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Local expression and distribution of a storage protein in the ovary of Hyphantria cunea.

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Archives of Insect Biochemistry and Physiology 48:111–120 (2001)
Local Expression and Distribution of a Storage Protein
in the Ovary of Hyphantria cunea
Hyang-Mi Cheon,1 Hong-Ja Kim,1 Duck-Hwa Chung,2 Myeong-Ok Kim,1 Joong-Suk Park,1 Chi-Young Yun,3
and Sook-Jae Seo1*
1
2
Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, Korea
Division of Applied Chemistry and Food Science and Technology, College of Agriculture, Gyeongsang
National University, Chinju, Korea
3
Department of Biology, Taejon University, Taejon, Korea
Storage protein-1 (HcSP-1) is a major storage protein found in
the hemolymph and fat body of Hyphantria cunea. HcSP-1 has
a high methionine (6.0%) and low aromatic amino acid content
(8.5%) (Cheon et al., 1998). In this study, the accumulation and
expression of HcSP-1 in ovary was investigated using biochemical and immunocytochemical methods. HcSP-1 was detected
in the ovaries in 6-day-old pupae and accumulated toward the
end of pupal life, when HcSP-1 transcripts were detectable by
Northern blot analysis and RT-PCR. In situ hybridization
showed that the HcSP-1 mRNA was located in the nurse cells
and follicular epithelial cells, but not in the oocyte. Though
most of the HcSP-1 that is incorporated in the yolk bodies of
the oocyte is probably sequestered from the surrounding
hemolymph, HcSP-1 is an important yolk protein contributing
to early yolk body formation before the development of patency
by the follicular epithelium. Arch. Insect Biochem. Physiol.
48:111–120, 2001. © 2001 Wiley-Liss, Inc.
Key words: local expression; storage protein; ovary; Hyphantria cunea
INTRODUCTION
In holometabolous insects, storage proteins
represent a major protein component of the larval hemolymph (Wyatt and Pan, 1978; Levenbook,
1985; Roberts and Brock, 1981). These proteins
are synthesized in large quantities by the fat body
of actively feeding larvae and are released into
the hemolymph. Other larval cell types including
the midgut (Palli and Locke, 1987a), epidermis
(Palli and Locke, 1987b), pericardial cells (Fife
et al., 1987), and testis (Miller et al., 1990) also
synthesize and export storage proteins, but their
proportional contribution to the total hemolymph
complement is probably rather small.
The larval hemolymph of the fall webworm,
© 2001 Wiley-Liss, Inc.
Hyphantria cunea accumulates two forms of storage protein termed HcSP-1 and HcSP-2 (Kim et
al., 1989). HcSP-1 has a relatively high methionine (6.0%) and a low aromatic amino acid content (8.5%) (Cheon et al., 1998), but does not
Contract grant sponsor: Brain Korea 21 project.
Abbreviations used: RT-PCR = reverse transcription-polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate
polyacrylamide gel electrophoresis; SP = storage protein.
*Correspondence to: Sook-Jae Seo, Division of Life Science,
College of Natural Sciences, Gyeongsang National University, Chinju, 660-701, Korea. E-mail: sookjae@gshp.gsnu.ac.kr
Received 24 October 2000; Accepted 4 June 2001
112
Cheon et al.
exhibit sexual dimorphism (Kim et al., 1989, Song
et al., 1997). It has been suggested that high-methionine hexamers of lepidopterans play a role in
egg formation because of their greater abundance
in females than males (Bean and Silhacek, 1989;
Ryan et al., 1985; Tojo et al., 1980). The synthesis or expression of a storage protein in the ovary
has not been previously reported. Therefore, the
goal of the present study was to use Northern
blot hybridization, RT-PCR, Southern blot, and
in situ hybridization to determine whether the
HcSP-1 gene is locally expressed in the ovary. Finally, we considered the relationship between oogenesis and locally expressed SP-1 in H. cunea.
MATERIALS AND METHODS
Animals
Fall webworms, Hyphantria cunea, were
reared on artificial diet at 27°C and 75% relative
humidity with a photoperiod of 16 h light:8 h dark.
Ovary Preparation
Ovaries were dissected from 6-, 8-, and 10day-old female pupae and adults, and were
cleaned of attached fat body cells. It was not practical to analyse ovaries from stages earlier than
6-day-old pupae. The samples were homogenized
in Ringer’s solution (150 mM NaCl, 1.8 mM CaCl2,
1.3 mM KCl, 10 mM Tris, pH 7.0) using a Dounce
glass-Teflon homogenizer (Seo et al., 1998). The
homogenates were centrifuged at 10,000g for 30
min at 4°C. The supernatant was removed and
stored at –70°C.
Electrophoresis
SDS-PAGE of ovarian samples was performed according to the method of Laemmli (1970)
using a 12.5% separating slab gel. All samples
were heated at 90°C for 9 min in the presence of
2% SDS and 5% 2-mercaptoethanol. Gels were
stained with Coomassie blue following completion
of electrophoresis.
Western Blot
Following SDS-PAGE, proteins in the gel
were electrotransferred to a sheet of nitrocellulose (0.45 µm, Bio-Rad, Hercules, CA) according
to the procedure of Towbin et al. (1979). The blots
were blocked in 20 mM Tris-HCl pH 7.6, 137 mM
NaCl, and 0.2% Tween-20 (buffer A) containing
5% nonfat dry milk, and then incubated with antiserum against HcSP-1 (Seo et al., 1998) at a
1:1,500 dilution in buffer A. After washing in
buffer A, the blots were incubated with horseradish-peroxidase-conjugated goat anti-rabbit IgG
(1:3,000) in buffer A for 1 h. Immunoreactivity
was determined using the enhanced chemiluminescence (ECL) reaction (Amersham, Buckinghamshire, UK).
RNA Isolation, RT-PCR, and Southern Blot
Analysis
The temporal profiles of transcript abundance for the storage protein in ovaries from 6-,
8-, and 10-day-old pupae and adult were examined using RT-PCR followed by Southern blotting
with specific radioactive probes. Total RNA was
isolated from ovary and other organs by lysis
buffer, spin column, and wash buffer according
to the protocol recommended by the manufacturer
(Qiagen Inc. Chatsworth, CA). Five-microgram
aliquots of each RNA preparation were reversetranscribed by the Superscript II reverse transcriptase (Gibco BRL) using Oligo(dT)12–18 primers
(Gibco BRL) in a reaction volume of 25 µl. The
reverse transcription products were diluted to 50
µl with TE buffer (10 mM Tris–HCl, 1 mM EDTA,
pH 8.0) and stored at –20°C as a cDNA pool until
use. For developmental profile analysis, 0.5 µl
ovary equivalents from the cDNA pools were used
as PCR templates. A 539-bp specific fragment was
amplified with the forward primer, 5′-CTTCGGCCAGCGTCGTCAA-3′, and the reverse primer,
5′-TGCGGCTCTGGTCATTTTCATC-3′. Thermal
cycling conditions were as follows: the reaction
was incubated at 94°C for 5 min followed by 35
cycles at 94°C for 30 s, 55°C for 30 s, and 72°C
for 60 s. After PCR amplification, 10 µl each of
the total 50 µl reaction was fractionated on a 1.2%
agarose gel and transferred onto a Hybond N+
membrane (Amersham) under alkaline conditions.
Radioactive cDNA probes were prepared from 25
ng HcSP-1 cDNA (EcoRV digested 1.5-kb cDNA
fragment). The HcSP-1 cDNA fragments were labeled by a random-primer DNA labeling system
(Gibco BRL) to incorporate [α-32P]dATP (NEN).
The membrane was prehybridized with 1.5 ×
SSPE (20 × SSPE: 3 M NaCl, 0.2 M NaH2PO4,
0.02 M EDTA), 7% sodium dodecylsulfate (SDS),
Expression of Storage Protein in H. cunea Ovary
10% polyethylene glycol (PEG), 0.25 mg/ml bovine serum albumin (BSA), and 0.1 mg/ml denatured salmon sperm DNA (Gibco BRL) for 4 h at
65°C. Hybridization was performed for 18 h at
65°C in the prehybridization buffer with 5 × 105
cpm/ml 32P-labelled probes. The membrane was
washed twice at 65°C in 2 × SSC (10 × SSC: 1.5
M NaCl, 0.15 M sodium citrate), 0.1% SDS for 15
min, twice in 0.1 × SSC, 0.1% SDS for 15 min,
and then autoradiographed. Southern blotting of
the PCR-amplified total RNA preparations without reverse transcription did not result in any appreciable signals, indicating that contamination
of RNA preparations with genomic DNA fragments was negligible (not shown).
Northern Blotting
Ten and thirty micrograms of total RNA from
fat body and other tissues, respectively, were denatured and subjected to electrophoresis in a 1.2%
agarose gel containing 2.2 M formaldehyde. Following electrophoresis, gels were rinsed in 10 ×
SSC and transferred to nitrocellulose (Schleicher
and Schuell) in 10 × SSC. Blots were prehybridized with 1.5 × SSPE, 7% SDS, 10% PEG, 0.1
mg/ml sonicated denatured salmon sperm DNA,
and 0.25mg/ml BSA for 4 h at 65°. Hybridization
was performed for 18 h at 65°C in the prehybridization buffer with 5 × 105 cpm/ml 32P-labelled probes prepared according to the method
of random priming (Feinberg and Vogelstein,
1983). The filter was washed twice with 1 × SSC,
0.1% SDS at 65°C for 15 min, and twice subsequently for 15 min with 0.1 × SSC and 0.1% SDS
at 65°C before exposure to X-ray film at –70°C.
In Situ Hybridization
In situ hybridization was performed based
on the protocol reported by Petraglia et al. (1992).
Fixed ovary tissues were sectioned at 10 µm with
a cryostat at –20°C. The tissue sections were hybridized with a 35S-labelled HcSP-1 cRNA probe
for 1 day at 60°C in a humid chamber. Sections
were incubated with RNase A to exclude the possibility of mis-matched sequences. After washing
and drying, sections were apposed to NTB2 emulsion (Eastman Kodak) for 2 weeks. The signals
were observed under a microscope. The control
section was pretreated with RNase and showed
no signal.
113
Immunocytochemistry
Immunocytochemistry was performed as previously described (Miller et al., 1990). Ovaries
were fixed for 3 h in a mixture of 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium
phosphate buffer (pH 7.5) containing 0.15 mM
CaCl2 and 0.45 M sucrose (FM). Fixation was
completed by incubating the ovaries overnight in
pH 10.4 FM without glutaraldehyde. The tissues
were rinsed in 0.1 M sodium phosphate buffer
(pH 7.5), dehydrated in a graded ethanol series
(up to 95%), and embedded in Lowicryl K4M
(Polysciences, Warrington, PA). Ultrathin sections
mounted on formvar-coated nickel grids were
treated for 10 min with Tris-buffered saline (TBS;
0.02 M Tris-HCl, pH 7.5 containing 0.5 M NaCl).
The sections were etched with 3% H2O2 in double
distilled H2O for 5 min and then blocked with 3%
BSA in TBS for 30 min. The sections were incubated with 1:200 diluted antiserum against HcSP1 in TBS plus 1% Tween-20 (TBS/Tween) for 60
min. Following a wash in TBS/Tween 3 times for
15 min with gentle agitation, the sections were
exposed to gold-goat anti-rabbit IgG (20 nm:
Zymed, San Francisco, CA) diluted 1:5 in TBS/
Tween for 60 min. The grids were washed with
0.3% BSA in TBS, and the sections poststained
with 2% uranyl acetate followed by 0.2% Reynolds’
lead citrate (Reynolds, 1963). Ultrastructural examination was performed on a Hitachi H-600
transmission electron microscope operating at
75kV. Controls included: (1) substitution of preimmune serum for primary antiserum; (2) use of
secondary antibody in the absence of treatment
with primary antibody; and (3) treatment of thin
sections with colloidal gold alone.
RESULTS
Accumulation of SP-1 in the Ovary
Ovary extracts from female pupae and adult
were compared by SDS-PAGE and Coomassie blue
staining to determine quantitative changes in the
concentration of HcSP-1 (Fig. 1). HcSP-1 was detected in small amounts in the ovaries in 6-dayold pupae, and had accumulated in large amounts
in pupae by day 10. The accumulation of HcSP-1
in the ovary toward the end of the pupal stage
coincides with a slight decline in the hemolymph,
suggesting a redistribution of SP-1 from hemo-
114
Cheon et al.
Locke, 1987a), epidermis (Palli and Locke, 1987b),
and testis (Miller et al., 1990) also synthesize and
export arylphorin. We, therefore, decided to determine if the HcSP-1 gene is expressed in the
ovary. Accordingly, ovaries were dissected from
day-6 pupae to adult and were thoroughly cleaned
of all visible adhering fat body. Total RNA from
fat body, midgut, ovary, and testis were hybridized with HcSP-1 cDNA probe. Northern blot
analyses indicated that no transcript was present
in midgut, but that a substantial amount was
present in fat body, and a lesser amount in the
gonad (Fig. 2A). The HcSP-1 probe revealed that
the same 2.5-kb transcript accumulated in the
ovary as in the fat body.
Developmental Pattern of the HcSP-1
Transcript in Ovary and Fat Body
Fig. 1. SDS-PAGE of ovary extracts of female H. cunea.
Each lane was loaded with 20 µg of ovary proteins. Bottom: Immunoblot analysis probed with HcSP-1 antiserum.
Molecular weight standards (× 103) and three yolk proteins
are marked on the right (YP1 from Lee et al., 1988; YP2
from Lee et al., 1995; YP3 from Lee and Kim, 1991). HcSP1 is marked on the left. P6-P10, 6-, 8-, and 10-day-old pupae;
A0, newly eclosed adult.
lymph into the ovary or other adult tissues (Seo
et al., 1998). However, there was an abrupt decrease in HcSP-1 content in the ovaries after
adult emergence (Fig. 1). In a double immunodiffusion test, antiserum against HcSP-1 showed
very weak precipitation lines with an extract of
adult ovaries (Lee et al., 1990). HcSP-1 is probably present in the ovaries of pupae less than 6
days old, but they were too small to sample for
biochemical analysis.
It is noteworthy that ovaries selectively accumulated several polypeptides, including three
kinds of yolk proteins (Fig. 1). Yolk proteins were
accumulated in larger quantities by the end of
the pupal stage and in adults.
Distribution of SP-1 Transcript Among Organs
Ray et al. (1987) argued that the arylphorin
gene is expressed exclusively in fat body cells of
wax moth larvae. However, in some insects, other
larval cell types such as the midgut (Palli and
The developmental profile of the HcSP-1
transcript in H. cunea ovary could not be determined by Northern blot, because the HcSP-1 transcript was expressed at low levels. Therefore, we
performed RT-PCR analysis using HcSP-1 genespecific primers (Fig. 2B). 0.6-kb PCR products
were detected in ovary from all developmental
stages tested except adults. Adult ovaries appear
to be in the equivalent of a post-vitellogenic stage,
when follicles show no remaining nurse cell cap
indicating the termination of vitellogenesis (Zimowska et al., 1991).
The developmental profiles of HcSP-1 and
HcSP-2 transcripts, as determined by Northern
blot, were markedly different in female fat body
(Fig. 2C). In general, the relative abundance of
HcSP-1 transcript in the female fat body is considerably greater than that of the HcSP-2 transcript. The quantity of HcSP-1 transcript was
greatest in late 7th instar larvae, at a time
when HcSP-2 transcript was present only at a
trace level. HcSP-1 levels gradually declined
thereafter and remained low throughout pupal
development (Fig. 2C, SP-1). In contrast, the
HcSP-2 transcript was present at trace levels
before the prepupal stage, reached its peak during prepupal and early pupal stages, and then
drastically declined after 2-day-old pupae (Fig.
2C, SP-2). In contrast, no big differences in developmental profiles between the two storage
protein transcripts were observed in males
(data not shown).
Expression of Storage Protein in H. cunea Ovary
115
Fig. 2. Presence of HcSP-1 transcript among tissue types
revealed by Northern blot (A). Developmental accumulation
of the HcSP-1 transcript from ovary (B) and fat body (C)
revealed by RT-PCR (B) and Northern blot (C), respectively.
Total RNA from fat body (10 µg) and other tissues (30 µg)
were separated and probed. For further details see Materials and Methods. Fb, fat body; Mg, midgut; Ov, ovary; Te,
testis; 7E-L, early, middle, and late 7th instar larvae; PP,
prepupae; P0-P10, pupae at days 0–10 of development; A0,
newly eclosed adult.
In Situ Hybridization and Localization of the
HcSP-1 Transcript in the Ovary
between the follicular epithelial cells surrounding the oocyte, but true patency was not yet evident. At this stage, a fair amount of HcSP-1 was
associated with the tunica propria and a few gold
particles labeling SP-1 were present in structures
that seemed to be the Golgi complex (Fig. 4A),
but the labeling was almost at background level.
The small yolk spheres containing HcSP-1 were
detectable at the periphery of the oocyte (Fig. 4B),
while the section labeled with antisera to YP2
showed a few gold particles in yolk spheres (Fig.
4C). Two antisera against YP1 and YP3 showed
no labeling in the same section (data not shown).
During the vitellogenic stage, follicular epithelial cells achieve patency, with large interfollicular spaces developing at the apical surfaces
(Seo et al., 1998). Once detected in the perioocytic
space, HcSP-1 was also detected within the transitional yolk bodies. Smaller transitional yolk bodies appeared to fuse with one another to form
The precise cellular localization of HcSP-1
mRNA in the ovary was examined by in situ hybridization. HcSP-1 specific labeling was observed in the nurse cells (Fig. 3A) and the
follicular epithelial cells (Fig. 3C–E) surrounding the oocyte, but not in the cytoplasm of the
oocyte where the protein granules were packed
(Fig. 4). In most follicles from the adult stage
(Fig. 3F), the follicular epithelial cells were engaged in prechoriogenic activity and the nurse
cell cap had disintegrated (data not shown). At
this stage, most follicular epithelial cells showed
no hybridization signal, but a few cells still gave
weak signals (Fig. 3F).
Immunocytochemistry
Just before vitellogenic (provitellogenic) ovaries, small interfollicular spaces were observed
116
Cheon et al.
Figure 3.
Expression of Storage Protein in H. cunea Ovary
117
Fig. 4. The localization of HcSP-1 associated the early yolk
body formation in provitellogenic ovary of H. cunea. Note
that HcSP-1 label is present on the tunica propria (Tp) in
considerable amounts. The follicular epithelium has small
intercellular spaces between adjacent plasma membranes
(A). Early yolk bodies containing HcSP-1 are observed
around the periphery of the oocyte (B). Immunogold labeling for HcSP-1 (B) is stronger than the minimal labeling for
YP2 (C). Magnification bar = 0.5 µm.
large mature yolk bodies that were heavily labeled with colloidal gold (Fig. 5).
hemolymph, and sequestered again by the fat
body at the end of the feeding period (Kim et al.,
1989). Storage protein can be used during metamorphosis and oogenesis as a source of amino acids (Tojo et al., 1980; Karpells et al., 1990). In B.
mori, the transcript and the protein are expressed
in both male and female fat bodies during the
fourth instar, but they are restricted to females
during the fifth instar (Izumi et al., 1988; Sakurai
et al., 1988). Ogawa and Tojo (1981) suggested
that the female-specific SP-1 of B. mori may supply amino acids for the formation of the egg yolk
protein precursor vitellogenin (Engelmann, 1979).
The relatively high methionine content of the B.
mori SP-1 may be metabolized to cystine for
chorion formation (Inokuchi, 1972; Sumioka and
DISCUSSION
SP-1 of Hyphantria cunea is a storage protein produced by the fat body, exported to the
Fig. 3. Localization of HcSP-1 mRNA in the ovarian follicle by in situ hybridization using a 35S-end labeled cRNA
fragment of HcSP-1. Hybridization signals (black dots) indicative of HcSP-1 mRNA are visible in the nurse cells
(A,C,E) and follicular epithelial cells (A,C,D,E,F). No signal
was identified in the control section pretreated with RNase
(B). A: Previtellogenic follicle. C: Early vitellogenic follicle.
D: Cross-section of oocyte in early vitellogenic stage, E,F:
Post-vitellogenic follicle. Nc, nurse cell; Fc, follicular epithelial cell; Oc, oocyte.
118
Cheon et al.
Fig. 5. The HcSP-1 internalization pathway in the H. cunea
oocyte during the vitellogenic stage. Once in the perioocytic
space (Ps), the storage protein (dark spots) is incorporated
into the transitional yolk body (*). Subsequently, the transi-
tional yolk body, which contains HcSP-1, is transformed into
a mature yolk body (4). Numbers 1–4 show the transition
from immature yolk body to mature yolk body. Ld, lipid droplet. Scale bar = 1 µm.
Yoshitake, 1974). SP-1 in Hyphantria cunea has
a somewhat higher content of methionine (6.0%),
indicating the possibility of contribution to egg
formation.
Larval hemolymph protein synthesis in lepidopterans occurs primarily, but not exclusively,
in larval fat body cells (Kanost et al., 1990). In
this report, we present evidence for storage protein transcription in the ovary of Hyphantria
cunea. However, the level of the HcSP-1 transcript
was much lower in the ovary than in fat body
cells. The size of the HcSP-1 transcript appeared
to be identical in the two cell types (Fig. 2A), suggesting that there are no tissue-specific differences associated with HcSP-1 RNA processing.
Presence in the follicle of a protein transcript considered up to now to be a storage protein is rather
unexpected. The persistence of the HcSP-1 transcript in the ovary of late pupae is similar to a
phenomenon in Heliothis virescens where the testes cells continue to transcribe the arylphorin
gene in the pharate adult and adult stages (Miller
et al., 1990). During the early stages of follicle
growth, HcSP-1 transcript is expected to present
in follicles, but it is almost impossible to identify
it by biochemical methods because of the minuteness of the follicles.
Just before the vitellogenic stage, HcSP-1 is
already present in small yolk bodies around periphery of the oocyte. This phenomenon is very
similar to the incorporation of YP2 into the early
yolk bodies during the provitellogenic stage of
Plodia interpunctella (Zimowska et al., 1994), in
which the interfollicular spaces are present but
are not yet truly patent (Zimowska et al., 1994).
The labeled HcSP-1 in the early yolk bodies seems
to be originated from follicular epithelial cells
rather than from nurse cells. Though HcSP-1
transcripts are observed in the nurse cell of H.
cunea ovary, it is very difficult to have evidence
for the transport of HcSP-1 mRNA or protein itself from nurse cells to oocyte. Even HcSP-1 was
distributed randomly in the cytoplasm of nurse
cells, it was not possible to determine whether
Expression of Storage Protein in H. cunea Ovary
the HcSP-1 in the nurse cells was from hemolymph or from de novo synthesis (Seo et al., 1998).
Immunocytochemical method was not sufficient
to make any identification of biosynthetic activity in the nurse cells.
Though the labeling of HcSP-1 in early
yolk bodies is not intense, its contribution to
the early yolk bodies is much greater than that
of yolk protein.
The HcSP-1 transcript is found in 6- through
10-day-old pupae, so HcSP-1 synthesis is presumably continuous from the previtellogenic to vitellogenic stage of H. cunea. Since t he immunogold
labeling of three Yps is not above background levels in early yolk bodies, their contribution to early
yolk bodies is apparently rather small.
During the vitellogenic stage, large protein
granules densely labeled with HcSP-1 were observed throughout the oocyte. At this stage, the
follicular epithelial cells exhibit patency with
large interfollicular spaces and no cell contacts
at the apical surfaces, permitting transport of
material in the hemolymph to the surface of the
oocyte (Seo et al., 1998). We observed that the H.
cunea storage protein is actively taken up into
the protein granules by the developing oocyte and
serves as a yolk protein during egg formation (Seo
et al., 1998). Though the incorporation of ovarian
HcSP-1 into yolk bodies is smaller than that of
the hemolymphatic HcSP-1, this transcript in follicles might contribute to the formation of early
yolk bodies before the vitellogenic stage.
ACKNOWLEDGMENTS
We are very grateful to Dr. Thomas W.
Sappington (USDA-ARS, IFNRRU) for a critical reading of the manuscript and to Dr. Seol
Kwang Youl (National Sericulture and Entomology Research Institute, RDA) for the fall webworm supply.
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expressions, local, cunea, distributions, ovary, protein, storage, hyphantria
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