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Variations of hydrogen peroxide and catalase expression in Bombyx eggs during diapause initiation and termination.

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A r t i c l e
VARIATIONS OF HYDROGEN
PEROXIDE AND CATALASE
EXPRESSION IN Bombyx EGGS
DURING DIAPAUSE INITIATION
AND TERMINATION
Yang-Hu Sima, Jin-Mei Yao, Yi-Sheng Hou, Li Wang,
and Lin-Chuan Zhao
Department of Life Science, Soochow University, Suzhou, P. R. China
For diapause eggs of the silkworm, Bombyx mori, diapause initiation is
prevented with hydrochloric acid (HCl) at around 20 h post-oviposition
while diapause status is terminated with chilling around 51C. To
investigate whether hydrogen peroxide (H2O2) and catalase expression
are involved in diapause initiation and termination, the concentration of
H2O2, relatively higher levels of catalase mRNA and activity of catalase
were compared between (1) 20-h-old diapause eggs and the HCl-treated
diapause eggs, and (2) 10-day-old diapause eggs and the 51C-chilled
diapause eggs. Compared to diapause eggs, the HCl-treated eggs had
significantly higher H2O2 concentrations (up from approximately
1–3 mmol/g fresh mass to 5–8 mmol/g fresh mass), higher relative level of
catalase mRNA (up from 0 to 35.2%) and higher catalase activity (up
from 2.51 units/mg protein to 4.97 units/mg protein) at 96 h posttreatment. On the other hand, the 51C chilling resulted in significant
increases of H2O2 concentration (up from 0.79 mmol/g fresh mass to
5.57 mmol/g fresh mass), relative level of catalase mRNA (up from 0 to
71.4%) and catalase activity (up from 0.88 units/mg protein to
3.42 units/mg protein) within 120 days. The results obtained in this work
suggest that variations of H2O2 and catalase expression in Bombyx eggs
C 2011 Wiley
are involved in diapause initiation and termination. Periodicals, Inc.
Keywords: diapause eggs; Bombyx mori; hydrogen peroxide; catalase
Grant sponsor: National Natural Science Foundation of China; Grant number: 30571406.
Correspondence to: Lin-Chuan Zhao, Department of Life Science, Soochow University, Suzhou 215123,
P. R. China. E-mail: sdzlc2008@126.com
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 77, No. 2, 72–80 (2011)
Published online in Wiley Online Library (wileyonlinelibrary.com).
& 2011 Wiley Periodicals, Inc. DOI: 10.1002/arch.20422
Hydrogen Peroxide and Bombyx Diapause
73
INTRODUCTION
Hydrogen peroxide (H2O2) functions as a signaling molecule controlling essential
processes, including gene expression, signal transduction and physiological regulation
(Bienert et al., 2006). On the other hand, catalase (H2O2:H2O2 oxidoreductase;
EC1.11.1.6) is solely responsible for the elimination of H2O2, which catalyzes the
degradation of H2O2 to water and oxygen (Felton and Summers, 1995; Switala and
Loewen, 2002). Catalase plays a central role in protecting the oocyte and early
embryos of the mosquito (Anopheles gambiae) from damage due to exposure to oxidized
compounds (Dejong et al., 2007). Moreover, exogenous H2O2 up-regulates catalase
expression of Protaetia brevitarsis, indicating catalase expression can be regulated in
response to variation of H2O2 (Kim et al., 2008).
The silkworm, Bombyx mori, is a typical insect with embryonic diapause (Yamashita,
1996). During diapause initiation, mitotic activity of embryonic cells in diapause eggs
slows down from 24 h and cell divisions finally stop at the G2 phase of cell cycle at
around 84 h at 251C (Nakagaki et al., 1991). However, diapause initiation can be
effectively prevented with hydrochloric acid (HCl) at around 20 h post-oviposition
(Yamashita and Hasegawa, 1985). On the other hand, diapause status is maintained
more than 300 days at around 251C, which can be terminated with chilling at
temperatures around 51C for 60–90 days (Yaginuma et al., 1990). In our previous
studies, we also found that diapause initiation in silkworm eggs was prevented with
exogenous H2O2. We also recorded significant increases in H2O2 concentrations and
in xanthine oxidase, but not catalase activity in diapause eggs chilled at 51C (Shen
et al., 2003; Zhao and Shi, 2010).
In our continuing investigations, we formed the hypothesis that H2O2 and catalase
expression are involved in diapause initiation and termination. Here we report on the
outcomes of experiments designed to test our hypothesis.
MATERIALS AND METHODS
Insects
The bivoltine strain (Dazao) of silkworm, Bombyx mori, was used in all experiments. The
diapause dynamics in this strain are completely controlled by the incubation conditions
experienced during maternal embryogenesis. Diapause eggs were obtained as
described by Morita et al. (2003) and Song et al. (2009). Eggs were incubated at
251C under continuous light throughout embryogenesis. The larvae were reared on
fresh mulberry leaves at 251C on a 12:12 L:D photoperiod. Pupae, adults and laid eggs
were kept under the same light and temperature conditions with larvae. The diapause
condition of eggs was checked 2 weeks after oviposition, by which time all nondiapause
eggs hatched.
Hydrochlorizing
The 20-h-old diapause eggs were hydrochlorized with 6 M HCl at 251C for 90 min to
prevent diapause initiation, as described by Morita et al. (2003). After hydrochlorizing,
the HCl-treated eggs were incubated 251C on a 12:12 L:D photoperiod for two weeks
to check the hatchability, in which three replicates (with an average of 100 eggs per
group) were run simultaneously for each regimen.
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, June 2011
Chilling
Ten-day-old diapause eggs were transferred into 51C for 120 days to terminate
diapause maintenance. After various duration of chilling, eggs were further
transferred into 251C on a 12:12 L:D photoperiod for two weeks to check the
hatchability, in which three replicates (with an average of 100 eggs per group) were
run simultaneously for each regimen.
Concentration of H2O2 Assay
Level of H2O2 in eggs was determined as described by Zhao and Shi (2009), based on
monitoring at the absorbance at 412 nm of the titanium-peroxide complex (binding
between Ti41and H2O2). The absorbance values were calibrated to a standard graph
generated with known content of H2O2.
Relative Amount of Catalase mRNA Assay
Total RNA from silkworm eggs was extracted with Trizol kit (Takara, Japan) and cDNA
was generated from 1 mg of total RNA using RT kit (Takara, Japan) according to the
manufacturer’s protocol. Products of the RT reaction were subjected to PCR
amplification using the forward primer for Bombyx catalase gene (Genebank accession
no.: AB164195): 50 -CTGATACTGTTCGTGACCCTC-30 and the reverse primer:
50 -GATGCCAGTTCACCTGCTTTG-30 . PCR was pre-denatured at 941C for 3 min,
30 cycles (each cycle was composed of incubations at 941C for 0.5 min, 571C for 1 min,
721C for 0.5 min), and extended at 721C for 10 min. Similar conditions were used to
amplify Bombyx actin A3 (Genebank accession no: X04507) using the forward primer:
50 -CAAGGCCAACAGAGAGAAGA-30 and the reverse primer: 50 -GATGGTGATGACCTGACCGT-30 . Linearity of amplification was confirmed up to 30 cycles from
1 mg of total RNA in the reverse transcription step. Specific PCR products obtained for
Bombyx catalase and actin A3 (397 and 415 bp, respectively) were analyzed on a 1.5%
agarose gel in Tris borate/EDTA buffer, visualized by staining with 1 mg/mL ethidium
bromide. The signal intensity of the bands was quantified by densitometric scanning.
Bombyx actin A3 cDNA product was used as a standard to equivalent levels of total RNA
subjected to RT-PCR for Bombyx catalase. To normalize the data obtained for catalase
mRNA, the amount of actin A3 mRNA was measured as internal standard in all
treatments (Niimi et al., 1993; Moribe et al., 2001).
Catalase Activity Assay
Catalase activity was determined as described by Zhao and Shi (2009), based on
monitoring the absorbance at 240 nm of H2O2. The decline of H2O2 was calculated
using an extinction coefficient of 0.043 mM1 cm1 ml1. One unit was defined as the
amount of soluble protein caused by the degradation of 1 mmol H2O2 per min at 251C.
Statistical Analysis
At least three independent biological replicates were performed for each experiment.
Data were expressed as the mean7SD and analyzed statistically using Student’s t-test.
Statistical significance was set at Po0.05.
Archives of Insect Biochemistry and Physiology
Hydrogen Peroxide and Bombyx Diapause
75
RESULTS
Hatchability of Diapause Eggs Treated with HCl and 51C
In this work, no hatching of laid eggs was observed within 14 days post-oviposition at
251C following incubation at 251C under continuous light, indicating that we
successfully obtained diapause eggs. Nearly 100% hatch was observed when 20-hold diapause eggs treated with 6 M HCl at 251C for 90 min. The influence of chilltreatments on hatchability as a function of chilling duration in 10-day-old diapause
eggs is shown in Figure 1. No hatching was observed in diapause eggs after chilling less
than 30 days. However, the hatchability increased from about 20% to nearly 90%
during 45 days to 75 days of chilling and remained at around 90% thereafter.
Variation of H2O2 Concentration
During diapause initiation at 251C, H2O2 concentrations in diapause eggs peaked at
24 h post-oviposition (Fig. 2A). However, relative to diapauses eggs, higher H2O2
concentrations (by approximately 2- to 3-fold) were recorded in the HCl-treated
diapause eggs following incubations from 24 to 96 h. Diapause eggs in the incubation
at 251C had very low H2O2 concentrations from 0 to 120 days, compared to
approximately 315% increases in chill-treated diapause eggs.
Variation of Catalase mRNA
Similar to H2O2, diapause eggs had the peak of catalase mRNA at 24 h (Fig. 3A).
Contrary to H2O2, compared to diapause eggs, catalase mRNA in the HCl-treated
diapause eggs was lower from 24 to 48 h post-oviposition (Fig. 3A). However, catalase
mRNA in the HCl-treated diapause eggs remained around 33% while that in diapause
eggs disappeared from 72 to 96 h post-oviposition. Catalase mRNA was undetectable
in diapause eggs in the incubation at 251C from 0 to 120 days, indicating no
transcription of catalase during diapause maintenance (Fig. 3B). Similar to H2O2, we
recorded marked increases in catalase mRNA in chill-treated diapause eggs (Fig. 3B).
Variation of Catalase Activity
Highest catalase activity was observed in diapause eggs at 24 h post-oviposition at 251C
(Fig. 4A). Compared to diapause eggs, the HCl-treated diapause eggs had lower
catalase activity from 24 to 72 h post-oviposition (Fig. 4A). Catalase activity significantly
Hatchability (%)
100
80
60
40
20
0
-20
-15
0
15
30
45
60
75
90
105
120
Days of chilling
Figure 1. Hatchability of diapause eggs transferred from 51C to 251C after various durations of chilling.
Hatchability was determined within two weeks at 251C after chilling of 51C. Each point represents the mean
values of three different experiments with the SD shown by a vertical bar.
Archives of Insect Biochemistry and Physiology
76
Archives of Insect Biochemistry and Physiology, June 2011
H2O2 (µmol/g fresh mass)
A
8.00
6.00
4.00
2.00
0.00
0
24
48
Hours after oviposition
72
96
H2O2 ( mol/g fresh mass)
B
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
-15
0
15
30
45
60
75
90
105
120
Days of chilling
Figure 2. Variation of H2O2 in diapause eggs of Bombyx mori, (A) diapause eggs (closed circles) and HCltreated eggs (open circles), (B) diapause eggs (closed circles) and chill-treated eggs (open circles).
Concentration of H2O2 was determined using spectrophotometry based on monitoring at the absorbance at
412 nm of the titanium-peroxide complex. Each point represents the mean values of three different
experiments with the SD shown by a vertical bar.
increased in the HCl-treated diapause eggs after 72 h post-oviposition. Catalase activity
in diapause eggs at 251C incubation remained low from 0 to 120 days. However,
catalase activity in chill-treated diapause eggs increased by around 292% within 45
days and remained at high levels (Fig. 4B).
DISCUSSION
In this work, we report that H2O2 concentrations in diapause eggs peaked at 24 h postoviposition. It has been reported that oxygen consumption of silkworm diapause eggs
abruptly decreases at 24 h post-oviposition at 251C (Yaginuma and Yamashita, 1999).
Therefore, the peak of H2O2 at 24 h may be due to the increase of oxygen
consumption. Catalase mRNA levels and catalase activity in diapause eggs peaked at 24
and 72 h post-oviposition, respectively. Catalase is responsible for the catalysis of H2O2
(Felton and Summers, 1995; Switala and Loewen, 2002). Exogenous H2O2 upregulates catalase expression in P. brevitarsis (Kim et al., 2008). We infer from this that
up-regulation of catalase in diapause eggs from oviposition to 24 h may be in response
to increased H2O2 while decline of H2O2 from 24 to 72 h is due to the combined effects
of declining oxygen consumption and enhanced catalase activity.
Time interval measuring enzyme (TIME) is an ATPase that can measure time intervals
by exhibiting a transitory burst of activation of the enzyme in accordance with diapause
Archives of Insect Biochemistry and Physiology
Hydrogen Peroxide and Bombyx Diapause
77
Relative amount of
catalase mRNA (%)
A
350
300
250
200
150
100
50
0
-50
0
24
B
Relative amount of
catalase mRNA (%)
48
72
96
Hours after oviposition
80
70
60
50
40
30
20
10
0
-10
-15
0
15
30
45
60
75
90
105 120
Days of chilling
Figure 3. Relative level of catalase mRNA in diapause eggs of Bombyx mori, (A) diapause eggs (closed circles)
and HCl-treated eggs (open circles), (B) diapause eggs (closed circles) and chill-treated eggs (open circles).
Relative level of catalase mRNA was determined using semi-quantitative RT-PCR. Expression levels were
normalized to the expression of the silkworm actin A3 gene. Each point represents the mean values of three
different experiments with the SD shown by a vertical bar.
development, which requires cold for resumption of embryonic development in the
silkworm. Peptidyl inhibitory needle (PIN) inhibits the ATPase activity of TIME (Kai et al.,
2008). Similar to cold, the treatment of HCl can cause the dissociation of the TIME-PIN
complex (Xu et al., 1999). The natural substrate and the function of TIME in diapause
eggs are not clear. In our previous study, we found that diapause initiation of silkworm
eggs could also be prevented with exogenous H2O2 (Shen et al., 2003). At present, HCl
resulted in significantly higher H2O2 coinciding with the prevention of diapause initiation.
It suggests that H2O2 in diapause eggs may play an important role in diapause initiation.
Oxygen consumption increases gradually five days after hydrochlorizing (Yaginuma and
Yamashita, 1999). Therefore, enhancement of H2O2 in the HCl-treated diapause eggs
from 24 to 72 h may not be due to an increase in oxygen consumption. However,
significantly lower catalase mRNA and catalase activity were observed in the HCl-treated
diapause eggs. It indicates that enhancement of H2O2 coinciding with the prevention of
diapause initiation might have resulted from down-regulation of catalase expression.
Diapause eggs had low H2O2, undetectable catalase mRNA and low catalase
activity during diapause maintenance. It indicates that no transcription of catalase in
diapause eggs during diapause maintenance may be in response to low H2O2. Oxygen
consumption of diapause eggs keeps at very low level (Yaginuma and Yamashita,
1999). Therefore, low level of H2O2 in diapause eggs during diapause maintenance is
related to low level of oxygen consumption.
Chill treatments resulted in the expression of Samui (a cold-inducible gene) and
sorbitol dehydrogenease (SDH) genes respectively at 5–6 days and 40–50 days postoviposition, activation of extracellular signal-regulated kinase (ERK) and mitogen-activated
Archives of Insect Biochemistry and Physiology
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Archives of Insect Biochemistry and Physiology, June 2011
A
Catalase activity(units)
6.00
4.00
2.00
0.00
0
24
48
72
Hours after oviposition
96
B
Catalase activity (units)
4.00
3.00
2.00
1.00
0.00
-15
0
15
30
45 60
75
Days of chilling
90
105
120
Figure 4. Variation of catalase activity in diapause eggs of Bombyx mori, (A) diapause eggs (closed circles) and
HCl-treated eggs (open circles), (B) diapause eggs (closed circles) and chill-treated eggs (open circles). Catalase
activity was determined using spectrophotometry based on monitoring at the absorbance at 240 nm of H2O2.
Each point represents the mean values of three different experiments with the SD shown by a vertical bar.
protein kinase (MAPK) in the yolk cells at 45 days, up-expression of a glycerol kinase-3
gene, and increased phosphorylation of glycogen synthase kinase-3b in diapause eggs
(Niimi et al., 1993; Moribe et al., 2001; Iwata et al., 2005; Fujiwara et al., 2006; Kihara
et al., 2009; Lin et al., 2009). It has been hypothesized that the chill treatments induce a
development-promoting signal and terminates diapause possibly via ROS, which act as
second messengers to activate various signal transduction cascades (Gabbita et al., 2000;
Chiarugi and Cirri, 2003). In this work, chill-treatments resulted in marked increase of
H2O2 in diapause eggs prior to diapause termination. Therefore, increase of H2O2 in chilltreated diapause eggs may play a role in diapause termination. Moreover, marked
increases of catalase mRNA and catalase activity were also observed in chill-treated
diapause eggs. It indicates that up-regulation of catalase in diapause eggs may be in
response to enhancement of H2O2 during chilling. As oxygen consumption does not
significantly increase during chilling (Yaginuma and Yamashita, 1999), the mechanism
underlying increase of H2O2 in chilled diapause eggs remains unclear.
Taken together, the results obtained in this work suggest that variations of H2O2
and catalase expression in silkworm eggs are involved in diapause initiation and
termination.
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