The winter eggs of Daphnia pulex, after passing safely through the winter , develop and hatch in the spring, multiplying by themselves, while some males emerging among them with the changes in environment produce fertile eggs, which are universally known as winter eggs . This study researches the factors governing the development of winter eggs through experiments

Asai, T.

Nakai, H.

Ogi, K.

English

Aquatic Commons

OGI Kenichi, ASAI Toshio, & NAKAI H. 1951 
Factors influencing the start of development in Daphnia pulex 
winter eggs. 
Biological Reviews Vol. 13, pp. 24 - 26 
Translated by the Central Office of Information. 
I. INTRODUCTION 
The winter eggs of Daphnia pulex, after passing safely through 
the winter , develop and hatch in the spring in numerous ditches and 
pools and subsequently lay summer eggs several times, multiplying by 
themselves, while some males emerging among them wi th the changes in 
environment produce fe r t i l e eggs, which are universally known as winter 
eggs (lasting eggs). These eggs, however, do not always pass safely 
through the winter. Given a suitable environment in the autumn, they 
hatch out in a very Short time, again producing f e r t i l e eggs (winter 
eggs). Occasionally, at this period of varied generations, a multitude of 
Daphnia pulex may be seen in quite & small pool of water. So f a r , 
however, very l i t t l e investigation has been done in connection with the 
factors governing the development of each separate generation of Daphnia pulex. 
As regards a r t i f i c ia l methods of treatment for in i t ia t ing 
development i n winter eggs, the drying process, temperature treatment 
or sunlight method are said to be the most effective. There are also 
reports by Banta (1921), Geltsoff (1937), Obreshkove (1940) and 
Matsudaira (1943), on the morphological changes during development 
and the breeding of l iving creatures; not one of these, however, gives 
a clear explanation of the factors influencing the s tar t of development 
2. 
in winter eggs. The writers attempted to investigate these factors, by 
giving the eggs various a r t i f ic ia l stimuli, through the spring and 
autumn season of 1950. The methods used for the experiments varied from 
centrifugal force, immersion in water, needle pr ick stimulation, 
drying process and sunlight radiation to chemical stimulation with a 
variety of substances. These experiments, though numerous, have not 
yet produced conclusive resul ts . In the case of the temperature treatment, 
however, a number of interesting resul ts have been obtained which are 
reported below. 
I I . Material and Methods. 
Daphnia pulex winter eggs collected from pools in the I n s t i t u t e ' s 
compound, were used as material. The hard protective shells of the eggs 
were f i r s t removed with a needle and then the eggs placed in a glass 
container for the experiments. The eggs were subjected to 
(1) sudden changes of temperatures (2° - 60°C); 
(2) cold conditions by putting them i n the refrigerator 
(3) maintenance at different temperatures (9° - 33°C) 
within the scale suitable for living creatures, and 
(4) treatment with alternating temperatures. 
The apparatus used for the experiment consisted of a thermostatically 
controlled water tank and a refrigerator. All the eggs that began 
to develop were identified within 2 or 3 days after treatment. The 
water temperature at the pools from which the eggs had been collected 
was constantly taken during the experiments for comparative reference 
3. 
in assessing the results of the experiments. 
III. Results of the experiments 
A) Sudden changes of temperature: the eggs collected from the 
pool (water temperature about 25°C) were put in the water in the glass 
container, in groups of 40 and were immersed in water at 40°C, 50°C., 
and 60°C,. for 1, 2 , 3 , 4 and 5 seconds respectively and then released 
into an indoor wa te r tank ( a t approx. 25°C) and examined. Although 
the experiments were repeated several times, the resu l t s were almost negative. 
Only a few among those treated for 2 or 3 seconds a t 50°C showed any 
signs of development and no significant differences were recognised 
compared with the control group (untreated eggs). Experiments were 
made with groups of 40-120 eggs, the treatment being of the same type 
as in the previous case, with the sole difference of immersion in 5 
stages a t 11.6°C ± 1.5°C, 21°C ± 0.8°C, 25°C ± 0 .2°C, 28°C ± 0 .2°C, 
35°C ± 0.2°C for 3 to 4 days; the results showed no development 
i n any of the contrasting groups. The l a t t e r experiment was carried 
out in the beginning of May (water temperature about 17°C) and the 
eggs especial ly selected for th i s experiment were res t r ic ted entirely to 
those produced in the spring of the year. 
B) Low temperature treatment: eggs collected in the middle of 
June (water temperature about 19°C) were placed in the refrigerator at 
2°C-5°C for 5 - 1 5 days and subsequent ly in the t h e r m o s t a t i c water tank 
at 23°C. This experiment was repeated 5 times, but the result only 
showed a few cases of i n i t i a l development and no significant difference was 
observed on comparison with untreated eggs. No greater success was 
achieved with the treatments given in reverse order, i.e.20° - 25°C followed 
4. 
by cold. 
c) Alternate low and high temperature treatment: the eggs were 
treated alternately at low (2°C - 5°C) and high temperatures (23°C) 
respectively for 1-15 days. For comparative purposes others were kept 
a t 2°C - 5°C and 23°C respectively throughout the whole period of the 
exper iments . These experiments were repeated several times. As a result 
although the eggs remained undeveloped at both the low and high temperatures, 
some degree of development was observed in the case of alternate treatment. 
Table I shows the test results . In th is Table, the number of developed 
eggs, indicates those identified af ter the second alternate low and 
high t e m p e r a t u r e treatment. No change was noticed, however, i n those 
returned, after treatment, to the outside pools. 
Table 1. Experiment with low and high 
temperatures repeated every 48 hours. 
D) Experiment at optimum temperature for development af ter . 
low temperature t reatment : 
As approximately 10% development was observed in the previous 
experiments, especially among eggs that received treatment a t various 
5. 
temperatures after low temperature treatment and including those 
accidentally developed through setae defects in the arrangement dating the 
low temperature treatment of B, i t appears possible that temperature has 
some effect upon the development of the eggs. I t i s furthermore now 
known from the resu l t s that there i s an optimum temperature for this 
development and, that th is optimum temperature i s in the region of 
12°C. Accordingly experiments were carried out on the basis of this 
hypothesis; i.e., the eggs treated a t low temperature (2° - 5°C) for 5-7 
days were kept a t 9° - 14°C for 1 - 4 days. The resul ts were 
remarkably good. Table 2 shows the r e s u l t of the experiment when 
the eggs were placed a t the optimum temperature (9° - 14°C) after 
5 days of low temperature treatment. In the case of those receiving the 
low temperature treatment for 6 - 7 days, the result proved to be almost 
the same as i n Table 2. The number of developed eggs shown in the Table 
was based on observations up to the second day after treatment and no 
change was noticed for several days afterwards. 
Table 2, 
Low temp. treatment only . . . . . . . Development ra t io 0% 
I ) Relative comparison of low optimum and high temperature treatments. 
As the eggs used in the previous experiments were almost a l l produced 
naturally i n the pools and collected from them, i t may be reasonably 
assumed that some might well have been produced this spring and had 
therefore, not passed a winter, or some might have been produced 
last year and have passed a winter but were unable t o develop in the 
s p r i n g . Would i t have been possible to separate these and, 
i f so would i t have made any difference to the results when they were 
treated at various temperatures? To se t t l e such queries some 
experiments were made towards the middle and end of September with 
eggs placed at the optimum temperature after the low temperature treatment 
for 4 - 6 days and with others placed at the optimum, high and low 
temperatures only. the resul ts are shown i n Table 3. An experiment, 
apart from these, t o apply the optimum temperature only was made a t the -
end of July, when treatment for 1 - 4 days at 9° -14°C was t r ied . 
All produced similar good resul ts . 
6. 
For the a r t i f i c i a l incubation of the winter eggs of Daphnia pulex, 
the drying process i s reported by many exper t s as giving the best resul t s . 
In the case of insect eggs, sunlight radiation i s also said to promote 
development. There i s furthermore a method based on the s ta te of 
inertia; taking those and other a r t i f i c ia l self-procreative processes 
IV. Observations. 
Developed eggs observed on the 3rd day or the optimum 
temperature treatment. 
No change in undeveloped eggs for several weeks afterwards. 
TABLE 3. 
7. 
applicable to many other forms of animal l i f e into consideration, the 
w r i t e r s attempted many and various mechanical, chemical and physical 
methods of stimulation, but all in vain as was stated before. I t i s 
nevertheless fe l t that there are many aspects which require further 
research. On the point of temperature, however, some conclusive 
resul ts have been obtained, as reported previously. That an 
optimum temperature i s necessary in order to stimulate development and 
that this op t imum temperature i s in the region of 12°C i s in agreement 
with the natural development conditions of the winter eggs. In 
experiment A, the eggs were places a t temperatures in the 11° - 60°C 
range, but scarcely any showed signs of d e v e l o p m e n t . This shows that 
as a direct stimulus for the development of the eggs, a simple application 
of temperature variation has no effect on the eggs. i t also supports the 
fact t h a t , even if the eggs from wa te r a t 25° - 27°C in July, August and 
September are placed at a temperature as low as 2° - 5°C , this does not 
necessarily constitute a s t i m u l u s for their development. Again, it i s 
scarcely probable that a mere process of refrigerating - wintering 
under natural conditions - would promote development or that direct 
transfer of wintered eggs into a high temperature such as the summer water 
temperatures of 23° - 27°C would have such an effect. In considering the 
resul t s of the alternative treatment of low and high temperatures i n 
experiment 0 , although, i t i s true that climatic conditions in the spring 
and autumn to the natural world change constantly and water temperatures 
go up and down in these seasons, such variations i n temperature ere 
not thought to be in the direct cause for the development of winter eggs. 
8. 
On the other hand, the experimental resul ts showed a small number of 
undeveloped eggs to have been, stimulated by one cause or anothar 
during the experiments. Such development may have been stimulated by 
the optimum temperature conditions for development (stated below) which 
were by chance created during the al ternate treatment. In the case of 
experiment D, the two stage treatment, f i r s t at low temperature and 
then a t optimum temperature of about 12°C, i s the direct and important 
factor for the development of winter eggs. Furthermore, in th is 
experiment, when the eggs were treated at low temperature for 5 days 
and regained at the optimum temperature for over 2 days, the results 
showed a great increase in the number of developed eggs, almost 100%. 
Exac t l y the same tendency was found in the case of low temperature 
treatment for 6 or 7 days in the experiments contacted in similar manner, 
so that a l i t t l e over one day may be considered as the (shortest period 
for the duration of the optimum temperature treatment. Moreover, 
there was an instance i n one experiment when only a small number of 
eggs developed when the period of the optimum temperature treatment was less 
than one day. For the duration of the optimum temperature treatment, 
therefore, over one day is considered to be necessary, but a minimum 
two day period i s probably the b e s t . On th is question of the basic 
time limits for low temperature and optimum temperature treatment, 
further investigation requires to be made. In experiment E, no 
difference in development was observed between eggs that received the 
two stage treatment of low and optimum temperatures and those receiving 
the optimum temperature treatment only. In each case, the development 
was 100%. As s i m i l a r experiments since July produced exactly the same 
9. 
10. 
resu l t s , the low temperature might be considered unnecessary. But it 
cannot be definitely discarded as unnecessary, in view of the case in 
experiment A (early May) in which eggs treated at 11.6° - 1.5°C 
temperatures did not develop a t a l l . The difference between the two 
cases was probably influenced by the season of the experiments. That 
i s to say, the A experiment was i n early May and the eggs used were a l l 
winter eggs, definitely produced that spring in the experimental room 
and consequently these winter eggs, immediately after production, may 
have required a period of dormancy. The existence of such a dormant 
period has often been discussed and the o p t i m u m temperature treatment 
after the dormant period may probably cause the ini t ia t ion of development. 
If so , can the low temperature treatment be substituted for the dormant 
period? To se t t l e these and o t te r points , experiments are in progress 
and reports will be made at a l a t e r date. 
1) Naturally produced winter eggs of Daphnia pulex are placed in 
w a t e r i n a glass container, after removal of the protective she l l s , 
and treated a t varying temperatures. The following are the results 
obtained. 
2) No development i s observed in the winter eggs after temperature 
variation treatment only. 
3) No development after low temperature (2° - 5°C) treatment only. 
4) 100% development i s obtained with eggs treated a t o p t i m u m 
temperature (about 12°C) for more than two days after low temperature 
treatment. 
5) Eggs develop with o p t i m u m temperature treatment only after the 
lapse of a definite period of time. 
6) Whether the low temperature treatment can be substituted for the 
lapse of a definite period) remains to be set t led by farther research. 
(Toyokawa Branch, Nagoya University; 
Okazaki Higher Normal School) 
11. 
Reference Literature 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Notice 
 
 
Please note that these translations were produced to assist the scientific staff of the 
FBA (Freshwater Biological Association) in their research. These translations were 
done by scientific staff with relevant language skills and not by professional 
translators. 
 
 
 


Factors influencing the start of development in Daphnia pulex winter eggs [Translation from: Biological Reviews Vol. 13, 24-26, 1951]

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Factors influencing the start of development in Daphnia pulex winter eggs [Translation from: Biological Reviews Vol. 13, 24-26, 1951]

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