66 Chech»acz et al.

Archives of Insect Biochemistry and Physiology 38:66–73 (1998)

Suboptimal Temperature-Dependent Changes in the Brain

Development and Activity in Galleria mellonella Larvae
Magdalena Chech»acz,1 Joanna Michalik,2 and Bronis»aw Cymborowski3*
1
Department of Neurophysiology, Nencki Institute of Experimental Biology,
Polish Academy of Sciences, Warsaw, Poland
2
Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, Warsaw, Poland
3
Department of Invertebrate Physiology, University of Warsaw, Warsaw, Poland

The development of Galleria mellonella larvae is strongly af-

fected by suboptimal temperature (18°C). One-day-old last-in-
star larvae react to 18°C with the arrest of further development
for several months described as facultative larval diapause. The
aim of this study was to find what type of changes, if any, in
the brain correlate with the larval diapause induced by sub-
optimal temperature. Morphological analysis demonstrated the
gradual inhibition of brain development. Paraldehyde-fuchsin
(PAF) staining revealed cyclicity in the activity of the medial
neurosecretory cells (M-NSC) in the larval brain. SDS-PAGE
was used to examine the brain proteins of larvae reared at
30°C and at 18°C. The rate of protein synthesis in the brain of
the last instar larvae kept at 18°C, measured as L-[35S]methio-
nine incorporation during 2-h incubation in vitro, was only
about 40% of the value characteristic for this tissue during nor-
mal development (at 30°C). Despite decrease in the rate of to-
tal protein synthesis, suboptimal temperature induced an
increase in the level of two major brain proteins: 112 and 84
kDa. In SDS-PAGE analysis, these two proteins appear 21–28
days after transfer to the lower temperature. Whether these
proteins are specific for induction of larval diapause of Galle-
ria mellonella remains to be further investigated. Arch. In-
sect Biochem. Physiol. 38:66–73, 1998. © 1998 Wiley-Liss, Inc.

Key words: Galleria mellonella; suboptimal temperature; developmental ar-

rest; larval diapause; neurosecretory cells; brain proteins

INTRODUCTION Contract grant sponsor: State Committee for Scientific Re-

The effect of temperature on insect growth
and development is one of the most interesting
and thoroughly studied aspects of insect physiol-
ogy. Numerous experiments have demonstrated
that the wax moth Galleria mellonella (Lepi-
doptera, Pyralidae) is a very convenient model for
studying temperature-mediated changes in insect
biology. The special role of temperature as one of
the strongest factors affecting development and
search; Contract grant number: 6P04B01113 (to J.M.); Con-
tract grant number: 6PO4C03815 (to B.C.).
Abbreviations used: PAF, paraldehyde fuchsin; JH, juvenile
hormone; M-NSC, medial neurosecretory cells; L-NSC, lat-
eral neurosecretory cells
*Correspondence to: Bronis»aw Cymborowski, Department
·
of Invertebrate Physiology, Warsaw University, 93 Zwirki i
Wigury St, 02-089 Warsaw, Poland; E-mail: bron@asp.
biogeo.uw.edu.pl.
Received 14 July 1997; accepted 16 February 1998

© 1998 Wiley-Liss, Inc.


metamorphosis of G. mellonella is the result of
its unusual habitat in beehives characterized by
lack of great fluctuations in the ambient tempera-
ture during the year. Furthermore, G. mellonella,
a species reared at a stable and optimal tempera-
ture (30°C) during long years of laboratory selec-
tion, became very sensitive to any temperature
changes. Previous experiments have demon-
strated that exposure of G. mellonella last-instar
larvae to low temperature (0°C) and to subopti-
mal temperature (18°C) cause different effects
(Cymborowski, 1991). More detailed studies have
indicated that two separate endocrine mecha-
nisms affecting development are induced by low
temperature stress (chilling stress) and by subopti-
mal temperature. It has been shown that exposure
of the early last instar larvae to low temperature
stress causes supernumerary moltings (Cymborow-
ski and BoguÑ, 1976), induced by rapid and persis-
tent increase in the juvenile hormone (JH) titer
(BoguÑ and Cymborowski, 1981). Further studies
showed that the rapid increase in the JH titer is
due to high allatotropic activity exhibited by the
brain of chilled larvae (BoguÑ and Cymborowski,
1984). Although chilling stress can cause super-
numerary moultings, suboptimal temperature
causes prolongation of the last instar, depending
on the age at which the larvae were placed at
18°C (Miko»ajczyk and Cymborowski, 1993). The
most strong and profound impact of suboptimal
temperature has been observed when 1-day-old
last instar larvae were transferred from optimal
conditions to 18°C. When G. mellonella larvae are
reared at 30°C, the last instar lasts about 8 days,
and pupation occurs on the ninth day, but at 18°C
the last instar can persist for more than 1 year.
However, some percentage of the larvae break
spontaneously their diapause after 3–4 weeks at
the lower temperature (Ðmietanko et al., 1989).
Transfer of 1-day-old last instar larvae to 18°C
results in developmental arrest at the stage of
the spinning larva and causes a marked delay in
pupation. This developmental arrest, described as
facultative larval diapause (Miko»ajczyk and
Cymborowski, 1993), is the consequence of low
ecdysteroid titer and high titer of JH in the lar-
val hemolymph due to changes in the neuroendo-
crine system (Muszy½ska-Pytel et al., 1993). The
transfer of these larvae from 18°C back to the
optimal temperature of 30°C results in the re-
sumption of development and synchronous pupa-
tion within 4–7 days (Ðmietanko et al., 1989). G.
mellonella is not the only known species in which
development can be affected by suboptimal tem-
Brain Response to Suboptimal Temperature 67
perature. Prolongation of the instar length caused
by temperature changes has been previously de-
scribed in other insects species: Dermestes lar-
darius (Jacob and Fleming, 1980), Manduca sexta
(Reynolds and Nottingham, 1985). In these cases
the observed delay in development could be ex-
plained by the effect of lower temperature on the
rate of growth and metabolic processes.
Because the brain is the center of the neu-
roendocrine regulatory system, as well as the di-
rect recipient of environmental stimuli, we decided
to find what type of changes in brain development
and activity correlate with the arrest of develop-
ment (larval diapause) observed at suboptimal
temperature.
MATERIALS AND METHODS
Galleria mellonella larvae were reared in
constant darkness at 30°C (optimal temperature)
on an artificial diet (Sehnal, 1966). Larvae were
kept at 30°C up to the first day of the last larval
instar. Thereafter, they were transferred from
30°C to 18°C (suboptimal temperature) and used
for experiments after different times spent at this
temperature (from 1 day to 84 days). Some ex-
periments were performed on last-instar larvae
reared continuously at 30°C (controls). The brains
of water-anesthetized larvae were dissected out
into an insect saline and used for experiments
described below.
Morphological and Histological Analysis
The maximal length and width of the larval
brain were determined by light microscopy using
an ocular micrometer. Shape of the brain was cal-
culated by subtraction of its maximal length from
its maximal width. Each determination was per-
formed on 10 individuals and repeated 3–4 times.
The paraldehyde-fuchsin (PAF) histological tech-
nique (Dogra and Tandan, 1964) was applied to
demonstrate the degree of accumulation of the
neurosecretory material in the medial neurosecre-
tory cells of larval brain.
Measurement of Protein Synthesis In Vitro
Brains (10 for each determination) washed
with saline were placed in 50 µl Grace’s medium
(Grace’s insect T.C. Medium; Gibco-BRL, Paisley,
Scotland) supplemented with 49.5 µCi L-[35S]-
methionine (specific activity 1175.0 Ci/mmol;
NEN Products, Boston, MA) and incubated for 2
h at 30°C. After incubation, the medium was
collected, brains were homogenized and both
68 Chech»acz et al.
samples were used for measuring incorporation
into proteins. The amount of [35S]methionine in-
corporated into protein was determined using the
boiling trichloroacetic acid method (Mans and
Novelli, 1961). The rate of protein synthesis was
calculated from the radioactivity incorporated into
TCA precipitable proteins. Each measurement of
protein synthesis was repeated 2–3 times.
Protein Electrophoresis and Densitometry
Dissected brains (20 brains for each sample)
were homogenized, mixed with buffer pH 6.8
(0.125 M Tris-HCl, 10% β-mercaptoethanol, 20%
glycerol, 4% sodium dodecyl sulfate (SDS), 0.05%
bromophenol blue) and placed in a boiling water
bath for 5 min. Proteins were separated by 10%
SDS-polyacrylamide gel electrophoresis (PAGE)
(Laemmli, 1970). Gels were run at 150 V/gel,
stained with Coomassie blue, destained with ace-
tic acid/methanol. Molecular weights of proteins
were calculated by comparison to protein stan-
dards (Low Range, Bio-Rad Prestained SDS-
PAGE standards; Hercules, CA). The relative
amount of individual brain proteins was deter-
mined by scanning the protein bands by Phar-
macia LKB densitometer and using GelScan XL
2.1 computer analysis system.
RESULTS
Effect of Suboptimal Temperature on Brain
Development
The exposure of 1-day-old last instar larvae
to 18°C causes the developmental arrest at the
stage of the spinning larva referred to as larval
diapause. Therefore, we wanted to determine how
suboptimal temperature influences the brain de-
velopment, with respect to its size and shape. We
found that suboptimal temperature disturbed the
normal progression of brain development. When
the last-instar larvae were reared at the optimal
temperature (30°C), the brain increases in size
(about 80%) and also undergoes profound changes
in its shape (Fig. 1A). The brain increases in length
from about 0.1 mm at day 1 to about 0.8 mm at
day 8, whereas its length increases only margin-
ally when the larvae were reared at 18°C (Fig. 1B).
In addition to that, only insignificant changes in
size (about 20%) of the brain was observed when
the larvae were kept at 18°C. The most intriguing
finding was that the brain of larvae kept for 28 days
at suboptimal temperature had the same size and
shape as the brain of 5-day-old control larvae reared
at optimal temperature. It seemed that only at the
end of fourth week at 18°C further development of
the brain occurred in these larvae. There appears
to be a correlation with change in brain shape and
levels of 84- and 112-kDa proteins (see below and
cf. Figs. 1 and 4). It should be also stressed that at
the same time 15–20% of diapausing larvae re-
sumed spontaneously their development and pu-
pated (data not shown).
Amount and Distribution of Neurosecretory
Material in the Neurosecretory Cells
In both groups of last instar larvae kept at
30°C (1-day-old and 7-day-old controls) and the
experimentals (kept at 18°C for 7, 28, and 84
days), eight cells situated in the pars inter-
cerebralis (i.e., medial neurosecretory cells M-
NSC) were visible after PAF staining. Using the
histochemical method of Dogra and Tandan (1964)
we demonstrated the changes induced by subop-
timal temperature in the neurosecretory material
in the M-NSC (Fig. 2). During development at op-
timal temperature, the amount of stainable ma-
terial increased gradually and the spherical
neurosecretory granules, characteristic for the
brain of 1-day-old last-instar larvae, formed
larger, spherical groups in the cytoplasm of M-
NSC. In the brains of larvae maintained at the
suboptimal temperature the neurosecretory
material accumulated into large masses. The
cytoplasm of medial neurosecretory cells of lar-
vae kept for 7 days at 18°C contained various
amounts of highly concentrated and randomly dis-
tributed neurosecretory granules within the cell.
In larvae that spent 28 and 84 days at subopti-
mal temperature, the cytoplasm of these cells
was completely filled with dark neurosecretory
masses. The neurosecretory cells of larvae reared
at 18°C with different degrees of accumulation of
stainable material are shown in Figure 2C–E. It is
very interesting to note that in some larvae main-
tained for 84 days at suboptimal temperature, the
amount of neurosecretory material decreased to very
low level (Fig. 2F). The lateral neurosecretory cells
(L-NSC) were very weakly stained and visible only
in the brain of 1-day-old last instar larvae (controls)
and larvae which spent 28, 56, and 84 days at 18°C
(data not shown).
Effect of Suboptimal Temperature on Protein
Synthesis In Vitro
Transfer of 1-day-old last-instar larvae from
optimal temperature to suboptimal temperature
resulted in a gradual decrease in the rate of pro-

tein synthesis in vitro (Fig. 3). At the end of the
first day after placing them at the suboptimal
temperature (18°C), the amount of protein syn-
thesis by the larval brain was much lower (22.3%)
than in larvae growing at optimal temperature
(30°C). The amount of protein synthesized in
brain tissue after 28 days at 18°C was only about
40% of the value characteristic for normal devel-
opment. The rate of protein synthesis was same
on day 56 also, i.e., after spending an additional
4 weeks under conditions that caused develop-
mental arrest (Fig. 3).
Electrophoretic and Densitometric Analyses
of Brain Proteins
The proteins in the homogenates of brains
from larvae kept at 30°C and 18°C were sepa-
Brain Response to Suboptimal Temperature 69
Fig. 1. Changes in size (line ± SD) and
shape (columns) of the brain of Galleria
mellonella last-instar larvae during devel-
opment. A: At 30°C (control). B: At 18°C
(suboptimal temperature). Size, maximal
width of the brain (mm); shape, expressed
as the result of subtraction of the maxi-
mal length (mm) from the maximal width
(mm) of the brain. Each determination was
performed on 10 individuals. Asterisks (*),
statistical significance determined by
Student’s t-test (P < 0.05); 0, first day of
last larval instar, i.e., the time of transfer
from 30°C to 18°C.
rated by SDS-PAGE. There was no significant
difference in the profiles of the majority of the
brain proteins between larvae reared at opti-
mal and suboptimal temperature (Fig. 4A). Two
bands (112 kDa and 84 kDa) were the major
brain protein in both groups of larvae. Profound
changes occurred in the level of these proteins
in normal, as well as in arrested larvae (Fig.
4B). In controls, these two proteins showed two-
fold increase on day 5 and day 7 of the last lar-
val instar. Whereas in diapausing larvae a
similar increase was observed on day 21 and
day 28 after transfer of the last instar larvae
to suboptimal temperature. There were also mi-
nor changes in the relative levels of a 53-kDa
proteins (not shown). The densitometric and
computer analysis of the intensity of staining
70 Chech»acz et al.
Fig. 2. Medial neurosecretory cells (M-NSC) of Galleria
mellonella brain stained with paraldehyde-fuchsin (PAF). M-
NSC in the brain of 1-day-old (A) and 7-day-old (B) last-
instar larvae reared at 30°C (controls), the last-instar larvae,
which spent at 18°C (suboptimal temperature) 7 days (C),
of 112- and 84-kDa protein bands showed that
transfer of larvae from 30°C to 18°C caused an
increase in the level of these proteins on days
21–28 (Fig. 4B). Between days 21 and 28, about
28 days (D), and 84 days (E). (F): Medial neurosecretory
cells in the brain of larvae kept at 18°C for 84 days, which
spontaneously break diapause. Arrows, M-NSC. A–F: Scale
bar = 25 µm.
15–20% of larvae resumed their development
spontaneously and pupated in spite of influence
of the diapause-promoting temperature condi-
tions (data not shown).

Fig. 3. Rate of protein synthesis in vitro by the brain of
Galleria mellonella last-instar larvae reared at 18°C for dif-
ferent periods. Data are presented as percentage of the value
measured for 1-day-old last instar larvae kept at 30°C, i.e.,
before the transfer of the larvae from optimal to suboptimal
temperature (±SD). The tissue (10 brains for each determi-
nation) was incubated in 50 µl Grace’s medium with 49.5 µCi
35
L-[ S]methionine for 2 h at 30°C. Larval age is indicated by
the number of days spent by larvae at 18°C. The intensity of
protein synthesis calculated for the last instar larvae at 30°C
for 3 days (*) and for 7 days (**) is statistically significant (P <
0.05) as determined by Student’s t-test.
DISCUSSION
According to earlier studies the suboptimal
temperature has a very strong effect on the de-
velopment of Galleria mellonella (Ðmietanko et
al., 1989). When day-one-last-instar larvae were
transferred to 18°C, further development was ar-
rested for several months (Miko»ajczyk and
Cymborowski, 1993).
The aim of this study was to determine the
nature of the changes in development and activ-
ity of the brain, i.e., the center of neuroendocrine
system correlated with the arrest of larval devel-
opment. The results reported here show that the
brain reacted to suboptimal temperature with
gradual arrest of its activity and development.
These gradual changes included a decrease in the
rate of brain protein synthesis in vitro. The rate
of protein synthesis in brain 28 days, after plac-
ing the larvae at 18°C, was only about 40% of
the level characteristic for normal development
(30°C). The decreased rate of fat body protein syn-
thesis, measured in vitro, was previously de-
scribed in many diapausing insects (Venkatesh
and Chippendale, 1986; Chippendale, 1988). Also,
the normal progression of brain development was
blocked by suboptimal temperature. Our studies
on brain development confirmed that exposure to
18°C causes developmental arrest at the stage of
the spinning larva (Miko»ajczyk and Cymborow-
Brain Response to Suboptimal Temperature 71
ski, 1993). We have also found that the brain of
the larvae that spent 28 days (the moment of the
switch over to further development) at 18°C had
the same size and shape as the brain of the nor-
mal five-day-old spinning larvae reared at 30°C.
In controls, the size of the larval brain during
the last instar is associated with the development
of optic lobes that causes marked changes in brain
shape (Fig. 1A). Thus, data presented in Figure
1B indicate that suboptimal temperature halts
the development of optic lobes since no signifi-
cant changes in the brain shape of the larvae
reared at 18°C was observed.
Further, 112-kDa protein and 84-kDa pro-
tein are the major brain proteins in larvae reared
at optimal as well as at suboptimal temperature.
The nature of these proteins and their physiologi-
cal role are unknown and will be further investi-
gated. However, it has been found that transfer
of the larvae from 30°C to 18°C caused a marked
increase in the level of these two major brain pro-
teins on days 21–28. Therefore, it appears that
during diapause the activity of the larval brain
is not completely blocked. Our studies on PAF
staining show that activity of medial neurosecre-
tory cells (M-NSC) induced by suboptimal tem-
perature is manifested by accumulation of the
neurosecretory material during larval diapause
and disappearance of this material after resump-
tion of development (Fig. 2). It is important to
note that in all our experiments 15–20% of dia-
pausing larvae spontaneously escape from dia-
pause 21–28 days after placing them at 18°C, and
resume development and pupate about 10 days
later. The cyclic changes in activity of the brain
neurosecretory cells were also observed during
diapause in Manduca sexta (Denlinger, 1985;
Hartfelder et al., 1994). The cyclicity observed in
activity of the medial neurosecretory cells of lar-
val brain are especially interesting because these
cells are responsible for synthesis and accumula-
tion of neuropeptides, i.e., they are involved in
the control of several physiological processes con-
nected with development and metamorphosis.
Some of the medial neurosecretory cells of G.
mellonella larval brain are known to be the source
of allatotropic hormone (Muszy½ska-Pytel, 1987).
However, the significance of the cyclic changes in
the activity of M-NSC remains obscure until the
function of all these cells is fully elucidated.
Our study demonstrated that the switch over
in the development of G. mellonella larvae, caused
by suboptimal temperature, results in the induc-
tion of brain activity and not in a complete ar-
72 Chech»acz et al.
rest of its development and function. It appears
that the main regulatory mechanism of larval dia-
pause in this species is to maintain a specific
physiological state, which permits a rapid re-
sumption of development within a few days after
raising the temperature from suboptimal to opti-
mal. Furthermore, the cyclic changes found in the
activity of M-NSC and simultaneous changes in
the level of two major brain proteins (112 kDa
and 84 kDa) might be under the control of the
circadian clock as it was postulated for Acheta
domesticus (Cymborowski, 1983), as well as for
Fig. 4. A: SDS-PAGE of brain proteins of
Galleria mellonella last-instar larvae reared
at 30°C (controls) and 18°C (suboptimal tem-
perature). Lanes A–C, last-instar larvae
reared at 30°C: A: 1-day-old larvae. B: 5-
day-old larvae. C: 7-day-old larvae. Lanes
D–J, last-instar larvae reared at 18°C. D: 1
day; E: 3 days; F: 7 days; G: 14 days; H: 21
days; I: 28 days; J: 56 days. Lane S, stan-
dard proteins. Arrowheads, two major brain
proteins: 112 kDa and 84 kDa. The homo-
genate of 20 brains was used for each lane.
B: Changes in the level of 112-kDa and 84-
kDa proteins in the brain of Galleria mel-
lonella last-instar larvae reared at 30°C
(controls) and 18°C (suboptimal tempera-
ture). Content of proteins determined by
scanning the protein bands (proteins were
separated by SDS-PAGE) by Pharmacia-
LKB densitometer, and GelScan XL com-
puter analysis system was used for
quantitation of the intensity of stained
bands. AU*mm- GelScan XL unit denotes
the intensity of staining, i.e., protein con-
tent (±SD).
Galleria mellonella larvae (Cymborowski et al.,
1989, 1991).
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