Báo cáo khoa học: Cause of mortality in insects under severe stress Hitoshi Matsumoto, Kohjiro Tanaka, Hirofumi Noguchi and Yoichi Hayakawa pdf

Cause of mortality in insects under severe stress
Hitoshi Matsumoto, Kohjiro Tanaka, Hirofumi Noguchi and Yoichi Hayakawa
Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan
Mortality in the host armyworm larvae Pseudaletia separata
parasitized by the parasitic wasp Cotesia kariyai was dra-
matically increased when they were simultaneously infected
by the entomopathogen Serratia marcescens. Previous
studies have shown that this strong insecticidal effect is due
to a metalloprotease-like insecticide (MPLI) released from
S. marcescens enterobacter. This study was conducted to
elucidate the exact cause of the mortality resulting from
MPLI.InjectionofMPLIcausedasharpincreasein
hemolymph dopamine concentration followed by elevated
levels of brain dopamine in armyworm larvae. [
3
H]Dop-
amine injected into the hemocoel, was incorporated into the
brains of MPLI-injected larvae to a level eight times greater
than in BSA-injected control larvae. Transmission electron
microscopy showed an obvious decrease in thickness and
density of the brain sheath in insects injected with MPLI.
This was probably due to the MPLI-induced elevation of
hemocyte metalloprotease activities. Further, electron
microscopic and TUNEL staining analyses showed a signi-
ficant increase in apoptotic cells in the brain 12 h after the
injection. Injection of 3-iodotyrosine (a tyrosine hydroxylase
inhibitor) before MPLI completely prevented the increase in
hemolymph dopamine in test larvae and their following
death. From these observations, we conclude that MPLI-
injected larvae may have suffered mortal damage through
increased apoptosis of brain cells caused by an influx of

dopamine from the hemolymph.
Keywords: apoptosis; brain; dopamine; insect; stress.
Most insects are generally short-lived. They may die from
a slight accident or injury. Intense external stress such
as mechanical immobilization or enforced activity some-
times triggers autointoxication, culminating in paralysis and
death [1–3]. For example, fighting between pairs of male
cockroaches (Nauphoeta cinerea) establishes a dominant–
subordinate relationship. Such interactions often kill the
subordinate insect without any visible external damage [4].
This is similar to the social stress found in mammals. It is
well known that male rats in particular show pronounced
dominant–subordinate behavior. Prolonged aggression
produces stress in the subordinate, and ultimately a diseased
state, characterized by a stress syndrome eventually leading
to death. These deaths cannot be attributed to external
damage [5]. Subordinate cockroaches may also die from
physiological changes comparable to those accompanying
stress syndromes in mammals. However, it is worth
emphasizing some notable differences between insects and
mammals: insects (cockroaches) die much more readily than
mammals. This is probably not only due to the difference in
body size but also to something unique in the physiological
systems of insects; they must possess a mechanism that
renders them particularly susceptible to intense stress.
To clarify the mechanism that controls mortality in insects,
we focused on dying parasitized host insects.
Parasitoid wasps never kill their host insects before their
larvae emerge from them. However, when host insects are
infected with entomopathogens such as baculoviruses and

microsporidia before or after parasitization, their premature
deaths have been observed before the wasp larvae have
completed their development [6]. In fact, most host Pseu-
daletia separata larvae die within 3 days of parasitization
by the wasp Cotesia kariyai when they are simultaneously
infected by the enterobacter Serratia marcescens. Previous
studies have shown that this mortality is mainly due to
metalloprotease-like insecticide (MPLI) released by S. mar-
cescens enterobacter [7]. Purified MPLI showed a strong
insecticidal effect with a median lethal dosage (LD
50
) of
13 pmol per larva. In preliminary experiments, we injected
purified MPLI into mice with the same dose per weight to
that used for the armyworm larvae, but we did not observe
any symptoms or disorders.
In this study, we tried to extended these experiments to
elucidate the mechanism by which MPLI kills armyworm
larvae within a few days of injection. Our results indicate
that the dopamine concentration in the hemolymph was
elevated by the injection of MPLI, resulting in influx of
dopamine into the brain through the externally damaged
sheath. At the same time, apoptosis of brain cells was
observed in the test larvae.
Materials and methods
Animals
P. separata larvae were reared on an artificial diet at
25 ± 1 °C with a photoperiod of 16-h light : 8-h dark.
Correspondence to Y. Hayakawa, Institute of Low Temperature
Science, Hokkaido University, Sapporo, Japan 060-0819.

Fax: 011 706 7142, Tel.: 011 706 6880,
E-mail:
Abbreviations: MPLI, metalloprotease-like insecticide; NH
2
-Mec,
7-amino-4-methylcoumarin; A2pr, 2,3-diaminopropionyl; ECD,
electrochemical detection; TUNEL, terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling.
(Received 14 May 2003, revised 25 June 2003, accepted 7 July 2003)
Eur. J. Biochem. 270, 3469–3476 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03745.x
Penultimate-instar larvae undergoing ecdysis 2–2.5 h after
lights on were designated as day-0 last-instar larvae. The
weight difference between P. separata larvae used for
bioassay was limited to within 0.02 g [8].
Chemicals
3,4-[7-
3
H]Dopamine (21.5 CiÆmmol
)1
) was purchased from
Dupont-NEN. Dopamine hydrochloride and 3-iodotyro-
sine were obtained from Nacalai Tesque Inc., Kyoto, Japan.
Fluorescent substrates (NH
2
-Mec-Ac-substrates) were pur-
chased from the Peptide Institute Inc., Minoh, Japan.
Experimental injection
MPLI used for injection experiments was purified as
described previously [7]. MPLI and BSA were diluted with
NaCl/P

i
(8 m
M
NaH
2
PO
4
,1.5m
M
KH
2
PO
4
, 137 m
M
NaCl, 2.7 m
M
KCl, pH 7.2) to 0.1 lgÆlL
)1
(final volume
10 lL) and injected into day-1 last-instar larvae of the
armyworm 8 h after lights on.
A 3-iodotyrosine-saturated solution was made by mixing
 5 mg 3-iodotyrosine with 1 mL NaCl/P
i
. Before injection
of MPLI, 10 lL of the 3-iodotyrosine solution was injected
twice into day-2 penultimate-instar and day-0 last-instar
larvae 6 h after lights on. MPLI was injected 2 h after the
second injection of 3-iodotyrosine.

Biogenic amine assay
The hemolymph sample (20 lL) was collected into a 1.5-mL
microtest tube containing 200 lLice-cold0.2
M
perchloric
acid and homogenized in an Artek Sonic Dismembrator
(10 pulses at 40 W). The supernatant after centrifugation at
20 000 g for 10 min at 4 °C was collected, and a 1-lL aliquot
was analyzed byHPLC–electrochemical detection (ECD) [9].
A dissected brain was placed in a 1.5-mL microtest tube
containing 70 lL0.2
M
perchloric acid, sonicated, and
centrifuged at 20 000 g for 10 min. A 50-lL aliquot of the
resulting supernatant was analysed by HPLC–ECD [9].
The HPLC–ECD system comprised an RP-HPLC C
18
column (Capcell Pak C
18
UG120; 4.6 · 150 mm; Shiseido
Co., Tokyo, Japan) and a coulometric electrochemical
detection system (ESA 5100 A, Bedford, MA, USA) [10].
Radiolabeled dopamine incorporation into brains
3,4-[7-
3
H]Dopamine was diluted with NaCl/P
i
to
10 lCiÆlL
)1

, and injected into larvae 6 h after injection of
BSAorMPLI.After1h,thebrainwasdissected,washed
three times with NaCl/P
i
, and immediately homogenized
with 100 lL0.2
M
perchloric acid. Dopamine was separated
by paper chromatography, and its radioactivity counted
using a liquid-scintillation counter (Aloka LSC-5100).
Microscopic observation
Brains were dissected from test armyworm larvae and fixed
with 2.5% glutaraldehyde and 1% paraformaldehyde in
Pipes buffer (0.1
M
Pipes, 0.05
M
sucrose, pH 7.4) at 4 °C.
Post-fixation and staining was performed in 2% aqueous
OsO4 and 2% uranyl acetate, respectively. The tissue was
embedded in Epon 812 (TAAB Laboratories Equipment
Ltd, Aldermaston, Berkshire, UK) after dehydration. Thin
sections were cut on an Ultracut (Reichert-Jung, Wien,
Austria). For electron microscopy, thin sections were briefly
stained in 2% aqueous uranyl acetate and 0.1% lead citrate
[11]. Micrographs were taken with a JEM-1200EX (Jeol
Ltd) electron microscope.
Assay of metalloprotease activity
Hemolymph was collected into a chilled microtest tube
containing NaCl/P

i
with 0.05% phenylthiourea, and imme-
diately centrifuged at 4 °C for 10 min at 500 g.The
collected supernatant was used as a plasma sample. The
remaining pellet was washed with NaCl/P
i
by gentle
suspension and centrifugation. The pellet was then homo-
genized, centrifuged at 20 000 g for 10 min at 4 °C, and
washed three times with NaCl/P
i
containing 0.05% phenyl-
thiourea. The precipitate after centrifugation was suspended
in NaCl/P
i
and used as the hemocyte membrane sample.
Dissected brains and fat body were homogenized in ice-cold
NaCl/P
i
containing 0.05% phenylthiourea by sonication,
and centrifuged at 20 000 g for 10 min at 4 °C. The pellets
Fig. 1. Dopamine levels in hemolymph (A) and brains (B) of MPLI-
injected larvae. Day-0 last-instar larvae of the armyworm were injected
with 17.4 pmol per larva of MPLI (s)(n ¼ 5–7), or BSA as a control
(d)(n ¼ 5–6), 6–7 h after ecdysis. *Significantly different from control
larval value (P <0.01:Student’st-test). **Significantly different from
control value (P <0.05:Student’st-test). Each point represents the
mean ± SD from the number of determinations in parentheses.
3470 H. Matsumoto et al.(Eur. J. Biochem. 270) Ó FEBS 2003
were suspended in NaCl/P

i
andusedasbrainandfatbody
samples, respectively.
Three metalloprotease substrates (1,NH
2
-Mec-Ac-
Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH
2
; 2,
NH
2
-Mec-Ac-Asp-Glu-Val-Asp-Ala-Pro-Lys(Dnp)-NH
2
; 3,
NH
2
-Mec-Ac-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH
2
)
were solubilized in dimethyl sulfoxide (final concentration
10 m
M
) and used as stock solutions. The reaction mixture
(total volume 190 lL) consisted of 50 m
M
Tris/HCl buffer
(pH 7.5), 0.1
M
NaCl, 10 m
M

CaCl
2
, 0.05% Brij35 and one
of the substrates (10 l
M
) [12,13]. The mixture, without the
tissue samples, was equilibrated at 37 °C for 10 min, and
the reaction was started by adding the tissue sample. The
reaction was terminated after 30 min by adding 20 lLice-
cold 50% (v/v) acetic acid. The release of fluorescent
product was detected at k
ex
328 nm and k
em
393 nm using
a fluorescence spectrophotometer (Shimadzu Co.) [14].
Analysis of DNA fragmentation by terminal
deoxynucleotidyl transferase-mediated dUTP nick end
labeling (TUNEL)
To detect apoptotic neural cells on sectioned preparations,
brains dissected from test larvae were fixed with 4%
paraformaldehyde in NaCl/P
i
and embedded in paraffin.
Apoptotic cells were detected on sections with the In situ
Cell Death Detection Kit POD (Boehringer-Mannheim)
according to the manufacturer’s instructions. All sections
were counterstained with hematoxylin [15].
Results
Hemolymph and brain dopamine levels in MPLI-treated

larval brains
Dopamine is the most abundant catecholamine in the insect
hemolymph and nervous tissues, where it may serve as a
hormone, neuromodulator and neurotransmitter [16,17].
Fig. 3. Morphological changes of the neural
sheath layer of MPLI-injected larval brains.
Brains were dissected from day-0 last-instar
larvae of the armyworm 12 h after injection of
17.4 pmol per larva of BSA (A,C) or MPLI
(B,D), and observed at a magnification
of ·5000 (A,B) and · 20 000 (C,D). Note that
the neurilemma of MPLI-injected larva is
thinner (indicated with bars shown in A and
B) and less dense (indicated with stars as
shown in C and D) than that of control BSA-
injected larva. Further, a gap between the
neurilemma and perineural cells is visible in
MPLI-injected larval brain (as indicated in D
with an arrow).
Fig. 2. [
3
H]Dopamine incorporation into MPLI-treated larval brains.
Brains were dissected from day-0 last-instar larvae of the armyworm
which had been injected with 17.4 pmol per larva of MPLI (closed
bar)orBSA(openbar)andtheninjectedwith10lCi [
3
H]dopamine
per larva. Radioactive dopamine was quantified as described in
Materials and methods. Each bar represents the mean ± SD from
four independent determinations.

Ó FEBS 2003 Death from stress in insects (Eur. J. Biochem. 270) 3471
Because it is also an essential intermediate of N-acetyldop-
amine and N-b-alanyldopamine, which function as tanning
agents for insect cuticle, the integument also contains
dopamine in high concentration. Previous studies have
shown that parasitization by C. kariyai wasps results in
increased dopamine levels in the integument, and that this
tissue secretes dopamine into the hemolymph, thereby
raising dopamine levels there [18,19]. Based on these
observations, we speculated that dopamine concentration
may be increased in the dying armyworm larvae injected
with MPLI. Dopamine levels were measured in the hemo-
lymph and brains of the larvae after injection of MPLI
(Fig. 1). As expected, hemolymph dopamine was increased
within 3 h of injection and reached levels 20–23 times higher
than those observed in control BSA-injected larvae. Brain
dopamine levels were also elevated by the injection and
reached the maximal level  9 h after the injection.
Dopamine incorporation into MPLI-treated larval brains
The delay in reaching maximal brain dopamine level
compared with maximal hemolymph dopamine level sug-
gested that dopamine flows into the brain from the
hemolymph. This was confirmed by the influx of [
3
H]dop-
amine into the brain after its injection into the hemocoel of
MPLI-injected larvae (Fig. 2). Radiolabeled dopamine
incorporated into MPLI-injected larval brain was 7–8 times
more abundant than that observed in control larvae.
Structural changes in MPLI-treated larval brains

The dopamine influx into the MPLI-injected larval brain
suggested that brains in the larvae may be damaged by the
MPLI treatment. To address this, brains of the MPLI-
injected larvae were analyzed by transmission electron
microscopy. Differences in the thickness and density of the
neurilemma are evident in Fig. 3: the neurilemma of the
MPLI-injected larvae is thinner and less dense than that of
the control larvae. Further, the perineural cells, which are
tightly attached to the neurilemma in the control brains, are
slightly separated from the neurilemma in the MPLI-treated
larval brains.
MPLI-induced enhancement of metalloprotease activity
in hemocytes
The structural changes in the brain sheath caused by the
MPLI treatment suggested that the neurilemma matrix
may be degraded by MPLI-induced proteolysis. To test this,
we first confirmed metalloprotease activity of MPLI using
three commercially available fluorogenic peptide substrates
[1,NH
2
-Mec-Ac-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-
Lys(Dnp)-NH
2
; 2,NH
2
-Mec-Ac-Asp-Glu-Val-Asp-Ala-
Pro-Lys(Dnp)-NH
2
; 3,NH
2

-Mec-Ac-Pro-Leu-Gly-Leu-A2pr
(Dnp)-Ala-Arg-NH
2
] [12,13]. Although MPLI hydrolyzed
all three, substrate 2 was hydrolyzed most rapidly (Fig. 4A).
This substrate specificity is similar to that of matrix
metalloproteinase stromelysin 1 [12]. As only 17.4 pmol
MPLI was injected into each test armyworm larva, it is
unreasonable to expect that the injected MPLI could
directly degrade the neurilemmal matrix. Instead, we specu-
lated that the injected MPLI activated other metallopro-
teases. To confirm this, metalloprotease activities in the
hemolymph (plasma and hemocytes), fat body and brain
were determined after injection of MPLI. Unexpectedly, the
hemocyte enzyme activity with substrate 2 was significantly
elevated 6 h after injection of MPLI, but the enzyme
Fig. 4. Metalloprotease activities of MPLI and extracts of various tis-
sues. (A) Enzyme activities of MPLI for three substrates (n ¼ 6); (B)
enzyme activities for substrate 2 in brains, fat body, hemocytes and
plasma 6 h after injection of MPLI (n ¼ 4); (C) time course of enzyme
activity with substrate 2 in hemocytes after injection of 17.4 pmol per
larva of MPLI (s) or BSA (d) into test larvae (n ¼ 4). Each point and
bar represents the mean ± SD for the number of determinations in
parentheses.
3472 H. Matsumoto et al.(Eur. J. Biochem. 270) Ó FEBS 2003
activities in the other tissues were not changed (Fig. 4B).
The substrate 2 hydrolyzing activity was increased approxi-
mately twofold in hemocytes 9 h after injection of MPLI
(Fig. 4C). Further, hemocytes showed broadly equivalent
hydrolyzing activities with all three substrates (data not

shown), indicating that MPLI did not stimulate the sole
metaloprotease in hemocytes after injection of MPLI.
However, there was no increase in the hydrolyzing activity
with synthetic substrates for serine proteases such as Var-
Pro-Arg-NH
2
-Mec, Ile-Glu-Gly-Arg- NH
2
-Mec, Phe-Ser-
Arg- NH
2
-Mec or Gln-Arg-Pro- NH
2
-Mec (data not
shown). Thus, as we speculated, injection of MPLI activated
hemocyte metalloproteases, which contributed to the deg-
radation of the neurilemmal matrix.
MPLI-induced apoptosis of brain cells
The final question is whether MPLI-induced elevation of
brain dopamine levels results in changes in the brain cells.
To examine this, brains dissected from the larvae 20 h after
injection of MPLI were studied by transmission electron
microscopy. Condensed chromatin was observed in the
brain cells of the MPLI-injected larvae (Fig. 5A), suggesting
that the brain cells were undergoing apoptosis as the result
of the MPLI injection. This was substantiated by evidence
that the number of TUNEL-positive cells was increased in
the brains of the larvae 12 h after injection of MPLI.
Prevention of the MPLI-induced dopamine elevation
decreased the insecticidal effect of MPLI

To confirm the contribution of dopamine to the mortality of
insects, we tried to avoid elevating hemolymph dopamine
concentrations after the MPLI injection and observed the
lethality. Prior injection of 3-iodotyrosine, a competitive
inhibitor of tyrosine hydroxylase, completely blocked
the MPLI-induced increase in hemolymph dopamine
(Fig. 6A). We analyzed the effects of this pretreatment with
3-iodotyrosine on the mortality of MPLI-injected insects.
The survival rate of MPLI-injected larvae gradually
decreased soon after the injection, and was only 20% 72 h
after the injection. When the larvae were injected with
3-iodotyrosine before the MPLI injection, none of them had
died 72 h after the injection of MPLI (Fig. 6B). Further, the
number of TUNEL-positive cells in the brain of the
3-iodotyrosine-injected larvae was obviously decreased
12 h after injection of MPLI (Fig. 6C,D). However, the
surviving 3-iodotyrosine-pretreated larvae did not meta-
morphose normally to pupae and died before pupation
(data not shown).
Fig. 5. Transmission electron micrographs (A,B) and TUNEL-staining (C,D) of MPLI-injected larval brains. Electron microscopic observation of
brains of armyworm larvae 20 h after injection of 17.4 pmol per larva of MPLI (A) or BSA (B). Note that MPLI treatment induced obvious
condensation of chromatins (indicated with white arrows). TUNEL staining of brains of the armyworm larvae 12 h after injection of MPLI (C) or
BSA (D). Note that MPLI treatment induced TUNEL-positive neural cells (indicated with black arrows).
Ó FEBS 2003 Death from stress in insects (Eur. J. Biochem. 270) 3473
Discussion
Dopamine plays a fundamental role as a neurotransmitter
in the mammalian central and peripheral nervous systems.
It is closely involved in a variety of important physiological
and behavioral processes such as modulation of motor skills
and higher-order cognitive function [20]. Insects have two

separate pools of dopamine: nervous system and integu-
ment [16,17,21,22]. Dopamine is the most abundant mono-
amine in the nervous system and may serve as a
neurotransmitter and neuromodulator [15,16]. Extremely
high concentrations of dopamine are present in the integu-
ment where it is used as an essential intermediate of cross-
linking agents in cuticle formation throughout insect
Fig. 6. Insecticidal effect of dopamine in hemolymph. (A) Hemolymph dopamine levels in armyworm larvae treated with MPLI and/or
3-iodotyrosine. Day-0 last-instar larvae of the armyworm were injected with 17.4 pmol per larva of MPLI (n ¼ 4) or BSA (n ¼ 4). 3-Iodotyrosine
was administered previously to the test larvae as described in Materials and methods. Hemolymph was collected for dopamine measurement 6 h
after injection with MPLI or BSA. Each column represents the mean ± SD for the number of determinations in parentheses. (B) Survival of larvae
treated with MPLI and/or 3-iodotyrosine. Day-0 last-instar larvae injected with 17.4 pmol per larva of MPLI (n ¼ 10) (m). Larvae pretreated with
3-iodotyrosine before injection with 17.4 pmol per larva of MPLI (n ¼ 10) (h). Larvae injected with 17.4 pmol per larva of BSA (n ¼ 10) (d).
This result was a typical case from four independent experiments, but the probabilities of significant survival difference between 3-iodotyrosine-
treated and nontreated animals were 100%. (C, D) TUNEL-staining of brains of the armyworm larvae pretreated with BSA (C) or 3-iodotyrosine
(D) 12 h after injection of MPLI. Note that 3-iodotyrosine pretreatment decreased the number of TUNEL-positive neural cells. Other explanations
as in Fig. 5.
3474 H. Matsumoto et al.(Eur. J. Biochem. 270) Ó FEBS 2003
development [21,22]. Previous studies indicate that the
dopamine concentration in the integument is about 50 times
that found in the hemolymph. Further, we found that
integument dopamine was secreted into incubation medium
in vitro [19]. Therefore, it is reasonable to expect that
integument dopamine is released into the hemolymph. If
this is true, the large increase in hemolymph dopamine
concentrationinMPLI-injectedlarvae(showninFig.1)
would also be due to its release from the integument.
Even though the dopamine concentration is significantly
increased in the hemolymph, dopamine cannot normally
penetrate the hemolymph/brain barrier because it is thought

that the neural sheath cells comprising this barrier are
selective to the exchange of metabolites and ions between
the blood and the underlying brain in healthy insects
[23–26]. However, once the neural sheath is damaged, as
seen in the MPLI-treated larval brain (Fig. 3), dopamine
can enter the brain through the neural sheath. Therefore, it
is plausible that the increased dopamine in the brain of
MPLI-injected larvae shown in Fig. 1 is due to influx from
the hemolymph. The MPLI-induced increase in brain
dopamine is smaller than the increase in hemolymph
dopamine. However, this difference may be mostly due to
the difference in the rate of dopamine metabolism in the two
tissues: dopamine is metabolized more rapidly in the brain
than in the hemolymph, therefore more dopamine may have
passed into the brain than was measured.
Many studies on dopamine-induced apoptosis of neural
cells as well as culture cells have been published over the last
few years [27–30]. To our knowledge this is the first to
provide evidence of this phenomenon occurring in insects.
The increased numbers of apoptotic neural cells after
injection of MPLI suggests that apoptosis of brain cells may
be the cause of MPLI-induced mortality.
As mentioned above, insects have a vast dopamine pool
in their integuments. These amounts, it would appear, are
enough to actually kill the insects. Therefore, given that
insects can be killed much more readily than mammals by
stress, it is reasonable to propose that the dopamine pool in
insect integument at least partly contributes to this mortal-
ity. We believe that the mechanism of death in insects
injected with MPLI does not only apply to particular cases

such as parasitized insects simultaneously infected with the
entomopathogen, but also to dying insects under severe
stress. Further studies should improve our understanding of
the fundamental role of dopamine in insects as well as the
molecular mechanism of their death.
Acknowledgements
This work was supported by the Program for Promotion of Basic
Research Activities for Innovative Biosciences (Japan).
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