J. Insect Physiol. Vol. 38, No. 11, pp. 877-883,
Printed in Great Britain. All rights reserved
1992
Copyright
0
0022-1910/92 $5.00 + 0.00
1992 PergamonPressLtd
THE EFFECT OF HIGH TEMPERATURE ON
METABOLISM OF MORZMUS
FUNEREUS LARVAE
DURING AN INTERMOULT PERIOD
J. IVANOVI&*M. JANKOVICHLADNI,S. DJORDJEVIC,S. STAMENOVIC
and J. LAZAREVIC
Laboratory of Insect Physiology and Biochemistry, Institute for Biological Research “Siniga Stankovi?,
Belgrade, 29 November 142, Yugoslavia
(Received 6 March 1992; revised 24 April 1992)
Abstract-The
effect of high temperature (35°C) on the metabolism (digestive enzyme
activities, fat body glycogen content, haemolymph trehalose and total lipid concentrations) in
Sth-instar larvae of Morimus funereus reared from hatching on an artificial diet at 23°C
(control) during the intermoult period (7-15 days) has been studied. The high temperature
provoked disturbances in the metabolism (reverisible thermal stress) of larvae. The midgut
amylolytic activity was significantly increased 6 h after treatment. Its fluctuations disappeared
after 72 h and the activity decreased to a negligible value. Up to 24 h, the glycogen content
of the controls showed die1 changes, disrupted at 35°C. The glycogen concentration which was
in inverse correlation with the trehalose concentration, increased from 6 to 72 h of exposure.
Thereafter, glycogen reserves were almost exhausted. With the exception of a decrease in total
lipid concentration observed in treated larvae after 48 h, other results were similar to those
of the controls. The results show that during thermal stress, larvae of M. funereus utilize
carbohydrates prior to lipids. Similar midgut proteolytic activities were observed in both
groups of larvae but in the treated larvae the enzyme activity was significantly lower. The
results are discussed with particular consideration of the modifying effect of die1 rhythms and
developmental processes.
Key Word Index: Thermal stress; Morimus fwereus;
intermoult period; midgut amylase;
midgut protease; fat body glycogen; haemolymph trehalose; haemolymph lipids
INTRODUCTION
Insects, like other living organisms, live under
changeable environmental conditions in which the
intensity of dominant factors and their combinations
vary to different degrees causing reversible or irreversible changes in the metabolism of insects. These
changes cause survival or death of an organism,
affecting the ecological distribution and population
dynamics of insects (Fry, 1971; Calow and Berry,
1989). Regardless of the fact that Selye (1950, 1973)
defined stress as a syndrome of physiological reactions elicited in an organism as a response to the effect
of environmental factors, the mode of action of stress
is yet ,only partly elucidated. Concerning this problem, a small number of studies were carried out on
organisms with wide physiological plasticity such as
plants (Bradshaw and Hardwick, 1989) and insects
(Stemburg, 1963; Cook and Holt, 1974; Davenport
*To whom all correspondence should be addressed.
and Evans, 1984; Woodring et al., 1988; IvanoviC and
JankoviC-Hladni, 1991). Up to now, only sporadic
studies on the differences in the system of physiological responses (hormones, metabolism) of developmental phases of insects to stressful factors have been
conducted (Pipa, 1976; Van Marrewijk et al., 1984;
Woodring et al., 1988; Rauschenbach, 1991; Cymborowski, 1991; Czernysh, 1991).
Our previous investigations on thermal stress in
larvae of the cerambycid beetle Morimus funereus,
derived from a natural habitat, have shown that the
responses of larvae at the level of the neuroendocrine
system and metabolism depend not only on the
intensity of the stress or and the duration of exposure
to it, as suggested by Selye (1973) on a modified
model of general adaptation syndrome, but also on
the phase of development (IvanoviC et al., 1975b), the
phase of the annual cycle (Ivanovii: et al., 1979, 1982)
and the composition of the diet (Ivanovi& et at., 1989;
JankoviC-Hladni et al., 1992).
877
J. IVANOVICet al.
878
TO elucidate the mechanisms of thermal stress in
M. funereus larvae bred from hatching on an artificial
diet at 23°C the system of metabolic responses of
these larvae have been investigated in short-lasting
stress (7 days) induced by a high temperature (35°C)
during the intermoult period (5th instar). Since, after
a 7-day exposure to 37°C all experimental larvae of
M. funereus fed an artificial diet survived (JankovicHladni et al., 1992) it was believed that a temperature
of 35°C would be favourable for conducting the
above mentioned studies.
MATERIALS
AND METHODS
as Specific activities, i.e. for amylase, it was OD
550/750 rim/h and for protease,
it was OD
2801750 rim/h..
Fat body glycogen and haemolymph trehalose
The concentrations
of fat body glycogen and
haemolymph trehalose were determined in individual
larvae spectrophotometrically at 620 nm and 630 nm,
respectively, using the anthrone reaction (Wyatt and
Kalf, 1957).
The glycogen content is expressed in mg glucose/
100 mg fat body, and the concentration of trehalose
in mg trehalose/ml haemolymph.
Insects and treatments
Lipid content
Adults of the cerambycid beetle M. funereus collected from oak in a mixed deciduous forest were
transferred to the laboratory for oviposition. Owing
to the cannibalistic behaviour of the larvae, newlyhatched larvae were kept singly in Petri dishes at
23°C in the dark. In a modified Drosophila diet
(Roberts, 1986) the sucrose content was reduced by
half (i.e. to 3.1%) and 0.5% of powdered multivitamin tablets was incorporated into the diet. Every 3
days the diet was replaced by fresh diet.
Larvae reared at 23°C till the 7th day of the 5th
instar were then divided into three experimental
groups: (a) larvae kept at 23°C (control); (b) larvae
exposed to 35°C from days 7 to 15 after ecdysis; (c)
larvae exposed to 35°C from days 7 to 11 and then
transferred to 23°C for 4 days (15 days after ecdysis).
The larvae were killed 1, 6, 12, 24, 48, 72, 96, 168
and 192 h after the temperature treatment, always in
the morning. Midgut homogenates, fat body and
haemolymph of individual larvae were stored at
-20°C and used in the study of the following biochemical parameters: digestive enzyme activities
(protease, amylase), fat body glycogen, haemolymph
trehalose, and lipid concentrations. For each experimental condition five larvae were employed.
The larval survival rate and the duration of the 5th
larval instar at the above experimental temperatures
were investigated in two additional groups of larvae:
(a) 10 larvae continuously exposed to 23°C (control)
and (b) 10 larvae continuously exposed to 35°C.
Total lipid content was measured in the haemolymph spectrophotometrically
using the vanillinphosphoric acid method (Stone and Mordue, 1980).
It is expressed in pg lipid/p1 haemolymph.
Digestive
Statistical
analysis
All data are presented as mean f SEM. Statistical
significance was determined using the Student’s ttest.
Spline production
The graphs are expressed as splines (slidewrite plus
version 3.10) using a PC.
RESULTS
Survival, duraton of the 5th instar
Survival of the 5th larval instar of M. funereus
exposed to 23°C (control) during the intermoult
period was 100%. When 7-day-old, Sth-instar larvae
were transferred from 23 to 35°C for the next 7 days
(from day 7 to day 15 of the intermoult period) they
also all survived. The intermoult period of the control
larvae lasted 18.4 k 1.7 days. On the other hand, 90%
of larvae exposed to 35°C more than 7 days died
successively between 36 to 52 days without moulting.
Only one larva out of 10 was able to moult into the
6th instar, but died on day 16 after ecdysis. The above
results show that a constant temperature of 35°C was
stressful for the developmental phase studied.
enzyme activities
The amylolytic
and proteolytic activities in the
aqueous midgut homogenates (each midgut separately) were determined spectrophotometrically
according to Bernfeld (1955) and Kunitz (1947)
respectively, under previously described optimal conditions in vitro (Ivanovic et al., 1975a). The total
protein content was measured by the method of
Lowry et al. (1951). Enzyme activities were expressed
Activities
of the digestive enzymes
Amylase. The dynamics of midgut amylolytic activity in Sth-instar M. funereus larvae exposed to 23°C
(control) and/or 35°C from days 7-15 of the intermoult period is shown in Fig. 1. The results indicate
that, in the control larvae, the enzyme activity fluctuated and showed three peaks of activity approximately on the same level (on days 8, 11 and 15). It is
M. funereus thermal
Intermoult
225’
6
,
v,
%!
9
,
OD 560/750nm/l
10
,
11
,
period
12
,
h
stress
879
(days)
Intermoult
13
14
15
I
I
I
16
;i
7
6
S
“’
0
24
46
10
period
(days)
11
12
13
14
15
16
96
120
144
168
192
216
0 23’C Icontrol)
.35’C
0-J
0
24
46
72
Hours
96
120
144
166
192
216
72
Hours
after treatment
Fig. 1. The effect of a stressful temperature (35°C) on
specific midgut amylolytic activity (SAA) in 5th~instar larvae of M. finereus from days 7 to 15 of the intermoult
period. The effect of 35” vs 23°C after 12 h-*(P < 0.05); 1,
72 h-**(I’ < 0.01); 6 h-***(P < 0.001). Each point represents the mean k SEM (n = 5).
after treatment
Fig. 2. The effect of a stressful temperature (35°C) on
snecific midgut uroteolvtic activity (SPA) in Sth-instar larvae of M. &eks
from days 7- to 15 .of the intermoult
period. The effect of 35 vs 23°C after 48 h-*; 12,72 h-**;
168 h-***. Other designations as in Fig. 1.
Fat body glycogen concentration
important to point out that during the first 24 h of the
experiment the amylolytic activity had its highest
value in the evening (20 h). In larvae exposed to 35°C
two peaks of amylolytic activity can be distinguished.
The first peak appeared 6 h after treatment, while the
second one was observed on day 8. Both maxima
were nearly on the same level. It is obvious that the
amylolytic activity of larvae exposed to 35°C shows
the first peak 6 h after treatment which presumably
represents a short-term increase caused by the stressful temperature. The second maximum appearing on
day 8 might be the consequence of a dual effect: the
effects of temperature and developmental processes
since a similar maximum, but at a lower level,
occurred in control larvae on the same day of the
experiment. Afterwards, on day 10 of the intermoult
period the amylolytic activity decreased to a negligible value and it remained at a low level till the end
of the experiment.
Protease. Negligible changes in proteolytic activity
were observed in the control larvae, under identical
experimental conditions as above (Fig. 2). During the
first 24 h of the experiment, the proteolytic activity
decreased gradually, reaching its minimal activity in
the evening (20 h).
It is interesting to mention that in two larvae out
of five, the highest enzyme activities were clearly
expressed on days 10 and 11. On day 15 of the
intermoult period, the proteolytic activity was significantly decreased. During the first 24 h, the stressful
temperature of 35°C also provoked a linear decrease
in proteolytic activity. Under the effect of the above
temperature the levels of proteolytic activity seen on
days 7-11 were lowered when compared to the controls. A decreasing tendency in proteolytic activity
was observed till day 14 of the intermoult period.
In the control larvae (Fig. 3), glycogen concentrations from days 8 to 10 were approximately the
same, but a significant rise in glycogen content was
detected on day 11. This rise was followed by a drop
on days 14 and 15. A 6 h-exposure of larvae to 35°C
resulted in an increase in glycogen concentraton.
Between days 8 and 10, the glycogen content remained almost at the same level. On day 11, the
glycogen concentration significantly decreased and
continued to decrease till day 14 of the experimental
period (Fig. 3).
Haemolymph
trehaiose concentration
In the control larvae (Fig. 4) the haemolymph
trehalose concentration was significantly increased
on day 14. Between days 8 and 14 the concentration
of this sugar was not altered. In the first 24 h of
the experiment, the trehalose concentration of the
Intermoult
7
6
9
10
period
11
I
I
I
I
mg glucose/lOQmg fat body
(days)
12
13
14
15
I
I
I
I
0 23T
16
(COntrOl)
35T
.
;(j--
P
0
I
24
I
I
46
72
Hours
‘\
I ‘\ _I
96
120
I
14%
_-*
**
168
I
192
216
after treatment
Fig. 3. The effect of a stressful temperature (35°C) on fat
body glycogen concentration in Sth-instar larvae of M.
funereus from day 7 to 15 of the intermoult period. The
effect of 35 vs 23°C after 96 h and 168 h-**.
Other
designations as in Fig. 1.
880
J.
Intermoult
7
period
(days)
8
9
10
11
12
13
14
15
I
I
I
I
I
I
I
I
70
mghl
IVANOVI~
16
A 48 h temperature
significant
drop
treatment of larvae provoked a
in lipid content.
A rise in lipid
concentration
was observed at the end of the investigation period, e.g. 96 h after the temperature
treatment (Fig. 5).
0 23’C (control)
.35*c
60 -
et al.
The effect of reexposure to the control temperature
20
1
0
I
I
I
I
I
I
I
I
I
24
46
72
96
120
144
168
192
216
Hours after treatment
Fig. 4. The effect of a stressful temperature (35°C) on
haemolymph trehalose concentraton in Sth-instar larvae of
M. ji&re& from days 7 to 15 of intermoult period. Other
designations as in Fig. 1.
control larvae attained its highest value at 14 h. On
the other hand, the stressful temperature
increased
the trehalose
concentration
in the larvae in the
evening (20 h). Under the effect of the above temperature the trehalose
content
was raised on days 8, 11
and 14, but on day 9 it decreased (Fig. 4).
Haemolymph lipid concentration
In the course of the first 24 h of the experiment,
the
highest lipid concentration
in control larvae (Fig. 5)
observed in the evening (20 h) suggests the existence
of die1 changes in this parameter.
In the above
fluctuations in larval lipid concentration with two
peaks, the lower on day 9, the higher on day 14, were
observed from day 7 to day 15 of the experimental
period. In larvae exposed to 35°C in the first 24 h of
treatment haemolymph lipid concentration was increased in the evening (20 h) also. On days 10 and 11
two similar higher values of lipid concentration were
detected while the highest peak appeared on day 14.
Intermoult
period
In larvae exposed to 35°C until day 11 and then
transferred once again to the control temperature
(23°C) for 4 days, all the metabolic parameters
studied attained the control level except the amylolytic activity which remained significantly decreased
(Fig. 6).
DISCUSSION
The results obtained from a 7-day exposure of the
Sth-instar larvae of M. funereus to a high temperature
(35°C) show significant changes in the metabolic
parameters studied, indicating disturbances in metabolism during the thermal stress. The observed
changes were dependent upon the exposure time to
the stressor: (a) in the course of 24 h of exposure the
high temperature affects mostly the die1 metabolic
changes by shifting the maxima and the minima of
the metabolic parameters studied; (b) during further
exposure to 35°C the changes observed in the
metabolic parameters are linked to the processes of
development.
Data from the literature reveal that during the
primary reactions to stress, either in insects or in
vertebrates, a non-specific reaction takes place
concerning mobilization of energy sources such as
sugars and lipids (Davenport and Evans, 1984; Van
Marrewijk et al., 1986).
(days)
VJ;j
$
0
I
0
0
I
I
I
I
I
I
I
I
I
24
46
72
96
120
144
166
192
216
Hours after treatment
Fig. 5. The effect of stressful temperature (35°C) on haemolymph total lipid concentration ;n 5th~i&tar larvae of M.
finerelcs from days 7 to 15 of the intermoult period. The
effect of 35 vs 23°C after 48 h, 96 h-*. Other designations
as in Fig. 1.
SAA
SPA
GLYC
TREH
LIP
Fig. 6. The effect of reexposure (35”C-11 days, transfer to
23T4
days) on metabolic parameters in Sth-instar larvae
of M. funereus. The response of SAA in larvae transferred
from 35 to 23°C h-*. Other designations as in Fig. 1.
881
M. funereus thermal stress
The results concerning changes in trehalose and
glycogen concentrations at the beginning of thermal
stress in Sth-instar M. funereus larvae show that the
metabolites of the carbohydrate metabolism play a
dominant role during this phase. After a 6 h-exposure
to 35°C larval fat body glycogen content was elevated. This rise was preceded by an increase in haemolymph trehalose concentration
observed in the
evening, 12 h after the exposure of larvae to the
stressor. Hence, it can be concluded that this high
temperature provokes disturbances in the die1 rhythm
of glycogen and trehalose concentrations which are
mutually correlated. This correlation was revealed in
M. funereus larvae during the winter (Ivanovic, 1991)
as well as in some other insect species (Baust and Lee,
1981; Rojas et al., 1992).
An increase in total haemolymph lipid concentration and simultaneously a decrease in fat body
glycogen content was detected at the end of the
experimental period, i.e. on day 14 of the intermoult
period. The rise in haemolymph trehalose and lipid
concentrations and the drop in the fat body glycogen
content were found in both groups of experimental
larvae, the controls and the temperature-treated
larvae. Due to the fact that during this phase of the
intermoult period the changes in the above parameters were similar in both groups of experimental
larvae, one can suppose that they are linked to
developmental processes of the insect.
The significant decrease in haemolymph total lipid
concentration after 48 h exposure of larvae to 35°C
indicates that the lipids are utilized as an energy
source after the carbohydrates. A decline in lipid
concentration was also found in head-ligated Acheta
domesticus during handling stress (Woodring et al.,
1989). In this insect, an increase in lipid concentration
was seen 15 and 120 min after handling. It must be
mentioned that in both experimental groups of M.
_@rereus larvae the hyperlipaemia and the hypertrehalosaemia occurred simultaneously at both temperatures in the evening on days 7 (up to 24 h) and on day
14 of the 5th instar. On the contrary, in starved
Periplaneta americana the total lipid concentration
correlated
negatively
with the trehalose level
(Downer, 1985). A link between changes in midgut
amylolytic activity and those in glycogen and trehalose concentrations
was found in M. jiiereus
larvae during thermal stress. After 1 h exposure to
35°C the amylolytic activity of the larvae was significantly increased reaching the maximal activity of this
enzyme 6 h after treatment. At present, it still remains
an open question as to what might be the biological
significance of the decrease in the activity of both
digestive enzymes in larvae of M. funereus exposed
more than 24 h to the stressful temperature.
A short exposure to 35°C of Sth-instar M. funereus
larvae fed a nutritious artificial diet provoked
changes in the metabolic parameters investigated.
During the return of larvae of M. funereus from
35 to 23°C it was shown that with the exception
of the amylolytic activity, all other metabolic parameters tested approached the control level. Prolonged exposure of larvae to 35°C (after day 14)
resulted in stress-induced suppression of larval development.
It was suggested that in larvae of Aedes aegypti
and Cephus cinctus, the high temperature provoked
a hormonal imbalance caused by the absence of
ecdysone (Mellanby, 1954; Church, 1955). As reported previously, under the prolonged effect of high
temperatures, the Al cell type of protocerebral
neurosecretory cells in larvae M. funereus and other
insect species showed decreased activities, i.e. lower
rates of neurohormone(s)
synthesis and release
(O’Kasha, 1964, 1968; Ivanovic et al., 1975a, 1980;
Jankovic-Hladni et al., 1983; Rauschenbach, 1983,
1991). The capabiity of one larva to moult to the 6th
instar under the effect of high temperature indicates
that there are individual differences in the thermoresistance within the species studied. Many authors
have discovered a polymorphism in the response of
numerous insect species to the effects of unfavourable
factors
(Wigglesworth,
1952; O’Kasha,
1964;
Rauschenbach,
1990). Individual
differences in
the response of M. funereus larvae to the effect of
a high temperature were found at the level of
the organism (thermoresistancy) and at the level
of some metabolic parameters studied (amylolytic
activity, proteolytic activity, glycogen). Furthermore,
in previous studies concerning the characteristics of
midgut amylase (pH) in M. funereus larvae, Ivanovic
and Marinkovic (1970) described individual differences in this parameter in larvae from a natural
habitat.
On the basis of the results obtained it can be
suggested that the complex metabolic response system enables the populations of M. funereus to survive
under extreme natural conditions. Individual variability observed at all levels of biological organization plays a decisive role in the evolution of this
species.
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