EFFECTS OF TEMPERATURE ON FERTILIZATION
I N HABROBRACON
R. L. ANDERSON
Johnson C. Smith University, Charlotte, North Carolina and
The Marine Biological Laboratory, Woods Hole, Massachusetts
Received February
XI,
1936
INTRODUCTION
I
N THE parasitic wasp Habrobracon juglandis (Ashm.), when a cross of
unrelated stocks is made, males (haploid) come from unfertilized eggs
and females (diploid) from fertilized eggs. When the parental stocks are
closely related, not only males from unfertilized eggs but also males (diploid) from fertilized eggs appear. That such biparental males should be
produced was puzzling until WHITINGbrought forth his theory of sex
determination in the Hymenoptera (1933a, 1933b, 1935).
According to this theory females have the sex chromosomes X and Y,
while haploid males have either X or Y and biparental males either two
X's or two Y's. One might then expect equal numbers of biparental male
and female offspring according to the principle of random fertilization.
However the number of biparental males is always much smaller than
that of the females. This deviation from equality WHITINGhas explained
in part by differential mortality, in part by differential maturation. When
parental stocks are unrelated differential maturation is complete, all
zygotes being XY and resulting in females only. Due to the greater viability of females in contrast to that of diploid males, hatchability of eggs
and viability of offspring is much greater when parents are unrelated. This
greater fecundity together with lack of biparental males, necessitates the
supplementary hypothesis of differential maturation.
It has already been shown (WHITINGand ANDERSON
1932) that temperature also affects the production of biparental males among progeny
from crosses of related stocks. A much higher percentage of males among
biparentals was produced when mothers were set a t high temperature
(3oOC) than a t low (2ooC), while the percentage of biparentals, males
and females, among total offspring was decreased.
After BOSTIAN(1934) produced an orange-eyed stock ( I 1-0) closely
related by grading to a certain wild-type stock (11) and giving very high
percentages of males among the biparentals, it was shown (ANDERSON
1935) that if stock 11 males were reared a t different temperatures ( 2 0 ° , 30°,
36°C) even though the stock 11-0 females were kept a t constant temperature (3ooC), significant differences appeared in percentages both of males
among biparentals and of biparentals among total offspring. In this case
GENETICS21: 467 July 1936
468
R. L. ANDERSON
the differences must have been due, not to differential viability of heterosyngamic (XY), homeosyngamic (XX and YY) and unfertilized (X and
U) eggs, but to change in percentage of eggs fertilized and of type of
fertilization.
EFFECTS OF CULTURE TEMPERATURES
Preliminary experiment. Females of stock 11-0 and males of stock 11
were mated a t room temperature and the females were then divided into
three groups. One group was set at 32'C, a second a t 27'C, and a third
at 2oOC. As usual the females were transferred through vials a, b, c, d
and e when larvae appeared. This was every four days for the 32OC and
the 27°C groups, but every ten or twelve days for those a t 2oOC. No transfer was made to a vial e in the low temperature group.
As in our previous experiments, the higher the temperature the greater
is the production of males among biparentals while percentage of biparentals is reduced (table I). I n all three groups (table 2) there is a
gradual rise of males among biparentals from vials a through b to c and
a slight drop from c to d. I n the high temperature groups a very significant
rise occurs from vials d to e.
TABLEI
Stock
11-0females
by stock
males: preliminary experiment. Totals and percentages *withstandard
errors for the different culture temperatures.
II
PERCENTAQE OF
GROUP
TEMPERATIJRES
BIPARENTAL
00
$8
IMPATERNATE
$3
PERCENT-
MALES
AMONQ
DIFFERENCES
RIPAR-
AQE OF
BIPAR-
DIFFERENCES
ENTAL0
ENTAM
A
B
c
32OC
2%
2ooc
703
1112
356
242
281
51
489
25.615
1.42
792
20.17t
155
1.07
12.565
1.64
A-B 5 . 4 4 5
1.78
B-C 7 . 6 2 5
1.97
A-C13.05+
2.22
63.75t
1.27
65.90+
1.01
72.445
1.88
A-B 2 . 1 5 k
1.62
B-C 6 . 5 4 5
2.14
A-C 8 . 6 9 k
2.27
Egg-counting experiment. As a further check on temperature effects, an
experiment was undertaken in which eggs were counted according to
methods developed by ANNAR. WHITINGand by C.H. BOSTIAN.In much of
the tedious technical work involved in this research the writer was greatly
assisted by Miss Roger Young, to whom he wishes to express his sincere
gratitude.
Stocks 11 and 11-0 were used and matings were made at room temperature of males and females reared a t 30°C. Sister females weremated
to the same males, half of the females mated to each male being set at
469
FERTILIZATION I N HABROBRACON
lower temperature (18~--zo~C)
and half a t higher temperature (3z0-33’C).
The females were given caterpillars that had been stung by wasps with
mutant factors other than orange and the paralyzed caterpillars were also
carefully examined and freed from any eggs observed that were laid by
the “foster mothers.” The females were then set in the incubators.
For the first four days each wasp was given one stung caterpillar each
day. On the fifth day each female was given two caterpillars. The number
of eggs laid by the females a t high temperature was greatly increased by
this increased feeding, but the additional caterpillar had no effect a t the
TABLE
2
Biparental ojspring from stock 11-0females by stock 11 males: preliminary experiment. Summaries
and percentages according to culture temperatures and ages of mothers (vials).
TEMPERATURE
VIALS
3 2OC
a
b
‘87
d
I49
63
e
52
22
267
288
299
I 66
92
55
62
87
46
31
a
I12
12
b
I37
41
66
16
9
I4
27°C
C
d
Totals
6767
56
86
55
23
C
20°C
BIPARENTAL OFFSPRINQ
99
252
566
676
489
295
I44
123
I 64
151
83
53
PERCENTAQE OF MALES
AMONQ BIPARENTAL8
23.04
25.44
26.96
26.74
29.80
17.08
17.7’
22.52
21.69
25.20
9.64
10.46
18.00
17.50
17.85
19.52
23.59
21.96
26.90
lower temperature. An increase to three caterpillars made no noticeable
difference in the number of eggs laid at either temperature. Each caterpillar was placed on a glass slide in the desired position and covered by
a small dish containing the female wasp. When observations were made
the slide was put on a glass stage of a low power binocular microscope
under which was placed a mirror so that the eggs on the under surface could
be counted by focussing down on the image. I n no case was it necessary
to move the caterpillar and thus endanger the results by shaking off
the eggs. All egg counts were made in rooms held a t temperatures to correspond to those of the incubators in which the eggs were being developed.
470
R. L. ANDERSON
The eggs and the larvae for the high temperature group were counted
every day until pupation occurred. Eclosion at this temperature took
place seven and a half days after the wasps were set with the caterpillars.
For the cool temperature group the eggs were counted on the first day
after setting and the females were transferred to new caterpillars. At this
low temperature the larvae did not begin to appear until the fourth day.
In general if the eggs had not developed into larvae by the sixth day, they
failed to develop at all. The larvae were then counted on the fifth and
sixth days and checks were made on them every other day until eclosion.
Eclosion began about the twenty-seventh day.
In the cool temperature material after the seventh or eighth day, there
were noticed several larvae which failed to increase in size and soon died.
Although such inviable larvae were not found in the high temperature
material developing at a rate almost four times as fast, they may nevertheless have been present. The higher percentage of eggs producing larvae
and the lower percentage of larvae producing pupae recorded for the low
temperature material (table 3) may be thus explained.
TABLE
3
Stock 11-0females by stock 11 males: egg-counting experiment. Totals, fecundity averages and viability
percentages with standard errors at different culture temperatures.
Temperatures
Number of females
Days set
Eggs
Larvae
Pupae
Biparental females
Biparental males
Impatemate males
Egg production per day
Percentage of eggs producing larvae
Percentage of larvae producing pupae
Percentage of pupae producing adults
Percentage of eggs producing adults
Percentage of eggs producing females
Percentage of eggs producing biparental males
Percentage of eggs producing impaternate males
Percentage of males among biparentals
Percentage of biparentals
32-33OC
I1
92
I839
I 284
1048
428
152
32'
20
-
69.82 k I . 0 7
81.62 f I . 78
84.06f I . 13
48.98f I . 16
23.22 f0.97
8.26f0.72
I 7.45 0.87
26.21k1.83
64.37 1.59
*
18-20°C
14
284
I990
1624
1227
532
67
508
7+
81.61f0.87
7 5 . 2 5 k I .OS
90.59 f0 . 85
55.63+ I . 1 1
26.73 f0.98
3.37 f0.47
25.53 f 0 . 9 8
11.18f1.28
54. I1 fI . 50
In this experiment we find the percentages of males among biparentals
at high and at low temperatures to be approximately the same as in our
preliminary experiment. The percentages of biparentals are reversed,
however, being higher in the high temperature group than in the low.
This is probably due to the smaller number of impaternate males surviv-
FERTILIZATION IN HABROBRACON
471
ing a t the higher temperature, percentage of eggs producing impaternate
males. The high temperature, being slightly higher than in the preliminary
experiment, may have reduced the viability of the males as has been previously shown (WHITINGand ANDERSON
1932).
The percentage of eggs developing into biparental adults, both males
ana females, is but slightly higher (31.48 percent) in the high temperature
material than in the low (30.10 percent). Nevertheless the percentage
developing into biparental males is almost two and one half times as great.
Evidently homeosyngamic fertilization rather than heterosyngamic is
favored by higher temperature. If it be supposed that higher temperature
is differentially deleterious to the males among the biparentals, then
homeosyngamy must be favored even more than the percentages indicate.
WHITING(1935) suggested sex-reversal of the XX or YY combinations
into females as an alternative to the hypothesis of differential maturation
bukdid not think this at all probable, nor did he suggest any explanation
for such a reversal. SNELL(1935) proposed a multiple chromosome hypothesis according to which XX or YY might develop into females if
heterozygous for any other sex-chromosome pair, ZW for example. The
facts herewith reported cannot be explained by the multiple chromosome
hypothesis since the material used was genetically homogeneous.
SUMMARY
A preliminary experiment with crosses of closely related stocks of
Habrobracon showed an increase in males among biparental offspring but
a decrease in biparentals as culture temperatures were increased. At all
temperatures percentages of males among biparentals changed significantly with increasing age of mothers.
An experiment in which eggs were counted showed that a t the higher
temperature the percentage of eggs producing impaternate males was
decreased. Among biparental off spring males were increased and females
correspondingly decreased.
The conclusion is drawn that increased temperature increases mortality
of males and increases homeosyngamy (male-producing combinations,
X with X or Y with U) a t the expense of heterosyngamy (female-producing, X with U).
LITERATURE CITED
ANDERSON,R. L., 1935 Offspring obtained from males reared a t difEerent temperatures in Habrobracon. Amer. Nat. 69:183-187.
1934 Biparental males and biparental ratios in Habrobracon. Biol. Bull. 66:166BOSTIAN,C. H.,
181.
SNELL,GEORGED.,1935 The determination of sex in Habrobracon. Proc. Nat. Acad. Sci. Wash.
21; 446-453.
472
R. L. ANDERSON
WHITING,P. W., 1933a Sex determination in Hymenoptera. The Collecting Net, Woods Hole, 8:
113-121,122.
1933b Selective fertilization and sex determination in Hymenoptera, Science 78: 537-538.
1935 Sex determination in bees and wasps. J. Hered. 26: 263-272.
WHITING,P. W. and ANDERSON,
R. L., 1932 Temperature and other factors concerned in male
biparentalism in Habrobracon. Amer. Nat. 66: 420-432.