable cultural conditions. The numbers he gives correspond closely in viability to the values obtained here for vials when the egg laying was of four or more days' duration. The difference is due to the fact that his vials were smaller than the 1-X-4-inch vials used in these experiments, and the amount of food still less in proportion.

Bridges ('21) has pointed out the necessity of having the environmental conditions as nearly optimum as possible to eliminate distortion in the ratios in experimental work.


D. Selective e fec t of comisiency The quotients for the various percentages of agar-agar are

given in figure 2 and table 3. The viability of vestigial ranged from 0.77 for 0.7 per cent agar to 0.89 for 1.96 per cent agar and down again to 0.82 for 4.76 per cent agar. It appears that soft food or extremely hard food was selective against homozygous vestigial larvae as compared with the wild type heterozygous for vestigial.

E. Selective efect of paper The surface acting agent paper had a marked effect in

improving the conditions of survival of vestigial. The results are indicated in figure 2 and table 4. The viability of vestigial improved progressively in this experiment from 0.81 for no paper to 0.91 for 1 gram of paper. It appears from the results that a plateau was probably reached and no further improvement should be expected from the addition of more paper than was used in these experiments.

EFFECTS OF THE MUTANT GENE FOR VESTIGIAL

There is a markedly lower ratio of vestigial to wild flies on the first day of a count. This is followed by a rapid rise in the ratio on the second and third days, for all depths, areas, consistencies, and amounts of paper. If this relation were due to a difference in the available supply of food at the beginning of the hatching period, then starting earlier the yeast culture on the food in the bottle should reduce the difference between the number of wild flies and vestigial flies in the first day's emergence. The results were negative when the food was inoculated with yeast forty-eight hours before the entrance of the flies (table 6).

Egg transfers Since the peculiar ratio on the first day was apparently not

due to a selective environmental condition, an experiment was planned to test whether it was due to a difference in the

THE JOURNAL or EXPERIYENTAL Z O ~ L O O Y , VOL. 53, NO. 2


hatching time of the eggs or to a lag through the larval and pupal period, the wild flies.emerging from the pupal cases earlier than the vestigial flies. The F, females used were of approximately the same age, having emerged within a six- or a three-hour period. These virgin females were back- Grossed to vestigial males and placed in vials. Slips of green blotting-paper soaked in fermenting banana water and partly covered with a film of food were placed on strips of glass and put in the vials (Li, '27). Usually the eggs were laid by the female at random over the surface of the strip of blotting- paper and in the food upon it, but if one end of the strip is pricked repeatedly with a needle, making the surface rough

TABLE 6

The non-efect of early yeasting

Day 9 ' Total Wild-type 1 2146 ~ 3i9 1 3376 1 2:s 1 2i1 16g3 I :g: 1 ::: 1 107 1 1939 Vestigial 115 253 308 161 174 151 , 81 I 1514 Viability 1 .47 1 .79 1 -82 I .75 I .79 1 .99 I .95 1 .86 1 .76 1 .78

and fibrous, the female lays practically every egg in this small area, where the white eggs stand out distinctly against the dark background. The egg laying commenced when the females were forty-eight hours old. During the laying period the slips were removed every three hours and fresh strips introduced. It was necessary to examine the edge of the blotting-paper with a lens before it was removed from the glass. slide, because occasionally an egg was laid in the angle formed by the junction of the paper with the glass. The eggs were counted on the old strip and it was placed on end in a vial containing food. Table 7 shows the eggs produced in each of seven trials. Beginning shortly before the eggs started hatching, these slips were examined hourly and a record made of the number of eggs hatched during the hour. The !&mite, being positively geotropic, crawled down the strip of mtnpaper onto the food in the vial shortly after hatching. The yeast on the paper seeded the food in the vial. In six


124 8

51 8

92 8

109 8

53 7

of the seven trials a record was made of the number of pupae formed. When the flies started emerging, they were counted every twenty-four hours.

466 52

375 60

588 60

678 60

479 59

TABLE 7

Number of females and the eggs they laid in suecersive three-hour periods

4 8

22 8

51 8

74 8

56 7

43 7

41 7

59 7

61 7

67 7

56 9

83 9

109 9

122 9

81 9

65 9

48 9

44 9

107 9

69 9

86 9

196 9

105 9

102 9

113 9

128 9

77 9

88 9

78 9

78 9

88 9

160 9

21 8

19 8

53 8

53 8

10 8

24 8

39 8

83 8

90 8

97 8

124 8

94 8

49 8 89 8

165 8

177 8

96 8

104 7

101 7

99 7

109

101 7

136 7

110 7

r

Table 8 shows the number of hours required for hatching and the number of eggs that hatched during each hour of five trials carried at 25°C. and of one trial at approximately 29°C. The number of hours required for the hatching of an egg laid ddring a three-hour period was calculated as start-


ing at the point an hour and a half after the transfer was made. At the higher temperature the mode was seventeen hours instead of twenty-one hours-a difference of four hours. Whether the higher temperature speeded up one type of egg more than the other is not certain. The distributions are similar and do not show any bimodal indications such as might be expected from a mixed population. From table 8, trials 1 to 5, it is seen that 2794 out of 3754 eggs, or 74.4 per cent, hatched into larvae during a three-hour period (twenty,

TABLE 8

Time in hours between laying of eggs and hatching of larvae

TRIAL

H O U T S

14 15 16 17 18 19 20 21 22 23 24 25 27 30

1

2 7 30 99 219 80 30 4

Total 1 471

~

2

1 2 5 49 160 264 278 83 9 1

3

3

1 40 202 328 430 162 26

2

1

4

4 2 55 153 192 66 11 1

5

5 7 34 141 309 195 48 13

852 1 1195 1 484 1 752

TCYI'AL

8 14 46 271 812 1094 888 494 114 11 1 1

3754

6

2 10 98 435 327 239 119 39 4 2

1275

twenty-one, and twenty-two hours, inclusive) and 3559 out of 3754 eggs, or 94.8 per cent, hatched during a five-hour period. In the case of the eggs hatching at a higher temperature (trial 6, table 8), 1218 out of 1275 eggs, or 95 per cent, hatched within a five-hour period.

Records were kept on the time of emergence of the adult flies, the growth of the larvae being in 1-X-4-inch vials, table 9. On the first day of emergence 2286 flies appeared; on the second, 838, and on the third, only 18 flies. That is, of 3142 flies from eggs laid within a three-hour period, 72.7 per cent


emerged in a single day and 26.7 per cent were delayed a day. The ratio was 1 wild to 0.59 vestigial on the first day and 1 wild to 2.44 vestigial on the second day. This result shows a decided lag in the development of vestigial, sufficient to ac- count for the ratios observed in the daily counts in the cultures of the previous experiments.

TABLE 9

Difference an emergence time of wild and vestigial pies, all eggs laid within a three-hwr period

PIBSTDAY I SECONDBAY 1 TEIBDDAY 1 TOTAL

+ + + + 1442 I 2 1 243 I 5% 1 2 1: 1 1687 I lI!5

1: 0.59 1 1: 2.44 I I 1: 0.86

Total

EWE

707 478 870

1213 707

1336 783

TABLE 10

Summary of egg-tranafer result8

LARVAE - ~-

686 471 852

1195 616

1275 752

___

PUPAE

464 841

1165 601

1261 745

+

310 250 412 556 277 625 3 73

VC4

257 189 292 409 267 553 343

6094 1 5847 I 5077 I 2803 1 2310

V I A B I r n Y

.83

.76

.71

.74

.96

.88

.92

.82

Table 10 summarizes the results of these egg transfers: 96 per cent of the eggs hatched, 94 per cent of the eggs resulted in pupae, and 84 per cent of the eggs produced flies; 46 per cent of the eggs produced wild flies and 38 per cent of the eggs produced vestigial, the ratio being 1 wild to 0.83 vestigial.

Vestigial and the rate of development Further egg-transfer experiments were performed to

determine whether this lag in the development of vestigial


occurred prior to hatching of the egg, during the larval period, in the pupal period, or throughout the entire growth period before the emergence of the adult. A one-hour laying period was used and hourly examinations were made during the hatching period (table 11). With the onset of puparium formation, hourly inspections were made and the puparia recorded (table 12) and removed, each being isolated on a block of gelatin in a separate vial (Bliss, '26). The mean time to the first appearance of abdominal bristles is shown in table 13; and to emergence is shown in table 14.

TABLE 11

Number of eggs hatching during a given hour after laying

HOUR

16 17 19 20 21 22 23 24 25 26 27 28

TBIAL 1

1 1 1

10 93

203 62 1 1 1 1 -

TRIAL 2 TBIAL 3

- - 1

21 253

29 3 1 4 1 1 1

TOTAL

1 1 2

37 522 260

66 3 5 2 2 1

There was no appreciable lag of vestigial during the egg stage. The lag at the time of puparium formation was five hours in trial 2 and six hours in trial 3; to the appearance of abdominal bristles was seven hours in both cases; and to emergence, six hours in trial 1, five hours in trial 2, and seven hours in trial 3. There was, then, an average lag of five and one-half hours at the end of the larval period and only a six-hour difference at the time the adult flies emerged from the pupal cases. It is evident that the retarding action of the mutant gene for vestigial on the rate of development is primarily operative during the larval period, with only a


possible slight effect upon embryonic development and pupal development.

If this effect were limited to the larval period, it might indicate, instead of an action of the vestigial gene on the

TABLE 12

H o w a from laying of egg t o puparium formation

HOUBS

94 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 124 127

Means

Trial 2

+ -

Trial 3

1 1 3 6 22 29 27 20 9 17 11, 5 1 1

1 -

- - - - - - - - -

108.1 1 105.3

VQ

Trial 2 _ _ _ ~

- - - - - - 2 2 2 3 7 5 6 6 10 6 13 12 5 4 2 1

1 -

-

Rial 3

2 - - - - 3 3 8 3 11 30 18 14 17 9 6 9 9 5

2 4 1

1

-

-

I 113.2 I 111.0

rate of growth, a weaker larval type slowed up by too heavy competition for food. The mean time for the completion of the larval period for the 128 individuals where only six or less larvae were in a vial was computed. Their mean time was identical with that of the entire trial.

164 MORRIS HENRY HARNEY

It was found that 97 per cent of the eggs hatched, 95 per cent produced a puparium and abdominal bristles on the

TABLE 13

Hours from hying of e g g to formation of abdominal bristles

HOUBB

171 174 176 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 206 207

Means

- - - - 1 2 10 11 3 4 3 6 10 14 10 10 3 9 5 12 2 4 1 1 1 - - - - - - - 188.2

+ -

Trial 3

1 - - 1 3 4 8 11 9 14 16 12 16 14 18 4 5 6 6 2 1 - - 1 1 1 - - - - - -

186.3

Trial 2

- - -- - - - - 2 1

1 4 2 1 4 2 1 3 8 12 6 8 3 6 9 5 6 1 1

-

- - 1

194.7

.-

Trial 3

- 1 1 - - - - 1 1 2 2 2 5 8 13 13 18 15 9 14 8 5 4 7 10 1 4 2 4 3 1 -

192.9

pupae, and 93 per cent resulted in adult emerged flies. The pupal cases from which flies had not emerged were opened and examined. Three flies had died after pupation and

SELECTION AND POPULATION

- - 1 2 3

1 3

3 1 4 5 9 19 12 17 15 8 7 17 8 6 6 12 7 4 6 5 4 4

1 1 2

-

-

-

- - - - 1 - -

I 214.6

165

VQ

- - - - 1 3 3

3 3 1 7 1 3 10 8 3 6 5

3 4 10 - 3 4 6

2 7 1 1 8 10 1 3 1

9 11 21 5 14 1 1 10 11 12 - 5 10 10 10 3 1 16 9 2 5 7 14 7 10 4 7 11 7 6 3 6 16 9 3 6 4 6 5 ' 2 8 8 3 3 3 14 9 8 - 1 7 2 5 4 - 8 3 10 2 1 9 6 2 - - 11 4 9 - 1 7 5

8 1 3 7 4 4

1 8 3 5

6 1 7 4 1 1 1 5 2 2 4 1 1

1

1

- - - - - - - - -

- - - - -

1 2

- - - -

- - - -

- -

- - - - -

- - - - - -

- - - - - - - - - - -

- - - - - - - - - - - - - - - - - - -

1 - - - - _- - - - - - -

I 209.6 1 207.3 1 221.4 1 215.3 I 214.8

TABLE 14

Eoura from laying of egg to emergence of imugoea

HOUBB

188 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237

Means


before the first appearance of the eye color; in one case failure occurred between the appearance of the head bristles and thoracic bristles; and in nine cases the fly appeared to have completed its development. In four of these nine the pupal case had been broken and at least the head protruded, and, with some individuals, part of the thorax was also freed from the case. In all these cases of dead pupae the fly was found to be a vestigial.

Figure 4

Rate of egg 2ayin.g

The rate at which a female lays eggs is ascertainable from the egg-transfer experiments, where each forty-eight-hour- old female was placed in a vial in which she could lay her eggs on a strip of blotting-paper soaked in fermenting banana juice and partially covered with banana-agar medium. At three-hour intervals this strip was removed, the eggs were counted, and a fresh strip was provided. Figure 4 shows the average yield of eggs for all the forty-eight-hour females during twelve successive three-hour periods. Evidently, the full egg-laying power had not yet been reached, for the curve of


output has risen to fully double the number of eggs per hour at the end of the thirty-six-hour test period when the females were three and a half days old. A second test period of thirty-six hours was started with these same females when they were six days old (fig. 4). The output averaged still higher than in the first period. A third test was started when the females were ten days old. In this last test the yield was on the decline (fig. 4). The total output for a twenty-four- hour period, from 3 P.M. to 3 P.M., was 56, 128, and 64 eggs, for the twoday-, six-day-, and ten-day-old females. The total average output for a ten-day period, from two to twelve days,

. may be estimated from these curves as about 980. I n the egg- transfer experiments 84 per cent of the eggs produced flies.

This value of nearly a thousand flies per ten-day laying period is far in excess of the best output where a female was allowed to lay in a culture bottle for ten consecutive days. But since the viability of vestigial in these egg-transfer experiments was 0.82, the distortion of the observed ratio from the gametic ratio was as great in the transfers as in the ten- day cultures. The optimum relation between area and duration of egg-laying period found was 4-ounce bottles with a two-day egg-laying period, since this condition (fig. 3) gave the least departure from the expected equality of vestigial with the wild type. Judging from figure 2, this distortion would have been even less had the percentage of agar been raised to about 2 or had an amount of paper corresponding to 1 gram per 24 sqxm. been employed.

CONCLUSIONS

1. Increase in the food volume (depth), with a constant area (24 sq.cm.) followed the law of diminishing returns- marked increases in the supportable population followed by minor increases until the optimum was reached (at 22 to 26 mm.). Beyond that point additional food had no effect upon the total population supportable on a given area.

2. The major increases in the total yield due to a greater depth of food were caused by the added reserve supply of

168 MORRIS HEXRY HARNLY

food which carried to full development a greater proportion of the larvae hatching from eggs produced during the latter part of the ten-day period of egg laying. The minor increases were due to a few more flies produced from the early laid eggs as well.

3. The larger the area (depth constant at 25 mm.), the larger the yield up to the optimum at 40 sq.cm. For standard experimental conditions the optimum area per single mated female seems to be closely approximated in the half- pint bottle, since with greater areas mechanical and other difficulties become rather large.

4. With the optimum area of 24 sq.cm. and depth or volume of food at 25 mm., the yield was increased 46 per cent by further stiffening with agar the medium in which the larvae lived. The optimum was at 3.9 per cent agar. A rise of 26 per cent occurred beyond the consistency (1.5 per cent agar) that precluded drowning.

5. In the consistency experiments there was a high yield the first few days. This was followed by a sudden and marked drop to a low yield maintained about two days. There was a subsequent rise to a low plateau which was maintained until the cultures started to run out. A possible explanation of this drop followed by a rise is found in Pearl and Penniman’s ( ’26) observations on pH in the banana-agar medium.

6. Paper worked into the surface of the food by the larvae increased the yield from a given area (24 sq.cm.), volume of food (25 mm. in depth), and consistency (1 per cent agar) by 60 per cent. The optimum was at about 0.75 gram per culture. This combination gave the highest output of flies (446 per culture) of any conditions tried. This, as in the case of consistency, was a physical or mechanical factor in the environment of the larvae.

7. With a constant area (24 sq.cm.) the volume of food had no primary selective power with respect to the two types in the mixed population. Secondarily, due to gassing or other changes in the conditions resulting from an increase in the depth of the food, there was a slight selective action of the greatest depths against the vestigial larvae.


8. A pronounced selective effect of crowding, produced by limiting the area, was found. Shorter periods of egg laying on the same areas demonstrated that this selective mortality was due to larval crowding.

9. Changes in the consistency or hardness of the food had a slight selective effect between heterozygous wild-vestigial and homozygous vestigial larvae. The closest approach to the expected 1: 1 ratio was reached at about 2 per cent agar. Both the softer and the extremely firm food gave slightly greater mortality effects upon vestigial.

10. Paper in the surface of the food improved the viability of the vestigial larvae to a considerable extent. The optimum was at 1 gram per culture, but 0.25 gram gave a marked improvement over none.

11. Temperature had a marked effect upon the length of time required for eggs to develop to the hatching point. At 25°C. the mode for development to hatching of the eggs was twenty-one hours. At approximately 29OC. the mode was seventeen hours.

12. Development of the homozygous vestigial was found to be retarded 5.5 hours during the larval period when compared with the heterozygous wild-vestigial larvae. There was practically no retardation during the embryonic and pupal stages.

13. The mutant gene for vestigial appeared to have a weak- ening effect upon the homozygous vestigial pupae, causing death in a small proportion of cases. The greater part of the mortality of vestigial occurred in the larval stage.


BIBLIOGRAPHY

BLISS, CHFXTER I. 1926 Temperature characteristics for pre-pupal development in Drosophila melanogaster. Jour. Gener. Physiol., vol. 9, pp. 467495.

BRIDOES, CALVIN B. 1921 Gametic and observed ratios in Drosophila. Amer. Natur., vol. 55, no. 636.

CLAUSEN, ROY ELWOOD 1924 The inheritance of cinnabar eye-color in Dro- sophila melanogaster, including data on the locus of jaunty. Jour. Exp. Zool., vol. 38, no. 4.

LI, Ju-CHI 1927 The effect of chromosome aberrations on development in Drosophila melanogaster.

PEARL, RAYMOND 1925 The biology of population growth. Alfred A. Knopf, Inc.

PEARL, RAYMOND, A ~ D W. B. D. PENNIMAN 1926 Culture media for Drosophila. I. Changes in hydrogen ion concentration of the medium. Amer. Natur., vol. 60, pp. 347-357.

Genetics, vol. 12, pp. 1-58.

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An experimental study of environmental factors in selection and population