56 P. Kalushkov et al. Journal of Insect Physiology 47 2001 55–61
been already recorded how much P. apterus responds to only short exposures to cold 5–12
° C; diapause was
induced in 30 to 75 of the females in spite of diapause- preventing long days Hodek, 1971b. Similar to
diapause incidence, the intensity of diapause could be affected by low temperatures in this insect.
Our aim was to determine whether a more intense diapause can be induced under conditions simulating
outdoor temperatures, i.e. thermoperiod with low scoto- phase temperature, than at comparable constant tempera-
tures.
Varjas and Sa´ringer 1998 suggest that the level of oxygen consumption is a better criterion of diapause
intensity. Thus the rate of oxygen consumption was also recorded, together with several other parameters: dur-
ation of pre-oviposition period, incidence of oviposition, fecundity, and parameters of cold hardiness.
2. Material and methods
2.1. Experimental animals and treatment The above mentioned question was repeatedly
addressed in two series of experiments I and II. The animals in both series were F1 offspring of adults col-
lected near C ˇ eske´ Budeˇjovice 49
° N, 14
° E, South
Bohemia. The larvae were reared under long day con- ditions 18L:6D, at 25
° C until the mid 3rd instar series
I or mid 4th instar II. They were subsequently placed into diapause-inducing short day photoperiod 12L:12D
and experimental temperatures where they completed larval development and spent four weeks of adult life
before measurements or further transfer see Section 2.2. The highest diapause intensity was recorded at this
age in earlier studies Hodek, 1983. Experimental tem- peratures were: constant temperatures 25 and 20
° C, and
thermoperiod 2515 °
C. 2.2. Preoviposition period and fecundity
After the diapause-inducing treatment, a portion of the diapausing adults were transferred to long day conditions
and constant temperature 25 °
C for photoperiodic acti- vation i.e. diapause development accelerated by
diapause averting photoperiods. Fifty individual pairs were observed daily in series I 35 in series II to record
the time until first oviposition. In series I, the total num- ber of eggs laid until death was counted. The incidence
of ovipositing females was calculated.
2.3. Oxygen consumption The metabolic rate was measured as oxygen consump-
tion manometrically with a Warburg respirometer Slama, 1960. Respiratory vessels of about 10 ml vol-
ume were employed with sodium calcite to absorb car- bon dioxide and with a small piece of wet cotton to
maintain constant pressure of water vapour. The recorded value of oxygen consumption of each individ-
ual at rest represented an average from three 0.5 h read- ings.
Oxygen consumption was measured at experimental temperatures 20 or 25
° C during photophase and 15, 20
or 25 °
C during scotophase one day before transfer to constant 25
° C and long days or short days series II.
After the transfer, both photophase both series and sco- tophase series II measurements were performed at
25 °
C on the day of transfer both series, two days after transfer and at subsequent seven-day intervals series II.
The oxygen consumption was measured 4–8 h after lights on in both series and at subjective midnight in
series II.
2.4. Cold hardiness In adults reared at three diapause-inducing conditions
series I supercooling points were measured according to Nedveˇd et al. 1995 in 16 individuals per treatment.
Survival was observed after exposure to 28.5 °
C for 5 days 30 individuals. Mortality caused by freezing was
then estimated as the proportion of individuals having a supercooling point above 28.5
° C, the rest was attributed
to non-freezing mortality. In series II, cold hardiness was measured as survival
after exposure to subzero temperatures above mean supercooling point. Thirty individuals were exposed to
constant temperatures of 25, 27, or 210 °
C; after 10 days they were transferred to laboratory temperatures
and survival was calculated after 24 h. Lethal tempera- ture for 50 of the animals was computed using logistic
regression Statistica software, nonlinear estimation, Statsoft, 1997; see Nedveˇd et al., 1998. The pairs of
values with their standard error were compared using t
-test.
3. Results
3.1. Pre-oviposition period and fecundity Duration of the pre-oviposition period was signifi-
cantly longer in females induced to diapause by thermo- period than by constant temperatures Table 1, Table 2.
The difference was smaller in series II, where the experi- mental treatment began in older larvae and was thus
shorter. The females reared during diapause-inducing conditions at a lower constant temperature of 20
° C
showed in both series significantly shorter pre-ovi- position periods than those reared at 25
° C.
The incidence of ovipositing females was high and similar in all experiments Table 2 in series II. In series
57 P. Kalushkov et al. Journal of Insect Physiology 47 2001 55–61
Table 1 Parameters of diapause intensity and cold hardiness in Pyrrhocoris apterus, measured after transfer to long days and 25
° C series I
a
Experimental Incidence of
Pre- Fecundity
Oxygen consumption Supercooling Survival Freeze Non-freeze
temperature ovipositing
oviposition eggs
on day 0 µ
lgh point
° C
mortality mortality
° C
females IO period days
25 82
22 ±
3.2 a 234
± 140
279 ±
91 a 211.7
± 4 a
37 23
40 20
62 17
± 4.1 b
152 ±
83 404
± 80 b
212.6 ±
4 a 67
12 21
2515 38
30 ±
4.1 c 271
± 195
446 ±
99 bc 212.4
± 4 a
60 27
13 n
50 50.IO100
50.IO100 20
48 35
a
Duration of pre-oviposition period, incidence of ovipositing females, fecundity, oxygen consumption rate on the day of transfer, supercooling point, survival after 5 days at 28.5
° C, freeze mortality and non-freeze mortality. Standard deviations and indication of different values letters a–
c; α=
0.05 according to Tukey’s HSD test are given. Table 2
Parameters of diapause intensity and cold hardiness in Pyrrhocoris apterus
, measured after transfer to long days and 25 °
C series II
a
Experimental Incidence of
Pre-oviposition Lethal
temperature ovipositing females period days
temperature °
C IO
LT
50
° C
25 62
23 ±
4 b 27.2
± 0.2
20 62
19 ±
4 a 27.6
± 0.1
2515 60
26 ±
3 c 28.1
± 0.2
n 35
50.IO100 3
× 30
a
Duration of pre-oviposition period ±
S.D., incidence of ovi- positing females, lower lethal temperature after 10 days exposure
LT
50
± S.E.M. Indication of different values letters a–c;
α= 0.05
according to Tukey’s HSD test is given.
I, the insects had lower viability. Some of the females reared at the thermoperiod died early after the transfer
into LD, and thus only a lower oviposition incidence was recorded Table 1; in insects from constant temperatures
the incidence was higher.
A great variance in fecundity of P. apterus has always been recorded e.g. Hodek, 1988, and thus the differ-
ences between individual samples are rarely significant. This was also the case here: although the average fec-
undity values were much higher in females reared at higher temperatures Table 1, the difference was not
significant ANOVA.
3.2. Oxygen consumption 3.2.1. Oxygen consumption before transfer Series II
Oxygen consumption rate was measured one day before transfer from experimental temperatures and
short-day conditions. The oxygen consumption rate was always higher at photophase than at scotophase Fig. 1A,
B values on day 21. The scotophase oxygen consump- tion rate was similar in all temperature regimens,
although scotophase temperatures were different 15, 20, 25
° CFig. 1B, values on day 21. In contrast, the pho-
tophase oxygen consumption rate was dependent on the temperature regimen Fig. 1A, values on day 21. Thus
the photophase oxygen consumption rate was higher in bugs reared at a thermoperiod of 2515
° C than in bugs
reared at constant 25 °
C, although the photophase tem- perature was 25
° C in both cases. At constant 20
° C, the
oxygen consumption was similar to that at constant 25
° C.
3.2.2. Oxygen consumption after transfer to constant 25
° C
The insects were transferred from experimental tem- peratures and short-day conditions to 25
° C and either
short-day or long-day conditions. 3.2.2.1. Short-day conditions Series II
The control group kept permanently at constant 25
° C maintained a
similar oxygen rate consumption during the whole experiment Fig. 1A, B. Insects tranferred from constant
20 °
C to constant 25 °
C showed an important transient increase in photophase oxygen consumption rate on the
day of transfer Fig. 1A, values on day 0. On day 2 after transfer, the oxygen consumption rate decreased to
the original level found before transfer Fig. 1A, B. The photophase oxygen consumption rate of insects trans-
ferred from the thermoperiod of 2515
° C did not change
on the day of transfer, but it decreased on day 2 after transfer Fig. 1A. The scotophase oxygen consumption
rate did not change after transfer Fig. 1B. From day 2 after transfer, the oxygen consumption rate remained low
and similar in all groups. The scotophase values Fig. 1B were always lower than the day values Fig. 1A.
3.2.2.2. Long-day conditions Series II Conditions on
days 21 and 0 were identical for insects transferred to short-day and long-day conditions. Therefore the oxygen
consumption rate shown for days 21 and 0 in Fig. 1 transfer to short-day conditions are identical with those
given in Fig. 2 transfer to long-day conditions. The oxygen consumption rate in all groups gradually
increased under long-day conditions. The increase was
58 P. Kalushkov et al. Journal of Insect Physiology 47 2001 55–61
Fig. 1. Oxygen consumption rates in females of P. apterus measured
under short-day conditions and experimental temperatures and after transfer to short-day conditions and 25
° C series II. Measurement at
photophase A, at scotophase B. Experimental temperatures: con- stant 25
° C squares, 20
° C circles, thermoperiod of 2515
° C
triangles. Arrows indicate the day of transfer. Females were trans- ferred during photophase or scotophase from 20
° C and during photo-
phase from 2515 °
C. Each point represents mean ±
S.E.M., n =
20. Twelve comparisons were made among the data plotted in Fig. 1 and
Fig. 2. The significance level was thus adjusted from 0.05 to 0.0042. There was a significant difference in the starting values day 21, or
day 0 in bugs from 25 °
C measured during the day photophase, uncovered by ANOVA F
= 7.4, P
= 0.0014. Tukey’s HSD test indicated
the value in the bugs from thermoperiod 2515 °
C as different from the other two conditions 20
° C, 25
° C. There was no such difference
in the starting values measured during night scotophase F =
1.89, P
= 0.16. There was a significant increase in oxygen consumption rate
from day 21 to day 0 in the bugs from 20 °
C in measurements held during photophase F
= 29, P
= 4.10
26
but not during scotophase F =
7, P
= 0.01. There was a significant decrease in oxygen consumption rate
from day 21 to day 2 in the bugs from thermoperiod 2515 °
C in measurements undertaken during the photophase in short day: F
= 20,
P =
5.10
25
; in long day: F =
26, P =
10
25
, but not during scotophase in short day: F
= 4.3, P
= 0.045; in long day: F
= 1.6, P
= 0.22. There was
not a significant difference between the three treatments on the day 2, in both photoperiods and day phases in short day, photophase: F
= 0.4,
P =
0.68; scotophase: F =
0.3, P =
0.77; in long day, photophase: F =
2.6, P
= 0.08; scotophase: F
= 5.8, P
= 0.0052.
most delayed in the group reared at the thermoperiod of 2515
° C Fig. 2A, B.
In series I, the oxygen consumption rate was only measured on the day of transfer to long-day conditions
and constant 25 °
C. The oxygen consumption rate was lowest in insects kept continuously at 25
° C Table 1.
Fig. 2. Oxygen consumption rates in females of P. apterus measured
under short-day conditions and experimental temperatures and after transfer to long-day conditions and 25
° C series II. For explanations
and statistics see Fig. 1.
3.3. Cold hardiness The supercooling point was similar in all three treat-
ments of series I ANOVA. Survival after 5-day cold exposure differed among treatments but there was no
clear relation to the intensity of diapause measured by the other variables. The samples reared under the therm-
operiod or constant 20
° C had lower proportions of non-
freeze mortality compared to those from constant 25 °
C Table 1.
The lethal temperature, LT
50
, was similar Table 2 in all three conditions of series II. However, the values
were different at the 5 level t-test of pairs of treatments. The lethal temperature of diapausing insects
was correlated P =
0.04 with the night temperature dur- ing diapause induction.
4. Discussion
